Screening for porcine endogenous retrovirus - C (PERV-C) in pigs

Screening for porcine endogenous retrovirus - C (PERV-C) in pigs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Screening for porcine endogenous retrovirus - C (PERV-C) in pigs Hina Jhelum, Dusan Kunec, Vasileios Papatsiros, Benedikt Kaufer, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4807994/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Porcine endogenous retrovirus C (PERV-C) is a gammaretrovirus present in the genome of many, but not all pigs. PERV-C is an ecotropic virus, able to infect only pig cells. In contrast, PERV-A and PERV-B are present in all pigs, they can infect cells of numerous host species including humans, and therefore pose a risk for xenotransplantation using pigs as donor animals. Intriguingly, PERV-C can recombine with PERV-A and the resulting PERV-A/C recombinants can also infect human cells and replicate to higher titers compared to the paternal PERV-A. Therefore, only PERV-C-free animals should be used for xenotransplantation. When screening wild boars and German landrace pigs for PERV-C, proviral variants were detected, and to detect these variants, new PCR methods were developed. Furthermore, PERVs are known to be active in living pigs as demonstrated by recombination and by different copy numbers in various organs and different parts of an organ. Copy numbers also increased with the age of the animals. This data was obtained by PCR using primers amplifying the highly conserved polymerase sequence which is identical in PERV-A, PERV-B and PERV-C. Here we screened indigenous Greek black pigs, Auckland Island pigs and German slaughterhouse pigs for the presence of PERV-C, using different PCR-based methods and optimized these detection methods. Auckland Island pigs were PERV-C-negative and represent the optimal donor pigs after genetic modifications. porcine endogenous retroviruses (PERVs) PERV-C xenotransplantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs. PERV-A and PERV-B are present in all pigs, whereas PERV-C is not present in all, but most pigs [ 1 ]. This correlates with the fact that PERV-C is phylogenetically younger compared to PERV-A and PERV-B [ 2 – 4 ]. Since PERV-A and PERV-B can infect human cells (human-tropic virus), they pose a risk for xenotransplantation using pig cells, tissues or organs [ 5 ]. PERV-A and PERV-B infect also cells from numerous other species (polytropic viruses). PERV-C infects only pig cells (ecotropic virus) and therefore, theoretically does not pose a risk for xenotransplantation. However, PERV-C can recombine with PERV-A, acquiring the receptor binding site for the receptor on cells of humans and other species, enabling the virus to efficiently replicate in human cells [ 6 , 7 ]. In addition, the replication rate of the PERV-A/C recombinants is higher compared to paternal PERV-A [ 8 , 9 ]. Therefore, it is recommended to use PERV-C-free animals as donor pigs for xenotransplantation [ 10 ]. To ensure that the animals are PERV-C-free, sensitive and specific conventional and real-time PCRs were developed [ 1 , 11 – 14 ]. When we screened wild boars and German landrace pigs for PERV-C using the commonly used PCR developed by Takeuchi et al. [ 1 ], we observed mutations in the primer binding sites giving a false negative result in some pigs. Therefore, we developed new PCRs to detect PERV-C variants in such animals [ 15 ]. It is well known that PERVs are active in living pigs [ 16 ]. This was demonstrated by different copy numbers of integrated proviruses in different organs and in different parts of one organ and by an increase of the copy number by age [ 17 – 21 ]. The lowest level of PERV-A and PERV-B proviral DNA was shown in muscles [ 20 ]. These investigations were performed using primer and probes binding to a highly conserved region in PERV's polymerase sequence (pol), not discriminating between PERV-A, PERV-B and PERV-C. Furthermore, there is good evidence that PERV-C is active in living pigs: Recombinants with PERV-A were found in somatic cells but such PERV-A/C were never found in the germ line [ 6 , 7 ]. When we screened indigenous Greek black pigs for PERV-C using another PCR, designated PCR4 [ 12 ], 11 of 21 animals were found positive [ 22 ]. None of the PERV-C-positive animals were positive for PERV-A/C. Indigenous Greek black pigs live in mountainous areas, they are resistant to weather conditions and to diseases even though numerous viruses including porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpes virus − 3 (PLHV-3), and porcine circovirus 3 (PCV3) were found in most of the animals [ 22 ]. Auckland Island pigs are ideal organ donors because of the optimal organ size for humans [ 23 ]. Therefore, there is no need for a knock-out of the porcine growth factor receptor as done with other pig breeds [ 24 ]. The second important advantage is the low number of porcine microorganisms. These animals lived for a hundred years on the remote Auckland Island, they were thoroughly studied in New Zealand [ 25 – 27 ] and used as donors of pig islets in the first clinical trials treating diabetes patients in New Zealand and Argentina. In these trials no transmission of porcine viruses, including PERV to the patients was observed [ 28 , 29 ]. Since more than 50% of the animals in New Zealand were PERV-C positive, we selected PERV-C negative animals from which cells were delivered to Germany and piglets were obtained by somatic cell nuclear transfer (SCNT) [ 21 ]. Genetic diversity, growth and heart function of these Auckland Island pigs at the Center for Innovative Medical Models (CiMM) in Munich as well as the absence of PERV-C demonstrate that they are excellent donor animals [ 23 ]. To evaluate our methods developed for the detection of numerous porcine viruses we recently screened German slaughterhouse pigs [ 30 ]. Nine of ten animals were PERV-C positive and therefore we included them in this study using different PCR approaches and sequencing methods to analyze PERV-C in indigenous Greek black pigs, Auckland Island pigs and pigs from a German slaughterhouse. Results PCR methods for the detection of PERV-C To screen pigs for the presence of PERV-C, a PCR developed by Takeuchi et al. [1] is commonly used, designated here PCR1 (Figure 1A). Later Dieckhoff et al. developed a new PCR [31], designated PCR4 by Kaulitz et al. [12] using primers based on a PERV-C sequence (accession number EU440732.1, [32]). In addition, PCR2, PCR3 and a real-time PCR were developed [12]. The PCR1 detected up to 1.1x10 3 molecules of a PERV-C plasmid, and the real-time PCR performed as duplex PCR simultaneously detecting PERV-C and porcine cyclophilin detected 100 copies/reaction PERV-C [12]. PCR1, PCR2 and PCR3 detected equally good PERV-C in German landrace pigs [12]. Later, when we detected new variants of PERV-C in German wild boars, we added two new PCRs, PCR6 and PCR7 [15]. All primer and probe sequences were found in the PERV-C reference sequence (accession number AM229312, [33]), but not in the PERV-A reference sequence (accession number AY288779.1, [34]) (Table 1). In this manuscript, the real-time PCR was performed as a duplex real-time PCR detecting in parallel porcine GAPDH and the sensitivity was 10 copies PERV-C /100ng DNA [21]. To evaluate the specificity of the developed PCRs and to determine the presence or absence of PERV-C, we screened indigenous Greek black pigs, Auckland Island pigs and pigs from a German slaughterhouse. In this manuscript, three new PCRs, PCR8, PCR9 and PCR10, were added, which partially were designed to obtain larger amplicons for sequencing purposes (Figure 1A). To stress it again, all primer and probe sequences were found in our PERV-C reference sequence, but not in the PERV-A reference sequence (Table 1). Detection of PERV-C in indigenous Greek black pigs Initially, 21 indigenous Greek black pigs were screened for PERV-C using PCR4. This screening revealed that 11 of 21 animals (52.4%) were positive [22]. To further investigate the presence of PERV-C in these animals, four animals from farm 1 were analyzed using PCR1. Screening with this PCR1 reconfirmed the presence of PERV-C in all pigs, except pig 3 (Figure 2A). The same result had been obtained for PCR4 [22]. The absence of PERV-C specific bands in PCR1 and PCR4 for animal 3 suggests that PERV-C is either absent in this animal or that the primer-binding sites are mutated, making them unrecognizable by one or both primers. To test these possibilities, PCR5 and PCR6 were performed (Figure 2A). Testing DNA from the liver and spleen of animals 2 and 4 with PCR5 and PCR6 produced strong amplicons identical in size to that of the positive control. No amplification was observed with PCR 5 and PCR6 when DNA from both tissues of animal 3 was used. Interestingly, no amplification was detected in animal 1 with PCR 5, and only a faint band was observed with PCR6 using DNA isolated from the spleen, but not from the liver. Given the importance of this PCR6 result, the PCR was repeated several times to ensure reproducibility, and the results of two experiments are presented in Figure 2A. The difference between liver and spleen suggests that there is a mutation in one or both primer binding sites in the provirus in the liver, but not in the spleen, indicating different proviruses in these organs. To obtain a larger amplicon for sequencing purposes, PCR8 was performed, using the forward primer of PCR6 and the reverse primer of PCR5. To our surprise, DNA extracted from all tissues from all four animals yielded amplicons, also from pig 3. Most interestingly, the amplicons from the liver and spleen of animal 3 were larger compared to the amplicons from animals 1, 2 and 4, indicating a potential insertion (Figure 2A). PCR7 (the combination of the forward primer of PCR5 and the reverse primer of the real-time PCR) and PCR9 (using the forward primer of the real-time PCR and the reverse primer of PCR5) were also positive for all four animals. Detection of PERV-C in other pigs When the DNA from the German slaughterhouse pigs were screened using a real-time PCR [12], all animals were also positive (Table 2). In contrast, the Auckland Island pigs and the PK15 cells were negative in the real-time PCR (Table 2). The Auckland Island pigs [21] and the pig cell line PK15 [35] are well known to be PERV-C negative. They were also PERV-C negative in this study in PCR4 (Figure 2B) and PCR8, which was using the forward primer of PCR6 and the reverse primer of PCR5 in order to obtain extended amplicons (Figure 2C, 2D). Sequence analysis of PERV-C from indigenous Greek black pigs Sanger sequencing of the PCR8 amplicon from pig 4 resulted in a high-quality sequence and an alignment of this sequence with a PERV-C reference sequence AM229312 [33] revealed that the sequences were nearly identical (Figure S1). However, since the Sanger sequencing of the other amplicons did not produce satisfactory results, possibly due to the simultaneous amplification of different proviruses present in the pig genome with differences in the sequence, all amplicons were sequenced using the Nanopore method. As expected, putative insertions were detected in the sequence from animal 3 when compared with the sequences from animals 2 and 4 (Figure 1B). The largest insertion disrupts the binding sites of the forward primers of PCR1 and PCR4, while the other disrupts the binding sites of the reverse primers of PCR1 and PCR4. The smallest insertion disrupts the binding site for the forward primer of PCR5. Consequently, none of these three PCRs produced amplicons when DNA from animal 3 was tested (Figure 2A). However, when the large insert and the whole amplicon sequence from animal 3 were analyzed using nucleotide BLAST (nBLAST), a high sequence similarity PERV-A was observed (Figure S2), indicating that the provirus amplified with PCR8 in pig 3 is PERV-A. Based on the nanopore sequences of the PCR8 amplicons from animals 3 and 4 a dendrogram was built, showing that the amplicon from animals 4 is closer to the PERV-C reference sequence and the amplicon from animal 3 is closer to the PERV-A reference sequence (Figure 3). Comparative analysis of the primer binding sites The sequences obtained by nanopore sequencing of the 692bp amplicons obtained by PCR8 were used to analyze the primer binding sites of PCR1, PCR4, PCR5, PCR6, PCR7, PCR8 and PCR 9 as well as the binding sites for primers and probes of the real-time PCR (Table 3). The nanopore sequences were of high quality and the sequences of the amplicons derived from the liver DNA and the spleen DNA were 100% identical. When the sequences of the primers and the corresponding sequences in the amplicons were compared, it was confirmed that the forward and reverse primers of PCR 1 and PCR4 are highly specific, because in the PERV-A sequence (amplicon of PCR8 from animal 4) the primer binding sites were disrupted by larger sequences absent in PERV-C. The sequences of the PCR5 primer binding sites in pig 3 had mutations, in the case of the forward primer an insertion of sequence “AAC” disrupted the primer binding site (Table 3), explaining the absence of amplicons when PCR5 was performed (Figure 2A). The real-time PCR is not PERV-C-specific In addition, a real-time PCR as described by Kaulitz et al. [12] was performed. Primers and the probe had been designed using sequences found in the PERV-C reference genome, but not in the PERV-A reference genome (Table 1). An identical copy number in the spleen and liver of animals 2 and 4 (21 Ct) was found, but a lower copy number in both tissues of animals 1 and 3 (26 Ct) (Table 2). Interestingly, no significant differences in the PERV copy numbers were observed between the liver and spleen of all animals. When the real-time PCR was used to screen the four indigenous Greek black pigs from farm 1, all four animals including pig 3, which was found PERV-C negative on the basis of PCR1, PCR4, PCR5 and PCR6 (Figure 2A) were found to be positive in the real-time PCR (Table 2). In contrast, the Auckland Island pigs, which were also found PERV-C negative using the mentioned PCRs, were negative in the real-time PCR (Table 2). All ten German slaughterhouse pigs were positive in the real-time PCR, including pig 3, which was found to be PERV-C negative using PCR1 and PCR4 [30] (Table 2). Sequences obtained by PCR10 In order to obtain an even larger sequence for extended sequencing, PCR10 was developed using the forward primer of PCR6 and a new primer downstream of the reverse primer of PCR5 (Table 1). PCR10 was positive for all tissues of all four indigenous Greek black pigs (Figure 2A), including pig 3. The amplicons from the liver and spleen of pig 3 and pig 4 were sequenced by the nanopore method and were compared with the PERV-C reference gene (Figure S3). There were significant differences, indicating that different proviruses had been amplified. When these sequences were analyzed using nBLAST, the sequences from pig 3 corresponded to PERV-B and that from pig 4 to PERV-A, indicating that the primer amplified non-PERV-C proviruses. PCR10 was also positive for the German slaughterhouse pig 3, the sequence of the amplicon was also identified as PERV-A. Therefore, a comparison of the primers of PCR1, PCR4, PCR5 and the primers and the probe of the real-time PCR with the corresponding binding sites in the sequences was not conductive (Figure S3). Interestingly, PCR10 was negative for an Auckland Island pig and positive for PK15 cells (Figure 2E). Screening for PERV-A/C To detect PERV-A/C recombinants, primer pairs were used which had been previously validated for identification of different types of recombinants [7, 36-38]. Despite comprehensive screening with these primer pairs, no PERV-A/C sequences could be detected in the genome of indigenous Greek black pigs (Figure 4). Discussion Here we analyzed for the first time integrated PERV-C proviruses in indigenous Greek black pigs, German landrace pigs and Auckland Island pigs and confirmed that Auckland Island pigs from the CiMM in Munich are PERV-C-negative. Furthermore, we tested several PCR assays to determine whether they correctly detect PERV-C. Eleven of 21 indigenous Greek black pigs in four different farms in Greece were found to carry PERV-C proviruses using PCR4. This confirms that not all pigs carry PERV-C proviruses as reported in various previous publications. For example, 113 of 348 (30%) Chinese miniature pigs [ 39 ], 176 of 181 (97%) German transgenic and non-transgenic pigs [ 31 ], and 25 of 98 (25.5%) US farm animals [ 11 ] carry PERV-C. The absence of PERV-C in several animals correlates with the fact that PERV-C is phylogenetically younger compared to PERV-A and PERV-B, which are present in all pigs [ 2 – 4 ]. Three of four indigenous Greek black pigs in farm 1 were found to be PERV-C-positive using both PCR1 and PCR4 [ 22 ]. Nine out of ten German slaughterhouse pigs were positive using these PCRs, while the porcine cell line PK15 was negative, as expected. Most importantly, all Auckland Island pigs from the CiMM in Munich, which were derived from cell lines from selected, PERV-C-negative, animals [ 21 ] were confirmed to be PERV-C negative. Although PERV-C does not infect human cells, it can recombine with the ubiquitous PERV-A present in all pig genomes. These PERV-A/C recombinants are able to infect human cells and replicate to higher titers compared to the paternal PERV-A [ 8 , 9 ]. Therefore, only PERV-C-negative animals should be used for preclinical and clinical xenotransplantation [ 10 ]. Sequencing of the amplicon produced by PCR8 using the Sanger sequencing showed that the sequence found in pigs 2 and 4 from farm 1 is nearly identical to the PERV-C reference sequence AM229312 [ 33 ]. Since the Sanger sequencing of the amplicons obtained by PCR8 using the DNA from pig 3 was not successful, possibly due to the presence of amplicons generated from different proviruses in the genome, all amplicons were sequenced using the Nanopore method. This sequencing showed insertions in the PERV sequences of animal 3. This is in agreement with the finding that the amplicons obtained when performing PCR8 using DNA from animal 3 was larger compared with the amplicons of the other three pigs. As a matter of fact, three putative insertions were found when the amplicons of PCR8 were sequenced and compared. These insertions disrupted the primer binding sites of PCR1, PCR4 and PCR5, explaining the negative results. The sequence “ATTGGCAACAGCGGGTACAAAAAGAT”, which is PERV-A specific, is absent in the PERV-C sequence of animals 1 and 4, suggesting it may be a sequence derived from PERV-A that is inserted in the PERV-C sequence in animal 3. However, sequence analysis of the sequence of the amplicon from pig 3 indicated, that the entire sequence corresponded to PERV-A. Therefore, the so-called insertions are not insertions into PERV-C. Insertions of shorter and larger PERV-A sequences into PERV-C sequences had been observed when recombinant PERV-A/C isolated from peripheral blood mononuclear cells (PBMCs) were analyzed [ 36 , 37 ], however such PERV-A/C sequences were never found in the germ line, but only in somatic cells, indicating that the recombinants are the result of recombination and de novo integration [ 6 ]. Of interest is the result of PCR8. The forward primer of PCR8 is identical to the forward primer of PCR6 and the reverse primer of PCR8 is identical to the reverse primer of PCR5. PCR5 and PCR6 did not give an amplicon when DNA from the Auckland Island pigs, PK15 and Greek animal 3 were tested. PCR5 and PCR6 were only positive with DNA from animals 1, 2 and 4 which were also positive with other PERV-C-specific PCRs such as PCR1 and PCR4 (Fig. 2 ). The real-time PCR also gave equivocal results (Table 2). Whereas the real-time PCR is negative in the case of Auckland Island pigs, it was unexpectedly positive in the case of animal 3, which was tested PERV-C negative by PCR1, PCR4, PCR5 and PCR6. The fact that PCR6 gave a positive result when analyzing DNA from the spleen of animal 1, but not from the liver of animal 1 is of interest. The negative result with the liver DNA indicates that the sequence of one or both primer binding sites is absent or mutated. This data indicates that different proviruses are integrated in the DNA of two tissues, the spleen and liver, of a single animal. The methods characterized here are required to identify PERV-C-negative pigs for xenotransplantation to prevent a recombination with PERV-A. However, we have to consider, that in addition to recombination with PERV-A, resulting in PERV-A/C recombinants, there are also other mechanisms able to transform the ecotropic PERV-C into a virus which can infect human cells. Gemeniano et al. [ 32 ] have shown that the C-terminal proline-rich region (PRR) in the envelope protein region is required in addition to the putative variable region A (VRA) and VRB for binding to cells. They showed that mutations in the PRR allowed infection of human cells. They also showed that the cellular receptor molecule binding the PERV-C with the mutated C-terminal region of the surface envelope protein is different from the receptor molecule when PERV-A is binding and infecting human cells. In conclusion, screening pigs for the presence of PERV-C is easy but requires some precautions. Our experience shows that PCR1 and PCR4 always gave clear results, showing that indigenous Greek black pig 3, all Auckland Island pigs, German slaughterhouse pig 3 and PK15 cells are PERV-C negative. PCR5 and PCR6 gave false-negative results and all others including the real-time PCR gave false-positive results. On the other hand, PCR1, PCR4, PCR5, PCR6, PCR8 and the real-time PCR were negative in the case of the Auckland Island pigs. Therefore, PCR1 and PCR4 should be used for routine screening for PERV-C and all other PCRs may help to characterize the sequences in the receptor binding domain of PERV. Materials and Methods Animals and tissues Liver and spleen tissues were obtained from 4 animals in Farm 1 located near Drama, North Greece [ 22 ]. These animals had been screened for different porcine viruses using real-time PCR (PCMV/PRV, PCV2, PCV3, PCV4, PLHV-1, PLHV-2 and PLHV-3), as well as real-time RT-PCR (HEV genotype 3). Blood samples from the Auckland Island pigs were obtained from Prof. Eckhard Wolf and Dr. Barbara Keßler, Chair for Molecular Animal Breeding and Biotechnology and CiMM, Munich. The three female animals were born in June 2023, they are the F1 generation of the animals born in April 2019 and were obtained by somatic cell nuclear transfer (SCNT) using PERV-C-negative kidney cells [ 21 ]. PBMCs were isolated by gradient centrifugation as described [ 40 ]. Spleen and liver samples from German pigs were obtained from a slaughterhouse near Berlin, their age was 6 months. PK15 (ACC 640) cells were ordered from the Leibniz Institute DSMZ German Collection of Microorganisms and Cell lines, Braunschweig, Germany. Using droplet digital PCR (ddPCR) 55 (52.5–60.1) PERV copies were detected in these cells using porcine GAPDH as a reference gene or 37 (35.0-39.1) copies using porcine beta actin (ACTB) as a reference [ 17 ]. DNA isolation DNA was isolated from the tissues according to the manufacturer´s instructions using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). DNA concentrations were determined using NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Worcester, MA, USA) or Qubit (Invitrogen, Thermo Fisher Scientific Inc., Worcester, MA, USA). PCR and real-time PCR Conventional PCR was performed to determine the presence of PERV-C using a set of primers (Table 1 ) [ 1 , 7 , 12 , 15 , 41 ]. Part of the reactions were carried out with DreamTaq DNA polymerase (Thermo Fisher Scientific, Worcester, MA, USA) and part with high-fidelity HiFi polymerase (PCRBiosystems, London, UK). All reactions were set up with a Biometra TRIO cycler (Analytik Jena, Jena, Germany). Each sample was subjected to an initial denaturation of 10 min at 95°C (one min for HiFi polymerase), followed by 45 amplification cycles of 95°C for 15 s (35 cycles for HiFi polymerase), the annealing temperatures are shown below (30 s for DreamTaq, 15 s for HiFi Polymerase). The corresponding extension times at 72°C for each PCR are shown below. In the case of DreamTaq DNA polymerase, but not HiFi polymerase, a final extension at 72°C for 5 min was added. The annealing temperature for PCR1 was 55°C, for PCR4 60°C, for PCR5, PCR6, PCR7, and PCR8 58°C, for PCR9 55°C and for PCR10 58°C. The extension times used were 9 s for PCR1 and PCR4, 10 s for PCR5, 16 s for PCR6, 5 s for PCR7, 21 s for PCR8, 9 s for PCR9 and 24 s for PCR10 using HiFi polymerase. In addition, a real-time PCR was established using specific primers and probe (Table 1 ) [ 12 , 15 ]. 100 ng DNA and the SensiFAST Probe No-ROX kit (Meridian Bioscience Cincinnati, OH, USA) in a 20 µL reaction volume. The cycling conditions used were initial denaturation 5 minutes at 95°C, followed by 45 amplification cycles of 95°C for 15 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s in a qTOWER3 G qPCR cycler (Analytik Jena, Jena, Germany). A standard curve was produced using the 510 bp amplicon of previously described PCR6 [ 21 ] as template. This PCR was performed as follows: 100 ng of DNA template, PCR buffer I containing MgCl 2 , 0.2 mM dNTPs, and one unit of DreamTaq DNA polymerase (Thermo Fisher Scientific, Worcester, MA, USA). In case of PCRBIO HiFi polymerase, PCRBIO reaction buffer along with one-unit HiFi polymerase was used. As positive control DNA from the PERV-C positive pig (#6249) was used. The sensitivity of the real-time PCR was 10 copies/100ng DNA [ 21 ] Sanger sequencing PCR8 amplicons from pigs 2 and 4 were gel purified using a DNA gel extraction kit (Monarch, New England Biolabs) and sent for sequencing (LGC Genomics, Berlin, Germany). Sequences were analyzed using ApE software. Nanopore sequencing PCR8 amplicons from pig 1, 3 and 4 were gel purified using DNA gel extraction kit (Monarch, New England Biolabs) and sent for sequencing (Plasmidsaurus, Eugene, OR, USA). Sequences were analyzed using SnapGene (San Diego, CA, USA). Sequence alignment and phylogenetic tree Sequence alignment and phylogenetic tree generation were done using Clustal Omega using sequences obtained from nanopore sequencing. ApE plasmid editor was used to align the sequences obtained from Sanger sequencing. Declarations Acknowledgements We thank Prof. Eckhard Wolf and Dr. Barbara Keßler, Chair for Molecular Animal Breeding and Biotechnology and CiMM, Munich, for blood from the Munich Auckland Island pigs. We thank Dr. Kostja Renko, German Federal Institute for Risk Assessment, Unit Strategies for Toxicological Assessments, Department Experimental Toxicology and ZEBET, Berlin, for the help when acquiring the organs from the German slaughterhouse pigs. Author contributions Conceptualization, writing, and supervision was performed by JD. Methodology, validation, formal analysis, and visualization was conducted by HJ, VP, BK, DK and JD. Investigation was conducted by HJ and DK. Funding acquisition was performed by BK and JD. Editing of the manuscript was performed by HJ, VP, BK, DK and JD. Funding This work was supported by Deutsche Forschungsgemeinschaft, TRR 127. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Ethics approval and consent to participate All animal procedures regarding animal care, handling and welfare concerning the indigenous Greek black pigs were approved by the Ethical Research Committee of the University of Thessaly (Ethical Approval Code: 104/16.11.2021). Investigations on the German slaughterhouse animals were performed using meat samples obtained from the slaughterhouse. All work with the Auckland Island pigs was performed with the permission of the local regulatory authority, Regierung von Oberbayern (ROB). Applications were reviewed by the ethics committee according to §15 TSchG (German Animal Welfare Act). Consent to publish Not applicable. Competing interests The authors declare no competing interests References Takeuchi Y, Patience C, Magre S, Weiss RA, Banerjee PT, Le Tissier P, Stoye JP. Host range and interference studies of three classes of pig endogenous retrovirus. J Virol. 1998;72(12):9986–91. Tönjes RR, Niebert M. 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Denner J. How Active Are Porcine Endogenous Retroviruses (PERVs)? Viruses. 2016;8(8):215. Fiebig U, Fischer K, Bähr A, Runge C, Schnieke A, Wolf E, Denner J. Porcine endogenous retroviruses: Quantification of the copy number in cell lines, pig breeds, and organs. Xenotransplantation. 2018;25(4):e12445. Krüger L, Stillfried M, Prinz C, Schröder V, Neubert LK, Denner J. Copy Number and Prevalence of Porcine Endogenous Retroviruses (PERVs) in German Wild Boars. Viruses. 2020;12(4):419. Sypniewski D, Machnik G, Mazurek U, Wilczok T, Smorag Z, Jura J, Gajda B. Distribution of porcine endogenous retroviruses (PERVs) DNA in organs of a domestic pig. Ann Transpl. 2005;10:46–51. Mazurek U, Kimsa MC, Strzalka-Mrozik B, Kimsa MW, Adamska J, Lipinski D, Zeyland J, Szalata M, Slomski R, Jura J, et al. Quantitative analysis of porcine endogenous retroviruses in different organs of transgenic pigs generated for xenotransplantation. Curr Microbiol. 2013;67:505–14. Fiebig U, Krüger L, Denner J. Determination of the Copy Number of Porcine Endogenous Retroviruses (PERV) in Auckland Island Pigs Repeatedly Used for Clinical Xenotransplantation and Elimination of PERV-C. Microorganisms. 2024;12(1):98. Jhelum H, Papatsiros V, Papakonstantinou G, Krabben L, Kaufer B, Denner J. Screening for viruses in indigenous Greek black pigs. Microorganisms. 2024;12(2):315. Lange A, Medugorac I, Ali A, Kessler B, Kurome M, Zakhartchenko V, Hammer S, Hauser A, Denner J, Dobenecker B, Wess G, Tan PLJ, Garkavenko O, Reichart B, Wolf E, Kemter E. Genetic diversity, growth and heart function of Auckland Island pigs, a potential source for organ xenotransplantation. Xenotransplantation. 2024;31(2):e12858. Hinrichs A, Riedel EO, Klymiuk N, Blutke A, Kemter E, Längin M, Dahlhoff M, Keßler B, Kurome M, Zakhartchenko V, Jemiller EM, Ayares D, Bidlingmaier M, Flenkenthaler F, de Hrabĕ M, Arnold GJ, Reichart B, Fröhlich T, Wolf E. Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies. Xenotransplantation. 2021;28(2):e12664. Fan B, Gongora J, Chen Y, Garkavenko O, Li Moran C. Population genetic variability and origin of Auckland Island feral pigs. J R Soc N Z. 2005;35:279–85. Garkavenko O, Muzina M, Muzina Z, Powels K, Elliott RB, Croxson MC. Monitoring for potentially xenozoonotic viruses in New Zealand pigs. J Med Virol. 2004;72:338–44. Garkavenko O, Dieckhoff B, Wynyard S, Denner J, Elliott RB, Tan PL, Croxson MC. Absence of transmission of potentially xenotic viruses in a prospective pig to primate islet xenotransplantation study. J Med Virol. 2008;80:2046–52. Wynyard S, Nathu D, Garkavenko O, Denner J, Elliott R. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand. Xenotransplantation. 2014;21:309–23. Morozov VA, Wynyard S, Matsumoto S, Abalovich A, Denner J, Elliott R. No PERV transmission during a clinical trial of pig islet cell transplantation. Virus Res. 2017;227:34–40. Jhelum H, Kaufer B, Denner J. Application of methods detecting xenotransplantation-relevant viruses for screening German slaughterhouse pigs. Viruses. 2024;16:1119. Dieckhoff B, Kessler B, Jobst D, Kues W, Petersen B, Pfeifer A, Kurth R, Niemann H, Wolf E, Denner J. Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation. Xenotransplantation. 2009;16(2):64–73. Gemeniano M, Mpanju O, Salomon DR, Eiden MV, Wilson CA. The infectivity and host range of the ecotropic porcine endogenous retrovirus, PERV-C, is modulated by residues in the C-terminal region of its surface envelope protein. Virology. 2006;346:108–17. Preuss T, Fischer N, Boller K, Tonjes RR. Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C. J Virol. 2006;80(20):10258–61. Scobie L, Taylor S, Wood JC, Suling KM, Quinn G, Meikle S, Patience C, Schuurman HJ, Onions DE. Absence of replication-competent human-tropic porcine endogenous retroviruses in the germ line DNA of inbred miniature Swine. J Virol. 2004;78(5):2502–9. Dieckhoff B, Karlas A, Hofmann A, Kues WA, Petersen B, Pfeifer A, Niemann H, Kurth R, Denner J. Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors. Arch Virol. 2007;152(3):629–34. Krüger L, Kristiansen Y, Reuber E, Möller L, Laue M, Reimer C, Denner J. A Comprehensive Strategy for Screening for Xenotransplantation-Relevant Viruses in a Second Isolated Population of Göttingen Minipigs. Viruses. 2019;12(1):38. Halecker S, Krabben L, Kristiansen Y, Krüger L, Möller L, Becher D, Laue M, Kaufer B, Reimer C, Denner J. Rare isolation of human-tropic recombinant porcine endogenous retroviruses PERV-A/C from Göttingen minipigs. Virol J. 2022;19(1):30. Karlas A, Irgang M, Votteler J, Specke V, Ozel M, Kurth R, Denner J. Characterisation of a human cell-adapted porcine endogenous retrovirus PERV-A/C. Ann Transpl. 2010;15(2):45–54. Wu J, Ma Y, Lv M, Yang Y, Guo Y, Yu X, Tian K, Zhang J. Large-scale survey of porcine endogenous retrovirus in Chinese miniature pigs. Comp Immunol Microbiol Infect Dis. 2008;31(4):367–71. Semaan M, Rotem A, Barkai U, Bornstein S, Denner J. Screening pigs for xenotransplantation: prevalence and expression of porcine endogenous retroviruses in Göttingen minipigs. Xenotransplantation. 2013;20(3):148–56. Duvigneau J, Hartl R, Groiss S, Gemeiner M. Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines. J Immunol Methods. 2005;306:16–27. Tables Table 1 Primers (fw, forward; rev, reverse) and probes used for the detection of PERV sequences (location according to Accession Nr. AM229312) and porcine GAPDH Primer/probe Sequence 5’-3’ Location a PERV-C b PERV-A c PERV-C PCR1 fwd d CTGACCTGGATTAGAACTGG 6606..6625 yes no PCR1 rev CCAGGACCATCCTCTAACAT 6867..6886 yes no PCR4 fwd GATTAGAACTGGAAGCCCCAAGTGCTCT 6614..6641 yes no PCR4 rev ACCATCCTCTAACATAACTTCTGGATCAGA 6872..6901 yes no PCR5 fwd CTATTCGCCTCAAAATAAACCAG 6778..6800 yes no PCR5 rev = PCR8 rev = PCR9 rev CATAGAGACCAATGCACATG 7086..7105 yes no PCR6 fwd= PCR8 fwd=PCR10 fwd CCAGGACCACCAAATAATGG 6435..6454 yes no PCR6 rev=envC real time rev ACTAAAATGGGGGCAAAACTT 6924..6944 yes no envC real-time fwd=PCR9 fwd CCCCAACCCAAGGACCAG 6853..6870 yes no envC real-time probe FAM-CTCTAACATAACTTCTGGATCAGACCC-BHQ1 6878..6904 Yes no PCR10 rev CACTGAAGCCTTTAATCAAACC 7183..7205 yes no PERV-A/C (380 bp amplicon) PERV-A-VRB e fwd CCTACCAGTTATAATCAATTTAATTATGGC 6129–6158 no yes PERV-C rev TATGTTAGAGGATGGTCCTGGTC 6451–6473 yes no PERV-A/C (1260 bp amplicon) PERV-A-VRB—fwd PERV-C-TMR rev CCTACCAGTTATAATCAATTTAATTATGGC CTCAAACCACCCTTGAGTAGTTTCC 6129–6158 7370–7395 no yes yes no pGAPDH pGAPDH-fwd pGAPDH-rev pGAPDH-probe ACATGGCCTCCAAGGAGTAAGA TCAGTGTCGGGGTTGAGCTAG HEX-CCA CCA ACC CCA GCA AGA G-BHQ a location of the PERV-C primer/probe according to accession number AM229312 [33], of the GAPDH primer/probe according to Duvigneau et al., 2005 [41], primers to detect PERV-A/C as described by Wood et al. [7] b primer/probe sequence is present in PERV-C sequence with accession number AM229312, clone1312 [33] c primer/probe sequence is present in PERV-A sequence with accession number AY288779.1 [34] d designed by Takeuchi et al. 1998 [1] e VRB, variable region B Table 2 Duplex real-time PCR for the detection of PERV-C and GAPDH in indigenous Greek black pigs from farm 1, Auckland Island pigs and German slaughterhouse pigs Mean ct 260/280 nm values a PERV-C pGAPDH Indigenous Greek black pigs, farm 1 Pig 1 spleen 25.07 19.16 1.85 liver 26.38 19.86 1.89 Pig 2 spleen 21.06 19.14 1.90 liver 21.47 19.92 1.80 Pig 3 spleen 26.92 19.93 1.87 liver 26.23 19.98 1.82 Pig 4 spleen 21.29 19.92 1.91 liver 20.70 19.20 1.86 Auckland Island pigs 13947 n.d. 19.53 1.81 13980 n.d. 19.03 1.89 13983 n.d. 19.19 1.90 German slaughterhouse pigs Pig 1 spleen 24.57 18.52 1.83 liver 28.66 20.12 1.91 Pig 2 spleen 21.67 17.68 1.80 liver 22.00 18.30 1.83 Pig 3 spleen 26.43 17.51 1.92 liver 31.05 20.08 1.84 Pig 4 spleen 25.11 18.20 1.86 liver 26.46 19.24 1.90 Pig 5 spleen 25.78 19.02 1.87 liver 28.90 20.00 1.93 Pig 6 spleen 26.36 19.39 1.89 liver 26.19 19.97 1.90 Pig 7 spleen 25.78 19.22 1.81 liver 27.42 20.17 1.82 Pig 8 spleen 23.49 19.59 1.85 liver 23.05 20.10 1.92 Pig 9 spleen 29.40 19.06 1.84 liver 31.68 19.84 1.93 Pig 10 spleen 27.12 19.12 1.87 liver 33.57 20.25 1.91 a indicates the purity of the DNA; n.d., not detected Table 3 Comparison if the sequences of the primers and probes used for the PCRs and real-time PCRs with the sequences found in the amplicons obtained by PCR8 using DNA from indigenous Greek black pigs 3 and 4 PCR Fwd/rev 1 fwd Primer CTGACCTGGATTAGAACTGG Pig 3 disrupted PCR8 Pig 4 CTGACCTGGATTAGAACTGG PCR8 1 rev Primer CCAGGACCATCCTCTAACAT Pig 3 disrupted PCR8 Pig 4 CCAGGACCATCCTCTAACAT PCR8 4 fwd Primer GATTAGAACTGGAAGCCCCAAGTGCTCT Pig 3 disrupted PCR8 Pig 4 GATTAGAACTGGAAGCCCCAAGTGCTCT PCR8 rev Primer ACCATCCTCTAACATAACTTCTGGATCAGA Pig 3 disrupted PCR8 Pig 4 ACCATCCTCTAACATAACTTCTGGATCAGA PCR8 5 fwd Primer CTATTCGCCTCAAAATAAACCAG Pig 3 CTATTCGCCTCA G AATA G A AACT CAG PCR8 Pig 4 CTATTCGCCTCAAAATAAACCAG PCR8 rev Primer CATAGAGACCAATGCACATG 6 fwd Primer CCAGGACCACCAAATAATGG Pig 3 not available PCR8 Pig 4 not available PCR8 rev Primer ACTAAAATGGGGGCAAAACTT Pig 3 A T TAAAA CA GGGGCGAAACTT PCR8 Pig 4 ACTAAAATGGGGGCAAAACTT PCR8 Real-time PCR fwd Primer CCCCAACCCAAGGACCAG Pig 3 C T CCAA T CCAAG A ACC GA PCR8 Pig 4 CCCCAACCCAAGGACCAG PCR8 probe Probe CTCTAACATAACTTCTGGATCAGACCC Pig 3 T T AC AA T A C AAC C TCTGGATCAGTCCC PCR8 Pig 4 CTCTAACATAACTTCTGGATCAGACCC PCR8 rev Primer ACTAAAATGGGGGCAAAACTT Pig 3 A T TAAAA CA GGGGC G AAACTT PCR8 Pig 4 ACTAAAATGGGGGCAAAACTT PCR8 7 fwd corresponds to fwd primer of PCR5 rev corresponds to the rev primer of the real-time PCR 8 fwd corresponds to fwd primer of PCR6 rev corresponds to the rev primer of PCR5 9 fwd corresponds to the fwd primer of the real-time PCR rev corresponds to the reverse primer of PCR5 fwd, forward primer, rev, reverse primer; mutations in comparison to the primer sequence were marked bold and grey. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Table3.docx Figure2uncropped.pptx Figure4uncropped.pptx Supplementaryfigure1.docx Supplementaryfigure2.docx Supplementaryfigure3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4807994","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":343320725,"identity":"69b8054f-a09c-4794-a1db-3fc3f90b16ca","order_by":0,"name":"Hina Jhelum","email":"","orcid":"","institution":"Free University Berlin","correspondingAuthor":false,"prefix":"","firstName":"Hina","middleName":"","lastName":"Jhelum","suffix":""},{"id":343320726,"identity":"db81ffd5-c500-455c-a6bb-eb48208d4e82","order_by":1,"name":"Dusan Kunec","email":"","orcid":"","institution":"Free University Berlin","correspondingAuthor":false,"prefix":"","firstName":"Dusan","middleName":"","lastName":"Kunec","suffix":""},{"id":343320727,"identity":"0078c57b-26f7-478c-ae75-8aa6219d8082","order_by":2,"name":"Vasileios Papatsiros","email":"","orcid":"","institution":"University of Thessaly","correspondingAuthor":false,"prefix":"","firstName":"Vasileios","middleName":"","lastName":"Papatsiros","suffix":""},{"id":343320728,"identity":"a04749b3-9705-45fe-bd39-44603bf69f30","order_by":3,"name":"Benedikt Kaufer","email":"","orcid":"","institution":"Free University Berlin","correspondingAuthor":false,"prefix":"","firstName":"Benedikt","middleName":"","lastName":"Kaufer","suffix":""},{"id":343320729,"identity":"e697520e-370f-4064-ab9e-182924fc8369","order_by":4,"name":"Joachim Denner","email":"data:image/png;base64,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","orcid":"","institution":"Free University Berlin","correspondingAuthor":true,"prefix":"","firstName":"Joachim","middleName":"","lastName":"Denner","suffix":""}],"badges":[],"createdAt":"2024-07-26 12:18:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4807994/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4807994/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63062026,"identity":"a468a9b4-8090-4b50-9cd0-39b8aedf4a20","added_by":"auto","created_at":"2024-08-22 16:27:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":223736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Schematic presentation of the used PCRs, the localization of the primer binding sites and of the probe and the length of the amplicons is shown. \u003cstrong\u003eB\u003c/strong\u003e, results of the nanopore sequencing of amplicons from the PCR8 of DNA from spleen and liver of animals 1, 3 and 4. The primer binding sites and the probe of the different PCR methods are indicated. P, pig; fwd, forward primer; rev, reverse primer. VRA, variable region A; VRB, variable region B, PRR, proline-rich region.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/bda4295031b594bc2e12a578.png"},{"id":63062025,"identity":"9a117013-591b-4458-b4e9-ffeb4a9811a4","added_by":"auto","created_at":"2024-08-22 16:27:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Results of different PCRs using DNA from liver and spleen from 4 indigenous Greek black pigs (animals 1, 2, 3,4) from farm 1. GAPDH was used to demonstrate identical DNA loading. \u003cstrong\u003eB\u003c/strong\u003e, results of PCR4 and \u003cstrong\u003eC\u003c/strong\u003e, of PCR8 using DNA from three Auckland Island pigs (13947, 13980, 13982) and from PK15 cells, \u003cstrong\u003eD\u003c/strong\u003e, results of PCR8 using DNA from spleen and liver of Greek black pigs 3 and 4 and PK15 cells, \u003cstrong\u003eE\u003c/strong\u003e, results of PCR10 using DNA from the Auckland Island pig 10377 and PK15 cells. S, spleen; L, liver; P, positive control, a lung tissue from a PERV-C positive animal; N, negative control; M, marker.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/ce13811c50474e499b4d1bc7.png"},{"id":63061447,"identity":"1f060e9e-a985-4c2b-adca-6cfdc6262a14","added_by":"auto","created_at":"2024-08-22 16:19:33","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36340,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of the nanopore sequences of the amplicons of PCR8 from indigenous Greek black pig 3 and 4 in comparison with the PERV-C reference genome (accession number AM229312 [32]) and the PERV-A reference genome (accession number AY288779.1 [34]). Scale bar refers to a phylogenetic distance of 0.05 nucleotide substitutions per site.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/b2bec051d197447a8a08de41.jpeg"},{"id":63061449,"identity":"2bba3285-a516-476c-a630-9b5156caf4c7","added_by":"auto","created_at":"2024-08-22 16:19:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Schematic presentation of the location of the primers used to detect PERV-A/C recombinants. \u003cstrong\u003eB\u003c/strong\u003e, Results of the PCR screening for PERV-A/C recombinants, in the upper part the primer pair PERV-A VRB fw and PERV-C TMR rev (1260 bp amplicon), in the lower part the primers PERV-A VRB fw and PERV-C rev (350 bp amplicon) were used. DNA from spleen (S) and liver (L) from four indigenous Greek black pigs from farm 1 were analyzed, in the upper part a 1 kb plus ladder (M), in the lower part a 100 bp plus ladder were used. DNA from 293 cells infected with a PERV-A/C released from PBMCs from a Göttingen minipig [37] was used as positive control (P), negative control (N) was water.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/a9ff81f89984eece7c7661da.png"},{"id":64594547,"identity":"dcc0f46a-4691-4bfe-8fd6-fbba330a53dc","added_by":"auto","created_at":"2024-09-16 10:25:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1337437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/56df1c99-07ca-4604-a484-78cfd28e4319.pdf"},{"id":63062024,"identity":"b61eba19-fdb5-429f-a499-a349132acd15","added_by":"auto","created_at":"2024-08-22 16:27:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18570,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/796715d81f73580312ce109b.docx"},{"id":63061444,"identity":"ceb38852-09c9-420a-b094-b7bf0e4b64ab","added_by":"auto","created_at":"2024-08-22 16:19:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16919,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/f971614f7c692137e216ffc4.docx"},{"id":63061446,"identity":"e237f0f3-b6ce-4ce6-b16a-9bb89150cb1a","added_by":"auto","created_at":"2024-08-22 16:19:33","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18232,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/5f69277eceeb9154638e82a7.docx"},{"id":63061458,"identity":"bd997587-7b91-4c1e-9b2b-a7e93393fb76","added_by":"auto","created_at":"2024-08-22 16:19:37","extension":"pptx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":150316916,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2uncropped.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/450d8eb9e466dde86bd2e214.pptx"},{"id":63061456,"identity":"88ad48a7-a1db-4788-8382-3c6f3a9d7651","added_by":"auto","created_at":"2024-08-22 16:19:34","extension":"pptx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":2591136,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4uncropped.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/7b68b628e8d52683481a8d28.pptx"},{"id":63061451,"identity":"7d225367-81cb-4cd9-9426-ae0d4113b258","added_by":"auto","created_at":"2024-08-22 16:19:33","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":35934,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/91f0e016f0dc8e5a69a94890.docx"},{"id":63061454,"identity":"e730c360-4b66-4e68-a79c-d1346cdc5845","added_by":"auto","created_at":"2024-08-22 16:19:34","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":14832,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/77be2523ec8d81f81575d15a.docx"},{"id":63062027,"identity":"c9ee3fd4-c661-4374-a25e-fc32e5869ff2","added_by":"auto","created_at":"2024-08-22 16:27:33","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":60635,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4807994/v1/280e62afe46ce735ba995c75.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening for porcine endogenous retrovirus - C (PERV-C) in pigs","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePorcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs. PERV-A and PERV-B are present in all pigs, whereas PERV-C is not present in all, but most pigs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This correlates with the fact that PERV-C is phylogenetically younger compared to PERV-A and PERV-B [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Since PERV-A and PERV-B can infect human cells (human-tropic virus), they pose a risk for xenotransplantation using pig cells, tissues or organs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. PERV-A and PERV-B infect also cells from numerous other species (polytropic viruses). PERV-C infects only pig cells (ecotropic virus) and therefore, theoretically does not pose a risk for xenotransplantation. However, PERV-C can recombine with PERV-A, acquiring the receptor binding site for the receptor on cells of humans and other species, enabling the virus to efficiently replicate in human cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, the replication rate of the PERV-A/C recombinants is higher compared to paternal PERV-A [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, it is recommended to use PERV-C-free animals as donor pigs for xenotransplantation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To ensure that the animals are PERV-C-free, sensitive and specific conventional and real-time PCRs were developed [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. When we screened wild boars and German landrace pigs for PERV-C using the commonly used PCR developed by Takeuchi et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], we observed mutations in the primer binding sites giving a false negative result in some pigs. Therefore, we developed new PCRs to detect PERV-C variants in such animals [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is well known that PERVs are active in living pigs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This was demonstrated by different copy numbers of integrated proviruses in different organs and in different parts of one organ and by an increase of the copy number by age [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The lowest level of PERV-A and PERV-B proviral DNA was shown in muscles [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These investigations were performed using primer and probes binding to a highly conserved region in PERV's polymerase sequence (pol), not discriminating between PERV-A, PERV-B and PERV-C. Furthermore, there is good evidence that PERV-C is active in living pigs: Recombinants with PERV-A were found in somatic cells but such PERV-A/C were never found in the germ line [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen we screened indigenous Greek black pigs for PERV-C using another PCR, designated PCR4 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], 11 of 21 animals were found positive [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. None of the PERV-C-positive animals were positive for PERV-A/C. Indigenous Greek black pigs live in mountainous areas, they are resistant to weather conditions and to diseases even though numerous viruses including porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpes virus \u0026minus;\u0026thinsp;3 (PLHV-3), and porcine circovirus 3 (PCV3) were found in most of the animals [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAuckland Island pigs are ideal organ donors because of the optimal organ size for humans [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, there is no need for a knock-out of the porcine growth factor receptor as done with other pig breeds [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The second important advantage is the low number of porcine microorganisms. These animals lived for a hundred years on the remote Auckland Island, they were thoroughly studied in New Zealand [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and used as donors of pig islets in the first clinical trials treating diabetes patients in New Zealand and Argentina. In these trials no transmission of porcine viruses, including PERV to the patients was observed [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Since more than 50% of the animals in New Zealand were PERV-C positive, we selected PERV-C negative animals from which cells were delivered to Germany and piglets were obtained by somatic cell nuclear transfer (SCNT) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Genetic diversity, growth and heart function of these Auckland Island pigs at the Center for Innovative Medical Models (CiMM) in Munich as well as the absence of PERV-C demonstrate that they are excellent donor animals [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo evaluate our methods developed for the detection of numerous porcine viruses we recently screened German slaughterhouse pigs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Nine of ten animals were PERV-C positive and therefore we included them in this study using different PCR approaches and sequencing methods to analyze PERV-C in indigenous Greek black pigs, Auckland Island pigs and pigs from a German slaughterhouse.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePCR methods for the detection of PERV-C\u003c/strong\u003e To screen pigs for the presence of PERV-C, a PCR developed by Takeuchi et al. [1] is commonly used, designated here PCR1 (Figure 1A). Later Dieckhoff et al. developed a new PCR [31], designated PCR4 by Kaulitz et al. [12] using primers based on a PERV-C sequence (accession number\u0026nbsp;EU440732.1, [32]). In addition, PCR2, PCR3 and a real-time PCR were developed [12]. The PCR1 detected up to 1.1x10\u003csup\u003e3\u003c/sup\u003e molecules of a PERV-C plasmid, and the real-time PCR performed as duplex PCR simultaneously detecting PERV-C and porcine cyclophilin detected 100 copies/reaction PERV-C [12]. PCR1, PCR2 and PCR3 detected equally good PERV-C in German landrace pigs [12]. Later, when we detected new variants of PERV-C in German wild boars, we added two new PCRs, PCR6 and PCR7 [15]. All primer and probe sequences were found in the PERV-C reference sequence (accession number\u0026nbsp;AM229312, [33]), but not in the PERV-A reference sequence (accession number AY288779.1, [34]) (Table 1). In this manuscript, the real-time PCR\u0026nbsp;was performed as a duplex real-time PCR detecting in parallel porcine GAPDH and the sensitivity was 10 copies PERV-C /100ng DNA [21]. To evaluate the specificity of the developed PCRs and to determine the presence or absence of PERV-C, we screened\u0026nbsp;indigenous Greek black pigs, Auckland Island pigs and pigs from a German slaughterhouse. In this manuscript, three new PCRs, PCR8, PCR9 and PCR10, were added, which partially were designed to obtain larger amplicons for sequencing purposes (Figure 1A). To stress it again, all primer and probe sequences were found in our PERV-C reference sequence, but not in the PERV-A reference sequence (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of PERV-C in indigenous Greek black pigs\u003c/strong\u003e Initially, 21\u0026nbsp;indigenous Greek black pigs were screened for PERV-C using PCR4. This screening revealed that 11 of 21 animals (52.4%) were positive [22]. To further investigate the presence of PERV-C in these animals, four animals from farm 1 were analyzed using PCR1. Screening with this PCR1 reconfirmed the presence of PERV-C in all pigs, except pig 3 (Figure 2A). The same result had been obtained for PCR4 [22]. The absence of PERV-C specific bands in PCR1 and PCR4 for animal 3 suggests that PERV-C is either absent in this animal or that the primer-binding sites are mutated, making them unrecognizable by one or both primers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo test these possibilities, PCR5 and PCR6 were performed (Figure 2A). Testing DNA from the liver and spleen of animals 2 and 4 with PCR5 and PCR6 produced strong amplicons identical in size to that of the positive control. No amplification was observed with PCR 5 and PCR6 when DNA from both tissues of animal 3 was used. Interestingly, no amplification was detected in animal 1 with PCR 5, and only a faint band was observed with PCR6 using DNA isolated from the spleen, but not from the liver. Given the importance of this PCR6 result, the PCR was repeated several times to ensure reproducibility, and the results of two experiments are presented in Figure 2A. The difference between liver and spleen suggests that there is a mutation in one or both primer binding sites in the provirus in the liver, but not in the spleen, indicating different proviruses in these organs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo obtain a larger amplicon for sequencing purposes, PCR8 was performed, using the forward primer of PCR6 and the reverse primer of PCR5. To our surprise, DNA extracted from all tissues from all four animals yielded amplicons, also from pig 3. Most interestingly, the amplicons from the liver and spleen of animal 3 were larger compared to the amplicons from animals 1, 2 and 4, indicating a potential insertion (Figure 2A). PCR7 (the combination of the forward primer of PCR5 and the reverse primer of the real-time PCR) and PCR9 (using the forward primer of the real-time PCR and the reverse primer of PCR5) were also positive for all four animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of PERV-C in other pigs\u003c/strong\u003e When the DNA from the German slaughterhouse pigs were screened using a real-time PCR [12], all animals were also positive (Table 2). In contrast, the Auckland Island pigs and the PK15 cells were negative in the real-time PCR (Table 2). The Auckland Island pigs [21] and the pig cell line PK15 [35] are well known to be PERV-C negative. They were also PERV-C negative in this study in PCR4 (Figure 2B) and PCR8, which was using the forward primer of PCR6 and the reverse primer of PCR5 in order to obtain extended amplicons (Figure 2C, 2D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence analysis of PERV-C from indigenous Greek black pigs\u003c/strong\u003e Sanger sequencing of the PCR8 amplicon from pig 4 resulted in a high-quality sequence and an alignment of this sequence with a PERV-C reference sequence AM229312 [33] revealed that the sequences were nearly identical (Figure S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, since the Sanger sequencing of the other amplicons did not produce satisfactory results, possibly due to the simultaneous amplification of different proviruses present in the pig genome with differences in the sequence, all amplicons were sequenced using the Nanopore method. As expected, putative insertions were detected in the sequence from animal 3 when compared with the sequences from animals 2 and 4 (Figure 1B). The largest insertion disrupts the binding sites of the forward primers of PCR1 and PCR4, while the other disrupts the binding sites of the reverse primers of PCR1 and PCR4. The smallest insertion disrupts the binding site for the forward primer of PCR5. Consequently, none of these three PCRs produced amplicons when DNA from animal 3 was tested (Figure 2A). However, when the large insert and the whole amplicon sequence from animal 3 were analyzed using nucleotide BLAST (nBLAST), a high sequence similarity PERV-A was observed (Figure S2), indicating that the provirus amplified with PCR8 in pig 3 is PERV-A.\u003c/p\u003e\n\u003cp\u003eBased on the nanopore sequences of the PCR8 amplicons from animals 3 and 4 a dendrogram was built, showing that the amplicon from animals 4 is closer to the PERV-C reference sequence and the amplicon from animal 3 is closer to the PERV-A reference sequence (Figure 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative analysis of the primer binding sites\u0026nbsp;\u003c/strong\u003eThe sequences obtained by nanopore sequencing of the 692bp amplicons obtained by PCR8 were used to analyze the primer binding sites of PCR1, PCR4, PCR5, PCR6, PCR7, PCR8 and PCR 9 as well as the binding sites for primers and probes of the real-time PCR (Table 3). The nanopore sequences were of high quality and the sequences of the amplicons derived from the liver DNA and the spleen DNA were 100% identical. When the sequences of the primers and the corresponding sequences in the amplicons were compared, it was confirmed that the forward and reverse primers of PCR 1 and PCR4 are highly specific, because in the PERV-A sequence (amplicon of PCR8 from animal 4) the primer binding sites were disrupted by larger sequences absent in PERV-C. The sequences of the PCR5 primer binding sites in pig 3 had mutations, in the case of the forward primer an insertion of sequence \u0026ldquo;AAC\u0026rdquo; disrupted the primer binding site (Table 3), explaining the absence of amplicons when PCR5 was performed (Figure 2A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe real-time PCR is not PERV-C-specific\u0026nbsp;\u003c/strong\u003eIn addition, a real-time PCR as described by Kaulitz et al. [12] was performed.\u0026nbsp;Primers and the probe had been designed using sequences found in the PERV-C reference genome, but not in the PERV-A reference genome (Table 1).\u0026nbsp;An identical copy number in the spleen and liver of animals 2 and 4 (21 Ct) was found, but a lower copy number in both tissues of animals 1 and 3 (26 Ct) (Table 2). Interestingly, no significant differences in the PERV copy numbers were observed between the liver and spleen of all animals.\u0026nbsp;When the real-time PCR was used to screen the four indigenous Greek black pigs from farm 1, all four animals including pig 3, which was found PERV-C negative on the basis of PCR1, PCR4, PCR5 and PCR6 (Figure 2A) were found to be positive in the real-time PCR (Table 2). In contrast, the Auckland Island pigs, which were also found PERV-C negative using the mentioned PCRs, were negative in the real-time PCR (Table 2). All ten German slaughterhouse pigs were positive in the real-time PCR, including pig 3, which was found to be PERV-C negative using PCR1 and PCR4 [30] (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequences obtained by PCR10\u0026nbsp;\u003c/strong\u003eIn order to obtain an even larger sequence for extended sequencing, PCR10 was developed using the forward primer of PCR6 and a new primer downstream of the reverse primer of PCR5 (Table 1). PCR10 was positive for all tissues of all four indigenous Greek black pigs (Figure 2A), including pig 3. The amplicons from the liver and spleen of pig 3 and pig 4 were sequenced by the nanopore method and were compared with the PERV-C reference gene (Figure S3). There were significant differences, indicating that different proviruses had been amplified. When these sequences were analyzed using nBLAST, the sequences from pig 3 corresponded to PERV-B and that from pig 4 to PERV-A, indicating that the primer amplified non-PERV-C proviruses. PCR10 was also positive for the German slaughterhouse pig 3, the sequence of the amplicon was also identified as PERV-A. Therefore, a comparison of the primers of PCR1, PCR4, PCR5 and the primers and the probe of the real-time PCR with the corresponding binding sites in the sequences was not conductive (Figure S3).\u0026nbsp;Interestingly, PCR10 was negative for an Auckland Island pig and positive for PK15 cells (Figure 2E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening for PERV-A/C\u0026nbsp;\u003c/strong\u003eTo detect PERV-A/C recombinants, primer pairs were used which had been previously validated for identification of different types of recombinants [7, 36-38]. Despite comprehensive screening with these primer pairs, no PERV-A/C sequences could be detected in the genome of indigenous Greek black pigs (Figure 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we analyzed for the first time integrated PERV-C proviruses in indigenous Greek black pigs, German landrace pigs and Auckland Island pigs and confirmed that Auckland Island pigs from the CiMM in Munich are PERV-C-negative. Furthermore, we tested several PCR assays to determine whether they correctly detect PERV-C. Eleven of 21 indigenous Greek black pigs in four different farms in Greece were found to carry PERV-C proviruses using PCR4. This confirms that not all pigs carry PERV-C proviruses as reported in various previous publications. For example, 113 of 348 (30%) Chinese miniature pigs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], 176 of 181 (97%) German transgenic and non-transgenic pigs [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and 25 of 98 (25.5%) US farm animals [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] carry PERV-C. The absence of PERV-C in several animals correlates with the fact that PERV-C is phylogenetically younger compared to PERV-A and PERV-B, which are present in all pigs [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThree of four indigenous Greek black pigs in farm 1 were found to be PERV-C-positive using both PCR1 and PCR4 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Nine out of ten German slaughterhouse pigs were positive using these PCRs, while the porcine cell line PK15 was negative, as expected. Most importantly, all Auckland Island pigs from the CiMM in Munich, which were derived from cell lines from selected, PERV-C-negative, animals [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] were confirmed to be PERV-C negative. Although PERV-C does not infect human cells, it can recombine with the ubiquitous PERV-A present in all pig genomes. These PERV-A/C recombinants are able to infect human cells and replicate to higher titers compared to the paternal PERV-A [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, only PERV-C-negative animals should be used for preclinical and clinical xenotransplantation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSequencing of the amplicon produced by PCR8 using the Sanger sequencing showed that the sequence found in pigs 2 and 4 from farm 1 is nearly identical to the PERV-C reference sequence AM229312 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Since the Sanger sequencing of the amplicons obtained by PCR8 using the DNA from pig 3 was not successful, possibly due to the presence of amplicons generated from different proviruses in the genome, all amplicons were sequenced using the Nanopore method. This sequencing showed insertions in the PERV sequences of animal 3. This is in agreement with the finding that the amplicons obtained when performing PCR8 using DNA from animal 3 was larger compared with the amplicons of the other three pigs. As a matter of fact, three putative insertions were found when the amplicons of PCR8 were sequenced and compared. These insertions disrupted the primer binding sites of PCR1, PCR4 and PCR5, explaining the negative results. The sequence \u0026ldquo;ATTGGCAACAGCGGGTACAAAAAGAT\u0026rdquo;, which is PERV-A specific, is absent in the PERV-C sequence of animals 1 and 4, suggesting it may be a sequence derived from PERV-A that is inserted in the PERV-C sequence in animal 3. However, sequence analysis of the sequence of the amplicon from pig 3 indicated, that the entire sequence corresponded to PERV-A. Therefore, the so-called insertions are not insertions into PERV-C. Insertions of shorter and larger PERV-A sequences into PERV-C sequences had been observed when recombinant PERV-A/C isolated from peripheral blood mononuclear cells (PBMCs) were analyzed [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], however such PERV-A/C sequences were never found in the germ line, but only in somatic cells, indicating that the recombinants are the result of recombination and de novo integration [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOf interest is the result of PCR8. The forward primer of PCR8 is identical to the forward primer of PCR6 and the reverse primer of PCR8 is identical to the reverse primer of PCR5. PCR5 and PCR6 did not give an amplicon when DNA from the Auckland Island pigs, PK15 and Greek animal 3 were tested. PCR5 and PCR6 were only positive with DNA from animals 1, 2 and 4 which were also positive with other PERV-C-specific PCRs such as PCR1 and PCR4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe real-time PCR also gave equivocal results (Table\u0026nbsp;2). Whereas the real-time PCR is negative in the case of Auckland Island pigs, it was unexpectedly positive in the case of animal 3, which was tested PERV-C negative by PCR1, PCR4, PCR5 and PCR6.\u003c/p\u003e \u003cp\u003eThe fact that PCR6 gave a positive result when analyzing DNA from the spleen of animal 1, but not from the liver of animal 1 is of interest. The negative result with the liver DNA indicates that the sequence of one or both primer binding sites is absent or mutated. This data indicates that different proviruses are integrated in the DNA of two tissues, the spleen and liver, of a single animal.\u003c/p\u003e \u003cp\u003eThe methods characterized here are required to identify PERV-C-negative pigs for xenotransplantation to prevent a recombination with PERV-A. However, we have to consider, that in addition to recombination with PERV-A, resulting in PERV-A/C recombinants, there are also other mechanisms able to transform the ecotropic PERV-C into a virus which can infect human cells. Gemeniano et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] have shown that the C-terminal proline-rich region (PRR) in the envelope protein region is required in addition to the putative variable region A (VRA) and VRB for binding to cells. They showed that mutations in the PRR allowed infection of human cells. They also showed that the cellular receptor molecule binding the PERV-C with the mutated C-terminal region of the surface envelope protein is different from the receptor molecule when PERV-A is binding and infecting human cells.\u003c/p\u003e \u003cp\u003eIn conclusion, screening pigs for the presence of PERV-C is easy but requires some precautions. Our experience shows that PCR1 and PCR4 always gave clear results, showing that indigenous Greek black pig 3, all Auckland Island pigs, German slaughterhouse pig 3 and PK15 cells are PERV-C negative. PCR5 and PCR6 gave false-negative results and all others including the real-time PCR gave false-positive results. On the other hand, PCR1, PCR4, PCR5, PCR6, PCR8 and the real-time PCR were negative in the case of the Auckland Island pigs. Therefore, PCR1 and PCR4 should be used for routine screening for PERV-C and all other PCRs may help to characterize the sequences in the receptor binding domain of PERV.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and tissues\u003c/h2\u003e \u003cp\u003eLiver and spleen tissues were obtained from 4 animals in Farm 1 located near Drama, North Greece [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These animals had been screened for different porcine viruses using real-time PCR (PCMV/PRV, PCV2, PCV3, PCV4, PLHV-1, PLHV-2 and PLHV-3), as well as real-time RT-PCR (HEV genotype 3). Blood samples from the Auckland Island pigs were obtained from Prof. Eckhard Wolf and Dr. Barbara Ke\u0026szlig;ler, Chair for Molecular Animal Breeding and Biotechnology and CiMM, Munich. The three female animals were born in June 2023, they are the F1 generation of the animals born in April 2019 and were obtained by somatic cell nuclear transfer (SCNT) using PERV-C-negative kidney cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. PBMCs were isolated by gradient centrifugation as described [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Spleen and liver samples from German pigs were obtained from a slaughterhouse near Berlin, their age was 6 months. PK15 (ACC 640) cells were ordered from the Leibniz Institute DSMZ German Collection of Microorganisms and Cell lines, Braunschweig, Germany. Using droplet digital PCR (ddPCR) 55 (52.5\u0026ndash;60.1) PERV copies were detected in these cells using porcine GAPDH as a reference gene or 37 (35.0-39.1) copies using porcine beta actin (ACTB) as a reference [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDNA isolation\u003c/h2\u003e \u003cp\u003eDNA was isolated from the tissues according to the manufacturer\u0026acute;s instructions using the DNeasy Blood \u0026amp; Tissue kit (Qiagen, Hilden, Germany). DNA concentrations were determined using NanoDrop ND-1000 (Thermo Fisher Scientific Inc., Worcester, MA, USA) or Qubit (Invitrogen, Thermo Fisher Scientific Inc., Worcester, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePCR and real-time PCR\u003c/h2\u003e \u003cp\u003eConventional PCR was performed to determine the presence of PERV-C using a set of primers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Part of the reactions were carried out with DreamTaq DNA polymerase (Thermo Fisher Scientific, Worcester, MA, USA) and part with high-fidelity HiFi polymerase (PCRBiosystems, London, UK). All reactions were set up with a Biometra TRIO cycler (Analytik Jena, Jena, Germany).\u003c/p\u003e \u003cp\u003eEach sample was subjected to an initial denaturation of 10 min at 95\u0026deg;C (one min for HiFi polymerase), followed by 45 amplification cycles of 95\u0026deg;C for 15 s (35 cycles for HiFi polymerase), the annealing temperatures are shown below (30 s for DreamTaq, 15 s for HiFi Polymerase). The corresponding extension times at 72\u0026deg;C for each PCR are shown below. In the case of DreamTaq DNA polymerase, but not HiFi polymerase, a final extension at 72\u0026deg;C for 5 min was added. The annealing temperature for PCR1 was 55\u0026deg;C, for PCR4 60\u0026deg;C, for PCR5, PCR6, PCR7, and PCR8 58\u0026deg;C, for PCR9 55\u0026deg;C and for PCR10 58\u0026deg;C. The extension times used were 9 s for PCR1 and PCR4, 10 s for PCR5, 16 s for PCR6, 5 s for PCR7, 21 s for PCR8, 9 s for PCR9 and 24 s for PCR10 using HiFi polymerase.\u003c/p\u003e \u003cp\u003eIn addition, a real-time PCR was established using specific primers and probe (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. 100 ng DNA and the SensiFAST Probe No-ROX kit (Meridian Bioscience Cincinnati, OH, USA) in a 20 \u0026micro;L reaction volume. The cycling conditions used were initial denaturation 5 minutes at 95\u0026deg;C, followed by 45 amplification cycles of 95\u0026deg;C for 15 s, annealing at 58\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 30 s in a qTOWER3 G qPCR cycler (Analytik Jena, Jena, Germany). A standard curve was produced using the 510 bp amplicon of previously described PCR6 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] as template. This PCR was performed as follows: 100 ng of DNA template, PCR buffer I containing MgCl\u003csub\u003e2\u003c/sub\u003e, 0.2 mM dNTPs, and one unit of DreamTaq DNA polymerase (Thermo Fisher Scientific, Worcester, MA, USA). In case of PCRBIO HiFi polymerase, PCRBIO reaction buffer along with one-unit HiFi polymerase was used. As positive control DNA from the PERV-C positive pig (#6249) was used. The sensitivity of the real-time PCR was 10 copies/100ng DNA [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSanger sequencing\u003c/h2\u003e \u003cp\u003ePCR8 amplicons from pigs 2 and 4 were gel purified using a DNA gel extraction kit (Monarch, New England Biolabs) and sent for sequencing (LGC Genomics, Berlin, Germany). Sequences were analyzed using ApE software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eNanopore sequencing\u003c/h2\u003e \u003cp\u003ePCR8 amplicons from pig 1, 3 and 4 were gel purified using DNA gel extraction kit (Monarch, New England Biolabs) and sent for sequencing (Plasmidsaurus, Eugene, OR, USA). Sequences were analyzed using SnapGene (San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSequence alignment and phylogenetic tree\u003c/h2\u003e \u003cp\u003eSequence alignment and phylogenetic tree generation were done using Clustal Omega using sequences obtained from nanopore sequencing. ApE plasmid editor was used to align the sequences obtained from Sanger sequencing.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Eckhard Wolf and Dr. Barbara Ke\u0026szlig;ler, Chair for Molecular Animal Breeding and Biotechnology\u003c/p\u003e\n\u003cp\u003eand CiMM, Munich, for blood from the Munich Auckland Island pigs. We thank Dr. Kostja Renko, German Federal Institute for Risk Assessment, Unit Strategies for Toxicological Assessments, Department Experimental Toxicology and ZEBET, Berlin, for the help when acquiring the organs from the German slaughterhouse pigs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, writing, and supervision was performed by JD. Methodology, validation, formal analysis, and visualization was conducted by HJ, VP, BK, DK and JD. Investigation was conducted by HJ and DK. Funding acquisition was performed by BK and JD. Editing of the manuscript was performed by HJ, VP, BK, DK and JD.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Deutsche Forschungsgemeinschaft, TRR 127.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures regarding animal care, handling and welfare concerning the indigenous Greek black pigs were approved by the Ethical Research Committee of the University of Thessaly (Ethical Approval Code: 104/16.11.2021). Investigations on the German slaughterhouse animals were performed using meat samples obtained from the slaughterhouse. All work with the Auckland Island pigs was performed with the permission of the local regulatory authority, Regierung von Oberbayern (ROB). Applications were reviewed by the ethics committee according to \u0026sect;15 TSchG (German Animal Welfare Act).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTakeuchi Y, Patience C, Magre S, Weiss RA, Banerjee PT, Le Tissier P, Stoye JP. Host range and interference studies of three classes of pig endogenous retrovirus. 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Application of methods detecting xenotransplantation-relevant viruses for screening German slaughterhouse pigs. Viruses. 2024;16:1119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDieckhoff B, Kessler B, Jobst D, Kues W, Petersen B, Pfeifer A, Kurth R, Niemann H, Wolf E, Denner J. Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation. Xenotransplantation. 2009;16(2):64\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGemeniano M, Mpanju O, Salomon DR, Eiden MV, Wilson CA. The infectivity and host range of the ecotropic porcine endogenous retrovirus, PERV-C, is modulated by residues in the C-terminal region of its surface envelope protein. Virology. 2006;346:108\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePreuss T, Fischer N, Boller K, Tonjes RR. Isolation and characterization of an infectious replication-competent molecular clone of ecotropic porcine endogenous retrovirus class C. J Virol. 2006;80(20):10258\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScobie L, Taylor S, Wood JC, Suling KM, Quinn G, Meikle S, Patience C, Schuurman HJ, Onions DE. Absence of replication-competent human-tropic porcine endogenous retroviruses in the germ line DNA of inbred miniature Swine. J Virol. 2004;78(5):2502\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDieckhoff B, Karlas A, Hofmann A, Kues WA, Petersen B, Pfeifer A, Niemann H, Kurth R, Denner J. Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors. Arch Virol. 2007;152(3):629\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKr\u0026uuml;ger L, Kristiansen Y, Reuber E, M\u0026ouml;ller L, Laue M, Reimer C, Denner J. A Comprehensive Strategy for Screening for Xenotransplantation-Relevant Viruses in a Second Isolated Population of G\u0026ouml;ttingen Minipigs. Viruses. 2019;12(1):38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalecker S, Krabben L, Kristiansen Y, Kr\u0026uuml;ger L, M\u0026ouml;ller L, Becher D, Laue M, Kaufer B, Reimer C, Denner J. Rare isolation of human-tropic recombinant porcine endogenous retroviruses PERV-A/C from G\u0026ouml;ttingen minipigs. Virol J. 2022;19(1):30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarlas A, Irgang M, Votteler J, Specke V, Ozel M, Kurth R, Denner J. Characterisation of a human cell-adapted porcine endogenous retrovirus PERV-A/C. Ann Transpl. 2010;15(2):45\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu J, Ma Y, Lv M, Yang Y, Guo Y, Yu X, Tian K, Zhang J. Large-scale survey of porcine endogenous retrovirus in Chinese miniature pigs. Comp Immunol Microbiol Infect Dis. 2008;31(4):367\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSemaan M, Rotem A, Barkai U, Bornstein S, Denner J. Screening pigs for xenotransplantation: prevalence and expression of porcine endogenous retroviruses in G\u0026ouml;ttingen minipigs. Xenotransplantation. 2013;20(3):148\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuvigneau J, Hartl R, Groiss S, Gemeiner M. Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines. J Immunol Methods. 2005;306:16\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003eTable 1 Primers (fw, forward; rev, reverse) and probes used for the detection of PERV sequences (location according to Accession Nr.\u0026nbsp;AM229312)\u0026nbsp;and porcine GAPDH\u0026nbsp;\u003c/p\u003e\n\u003ctable style=\"width: 4.7e+2pt;border: none;margin-left:3.5pt;background:#CCCCCC;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;background: white;padding: 0in 3.5pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003ePrimer/probe\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;background: white;padding: 0in 3.5pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eSequence 5\u0026rsquo;-3\u0026rsquo;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;background: white;padding: 0in 3.5pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003eLocation\u003csup\u003ea\u003c/sup\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;background: white;padding: 0in 3.5pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003ePERV-C\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;background: white;padding: 0in 3.5pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003ePERV-A\u003csup\u003ec\u003c/sup\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003ePERV-C\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePCR1 fwd\u003cstrong\u003e\u003csup\u003ed\u003c/sup\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCTGACCTGGATTAGAACTGG\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6606..6625\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePCR1 rev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCCAGGACCATCCTCTAACAT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6867..6886\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePCR4 fwd\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eGATTAGAACTGGAAGCCCCAAGTGCTCT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6614..6641\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: black;\"\u003ePCR4 rev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eACCATCCTCTAACATAACTTCTGGATCAGA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6872..6901\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePCR5 fwd\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCTATTCGCCTCAAAATAAACCAG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6778..6800\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color: black;\"\u003ePCR5 rev = PCR8 rev = PCR9 rev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCATAGAGACCAATGCACATG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e7086..7105\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePCR6 fwd= PCR8 fwd=PCR10 fwd\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCCAGGACCACCAAATAATGG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6435..6454\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePCR6 rev=envC real time rev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eACTAAAATGGGGGCAAAACTT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6924..6944\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eenvC real-time fwd=PCR9 fwd\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCCCCAACCCAAGGACCAG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6853..6870\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eenvC real-time probe\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eFAM-CTCTAACATAACTTCTGGATCAGACCC-BHQ1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6878..6904\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eYes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePCR10 rev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCACTGAAGCCTTTAATCAAACC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e7183..7205\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 471.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003ePERV-A/C\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;(380 bp amplicon)\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePERV-A-VRB\u003c/span\u003e\u003csup\u003e\u003cspan style=\"font-size:19px;color:black;\"\u003ee\u003c/span\u003e\u003c/sup\u003e\u003csup\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/sup\u003e\u003cspan style=\"color:black;\"\u003efwd\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCCTACCAGTTATAATCAATTTAATTATGGC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6129\u0026ndash;6158\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border: none;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePERV-C rev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eTATGTTAGAGGATGGTCCTGGTC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6451\u0026ndash;6473\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 471.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003ePERV-A/C\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;(1260 bp amplicon)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePERV-A-VRB\u0026mdash;fwd\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003ePERV-C-TMR rev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCCTACCAGTTATAATCAATTTAATTATGGC\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eCTCAAACCACCCTTGAGTAGTTTCC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e6129\u0026ndash;6158\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003e7370\u0026ndash;7395\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eyes\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"color:black;\"\u003eno\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"color:black;\"\u003epGAPDH\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 109.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003epGAPDH-fwd\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003epGAPDH-rev\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003epGAPDH-probe\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 198.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eACATGGCCTCCAAGGAGTAAGA\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eTCAGTGTCGGGGTTGAGCTAG\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"color:black;\"\u003eHEX-CCA CCA ACC CCA GCA AGA G-BHQ\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;background: white;padding: 0in 3.5pt;height: 11.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003elocation of the PERV-C primer/probe according to accession number\u0026nbsp;AM229312 [33], of the GAPDH primer/probe according to Duvigneau et al., 2005 [41], primers to detect PERV-A/C\u0026nbsp;as described by\u0026nbsp;Wood et al. [7]\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eprimer/probe sequence is present in PERV-C sequence with accession number AM229312, clone1312 [33]\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eprimer/probe sequence is present in PERV-A sequence with accession number AY288779.1 [34]\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003csup\u003ed\u0026nbsp;\u003c/sup\u003edesigned by Takeuchi et al. 1998 [1]\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003csup\u003ee\u0026nbsp;\u003c/sup\u003eVRB, variable region B\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003eTable 2 Duplex real-time PCR for the detection of PERV-C\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003eand GAPDH in indigenous Greek black pigs from farm 1,\u0026nbsp;\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003eAuckland Island pigs and German slaughterhouse pigs\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 106.3pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Mean ct\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 63.8pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e260/280 nm values\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003ePERV-C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003epGAPDH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 251.35pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003eIndigenous Greek black pigs, farm 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 1 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e25.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e26.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 2 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e21.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e21.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 3 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e26.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e26.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 4 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e21.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e20.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 187.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003eAuckland Island pigs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e13947\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e13980\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 81.25pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e13983\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;padding:0in 0in 0in 0in;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 187.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003eGerman slaughterhouse pigs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"border:none;border-bottom:solid windowtext 1.0pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 1 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e24.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e18.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e28.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e20.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 2 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e21.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e17.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e22.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e18.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 3 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e26.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e17.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e31.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e20.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 4 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e25.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e18.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e26.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 5 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e25.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e28.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e20.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 6 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e26.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e26.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 7 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e25.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e27.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e20.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 8 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e23.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e23.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e20.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 9 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e29.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e31.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003ePig 10 spleen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e27.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e19.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e33.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e20.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e1.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003ea\u003c/sup\u003eindicates the purity of the DNA; n.d., not detected\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003eTable 3 Comparison if the sequences of the primers and probes used for the PCRs and real-time PCRs with the sequences found in the amplicons obtained by PCR8 using DNA from indigenous Greek black pigs 3 and 4\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eFwd/rev\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003efwd\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePrimer\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eCTGACCTGGATTAGAACTGG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePig 3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003edisrupted\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePig 4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eCTGACCTGGATTAGAACTGG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp 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top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePrimer\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eGATTAGAACTGGAAGCCCCAAGTGCTCT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003edisrupted\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePig 4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eGATTAGAACTGGAAGCCCCAAGTGCTCT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003erev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePrimer\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eACCATCCTCTAACATAACTTCTGGATCAGA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePig 3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003edisrupted\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePig 4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eACCATCCTCTAACATAACTTCTGGATCAGA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eProbe\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eCTCTAACATAACTTCTGGATCAGACCC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePig 3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";background:lightgrey;'\u003eT\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eT\u003cstrong\u003e\u003cspan style=\"background:lightgrey;\"\u003eAC\u003c/span\u003e\u003c/strong\u003eAA\u003cstrong\u003e\u003cspan style=\"background:lightgrey;\"\u003eT\u003c/span\u003e\u003c/strong\u003eA\u003cstrong\u003e\u003cspan style=\"background:lightgrey;\"\u003eC\u003c/span\u003e\u003c/strong\u003eAAC\u003cstrong\u003e\u003cspan style=\"background:lightgrey;\"\u003eC\u003c/span\u003e\u003c/strong\u003eTCTGGATCAGTCCC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePig 4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eCTCTAACATAACTTCTGGATCAGACCC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003erev\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePrimer\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eACTAAAATGGGGGCAAAACTT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePig 3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eA\u003cstrong\u003e\u003cspan style=\"background:lightgrey;\"\u003eT\u003c/span\u003e\u003c/strong\u003eTAAAA\u003cstrong\u003e\u003cspan style=\"background:lightgrey;\"\u003eCA\u003c/span\u003e\u003c/strong\u003eGGGGC\u003cstrong\u003e\u003cspan style=\"background:lightgrey;\"\u003eG\u003c/span\u003e\u003c/strong\u003eAAACTT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePig 4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003eACTAAAATGGGGGCAAAACTT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003ePCR8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 290.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003efwd corresponds to fwd primer of PCR5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 290.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003erev corresponds to the rev primer of the real-time PCR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 290.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003efwd corresponds to fwd primer of PCR6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 290.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003erev corresponds to the rev primer of PCR5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 290.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003efwd corresponds to the fwd primer of the real-time PCR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 42.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 290.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:11px;font-family:\"Courier New\";'\u003erev corresponds to the reverse primer of PCR5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:13px;\"\u003efwd, forward primer, rev, reverse primer; mutations in comparison to the primer sequence were marked bold and grey.\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"porcine endogenous retroviruses (PERVs), PERV-C, xenotransplantation","lastPublishedDoi":"10.21203/rs.3.rs-4807994/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4807994/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePorcine endogenous retrovirus C (PERV-C) is a gammaretrovirus present in the genome of many, but not all pigs. PERV-C is an ecotropic virus, able to infect only pig cells. In contrast, PERV-A and PERV-B are present in all pigs, they can infect cells of numerous host species including humans, and therefore pose a risk for xenotransplantation using pigs as donor animals. Intriguingly, PERV-C can recombine with PERV-A and the resulting PERV-A/C recombinants can also infect human cells and replicate to higher titers compared to the paternal PERV-A. Therefore, only PERV-C-free animals should be used for xenotransplantation. When screening wild boars and German landrace pigs for PERV-C, proviral variants were detected, and to detect these variants, new PCR methods were developed. Furthermore, PERVs are known to be active in living pigs as demonstrated by recombination and by different copy numbers in various organs and different parts of an organ. Copy numbers also increased with the age of the animals. This data was obtained by PCR using primers amplifying the highly conserved polymerase sequence which is identical in PERV-A, PERV-B and PERV-C. Here we screened indigenous Greek black pigs, Auckland Island pigs and German slaughterhouse pigs for the presence of PERV-C, using different PCR-based methods and optimized these detection methods. Auckland Island pigs were PERV-C-negative and represent the optimal donor pigs after genetic modifications.\u003c/p\u003e","manuscriptTitle":"Screening for porcine endogenous retrovirus - C (PERV-C) in pigs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-22 16:19:28","doi":"10.21203/rs.3.rs-4807994/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd433bfd-3101-4a1e-b5f6-137d86be566d","owner":[],"postedDate":"August 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T10:17:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-22 16:19:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4807994","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4807994","identity":"rs-4807994","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}