Porcine circovirus type 2 (PCV2) in Brazil in the past 55 years (1967-2022): Evolutionary rates and patterns

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L.F. Rodrigues, A. C.M. Cruz, A. E. Souza, F. B. Knackfuss, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4320085/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 circovirus type 2 (PCV2) is a common viral agent frequently described in herds, with a marked genetic variability impacted by factors such as vaccination and commercial trade. In this study, a retrospective analysis of PCV2 was performed based on molecular characterization, Bayesian inference, and the haplotype network in tissue samples obtained from Brazilian herds from 1967 to 2018. A nested PCR targeting a 225-base pair (bp) fragment of the PCV2 capsid gene was performed followed by a bi-directional sequencing. A total of 39/178 (21.9%) samples tested positive for PCV2. Genotype 2d was the major genotype described, and this genotype was detected before the introduction of the PCV2 vaccination. The mean substitution rate was 1.54 × 10 − 3 substitutions per site per year (s/s/y). The haplotype diversity of PCV2 was 0.98, which shows a high mutational rate compatible with the topology found in the haplotype network. In conclusion, most of the Brazilian samples were genotyped as PCV2d, even those obtained before the introduction of the PCV2 vaccine in Brazilian herds. The PCV2 sequence from 1967 represented the oldest description in America, and the second oldest description in the world; phylogenetic analysis and haplotype network indicated the high diversity of PCV2 as well as its high evolution rate. Our results may increase knowledge about the genetic diversity, evolution, and dispersal patterns of PCV2 in Brazil. Genotype Bayesian inference Haplotype network Pig Formalin-fixed paraffin-embedded (FFPE) tissue Introduction Porcine circovirus type 2 (PCV2) is commonly described in high-sanitary standard herds, and in continuous-flow, full-cycle, and multi-source termination pig farms. The marked genetic diversity and peculiar evolutionary pathways of the virus are related to its geographical distribution and its globalized presence due to livestock movements and trade routes [ 1 ]. PCV2a or PCV2b-based current vaccines are adequate in preventing clinical disease in most instances and may be able to minimize other genotypes’ replication but not abolish it [ 2 ]. Due to the introduction of inactivated PCV2 vaccines in Europe, North America, and in other areas, a continuous change in the prevalence of PCV2 genotypes has been observed [ 3 , 4 ]. Genotyping of Brazilian PCV2-positive samples was performed for the first time in 2012 [ 5 ] which demonstrated the circulation of genotype 2a and 2b in the country. In 2016, in a molecular study performed in 31 Brazilian swine herds [ 6 ], detected PCV2a in 73.3% of the plasma samples, followed by PCV2b in 26.7% of samples. Although the circulation of the virus is well-described in Brazil [ 6 , 7 , 8 ], the circulation of PCV2 genotypes before and after vaccination, and the origin of PCV2 genotypes in Brazilian herds, is not known. Here, we performed for the first time a 55-year retrospective study of PCV2 in swine populations from Brazil, addressing the molecular features of circulating genotypes in samples obtained from 1967 to 2018. In addition, the evolutionary history of PCV2 in Brazil was inferred using Bayesian inference and haplotype network analysis. Materials and methods Sample collection, tissue processing, and DNA extraction For this study, 178 frozen or formalin-fixed paraffin-embedded (FFPE) fragments of tissue from pigs from Southeastern Brazil obtained between 1967 and 2018 were selected. First, FFPE tissue from 143 pigs with different clinical signs, submitted to necropsy from 1967 to 2016 in Rio de Janeiro, were kindly provided by Laboratório de Anatomia Patológica do Centro Estadual de Pesquisa em Sanidade Animal Geraldo Manhães Carneiro (PESAGRORIO). Slices from each FFPE block were pooled by animal, identified, and kept in 2 mL microtubes (approximately 2 grams). The Deparaffinization Solution (QIAGEN®, USA) was used for deparaffinization, followed by DNA extraction using a commercial QIAamp DNA FFPE Tissue Kit ® (QIAGEN®, USA) according to manufacturer instructions. In addition, lung, kidney, liver, and lymph node samples from another 35 pigs were collected through an evisceration process performed in the daily routine of a slaughterhouse in the state of Espírito Santo in 2017 and 2018 [ 8 ]. Tissue fragments were stored at -20ºC until processing, pooled by animal, identified, and kept in 2 mL microtubes until DNA extraction. The extraction was performed with guanidine isothiocyanate and silica [ 9 ]. The study was approved by the Animal Use Ethics Committee of Fluminense Federal University under Protocol no.1013/2018. PCV2 nested PCR and sequencing Nested PCR was performed by targeting a 225 bp fragment of the PCV2 capsid gene [ 10 ]. Bi-directional sequencing was performed by Myleus Biotechnology (Belo Horizonte, Brazil) using an ABI3130 automated sequencer (Applied Biosystems, Foster City, CA). Sequence selection, edition, and phylogenetic analysis For genetic analysis, a dataset of complete PCV2 ORF2 sequences (VP2) up to the year 2022, from NCBI website was assembled using their reference numbers through GenBank or using the Basic Local Aligment Search Tool (BLAST). To build phylogenetic networks, identical prototype sequences were selected in DAMBE software [ 11 ], grouped, and the first sequence from each group was used (Supplementary Table S1 ). Sequences were edited and aligned using the MEGA 11 program [ 12 ]. Hasegawa, Kishino and Yano (HKY) model was applied in a maximimum likelihood analysis with 1000 bootstraps replicates. Recombination analysis All PCV2-positive samples were submitted to Bootscan [ 13 , 14 ], Chimaera [ 15 ], and 3SEQ [ 16 ] analysis (P value < 0.01) implemented in the Recombination Detection Program (RDP4) [ 17 ]. Evolutionary and Bayesian coalescent inference studies Estimation of evolutionary rates and the time of most common recent ancestor (tMRCA) of PCV2 sequences were determined by a coalescent Bayesian Markov chain Monte Carlo (MCMC) available in the BEAST 1.10.4 package [ 18 ]. Using the GTR and 200 million steps of MCMC, different molecular clock models (Strict and relaxed uncorrelated lognormal), as well as different population dynamics models (constant population size; exponential, expansional, and logistic population growth; and the Bayesian Skyline coalescent model) were tested. The marginal maximum likelihood estimation (MLE) was used to choose the best-fit parameters. The GTR model, relaxed lognormal clock, and the Bayesian Skyline coalescent model presented stronger evidence over the other parameters. Statistical uncertainty in the data was reflected by the 95% highest probability density (HPD) values. MCMC analysis was run for 200 × 10 6 generations to achieve the convergence of parameters, which was assessed after 25% burn-in and calculation of effective sample size (ESS). Trees with the largest product of posterior clade probabilities were selected from the posterior tree distribution after a 50 million steps (25%) burn-in using TreeAnnotator program [ 19 ]. The final tree was visualized with the FigTree v1.4.4 software. Genetic diversity and haplotype network analysis The p-distance between PCV2 genotype groups and within each genotype group was calculated using the neighbor-joining p-distance model with 1000 bootstrap replicates and the MEGA 11 software [ 12 ]. The data set was first converted to the DNAsp program, then the RDF file extension was used for median-joining (MJ) analysis with the NETWORK 10.20.0.0 program, adopting the criterion of parsimony [ 20 ]. Results A total of 39/178 (21.9%) of pooled swine samples tested positive for PCV2. Of these, 9/35 (25%) came from frozen samples, and 30/143 (20.9%) from FFPE samples. It was possible to identify positive samples over the 55 years included in this retrospective study (Fig. 1 ), and the sample RJ018/1967 is the oldest positive sample sequenced to date. After sequencing, 25 PCV2 sequences presented electropherograms of sufficient quality for sequence analysis and phylogeographic studies (24 from Rio de Janeiro and one from Espírito Santo state). The recovered partial ORF2 sequences of PCV2 from pooled organs in this study were deposited in GenBank (accession numbers: MN695256-MN695280). Based on the motif 5’-TCA/AAC/CCC/CTC/ACT/GTG-3’ (positions 86, 87, 88, 89, 90, 91) present in the ORF2 region corresponding to the AA sequence SNPLTV [ 3 ], 23 sequences were typed as PCV2d. For 2 samples (RJ071/1974 and RJ033/1981), with amino acid (AA) substitutions in positions 86 and 89, it was not possible to determine the type by this criterion. In addition to the AA determinant changes linked to genotype classification (P-S in samples RJ071/1974 and RJ033/1981), another 7 substitutions were found in 4 AAs (116, 125, 132, and 134). No evidence of recombination was detected in the samples from this study (Table 1 ). Table 1 Representation of non-synonymous substitutions found from partial sequence alignment of the PCV2 capsid protein coding region. The dots indicate the identity of amino acids with the reference sequences. The classification of PCV2 types in circulation in Southeast region of Brazil was performed according to Xiao et al., 2012. Reference sequences genotype 86 87 88 89 90 91 106 116 121 125 131 132 134 KC620521 PCV2a T N K I S I W R T L P K K MF150189 PCV2b S N P R S V W R S . T . T EU148503 PCV2c S N P L T V F . . . T . T KP698398 PCV2d S N P L T V W . . . T . N KT795287 PCV2e T N P L S V F . . . N . N Sequences from this study RJ018/1967 PCV2d S N P L T V W R T L T K K RJ019/1968 PCV2d . . . . . . . . . . . . . RJ020/1970 PCV2d . . . . . . . . . . . . N RJ105/1970 PCV2d . . . . . . . . . . . . N RJ116/1971 PCV2d . . . . . . . . . . . . N RJ124/1971 PCV2d . . . . . . . . . . . . . RJ131/1972 PCV2d . . . . . . . . . . . . N RJ044/1972 PCV2d . . . . . . . . . . . . N RJ071/1974 UG* P . . . . . . . . . . . . RJ155/1974 PCV2d . . . . . . . . . . . . N RJ065/1975 PCV2d . . . . . . . . . . . . N RJ110/1975 PCV2d . . . . . . . W . V . . . RJ113/1975 PCV2d . . . . . . . . . . . . N RJ069/1976 PCV2d . . . . . . . . . . . . N RJ059/1978 PCV2d . . . . . . . . . . . . N RJ137/1980 PCV2d . . . . . . . . . . . N D RJ149/1980 PCV2d . . . . . . . W . V . . N RJ033/1981 UG* . . . P . . . . . . . . Y RJ038/1982 PCV2d . . . . . . . . . . . . N RJ005/1990 PCV2d . . . . . . . . . . . . . RJ042/1990 PCV2d . . . . . . . . . . . . N RJ031/2008 PCV2d . . . . . . . . . . . . N RJ022/2011 PCV2d . . . . . . . . . . . . N RJ030/2011 PCV2d . . . . . . . . . . . . N ES011/2017 PCV2d . . . . . . . . . . . . . UG* undetermined genotype The within and between-group mean distance were calculated for each different PCV2 genotype, and the within group mean distance values ​​ranged from 0.009 (PCV2h) to 0.057 (PCV2a). The 25 sequences from this study were classified as PCV2d genotype, with an intragroup diversity of 0.016. The mean distance between PCV2d and other genotypes ranged from 0.031 (PCV2g) to 0.222 (PCV2e). Notably, the lowest genetic distances were observed between 2d to 2g (0.031), 2d to 2f (0.041), and 2d to 2b (0.054) (Supplementary Table S2 ). From the 147 PCV2 prototypes available in the GenBank, 66 unique sequences were used in evolutionary analysis along with the 25 sequences from this study. The phylogenetic tree showed the formation of 7 subclades, corresponding to the 7 genotypes of PCV2 already described. This PCV2d clade covered all samples from this study, including samples RJ033 and RJ071 that were not classified by amino acid substitutions and reference sequences from China, Korea, Russia, and Brazil (Fig. 2 ). Based on the fragment analyzed and the available dataset, no association was observed between geographic origin and genotypic distribution. For Bayesian analysis, identical study sequences were removed to avoid methodological bias due to an over-representation of Brazilian samples. A Bayesian tree was constructed based on a more stringent dataset composed of 13 PCV2 sequences from this study and 66 PCV2 prototypes. A mean substitution rate of 1.54 × 10 − 3 s/s/y was found for this region of capsid protein, with a 95% HPD interval between 9.32 × 10 − 4 and 2.18 × 10 − 3 s/s/y (Fig. 3 ). Based on the fragment analyzed, the origin of PCV2d dated back to the 60s, with some of our samples clustering on a separate branch with posterior probability (pp) = 1. The Bayesian analysis evidenced clades that were constituted by different genotypes. The formation of new clades was observed every 10 years approximately (1970, 1982, 1992, 2002), with a division into 2 large clades composed of samples 2a and 2b in the early 1970s, and the emergence of genotype 2e from 2b in 1975. A further division was observed in at the beginning of 1980s involving each large clade, generating 2 new subclades with no genotype changes. New divisions were then observed in 1992 and 2002, including a few samples from that study and 2d prototypes. The 91 sequences used in the haplotype network grouped into 78 haplotypes, with 34 median vectors (Supplementary Table S3 ). The haplotype diversity of PCV2 was 0.9878, a high mutational rate that is compatible with the topology found in the haplotype network (Fig. 4 ). The five clusters were represented in the haplotype network: two with genotypes PCV2a, one with genotype 2h, one with 2c, one with 2e; and a bigger central one with genotypes 2b, 2d, 2f, and 2g. Haplotypes 1 to 13 include all sequences from this study and are clustered together with haplotypes H17 (reference sequences from Japan, United States, South Korea, China, and Taiwan) and H18 (Brazil), all previously classified as PCV2d. The haplotype network showed a star-like topology, with most of the unique haplotypes closely related to central haplotypes and constituted by PVC2d genotypes: H3, composed of 10 Brazilian strains from this study, H74 (KX828226_KOR_2015), and 1 PCV2b genotype H77 (HQ831530_PRT_2003). The central haplotypes were the ancestral ones. In fact, the highest number of median vectors was observed in the cluster referring to genotypes 2b, 2d, 2f, and 2g (15 vectors), which comprised the oldest PCV2 samples (including those characterized in this study), followed by the cluster PCV2a (12 vectors) and PCV2c (6 vectors) (Supplementary File 3). The PCV2a genotype showed a dispersion in 2 clades which is explained by its high intragroup diversity. The haplotype that most differed from the central haplotype was H50 (KT867797_USA_2006), belonging to genotype 2e. As observed in phylogenetic and Bayesian analysis, no association was observed between geographical distribution and haplotypes (Fig. 4 ). Discussion One of the biggest challenges of this study was the use of archival swine tissue, stored for up to 50 years, in the molecular diagnosis and characterization of PCV2. Many clinical specimens are kept in collections world-wide, and the genetic information that this type of material may provide is extremely important for research related to human and animal health [ 21 ]. Tissue treatment with 10% formaldehyde often leads to DNA fragmentation, resulting in false negative results by degradation of target nucleic acid, and/or presence of substances that could inhibit the PCR reaction [ 22 , 23 ]. For this reason, the choice of the target fragment was important in DNA amplification of ancient FFPE tissues. The earliest detection of PCV2 from archived tissues/cell lines is from Germany in 1962, but the fragment (only 66 bp) was unfortunately outside of the target region for genotyping. In this study, PCV2 was detected in 25 samples, including 1 sample from 1967 and several samples from the late 60s and early 70s, which were the oldest FFPE tissue fragments available in the PESAGRO-RIO collection. RJ018/1967 is the second oldest positive sample in the world, and the oldest in the Americas. These results corroborate a retrospective study suggesting that PCV2 has possibly caused isolated episodes of systemic disease since 1962, long before the major epidemic from the 1990s [ 24 ]. In 2008, a genotype classification based on the nucleotide diversity in the ORF2 was accepted, and so far, 8 PCV2 genotypes have been described (a-h) based on analysis of the genomic region encoding the ORF2 capsid protein (nt 1486 − 1473) [ 25 , 26 , 27 ]. Among these, PCV2a, PCV2b, and PCV2d (earlier known as mutant PCV2b) are the major genotypes, with marked differences in virulence among them [ 2 , 28 ]. To date, there is no PCV2 evolutionary study in Brazil, just the analysis of the genotype of small groups of animals. Of the genotypes circulating in Brazil, Vidigal et al. [ 5 ] found in 9 samples the co-circulation of PCV2a (5 samples) and PCV2b (4 samples). Cruz et al. in 2016 [ 6 ], studying 31 Brazilian swine herds, detected the genotype PCV2a in 73.3% of the plasma samples, followed by PCV2b (26.7%). To the best of our knowledge, this retrospective study is the first description of PCV2d in Brazil, and its predominance comparing to 2a and 2b. PCV2 vaccine development has been in a state of continuous updating because of the high mutation rate of the PCV2 genome, and the emergence of new PCV2 subtypes due to selective pressures promoted also by vaccination-induced immunity escape [ 29 ]. PCV2 vaccination started in 2004 in Europe and in 2006 in the Americas [ 2 ]. Based on the criteria previously described [ 27 ] and phylogenetic analyses, the PCV2d genotype was the only genotype detected in this study, which included samples collected prior to the introduction of vaccines in Brazil (2007), demonstrating the circulation of PCV2d genotype in Brazilian herds many years before the introduction of vaccination. Similar results were obtained by Tsai et al. [ 30 ] who detected PCV2a, PCV2b, and even PCV2d genotypes in Taiwan in 2000, 2001, and 2002, also before the introduction of PCV2 vaccines in this country. Although the DNA fragmentation of ancient FFPE has led to a limited fragment, the mutation rate found in the samples of this study (1.54 × 10 − 3 ) are very close to those obtained by other authors based on complete capsid sequences (1.2 × 10 − 3 ) [ 2 , 3 , 29 ]. Our study demonstrated the circulation of this genotype since 1967 (the oldest sequence detected in Brazil), and our results suggest that it still represents the main circulating genotype even 50 years later. Bayesian inference has become the standard for PCV2 phylogenetic analyses [ 31 ]. In this study, the topology of the phylogeographic tree was also recovered by the network analysis. The high haplotypic diversity of PCV2 observed in this study is related to its high mutational rate, as described by several authors [ 29 , 32 , 33 ], even considering that the sequences obtained were partial. The large number of median vectors in the PCV2a and PCV2c clusters can be justified since PCV2a is the first reported genotype [ 2 ] and because PCV2c was first identified from pig sera collected in 1980, 1987, and 1990 in Denmark, and not long reported since then [ 34 ]. The larger number of median vectors (15 vectors) in clusters with genotypes 2b, 2d, 2f, and 2g was an interesting finding, and suggests that these genotypes share a common origin. This hypothesis is supported by the low intergroup genetic distances among these genotypes (Supplementary File 2), suggesting once again a close evolutionary relationship. As demonstrated by the phylogeographic tree, PCV2b and PCV2d share a common ancestor, with the emergence of PCV2d possibly predating PCV2b. This is unlike what was previously assumed [ 3 , 35 ]. In our analysis, the origin of PCV2d dated back to the 60s. Furthermore, some of our samples clustered on a distinct branch of the Bayesian tree (pp = 1), preceding the origin of the "modern" 2d. We are aware that this surprising topology may be a methodological bias due to the short length of our fragment and the lack of old reference samples available in the databases. However, it is worth noting that several of the samples described in this study are the oldest sequences to date, and the discovery of PCV2 circulation in the 1960s and 1970s could completely change what is known about the evolutionary history of this virus. Therefore, further studies using the whole PCV2 genome, and a more comprehensive database are needed to confirm or refute this hypothesis. In conclusion, the technique of DNA extraction in ancient tissue made it possible to obtain PCV2 genetic material from FFPE samples up to 50 years old, expanding the information about the evolution of PCV2 in Brazil and the positive PCV2 sample from 1967, which represented the second oldest description in the world. All PCV2 samples from this study were genotyped as PCV2d, even those obtained before the outbreak of circoviruses and introduction of PCV2 vaccine in Brazilian herds. Phylogenetic analysis and haplotype network confirmed the high diversity of PCV2, as well as its high mutation rate. This information may guide further studies to clarify the routes of entry and the spread of PCV2 in Brazil and contributes to increased knowledge about the evolution of the virus. Declarations Author Contribution I.L. Rodrigues and C.H.C. Costa performed the sample collection; I.L. Rodrigues, A.E. Souza and A.C.M. Cruz carried out the molecular experiment; F.B. 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Vet Microbiol 208:239–246. https://doi:10.1016/j.vetmic.2017.08.006 Tsai GT, Lin YC, Lin WH et al (2019) Phylogeographic and genetic characterization of porcine circovirus type 2 in Taiwan from 2001–2017. Sci Rep 9(1):10782. https://doi 10.1038/s41598-019-47209-1 Additional Declarations No competing interests reported. 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-4320085","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":297591166,"identity":"10a1020a-c1ad-41f9-ba8b-8416e7c15f12","order_by":0,"name":"I. L.F. Rodrigues","email":"","orcid":"","institution":"Fluminense Federal University","correspondingAuthor":false,"prefix":"","firstName":"I.","middleName":"L.F.","lastName":"Rodrigues","suffix":""},{"id":297591171,"identity":"92fde2ed-9e8a-4da3-a9a7-edf230713ea0","order_by":1,"name":"A. C.M. Cruz","email":"","orcid":"","institution":"Fluminense Federal University","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"C.M.","lastName":"Cruz","suffix":""},{"id":297591176,"identity":"d4345817-cffc-455b-b1ad-c91bb6ca7c0b","order_by":2,"name":"A. E. Souza","email":"","orcid":"","institution":"Fluminense Federal University","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"E.","lastName":"Souza","suffix":""},{"id":297591182,"identity":"304fa58d-59c3-49fb-8555-75ec2c815d7a","order_by":3,"name":"F. B. Knackfuss","email":"","orcid":"","institution":"Universidade do Grande Rio/ UNIGRANRIO","correspondingAuthor":false,"prefix":"","firstName":"F.","middleName":"B.","lastName":"Knackfuss","suffix":""},{"id":297591187,"identity":"f13e16ba-3170-43f8-9bb1-87ab9cec6fb5","order_by":4,"name":"C. H.C. Costa","email":"","orcid":"","institution":"Agricultiural Research Company of the State of Rio de Janeiro-Pesagro-Rio","correspondingAuthor":false,"prefix":"","firstName":"C.","middleName":"H.C.","lastName":"Costa","suffix":""},{"id":297591191,"identity":"0aeeda6b-e91b-47da-a0e8-496f52e0bdb9","order_by":5,"name":"B. V. Lago","email":"","orcid":"","institution":"Oswaldo Cruz Foundation","correspondingAuthor":false,"prefix":"","firstName":"B.","middleName":"V.","lastName":"Lago","suffix":""},{"id":297591195,"identity":"79ad2efe-5290-4c9c-88af-1a4e93ba7344","order_by":6,"name":"R. L. Silveira","email":"","orcid":"","institution":"Fluminense Federal University","correspondingAuthor":false,"prefix":"","firstName":"R.","middleName":"L.","lastName":"Silveira","suffix":""},{"id":297591198,"identity":"9de8643f-35e5-467b-b26b-926b4ec3c023","order_by":7,"name":"T. X. Castro","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDADfmYGAwYGGzkQiR/wgMkEBgbJZpDiNGMStBgcIFaLvUTyw48/f9yTMz7OvIGZJ8GAwVz6AAFbeI4ZS/MkFBubHWYrAGux7EsgoIW9h0GaISEhcdthHgNm3h9/GAzOEPILMw/zzx9ALZubgVpAthDWwt7DJsED1AL0CLFazhwzs+ZJSzCWAPrl4JwEAx7LHgJa2GckP775wyZBjr//8MYHbxIM5Mx5CGhBAQcYYBE1CkbBKBgFo4AyAADnfjXc3khvugAAAABJRU5ErkJggg==","orcid":"","institution":"Fluminense Federal University","correspondingAuthor":true,"prefix":"","firstName":"T.","middleName":"X.","lastName":"Castro","suffix":""}],"badges":[],"createdAt":"2024-04-24 19:10:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4320085/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4320085/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56718309,"identity":"5c4aeb86-e428-4bf5-86c7-83226d5f7437","added_by":"auto","created_at":"2024-05-19 00:01:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":643544,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4320085/v1/38be3f90-f6c4-4265-ba91-eb22bf8d6028.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Porcine circovirus type 2 (PCV2) in Brazil in the past 55 years (1967-2022): Evolutionary rates and patterns","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePorcine circovirus type 2 (PCV2) is commonly described in high-sanitary standard herds, and in continuous-flow, full-cycle, and multi-source termination pig farms. The marked genetic diversity and peculiar evolutionary pathways of the virus are related to its geographical distribution and its globalized presence due to livestock movements and trade routes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. PCV2a or PCV2b-based current vaccines are adequate in preventing clinical disease in most instances and may be able to minimize other genotypes\u0026rsquo; replication but not abolish it [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to the introduction of inactivated PCV2 vaccines in Europe, North America, and in other areas, a continuous change in the prevalence of PCV2 genotypes has been observed [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Genotyping of Brazilian PCV2-positive samples was performed for the first time in 2012 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] which demonstrated the circulation of genotype 2a and 2b in the country. In 2016, in a molecular study performed in 31 Brazilian swine herds [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], detected PCV2a in 73.3% of the plasma samples, followed by PCV2b in 26.7% of samples. Although the circulation of the virus is well-described in Brazil [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the circulation of PCV2 genotypes before and after vaccination, and the origin of PCV2 genotypes in Brazilian herds, is not known.\u003c/p\u003e \u003cp\u003eHere, we performed for the first time a 55-year retrospective study of PCV2 in swine populations from Brazil, addressing the molecular features of circulating genotypes in samples obtained from 1967 to 2018. In addition, the evolutionary history of PCV2 in Brazil was inferred using Bayesian inference and haplotype network analysis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection, tissue processing, and DNA extraction\u003c/h2\u003e \u003cp\u003eFor this study, 178 frozen or formalin-fixed paraffin-embedded (FFPE) fragments of tissue from pigs from Southeastern Brazil obtained between 1967 and 2018 were selected. First, FFPE tissue from 143 pigs with different clinical signs, submitted to necropsy from 1967 to 2016 in Rio de Janeiro, were kindly provided by Laborat\u0026oacute;rio de Anatomia Patol\u0026oacute;gica do Centro Estadual de Pesquisa em Sanidade Animal Geraldo Manh\u0026atilde;es Carneiro (PESAGRORIO). Slices from each FFPE block were pooled by animal, identified, and kept in 2 mL microtubes (approximately 2 grams). The Deparaffinization Solution (QIAGEN\u0026reg;, USA) was used for deparaffinization, followed by DNA extraction using a commercial QIAamp DNA FFPE Tissue Kit \u0026reg; (QIAGEN\u0026reg;, USA) according to manufacturer instructions.\u003c/p\u003e \u003cp\u003eIn addition, lung, kidney, liver, and lymph node samples from another 35 pigs were collected through an evisceration process performed in the daily routine of a slaughterhouse in the state of Esp\u0026iacute;rito Santo in 2017 and 2018 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Tissue fragments were stored at -20\u0026ordm;C until processing, pooled by animal, identified, and kept in 2 mL microtubes until DNA extraction. The extraction was performed with guanidine isothiocyanate and silica [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The study was approved by the Animal Use Ethics Committee of Fluminense Federal University under Protocol no.1013/2018.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePCV2 nested PCR and sequencing\u003c/h2\u003e \u003cp\u003eNested PCR was performed by targeting a 225 bp fragment of the PCV2 capsid gene [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Bi-directional sequencing was performed by Myleus Biotechnology (Belo Horizonte, Brazil) using an ABI3130 automated sequencer (Applied Biosystems, Foster City, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSequence selection, edition, and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eFor genetic analysis, a dataset of complete PCV2 ORF2 sequences (VP2) up to the year 2022, from NCBI website was assembled using their reference numbers through GenBank or using the \u003cem\u003eBasic Local Aligment Search Tool\u003c/em\u003e (BLAST). To build phylogenetic networks, identical prototype sequences were selected in DAMBE software [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], grouped, and the first sequence from each group was used (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Sequences were edited and aligned using the MEGA 11 program [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Hasegawa, Kishino and Yano (HKY) model was applied in a maximimum likelihood analysis with 1000 bootstraps replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRecombination analysis\u003c/h2\u003e \u003cp\u003eAll PCV2-positive samples were submitted to Bootscan [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], Chimaera [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and 3SEQ [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] analysis (P value\u0026thinsp;\u0026lt;\u0026thinsp;0.01) implemented in the Recombination Detection Program (RDP4) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEvolutionary and Bayesian coalescent inference studies\u003c/h2\u003e \u003cp\u003eEstimation of evolutionary rates and the time of most common recent ancestor (tMRCA) of PCV2 sequences were determined by a coalescent Bayesian Markov chain Monte Carlo (MCMC) available in the BEAST 1.10.4 package [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Using the GTR and 200\u0026nbsp;million steps of MCMC, different molecular clock models (Strict and relaxed uncorrelated lognormal), as well as different population dynamics models (constant population size; exponential, expansional, and logistic population growth; and the Bayesian Skyline coalescent model) were tested. The marginal maximum likelihood estimation (MLE) was used to choose the best-fit parameters. The GTR model, relaxed lognormal clock, and the Bayesian Skyline coalescent model presented stronger evidence over the other parameters. Statistical uncertainty in the data was reflected by the 95% highest probability density (HPD) values. MCMC analysis was run for 200 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e generations to achieve the convergence of parameters, which was assessed after 25% burn-in and calculation of effective sample size (ESS). Trees with the largest product of posterior clade probabilities were selected from the posterior tree distribution after a 50\u0026nbsp;million steps (25%) burn-in using TreeAnnotator program [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The final tree was visualized with the FigTree v1.4.4 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenetic diversity and haplotype network analysis\u003c/h2\u003e \u003cp\u003eThe p-distance between PCV2 genotype groups and within each genotype group was calculated using the neighbor-joining p-distance model with 1000 bootstrap replicates and the MEGA 11 software [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The data set was first converted to the DNAsp program, then the RDF file extension was used for median-joining (MJ) analysis with the NETWORK 10.20.0.0 program, adopting the criterion of parsimony [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 39/178 (21.9%) of pooled swine samples tested positive for PCV2. Of these, 9/35 (25%) came from frozen samples, and 30/143 (20.9%) from FFPE samples. It was possible to identify positive samples over the 55 years included in this retrospective study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and the sample RJ018/1967 is the oldest positive sample sequenced to date.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter sequencing, 25 PCV2 sequences presented electropherograms of sufficient quality for sequence analysis and phylogeographic studies (24 from Rio de Janeiro and one from Esp\u0026iacute;rito Santo state). The recovered partial ORF2 sequences of PCV2 from pooled organs in this study were deposited in GenBank (accession numbers: MN695256-MN695280). Based on the motif 5\u0026rsquo;-TCA/AAC/CCC/CTC/ACT/GTG-3\u0026rsquo; (positions 86, 87, 88, 89, 90, 91) present in the ORF2 region corresponding to the AA sequence SNPLTV [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], 23 sequences were typed as PCV2d. For 2 samples (RJ071/1974 and RJ033/1981), with amino acid (AA) substitutions in positions 86 and 89, it was not possible to determine the type by this criterion.\u003c/p\u003e \u003cp\u003eIn addition to the AA determinant changes linked to genotype classification (P-S in samples RJ071/1974 and RJ033/1981), another 7 substitutions were found in 4 AAs (116, 125, 132, and 134). No evidence of recombination was detected in the samples from this study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRepresentation of non-synonymous substitutions found from partial sequence alignment of the PCV2 capsid protein coding region. The dots indicate the identity of amino acids with the reference sequences. The classification of PCV2 types in circulation in Southeast region of Brazil was performed according to Xiao et al., 2012.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"15\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" 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colname=\"c15\"\u003e \u003cp\u003e134\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKC620521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMF150189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e 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colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKP698398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKT795287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSequences from this study\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ018/1967\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ019/1968\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ020/1970\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ105/1970\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e 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colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ124/1971\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e 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colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ044/1972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e 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colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e 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align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ069/1976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e 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align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ137/1980\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e 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colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ022/2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRJ030/2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eES011/2017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCV2d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eUG* undetermined genotype\u003c/p\u003e \u003cp\u003eThe within and between-group mean distance were calculated for each different PCV2 genotype, and the within group mean distance values ​​ranged from 0.009 (PCV2h) to 0.057 (PCV2a). The 25 sequences from this study were classified as PCV2d genotype, with an intragroup diversity of 0.016. The mean distance between PCV2d and other genotypes ranged from 0.031 (PCV2g) to 0.222 (PCV2e). Notably, the lowest genetic distances were observed between 2d to 2g (0.031), 2d to 2f (0.041), and 2d to 2b (0.054) (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFrom the 147 PCV2 prototypes available in the GenBank, 66 unique sequences were used in evolutionary analysis along with the 25 sequences from this study. The phylogenetic tree showed the formation of 7 subclades, corresponding to the 7 genotypes of PCV2 already described. This PCV2d clade covered all samples from this study, including samples RJ033 and RJ071 that were not classified by amino acid substitutions and reference sequences from China, Korea, Russia, and Brazil (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on the fragment analyzed and the available dataset, no association was observed between geographic origin and genotypic distribution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor Bayesian analysis, identical study sequences were removed to avoid methodological bias due to an over-representation of Brazilian samples. A Bayesian tree was constructed based on a more stringent dataset composed of 13 PCV2 sequences from this study and 66 PCV2 prototypes. A mean substitution rate of 1.54 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e s/s/y was found for this region of capsid protein, with a 95% HPD interval between 9.32 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e and 2.18 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e s/s/y (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the fragment analyzed, the origin of PCV2d dated back to the 60s, with some of our samples clustering on a separate branch with posterior probability (pp)\u0026thinsp;=\u0026thinsp;1. The Bayesian analysis evidenced clades that were constituted by different genotypes. The formation of new clades was observed every 10 years approximately (1970, 1982, 1992, 2002), with a division into 2 large clades composed of samples 2a and 2b in the early 1970s, and the emergence of genotype 2e from 2b in 1975. A further division was observed in at the beginning of 1980s involving each large clade, generating 2 new subclades with no genotype changes. New divisions were then observed in 1992 and 2002, including a few samples from that study and 2d prototypes.\u003c/p\u003e \u003cp\u003eThe 91 sequences used in the haplotype network grouped into 78 haplotypes, with 34 median vectors (Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The haplotype diversity of PCV2 was 0.9878, a high mutational rate that is compatible with the topology found in the haplotype network (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The five clusters were represented in the haplotype network: two with genotypes PCV2a, one with genotype 2h, one with 2c, one with 2e; and a bigger central one with genotypes 2b, 2d, 2f, and 2g. Haplotypes 1 to 13 include all sequences from this study and are clustered together with haplotypes H17 (reference sequences from Japan, United States, South Korea, China, and Taiwan) and H18 (Brazil), all previously classified as PCV2d.\u003c/p\u003e \u003cp\u003eThe haplotype network showed a star-like topology, with most of the unique haplotypes closely related to central haplotypes and constituted by PVC2d genotypes: H3, composed of 10 Brazilian strains from this study, H74 (KX828226_KOR_2015), and 1 PCV2b genotype H77 (HQ831530_PRT_2003). The central haplotypes were the ancestral ones. In fact, the highest number of median vectors was observed in the cluster referring to genotypes 2b, 2d, 2f, and 2g (15 vectors), which comprised the oldest PCV2 samples (including those characterized in this study), followed by the cluster PCV2a (12 vectors) and PCV2c (6 vectors) (Supplementary File 3). The PCV2a genotype showed a dispersion in 2 clades which is explained by its high intragroup diversity. The haplotype that most differed from the central haplotype was H50 (KT867797_USA_2006), belonging to genotype 2e. As observed in phylogenetic and Bayesian analysis, no association was observed between geographical distribution and haplotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOne of the biggest challenges of this study was the use of archival swine tissue, stored for up to 50 years, in the molecular diagnosis and characterization of PCV2. Many clinical specimens are kept in collections world-wide, and the genetic information that this type of material may provide is extremely important for research related to human and animal health [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Tissue treatment with 10% formaldehyde often leads to DNA fragmentation, resulting in false negative results by degradation of target nucleic acid, and/or presence of substances that could inhibit the PCR reaction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For this reason, the choice of the target fragment was important in DNA amplification of ancient FFPE tissues.\u003c/p\u003e \u003cp\u003eThe earliest detection of PCV2 from archived tissues/cell lines is from Germany in 1962, but the fragment (only 66 bp) was unfortunately outside of the target region for genotyping. In this study, PCV2 was detected in 25 samples, including 1 sample from 1967 and several samples from the late 60s and early 70s, which were the oldest FFPE tissue fragments available in the PESAGRO-RIO collection. RJ018/1967 is the second oldest positive sample in the world, and the oldest in the Americas. These results corroborate a retrospective study suggesting that PCV2 has possibly caused isolated episodes of systemic disease since 1962, long before the major epidemic from the 1990s [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn 2008, a genotype classification based on the nucleotide diversity in the ORF2 was accepted, and so far, 8 PCV2 genotypes have been described (a-h) based on analysis of the genomic region encoding the ORF2 capsid protein (nt 1486\u0026thinsp;\u0026minus;\u0026thinsp;1473) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Among these, PCV2a, PCV2b, and PCV2d (earlier known as mutant PCV2b) are the major genotypes, with marked differences in virulence among them [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To date, there is no PCV2 evolutionary study in Brazil, just the analysis of the genotype of small groups of animals. Of the genotypes circulating in Brazil, Vidigal et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] found in 9 samples the co-circulation of PCV2a (5 samples) and PCV2b (4 samples). Cruz et al. in 2016 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], studying 31 Brazilian swine herds, detected the genotype PCV2a in 73.3% of the plasma samples, followed by PCV2b (26.7%). To the best of our knowledge, this retrospective study is the first description of PCV2d in Brazil, and its predominance comparing to 2a and 2b.\u003c/p\u003e \u003cp\u003ePCV2 vaccine development has been in a state of continuous updating because of the high mutation rate of the PCV2 genome, and the emergence of new PCV2 subtypes due to selective pressures promoted also by vaccination-induced immunity escape [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. PCV2 vaccination started in 2004 in Europe and in 2006 in the Americas [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Based on the criteria previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and phylogenetic analyses, the PCV2d genotype was the only genotype detected in this study, which included samples collected prior to the introduction of vaccines in Brazil (2007), demonstrating the circulation of PCV2d genotype in Brazilian herds many years before the introduction of vaccination. Similar results were obtained by Tsai et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] who detected PCV2a, PCV2b, and even PCV2d genotypes in Taiwan in 2000, 2001, and 2002, also before the introduction of PCV2 vaccines in this country.\u003c/p\u003e \u003cp\u003eAlthough the DNA fragmentation of ancient FFPE has led to a limited fragment, the mutation rate found in the samples of this study (1.54 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) are very close to those obtained by other authors based on complete capsid sequences (1.2 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our study demonstrated the circulation of this genotype since 1967 (the oldest sequence detected in Brazil), and our results suggest that it still represents the main circulating genotype even 50 years later.\u003c/p\u003e \u003cp\u003eBayesian inference has become the standard for PCV2 phylogenetic analyses [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, the topology of the phylogeographic tree was also recovered by the network analysis. The high haplotypic diversity of PCV2 observed in this study is related to its high mutational rate, as described by several authors [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], even considering that the sequences obtained were partial. The large number of median vectors in the PCV2a and PCV2c clusters can be justified since PCV2a is the first reported genotype [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and because PCV2c was first identified from pig sera collected in 1980, 1987, and 1990 in Denmark, and not long reported since then [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The larger number of median vectors (15 vectors) in clusters with genotypes 2b, 2d, 2f, and 2g was an interesting finding, and suggests that these genotypes share a common origin. This hypothesis is supported by the low intergroup genetic distances among these genotypes (Supplementary File 2), suggesting once again a close evolutionary relationship.\u003c/p\u003e \u003cp\u003eAs demonstrated by the phylogeographic tree, PCV2b and PCV2d share a common ancestor, with the emergence of PCV2d possibly predating PCV2b. This is unlike what was previously assumed [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In our analysis, the origin of PCV2d dated back to the 60s. Furthermore, some of our samples clustered on a distinct branch of the Bayesian tree (pp\u0026thinsp;=\u0026thinsp;1), preceding the origin of the \"modern\" 2d. We are aware that this surprising topology may be a methodological bias due to the short length of our fragment and the lack of old reference samples available in the databases. However, it is worth noting that several of the samples described in this study are the oldest sequences to date, and the discovery of PCV2 circulation in the 1960s and 1970s could completely change what is known about the evolutionary history of this virus. Therefore, further studies using the whole PCV2 genome, and a more comprehensive database are needed to confirm or refute this hypothesis.\u003c/p\u003e \u003cp\u003eIn conclusion, the technique of DNA extraction in ancient tissue made it possible to obtain PCV2 genetic material from FFPE samples up to 50 years old, expanding the information about the evolution of PCV2 in Brazil and the positive PCV2 sample from 1967, which represented the second oldest description in the world. All PCV2 samples from this study were genotyped as PCV2d, even those obtained before the outbreak of circoviruses and introduction of PCV2 vaccine in Brazilian herds. Phylogenetic analysis and haplotype network confirmed the high diversity of PCV2, as well as its high mutation rate. This information may guide further studies to clarify the routes of entry and the spread of PCV2 in Brazil and contributes to increased knowledge about the evolution of the virus.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eI.L. Rodrigues and C.H.C. Costa performed the sample collection; I.L. Rodrigues, A.E. Souza and A.C.M. Cruz carried out the molecular experiment; F.B. Knackfuss performed the haplotypes analysis; B. V. Lagos and T.X. Castro performed the phylogenetic and Bayesians analysis. R.L.Silveira and T.X. Castro conceived of the presented idea and supervised the project. All authors provided critical feedback and helped shape the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the Plataforma de Sequenciamento de DNA of the Laboratorio Multiusuarios ́ de Microbiologia e Parasitologia (LMMP) of the Universidade Federal Fluminense.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eOlvera A, Cortey M, Segal\u0026eacute;s J (2007) Molecular evolution of porcine circovirus type 2 genomes: Phylogeny and clonality. Virology 357:175\u0026ndash;185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.virol.2006.07.047\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKaruppannan AN, Opriessnig T (2017) Porcine circovirus type 2 (PCV2) vaccines in the context of current molecular epidemiology. Viruses 9:1\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.3390/v9050099\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXiao C, Halbur PG, Opriessnig T (2015) Global molecular genetic analysis of porcine circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d. J Gen Virol 96:1830\u0026ndash;1841. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1099/vir.0.000100\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKwon T, Lee DU, Yoo SJ et al (2017) Genotypic diversity of porcine circovirus type 2 (PCV2) and genotype shift to PCV2d in Korean pig population. Virus Res 228:24\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.virusres.2016.11.015\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eVidigal PMP, Mafra CL, Silva FMF (2012) Tripping over emerging pathogens around the world: A phylogeographical approach for determining the epidemiology of Porcine circovirus-2 (PCV-2), considering global trading. Virus Res 163:320\u0026ndash;327. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.virusres.2011.10.019\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCruz ACM, Silveira RL, Baez CF et al (2016) Clinical aspects and weight gain reduction in swine infected with porcine circovirus type 2 and torque teno sus virus in Brazil. 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Sci Rep 9(1):10782. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi 10.1038/s41598-019-47209-1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\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":"Genotype, Bayesian inference, Haplotype network, Pig, Formalin-fixed paraffin-embedded (FFPE) tissue","lastPublishedDoi":"10.21203/rs.3.rs-4320085/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4320085/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePorcine circovirus type 2 (PCV2) is a common viral agent frequently described in herds, with a marked genetic variability impacted by factors such as vaccination and commercial trade. In this study, a retrospective analysis of PCV2 was performed based on molecular characterization, Bayesian inference, and the haplotype network in tissue samples obtained from Brazilian herds from 1967 to 2018. A nested PCR targeting a 225-base pair (bp) fragment of the PCV2 capsid gene was performed followed by a bi-directional sequencing. A total of 39/178 (21.9%) samples tested positive for PCV2. Genotype 2d was the major genotype described, and this genotype was detected before the introduction of the PCV2 vaccination. The mean substitution rate was 1.54 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e substitutions per site per year (s/s/y). The haplotype diversity of PCV2 was 0.98, which shows a high mutational rate compatible with the topology found in the haplotype network. In conclusion, most of the Brazilian samples were genotyped as PCV2d, even those obtained before the introduction of the PCV2 vaccine in Brazilian herds. The PCV2 sequence from 1967 represented the oldest description in America, and the second oldest description in the world; phylogenetic analysis and haplotype network indicated the high diversity of PCV2 as well as its high evolution rate. Our results may increase knowledge about the genetic diversity, evolution, and dispersal patterns of PCV2 in Brazil.\u003c/p\u003e","manuscriptTitle":"Porcine circovirus type 2 (PCV2) in Brazil in the past 55 years (1967-2022): Evolutionary rates and patterns","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 09:57:36","doi":"10.21203/rs.3.rs-4320085/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":"81a1f15f-3261-4faf-ba01-d7e4ac81c1de","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-18T21:31:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-02 09:57:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4320085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4320085","identity":"rs-4320085","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}