Effect of stabilizers on the detection of swine influenza A virus (swIAV) in spiked oral fluids over time

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Grau, K. Lillie-Jaschniski, A. Graaf-Rau, T. Harder, M. Eddicks, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4486513/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Nov, 2024 Read the published version in Porcine Health Management → Version 1 posted 7 You are reading this latest preprint version Abstract Background Aggregated samples such as oral fluids (OFs) display an animal friendly and time and cost-efficient sample type for swine Influenza A virus (swIAV) monitoring. However, further molecular and biological characterization of swIAV is of particular significance. The reportedly inferior suitability of aggregated samples for subtyping of swIAV presents a major drawback compared to nasal swabs, still considered the most appropriate sample type for this purpose (1). In addition, the viral load in the original sample, storage conditions and characteristics of different swIAV strains might further compromise the eligibility of aggregated samples for molecular detection and subtyping. Therefore, the present study aimed to evaluate the suitability of stabilizing media to minimize the degradation of viral RNA and thus increase the detection and subtyping rate of swIAV by RT-qPCR in spiked OFs under different conditions (virus strain, storage temperature and viral load in the original sample) over a time span of 14 days. Results The use of stabilizing media in spiked OFs resulted in a significant higher probability to detect swIAV RNA compared to OFs without stabilizers (OR = 46.1, p < 0.001). In addition, the probability of swIAV degradation over time was significantly reduced in samples suspended with stabilizer (OR = 5.80, p < 0.001), in samples stored at 4°C (OR = 2.53, p < 0.001) and in samples spiked with the avian derived H1N1 subtype (OR = 2.26, p < 0.01). No significant differences in swIAV RNA detection and degradation of swIAV RNA in spiked OFs over time were observed between the three different stabilizing media. Conclusion Addition of stabilizers and storage of samples at cooled conditions highly significantly improved detection and subtyping of swIAV in spiked OFs. swine influenza A virus subtyping surveillance stabilizing media RT-qPCR Figures Figure 1 Figure 2 Figure 3 Background Economic losses due to swine Influenza A virus (swIAV) impacting the swine industry and their potential threat to human health highlight the need for rapid and accurate identification and subtyping of these viruses (2, 3). Commonly more than one swIAV subtype circulates in one herd (4). Frequent spill over infections of human A (H1N1) 2009 pandemic virus into swine populations since 2009 established enzootically infected herds with highly variable clinical signs (5). As a result, the already sophisticated diagnosis of swIAV is further complicated (6–8). Whereas diagnostics for individual animals are both, time-consuming and cost-intensive, sampling techniques assessing groups of pigs have gained increasing interest concerning the detection and monitoring of various pathogens. One common aggregate non-invasive sampling method is the collection of oral fluids (OFs) (9–11), which can be collected also by staff with little previous experience. However, those specimens have shown shortcomings concerning their suitability for the subtyping of swIAV strains (9) due to lower viral loads in aggregate OF samples compared to individual samples, i.e. nasal swabs (12). In addition, the presence of salivary enzymes, proteins (9, 13), cellular debris (14) and high bacterial loads in oral fluids as well as inadequate transport conditions (15) can contribute to further degradation of viral RNA. To avoid degradation of intact virus or viral nucleic acids different stabilizers have been described for the molecular detection of PRRSV (16), avian influenza virus and New castle Disease virus (17), SARS-CoV-2 (18), herpes simplex viruses, enteroviruses, and adenoviruses (19). However, the stability of swIAV in aggregated samples is poorly investigated. Therefore, the aim of the present study was to assess under cooled conditions whether the addition of stabilizing media to OFs, spiked with two different swIAV strains, improve the rate of detection and the suitability of subtyping by RT-qPCR under different storage conditions and viral loads in the original sample over a time period of 14 days. Material and methods A minimum of 50mL OF was collected from 24 pigs on a farm which was considered negative for swIAV through regular monitoring by RT-qPCR, ELISA and hemagglutination inhibition (HI) assays, all with negative results. Until further analysis the samples were stored at -80°C. Before further investigations the samples were centrifuged at 2000g for 15 minutes. The samples were spiked with swIAV isolates A/swine/Germany/2022AI04470/2022 (H1avN2, clade 1C) and A/swine/Germany/2022AI03601/2022 (H1pdmN2, clade 1A) propagated in swine testicle cells for two passages (FLI Collection of Cell Lines in Veterinary Medicine CCLV-RIE 0606), respectively to achieve each threshold cycle values (Ct) of 25 and 32, respectively. And additionally, stabilizing media was added to the spiked OF specimens at a ratio of 3:1. Three different media were used (i) Sigma-Virocult® MW950S (V) (Check Diagnostics GmbH, Westerau, Germany), (ii) PrimeStore® MTM (P) (Longhorn Vaccines & Diagnostics LLC, Bethesda, USA, P) and (iii) NucleoProtect VET Reagent® (N) (MACHEREY-NAGEL GmbH & Co. KG, Dueren, Germany, M&N). Aliquots of the spiked and diluted oral fluids were stored at either 4°C or at room temperature (22 ± 2°C) and replica of supernatants were taken for analysis at 72 h, 7 days (168 h) and 14 days (336 h). RNA was extracted from the supernatants at the indicated time points by using 100 µL volume within the NucleoMag®VET Kit (Macherey-Nagel GmbH & Co. KG, Dueren, Germany) according to the manufacturer’s instructions (MACHEREY-NAGEL GmbH & Co. KG, Dueren, Germany) and kept frozen at -80°C until all extractions were finalized. All RNAs were investigated in a modified generic Matrix-protein specific one step RT-qPCR for the detection of Influenza A virus (20). Samples with a Ct-value of < 40 were rated as swIAV positive. Based on previous publications and due to lower detection limits of subtype- and lineage-specific RT-qPCRs (7, 21), samples with Ct-values of ≤ 33 were selected for subtyping as described elsewhere (22). Statistical analysis The temperature, viral load, time, subtype, stabilizer (with, without) and stabilizer medium (V, P, N) were considered as potential influential factors (predictors) on swIAV positivity in a multivariable logistic regression. The backwards variable selection was used to reduce the number of predictors to only important ones. The stabilizer (with, without), viral load and time remained in the final model after backwards selection. The temperature, viral load, time, subtype, stabilizer (with, without) and stabilizer medium (V, P, N) were considered as potential influential factors (predictors) on degradation of viral RNA in a multivariable linear regression. The backwards variable selection was used to reduce the number of predictors to only important ones. The stabilizer medium (V, P, N), subtype, temperature, and time remained in the final model after backwards selection. The normality of residuals, homogeneity of variance of residuals, linearity of residuals, the existence of influential points and collinearity of the final multivariable linear model were checked visually via the performance R-package. The assumptions were found to be satisfied. All contrasts (differences) between categories of predictors for both logistic and linear models were assessed after model-fitting by the estimated marginal means (R package - emmeans) with Tukey p-value correction for multiple comparisons. Results with a P-value < 0.05 were considered statistically significant. Data analysis was performed using R 4.3.3 (2024-02-29). Results Laboratory investigation Detection of swIAV RNA in spiked oral fluids In general, swIAV could be detected by RT-qPCR in 94% of the spiked samples with stabilizing media and 59% of the control samples. Additionally, Ct-values ≤ 33 were found in 67% of the spiked samples with stabilizing media and in 38% of the control samples. Details on the swIAV detection and subtyping rate (Ct-values ≤ 33) in association with the different stabilizing media, viral loads, storage temperature and time after spiking are displayed in Table 1 . Table 1 Number (n) and percentage (%) of swIAV positive (Ct < 40) samples and samples with Ct-values (Ct ≤ 33) by influential factors viral load, swIAV subtype, stabilizer type, time after spiking and temperature. Influential factors Parameter swIAV RNA positive total n = 109 Ct-values ≤ 33 total n = 76 Viral load High 63/64 (98%) 53/64 (83%) low 46/64 (72%) 23/64 (36%) swIAV subtype H1avN2 56/64 (88%) 40/64 (63%) H1pdmN2 53/64 (83%) 36/64 (56%) Stabilizer Yes 90/96 (94%) 64/96 (67%) No (Control) 19/32 (59%) 12/32 (38%) NucleoProtect VET Reagent® 29/32 (91%) 20/32 (63%) Primestore® 29/32 (91%) 21/32 (66%) Sigma-Virocult® 32/32 (100%) 23/32 (72%) Time after spiking 0 h 32/32 (100%) 32/32 (100%) 72 h 27/32 (84%) 17/32 (53%) 168 h (7 d) 27/32 (84%) 15/32 (45%) 336 h (14 d) 23/32 (72%) 12/32 (38%) Temperature 4°C 57/64 (89%) 42/64 (66%) Room temperature (22°C ± 2°C) 52/64 (81%) 34/64 (53%) In Table 2 details on swIAV positive samples and potentially subtypeable samples with Ct-values ≤ 33 over the study period for the different stabilizing media and conditions (storage, swIAV subtypes, viral loads) are presented. Regardless of the treatment and the storage conditions all samples spiked with high viral loads (Ct-25) of H1avN2 yielded RT-qPCR positive results until the end of the study on day 14. However, only samples with stabilizers showed Ct-values ≤ 33 over the entire study period. Samples spiked at lower viral loads (Ct-32) of H1avN2 were only positive until day 14 when stored with Sigma-Virocult® and PrimeStore®. Here, cooling of Sigma-Virocult® suspended samples resulted in Ct-values ≤ 33 until day 7, whereas cooling did not affect the subtyping rate of the other samples spiked at lower viral loads (Ct-32) of H1avN2. All samples spiked with high viral loads of H1pdmN2 were positive until 14 days, when suspended with media. In contrast, only control group samples stored under refrigerated conditions yielded swIAV positive results over the entire study period. Cooling resulted in a longer subtyping rate for samples suspended with Sigma-Virocult®. In case of low viral loads of H1pdmN2 only samples suspended with media and stored at 4°C were positive until day 14. Here cooling also prolonged the subtyping rate of samples suspended with Sigma-Virocult® and PrimeStore®. Table 2 Overview of swIAV detection by RT-qPCR in oral fluids spiked with high or low viral loads (H1avN2 or H1pdmN2) and suspended in three different stabilizer media or without stabilizer media at different storage conditions, detected Ct-values ≥ 40 were evaluated as negative (neg.), Ct-values > 33 were not subtyped. TP = time after spiking, C = control, V = Sigma-Virocult®, P = Primestore®, N= NucleoProtect VET Reagent®; neg. = negative. Probability to detect swIAV RNA in spiked oral fluid samples Univariate Analysis Univariate analysis revealed no significant differences in the probability to detect swIAV between samples spiked with H1avN2 and samples spiked with H1pdmN2 (p = 0.456) and samples stored at 4°C compared to samples stored at room temperature (p = 0.213), respectively. In addition, detection of swIAV did not differ significantly between samples suspended with Sigma-Virocult® and PrimeStore® (p = 0.5), Sigma-Virocult® and NucleoProtect VET Reagent® (p = 0.5), and NucleoProtect VET Reagent® and PrimeStore® (p > 0.9), respectively. Multivariable Analysis The probability to detect swIAV RNA by RT-qPCR in spiked OFs was subsequently investigated by multivariable analysis. The use of stabilizers in the spiked OFs resulted in a significant higher probability to detect swIAV RNA compared to samples without stabilizers (OR = 46.1, p < 0.001). In addition, regardless of the swIAV subtype that was used, the chance to detect swIAV RNA was significantly lower when low viral loads were used (OR = 0.01, p < 0.001). Long time of storage (336h, 14 d) reduced the probability to detect swIAV compared to immediate analysis (0h) (OR = 0.01, p = 0.002) (Fig. 1 , Table 3 ). Table 3 Odds Ratio (OR) with 95% Confidence Interval (CI) and p-value for the detection of swIAV RNA in spiked OFs in dependency of the factors stabilizer, time, and viral load estimated by the multivariable logistic regression. Influential factors Parameter OR 95% CI p-value Multivariable analysis Viral load Low / High 0.01 0.00, 0.11 0.999 336h / 0h 0.01 0.00, 0.30 0.002 336h / 72h 0.21 0.02, 2.26 0.330 336h / 168h 0.21 0.02, 2.26 0.330 Stabilizer Without / With 0.02 0.00, 0.12 < 0.001 Degradation of swIAV RNA over time The degradation of swIAV RNA over time was calculated by subtracting the Ct-values measured at the beginning (0h) of the study from the respective subsequently measured Ct-values. The results represented as increase in Ct-values, are depicted in Fig. 2 for each stabilizer and control group samples with respect to different storage conditions and the viral loads. Univariate Analysis In the univariate analysis no significant differences in the increase of the Ct-value was found between samples spiked with high viral loads and samples spiked with low viral loads (p = 0.42). In accordance time did not have a significant effect on the degradation of swIAV RNA (p = 0.27). Multivariable Analysis The degradation of swIAV RNA over time was subsequently measured through a multivariable analysis. Adding Sigma-Virocult®, PrimeStore® or NucleoProtect VET Reagent® to OF samples resulted in a significant lower increase of the Ct-value (p < 0.001) compared to samples without medium. In addition, degradation of swIAV RNA over time was significantly lower in samples spiked with H1avN2 compared to samples spiked with H1pdmN1 (p < 0.001) and samples kept refrigerated at 4°C compared to samples stored at room temperature (p < 0.001), respectively (Table 4 ). The degradation of swIAV RNA, depicted as increase of Ct-values, is shown in Fig. 3 . Table 4 Slopes (Beta), Confidence Intervals (CIs) and p-value of the factors (swIAV subtype, stabilizers, time after spiking, and temperature) influencing Ct-values using a multivariable linear analysis (N = NucleoProtect VET Reagent®, C = Control, P = Primestore®, V = Virocult®). Influential factors Differences between categories Beta 95% CIs p-value swIAV subtype H1avN2 – H1pdmN2 2.26 1.27, 3.25 < 0.001 Stabilizers C – N -5.98 -8.27, -3.69 < 0.001 C - P -5.22 -7.51, -2.94 < 0.001 N - P 0.76 -1.53, 3.05 0.8 C - V -6.09 -8.38, -3.80 0.9 P - V -0.86 -3.15, 1.43 0.8 Time after spiking 72h − 168h -0.04 -1.50, 1.42 0.998 72h − 336h 1.35 -0.10, 2.81 0.074 168h − 336h 1.39 -0.07, 2.85 0.065 Temperature 4°C – Room temperature 22°C ± 2°C) 2.53 1.54, 3.52 < 0.001 Table 4 Slopes (Beta), Confidence Intervals (CIs) and p-value of the factors (swIAV subtype, stabilizers, time after spiking, and temperature) influencing Ct-values using a multivariable linear analysis (N = NucleoProtect VET Reagent®, C = Control, P = Primestore®, V = Sigma-Virocult®). Influential factors Differences between categories Beta 95% CIs p-value swIAV subtype H1avN2 – H1pdmN2 2.26 1.27, 3.25 < 0.001 Stabilizers C – N -5.98 -8.27, -3.69 < 0.001 C - P -5.22 -7.51, -2.94 < 0.001 N - P 0.76 -1.53, 3.05 0.8 C - V -6.09 -8.38, -3.80 0.9 P - V -0.86 -3.15, 1.43 0.8 Time after spiking 72h − 168h -0.04 -1.50, 1.42 0.998 72h − 336h 1.35 -0.10, 2.81 0.074 168h − 336h 1.39 -0.07, 2.85 0.065 Temperature 4°C – Room temperature 22°C ± 2°C) 2.53 1.54, 3.52 < 0.001 Discussion Since swIAV are enzootically circulating in pig populations and new subtypes with prepandemic potential might evolve, surveillance of swIAV in pig herds is of considerable importance (3, 23). However, active surveillance of individual animals using nasal swabs is hampered by (i) the short infectious period enabling virus detection only for a couple of days and (ii) the high costs. Particularly, herds with low prevalence of swIAV require a high sample size (24). Therefore, aggregate samples as OFs, USW or environmental samples have gained increasing interest as cost-effective, non-invasive animal friendly sampling procedures (1, 25). However, deterioration of virus and viral RNA resulting from degradative bacterial and salivary enzymes and inhibitors (13) such as Glycoprotein-340 and MUC5B (9) might resemble a major drawback of aggregate samples like OFs and USW (26, 27), as low viral loads reduce the probability to characterize swIAV subtypes by multiplex RT-qPCR or sequencing (21, 22). However, the identification of different strains circulating in a pig herd is of particular importance to implement prophylactic measures like vaccination as cross protection between different swIAV strains is limited (28–32). Therefore, the effect of different stabilizers on improving the detection and subtyping rate of aggregate samples was assessed under laboratory conditions as samples with controlled laboratory swIAV contamination may not reflect samples from naturally infected pigs. Due to suitability of spiking, OFs were used for the laboratory study. Currently, three main swIAV subtypes (H1N1, H1N2, and H3N2) with a large abundance of genotypes and variants, that differ genetically and antigenically between regions, are circulating in the European pig population (8, 23, 33). In our experiment, the subtypes H1avN2 and H1pdmN2 (clade 1C and 1A, respectively) were selected. A pandemic strain was chosen, due to the increasing detection rate of pandemic strains or reassortants containing internal genes of human pandemic origin in several European countries (3, 5, 23, 33). The H1avN2 subtype, which emerged 2003 in Denmark is now predominantly found, in Denmark (8, 34), and has also been detected in e.g. France (35), Sweden, Germany (23), Sapin (36), Italy (37), Belgium and the Netherlands (33). The results of the laboratory study clearly show that cooled storage (4°C) of OFs even without a stabilizer medium facilitated swIAV RNA detection up to 14 days, at least at high viral loads. According to Hanáo-Diaz (38) the most important aspects in handling of OFs includes immediate cooling of the samples (4°C) after collection and during storage. Similar recommendations can be obtained from various authors on the management of OFs for the detection of Porcine Reproductive Respiratory Syndrome Virus (PRRSV) (38–40). However, it should be noted that in case of low viral loads, as often only detected in enzootically infected farms, even at 4°C the degradation of RNA is high and the detection rate is highly reduced. However, by adding stabilizing media and simultaneous cooling of the samples, swIAV RNA can still be detected even at low viral loads for at least 14 days. The addition of a stabilizer did not only enable prolonged detectability of swIAV by PCR, but also increases the probability to subtype swIAV, at least in cases where high viral loads are present in the original sample. Also, for PRRSV, another RNA virus, Decorté (41) highlighted the positive effect of Oragene RNA stabilizer medium on the duration of PRRSV detection in oral fluids. This RNA stabilizer was the only one out of three (Aware Messenger, Saliva Gene Collection Module) which had a positive impact on detection of PRRSV at room temperature after 168h (41). However, no significant impact through the addition of GTP on the detection of PRRS in oral fluids were obtained by Weiser (40). In addition, an antimicrobial treatment of oral fluid samples showed no significant improvement concerning the detection of PRRSV by RT-qPCR (39). In our experiment the performance of three different stabilizers was compared. It should be noted that molecular transport media or virus transport media have different characteristics. Whereas molecular transport media inactivates all germs in the collected sample and thus, is not appropriate for cultivation of viruses, but most suitable for the transport of viruses with a high risk of infection as e.g. such as SARS-CoV-2 (42), virus transport media still allows further diagnostics based on virus propagation (15). In the present study, PrimeStore® and NucleoProtect VET Reagent®, were used as example of molecular transport medium. Primestore®, contains guanidine thiocyanate which inactivates germs, but still enables the detection of nucleic acids. According to the manufacturer’s instructions, the adding of NucleoProtect Vet Reagent® enables the detection of RNA for one week at RT and up to one month at 4°C. However, in the present study OFs supplemented with NucleoProtect VET Reagent® revealed swIAV negative PCR results as soon as seven days after spiking with low viral for subtype H1avN2 stored at 4°C. Sigma-Virocult® is a virus transport media which does not inactivate viruses. Consequently, it could be used to enable further diagnostic such as virus isolation in cell culture (15). According to Rudsdale (43), detection of influenza A virus in samples suspended with Sigma-Virocult® is possible up to at least 8 days at both room temperature and under cooled conditions. In the present study, swIAV RNA was detectable in samples spiked with Sigma-Virocult® after 14 days (RT) regardless of the subtype. Based on the conditions of our study no statistically significant differences between the three investigated stabilising media were detected. However, Sigma-Virocult® has yielded the highest percentage of positive and subtypeable samples under laboratory conditions. Conclusion Addition of stabilizers improved detection and subtyping of swIAV in spiked oral fluids during 14 days of storage. However, it is evident that even with the addition of stabilizing media, molecular swIAV diagnostics can be improved by refrigerated shipping and storage of the samples. Under the conditions of our study no significant differences between the three tested media were found. Abbreviations av avian Ct Cycle threshold FLI Friedrich-Loeffler-Institute HA hemagglutinin inhibition LMU Ludwig-Maximilians-Universität OF oral fluid(s) RT-qPCR real-time quantitative PCR swIAV swine Influenza A virus SARS-CoV-2 severe acute respiratory syndrome coronavirus type 2 V Sigma-Virocult® R Primestore® N NucleoProtect Vet Reagent® C Control GTP Guanosin-5-triphosphate Declarations Ethics approval and consent to participate The sampling protocol and procedures were approved by the Ethic commission of the LMU Munich, accession number 403-27-05-2024 . Consent for publication Not applicable. Availability of data and materials The dataset is available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding This study was financially supported by Ceva Tiergesundheit GmbH, Germany. Authors’ Contributions Conceptualization: JS, TH, KLJ, MR. Data curation: AGR, KG, JS, YZ, ME, SZ. Formal analysis: AGR, JS, ME, KG, SZ. Funding acquisition: JS, KLJ. Project administration: JS, TH, MR. Resources: KLJ. Software: YZ. Supervision: JS, TH, MR. Validation: TH, ME, YZ, SZ. Visualization: JS, YZ, KG, ME, SZ. Writing – original draft: JS, KG, ME. All authors read and approved the final manuscript. Acknowledgements We gratefully thank the participating farmers and veterinarians for their valuable support. Authors’ information Clinic for Swine at the Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-Universität München, Oberschleißheim, Germany CEVA Tiergesundheit, Germany Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany. References Garrido-Mantilla J, Alvarez J, Culhane M, Nirmala J, Cano JP, Torremorell M. Comparison of individual, group and environmental sampling strategies to conduct influenza surveillance in pigs. BMC Vet Res. 2019;15(1):61. Fouchier RA, Osterhaus AD, Brown IH. Animal influenza virus surveillance. 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Evaluation of the Pathogenicity and the Escape from Vaccine Protection of a New Antigenic Variant Derived from the European Human-Like Reassortant Swine H1N2 Influenza Virus. Viruses. 2020;12(10). Everett HE, Aramouni M, Coward V, Ramsay A, Kelly M, Morgan S, et al. Vaccine-mediated protection of pigs against infection with pandemic H1N1 2009 swine influenza A virus requires a close antigenic match between the vaccine antigen and challenge virus. Vaccine. 2019;37(17):2288-93. Tamura S, Tanimoto T, Kurata T. Mechanisms of broad cross-protection provided by influenza virus infection and their application to vaccines. Jpn J Infect Dis. 2005;58(4):195-207. Chepkwony S, Parys A, Vandoorn E, Stadejek W, Xie J, King J, et al. Genetic and antigenic evolution of H1 swine influenza A viruses isolated in Belgium and the Netherlands from 2014 through 2019. Sci Rep. 2021;11(1):11276. Trebbien R, Bragstad K, Larsen LE, Nielsen J, Bøtner A, Heegaard PM, et al. Genetic and biological characterisation of an avian-like H1N2 swine influenza virus generated by reassortment of circulating avian-like H1N1 and H3N2 subtypes in Denmark. Virol J. 2013;10:290. Rose N, Hervé S, Eveno E, Barbier N, Eono F, Dorenlor V, et al. Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events. Vet Res. 2013;44(1):72. Sosa Portugal S, Cortey M, Tello M, Casanovas C, Mesonero-Escuredo S, Barrabés S, et al. Diversity of influenza A viruses retrieved from respiratory disease outbreaks and subclinically infected herds in Spain (2017-2019). Transbound Emerg Dis. 2021;68(2):519-30. Beato MS, Tassoni L, Milani A, Salviato A, Di Martino G, Mion M, et al. Circulation of multiple genotypes of H1N2 viruses in a swine farm in Italy over a two-month period. Vet Microbiol. 2016;195:25-9. Henao-Diaz A, Giménez-Lirola L, Baum DH, Zimmerman J. Guidelines for oral fluid-based surveillance of viral pathogens in swine. Porcine Health Manag. 2020;6:28. J.R. Prickett SC, J.M. Kinyon. Stability of porcine reproductive and respiratory syndrome virus and antibody in swine oral fluid. Journal of Swine Health and Production. 2010;18:187-95. Weiser AC, Poonsuk K, Bade SA, Gauger PC, Rotolo M, Harmon K, et al. Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR. J Vet Diagn Invest. 2018;30(6):807-12. Decorte I, Van der Stede Y, Nauwynck H, De Regge N, Cay AB. Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR. Vet J. 2013;197(2):224-8. Welch SR, Davies KA, Buczkowski H, Hettiarachchi N, Green N, Arnold U, et al. Analysis of Inactivation of SARS-CoV-2 by Specimen Transport Media, Nucleic Acid Extraction Reagents, Detergents, and Fixatives. J Clin Microbiol. 2020;58(11). A. Rudsdale DS. Investigation of the Suitability of the Virocult® Swab Transport Device for Influenza A Specimens Which Are to be Analyzed by Cell Culture or Molecular Techniques. Clinical Virology Symposium; 2009. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 11 Nov, 2024 Read the published version in Porcine Health Management → Version 1 posted Editorial decision: Revision requested 03 Jul, 2024 Reviews received at journal 26 Jun, 2024 Reviewers agreed at journal 25 Jun, 2024 Reviewers invited by journal 10 Jun, 2024 Editor assigned by journal 10 Jun, 2024 Submission checks completed at journal 10 Jun, 2024 First submitted to journal 27 May, 2024 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-4486513","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":315103832,"identity":"02eaac2f-1cac-49a9-80c7-87ecd70469e2","order_by":0,"name":"K. Grau","email":"data:image/png;base64,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","orcid":"","institution":"Ludwig-Maximilians-Universität München","correspondingAuthor":true,"prefix":"","firstName":"K.","middleName":"","lastName":"Grau","suffix":""},{"id":315103834,"identity":"78fd9e93-d36a-4705-a8d3-e91e1271d80b","order_by":1,"name":"K. Lillie-Jaschniski","email":"","orcid":"","institution":"CEVA Tiergesundheit","correspondingAuthor":false,"prefix":"","firstName":"K.","middleName":"","lastName":"Lillie-Jaschniski","suffix":""},{"id":315103835,"identity":"c15ea1b7-05ba-48ed-9139-68d58a3cbfbe","order_by":2,"name":"A. Graaf-Rau","email":"","orcid":"","institution":"Friedrich-Loeffler-Institut","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"","lastName":"Graaf-Rau","suffix":""},{"id":315103836,"identity":"29cbd1e3-9b27-42c0-99c2-e7652d019b04","order_by":3,"name":"T. Harder","email":"","orcid":"","institution":"Friedrich-Loeffler-Institut","correspondingAuthor":false,"prefix":"","firstName":"T.","middleName":"","lastName":"Harder","suffix":""},{"id":315103837,"identity":"fb12bd5d-169e-4b81-b496-f2bffdda7e75","order_by":4,"name":"M. Eddicks","email":"","orcid":"","institution":"Ludwig-Maximilians-Universität München","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"","lastName":"Eddicks","suffix":""},{"id":315103838,"identity":"341c124e-1e32-4cc0-b9f2-15cab941bfda","order_by":5,"name":"S. Zöls","email":"","orcid":"","institution":"Ludwig-Maximilians-Universität München","correspondingAuthor":false,"prefix":"","firstName":"S.","middleName":"","lastName":"Zöls","suffix":""},{"id":315103839,"identity":"9a72e3d9-142e-4e0c-acdd-0de6328593fc","order_by":6,"name":"Y. Zablotski","email":"","orcid":"","institution":"Ludwig-Maximilians-Universität München","correspondingAuthor":false,"prefix":"","firstName":"Y.","middleName":"","lastName":"Zablotski","suffix":""},{"id":315103840,"identity":"b04abed9-4770-4323-a188-5f18d4c0648b","order_by":7,"name":"M. Ritzmann","email":"","orcid":"","institution":"Ludwig-Maximilians-Universität München","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"","lastName":"Ritzmann","suffix":""},{"id":315103841,"identity":"d80a65e2-c722-4c2f-90b5-3dd4e8cd3ecd","order_by":8,"name":"J. Stadler","email":"","orcid":"","institution":"Ludwig-Maximilians-Universität München","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"","lastName":"Stadler","suffix":""}],"badges":[],"createdAt":"2024-05-27 17:38:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4486513/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4486513/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40813-024-00386-6","type":"published","date":"2024-11-11T15:58:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59004821,"identity":"3cdf31f2-2ebf-423a-9a2e-3d53b37e6d0a","added_by":"auto","created_at":"2024-06-25 07:52:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32720,"visible":true,"origin":"","legend":"\u003cp\u003eProbability (%) to detect swIAV by qRT-PCR in spiked oral fluids with the addition of stabilizers or without (A), collected at different time points (start (0), 72h, 168h (7 d), and 336h (14 d) after spiking) (B) and with either high (H; Ct-25) or low (L; Ct-32) viral loads of swIAV (C).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4486513/v1/c59db222210c1c6cefcf81e8.jpg"},{"id":59004820,"identity":"a8b29bf4-bb19-42a7-8a7d-97f5a94bd185","added_by":"auto","created_at":"2024-06-25 07:52:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80918,"visible":true,"origin":"","legend":"\u003cp\u003eStability of swIAV RNA in spiked oral fluids. Oral fluids from swIAV negative pigs were spiked with H1avN1 or H1pdmN2 with either high (H; Ct-25) or low (L; Ct-32) viral loads of swIAV and stored at different temperatures (room temperature, (RT,) or refrigerated (4°C), with the addition of stabilizers (V = Sigma-Virocult®, R = Primestore®, N = NucleoProtect VET Reagent®) or without (C= Control). Samples were collected at predetermined intervals (start (0), 72h, 168h (7 d), and 336h (14 d) after spiking) and analyzed for swIAV by RT-qPCR. The red line indicates the limit for positive samples (CT \u0026lt; 40) and the green line for subtypeable samples.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4486513/v1/6530981ec1493162713a62e0.jpg"},{"id":59004823,"identity":"0a3bed73-f2ca-4fff-ad9e-92c06583d5e2","added_by":"auto","created_at":"2024-06-25 07:52:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59053,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of the final multivariable linear regression after backwards variable selection showing the degradation of swIAV RNA, depicted as increase of Ct-values by qRT-PCR in spiked oral fluids with the addition of the different stabilizer media or without (A), with either H1avN1 or H1pdmN2 (B), collected at different time points (start (0), 72h, 168h (7 d), and 336h (14 d) after spiking) (C) and stored at either room temperature, (RT,) or refrigerated (4°C) (D).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4486513/v1/8fdf13ea43ebc3c6e11a8712.jpg"},{"id":69284852,"identity":"47294896-4a12-40f7-8640-3a4f11c9df3b","added_by":"auto","created_at":"2024-11-18 19:23:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":821385,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4486513/v1/1b3a86e2-bd3c-4bb6-b3fe-917467a255df.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of stabilizers on the detection of swine influenza A virus (swIAV) in spiked oral fluids over time","fulltext":[{"header":"Background","content":"\u003cp\u003eEconomic losses due to swine Influenza A virus (swIAV) impacting the swine industry and their potential threat to human health highlight the need for rapid and accurate identification and subtyping of these viruses (2, 3).\u003c/p\u003e \u003cp\u003eCommonly more than one swIAV subtype circulates in one herd (4). Frequent spill over infections of human A (H1N1) 2009 pandemic virus into swine populations since 2009 established enzootically infected herds with highly variable clinical signs (5). As a result, the already sophisticated diagnosis of swIAV is further complicated (6\u0026ndash;8). Whereas diagnostics for individual animals are both, time-consuming and cost-intensive, sampling techniques assessing groups of pigs have gained increasing interest concerning the detection and monitoring of various pathogens. One common aggregate non-invasive sampling method is the collection of oral fluids (OFs) (9\u0026ndash;11), which can be collected also by staff with little previous experience. However, those specimens have shown shortcomings concerning their suitability for the subtyping of swIAV strains (9) due to lower viral loads in aggregate OF samples compared to individual samples, i.e. nasal swabs (12). In addition, the presence of salivary enzymes, proteins (9, 13), cellular debris (14) and high bacterial loads in oral fluids as well as inadequate transport conditions (15) can contribute to further degradation of viral RNA. To avoid degradation of intact virus or viral nucleic acids different stabilizers have been described for the molecular detection of PRRSV (16), avian influenza virus and New castle Disease virus (17), SARS-CoV-2 (18), herpes simplex viruses, enteroviruses, and adenoviruses (19). However, the stability of swIAV in aggregated samples is poorly investigated.\u003c/p\u003e \u003cp\u003eTherefore, the aim of the present study was to assess under cooled conditions whether the addition of stabilizing media to OFs, spiked with two different swIAV strains, improve the rate of detection and the suitability of subtyping by RT-qPCR under different storage conditions and viral loads in the original sample over a time period of 14 days.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eA minimum of 50mL OF was collected from 24 pigs on a farm which was considered negative for swIAV through regular monitoring by RT-qPCR, ELISA and hemagglutination inhibition (HI) assays, all with negative results. Until further analysis the samples were stored at -80\u0026deg;C. Before further investigations the samples were centrifuged at 2000g for 15 minutes. The samples were spiked with swIAV isolates A/swine/Germany/2022AI04470/2022 (H1avN2, clade 1C) and A/swine/Germany/2022AI03601/2022 (H1pdmN2, clade 1A) propagated in swine testicle cells for two passages (FLI Collection of Cell Lines in Veterinary Medicine CCLV-RIE 0606), respectively to achieve each threshold cycle values (Ct) of 25 and 32, respectively. And additionally, stabilizing media was added to the spiked OF specimens at a ratio of 3:1. Three different media were used (i) Sigma-Virocult\u0026reg; MW950S (V) (Check Diagnostics GmbH, Westerau, Germany), (ii) PrimeStore\u0026reg; MTM (P) (Longhorn Vaccines \u0026amp; Diagnostics LLC, Bethesda, USA, P) and (iii) NucleoProtect VET Reagent\u0026reg; (N) (MACHEREY-NAGEL GmbH \u0026amp; Co. KG, Dueren, Germany, M\u0026amp;N). Aliquots of the spiked and diluted oral fluids were stored at either 4\u0026deg;C or at room temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and replica of supernatants were taken for analysis at 72 h, 7 days (168 h) and 14 days (336 h). RNA was extracted from the supernatants at the indicated time points by using 100 \u0026micro;L volume within the NucleoMag\u0026reg;VET Kit (Macherey-Nagel GmbH \u0026amp; Co. KG, Dueren, Germany) according to the manufacturer\u0026rsquo;s instructions (MACHEREY-NAGEL GmbH \u0026amp; Co. KG, Dueren, Germany) and kept frozen at -80\u0026deg;C until all extractions were finalized. All RNAs were investigated in a modified generic Matrix-protein specific one step RT-qPCR for the detection of Influenza A virus (20). Samples with a Ct-value of \u0026lt;\u0026thinsp;40 were rated as swIAV positive. Based on previous publications and due to lower detection limits of subtype- and lineage-specific RT-qPCRs (7, 21), samples with Ct-values of \u0026le;\u0026thinsp;33 were selected for subtyping as described elsewhere (22).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe temperature, viral load, time, subtype, stabilizer (with, without) and stabilizer medium (V, P, N) were considered as potential influential factors (predictors) on swIAV positivity in a multivariable logistic regression. The backwards variable selection was used to reduce the number of predictors to only important ones. The stabilizer (with, without), viral load and time remained in the final model after backwards selection.\u003c/p\u003e \u003cp\u003eThe temperature, viral load, time, subtype, stabilizer (with, without) and stabilizer medium (V, P, N) were considered as potential influential factors (predictors) on degradation of viral RNA in a multivariable linear regression. The backwards variable selection was used to reduce the number of predictors to only important ones. The stabilizer medium (V, P, N), subtype, temperature, and time remained in the final model after backwards selection. The normality of residuals, homogeneity of variance of residuals, linearity of residuals, the existence of influential points and collinearity of the final multivariable linear model were checked visually via the performance R-package. The assumptions were found to be satisfied.\u003c/p\u003e \u003cp\u003eAll contrasts (differences) between categories of predictors for both logistic and linear models were assessed after model-fitting by the estimated marginal means (R package - emmeans) with Tukey p-value correction for multiple comparisons. Results with a P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Data analysis was performed using R 4.3.3 (2024-02-29).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eLaboratory investigation\u003c/h2\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003eDetection of swIAV RNA in spiked oral fluids\u003c/h2\u003e\n \u003cp\u003eIn general, swIAV could be detected by RT-qPCR in 94% of the spiked samples with stabilizing media and 59% of the control samples. Additionally, Ct-values\u0026thinsp;\u0026le;\u0026thinsp;33 were found in 67% of the spiked samples with stabilizing media and in 38% of the control samples. Details on the swIAV detection and subtyping rate (Ct-values\u0026thinsp;\u0026le;\u0026thinsp;33) in association with the different stabilizing media, viral loads, storage temperature and time after spiking are displayed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber (n) and percentage (%) of swIAV positive (Ct\u0026thinsp;\u0026lt;\u0026thinsp;40) samples and samples with Ct-values (Ct\u0026thinsp;\u0026le;\u0026thinsp;33) by influential factors viral load, swIAV subtype, stabilizer type, time after spiking and temperature.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInfluential factors\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eswIAV RNA positive\u003c/p\u003e\n \u003cp\u003etotal n\u0026thinsp;=\u0026thinsp;109\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCt-values\u0026thinsp;\u0026le;\u0026thinsp;33\u003c/p\u003e\n \u003cp\u003etotal n\u0026thinsp;=\u0026thinsp;76\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eViral load\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63/64 (98%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53/64 (83%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46/64 (72%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23/64 (36%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eswIAV subtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1avN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56/64 (88%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40/64 (63%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1pdmN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53/64 (83%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36/64 (56%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eStabilizer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90/96 (94%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64/96 (67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo (Control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19/32 (59%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12/32 (38%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleoProtect VET Reagent\u0026reg;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29/32 (91%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20/32 (63%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePrimestore\u0026reg;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29/32 (91%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21/32 (66%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSigma-Virocult\u0026reg;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32/32 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23/32 (72%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eTime after spiking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32/32 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32/32 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27/32 (84%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17/32 (53%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e168 h (7 d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27/32 (84%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15/32 (45%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336 h (14 d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23/32 (72%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12/32 (38%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57/64 (89%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42/64 (66%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRoom temperature (22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52/64 (81%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34/64 (53%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eIn Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e details on swIAV positive samples and potentially subtypeable samples with Ct-values\u0026thinsp;\u0026le;\u0026thinsp;33 over the study period for the different stabilizing media and conditions (storage, swIAV subtypes, viral loads) are presented. Regardless of the treatment and the storage conditions all samples spiked with high viral loads (Ct-25) of H1avN2 yielded RT-qPCR positive results until the end of the study on day 14. However, only samples with stabilizers showed Ct-values\u0026thinsp;\u0026le;\u0026thinsp;33 over the entire study period. Samples spiked at lower viral loads (Ct-32) of H1avN2 were only positive until day 14 when stored with Sigma-Virocult\u0026reg; and PrimeStore\u0026reg;. Here, cooling of Sigma-Virocult\u0026reg; suspended samples resulted in Ct-values\u0026thinsp;\u0026le;\u0026thinsp;33 until day 7, whereas cooling did not affect the subtyping rate of the other samples spiked at lower viral loads (Ct-32) of H1avN2. All samples spiked with high viral loads of H1pdmN2 were positive until 14 days, when suspended with media. In contrast, only control group samples stored under refrigerated conditions yielded swIAV positive results over the entire study period. Cooling resulted in a longer subtyping rate for samples suspended with Sigma-Virocult\u0026reg;. In case of low viral loads of H1pdmN2 only samples suspended with media and stored at 4\u0026deg;C were positive until day 14. Here cooling also prolonged the subtyping rate of samples suspended with Sigma-Virocult\u0026reg; and PrimeStore\u0026reg;.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eOverview of swIAV detection by RT-qPCR in oral fluids spiked with high or low viral loads (H1avN2 or H1pdmN2) and suspended in three different stabilizer media or without stabilizer media at different storage conditions, detected Ct-values \u0026ge; 40 were evaluated as negative (neg.), Ct-values \u0026gt; 33 were not subtyped. TP = time after spiking, C = control, V = Sigma-Virocult\u0026reg;, P = Primestore\u0026reg;, N= NucleoProtect VET Reagent\u0026reg;; neg. = negative.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" width=\"694\" height=\"748\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eProbability to detect swIAV RNA in spiked oral fluid samples\u003c/h2\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003eUnivariate Analysis\u003c/h2\u003e\n \u003cp\u003eUnivariate analysis revealed no significant differences in the probability to detect swIAV between samples spiked with H1avN2 and samples spiked with H1pdmN2 (p\u0026thinsp;=\u0026thinsp;0.456) and samples stored at 4\u0026deg;C compared to samples stored at room temperature (p\u0026thinsp;=\u0026thinsp;0.213), respectively. In addition, detection of swIAV did not differ significantly between samples suspended with Sigma-Virocult\u0026reg; and PrimeStore\u0026reg; (p\u0026thinsp;=\u0026thinsp;0.5), Sigma-Virocult\u0026reg; and NucleoProtect VET Reagent\u0026reg; (p\u0026thinsp;=\u0026thinsp;0.5), and NucleoProtect VET Reagent\u0026reg; and PrimeStore\u0026reg; (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9), respectively.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eMultivariable Analysis\u003c/h2\u003e\n \u003cp\u003eThe probability to detect swIAV RNA by RT-qPCR in spiked OFs was subsequently investigated by multivariable analysis. The use of stabilizers in the spiked OFs resulted in a significant higher probability to detect swIAV RNA compared to samples without stabilizers (OR\u0026thinsp;=\u0026thinsp;46.1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In addition, regardless of the swIAV subtype that was used, the chance to detect swIAV RNA was significantly lower when low viral loads were used (OR\u0026thinsp;=\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Long time of storage (336h, 14 d) reduced the probability to detect swIAV compared to immediate analysis (0h) (OR\u0026thinsp;=\u0026thinsp;0.01, p\u0026thinsp;=\u0026thinsp;0.002) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eOdds Ratio (OR) with 95% Confidence Interval (CI) and p-value for the detection of swIAV RNA in spiked OFs in dependency of the factors stabilizer, time, and viral load estimated by the multivariable logistic regression.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInfluential factors\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"8\"\u003e\n \u003cp\u003eMultivariable analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eViral load\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLow / High\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00, 0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eTime after spiking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72h / 0h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00, 1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.094\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e168h / 0h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00, 1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.094\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72 / 168h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.07, 13.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336h / 0h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00, 0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336h / 72h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02, 2.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.330\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336h / 168h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02, 2.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.330\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStabilizer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWithout / With\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00, 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eDegradation of swIAV RNA over time\u003c/h2\u003e\n \u003cp\u003eThe degradation of swIAV RNA over time was calculated by subtracting the Ct-values measured at the beginning (0h) of the study from the respective subsequently measured Ct-values. The results represented as increase in Ct-values, are depicted in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e for each stabilizer and control group samples with respect to different storage conditions and the viral loads.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eUnivariate Analysis\u003c/h2\u003e\n \u003cp\u003eIn the univariate analysis no significant differences in the increase of the Ct-value was found between samples spiked with high viral loads and samples spiked with low viral loads (p\u0026thinsp;=\u0026thinsp;0.42). In accordance time did not have a significant effect on the degradation of swIAV RNA (p\u0026thinsp;=\u0026thinsp;0.27).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eMultivariable Analysis\u003c/h2\u003e\n \u003cp\u003eThe degradation of swIAV RNA over time was subsequently measured through a multivariable analysis. Adding Sigma-Virocult\u0026reg;, PrimeStore\u0026reg; or NucleoProtect VET Reagent\u0026reg; to OF samples resulted in a significant lower increase of the Ct-value (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to samples without medium. In addition, degradation of swIAV RNA over time was significantly lower in samples spiked with H1avN2 compared to samples spiked with H1pdmN1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and samples kept refrigerated at 4\u0026deg;C compared to samples stored at room temperature (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The degradation of swIAV RNA, depicted as increase of Ct-values, is shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSlopes (Beta), Confidence Intervals (CIs) and p-value of the factors (swIAV subtype, stabilizers, time after spiking, and temperature) influencing Ct-values using a multivariable linear analysis (N\u0026thinsp;=\u0026thinsp;NucleoProtect VET Reagent\u0026reg;, C\u0026thinsp;=\u0026thinsp;Control, P\u0026thinsp;=\u0026thinsp;Primestore\u0026reg;, V\u0026thinsp;=\u0026thinsp;Virocult\u0026reg;).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInfluential factors\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDifferences between categories\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBeta\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CIs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eswIAV subtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1avN2 \u0026ndash; H1pdmN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.27, 3.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eStabilizers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC \u0026ndash; N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-5.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.27, -3.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC - P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-5.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-7.51, -2.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN - P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.53, 3.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC - V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-6.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.38, -3.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN - V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.40, 2.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP - V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.15, 1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eTime after spiking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72h \u0026minus;\u0026thinsp;168h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.50, 1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.998\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72h \u0026minus;\u0026thinsp;336h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.10, 2.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e168h \u0026minus;\u0026thinsp;336h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.07, 2.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.065\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u0026deg;C \u0026ndash; Room temperature 22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.54, 3.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSlopes (Beta), Confidence Intervals (CIs) and p-value of the factors (swIAV subtype, stabilizers, time after spiking, and temperature) influencing Ct-values using a multivariable linear analysis (N\u0026thinsp;=\u0026thinsp;NucleoProtect VET Reagent\u0026reg;, C\u0026thinsp;=\u0026thinsp;Control, P\u0026thinsp;=\u0026thinsp;Primestore\u0026reg;, V\u0026thinsp;=\u0026thinsp;Sigma-Virocult\u0026reg;).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInfluential factors\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDifferences between categories\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBeta\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CIs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eswIAV subtype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1avN2 \u0026ndash; H1pdmN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.27, 3.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eStabilizers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC \u0026ndash; N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-5.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.27, -3.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC - P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-5.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-7.51, -2.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN - P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.53, 3.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC - V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-6.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-8.38, -3.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN - V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.40, 2.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP - V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.15, 1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eTime after spiking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72h \u0026minus;\u0026thinsp;168h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.50, 1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.998\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72h \u0026minus;\u0026thinsp;336h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.10, 2.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e168h \u0026minus;\u0026thinsp;336h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.07, 2.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.065\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u0026deg;C \u0026ndash; Room temperature 22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.54, 3.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince swIAV are enzootically circulating in pig populations and new subtypes with prepandemic potential might evolve, surveillance of swIAV in pig herds is of considerable importance (3, 23). However, active surveillance of individual animals using nasal swabs is hampered by (i) the short infectious period enabling virus detection only for a couple of days and (ii) the high costs. Particularly, herds with low prevalence of swIAV require a high sample size (24). Therefore, aggregate samples as OFs, USW or environmental samples have gained increasing interest as cost-effective, non-invasive animal friendly sampling procedures (1, 25). However, deterioration of virus and viral RNA resulting from degradative bacterial and salivary enzymes and inhibitors (13) such as Glycoprotein-340 and MUC5B (9) might resemble a major drawback of aggregate samples like OFs and USW (26, 27), as low viral loads reduce the probability to characterize swIAV subtypes by multiplex RT-qPCR or sequencing (21, 22). However, the identification of different strains circulating in a pig herd is of particular importance to implement prophylactic measures like vaccination as cross protection between different swIAV strains is limited (28\u0026ndash;32). Therefore, the effect of different stabilizers on improving the detection and subtyping rate of aggregate samples was assessed under laboratory conditions as samples with controlled laboratory swIAV contamination may not reflect samples from naturally infected pigs. Due to suitability of spiking, OFs were used for the laboratory study. Currently, three main swIAV subtypes (H1N1, H1N2, and H3N2) with a large abundance of genotypes and variants, that differ genetically and antigenically between regions, are circulating in the European pig population (8, 23, 33). In our experiment, the subtypes H1avN2 and H1pdmN2 (clade 1C and 1A, respectively) were selected. A pandemic strain was chosen, due to the increasing detection rate of pandemic strains or reassortants containing internal genes of human pandemic origin in several European countries (3, 5, 23, 33). The H1avN2 subtype, which emerged 2003 in Denmark is now predominantly found, in Denmark (8, 34), and has also been detected in e.g. France (35), Sweden, Germany (23), Sapin (36), Italy (37), Belgium and the Netherlands (33).\u003c/p\u003e \u003cp\u003eThe results of the laboratory study clearly show that cooled storage (4\u0026deg;C) of OFs even without a stabilizer medium facilitated swIAV RNA detection up to 14 days, at least at high viral loads. According to Han\u0026aacute;o-Diaz (38) the most important aspects in handling of OFs includes immediate cooling of the samples (4\u0026deg;C) after collection and during storage. Similar recommendations can be obtained from various authors on the management of OFs for the detection of Porcine Reproductive Respiratory Syndrome Virus (PRRSV) (38\u0026ndash;40). However, it should be noted that in case of low viral loads, as often only detected in enzootically infected farms, even at 4\u0026deg;C the degradation of RNA is high and the detection rate is highly reduced. However, by adding stabilizing media and simultaneous cooling of the samples, swIAV RNA can still be detected even at low viral loads for at least 14 days. The addition of a stabilizer did not only enable prolonged detectability of swIAV by PCR, but also increases the probability to subtype swIAV, at least in cases where high viral loads are present in the original sample. Also, for PRRSV, another RNA virus, Decort\u0026eacute; (41) highlighted the positive effect of Oragene RNA stabilizer medium on the duration of PRRSV detection in oral fluids. This RNA stabilizer was the only one out of three (Aware Messenger, Saliva Gene Collection Module) which had a positive impact on detection of PRRSV at room temperature after 168h (41). However, no significant impact through the addition of GTP on the detection of PRRS in oral fluids were obtained by Weiser (40). In addition, an antimicrobial treatment of oral fluid samples showed no significant improvement concerning the detection of PRRSV by RT-qPCR (39).\u003c/p\u003e \u003cp\u003eIn our experiment the performance of three different stabilizers was compared. It should be noted that molecular transport media or virus transport media have different characteristics. Whereas molecular transport media inactivates all germs in the collected sample and thus, is not appropriate for cultivation of viruses, but most suitable for the transport of viruses with a high risk of infection as e.g. such as SARS-CoV-2 (42), virus transport media still allows further diagnostics based on virus propagation (15). In the present study, PrimeStore\u0026reg; and NucleoProtect VET Reagent\u0026reg;, were used as example of molecular transport medium. Primestore\u0026reg;, contains guanidine thiocyanate which inactivates germs, but still enables the detection of nucleic acids. According to the manufacturer\u0026rsquo;s instructions, the adding of NucleoProtect Vet Reagent\u0026reg; enables the detection of RNA for one week at RT and up to one month at 4\u0026deg;C. However, in the present study OFs supplemented with NucleoProtect VET Reagent\u0026reg; revealed swIAV negative PCR results as soon as seven days after spiking with low viral for subtype H1avN2 stored at 4\u0026deg;C. Sigma-Virocult\u0026reg; is a virus transport media which does not inactivate viruses. Consequently, it could be used to enable further diagnostic such as virus isolation in cell culture (15). According to Rudsdale (43), detection of influenza A virus in samples suspended with Sigma-Virocult\u0026reg; is possible up to at least 8 days at both room temperature and under cooled conditions. In the present study, swIAV RNA was detectable in samples spiked with Sigma-Virocult\u0026reg; after 14 days (RT) regardless of the subtype. Based on the conditions of our study no statistically significant differences between the three investigated stabilising media were detected. However, Sigma-Virocult\u0026reg; has yielded the highest percentage of positive and subtypeable samples under laboratory conditions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAddition of stabilizers improved detection and subtyping of swIAV in spiked oral fluids during 14 days of storage. However, it is evident that even with the addition of stabilizing media, molecular swIAV diagnostics can be improved by refrigerated shipping and storage of the samples. Under the conditions of our study no significant differences between the three tested media were found.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eav\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;avian\u003c/p\u003e\n\u003cp\u003eCt\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cycle threshold\u003c/p\u003e\n\u003cp\u003eFLI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Friedrich-Loeffler-Institute\u003c/p\u003e\n\u003cp\u003eHA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;hemagglutinin inhibition\u003c/p\u003e\n\u003cp\u003eLMU\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ludwig-Maximilians-Universit\u0026auml;t\u003c/p\u003e\n\u003cp\u003eOF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;oral fluid(s)\u003c/p\u003e\n\u003cp\u003eRT-qPCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;real-time quantitative PCR\u003c/p\u003e\n\u003cp\u003eswIAV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;swine Influenza A virus\u003c/p\u003e\n\u003cp\u003eSARS-CoV-2\u0026nbsp; \u0026nbsp;severe acute respiratory syndrome coronavirus type 2\u003c/p\u003e\n\u003cp\u003eV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Sigma-Virocult\u0026reg;\u003c/p\u003e\n\u003cp\u003eR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Primestore\u0026reg;\u003c/p\u003e\n\u003cp\u003eN\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;NucleoProtect Vet Reagent\u0026reg;\u003c/p\u003e\n\u003cp\u003eC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Control\u003c/p\u003e\n\u003cp\u003eGTP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Guanosin-5-triphosphate\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sampling protocol and procedures were approved by the Ethic commission of the LMU Munich, accession number \u003cstrong\u003e403-27-05-2024\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset is available from the corresponding author on reasonable request.\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\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by Ceva Tiergesundheit GmbH, Germany.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization:\u0026nbsp;JS, TH, KLJ, MR. Data curation: AGR, KG, JS, YZ, ME, SZ. Formal analysis: AGR, JS, ME, KG, SZ. Funding acquisition: JS, KLJ. Project administration: JS, TH, MR. Resources: KLJ. Software: YZ. Supervision: JS, TH, MR. Validation: TH, ME, YZ, SZ. Visualization: JS, YZ, KG, ME, SZ. Writing \u0026ndash; original draft: JS, KG, ME. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully thank the participating farmers and veterinarians for their valuable support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinic for Swine at the Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-Universit\u0026auml;t M\u0026uuml;nchen, Oberschlei\u0026szlig;heim, Germany\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCEVA Tiergesundheit, Germany\u003c/p\u003e\n\u003cp\u003eInstitute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGarrido-Mantilla J, Alvarez J, Culhane M, Nirmala J, Cano JP, Torremorell M. 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Vaccines against influenza A viruses in poultry and swine: Status and future developments. Vaccine. 2015;33(21):2414-24.\u003c/li\u003e\n\u003cli\u003eDeblanc C, Qu\u0026eacute;guiner S, Gorin S, Chastagner A, Herv\u0026eacute; S, Paboeuf F, et al. Evaluation of the Pathogenicity and the Escape from Vaccine Protection of a New Antigenic Variant Derived from the European Human-Like Reassortant Swine H1N2 Influenza Virus. Viruses. 2020;12(10).\u003c/li\u003e\n\u003cli\u003eEverett HE, Aramouni M, Coward V, Ramsay A, Kelly M, Morgan S, et al. Vaccine-mediated protection of pigs against infection with pandemic H1N1 2009 swine influenza A virus requires a close antigenic match between the vaccine antigen and challenge virus. Vaccine. 2019;37(17):2288-93.\u003c/li\u003e\n\u003cli\u003eTamura S, Tanimoto T, Kurata T. Mechanisms of broad cross-protection provided by influenza virus infection and their application to vaccines. Jpn J Infect Dis. 2005;58(4):195-207.\u003c/li\u003e\n\u003cli\u003eChepkwony S, Parys A, Vandoorn E, Stadejek W, Xie J, King J, et al. Genetic and antigenic evolution of H1 swine influenza A viruses isolated in Belgium and the Netherlands from 2014 through 2019. Sci Rep. 2021;11(1):11276.\u003c/li\u003e\n\u003cli\u003eTrebbien R, Bragstad K, Larsen LE, Nielsen J, B\u0026oslash;tner A, Heegaard PM, et al. Genetic and biological characterisation of an avian-like H1N2 swine influenza virus generated by reassortment of circulating avian-like H1N1 and H3N2 subtypes in Denmark. Virol J. 2013;10:290.\u003c/li\u003e\n\u003cli\u003eRose N, Herv\u0026eacute; S, Eveno E, Barbier N, Eono F, Dorenlor V, et al. Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events. Vet Res. 2013;44(1):72.\u003c/li\u003e\n\u003cli\u003eSosa Portugal S, Cortey M, Tello M, Casanovas C, Mesonero-Escuredo S, Barrab\u0026eacute;s S, et al. Diversity of influenza A viruses retrieved from respiratory disease outbreaks and subclinically infected herds in Spain (2017-2019). Transbound Emerg Dis. 2021;68(2):519-30.\u003c/li\u003e\n\u003cli\u003eBeato MS, Tassoni L, Milani A, Salviato A, Di Martino G, Mion M, et al. Circulation of multiple genotypes of H1N2 viruses in a swine farm in Italy over a two-month period. Vet Microbiol. 2016;195:25-9.\u003c/li\u003e\n\u003cli\u003eHenao-Diaz A, Gim\u0026eacute;nez-Lirola L, Baum DH, Zimmerman J. Guidelines for oral fluid-based surveillance of viral pathogens in swine. Porcine Health Manag. 2020;6:28.\u003c/li\u003e\n\u003cli\u003eJ.R. Prickett SC, J.M. Kinyon. Stability of porcine reproductive and respiratory syndrome virus and antibody in swine oral fluid. Journal of Swine Health and Production. 2010;18:187-95.\u003c/li\u003e\n\u003cli\u003eWeiser AC, Poonsuk K, Bade SA, Gauger PC, Rotolo M, Harmon K, et al. Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR. J Vet Diagn Invest. 2018;30(6):807-12.\u003c/li\u003e\n\u003cli\u003eDecorte I, Van der Stede Y, Nauwynck H, De Regge N, Cay AB. Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR. Vet J. 2013;197(2):224-8.\u003c/li\u003e\n\u003cli\u003eWelch SR, Davies KA, Buczkowski H, Hettiarachchi N, Green N, Arnold U, et al. Analysis of Inactivation of SARS-CoV-2 by Specimen Transport Media, Nucleic Acid Extraction Reagents, Detergents, and Fixatives. J Clin Microbiol. 2020;58(11).\u003c/li\u003e\n\u003cli\u003eA. Rudsdale DS. Investigation of the Suitability of the Virocult\u0026reg; Swab Transport Device for Influenza A Specimens Which Are to be Analyzed by Cell Culture or Molecular Techniques. Clinical Virology Symposium; 2009.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"porcine-health-management","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phmj","sideBox":"Learn more about [Porcine Health Management](http://porcinehealthmanagement.biomedcentral.com/)","snPcode":"40813","submissionUrl":"https://submission.nature.com/new-submission/40813/3","title":"Porcine Health Management","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"swine, influenza A virus, subtyping, surveillance, stabilizing media, RT-qPCR","lastPublishedDoi":"10.21203/rs.3.rs-4486513/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4486513/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAggregated samples such as oral fluids (OFs) display an animal friendly and time and cost-efficient sample type for swine Influenza A virus (swIAV) monitoring. However, further molecular and biological characterization of swIAV is of particular significance. The reportedly inferior suitability of aggregated samples for subtyping of swIAV presents a major drawback compared to nasal swabs, still considered the most appropriate sample type for this purpose (1). In addition, the viral load in the original sample, storage conditions and characteristics of different swIAV strains might further compromise the eligibility of aggregated samples for molecular detection and subtyping.\u003c/p\u003e\n\u003cp\u003eTherefore, the present study aimed to evaluate the suitability of stabilizing media to minimize the degradation of viral RNA and thus increase the detection and subtyping rate of swIAV by RT-qPCR in spiked OFs under different conditions (virus strain, storage temperature and viral load in the original sample) over a time span of 14 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of stabilizing media in spiked OFs resulted in a significant higher probability to detect swIAV RNA compared to OFs without stabilizers (OR = 46.1, p \u0026lt; 0.001). In addition, the probability of swIAV degradation over time was significantly reduced in samples suspended with stabilizer (OR = 5.80, p \u0026lt; 0.001), in samples stored at 4°C (OR = 2.53, p \u0026lt; 0.001) and in samples spiked with the avian derived H1N1 subtype (OR = 2.26, p \u0026lt; 0.01). No significant differences in swIAV RNA detection and degradation of swIAV RNA in spiked OFs over time were observed between the three different stabilizing media.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAddition of stabilizers and storage of samples at cooled conditions highly significantly improved detection and subtyping of swIAV in spiked OFs.\u003c/p\u003e","manuscriptTitle":"Effect of stabilizers on the detection of swine influenza A virus (swIAV) in spiked oral fluids over time","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-25 07:52:09","doi":"10.21203/rs.3.rs-4486513/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-03T15:52:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-26T14:42:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121315226909520557470879914273753466346","date":"2024-06-25T17:05:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-10T20:52:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-10T09:36:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-10T09:34:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Porcine Health Management","date":"2024-05-27T17:37:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"porcine-health-management","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phmj","sideBox":"Learn more about [Porcine Health Management](http://porcinehealthmanagement.biomedcentral.com/)","snPcode":"40813","submissionUrl":"https://submission.nature.com/new-submission/40813/3","title":"Porcine Health Management","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"932657b4-7763-46cb-afe8-25d985389d37","owner":[],"postedDate":"June 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-18T19:16:49+00:00","versionOfRecord":{"articleIdentity":"rs-4486513","link":"https://doi.org/10.1186/s40813-024-00386-6","journal":{"identity":"porcine-health-management","isVorOnly":false,"title":"Porcine Health Management"},"publishedOn":"2024-11-11 15:58:05","publishedOnDateReadable":"November 11th, 2024"},"versionCreatedAt":"2024-06-25 07:52:09","video":"","vorDoi":"10.1186/s40813-024-00386-6","vorDoiUrl":"https://doi.org/10.1186/s40813-024-00386-6","workflowStages":[]},"version":"v1","identity":"rs-4486513","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4486513","identity":"rs-4486513","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}