lunes, 25 de mayo de 2020

First report of Porcine Respirovirus 1 in South America Author links open overlay panelB.AgüeroaJ.MenaaF.BerriosaR.TapiaaC.SalinasaJayeetaDuttabHarm vanBakelbS.K.MorcB.BritodR.A.MedinaefV.Neiraa https://doi.org/10.1016/j.vetmic.2020.108726Get rights and content Under a Creative Commons licenseopen access Highlights • The manuscript described the first report of Porcine Respirovirus in Latin America, specifically in Chile. The virus was genetically characterized and is related with viruses previously reported in USA and China. Up to date, there is scarce studies in other countries. Due its zoonotic potential is still unknown, is important know more about it. • We collected 164 samples between 2015 and 2019 during an influenza surveillance program and include nasal swabs (121), oral fluids (8) and lungs (25); from 6 different commercial swine farm companies, located in 5 administrative regions of Chile (Valparaiso, Metropolitan, O’Higgins, Maule and Ñuble), representing >80% of Chilean swine production. • We tested these samples by RT-PCR to identify PRV1. From the positive RT-PCR samples (31 positives), 11 were selected to attempt viral isolation in African green monkey kidney (Vero) cells, but this attempt was unsuccessful. Eight positive samples were submitted for whole-genome sequencing using the Illumina. We were able to obtain two near to complete genomes from two different farms. • The phylogenetic results suggest that the Chilean PRV1 are genetically related to RPV1 strains from USA and China, but due to the scarce information it is not possible to determine actual the origin. Abstract Porcine respirovirus 1 (PRV1) is an emerging virus in pigs that has been previously described in the USA and China. There are no reports of its presence in the rest of the world. The objective of this study was to determine the occurrence of PRV1 in Chile and to determine its phylogeny. Thus, we collected samples (oral fluids, nasal swabs, and lungs) from a swine influenza A virus (IAV) surveillance program, most of which belonged to pigs with respiratory disease. The samples were analyzed by RT-PCR, and the viral sequencing was obtained using RNA whole-genome sequencing approach. Maximum likelihood phylogeny was constructed with the available references. Thirty-one of 164 samples (18.9%) were RT-PCR positive for PRV1: 62.5% oral fluids, 19.0% nasal swabs, and 8.6% lungs. All 6 farms in this study had at least one positive sample, with 6-40% of positive results per farm, which suggests that PRV1 is disseminated in Chilean swine farms. Twenty-one of 31 (67,7%) PRV1-positive samples were also positive for IAV, so the role of PRV1 as secondary pathogen in respiratory disease needs to be further evaluated. Near to complete genome of two PRV1s were obtained from two farms. The phylogenies, in general, showed low bootstrap support, except the concatenated genome and the L gene trees which showed clustering of the Chilean PRV1 with Asian sequences, suggesting a close genetic relationship. This is the first report of PRV1 in the Southern Hemisphere. Further studies are necessary to determine the genetic diversity of this virus in Chile. Keywords Porcine respirovirus 1porcine parainfluenza virus 1SwineSouth AmericaChile 1. Introduction Genus Respirovirus belongs to the family Paramyxoviridae, order Mononegavirales, and including 6 viral species: Bovine respirovirus 3, Caprine respirovirus 3, Human respirovirus 1, Human respirovirus 3, Murine respirovirus, and Porcine respirovirus 1 (PRV1) (Amarasinghe et al., 2018, 2019). Historically, these viruses have been related to respiratory infections in human and domestic animals (Li et al. 2016; Maclachlan et al. 2017; Park et al. 2019). PRV1, also known as porcine parainfluenza virus 1 (PPIV-1), was first described in 2013 in nasopharyngeal and rectal swab samples of deceased pigs collected from a slaughterhouse in Hong Kong (Lau et al., 2013). Although its pathogenicity is uncertain, this emerging virus has been associated with respiratory disease in pigs, evidenced by clinical signs that may include coughing, minor sneezing, and serous nasal discharge (Palinski et al., 2016). PRV1 has also been found in co-infections with porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV), suggesting that it may participate in the porcine respiratory disease complex (PRDC), increasing the severity of respiratory disease (Welch et al., 2017). Although PRV1 is genetically related to Human respirovirus 1 (Lau et al., 2013), its zoonotic potential is still unknown. PRV1 has a single-stranded, negative-sense linear RNA genome of ∼15 kb in length, which encodes 6 major proteins: nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large (L) protein (Palinski et al., 2016; Park et al., 2017, 2019). The F and HN are the major envelope glycoproteins on the viral surface, which are crucial for receptor binding, entry, and fusion between the viral envelope and the host cell membrane. They are responsible for inducing the production of neutralizing antibodies (Henrickson, 2003), so they are subject to humoral immune pressure with the consequent genetic variability. Therefore, phylogenetic analysis of the F and HN genes may be useful to carry out molecular epidemiological studies of this virus (Park et al., 2019). PRV1 has been detected in several states of the United States of America (USA), widespread in commercial swine farms (Palinski et al. 2016; Park et al. 2019). To date, PRV1 has only been described in the USA and China, and only 9 whole or near-complete genomes are available in public repositories (Park et al., 2019). Little is known about the epidemiology and distribution of PRV1 in the rest of the world. In this study, we report the first detection of PRV1 in South America and its genetic comparison with other previously described porcine respiroviruses. 2. Materials and Methods One hundred and sixty-four samples were obtained from 6 swine production companies, located in 5 administrative regions of Chile (Valparaiso, Metropolitan, O’Higgins, Maule, and Ñuble), where >80% of the Chilean intensive swine production is located. Samples were collected between 2015 and 2019 during a swine influenza surveillance program, including nasal swabs (121), oral fluids (8) and, lungs (35). In general, the samples were collected in grower pigs between 3 to 11-weeks of age, specifically from pens where clinical symptoms of respiratory disease including sneezing and coughing was observed. The details of the sample collection are provided in Table 1 and supplemental information Table 1. Table 1. Overall results of RT-PCR of Porcine Respirovirus 1 (PRV1) and Influenza A (IAV) in samples of 6 industrial swine farms of Chile. Region Farm Sample Year Total Samples RT-PCR PRV1 positive RT-PCR IAV positive PRV1 & IAV positive Maule Farm 1 Nasal Swab 2018 5 3 (60%) 1 (20%) 1 (20%) Maule Farm 1 Oral Fluid 2018 4 3 (75%) 4 (100%) 3 (75%) Maule Farm 1 Nasal Swab 2015 66 9 (14%) 52 (79%) 8 (12%) Maule Farm 1 Oral Fluid 2015 4 2 (50%) 2 (50%) 2 (50%) Ñuble Farm 2 Nasal Swab 2019 23 6 (26%) 4 (17%) 2 (9%) O'Higgins Farm 3 Nasal Swab 2019 8 2 (25%) 6 (75%) 2 (25%) O'Higgins Farm 3 Lung 2019 4 1 (25%) 3 (75%) 1 (25%) Santiago Farm 4 Nasal Swab 2019 4 0 (0%) 3 (75%) 0 (0%) Santiago Farm 4 Lung 2019 31 2 (6%) 17 (55%) 1 (3%) Valparaiso Farm 5 Nasal Swab 2019 5 2 (40%) 2 (40%) 1 (20%) O'Higgins Farm 6 Nasal Swab 2019 10 1 (10%) 4 (40%) 0 (0%) Total 164 31 (18,9%) 98 (59,8%) 21(12,8%) The PRV1 was detected by a conventional RT-PCR for specific amplification of PRV1, which was inspired by a RT-PCR with degenerated primers to amplify paramyxoviruses (van Boheemen et al., 2012; Neira et al., 2017). Thus, we designed the forward PRV1 F 5’-GAR GGT TAT TGT CAR AAA CTT TGG AC-3’ and reverse PRV1 F 5’-TCA CRG CAA TTG CTT GGT TRT CR CC-3’ primers, which amplified a 120 bp fragment (nt positions 10734 to 10854). Briefly, RNA was purified using Chomczynski-phenol solution (Winkler, BM-1755, Chile) and the RT-PCR was then carried out using the AgPath-ID™ One-Step RT-PCR Reagents kit (Life Technologies, 4387391, USA), following the manufacturer’s instructions. The RT-PCR master mix contained 12.5 µL of 2X RT-PCR Buffer, 1 µL of RT-PCR Enzyme Mix, 0.4 µM of each primer, and nuclease-free water until complete 20 µL. Five µL of template RNA (or nuclease free-water for a negative control) were added to complete a final reaction volume of 25 µL. RT-PCR was initiated at 45 °C for 10 min (reverse transcription), followed by 95 °C for 10 min (initial denaturation) and 40 cycles of 2-step cycling comprising denaturation at 95 °C for 15 s and annealing-extension at 60 °C for 45 s. The PCR products were run in 2% agarose gel at 120 V for 30 min. Eleven positive RT-PCR samples were selected to attempt viral isolation in African green monkey kidney (Vero) cells (ATCC® CCL-81™, USA). This is a previously used cell line to grow other parainfluenza viruses, including respiroviruses (Henrickson, 2003; Eberle et al., 2016). Briefly, cells were grown in minimum essential medium (MEM) with Earle’s salts and L-glutamine (Corning, 10-010-CMR, USA) supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences, SH30071.03, USA) and 1% antibiotic-antimycotic solution (Biological Industries, 03,033-1B, Israel). The inoculum was incubated for 1 h at 37 °C and 5% CO2. Then, the inoculum was removed, and MEM supplemented with 1% antibiotic-antimycotic solution and 1 μg/mL of trypsin treated with N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) was added. Cells were incubated at 37 °C and observed for cytopathic effect (CPE) daily for 5 days. Complementarily, the supernatant was tested by the previously described RT-PCR to confirm the presence of PRV1, after 2 passages. A selection of 8 samples with strong PCR amplification was submitted for RNA sequencing at Icahn School of Medicine at Mount Sinai, NY, USA, and Veterinary Diagnostic Lab, College of Veterinary Medicine, University of Minnesota. Library preparation was performed using the Illumina TruSeq kit, after depletion of ribosomal RNAs with the Ribo-Zero™ Magnetic Gold Kit Human/Mouse/Rat (Illumina). Following paired-end (2 × 100 nt) sequencing on the Illumina HiSeq platform, raw reads were trimmed for quality using Trimommatic (Bolger et al., 2014). A fasta file containing all complete or near-complete genomes (>14k bp long) of respiroviruses (n = 641) was obtained from GenBank and used as a reference to extract all reads mapped to these sequences using the Burrows-Wheeler Aligner (BWA-MEM) and Samtools (Li and Durbin, 2009). The mapped reads were then assembled into contigs using SPAdes (Nurk et al., 2013). The contigs were aligned to an annotated reference (JX857409) and visualized using Icarus (Mikheenko et al., 2016). The contigs were further queried in BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine their identity. The closest reference sequence (accession number JX857409) was used to create a final reference assembly with BWA-MEM and to create the final consensus sequences using Samtools mpileup. The consensus sequences were annotated using GATU (Tcherepanov et al., 2006). Phylogenetic analyzes were performed using the complete concatenated genome and all genes independently, using both nucleotide and amino acid sequences. All PRV1 reference sequences available in NCBI GenBank were used to construct the phylogeny (https://www.ncbi.nlm.nih.gov/genbank/). An outgroup for each protein-coding region phylogeny was obtained by querying the study sequence in BLASTX, and the closest Human respirovirus 1 sequence was selected. The sequences were aligned using MUSCLE v3.8.3 (Edgar, 2004). The phylogenetic trees were constructed by the maximum likelihood method using IQ-TREE with substitution model selection (ModelFinder implemented in IQ-TREE) option and 1000 bootstrap (Nguyen et al., 2014). Also, phylogenetic analysis using Bayesian inference in mrBayes 3.2.7 was performed to support the ML analyzes (Ronquist et al., 2012). This study was performed at Animal Virology Laboratory, Department of Animal Preventive Medicine, Faculty of Veterinary and Animal Sciences, University of Chile. The protocols for animal sampling were approved by the Ethical Scientific Committee for Animals and Environment Care from the University of Chile, certificate number 17077 VET-UCH. 3. Results and Discussion Thirty-one of 164 total samples (18.9%) were RT-PCR positive for PRV1. The overall diagnostic results are included in Table 1 and supplemental information Table 1. From all the six evaluated farms at least one positive sample was obtained, and the within-farm percentage of positive samples ranged between 6 and 40%. These results suggest that PRV1 is disseminated in Chilean swine farms, similar to the results found in the USA (Palinski et al., 2016). However, more studies with a higher number of samples and farms are needed to confirm the widespread of PRV1 in Chile. The virus was detected in all types of samples analyzed in this study: oral fluids (5/8, 62.5%), nasal swabs (23/121, 19.0%), and lungs (3/35, 8.6%). This is consistent with a previous study that also detected a higher percentage of positive samples in oral fluids and nasal swabs than in the lungs (Park et al., 2019). Palinski et al. (2016) showed that PRV1 replicates in epithelial cells of the upper respiratory tract, resulting in nasal shedding (Palinski et al., 2016). Therefore, oral fluids and nasal swabs are likely to be the best samples for detecting the virus. Twenty-one of 31 (67,7%) PRV1-positive samples were also positive for IAV, by RT-PCR. No clinical symptoms have been associated directly with PRV1 during pathogenesis studies, raising the possibility that PRV1 infection is asymptomatic in the absence of additional factors, such as coinfections (Palinski et al., 2016). PRV1 has been previously detected in coinfections with IAV and PRRSV (Welch et al., 2017), although its relevance in the PRDC has not yet been evaluated. Due to the high percentage of samples positive for both PRV1 and IAV in this study, the role of PRV1 as a secondary pathogen in respiratory disease warrants further evaluation. We were not able to isolate PRV1 in Vero cells. The supernatant was positive for PRV1 by RT-PCR in the first passage but became negative in the second passage. Previous studies described the difficulty of isolating PRV1 (Palinski et al., 2016). Recently, PRV1 was successfully isolated in LLC-MK2 cells (ATCC® CCL-7™) (Park et al., 2019). This cell line should be used in further studies to isolate Chilean PRV1 but is not yet available in the country. We obtained next-generation sequencing reads of PRV1 in 7 of 8 samples. However, near-complete genomes were obtained only from 2 samples (Chile/VN140126763/2018 and Chile/VN14014564/2019) (Accession numbers pending), which were used for phylogeny. The final consensus sequences for both samples obtained by reference assembly represents ∼97% of the PRV1 genome. The Chile/VN140126763/2018 sample was obtained from 1,066 reads and assembled in 2 contigs with coverage of 7 × . On the other hand, the Chile/VN14014564/2019 sample was obtained from 843 reads and assembled in 5 contigs with coverage of 8 × . Even though some gaps were observed due to partial sequencing, the genomic organization is typical of viruses belonging to the family Paramyxoviridae containing all representative genes (N, P, M, F, HN, and L). For the Chile/VN14012673/2018 sample, the M, F, and HN genes were completely sequenced, and N (91,2%), P (97,2%), and L (99,8%) genes were partially sequenced. Nucleotide positions for the partial genes cover are detailed in supplemental information Table 2. For the Chile/VN14014564/2019 sample, the N, P, and M genes were completely sequences, and F (86,8%), HN (94,7%), and L (95%) were partially sequenced. The two near-complete genomes were obtained from samples belonged to 2 different farms with no geographical or epidemiological relationship. On the other hand, no other paramyxoviruses were obtained from the samples but reads related to Porcine Astrovirus, Porcine circovirus, and Suid Herpesvirus 2 were commonly found in all samples analyzed. The Chilean PRV1s showed >95% nucleotide identity with PRV1 strains detected in China ( GenBank accession numbers: JX857409.1 - JX857411.1). Blastn results are included in supplemental information. We used all PRV1 available sequences for the phylogeny. For the genome, N, P, M, F and L genes the trees were constructed with 9 PRV1 reference sequences, while the phylogeny of the HN gene was constructed with 19 PRV1 reference sequences. An outgroup with a Human respirovirus 1 sequence was always included. The details of the substitution models used are described in Fig. 1 and supplemental information Figure 2. In general, the Chilean PRV1s were grouped into a sub-clade among Chinese PRV1 sequences, and the North American PRV1 sequences were grouped into another monophyletic cluster. The trees did not have high bootstrap values to determine a clear relationship of Chilean viruses to Asian or North American viruses (Fig. 1, Supplemental information Figure 2). However, the complete genome and the L gene trees had the highest bootstrap support, and the topology of the amino acid and nucleotide trees were more consistent and similar to the Bayesian analysis. The topology of these trees supports the close relationship between Chilean and Chinese PRV1 sequences and the presence of a North American monophyletic cluster. Overall, these results suggest that Chilean PRV1s are genetically related to strains from the USA and China, but, due to the limited information available, their origin cannot be determined. Fig. 1 Download : Download high-res image (753KB)Download : Download full-size image Fig. 1. Phylogenetic analysis of amino acid sequences of PRV1. Phylogenetic trees were constructed using the sequences obtained in this study and all PRV1 sequences available in GenBank. Human respirovirus 1 was used as outgroup. The phylogenetic trees were inferred using maximum likelihood method in IQ-TREE based on the model FLU + G4 for the genome, fusion and HN protein and JTT + G4 for L protein with 1000 bootstrap replicates. Bootstrap values are indicated at each node. The Chilean strain is represented in red, purple for Chinese and blue for the USA strains. GenBank accession numbers for each sequence are given next to strain name 4. Conclusion This is the first report of PRV1 in the Southern Hemisphere. To date, PRV1 has only been reported in China and the USA. The results suggest that PRV1 is a ubiquitous virus in Chilean swine farms and that it is highly geographically distributed. It should be noted that the whole-genome sequencing was attempted in 8 samples and two PRV1 near to complete genome were obtained. Therefore, using only two sequenced strains, it was not possible to determine the genetic diversity of PRV1 in Chile. However, the results confirm the genetic relationship between Chilean and other PRV1s identified in China and the USA. Further studies are necessary to obtain the complete genome of Chilean PRV1 strains, determine the genetic diversity of this virus in the country, and its implication and importance in swine respiratory disease. Data Availability statement All the data that support this study are available upon request. Funding This study was partly funded by the Programa Fondecyt de Iniciación N° 11170877 to V.N; the Programa Beca Doctorado Nacional de CONICYT N° 3344/2016 to J.M; Programa de Investigación Asociativa from the Comisión Nacional de Investigación Científica y Tecnológica, project CONICYT-PIA Anillo ACT 1408 to R.A.M. and V.N. and the Center for Research in Influenza Pathogenesis (CRIP), a National Institute of Allergy and Infectious Diseases–funded Center of Excellence in Influenza Research and Surveillance (CEIRS), contract number HHSN272201400008C to H.V.B. and R.A.M. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments We are grateful to the Chilean swine veterinarians for their support in sample collection. We also thank the staff of the Animal Virology Laboratory of University of Chile for its support processing samples. Appendix A. Supplementary data Download all supplementary files included with this articleHelp The following are Supplementary data to this article: Download : Download zip file (333B) Download : Download Word document (640KB) References Amarasinghe et al., 2018 G.K. Amarasinghe, N.G. Aréchiga Ceballos, A.C. Banyard, C.F. Basler, S. Bavari, A.J. Bennett, K.R. Blasdell, T. Briese, A. Bukreyev, Y. Caì, C.H. Calisher, C. Campos Lawson, K. Chandran, C.A. Chapman, C.Y. Chiu, K.-S. Choi, P.L. Collins, R.G. Dietzgen, V.V. Dolja, O. Dolnik, L.L. Domier, R. Dürrwald, J.M. Dye, A.J. Easton, H. Ebihara, J.E. Echevarría, A.R. Fooks, P.B.H. Formenty, R.A.M. Fouchier, C.M. Freuling, E. Ghedin, T.L. Goldberg, R. Hewson, M. Horie, T.H. Hyndman, D. Jiāng, R. Kityo, G.P. Kobinger, H. Kondō, E.V. Koonin, M. Krupovic, G. Kurath, R.A. Lamb, B. Lee, E.M. Leroy, P. Maes, A. Maisner, D.A. Marston, S.K. Mor, T. Müller, E. Mühlberger, V.M.N. Ramírez, S.V. Netesov, T.F.F. Ng, N. Nowotny, G. Palacios, J.L. Patterson, J.T. Pawęska, S.L. Payne, K. Prieto, B.K. Rima, P. Rota, D. Rubbenstroth, M. Schwemmle, S. Siddell, S.J. Smither, Q. Song, T. Song, M.D. Stenglein, D.M. Stone, A. Takada, R.B. Tesh, L.M. Thomazelli, K. Tomonaga, N. Tordo, J.S. Towner, N. Vasilakis, S. Vázquez-Morón, C. Verdugo, V.E. Volchkov, V. Wahl, P.J. Walker, D. Wang, L.-F. Wang, J.F.X. Wellehan, M.R. Wiley, A.E. Whitfield, Y.I. Wolf, G. Yè, Y.-Z. Zhāng, J.H. Kuhn Taxonomy of the order Mononegavirales: update 2018 Arch. Virol. (2018), pp. 1-12, 10.1007/s00705-018-3814-x View Record in ScopusGoogle Scholar Amarasinghe et al., 2019 G.K. Amarasinghe, M.A. Ayllón, Y. Bào, C.F. Basler, S. Bavari, K.R. Blasdell, T. Briese, P.A. Brown, A. Bukreyev, A. Balkema-Buschmann, U.J. Buchholz, C. Chabi-Jesus, K. Chandran, C. Chiapponi, I. Crozier, R.L. de Swart, R.G. Dietzgen, O. Dolnik, J.F. Drexler, R. Dürrwald, W.G. Dundon, W.P. Duprex, J.M. Dye, A.J. Easton, A.R. Fooks, P.B.H. Formenty, R.A.M. Fouchier, J. Freitas-Astúa, A. Griffiths, R. Hewson, M. Horie, T.H. Hyndman, D. Jiāng, E.W. Kitajima, G.P. Kobinger, H. Kondō, G. Kurath, I.V. Kuzmin, R.A. Lamb, A. Lavazza, B. Lee, D. Lelli, E.M. Leroy, J. Lǐ, P. Maes, S.Y.L. Marzano, A. Moreno, E. Mühlberger, S.V. Netesov, N. Nowotny, A. Nylund, A.L. Økland, G. Palacios, B. Pályi, J.T. Pawęska, S.L. Payne, A. Prosperi, P.L. Ramos-González, B.K. Rima, P. Rota, D. Rubbenstroth, M. Shī, P. Simmonds, S.J. Smither, E. Sozzi, K. Spann, M.D. Stenglein, D.M. Stone, A. Takada, R.B. Tesh, K. Tomonaga, N. Tordo, J.S. Towner, B. van den Hoogen, N. Vasilakis, V. Wahl, P.J. Walker, L.F. Wang, A.E. Whitfield, J.V. Williams, F.M. Zerbini, T. Zhāng, Y.Z. Zhang, J.H. Kuhn Taxonomy of the order Mononegavirales: update 2019 Arch. Virol., 164 (2019), pp. 1967-1980, 10.1007/s00705-019-04247-4 CrossRefView Record in ScopusGoogle Scholar Bolger et al., 2014 A.M. Bolger, M. Lohse, B. Usadel Trimmomatic: a flexible trimmer for Illumina sequence data Bioinformatics, 30 (2014), pp. 2114-2120, 10.1093/bioinformatics/btu170 CrossRefView Record in ScopusGoogle Scholar Eberle et al., 2016 K.C. Eberle, J.L. McGill, T.A. Reinhardt, R.E. Sacco Parainfluenza Virus 3 Blocks Antiviral Mediators Downstream of the Interferon Lambda Receptor by Modulating Stat1 Phosphorylation J. Virol., 90 (2016), pp. 2948-2958, 10.1128/jvi.02502-15 View Record in ScopusGoogle Scholar Edgar, 2004 R.C. Edgar MUSCLE: Multiple sequence alignment with high accuracy and high throughput Nucleic Acids Res., 32 (2004), pp. 1792-1797, 10.1093/nar/gkh340 CrossRefView Record in ScopusGoogle Scholar Henrickson, 2003 K.J. Henrickson Parainfluenza viruses Clin. Microbiol. Rev., 16 (2003), pp. 242-264, 10.1128/cmr.16.2.242-264.2003 View Record in ScopusGoogle Scholar Lau et al., 2013 S.K.P. Lau, P.C.Y. Woo, Y. Wu, A.Y.P. Wong, B.H.L. Wong, C.C.Y. Lau, R.Y.Y. Fan, J.P. Cai, H.W. Tsoi, K.H. Chan, K.Y. Yuen Identification and characterization of a novel paramyxovirus, porcine parainfluenza virus 1, from deceased pigs J. Gen. Virol., 94 (2013), pp. 2184-2190, 10.1099/vir.0.052985-0 CrossRefView Record in ScopusGoogle Scholar Li and Durbin, 2009 H. Li, R. Durbin Fast and accurate short read alignment with Burrows-Wheeler transform Bioinformatics, 25 (2009), pp. 1754-1760, 10.1093/bioinformatics/btp324 CrossRefView Record in ScopusGoogle Scholar Li et al., 2016 W. Li, F. Hao, L. Mao, Z. Wang, T. Zhou, J. Deng, J. Li, W. Zhang, L. Yang, Y. Lv, J. Jiang Pathogenicity and horizontal transmission studies of caprine parainfluenza virus type 3 JS2013 strain in goats Virus Res., 223 (2016), pp. 80-87, 10.1016/j.virusres.2016.06.021 ArticleDownload PDFView Record in ScopusGoogle Scholar Maclachlan et al., 2017 N. Maclachlan, E. Dubovi, S. Barthold, D. Swayne, J. Winton Fenner’s Veterinary Virology Fenner’s Veterinary Virology (2017), pp. 327-356, 10.1016/B978-0-12-800946-8.00017-9 Google Scholar Mikheenko et al., 2016 A. Mikheenko, G. Valin, A. Prjibelski, V. Saveliev, A. Gurevich Icarus: visualizer for de novo assembly evaluation Bioinformatics, 32 (2016), pp. 3321-3323, 10.1093/bioinformatics/btw379 CrossRefView Record in ScopusGoogle Scholar Neira et al., 2017 V. Neira, R. Tapia, C. Verdugo, G. Barriga, S. Mor, T.F.F. Ng, V. García, Río J. Del, P. Rodrigues, C. Briceño, R.A. Medina, D. González-Acuña Novel Avulaviruses in Penguins Antarctica. Emerg. Infect. Dis. J., 23 (2017), 10.3201/eid2307.170054 Google Scholar Nguyen et al., 2014 L.-T. Nguyen, H.A. Schmidt, A. von Haeseler, B.Q. Minh IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies Mol. Biol. Evol., 32 (2014), pp. 268-274, 10.1093/molbev/msu300 View Record in ScopusGoogle Scholar Nurk et al., 2013 S. Nurk, A. Bankevich, D. Antipov, A. Gurevich, A. Korobeynikov, A. Lapidus, A. Prjibelsky, A. Pyshkin, A. Sirotkin, Y. Sirotkin, R. Stepanauskas, J. McLean, R. Lasken, S.R. Clingenpeel, T. Woyke, G. Tesler, M.A. Alekseyev, P.A. Pevzner Assembling genomes and mini-metagenomes from highly chimeric reads Lecture Notes in Computer Science (Including Subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) (2013), pp. 158-170, 10.1007/978-3-642-37195-0_13 CrossRefView Record in ScopusGoogle Scholar Palinski et al., 2016 R.M. Palinski, Z. Chen, J.N. Henningson, Y. Lang, R.R.R. Rowland, Y. Fang, J. Prickett, P.C. Gauger, B.M. Hause Widespread detection and characterization of porcine parainfluenza virus 1 in pigs in the USA J. Gen. Virol., 97 (2016), pp. 281-286, 10.1099/jgv.0.000343 CrossRefView Record in ScopusGoogle Scholar Park et al., 2017 J.Y. Park, M. Welch, K.M. Harmon, J. Zhang, P.E. Piñeyro, G. Li, P.C. Gauger Complete genome sequence of Porcine respirovirus 1 strain USA/MN25890NS/2016, isolated in the United States Genome Announc., 5 (2017), 10.1128/genomeA.01139-17 Google Scholar Park et al., 2019 J.Y. Park, M.W. Welch, K.M. Harmon, J. Zhang, P.E. Piñeyro, G. Li, B.M. Hause, P.C. Gauger Detection, isolation, and in vitro characterization of porcine parainfluenza virus type 1 isolated from respiratory diagnostic specimens in swine Vet. Microbiol., 228 (2019), pp. 219-225, 10.1016/j.vetmic.2018.12.002 ArticleDownload PDFCrossRefView Record in ScopusGoogle Scholar Ronquist et al., 2012 F. Ronquist, M. Teslenko, P. Van Der Mark, D.L. Ayres, A. Darling, S. Höhna, B. Larget, L. Liu, M.A. Suchard, J.P. Huelsenbeck Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space Syst. Biol., 61 (2012), pp. 539-542, 10.1093/sysbio/sys029 CrossRefView Record in ScopusGoogle Scholar Tcherepanov et al., 2006 V. Tcherepanov, A. Ehlers, C. Upton Genome Annotation Transfer Utility (GATU): rapid annotation of viral genomes using a closely related reference genome BMC Genomics, 7 (2006), p. 150, 10.1186/1471-2164-7-150 Google Scholar van Boheemen et al., 2012 S. van Boheemen, T.M. Bestebroer, J.H. Verhagen, A.D.M.E. Osterhaus, S.D. Pas, S. Herfst, R.A.M. Fouchier A family-wide rt-pcr assay for detection of paramyxoviruses and application to a large-scale surveillance study PLoS One, 7 (2012), 10.1371/journal.pone.0034961 Google Scholar Welch et al., 2017 M. Welch, J. Park, P. Gauger, K. Harmon, K. Lin Animal Industry Report Animal Industry Report Animal Industry Report Summary of Veterinary Diagnostic Laboratory Data Summary of Veterinary Diagnostic Laboratory Data Recommended Citation Recommended Citatio

First report of Porcine Respirovirus 1 in South America

Under a Creative Commons license
open access

Highlights

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The manuscript described the first report of Porcine Respirovirus in Latin America, specifically in Chile. The virus was genetically characterized and is related with viruses previously reported in USA and China. Up to date, there is scarce studies in other countries. Due its zoonotic potential is still unknown, is important know more about it.
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We collected 164 samples between 2015 and 2019 during an influenza surveillance program and include nasal swabs (121), oral fluids (8) and lungs (25); from 6 different commercial swine farm companies, located in 5 administrative regions of Chile (Valparaiso, Metropolitan, O’Higgins, Maule and Ñuble), representing >80% of Chilean swine production.
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We tested these samples by RT-PCR to identify PRV1. From the positive RT-PCR samples (31 positives), 11 were selected to attempt viral isolation in African green monkey kidney (Vero) cells, but this attempt was unsuccessful. Eight positive samples were submitted for whole-genome sequencing using the Illumina. We were able to obtain two near to complete genomes from two different farms.
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The phylogenetic results suggest that the Chilean PRV1 are genetically related to RPV1 strains from USA and China, but due to the scarce information it is not possible to determine actual the origin.

Abstract

Porcine respirovirus 1 (PRV1) is an emerging virus in pigs that has been previously described in the USA and China. There are no reports of its presence in the rest of the world. The objective of this study was to determine the occurrence of PRV1 in Chile and to determine its phylogeny. Thus, we collected samples (oral fluids, nasal swabs, and lungs) from a swine influenza A virus (IAV) surveillance program, most of which belonged to pigs with respiratory disease. The samples were analyzed by RT-PCR, and the viral sequencing was obtained using RNA whole-genome sequencing approach. Maximum likelihood phylogeny was constructed with the available references. Thirty-one of 164 samples (18.9%) were RT-PCR positive for PRV1: 62.5% oral fluids, 19.0% nasal swabs, and 8.6% lungs. All 6 farms in this study had at least one positive sample, with 6-40% of positive results per farm, which suggests that PRV1 is disseminated in Chilean swine farms. Twenty-one of 31 (67,7%) PRV1-positive samples were also positive for IAV, so the role of PRV1 as secondary pathogen in respiratory disease needs to be further evaluated. Near to complete genome of two PRV1s were obtained from two farms. The phylogenies, in general, showed low bootstrap support, except the concatenated genome and the L gene trees which showed clustering of the Chilean PRV1 with Asian sequences, suggesting a close genetic relationship. This is the first report of PRV1 in the Southern Hemisphere. Further studies are necessary to determine the genetic diversity of this virus in Chile.

Keywords

Porcine respirovirus 1
porcine parainfluenza virus 1
Swine
South America
Chile

1. Introduction

Genus Respirovirus belongs to the family Paramyxoviridae, order Mononegavirales, and including 6 viral species: Bovine respirovirus 3, Caprine respirovirus 3, Human respirovirus 1, Human respirovirus 3, Murine respirovirus, and Porcine respirovirus 1 (PRV1) (Amarasinghe et al., 2018, 2019). Historically, these viruses have been related to respiratory infections in human and domestic animals (Li et al. 2016; Maclachlan et al. 2017; Park et al. 2019).
PRV1, also known as porcine parainfluenza virus 1 (PPIV-1), was first described in 2013 in nasopharyngeal and rectal swab samples of deceased pigs collected from a slaughterhouse in Hong Kong (Lau et al., 2013). Although its pathogenicity is uncertain, this emerging virus has been associated with respiratory disease in pigs, evidenced by clinical signs that may include coughing, minor sneezing, and serous nasal discharge (Palinski et al., 2016). PRV1 has also been found in co-infections with porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV), suggesting that it may participate in the porcine respiratory disease complex (PRDC), increasing the severity of respiratory disease (Welch et al., 2017). Although PRV1 is genetically related to Human respirovirus 1 (Lau et al., 2013), its zoonotic potential is still unknown.
PRV1 has a single-stranded, negative-sense linear RNA genome of ∼15 kb in length, which encodes 6 major proteins: nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large (L) protein (Palinski et al., 2016; Park et al., 2017, 2019). The F and HN are the major envelope glycoproteins on the viral surface, which are crucial for receptor binding, entry, and fusion between the viral envelope and the host cell membrane. They are responsible for inducing the production of neutralizing antibodies (Henrickson, 2003), so they are subject to humoral immune pressure with the consequent genetic variability. Therefore, phylogenetic analysis of the F and HN genes may be useful to carry out molecular epidemiological studies of this virus (Park et al., 2019).
PRV1 has been detected in several states of the United States of America (USA), widespread in commercial swine farms (Palinski et al. 2016; Park et al. 2019). To date, PRV1 has only been described in the USA and China, and only 9 whole or near-complete genomes are available in public repositories (Park et al., 2019). Little is known about the epidemiology and distribution of PRV1 in the rest of the world. In this study, we report the first detection of PRV1 in South America and its genetic comparison with other previously described porcine respiroviruses.

2. Materials and Methods

One hundred and sixty-four samples were obtained from 6 swine production companies, located in 5 administrative regions of Chile (Valparaiso, Metropolitan, O’Higgins, Maule, and Ñuble), where >80% of the Chilean intensive swine production is located. Samples were collected between 2015 and 2019 during a swine influenza surveillance program, including nasal swabs (121), oral fluids (8) and, lungs (35). In general, the samples were collected in grower pigs between 3 to 11-weeks of age, specifically from pens where clinical symptoms of respiratory disease including sneezing and coughing was observed. The details of the sample collection are provided in Table 1 and supplemental information Table 1.
Table 1. Overall results of RT-PCR of Porcine Respirovirus 1 (PRV1) and Influenza A (IAV) in samples of 6 industrial swine farms of Chile.
RegionFarmSampleYearTotal SamplesRT-PCR PRV1 positiveRT-PCR IAV positivePRV1 & IAV positive
MauleFarm 1Nasal Swab201853 (60%)1 (20%)1 (20%)
MauleFarm 1Oral Fluid201843 (75%)4 (100%)3 (75%)
MauleFarm 1Nasal Swab2015669 (14%)52 (79%)8 (12%)
MauleFarm 1Oral Fluid201542 (50%)2 (50%)2 (50%)
ÑubleFarm 2Nasal Swab2019236 (26%)4 (17%)2 (9%)
O'HigginsFarm 3Nasal Swab201982 (25%)6 (75%)2 (25%)
O'HigginsFarm 3Lung201941 (25%)3 (75%)1 (25%)
SantiagoFarm 4Nasal Swab201940 (0%)3 (75%)0 (0%)
SantiagoFarm 4Lung2019312 (6%)17 (55%)1 (3%)
ValparaisoFarm 5Nasal Swab201952 (40%)2 (40%)1 (20%)
O'HigginsFarm 6Nasal Swab2019101 (10%)4 (40%)0 (0%)
Total16431 (18,9%)98 (59,8%)21(12,8%)
The PRV1 was detected by a conventional RT-PCR for specific amplification of PRV1, which was inspired by a RT-PCR with degenerated primers to amplify paramyxoviruses (van Boheemen et al., 2012; Neira et al., 2017). Thus, we designed the forward PRV1 F 5’-GAR GGT TAT TGT CAR AAA CTT TGG AC-3’ and reverse PRV1 F 5’-TCA CRG CAA TTG CTT GGT TRT CR CC-3’ primers, which amplified a 120 bp fragment (nt positions 10734 to 10854). Briefly, RNA was purified using Chomczynski-phenol solution (Winkler, BM-1755, Chile) and the RT-PCR was then carried out using the AgPath-ID™ One-Step RT-PCR Reagents kit (Life Technologies, 4387391, USA), following the manufacturer’s instructions. The RT-PCR master mix contained 12.5 µL of 2X RT-PCR Buffer, 1 µL of RT-PCR Enzyme Mix, 0.4 µM of each primer, and nuclease-free water until complete 20 µL. Five µL of template RNA (or nuclease free-water for a negative control) were added to complete a final reaction volume of 25 µL. RT-PCR was initiated at 45 °C for 10 min (reverse transcription), followed by 95 °C for 10 min (initial denaturation) and 40 cycles of 2-step cycling comprising denaturation at 95 °C for 15 s and annealing-extension at 60 °C for 45 s. The PCR products were run in 2% agarose gel at 120 V for 30 min.
Eleven positive RT-PCR samples were selected to attempt viral isolation in African green monkey kidney (Vero) cells (ATCC® CCL-81™, USA). This is a previously used cell line to grow other parainfluenza viruses, including respiroviruses (Henrickson, 2003; Eberle et al., 2016). Briefly, cells were grown in minimum essential medium (MEM) with Earle’s salts and L-glutamine (Corning, 10-010-CMR, USA) supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences, SH30071.03, USA) and 1% antibiotic-antimycotic solution (Biological Industries, 03,033-1B, Israel). The inoculum was incubated for 1 h at 37 °C and 5% CO2. Then, the inoculum was removed, and MEM supplemented with 1% antibiotic-antimycotic solution and 1 μg/mL of trypsin treated with N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) was added. Cells were incubated at 37 °C and observed for cytopathic effect (CPE) daily for 5 days. Complementarily, the supernatant was tested by the previously described RT-PCR to confirm the presence of PRV1, after 2 passages.
A selection of 8 samples with strong PCR amplification was submitted for RNA sequencing at Icahn School of Medicine at Mount Sinai, NY, USA, and Veterinary Diagnostic Lab, College of Veterinary Medicine, University of Minnesota. Library preparation was performed using the Illumina TruSeq kit, after depletion of ribosomal RNAs with the Ribo-Zero™ Magnetic Gold Kit Human/Mouse/Rat (Illumina). Following paired-end (2 × 100 nt) sequencing on the Illumina HiSeq platform, raw reads were trimmed for quality using Trimommatic (Bolger et al., 2014). A fasta file containing all complete or near-complete genomes (>14k bp long) of respiroviruses (n = 641) was obtained from GenBank and used as a reference to extract all reads mapped to these sequences using the Burrows-Wheeler Aligner (BWA-MEM) and Samtools (Li and Durbin, 2009). The mapped reads were then assembled into contigs using SPAdes (Nurk et al., 2013). The contigs were aligned to an annotated reference (JX857409) and visualized using Icarus (Mikheenko et al., 2016). The contigs were further queried in BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine their identity. The closest reference sequence (accession number JX857409) was used to create a final reference assembly with BWA-MEM and to create the final consensus sequences using Samtools mpileup. The consensus sequences were annotated using GATU (Tcherepanov et al., 2006). Phylogenetic analyzes were performed using the complete concatenated genome and all genes independently, using both nucleotide and amino acid sequences. All PRV1 reference sequences available in NCBI GenBank were used to construct the phylogeny (https://www.ncbi.nlm.nih.gov/genbank/). An outgroup for each protein-coding region phylogeny was obtained by querying the study sequence in BLASTX, and the closest Human respirovirus 1 sequence was selected. The sequences were aligned using MUSCLE v3.8.3 (Edgar, 2004). The phylogenetic trees were constructed by the maximum likelihood method using IQ-TREE with substitution model selection (ModelFinder implemented in IQ-TREE) option and 1000 bootstrap (Nguyen et al., 2014). Also, phylogenetic analysis using Bayesian inference in mrBayes 3.2.7 was performed to support the ML analyzes (Ronquist et al., 2012).
This study was performed at Animal Virology Laboratory, Department of Animal Preventive Medicine, Faculty of Veterinary and Animal Sciences, University of Chile. The protocols for animal sampling were approved by the Ethical Scientific Committee for Animals and Environment Care from the University of Chile, certificate number 17077 VET-UCH.

3. Results and Discussion

Thirty-one of 164 total samples (18.9%) were RT-PCR positive for PRV1. The overall diagnostic results are included in Table 1 and supplemental information Table 1. From all the six evaluated farms at least one positive sample was obtained, and the within-farm percentage of positive samples ranged between 6 and 40%. These results suggest that PRV1 is disseminated in Chilean swine farms, similar to the results found in the USA (Palinski et al., 2016). However, more studies with a higher number of samples and farms are needed to confirm the widespread of PRV1 in Chile. The virus was detected in all types of samples analyzed in this study: oral fluids (5/8, 62.5%), nasal swabs (23/121, 19.0%), and lungs (3/35, 8.6%). This is consistent with a previous study that also detected a higher percentage of positive samples in oral fluids and nasal swabs than in the lungs (Park et al., 2019). Palinski et al. (2016) showed that PRV1 replicates in epithelial cells of the upper respiratory tract, resulting in nasal shedding (Palinski et al., 2016). Therefore, oral fluids and nasal swabs are likely to be the best samples for detecting the virus.
Twenty-one of 31 (67,7%) PRV1-positive samples were also positive for IAV, by RT-PCR. No clinical symptoms have been associated directly with PRV1 during pathogenesis studies, raising the possibility that PRV1 infection is asymptomatic in the absence of additional factors, such as coinfections (Palinski et al., 2016). PRV1 has been previously detected in coinfections with IAV and PRRSV (Welch et al., 2017), although its relevance in the PRDC has not yet been evaluated. Due to the high percentage of samples positive for both PRV1 and IAV in this study, the role of PRV1 as a secondary pathogen in respiratory disease warrants further evaluation.
We were not able to isolate PRV1 in Vero cells. The supernatant was positive for PRV1 by RT-PCR in the first passage but became negative in the second passage. Previous studies described the difficulty of isolating PRV1 (Palinski et al., 2016). Recently, PRV1 was successfully isolated in LLC-MK2 cells (ATCC® CCL-7™) (Park et al., 2019). This cell line should be used in further studies to isolate Chilean PRV1 but is not yet available in the country.
We obtained next-generation sequencing reads of PRV1 in 7 of 8 samples. However, near-complete genomes were obtained only from 2 samples (Chile/VN140126763/2018 and Chile/VN14014564/2019) (Accession numbers pending), which were used for phylogeny. The final consensus sequences for both samples obtained by reference assembly represents ∼97% of the PRV1 genome. The Chile/VN140126763/2018 sample was obtained from 1,066 reads and assembled in 2 contigs with coverage of 7 × . On the other hand, the Chile/VN14014564/2019 sample was obtained from 843 reads and assembled in 5 contigs with coverage of 8 × . Even though some gaps were observed due to partial sequencing, the genomic organization is typical of viruses belonging to the family Paramyxoviridae containing all representative genes (N, P, M, F, HN, and L). For the Chile/VN14012673/2018 sample, the M, F, and HN genes were completely sequenced, and N (91,2%), P (97,2%), and L (99,8%) genes were partially sequenced. Nucleotide positions for the partial genes cover are detailed in supplemental information Table 2. For the Chile/VN14014564/2019 sample, the N, P, and M genes were completely sequences, and F (86,8%), HN (94,7%), and L (95%) were partially sequenced. The two near-complete genomes were obtained from samples belonged to 2 different farms with no geographical or epidemiological relationship. On the other hand, no other paramyxoviruses were obtained from the samples but reads related to Porcine Astrovirus, Porcine circovirus, and Suid Herpesvirus 2 were commonly found in all samples analyzed.
The Chilean PRV1s showed >95% nucleotide identity with PRV1 strains detected in China ( GenBank accession numbers: JX857409.1 - JX857411.1). Blastn results are included in supplemental information. We used all PRV1 available sequences for the phylogeny. For the genome, N, P, M, F and L genes the trees were constructed with 9 PRV1 reference sequences, while the phylogeny of the HN gene was constructed with 19 PRV1 reference sequences. An outgroup with a Human respirovirus 1 sequence was always included. The details of the substitution models used are described in Fig. 1 and supplemental information Figure 2. In general, the Chilean PRV1s were grouped into a sub-clade among Chinese PRV1 sequences, and the North American PRV1 sequences were grouped into another monophyletic cluster. The trees did not have high bootstrap values to determine a clear relationship of Chilean viruses to Asian or North American viruses (Fig. 1, Supplemental information Figure 2). However, the complete genome and the L gene trees had the highest bootstrap support, and the topology of the amino acid and nucleotide trees were more consistent and similar to the Bayesian analysis. The topology of these trees supports the close relationship between Chilean and Chinese PRV1 sequences and the presence of a North American monophyletic cluster. Overall, these results suggest that Chilean PRV1s are genetically related to strains from the USA and China, but, due to the limited information available, their origin cannot be determined.
Fig. 1
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Fig. 1. Phylogenetic analysis of amino acid sequences of PRV1. Phylogenetic trees were constructed using the sequences obtained in this study and all PRV1 sequences available in GenBank. Human respirovirus 1 was used as outgroup. The phylogenetic trees were inferred using maximum likelihood method in IQ-TREE based on the model FLU + G4 for the genome, fusion and HN protein and JTT + G4 for L protein with 1000 bootstrap replicates. Bootstrap values are indicated at each node. The Chilean strain is represented in red, purple for Chinese and blue for the USA strains. GenBank accession numbers for each sequence are given next to strain name

4. Conclusion

This is the first report of PRV1 in the Southern Hemisphere. To date, PRV1 has only been reported in China and the USA. The results suggest that PRV1 is a ubiquitous virus in Chilean swine farms and that it is highly geographically distributed. It should be noted that the whole-genome sequencing was attempted in 8 samples and two PRV1 near to complete genome were obtained. Therefore, using only two sequenced strains, it was not possible to determine the genetic diversity of PRV1 in Chile. However, the results confirm the genetic relationship between Chilean and other PRV1s identified in China and the USA. Further studies are necessary to obtain the complete genome of Chilean PRV1 strains, determine the genetic diversity of this virus in the country, and its implication and importance in swine respiratory disease.

Data Availability statement

All the data that support this study are available upon request.

Funding

This study was partly funded by the Programa Fondecyt de Iniciación N° 11170877 to V.N; the Programa Beca Doctorado Nacional de CONICYT N° 3344/2016 to J.M; Programa de Investigación Asociativa from the Comisión Nacional de Investigación Científica y Tecnológica, project CONICYT-PIA Anillo ACT 1408 to R.A.M. and V.N. and the Center for Research in Influenza Pathogenesis (CRIP), a National Institute of Allergy and Infectious Diseases–funded Center of Excellence in Influenza Research and Surveillance (CEIRS), contract number HHSN272201400008C to H.V.B. and R.A.M.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

We are grateful to the Chilean swine veterinarians for their support in sample collection. We also thank the staff of the Animal Virology Laboratory of University of Chile for its support processing samples.

Appendix A. Supplementary data

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