Showing posts with label pigs. Show all posts
Showing posts with label pigs. Show all posts
Friday, September 16, 2016
Pegivirus Infection in Domestic Pigs Germany Volume 22 Number 7—July 2016 Emerging Infectious Disease journal CDC
Pegivirus Infection in Domestic Pigs Germany Volume 22 Number 7—July 2016 Emerging Infectious Disease journal CDC
Pegivirus Infection in Domestic Pigs, Germany - Volume 22, Number 7—July 2016 - Emerging Infectious Disease journal - CDC
Volume 22, Number 7—July 2016
Letter
Pegivirus Infection in Domestic Pigs, Germany
On This Page
- Letter
- Suggested Citation
Suggested citation for this article
To the Editor: The family Flaviviridae includes many human and animal virus pathogens. Recently, in addition to the genera Flavivirus, Hepacivirus, and Pestivirus, a fourth genus, Pegivirus, has been identified (1). In addition to human pegiviruses, a range of phylogenetic, highly divergent pegiviral sequences have been identified in various animal species, including primates, bats, rodents, and horses (2). We report the detection of a porcine pegivirus (PPgV) in serum samples from pigs.
Initially, we investigated pooled serum samples by using high-throughput sequencing methods and isolated RNA from individual porcine serum samples by using the QIAmp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). We prepared libraries compatible with Illumina (San Diego, CA, USA) sequencing from pooled samples and individual serum samples by using the ScriptSeq version 2 RNA-Seq Library Preparation Kit (Epicenter, Madison, WI, USA) and sequenced them by using a HiSeq 2500 (2 × 150 cycles paired-end; Illumina) for pooled samples and MiSeq (2 × 250 cycles paired-end; Illumina) for individual samples (3).
We conducted quantitative reverse transcription PCR (RT-PCR) by using a Quantitect-SYBR Green Assay (QIAGEN) and primers PPgV_fwd: 5?-CTGTCTATGCTGGTCACGGA-3? and PPgV_rev: 5?-GCCATAGAACGGGAAGTCGC-3?. By using high-throughput sequencing of the pooled serum sample library (23,167,090 reads), we identified 1 contig (4,582 bp) that had distant nucleotide sequence simi-larity to bat pegivirus (69% and 4% sequence coverage) and 2 contigs (2,683 bp and 665 bp) that had 73% sequence coverage, thereby covering 8% and 37% of the identified sequence. RT-PCR with primers designed on basis of recovered sequences identified the sample containing pegivirus sequences. Subsequent MiSeq analysis (7,085,595 reads) of an RNA library prepared from a sample from 1 animal identified 1 contig (9,145 nt) with sequence similarity to pe-givirus sequences.
We performed 3? end completion of the viral genome by rapid amplification of cDNA ends and identified the entire open reading frame of PPgV_903 encoding 2,972 aa (GenBank accession no. KU351669). Analysis of the pegivirus 5?-untranslated region identified a highly structured internal ribosome entry site motif (Technical Appendix[PDF - 302 KB - 5 pages]), which was similar in structure to previously described 5? untranslated region structures of other pegiviruses (4,5).
Pegiviruses do not encode a protein homologous to the capsid protein of other viruses of the family Flaviviridae, another common feature of pegiviruses (6). The presence of cleavage sites for cellular signal peptidases and viral proteases indicates that, similar to polyproteins of other pegiviruses and members of the genus Hepacivirus, the pegivirus polyprotein NH2-E1- E2-Px-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (E [envelope], NS [nonstructural], and Px [protein X]) is cleaved co-translationally and posttranslationally.
We tested 3 additional animals from the same breeding cohort for virus RNA at irregular intervals for 22 months. One animal was positive for pegivirus RNA for 7 months, and the other 2 animals had pegivirus RNA in serum for 16 and 22 months. None of these animals showed obvious clinical signs attributable to virus infection. Follow-up investigation of 455 serum samples from 37 swine holdings from Germany identified 10 (2.2%) samples from 6 pig holdings that contained pegivirus RNA. We obtained 2 additional near full-length genomic sequences (PPgV_80F and PPgV_S8-7) from 2 animals in different herds by high-throughput sequencing, RT-PCR, and Sanger sequencing (GenBank accession nos. KU351670 and KU351671).
Figure. Phylogenetic analysis of human and animal pegiviruses. We constructed a maximum-likelihood tree on the basis of the complete coding region and used the general time reversible model for modeling of substitutions....
Phylogenetic analyses of complete coding regions showed the close relationship of the 3 pegivirus sequences from Germany. These 3 sequences formed a separate clade within the genus Pegivirus (Figure). Pairwise comparison between PPgV_903 and the other 2 pegivirus sequences showed strong nucleotide identities (96.0%–98.4%). A distance scan over the entire polyprotein showed genetic distance to other pegiviruses and demonstrated that NS3 and NS5B contain the most conserved regions among pegivirus polyproteins (Technical Appendix[PDF - 302 KB - 5 pages]).
In horses, 2 distinct pegiviruses that had different potentials to cause clinical disease in infected animals have been described (4,7). No obvious clinical effects were observed in pegivirus-infected animals during our study. However, potential consequences of viral infection for animal health and food production need to be explored more closely under field and experimental conditions. Pegiviruses can interact with the immune system of the host. Co-infection with human pegivirus and HIV can have beneficial effects, which result in decreased retroviral loads and delayed disease progression (8).
It will be useful to investigate whether co-infections with pegiviruses can influence clinical manifestations of infectious diseases of swine, including multifactorial diseases such as postweaning multisystemic wasting syndrome, in which unknown immune modulating virus infections have been suggested to influence the degree of clinical illness (9). RNA viruses have considerable potential to adapt to new environmental conditions and to overcome host restrictions (10). Until now, the host tropism of PPgV has not been investigated in detail. Therefore, additional studies will be required to elucidate whether the spectrum of potential hosts might include other farm or companion animals, and whether the virus might be able to infect humans.
Christine Baechlein1, Adam Grundhoff1, Nicole Fischer, Malik Alawi, Doris Hoeltig, Karl-Heinz Waldmann, and Paul Becher
Acknowledgments
We thank Jens Böttcher, Thomas Große Beilage, Diana Meemken, Alexandra von Altrock, and Cornelia Schwennen for collecting serum samples; Polina Parfentev for providing excellent technical assistance; and Daniela Indenbirken for providing support in preparation of the RNA library.
This study was supported by the German Center for Infection Research/Thematic Translational Unit Emerging Infections.
References
- Stapleton JT, Bukh J, Muerhoff AS, Foung S, Simmonds P. Assignment of human, simian and bat pegiviruses (previously described as GBV-A, GBV-C, and GBV-D) as members of a new genus (Pegivirus) within the Flaviviridae [cited 2015 Oct 21]. http://www.ictvonline.org/proposals/2012.011a-dV.A.v2.Pegivirus.pdf
- Thézé J, Lowes S, Parker J, Pybus OG. Evolutionary and phylogenetic analysis of the hepaciviruses and pegiviruses. Genome Biol Evol.2015;7:2996–3008 .DOIPubMed
- Baechlein C, Fischer N, Grundhoff A, Alawi M, Indenbirken D, Postel A, Identification of a novel hepacivirus in domestic cattle from Germany. J Virol.2015;89:7007–15. DOIPubMed
- Kapoor A, Simmonds P, Cullen JM, Scheel TK, Medina JL, Giannitti F, Identification of a pegivirus (GB virus-like virus) that infects horses. J Virol.2013;87:7185–90. DOIPubMed
- Simons JN, Desai SM, Schultz DE, Lemon SM, Mushahwar IK. Translation initiation in GB viruses A and C: evidence for internal ribosome entry and implications for genome organization. J Virol. 1996;70:6126–35.PubMed
- Stapleton JT, Foung S, Muerhoff AS, Bukh J, Simmonds P. The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae. J Gen Virol. 2011;92:233–46 .DOIPubMed
- Chandriani S, Skewes-Cox P, Zhong W, Ganem DE, Divers TJ, Blaricum AJ, Identification of a previously undescribed divergent virus from theFlaviviridae family in an outbreak of equine serum hepatitis. Proc Natl Acad Sci U S A. 2013;110:E1407–15. DOIPubMed
- Schwarze-Zander C, Blackard JT, Rockstroh JK. Role of GB virus C in modulating HIV disease. Expert Rev Anti Infect Ther. 2012;10:563–72 .DOIPubMed
- Grau-Roma L, Fraile L, Segalés J. Recent advances in the epidemiology, diagnosis and control of diseases caused by porcine circovirus type 2. Vet J.2011;187:23–32. DOIPubMed
- Rosenberg R. Detecting the emergence of novel, zoonotic viruses pathogenic to humans. Cell Mol Life Sci. 2015;72:1115–25. DOIPubMed
Figure
Technical Appendix
Suggested citation for this article: Baechlein C, Grundhoff A, Fischer N, Alawi M, Hoeltig D, Waldmann K-H, et al. Pegivirus infection in domestic pigs, Germany [letter]. Emerg Infect Dis. 2016 Jul [date cited]. http://dx.doi.org/10.3201/eid2207.160024
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Wednesday, September 14, 2016
Senecavirus A in Pigs United States 2015 Volume 22 Number 7—July 2016 Emerging Infectious Disease journal CDC
Senecavirus A in Pigs United States 2015 Volume 22 Number 7—July 2016 Emerging Infectious Disease journal CDC
Senecavirus A in Pigs, United States, 2015 - Volume 22, Number 7—July 2016 - Emerging Infectious Disease journal - CDC
Volume 22, Number 7—July 2016
Letter
Senecavirus A in Pigs, United States, 2015
On This Page
- Letter
- Suggested Citation
Suggested citation for this article
To the Editor: Senecavirus A (SVA) has been sporadically identified in pigs with idiopathic vesicular disease in the United States and Canada (1–3). Clinical symptoms observed include ruptured vesicles and erosions on the snout and lameness associated with broken vesicles along the coronary band. A recent report characterized SVA in pigs in Brazil with similar clinical symptoms in addition to a higher proportion of deaths than would be expected in pigs 1–4 days of age (4,5). Several outbreaks of this infection in pigs were reported in the summer of 2015 in the United States; the more severe clinical features resembled those seen in outbreaks in Brazil (6). Subsequent testing by PCR of 2,033 oral fluid samples from material submitted during 441 routine diagnostic testing procedures (from 25 states) identified 5 SVA-positive cases (1%) (7). Besides affecting animal health, SVA infection is notable because its clinical symptoms resemble those caused by foot-and-mouth disease and vesicular stomatitis viruses. When vesicular disease is observed in US swine, mandatory reporting and testing of animals for foreign animal diseases are required.
In June 2015, we collected 25 nasal and 25 rectal swab specimens from healthy pigs at 5 pig markets in North Carolina (250 total samples), representing pigs from 5 producers per market; the pigs were commingled for <12 hours. Primary markets 1 and 2 were slaughterhouses that purchased top quality pigs. Secondary market 3 was a slaughterhouse that purchased lower quality pigs (primarily underweight or herniated pigs). Market 4 was a broker that purchased pigs for culling and resold them for slaughter. Market 5 was a culled pig slaughterhouse. At markets 1–4, animals were ?20 weeks of age; at market 5, animals were >10 weeks of age.
We sampled the same sites a second time in August 2015. Again, we performed metagenomic sequencing on swab specimens pooled by producer (5 specimens per pool, 50 total pools per sampling) (8). Reads most similar to SVA were identified in numerous pools from samplings and at 4 different markets. Quantitative reverse transcription PCR (qRT-PCR) was performed at the Kansas State Veterinary Diagnostic Laboratory (Manhattan, KS, USA) on the original pooled samples and was positive for SVA (cycle threshold [Ct] <37) for 26 (52%) pools from June and 18 (36%) pools from August. Sites 2 (n = 1 pool positive), 3 (n = 10), 4 (n = 5), and 5 (n = 10) had positive results in June, and sites 3 (n = 10), 4 (n = 1) and 5 (n = 7) had positive results in August. Both specimen types had an approximately equal number of positive results. We carried out virus isolation on swine testicle cells (positive samples from the second sampling), and 100% cytopathic effects were observed for 5 samples that tested positive for SVA by qRT-PCR with Ct values 16–21.
Figure. Phylogenetic tree of Senecavirus A P1 sequences. Maximum-likelihood analysis in combination with 1,000 bootstrap replicates as implemented in MEGA 6.06 (http://www.megasoftware.net) was used to derive the tree on the basis...
Templated assembly of the metagenomic sequencing reads with the SVA prototype strain SVV-001 genome (GenBank accession no. DQ641257) yielded near complete genomes from 5 pools (GenBank nos. KT827249–KT827253). The polyprotein-encoding region of the genomes showed >99% pairwise identity to each other and were most similar to sequences determined from recent outbreaks in Brazil (97%–98% nucleotide and >99% amino acid identity). Analysis of the P1 region of the genome found >99% nucleotide identity between 2015 US SVA sequences and 97% identity to SVA from Brazil. The contemporary US SVA sequences were more distantly related to SVA from an outbreak in Canada in 2011 (95% identity) and to historical US sequences (87%–92% identity). To investigate SVA phylogeny, we performed ClustalW (http://www.genome.jp/tools/clustalw/) alignment of P1 nucleotide sequences, followed by maximum-likelihood analysis using the best-fitting Kimura 2-parameter plus gamma distribution model of evolution. The 2015 US SVA sequences were most closely related to SVA sequences from Brazil; these sequences shared a common ancestor in Canada/11-55910-2011 (Figure).
Our results suggest that SVA commonly circulates in secondary and culled swine markets in North Carolina and that these strains are most similar to strains characterized in 2014–2015 in Brazil, which were associated with idiopathic vesicular disease and neonatal death. Little diagnostic testing is performed on culled animals, which may in part explain the discrepancy between 1% of oral fluids submitted for diagnostic testing being positive for SVA (7), compared with 72% of culled swine swab specimen pools in this study . The sole sample from primary markets that was positive for SVA by qRT-PCR had a Ct of 36.9, just below the negative cutoff of 37.
Further research is needed to address possible correlation between SVA and health status of animals sold at lower value to cull markets. A notable distinction between contemporary SVA in the United States and Brazil, however, is that all the US samples originated from healthy animals that showed no clinical symptoms. Given the high genetic similarity between contemporary US SVA sequences and those from Brazil, additional cofactors likely affect clinical disease.
Ben M. Hause , Olivia Myers, Joshua Duff, and Richard A. Hesse
, Olivia Myers, Joshua Duff, and Richard A. HesseAcknowledgment
Financial support for this study was provided by Zoetis and Boehringer Ingelheim.
References
- Singh K, Corner S, Clark SG, Scherba G, Fredrickson R. Seneca valley virus and vesicular lesions in a pig with idiopathic vesicular disease. J Vet Sci Technol. 2012;3:123 [2015 Sep 2015]. http://www.omicsonline.org/seneca-valley-virus-and-vesicular-lesions-in-a-pig-with-idiopathic-vesicular-disease-2157-7579.1000123.php?aid=9061
- Pasma T, Davidson S, Shaw SL. Idiopathic vesicular disease in swine in Manitoba. Can Vet J. 2008;49:84–5.PubMed
- Hales LM, Knowles NJ, Reddy PS, Xu L, Hay C, Hallenbeck PL. Complete genome sequence analysis of Seneca valley virus-001, a novel oncolytic picornavirus. J Gen Virol. 2008;89:1265–75. DOIPubMed
- Vannucci FA, Linhares DCL, Barcellos DESN, Lam HC, Collins J, Marthaler D. Identification and complete genome of Seneca valley virus in vesicular fluid and sera of pigs affected with idiopathic vesicular disease, Brazil. Transbound Emerg Dis. 2015;62:589–93. DOIPubMed
- Leme RA, Zotti E, Alcântara BK, Oliveira MV, Freitas LA, Alfieri AF, Senecavirus A: an emerging vesicular infection in Brazilian pig herds. Transbound Emerg Dis. 2015;62:603–11. DOIPubMed
- American Association of Swine Veterinarians. Senecavirus A (Seneca Valley virus) in swine-breeding herd cases [cited 2015 Sep 25].https://www.aasv.org/SVV/SVA_Sow_ISU_9215.pdf
- American Association of Swine Veterinarians. SVV oral fluids testing sheds light on distribution [cited 2015 Sep 25].https://www.aasv.org/news/story.php?id=8361
- Hause BM, Collin EA, Anderson J, Hesse RA, Anderson G. Bovine rhinitis viruses are common in U.S. cattle with bovine respiratory disease. PLoS One. 2015;10:e0121998. DOIPubMed
Figure
Suggested citation for this article: Hause BM, Myers O, Duff J, Hesse RA. Senecavirus A in pigs, United States, 2015. Emerg Infect Dis. 2016 Jul [date cited].http://dx.doi.org/10.3201/eid2207.151591
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