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      Complete Genome Sequences of Two Novel Human-Like H3N2 Influenza A Viruses, A/swine/Oklahoma/65980/2017 (H3N2) and A/Swine/Oklahoma/65260/2017 (H3N2), Detected in Swine in the United States

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          Abstract

          Two novel human-like H3N2 influenza A virus strains, A/swine/Oklahoma/65980/2017 (H3N2) and A/swine/Oklahoma/65260/2017 (H3N2), were isolated from porcine samples submitted to the Iowa State University Veterinary Diagnostic Laboratory in the United States.

          ABSTRACT

          Two novel human-like H3N2 influenza A virus strains, A/swine/Oklahoma/65980/2017 (H3N2) and A/swine/Oklahoma/65260/2017 (H3N2), were isolated from porcine samples submitted to the Iowa State University Veterinary Diagnostic Laboratory in the United States.

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          Reassortment between Swine H3N2 and 2009 Pandemic H1N1 in the United States Resulted in Influenza A Viruses with Diverse Genetic Constellations with Variable Virulence in Pigs

          Repeated spillovers of the H1N1 pandemic virus (H1N1pdm09) from humans to pigs resulted in substantial evolution of influenza A viruses infecting swine, contributing to the genetic and antigenic diversity of influenza A viruses (IAV) currently circulating in swine. The reassortment with endemic swine viruses and maintenance of some of the H1N1pdm09 internal genes resulted in the circulation of different genomic constellations in pigs. Here, we performed a whole-genome phylogenetic analysis of 368 IAV circulating in swine from 2009 to 2016 in the United States. We identified 44 different genotypes, with the most common genotype (32.33%) containing a clade IV-A HA gene, a 2002-lineage NA gene, an M-pdm09 gene, and remaining gene segments of triple reassortant internal gene (TRIG) origin. To understand how different genetic constellations may relate to viral fitness, we compared the pathogenesis and transmission in pigs of six representative genotypes. Although all six genotypes efficiently infected pigs, they resulted in different degrees of pathology and viral shedding. These results highlight the vast H3N2 genetic diversity circulating in U.S. swine after 2009. This diversity has important implications in the control of this disease by the swine industry, as well as a potential risk for public health if swine-adapted viruses with H1N1pdm09 genes have an increased risk to humans, as occurred in the 2011-2012 and 2016 human variant H3N2v cases associated with exhibition swine. IMPORTANCE People continue to spread the 2009 H1N1 pandemic (H1N1pdm09) IAV to pigs, allowing H1N1pdm09 to reassort with endemic swine IAV. In this study, we determined the 8 gene combinations of swine H3N2 IAV detected from 2009 to 2016. We identified 44 different genotypes of H3N2, the majority of which contained at least one H1N1pdm09 gene segment. We compared six representative genotypes of H3N2 in pigs. All six genotypes efficiently infected pigs, but they resulted in different degrees of lung damage and viral shedding. These results highlight the vast genetic diversity of H3N2 circulating in U.S. swine after 2009, with important implications for the control of IAV for the swine industry. Because H1N1pdm09 is also highly adapted to humans, these swine viruses pose a potential risk to public health if swine-adapted viruses with H1N1pdm09 genes also have an increased risk for human infection.
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            Metagenomic analysis of the RNA fraction of the fecal virome indicates high diversity in pigs infected by porcine endemic diarrhea virus in the United States

            Background Emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) in North America, Asia and Europe has caused severe economic loss to the global swine industry. However, the virome of PEDV infected pigs and its effect on disease severity remains unknown. The advancements of sequencing technology have made it possible to characterize the entire microbiome of different body sites for any host. Methods The objective of this study was to characterize the RNA virome in PEDV-positive pigs using the hypothesis-free metagenomics approach based on next-generation sequencing. Specifically, 217 PEDV-positive swine fecal swab samples collected from diarrheic piglets over 17 US states during 2015–2016 were analyzed. Results A Kraken algorithm-based bioinformatics analysis revealed the presence of up to 9 different RNA genera besides PEDV (Alphacoronavirus genus), including Mamastrovirus (52%, 113/217), Enterovirus (39%, 85/217), Sapelovirus (31%, 67/217), Posavirus (30%, 66/217), Kobuvirus (23%, 49/217), Sapovirus (13%, 28/217), Teschovirus (10%, 22/217), Pasivirus (9%, 20/217), and Deltacoronavirus (3%, 6/217). There were 58 out of 217 piglets (27%) have PEDV infection alone whereas the remaining 159 (73%) shed 2 up to 9 different viruses. Conclusion These findings demonstrated that PEDV infected diarrheic pigs had an extensive RNA viral flora consisting of four different families: Astroviridae, Picornaviridae, Caliciviridae, and Coronaviridae.
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              High-throughput whole genome sequencing of Porcine reproductive and respiratory syndrome virus from cell culture materials and clinical specimens using next-generation sequencing technology.

              Next-generation sequencing (NGS) technologies have increasingly played crucial roles in biological and medical research, but are not yet in routine use in veterinary diagnostic laboratories. We developed and applied a procedure for high-throughput RNA sequencing of Porcine reproductive and respiratory syndrome virus (PRRSV) from cell culture-derived isolates and clinical specimens. Ten PRRSV isolates with known sequence information, 2 mixtures each with 2 different PRRSV isolates, and 51 clinical specimens (19 sera, 16 lungs, and 16 oral fluids) with various PCR threshold cycle (Ct) values were subjected to nucleic acid extraction, cDNA library preparation (24-plexed), and sequencing. Whole genome sequences were obtained from 10 reference isolates with expected sequences and from sera with a PRRSV real-time reverse transcription PCR Ct ≤ 23.6, lung tissues with Ct ≤ 21, and oral fluids with Ct ≤ 20.6. For mixtures with PRRSV-1 and -2 isolates (57.8% nucleotide identity), NGS was able to distinguish them as well as obtain their respective genome sequences. For mixtures with 2 PRRSV-2 isolates (92.4% nucleotide identity), sequence reads with nucleotide ambiguity at numerous sites were observed, indicating mixed infection; however, individual virus sequences could only be separated when 1 isolate identity and sequence in the mixture is known. The NGS approach described herein offers the prospect of high-throughput sequencing and could be adapted to routine workflows in veterinary diagnostic laboratories, although further improvement of sequencing outcomes from clinical specimens with higher Ct values remains to be investigated.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                Microbiol Resour Announc
                Microbiol Resour Announc
                ga
                mra
                MRA
                Microbiology Resource Announcements
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                2576-098X
                21 November 2018
                November 2018
                : 7
                : 20
                : e01203-18
                Affiliations
                [a ]Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA
                [b ]Virus and Prion Research Unit, National Animal Disease Center, USDA-ARS, Ames, Iowa, USA
                KU Leuven, Department of Microbiology and Immunology, Rega Institute, Laboratory of Viral Metagenomics, Leuven, Belgium
                Author notes
                Address correspondence to Phillip C. Gauger, pcgauger@ 123456iastate.edu .

                Citation Zeller MA, Li G, Harmon KM, Zhang J, Vincent AL, Anderson TK, Gauger PC. 2018. Complete genome sequences of two novel human-like H3N2 influenza A viruses, A/swine/Oklahoma/65980/2017 (H3N2) and A/swine/Oklahoma/65260/2017 (H3N2), detected in swine in the United States. Microbiol Resour Announc 7:e01203-18. https://doi.org/10.1128/MRA.01203-18.

                Author information
                https://orcid.org/0000-0002-3138-5535
                https://orcid.org/0000-0003-2540-8769
                Article
                MRA01203-18
                10.1128/MRA.01203-18
                6256625
                30533826
                f447b9b1-15f6-4da6-a464-457e4bd0942d

                This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

                History
                : 29 August 2018
                : 24 October 2018
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 11, Pages: 2, Words: 1416
                Funding
                Funded by: Presidential Interdisciplinary Research Initiative;
                Award ID: 721-18-17-IR-1710
                Award Recipient :
                Funded by: Iowa State University (ISU), https://doi.org/10.13039/100009227;
                Award Recipient :
                Categories
                Genome Sequences
                Custom metadata
                November 2018

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