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.

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.

ANNOUNCEMENT

Influenza A virus (IAV) causes respiratory disease in swine and is a pathogen shared between humans and pigs. Genetic drift and spillover of human IAV with subsequent reassortment may result in human-like IAV strains novel to swine. We describe here two novel IAV strains detected in swine in 2017 that contain human seasonal influenza virus gene segments potentially transmitted through reverse zoonosis.

Nasal swabs originating from one swine production system, but separate locations in Oklahoma, were submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) in 2017. An H3N2 virus with a novel human-like hemagglutinin (HA) sequence was detected using the VetMAX Gold swine influenza virus (SIV) subtyping real-time PCR (Thermo Fisher Scientific, Waltham, MA). Whole-genome sequencing was performed on strains A/swine/Oklahoma/65980/2017 (H3N2) and A/swine/Oklahoma/65260/2017 (H3N2), isolated in Madin-Darby canine kidney cells. Nucleic acids were extracted using the MagMAX pathogen RNA/DNA kit (catalog number 4462359) and a KingFisher Flex system (both Thermo Fisher Scientific) to construct sequencing libraries using TruSeq (Illumina, Inc., San Diego, CA). Sequencing was performed on a MiSeq system (Illumina, Inc.) following standard Illumina protocols at the ISU VDL (1, 2). Approximately 2,000,000 raw sequencing reads per sample were preprocessed using Trimmomatic version 0.36 and subjected to sequencing quality analysis with FastQC (3, 4). Quality-trimmed total reads were mapped against reference sequences downloaded from the NCBI Influenza Sequence Database (ftp://ftp.ncbi.nih.gov/genomes/INFLUENZA/) using BWA-MEM (5). Mapped reads were extracted using SAMtools (6) and used for de novo assembly. For each segment, contigs were assembled using ABySS (7) and SPAdes (8). The resulting contigs were manually curated in SeqMan Pro to remove contamination and trim chimeric contigs, thus generating a consensus sequence per segment.

A comparison of the nucleotide sequences of both strains demonstrated that the HA, NA, and M sequences were 99.5%, 99.9%, and 99.5% similar, respectively. The NS1 sequences were identical, and the PB1, PB2, and PA sequences had greater than 99.7% identity. The HA sequences of both isolates demonstrated 99% nucleotide (nt) identity to that of the human IAV strain A/Baltimore/0294/2017 (H3N2) (GenBank accession number KY949654). Each isolate had 10 amino acid substitutions in the HA compared to the most similar human strain. The NA sequence of A/swine/Oklahoma/65980/2017 was most similar (99% nt identity) to that of human IAV strain A/Tennessee/06/2017 (H3N2) (GenBank accession number CY226641), and the NA sequence of A/swine/Oklahoma/65260/2017 was most similar (99% nt identity) to that of human IAV strain A/Baltimore/0223/2017 (H3N2) (GenBank accession number KY950122). PB2, PB1, PA, NP, and NS genes were similar to swine-origin triple-reassortant IAV. Phylogenetic analysis indicates that the M gene was derived from the 2009 H1N1 pandemic matrix circulating in swine. The HA and NA were nested within a monophyletic clade of 2016 to 2017 human seasonal H3 IAV, suggesting novel human-to-swine transmission (9).

This study documents a human-to-swine spillover and the potential for human seasonal IAV to cross the species barrier and infect swine. The ISU VDL has detected 21 genetically similar human-like H3 strains since the fall of 2016 (http://influenza.cvm.iastate.edu/correlation.php). The viruses have acquired a swine internal gene constellation through reassortment (10, 11), with at least 10 amino acid mutations in the HA suggesting adaptation and transmission in swine. The USDA swine surveillance system also reported similar human seasonal IAV designated “human-like 2016” in the Influenza A Virus in Swine Surveillance report (http://www.aphis.usda.gov/animal_health/animal_dis_spec/swine/downloads/fy2018quarter1swinereport.pdf).

Data availability.

The complete genome sequence of A/swine/Oklahoma/65980/2017 (H3N2) has been deposited in GenBank under the accession numbers MG720213 through MG720220 and that of A/swine/Oklahoma/65260/2017 (H3N2) under the accession numbers MG720221 through MG720228.