Aerosol Transmission from Infected Swine to Ferrets of an H3N2 Virus Collected from an Agricultural Fair and Associated with Human Variant Infections

Abstract

Influenza A viruses (IAV) sporadically transmit from swine to humans, typically associated with agricultural fairs in the United States. A human seasonal H3 virus from the 2010-2011 IAV season was introduced into the U.S. swine population and termed H3.2010.1 to differentiate it from the previous swine H3 virus. This H3N2 lineage became widespread in the U.S. commercial swine population, subsequently spilling over into exhibition swine, and caused a majority of H3N2 variant (H3N2v) cases in humans in 2016 and 2017. A cluster of human H3N2v cases were reported at an agricultural fair in 2017 in Ohio, where 2010.1 H3N2 IAV was concurrently detected in exhibition swine. Genomic analysis showed that the swine and human isolates were nearly identical. In this study, we evaluated the propensity of a 2010.1 H3N2 IAV (A/swine/Ohio/A01354299/2017 [sw/OH/2017]) isolated from a pig in the agricultural fair outbreak to replicate in ferrets and transmit from swine to ferret. sw/OH/2017 displayed robust replication in the ferret respiratory tract, causing slight fever and moderate weight loss. Further, sw/OH/2017 was capable of efficient respiratory droplet transmission from infected pigs to contact ferrets. These findings establish a model for evaluating the propensity of swine IAV to transmit from pig to ferret as a measure of risk to the human population. The identification of higher-risk swine strains can then be targeted for control measures to limit the dissemination at human-swine interfaces to reduce the risk of zoonotic infections and to inform pandemic planning.IMPORTANCE A recently emerged lineage of human-like H3N2 (H3.2010.1) influenza A virus (IAV) from swine has been frequently detected in commercial and exhibition swine in recent years and has been associated with H3N2 variant cases in humans from 2016 and 2017. To demonstrate a model for characterizing the potential for zoonotic transmission associated with swine IAV, we performed an in vivo study of transmission between pigs infected with an H3.2010.1 H3N2 IAV and aerosol contact ferrets. The efficient interspecies transmission demonstrated for the H3.2010.1 IAV in swine emphasizes the need for further characterization of viruses circulating at the swine-human interface for transmission potential prior to human spillover and the development and implementation of more robust vaccines and control strategies to mitigate human exposure to higher-risk swine strains.

Keywords: H3N2; ferrets; human-like; influenza; swine; transmission.

FIG 1

Phylogenetic relationships of H3N2 IAV collected from exhibition swine at agricultural fairs and H3N2v from 2016 to 2017. Maximum likelihood phylogenies of HA (A) and NA (B) were inferred and included 19 swine IAV and 29 H3N2v viruses (n = 26 collected between 2016 and 2017) associated with swine exhibitions at agricultural fairs. A/swine/Ohio/A01354299/2017 (sw/OH/2017) is indicated by a red square, and all H3N2v branches are colored red. HA and NA genes collected from 2017 agricultural fairs (cyan) and 2016 agricultural fairs (yellow) are included with a random sample of contemporary commercial swine IAV (2010.1 “human-like” swine H3 HA in blue, clade IV swine HA in black; 2002 N2 NA in blue, 1998 N2 NA in black). Human seasonal H3 or N2 IAV (gray) is included for context. The trees are midpoint rooted for clarity, branch lengths are drawn to scale, the scale bar indicates the number of nucleotide substitutions per site, and statistical support (SH-aLRT) is presented for branches of interest. Trees with tip labels and statistical support are provided in Fig. S1 and S2 in the supplemental material.

FIG 2

Replication of A/swine/Ohio/A01354299/2017 in ferrets. Four ferrets were inoculated intranasally with 10⁶ TCID₅₀ of sw/OH/2017. Nasal wash samples were collected at 1, 3, and 5 dpi and bronchoalveolar lavage (BALF) was collected at 5 dpi and titrated in MDCK cells via TCID₅₀ assay (A). The limit of detection was 10¹ TCID₅₀/ml and is indicated by a dotted line. Clinical signs were assessed by weight loss (B) and increased body temperature (C) compared to baseline calculated from values collected at −2 to 0 dpi.

FIG 3

Formalin-fixed ferret lung tissue sections were collected at 5 dpi, stained with hematoxylin and eosin, and evaluated for pathology. (A) Nonchallenged ferrets demonstrated normal bronchiolar epithelium and adjacent alveoli; (B) severe bronchiolar epithelial necrosis with abundant neutrophils and marked peribronchiolar lymphocytic cuffing was observed in ferrets challenged with A/swine/Ohio/A01354299/2017; (C) normal blood vessel from a nonchallenged ferret; (D) severe perivascular edema and hemorrhage admixed with large numbers of lymphocytes and neutrophils; (E) normal submucosal glands; (F) affected submucosal glands are necrotic and effaced by large numbers of neutrophils and cellular debris. Magnifications for all panels, ×200.

FIG 4

Experimental design for assessment of swine-to-ferret transmission. Five pigs were housed in a standard, raised pig deck and inoculated intranasally with 2 ml of 10⁶ TCID₅₀ A/swine/Ohio/A01354299/2017. Four naive ferrets, individually housed in isolator cages, were placed adjacent to the pig deck with approximately 7.5 cm of separation between the front of the ferret cages and the pig deck. The impermeable outer doors of the isolators were removed on day 1 postinfection to facilitate exposure of ferrets to infectious porcine respiratory droplets.

FIG 5

Transmission of sw/OH/2017 from inoculated pigs to contact ferrets. Virus replication was assessed via collection of nasal swabs and nasal washes for pigs and ferrets, respectively. Samples were titrated on MDCK cells by TCID₅₀ assay (A). The limit of detection was 10¹ TCID₅₀/ml and is indicated by a dotted line. Ferrets were monitored for changes in body weight (B) and body temperature (C) compared to baseline calculated from values collected at −2 to 0 dpi, until termination of the study from 14 dpc.