A Novel A(H7N2) Influenza Virus Isolated from a Veterinarian Caring for Cats in a New York City Animal Shelter Causes Mild Disease and Transmits Poorly in the Ferret Model

Abstract

In December 2016, a low-pathogenic avian influenza (LPAI) A(H7N2) virus was identified to be the causative source of an outbreak in a cat shelter in New York City, which subsequently spread to multiple shelters in the states of New York and Pennsylvania. One person with occupational exposure to infected cats became infected with the virus, representing the first LPAI H7N2 virus infection in a human in North America since 2003. Considering the close contact that frequently occurs between companion animals and humans, it was critical to assess the relative risk of this novel virus to public health. The virus isolated from the human case, A/New York/108/2016 (NY/108), caused mild and transient illness in ferrets and mice but did not transmit to naive cohoused ferrets following traditional or aerosol-based inoculation methods. The environmental persistence of NY/108 virus was generally comparable to that of other LPAI H7N2 viruses. However, NY/108 virus replicated in human bronchial epithelial cells with an increased efficiency compared with that of previously isolated H7N2 viruses. Furthermore, the novel H7N2 virus was found to utilize a relatively lower pH for hemagglutinin activation, similar to human influenza viruses. Our data suggest that the LPAI H7N2 virus requires further adaptation before representing a substantial threat to public health. However, the reemergence of an LPAI H7N2 virus in the northeastern United States underscores the need for continuous surveillance of emerging zoonotic influenza viruses inclusive of mammalian species, such as domestic felines, that are not commonly considered intermediate hosts for avian influenza viruses.IMPORTANCE Avian influenza viruses are capable of crossing the species barrier to infect mammals, an event of public health concern due to the potential acquisition of a pandemic phenotype. In December 2016, an H7N2 virus caused an outbreak in cats in multiple animal shelters in New York State. This was the first detection of this virus in the northeastern United States in over a decade and the first documented infection of a felid with an H7N2 virus. A veterinarian became infected following occupational exposure to H7N2 virus-infected cats, necessitating the evaluation of this virus for its capacity to cause disease in mammals. While the H7N2 virus was associated with mild illness in mice and ferrets and did not spread well between ferrets, it nonetheless possessed several markers of virulence for mammals. These data highlight the promiscuity of influenza viruses and the need for diligent surveillance across multiple species to quickly identify an emerging strain with pandemic potential.

Keywords: H7N2; cats; ferret; influenza; low-pathogenic avian influenza virus; pathogenesis; transmission.

FIG 1

Pathogenicity and transmissibility of NY/108 virus following intranasal inoculation in ferrets and mice. (A and B) Six ferrets were inoculated i.n. with 10⁶ EID₅₀ of virus, and nasal wash specimens were collected from each ferret on alternate days postinoculation (p.i.) to assess viral replication (left sets of bars). A naive ferret was placed in the same cage at 24 h p.i. to assess transmission via DC (A) or in an adjacent cage with perforated sidewalls to assess transmission via RD (B), and nasal washes were collected on alternate days postcontact (p.c.) to assess virus transmission in the presence of direct contact or via respiratory droplets (right sets of bars). (C) Tissues were collected at day 3 p.i. from three ferrets that had been inoculated i.n. with 10⁶ EID₅₀ of virus for viral titration. Bars represent individual ferrets. NT, nasal turbinates; Tr, tracheal tissue; Lg, lung tissue; OB, olfactory bulb tissue; Bn, brain tissue (pooled anterior and posterior); Int, intestinal tissue (pooled duodenum, jejunoileal loop, and descending colon); Eye, pooled left and right eye tissue; Conj, conjunctival tissue (pooled left and right). The titers in all tissues are expressed as the mean titer per gram of tissue, with the exception of nasal turbinates and eye and conjunctival tissues, for which the titers are expressed as the mean titer per milliliter of tissue homogenate. (D) Groups of 3 mice each were inoculated i.n. with virus (serial dilution range, 10⁶ to 10² EID₅₀ of virus) and euthanized at day 3 or 6 p.i. for collection of nose or lung tissue for virus titration. Titers are presented as the mean titer per milliliter of tissue plus the standard deviation and are inclusive for 3 mice unless otherwise specified. The limit of virus detection was 10^1.5 EID₅₀.

FIG 2

Infectivity and transmissibility of NY/108 virus following aerosol inoculation in ferrets. Ferrets were inoculated by the aerosol inhalation (AR) route with a high (A) or low (B) dose of virus, and nasal washes were collected from each ferret on alternate days p.i. to assess viral replication (left sets of bars). At 24 h p.i., a naive ferret was placed in the same cage as each inoculated ferret, and nasal washes were collected on alternate days p.c. to assess virus transmission (right sets of bars). (C) Ferrets were inoculated by the ocular aerosol (OA) route, and nasal washes were collected from each ferret on alternate days p.i. to assess viral replication. Bars represent individual ferrets. The limit of virus detection was 10^1.5 EID₅₀. Inoculum doses are listed in the table. ND, the ferret ocular surface was exposed to aerosolized virus during inhalation exposure, but ocular doses were not specifically determined.

FIG 3

Kinetics of H7N2 and pandemic 2009 H1N1 virus replication in human and ferret respiratory tract cells. Human bronchial epithelial (Calu-3) cells (A to C) and ferret differentiated primary tracheal epithelial cells (FTECs) (D) were grown on transwell inserts and infected apically in triplicate at a multiplicity of infection (MOI) of approximately 0.01 (10^3.7 EID₅₀/well) or 0.001, as specified, with A/New York/108/16, A/New York/107/03, A/Tky/VA/4529/02 (LPAI H7N2), or A/Mexico/4482/09 (pandemic H1N1) virus. (D) NY/108 virus replicated in only 1/3 cultures, so only the results for this replicate are shown. Cells were incubated at 37°C or 33°C, as specified. Culture supernatants were collected at the indicated times p.i. for titration in eggs. The limit of virus detection was 10^1.5 EID₅₀. Samples with no virus detection were assigned a value of 10^1.5. *, P < 0.05 between Mex/4482 virus and each of the H7N2 viruses; **, P < 0.05 between NY/108 and NY/107 viruses.

FIG 4

Influenza virus fusion threshold measured by syncytium formation assay. Vero cells were infected with H7 influenza viruses for 16 h before treatment with 5 μg/ml of TPCK-trypsin for 15 min, and then the cells were incubated with fusion buffer with pH values ranging from 4.8 to 7.4 at 37°C for 5 min, followed by incubation with cell culture medium containing 10% FBS for three additional hours. NP-positive cells were identified by immunofluorescence microscopy with anti-NP antibody after fixation. The syncytia among NP-positive cells at the highest pH are shown on the left, and the syncytia among NP-positive cells upon fusion induction at a pH 0.1 unit higher are shown on the right.