Ferret model to mimic the sequential exposure of humans to historical H3N2 influenza viruses.

Mutations accumulate in influenza A virus proteins, especially in the main epitopes on the virus surface glycoprotein hemagglutinin (HA). For influenza A(H3N2) viruses, in particular, the antigenicity of their HA has altered since their emergence in 1968, requiring changes of vaccine strains every few years. Most adults have been exposed to several antigenically divergent H3N2 viruses through infection and/or vaccination, and those exposures affect the immune responses of those individuals. However, animal models reflecting this 'immune history' in humans are lacking and naïve animals are generally used for vaccination and virus challenge studies. Here, we describe a ferret model to mimic the serial exposure of humans to antigenically different historical H3HA proteins. In this model, ferrets were sequentially immunized with adjuvanted recombinant H3HA proteins from two or three different H3HA antigenic clusters in chronological order, and serum neutralizing antibody titers were examined against the homologous virus and viruses from different antigenic clusters. For ferrets immunized with a single HA antigen, serum neutralizing antibody titers were elevated specifically against the homologous virus. However, after immunization with the second or third antigenically distinct HA antigen in chronological order, the ferrets showed an increase in more broadly cross-reactive neutralizing titers against the antigenically distinct viruses and against the homologous virus. Sequentially immunized animals challenged with an antigenically advanced H3N2 virus showed attenuated virus growth and less body temperature increase compared with naïve animals. These results suggest that sequential exposure to antigenically different HAs elicits broader neutralizing activity in sera and enhances immune responses against more antigenically distinct viruses Our findings may partly explain why adults who have been exposed to antigenically divergent HAs are less likely to be infected with influenza virus and have severe symptoms than children.

Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccine-mediated protection from disease. To ascertain and rank the risk of VOCs and VOIs, we analyze the ability of 14 variants (614G, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, Mu, and Omicron) to escape from mRNA vaccine-induced antibodies. The variants show differential reductions in neutralization and replication by post-vaccination sera. Although the Omicron variant (BA.1, BA.1.1, and BA.2) shows the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retain moderate neutralizing activity against that variant. Therefore, vaccination remains an effective strategy during the COVID-19 pandemic.

Robustness of the Ferret Model for Influenza Risk Assessment Studies: a Cross-Laboratory Exercise.

Past pandemic influenza viruses with sustained human-to-human transmissibility have emerged from animal influenza viruses. Employment of experimental models to assess the pandemic risk of emerging zoonotic influenza viruses provides critical information supporting public health efforts. Ferret transmission experiments have been utilized to predict the human-to-human transmission potential of novel influenza viruses. However, small sample sizes and a lack of standardized protocols can introduce interlaboratory variability, complicating interpretation of transmission experimental data. To assess the range of variation in ferret transmission experiments, a global exercise was conducted by 11 laboratories using two common stock H1N1 influenza viruses with different transmission characteristics in ferrets. Parameters known to affect transmission were standardized, including the inoculation route, dose, and volume, as well as a strict 1:1 donor/contact ratio for respiratory droplet transmission. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored and analyzed. The overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. Among environmental parameters that varied across laboratories, donor-to-contact airflow directionality was associated with increased transmissibility. To attain high confidence in identifying viruses with moderate to high transmissibility or low transmissibility under a smaller number of participating laboratories, our analyses support the notion that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment. IMPORTANCE Following detection of a novel virus, rapid characterization efforts (both in vitro and in vivo) are undertaken at numerous laboratories worldwide to evaluate the relative risk posed to human health. Aggregation of these data are critical, but the use of nonstandardized protocols can make interpretation of divergent results a challenge. For evaluation of virus transmissibility, a multifactorial trait which can only be evaluated in vivo, identifying intrinsic levels of variability between groups can improve the utility of these data, as well as ensure that experiments are performed with sufficient replication to ensure high confidence in compiled results. Using the ferret transmission model and two influenza A viruses, we conducted a multicenter standardization exercise to improve the interpretation of transmission data generated during risk assessment activities; this exercise serves as a model for future efforts employing both in vitro and in vivo models against possible pandemic pathogens.

Pulmonary lesions induced by SARS-CoV-2 infection in domestic cats.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019, which ranges from fatal disease in some to mild or subclinical in most affected individuals. Many recovered human patients report persistent respiratory signs; however, lung disease in post-acute infection is poorly understood. Our objective was to describe histologic lung lesions and viral loads following experimental SARS-CoV-2 infection in 11 cats. Microscopic evaluation at 3, 6, 10, or 28 days postinoculation (DPI) identified mild to moderate patchy interstitial pneumonia, bronchiolar epithelial damage, and occlusive histiocytic bronchiolitis. Based on immunohistochemistry, alveolar septal thickening was due to CD204-positive macrophages, fewer B and T lymphocytes, type II pneumocytes, and capillary proliferation with a relative dearth of fibrosis. In blood vessel endothelium, there was reactive hypertrophy or vacuolar degeneration and increased MHC II expression at all time points. Unexpectedly, one cat from the 28 DPI group had severe subacute regionally extensive lymphohistiocytic pneumonia with multifocal consolidation, vasculitis, and alveolar fibrin. Reverse transcriptase-quantitative polymerase chain reaction identified SARS-CoV-2 RNA within the lung at 3 and 6 DPI, and viral RNA was below the limit of detection at 10 and 28 DPI, suggesting that pulmonary lesions persist beyond detection of viral RNA. These findings clarify our comparative understanding of disease induced by SARS-CoV-2 and suggest that cats can serve as an informative model to study post-acute pulmonary sequelae.

Characterization of a new SARS-CoV-2 variant that emerged in Brazil.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems.

Plasticity of the Influenza Virus H5 HA Protein.

Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants.IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

Protective Immunity and Persistent Lung Sequelae in Domestic Cats after SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 readily transmits between domestic cats. We found that domestic cats that recover from an initial infection might be protected from reinfection. However, we found long-term persistence of inflammation and other lung lesions after infection, despite a lack of clinical symptoms and limited viral replication in the lungs.

Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems.

Programming Multifaceted Pulmonary T Cell Immunity by Combination Adjuvants.

Induction of protective mucosal T cell memory remains a formidable challenge to vaccinologists. Using a combination adjuvant strategy that elicits potent CD8 and CD4 T cell responses, we define the tenets of vaccine-induced pulmonary T cell immunity. An acrylic-acid-based adjuvant (ADJ), in combination with Toll-like receptor (TLR) agonists glucopyranosyl lipid adjuvant (GLA) or CpG, promotes mucosal imprinting but engages distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of TH1/TC1/TH17/TC17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampens T cell receptor (TCR) signaling, mitigates terminal differentiation of effectors, and enhances the development of CD4 and CD8 TRM cells that protect against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells play a vital role in optimal programming of CD8 TRM and viral control. Taken together, these findings provide further insights into vaccine-induced multifaceted mucosal T cell immunity with implications in the development of vaccines against respiratorypathogens, including influenza virus and SARS-CoV-2.

Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development.

At the end of 2019, a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was detected in Wuhan, China, that spread rapidly around the world, with severe consequences for human health and the global economy. Here, we assessed the replicative ability and pathogenesis of SARS-CoV-2 isolates in Syrian hamsters. SARS-CoV-2 isolates replicated efficiently in the lungs of hamsters, causing severe pathological lung lesions following intranasal infection. In addition, microcomputed tomographic imaging revealed severe lung injury that shared characteristics with SARS-CoV-2-infected human lung, including severe, bilateral, peripherally distributed, multilobular ground glass opacity, and regions of lung consolidation. SARS-CoV-2-infected hamsters mounted neutralizing antibody responses and were protected against subsequent rechallenge with SARS-CoV-2. Moreover, passive transfer of convalescent serum to naïve hamsters efficiently suppressed the replication of the virus in the lungs even when the serum was administrated 2 d postinfection of the serum-treated hamsters. Collectively, these findings demonstrate that this Syrian hamster model will be useful for understanding SARS-CoV-2 pathogenesis and testing vaccines and antiviral drugs.

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.

Mutations in the Neuraminidase-Like Protein of Bat Influenza H18N11 Virus Enhance Virus Replication in Mammalian Cells, Mice, and Ferrets.

To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets.IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals.

Isolation of Highly Pathogenic H5N1 Influenza Viruses in 2009-2013 in Vietnam.

Routine surveillance and surveillance in response to influenza outbreaks in avian species in Vietnam in 2009-2013 resulted in the isolation of numerous H5N1 influenza viruses of clades 1.1.2, 2.3.2.1a, 2.3.2.1b, 2.3.2.1c, and 2.3.4.1. Consistent with other studies, we found that viruses of clade 2.3.2.1c were dominant in Vietnam in 2013 and circulated in the northern, central, and southern parts of the country. Phylogenetic analysis revealed reassortment among viruses of clades 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c; in contrast, no reassortment was detected between clade 2.3.2.1 viruses and viruses of clades 1.1.2 or 2.3.4.1, respectively. Deep-sequencing of 42 of the 53 isolated H5N1 viruses revealed viral subpopulations encoding variants that may affect virulence, host range, or sensitivity to antiviral compounds; virus isolates containing these subpopulations may have a higher potential to transmit and adapt to mammals. Among the viruses sequenced, a relatively high number of non-synonymous nucleotide polymorphisms was detected in a virus isolated from a barn swallow, possibly suggesting influenza virus adaption to this host.

Effective mosaic-based nanovaccines against avian influenza in poultry.

Avian influenza virus (AIV) is an extraordinarily diverse pathogen that causes significant morbidity in domesticated poultry populations and threatens human life with looming pandemic potential. Controlling avian influenza in susceptible populations requires highly effective, economical and broadly reactive vaccines. Several AIV vaccines have proven insufficient despite their wide use, and better technologies are needed to improve their immunogenicity and broaden effectiveness. Previously, we developed a "mosaic" H5 subtype hemagglutinin (HA) AIV vaccine and demonstrated its broad protection against diverse highly pathogenic H5N1 and seasonal H1N1 virus strains in mouse and non-human primate models. There is a significant interest in developing effective and safe vaccines against AIV that cannot contribute to the emergence of new strains of the virus once circulating in poultry. Here, we report on the development of an H5 mosaic (H5M) vaccine antigen formulated with polyanhydride nanoparticles (PAN) that provide sustained release of encapsulated antigens. H5M vaccine constructs were immunogenic whether delivered by the modified virus Ankara (MVA) strain or encapsulated within PAN. Both humoral and cellular immune responses were generated in both specific-pathogen free (SPF) and commercial chicks. Importantly, chicks vaccinated by H5M constructs were protected in terms of viral shedding from divergent challenge with a low pathogenicity avian influenza (LPAI) strain at 8 weeks post-vaccination. In addition, protective levels of humoral immunity were generated against highly pathogenic avian influenza (HPAI) of the similar H5N1 and genetically dissimilar H5N2 viruses. Overall, the developed platform technologies (MVA vector and PAN encapsulation) were safe and provided high levels of sustained protection against AIV in chickens. Such approaches could be used to design more efficacious vaccines against other important poultry infections.

Single-replication BM2SR vaccine provides sterilizing immunity and cross-lineage influenza B virus protection in mice.

Both influenza A and B viruses cause outbreaks of seasonal influenza resulting in significant morbidity and mortality. There are two antigenically distinct lineages of influenza B virus, Yamagata lineage (YL) and Victoria lineage (VL). Since both B lineages have been co-circulating for years, more than 70% of influenza vaccines currently manufactured are quadrivalent consisting of influenza A (H1N1), influenza A (H3N2), influenza B (YL) and influenza B (VL) antigens. Although quadrivalent influenza vaccines tend to elevate immunity to both influenza B lineages, estimated overall vaccine efficacy against influenza B is still only around 42%. Thus, a more effective influenza B vaccine is needed. To meet this need, we generated BM2-deficient, single-replication (BM2SR) influenza B vaccine viruses that encode surface antigens from influenza B/Wisconsin/01/2010 (B/WI01, YL) and B/Brisbane/60/2008 (B/Bris60, VL) viruses. The BM2SR-WI01 and BM2SR-Bris60 vaccine viruses are replication-deficient in vitro and in vivo, and can only replicate in a cell line that expresses the complementing BM2 protein. Both BM2SR viruses were non-pathogenic to mice, and vaccinated animals showed elevated mucosal and serum antibody responses to both Yamagata and Victoria lineages in addition to cellular responses. Serum antibody responses included lineage-specific hemagglutinin inhibition antibody (HAI) responses as well as responses to the stem region of the hemagglutinin (HA). BM2SR vaccine viruses provided apparent sterilizing immunity to mice against intra- and inter-lineage drifted B virus challenge. The data presented here support the feasibility of BM2SR as a platform for next-generation trivalent influenza vaccine development.

Effectiveness of Whole, Inactivated, Low Pathogenicity Influenza A(H7N9) Vaccine against Antigenically Distinct, Highly Pathogenic H7N9 Virus.

The recent emergence of highly pathogenic influenza A(H7N9) variants poses a great risk to humans. We show that ferrets vaccinated with low pathogenicity H7N9 virus vaccine do not develop severe symptoms after infection with an antigenically distinct, highly pathogenic H7N9 virus. These results demonstrate the protective benefits of this H7N9 vaccine.

Diversity of Influenza A(H5N1) Viruses in Infected Humans, Northern Vietnam, 2004-2010.

Influenza viruses exist in each host as a collection of genetically diverse variants, which might enhance their adaptive potential. To assess the genetic and functional diversity of highly pathogenic avian influenza A(H5N1) viruses within infected humans, we used deep-sequencing methods to characterize samples obtained from infected patients in northern Vietnam during 2004-2010 on different days after infection, from different anatomic sites, or both. We detected changes in virus genes that affected receptor binding, polymerase activity, or interferon antagonism, suggesting that these factors could play roles in influenza virus adaptation to humans. However, the frequency of most of these mutations remained low in the samples tested, implying that they were not efficiently selected within these hosts. Our data suggest that adaptation of influenza A(H5N1) viruses is probably stepwise and depends on accumulating combinations of mutations that alter function while maintaining fitness.

Characterization of a Feline Influenza A(H7N2) Virus.

During December 2016-February 2017, influenza A viruses of the H7N2 subtype infected ≈500 cats in animal shelters in New York, NY, USA, indicating virus transmission among cats. A veterinarian who treated the animals also became infected with feline influenza A(H7N2) virus and experienced respiratory symptoms. To understand the pathogenicity and transmissibility of these feline H7N2 viruses in mammals, we characterized them in vitro and in vivo. Feline H7N2 subtype viruses replicated in the respiratory organs of mice, ferrets, and cats without causing severe lesions. Direct contact transmission of feline H7N2 subtype viruses was detected in ferrets and cats; in cats, exposed animals were also infected via respiratory droplet transmission. These results suggest that the feline H7N2 subtype viruses could spread among cats and also infect humans. Outbreaks of the feline H7N2 viruses could, therefore, pose a risk to public health.