Selection and antigenic characterization of immune-escape mutants of H7N2 low pathogenic avian influenza virus using homologous polyclonal sera.

Understanding the dynamics of the selection of influenza A immune escape variants by serum antibody is critical for designing effective vaccination programs for animals, especially poultry where large populations have a short generation time and may be vaccinated with high frequency. In this report, immune-escape mutants of A/turkey/New York/4450/1994 H7N2 low pathogenic avian influenza virus, were selected by serially passaging the virus in the presence of continuously increasing concentrations of homologous chicken polyclonal sera. Amino acid mutations were identified by sequencing the parental hemagglutinin (HA) gene and every 10 passages by both Sanger and deep sequencing, and the antigenic distance of the mutants to the parent strain was determined. Progressively, a total of five amino acid mutations were observed over the course of 30 passages. Based on their absence from the parental virus with deep sequencing, the mutations appear to have developed de novo. The antigenic distance between the selected mutants and the parent strain increased as the number of amino acid mutations accumulated and the concentration of antibodies had to be periodically increased to maintain the same reduction in virus titer during selection. This selection system demonstrates how H7 avian influenza viruses behave under selection with homologous sera, and provides a glimpse of their evolutionary dynamics, which can be applied to developing vaccination programs that maximize the effectiveness of a vaccine over time.

Pathogenicity and Transmissibility of North American H7 Low Pathogenic Avian Influenza Viruses in Chickens and Turkeys.

Low pathogenic avian influenza (LPAI) viruses can silently circulate in poultry and wild aquatic birds and potentially mutate into highly pathogenic avian influenza (HPAI) viruses. In the U.S., recent emergence and spread of H7N8 and H7N9 HPAI viruses not only caused devastating losses to domestic poultry but also underscored the capability of LPAI viruses to mutate into HPAI viruses. Therefore, in this study, we evaluated pathogenicity and transmissibility of H7N8 and H7N9 LPAI viruses (the progenitors of HPAI viruses) in chickens and turkeys. We also included H7N2 isolated from an outbreak of LPAI in commercial chickens. H7 viruses replicated more efficiently in the respiratory tract than in the gastrointestinal tract, suggesting that their replication is restricted to the upper respiratory tract. Specifically, H7N2 replicated most efficiently in two-week-old chickens and turkeys. In contrast, H7N8 replicated least efficiently in those birds. Further, replication of H7N2 and H7N9 was restricted in the upper respiratory tract of four-week-old specific-pathogen-free (SPF) and broiler chickens. Despite their restricted replication, the two viruses efficiently transmitted from infected to naïve birds by direct contact, leading to seroconversion of contacted chickens. Our findings suggest the importance of continuous monitoring and surveillance of LPAI viruses in the fields.

Differentiated human airway organoids to assess infectivity of emerging influenza virus.

Novel reassortant avian influenza H7N9 virus and pandemic 2009 H1N1 (H1N1pdm) virus cause human infections, while avian H7N2 and swine H1N1 virus mainly infect birds and pigs, respectively. There is no robust in vitro model for assessing the infectivity of emerging viruses in humans. Based on a recently established method, we generated long-term expanding 3D human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. We report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. In addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. We also established improved 2D monolayer culture conditions for the differentiated airway organoids. To demonstrate the ability of differentiated airway organoids to identify human-infective virus, 3D and 2D differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, H7N9/Ah versus H7N2 and H1N1pdm versus an H1N1 strain isolated from swine (H1N1sw). The human-infective H7N9/Ah virus replicated more robustly than the poorly human-infective H7N2 virus; the highly human-infective H1N1pdm virus replicated to a higher titer than the counterpart H1N1sw. Collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. These differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human.

Mice with type 1 diabetes exhibit increased susceptibility to influenza A virus.

Type 1 diabetes (T1D) is a metabolic disease induced by abnormal insulin secretions from damaged islet B cells. Clinical observations have shown that T1D patients are more easily infected by influenza A virus (IAV) and suffer more serious symptoms than non-T1D patients. To investigate the susceptibility of T1D mice to IAV, a T1D mouse model was built by intraperitoneal injection of diluted streptozotocin (STZ) over 5 consecutive days, followed by infection with three subtypes of IAV (H1N1/H5N1/H7N2). The T1D-infected mice showed more serious clinical symptoms and lower survival rates than the non-T1D infected mice. The hematoxylin and eosin (H&E) staining results revealed an increase in serious pathological damage to the lung and pancreas in T1D-infected mice. Immunohistochemistry results indicated higher IAV loads and a more extensive distribution of positive signals in the lungs and pancreas of T1D-infected mice than in those of non-T1D infected mice. Furthermore, according to real-time quantitative polymerase chain reaction (PCR) results, viral replication appeared to occur more easily in the lungs of T1D-infected mice. Thus, T1D-infected mice exhibited higher susceptibility to IAV than did normal mice. This study contributes a mouse model suitable for T1D research as well as valuable information about the mechanism underlying T1D patients' increased susceptibility to IAV.

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.

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.

Characterization of H7N2 Avian Influenza Virus in Wild Birds and Pikas in Qinghai-Tibet Plateau Area.

Qinghai Lake is a major migrating bird breeding site that has experienced several recent highly pathogenic avian influenza virus (HPAIV) epizootics. From 2006 to 2009 we studied Qinghai's wild birds and pikas for evidence of AIV infections. We sampled 941 healthy wild animals and isolated seventeen H7N2 viruses (eight from pikas and nine from wild birds). The H7N2 viruses were phylogenetically closely related to each other and to viruses isolated in Hong Kong in the 1970s. We determined the pathogenicity of the H7N2 viruses by infecting chickens and mice. Our results suggest that pikas might play an important role in the ecology of AIVs, acting as intermediate hosts in which viruses become more adapted to mammals. Our findings of AI infection in pikas are consistent with previous observations and raise the possibility that pikas might play a previously unrecognized role in the ecology of AIVs peridomestic aquatic environments.

Intranasal vaccination with H5, H7 and H9 hemagglutinins co-localized in a virus-like particle protects ferrets from multiple avian influenza viruses.

Avian influenza H5, H7 and H9 viruses top the World Health Organization's (WHO) list of subtypes with the greatest pandemic potential. Here we describe a recombinant virus-like particle (VLP) that co-localizes hemagglutinin (HA) proteins derived from H5N1, H7N2, and H9N2 viruses as an experimental vaccine against these viruses. A baculovirus vector was configured to co-express the H5, H7, and H9 genes from A/Viet Nam/1203/2004 (H5N1), A/New York/107/2003 (H7N2) and A/Hong Kong/33982/2009 (H9N2) viruses, respectively, as well as neuraminidase (NA) and matrix (M1) genes from A/Puerto Rico/8/1934 (H1N1) virus. Co-expression of these genes in Sf9 cells resulted in production of triple-subtype VLPs containing HA molecules derived from the three influenza viruses. The triple-subtype VLPs exhibited hemagglutination and neuraminidase activities and morphologically resembled influenza virions. Intranasal vaccination of ferrets with the VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with H5N1, H7N2, and H9N2 viruses.

Exposure to a low pathogenic A/H7N2 virus in chickens protects against highly pathogenic A/H7N1 virus but not against subsequent infection with A/H5N1.

Recent evidences have demonstrated that the presence of low pathogenic avian influenza viruses (LPAIV) may play an important role in host ecology and transmission of avian influenza viruses (AIV). While some authors have clearly demonstrated that LPAIV can mutate to render highly pathogenic avian influenza viruses (HPAIV), others have shown that their presence could provide the host with enough immunological memory to resist re-infections with HPAIV. In order to experimentally study the role of pre-existing host immunity, chickens previously infected with H7N2 LPAIV were subsequently challenged with H7N1 HPAIV. Pre-infection of chickens with H7N2 LAPIV conferred protection against the lethal challenge with H7N1 HPAIV, dramatically reducing the viral shedding, the clinical signs and the pathological outcome. Correlating with the protection afforded, sera from chickens primed with H7N2 LPAIV reacted with the H7-AIV subtype in hemagglutination inhibition assay and specifically with the N2-neuraminidase antigen. Conversely, subsequent exposure to H5N1 HPAIV resulted in a two days-delay on the onset of disease but all chickens died by 7 days post-challenge. Lack of protection correlated with the absence of H5-hemagglutining inhibitory antibodies prior to H5N1 HPAIV challenge. Our data suggest that in naturally occurring outbreaks of HPAIV, birds with pre-existing immunity to LPAIV could survive lethal infections with HA-homologous HPAIV but not subsequent re-infections with HA-heterologous HPAIV. These results could be useful to better understand the dynamics of AIV in chickens and might help in future vaccine formulations.

Pathobiology and transmission of highly and low pathogenic avian influenza viruses in European quail (Coturnix c. coturnix).

European quail (Coturnix c. coturnix) may share with Japanese quail (Coturnix c. japonica) its potential as an intermediate host and reservoir of avian influenza viruses (AIV). To elucidate this question, European quail were experimentally challenged with two highly pathogenic AIV (HPAIV) (H7N1/HP and H5N1/HP) and one low pathogenic AIV (LPAIV) (H7N2/LP). Contact animals were also used to assess the viral transmission among birds. Severe neurological signs and mortality rates of 67% (H7N1/HP) and 92% (H5N1/HP) were observed. Although histopathological findings were present in both HPAIV-infected groups, H5N1/HP-quail displayed a broader viral antigen distribution and extent of microscopic lesions. Neither clinical nor pathological involvement was observed in LPAIV-infected quail. Consistent long-term viral shedding and effective transmission to naive quail was demonstrated for the three studied AIV. Drinking water arose as a possible transmission route and feathers as a potential origin of HPAIV dissemination. The present study demonstrates that European quail may play a major role in AI epidemiology, highlighting the need to further understand its putative role as an intermediate host for avian/mammalian reassortant viruses.

Serum and egg yolk antibody detection in chickens infected with low pathogenicity avian influenza virus.

Surveillance for low pathogenicity avian influenza virus (LPAIV) infections has primarily relied on labor-intensive collection and serological testing of serum, but for many poultry diseases, easier-to-collect yolk samples have replaced serum for surveillance testing. A time-course LPAIV infection study in layers was performed to evaluate the utility of antibody detection in serum vs. egg yolk samples. Layers inoculated with the LPAIV A/Bobwhite Quail/Pennsylvania/20304/98 (H7N2) were tested for antibody levels in the serum and egg yolk by using the agar gel immunodiffusion test (AGID), hemagglutination-inhibition test (HI), and a commercially available enzyme-linked immunosorbent assay (ELISA). Anti-influenza specific antibodies were detected in the serum as early as 7 days postinoculation (DPI), and the majority of the hens remained positive until 42 DPI. Antibodies in the egg yolk were first detected by AGID at 7 DPI, which was also the first day of detection in serum. However, the majority of the eggs were positive by all techniques at 11 DPI and remained positive until 42 DPI, at which time the number of AGID+ and HI+ samples declined slightly as compared to ELISA+ samples. These results suggest that egg yolk can be an alternative to serum for flock serological surveillance against LPAIV infections, and the three methods (AGID, HI, and ELISA) will give similar results for first 42 days after infection, although AGID may give earlier positive response.

Low pathogenic avian influenza A (H7N2) virus infection in immunocompromised adult, New York, USA, 2003.

In 2003, infection with low pathogenic avian influenza A (H7N2) virus was identified in an immunocompromised man with fever and community-acquired pneumonia in New York, USA. The patient recovered. Although the source of the virus was not identified, this case indicates the usefulness of virus culture for detecting novel influenza A viruses.

Highly (H5N1) and low (H7N2) pathogenic avian influenza virus infection in falcons via nasochoanal route and ingestion of experimentally infected prey.

An experimental infection with highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) viruses was carried out on falcons in order to examine the effects of these viruses in terms of pathogenesis, viral distribution in tissues and viral shedding. The distribution pattern of influenza virus receptors was also assessed. Captive-reared gyr-saker (Falco rusticolus x Falco cherrug) hybrid falcons were challenged with a HPAI H5N1 virus (A/Great crested grebe/Basque Country/06.03249/2006) or a LPAI H7N2 virus (A/Anas plathyrhynchos/Spain/1877/2009), both via the nasochoanal route and by ingestion of previously infected specific pathogen free chicks. Infected falcons exhibited similar infection dynamics despite the different routes of exposure, demonstrating the effectiveness of in vivo feeding route. H5N1 infected falcons died, or were euthanized, between 5-7 days post-infection (dpi) after showing acute severe neurological signs. Presence of viral antigen in several tissues was confirmed by immunohistochemistry and real time RT-PCR (RRT-PCR), which were generally associated with significant microscopical lesions, mostly in the brain. Neither clinical signs, nor histopathological findings were observed in any of the H7N2 LPAI infected falcons, although all of them had seroconverted by 11 dpi. Avian receptors were strongly present in the upper respiratory tract of the falcons, in accordance with the consistent oral viral shedding detected by RRT-PCR in both H5N1 HPAI and H7N2 LPAI infected falcons. The present study demonstrates that gyr-saker hybrid falcons are highly susceptible to H5N1 HPAI virus infection, as previously observed, and that they may play a major role in the spreading of both HPAI and LPAI viruses. For the first time in raptors, natural infection by feeding on infected prey was successfully reproduced. The use of avian prey species in falconry husbandry and wildlife rehabilitation facilities could put valuable birds of prey and humans at risk and, therefore, this practice should be closely monitored.

Improved hatchability and efficient protection after in ovo vaccination with live-attenuated H7N2 and H9N2 avian influenza viruses.

Mass in ovo vaccination with live attenuated viruses is widely used in the poultry industry to protect against various infectious diseases. The worldwide outbreaks of low pathogenic and highly pathogenic avian influenza highlight the pressing need for the development of similar mass vaccination strategies against avian influenza viruses. We have previously shown that a genetically modified live attenuated avian influenza virus (LAIV) was amenable for in ovo vaccination and provided optimal protection against H5 HPAI viruses. However, in ovo vaccination against other subtypes resulted in poor hatchability and, therefore, seemed impractical. In this study, we modified the H7 and H9 hemagglutinin (HA) proteins by substituting the amino acids at the cleavage site for those found in the H6 HA subtype. We found that with this modification, a single dose in ovo vaccination of 18-day old eggs provided complete protection against homologous challenge with low pathogenic virus in ≥ 70% of chickens at 2 or 6 weeks post-hatching. Further, inoculation of 19-day old egg embryos with 106 EID50 of LAIVs improved hatchability to ≥ 90% (equivalent to unvaccinated controls) with similar levels of protection. Our findings indicate that the strategy of modifying the HA cleavage site combined with the LAIV backbone could be used for in ovo vaccination against avian influenza. Importantly, with protection conferred as early as 2 weeks post-hatching, with this strategy birds would be protected prior to or at the time of delivery to a farm or commercial operation.

Structures of receptor complexes of a North American H7N2 influenza hemagglutinin with a loop deletion in the receptor binding site.

Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type (alpha2-3) receptor binding profile, with only moderate binding to human-type (alpha2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.