Surveillance of avian influenza viruses in Hebei Province of China from 2021 to 2023: identification of a novel reassortant H3N3.

Avian influenza remains a global public health concern for its well-known point mutation and genomic segment reassortment, through which plenty of serum serotypes are generated to escape existing immune protection in animal and human populations. Some occasional cases of human infection of avian influenza viruses (AIVs) since 2020 posed a potential pandemic risk through human-to-human transmission. Both east-west and north-south migratory birds fly through and linger in the Hebei Province of China as a stopover habitat, providing an opportunity for imported AIVs to infect the local poultry and for viral gene reassortment to generate novel stains. In this study, we collected more than 6,000 environmental samples (mostly feces) in Hebei Province from 2021 to 2023. Samples were screened using real-time RT-PCR, and virus isolation was performed using the chick embryo culture method. We identified 10 AIV isolates, including a novel reassortant H3N3 isolate. Sequencing analysis revealed these AIVs to be highly homologous to those isolated in the Yellow River Basin. Our findings supported that AIVs keep evolving to generate new isolates, necessitating a continuous risk assessment of local avian influenza in wild waterfowl in Hebei, China.

Effects of different HA and NA gene combinations on the growth characteristics of the H3N8 influenza candidate vaccine virus.

Since 2022, three human cases of a novel H3N8 avian influenza virus infection have been reported in three provinces in China. Specific vaccines are important means of preparing for the potential influenza pandemic. Thus, H3N8 viruses [A/Henan/cnic410/2022 (HN410) and A/Changsha/1000/2022(CS1000)] were isolated from the infected patients as prototype viruses to develop candidate vaccine viruses (CVVs) using the reverse genetics (RG) technology. Five reassortant viruses with different HA and NA combinations were constructed based on the two viruses to get a high-yield and safe CVV. The results showed that all viruses had similar antigenicity but different growth characteristics. Reassortant viruses carrying NA from CS1000 exhibited better growth ability and NA enzyme activity than the ones carrying HN410 NA. Furthermore, the NA gene of CS1000 had one more potential N-glycosylation site at position 46 compared with HN410. The substitution of position 46 showed that adding or removing N-glycosylation sites to different reassortant viruses had different effects on growth ability. A reassortant virus carrying HN410 HA and CS1000 NA with high growth ability was selected as a CVV, which met the requirements for a CVV. These data suggest that different surface gene combinations and the presence or absence of potential N-glycosylation sites on position 46 in the NA gene affect the growth characteristics of H3N8 CVVs.

CRISPR/Cas13a-based genome editing for establishing the detection method of H9N2 subtype avian influenza virus.

Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/µL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.

Cocirculation of Genetically Distinct Highly Pathogenic Avian Influenza H5N5 and H5N1 Viruses in Crows, Hokkaido, Japan.

We isolated highly pathogenic avian influenza (HPAI) H5N5 and H5N1 viruses from crows in Hokkaido, Japan, during winter 2023-24. They shared genetic similarity with HPAI H5N5 viruses from northern Europe but differed from those in Asia. Continuous monitoring and rapid information sharing between countries are needed to prevent HPAI virus transmission.

The amino acid variation at hemagglutinin sites 145, 153, 164 and 200 modulate antigenicity andreplication of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes circulating in the poultry industry, inflicts substantial economic damage. Mutations in the hemagglutinin (HA) and neuraminidase (NA) proteins of H9N2 frequently alter viral antigenicity and replication. In this paper, we analyzed the HA genetic sequences and antigenic properties of 26 H9N2 isolates obtained from chickens in China between 2012 and 2019. The results showed that these H9N2 viruses all belonged to h9.4.2.5, and were divided into two clades. We assessed the impact of amino acid substitutions at HA sites 145, 149, 153, 164, 167, 168, and 200 on antigenicity, and found that a mutation at site 164 significantly modified antigenic characteristics. Amino acid variations at sites 145, 153, 164 and 200 affected virus's hemagglutination and the growth kinetics in mammalian cells. These results underscore the critical need for ongoing surveillance of the H9N2 virus and provide valuable insights for vaccine development.

Evaluating the epizootic and zoonotic threat of an H7N9 low-pathogenicity avian influenza virus (LPAIV) variant associated with enhanced pathogenicity in turkeys.

Between 2013 and 2017, the A/Anhui/1/13-lineage (H7N9) low-pathogenicity avian influenza virus (LPAIV) was epizootic in chickens in China, causing mild disease, with 616 fatal human cases. Despite poultry vaccination, H7N9 has not been eradicated. Previously, we demonstrated increased pathogenesis in turkeys infected with H7N9, correlating with the emergence of the L217Q (L226Q H3 numbering) polymorphism in the haemagglutinin (HA) protein. A Q217-containing virus also arose and is now dominant in China following vaccination. We compared infection and transmission of this Q217-containing 'turkey-adapted' (ty-ad) isolate alongside the H7N9 (L217) wild-type (wt) virus in different poultry species and investigated the zoonotic potential in the ferret model. Both wt and ty-ad viruses demonstrated similar shedding and transmission in turkeys and chickens. However, the ty-ad virus was significantly more pathogenic than the wt virus in turkeys but not in chickens, causing 100 and 33% mortality in turkeys respectively. Expanded tissue tropism was seen for the ty-ad virus in turkeys but not in chickens, yet the viral cell receptor distribution was broadly similar in the visceral organs of both species. The ty-ad virus required exogenous trypsin for in vitro replication yet had increased replication in primary avian cells. Replication was comparable in mammalian cells, and the ty-ad virus replicated successfully in ferrets. The L217Q polymorphism also affected antigenicity. Therefore, H7N9 infection in turkeys can generate novel variants with increased risk through altered pathogenicity and potential HA antigenic escape. These findings emphasize the requirement for enhanced surveillance and understanding of A/Anhui/1/13-lineage viruses and their risk to different species.

Avian influenza viruses in wild birds in Canada following incursions of highly pathogenic H5N1 virus from Eurasia in 2021-2022.

Following the detection of novel highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in Newfoundland, Canada, in late 2021, avian influenza virus (AIV) surveillance in wild birds was scaled up across Canada. Herein, we present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds during the first year (November 2021-November 2022) following the incursions of HPAIV from Eurasia. The key objectives of the surveillance program were to (i) identify the presence, distribution, and spread of HPAIV and other AIVs; (ii) identify wild bird morbidity and mortality associated with HPAIV; (iii) identify the range of wild bird species infected by HPAIV; and (iv) genetically characterize detected AIV. A total of 6,246 sick and dead wild birds were tested, of which 27.4% were HPAIV positive across 12 taxonomic orders and 80 species. Geographically, HPAIV detections occurred in all Canadian provinces and territories, with the highest numbers in the Atlantic and Central Flyways. Temporally, peak detections differed across flyways, though the national peak occurred in April 2022. In an additional 11,295 asymptomatic harvested or live-captured wild birds, 5.2% were HPAIV positive across 3 taxonomic orders and 19 species. Whole-genome sequencing identified HPAIV of Eurasian origin as most prevalent in the Atlantic Flyway, along with multiple reassortants of mixed Eurasian and North American origins distributed across Canada, with moderate structuring at the flyway scale. Wild birds were victims and reservoirs of HPAIV H5N1 2.3.4.4b, underscoring the importance of surveillance encompassing samples from sick and dead, as well as live and harvested birds, to provide insights into the dynamics and potential impacts of the HPAIV H5N1 outbreak. This dramatic shift in the presence and distribution of HPAIV in wild birds in Canada highlights a need for sustained investment in wild bird surveillance and collaboration across interagency partners. IMPORTANCE: We present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds in the year following the first detection of highly pathogenic avian influenza virus (HPAIV) H5N1 on the continent. The surveillance program tested over 17,000 wild birds, both sick and apparently healthy, which revealed spatiotemporal and taxonomic patterns in HPAIV prevalence and mortality across Canada. The significant shift in the presence and distribution of HPAIV in Canada's wild birds underscores the need for sustained investment in wild bird surveillance and collaboration across One Health partners.

In turkeys, unlike chickens, the non-structural NS1 protein does not play a significant role in the replication and tissue tropism of the H7N1 avian influenza virus.

The economic losses caused by high pathogenicity (HP) avian influenza viruses (AIV) in the poultry industry worldwide are enormous. Although chickens and turkeys are closely related Galliformes, turkeys are thought to be a bridging host for the adaptation of AIV from wild birds to poultry because of their high susceptibility to AIV infections. HPAIV evolve from low pathogenicity (LP) AIV after circulation in poultry through mutations in different viral proteins, including the non-structural protein (NS1), a major interferon (IFN) antagonist of AIV. At present, it is largely unknown whether the virulence determinants of HPAIV are the same in turkeys and chickens. Previously, we showed that mutations in the NS1 of HPAIV H7N1 significantly reduced viral replication in chickens in vitro and in vivo. Here, we investigated the effect of NS1 on the replication and virulence of HPAIV H7N1 in turkeys after inoculation with recombinant H7N1 carrying a naturally truncated wild-type NS1 (with 224 amino-acid "aa" in length) or an extended NS1 with 230-aa similar to the LP H7N1 ancestor. There were no significant differences in multiple-cycle viral replication or in the efficiency of NS1 in blocking IFN induction in the cell culture. Similarly, all viruses were highly virulent in turkeys and replicated at similar levels in various organs and swabs collected from the inoculated turkeys. These results suggest that NS1 does not play a role in the virulence or replication of HPAIV H7N1 in turkeys and further indicate that the genetic determinants of HPAIV differ in these two closely related galliform species.

An avian-origin internal backbone effectively increases the H5 subtype avian influenza vaccine candidate yield in both chicken embryonated eggs and MDCK cells.

Inactivated vaccines play an important role in preventing and controlling the epidemic caused by the H5 subtype avian influenza virus. The vaccine strains are updated in response to alterations in surface protein antigens, while an avian-derived vaccine internal backbone with a high replicative capacity in chicken embryonated eggs and MDCK cells is essential for vaccine development. In this study, we constructed recombinant viruses using the clade 2.3.4.4d A/chicken/Jiangsu/GY5/2017(H5N6, CkG) strain as the surface protein donor and the clade 2.3.4.4b A/duck/Jiangsu/84512/2017(H5N6, Dk8) strain with high replicative ability as an internal donor. After optimization, the integration of the M gene from the CkG into the internal genes from Dk8 (8GM) was selected as the high-yield vaccine internal backbone, as the combination improved the hemagglutinin1/nucleoprotein (HA1/NP) ratio in recombinant viruses. The r8GMΔG with attenuated hemagglutinin and neuraminidase from the CkG exhibited high-growth capacity in both chicken embryos and MDCK cell cultures. The inactivated r8GMΔG vaccine candidate also induced a higher hemagglutination inhibition antibody titer and microneutralization titer than the vaccine strain using PR8 as the internal backbone. Further, the inactivated r8GMΔG vaccine candidate provided complete protection against wild-type strain challenge. Therefore, our study provides a high-yield, easy-to-cultivate candidate donor as an internal gene backbone for vaccine development.

Prevalence and risk factor for H9N2 avian influenza virus in poultry retail shops of Madhya Pradesh.

H9N2 avian Influenza virus subtype is highly neglected but have the potential to emerge as a next pandemic influenza virus, by either itself evolution or through the donation of genes to other subtype. So to understand the extent of H9N2 virus prevalence and associated risk factors in poultry of retail shops and their surrounding environment a cross sectional study was carried out. A total of 500 poultry tissue and 700 environmental samples were collected from 20 district of Madhya Pradesh. Virus isolation was carried out in egg inoculation and harvested allantoic fluid was tested for HA and further molecular confirmation of subtypes by RT-PCR using H9 specific primers. Prevalence was calculated and positive samples were statistically associated with observed risk factors using univariate and multivariate logistic regression analysis. A total of 9.4% and 9.7% prevalence in tissue samples and environmental samples has been reported respectively and out of 20 districts 10 (50%) were found positive for the virus. Out of 21 studied risk factors only two risk factors named as "keeping total number birds slaughtered per day" and "procuring birds from wholesaler" were found significantly associated with the H9N2 positivity in multivariate logistic regression analysis. This high level of H9N2 positivity in birds with no clinical manifestations providing a great opportunity for avian influenza virus for amplification, co-infection in other animals like dogs, cats, pigs and in human through genetic re-assortment that may lead to emergence of a novel influenza virus with high zoonotic potential. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-024-00865-y.

Proteomics Analysis of Duck Lung Tissues in Response to Highly Pathogenic Avian Influenza Virus.

Domestic ducks (Anas platyrhynchos domesticus) are resistant to most of the highly pathogenic avian influenza virus (HPAIV) infections. In this study, we characterized the lung proteome and phosphoproteome of ducks infected with the HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala) at 12 h, 48 h, and 5 days post-infection. A total of 2082 proteins were differentially expressed and 320 phosphorylation sites mapping to 199 phosphopeptides, corresponding to 129 proteins were identified. The functional annotation of the proteome data analysis revealed the activation of the RIG-I-like receptor and Jak-STAT signaling pathways, which led to the induction of interferon-stimulated gene (ISG) expression. The pathway analysis of the phosphoproteome datasets also confirmed the activation of RIG-I, Jak-STAT signaling, NF-kappa B signaling, and MAPK signaling pathways in the lung tissues. The induction of ISG proteins (STAT1, STAT3, STAT5B, STAT6, IFIT5, and PKR) established a protective anti-viral immune response in duck lung tissue. Further, the protein-protein interaction network analysis identified proteins like AKT1, STAT3, JAK2, RAC1, STAT1, PTPN11, RPS27A, NFKB1, and MAPK1 as the main hub proteins that might play important roles in disease progression in ducks. Together, the functional annotation of the proteome and phosphoproteome datasets revealed the molecular basis of the disease progression and disease resistance mechanism in ducks infected with the HPAI H5N1 virus.

Role of miRNA in Highly Pathogenic H5 Avian Influenza Virus Infection: An Emphasis on Cellular and Chicken Models.

The avian influenza virus, particularly the H5N1 strain, poses a significant and ongoing threat to both human and animal health. Recent outbreaks have affected domestic and wild birds on a massive scale, raising concerns about the virus' spread to mammals. This review focuses on the critical role of microRNAs (miRNAs) in modulating pro-inflammatory signaling pathways during the pathogenesis of influenza A virus (IAV), with an emphasis on highly pathogenic avian influenza (HPAI) H5 viral infections. Current research indicates that miRNAs play a significant role in HPAI H5 infections, influencing various aspects of the disease process. This review aims to synthesize recent findings on the impact of different miRNAs on immune function, viral cytopathogenicity, and respiratory viral replication. Understanding these mechanisms is essential for developing new therapeutic strategies to combat avian influenza and mitigate its effects on both human and animal populations.

Evaluating the Impact of Low-Pathogenicity Avian Influenza H6N1 Outbreaks in United Kingdom and Republic of Ireland Poultry Farms during 2020.

In January 2020, increased mortality was reported in a small broiler breeder flock in County Fermanagh, Northern Ireland. Gross pathological findings included coelomitis, oophoritis, salpingitis, visceral gout, splenomegaly, and renomegaly. Clinical presentation included inappetence, pronounced diarrhoea, and increased egg deformation. These signs, in combination with increased mortality, triggered a notifiable avian disease investigation. High pathogenicity avian influenza virus (HPAIV) was not suspected, as mortality levels and clinical signs were not consistent with HPAIV. Laboratory investigation demonstrated the causative agent to be a low-pathogenicity avian influenza virus (LPAIV), subtype H6N1, resulting in an outbreak that affected 15 premises in Northern Ireland. The H6N1 virus was also associated with infection on 13 premises in the Republic of Ireland and six in Great Britain. The close genetic relationship between the viruses in Ireland and Northern Ireland suggested a direct causal link whereas those in Great Britain were associated with exposure to a common ancestral virus. Overall, this rapidly spreading outbreak required the culling of over 2 million birds across the United Kingdom and the Republic of Ireland to stamp out the incursion. This report demonstrates the importance of investigating LPAIV outbreaks promptly, given their substantial economic impacts.

Inactivation of Avian Influenza Virus Inoculated into Ground Beef Patties Cooked on a Commercial Open-Flame Gas Grill.

With the emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (AIV) infection of dairy cattle and its subsequent detection in raw milk, coupled with recent AIV infections affecting dairy farm workers, experiments were conducted to affirm the safety of cooked ground beef related to AIV because such meat is often derived from cull dairy cows. Specifically, retail ground beef (percent lean:fat = ca. 80:20) was inoculated with a low pathogenic AIV (LPAIV) isolate to an initial level of 5.6 log10 50% egg infectious doses (EID50)  per 300 g patty. The inoculated meat was pressed into patties (ca. 2.54 cm thick, ca. 300 g each) and then held at 4 °C for up to 60 min. In each of the two trials, two patties for each of the following three treatments were cooked on a commercial open-flame gas grill to internal instantaneous temperatures of 48.9 °C (120°F), 62.8 °C (145°F), or 71.1 °C (160°F), but without any dwell time. Cooking inoculated ground beef patties to 48.9 °C (ave. cooking time of ca. 15 min) resulted in a mean reduction of ≥2.5 ± 0.9 log10 EID50 per 300 g of ground beef as assessed via quantification of virus in embryonating chicken eggs (ECEs). Likewise, cooking patties on a gas grill to 62.8 °C (ave. cooking time of ca. 21 min) or to the USDA FSIS recommended minimum internal temperature for ground beef of 71.1 °C (ave. cooking time of ca. 24 min) resulted in a reduction to nondetectable levels from initial levels of ≥5.6 log10 EID50 per 300 g. These data establish that levels of infectious AIV are substantially reduced within inoculated ground beef patties (20% fat) using recommended cooking procedures.

High Prevalence of Highly Pathogenic Avian Influenza: A Virus in Vietnam's Live Bird Markets.

Background: In recent years, Vietnam has suffered multiple epizootics of influenza in poultry. Methods: From 10 January 2019 to 26 April 2021, we employed a One Health influenza surveillance approach at live bird markets (LBMs) and swine farms in Northern Vietnam. When the COVID-19 pandemic permitted, each month, field teams collected oral secretion samples from poultry and pigs, animal facility bioaerosol and fecal samples, and animal worker nasal washes at 4 LBMs and 5 swine farms across 5 sites. Initially samples were screened with molecular assays followed by culture in embryonated eggs (poultry swabs) or Madin-Darby canine kidney cells (human or swine swabs). Results: Many of the 3493 samples collected had either molecular or culture evidence for influenza A virus, including 314 (37.5%) of the 837 poultry oropharyngeal swabs, 144 (25.1%) of the 574 bioaerosol samples, 438 (34.9%) of the 1257 poultry fecal swab samples, and 16 (1.9%) of the 828 human nasal washes. Culturing poultry samples yielded 454 influenza A isolates, 83 of which were H5, and 70 (84.3%) of these were highly pathogenic. Additionally, a positive human sample had a H9N2 avian-like PB1 gene. In contrast, the prevalence of influenza A in the swine farms was much lower with only 6 (0.4%) of the 1700 total swine farm samples studied, having molecular evidence for influenza A virus. Conclusions: This study suggests that Vietnam's LBMs continue to harbor high prevalences of avian influenza A viruses, including many highly pathogenic H5N6 strains, which will continue to threaten poultry and humans.

Co-infection of H9N2 subtype avian influenza virus and QX genotype live attenuated infectious bronchitis virus increase the pathogenicity in SPF chickens.

Avian influenza virus (AIV) infection and vaccination against live attenuated infectious bronchitis virus (aIBV) are frequent in poultry worldwide. Here, we evaluated the clinical effect of H9N2 subtype AIV and QX genotype aIBV co-infection in specific-pathogen-free (SPF) white leghorn chickens and explored the potential mechanisms underlying the observed effects using by 4D-FastDIA-based proteomics. The results showed that co-infection of H9N2 AIV and QX aIBV increased mortality and suppressed the growth of SPF chickens. In particular, severe lesions in the kidneys and slight respiratory signs similar to the symptoms of virulent QX IBV infection were observed in some co-infected chickens, with no such clinical signs observed in single-infected chickens. The replication of H9N2 AIV was significantly enhanced in both the trachea and kidneys, whereas there was only a slight effect on the replication of the QX aIBV. Proteomics analysis showed that the IL-17 signaling pathway was one of the unique pathways enriched in co-infected chickens compared to single infected-chickens. A series of metabolism and immune response-related pathways linked with co-infection were also significantly enriched. Moreover, co-infection of the two pathogens resulted in the enrichment of the negative regulation of telomerase activity. Collectively, our study supports the synergistic effect of the two pathogens, and pointed out that aIBV vaccines might increased IBV-associated lesions due to pathogenic co-infections. Exacerbation of the pathogenicity and mortality in H9N2 AIV and QX aIBV co-infected chickens possibly occurred because of an increase in H9N2 AIV replication, the regulation of telomerase activity, and the disturbance of cell metabolism and the immune system.

Detection and characterization of H5N1 HPAIV in environmental samples from a dairy farm.

The recent expansion of HPAIV H5N1 infections in terrestrial mammals in the Americas, most recently including the outbreak in dairy cattle, emphasizes the critical need for better epidemiological monitoring of zoonotic diseases. In this work, we detected, isolated, and characterized the HPAIV H5N1 from environmental swab samples collected from a dairy farm in the state of Kansas, USA. Genomic sequencing of these samples uncovered two distinctive substitutions in the PB2 (E249G) and NS1 (R21Q) genes which are rare and absent in recent 2024 isolates of H5N1 circulating in the mammalian and avian species. Additionally, approximately 1.7% of the sequence reads indicated a PB2 (E627K) substitution, commonly associated with virus adaptation to mammalian hosts. Phylogenetic analyses of the PB2 and NS genes demonstrated more genetic identity between this environmental isolate and the 2024 human isolate (A/Texas/37/2024) of H5N1. Conversely, HA and NA gene analyses revealed a closer relationship between our isolate and those found in other dairy cattle with almost 100% identity, sharing a common phylogenetic subtree. These findings underscore the rapid evolutionary progression of HPAIV H5N1 among dairy cattle and reinforces the need for more epidemiological monitoring which can be done using environmental sampling.

Highly Pathogenic Avian Influenza Virus A(H5N1) Clade 2.3.4.4b Infection in Free-Ranging Polar Bear, Alaska, USA.

We report a natural infection with a Eurasian highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in a free-ranging juvenile polar bear (Ursus maritimus) found dead in North Slope Borough, Alaska, USA. Continued community and hunter-based participation in wildlife health surveillance is key to detecting emerging pathogens in the Arctic.

H19 influenza A virus exhibits species-specific MHC class II receptor usage.

Avian influenza A virus (IAV) surveillance in Northern California, USA, revealed unique IAV hemagglutinin (HA) genome sequences in cloacal swabs from lesser scaups. We found two closely related HA sequences in the same duck species in 2010 and 2013. Phylogenetic analyses suggest that both sequences belong to the recently discovered H19 subtype, which thus far has remained uncharacterized. We demonstrate that H19 does not bind the canonical IAV receptor sialic acid (Sia). Instead, H19 binds to the major histocompatibility complex class II (MHC class II), which facilitates viral entry. Unlike the broad MHC class II specificity of H17 and H18 from bat IAV, H19 exhibits a species-specific MHC class II usage that suggests a limited host range and zoonotic potential. Using cell lines overexpressing MHC class II, we rescued recombinant H19 IAV. We solved the H19 crystal structure and identified residues within the putative Sia receptor binding site (RBS) that impede Sia-dependent entry.