Genome sequences of haemagglutinin cleavage site predict the pathogenicity phenotype of avian influenza virus: statistically validated data for facilitating rapid declarations and reducing reliance on in vivo testing.

Based on the pathogenicity in chickens, most H1-H16 avian influenza viruses (AIV) cause mild diseases, whereas some of the H5 and H7 AI viruses cause severe, systemic disease. The number of basic amino acids in the haemagglutinin (HA) cleavage site of AIV plays a critical role in pathogenicity. As we gain a greater understanding of the molecular mechanisms of pathogenicity, genome sequencing of the HA0 cleavage site has assumed a greater role in assessment of the potential pathogenicity of H5 and H7 viruses. We validated the use of HA cleavage site motif analysis by comparing molecular pathotyping data against experimental in vivo (intravenous pathogenicity index [IVPI] and lethality) data for determination of both low pathogenicity and high pathogenicity AI virus declaration with the goal of expediting pathotype confirmation and further reducing the reliance on in vivo testing. Our data provide statistical support to the continued use of molecular determination of pathotype for AI viruses based on the HA cleavage site sequence in the absence of an in vivo study determination. This approach not only expedites the declaration process of highly pathogenic AIV (HPAIV) but also reduces the need for experimental in vivo testing of H5 and H7 viruses.

Agricultural and geographic factors shaped the North American 2015 highly pathogenic avian influenza H5N2 outbreak.

The 2014-2015 highly pathogenic avian influenza (HPAI) H5NX outbreak represents the largest and most expensive HPAI outbreak in the United States to date. Despite extensive traditional and molecular epidemiological studies, factors associated with the spread of HPAI among midwestern poultry premises remain unclear. To better understand the dynamics of this outbreak, 182 full genome HPAI H5N2 sequences isolated from commercial layer chicken and turkey production premises were analyzed using evolutionary models able to accommodate epidemiological and geographic information. Epidemiological compartmental models embedded in a phylogenetic framework provided evidence that poultry type acted as a barrier to the transmission of virus among midwestern poultry farms. Furthermore, after initial introduction, the propagation of HPAI cases was self-sustainable within the commercial poultry industries. Discrete trait diffusion models indicated that within state viral transitions occurred more frequently than inter-state transitions. Distance and sample size were very strongly supported as associated with viral transition between county groups (Bayes Factor > 30.0). Together these findings indicate that the different types of midwestern poultry industries were not a single homogenous population, but rather, the outbreak was shaped by poultry industries and geographic factors.

Cross-Protection by Inactivated H5 Prepandemic Vaccine Seed Strains against Diverse Goose/Guangdong Lineage H5N1 Highly Pathogenic Avian Influenza Viruses.

The highly pathogenic avian influenza virus (HPAIV) H5N1 A/goose/Guangdong/1996 lineage (Gs/GD) is endemic in poultry across several countries in the world and has caused sporadic lethal infections in humans. Vaccines are important in HPAIV control both for poultry and in prepandemic preparedness for humans. This study assessed inactivated prepandemic vaccine strains in a One Health framework across human and agricultural and wildlife animal health, focusing on the genetic and antigenic diversity of field H5N1 Gs/GD viruses from the agricultural sector and assessing cross-protection in a chicken challenge model. Nearly half (47.92%) of the 48 combinations of vaccine and challenge viruses examined had bird protection of 80% or above. Most vaccinated groups had prolonged mean death times (MDT), and the virus-shedding titers were significantly lower than those of the sham-vaccinated group (P ≤ 0.05). The antibody titers in the prechallenge sera were not predictive of protection. Although vaccinated birds had higher titers of hemagglutination-inhibiting (HI) antibodies against the homologous vaccine antigen, most of them also had lower or no antibody titer against the challenge antigen. The comparison of all parameters and homologous or closely related vaccine and challenge viruses gave the best prediction of protection. Through additional analysis, we identified a pattern of epitope substitutions in the hemagglutinin (HA) of each challenge virus that impacted protection, regardless of the vaccine used. These changes were situated in the antigenic sites and/or reported epitopes associated with virus escape from antibody neutralization. As a result, this study highlights virus diversity, immune response complexity, and the importance of strain selection for vaccine development to control H5N1 HPAIV in the agricultural sector and for human prepandemic preparedness. We suggest that the engineering of specific antigenic sites can improve the immunogenicity of H5 vaccines.IMPORTANCE The sustained circulation of highly pathogenic avian influenza virus (HPAIV) H5N1 A/goose/Guangdong/1996 (Gs/GD) lineage in the agricultural sector and some wild birds has led to the evolution and selection of distinct viral lineages involved in escape from vaccine protection. Our results using inactivated vaccine candidates from the human pandemic preparedness program in a chicken challenge model identified critical antigenic conformational epitopes on H5 hemagglutinin (HA) from different clades that were associated with antibody recognition and escape. Even though other investigators have reported epitope mapping in the H5 HA, much of this information pertains to epitopes reactive to mouse antibodies. Our findings validate changes in antigenic epitopes of HA associated with virus escape from antibody neutralization in chickens, which has direct relevance to field protection and virus evolution. Therefore, knowledge of these immunodominant regions is essential to proactively develop diagnostic tests, improve surveillance platforms to monitor AIV outbreaks, and design more efficient and broad-spectrum agricultural and human prepandemic vaccines.

Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses.

The panzootic caused by A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) A(H5) viruses has occurred in multiple waves since 1996. From 2013 onwards, clade 2.3.4.4 viruses of subtypes A(H5N2), A(H5N6), and A(H5N8) emerged to cause panzootic waves of unprecedented magnitude among avian species accompanied by severe losses to the poultry industry around the world. Clade 2.3.4.4 A(H5) viruses have expanded in distinct geographical and evolutionary pathways likely via long distance migratory bird dispersal onto several continents and by poultry trade among neighboring countries. Coupled with regional circulation, the viruses have evolved further by reassorting with local viruses. As of February 2019, there have been 23 cases of humans infected with clade 2.3.4.4 H5N6 viruses, 16 (70%) of which had fatal outcomes. To date, no HPAI A(H5) virus has caused sustainable human-to-human transmission. However, due to the lack of population immunity in humans and ongoing evolution of the virus, there is a continuing risk that clade 2.3.4.4 A(H5) viruses could cause an influenza pandemic if the ability to transmit efficiently among humans was gained. Therefore, multisectoral collaborations among the animal, environmental, and public health sectors are essential to conduct risk assessments and develop countermeasures to prevent disease and to control spread. In this article, we describe an assessment of the likelihood of clade 2.3.4.4 A(H5) viruses gaining human-to-human transmissibility and impact on human health should such human-to-human transmission occur. This structured analysis assessed properties of the virus, attributes of the human population, and ecology and epidemiology of these viruses in animal hosts.

Highly Pathogenic Avian Influenza A(H7N3) Virus in Poultry, United States, 2020.

An outbreak of low-pathogenicity avian influenza A(H7N3) virus of North American wild bird lineage occurred on commercial turkey farms in North Carolina and South Carolina, USA, during March-April 2020. The virus mutated to the highly pathogenic form in 1 house on 1 farm via recombination with host 28S rRNA.

Lack of Susceptibility to SARS-CoV-2 and MERS-CoV in Poultry.

We challenged chickens, turkeys, ducks, quail, and geese with severe acute respiratory syndrome coronavirus 2 or Middle East respiratory syndrome coronavirus. We observed no disease and detected no virus replication and no serum antibodies. We concluded that poultry are unlikely to serve a role in maintenance of either virus.

Low Pathogenicity Avian Influenza (H5N2) Viruses, Dominican Republic.

Low pathogenicity avian influenza (H5N2) virus was detected in poultry in the Dominican Republic in 2007 and re-emerged in 2017. Whole-genome sequencing and phylogenetic analysis show introduction of an H5N2 virus lineage from Mexico into poultry in the Dominican Republic, then divergence into 3 distinct genetic subgroups during 2007-2019.

Loss of Fitness of Mexican H7N3 Highly Pathogenic Avian Influenza Virus in Mallards after Circulating in Chickens.

Outbreaks of highly pathogenic avian influenza (HPAI) virus subtype H7N3 have been occurring in commercial chickens in Mexico since its first introduction in 2012. In order to determine changes in virus pathogenicity and adaptation in avian species, three H7N3 HPAI viruses from 2012, 2015, and 2016 were evaluated in chickens and mallards. All three viruses caused high mortality in chickens when given at medium to high doses and replicated similarly. No mortality or clinical signs and similar infectivity were observed in mallards inoculated with the 2012 and 2016 viruses. However, the 2012 H7N3 HPAI virus replicated well in mallards and transmitted to contacts, whereas the 2016 virus replicated poorly and did not transmit to contacts, which indicates that the 2016 virus is less adapted to mallards. In vitro, the 2016 virus grew slower and to lower titers than did the 2012 virus in duck fibroblast cells. Full-genome sequencing showed 115 amino acid differences between the 2012 and the 2016 viruses, with some of these changes previously associated with changes in replication in avian species, including hemagglutinin (HA) A125T, nucleoprotein (NP) M105V, and NP S377N. In conclusion, as the Mexican H7N3 HPAI virus has passaged through large populations of chickens in a span of several years and has retained its high pathogenicity for chickens, it has decreased in fitness in mallards, which could limit the potential spread of this HPAI virus by waterfowl.IMPORTANCE Not much is known about changes in host adaptation of avian influenza (AI) viruses in birds after long-term circulation in chickens or other terrestrial poultry. Although the origin of AI viruses affecting poultry is wild aquatic birds, the role of these birds in further dispersal of poultry-adapted AI viruses is not clear. Previously, we showed that HPAI viruses isolated early from poultry outbreaks could still infect and transmit well in mallards. In this study, we demonstrate that the Mexican H7N3 HPAI virus after four years of circulation in chickens replicates poorly and does not transmit in mallards but remains highly pathogenic in chickens. This information on changes in host adaptation is important for understanding the epidemiology of AI viruses and the role that wild waterfowl may play in disseminating viruses adapted to terrestrial poultry.

Spotlight on avian pathology: can we reduce the pandemic threat of H9N2 avian influenza to human and avian health?

COVID-19 should be a "call to arms" for the poultry industry to reassess containment of the H9N2 subtype of low pathogenicity avian influenza viruses. Strains of this virus are a human pandemic threat and a severe economic burden on poultry production. Over the past 20 years they have spread throughout Asia, Africa, Middle East and parts of Europe. As a global industry, a critical need is to re-imagine production and marketing chains, especially in low and middle-income countries, where the structure of much of the industry facilitates virus transmission, especially, but not only, in improperly managed live poultry markets and related value chains. Better, appropriately matched vaccines are needed to support this process but such vaccines cannot, alone, overcome the existing defects in biosecurity, including high farm densities. None of this will occur unless the threat posed by this virus to global health security is recognized.

Pathobiology and innate immune responses of gallinaceous poultry to clade 2.3.4.4A H5Nx highly pathogenic avian influenza virus infection.

In the 2014-2015 Eurasian lineage clade 2.3.4.4A H5 highly pathogenic avian influenza (HPAI) outbreak in the U.S., backyard flocks with minor gallinaceous poultry and large commercial poultry (chickens and turkeys) operations were affected. The pathogenesis of the first H5N8 and reassortant H5N2 clade 2.3.4.4A HPAI U.S. isolates was investigated in six gallinaceous species: chickens, Japanese quail, Bobwhite quail, Pearl guinea fowl, Chukar partridges, and Ring-necked pheasants. Both viruses caused 80-100% mortality in all species, except for H5N2 virus that caused 60% mortality in chickens. The surviving challenged birds remained uninfected based on lack of clinical disease and lack of seroconversion. Among the infected birds, chickens and Japanese quail in early clinical stages (asymptomatic and listless) lacked histopathologic findings. In contrast, birds of all species in later clinical stages (moribund and dead) had histopathologic lesions and systemic virus replication consistent with HPAI virus infection in gallinaceous poultry. These birds had widespread multifocal areas of necrosis, sometimes with heterophilic or lymphoplasmacytic inflammatory infiltrate, and viral antigen in parenchymal cells of most tissues. In general, lesions and antigen distribution were similar regardless of virus and species. However, endotheliotropism was the most striking difference among species, with only Pearl guinea fowl showing widespread replication of both viruses in endothelial cells of most tissues. The expression of IFN-γ and IL-10 in Japanese quail, and IL-6 in chickens, were up-regulated in later clinical stages compared to asymptomatic birds.

Transmission Dynamics of Highly Pathogenic Avian Influenza Virus A(H5Nx) Clade 2.3.4.4, North America, 2014-2015.

Eurasia highly pathogenic avian influenza virus (HPAIV) H5 clade 2.3.4.4 emerged in North America at the end of 2014 and caused outbreaks affecting >50 million poultry in the United States before eradication in June 2015. We investigated the underlying ecologic and epidemiologic processes associated with this viral spread by performing a comparative genomic study using 268 full-length genome sequences and data from outbreak investigations. Reassortant HPAIV H5N2 circulated in wild birds along the Pacific flyway before several spillover events transmitting the virus to poultry farms. Our analysis suggests that >3 separate introductions of HPAIV H5N2 into Midwest states occurred during March-June 2015; transmission to Midwest poultry farms from Pacific wild birds occurred ≈1.7-2.4 months before detection. Once established in poultry, the virus rapidly spread between turkey and chicken farms in neighboring states. Enhanced biosecurity is required to prevent the introduction and dissemination of HPAIV across the poultry industry.

New Reassortant Clade 2.3.4.4b Avian Influenza A(H5N6) Virus in Wild Birds, South Korea, 2017-18.

We isolated new reassortant avian influenza A(H5N6) viruses from feces of wild waterfowl in South Korea during 2017-18. Phylogenetic analysis suggested that reassortment occurred between clade 2.3.4.4b H5N8 and Eurasian low pathogenicity avian influenza viruses circulating in wild birds. Dissemination to South Korea during the 2017 fall migratory season followed.

Reassortant Clade 2.3.4.4 of Highly Pathogenic Avian Influenza A(H5N6) Virus, Taiwan, 2017.

A highly pathogenic avian influenza A(H5N6) virus of clade 2.3.4.4 was detected in a domestic duck found dead in Taiwan during February 2017. The endemic situation and continued evolution of various reassortant highly pathogenic avian influenza viruses in Taiwan warrant concern about further reassortment and a fifth wave of intercontinental spread.

Pathobiology of Clade 2.3.4.4 H5Nx High-Pathogenicity Avian Influenza Virus Infections in Minor Gallinaceous Poultry Supports Early Backyard Flock Introductions in the Western United States in 2014-2015.

In 2014 and 2015, the United States experienced an unprecedented outbreak of Eurasian clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus. Initial cases affected mainly wild birds and mixed backyard poultry species, while later outbreaks affected mostly commercial chickens and turkeys. The pathogenesis, transmission, and intrahost evolutionary dynamics of initial Eurasian H5N8 and reassortant H5N2 clade 2.3.4.4 HPAI viruses in the United States were investigated in minor gallinaceous poultry species (i.e., species for which the U.S. commercial industries are small), namely, Japanese quail, bobwhite quail, pearl guinea fowl, chukar partridges, and ring-necked pheasants. Low mean bird infectious doses (<2 to 3.7 log10) support direct introduction and infection of these species as observed in mixed backyard poultry during the early outbreaks. Pathobiological features and systemic virus replication in all species tested were consistent with HPAI virus infection. Sustained virus shedding with transmission to contact-exposed birds, alongside long incubation periods, may enable unrecognized dissemination and adaptation to other gallinaceous species, such as chickens and turkeys. Genome sequencing of excreted viruses revealed numerous low-frequency polymorphisms and 20 consensus-level substitutions in all genes and species, but especially in Japanese quail and pearl guinea fowl and in internal proteins PB1 and PB2. This genomic flexibility after only one passage indicates that influenza viruses can continue to evolve in galliform species, increasing their opportunity to adapt to other species. Our findings suggest that these gallinaceous poultry are permissive for infection and sustainable transmissibility with the 2014 initial wild bird-adapted clade 2.3.4.4 virus, with potential acquisition of mutations leading to host range adaptation.IMPORTANCE The outbreak of clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus that occurred in the United States in 2014 and 2015 represents the worst livestock disease event in the country, with unprecedented socioeconomic and commercial consequences. Epidemiological and molecular investigations can identify transmission pathways of the HPAI virus. However, understanding the pathogenesis, transmission, and intrahost evolutionary dynamics of new HPAI viruses in different avian species is paramount. The significance of our research is in examining the susceptibility of minor gallinaceous species to HPAI virus, as this poultry sector also suffers from HPAI epizootics, and identifying the biological potential of these species as an epidemiological link between the waterfowl reservoir and the commercial chicken and turkey populations, with the ultimate goal of refining surveillance in these populations to enhance early detection, management, and control in future HPAI virus outbreaks.

Pathobiology of Tennessee 2017 H7N9 low and high pathogenicity avian influenza viruses in commercial broiler breeders and specific pathogen free layer chickens.

In March 2017, H7N9 highly pathogenic avian influenza (HPAI) virus was detected in 2 broiler breeder farms in the state of Tennessee, USA. Subsequent surveillance detected the low pathogenicity avian influenza (LPAI) virus precursor in multiple broiler breeder farms and backyard poultry in Tennessee and neighboring states. The pathogenesis of the H7N9 LPAI virus was investigated in commercial broiler breeders, the bird type mostly affected in this outbreak. Infectivity, transmissibility, and pathogenesis of the H7N9 HPAI and LPAI viruses were also studied in 4-week-old specific pathogen free (SPF) leghorn chickens. The mean bird infectious doses (BID50) for the LPAI isolate was 5.6 log10 mean egg infectious dose (EID50) for broiler breeders and 4.3 log10 EID50 for SPF layer chickens, and no transmission to contact-exposed birds was observed. In both bird types, virus shedding was almost exclusively from the oropharyngeal route. These findings suggest sub-optimal adaptation for sustained transmission with the H7N9 LPAI isolate, indicating that factors other than the birds genetic background may explain the epidemiology of the outbreak. The BID50 for the HPAI isolate in SPF layer chickens was more than 2 logs lower (<2 log10 EID50) than the LPAI isolate. Also, the HPAI virus was shed by both the oropharyngeal and cloacal routes and transmitted to contacts. Greater susceptibility and easier transmission of the H7N9 HPAI virus are features of the HP phenotype that could favor the spread of HPAI over LPAI viruses during outbreaks.

Highly Pathogenic Avian Influenza A(H7N9) Virus, Tennessee, USA, March 2017.

In March 2017, highly pathogenic avian influenza A(H7N9) was detected at 2 poultry farms in Tennessee, USA. Surveillance data and genetic analyses indicated multiple introductions of low pathogenicity avian influenza virus before mutation to high pathogenicity and interfarm transmission. Poultry surveillance should continue because low pathogenicity viruses circulate and spill over into commercial poultry.

Airborne Transmission of Highly Pathogenic Influenza Virus during Processing of Infected Poultry.

Exposure to infected poultry is a suspected cause of avian influenza (H5N1) virus infections in humans. We detected infectious droplets and aerosols during laboratory-simulated processing of asymptomatic chickens infected with human- (clades 1 and 2.2.1) and avian- (clades 1.1, 2.2, and 2.1) origin H5N1 viruses. We detected fewer airborne infectious particles in simulated processing of infected ducks. Influenza virus-naive chickens and ferrets exposed to the air space in which virus-infected chickens were processed became infected and died, suggesting that the slaughter of infected chickens is an efficient source of airborne virus that can infect birds and mammals. We did not detect consistent infections in ducks and ferrets exposed to the air space in which virus-infected ducks were processed. Our results support the hypothesis that airborne transmission of HPAI viruses can occur among poultry and from poultry to humans during home or live-poultry market slaughter of infected poultry.

Novel Reassortant Clade 2.3.4.4 Avian Influenza A(H5N8) Virus in Wild Aquatic Birds, Russia, 2016.

The emergence of novel avian influenza viruses in migratory birds is of concern because of the potential for virus dissemination during fall migration. We report the identification of novel highly pathogenic avian influenza viruses of subtype H5N8, clade 2.3.4.4, and their reassortment with other avian influenza viruses in waterfowl and shorebirds of Siberia.

Reoccurrence of Avian Influenza A(H5N2) Virus Clade 2.3.4.4 in Wild Birds, Alaska, USA, 2016.

We report reoccurrence of highly pathogenic avian influenza A(H5N2) virus clade 2.3.4.4 in a wild mallard in Alaska, USA, in August 2016. Identification of this virus in a migratory species confirms low-frequency persistence in North America and the potential for re-dissemination of the virus during the 2016 fall migration.

Reassortant Clade 2.3.4.4 Avian Influenza A(H5N6) Virus in a Wild Mandarin Duck, South Korea, 2016.

A reassortant clade 2.3.4.4 avian influenza A(H5N6) virus was isolated from a fecal sample of a Mandarin duck (Aix galericulata) in South Korea during October 2016. This virus was genetically similar to H5N6 subtype virus isolates from China, Vietnam, Laos, and Hong Kong, including human isolates.