Zoonotic Avian Influenza Viruses Research Articles

The host tropism of current zoonotic H7N9 viruses depends mainly on an acid-labile hemagglutinin with a single amino acid mutation in the stalk region.

The incidence of human infection by zoonotic avian influenza viruses, especially H5N1 and H7N9 viruses, has increased. Current zoonotic H7N9 avian influenza viruses (identified since 2013) emerged during reassortment of viruses belonging to different subtypes. Despite analyses of their genetic background, we do not know why current H7N9 viruses are zoonotic. Therefore, there is a need to identify the factor(s) responsible for the extended host tropism that enables these viruses to infect humans as well as birds. To identify H7N9-specific amino acids that confer zoonotic properties on H7N9 viruses, we performed multiple alignment of the hemagglutinin (HA) amino acid sequences of A/Shanghai/1/2013 (H7N9) and A/duck/Zhejiang/12/2011(H7N3) (a putative, non- or less zoonotic HA donor to the zoonotic H7N9 virus). We also analyze the function of an H7N9 HA-specific amino acid with respect to HA acid stability, and evaluated the effect of acid stability on viral infectivity and virulence in a mouse model. HA2-116D, preserved in current zoonotic H7N9 viruses, was crucial for loss of HA acid stability. The acid-labile HA protein in H7 viruses played an important role in infection of human airway epithelial cells; HA2-116D contributed to infection and replication of H7 viruses. Finally, HA2-116D served as a H7 virulence factor in mice. These results suggest that acid-labile HA harboring HA2-116D confers zoonotic characteristics on H7N9 virus and that future novel zoonotic avian viruses could emerge from non-zoonotic H7 viruses via acquisition of mutations that remove HA acid stability.

Genetic insights of H9N2 avian influenza viruses circulating in Mali and phylogeographic patterns in Northern and Western Africa.

Avian influenza viruses (AIVs) of the H9N2 subtype have become widespread in Western Africa since their first detection in 2017 in Burkina Faso. However, the genetic characteristics and diffusion patterns of the H9N2 virus remain poorly understood in Western Africa, mainly due to limited surveillance activities. In addition, Mali, a country considered to play an important role in the epidemiology of AIVs in the region, lacks more comprehensive data on the genetic characteristics of these viruses, especially the H9N2 subtype. To better understand the genetic characteristics and spatio-temporal dynamics of H9N2 virus within this region, we carried out a comprehensive genetic characterization of H9N2 viruses collected through active surveillance in live bird markets in Mali between 2021 and 2022. We also performed a continuous phylogeographic analysis to unravel the dispersal history of H9N2 lineages between Northern and Western Africa. The identified Malian H9N2 virus belonged to the G1 lineage, similar to viruses circulating in both Western and Northern Africa, and possessed multiple molecular markers associated with an increased potential for zoonotic transmission and virulence. Notably, some Malian strains carried the R-S-N-R motif at their cleavage site, mainly observed in H9N2 strains in Asia. Our continuous phylogeographic analysis revealed a single and significant long-distance lineage dispersal event of the H9N2 virus to Western Africa, likely to have originated from Morocco in 2015, shaping the westward diffusion of the H9N2 virus. Our study highlights the need for long-term surveillance of H9N2 viruses in poultry populations in Western Africa, which is crucial for a better understanding of virus evolution and effective management against potential zoonotic AIV strain emergence.

The Origin of Internal Genes Contributes to the Replication and Transmission Fitness of H7N9 Avian Influenza Virus.

H9N2 avian influenza viruses (AIVs) have donated internal gene segments during the emergence of zoonotic AIVs, including H7N9. We used reverse genetics to generate A/Anhui/1/13 (H7N9) and three reassortant viruses (2:6 H7N9) which contained the hemagglutinin and neuraminidase from Anhui/13 (H7N9) and the six internal gene segments from H9N2 AIVs belonging to (i) G1 subgroup 2, (ii) G1 subgroup 3, or (iii) BJ94 lineages, enzootic in different regions throughout Asia. Infection of chickens with the 2:6 H7N9 containing G1-like H9N2 internal genes conferred attenuation in vivo, with reduced shedding and transmission to contact chickens. However, possession of BJ94-like H9N2 internal genes resulted in more rapid transmission and significantly elevated cloacal shedding compared to the parental Anhui/13 H7N9. In vitro analysis showed that the 2:6 H7N9 with BJ94-like internal genes had significantly increased replication compared to the Anhui/13 H7N9 in chicken cells. In vivo coinfection experiments followed, where chickens were coinfected with pairs of Anhui/13 H7N9 and a 2:6 H7N9 reassortant. During ensuing transmission events, the Anhui/13 H7N9 virus outcompeted 2:6 H7N9 AIVs with internal gene segments of BJ94-like or G1-like H9N2 viruses. Coinfection did lead to the emergence of novel reassortant genotypes that were transmitted to contact chickens. Some of the reassortant viruses had a greater replication in chicken and human cells compared to the progenitors. We demonstrated that the internal gene cassette determines the transmission fitness of H7N9 viruses in chickens, and the reassortment events can generate novel H7N9 genotypes with increased virulence in chickens and enhanced zoonotic potential. IMPORTANCE H9N2 avian influenza viruses (AIVs) are enzootic in poultry in different geographical regions. The internal genes of these viruses can be exchanged with other zoonotic AIVs, most notably the A/Anhui/1/2013-lineage H7N9, which can give rise to new virus genotypes with increased veterinary, economic and public health threats to both poultry and humans. We investigated the propensity of the internal genes of H9N2 viruses (G1 or BJ94) in the generation of novel reassortant H7N9 AIVs. We observed that the internal genes of H7N9 which were derivative of BJ94-like H9N2 virus have a fitness advantage compared to those from the G1-like H9N2 viruses for efficient transmission among chickens. We also observed the generation of novel reassortant viruses during chicken transmission which infected and replicated efficiently in human cells. Therefore, such emergent reassortant genotypes may pose an elevated zoonotic threat.

Adaptation of Two Wild Bird-Origin H3N8 Avian Influenza Viruses to Mammalian Hosts.

Wild birds play an important role in the emergence, evolution, and spread of zoonotic avian influenza viruses (AIVs). However, there are few studies on the cross-species transmission of the H3N8 AIV originating from wild birds. In this study, we investigated the transmissibility and pathogenicity of two H3N8 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds, GZA1 and XJ47, to mammals. The HA genes of both strains belonged to Eurasian isolates, while the other genes were derived from a variety of other subtypes of AIVs. Both strains can infect specific-pathogen-free (SPF) chickens, BALB/c mice, and guinea pigs. The XJ47 strain spread horizontally in SPF chickens and guinea pigs. The GZA1 strain did not spread horizontally but caused higher weight loss and mild lung inflammation in mice. P12-GZA1- and P12-XJ47-adapted strains obtained after 12 passages in the lung of mice showed enhanced pathogenicity in mice, which led to obvious clinical symptoms, lung inflammation, and 100% death. Both adapted strains have the reported mutation T97I in the PA, and the reported mutation D701N in PB2 has been found in the P12-GZA1-adapted strain. This study provides an important scientific basis for the continuous monitoring of wild AIVs and the mechanism underlying AIV cross-species transmission.

Serologic Evidence of Occupational Exposure to Avian Influenza Viruses at the Wildfowl/Poultry/Human Interface.

Ecological interactions between wild aquatic birds and outdoor-housed poultry can enhance spillover events of avian influenza viruses (AIVs) from wild reservoirs to domestic birds, thus increasing the related zoonotic risk to occupationally exposed workers. To assess serological evidence of AIV infection in workers operating in Northern Italy at the wildfowl/poultry interface or directly exposed to wildfowl, serum samples were collected between April 2005 and November 2006 from 57 bird-exposed workers (BEWs) and from 7 unexposed controls (Cs), planning three sample collections from each individual. Concurrently, AIV surveillance of 3587 reared birds identified 4 AIVs belonging to H10N7, H4N6 and H2N2 subtypes while serological analysis by hemagglutination inhibition (HI) assay showed recent infections caused by H1, H2, H4, H6, H10, H11, H12, and H13 subtypes. Human sera were analyzed for specific antibodies against AIVs belonging to antigenic subtypes from H1 to H14 by using HI and virus microneutralization (MN) assays as a screening and a confirmatory test, respectively. Overall, antibodies specific to AIV-H3, AIV-H6, AIV-H8, and AIV-H9 were found in three poultry workers (PWs) and seropositivity to AIV-11, AIV-H13—still detectable in October 2017—in one wildlife professional (WP). Furthermore, seropositivity to AIV-H2, accounting for previous exposure to the “extinct” H2N2 human influenza viruses, was found in both BEWs and Cs groups. These data further emphasize the occupational risk posed by zoonotic AIV strains and show the possible occurrence of long-lived antibody-based immunity following AIV infections in humans.

Glycosylation generates an efficacious and immunogenic vaccine against H7N9 influenza virus.

Zoonotic avian influenza viruses pose severe health threats to humans. Of several viral subtypes reported, the low pathogenic avian influenza H7N9 virus has since February 2013 caused more than 1,500 cases of human infection with an almost 40% case-fatality rate. Vaccination of poultry appears to reduce human infections. However, the emergence of highly pathogenic strains has increased concerns about H7N9 pandemics. To develop an efficacious H7N9 human vaccine, we designed vaccine viruses by changing the patterns of N-linked glycosylation (NLG) on the viral hemagglutinin (HA) protein based on evolutionary patterns of H7 HA NLG changes. Notably, a virus in which 2 NLG modifications were added to HA showed higher growth rates in cell culture and elicited more cross-reactive antibodies than did other vaccine viruses with no change in the viral antigenicity. Developed into an inactivated vaccine formulation, the vaccine virus with 2 HA NLG additions exhibited much better protective efficacy against lethal viral challenge in mice than did a vaccine candidate with wild-type (WT) HA by reducing viral replication in the lungs. In a ferret model, the 2 NLG-added vaccine viruses also induced hemagglutination-inhibiting antibodies and significantly suppressed viral replication in the upper and lower respiratory tracts compared with the WT HA vaccines. In a mode of action study, the HA NLG modification appeared to increase HA protein contents incorporated into viral particles, which would be successfully translated to improve vaccine efficacy. These results suggest the strong potential of HA NLG modifications in designing avian influenza vaccines.

“Genetic tuning” of avian influenza virus host adaptation from birds to humans

Abstract Avian influenza viruses can adapt to infect humans by accumulating mutations or reassortments, potentially leading to a pandemic. However, the adaptation dynamics of zoonotic avian influenza viruses in their transition from animal hosts to human hosts remains largely unknown. In a recent PNAS report, Liu et al. have presented deep sequencing data showing host adaptation by “genetic tuning” of a zoonotic H7N9 avian influenza virus to cross the species barrier to infect humans. This provides the first in-depth data on avian influenza virus adaptation at the bird-human interface. As a one-health approach, the deep sequencing approach as implemented by Liu et al. is applicable to the study of emerging zoonotic viruses and provides valuable data on “genetic tuning” in virus-host adaptation. This experimental design should be useful for studying other zoonotic viruses, including SARS-CoV-2 and the novel swine influenza virus, and helpful for mitigating future pandemic public health risks.

Active virological surveillance in backyard ducks in Bangladesh: detection of avian influenza and gammacoronaviruses

ABSTRACT Domestic waterfowl play an important role in the perpetuation and transmission of avian pathogens including avian influenza viruses (AIV) of low and high pathogenicity, which pose severe economic and public health concerns in Bangladesh. This study focused on active surveillance of several avian viral pathogens with a special reference to AIV in selected backyard duck populations in Bangladesh. A total of 500 pooled oropharyngeal and cloacal samples from individual ducks of four districts were tested by real time PCRs for the presence of AIV, avian avulavirus-1, anatid herpesvirus-1, avian parvovirus, avian bornavirus and avian coronavirus. The investigation identified 27 (5.4%) ducks positive for AIV and 12 (2.4%) positive for avian coronavirus. In 13 samples, RNA specific for AIV H4N6 was detected. Phylogenetic analysis of the AIV haemagglutinin H4 and neuraminidase N6 genes suggested a clustering of Bangladeshi AIV H4N6 in Eurasian lineage group 2. Other AIV positive samples had very low virus loads (Cq > 36) and were not subtyped. Coronaviral sequences of a fragment of the polymerase gene were related to Eurasian-Australian duck gamma-coronaviruses. Our current active surveillance in free-range domestic backyard ducks in Bangladesh failed to detect highly pathogenic (HP) AIV in contrast to our previous passive monitoring study. Nevertheless, active monitoring of domestic duck populations may be important to highlight presence and transmission dynamics of economically less important AIV that still may serve as reassortment partners for the generation of new HP and zoonotic AIV. RESEARCH HIGHLIGHTS Active surveillance for viral pathogens in domestic free-range backyard ducks. Detection of avian influenza virus subtype H4N6. First identification of avian gammacoronavirus in ducks in Bangladesh.

Avian influenza viruses in humans: lessons from past outbreaks.

Human infections with avian influenza viruses (AIV) represent a persistent public health threat. The principal risk factor governing human infection with AIV is from direct contact with infected poultry and is primarily observed in Asia and Egypt where live-bird markets are common. Changing patterns of virus transmission and a lack of obvious disease manifestations in avian species hampers early detection and efficient control of potentially zoonotic AIV. Despite extensive studies on biological and environmental risk factors, the exact conditions required for cross-species transmission from avian species to humans remain largely unknown. The development of a universal ('across-subtype') influenza vaccine and effective antiviral therapeutics are a priority. Sustained virus surveillance and collection of ecological and physiological parameters from birds in different environments is required to better understand influenza virus ecology and identify risk factors for human infection.

Co-subsistence of avian influenza virus subtypes of low and high pathogenicity in Bangladesh: Challenges for diagnosis, risk assessment and control

Endemic co-circulation of potentially zoonotic avian influenza viruses (AIV) of subtypes H5N1 and H9N2 (G1 lineage) in poultry in Bangladesh accelerated diversifying evolution. Two clinical samples from poultry obtained in 2016 yielded five different subtypes (highly pathogenic [HP] H5N1, HP H5N2, HP H7N1, HP H7N2, H9N2) and eight genotypes of AIV by plaque purification. H5 sequences grouped with clade 2.3.2.1a viruses while N1 was related to an older, preceding clade, 2.2.2. The internal genome segments of the plaque-purified viruses originated from clade 2.2.2 of H5N1 or from G1/H9N2 viruses. H9 and N2 segments clustered with contemporary H9N2 strains. In addition, HP H7 sequences were detected for the first time in samples and linked to Pakistani HP H7N3 viruses of 2003. The unexpected findings of mixtures of reassorted HP H5N1 and G1-like H9N2 viruses, which carry genome segments of older clades in association with the detection of HP H7 HA segments calls for confirmation of these results by targeted surveillance in the area of origin of the investigated samples. Hidden niches and obscured transmission pathways may exist that retain or re-introduce genome segments of older viruses or reassortants thereof which causes additional challenges for diagnosis, risk assessment and disease control.

From H5N1 to HxNy: An epidemiologic overview of human infections with avian influenza in the Western Pacific Region, 2003–2017

Since the first confirmed human infection with avian influenza A(H5N1) virus was reported in Hong Kong Special Administrative Region in 1997, sporadic zoonotic avian influenza viruses causing illness in humans have been identified globally, with the WHO Western Pacific Region as one of the hotspots. From November 2003, when a resurgence of H5N1 virus activity in humans and animals occurred, through September 2017, 1,838 human infections with avian influenza viruses in the region were reported to WHO. Viruses infecting humans were A(H5N1), A(H5N6), A(H6N1), A(H7N9), A(H9N2) and A(H10N8). The vast majority of infections were with H7N9 (n=1,562) and H5N1 (n=238) viruses, and most (n=1583, 86%) were reported from December through April. In poultry and wild birds, H5N1 and H5N6 subtypes were the most widely distributed, with outbreaks reported from ten and eight countries and areas, respectively. Regional analyses of human infections with avian influenza subtypes revealed distinct patterns and variations in epidemiology across countries, age, and time. Such epidemiologic patterns may not be apparent from aggregated global summaries or isolated country reports; regional assessment can offer additional insight that can inform risk assessment and response efforts. As infected animals and contaminated environments are the primary source of human infections, regional analyses that bring together surveillance data from human and animal health sectors are an important basis for exposure and transmission risk assessment and public health action. A One Health approach combining sustained event-based surveillance with enhanced collaboration between the human, animal (domestic and wildlife), and environmental sectors will provide a basis to inform joint risk assessment and coordinated response capacities

The Multifaceted Zoonotic Risk of H9N2 Avian Influenza.

Poultry-adapted H9N2 avian influenza viruses (AIVs) are commonly found in many countries in Asia, the Middle East, Africa, and Europe, and although classified as low pathogenic viruses, they are an economically important disease. Besides the importance of the disease in the poultry industry, some H9N2 AIVs are also known to be zoonotic. The disease in humans appears to cause primarily a mild upper respiratory disease, and doesn’t cause or only rarely causes the severe pneumonia often seen with other zoonotic AIVs like H5N1 or H7N9. Serologic studies in humans, particularly in occupationally exposed workers, show a large number of people with antibodies to H9N2, suggesting infection is commonly occurring. Of the four defined H9N2 poultry lineages, only two lineages, the G1 and the Y280 lineages, are associated with human infections. Almost all of the viruses from humans have a leucine at position 226 (H3 numbering) of the hemagglutinin associated with a higher affinity of binding with α2,6 sialic acid, the host cell receptor most commonly found on glycoproteins in the human upper respiratory tract. For unknown reasons there has also been a shift in recent years of poultry viruses in the G1 and Y280 lineages to also having leucine instead of glutamine, the amino acid found in most avian viruses, at position 226. The G1 and Y280 poultry lineages because of their known ability to infect humans, the high prevalence of the virus in poultry in endemic countries, the lack of antibody in most humans, and the shift of poultry viruses to more human-like receptor binding makes these viruses a human pandemic threat. Increased efforts for control of the virus, including through effective vaccine use in poultry, is warranted for both poultry and public health goals.

Natural Reassortants of Potentially Zoonotic Avian Influenza Viruses H5N1 and H9N2 from Egypt Display Distinct Pathogenic Phenotypes in Experimentally Infected Chickens and Ferrets.

The cocirculation of zoonotic highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 and avian influenza virus (AIV) of subtype H9N2 among poultry in Egypt for at least 6 years should render that country a hypothetical hot spot for the emergence of reassortant, phenotypically altered viruses, yet no reassortants have been detected in Egypt. The present investigations proved that reassortants of the Egyptian H5N1 clade 2.2.1.2 virus and H9N2 virus of the G1-B lineage can be generated by coamplification in embryonated chicken eggs. Reassortants were restricted to the H5N1 subtype and acquired between two and all six of the internal segments of the H9N2 virus. Five selected plaque-purified reassortant clones expressed a broad phenotypic spectrum both in vitro and in vivo Two groups of reassortants were characterized to have retarded growth characteristics in vitro compared to the H5N1 parent virus. One clone provoked reduced mortality in inoculated chickens, although the characteristics of a highly pathogenic phenotype were retained. Enhanced zoonotic properties were not predicted for any of these clones, and this prediction was confirmed by ferret inoculation experiments: neither the H5N1 parent virus nor two selected clones induced severe clinical symptoms or were transmitted to sentinel ferrets by contact. While the emergence of reassortants of Egyptian HPAIV of subtype H5N1 with internal gene segments of cocirculating H9N2 viruses is possible in principle, the spread of such viruses is expected to be governed by their fitness to outcompete the parental viruses in the field. The eventual spread of attenuated phenotypes, however, would negatively impact syndrome surveillance on poultry farms and might foster enzootic virus circulation.IMPORTANCE Despite almost 6 years of the continuous cocirculation of highly pathogenic avian influenza virus H5N1 and avian influenza virus H9N2 in poultry in Egypt, no reassortants of the two subtypes have been reported. Here, the principal compatibility of the two subtypes is shown by forcing the reassortment between copassaged H5N1 und H9N2 viruses in embryonated chicken eggs. The resulting reassortant viruses displayed a wide range of pathogenicity including attenuated phenotypes in chickens, but did not show enhanced zoonotic propensities in the ferret model.

Monitoring Avian Influenza Viruses from Chicken Carcasses Sold at Markets, China, 2016.

During 2016 in Guangzhou, China, we detected infectious avian influenza viruses (AIVs) in 39.8% of samples from chicken carcasses slaughtered at live poultry markets but none from carcasses supplied to supermarkets by facilities bypassing live poultry markets. Promoting supply chains with high biosecurity may reduce the risk for zoonotic AIV transmission.

Evolutionary trajectories and diagnostic challenges of potentially zoonotic avian influenza viruses H5N1 and H9N2 co-circulating in Egypt

In Egypt, since 2006, descendants of the highly pathogenic avian influenza virus (HP AIV) H5N1 of clade 2.2 continue to cause sharp losses in poultry production and seriously threaten public health. Potentially zoonotic H9N2 viruses established an endemic status in poultry in Egypt as well and co-circulate with HP AIV H5N1 rising concerns of reassortments between H9N2 and H5N1 viruses along with an increase of mixed infections of poultry.Nucleotide sequences of whole genomes of 15 different isolates (H5N1: 7; H9N2: 8), and of the hemagglutinin (HA) and neuraminidase (NA) encoding segments of nine further clinical samples (H5N1: 2; H9N2: 7) from 2013 and 2014 were generated and analysed. The HA of H5N1 viruses clustered with clade 2.2.1 while the H9 HA formed three distinguishable subgroups within cluster B viruses. BEAST analysis revealed that H9N2 viruses are likely present in Egypt since 2009. Several previously undescribed substituting mutations putatively associated with host tropism and virulence modulation were detected in different proteins of the analysed H9N2 and H5N1 viruses.Reassortment between HP AIV H5N1 and H9N2 is anticipated in Egypt, and timely detection of such events is of public health concern. As a rapid tool for detection of such reassortants discriminative SYBR-Green reverse transcription real-time PCR assays (SG-RT-qPCR), targeting the internal genes of the Egyptian H5N1 and H9N2 viruses were developed for the rapid screening of viral RNAs from both virus isolates and clinical samples. However, in accordance to Sanger sequencing, no reassortants were found by SG-RT-qPCR.Nevertheless, the complex epidemiology of avian influenza in poultry in Egypt will require sustained close observation. Further development and continuing adaptation of rapid and cost-effective screening assays such as the SG-RT-qPCR protocol developed here are at the basis of efforts for improvement the currently critical situation.

Influenza viruses

In the last decade, sporadic and lethal human disease caused by zoonotic avian influenza viruses, and the seasonal activity of human H1N1 2009 pandemic type have driven intense epidemiological and laboratory studies into the virus life cycle. This article highlights major developments from mid-2012 to early 2014. Advances in molecular techniques and efficient rollout of diagnostic tests have enabled the rapid identification of clinical cases and detailed genetic sequencing of viral genomes. Studies have contributed widely to the understanding of how and when influenza viruses circulate, what determines their innate pathogenicity in particular hosts and whether host cofactors influence disease severity. Other imperatives include investigations into how influenza can be better prevented by vaccination, or treated with antiviral drugs. Avian influenza viruses present a continuous threat to human populations. There is a need for sustained surveillance and downstream research to evaluate the potential for future pandemics.