Development of a nucleoside-modified mRNA vaccine against clade 2.3.4.4b H5 highly pathogenic avian influenza virus.

mRNA lipid nanoparticle (LNP) vaccines would be useful during an influenza virus pandemic since they can be produced rapidly and do not require the generation of egg-adapted vaccine seed stocks. Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. Here, we generate an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. The H5 mRNA-LNP vaccine elicits strong T cell and antibody responses in female mice, including neutralizing antibodies and broadly-reactive anti-HA stalk antibodies. The H5 mRNA-LNP vaccine elicits antibodies at similar levels compared to whole inactivated vaccines in female mice with and without prior H1N1 exposures. Finally, we find that the H5 mRNA-LNP vaccine is immunogenic in male ferrets and prevents morbidity and mortality of animals following 2.3.4.4b H5N1 challenge. Together, our data demonstrate that a monovalent mRNA-LNP vaccine expressing 2.3.4.4b H5 is immunogenic and protective in pre-clinical animal models.

Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Emergence of a new genotype of clade 2.3.4.4b H5N1 highly pathogenic avian influenza A viruses in Bangladesh.

Influenza virological surveillance was conducted in Bangladesh from January to December 2021 in live poultry markets (LPMs) and in Tanguar Haor, a wetland region where domestic ducks have frequent contact with migratory birds. The predominant viruses circulating in LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses. Additional LPAIs were found in both LPM (H4N6) and Tanguar Haor wetlands (H7N7). Genetic analyses of these LPAIs strongly suggested long-distance movement of viruses along the Central Asian migratory bird flyway. We also detected a novel clade 2.3.4.4b H5N1 virus from ducks in free-range farms in Tanguar Haor that was similar to viruses first detected in October 2020 in The Netherlands but with a different PB2. Identification of clade 2.3.4.4b HPAI H5N1 viruses in Tanguar Haor provides continued support of the role of migratory birds in transboundary movement of influenza A viruses (IAV), including HPAI viruses. Domestic ducks in free range farm in wetland areas, like Tangua Haor, serve as a conduit for the introduction of LPAI and HPAI viruses into Bangladesh. Clade 2.3.4.4b viruses have dominated in many regions of the world since mid-2021, and it remains to be seen if these viruses will replace the endemic clade 2.3.2.1a H5N1 viruses in Bangladesh.

Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer.

The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

Development of a nucleoside-modified mRNA vaccine against clade 2.3.4.4b H5 highly pathogenic avian influenza virus.

Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. We generated an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. We show that the vaccine is immunogenic in mice and ferrets and prevents morbidity and mortality of ferrets following 2.3.4.4b H5N1 challenge.

Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer.

While SARS-CoV-2 has sporadically infected a wide range of animal species worldwide1, the virus has been repeatedly and frequently detected in white-tailed deer in North America2â€"7. The zoonotic origins of this pandemic virus highlight the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, which disseminated across hundreds of kilometers. We discovered that alpha and delta variants evolved in white-tailed deer at three-times the rate observed in humans. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only faster in white-tailed deer but driven by different mutational biases and selection pressures. White-tailed deer are not just short-term recipients of human viral diversity but serve as reservoirs for alpha and other variants to evolve in new directions after going extinct in humans. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal model experiments using viruses isolated from white-tailed deer. Still, SARS-CoV-2 viruses have transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

Defining the risk of SARS-CoV-2 variants on immune protection.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters.

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

Distinct but connected avian influenza virus activities in wetlands and live poultry markets in Bangladesh, 2018-2019.

From April 2018 to October 2019, we continued active surveillance for influenza viruses in Bangladeshi live poultry markets (LPMs) and in Tanguar Haor, a wetland region of Bangladesh where domestic ducks have frequent contact with migratory birds. The predominant virus subtypes circulating in the LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses of the H5N1-R1 genotype, like those found in previous years. Viruses of the H5N1-R2 genotype, which were previously reported as co-circulating with H5N1-R1 genotype viruses in LPM, were not detected. In addition to H9N2 viruses, which were primarily found in chicken and quail, H2N2, H3N8 and H11N3 LPAI viruses were detected in LPMs, exclusively in ducks. Viruses in domestic ducks and/or wild birds in Tanguar Haor were more diverse, with H1N1, H4N6, H7N1, H7N3, H7N4, H7N6, H8N4, H10N3, H10N4 and H11N3 detected. Phylogenetic analyses of these LPAI viruses suggested that some were new to Bangladesh (H2N2, H7N6, H8N4, H10N3 and H10N4), likely introduced by migratory birds of the Central Asian flyway. Our results show a complex dynamic of viral evolution and diversity in Bangladesh based on factors such as host populations and geography. The LPM environment was characterised by maintenance of viruses with demonstrated zoonotic potential and H5N1 genotype turnover. The wetland environment was characterised by greater viral gene pool diversity but a lower overall influenza virus detection rate. The genetic similarity of H11N3 viruses in both environments demonstrates that LPM and wetlands are connected despite their having distinct influenza ecologies.

Molecular detection of influenza A viruses and H5 subtype among migratory Amur falcons (Falco amurensis) and captive birds of prey.

Influenza A viruses (IAVs) and Newcastle disease viruses (NDVs) are major human and animal health threats with geographic differences in prevalence, characteristics and host populations. Currently, there is sparse information on IAVs and NDVs in avian species in South Africa. Because raptors feed on live wild birds which are the reservoir hosts of IAVs and NDVs, we considered them a good sentinel for surveillance. Therefore, in addition to other resident birds of prey, migratory Amur falcons (Falco amurensis) were screened for IAVs and NDVs. Oropharyngeal and cloacal samples were collected from raptor species at three sampling sites in KwaZulu-Natal Province and samples were screened for IAVs and NDVs using molecular and virus isolation methods. IAV-positive samples were further screened for the presence of H5, H7 and H9 viruses. A total of 14 samples from 11 birds (45.8% of all sampled birds) were IAV positive with Ct lower than 36 in duplicate tests. Five out of 24 birds (20.8%) were positive for IAV RNA in duplicate testing, albeit at low concentrations. Among raptor samples, three out of 24 birds (12.5%) were positive for IAVs with viral RNA detected in both cloacal and oropharyngeal swabs. One IAV-positive sample was also positive for H5 subtype (4.1%); all other samples were H5, H7 and H9 negative. Besides, all samples were NDV negative. Overall, our results support the development of more intensive and expanded influenza and other emerging virus studies in raptor species.

It's the Wrong Virus: Rapid Adaptation of Operations Inside a Highly Pathogenic Avian Influenza Select Agent Laboratory in Response to the 2019 SARS-CoV-2 Pandemic.

Background: The Animal Biosafety Level 3 Enhanced (ABSL-3+) laboratory at St. Jude Children's Research Hospital has a long history of influenza pandemic preparedness. The emergence of SARS-CoV-2 and subsequent expansion into a pandemic has put new and unanticipated demands on laboratory operations since April 2020. Administrative changes, investigative methods requiring increased demand for inactivation and validation of sample removal, and the adoption of a new animal model into the space required all arms of our Biorisk Management System (BMS) to respond with speed and innovation. Results: In this report, we describe the outcomes of three major operational changes that were implemented to adapt the ABSL-3+ select agent space into a multipathogen laboratory. First were administrative controls that were revised and developed with new Institutional Biosafety Committee protocols, laboratory space segregation, training of staff, and occupational health changes for potential exposure to SARS-CoV-2 inside the laboratory. Second were extensive inactivation and validation experiments performed for both highly pathogenic avian influenza and SARS-CoV-2 to meet the demands for sample removal to a lower biosafety level. Third was the establishment of a new caging system to house Syrian Golden hamsters for SARS-CoV-2 risk assessment modeling. Summary: The demands placed on biocontainment laboratories for response to SARS-CoV-2 has highlighted the importance of a robust BMS. In a relatively short time, the ABSL-3+ was able to adapt from a single select agent space to a multipathogen laboratory and expand our pandemic response capacity.

Detection of a Novel Reassortant H9N9 Avian Influenza Virus in Free-Range Ducks in Bangladesh.

Wild aquatic birds are the primary natural reservoir for influenza A viruses (IAVs). In this study, an A(H9N9) influenza A virus (A/duck/Bangladesh/44493/2020) was identified via routine surveillance in free-range domestic ducks in Bangladesh. Phylogenetic analysis of hemagglutinin showed that the H9N9 virus belonged to the Y439-like lineage. The HA gene had the highest nucleotide identity to A/Bean Goose (Anser fabalis)/South Korea/KNU 2019-16/2019 (H9N2). The other seven gene segments clustered within the Eurasian lineage.

Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs.

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979-1983, 1984-1987 and 1988-1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor-binding specificity of the EA swine influenza viruses-that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979-1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

Highly Pathogenic Avian Influenza A(H5N6) Virus Clade 2.3.4.4h in Wild Birds and Live Poultry Markets, Bangladesh.

Migratory birds play a major role in spreading influenza viruses over long distances. We report highly pathogenic avian influenza A(H5N6) viruses in migratory and resident ducks in Bangladesh. The viruses were genetically similar to viruses detected in wild birds in China and Mongolia, suggesting migration-associated dissemination of these zoonotic pathogens.

Continued Evolution of H5Nx Avian Influenza Viruses in Bangladeshi Live Poultry Markets: Pathogenic Potential in Poultry and Mammalian Models.

The genesis of novel influenza viruses through reassortment poses a continuing risk to public health. This is of particular concern in Bangladesh, where highly pathogenic avian influenza viruses of the A(H5N1) subtype are endemic and cocirculate with other influenza viruses. Active surveillance of avian influenza viruses in Bangladeshi live poultry markets detected three A(H5) genotypes, designated H5N1-R1, H5N1-R2, and H5N2-R3, that arose from reassortment of A(H5N1) clade 2.3.2.1a viruses. The H5N1-R1 and H5N1-R2 viruses contained HA, NA, and M genes from the A(H5N1) clade 2.3.2.1a viruses and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. H5N2-R3 viruses contained the HA gene from circulating A(H5N1) clade 2.3.2.1a viruses, NA and M genes from concurrently circulating A(H9N2) influenza viruses, and PB2, PB1, PA, NP, and NS genes from other Eurasian influenza viruses. Representative viruses of all three genotypes and a parental clade 2.3.2.1a strain (H5N1-R0) infected and replicated in mice without prior adaptation; the H5N2-R3 virus replicated to the highest titers in the lung. All viruses efficiently infected and killed chickens. All viruses replicated in inoculated ferrets, but no airborne transmission was detected, and only H5N2-R3 showed limited direct-contact transmission. Our findings demonstrate that although the A(H5N1) viruses circulating in Bangladesh have the capacity to infect and replicate in mammals, they show very limited capacity for transmission. However, reassortment does generate viruses of distinct phenotypes.IMPORTANCE Highly pathogenic avian influenza A(H5N1) viruses have circulated continuously in Bangladesh since 2007, and active surveillance has detected viral evolution driven by mutation and reassortment. Recently, three genetically distinct A(H5N1) reassortant viruses were detected in live poultry markets in Bangladesh. Currently, we cannot assign pandemic risk by only sequencing viruses; it must be conducted empirically. We found that the H5Nx highly pathogenic avian influenza viruses exhibited high virulence in mice and chickens, and one virus had limited capacity to transmit between ferrets, a property considered consistent with a higher zoonotic risk.

Continuing evolution of highly pathogenic H5N1 viruses in Bangladeshi live poultry markets.

Since November 2008, we have conducted active avian influenza surveillance in Bangladesh. Clades 2.2.2, 2.3.4.2, and 2.3.2.1a of highly pathogenic avian influenza H5N1 viruses have all been identified in Bangladeshi live poultry markets (LPMs), although, since the end of 2014, H5N1 viruses have been exclusively from clade 2.3.2.1a. In June 2015, a new reassortant H5N1 virus (H5N1-R1) from clade 2.3.2.1a was identified, containing haemagglutinin, neuraminidase, and matrix genes of H5N1 viruses circulating in Bangladesh since 2011, plus five other genes of Eurasian-lineage low pathogenic avian influenza A (LPAI) viruses. Here we report the status of circulating avian influenza A viruses in Bangladeshi LPMs from March 2016 to January 2018. Until April 2017, H5N1 viruses exclusively belonged to H5N1-R1 clade 2.3.2.1a. However, in May 2017, we identified another reassortant H5N1 (H5N1-R2), also of clade 2.3.2.1a, wherein the PA gene segment of H5N1-R1 was replaced by that of another Eurasian-lineage LPAI virus related to A/duck/Bangladesh/30828/2016 (H3N8), detected in Bangladeshi LPM in September 2016. Currently, both reassortant H5N1-R1 and H5N1-R2 co-circulate in Bangladeshi LPMs. Furthermore, some LPAI viruses isolated from LPMs during 2016-2017 were closely related to those from ducks in free-range farms and wild birds in Tanguar haor, a wetland region of Bangladesh where ducks have frequent contact with migratory birds. These data support a hypothesis where Tanguar haor-like ecosystems provide a mechanism for movement of LPAI viruses to LPMs where reassortment with poultry viruses occurs adding to the diversity of viruses at this human-animal interface.

H9N2 influenza viruses from Bangladesh: Transmission in chicken and New World quail.

The H9N2 influenza viruses that have become established in Bangladeshi live poultry markets possess five gene segments of the highly pathogenic H7N3 avian influenza virus. We assessed the replication, transmission, and disease potential of three H9N2 viruses in chickens and New World quail. Each virus replicated to high titers and transmitted by the airborne route to contacts in both species. Infected chickens showed no disease signs, and the viruses differed in their disease potential in New World quail. New World quail were more susceptible than chickens to H9N2 viruses and shed virus after airborne transmission for 10 days. Consequently, New World quail are a potential threat in the maintenance and spread of influenza virus in live poultry markets.

Genetic characterization and pathogenic potential of H10 avian influenza viruses isolated from live poultry markets in Bangladesh.

Fatal human cases of avian-origin H10N8 influenza virus infections have raised concern about their potential for human-to-human transmission. H10 subtype avian influenza viruses (AIVs) have been isolated from wild and domestic aquatic birds across Eurasia and North America. We isolated eight H10 AIVs (four H10N7, two H10N9, one H10N1, and one H10N6) from live poultry markets in Bangladesh. Genetic analyses demonstrated that all eight isolates belong to the Eurasian lineage. HA phylogenetic and antigenic analyses indicated that two antigenically distinct groups of H10 AIVs are circulating in Bangladeshi live poultry markets. We evaluated the virulence of four representative H10 AIV strains in DBA/2J mice and found that they replicated efficiently in mice without prior adaptation. Moreover, H10N6 and H10N1 AIVs caused high mortality with systemic dissemination. These results indicate that H10 AIVs pose a potential threat to human health and the mechanisms of their transmissibility should be elucidated.

Role of domestic ducks in the emergence of a new genotype of highly pathogenic H5N1 avian influenza A viruses in Bangladesh.

Highly pathogenic avian influenza H5N1 viruses were first isolated in Bangladesh in February 2007. Subsequently, clades 2.2.2, 2.3.4.2 and 2.3.2.1a were identified in Bangladesh, and our previous surveillance data revealed that by the end of 2014, the circulating viruses exclusively comprised clade 2.3.2.1a. We recently determined the status of circulating avian influenza viruses in Bangladesh by conducting surveillance of live poultry markets and waterfowl in wetland areas from February 2015 through February 2016. Until April 2015, clade 2.3.2.1a persisted without any change in genotype. However, in June 2015, we identified a new genotype of H5N1 viruses, clade 2.3.2.1a, which quickly became predominant. These newly emerged H5N1 viruses contained the hemagglutinin, neuraminidase and matrix genes of circulating 2.3.2.1a Bangladeshi H5N1 viruses and five other genes of low pathogenic Eurasian-lineage avian influenza A viruses. Some of these internal genes were closely related to those of low pathogenic viruses isolated from ducks in free-range farms and wild birds in a wetland region of northeastern Bangladesh, where commercially raised domestic ducks have frequent contact with migratory birds. These findings indicate that migratory birds of the Central Asian flyway and domestic ducks in the free-range farms in Tanguar haor-like wetlands played an important role in the emergence of this novel genotype of highly pathogenic H5N1 viruses.