Disruption of influenza virus packaging signals results in various misassembled genome complexes.

IMPORTANCE: The influenza A virus genome consists of eight distinct viral RNAs (vRNAs) that are typically packaged into a single virion as an octameric complex. How this genome complex is assembled and incorporated into the virion is poorly understood, but previous research suggests a coordinative role for packaging signals present in all vRNAs. Here, we show that disruption of two packaging signals in a model H7N7 influenza A virus results in a mixture of virions with unusual vRNA content, including empty virions, virions with one to four vRNAs, and virions with octameric complexes composed of vRNA duplicates. Our results suggest that (i) the assembly of error-free octameric complexes proceeds through a series of defined vRNA sub-complexes and (ii) virions can bud without incorporating complete octameric complexes.

Novel H7N7 avian influenza viruses detected in migratory wild birds in eastern China between 2018 and 2020.

Wild birds are the natural reservoirs of avian influenza viruses, and surveillance and assessment of these viruses in wild birds provide valuable information for early warning and control of animal diseases. In this study, we isolated 19 H7N7 avian influenza viruses from wild bird between 2018 and 2020. Full genomic analysis revealed that these viruses bear a single basic amino acid in the cleavage site of their hemagglutinin gene, and formed four different genotypes by actively reassorting other avian influenza viruses circulating in wild birds and ducks. The H7N7 viruses bound to both avian-type and human-type receptors, although their affinity for human-type receptors was markedly lower than that for avian-type receptors. Moreover, we found that the H7N7 viruses could replicate efficiently in the upper respiratory tract and caecum of domestic ducks, and that the H5/H7 inactivated vaccine used in poultry in China provided complete protection against H7N7 wild bird virus challenge in ducks. Our findings demonstrate that wild bird H7N7 viruses pose a substantial threat to the poultry industry across the East Asian-Australian migratory flyway, emphasize the importance of influenza virus surveillance in both wild and domestic birds, and support the development of active control strategies against H7N7 virus.

Emergence, Evolution, and Pathogenicity of Influenza A(H7N4) Virus in Shorebirds in China.

A 2-year surveillance study of influenza A viruses in migratory birds was conducted to understand the subsequent risk during the migratory seasons in Dandong Yalu River Estuary Coastal Wetland National Nature Reserve, Liaoning Province, China, a major stopover site on the East Asian-Australasian flyway. Overall, we isolated 27 influenza A viruses with multiple subtypes, including H3N8 (n = 2), H4N6 (n = 2), H4N7 (n = 2), H7N4 (n = 9), H7N7 (n = 1), H10N7 (n = 7), and H13N6 (n = 4). Particularly, a novel reassortant influenza A(H7N4) virus was first identified in a woman and her backyard poultry flock in Jiangsu Province, China, posing a serious threat to public health. Here, we describe the genetic characterization and pathogenicity of the nine influenza A(H7N4) isolates. Phylogenetic analysis indicated that complex viral gene flow occurred among Asian countries. We also demonstrated a similar evolutionary trajectory of the surface genes of the A(H7N4) isolates and Jiangsu human-related A(H7N4) viruses. Our A(H7N4) isolates exhibited differing degrees of virulence in mice, suggesting a potential risk to other mammalian species, including humans. We revealed multiple mutations that might affect viral virulence in mice. Our report highlights the importance and need for the long-term surveillance of avian influenza virus in migratory birds combined with domestic poultry surveillance along migratory routes and flyways and, thereby, the development of measures to manage potential health threats. IMPORTANCE The H7 subtype avian influenza viruses, such as H7N2, H7N3, H7N4, H7N7, and H7N9, were documented as being capable of infecting humans, and the H7 subtype low pathogenicity avian influenza viruses are capable of mutating into highly pathogenic avian influenza; therefore, they pose a serious threat to public health. Here, we investigated the evolutionary history, molecular characteristics, and pathogenicity of shorebird-origin influenza A(H7N4) viruses, showing a similar evolutionary trajectory with Jiangsu human A(H7N4) viruses in HA and NA genes. Moreover, our isolates exhibited variable virulence (including moderate virulence) in mice, suggesting a potential risk to other mammalian species, including humans.

Genetic Characterization and Pathogenicity of H7N7 and H7N9 Avian Influenza Viruses Isolated from South Korea.

H7 low pathogenic avian influenza viruses (LPAIVs) can mutate into highly pathogenic avian influenza viruses (HPAIVs). In addition to avian species, H7 avian influenza viruses (AIVs) also infect humans. In this study, two AIVs, H7N9 (20X-20) and H7N7 (34X-2), isolated from the feces of wild birds in South Korea in 2021, were genetically analyzed. The HA cleavage site of the two H7 Korean viruses was confirmed to be ELPKGR/GLF, indicating they are LPAIVs. There were no amino acid substitutions at the receptor-binding site of the HA gene of two H7 Korean viruses compared to that of A/Anhui/1/2013 (H7N9), which prefer human receptors. In the phylogenetic tree analysis, the HA gene of the two H7 Korean viruses shared the highest nucleotide similarity with the Korean H7 subtype AIVs. In addition, the HA gene of the two H7 Korean viruses showed high nucleotide similarity to that of the A/Jiangsu/1/2018(H7N4) virus, which is a human influenza virus originating from avian influenza virus. Most internal genes (PB2, PB1, PA, NP, NA, M, and NS) of the two H7 Korean viruses belonged to the Eurasian lineage, except for the M gene of 34X-2. This result suggests that active reassortment occurred among AIVs. In pathogenicity studies of mice, the two H7 Korean viruses replicated in the lungs of mice. In addition, the body weight of mice infected with 34X-2 decreased 7 days post-infection (dpi) and inflammation was observed in the peribronchiolar and perivascular regions of the lungs of mice. These results suggest that mammals can be infected with the two H7 Korean AIVs. Our data showed that even low pathogenic H7 AIVs may infect mammals, including humans, as confirmed by the A/Jiangsu/1/2018(H7N4) virus. Therefore, continuous monitoring and pathogenicity assessment of AIVs, even of LPAIVs, are required.

The Effector Domain of the Influenza A Virus Nonstructural Protein NS1 Triggers Host Shutoff by Mediating Inhibition and Global Deregulation of Host Transcription When Associated with Specific Structures in the Nucleus.

Host shutoff in influenza A virus (IAV) infection is a key process contributing to viral takeover of the cellular machinery and resulting in the downregulation of host gene expression. Analysis of nascently transcribed RNA in a cellular model that allows the functional induction of NS1 demonstrates that NS1 suppresses host transcription. NS1 inhibits the expression of genes driven by RNA polymerase II as well as RNA polymerase I-driven promoters, but not by the noneukaryotic T7 polymerase. Additionally, transcriptional termination is deregulated in cells infected with wild-type IAV. The NS1 effector domain alone is able to mediate both effects, whereas NS1 mutant GLEWN184-188RFKRY (184-188) is not. Overexpression of CPSF30 counteracts NS1-mediated inhibition of RNA polymerase II-driven reporter gene expression, but knockdown of CPSF30 expression does not attenuate gene expression. Although NS1 is associated with nuclear chromatin, superresolution microscopy demonstrates that NS1 does not colocalize with genomic DNA. Moreover, NS1 mutants and NS1 fusion proteins, unable to associate with nuclear chromatin and displaying an altered subcellular distribution are still able to attenuate reporter gene expression. However, tethering NS1 artificially to the cytoskeleton results in the loss of reporter gene inhibition. A NS1 deficient in both native nuclear localization signals (NLS) is able to inhibit gene expression as effective as wild-type NS1 when a synthetic NLS relocates it to specific structures of the nucleus. Colocalization experiments and reporter gene cotransfection experiments with a NS1 fusion guiding it to nuclear speckles suggest that the presence of NS1 in nuclear speckles seems to be essential for host shutoff. IMPORTANCE We investigated the role of IAV nonstructural protein 1 NS1 in host gene shutoff-a central feature of IAV replication. We demonstrate that the effector domain of NS1 alone mediates host gene shutoff by inhibition of host transcription and by deregulation of the polyadenylation (polyA) signal-mediated 3' termination of host transcription. NS1 mutated in amino acids 184 to 188 fails to shut off host gene expression. Knockdown of CPSF30 does not result in transcriptional attenuation. By analyzing the subcellular localization of modified NS1 proteins and relating these data to their ability to inhibit reporter gene expression, we show for the first time that the presence of NS1 in granular structures of the nucleus-representing most likely nuclear speckles-seems to be essential to mediate host gene shutoff. Thus, our data present so far unknown insights into the molecular and spatial requirements needed for IAV-NS1-mediated host shutoff.

Equine Influenza Virus and Vaccines.

Equine influenza virus (EIV) is a constantly evolving viral pathogen that is responsible for yearly outbreaks of respiratory disease in horses termed equine influenza (EI). There is currently no evidence of circulation of the original H7N7 strain of EIV worldwide; however, the EIV H3N8 strain, which was first isolated in the early 1960s, remains a major threat to most of the world's horse populations. It can also infect dogs. The ability of EIV to constantly accumulate mutations in its antibody-binding sites enables it to evade host protective immunity, making it a successful viral pathogen. Clinical and virological protection against EIV is achieved by stimulation of strong cellular and humoral immunity in vaccinated horses. However, despite EI vaccine updates over the years, EIV remains relevant, because the protective effects of vaccines decay and permit subclinical infections that facilitate transmission into susceptible populations. In this review, we describe how the evolution of EIV drives repeated EI outbreaks even in horse populations with supposedly high vaccination coverage. Next, we discuss the approaches employed to develop efficacious EI vaccines for commercial use and the existing system for recommendations on updating vaccines based on available clinical and virological data to improve protective immunity in vaccinated horse populations. Understanding how EIV biology can be better harnessed to improve EI vaccines is central to controlling EI.

H7N7 Avian Influenza Virus Mutation from Low to High Pathogenicity on a Layer Chicken Farm in the UK.

Avian influenza virus (AIV) subtypes H5 and H7 are capable of mutating from low to high pathogenicity strains, causing high mortality in poultry with significant economic losses globally. During 2015, two outbreaks of H7N7 low pathogenicity AIV (LPAIV) in Germany, and one each in the United Kingdom (UK) and The Netherlands occurred, as well as single outbreaks of H7N7 high pathogenicity AIV (HPAIV) in Germany and the UK. Both HPAIV outbreaks were linked to precursor H7N7 LPAIV outbreaks on the same or adjacent premises. Herein, we describe the clinical, epidemiological, and virological investigations for the H7N7 UK HPAIV outbreak on a farm with layer chickens in mixed free-range and caged units. H7N7 HPAIV was identified and isolated from clinical samples, as well as H7N7 LPAIV, which could not be isolated. Using serological and molecular evidence, we postulate how the viruses spread throughout the premises, indicating potential points of incursion and possible locations for the mutation event. Serological and mortality data suggested that the LPAIV infection preceded the HPAIV infection and afforded some clinical protection against the HPAIV. These results document the identification of a LPAIV to HPAIV mutation in nature, providing insights into factors that drive its manifestation during outbreaks.

Isolation and characterization of low pathogenic H7N7 avian influenza virus from a red-crowned crane in a zoo in South Korea.

BACKGROUND: South Korea conducts annual national surveillance programs to detect avian influenza (AI) in domestic poultry, live bird markets, and wild birds. In March 2017, an AIV was isolated from fecal samples in an outdoor aviary flight cage in a zoo in Korea. RESULTS: Nucleotide sequencing identified the isolate as low pathogenic avian influenza virus (LPAIV) H7N7, and DNA barcoding analysis identified the host species as red-crowned crane. This isolate was designated A/red-crowned crane/Korea/H1026/2017 (H7N7). Genetic analysis and gene constellation analysis revealed that A/red-crowned crane/Korea/H1026/2017 (H7N7) showed high similarity with four H7N7 LPAIVs isolated from wild bird habitats in Seoul and Gyeonggi in early 2017. CONCLUSIONS: Considering the genetic similarity and similar collection dates of the viruses, and the fact that zoo bird cages are vulnerable to AIV, it is likely that fecal contamination from wild birds might have introduced LPAIV H7N7 into the red-crowned crane at the zoo. Therefore, our results emphasize that enhanced biosecurity measures should be employed during the wild bird migration season, and that continued surveillance should be undertaken to prevent potential threats to avian species in zoos and to humans.

Low pathogenic avian influenza virus isolates with different levels of defective genome segments vary in pathogenicity and transmission efficiency.

Defective interfering particles (DIPs) of influenza virus are generated through incorporation of highly truncated forms of genome segments, mostly those coding polymerase complex proteins (PB2, PB1, PA). Such particles are able to replicate only in the presence of a virus with the complete genome, thus DIPs may alter the infection outcome by suppressing production of standard virus particles, but also by stimulating the immune response. In the present study we compared the clinical outcome, mortality and transmission in chickens and turkeys infected with the same infectious doses of H7N7 low pathogenic avian influenza virus containing different levels of defective gene segments (95/95(DVG-high) and 95/95(DVG-low)). No clinical signs, mortality or transmission were noted in SPF chickens inoculated with neither virus stock. Turkeys infected with 95/95(DVG-high) showed only slight clinical signs with no mortality, and the virus was transmitted only to birds in direct contact. In contrast, more severe disease, mortality and transmission to direct and indirect contact birds was observed in turkeys infected with 95/95(DVG-low). Apathy, lower water and food intake, respiratory system disorders and a total mortality of 60% were noted. Shedding patterns in contact turkeys indicated more efficient within- and between-host spread of the virus than in 95/95(DVG-high) group. Sequencing of virus genomes showed no mutations that could account for the observed differences in pathogenicity. The results suggest that the abundance of DIPs in the inoculum was the factor responsible for the mild course of infection and disrupted virus transmission.

The Emergence of H7N7 Highly Pathogenic Avian Influenza Virus from Low Pathogenicity Avian Influenza Virus Using an in ovo Embryo Culture Model.

Outbreaks of highly pathogenic avian influenza virus (HPAIV) often result in the infection of millions of poultry, causing up to 100% mortality. HPAIV has been shown to emerge from low pathogenicity avian influenza virus (LPAIV) in field outbreaks. Direct evidence for the emergence of H7N7 HPAIV from a LPAIV precursor with a rare di-basic cleavage site (DBCS) was identified in the UK in 2008. The DBCS contained an additional basic amino acid compared to commonly circulating LPAIVs that harbor a single-basic amino acid at the cleavage site (SBCS). Using reverse genetics, outbreak HPAIVs were rescued with a DBCS (H7N7DB), as seen in the LPAIV precursor or an SBCS representative of common H7 LPAIVs (H7N7SB). Passage of H7N7DB in chicken embryo tissues showed spontaneous evolution to a HPAIV. In contrast, deep sequencing of extracts from embryo tissues in which H7N7SB was serially passaged showed retention of the LPAIV genotype. Thus, in chicken embryos, an H7N7 virus containing a DBCS appears naturally unstable, enabling rapid evolution to HPAIV. Evaluation in embryo tissue presents a useful approach to study AIV evolution and allows a laboratory-based dissection of molecular mechanisms behind the emergence of HPAIV.

Amino acid substitutions involved in the adaptation of a novel H7N7 avian influenza virus in mice.

The H7N7 avian influenza viruses can infect humans and poses a great threat to human health. To identify the amino acid substitutions that are associated with adaptation of avian-origin H7N7 virus to mammals, adaptation of the H7N7 virus was carried out by serial lung-to-lung passage in mice. Genomic analysis of the mouse-adapted virus revealed amino acid changes in the PB2 (E525G, M645I, and D701N), NP (I475V), HA(D103N), and NA(K142E) proteins. The adapted H7N7 virus was more virulent in mice than the wild-type virus. Our results suggest that continued surveillance of poultry populations for these substitutions in the H7N7 virus is required.

Genetic and antigenic characterization of the first H7N7 low pathogenic avian influenza viruses isolated in Vietnam.

During the annual surveillance of avian influenza viruses (AIVs) in Vietnam in 2018, three H7N7 AIV isolates were identified in domestic ducks in a single flock in Vinh Long province. The present study is the first documented report of H7N7 virus isolates in Vietnam and aimed to characterize these viruses, both genetically and antigenically. Deduced amino acid sequences for the hemagglutinins (HAs) indicated a low pathogenicity of these viruses in chickens. Phylogenetic analysis revealed that the H7 HA genes of these isolates were closely related to each other and belonged to the European-Asian sublineage, together with those of H7N3 viruses isolated from ducks in Cambodia during 2017. They were not genetically related to those of Chinese H7N9 or H7N1 viruses that were previously detected in Vietnam during 2012. Interestingly, the M genes of the two H7N7 virus isolates were phylogenetically classified into distinct groups, suggesting an ongoing reassortment event in domestic ducks because they were isolated from the same flock. These H7N7 viruses exhibited somewhat different antigenic characteristics compared with other representative H7 low pathogenic AIVs. Surprisingly, the antigenicity of Vietnamese H7N7 viruses is similar to Chinese H7N9 highly pathogenic AIV. The findings of this study suggest that H7N7 viruses may be undergoing reassortment and antigenic diversification in poultry flocks in Vietnam. The silent spread of Vietnamese H7N7 viruses in chickens may lead to acquire high pathogenicity in chickens although the zoonotic potential of the viruses seems to be low since these viruses retain typical avian-specific motifs in the receptor-binding site in the HA and there is no mutation related to mammalian adaptation in PB2 gene. Thus, these results highlight the need for continuous and intensive surveillance of avian influenza in Vietnam, targeting not only highly pathogenic AIVs but also low pathogenic viruses.

Mutations in the H7 HA and PB1 genes of avian influenza a viruses increase viral pathogenicity and contact transmission in guinea pigs.

Avian influenza A viruses (AIV) of the H7 subtype continue to evolve posing a pandemic threat. However, molecular markers of H7N7 AIV pathogenicity and transmission in mammals remain poorly understood. In this study, we performed a systematic in vitro and in vivo analysis by comparing an H7N7 highly pathogenic AIV and its ferret adapted variant. Passaging an H7N7 AIV in ferrets led to six mutations in genes encoding the viral polymerase complex and the viral surface proteins. Here, we show that mutations in the H7 hemagglutinin gene cause increased pathogenicity in mice. Contact transmission between guinea pigs required additional mutations in the gene encoding the polymerase subunit PB1. Thus, particular vigilance is required with respect to HA and PB1 mutations as predictive molecular markers to assess the pandemic risk posed by emerging H7 avian influenza viruses.

Packaging of the Influenza Virus Genome Is Governed by a Plastic Network of RNA- and Nucleoprotein-Mediated Interactions.

The genome of influenza A virus is organized into eight ribonucleoproteins, each composed of a distinct RNA segment bound by the viral polymerase and oligomeric viral nucleoprotein. Packaging sequences unique to each RNA segment together with specific nucleoprotein amino acids are thought to ensure the precise incorporation of these eight ribonucleoproteins into single virus particles, and yet the underlying interaction network remains largely unexplored. We show here that the genome packaging mechanism of an H7N7 subtype influenza A virus widely tolerates the mutation of individual packaging sequences in three different RNA segments. However, combinations of these modified RNA segments cause distinct genome packaging defects, marked by the absence of specific RNA segment subsets from the viral particles. Furthermore, we find that combining a single mutated packaging sequence with sets of specific nucleoprotein amino acid substitutions greatly impairs the viral genome packaging process. Along with previous reports, our data propose that influenza A virus uses a redundant and plastic network of RNA-RNA and potentially RNA-nucleoprotein interactions to coordinately incorporate its segmented genome into virions.IMPORTANCE The genome of influenza A virus is organized into eight viral ribonucleoproteins (vRNPs); this provides evolutionary advantages but complicates genome packaging. Although it has been shown that RNA packaging sequences and specific amino acids in the viral nucleoprotein (NP), both components of each vRNP, ensure selective packaging of one copy of each vRNP per virus particle, the required RNA-RNA and RNA-NP interactions remain largely elusive. We identified that the genome packaging mechanism tolerates the mutation of certain individual RNA packaging sequences, while their combined mutation provokes distinct genome packaging defects. Moreover, we found that seven specific amino acid substitutions in NP impair the function of RNA packaging sequences and that this defect is partially restored by another NP amino acid change. Collectively, our data indicate that packaging of the influenza A virus genome is controlled by a redundant and plastic network of RNA/protein interactions, which may facilitate natural reassortment processes.

A viral race for primacy: co-infection of a natural pair of low and highly pathogenic H7N7 avian influenza viruses in chickens and embryonated chicken eggs.

Highly pathogenic avian influenza virus (HPAIV) infection in poultry caused devastating mortality and economic losses. HPAIV of subtypes H5 and H7 emerge from precursor viruses of low pathogenicity (LP) by spontaneous mutation associated with a shift in the susceptibility of the endoproteolytic cleavage site of the viral hemagglutinin protein from trypsin- to furin-like proteases. A recently described natural pair of LP/HP H7N7 viruses derived from two spatio-temporally linked outbreaks in layer chickens was used to study how a minority of mutated HP virions after de novo generation in a single host might gain primacy. Co-infection experiments in embryonated eggs and in chickens were conducted to investigate amplification, spread and transmissionof HPAIV within a poultry population that experiences concurrent infection by an antigenically identical LP precursor virus. Simultaneous LPAIV co-infection (inoculum dose of 106 egg-infectious dose 50% endpoint (EID50)/0.5 mL) withincreasing titers of HPAIV from 101 to 105.7 EID50/0.5 mL) had a significant impeding impact on HP H7 replication, viral excretion kinetics, clinical signs and histopathological lesions (in vivo) and on embryo mortality (in ovo). LP/HP co-infected chickens required a hundredfold higher virus dose (HPAIV inoculum of 105 EID50) compared to HPAIV mono-infection (HPAIV inoculum of 103 EID50) to develop overt clinical signs, mortality and virus spread to uninfected sentinels. Escape and spread of HP phenotypes after de novo generation in an index host may therefore be highly precarious due to significant competition with co-circulating LP precursor virus.

Pathogenesis and genetic characteristics of novel reassortant low-pathogenic avian influenza H7 viruses isolated from migratory birds in the Republic of Korea in the winter of 2016-2017.

In this study, we characterized H7 subtype low-pathogenicity (LP) influenza A viruses (IAVs) isolated from wild bird habitats in the Republic of Korea from 2010 to early 2017. Through national surveillance, 104 H7 IAVs were isolated, accounting for an average of 14.9% of annual IAV isolations. In early 2017, H7 subtypes accounted for an unusually high prevalence (43.6%) of IAV detections in wild birds. Phylogenetic analysis revealed that all the viruses isolated in the winter of 2016-2017 fell within cluster II of group C, belonging to the Eurasian lineage of H7 IAVs. Notably, cluster II of group C included the H7 gene from the highly pathogenic H7N7 IAV that was detected in northeastern Italy in April of 2016. Through a gene-constellation analysis, the H7 LPIAVs that we isolated constituted ≥11 distinct genotypes. Because the viruses belonging to the genotypes G2.1 and G1 were observed most frequently, we compared the replication and transmission of representative viruses to these genotypes in specific-pathogen-free chickens. Notably, the representative G2.1 strain was capable of systemic replication and efficient transmission in chickens (as evidenced by virus isolation and histopathological examination) without any clinical signs except mortality (in one infected chicken). The efficient subclinical viral replication and shedding of the G2.1 virus in chickens may facilitate its silent spread among poultry after introduction. Given that wild birds harbor novel strains that could affect poultry, our results highlight the need for enhanced IAV surveillance in both wild birds and poultry in Eurasia.

Direct evidence of H7N7 avian influenza virus mutation from low to high virulence on a single poultry premises during an outbreak in free range chickens in the UK, 2008.

H5 and H7 subtypes of low pathogenicity avian influenza viruses (LPAIVs) have the potential to evolve into highly pathogenic avian influenza viruses (HPAIVs), causing high mortality in galliforme poultry with substantial economic losses for the poultry industry. This study provides direct evidence of H7N7 LPAIV mutation to HPAIV on a single poultry premises during an outbreak that occurred in June 2008 in free range laying hens in Oxfordshire, UK. We report the first detection of a rare di-basic cleavage site (CS) motif (PEIPKKRGLF), unique to galliformes, that has previously been associated with a LPAIV phenotype. Three distinct HPAIV CS sequences (PEIPKRKKRGLF, PEIPKKKKRGLF and PEIPKKKKKKRGLF) were identified in the infected sheds suggesting molecular evolution at the outbreak premises. Further evidence for H7N7 LPAIV preceding mutation to HPAIV was derived by examining clinical signs, epidemiological descriptions and analysing laboratory results on the timing and proportions of seroconversion and virus shedding at each infected shed on the premises. In addition to describing how the outbreak was diagnosed and managed via statutory laboratory testing, phylogenetic analysis revealed reassortant events during 2006-2008 that suggested likely incursion of a wild bird origin LPAIV precursor to the H7N7 HPAIV outbreak. Identifying a precursor LPAIV is important for understanding the molecular changes and mechanisms involved in the emergence of HPAIV. This information can lead to understanding how and why only some H7 LPAIVs appear to readily mutate to HPAIV.

Two genetically diverse H7N7 avian influenza viruses isolated from migratory birds in central China.

After the emergence of H7N9 avian influenza viruses (AIV) in early 2013 in China, active surveillance of AIVs in migratory birds was undertaken, and two H7N7 strains were subsequently recovered from the fresh droppings of migratory birds; the strains were from different hosts and sampling sites. Phylogenetic and sequence similarity network analyses indicated that several genes of the two H7N7 viruses were closely related to those in AIVs circulating in domestic poultry, although different gene segments were implicated in the two isolates. This strongly suggested that genes from viruses infecting migratory birds have been introduced into poultry-infecting strains. A Bayesian phylogenetic reconstruction of all eight segments implied that multiple reassortments have occurred in the evolution of these viruses, particularly during late 2011 and early 2014. Antigenic analysis using a hemagglutination inhibition test showed that the two H7N7 viruses were moderately cross-reactive with H7N9-specific anti-serum. The ability of the two H7N7 viruses to remain infectious under various pH and temperature conditions was evaluated, and the viruses persisted the longest at near-neutral pH and in cold temperatures. Animal infection experiments showed that the viruses were avirulent to mice and could not be recovered from any organs. Our results indicate that low pathogenic, divergent H7N7 viruses circulate within the East Asian-Australasian flyway. Virus dispersal between migratory birds and domestic poultry may increase the risk of the emergence of novel unprecedented strains.

Inactivated H7 Influenza Virus Vaccines Protect Mice despite Inducing Only Low Levels of Neutralizing Antibodies.

Avian influenza viruses of the H7 hemagglutinin (HA) subtype present a significant public health threat, as evidenced by the ongoing outbreak of human A(H7N9) infections in China. When evaluated by hemagglutination inhibition (HI) and microneutralization (MN) assays, H7 viruses and vaccines are found to induce lower level of neutralizing antibodies (nAb) than do their seasonal counterparts, making it difficult to develop and evaluate prepandemic vaccines. We have previously shown that purified recombinant H7 HA appear to be poorly immunogenic in that they induce low levels of HI and MN antibodies. In this study, we immunized mice with whole inactivated reverse genetics reassortant (RG) viruses expressing HA and neuraminidase (NA) from 3 different H7 viruses [A/Shanghai/2/2013(H7N9), A/Netherlands/219/2003(H7N7), and A/New York/107/2003(H7N2)] or with human A(H1N1)pdm09 (A/California/07/2009-like) or A(H3N2) (A/Perth16/2009) viruses. Mice produced equivalent titers of antibodies to all viruses as measured by enzyme-linked immunosorbent assay (ELISA). However, the antibody titers induced by H7 viruses were significantly lower when measured by HI and MN assays. Despite inducing very low levels of nAb, H7 vaccines conferred complete protection against homologous virus challenge in mice, and the serum antibodies directed against the HA head region were capable of mediating protection. The apparently low immunogenicity associated with H7 viruses and vaccines may be at least partly related to measuring antibody titers with the traditional HI and MN assays, which may not provide a true measure of protective immunity associated with H7 immunization. This study underscores the need for development of additional correlates of protection for prepandemic vaccines.IMPORTANCE H7 avian influenza viruses present a serious risk to human health. Preparedness efforts include development of prepandemic vaccines. For seasonal influenza viruses, protection is correlated with antibody titers measured by hemagglutination inhibition (HI) and virus microneutralization (MN) assays. Since H7 vaccines typically induce low titers in HI and MN assays, they have been considered to be poorly immunogenic. We show that in mice H7 whole inactivated virus vaccines (WIVs) were as immunogenic as seasonal WIVs, as they induced similar levels of overall serum antibodies. However, a larger fraction of the antibodies induced by H7 WIV was nonneutralizing in vitro Nevertheless, the H7 WIV completely protected mice against homologous viral challenge, and antibodies directed against the HA head were the major contributor toward immune protection. Vaccines against H7 avian influenza viruses may be more effective than HI and virus neutralization assays suggest, and such vaccines may need other methods for evaluation.

H7N7 Highly Pathogenic Avian Influenza in Poultry Farms in Italy in 2016.

After the H7N7 highly pathogenic (HP) avian influenza (AI) outbreak in 2013, and a single case of H5N8 HPAI in 2014, in April 2016, a H7N7 HPAI virus was detected in northeastern Italy. The case occurred in an organic free-range laying hen farm located in proximity with one of the highest densely populated poultry areas (DPPAs) in Italy. Control measures provided by the Council of the European Union in directive 2005/94/CE were promptly applied, and enhanced surveillance activities were implemented in the DPPAs. On May 16, 2016, a second case was confirmed in a fattening turkey farm within the protection zone of the previous outbreak. Following an epidemiologic inquiry, another turkey farm was considered at risk of transmission and was subjected to preemptive culling. Epidemiologic data and phylogenetic analyses indicated that the virus was likely introduced from wild birds as a low pathogenicity AI strain, through direct contact. The rapid containment of the outbreak proves the level of preparedness of the veterinary public health sector in Italy. Nevertheless, the recurrent introductions from wild birds indicate the need of improving both the biosecurity levels in the DPPA and the surveillance activities in wild birds to quickly detect the presence of AI in the territory.