Pathogenic H5N1 Virus Research Articles

Infection of South American coatis (Nasua nasua) with highly pathogenic avian influenza H5N1 virus displaying mammalian adaptive mutations

Deadly outbreaks among poultry, wild birds, and carnivorous mammals by the highly pathogenic H5N1 virus of the clade 2.3.4.4b have been reported in South America. The increasing virus incidence in various mammal species poses a severe zoonotic and pandemic threat. In Uruguay, the clade 2.3.4.4b viruses were first detected in February 2023, affecting wild birds and backyard poultry. Three months after the first reported case in Uruguay, the disease affected a population of 23 coatis (Nasua) in an ecological park. Most animals became infected, likely directly or indirectly from wild birds in the park, and experienced sudden death. Five animals from the colony survived, and four of them developed antibodies. The genomes of the H5N1 strains infecting coatis belonged to the B3.2 genotype of the clade 2.3.4.4b. Genomes from coatis were closely associated with those infecting backyard poultry, but transmission likely occurred through wild birds. Notable, two genomes have a 627K substitution in the RNA polymerase PB2 subunit, a hallmark amino acid linked to mammalian adaptation. Our findings support the ability of the avian influenza virus of the 2.3.4.4b clade to infect and transmit among terrestrial mammals with high pathogenicity and undergo rapid adaptive changes. It also highlights the coatis' ability to develop immunity and naturally clear the infection.

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

We present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds in the year following the first detection of highly pathogenic avian influenza virus (HPAIV) H5N1 on the continent. The surveillance program tested over 17,000 wild birds, both sick and apparently healthy, which revealed spatiotemporal and taxonomic patterns in HPAIV prevalence and mortality across Canada. The significant shift in the presence and distribution of HPAIV in Canada's wild birds underscores the need for sustained investment in wild bird surveillance and collaboration across One Health partners.

A synthetic TLR4 agonist significantly increases humoral immune responses and the protective ability of an MDCK-cell-derived inactivated H7N9 vaccine in mice.

Antigenically divergent H7N9 viruses pose a potential threat to public health, with the poor immunogenicity of candidate H7N9 vaccines demonstrated in clinical trials underscoring the urgent need for more-effective H7N9 vaccines. In the present study, mice were immunized with various doses of a suspended-MDCK-cell-derived inactivated H7N9 vaccine, which was based on a low-pathogenic H7N9 virus, to assess cross-reactive immunity and cross-protection against antigenically divergent H7N9 viruses. We found that the CRX-527 adjuvant, a synthetic TLR4 agonist, significantly enhanced the humoral immune responses of the suspended-MDCK-cell-derived H7N9 vaccine, with significant antigen-sparing and immune-enhancing effects, including robust virus-specific IgG, hemagglutination-inhibiting (HI), neuraminidase-inhibiting (NI), and virus-neutralizing (VN) antibody responses, which are crucial for protection against influenza virus infection. Moreover, the CRX-527-adjuvanted H7N9 vaccine also elicited cross-protective immunity and cross-protection against a highly pathogenic H7N9 virus with a single vaccination. Notably, NI and VN antibodies might play an important role in cross-protection against lethal influenza virus infections. This study showed that a synthetic TLR4 agonist adjuvant has a potent immunopotentiating effect, which might be considered worth further development as a means of increasing vaccine effectiveness.

Detection of clade 2.3.4.4b highly pathogenic H5N1 influenza virus in New York City.

Highly pathogenic avian influenza viruses of the H5N1 clade 2.3.4.4b were detected in North America in the winter of 2021/2022. These viruses have spread across the Americas, causing morbidity and mortality in both wild and domestic birds as well as some mammalian species, including cattle. Many surveillance programs for wildlife as well as commercial poultry operations have detected these viruses. In this study, we conducted surveillance of avian species in the urban environment in New York City. We detected highly pathogenic H5N1 viruses in six samples from four different bird species and performed whole-genome sequencing. Sequencing analysis showed the presence of multiple different genotypes. Our work highlights that the interface between animals and humans that may give rise to zoonotic infections or even pandemics is not limited to rural environments and commercial poultry operations but extends into the heart of our urban centers.IMPORTANCEWhile surveillance programs for avian influenza viruses are often focused on migratory routes and their associated stop-over locations or commercial poultry operations, many bird species-including migratory birds-frequent or live in urban green spaces and wetlands. This brings them into contact with a highly dense population of humans and pets, providing an extensive urban animal-human interface in which the general public may have little awareness of circulating infectious diseases. This study focuses on virus surveillance of this interface, combined with culturally responsive science education and community outreach.

Molecular modeling and phylogenetic analyses highlight the role of amino acid 347 of the N1 subtype neuraminidase in influenza virus host range and interspecies adaptation.

The N1 neuraminidases (NAs) of avian and pandemic human influenza viruses contain tyrosine and asparagine, respectively, at position 347 on the rim of the catalytic site; the biological significance of this difference is not clear. Here, we used molecular dynamics simulation to model the effects of amino acid 347 on N1 NA interactions with sialyllacto-N-tetraoses 6'SLN-LC and 3'SLN-LC, which represent NA substrates in humans and birds, respectively. Our analysis predicted that Y347 plays an important role in the NA preference for the avian-type substrates. The Y347N substitution facilitates hydrolysis of human-type substrates by resolving steric conflicts of the Neu5Ac2-6Gal moiety with the bulky side chain of Y347, decreasing the free energy of substrate binding, and increasing the solvation of the Neu5Ac2-6Gal bond. Y347 was conserved in all N1 NA sequences of avian influenza viruses in the GISAID EpiFlu database with two exceptions. First, the Y347F substitution was present in the NA of a specific H6N1 poultry virus lineage and was associated with the substitutions G228S and/or E190V/L in the receptor-binding site (RBS) of the hemagglutinin (HA). Second, the highly pathogenic avian H5N1 viruses of the Gs/Gd lineage contained sporadic variants with the NA substitutions Y347H/D, which were frequently associated with substitutions in the HA RBS. The Y347N substitution occurred following the introductions of avian precursors into humans and pigs with N/D347 conserved during virus circulation in these hosts. Comparative evolutionary analysis of site 347 revealed episodic positive selection across the entire tree and negative selection within most host-specific groups of viruses, suggesting that substitutions at NA position 347 occurred during host switches and remained under pervasive purifying selection thereafter. Our results elucidate the role of amino acid 347 in NA recognition of sialoglycan substrates and emphasize the significance of substitutions at position 347 as a marker of host range and adaptive evolution of influenza viruses.

Influenza A viruses in gulls in landfills and freshwater habitats in Minnesota, United States.

Introduction: The unpredictable evolution of avian influenza viruses (AIVs) presents an ongoing threat to agricultural production and public and wildlife health. Severe outbreaks of highly pathogenic H5N1 viruses in US poultry and wild birds since 2022 highlight the urgent need to understand the changing ecology of AIV. Surveillance of gulls in marine coastal environments has intensified in recent years to learn how their long-range pelagic movements potentially facilitate inter-hemispheric AIV movements. In contrast, little is known about inland gulls and their role in AIV spillover, maintenance, and long-range dissemination. Methods: To address this gap, we conducted active AIV surveillance in ring-billed gulls (Larus delawarensis) and Franklin's gulls (Leucophaeus pipixcan) in Minnesota's natural freshwater lakes during the summer breeding season and in landfills during fall migration (1,686 samples). Results: Whole-genome AIV sequences obtained from 40 individuals revealed three-lineage reassortants with a mix of genome segments from the avian Americas lineage, avian Eurasian lineage, and a global "Gull" lineage that diverged more than 50 years ago from the rest of the AIV global gene pool. No poultry viruses contained gull-adapted H13, NP, or NS genes, pointing to limited spillover. Geolocators traced gull migration routes across multiple North American flyways, explaining how inland gulls imported diverse AIV lineages from distant locations. Migration patterns were highly varied and deviated far from assumed "textbook" routes. Discussion: Viruses circulating in Minnesota gulls during the summer breeding season in freshwater environments reappeared in autumn landfills, evidence of AIV persistence in gulls between seasons and transmission between habitats. Going forward, wider adoption of technological advances in animal tracking devices and genetic sequencing is needed to expand AIV surveillance in understudied hosts and habitats.

Highly Pathogenic H5 Influenza Viruses Isolated between 2016 and 2017 in Vietnamese Live Bird Markets.

Routine surveillance in live poultry markets in the northern regions of Vietnam from 2016 to 2017 resulted in the isolation of 27 highly pathogenic avian H5N1 and H5N6 viruses of 3 different clades (2.3.2.1c, 2.3.4.4f, and 2.3.4.4g). Sequence and phylogenetic analysis of these viruses revealed reassortment with various subtypes of low pathogenic avian influenza viruses. Deep-sequencing identified minor viral subpopulations encoding variants that may affect pathogenicity and sensitivity to antiviral drugs. Interestingly, mice infected with two different clade 2.3.2.1c viruses lost body weight rapidly and succumbed to virus infection, whereas mice infected with clade 2.3.4.4f or 2.3.4.4g viruses experienced non-lethal infections.

Comparative Surface Electrostatics and Normal Mode Analysis of High and Low Pathogenic H7N7 Avian Influenza Viruses.

Influenza A viruses are rarely symptomatic in wild birds, while representing a higher threat to poultry and mammals, where they can cause a variety of symptoms, including death. H5 and H7 subtypes of influenza viruses are of particular interest because of their pathogenic potential and reported capacity to spread from poultry to mammals, including humans. The identification of molecular fingerprints for pathogenicity can help surveillance and early warning systems, which are crucial to prevention and protection from such potentially pandemic agents. In the past decade, comparative analysis of the surface features of hemagglutinin, the main protein antigen in influenza viruses, identified electrostatic fingerprints in the evolution and spreading of H5 and H9 subtypes. Electrostatic variation among viruses from avian or mammalian hosts was also associated with host jump. Recent findings of fingerprints associated with low and highly pathogenic H5N1 viruses, obtained by means of comparative electrostatics and normal modes analysis, prompted us to check whether such fingerprints can also be found in the H7 subtype. Indeed, evidence presented in this work showed that also in H7N7, hemagglutinin proteins from low and highly pathogenic strains present differences in surface electrostatics, while no meaningful variation was found in normal modes.

Inactivation of Avian Influenza Viruses by Chemical Disinfectants and the Influence of Faecal Matter

Inactivation of avian influenza viruses (AIVs) using chemical disinfectants is an indispensable biosecurity measure during outbreaks. The efficiency of disinfection process depends on disinfectant type and presence or absence of organic matter. It is also not clear whether AIV pathotype could influence its tolerability to a given disinfectant. Here, we assessed the efficiency of formalin and caustic soda against high (H5N1) and low (H9N2) pathogenic AIVs in the presence and absence of wet faeces. Our results showed that the effectiveness of both disinfectants depends on the target virus with the low pathogenic H9N2 virus being generally more susceptible to disinfection than the high pathogenic H5N1 virus. Wet faeces slightly influenced disinfection action in a virus- and contact time- dependent manner. This study highlights the importance of eliminating faecal matter before initiating a disinfection process and indicates the influence of disinfectant type and AIVs pathotype when selecting the appropriate disinfectant.

Generation of an avian influenza DIVA vaccine with a H3-peptide replacement located at HA2 against both highly and low pathogenic H7N9 virus

ABSTRACT A differentiating infected from vaccinated animals (DIVA) vaccine is an ideal strategy for viral eradication in poultry. Here, according to the emerging highly pathogenic H7N9 avian influenza virus (AIV), a DIVA vaccine strain, named rGD4HALo-mH3-TX, was successfully developed, based on a substituted 12 peptide of H3 virus located at HA2. In order to meet with the safety requirement of vaccine production, the multi-basic amino acid located at the HA cleavage site was modified. Meanwhile, six inner viral genes from a H9N2 AIV TX strainwere introduced for increasing viral production. The rGD4HALo-mH3-TX strain displayed a similar reproductive ability with rGD4 and low pathogenicity in chickens, suggesting a good productivity and safety. In immuned chickens, rGD4HALo-mH3-TX induced a similar antibody level with rGD4 and provided 100% clinical protection and 90% shedding protection against highly pathogenic virus challenge. rGD4HALo-mH3-TX strain also produced a good cross-protection against low pathogenic AIV JD/17. Moreover, serological DIVA characteristics were evaluated by a successfully established competitive inhibition ELISA based on a 3G10 monoclonal antibody, and the result showed a strong reactivity with antisera of chickens vaccinated with H7 subtype strains but not rGD4HALo-mH3-TX. Collectedly, rGD4HALo-mH3-TX is a promising DIVA vaccine candidate against both high and low pathogenic H7N9 subtype AIV.

Characterization of two chicken origin highly pathogenic H7N9 viruses isolated in northern China

Since the national vaccination program was implemented with the H5/H7 bivalent vaccine in poultry in September 2017, the prevalence of H7N9 avian influenza viruses (AIVs) has been controlled effectively in China. However, highly pathogenic H7N9 viruses still exist, causing sporadic outbreaks especially in some regions of northern China. During our routine surveillance in poultry in 2020, we isolated two strains of H7N9 subtype AIV from breeder layer farms in northern China. We found that these two chicken-origin H7N9 isolates were both highly pathogenic (HP) with a four-amino-acid (KRTA) insertion and an I326V mutation (H3 numbering) in the cleavage site of HA to make the motif PEVPKRKRTAR↓GLF. Molecular markers associated with antigenic drift and enhanced pathogenicity in mammals and interspecies transmission were detected in both isolates. Remarkably, both strains gained the F102V and N157D mutations in their HA genes, which have never been reported before. Solid-phase direct binding assay showed that these two isolates both had dual-receptor binding characteristics, while thermal and acid stability assays indicated that they were relatively stable in high-temperature or acidic conditions. In addition, the animal experiments demonstrated that both strains were highly pathogenic to chickens but low pathogenic to mice. These results suggested that the evolution of H7N9 subtype AIV is still continuing, and they pose a potential threat to poultry and public health. Thus, attention should be paid to the importance of continual surveillance of the H7N9 AIVs.

Impact of the R292K Mutation on Influenza A (H7N9) Virus Resistance towards Peramivir: A Molecular Dynamics Perspective.

In March 2013, a novel avian influenza A (H7N9) virus emerged in China. By March 2021, it had infected more than 1500 people, raising concerns regarding its epidemic potential. Similar to the highly pathogenic H5N1 virus, the H7N9 virus causes severe pneumonia and acute respiratory distress syndrome in most patients. Moreover, genetic analysis showed that this avian H7N9 virus carries human adaptation markers in the hemagglutinin and polymerase basic 2 (PB2) genes associated with cross-species transmissibility. Clinical studies showed that a single mutation, neuraminidase (NA) R292K (N2 numbering), induces resistance to peramivir in the highly pathogenic H7N9 influenza A viruses. Therefore, to evaluate the risk for human public health and understand the possible source of drug resistance, we assessed the impact of the NA-R292K mutation on avian H7N9 virus resistance towards peramivir using various molecular dynamics approaches. We observed that the single point mutation led to a distorted peramivir orientation in the enzyme active site which, in turn, perturbed the inhibitor’s binding. The R292K mutation induced a decrease in the interaction among neighboring amino acid residues when compared to its wild-type counterpart, as shown by the high degree of fluctuations in the radius of gyration. MM/GBSA calculations revealed that the mutation caused a decrease in the drug binding affinity by 17.28 kcal/mol when compared to the that for the wild-type enzyme. The mutation caused a distortion of hydrogen bond-mediated interactions with peramivir and increased the accessibility of water molecules around the K292 mutated residue.

Immune Enhancement by the Tetra-Peptide Hydrogel as a Promising Adjuvant for an H7N9 Vaccine against Highly Pathogenic H7N9 Virus.

Background: Short peptide hydrogel was reported as a possible adjuvant for vaccines. In order to evaluate whether the Tetra-Peptide Hydrogel can be a promising adjuvant for an H7N9 vaccine against the highly pathogenic H7N9 virus, we conducted this study. Methods: Tetra-Peptide Hydrogels (D and L conformations) were prepared by a self-assembly system using a Naproxen acid modified tetra peptide of GFFY (Npx-GFFY). Mice received two immunizations with the D-Tetra-Peptide Hydrogel adjuvant vaccine, the L-Tetra-Peptide Hydrogel adjuvant vaccine, or the split vaccine. Fourteen days following the second dose, the mice were challenged with the highly pathogenic A/Guangdong/GZ8H002/2017(H7N9) virus. The mice were observed for signs of illness, weight loss, pathological alterations of the lung tissues and immune responses in the following 2 weeks. Results: The D/L-Tetra-Peptide Hydrogels resembled long bars with hinges on each other, with a diameter of ~10 nm. The H7N9 vaccine was observed to adhere to the hydrogel. All the unvaccinated mice were dead by 8 days post infection with H7N9. The mice immunized by the split H7N9 vaccine were protected against infection with H7N9. Mice immunized by D/L-Tetra-Peptide Hydrogel adjuvant vaccines experienced shorter symptomatic periods and their micro-neutralization titers were higher than in the split H7N9 vaccine at 2 weeks post infection. The hemagglutinating inhibition (HI) titer in the L-Tetra-Peptide Hydrogel adjuvant vaccine group was higher than that in the split H7N9 vaccine 1 week and 2 weeks post infection. The HI titer in the D-Tetra-Peptide Hydrogel adjuvant vaccine group was higher than that in the split H7N9 vaccine at 2 weeks post infection. Conclusion: The D/L Tetra-Peptide Hydrogels increased the protection of the H7N9 vaccine and could be promising adjuvants for H7N9 vaccines against highly pathogenic H7N9 virus.

Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice.

Since mid-2016, the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic (HP) phenotype in China, raising many concerns about public health and poultry industry. The insertion of a “KRTA” motif at hemagglutinin cleavage site (HACS) occurred in the early stage of HP H7N9 variants. During the co-circulation, the HACS of HP-H7N9 variants were more polymorphic in birds and humans. Although HP-H7N9 variants, unlike the H5 subtype virus, exhibited the insertions of basic and non-basic amino acids, the underlying function of those insertions and substitutions remains unclear. The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China. Then, we generated six H7N9 viruses bearing the PEIPKGR/G, PEVPKGR/G, PEVPKRKRTAR/G, PEVPKGKRTAR/G, PEVPKGKRIAR/G, and PEVPKRKRR/G motifs. Interestingly, after the deletion of threonine and alanine (TA) at HACS, the H7N9 viruses manifested decreased thermostability and virulence in mice, and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence. By contrast, the insertion of non-basic amino acid isoleucine and alanine (IA) decreased the transmissibility in chickens and virulence in mice. Remarkably, the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens, suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice. The ongoing evolution of H7N9 increasingly threatens public health and poultry industry, so, its comprehensive surveillance and prevention of H7N9 viruses should be pursued.

Influenza A and D Viruses in Non-Human Mammalian Hosts in Africa: A Systematic Review and Meta-Analysis.

We conducted a systematic review and meta-analysis to investigate the prevalence and current knowledge of influenza A virus (IAV) and influenza D virus (IDV) in non-human mammalian hosts in Africa. PubMed, Google Scholar, Wiley Online Library and World Organisation for Animal Health (OIE-WAHIS) were searched for studies on IAV and IDV from 2000 to 2020. Pooled prevalence and seroprevalences were estimated using the quality effects meta-analysis model. The estimated pooled prevalence and seroprevalence of IAV in pigs in Africa was 1.6% (95% CI: 0–5%) and 14.9% (95% CI: 5–28%), respectively. The seroprevalence of IDV was 87.2% (95% CI: 24–100%) in camels, 9.3% (95% CI: 0–24%) in cattle, 2.2% (95% CI: 0–4%) in small ruminants and 0.0% (95% CI: 0–2%) in pigs. In pigs, H1N1 and H1N1pdm09 IAVs were commonly detected. Notably, the highly pathogenic H5N1 virus was also detected in pigs. Other subtypes detected serologically and/or virologically included H3N8 and H7N7 in equids, H1N1, and H3N8 and H5N1 in dogs and cats. Furthermore, various wildlife animals were exposed to different IAV subtypes. For prudent mitigation of influenza epizootics and possible human infections, influenza surveillance efforts in Africa should not neglect non-human mammalian hosts. The impact of IAV and IDV in non-human mammalian hosts in Africa deserves further investigation.

The viral distribution and pathological characteristics of BALB/c mice infected with highly pathogenic Influenza H7N9 virus

BackgroundThe highly pathogenic Influenza H7N9 virus is believed to cause multiple organ infections. However, there have been few systematic animal experiments demonstrating the virus distribution after H7N9 virus infection. The present study was carried out to investigate the viral distribution and pathological changes in the main organs of mice after experimental infection with highly pathogenic H7N9 virus.MethodsInfection of mice with A/Guangdong/GZ8H002/2017(H7N9) virus was achieved via nasal inoculation. Mice were killed at 2, 3, and 7 days post infection. The other mice were used to observe their illness status and weight changes. Reverse transcription polymerase chain reaction and viral isolation were used to analyse the characteristics of viral invasion. The pathological changes of the main organs were observed using haematoxylin and eosin staining and immunohistochemistry.ResultsThe weight of H7N9 virus-infected mice increased slightly in the first two days. However, the weight of the mice decreased sharply in the following days, by up to 20%. All the mice had died by the 8th day post infection and showed multiple organ injury. The emergence of viremia in mice was synchronous with lung infection. On the third day post infection, except in the brain, the virus could be isolated from all organs (lung, heart, kidney, liver, and spleen). On the seventh day post infection, the virus could be detected in all six organs. Brain infection was detected in all mice, and the viral titre in the heart, kidney, and spleen infection was high.ConclusionAcute diffuse lung injury was the initial pathogenesis in highly pathogenic H7N9 virus infection. In addition to lung infection and viremia, the highly pathogenic H7N9 virus could cause multiple organ infection and injury.

Single Dose of Bivalent H5 and H7 Influenza Virus-Like Particle Protects Chickens Against Highly Pathogenic H5N1 and H7N9 Avian Influenza Viruses.

Both H5N1 and H7N9 subtype avian influenza viruses cause enormous economic losses and pose considerable threats to public health. Bivalent vaccines against both two subtypes are more effective in control of H5N1 and H7N9 viruses in poultry and novel egg-independent vaccines are needed. Herein, H5 and H7 virus like particle (VLP) were generated in a baculovirus expression system and a bivalent H5+H7 VLP vaccine candidate was prepared by combining these two antigens. Single immunization of the bivalent VLP or commercial inactivated vaccines elicited effective antibody immune responses, including hemagglutination inhibition, virus neutralizing and HA-specific IgG antibodies. All vaccinated birds survived lethal challenge with highly pathogenic H5N1 and H7N9 viruses. Furthermore, the bivalent VLP significantly reduced viral shedding and virus replication in chickens, which was comparable to that observed for the commercial inactivated vaccine. However, the bivalent VLP was better than the commercial vaccine in terms of alleviating pulmonary lesions caused by H7N9 virus infection in chickens. Therefore, our study suggests that the bivalent H5+H7 VLP vaccine candidate can serve as a critical alternative for the traditional egg-based inactivated vaccines against H5N1 and H7N9 avian influenza virus infection in poultry.

Deciphering transmission dynamics and spillover of avian influenza viruses from avian species to swine populations globally

Genome sequences of eleven avian influenza virus (AIV) subtypes have been reported in swine populations from seven countries until August 2020. To unravel the transmission dynamics and spillover events of AIVs from avian reservoirs to swine, full-length hemagglutinin (HA) sequences of AIV subtypes (n = 11) reported from various avian species and swine were retrieved from the ‘Influenza Research Database’. Phylogenetic analysis identified closely related avian and swine AIV sequences suggesting potential spillover events from multiple domestic and wild avian species, including chicken, duck, pigeon, goose, quail, and aquatic birds to swine. Furthermore, N-linked glycosylation analysis of these closely related AIV sequences supported the possibility of multiple spillover events of highly pathogenic H5N1 and low pathogenic H9N2 viruses from various avian species to swine. The principal coordinate analysis further validated these findings for H5N1 and H9N2 viruses; however, spillover events of the other nine AIV subtypes were limited. Interestingly, the presence of potential mammalian adaptation markers, particularly in some of the swine H5N1, H7N9, and H9N2 viruses, suggested that these viruses may have already adapted in swine. The occurrence and circulation of these AIVs in swine, especially the H5N1 and H9N2 viruses with numerous spillover events from the avian reservoirs to swine, pose a significant threat in terms of their reassortment with endemic swine viruses or circulating human influenza viruses within the swine which may facilitate the emergence of a novel influenza virus strain with pandemic potential.

H7N9 influenza virus-like particle based on BEVS protects chickens from lethal challenge with highly pathogenic H7N9 avian influenza virus

H7N9 avian influenza virus poses a dual threat to both poultry industry and public health. Therefore, it is highly urgent to develop an effective vaccine to reduce its pandemic potential. Virus-like particles (VLP) represent an effective approach for pandemic vaccine development. In this study, a recombinant baculovirus co-expressing the HA, NA and M1 genes of the H7N9 virus was constructed for generation of H7N9 VLP. Single immunization of chickens with 15 μg of the VLP or the commercial whole virus inactivated vaccine stimulates high hemagglutination inhibition, virus neutralizing and HA-specific IgY antibodies. Moreover, the antiserum had a good cross-reactivity with H7N9 field strains isolated in different years. Within 14 days after a lethal challenge with highly pathogenic (HP) H7N9 virus, no clinical symptoms and death were observed in the vaccinated chickens, and no virus was recovered from the organs. Compared to the non-vaccinated chickens, H7N9 VLP significantly reduced the proportion of animals shedding virus. Only 30 % of the VLP-vaccinated birds shed virus, whereas virus shedding was detected in 50 % of the chickens immunized with the commercial vaccine. Moreover, both vaccines dramatically alleviated pulmonary lesions caused by HP H7N9 virus, with a greater degree observed for the VLP. Altogether, our results indicated that the H7N9 VLP vaccine candidate confers a complete clinical protection against a lethal challenge with HP H7N9 virus, significantly inhibits virus shedding and abolishes viral replication in chickens. The VLP generated in this study represents a promising alternative strategy for the development of novel H7N9 avian influenza vaccines for chickens.

Optimizing the early detection of low pathogenic avian influenza H7N9 virus in live bird markets.

In Southeast Asia, surveillance at live bird markets (LBMs) has been identified as crucial for detecting avian influenza viruses (AIV) and reducing the risk of human infections. However, the design of effective surveillance systems in LBMs remains complex given the rapid turn-over of poultry. We developed a deterministic transmission model to provide guidance for optimizing AIV surveillance efforts. The model was calibrated to fit one of the largest LBMs in northern Vietnam at high risk of low pathogenic H7N9 virus introduction from China to identify the surveillance strategy that optimizes H7N9 detection. Results show that (i) using a portable diagnostic device would slightly reduce the number of infected birds leaving the LBM before the first detection, as compared to a laboratory-based diagnostic strategy, (ii) H7N9 detection could become more timely by sampling birds staying overnight, just before new susceptible birds are introduced at the beginning of a working day, and (iii) banning birds staying overnight would represent an effective intervention to reduce the risk of H7N9 spread but would decrease the likelihood of virus detection if introduced. These strategies should receive high priority in Vietnam and other Asian countries at risk of H7N9 introduction.