Pathogenic Avian Influenza H5N1 Research Articles

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.

Impact of Individual Viral Gene Segments from Influenza A/H5N8 Virus on the Protective Efficacy of Inactivated Subtype-Specific Influenza Vaccine

Since its emergence in 2014, the highly pathogenic avian influenza H5N8 virus has continuously and rapidly spread worldwide in the poultry sector resulting in huge economic losses. A typical inactivated H5N8 vaccine is prepared using the six internal genes from A/PR8/1934 (H1N1) and the two major antigenic proteins (HA and NA) from the circulating H5N8 strain with the HA modified to a low pathogenic form (PR8HA/NA-H5N8). The contribution of the other internal proteins from H5N8, either individually or in combination, to the overall protective efficacy of PR8-based H5N8 vaccine has not been investigated. Using reverse genetics, a set of PR8-based vaccines expressing the individual proteins from an H5N8 strain were rescued and compared to the parent PR8 and low pathogenic H5N8 strains and the commonly used PR8HA/NA-H5N8. Except for the PR8-based vaccine strains expressing the HA of H5N8, none of the rescued combinations could efficiently elicit virus-neutralizing antibodies. Compared to PR8, the non-HA viral proteins provided some protection to infected chickens six days post infection. We assume that this late protection was related to cell-based immunity rather than antibody-mediated immunity. This may explain the slight advantage of using full low pathogenic H5N8 instead of PR8HA/NA-H5N8 to improve protection by both the innate and the humoral arms of the immune system.

Multiple Reassortants of H5N8 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Viruses Detected in South Korea during the Winter of 2020-2021.

During October 2020–January 2021, we isolated a total of 67 highly pathogenic avian influenza (HPAI) H5N8 viruses from wild birds and outbreaks in poultry in South Korea. We sequenced the isolates and performed phylogenetic analysis of complete genome sequences to determine the origin, evolution, and spread patterns of these viruses. Phylogenetic analysis of the hemagglutinin (HA) gene showed that all the isolates belong to H5 clade 2.3.4.4 subgroup B (2.3.4.4b) and form two distinct genetic clusters, G1 and G2. The cluster G1 was closely related to the 2.3.4.4b H5N8 HPAI viruses detected in Europe in early 2020, while the cluster G2 had a close genetic relationship with the 2.3.4.4b H5N8 viruses that circulated in Europe in late 2020. A total of seven distinct genotypes were identified, including five novel reassortants carrying internal genes of low pathogenic avian influenza viruses. Our Bayesian discrete trait phylodynamic analysis between host types suggests that the viruses initially disseminated from migratory waterfowl to domestic duck farms in South Korea. Subsequently, domestic duck farms most likely contributed to the transmission of HPAI viruses to chicken and minor poultry farms, highlighting the need for enhanced, high levels of biosecurity measures at domestic duck farms to effectively prevent the introduction and spread of HPAI.

The virulence modulator PA-X protein has minor effect on the pathogenicity of the highly pathogenic H7N9 avian influenza virus in mice

PA-X is a novel discovered accessory protein encoded by the PA mRNA of the influenza A virus. Accumulated studies have demonstrated the crucial role of this protein in regulating the virulence of various subtypes of influenza virus, including H1N1, H5N1, H9N2, H1N2, H3N8 and H3N2 virus. However, the role of PA-X protein in regulating the virulence of the highly pathogenic avian H7N9 virus was unknown. In this study, we firstly generated two recombinant H7N9 viruses which have lower PA-X expression level than the parental H7N9 virus. We then systematically compared their difference in virus replication, polymerase activity, virulence and virus-induced host immune responses in mice. The results showed that the PA-X deficient viruses significantly increased viral replication in madin darby canine kidney cells and slightly increased viral replication in mouse lung. In addition, loss of PA-X expression significantly increased viral polymerase activity and alleviated the host-shutoff activity mediated by the parental PA protein. However, in contrast with the usual function of PA-X in regulating the virulence in different subtype influenza virus, no obvious effect on viral virulence in mice was observed by H7N9 PA-X protein. Furthermore, among the 12 kinds of cytokines and 2 kinds of complement derived components that we tested, the PA-X deficiency viruses only induced significantly higher expression levels of MX1 than the parental virus. Altogether, these results showed that PA-X has little effect on viral virulence and viral induced innate immune response of the H7N9 subtype virus. Our study adds further information for the growing understanding of the complexity of PA-X in regulating viral virulence and host innate immune response of different influenza virus.

Aerosolized Exposure to H5N1 Influenza Virus Causes Less Severe Disease Than Infection via Combined Intrabronchial, Oral, and Nasal Inoculation in Cynomolgus Macaques.

Infection with highly pathogenic avian H5N1 influenza virus in humans often leads to severe respiratory disease with high mortality. Experimental infection in non-human primates can provide additional insight into disease pathogenesis. However, such a model should recapitulate the disease symptoms observed in humans, such as pneumonia and inflammatory cytokine response. While previous studies in macaques have demonstrated the occurrence of typical lesions in the lungs early after infection and a high level of immune activation, progression to severe disease and lethality were rarely observed. Here, we evaluated a routinely used combined route of infection via intra-bronchial, oral, and intra-nasal virus inoculation with aerosolized H5N1 exposure, with or without the regular collection of bronchoalveolar lavages early after infection. Both combined route and aerosol exposure resulted in similar levels of virus replication in nose and throat and similar levels of immune activation, cytokine, and chemokine release in the blood. However, while animals exposed to H5N1 by combined-route inoculation developed severe disease with high lethality, aerosolized exposure resulted in less lesions, as measured by consecutive computed tomography and less fever and lethal disease. In conclusion, not virus levels or immune activation, but route of infection determines fatal outcome for highly pathogenic avian H5N1 influenza infection.

Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

BackgroundBoth the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections.MethodsAccording to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction.ResultsIn the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism.ConclusionsCompared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

Genetic characterization of highly pathogenic avian Influenza H5Nx clade 2.3.4.4b reveals independent introductions in nigeria.

Among recurrent sanitary emergencies able to spread rapidly worldwide, avian influenza is one of the main constraints for animal health and food security. In West Africa, Nigeria has been experiencing repeated outbreaks of different strains of avian influenza virus (AIV) since 2006 and is also recognized as a hot spot in the region for the introduction of emerging strains by migratory wild birds. Here, we generated complete genomes of 20 highly pathogenic avian influenza (HPAI) H5N8 viruses collected during active surveillance in Nigerian live bird markets (LBM) and from outbreaks reported in the country between 2016 and 2019. Phylogenetic analysis reveals that the Nigerian viruses cluster into four separate genetic groups within HPAI H5 clade 2.3.4.4b. The first group includes 2016-2017 Nigerian viruses with high genetic similarity to H5N8 viruses detected in Central African countries, while the second includes Nigerian viruses collected both in LBM and poultry farms (2018-2019), as well as in Cameroon, Egypt and Siberia. A natural reassortant strain identified in 2019 represents the third group: H5N8 viruses with the same gene constellation were identified in 2018 in South Africa. Finally, the fourth introduction represents the first detection in the African continent of the H5N6 subtype, which is related to European viruses. Bayesian phylogeographic analyses confirmed that the four introductions originated from different sources and provide evidence of the virus spread within Nigeria, as well as diffusion beyond its borders. The multiple epidemiological links between Nigeria, Central and Southern African countries highlight the need for harmonized and coordinated surveillance system to control AIV impact. Improved surveillance at the Wetlands, LBMs and early warning of outbreaks are crucial for prevention and control of AIV, which can be potentially zoonotic and be a threat to human health.

Elicitation of Highly Pathogenic Avian Influenza H5N1 M2e and HA2-Specific Humoral and Cell-Mediated Immune Response in Chicken Following Immunization With Recombinant M2e-HA2 Fusion Protein.

The study was aimed to evaluate the elicitation of highly pathogenic avian influenza (HPAI) virus (AIV) M2e and HA2-specific immunity in chicken to develop broad protective influenza vaccine against HPAI H5N1. Based on the analysis of Indian AIV H5N1 sequences, the conserved regions of extracellular domain of M2 protein (M2e) and HA2 were identified. Synthetic gene construct coding for M2e and two immunodominant HA2 conserved regions was designed and synthesized after codon optimization. The fusion recombinant protein (~38 kDa) was expressed in a prokaryotic system and characterized by Western blotting with anti-His antibody and anti-AIV polyclonal chicken serum. The M2e–HA2 fusion protein was found to be highly reactive with known AIV-positive and -negative chicken sera by ELISA. Two groups of specific pathogen-free (SPF) chickens were immunized (i/m) with M2e synthetic peptide and M2e–HA2 recombinant protein along with one control group with booster on the 14th day and 28th day with the same dose and route. Pre-immunization sera and whole blood were collected on day 0 followed by 3, 7, 14, 21, and 28 days and 2 weeks after the second booster (42 day). Lymphocyte proliferation assay by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method revealed that the stimulation index (SI) was increased gradually from days 0 to 14 in the immunized group (p < 0.05) than that in control chicken. Toll-like receptor (TLR) mRNA analysis by RT-qPCR showed maximum upregulation in the M2e–HA2-vaccinated group compared to M2e- and sham-vaccinated groups. M2e–HA2 recombinant protein-based indirect ELISA revealed that M2e–HA2 recombinant fusion protein has induced strong M2e and HA2-specific antibody responses from 7 days post-primary immunization, and then the titer gradually increased after booster dose. Similarly, M2e peptide ELISA revealed that M2e–HA2 recombinant fusion protein elicited M2e-specific antibody from day 14 onward. In contrast, no antibody response was detected in the chicken immunized with synthetic peptide M2e alone or control group. Findings of this study will be very useful in future development of broad protective H5N1 influenza vaccine targeting M2e and HA2.

Molecular Characterizations of a High Pathogenic Avian Influenza H5N8 in Iraq

High pathogenicity avian influenza subtype H5N8 viruses were detected in different areas in Iraq at thelast 2017 and early 2018 and 2019 and the disease was detected clinically. This disease is responsible forhigh economic losses for poultry industry and threat human health, so that, this study was conducted formolecular detection, characterization and phylogenic analysis of avian influenza in Iraq. AI subtype H5N8 isan infectious disease primarily in birds and responsible for severe respiratory illness which associated witha high percentage of morbidity and mortality in wild and domestic birds. During this study one hundred fiftydifferent samples including (trachea, larynx and lung) were collected from different areas of broiler chickenfrom Baghdad and its surrounding regions during winter 2019. Avian influenza virus subtype H5N8 wasdetected by using real time RT-PCR technique, and specific kits (KyltR Germany) for AIV subtypes H5 andN8 were used respectively. The results revealed that (15) samples out of (150) collected samples were gavepositive results for avian influenza H5 these positive samples were prepared for a second step of detectionby RT-PCR test specific for N8 subtype the results revealed that only (12) out of (15) tested samples werepositive for H5N8 (8%).Genetic sequencing of isolates and phylogenic analysis of three selected isolates of(H5N8) bellowing to different areas indicate that all strains bellowing to H5 class (2.3.4.4) high pathogenicavian influenza revealed that they are closely related to Egyptian strain (A/ duck/ Egypt/ F446/2017.H5N8-MH893737.1) (with 97.6% identity). Analysis of the mono-basic amino acid (PQIEPR / GLF) atthe hemagglutinin cleavage site revealed there is no deletion of the stalk region with the neuraminidaseindicated that the isolates is a typical HPAI strain (A / duck / Egypt / F446/2017). The similarity of thenucleotide sequence analysis of hemagglutinin gene revealed that there was a high homology (97.6%) to thatof A/duck/Egypt/F446/2017 H5N8 .

Molecular characterization of low pathogenic avian influenza H9N2 virus during co-circulation with newly-emerged highly pathogenic avian influenza H5N8 virus in Iran.

Identification of molecular characteristics of low pathogenic avian influenza (LPAI) H9N2 virus provides insights into the evolution of this subtype due to the modulation of genomic characteristics in co-circulation with another subtype. The present study aimed to analyze the molecular and phylogenetic characteristics of the current LPAI H9N2 virus in characteristics of internal proteins are crucial for the adaptations of AIVs viruses to a new host. Since H9N2 is indigenous among poultry, continuous monitoring of viral genetic changes is needed for risk assessment of potential transmissibility to human population and emergence of new reassortant virus. domestic poultry during the emergence of new highly pathogenic avian influenza (HPAI) H5N8 virus in Iran. To this end, deep sequencing of LPAI H9N2 virus was performed on Illumina MiSeq sequencing platform and the complete sequences of avian influenza viruses were obtained from GISAID EpiFlu database. Phylogenetic analysis of the surface and internal gene segments showed that the H9N2 2018 virus was closely related to Pakistani H9N2 isolates. HA cleavage site motif sequence of the Iranian isolate was 317KSSR GLF323. The A/chicken/Iran/1/2018 H9N2 strain carried the amino acid substitution (Q216L), which is a mutation correlated with a shift in the affinity of the HA from avian type sialic receptors to human type. Besides surface glycoproteins, molecular. Keywords: A/H9N2; molecular characterization; A/H5N8; co-circulation; A/H5N1; Illumina MiSeq.

Phylogenetic analysis of HPAI H5N1 virus from duck swab specimens in Indonesia.

Objective:A phylogenetic study was carried out on the avian influenza virus (AIV) isolated from a disease outbreak in Sidenreng Rappang Regency, South Sulawesi, Indonesia, in 2018.Material and Methods:Oropharyngeal swabs and organ samples were obtained from ducks that showed clinical symptoms: torticollis, fascial edema, neurological disorders, the corneas appear cloudy, and death occurs less than 1 day after symptoms appear. In this study, isolate A/duck/Sidenreng Rappang/07180110-11/2018 from duck was sequenced and characterized.Results:It was found that each gene segment of the virus has the highest nucleotide homology to the Indonesian highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.2.1c. Multiple alignments of the sample Hemagglutinin (HA) gene with the avian influenza references virus showed that the pattern of amino acid arrangement in the cleavage site PQRERRRK-RGLF is the characteristic of the HPAI virus. In addition, the HA gene contained Q222 (glutamine) and G224 (glycine), signifying a high affinity to avian receptor binding specificity (SA α2,3 Gal). Furthermore, there was no genetic reassortment of this virus based on the phylogenetic analysis of HA, NA, PB1, PB2, PA, NP, M, and NS genes.Conclusion:The HPAI H5N1 clade 2.3.2.1c virus was identified in duck farms in South Sulawesi, Indonesia.

Evaluation of the avian influenza surveillance system in Enugu State Nigeria, 2015-2017

Introduction: the resurgence of highly pathogenic avian influenza (HPAI) H5N1 was reported in Nigeria in 2014. The isolation of a reassortants strain of influenza A subtype H5N8 in 2017 fueled speculation of a possible the emergence of a novel influenza strain with no prior human or animal immunity. About 3.4% of poultry in Enugu State were affected between 2015-2017. Hence, the need for a comprehensive reviews of the avian influenza (AI) surveillance system in Enugu State, Nigeria.

Avian influenza H5N1: still a pandemic threat?

Highly pathogenic avian influenza H5N1 viruses have become endemic in global poultry populations over the past 25 years and pose an ongoing public health threat. Although the incidence of human cases has declined, viruses from the H5N1 lineage can now be found in poultry throughout Asia, the Middle East and Africa, in addition to causing outbreaks in Europe and the Americas. The recent emergence and spread of reassortant H5Nx viruses, resulting in regional poultry outbreaks, has increased the risk for further evolution of these viruses and possible avian-to-human transmission. Ongoing surveillance and pandemic preparedness for H5N1 and other avian influenza viruses of public health concern are warranted.

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A reassortant highly pathogenic avian influenza H5N6 virus originating from the wildbird-origin H5N6 and the poultry H9N2/H7N9 viruses in Xinjiang, China

The asian lineage of H5, H7, and H9 avian influenza viruses (AIVs) is the most prevalent in the world, and the co-existence of H5, H7, and H9 viruses provide an opportunity to generate some novel reassortants. Herein we report our isolation of two H5N6 virus strains (named XJ-H5N6) from domestic geese in live poultry markets (LPMs) in Urumqi during the winter of 2016-2017, and analysis of the viral genetic mutations, pathogenicity, reassortant, and origin. BLAST analysis revealed that the HA, NA, MP, NP, and NS of XJ-H5N6 had the highest sequence identity (98.5-99.5%) with those 5 genes from H5N6 highly pathogenic avian influenza virus (HPAIV) isolated from wild birds in Ningxia (NX-WB-H5N6); the viral PA and PB1 had the highest sequence identity (98.6% and 98.9%) with those 2 genes from HPAIV strain A/Environment/Chongqing/45373/2015 (H5N6) (CQ-EN-H5N6); while the viral PB2 had the highest sequence identity (99.0%) with this gene from low pathogenic avian influenza virus (LPAIV) strain A/environment/Jiangsu/01.20 TCCX004/2015 (H9N2). Except for the PB2, phylogenetic analysis showed that the other 7 viral genes were clustered together with NX-WB-H5N6 and CQ-EN-H5N6, while the viral PB2 was clustered together with the poultry H9N2/H7N9 viruses. The viral HA protein contained multiple basic amino acid residues (RRRKR/GL) at the cleavage site which represents HPAIV. Additionally, the viral multiple mutations could enhance the viral virulence and the capacity of cross-species transmission. Our data indicate that XJ-H5N6 has been generated by the reassortant of H5N6 HPAIV from wild birds in Ningxia and H9N2/H7N9 LPAIV in poultry. The results of this study may help us understand the viral adaptation and transmission patterns from wild birds to poultry.

A single-amino-acid mutation at position 225 in hemagglutinin attenuates H5N6 influenza virus in mice

The highly pathogenic avian influenza H5N6 viruses are widely circulating in poultry and wild birds, and have caused 38 human infections including 21 deaths; however, the key genetic determinants of the pathogenicity of these viruses have yet to be fully investigated. Here, we characterized two H5N6 avian influenza viruses––A/duck/Guangdong/S1330/2016 (GD/330) and A/environment/Fujian/S1160/2016 (FJ/160)––that have similar viral genomes but differ markedly in their lethality in mice. GD/330 is highly pathogenic with a 50% mouse lethal dose (MLD50) of 2.5 log10 50% egg infectious doses (EID50), whereas FJ/160 exhibits low pathogenicity with an MLD50 of 7.4 log10 EID50. We explored the molecular basis for the difference in virulence between these two viruses. By using reverse genetics, we created a series of reassortants and mutants in the GD/330 background and assessed their virulence in mice. We found that the HA gene of FJ/160 substantially attenuated the virulence of GD/330 and that the mutation of glycine (G) to tryptophan (W) at position 225 (H3 numbering) in HA played a key role in this function. We further found that the amino acid mutation G225W in HA decreased the acid and thermal stability and increased the pH of HA activation, thereby attenuating the H5N6 virus in mice. Our study thus identifies a novel molecular determinant in the HA protein and provides a new target for the development of live attenuated vaccines and antiviral drugs against H5 influenza viruses.

INFLUENZA : A COMPLETE OVERVIEW

● At first haemophilus influenza was considered as the causative agent for influenza but after the research it has been found that it caused various other types of infections but not influenza. Influenza was caused by some flu virus which was first isolated from pigs in 1931 and from humans in 1933.(4) ● The 1918 influenza pandemic was the most severe pandemic in recent history it was caused by an H1N1 virus with the genes of avian origin although there is not universal consensus regarding where the virus originated it spread worldwide during 1918 and 1919. (3) ● It was first identified in military personnel in spring 1980 it is estimated that about 500 million people or one third of the world's population became infected with this virus.(3) ● The number of deaths was estimated to be at least 50 million worldwide with about 6,75,000 deaths occurring in the United States. (3) ● Mortality was high in people younger than five years old ,20 to 40 years old and in 65 years and older. The high mortality in healthy people including those in the 20 to 40 year age group was a unique feature of this pandemic. (3) ● There was no vaccine to protect against influenza infection and no antibiotics to treat secondary bacterial infections that can be associated with influenza infection control efforts worldwide were limited to non-pharmaceutical intervention such as isolation quarantine good personal hygiene use of disinfectants and limitations of public gathering which were applied unevenly.(3) ● In the northern and southern parts of the world outbreaks occur mainly in the winter while around the equator outbreaks may occur at any time of the year .In the northern and southern parts of the world outbreaks occur mainly in the winter while around the equator outbreaks may occur at any time of the year.(7) ● In the 20th century three influenza pandemics occurred Spanish influenza in 1918 where the death toll ranged from 17- 200 million deaths, Asian influenza in 1957- 2 million deaths and Hong Kong influenza in 1968 -1 million deaths. ● The world health organisation declared an outbreak of a new type of influenza A or H1N1 to be a pandemic in June 2009. ● influenza may also affect other animals including pig horses and birds.(9) ● The name “influenza” originated in 15th century Italy, from an epidemic attributed to “influence of the stars.” The first pandemic that fits the description of influenza was in 1580. At least four pandemics of influenza occurred in the 19th century, and three occurred in the 20th century. The pandemic of “Spanish” influenza in 1918–1919 caused an estimated 21 million deaths worldwide. The first pandemic of the 21st century occurred in 2009–2010. Historically, influenza viruses of three HA subtypes (H1, H2 and H3) have acquired the ability to be transmitted efficiently between humans. Currently, influenza viruses of the H1 and H3 subtype co-circulate in humans, however influenza viruses of the H2, H5, H6, H7 and H9 subtype are also considered to represent a pandemic threat. In 1997, a large outbreak of highly pathogenic avian influenza (HPAI) H5N1 virus in poultry in Hong Kong resulted in the first documented cases of direct transmission of HPAI H5N1 virus from poultry to humans, with a fatal outcome in 6 out of 18 cases [17]. As a result, this outbreak warranted the mass culling of 1.5 million chickens. In 2003, a large outbreak of an HPAI H7N7 virus in poultry in the Netherlands resulted in 89 cases of human infections, one of which was fatal [21]. HPAI H7N7 virus displayed an unusual tissue tropism; the virus targeted the conjunctiva, resulting in conjunctivitis, a symptom rarely reported for other influenza virus subtypes.(1)

Highly Pathogenic Avian Influenza H5N8 in Poland in 2019-2020.

IntroductionRepeated incursions of highly pathogenic avian influenza virus (HPAIV) H5 subtype of Gs/GD lineage pose a serious threat to poultry worldwide. We provide a detailed analysis of the spatio-temporal spread and genetic characteristics of HPAIV Gs/GD H5N8 from the 2019/20 epidemic in Poland.Material and methodsSamples from poultry and free-living birds were tested by real-time RT-PCR. Whole genome sequences from 24 (out of 35) outbreaks were generated and genetic relatedness was established. The clinical status of birds and possible pathways of spread were analysed based on the information provided by veterinary inspections combined with the results of phylogenetic studies.ResultsBetween 31 December 2019 and 31 March 2020, 35 outbreaks in commercial and backyard poultry holdings and 1 case in a wild bird were confirmed in nine provinces of Poland. Most of the outbreaks were detected in meat turkeys and ducks. All characterised viruses were closely related and belonged to a previously unrecognised genotype of HPAIV H5N8 clade 2.3.4.4b. Wild birds and human activity were identified as the major modes of HPAIV spread.ConclusionThe unprecedentedly late introduction of the HPAI virus urges for re-evaluation of current risk assessments. Continuous vigilance, strengthening biosecurity and intensifying surveillance in wild birds are needed to better manage the risk of HPAI occurrence in the future.

Virulence and transmissibility of highly pathogenic avian influenza H5N6 viruses in the ferret model

Background: Highly pathogenic avian influenza (HPAI) H5 viruses have exhibited substantial geographic spread throughout Asia, Europe, Africa, and recently North America since their first detection in 1996. Accumulation of mutations in the HA gene has resulted in several phylogenetic clades, while reassortment with other avian influenza viruses has led to the emergence of new virus subtypes (H5Nx), notably H5N2, H5N6, and H5N8. H5Nx viruses represent a threat to both the poultry industry and human health. As reassortment and accumulation of mammalian adaptations may increase virus fitness and virulence, continuous in vivo evaluation of H5Nx viruses is necessary in aiding pandemic preparedness efforts. Methods and materials: Ferrets were inoculated with HPAI H5N6 virus (A/Sichuan/26221/2014, A/Yunnan/14563/2015, or A/duck/Bangladesh/19D770/2017) and evaluated for clinical signs and symptoms of infection, replication throughout the respiratory tract, extra pulmonary virus spread, and virus transmission. A North American HPAI H5N2 virus (A/turkey/Minnesota/10915/2015) was used for comparison. Levels of virus released into the air by infected ferrets was evaluated using a cyclone bioaerosol sampler. Replication kinetics of all viruses in the human bronchial epithelial cell line, Calu-3, was evaluated. Results: Tested H5N6 viruses were more pathogenic in ferrets compared to the H5N2 virus. However, pathogenicity profiles of the H5N6 viruses varied by strain, from a mild infection with only sporadic virus dissemination beyond the respiratory tract, to a severe and fatal infection. In vitro evaluation of H5Nx virus replication in Calu-3 cells and the presence of mammalian adaptation markers in key genes supported these findings. Limited transmission between co-housed ferrets was observed with the H5N6 viruses but not the H5N2 virus, which is in agreement with generally low levels of aerosolized virus exhaled from virus-inoculated ferrets. Conclusion: HPAI H5 viruses bearing the N6 subtype neuraminidase and mammalian adaptation markers in the polymerase genes are capable of causing lethal infection in ferrets as well as transmitting to uninfected animals in a direct contact setting. The heterogeneity in mammalian virulence among H5N6 viruses underscores the necessity of in vivo evaluation of H5Nx viruses as they continue to evolve and pose a threat to human health.

Type IIb Heat Labile Enterotoxin B Subunit as a Mucosal Adjuvant to Enhance Protective Immunity against H5N1 Avian Influenza Viruses.

Human infections with highly pathogenic avian influenza H5N1 viruses persist as a major global health concern. Vaccination remains the primary protective strategy against H5N1 and other novel avian influenza virus infections. We investigated the use of E. coli type IIb heat labile enterotoxin B subunit (LTIIb-B5) as a mucosal adjuvant for intranasal immunizations with recombinant HA proteins against H5N1 avian influenza viruses. Use of LTIIb-B5 adjuvant elicited more potent IgG, IgA, and neutralizing antibody titers in both sera and bronchoalveolar lavage fluids, thus increasing protection against lethal virus challenges. LTIIb-B5 mucosal adjuvanticity was found to trigger stronger Th17 cellular response in spleen lymphocytes and cervical lymph nodes. Studies of anti-IL-17A monoclonal antibody depletion and IL-17A knockout mice also suggest the contribution from Th17 cellular response to anti-H5N1 protective immunity. Our results indicate a link between improved protection against H5N1 live virus challenges and increased Th17 response due to the use of LTIIb-B5 mucosal adjuvant with HA subunit proteins.