Structural and Functional Studies of Influenza Virus A/H6 Hemagglutinin.
ABSTRACT: In June 2013, the first human infection by avian influenza A(H6N1) virus was reported in Taiwan. This incident raised the concern for possible human epidemics and pandemics from H6 viruses. In this study, we performed structural and functional investigation on the hemagglutinin (HA) proteins of the human-infecting A/Taiwan/2/2013(H6N1) (TW H6) virus and an avian A/chicken/Guangdong/S1311/2010(H6N6) (GD H6) virus that transmitted efficiently in guinea pigs. Our results revealed that in the presence of HA1 Q226, the triad of HA1 S137, E190 and G228 in GD H6 HA allows the binding to both avian- and human-like receptors with a slight preference for avian receptors. Its conservation among the majority of H6 HAs provides an explanation for the broader host range of this subtype. Furthermore, the triad of N137, V190 and S228 in TW H6 HA may alleviate the requirement for a hydrophobic residue at HA1 226 of H2 and H3 HAs when binding to human-like receptors. Consequently, TW H6 HA has a slight preference for human receptors, thus may represent an intermediate towards a complete human adaptation. Importantly, the triad observed in TW H6 HA is detected in 74% H6 viruses isolated from Taiwan in the past 14 years, suggesting an elevated threat of H6 viruses from this region to human health. The novel roles of the triad at HA1 137, 190 and 228 of H6 HA in binding to receptors revealed here may also be used by other HA subtypes to achieve human adaptation, which needs to be further tested in laboratory and closely monitored in field surveillance.
Project description:The receptor-binding specificity of influenza A viruses is a major determinant for the host tropism of the virus, which enables interspecies transmission. In 2013, the first human case of infection with avian influenza A (H6N1) virus was reported in Taiwan. To gather evidence concerning the epidemic potential of H6 subtype viruses, we performed comprehensive analysis of receptor-binding properties of Taiwan-isolated H6 HAs from 1972 to 2013. We propose that the receptor-binding properties of Taiwan-isolated H6 HAs have undergone three major stages: initially avian receptor-binding preference, secondarily obtaining human receptor-binding capacity, and recently human receptor-binding preference, which has been confirmed by receptor-binding assessment of three representative virus isolates. Mutagenesis work revealed that E190V and G228S substitutions are important to acquire the human receptor-binding capacity, and the P186L substitution could reduce the binding to avian receptor. Further structural analysis revealed how the P186L substitution in the receptor-binding site of HA determines the receptor-binding preference change. We conclude that the human-infecting H6N1 evolved into a human receptor preference.
Project description:Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from ?2-3-linked (avian-like) to ?2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds ?2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and human receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. This binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics.
Project description:H6 avian influenza viruses (AIVs) have a worldwide distribution, and they pose a potential concern for public health. In Taiwan, H6 AIVs have circulated in domestic chickens for more than 40 years, and certain strains have crossed the species barrier to infect mammals. With the goal of containing the disease, there is a pressing need to develop a safe and effective vaccine for pandemic preparedness. In this study, we prepared a virus-like particle (VLP) that consisted of the hemagglutinin (HA) and matrix protein 1 (M1) derived from a H6 AIV as a vaccine antigen, and we examined the immunogenicity and protective efficacy when combined with an adjuvant in a chicken model. Full-length HA and M1 protein genes were cloned and expressed using a baculovirus expression system, and VLPs were purified from the supernatant of insect cell cultures. We performed nanoparticle-tracking analysis and transmission electron microscopy to validate that the particle structure and properties resembled the native virions. In animal experiments, specific-pathogen-free chickens that received the H6 VLPs in combination with an adjuvant showed superior H6N1 virus-specific serum IgG and hemagglutination-inhibition antibody responses, which lasted more than 112 days. Following the H6N1 viral challenge, the vaccinated chickens showed reduced viral replication in the lungs, kidneys and conjunctival/cloacal shedding. The antibodies induced in the chickens by the vaccine were able to cross-react with the H6N1 human isolate and drifted avian H6N1 isolates. In summary, the H6 VLP vaccine elicited superb immunogenicity in vivo, and the use of an adjuvant further enhanced the antiviral protective efficacy. This vaccine formulation could potentially be used to manage H6 influenza virus infections in chickens.
Project description:A bi-cistronic baculovirus expression vector was constructed to facilitate the expression, detection, and isolation of the hemagglutinin (HA) fragment HA1 of H6N1 avian influenza virus (AIV) in an insect and a culture of its cells. In this construct, the GP67sp signal peptide promoted the secretion of the recombinant protein into the culture medium, and improved protein expression and purification. Enhanced green fluorescent protein, co-expressed through an internal ribosome entry site, served as a visible reporter for protein expression detection. The hemolymph of Spodoptera litura larvae infected with the bi-cistronic baculovirus was collected for the purification of the recombinant HA1, which was found to be glycosylated, and monomeric and trimeric forms of the recombinant HA1 were identified. Proteins expressed in both the cell culture and larvae served as effective subunit vaccines for the production of antiserum against HA. The antiserum recognized the H6 subtype of AIV but not the H5 subtype.
Project description:The A/teal/Hong Kong/W312/97 (H6N1) influenza virus and the human H5N1 and H9N2 influenza viruses possess similar genes encoding internal proteins, suggesting that H6N1 viruses could become novel human pathogens. The molecular epidemiology and evolution of H6 influenza viruses were characterized by antigenic and genetic analyses of 29 H6 influenza viruses isolated from 1975 to 1981 and 1997 to 2000. Two distinct groups were identified on the basis of their antigenic characteristics. Phylogenetic analysis revealed that all H6N1 viruses isolated from terrestrial poultry in 1999 and 2000 are closely related to A/teal/Hong Kong/W312/97 (H6N1), and the nucleotide sequences of these viruses and of A/Hong Kong/156/97 (H5N1) were more than 96% homologous. The hemagglutinin (HA) of the 1999 and 2000 terrestrial viruses does not have multiple basic amino acids at the site of cleavage of HA1 to HA2; however, a unique insertion of aspartic acid in HA1 between positions 144 and 145 (H3 numbering) was found. The neuraminidase of these terrestrial H6N1 viruses has a deletion of 19 amino acids characteristic of A/Hong Kong/156/97 (H5N1). Evolutionary analysis suggested that these H6N1 viruses coevolved with A/quail/Hong Kong/G1/97-like H9N2 viruses and became more adapted to terrestrial poultry. These terrestrial 1999 and 2000 A/teal/Hong Kong/W312/97 (H6N1)-like viruses, along with the H9N2 viruses, could have been involved in the genesis of the pathogenic H5N1 influenza viruses of 1997. The presence of H6N1 viruses in poultry markets in Hong Kong that possess seven of the eight genes of the A/Hong Kong/156/97 (H5N1) virus raises the following fundamental questions relevant to influenza pandemic preparedness: could the pathogenic H5N1 virus reemerge and could the H6N1 viruses directly cross the species barrier to mammals?
Project description:The H6N1 avian influenza virus has circulated in Taiwan for more than 40 years. The sporadic activity of low pathogenic H5N2 virus has been noted since 2003, and highly pathogenic H5N2 avian influenza virus has been detected since 2008. Ressortant viruses between H6N1 and H5N2 viruses have become established and enzootic in chickens throughout Taiwan. Outbreaks caused by Novel highly pathogenic H5 avian influenza viruses whose HA genes were closely related to that of the H5N8 virus isolated from ducks in Korea in 2014 were isolated from outbreaks in Taiwan since early 2015. The avian influenza virus infection status is becoming much more complicated in chickens in Taiwan. This necessitates a rapid and simple approach to detect and differentiate the viruses that prevail. H6N1, H5N2 and novel H5 viruses were simultaneously subtyped and pathotyped in this study using reverse transcription loop-mediated isothermal amplification and microarray, with detection limits of 10°, 10(1) and 10° viral copy numbers, respectively. The microarray signals were read by the naked eye with no expensive equipment needed. The method developed in this study could greatly improve avian influenza virus surveillance efficiency.
Project description:BACKGROUND:We describe the results of an open label Phase I trial of a live attenuated H6N1 influenza virus vaccine (ClinicalTrials.gov Identifier: NCT00734175). METHODS AND FINDINGS:We evaluated the safety, infectivity, and immunogenicity of two doses of 10(7) TCID(50) of the H6N1 Teal HK 97/AA ca vaccine, a cold-adapted and temperature sensitive live, attenuated influenza vaccine (LAIV) in healthy seronegative adults. Twenty-two participants received the first dose of the vaccine, and 18 received the second dose of vaccine 4 weeks later. The vaccine had a safety profile similar to that of other investigational LAIVs bearing avian hemagglutinin (HA) and neuraminidase (NA) genes. The vaccine was highly restricted in replication: two participants had virus detectable by rRT-PCR beyond day 1 after each dose. Antibody responses to the vaccine were also restricted: 43% of participants developed a serum antibody response as measured by any assay: 5% by hemagglutination-inhibition assay, 5% by microneutralization assay, 29% by ELISA for H6 HA-specific IgG and 24% by ELISA for H6 HA specific IgA after either 1 or 2 doses. Following the second dose, vaccine specific IgG and IgA secreting cells as measured by ELISPOT increased from a mean of 0.6 to 9.2/10(6) PBMCs and from 0.2 to 2.2/10(6) PBMCs, respectively. CONCLUSION:The H6N1 LAIV had a safety profile similar to that of LAIV bearing other HA and NA genes, but was highly restricted in replication in healthy seronegative adults. The H6N1 LAIV was also not as immunogenic as the seasonal LAIV.
Project description:In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding.
Project description:H6N1 influenza A is an avian virus but in 2013 infected a human in Taiwan. We studied the phylogeography of avian origin H6N1 viruses in the Influenza Research Database and the Global Initiative on Sharing Avian Influenza Data EpiFlu Database in order to characterize their recent evolutionary spread. Our results suggest that the H6N1 virus that infected a human in Taiwan is derived from a diversity of avian strains of H6N1 that have circulated for at least seven years in this region. Understanding how geography impacts the evolution of avian influenza could allow disease control efforts to focus on areas that pose the greatest risk to humans. The serious human infection with a known avian influenza virus underscores the zoonotic potential of diverse avian strains of influenza, and the need for comprehensive influenza surveillance in animals and the value of public sequence databases including GISAID and the IRD.
Project description:Sporadic activity by H5N2 influenza viruses has been observed in chickens in Taiwan from 2003 to 2012. The available information suggests that these viruses were generated by reassortment between a Mexican-like H5N2 virus and a local enzootic H6N1 virus. Yet the origin, prevalence, and pathogenicity of these H5N2 viruses have not been fully defined. Following the 2012 highly pathogenic avian influenza (HPAI) outbreaks, surveillance was conducted from December 2012 to July 2013 at a live-poultry wholesale market in Taipei. Our findings showed that H5N2 and H6N1 viruses cocirculated at low levels in chickens in Taiwan. Phylogenetic analyses revealed that all H5N2 viruses had hemagglutinin (HA) and neuraminidase (NA) genes derived from a 1994 Mexican-like virus, while their internal gene complexes were incorporated from the enzootic H6N1 virus lineage by multiple reassortment events. Pathogenicity studies demonstrated heterogeneous results even though all tested viruses had motifs (R-X-K/R-R) supportive of high pathogenicity. Serological surveys for common subtypes of avian viruses confirmed the prevalence of the H5N2 and H6N1 viruses in chickens and revealed an extraordinarily high seroconversion rate to an H9N2 virus, a subtype that is not found in Taiwan but is prevalent in mainland China. These findings suggest that reassortant H5N2 viruses, together with H6N1 viruses, have become established and enzootic in chickens throughout Taiwan and that a large-scale vaccination program might have been conducted locally that likely led to the introduction of the 1994 Mexican-like virus to Taiwan in 2003.H5N2 avian influenza viruses first appeared in chickens in Taiwan in 2003 and caused a series of outbreaks afterwards. Phylogenetic analyses show that the chicken H5N2 viruses have H5 and N2 genes that are closely related to those of a vaccine strain originating from Mexico in 1994, while the contemporary duck H5N2 viruses in Taiwan belong to the Eurasian gene pool. The unusually high similarity of the chicken H5N2 viruses to the Mexican vaccine strain suggests that these viruses might have been introduced to Taiwan by using inadequately inactivated or attenuated vaccines. These chicken H5N2 viruses are developing varying levels of pathogenicity that could lead to significant consequences for the local poultry industry. These findings emphasize the need for strict quality control and competent oversight in the manufacture and usage of avian influenza virus vaccines and indicate that alternatives to widespread vaccination may be desirable.