Identification, sequence analysis, and infectivity of H9N2 avian influenza viruses isolated from geese.
ABSTRACT: The subtype H9N2 avian influenza virus greatly threatens the Chinese poultry industry, even with annual vaccination. Waterfowl can be asymptomatically infected with the H9N2 virus. In this study, three H9N2 virus strains, designated A/Goose/Jiangsu/YZ527/2011 (H9N2, Gs/JS/YZ527/11), A/Goose/Jiangsu/SQ119/2012 (H9N2, Gs/JS/SQ119/12), and A/Goose/Jiangsu/JD564/2012 (H9N2, Gs/JS/JD564/12), were isolated from domestic geese. Molecular characterization of the three isolates showed that the Gs/JS/YZ527/11 virus is a double-reassortant virus, combining genes of A/Quail/Hong Kong/G1/97 (H9N2, G1/97)-like and A/Chicken/Shanghai/F/98 (H9N2, F/98)-like; the Gs/JS/SQ119/12 virus is a triple-reassortant virus combining genes of G1/97-like, F/98-like, and A/Duck/Shantou/163/2004 (H9N2, ST/163/04)-like. The sequences of Gs/JS/JD564/12 share high homology with those of the F/98 virus, except for the neuraminidase gene, whereas the internal genes of Gs/JS/YZ527/11 and Gs/JS/SQ119/12 are closely related to those of the H7N9 viruses. An infectivity analysis of the three isolates showed that Gs/JS/SQ119/12 and Gs/JS/YZ527/11 replicated well, with seroconversion, in geese and chickens, the Gs/JS/JD564/12 did not infect well in geese or chickens, and the F/98 virus only infected chickens, with seroconversion. Emergence of these new reassortant H9N2 avian influenza viruses indicates that these viruses can infect both chicken and goose and can produce different types of lesions in each species.
Project description:During a study on high mortality cases of goose embryo in Shandong Province, China (2014-2015), we isolated an H9N2 avian influenza virus (AIV) strain (A/goose/Shandong/DP01/2014, DP01), which was supposedly the causative agent for goose embryo death. Sequence analysis revealed that DP01 shared 99.9% homology in the HA gene with a classic immune suppression strain SD06. To study the potential vertical transmission ability of the DP01 strain in breeder goose, a total of 105 Taizhou breeder geese, which were 360 days old, were equally divided into five groups (A, B, C, D, and E) for experimental infection. H9N2 AIV (DP01) was used for inoculating through intravenous (group A), intranasal instillation (group B), and throat inoculation (group C) routes, respectively. The geese in group D were inoculated with phosphate buffer solution (PBS) and those in group E were the non-treated group. At 24 h post inoculation, H9N2 viral RNA could be detected at vitelline membrane, embryos, and allantoic fluid of goose embryos from H9N2 inoculated groups. Furthermore, the HA gene of H9N2 virus from vitelline membrane, embryo, allantoic fluid, and gosling shared almost 100% homology with an H9N2 virus isolated from the ovary of breeder goose, which laid these eggs, indicating that H9N2 AIV can be vertically transmitted in goose. The present research study provides evidence that vertical transmission of H9N2 AIV from breeding goose to goslings is possible.
Project description:H9N2 subtype influenza A virus (IAV) has more than 20 genotypes that are able to cross species barriers and expand from birds to mammals and humans. To better understand the impact of different H9N2 genotypes and their characteristics, five H9N2 viruses from different hosts including chickens, geese, pigs, mink, and humans representing the B69 88(Gs/14, Ck/15, and Mi/14), B35 (Sw/08) and G9 genotypes (Hu/04) were infected in chicken and mice. In mice, mammal-origin viruses replicated at higher levels in the lungs compared to avian viruses. The goose-virus replicated at the lowest levels indicating poor adaptation. Increased pro-inflammatory cytokines were positively correlated with viral loads in the lung. In chickens, all viruses were excreted from cloacal and/or oropharyngeal swabs. Interestingly, Mink-origin virus exhibited higher virulence and replication in mice and chickens. Our data indicate that mammal-origin H9N2 viruses are more adapted and virulent in mice than the avian-origin viruses.
Project description:The H5N1 influenza virus, which killed humans and poultry in 1997, was a reassortant that possibly arose in one type of domestic poultry present in the live-poultry markets of Hong Kong. Given that all the precursors of H5N1/97 are still circulating in poultry in southern China, the reassortment event that generated H5N1 could be repeated. Because A/goose/Guangdong/1/96-like (H5N1; Go/Gd) viruses are the proposed donors of the hemagglutinin gene of the H5N1 virus, we investigated the continued circulation, host range, and transmissibility of Go/Gd-like viruses in poultry. The Go/Gd-like viruses caused weight loss and death in some mice inoculated with high virus doses. Transmission of Go/Gd-like H5N1 viruses to geese by contact with infected geese resulted in infection of all birds but limited signs of overt disease. In contrast, oral inoculation with high doses of Go/Gd-like viruses resulted in the deaths of up to 50% of infected geese. Transmission from infected geese to chickens occurred only by fecal contact, whereas transmission to quail occurred by either aerosol or fecal spread. This difference is probably explained by the higher susceptibility of quail to Go/Gd-like virus. The high degree of susceptibility of quail to Go/Gd (H5N1)-like viruses and the continued circulation of H6N1 and H9N2 viruses in quail support the hypothesis that quail were the host of origin of the H5N1/97 virus. The ease of transmission of Go/Gd (H5N1)-like viruses to land-based birds, especially quail, supports the wisdom of separating aquatic and land-based poultry in the markets in Hong Kong and the need for continued surveillance in the field and live-bird markets in which different types of poultry are in contact with one another.
Project description:Over the course of two waves of infection, H7N9 avian influenza A virus has caused 436 human infections and claimed 170 lives in China as of July 2014. To investigate the prevalence and genetic diversity of H7N9, we surveyed avian influenza viruses in poultry in Jiangsu province within the outbreak epicenter. We found frequent occurrence of H7N9/H9N2 coinfection in chickens. Molecular clock phylogenetic analysis confirms coinfection by H7N9/H9N2 viruses and also reveals that the identity of the H7N9 outbreak lineage is confounded by ongoing reassortment between outbreak viruses and diverse H9N2 viruses in domestic birds. Experimental inoculation of a coinfected sample in cell culture yielded two reassortant H7N9 strains with polymerase segments from the original H9N2 strain. Ongoing reassortment between the H7N9 outbreak lineage and diverse H9N2 viruses may generate new strains with the potential to infect humans, highlighting the need for continued viral surveillance in poultry and humans.We found frequent occurrence of H7N9/H9N2 coinfection in chickens. The H7N9 outbreak lineage is confounded by ongoing reassortment between H7N9 and H9N2 viruses. The importance of H9N2 viruses as the source of novel avian influenza virus infections in humans requires continuous attention.
Project description:In 2014, a sentinel chicken surveillance for avian influenza viruses was conducted in aquatic bird habitat near Wuxi City, Jiangsu Province, China. Two H7N2, one H5N6, and two H9N2 viruses were isolated. Sequence analysis revealed that the H7N2 virus is a novel reassortant of H7N9 and H9N2 viruses and H5N6 virus is a reassortant of H5N1 clade 2.3.4 and H6N6 viruses. Substitutions V186 and L226 (H3 numbering) in the hemagglutinin (HA) gene protein was found in two H7N2 viruses but not in the H5N6 virus. Two A138 and A160 mutations were identified in the HA gene protein of all three viruses but a P128 mutation was only observed in the H5N6 virus. A deletion of 3 and 11 amino acids in the neuraminidase stalk region was found in two H7N2 and H5N6 viruses, respectively. Moreover, a mutation of N31 in M2 protein was observed in both two H7N2 viruses. High similarity of these isolated viruses to viruses previously identified among poultry and humans, suggests that peridomestic aquatic birds may play a role in sustaining novel virus transmission. Therefore, continued surveillance is needed to monitor these avian influenza viruses in wild bird and domestic poultry that may pose a threat to poultry and human health.
Project description:Low pathogenic avian influenza virus can mutate to a highly pathogenic strain that causes severe clinical signs in birds and humans. Migratory waterfowl, especially ducks, are considered the main hosts of low pathogenic avian influenza virus, but the role of geese in dispersing the virus over long-distances is still unclear. We collected throat and cloaca samples from three goose species, Bean goose (Anser fabalis), Barnacle goose (Branta leucopsis) and Greater white-fronted goose (Anser albifrons), from their breeding grounds, spring stopover sites, and wintering grounds. We tested if the geese were infected with low pathogenic avian influenza virus outside of their wintering grounds, and analysed the spatial and temporal patterns of infection prevalence on their wintering grounds. Our results show that geese were not infected before their arrival on wintering grounds. Barnacle geese and Greater white-fronted geese had low prevalence of infection just after their arrival on wintering grounds in the Netherlands, but the prevalence increased in successive months, and peaked after December. This suggests that migratory geese are exposed to the virus after their arrival on wintering grounds, indicating that migratory geese might not disperse low pathogenic avian influenza virus during autumn migration.
Project description:Avian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells.To investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens.We observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding.Our findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production.
Project description:Vaccination is one of the leading methods of controlling the spread of the Avian Influenza (AI) viruses in Indonesia. The variety of circulating viruses and their ability to mutate must be followed by updating the vaccine master seed used in the field. In this study, we identified the reassortant H9N2 viruses in chicken farms that showed significant problems in decreased egg production with high mortality. The reassortant H9N2 viruses derived the PB2 gene from the H5N1 virus. The pathogenicity test results of the reassortant virus showed various clinical signs of illness, a high mortality rate (10%), and decreased egg production down to 63.12% at two weeks post-infection. In a vaccine efficacy test, the vaccinated groups showed minimally decreased egg production that started to increase to more than 80% at 4-7 weeks post-challenge. Our study showed that inactivated bivalent and monovalent reassortant H9N2 vaccines can induce antibody response, reducing the mortality and virus shedding caused by reassortant H9N2 virus infection. The reassortant H9N2 virus is a threat that requires vigilance in poultry farms and the industry. The vaccines used in this study can be one of the options for control or prevention measures on farms infected with the reassortant H9N2 viruses.
Project description:We conducted a 3-year longitudinal serologic survey on an open cohort of poultry workers, swine workers, and general population controls to assess avian influenza A virus (AIV) seroprevalence and seroincidence and virologic diversity at live poultry markets (LPMs) in Wuxi City, Jiangsu Province, China. Of 964 poultry workers, 9 (0.93%) were seropositive for subtype H7N9 virus, 18 (1.87%) for H9N2, and 18 (1.87%) for H5N1. Of 468 poultry workers followed longitudinally, 2 (0.43%), 13 (2.78%), and 7 (1.5%) seroconverted, respectively; incidence was 1.27, 8.28, and 4.46/1,000 person-years for H7N9, H9N2, and H5N1 viruses, respectively. Longitudinal surveillance of AIVs at 9 LPMs revealed high co-circulation of H9, H7, and H5 subtypes. We detected AIVs in 726 (23.3%) of 3,121 samples and identified a high diversity (10 subtypes) of new genetic constellations and reassortant viruses. These data suggest that stronger surveillance for AIVs within LPMs and high-risk populations is imperative.
Project description:Outbreaks of pandemic H1N1 2009 (pH1N1) in turkeys have been reported in several countries. Co-infection of pH1N1 and avian H9N2 influenza viruses in turkeys provide the opportunity for their reassortment, and novel reassortant viruses might further be transmitted to other avian species. However, virulence and transmission of those reassortant viruses in poultry remain unclear. In the present study, we generated 16 single-gene reassortant influenza viruses including eight reassortants on the pH1N1 background by individual replacement with a corresponding gene segment from H9N2 and eight reassortants on the H9N2 background replaced individually with corresponding gene from pH1N1, and characterized reassortants viruses in turkeys and chickens. We found that the pH1N1 virus dramatically increased its infectivity and transmissibility in turkeys and chickens after introducing any gene (except for PB2) from H9N2 virus, and H9N2 virus acquired single gene (except for HA) of pH1N1 almost did not influence its replication and transmission in turkeys and chickens. Additionally, 13 reassortant viruses transmitted from turkeys to chickens. Our results indicate that turkeys and chickens are susceptible to pH1N1-H9N2 reassortant viruses, and mixing breeding of different avian species would facilitate the transmission of these reassortant viruses.