We report the serological evidence of low-pathogenic avian influenza (LPAI) H9N2 infection in an occupational poultry-exposed population and a general population. A serological survey of an occupational poultry-exposed population and a general population was conducted using a haemagglutinin-inhibiting (HI) assay in Shanghai, China, from January 2008 to December 2010. Evidence of higher anti-H9 antibodies was found in serum samples collected from poultry workers. During this period, 239 H9N2 avian influenza viruses (AIVs) were isolated from 9297 tracheal and cloacal paired specimens collected from the poultry in live poultry markets. In addition, a total of 733 influenza viruses were isolated from 1569 nasal and throat swabs collected from patients with influenza-like symptoms in a sentinel hospital, which include H3N2, H1N1, pandemic H1N1 and B, but no H9N2 virus was detected. These findings highlight the need for long-term surveillance of avian influenza viruses in occupational poultry-exposed workers.

BackgroundVaccines constitute a unique selective pressure, different from natural selection, drives the evolution of influenza virus. In this study, A/Chicken/Shanghai/F/1998 (H9N2) was continually passaged in specific pathogen-free embryonated chicken eggs with or without selective pressures from antibodies induced by homologous maternal antibodies. Genetic mutations, antigenic drift, replication, and pathogenicity of the passaged virus were evaluated.ResultsAntigenic drift of the passaged viruses occurred in the 47th generation (vF47) under selective pressure on antibodies and in the 52nd generation (nF52) without selective pressure from antibodies. Seven mutations were observed in the vF47 virus, with three in PB2 and four in HA, whereas 12 mutations occurred in the nF52 virus, with three in PB2, two in PB1, four in HA, one in NP, one in NA, and one in NS. Remarkably, the sequences of the HA segment from vF47 were 100% homologous with those of the nF52 virus. Both the vF47 and nF52 viruses showed enhanced replication compared to the parental virus F/98, but higher levels of replication and pathogenicity were displayed by nF52 than by vF47. An inactive vaccine derived from the parental virus F/98 did not confer protection against challenges by either the vF47 or nF52 virus, but inactive vaccines derived from the vF47 or nF52 virus were able to provide protection against a challenge using F/98.ConclusionTaken together, the passage of H9N2 viruses with or without selective pressure of the antibodies induced by homologous maternal antibodies showed genetic variation, enhanced replication, and variant antigenicity. Selective pressure of the antibody does not seem to play a key role in antigenic drift in the egg model but may impact the genetic variation and replication ability of H9N2 viruses. These results improve understanding of the evolution of the H9N2 influenza virus and may aid in selecting appropriate vaccine seeds.

H9N2 avian influenza virus has spread worldwide, and vaccination with an inactivated virus is currently the major prevention method in China. To further understand the effect of the selection pressure from antibodies on the evolution of H9N2 avian influenza virus, F/98 (A/Chicken/Shanghai/F/98), which is the vaccine representative of H9N2 avian influenza virus in East China, was used for serial passaging for 20 generations in chickens with and without vaccination. After plaque purification from trachea and lung tissues, 390 quasispecies were obtained. The second-generation quasispecies under the selection pressure of vaccine antibodies had undergone 100% antigen variation, while after passaging to the fifth generation, only 30–40% of the quasispecies displayed antigen variation when there was no selection pressure of vaccine antibodies, implying that the selection pressure of vaccine antibodies promotes the antigen variation of F/98. We found for the first time that there were three mutation hotspots in the HA genes of the quasispecies under the selection pressure of vaccine antibodies, which were K131R, A168T, and N201D. Moreover, under the selection pressure of vaccine antibodies, 10 amino acids (67–76) of the NA protein of all quasispecies were deleted, and PB2 of the quasispecies had undergone a high-frequency R355K mutation. However, without selection pressure of vaccine antibodies, NP had undergone two high-frequency mutations, namely, V186I and L466I, and a high-frequency mutation of L77I appeared in the NS gene. This result shows that the vaccine antibody selection pressure could control and regulate gene variation of the F/98 virus. Compared to that of the parental virus F/98, the EID50 of the twentieth passaged virus under the selection pressure of vaccine antibodies did not change, while the EID50 of the twentieth passaged virus without selection pressure of vaccine antibodies was significantly enhanced by 794 times. Furthermore, the twentieth passaged virus with selection pressure from vaccine antibodies lost its lethal ability in embryonated chicken eggs, whereas the EID50 of the twentieth passaged virus without selection pressure of vaccine antibodies increased to 6.3 times that of the F/98 strain. All the above results show that the selection pressure of vaccine antibodies promotes the antigen variation of H9N2 avian influenza virus and plays a role in regulating and controlling gene mutation of H9N2 avian influenza virus.

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H9N2 avian influenza virus has spread worldwide, and vaccination with an inactivated virus is currently the major prevention method in China. To further understand the effect of the selection pressure from antibodies on the evolution of H9N2 avian influenza virus, F/98 (A/Chicken/Shanghai/F/98), which is the vaccine representative of H9N2 avian influenza virus in East China, was used for serial passaging for 20 generations in chickens with and without vaccination. After plaque purification from trachea and lung tissues, 390 quasispecies were obtained. The second-generation quasispecies under the selection pressure of vaccine antibodies had undergone 100% antigen variation, while after passaging to the fifth generation, only 30-40% of the quasispecies displayed antigen variation when there was no selection pressure of vaccine antibodies, implying that the selection pressure of vaccine antibodies promotes the antigen variation of F/98. We found for the first time that there were three mutation hotspots in the HA genes of the quasispecies under the selection pressure of vaccine antibodies, which were K131R, A168T, and N201D. Moreover, under the selection pressure of vaccine antibodies, 10 amino acids (67-76) of the NA protein of all quasispecies were deleted, and PB2 of the quasispecies had undergone a high-frequency R355K mutation. However, without selection pressure of vaccine antibodies, NP had undergone two high-frequency mutations, namely, V186I and L466I, and a high-frequency mutation of L77I appeared in the NS gene. This result shows that the vaccine antibody selection pressure could control and regulate gene variation of the F/98 virus. Compared to that of the parental virus F/98, the EID 50 of the twentieth passaged virus under the selection pressure of vaccine antibodies did not change, while the EID 50 of the twentieth passaged virus without selection pressure of vaccine antibodies was significantly enhanced by 794 times. Furthermore, the twentieth passaged virus with selection pressure from vaccine antibodies lost its lethal ability in embryonated chicken eggs, whereas the EID 50 of the twentieth passaged virus without selection pressure of vaccine antibodies increased to 6.3 times that of the F/98 strain. All the above results show that the selection pressure of vaccine antibodies promotes the antigen variation of H9N2 avian influenza virus and plays a role in regulating and controlling gene mutation of H9N2 avian influenza virus.