Antigenic evolution of H9N2 chicken influenza viruses isolated in China during 2009–2013 and selection of a candidate vaccine strain with broad cross-reactivity
Introduction
H9N2 influenza virus, characterized as a low-pathogenicity pathogen, has been circulating worldwide in multiple avian species, resulting in great economic losses due to reduced egg production or high mortality associated with co-infection with other pathogens (Sun and Liu, 2015, Webster et al., 1992, Xu et al., 2007). In China, H9N2 viruses were first isolated from diseased chickens in 1994 (Chen et al., 1994); they have since become widespread in these populations (Li et al., 2005, Pu et al., 2014, Sun et al., 2010, Zhang et al., 2009). H9N2 viruses also pose a threat to public health, having caused repeated human infections in China since 1998 (Perdue and Swayne, 2005, Sun and Liu, 2015, Webster et al., 1992, Xu et al., 2007). Serologic surveillance indicates that a certain proportion (4.6–37.2%) of people in China might be seropositive for H9N2 viruses (Liang et al., 2003, Yu et al., 2013). Since 2013, all 6 internal genes of the novel H7N9 and H10N8 viruses isolated from humans have been found in H9N2 viruses circulating in China (Chen et al., 2014, Gao et al., 2013, Pu et al., 2014). Thus, H9N2 influenza viruses are high on the list of candidates that could cause another human influenza pandemic.
To prevent H9N2 AIV infections in chickens, farms in China have administered commercially inactivated vaccines such as A/chicken/Shandong/6/1996 (Ck/SD/6/96) and A/chicken/Shanghai/F/1998 (Ck/SH/F/98) to their flocks since 1998 (Li et al., 2005, Zhang et al., 2008). However, the antigenic drift of H9N2 AIVs continues to occur and induce sporadic disease outbreaks in vaccinated chicken flocks (Huang et al., 2010, Li et al., 2005, Pu et al., 2014, Sun et al., 2012, Zhang et al., 2008, Zhang et al., 2012). Since 2010, antigenic variants of H9N2 viruses have possessed increased fitness to escape immunization pressure and caused the country-wide outbreaks in chickens that contributed to the generation of the novel H7N9 viruses (Pu et al., 2014). These facts emphasize that understanding the antigenicity of circulating viruses and updating matched vaccine strains are extremely urgent actions.
Although the antigenic drift of H9N2 chicken influenza viruses from vaccine strains was fully addressed (Li et al., 2005, Pu et al., 2014, Sun et al., 2012), the antigenic evolution of prevailing viruses isolated since 2009 is not well known. We previously investigated the H9N2 chicken influenza viruses isolated in China from 1996 to 2008 and identified 3 major antigenic groups (C, D, and E) (Sun et al., 2010). In the present study, we performed hemagglutination inhibition (HI) and neutralization assays to further investigate the antigenic evolution of the chicken H9N2 viruses isolated during 2009–2013. Additionally, we analyzed the genetic characterization of the HA gene, in which crucial substitutions might result in different antigenic properties (Boni, 2008, McHardy and Adams, 2009, Rambaut et al., 2008, Webster et al., 1992). On the basis of these data, we selected and evaluated a vaccine candidate.
Section snippets
Ethics statement
All animal research was approved by the Beijing Association for Science and Technology (approval ID SYXK, Beijing, 2007-0023) and performed in compliance with the Beijing Laboratory Animal Welfare and Ethics guide lines issued by the Beijing Administration Committee of Laboratory Animals and in accordance with the China Agricultural University (CAU) Institutional Animal Care and Use Committee guidelines (ID: SKLAB-B-2010-003) approved by the Animal Welfare Committee of CAU.
Virus
On the basis of their
Antigenic analysis of H9N2 chicken influenza viruses
Among the 27 newly identified viruses isolated in 2009–2013, 3 belonged to the previously identified antigenic groups D and E, and all others formed a novel antigenic group, F (Fig. 2 and Table S1). Most group F viruses had low or moderate reactivity to group C and E antisera but reacted well with group D and F antisera (Table S1). Further comparison of groups D and F showed that group F viruses have a different antigenic profile in reactions with antisera to the recent viruses Ck/HeB/YT/10,
Discussion
H9N2 viruses isolated during a certain period or in a certain region were previously believed to share similar antigenicity profiles (Webster et al., 1992, Xu et al., 2007, Zhang et al., 2008). Therefore, vaccine strains isolated during the late 20th century were used in chicken flocks throughout China for more than 10 years (Li et al., 2005, Sun et al., 2012). However, our and other previous studies revealed that some H9N2 chicken influenza viruses isolated especially since 2007 underwent
Conclusion
Taken together, our findings demonstrate the newly antigenic changes of H9N2 chicken influenza viruses in China from 2009–2013. The tendency of the novel group F viruses toward antigenic diversity highlights the necessity of monitoring antigenic variants in chickens. Vaccine strains should be systematically evaluated and regularly updated to achieve optimal protection against AIV infections.
Conflicts of interest
None
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 31430086); the National High Technology Research and Development Program (863 Program) of China (Grant No.2012AA101303); the China Agriculture Research System, Poultry-Related Science and Technology Innovation Team of Peking; and the Chinese Universities Scientific Fund (2015DY004). This work was also funded in part by Contract numbers HHSN266200700005C and HHSN272201400006C from the National Institute of
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