Antigenic Variation of Avian Influenza A(H5N6) Viruses, Guangdong Province, China, 2014–2018

Market surveillance showed continuing circulation of avian influenza A(H5N6) virus in live poultry markets in Guangdong Province in 2017, despite compulsory vaccination for avian influenza A(H5Nx) and A(H7N9). We analyzed H5N6 viruses from 2014–2018 from Guangdong Province, revealing antigenic drift and decreased antibody response against the vaccine strain in vaccinated chickens.

viruses in Guangdong Province cluster separately from the A/chicken/Guizhou/4/2013 (Re-8) vaccine strain, based on HA sequences ( Figure 2). All N6 genes belong to the Eurasian lineage. Both HA and NA genes of the human H5N6 virus clustered with the H5N6 viruses found in the environment and poultry in our study (Appendix 1 Figure).
We classified both surface and internal genes of HPAI H5N6 viruses from Guangdong Province into different sublineages according to tree topology and bootstrap values of >85% and further classified the HA and NA genes into 4 subgroups ( Figure 2). Phylogeny of the internal genes of the recent clade 2.3.4.4 H5N6 viruses showed they evolved from H5N1 viruses from 2013-2014, in which, from 2015 onward, almost all PB2 genes were replaced by H6 subtype-origin PB2 genes. Substitution of the PB2 gene can change the virulence and pathogenicity in mammals and in different bird species (12). Moreover, from 2016 onward, H5N6 acquired PB1 and PA genes from (avian) H3-like or LPAI gene pools. In 2017, NP, M, and NS genes from H3-like viruses and local LPAI gene pools were first detected in circulating H5N6 viruses (Figure 2). Closely related H5N6 viruses from China with similar internal gene composition did not show any intravenous pathogenicity in ducks and lower intravenous pathogenicity in chickens (13), which could explain the widespread circulation of H5N6 viruses in Guangdong Province.
We determined HI titers in serum of H5 A/chicken/ Guizhou/4/2013 (Re-8)-vaccinated chickens (n = 5) and serum from the H5N6 virus-infected human patient to human and environmental H5N6 viral isolates from different time points using a standard protocol (14). Serum from chickens vaccinated with H5 A/chicken/Guizhou/4/2013 (Re-8) showed high titers (8-10 log 2 ) to the human H5N6 isolates from 2014-2017 and lower titers (4-6 log 2 ) to the human H5N6 isolate from 2018. We observed a similar trend when using environmental isolates for the HI assays. Conversely, serum from the H5N6-infected human in 2018 showed higher titers to human H5N6 isolates in 2017 and 2018 (6 log 2 ) than to those from 2014 and 2015 (4 log 2 ) (Table).

Conclusions
Compulsory vaccination of the combined inactivated influenza vaccine was implemented in Guangdong Province in July 2017. Although the prevalence of H7N9 in LPMs decreased abruptly, we revealed uninterrupted circulation of H5N6 viruses in LPMs after implementation of the . Moreover, the World Health Organization proposed a new A/Guangdong/18SF020/2018-like H5N6 candidate vaccine virus, which was partly based on strain A/Guangdong/18SF020/2018 reported in this study (15).
Vaccine escape variants remain a risk for human and animal health. Therefore, future policy should focus on preventing the spread of avian influenza viruses along the market chain by strengthening farm-level surveillance and biosecurity, as well as implementing measures to monitor and prevent the spread of avian influenza viruses that have zoonotic potential in the market chain.

About the Author
Dr. Bai is a postdoctoral researcher at the Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China, whose research focuses on the epidemiology, evolution, and transmission of avian influenza viruses in Guangdong Province. Ms. Sikkema is a PhD candidate at the Erasmus Medical Centre, Rotterdam, the Netherlands, whose research focuses on the risk-based surveillance of influenza viruses in the market chain.