BACKGROUND : The threat of poultry-origin H6 avian influenza viruses to human health emphasizes the importance
of monitoring their evolution. South Africa’s H6N2 epidemic in chickens began in 2001 and two co-circulating
antigenic sub-lineages of H6N2 could be distinguished from the outset. The true incidence and prevalence of H6N2
in the country has been difficult to determine, partly due to the continued use of an inactivated whole virus H6N2
vaccine and the inability to distinguish vaccinated from non-vaccinated birds on serology tests. In the present
study, the complete genomes of 12 H6N2 viruses isolated from various farming systems between September 2015
and February 2019 in three major chicken-producing regions were analysed and a serological experiment was used
to demonstrate the effects of antigenic mismatch in diagnostic tests.
RESULTS : Genetic drift in H6N2 continued and antigenic diversity in sub-lineage I is increasing; no sub-lineage II viruses
were detected. Reassortment patterns indicated epidemiological connections between provinces as well as different
farming systems, but there was no reassortment with wild bird or ostrich influenza viruses. The sequence mismatch
between the official antigens used for routine hemagglutination inhibition (HI) testing and circulating field strains has
increased steadily, and we demonstrated that H6N2 field infections are likely to be missed. More concerning, sublineage
I H6N2 viruses acquired three of the nine HA mutations associated with human receptor-binding preference
(A13S, V187D and A193N) since 2002. Most sub-lineage I viruses isolated since 2015 acquired the K702R mutation in
PB2 associated with the ability to infect humans, whereas prior to 2015 most viruses in sub-lineages I and II contained
the avian lysine marker. All strains had an unusual HA0 motif of PQVETRGIF or PQVGTRGIF.
CONCLUSIONS : The H6N2 viruses in South African chickens are mutating and reassorting amongst themselves but have
remained a genetically pure lineage since they emerged more than 18 years ago. Greater efforts must be made by
government and industry in the continuous isolation and characterization of field strains for use as HI antigens, new
vaccine seed strains and to monitor the zoonotic threat of H6N2 viruses.
Additional file 1: Table S1a. Percentage nucleotide sequence identity
in the HA genes of sub-lineage I viruses isolated since 2015.
Additional file 2: Table S1b. Percentage amino sequence identity in
the HA proteins of all sub-lineage I viruses.