Continuing evolution of H6N2 influenza A virus in South African chickens and the implications for diagnosis and control

Abstract

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 twelve 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, sub-lineage 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 HA 0 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.

The Evolution, Spread and Global Threat of H6Nx Avian Influenza Viruses

“…ST339-like viruses and ST2853-like viruses have both been interchangeably termed Group I and Group II (e.g., [ 17 , 23 ]). In addition, several well-supported clades have been identified and named within the literature, for example South African sub-lineages I and II ( Figure 4 ) [ 32 , 33 , 34 ]. Sporadic and conflicting use of clade nomenclature between studies complicates scientific discussion of H6 evolution and diversity.…”

“…In the East Asian sub-region, H6N1, H6N2, H6N5, H6N6, H6N8 and H6N9 co-circulate in chicken and duck populations, undergoing frequent reassortment with other subtypes [ 23 , 39 , 40 ]. The migratory waterfowl reservoir is the most common source of H6 introduction into domestic poultry but is typically restricted in their ability to spread further [ 33 ]. H6 viruses typically cause asymptomatic infections in waterfowl but infection of chickens with H6 viruses are often associated with reduced egg yield, upper respiratory tract infection, morbidity and increased mortality [ 20 ].…”

“…Clinical signs of H6N2 in layers range from nearly inapparent to relatively severe respiratory signs with egg yield reductions up to 30%. Weekly mortality increases between 1.6 and 5.1-fold with severe losses in broiler breeders reporting production losses up to 60% with further mortalities of up to 37% [ 33 ]. Some histological features present in chickens infected with H6N2 are fibrinous yolk peritonitis, salpingitis, oophoritis, nephritis and swollen kidneys [ 41 ].…”

“…South Africa’s H6N2 epizootic in chickens began in 2001 but was traced to a progenitor originating from a reassortment between viruses that infected commercial ostriches in the Western Cape Province in the mid to late 1990s, notably an H6N8 and an H9N2 virus [ 33 ]. The virus likely derived its M, NP, PB1 and PA genes from the H9N2 1995 ostrich virus A/Ostrich/South Africa/9508103 and the H6N8 virus, (A/Ostrich/South Africa/KK98/98) isolated in 1998 [ 32 ].…”

“…The virus likely derived its M, NP, PB1 and PA genes from the H9N2 1995 ostrich virus A/Ostrich/South Africa/9508103 and the H6N8 virus, (A/Ostrich/South Africa/KK98/98) isolated in 1998 [ 32 ]. H6N2 is a controlled disease within South Africa and thus requires compulsory serological testing of registered compartmentalised commercial poultry farms and operations with significant contributions from non-commercial poultry flocks (e.g., backyard chickens) [ 33 ].…”