Biological Characterization Of HA Mutants Of Different H3N2 Avian Influenza Viruses

In the last century,H1N1,H2N2,and H3N2 subtype influenza viruses caused human influenza pandemics in 1918,1957 and 1968 respectively.In 2009,a new H1N1 subtype influenza virus broke the interspecies barrier and spread from pigs to humans,which caused the fourth influenza pandemic.Among them,the H3N2 subtype influenza virus has a wide host range.If the subtype influenza virus crosses from other hosts to humans,it may cause a new human influenza outbreak and bring serious disasters to humans.The laboratory has previously evaluated the droplet transmissibility of three H3N2 subtype avian influenza viruses(AIV)isolated from the Chinese live poultry markets on ferrets.The three viruses are A/duck/Fujian/S2186/2011(H3N2),referred to as DK/FJ/S2186/11;A/duck/Guangxi/S4011/2014(H3N2),referred to as DK/GX/S4011/14;A/duck/Guangxi/ S4234/2014(H3N2)is referred to as DK/GX/S4234/14.The results of transmission experiments show that these three strains can be spread by droplets among ferrets,among which DK/GX/S4011/14 can efficiently spread by droplets among ferrets.Isolation,identification and sequencing of viruses in ferret nasal washes,organs and tissues that collected and preserved in previous experiments showed that the above three viruses had mutations during replication in ferrets,and mutations in the HA proteins of these three viruses are A138 S,Q226R,Q226 L and G228 S,and the mutations in PB2 protein are A588 T and D701 N.Some of these mutations have been proven to enhance the transmissibility of influenza viruses.In order to explore the effects of the above mutations on the biological characteristics of H3N2 subtype AIVs.The mutant strains in ferrets were separated and purified.Two mutant strains derived from DK/FJ/S2186/11 has mutations of Q226 R and G228 S in HA,(DK /FJ/S2186/11-HA/Q226 R and DK/FJ/S2186/11-HA/G228S).Three mutant strains derived from DK/GX/S4011/14 has mutations of A138 S and G228 S in HA and mutation of A588 T in PB2,(DK /GX/S4011/14-HA/A138 S,DK/GX/S4011/14-HA/G228 S and DK/GX/S4011/14-HA/G228S+PB2/A588T).Two mutants derived from DK/GX/S4234/14 has mutations of Q226 L and has the mutation of D701 N in PB2,(DK/GX/S4234/14-HA/Q226 L and DK/GX/S4234/14-HA/R224K+PB2/D701N).Solid-phase binding ELISA method was used to detect the receptor binding properties of 3wild-type strains and 7 mutants.It was found that mutation of A138 S in HA can improve the binding ability of H3N2 AIV to ?-2,6 SA,which is the human type receptor,so that the virus preferentially binds to ?-2,3-SA.And the avian receptor is converted to bind avian and human receptors at the same level.The mutations of Q226 R,Q226L and G228 S in HA can change the receptor binding properties of H3N2 AIVs from avian receptors to human type receptors preferentially.We analyzed the amino acids at positions 226 and 228 of H3N2 avian and human influenza viruses detected since 1970.Among 512 avian isolates,493 have 226 Q in HA,and 492 have 228 G.In human H3N2 isolates,the amino acid at 226 differed at different stages: 226 Q was detected in a few strains isolated from 1970–1990.After 1990,the amino acid at 226 in HA of the viruses experienced a transition from L to V and then to I.However,over the past 50 years,99.99% of H3N2 subtype human influenza isolates have 228 S in their HA.The avian-like 226 Q in HA was detected in many of the early H3N2 human viruses suggesting that mutation at 226 may not be an absolute requirement for H3N2 virus to cause a pandemic,whereas the 228 S mutation is important and necessary for a pandemic H3N2 virus.Infection study in mice showed that all of mutants of H3N2 AIVs can replicate effectively in the respiratory system of mice.There is no significant change in the pathogenicity of the virus to BALB/c mice after mutation.Ferrets were used to evaluate the replication and transmission of DK/FJ/S2186/11-HA/Q226 R,DK/FJ/S2186/11-HA/G228 S,DK/GX/S4234/14-HA/Q226 L and DK/GX/S4011/14-G228 S.We found that all four viruses replicated efficiently in ferrets.Three of the four H3N2 mutants that bear 226 L or228S in HA are highly transmissible in ferrets.These results indicate that the occurrence of Q226 R,Q226L and G228 S in HA can enhance the droplet transmission ability of the virus.The above results indicated that H3N2 subtype AIVs can effectively replicate in ferrets after being infected,and easily to obtain key amino acid mutations when it spreads among ferrets.After mutation,the receptor binding properties of the viruses can be changed,and it can transmit efficiently through respiratory droplets among ferrets,suggesting that this subtype of AIVs is a potential threat to humans.The monitoring,prevention and control of this subtype of AIVs should be further strengthened.