Molecular Mechanism Of The Evolution And Pathogenicity Of Highly Pathogenic H7N9 Avian Influenza Viruses

H7N9 highly pathogenic avian influenza virus?HPAIV?,emerged in China in 2017,obtained an insertion of four amino acids in their hemagglutinin cleavage site and were lethal in chickens,leading to disastrous consequences for the poultry industry.More importantly,H7N9 HPAIVs have caused severe human infections,posing an increased threat to public health.We systematically characterized the genetic evolution,receptor binding preference,transmission in ferrets,pathogenicity in mammals and waterfowl,and the molecular mechanisms of the virulent difference in mammals of these viruses.Our study provides a picture of the evolution of H7N9 and sheds light on the control and basic research of H7N9 in China.Here,we evaluated the evolution of 81 HPAIVs isolated from avian species and humans between2017 and 2018.H7N9 HPAIVs evolved quickly and formed 8 different genotypes since its first emergence in Guangdong province.G2 viruses were predominant and they were detected in 13 provinces.All H7N9HPAIVs could bind to the SA?-2,3 Gal and SA?-2,6 Gal receptors simultaneously.Although CK/SD008virus replicated efficiently in the nasal turbinates,tonsils,trachea,lungs,and brains in ferrets,the virus transmitted in ferrets by respiratory droplet with low efficiency.But CK/SD008 readily acquired the PB2E627K or PB2 D701N mutation when replicated in ferrets.We purifed the mutants from the ferret lung samples by limited dilution in eggs.The mutant viruses,SD008-PB2/627K and CK/SD008-PB2/701N,were both lethal and efficiently transmissible in ferrets.H7N9 HPAIVs could replicate efficiently in the turbinates and lungs of mice,although the mortality rate varied among different viruses.The 50%mouse-lethal dose(MLD₅₀)of H7N9 HPAIVs ranged from 3.2 log₁₀0 EID₅₀ to 4.8 log₁₀0 EID₅₀.These results indicated that,after circulating in poultry for only a few months,some H7N9 highly pathogenic viruses have become virulent in mice.Some H7N9 HPAIVs,which were reassortants of the chicken H7N9 viruses and some unknown duck viruses,replicated systemically in ducks and were lethal in ducks.We compared two genetically similar H7N9 HPAIVs,CK/S1220 and CK/SD098,which both belonged to genotype 2 but differ in their pathogenicity in mice.To assess the genetic basis of the difference in virulence in mice,we generated a series of reassortants and mutants by reverse genetics and tested their virulence in mice.We found that NP gene of CK/SD098 virus dramatically attenuated the virulence of CK/S1220 virus and that the amino acids at positions 286 and 437 in NP were both important determinants for pathogenicity in mice.We further demonstrated that these two amino acids affected the import and export of NP into the nucleus,thereby dramatically decreased the synthesis of cRNA,mRNA and vRNA,thus impaired the viral life cycle and attenuating the virulence of H7N9 HPAIVs in mice.In summary,our results provide strong evidences that H7N9 HPAIVs evolved rapidly and some viruses were lethal in mammals and ducks.H7N9 HPAIV readily acquired PB2 E627K or D701N mutation and these mutant viruses were lethal and efficiently transmissible in ferrets.Additionally,NP subunit is an important virulence factor for H7N9 HPAIVs in mice.These results can provide references for the future monitoring and prevention of H7N9 HPAIVs.