| H9N2 avian influenza virus(AIV)has been circulating widely in poultry in China,which is highly adapted to poultry and has become one of the predominant subtypes.H9N2 AIV has not received sufficient attention as it is a low pathogenic pathogen.However,H9N2 AIV can infect a wide range of host species and provides internal genes for emerging influenza viruses such as H7N9,H5N6 and H10N8.H9N2 AIV poses a potential risk to animal and public health.It is particularly important to study the mechanisms for epidemiology,pathogenicity and transmission of H9N2 AIV.In this study,ten H9N2 AIV strains were isolated during monitoring for AIVs epidemics and evolution in Liaoning Province,China.The hemagglutinin(HA),neuraminidase(NA),polymerase basic 1(PB1),polymerase acidic(PA),nucleoprotein(NP)and nonstructual protein(NS)of these ten isolates were all from the F98 subline of the Eurasian lineage of H9N2 AIVs.The nucleotide sequence identity was 93.8～100.0%.The polymerase basic 2(PB2)and matrix protein(M)genes were from G1 subline of the Eurasian lineage of H9N2 AIVs and showed 93.6～100.0% homology.Phylogenetic analysis of ten H9N2 isolates indicated that they all belonged to genotype S,which was genotype with more recombination with H7N9 and other viruses.The study of biological characteristics found that ten H9N2 isolates showed different lethality to embryonated chicken eggs.Two H9N2 isolates with different lethality to embryonated chicken eggs but similar genetic backgrounds were selected as model viruses to explore the molecular mechanism determining the pathogenicity and transmissibility of H9N2 AIVs,namely A/chicken/Liaoning/07/2016(CKLN/07)with lethality to embryonated chicken eggs and A/chicken/Liaoning/17/2016(CKLN/17)without lethality to embryonated chicken eggs,separately.By means of the reverse genetics system,single gene substitution and site-directed mutation,the HA protein was identified as the key protein affecting the pathogenicity of the two H9N2 isolates.There was only one different amino acid residue at the position 137(H3numbering)in HA protein of the two isolates,so the HA-137 isoleucine(I)was the key molecular marker enhancing the pathogenicity of H9N2 AIVs.We evaluated the transmission of the two H9N2 isolates in guinea pigs.The results showed that CKLN/07 was limitedly transmitted among guinea pigs,and two of three guinea pigs were infected in contacted group,while CKLN/17 could not be transmitted among guinea pigs.When the threonine(T)at the position 137 in HA protein of CKLN/17 was mutated to I(HA-T137I),CKLN/17 obtained the efficient transmissibility,and all three guinea pigs were infected in contacted group.We also found that the polymerase activity was enhanced when the position 352 in NP protein was replaced by methionine(M)from valine(V),wnich explained why CKLN/17 with HA-T137 I mutation and NP-352 M had stronger transmissibility.Further studies showed that the HA-T137 I mutation changed the receptor-binding characteristics of the H9N2 AIVs from α2,3 sialic acid receptors to α2,6sialic acid receptors.HA-T137 I mutation also improved heat stability and reduced the p H value of HA protein activation of H9N2 AIVs,so that H9N2 AIVs had stronger environmental resistance and were more likely to infect mammals and spread among mammals.H9N2 AIVs can infect a variety of wild birds.The transmission mechanism of H9N2 AIVs between wild birds is still far from being fully understood.A large number of wild birds inhabit in the wetland ecological environment of Liaoning Province,and the freshwater crab is an important biological link in the wetland ecosystem.In this study,the freshwater crab was used as the representative model of wetland biology to explore the transmission mechanism of H9N2 AIVs in multi host wetland ecosystem.We analyzed the effects of freshwater crabs on the bioaccumulation and transmission of H9N2 AIVs.The study found that freshwater crabs could bioaccumulate H9N2 AIVs and prolong the survival time of H9N2 AIVs in water for up to 12 hours.Freshwater crabs could be used as biological vectors for the transmission of H9N2 AIVs to spread influenza virus to other freshwater crabs.The above results enriched the pathogenic ecology of H9N2 AIVs,and classified the freshwater crab as a new biological vector in AIV ecosystem.In conclusion,we identified the important roles of the position 137 in HA protein and the position 352 in NP protein in the replication and transmission of H9N2 AIVs.The synergistic effect of HA and NP proteins could enhance the adaptability of H9N2 AIVs to mammalian host.Freshwater crab could be used as the biological transmission vector of AIV in nature,which provided a new research idea for the monitoring,prevention and control of AIV. |
