| H9N2 subtype avian influenza is an infectious disease that causes serious damage to poultry and is closely related to human public health.Epidemiological studies have shown that there are many different genotypes of H9N2 subtype AIV in domestic poultry in recent years.In order to reveal the epidemic pattern of H9N2 subtype AIV in Guangdong and its pathogenicity to waterfowl and mammals,in this study,a total genetic evolution analysis,antigenic variation,and pathogenicity in goslings and Balb/c mice were performed on 7 H9N2 subtype AIV isolated from poultry in Guangdong province from 2016 to 2017.The genetic evolution analysis showed that the HA genes of the seven strains of H9N2 subtype AIV were all distributed on the BJ/94-like branch,that is,the h9.4.2.2 branch and belonged to the same strain as the vaccine strain A/Chicken/Shanghai /F/98.The 7 isolates were all located in the BJ/94-like branch of the NA gene,of which 5 were closely related to the Wuhan isolate A/Chicken/Wuhan/JXQL01/2015.The PB1,PA,NP and NS genes of 7 isolates were located in the F/98-like branch.The PB2 gene is located on the new branch represented by the strain A/Chicken/Shandong/SIC39/2015.6 strains of the M gene are located on the G1-like branch represented by the strain A/Quail/HongKong/G1/97,in which a strain CK/GD/17126/2017 is located on the TW/66-like branch of the North American lineage,indicates that the gene of the H9N2 subtype AIV presents a diverse rearrangement or recombination.The analysis of the sequence of the gene segments,the amino acid sequence and the variation of the molecular characteristics found that the amino acid sequences of the cleavage sites of the amino acid sequences 333-338 of HA of the 7 H9N2 avian influenza strains in this study were only PSRSSR,lacking of basic amino acid insertions,in line with the sequence characteristics of low pathogenic avian influenza.All amino acids at position 234 are capable of binding to mammalian a-2,6-galactoside sialic acid receptors and are resulted in infection to mammals.At position 315,the amino acid mutation from P to S caused a glycosylation site at position 313,resulting in the original eight glycosylation sites becoming nine.The seven viruses in this study were missed 3 amino acids T,E,and I in positions 63-65 of the neck of the NA protein.The amino acid deletion at this site is considered to be a unique molecular marker for avian influenza of the Chinese chicken H9N2 subtype.The loss of this site also results in the loss of one glycosylation site of the virus at position 61.Except the CK/GD/17126/2017 strain which belong to the TW/66-like branch,was not mutated at 31 in the this study,and other strains were mutated from S to N at position 31.This mutation was consistent with the molecular characteristics of avian influenza virus resistance to amantadine.Other internal genes were not found to enhance the virulence of the virus or extend the host range of mutation characteristics.The pathogenicity test of goslings on the goslings showed that: after 4 isolates were intramuscularly inoculated with 106 EID50/200 μl of virus to inoculate 7-day-old geese,all attacking geese had no obvious symptoms.No virus was detected from the goose’s throat or cloacal swab.Four days after the challenge,no virus was detected in the heart,liver,spleen,lung,kidney,and brain tissues of the attacking goose.The strain CK/GD/17549/2017 can detect the virus in the brain and heart,but the virus titer is low.It was shown that the four strains of H9N2 subtype AIV tested did not cause systemic infection and clinical disease in goslings by intramuscular injection.The pathogenicity test of the strains to Balb/c mice showed that 4 isolates were inoculated intranasally with a dose of 106 EID50/4 weeks old Balb/c mice,and each challenged group of mice appeared the weight loss situation.The weight loss of mice in the PG/GD/ZG12/2016 challenge group was the most obvious,but none of the mice died in each group.All 4 isolates were able to replicate in the lungs of mice with a virus titer of up to 6.4 log10 EID50/200μl.The strain CK/GD/16719/2016 detected virus in the lungs,turbinate,brain and kidneys after infection in mice.In addition,the virus was also detected in the heart of mice infected with the strain CK/GD/17530/2017.It showed that some of the H9N2 subtype AIV isolates could be replicated in multiple tissue organs in Balb/c mice after infection,and had certain pathogenicity. |
