| H9N2 subtype avian influenza,whose genetic structure is complex,is difficult to control and has become a serious threat to Chinese poultry industry.In this study,three H9N2 subtype influenza virus strains were isolated from the live poultry market in Jiangsu,Yangzhou province and the animal hospital of Yangzhou University,named A/Goose/Jiangsu/YZ527/2011(H9N2,Gs/JS/YZ527/11),A/Goose/Jiangsu/SQ119/2012(H9N2,Gs/JS/SQ119/12)and A/Goose/Jiangsu/JD564/2012(H9N2,Gs/JS/JD564/12),respectively.The genetic characteristics,infectivity and innate immune responses in chickens and geese for the three isolates were analyzed.The results of genetic characteristics showed that HA and PB2 genes of Gs/JS/SQ119/12 strains belongs to G9-like and G1-like lineage,HA and PB2 genes of Gs/JS/YZ527/11 and Gs/JS/JD564/12 belong to the F/98-like lineage,while NA,PB1,PA,NS and NP genes of Gs/JS/YZ527/11,Gs/JS/SQ119/12 and Gs/JS/JD564/12belong to the F/98-like lineage;M gene Gs/JS/YZ527/11 and Gs/JS/SQ119/12 belong to G1-like branch,M gene Gs/JS/JD564/12 belongs to the F/98-like branch.Following genotypic analysis,the three-amino-acid deletion at residues 63-65 of NA were observed in three isolates(Table 1).These results showed that Gs/JS/YZ527/11 virus was a double-reassortant virus including G1/97-like and F/98-like viruses;Gs/JS/JD564/12 virus almost was the prototype of F/98-like virus except NA gene;and Gs/JS/SQ119/12 virus was a triple-reassortant virus containing G1/97-like,F/98-like and ST/163/04-like viruses.The HA gene cleavage site of the three isolates were RSSR↓GLF,but there were differences in the receptor binding sites of 198 and 233-234 amino acid sites which consist of pouch-like structure of HA gene and glycosylation sites.Compared with the Ck/SH/F/98 strain,the red blood cell adsorption and glycosylation sites on the NA gene of the isolate were changed.The assay of pathogenicity to goose and chicken showed that Gs/JS/JD564/12 strain can only be pathogenic to a portion of chickens and geese and the two strains of Gs/JS/JD527/11 and Gs/JS/SQ119/12 are both pathogenic to chickens and geese.The results above showed that the three goose origin strains of H9N2 subtype avian influenza virus were complex,and there was significant difference in the infection rate between chickens and geese.Chickens and geese were respectively terrestrial and aquatic hosts of avian influenza virus,which had their specific responses to avian influenza virus.Chickens were usually susceptible to the infection of avian influenza virus,but geese not.In this study,we compared the three isolates of H9N2 which have the differences in the innate immune response in chickens and geese by using RT-qPCR.In chicken,the TLR-7 and IFN-γ gene expression induced by Gs/JS/SQ119/12were significantly upregulated 3.7 times(P<0.05)and 2.2 times(P<0.05)than those of the empty control group,respectively,on day 3 after inoculation.But in geese,the level of TLR-7 gene expression induced by Gs/JS/SQ119/12,was strongly upregulated 4.0 times(P<0.05)compared with the controls(P<0.01)at 1 dpi.Only IFN-y titer in the chickens inoculated with Gs/JS/YZ527/11 was upregulated 2.1 times(P<0.05)than that of controls at 3 dpi,implying that the antivirus programs of IFN were initiated by Gs/JS/YZ527/11 in chickens.In geese,The TLR-7 gene expression induced by Gs/JS/YZ527/11 was increased 2.3-fold higher than that of controls at 3 dpi(P<0.05).The above results showed that both Gs/JS/SQ119/12 wand Gs/JS/YZ527/11 viruses could quickly start the Toll-like receptor recognition in the geese,but were slow in chicken.The TLR-7 gene expression induced by Gs/JS/JD564/12 was higher 2.7 times(P<0.05)than that of controls at 1 dpi,similarly at 1 dpi,the TLR-7 gene expression induced by CK/SH/F/98 was higher 6.1-fold(P<0.01)than that of controls.Indicating that the level of TLR-7 gene expression induced by CK/SH/F/98 was apparently higher than that of the Gs/JS/JD564/12(P<0.05).In geese,the TLR-21 and RIG-I expression induced by Gs/JS/JD564/12 was upregulated 4.2 times(P<0.01)and 4.9 times(P<0.01)compared to that of controls.It shows that the Gs/JS/JD564/12 virus could also quickly start the innate immune response in the host goose.In chickens,the level of IL-1β induced by Gs/JS/JD564/12 was significantly higher 5.5 times(P<0.01)than that of controls at 1 dpi,while the IL-1β expression from CK/SH/F/98-infected group was significantly upregulated 1.9 times(P<0.05)than that of controls.It is shown that the Gs/JS/JD564/12 virus was cleaned more quickly than the CK/SH/F/98 virus in the host chicken.In addition,the level of the innate immune response factor expression in the host goose inoculated with Gs/JS/JD564/12 was apparently higher than that in the host chicken,which reached a peak at 5 dpi.For example,in the groups infected with Gs/JS/JD564/12 at 5 dpi,the TLR-21,IFN-α,IFN-γ,IL-1β and IL-6 gene expression in host goose was 34.5 times(P<0.01),46 times(P<0.01),21.6 times(P<0.01),14 times(P<0.01)and 13.7 times(P<0.01)higher than that in host chicken,respectively.This results indicated that Gs/JS/JD564/12 strain not only could be quickly recognized by the geese,but also could induce a stronger innate immune response in geese than that in the chicken host.In contrast,the innate immune response induced by CK/SH/F/98 strain in chickens could be stronger than that in geese.Sequence analysis revealed that,the Gs/JS/JD564/12 and the CK/SH/F/98 genes shared high homology in HA,PB2,PB1,PA,NS,NP and M genes,only the NA gene of Gs/JS/JD564/12 shared 92.7%homology with that of CK/SH/F/98,and had additional a new glycosylation site at position 264,which effect on pathogenicity and innate immune responses in chicken and geese were required further study.In summary,the innate immune responses induced by the three isolates in geese could stronger and faster than those in chickens. |
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