| Avian influenza(AI)is an important disease caused by the avian influenza virus(AIV)that seriously endangers the development of poultry industry and human health.The H9N2 subtype AI is endemic in China,which has important economic and public health significance.Immunization with inactivated vaccines is the main prevention strategy of H9N2 subtype AIV in China.However,inactivated vaccines have some problems such as the narrow antigen-spectrum and a single type of immune response,which is difficult to deal with rapid antigen mutations.Therefore,the poultry farms that are vaccinated still outbreak H9N2 subtype AI.Live attenuated vaccines have multiple advantages,inculding high security,easy vaccination,long-term protection and effective mucosal,cellular,and humoral immunity.Therefore,our laboratory developed a recombinant cold-adapted vaccine candidate rTX-HA-NA.In this study,the biological,physical,and chemical properties of rTX-HA-NA strains were determined.Meanwhile,the immune efficacy was further evaluated.Our study will lay a theoretical foundation for further development of vaccines.1 Evaluation of genetic stability and physical and chemical properties of recombinant cold-adapted H9N2 subtype live attenuated vaccineFirst,the purity of rTX-HA-NA strain was determined,Next,rTX-HA-NA strain was continuously cultured on SPF chicken embryos and SPF chickens for evaluation of genetic stability in vitro and in vivo.Finally,rTX-HA-NA was treated with different physical and chemical conditions,and then inoculated into chicken embryo fibroblast cells(CEF),the physicochemical properties of rTX-HA-NA were explored by measuring the 50%tissue culture infective dose(TCID50)and the hemagglutinationassay(HA).The results showed that the vaccine was free of bacteria and mycoplasma contamination.There were no genetic mutations and no change of the biological characteristics and cold-adapted phenotype after serial passages in the chicken embryos.After continuous passage in the SPF chickens,the viral detection rate of the vaccine was significantly reduced,and there was no risk of virulence returning.Both the rTX-HA-NA and the parent virus rTX were sensitive to high temperature,ether,and chloroform,but had strong tolerance against 1%pancreatin.The HA change of rTX-HA-NA in the range of pH 3.0-9.0 was not significant.2 Safety evaluation of recombinant cold-adapted H9N2 subtype live attenuated vaccineTo systematically evaluate the safety of the cold-adapted vaccinein the target and non-target animals,inoculated SPF chickens and mallard ducks infected the virus via the ocular and intranasal routes,the clinical manifestations,local symptoms,and histopathological change were observed.Meanwhile,physiochemical indexes of blood,the viral distribution in vivo,and the ability of environmental transmission and the contact transmission were detected.The results showed that after inoculation rTX-HA-NA in chickens,individual blood indexes changed slightly,and there was no effect on weight gain or body temperature.The results of histopathological observation and the viral distribution of the virus in vivo showed that lung congestion and bleeding of rTX-HA-NA group were milder than that of rTX group,other organ symptoms were not obvious.Throat shedding rate of rTX-HA-NA group was significantly lower than that of rTX group,and cloaca does not shed.Furthermore,TX-HA-NA had no ability of contact transmission.rTX-HA-NA shedding rate of feed,drinking water and fecal swabs was lower compared with the parent virus,and it is safe to the surrounding environment.After inoculatedion rTX-HA-NA in mallard ducks the strain did not cause the changes of clinical manifestations,histopathology and blood physiochemical indexes in the non-target animals.3 The immune efficacy evaluation of recombinant cold-adapted H9N2 subtype live attenuated vaccineIn this study,the levels of humoral,cellular,and mucosal immunity after immunization with rTX-HA-NA in SPF chickens were measured.Serum detection results showed that one week after immunization the HI titer of rTX-HA-NA reached 5.2 log2,and reached a peak at five weeks after immunization(8.75 log2).The cytokine detection results showed that the cytokine transcription levels of rTX-HA-NA group were significantly higher than that of rTX on day 3 and 5 after immunization.The results of local mucosal immue response showed that the IgA secreted by the nasal and tracheal mucosa in the rTX-HA-NA group was higher than the inactivated rTX group,and the IgG level in the nasal and tracheal of the inactivated rTX group was higher than that in the rTX-HA-NA group immuned in 7 d.The results of the immune efficacy assay showed that,P1 and P15 of rTX-HA-NA can provide throat shedding protection rates of 80%and 90%after challenging homologous virus TX,respectively,and the cloaca shedding protection rate was both 100%.The inactivated rTX group provided a 80%throat shedding protection,and a 100%cloaca shedding protection.However,rTX-HA-NA can only provide a certain degree of cross-protection against heterologous strain YZ-C. |
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