| Avian influenza(AI)is an important disease that seriously harms the healthy development of chicken industry and human health in China,in which H9N2 subtype avian influenza is endemic and has economic and public health significance.The existing H9N2 subtype avian influenza vaccine mainly uses oil-inactivated vaccines developed by prevalent strains,but such inactivated vaccines have a narrow antigenic spectrum and cannot induce cellular immunity and mucosal immunity.It is difficult to deal with the rapid antigen shift of avian influenza virus(AIV).Some studies in recent years have found that H9N2 subtype avian influenza virus infection is still frequent in some livestock farms that have been vaccinated.Subsequent studies have further confirmed that this phenomenon is caused by the variability of the H9N2 subtype AIV,resulting in changes in antigenicity.Therefore,it is necessary to update the vaccine strain of H9N2 subtype avian influenza virus and carry out a new vaccine development.In this study,a H9N2 subtype cold-adapted strain cultivated in our laboratory was used as the master donor virus,and the HA and NA genes were replaced with the parental strain,and a cold-adapted attenuated recombinant strain of "6+2" model was constructed.In addition,The NS gene was replaced with a fragment of NS1 truncation mutants,and a double-attenuated strain combining cold adaptation with NS1 gene deletion was constructed.The biological characteristics of the constructed strain were evaluated,and the immune efficacy was also tested.The preliminary evaluation provided new ideas for the development of avian influenza vaccine.1 Identification of cold-adapted and temperature-sensitive phenotype related genes in a cold-adapted strain of H9N2 subtype avian influenza virusIn order to identify the key genes related to cold adaptation(ca)and temperature sensitivity(ts)of cold-adapted H9N2 subtype AIV strain cultivated in the laboratory.Using PR8 as backbone,the expression plasmids of each gene of cold-adapted strain were replaced to the corresponding genes of PR8,and 293T and MDCK co-cultured cells were transfected,then 8 recombinant viruses were obtained.The result of cold adaptation characterization of the recombinant virus showed that the NP fragment played the most important role in the cold adaptation characteristics,and the NA fragment had a weaker effect.The result of ts characterization of the recombinant virus showed that the PB2 and NP fragment played the most important role in the temperature sensitive characteristics,meanwhile the PB1 and M fragment had a weaker effect.Using TX as the backbone,recombinant virus rTX-25-NP was contracted by replacing the NP gene with the cold-adapted strain’s NP segment.In addition,recombinant virus rTX-25-NP-NS1-73 and rTX-25-NP-NS1-128 were constructed by replacing the NS gene with truncated NS1 gene.These recombinant viruses with the NP gene from cold-adapted strain TX-25-CE30 did not acquire ca and ts phenotype.Therefore,multiple genes segments synergistically control the ca and ts phenotype of TX-25-CE30.2 Exploration on rescue conditions of cold adapted strain of H9N2 subtype avian influenza virusThe replication ability of cold-adapted strain TX-25-CE30 on several cell types was determined.It was found that the replication of the cold-adapted strain on the common transfected cells like 293T,MDCK,Vero and COS-1 was poor.It has good replication ability on chicken embryo fibroblast,and also has a certain ability to replicate on the passaged chicken fibroblast cell line DF-1.TX-25-CE30 had the best replication ability on 293T and DF-1 at 33℃under the concentration of 2 μg/mL TPCK-treated trypsin.The cold-adapted strain had the highest replication titer in chicken embryos at 33℃.Eight plasmids expressing the genes of H9N2 subtype cold-adapted attenuated strain TX-25-CE30 were co-transfected into 293T and DF-1 co-cultured cells,and the recombinant strain rTX-25-CE30 was successfully obtained,which has cold adaptation and temperature sensitivity characteristics as the parental strain.3 Construction of cold-adapted and NS truncated attenuated recombinant H9N2 subtype avian influenza virus and evaluation of immune efficacyUsing the internal genes of cold-adapted strain TX-25-CE30 as the backbone,the recombinant strain with HA and NA gene replaced by TX strain was successfully rescued through the reverse genetic platform established in Chapter 2 and was named as rTX Ca-HA-NA.A dual-attenuated virus which combined the cold-adapted attenuation with the truncated NS gene was successfully rescued and named as rTX ca-HA-NA-NS73,in which the HA and NA genes were derived from the parental strain TX,and NS was the segment retaining the 73 amino acids at the N-terminus of the NS 1 gene,and the remaining 5 genes were derived from the cold-adapted strain TX-25-CE30.rTX ca-HA-NA-NS73 could not be detected in tracheal and lung tissues after vaccination.In the group vaccinated with rTX ca-HA-NA,only one chicken could be detected a low level of viral replication in lung tissue on day 5 after infection,and the remaining chickens were unable to be detected for virus in the trachea and lungs.rTX-NS1-73 was detected in the trachea with a low level of replication.In the lungs,only a low level of replication was detected in 1 chicken on 3 day post inoculation,and the viruses in the remaining chickens were not detectable.The parental strain rTX was detected the highest replication titers from the trachea and lungs.Two types of viruses rTX ca-HA-NA and rTX ca-HA-NA-NS73 vaccination groups could shed virus through the throat,but hardly through the cloaca,and both viruses lost contact transmission ability.Chickens in the rTX-NS1-73 vaccinated group could shed virus only through the throat.rTX-NS1-73 did not lose contact transmission ability.The exposed chickens could shed virus through the throat on the 3rd and 5th day after exposure,but they could not shed virus through the cloaca.After 21 days of exposure,the chickens were becoming seroconversion.In the parental rTX group,the inoculated chickens could shed virus through the throat and cloaca.The parental rTX group showed the ability to spread through contact,and the exposed chickens could shed virus through the throat and cloaca.The three attenuated strains rTX-NS1-73,rTX ca-HA-NA,and rTX ca-HA-NA-NS73 all induced high HI antibody levels in chickens after immunization.rTX ca-HA-NA provides a 100%protection rate against challenge with rTX.rTX ca-HA-NA-NS73 provides a 90%protection rate against viral shedding in throat,and 100%protection against viral shedding in cloaca.rTX-NS1-73 provided 100%protection against viral shedding in throat and 75%protection against viral shedding in cloaca.When challenged with antigen shift strain YZ-C,rTX ca-HA-NA,rTX ca-HA-NA-NS73,rTX-NS1-73 could not effectively prevent the virus shedding from the throat,but could significantly reduce virus shedding through cloaca. |
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