Study On The Safety And Protective Efficacy In Piglets With A Porcine Pseudorabies Virus Vaccine Strain Carrying Deletion Of TK/gE/gI Genes

Pseudorabies virus（PRV）is an alpha-herpes virus that mainly causes reproductive disorders in sows and neurological diseases in piglets.In 2011,the gene changes of the virus occurred in China,and outbreaks occurred in large-scale pig farms in many provinces,causing serious economic losses.Our laboratory isolated and identified a PRV high virulent strain ZJ01,and constructed a strain with deletion of TK/g E/g I gene,named as ZJ01R.In this study,ZJ01R was serially passaged by 35 times in ST cells,and the virus titer,purity,immunogenicity and virulence stability of the generation were systematically studied.And the possibility of virulence retun,and the genes recombination between the virus strain and parental strains and traditional strains were examined.Furthermore,immune efficacy of ZJ01R was studied by comparison with three commercial PRV gene-deleted live vaccines.The results showed that PRV ZJ01R strain has high purity and stablility of avarulent and immuncity.And it has no risk of the retuning of virulence and recombination of the virus with the wild PRV genes.The ZJ01R vaccine also provides excellent protection efficacy against PRV variant strain challenge.It makes an important foundation for the development of PRV new vaccine.The main research contents including 5 parts as following:1.Passage and purity identification of PRV TK/g E/g I gene deletion strain ZJ01RIn this study,PRV ZJ01R strain was continuously passaged by 35 times in ST cells,and the virus TCID₅₀ and purity were determined respectively by using the different passages virus.The results showed that the TCID₅₀ of the F1,F5,F20,F30 and F35 passages was 10^8.0-10^8.5/m L.PCR detection of the F1,F5,F20 and F35 passages was positive for the g B gene,and negative for both TK and g E/g I genes of PRV.And there is no bacterial contamination,no mycoplasma contamination,and no exogenous virus contamination in different generations of the virus liquids.The results show that the ZJ01R strain has a stable virus titer,no bacterial,mycoplasma and foreign virus contamination,and high purity within 35passages.It can be used as a candidate virus for developing a new PRV genes deletion live vaccine.2.The immunogenicity stability of a PRV live vaccine strain ZJ01R in seed batchThe 28-30 days-old piglets free of PRV infection were selected and individually injected intramuscularly with the F1,F5,F20,F30 and F35 generation of PRV ZJ01R strain at dose of 10^6.0TCID₅₀ for one piece.And DMEM was inoculated as negative control.The immunological efficacy was observed by challenge with PRV ZJ01 strain at 28 days post vaccination.The results showed that all the piglets immunized with the ZJ01R strain produced higher levels of anti-g B and no anti-g E antibodies detected with ELISA.And anti-PRV ZJ01 neutralizing antibodies titers were more than 1:32.After challenge with the virulent strain ZJ01 nasally,all the piglets in the no vaccination control group showed serve respiratory clinical signs and neurological symptoms,and 3 out of 5 piglets died.But the piglets in the 5 ZJ01R immunized groups had no abnormal clinical symptoms and could resist the challeng with the virulent strain effectively.The viral loads in the brains of pigs were significantly lower than those in the challenge control group（P<0.05）,and the protective effect was 100%.It indicted that PRV ZJ01R strain is stable in immunogenicity,and can be used for the development of vaccine in the future.3.The distribution and virulence stability of PRV vaccine strain ZJ01R in pigletsIn order to clarify the safety of PRV strain ZJ01R in piglets,21 to 28-day-old piglets free of PRV infection were selected and injected intramuscularly with ZJ01R strain（F5）.And the distribution of the virus in piglets after vaccination was observed.The results showed that the pigs had no obvious clinical symptoms after inoculation with ZJ01R.PRV ZJ01R could be detected in the pig brain tissue,but not in the liver,lung,spleen,tonsil and lymph nodes at 7,14 and 21 days post inoculation（dpi）.It indicates that the ZJ01R can infect in the central nervous system with no pathogensis in piglets.In second experiment,21 to 28-day-old piglets free of PRV infection were inoculated with ZJ01R of F5,F30 and F35 generations,respectively,and observed for 14 days.The results showed that the piglets inoculated with the different virus generations had no fever and no obvious clinical symptoms.The secreting of the virus occurred only at 3-6 dpi.The piglets had no macroscopic and histopathological lesions at 7 and 14 dpi.And the ZJ01R could not be detected in the liver and lung tissues,but could be detected in the brain tissue by PCR.Meanwhile,ant-PRV g B antibody could be detected with ELISA,but anti-g E could not.In addition,60 8-weeks old BALB/c mice were selected and inoculated with F5,F30 and F35 generation of ZJ01R and observed for 14 days.The results showed that the mice inoculated with the virus showed no clinical signs or death.And ZJ01R could be detected in the brain tissues of 6～7 out of 10 mice by PCR,but not in other tissues.Meanwhile,all the mice in the ZJ01 challenge control group died,and the virus could be detected by PCR.Those results show that the piglets immunized with the different passages of PRV ZJ01R strain showed no clinical symptoms,and excreting virus shortly time,and the virus existing and low injury in the brain tissue.The ZJ01R was not pathogenic to piglets and it has avirulent stability.4.The possibility of the virulence recovery and the gene recombination of ZJ01R with other virulent strainsIn order to further clarify the safety of PRV strain ZJ01R,21-28-day-old PRV-negative healthy piglets were used to passage ZJ01R.The piglets were inoculated with ZJ01R（F1generation）intramuscularly.And the brain tissue homogenate containing the virus was prepared at 14 dpi,and then be used to inoculate into 5 susceptible piglets according to the same method.As this method,the virus was passaged in piglets by 5 times.After each inoculation,the animals were kept in isolation for 14 days,and their body temperature was measured every day.On the 14th day,they were culled.Pathological observation and ZJ01R detection of brain,lung and liver tissues were carried out.The results showed that no clinical symptoms,macroscopic lesions and histopathological changes were observed in the piglets inoculated with the virus tissues,comparing with those in negative control group.During 3-6 days after inoculation,nasal swabs were positive for PRV.At 14 days after inoculation,ZJ01R was positive in brain tissue,and negative in liver and lung tissue.At fifth passage,ZJ01R g E/g I and TK gene tests were negative,and the g B gene sequence was consistent with the F1 generation virus.It indicats that the PRV ZJ01R strain had no virulence reversion after being passaged in piglets by 5 times.Meanwhile,the possibility of the genes recombination between ZJ01R and other PRV strains were examined.ZJ01R and its parental strain ZJ01were mixed in equal amounts,and passaged by 3 times,and 3 clones were selected in the plaque test.The results of PCR for the TK,g I/g E and g B genes of PRV showed that 2 of 3clones were PRV ZJ01R and 1 of 3 clones was PRV ZJ01,indicating that the genes recombinant did not occurred in the mix cultures of those two viruses strain.In addition,ZJ01R and LA strains were mixed in equal amounts and passaged by 10 times,and 10 plaques were selected in the plaque test.The identification results showed that 6 plaques were cloned as ZJ01R,and 4 plaques were LA strains.Meanwhile,no recombinant virus appear in the mix cultures.The above results showed that the PRV ZJ01R strain did not return to strong virulence after 5 consecutive passages in pigs,and there was no genetic recombination in the mixed culture with the wild ZJ01 strain and the LA strain.It had high safety in pigs.5.Comparison of immune efficacy of ZJ01R strain vaccine with those of the commercial PRV vaccinesIn this study,the PRV live vaccine（ZJ01R strain）was prepared by freeze-drying of ZJ01R virus culture.And the sterility test of the vaccine was qualified.The virus content in the vaccine was 10^6.5TCID₅₀/ml.PRV-negative piglets aged 28-30 days were inoculated with ZJ01R vaccine and 3 commercial PRV vaccines with strains of Bartha-K61,HB2000 and SA215,respectively,to compare their immune protection efficacy.The results showed that the anti-g B antibody could be induced at 7 dpi and increased until 28 dpi.Anti-PRV neutralizing antibodies also gradually increased from 14 to 28 days after immunization.The neutralizing antibody titers against PRV LA strain in the sera of pigs vaccinated with the 4strains vaccine were 1:38.4～1:44.8.However,the anti-PRV ZJ01 neutralizing antibody titers in the pigs inoculated with ZJ01R,Bartha K61,HB2000 and SA215 strain were 1:44.8,1:11.2,1:20.8,and 1:20.8,respectively.At 28 dpi,the pigs were challenged with PRV ZJ01 intranasal and observed for 14 days.The results showed that the pigs in the challenge control group developed severe clinical symptoms,and 3/5 pigs died.But all pigs in the ZJ01R,HB2000and SA215 vaccines groups had no obvious clinical symptoms,and the immune protection rate was 100%.Meanwhile,the immune protection rates in Bartha-K61 vaccine group was only 60%.From 1 to 14 days after challenge,the viral nasal cavity excretion amount and excretion time of ZJ01 in ZJ01R vaccine group were significantly lower than those in Bartha-K61,HB2000 and SA215 vaccines groups.At 14 day post challenge,the PRV loads in the lung tissue in ZJ01R vaccine group were significantly lower than those in Bartha-K61,HB2000 and SA215 groups（P<0.05）and the challenge control group（P<0.01）.It indicates that the ZJ01R strain live vaccine could provide high protective efficacy against PRV virulent ZJ01 challenge.It has high value be used to develop new PRV vaccine against the epidemic PRV variant strains in the future.