Pathogenicity Of DHAV-3 Isolated From Guangdong Province And Immune Effection Of Recombinant DEV Expressing VP1 Gene Of DHAV-3

Duck virus hepatitis(DVH)causes a highly fatal,contagious and rapidly spreading viral infection of young ducklings at approximately 1 week old;in mainland China,DVH is primarily caused by DHAV-1 and DHAV-3.The emergence of DHAV-3 and co-infection with DHAV-1 and DHAV-3 caused DVH difficultly to control.Duck viral enteritis,which is caused by virulent duck enteritis virus(DEV),is a highly serious infectious disease in ducks,geese,and wild waterfowls.DEV,also called anatidherpes virus 1,is a member of the Mardivirus genus in the Alphaherpesvirinae subfamily of the Herpesviridae family in the order Herpesvirales.As a recombinant viral vector,duck enteritis virus has been thoroughly studied,because it has a huge genome and a narrow host range.In this study,the main contents included two aspects,on the one hand,we systematic resesrched on the pathogenicity of DHAV-3 isolated from Guangdong province;on the other hand,we conducted recombinant DEV expressing DHAV-3 VP1 and evaluated its immune response effect and protective effect.1.Pathogenicity research of DHAV-3(1)Virulence identification of DHAV-3We observed and detected clinical symptoms,gross lesions,histopathological analysis and viral RNA loads in different tissues of DHAV-3-infected dead ducklings and measured ELD50 and LD50 of DHAV-3.The results showed that DHAV-3 could cause the death of duck embryo and ducklings quickly,and the mortalities were extremely high.Anatomical analysis revealed that dead ducklings had typical visible lesions in liver,spleen and gallbladder.And heart,liver,spleen,lung,kidney,intestine and brain had different degree pathological damage with DHAV-3 infected.The highest viral titer was observed in the liver.DHAV-3 also replicated rapidly in the spleen,to a higher titer and the viral titer in thymus and muscle was the least.LD50 of 11-day-old duck embryo was 10-5.167/0.2 mL and LD50 of 5-day-old ducklings was 10-6.375/0.2 mL.(2)Transcriptome analysis of ducklings livers post DHAV-3 infection5-days old ducklings were infected with 10 LD50 DHAV-3,and livers were collected at 12 and 48 hpi.Total RNAs were extracted and sequenced by RNA-Seq technology.The sequenced segments were compared and noted for screening of the differentially expressed genes by GO and KEGG database in NCBI.The results showed genes expression pattern in livers at 12 and 48 hpi were significant differences.At 48 hpi,large numbers of immune genes were up-regulated and mainly enriched in the signaling pathways as Cytokine-cytokine receptor interaction,Toll-like receptor signaling pathway,RIG-I-like receptor signaling pathway,Apoptosis and Jak-STAT signaling pathway.(3)Detection of innate immune response related molecular of DHAV-3 infection5-days old ducklings were infected with 10 LD50 DHAV-3.Viral loading and the relative expression of immune-related genes of livers and spleens at 3,6,9,12,24,36 and 48 hours post infection were quantified with RT-qPCR.The results showed that low copies virus was detected in the livers and spleens at 3 hpi and the expressions of 12 kinds of immune response genes was up-regulated at different degrees in the livers,but those presented diversity in the spleen.At 6-24 hpi,the expression pattern were different between livers and spleens.With the time extension,the DHAV-3 copies was accumulated,the expression of immune response genes were significant up-regulated at 36?48 hpi,but failed to resist DHAV-3 infection.All ducklings were dead,eventually.2.Construction of recombinant duck enteritis virus expressing VP1 gene from DHAV-3(1)Cloning the full lenth gene of DHAV-3 and identification of VP1 antigen advantage domainAccording to DHAV-3 reference sequence was published on GenBank,the genome of DHAV-3 was divided into six overlapping fragments.Using synthetic degenerate primers,the full length gene of DHAV-3 was cloned.Specific primers for DHAV-3 VP1 gene were designed.Then,we obtained VP1 gene by RT-PCR,and VP1 was truncated and expressed.Mouse anti-DHAV-3 pAb and chicken anti-DHAV-3 yolk antibody were used to identify antigen advantage domain of VP1.The result showed that its antigen advantage domain was 90?175 aa.(2)Selection,purification and identification of recombinant virus with gpt select markerFirst of all,VP1 was cloned into pcDNA3.1(+),and the VP1 expression cassette was amplified,which contains the CMV promotor,VP1 gene and BHG pA.Using pMD18T-gpt expression cassette as the templet and the loxP site was on both ends of gpt expression cassette with PCR.The gpt-loxP expression cassette gene was amplified and inserted into pEASY-Blunt simple to obtain pEASY-Blunt-simple-gpt-loxP expression cassette.By a series of enzymatic cleavage and ligation,recombinant virus transfer vector pEASY-?Us10-gpt-loxP-VP1 was constructed successfully.The transfer vector pEASY-?Us10-gpt-loxP-VP1 and total DNA of DEV C-KCE were co-transfected in DEFs.After 11 rounds of MPA pressure screening and 4 rounds of plaque purification,the purified recombinant virus containing the selection marker was obtained and it was correct by PCR identification.It was named as rDEV-?Us10-gpt-loxP-3-VP1.(3)Selection and purification of recombinant virus without gpt select markerTotal DNA of rDEV-?Us10-gpt-loxP-3-VP1 and the recombinant plasmid pKozak-NLS-Cre were co-transfected in DEFs.Under the action of Cre recombinant enzymes,the gpt expression cassette between two loxP sites which were in the same direction was removed and remained the only one loxP site.Identification results showed that recombinant virus without the selection marker gpt gene was detected in mixed virus by PCR.After 5 rounds of plaque purification,the purified recombinant virus only containing DHAV-3 VP1 gene and one loxP site were identified by PCR,RT-PCR,Western blot and IFA and named as rDEV-AUs10-3-VP1.Total DNA of the first,fifth,tenth,fifteenth and twentieth generation rDEV-AUs10-3-VP1 was extracted and VP1 was amplified with specific primers for DHAV-3 VP1.The results demonstrated that VP1 gene was stable in the genome of rDEV-AUs10-3-VP11.After overspeed centrifugal concentration and stain by phosphotungstic acid solution,we observed typical duck pestilence virus particles that containing capsule membrane and nuclear capsid form under EM.3.Animals immune and challenge(1)Laying ducks immuneLaying ducks were vaccinated intramuscularly with rDEV-?Us10-3-VP1 at three doses,a dose of 2×105,5×105 and 1×106 PFU,DEV C-KCE at a dose of 2×105 PFU,and PBS as a control,respectively.Laying ducks were immuned twice.Boost immune was conducted three weeks after the first immunization.The results showed that the titer of specific antibody to DEV C-KCE and DHAV-3 VP1 reached peak at 7 d after the boost immunization in serum.In yolk,the titer of specific antibody reached its peak 14 d after the boost immunization.Specific antibody in serum and yolk presented a dose dependent with immune dose.The kinetic level of NT antibody titer against DEV in serum and yolk had the same trend with the specific antibody.And it also presented a dose dependent,but NT antibody titer against DHAV-3 was very low.The numbers of CD4+ and CD8+ T-lymphocytes were both increases,but the numbers of CD8+ T-lymphocytes was more than that of CD4+.The results showed the ratio of CD4+/CD8+ T-lymphocytes was decreased at 7 d post vaccinated,and continued to decline at 14 d post vaccinated and recover to normal levels at 21 d post vaccinated.(2)Ducklings Immune and challenge3-day-old ducklings were immuned intramuscularly with rDEV-?Us10-3-VP1 and DEV C-KCE at a dose of 5?105 PFU and PBS as a control and only with a single immune.And ducklings were challenge with 100 LD50 virulent DEV AV1222 strain and virulent DHAV-3 at 7 days after immunization.Observed daily and recorded the clinical symptoms and death until 2 weeks after attack.Survival rates in the rDEV-?Us10-3-VP1 group and DEV C-KCE group were 80%and control group ducklings were dead,which suggested that DHAV-3 VP1 insert and DEV US 10 deletion did not affect DEV C-KCE provide the protection to resistant DEV AV1222 infection.But all ducklings in the DEV C-KCE and control group were dead and only one in the rDEV-?Us10-3-VP1 group was alive at the fourth day after challenge with lethal DHAV-3 and it was dead at the eighth day after challenge with DHAV-3.Above results showed that rDEV-AUs 10-3-VP1 could not provide effective protection for ducklings to resistant DHAV-3 infection.Our studies provide the data for exploring DHAV-3 infection mechanism and duckling resistance DHAV-3 infection.And it provide theoretical basis and technology platform for constructing recombainant virus live vector vaccine and perfect the reverse genetic operation platform of DEV in our lab,and provide technology and material basis for further explore the candidate insertional sites of recombinant DEV and the biological function of US 10 protein.