DNA Vaccines Which Express σC,σB Proteins Against DRV In Muscovy Duck

Muscovy duck (Cairina moschata) reovirus (DRV) is the causative agent of Muscovy duck liver white spots disease,a newly recognized Muscovy duck disease of economic importance, characterized by apathy,weak foot, diarrhea and tenosynovitis/viral arthritis.Muscovy ducks that recover from infection are greatly stunted in growth.At necropsy,in most cases,liver and spleen are swollen and show small white necrotic foci,kidney is hemorrhagic.During the infection of DRV,young Muscovy ducklings are the most susceptible,the mortality is high.Muscovy duck reovirus belongs to Reovirus genus and is the member of Avian reovirus which is in the second subgroup.It shares common characters with Avian reovirus.The virus mainly infect Muscovy duck with the the age of 4-45days during summertime.The mortality rate is about 20%～90% with great diversity at about 10%～30%, and will reach above 90% when commix infected or stimulaled.Survivals are stunted in growth,and become stiff ducks,which affect the the development of Muscovy duck industry .There were great losses in 2000,when 20million Muscovy ducks died in Putian county of Fujian province .S3 (1104bp) and S40RF2 (810bp)gene fragments of YB isolated in Fujian express proteinσB and proteinσC of the virus respectively. Immunized with these two genes will elicite specific immune responses and provide protection against DRV, as a result , a DNA vaccine which contains S3 gene and S4ORF2 gene may provide a new strategy for protection against this virus.In order to study S3 gene and S4ORF2 gene DNA vaccines,we subcloned these two genes into eukaryotic expressing vectors pCI which contains the CMV promoter ,and designate them as pCIS3 and pCIS4ORF2 respectively. PCR and enzyme results show pCIS3 gene DNA vaccine and pCIS4ORF2 gene DNA vaccine were successfully constructed.We use ELISA to detect the antibody titer of DRV therefore an indirect enzymelinked immunosorbent assay (ELISA) was developed to detect the antibody titer of DRV.Negative serum, positive serum (its agar gel precipitation titer is 1: 16) , rabbit-duck serum (its AGP titer is 1: 128) ,HRP enzyme-labeled rabbit-duck IgG were also produced. The optimal reaction conditions were determined. It was shown that the diluting antigen to 1:40, the enzyme-labeled rabbit-duck serum to 1:400, the serum sample to 1:40 were their best working concentrations and the critical OD value was calculated as 0．5 between positive and negative. The highest-level extent of dilution of serum was determined to be its terminal point antibody titer if the diluted serum could produce positive reaction.To detect the vaccine efficacy, groups of 2days Muscovy ducks were intramuscular inoculated with two doses of 100μg of pCIS3,pCIS4ORF2,pCIS3+pCIS4ORF2 respectively, A group of Muscovy ducks were injected with PBS as control,in one week interval. One week after the second dose, all Muscovy ducks were challenged with TCID₅₀ =10^-7．1934 /0．1ml virus .Sera were collected every week after vaccination for detecting of antibody titers, and Muscovy ducks were observed daily for disease signs and deaths for 2 weeks.Results shows that all Muscovy ducks generated antibodies after the first dose and before the challenge, of which the average titers is higher than 1: 20 on the average one week after the first dose.Titers can reach the hightest point at 1:320 two weeks after challenge in the pCIS3 + pCIS4ORF2 immunized groups. While the other groups were 1:160．Muscovy ducks in the pCIS3 +pCIS4ORF2 immunized groups were completely protected from virus challenge (with few disease signs and no deaths), while pCIS3 immunized groups only provided 80 % protection(with more disease signs); Muscovy ducks in the pCIS4ORF2 immunized groups were 90 % protected from virus challenge (with few disease signs). These results indicate that pCIS3+pCIS4ORF2 immunized has better effect in the protection compared with the other two immunized groups.To detect the vaccine efficacy with adjuvants, groups of 2days Muscovy ducks were intramuscular inoculated with two doses of 100μg of pCIS3+pCIS4ORF2 with whiteruss adjuvant,pCIS3+pCIS4ORF2 with glycerine adjuvant,pCIS3+pCIS4ORF2 with aluminium hydroxide adjuvan,pCIS3+pCIS4ORF2 respectively,immunity processes were the same with the first animal experiment.Results shows that all Muscovy ducks generated antibodies after the first dose and before the challenge, of which the average titers is higher than 1: 40 on the average one week after the first dose.Titers can reach the hightest point at 1:640 two weeks after challenge in pCIS3+ pCIS4ORF2 with whiteruss adjuvant,pCIS3+pCIS4ORF2 with glycerine adjuvant groups. While pCIS3+pCIS4ORF2 with aluminium hydroxide adjuvant,pCIS3+pCIS4ORF2 groups were 1:160 and 1:320 respectively. Muscovy ducks in pCIS3 +pCIS4ORF2 with whiteruss adjuvant,pCIS3+pCIS4ORF2 with glycerine adjuvant and pCIS3 + pCIS4ORF2 groups were completely protected from virus challenge (with few signs but no deaths), while pCIS3+pCIS4ORF2 with aluminium hydroxide adjuvant immunized groups only provided 70% protection; These results indicate that pCIS3 +pCIS4ORF2 immunized with whiteruss adjuvant or glycerine adjuvant has better effect in the protection compared with the other two immunizedgroups and antibody titers were higher with adjuvants than without adjuvants.