Construction Of Recombinant Marek’s Disease Viruses Expressing The σB And σC Proteins Of Avian Reovirus

Chickens infected with avian reoviruses (ARV) can show various clinical signs including arthritis, gastroenteritis, hepatitis, tenosynovitis, myoearditis, malnutrition syndrome, chronic respiratory diseases and the increase of death rate, the decrease of egg laying. In addtion, this disease can also transmits by vertical transmission thus provoking considerabl economic losses in poultry industry. The research shows that the attenuated vaccine S1133 currently in use may have some toxicity. This situation has encouraged the development of a safe and effective new vaccine to prevent and control ARV.In this research, we firstly amplified σB and aC gene from the ARV GX/2010/1 cDNA, the σB or aC gene was directional cloned into pEASY-M1 expression vector, then, the expression cassettes for σB or aC was amplified by PCR. The product was cloned into the pU/D vector which contained the homologous sequence to the sorfl-sorf2 junction region of the CVI988 genome. The transfer plasmid, pU/D-aB and pU/D-σC, was constructed. The pU/D-aB or pU/D-aC plasmid was transfected into chicken embryo fibroblast cells (CEF) along with CVI988-DNA which containing the green fluoresence protein gene(rMDV-GFP). These new genomic DNAs were used for subsequent transfection experiments, and the final recombinant viruses rMDV-σB and rMDV-aC were acquired after screening and purifying.In this study, we evaluated the protection efficacy of rMDV-σB and rMDV-σC against both ARV GX/2010/1 and vvMDV RB1B by vaccinating 1-day-old SPF chickens. SPF chickens were divided into four groups:rMDV-σB vaccined challenged group (rMDV-σB), rMDV-aC vaccined challenged group (rMDV-aC), S1133 vaccined challenged group, non-vaccinated challenged group (NV-ch, positive control) and non-vaccinated, non-challenged group (NV-Nch, negative control). The protection against ARV was evaluated on morbidity, mortality, clinical symptoms, histopathological, alterations, weight gain after challenge and serology. The first two groups were immunized with 5000 plaque forming unit (PFU) of the rMDV-σB or rMDV-σC virus; the third group were inoculated with S1133 attenuated vaccine when chichens were 7days old. All the chichens were challenged with ARV virulent strain when chickens were 21 days old by the palmula route,104 0EID50 per chicken. The morbidity of chickens vaccined with rMDV-σB (40%) or rMDV-σC(26.7%) was significantly lower (p<0.05) than the NV-ch group(100%), respectively. The average body weight gain was recorded between 21 and 35 days of age (14 days post challenge, d.p.c.). Among the vaccinated groups, there is no differences observed compared with the other groups (p>0.05), but the rMDV-aC group is heavier than rMDV-aB and S1133 group. The histopathological alterations in the spleen of the rMDV-σC group is slighter than the S1133 and rMDV-aB and S1133 group.SPF chickens were immunized with rMDV-σB or rMDV-σC to test the protective efficacy against MDV. Chickens were randomly divided into CVI988/Rispens group, rMDV-aB group, rMDV-σC group, and challenges control group, each group contains 20 SPF chickens. The rMDV-σB or rMDV-σC group was subcutaneously inoculated with 5000 PFU of rMDV-aB or rMDV-σC virus, while the CVI988/Rispens group inoculated with 5000 PFU of MDV CVI988/Rispens vaccine strains. All of the SPF chickens were challenged with 500 PFU of vvMDV RB1B strain at 7 days post-immunization. After challenged with MDV, we observe the chickens for 90 days. The result showed that protection rate of rMDV-σB and rMDV-σC group was 100% just as the CVI988/Rispens strain group. All chickens of control group showed clinical signs. Overall this study demonstrates that the recombinant virus of rMDV-aC is a suitable candidate vaccine strain of ARV.