| Chicken Marek’s disease (MD) is a kind of chicken infectious oncosis, characterized by the proliferation of lymphoid tissue, paralysis of peripheral nerve, and tumor in organs, muscles and skin, is one of the most serious infectious diseases for poultry industry worldwide. In recent years, the virulence of Marek’s disease virus (MDV) evolved from mild virulent to virulent, and to very virulent. Conventional vaccines are not effective to protect chickens against very virulent MDV. It is necessary to develop new vaccine and the researches majorly focus on development of recombinant vaccine by genetic engineering. REV LTR has strong promoter and enhancer, which can accelerate the replication of the virus, but does not enhance the virulence of the parent strains. In this study, to develop a more effective and safe MDV vaccine or virus vector, the biosafety of recombinant Marek’s disease virus containing REV LTR in CVI988/Rispens strain was evaluated.1 The biological properties and viral shedding in environment of recombinant MDVIn order to determine the stability of rMDV-LTR, recombinant MDV was serially passaged in CEF and SPF chickens. The insertion of LTR gene in IRS and TRS was identified by PCR. After 1-day-old SPF chickens were inoculated with rMDV-LTR, non-immune chickens were raised with the infected chicken for evaluation of contact transmission. The blood cells and feather pulps were collected for virus isolation and PCR detection. The feed, water, manure,and litter were also collected for virus isolation and PCR detection.The results showed that rMDV-LTR and parent virus CV1988/Rispens grew well in CEF cells and reached the peak titre on day 7 postinfection. However, the growth rate of parent virus was much quick than that of rMDV-LTR. There was no change in the insertion sequence even the virus was passaged in CEF and chickens. The 5 generation of rMDV-LTR produce no significant pathological change in histopathological observation. The result of virus isolation combined with IFA and tissue PCR test showed that the rMDV-LTR could transmit from infected chickens to non-immunized birds by horizontal transmission. The rMDV-LTR was not detectable in feed, manure, water and cotton swabs, indicating that it has same biosafety as CVI988/Rispens.2 The immune efficacy of recombinant MDVA total of 180 1-day-old SPF chicken were divided randomly into 8 groups,20 chicken/group. The chickens were immunized with 3000 PFU and 2000 PFU (0.2 mL per chicken) of CVI988/Rispens,500 PFU、750 PFU、1000 PFU、2000 PFU、3000 PFU (0.2 mL per chicken) of rMDV-LTR, and 0.2 mL of sterile PBS, respectively. After 10 days, all chickens were challenged with 500 PFU (0.2mL per chicken) vvMDV rMd5. The result showed that about 85% chicken in control group died within 60 days, and all chicken in immunization groups with rMDV-LTR or CVI988/Rispens survived. There was no pathological change in histopathological observation in the survived chickens. These data indicated that rMDV-LTR could provide 100% protection against MDV challenge, which is same as the protection rate of CVI988/Rispens.3 The biosafety evaluation of recombinant MDV in immunized target animals, non-target animalsThe target animals of one-day-old chickens were immunized with rMDV-LTR and CVI988/ Rispens in 0.2 mL at one dose (3×103 PFU/0.2 mL) for once, one dose (3×103 PFU/0.2 mL) for twice, and lager dose (6×104PFU/0.2 mL) for once, sterile PBS was set as negative control. Non-target animals of ducks and geese were immunized with 0.2 ml of rMDV-LTR and PBS, and ICR mice were immunized with 0.2 ml,0.4 ml of rMDV-LTR and 0.2 ml of PBS. Five target animals, non-target animals were killed randomly to analyze the distribution of viruses and histopathological lession. The result showed that there were no significant pathological changes in histopathological section, and no effect on body weight gain in target animals and non-target animals... |
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