Construction Of RHVT Expressing The VP2Protein Of IBDV And Evaluation Of Immune Effects

Infectious bursal disease (IBD), caused by an avibirnavirus, has been aneconomically significant, widely distributed condition affecting immature chickenssince1960. The classical type1conventional strain is responsible for up to5%mortality in susceptible flocks. As a result of immunosuppression, growth rate,liveability, and productivity may be adversely affected by subsequent exposure to awide range of viral, bacterial, and protozoal agents. Since the disease was reported,ithad been an important threat for the poultry industry.Before the emergence of variantand very virulent IBDV,IBD had been under well control with the vaccine for a longtime.Vaccination is the primary means to prevent vvIBDV,and inactivated virusvaccine and attenuated vaccine play a great role to control IBD，but vvIBDV ispathogenic and not suitable for cell culture.To obtain the traditional livevaccine,vvIBDV were attenuated by passaging blindly in CEF cells or chickembryo,which waste a lot of time and effort and the disadvantage of hardlydifferentiation vaccinated from infected chickens still exist. However,researches ongene engineering vaccine is a good solution to the problem,especially on virus vectorvaccine.Herpesvirus of turkeys (HVT) is widely used as a vaccine in immunizationagainst Marek’s disease has been over40years,and is considered to be an idealmultivalent vaccine vectors. For reason which are HVT is not pathogenic and goodsafety for chickens and other animals and can continue to exist in chickens forlifetime to stimulate the body to produce higher levels of immune response andhumoral immune and cell immune. Furthermore, HVT can be prepared as alyophilized vaccine thereby facilitating storage and transportationHere, we first established a system to generate the HVT vaccine strain by usingthe transfection of overlapping fosmid DNAs. Using this system,we constructrecombinant herpesvirus of turkeys (rHVT) expressing the VP2gene of infectiousbursal disease virus (IBDV),the vp2gene of IBDV strain HLJ-0504was amplified by RT-PCR and cloned into expression vector pCAGGs. The vp2expression cassettewhich containing the Pec promoter and poly(A) signal sequence was obtained byendonuclease digestion and cloned into pENTR plasmid to yield the entry clonepENTR-VP2. The insertion of Pec-VP2cassette into the desired destination fosmidF5354ccdBK+was done by mixing the destination fosmid with entry clone and treatedwith LR Clonase II enzyme. The recombinant virus, designated rHVT-VP2, wasgenerated by co-transfection of5overlapping fosmid DNAs which cover thecomplete HVT genome. PCR, Western blot and immunofluorenscence assaysindicated that the IBDV VP2gene was stable existed and expressed over20passages.These data showed that the recombinant HVT expressing the VP2gene of IBDV wassuccessfully constructed.Then the recombinant virus was immunized one day-old SPF chickens, toevaluate their immune protective effects.The results showed that100%serum werepositive at14days p.i.,chanllenged with vvIBDV at3weeks p.i.and5weeks p.i.,therecombinant viruses provides both100%protection against vvIBDV, indicating thatthe recombinant virus rHVT-VP2can be used as an alternative vaccine to prevent IBDand provided a basis for evaluation in chicks for immune protection against IBDV.