Establishment Of PCR Differential Detection Method For Marek’s Disease Vaccine Virus And Wild Virus And Construction Of Feline Herpesvirus Recombinant Virus

Marek’s s disease(MD)is a tumor disease caused by Marek’s disease virus(Marek’s disease virus,MDV),which has a high incidence rate and mortality rate,which seriously endangers the poultry breeding industry.Vaccination is a powerful means to prevent and control Marek’s disease,but the virulence of Marek’s disease virus is increasing,and the existing vaccines can’t provide complete protection.Although the newly developed vaccine can effectively prevent and control the infection of super virulent strains,the current detection technology can’t distinguish the newly developed vaccine virus from wild virus.There is an urgent need for new detection technology in the market to provide support for the diagnosis of Marek’s disease virus and auxiliary purification of chickens.Feline herpesvirus(FHV-I)caused by feline upper respiratory tract disease is a serious and highly contagious disease in clinic.It often endangers the life safety of felines together with other feline diseases.At present,there is no vaccine with good immune effect for most feline diseases in clinic.As a tool carrier for the research and development of new vaccines,herpesvirus has good immune protection effect and market application prospect.Therefore,the construction of cat herpesvirus recombinant vaccine is of great significance to the research and development of cat vaccines.Based on this,this study established a PCR differential detection technology to distinguish Marek’s disease vaccine virus from MDV-I,and constructed a recombinant feline herpesvirus virus with bacterial artificial chromosome.The specific research results are as follows:1.Establishment of PCR identification and detection method for MD vaccine virus and MDV-IIn this study,genome-wide analysis was conducted on the sequences of more than 140Marek’s disease virus strains published in NCBI Gen Bank database.It was determined that Marek’s disease CVI988 and 814 vaccine viruses had 177 bp base insertion at the position of meq gene compared with MDV-I.The newly developed vaccine strain SC9-I lost meq gene compared with MDV-I,and r MDV-MS-△meq lost 468 bp base sequence of the first half of meq gene compared with MDV-I.Based on this,the primers were designed and the reaction conditions were optimized.Finally,the PCR detection method for distinguishing Marek’s disease CVI988,814,SC9-1,r MDV-MS-△meq vaccine virus and MDV-I was established.Using the genomes of MDV-I and 814 vaccine viruses,CIAV,IBV,IBDV,ALV,IBH,IAV,NDV and CEF as templates,the results showed that only MDV-I and 814 vaccine viruses had specific amplification;MDV-I and 814 vaccine viruses were used as templates and negative controls were set for sensitivity detection,the results showed that the sensitivity of this method was 0.01ng/μL;MDV-I and 814 vaccine viruses were used as templates and negative controls were set for repeated detection,the results showed that the results of three repeated amplification were stable;The coincidence rate of clinical samples was tested by this method,the results showed that the coincidence rate of positive samples and negative samples was 100%.The above results show that the PCR detection method established in this study to distinguish MDV-I and vaccine virus has strong specificity,good sensitivity,stable amplification results and high coincidence rate of clinical samples.A 3-week-old chicken in a breeding farm in Hunan Province still showed neurological symptoms suspected of Marek’s disease after being immunized at the age of 1 day,but it was uncertain whether it was caused by MDV-I or vaccine virus.This method was used to diagnose dead chickens,and the amplified target fragments of MDV-I and vaccine virus were sent to sequencing to verify the accuracy of this method.Sequencing results showed that the amplified target fragments were consistent with MDV-I and vaccine strains respectively,indicating that this method can be used to diagnose MDV-I and distinguish it from CVI988 and 814 vaccine strains and SC9-1 and r MDV-MS-△meq newly developed vaccine strains.2.Construction of recombinant virus rFHV-g G~-/EGFP~+The purpose of this study is to accurately cut the virus g G gene by CRISPR-Cas9technology and replace the g G with BAC related elements by homologous recombination to obtain the recombinant cat herpesvirus.Firstly,CRFK-Cas9 cell line(CRFK-Cas9-sg G)expressing sg RNA targeting g G gene was constructed by lentivirus packaging system.After BAC transfer vector containing homologous arms at both ends of g G gene was transfected into the cell,FHV-I was infected with MOI=0.1 at the same time.After cell blind transmission,the recombinant virus with green fluorescence was selected under Eco-gpt pressure.The results showed that the recombinant virus was obtained in the second blind transmission under the synergy of CRISPR-Cas9 technology.In order to determine the correctness of the recombinant virus,the recombinant virus genome was identified by PCR and transfected into eukaryotic cells,and the growth characteristics and genetic stability of the recombinant virus in vitro were further analyzed.The results showed that the target bands were amplified by PCR,and the sequencing results were consistent with the reference sequence.Transfected eukaryotic cells could save infectious virus particles,indicating that the recombinant virus was correct;The growth characteristics of the recombinant virus in vitro are not significantly different from those of wild virus,and the proliferation in vitro also has genetic stability,indicating that the insertion of BAC element between feline herpesvirus genome US3 and US6 does not affect the basic characteristics of its culture and proliferation in vitro.In conclusion,this study established a PCR detection method to distinguish Marek’s disease CVI988,814,SC9-1,r MDV-MS-△meq vaccine virus and wild virus,and preliminarily applied it,which can be further developed and utilized as a new detection technology to provide technical support for the diagnosis of Marek’s disease and chicken purification.At the same time,the recombinant virus of feline herpesvirus with bacterial artificial chromosome(BAC)was constructed,which laid the foundation for the research and development of feline vaccine and the establishment of reverse genetic operation platform.