Clone Of Columbid Herpesvirus Type 1 Genome And The Preparation Of DNA Vaccines

Pigeon herpesvirus(PiHV),also named Columbid herpesvirus-1(Co HV-1),was a member of the Mardivirus genus within the subfamily Alphaherpesvirinae and the family Herpesviridae.The significant clinical signs of sick pigeons were anorexia,lassitude,a few signs presented conjunctivitis and neurological symptoms.Pigeon,host of the virus,was one of our country economic animals,and CoHV-1 brings serious economic losses to pigeons industry.There is no special effects drugs to prevent the recurrence of the disease and prevent the transmission of the virus,therefore,the development of safe and long-term effective CoHV-1 vaccine has become the key to prevent virus infection.DNA vaccine,also known as gene vaccine,is a new type of vaccine,and it has improved the prevention and treatment of some kinds of herpes virus disease.There are many herpes virus DNA vaccine immunization cases,such as Marek's disease virus and infectious laryngotracheitis virus,etc.In 2013,CoHV-1 Heilongjiang strain(CoHV-1 HLJ strain)was isolated from the infected feral pigeon in Harbin Kangda Poultry Disease Technical Service Center.In recent years,numerous studies reported that CoHV-1 isolated from pigeons and some birds of prey,however,rare to CoHV-1 genome sequence.A total of 95 pairs of primers were designed according to other avain alphaherpesviruses to amplify the CoHV-1 genome.The CoHV-1 HLJ strain was cloned by conventional PCR and single oligonucleotide nested PCR(SON-PCR).CoHV-1 HLJ genome sequence was assembled and analyzed by bioinformatics software such as DNAStar,ORF Finder and MrBayes,etc.The CoHV-1-HLJ genome was 204,237 bp in length(GenBank accession number: KX589235),which has a class E structural characteristics,encoding 130 putative protein.Phylogenetically,CoHV-1 HLJ shared common genetic properties with other avian alphaherpesviruses,moreover,the complete CoHV-1-HLJ genome resided in a monophyletic group with the Falconid herpesvirus type 1(Fa HV-1)genome,represented a more distance from other alphaherpesviruses.The multiple alignment of nucloetide sequences of Fa HV-1 genome(130 gene sequences)along with CoHV-1 genome was up to 99.4%,and 74 amino acid sequences encoding by these genes were inconsistent,including envelope glycoprotein C(gC),envelope glycoprotein D(gD),and envelope glycoprotein E(gE)etc,which may affect the process of virus invasion,adsorption and propagation among host cells.Three pairs of primers of gC,gD and gE gene were designed according the acquired complete CoHV-1-HLJ genome.Then the three genes were inserted into eukaryotic expression vector pcDNA3.1(+)to build eukaryotic expression plasmids pgC,pgD and pgE,respectively.The recombinant plasmids were emulsified by Freund's adjuvant,then immunized pigeons,and the inactivated virus group,empty vector group and sterile PBS group were the control groups.The results of animal experiment indicated that the three DNA vaccines,pgC,pgD and pgE,could induce a higher level of humoral and cellular immune responses,and the immune effect of pgD DNA vaccines was better than others.In this study,the primary complete sequence of the feral CoHV-1-HLJ was cloned and analyzed,which can provide the reference for the evolution of the virus between different organisms.Meanwhile,the recombinant eukaryotic expression plasmids were built successfully,which could be used to prevent CoHV-1 effectively in pigeons,and provided the reference for preventing and controlling CoHV-1.