| Adenovirus was widely applied into virus vector construction because of its advantageous mucous immunity. Duck adenovirus type 1, including egg drop syndrome virus (EDSV) or EDSV-alike fowl adenovirus, have large range of host, and the virulence of the isolates or strains varied. Then apathogenic duck adenovirus type 1 virus is a promising delivery vector in gene pharmacy for poultry. Genetic marker vaccine against avian influenza virus (AIV) is a trend, because poultry inoculating with-inactivated AIV whole virus vaccine can produce slow antibody and have no help for infection information by surveillance of antibody. The immune efficacy of vaccine are decided mainly by surface glycoprotein of hemagglutinin (HA) and neuraminase of AIV, apparently, genetic marker vaccine is a potential one for control AIV. Now, in this paper, one low virulent duck adenovirus type 1 virus strain was screened through pathogenicity analysis and fiber protein gene sequencing, and then its non-essential region for replication was decided by genome mutation assay. The recombinant adenovirus vector was constructed with insertion of the protective antigen Hemagglutinin gene of AIV subtype H5 and the enhance green fluorescence protein gene. The recombinant vaccine only expressing H5 HA of AIV was obtained by site-specific recombination of Cre-loxP system to self-excise the GFP reporter gene in recombinant virus genome. In addition, the nucleotide detection methods were developed for distinction of the recombinant vaccine from field infection of AIV or EDSV causing egg drop.These studies include:1.The biological feature and fiber protein analysis of fowl adenovirus QUstrainThe fiber protein gene of quail-original adenovirus QU strain was amplified by polyerase chain reaction and sequenced, the result showed that the target fragment is highly homologous with those of chicken original HS strain and AV127 strain. The young specific-pathogen-free chicken, oro-nasally inoculated with the QU strain, presented no clinical sign and growth withdrawing, no lethal to chicken embryo and lower motility rate to duck embryo than HS strain that causes egg drop of laying chickens. The QU strain adapted to chicken embryos liver cell, duck embryos fibroblast cell and chicken embryos fibroblast cell, and the virus titer was highest in chicken embryos liver cell. Above data suggested that the QU strain was apathogenic adenovirus in chicken and some characteristic of the virus assembles egg drop syndrome virus. So, the fowl adenovirus virus may be a candidate vector for poultry gene engineering vaccine or gene therapy.2. Mutation analysis of genome of fowl adenovirus QU strainThe primers were designed for amplification of 2.7kb of fragment on the basis of E4-closly region sequence of duck adenovirus type 1 genome, pADGFP was constructed, in which enhance green fluorescence protein gene cassette of pEGFP-C1 plasmid digested with mluâ… and Aseâ… cutter, and inserted into the amplified fragment through the Bglâ…¡and Stuâ… endonuclease. The rQUGFP recombinant virus was gained by co-transfection of QU virus and pADGFP plasmid. The one step growth curve of the rQUGFP virus was identical with that of parent QU strain and the titers of serially-passenging recombinant virus were stable. These data suggested that the E4 nearby region of QU strain genome is nonessential for virus replication, and the inserted foreign gene into virus genome could be expressed efficiently.3. Development of Detection Method for Hemagglutinin Gene of Avian Influenza Virus Subtype H5One pair of primers were designed on the basis of hemagglutinin (HA) gene of avian influenza virus(AIV), allowing detection of AIV subtype H5, and the specific amplicon was 176bp in length. The primers were tested to be specific for AIV with no cross-reaction to RNA from the chicken muscle and other chicken pathogens such as Newcastle disease virus etc. The sensitivity of this assay was about 50pg of total RNA of AIV subtype H5. Our data showed that the establishment of this method was simple and feasible for screening and purification of the following AIV HA-expressing recombinant fowl adenovirus vaccine. In addition, the primer was specific for 579bp of fragment of AIV subtype H9,and available for simultaneous subtyping these two subtype AIV by one-step reverse transcription-polyerase chain reaction.4. Construction of recombinant fowl aflenovirus expressing hemagglutinin of avian influenza virus subtype H5The amplicon of 2.7kb in length was cloned as pAD16 according to E4-close region of duck adenovirus type 1 QU strain genome. The pHAE plasmid was constructed, the hemagglutinin gene of avian influenza virus type H5 was amplified by RT-PCR and inserted in pEGFP-C1 for its CMV promoter and polyA singal tail. The loxP-GFP-loxP fragment from ploxH, which constructed through GFP cassette gent inserted the double loxP sequence using PCR extended, and the H5 hemagglutinin cassette gene from pHAE plasmid were inserted in pAD16 plasmid, the delivery vector pHAGFP was constructed. The redcombinant virus rQUHAGFP was gained through co-trnasfection of pHAGFP with genome DNA of QU strain. After second co-transfeetion of Cre recombinase plasmid with rQUHAGFP, the GFP reporter gene in the recombinant virus genome was removed self-excisely and the recombinant rQUHA virus only involving H5 hemagglutinin gene cassette was obtained finally. The resulted showed that the recombinant virus was stable for replication by one step growth curve test and. also serially passenging recombinant vaccine can consistently expressed the H5 hemagglutinin.5. Immunization and surveillance of avian influenza virus H5 hemagglutinin-expressing recombinant fowl adenovirusThe immune efficacy of a recombinant fowl adenovirus vaccine expressing H5 bemagglutinin(HA) of avian influenza virus was confirmed. The specific pathogen flee chickens were inoculated oro-nasally with recombinant rQUHA virus and fowl adenovirus QU strain. The antibodies against H5 HA of avian influenza virus and duck adenovirus virus increased six days after immunization and the antibodies arrived at the level of 5.31og2 and 7.5log2 respectively after 12 days. At the time the antibodies to QU strain were 7.53log2 HI titer. The results demonstrated that the recombinant vaccine might activate chicken to produce antibodies against H5 HA as well as QU virus antigen. According to the nucleoprotein gene of avian influenza virus and the deletion region of duck adenovirus type 1 virus genome through insertion of target gene, two oligonucleotide primers were designed, the 219bp of fragment specific for AIV and 151 bp for duck adenovirus type 1. The development of detection method of both one step RT-PCR for AIV and PCR for duck adenovirus type 1 were useful for differentiation of the recombinant vaccine from field egg drop syndrome virus or avian influenza virus infection in poultry. |
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