Isolation And Identification Of Canine Adenovirus And Construction Of Canine Adenovirus Genomic Library

Canine adeno(CA)is a infectious disease,caused by Canine adeno virus(CAV).And the disease is widely popular in many animals,such as domestic dogs,foxes,bears,coyotes,raccoons and so on.Generally,over 50%dogs can be infected by CAV,among which the death rate can reach 25-40%.Besides,CAV often co-infect animals with Canine distemper virus(CDV)Canine parvovirus type(CPV)and Canine parainfluenza virus(CPIV),which makes the clinical symptoms more complicated.Our study focused on isolation and identification of virus from dogs which had the clinical symptoms of CA,CD,CP or CPI.After isolation and identification,we got 4 strains of CAV-?,one strain CPV.And then we constructed the whole genome library of one of the 4 strains of CAV-?.The main content of our research can be described as follow1.Isolation and identification of virusWe collected 410 samples from several pet hospitals,and 83 of the samples were chosen to inoculate on MDCK cells.After inoculation,we found obvious and stable cytopathic effects(CPE)from 13 samples.We kept on inoculating the 13 samples on MDCK cells until the typical grape bunch-like pathological emerged.Then the samples were identified via PCR by specific primers of CAV,CDV,CPV and CPIV respectively.Four 1030bp specific PCR products were got from 4 samples(number:27/162/201/208).After purification of those 4 samples,We sequenced the virus and compared the complete genomes with standard strain CAV-?.The results of multiple alignments showed that homology between 27/162 standard strain CAV-? reached 99%on nucleotide level,while the 201 and 208 to 97%.So,we got 4 strains of CAV-? and designated the 4 strains as WH-27,WH-162,WH-201 and WH-208 respectively.Besides,a 576bp specific PCR product got from another sample(number 182)was the same as that derived from Canine parvovirus 2b strain CPV-BM(11).We sequenced the segment and compared it with VP2 gene of Canine parvovirus 2b strain CPV-BM(11),and the results showed that the homology reached 100%.So,we got a strain CPV 2b and designated as WH-182.After all,it turned out to be negative by using specific primers of CDV or CPIV for PCR operation.Electron microscope observation revealed that the diameter of virus particle of CAV-? WH-27 was 60.67 nm,and that the viron involved a form of a hexagon and a cubic symmetrical icosabedron structure with significant spikes.From the observation,we could also see that the diameter of particle of WH-182 was between 20 to 26nm,the shape of the particles appeared to be round,of which majority were solid-core while the left hollow.2.Construction of the whole genome library of CAV-? WH-27Firstly,both the DNA of CAV-? WH-27 and pBlueScript ? SK(+)vector were cut with the single restriction enzyme Hind ?.Then,we ligated multiple sizes of DNA segments with the vector.After transformation into the competent E.coli(DH5a),we random selected 100 single colonies of ligated products.After confirmation by cutting with Hind ?,we sequenced different sizes of DNA segments and got the majority of the virus genome.At last,we got the left of the genome by PCR and constructed the whole genome library of CAV-? WH-27.