Analysis Of Genomic Characteristics And Identification Of The Genes Dispensable For Replication Of Porcine Cytomegalovirus

Porcine cytomegalovirus (PCMV), a member of Betaherpesvirinae, causes characteristic inclusion body rhinitis in newborn piglets, with25%mortality. PCMV infection often displays latent in pigs with a worldwide distribution, and impacts pig industry to some extent. In this study, to find out the situation of PCMV infection in China, PCR was applied to detect PCMV in the clinical lung samples and ELISA was established using the expressed glycoprotein B (gB) of PCMV to detect the antibodies to PCMV in the serum samples. To provide a scientific basis for the taxonomy of PCMV, an isolate of PCMV was sequenced and its genomic characteristics were analyzed. Meanwhile, the genes dispensable for replication of PCMV were identified in order to lay the foundation for the research associated with the gene function and molecular etiology of PCMV.Eight hundred and twelve lung samples collected from49intensive pig farms of China during2006to2010were detected by using a PCR. The results showed that317samples were positive for PCMV with a positive rate of39.04%, and the samples from97.96%of pig farms were positive.142amplified products were randomly selected and subjected to sequencing. The results showed that there was97.6to100percent of nucleotide identity and95.6to100percent similarity of deduced amino acid among these amplified fragments. Based on the phylogentic analysis, PCMV strains in China were mainly clustered in subgroup2and3. The above results indicate that PCMV infection is widespread in China and the DPOL genes of PCMV share minor viaration.The gB gene of PCMV was prokaryotically expressed and the expressed gB was used to establish indirect ELISA for the detection of PCMV antibodies.1234serum samples collected from27pig farms in14areas during2009to2011were detected by ELISA. The results showed that the positive rate of antibody to PCMV was84.28percent and all the detected pig farms were positive for PCMV infection; the positive rate increased as pigs grown up, and there was a positive rate of99.46percent in sows, indicating that PCMV infection is extensively common in pig herds in China.By co-culturing PT-K75cells with pulmonary alveolar macrophage (PAM) with PCMV, a strain of PCMV was isolated and designated BJ09. Following PCMV infection, PT-K75cells exhibited the cytopathetic effect (CPE) with occasional syncytium and giant cells. Typical virons of PCMV could be observed by transmission electron microscope (TEM).Genomic DNA of PCMV was extracted from the PT-K75cells infected with BJ09. By the method of high throughput sequencing, the complete genome sequence was obtained, and the genomic characteristics were analyzed. The results indicated that the PCMV genome is128367bp, containing79predicted open reading frames (ORFs). Of these ORFs,69have counterparts in human herpesvirus6A (HHV-6A),6B (HHV-6B) and7(HHV-7), and two ORFs are homologous to other members in Betaherpesvirinae. And the rest eight ORFs have no homologs in herpesvirus. All the homologs have higher identity and possess similar orientation and location to roseoloviruses. The PCMV genome is a DR-U-DR type, similar to HHV-6A, HHV-6B and HHV-7. Phylogenetic analyses of several core genes indicate that PCMV could be clustered in a branch with roseoloviruses. Based on the above results, we suggest that PCMV could be classified into the genus Roseolovirus of the subfamily Betaherpesvirinae as a new member.Twenty-two genes of PCMV were chosen to construct the plasmids with homologous arms. By the method of homologous recombination, an EGFP gene was inserted into the initiation codons of the22genes, respectively. The replication ability of the obtained recombinant viruses was analyzed. The results showed that five recombinant viruses (rPCMV-U24p, rPCMV-U26p, rPCMV-U28, rPCMV-U70and rPCMV-U81) were obtained after the plasmid with U24p, U26p, U28, U70or U81were recombined with PCMV, suggesting that the U24p, U26p, U28, U70and U81of PCMV were dispensable for replication of PCMV.In conclusion, our findings indicate that PCMV infection are widespread in pig herds in China; PCMV could be classified into the genus Roseolovirus of the subfamily Betaherpesvirinae; and the U24p, U26p, U28, U70and U81of PCMV are dispensable for replication of the virus. Our study provides valuable epidemiology data for PCMV infection and important scientific basis for the taxonomy of PCMV.