| Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family Arteriviridae,which shares a marked tropism for the monocyte/macrophage lineage in vivo. Heparin sulphate (HS)and Sialoadhesin (Sn) and CD163 were considered to be the three receptors involved in the process ofPRRSV infection to porcine alveolar macrophages (PAM). HS can mediate virus attachment but nointernalization; Sn is sufficient for both PRRSV attachment and internalization; CD163 maybe paly arole in viral uncoating and genomic RNA releasing. It was reported that the viral M protein on itself, oras a complex with GP5, was identified as a HS binding protein. But the viral proteins involved in theinteraction with Sn are unknown. And the counterpart on the Sn, which attaches to the viral proteins, isalso unclear. The aim of the present study is to investigate the interaction between Sn and viral proteins.The gene of Sn was generated by RT-PCR from PAM cells, and inserted into a eukaryoticexpressional vector pcDNA3.1 to yield recombinant plasmid pSn. PK15 cell, a PRRSV no-permissivecell line, was transfected by purified pSn. Then the cells were exposured to PRRSV CH-1a strain for thebinding of virus after 48hpt. In IFA test, the PRRSV-attached cells could be detectable by anti-Nmonoclonal antibody. Subsequently, the full-length of Sn was divided into a series of fragments andtransfected into PK15 cells for the viral binding test, until one single domain was embodied in a shorterfragment. Anti-Sn-polyclonal serum was generated and employed in the process of viral binding to thetransfected-PK15 cells or PAM cells. In the study, the cells transfected by pSn take an ability ofadhesion the virus, and the fluorescence was focus on the cellular membranes of PK15. Among theseveral fragments, only the pSialA (at the N-tennini of Sn) was discovered to be fluorescence positive.Finally, the sequence coded the amino acid from 19 to 150 of the Sn, in which the first domain(20-136aa) contained only, was detected to be positive in attaching the virus. The attachment totransfected PK15 cells was effectively inhibited by anti-Sn-polyclonal serum, and the similar inhibitionon PAM cells was observed. By analysis with FAC, we found the inhibition of anti-serum was in adose-dependent manner.Heptad repeat motif is the necessary configuration for viruses if the viral proteins be inosculatedwith their host cellar membrane by the rule of I-type virus-membrane fusion mechanism. But there is noheptad repeat motif in all the structural proteins of PRRSV, suggesting the PRRSV-PAM fusion do notfollow the I-type virus-membrane fusion mechanism. The envelope proteins of PRRSV were extractedfrom semipurified viruses and permitted to incubate with PAM cells, followed by detection with a serialof monoclonal antibodies specific against different proteins of PRRSV in IFA. Among those viralproteins, only GP5 was found to be positive in IFA, suggesting the protein was involved into theattachment of PRRSV to PAM. For other viral proteins, whether or not related to the process, could notbe known clearly until.GP5, the most important neutralizing antigen of PRRSV, has the highest genetic diversity amongisolates. To more fully understand the extent of genetic diversity, we analyzed and compared the GP5 sequences of PRRSVs which were isolated from 1996 to 2006 in mainland China. Fifteen GP5sequences collected from the clinical samples by our lab were compared with all the 27 sequences frommainland China available in GenBank, together with 260 representative sequences from North America,Europe, and other Asian countries. As a result, all the Chinese isolates examined belong to the NA-type.Two subgroups with great diversity were classified from the 42 Chinese NA-type isolates. In thephylogenetic tree, the two Chinese subgroups separated on the two polar of the NA-type cluster. Theidentity of 85.0-89.5% was observed between the two subgroups. All the sequences of subgroup 1 werehighly variable in the antibody binding site of the primary neutralizing epitope of PRRSV, while therewas no mutation at the same position in subgroup 2. In subgroup 1, two key amino acids, which wereconsidered to be involved in PRRSV attenuation, were accordant with the VR-2332 strain. On the otherhand, the subgroup 2 isolates were identical to the RespPRRS/Repro vaccine strain. As for their origins,the subgroup 1 was restricted to regions in southeast China. Taken together, these large differencesamong PRRSVs need to be taken into consideration for control and preventive measures.A highly pathogenic disease in pigs, characterized by high-fever, high morbidity and mortality rate,was outbreaked in South China in April 2006, and then spread quickly in most pig-fanning provinces.PRRSV mutant was considered to be one of the etiologies. In the present study, 5 full-length highpathogenic PRRSV isolated in the "high-fver disease" were examined. Phylogenetic analysis and aminoacid homogeneity showed that the high pathogenic PRRSV isolates shared the same origin. A deletionof 30 amino acids was found in the NSP2 protein, but the deletion maybe not involved into thevirulence of PRRSV. The changement of positions and number of N-glycosylated sites of GP5 maybeparticipate in the virulence of PRRSV. |
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