Simian virus 40 (SV40) is a member of the Polyomaviridae family which infects rhesus monkeys. In 1960,Sweet and Hilleman isolated SV40 from poliovirus vaccine produced in monkey kidney cells,and from rhesus monkey kidney cells (11). Soon afterwards,it was discovered that SV40 was able to induce tumors in newborn hamsters and transform many human cultured cell lines (3). Subsequently,SV40 was isolated from many human tumor tissues,including encephaloma,osteosarcoma and lymphoma (6, 9, 12). The SV40 genome can be sequenced by PCR from many human tumor tissues (5) ,suggesting that SV40 has pathogen representing an increasing risk to human health. Although it is uncertain whether SV40 directly induces these tumors,more and more data demonstrate that SV40 can transform cells by multiple cell signal transduction pathways via large T and small t proteins (1). To our knowledge,SV40 contamination of the polio vaccine during production and its close relationship with tumors has aroused scientific interest in conducting further studies.
The SV40 genome is a circular covalently closed dsDNA molecule with a length of about 5 200bp,consisting of a regulatory region,and early and late gene regions. The regulatory region promotes transcription and replication of the viral DNA. The early gene region encodes regulatory proteins for large T antigen (T-ag),small t antigen (t-ag) and early leader protein. The late gene region encodes structural proteins VP1,VP2,VP3 and agnoprotein,among which VP1 is the main structural protein,and VP2 and VP3 are the se-cond most important structural proteins. The agnoprotein is an SV40-specific protein in the Polyomaviri-dae family but its functions are not clear at the moment.
Despite the extreme conservation of the SV40 genome,recent studies indicate that heterogeneity exists in different isolates and is represented principally in the form of variations in the regulatory region and the C-terminus of the T antigen (4). In order to determine the epidemic SV40 genotype in Yunnan rhesus monkeys,data that is important for further studies,an SV40 strain was isolated from a captured Yunnan rhesus monkey. Its complete genome was sequenced and analyzed. Our findings demonstrate that this is a new virus isolate.
The virus was obtained from the isolated monkey kidney cell culture of a SV40 infected Yunnan rhesus monkey,and named as SV-IMB. The primes were synthesized and DNA was sequenced by Sangon Corporation. The Taq enzyme and clone vector PMD18-T were purchased from Takara Corporation.
The primers were designed based upon the complete sequence of the SV40 -776 strain.
The DNAs to be used as the template were abstracted via the conventional phenol-chloroform approach from cultures with obvious cellular patholo-gical changes. PCR amplification was performed on the virus genome by using the primers listed above subject to the follo-wing reaction program: 1cycle of denaturing at 95℃ for 1 min; 35 cycles of denaturing at 94℃ for 30s; annealing at 52℃ for 30s; 72℃ elongating at 72℃ for 45s; 1 elongating at 72℃ for 7min.The PCR products were cloned into a PMD-18 vector and transformed to E coli. DH5α after harvest and purification. Plasmids were abstracted from the screened clones,and the positive clones identified by PCR were sequenced by Sangon Corporation.
In order to analyze the homology and variations between SV-IMB strain and other 12 SV40 isolates,we compared their sequences by using the Clustal X 1.83 and Vector NTI 6.0 programs. The information on these isolates are presented in Table 2.The complete sequence of SV-IMB was submitted to the GenBank under accession number DQ660375.
Table 1. Primers for PCR Amplification
Table 2. History of SV40 isolates used in this study
Table 3. Nucleotide and amino acid homology comparison between SV-IMB and other SV40 isolates