-
The Sindbis-like virus is a representative Alphavirus in the Togaviridae family, which infects the host cells of insects, birds and mammals via mosquitoes and insects; infected humans develop fever, rash, arthritis and on occasion, encephalitis (2, 4). The whole or partial genomic sequences of many Sindbis-like viral strains have been reported worldwide (3). In recent years, epidemics of encephalitis have given the genomic analysis of Sindbis-like viruses an elevated importance and extensive epidemiological significance. The YN87448 strain was first isolated in 1986 from the blood of a patient with fever in Yunnan Province (7). Its genome contains a 5'-NTR in the non-structural region, nsP1, nsP2, nsP3, nsP4 regions, capsids in conserved, non-conserved and structural regions, E1, E2, E3, 6K structural regions and a 3'-NTR in non-transcriptional region (4), revealing a high homology to Sindbis-like isolates and in particular, a 99% homology to the SAAR86 strain isolated from Africa. In order to investigate the epidemics of Sindbis-like virus infections in Yunnan and the molecular differences between the YN87448 strain and other Sindbis-like viral isolates, we isolated a Sindbis-IMB strain from the blood of a suspected encephalitis patient in Yunnan Province. The results of virus identification demonstrated a high homology to the YN87448 strain. The sequencing of the whole genome was performed and compared with other isolates.
HTML
-
24 serum samples were collected from patients with unexplained fever in June, 2006 at Medical Institutions in Eryuan County of Yunnan Province. Serum samples were inoculated into Vero cells (Vero cells were provided by the Viral Immunology Dept. of the Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS), and samples with obvious cytopathic effect (CPE) observed in 3 continuous passages were inoculated into mice with an age of 1 – 3 days. Each of the samples with obvious CPE observation was inoculated intracranially into 6 to 8 mice for observing fever development in mice. Brain tissues of mice with fever were triturated to 10% suspension for passage amplification in Vero cells. The virus was identified by blood clotting and blood clotting inhibition tests (the Sindbis-like virus blood clotting antigen was provided by Yunnan Institute for Endemic Diseases Control and Prevention).
-
Based upon the complete sequence of the YN87448 strain, 12 primers were designed (Table 1) and synthesized by Shanghai Sangon Biological Eng-ineering Technology & Service Co.Ltd..
Table 1. Primers for RT-PCR Amplification
-
Viral supernatant in infected cells was collected. Total RNA was extracted using the QIAmp Viral RNA reagent kit. cDNA was amplified by Promega Access Quick RT-PCR under the following reaction conditions: 45℃ reverse transcription 40 min; 95℃ predenaturating 2 min; 94℃ denaturating 40 sec; 56℃ annealing 40 sec; 72℃ extension 1 min; 35 cycles; 72℃ extension 10 min. The specific PCR products were cloned into PMD-18T vector (purchased from TaKaRa Corp.) 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 by using RV-M and M13-47 primers in two directions. Sequencing results were analyzed by the OMEGA(Rainbow Techologies, inc.) software package. The cloning approach is shown in Fig. 1. The sequences of other Sindbis-like isolates YN87448, Girdwood, SAAR 86, SINOCK82 and SJ160 to be compared with Sindbis-IMB were obtained from GenBank (Table 2).
Table 2. Homology Comparison Between Sindbis-IMB and Other Isolates
-
The data of whole genomic sequence from different strains were aligned using MEGA package (version 3.1), and phylogenetic trees were constructed using the neighbor-joining program, from a distance matrix corrected for nucleotide substitutions by the Kimura two-parameter model. Tree stability was assessed via bootstrapping over 500 replicates. The cluster was regarded as strongly supported if the bootstrap value exceeded 95%.
Virus isolation
Primers design
Extraction of viral RNA, Synthesis and cloning of cDNA
Sequencing and analysis
Construction of phylogenetic trees
-
One viral isolate that could cause CPE in Vero cells and death of mice was taken from 24 samples and named Sindbis-IMB.
-
The whole genomic sequence of Sindbis-IMB was 11 717 bp encoding 3 773 amino acids, of which 7 622 nucleotides were in non-structural regions (excluding 5'-NTR): nsP1, nsP2, nsP3 and nsP4 regions, conserved and non-conserved regions, and capsids in the structural region. The 4059 nucleotides in the structural region (excluding 3'-NTR) were E1, E2, E3 and 6K. The sequencing fragments in two directions were assembled to obtain the whole genomic sequence. After analysis, the ratio of four nucleotides in the whole genomic sequence was determined to be: A: 28.70%; T: 21.22%; G: 25.42%; C: 25.88%.
-
The homology of the whole genomic sequence of Sindbis-IMB was 98.6% as compared with the other five isolates. Substantial homology was also found to other strains, such as SAAR86 and Girdwood isolates from South Africa, the XJ160 isolate from Xinjiang in China and the Ockelbo isolate from Sweden (Table 2). In the constructed phylogenetic tree, the closest genetic relationship was to the YN87448 strain compared with the whole genomic sequence of Sindbis-IMB (Fig. 2).
-
The results of nucleotides and encoding amino acids comparison between Sindbis-IMB and other isolates showed the highest homology to the YN87448 isolate and the second highest to the SAAR86 isolate (Table 3). The main variations were observed in the non-structural region, while the structural region was relatively stable with a mere additional three nucleotides (AAA) at the 5'-terminus in E2 gene.
Table 3. Whole Genomic Nucleotides and Amino Acids Comparison Between Sindbis-IMB and Other Isolates
-
The sequence comparison indicated a greatest variation of nucleotide sequences in the non-structural region, amino acids were noted in positions of 230, 231, 443, 781, 1582, 1746, termed K, E, N, R, H, L; and 3 resulting in some variations of amino acids sequences. The sequences of completely different additional amino acids termed Q, S, A were found in the nsP4 terminus (Table 4).
Table 4. Non-structural protein comparison between Sindbis-IMB and other isolates