| GB virus C is discovered in the middle of 1990s by two independent groups. As a member of the Flaviviridae family, GBV-C is an RNA positive single linear strand virus with approximately 9400 nucleotides. Due to the shared transmission routes, GBV-C co-infection was frequent among individuals with HIV-1/HCV mono or dual infection. Up to data, GBV-C could phylogenetically claffied as six major genotypes with a distinct regional distribution. Although GBV-C infection has not been associated with any typical syndrom, it is notable that co-infection with GBV-C and HIV-1 could give a more favorable outcome for patients with higher CD4 cell counts and lower HIV-1 serum viral loads. Futhermore, GBV-C genotypes influences on the AIDS disease progress has been proved in the case of GBV-C/HIV co-infection. GBV-C genotype 2 and 5 seems to give more favorable results than other genotypes. These findings raise the interest on GBV-C epidemiological characters in the context of co-infection with HIV-1.The Yunnan province is situated in southwest China, borders Southeast Asia countries of Laos, Vietnam and Myanmar to the south. As a hub on the trans-continental drug trafficking road, Yunnan has played a critical role in spreading many blood-borne infections in China. For example, HIV-1 and HCV are introduced from the Golden Triangle to Yunnan and then spread to other provinces of the country. Moreover, in this region, the general population has been demonstrated to be threated with the more frequent HIV-1 spreading from IDUs through sex transmission. However, there are no reports on the epidemiology of GBV-C among intravenous drug users (IDUs) and no-IDUs in this region.In order to investigate the infection rate of GBV-C in Yunnan China,231 Injecting Drug Users (IDUs) and no-IDUs were recruited from this region. Among them, GBV-C sero-positive rate of 25.97%(60/231) was revealed of GBV-C with anti-E2 antibody detection. GBV-C RNA positive rate was 32.04%(74/231). Only five cases (2.16%) with clinically confirmed AIDS were positive for both GBV-C anti-E2 and GBV-C RNA. Moreover, chi-square test showed that the rate of GBV-C/HIV-1/HCV triple infection (21.38%) was significantly higher than those of GBV-C/HIV-1 (0%) and GBV-C/HCV (7.55%) dual-infections among IDUs (P<0.05). This revealed 33.33% of no-IDUs having GBV-C/HCV dual-infection, which significantly higher than 4.17% of GBV-C/HIV-1/HCV triple infection rates (P<0.05). It showed that the rate of GBV-C in individuals with serious and mild AIDS syndroms (47.83% and 31.42%) was significantly higher than those (12.24%) with medium syndroms (P<0.05). Unfortunately, there was no significant difference between GBV-C+/-pair groups in terms of ALT levels, CD4+ count, HIV and HCV viral loa, and HCV genotype/subtype.To describe GBV-C genotype distribution in Yunnan, China, GBV-C 5'UTR, E2 and full-length genome was amplifiled and sequenced, the phylogenetic tree were furtherly reconstructed. The obtained sequences could be divided into three phylogenetic groups. A sequence (2.3%) from Kunming (NK07) was classified into genotype 3, the reported predominant genotype in China. Two sequences (4.7%) from Dehong (DH019 and DH021) were classified into genotype 4, which was common in Southeast Asia. Unexpectedly, the remaining 40 sequences (93%) formed a solid cluster with 97%-99% of bootstrap support. Among the three genomes, KY117, DL185, and DH028, the nucleotide similarities ranged from 91.2 to 99.2%(mean 95.2%) over the complete genome length. Comparing KY117, DL185, and DH028 with the six references, the nucleotide similarities were of 86.2 to 89.0%(mean 87.6%) over the complete genome length. To exclude the potential recent events of viral recombination, similarity plotting was performed. Comparing the three full-length GBV-C genome sequences with a series of references, representing the known six GBV-C genotypes, no meaningful finding was obtained. These results may support a designation of the three full-length sequences as a new GBV-C genotype we temporarily assigned genotype 7. Based on the finding of GBV-C genotype 7, GBV-C genotype 7 was the most predominant in Yunnan region, accounting for 93.02%(40/43). Variants of genotype 4 for 4.65%(2/43) were the second most predominant. They included only 1 (2.33%) isolates of genotype 3. To formulate the novel nomenclature standards of GBV-C genotype, the distributions of intra-and inter-genotype pair wise genetic distances were analyzed based on GBV-C E2 sequences. The dispersal of the pair wise genetic distances that fell in 0.0345-0.0900 were considered as the same genotype. This distribution fell in 0.1282～0.1438 were considered as the different genotype. While it fell in 0.0900～0.1282 were considered as the same genotype or recombination genotype (need to recombination plot analysis).With the development of analysis technique, it is possible to deduce the origin and evolutionary history of GBV-C. Here, evolutionary rate of GBV-C were calculated based on the sequences of 5'NCR,E1,E2,NS5B and full-length genome respectively. It was revealed that GBV-C evolutionary rate fell in the range of 10-5～10-2sub/site/year. Using these rate of evolutionary rate, the estimated most recent common ancestor (tMRCA) were in the range of 152.96～1798.76 years. Through intergenotype analysis of GBV-C nucleotide sequence similarity on different genes, E1/E2 (nucleotides 900～1250) was proved to be more suitable to deduce GBV-C origin and molecular evolution. Thus, evolutionary rates of GBV-C five genotypes were estimated using the E1/E2 sequences. The tMRCA estimates of the root for each dated tree showed that GBV-C genotype 1 originated in 1238 from West Africa, genotype 2 originated in 1928 from Europe, genotype 3 originated in 1987 from Japan, genotype 41981 from East Asian, genotype 51975 from Southern Africa. To further investigate the evolutionary origins of GBV-C major strains circulating in China, we performed phylogeographic analysis using recently developed Bayesian phylogeographic inference framework. It was deduced that GBV-C genotype 3 was origined from Japan, and GBV-C 4 from East Southren Asian countries, and then further spread to other provinces of China.In the immunocompetent subjects, GBV-C clearance is common which occurring in approximately 60 to 75% of GBV-C-infected persons. The virus clearance and unsyndrom mechanism was far undescried, the existence of microRNA in host cells was considered as one of the reasons. To search the miRNA with complementarity to the GBV-C 3'NCR genome, we performed in silico analysis with ViTa algorithms.12 microRNA were designed and screened, microRNA Has-miR148a,Has-miR-152 and Has-miR-301 were sorted according to their host specificity and "Seed regions" conservation. Furthermore, we developed the stem loop RT-PCR,fluorescence quantitative PCR and eukaryotic cells express system of microRNA Has-miR148a and its targeting sequence, GBV 3'NCR genome. It was found overexpression of Has-miR148a could inhibite GBV-C 3'NCR genome expression in Huh7.5.1 cell. However, the inhibition of miR-148a expression with specific antisense oligonucleotide (ASO) could inverse this inhibition. In conclusion, the infection rate of GBV-C, distribution of its genotypes, origin and evolution characters of GBV-C, and the microRNA related machanism of GBV-C self-clearence were investigated. The high frequency of GB Virus C among IDU and on-IDU in Yunnan china, the predominance of a novel GBV-C genotype (GBV-C 7) was firstly described. Moreover, the analysis of origin and evolutionary history of GBV-C gives an outline on GBV-C spreed and migeration in Yunna region and the whole country. It was proved the correlation between GBV-C 3'NCR genome and Has-miR-148a. |
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