| 1. Seroincidence and Correlates of Herpes Simplex Virus Type 2 among HIV Serodiscordant Couples in Dehong Prefecture, Yunnan ProvinceObjective:To determine the seroincidence of herpes simplex virus type 2 (HSV-2) and its correlates among HIV serodiscordant couples in Dehong prefecture, Yunnan province and provide the basis for the prevention and control of HSV-2 infection in China.Methods:All 34 villages from 5 counties of Dehong prefecture, Yunnan province which have 20 or more HIV/AIDS patients when the investigation started were included in this study. HIV serodiscordant couples were recruited, demographic information and blood samples were collected and HSV-2 seropositivity were determined by the ELISA test at the baseline survey. Follow-up investigation was constructed one year later among initially HSV-2 negative subjects. HSV-2 seroincidence was acquired and predictors of incident infection were examined using Cox proportional hazards model.Results:(1) A total of 1048 HIV/AIDS patients and 1022 HIV negative spouses were recruited in our study. Baseline seroprevalence of HSV-2 for HIV/AIDS patients were 36.4%,29.4% for male and 61.1% for female. Baseline HSV-2 seroprevalence for HIV negative spouse were 28.9%,25.7% for men and 29.7% for women. (2) By the end of the baseline survey, a total of 667 HIV/AIDS patients and 727 HIV negative spouse were tested HSV-2 negative. At the final survey started December,2010,248 HIV/AIDS patients loss to follow up. There was no significant difference of sex, age, education, profession between subjects followed up and loss to follow up while proportion of Dai Nationality and subjects from Yingjiang County were significantly higher in those who loss to follow up than follow up. By the end of final survey,194 HIV negative spouses were loss to follow up. There was no significant difference of sex, age, education, profession between subjects followed up and loss to follow up while proportion of Han Nationality and subjects from Yingjiang County were significantly higher in those who loss to follow up than follow up. (3) HSV-2 incidence for HIV/AIDS patients was 2.98 cases/100 person-years,2.83 cases/100 person-years for male patients and 3.96 cases/100 person-years for female patients. HSV-2 incidence was 4.17 cases/100 person-years for HIV negative spouses,8.22 and 3.18 cases/100 person-years for male and female respectively. Incident HSV-2 infection among HIV negative spouses was independently associated with Han Nationality and mate's baseline HSV-2 infection while incident HSV-2 infection among HIV/AIDS patients was independently associated with counterpart's baseline HSV-2 infection.Conclusions:seroprevalence and seroincidence among HIV serodiscordant couples were high. Baseline HSV-2 seroprevalence of HIV/AIDS patients was higher among female than male while HSV-2 seroincidence of HIV negative spouse was higher among male than female. HSV-2 seroincidence among HIV serodiscordant couples was correlated with counterpart's baseline HSV-2 serostatus. The transmission of HSV-2 between couples existed. It is most likely that HSV-2 is transmitted from HIV/AIDS patients to their spouses for the reason that baseline HSV-2 seroprevalence of was higher among HIV/AIDS patients. HIV negative spouses were under the dual risk of HIV and HSV-2 infection. Public health and clinical care programs should be launched as soon as possible among HSV-2 serodiscordant couples, which is also meaningful for the prevention and control of HIV infection.2. Phylogenetic Analysis of Clinical Herpes Simplex Virus Type 2 Isolates from Shanghai and Taizhou, Zhejiang ProvinceObjective:To explore genetic variability of clinical HSV-2 isolates and determined circulating wild types strains of Shanghai and Taizhou, Zhejiang province, providing basis for further molecular epidemiology research.Methods:A total of 43 herpes fluid samples were consecutively collected from STD clinics of Shanghai and Zhejiang. Propagation of virus was performed by infecting Vero cells. Cells were collected until 80% of CPE was observed and supernatant was gathered for DNA extraction. Two regions of HSV-2 genome, US4 gene and US6 gene, were amplified with primers been published previously and designed according to HSV-2 reference strain HG52. Purification and sequencing of PCR products were finished by Genscript and Takara Companies. Alignments of the sequences were constructed by ClustalX2.0, recombination network included in the Splits Tree4.0 program was used to detect the recombination events. Bootscan method included in the SimPlot3.5.1 program was performed to validate the presence of recombination. Isolates presenting conflicting signals were removed from the dataset and compared with 48 isolates of US4 encoding sequences and 6 isolates of US6 encoding sequences downloaded from GenBank. Trees were constructed using Maximum-Likelihood method included in PhyML3.0. Bootstrap of 100 replications was carried out to estimate the robustness of the trees.Results:(1) CPE was observed among infected Vero cells, indicating the propagation of HSV-2 virus. Virus isolation rate was 66.7% for samples collected from Shanghai and 92.3% for samples collected from Taizhou, Zhejiang province. (2) US4 and US6 gene were amplified and PCR products were visualized by electrophoresis. The size of PCR products were 2180bp and 1200bp respectively, which was conform to the target gene length.32 CPE positive samples were all successfully amplified. (3) Multiple alignments shown high homology between US4 and US6 gene. A total of 34 variable sites were detected in the US4 alignment,15(45.7%) of which were synonymous and 19(54.3%) of which were nonsynonymous substitution; 10 variable sites were detected in the alignment, within which 6(60%) were synonymous and 4(40%) were nonsynonymous substitution (4) Recombination network based on US4 gene presented a complex reticulated topology while network based on US6 gene appeared tree-like branches. The bootscan test included in Simplot3.5.1 confirmed the presence of recombination. Network was reconstructed after removing isolates presenting conflicting signals, presenting tree-like topologies. Both networks based on US4 and US6 gene without recombination signals could be classified into at least two main branches. (5) Maximum-Likelihood (ML) trees based on isolates from our study and other 48 isolates of US4 encoding sequences previously described showed that The eight Tanzania isolates classified as clade A in our study was in line with previous studies of phylogenetic analysis of gG encoding sequences. The HSV-2 reference strain HG52, as well as other isolates from Tanzania, Norway and Sweden previously genotyped as clade B, was shown still to be classified into the same strain.7 isolates from Shanghai and 3 isolates from Taizhou, Zhejiang province had been classified as clade B as well. Another 12 isolates from China (7 from Shanghai and 5 from Zhejiang) were separated from clade A and clade B, designated as clade B'. Maximum-Likelihood (ML) trees based on isolates from our study and other 6 isolates of US4 encoding sequences downloaded from GenBank showed that 16 isolates from Shanghai,7 isolates from Zhejiang,2 Swedish isolates and 3 USA isolates as well as strain IIG52 were clustered into clade B. Other 8 isolates from China (4 from Shanghai and 4 from Zhejiang) and 1 isolates from Iran were clustered into clade B'. All main branches were supported by high boostrap value.Conclusions:A novel clade B'of HSV-2, probably the transitionary subclade between clade A and clade B, was first found in our study. As for now, cladeB and clade B'is prevalent in China. Based on current data, Clade A mainly circulated in Africa and parts of Europe, clade B extensively distributed in Africa, Europe, USA and Asia, while clade B'detected in the isolates from China and Iran. As HSV-2 is assumed to cospeciated with its host, the pattern of HSV-2 phylogeny probably was the result of ancient human migration. Inter and intro recombination could also be crucial to the evolution of HSV-2. |
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