Analysis on gene sequence of HIV isolated from men who have sex with men in Beijing

To clarify the genetic characteristics of human immunodeficiency virus (HIV) circulated in the population of men who have sex with men (MSM) in Zhengzhou, Henan and to analyze its relationship with HIV-1 prevailing in the paid blood donors (PBDs). Thirty-one MSM who were confirmed as HIV positive individuals in 2010 together with 41 HIV-positive former PBDs were enrolled in the study. Information on related epidemiological characteristics and their plasma were collected. RT-PCR was used to amplify HIV-1 full length gag (1584 bp), pol (3147 bp) genes and partial env gene (C2V3 segment, 558 bp) followed by sequencing on those subjects. Online software available at LosAlamos HIV Database was used to identify the HIV subtypes based on the findings of the sequences. Phylogenetic tree was used to identify the possible relationship of transmission. Fifty-three full length gag, 38 full length pol and 48 partial env (C2V3) genes were collected from 72 participants. Among the 31 HIV(+) MSM individuals, 14 CRF01_AE strains, 5 CRF07_BC strains and 12 subtype B (1 subtype B and 11 B') strains were identified respectively. All of the 41 strains identified from former PBDs were infected by B' strains. The CRF01_AE strains identified in MSM showed a close relationship to those identified from both Hebei and Liaoning provinces. The CRF07_BC strains showed a close relationship with those from Shijiazhuang and Beijing cities. Among the 12 subtype B strains, 8 sequences grouped into 1 cluster with 1 sequence from the former PBDs. Two sequences grouped with 02HNseq4 suggested that B' had been prevailed in the MSM population might come from the former PBDs and were closely related to the strains identified in the MSM population. Complicated genetic background and multiple introductions of HIV in the MS population in Zhengzhou, were found. This was also the first report which noticed that the subtype B epidemic among Zhengzhou MSM was mainly originated from the B' among the former PBDs.

Objective To understand the molecular epidemiological characterization of human immunodeficiency virus (HIV)-1 CRF01_AE strains in China. Methods Data were extracted by a systematic search in the databases combined with literature review. Data were then grouped according to the sites and risk groups for a Meta-analysis. Sequences of CRF01_AE pol genes from China were downloaded from Los Alamos database to build phylogenetic trees by means of FastTree2.1. The Bayes factor test was calculated by BEAST V1.6.2 package and Spread to explore spatial transmission links. Results Meta-analysis demonstrated that CRF01_AE strains accounted for more than 45.0% of all subtypes among men who have sex with men (MSM) in six areas of China. Among heterosexuals in eastern, northern, southwestern and south-central China, the proportions of CRF01_AE strains exceeded 30.0%. The strains were also prevalent among intravenous drug users in south-central regions, accounting for 57.3% (95%CI: 35.1%-79.6%), and were only detected among blood donors in south-central China (10.6%, 95%CI: 6.2%-14.9%). Seven distinct phylogenetic clusters of CRF01_AE strains which were transmitted independently were identified. Clusters 1, 3 and 5 were prevalent among heterosexuals, while clusters 2 and 6 were found primarily among MSM, and clusters 4 was detected chiefly among sexual contact people. Geographically, clusters 1, 2, 3, 4 and 5 were prevalent in southwestern areas. Clusters 1 and 7 were circulating in south-central areas. Clusters 2 and 6 were prevalent in northern areas, while clusters 2 and 4 were dominant in eastern regions and cluster 6 was prevalent in northeastern China. The Bayes factor test reveals the complexity of transmission links among eastern, south-central, southwestern, northern, and northeastern provinces. Conclusion CRF01_AE strains are prevalent in most high risk groups in multiple regions of China and the transmission between different regions is complicated. Key words: CRF01_AE strains; Epidemiology, molecular; Meta-analysis; Phylogenetic analysis; Bayes theorem

To investigate the dynamic change and associated risk factors of HIV sero-conversion rate in Beijing. 809 sero-negative men who have sex with men (MSM) were recruited in the cohort from August to December in 2009. HIV sero-antibody, medicinal examination and behavior questionnaire interview were carried out every six months. 962 MSM with overall baseline prevalence of HIV infection as 6.34% (61/962) together with 809 sero-negative MSM, were enrolled in the cohort. Of the 809 sero-negative participants, 95.1% (769/ 809), 85.5% (692/809) and 71.0% (574/809) of them were retained in the 6-month, 12-month and 18-month follow-up visits, with 19, 29 and 17 of them became HIV sero-conversion at 6-month, 12-month, and 18-month follow-up visits and the HIV incidence rates appeared to be 5.47, 12.37 and 6.86 per 100 person-years respectively. The HIV incidence was 7.59 per 100 person-years in the 18 months follow-up visit. Factors including: younger than 25-years old (HR = 2.32, 95%CI: 1.39 - 3.87), having more than 8 MSM partners (HR = 2.50, 95%CI: 1.49 - 4.20), less than 2000¥ every month income (HR = 1.76, 95%CI: 1.05 - 2.95), having more than 4 homosexual partners in the last six months (HR = 3.50, 95%CI: 2.11 - 5.81), showing phimosis and redundant prepuce (HR = 2.47, 95%CI: 1.50 - 4.07) as well as positive syphilis test (HR = 2.62, 95%CI: 1.53 - 4.49) etc., were significantly associated with HIV incidence. High HIV incidence was shown among MSM in Beijing and had spread fast in this population, calling for more favorable prevention measures to be taken.

Objective To investigate the genetic characteristics of human norovirus (NoV) among infants under 5 years of age with diarrhea in Chaoyang District, Beijing from 2011 to 2017. Methods NoV-positive stool samples were collected from 2011 to 2017 in this region. The partial RdRp and VP1 genes were amplified and sequenced. Multi-sequence alignment was performed and phylogenetic tree was constructed using Mega software. Results A total of 151 samples were sequenced and analyzed. The ratio of male and female was 2.28∶1 with mean age of 1.72 years. Fourteen NoV subtypes were detected, including GII.Pe/GII.4 (47.68%), GII.P12/GII.3 (20.53%), GII.P4/GII.4 (17.22%), GII.P16/GII.2 (3.31%), GII.P12/GII.12 (1.99%), GII.P17/GII.17 (1.99%), GII.P16/GII.13 (1.32%), GII.P7/GII.7 (1.32%), GII.P7/GII.6 (1.32%), GII.P2/GII.2 (0.66%), GII.P21/GII.21 (0.66%), GII.Pg/GII.12 (0.66%), GI.Pa/GI.3 (0.66%) and GI.P6/GI.6 (0.66%). Conclusions NoV genetic diversity was found among infants under 5 with diarrhea in Chaoyang district, Beijing. The subtypes from surveillance and those from epidemics occurred in chronological order. The surveillance should be strengthened for early detection of new subtype for monitoring the epidemic and vaccine design. Key words: Norovirus; Viral diarrhea; Genotype; Phylogenetic analysis

Origin and Outbreak of HIV-1 CRF55_01B Among MSM in Shenzhen, China

Objective To investigate the genetic characteristics of VP1 genes carried by coxsackievirus A16 strains isolated from cases of hand foot and mouth disease (HFMD) in Shenzhen during 2016 to 2017. Methods Fecal and anal swab specimens were collected from patients with mild HFMD in four sentinel hospitals and the Institute of Pathogen Biology, Shenzhen Center for Disease Control and Prevention, China during 2016 to 2017. All specimens were tested for CVA16 viral RNA using real-time RT-PCR. The VP1 genes of 51 randomly selected CVA16 strains were amplified by RT-PCR and then sequenced using TaKaRa Biomedical Technology (Dalian). Bioinformatics software, including Mega6.02, BioEdit and DNAStar, was used for comparison and analysis of the VP1 genes. Results CVA16 strains in Shenzhen during 2016 to 2017 mainly belonged to B1a and B1b subtypes as well as an emerging subtype B3. The epidemic of B1b subtype was found in both 2016 (28 strains) and 2017 (19 strains), while the B1a subtype (two strains) was only detected in 2017. Two B3 subtype strains were detected in 2017. The strains of B1b subtype were closely related to the strains isolated in Shanghai (JQ314149), Wenzhou (KP289416) and Beijing (KU254598), while the B1a subtype strains were closely related to the strains isolated in Kunming (JQ316639) and Tailand (GQ184139). The B3 subtype strain was an emerging CVA16 epidemic strain in mainland China. Further comparison of the CVA16 epidemic strains in Shenzhen area during 2016 to 2017 with the CVA16 strains causing severe neurological symptoms showed that two amino acid mutations (S14N and M23L) were found in VP1 protein. Conclusions The epidemic strains of CVA16 were B1b subtype in Shenzhen area in 2016. However, B1a, B1b and the emerging B3 subtype strains were prevalent in 2017. Compared with the CVA16 strains causing severe neurological symptoms, the CVA16 strains circulating in Shenzhen during 2016 to 2017 carried two amino acid mutations inVP1 protein. Key words: Coxsackievirus A16; Hand, foot and mouth disease; Phylogenetic analysis

Using methodology of molecular genetics to explore the origin, phylogen, and gene flow of Mycobacterium tuberculosis (MTB) Beijing lineage in the five provinces from northern China, including Heilongjiang, Jilin, Liaoning, Neimenggu and Ningxia. 234 MTB Beijing lineage strains were genotyped by 24 Variable Number Tandem Repeat (VNTR), and the h (the allelic diversity) value of each VNTR locus was calculated. On individual level of phylogeny, it was constructed Neighbor-Joining (N-J) tree and minimum spanning tree (MST). Phylogenetic tree was built at the population level, and the most recent common ancestor (TMRCA) was estimated through Bayesian model. Molecular variance (AMOVA) was used to understand the gene flow among strains discovered from the five provinces. Allelic diversities of the 24 VNTR loci were low (h: 0.000 - 0.744). 234 strains of MTB Beijing lineage were dispersed in individual branch of the N-J tree, with 62.0% (145) of them grouped to the same "colonial complexes" in MST. At the population level, the evolution relationship of 234 strains appeared the closest to Beijing lineage, which was from MIRU-VNTRplus database, and the bootstrap was 100. The TMRCA was 5308 (95%CI: 4263 - 6470) years. Differences of pairwise Fst values acquired by AMOVA between Jilin and Heilongjiang, Liaoning, Neimenggu and Ningxia, were not statistically significant (P > 0.05). The genetic similarity of Beijing lineage MTB from the five provinces of northern China was high. The phylogeny branches had no characteristic dispersal in each province. It was speculated that these strains showed an evolution from a clone of MTB Beijing lineage (about 5000 years ago). The gene flow was taking place between neighboring zones.

Objective To investigate the genetic evolution of VP1 gene of pathogenic coxsackievirus A16 (CV-A16) strain isolated from clinical hand, foot, and mouth disease (HFMD) patients. Methods A total of 160 HFMD cases with CV-A16-positive results were collected from hospitals in Kunming during January 2015 to June 2017. Fecal samples were collected. Real-time polymerase chain reaction (PCR) was used to detect the CV-A16 virus nucleic acid. The VP1 genes of CV-A16-positive samples were amplified by reverse transcription-PCR. The amplified positive products were sequenced and aligned. The homologies were identified and their subgenotypes were determined. The phylogenetic tree was constructed and homology modeling was conducted. Results All the 160 CV-A16 isolates were B2 subtypes. The genetic distance between detected strains of CV-A16 and the strains in Fujian, Beijing, Nanjing was 0.76. The genetic distance to the strains in Malaysia was 0.78, and to the strains in Australia was 1.86. Homologous modeling revealed that the amino acid sequence of the VP1 gene of the strain had a G227R mutation. Conclusions There is no major genetic variation in the CV-A16 strains during 3 years. CV-A16 isolates are close to those of epidemic strains in Beijing, Fujian and Malaysia, but are far fram the strains from Australia. Key words: Hand, foot and mouth disease; Coxsackievirus A16; VP1 gene; Phylogenetic tree; Homology modeling

To analyze the molecular epidemiological characteristic of HIV-1 B'/C strains prevalent in Beijing. Plasma samples were collected from 200 newly diagnosed HIV-1 B'/C individuals reported during 2006 to 2010 in Beijing. The gag gene fragments were amplified from RNA template extracted from plasma with reverse transcription (RT) and nested polymerase chain reaction (PCR). And the sequences were analyzed by phylogenetic methods and Entropy analysis. A total of 159 sequences were successfully amplified from the gag genes of which 147 was CRF07_BC and 12 CRF08_BC. There were 3 main sub-clusters in CRF07_BC phylogenetic tree and they were named as sub-cluster IDU-Max (89 sequences), sub-cluster IDU-Min (22 sequences) and sub-cluster MSM (34 sequences) based on transmission.No international reference strain was closely related with these three sub-clusters except for one strain identified in Taiwan. All CRF07_BC recombinant strains were remarkable for their low interpatient diversity in gag genes (3.7%, 3.3% and 2.0% for isolates from IDU-Max, IDU-Min and MSM respectively).When compared with sub-cluster IDU-Max, there were 32 and 41 significantly different sites of nucleotide polymorphism compositions in sub-clusters IDU-Min and MSM. This is the first report of describing the existence of three main epidemic sub-clusters in CRF07_BC strains prevalent in Beijing. And IDU-Max sub-cluster is the dominant strain. The CRF07_BC in Beijing are less diverse than other strains and may be derived from a common ancestor.

To explore the population-genetics characteristics of Mycobacterium tuberculosis (MTB) prevailing at the Sichuan basin of China. A total of 413 MTB strains collected from Sichuan basin were genotyped by large sequence polymorphism (LSP) and 15 loci variable number tandem repeat (VNTR). Difference between the distribution of lineage population was analyzed by χ(2)-test and the discriminatory ability of each VNTR locus was evaluated under the Hunter-Gaston Discriminatory Index (HGI). Both phylogeny on population level and genetic structure were demonstrated through N-J tree and the Minimal Spanning Tree (MST). Genetic differentiation of different lineage strains was analyzed by Analysis of Molecular Variance (AMOVA). Time of the Most Recent Common Ancestor (TMRCA) was calculated based on the Bayesian model. Four hundred and thirteen MTBs were divided into two major lineages, in which the Beijing lineage accounted for 56.2% (232/413) and the Euro-American lineage for 43.8% (181/413). There was no significant difference of population distribution between the two lineages (P > 0.05). The N-J tree of Beijing lineage MTB presented distinctly "star-like" and 72.4% strains were grouped to one clonal complex in MST. The Euro-American lineages MTB presented "branch-like" in N-J tree and were grouped into multiple clonal complexes in MST. There was significant genetic differentiation in Beijing lineage MTBs between Chongqing and Sichuan (FST = 0.018 91, P < 0.05), but not in the Euro-American lineage MTB (FST = 0.005 19, P > 0.05). TMRCA of the largest clonal complex in Euro-American lineage MTB appeared to be 723 (95% CI: 517-946) years. Both Beijing lineage and Euro-American lineage MTBs were competitively prevalent in the Sichuan basin. There was some difference noticed between the two lineages referring to genetic differentiation. The invasion of Euro-American lineages MTB to the basin area might be associated with a war occurred in this area about 720 years ago.

Objective To analyze the variation characteristics of HIV-1 Gp120 sequences in men who have sex with men (MSM) in Guangzhou. Methods Plasma samples were collected from HIV-1 infected MSM before antiretroviral treatment. Viral RNA was extracted from plasma. Gp120 gene sequences were amplified by reverse transcription and nested-PCR using specific primers. Phylogenetic tree, length polymorphism, amino acid characteristics of V3 loop, co-receptors and signature amino acids were analyzed. Results The phylogenetic tree were divided into 4 clusters, and the most prevalent subtypes were CRF07_BC (34/61, 55.74%) and CRF01_AE (24/61, 39.34%). Majority of HIV-1 Gp120 sequences had 496-515 amino acids. Among five hypervariable regions, the V1 region had the highest levels of length polymorphism and V3 region had the lowest. The top four peptide of V3 loop were GPGQ (56/58, 96.55%). Most of the co-receptors HIV-1 strains used was CCR5(50/58, 86.21%)according to four methods of comprehensive prediction. There are four signature amino acids in CRF01_AE subtype strains, and the frequency of occurrence was 0.75-0.83; there are eight signature amino acids in CRF07_BC subtype strains, and the frequency was 0.74-0.94. Conclusions The length of Gp120 sequences in MSM in Guangzhou has a high polymorphism. The top four peptide of V3 loop, co-receptor and signature amino acid of V3 ring have formed unique patterns. Key words: HIV-1; Gp120; V3 loop; Signature amino acid

Phylogenetic Analysis of HIV-1 CRF65_CPX Reveals Yunnan Province Is Still a Source Contributing to the Spread of HIV-1 in China

Objective To confirm the death of a child injured by a dog was due to rabies and to understand the molecular biologic features of rabies virus in Kaili, Guizhou province. Methods Brain tissue samples of patient and dog were collected to detect the rabies virus by direct immunofluorescence assay （DFA） and RT-nested PCR assay. Homology and phylogenetic tree were analyzed based on the whole nueleotide and deduced amino acid sequence of N gene of rabies virus followed by molecular epidemiological analysis. Results Both the human and dog brain tissue samples were confirmed positive by DFA and RT-nested PCR assay. The homology analysis of N gene sequences among GZH, GZD and other epidemic and vaccine rabies strains isolated from other provinces and other countries indicated that the detected samples shared the highest homology with the strain detected in Anlong prefecture in Guizhou in the year of 2006, and the homology between GZH and GZD was as high as 100%. Besides, among the vaccine strains, GZH and GZD showed the highest homology with strain CNT. Phylogenetic analysis indicated that the two samples were very close and belonged to genetype 1 lyssavirus, with the closest relationship between samples reported in Guizhou and Beijing. Conclusion It was confirmed on the viral molecular level that both the human and dog in Kaili were suffered from rabies, and the pathogens were genetype 1 lyssavirus. The prevalent strains in Kaili city was probably imported from other prefectures of Guizhou province, suggesting that prevention and control measures on rabies in Guizhou province should be strengthened. Key words: Rabies/virus ; N gene ; Homology ; Phylogenetic tree

Objectived：The main purposes of this study were to surveillance the HIV transmission and try to prevent it in Taiwan. (1) For the subtype surveillance purpose, this study was try to investigate the distribution of HIV-1 subtypes among different high-risk groups、spread pathways and evolution time period in Taiwan. (2)For the prevention purpose, this study wish to develop the HIV-1 infection risk prediction model for IDUs by using the questionnaire of risk factor investigation. This risk prediction model would aim to be a tool for predict their probable risk of HIV infection and increase their self-awareness HIV-1 infection. Methods：The study population came from a Taiwan HIV-1 surveillance network which builded from 2004. This network established a database which included HIV-1 infection risk factor questionnaire collection and HIV-1 seropositive blood samples from IDUs of jails and HIV/AIDS Outpatient department of China medical university hospital in Taiwan annually. (1) About HIV-1subtype surveillance, we collected 2007-2009 HIV-1 seropositive blood samples from HIV-1 surveillance network. Their subtypes were examined by using DNA sequencing and phylogenetic analysis, and applyed coalescent theory, molecular clock analysis to estimated evolution rate and time of the most recent common ancestors (TMRCA). And, the risk factor characteristics of different subtypes were calculated by the biostatistic method. (2) Using 2004 to 2005 risk factor questionnaire database in HIV-1 surveillance network for building up HIV-1 infection risk prediction model for IDUs, we investigated risk factors of HIV-1 infection by case-control study. The risk prediction model was established by logistic regression, and odds ratios (ORs) for each risk factor were summed. Then, we used ROC curve (Receiver operating characteristic curve) to know HIV-1 risk threshold of IDUs. Next, we collected 2007 -2008 data of risk factor questionnaire database to validate the accuracy of this risk prediction model. Result：(1) HIV-1 subtype surveillance : HIV-1 subtype determined by phylogenetic analysis and nested multiplex PCR. After subtyping, we found three HIV-1 subtypes in our cases, including CRF07_BC, subtype B and CRF01_AE, most of these is CRF07_BC. CRF07_BC originated in 1993.6-1997.3 in China. TMRCA of CRF07_BC from southern, middle and northern were dated to 1998.3-2003.3, 2003.4-2004.4 and 2002.7-2004.3. In HIV-1 subtype and related HIV-1risk factor analysis showed, demographic and risk behavior factos among HIV-1subtype were differenct. (2) For risk factor prevention purpose: In the risk prediction model of IDUs, the significant variables among men were education, syringe sharing, heroin diluention sharing, and knowledge of AIDS- persons often had the venereal diseases, they easier had AIDS, and among women was syringe sharing. The risk threshold value of ORs in men is 7.435 (sensitivity: 0.96; specificity: 0.52). Because only one significant variable among women, the risk threshold value of ORs was not calculated. Finally, we compared with the sum of total ORs for each risk factor and the risk threshold of ORs, then, we could identify the risk of HIV infection. Furthermore, we collected questionnaire in 2007 ~ 2008, and tested this prediction model. The test result is : sensitivity – 72.2% ; specificity – 60.5%.

To explore the distribution characteristics of the types of M protein gene (emm) in group A streptococcus (GAS) isolated from children in Beijing in the year 2011. During May to July in 2011, a total of 3315 patients who were diagnosed scarlet fever or pharyngeal infection by doctors in pediatric outpatient and emergency units of 36 hospitals, were selected as subjects. Their throat swab samples were collected and isolated the strains of GAS. Gene emm was then amplified and sequenced by PCR method, and the differences in types of gene emm between different populations and diseases were compared. A total of 633 strains of GAS were isolated from the 3315 throat swab samples, 610 strains out of which were gene emm positive and were recruited in the study. Out of the 610 recruited strains, 448 (73.4%) were isolated from scarlet fever patients, the other 162 (26.6%) were isolated from pharyngeal infection patients; 397 (65.1%) were from urban, the other 213 (34.9%) were from suburb; 240 (39.4%) were from patients aging between 1 - 5 years old, the other 369(60.6%) were from patients aging 6 - 18 years old. A total of 8 types of gene emm (scarlet fever: 6 types, pharyngeal infection: 4 types) and 21 subtypes of gene emn (scarlet fever: 16 subtypes, pharyngeal infection: 10 subtypes) were identified. Three new subtypes were found in the study, naming emm1.63, emm12.62 and st5144.20. Among them, emm1.63 was found both in scarlet fever and pharyngeal infection patients, while emm12.62 and st5144. 20 were only found in pharyngeal infection patients. Among all the types of gene-emm, emm12 accounted for the highest percentage as 80.5% (491/610) and then followed by emm1 (18.0% (110/610)). Among all the subtypes, the dominant subtype was emm12.00, accounting for 69.0% (421/610), following by emm1.00 (16.9% (103/610)) and emm12.19 (6.1% (37/610)). All the above types and subtypes of gene emm were the most prevalent strains in scarlet fever patients and pharyngeal infection patients. Significant differences in the distribution of prevalent strains were observed among various aging patients and regions. The constituent ratios of emm1, emm1.00 and emm12.19 were higher in patients from suburb (emm1: 22.1% (47/213), emm1.00: 19.2% (40/213), emm12.19: 8.0% (17/213)) than those in urban areas (emm1: 15.9% (63/397), emm1.00: 15.6% (62/397), emm12.19: 5.0% (20/397)). The difference showed statistical significance (P < 0.05). The constituent ratio of emm1.00 was higher among patients aging 6-18 years old (19.2% (71/369)) than those aging 1 - 5 years old (13.3% (32/240)). The difference also showed statistical significance (χ(2) = 8.45, P < 0.05). Among the types of gene emm in GAS isolated from children in Beijing in year 2011, the most prevalent two were emm12 and emm1, and the most prevalent emm subtypes were emm12.00, emm1.00 and emm12.19. A significant difference in their distribution between various aging patients and isolated places can be obviously found.