In the last decade, next-generation sequencing (NGS) technology, which is characterized by being high-throughput, rapid, sensitive, and accurate, has developed rapidly. Main components of NGS are platforms: 454 sequencing; illumina sequencing; ion torrent sequencing; SOLID sequencing. NGS is used widely for the human immunodeficiency virus (HIV)-1. In this review, we focus on applications of the dynamics of HIV-1 quasispecies.
Animals ; HIV Infections ; virology ; HIV-1 ; classification ; genetics ; isolation & purification ; High-Throughput Nucleotide Sequencing ; methods ; Humans

OBJECTIVETo develop a rapid test kit for antibody to HIV by nano immunomagnetic lateral flow method.

METHODSA rapid test kit was developed by conjugation of the HIV antigen gp41 and gp36 to 200nm super paramagnetic particles by carbodiimide (EDC) and coating of the HIV antigen gp41 and gp36 to nitrocellulose membrane. Then the kit was evaluated with serials of experiments.

RESULTSThe kit was qualified with examination of national reference panel of anti-HIV antibody for colloidal gold diagnostic kit. The sensitivity was 100% by tested with 20 HIV antibody positive sera, the specificity was 98.5% by tested with 600 HIV antibody negative sera, respectively. The stability of the kit was over 12 month by storage at room temperature.

CONCLUSIONA diagnostic kit for antibody to HIV was developed with the advantages of convenience, rapid test, good stability and point of care.

Antibodies, Anti-Idiotypic ; immunology ; Gold Colloid ; chemistry ; HIV ; immunology ; isolation & purification ; HIV Antibodies ; HIV Envelope Protein gp41 ; HIV Infections ; diagnosis ; HIV Seropositivity ; blood ; HIV-1 ; immunology ; isolation & purification ; Immunomagnetic Separation ; methods ; Molecular Biology ; methods ; Nanotechnology ; Reagent Kits, Diagnostic

OBJECTIVETo compare the results of detecting HIV-1 load by using NucliSens HIV-1 QT and Amplicor HIV-1 monitor 1.5 assays.

METHODSEighty-two clinical samples were collected and HIV viral load was determined with the above-mentioned two methods.

RESULTSThe number of samples in which values obtained by NucliSens HIV-1 QT and Amplicor HIV-1 monitor 1.5 differed by <0.5 log10 RNA copies/ml and in which the viral load was undetectable accounted for 88.9 percent of the measures. The correlation coefficient between the two methods was 0.956 in 56 samples of Deltalog10 VL<0.5.

CONCLUSIONThe results of HIV-1 viral load determination with the two methods are highly comparable.

HIV Infections ; virology ; HIV-1 ; genetics ; isolation & purification ; Humans ; Nucleic Acid Amplification Techniques ; instrumentation ; methods ; RNA, Viral ; genetics ; Viral Load

OBJECTIVETo analyze the geographical distribution and risk factors of HIV-1 subtypes in Yunnan province.

METHODSBlood samples from 1319 HIV positives were collected in Yunnan Province from 2001 to 2006. The nested polymerase chain reaction was used to amplify the gag (p24)-protease fragments from RNA extracted from plasma or sera. The sequences were used for subtype determination by phylogenetic tree analysis.

RESULTSAmong 1319 samples studied, the subtypes has been successfully obtained from 644 samples that were constituted of seven subtypes: CRF08_BC, CRF07_BC, CRF07/08_BC, CRF01_AE, C, B' and URFB/C. C/CRF07_BC/CRF08_BC were distributed in the whole province, but CRF01_AE were mainly distributed in the boarding areas with Myanmar such as Dehong, Baoshan, Xishuangbanna and Puer. Moreover, injecting drugs users accounted for 61.6% (270/438) among C/CRF07_BC/CRF08_BC infections, while only 8.5% (15/177) among CRF01_AE infections.

CONCLUSIONOur data indicated that at least seven subtypes were identified in Yunnan province, the relationship between subtypes and transmission routes were analyzed, and the geographic difference of subtypes was also observed.

China ; DNA, Viral ; Genotype ; HIV Infections ; transmission ; virology ; HIV-1 ; classification ; isolation & purification ; Humans ; Sequence Analysis, DNA

Objective: To understand the transmission patterns and risk factors of HIV-1 strain CRF01_AE subtypes in China, and to provide guidance for the implementation of precise intervention. Methods: A total of 2 094 CRF01_AE pol sequences were collected in 19 provinces in China between 1996 and 2014. Phylogenetic tree was constructed by PhyML 3.0 software to select the transmission clusters. Transmission network was constructed by Cytoscape 3.6.0, which was further used for exploring of the major risk factors. Results: Of the 2 094 sequences, 12.18% (255/2 094) were in clusters. A total of 82 transmission clusters were identified. The numbers of clusters and contained sequences in intra-provincial transmission (61, 173) were significantly more than those in inter-provincial transmission (21, 82). The ratio of transmission clustering in MSM increased over time from 2.41% (2/83) during 1996-2008 to 23.61% (72/305) during 2013-2014, showing a significant upward trend (χ(2)=27.800, df=1, P=0.000). The proportion of MSM with inter-provincial transmission clusters were higher than those with intra-provincial transmission clusters, which increased from 0.67% (2/297) during 1996-2008 to 6.36%(30/472) during 2013-2014, showing a significant upward trend (χ(2)=20.276, df=1, P=0.000). The transmission rate in homosexuals of the inter-transmission clusters (86.59%, 71/82) was higher than that of intra-provincial transmission clusters (56.65%, 98/173), and the difference was statistically significant (χ(2)=22.792, P=0.000). The proportion of inter-provincial transmission clusters with more than 2 transmission routes (33.33%, 7/21) was higher than that of intra-provincial clusters (13.11%, 8/61), and the difference was statistically significant (χ(2)=4.273, P=0.039). Results from the transmission network analysis indicated that the proportion of high risk population (degree≥4) with inter-provincial transmission clusters (51.22%, 42/82) was significantly higher than that with intra-provincial transmission clusters (26.59%, 46/173), and the difference was statistically significant (χ(2)=14.932, P=0.000). Inter-provincial clusters were mainly detected in and and MSM. Conclusions: Complex transmission networks were found for HIV-1 CRF01_AE strains in the mainland of China. Inter-provincial transmission clusters increased rapidly, MSM played an important role in the wide spread of the strain. More researches in transmission networks are needed to guide the precision intervention.
China/epidemiology* ; HIV Infections/virology* ; HIV-1/isolation & purification* ; Homosexuality, Male/statistics & numerical data* ; Humans ; Male ; Phylogeny

OBJECTIVETo establish the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methods for on-site HIV-1 detection.

METHODSAs for the real-time fluorescent RT-LAMP, we firstly tested the specificity and sensitivity, then explored its quantitative determination, and finally applied the method to the detection of 35 HIV-1 positive samples. For colorimetric judgment, after choosing different ameliorates to modify Hydroxynaphthol blue (HNB), we tested their real effects on coloration, and then picked out the modified dyes with obvious color change to test the sensitivity and the detection of the 35 HIV-1-positive samples.

RESULTSThe real-time fluorescent RT-LAMP showed great specificity of HIV-1, and the sensitivity to detect HIV-1 RNA was between 10 and 100 copies per reaction. On testing 35 HIV-1-positive samples, the method could reach 100 percent detection rate. However, for the quantitative determination, the quantitative relation was not observed regarding the HIV-1 RNA of below 10(3) copies per reaction. Three modified HNB dyes with clear color variation between the reaction tubes of the negative and the positive were got in the study, and their sensitivities equaled to the level of agarose gel electrophoresis. Similarly, 100% (35/35) detection rate was reached when the colorimetric RT-LAMP with the modified dyes was applied to detect 35 HIV-1-positive samples.

CONCLUSIONThe established real-time fluorescence method and the modified color judgment of RT-LAMP could be helpful for truly achieving rapid, accurate, and sensitive on-site detection of HIV-1.

HIV-1 ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods ; Reverse Transcriptase Polymerase Chain Reaction

To investigate HIV-1 subtype distribution and prevalence of HIV-1 drug resistance in Liuzhou and Nanning, a total of 304 HIV-infected subjects or AIDS patients from Liuzhou and Nanning were recruited. Whole blood was withdrawn from a peripheral vein of each subject. HIV RNA were extracted from plasma, and subjected to PCR amplification targeting HIV pol gene fragment and DNA sequencing. Sequences obtained were subtyped by phylogenetic analysis. Two subtypes, CRF01_AE and CRF07_BC, were found in subjects from Liuzhou, accounting for 75.2% and 24.8%, respectively. Subtype CRF01 AE, CRFO8_BC, B, and C were found in subjects from Nanning. CRF01_AE and CRF08 BC were still the dominant strains in Nanning, accounting for 85.8% and 11.5%, respectively. Sequences were also analyzed for drug resistance mutations, and rates of drug resistance were calculated. The rate of drug resistance was 3.3% in ART-naive subjects from Liuzhou, and 8.7% in the ART-experienced. For patients from Nanning, the rate was 1.4% in ART-naive subjects, and 27.5% in ART-experienced subjects.
Anti-HIV Agents ; pharmacology ; China ; epidemiology ; Drug Resistance, Viral ; Genotype ; HIV Infections ; epidemiology ; virology ; HIV-1 ; classification ; drug effects ; genetics ; isolation & purification ; Humans ; Molecular Sequence Data ; Phylogeny

Objective: To understand the characteristics and dynamic of HIV-1 subtype distribution in men who have sex with men (MSM) in Guangzhou between 2008 and 2015. Methods: HIV-1 RNAs were extracted from serum samples of the individuals newly diagnosed with HIV-1 infection among MSM living in Guangzhou between 2008 and 2015. The pol gene segments of HIV-1 genome from these RNA samples were amplified by nested reverse transcription polymerase chain reaction (nested-PCR) and were sequenced. Subsequently, the phylogenetic tree was reconstructed using pol sequences of samples and references together and the subtype of HIV-1 was determined. The distributions of HIV-1 subtypes detected in MSM with different demographic characteristics in different years were compared. Results: A total of 2 210 pol gene segments were successfully obtained from 2 473 serum samples of the MSM. The average age of 2 210 MSM was 30.19 years with standard deviation of 8.22 years, the unmarried MSM and those in Han ethnic group accounted for 73.39% and 90.81%, respectively. The proportion of subtype CRF07_BC (38.10%) was highest, followed by CRF01_AE (34.84%), CRF55_01B (14.62%), B (6.06%), URFs (3.58%), CRF59_01B (2.17%) and other subtypes (0.63%). The annual proportions of subtype B (P=0.000, 99%CI:0.000-0.000), CRF07_BC (χ(2)=14.965, P=0.036), CRF55_01B (χ(2)=18.161, P=0.011) and URFs (P=0.001, 99% CI: 0.000-0.001) were significantly different. The proportion of subtype B showed a gradual decrease from 14.08% to 4.33% (P=0.000, 99%CI: 0.000-0.000), while the proportion of URFs rapidly increased from 0% to 6.40% (P=0.000, 99% CI: 0.000-0.000). The rate of URFs was significantly higher in farmers and migrant workers than in other groups (P=0.017, 99%CI: 0.014- 0.020) and the rate of URFs was higher in individuals who had multi sexual partners (χ(2)=5.733, P=0.017). Conclusions: CRF07_BC and CRF01_AE were the predominant HIV-1 subtypes and multiple subtypes co-circulated among MSM in Guangzhou between 2008 and 2015. The recombinations of HIV-1 continue to occur in MSM. Strengthening behavioral intervention for farmers, migrant workers and individuals who have multi sexual partners has the important epidemiological significance against the emerging and circulating of the novel recombinant virus among MSM in Guangzhou.
China/epidemiology* ; Genes, pol ; Genotype ; HIV Infections/virology* ; HIV Seropositivity/genetics* ; HIV-1/isolation & purification* ; Homosexuality, Male ; Humans ; Male ; Phylogeny ; Polymerase Chain Reaction ; RNA, Viral/blood* ; Sexual Behavior

OBJECTIVETo establish a specific and sensitive Enzyme-linked immunosorbent Assay (ELISA) kit for detection of HIV-1 antibody in urine using Escherichia coli expression products as coating antigen.

METHODSThe truncated HIV-1 gp41 gene fragment of major antigenic epitopes was inserted into the plasmid pET22b to obtain expression plasmid pET22b-mgp41. The recombinant antigen was expressed in BL21 (DE3) strains of Escherichia coli and was purified by immobilized metal chelation and gel filtration chromatography. Using this antigen as coating antigen, a HIV-1 urine antibody ELISA kit was developed. In order to examine the clinical utility of the kit, 5437 urine samples were assayed, which consisted of 641 urine samples from HIV infected patients and 4796 samples from normal subjects. Results The purity of purified antigen is up to 95%. Anti-HIV antibodies were detected in all the urine samples from HIV infected patients, and the diagnostic sensitivity for HIV-1 infection was 100%. In healthy control group, 71 cases showed false positive, the specificity was 98.52%.

CONCLUSIONThe HIV-1 urine antibody kit can be used in screening and diagnosing for HIV-1 infection.

Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; HIV Antibodies ; urine ; HIV Core Protein p24 ; genetics ; immunology ; isolation & purification ; HIV Infections ; immunology ; urine ; HIV-1 ; genetics ; immunology ; Humans ; Recombinant Proteins ; genetics ; immunology ; isolation & purification

OBJECTIVETo develop a rapid assay for simultaneous detection of HIV p24 antigen (Ag) and anti-HIV antibody (Ab).

METHODSHIV-1 gp41 antigen and HIV-2 gp36 antigen were expressed by recombinant baculovirus insect system and purified by immunochromatography. p24 monoclonal antibody (mAb) was obtained from p24 hybridoma cell line. Purified antigen and mAb were dot blotted to nitrocellular membrane; 20 nm colloidal gold-anti-human IgG ab and p24 ab complex were used for this test. Previously detected 39 sera specimens were tested in this study to compare with the result of HIV test with commercial HIV test kit.

RESULTS20 mg/L purified gp41 Ag and gp36 Ag were obtained from recombinant baculovirus-insect cell system; 1.5 mg/L p24 mAb was obtained from p24 mAb hybridoma cell line. Compared the test result of 39 sera with commercial HIV test kits, consistency rate was 100%.

CONCLUSIONSThe rapid assay for simultaneous detection of HIV p24 antigen and anti-HIV antibody provides a simple, sensitive and reliable test for HIV diagnosis.

AIDS Serodiagnosis ; Gene Products, env ; biosynthesis ; isolation & purification ; HIV Antibodies ; blood ; HIV Antigens ; biosynthesis ; isolation & purification ; HIV Core Protein p24 ; blood ; HIV Envelope Protein gp41 ; biosynthesis ; isolation & purification ; HIV Infections ; diagnosis ; HIV-1 ; immunology ; HIV-2 ; immunology ; Humans ; Reagent Kits, Diagnostic ; standards ; env Gene Products, Human Immunodeficiency Virus