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 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 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 the genomic properties of HIV-1, we collected 3,081 sequences from the HIV Sequence Database. The sequences were categorized according to sampling region, country, year, subtype, gene name, and sequence and were saved in a database constructed for this study. The relative synonymous codon usage (RSCU) values of matrix, capsid, and gp120 and gp41 genes were calculated using correspondence analysis. The synonymous codon usage patterns based on the geographical regions of African countries showed broad distributions; when all the other regions, including Asia, Europe, and the Americas, were taken into account, the Asian countries tended to be divided into two groups. The sequences were clustered into nine non-CRF subtypes. Among these, subtype C showed the most distinct codon usage pattern. To determine why the codon usage patterns in Asian countries were divided into two groups for four target genes, the sequences of the isolates from the Asian countries were analyzed. As a result, the synonymous codon usage patterns among Asian countries were divided into two groups, the southern Asian countries and the other Asian countries, with subtype 01_AE being the most dominant subtype in southern Asia. In summary, the synonymous codon usage patterns among the individual HIV-1 subtypes reflect genetic variations, and this bioinformatics technique may be useful in conjunction with phylogenetic methods for predicting the evolutionary patterns of pandemic viruses.
HIV-1/*genetics/*isolation & purification ; Gene Expression Regulation, Viral/*genetics ; Europe/epidemiology ; Codon/*genetics ; Asia/epidemiology ; Americas/epidemiology ; Africa/epidemiology

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

HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naive Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.
Anti-HIV Agents/pharmacology ; Drug Resistance, Viral/*genetics ; HIV Envelope Protein gp41/*genetics/metabolism/pharmacology ; HIV Infections/virology ; HIV-1/*genetics/isolation & purification/*metabolism ; Humans ; Peptide Fragments/pharmacology ; *Polymorphism, Genetic ; Protein Structure, Tertiary/genetics ; Republic of Korea ; Virus Internalization

OBJECTIVETo express the Gag protein of HIV-1 strain CN54 in Pichia pastoris (P.pastoris), optimize fermentation parameters and purify Gag antigen.

METHODSThe Gag gene was subcloned into downstream of aox1 promoter of Pichia expression vector pPS1.0, an integrative vector which possesses an identical 5' untranslated region as the natural aox1 gene and employs both in vitro construction and in vivo selection for multi-copy integrants. The recombinant vector was introduced into P.pastoris strain GS115 by electroporation and selected with G418 for Gag gene integration. Super G418 resistant clones were selected and screened for Gag expression. The engineered P.pastoris was cultured to high cell density (>300 A600 Units/ml) in a 5L fermentor. Through methanol induction, the expression level of Gag reached 120 mg/L. Intracellularly expressed Gag was released by high-pressure homogenization and purified through Sepharose FF and DEAE Sepharose FF column chromatography, the purity of Gag reached up to 90%.

RESULTSWestern-blotting suggested that purified Gag expressed in P.pastoris could react specifically with serum from HIV infected individual.

CONCLUSIONGag antigen expressed in P.pastoris has provided a good basis for the development of a new generation of HIV vaccine candidates against some Chinese prevalent strains.

Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Fermentation ; Gene Expression ; HIV Core Protein p24 ; biosynthesis ; genetics ; isolation & purification ; HIV-1 ; genetics ; metabolism ; Humans ; Pichia ; genetics ; metabolism

OBJECTIVETo study the distribution of human immunodeficiency virus-1 (HIV-1) strains subtypes in Shandong province and to study their source in order to predict the epidemic trend.

METHODSEpidemiological investigation was made and 93 DNA fragments of HIV-1 env, gag, tat gene were amplified by nested polymerase chain reaction from people infected with HIV-1, in 2002 - 2003. Their C2-V3, P17/P24, 1st exon of tat and adjacent region were sequenced.

RESULTSSequence analysis showed that there were 7 HIV-1 strains or circulating recombinant forms (CRFs), B' (n = 71), CRF01-AE (n = 9), CRF07-BC (n = 3), CRF08-BC (n = 3), B (n = 2), C (n = 2), CRF02-AG (n = 2). B' strains was the predominant which, covered 10 cities and 4 kinds of population including blood donors, blood receivers, spouses of the infected people and clients of the sex workers. CRF07-BC, CRF08-BC strains were identified in 5 cities, mainly from injecting drug users. CRF01-AE and other strains were found distributed in developed cities, among sex workers.

CONCLUSIONThere were many kinds of subtypes and CRFs of HIV and their genomes which generated obvious variation in Shandong province, suggesting that they might facilitate the spread of the disease in Shandong province.

Adolescent ; Adult ; Aged ; China ; epidemiology ; DNA, Viral ; genetics ; Female ; HIV Infections ; epidemiology ; genetics ; HIV-1 ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Molecular Epidemiology

OBJECTIVETo compare the concordance in predicting genotype HIV-1 drug resistance between In-house method and TRUGENE(TM) or ViroSeq(TM) method.

METHODS25 international proficiency testing (PT) samples received from 2009 to 2013 were detected by three methods, then pairwise comparison results was analyzed to validate their concordance on drug resistance mutation and drug resistance report. To further confirm the results, another 15 serum specimens were detected by In-house and TRUGENE(TM) methods, then compared their results concordance.

RESULTSThe evaluation of the consistency of resistance-associated mutations showed that for 25 PT samples, the consistency was 99.42% (2933/2950) in testing drug resistance mutation sites among the three methods; all pairwise comparison Kappa values >0.81(P < 0.01). The evaluation of the consistency of drug resistance test showed that inconsistent comparison results mainly concentrated in the four nucleoside reverse transcriptase inhibitors zidovudine (AZT), didanosine (ddI), stavudine (d4T), abacavir (ABC), and the inconsistencies were mainly minor. Where the minor inconsistencies between In-house and ViroSeq(TM) methods were 28% (7/25), 28% (7/25), 16% (4/25) and 20% (5/25), respectively. And the inconsistencies between In-house and TRUGENE(TM) method were separately 44% (11/25), 28% (7/25), 36% (9/25) and 28% (7/25);while the inconsistencies between TRUGENE(TM) and ViroSeq(TM) method were separately 24% (6/25), 8% (2/25), 28% (7/25) and 8% (2/25). AZT in the comparison between In-house and ViroSeq(TM) methods, ddI, d4T, ABC and TDF in the comparison between In-house and TRUGENE(TM) methods were moderately consistent (weighted Kappa values were separately 0.54, 0.44, 0.52, 0.42, 0.59, all the P value <0.01). The other compared results were all highly-consistent (weighted Kappa values were 0.61-0.80) or extremely high-consistent (weighted Kappa values were >0.80). For 15 serum specimens, 99.55% (1762/1770) drug resistance mutation sites could be detected by the two methods, the difference about drug results concentrated in AZT, ddI, d4T and ABC.

CONCLUSIONThe In-house genotyping system had a high concordance with commercial TRUGENE(TM) or ViroSeq(TM) genotyping system.

Drug Resistance, Viral ; genetics ; Genes, Viral ; Genotype ; HIV-1 ; drug effects ; genetics ; isolation & purification ; Humans ; Reagent Kits, Diagnostic