BACKGROUNDAlthough it was widely accepted that full-length HIV genome sequences is important in studying virus genetic evolution and variation as well as developing vaccine candidate, to directly sequencing HIV-1 genome of virion RNA remains as a challenge worldwide. Up to date, no published genomic sequences from virion RNA are available for Chinese prevalent HIV-1 strains due to the absence of specialized protocol and appropriate lab equipments. In this study we developed a straightforward approach for amplifying and sequencing HIV virion RNA from plasma by modifying published protocols and further confirmed it is suitable to process Chinese samples.

METHODSThe methods for viral RNA extraction and gene amplification was modified and optimized as could be widely used in most Chinese labs. Gene alignment of Chinese HIV-1 strains was employed for designing specialized primer sets for Thai-B and BC recombinant strains. Based on comprehensively consideration of high variable gene region and recombinant breakpoints in BC recombinant strains, a three-amplicon strategy (including 4.3-kb gag-pol, 2.9-kb pol-env and 2.7-kb env-nef) was developed. In addition, one amplicon (9 kb near full-length genome) was also used in 32 samples with varied viral loads. All amplicons were directly sequenced by DNA automated sequencer.

RESULTSTwenty-five percent (8/32) amplification efficiency was achieved by the one-amplicon strategy and 65.6% (21/32) by three-amplicon strategy. For one amplicon strategy, none of complete near full-length genome sequences was obtained by DNA sequencing. For three-amplicon strategy, 75% sequences were achieved in DNA sequencing. Amplification efficiency but not sequencing efficiency was closely associated with viral loads.

CONCLUSIONThree-amplicon strategy covering all encoding regions of HIV-1 is suitable for Thai-B and BC recombinant strains and could be potentially employed in less-well equipped Chinese labs.

Genome, Viral ; genetics ; HIV-1 ; genetics ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Virion ; genetics

Human immunodeficiency virus (HIV) is a retrovirus, belongs to Lentiviridae family. As long as viral genetic material entering into host cytoplasm, double-strand DNAs synthesis occurs which is catalyzed by reverse transcriptase (RT) with viral plus-strand RNA as template. This reverse transcription is a key link of HIV-1 life cycle and an important target for anti-HIV drug development. The process of reverse transcription can be divided into several steps: formation of minus-strand strong-stop DNA; the first translocation; initiation of plus-strand DNA synthesis; and, the second translocation and the completion of both strands. These steps can be detected individually by using polymerase chain reaction (PCR) according to the amplified products on the region of R/U5, U3, U5/PBS and the sequence between LTR and Gag. In this review, we summarize the principle for detecting stages of HIV-1 reverse transcription by using PCR.
DNA Replication ; genetics ; DNA, Viral ; biosynthesis ; genetics ; HIV Reverse Transcriptase ; genetics ; metabolism ; HIV-1 ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcription

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

Nucleoside reverse transcriptase inhibitors which act as a major component of highly active antiretroviral therapy regimens are widely used in treatment of Acquired Immune Deficiency Syndrome. However, the emergence of drug-resistant variants of HIV-1 severely limits the effectiveness of these drugs. Many drug resistance mutations confer a fitness cost, which can be partially overcome by compensatory mutations or other molecular mechanisms. This review focuses on the impacts of resistance mutations emerging during treatment with nucleoside reverse transcriptase inhibitors on viral fitness, and inter actions between these mutations.
Animals ; Drug Resistance, Viral ; HIV Infections ; drug therapy ; virology ; HIV Reverse Transcriptase ; antagonists & inhibitors ; genetics ; metabolism ; HIV-1 ; drug effects ; enzymology ; genetics ; physiology ; Humans ; Mutation ; Nucleosides ; therapeutic use ; Reverse Transcriptase Inhibitors ; therapeutic use

JB25 and JB26 are new HIV-1 nonnucleoside reverse transcriptase inhibitors, and show potent anti-HIV activities. Sequential passage experiments with wild-type virus were performed to select and identify mutations induced by these two compounds in vitro. For the initial passage, compounds were present at approximately 2-fold IC50 in MT-2 cells. When cytopathic effect (CPE) was observed in more than 75% of the cells, the culture supernatants were collected. For the subsequent passages, fresh MT-2 cells were infected with 1 mL supernatants from the previous passage (regardless of the virus titer) and cultured in the presence of the compounds at concentrations that were increased 2-fold compared with that in the previous passage. This procedure was repeated with increasing concentrations for 12 passages. JB25 had amino acid substitution L100I (TTA-->ATA) at passage 6, and then changed into 100 M (ATA-->ATG) at passage 12, which was rare mutation form and had not been reported. At the same time, Y188C (TAT-->TGT) mutation appeared at passage 10. For JB26, there was a L100I (TTA-->ATA) mutation at passage 10. In a word, JB25 and JB26 showed a low genetic barrier to the development of resistance, and the resistance to JB26 developed slower than JB25. The mutations selected by JB25 and JB26 were mainly associated with codons 188 and 100 of HIV-1 reverse transcriptase.
Amino Acid Sequence ; Amino Acid Substitution ; Anti-HIV Agents ; pharmacology ; Cell Line ; Codon ; Drug Resistance, Viral ; HIV Reverse Transcriptase ; antagonists & inhibitors ; genetics ; HIV-1 ; drug effects ; enzymology ; genetics ; Humans ; Mutation ; Reverse Transcriptase Inhibitors ; pharmacology

OBJECTIVETo collect background information on drug resistance mutations in treatment-naïve HIV-1 infected individuals in Liaoning Province.

METHODSSamples from 91 antiretroviral therapy-naïve patients were collected. The entire protease gene and 1-290 amino acids of the reverse transcriptase gene were amplified by nested PCR from provirus DNA and sequenced. The results were analyzed with HIVdb-Drug Resistance Algorithm, and genotypic resistance mutations were determined to particular anti-HIV drugs.

RESULTSTotally 91 sequences were obtained, 3 of which displayed M46I mutations in the protease gene. Minor resistance mutation rate to protease inhibitors was 100%, including types of L63P (60.4%), V77I (60.4%), M36I/V (31.9%), A71V/T (22.0%), L10I (8.8%), and K20R (6.6%). Only one sequence carried reverse transcriptase related resistance mutations M184I.

CONCLUSIONSAbout 4.4% of HIV-1 infected individuals in Liaoning Province carried strains with drug resistance mutations. Most treatment-naïve HIV-1 infected individuals in Liaoning Province were sensitive to the currently available antiviral medicines, but antiviral treatment must be in accordance with the strict procedures to keep better adherence and avoid the prevalence of drug-resistant strains.

Adult ; China ; epidemiology ; Drug Resistance, Viral ; genetics ; Female ; HIV Infections ; drug therapy ; epidemiology ; HIV Protease ; genetics ; HIV Reverse Transcriptase ; genetics ; HIV-1 ; genetics ; Humans ; Male ; Molecular Epidemiology ; Mutation ; Sequence Analysis, DNA

OBJECTIVETo collect background information on drug-resistant HIV-1 strains in various regions before the start of nation-wide antiretroviral therapy in China.

METHODSTwenty percent of the 2,000 blood samples from antiretroviral therapy naive patients collected for the 2nd national HIV molecular epidemiology survey (NHMES) in 2002 were randomly sampled for this study. The entire protease gene and 20-230 amino acids of the reverse transcriptase gene were amplified by PCR from provirus DNA and sequenced. The results were analyzed with HIV db-Drug Resistance Algorithm and genotypic resistance mutations were determined to particular anti-HIV drugs.

RESULTSTotally 164 protease gene sequences and 138 reverse transcriptase gene sequences were obtained from patients; 0.61% of 164 sequences displayed primary resistance mutations in the protease gene, whereas 99.39% carried 1 or more secondary mutations. Genotypic resistance to at least one nucleoside reverse transcriptase inhibitors (NRTI) was present in 5.80%,and resistance to at least one non-nucleo side reverse transcriptase inhibitors (NNRTI) was present in 1.45% of samples.

CONCLUSIONThe prevalence of genotypic drug resistance is very low in drug-naive HIV infected patients from 21 provinces of China tested in this study. Laboratories participated in the NHMES have organized a network to provide drug resistance monitoring service in the current nation-wide antiviral treatment program in China.

Anti-HIV Agents ; therapeutic use ; China ; epidemiology ; Drug Resistance, Viral ; Genotype ; HIV Infections ; drug therapy ; epidemiology ; virology ; HIV Protease ; genetics ; HIV Protease Inhibitors ; therapeutic use ; HIV Reverse Transcriptase ; genetics ; HIV-1 ; genetics ; Humans ; Mutation ; Reverse Transcriptase Inhibitors ; therapeutic use ; Sentinel Surveillance

OBJECTIVETo study the drug resistance status on HIV-1 patients who had been treated with highly active antiretroviral therapy (HAART) and those treatment-naive ones in Hubei province.

METHODSNested polymerase chain reaction (PCR) was used to amplify 2 kb DNA fragment in HIV pol gene from peripheral blood of the HIV infected patients and the PCR products were sequenced. The sequences were compared to the Stanford HIV drug resistance database.

RESULTSNineteen patients were treated with regimens composed of two Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and one Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI), with 25 patients as treatment-naive. Some protease (PR) drug-resistant mutations were found in these samples, such as D30N (2.27%), D30G (2.27%), M46I (4.55%), M46N (2.27%), I47V (4.55%), I84V (4.55%), I84L (2.27%), N88S (2.27%) and L90S (2.27%) that all belonged to major drug-resistant but A71T (29.55%) belonged to minor resistance mutations Five treated patients were detected having mutations associated RT drug resistance: M41L (5.26%), A62V (5.26%),D67N (5.26%), L210W (5.26%), T215Y (15.79%); K103E (5.26%), K103N (10.53%), Y181C (5.26%), G190A (5.26%), K238N (5.26%), while five treatment-naive patients were detected to have had mutations associated RT drug resistance M184V (4%), K65N (4%), Y115M (4%), F116L (4%), M184I (4%), V179D (4%), G190R (4%).Some additional mutations were detected in RT whose role involve in drug resistance still remained unknown. F214L was positively associated with HAART treatment (P = 0.03).

CONCLUSIONSignificant differences were found between drug resistance mutations to RTIs in treated and treat-naive patients in Hubei province,indicating that drugs had affected the occurrence of drug resistance mutations. At the same time, novel RT mutations F214L might be associated with HAART or some other drugs.

Antiretroviral Therapy, Highly Active ; China ; Drug Resistance, Viral ; genetics ; Genotype ; HIV Infections ; drug therapy ; HIV-1 ; drug effects ; genetics ; Humans ; Mutation ; Reverse Transcriptase Inhibitors ; therapeutic use

Objective To analyze the genetic subtypes of human immunodeficiency virus (HIV) and the prevalence of pretreatment drug resistance in the newly reported HIV-infected men in Guangxi. Methods The stratified random sampling method was employed to select the newly reported HIV-infected men aged≥50 years old in 14 cities of Guangxi from January to June in 2020.The pol gene of HIV-1 was amplified by nested reverse transcription polymerase chain reaction and then sequenced.The mutation sites associated with drug resistance and the degree of drug resistance were then analyzed. Results A total of 615 HIV-infected men were included in the study.The genetic subtypes of CRF01_AE,CRF07_BC,and CRF08_BC accounted for 57.4% (353/615),17.1% (105/615),and 22.4% (138/615),respectively.The mutations associated with the resistance to nucleoside reverse transcriptase inhibitors (NRTI),non-nucleoside reverse transcriptase inhibitors (NNRTI),and protease inhibitors occurred in 8 (1.3%),18 (2.9%),and 0 patients,respectively.M184V (0.7%) and K103N (1.8%) were the mutations with the highest occurrence rates for the resistance to NRTIs and NNRTIs,respectively.Twenty-two (3.6%) patients were resistant to at least one type of inhibitors.Specifically,4 (0.7%),14 (2.3%),4 (0.7%),and 0 patients were resistant to NRTIs,NNRTIs,both NRTIs and NNRTIs,and protease inhibitors,respectively.The pretreatment resistance to NNRTIs had much higher frequency than that to NRTIs (2.9% vs.1.3%;χ²=3.929,P=0.047).The prevalence of pretreatment resistance to lamivudine,zidovudine,tenofovir,abacavir,rilpivirine,efavirenz,nevirapine,and lopinavir/ritonavir was 0.8%, 0.3%, 0.7%, 1.0%, 1.3%, 2.8%, 2.9%, and 0, respectively. Conclusions CRF01_AE,CRF07_BC,and CRF08_BC are the three major strains of HIV-infected men≥50 years old newly reported in Guangxi,2020,and the pretreatment drug resistance demonstrates low prevalence.
Male ; Humans ; Middle Aged ; Reverse Transcriptase Inhibitors/therapeutic use* ; HIV Infections/drug therapy* ; Drug Resistance, Viral/genetics* ; China/epidemiology* ; Mutation ; HIV-1/genetics* ; Protease Inhibitors/therapeutic use* ; Genotype

OBJECTIVETo evaluate the amplification rate and the lowestlower detection limit of an in-house HIV-1 Drug resistant (HIVDR) genotyping test.

METHODSA total of 30 plasma samples were selected, which covered all major HIV-1 subtypes predominating prevailing in China (B', CRF07_BC, CRF01 _AE). The viral loads of the 30 selected samples were detected in triplicate by Easy Q method and the average values were taken as the viral loads of the samples. Each sample was diluted to the concentration of > 1000 copies/ml, 401-1000 copies/ml, 101-400 copies/ml, 50-100 copies/ml and < 50 copies/ml with HIV-negative plasma. After extraction of nucleic acids, RT-PCR and nested PCR amplification were performed, the efficiency of amplification of each subtype and the minimum detection limit were determined statistically based on the PCR results.

RESULTSThe viral loads of the selected samples ranged from 2.03 x 10(2)-5.92 x 10(4) copies/ml. The sample of 50-1000 copies/ml have a high amplification rate (86%).

CONCLUSIONThe In-house method for HIV-1 drug resistance genotyping has a high sensitivity with a high successful amplification rate, especially in the samples with low viral load. This method can be used to the detection of drug-resistant virus and to provide scientific data to treatment options for patients.

China ; Drug Resistance, Viral ; Genotype ; HIV-1 ; classification ; drug effects ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Viral Load