Sequence analysis of C2-V4 of HIV-1 env genes in Fuxin / 预防医学

Objective@#To learn the sequence characteristics of C2-V4 of HIV-1 env genes and the epitope variation of representative broadly monoclonal neutralizing antibody in Fuxin, so as to provide evidence for the HIV-1 variation trend and the biological characteristics of V3 loop.@*Methods @#The whole blood samples of 112 HIV-1 cases in Fuxin Health Service Center from 2018 to 2019 were collected and the DNA was extracted. The C2-V4 of env genes were amplified by nested-PCR and the PCR products were subjected to sequencing. The bioinformatics analysis was carried out using MEGA software, and the V3-tip motifs, co-receptors, net charge and characteristic amino acids were analyzed using HIV Database. @*Results@#Totally 101 effective gene sequences were obtained, and 5 types of V3-tip motifs were found. Among them, 77 pieces of GPGQ ( 76.24% ) were found in CRF01_AE, CRF07_BC, CRF65_cpx and G subtypes; 19 pieces of GPGR ( 18.81% ) were found in CRF01_AE, CRF07_BC and B subtypes; 3 pieces of GPGH, 1 piece of GPGK and 1 piece of GPGA were only found in CRF01_ AE subtype. The co-receptor was mainly CCR5 ( 84, 83.17% ) . The net charge numbers of V3 loops in CRF01_ AE, CRF07_ BC, B, CRF65_cpx and G were 3.28±1.17, 3.22±0.92, 4.25±0.83, 2.50±0.50 and 3, respectively. The mutation rates of neutralizing antibodies binding b12 and VRC01 were 0-9.90%. The deletion rates of N-glycosylation sites of 295 and 332 were 18.81% and 14.85%, without the loss of both sites.@*Conclusions @#The HIV-1 strains in Fuxin from 2018 to 2019 are macrophage-tropic and non-syncytium-inducing, with GPGQ as the main type of V3-tip motif, CCR5 as the main co-receptor, slow replication and low ability to escape neutralizing antibodies.

Analysis on variation characteristics of HIV-1 Gp120 sequences in men who have sex with men in Guangzhou / 中华实验和临床病毒学杂志

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.

Determination o viral tropism by genotyping an phenotyping assays in Braziliaan hiv-1-infected patients / Determinação do tropismo viral por ensaios genotípicos e fenotípicos em pacientes brasileiros infectados por HIV-1

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain. Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno[coreceptor] analysis with a false positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5% and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of coreceptor usage tests in clinical practice.

A aplicação clínica dos antagonistas de CCR5 envolve em primeiro lugar determinar o uso de co-receptor pela cepa viral infectante. Programas de bioinformática que prevêem o uso co-receptor poderiam fornecer um método alternativo para selecionar candidatos para o tratamento com os antagonistas do CCR5, particularmente em países com poucos recursos financeiros. Assim, o presente estudo teve por objetivo identificar a melhor abordagem utilizando ferramentas de bioinformática para determinar qual o tipo de co-receptor do HIV-1 que poderia ser usado na prática clínica. Sequências de DNA proviral e Trofile resultados a partir de 99 pacientes infectados pelo HIV-1 sob monitorização clínica foram avaliadas. Com base nos resultados do Teste Trofile, as variantes virais presentes eram R5 (81,1%), R5X4 (21,4%) e X4 (1,8%). Determinação do tropismo pela análise do Geno2pheno, com taxa de falso positivos de 10% apresentou desempenho mais adequado para esta amostragem: as cepas R5 e X4 foram encontradas em frequências de 78,5% e 28,4%, respectivamente, e foi de 78,6% a concordância entre os resultados fenotípicos e genotípicos. Mais estudos são necessários para esclarecer como a diversidade genética entre as cepas do vírus afeta abordagens baseadas na determinação do tropismo pelas ferramentas de bioinformática. Embora esta estratégia possa ser útil para o rastreio de pacientes em países em desenvolvimento, permanecem algumas limitações que restringem a aplicação mais ampla para utilização de testes de co-receptor na prática clínica.

Variability of the conserved V3 loop tip motif in HIV-1 subtype B isolates collected from Brazilian and French patients

The diversity of the V3 loop tip motif sequences of HIV-1 subtype B was analyzed in patients from Botucatu (Brazil) and Montpellier (France). Overall, 37 tetrameric tip motifs were identified, 28 and 17 of them being recognized in Brazilian and French patients, respectively. The GPGR (P) motif was predominant in French but not in Brazilian patients (53.5 percent vs 31.0 percent), whereas the GWGR (W) motif was frequent in Brazilian patients (23.0 percent) and absent in French patients. Three tip motif groups were considered: P, W, and non-P non-W groups. The distribution of HIV-1 isolates into the three groups was significantly different between isolates from Botucatu and from Montpellier (P < 0.001). A higher proportion of CXCR4-using HIV-1 (X4 variants) was observed in the non-P non-W group as compared with the P group (37.5 percent vs 19.1 percent), and no X4 variant was identified in the W group (P < 0.001). The higher proportion of X4 variants in the non-P non-W group was essentially observed among the patients from Montpellier, who have been infected with HIV-1 for a longer period of time than those from Botucatu. Among patients from Montpellier, CD4+ cell counts were lower in patients belonging to the non-P non-W group than in those belonging to the P group (24 cells/µL vs 197 cells/µL; P = 0.005). Taken together, the results suggest that variability of the V3 loop tip motif may be related to HIV-1 coreceptor usage and to disease progression. However, as analyzed by a bioinformatic method, the substitution of the V3 loop tip motif of the subtype B consensus sequence with the different tip motifs identified in the present study was not sufficient to induce a change in HIV-1 coreceptor usage.

The binding ability of V_3 region derived from diverse human immunodeficience type 1(HIV-1) to different cells / 中国免疫学杂志

Objective:To determine the binding ability of ~V-1 V3 loop to target cells.Methods:V3 loop peptides(V3-HBIO,V3-ADA,V3-89.6)derived from different HIV-1 strains LIIB(X4-nopic),ADA(R5-tmpic),89.6(R5X4-tropic) and the biotinylated V3-BH1O(biotin-BHIO) and V3 -ADA( biotin-ADA) were synthesized. The binding of the biotinylated V3 peptides to cells and the binding targets were analyzed using flow cytometry. Results:V3 BH10 can bind to a wide range of cell lines, while bio-ADA scarcely binds to the monocytes derived from periperal blood mononuclear cells.Antibody anti-CXCR4 binding to cells blocked by V3 -BH10 and biotin-BH1O binding was blocked by protease inhibitors. The binding of V3-BH10 could be significantly enhanced by V3-BHlO but by neither V3-89.6 nor V3-ADA.Conclusion:The binding ability of the V3 loop derived from diverse HIV- I strains are different. The V3 loop derived from HIV-1 X4-tropic strain directly binds to a wide range of cell lines, the binding targets are multiple including at least coreceptor and proteases. The V3 loop derived from R5 -tropic strain ADA does scarcely.