Análisis filogenético de variantes de VIH-1, mediante la subtipificación de las secuencias de la región pol de VIH-1, período 2010-2015 / Phylogenetic analysis of HIV-1 variants of a comprehensive HIV clinic in Guatemala, through the subtyping of the pol-HIV-1 region sequences obtained during the period 2010-2015

El genoma del VIH contiene nueve genes, tres de estos genes (gag, pol y env) codifican proteínas estructurales. Existen dos variantes principales de este virus, VIH-1 y VIH-2. El primero es el causante de la mayoría de las infecciones a nivel mundial, actualmente se han identificado nueve subtipos de VIH-1 y 58 formas recombinantes circulantes (FRC). En Centroamérica, el subtipo B del VIH-1 es el causante de la mayoría de los casos de VIH positivo; en Guatemala se ha reportado la presencia de subtipo B, de formas recombinantes BF1 y del subtipo C; sin embargo, actualmente no existen análisis filogenéticos que indiquen las variantes de este subtipo. Debido a lo anterior, el objetivo del estudio fue llevar a cabo la subtipificación de 400 secuencias de la región pol del VIH-1 obtenidas de 400 pacientes VIH-1 positivos, en una clínica de atención integral de Guatemala del 2010 al 2015. Para determinar los distintos subtipos de VIH-1 presentes en Guatemala se realizó la subtipificación de las secuencias obtenidas por la prueba de genotipo en formato FASTA, con la herramienta REGA HIV-1 Subtyping Tool Version 3.0. Con el fin de determinar la relación entre las variantes de VIH-1, se realizó un alineamiento de secuencias y árboles filogenéticos utilizando el método Neighbor Joining y Máxima Verosimilitud con 100 réplicas bootstrap, con el programa MEGA 7.0.21. Se determinó que el subtipo con mayor frecuencia de las secuencias analizadas es el subtipo B con un 71.5 %, seguido de la forma recombinante BD (16.75 %) y el subtipo B-like (7.75 %)

The HIV genome contains nine genes, three of these genes (gag, pol, and env) encode structural proteins. There are two main variants of this virus, HIV-1 and HIV-2. The first one (HIV-1) is the cause of most infections worldwide, of which nine subtypes and 58 circulating recombinant forms (CRF) have been identified. In Central America, subtype B of HIV-1 is the cause of the majority of HIV positive cases. In Guatemala, it has been reported the presence of subtype B, recombinant forms BF1 and subtype C. However, no phylogenetic analysis has been performed to indicate the variants of this subtype. The aim of the study was to subtype 400 sequences of the pol region of HIV-1, of samples that were obtained from a care clinic during the period 2010 to 2015. To determine the different subtypes of HIV-1 present in Guatemala, the subtyping of the sequences obtained by the genotype test, in FASTA format, was performed with REGA HIV-1 Subtyping Tool - Version 3.0. In order to determine the relationship between HIV-1 variants, an alignment of sequences and phylogenetic trees was performed using the Neighbor Union and Maximum Likelihood method with 100 bootstrap replicas, with the MEGA 7.0.21 program. It was determined that the subtypes with the highest prevalence of the studied sequences are the subtype B (71.5 %), recombinant BD (16.75 %), and subtype B-like (7.75 %)

Preparation and identification of Env-pseudotyped subtype B HIV-1 / 军事医学

Objective To prepare the Env-pseudotyped subtype B HIV-1 with enhanced green fluorescent protein ( EG-FP) gene,explore HIV-1 infection mechanisms and develop feasible methods of identification .Methods The Env-pseudo-typed viruses were packaged in HEK293T cells by cotransfection, and the reporter gene and P24 protein were detected by PCR, Western blot and ELISA .Reporter gene amplification , viral titration assay and a single round of infection assay were performed after the env-pseudotyped viruses infected HIV-1 permissive cell .Results and Conclusion A generation and identification method of the pseudotyped HIV-1 was established . The Env-pseudotyped subtype B HIV-1 has been prepared, which is able to infect SupT1 and TZM-bl cells through infection assay .

Detection of the B"-GWGR variant in the southernmost region of Brazil: unveiling the complexity of the human immunodeficiency virus-1 subtype B epidemic

Typical human immunodeficiency virus-1 subtype B (HIV-1B) sequences present a GPGR signature at the tip of the variable region 3 (V3) loop; however, unusual motifs harbouring a GWGR signature have also been isolated. Although epidemiological studies have detected this variant in approximately 17-50% of the total infections in Brazil, the prevalence of B"-GWGR in the southernmost region of Brazil is not yet clear. This study aimed to investigate the C2-V3 molecular diversity of the HIV-1B epidemic in southernmost Brazil. HIV-1 seropositive patients were ana-lysed at two distinct time points in the state of Rio Grande do Sul (RS98 and RS08) and at one time point in the state of Santa Catarina (SC08). Phylogenetic analysis classified 46 individuals in the RS98 group as HIV-1B and their molecular signatures were as follows: 26% B"-GWGR, 54% B-GPGR and 20% other motifs. In the RS08 group, HIV-1B was present in 32 samples: 22% B"-GWGR, 59% B-GPGR and 19% other motifs. In the SC08 group, 32 HIV-1B samples were found: 28% B"-GWGR, 59% B-GPGR and 13% other motifs. No association could be established between the HIV-1B V3 signatures and exposure categories in the HIV-1B epidemic in RS. However, B-GPGR seemed to be related to heterosexual individuals in the SC08 group. Our results suggest that the established B"-GWGR epidemics in both cities have similar patterns, which is likely due to their geographical proximity and cultural relationship.

Screening program on novel drug resistance mutations of subtype B' in human immunodeficiency virus type 1 in China / 中华流行病学杂志

Objective To screen the level of novel drug resistance mutations in subtype B' in China. Methods 451 pol sequences collected from the previous study, which including 354 AIDS patients who had received antiretroviral treatment(ART)and 97 the untreated patients. Entire protease gene(codous 1-99)and full-length reverse transcriptase gene(codons 1-560)were included.Variation of mutations between the treated and the untreated patients with consensus/ancestral sequences were compared and the mutations with higher frequencies in the treated patients than in the untreated patients were screened before submitting the mutations to the Stanford HIV Drug Resistance Database(SHDB)(http://hivdb.stanford.edu/). Relation between the mutations and resistance preliminarily was then analyzed, according to the information including SHDB. Results Frequencies of 7 mutations at 6 positions, DI23E, V292I, K366R, T369A, T369V, A371V and 1375V, 2 at DNA polymerase domain and 5 at connection domain of reverse transcriptase(RT)were higher in the treated patients than in the untreated patients. The information of 7 mutations including the SHDB showed that 7 mutations were major variants at corresponding positions, and theirs frequencies were higher in the treated patients using some drugs, than in the untreated patients. Conclusion 7mutations being screened from the China subtype B were possibly associated with the resistance,which called for the construction of mutated viruses by site-directed mutagenesis to identify their effects on the susceptivity of different drugs.

HIV subtype, epidemiological and mutational correlations in patients from Paraná, Brazil

OBJECTIVE: Analyze patients with HIV infection from Curitiba, Paraná, their epidemiological characteristics and HIV RAM. METHODS: Patients regularly followed in an ID Clinic had their medical data evaluated and cases of virological failure were analyzed with genotypic report. RESULTS: Patients with complete medical charts were selected (n = 191). Demographic and clinical characteristics were compared. One hundred thirty two patients presented with subtype B infection (69.1 percent), 41 subtype C (21.5 percent), 10 subtype F (5.2 percent), 7 BF (3.7 percent) and 1 CF (0.5 percent). Patients with subtype B infection had been diagnosed earlier than patients with subtype non-B. Also, subtype B infection was more frequent in men who have sex with men, while non-B subtypes occurred more frequently in heterosexuals and women. Patients with previous history of three classes of ARVs (n = 161) intake were selected to evaluate resistance. For RT inhibitors, 41L and 210W were more frequently observed in subtype B than in non-B strains. No differences between subtypes and mutations were observed to NNTRIs. Mutations at 10, 32 and 63 position of protease were more observed in subtype B viruses than non-B, while positions 20 and 36 of showed more amino acid substitutions in subtype non-B viruses. Patients with history of NFV intake were evaluated to resistance pathway. The 90M pathway was more frequent in subtypes B and non-B. Mutations previously reported as common in non-B viruses, such as 65R and 106M, were uncommon in our study. Mutations 63P and 36I, previously reported as common in HIV-1 subtypes B and C from Brazil, respectively, were common. CONCLUSION: There is a significant frequency of HIV-1 non-B infections in Paraná state, with isolates classified as subtypes C, F, BF and BC. Patients with subtype C infection were more frequently female, heterosexual and had a longer average time of HIV diagnosis.

Techniques used to identify the Brazilian variant of HIV-1 subtype B

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70 percent, compared to 64 percent for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85 percent, compared to 88 percent for GWGR. The assessment of RFLP revealed 68 percent sensitivity and 94 percent specificity for the B-GPGR strain compared to 84 and 90 percent for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.