Aurora Fernández-García

HIV-1 transmission cluster with T215D revertant mutation among newly diagnosed patients from the Basque Country, Spain.

Objective:To determine the introduction of HIV-1 genetic forms and to examine transmission clusters and resistance to antiretroviral inhibitors among newly diagnosed patients from the Basque Country, Spain, during 2004-2007. Methods:A total of 261 samples, corresponding to 47.5% heterosexuals, 37.9% men who have sex with men (MSM), and 11.1% intravenous drug users were analyzed in protease and reverse transcriptase to examine phylogenetic relationships and drug resistance-associated mutations. Results:Subtype B was detected in 220 (84.3%) samples and non-B subtype variants in 41 (15.7%) samples. Nearly half (47%) of the sequences grouped in transmission clusters. One of these comprised 14 individuals, 12 of them MSM, with the T215D revertan mutation. In largest transmission clusters, the percentage of MSM was higher than heterosexuals (P < 0.001). Resistance mutations were detected in 29 (11.1%) patients: 20 (7.6%) of them to nucleoside reverse transcriptase inhibitor; 6 (2.3%) to nonnucleoside reverse transcriptase inhibitor (NNRTI); and 1 each to protease inhibitors, protease inhibitor plus NNRTI, and nucleoside reverse transcriptase inhibitor plus NNRTI, respectively. Conclusions:Our findings underscore recommendations for HIV-1 genotyping in newly diagnosed patients not only to provide information on transmitted drug resistance as an issue in public health and as a guide to future therapy but also to document transmission clusters and to increase the necessary preventive measures.

Evaluation of genotypic tropism prediction tests compared with in vitro co-receptor usage in HIV-1 primary isolates of diverse subtypes

OBJECTIVES To evaluate the sensitivity and specificity of genotypic methods for predicting the co-receptor usage of subtypes B and non-B HIV-1 primary isolates, using as gold standard the infectivity of each primary isolate in GHOST cells stably expressing HIV-1 co-receptors. METHODS Primary isolates were obtained by co-culturing either patients peripheral blood mononuclear cells (PBMCs) or ultracentrifuged plasma with donor-activated PBMCs. In vitro co-receptor usage was determined by infecting GHOST cells. Tropism prediction, based on V3 sequences, was determined with simple rules and bioinformatic tools (Geno2pheno[coreceptor] and WebPSSM). RESULTS This study includes 102 HIV-1 primary isolates; 23 (22.5%) subtype B and 79 (77.5%) non-B genetic forms. V3 sequences were classified into six subtypes (A-G), although 32 (31.4%) were circulating recombinant forms and 21 (20.6%) were unique recombinant forms. Sixty-nine isolates were R5, 27 R5X4 and 6 X4. The highest levels of sensitivity and specificity for the detection of X4 strains among V3 sequences, between 91% and 100%, were obtained by using PSSM(x4r5), PSSM(si/nsi) and the 11/25 rule for sequences of subtypes A, B and G, but not for subtype F. Establishing the recommended cut-off for clinical settings of a 10% false positive rate for Geno2pheno, we obtained 93% specificity and 97% sensitivity. CONCLUSIONS Comparing genotypic assays for HIV-1 co-receptor use with a cell-culture phenotypic assay could provide more reliable results of sensitivity and specificity for the detection of X4 strains than comparing them with recombinant assays, considered as gold standard. In general, except for subtype F isolates, there is a good correlation for tropism prediction.

Identification of a new HIV type 1 circulating BF intersubtype recombinant form (CRF47_BF) in Spain.

We report the identification of a new HIV-1 circulating recombinant form (CRF47_BF) derived from subtypes B and F. It was initially identified in protease-reverse transcriptase sequences from nine individuals from three separate regions of Spain who acquired HIV-1 infection via sexual contact. All nine sequences formed a strongly supported phylogenetic cluster, branching apart from all known CRFs, and in bootscan analyses were BF mosaics with two coincident breakpoints. Two epidemiologically unlinked viruses were sequenced in near full-length genomes, which exhibited identical mosaic structures, with 16 intersubtype breakpoints in a genome predominantly of subtype B. Subtype F segments of the new CRF failed to cluster with any of the near full-length genome subtype F sequences available in public databases. Recent dates of HIV-1 diagnoses and short genetic distances suggest a recent origin of this CRF. This is the tenth reported CRF_BF, the first apparently having originated outside of South America.

Development of a Panel of Well-Characterized Human Immunodeficiency Virus Type 1 Isolates from Newly Diagnosed Patients Including Acute and Recent Infections

The aim of this study was the development of a panel constituted by well-defined HIV-1 strains of different genetic forms, with a particular focus on isolates from acute and recent infections. Fourteen HIV-1 isolates, including four from acute and five from recent infections, were expanded in peripheral blood mononuclear cells. SI phenotype, coreceptors use, and TCID(50)/ml were determined. V3 net charge was calculated. Near full-length genomes were amplified by RT-nested PCR in four overlapping segments. Phylogenetic analyses were performed with neighbor-joining trees and bootscanning. Analysis of cysteine residues, lengths of variable regions, and potential N-linked glycosylation sites in gp120 and gp41 was performed. Viral stocks were produced. Thirteen strains were NSI/R5 and one SI/R5,X4. TCID(50)/ml ranged between 10(4.6) and 10(6). V3 net charge was <+5 in 12 sequences and +5 in two sequences. Near full-length HIV-1 genomes analysis identified viruses of the following genetic forms: eight subtype B, three subtype C, two CRF02_AG, and one subtype G. Cysteine residues that form the V1,V2,V3, and V4 loops were highly conserved. The number of potential N-linked glycosylation sites in gp120 and gp41 ranged between 24-29 and 4-6, respectively. Seven potential N-linked glycosylation sites in gp120 and three in gp41 were conserved. V1, V2, V4, and V5 variable regions exhibited substantial length variation. In addition, an analysis of transmitted and natural resistance to current antiretroviral drugs in these strains was performed. It is worth mentioning that the 13S mutation in the V3 sequence, associated with resistance to maraviroc, was observed in a subtype B strain that harbored resistance mutations to nucleoside reverse transcriptase inhibitors and to T20. The availability of a panel including strains from acute and recent infections should be a valuable resource for optimizing and standardizing vaccine candidate assessment. Near full-length genome characterization may be necessary for evaluating clade-specific reactivities.

Epidemiological Surveillance of HIV-1 Transmitted Drug Resistance in Spain in 2004-2012: Relevance of Transmission Clusters in the Propagation of Resistance Mutations

Our objectives were to carry out an epidemiological surveillance study on transmitted drug resistance (TDR) among individuals newly diagnosed of HIV-1 infection during a nine year period in Spain and to assess the role of transmission clusters (TC) in the propagation of resistant strains. An overall of 1614 newly diagnosed individuals were included in the study from January 2004 through December 2012. Individuals come from two different Spanish regions: Galicia and the Basque Country. Resistance mutations to reverse transcriptase inhibitors (RTI) and protease inhibitors (PI) were analyzed according to mutations included in the surveillance drug-resistance mutations list updated in 2009. TC were defined as those comprising viruses from five or more individuals whose sequences clustered in maximum likelihood phylogenetic trees with a bootstrap value ≥90%. The overall prevalence of TDR to any drug was 9.9%: 4.9% to nucleoside RTIs (NRTIs), 3.6% to non-nucleoside RTIs (NNRTIs), and 2.7% to PIs. A significant decrease of TDR to NRTIs over time was observed [from 10% in 2004 to 2% in 2012 (p=0.01)]. Sixty eight (42.2%) of 161 sequences with TDR were included in 25 TC composed of 5 or more individuals. Of them, 9 clusters harbored TDR associated with high level resistance to antiretroviral drugs. T215D revertant mutation was transmitted in a large cluster comprising 25 individuals. The impact of epidemiological networks on TDR frequency may explain its persistence in newly diagnosed individuals. The knowledge of the populations involved in TC would facilitate the design of prevention programs and public health interventions.

Short communication: Biological and genetic characterization of HIV type 1 subtype B and nonsubtype B transmitted viruses: usefulness for vaccine candidate assessment.

Due to the extraordinary degree of genetic diversity of HIV-1 and the structural complexity of its envelope glycoproteins, designing an effective vaccine is difficult, requiring the development of viral reagents to assess vaccine-elicited neutralizing antibodies. The aim of this study was to improve on our previously developed panel of HIV-1 strains of different genetic forms, focusing on strains from acute and recently acquired infections as the most representative of the transmitted viruses. HIV-1 primary isolates were expanded in peripheral blood mononuclear cells. Viral stocks of 40 ml each were produced. Syncytium-inducing (SI) phenotype, coreceptor use, and TCID(50)/ml were determined. Near full-length HIV-1 genomes were amplified by RT-nested PCR in four overlapping segments. Phylogenetic analyses were performed with neighbor-joining trees and bootscanning. Forty-four HIV-1 strains were included in the panel. Twenty-four (54.1%) strains were from early infections (16 acute and 8 recent); of them, 21 (87%) were sexually transmitted. NSI/R5 phenotype was detected in 37 (84.1%) viruses and SI/R5,X4 in another 7 (15.9%). TCID(50)/ml ranged between 10(4) and 10(6.6). Twelve different genetic forms constituted this panel: subtypes A1, B, C, F1, and G; circulating recombinant forms CRF02_AG, CRF14_BG, and CRF24_BG; and unique recombinant forms CRF02_AG/A3, BF1, CRF12_BF/B, and DF1G. In conclusion, in this study, we report the development of a comprehensive and well-characterized panel of HIV-1 isolates for assessing neutralization in HIV vaccine research. This panel is available for distribution through the Programme EVA Centre for AIDS Reagents, National Institute for Biological Standard and Control (NIBSC).

Phylogenetic structure in African HIV-1 subtype C revealed by selective sequential pruning

Subtype C is the most prevalent clade in the HIV-1 pandemic. Previous studies suggested that African HIV-1 subtype C lacks a well-defined phylogenetic structure. Here we show that, by sequential pruning of ambiguously positioned taxa, a well-defined intrasubtype C phylogenetic structure becomes apparent, with 52% African HIV-1 subtype C isolates analyzed in envelope sequences branching within 11 clusters, also supported in a tree of full-length genomes, and all with geographical associations. Among 46 viruses recently transmitted in South Africa, 70% branched within 7 clusters (41% in the largest one) and 15% additional isolates were intercluster recombinants. Choice of the outgroup sequence and inclusion of intrasubtype recombinant viruses in the analyses could greatly affect support of clusters. The identification of clusters comprising a large proportion of African HIV-1 subtype C viruses may have implications for the design of vaccines intended for use in areas where subtype C is prevalent.

Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites

Neospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.

Near Full-Length Genome Characterization of a Newly Identified HIV Type 1 Subtype F Variant Circulating in St. Petersburg, Russia

We report the near full-length genome characterization of an HIV-1 subtype F virus (D88_845) collected in St. Petersburg, Russia, from a 25-year-old Russian woman perinatally infected in 1982. In a Bayesian phylogenetic analysis, the genome sequence branched basally to the subsubtype F1 clade. In partial sequences, D88_845 clustered with 13 other subtype F sequences from Russia, corresponding to gag (n = 2), pol (n = 3), and env (n = 8) segments. At least 11 of these sequences are from samples collected in St. Petersburg from heterosexually infected Russian individuals. In each of these segments, the Russian viruses formed a monophyletic cluster that branched as a sister clade of the F1 subsubtype. One sequence from Belgium branched with D88_845 with a posterior probability of 0.99. This is the first report on the identification and near full-length genome characterization of the subtype F variant circulating in St. Petersburg, which is closely related to, but distinct from, the F1 subsubtype.

Improvement of HIV-1 coreceptor tropism prediction by employing selected nucleotide positions of the env gene in a Bayesian network classifier

OBJECTIVES This study aimed to develop a genotypic method to predict HIV-1 coreceptor usage by employing the nucleotide sequence of the env gene in a tree-augmented naive Bayes (TAN) classifier, and to evaluate its accuracy in prediction compared with other available tools. METHODS A wrapper data-mining strategy interleaved with a TAN algorithm was employed to evaluate the predictor value of every single-nucleotide position throughout the HIV-1 env gene. Based on these results, different nucleotide positions were selected to develop a TAN classifier, which was employed to predict the coreceptor tropism of all the full-length env gene sequences with information on coreceptor tropism currently available at the Los Alamos HIV Sequence Database. RESULTS Employing 26 nucleotide positions in the TAN classifier, an accuracy of 95.6%, a specificity (identification of CCR5-tropic viruses) of 99.4% and a sensitivity (identification of CXCR4/dual-tropic viruses) of 80.5% were achieved for the in silico cross-validation. Compared with the phenotypic determination of coreceptor usage, the TAN algorithm achieved more accurate predictions than WebPSSM and Geno2pheno [coreceptor] (P<0.05). CONCLUSIONS The use of the methodology presented in this work constitutes a robust strategy to identify genetic patterns throughout the HIV-1 env gene differently present in CCR5-tropic and CXCR4/dual-tropic viruses. Moreover, the TAN classifier can be used as a genotypic tool to predict the coreceptor usage of HIV-1 isolates reaching more accurate predictions than with other widely used genotypic tools. The use of this algorithm could improve the correct prescribing of CCR5 antagonist drugs to HIV-1-infected patients.