24 Hours in the Life of HIV-1 in a T Cell Line.

HIV-1 infects CD4+ T cells and completes its replication cycle in approximately 24 hours. We employed repeated measurements in a standardized cell system and rigorous mathematical modeling to characterize the emergence of the viral replication intermediates and their impact on the cellular transcriptional response with high temporal resolution. We observed 7,991 (73%) of the 10,958 expressed genes to be modulated in concordance with key steps of viral replication. Fifty-two percent of the overall variability in the host transcriptome was explained by linear regression on the viral life cycle. This profound perturbation of cellular physiology was investigated in the light of several regulatory mechanisms, including transcription factors, miRNAs, host-pathogen interaction, and proviral integration. Key features were validated in primary CD4+ T cells, and with viral constructs using alternative entry strategies. We propose a model of early massive cellular shutdown and progressive upregulation of the cellular machinery to complete the viral life cycle.

Human immunodeficiency virus type 1 (HIV-1) genotyping in Rio de Janeiro, Brazil: assessing subtype and drug-resistance associated mutations in HIV-1 infected individuals failing highly active antiretroviral therapy

In order to assess the human immunodeficiency virus type 1 (HIV-1) drug resistance mutation profiles and evaluate the distribution of the genetic subtypes in the state of Rio de Janeiro, Brazil, blood samples from 547 HIV-1 infected patients failing antiretroviral (ARV) therapy, were collected during the years 2002 and 2003 to perform the viral resistance genotyping at the Renageno Laboratory from Rio de Janeiro (Oswaldo Cruz Foundation). Viral resistance genotyping was performed using ViroSeqTM Genotyping System (Celera Diagnostic-Abbott, US). The HIV-1 subtyping based on polymerase (pol) gene sequences (protease and reverse transcriptase-RT regions) was as follows: subtype B (91.2%), subtype F (4.9%), and B/F viral recombinant forms (3.3%). The subtype C was identified in two patients (0.4%) and the recombinant CRF_02/AG virus was found infecting one patient (0.2%). The HIV-1 genotyping profile associated to the reverse transcriptase inhibitors has shown a high frequency of the M184V mutation followed by the timidine-associated mutations. The K103N mutation was the most prevalent to the non-nucleoside RT inhibitor and the resistance associated to protease inhibitor showed the minor mutations L63P, L10F/R, and A71V as the more prevalent. A large proportion of subtype B was observed in HIV-1 treated patients from Rio de Janeiro. In addition, we have identified the circulation of drug-resistant HIV-1 subtype C and are presenting the first report of the occurrence of an African recombinant CRF_02/AG virus in Rio de Janeiro, Brazil. A clear association between HIV-1 subtypes and protease resistance mutations was observed in this study. The maintenance of resistance genotyping programs for HIV-1 failing patients is important to the management of ARV therapies and to attempt and monitor the HIV-1 subtype prevalence in Brazil.

Polymerase chain reaction genotyping of Epstein-Barr virus in scraping samples of the tongue lateral border in HIV-1 seropositive patients

The Epstein-Barr virus (EBV) is the etiological agent of oral hairy leukoplakia (OHL), an oral lesion with important diagnostic and prognostic value in acquired immunodeficiency disease syndrome. The two EBV genotypes, EBV-1 and EBV-2, can be distinguished by divergent gene sequences encoding the EBNA-2, 3A, 3B, and 3C proteins. The purpose of this study was to identify the EBV genotype prevalent in 53 samples of scrapings from the lateral border of the tongue of HIV-1 seropositive patients, with and without OHL, and to correlate the genotypes with presence of clinical or subclinical OHL with the clinic data collected. EBV-1 and EBV-2 were identified through PCR and Nested-PCR based on sequence differences of the EBNA-2 gene. EBV-1 was identified in the 31 samples (15 without OHL, 7 with clinical OHL and 9 with subclinical OHL), EBV-2 in 12 samples (10 without OHL, 1 with clinical and 1 subclinical OHL), and a mixed infection in 10 samples (2 without OHL, 3 with clinical and 5 with subclinical OHL). The presence of EBV-1 was higher in women, but a significant statistical result relating one the EBV genotypes to the development of OHL was not found. We conclude that the oral epithelium in HIV-1 seropositive patients can be infected by EBV-1, EBV-2 or by a mixed viral population.

Determinants and trends in perinatal human immunodeficiency virus type 1 (HIV-1) transmission in the metropolitan area of Belo Horizonte, Brazil: 1998 - 2005

Significant decrease in human immunodeficiency virus type 1 (HIV-1) vertical transmission has been observed worldwide in centers where interventions such as antiretroviral therapy (ART), elective cesarean section, and avoidance of breastfeeding have been implemented. This prospective cohort study aimed to assess the determinants of and the temporal trends in HIV-1 vertical transmission in the metropolitan area of Belo Horizonte, Brazil from January 1998 to December 2005. The rate of HIV-1 vertical transmission decreased from 20% in 1998 to 3% in 2005. This decline was associated with increased use of more complex ART regimens during pregnancy. Multivariate analysis restricted to clinical variables demonstrated that non ART, neonatal respiratory distress/sepsis and breastfeeding were independently associated with HIV-1 vertical transmission. When laboratory parameters were included in the model, high maternal viral load and non maternal ART were associated with HIV-1 vertical transmission. The results from this study confirm the impact of ART in the reduction of HIV-1 vertical transmission and indicate the need for improvement in the care and monitoring of mother and infant pairs affected by HIV-1.

Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate

The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. The inhibitory effects of GA and 15 gallates on Herpes Simplex Virus type 1 (HSV-1) and Human Immunodeficiency Virus (HIV-1) replication were investigated here. After a preliminary screening of these compounds, GA and pentyl gallate (PG) seemed to be the most active compounds against HSV-1 replication and their mode of action was characterized through a set of assays, which attempted to localize the step of the viral multiplication cycle where impairment occurred. The detected anti-HSV-1 activity was mediated by the inhibition of virus attachment to and penetration into cells, and by virucidal properties. Furthermore, an anti-HIV-1 activity was also found, to different degrees. In summary, our results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.

Improved Innate and Adaptive Immunostimulation by Genetically Modified HIV-1 Protein Expressing NYVAC Vectors.

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

Molecular characterisation of newly identified HIV-1 infections in Curitiba, Brazil: preponderance of clade C among males with recent infections

As in many areas of Brazil, the AIDS epidemic in Curitiba is relatively stable, but surveillance is important to support public policy. The molecular characteristics of HIV may be instrumental for monitoring epidemic trends. We evaluated plasma HIV-1 RNA (n = 37) from 38 cases presenting with positive serology, who were among 820 consenting volunteers visiting the downtown counselling and serology testing centre. Seroprevalence was 4.6% (CI 95% 3.2-6.3) and the estimated HIV incidence, as defined by the BED assay, was 2.86 persons/years (CI 95% 1.04-4.68). An additional set of contemporaneous, anonymous samples from a local laboratory was also analysed (n = 20). Regions of the HIV-1 polymerase (n = 57) and envelope (n = 34) were evaluated for subtyping, determination of mosaic structure, primary drug resistance mutations (pDRM), envelope V3 loop motifs and amino acid signatures related to viral tropism. HIV-1 clade B was observed in 53% of cases; HIV-1C in 30% and BC mosaics in 14%, with one F genome and one CF mosaic. Clade C infection was associated with recent infections among males (p < 0.03). Stanford surveillance pDRM was observed in 8.8% of sequences, with 7% showing high level resistance to at least one antiretroviral drug. Tropism for CXCR4 co-receptor was predicted in 18% of envelope sequences, which were exclusively among clade B genomes and cases with serological reactivity to chronic infection.

Analysis of HIV-1 expression level and sense of transcription by high-throughput sequencing of the infected cell.

Next-generation sequencing offers an unprecedented opportunity to jointly analyze cellular and viral transcriptional activity without prerequisite knowledge of the nature of the transcripts. SupT1 cells were infected with a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped HIV vector. At 24 h postinfection, both cellular and viral transcriptomes were analyzed by serial analysis of gene expression followed by high-throughput sequencing (SAGE-Seq). Read mapping resulted in 33 to 44 million tags aligning with the human transcriptome and 0.23 to 0.25 million tags aligning with the genome of the HIV-1 vector. Thus, at peak infection, 1 transcript in 143 is of viral origin (0.7%), including a small component of antisense viral transcription. Of the detected cellular transcripts, 826 (2.3%) were differentially expressed between mock- and HIV-infected samples. The approach also assessed whether HIV-1 infection modulates the expression of repetitive elements or endogenous retroviruses. We observed very active transcription of these elements, with 1 transcript in 237 being of such origin, corresponding on average to 123,123 reads in mock-infected samples (0.40%) and 129,149 reads in HIV-1-infected samples (0.45%) mapping to the genomic Repbase repository. This analysis highlights key details in the generation and interpretation of high-throughput data in the setting of HIV-1 cellular infection.

Highly pathogenic adapted HIV-1 strains limit host immunity and dictate rapid disease progression.

OBJECTIVE:: The study of HIV-1 rapid progressors has been limited to specific case reports. Nevertheless, identification and characterization of the viral and host factors involved in rapid progression are crucial when attempting to uncover the correlates of rapid disease outcome. DESIGN:: We carried out comparative functional analyses in rapid progressors (n = 46) and standard progressors (n = 46) early after HIV-1 seroconversion (≤1 year). The viral traits tested were viral replicative capacity, co-receptor usage, and genomic variation. Host CD8 T-cell responses, humoral activity, and HLA immunogenetic markers were also determined. RESULTS:: Our data demonstrate an unusual convergence of highly pathogenic HIV-1 strains in rapid progressors. Compared with standard progressors, rapid progressor viral strains show higher in-vitro replicative capacity (81.5 vs. 67.9%; P = 0.025) and greater X4/DM co-receptor usage (26.3 vs. 2.8%; P = 0.006) in early infection. Limited or absent functional HIV-1 CD8 T-cell responses and neutralizing activity were measured in rapid progressors. Moreover, the increase in common HLA allele-restricted CD8 T-cell escape mutations in rapid progressors acts as a signature of uncontrolled HIV-1 replication and early impairment of adaptive cellular responses. CONCLUSION:: Our data support a dominant role for viral factors in rapid progressors. Robust HIV-1 replication and intrinsic viral properties limit host adaptive immune responses, thus driving rapid disease progression.

Added Value of Deep Sequencing Relative to Population Sequencing in Heavily Pre-Treated HIV-1-Infected Subjects.

OBJECTIVE: To explore the potential of deep HIV-1 sequencing for adding clinically relevant information relative to viral population sequencing in heavily pre-treated HIV-1-infected subjects. METHODS: In a proof-of-concept study, deep sequencing was compared to population sequencing in HIV-1-infected individuals with previous triple-class virological failure who also developed virologic failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Viral susceptibility was inferred before salvage therapy initiation and at virological failure using deep and population sequencing genotypes interpreted with the HIVdb, Rega and ANRS algorithms. The threshold level for mutant detection with deep sequencing was 1%. RESULTS: 7 subjects with previous exposure to a median of 15 antiretrovirals during a median of 13 years were included. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively. Self-reported treatment adherence was adequate in 4 and partial in 2; one individual underwent treatment interruption during follow-up. Deep sequencing detected all mutations found by population sequencing and identified additional resistance mutations in all but one individual, predominantly after virological failure to deep salvage therapy. Additional genotypic information led to consistent decreases in predicted susceptibility to etravirine, efavirenz, nucleoside reverse transcriptase inhibitors and indinavir in 2, 1, 2 and 1 subject, respectively. Deep sequencing data did not consistently modify the susceptibility predictions achieved with population sequencing for darunavir, tipranavir or raltegravir. CONCLUSIONS: In this subset of heavily pre-treated individuals, deep sequencing improved the assessment of genotypic resistance to etravirine, but did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility. These data may inform the design of future studies addressing the clinical value of minority drug-resistant variants in treatment-experienced subjects.

HIV-1 reverse transcriptase connection domain mutations: dynamics of emergence and implications for success of combination antiretroviral therapy.

BACKGROUND: Factors promoting the emergence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) connection domain mutations and their effect on antiretroviral therapy (ART) are still largely undetermined. We investigated this matter by analyzing genotypic resistance tests covering 400 amino acid positions in the RT of HIV-1 subtype B viruses and corresponding treatment histories and laboratory measurements. METHODS: The emergence of connection domain mutations was studied in 334 patients receiving monotherapy or dual therapy with thymidine analogues at the time of the genotypic resistance test. Response to subsequent combination ART (cART) was analyzed using Cox regression for 291 patients receiving unboosted protease inhibitors. Response was defined by ever reaching an HIV RNA level <50 copies/mL during the first cART. RESULTS: The connection domain mutations N348I, R356K, R358K, A360V, and A371V were more frequently observed in ART-exposed than ART-naive patients, of which only N348I and A360V were nonpolymorphic (with a prevalence of <1.5% in untreated patients). N348I correlated with M184V and predominantly occurred in patients receiving lamivudine and zidovudine concomitantly. A360V was not associated with specific drug combinations and was found to emerge later than M184V or thymidine analogue mutations. Nonpolymorphic connection domain mutations were rarely detected in the absence of established drug resistance mutations in ART-exposed individuals (prevalence, <1%). None of the 5 connection domain mutations associated with treatment showed a statistically significant effect on response to cART. CONCLUSIONS: Despite their frequent emergence, connection domain mutations did not show large detrimental effects on response to cART. Currently, routine implementation of connection domain sequencing seems unnecessary for developed health care settings.

Effectiveness and safety of isoniazid chemoprophylaxis for HIV-1 infected patients from Rio de Janeiro

The clinical and epidemiological characteristics, adverse events, treatment adherence and effectiveness of isoniazid chemoprophylaxis were analyzed in a cohort of 138 tuberculosis/HIV-coinfected patients. An open, non-randomized, pragmatic prophylactic trial was conducted on adult patients with a normal chest X-ray and positive tuberculin skin test (> 5 mm) who received isoniazid chemoprophylaxis (300 mg/day) for six months. The mean of follow up was 2.8 years (SD 1.3). Adherence to chemoprophylaxis was 87.7% (121/138). Only one patient presented tuberculosis after the end of chemoprophylaxis, corresponding to 0.3 cases per 100 persons per year. The relative risk of some adverse effects was 4.6 times higher (95% CI: 1.9-11.5) in patients with positive anti-HCV serology (4/9, 44.4%) compared to those with negative serology (12/129, 9.6%) (p = 0.002). This study provides evidence regarding the effectiveness and safety of a short and self-administered isoniazid regimen. We recommend the implementation of this routine by health service practitioners.

Prevalence and resistance mutations of non-B HIV-1 subtypes among immigrants in Southern Spain along the decade 2000-2010.

Most of the non-B HIV-1 subtypes are predominant in Sub-Saharan Africa and India although they have been found worldwide. In the last decade, immigration from these areas has increased considerably in Spain. The objective of this study was to evaluate the prevalence of non-B subtypes circulating in a cohort of HIV-1-infected immigrants in Seville, Southern Spain and to identify drug resistance-associated mutations. METHODS: Complete protease and first 220 codons of the reverse transcriptase coding regions were amplified and sequenced by population sequencing. HIV-1 subtypes were determined using Stanford University Drug Resistance Database, and phylogenetic analysis was performed comparing multiple reported sequences. Drug resistance mutations were defined according to the International AIDS Society-USA. RESULTS: From 2000 to 2010 a total of 1,089 newly diagnosed HIV-1-infected patients were enrolled in our cohort. Of these, 121 were immigrants, of which 98 had ethical approval and informed consent to include in our study. Twenty-nine immigrants (29/98, 29.6%) were infected with non-B subtypes, of which 15/29 (51.7%) were CRF02-AG, mostly from Sub-Saharan Africa, and 2/29 (6.9%) were CRF01-AE from Eastern Europe. A, C, F, J and G subtypes from Eastern Europe, Central-South America and Sub-Saharan Africa were also present. Some others harboured recombinant forms CRF02-AG/CRF01-AE, CRF2-AG/G and F/B, B/C, and K/G, in PR and RT-coding regions. Patients infected with non-B subtypes showed a high frequency of minor protease inhibitor resistance mutations, M36I, L63P, and K20R/I. Only one patient, CRF02_AG, showed major resistance mutation L90M. Major RT inhibitor resistance mutations K70R and A98G were present in one patient with subtype G, L100I in one patient with CRF01_AE, and K103N in another patient with CRF01_AE. Three patients had other mutations such as V118I, E138A and V90I. CONCLUSIONS: The circulation of non-B subtypes has significantly increased in Southern Spain during the last decade, with 29.6% prevalence, in association with demographic changes among immigrants. This could be an issue in the treatment and management of these patients. Resistance mutations have been detected in these patients with a prevalence of 7% among treatment-naïve patients compared with the 21% detected among patients under HAART or during treatment interruption.

Consensus statement of the European guidelines on clinical management of HIV-1 tropism testing

Introduction: Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. To date, in most European countries HIV tropism is determined using a phenotypic test. Recently, new data have emerged supporting the use of a genotypic HIV V3-loop sequence analysis as the basis for tropism determination. The European guidelines group on clinical management of HIV-1 tropism testing was established to make recommendations to clinicians and virologists. Methods: We searched online databases for articles from Jan 2006 until March 2010 with the terms: tropism or CCR5-antagonist or CCR5 antagonist or maraviroc or vicriviroc. Additional articles and/or conference abstracts were identified by hand searching. This strategy identified 712 potential articles and 1240 abstracts. All were reviewed and finally 57 papers and 42 abstracts were included and used by the panel to reach a consensus statement. Results: The panel recommends HIV-tropism testing for the following indications: i) drug-naïve patients in whom toxicity or limited therapeutic options are foreseen; ii) patients experiencing therapy failure whenever a treatment change is considered. Both the phenotypic Enhanced Trofile assay (ESTA) and genotypic population sequencing of the V3-loop are recommended for use in clinical practice. Although the panel does not recommend one methodology over another it is anticipated that genotypic testing will be used more frequently because of its greater accessibility, lower cost and shorter turnaround time. The panel also provides guidance on technical aspects and interpretation issues. If using genotypic methods, triplicate PCR amplification and sequencing testing is advised using the G2P interpretation tool (clonal model) with an FPR of 10%. If the viral load is below the level of reliable amplification, proviral DNA can be used, and the panel recommends performing triplicate testing and use of an FPR of 10%. If genotypic DNA testing is not performed in triplicate the FPR should be increased to 20%. Conclusions: The European guidelines on clinical management of HIV-1 tropism testing provide an overview of current literature, evidence-based recommendations for the clinical use of tropism testing and expert guidance on unresolved issues and current developments. Current data support both the use of genotypic population sequencing and ESTA for co-receptor tropism determination. For practical reasons genotypic population sequencing is the preferred method in Europe.