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1.  Trends in Prevalence of HIV-1 Drug Resistance in a Public Clinic in Maputo, Mozambique 
PLoS ONE  2015;10(7):e0130580.
Background
An observational study was conducted in Maputo, Mozambique, to investigate trends in prevalence of HIV drug resistance (HIVDR) in antiretroviral (ART) naïve subjects initiating highly active antiretroviral treatment (HAART).
Methodology/Principal Findings
To evaluate the pattern of drug resistance mutations (DRMs) found in adults on ART failing first-line HAART [patients with detectable viral load (VL)]. Untreated subjects [Group 1 (G1; n=99)] and 274 treated subjects with variable length of exposure to ARV´s [6–12 months, Group 2 (G2;n=93); 12-24 months, Group 3 (G3;n=81); >24 months (G4;n=100)] were enrolled. Virological and immunological failure (VF and IF) were measured based on viral load (VL) and T lymphocyte CD4+ cells (TCD4+) count and genotypic resistance was also performed. Major subtype found was C (untreated: n=66, 97,06%; treated: n=36, 91.7%). Maximum virological suppression was observed in G3, and significant differences intragroup were observed between VF and IF in G4 (p=0.022). Intergroup differences were observed between G3 and G4 for VF (p=0.023) and IF between G2 and G4 (p=0.0018). Viral suppression (<50 copies/ml) ranged from 84.9% to 90.1%, and concordant VL and DRM ranged from 25% to 57%. WHO cut-off for determining VF as given by 2010 guidelines (>5000 copies/ml) identified 50% of subjects carrying DRM compared to 100% when lower VL cut-off was used (<50 copies/ml). Length of exposure to ARVs was directly proportional to the complexity of DRM patterns. In Mozambique, VL suppression was achieved in 76% of individuals after 24 months on HAART. This is in agreement with WHO target for HIVDR prevention target (70%).
Conclusions
We demonstrated that the best way to determine therapeutic failure is VL compared to CD4 counts. The rationalized use of VL testing is needed to ensure timely detection of treatment failures preventing the occurrence of TDR and new infections.
doi:10.1371/journal.pone.0130580
PMCID: PMC4494809  PMID: 26151752
2.  Genetic Analysis and Natural Polymorphisms in HIV-1 gp41 Isolates from Maputo City, Mozambique 
Abstract
Enfuvirtide was the first fusion inhibitor approved by the Food and Drug Administration (FDA) in 2003 for HIV-1 infection in treatment-experienced patient. It is the first approved antiviral agent to attack the HIV life cycle in its early stages. For HIV fusion to occur, the HR1 and HR2 domains in the gp41 region need to interact. Enfuvirtide is a synthetic peptide that corresponds to 36 amino acids of the HR2, which competitively binds to HR1 inhibiting the interaction with the HR2 domain thus preventing fusogenic conformation and inhibiting viral entry into host cells. Resistance to enfuvirtide is conferred by mutations occurring in the HR1 region involving residues 36–45. Mozambique, a sub-Saharan country, with an HIV prevalence of 11.5%, provides first line and second line antiretroviral therapy (ART)-based treatment. In poor resource settings such as Mozambique the lack of adequate infrastructures, the high costs of viral load tests, and the availability of salvage treatment have hindered the intended objective of monitoring HIV treatment, suggesting an important concern regarding the development of drug resistance. The general aim of this study was to evaluate naturally occurring polymorphisms and resistance-associated mutations in the gp41 region of HIV-1 isolates from Mozambique. The study included 78 patients naive to ARV treatment and 28 patients failing first line regimen recruited from Centro de Saúde Alto-Maé situated in Maputo. The gp41 gene from 103 patients was sequenced and resistance-associated mutations for enfuvirtide were screened. Subtype analysis revealed that 96% of the sequences were classified as subtype C, 2% as subtype G, 1% as subtype A1, and the other 1% as a mosaic form composed of A1/C. No enfuvirtide resistance-associated mutations in HR1 of gp41 were detected. The major polymorphisms in the HR1 were N42S, L54M, A67T, and V72I. This study suggests that this new class of antiviral drug may be effective as a salvage therapy in patients failing first line regimens in Mozambique. However, further phenotypic studies are required to determine the clinical relevance of the polymorphisms detected in this study.
doi:10.1089/aid.2013.0244
PMCID: PMC4046211  PMID: 24188582
3.  Correction: Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis 
Rhee, Soo-Yon | Blanco, Jose Luis | Jordan, Michael R. | Taylor, Jonathan | Lemey, Philippe | Varghese, Vici | Hamers, Raph L. | Bertagnolio, Silvia | de Wit, Tobias F. Rinke | Aghokeng, Avelin F. | Albert, Jan | Avi, Radko | Avila-Rios, Santiago | Bessong, Pascal O. | Brooks, James I. | Boucher, Charles A. B. | Brumme, Zabrina L. | Busch, Michael P. | Bussmann, Hermann | Chaix, Marie-Laure | Chin, Bum Sik | D’Aquin, Toni T. | De Gascun, Cillian F. | Derache, Anne | Descamps, Diane | Deshpande, Alaka K. | Djoko, Cyrille F. | Eshleman, Susan H. | Fleury, Herve | Frange, Pierre | Fujisaki, Seiichiro | Harrigan, P. Richard | Hattori, Junko | Holguin, Africa | Hunt, Gillian M. | Ichimura, Hiroshi | Kaleebu, Pontiano | Katzenstein, David | Kiertiburanakul, Sasisopin | Kim, Jerome H. | Kim, Sung Soon | Li, Yanpeng | Lutsar, Irja | Morris, Lynn | Ndembi, Nicaise | Kee, Peng NG | Paranjape, Ramesh S. | Peeters, Martine | Poljak, Mario | Price, Matt A. | Ragonnet-Cronin, Manon L. | Reyes-Terán, Gustavo | Rolland, Morgane | Sirivichayakul, Sunee | Smith, Davey M. | Soares, Marcelo A. | Soriano, Vincent V. | Ssemwanga, Deogratius | Stanojevic, Maja | Stefani, Mariane A. | Sugiura, Wataru | Sungkanuparph, Somnuek | Tanuri, Amilcar | Tee, Kok Keng | Truong, Hong-Ha M. | van de Vijver, David A. M. C. | Vidal, Nicole | Yang, Chunfu | Yang, Rongge | Yebra, Gonzalo | Ioannidis, John P. A. | Vandamme, Anne-Mieke | Shafer, Robert W.
PLoS Medicine  2015;12(6):e1001845.
doi:10.1371/journal.pmed.1001845
PMCID: PMC4452696  PMID: 26030872
4.  Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis 
Rhee, Soo-Yon | Blanco, Jose Luis | Jordan, Michael R. | Taylor, Jonathan | Lemey, Philippe | Varghese, Vici | Hamers, Raph L. | Bertagnolio, Silvia | de Wit, Tobias F. Rinke | Aghokeng, Avelin F. | Albert, Jan | Avi, Radko | Avila-Rios, Santiago | Bessong, Pascal O. | Brooks, James I. | Boucher, Charles A. B. | Brumme, Zabrina L. | Busch, Michael P. | Bussmann, Hermann | Chaix, Marie-Laure | Chin, Bum Sik | D’Aquin, Toni T. | De Gascun, Cillian F. | Derache, Anne | Descamps, Diane | Deshpande, Alaka K. | Djoko, Cyrille F. | Eshleman, Susan H. | Fleury, Herve | Frange, Pierre | Fujisaki, Seiichiro | Harrigan, P. Richard | Hattori, Junko | Holguin, Africa | Hunt, Gillian M. | Ichimura, Hiroshi | Kaleebu, Pontiano | Katzenstein, David | Kiertiburanakul, Sasisopin | Kim, Jerome H. | Kim, Sung Soon | Li, Yanpeng | Lutsar, Irja | Morris, Lynn | Ndembi, Nicaise | NG, Kee Peng | Paranjape, Ramesh S. | Peeters, Martine | Poljak, Mario | Price, Matt A. | Ragonnet-Cronin, Manon L. | Reyes-Terán, Gustavo | Rolland, Morgane | Sirivichayakul, Sunee | Smith, Davey M. | Soares, Marcelo A. | Soriano, Vincent V. | Ssemwanga, Deogratius | Stanojevic, Maja | Stefani, Mariane A. | Sugiura, Wataru | Sungkanuparph, Somnuek | Tanuri, Amilcar | Tee, Kok Keng | Truong, Hong-Ha M. | van de Vijver, David A. M. C. | Vidal, Nicole | Yang, Chunfu | Yang, Rongge | Yebra, Gonzalo | Ioannidis, John P. A. | Vandamme, Anne-Mieke | Shafer, Robert W.
PLoS Medicine  2015;12(4):e1001810.
Background
Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes.
Methods and Findings
We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling.
Conclusions
Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.
In this individual patient and sequence-level meta-analysis, Soo-Yon Rhee and colleagues measure regional trends in HIV-1 transmitted drug resistance prevalence and investigate the specific mutations responsible for TDR in different regions and in different virus subtypes.
Editors' Summary
Background
About 35 million people are currently infected with HIV, the virus that causes AIDS by destroying immune system cells and leaving infected individuals susceptible to other infections. Early in the AIDS epidemic, most HIV-infected individuals died within ten years of infection. Then, in 1996, effective antiretroviral (ARV) therapy—drug combinations that suppress HIV replication by inhibiting reverse transcriptase and other essential viral enzymes—became available. For people living in affluent countries, HIV/AIDS became a chronic condition, but because ARV therapy was expensive, HIV/AIDS remained fatal in low- and middle-income countries (LMICs). In 2003, the international community began to work towards achieving universal access to ARV therapy. Now, more than 10 million HIV-positive individuals in LMICs receive ARV therapy, usually as a fixed-dose combination of two nucleoside reverse transcriptase inhibitors (NRTIs), such as tenofovir and lamivudine, plus a non-nucleoside reverse transcriptase inhibitor (NNRTI), such as efavirenz or nevirapine.
Why Was This Study Done?
The global scale-up of ARV therapy has reduced deaths from HIV/AIDS and the incidence of HIV infection in LMICs, but the development of resistance to ARV therapy is threatening these advances. HIV rapidly accumulates genetic changes (mutations), some of which make HIV resistant to ARV therapy. Up to 30% of patients receiving a fixed-dose NRTI/NNRTI combination develop virological failure, and a high proportion of these patients develop mutations associated with resistance to the ARVs in their regimen. Moreover, the proportion of newly infected, ARV-naïve individuals with transmitted drug resistance (TDR) is also increasing. Organizations involved in HIV/AIDS control need to understand the regional and temporal mutational patterns of TDR to inform the development of guidelines for first-line ARV therapy and of inexpensive resistance mutation assays for use in LMICs. Here, using a statistical approach called meta-analysis to combine information from individual patients about the resistance mutations they carry, the researchers investigate the molecular epidemiology of TDR (the patterns of molecular changes underlying TDR in populations) and identify the HIV drug-resistance mutations most responsible for TDR in different world regions.
What Did the Researchers Do and Find?
The researchers identified 287 studies published between 2000 and 2013 from 111 countries that included the reverse transcriptase sequences of HIV viruses from 50,870 ARV-naïve, HIV-positive individuals. The researchers analyzed each virus sequence for the presence of 93 surveillance drug-resistance mutations (SDRMs) previously shown to be specific indicators of TDR. Meta-analysis of these data indicated that the average overall prevalence of TDR (the proportion of ARV-naïve, HIV-positive individuals infected with a virus carrying one or more SDRMs) ranged from 2.8% in sub-Saharan Africa to 11.5% in North America. In sub-Saharan Africa, the odds (chance) of TDR increased 1.09-fold per year following national ARV scale-up; this increase was attributable to an increase in NRTI- and NNRTI-associated resistance. By contrast, in LMICs in south/southeast Asia, the odds of TDR remained unchanged following ARV scale-up. In Latin America/Caribbean, North America, Europe, and upper-income Asian countries, the odds of TDR have increased by around 1.10-fold per year since 1995, mainly as a result of increased NNRTI resistance. Four NNRTI-associated and 16 NRTI-associated SDRMs accounted for most NNRTI- and NRTI-associated TDR, respectively, in all regions. Notably, in sub-Saharan Africa and south/southeast Asia, most of the NNRTI-associated SDRMs detected were associated with high-level resistance to nevirapine or efavirenz. Finally, the researchers report that 95% of TDR viruses in sub-Saharan Africa and south/southeast Asia were unrelated and had therefore arisen independently.
What Do These Findings Mean?
Because many drug-resistance mutations reduce HIV’s fitness and tend to be lost rapidly in individuals not exposed to ARV therapy, differences among the datasets used in this meta-analysis with respect to how long each ARV-naïve patient had been infected with HIV before virus sampling may limit the accuracy of these findings. Nevertheless, the finding that most of the TDR strains detected in sub-Saharan Africa and south/southeast Asia arose independently suggests that improved patient adherence to ARV therapy and the use of ARV regimens that contain drugs to which HIV rarely develops resistance (regimens with a high genetic barrier to resistance) should reduce the generation of new ARV-resistant strains and mitigate TDR increases. In addition, the finding that a few NNRTI-resistance mutations were responsible for most cases of transmitted high-level resistance suggests that an inexpensive assay that detects these specific mutations may be useful for pre-therapy screening in LMICs with high TDR levels.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001810.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS, summaries of recent research findings on HIV care and treatment, and personal stories about living with HIV/AIDS
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on antiretroviral drugs and on universal access to ARV therapy; Avert also provides personal stories about living with HIV/AIDS
The World Health Organization provides information on all aspects of HIV/AIDS (in several languages), including its guidelines on the use of antiretroviral therapy for treating and preventing HIV infection
The UNAIDS World AIDS Day Report 2014 provides up-to-date information about the AIDS epidemic and efforts to halt it, including progress towards universal access to antiretroviral therapy
The Stanford University HIV Drug Resistance Database includes information about surveillance drug-resistant mutations (SDRMs) and an interactive map displaying HIV drug resistance in ARV-naïve populations
doi:10.1371/journal.pmed.1001810
PMCID: PMC4388826  PMID: 25849352
5.  Reactivation of latent HIV-1 by new semi-synthetic ingenol esters 
Virology  2014;0:328-339.
The ability of HIV to establish long-lived latent infection is mainly due to transcriptional silencing of viral genome in resting memory T lymphocytes. Here, we show that new semi-synthetic ingenol esters reactivate latent HIV reservoirs. Amongst the tested compounds, 3-caproyl-ingenol (ING B) was more potent in reactivating latent HIV than known activators such as SAHA, ingenol 3,20-dibenzoate, TNF-α, PMA and HMBA. ING B activated PKC isoforms followed by NF-κB nuclear translocation. As virus reactivation is dependent on intact NF-κB binding sites in the LTR promoter region ING B, we have shown that. ING B was able to reactivate virus transcription in primary HIV-infected resting cells up to 12 fold and up to 25 fold in combination with SAHA. Additionally, ING B promoted up-regulation of P-TEFb subunits CDK9/Cyclin T1. The role of ING B on promoting both transcription initiation and elongation makes this compound a strong candidate for an anti-HIV latency drug combined with suppressive HAART.
doi:10.1016/j.virol.2014.05.033
PMCID: PMC4383768  PMID: 25014309
HIV; Latency; Ingenol; PKC; NF-kB; Resting cells; P-TEFb
6.  Genetic Diversity in HIV-1 Subtype C LTR from Brazil and Mozambique Generates New Transcription Factor-Binding Sites 
Viruses  2014;6(6):2495-2504.
The HIV-1 subtype C has been substituting the subtype B population in southern Brazil. This phenomenon has been previously described in other countries, suggesting that subtype C may possess greater fitness than other subtypes. The HIV-1 long-terminal repeat (LTR) is an important regulatory region critical for the viral life cycle. Sequence insertions immediately upstream of the viral enhancer are known as the most frequent naturally occurring length polimorphisms (MFNLP). Previous reports demonstrated that the MFNLP could lead to the duplication of transcription factor binding sites (TFBS) enhancing the activity of the HIV-1 subtype C LTR. Here, we amplified and sequenced the LTR obtained from proviral DNA samples collected from patients infected with subtype C from the Southern Region of Brazil (naïve or treatment failure) and Mozambique (only naïve). We confirm the presence of different types of insertions in the LTR sequences of both the countries leading to the creation of additional TFBS. In the Brazilian clinical samples, the frequency of the sequence insertion was significantly higher in subjects experiencing treatment failure than in antiretroviral naïve patients.
doi:10.3390/v6062495
PMCID: PMC4074939  PMID: 24960272
HIV-1; Subtype C; LTR; insertion; NFkB; RBEIII; Brazil; Rio Grande do Sul; Paraná; Mozambique; MFNLP
7.  Dual Role of Novel Ingenol Derivatives from Euphorbia tirucalli in HIV Replication: Inhibition of De Novo Infection and Activation of Viral LTR 
PLoS ONE  2014;9(5):e97257.
HIV infection is not cleared by antiretroviral drugs due to the presence of latently infected cells that are not eliminated with current therapies and persist in the blood and organs of infected patients. New compounds to activate these latent reservoirs have been evaluated so that, along with HAART, they can be used to activate latent virus and eliminate the latently infected cells resulting in eradication of viral infection. Here we describe three novel diterpenes isolated from the sap of Euphorbia tirucalli, a tropical shrub. These molecules, identified as ingenols, were modified at carbon 3 and termed ingenol synthetic derivatives (ISD). They activated the HIV-LTR in reporter cell lines and human PBMCs with latent virus in concentrations as low as 10 nM. ISDs were also able to inhibit the replication of HIV-1 subtype B and C in MT-4 cells and human PBMCs at concentrations of EC50 0.02 and 0.09 µM respectively, which are comparable to the EC50 of some antiretroviral currently used in AIDS treatment. Control of viral replication may be caused by downregulation of surface CD4, CCR5 and CXCR4 observed after ISD treatment in vitro. These compounds appear to be less cytotoxic than other diterpenes such as PMA and prostratin, with effective dose versus toxic dose TI>400. Although the mechanisms of action of the three ISDs are primarily attributed to the PKC pathway, downregulation of surface receptors and stimulation of the viral LTR might be differentially modulated by different PKC isoforms.
doi:10.1371/journal.pone.0097257
PMCID: PMC4020785  PMID: 24827152
8.  HIV-1 Nef Inhibits Protease Activity and Its Absence Alters Protein Content of Mature Viral Particles 
PLoS ONE  2014;9(4):e95352.
Nef is an important player for viral infectivity and AIDS progression, but the mechanisms involved are not completely understood. It was previously demonstrated that Nef interacts with GagPol through p6*-Protease region. Because p6* and Protease are involved in processing, we explored the effect of Nef on viral Protease activity and virion assembly. Using in vitro assays, we observed that Nef is highly capable of inhibiting Protease activity. The IC50 for nef-deficient viruses in drug susceptibility assays were 1.7- to 3.5-fold higher than the wild-type counterpart varying with the type of the Protease inhibitor used. Indicating that, in the absence of Nef, Protease is less sensitive to Protease inhibitors. We compared the protein content between wild-type and nef-deficient mature viral particles by gradient sedimentation and observed up to 2.7-fold reduction in the Integrase levels in nef-deficient mature particles. This difference in levels of Integrase correlated with the difference in infectivity levels of wild type and nef-deficient viral progeny. In addition, an overall decrease in the production of mature particles was detected in nef-deficient viruses. Collectively, our data support the hypothesis that the decreased infectivity typical of nef-deficient viruses is due to an abnormal function of the viral Protease, which is in turn associated with less mature particles being produced and the loss of Integrase content in these particles, and these results may characterize Nef as a regulator of viral Protease activity.
doi:10.1371/journal.pone.0095352
PMCID: PMC3991643  PMID: 24748174
9.  Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases 
Genetics and Molecular Biology  2013;37(1):120-126.
Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.
PMCID: PMC3958318  PMID: 24688299
CCR5; genome editing; gene knockout; TALEN; HMA
10.  Synthetic 1,4-Pyran Naphthoquinones Are Potent Inhibitors of Dengue Virus Replication 
PLoS ONE  2013;8(12):e82504.
Dengue virus infection is a serious public health problem in endemic areas of the world where 2.5 billion people live. Clinical manifestations of the Dengue infection range from a mild fever to fatal cases of hemorrhagic fever. Although being the most rapidly spreading mosquito-borne viral infection in the world, until now no strategies are available for effective prevention or control of Dengue infection. In this scenario, the development of compounds that specifically inhibit viral replication with minimal effects to the human hosts will have a substantial effect in minimizing the symptoms of the disease and help to prevent viral transmission in the affected population. The aim of this study was to screen compounds with potential activity against dengue virus from a library of synthetic naphthoquinones. Several 1,2- and 1,4-pyran naphthoquinones were synthesized by a three-component reaction of lawsone, aldehyde (formaldehyde or arylaldehydes) and different dienophiles adequately substituted. These compounds were tested for the ability to inhibit the ATPase activity of the viral NS3 enzyme in in vitro assays and the replication of dengue virus in cultured cells. We have identified two 1,4-pyran naphthoquinones, which inhibited dengue virus replication in mammal cells by 99.0% and three others that reduced the dengue virus ATPase activity of NS3 by two-fold in in vitro assays.
doi:10.1371/journal.pone.0082504
PMCID: PMC3869945  PMID: 24376541
11.  Genetic Diversity and Naturally Polymorphisms in HIV Type 1 Integrase Isolates from Maputo, Mozambique: Implications for Integrase Inhibitors 
AIDS Research and Human Retroviruses  2012;28(12):1788-1792.
Abstract
HIV proviral DNA integration into the host chromosome is carried out by integrase becoming an important target antiretroviral therapy. Raltegravir was the first integrase inhibitor approved for use in HIV therapy and elvitegravir is in the late phase of clinical development; both show good results in monotherapy studies and may be used worldwide for rescue therapy. In this work we analyzed 57 integrase sequences obtained from samples from drug-naive and first line regime-failing patients from Maputo, Mozambique, to evaluate the presence of natural polymorphisms and resistance mutations associated with raltegravir and elvitegravir. No major mutations conferring resistance to integrase inhibitors were found and polymorphic accessory mutations were solely observed in low frequency among subtype C sequences—L74M (3.4%), T97A (1.8%), and E157Q (1.8%)—suggesting that this new antiretroviral drug class will be effective in Mozambique providing a good perspective to the introduction of this class of drugs in that country.
doi:10.1089/aid.2012.0058
PMCID: PMC3505052  PMID: 22497664
12.  In Vitro–Reduced Susceptibility to Artemether in P. falciparum and Its Association With Polymorphisms on Transporter Genes 
The Journal of Infectious Diseases  2012;206(3):324-332.
Plasmodium falciparum with reduced sensitivity to artemisinin derivatives has been observed in endemic areas, but the molecular mechanisms for this reduced sensitivity remain unclear. We evaluated the association between in vitro susceptibility of P. falciparum isolates obtained from southwest Nigeria and polymorphisms in selected putative transporter genes (PFE0775C, PF13_0271, pfmrp1, pfcrt, and pfmdr1). Modified schizont inhibition assay was used to determine the in vitro parasite susceptibility to artemether (ATH). Polymorphisms in selected genes were detected by polymerase chain reaction followed by direct DNA sequencing. The half-maximal inhibitory concentration (IC50) geometric mean (GM) for all P. falciparum isolates was 1.78 nM (range, 0.03–10.43 nM). Polymorphisms at codons 241, 86, and 76 of PFE0775C, pfmdr1, and pfcrt genes, respectively, were associated with reduced susceptibility to ATH. A new S263P single-nucleotide polymorphism on the PFE0775C gene was also detected in 27% of the isolates. Patient isolates harboring V241L or S263P polymorphisms on the PFE0775C gene showed increased IC50 (GM: 3.08 nM and 1.79 nM, respectively). Plasmodium falciparum isolates harboring mutant Y86 pfmdr1 and P263 PFE0775C alleles showed a 2.5–5.5-fold increase in ATH IC50. This study shows that polymorphisms on the PFE0775C and pfmdr1 genes are associated with reduced sensitivity to ATH in fresh isolates of P. falciparum from Nigeria.
doi:10.1093/infdis/jis359
PMCID: PMC3490696  PMID: 22615315
13.  Evolution of Primary HIV Drug Resistance in a Subtype C Dominated Epidemic in Mozambique 
PLoS ONE  2013;8(7):e68213.
Objective
In Mozambique, highly active antiretroviral treatment (HAART) was introduced in 2004 followed by decentralization and expansion, resulting in a more than 20-fold increase in coverage by 2009. Implementation of HIV drug resistance threshold surveys (HIVDR-TS) is crucial in order to monitor the emergence of transmitted viral resistance, and to produce evidence-based recommendations to support antiretroviral (ARV) policy in Mozambique.
Methods
World Health Organization (WHO) methodology was used to evaluate transmitted drug resistance (TDR) in newly diagnosed HIV-1 infected pregnant women attending ante-natal clinics in Maputo and Beira to non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI). Subtypes were assigned using REGA HIV-1 subtyping tool and phylogenetic trees constructed using MEGA version 5.
Results
Although mutations associated with resistance to all three drug were detected in these surveys, transmitted resistance was analyzed and classified as <5% in Maputo in both surveys for all three drug classes. Transmitted resistance to NNRTI in Beira in 2009 was classified between 5–15%, an increase from 2007 when no NNRTI mutations were found. All sequences clustered with subtype C.
Conclusions
Our results show that the epidemic is dominated by subtype C, where the first-line option based on two NRTI and one NNRTI is still effective for treatment of HIV infection, but intermediate levels of TDR found in Beira reinforce the need for constant evaluation with continuing treatment expansion in Mozambique.
doi:10.1371/journal.pone.0068213
PMCID: PMC3728366  PMID: 23935858
14.  Dynamics of resistance mutations to NS3 protease inhibitors in a cohort of Brazilian patients chronically infected with hepatitis C virus (genotype 1) treated with pegylated interferon and ribavirin: a prospective longitudinal study 
Virology Journal  2013;10:57.
Abstract
About sixty thousand new cases of Hepatitis C virus (HCV) infection are recorded in Brazil each year. These cases are currently treated with pegylated interferon (PEG-IFN) and ribavirin (RBV) with an overall success rate of 50%. New compounds for anti-HCV therapy targeted to the HCV NS3 protease are being developed and some already form the components of licensed therapies. Mapping NS3 protease resistance mutations to protease inhibitors or anti-viral drug candidates is important to direct anti-HCV drug treatment.
Methods
Sequence analysis of the HCV NS3 protease was conducted in a group of 68 chronically infected patients harboring the HCV genotype 1. The patients were sampled before, during and after a course of PEG-IFN-RBV treatment.
Results
Resistance mutations to the protease inhibitors, Boceprevir and Telaprevir were identified in HCV isolated from three patients (4.4%); the viral sequences contained at least one of the following mutations: V36L, T54S and V55A. In one sustained virological responder, the T54S mutation appeared during the course of PEG-IFN and RBV therapy. In contrast, V36L and V55A mutations were identified in virus isolated from one relapsing patient before, during, and after treatment, whereas the T54S mutation was identified in virus isolated from one non-responding patient, before and during the treatment course.
Conclusions
The incidence and persistence of protease resistance mutations occurring in HCV from chronically infected patients in Brazil should be considered when using protease inhibitors to treat HCV disease. In addition, patients treated with the current therapy (PEG-IFN and RBV) that are relapsing or are non-responders should be considered candidates for protease inhibitor therapy.
doi:10.1186/1743-422X-10-57
PMCID: PMC3599441  PMID: 23409973
HCV NS3 protease; Drug resistance persistency; Selection pressure; Antiviral drugs; Chronic Hepatitis C infection
15.  Differential In Vitro Kinetics of Drug Resistance Mutation Acquisition in HIV-1 RT of Subtypes B and C 
PLoS ONE  2012;7(10):e46622.
Background
HIV-1 subtype B is the most prevalent in developed countries and, consequently, it has been extensively studied. On the other hand, subtype C is the most prevalent worldwide and therefore is a reasonable target for future studies. Here we evaluate the acquisition of resistance and the viability of HIV-1 subtype B and C RT clones from different isolates that were subjected to in vitro selection pressure with zidovudine (ZDV) and lamivudine (3TC).
Methods/Principal Findings
MT4 cells were infected with chimeric virus pseudotyped with RT from subtype B and C clones, which were previously subjected to serial passage with increasing concentrations of ZDV and 3TC. The samples collected after each passage were analyzed for the presence of resistance mutations and VL. No differences were found between subtypes B and C in viral load and resistance mutations when these viruses were selected with 3TC. However, the route of mutations and the time to rebound of subtype B and C virus were different when subjected to ZDV treatment. In order to confirm the role of the mutations detected, other clones were generated and subjected to in vitro selection. RT subtype B virus isolates tended to acquire different ZDV resistance mutations (Q151M and D67N or T215Y, D67D/N and F214L) compared to subtype C (D67N, K70R, T215I or T215F).
Conclusions/Significance
This study suggests that different subtypes have a tendency to react differently to antiretroviral drug selection in vitro. Consequently, the acquisition of resistance in patients undergoing antiretroviral therapy can be dependent on the subtypes composing the viral population.
doi:10.1371/journal.pone.0046622
PMCID: PMC3463560  PMID: 23056372
16.  Natural transmission of feline immunodeficiency virus from infected queen to kitten 
Virology Journal  2012;9:99.
Background
Feline immunodeficiency virus (FIV) is a naturally occurring lentivirus that infects cats. The primary mode of transmission occurs through bite wounds, and other routes are difficult to observe in nature.
Findings
The purpose of this study was to evaluate FIV transmission from queen to kitten in a colony of naturally infected stray cats. With this aim, a queen was monitored over a period of three years. A blood sample was taken to amplify and sequence gag, pol and env regions of the virus from the queen, two kittens and other cats from the colony.
Conclusion
Phylogenetic analysis showed evidence of queen to kitten transmission.
doi:10.1186/1743-422X-9-99
PMCID: PMC3439265  PMID: 22632459
Vertical transmission; FIV; Stray cats
17.  Frequency of human immunodeficiency virus type-2 in hiv infected patients in Maputo City, Mozambique 
Virology Journal  2011;8:408.
The HIV/AIDS pandemic is primarily caused by HIV-1. Another virus type, HIV-2, is found mainly in West African countries. We hypothesized that population migration and mobility in Africa may have facilitated the introduction and spreading of HIV-2 in Mozambique. The presence of HIV-2 has important implications for diagnosis and choice of treatment of HIV infection. Hence, the aim of this study was to estimate the prevalence of HIV-2 infection and its genotype in Maputo, Mozambique.
HIV-infected individuals (N = 1,200) were consecutively enrolled and screened for IgG antibodies against HIV-1 gp41 and HIV-2 gp36 using peptide-based enzyme immunoassays (pepEIA). Specimens showing reactivity on the HIV-2 pepEIA were further tested using the INNO-LIA immunoblot assay and HIV-2 PCR targeting RT and PR genes. Subtype analysis of HIV-2 was based on the protease gene.
After screening with HIV-2 pepEIA 1,168 were non-reactive and 32 were reactive to HIV-2 gp36 peptide. Of this total, 30 specimens were simultaneously reactive to gp41 and gp36 pepEIA while two samples reacted solely to gp36 peptide. Only three specimens containing antibodies against gp36 and gp105 on the INNO-LIA immunoblot assay were found to be positive by PCR to HIV-2 subtype A.
The proportion of HIV-2 in Maputo City was 0.25% (90%CI 0.01-0.49). The HIV epidemic in Southern Mozambique is driven by HIV-1, with HIV-2 also circulating at a marginal rate. Surveillance program need to improve HIV-2 diagnosis and consider periodical survey aiming to monitor HIV-2 prevalence in the country.
doi:10.1186/1743-422X-8-408
PMCID: PMC3179751  PMID: 21849066
HIV-2; laboratory diagnosis; sub-Saharan Africa; Mozambique
18.  Interactions between SIVNef, SIVGagPol and Alix correlate with viral replication and progression to AIDS in rhesus macaques 
Virology  2009;394(1):47-56.
Infection with Simian Immunodeficiency Virus (SIV) leads to high viral loads and progression to Simian AIDS (SAIDS) in rhesus macaques. The viral accessory protein Nef is required for this phenotype in monkeys as well as in HIV-infected humans. Previously, we determined that HIVNef binds HIVGagPol and Alix for optimal viral replication in cells. In this study, we demonstrated that these interactions could correlate with high viral loads leading to SAIDS in the infected host. By infecting rhesus macaques with a mutant SIVmac239, where sequences in the nef gene that are required for these interactions were mutated, we observed robust viral replication and disease in two out of four monkeys, where they reverted to the wild type genotype and phenotype. These two rhesus macaques also died of SAIDS. Two other monkeys did not progress to disease and continued to harbor mutant nef sequences. We conclude that interactions between Nef, GagPol and Alix contribute to optimal viral replication and progression to disease in the infected host.
doi:10.1016/j.virol.2009.08.024
PMCID: PMC2767429  PMID: 19748111
SIV; HIV; Nef; monkey infection; pathogenesis; disease progression; GagPol; Alix
19.  Two Lineages of Dengue Virus Type 2, Brazil 
Emerging Infectious Diseases  2010;16(3):576-578.
doi:10.3201/eid1603.090996
PMCID: PMC3322019  PMID: 20202456
Dengue viruses; DENV-2; vector-borne; infections; outbreak; viruses; dengue; Brazil; letter
20.  Brazilian Network for HIV Drug Resistance Surveillance: a survey of individuals recently diagnosed with HIV 
Use of antiretrovirals is widespread in Brazil, where more than 200,000 individuals are under treatment. Although general prevalence of primary antiretroviral resistance in Brazil is low, systematic sampling in large metropolitan areas has not being performed.
The HIV Threshold Survey methodology (HIV-THS, WHO) was utilized, targeting Brazil's four major regions and selecting the six most populated state capitals: Sao Paulo, Rio de Janeiro, Salvador, Porto Alegre, Brasilia and Belem. We were able to sequence samples from 210 individuals with recent HIV diagnosis, 17 of them (8.1%) carrying HIV isolates with primary antiretroviral resistance mutations. Five, nine and four isolates showed mutations related to resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs), respectively. Using HIV-THS, we could find an intermediate level of transmitted resistance (5% to 15%) in Belem/Brasilia, Sao Paulo and Rio de Janeiro. Lower level of transmitted resistance (<5%) were observed in the other areas. Despite the extensive antiretroviral exposure and high rates of virologic antiretroviral failure in Brazil, the general prevalence of primary resistance is still low. However, an intermediate level of primary resistance was found in the four major Brazilian cities, confirming the critical need to start larger sampling surveys to better define the risk factors associated with transmission of resistant HIV.
doi:10.1186/1758-2652-12-20
PMCID: PMC2759910  PMID: 19765271
21.  Differential Drug Resistance Acquisition in HIV-1 of Subtypes B and C 
PLoS ONE  2007;2(8):e730.
Background
Subtype C is the most prevalent HIV-1 subtype in the world, mainly in countries with the highest HIV prevalence. However, few studies have evaluated the impact of antiretroviral therapy on this subtype. In southern Brazil, the first developing country to offer free and universal treatment, subtypes B and C co-circulate with equal prevalence, allowing for an extensive evaluation of this issue.
Methods and Findings
Viral RNA of 160 HIV-1+ patients was extracted, and the protease and reverse transcriptase genes were sequenced, subtyped and analyzed for ARV mutations. Sequences were grouped by subtype, and matched to type (PI, NRTI and NNRTI) and time of ARV exposure. Statistical analyses were performed to compare differences in the frequency of ARV-associated mutations. There were no significant differences in time of treatment between subtypes B and C groups, although they showed distinct proportions of resistant strains at different intervals for two of three ARV classes. For PI, 26% of subtype B strains were resistant, compared to only 8% in subtype C (p = 0.0288, Fisher's exact test). For NRTI, 54% of subtype B strains were resistant versus 23% of subtype C (p = 0.0012). Differences were significant from 4 years of exposure, and remained so until the last time point analyzed. The differences observed between both subtypes were independent of time under rebound viremia in cases of virologic failure and of the number of HAART regimens used by treated patients.
Conclusions
Our results pointed out to a lower rate of accumulation of mutations conferring resistance to ARV in subtype C than in subtype B. These findings are of crucial importance for current initiatives of ARV therapy roll-out in developing countries, where subtype is C prevalent.
doi:10.1371/journal.pone.0000730
PMCID: PMC1939879  PMID: 17710130
22.  Development of a New Methodology for Screening of Human Immunodeficiency Virus Type 1 Microbicides Based on Real-Time PCR Quantification▿  
Potential topical retrovirucides or vaginal microbicides against human immunodeficiency virus type 1 (HIV-1) include nonnucleoside reverse transcriptase inhibitors (NNRTIs). To be successful, such agents have to be highly active against cell-free virions. In the present study, we developed a new real-time PCR-based assay to measure the natural endogenous reverse transcription (NERT) activity directly on intact HIV-1 particles in the presence of reverse transcriptase (RT) inhibitors. We further evaluated the permeability to nevirapine (NVP) and efavirenz (EFV) and their retention within nascent viral particles. We also demonstrated the NVP and EFV inhibitory effects on NERT activity and the impact of resistance mutations measured directly by this new strategy. Furthermore, the results showed a clear correlation between NERT activity and classical infectivity assays. The 50% inhibitory concentrations (IC50s) of NVP and EFV were demonstrated to be up to 100-fold higher for cell-free than for cell-associated virions, suggesting that cell-free virions are less permeable to these drugs. Our results suggest that NVP and EFV penetrate both the envelope and the capsid of HIV-1 particles and readily inactivate cell-free virions. However, the characteristics of these NNRTIs, such as lower permeability and lower retention during washing procedures, in cell-free virions reduce their efficacies as microbicides. Here, we demonstrate the usefulness of the NERT real-time PCR as an assay for screening novel antiretroviral compounds with unique mechanisms of action.
doi:10.1128/AAC.00749-06
PMCID: PMC1797782  PMID: 17116672
23.  Interactions between Nef and AIP1 proliferate multivesicular bodies and facilitate egress of HIV-1 
Retrovirology  2006;3:33.
Background
Nef is an accessory protein of primate lentiviruses, HIV-1, HIV-2 and SIV. Besides removing CD4 and MHC class I from the surface and activating cellular signaling cascades, Nef also binds GagPol during late stages of the viral replicative cycle. In this report, we investigated further the ability of Nef to facilitate the replication of HIV-1.
Results
To this end, first the release of new viral particles was much lower in the absence of Nef in a T cell line. Since the same results were obtained in the absence of the viral envelope using pseudo-typed viruses, this phenomenon was independent of CD4 and enhanced infectivity. Next, we found that Nef not only possesses a consensus motif for but also binds AIP1 in vitro and in vivo. AIP1 is the critical intermediate in the formation of multivesicular bodies (MVBs), which play an important role in the budding and release of viruses from infected cells. Indeed, Nef proliferated MVBs in cells, but only when its AIP1-binding site was intact. Finally, these functions of Nef were reproduced in primary macrophages, where the wild type but not mutant Nef proteins led to increased release of new viral particles from infected cells.
Conclusion
We conclude that by binding GagPol and AIP1, Nef not only proliferates MVBs but also contributes to the egress of viral particles from infected cells.
doi:10.1186/1742-4690-3-33
PMCID: PMC1526754  PMID: 16764724
24.  Discordances between Interpretation Algorithms for Genotypic Resistance to Protease and Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Are Subtype Dependent 
The major limitation of drug resistance genotyping for human immunodeficiency virus remains the interpretation of the results. We evaluated the concordance in predicting therapy response between four different interpretation algorithms (Rega 6.3, HIVDB-08/04, ANRS [07/04], and VGI 8.0). Sequences were gathered through a worldwide effort to establish a database of non-B subtype sequences, and demographic and clinical information about the patients was gathered. The most concordant results were found for nonnucleoside reverse transcriptase (RT) inhibitors (93%), followed by protease inhibitors (84%) and nucleoside RT inhibitor (NRTIs) (76%). For therapy-naive patients, for nelfinavir, especially for subtypes C and G, the discordances were driven mainly by the protease (PRO) mutational pattern 82I/V + 63P + 36I/V for subtype C and 82I + 63P + 36I + 20I for subtype G. Subtype F displayed more discordances for ritonavir in untreated patients due to the combined presence of PRO 20R and 10I/V. In therapy-experienced patients, subtype G displayed a lot of discordances for saquinavir and indinavir due to mutational patterns involving PRO 90 M and 82I. Subtype F had more discordance for nelfinavir attributable to the presence of PRO 88S and 82A + 54V. For the NRTIs lamivudine and emtricitabine, CRF01_AE had more discordances than subtype B due to the presence of RT mutational patterns 65R + 115 M and 118I + 215Y, respectively. Overall, the different algorithms agreed well on the level of resistance scored, but some of the discordances could be attributed to specific (subtype-dependent) combinations of mutations. It is not yet known whether therapy response is subtype dependent, but the advice given to clinicians based on a genotypic interpretation algorithm differs according to the subtype.
doi:10.1128/AAC.50.2.694-701.2006
PMCID: PMC1366873  PMID: 16436728
25.  Impact of HIV-1 Subtype and Antiretroviral Therapy on Protease and Reverse Transcriptase Genotype: Results of a Global Collaboration 
PLoS Medicine  2005;2(4):e112.
Background
The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate.
Methods and Findings
To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates.
Conclusion
Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.
Most research has focused on HIV-1 subtype B, although most infections worldwide are non-B. However, this paper suggests that the same drug resistance mutations occur across subtypes
doi:10.1371/journal.pmed.0020112
PMCID: PMC1087220  PMID: 15839752

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