Drug-resistant Virus Research Articles

Rapid Initiation of Antiretroviral Therapy after HIV Diagnosis.

Roughly, 38 million people are living with HIV worldwide. Despite the success of antiretroviral therapy (ART) for suppressing virus replication and restore immunity in infected persons, the HIV epidemics are not controlled globally. Each year 1.8 million new HIV infections occur. This rate has declined only slightly during the past decade, despite huge efforts for expanding ART coverage, pre- and post-exposure prophylaxis, and stopping vertical transmission. To achieve the United Nations Programme on HIV/AIDS goals of 95-95-95 by 2030, renewed efforts and innovative strategies must be undertaken. The source of most new HIV infections is people unaware of their HIV-positive status and/or not linked to care. Thus, efforts for unveiling HIV positives and, especially, rapid initiation of ART and retention in care would be the most effective interventions for halting HIV spreading globally. In certain settings, access to point-of-care diagnostic tests and immediate start of ART (even the same day) must be implemented at large scale. Selection of the most convenient ART to be prescribed empirically is an important caveat to minimize the risks of treatment failure. Ideally, it must be easy to take, coformulated as single-tablet regimen (STR), well tolerated, with no requests for prior human leukocyte antigen testing, depict few drug interactions, keep activity against transmitted drug-resistant viruses, remain efficacious in patients with elevated HIV-RNA, and/or low CD4 counts, and when present, suppress hepatitis B coinfection. At this time, the coformulation of darunavir, cobicistat, emtricitabine, and tenofovir alafenamide (Symtuza®) is the only regimen that has been evaluated in a Phase 3 trial as "test-and-treat" strategy. Results at 48 weeks in the DIAMOND study are reassuring, as more than 90% of individuals achieve undetectable viremia.

A5 Near full-length HIV-1 genome sequencing in newly diagnosed individuals in Sweden

The Swedish HIV-1 epidemic is characterized by a high diversity in HIV subtypes and recombinants as a result of migration. To study the time from infection through viral diversification, transmission patterns, and drug resistance in minor quasispecies, a robust protocol for pan-genotypic near full-length HIV-1 genome (HIV-NFLG) next-generation sequencing (NGS) is key. Our group has established two protocols for HIV-NFLG on the Illumina platform that we aim to compare and, if necessary, modify to find a method with optimized coverage, depth, and subtype inclusivity. Zanini et al. (https://doi.org/10.7554/eLife.11282.001) have developed a method with one-step RT-PCR with six overlapping primer sets, followed by NGS and quality filtering and assembly with in-house methods. Aralaguppe et al. (https://doi.org/10.1016/j.jviromet.2016.07.010) have designed amplification in two fragments, followed by multiplexed NGS and quality control and assembly with Iterative Virus Assembler and VICUNA. Both methods have high coverage per nucleotide and low error rates in amplification and sequencing and can reliably identify SNPs at 1 per cent of the viral population with linkage within the quasispecies. Subtype inclusivity remains a challenge even though both methods show success in amplifying and sequencing subtypes B, C, and the common recombinants 01_AE and 02_AG. Therefore, we aim to evaluate and optimize our NFLG NGS methods on a panel of patient samples that more completely reflects HIV-1 diversity in Sweden. Patient samples from fifty treatment-naïve viremic individuals representing the genotypic HIV-1 panorama in Sweden, including CRFs, are being amplified and sequenced by both protocols. Coverage of the genome, error rate, and possible depth of quasispecies analysis is being evaluated. We will compare number of reads, coverage across the HIV genome, and representation of minor single nucleotide variants as well as subtype inclusivity and impact of plasma RNA levels. To do this we will use an in-house bioinformatic pipeline. The NFLG sequences will also be analyzed with phylogenetic tools for determination of subtypes including CRFs and URFs.

An Evolutionary Model-Based Approach To Quantify the Genetic Barrier to Drug Resistance in Fast-Evolving Viruses and Its Application to HIV-1 Subtypes and Integrase Inhibitors.

Viral pathogens causing global disease burdens are often characterized by high rates of evolutionary changes. The extensive viral diversity at baseline can shorten the time to escape from therapeutic or immune selective pressure and alter mutational pathways. The impact of genotypic background on the barrier to resistance can be difficult to capture, particularly for agents in experimental stages or that are recently approved or expanded into new patient populations. We developed an evolutionary model-based counting method to quickly quantify the population genetic potential to resistance and assess population differences. We demonstrate its applicability to HIV-1 integrase inhibitors, as their increasing use globally contrasts with limited availability of non-B subtype resistant sequence data and corresponding knowledge gap. A large sequence data set encompassing most prevailing HIV-1 subtypes and resistance-associated mutations of currently approved integrase inhibitors was investigated. A complex interplay between codon predominance, polymorphisms, and associated evolutionary costs resulted in a subtype-dependent varied genetic potential for 15 resistance mutations against integrase inhibitors. While we confirm the lower genetic barrier of subtype B for G140S, we convincingly discard a similar effect previously suggested for G140C. A supplementary analysis for HIV-1 reverse transcriptase inhibitors identified a lower genetic barrier for K65R in subtype C through differential codon usage not reported before. To aid evolutionary interpretations of genomic differences for antiviral strategies, we advanced existing counting methods with increased sensitivity to identify subtype dependencies of resistance emergence. Future applications include novel HIV-1 drug classes or vaccines, as well as other viral pathogens.

HIV Co-Receptor Usage, Broadly Neutralising Antibodies, and Treatment

The discovery of a new generation of highly potent broadly neutralising antibodies (bnAb) has provided a new weapon in the fight against HIV-1. It is envisioned that multiple bnAb or a single bnAb in conjunction with antiretrovirals (ARV) can be used to treat HIV infection, especially individuals harbouring extensively drug-resistant virus or those that require regimen simplification. Furthermore, it is believed that bnAb may eliminate latently infected cells through antibody-mediated cellular cytotoxicity, and this functionality may induce virus remission. BnAb epitopes and HIV envelope determinants for CCR5 and CXCR4 usage often overlap, and this provides the basis for believing that there is a relationship between receptor utilisation and bnAb sensitivity. This review highlights the important intersection between HIV co-receptor usage and bnAb therapy. Compared to CCR5-using strains, CXCR4 strains are generally more resistant to bnAb that target the V1-V2 apex and V3 N332 glycan, but not the other envelope domains. This association between bnAb sensitivity and co-receptor usage can be leveraged both to develop pre-treatment assays to identify resistant strains, as well as to anticipate potential adverse outcomes with future HIV antibody-based therapeutics.

Azithromycin, a 15-membered macrolide antibiotic, inhibits influenza A(H1N1)pdm09 virus infection by interfering with virus internalization process.

The pandemic influenza 2009 (A(H1N1)pdm09) virus currently causes seasonal and annual epidemic outbreaks. The widespread use of anti-influenza drugs such as neuraminidase and matrix protein 2 (M2) channel inhibitors has resulted in the emergence of drug-resistant influenza viruses. In this study, we aimed to determine the anti-influenza A(H1N1)pdm09 virus activity of azithromycin, a re-positioned macrolide antibiotic with potential as a new anti-influenza candidate, and to elucidate its underlying mechanisms of action. We performed in vitro and in vivo studies to address this. Our in vitro approaches indicated that progeny virus replication was remarkably inhibited by treating viruses with azithromycin before infection; however, azithromycin administration after infection did not affect this process. We next investigated the steps inhibited by azithromycin during virus invasion. Azithromycin did not affect attachment of viruses onto the cell surface, but blocked internalization into host cells during the early phase of infection. We further demonstrated that azithromycin targeted newly budded progeny virus from the host cells and inactivated their endocytic activity. This unique inhibitory mechanism has not been observed for other anti-influenza drugs, indicating the potential activity of azithromycin before and after influenza virus infection. Considering these in vitro observations, we administered azithromycin intranasally to mice infected with A(H1N1)pdm09 virus. Single intranasal azithromycin treatment successfully reduced viral load in the lungs and relieved hypothermia, which was induced by infection. Our findings indicate the possibility that azithromycin could be an effective macrolide for the treatment of human influenza.

A viral genome wide association study and genotypic resistance testing in patients failing first line antiretroviral therapy in the first large countrywide Ethiopian HIV cohort

BackgroundAntiretroviral therapy (ART) was rolled-out in Ethiopia in 2005, but there are no reports on outcome of ART and human immunodeficiency virus drug resistance (HIVDR) at national level. We described acquired drug resistance mutations in pol gene and performed a viral genome wide association study in virologic treatment failure patients who started first line ART during 2009–2011 in the first large countrywide HIV cohort in Ethiopia.MethodsThe outcome of tenofovir (TDF)- and zidovudine (ZDV)-based ART was defined in 874 ART naïve patients using the on-treatment (OT) and intention-to-treat (ITT) analyses. Genotypic resistance testing was done in patients failing ART (> 1000 copies/ml) at month 6 and 12. Near full-length genome sequencing (NFLG) was used to assess amino acid changes in HIV-1 gag, pol, vif, vpr, tat, vpu, and nef genes between paired baseline and month 6 samples.ResultsHigh failure rates were found in ITT analysis at month 6 and 12 (23.3%; 33.9% respectively). Major nucleoside and non-nucleoside reverse transcriptase (NRTI/NNRTI) drug resistance mutations were detected in most failure patients at month 6 (36/47; 77%) and month 12 (20/30; 67%). A high rate of K65R was identified only in TDF treated patients (35.7%; 50.0%, respectively). No significant difference was found in failure rate or extent of HIVDR between TDF- and ZDV- treated patients. All target regions of interest for HIVDR were described by NFLG in 16 patients tested before initiation of ART and at month 6.ConclusionIn this first Ethiopian national cohort, a high degree of HIVDR was seen among ART failure patients, independent on whether TDF- or ZDV was given. However, the major reason to ART failure was lost-to-follow-up rather than virologic failure. Our NFLG assay covered all relevant target genes for antiretrovirals and is an attractive alternative for HIVDR surveillance.

Discovery and Development of Anti-HIV Therapeutic Agents: Progress Towards Improved HIV Medication.

The history of the human immunodeficiency virus (HIV)/AIDS therapy, which spans over 30 years, is one of the most dramatic stories of science and medicine leading to the treatment of a disease. Since the advent of the first AIDS drug, AZT or zidovudine, a number of agents acting on different drug targets, such as HIV enzymes (e.g. reverse transcriptase, protease, and integrase) and host cell factors critical for HIV infection (e.g. CD4 and CCR5), have been added to our armamentarium to combat HIV/AIDS. In this review article, we first discuss the history of the development of anti-HIV drugs, during which several problems such as drug-induced side effects and the emergence of drug-resistant viruses became apparent and had to be overcome. Nowadays, the success of Combination Antiretroviral Therapy (cART), combined with recently-developed powerful but nonetheless less toxic drugs has transformed HIV/AIDS from an inevitably fatal disease into a manageable chronic infection. However, even with such potent cART, it is impossible to eradicate HIV because none of the currently available HIV drugs are effective in eliminating occult “dormant” HIV cell reservoirs. A number of novel unique treatment approaches that should drastically improve the quality of life (QOL) of patients or might actually be able to eliminate HIV altogether have also been discussed later in the review.

HIV-1 drug resistance surveillance among parturient women on anti-retroviral therapy in the Eastern Cape, South Africa: Implications for elimination of mother-to-child transmission

BackgroundThe emergence of HIV drug resistance poses a significant threat to achieving the goal of elimination of mother-to-child transmission. ObjectivesWe assessed the extent and patterns of HIV-1 drug resistance mutations (DRMs) within the context of the public sector prevention of mother-to-child transmission (PMTCT) programme in the Eastern Cape, South Africa. Study designWe conducted analysis of the Pol sub-genomic sequence of RNA extracted from plasma samples of women with probable virological failure at delivery between January and May 2018 from two large maternity centres in the Eastern Cape using standard protocols. Partial pol gene covering 1030bp were amplified and sequenced according to previously reported protocol. DRMs were determined by submitting the generated partial pol sequences to the Stanford drug resistance database for query on mutations associated with drug resistance in HIV viruses. We examined the correlates of DRMs using bivariate analysis. ResultsThe age of parturient women ranged from 16 to 43 years. The majority of the parturient women were currently on Efavirenz-based regimen (first line ART) (82.5%) and had been on ART for more than 12 months (65.0%). The prevalence of DRMs was 72.5% (n = 58). The CD4 count demonstrated a negative linear association with the DRMs (p = 0.002). The predominant DRMs were K103 N (n = 43; 74.1%), M184 V (n = 28; 48.3%) and K65R (n = 11; 19%). Among the parturient women on EFV-based regimen treatment; 79.1% already had K103 N while nine patients on protease inhibitor-based regimen still harboured K103 N. The majority of the M184 V mutations were observed in parturient women on first line regimen (n = 23; 82.1%). ConclusionsWe found a high prevalence of DRMs in women delivering their index babies at high viral loads in the study settings. Drug resistance surveillance using point-of-care reverse transcriptase-PCR strategies for the screening of pregnant women on ART could be a game-changer in the resource-constrained settings.

Discovery of Small-Molecule Inhibitors Targeting the E3 Ubiquitin Ligase Activity of the Herpes Simplex Virus 1 ICP0 Protein Using an In Vitro High-Throughput Screening Assay.

Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-β)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.

Analysis of hepatitis B virus (HBV) drug resistance variations in HBV carriers of Chinese blood donors

Background: To analyze hepatitis B virus (HBV) mutations of nucleoside drug resistance in blood donors of Shantou Central Blood Station. Methods: A total of 22,539 blood donors in Shantou City in 2014 were recruited in the study. Two hundred and one blood samples were detected positive by two types of enzyme-linked immunosorbent assay (ELISA) and 99 cases of HBV positive samples were detected by fluorescence quantitative polymerase chain reaction (PCR). Among them, 45 cases with viral loads greater than 1,000 IU/mL were sequenced by PCR sequencing method. Drug resistance mutation sites of sample DNA sequences were analyzed. Results: All known 10 drug resistant sites of HBV were not detected from these 45 cases of HBV blood donor carriers. Conclusions: The prevalence of HBV strains from blood donors in Shantou were wild-type.

HIV drug resistance: Insights from mathematical modelling

In 2013 the World Health Organization recommended the initiation of antiretroviral therapy (ART) to any person who tests HIV positive irrespective of his/her CD4+ count. However, implementation of the new guidelines poses a lot of challenges especially in Sub-Sahara Africa such as: drug side effects, drug resistance-mutations and significant financial burdens. Most importantly, it has been established that HIV resistance and subsequent virologic failure occur in a substantial proportion of HIV-infected patients receiving HAART. This study therefore, seeks to investigate the emergence of drug resistant HIV virus during treatment with the aim of determining the proper use of HIV therapy that would lessen drug resistance. To carry out the analysis a ten dimensional in-vivo mathematical model is proposed for HIV dynamics. The model is formulated in such away that it takes into account two virus strain, that is, the wild type as well as the naive type HIV virus. The in-vivo model is shown to be both biologically meaningful and mathematically well posed. The existence of unique infection-free equilibrium point is determined and both its local and global stability investigated. In addition, the basic reproduction number for each viral strain is computed using the next generation matrix method. An optimal control model is proposed and analysed by applying Pontryagin maximum principle, to obtain the optimal drug combination for HIV treatment. Here two drugs, that is, Reverse Transcriptase inhibitor and Protease inhibitor are used as the controls in the model. We provide an objective function for the minimisation of the number of wild type HIV virus and the drug resistant virus as well as the costs associated with the use of Reverse Transcriptase inhibitor and protease inhibitor. The forward backward sweep method is applied to numerically solve the optimality system. From the numerical simulations, it is evident that protease inhibitor is the most effective drug in controlling HIV infection. The results suggest that prolonged use of HAART leads to development of drug resistant and that people with drug-resistant infection could play a core role in the epidemic of HIV.

Highly Drug-Resistant HIV-1 Protease Mutant PRS17 Shows Enhanced Binding to Substrate Analogues.

We report the structural analysis of highly drug-resistant human immunodeficiency virus protease (PR) variant PRS17, rationally selected by machine learning, in complex with substrate analogues. Crystal structures were solved of inhibitor-free inactive PRS17-D25N, wild-type PR/CA-p2 complex, and PRS17 in complex with substrate analogues, CA-p2 and p2-NC. Peptide analogues p2-NC and CA-p2 exhibit inhibition constants of 514 and 22 nM, respectively, for PRS17 or approximately 3-fold better than for PR. CA-p2 is a better inhibitor of PRS17 than are clinical inhibitors (Ki = 50–8390 nM) except for amprenavir (Ki = 11 nM). G48V resistance mutation induces curled flap tips in PRS17-D25N structure. The inner P2–P2′ residues of substrate analogues in PRS17 complexes maintain similar conformations to those of wild-type complex, while significant conformational changes are observed in the peripheral residues P3, P4′ of CA-p2 and P3, P4, and P3′ of p2-NC. The loss of β-branched side chain by V82S mutation initiates a shift in 80’s loop and reshapes the S3/S3′ subsite, which enhances substrate binding with new hydrogen bonds and van der Waals interactions that are absent in the wild-type structures. The steric hindrance caused by G48V mutation in the flap of PRS17 contributes to altered binding interactions of P3 Arg, P4′ norleucine of CA-p2, and P4 and P3′ of p2-NC with the addition of new hydrogen bonds and van der Waals contacts. The enhanced interaction of PRS17 with substrate analogues agrees with their relative inhibition, suggesting that this mutant improves substrate binding while decreasing affinity for clinical inhibitors.

Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)

Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-length virus genome data. Here we describe advances to a protocol that exploits the circular HBV genome structure, using isothermal rolling-circle amplification to enrich HBV DNA, generating concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by analysing reads generated from plus and minus strands. With this approach, we can achieve an improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. Thus while Illumina sequencing may still be the most accurate way to capture within-sample diversity, Nanopore data can contribute to an understanding of linkage between polymorphisms within individual virions. The combination of isothermal amplification and Nanopore sequencing also offers appealing potential to develop point-of-care tests for HBV, and for other viruses.

Determination of reverse transcriptase inhibitor nucleoside analogue resistance profile in pretreatment phase of patients with viral hepatitis B

HBV is a DNA virus and the causative agent of hepatitis B infection. Hepatitis B is a contagious disease and is still a major health problem all over the world. When the infection become chronic, it may cause serious diseases such as fibrosis, cirrhosis and/or hepatocellular carcinoma. Interferon/pegylated interferon by intravenous route and nucleoside/nucleotide (NA) analogues such as lamivudine, adefovir, entecavir, telbivudine and tenofovir given by oral route are used in the treatment. Antivirals given by oral route are mostly preferred in the treatment. However, because of the replication strategy and biological properties of HBV, mutations that cause antiviral resistance against these drugs can occur at different rates, although they can vary from drug to drug over time. It is possible that drug resistant virus may transmit from patient to healthy individuals. Therefore, there is a possibility of infection with drug-resistant HBV before treatment. Antiviral resistance mutations are divided into four categories; i) Nucleos(t)ide analog resistance (NAr)-related mutations, ii) primary drug resistance mutations, iii) secondary/compensatory mutations, iv) putative antiviral resistance mutations and pre-treatment variations. Recent studies have focused particularly on putative mutations and pre-treatment variations. The aim of this study was to better understanding of the antiviral resistance profiles of chronic hepatitis B (CHB) patients treated and untreated with NA, and help to preventunnecessary drug use, minimize the side effects and economic damages. A total of 124 patients who have received nucleoside analog (NA) drug treatments (n= 72) and patients without NA treatment (n= 52) were included in the study. Viral DNA was isolated from the plasma samples of the patients. A DNA fragment, which is 551 bp, was amplified and sequenced including the binding side of all nucleoside analogs containing the B, C and D domains located in the reverse transcriptase region in the HBV genome. Different types of mutations were detected in 13 (18.05%) of 72 treated patients and in 18 (34.61%) of 52 untreated patients (p< 0.05). Primary drug resistance mutations such as rtI169T, rtA181T/V, rtT184A/C/F/G/I/L/M/S, rtA194T, rtS202C/G/I, rtM204I/V/S, rtN236T, rt M250I/L/V and rtV173L were not detected in any of the patient samples. However, potential drug resistance mutations such as rtR164R, rtG165D/A, rtG172Q, rtS176N, rtF178V, rtA181G, rtS185N/G/C, rtV207M, rtQ215H/S, rtL231V, rtI233K, rtN238S, rtV253T, rtC256G/S and rtI266R/V were detected in untreated patient samples in B, C, D and D domains of reverse transcriptase region. Our results have suggested that the detection of pretreatment variations could be helpful for choosing the correct antiviral drug for the better treatment management.

Detection of Hepatitis B Virus Drug Resistance Mutations by Pyrosequencing Method

Detection of Hepatitis B Virus Drug Resistance Mutations by Pyrosequencing Method

Neurocognitive Impairment Risk Among Individuals With Multiple Drug-Resistant Tuberculosis and Human Immunodeficiency Virus Coinfection: Implications for Systematic Linkage to and Retention of Care in Tuberculosis/Human Immunodeficiency Virus Treatment.

Although neurocognitive impairment (NCI) is a well-recognized challenge in human immunodeficiency virus (HIV), there is little evidence regarding it among individuals with multiple drug-resistant tuberculosis (MDR-TB) within HIV endemic sub-Saharan Africa. The extent of NCI risk, particularly HIV-associated neurocognitive disorders (HAND) risk, was investigated in 200 microbiologically confirmed inpatients with MDR-TB at a TB-specialist hospital in KwaZulu-Natal Province, South Africa. Within this population, the prevalence of HIV coinfection, major depressive episode, and substance use disorder was 89.50%, 10.50%, and 7.00%, respectively. After excluding individuals with major depressive episode/substance use disorder and monoinfection (i.e., MDR-TB without HIV), the prevalence of HAND risk was 43.5%. Older and low-income individuals had significantly greater odds of HAND risk, whereas those with family members/relatives who work(ed) in the health services had lower odds. The role of timely linkage to and retention of care in TB/HIV treatment to offset cognitive decline in MDR-TB/HIV coinfected individuals needs to be investigated further.

Role of co-expressed APOBEC3F and APOBEC3G in inducing HIV-1 drug resistance

The APOBEC3 enzymes can induce mutagenesis of HIV-1 proviral DNA through the deamination of cytosine. HIV-1 overcomes this restriction through the viral protein Vif that induces APOBEC3 proteasomal degradation. Within this dynamic host-pathogen relationship, the APOBEC3 enzymes have been found to be beneficial, neutral, or detrimental to HIV-1 biology. Here, we assessed the ability of co-expressed APOBEC3F and APOBEC3G to induce HIV-1 resistance to antiviral drugs. We found that co-expression of APOBEC3F and APOBEC3G enabled partial resistance of APOBEC3F to Vif-mediated degradation with a corresponding increase in APOBEC3F-induced deaminations in the presence of Vif, in addition to APOBEC3G-induced deaminations. We recovered HIV-1 drug resistant variants resulting from APOBEC3-induced mutagenesis, but these variants were less able to replicate than drug resistant viruses derived from RT-induced mutations alone. The data support a model in which APOBEC3 enzymes cooperate to restrict HIV-1, promoting viral inactivation over evolution to drug resistance.

OC 8450 ABSENCE OF MINORITY HIV-1 DRUG-RESISTANT VARIANTS FOLLOWING MOTHER-TO-CHILD TRANSMISSION DOES NOT PREDICT VIROLOGIC SUCCESS OF FIRST-LINE ANTIRETROVIRAL THERAPY

BackgroundAlthough minority HIV-1 drug-resistant HIV-1 variants may be selected under antiretroviral pressure, leading to therapy failure, their clinical significance remains controversial. This is particularly relevant in the case of prevention of mother-to-child transmission (MTCT), where transmitted drug resistance can affect treatment outcomes.MethodsAn ultrasensitive HIV-1 genotyping assay based on deep sequencing (DEEPGENHIV) with a 1% mutation frequency sensitivity, was used to quantify MTCT drug-resistant variants in 38 prenatally HIV-infected children experiencing (Group I, n=27) or not (Group II, n=11) virologic failure 12 months after initiating first-line antiretroviral therapy (ART) as part of a paediatric cohort in Uganda.ResultsInfants were infected with subtype A(n=20), D(n=16) or C(n=2) HIV-1 strains, distributed equally between both patients’ groups. Similarly, no significant difference was observed in intra-patient HIV-1 diversity among viruses obtained from Group I or II individuals at baseline. DEEPGENHIV was able to detect all the mutations originally detected in samples obtained from four control patients in Group II, where drug resistance was identified at baseline using Sanger sequencing, e.g. K65R (78% mutation frequency), K103N (47%), or M184V (85%). More importantly, a series of low abundance (&lt;20% detection limit of Sanger) primary and compensatory mutations associated with resistance to PIs (D30N, Q48V), NRTIs (D67N, K219Q), or NNRTIs (L100I, K103N) were identified in both groups of patients, although just a few seem to have been selected and became majority variants after 12 or 24 months of ART.ConclusionDEEPGENHIV improves the detection of minority viral variants in infants following MTCT; however, most of the emergent HIV-1 drug resistance mutations were not present at low frequency at baseline in subjects failing ART, most likely being generated and selected following exposure to treatment. Further studies, using this or other ultrasensitive assays, are needed to better understand the transmission, dynamics and overall evolution of minority drug-resistant viruses in MTCT.

Preparation of a monoPEGylated derivative of cyanovirin-N and its virucidal effect on acyclovir-resistant strains of herpes simplex virus type 1.

The long-term administration of acyclovir (ACV) for therapy against herpes simplex virus type 1 (HSV-1) infections can result in the emergence of ACV-resistant HSV strains. It is therefore urgent to develop new anti-herpetic compounds with mechanisms that differ from that of ACV. Cyanovirin-N (CV-N) is an antiviral agent that has an inhibitory effect on HSV-1 infections, and PEGylation of CV-N is potentially useful for pharmaceutical applications. Here, a (Gly4Ser)3 linker molecule was attached to the N-terminus of CV-N, and the resulting compound, linker-CV-N (LCV-N), was produced on a pilot scale with purity up to 95%. Then, PEG10k-LCV-N was synthesized by modifying at the α-amine group of the N-terminus of LCV-N with 10-kDa polyethylene glycol propionaldehyde (mPEG-ALD). CV-N, LCV-N and PEG10k-LCV-N were all found to have potent inhibitory activity against ACV-resistant HSV strains with IC50 values in the nM range. LCV-N was the most potent of these three compounds against both normal and ACV-resistant HSV strains. Although PEG10k-LCV-N showed less antiviral activity than CV-N and LCV-N, it still exhibited significant and universal virucidal activity against drug-resistant viruses. The toxicity and immunogenicity of PEG10k-LCV-N were dramatically lower than those of CV-N and LCV-N. In conclusion, we suggest that LCV-N and PEG10k-LCV-N are promising and safe microbicides for the control and/or treatment of ACV-resistant HSV infection.

English

Tuberculosis (TB) has become a global health challenge; it is a serious problem in sub-Saharan Africa, where rates of Human Immunodeficiency Virus (HIV) co-infection and drug resistance are high. The prevalence and rifampicin (RIF) resistance of Mycobacterium tuberculosis (MTB) among patients attending Federal Medical Centre Makurdi, Benue State was investigated. Sputum samples collected from 200 patients were tested for acid-fast Bacilli (AFB) using Ziehl-Neelsen stain. GeneXpert machine was used to test the resistance of the AFB to rifampicin. The prevalence of TB was 25.5% and the infection rate was higher in males (28.3%) than in females (23.1%), even though the difference was not statistically significant (2=0.684; p&gt;0.05). The prevalence of TB among HIV patients was also investigated. Prevalence of TB in HIV sero-positive patients was 22.2 and 28.9% in HIV sero-negative patients. This difference, however, was not statistically significant (2=4.453; p&gt;0.05). Most (70.6%) of the AFB positive specimens were susceptible to rifampicin; only a few (29.4%) were resistant to it (2=43.377; p&lt;0.05). With the high prevalence of TB and the high rifampicin resistance (MDR-TB) in Benue State, policy makers and government should increase budgetary allocations for TB control and prevention through sourcing or appealing to international organizations for finance. In addition, the government should encourage research for the development of new and better TB vaccines by financing the project. Key words: Prevalence, resistance, Mycobacterium tuberculosis, Makurdi. &nbsp