Validity of Self-Reported Hepatitis C Virus Status Among Criminal Justice-Involved Persons Living With HIV.

The prevalence of HIV/hepatitis C virus (HCV) coinfection among justice-involved persons is high. The validity of self-reported HCV status in this population has important public health implications, yet has not been studied. Justice-involved persons with HIV from Washington, DC, were enrolled in a study that investigated a mobile health intervention to support HIV treatment. Self-reported and laboratory-confirmed HCV status was compared. Among 103 participants, chronic HCV prevalence was 13%. Positive predictive value of self-reported positive chronic HCV status was low at 55%, and negative predictive value was 98%. Cohen's kappa statistic was 0.60 for agreement. Two women who reported negative HCV status were found to have HCV.

Performance comparison of next generation sequencing analysis pipelines for HIV-1 drug resistance testing.

Next generation sequencing (NGS) is a trending new standard for genotypic HIV-1 drug resistance (HIVDR) testing. Many NGS HIVDR data analysis pipelines have been independently developed, each with variable outputs and data management protocols. Standardization of such analytical methods and comparison of available pipelines are lacking, yet may impact subsequent HIVDR interpretation and other downstream applications. Here we compared the performance of five NGS HIVDR pipelines using proficiency panel samples from NIAID Virology Quality Assurance (VQA) program. Ten VQA panel specimens were genotyped by each of six international laboratories using their own in-house NGS assays. Raw NGS data were then processed using each of the five different pipelines including HyDRA, MiCall, PASeq, Hivmmer and DEEPGEN. All pipelines detected amino acid variants (AAVs) at full range of frequencies (1~100%) and demonstrated good linearity as compared to the reference frequency values. While the sensitivity in detecting low abundance AAVs, with frequencies between 1~20%, is less a concern for all pipelines, their specificity dramatically decreased at AAV frequencies <2%, suggesting that 2% threshold may be a more reliable reporting threshold for ensured specificity in AAV calling and reporting. More variations were observed among the pipelines when low abundance AAVs are concerned, likely due to differences in their NGS read quality control strategies. Findings from this study highlight the need for standardized strategies for NGS HIVDR data analysis, especially for the detection of minority HIVDR variants.

Measurement error and variant-calling in deep Illumina sequencing of HIV.

MOTIVATION: Next-generation deep sequencing of viral genomes, particularly on the Illumina platform, is increasingly applied in HIV research. Yet, there is no standard protocol or method used by the research community to account for measurement errors that arise during sample preparation and sequencing. Correctly calling high and low-frequency variants while controlling for erroneous variants is an important precursor to downstream interpretation, such as studying the emergence of HIV drug-resistance mutations, which in turn has clinical applications and can improve patient care. RESULTS: We developed a new variant-calling pipeline, hivmmer, for Illumina sequences from HIV viral genomes. First, we validated hivmmer by comparing it to other variant-calling pipelines on real HIV plasmid datasets. We found that hivmmer achieves a lower rate of erroneous variants, and that all methods agree on the frequency of correctly called variants. Next, we compared the methods on an HIV plasmid dataset that was sequenced using Primer ID, an amplicon-tagging protocol, which is designed to reduce errors and amplification bias during library preparation. We show that the Primer ID consensus exhibits fewer erroneous variants compared to the variant-calling pipelines, and that hivmmer more closely approaches this low error rate compared to the other pipelines. The frequency estimates from the Primer ID consensus do not differ significantly from those of the variant-calling pipelines. AVAILABILITY AND IMPLEMENTATION: hivmmer is freely available for non-commercial use from https://github.com/kantorlab/hivmmer. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

HIV-1 second-line failure and drug resistance at high-level and low-level viremia in Western Kenya.

OBJECTIVE: Characterize failure and resistance above and below guidelines-recommended 1000 copies/ml virologic threshold, upon second-line failure. DESIGN: Cross-sectional study. METHODS: Kenyan adults on lopinavir/ritonavir-based second-line were enrolled at AMPATH (Academic Model Providing Access to Healthcare). Charts were reviewed for demographic/clinical characteristics and CD4/viral load were obtained. Participants with detectable viral load had a second visit and pol genotyping was attempted in both visits. Accumulated resistance was defined as mutations in the second, not the first visit. Low-level viremia (LLV) was detectable viral load less than 1000 copies/ml. Failure and resistance associations were evaluated using logistic and Poisson regression, Fisher Exact and t-tests. RESULTS: Of 394 participants (median age 42, 60% women, median 1.9 years on second-line) 48% had detectable viral load; 21% had viral load more than 1000 copies/ml, associated with younger age, tuberculosis treatment, shorter time on second-line, lower CD4count/percentage, longer first-line treatment interruption and pregnancy. In 105 sequences from the first visit (35 with LLV), 79% had resistance (57% dual-class, 7% triple-class; 46% with intermediate-to-high-level resistance to ≥1 future drug option). LLV was associated with more overall and NRTI-associated mutations and with predicted resistance to more next-regimen drugs. In 48 second-visit sequences (after median 55 days; IQR 28-33), 40% accumulated resistance and LLV was associated with more mutation accumulation. CONCLUSION: High resistance upon second-line failure exists at levels above and below guideline-recommended virologic-failure threshold, impacting future treatment options. Optimization of care should include increased viral load monitoring, resistance testing and third-line ART access, and consideration of lowering the virologic failure threshold, though this demands further investigation.

Gp41 and Gag amino acids linked to HIV-1 protease inhibitor-based second-line failure in HIV-1 subtype A from Western Kenya.

INTRODUCTION: Failure of protease-inhibitor (PI)-based second-line antiretroviral therapy (ART) with medication adherence but no protease drug resistance mutations (DRMs) is not well understood. This study investigated the involvement of gp41 and gag as alternative mechanisms, not captured by conventional resistance testing and particularly relevant in resource-limited settings where third-line ART is limited. METHODS: We evaluated gp41 and gag for unique amino acids in seven subtype A infected Kenyans failing second-line therapy with no PI resistance yet detectable lopinavir (query dataset), compared to seven similar-setting patients with PI resistance or undetectable lopinavir and 69 publically available subtype A Kenyan whole-genomes sequences. RESULTS: Three gp41 (607T, 641L, 721I) and four gag (124S, 143V, 339P, 357S) amino acids were significantly more frequent in the query dataset compared to the other datasets, with significantly high co-occurrence. CONCLUSION: The genotypic analysis of a unique group of HIV-1 subtype A infected patients, identified seven amino acids that could potentially contribute to a multi-gene mechanism of PI-based ART failure in the absence of PI DR mutations.

Improved HIV-1 Viral Load Monitoring Capacity Using Pooled Testing With Marker-Assisted Deconvolution.

OBJECTIVE: Improve pooled viral load (VL) testing to increase HIV treatment monitoring capacity, particularly relevant for resource-limited settings. DESIGN: We developed marker-assisted mini-pooling with algorithm (mMPA), a new VL pooling deconvolution strategy that uses information from low-cost, routinely collected clinical markers to determine an efficient order of sequential individual VL testing and dictates when the sequential testing can be stopped. METHODS: We simulated the use of pooled testing to ascertain virological failure status on 918 participants from 3 studies conducted at the Academic Model Providing Access to Healthcare in Eldoret, Kenya, and estimated the number of assays needed when using mMPA and other pooling methods. We also evaluated the impact of practical factors, such as specific markers used, prevalence of virological failure, pool size, VL measurement error, and assay detection cutoffs on mMPA, other pooling methods, and single testing. RESULTS: Using CD4 count as a marker to assist deconvolution, mMPA significantly reduces the number of VL assays by 52% [confidence interval (CI): 48% to 57%], 40% (CI: 38% to 42%), and 19% (CI: 15% to 22%) compared with individual testing, simple mini-pooling, and mini-pooling with algorithm, respectively. mMPA has higher sensitivity and negative/positive predictive values than mini-pooling with algorithm, and comparable high specificity. Further improvement is achieved with additional clinical markers, such as age and time on therapy, with or without CD4 values. mMPA performance depends on prevalence of virological failure and pool size but is insensitive to VL measurement error and VL assay detection cutoffs. CONCLUSIONS: mMPA can substantially increase the capacity of VL monitoring.

Treatment failure and drug resistance in HIV-positive patients on tenofovir-based first-line antiretroviral therapy in western Kenya.

INTRODUCTION: Tenofovir-based first-line antiretroviral therapy (ART) is recommended globally. To evaluate the impact of its incorporation into the World Health Organization (WHO) guidelines, we examined treatment failure and drug resistance among a cohort of patients on tenofovir-based first-line ART at the Academic Model Providing Access to Healthcare, a large HIV treatment programme in western Kenya. METHODS: We determined viral load (VL), drug resistance and their correlates in patients on ≥six months of tenofovir-based first-line ART. Based on enrolled patients' characteristics, we described these measures in those with (prior ART group) and without (tenofovir-only group) prior non-tenofovir-based first-line ART using Wilcoxon rank sum and Fisher's exact tests. RESULTS: Among 333 participants (55% female; median age 41 years; median CD4 336 cells/µL), detectable (>40 copies/mL) VL was found in 18%, and VL>1000 copies/mL (WHO threshold) in 10%. Virologic failure at both thresholds was significantly higher in 217 participants in the tenofovir-only group compared with 116 in the prior ART group using both cut-offs (24% vs. 7% with VL>40 copies/mL; 15% vs. 1% with VL>1000 copies/mL). Failure in the tenofovir-only group was associated with lower CD4 values and advanced WHO stage. In 35 available genotypes from 51 participants in the tenofovir-only group with VL>40 copies/mL (69% subtype A), any resistance was found in 89% and dual-class resistance in 83%. Tenofovir signature mutation K65R occurred in 71% (17/24) of the patients infected with subtype A. Patients with K65R had significantly lower CD4 values, higher WHO stage and more resistance mutations. CONCLUSIONS: In this Kenyan cohort, tenofovir-based first-line ART resulted in good (90%) virologic suppression including high suppression (99%) after switch from non-tenofovir-based ART. Lower virologic suppression (85%) and high observed resistance levels (89%) in the tenofovir-only group impact future treatment options, support recommendations for widespread VL monitoring in such resource limited settings to identify early treatment failure and suggest consideration of individualized resistance testing to design effective subsequent regimens.

One-Step Ligation on RNA Amplification for the Detection of Point Mutations.

The detection of point mutations is required in the diagnosis of many human diseases. The conformal specificity of DNA ligases was elegantly used to distinguish single-nucleotide mismatches. However, to detect point mutations in RNA retroviruses, conventional ligase-mediated approaches require the reverse transcription of viral genomes before separate ligation and amplification steps. We developed one-step ligation on RNA amplification (LRA) for the direct detection of RNA point mutations. The process combines the ligase-mediated joining of two oligonucleotides and subsequent hot start amplification into a single-tube reaction. We report that modifications to the structure of the oligonucleotide ligation probes improve the rate of ligation and the specificity of mutation detection on RNA. We applied LRA to the detection of a common, clinically relevant HIV-1 reverse transcriptase drug-resistant point mutation, K103N, and compared it with allele-specific PCR and pyrosequencing. LRA achieved a limit of specific quantitation of 1:100 (1%), and a limit of specific detection for mutant K103N RNA transcripts among excess wild-type strands of 1:10,000 (0.01%). LRA also exhibited good detection threshold of 5 × 10(2) copies/µL K103N RNA transcripts. LRA is a novel point mutation detection method, with potential utilization in HIV drug resistance detection and early diagnostics of genetic disorders associated with other infectious diseases and cancer.

Extensive drug resistance in HIV-infected Cambodian children who are undetected as failing first-line antiretroviral therapy by WHO 2010 guidelines.

Antiretroviral therapy in resource-limited settings is monitored clinically and immunologically according to WHO guidelines. Frequent misclassification of virologic failure is reported, mostly in adults, leading to early therapy switch or late failure diagnosis. Pediatric treatment monitoring and resistance data upon first-line failure are limited, particularly when the 2010-WHO pediatric guidelines are used without routine viral load monitoring. We previously reported high treatment failure misclassification rates by pediatric 2010 guidelines in Cambodian children on first-line therapy. Here we determine the extent and patterns of resistance, with yearly viral load and 6-monthly CD4. Drug resistance mutations were determined using the IAS-USA 2011 list. Predicted resistance interpretation was determined with Stanford Database tools. Fifty-one children with available genotypes met inclusion criteria. All but one (subtype B) were CRF01_AE. The most common regimen was stavudine, lamivudine, and nevirapine (96%), taken for a median of 2.2 years. Resistance was seen in 98%; 96% to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs); 51% with ≥4 mutations. The most common NRTI mutations were 184V/I and 67N and the most common NNRTI mutations were 181C/Y/I/V and 190A/S. A total of 22% had multiresistant mutations and 18% had predicted high-level resistance to subsequent therapy options didanosine, abacavir, etravirine, and tenofovir. In 98% of Cambodian children misclassified as nonfailing first-line therapy by 2010 guidelines, 51% had extensive drug resistance to current and 18% to subsequent antiretroviral therapy. Affordable routine viral load monitoring allowing for early and more accurate treatment failure diagnosis is desperately needed in resource-limited settings.

CCR5 mutations distinguish N-terminal modifications of RANTES (CCL5) with agonist versus antagonist activity.

CCR5 is the major HIV-1 entry coreceptor. RANTES/CCL5 analogs are more potent inhibitors of infection than native chemokines; one class activates and internalizes CCR5, one neither activates nor internalizes, and a third partially internalizes without activation. Here we show that mutations in CCR5 transmembrane domains differentially impact the activity of these three inhibitor classes, suggesting that the transmembrane region of CCR5, a key interaction site for inhibitors, is a sensitive molecular switch, modulating receptor activity.

Resistance to the CCR5 inhibitor 5P12-RANTES requires a difficult evolution from CCR5 to CXCR4 coreceptor use.

Viral resistance to small molecule allosteric inhibitors of CCR5 is well documented, and involves either selection of preexisting CXCR4-using HIV-1 variants or envelope sequence evolution to use inhibitor-bound CCR5 for entry. Resistance to macromolecular CCR5 inhibitors has been more difficult to demonstrate, although selection of CXCR4-using variants might be expected. We have compared the in vitro selection of HIV-1 CC1/85 variants resistant to either the small molecule inhibitor maraviroc (MVC) or the macromolecular inhibitor 5P12-RANTES. High level resistance to MVC was conferred by the same envelope mutations as previously reported after 16-18 weeks of selection by increasing levels of MVC. The MVC-resistant mutants were fully sensitive to inhibition by 5P12-RANTES. By contrast, only transient and low level resistance to 5P12-RANTES was achieved in three sequential selection experiments, and each resulted in a subsequent collapse of virus replication. A fourth round of selection by 5P12-RANTES led, after 36 weeks, to a "resistant" variant that had switched from CCR5 to CXCR4 as a coreceptor. Envelope sequences diverged by 3.8% during selection of the 5P12-RANTES resistant, CXCR4-using variants, with unique and critical substitutions in the V3 region. A subset of viruses recovered from control cultures after 44 weeks of passage in the absence of inhibitors also evolved to use CXCR4, although with fewer and different envelope mutations. Control cultures contained both viruses that evolved to use CXCR4 by deleting four amino acids in V3, and others that maintained entry via CCR5. These results suggest that coreceptor switching may be the only route to resistance for compounds like 5P12-RANTES. This pathway requires more mutations and encounters more fitness obstacles than development of resistance to MVC, confirming the clinical observations that resistance to small molecule CCR5 inhibitors very rarely involves coreceptor switching.

Extreme genetic divergence is required for coreceptor switching in HIV-1 subtype C.

BACKGROUND: Coreceptor switching from CCR5 to CXCR4 is less common in subtype C HIV-1 infection than in subtype B for reasons that are unclear. We have examined sequential virus samples from a subtype C-infected child who had evidence of coreceptor switching. METHODS: To examine HIV-1 envelope evolution towards CXCR4 usage, env sequences were correlated with phenotypic characteristics determined by entry assays, as well as the ability to use alternative coreceptors such as FPRL1, CCR3, CCR8 and others. The value of a phenotype predictor based on V3 sequences was also assessed. RESULTS: Ninety-three sequences revealed 3 distinct coexistent virus lineages and only some members of one lineage evolved to use CXCR4. These lineages also had diverse alternative coreceptor patterns including the ability to use FPRL1, CCR3, CCR8, APJ, CMKLR1, RDC-1, CXCR6, CCR1, GPCR1, GPR15 and CCR6. Coreceptor switching was associated with extensive and rapid sequence divergence in the V1/V2 region in addition to V3 changes. Furthermore, interlineage recombination within the C2 region resulted in low predictability of a V3 sequence-based phenotype algorithm, and highlighted the importance of V1/V2 and V3 sequences in coreceptor usage. CONCLUSION: These results suggest that the evolution to coreceptor switching in subtype C infection requires more mutations than other subtypes, and this contributes to the reduced incidence of R5X4 viruses.

"Resistance" to PSC-RANTES revisited: two mutations in human immunodeficiency virus type 1 HIV-1 SF162 or simian-human immunodeficiency virus SHIV SF162-p3 do not confer resistance.

Resistance of human immunodeficiency virus type 1 (HIV-1) to small-molecule CCR5 inhibitors is well demonstrated, but resistance to macromolecular CCR5 inhibitors (e.g., PSC-RANTES) that act by both CCR5 internalization and receptor blockade had not been reported until recently (3). The report of a single simian-human immunodeficiency virus SHIV(SF162-p3) variant with one V3 and one gp41 sequence change in gp160 that conferred both altered replicative fitness and resistance to PSC-RANTES was therefore surprising. We introduced the same two mutations into both the parental HIV-1(SF162) and the macaque-adapted SHIV(SF162-p3) and found minor differences in entry fitness but no changes in sensitivity to inhibition by either PSC-RANTES or the small-molecule allosteric inhibitor TAK-779. We attribute the earlier finding to confounding fitness effects with inhibitor sensitivity.

Fitness epistasis and constraints on adaptation in a human immunodeficiency virus type 1 protein region.

Fitness epistasis, the interaction among alleles at different loci in their effects on fitness, has potentially important consequences for adaptive evolution. We investigated fitness epistasis among amino acids of a functionally important region of the human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein (gp120). Seven mutations putatively involved in the adaptation of the second conserved to third variable protein region (C2-V3) to the use of an alternative host-cell chemokine coreceptor (CXCR4) for cell entry were engineered singly and in combinations on the wild-type genetic background and their effects on viral infectivity were measured. Epistasis was found to be common and complex, involving not only pairwise interactions, but also higher-order interactions. Interactions could also be surprisingly strong, changing fitness by more than 9 orders of magnitude, which is explained by some single mutations being practically lethal. A consequence of the observed epistasis is that many of the minimum-length mutational trajectories between the wild type and the mutant with highest fitness on cells expressing the alternative coreceptor are selectively inaccessible. These results may help explain the difficulty of evolving viruses that use the alternative coreceptor in culture and the delayed evolution of this phenotype in natural infection. Knowledge of common, complex, and strong fitness interactions among amino acids is necessary for a full understanding of protein evolution.

Evolution of CCR5 use before and during coreceptor switching.

The envelope gene (env) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1- to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants.

Longitudinal analysis of HIV type 1 subtype C envelope sequences from South Africa.

The envelope genes of 23 subtype C viral isolates from five individuals with early HIV-1 infection, followed for 2-4 years, were sequenced, analyzed, and correlated to coreceptor usage. Isolates from three participants used the CCR5 coreceptor at all time points, with no significant adaptations in the variable loop lengths, predicted N-linked glycosylation sites, or predicted change in sensitivity to monoclonal antibodies with disease progression. However, two individuals, Du151 and Du179, who had previously been shown to be dually infected with two phylogenetically distinct subtype C strains, were able to use CXCR4 with disease progression. The intraperson genetic diversity was 9% for Du151 and 3% for Du179 compared to <2% for participants who did not undergo a coreceptor switch. In both cases this coreceptor switch was associated with specific amino acid changes in the crown, an increased net amino acid charge in the V3 loop, and an increase in the length of the V1 region.

Genetic characteristics of the V3 region associated with CXCR4 usage in HIV-1 subtype C isolates.

CXCR4 coreceptor usage appears to occur less frequently among HIV-1 subtype C viruses. The aim of this study was to investigate the genetic determinants within the V3 region of subtype C isolates able to use CXCR4. Thirty-two subtype C isolates with known phenotypes (16 R5, 8 R5X4 and 8 X4 isolates) were assessed. A subtype C-specific V3 heteroduplex tracking assay (HTA) was used to determine sample complexity, and nucleotide sequencing analysis was used to compare characteristics associated with CCR5 and CXCR4-using isolates. There were sufficient genetic differences to discriminate between R5 viruses and those able to use CXCR4. In general, R5 isolates had an HTA mobility ratio >0.9 whereas CXCR4-using isolates were usually <0.9. Multiple bands were more frequently seen among the dualtropic isolates. Sequence analysis of the V3 region showed that CXCR4-using viruses were often associated with an increased positive amino acid charge, insertions and loss of a glycosylation site, similar to HIV-1 subtype B. In contrast, where subtype B consensus V3 has a GPGR crown motif irrespective of coreceptor usage, all 16 subtype C R5 viruses had a conserved GPGQ sequence at the tip of the loop, while 12 of the 16 (75%) CXCR4-using viruses had substitutions in this motif, most commonly arginine (R). These findings were confirmed using a larger published data set. We therefore suggest that changes within the crown motif of subtype C viruses might be an additional pathway to utilise CXCR4 and thus GPGQ may limit the potential for the development of X4 viruses.

A reliable phenotype predictor for human immunodeficiency virus type 1 subtype C based on envelope V3 sequences.

In human immunodeficiency virus type 1 (HIV-1) subtype B infections, the emergence of viruses able to use CXCR4 as a coreceptor is well documented and associated with accelerated CD4 decline and disease progression. However, in HIV-1 subtype C infections, responsible for more than 50% of global infections, CXCR4 usage is less common, even in individuals with advanced disease. A reliable phenotype prediction method based on genetic sequence analysis could provide a rapid and less expensive approach to identify possible CXCR4 variants and thus increase our understanding of subtype C coreceptor usage. For subtype B V3 loop sequences, genotypic predictors have been developed based on position-specific scoring matrices (PSSM). In this study, we apply this methodology to a training set of 279 subtype C sequences of known phenotypes (228 non-syncytium-inducing [NSI] CCR5(+) and 51 SI CXCR4(+) sequences) to derive a C-PSSM predictor. Specificity and sensitivity distributions were estimated by combining data set bootstrapping with leave-one-out cross-validation, with random sampling of single sequences from individuals on each bootstrap iteration. The C-PSSM had an estimated specificity of 94% (confidence interval [CI], 92% to 96%) and a sensitivity of 75% (CI, 68% to 82%), which is significantly more sensitive than predictions based on other methods, including a commonly used method based on the presence of positively charged residues (sensitivity, 47.8%). A specificity of 83% and a sensitivity of 83% were achieved with a validation set of 24 SI and 47 NSI unique subtype C sequences. The C-PSSM performs as well on subtype C V3 loops as existing subtype B-specific methods do on subtype B V3 loops. We present bioinformatic evidence that particular sites may influence coreceptor usage differently, depending on the subtype.

In vitro generation of HIV type 1 subtype C isolates resistant to enfuvirtide.

Enfuvirtide (ENF) is the first in a new class of antiretroviral agents targeting the fusion process of the viral life cycle. ENF is a synthetic 36-amino acid peptide that binds to the HR-1 region of gp41 preventing fusion of viral and cellular membranes. With the introduction of ENF there are now four classes of antiretrovirals each with distinct and different resistance pathways. Resistance to ENF among subtype B HIV-1 isolates is associated with amino acid changes mainly in the HR-1 region, although other regions of envelope have also been implicated. To determine whether subtype C viruses developed resistance mutations similar to subtype B viruses, 11 subtype C and 4 subtype B viruses were passaged in the presence of increasing concentrations of ENF. The subtype C isolates showed varying levels of replication at 1 microg/ml ENF by day 18, but by day 29 all replicated efficiently at 10 microg/ml ENF. All subtype C isolates showed evidence of genotypic changes in gp41 HR-1 following exposure to ENF that included G36S/E/D, I37T, V38M/A/L/E, N/S42D, N43K, L45R/M, and A50T/V. Three subtype C viruses had compensatory changes in the HR-2 region, which corresponds to the ENF sequence, and two isolates had changes in the V3 region. Mutational patterns among the four subtype B viruses were similar to those for subtype C and those previously published in the literature. These data indicate that in vitro resistance to ENF develops rapidly among HIV-1 subtype C isolates. In general, mutational patterns for subtype C were similar to those described for subtype B, suggesting that the mechanism of action for ENF is similar for HIV-1 subtype B and C isolates.

Use of alternate coreceptors on primary cells by two HIV-1 isolates.

Two HIV-1 isolates (CM4 and CM9) able to use alternate HIV-1 coreceptors on transfected cell lines were tested for their sensitivity to inhibitors of HIV-1 entry on primary cells. CM4 was able to use CCR5 and Bob/GPR15 efficiently in transfected cells. The R5 isolate grew in Delta32/Delta32 CCR5 PBMC in the absence or presence of AMD3100, a CXCR4-specific inhibitor, indicating that it uses a receptor other than CCR5 or CXCR4 on primary cells. It was insensitive to the CCR5 entry inhibitors RANTES and PRO140, but was partially inhibited by vMIP-1, a chemokine that binds CCR3, CCR8, GPR15 and CXCR6. The coreceptor used by this isolate on primary cells is currently unknown. CM9 used CCR5, CXCR4, Bob/GPR15, CXCR6, CCR3, and CCR8 on transfected cells and was able to replicate in the absence or presence of AMD3100 in Delta32/Delta32 CCR5 PBMC. It was insensitive to eotaxin, vMIP-1 and I309 when tested individually, but was inhibited completely when vMIP-1 or I309 was combined with AMD3100. Both I309 and vMIP-1 bind CCR8, strongly suggesting that this isolate can use CCR8 on primary cells. Collectively, these data suggest that some HIV-1 isolates can use alternate coreceptors on primary cells, which may have implications for strategies that aim to block viral entry.