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1.  Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay 
Journal of Clinical Microbiology  2013;51(11):3666-3674.
High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5′ end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring.
PMCID: PMC3889749  PMID: 23985909
2.  Molecular Characterization of Ambiguous Mutations in HIV-1 Polymerase Gene: Implications for Monitoring HIV Infection Status and Drug Resistance 
PLoS ONE  2013;8(10):e77649.
Detection of recent HIV infections is a prerequisite for reliable estimations of transmitted HIV drug resistance (t-HIVDR) and incidence. However, accurately identifying recent HIV infection is challenging due partially to the limitations of current serological tests. Ambiguous nucleotides are newly emerged mutations in quasispecies, and accumulate by time of viral infection. We utilized ambiguous mutations to establish a measurement for detecting recent HIV infection and monitoring early HIVDR development. Ambiguous nucleotides were extracted from HIV-1 pol-gene sequences in the datasets of recent (HIVDR threshold surveys [HIVDR-TS] in 7 countries; n=416) and established infections (1 HIVDR monitoring survey at baseline; n=271). An ambiguous mutation index of 2.04×10-3 nts/site was detected in HIV-1 recent infections which is equivalent to the HIV-1 substitution rate (2×10-3 nts/site/year) reported before. However, significantly higher index (14.41×10-3 nts/site) was revealed with established infections. Using this substitution rate, 75.2% subjects in HIVDR-TS with the exception of the Vietnam dataset and 3.3% those in HIVDR-baseline were classified as recent infection within one year. We also calculated mutation scores at amino acid level at HIVDR sites based on ambiguous or fitted mutations. The overall mutation scores caused by ambiguous mutations increased (0.54×10-23.48×10-2/DR-site) whereas those caused by fitted mutations remained stable (7.50-7.89×10-2/DR-site) in both recent and established infections, indicating that t-HIVDR exists in drug-naïve populations regardless of infection status in which new HIVDR continues to emerge. Our findings suggest that characterization of ambiguous mutations in HIV may serve as an additional tool to differentiate recent from established infections and to monitor HIVDR emergence.
PMCID: PMC3798419  PMID: 24147046
3.  Comparison of HIV-1 resistance profiles in plasma RNA versus PBMC DNA in heavily treated patients in Honduras, a resource-limited country 
The World Health Organization currently does not recommend the use of dried blood spot specimens for drug resistance testing in patients undergoing antiretroviral therapy (ART). Therefore, HIV-1 resistance testing using peripheral blood mononuclear cells (PBMCs) may be of value in resource-limited settings. We compared genotypic resistance profiles in plasma and PBMCs from patients failing ART in two cities of Honduras (Tegucigalpa and San Pedro Sula), a resource-limited country. One hundred patients failing ART were randomly selected from a longitudinal patient monitoring cohort. Plasma and PBMC samples without patient identifier were used for genotypic resistance testing. Sequence data were analyzed, resistance profiles were determined and compared using Stanford HIV Drug Resistance Database algorithm. Specimens with concordant resistance profiles between the two compartments were 88% (95% CI: 80.3% - 94.5 %). Nine specimens (12%, 95% CI: 6.5% - 21.3%) had discordant resistance profiles of clinical significance. Logistic regression analyses indicated that patients on triple therapy were 17.24 times more likely to have concordant drug resistance profile than those on non-triple therapies (OR=17.24, 95% CI: 3.48, 83.33), while patients with increasing number of regimens and years on ART have a decreased rate of concordance (OR = 0.59, 95% CI: 0.32, 1.09 and OR = 0.62, 95% CI: 0.43, 0.88), respectively, than those with less number of regimens and years on ART. Our results show high level of concordance between plasma and PBMC resistance profiles, indicating the possibility of using PBMCs for drug resistance testing in resources-limited settings.
PMCID: PMC3316452  PMID: 22493752
HIV-1 drug resistance; RNA; PBMCs; concordance; discordance; resistance profile; resource-limited setting
4.  Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings 
PLoS ONE  2011;6(11):e28184.
Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX.
The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE® and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.
PMCID: PMC3223235  PMID: 22132237
5.  Development and Application of a Broadly Sensitive Dried-Blood-Spot-Based Genotyping Assay for Global Surveillance of HIV-1 Drug Resistance ▿  
Journal of Clinical Microbiology  2010;48(9):3158-3164.
As antiretroviral therapy (ART) is scaled up in resource-limited countries, surveillance for HIV drug resistance (DR) is vital to ensure sustained effectiveness of first-line ART. We have developed and applied a broadly sensitive dried-blood-spot (DBS)-based genotyping assay for surveillance of HIV-1 DR in international settings. In 2005 and 2006, 171 DBS samples were collected under field conditions from newly diagnosed HIV-1-infected individuals from Malawi (n = 58), Tanzania (n = 60), and China (n =53). In addition, 30 DBS and 40 plasma specimens collected from ART patients in China and Cameroon, respectively, were also tested. Of the 171 DBS analyzed at the protease and RT regions, 149 (87.1%) could be genotyped, including 49 (81.7%) from Tanzania, 47 (88.7%) from China, and 53 (91.4%) from Malawi. Among the 70 ART patient samples analyzed, 100% (30/30) of the Chinese DBS and 90% (36/40) of the Cameroonian plasma specimens were genotyped, including 8 samples with a viral load of <400 copies/ml. The results of phylogenetic analyses indicated that the subtype, circulating recombinant form (CRF), and unique recombinant form (URF) distribution was as follows: 73 strains were subtype C (34%), 37 were subtype B (17.2%), 24 each were CRF01_AE or CRF02_AG (11.2% each), 22 were subtype A1 (10.2%), and 9 were unclassifiable (UC) (4.2%). The remaining samples were minor strains comprised of 6 that were CRF07_BC (2.8%), 5 that were CRF10_CD (2.3%), 3 each that were URF_A1C and CRF08_BC (1.4%), 2 each that were G, URF_BC, and URF_D/UC (0.9%), and 1 each that were subtype F1, subtype F2, and URF_A1D (0.5%). Our results indicate that this broadly sensitive genotyping assay can be used to genotype DBS collected from areas with diverse HIV-1 group M subtypes and CRFs. Thus, the assay is likely to become a useful screening tool in the global resistance surveillance and monitoring of HIV-1 where multiple subtypes and CRFs are found.
PMCID: PMC2937690  PMID: 20660209
7.  Diminished RNA Primer Usage Associated with the L74V and M184V Mutations in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Provides a Possible Mechanism for Diminished Viral Replication Capacity†  
Journal of Virology  2003;77(16):8621-8632.
The emergence of drug resistance-conferring mutations can severely compromise the success of chemotherapy directed against human immunodeficiency virus type 1 (HIV-1). The M184V and/or L74V mutation in the reverse transcriptase (RT) gene are frequently found in viral isolates from patients treated with the nucleoside RT inhibitors lamivudine (3TC), abacavir (ABC), and didanosine (ddI). However, the effectiveness of combination therapy with regimens containing these compounds is often not abolished in the presence of these mutations; it has been conjectured that diminished fitness of HIV-1 variants containing L74V and M184V may contribute to sustained antiviral effects in such cases. We have determined that viruses containing both L74V and M184V are more impaired in replication capacity than viruses containing either mutation alone. To understand the biochemical mechanisms responsible for this diminished fitness, we generated a series of recombinant mutated enzymes containing either or both of the L74V and M184V substitutions. These enzymes were tested for their abilities to bypass important rate-limiting steps during the complex process of reverse transcription. We studied both the initiation of minus-strand DNA synthesis with the cognate replication primer human tRNA3Lys and the initiation of plus-strand DNA synthesis, using a short RNA primer derived from the viral polypurine tract. We observed that the efficiencies of both reactions were diminished with enzymes containing either L74V or M184V and that these effects were significantly amplified with the double mutant. We also show that release from intrinsic pausing sites during reverse transcription appears to be a major obstacle that cannot be efficiently bypassed. Our data suggest that the efficiency of RNA-primed DNA synthesis represents an important consideration that can affect viral replication kinetics.
PMCID: PMC167213  PMID: 12885880
8.  The M184V Substitution in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Delays the Development of Resistance to Amprenavir and Efavirenz in Subtype B and C Clinical Isolates† 
The M184V substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), encoding high-level resistance to lamivudine (3TC), results in decreased HIV-1 replicative capacity, diminished RT processivity, and increased RT fidelity in biochemical assays. We assessed the effect of M184V on the development of resistance to the nonnucleoside RT inhibitors efavirenz (EFV) and nevirapine, and to the protease inhibitor amprenavir (APV) in tissue culture. Genotypic analysis revealed differences in EFV resistance-conferring mutations in subtype B (K103N) versus subtype C (V106 M), and the appearance of both was significantly delayed in the M184V-containing variants compared with the wild type (WT). Similarly, there was a marked delay in the emergence of mutations associated with APV resistance (I54 M/L/V) in subtype B viruses harboring M184V compared with paired WT viral isolates.
PMCID: PMC161854  PMID: 12821504
9.  Pressure of Zidovudine Accelerates the Reversion of Lamivudine Resistance-Conferring M184V Mutation in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 
We cultured lamivudine-resistant human immunodeficiency virus type 1 (HIV-1) variants over an extended period of time in the presence of zidovudine and observed a premature reversion of the resistance-conferring M184V mutation. These data suggest that the presence of ZDV amplifies differences in replication capacity between wild-type HIV-1 and the mutant variant.
PMCID: PMC127311  PMID: 12069983
10.  An Intact U5-Leader Stem Is Important for Efficient Replication of Simian Immunodeficiency Virus 
Journal of Virology  2001;75(23):11924-11929.
Previous work has shown that four deletions in simian immunodeficiency virus (SIV), termed SD1a, SD1b, SD1c, and SD6, which eliminated sequences at nucleotide positions 322 to 362, 322 to 370, 322 to 379, and 371 to 379, respectively, located downstream of the primer binding site, impaired viral replication capacity to different extents. Long-term culturing of viruses containing the SD1a, SD1b, and SD6 deletions led to revertants that possessed wild-type replication kinetics. We now show that these revertants retained the original deletions in each case but that novel additional mutations were also present. These included a large deletion termed D1 (nt +216 to +237) within the U5 region that was shown to be biologically relevant to reversion of both the SD1a and SD1b constructs. In the case of SD6, two compensatory point mutations, i.e., A+369G, termed M1, located immediately upstream of the SD6 deletion, and C+201T, termed M2, within U5, were identified and could act either singly or in combination to restore viral replication. Secondary structure suggests that an intact U5-leader stem is important in SIV for infectiousness and that the additional mutants described played important roles in restoration of this motif.
PMCID: PMC114783  PMID: 11689678
11.  Partial Restoration of Replication of Simian Immunodeficiency Virus by Point Mutations in either the Dimerization Initiation Site (DIS) or Gag Region after Deletion Mutagenesis within the DIS 
Journal of Virology  2001;75(23):11920-11923.
We used the simian immunodeficiency virus (SIV) molecular clone SIVmac239 to generate a deletion construct, termed SD2, in which we eliminated 22 nucleotides at positions +398 to +418 within the putative dimerization initiation site (DIS) stem. This SD2 deletion severely impaired viral replication, due to adverse effects on the packaging of viral genomic RNA, the processing of Gag proteins, and viral protein patterns. However, long-term culture of SD2 in either C8166 or CEMx174 cells resulted in restoration of replication capacity, due to two different sets of three compensatory point mutations, located within both the DIS and Gag regions. In the case of C8166 cells, both a K197R and a E49K mutation were identified within the capsid (CA) protein and the p6 protein of Gag, respectively, while the other point mutation (A423G) was found within the putative DIS loop. In the case of CEMx174 cells, two compensatory mutations were present within the viral nucleocapsid (NC) protein, E18G and Q31K, in addition to the same A423G substitution as observed with C8166 cells. A set of all three mutations was required in each case for restoration of replication capacity, and either set of mutations could be substituted for the other in both the C8166 and CEMx174 cell lines.
PMCID: PMC114782  PMID: 11689677
12.  Leader Sequences Downstream of the Primer Binding Site Are Important for Efficient Replication of Simian Immunodeficiency Virus 
Journal of Virology  2000;74(19):8854-8860.
Simian immunodeficiency virus (SIV) infection of macaques is remarkably similar to that of human immunodeficiency virus type 1 (HIV-1) in humans, and the SIV-macaque system is a good model for AIDS research. We have constructed an SIV proviral DNA clone that is deleted of 97 nucleotides (nt), i.e., construct SD, at positions (+322 to +418) immediately downstream of the primer binding site (PBS) of SIVmac239. When this construct was transfected into COS-7 cells, the resultant viral progeny were severely impaired with regard to their ability to replicate in C8166 cells. Further deletion analysis showed that a virus termed SD1, containing a deletion of 23 nt (+322 to +344), was able to replicate with wild-type kinetics, while viruses containing deletions of 21 nt (+398 to +418) (construct SD2) or 53 nt (+345 to +397) (construct SD3) displayed diminished capacity in this regard. Both the SD2 and SD3 viruses were also impaired with regard to ability to package viral RNA, while SD1 viruses were not. The SD and SD3 constructs did not revert to increased replication ability in C8166 cells over 6 months in culture. In contrast, long-term passage of the SD2 mutated virus resulted in a restoration of replication capacity, due to the appearance of four separate point mutations. Two of these substitutions were located in leader sequences of viral RNA within the PBS and the dimerization initiation site (DIS), while the other two were located within two distinct Gag proteins, i.e., CA and p6. The biological relevance of three of these point mutations was confirmed by site-directed mutagenesis studies that showed that SD2 viruses containing each of these substitutions had regained a significant degree of viral replication capacity. Thus, leader sequences downstream of the PBS, especially the U5-leader stem and the DIS stem-loop, are important for SIV replication and for packaging of the viral genome.
PMCID: PMC102079  PMID: 10982327

Results 1-12 (12)