First cases of Zika virus-infected US blood donors outside states with areas of active transmission.

BACKGROUND: Zika virus (ZIKV) is transmitted by Aedes mosquitos and can result in severe congenital and adult neurologic abnormalities. ZIKV has rapidly spread northward through Central America and the Caribbean and autochthonous cases have been identified in the continental United States. High rates of ZIKA RNA positivity were detected in blood donors during previous epidemics. ZIKV transmission by transfused blood from healthy donor components has been a growing concern. STUDY DESIGN AND METHODS: Individual-donation aliquots of plasma from volunteer blood donors were tested individually with an investigational Procleix ZIKV assay. Initially reactive samples were tested for ZIKV RNA in plasma and red blood cells (RBCs) and for ZIKV-specific antibodies in serum. A confirmed positive classification required confirmation of RNA and/or detection of ZIKV antibodies in index and/or follow-up samples. RESULTS: Between September 19 and November 30, 2016, a total of 466,834 donations were screened for ZIKV RNA. Five donors (one in approx. 93,000) were reactive for ZIKV RNA by both the Procleix ZIKV assay and supplemental testing. The donations were collected outside areas considered as having active transmission, and all five donors had travel exposures. A lookback case demonstrated no infection despite transfusion of a Zika IgG-positive platelet (PLT) component with probable low levels of ZIKV RNA. CONCLUSIONS: This report describes the first ZIKV-positive donors detected outside areas with active transmission. These donors most likely represent travel-acquired "tail-end infections" with prolonged RBC-associated ZIKV RNA. The lack of transmission to the recipient of an apheresis PLT may suggest that these units are not infectious.

Analytical and clinical performance evaluation of the cobas TaqScreen MPX Test for use on the cobas s 201 system.

Clinical specificity, analytical and clinical sensitivities, reproducibility, and subtype/genotype coverage of the cobas TaqScreen MPX Test, a multiplex nucleic acid test with expanded coverage of HIV variants, were determined. A total of 72,281 blood donations were evaluated. The 95% limit of detection (LOD) for the MPX Test inclusive viruses was determined by testing six dilutions of WHO or Roche standards. Over 3000 high-risk and confirmed seropositive specimens were tested with the MPX and COBAS AmpliScreen Tests. Ten subtypes of HIV-1 Group M, HIV-1 Group O, HIV-2 A and B, HBV genotypes A-H, and HCV genotypes 1-6 were tested with the MPX Test. Reproducibility panels were evaluated at three testing sites across three lots. Clinical specificity in pools was 99.99%. There was one HBV yield case. The LODs for HIV-1 Group M, HCV, and HBV were 49, 11, and 3.8 IU/mL, respectively, and 89 and 59.3 copies/mL for HIV-1 Group O and HIV-2, respectively. Concordance between the MPX and the AmpliScreen Tests was 94.9%. Clinical sensitivity based on AmpliScreen comparison was 97.8-99.5%. All genotype/subtype replicates were detected at three times the LOD. Reproducibility was 98.3-100%. In conclusion, the MPX Test is robust and covers HIV-1 Group O and HIV-2.

PROCLEIX West Nile virus assay based on transcription-mediated amplification.

The PROCLEIX West Nile virus (WNV) assay is based on transcription-mediated amplification and is the most sensitive nucleic acid test commercially available today for blood screening. Since 2003, in the USA, the assay has been used for year-round screening of blood donations in minipools of 16 and in an individual-donation testing format, when appropriate triggering guidelines for such switch become effective. The test can be run on the semiautomated PROCLEIX modular platform or the fully-automated PROCLEIX TIGRIS System. This assay and the corresponding platforms have been developed and are manufactured by San Diego-based Gen-Probe, Inc., while they are marketed and distributed by Chiron, a Novartis business. This review covers some of the epidemiological and virological aspects of WNV, as well as clinical questions and technological assessment of the transcription-mediated amplification and alternative technologies used in WNV blood screening.

A multi-Chinese blood center study testing serologic-negative donor samples for hepatitis C virus and human immunodeficiency virus with nucleic acid testing.

BACKGROUND: A multi-blood center study was conducted to evaluate a human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) multiplex nucleic acid testing (NAT) donor screening test and to determine the residual risk for HIV-1 and HCV infection. STUDY DESIGN AND METHODS: A commercially available HIV-1 and HCV assay (Procleix, Chiron Corp.) was used for simultaneous detection of HIV-1 RNA and HCV RNA on 89,647 unlinked donor samples. NAT was performed with pools of 16 samples that had passed all routine screening tests. Single-donor NAT was performed for samples that had been disqualified by any reactive screening test result(s). Anti-HCV (Ortho third-generation HCV enzyme immunoassay [EIA]), alanine aminotransferase, and HCV NAT (Roche COBAS Amplicor HCV test) confirmatory tests were used for HCV EIA-nonreactive, HCV NAT-reactive samples. RESULTS: Three HCV NAT yield cases and no HIV-1 yield cases were detected. The yield rate for HCV NAT was 3.4 per 10(5) (95 percent confidence interval [CI], 0.7-9.8). The estimated incidence rate for HCV is 24.2 per 100,000 person-years (95% CI, 3.4-88.0). If minipool NAT is added to routine donor screening, the residual risk for HCV is estimated to be reduced to 1 in 20.4x10(4) (95% CI, 1 in 5.2x10(4)-1 in 165.5x10(4)). CONCLUSION: The residual risk for transfusion-transmitted HCV infection is still relatively high in China. Incorporating NAT technology into blood donor screening would be estimated to reduce the residual risk of HCV infections eightfold over current EIA screening.

Evaluation of a multiplex human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus nucleic acid testing assay to detect viremic blood donors in northern Thailand.

BACKGROUND: Screening of blood donors with nucleic acid testing (NAT) for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) has been implemented recently in the United States. There are limited data, however, on the additional NAT yield of donors in developing countries in Asia where the prevalence of infection is higher. In addition, data on hepatitis B virus (HBV) NAT in high prevalence areas are minimal. STUDY DESIGN AND METHODS: A total of 5083 whole-blood donors at the Chiang Mai University Hospital, Thailand, blood bank were evaluated with a commercially available NAT assay (Procleix Ultrio, Gen-Probe, Inc.) to screen individual donations. RESULTS: No NAT yield cases were found for HIV-1 or HCV. There were 17 samples with discrepant HBV DNA NAT and hepatitis B surface antigen (HBsAg) tests, however. Seven of these were HBV DNA NAT-positive, HBsAg-negative; of these 7, 1 was NAT-positive at baseline, but negative on follow-up, and considered a false-positive, 1 had an acute infection, and 5 had chronic prevalent HBV infections, for a NAT yield of 6 in 4798 HBsAg negative donors (1:800). In addition there were 10 NAT-negative, HBsAg-positive serum samples. All were anti-hepatitis B core antigen immunoglobulin G-positive; on testing with a more sensitive NAT target capture assay, 5 were positive (1.8-20.6 IU/mL) and 5 were negative. CONCLUSION: Multiplex NAT screening of individual-donor serum samples in Northern Thailand detected approximately 1 per 800 HBV NAT-positive, HBsAg-negative donors. The especially high prevalence of HBV infection in Thailand and other Asian countries suggests that HBV NAT screening of donors will be more cost-effective than in other areas.

Performance of human immunodeficiency virus type 1 gp41 assays for detecting enfuvirtide (T-20) resistance mutations.

The performance of a gp41 assay, created using reagents designed for use with the OpenGene DNA Sequencing System, was evaluated using a panel of plasma samples obtained from enfuvirtide-naive and -experienced human immunodeficiency virus type 1-infected subjects. Resulting sequence data were highly accurate compared to a "home brew" assay and clonal sequence analysis.

Blinded, multicenter comparison of methods to detect a drug-resistant mutant of human immunodeficiency virus type 1 at low frequency.

We determined the abilities of 10 technologies to detect and quantify a common drug-resistant mutant of human immunodeficiency virus type 1 (lysine to asparagine at codon 103 of the reverse transcriptase) using a blinded test panel containing mutant-wild-type mixtures ranging from 0.01% to 100% mutant. Two technologies, allele-specific reverse transcriptase PCR and a Ty1HRT yeast system, could quantify the mutant down to 0.1 to 0.4%. These technologies should help define the impact of low-frequency drug-resistant mutants on response to antiretroviral therapy.

Comparison of the COBAS TAQMAN HIV-1 HPS with VERSANT HIV-1 RNA 3.0 assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes.

Quantitation of HIV-1 RNA levels in plasma has an undisputed prognostic value and is extremely important for evaluating response to antiretroviral therapy. The purpose of this study was to evaluate the performance of the real-time PCR COBAS TaqMan 48 analyser, comparing it to the existing VERSANT 3.0 (bDNA) for HIV-1 RNA quantitation in plasma of individuals infected with different HIV-1 subtypes (104 blood samples). A positive linear correlation between the two tests (r2 = 0.88) was found. Quantitation by the COBAS TaqMan assay was approximately 0.32log10 higher than by bDNA. The relationship between the two assays was similar within all subtypes with a Deming regression of <1 and <0 for the Bland-Altman plots. Overall, no significant differences were found in plasma viral load quantitation in different HIV-1 subtypes between both assays; therefore these assays are suitable for viral load quantitation of highly genetically diverse HIV-1 plasma samples.

Genotypic and phenotypic analyses of human immunodeficiency virus type 1 in antiretroviral drug-naive Nigerian patients.

We analyzed the subtypes and genotypic and phenotypic drug susceptibility profiles of 18 HIV-1 isolates from treatment-naive patients in Nigeria. A modified gp41-based heteroduplex mobility assay was used to determine the clade designation based on the envelope gene. The protease and most of the reverse transcriptase regions were cloned into a retroviral expression vector and sequenced. Samples were also analyzed phenotypically using a rapid phenotypic assay (PhenoSense HIV, ViroLogic, Inc.). According to the modified gp41-based heteroduplex mobility assay, the patients were infected with either clade G (17 specimens) or clade A (one specimen) isolates. From phylogenetic analyses of 1212 nucleotides of the polymerase gene, 14 of the 18 isolates were strongly grouped with subtype G reference strains. The remaining four isolates were grouped with the CRF_02_AG clade. Within the protease region, all 18 isolates had mutations/polymorphic substitutions at six locations compared to the HIV-1 NL4-3 reference sequence, two of which have been associated with resistance to protease inhibitors (K20I and M36I). At least half of the isolates had mutations/polymorphic substitutions at an additional five positions in the protease region. Within the reverse transcriptase (RT) region, all 18 isolates showed an E291D mutation/polymorphic substitution. Mutations/polymorphic substitutions were also found in at least half of the isolates at 21 positions. The phenotypic profiles of the viruses correlated well with the observed genotypes. Two isolates showed slightly reduced susceptibility to one or two of the five PIs assessed (ritonavir and ritonavir/nelfinavir) and all 18 viruses were susceptible to all NRTIs and NNRTIs analyzed.

TaqMan RT-PCR and VERSANT HIV-1 RNA 3.0 (bDNA) assay Quantification of HIV-1 RNA viral load in breast milk.

BACKGROUND: Transmission of HIV via breast milk is a primary cause of pediatric HIV infection in developing countries. Reliable methods to detect breast milk viral load are important. OBJECTIVE: To correlate the ability of the VERSANT HIV 3.0 (bDNA) assay to real-time (RT) TaqMan PCR in quantifying breast milk HIV-1 RNA. STUDY DESIGN: Forty-six breast milk samples that had been spiked with cell-free HIV-1 and eight samples spiked with cell-associated HIV-1 were assayed for HIV-1 RNA by both VERSANT HIV 3.0 and TaqMan RNA assays. RESULTS: Only assays on the cell-free samples were statistically compared. Both a Deming regression slope and a Bland-Altman slope indicated a linear relationship between the two assays. TaqMan quantitations were on average 2.6 times higher than those of HIV 3.0. A linear relationship was observed between serial dilutions of spiked cell-free HIV-1 and both the VERSANT HIV 3.0 and the TaqMan RNA assays. CONCLUSION: The two methods correlated well although the VERSANT HIV 3.0 research protocol quantified HIV-1 RNA slightly lower than TaqMan.

Comprehensive comparison of the VERSANT HIV-1 RNA 3.0 (bDNA) and COBAS AMPLICOR HIV-1 MONITOR 1.5 assays on 1,000 clinical specimens.

BACKGROUND: Plasma human immunodeficiency virus type 1 (HIV-1) RNA level is an important parameter for patient management, yet viral load assays from different manufacturers are not standardized. OBJECTIVES AND STUDY DESIGN: In this study, we evaluated the concordance between test results obtained for 1,000 plasma specimens collected from HIV-1-infected individuals measured with the VERSANT HIV-1 RNA 3.0 assay (bDNA) and the COBAS AMPLICOR HIV-1 MONITOR 1.5 test (PCR). We compared viral load values obtained by each of these assays throughout their dynamic ranges, with particular focus on samples with low viral load (i.e. 50-250 copies/mL), and calculated the estimated distribution of distinct plasma viral load levels for the entire study population modeled from the data observed in the study. RESULTS: We found that these two assays show excellent agreement, with a correlation (R(2)) of 0.957 and a slope of 1.004. The mean difference in viral load values between the two assays was less than 0.10-log(10) throughout the dynamic range and 98.2% of all samples had bDNA and PCR results within 0.5-log(10) of each other, a difference that is within the range considered to be a minimal change in plasma viremia. Moreover, the two assays show very similar results across all assay ranges tested. The estimated prevalence of samples with results <50 copies/mL, 50-250 copies/mL, and 250-500,000 copies/mL were 41.6%, 7.7%, and 49.7%, respectively, by the bDNA assay, and 42.4%, 6.9%, and 50.7%, respectively, by the PCR assay. CONCLUSION: Based on our findings from 1,000 clinical specimens, we do not see the need to re-establish a baseline value or apply a conversion factor when switching from one assay to the other. Since the majority of our patient population likely is infected with subtype B virus, it is unclear if our findings will apply to other patient populations with a greater incidence of infection with non-B subtypes.

Prevalence of HCV coinfection in HIV-infected individuals in Nigeria and characterization of HCV genotypes.

BACKGROUND: Coinfection with hepatitis C virus (HCV) in individuals infected with HIV is associated with a higher incidence of liver injury, hepatic decompensation, and decreased survival time than that seen in an HIV-monoinfected population. While prevalence studies on HIV/HCV coinfection have been performed in the U.S. and in some European countries, little is known about coinfection rates in Africa. DESIGN: Retrospectively collected specimens from 146 confirmed HIV-positive individuals in Nigeria who had access to antiretroviral therapy (ART) were tested for HCV RNA, using the VERSANT HCV RNA qualitative assay (TMA), and, if HCV RNA-positive, for HCV genotype using the VERSANT HCV genotype assay (LiPA). RESULTS: Twelve out of the 146 individuals tested (8.2%) were HCV positive. Nine of the 12 HCV-positive individuals were infected with HCV genotype 1 (five 1a, three 1b, one non-subtypable) and three were infected with HCV genotype 2 (all non-subtypable). Coinfected individuals were more likely to be male, older, and have lower CD4+ cell counts than HIV-monoinfected individuals, although none of the differences reached statistical significance. CONCLUSION: The results highlight the potential public health impact of HCV infection in Nigeria, where anti-HCV testing is generally not performed in HIV-infected populations or in most blood transfusion centers.

Virologic therapy response significantly correlates with the number of active drugs as evaluated using a LiPA HIV-1 resistance scoring system.

BACKGROUND: Resistance testing is increasingly accepted as a tool in guiding the selection of human immunodeficiency virus type 1 (HIV-1) antiretroviral therapy in HIV-1 infected individuals who fail their current regimen. OBJECTIVES: To descriptively compare the correlation between virologic treatment response and results using three genotypic HIV-1 drug resistance interpretation systems: the VERSANT HIV-1 Resistance Assay (LiPA) system and two sequence-based interpretation systems. STUDY DESIGN: Specimens from 213 HIV-1-infected subjects, either starting (n=104) or switching to (n=109) a regimen of three or four antiretroviral drugs, were collected retrospectively at baseline and after 3 months of uninterrupted therapy. The correlation between viral load change and the number of predicted active drugs in the treatment regimen was assessed. An interpretation algorithm was recently developed to process VERSANT HIV-1 Resistance Assay (LiPA) data. The number of active drugs predicted using this algorithm was rank correlated with the viral load change over a 3-month treatment period. For comparison, a similar calculation was made using two sequence-based algorithms (REGA version 5.5 and VGI GuideLines Rules 4.0), both applied on the same sequences. RESULTS: Statistically significant (p<0.05) correlation coefficients for each of the three HIV-1 drug resistance interpretation systems were observed in the treatment-experienced subjects on a 3-drug regimen (-0.39, -0.38, and -0.42, respectively) as well as on a 4-drug regimen (-0.33, -0.31, and -0.37, respectively). However, no significant correlation was observed in treatment-naive subjects, probably due to the very low frequency of drug resistance in these subjects. CONCLUSION: All three genotypic drug resistance interpretation systems (LiPA version 1, REGA version 5.5, and VGI GuideLines Rules 4.0) were statistically significantly correlated with virologic therapy response as measured by viral load testing.

Multicenter evaluation of the VERSANT hepatitis B virus DNA 3.0 assay.

The VERSANT hepatitis B virus (HBV) 3.0 Assay (branched DNA [bDNA]) (referred to herein as VERSANT 3.0) was evaluated at four external sites for analytical sensitivity, specificity, reproducibility, linearity of quantification, and limits of detection. In addition, each of the test evaluation sites provided HBV DNA-positive clinical samples that were previously analyzed by one of three commercially available HBV DNA quantitative tests: Digene Hybrid Capture II HBV DNA Test (Digene); VERSANT HBV DNA 1.0 Assay (bDNA) (VERSANT 1.0); and COBAS AMPLICOR HBV Monitor Test (COBAS AMPLICOR). These samples were reexamined using VERSANT 3.0. The results from these studies showed that VERSANT 3.0 has high specificity (99.3%), excellent reproducibility (between-run coefficient of variation [CV] = 1.6 to 9.4%; within-run CV = 6.5 to 20.7%), and a broad linear range of quantification (2.0 x 10(3) to 1.0 x 10(8) HBV DNA copies/ml) that facilitate the monitoring of HBV DNA levels at diagnosis and throughout the course of treatment. In general, correlation was very good between results obtained from clinical samples analyzed by VERSANT 3.0 and the comparative HBV DNA quantitative assays (VERSANT 1.0, R(2) = 0.900; Digene, R(2) = 0.985; COBAS AMPLICOR, R(2) = 0.771). The greatest differences in comparative quantitation occurred at HBV DNA levels approaching the limits of the dynamic ranges for the comparative assays. The performance characteristics of the new VERSANT 3.0 assay demonstrated that it provides a reliable and robust method for routinely monitoring serum HBV DNA levels in assessing disease activity and determining response to antiviral treatment.

Successful HCV genotyping of previously failed and low viral load specimens using an HCV RNA qualitative assay based on transcription-mediated amplification in conjunction with the line probe assay.

BACKGROUND: Hepatitis C virus (HCV) genotyping is a critical part of the diagnostic work-up for chronic hepatitis C. The VERSANT HCV line probe assay (LiPA) marketed by Bayer Corporation requires PCR-derived amplicons for genotyping usually obtained from commercial assays, including Amplicor HCV 2.0 (Amplicor 2.0), Amplicor HCV Monitor 2.0, or SuperQuant. Occasionally, PCR-based methods in conjunction with LiPA fail to give a genotyping result. Although most genotyping failures occur among low viral load specimens, some occur in specimens with relatively high viral loads. The Bayer HCV RNA Qualitative assay (HCV TMA), with a limit of detection of approximately 5-10 IU/ml, is more sensitive than other commercial assays. OBJECTIVES: An HCV genotyping protocol using HCV TMA linked with LiPA (TMA-LiPA) was developed and tested for ability to genotype samples that had previously failed genotyping by PCR-based methods in conjunction with LiPA. STUDY DESIGN: Clinical specimens were obtained from eight independent laboratories in Canada and the US and tested with TMA-LiPA at the Bayer Reference Testing Laboratory. Specimens included those that failed to produce a genotype result when a PCR-based assay was used in conjunction with LiPA and specimens for which genotyping was not attempted because the viral load was below the validated cut-off determined in the laboratory of origin. RESULTS AND CONCLUSIONS: TMA-LiPA successfully genotyped 68 of 75 (90.7%) specimens that had failed genotyping by PCR-based methods used in conjunction with LiPA and 36 of 40 (90.0%) specimens that were rejected for genotyping due to low viral load. Moreover, TMA-LiPA assigned subtype for 79 of 107 (73.8%) specimens. Our TMA-LiPA results reflected the distribution of HCV genotypes found in North America, and were 100% concordant with those of Amplicor 2.0 in conjunction with LiPA for control specimens genotyped by both assays. TMA-LiPA may prove useful both in optimizing LiPA performance and genotyping patient specimens.

Nelfinavir-resistant, amprenavir-hypersusceptible strains of human immunodeficiency virus type 1 carrying an N88S mutation in protease have reduced infectivity, reduced replication capacity, and reduced fitness and process the Gag polyprotein precursor aberrantly.

The evolution of human immunodeficiency virus type 1 (HIV-1) strains with reduced susceptibility to protease inhibitors (PIs) is a major cause of PI treatment failure. A subset of subjects failing a therapy regimen containing the PI nelfinavir developed mutations at position 88 in the protease region. The N88S mutation occurring in some of these subjects induces amprenavir hypersusceptibility and a reduction of fitness and replication capacity. Here we demonstrate that substitutions L63P and V77I in protease, in combination, partially compensate for the loss of fitness, loss of replication capacity, loss of specific infectivity, and aberrant Gag processing induced by the N88S mutation. In addition, these mutations partially ablate amprenavir hypersusceptibility. Addition of mutation M46L to a strain harboring mutations L63P, V77I, and N88S resulted in a reduction of fitness and infectivity without changing Gag-processing efficiency, while amprenavir hypersusceptibility was further diminished. The ratio of reverse transcriptase activity to p24 protein was reduced in this strain compared to that in the other variants, suggesting that the M46L effect on fitness occurred through a mechanism different from a Gag-processing defect. We utilized these mutant strains to undertake a systematic comparison of indirect, single, cycle-based measures of fitness with direct, replication-based fitness assays and demonstrated that both yield consistent results. However, we observed that the magnitude of the fitness loss for one of the mutants varied depending on the assay used.

Qualitative detection of hepatitis C virus RNA: comparison of analytical sensitivity, clinical performance, and workflow of the Cobas Amplicor HCV test version 2.0 and the HCV RNA transcription-mediated amplification qualitative assay.

The qualitative Cobas Amplicor hepatitis C virus (HCV) version 2.0 assay (HCV PCR) and the Bayer Reference Testing Laboratory HCV RNA transcription-mediated amplification assay (HCV TMA) were compared for analytical sensitivity, clinical performance, and workflow. Limits of detection were determined by testing dilutions of the World Health Organization HCV standard in replicates of 15 at concentrations of from 1.0 to 70 IU/ml. The limit of detection of the HCV PCR assay was calculated to be 45 IU/ml on initial testing and 32 IU/ml after resolution of gray zone results. The calculated limit of detection for HCV TMA was 6 IU/ml. To compare clinical performance, 300 specimens, grouped as follows, were evaluated: 112 samples that were indeterminate in an anti-HCV enzyme immunoassay (EIA) and for which HCV RNA was not detected by HCV PCR; 79 samples that were EIA positive and for which HCV RNA was not detected by HCV PCR; and 105 samples that were both EIA and HCV PCR positive. For these groups, interassay concordance ranged from 96.2% to 100%. In addition, three HCV PCR gray zone specimens and one neonatal specimen were also evaluated. A 64-sample run (full run, 91 specimens) required 5 h for testing by HCV TMA, whereas almost 8 h were required to test a full run of 22 specimens by HCV PCR. HCV TMA demonstrated excellent concordance with HCV PCR when clinical samples were tested. However, HCV TMA was more sensitive than HCV PCR, required less time for test result completion, and had a greater throughput.

Altered substrate specificity of drug-resistant human immunodeficiency virus type 1 protease.

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV.