Protease Sequences Research Articles

Diversity of Mexican HIV-1 Protease Sequences.

Protease is one of three enzymes encoded within HIV's pol gene and a retroviral aspartyl-protease which unlike other aspartyl-proteases form a 22kDa homodimer to exert its activity. Protease is responsible for the cleavage of viral Gag-Pol polypeptide into mature viral proteins and a target of current anti-retroviral therapy (ART). Saquinavir was the first FDA-approved protease inhibitor. This image maps Shannon entropy levels of variable sites as derived from the study of 777 Mexican protease nucleotide sequences. Shannon entropy was calculated employing amino acid equivalents representing increases in entropy with regards to HXB2 reference. Entropy levels were arbitrarily classified as high (> 0.6), mid (between 0.6 and 0.2) and low (below 0.2). Protease sites having high and mid entropy levels were mapped into an X-ray diffraction crystal of HIV-1 protease dimer coupled to the anti-retroviral drug saquinavir. All sites exhibiting high and mid entropy affected the surface of the protein and were confined to an area located between the elbow and the 60's loop. High levels of entropy were observed in residues surrounding the 10's loop, the elbow region, the 60's loop and in the first half of the C-terminal domain. Protease sequence diversity is mostly a consequence of ART-driven selection for mutations that lower affinity for a drug, enabling viral replication in spite of continued ART use. Genetic variation continues to represent a major challenge for HIV prevention and treatment.

Vacuolar targeting of aldehyde dehydrogenase 6 tagging with signal peptide of proteinase A.

Vacuoles are useful materials with antimicrobial and anticancerous properties. Vacuolar proteins can discompose macromolecules from the outside of yeast cells. The objective of this study was to determine the function of a protein transported into a vacuole. Specifically, cytosolic protein aldehyde dehydrogenase 6 (ALD6) was used for the delivery to the vacuole. To transport cytosolic protein to the vacuole in this study, a transfer vector including a signal peptide sequence isolated from vacuolar protein proteinase A was designed. A signal peptide is an amino acid sequence in front of the transported protein. Signal peptides have various delivery pathways according to the kind of signal sequence they contain. They play important roles in transporting proteins to organelles, in cellular mechanisms, and the transfer of protein outside and inside eukaryotes. Thus, we focused on the design of a transfer vector containing a signal peptide sequence isolated from the DNA sequence of proteinase A (PEP4). In addition, this study evaluated the expression level of cytosolic ALD6 after being transported into the yeast vacuole. Our results showed that the developed transfer vector was useful for delivering proteins to vacuole by using signal peptide sequence. Therefore, this transfer vector might be used as a tool to deliver target proteins to organelles of interest in eukaryotes.

Expanded Spectrum of Antiretroviral-Selected Mutations in Human Immunodeficiency Virus Type 2.

HIV-1 and HIV-2 differ in their antiretroviral (ARV) susceptibilities and drug resistance mutations (DRMs). We analyzed published HIV-2 pol sequences to identify HIV-2 treatment-selected mutations (TSMs). Mutation prevalences were determined by HIV-2 group and ARV status. Nonpolymorphic mutations were those in <1% of ARV-naive persons. TSMs were those associated with ARV therapy after multiple comparisons adjustment. We analyzed protease (PR) sequences from 483 PR inhibitor (PI)-naive and 232 PI-treated persons; RT sequences from 333 nucleoside RT inhibitor (NRTI)-naive and 252 NRTI-treated persons; and integrase (IN) sequences from 236 IN inhibitor (INSTI)-naive and 60 INSTI-treated persons. In PR, 12 nonpolymorphic TSMs occurred in ≥11 persons: V33I, K45R, V47A, I50V, I54M, T56V, V62A, A73G, I82F, I84V, F85L, L90M. In RT, 9 nonpolymorphic TSMs occurred in ≥10 persons: K40R, A62V, K70R, Y115F, Q151M, M184VI, S215Y. In IN, 11 nonpolymorphic TSMs occurred in ≥4 persons: Q91R, E92AQ, T97A, G140S, Y143G, Q148R, A153G, N155H, H156R, R231 5-amino acid insertions. Nine of 32 nonpolymorphic TSMs were previously unreported. This meta-analysis confirmed the ARV association of previously reported HIV-2 DRMs and identified novel TSMs. Genotypic and phenotypic studies of HIV-2 TSMs will improve approaches to predicting HIV-2 ARV susceptibility and treating HIV-2-infected persons.

High levels of pretreatment and acquired HIV drug resistance in Nicaragua: results from the first nationally representative survey, 2016.

IntroductionA nationally representative HIV drug resistance (HIVDR) survey in Nicaragua was conducted to estimate the prevalence of pretreatment HIVDR (PDR) among antiretroviral therapy (ART) initiators and acquired HIVDR among people living with HIV (PLHIV) who had received ART for 12 ± 3 months (ADR12) and ≥48 months (ADR48).MethodsA nationwide cross‐sectional survey with a two‐stage cluster sampling was conducted from March to November 2016. Nineteen of 45 total ART clinics representing >90% of the national cohort of adults on ART were included. ART initiators were defined as PLHIV initiating or reinitiating first‐line ART. HIVDR was assessed for protease, reverse transcriptase and integrase Sanger sequences using the Stanford HIVdb algorithm. Viral load (VL) suppression was defined as <1000 copies/mL. Results were weighted according to the survey design.Results and discussionA total of 638 participants were enrolled (PDR: 171; ADR12: 114; ADR48: 353). The proportion of ART initiators with prior exposure to antiretrovirals (ARVs) was 12.3% (95% CI: 5.8% to 24.3%). PDR prevalence to any drug was 23.4% (95% CI: 14.4% to 35.6%), and 19.3% (95% CI: 12.2% to 29.1%) to non‐nucleoside reverse transcriptase inhibitors (NNRTI). NNRTI PDR was higher in ART initiators with previous ARV exposure compared with those with no exposure (76.2% vs. 11.0%, p < 0.001). Protease inhibitors (PI) and integrase strand transfer inhibitors PDR was not observed. VL suppression rate was 77.8% (95% CI: 67.1% to 85.8%) in ADR12 and 70.3% (95% CI: 66.7% to 73.8%) in ADR48. ADR12 prevalence to any drug among PLHIV without VL suppression was 85.1% (95% CI: 66.1% to 94.4%), 82.4% to NNRTI and 70.2% to nucleoside reverse transcriptase inhibitors (NRTI). ADR48 prevalence to any drug among PLHIV without VL suppression was 75.5% (95% CI: 63.5% to 84.5 %), 70.7% to NNRTI, 59.4% to NRTI and 4.6% to PI.ConclusionsDespite implementation challenges yielding low‐precision HIVDR estimates, high rates of NNRTI PDR were observed in Nicaragua, suggesting consideration of non‐NNRTI‐based first‐line regimens for ART initiators. Strengthened HIVDR monitoring, systematic VL testing, and improved ART adherence support are also warranted.

Mexican HIV-1 Protease Sequence Diversity.

Protease is one of three enzymes encoded within HIV's pol gene, responsible for the cleavage of viral Gag-Pol polypeptide into mature viral proteins and a target of current anti-retroviral therapy. Protease diversity analysis in Latin America has been lacking in spite of extensive studies of protease-inhibitor resistance mutations. We studied the diversity of 777 Mexican protease sequences and found that all were subtype B except one (CRF02_AG). Phylogenetic analysis suggested the existence of six different clades with geospecific contributions. Thirty-three percent of sites were conserved, 25% had conservative substitutions, and 41% exhibited physicochemical changes. The most conserved regions surrounded the active site, most of the flap domain, and a region between the 60's loop and C-terminal triad. A single sequence exhibited an active site mutation (T26S). Variable sites were mapped to a crystallographic structure, providing further insight into the distribution and functional relevance of variable sites among Mexican isolates.

Accessory mutations balance the marginal stability of the HIV-1 protease in drug resistance.

The HIV-1 protease is a major target of inhibitor drugs in AIDS therapies. The therapies are impaired by mutations of the HIV-1 protease that can lead to resistance to protease inhibitors. These mutations are classified into major mutations, which usually occur first and clearly reduce the susceptibility to protease inhibitors, and minor, accessory mutations that occur later and individually do not substantially affect the susceptibility to inhibitors. Major mutations are predominantly located in the active site of the HIV-1 protease and can directly interfere with inhibitor binding. Minor mutations, in contrast, are typically located distal to the active site. A central question is how these distal mutations contribute to resistance development. In this article, we present a systematic computational investigation of stability changes caused by major and minor mutations of the HIV-1 protease. As most small single-domain proteins, the HIV-1 protease is only marginally stable. Mutations that destabilize the folded, active state of the protease therefore can shift the conformational equilibrium towards the unfolded, inactive state. We find that the most frequent major mutations destabilize the HIV-1 protease, whereas roughly half of the frequent minor mutations are stabilizing. An analysis of protease sequences from patients in treatment indicates that the stabilizing minor mutations are frequently correlated with destabilizing major mutations, and that highly resistant HIV-1 proteases exhibit significant fractions of stabilizing mutations. Our results thus indicate a central role of minor mutations in balancing the marginal stability of the protease against the destabilization induced by the most frequent major mutations.

Expression of Lon-like Protease Gene from Lactobacillus plantarum IIA-1A5 in Escherichia coli BL21(DE3)

Proteases are one of most important and abundant enzymes produced by the biotechnology industry, for scientific, physiological and industrial application and dominates of the whole enzyme market. Lactobacillus plantarum IIA-1A5 is an Indonesian lactic acid bacteria (LAB) isolated from beef Peranakan Ongole cattle. Preliminary analysis on its whole genome sequence indicated that this strain harbours some genes involved in protein degradation and might be promising to be further applied. This study aims to optimize the gene sequence of a lon-like protease of L. plantarum IIA-1A5 for heterologous expression system. The Lon-like gene expression system is made using genes that have been optimized first in silico. pET-28a(+), E. coli BL21(DE3), Nde1 and BamH1 were used in this study as a expression vector, a host and retriction enzyme, respectively. Molecular weight was validated using SDS-PAGE and expasy.org software. The results showed that optimization increased codon adaptation index value (CAI) and GC content to 0.97 and 56.57%, respectively, which were suitable for the E. coli expression system. The Lon-like IIA gene was successfully expressed in the cell cytoplasm by induction of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C. As many as 88% of Lon-like IIA codons were distributed in the 91-100 quality group. Lon-like IIA was successfully expressed in a host cell induced with 1 mM IPTG at 37oC . IPTG induction was performed at the 3rd hour of incubation with OD600 0.59. In addition, Lon-like IIA molecular weight was detected approximately 43 kDa.

Transmitted drug resistance mutations and trends of HIV-1 subtypes in treatment-naïve patients: A single-centre experience.

Transmitted drug resistance (TDR) and HIV-1 genetic diversity may affect treatment efficacy and clinical outcomes. Here we describe the circulating viral subtypes and estimate the prevalence of drug resistance among antiretroviral therapy (ART)-naïve patients attending Sapienza University Hospital (Rome, Italy) from 2006-2017. Genotypic resistance testing (GRT) was performed on 668 ART-naïve patients for integrase (n = 52), protease and reverse transcriptase (n = 668) sequences. Twenty-one different HIV-1 subtypes and circulating recombinant forms (CRFs) were identified. Subtype B was the most common (67.1%), followed by CRF02_AG (8.4%), and subtypes C and F (both 6.0%). A significantly increase in the proportion of non-B strains (P < 0.001) and the rate of non-Italian patients was observed over time. The overall prevalence of TDR was 9.4% (NRTI, 4.2%; NNRTI, 5.8%; and PI, 1.0%) and was higher in subtype B strains. Transmitted INSTI mutations (Q148H and G140S) responsible for high-level resistance to raltegravir and elvitegravir and intermediate resistance to dolutegravir and bictegravir were found, for the first time, in two individuals. Minor or accessory INSTI mutations were detected in 17.3% of patients. No significant decrease in the prevalence of TDR was documented over time. The significant increase in non-B subtypes suggests that the molecular epidemiology of HIV-1 is changing. Detection of a major INSTI mutation in two ART-naïve patients highlights the importance of performing GRT before commencing treatment. This finding and the lack of a significant reduction in TDRs underline the importance of continuous surveillance of resistance mutations.

Identification and enzymatic characterization of clip domain serine protease in the digestive fluid of the sea hare, Aplysia kurodai.

Clip domain serine proteases (CDSPs) participate in the extracellular signaling cascades of various biological processes such as innate immune responses in invertebrates. CDSP genes have been isolated from numerous invertebrates. Nevertheless, the enzymatic properties of mollusk CDSPs are poorly understood. In the present study, we demonstrated that the amino acid sequences of the trypsin-like serine protease purified from the digestive fluid of the sea hare, Aplysia kurodai resemble those of the unidentified CDSP-type protein (TPS3) of Aplysia californica predicted by genome analysis. The purified enzyme produced single 34 and 26.5 kDa bands on SDS-PAGE under non-reducing and reducing conditions, respectively. The 34-kDa band generated two amino-terminal sequences that were similar to the deduced sequences of the clip and catalytic domains of TPS3. The single amino-terminal sequence of the 26.5 kDa band showed a single sequence homologous to the catalytic domain. Thus, the purified enzyme consists of clip and catalytic domains bridged by disulfide linkage(s). The subsite specificity and inhibitor sensitivity of the purified enzyme were clearly distinct from those of horseshoe crab and silkworm CDSPs. A good substrate for the sea hare enzyme was pyroglutamyl-Arg-Thr-Lys-Arg-4-methyl-7-coumarylamide. The enzyme activity was strongly inhibited by aprotinin but not leupeptin. The physiological function of the enzyme in the digestive fluid remains to be determined.

A8 Epidemiological study of transmission clusters in a local HIV-1 cohort

Integration of molecular, clinical, and demographic data represents a powerful tool to understand the dynamics of local HIV-1 transmission chains (TCs). The aim of our study was the phylogenetic analysis of the TCs within a HIV-1 cohort and the description of the relevant patient data within a TC. We performed a phylogenetic analysis of 757 sequences from newly HIV-1 diagnosed patients in Málaga (Southern Spain) during the period 2004–15. We used partial pol gene sequences in a preliminary phylogenetic reconstruction using the Neighbour Joining method (MEGA v6.06 program). After eliminating branches with bootstrap values <80 per cent, we constructed a new phylogeny by Maximum likelihood method (FastTree program). We considered as TC any cluster with bootstrap values ≥ 90 per cent. Patient sequences within and outside TCs were compared. Resistance mutations in the protease (PR) and reverse transcriptase (RT) sequences were analyzed using the Stanford algorithm. Four hundred and fifty-one out of 757 patients (59.6%) were grouped into fifty-three TCs, seventeen of them with five or more subjects. The largest number of patients associated within a TC was ninety. Patients younger than 40 years [odds ratio (OR) 1.75, 95% confidence interval (95% CI) 1.2–2.4, P = 0.002], men who have sex with men (MSM) (OR 2.14, 95% CI 1.3–3.2, P < 0.0001), non-Spanish (OR 1.48, 95% CI 1.0–2.1, P = 0.038), with a non-B subtype HIV-1 (OR 3.12, 95% CI 2.0–4.8, P < 0.0001), and presenting primary resistance mutations (OR 14.1, 95% CI 3.1–62.6, P = 0.001) were more likely to be associated within a cluster. Ninety-four out of 118 patients (79.6%) with transmission resistance mutations were included in some TC. The most frequent mutations associated with clusters were T69D/N, L210W, and K219E/Q, for NRTIs, K103N, and G190A/S for NNRTIs, and the I54L/M and L90M mutations for PIs. The prevalence for resistance to NNRTIs in TCs was 13.7 per cent. There were two TCs of rarer non-B subtypes: CRF19_cpx, with twenty-one individuals, sixteen of them (76.2%) with mutation G190A; and CRF51_01B with thirty-nine patients, twenty of them with the K103N mutation. Approximately 60 per cent of newly HIV-1 diagnosed patients were included in a TC. Younger patients, MSM, non-Spanish, with non-B subtype HIV-1 and primary resistance mutations were more likely to belong to a cluster. NNRTI mutations were the most frequent ones among patients in TCs. We observed two TCs represented by infrequent non-B subtypes in our area—CRF19_cpx and CRF51_01B—both of which were associated to the transmission of primary resistance.

The Investigation of Drug Resistance Substitutions in NS3 Protease Sequence of Hepatitis C Virus from Non-Responder Patients.

Background:Even with the fantastic successes of direct-acting antivirals (DAA) in the treatment of Hepatitis C Virus (HCV) infection, natural drug resistance remains a challenging obstacle for their impacts. The data regarding protease inhibitors (PIs) resistance in Iran population are limited. The aim of this study was to investigate the variations in NS3 protease of HCV from non-responder patients.Methods:In this cross-sectional study, 14 HCV infected patients with genotype 1(N=5) and 3(N=9) who have not responded to Interferon-related regime were enrolled from Liver Clinic, Shiraz. The NS3 protease region was amplified by Nested-PCR followed by product gel extraction. Besides, some amplified protease regions were cloned into a cloning vector to improve the sensitivity of mutation detection. Both crude and cloned sequences were then introduced into sequencing. The obtained sequences were compared with the NS3 reference sequences and analyzed by Geno2pheno available software to find possible substitutions. In the end, the phylogenetic tree was constructed. Results:Among variations responsible for PIs resistance, only one out of 14 (7%) sample who was infected with genotype 1a, harbored R117C+N174S double mutation, which causes reduced susceptibility to Telaprevir. Any another resistance mutation was not found among the studied population. The most frequent substitutions were determined as I52M(N=9), S102A(N=9), S166A(8) and V170I(8) for genotype 3a, and F147S/A(4) for genotype 1. However, some uncharacterized substitutions on scored position, including I132L(N=1), I170V(N=3) and N174S(N=2) were also determined among sequences. Phylogenetic analysis demonstrated that the protease region has enough power to correctly classify enrolled samples into relevant clusters on the tree. There were 2, 3 and 9 cases of sub-genotypes 1a, 1b, and 3a, respectively.Conclusion:A low frequency of PIs resistance mutations in our HCV infected population is a hopeful point of starting these drugs in HCV infected patients.

Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, includingneo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

Bio-enzymes for inhibition and elimination of Escherichia coli O157:H7 biofilm and their synergistic effect with sodium hypochlorite

Escherichia coli O157:H7 is one of the most important pathogens worldwide. In this study, three different kinds of enzymes, DNase I, proteinase K and cellulase were evaluated for inhibitory or degrading activity against E. coli O157:H7 biofilm by targeting extracellular DNA, proteins, and cellulose, respectively. The cell number of biofilms formed under proteinase K resulted in a 2.43 log CFU/cm2 reduction with an additional synergistic 3.72 log CFU/cm2 reduction after NaClO post-treatment, while no significant reduction occurred with NaClO treatment alone. It suggests that protein degradation could be a good way to control the biofilm effectively. In preformed biofilms, all enzymes showed a significant reduction of 16.4–36.7% in biofilm matrix in 10-fold diluted media (p < 0.05). The sequential treatment with proteinase K, cellulase, and NaClO showed a significantly higher synergistic inactivation of 2.83 log CFU/cm2 compared to 1.58 log CFU/cm2 in the sequence of cellulase, proteinase K, and NaClO (p < 0.05). It suggests that the sequence of multiple enzymes can make a significant difference in the susceptibility of biofilms to NaClO. This study indicates that the combination of extracellular polymeric substance-degrading enzymes with NaClO could be useful for the efficient control of E. coli O157:H7 biofilms.

PREVALENCE AND STRUCTURE OF HIV-1 DRUG RESISTANCE AMONG TREATMENT NAÏVE PATIENTS SINCE THE INTRODUCTION OF ANTIRETROVIRAL THERAPY IN THE RUSSIAN FEDERATION

Aim:to analyze the prevalence, structure of drug resistance and drug resistance mutations in the protease and reverse transcriptase genes of HIV-1 among treatment naïve patients.Materials and methods.We analyzed protease and reverse transcriptase sequences from 1560 treatment naïve HIV-infected patients from all Federal Districts of the Russian Federation with the first positive immune blot during 1998–2017. Sequences were analyzed for the presence of drug resistance mutations and predicted drug resistance to antiretroviral drugs using two algorithms — Stanford HIVDR Database (HIVdb) and the 2009 SDRM list (CPR).Results.The prevalence of drug resistance mutations was 11,1%. More often the prevalence of drug resistance was found for non-nucleoside reverse transcriptase inhibitor drugs (rilpivirine, nevirapine, efavirenz). The prevalence of transmitted drug resistance associated with mutations from the SDRM list was 5,3%, which is classified by the WHO as a moderate level. However, it should be noted that since the large-scale use of antiretroviral drugs in the Russian Federation, there has been a trend towards a gradual increase in the level of the transmitted drug resistance, and in 2016 it has already reached 6,1%.Conclusion.The results demonstrate the need for regular surveillance of the prevalence of HIV drug resistance to antiretroviral drugs among treatment naïve patients in the Russian Federation.

New Promising Targets for Synthetic Omptin-Based Peptide Vaccine against Gram-Negative Pathogens.

Omptins represent a family of proteases commonly found in various Gram-negative pathogens. These proteins play an important role in host–pathogen interaction and have been recognized as key virulence factors, highlighting the possibility of developing an omptin-based broad-spectrum vaccine. The prototypical omptin, His-tagged recombinant Pla, was used as a model target antigen. In total, 46 linear and 24 conformational epitopes for the omptin family were predicted by the use of ElliPro service. Among these we selected highly conserved, antigenic, non-allergenic, and immunogenic B-cell epitopes. Five epitopes (2, 6, 8, 10, and 11 corresponding to Pla regions 52–60, 146–150, 231–234, 286–295, and 306–311, respectively) could be the first choice for the development of the new generation of target-peptide-based vaccine against plague. The partial residues of omptin epitopes 6, 8, and 10 (regions 136–145, 227–230, and 274–285) could be promising targets for the multi-pathogen vaccine against a group of enterobacterial infections. The comparative analysis and 3D modeling of amino acid sequences of several omptin family proteases, such as Pla (Yersinia pestis), PgtE (Salmonella enterica), SopA (Shigella flexneri), OmpT, and OmpP (Escherichia coli), confirmed their high cross-homology with respect to the identified epitope clusters and possible involvement of individual epitopes in host–pathogen interaction.

Validation of publicly-available software used in analyzing NGS data for HIV-1 drug resistance mutations and transmission networks in a Washington, DC, Cohort.

The DC Cohort is an ongoing longitudinal observational study of persons living with HIV. To better understand HIV-1 drug resistance and potential transmission clusters among these participants, we performed targeted, paired-end next-generation sequencing (NGS) of protease, reverse transcriptase and integrase amplicons. We elected to use free, publicly-available software (HyDRA Web, Stanford HIVdb and HIV-TRACE) for data analyses so that laboratory personnel without extensive bioinformatics expertise could use it; making the approach accessible and affordable for labs worldwide. With more laboratories transitioning away from Sanger-based chemistries to NGS platforms, lower frequency drug resistance mutations (DRMs) can be detected, yet their clinical relevance is uncertain. We looked at the impact choice in cutoff percentage had on number of DRMs detected and found an inverse correlation between the two. Longitudinal studies will be needed to determine whether low frequency DRMs are an early indicator of emerging resistance. We successfully validated this pipeline against a commercial pipeline, and another free, publicly-available pipeline. RT DRM results from HyDRA Web were compared to both SmartGene and PASeq Web; using the Mantel test, R2 values were 0.9332 (p<0.0001) and 0.9097 (p<0.0001), respectively. PR and IN DRM results from HyDRA Web were then compared with PASeq Web only; using the Mantel test, R2 values were 0.9993 (p<0.0001) and 0.9765 (p<0.0001), respectively. Drug resistance was highest for the NRTI drug class and lowest for the PI drug class in this cohort. RT DRM interpretation reports from this pipeline were also highly correlative compared to SmartGene pipeline; using the Spearman’s Correlation, rs value was 0.97757 (p<0.0001). HIV-TRACE was used to identify potential transmission clusters to better understand potential linkages among an urban cohort of persons living with HIV; more individuals were male, of black race, with an HIV risk factor of either MSM or High-risk Heterosexual. Common DRMs existed among individuals within a cluster. In summary, we validated a comprehensive, easy-to-use and affordable NGS approach for tracking HIV-1 drug resistance and identifying potential transmission clusters within the community.

Low Frequency of Protease Inhibitor Resistance Mutations and Insertions in HIV-1 Subtype C Protease Inhibitor-Naïve Sequences.

Human immunodeficiency virus-1 (HIV-1) protease sequences from 2,225 protease inhibitor (PI)-naïve HIV-1 subtype C-infected individuals collected over a 14-year period were analyzed for polymorphisms. Over 50% of sequences differed from an HIV-1 subtype B consensus sequence at 8 of the 99 amino acids at residues 12, 15, 19, 36, 41, 69, 89, and 93, but not in the functionally important regions. The frequency of primary resistance and accessory mutations occurred in <1% of the sequences. Of note, 11 sequences (0.5%) harbored amino acid insertions between residues 36 and 39, located in the elbow of the flap region. The insertions were found throughout the 13-year period. Occurrence of insertions in subtype C viruses is rare and viruses remain sensitive to currently used PIs (lopinavir/r, atazanavir/r, and darunavir/r). However, ongoing characterization of isolates is required to identify changes that may impact PI treatment since PIs are part of standard SA regimens.

Prevalence of antiretroviral drug resistance in treatment-naive injecting drug users infected with HIV-1 in Guangzhou, 2008-2015

Objective: To understand the prevalence of drug resistance in treatment-naive injecting drug users (IDUs) infected with HIV-1 in Guangzhou. Methods: HIV-1 RNA were extracted from the serum specimens of the newly confirmed HIV-1 positive IDUs living in Guangzhou, being infected through injecting drug use and receiving no antiretroviral therapy at the time of confirmation during 2008-2015. Full sequence of pol protease (PR) gene and partial sequence of reverse transcriptase (RT) gene were amplified by nested reverse transcription polymerase chain reaction (nested-PCR) and sequenced. After that, data were submitted to the HIV resistance database of Stanford University for drug resistance analysis. Results: Among the 518 HIV-1 infected IDUs, HIV-1pol gene segments were successfully obtained from the serum samples of 407 HIV-1 infected IDUs (78.57%) aged 18-64 (37.44±8.14) years. Among them, males accounted for 89.68% (365/407), those of Han ethnic group accounted for 89.93% (366/407), the unmarried accounted for 55.28% (225/407), and those with education level of junior high school or below accounted for 83.78% (341/407). The distribution of subtypes was predominated by CRF07_BC (47.18%, 192/407), followed by CRF01_AE (23.83%, 97/407), CRF08_BC (22.85%, 93/407), and other subtypes (6.14%, 25/407). The overall prevalence of drug resistance was 3.44% (14/407). The prevalence of drug resistance to protease inhibitors, nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors were 1.47%(6/407), 0.25% (1/407) and 1.72% (7/407) respectively. The mutation rate was 12.29% (50/407). No major drug resistance mutation was detected in protease and nucleoside reverse transcriptase regions. Higher rate of V179E mutation in the non-nucleoside reverse transcriptase region was detected in other subtypes and subtype CRF07_BC. Mutation seemed to have occurred in all 8 cases of subtype CRF55_01B in other subtypes. The highest mutation rate of E138A was detected in subtype CRF08_BC (3.23%). Two cases were resistant to all four drugs of NNRTIs. Conclusions: The prevalence of drug resistance in treatment-naive HIV-1 positive IDUs remained at a relatively low level during 2008-2015, in Guangzhou. Most infections were sensitive to existing antiviral drugs. However, drug resistance surveillance in IDUs infected with HIV should be strengthened to prevent the prevalence of multi-drug resistance and cross drug resistance.

Sexual intermingling of Arab and Jewish MSM in Israel: results of a molecular epidemiology study.

MSM comprise ∼30% of new HIV infections in Israel, a country with mixed Jewish and Arab populations. We molecularly characterized HIV-1 in the Arab and Jewish MSM (AMSM, JMSM) populations to reveal possible interethnical connections. Cross-sectional study. All Israeli-born, HIV-1-infected MSM diagnosed between 2005 and 2016 (n = 1143) were cross-matched with the National Civil Registry to identify religion (Jews/Muslims/Christians). Transmitted drug-resistance mutations (TDRM) and HIV-1 subtypes were determined on the first partial protease and reverse transcriptase sequences from treatment-naive patients and phylogenetic trees were constructed. Among MSM, 6.4% (73/1143) were Arabs and 93.6% (1070/1143) were Jews. Interestingly, a higher proportion of Arabs was identified among non-MSM (19%, 46/247 versus 6.4%, 73/1143, P < 0.01). Subtype analysis of 62 HIV-1 AMSM and 440 randomly selected HIV-1 JMSM sequences revealed 80.6, 8.1, 4.8 and 6.5% of AMSM and 82.3, 9.5, 4.1 and 4.1% of JMSM had B, A, C and non-A/B/C, respectively. Overall, 13.1% (66/502) had TDRM; reverse transcriptase-K103N/S, M184 V, T215S and protease-L90M were the most common. TDRM prevalence was not significantly higher in JMSM compared to AMSM (P = 0.1) and no temporal changes were observed in their frequency. Phylogenetic analysis demonstrated AMSM and JMSM clusters including L90M, K103N/S or T215S TDRM. Intermingling of AMSM and JMSM HIV-1 in clusters of HIV-1 sequences suggest interethnical sexual contacts among these MSM. Interventions aiming to prevent HIV-transmission in MSM should similarly address both populations groups. The high TDRM frequency requires continuation of resistance testing.

HIV Drug Resistance Mutations in Patients with HIV and HIV-TB Coinfection After Failure of First-Line Therapy: A Prevalence Study in a Resource-Limited Setting

Introduction:The present study aimed to report the prevalent HIV-1 drug-resistant mutations in patients with HIV-1 alone and tuberculosis (TB) coinfection alone to improve our understanding of the mutation patterns and aid treatment decisions.Methods:Patients with HIV-1 and HIV-TB on treatment for more than 1 year with suspected failure were recruited. Sequencing of protease and two-thirds of the region of reverse transcriptase gene was done for drug-resistant mutations.Results:In the HIV-TB group (n = 25), 88%, 92%, and 12% had mutations to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs), respectively. In the HIV-alone group (n = 25), 84%, 100%, and 4% had mutations to NRTIs, NNRTIs, and PIs, respectively. M184V, M41L, D67N, G190A, A98G, and K103N were the most common mutations seen.Conclusion:There is a high prevalence of drug-resistant mutations in HIV and HIV-TB coinfected patients.