High conservation of herpes simplex virus UL5/UL52 helicase-primase complex in the era of new antiviral therapies.

The emergence of herpes simplex virus (HSV) resistance to current antiviral drugs, that all target the viral DNA polymerase, constitutes a major obstacle to antiviral treatment effectiveness of HSV infections, especially in immunocompromised patients. A novel and promising class of inhibitors of the HSV UL5/UL52 helicase-primase (HP) complex has been reported to hinder viral replication with a high potency. In this study, we describe the low natural polymorphism (interstrain identity >99.1% at both nucleotide and amino acid levels) of HSV HP complex subunits pUL5 and pUL52 among 64 HSV (32 HSV-1 and 32 HSV-2) clinical isolates, and we show that the HSV resistance profile to the first-line antiviral drug acyclovir (ACV) does not impact on the natural polymorphism of HSV HP complex. Genotypic tools and polymorphism data concerning HSV HP complex provided herein will be useful to detect drug resistance mutations in a relevant time frame when HP inhibitors (HPIs), i.e., amenamevir and pritelivir, will be available in medical practice.

Validation of the QIAsymphony RGQ system for DNA quantitation of different BK virus genotypes in whole blood samples.

Human polyomavirus BK (BKV) is increasingly recognized as an opportunistic pathogen in transplant recipients. The aim of this work was to evaluate the artus(®) BK Virus QS-RGQ assay on the QIAsymphony RGQ system in whole blood (WB) samples (tests performed in an off-label capacity) according to different BKV genotypes by comparison with a laboratory-developed assay. BKV loads were measured in 111 WB samples and BKV genotype was determined by sequencing the full-length VP1 gene. The artus(®) assay exhibited a limit of detection of 77copies/mL, a linearity range from 3.0 to 6.0log10copies/mL, intra-assay and inter-assay coefficients of variation ranging from 0.65% to 5.18%. Regarding BKV quantitation, artus(®) and laboratory-developed assays were highly correlated (Spearman correlation coefficient Rho=0.79; P<0.0001) with an excellent overall agreement (96.4%) and no significant quantitative difference according to Bland-Altman analysis (mean difference: -0.34log10copies/mL). The results did not show any influence of BKV genotype on BKV quantitation by the artus(®) assay, except a potential underquantitation of BKV subtype Ia which deserves further confirmation. In conclusion, the QIAsymphony RGQ system appears to be appropriate for the quantitation of BKV load in WB samples.

Routine use of duplex real-time PCR assays including a commercial internal control for molecular diagnosis of opportunistic DNA virus infections.

The aim of this work was to improve the validity of laboratory-developed real-time PCR protocols implemented in the laboratory for molecular diagnosis of opportunistic DNA virus infections using the Simplexa™ extraction and amplification control (SEAC) which allows the monitoring of the whole extraction and amplification process. Herpes simplex virus (HSV), varicella-zoster virus (VZV), human cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK virus (BKV), and adenovirus (AdV) genomes were investigated in 152 different clinical specimens. The use of the SEAC did not influence the results of the different virus-specific PCRs. The SEAC results showed high reproducibility with a mean Cp value of 31.08±1.44, and were not influenced by the virus-specific PCR performed or the type of clinical specimen tested. The SEAC in the DNA extracts showed high stability during storage at both +4°C and -20°C. These data allowed establishing a new procedure for the validation of viral PCR results. In conclusion, the SEAC provides a reliable option for improving the diagnosis of opportunistic viral infections by laboratory-developed real-time PCR assays in quality assurance programs.

Presence of HIV-1 R5 viruses in cerebrospinal fluid even in patients harboring R5X4/X4 viruses in plasma.

BACKGROUND: HIV-1 viruses have the ability to use CCR5 or CXCR4 coreceptors either solely (R5 or X4) or in combination (R5X4). The CCR5 antagonists block HIV entry into the cell and are specifically active against HIV-1 R5 strains. The objectives of this study were to investigate the predicted tropism of viruses present in paired cerebrospinal fluid (CSF) and plasma in a group of 22 HIV-1-infected patients with neurological disorders and to search for eventual discordance of predicted virus tropism between both compartments. METHODS: Paired CSF and plasma samples were selected from subjects harboring neurological disorders. V3 env was amplified and bulk sequenced, and HIV-1 coreceptor usage was determined from the V3 env region sequence by Geno2Pheno and position-specific scoring matrices (PSSM) algorithms. RESULTS: The majority of subjects (19 of 22) had concordant virus predicted tropism in both compartments. All patients having R5-tropic viruses in plasma had R5-tropic viruses in CSF (17 of 22). Patients having R5X4/X4-tropic viruses in plasma could have R5X4/X4-using (2 of 22) or R5-tropic viruses (3 of 22) in CSF. The case of R5-tropic viruses in plasma and R5X4/X4-tropic viruses in CSF was never observed. CONCLUSIONS: The majority of these patients have R5-using viruses in CSF that is mainly concordant with the predicted tropism in plasma. However, R5X4/X4 tropism in plasma does not necessarily mean the same predicted tropism in CSF compartment. Then, clinical therapeutic trials testing the clinical response to the CCR5 antagonists in patients with neurological disorders could be envisaged to analyze the effects of this therapeutic class in this setting.

Antiretroviral combinations implicated in emergence of the L74I and L74V resistance mutations in HIV-1-infected patients.

BACKGROUND: Very little is known about the alternative L74I mutation. This lack of knowledge has led to contradictory and confusing attitudes to L74I; although this mutation is not listed by the International AIDS Society - USA, it is increasingly included in several resistance algorithms. OBJECTIVE: To compare and clarify the role of each antiretroviral compound and the resistance background in the emergence of the L74I and L74V mutations. METHODS: We focused on the treatment used at the exact time of any L74V or L74I emergences in 74 patients, and we compared the use of each nucleoside reverse transcriptase inhibitor (NRTI) separately and in combination between the 74I and the 74V groups. The distribution of other NRTI and non-NRTI mutations between the two groups was also analysed. RESULTS: A majority of L74I mutations is selected under the zidovudine plus abacavir combination or under tenofovir with thymidine analogue mutations in the resistance background. The K103N substitution also plays an important role in the L74I emergence when not associated with the other non-NRTI mutations seen in this study: L100I, G190A and Y181C. Didanosine plays the principal role in the L74V emergence. CONCLUSIONS: This study shows that the L74I and the L74V correspond to two different mutation pathways, conferring probably different resistance and replication advantages on HIV depending on the context. Taking into account more systematically the L74I mutation, whose impact is certainly currently underestimated, would increase our understanding of this substitution and its effects on the drug activity in vivo.

Impact of gag mutations on selection of darunavir resistance mutations in HIV-1 protease.

OBJECTIVES: To search for genetic factors in the protease and gag regions (NC-p1/TFP-p6/p6pol) involved in selection of darunavir resistance mutations. PATIENTS AND METHODS: We analysed 48 protease inhibitor (PI)-experienced HIV-infected patients experiencing darunavir treatment failure. Viral genotyping at baseline and months 3 and 6 was used to assess the selection of mutations in the protease and gag regions conferring resistance to PIs. RESULTS: There were no genotypic differences in the studied gag region between baseline and the latest available rebound isolates. There was an association between the presence of the mutation A431V in the gag sequence and the selection of the L76V mutation in the protease sequence in the latest available rebound. The I437T/V mutation in gag and the L76V mutation in the protease were associated with a lower risk of selecting darunavir resistance mutations. CONCLUSIONS: In these PI-treated patients experiencing treatment failure of a darunavir-containing regimen, we showed that mutations in the gag region NC-p1/TFP-p6/p6pol may influence the selection of darunavir resistance mutations; in particular, the I437T/V gag mutation that confers resistance to PIs reduces the selection of such mutations. Virus with L76V in protease or I437T/V in gag may be already resistant to darunavir and, therefore, no additional resistance mutations need to be selected.