Humans encode seven APOBEC3 (A3A-A3H) cytidine deaminase proteins that differ in their expression profiles, preferred nucleotide recognition sequence and capacity for restriction of RNA and DNA viruses. We identified APOBEC3 hotspots in numerous herpesvirus genomes. To determine the impact of host APOBEC3 on herpesvirus biology in vivo, we examined whether murine APOBEC3 (mA3) restricts murine gammaherpesvirus 68 (MHV68). Viral replication was impaired by several human APOBEC3 proteins, but not mA3, upon transfection of the viral genome. The restriction was abrogated upon mutation of the A3A and A3B active sites. Interestingly, virus restriction by A3A, A3B, A3C, and A3DE was lost if the infectious DNA was delivered by the virion. MHV68 pathogenesis, including lung replication and splenic latency, was not altered in mice lacking mA3. We infer that mA3 does not restrict wild type MHV68 and restriction by human A3s may be limited in the herpesvirus replication process.

Human Immunodeficiency Virus-1 (HIV-1) drug resistance genotyping assay is a part of clinical management of HIV-1 positive individuals under treatment with highly active antiretroviral therapy (HAART). Routine monitoring of drug resistance mutations in resource limited settings like India is not possible due to high cost of commercial drug resistance assays. In this study we developed an in-house, cost effective HIV-1 drug resistance genotyping assay for Indian patients and validated it against the US-FDA-approved ViroSeq HIV-1 drug resistance testing system. A reference panel of 20 clinical samples was used to develop and validate the assay against ViroSeq HIV-1 drug resistance testing system which was subsequently used to genotype a clinical panel of 225 samples. The Stanford HIV database was used to identify drug resistant mutations. The analytical sensitivity of the assay was 1000 HIV-1 RNA copies/ml of plasma sample while precision and reproducibility was 99.68±0.16% and 99.76±0.18% respectively. One hundred and one drug resistant mutations were detected by the in-house assay compared to 104 by ViroSeq system in the reference panel. The assay had 91.55% success rate in genotyping the clinical panel samples and was able to detect drug resistant mutations related to nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse-transcriptase inhibitor (NNRTI) as well as protease inhibitor (PI) classes of antiretroviral drugs. It was found to be around 71.9% more cost effective compared to ViroSeq genotyping system. This evaluation of the assay on the clinical panel demonstrates its potential for monitoring clinical HIV-1 drug resistance mutations and population-based surveillance in resource limited settings like India.

BackgroundHuman Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India.ResultsLinear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p < 0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p = 0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000–25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians.ConclusionThe HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

BACKGROUNDMother-to-child transmission of HIV is a major cause of HIV in children. Detection of virus by DNA PCR is of utmost importance in <6 months of age as antibody detection is not helpful. Dried blood spot (DBS) method is a cheap, less invasive, easy and feasible method as compared to whole blood collection. Followup of babies up to 18 months is needed before declaring them negative. The study was undertaken to know the HIV transmission rates in babies up to 18 months.