High Level of Pre-Treatment HIV-1 Drug Resistance and Its Association with HLA Class I-Mediated Restriction in the Pumwani Sex Worker Cohort.

BACKGROUND: We analyzed the prevalence of pre-antiretroviral therapy (ART) drug resistance mutations (DRMs) in a Kenyan population. We also examined whether host HLA class I genes influence the development of pre-ART DRMs. METHODS: The HIV-1 proviral DNAs were amplified from blood samples of 266 ART-naïve women from the Pumwani Sex Worker cohort of Nairobi, Kenya using a nested PCR method. The amplified HIV genomes were sequenced using next-generation sequencing technology. The prevalence of pre-ART DRMs was investigated. Correlation studies were performed between HLA class I alleles and HIV-1 DRMs. RESULTS: Ninety-eight percent of participants had at least one DRM, while 38% had at least one WHO surveillance DRM. M184I was the most prevalent clinically important variant, seen in 37% of participants. The DRMs conferring resistance to one or more integrase strand transfer inhibitors were also found in up to 10% of participants. Eighteen potentially relevant (p < 0.05) positive correlations were found between HLA class 1 alleles and HIV drug-resistant variants. CONCLUSIONS: High levels of HIV drug resistance were found in all classes of antiretroviral drugs included in the current first-line ART regimens in Africa. The development of DRMs may be influenced by host HLA class I-restricted immunity.

The Biosafety Level 4 Zoonotic Laboratory Network (BSL4ZNet): Report of a workshop on live animal handling.

The Biosafety Level 4 Zoonotic Laboratory Network (BSL4ZNet) was established in 2016, to provide a means of communication and support for the global high-containment laboratory community. Its working groups focus on international response, institutional cooperation and knowledge sharing, scientific excellence and training. In the latter role, BSL4ZNet sponsored its first international workshop in February 2018, held at the USDA National Centers for Animal Health, Ames, Iowa, USA, focused on necropsy procedures in high-containment laboratories. A second workshop, in November 2018, was held at the National Microbiology Laboratories (CFIA/PHAC) in Winnipeg, Canada, and focused on decontamination. A third workshop, held at the Australian Animal Health Laboratory in Geelong, Australia, in February 2019, was devoted to handling methods and ethical concerns for live animals in high-containment laboratories. The third workshop brought together 12 laboratorians from seven partner organizations in Australia, Canada, Germany, the United Kingdom and the United States. It included both discussion-based and hands-on training sessions on animal welfare, animal models, site-specific infrastructure constraints, health monitoring and humane endpoints, sampling procedures, and carcass disposal. This report summarizes the inception, development, and structure of the BSL4ZNet, and highlights the aims and results of the Geelong workshop.

Identification and Characterization of Positively Selected Mutations in Nef of Four HIV-1 Major Subtypes from Los Alamos National Laboratory.

BACKGROUND: Human immunodeficiency virus-1 (HIV-1) mutates rapidly to escape host immune pressure. This results in the generation of positively selected mutations (PSM) throughout the viral genome. Escape mutations in Nef, one of the accessory proteins of HIV-1, which plays an important role in viral pathogenicity have previously been identified in several large cohort studies, but the evolution of PSMs overtime in various HIV-1 subtypes remains unknown. METHODS: 161 clade A1, 3093 clade B, 647 clade C and 115 clade D HIV-1 nef sequences were obtained from the HIV Database of Los Alamos National Laboratory and aligned using MEGA 6.0. The sequences from each clade were grouped based on the year of collection. Quasi analysis was used to identify PSMs and the number and locations of PSMs were compared among different subtypes. RESULTS: PSMs for all four subtypes were distributed across the sequence of Nef, and conserved residues F90, W113, PxxPxR (a.a 72-77) remain unaltered overtime. The frequency of PSMs was stable among subtype B sequences but increased overtime for other subtypes. Phylogenetic analysis shows that sequences containing PSMs tend to cluster together at both inter and intra- subtype levels. CONCLUSION: Identification of PSMs and their changes overtime within various subtypes of HIV-1 is important in defining global viral evolutionary patterns that can provide insights for designing therapeutic strategies.

Interplay between HIV-1 and Host Genetic Variation: A Snapshot into Its Impact on AIDS and Therapy Response.

As of February 2012, 50 circulating recombinant forms (CRFs) have been reported for HIV-1 while one CRF for HIV-2. Also according to HIV sequence compendium 2011, the HIV sequence database is replete with 414,398 sequences. The fact that there are CRFs, which are an amalgamation of sequences derived from six or more subtypes (CRF27_cpx (cpx refers to complex) is a mosaic with sequences from 6 different subtypes besides an unclassified fragment), serves as a testimony to the continual divergent evolution of the virus with its approximate 1% per year rate of evolution, and this phenomena per se poses tremendous challenge for vaccine development against HIV/AIDS, a devastating disease that has killed 1.8 million patients in 2010. Here, we explore the interaction between HIV-1 and host genetic variation in the context of HIV/AIDS and antiretroviral therapy response.

Effect of size, quaternary structure and translational error on the static and dynamic heterogeneity of beta-galactosidase and measurement of electrophoretic dynamic heterogeneity.

Single enzyme molecule assays were performed using capillary electrophoresis-based protocols on beta-galactosidase from Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus helveticus and Bacillus circulans. The enzyme was found to show static heterogeneity with respect to catalytic rate and the variance in rate increased with protein size. This is consistent with the proposal that random errors in translation may be an important underlying component of enzyme heterogeneity. Additionally these enzymes were found to show static heterogeneity with respect to electrophoretic mobility. Comparison of wild-type and rpsL E. coli beta-galactosidase expressed in the presence and absence of streptomycin suggested that increases in error do not result in detectable increases in the dynamic heterogeneity of activity with increasing temperature. Finally, a method was developed to measure the dynamic heterogeneity in electrophoretic mobility.

Effect of alteration of translation error rate on enzyme microheterogeneity as assessed by variation in single molecule electrophoretic mobility and catalytic activity.

The role of translation error for Escherichia coli individual beta-galactosidase molecule catalytic and electrophoretic heterogeneity was investigated using CE-LIF. An E. coli rpsL mutant with a hyperaccurate translation phenotype produced enzyme molecules that exhibited significantly less catalytic heterogeneity but no reduction of electrophoretic heterogeneity. Enzyme expressed with streptomycin-induced translation error had increased thermolability, lower activity, and no significant change to catalytic or electrophoretic heterogeneity. Modeling of the electrophoretic behaviour of beta-galactosidase suggested that variation of the hydrodynamic radius may be the most significant contributor to electrophoretic heterogeneity.