Tissue-specific metabolic enzyme levels covary with whole-animal metabolic rates and life-history loci via epistatic effects.

Metabolic rates, including standard (SMR) and maximum (MMR) metabolic rate have often been linked with life-history strategies. Variation in context- and tissue-level metabolism underlying SMR and MMR may thus provide a physiological basis for life-history variation. This raises a hypothesis that tissue-specific metabolism covaries with whole-animal metabolic rates and is genetically linked to life history. In Atlantic salmon (Salmo salar), variation in two loci, vgll3 and six6, affects life history via age-at-maturity as well as MMR. Here, using individuals with known SMR and MMR with different vgll3 and six6 genotype combinations, we measured proxies of mitochondrial density and anaerobic metabolism, i.e. maximal activities of the mitochondrial citrate synthase (CS) and lactate dehydrogenase (LDH) enzymes, in four tissues (heart, intestine, liver, white muscle) across low- and high-food regimes. We found enzymatic activities were related to metabolic rates, mainly SMR, in the intestine and heart. Individual loci were not associated with the enzymatic activities, but we found epistatic effects and genotype-by-environment interactions in CS activity in the heart and epistasis in LDH activity in the intestine. These effects suggest that mitochondrial density and anaerobic capacity in the heart and intestine may partly mediate variation in metabolic rates and life history via age-at-maturity. This article is part of the theme issue 'The evolutionary significance of variation in metabolic rates'.

Clinical evaluation of a low cost, in-house developed real-time RT-PCR human immunodeficiency virus type 1 (HIV-1) quantitation assay for HIV-1 infected patients.

OBJECTIVES: HIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access. MATERIALS AND METHODS: A real-time competitive RT-PCR in house assay (Sing-IH) was developed in Singapore. Using HXB2 as reference, the assay's primers and probes were designed to generate a 183-bp product that overlaps a portion of the LTR region and gag region. A competitive internal control (IC) was included in each assay to monitor false negative results due to inhibition or human error. Clinical evaluation was performed on 249 HIV-1 positive patient samples in comparison with the commercially available Generic HIV Viral Load assay. Correlation and agreement of results were assessed for plasma HIV-1 quantification with both assays. RESULTS: The assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100-1000000 copies/mL. Comparative analysis with reference to the Generic assay demonstrated good agreement between both assays with a mean difference of 0.22 log10 copies/mL and 98.8% of values within 1 log10 copies/mL range. Furthermore, the Sing-IH assay can quantify HIV-1 group M subtypes A-H and group N isolates adequately, making it highly suitable for our region, where subtype B and CRF01_AE predominate. CONCLUSIONS: With a significantly lower running cost compared to commercially available assays, the broadly sensitive Sing-IH assay could help to overcome the cost barriers and serve as a useful addition to the currently limited HIV viral load assay options for resource-limited settings.

Phylodynamic profile of HIV-1 subtype B, CRF01_AE and the recently emerging CRF51_01B among men who have sex with men (MSM) in Singapore.

HIV-1 subtype B and CRF01_AE are the predominant infecting subtypes among men who have sex with men (MSM) in Singapore. The genetic history, population dynamics and pattern of transmission networks of these genotypes remain largely unknown. We delineated the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and the recently characterized CRF51_01B strains circulating among the MSM population in Singapore. A total of 105 (49.5%) newly-diagnosed treatment-naïve MSM were recruited between February 2008 and August 2009. Phylogenetic reconstructions of the protease gene (HXB2: 2239 - 2629), gp120 (HXB2: 6942 - 7577) and gp41 (HXB2: 7803 - 8276) of the env gene uncovered five monophyletic transmission networks (two each within subtype B and CRF01_AE and one within CRF51_01B lineages) of different sizes (involving 3 - 23 MSM subjects, supported by posterior probability measure of 1.0). Bayesian coalescent analysis estimated that the emergence and dissemination of multiple sub-epidemic networks occurred between 1995 and 2005, driven largely by subtype B and later followed by CRF01_AE. Exponential increase in effective population size for both subtype B and CRF01_AE occurred between 2002 to 2007 and 2005 to 2007, respectively. Genealogical estimates suggested that the novel CRF51_01B lineages were probably generated through series of recombination events involving CRF01_AE and multiple subtype B ancestors. Our study provides the first insight on the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and CRF51_01B viral strains circulating among MSM in Singapore.

High prevalence of CXCR4 usage among treatment-naive CRF01_AE and CRF51_01B-infected HIV-1 subjects in Singapore.

BACKGROUND: Recent studies suggest HIV-1 inter-subtype differences in co-receptor usage. We examined the correlation between HIV-1 subtype and co-receptor usage among treatment-naïve HIV-1 subjects in Singapore. Additionally, we investigated whether the subtype co-receptor association was influenced by stage of infection. METHODS: V3 sequences of HIV-1 envelope protein gp120 were obtained from 110 HIV treatment-naïve patients and genotypic co-receptor tropism determination was performed using Geno2pheno. Two false-positive rate (FPR) cut-offs, 10% and 5.75% were selected for tropism testing. RESULTS: Subtype assignment of viral strains from 110 HIV-infected individuals based on partial sequencing of HIV-1 pol, gp120 and gp41 were as follows: 27 subtype B, 64 CRF01_AE, 10 CRF51_01B, and 9 other subtypes. At FPR=10%, 10 (100%) CRF51_01B-infected subjects and 26 (40.6%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 7 (25.9%) subtype B subjects and 1 (11.1%) CRF33_01B-infected subject (P < 0.001). At FPR=5.75%, 10 (100%) CRF51_01B-infected subjects and 20 (31.3%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 4 (14.8%) subtype B and 1 (11.1%) CRF33_01B-infected subjects (P < 0.001). Among those with evidence of seroconversion within 2 years prior to study enrolment, 100% of CRF51_01B-infected subjects had CXCR4-using virus, independent of Geno2pheno FPR. CONCLUSION: CRF51_01B and CRF01_AE-infected individuals have higher prevalence of CXCR4-usage compared to subtype B infected individuals. Further studies examining these differences could help optimise the use of CCR5-antagonist in populations with these subtypes, and increase our understanding of HIV-1 biology.

Identification of new CRF51_01B in Singapore using full genome analysis of three HIV type 1 isolates.

A recent HIV-1 molecular epidemiology survey in Singapore identified a novel CRF01_AE/B recombinant form, which accounted for 13 (11.9%) of 109 patient samples. Peripheral blood mononuclear cell DNA from three of these 13 patients was used to generate near full-length sequences to characterize the novel CRF01_AE/B recombinant form. The three isolates had a recombinant structure composed of CRF01_AE and subtype B, and shared identical breakpoints. As the three patients were not epidemiologically linked, this recombinant form has been designated CRF51_01B. Identification of the novel recombinant forms indicates ongoing active HIV-1 transmission in Singapore.

Clinical evaluation of an in-house human immunodeficiency virus (HIV) genotyping assay for the detection of drug resistance mutations in HIV-1 infected patients in Singapore.

INTRODUCTION: Human immunodeficiency virus type 1 (HIV-1) genotyping resistance test (GRT) is essential for monitoring HIV-1 drug resistance mutations (DRMs). High cost and HIV-1 genetic variability are challenges to assay availability in Singapore. An in-house Sanger sequencing-based GRT method was developed at the Communicable Disease Centre (CDC), Singapore's HIV national treatment reference centre for both subtype B and non-subtype B HIV-1. MATERIALS AND METHODS: The in-house GRT sequenced the fi rst 99 codons of protease (PR) and 244 codons of reverse transcriptase (RT) in the pol gene. The results were compared with the Food and Drug Administration (FDA)-approved ViroSeq™ HIV-1 Genotyping System. RESULTS: Subtype assignment for the 46 samples were as follows: 31 (67.4%) CRF01_AE, 14 (30.5%) subtype B and 1 (2.1%) subtype C. All 46 samples had viral load of ≥500 copies/mL, and were successfully amplified by the in-house primer sets. Compared to the ViroSeq™ test, our in-house assay showed drug-resistance conferring codon concordance of 99.9% at PR and 98.9% at RT, and partial concordance of 0.1% at PR and 1.1% at RT. No discordant result was observed. CONCLUSION: The assay successfully identified DRMs in both subtype AE and B, making it suitable for the efficient treatment monitoring in genetically diverse population. At less than half of the running cost compared to the ViroSeq™ assay, the broadly sensitive in-house assay could serve as a useful addition to the currently limited HIV genotyping assay options for resource-limited settings, thereby enhancing the DRM surveillance and monitoring in the region.

Molecular epidemiology of HIV type 1 in Singapore and identification of novel CRF01_AE/B recombinant forms.

To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).