Molecular evolution of attachment glycoprotein (G) gene in human respiratory syncytial virus detected in Japan 2008–2011

We investigated the evolution of the C-terminal 3rd hypervariable region of G gene in the prevalent human respiratory syncytial virus (RSV) subgroups A (RSV-A) and B (RSV-B) in Japan in 2008–2011. Phylogenetic analysis and the evolutionary timescale was obtained by the Bayesian Markov Chain Monte Carlo method. All 38 RSV-A strains detected were classified into genotype NA1 and the 17 RSV-B strains detected belonged to genotypes BA and GB2. NA1 subdivided around 1998 in the present phylogenetic tree. Genotype BA subdivided around 1994. The evolutionary rates for RSV-A and RSV-B were estimated at 3.63 × 10⁻³ and 4.56 × 10⁻³ substitutions/site/year, respectively. The mean evolutionary rate of RSV-B was significantly faster than that of RSV-A during all seasons. The pairwise distance was relatively short (less than 0.06). In addition, some unique sites under positive selection were found. The results suggested that this region of the RSV strains rapidly evolved with some unique amino acid substitutions due to positive pressure.     

Molecular evolution of human respiratory syncytial virus attachment glycoprotein (G) gene of new genotype ON1 and ancestor NA1

We conducted a comprehensive genetic analysis of the C-terminal 3rd hypervariable region of the attachment glycoprotein (G) gene in human respiratory syncytial virus subgroup A (HRSV-A) genotype ON1 (93 strains) and ancestor NA1 (125 strains). Genotype ON1 contains a unique mutation of a 72 nucleotide tandem repeat insertion (corresponding to 24 amino acids) in the hypervariable region. The Bayesian Markov chain Monte Carlo (MCMC) method was used to conduct phylogenetic analysis and a time scale for evolution. We also calculated pairwise distances (p-distances) and estimated the selective pressure. Phylogenetic analysis showed that the analyzed ON1 and NA1 strains formed 4 lineages. A strain belonging to lineage 4 of ON1 showed wide genetic divergence (p-distance, 0.072), which suggests that it might be a candidate new genotype, namely ON2. The emergence of genotype NA1 was estimated to have occurred in 2000 (95% of highest probability density, HPD; 1997–2002) and that of genotype ON1 in 2005 (95% HPD; 2000–2010) based on the time-scaled phylogenetic tree. The evolutionary rate of genotype ON1 was higher than that of ancestral genotype NA1 (6.03 × 10⁻³ vs. 4.61 × 10⁻³ substitutions/site/year, p < 0.05). Some positive and many negative selection sites were found in both ON1 and NA1 strains. The results suggested that the new genotype ON1 is rapidly evolving with antigenic changes, leading to epidemics of HRSV infection in various countries.     

The genetic variability of glycoproteins among respiratory syncytial virus subtype A in China between 2009 and 2013

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and children under 5 years of age. The novel genotype ON1 has a 72-nucleotide duplication, which is the largest duplicated genome portion of RSV. Whether the ON1 genotype will follow the pattern of the BA genotype, which has a 60-nucleotide duplication, and become the predominant RSV-A strain is a global concern. To obtain information regarding the prevalence of the ON1 genotype in Chongqing in Southwestern China, we examined the circulation pattern of RSV-A identified over four consecutive years (June 2009 to August 2013). In this study, 312 (12%) RSV-A strains were isolated from 2601 nasopharyngeal aspirates, and partial G gene was sequenced successfully in 250 isolates. Of the sequenced Chongqing RSV-A isolates, 237 (94.8%) strains were the NA1 genotype, 4 (1.6%) strains were the NA3 genotype, 4 (1.6%) strains were the NA4 genotype, 1 (0.4%) strain was the GA1 genotype, and 4 (1.6%) strains were identified as the ON1 genotype. Analysis of the distribution, phylogeny, and evolution of the ON1 strains that were collected globally until December 2013 revealed that the ON1 genotype has rapidly disseminated across the world under positive selection pressures. Future studies will determine whether this new genotype will continue to spread and become the dominant strain of RSV-A worldwide. These findings may contribute to the understanding of RSV evolution and to the potential development of a vaccine against RSV.     

Displacement of predominant respiratory syncytial virus genotypes in Malaysia between 1989 and 2011

From 1989 to 2011 in Kuala Lumpur, Malaysia, multiple genotypes from both respiratory syncytial virus (RSV) subgroups were found co-circulating each year. RSV-A subgroup predominated in 12 out of 17 years with the remaining years predominated by RSV-B subgroup. Local RSV strains exhibited temporal clustering with RSV strains reported in previous epidemiological studies. Every few years, the existing predominant genotype was replaced by a new genotype. The RSV-A genotypes GA2, GA5 and GA7 were replaced by NA1 and NA2, while BA became the predominant RSV-B genotype. A unique local cluster, BA12, was seen in 2009, and the recently-described ON1 genotype with 72-nt duplication emerged in 2011. Our findings will have important implications for future vaccine intervention.     

Molecular evolution of the hypervariable region of the attachment glycoprotein gene in human respiratory syncytial virus subgroup B genotypes BA9 and BA10

We studied the molecular evolution of the C-terminal 3rd hypervariable region in the attachment glycoprotein gene of human respiratory syncytial virus subgroup B (HRSV-B) genotypes BA9 and BA10. We performed time-scaled phylogenetic analyses using Bayesian Markov chain Monte Carlo methods. We also performed a genetic distance analysis (p-distance analysis), positive and negative selection analyses, and a Bayesian skyline plot (BSP) analysis. We found that genotype BA9 diverged from the common ancestor of genotypes BA7, BA8, and BA10, while genotype BA10 diverged from the ancestor of genotypes BA7 and BA8. Strains of both genotypes were distributed worldwide. BA9 and BA10 diverged between 1999 and 2001. Both BA9 and BA10 evolved rapidly (about 4.8 × 10^− 3 substitutions/site/year) and formed three distinct lineages in a 10-year period. BA10 strains belonging to lineage 3 had large genetic distances (p-distance > 0.07). Thus, it may be possible to classify these strains as a new genotype, BA11. No positive selection site was detected in either genotype. Phylodynamic analyses showed that the effective population size of BA10 decreased gradually since 2010 and BA9 slightly decreased since 2009. The results suggested that the recently prevalent HRSV-B genotypes BA9 and BA10 evolved uniquely, leading to epidemics of HRSV-B worldwide over a 15-year period.     

Genotyping of Helicobacter pylori shows high diversity of strains circulating in central Vietnam

In Vietnam, the high prevalence of Helicobacter pylori infection represents a serious health problem. Virulence genes of H. pylori have been associated to increased risk of severe gastrointestinal diseases and the genetic background differs in geographical areas. We investigated cagA and vacA genotypes of H. pylori from dyspeptic patients from central Vietnam and the correlation with clinical outcomes; we also performed sequencing analysis of partial cagA gene.Overall, 84% of strains were cagA-positive, 75% were East-Asian type with a prevalence of vacAs1i1m1 and vacAs1i1m2 genotypes (66.7% and 33.3%, respectively) and 9% were Western type vacAs1i1m1 (n = 4) and vacAs1i1m2 (n = 4); vacAs1i2m2 (n = 4) and vacAs2i2m2 (n = 2) genotypes were associated to cagA-negative. Strains from gastric ulcer and cancer were of East-Asian type, while cagA-negative or Western strains were from gastritis and duodenal ulcer. H. pylori strains from gastric ulcer patients were predominantly vacAs1i1m1 compared to other vacA genotypes (p < 0.05). East-Asian type strains vacAs1i1m1 or vacAs1i1m2 were found in gastric cancer patients and also in less severe disease. Phylogenetic tree analysis of CagA sequences showed the co-circulation of H. pylori of different geographical origins with Western sequences closer related to Cambodia, one of the entry of Western strains in Southeast-Asia through human migrations. Sequence analysis revealed in two Western type strains a chimeric CagA-3′ region with identity with East-Asian CagA suggesting recombination event in the process of evolution among East-Asian and Western H. pylori strains. Moreover, polymorphism in CagA multimerization (CM) motif was observed including new East-Asian CM motifs. In conclusion, we have found in central Vietnam a geographically dependent diversity of cagA genotype, with higher rates of cagA-negative and Western-type strains compared with other nation's parts that can partly explain the lower risk of gastric cancer. The polymorphism of CM motifs may explain the variability of disease manifestations of vacAs1i1m1 and s1i1m2 East-Asian isolates.     

Rapid replacement of human respiratory syncytial virus A with the ON1 genotype having 72 nucleotide duplication in G gene

Human respiratory syncytial virus (HRSV) is the main cause of severe respiratory illness in young children and elderly people. We investigated the genetic characteristics of the circulating HRSV subgroup A (HRSV-A) to determine the distribution of genotype ON1, which has a 72-nucleotide duplication in attachment G gene. We obtained 456 HRSV-A positive samples between October 2008 and February 2013, which were subjected to sequence analysis. The first ON1 genotype was discovered in August 2011 and 273 samples were identified as ON1 up to February 2013. The prevalence of the ON1 genotype increased rapidly from 17.4% in 2011–2012 to 94.6% in 2012–2013. The mean evolutionary rate of G protein was calculated as 3.275 × 10⁻³ nucleotide substitution/site/year and several positively selected sites for amino acid substitutions were located in the predicted epitope region. This basic and important information may facilitate a better understanding of HRSV epidemiology and evolution.     

Genetic analysis of human rhinovirus species A to C detected in patients with acute respiratory infection in Kumamoto prefecture,Japan 2011–2012

We performed detailed genetic analysis of the VP4/VP2 coding region in human rhinovirus species A to C (HRV-ABC) strains detected in patients with a variety of acute respiratory infections in Kumamoto, Japan in the period 2011–12. The phylogenetic tree and evolutionary timescale were obtained by the Bayesian Markov chain Monte Carlo method. Phylogenetic analyses showed that the present HRV-A, -B, and -C strains belonged to 25, 4, and 18 genotypes, respectively. Some new genotypes were confirmed as prevalent strains of HRV-C. An ancestor of the present HRV-ABCs could be dated back to about 20,000 years ago. The present HRV-A and -C strains have wide genetic divergence (pairwise distance >0.2) with rapid evolutionary rates (around 7 × 10⁻⁴ to 4 × 10⁻³ substitutions/site/year). Over 100 sites were found to be under negative selection, while no positively selected sites were found in the analyzed region. No evidence of recombination events was found in this region of the present strains. Our results indicate that the present HRV strains have rapidly evolved and subsequently diverged over a long period into multiple genotypes.     

Molecular evolution of the fusion protein gene in human respiratory syncytial virus subgroup A

We studied the molecular evolution of the fusion protein (F) gene in the human respiratory syncytial virus subgroup A (HRSV-A). We performed time-scaled phylogenetic analyses using the Bayesian Markov chain Monte Carlo (MCMC) method. We also conducted genetic distance (p-distance), positive/negative selection, and Bayesian skyline plot analyses. Furthermore, we mapped the amino acid substitutions of the protein. The MCMC-constructed tree indicated that the HRSV F gene diverged from the bovine RSV (BRSV) gene approximately 550 years ago and had a relatively low substitution rate (7.59 × 10^− 4 substitutions/site/year). Moreover, a common ancestor of HRSV-A and -B diverged approximately 280 years ago, which has since formed four distinct clusters. The present HRSV-A strains were assigned six genotypes based on F gene sequences and attachment glycoprotein gene sequences. The present strains exhibited high F gene sequence similarity values and low genetic divergence. No positive selection sites were identified; however, 50 negative selection sites were identified. F protein amino acid substitutions at 17 sites were distributed in the F protein. The effective population size of the gene has remained relatively constant, but the population size of the prevalent genotype (GA2) has increased in the last 10 years. These results suggest that the HRSV-A F gene has evolved independently and formed some genotypes.     

Evaluation of the attenuation,immunogenicity, and efficacy of a live virus vaccine generated by codon-pair bias de-optimization of the 2009 pandemic H1N1 influenza virus,in ferrets

Codon-pair bias de-optimization (CPBD) of viruses involves re-writing viral genes using statistically underrepresented codon pairs, without any changes to the amino acid sequence or codon usage. Previously, this technology has been used to attenuate the influenza A/Puerto Rico/8/34 (H1N1) virus. The de-optimized virus was immunogenic and protected inbred mice from challenge. In order to assess whether CPBD could be used to produce a live vaccine against a clinically relevant influenza virus, we generated an influenza A/California/07/2009 pandemic H1N1 (2009 pH1N1) virus with de-optimized HA and NA gene segments (2009 pH1N1-(HA + NA)^Min), and evaluated viral replication and protein expression in MDCK cells, and attenuation, immunogenicity, and efficacy in outbred ferrets. The 2009 pH1N1-(HA + NA)^Min virus grew to a similar titer as the 2009 pH1N1 wild type (wt) virus in MDCK cells (∼10⁶ TCID₅₀/ml), despite reduced HA and NA protein expression on western blot. In ferrets, intranasal inoculation of 2009 pH1N1-(HA + NA)^Min virus at doses ranging from 10³ to 10⁵ TCID₅₀ led to seroconversion in all animals and protection from challenge with the 2009 pH1N1 wt virus 28 days later. The 2009 pH1N1-(HA + NA)^Min virus did not cause clinical illness in ferrets, but replicated to a similar titer as the wt virus in the upper and lower respiratory tract, suggesting that de-optimization of additional gene segments may be warranted for improved attenuation. Taken together, our data demonstrate the potential of using CPBD technology for the development of a live influenza virus vaccine if the level of attenuation is optimized.     

Gene polymorphisms in CCR5, CCR2, SDF1 and RANTES among Chinese Han population with HIV-1 infection

Chemokines and chemokine receptors are crucial for immune response in HIV-1 infection. Although many studies have been done to investigate the relationship between chemokines and chemokine receptor gene polymorphisms and host’s susceptibility to HIV-1 infection, the conclusions are under debate. In the present study, a cohort of 287 HIV-1 seropositive patients, 388 ethnically age-matched healthy controls and 49 intravenous drug users (IDUs) HIV-1 exposed seronegative individuals (HESN) from Chinese Han population were enrolled in order to determine the influence of host genetic factors on HIV-1 infection. Seven polymorphisms on four known chemokines/chemokine receptor genes (CCR5Δ32, CCR5 m303, CCR5 59029A/G, CCR2 64I, RANTES −403A/G, RANTES −28C/G and SDF1 3′-A) were screened. CCR5Δ32 and CCR5 m303 were absent or infrequent in Chinese Han population, which may not be hosts’ genetic protective factors for HIV-1 infection. Our results showed the CCR5 59029A/G, CCR2 64I and SDF1 3′-A were not associated with host’s resistance to HIV-1 infection. The frequency of RANTES −403A allele was significantly lower in HIV-1 patients than in healthy blood donors (p = 0.0005) and HESN group (p = 0.035), which implied the association between A allele and reduced HIV-1 infection risk. Different genetic models were assessed to investigate this association (AA vs. GG + AG, OR = 0.38 95% CI, 0.22–0.65 p = 0.0004; A vs. G, OR = 0.66 95% CI, 0.52–0.84 p = 0.0006), which supported this association, either. The genotype and allele distribution of RANTES −28 between HIV-1 patients and healthy controls (genotype profile: p = 0.072; allele profile: p = 0.027) or HIV-1 seronegative group (genotype profile: p = 0.036; allele profile: p = 0.383) were both at the marginal level of significance, which were not observed after Bonferroni correction. All these results suggest the RANTES −403A may be associated with reduced susceptibility to HIV-1 infection, while the RANTES −28 locus not. By lack of the patients’ clinical information, whether these polymorphisms affect AIDS disease progression and their role in different HIV-1 infection routes could not performed in present study and needs to be assessed in ongoing studies.     

Mucoviscous characteristics of Klebsiella pneumoniae strains: A factor of clinical severity?

IntroductionHypermucoviscous Klebsiella pneumoniae (KP) strains are responsible for complicated bacteremia with multiple septic sites (liver, central nervous system, muscles). We aimed to compare the clinical severity of patients presenting with KP bacteremia based on the hypermucoviscous or non-hypermucoviscous characteristic of the strains.MethodsObservational retrospective study successively including all patients with KP bacteremia from May 2013 to March 2015 at the tertiary medical center of New Caledonia. The hypermucoviscous characteristic was defined by the string test results and molecular analysis to determine the capsular serotype.ResultsA total of 55 bacteremic patients were included in the study; 27% of isolated strains were hypermucoviscous. Hypermucoviscous strains accounted for two-thirds of community-acquired infections (72.5% vs. 33.4%, p = 0.01). The rate of intensive care hospitalization was high (hypermucoviscous 46.7%; standard 52.5%) without any difference between the two groups. No significant difference was observed in case fatality (hypermucoviscous 46.7% vs. standard 15%, p = 0.07) but patients with hypermucoviscous strains had longer hospital stays (73.5 days versus 50.7 days, p = 0.04) and longer persistence of positive blood cultures despite an appropriate treatment (OR 1.41, 95% CI: 1.0–1.96, p = 0.045).ConclusionHypermucoviscous KP bacteremia account for most community-acquired Klebsiella infections in New Caledonia and are associated with longer hospital stay and persistence of positive blood cultures despite the implementation of an appropriate treatment.     

Genetic characterization of circulating seasonal Influenza A viruses (2005–2009) revealed introduction of oseltamivir resistant H1N1 strains during 2009 in eastern India

Influenza surveillance was implemented in Kolkata, eastern India in 2005 to identify the circulating subtypes and characterize their genetic diversity. Throat and nasal swabs were collected from outpatients with influenza-like illness (ILI). Of 2844 ILI cases identified at two referral hospitals during October 2005–September 2009, 309 (10.86%) were positive for Influenza A by real time RT-PCR, of which 110 (35.60%) were subtyped as H1N1 and 199 (64.40%) as H3N2. Comparison of the nucleotide (nt) and amino acid (aa) sequences of the HA1 gene for H1N1 and H3N2 strains showed that a subset of strains precede WHO recommended contemporary strains by 1–2 years. The Kolkata H1N1 strains clustered in Clade II, subgroup 2B with A/Brisbane/59/2007 but were distant from the corresponding vaccine strains (New Caledonia/20/99 and A/Solomon Island/3/06). The 2005–06 and 2007 H3N2 strains (15/17) clustered either A/Brisbane/10/2007-like (n = 8) or A/Nepal/921/2006 like (n = 7) strains, whereas 2008 strains (8/12) and 2009 strains (4/4) were similar to the 2010–11 vaccine strain A/Perth/16/2009. More aa substitutions were found in HA or NA genes of H3N2 than in H1N1 strains. No mutation conferring neuraminidase resistance was observed in any of the strain during 2005–08, however in 2009, drug resistant marker (H275Y) was present in seasonal H1N1, but not in co-circulating H3N2 strains. This is the first report of genetic characterization of circulating Influenza A strains from India. The results also highlight the importance of continuing Influenza surveillance in developing countries of Asia for monitoring unusual strains with pandemic potential and mutations conferring antiviral resistance.     

Transmission dynamics of hepatitis C virus among intra venous drug users in the border state of Manipur,India

Intra venous drug users (IVDUs) are at high risk for hepatitis C virus (HCV) infection owing to their high rate of drug abuses. The north-eastern part of India has a high prevalence of IVDUs with Manipur being the worst hit state. The aim of the study was to document the molecular epidemiology, the patterns of HCV transmission, genomic variation and recombination events within HCV genome among IVDUs of Manipur, India. 91 anti-HCV sero-reactive blood samples were collected from IVDUs in Manipur. The samples were processed for RNA extraction, nested RT-PCR, sequencing and quantitative viral RNA estimation. Phylogeographic analysis of the sequenced core and NS5B regions of HCV genome was performed to determine the probable transmission route and recombinant HCV strains. 83 out of 91 anti-HCV seropositive samples were RNA positive (91.20%) based on 5′UTR of HCV genome by nested RT-PCR. Of the RNA positive samples, 73 paired partial core and NS5B gene were sequenced. Three major genotype and eight subtypes were detected while no recombinant strains were found. Individuals with genotype 1 had the mean viral load (5.94 ± 0.705 log₁₀IU/ml) followed by genotype 3 (4.91 ± 0.49 log₁₀IU/ml) and 6 (3.96 ± 0.32 log₁₀IU/ml). The viral load was statistically significant among the male individuals at 4.822 ± 1.36 log₁₀IU/ml compared to 4.767 ± 0.49 log₁₀IU/ml for females (t = 3.249, p < 0.005). The phylogeographic results indicated 3b, 6h originated from Vietnam, 1a had Indian origin, 3a, 6k originated from southern China while 1b originated from Myanmar, respectively. The incidence of eight different subtypes in Manipur reflects the transmission of these strains from the “Golden Triangle” drug trafficking regions. Sequence analysis confirmed the transmission routes of HCV, which is linked to China and Vietnam for the newly emergent genotype 6 in north-eastern India.     

Genetic variability of human respiratory syncytial virus in Pune,Western India

Human respiratory syncytial virus (RSV) is one of the most important respiratory viruses causing acute respiratory tract infections amongst children. Based on genotyping of the attachment glycoprotein (G) gene, it is divided into two groups, RSV-A and RSV-B. Infection with one group does not confer immunity against the other and children infected with one antigenic group are more likely to be reinfected with the heterologous group. We tested 854 samples of patients with influenza like illness (ILI)/severe respiratory illness (SARI) during the period 2009–2012 for RSV using a conventional multiplex RT-PCR and found 159 (18.61%) samples to be positive for RSV of which 130 (15.22%) were positive for RSV-B and 29 (3.39%) for RSV-A suggesting that RSV-B was the predominant group circulating in Western India during the study period. Seasonal RSV outbreaks were observed in the monsoon and winter months. RSV was more prevalent amongst children in the 0–24 month age group (21.53%) in comparison to children in the 24–60 month age group (13.01%). Phylogenetic analysis using the G gene of 27 representative RSV-A positive samples revealed that all sequences belonged to the NA1 genotype. Of these, 5 sequences exhibited the novel 72 nucleotide duplication in the C-terminal of the G gene first reported from Ontario, Canada and clustered in the newly designated ON1 genotype. Also, 32 of the 33 RSV-B sequences exhibited the 60 nucleotide duplication associated with genotype BA and phylogenetic analysis showed that these sequences belonged to the genotype BA9 and BA12. We also found one RSV-B sequence belonging to genotype GB2, which has not been previously reported in India.     

Association of toll-like receptors with susceptibility to tuberculosis suggests sex-specific effects of TLR8 polymorphisms

Background: Toll-like receptors (TLRs) are involved in the recognition of conserved microbial structures, leading to activation of an inflammatory response and formation of an adaptive immune response. Methods: Twenty-three polymorphisms in five TLR genes were genotyped in 729 tuberculosis cases and 487 healthy controls in a population-based case-control association study in a South African population. Results: We detected sex-specific associations for TLR8 polymorphisms, with rs3761624 (OR = 1.54, p < 0.001), rs3764879 (OR = 1.41, p = 0.011) and rs3764880 (OR = 1.42, p = 0.011) associated in females and rs3764879 (OR = 0.72, p = 0.013) and rs3764880 (OR = 0.75, p = 0.036) associated in males. Epistatic interactions between the TLR genes were investigated and the TLR1_rs4833095 polymorphism was shown to interact with TLR2_rs3804100 and (GT)_n microsatellite (p = 0.002) and alter susceptibility to TB. We also studied the role of TLRs in disease caused by different Mycobacterium tuberculosis genotypes in 257 tuberculosis cases, and identified associations between specific TLR polymorphisms and disease caused by specific strains. Conclusion: This study provides further evidence that the TLRs play an important role in the outcome of tuberculosis disease, and suggests a partial explanation for the male bias in tuberculosis ratios.     

NRAMP1 D543N and INT4 polymorphisms in susceptibility to pulmonary tuberculosis: A meta-analysis

The association of natural resistance associated macrophage protein 1 (NRAMP1) polymorphisms (D543N, INT4) with pulmonary tuberculosis (PTB) risk have been widely reported. However, the findings of previous studies were inconsistent. To clarify the role of these polymorphisms in PTB, we performed a meta-analysis of all available and relevant published studies. Based on comprehensive searches of the PubMed, Medline, Embase, Web of Science, Elsevier Science Direct and Cochrane Library database, we identified outcome data from all articles estimating the association between NRAMP1 polymorphisms and PTB risk. For D543NA/G polymorphism, no associations were found in all genetic models. For INT4C/G polymorphism, significant increased PTB risk was observed in recessive model (CC vs. GC + GG: P = 0.025, OR = 1.35, 95% CI = 1.04–1.75). In the subgroup analysis by ethnicity, significantly increased risk were observed for D543NA/G polymorphism in Americans (GA vs. GG: P = 0.03, OR = 1.31, 95% CI = 1.03–1.67; AA + AG vs. GG: P = 0.032, OR = 1.29, 95% CI = 1.02–1.63). Moreover, the INT4C/G polymorphism was also associated with increased risk of TB for Africans in allele model (A vs. G: P = 0.012, OR = 1.41, 95% CI = 1.08–1.85), heterozygous model (GA vs. GG: P = 0.004, OR = 1.53, 95% CI = 1.14–2.04) and dominant model (AA + AG vs. GG: P = 0.007, OR = 1.49, 95% CI = 1.12–1.98). This meta-analysis provides evidences that INT4C/G was associated with increased susceptibility to pulmonary tuberculosis in overall population in recessive model. D543NA/G polymorphism was associated with PTB increased risk in Americans, while INT4C/G polymorphism in Africans. Further well-designed, large scale studies are required to confirm this conclusion.     

Detection of serum AFB1-lysine adduct in Malaysia and its association with liver and kidney functions

Aflatoxin is ubiquitously found in many foodstuffs and produced by Aspergillus species of fungi. Of many aflatoxin metabolites, AFB₁ is classified by the International Agency for Research on Cancer (IARC) as group one carcinogen and linked to the development of hepatocellular carcinoma (HCC). The study on molecular biomarker of aflatoxin provides a better assessment on the extent of human exposure to aflatoxin. In Malaysia, the occurrences of aflatoxin-contaminated foods have been documented, but there is a lack of data on human exposure to aflatoxin. Hence, this study investigated the occurrence of AFB₁-lysine adduct in serum samples and its association with liver and kidney functions. 5 ml fasting blood samples were collected from seventy-one subjects (n = 71) for the measurement of AFB₁-lysine adduct, albumin, total bilirubin, AST (aspartate aminotransferase), ALT (alanine transaminase), ALP (alkaline phosphatase), GGT (gamma-glutamyl transpeptidase), creatinine and BUN (blood urea nitrogen). The AFB₁-lysine adduct was detected in all serum samples (100% detection rate) with a mean of 6.85 ± 3.20 pg/mg albumin (range: 1.13–18.85 pg/mg albumin). Male subjects (mean: 8.03 ± 3.41 pg/mg albumin) had significantly higher adduct levels than female subjects (mean: 5.64 ± 2.46 pg/mg albumin) (p < 0.01). It was noteworthy that subjects with adduct levels greater than average (>6.85 pg/mg albumin) had significantly elevated level of total bilirubin (p < 0.01), GGT (p < 0.05) and creatinine (p < 0.01). Nevertheless, only the level of total bilirubin, (r = 0.347, p-value = 0.003) and creatinine (r = 0.318, p-value = 0.007) showed significant and positive correlation with the level of AFB₁-lysine adduct. This study provides a valuable insight on human exposure to aflatoxin in Malaysia. Given that aflatoxin can pose serious problem to the health, intervention strategies should be implemented to limit/reduce human exposure to aflatoxin. Besides, a study with a big sample size should be warranted in order to assess aflatoxin exposure in the general population of Malaysia.     

Intra-host evolutionary rates in HIV-1C env and gag during primary infection

BackgroundHIV-1 nucleotide substitution rates are central for understanding the evolution of HIV-1. Their accurate estimation is critical for analysis of viral dynamics, identification of divergence time of HIV variants, inference of HIV transmission clusters, and modeling of viral evolution.MethodsIntra-patient nucleotide substitution rates in HIV-1C gag and env gp120 V1C5 were analyzed in a longitudinal cohort of 32 individuals infected with a single viral variant. Viral quasispecies were derived by single genome amplification/sequencing from serially sampled blood specimens collected at median (IQR) of 5 (4–6) times per subject from enrollment (during Fiebig stages II to V) over a median (IQR) of 417 (351–471) days post-seroconversion (p/s). HIV-1C evolutionary rates were estimated by BEAST v.1.7 using a relaxed lognormal molecular clock model. The effect of antiretroviral therapy (ART) on substitution rates in gag and env was assessed in a subset of six individuals who started ARV therapy during the follow-up period.ResultsDuring primary HIV-1C infection, the intra-patient substitution rates were estimated at a median (IQR) of 5.22E-03 (3.28E-03-7.55E-03) substitutions per site per year of infection within gag, and 1.58E-02 (9.99E-03-2.04E-02) substitutions per site per year within env gp120 V1C5. The substitution rates in env gp120 V1C5 were higher than in gag (p < 0.001, Wilcoxon signed rank test). The median (IQR) relative rates of evolution at codon positions 1, 2, and 3 were 0.73 (0.48–0.84), 0.67 (0.52–0.86), and 1.54 (1.21–1.71) in gag, and 1.01 (0.86–1.15), 1.05 (0.99–1.21), and 0.86 (0.67–0.94) in env gp120 V1C5, respectively. A first to the third position codon rate ratio >1.0 within env was found in 25 (78.1%) cases, but only in 4 (12.5%) cases in gag, while a second to the third position codon rate ratio >1.0 in env was observed in 26 (81.3%) cases, but in gag only in 2 (6.3%) cases (p < 0.001 for both comparisons, Fisher’s exact test). No ART effect on substitution rates in gag and env was found, at least within the first 3–4 months after ART initiation. Individuals with early viral set point ⩾4.0 log₁₀ copies/ml had higher substitution rates in env gp120 V1C5 (median (IQR) 1.88E-02 (1.54E-02–2.46E-02) vs. 1.04E (7.24E-03–1.55E-02) substitutions per site per year; p = 0.017, Mann–Whitney sum rank test), while individuals with early viral set point ⩾3.0 log₁₀ copies/ml had higher substitution rates in gag (median (IQR) 5.66E-03 (3.45E-03–7.94E-03) vs. 1.78E-03 (4.57E-04–5.15E-03); p = 0.028; Mann–Whitney sum rank test).ConclusionsThe results suggest that in primary HIV-1C infection, (1) intra-host evolutionary rates in env gp120 V1C5 are about 3-fold higher than in gag; (2) selection pressure in env is more frequent than in gag; (3) initiation of ART does not change substitution rates in HIV-1C env or gag, at least within the first 3–4 months after starting ART; and (4) intra-host evolutionary rates in gag and env gp120 V1C5 are higher in individuals with elevated levels of early viral set point.     

Transmitted drug resistance and phylogenetic analysis of HIV CRF01_AE in Northern Vietnam

The HIV epidemic in Vietnam began in injecting drug users (IDUs), but increasingly affects the general population. It is therefore important to monitor the spread of infection and, since antiretroviral therapy (ART) is now used more frequently, the prevalence of transmitted drug resistance. Sixty-three 1000 bp pol-gene sequences were generated from treatment-naive HIV-1 CRF01_AE infected patients from four clinics in Northern Vietnam. Four drug resistance mutations; Y181C, L210W, L74I and V75M, were found in four different patients, giving a prevalence of 6.3% (4/63). Earlier studies have shown a lower prevalence and the transmission rate should be regularly monitored prospectively in Vietnam. Additional CRF01_AE (N  =  190) and outgroup subtype B sequences (N  =  4) were retrieved from databases and included for phylogenetic analysis and calculations of the time of the most recent common ancestor (tMRCA). The 63 samples from our study clustered into two distinct groups; one small clade (N  =  3) that had a tMRCA in year 1997.5 and a larger group with an estimated tMRCA in 1989.8. The Vietnamese samples in the large group were distinct from CRF01_AE sequences from Thailand, but closely related to previously sequenced isolates from Vietnam, southern China and the Czech Republic, while the samples in the smaller clade appeared to represent a more recent introduction from Southern Vietnam. Our results showed that sequences from IDUs were intermingled with sequences from sexually infected patients, indicating frequent exchange of virus between the transmission risk groups in Northern Vietnam.