Sex-specific selective pressures on body mass in the greater white-toothed shrew, Crocidura russula.

The direction, intensity and shape of viability-, sexual- and fecundity selection on body mass were investigated in a natural population of the greater white-toothed shrew (Crocidura russula), combining parentage assignment through molecular techniques and mark-recapture data over several generations. A highly significant stabilizing viability selection was found in both sexes, presumably stemming from the constraints imposed by their insectivorous habits and high metabolic costs. Sexual selection, directional in both sexes, was twice as large in males than in females. Our results suggest that body mass matters in this context by facilitating the acquisition and defense of a breeding territory. No fecundity selection could be detected. The direction of sexual size dimorphism (SSD) was in agreement with the observed pattern of selective pressures: males were heavier than females, because of stronger sexual selection. SSD intensity, however, was low compared with other mammals, because of the low level of polygyny, the active role of females in territory defense and the intensity of stabilizing viability selection.

Human immunodeficiency virus type 1 neutralization by plasma from B or F genotype infected individuals

Anti-human immunodeficiency virus type 1 (HIV-1) "binding antibodies" (antibodies capable of binding to synthetic peptides or proteins) occur throughout HIV-1 infection, are high-titered and highly cross-reactive, as confirmed in this study by analyzing plasma from B and F genotype HIV-1 infected individuals. Plasma from individuals infected with clade F HIV-1 displayed the most frequent cross-reactivity, in high titers, while Bbr plasma showed much higher specificity. Similarly, neutralization of a reference HIV-1 isolate (HIV-1 MN) was more frequently observed by plasma from F than B genotype infected individuals. No significant difference was seen in neutralization susceptibility of primary B, Bbr or F clade HIV-1 by plasma from individuals infected with the classical B (GPGR) or F HIV-1, but Bbr (GWGR) plasma were less likely to neutralize the F genotype primary HIV-1 isolates. The data indicate that both B and F genotype derived vaccines would be equally effective against B and F HIV-1 infection, with a slightly more probable effectiveness for F than B genotype. Although the Bbr variant appears to induce a much more specific humoral immune response, the susceptibility in neutralizing the Brazilian HIV-1 B genotype Bbr variant is similar to that observed with the classical B genotype HIV-1.

Evolution of C(4) phosphoenolpyruvate carboxykinase in grasses, from genotype to phenotype.

C(4) photosynthesis is an adaptation over the classical C(3) pathway that has evolved multiple times independently. These convergences are accompanied by strong variations among the independent C(4) lineages. The decarboxylating enzyme used to release CO(2) around Rubisco particularly differs between C(4) species, a criterion used to distinguish three distinct biochemical C(4) subtypes. The phosphoenolpyruvate carboxykinase (PCK) serves as a primary decarboxylase in a minority of C(4) species. This enzyme is also present in C(3) plants, where it is responsible for nonphotosynthetic functions. The genetic changes responsible for the evolution of C(4)-specific PCK are still unidentified. Using phylogenetic analyses on PCK sequences isolated from C(3) and C(4) grasses, this study aimed at resolving the evolutionary history of C(4)-specific PCK enzymes. Four independent evolutions of C(4)-PCK were shown to be driven by positive selection, and nine C(4)-adaptive sites underwent parallel genetic changes in different C(4) lineages. C(4)-adaptive residues were also observed in C(4) species from the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) subtype and particularly in all taxa where a PCK shuttle was previously suggested to complement the NADP-ME pathway. Acquisitions of C(4)-specific PCKs were mapped on a species tree, which revealed that the PCK subtype probably appeared at the base of the Chloridoideae subfamily and was then recurrently lost and secondarily reacquired at least three times. Linking the genotype to subtype phenotype shed new lights on the evolutionary transitions between the different C(4) subtypes.

Comparison of HPV genotyping by type-specific PCR and sequencing

Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide and there is a strong link between certain high-risk viral types and cervical carcinogenesis. Although there are several typing methods, it is still unclear which test is the best. This study compared the effectiveness of type-specific PCR (TS-PCR) and sequencing, with a focus on their clinical application. A total of 260 cervical samples from HPV-positive patients were tested for types 6, 11, 16, 18, 31, 33 and 35 using TS-PCR and sequencing. The genotype was identified in 36% of cases by TS-PCR and in 75% by sequencing. Sequencing was four times more likely to identify the viral type in positive samples than TS-PCR (p = 0.00). Despite being more effective for virus genotyping, sequencing was unable to identify viral types in multiple infections. Combining both techniques resulted in highly sensitive detection (87% of cases), showing that they are complementary methods. HPV genotyping is an important step in HPV management, helping to identify patients with a higher risk of developing cervical cancer and contributing to the development of type-specific vaccines.

Sequencing of E2 and NS5A regions of HCV genotype 3a in Brazilian patients with chronic hepatitis

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.

DRB1*03:01 haplotypes: differential contribution to multiple sclerosis risk and specific association with the presence of intrathecal IgM bands.

BACKGROUND Multiple sclerosis (MS) is a multifactorial disease with a genetic basis. The strongest associations with the disease lie in the Human Leukocyte Antigen (HLA) region. However, except for the DRB1*15:01 allele, the main risk factor associated to MS so far, no consistent effect has been described for any other variant. One example is HLA-DRB1*03:01, with a heterogeneous effect across populations and studies. We postulate that those discrepancies could be due to differences in the diverse haplotypes bearing that allele. Thus, we aimed at studying the association of DRB1*03:01 with MS susceptibility considering this allele globally and stratified by haplotypes. We also evaluated the association with the presence of oligoclonal IgM bands against myelin lipids (OCMB) in cerebrospinal fluid. METHODS Genotyping of HLA-B, -DRB1 and -DQA1 was performed in 1068 MS patients and 624 ethnically matched healthy controls. One hundred and thirty-nine MS patients were classified according to the presence (M+, 58 patients)/absence (M-, 81 patients) of OCMB. Comparisons between groups (MS patients vs. controls and M+ vs. M-) were performed with the chi-square test or the Fisher exact test. RESULTS Association of DRB1*03:01 with MS susceptibility was observed but with different haplotypic contribution, being the ancestral haplotype (AH) 18.2 the one causing the highest risk. Comparisons between M+, M- and controls showed that the AH 18.2 was affecting only M+ individuals, conferring a risk similar to that caused by DRB1*15:01. CONCLUSIONS The diverse DRB1*03:01-containing haplotypes contribute with different risk to MS susceptibility. The AH 18.2 causes the highest risk and affects only to individuals showing OCMB.

Gender-Specific Effects of Genetic Variants within Th1 and Th17 Cell-Mediated Immune Response Genes on the Risk of Developing Rheumatoid Arthritis.

The present study was conducted to explore whether single nucleotide polymorphisms (SNPs) in Th1 and Th17 cell-mediated immune response genes differentially influence the risk of rheumatoid arthritis (RA) in women and men. In phase one, 27 functional/tagging polymorphisms in C-type lectins and MCP-1/CCR2 axis were genotyped in 458 RA patients and 512 controls. Carriers of Dectin-2 rs4264222T allele had an increased risk of RA (OR = 1.47, 95%CI 1.10-1.96) whereas patients harboring the DC-SIGN rs4804803G, MCP-1 rs1024611G, MCP-1 rs13900T and MCP-1 rs4586C alleles had a decreased risk of developing the disease (OR = 0.66, 95%CI 0.49-0.88; OR = 0.66, 95%CI 0.50-0.89; OR = 0.73, 95%CI 0.55-0.97 and OR = 0.68, 95%CI 0.51-0.91). Interestingly, significant gender-specific differences were observed for Dectin-2 rs4264222 and Dectin-2 rs7134303: women carrying the Dectin-2 rs4264222T and Dectin-2 rs7134303G alleles had an increased risk of RA (OR = 1.93, 95%CI 1.34-2.79 and OR = 1.90, 95%CI 1.29-2.80). Also five other SNPs showed significant associations only with one gender: women carrying the MCP-1 rs1024611G, MCP-1 rs13900T and MCP-1 rs4586C alleles had a decreased risk of RA (OR = 0.61, 95%CI 0.43-0.87; OR = 0.67, 95%CI 0.47-0.95 and OR = 0.60, 95%CI 0.42-0.86). In men, carriers of the DC-SIGN rs2287886A allele had an increased risk of RA (OR = 1.70, 95%CI 1.03-2.78), whereas carriers of the DC-SIGN rs4804803G had a decreased risk of developing the disease (OR = 0.53, 95%CI 0.32-0.89). In phase 2, we genotyped these SNPs in 754 RA patients and 519 controls, leading to consistent gender-specific associations for Dectin-2 rs4264222, MCP-1 rs1024611, MCP-1 rs13900 and DC-SIGN rs4804803 polymorphisms in the pooled sample (OR = 1.38, 95%CI 1.08-1.77; OR = 0.74, 95%CI 0.58-0.94; OR = 0.76, 95%CI 0.59-0.97 and OR = 0.56, 95%CI 0.34-0.93). SNP-SNP interaction analysis of significant SNPs also showed a significant two-locus interaction model in women that was not seen in men. This model consisted of Dectin-2 rs4264222 and Dectin-2 rs7134303 SNPs and suggested a synergistic effect between the variants. These findings suggest that Dectin-2, MCP-1 and DC-SIGN polymorphisms may, at least in part, account for gender-associated differences in susceptibility to RA.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

Recurrent mutations in the CDKL5 gene: Genotype-phenotype relationships.

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been described in epileptic encephalopathies in females with infantile spasms with features that overlap with Rett syndrome. With more than 80 reported patients, the phenotype of CDKL5-related encephalopathy is well-defined. The main features consist of seizures starting before 6 months of age, severe intellectual disability with absent speech and hand stereotypies and deceleration of head growth, which resembles Rett syndrome. However, some clinical discrepancies suggested the influence of genetics and/or environmental factors. No genotype-phenotype correlation has been defined and thus there is a need to examine individual mutations. In this study, we analyzed eight recurrent CDKL5 mutations to test whether the clinical phenotype of patients with the same mutation is similar and whether patients with specific CDKL5 mutations have a milder phenotype than those with other CDKL5 mutations. Patients bearing missense mutations in the ATP binding site such as the p.Ala40Val mutation typically walked unaided, had normocephaly, better hand use ability, and less frequent refractory epilepsy when compared to girls with other CDKL5 mutations. In contrast, patients with mutations in the kinase domain (such as p.Arg59X, p.Arg134X, p.Arg178Trp/Pro/Gln, or c.145 + 2T > C) and frameshift mutations in the C-terminal region (such as c.2635_2636delCT) had a more severe phenotype with infantile spasms, refractory epileptic encephalopathy, absolute microcephaly, and inability to walk. It is important for clinicians to have this information when such patients are diagnosed. © 2012 Wiley Periodicals, Inc.

Genotype 3 is associated with accelerated fibrosis progression in chronic hepatitis C.

BACKGROUND/AIMS: While several risk factors for the histological progression of chronic hepatitis C have been identified, the contribution of HCV genotypes to liver fibrosis evolution remains controversial. The aim of this study was to assess independent predictors for fibrosis progression. METHODS: We identified 1189 patients from the Swiss Hepatitis C Cohort database with at least one biopsy prior to antiviral treatment and assessable date of infection. Stage-constant fibrosis progression rate was assessed using the ratio of fibrosis Metavir score to duration of infection. Stage-specific fibrosis progression rates were obtained using a Markov model. Risk factors were assessed by univariate and multivariate regression models. RESULTS: Independent risk factors for accelerated stage-constant fibrosis progression (>0.083 fibrosis units/year) included male sex (OR=1.60, [95% CI 1.21-2.12], P<0.001), age at infection (OR=1.08, [1.06-1.09], P<0.001), histological activity (OR=2.03, [1.54-2.68], P<0.001) and genotype 3 (OR=1.89, [1.37-2.61], P<0.001). Slower progression rates were observed in patients infected by blood transfusion (P=0.02) and invasive procedures or needle stick (P=0.03), compared to those infected by intravenous drug use. Maximum likelihood estimates (95% CI) of stage-specific progression rates (fibrosis units/year) for genotype 3 versus the other genotypes were: F0-->F1: 0.126 (0.106-0.145) versus 0.091 (0.083-0.100), F1-->F2: 0.099 (0.080-0.117) versus 0.065 (0.058-0.073), F2-->F3: 0.077 (0.058-0.096) versus 0.068 (0.057-0.080) and F3-->F4: 0.171 (0.106-0.236) versus 0.112 (0.083-0.142, overall P<0.001). CONCLUSIONS: This study shows a significant association of genotype 3 with accelerated fibrosis using both stage-constant and stage-specific estimates of fibrosis progression rates. This observation may have important consequences for the management of patients infected with this genotype.

Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC).

BACKGROUND: Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. METHODOLOGY/FINDINGS: We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. CONCLUSION/SIGNIFICANCE: Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.

HLA Class I alleles associated with HCV polymorphisms and response to antiviral therapy in HCV genotype 1-infected patients

Aims: The adaptive immune response against hepatitis C virus (HCV) is significantly shaped by the host's composition of HLA alleles. Thus, the HLA phenotype is a critical determinant of viral evolution during adaptive immune pressure. Potential associations of HLA class I alleles with polymorphisms of HCV immune escape variants are largely unknown. Methods: Direct sequence analysis of the genes encoding the HCV proteins E2, NS3 and NS5B in a cohort of 159 patients with chronic HCV genotype 1 infection who were treated with pegylated interferon-alfa 2b and ribavirin in a prospective controlled trial for 48 weeks was exhibited. HLA class I genotyping was performed by strand-specific reverse hybridization with the INNO-LiPA line probe assays for HLA-A and HLA-B and by strand-specific PCR-SSP. We analyzed each amino acid position of HCV proteins using an extension of Fisher's exact test for associations with HLA alleles. In addition, associations of specific HLA alleles with inflammatory activity, liver fibrosis, HCV RNA viral load and virologic treatment outcome were investigated. Results: Separate analyses of HCV subtype 1a and 1b isolates revealed substantially different patterns of HLA-restricted polymorphisms between subtypes. Only one polymorphism within NS5B (V2758x) was significantly associated with HLA B*15 in HCV genotype 1b infected patients (adjusted p=0,048). However, a number of HLA class I-restricted polymorphisms within novel putative HCV CD8+ T cell epitopes (genotype 1a: HLA-A*11 GTRTIASPK1086-1094 [NS3], HLA-B*07 WPAPQGARSL1111-1120 [NS3]; genotype 1b: HLA-A*24 HYAPRPCGI488-496 [E2], HLA-B*44 GENETDVLL530-538 [E2], HLA-B*15 RVFTEAMTRY2757-2766 [NS5B]) were observed with high predicted epitope binding scores assessed by the web-based software SYFPEITHI (>21). Most of the identified putative epitopes were overlapping with already otherwise published epitopes, indicating a high immunogenicity of the accordant HCV protein region. In addition, certain HLA class I alleles were associated with inflammatory activity, stage of liver fibrosis, and sustained virologic response to antiviral therapy. Conclusions: HLA class I restricted HCV sequence polymorphisms are rare. HCV polymorphisms identified within putative HCV CD8+ T cell epitopes in the present study differ in their genomic distribution between genotype 1a and 1b isolates, implying divergent adaptation to the host's immune pressure on the HCV subtype level.

Extended treatment of treatment-naive patients with chronic hepatitis c virus (HCV) genotype 1-infection and slow response to pegylated interferon-alfa and ribavirin: a meta-analysis

Aims: Recently, several clinical trials analyzed if extended duration of treatment with pegylated interferon-alfa and ribavirin over 48 weeks can improve sustained virologic response (SVR) rates in HCV genotype 1-infected patients with slow virologic response. Because results of these clinical trials are conflicting, we performed a metaanalysis to determine the overall impact of extended treatment compared to standard treatment on virologic response rates in treatment-naive HCV genotype 1 slow responders. Methods: Literature search was performed independently by two observers using Pub Med, EMBASE, CENTRAL and abstracts presented in English at international liver and gastroenterology meetings. Randomized controlled clinical trials (RCTs; but studies that re-analyzed data retrospectively RCTs were also allowed) were considered if they included monoinfected treatment-naive HCV genotype 1 patients and compared treatment with pegIFN-alfa 2a or 2b in combination with ribavirin for 48 weeks versus extended treatment (up to 72 weeks) in slow responders. Primary and secondary end points were SVR rates and end-of-treatment (EOT) and relapse rates, respectively. In the present meta-analysis, study endpoints were summarized with a DerSimonian-Laird estimate for binary outcome basing on a random effects model. Results: Literature search yielded seven RTCs addressing the benefit of extended treatment with pegylated interferon-alfa and ribavirin in treatment-naive HCV genotype 1 slow responders. In total, 1330 slow responders were included in our meta-analysis. We show that extended treatment duration compared to the standard of care significantly improves SVR rates in HCV genotype 1 slow responders (12.4% improvement of overall SVR rate, 95% CI 0.055- 0.193, P = 0.0005). In addition, we show that rates of viral relapse were significantly reduced by extended treatment (24.1% reduction of relapse, 95% CI −0.3332 to −0.1487, P < 0.0001), whereas no significant impact of extended treatment on EOT response rates was found. Though extended treatment was burdened with an enhanced rate of premature treatment discontinuation due to interferonalfa- and ribavirin-related side effects, the frequency of serious adverse events was not increased. Conclusions: Treatment extension in HCV genotype 1 slow responders can improve SVR rates in difficult to treat patients and should be considered in patients who need to be treated before specific antivirals will be approved.

MHC-genotype of progeny influenced by parental infection.

In a previous series of in vitro fertilization experiments with mice we found non-random combination of major histocompatibility complex (MHC) haplotypes in the very early embryos. Our results suggested that two selection mechanisms were operating: (i) the eggs selected specific sperm; and (ii) the second meiotic division in the eggs was influenced by the type of sperm that entered the egg. Furthermore, the proportion of MHC-heterozygous embryos varied over time, suggesting that non-random fertilization was dependent on an external factor that changed over time. As a higher frequency of heterozygous individuals correlated with an uncontrolled epidemic by MHV (mouse hepatitis virus), we suggested that MHV-infection might have influenced the outcome of fertilization. Here, we present an experiment that tests this hypothesis. We infected randomly chosen mice with MHV and sham-infected control mice five days before pairing. We recovered the two-cell embryos from the oviduct, cultured them until the blastocyst stage, and determined the genotype of each resulting blastocyst by polymerase chain reaction. We found the pattern that we expected from our previous experiments: virus-infected mice produced more MHC-heterozygous embryos than sham-infected ones. This suggests that parents are able to promote specific combinations of MHC-haplotypes during fertilization according to the presence or absence of a viral infection.

Methicillin-susceptible Staphylococcus aureus endocarditis isolates are associated with clonal complex 30 genotype and a distinct repertoire of enterotoxins and adhesins.

BACKGROUND: Using multinational collections of methicillin-susceptible Staphylococcus aureus (MSSA) isolates from infective endocarditis (IE) and soft tissue infections (STIs), we sought to (1) validate the finding that S. aureus in clonal complex (CC) 30 is associated with hematogenous complications and (2) test the hypothesis that specific genetic characteristics in S. aureus are associated with infection severity. METHODS: IE and STI isolates from 2 cohorts were frequency matched by geographic origin. Isolates underwent spa typing to infer CC and multiplex polymerase chain reaction for presence of virulence genes. RESULTS: 114 isolate pairs were genotyped. IE isolates were more likely to be CC30 (19.5% vs 6.2%; P = .005) and to contain 3 adhesins (clfB, cna, map/eap; P < .0001 for all) and 5 enterotoxins (tst, sea, sed, see, and sei; P ≤ .005 for all). CC30 isolates were more likely to contain cna, tst, sea, see, seg, and chp (P < .05 for all). CONCLUSIONS: MSSA IE isolates were significantly more likely to be CC30 and to possess a distinct repertoire of virulence genes than MSSA STI isolates from the same region. The genetic basis of this association requires further study.