Validation and further characterization of a major quantitative trait locus associated with host response to experimental infection with porcine reproductive and respiratory syndrome virus

Infectious diseases are costly to the swine industry; porcine reproductive and respiratory syndrome (PRRS) is the most devastating. In earlier work, a quantitative trait locus associated with resistance/susceptibility to PRRS virus was identified on Sus scrofa chromosome 4 using approximately 560 experimentally infected animals from a commercial cross. The favorable genotype was associated with decreased virus load and increased weight gain (WG). The objective here was to validate and further characterize the association of the chromosome 4 region with PRRS resistance using data from two unrelated commercial crossbred populations. The validation populations consisted of two trials each of approximately 200 pigs sourced from different breeding companies that were infected with PRRS virus and followed for 42 days post‐infection. Across all five trials, heritability estimates were 0.39 and 0.34 for viral load (VL; area under the curve of log‐transformed viremia from 0 to 21 days post‐infection) and WG to 42 days post‐infection respectively. Effect estimates of SNP WUR10000125 in the chromosome 4 region were in the same directions and of similar magnitudes in the two new trials as had been observed in the first three trials. Across all five trials, the 1‐Mb region on chromosome 4 explained 15 percent of genetic variance for VL and 11 percent for WG. The effect of the favorable minor allele at SNP WUR10000125 was dominant. Ordered genotypes for SNP WUR10000125 showed that the effect was present irrespective of whether the favorable allele was paternally or maternally inherited. These results demonstrate that selection for host response to PRRS virus infection could reduce the economic impact of PRRS.     

Relationship of Glucocorticoid Receptor Expression in Peripheral Blood Mononuclear Cells and the Cochlea of Guinea Pigs and Effects of Dexamethasone Administration

Background

Glucocorticoids (GCs) are widely used to treat sudden sensorineural hearing loss (SSNHL) and significantly improve hearing. However, GC insensitivity has been observed in some patients of SSNHL.

Objective

To study the correlation between GR expression in peripheral blood mononuclear cells (PBMCs) and in the cochlea of guinea pigs at mRNA and protein levels.

Methods

One group of guinea pigs received dexamethasone (10 mg/kg/day) intraperitoneally for 7 consecutive days (dexamethasone group), and another group of guinea pigs received normal saline (control group). Real time PCR and Western blotting were used to detect the expression of GR mRNA and GR protein in PBMCs and the cochleae.

Results

The GR mRNA and GR protein were detected in both PBMCs and the cochlear tissue of guinea pigs. GR mRNA and GR protein levels in PBMCs were positively correlated with those in the cochlea. The expression of GR mRNA and GR protein was significantly increased in the dexamethasone group compared to the control group.

Conclusions

Levels of GR mRNA and GR protein in the PBMCs were positively correlated with those in the cochlea of guinea pigs. Systemic dexamethasone treatment can significantly up-regulate GR expression in PBMCs and in the cochlea. Measurement of the GR level in PBMCs could be used as an indicator of GR level in the cochlea.     

Genome-wide eQTLs and heritability for gene expression traits in unrelated individuals

Background

While the possible sources underlying the so-called ‘missing heritability’ evident in current genome-wide association studies (GWAS) of complex traits have been actively pursued in recent years, resolving this mystery remains a challenging task. Studying heritability of genome-wide gene expression traits can shed light on the goal of understanding the relationship between phenotype and genotype. Here we used microarray gene expression measurements of lymphoblastoid cell lines and genome-wide SNP genotype data from 210 HapMap individuals to examine the heritability of gene expression traits.

Results

Heritability levels for expression of 10,720 genes were estimated by applying variance component model analyses and 1,043 expression quantitative loci (eQTLs) were detected. Our results indicate that gene expression traits display a bimodal distribution of heritability, one peak close to 0% and the other summit approaching 100%. Such a pattern of the within-population variability of gene expression heritability is common among different HapMap populations of unrelated individuals but different from that obtained in the CEU and YRI trio samples. Higher heritability levels are shown by housekeeping genes and genes associated with cis eQTLs. Both cis and trans eQTLs make comparable cumulative contributions to the heritability. Finally, we modelled gene-gene interactions (epistasis) for genes with multiple eQTLs and revealed that epistasis was not prevailing in all genes but made a substantial contribution in explaining total heritability for some genes analysed.

Conclusions

We utilised a mixed effect model analysis for estimating genetic components from population based samples. On basis of analyses of genome-wide gene expression from four HapMap populations, we demonstrated detailed exploitation of the distribution of genetic heritabilities for expression traits from different populations, and highlighted the importance of studying interaction at the gene expression level as an important source of variation underlying missing heritability.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-13) contains supplementary material, which is available to authorized users.     

Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor β 1 (TGF-β 1) Axis in Hepatitis C Virus-Expressing Hepatocytes

Background

The pro-fibrogenic cytokine connective tissue growth factor (CTGF) plays an important role in the development and progression of fibrosis in many organ systems, including liver. However, its role in the pathogenesis of hepatitis C virus (HCV)-induced liver fibrosis remains unclear.

Methods

In the present study, we assessed CTGF expression in HCV-infected hepatocytes using replicon cells containing full-length HCV genotype 1 and the infectious HCV clone JFH1 (HCV genotype 2) by real-time PCR, Western blot analysis and confocal microscopy. We evaluated transforming growth factor β1 (TGF-β1) as a key upstream mediator of CTGF production using neutralizing antibodies and shRNAs. We also determined the signaling molecules involved in CTGF production using various immunological techniques.

Results

We demonstrated an enhanced expression of CTGF in two independent models of HCV infection. We also demonstrated that HCV induced CTGF expression in a TGF-β1-dependent manner. Further dissection of the molecular mechanisms revealed that CTGF production was mediated through sequential activation of MAPkinase and Smad-dependent pathways. Finally, to determine whether CTGF regulates fibrosis, we showed that shRNA-mediated knock-down of CTGF resulted in reduced expression of fibrotic markers in HCV replicon cells.

Conclusion

Our studies demonstrate a central role for CTGF expression in HCV-induced liver fibrosis and highlight the potential value of developing CTGF-based anti-fibrotic therapies to counter HCV-induced liver damage.     

Filaggrin genotype determines functional and molecular alterations in skin of patients with atopic dermatitis and ichthyosis vulgaris

Background

Several common genetic and environmental disease mechanisms are important for the pathophysiology behind atopic dermatitis (AD). Filaggrin (FLG) loss-of-function is of great significance for barrier impairment in AD and ichthyosis vulgaris (IV), which is commonly associated with AD. The molecular background is, however, complex and various clusters of genes are altered, including inflammatory and epidermal-differentiation genes.

Objective

The objective was to study whether the functional and molecular alterations in AD and IV skin depend directly on FLG loss-of-function, and whether FLG genotype determines the type of downstream molecular pathway affected.

Methods and Findings

Patients with AD/IV (n = 43) and controls (n = 15) were recruited from two Swedish outpatient clinics and a Swedish AD family material with known FLG genotype. They were clinically examined and their medical history recorded using a standardized questionnaire. Blood samples and punch biopsies were taken and trans-epidermal water loss (TEWL) and skin pH was assessed with standard techniques. In addition to FLG genotyping, the STS gene was analyzed to exclude X-linked recessive ichthyosis (XLI). Microarrays and quantitative real-time PCR were used to compare differences in gene expression depending on FLG genotype. Several different signalling pathways were altered depending on FLG genotype in patients suffering from AD or AD/IV. Disease severity, TEWL and pH follow FLG deficiency in the skin; and the number of altered genes and pathways are correlated to FLG mRNA expression.

Conclusions

We emphasize further the role of FLG in skin-barrier integrity and the complex compensatory activation of signalling pathways. This involves inflammation, epidermal differentiation, lipid metabolism, cell signalling and adhesion in response to FLG-dependent skin-barrier dysfunction.     

Global copy number analyses by next generation sequencing provide insight into pig genome variation

Background

Copy number variations (CNVs) confer significant effects on genetic innovation and phenotypic variation. Previous CNV studies in swine seldom focused on in-depth characterization of global CNVs.

Results

Using whole-genome assembly comparison (WGAC) and whole-genome shotgun sequence detection (WSSD) approaches by next generation sequencing (NGS), we probed formation signatures of both segmental duplications (SDs) and individualized CNVs in an integrated fashion, building the finest resolution CNV and SD maps of pigs so far. We obtained copy number estimates of all protein-coding genes with copy number variation carried by individuals, and further confirmed two genes with high copy numbers in Meishan pigs through an enlarged population. We determined genome-wide CNV hotspots, which were significantly enriched in SD regions, suggesting evolution of CNV hotspots may be affected by ancestral SDs. Through systematically enrichment analyses based on simulations and bioinformatics analyses, we revealed CNV-related genes undergo a different selective constraint from those CNV-unrelated regions, and CNVs may be associated with or affect pig health and production performance under recent selection.

Conclusions

Our studies lay out one way for characterization of CNVs in the pig genome, provide insight into the pig genome variation and prompt CNV mechanisms studies when using pigs as biomedical models for human diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-593) contains supplementary material, which is available to authorized users.     

Dynamics of Japanese Encephalitis Virus Transmission among Pigs in Northwest Bangladesh and the Potential Impact of Pig Vaccination

Background

Japanese encephalitis (JE) virus infection can cause severe disease in humans, resulting in death or permanent neurologic deficits among survivors. Studies indicate that the incidence of JE is high in northwestern Bangladesh. Pigs are amplifying hosts for JE virus (JEV) and a potentially important source of virus in the environment. The objectives of this study were to describe the transmission dynamics of JEV among pigs in northwestern Bangladesh and estimate the potential impact of vaccination to reduce incidence among pigs.

Methodology/Principal Findings

We conducted a comprehensive census of pigs in three JE endemic districts and tested a sample of them for evidence of previous JEV infection. We built a compartmental model to describe JEV transmission dynamics in this region and to estimate the potential impact of pig vaccination. We identified 11,364 pigs in the study area. Previous JEV infection was identified in 30% of pigs with no spatial differences in the proportion of pigs that were seropositive across the study area. We estimated that JEV infects 20% of susceptible pigs each year and the basic reproductive number among pigs was 1.2. The model suggest that vaccinating 50% of pigs each year resulted in an estimated 82% reduction in annual incidence in pigs.

Conclusions/Significance

The widespread distribution of historic JEV infection in pigs suggests they may play an important role in virus transmission in this area. Future studies are required to understand the contribution of pig infections to JE risk in humans and the potential impact of pig vaccination on human disease.     

Occult Hepatitis B Virus Infection in Anti-HBs-Positive Infants Born to HBsAg-Positive Mothers in China

Objective

To investigate the prevalence of occult HBV infection (OBI) among children and to characterize virology of occult HBV, we conducted an epidemiological survey.

Methods

186 HB-vaccinated infants born to HBsAg-positive mothers were included in the study. Serological tests for HBV markers were performed using commercial ELISA kits. Real-time quantitative PCR and nested PCR were used to detect HBV DNA. PCR products of the C and pre-S/S regions were sequenced and analyzed.

Results

1.61% (3/186) infants were HBsAg positive, and 4.92% (9/183) infants were considered as occult infection. The viral load of mothers was associated with occult infection (P = 0.020). Incomplete three-dose injections of HB vaccine was associated with HBV infection (P = 0.022). Six OBI infants were positive for anti-HBs, but their titers were not greater than 100 mIU/mL. Seven isolated HBV pre-S/S sequences were obtained from nine OBI infants. Three of the sequences were genotype C, and four of the sequences were genotype C/D. Escape mutation S143L was found in the four sequences of genotype C/D. All seven sequences lacked G145R and other escape mutation in S region.

Conclusions

Occult HBV infection was detected in anti-HBs positive infants born to HBsAg-positive mothers in China. Occult infection was associated with absent anti-HBs or with low anti-HBs level, high maternal viral loads and escape mutations in the S gene.     

Prioritizing genes responsible for host resistance to influenza using network approaches

Background

The genetic make-up of humans and other mammals (such as mice) affects their resistance to influenza virus infection. Considering the complexity and moral issues associated with experiments on human subjects, we have only acquired partial knowledge regarding the underlying molecular mechanisms. Although influenza resistance in inbred mice has been mapped to several quantitative trait loci (QTLs), which have greatly narrowed down the search for host resistance genes, only few underlying genes have been identified.

Results

To prioritize a list of promising candidates for future functional investigation, we applied network-based approaches to leverage the information of known resistance genes and the expression profiles contrasting susceptible and resistant mouse strains. The significance of top-ranked genes was supported by different lines of evidence from independent genetic associations, QTL studies, RNA interference (RNAi) screenings, and gene expression analysis. Further data mining on the prioritized genes revealed the functions of two pathways mediated by tumor necrosis factor (TNF): apoptosis and TNF receptor-2 signaling pathways. We suggested that the delicate balance between TNF’s pro-survival and apoptotic effects may affect hosts’ conditions after influenza virus infection.

Conclusions

This study considerably cuts down the list of candidate genes responsible for host resistance to influenza and proposed novel pathways and mechanisms. Our study also demonstrated the efficacy of network-based methods in prioritizing genes for complex traits.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-816) contains supplementary material, which is available to authorized users.