First report of bovine viral diarrhea virus subgenotypes 1d and 1e in southern Chile.

Bovine viral diarrhea virus (BVDV) affects cattle worldwide causing severe productive and economic loss. In this study, we investigated the subgenotypes of BVDV circulating in cattle samples from the Aysén region, an active cattle breeding area located in southern Chile. Partial amplification of the 5' untranslated region (UTR) was performed by polymerase chain reaction (PCR), and twelve samples were analyzed by Sanger sequencing and phylogenetic analysis. Eight samples were identified as belonging to Pestivirus bovis subgenotype 1e, three to 1-b, and one to 1-d. The phylogenetic analyses performed revealed a marked distance between these now-identified strains and those previously reported in the country. These findings support the need to continually expand the analysis of the variability of the viral phylogeny for the currently circulating BVDV strains and to update the vaccines recommended for this livestock area and surrounding areas.

Epithelial TLR4 Signaling Activates DUOX2 to Induce Microbiota-Driven Tumorigenesis.

BACKGROUND & AIMS: Chronic colonic inflammation leads to dysplasia and cancer in patients with inflammatory bowel disease. We have described the critical role of innate immune signaling via Toll-like receptor 4 (TLR4) in the pathogenesis of dysplasia and cancer. In the current study, we interrogate the intersection of TLR4 signaling, epithelial redox activity, and the microbiota in colitis-associated neoplasia. METHODS: Inflammatory bowel disease and colorectal cancer data sets were analyzed for expression of TLR4, dual oxidase 2 (DUOX2), and NADPH oxidase 1 (NOX1). Epithelial production of hydrogen peroxide (H2O2) was analyzed in murine colonic epithelial cells and colonoid cultures. Colorectal cancer models were carried out in villin-TLR4 mice, carrying a constitutively active form of TLR4, their littermates, and villin-TLR4 mice backcrossed to DUOXA-knockout mice. The role of the TLR4-shaped microbiota in tumor development was tested in wild-type germ-free mice. RESULTS: Activation of epithelial TLR4 was associated with up-regulation of DUOX2 and NOX1 in inflammatory bowel disease and colorectal cancer. DUOX2 was exquisitely dependent on TLR4 signaling and mediated the production of epithelial H2O2. Epithelial H2O2 was significantly increased in villin-TLR4 mice; TLR4-dependent tumorigenesis required the presence of DUOX2 and a microbiota. Mucosa-associated microbiota transferred from villin-TLR4 mice to wild-type germ-free mice caused increased H2O2 production and tumorigenesis. CONCLUSIONS: Increased TLR4 signaling in colitis drives expression of DUOX2 and epithelial production of H2O2. The local milieu imprints the mucosal microbiota and imbues it with pathogenic properties demonstrated by enhanced epithelial reactive oxygen species and increased development of colitis-associated tumors. The inter-relationship between epithelial reactive oxygen species and tumor-promoting microbiota requires a 2-pronged strategy to reduce the risk of dysplasia in colitis patients.

Expression and purification of the surface proteins from Andes virus.

Andes virus is the main causative agent of Hantavirus cardiopulmonary syndrome in South America. There are currently no vaccines or treatments against Andes virus. However, there are several evidences suggesting that antibodies against Andes virus envelope glycoproteins may be enough to confer full protection against Hantavirus cardiopulmonary syndrome. The goal of the present work was to express, purify and characterize the extracellular domains of Andes virus glycoproteins Gn and Gc. We generated two adenoviral vectors encoding the extracellular domains of Andes virus glycoproteins Gn and Gc. Both molecules were expressed by adenoviral transduction in SiHa cells. We found that sGc ectodomain was mainly secreted into the culture medium, whereas sGn was predominantly retained inside the cells. Both molecules were expressed at very low concentrations (below 1 µg/mL). Treatment with the proteasome inhibitor ALLN raised sGc concentration in the cell culture medium, but did not affect expression levels of sGn. Both ectodomains were purified by immobilized metal ion affinity chromatography, and were recognized by sera from persons previously exposed to Andes virus. To our knowledge, this is the first work that addresses the expression and purification of Andes virus glycoproteins Gn and Gc. Our results demonstrate that sGn and sGc maintain epitopes that are exposed on the surface of the viral envelope. However, our work also highlights the need to explore new strategies to achieve high-level expression of these proteins for development of a vaccine candidate against Andes virus.

Intestinal Epithelial Inactivity of Dual Oxidase 2 Results in Microbiome-Mediated Metabolic Syndrome.

BACKGROUND & AIMS: Metabolic syndrome (MetS) is characterized by obesity, glucose intolerance, and hepatic steatosis. Alterations in the gut microbiome play important roles in the development of MetS. However, the mechanisms by which this occurs are poorly understood. Dual oxidase 2 (DUOX2) is an antimicrobial reduced nicotinamide adenine dinucleotide phosphate oxidase expressed in the gut epithelium. Here, we posit that epithelial DUOX2 activity provides a mechanistic link between the gut microbiome and the development of MetS. METHODS: Mice carrying an intestinal epithelial-specific deletion of dual oxidase maturation factor 1/2 (DA IEC-KO), and wild-type littermates were fed a standard diet and killed at 24 weeks. Metabolic alterations were determined by glucose tolerance, lipid tests, and body and organ weight measurements. DUOX2 activity was determined by Amplex Red. Intestinal permeability was determined by fluorescein isothiocyanate-dextran, microbial translocation assessments, and portal vein lipopolysaccharide measurements. Metagenomic analysis of the stool microbiome was performed. The role of the microbiome was assessed in antibiotic-treated mice. RESULTS: DA IEC-KO males showed increased body and organ weights accompanied by glucose intolerance and increased plasma lipid and liver enzyme levels, and increased adiposity in the liver and adipose tissue. Expression of F4/80, CD68, uncoupling protein 1, carbohydrate response element binding protein, leptin, and adiponectin was altered in the liver and adipose tissue of DA IEC-KO males. DA IEC-KO males produced less epithelial H2O2, had altered relative abundance of Akkermansiaceae and Lachnospiraceae in stool, and showed increased portal vein lipopolysaccharides and intestinal permeability. Females were protected from barrier defects and MetS, despite producing less H2O2. Antibiotic depletion abrogated all MetS phenotypes observed. CONCLUSIONS: Intestinal epithelial inactivity of DUOX2 promotes MetS in a microbiome-dependent manner.

Distinct genotypic distribution of cytomegalovirus (CMV) envelope glycoprotein B (gB) in a Cuban cohort of patients with different CMV diseases.

To investigate the association between human CMV glycoprotein B (gB) genotypes and CMV disease, we retrospectively analysed 73 biological samples from 56 Cuban patients with different CMV-related diseases using a multiplex nested PCR for detection of the reported 5 CMV gB genotypes. All 4 main genotypes 1 to 4 were found in the clinical samples while no genotype 5 was detected. Among the individuals analysed, genotype gB-2 was the most prevalent (38%) followed by gB-1 (30%) and mixed infections (16%) being mainly detected among immunosuppressed patients (7 out of 9), although there was no association between mixed infections and CMV rejection in transplant recipients. Genotype gB-4 was the least frequent (5 patients), which was almost exclusively detected in mixed infections (4 out of 5, p<0.0001). Genotype gB-1 was more frequently detected in AIDS patients (47%) although it was not statistically significant, while 68% of transplant patients showed mixed infections (p<0.05). This study represents the first report of human CMV gB genotypes in Cuban patients; however, the study is limited by the small number patients, thus making it difficult to draw firm conclusions about the distribution of CMV genotypes in Cuba. Nevertheless, this preliminary report has allowed us to identify that the main 4 CMV genotypes are present in the Cuban population, with genotypes 2 and 1 being the most frequent strains.