Victoria Ley

Bovine Enteroviruses as Indicators of Fecal Contamination

ABSTRACT Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.

Survey of Bovine Enterovirus in Biological and Environmental Samples by a Highly Sensitive Real-Time Reverse Transcription-PCR

ABSTRACT Animal enteroviruses shed in the feces of infected animals are likely environmental contaminants and thus can be used as indicators of animal fecal pollution. Previous work has demonstrated that bovine enterovirus (BEV) present in bovine feces contaminates waters adjacent to cattle herds and that BEV-like sequences are also present in shellfish and in deer feces from the same geographical area. However, little information is available about the prevalence, molecular epidemiology, and genomic sequence variation of BEV field isolates. Here we describe an optimized highly sensitive real-time reverse transcription-PCR method to detect BEV RNA in biological and environmental samples. A combination of the amplification procedure with a previously described filtration step with electropositive filters allowed us to detect up to 12 BEV RNA molecules per ml of water. The feasibility of using the method to detect BEV in surface waters at a high risk of fecal pollution was confirmed after analysis of water samples obtained from different sources. The method was also used to study the prevalence of BEV in different cattle herds around Spain, and the results revealed that 78% (78 of 100) of the fecal samples were BEV positive. BEV-like sequences were also detected in feces from sheep, goats, and horses. Nucleotide sequence analyses showed that BEV isolates are quite heterogeneous and suggested the presence of species-specific BEV-like variants. Detection of BEV-like sequences may help in the differentiation and characterization of animal sources of contamination.

Teschoviruses as Indicators of Porcine Fecal Contamination of Surface Water

ABSTRACT Teschoviruses specifically infect pigs and are shed in pig feces. Hence, their presence in water should indicate contamination with pig fecal residues. To assess this hypothesis, we have developed a real-time reverse transcriptase PCR (RT-PCR) method that allows the quantitative detection of pig teschovirus (PTV) RNA. The method is able to detect 92 fg of PTV RNA per ml of sample. Using this method, we have detected the presence of PTV RNA in water and fecal samples from all pig farms examined (n = 5). Feces from other animal species (cattle, sheep, and goats) were negative in this test. To compare the PTV RNA detection method with conventional chemical determinations currently in use for evaluation of water contamination, we analyzed water samples collected downstream from a pig slurry spillage site. We have found a positive correlation within both types of determinations. The sensitivity of the PTV detection assay was similar to that achieved by unspecific organic matter determination and superior to all other conventional chemical analyses performed. Furthermore, the new method is highly specific, revealing the porcine origin of the contamination, a feature that is lacking in currently available methods for the assessment of water contamination.

Identification of T-Cell Epitopes in Nonstructural Proteins of Foot-and-Mouth Disease Virus

ABSTRACT Porcine T-cell recognition of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP) was tested using in vitro lymphoproliferative responses. Lymphocytes were obtained from outbred pigs experimentally infected with FMDV. Of the different NSP, polypeptides 3A, 3B, and 3C gave the highest stimulations in the in vitro assays. The use of overlapping synthetic peptides allowed the identification of amino acid regions within these proteins that were efficiently recognized by the lymphocytes. The sequences of some of these antigenic peptides were highly conserved among different FMDV serotypes. They elicited major histocompatibility complex-restricted responses with lymphocytes from pigs infected with either a type C virus or reinfected with a heterologous FMDV. A tandem peptide containing the T-cell peptide 3A[21–35] and the B-cell antigenic site VP1[137–156] also efficiently stimulated lymphocytes from infected animals in vitro. Furthermore, this tandem peptide elicited significant levels of serotype-specific antiviral activity, a result consistent with the induction of anti-FMDV antibodies. Thus, inclusion in the peptide formulation of a T-cell epitope derived from the NSP 3A possessing the capacity to induce T helper activity can allow cooperative induction of anti-FMDV antibodies by B cells.

Infection with foot-and-mouth disease virus results in a rapid reduction of MHC class I surface expression.

The modulation of MHC class I molecule expression on the surface of cells as a consequence of foot-and-mouth disease virus (FMDV) infection has been examined. On cells infected with FMDV, class I expression was reduced to approximately 70% of the initial value 3 h after the infection and to 53% after 6 h. On cells depleted of surface class I complexes by acid treatment, the appearance of newly assembled class I-peptide complexes on the cell surface of non-infected cells increased immediately upon neutralization and original class I levels were recovered in about 20 h. In contrast, the appearance of new peptide-bound class I molecules on the cell surface was inhibited as early as 30 min after FMDV infection. Since the shut-down of FMDV-mediated host protein synthesis occurs approximately 2-3 h post-infection, this result suggests that an earlier event, which prevents the surface expression of newly synthesized complexes, is induced following FMDV infection. Thus, FMDV-infected cells rapidly become unable to present viral peptides in association with MHC class I molecules to T lymphocytes. Such a mechanism would assist virus evasion of the cytotoxic immune response of the host.

Restriction site map of African swine fever virus DNA

Treatment of African swine fever virus DNA (about 170 kbp) with the restriction endonucleases SalI, EcoRI, KpnI, PvuI, and SmaI yielded 14, 31, 17, 13, and 11 fragments, respectively. The order of the restriction fragments produced by each nuclease was established by identifying the crosslinked EcoRI and SalI terminal fragments and then finding overlapping fragments. The five restriction fragment maps were integrated into a single map by locating SalI, KpnI, PvuI, and SmaI sites in cloned EcoRI fragments, and orienting each fragment in the overall map.

Structure of Swine Vesicular Disease Virus: Mapping of Changes Occurring during Adaptation of Human Coxsackie B5 Virus To Infect Swine

ABSTRACT The structure of swine vesicular disease virus (SVDV) was solved and refined at a 3.0-Å resolution by X-ray crystallography to gain information about the role of sequence changes that occurred as this virus evolved from the parental human pathogen coxsackievirus B5 (CVB5). These amino acid substitutions can be clustered in five distinct regions: (i) the antigenic sites, (ii) the hydrophobic pocket of the VP1 β-sandwich, (iii) the putative CAR binding site, (iv) the putative heparan sulfate binding site, and (v) the fivefold axis. The VP1 pocket is occupied by a branched pocket factor, apparently different from that present in the closely related virus CVB3 and in other picornaviruses. This finding may be relevant for the design of new antiviral compounds against this site. Density consistent with the presence of ions was observed on the fivefold and threefold axes. The structure also provided an accurate description of the putative receptor binding sites.

A porcine CD8+ T Cell Clone with Heterotypic Specificity for Foot-and-mouth Disease Virus

Foot-and-mouth disease virus (FMDV)-specific T cell lines and clones have been obtained from a swine lymphocyte antigen (SLA) inbred miniature pig vaccinated with chemically inactivated virus. One of the clones obtained, CE3, showed a specific and heterotypic proliferation against infectious but not inactivated FMDV in the presence of syngeneic peripheral blood mononuclear cells (PBMC). Adherent cells from PBMC were sufficient to support specific activation of the clone and the proliferation was abolished when the contact between CE3 and adherent cells was prevented. Phenotypic characterization of CE3 cells revealed expression of CD2, CD25 (interleukin-2 receptor), SLA class I and SLA class II. Furthermore, the cells were highly CD8 positive but showed low expression of CD4. The expression of T cell receptor (TCR) alpha and beta genes confirmed their T cell nature. Consistent with the CD8 phenotype, the proliferative response of CE3 was inhibited with MAbs to SLA class I and CD8. Altogether, these results indicate that CE3 is a porcine SLA class I-restricted CD8+ T cell clone, that recognizes a heterotypic FMDV antigen.

Isolation and characterization of the main allergen of Dermatophagoides farinae by monoclonal antibodies that recognize IgE related epitopes

Mouse monoclonal antibodies (MAbs) with different specificities against Dermatophagoides farinae (D. farinae) extract have been obtained. Fifteen of these antibodies reacted with allergen molecules contained in D. farinae and D. pteronyssinus extracts, immunoprecipitating the main allergen of D. farinae (DF29) and homologous allergen of D. pteronyssinus (DP28). In addition, the monoclonal antibody MADF2 immunoprecipitated DF29 together with two other polypeptides (mol. wt 20,000 and 40,000) from D. farinae extracts. Five monoclonal antibodies (MADF2, MADF5, MADF9, MADF10 and MADF13) were selected to study their epitope specificity and the relationship of the epitope location on the allergen with the IgE binding site. By cross-inhibition studies two different epitopes and two partly overlapping determinants were found. In addition, two of these epitopes, those defined by MADF13 and MADF5, are close to, or overlapping, IgE binding site(s) on the allergen molecule. DF29 allergen from D. farinae extract was purified by affinity chromatography using MADF5 coupled to Sepharose. The purified allergen had capacity to bind mite specific human IgE and demonstrated an allergenic activity of up to 70% of total extract of D. farinae. These results indicate that DF29 molecule is the main allergen from D. farinae extracts.

Swine vesicular disease virus. Pathology of the disease and molecular characteristics of the virion.

Abstract Swine vesicular disease is a highly contagious disease of pigs that is caused by an enterovirus of the family Picornaviridae. The virus is a relatively recent derivative of the human coxsackievirus B5, with which it has high molecular and antigenic homology. The disease is not severe, and affected animals usually show moderate general weakening and slight weight loss that is recovered in few days, as well as vesicular lesions in the mucosa of the mouth and nose and in the interdigital spaces of the feet. However, the similarity of these lesions to those caused by foot-and-mouth disease virus has led to the inclusion of this virus in list A of the Office International des Epizooties. The disease has been eradicated in the European Union except in Italy, where it is considered endemic in the south. Nevertheless, as occasional outbreaks still appear and must be eliminated rapidly, European countries are on the alert and farms are monitored routinely for the presence of the virus. This circumstance has led to a considerable effort to study the pathology of the disease and the molecular biology and antigenicity of the virus, and to the development of optimized methods for the diagnosis of the infection.