Genetic and antigenic characterization of Bungowannah virus, a novel pestivirus.

Bungowannah virus, a possible new species within the genus Pestivirus, has been associated with a disease syndrome in pigs characterized by myocarditis with a high incidence of stillbirths. The current analysis of the whole-genome and antigenic properties of this virus confirms its unique identity, and further suggests that this virus is both genetically and antigenically remote from previously recognized pestiviruses. There was no evidence of reactivity with monoclonal antibodies (mAbs) that are generally considered to be pan-reactive with other viruses in the genus, and there was little cross reactivity with polyclonal sera. Subsequently, a set of novel mAbs has been generated which allow detection of Bungowannah virus. The combined data provide convincing evidence that Bungowannah virus is a member of the genus Pestivirus and should be officially recognized as a novel virus species.

Broad spectrum reactivity versus subtype specificity-trade-offs in serodiagnosis of influenza A virus infections by competitive ELISA.

Avian influenza viruses (AIVs) of the H5 and H7 subtypes can cause substantial economic losses in the poultry industry and are a potential threat to public health. Serosurveillance of poultry populations is an important monitoring tool and can also be used for control of vaccination campaigns. The purpose of this study was to develop broadly reactive, yet subtype-specific competitive ELISAs (cELISAs) for the specific detection of antibodies to the notifiable AIV subtypes H5 and H7 as an alternative to the gold standard haemagglutination inhibition assay (HI). Broadly reacting monoclonal competitor antibodies (mAbs) and genetically engineered subtype H5 or H7 haemagglutinin antigen, expressed and in vivo biotinylated in insect cells, were used to develop the cELISAs. Sera from galliform species and water fowl (n=793) were used to evaluate the performance characteristics of the cELISAs. For the H5 specific cELISA, 98.1% test sensitivity and 91.5% test specificity (97.7% and 90.2% for galliforms; 98.9% and 92.6% for waterfowl), and for the H7 cELISA 97.3% sensitivity and 91.8% specificity (95.3% and 98.9% for galliforms; 100% and 82.7% for waterfowl) were reached when compared to HI. The use of competitor mAbs with broad spectrum reactivity within an AIV haemagglutinin subtype allowed for homogenous detection with high sensitivity of subtype-specific antibodies induced by antigenically widely distinct isolates including antigenic drift variants. However, a trade-off regarding sensitivity versus nonspecific detection of interfering antibodies induced by phylo- and antigenically closely related subtypes, e.g., H5 versus H2 and H7 versus H15, must be considered. The observed intersubtype antibody cross-reactivity remains a disturbance variable in AIV subtype-specific serodiagnosis which negatively affects specificity.

Detection of European porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridization-immunohistochemistry double labelling.

Two groups of five pigs aged 6 weeks were each infected oronasally with one of two different European isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The animals were killed sequentially at 4, 7, 14 or 21 days post-inoculation for examination. The methods used consisted of histopathology, and mono- and double-labelling techniques based on in-situ hybridization, immunofluorescence and immunohistochemistry. Porcine alveolar macrophages (PAMs) contained large amounts of PRRSV antigen and PRRSV RNA, as shown by double labelling with (1) either PRRSV immunofluorescence or PRRSV-specific in-situ hybridization with digoxigenin-labelled riboprobes, and (2) immunolabelling with Mac 387 antibody for calprotectin. Expression of PRRSV-RNA was not detectable in cytokeratin-positive hypertrophic and proliferating pneumocytes or in cells of alveolar ducts or bronchiolar epithelium. The use of two-colour immunofluorescence with confocal laser scanning microscopy and double labelling with in-situ hybridization-immunohistochemistry showed that PAMs were the only pulmonary target cells. This contradicts earlier reports that epithelial pulmonary cells may also be infected by PRRSV.

Porcine teschoviruses comprise at least eleven distinct serotypes: molecular and evolutionary aspects.

Nucleotide sequencing and phylogenetic analysis of 10 recognized prototype strains of the porcine enterovirus (PEV) cytopathic effect (CPE) group I reveals a close relationship of the viral genomes to the previously sequenced strain F65, supporting the concept of a reclassification of this virus group into a new picornavirus genus. Also, nucleotide sequences of the polyprotein-encoding genome region or the P1 region of 28 historic strains and recent field isolates were determined. The data suggest that several closely related but antigenically and molecular distinct serotypes constitute one species within the proposed genus Teschovirus. Based on sequence data and serological data, we propose a new serotype with strain Dresden as prototype. This hitherto unrecognized serotype is closely related to porcine teschovirus 1 (PTV-1, former PEV-1), but induces type-specific neutralizing antibodies. Sequencing of field isolates collected from animals presenting with neurological disorders prove that other serotypes than PTV-1 may also cause polioencephalomyelitis of swine.

Detection of porcine enteroviruses by nRT-PCR: differentiation of CPE groups I-III with specific primer sets.

Porcine enteroviruses (PEV) comprising at least 13 serotypes grouped into three species are described as causative agents of neurological disorders, fertility disorders, and dermal lesions of swine. Despite their well-documented acid stability, enteric infection route, and similarity of clinical symptoms, most of the porcine enterovirus (PEV) serotypes are set apart from the genus Enterovirus of the Picornaviridae. Hence, PCR procedures used commonly to detect enteroviruses are not applicable to epizootic relevant PEV serotypes. A nested RT-PCR protocol is described now suited to detect all known porcine enterovirus serotypes using three sets of primer pairs. These primer pairs were designed to amplify either highly conserved sequences of the 5'-nontranslated region (5'-NTR) or the polymerase gene region of the relevant virus species. All 13 acknowledged serotypes of three PEV species and several field isolates of clinical specimens were detectable. The specificity of the PCR procedure is supported by the observation that RT-PCR-positive field isolates coincide with serological PEV classification. PEV PCR is more rapid and less laborious than the time-consuming virus isolation by tissue culture techniques over several passages and serotyping. Because other viruses such as classical swine fever virus, pseudorabies virus, porcine parvovirus, swine vesicular disease virus, and foot-and-mouth disease virus may cause diseases with similar clinical symptoms, PCR detection of all PEVs closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the diagnosis of all relevant viruses causing such symptoms.

Identification of group I porcine enteroviruses by monoclonal antibodies in cell culture.

A panel of monoclonal antibodies (mAb) against porcine enteroviruses (PEV) was established. One of these mAbs reacts group-specifically with PEV of serotype group I in the indirect immunofluorescence assay (IIF). This mAb is very well suited for diagnosis of PEV infections. However, the mAb neither neutralizes virus nor does it react with virus particles in immuno electron microscopy (IEM). Another mAb is PEV-1 specific in IIF, neutralizes virus, and is suited for IEM. Both mAbs presumably recognize conformation-dependent epitopes of the virus.

Early diagnosis and treatment of blunt diaphragmatic injury.

Diaphragmatic rupture following blunt torso trauma is an infrequent injury often posing a considerable diagnostic challenge. Between June 1984 and July 1986, nine patients sustaining rupture of the diaphragm due to blunt trauma were treated. All were injured in high-speed motor-vehicle accidents. Six patients arrived in shock (SBP less than or equal to 95 mm Hg). All patients had multiple associated injuries; mean ISS = 41.5. Eight patients (89%) had associated intra-abdominal injuries, the spleen being injured most frequently (63%). Associated thoracic injuries occurred in six patients (67%). The admission CXR was abnormal in eight patients (89%) but diagnostic of diaphragmatic rupture in only four. DPL was positive in four of five patients (80%) and falsely negative in one. Seven ruptures were left-sided, one right-sided and one involved the central tendon. All injuries were diagnosed shortly after admission and successfully repaired via a transabdominal approach. One patient died as a result of a massive subdural hematoma. Blunt diaphragmatic rupture is an indication of a high-energy insult and is usually associated with other major organ system injuries. An aggressive approach to the management of the multiply injured patient can result in early recognition and successful treatment of this injury.

Predicting medical specialty choice: a model based on students' records.

A discriminant analysis of objective and subjective measures from the records of 628 students who graduated from the University of Medicine and Dentistry of New Jersey-New Jersey Medical School over a six-year period was used to generate a model for the prediction of medical specialty choice. The authors found that National Board of Medical Examiners Part II examination scores, sex, race, grades given by preceptors, and a score derived from narrative comments by preceptors during clinical experiences in psychiatry contained information for predicting such a choice. With this model, the correct prediction rate for all specialties was 41 percent. The correct prediction rate for individual specialties ranged from a low of 28 percent for family practice to a high of 68 percent for psychiatry.