Development of PCR tests for the detection of bovine herpesvirus-1, bovine respiratory syncytial viruses and pestiviruses

The development of PCR assays for detection of BHV-1, BRSV, BVDV and another pestiviruses is summarized. A polymerase chain reaction assay based on primers selected from the viral gI glycoprotein gene detected 3 fg pure BHV-1 DNA, 0.1-1.0 TCID50 or a single infected cell. No amplification was observed with DNA from BHV-2, BHV-3, BHV-4, OHV-1 or OHV-2. However, a fragment of the correct size (468 bp) was amplified using DNA from herpesviruses isolated from reindeer, red deer and goat. The PCR assay was able to detect virus in nasal swabs 1-14 days after experimental infection of cattle and there was a good correlation when PCR was compared to virus isolation for the detection of BHV-1 in clinical field samples. Detection of BHV-1 in fetal bovine serum and semen samples was also successful. PCR detecting a broad range of BVDV, BDV and HCV was developed. Of six sets of primers selected from different parts of the pestivirus genome the best results were provided by a pair 324/326 from the highly conserved 5'-non-coding region which gave an amplification with all 129 isolates tested. This panel consisted of 79 isolates from cattle, 33 from pigs and 17 from sheep. Differentiation between viruses was achieved by cleavage of the PCR-amplified products (288 bp) with the restriction endonucleases AvaI and BglI. The BVDV products were cleaved by AvaI, HCV by BglI and AvaI. Both enzymes, AvaI and BglI, did not cut the BDV products. A nested polymerase chain reaction assay was developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein. The sensitivity of PCR assay was 0.1 TCID50. No cross reaction was observed with nine heterologous respiratory viruses. PCR products of bovine and human RSV strains were discriminated using endonuclease ScaI, which specifically cleaved products of BRSV. PCR assay detected BRSV in nasal swabs collected from cattle in the acute stage of respiratory disease. In vitro amplification detected 31 positive samples of 35 while immunofluorescence only 23 samples.     

Immunobiology of bovine respiratory syncytial virus infections

This paper describes recent findings on the immunobiology of bovine respiratory syncytial virus (BRSV) infections. The pathobiology of alveolar macrophages and BRSV, and the immunological reaction of cattle to the virus after natural or experimental infection, or vaccination, were studied. Because in severe cases BRSV infection leads to lower respiratory tract disease, replication of BRSV in alveolar macrophages was studied. Alveolar macrophages, which are important aspecific defense cells in the lower respiratory tract, exhibited a high intrinsic resistance to BRSV. Furthermore, BRSV-infected alveolar macrophages produced significantly less nitric oxide (which has a bacteriocidal effect) than uninfected macrophages. The kinetics of antibody titres against the envelope protein G were different from those of antibody titres against the envelope protein F. For example, many animals that are reinfected do not possess antibodies against the G protein. After vaccination or after natural infection, antibody titres against the F and G protein, and against epitopes on the F protein, differed markedly, and also in animals with different MHC haplotypes. These findings may be related to differences in protection. The strains of BRSV that circulate in the Netherlands belong to the subgroups A and AB. There was no evidence for differences in virulence between these subgroups. BRSV could be detected in 30% of lungs obtained from calves suffering from severe lower respiratory tract disease. Based on cross-protection studies, calves that were infected with a virus from a particular BRSV subgroup were protected against reinfection with a virus from a different subgroup. A recombinant gE-protein negative bovine herpesvirus 1 vaccine carrying a gene encoding the G protein of BRSV, and a DNA vaccine encoding the same protein afforded protection after experimental challenge of calves. This offers the possibility to develop effective multivalent (gE-negative BHV1) marker vaccines in the future.     

A bovine model of vaccine enhanced respiratory syncytial virus pathophysiology

A critical issue has been the observation that vaccination of children with a formalin-inactivated respiratory syncytial virus (RSV) vaccine is associated with disease enhancement. We have taken advantage of bovine RSV and our experience with this disease in calves to develop a natural model that parallels human disease. Using formalin-inactivated bovine RSV vaccine calves were either sham-vaccinated/infected, vaccinated/infected, or vaccinated/sham-infected and their clinical signs, pulmonary function, and histological lung lesions quantitatively scored. Interestingly there was significantly greater disease in vaccinated/infected calves and histological lesions in calves were similar to those of affected children. Finally, we note that vaccination did not induce neutralizing antibodies, but IgG antibodies were detected by ELISA. Our model of RSV enhanced disease is important because it provides quantifiable evidence of disease severity that can be applied to evaluate the mechanisms of immunopathology and the safety of candidate RSV vaccines.     

Serological indication for persistence of bovine respiratory syncytial virus in cattle and attempts to detect the virus

To identify putative persistent bovine respiratory syncytial virus (BRSV) infections in cattle, seven cattle that had experienced BRSV infections were treated with corticosteroids for two periods of 5 days. During the 5-day periods and the 3 weeks after treatment, attempts were made to isolate BRSV from lung lavage fluid and nasal swab specimens. Fluorescent antibody tests were used to detect BRSV antigen in lung lavage cells. A BRSV specific polymerase chain reaction (PCR) assay was developed, and was performed on lung lavage samples of all seven cattle as well as on various tissues of five of the cattle. In addition, nasal swabs of 74 over-one-year-old cattle, in a closed dairy herd were also assayed by PCR. The virus or its RNA was not detected in putative carriers, by any of the methods used, whereas all positive controls were positive. After corticosteroid treatment, three of the seven cattle showed a fourfold rise in antibody titre, suggesting induction of virus replication. BRSV-seronegative sentinel calves, that were housed together with each corticosteroid-treated animal, did not develop antibodies to BRSV indicating that BRSV was not shed by corticosteroid-treated cattle, or was shed at a very low level. In addition BRSV was not detected in seropositive cattle in a closed farm in summer. Although we consider the rises in antibody titres against BRSV an indication for persistence of BRSV in cattle, BRSV or its RNA was not detected in infected cattle.     

Molecular heterogeneity for bovine alpha-mannosidosis: PCR based assays for detection of breed-specific mutations

Given the elective nature of photorefractive keratectomy (PRK), the high expectations of patients, and misconceptions of the general public about refractive surgery, the use of the excimer laser for PRK opens the door to new liability risks for ophthalmologists and, in the comanagement environment, referring optometrists. The authors discuss informed consent, marketing, comanagement, and off-label use guidelines and protocols to help protect ophthalmologists against claims and better defend those that might arise.     

Cell-mediated immune responses of lambs to challenge with bovine respiratory syncytial virus

(-)-Indolactam V (ILV) is a synthetic analog of the teleocidins, a newly described class of tumor promoters. Here we describe the mitogenic and comitogenic effect of (-)-ILV on human adult and fetal/neonatal (cord) lymphocytes. (-)-ILV induced proliferation in a dose-dependent manner, with optimal concentrations between 2.6 and 5.2 microM. Cord lymphocytes gave significantly lower (-)-ILV responses compared to the corresponding a2ult cells (50-60% lower at optimal mitogenic doses). The mitogenic action of (-)-ILV was apparently independent of the presence of adherent monocytes. H-7, a relatively specific protein kinase (PK) C inhibitor, and H-8, a less specific PK inhibitor, suppressed (-)-ILV-induced proliferation by 54 and 36%, respectively. Suboptimal concentrations of (-)-ILV were strongly mitogenic for both adult and cord lymphocytes when added together with calcium ionophore A23187, phytohemagglutinin, or the anti-T cell receptor monoclonal antibodies OKT3 and TCR1. In adult cells, the synergistic effect decreased with increasing concentrations of (-)-ILV or/and the other comitogens, and was, in most (> 60%) cases, converted to inhibition at the combination of optimal doses. The inhibition was invariably reversed by depletion of monocytes. In contrast, among cord lymphocytes (-)-ILV and OKT3 or TCR-1 showed strong cooperative effect at all concentrations tested. The (-)-ILV hyporesponsiveness and distinct comitogenic pattern of cord lymphocytes, together with the previously described lower cord cell response to phorbol ester TPA, suggest functional differences in signal transduction mechanisms between cord (naive) and adult (memory) T cells, possibly at the level of monocytes.     

Immune response of calves experimentally infected with non-cell-culture-passaged bovine respiratory syncytial virus

The immune response of calves was studied following infection with non-cell-passaged Bovine respiratory syncytial virus (BRSV). Two groups of 6 specific pathogen free (SPF) calves were housed in separate isolation rooms. One group was inoculated intranasally with a non-cell-passaged BRSV strain and the control group was mock-infected. A BRSV specific antibody response was observed for all the BRSV infected calves. These antibodies were shown to have neutralizing activity. No lymphocyte proliferation response was detected in the mock-infected group whereas three animals in the infected group were positive three weeks after the infection. All BRSV-infected calves, except one, produced interferon-gamma (IFN-gamma) one week post-infection and IFN-gamma was observed in all six infected calves after three weeks. The control group showed no IFN-gamma synthesis. In spite of the limits of the BRSV infection model, humoral and cellular immune responses were actively developed by all the calves against this pathogen.     

Diagnosis of meningoencephalitis caused by herpes simplex virus type 1 using nested PCR in CSF samples

BACKGROUND: The aim of this study was to achieve the early diagnosis of the neurologic alteration caused by the Herpes Simplex virus type 1 (HSV-1) with the nested PCR technique in CSF. PATIENTS AND METHODS: From January, 1994 to October, 1995, 140 CSF from 140 patients were studied in our laboratory. Ninety-five were diagnosed with viral meningoencephalitis (Group A) and 45 with other neurologic diseases (Group B). Nested PCR of HSV-1 and conventional viral cultures were carried out in all the cases. RESULTS: Laboratory diagnosis was achieved in 13 (13.68%) of Group A patients: in 12 (12.63%) HSV-1 genome was detected by nested PCR and in one patient adenovirus was isolated. In Group B, the HSV-1 was detected by nested PCR in 2 patients (4.44%). CONCLUSIONS: The results obtained demonstrate the usefulness of nested PCR in HSV-1 infection for the diagnosis of herpetic meningoencephalitis in initial stages of the disease, from a single CSF sample.     

Preparation of respiratory syncytial virus subgroup A and B antigens for enzyme immunoassay antibody detection

A simplified method was described for purification of respiratory syncytial virus (RSV) subgroup A and B aimed to be used as antigens in enzyme immunoassay (EIA). The titer of each RSV subgroup and the amount of protein was determined from the visible band in 45% sucrose gradient. The quality of prepared RSV subgroup antigens for EIA was described in terms of the achievable final titer, the amount of protein, and EIA criss-cross titration. The RSV subgroup A and B antigens, diluted as 1:100 (low opalescent band in 45% sucrose layer) or 1:800 (high opalescent band in 45% sucrose layer) produced a positive reaction in EIA criss-cross titration with IgG antibodies from the patient's serum (convalescent phase) diluted as 1:25,600 (for RSV A) and 1:6,400 (for RSV B). This method offers shorter and more simplified steps of viral antigen purification, and provides acceptable quantity and quality of viral antigens appropriate for use in EIA.     

Replication of bovine respiratory syncytial virus in bovine and ovine peripheral blood lymphocytes and monocytes and monocytic cell lines

The present study compared the replication of bovine respiratory syncytial virus (BRSV) in bovine and ovine peripheral blood mononuclear cells, ovine and bovine monocytic cell lines and ovine alveolar macrophages. Low titres of virus were detected in ovine and bovine lymphocytes and monocytes 24-96 h post-exposure to the virus but there was no apparent replication of the virus in ovine alveolar macrophages during the culture period. The virus replicated to higher but statistically insignificant titres in ovine and bovine peripheral blood monocytes than in lymphocytes, with lymphocytes yielding peak titres significantly earlier. The secondary cell lines obtained from ovine liver and bone marrow also supported the replication of BRSV to high titres. The titres of BRSV in ovine and bovine lymphocytes and monocytes were significantly lower than in secondary cell lines. The addition of human recombinant tumour necrosis factor alpha after exposure to the virus or pre-incubation of ovine or bovine monocytic cells with either human recombinant interleukin 2 or phorbol myristate acetate before exposure to BRSV, did not significantly affect virus titre. Pre-incubation of cells with indomethacin or actinomycin significantly lowered virus titre (p < 0.05).     

Antigenic structure of the central conserved region of protein G of bovine respiratory syncytial virus

Epitopes were resolved at the amino acid level for nine monoclonal antibodies (MAbs) directed against the central conserved region of protein G of bovine respiratory syncytial virus (BRSV-G). Peptide binding studies showed which amino acids in the epitope contributed to antibody binding. The details of the epitopes were compared with the high-resolution structure of a synthetic peptide corresponding to the central conserved region of BRSV-G, and this indicated which face of the central conserved region is the antigenic structure. The major linear epitope of the central conserved region of BRSV-G is located at the tip of the loop, overlapping a relatively flat surface formed by a double disulfide-bonded cystine noose. At least one, but possibly two sulfur atoms of a disulfide bridge that line the conserved pocket at the center of the flat surface, is a major contributor to antibody binding. Some of the residue positions in the epitope have mutated during the evolution of RSV-G, which suggests that the virus escaped antibody recognition with these mutations. Mutations that occur at positions 177 and 180 may have only a local effect on the antigenic surface, without influencing the structure of the backbone, whereas mutations at positions 183 and 184 will probably have major structural consequences. The study explains the antigenic, structural, and functional importance of each residue in the cystine noose which provides information for peptide vaccine design. Additionally, analysis of the epitopes demonstrated that two point mutations at positions 180 and 205 define the preliminary classification of BRSV subgroups.     

Animal models of respiratory syncytial virus infection

Over the past two decades, animal models of respiratory syncytial virus (RSV) infection have been developed using primates, cotton rats, mice, calves, guinea pigs, ferrets, and hamsters. Use of these models has shed light on the mechanisms of vaccine-enhanced disease seen in clinical trials of a formalin-inactivated RSV vaccine and has provided a means for testing efficacy and safety of candidate prophylactic and therapeutic strategies. The development of multiple animal models has coincided with the realization that RSV disease in humans is a multifaceted disease whose clinical manifestations and sequelae depend upon age, genetic makeup, immunologic status, and concurrent disease within subpopulations. There is no single human subpopulation in whom all forms of RSV disease manifest, nor is there a single animal model that duplicates all forms of RSV disease. The choice of an experimental model will be governed by the specific manifestation of disease to be studied.     

Analysis of the bovine respiratory syncytial virus fusion protein (F) using monoclonal antibodies

Seven monoclonal antibodies (MAbs) directed against bovine respiratory syncytial virus (BRSV) fusion (F) protein were produced and characterized by radioimmunoprecipitation and immunofluorescence assays. These seven MAbs together with the previously described MAbs (Beeler and Van Wyke Coelingh, 1989) to the F protein of human respiratory syncytial virus (HRSV) were used to study the antigenic variation of 12 strains of ungulate RSV. All except one MAbs specific for the HRSV-F protein reacted with ungulate RSV strains less efficiently, indicating that some epitopes are conserved, and others are not conserved on the F proteins of HRSV and BRSV strains. Three MAbs specific to the BRSV-F protein neutralized virus infectivity and reacted with all the ungulate RSV strains, suggesting that these epitopes are well conserved. Based on the reactivity of three other MAbs specific to the BRSV-F protein, ungulate RSVs could be grouped into two subgroups. The results indicated that there are antigenic variations in the F protein among ungulate RSV strains.     

Milk production and reproductive performance in dairy cows given bovine respiratory syncytial virus vaccine prior to parturition

OBJECTIVE: To assess the effect of vaccination against bovine respiratory syncytial virus on milk production, reproductive performance, and health in lactating dairy cows. DESIGN: Prospective randomized block design. ANIMALS: 385 Holstein dairy cows and heifers. PROCEDURE: Cows were grouped by lactation number, season of calving, and previous mature equivalent 305-day milk production (where appropriate). Prior to parturition, cows and heifers were randomly assigned to be vaccinated i.m. against infectious bovine rhinotracheitis, bovine viral diarrhea, and parainfluenza 3 viruses by use of a three-way vaccine, or to be vaccinated against those viruses as well as bovine respiratory syncytial virus, using a four-way vaccine. Milk production was measured daily through 305 days of lactation. Reproductive and medical records were reviewed to obtain insemination dates and record medical problems of cows in each vaccine treatment group. RESULTS: Compared with the three-way vaccine, administration of the four-way vaccine was associated with higher milk production (1.39 kg [3.06 lb] more milk/d) in first-parity cows during the first 21 weeks of lactation. Vaccination did not have any effect on milk production after the first 21 weeks of lactation in cows of any parity. Conception rates at first insemination were higher for four-way vaccinated first-parity cows than for three-way vaccinated first-parity cows (54.6 vs 32.7%). Compared with second-parity cows that received the three-way vaccine, first insemination conception rate was improved for second-parity cows vaccinated with the four-way vaccine (28.9 vs 47.8%, respectively). In cows of third or greater parity, first insemination conception rate was not different between the 2 vaccine treatment groups. CLINICAL IMPLICATIONS: Vaccination of heifers against bovine respiratory syncytial virus prior to partrition may increase milk production and first insemination conception rates.     

Quantification of Bordetella pertussis in clinical samples by colorimetric detection of competitive PCR products

Quantification of microorganisms is an important part of the normal diagnostic work of a clinical microbiology laboratory. Traditionally the diagnosis of pertussis is subject to a yes or no approach with no quantitative dimension. This can, however, be of interest as a factor when judging the risk of a patient spreading the bacterium and as a research tool. The aim of the present study was to develop a PCR-based quantitative assay for Bordetella pertussis DNA in clinical nasopharyngeal aspirates by combining a quantitative PCR with a colorimetric detection principle, DIANA (detection of immobilised amplified nucleic acid). A competitor to the PCR target sequence in IS-481, containing a lac-operator, was constructed and calibrated, and a test protocol prepared. A total of 46 clinical nasopharyngeal aspirates, previously diagnosed using a standard nested PCR assay and quantified by culture, were analysed by the quantitative PCR. The method showed acceptable precision and accuracy considering that it estimates the total number of bacterial genomes while culture detects viable bacteria. Recognised advantages were the simple colorimetric detection, the inborn indication of a working PCR assay, and the possibility of obtaining results even when partial inhibition of the PCR assay was seen. In addition, the quantitative PCR result can be obtained within one day compared to 3-10 days for culture. The present results and the qualities of the quantitative PCR suggest that this assay will be a useful complement in routine diagnostics and in research.     

A model for respiratory syncytial virus (RSV) infection based on experimental aerosol exposure with bovine RSV in calves

Five conventionally kept calves aged between 17 and 24 days were experimentally infected with bovine respiratory syncytial virus (BRSV) by aerosol in order to mimic the natural infection route. The calves were killed and autopsies performed 7 days after the first virus challenge. The BRSV isolate used induced tracheitis, bronchitis and atelectasis in infected calves. The only virus which could be isolated from the lungs of the calves was BRSV. In addition, Mycoplasma bovirhinis was isolated from the lungs or/and trachea of two calves. The clinical and histopathological findings, as well as the detection of BRSV antigens by immunofluorescence in the epithelial cells of lung and trachea, and the reisolation of the virus from bronchoalveolar lavage fluids of all inoculated calves, provided confirmation of successful infection with BRSV.     

A monoclonal antibody pool for routine immunohistochemical detection of human respiratory syncytial virus antigens in formalin-fixed, paraffin-embedded tissue

Four monoclonal antibodies (MAbs) with specificities for epitopes on human respiratory syncytial virus (RSV) proteins preserved after formalin fixation and paraffin embedding were identified in fixed and embedded virus-infected HEp-2 cell pellets. The MAbs bound epitopes on the fusion protein, the nucleoprotein, the phosphoprotein, and the M2 protein of the virus. Following high-temperature antigen unmasking, immunohistochemical staining revealed RSV antigens in the lungs of five of seven children who died with confirmed RSV infection and in none of nine children who died for other reasons, with no evidence of RSV infection. Staining was cytoplasmic, granular, and confined to epithelial cells. Intense staining was seen at the apex of ciliated bronchial and bronchiolar epithelial cells in all five positive cases. In one case, of pneumonitis, infected pneumocytes were present in the alveoli and in several cases, CD68-positive, cytokeratin-negative alveolar macrophages stained for viral antigens. These antibodies may prove useful in studies of the pathogenesis of RSV infection.     

An experimental study of a concurrent primary infection with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) in calves

Experimental infections with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) were performed to study the effect of concurrent BRSV and BVDV infections. Twelve seronegative calves, in 3 groups, were inoculated on a single occasion with pure BRSV (group A), BRSV and noncytopathogenic BVDV (group B) or mock infected (group C). Mild respiratory symptoms were recorded 4 to 5 days post inoculation (dpi) in group A and group B calves. One calf in group A was severely affected and required medical treatment. In group B, fever (40.7-41.4 degrees C) was prominent 7 to 8 dpi. Only calves in group B were BVDV positive in purified lymphocytes at 2 to 14 dpi and showed increased serum interferon levels, with a peak at 4 dpi, indicating BVDV to be responsible for inducing the rise. BRSV was detected in lung lavage fluids up to 7 dpi for group A calves, compared to 11 dpi for group B and calves in this group also seroconverted later displaying lower BRSV titers. The time lag before an antibody response and the titers recorded in group B, indicated that the duration of BVDV infection in lymphocytes negatively influenced the capacity to mount a BRSV antibody response.