Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection.

Canine distemper virus (CDV) infection of ferrets causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system (CNS). We have tested candidate CDV vaccines incorporating the fusion (F) and hemagglutinin (HA) proteins in the highly attenuated NYVAC strain of vaccinia virus and in the ALVAC strain of canarypox virus, which does not productively replicate in mammalian hosts. Juvenile ferrets were vaccinated twice with these constructs, or with an attenuated live-virus vaccine, while controls received saline or the NYVAC and ALVAC vectors expressing rabies virus glycoprotein. Control animals did not develop neutralizing antibody and succumbed to distemper after developing fever, weight loss, leukocytopenia, decreased activity, conjunctivitis, an erythematous rash typical of distemper, CNS signs, and viremia in peripheral blood mononuclear cells (as measured by reverse transcription-PCR). All three CDV vaccines elicited neutralizing titers of at least 1:96. All vaccinated ferrets survived, and none developed viremia. Both recombinant vaccines also protected against the development of symptomatic distemper. However, ferrets receiving the live-virus vaccine lost weight, became lymphocytopenic, and developed the erythematous rash typical of CDV. These data show that ferrets are an excellent model for evaluating the ability of CDV vaccines to protect against symptomatic infection. Because the pathogenesis and clinical course of CDV infection of ferrets is quite similar to that of other Morbillivirus infections, including measles, this model will be useful in testing new candidate Morbillivirus vaccines.

Identification of a putative cellular receptor for bovine herpesvirus 1.

Recognition of a host cell receptor by a virus is the first and perhaps the most crucial step in initiating the disease process. This study was undertaken to identify the cellular receptor(s) for bovine herpesvirus 1 (BHV-1). Previously, we reported the development and characterization of bovine anti-idiotypic antibodies (anti-ids) that induce neutralizing antibodies to BHV-1. These anti-ids inhibit BHV-1 penetration of permissive cells. We have used these anti-ids, which mimic an epitope on the virus glycoprotein IV (gIV), and gradient-purified virus in immunoprecipitation (IP) as well as photoaffinity labelling (PAL) assays. In the IP assays, both bovine anti-ids and BHV-1 virions coupled to Sepharose precipitated a 60K protein from 125I-labelled BHV-1 permissive cell membrane extracts. Normal bovine IgG or an irrelevant virus, transmissible gastroenteritis virus (TGEV), used as negative controls failed to precipitate this protein. Similarly, in the PAL assays, the 60K cell surface protein was identified on cells permissive for BHV-1 infection, but not on non-permissive cells when 125I-labelled ligands, the anti-ids or BHV-1 were used as probes. The iodinated ligands failed to identify the 60K protein if they had been pretreated with the antibody 1. Pretreatment of the iodinated ligands with an isotype-matched control antibody had no effect on the identification of the 60K protein present on cells permissive for BHV-1 infection. The negative controls, i.e. normal bovine IgG and TGEV, failed to identify this 60K protein on permissive or non-permissive cells. These results suggest that the 60K protein is a cellular receptor recognized by BHV-1 during the infection process.

Detection of c-sis proto-oncogene transcripts by direct enzyme-labeled cDNA probes and in situ hybridization.

Using in situ hybridization and platelet-derived growth factor (PDGF) cDNA probes labeled with horseradish peroxidase, PDGF-A and -B (c-cis proto-oncogene) mRNA transcripts were identified and localized in proliferating cultures. A human retinal pigment epithelial (RPE) cell line and a glial cell line were treated with either transforming growth factor beta-1 (TGFB1), phorbol-12-myristate-13-acetate (PMA), or thrombin from human plasma and compared for their ability to stimulate the production of PDGF-A and -B. Expression of both PDGF-A and -B transcripts were found to be localized predominantly in the cytoplasm of TGFB1-treated RPE cells, with a portion of these cells displaying a hybridization response in the nuclear region. When compared to PMA- and thrombin-treated cells, TGFB1 stimulated the RPE cell line to yield the greatest amount of detectable PDGF mRNA. In addition, the hybridization response observed in TGFB1-treated cells was shown to be RNA dependent.

Culture confirmation of cytomegalovirus and herpes simplex virus by direct enzyme-labeled DNA probes and in situ hybridization.

Using probes consisting of horseradish peroxidase (HRP) directly attached to DNA, scrapings or trypsinized cells from 217 adequate clinical samples were cultured and analyzed in 3 blind studies by in situ hybridization for the presence of cytomegalovirus (CMV) and herpes simplex virus (HSV). Sixty samples were judged inadequate due to insufficient cell numbers; however, this problem was significantly decreased during the course of the study. One hundred and eighteen samples were found positive and 70 samples were found negative for CMV. Scrapings of cultured cells from 29 clinical samples revealed 9 samples which were positive and 20 samples which were negative for HSV. Forty-two additional samples, containing either uninfected cells or cells infected with various strains of CMV, were analyzed for the ability of the HRP-DNA CMV probe to detect such isolates. Twenty samples were positive and 22 negative for CMV. No false-negatives or false-positives were observed for either CMV or HSV. In addition to the specificity noted above neither the CMV nor the HSV DNA probe hybridized to potential contaminants found in clinical specimens.

DNA restriction endonuclease cleavage pattern and protein antigen profile of Ehrlichia risticii.

Molecular characterization of Ehrlichia risticii, the etiological agent of Potomac horse fever, was performed. Restriction endonuclease cleavage of E. risticii DNA generated distinct patterns by different enzymes. The DNA cleavage patterns of E. risticii isolates obtained from different geographic regions were similar. Protein analysis identified thirty-five distinct proteins with molecular weights ranging from 160 to 16 kilodalton (kDa). Antigenic analysis by radioimmunoprecipitation using 125I surface labeled E. risticii and by Western blotting determined the presence of eighteen antigens (160, 110, 86, 84, 81, 70, 55, 51, 49, 44, 41, 36, 33, 31, 28, 24, 22 and 16 kDa) of which nine (110, 86, 70, 55, 51, 49, 44, 33, and 28 kDa) were major antigens. Fourteen of these antigens, which included the major antigens, were apparent surface components. There were no heat-modifiable proteins but lipopolysaccharide components of 245 and 14 kDa, resistant to proteinase K and of non-antigenic character, were detected in the organism.

Molecular cloning of Ehrlichia risticii and development of a gene probe for the diagnosis of Potomac horse fever.

A gene bank of Ehrlichia risticii was constructed in plasmid vector pUC13. Five clones representing discrete regions of the E. risticii genome were tested for their ability to hybridize specifically to E. risticii DNA. None of the clones cross-hybridized with Ehrlichia equi DNA, whereas four of these clones cross-hybridized with Ehrlichia canis and Ehrlichia sennetsu DNAs. However, one clone carrying a 1-kilobase HindIII fragment of E. risticii DNA failed to cross-react with the genomes of E. sennetsu, E. canis, and E. equi in dot blot hybridization assays. The sensitivity of this probe for the detection of E. risticii DNA was approximately 0.5 pg. By using this probe, the E. risticii DNA was detected in the peripheral blood mononuclear cells of 30 experimentally infected horses by 7 days postinfection (p.i.); the detection of E. risticii DNA peaked between 14 and 17 days p.i., a period immediately after the peak of the second rise in body temperature, during leukopenia and at the onset of diarrhea. E. risticii DNA was not detectable by 25 to 30 days p.i. E. risticii DNA was not detected in noninfected control horses.

Comparison of in situ hybridization and monoclonal antibodies for early detection of cytomegalovirus in cell culture.

The abilities of each of four diagnostic tests--direct fluorescent monoclonal antibody (direct FA) staining, indirect fluorescent monoclonal antibody (indirect FA) staining, in situ hybridization with biotinylated DNA probes, and in situ hybridization with DNA probes directly linked to enzymatically active horseradish peroxidase-to detect cytomegalovirus soon after culture were compared. Only the indirect FA test and the in situ hybridization method with directly linked HRP-DNA probes provided consistent and reliable cytomegalovirus detection as early as 15 h postinfection.

In vitro interference between equine herpesvirus types 1 and 2.

Interference between equine herpesvirus types 1 (EHV-1) and 2 (EHV-2) was studied in equine dermis (ED) monolayer cell cultures and equine lymphocyte cultures. Cell cultures were infected with EHV-2, and after a short incubation period, the cultures were superinfected with EHV-1. At various intervals, different measurements of EHV-1 expression in dually infected cultures, compared with those in cultures infected with EHV-1 alone, were studied. In dually infected ED cell cultures, the EHV-1 cytopathic effect, EHV-1 titer, and EHV-1 enzyme-linked immunosorbent assay antigen titer were maximally reduced to values of 40%, 58.5%, and 54.9%, respectively, at postsuperinfection hour (PSIH) 36. Values of these EHV-1 expressions were subsequently increased at PSIH 48. However, thymidine kinase activity was reduced to a maximum of 67.3% reduction at PSIH 48. In dually infected lymphocyte cultures, the EHV-1 titer, EHV-1 infective centers, EHV-1 enzyme-linked immunosorbent assay antigen titer, and thymidine kinase activity were maximally reduced to values of 77.4%, 78.7%, 98.3%, and 72.9%, respectively, at PSIH 24. These reductions of EHV-1 expressions were completely abrogated at PSIH 48 to 72. In both cell culture systems, a marked interference of EHV-1 by EHV-2 was observed; this was transient in the lymphocyte cultures, but was more prolonged in ED cell cultures. This interference appeared not to be interferon mediated. The multiplication of EHV-2 in the dually infected ED cell cultures appeared unaffected.