Dirk Deregt

Bovine viral diarrhea virus in alpaca: abortion and persistent infection

An alpaca herd in eastern Ontario experienced vague signs of illness, including anorexia and lethargy in 9 animals, 2.5 months after the addition of a chronically ill cria and his dam to the farm. Subsequently 2 alpaca had early pregnancy loss; one aborted at 5.5 months gestation and the other at 7 months gestation. Seventeen were found to have serum antibody to bovine viral diarrhea virus (BVDV), with highest titers to BVDV type 1. The fetus that was aborted at 5.5 months gestation, 3 months after the clinical outbreak, was found to be positive for BVDV on immunohistochemical staining, and noncytopathic BVDV type 1b was isolated. Of the 13 cria born alive that season, a single male underweight alpaca cria, born 9 months after the clinical illnesses, was infected with BVDV type 1b. The cria was positive for BVDV at birth, at 3 and 26 days of age and continued to be positive for noncytopathic BVDV using virus isolation, nested reverse transcription PCR, antigen detection ELISA, and immunohistochemical staining until euthanasia at 46 days of age. The cria remained serum antibody negative to both BVDV type 1 and type 2. A diagnosis of persistent infection was made. This is the first report describing persistent infection with BVDV in an alpaca cria.

Structural proteins of bovine coronavirus and their intracellular processing.

The Quebec isolate of bovine coronavirus (BCV) was found to contain four unique major structural proteins. These proteins consisted of the peplomeric protein (gp190/E2, gp100/E2), the nucleocapsid protein (p53/N) and its apparent trimer (p160/N), a family of small matrix glycoproteins (gp26/E1, gp25/E1 and p23/E1) and the putative haemagglutinin (gp124/E3). Pulse-chase experiments utilizing polyclonal antiserum and monoclonal antibodies indicated that the unique BCV E3 protein had its primary precursor an N-linked glycoprotein with an Mr of 59,000 (gp59) which underwent rapid dimerization by disulphide bond formation to yield gp118. Further glycosylation of gp118 produced gp124/E3 which incorporated fucose. Thus gp124/E3 was probably a homodimer. The processing of the E2 and E1 proteins of BCV was similar to that shown previously for mouse hepatitis virus. A large N-linked precursor glycoprotein, gp170, underwent further glycosylation to yield gp190/E2 before subsequent proteolytic cleavage to yield gp100/E2. The glycosylated E1 (gp26, gp25) proteins arose as a result of O-linked glycosylation of p23/E1 as indicated by the resistance of these species to tunicamycin.

Mapping of a type 1-specific and a type-common epitope on the E2 (gp53) protein of bovine viral diarrhea virus with neutralization escape mutants.

Bovine viral diarrhoea viruses (BVDV) have recently been segregated into two genotypes, BVDV 1 and BVDV 2. However, the antigenic differences and similarities of BVDV 1 and BVDV 2 remain poorly defined. In this study, the E2 epitopes of two neutralizing monoclonal antibodies (mAbs) produced against an isolate of BVDV 1 were mapped. The mAb 157, previously determined to be broadly cross-reactive to BVDV, was discovered to be BVDV 1-specific, whereas mAb 348 bound to and neutralized BVDV 2. Both mAbs bound to epitopes within the first 192 amino acids of the E2 protein as determined by reactions with a C-terminally truncated E2. To identify critical amino acids affecting these epitopes, mAb escape mutants were selected for sequencing from BVDV 1 and BVDV 2 strains with different (wild-type) mAb binding phenotypes. In addition, the E2 gene of several BVDV were sequenced and the sequences were compared with amino acid changes in mutant viruses. Single nucleotide changes in escape mutants selected with mAb 157 resulted in deduced amino acid changes at E2 positions 9, 32 or 72. Amino acid changes at position 72 also affected the epitope of mAb 348. Alignment of E2 nucleotide sequences revealed that BVDV 2 are missing six nucleotides encoding the equivalent of amino acids 31 and 32 of BVDV 1 and thus, this difference can account for the BVDV 1-specificity of mAb 157. Single nucleotide mutations in mAb 348 escape mutants of BVDV 1 and BVDV 2 resulted in changes in 3 amino acids in the previously described immunodominant 71-74 region (Virology 190, 763-772). A fourth amino acid change observed in a mutant of BVDV 2 extended this region to position 77. Thus, the amino acid changes affecting the conserved epitope of mAb 348 occurred in a short spatial array over only seven amino acids, unlike the described composite epitopes previously mapped to this region.

Monoclonal antibodies to the E2 protein of a new genotype (type 2) of bovine viral diarrhea virus define three antigenic domains involved in neutralization.

Bovine viral diarrhea virus (BVDV) has recently been segregated into two genotypes, namely, BVDV 1 and BVDV 2. Viruses of the BVDV 2 genotype are a cause of hemorrhagic and acute fatal disease in cattle in the US and Canada. In this study, monoclonal antibodies (mAbs) to the newly described BVDV 2 were produced after immunization with virus or a combination of virus and E2 peptide. From an original panel of 17 mAbs, 13 mAbs were identified as E2-specific by reactivity with a BVDV 2 recombinant E2 protein expressed in insect cells. Nine E2 mAbs were observed to be virus-neutralizing. The E2 epitopes represented by the mAbs were found to be highly conserved among BVDV 2 isolates associated with hemorrhagic or severe disease in cattle. Except for one virus-neutralizing E2 mAb, the mAbs showed few or relatively weak cross-reactions with BVDV 1. Two non-neutralizing E2 mAbs were BVDV 2-specific. In contrast to BVDV 1 for which conserved neutralizing epitopes have been mapped in one immunodominant domain, the virus-neutralizing E2 mAbs produced to BVDV 2 were found to bind to highly conserved epitopes in three antigenic domains.

DNA prime protein boost strategies protect cattle from bovine viral diarrhea virus type 2 challenge.

At present, infections with bovine viral diarrhea virus (BVDV) type 2 occur nearly as frequently as those with BVDV type 1, so development of vaccines that protect cattle from both type 1 and type 2 BVDV has become critical. In this study, we compared various DNA prime-protein boost vaccination strategies to protect cattle from challenge with BVDV-2 using the major protective antigen of BVDV, glycoprotein E2. Calves were immunized with a plasmid encoding either type 1 E2 (E2.1) or type 2 E2 (E2.2) or with both plasmids (E2.1+E2.2). This was followed by a heterologous boost with E2.1, E2.2 or E2.1 and E2.2 protein formulated with Emulsigen and a CpG oligodeoxynucleotide. Subsequently, the calves were challenged with BVDV-2 strain 1373. All vaccinated calves developed both humoral and cell-mediated immune responses, including virus-neutralizing antibodies and IFN-gamma-secreting cells in the peripheral blood. Depletion studies showed that CD4+ T cells were responsible for IFN-gamma production. Furthermore, the calves vaccinated with either the E2.2 or the E2.1+E2.2 vaccines were very well protected from challenge with BVDV-2, having little leukopenia and showing no weight loss or temperature response. In addition, the animals vaccinated with the E2.1 vaccine were partially protected, so there was a certain level of cross-protection. These data demonstrate that a vaccination strategy consisting of priming with E2.2 or E2.1+E2.2 DNA and boosting with E2.2 or E2.1+E2.2 protein fully protects cattle from BVDV-2 challenge.

A Comparison of Polymerase Chain Reaction with and without RNA Extraction and Virus Isolation for Detection of Bovine Viral Diarrhea Virus in Young Calves

Previously, the authors described a multiplex reverse transcriptase–polymerase chain reaction (PCR) assay for detection and typing of bovine viral diarrhea virus (BVDV) from blood of persistently infected (PI) cattle that could be used with or without RNA extraction. In the present study, the PCR assay was evaluated for its ability to detect BVDV in young calves as a screening tool for detection of persistent infections. Both methods, PCR after RNA extraction (rPCR) and the direct method without RNA extraction (dPCR) were applied and compared with virus isolation (VI) with diagnostic specimens. From 450 whole blood samples from Ontario calves, 47 and 39 samples were positive by rPCR and VI, respectively. From the 47 samples positive by rPCR, 45 (96%) also were positive by dPCR when samples were tested both undiluted and diluted 1:10. In comparison to VI, the relative sensitivities of both PCR assays were 100%. Examination of the results indicates that both PCR assays can be used for screening calves for persistent infection with BVDV.

Structural analysis of the conformational domains involved in neutralization of bovine coronavirus using deletion mutants of the spike glycoprotein S1 subunit expressed by recombinant baculoviruses

Abstract Two conformation-dependent neutralizing epitopes, A and B, have been mapped to the S1 subunit of the S spike glycoprotein of bovine coronavirus (BCV). In order to characterize the structure of these antigenic sites, we constructed a series of cDNA clones encoding deleted or truncated S1 derivatives and expressed the modified genes in insect cells using recombinant baculoviruses. Monoclonal antibodies directed against epitopes A and B recognized only the mutant S1 polypeptides containing amino acids 324–720, as demonstrated by immunoprecipitation and Western blot analysis in the absence of β-mercaptoethanol. In addition, two domains within this region were identified and only mutants containing both domains were immunoreactive, indicating that both were critical in the formation of the antigenic determinants. One domain was localized between residues 324 and 403 and the other at residues 517–720. Deletion of either domain inhibited extracellular secretion of the mutant proteins whereas mutants containing both or none of the domains were secreted efficiently. This observation suggests a vital function of the native conformation of the S1 protein in both antigenic structure and intracellular transport. Antigenic determinants A and B were not distinguished, but these determinants appeared to require both domains for epitope formation. Our results suggest that the antigenic determinants formed by two domains are likely associated with the probable polymorphic region of the BCV S1 subunit.

Isolation of bovine viral diarrhoea viruses from bison

intranasally with 2 x 107 TCID50. The calves were monitored daily for signs of disease, and blood samples and nasal and rectal swabs were collected on the day of inoculation and two, four, seven, nine, 11, 14, 17, 21, 24 and 28 days later. All procedures complied with the guidelines of the Canadian Council on Animal Care. Virus isolation was performed as described by Tessaro and others (1999). Two of the three calves developed fever (40·5 and 42·0°C), but none showed any other signs of disease. Virus was only isolated from serum and/or leucocytes four and seven days after inoculation. All three animals showed a decrease in total leucocyte counts of 50 per cent or more by day 7, and had seroconverted to BVDV at day 11, as determined by a serum neutralisation assay (Deregt and others 1992). Thus, although the isolate could infect cattle, it appeared to be innocuous, at least for calves of this age group. Both of the bison isolates were typed as BVDV-1 using a multiplex PCR that distinguishes between BVDV-1 and BVDV-2 strains (Gilbert and others 1999). This PCR uses primers to amplify sequences of the NS5B polymerase gene. Since this gene has not been used for phylogenetic analysis, as have the Npro and E2 genes, the entire E2 gene of each isolate was sequenced by the methods described by Deregt and others (1998) (Fig 1). The phylogenetic relationships between the E2 genes of pestiviruses representing all five recognised species were determined using the distance matrix programs PRODIST and FITCH (PHYLIP). Bootstrap resampling of the phylogenetic trees was carried out to test the robustness of the observed clades. The phylogenetic relationships of the two bison isolates to other pestiviruses are shown in Fig 2. Both bison isolates clustered in the species BVDV-1, with isolate V1127 in the BVDV Isolation of bovine viral diarrhoea viruses from bison

Multiplex RT-PCR detection and microarray typing of vesicular disease viruses.

A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.

Mapping of neutralizing epitopes to fragments of the bovine coronavirus E2 protein by proteolysis of antigen-antibody complexes.

Neutralizing antigenic domains on bovine coronavirus gp100/E2 were mapped to fragments of this protein by proteolytic cleavage and fragment analysis. The procedure involved analysis of fragments generated after incubation of E2-monoclonal antibody complexes with various proteases. The smallest antibody-bound fragments obtained were a 50K fragment following Staphylococcus aureus V8 protease and submaxillary protease digestion, and a 37K fragment following trypsin digestion. Trypsin also produced a transient antibody-bound 50K fragment. A 40K fragment which was not bound by antibody was observed following digestions with all three proteases. The 50K fragments generated by V8, submaxillary protease and trypsin comigrated on gels and displayed the same altered mobility under non-reducing conditions, suggesting identity of these fragments and indicating the presence of disulphide linkages in these fragments. The 40K fragments generated by these three enzymes also comigrated and displayed the same altered mobility under non-reducing conditions. The 37K trypsin fragment contained both neutralizing domains, A and B.