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.

Herpesvirus infection in woodland caribou in Alberta, Canada.

Sera and genital swabs collected from 121 adult woodland caribou (Rangifer tarandus caribou) in five subpopulations in northern Alberta, Canada, between December 1997 and October 1999, were examined for evidence of infection with herpesviruses or pestiviruses. No virus was isolated from sera or swabs, and no antibodies against bovine viral diarrhea virus were detected. However, 63 (52%) of the 121 animals had neutralizing antibody titers against bovine herpesvirus 1. There was sufficient serum from 37 of the 121 caribou to allow parallel testing for antibodies against a new alphaherpesvirus isolated from an elk (Cervus elaphus nelsoni), and 20 animals had antibodies against this virus. Paired sera collected 11 mo apart from 14 caribou showed seroconversion in seven animals, indicating that an active herpesvirus infection was present. Virus neutralization data suggest that these caribou are infected with a distinct alphaherpesvirus.

Attenuation of a virulent type 2 bovine viral diarrhea virus.

The purpose of this study was to produce an attenuated bovine viral diarrhea virus (BVDV) type 2 strain as a tool for identifying potential virulence markers in the BVDV2 genome. The attenuation of the virulent strain, BVDV2-24515, was accomplished by in vivo and in vitro passage. The strain was initially used to infect an elk (Cervus elaphus) [J. Wildl. Dis. 35 (1999) 671], re-isolated at 7 days post-inoculation from serum, and then subsequently passaged 56 times in cell culture. Two groups of calves were inoculated intranasally with either BVDV2-24515 or the putative attenuated virus, designated BVDV2-LATT. Calves inoculated with BVDV2-24515 had cumulative clinical scores which ranged from 6 to 53. Clinical signs in these calves consisted of anorexia, depression, dehydration, diarrhea (+/-bloody), and pneumonia. Several calves developed leukocytopenia, primarily a neutrocytopenia, and presented lesions of enteritis or pneumonia at necropsy. In contrast, cattle inoculated with BVDV2-LATT had cumulative clinical scores which ranged from 0 to 2. This was not significantly different from that of controls which received no virus (range: 0-1). Calves inoculated with BVDV2-LATT produced high neutralizing antibody titers against BVDV2. Thus, in addition to its potential use as a tool for identifying virulence markers, the attenuated virus is also worthy of further study as a candidate virus for inclusion in a modified-live vaccine.

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.

A single amino acid change within antigenic domain II of the spike protein of bovine coronavirus confers resistance to virus neutralization.

The spike glycoprotein is a major neutralizing antigen of bovine coronavirus (BCV). Conformational neutralizing epitopes of group A and group B monoclonal antibodies (MAbs) have previously been mapped to two domains at amino acids 351 to 403 (domain I) and amino acids 517 to 621 (domain II). To further map antigenic sites, neutralization escape mutants of BCV were selected with a group A MAb which has both in vitro and in vivo virus-neutralizing ability. The escape mutants were demonstrated to be neutralization resistant to the selecting group A MAb and remained sensitive to neutralization by a group B MAb. In radioimmunoprecipitation assays, the spike proteins of neutralization escape mutants were shown to have lost their reactivities with the selecting group A MAb. Sequence analysis of the spike protein genes of the escape mutants identified a single nucleotide substitution of C to T at position 1583, resulting in the change of alanine to valine at amino acid position 528 (A528V). The mutation occurs in domain II and in a location which corresponds to the hypervariable region of the spike protein of the coronavirus mouse hepatitis virus. Experimental introduction of the A528V mutation into the wild-type spike protein resulted in the loss of MAb binding of the mutant protein, confirming that the single point mutation was responsible for the escape of BCV from immunological selective pressure.

Recombinant bovine adenovirus type 3 expressing bovine viral diarrhea virus glycoprotein E2 induces an immune response in cotton rats.

Recombinant bovine adenovirus is being developed as a live vector for animal vaccination and for human gene therapy. In this study, two replication-competent bovine adenovirus 3 (BAV-3) recombinants (BAV331 and BAV338) expressing bovine viral diarrhea virus (BVDV) glycoprotein E2 in the early region 3 (E3) of BAV-3 were constructed. Recombinant BAV331 contains chemically synthesized E2 gene (nucleotides modified to remove internal cryptic splice sites) under the control of BAV-3 E3/major late promoter (MLP), while recombinant BAV338 contains original E2 gene under the control of human cytomegalovirus immediate early promoter. Since E2, a class I membrane glycoprotein, does not contain its own signal peptide sequence at the 5' end, the bovine herpesvirus 1 (BHV-1) glycoprotein D signal sequence was fused in frame to the E2 open reading frame (ORF) for proper processing of the E2 glycoprotein in both the recombinant viruses. Recombinant E2 protein expressed by BAV331 and BAV338 recombinant viruses was recognized by E2-specific monoclonal antibodies as a 53-kDa protein, which also formed dimer with an apparent molecular weight of 94 kDa. Insertion of an E2-expression cassette in the E3 region did not effect the replication of recombinant BAV-3s. Intranasal immunization of cotton rats with these recombinant viruses generated E2-specific IgA and IgG responses at the mucosal surfaces and in the serum. In summary, these results show that the pestivirus glycoprotein can be expressed efficiently by BAV-3. In addition, mucosal immunization with replication-competent recombinant bovine adenovirus 3 can induce a specific immune response against the expressed antigen.

Antigenic and molecular characterization of a herpesvirus isolated from a North American elk.

OBJECTIVE: To determine whether a herpesvirus isolated from the semen of a North American elk was related to bovine herpesvirus 1 (BHV-1). SAMPLE POPULATION: Semen from 1 healthy bull elk and 2 subtypes of BHV-1 (BHV-1.1 and BHV-1.2). PROCEDURES: A virus with cytopathic and electron microscopic characteristics consistent with an alpha-herpesvirus was isolated from elk semen, using fetal bovine kidney cells. Cross-neutralization assays were performed with antisera against BHV-1 and the elk herpesvirus (EIkHV). Restriction endonuclease digests of EIkHV DNA were compared with digests of BHV-1.1 and BHV-1.2 DNA. A portion of the ElkHV DNA polymerase gene was amplified with consensus primers by use of the polymerase chain reaction and sequenced. Sequence was compared with known sequences of other herpesviruses. An immunoperoxidase monolayer assay was used to determine reactivities of 22 BHV-1-specific monoclonal antibodies (mAb) against ElkHV. In vitro neutralizing activities of the reactive mAb were determined by use of a microneutralization assay. RESULTS: Results of cross-neutralization assays indicated that ElkHV was serologically related to BHV-1. Endonuclease digestion of ElkHV DNA generated fragments that were distinct from those of BHV-1. Nucleotide sequencing confirmed that ElkHV is an alphaherpesvirus closely related to but distinct from BHV-1. Six of 22 BHV-1-specific mAb reacted against ElkHV; 2 of these 6 also neutralized in vitro infectivity of ElkHV. CONCLUSIONS AND CLINICAL RELEVANCE: ElkHV is antigenically and genetically distinguishable from BHV-1. However, the viruses are serologically related and share at least 6 antigenic determinants, one of which is a major neutralizing determinant.

Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.

A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera.

Viremia and virus shedding in elk infected with type 1 and virulent type 2 bovine viral diarrhea virus.

In order to determine whether elk (Cervus elaphus) could be infected with and shed bovine viral diarrhea virus (BVDV) and to determine whether BVDV could cause disease in elk, two groups of five yearling elk each and two control cattle were experimentally inoculated intranasally with type 1 Singer strain or a virulent type 2 isolate of BVDV, strain 24515. Virulence of the type 2 isolate was confirmed by inoculation of a control bovine cow which developed diarrhea, dehydration, severe thrombocytopenia, hemorrhages, and enteritis with intestinal necrosis. None of the elk inoculated with type 1 or type 2 BVDV developed clinical signs of illness. However, all elk became infected as demonstrated by viremia, nasal shedding, and/or seroconversion. One uninoculated, in-contact elk contracted type 1 BVDV and seroconverted. Thus, although BVDV does not appear capable of producing disease in nonpregnant elk, the species is susceptible to infection and can shed and transmit BVDV.

Typing of bovine viral diarrhea viruses directly from blood of persistently infected cattle by multiplex PCR.

A nested multiplex PCR was developed for genotyping of bovine viral diarrhea viruses (BVDVs). The assay could detect as little as 3 50% tissue culture infective doses of BVDV per ml and typed 42 out of 42 cell culture isolates. BVDV was also successfully typed, with or without RNA extraction, from all 27 whole-blood samples examined from 22 carriers or probable carriers and 5 experimentally infected cattle.

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.

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.

A monoclonal antibody-based immunoperoxidase monolayer (micro-isolation) assay for detection of type 1 and type 2 bovine viral diarrhea viruses.

A monoclonal antibody (mAb)-based immunoperoxidase monolayer assay (IPMA) for detection of bovine viral diarrhea virus (BVDV) was developed and compared with an existing bovine polyclonal antibody (pAb)-based IPMA. A pool of 5 mAbs, 4 mAbs produced to a type 1 BVDV and 1 mAb produced to a type 2 BVDV, was utilized in the mAb-IPMA. The mAbs were chosen for inclusion in the pool because of their broad cross-reactivities with type 1 and/or type 2 BVDV, their apparent avidities for antigen, their reactivity to different BVDV proteins, and their lack of competition for binding sites or their binding to unusual BVDV isolates. The mAb-IPMA outperformed the pAb-IPMA in staining, ease of reading test results, and relative sensitivity with a panel of known BVDV positive and negative sera. The relative sensitivities of the mAb-IPMA and pAb-IPMA were 100% and 93.5%, respectively, for 62 positive samples including several that were known to contain type 2 BVDV. With retesting, the pAb-IPMA gave a similar level of sensitivity as that of the mAb-IPMA. Both tests gave a specificity of 100% for 40 negative serum samples obtained from a BVDV-free herd.

Detection of equine arteritis virus in the semen of carrier stallions by using a sensitive nested PCR assay.

A nested PCR, developed for the detection of equine arteritis virus (EAV) in semen, detected less than 2.5 PFU of EAV per ml of naturally infected seminal plasma. Based on results from testing 88 semen samples from 70 stallions, the sensitivity and specificity of the test were 100 and 97%, respectively.

Typing of porcine reproductive and respiratory syndrome viruses by a multiplex PCR assay.

A rapid multiplex PCR assay was developed to distinguish between North American and European genotypes of porcine reproductive and respiratory syndrome (PRRS) virus after a portion of the polymerase gene (open reading frame 1b) was sequenced for two North American PRRS virus strains. DNA products with unique sizes characteristic of each genotype were obtained.

Rapid detection of bovine herpesvirus 1 in the semen of infected bulls by a nested polymerase chain reaction assay.

A nested polymerase chain reaction (PCR) assay was developed for the detection of bovine herpesvirus 1 (BHV-1) in bovine semen and compared with the virus isolation method. When extended semen, commonly used in the bovine artificial insemination industry, was inoculated with BHV-1, the PCR assay detected BHV-1 DNA in semen inoculated at 0.25-2.5 TCID50 per 0.5 mL. In contrast, the lower limit of detection for virus isolation was 250 TCID50 of BHV-1 inoculated in 0.5 mL of extended semen. These methods were also used to detect BHV-1 in the semen of four bulls which were experimentally infected with BHV-1. All infected bulls demonstrated balanitis at 3 d post-inoculation (DPI) and severe balanoposthitis at 4 DPI. BHV-1 was detected in raw semen by virus isolation and PCR at 2 DPI, before balanitis was evident. For virus isolation, the last day that BHV-1 was detected during primary infection was 7 DPI for two bulls and 9 and 11 DPI for the other two bulls. In contrast, PCR detected BHV-1 in the bulls' semen until 14 or 18 DPI. For individual animals, PCR detected BHV-1 during primary infection for at least 1-10 d longer than virus isolation. Reactivation of BHV-1 from latency without the presence of visible lesions was promoted twice by two series of 5 d dexamethasone injections. For the first series of dexamethasone treatments, a positive virus isolation result was obtained on the 5th d of treatment for only one bull. In contrast, two bulls demonstrated evidence of viral reactivation on this day by PCR. All bulls shed BHV-1 in semen on d 4 after dexamethasone treatment, as evidenced by positive virus isolation and PCR results. One bull was still PCR positive 13 d later. For the second series of dexamethasone treatments, a small amount of virus was isolated from semen collected on d 3 or 4 after treatment for two bulls but not from the other two bulls. In contrast, semen samples from all bulls were PCR positive for either or both of these 2 d. In total, from 80 semen samples, 45 were PCR positive and 26 were virus isolation positive. Thus, the PCR assay detected BHV-1 shedding in bulls earlier, more often, and for a longer duration, than did the virus isolation method.

An ELISA for porcine reproductive and respiratory syndrome: production of antigen of high quality.

A reliable method was developed to produce a viral antigen preparation from porcine reproductive and respiratory syndrome virus (PRRSV) infected MARC-145 cells by solubilizing the virus with Triton X-100. This method eliminated problems previously encountered with high background reactions associated with PRRSV antigen or cell control antigen. With this new antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was adapted to detect swine serum anti-body against PRRSV. In the ELISA, non-specific reactions associated with test serum samples have been eliminated by utilizing an effective blocking serum diluent. The ELISA is more sensitive than an indirect immunofluorescent assay (IFA), particularly with late-infection sera, while maintaining a high diagnostic specificity. In a comparison of IFA and ELISA using sera collected from 250 pigs of various ages from 5 herds that had PRRS histories, IFA revealed 178 positive samples and 72 negative samples. All of the IFA-positive sera were proven to be ELISA reactors. However, nearly one-half (34/72) of the IFA-negative samples were also ELISA reactors. The diagnostic specificity and sensitivity of the ELISA were 100% and 96.6% with 257 serum samples collected from known healthy PRRS-negative swine herds and 57 sera collected from infected swine at 6 to 56 days after infection, respectively. The ELISA is technically superior to IFA, time-efficient and cost-effective, and suitable for testing of a large number of samples over a short period of time.

Bovine viral diarrhea virus: biotypes and disease.

Bovine viral diarrhea virus continues to produce significant economic losses for the cattle industry and challenges investigators with the complexity of diseases it produces and the mechanisms by which it causes disease. This paper updates and attempts to clarify information regarding the roles of noncytopathic and cytopathic bovine viral diarrhea viruses in persistent infections and mucosal disease. It also covers, in brief, what is known of the new diseases: thrombocytopenia and hemorrhagic disease, and a disease resembling mucosal disease that is apparently caused solely by noncytopathic virus. Although a good understanding of the roles of the 2 biotypes in the production of persistent infections and the precipitation of mucosal disease has been obtained, there are still unanswered questions regarding the origin of cytopathic viruses and the mechanism by which they cause pathological changes in cells. It is apparent, however, that cytopathic bovine viral diarrhea viruses arise by mutation of noncytopathic viruses, and it is known that p80 is the marker protein for cytopathic viruses. The previous distinction between mild bovine viral diarrhea and fatal mucosal disease has been eroded with the emergence of new virulent bovine viral diarrhea viruses. The new diseases pose a threat to the cattle industry and present a new challenge for investigators. Index Veterinarius (1984-1994) and Medline (1985-1994) databases and personal files updated since 1987 from BIOSIS Previews and Biosciences Information Services were used to search the literature.