Attenuated porcine-derived type 2 bovine viral diarrhea virus as vector stably expressing viral gene.

Infectious bovine viral diarrhea virus (BVDV) cDNA clones have been used for the expression of classical swine fever virus (CSFV) genes for immune prevention and control. However, can it be used for the expression of an allogenetic fragment? To answer this question, a BVDV chimeric virus expressing the spike (S) antigen fragment of porcine epidemic diarrhea virus (PEDV) was constructed. Antigen S499-602 was inserted into pig-derived BVDV-2 infectious cDNA clone pASH28 in tandem by overlapping PCR, located between the seventh and eighth amino acids at the N-terminus of the capsid (C) protein of BVDV. Indirect immunofluorescence assay confirmed that the chimeric virus vASH-dS312 containing double S499-602 sequences was successfully assembled, which could react with the monoclonal antibody (MAb) against BVDV E2 and PEDV S proteins. Further western blot analysis confirmed that the exogenous S499-602 double protein could be stably expressed. Next, the chimeric virus vASH-dS312 was administered to BALB/C mice either orally or by intramuscular injection. The immunized mice were healthy and showed no signs of toxicity. IgG against BVDV and PEDV antibodies could be detected in the mice administered vASH-dS312 by intramuscular injection, which had neutralization activity against BVDV and PEDV. Thus, this study reported a new insertion site in the BVDV infectious cDNA clone that could successfully express an allogenetic antigen.

N pro of Bungowannah virus exhibits the same antagonistic function in the IFN induction pathway than that of other classical pestiviruses.

Bungowannah virus is the most divergent atypical pestivirus that had been detected up to now, and does not fit into any of the four approved species: Bovine viral diarrhea virus type 1 (BVDV-1) and type 2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV). However, the presence of N(pro) and E(rns) coding regions, which are unique to pestiviruses, provides clear evidence of a pestivirus. Nevertheless, the amino acid identity of Bungowannah virus N(pro) and BVDV-1 N(pro) (strain CP7) is only 51.5%. By using a BVDV-1 backbone, a novel chimeric construct was generated, in which the genomic region encoding the non-structural protein N(pro) was replaced by that of Bungowannah virus (CP7_N(pro)-Bungo). In vitro studies of CP7_N(pro)-Bungo revealed autonomous replication with the same efficacy as the BVDV backbone CP7 and infectious high-titer virus could be collected. In order to compare the ability of interferon (IFN) suppression, two reporter gene assays, specific for type-I IFN, were carried out. In virus-infected cells, no significant difference in blocking of IFN expression between the parental virus CP7, Bungowannah virus and the chimeric construct CP7_N(pro)-Bungo could be detected. In contrast, an N(pro) deletion mutant showed an impaired replication in bovine cells and a marked type-I IFN response. Taken together, our findings reveal the compatibility of non-structural protein N(pro) of atypical Bungowannah virus with a BVDV type 1 backbone and its characteristic feature as an inhibitor of type-I IFN induction with an inhibitor-activity comparable to other pestiviruses.

Experimental infection of calves with bovine viral diarrhoea virus type-2 (BVDV-2) isolated from a contaminated vaccine.

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100%, homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.

Experimental infection of pregnant ewes with bovine viral diarrhea virus type-2 (BVDV-2): effects on the pregnancy and fetus.

The reproduction effects of bovine viral diarrhea virus type-2 (BVDV-2) infection were investigated in ewes inoculated with a non-cytopathic BVDV-2 isolate at three stages of gestation. Virus inoculation was followed by a transient viremia, accompanied by a transient and mild hyperthermia and nasal discharge in a few animals. Some ewes were sacrificed at different time-points after virus inoculation to study the kinetics of fetal infection. Infectivity and viral antigens were detected in placentomes from day 7 to 36 post-inoculation (pi) and in fetal fluids and tissues between days 10 and 28 pi. Cardiac petechial hemorrhages and hemoperitoneum accompanied by a severe fibrinous ulcerative placentitis were observed in fetuses examined at days 21, 28 and 36 pi. Inoculation of ewes at days 55-60 of gestation resulted in a prolonged virus replication in placentomes and fetal tissues; ewes that were allowed to proceed with pregnancy had 77% of abortions or fetal and perinatal deaths. Seven stillbirths, unviable and viable lambs born to these ewes were virus-positive at birth. Infectious virus was repeatedly isolated from leukocytes of two lambs up to 2 and 6 months of age, indicating they were persistently infected. Ewes inoculated at days 65-70 of gestation had 66.6% of fetal and perinatal losses. Three viable lambs born to these ewes were healthy, BVDV antibody-positive and virus-negative. A transient viral replication in placentomes and in a few fetal tissues, followed by the rise of fetal neutralizing antibodies and virus clearance was the result of inoculating ewes at days 120-125 of gestation. Lambs born to these ewes were healthy, antibody-positive and virus-negative. These results demonstrate that the biology of BVDV-2 infection in pregnant sheep is essentially similar to that of BVDV-1 in pregnant cattle and sheep. These features make this species an attractive animal model for studying the pathogenesis of congenital BVDV-2 infection.