To characterize the genetic diversity of bovine viral diarrhea viruses (BVDV) circulating in Korea, 11 BVDV isolates were obtained from 467 field samples collected during 2005~2006 in Korea. All of the BVDV isolates were identified as non-cytopathic (non-cp) BVDV biotypes. The 5' noncoding region (NCR) genes of the isolates were sequenced and analyzed. In total, ten BVDV isolates were typed as BVDV-1 by comparing the genomic sequences to the 5' NCR. One isolate (05R169) showed 98.6% nucleotide sequence identity with the BVDV-2 reference strain and was therefore typed as BVDV-2. Our results indicate that BVDV-1 is the main genotype circulating in the cattle population of Korea.
Animals ; Base Sequence ; Cattle ; Diarrhea Virus 1, Bovine Viral ; Diarrhea Virus 2, Bovine Viral ; Diarrhea Viruses, Bovine Viral* ; Genetic Variation ; Genotype ; Korea*

In order to obtain virus-like particles (VLPs) for prevention of bovine viral diarrhea virus 1 (BVDV-1), the C-E^rns-E1-E2 region was cloned into a pFastBacDaul vector for generating the recombinant Bacmid-BVDV-1 in DH10Bac Escherichia coli. The recombinant baculovirus Baculo-BVDV-1 was produced by transfecting the Sf9 cells with Bacmid-BVDV-1. The expressed protein and the assembled VLPs were determined by immunofluorescence, Western blotting and electron microscopy. Guinea pigs were immunized with inactivated VLPs coupled with the Montanide ISA-201 adjuvant. The immunogenicity of VLPs was evaluated by monitoring the humoral immune response with neutralizing antibody titer determination, as well as by analyzing the cell-mediated immune response with lymphocyte proliferation assay. The protective efficacy of VLPs was evaluated by challenging with 10⁶ TCID₅₀ virulent BVDV-1 strain AV69. The results showed that the recombinant Baculo-BVDV-1 efficiently expressed BVDV structural protein and form VLPs in infected Sf9 cells. The immunization of guinea pigs with VLPs resulted in a high titer (1:144) of neutralizing antibody, indicating an activated cellular immunity. Significantly lower viral RNA in the blood of the post-challenged immunized guinea pigs was observed. The successful preparation of BVDV VLPs with insect cell expression system and the observation of the associated immunogenicity may facilitate further development of a VLPs-based vaccine against BVD.
Animals ; Antibodies, Viral ; Diarrhea ; Diarrhea Virus 1, Bovine Viral ; Guinea Pigs ; Mineral Oil ; Viral Envelope Proteins ; Viral Vaccines

Bovine viral diarrhea virus (BVDV) of the genus Pestivirus is known as a common contaminant of cell culture-derived vaccines. Hog cholera virus (HCV), which is also of the genus Pestivirus and an important livestock disease in Korea, is recognized as a potential contaminant of vaccines produced in porcine cells. However, it is difficult for the National Biological Assays of korea to adequately detect contamination of these agents in biological products. For these reasons, we established rapid and sensitive methods for the detection of BVDV and HCV contamination in cell cultures and veterinary biologicals by using RT-PCR and nested PCR assays. We designed a Pestivirus primer amplifying 152 bp to detect both BVDV and HCV and a common primer amplifying 237 bp to detect only BVDV. Also, for the differentiation between BVDV type 1 and type 2, nested PCR was conducted using the amplified 237 bp PCR product, to amplify 179 bp in BVDV type 2 genome. The sensitivity of the PCR using common primer for the detection of BVDV was 400 TCID50/ml. All 6 strains of Korean BVDV isolates, 5 vaccines strains and the standard strain NADL could be detected. No reactions were observed when testing 5 types of viruses infecting pigs (HCV, TGEV, PEDV, JEV, PRRSV), 4 types infecting cattle (Akabane virus, BEFV, BCV, BRV) and 4 types infecting cats (FIP, FPL, FCV, FVR). Using this RT-PCR assay, commercial vaccines were tested and, 55 lots from 12 vaccine companies, were negative for BVDV contaminations. Same results were obtained by the immunoflourescence assay. The newly developed PCR or RT-PCR assays can be used as rapid, reliable, sensitive, and simple methods for the detection of BVDV (Pestivirus) in cell cultures, master seeds, and live viral vaccines.
Animals* ; Biological Assay ; Biological Products ; Cats ; Cattle ; Cell Culture Techniques ; Classical swine fever virus ; Diarrhea Virus 1, Bovine Viral ; Diarrhea Virus 2, Bovine Viral ; Diarrhea* ; Genome ; Korea ; Livestock ; Pestivirus ; Polymerase Chain Reaction* ; Swine ; Vaccines ; Viral Vaccines*

The purpose of this study was to determine whether manually plucked hairs might serve as an alternative sample for a quantitative real time polymerase chain reaction (qRT-PCR) testing. Twenty three, 1~3 week old, non-bovine viral diarrhea virus (BVDV) vaccinated calves, found to be positive for BVDV by immunohistochemical staining, were selected and hairs were manually plucked from the ear. qRT-PCR was performed on samples consisting of more than 30 hairs (30~100) and whole blood. All 23 animals were positive for the virus by qRT-PCR performed on the whole blood and when samples of more than 30 hairs were assayed. Additionally, qRT-PCR was performed on groups of 10 and 20 hairs harvested from 7 out of 23 immunohistochemical staining-positive calves. When groups of 20 and 10 hairs were tested, 6 and 4 animals, respectively, were positive for the virus.
Animals ; Antibodies, Viral/analysis/diagnostic use ; Bovine Virus Diarrhea-Mucosal Disease/blood/*diagnosis/virology ; Cattle ; Diarrhea Virus 1, Bovine Viral/genetics/*isolation & purification ; Diarrhea Virus 2, Bovine Viral/genetics/*isolation & purification ; Hair/virology ; RNA, Viral/chemistry/genetics ; Real-Time Polymerase Chain Reaction/methods/*veterinary