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*

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*

To select a less pathogenic bovine viral diarrhea virus (BVDV) strain for the construction of chimeric pestivirus harboring classical swine fever virus (CSFV) E2 gene, five Korean BVDV isolates (four type 1 isolates and a type 2 isolate) were evaluated for their pathological and biological properties with respect to porcine infection. Each of five groups of 100-day-old pigs was inoculated intranasally with one of the five BVDV isolates. No clinical sign or leukopenia was observed in any pig throughout the duration of the experiment, but viruses were detected in blood, nasal discharges and postmortem samples using RT-PCR. These results indicated that although the five BVD viruses could infect pigs, they did not cause clinically apparent symptoms. Because of its proper infection dynamics shown in this preliminary animal study and its fast growth rate and quick CPE in cell culture, one isolate (KD26-1) was chosen among the five isolates to test its virulence and immunogenic properties in 40-day-old piglets. Neither clinical sign nor pathological lesion was observed in 40-day-old piglets during the course of infection of isolate KD26-1. The first neutralizing antibodies were detectable 14 days post-inoculation (PI) and increased to 1:128~1:256 28 days PI. A BVDV specific gene was detectable by RT-PCR in tonsil, spleen, inguinal lymph node and brain until 14 days PI. According to this study, it can be concluded that isolate KD26-1 has little pathological effect in pigs and is a candidate for construction of chimeric pestivirus harboring CSFV E2 gene.
Animals ; Antibodies, Neutralizing ; Brain ; Cell Culture Techniques ; Classical swine fever virus ; Diarrhea ; Leukopenia ; Lymph Nodes ; Palatine Tonsil ; Pestivirus ; Spleen ; Sprains and Strains ; Swine ; Viruses

To select a less pathogenic bovine viral diarrhea virus (BVDV) strain for the construction of chimeric pestivirus harboring classical swine fever virus (CSFV) E2 gene, five Korean BVDV isolates (four type 1 isolates and a type 2 isolate) were evaluated for their pathological and biological properties with respect to porcine infection. Each of five groups of 100-day-old pigs was inoculated intranasally with one of the five BVDV isolates. No clinical sign or leukopenia was observed in any pig throughout the duration of the experiment, but viruses were detected in blood, nasal discharges and postmortem samples using RT-PCR. These results indicated that although the five BVD viruses could infect pigs, they did not cause clinically apparent symptoms. Because of its proper infection dynamics shown in this preliminary animal study and its fast growth rate and quick CPE in cell culture, one isolate (KD26-1) was chosen among the five isolates to test its virulence and immunogenic properties in 40-day-old piglets. Neither clinical sign nor pathological lesion was observed in 40-day-old piglets during the course of infection of isolate KD26-1. The first neutralizing antibodies were detectable 14 days post-inoculation (PI) and increased to 1:128~1:256 28 days PI. A BVDV specific gene was detectable by RT-PCR in tonsil, spleen, inguinal lymph node and brain until 14 days PI. According to this study, it can be concluded that isolate KD26-1 has little pathological effect in pigs and is a candidate for construction of chimeric pestivirus harboring CSFV E2 gene.
Animals ; Antibodies, Neutralizing ; Brain ; Cell Culture Techniques ; Classical swine fever virus ; Diarrhea ; Leukopenia ; Lymph Nodes ; Palatine Tonsil ; Pestivirus ; Spleen ; Sprains and Strains ; Swine ; Viruses

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

Recently, a novel atypical porcine pestivirus (APPV) in pig was reported. In this study, two APPV strains, APPV-China/GZ01/2016 (GZ01) and APPV-China/GD-SD/2016 (GD-SD), were identified in two newborn piglet herds with congenital tremor from China. The open reading frame of the two strains shared an 83.5% nucleotide identity. Phylogenetically, the APPV strains were placed into two groups: GZ01 belonged to group I and GD-SD belonged to group II. A high viral load was detected in the cerebellum (quantification cycles < 26). Further studies should be carried out to thoroughly elucidate the development of congenital tremors caused by APPV.
Cerebellum ; China ; Genome ; Humans ; Infant, Newborn ; Open Reading Frames ; Pestivirus ; Tremor ; Viral Load

Upon infection, bovine viral diarrhea virus (BVDV), one of the pestiviruses in the Flaviviridae family, is divided into two biotypes, cytopathic (cp) and noncytopathic (ncp). The mechanism of cytopathogenicity, however, is not elucidated yet. In this study, we have investigated viral genetic element affecting the cytopathogenicity of BVDV by using an infectious cDNA molecular clone containing a dominant selective marker, puromycin acetyltransferase gene (pNADL/pac). From the recombinant cDNA clone pNADL/pac, viral RNA was synthesized by T7 RNA polymerase in vitro. By selecting the MDBK cells transfected with in vitro transcribed cp NADL/pac viral RNA with puromycin, we obtained the selected MDBK cells harboring cp NADL/pac viral RNA, which did not show cytopathic effect and could be passaged. Little cytopathic effect was observed upon infection of naive MDBK cells with the viral particles released from the selected cp NADL/pac-transfected MDBK cells. Complete nucleotide analysis of viral particles released from cp NADL/pac-selected MDBK cells revealed 5 substitutions in the viral open reading frame, but not in the 5' and 3'NTRs. Interestingly, only two point mutations (G8147A and T11343C) changed their amino acid code (M2588V in NS4B and T2653M in NSSB), and the other 3 substitutions (C1627T, T3097C, and T3376C) was silent. Therefore, our results suggest that NS4B and NSSB proteins of BVDV may play a role in cytopathogenicity.
Clone Cells ; Diarrhea ; DNA, Complementary ; DNA-Directed RNA Polymerases ; Flaviviridae ; Humans ; Open Reading Frames ; Pestivirus ; Point Mutation ; Puromycin ; RNA, Viral ; Virion

The aim of this study was to produce E^rns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified E^rns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-E^rns was constructed based on the gene sequence of BVDV-1 NADL strain. The E^rns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-E^rns into CHO cells. The expression and purification of the E^rns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the E^rns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified E^rns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the E^rns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the E^rns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log₁₀ was induced in rabbits. In this study, purified BVDV E^rns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.
Rabbits ; Animals ; Cricetinae ; Cricetulus ; CHO Cells ; Antibodies, Viral ; Diarrhea Viruses, Bovine Viral/genetics* ; Antibodies, Monoclonal/genetics* ; Diarrhea ; Viral Vaccines/genetics*

Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, Salmonella enterica ser. Typhimurium, and Neospora caninum; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.
Animals ; Birds ; Cattle ; Cockroaches ; Diarrhea Viruses, Bovine Viral ; Diarrhea ; Diptera ; Disease Reservoirs ; Disease Vectors ; Enterovirus ; Enterovirus, Bovine ; Genome ; Insects ; Neospora ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Rodentia ; Salmonella enterica ; Virulence Factors

In this study, we cloned the NS3 gene from bovine viral diarrhea virus (BVDV) VEDEVAC strain. The result showed that the average P-distance of Pestivirus NS3 amino acid sequence was 0.07 and the VEDEVAC strain was classified to BVDV type 1. Using pET-30a(+) as vector and Escherichia coli Rosetta (DE3) as host, we obtained purified recombinant NS3 protein by Ni-NTA affinity chromatography. Western blotting analysis demonstrated that both BVDV positive serum and classical swine fever virus (CSFV) positive serum were able to recognize the recombinant NS3 protein. Indirect-ELISA assay indicated that the protein could be used as detection antigen.
Animals ; Cattle ; Cloning, Molecular ; Diarrhea Viruses, Bovine Viral ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Peptide Hydrolases ; genetics ; immunology ; Phylogeny ; RNA Helicases ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Sequence Analysis, Protein ; Viral Nonstructural Proteins ; genetics ; immunology