Akio Fukusho

Classical swine fever: the global situation

A historical and current perspective is given of classical swine fever and its impact on pig production in different regions of the world. Data were obtained from a variety of sources including returns to the Office International des Epizooties, official government reports, other published material and local information through personal contacts. The disease has been recognized for about 170 years and efforts to control it by official intervention began in the nineteenth century. Despite this it remains a lingering problem in many parts of the world where it has both, an economic impact on swine production and a constraining effect on trade due to the measures necessary to prevent spread.

Induction of antibodies in mice by a recombinant baculovirus expressing pseudorabies virus glycoprotein B in mammalian cells

The glycoprotein gB of pseudorabies virus (PrV) was expressed in various mammalian cells by a recombinant baculovirus carrying the PrV gB gene under the control of the CAG promoter. When the recombinant baculovirus was inoculated into the stable porcine kidney cell line CPK, expression of PrV gB was detected by immunofluorescent antibody analysis and a 155 kDa of protein, which has the same molecular mass as the native PrV gB, was detected by Western blotting. High levels of expression of PrV gB were observed in BHK-21, HmLu-1 and SK-H cell lines. Furthermore, anti-PrV gB-specific antibodies against PrV gB protein were detected by the enzyme-linked immunosorbent assay in mice inoculated the recombinant baculovirus. The recombinant baculovirus containing the PrV glycoprotein gB gene under the CAG promoter could be a candidate for a pseudorabies vaccine.

ESTABLISHMENT AND CHARACTERIZATION OF A PORCINE KIDNEY CELL LINE, FS-L3, WHICH FORMS UNIQUE MULTICELLULAR DOMES IN SERUM-FREE CULTURE

SummaryA stable porcine kidney epithelial cell line, FS-L3, was established and maintained in Eagle’s minimum essential medium containing 0.295% tryptose phosphate broth, 0.5% Bacto Peptone, and 10 mM N, N-Bis (2-hydroxyethyl)-2-aminoethanesulfonic acid without any serum. The mode of chromosomes is 37 to 38. The FS-L3 cells formed fluid-filled, multicellular, three-dimensional domes on a single monolayer. The number of domes increased markedly after further cultivation. The origin of this cell line was confirmed as porcine by hybridization using PRE-1, which can be detected as a specific sequence in the porcine genome. It was also found that FS-L3 cells were free from possible adventitious viruses and mycoplasmas.

Establishment of a serum-free culture cell line, CPK-NS, which is useful for assays of classical swine fever virus

A stable porcine kidney cell line, CPK-NS, was established and maintained in serum-free culture. A cytopathic effect (CPE) was observed clearly in CPK-NS cells infected with some classical swine fever virus (CSFV) strains which did not show the exaltation of Newcastle disease virus (END) phenomenon. Chromosome condensation and DNA fragmentation, a marker for apoptosis, were detected in cells infected with END phenomenon-negative CSFV strains. By using the CPE induced by infection with an END phenomenon-negative CSFV strain in CPK-NS cells, assays of CSFV were established. The virus titer determined in CPK-NS cells shows a high correlation with the usual peroxidase-linked assay, dome disappearance method and END method. Furthermore, the antibody titer by neutralizing test with CPK-NS cells also correlated with that measured by the usual neutralizing peroxidase-linked assay and dome disappearance method. These stable CPK-NS cells have the great advantage that a clear CPE was caused by infection with END phenomenon-negative CSFV strains and bovine serum is not necessary for cell culture and virus assays.

Rapid detection of African horsesickness virus by the reverse transcriptase polymerase chain reaction (RT-PCR) using the amplimer for segment 3 (VP3 gene)

SummaryThe complete sequence of the major core protein (VP3) gene of African horsesickness virus serotype 4 (AHSV-4; vaccine strain) was determined by analysis of a complete cDNA clone representing segment 3. The RNA was 2 789 bp long and a comparison of its sequence with that of bluetongue virus serotype 10 (BTV-10) revealed 58% nucleotide similarity. Based on these data, the reverse transcriptase-polymerase chain reaction (RT-PCR) technique was applied to the specific detection of AHSV using a pair of primers designed for AHSV-4 VP3 gene. Approximately 230 bp of PCR products were amplified by RT-PCR from the total RNA extracts (mRNA and dsRNA) of Vero cells infected with eight serotypes of AHSV. No product was observed analogous to other orbiviruses. The supernatant of the infected cell culture fluid without any RNA purification was also suitable as a template for RT-PCR after being denatured at 94°C for 5 min. The sensitivity of this method was between 100 and 101 TCID50 when viral RNA from the supernatant of infected cell culture was subjected to RT-PCR. The whole procedure for detecting the virus RNA by RT-PCR could be carried out within 5h. The RT-PCR with AHSV VP3 gene as a target was found to be a simple, highly specific and sensitive assay for AHSV.

Methods to select suitable fetal bovine serum for use in quality control assays for the detection of adventitious viruses from biological products

Production of biological products, especially vaccines, usually requires materials derived from animals, and there are always risks that animal pathogens derived from these materials could contaminate the final products. Detection of adventitious agents is performed by quality control tests. In these biological assays, animal derived materials are also used and another problem arises, as fetal bovine serum (FBS) is used as an ingredient in tissue culture media. FBS contaminated with bovine viral diarrhea virus (BVDV) or other bovine pathogens, as well as antibodies against these pathogens may lead to false results in quality control assays. In this study, in order to determine the actual status of commercial FBS, we performed quality tests on various FBS samples. As a result, in 28 of 49 FBS samples (57.1%), pestivirus genes were detected by pan-pestivirus reverse transcription-polymerase chain reaction assay. Furthermore, two samples contained infectious BVDV. Neutralizing antibodies against BVDVs were detected in 48 of 49 samples (97.6%) by the virus neutralization test based on the serum-dilution or virus-dilution methods. Antibodies against other bovine pathogens were detected rarely in these samples. From our results, we recommend methods to select FBS that are focused on detection of BVDV and neutralizing antibodies against BVDV.

A new assay for classical swine fever virus based on cytopathogenicity in porcine kidney cell line FS-L3

A new assay termed the dome disappearance method for classical swine fever virus (CSFV) using FS-L3 cells with serum-free culture medium was developed. The CSFV live vaccine GPE- strain grows well and shows a slight cytopathic effect (CPE) in FS-L3 cells. This CPE results in the disappearance of the unique fluid-filled multicellular domes on a single monolayer of FS-L3 cells. By using this phenomenon, dome disappearance, as a marker of infection, it was possible to determine the titers of CSFV and its neutralizing antibody. The virus titer determined by this method shows a good correlation with that determined by immunochemical and interference methods. Furthermore, the amount of neutralizing antibody measured by this method also correlated with that measured by the Exaltation of Newcastle Disease Virus (END) neutralizing method. The dome disappearance method developed in this experiment is a simple and safe procedure and has the great advantage that bovine serum, which may contain antibody against bovine viral diarrhea virus, is not necessary for the cultivation of FS-L3 cells.

Isolation of different non-cytopathogenic bovine viral diarrhoea (BVD) viruses from cytopathogenic BVD virus stocks using reverse plaque formation method

Non-cytopathogenic (NCP) bovine viral diarrhoea (BVD) viruses were isolated from three cytopathogenic (CP) BVD virus stocks using the reverse plaque formation method, which was based on intrinsic interference. By means of an exaltation of Newcastle disease virus (END) test, these NCP BVD viruses were divided into two groups; END phenomenon positive (END+) and END phenomenon negative (END-) viruses. Additionally, the END+ NCP BVD viruses interfered only with CP BVD virus whereas the END- NCP BVD viruses interfered with vesicular stomatitis virus as well as CP BVD virus. Differences in antigenicity existed among the three CP strains, however, each group of parent CP BVD virus and derivative NCP BVD virus was antigenically indistinguishable.

Pathogenicity and kinetics of virus propagation in swine infected with the cytopathogenic classical swine fever virus containing defective interfering particles

Summary. To analyze the pathogenicity and in vivo kinetics of the cytopathogenic (cp) classical swine fever virus (CSFV) WB82 strain, which is composed of cp defective interfering (DI) particles and noncytopathogenic (noncp) helper virus (WB82/E+ strain), WB82 and WB82/E+ strains were administered separately to domestic pigs. After inoculation with either strain, all pigs showed typical symptoms of classical swine fever (CSF), such as leucopenia and high fever. There were few differences in clinical signs and survival times between each group. However, the appearance of some symptoms of CSF had a tendency to be delayed following infection with the WB82 strain, when compared with the WB82/E+ strain. Virus isolation and detection of subgenomic (sg) and full-length viral (flv) RNA by RT-PCR was carried out using sera, 10% homogenized organs and oral, nasal and rectal swabs. Both noncytopathogenic helper virus and cp DI particles were detected in samples from pigs infected with the WB82 strain, but only noncp phenotype virus was isolated from pigs infected with the WB82/E+ strain. Interestingly, the cp DI particles appeared six to seven days later than helper virus in sera from pigs infected with the WB82 strain. Although active cp DI particles could not be isolated from swabs, sg RNA as well as flv RNA was detected in swabs from animals infected with the WB82 strain. These results suggest that progeny cp DI particles are replicated from parent DI particles after noncp virus replication, and subsequently discharged from infected animals. Furthermore, propagation of DI particles or replication of sg RNA, following propagation of helper virus, appears to inhibit the appearance of CSF symptoms induced by virulent helper CSFV.

Field Distribution of END Phenomenon-Negative Bovine Viral Diarrhea Virus

Field isolates of BVDV which do not show the exaltation of Newcastle disease virus (END) phenomenon (END–) are rarely reported. In this study, 45 BVDV field isolates from cattle in Hokkaido prefecture in Japan were analyzed by the reverse plaque formation method, the END method and observation of cytopathic effects. END– virus was detected in 34 of 45 isolates (75.6%), although 35 of 45 field isolates contained END phenomenon positive virus as the predominant virus population. We propose that END– viruses are widely distributed in the field and that it is possible that the mixture of biologically distinct BVDV correlates with the appearance of disease in infected animals.