Classical swine fever is a highly contagious, notifiable disease of pigs and wild boars listed by the World Organisation for Animal Health (OIE). Therefore, methods employed in the diagnosis of CSF should be fast, sensitive and specific. The aim of this study was optimisation of the reverse transcription reaction to increase the sensitivity of real-time RT-PCR for the detection of classical swine fever virus, the aetiological agent of the disease. The efficiency of reverse transcription reaction was compared including a range of reverse transcriptases, thermal conditions and priming methods based on results obtained in the following realtime PCR. Depending on catalysis and the priming method used in the study a significant diversity of results was observed. The best efficacy of reverse transcription was obtained using SuperScript II reverse transcriptase and priming with random nonamers and reverse, gene-specific primer. This combination improved the sensitivity of RT-PCR nearly 1000 times as compared to the method with AMV reverse transcriptase coupled with random hexamers. In summary, this study has demonstrated that the optimisation of reverse transcription can contribute to a higher sensitivity of RT-PCR diagnostic methods.
The effects of classical swine fever (CSF) virus infection on the porcine leukocyte subsets were investigated by flow cytometry in acute, chronic and convalescent forms of the disease. The virus antigen could be first detected in the monocytes on postinfection (p.i.) day 10 while in the lymphocytes on p.i. day 13. It could be established that the ratio of CD6+ cells decreased until p.i. day 6, but afterwards it started to increase and reached different values. The CD4+CD8+, the CD8+ and the CD6- cells were obviously higher virus positive than the CD4+ and the CD4-CD8-subsets, but essentially all subsets could be infected. The ratio of CD8+ cells increased during the disease, while the number of double positive cells decreased, and that of the CD4+ cells was variable. The viral antigen could be detected in a lower percentage of the CD4+CD8+, CD8+, CD6+ and CD6- cells of the pigs affected with the chronic form of the disease than in those with the acute form. During the experiments no viral antigen could be detected in the leukocytes of the pig that became convalescent, though the changes in its leukocyte subsets were very similar to those seen in pigs in which the viral antigen could be detected. The studies have revealed that essentially all leukocyte subsets can be infected with the CSF virus, but in very different amounts.
The quasispecies nature of three animal pathogenic RNA viruses of field origin was examined by testing variants of classical swine fever virus (CSFV) originating from geographically different areas, feline coronavirus (FCoV) detected from the same animal by successive sampling, and rabbit haemorrhagic disease virus (RHDV) originating from successive outbreaks in the same geographic area. Clinical samples were investigated using reverse transcriptase polymerase chain reaction (RT-PCR) and ensuing single strand conformational polymorphism (SSCP) assay. By the combination of these methods even subtle differences could be detected among the amplified fragments of the same virus species of different origin. FCoV proved to comprise the most and CSFV the less heterogeneous virus quasispecies. The results show that the combination of RT-PCR and SSCP provides novel and highly sensitive means for the characterisation of RNA viruses, with special regard to genome composition, evolution, features of pathogenicity and molecular epizootiology.
The vaccine-induced maternal immunity against classical swine fever (CSF) was investigated in this study. Eight sows were vaccinated with the Chinese strain of classical swine fever virus (CSFV). The length of time between vaccination and farrowing was 167-217 days. Milk samples from the front, middle and back udder sections and blood samples were taken from the sows on days 3 and 14 after farrowing. Blood samples were obtained from the piglets at the age of 3, 6 and 10 weeks. The antibody level of the milk was examined by ELISA, and that of blood samples by the virus neutralization (VN) test as well. In all 3-week-old piglets and in 80% of the 6-week-old animals the neutralizing antibody level reached the titre of 1:40. In none of the 10-week-old piglets did the titre exceed the value of 1:20, but in about 25% of the piglets it reached 1:20; the half of these piglets came from two litters. In none of the piglets did the antibody level reach the negative threshold in the ELISA test during the study. No significant differences were found between the udder sections in milk antibody level by ELISA.
The biological properties of bovine viral diarrhoea virus (BVDV) strain Oregon C24V were studied after intranasal and subcutaneous infection of pregnant sows. This virus strain is widely used in Hungary for immunising cattle against bovine viral diarrhoea (BVD). Based upon the results of the clinical, gross pathological, histopathological and virological examinations it can be established that the given strain caused asymptomatic infection and serological conversion in sows that were in the second third of gestation. The virus caused clinically apparent disease in some of the piglets born at term, which indicates that it had crossed the placenta. More than half (57%) of the live-born piglets died within 60 days of birth. The sows and their progeny did not shed the virus. BVDV infection has great differential diagnostic importance in pigs, as classical swine fever (CSF) virus strains of reduced virulence cause similar clinical symptoms and gross and histopathological changes.
. ( 2000 ): Vaccination with a single dose of a recombinant porcine adenovirus expressing the classicalswinefevervirus gp55 (E2) gene protects pigs against classical swine fever . Vaccine 18 , 1040 – 1050
– 1549 . 10.1093/molbev/msy096 Li , C. , Zheng , H. , Wang , Y. , Dong , W. , Liu , Y. , Zhang , L. and Zhang , Y. ( 2019 ): Antiviral role of IFITM proteins in classicalswinefevervirus infection . Viruses 11 , 126 . 10.3390/v
influenza A virus (IAV), samples for the detection of IAV RNA were not taken. However, the presence of Japanese encephalitis virus, Aujeszky's disease (pseudorabies) virus, African and classicalswinefevervirus was not verified, since none of these