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PubMed1998

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.

Animals↗
PubMedDate not supplied

Prostaglandin A1 inhibits replication of classical swine fever virus.

Prostaglandins (Pgs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1) on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV) in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 micrograms/ml, which was nontoxic to the cells, PgA1 inhibitis virus production in 99%. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus decreased in amount.

Animals↗
PubMed2005

Monte Carlo simulation of classical swine fever epidemics and control. II. Validation of the model.

A stochastic and spatial simulation model was developed to simulate the spread of classical swine fever virus among herds in a certain area. A model is a simplification of a real system. The mechanisms and parameters are often not exactly known. Validation is necessary to gain insight into model behaviour and to identify risk factors with great impact on the response variables. Several risk factors such as incubation period, number of daily farm contacts, probability of detection, probability of infection after contact, probability of local spread and time from infection to infectivity were considered in the model as probability distributions in order to take the stochastic component of disease dynamics into account. In order to estimate the effects of the risk factors on the response variables mean size and duration of epidemics, a sensitivity analysis was performed. A fractional factorial design with two-level factors (2(7-2) design) was developed to gain the maximum strength with minimum demand on the calculating capacity. The main factors were unconfounded with any other main factor and also unconfounded with two-factor interactions. Apart from the time from infection to infectivity, all risk factors had a significant effect on the mean size and duration of epidemics (p<0.05). Eight two-factor interactions had a significant influence as well (p<0.05). Mainly, two-factor interactions with probability of detection were significant thus emphasising the impact of a rapid detection of outbreaks. The reaction of the simulation responses to changing of the parameter values was consistent with the expected reaction.

Animals↗
PubMedDate not supplied

[Prediction of recognition sites for genomic replication of classical swine fever virus with information analysis].

In order to explore the mechanism for the genomic replication of classical swine fever virus (CSFV), so as to make a basis for investigating its pathogenicity, an introduction of the information theory is presented in connection with the statistical mechanics, whence small-sample statistics appears naturally as a consequence of the Bayesian approach. Furthermore, a selection rule for identifying the pattern of a recognition site for an RNA-binding protein is proposed by means of the maximum entropy principle. Based on those, the information contents of 3'-untranslated regions (3'UTRs) of genomes of 20 CSFV strains and 5'-untranslated regions (5'UTRs) of genomes of 58 CSFV strains are analyzed with a computational algorithm in a reduction mode, and the 3'UTR sites of 20 strains and 5'UTR sites of 58 strains containing important motifs are extracted from the unaligned RNA sequences of unequal lengths. These sites, which have the patterns of sequence and structure similar to the putative cis elements related to the regulation of genomic replication, would be identified as the potential recognition sites in 3'UTRs and 5'UTRs for CSFV replicase responsible for classical swine fever virus genomic replication, and to some extent, this identification is supported by experimental evidence. Finally, information analysis allows a presumption to be made about the CSFV RNA replication initiation mechanism.

3' Untranslated Regions↗
PubMed2004

Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection.

The diagnostic properties of several assays on live animals were studied using data from different experiments. These experiments involved 128 classical swine fever virus (CSFV) infected pigs (weaner pigs, fatteners and sows). Since all pigs in the study were infected with CSFV, only the proportion of test positive results and the time until a test positive result is obtained were evaluated. The RT-nPCR detected the highest proportion of infected pigs (98.9%), whereas the Antigen ELISA gave the worst detection results (74.7%). Within the group of test positive animals, infection was detected earliest using the leukocyte count and latest using Antigen ELISA. Using the virus neutralisation test, antibodies against CSFV were detectable on average 7.6 days after the onset of viraemia in virus isolation in whole blood. Using survival analysis, the time until the first positive diagnosis and the proportion of detected animals were combined in one test. Results showed that RT-nPCR performed significantly better than either virus isolation in different blood fractions or antigen ELISA. It is concluded that the RT-nPCR technique is the best diagnostic tool available for early detection of a classical swine fever infection.

Animals↗
PubMed2000

Genetic grouping of classical swine fever virus by restriction fragment length polymorphism of the E2 gene.

A method for genetic grouping of classical swine fever viruses (CSFV) was developed based on the restriction fragment length polymorphism (RFLP) revealed by AvaII, BanII and PvuII digestion of RT-PCR amplified segments of the E2 gene. From inspection of the genetic sequences of Thai isolates and reference strains, the RFLP method was designed to be capable of differentiating all known genogroups and subgenogroups suggested by phylogenetic analysis of the CSFV E2 gene. The method was applied to 60 CSFV samples which included three genogroups and seven subgenogroups. Unlike previously described RFLP methods, the agarose gel patterns obtained from these samples were completely in agreement with the predicted RFLP patterns and enabled accurate genetic grouping of CSFV at the subgenogroup level. The simplicity of this method allows rapid CSFV genogrouping at diagnostic laboratories without sequencing facilities and provides a useful method for diagnosis as well as epidemiological investigation and control of classical swine fever outbreaks.

Animals↗
PubMedDate not supplied

[Immune reactions and mechanisms in classical swine fever].

The authors review published data on the immune response in classical swine fever infection. Characteristics of the neutralizing antibodies, cytolytic reactions directed at elimination of infected cells, lymphocyte proliferation, and delayed-type hypersensitivity are discussed. Problems of potential theoretical and practical interest are formulated.

Animals↗
PubMed2002

Influence of maternal antibodies on efficacy of a subunit vaccine: transmission of classical swine fever virus between pigs vaccinated at 2 weeks of age.

This study shows the effectiveness of vaccination with an E2 subunit vaccine against classical swine fever (CSF) in 2-week-old piglets. Half of the piglets were carrying maternally derived antibodies (MDAs) at the time of vaccination. Three and 6 months later, antibody levels were compared between the two treatments. Moreover, reduction of virus transmission was investigated at 3 and 6 months by doing transmission experiments. The vaccine was found to be capable of reducing virus transmission significantly at both time intervals. Maternal immunity reduced vaccination-induced antibody levels after 3 and 6 months and possibly led to a less effective protection against virus transmission after 6 months.

Animals↗
PubMed2000

Transient classical swine fever virus infection in wild boar piglets partially protected by maternal antibodies.

An experimental study was conducted to investigate the clinical course of classical swine fever (CSF) in wild boar piglets partially protected by maternal antibodies. Five healthy wild boar piglets with a low serum titre of colostral antibodies against CSF virus were challenged with virulent CSF virus at the age of three months. Apart of reduced food intake and diarrhoea no major clinical symptoms were noticed after challenge. These signs were seen during the second and third week of infection, afterwards the piglets recovered completely. CSF virus could be re-isolated from blood samples taken on day 12 and day 19 post challenge. From blood samples taken later on and from the organ material taken at post mortem examinations no CSF virus could be isolated anymore. It can be concluded that the presence of maternal antibodies influences the clinical course of CSF in terms that the outcome is rather transient than lethal. Such wild boar could play a crucial role in the spread of CSF virus and might contribute to the maintenance of long lasting epizootics.

Animals↗
PubMed2001

Classical swine fever: pathogenesis of glomerular damage and immunocharacterization of immunocomplex deposits.

Twenty-six pigs were inoculated with a virulent isolate (Quillota strain) of classical swine fever (hog cholera) virus to determine the chronological development of lesions in the renal glomeruli and the pathogenesis of glomerular damage and immunocomplex deposition. The study included the use of histopathological, ultrastructural and immunohistochemical (detection of viral antigen gp55, myeloid-histiocyte antigen, IgM, IgG and C1q) techniques. The main changes in glomerular structure were observed from 7 days post-inoculation (dpi) onwards, at which time the glomeruli showed macrophage infiltrations in the mesangium, and viral infection in circulating cells, glomerular endothelial cells and podocytes. Moreover, significant subcellular changes were detected in podocytes, which appeared swollen, with fusion of foot processes. Immunocomplex deposits immunoreactive for IgM, IgG and C1q were detected in mesangial, subepithelial and subendothelial areas from 10 dpi, but viral antigen was not detected as a component of these deposits; fusion of foot processes had increased in severity, especially near immunocomplex deposits. All these changes had increased still further in the final phase of the experiment (14 dpi), with neutrophil infiltrations in the mesangium.

Animals↗
PubMed2001

An experimental infection with classical swine fever virus in pregnant sows: transmission of the virus, course of the disease, antibody response and effect on gestation.

An experimental infection with classical swine fever (CSF) virus in 12 conventional gilts, housed in a sow-box housing system, was conducted in order to evaluate horizontal transmission, clinical, virological and serological response, and the effect on gestation. Two of the 12 gilts, of which 10 were pregnant, were experimentally inoculated. They became viraemic for the first time 6 days post-inoculation (dpi). The contact gilts became viraemic between 18 and 21 days post inoculation. On the basis of virological findings and the martingale estimate of R0 (13.0) it was concluded that the two experimentally inoculated gilts infected all contact gilts, although random contacts between gilts were not possible. The prescence of CSF infection could be diagnosed earlier and during a longer period when the leucocyte count or polymerase chain reaction were used in comparison with virus isolation in whole blood (P < 0.05). The observed clinical symptoms were atypical and highly variable between the gilts, which hampered clinical diagnosis. The pregnant gilts became infected between day 43 and 67 of gestation. In all cases vertical virus transmission occurred and this resulted partially in abortion and/or mummification.

Abortion, Veterinary↗
PubMed2001

Oral immunisation against classical swine fever (CSF): onset and duration of immunity.

In an experimental study, onset and duration of immunity after oral immunisation of pigs with a classical swine fever (CSF) live virus vaccine based on the strain "C" has been evaluated. Sixteen weaner piglets (group 1) were orally instilled by syringe with the content of one vaccine bait whereas eighteen piglets (group 2) were fed with one bait. Six unvaccinated piglets represented the control group (group 3). The pigs having 2, 4, 6 and 10 days post vaccination (p.v.) were challenged with the highly virulent CSF virus (CSFV) strain "Koslov" to detect onset of immunity. After oral instillation of vaccine (group 1) the pigs were protected from a clinical infection 4 days p.v. One of four piglets reacted for a short time with an increase of body temperature. In group 2, a partial protection was already detected on day 2 p.v. On day 10 p.v., all animals were resistant to an experimental challenge infection. No protective neutralising antibodies were elicited until day 10 p.v. in both groups. Three animals of each group vaccinated orally against CSF were challenged approximately 6 and 10 months p.v. to evaluate duration of immunity. All vaccinated pigs developed neutralising antibodies and showed a protective immunity against an infection with CSFV until 10 months p.v. Furthermore, no vaccinated animal developed a viraemia after challenge. Altogether, 5 of 34 vaccinated pigs as well as all controls died after infection and showed typical gross lesions for CSF. The tonsils of the surviving pigs were negative for viral antigen by immunofluorescence.

Administration, Oral↗
PubMed2005

Detection and quantitative pathogenesis study of classical swine fever virus using a real time RT-PCR assay.

A real time reverse transcription (RT) TaqMan PCR assay for the detection of classical swine fever virus (CSFV) previously described for use on a SmartCycler was validated on the Applied Biosystems AB 7700 Sequence Detection System using the Roche MagNA pure instrument for nucleic acid extraction and reaction set up. The primers and probe were specific for the CSFV strains (NSW, Baker and Weybridge) and did not react with other pestiviruses (BDV Tobias, BDV #327, BVDV non-CPE and BVDV C24V). Analysis of blood samples collected from pigs 1-6 and 8 days post-oronasal infection showed that over >10(6) range there was a linear relationship between log10TCID50ml-1 blood and the log10 normalised genetic load measured by quantitative TaqMan assay. The assay was used to assess CSFV shedding from infected pigs by quantitative TaqMan assay of virus genetic loads in tonsil, nasal and rectal swabs. Infection of tonsils was detected as early as 1 day post-inoculation. Shedding of virus detected by nasal and rectal swabs commenced on the third day post-inoculation. Quantitative TaqMan was used to analyse virus genetic load in tissues collected from pigs killed on days 1-3, 5 and 8 post-infection. Virus infection appeared first in tonsil (day 1), then submandibular lymph node, spleen, ileum and mesenteric lymph node (by day 3). Thereafter, virus spread to the visceral organs and finally to the pancreas and brain. Tonsil, nasal and rectal swabs as well as whole blood were found to be suitable samples for the rapid detection of CSFV using the TaqMan assay and automated nucleic acid extraction and reaction set up.

Animals↗
PubMed2005

Phylogenetic analysis of classical swine fever virus (CSFV) field isolates from outbreaks in South and Central America.

To date, there is little information concerning the epidemiological situation of classical swine fever (CSF) in the Americas. Besides summarizing the available data, genotyping of isolates from outbreaks in domestic pigs in several countries of South and Central America was performed. For this, a 190 base fragment of the E2 envelope glycoprotein gene was used. European strains and isolates, and historical isolates from the United States (US) were included for comparison. In contrast to the situation in most parts of Europe, where group 2 isolates predominate, it was found that all the isolates from the American continent analyzed belonged to group 1 and were further resolved into three subgroups. The Cuban isolates clustered in subgroup 1.2, whereas the isolates from Honduras and Guatemala clustered in subgroup 1.3. The remaining isolates from Argentina, Brazil, Colombia and Mexico generated four poorly resolved clusters in subgroup 1.1, together with the vaccine strains, with historical European and US isolates, and with a recent Russian isolate. While the vaccine strains and the historical European isolates formed a relatively distinct cluster, one of the US isolates clustered together with the Mexican, and another one with Colombian isolates. Historically, CSF (hog cholera) was observed almost simultaneously in the US and in Europe in the first half of the 19th century, and its origin remains a matter of discussion. Our results showed that the US isolates are closely related to isolates from South America, while appearance of isolates in Cuba on one hand and in Honduras and Guatemala on the other hand, seems to have been due to unrelated events. This allows to speculate that at least in the American continent, CSF virus may have appeared independently in several regions, and spreading may have been a secondary effect.

Animals↗
PubMedDate not supplied

[Synthesis and immunochemical properties of the recombinant major surface glycoprotein E2 of the classical swine fever virus].

Recombinant E2 protein from vaccine strain of classical swine fever virus (CSFV) and from SCFV virulent strain Shimen was synthesized in SF-21 and High-Five cell culture with baculovirus as the expressing vector. For secretion, hydrophobic C-terminal transmembrane domain was removed and N-terminal signal polypeptide of 38 amino acids was added. Maximum accumulation of recombinant products in SF-21 cells was observed after 48 h and in medium 96 h after infection with recombinant baculovirus. In High-Five cells and in culture medium the maximum accumulation of E2 was observed after 96 h. The level of E2 expression is 5-10 micrograms/106 cells. The products of expression were purified by affinity chromatography and their specificity confirmed in immunochemical tests with a series of reference monoclonal antibodies. The product can be used for detecting antibodies to SCFV by competitive enzyme immunoassay.

Animals↗
PubMed1998

A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs.

Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal "P" site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met-tRNAiMet, eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed.

Animals↗
PubMed2005

Candidate multi-peptide-vaccine against classical swine fever virus induced potent immunity with serological marker.

Our previous study proposed a protective multi-peptide-vaccine (MPV) with Freund's adjuvant against classical swine fever virus (CSFV). In this study, another candidate MPV, using aluminum adjuvant, was further examined. All immunized pigs kept healthy during the experimental period, while the control group rapidly showed clinical symptoms and died. Moreover, anti-sera from MPV-immunized pigs could interact with peptides involved in the MPV, in contrast to anti-sera from non-immunized or infected ones. This property permits MPV-immunized pigs to be easily differentiated from infected ones with simple serological method. Therefore, this new MPV is suitable to act as a candidate marker vaccine against CSFV.

Adjuvants, Immunologic↗