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PubMed1998

[Classical swine fever in 1993 in Switzerland: molecular-epidemiologic characterization of the virus isolate].

RT-PCR followed by direct nucleotide sequencing of the amplified cDNA was carried out to analyse most of the 5' nontranslated region (5'NTR) of classical swine fever virus (CSFV) isolates from the five 1993 disease outbreaks in Switzerland. Sequence data were compared to other CSFV strains, and dendrograms were constructed in order to determine the phylogenetic relationship of the Swiss virus strains. Dendrograms formed by the analysis of different parts of the 5'NTR were compared. It was shown that all Swiss isolates were related to other CSFV strains involved in disease outbreaks in Europe in the 1990s. Two of the isolates were indistinguishable from a CSFV strain isolated from wild boar meat imported from China into Austria in 1993. The risk of introducing classical swine fever by improperly treated swill and, in particular by importing wild boar meat is discussed.

Animals↗
PubMed2004

When can a veterinarian be expected to detect classical swine fever virus among breeding sows in a herd during an outbreak?

The herd sensitivity (HSe) and herd specificity (Hsp) of clinical diagnosis of an infection with classical swine fever (CSF) virus during veterinary inspection of breeding sows in a herd was evaluated. Data gathered from visits to herds during the CSF outbreak in 1997-1998 in The Netherlands were used for the analysis. Herds were visited one or more times by the same or by different veterinarians. On the basis of the veterinarians' reports, each visit was coded as 0 (negative clinical diagnosis) or 1 (positive clinical diagnosis). The HSe for clinical diagnosis of CSF was modelled as a function of days elapsed since introduction of the virus. The moment of introduction of the CSF virus in the CSF-positive herds was unknown, so for each herd, a probability distribution for the unknown number of days since introduction was derived from serum samples collected at depopulation. The information from the reports of the veterinarians and from the test results of the serum samples at depopulation was combined in a Bayesian analysis. Data from CSF-negative herds were analysed to estimate HSp of clinical diagnosis of CSF. The HSe of clinical diagnosis was 0.5 at 37 days after virus introduction (95% CI: 31, 45) and reached 0.9 at 47 days after virus introduction (95% CI: 41, 54). The estimated herd specificity was 0.72 (95% CI: 0.64, 0.79). Dependence of HSe and HSp on characteristics of the veterinarians and the herds also was studied. Specialisation of the veterinarian significantly, although not markedly, affected the HSe.

Animal Husbandry↗
PubMed2006

Detection of classical swine fever vaccine virus in blood and tissue samples of pigs vaccinated either with a conventional C-strain vaccine or a modified live marker vaccine.

Attenuated live classical swine fever (CSF) viruses are the most efficacious vaccines against the disease. However, little is known about the distribution and detection of CSF vaccine viruses in the host. We therefore compared the new recombinant attenuated marker vaccine virus CP7_E2alf with the conventional C-strain vaccine concerning virus isolation, antigen-, and genome-detection in different samples within the first 42 days post-vaccination (p.v.). Leukocytes and several organs such as tonsils, lymph nodes, spleen, thymus, parotis and kidney were also tested using highly sensitive real-time reverse transcription-polymerase chain reaction (RT-PCR) techniques. It was demonstrated that vaccine virus could be detected by live animal sampling only in a few leukocytes samples at very low titres and genome copy numbers within the first 14 days after immunisation. Vaccine virus could also be isolated from individual tonsil samples within the first 6 days after vaccine application. In contrast, vaccine virus genomes were consistently detected in the tonsils up to day 42 by real-time RT-PCR. Distribution, amount of virus and viral genome levels were similar for both tested vaccines. In conclusion, blood samples could be the sample material of choice for detecting CSF wild type virus infection even in vaccinated animals after more than 14 days p.v., while tonsil sampling provided appropriate material for long-term detection of both tested CSF vaccine viruses using real-time RT-PCR methods.

Animals↗
PubMed2000

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.

Animals↗
PubMed2001

[Detection of infection with classical swine fever virus in wild boar: a comparison of different laboratory diagnostic methods].

The aim of this study was to evaluate two commercially available ELISAs for routine diagnosis of classical swine fever virus (CSFV) in wild boar. For this, 222 tissue samples from wild boar were tested in the ELISAs and the results were compared to those obtained using standard methods. First, frozen spleen sections were examined by direct immunofluorescence, and organ suspensions were prepared and tested for CSFV antigen samples were simultaneously examined with the Chekit-ELISA (Dr. Bommeli AG) and the Herd-Chek-ELISA (IDEXX). From the 222 organ suspensions examined in cell culture 102 were positive for CSFV, while no virus could be isolated from the remaining 120 samples. Taking virus isolation as a standard, the Chekit-ELISAs showed a sensitivity of 97%, and the Herd-Chek-ELISA of 72.5%. Both ELISAs revealed high specificities ranging between 99 and 100%. No correlation was found between false negative results obtained in one or in both of the ELISAs with the positive findings in the immunofluorescence test and in the PLA, nor with the clinical reports. Due to the fact that a big number of samples can be processed in a short time with accurate results, the Chekit-ELISA may be considered useful for routine testing of wild boar samples for CSFV.

Animals↗
PubMed1995

Classical swine fever in wild boar (Sus scrofa)--experimental infections and viral persistence.

A pregnant wild boar and two wild boar weaners were inoculated intranasally with a field isolate of classical swine fever virus (CSFV) recently derived from a diseased domestic pig. The clinical, pathological and haematological findings noted in the young wild boars were comparable to those in domestic weaner pigs inoculated with the same virus isolate. Both wild boars showed the acute haemorrhagic form of CSF, one animal died 18 days post inoculation (p. i.) and the second one had to be euthanized when moribund two days later. The wild boar sow did not show any signs of illness p. i. but seroconversion was noticed. Twenty-eight days p. i. birth was given to six clinically healthy offsprings. One of the newborn proved to be viraemic until death when 39 days of age. Except for poor growth no other symptoms were noticed in this piglet. The non-viraemic litter mates remained healthy, although they had close contact to the persistently infected piglet. High titres of neutralizing antibodies against CSFV were measured in the serum samples of these offsprings. All findings were more or less in accordance with observations previously made in domestic pigs when infected with CSFV around 85 to 90 days of gestation. The wild boar was calculated to have been inoculated at about 87 to 92 days of gestation.

Animals↗
PubMed2002

Experimental non-transmissible marker vaccines for classical swine fever (CSF) by trans-complementation of E(rns) or E2 of CSFV.

Three mutants with deletions in the E2 gene of the infectious DNA copy of the classical swine fever virus (CSFV) strain-C were constructed: one missing the B/C domain of CSFV-E2 between amino acids (aa) 693 and 746, one missing the A domain between aa 800 and 864, and one missing the complete E2 between aa 689 and 1062. All three CSFV-E2 deletion mutants were unable to generate viable virus, indicating that each of the antigenic domains of E2 is essential for viability of CSFV. To rescue the CSFV-E2 deletion mutants SK6 cell lines constitutively expressing glycoprotein E2 of CSFV were generated. The rescued viruses infected and replicated in SK6 cells as demonstrated by expression of viral proteins, but this primary infection did not result in reproduction of infectious virus. Thus, these E2 complemented viruses are considered non-transmissible. In previous experiments, we showed that simultaneous injection of E(rns) complemented virus (Flc23) via intradermal (ID), intramuscular (IM) or intranasal (IN) routes conferred protection to pigs against a lethal challenge with CSFV [J. Virol. 74 (2000) 2973]. Here, we evaluate different routes of application (ID, IM or IN) with E(rns) complemented virus Flc23 in order to find the best route for complemented CSFVs. Intradermal injection with Flc23 protected pigs against a lethal CSFV challenge, whereas intramuscular injection induced partial protection, and intranasal injection did not mediate a protective immune response in pigs at all. We used the intradermal route of vaccination to test the E2 complemented viruses. Vaccination of pigs via the intradermal route with the E2 complemented CSFVs also resulted in the induction of antibodies and in (partial) protection against CSFV challenge. Pigs vaccinated with E2 complemented virus Flc4 (deletion B/C domain) survived a lethal CSFV challenge, whereas partial protection was induced in pigs vaccinated with Flc47 (deletion E2) or Flc48 (deletion A domain) E2 complemented viruses. Serological data demonstrate that these E2 complemented mutant viruses are, in combination with well known diagnostic tests based on E2, potential marker vaccines for CSF.

Animals↗
PubMed1999

Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus.

To determine the minimal requirements for autonomous RNA replication of classical swine fever virus (CSFV), genomes having in-frame deletions within the genes for structural and flanking nonstructural proteins were constructed, based on an infectious cDNA clone of CSFV Alfort/187. RNA was transcribed in vitro from the respective plasmids and transfected into SK-6 swine kidney cells. The replication competence of the RNA was determined by immunostaining transfected cells for CSFV NS3 protein and by analysis of cell extracts for viral RNA, as well as protein synthesis at different times after transfection. The genes encoding N(pro), C, E(rns), E1, E2, p7, and NS2 proved to be dispensable for RNA replication, but the efficiency of replication varied strongly between individual constructs. RNA replicons containing the complete NS2-NS3 gene persisted in transfected cells and continued to replicate without causing any obvious morphological or functional damage to the cells, whereas genomes lacking the NS2 gene replicated more efficiently and induced a cytopathic effect. These findings suggest that NS2, although it is not essential for pestivirus RNA replication, has a regulatory function therein. Both cytopathogenic and noncytopathogenic replicons were packaged into virus particles provided in trans by a cotransfected full-length helper virus genome.

Animals↗
PubMed1998

Inactivation of the RNase activity of glycoprotein E(rns) of classical swine fever virus results in a cytopathogenic virus.

Envelope glycoprotein E(rns) of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E(rns) are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E(rns) was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E(rns), the mutated proteins had lost their RNase activity. The mutated proteins reacted with E(rns)-specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E(rns). This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E(rns) (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E(rns) gene and by neutralizing polyclonal antibodies directed against E(rns), indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E(rns) plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.

Animals↗
PubMed1997

The presence of RNA splicing signals in the cDNA construct of the E2 gene of classical swine fever virus affected its expression.

E2 is the major neutralizing antigen for classical swine fever virus (CSFV) infection. Previously, we have cloned and sequenced the E2 cDNA of Taiwan strain p97 by the reverse transcription-polymerase chain reaction (RT-PCR) method from CSFV-infected tissue. The presence of RNA splicing donor and acceptor sites were found in the cDNA sequence. In this study, transfection of E2 cDNA into mammalian cells resulted in the production of a spliced RNA. Site-directed mutagenesis of the donor and acceptor sites prevented the RNA splicing event and generated a full length transcript in COS7 cells. Although the spliced E2 transcript has not been reported in natural infection of CSFV, this study suggested that the potential splicing sites affected the E2 gene expression when the plasmid-based E2 gene was introduced into mammalian cells.

Amino Acid Sequence↗
PubMedDate not supplied

In vitro effect of classical swine fever virus on a porcine aortic endothelial cell line.

The effect of classical swine fever (CSF) virus on some phenotypic and functional features of an established porcine aortic endothelial cell (AOC) line was investigated. AOC cells show most of the characteristics of primary endothelial cells, avoiding the alterations and senescence that these cells undergo after a few passages in culture. AOC cells were susceptible to CSF virus infection to a high degree, reaching 90% of CSF virus positive cells after 24 h of infection; however as with other porcine susceptible cells, no cytopathic effect could be observed. In these conditions none of the surface molecules studied, including SLA-II MHC antigens, adhesion or co-stimulatory molecules, were altered by virus infection after 24 or 48 h. Functionally CSF virus infection induced a decrease in the pro-coagulant activity of the AOC cells, determined by the increase in the clot formation time shown by the lysates of these cells. This contrasts with the increase observed in the expression of mRNA corresponding to IL-1 alpha and IL-6, two proinflammatory and pro-coagulant cytokines, in CSF virus-infected AOC cells.

Animals↗
PubMed2003

Detection of classical swine fever virus in boar semen by reverse transcription-polymerase chain reaction.

A seminested reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of classical swine fever virus (CSFV) in semen. Five boars were inoculated intranasally with CSFV isolate propagated in PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Semen and serum samples were collected twice weekly for 63 days postinoculation (dpi). Samples were tested for the presence of antibodies to CSFV by an enzyme-linked immunosorbent assay and for the presence of CSFV nucleic acid by seminested RT-PCR. Antibodies to CSFV could be detected as early as 7 dpi in 1 boar, and all 5 infected boars were found positive by 14 dpi. CSFV from boar semen was infrequently identified by virus isolation compared with seminested RT-PCR. CSFV nucleic acid was detected in semen by seminested RT-PCR as early as 7 dpi in 3 infected boars and persistently thereafter in all 5 infected boars until 63 dpi. When separated fractions of CSFV-contaminated semen were analyzed by the seminested RT-PCR, the CSFV nucleic acid was detected mainly in seminal fluid and occasionally in nonsperm cells. CSFV antigen was also detected in nonsperm cells from semen smear by immunohistochemistry. Thus, infection via semen, specially through CSFV-infected seminal fluid, seems to be a major route of transmission of CSFV.

Animals↗
PubMed2000

[Laboratory findings during the classic swine fever epidemic of 1997-1998].

The results of the laboratory tests carried out by the Institute for Animal Science and Health (ID-Lelystad), the Netherlands, on samples collected during the Classical Swine Fever (CSF) epidemic 1997-1998 are summarized in this article. The relevance of the different laboratory tests and various samples collected on the eradication of CSF during an outbreak is evaluated.

Animals↗
PubMed2004

Classical swine fever virus induces tumor necrosis factor-alpha and lymphocyte apoptosis.

The expression of tumor necrosis factor (TNF)-alpha and apoptosis was studied in lymph nodes from pigs infected with Classical swine fever virus (CSFV). Pigs were inoculated with CSFV and euthanized at 3, 5, 7, and 10 days postinoculation. An increase in TNF-alpha expression was detected in CSFV-infected lymph nodes using a reverse transcription-polymerase chain reaction, and TNF-alpha protein was detected in lymph nodes by immunohistochemistry. The majority of TNF-alpha-positive cells also expressed the SWC3a antigen, a specific marker for porcine leukocytes. By combined use of in situ hybridization and immunohistochemistry, CSFV infection was detected in lymph nodes macrophage. Lymphocytes death occurred by apoptosis that was characterized by condensed shrunken cells and the formation of apoptotic bodies, some of them contained pyknotic nuclear remnants. Apoptosis was detected in situ by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) reaction. A double-labeling experiment using immunohistochemistry and TUNEL reaction for the detection of CSFV and apoptosis demonstrated that the majority of labeled cells were positive for CSFV or apoptosis. This suggests that CSFV can induce apoptosis directly and indirectly. Apoptotic cells induced by viral infection were more abundant than CSFV-infected cells in all lymph nodes tested. A double-labeling experiment using immunohistochemistry and TUNEL reaction for the detection of TNF-alpha and apoptosis demonstrated that labeled cells were positive for either TNF-alpha or apoptosis, and both. The present study addressed two important issues regarding CSFV-induced apoptosis: (i) viral infection and apoptosis colocalize at the cell level; and (ii) one or more factors (e.g., TNF-alpha) released from macrophages may induce apoptosis in uninfected bystander cells.

Animals↗
PubMed2000

Rescue of infectious classical swine fever and foot-and-mouth disease virus by RNA transfection and virus detection by RT-PCR after extended storage of samples in Trizol.

A method for storing samples containing classical swine fever virus (CSFV) or foot-and-mouth disease virus (FMDV), respectively, was developed, which abolishes the infectivity of both plus strand RNA viruses, and allows storage of samples above 0 degrees C for an extended time, yet preserves the viral RNA in a state which allows its detection by reverse transcription-polymerase chain reaction (RT-PCR), and even rescue of infectious virus after transfection of the extracted RNA into susceptible cells. Supernatants from infected cell cultures as well as organs from diseased animals were stored in Trizol(R) for 1-4 weeks at -20 degrees C, 4 degrees C, room temperature, or 37 degrees C. RNA was then extracted and used subsequently for RT-PCR, as well as transfection into susceptible cells to initiate the replication of progeny virus. Formaldehyde-fixed samples were also included in this study. Storage up to 4 weeks at 37 degrees C in Trizol(R) still yielded positive RT-PCR results and rescue of infectious virus upon RNA transfection. In contrast, formaldehyde fixation reduced drastically the detectability of viral RNA. This method represents a safe and inexpensive alternative to -70 degrees C (dry ice) storage or transport of samples, and abolishes the biosafety risks involved in shipping deep-frozen infectious materials.

Animals↗
PubMed1999

Incidence of classical swine fever (CSF) in wild boar in a densely populated area indicating CSF virus persistence as a mechanism for virus perpetuation.

A virological survey was carried out to establish the distribution of classical swine fever (CSF) virus among wild boar in the Federal State of Brandenburg, Germany. Organ materials and blood samples were collected from 11,670 wild boar shot or found dead during the period March 1995 to December 1997. In total 211 (1.8%) wild boar were positive for CSF virus or antigen. The incidence of CSF-positive animals decreased continuously from 4.6% at the beginning of the epidemic in 1995 to 0.7% in 1997. The highest incidence of positive animals (22%) was found in wild-boar piglets younger than 3 months of age in 1995. The findings were indicative for the decisive role which young wild boar play in the epidemiology of CSF. Following intrauterine transfer some of the wild-boar piglets were probably persistently infected with CSF virus as experienced experimentally. Such piglets can be held responsible for CSF virus perpetuation within the wild-boar population. No CSF virus was isolated from adult wild boar weighing more than 75 kg. During 3 years of monitoring a sufficient number of susceptible wild boar, in particular young animals, was available to maintain the infection chain in that area. It was concluded that persistently infected piglets and the high population density of wild boar in the Brandenburg region offered optimal conditions for the establishment of an CSF epidemic.

Age Factors↗
PubMed2005

Statistical evaluation of classical swine fever surveillance plans in Italy (1995-2003).

Summary Surveillance plans were carried out in Italy from 1995 to 2003, with the main aim of controlling the possible circulation of low-virulence classical swine fever viral strains. The repetition of a serological monitoring programme over several years, with constantly negative results, can prove the absence of the infection while increasing the degree of confidence. In our case, in 2003, after eight repetitions of the surveillance plan, the probability that there was at least one infected farm reached 0.006%. Therefore, on the basis of the results of the sampling carried out from 1995 to 2003, we may state that there is no endemic infection, even because of a low virulence virus, in continental Italy.

Animals↗