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PubMed2005

Role of double-stranded RNA and Npro of classical swine fever virus in the activation of monocyte-derived dendritic cells.

Classical swine fever virus (CSFV) is a noncytopathogenic (ncp) positive-sense RNA virus that replicates in myeloid cells including macrophages and dendritic cells (DC). The virus does not induce type I interferon (IFN-alpha/beta), which in macrophages has been related to the presence of the viral Npro gene. In the present work, the role of viral double-stranded (ds)RNA and Npro in the virus-host cell interaction has been analyzed. Higher levels of detectable dsRNA were produced by a genetically engineered cytopathogenic (cp) CSFV compared with ncp CSFV, and cp CSFV induced IFN-alpha/beta in PK-15 cells. With DC, there was only a small difference in the levels of dsRNA between the cp and ncp viruses, and no IFN-alpha/beta was produced. However, the cp virus induced a higher degree of DC maturation, in terms of CD80/86 and MHC II expression. Npro deletion mutants induced an increase in DC maturation and IFN-alpha/beta production-for both ncp and cp viruses-despite reduced replication efficiency in the DC. Deletion of Npro did not influence dsRNA levels, indicating that the interference was downstream of dsRNA turnover regulation. In conclusion, the capacity of CSFV to replicate in myeloid DC, and prevent IFN-alpha/beta induction and DC maturation, requires both regulated dsRNA levels and the presence of viral Npro.

Animals↗
PubMed2005

Genetic typing of classical swine fever viruses from Lao PDR by analysis of the 5' non-coding region.

The 5' non-coding region (5'-NCR) of 27 classical swine fever virus (CSFV) isolates from Lao People's Democratic Republic (Lao PDR) during 1997 and 1999 were amplified by RT-PCR. A 150-bp region of the 5'-NCR amplicons was analysed and compared with reference CSFV of European and Asian origin and a phylogenetic dendrogram constructed. Following analysis, all viruses were determined to belong to genogroup 2. Viruses from Lao PDR grouped on a geographical basis with the majority of northern/central isolates falling into subgroup 2.1 and southern/central isolates falling into subgroup 2.2. These results concur with previous studies of CSF viruses from Lao PDR, although this study recognized the first occurrence of subgroup 2.1 in southern Lao PDR.

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PubMed1998

Porcine cells persistently infected with classical swine fever virus protected from pestivirus-induced cytopathic effect.

Cytopathogenicity of classical swine fever virus (CSFV) depends on the presence of defective particles containing a subgenomic (sg) RNA with a defined deletion. In a previous report we described the spontaneous generation of this sg RNA and therefore of cytopathogenic (cp) CSFV in porcine kidney cell cultures persistently infected with CSFV. Frequently, some cells survived the CPE and could be further propagated. They remained positive for viral antigen and continued to shed complete virus and in most cases also defective virus particles. SK-6 cells that had survived the CPE (CPE(surv)cells) were used to investigate these findings further. In contrast to persistently infected cells that had not experienced a CPE, CPE(surv) cells were protected from the CPE when superinfected with cp CSFV or with cp bovine viral diarrhoea virus. Similarly, cells which were rescued and further propagated after acute infection with cp CSFV also proved to be protected from the CSFV-induced CPE. When either virus obtained from CPE(surv) cells that had spontaneously lost the sg RNA or virus from which defective particles had been removed was used to establish persistently infected cells, these cells were also protected from the CPE after superinfection with cp CSFV. These findings suggest that the virus contained in CPE(surv) cells confers on the host cell the ability to resist the CSFV-induced CPE. However, when naive cells were infected with supernatants from CPE(surv)cells that contained defective virus particles, the CPE reappeared within three to five virus passages, indicating that the sg RNA retained its cytopathogenic potential.

Animals↗
PubMed2005

Negative impact of porcine reproductive and respiratory syndrome virus infection on the efficacy of classical swine fever vaccine.

Recent findings suggest that porcine reproductive and respiratory syndrome virus (PRRSV) possesses immunomodulatory properties. To investigate the effect of PRRSV infection on classical swine fever (CSF) vaccine efficacy, 17-day-old pigs were divided into five groups. The experimental group was infected with a Thai PRRSV (US genotype) a week before CSF vaccination and challenged with a virulent CSF virus (CSFV) 3 weeks following vaccination. The control groups received no PRRSV infection, no CSF vaccination, no CSF challenge, or in combination were included. The results demonstrated that PRRSV infection significantly inhibited host immune response that resulted in vaccination failure in the subsequent CSFV exposure. Following CSF challenge, the PRRSV-infected, vaccinated pigs exhibited clinical, virological and pathological features resembled to those of the non-vaccinated groups. The findings indicated that CSF immunization during an acute phase of PRRSV infection could result in vaccination failure.

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PubMed2002

Rate of inter-herd transmission of classical swine fever virus by different types of contact during the 1997-8 epidemic in The Netherlands.

In this study we quantified the rate at which classical swine fever had been transmitted by several different types of inter-herd contact during the 1997-8 epidemic in The Netherlands. During that epidemic 428 CSFV-infected pig herds were detected, 403 of which were include in this study. The estimated rates of transmission were 0.065 per shipment of live pigs, 0.011 per contact by a pig transportation lorry, 0.0068 per person contact, 0.0007 per dose of semen, 0.0065 per contact with a potentially contaminated pig assembly point, 0.027 per week per infected herd within a radius of 500 metres and 0.0078 per week per infected herd at a distance between 500 and 1000 metres. These transmission rates can be used to optimize the strategy to stop future epidemics of CSF in The Netherlands. In addition, the analysis demonstrated in this paper, can be used to quantify CSFV transmission rates from other epidemics.

Animal Husbandry↗
PubMed2005

Monte Carlo simulation of classical swine fever epidemics and control. I. General concepts and description of the model.

A Monte Carlo simulation has been developed to describe the spread of classical swine fever virus between farms within a certain region. The data of the farms can be imported and considered individually. Transmission occurs via the infection routes direct animal and indirect person and vehicle contact, as well as by contaminated sperm and local spread. Parameters, such as incubation period and probability of detection, can be varied by the user and their impact on disease spread can be studied. The control measures stamping-out, movement control and pre-emptive slaughter in circular restriction areas as well as contact tracing can be applied and their effect on disease spread can thus be analysed. The numbers of culled and restricted farms and animals per epidemic and per day within an epidemic, the epidemic duration and the total length of restrictions per restricted farm are given. In an example, simulation runs were performed under the condition of application of all four-control measures. Because no real farm data were available, a test area was generated stochastically with a farm density of 1.3 farms/km(2). The distributions of the number of infected farms per epidemic and the epidemic length are shown.

Animals↗
PubMed2000

Genetic typing of classical swine fever virus isolates from the territory of the Czech Republic.

Epizootiological studies based on genetic typing were performed using 14 isolates from outbreaks of classical swine fever (CSF) in domestic pigs and wild boar in the Czech Republic which occurred between 1991 and 1998. They were compared with Austrian, Slovakian, Hungarian, Polish and German isolates. The aim of this study was to characterise the CSF virus isolates and find out the possible relationships between the outbreaks in domestic pigs and wild boar, and to map the spread of the virus in the Czech Republic. For this, fragments of the 5' nontranslated region (5' NTR) and of the E2 glycoprotein gene were sequenced and used for genetic typing. The analysis of both fragments of the genome showed that the Czech isolates belong to two CSF subgroups within group 2, namely to subgroups 2.2 and 2.3. A close relation was found with Austrian isolates from 1992 and 1994, belonging to subgroup 2.2. The isolates in subgroup 2.3 formed a very homogeneous group, although they originated from different regions of the country. They seem identical to two Slovakian isolates from 1998, and differed from Hungarian isolates from 1992. Epizootiological links became evident when the epidemiological data were compared.

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PubMed2005

Susceptibility of in vivo- and in vitro-produced porcine embryos to classical swine fever virus.

The objective of this study was to investigate the susceptibility of in vivo- and in vitro-produced (IVP) porcine embryos to classical swine fever virus (CSFV). IVP zona pellucida (ZP)-intact porcine embryos (n = 721) were co-cultured with CSFV for 120 h. After washing according to the International Embryo Transfer Society guidelines (without trypsin) and transferring embryos to CSFV-susceptible porcine kidney cells (PK15 cell line), no virus was isolated. However, when 88 IVP ZP-intact porcine embryos were co-cultured with CSFV for only 48 h before being transferred to PK15 cells, virus was isolated in three of six replicates. Similarly, 603 in vivo-produced porcine embryos were co-cultured with CSFV for 120 h. Subsequently, CSFV was isolated in eight of 50 groups (16%) and the ability of these to form a blastocyst was significantly reduced when compared with the control group (68.2 +/- 19.9% vs 81.9 +/- 9.7%; p < or = 0.001). In contrast, the development of CSFV-exposed IVP porcine embryos was not affected when compared with control embryos (19.1 +/- 10.8% vs 18.9 +/- 10.6%; p > or = 0.05). After removal of the ZP of IVP embryos and subsequent co-culture with CSFV, the virus was isolated from all groups of embryos. These data suggest that virus replication had occurred in the embryonic cells. In conclusion, data indicate that in vivo- and in vitro-produced ZP-intact porcine embryos differ in their susceptibility to CSFV. Hatched or micro-manipulated embryos may increase the risk of transmission of CSFV by embryo transfer, which has to be confirmed by in vivo tests under isolation conditions.

Animals↗
PubMed2005

Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells.

Vaccination of pigs against Classical swine fever virus (CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3-NS4A were used to identify viral genes encoding CTL epitopes. Monocyte-derived dendritic cells (DCs) and fibrocytes served as the APCs. In vitro translation of the mRNA and microscopic analysis of transfected cells demonstrated that E2 and NS3-NS4A could be identified. APCs transfected with either of the mRNA molecules restimulated CSFV-specific T cells to produce gamma interferon and specific cytotoxic activity against CSFV-infected target cells. The presence of CTL epitopes on E2 was confirmed by using d/d-haplotype MAX cells expressing E2 constitutively as target cells in d/d-haplotype CTL assays. A potent CTL activity against E2 was detected early (1-3 weeks) after CSFV challenge. This work corroborates the existence of CTL epitopes within the non-structural protein domain NS3-NS4A of CSFV. Furthermore, epitopes on the E2 protein can also now be classified as targets for CTLs, having important implications for vaccine design, especially subunit vaccines. As for the use of mRNA-transfected APCs, this represents a simple and efficient method to identify viral genes encoding CTL epitopes in outbred populations.

Animals↗
PubMed1999

The 1997-1998 classical swine fever epidemic in The Netherlands--a survival analysis.

The aim of this analysis was to characterise the temporal pattern of infection during the 1997/98 classical swine fever (CSF) epidemic in The Netherlands and hence identify and quantify risk factors for infection in different enterprise types and areas. Survival analysis and Cox proportional hazards regression were used to describe the epidemic. Substantial differences in temporal survival patterns (herd breakdown rate) were found between areas where different control policies operated. Factors with a significant influence on the infection hazard of individual herds included: sow numbers as a percentage of total sows and fatteners (HR = 3.38 for mixed herds (0.1-60% sows) vs. fattening herds (0% sows) and HR = 2.74 for breeding herds (60-100% sows) vs. fattening herds), the number of 'transport contacts per month' (>0.3 vs. <0.3; HR = 4.11), pig density (pigs/km2) in the area (HR1000 pigs 1.48) and herd size (HR100 pigs = 1.01). Pre-emptive slaughter in an area appeared to be associated with lower subsequent disease levels. Higher frequency of transport contacts for welfare slaughter during the epidemic, however, well regulated and controlled, was associated with a substantially higher risk of becoming infected. The positive association of a higher pig density with CSF indicates the potential importance of local spread as a factor in disease transmission and emphasizes that dilution of the pig population can contribute to reduction in CSF occurrence. This analysis suggests however, that if pre-emptive slaughter can promptly be applied effectively in an area after initial diagnosis, pig density is then not a significant factor. Mixed and breeding herds had a higher probability of becoming infected than fattening herds, possibly due to different types and frequencies of inter-herd contacts. These contacts continue to some extent during the epidemic, despite the standstill of animal movements.

Animal Husbandry↗
PubMed1999

Quantification of the transmission of classical swine fever virus between herds during the 1997-1998 epidemic in The Netherlands.

In this study, we describe a method to quantify the transmission of Classical Swine Fever Virus (CSFV) between herds from data collected during the 1997-1998 epidemic in The Netherlands. From the contacts between infected herds and the serological findings shortly before depopulation, we estimated the week of virus introduction and the length of the period over which the herd emitted virus for each CSFV-infected herd. From these data, we estimated the infection-rate parameter beta (the average number of herds infected by one infectious herd during one week) and the herd reproduction ratio, Rh (the average total number of secondary outbreaks caused by one infectious herd, i.e. in its entire infectious period), using a SIR-model for different sets of CSF control measures. When Rh > 1, an epidemic continues to grow. On the other hand, when Rh < 1 an epidemic will fade out. During the phase before the first outbreak was diagnosed and no specific measures had been implemented, beta was estimated at 1.09 and Rh at 6.8. In the subsequent phase infected herds were depopulated, movement restrictions were implemented, infected herds were traced forward and backward and the herds in the protection and surveillance zones were clinically inspected by the veterinary authorities (regional screening). This set of measures significantly reduced beta to 0.38. However, Rh was 1.3 and thus still > 1. Consequently, the number of outbreaks continued to grow. After a number of additional measures were implemented, the value of Rh was reduced to 0.5 and the epidemic came to an end. These measures included pre-emptive slaughter of herds that had been in contact with infected herds or were located near an infected herd, increased hygienic procedures, replacement of transports of pigs for welfare reasons by killing of young piglets and a breeding ban, and regional screening for CSF-infected herds by local veterinary practitioners.

Animal Husbandry↗
PubMed2001

Application of competitive enzyme-linked immunosorbent assay for the serologic diagnosis of classical swine fever virus infection.

A competitive enzyme-linked immunosorbent assay (C-ELISA), based on a truncated E2 recombinant protein of the Alfort/187 strain of classical swine fever virus (CSFV) and a specific monoclonal antibody M1669, was evaluated using 2,000 sera from clinically healthy pigs in Canada (a CSFV-free country) and sera from experimentally infected pigs. The relative specificity and sensitivity of the C-ELISA were 100% and 86%, respectively, at a cutoff of 25% inhibition using negative and positive pig sera, as defined by the neutralizing peroxidase-linked assay (NPLA). A kappa value of 0.91 was obtained, indicating an excellent level of agreement between the NPLA and the C-ELISA. When sera from 120 infected pigs were used in the test at > or = 21 days postinfection, the sensitivity of the C-ELISA and the kappa value increased to 97% and 0.98, respectively. This C-ELISA will be useful when a large number of samples must be tested, as could occur during a disease outbreak or for surveillance or prevalence studies.

Animals↗
PubMed2000

[The leukocyte count is a valuable parameter for detecting classical swine fever].

In this paper we describe a study of the use of the white blood cell count (wbcc) as a parameter for detecting outbreaks of Classical Swine Fever (CSF). Meta-analysis of the results of challenge experiments revealed that oronasal infection of SPF-pigs with the virulent CSF virus (CSFV) strains Brescia or NL9201 resulted in a significant decrease in the average white blood cell count during the first week after inoculation of the virus. Challenge of conventional finishing pigs and sows with the moderately virulent strain Paderborn also resulted in a significant decrease in the average wbcc. However, this decrease was not observed after inoculation of SPF pigs with the mildly virulent CSFV strains Henken, Zoelen, or Bergen. The usefulness of clinical inspection in combination with wbcc to detect CSF outbreaks in the field was examined using the results of 214 EDTA blood specimens collected from 22 infected herds and 7250 EDTA blood specimens collected from 1450 non-infected herds. Half of the infected herds had been infected with the moderately virulent CSFV strain Venhorst (closely related to strain Paderborn) during the 1997-98 epidemic in the Netherlands. The other half had been infected with the moderately virulent CSFV strain Loraine. Using these data as a starting point, 1000 samples of one to ten specimens were generated by Monte Carlo simulation. These simulated samples and the samples of the non-infected herds were analysed by use of Receiver Operating Characteristic curves. On the basis of that analysis, the optimal number of animals whose wbcc needed to be determined to detect a CSF outbreak was five. With this number of animals, in conjunction with the threshold of 8000 white blood cells per mm3 (meaning that a herd is designated as CSF suspect if one or more of the five specimens has a white blood cell count of 8000 leukocytes/mm3 or less), the test procedure had a herd sensitivity (HSE) of 94.5% and a herd specificity (HSP) of 97.2%). The HSE is defined as the percentage of samples of infected herds with a positive result of the test procedure; HSP is defined as the percentage of uninfected herds with a negative result of the test procedure. We conclude that the wbcc can help the veterinary practitioner to detect outbreaks of CSF caused by (moderately) virulent CSFV strains. However, for the detection of outbreaks caused by mildly virulent CSFV strains, the contribution of the wbcc is doubtful. Development of additional tools that can improve the clinical diagnosis of the veterinary practitioner remains desirable.

Animals↗
PubMed2000

Classical swine fever virus: a second ring test to evaluate RT-PCR detection methods.

Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control samples. Two types of test were evaluated. One amplified a part of the 5'-non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested RT-PCR. The results of the laboratories were compared with one another, and with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al., Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed.

Animals↗
PubMed1996

Comparison of the protective efficacy of recombinant pseudorabies viruses against pseudorabies and classical swine fever in pigs; influence of different promoters on gene expression and on protection.

The glycoprotein E (gE) locus in the genome of pseudorabies virus (PRV) was used as an insertion site for the expression of glycoprotein E1 of classical swine fever virus (CSFV). Transcription of E1 in the recombinants M401, M402 or M403 was regulated by the gD promoter of PRV, the immediate early gene promoter of human cytomegalovirus, or the gE promoter of PRV, respectively. Groups of four pigs were vaccinated once intramuscularly with 10(6) plaque forming units (p.f.u.) of the recombinant viruses and challenged intranasally with 100 50% lethal doses of virulent CSFV and with 10(5) p.f.u. of virulent PRV. All pigs vaccinated with M402 were fully protected against both classical swine fever and pseudorabies.

Animals↗
PubMed1996

Production of monoclonal antibodies against classical swine fever virus and their use for antigenic characterization of Japanese isolates.

Twenty monoclonal antibodies (MAbs) were produced against the virulent ALD or vaccinal GPE- strains of classical swine fever (CSF) virus. The MAbs were divided into 7 groups on the basis of the difference in the reaction spectrum to various CSF and bovine viral diarrhea-mucosal disease (BVD-MD) viruses. The panel of the MAbs could classify 20 CSF viruses into 6 types, and could discriminate between not only CSF and BVD-MD viruses but also the vaccinal GPE- strain and the field isolates of CSF virus. The results indicated that the MAbs were useful for diagnosis and investigation of CSF viruses.

Animals↗
PubMedDate not supplied

Classical swine fever virus: clinical, virological, serological and hematological findings after infection of domestic pigs and wild boars with the field isolate "Spante" originating from wild boar.

A classical swine fever virus (CSFV) field isolate originating from wild boar was investigated on its virulence in domestic pigs and wild boar. Three weaner pigs and two wild boars (yearlings) were intranasally inoculated with the isolate "Spante" and tested for clinical, virological, hematological and serological findings until day 31 after infection (p. i.). One day p. i. the piglets were put in contact to three sentinel pigs. During a period of 31 d neither the domestic pigs nor the wild boars showed clinical signs specific for CSF. Two infected weaner pigs became transiently viraemic, transmitted CSFV in nasal secretions, showed a slight leukopenia and reacted serologically positive. The contact infection resulted in a viraemia in two sentinel piglets on day 30. Only one contact animal developed antibodies. None of the wild boars became viraemic, excreted CSFV in nasal secretions or developed antibodies. The CSFV isolate "Spante" represents a low virulent virus. Referring to a significant higher percentage of virologically positive tissue samples after nested PCR compared with the virus isolation, persistence of CSFV is discussed.

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