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At least 325 records · Page 18Linked to original sources
PubMed2003

Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA.

We performed experimental infection in 10-week-old pigs with the Paderborn isolate of classical swine fever virus (CSFV). Despite being epidemiologically linked to the major CSFV outbreak in The Netherlands in 1997, the in vivo replication kinetics of this isolate have to our knowledge not been described in detail previously. We found that oronasal infection with 10(4.7) TCID(50) produced mortality in three out of five pigs after 29-31 days, and severe clinical symptoms in one out of five pigs, while one out of five pigs exhibited no clinical symptoms. At this infection dose, pigs had viral RNA (monitored by quantitative reverse transcription (RT)-PCR) in serum as soon as 2 days post-infection, and excretion of infectious virus (monitored by sentinel pigs) appeared to be virtually concomitant with viremia onset. While virus RNA was cleared from the serum of most pigs after 1-2 weeks, some pigs had viral RNA in serum for more than 30 days, and exhibited only mild clinical symptoms. We observed an excellent correlation between clinical symptoms and viral RNA loads in serum, while serum antibody levels were low. Clinically affected pigs had up to 1000-fold higher serum viral RNA loads than did pigs without clinical symptoms. At this level of infection, and this age group, the Paderborn isolate exhibited a strikingly wide range of replication patterns, which might be relevant to the spread of the virus through susceptible pig populations, and the severity of the 1997-1998 outbreak.

Animals↗
PubMed2001

Adsorption of colostral antibodies against classical swine fever, persistence of maternal antibodies, and effect on response to vaccination in baby pigs.

OBJECTIVE: To determine kinetics of antibody absorption, persistence of antibody concentrations, and influence of titers on vaccination of baby pigs with a vaccine against classical swine fever (CSF). ANIMALS: 15 sows and their litters. PROCEDURE: Farrowings were supervised. Initial time of suckling was recorded. In the first experiment, blood samples were collected at farrowing, 2 and 4 hours after suckling, and hourly until 10 hours after initial suckling. Samples were assayed for CSF antibodies, using a serum neutralizing (SN) test. A second experiment included 33 baby pigs vaccinated as follows: 10 prior to ingestion of colostrum, 18 between 1 and 4 hours after ingestion of colostrum, and 5 at 12 hours after ingestion of colostrum. Fourteen pigs were vaccinated when 7 weeks old, and 15 pigs were not vaccinated. At 10 weeks of age, pigs were challenge-exposed with virulent CSF virus. Blood samples were collected and assayed for CSF antibodies and p125 antigen and p125 antibodies. RESULTS: CSF antibodies were detected in pigs beginning 2 hours after suckling. Colostral antibodies persisted for > 7 weeks (half-life, 79 days). Vaccination of pigs before suckling provided effective protection from severe disease after challenge-exposure. However, vaccination of neonates with antibody titers was not effective, because 19 of 23 (82%) pigs succumbed after challenge-exposure. All pigs vaccinated when 7 weeks old resisted challenge-exposure, whereas all unvaccinated control pigs succumbed. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination before ingestion of colostrum conferred good protection against CSF in baby pigs. Vaccination of 7-week-old pigs that had decreasing concentrations of passively acquired antibodies was efficacious.

Adsorption↗
PubMed2005

Evaluation of the epidemiological importance of classical swine fever infected, E2 sub-unit marker vaccinated animals with RT-nPCR positive blood samples.

It has been demonstrated that pigs that have been double vaccinated with an E2 sub-unit marker vaccine and that are infected with classical swine fever virus (CSFV) through a natural contact infection may react positive in a CSFV detecting RT-nPCR test, whereas no virus could be isolated by using the conventional virus isolation (VI) technique. To evaluate whether these vaccinated and infected pigs may spread the virus, three experiments were set up. In the first, susceptible pigs were inoculated with serum originating from vaccinated RT-nPCR positive pigs. In the second, vaccinated RT-nPCR positive pigs were brought into contact with sentinel animals. In the third, vertical transmission was evaluated in RT-nPCR positive vaccinated pregnant gilts. In the first two experiments, no proof of virus transmission was found, whereas in the third vertical transmission was observed. The conclusion is that in vaccinated pigs that are positive in RT-nPCR but negative in VI, the level of circulating virus is probably not high enough for horizontal transmission, whereas vertical transmission of the virus is possible.

Animals↗
PubMed2001

Classical swine fever virus (C strain) distribution in organ samples of inoculated piglets.

Enzyme linked immunosorbent assays (ELISAs) and a nested polymerase chain reaction after reverse transcription (RT-PCR) were used for the detection of the Chinese strain (C strain) of classical swine fever virus (CSFV) in blood and tissue samples of experimentally inoculated piglets. One group of 10 piglets was inoculated with C strain material from rabbits and a second one with material from infected minipig kidney (MPK) cell culture. Tested blood samples were taken on the day of inoculation as well as on days 2, 4, 6, 8, 10, 13 and 16. Samples of spleen, tonsil and brain tissue were collected from piglets on days 6, 8, 10, 13 and 16 and tested for glycoprotein E(RNS) and protein NS2-3 using commercially available ELISA kits. E(RNS) and NS2-3 were detected earlier in blood samples of piglets inoculated with the C strain propagated in a cell culture. Regardless of propagation the presence of the viral E(RNS) and NS2-3 was detected in spleen and tonsil samples simultaneously. The C strain propagated in a cell culture was found in only one brain sample, whereas, the virus propagated in rabbits was detected in 70% of the brain samples. For the detection of the CSFV RNA in blood samples, a part within the 5' non-coding region was amplified. The differences in the results gained by antigen detection in blood samples decreased when nested RT-PCR was used.

Animals↗
PubMed1996

Detection of antibodies against classical swine fever virus in swine sera by indirect ELISA using recombinant envelope glycoprotein E2.

Recombinant envelope protein E2 (gp55) of classical swine fever virus (CSFV) strain Alfort/187 was evaluated as an alternative to whole virus as ELISA antigen for the detection of antibodies against CSFV. A glycosylated and a non-glycosylated form of E2 was expressed in the baculovirus system. Six histidine residues added at the carboxy terminus of each of the recombinant proteins allowed purification by nickel-chelate affinity chromatography. Comparison of the antigenic properties of the two proteins in indirect and blocking ELISAs revealed that the glycosylated form resulted in both higher sensitivity and specificity. The indirect ELISA, using glycosylated E2, either derived from crude cell extract or affinity-purified, was validated by testing a total of 2719 porcine sera. Its final version proved to be as sensitive (98.3%) as the virus neutralization test when sera from infected pig herds were examined, and highly specific (99.6%) when applied to test negative sera. It is therefore suitable for large scale monitoring of classical swine fever.

Animals↗
PubMed2001

Factors associated with the introduction of classical swine fever virus into pig herds in the central area of the 1997/98 epidemic in The Netherlands.

A matched case-control study of 135 infected and 99 uninfected pig herds from the central area of the 1997 to 1998 epidemic of classical swine fever (CSF) in The Netherlands was undertaken to identify factors associated with the introduction of the virus. The herds were matched on the basis of herd type and the shortest geographical distance between pairs of herds. Data on management, hygienic measures, experiences during the depopulation of an infected nearest neighbour, and the frequency of contact with professionals and other agencies were collected by means of a questionnaire taken by personal interview. There were no significant differences between the infected and uninfected herds in the median total number of contacts per year with professionals and other agencies either with or without contact with the pigs. On the basis of a multivariable analysis, five variables were found to be significantly associated with an increased risk of infection: (1) the presence of commercial poultry on the premises; (2) visitors entering the pig units without wearing an overcoat or overalls and boots supplied by the farm; (3) the driver of the lorry transporting pigs for the Pig Welfare Disposal Scheme (PWDS) using his own boots instead of boots supplied by the farm; (4) herds of moderate size (500 to 1,000 animals) and very large herds (>7,000 animals) were at greater risk than small herds (<500 animals); and (5) an aerosol, produced during high-pressure cleaning of the electrocution equipment used to kill the pigs on a neighbouring infected herd less than 250 m away was carried by the wind on to the premises. Two variables were significantly associated with a decreased risk of CSFV-infection: (1) more than 30 years of experience in pig farming; and (2) additional cleaning of the lorries used to transport pigs for the PWDS before they were allowed on to the premises. In the opinion of the cooperating farmers, airborne transmission of the virus and its transmission during the depopulation of an infected neighbour were among the most important routes of infection.

Animal Husbandry↗
PubMed1998

Cloning, expression and sequence analysis of the classical swine fever virus nucleocapsid protein.

The DNA complementary to the 5'-terminal 1929 nucleotides of classical swine fever virus (CSFV; alias hog cholera virus, HCV) LPC vaccine strain RNA was cloned and sequenced. The sequence encompasses a 5'-noncoding region (NCR) of 264 nucleotides and an open reading frame (ORF) of 1665 nucleotides. The cloned sequence contains genes of four viral proteins, P23, nucleocapsid (core) protein, E0 and part of E1 proteins. Alignment of the 5'-terminal 1929 nucleotides of LPC strain with other strains of CSFV showed well conservation and a homology as high as 84-95% was found between these strains. The cDNA of CSFV-LPC core was cloned into an expression vector, and a fusion protein of 38.5 kDa was obtained which reacted strongly to CSFV antiserum. Purification of the core fusion protein was achieved by a single-step affinity chromatography and the purified product could be recognized by the sera of CSFV-infected swine in ELISA assay. Phylogenetic analysis of the 5'-terminal 1929 nucleotides between pestiviruses revealed that the 5'-end region seems to be suitable for differentiation of different strains of CSFV.

Amino Acid Sequence↗
PubMed2005

Classical swine fever (CSF) in wild boar: the role of the transplacental infection in the perpetuation of CSF.

Thirty-four pregnant wild sows and their unborn progeny derived from an endemically infected population in the district of Nordvorpommern (Mecklenburg-Western Pomerania) were investigated for classical swine fever virus (CSFV) and antibodies. During the last 2.5 years of the epidemic, 20 out of 34 pregnant wild sows investigated were serologically positive. No CSFV or viral RNA was detected in organs derived from these animals and their progeny. This indicates that young wild boars persistently infected by transplacental virus transmission do not play a crucial role in the perpetuation of CSFV in wild boar. Other factors seem to be more important for the establishment of CSF as well as for virus perpetuation in the population.

Animals↗
PubMed2003

Inactivation of classical swine fever virus: association of hydrostatic pressure and ultraviolet irradiation.

Reversible pressure-induced disassembly of several viruses has suggested the idea of using hydrostatic pressure to suppress virus infectivity. In this study, the effects of high hydrostatic pressure and ultraviolet (UV) irradiation were investigated on classical swine fever virus (CSFV) in an attempt to eliminate residual infectivity. The structural modifications were followed by intrinsic fluorescence and biological activity assays. The kinetics of CSFV inactivation showed that pressure-induced inactivation was not enough to eliminate viral infectivity. However, when pressure was applied in association with UV irradiation no infectious focus was observed. The application of these two methods against CSFV can be an attractive inactivation strategy for the development of a vaccine.

Animals↗
PubMed1999

[The morphological structure of classical swine fever virus and some characteristics of its multiplication].

Some characteristics of the multiplication of classical swine fever virus(CSFV) Thiverval strain were studied by means of the immunofluorescence technique. Under optimum culture conditions, the concentration of CSFV in the culture liquid multiplicated by MPK cells is ten times higher than by PK-15 cells. The half-life period of CSFV at 37 degrees C is 3 hour. The location of CSFV's replication in host cells is detected by the monoclonal antibodies of the structural protein E2 and the non-structural protein p120 of CSFV. Based on the above results, the ultrastructure of CSFV particles inside host cells was further studied using electron microscope and the changes of the ultrastructure of CSFV-infected cells are shown. The possible reasons for lower viral titre are also discussed in this paper.

Animals↗
PubMed1999

Detection of low-virulent classical swine fever virus in blood of experimentally infected animals: comparison of different methods.

The effectiveness of virus isolation, commercial antigen enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and flow cytometry in detection of a low-virulent classical swine fever virus (CSFV) in blood in the early period of infection was evaluated. Domestic pigs at the age of 6-8 weeks and young wild boars were inoculated with a low-virulent field isolate of CSFV originating from a wild boar. This virus induced serious clinical reaction in only one pig which was naturally infected with Pasteurella multocida. Nine of 13 infected domestic pigs showed viremia. All infected weanling pigs were found viraemic by virus isolation on day 6 post infection (p.i.) but virus-free by RT-PCR. The flow cytometry was apparently not as sensitive as the virus isolation. Two young wild boars infected with the virus were viremic only for the first 2 days p.i. Virus isolation and RT-PCR were of similar sensitivity. Three different commercial antigen ELISAs used were not able to detect viral antigen in any animal.

Animals↗
PubMed2000

Economic aspects of the control of classical swine fever outbreaks in the European Union.

A framework for assessing the economic impact of classical swine fever (CSF) is presented, including an analysis of direct and indirect costs. Direct costs are divided into calculated costs, expenditure on control measures (payable costs) and costs due to financial transfers. The economic impact of current control strategies is described using practical examples. In most cases, the largest part of the direct costs is associated with transport standstill measures, of which approximately 45% are calculated costs. Alternative strategies, still based on non-vaccination offer a potential for reducing these costs. Various economic aspects of emergency vaccination are described using as an example, a hypothetical optimistic CSF case. In order to explore the impact of applying emergency vaccination using marker vaccines, additional research is required using simulation modelling. This research should include an assessment of risk and uncertainty with respect to calculating the epidemiological impact and the direct costs.

Animals↗
PubMed2003

RNA-dependent RNA polymerase activity of Classical swine fever virus NS5B protein expressed in natural host cells.

The NS5B gene, cloned from Classical swine fever virus (CSFV) genome, was expressed in porcine kidney cells PK-15, natural host of CSFV. In purifying cytoplasmic extracts from these cells by means of different concentrations of salt, glycerol and detergent four fractions, namely crude supernatant (SC) and different purified supernatants (S1, S2 and S3) were obtained. Using Western blot analysis the NS5B protein was found in all these fractions, showing that it was soluble in both higher and lower concentrations of salt, glycerol and detergent. The NS5B protein present in the four different fractions exhibited RNA-dependent RNA polymerase (RdRp) activity, but it was unable to complete the whole process of RNA synthesis. Site-directed mutation analysis showed that Thy216 and Cyt228 were essential for RNA synthesis while Cyt219 was not, suggesting that CSFV RdRp was template-specific. We conclude that initiation of RNA synthesis by CSFV RdRp includes also template priming.

Animals↗
PubMed1999

[Comparison of laboratory diagnostic methods for the detection of infection with the virus of classical swine fever in the early inspection phase: an experimental study].

Virus isolation in the PK-15 cell culture, two commercial antigen ELISAs, reverse transcriptional-polymerase chain reaction (RT-PCR), and flow cytometry have been evaluated to detect viremic pigs in the early period of classical swine fever virus (CSFV) infection. Domestic pigs were experimentally inoculated with the virulent CSFV strain 'Alfort 187' and two field isolates. CSFV isolation and RT-PCR were found to be the most sensitive methods for the detection of highly virulent CSFV in the early period of infection which is characterized by the absence of clinical symptoms. Using antigen ELISAs and flow cytometry CSFV could be detected in infected animals after the onset of clinical signs. After infection with a less virulent CSFV field isolate originating from wild boar, viremic pigs could be identified by direct virus isolation. The reasons for the negative results of the RT-PCR still remain unknown. In conclusion we recommend to modify the procedure (antigen ELISA) for the detection of clinically healthy domestic pigs in accordance with the decision 98/413/EC.

Animals↗
PubMed2005

The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations following exposure to classical swine fever virus (CSFV).

Surface expression of IL-2R-alpha (CD25) is widely used to identify activated lymphocyte populations, while interferon-gamma (IFN-gamma) levels have been shown to be a good indicator of cell-mediated immunity (CMI) in pigs. To investigate the relationship between these two parameters, we developed an intracellular cytokine-staining assay and studied the kinetics of cytokine (IFN-gamma and interleukin-10, IL-10) production relative to CD25 expression in porcine lymphocyte subpopulations, following immunization with a classical swine fever (CSF) vaccine. The number of activated memory T cells (CD4(+)CD8(+)CD25(+) cells) increased slightly in the peripheral blood mononuclear cell (PBMC) population soon after vaccination, then diminished within a few weeks. The number of activated cytotoxic T cells (CD4(-)CD8(+)CD25(+) cells) peaked approximately 2 weeks after the memory population. Although the number of IFN-gamma producing cells detected in this experiment was relatively low, the CD4(+)CD8(+) T cells were major IFN-gamma producers in the PBMCs throughout the experiment. In another experiment, CSF-vaccinated pigs were challenged with a virulent classical swine fever virus (CSFV), and the kinetics of CD25 expression and cytokine productions were monitored. Following exposure to the virus, the number of IFN-gamma producing cells in the PBMCs increased markedly in both the vaccinated and unvaccinated groups. The CD4(-)CD8(+) cells were major IFN-gamma producing cells in vaccinated pigs, while both CD4(+)CD8(+) and CD4(-)CD8(+) populations contributed to the IFN-gamma production in the control group. Interestingly, the enhanced IFN-gamma production was not associated with the upregulation of CD25 expression following the CSFV challenge. In addition, exposure to the virulent CSFV significantly increased interleukin-10 production by the CD4(-)CD8(+) populations in PBMCs of the unvaccinated pigs. Taken together, our results indicated that CD25 expression and IFN-gamma production were not tightly associated in porcine lymphocytes. In addition, the CD4(-)CD8(+) lymphocytes of the PBMCs played a major role in cytokine productions following the CSFV challenge.

Animals↗
PubMed1998

Application of a computer program for genetic typing of classical swine fever virus isolates from Germany.

The commercial software program HLA SequiTyper (Amersham Pharmacia Biotech), designed originally for human leukocyte antigen typing, was adapted for rapid typing of classical swine fever (CSF) virus isolates. The program compares new sequence data with those stored in a database file and calculates the most probable assignment. For generating the CSF virus sequence database, 150 bp of the 5' nontranslated genomic region (5'-NTR) from 88 German classical swine fever virus isolates from outbreaks between 1984 and 1997 were solid-phase sequenced directly after RT-PCR amplification. Sequence alignments showed that they all belonged to the previously defined genetic group 2. Within this group, six different subgroups could be distinguished, and were designated according to the geographic location where they are either still endemic or where they appeared most commonly. The advantage of using the HLA SequiTyper program is that it reads directly the sequence files as generated by the ALF sequencer (Amersham Pharmacia Biotech), making any manipulations unnecessary. In addition, a constant quality control of the raw sequence data can be achieved, as more than one sequence from the same isolate can be evaluated at once. Using this approach, new CSF isolates can be typed within 2 days.

Animals↗
PubMed1999

Oral immunisation of swine with a classical swine fever vaccine (Chinese strain) and transmission studies in rabbits and sheep.

Seven experiments including a total of 47 pigs, 11 wild boars, 26 rabbits, 10 hares and 16 sheep were carried out to assess the efficacy, safety and transmission of the Chinese vaccine strain of the classical swine fever virus (CSFV) administrated by the oral route. Within 3 weeks after oral vaccination, a clear seroconversion occurred in the pigs. Six weeks after vaccination, vaccinated pigs were fully protected against a virulent challenge. The C-strain was not isolated from tonsils, spleen, lymph nodes, thymus, saliva, urine and faeces of pigs within 4 days after oral vaccination. In one experiment, susceptible pigs were placed in direct contact with vaccinated pigs. None of these contact-exposed pigs became serologically positive for CSFV antibodies. It is concluded that the C-strain induces protection in pigs when administrated by the oral route and is not shed by vaccinated pigs. Serum anti-CSFV antibodies developed in seven out of eight wild boars vaccinated by the oral route. No vaccine virus was detected in the spleen and tonsils of these animals. The results in wild boar were in accordance with those obtained in domestic pigs. Sheep did not show any clinical signs after oral vaccination while rabbits had moderate hyperthermia and growth retardation. No clinical response to oral immunisation in hares was detected. At the end of the experiment, no sheep had detectable serum antibodies against CSFV, whereas a few vaccinated rabbits and hares became seropositive. None of the contact-exposed rabbits and hares seroconverted. These data indicate that the C-strain is safe for sheep and as expected, moderately or not pathogenic for rabbits and hares. These efficacy and safety studies on oral vaccination with the C-strain under experimental conditions provide essential information for further studies in wild boars under experimental and field conditions, including assays with baits to control a CSF epidemic.

Administration, Oral↗
PubMed2003

Construction of an infectious chimeric classical swine fever virus containing the 5'UTR of bovine viral diarrhea virus, and its application as a universal internal positive control in real-time RT-PCR.

RT-PCR is used widely as a diagnostic method to detect and differentiate pestiviruses. The construction of two chimeric classical swine fever virus (CSFV) recombinants based on a marker virus constructed previously [J. Virol. 72 (1998) 5318-5322] is described. These viruses, termed vA187CAT_5UTRBVD and vA187CAT_IRESBVD, contain the entire 5' untranslated region (5'UTR) or the internal ribosome entry site (IRES) of bovine viral diarrhea virus (BVDV), respectively. Both chimeric viruses proved to be infectious in cell culture. Hence, the 5'UTR as well as the IRES element only of BVDV can substitute for the corresponding genome region of CSFV. Next, two sets of primers and corresponding dual-labeled TaqMan probes were designed; one detecting specifically a conserved but CSFV-specific area within the 5'UTR of wild-type CSFV, the other one targeting the CAT gene inserted in vA187CAT_5UTRBVD. The two primer/probe sets were combined in a closed-tube multiplex one-step RT-PCR. To monitor the entire extraction and detection process limited amounts of vA187CAT_5UTRBVD were added directly to clinical samples before RNA extraction. The multiplex RT-PCR proved to be as sensitive as the single primer/probe set method, but allowed the validation of each sample tested individually, based on the detection of the CAT marker gene. vA187CAT_5UTRBVD was also used successfully for foot-and-mouth disease virus (FMDV) TaqMan RT-PCR. Therefore, it is considered a universal internal positive control for RT-PCR assays to exclude loss of RNA during extraction, or failure of amplification due to inhibitory substances present in the sample.

5' Untranslated Regions↗