Transmission of encephalomyocarditis virus (EMCV) among pigs experimentally quantified.

Two types of transmission experiments were performed to estimate the basic reproduction ratio R(0), indicating the level of encephalomyocarditis virus (EMCV) transmission among pigs. In a first experimental set-up with nine separate pairs, one randomly chosen piglet per pair was inoculated with a Belgian (myocardial) EMCV strain (B279/95, 10(3)TCID(50)/ml oronasally) and placed back into the pen. In the second experiment with two separate groups of five piglets, two piglets in each group were inoculated at the start. During the experiments, viraemia in blood and excretions was measured as well as the serological response against EMCV antigen. After death or euthanasia, the piglets were checked for heart lesions and virus isolation was done on various tissues. In both the experiments, the majority of the inoculated piglets either died with typical heart lesions (five out of nine and three out of four resp.), or produced high levels of neutralising antibody. EMC virus was isolated from the hearts of all piglets that died during either one of the experiments. The pairwise experiment revealed a point estimate for R(0) of 2.0 (95% confidence interval (CI)=0.37-10.74), while the group experiment resulted in a R(0)-value of 0.71 (95% CI=0.08-4.93). Combining the information from both experiments results in an estimate for R(0) of 1.24 (95% CI=0.39-4.35). Since R(0) has values around the threshold value of 1, the spread of EMCV due to contacts between pigs will in most cases be limited, but due to chance processes may lead to large outbreaks as well.

Classical swine fever (CSF) marker vaccine. Trial II. Challenge study in pregnant sows.

The efficacy of two marker vaccines against classical swine fever (CSF) was tested in a large scale laboratory trial in several National Swine Fever Laboratories (NSFL) of the EU member states. The vaccines were: BAYOVAC CSF Marker (Vaccine A) from Bayer, Leverkusen, Germany and PORCILIS PESTI (Vaccine B) from Intervet, Boxmeer, The Netherlands. At the NSFL of Belgium, The Netherlands and Germany experiments were carried out to examine the ability of the vaccines to prevent transplacental transmission of CSF virus. In Belgium and The Netherlands pregnant sows were vaccinated once and challenged with virulent CSF virus 14 days later, which was around day 60 of gestation. At the NSFL in Germany sows were vaccinated twice, on days 25 and 46 of pregnancy and were challenged fourteen days after booster vaccination (day 60 of gestation). Apart from minor inflammatory reactions in some sows, no reactions post vaccination were noticed in either vaccine group. Sows vaccinated with Vaccine A were better protected against clinical CSF than sows vaccinated with Vaccine B. The antibody response after vaccination with Vaccine A was more pronounced than after vaccination with Vaccine B. After single vaccination six out of eight sows vaccinated with Vaccine A and all eight sows vaccinated with Vaccine B had viraemic piglets. After double vaccination one out of four litters from sows vaccinated with Vaccine A and four out of five litters from sows vaccinated with Vaccine B were found to be viraemic. However, both vaccines reduced the transmission probability significantly (Vaccine A: P=0.004, Vaccine B: P=0.024) after booster vaccination. However, Vaccine A appeared in this regard more potent as the estimated probability of fetal infections was lower. Nevertheless the risk of virus spreading after vaccination via transplacental transmission is still present and has to be addressed from an epidemiological point of view.

Molecular epidemiology of a large classical swine fever epidemic in the European Union in 1997-1998.

A big epidemic of classical swine fever (CSF) occurred in the European Community in 1997. The first case was reported at the beginning of January 1997 from Germany. The disease presumably spread to the Netherlands, and from there to Italy, Spain and eventually to Belgium. About 30 isolates from these outbreaks were analysed by comparison of the nucleotide sequence data generated from fragments of both the E2 glycoprotein gene (190 nucleotides) and from the 5'-nontranslated region (5'-NTR; 150 nucleotides). By combining epidemiological data with genetic typing, it was found that the outbreaks were related and caused by a virus belonging to the genetic subgroup 2.1. As this type of virus had been reported infrequently in Europe and not at all since 1993, we postulate that it was newly introduced into the European Union (EU).

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.

An experimental infection with classical swine fever in E2 sub-unit marker-vaccine vaccinated and in non-vaccinated pigs.

The clinical and virological protection induced by an E2 sub-unit marker-vaccine against Classical Swine Fever (CSF) was examined during an experimental infection in vaccinated and non-vaccinated pigs. Forty-five pigs were equally distributed over three adjacent pens of an isolation unit, there was only indirect (airborne) contact between pigs in the different pens. In pen 3 all pigs were vaccinated twice with 4 weeks interval. Pigs in pens 1 and 2 were not vaccinated. Two weeks after booster vaccination, one randomly selected pig in the middle pen was experimentally inoculated with CSF virus. After the initial virus spread in the infected pen, all pigs in the non-vaccinated adjacent pen were infected. In the vaccinated pen, seven out of 14 pigs became infected during the experiment. Survival analysis showed that virus transmission by direct and indirect contact was significantly (p<0.001) delayed in vaccinated pigs as compared to non-vaccinated pigs. In the non-vaccinated pens over 40% of the pigs died and typical clinical signs were noticed. In the vaccinated pen no mortality and no clinical symptoms were observed. Although double vaccination with an E2 sub-unit marker-vaccine was able to prevent the clinical course of the disease it was unable to prevent infection through indirect contact. This finding combined with the slow serological response after vaccination will complicate the possible use of the vaccine in emergency vaccination programmes.

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

Six laboratories participated in an exercise to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Two sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 34 samples of random primed cDNA. These had been synthesised from viral RNA representative of seven different genetic subtypes of CSFV. The other set comprised 40 clinical samples containing tonsil, spleen, whole blood or serum from a pig that had been experimentally infected with CSFV. Each laboratory tested the samples using one or more PCR/RT-PCR tests that they were accustomed to using. The methods and results of the laboratories were compared with one another. The RT-PCR results obtained from testing the clinical samples were also compared with those obtained by virus isolation and antigen ELISA.ELISA. Both RT-PCR and RT-nested PCR appeared to give some false positive results. Several of the PCR tests appear suitable in terms of specificity and sensitivity. Further trials are necessary to compare results when the same test is performed by different laboratories, and to show that improved control procedures can eliminate problems due to false positive reactions.A limited comparison of extraction and reverse transcription procedures showed similar results in each of three participating laboratories, even though the methods were not standardised.

Classical swine fever virus is genetically stable in vitro and in vivo.

Phylogenetic analyses of large numbers of classical swine fever strains have revealed a high degree of sequence conservation in the genomic regions examined, suggesting either a recent common ancestor or a low evolution rate. This low variability is in contrast to findings with other RNA viruses. To investigate the consequence of this apparent genetic stability on phylogenetic examinations, the Belgian field isolate Wingene'93 was passaged in pigs as well as in cell culture by various methods. Sequence analyses of viruses collected after various passages in three target regions proposed for phylogenetic studies (5' NTR, E2, and NS5B) revealed a complete sequence conservation. Only when the amount of passaged virus was lowered, mimicking a genetic bottleneck, a single point mutation was observed in the E2 gene. Additionally, only four nucleotide substitutions were observed when the genome of a virus obtained after 96 cell passages in persistently infected cells was compared with its parental virus, the recombinant virus derived from an infectious cDNA clone of CSFV strain Alfort/187. This low mutation frequency observed both in vitro and in vivo demonstrates that classical swine fever virus is genetically stable. Hence, even minor mutations can be considered significant in molecular epidemiological studies.

Phylogenetic analysis of European encephalomyocarditis viruses: comparison of two genomic regions.

The phylogenetic relationships of encephalomyocarditis (EMC) viruses isolated from pigs and rodents in Europe were determined by comparison of nucleotide sequences from two different regions of the virus genome, the VP3/VP1 gene junction (part of the capsid-coding region) and part of the 3D polymerase-coding region. Thirty-five European EMC viruses could be divided into two genetic groups, one which contained viruses from Greece isolated between 1986 and 1997 and from Belgium in 1991 and the other which contained viruses from Italy (1986-1996), Cyprus (1994-1995), France (1995) and Belgium (1995-1996).

Epidemiologic, pathogenic and molecular analysis of recent encephalomyocarditis outbreaks in Belgium.

In 1991 EMCV was isolated for the first time in Belgium from the offspring of a sow with reproductive failure. From August 1995 until December 1996, EMCV was diagnosed in 154 Belgian pig holdings in association with myocardial failure and sudden death in fatteners and suckling piglets or with reproductive failure in sows. To clarify some epidemiological aspects 3 EMCV isolates characteristic for the different clinical pictures and outbreaks were studied. Field observations and animal experiments indicated that the pathogenicity induced by each isolate is specific for one age category and that the spread of the virus is limited. The presented data also suggest that rodents may play a role in the transmission of EMCV but that pig-to-pig transmission is at least as important. Molecular analysis of two separate regions on the genomes of the respective EMCV isolates showed that the 1995-96 EMCV epizootic in Belgium was due to a new virus introduction. Furthermore, the VP1 coding gene is proposed as a marker of virulence.

Identification of encephalomyocarditis virus in clinical samples by reverse transcription-PCR followed by genetic typing using sequence analysis.

The objective of the present study was to gain a better understanding of the epidemiology of encephalomyocarditis virus (EMCV) infections in pigs by applying molecular techniques. The diagnostic potential of a reverse transcription-PCR (RT-PCR) targeting 286 nucleotides at the 3' end of the gene which encodes the viral polymerase was assessed with experimental and field samples. In addition, the use of the amplified sequences for an epidemiological study was evaluated. The heart was clearly shown to be the most suitable organ. The detection limit was determined to be 1 viral particle in 100 mg of heart tissue. The sensitivity and specificity of the assay on the basis of the results obtained in this study were 94 and 100%, respectively. Phylogenetic analysis of the amplified sequences classified EMCVs in two distinct lineages. Group A consists of the reference strain ATCC 129B, all isolates collected between 1991 and 1994 in Belgium in association with reproductive failure, and all Greek isolates. All Belgian isolates collected since the first isolation of EMCV in relation to myocardial failure in fatteners in Belgium group together with the isolates from Cyprus (1996 and 1997), Italy (1986 to 1996), and France (1995) in group B irrespective of their pathogenicity. The analyzed part of the 3D gene differed by 13.0% between Groups A and B. In contrast to the sequence homogeneity of the Belgian isolates collected between 1991 and 1994, molecular diversity, which ranged between 0 and 2%, was observed among the Belgian isolates collected in 1995 and 1996. Among all Greek isolates the diversity ranged between 1 and 8%. However, this diversity does not seem to reflect geographical links between the outbreaks. A RT-PCR for the rapid and specific diagnosis of EMCV in a variety of clinical samples followed by nucleotide sequence analysis proved to be valuable for molecular epidemiological studies.

Comparative study of the pathogenic properties of a Belgian and a Greek encephalomyocarditis virus (EMCV) isolate for sows in gestation.

Thirteen conventional sows were inoculated between 61 and 92 days of gestation with a Belgium or a Greek EMCV isolate to investigate the difference in pathogenicity of both strains for sows in gestation. The Belgian EMCV strain was isolated from the offspring of a sow with productive failure and without myocardial lesions. The Greek strain was isolated from a 3-month-old pig with prominent myocardial lesions. The present study demonstrates a transplacental virus transmission with fetal death following an infection of the sows in gestation with both isolates. The fetal pathogenicity was more severe with the Greek strain than with the Belgian isolate. No myocardial lesions were noticed.

Rapid diagnosis of encephalomyocarditis virus infections in pigs using a reverse transcription-polymerase chain reaction.

Encephalomyocarditis virus (EMCV) is widespread and the economic losses caused by an EMCV outbreak in pig holdings and the similarity between a foot-and-mouth disease virus (FMDV) and an EMCV infection in young piglets stress the need for a rapid, specific and broad diagnostic assay. An alternative to the time-consuming seroneutralisation assay, currently used for the characterisation of EMCV, is described. An EMCV specific reverse transcription-polymerase chain reaction (RT-PCR), using primers located in a conserved region of the 3D gene of the viral genome, was developed and tested on 114 different EMCV isolates. The identity of the respective amplicons was confirmed by sequencing. The potential of this assay for future diagnostic purposes was demonstrated by applying the RT-PCR on tissue samples collected from an experimentally infected piglet.

Comparison of the pathogenic, antigenic and molecular characteristics of two encephalomyocarditis virus (EMCV) isolates from Belgium and Greece.

The pathogenicity of two porcine encephalomyocarditis virus (EMCV) isolates for sows in gestation and young piglets was studied. One virus originated from a case of reproductive failure in pigs in Belgium and the other from a case of acute myocarditis in pigs in Greece. Sows in the mid-gestation period and one- to two-month old piglets were inoculated with each isolate. The molecular relationship between both isolates was studied by determining the nucleotide sequence located across the junction of the 1C and 1D capsid-coding genes. Antigenic analysis was performed using a panel of 35 monoclonal antibodies raised against an Italian field isolate of EMCV. All three approaches revealed differences between both isolates and also confirmed that there was no link between the two outbreaks of disease.