Development of an internal control for the detection of the African swine fever virus by PCR.

For the detection of African swine fever virus (ASFV) by polymerase chain reaction (PCR) in clinical samples, an internal control was constructed to identify false negative results in each reaction. The internal control was designed in such a way that the same primer pair was used to amplify the internal control and the target DNA which were differentiated by size. The lower detection limit was reached at about 30 internal control DNA copies and about 50 genomic ASFV DNA copies. The use of the internal control revealed that from a total of 12 uninfected samples, 10 samples contained inhibitors. After a one in two dilution, all ten of these samples amplified the internal control satisfactorily. From a total of 16 samples from pigs suspected of having ASF infection, nine contained inhibitors. After a one in two dilution, we observed internal control amplification and target DNA amplification for four samples.

Detection of encephalomyocarditis virus in clinical samples by immunomagnetic separation and one-step RT-PCR.

A method of immunomagnetic separation and one-step reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Encephalomyocarditis virus (EMCV). EMCV was captured from sample on magnetic beads with homologous monoclonal antibody and then heat denatured. The heated beads were used directly in one-step RT-PCR reaction to amplify a 285-bp PCR fragment at the 3' end of the genomic region that encodes the viral polymerase. This method detected as little as 3.5 TCID(50) of EMCV from infected cell culture. It was shown with this method that the sensitivity of RT-PCR increased when applied for the detection of EMCV added to fecal extract. Using this protocol EMCV was detected from heart homogenate samples containing less than 100 TCID(50)/ml. The amplified product was sequenced to ensure specificity. The immunomagnetic-RT/PCR procedure described here should be useful for the rapid, specific and sensitive detection of EMCV in clinical samples. This technique is rapid, reliable and can be readily adapted to detect EMCV from other clinical samples.

Isolation of a non-haemadsorbing, non-cytopathic strain of African swine fever virus in Madagascar.

African swine fever (ASF) suspected clinically in Madagascar (1998-9) was confirmed by polymerase chain reaction (PCR) and nucleotide sequencing, following virus isolation. No haemadsorption or cytopathic effect could be detected following leukocyte inoculation, but viral growth in cells was confirmed by PCR. Detection of ASF virus genome was carried out by amplification of a highly conserved region coding for the p72 protein. Nucleotide sequencing of the amplicon revealed 99.2% nucleotide identity between the recent Malagasy strains and a virus recovered from the 1994 outbreak in Mozambique (SPEC265). A serological survey performed on 449 sera, revealed that only 5.3% of the sera taken from pigs between 1998 and 1999 were positive.

Expression and characterization of part of hog cholera virus non-structural proteins.

In a preceding paper, the molecular cloning and partial nucleotide sequence of the Alfort strain of hog cholera virus (HCV) was described. To study the genetic organization of the 3'-end of the HCV genome, which encodes some of the non-structural proteins, a cDNA fragment (S2.20) of 849 nucleotides was subcloned into the bacterial expression vector pGEX-3X and expressed in Escherichia coli as a S2.20-glutathione-S-transferase fusion protein (S2.20-GST). This protein was used to produce HCV-specific monoclonal antibodies. Using Western immunoblotting, these antibodies could be used to identify a specific gene product of the HCV Alfort strain. Three proteins, with relative molecular weights of 76, 107 and 145 kDa, were detected. These proteins were also observed for eight other HCV strains. With the bovine viral diarrhoea virus (BVDV) NADL strain and the border disease virus (BDV) Aveyron strain, only one protein, with a relative molecular weight of 72 kDa, was detected. With the BVDV New York strain, two proteins, with relative molecular weights of 70 and 100 kDa, were recognized. The significance of these findings with respect to pestivirus genomic organization is discussed.

Partial sequencing of hog cholera virus Alfort strain genome and its comparison with other pestivirus strains.

After molecular RNA cloning of the Alfort strain (Alfort/LCRV) of hog cholera virus (HCV), the nucleotide sequence of about 70% of the total genome was determined. This sequence was compared with homologous parts of previously published pestivirus genomes. The average homology with another clone of the Alfort strain (Alfort/FRC) was found to be lower (86.1%) than with Brescia strain of HCV (94.3%), while, compared with NADL, Osloss and SD-1 (3 different strains of bovine viral diarrhea virus, BVDV), the average homology was 67%. Although the amino-acid sequences show a higher degree of conservation, they had a similar degree of homology (92.7, 96.7, 69, 68.2 and 69%, respectively). Partial sequence comparison also revealed that Alfort/LCRV strain was more closely related to Alfort 187 (98.6% for the nucleotides and amino acids) and Weybridge (97.3% for the nucleotides and 96.1% for the amino acids) strains of HCV than it was to Alfort/FRC. These results may indicate that the Alfort/FRC strain has undergone more genomic variations during its historical passage. Genomic relationships among the pestiviruses are discussed.

A rapid and sensitive chemiluminescence dot-immunobinding assay for screening hybridoma supernatants.

The present report describes a simple and rapid dot-immunobinding assay combined with a chemiluminescence detection system for screening hybridoma supernatants for specific monoclonal antibodies (MAbs). Small rectangular nitrocellulose filters dotted with either crude mixtures of antigens, or with control samples, were placed in six well plates, incubated with hybridoma supernatants, then stained with peroxidase-conjugated anti-mouse IgG. The reaction was performed with a chemiluminescence detection system. We used this method to screen hybridoma supernatants for MAbs against a 354 amino acid polypeptide of hog cholera virus (HCV) gp33-gp55 protein expressed as a fusion protein. We also extended it for the screening of MAbs against foot-and-mouth disease virus (FMDV). The chemiluminescence dot-immunobinding assay (CDIA) was compared with neutralization (N) and immunofluorescence (IF) screening tests and some FMDV seroneutralizing MAbs were found to be either poorly reactive or undetected by the IF test. The advantage of the present method is that it detects in only one step all MAbs detected in the IF and/or N tests together with some MAbs not detected by either of these methods. The present method is at least 356 times more sensitive than the IF test.

cDNA probes for the detection of pestiviruses.

Probes were prepared from genomic RNA of Hog Cholera Virus (HCV) after synthesis of cDNA and cloning. Six probes were selected according to their place on the viral genome determined by sequencing and comparison with BVDV sequence. These probes were hybridized with two strains of HCV (Alfort and Nord), two strains of Bovine Viral Diarrhea (BVDV) (NADL, New York) and four strains of Border Disease (BD) (Lyon 1, Lyon 2, Aveyron, IEMVT). This panel of six probes seem to be able to differentiate pestiviruses but some differences rely only on slight intensity of the hybridization.