D.J Paton

Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis

SummaryA polymerase chain reaction-based assay capable of detecting a broad range of pestiviruses from pigs, cattle, or sheep was developed. Of six sets of primers selected from different parts of the pestivirus genome, the best results were provided by a pair from the highly conserved 5′ non-coding region which gave amplification with all 129 isolates tested. This panel consisted of 33 isolates from pigs, 79 from cattle, and 17 from sheep. Differentiation between the viruses was achieved by cutting the PCR-amplified products with the restriction endonucleases AvaI and Bg1I. Using this procedure it was possible to distinguish at least 3 genogroups; group 1 (HCV) contained 32 of the pig isolates, group II (BVDV) contained all the cattle isolates tested plus 6 sheep isolates and group III (BDV) contained 11 sheep isolates and 1 pig isolate.

Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups

Summary. Seventy-eight bovine viral diarrhoea viruses (BVDV) recently collected in Austria, France, Hungary, Italy, Slovakia, Spain and UK were genetically typed in the 5′-untranslated (5′UTR) and autoprotease (Npro) regions of the pestivirus genome. Seventy-six of the isolates were BVDV-1 and two French isolates were of the BVDV-2 genotype. Phylogenetic analysis of the 5′UTR (245 nt), including additional BVDV-1 sequences from USA, Canada, Germany, New Zealand, Mozambique and Sweden, taken from GenBank and from our previous works, indicated that these viruses were clustered not only into the two generally accepted groups (BVDV-1a –“NADL like” and BVDV-1b –“Osloss like”), but altogether into 11 phylogenetic groups. Similar clustering was observed with Npro region sequences (385 nt) and the highest bootstrap values (over 95%) were obtained by phylogeny combining 5′UTR and Npro sequences. Some associations between the genetic grouping and the origin of the isolates were apparent, probably reflecting historical trade contacts. Considering the variability of isolates it is recommended that diagnostic PCR primers should be re-examined to ensure coverage of all BVDV-1 groups. The genogroups were less clearly differentiated by monoclonal antibody typing, suggesting significant antigenic similarities within the BVDV-1 genotype.

Closed one-tube reverse transcription nested polymerase chain reaction for the detection of pestiviral RNA with fluorescent probes.

An assay was developed in which reverse transcription (RT), nested polymerase chain reaction (PCR) and accumulation of amplicon-specific fluorescence could take place in a single, closed reaction tube. The assay, which was classical swine fever virus RNA-specific, was compared with other methods for detection of this virus, including various RT-PCR configurations, virus isolation and ELISA. The new method was very sensitive, and less prone to giving false positive results compared to nested PCR carried out in separate reaction tubes. Substitution of different fluorescent probes resulted in specific tests for border disease virus and for bovine viral diarrhoea type II (BVD-II), and one that could detect all pestiviruses except for some BVD-II viruses.

The detection of bovine viral diarrhoea virus in bulk milk samples by the use of a single-tube RT-PCR.

A single step, single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) test was developed to detect the presence of bovine viral diarrhoea virus (BVDV) in somatic cells from bulk milk samples. The test was configured using commercial kit-form RNA extraction and RT-PCR procedures. The test was validated by examining bulk milk samples from approximately 80 herds with a history of BVDV and comparing results with those obtained from samples from a similar-sized control group. The test proved highly specific, giving a positive result in 20.5% of herds with a history of BVDV, with no control herds positive. Its sensitivity was likewise high, detecting, at its maximum, one persistently infected (PI) cow in a herd of 162 lactating animals. In 19 herds where follow-up blood tests were performed, the RT-PCR gave a positive result in all ten herds where at least one lactating PI animals was present. In control involving the detection of PI cattle, the test provides a rapid and inexpensive alternative to individual animal testing for those cows in milk at the time of sampling.

Genetic variability of classical swine fever virus

The genetic variability of classical swine fever virus was studied by comparative nucleotide sequence analysis of 76 virus isolates, collected during a half century from three continents. Parts of the E2 (gp55) and the polymerase gene coding regions of the viral genome were amplified by RT-PCR and DNA fragments of 254 and 207 bp, respectively, were sequenced. The comparative sequence analysis of the E2 region revealed two main phylogenetic groups of CSFV, indicating that the virus apparently evolved from two ancestor nodes. Group I (represented by Brescia strain) consisted of old and recent American and Asian viruses, as well as old English isolates from the 1950s. This group was subdivided into three subgroups, termed I.A-I.C. Group II (represented by Alfort strain) consisted of relatively recent isolates from Europe, together with strain Osaka, which was isolated in Japan from a pig of European origin. Based on genetic distances the group was divided into subgroups II.A and II.B. Malaysian isolates were branched into both groups, indicating multiple origins for contemporaneous outbreaks in that country. All ten vaccine strains tested were branched in group I, implying a common ancestor. The Japanese Kanagawa strain, isolated in 1974, and the British Congenital Tremor strain from 1964 were the most distinct variants of CSFV in our collection. The comparison of the nucleotide sequences of the polymerase coding region of 32 European strains distinguished subgroups II.A and II.B which were similar to the corresponding subgroups of the E2 phylogenetic tree. Thus, the results revealed that the E2 region and the polymerase coding regions seem to be appropriate for the grouping of CSFV isolates from all over the world, distinguishing two major groups of the virus. The reliability of these regions for phylogenetic analysis is indicated by the similarity of the results obtained from the two separate parts of the CSFV genome.

Classical swine fever virus: a second 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.

Phylogenetic Comparison and Molecular Epidemiology of Classical Swine Fever Virus

The genetic diversity of classical swine fever virus (CSFV) was studied by RT-PCR amplification and sequencing of a 409 bp fragment of the NS5B polymerase region. A total of 106 viruses isolated from 20 countries over a period of 52 years (1945–1997) were included in the phylogenetic study. The results showed that the viruses could be divided into two main groups. Group 1 consisted of Asian and South American isolates from the 1980s, as well as of old European and American isolates. Group 2 consisted mostly of recent European viruses from the 1980s and 1990s, and was further divided into three subgroups largely according to geographic origin and/or year of isolation. Five 1997 CSFV isolates from Germany, Netherlands and Italy clustered together indicating a common origin for these outbreaks, but two other 1997 isolations in different regions of Germany are likely due to different epidemiological events. The results show that the NS5B region of the genome gives a good resolution for phylogenetic studies of CSFV. Molecular epidemiology based on nucleotide sequence diversity is a useful tool for tracing virus spread and for developing disease control strategies.

Genetic heterogeneity of classical swine fever virus in Central Europe

The aim of this work was to genetically characterize Central European isolates of classical swine fever virus (CSFV) and to evaluate the applicability of molecular analysis in the epizootiology of CSFV infections. Thirty four viruses, derived from Central European pigs or wild boar, were examined. All of these viruses were detected by each of three sets of oligonucleotide primers which had been designed for the specific RT-PCR amplification of different genomic regions. Comparative sequence analysis of the PCR products showed that they were of a genetic type common in Western Europe. Further discrimination of virus isolates was possible, into subgroups that largely coincided with their regions of origin in Poland, Slovakia, Hungary and Estonia. The discriminatory ability of the technique was improved by the analysis of a composite dataset consisting of all of the sequence data from all of the viruses. Using this approach we were able to distinguish between all of the viruses and to group them in a manner that precisely matched their geographical origins, apart from a single Estonian isolate which grouped with viruses from Eastern Poland.

Characterisation of a recent virulent transmissible gastroenteritis virus from Britain with a deleted ORF 3a

SummaryAnalyses of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) isolates have suggested that tropism and pathogenicity are influenced by the spike protein and ORF 3. In general, enteric viruses (TGEV) have been shown to contain intact spike and ORF 3 genes, whilst respiratory isolates (PRCV) have major deletions within both regions. Virulence has been correlated to a functional ORF 3. Here, sequence analysis of a recent isolate of virulent TGEV, revealed a variant with an intact spike gene, but a large deletion in ORF 3a. This suggests that ORF 3a is not essential for enteric virulence.

Foetal cross-protection experiments between type 1 and type 2 bovine viral diarrhoea virus in pregnant ewes.

A flock of 82 non-pregnant ewes was split into three immunisation groups and given an intranasal dose of either cell culture medium, or a type 1 or a type 2 bovine viral diarrhoea virus (BVDV-1 or BVDV-2). Two months later the flock was reconstituted and after a further three weeks, the ewes were bred to pestivirus negative rams after synchronisation of oestrus using progesterone sponges. Fifty-five ewes were segregated into three challenge groups, each of which comprised ewes from different immunisation groups. At 7 weeks gestation, one challenge group was given an intranasal dose of cell culture medium, whilst the other two were given intranasal doses of either BVDV-1 or BVDV-2, using the same inocula as for the immunisations. Three weeks later, the ewes were killed and their foetuses tested for the presence of BVDV-1 and BVDV-2. The results showed that immunisation of six ewes without subsequent challenge did not lead to infection of any of their 11 foetuses. Challenge with BVDV-1 or BVDV-2 in the absence of immunisation lead to 15 out of 15 or 11 out of 14 foetuses becoming infected, respectively. Immunisation with the homologous virus to that used for challenge resulted in complete protection of 32 foetuses from 15 ewes. Heterologous protection was one way. All 12 foetuses from ewes immunised with BVDV-1 were protected from challenge with BVDV-2, whereas 18 foetuses from ewes immunised with BVDV-2 were all infected after challenge with BVDV-1. This provides evidence that a recent exposure to infection with one pestivirus does not necessarily induce foetal protection against another. The one-way result suggests that factors other than antigenic differences are involved in cross-protection.