Stefan Vilcek

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.

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.

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.

Molecular Characterization of the 3′ Noncoding Region of Classical Swine Fever Virus Vaccine Strains

The genomes of classical swine fever virus (CSFV) vaccine strains are poorly characterized, and the mechanisms for their attenuation remain unknown. The aim of the present study was to characterize the 3′ noncoding region (3′ NCR) of a number of attenuated vaccine strains of CSFV in order to examine changes in the viral genome after attenuation. The results showed that the 3′ NCR:s of Porcivac, Rovac, Russian LK and original Chinese vaccine strain contain insertions very similar to that present in the published nucleotide sequence of the C-strain. Phylogenetic analysis showed that the vaccine strains Porcivac, Rovac and Russian LK vaccine were closely related to each other, but that these three strains showed a distant relationship with two tested variants of the Chinese vaccine strain (C-strain and original Chinese vaccine). The 3′ NCR insertion is not likely to be a marker for attenuation of the virus, since many CSFV vaccine strains do not contain the insertion. The fact that the insertions occur in lapinized vaccine strains suggests that these genetic segments were incorporated during the adaptation of the virus to the rabbit host system.

Genetic analysis of pestiviruses at the 3' end of the genome.

Specific PCR primers were selected for each pestivirus genotype which flanked the 3′-part of the NS5B gene and more than three quarters of the 3′-UTR. PCR products were sequenced in both directions using an automatic sequencing device and analyzed by computer package program DNASTAR. A comparative analysis of the 3′ untranslated region (3′-UTR) of 82 viruses, representing the four genotypes of the Pestivirus genus, provided a similar phylogenetic grouping as other genomic regions. Intertypic recombination was not observed, but Border disease virus (BDV) and Bovine viral diarrhoea virus type 1 (BVDV I) showed great intragenotypic variability. In most pestiviruses the stop codon is TGA, but BDV isolates were found to have either a TAG or a TAA stop codon. Various deletions and insertions were observed in the 3′-UTR region. Viruses of the BVDV Ib group contained a characteristic deletion of 41 nucleotides. Compared to the 5′-UTR, the 3′-UTR was less conserved. The first 50–60 nucleotides were particularly variable, whilst the most conserved part was found at the 3′ end of the studied region. All 82 viruses contained AT-rich stretches, the positions and sizes of which differed between the genotypes.

Establishment of serial persistent infections with bovine viral diarrhoea virus in cattle and sheep and changes in epitope expression related to host species

SummaryA pestivirus was transmitted by contact from a persistently infected (P.I.) bullock to pregnant sheep. This resulted in the birth of EI. lambs, one of which in turn was able to transmit virus by contact to pregnant cattle. Two of these animals gave birth to EI. calves, from one of which the virus was again transmitted by contact with pregnant sheep, leading to another generation of EI. lambs. The expression of one or more epitopes on the E2 glycoprotein of the viruses isolated from this series of alternate cattle-sheep transmissions appeared to depend on the host species. Thus, several monoclonal antibodies which bound strongly to, and neutralised, viruses isolated from the bovine hosts, failed to bind or neutralise in the case of sheep isolates. The viral consensus sequences of the E2 gene as well as parts of the 5’ untranslated region and of the NPro and capsid genes were compared between the different isolates. This revealed a high degree of genetic stability. However, a single codon change at amino acid position 9 of the E2 gene correlated with and was able to cause the loss of particular epitopes.

Storage of bovine viral diarrhoea virus samples on filter paper and detection of viral RNA by a RT-PCR method

Of four solid carriers tested, Whatman paper No 1 was the best for storing blood and serum samples for the diagnosis of bovine viral diarrhoea (BVD) by means of viral RNA detection. The filter papers were impregnated with 10 microl of blood or serum, followed by air drying. Samples collected in this way from persistently infected animals had lost infectivity within a few days, but viral RNA could still be detected by RT-PCR for up to 6 months. When investigated by RT-PCR, 12 blood and 10 serum samples selected at random from animals persistently infected with BVD virus showed the same results whether samples had been spotted onto filters or examined directly from the liquid state. The filters spotted with blood or serum are convenient for storage and transport of samples to a diagnostic laboratory without the need for cooling. Sequencing of amplified RNA can be used subsequently for genetic typing.

ORGANIZATION AND DIVERSITY OF THE 3'-NONCODING REGION OF CLASSICAL SWINE FEVER VIRUS GENOME

Specific PCR primers were selected to amplify a 359 bp DNA fragment flanking the 3′-part of the polymerase gene and the 3′-noncoding (3′-NC) region of the genome of classical swine fever virus (CSFV). In RT-PCR the selected fragment was amplified from the genomes of 27 viral strains collected from Europe, America and Asia over a period of a half century as well as from three vaccine strains of CSFV. Eight PCR products were sequenced using an automatic sequencing device. Nucleotide sequence analysis was performed by computer programs DNASTAR and PHYLIP. The comparative studies revealed that the 3′-NC region contains a variable region of nucleotides which is located immediately after the stop codon TGA or TAA. Furthermore, a strongly conserved constant region was identified near to the extreme 3′-terminus. Two imperfect repeated sequences were found both in the variable and in the constant regions. In addition, the variable region was characterized by the occurrence of a 50 bp long poly AT track. Phylogenetic analysis with different mathematical approaches (MegAlign, Neighbor-Joining method, Maximum Likelihood, Parsimony) revealed that the studied CSFV strains were clustered into two main phylogenetic groups. Group I was comprised of the reference strain Brescia, together with old American and European field isolates and a Brazilian vaccine strain. Group II included the reference strain Alfort (Tübingen) and recent European field strains. The Congenital Tremor strain formed a distinct lineage which, although being highly divergent, was more closely related to group I than to group II. In conclusion, the present phylogenetic grouping yielded very similar results as previous studies based on comparison of the E2 (gp55) region. The agreement of the phylogenetic analysis in the two distinct regions confirms the reliability of the genetic grouping of CSFV strains into two main genogroups.