GB2300419A - Detection of classical swine fever virus - Google Patents

Abstract

A method is provided for the determination of Classical Swine Fever Virus CSFV in a sample comprising exposing the sample to conditions under which, if CSFV is present, a region of its genome having at least 80% homology to bases 2218 to 2888 or bases 11149 to 12238 of the genome of the Alfort strain of CSFV is amplified and relating the amplification of said region to the presence of CSFV. The method permits the differentiation between CSFV of different strains and origins and the closely related pestiviruses Bovine Viral Diarrhea (BVDV) and Border Disease Virus (BDV). Oligonucleotides suitable for use as primers are disclosed.

Description

DETECTION OF SWINE FEVER VIRUSES The present invention relates to methods for the determination of the presence of Classical Swine Fever Virus (CSFV), methods of distinguishing CSFV from closely related viruses, methods of studying its epidemiology, to apparatus and kits for the performance of all the aforesaid and to oligonucleotides comprising sequences suitable for use in the methods, apparatus and kits.

CSFV, formerly known as Hog cholera virus, is a member of the genus Pestivirus, recently classified as part of the family Flaviviridae, which are economically important pathogens in veterinary medicine.

They are single-stranded positive polarity viruses which cause diseases of economic significance and are responsible for substantial losses in cattle, swine and sheep farming. Bovine viral diarrhea (BVDV) and border disease virus (BDV) are also members of this genus, but are both ruminant pathogens which only occasionally infect swine, whereas CSFV is highly virulent to swine, resulting in livestock loss throughout the world.

While the ruminant pestiviruses, BDV and BVDV, are found world-wide some nations have been able to fully eliminate CSFV from their national swine herds. In such countries it is important to maintain constant surveillance in order to detect re-entry of the disease at an early stage and to take any necessary action to prevent its spread.

Methods for the detection of CSFV which can differentiate it from the other, less harmful, pestiviruses are useful, particularly if they are capable of detecting very low levels of infection as this will allow action to be taken as early as possible.

During an outbreak of a viral disease it is also important to be able to rapidly deduce how the viral agent was introduced, how it is being transmitted and what are the potential risks of further infection.

Incorrect assumptions are often made as a result of miscalculating the number or origin of viral strains involved in an outbreak. This can result in unnecessary deployment of resources whilst infection continues unchecked. Methods which identify the origin of the disease are therefore of particular interest as they enable action to be directed to the point of entry and thus save on resources.

CSFV is difficult to distinguish from BDV and BVDV because all three types of virus are structurally and immunogenically similar. Meyer et al found the overall proportion of identical nucleotides between CSFV and BVDV to be approximately 70% (Meyer G, Rumenapf T and Thiel Heinz-Jurgen; J Virology 171, pp555-567 (1989)). CSFV are a homogenous group of viruses and it is difficult to differentiate between viruses of different strains or origins using standard immunological means.

The genomic RNA of two species of CSFV has been fully sequenced.

Meyer et al sequenced the genomic RNA of the Alfort strain of CSFV (Meyer G, Rumenapf T and Thiel Heinz-Jurgen; J Virology 171, pp555-567 (1989)) and Moormann et al have sequenced the genomic RNA of the Brescia strain of CSFV (Moormann R J B, Warmerdam P A M, Van Der Meer B, Schapper W M M, Wensvoort G and Hulst M M; J Virology 177 pp18-198 (1990)). Nucleic acid probes to CSFV have now been developed (US 5112753 (1989)) and PCR technology has been used to differentiate CSFV from other pestiviruses, ie. BDV and BVDV by Katz et al (nested amplification) (Katz J B, Ridpath J F and Bolin S R; J Clin Microbiol Vol 31 No 3 (1993) pp565-568) and by Wirz et al (reverse transcriptase PCR) (Wirz B, Tratschin J, Muller H K and Mitchell D B; J Clin Microbiol Vol 31 No 5 (1993) pp1148-1154).A further PCR based method recently used to differentiate CSFV from other pestiviruses has recently been described by Vilcek et al (Vilcek S, Herring A J, Herring J A, Nettleton P F, Lowings J P and Paton D J; Arch Virol (1994) 136: pp309-323). However, none of these methods can differentiate between CSFV of different origins and the epidemiological value of the assays is limited because the regions selected for amplification are highly conserved.

It is an aim of the present invention to provide methods of detecting CSFV which are preferably capable of distinguishing CSFV from other pestiviruses and more preferably capable of distinguishing CSFV of different strain or origin from one another. This will facilitate epidemiological studies of the disease and enable early action to be taken to contain the disease.

A first aspect of the present invention provides a method for the determination of CSFV in a sample comprising exposing the sample to conditions under which if CSF\' is present a region of its genome having at least 80% homology to bases 2218 to 2888 or bases 11149 to 12238 of the genome of the Alfort strain of CSFV is amplified and relating the amplification of said region to the presence or amount of CSFV present. Embodiments of this aspect of the invention include those in which a sequence having at least 85% homology with the bases described above is amplified, more preferably a sequence with at least 90% or at least 95% and most preferably having 100% homology. Thus a sequence downstream or upstream may also be amplified or a sequence with some variation which does not affect the efficacy of the method is also suitable.

The amplification of the sequence is preferably performed after a strand of nucleic acid complimentary to the region has been generated by reverse transcription. The resulting double stranded material may then be amplified. eg. by PCR, preferably utilising a pair of primers wherein the first primer is complimentary to any one of SEQ ID No.s 1 to 4 and the second primer is complimentary to SEQ ID No. 5 or 6 or alternatively utilising a pair of primers wherein the first primer is complimentary to any one of SEQ ID No.s 61 to 68 and the second primer is complimentary to SEQ ID No. 69.

Preferred primers which are complimentary to SEQ ID No.s 1 to 4 are SEQ ID No.s 31 to 34 and preferred primers which are complimentary to SEQ ID No.s 5 and 6 are SEQ ID No.s 35 and 36. Preferred primers which are complimentary to SEQ ID No.s 61 to 68 are SEQ ID NO.s 73 to 80 and a preferred primer which is complimentary to SEQ ID No. 69 is SEQ ID No. 81.

A second aspect of the invention comprises a method for the detection and identification of CSFV in a sample comprising: i) exposing the sample to conditions under which if CSFV is present a region of its genome having at least 80% homology to bases 2218 to 2888 of the genome of the Alfort strain of CSFV is amplified, and ii) sequencing a second region lying within the region amplified in step i) and having at least 80% homology to bases 2467 to 2738 of the genome of the Alfort strain of CSFV or i) exposing the sample to conditions under which if CSFV is present a region of its genome having at least 80; ; homology to bases 11149 to 12238 of the genome of the Alfort strain of CSFV is amplified, and ii) sequencing a second region lying within the region amplified in step i) and having at least 80% homology to bases 11498 to 11850 of the genome of the Alfort strain of CSFV and iii) relating the sequence of the second region in each case to the identity and/or origin of the virus.

As in the first aspect of the invention embodiments of this aspect preferably comprise the amplification of a sequence having at least 80% homology with the bases described above, more preferably at least 90% or at least 95% and most preferably having 100X homology. Thus a sequence downstream or upstream may also be amplified or a sequence with some variation which does not affect the efficacy of the method is also suitable.

In preferred embodiments step i) comprises the generation of a strand of nucleic acid complementary to the region by reverse transcription and the resulting double stranded material is then amplified by PCR.

More preferred embodiments are those in which PCR is performed utilising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 1 to 4 and a second primer is complimentary to SEQ ID No. 5 or 6 or alternatively utilising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 61 to 68 and a second primer is complimentary to SEQ ID No. 69.

Preferred primers which are complimentary to SEQ ID No.s 1 to 4 are SEQ ID No.s 31 to 34 and preferred primers which are complimentary to SEQ ID No.s 5 and 6 are SEQ ID No.s 35 and 36. Preferred primers which are complimentary to SEQ ID No.s 61 to 68 are SEQ ID No.s 73 to 80 and a preferred primer which is complimentary to SEQ ID No. 69 is SEQ ID No. 81.

In step ii) the second sequence region may be sequenced by any convenient method eg. by the chain termination dideoxy procedure (Sanger method) or by first cloning the nucleic material to be sequenced with the M13 mp series of single-stranded vectors as described in 'Principles of gene manipulation' ( R W Old & S B Primrose, 4th Edn, Blackwell Scientific Press, 1992). Kits are also available for sequencing nucleic acid, eg. Promega produce a fmol sequencing kit.

In preferred embodiments the second region is first amplified. This is suitably performed by the generation of a strand of nucleic acid complimentary to the region by reverse transcription and the resulting double stranded material is then amplified by PCR. More preferred embodiments are those in which PCR is performed utilising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 7 to 14 and a second primer is complimentary to any one of SEQ ID No.s 15 to 30 or alternatively utilising a pair of primers wherein a first primer is complimentary to SEQ ID No. 70 or 71 and a second primer is complimentary to SEQ ID No. 72.

Preferred primers which are complimentary to SEQ ID No.s 7 to 14 are SEQ ID No.s 37 to 44 and preferred primers which are complimentary to SEQ ID No.s 15 to 30 are SEQ ID No.s 45 to 60. Preferred primers which are complimentary to SEQ ID No.s 70 and 71 are SEQ ID No.s 82 and 83 and a preferred primer which is complimentary to SEQ ID No. 72 is SEQ ID No. 84.

Step iii) may be performed by comparing the sequence of the second region to corresponding sequences derived from viruses of known strain or origin. A direct match will enable the identification of the strain or origin of CSFV or a comparison with known sequences may be used to determine the closest related strain or origin. Alternatively the nucleic acid sequence may be compared to that of viruses of known origin and the relationships between the viruses determined with the aid of a computer. The information obtained can also be used to study the evolution of the virus.

A third aspect of the present invention provides oligonucleotides having at least 80Z homology to any one of SEQ ID No.s 1 to 84.

Preferred embodiments of this aspect of the invention include those having at least 85% homology more preferably a sequence having at least 90% or 95% homology and most preferably having 100% homology.

A fourth aspect of the invention provides the use of an oligonucleotide described in the third aspect in any one of the methods described in the first and second aspects.

A fifth aspect of the invention provides kits suitable for performing the methods described above. In particular kits are provided for the determination of CSFV in a sample comprising PCR primers capable of initiating the amplification of a sequence having at least 80% homology to bases 2218 to 2888 or bases 11149 to 12238 of the genome of the Alfort strain of CSFV. One embodiment of this aspect provides a kit comprising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 1 to 4 and a second primer is complimentary to SEQ ID No. 5 or 6. Preferably the first primer comprises one of SEQ ID No.s 31 to 34 and the second primer comprises SEQ ID No. 35 or 36. A further embodiment provides a kit comprising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 61 to 68 and a second primer is complimentary to SEQ ID No. 69.Preferably the first primer comprises one of SEQ ID No.s 73 to 80 and the second primer comprises SEQ ID No. 81.

This aspect also provides a kit for the detection and identification of CSFV in a sample comprising a pair of PCR primers capable of initiating the amplification of a sequence having at least 80% homology to bases 2218 to 2888 of the genome of the Alfort strain of CSFV and a second pair of PCR primers capable of initiating the amplification of a sequence having at least 80% homology to bases 2467 to 2738 of the genome of the Alfort strain of CSFV.

Preferably the first pair of primers comprises a first primer complimentary to any one of SEQ ID No.s 1 to 4 and a second primer complimentary to SEQ ID No. 5 or 6 and the second pair of primers comprises a first primer complimentary to any one of SEQ ID No.s 7 to 14 and a second primer complimentary to any one of SEQ ID No.s 15 to 30.

More preferably the first pair of primers comprises a first primer selected from of SEQ ID No.s 31 to 34 and a second primer selected from SEQ ID No.s 35 and 36 and the second pair of primers comprises a first primer selected from SEQ ID No.s 37 to 44 and a second primer selected from SEQ ID No.s 45 to 60.

This embodiment further provides a kit for the detection and identification of CSFV in a sample comprising a pair of PCR primers capable of initiating the amplification of a sequence having at least 80X homology to bases 11149 to 12238 of the genome of the Alfort strain of CSFV and a second pair of PCR primers capable of initiating the amplification of a sequence having at least 80% homology to bases 11498 to 11850 of the genome of the Alfort strain of CSFV.

Preferably the first pair of primers comprises a first primer complimentary to any one of SEQ ID No.s 61 to 68 and a second primer complimentary to SEQ ID No. 69 and the second pair of primers comprises a first primer complimentary to SEQ ID No. 70 or 71 and a second primer complimentary to SEQ ID No. 72.

More preferably the first pair of primers comprises a first primer selected from SEQ ID No.s 73 to 80 and the second primer comprises SEQ ID No. 81 and the second pair of primers comprises a first primer selected from SEQ ID No.s 82 and 83 and the second primer comprises SEQ ID No. 84.

FIGURES Figure 1 shows a phylogenic tree generated from the sequences of the gp fragments determined.

Figure 2 shows a phylogenic tree generated from the sequences of the pol fragments determined.

Figure 3 shows a phylogenic tree generated from the data obtained from the method of Example 1.

The methods and apparatus of the invention will now be illustrated by example only with reference to the following non-limiting Examples.

Further embodiments will occur to those skilled in the art in the light of these.

EXAMPLE 1: DETECTION OF CSFV USING PRIMERS TARGETED TO THE E1/E2 GROUP AND THE NSEB GROUP OF THE VIRAL GENOME Two groups of viral isolates were used in the following experiments.

The first consisted of 33 porcine isolates (including 32 CSFVs), 4 bovine isolates and 4 ovine isolates; these were used to determine the specificity of the primers. These isolates were antigenically diverse and had been isolated from many different countries.

Epidemiological studies were then performed using isolates obtained from domestic swine and free living or captive wild boar over a seven year period (1985 to 1992). These were isolated from six provinces in Italy, and named according to their geographical origin (north (n), central (c) or south (s)), chronology of isolation (1 to 8) and animal type (Wild (W) or Domestic (D)). Thus clW was the first virus to be isolated and was obtained from a wild boar (captive herd) in the central region of the study.

The first isolation from Livorno province in October 1985 was followed during June 1986 to August 1987 by five outbreaks in domestic pigs and 53 cases in free living wild boar. From September 1987 to May 1990 129 cases of CSFV were detected in free living wild boars (viruses c2W was one of these), five outbreaks occurred in captive herds of wild boar and nine outbreaks occurred in domestic pigs. These cases occurred in Livorno and the three adjoining central provinces of Pisa, Siena and Grosseto. In 1991 these provinces had three further outbreaks in domestic pigs (including c3D and c4D); in addition an isolated outbreak (n5W) occurred in a captive herd of wild boar in the more northerly province of Parma.In 1992 there was one further outbreak (n6W) in the northern province of Massa which adjoins Parma and the two further southern outbreaks (s7D and s8D) on the borders of Roma and Latina.

There was some evidence that the s7D and s8D isolates were of lower virulence than the viruses isolated earlier (Ferrari G, Rutili D, Autorino G L, Cardetti G & Amaddeo D; (1993), XLVII Convegno Nazionale S I S Vet Riccione (FO) Italy, 29 September to 2 October 1993). There was some anecdotal evidence that there had been wild boar/ domestic pig contact prior to the outbreaks caused by viruses c3D and c4D. All the CSFVs were passaged in the PK15 cell line, while the ruminant viruses were grown in bovine turbinate cells. None of the viruses used in this study were biologically cloned and none of the Italian isolates had received more than four in vitro culture passages.

RNA was extracted from cells 48 to 72 hours post infection using the acid phenol method (Stallcup M R & Washington L D; J. Biological Chemistry (1983) 258, 2802-2807). Reverse transcriptase PCR (RT-PCR) was performed on samples of the isolates, according to the method described by Sambrook et al, (Sambrook J, Fritsch E F, & Maniatis T; Molecular cloning: A Laboratory Manual, 2nd Ed. New York, Cold Spring Harbor Laboratory (1989) using primers targeted at the E1/E2 (gp33/gp55) gene junction (according to the nomenclature described by Rumenapf et al (Rumenapf T, Unger G, Strauss J H & Thiel H J, (1993), Journal of virology 67, 3288-3294). SEQ ID No. 31 was used as the forward primer and SEQ ID No. 35 as the reverse primer.The following buffers solutions were used: 60mM Tris-HCl pH 8.5 at 200C, 15mM ammonium sulphate and 2.5mM magnesium chloride. 35 cycles of amplification were performed, the denaturing step being performed at 950C for 45 seconds, the annealing step at 550C for 60 seconds and the final step at 720C for 5 minutes. The fragment amplified was termed the gp fragment.

RT-PCR was also performed on samples of the isolates using primers targeted to the polymerase gene NS5B using SEQ ID No. 73 as the forward primer and SEQ ID No.81 as the reverse primer. The buffer solutions and conditions under which the PCR was performed were the same as those used to amplify the gp fragment, except the annealing temperature which was reduced to 500C. The fragment amplified was termed the pol fragment.

The gp fragment was sequenced after pooling five RT-PCR reactions from each of the CSFV isolates and purifying them using agarose gel electrophoresis and Prepagene silica matrix (Bio-Rad). The purified products were cut with the restriction enzymes EcoR1 and Sall prior to cloning into similarly cut pBluescript KS(+) and KS(-) (Stratagene) plasmids. These constructs were used to transform E.Coli strain XL-blue (Stratagene) and DNA sequencing (sequenase USB) was performed from single stranded templates produced by helper phage phagemid rescue.

The pol fragment was directly sequenced after pooling five RT-PCR reactions which had been purified by passing through a Wizard PCR preparative column (Promega). These fragments were then directly sequenced by cycle sequencing using fmol sequencing kit (Promega).

SEQ ID Nos. 82 and 84 which had been kinase labelled with [alpha-32PjATP (Amersham) in accordance with the manufacturers recommendations were used as primers to generate 475 bases of unambiguous sequence.

Multiple alignments of the sequences determined together with published sequences of pestiviruses were carried out using the PILEUP program on the GCG package (Devereux J, Haeberli P, & Smithies O; (1984) Nucleic Acids Research 12, 387-1438). The data was analysed with the multiple programs of the PHYLIP package (Felsenstein J; (1989), Cladistics 5, 164-166). Analysis included maximum likelihood, least square, neighbour-joining, UPGMA, compatibility and multiple parsimony methods.

The primers SEQ ID No. 31 and 35 produced a specific 671 bp product from all the CSFV RNA samples. No products were detected with bovine or ovine isolates nor from the non-CSFV porcine virus. An additional band was also detected at 250 bp for some isolates. The primers SEQ ID No. 73 and 81 produced a product of 1090 bp from all CSFV infected RNA samples but were not assessed with ruminant viruses.

Multiple alignment for the gp fragments included sequences from two other strains of CSFV, the Alfort 187 strain (Muyldermans G, San Gabriel M C, Caij a, De Smet A & Hamers R; (1993) Archives of Virology, 132, 429-435) and the Weybridge strain (Yu M, McColl K A & BR< Gould A R; (1993) Virus Research, 28, 203-208). All sequences showed a high level of conservation with variation occurring mainly at wobble bases. Nucleotide and peptide distances between the viruses in the gp region are shown below in Table 1. Nucleic acid identities varied from 82.53% between Brescia and n5W (83 substitutions) and 99.79 % between clW and c3D (one substitution).

TABLE 1: NUCLEOTIDE AND PEPTIDE DISTANCES Nucleolide distance (%)

lSio &commat; fq Xe Alroz 1 e Ka if" ,(O Air., SEW .1W aw ,1D tiW '417 99.93 ,ttjo , gB.ii] 92.00 n4.e4 99.09 85.05 94.94 G4.t4 o.nJ 55.05 n4.Q4 94.94 94 21 F18 4 9'.' 4Z . G2.w1 94.94 15.05 as.05 e4.n D4.04 84.o4 n5.05 s4.n4 64 94 n4 93 llRt5Ci4 94 94 95.67 . to,96 gel.74 02.74 gel.95 01.53 93.37 or.50 tJ.J7 93 J7 n7 95 s7D B0.70 95.57 93 67 . 90.15 9Z.47 99.11 sizes (9.69 99.66 99.47 19.05 oo 93 snr) 15.57 94 94 93.04 99.37 . .2G 97.47 97.99 99.69 us.47 99.29 sn.t4 nn 42 Altor 90 20 96.9 93.97 100.00 99.37 99.96 99.11 91.59 91.31 91.we 90 74 99 99 nGW 96.20 96.57 93.67 i0.00 99.37 100.00 . 96.32 90.9) 90 37 90.11 99.69 6970 I,JW 99.97 94.94 93 O4 99.37 99.73 99.37 99.37 . | t9.09 99.66 09.47 99.05 99,63 clW 90.1O 95.92 Dszs7 96.10 91 47 96.10 96.10 97.42 . 99.76 99.16 99.16 c3IJ sn.n4 96 20 96.70 97.42 sG.ts 97.47 a7.t7 9994 99.37 !! J7 , 90.96 98 95 99 17 c7 99.97 94.94 94 9 99.73 99.10 99.73 99.73 99.10 99.37 99.73 . 99.16 1 99 37 ev4D 96.10 n4.4 94 97.47 7.te 974 7 9t.47 G8.64 99.37 99.73 1 90.73 .

91.?? 12.41 91.77 99.97 94,94 99.9? 9.97 94.54 94.94 44.30 66.97 94.30 3yline ptiet ct e ( Dgo ) Sequencing results indicated three subgroups, each having inter-group identities of greater than 97%. The first consists of Weybridge and Alfort 187 strains with an identity of 98.53X (seven substitutions).

the second (north/south) group contains s7D, s8D, Alfort, n6W and n5W with identities ranging from 97.53% to 98.95% (up to 13 substitutions). A third (central) group consists of clW, c3D, c2W and c4D and has identities ranging from 98.9% to 99.79%, corresponding to between one and four substitutions, making it the most homogenous of the subgroups. The remaining viruses, Brescia and s7D2, did not fall into any of the subgroups. The viruses s7D2 and s7D were isolated from the same stock of uncloned virus and could be a result of two distinct viruses existing within one animal or a result of in vitro cross-contamination.

Similar relationships to these were observed at the peptide level; identities being greater within the north/south group than within the central group. The presence of minor variants within some of the other virus stocks were also noted but all such mutations were unique to individual clones.

The sequences from the gp and pol regions were used as input for phylogenic analyses which provided group relationships essentially the same as those outlined above. Figures 1 and 2 show phylogenic trees from maximum likelihood analysis of each set of data. Statistical analysis using bootstrapping or delete half jack-knifing showed the confidence in these results to be high (P < 0.01 for most nodes). The high confidence levels in the groupings were confirmed by the minimal effects of altering the transition/transversion ratios from between 1:1 and 6:1. A similar grouping was obtained when the gp sequences were analysed in either of two sections, indicating that the results were not distorted by local selection (for example of a critical antigenic epitope).Analysis of derived amino acid sequences provided results which were less informative and group relationships were not as significant when bootstrapped, however the tree was essentially unchanged.

The experiments described above show the primers to be specific for CSFV, amplifying a product of 671 bp from every CSFV examined. The sensitivity was sufficient for all products to be clearly visible within ethidium stained agarose gels. The method could therefore be used in a laboratory to confirm a diagnosis of CSFV.

The trees shown in Figures 1 and 2 show similar groupings of the viruses (central group, north/south group and Brescia alone) which makes false grouping due to recombination events very unlikely. It appears that initial infection spread from captive (clW) to free living wild boar (c2W) and back to domestic swine (c3D and c4D) over a six year period. Prior to this study wild boar were suspected of being a reservoir for the virus for the following reasons: no captive swine outbreaks occurred between October 1985 and May 1986 but the disease reappeared in June 1986; infected free living wild boar were found dead or were shot near all the reported outbreaks; fencing around most of the infected premises was inadequate to avoid wild boar contact; actual wild boar contact was very likely in at least one of the 1991 outbreaks where the disease occurred 21 days after an escape incident.The analysis supports the presence of a CSFV reservoir in the free living population because viruses isolated from captive or free living wild boar or from domestic pigs appear nearly identical and the virus remained almost unchanged after the period October 1985 to May 1986 when no captive wild boar or domestic pig swine infection occurred. The results of the analysis correlate precisely with the known and suspected epidemiology.

The northern outbreaks (n5W and n6W) were thought to be distinct from the central outbreaks mainly because the provinces between the two areas were free from the disease and both natural and man made barriers separated the zones making natural movement of wild boar impossible. The results of the experiments described above confirms this; the northern isolates were found to be a separate subtype.

Little was known about the southern isolates (s7D and s8D), but the source of infection was suspected to be the importation of pigs from Eastern Europe. The experiments described above have confirmed that these viruses are similar to the northern group, but the variability of the north/south group supports the suspicion that these outbreaks may have resulted from separate introductions of similar viruses.

The viruses used in the study were also assessed for reactivity to a panel of 23 monoclonal antibodies. An indirect immunoperoxidase method was performed using previously optimised dilutions of antibody to probe fixed monolayers of cells that had been infected with approximately 300 TCID50 of each virus. The results confirmed that all the viruses were CSFVs and not the closely related BVD or BD pestiviruses. Nine of the antibodies showed some discrimination between isolates although in some cases the positive staining was very weak making interpretation difficult. All of the Italian isolates were clearly different from the Alfort and Brescia reference strains.

two groups of viruses (c2W/n6W/s7D and clW/c3D) appeared identical or very similar, whereas the patterns of staining for each of the other viruses were unique.

Thus monoclonal antibodies were able to show some grouping ability but produced a less likely epidemiological model, which when tested with bootstrapping resampling was shown to be unsound. This may be becasue the antibodies are reactive to only a small number of immunogenic sites and small changes elsewhere may not be indicated.

Claims (27)

1. A method for the determination of CSFV in a sample comprising exposing the sample to conditions under which if CSFV is present a region of its genome having at least 80% homology to bases 2218 to 2888 or bases 11149 to 12238 of the genome of the Alfort strain of CSFV is amplified and relating the amplification of said region to the presence or amount of CSFV present.

2. A method as claimed in Claim 1 wherein a strand of nucleic acid complimentary to the region is generated by reverse transcription and the resulting double stranded nucleic acid is amplified.

3. A method as claimed in Claim 2 wherein the double stranded nucleic acid is amplified by PCR utilising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 1 to 4 and a second primer is complimentary to SEQ ID No. 5 or 6.

4. A method as claimed in Claim 3 wherein the first primer comprises one of SEQ ID No.s 31 to 34 and the second primer comprises SEQ ID No.

35 or 36.

5. A method as claimed in Claim 3 wherein the double stranded nucleic acid is amplified by PCR utilising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 61 to 68 and a second primer is complimentary to SEQ ID No. 69.

6. A method as claimed in Claim 5 wherein the first primer comprises one of SEQ ID No.s 73 to 80 and the second primer comprises SEQ ID No.

81.

7. A method for the detection and identification of CSFV in a sample comprising: i) exposing the sample to conditions under which if CSFV is present a region of its genome having at least 80% homology to bases 2218 to 2888 of the genome of the Alfort strain of CSFV is amplified, and ii) sequencing a second region lying within the region amplified in step i) and having at least 80% homology to bases 2467 to 2738 of the genome of the Alfort strain of CSFV or i) exposing the sample to conditions under which if CSFV is present a region of its genome having at least 80% homology to bases 11149 to 12238 of the genome of the Alfort strain of CSFV is amplified, and ii) sequencing a second region lying within the region amplified in step i) and having at least 80% homology to bases 11498 to 11850 of the genome of the Alfort strain of CSFV and iii) relating the sequence of the second region in either case to the identity and/or origin of the virus.

8. A method as claimed in Claim 7 wherein step i) comprises the generation of a strand of nucleic acid complimentary to the region by reverse transcription and the resulting double stranded nucleic acid is then amplified by PCR.

9. A method as claimed in Claim 8 wherein the resulting double stranded nucleic acid is amplified by PCR utilising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 1 to 4 and a second primer is complimentary to SEQ ID No. 5 or 6 or alternatively utilising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 61 to 68 and a second primer is complimentary to SEQ ID No. 69.

10. A method as claimed in Claim 9 wherein the first primer comprises one of SEQ ID No.s 31 to 34 and the second primer comprises SEQ ID No.

35 or 36 or alternatively the first primer comprises one of SEQ ID No.s 73 to 80 and the second primer comprises SEQ ID No. 81.

11. A method as claimed in any one of Claims 7 to 10 wherein step ii) comprises the amplification of the second region.

12. A method as claimed in Claim 11 wherein step ii) comprises the generation of a strand of nucleic acid complimentary to the region by reverse transcription and the resulting double stranded nucleic acid is then amplified by PCR.

13. A method as claimed in Claim 12 wherein the resulting double stranded nucleic acid is amplified by PCR utilising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 7 to 14 and a second primer is complimentary to any one of SEQ ID No.s 15 to 30 or alternatively utilising a pair of primers wherein a first primer is complimentary to SEQ ID No. 70 or 71 and a second primer is complimentary to SEQ ID No. 72.

14. A method as claimed in Claim 13 wherein the first primer comprises one of SEQ ID No.s 37 to 44 and the second primer comprises one of SEQ ID No.s 45 to 60 or alternatively the first primer comprises one of SEQ ID No. 82 or 83 and the second primer comprises SEQ ID No. 84.

15. An oligonucleotide having at least 80Z homology to a sequence given in any one of SEQ ID No.s 1 to 84.

16. The use of an oligonucleotide having at least 80% homology to a sequence according to any one of SEQ ID No.s 1 to 84 in a method according to any one of Claims 1 to 12.

17. A kit for the determination of CSFV in a sample comprising PCR primers capable of initiating the amplification of a sequence having at least 80% homology to bases 2218 to 2888 or bases 11149 to 12238 of the genome of the Alfort strain of CSFV.

18. A kit as claimed in Claim 17 comprising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 1 to 4 and a second primer is complimentary to SEQ ID No. 5 or 6.

19. A kit as claimed in Claim 18 wherein the first primer comprises one of SEQ ID No.s 31 to 34 and the second primer comprises SEQ ID No.

35 or 36.

20. A kit as claimed in Claim 17 comprising a pair of primers wherein a first primer is complimentary to any one of SEQ ID No.s 61 to 68 and a second primer is complimentary to SEQ ID No. 69.

21. A kit as claimed in Claim 20 wherein the first primer comprises one of SEQ ID No.s 73 to 80 and the second primer comprises SEQ ID No.

81.

22. A kit for the detection and identification of CSFV in a sample copmprising a pair of PCR primers capable of initiating the amplification of a sequence having at least 80X homology to bases 2218 to 2888 of the genome of the Alfort strain of CSFV and a second pair of PCR primers capable of initiating the amplification of a sequence having at least 80% homology to bases 2467 to 2738 of the genome of the Alfort strain of CSFV.

23. A kit as claimed in claim 22 wherein the first pair of primers comprises a first primer complimentary to any one of SEQ ID No.s 1 to 4 and a second primer complimentary to SEQ ID No. 5 or 6 and the second pair of primers comprises a first primer complimentary to any one of SEQ ID No.s 7 to 14 and a second primer complimentary to any one of SEQ ID No.s 15 to 30

24. A kit as claimed in claim 23 wherein the first pair of primers comprises a first primer selected from of SEQ ID No.s 31 to 34 and a second primer selected from SEQ ID No.s 35 and 36 and the second pair of primers comprises a first primer selected from SEQ ID No.s 37 to 44 and a second primer selected from SEQ ID No.s 45 to 60

25.A kit for the detection and identification of CSFV in a sample copmprising a pair of PCR primers capable of initiating the amplification of a sequence having at least 80% homology to bases 11149 to 12238 of the genome of the Alfort strain of CSFV and a second pair of PCR primers capable of initiating the amplification of a sequence having at least 80X homology to bases 11498 to 11850 of the genome of the Alfort strain of CSFV.

26. A kit as claimed in Claim 25 wherein the first pair of primers comprises a first primer complimentary to any one of SEQ ID No.s 61 to 68 and a second primer complimentary to SEQ ID No. 69 and the second pair of primers comprises a first primer complimentary to SEQ ID No.

70 or 71 and a second primer complimentary to SEQ ID No. 72.

27. A kit as claimed in claim 26 wherein the first pair of primers comprises a first primer selected from SEQ ID No.s 73 to 80 and the second primer comprises SEQ ID No. 81 and the second pair of primers comprises a first primer selected from SEQ ID No.s 82 and 83 and the second primer comprises SEQ ID No. 84.

SEOUENCE LISTING SEQ ID 1: 3' TCT GGT CTG ACC GGN ATG CT 5' SEQ ID 2: 3' TCT GGT CAG ACC CGN ATA CT 5' SEQ ID 3: 3' TCC CGT CAG ACC GGN ATG CT 5' SEQ ID 4: 3' TCC GGT CAG ACC GGN ATA CT 5' SEQ ID 5: 3' AAA TGG TGA AGA CAA GAG T 5' SEQ ID 6: 3' AAG TGG TGA AGA CAA GAG T 5' SEQ ID 7: 3' AGC TGT TGG TTA CTC TAT CCC SEQ ID 8: 3' AGC TGT TGG TTG CTC TAT CCC SEQ ID 9: 3' AGC AGT TGG TTA CTC TAT CCC SEQ ID 10: 3' AGC AGT TGG TTG CTC TAT CCC SEQ ID 11: 3' AGT TGT TGG TTA CTC TAT CCC SEQ ID 12: 3' AGT TGT TGG TTG CTC TAT CCC SEQ ID 13: 3' AGT AGT TGG TTA CTC TAT CCC SEQ ID 14: 3' AGT AGT TGG TTG CTC TAT CCC SEQ ID 15: 3' GTG TCG GGT TTA GGT TTC AGT AG SEQ ID 16: 3' GTG TCG GGT TTG GGT TTC AGT AG SEQ ID 17: 3' GTG TCG GGC TTA GGT TTC AGT AG SEQ ID 18: 3' GTG TCG GGC TTG GGT TTC AGT AG SEQ ID 19: 3' GTG TCA GGT TTA GGT TTC AGT AG SEQ ID 20: 3' GTG TCA GGT TTG GGT TTC AGT AG SEQ ID 21: 3' GTG TCA GGC TTA GGT TTC AGT AG SEQ ID 22: 3' GTG TCA GGC TTG GGT TTC AGT AG SEQ ID 23: 3' GTG TCG GGT TTA GGC TTC AGT AG SEQ ID 24: 3' GTG TCG GGT TTG GGC TTC AGT AG SEQ ID 25: 3' GTG TCG GGC TTA GGC TTC AGT AG SEQ ID 26: 3' GTG TCG GGC TTG GGC TTC AGT AG SEQ ID 27: 3' GTG TCA GGT TTA GGC TTC AGT AG SEQ ID 28: 3' GTG TCA GGT TTG GGC TTC AGT AG SEQ ID 29: 3' GTG TCA GGC TTA GGC TTC AGT AG SEQ ID 30: 3' GTG TCA GGC TTG GGC TTC AGT AG SEQ ID 31: forward 5' AGA CCA GAC TGG CCN TAC GA 3' SEQ ID 32: forward 5' AGA CCA GAC TGG CCN TAT GA 3' SEQ ID 33: forward 5' AGG CCA GAC TGG CCN TAC GA 3' SEQ ID 34: forward 5' AGG CCA GAO TGG CCN TAT GA 3' SEQ ID 35: reverse 5 TTT ACC ACT TCT GTT CTC A 3' SEQ ID 36: reverse 5 TTC ACC ACT TCT GTT CTC A 3' SEQ ID 37: forward 5' TCG ACA ACC AAT GAG ATA GGG SEQ ID 38: forward 5' TCG ACA ACC AAC GAG ATA GGG SEQ ID 39: forward 5' TCG TCA ACC AAT GAG ATA GGG SEQ ID 40: forward 5' TCG TCA ACC AAC GAG ATA GGG SEQ ID 41: forward 5' TCA ACA ACC AAT GAG ATA GGG SEQ ID 42: forward 5' TCA ACA ACC AAC GAG ATA GGG SEQ ID 43: forward 5' TCA TCA ACC AAT GAG ATA GGG SEQ ID 44: forward 5' TCA TCA ACC AAC GAG ATA GGG SEQ ID 45: reverse 5' CAC AGC CCA AAT CCA AAG TCA TC SEQ ID 46: reverse 5' CAC AGC CCA AAC CCA AAG TCA TC SEQ ID 47: reverse 5' CAC AGC CCC AAT CCA AAG TCA TC SEQ ID 48: reverse 5' CAC AGC CCG AAC CCA AAG TCA TC SEQ ID 49: reverse 5' CAC ACT CCA AAC CCA AAG TCA TC SEQ ID 50: reverse 5' CAC AGT CCA AAT CCA AAG TCA TC SEQ ID 51: reverse 5' CAC AGT CCC AAC CCA AAG TCA TC SEQ ID 52: reverse 5' CAC AGT CCC AAT CCA AAG TCA TC SEQ ID 53: reverse 5' CAC AGC CCA AAT CCG AAG TCA TC SEQ ID 54: reverse 5' CAC AGC CCA AAC CCC AAG TCA TC SEQ ID 55: reverse 5' CAC AGC CCC AAT CCC AAG TCA TC SEQ ID 56: reverse 5' CAC AGC CCC AAC CCG AAG TCA TC SEQ ID 57: reverse 5' CAC AGT CCA AAC CCC AAG TCA TC SEQ ID 58: reverse 5' CAC AGT CCA AAT CCC AAG TCA TC SEQ ID 59: reverse 5' CAC AGT CCC AAC CCG AAG TCA TC SEQ ID 60: reverse 5' CAC AGT CCG AAT CCC AAG TCA TC SEQ ID 61: 3' TAC AAC TTA CAC AAT TGT TAC SEQ ID 62: 3' TAC AAC TTA CAC AAC TGT TAC SEQ ID 63: 3' TAC AAT TTA CAC AAT TGT TAC SEQ ID 64: 3' TAC AAT TTA CAC AAC TGT TAC SEQ ID 65: 3' TAC GAC TTA CAC AAT TGT TAC SEQ ID 66: 3' TAC GAC TTA CAC AAC TGT TAC SEQ ID 67: 3' TAC GAT TTA CAC AAT TGT TAC SEQ ID 68: 3' TAC GAT TTA CAC AAC TGT TAC SEQ ID 69: 3' GGA GGT CGA TTT CAC GAC A SEQ ID 70 3' GGT CAC CAC TCT CCC CAT G) SEQ ID 71: 3t GGT CAC CAC TCT CTC CAT G) SEQ ID 72: 3' ACG TAA CTG GTC GGT CAA CGG GAG AAA SEQ ID 73: forward 5' ATG TTG AAT GTG TTA ACA ATG SEQ ID 74: forward 5' ATG TTG AAT GTG TTG ACA ATG SEQ ID 75: forward 5' ATG TTA AAT GTG TTA ACA ATG SEQ ID 76: forward 5' ATG TTA AAT GTG TTG ACA ATG SEQ ID 77: forward 5' ATG CTG AAT GTG TTA ACA ATG SEQ ID 78: forward 5' ATG CTG AAT GTG TTG ACA ATG SEQ ID 79: forward 5' ATG CTA AAT GTG TTA ACA ATG SEQ ID 80: forward 5' ATG CTA AAT GTG TTG ACA ATG SEQ ID 81: reverse 5' CCT CCA GCT AAA GTG CTG T SEQ ID 82: forward 5' CCA GTG GTG AGA GGG GTA C SEQ ID 83: forward 5' CCA TGT GTG AGA GAG GTA C SEQ ID 84: reverse 5' TGC ATT GAC CAG CCA GTT GCC CTC TTT