Nucleotide and polypeptide sequences of pestivirus strains

Details Nucleotide and polypeptide sequences of pestivirus strains Nucleotide and polypeptide sequences of pestivirus strains

1996-12-24

2001-01-30

Stucker, Jeffrey (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Virus or component thereof

C424S085100, C530S350000, C530S395000, C435S005000, C435S007100

Reexamination Certificate

active

06180109

ABSTRACT:

FIELD OF THE INVENTION
The invention discloses a method for the construction of a full-length DNA copy of the genome of the C-strain, a classical swine fever vaccine strain, and transcription of RNA thereof which after transfection in cells gives rise to synthesis of infectious C-strain virus. The invention also comprises C-strain derived (pestivirus) vaccines, as well as subunit vaccines against pestivirus and diagnostic means and methods in relation to pestivirus infections. The invention furthermore provides a method of detecting an immunoactive substance in a sample by means of a competitive assay.
BACKGROUND OF THE INVENTION
Classical swine fever (CSF) or hog cholera is a highly contagious and often fatal disease of pigs which is characterised by fever and haemorrhages and can run an acute or chronic course (Van Oirschot. 1986. Hog cholera, p. 289-300. In Diseases of Swine. Iowa State University Press, Ames). Outbreaks of the disease occur intermittently in several European and other countries and can cause large economic losses.
Vaccination of pigs with a live attenuated Classical swine fever virus (CSFV) vaccine strain, the “Chinese” strain (C-strain), protects pigs against CSF (Terpstra and Wensvoort. 1988. Vet. Microbiol. 16: 123-128). A major drawback of vaccinating pigs with the conventional vaccines, of which the C-strain is one, is that these vaccinated pigs cannot be distinguished serologically from pigs infected with a CSFV field strain. The C-strain, however, is considered one of the most effective and safe live vaccines. Addition of a (serological) marker to the C-strain would be highly advantageous and would improve the vaccine.
CSFV is a member of the Pestivirus genus of the Flaviviridae (Francki, R. I. B. et al. 1991. Flaviviridae, p. 223-233. In Fifth report of the International Committee on Taxonomy of Viruses. Archiv. Virol. Suppl. 2, Springer Verlag, Vienna). The other two members of the Pestivirus genus, which are structurally, antigenically and genetically closely related to CSFV, are Bovine viral diarrhoea virus (BVDV) mainly affecting cattle, and Border disease virus (BDV) mainly affecting sheep (Moennig and Plagemann, 1992. Adv. Virus Res. 41: 53-98; Moormann et al., 1990. Virology 177: 184-198; Becher et al. 1994. Virology 198: 542-551).
The genomes of pestiviruses consist of a positive strand RNA molecule of about 12.5 kb (Renard et al. 1985. DNA 4: 429-438; Moormann and Hulst 1988. Virus Res. 11: 281-291; Becher et al. 1994. Virology 198: 542-551). The positive strand RNA genomes of several non-cytopathogenic BVDV strains, however, may be considerably larger (Meyers et al. 1991. Virology 180: 602-616; Meyers et al. 1992. Virology 191: 368-386; Qi et al. 1992. Virology 189: 285-292).
An inherent property of viruses with a positive strand RNA genome is that their genomic RNA is infectious, i.e. after transfection of this RNA in cells that support viral replication infectious virus is produced. As expected, the genomic (viral) RNA of pestiviruses is also infectious (Moennig and Plagemann, 1992. Adv. Virus Res. 41: 53-98).
For several years recombinant DNA technology has allowed in vitro transcription of cloned DNA. This possibility has opened the way to synthesize infectious RNA in vitro from a DNA copy of the genome of a positive strand RNA virus. It is well known in the field of molecular engineering that DNA, in contrast to RNA, is easily manipulated by site directed mutagenesis. Hence, the availability of the technique to produce synthetic infectious RNA has greatly enhanced the study of e.g. replication, virulence, pathogenesis, RNA recombination, vector development, and antiviral strategies of the positive strand RNA viruses. However, application of the technology may cause severe problems. The nature of these problems has been described in a recent review by Boyer and Haenni. 1994. (Virology 198: 415-426). In fact, the success or failure to construct a full-length DNA copy of the genome of a positive strand RNA virus and to produce synthetic infectious RNA from such a full-length DNA copy cannot be reliably predicted.
SUMMARY OF THE INVENTION
The invention provides nucleotide sequences corresponding to a CSFV genome which comprise at least a part of the nucleotide sequence of the CSFV C-strain depicted in SEQ ID No. 1, or a complement or RNA equivalent of such nucleotide sequence, or mutants thereof. Also provided are degenerate nucleotide sequences having different nucleotides but encoding the same amino acids. The invention also covers potypeptides encoded by these nucleotide sequences, and vaccine strains, the genome of which contains such a nucleotide sequence, in particular a recombinant virus strain based on transcripts of a full-length DNA copy of the genome of the CSFV C-strain.
Partial nucleotide sequences as indicated above are also useful, in particular those which contain a mutation in the structural region of the virus genome, i.e. in the nucleotide sequence encoding amino acids 1-1063 of the sequence depicted in SEQ ID No. 1. The mutation may be a substitution by a corresponding part of the genome of another pestivirus strain, a substitution of one or amino acids, or a deletion. The mutation may also be an inserted or substituted heterologous nucleotide sequence altering the translation strategy of the CSFV nucleotide sequence or altering the processing of a polypeptide encoded by the CSFV nucleotide sequence. Furthermore, the mutation may be an inserted or substituted heterologous nucleotide sequence encoding a polypeptide inducing immunity against another pathogen; in this case the CSFV sequence is used as a vector for heterologous immunogens.
The invention is also concerned with nucleotide sequences of a pestivirus genome in general or a part or a mutant thereof, which sequences contain a mutation in a subregion of the E1 protein, i.e. in the nucleotide sequence encoding amino acids corresponding to amino acids 691-750 or 785-870 of the sequence depicted in SEQ ID No. 1, as well as the polypeptides encoded by these nucleotide sequences. These polypeptides are particularly useful for protecting animals against a pestivirus infection in such a way so as to allow a diagnosis which distinguishes between animals infected with field strains of the pestivirus and vaccinated animals.
The invention is furthermore concerned with vaccines containing a nucleotide sequence, a polypeptide, or a vaccine strain as indicated above, as well as to diagnostic compositions containing a nucleotide sequence or a polypeptide as mentioned above, or an antibody raised against such polypeptide.
The invention also relates to methods and means for diagnosis of pestivirus infections, especially with such means and methods which distinguish between infected animals infected and vaccinated animals.
The invention also provides a method for determining test substances, such as an antibody or an antigen in an immunoassay, by means of a specific binding test, wherein a specifically binding reference substance in immobilised form and the same specifically binding reference substance in labeled form are used.
DETAILED DESCRIPTION OF THE INVENTON
The invention provides the complete cDNA sequence of the RNA genome of the “Chinese” strain (C-strain; EP-A-351901) of CSFV. This allows the construction of a full-length DNA copy of this sequence, of which synthetic RNA can be transcribed that after transfection in suitable cells, such as SK6-M cells (Kasza, L. et al. 1972. Res. Vet. Sci., 13: 46-51; EP-A-351901) gives rise to synthesis of infectious C-strain virus. The use of this finding for the development of modified C-strain vaccines, e.g. vaccines which contain a (serological) marker, is described. Although the invention is illustrated for one CSFV strain, it is also applicable and useful for other pestivirus strains by exchanging specific genomic segments, described below, between the other pestivirus and the CSFV C-strain, or by constructing an “infectious”, DNA copy of the other pestivirus.
The nucleotide sequence of a DNA copy of the genomic RNA of the C-stra

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