Non-spreading pestivirus Number:7,521,058 from the United States Patent and Trademark Office (PTO) owispatent The invention relates to vaccines used in the eradication or control of pestivirus infections, particularly those used in pigs or ruminants. The invention provides nucleic acid, pestivirus-like particles and a pestivirus vaccine, comprising the nucleic acid or particles, which is capable of eliciting a proper immune response without having the ability to spread throughout the vaccinated animal, thereby avoiding the negative consequences of viral spread. Preferably, the immune response allows for serological discrimination between vaccinated animals and wild-type pestivirus infected animals.<H pestivirus, spreading, Non, spreading, p, 7521058.html, Patent, pto, 7521058

Title: Non-spreading pestivirus

Abstract: The invention relates to vaccines used in the eradication or control of pestivirus infections, particularly those used in pigs or ruminants. The invention provides nucleic acid, pestivirus-like particles and a pestivirus vaccine, comprising the nucleic acid or particles, which is capable of eliciting a proper immune response without having the ability to spread throughout the vaccinated animal, thereby avoiding the negative consequences of viral spread. Preferably, the immune response allows for serological discrimination between vaccinated animals and wild-type pestivirus infected animals.

Patent Number: 7,521,058 Issued on 04/21/2009 to Widjojoatmodjo, et al.

Inventors: Widjojoatmodjo; Myra Noorely (Utrecht, NL), Moormann; Robertus Jacobus Maria (Dronten, NL), van Rijn; Petrus Antonius (Lelystad, NL)
Assignee: ID-Lelystad, Instituut voor Dierhouderij en Diergezondheid B.V. (Lelystad, NL)

Appl. No.: 11/087,067

Filed: March 21, 2005

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09948966	Aug., 2005	6923969
PCT/NL00/00153	Mar., 2000

Foreign Application Priority Data


Mar 08, 1999 [EP]			99200669

Current U.S. Class: 424/218.1 ; 424/199.1; 424/202.1; 435/70.1; 536/23.1

Current International Class: A61K 39/193 (20060101); A61K 39/12 (20060101); C12P 21/04 (20060101); C07H 21/02 (20060101); A61K 39/295 (20060101); A61K 39/275 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


5792462	August 1998	Johnston et al.
6033886	March 2000	Conzelmann
6168942	January 2001	Cao et al.
6296854	October 2001	Pushko et al.
6555346	April 2003	Kretzdorn et al.
6923969	August 2005	Widjojoatmodjo et al.
7179473	February 2007	Meyers

Foreign Patent Documents


0 389 034	Dec., 1990	EP
WO 95/34380	Jun., 1995	WO
WO 96/19498	Jun., 1996	WO
WO 96/25496	Aug., 1996	WO
WO 99/31257	Dec., 1998	WO
WO 97/28487	Jun., 1999	WO
WO 00/53766	Sep., 2000	WO

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Primary Examiner: Campell; Bruce
Assistant Examiner: Blumel; Benjamin P
Attorney, Agent or Firm: TraskBritt

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 09/948,966, filed Sep. 7, 2001, now U.S. Pat. No. 6,923,969, issued Aug. 2, 2005, which is a continuation of International Patent Application No. PCT/NL00/00153 filed on Mar. 8, 2000 designating the United States of America and published in English as PCT International Publication No. WO 00/53766 on Sep. 14, 2000, the contents of the entirety of both of which are incorporated by this reference.

Claims

What is claimed is:

1. A non-spreading pestivirus vaccine produced by a process comprising: obtaining a multitude of pestivirus-like particles produced by a process comprising: providing a cell permissive for pestiviral infection; introducing a first recombinant nucleic acid sequence comprising a pestiviral genomic sequence having at least one functional deletion, wherein the functional deletion is selected from the group consisting of a deletion of amino acids 381 to 422, 405 to 436, 273 to 488, 422 to 488, 436 to 488, 422 to 436, and combinations thereof in E.sup.rns corresponding to amino acids 118 to 161, 144 to 175, 6 to 229, 161 to 229, 175 to 229, 161 to 175, respectively, of SEQ ID NO: 22, and said first recombinant nucleic acid sequence encoding at least one structural protein or an immunodominant portion of the structural protein; introducing into said cell a nucleic acid construct, wherein a second nucleic acid sequence therein complements said at least one functional deletion in said first recombinant nucleic acid sequence; and replicating said first recombinant nucleic acid sequence in said cell, thus producing a pestivirus-like particle having the first replicated recombinant nucleic acid sequence; and preparing a suspension of said multitude of pestivirus-like particles in a suitable diluent.

2. The non-spreading pestivirus vaccine of claim 1, wherein said pestiviral genomic sequence is derived from a pestivirus vaccine.

3. The non-spreading pestivirus vaccine of claim 1, wherein said pestiviral genomic sequence is from a classical swine fever virus.

4. The non-spreading pestivirus vaccine of claim 1, wherein said pestiviral genomic sequence is from a bovine viral diarrhea virus.

5. The non-spreading pestivirus vaccine of claim 1, wherein said pestiviral genomic sequence is from a Border disease virus.

6. The non-spreading pestivirus vaccine of claim 1, wherein said pestiviral genomic sequence is from a C-strain swine fever virus.

7. The non-spreading pestivirus vaccine of claim 1, further comprising combining an adjuvant with the suspension.

8. A non-spreading pestivirus vaccine produced by a process comprising: obtaining a multitude of pestivirus-like particles produced by a process comprising: providing a cell permissive for pestiviral infection; introducing a first recombinant nucleic acid sequence comprising a pestiviral genomic sequence having at least one functional deletion, wherein the functional deletion is selected from the group consisting of a deletion of amino acids 422 to 488, 436 to 488, 422 to 436, and combinations thereof in E.sup.rns corresponding to amino acids 161 to 229, 175 to 229, 161 to 175, respectively, of SEQ ID NO: 22, and the first recombinant nucleic acid sequence encoding at least one structural protein or an immunodominant portion of the structural protein; introducing into the cell a nucleic acid construct, wherein a second nucleic acid sequence therein complements the at least one functional deletion in the first recombinant nucleic acid sequence; and replicating the first recombinant nucleic acid sequence in the cell, thus producing a pestivirus-like particle having the first replicated recombinant nucleic acid sequence; and preparing a suspension of the multitude of pestivirus-like particles in a suitable diluent.

9. A non-spreading pestivirus vaccine produced by a process comprising: obtaining a multitude of pestivirus-like particles produced by a process comprising: providing a cell permissive for pestiviral infection; introducing a first recombinant nucleic acid sequence comprising a pestiviral genomic sequence having at least one functional deletion in a fragment encoding at least a part of one pestiviral structural protein related to viral spread, and said first recombinant nucleic acid sequence encoding at least one structural protein or an immunodominant portion of the structural protein; introducing into said cell a nucleic acid construct, wherein a second nucleic acid sequence therein complements said at least one functional deletion in said first recombinant nucleic acid sequence; and replicating said first recombinant nucleic acid sequence in said cell, thus producing a pestivirus-like particle having the first replicated recombinant nucleic acid sequence; and preparing a suspension of said multitude of pestivirus-like particles in a suitable diluent; wherein the functional deletion is selected from the group consisting of a deletion of amino acids 422 to 488, 436 to 488, 422 to 436, and combinations thereof in E.sup.rns corresponding to amino acids 161 to 229, 175 to 229, 161 to 175, respectively, of SEQ ID NO: 22.

10. The non-spreading pestivirus vaccine of claim 9, wherein said pestiviral genomic sequence is derived from a pestivirus vaccine strain.

11. The non-spreading pestivirus vaccine of claim 9, wherein said pestiviral genomic sequence is derived from a classical swine fever virus.

12. The non-spreading pestivirus vaccine of claim 9, wherein said pestiviral genomic sequence is derived from a bovine viral diarrhea virus.

13. The non-spreading pestivirus vaccine of claim 9, wherein said pestiviral genomic sequence is derived from a Border disease virus.

14. The non-spreading pestivirus vaccine of claim 9, wherein said pestiviral genomic sequence is derived from a C-strain swine fever virus.

15. The non-spreading pestivirus vaccine of claim 9, wherein the second nucleic acid sequence is stably expressed.

16. The non-spreading pestivirus vaccine of claim 9, further comprising combining an adjuvant with said suspension.

17. The non-spreading pestivirus vaccine of claim 9, wherein said process further comprises harvesting said pestivirus-like particle.

18. The pestivirus-like particle of claim 9, wherein said first recombinant nucleic acid sequence encoding at least one pestiviral protein or substantial part thereof related to viral spread is related to viral infectivity.

19. A non-spreading pestivirus vaccine produced by a process comprising: obtaining a multitude of pestivirus-like particles produced by a process comprising: providing a cell permissive for pestiviral infection; introducing a first recombinant nucleic acid sequence comprising a pestiviral genomic sequence having at least one functional deletion, wherein the functional deletion is a cysteine to serine change at position 405 or 381 in E.sup.rns corresponding to a cysteine to serine change at position 144 or 118, respectively, of SEQ ID NO: 22, and the first recombinant nucleic acid sequence encoding at least one structural protein or an immunodominant portion of the structural protein; introducing into the cell a nucleic acid construct, wherein a second nucleic acid sequence therein complements the at least one functional deletion in the first recombinant nucleic acid sequence; and replicating the first recombinant nucleic acid sequence the cell, thus producing a pestivirus-like particle having the first replicated recombinant nucleic acid sequence; and preparing a suspension of the multitude of pestivirus-like particles in a suitable diluent.

20. A non-spreading pestivirus vaccine produced by a process comprising: obtaining a multitude of pestivirus-like particles produced by a process comprising: providing a cell permissive for pestiviral infection; introducing a first recombinant nucleic acid sequence comprising a pestiviral genomic sequence having at least one functional deletion in a fragment encoding at least a part of one pestiviral structural protein related to viral spread, and said first recombinant nucleic acid sequence encoding at least one structural protein or an immunodominant portion of the structural protein; introducing into said cell a nucleic acid construct, wherein a second nucleic acid sequence therein complements said at least one functional deletion in said first recombinant nucleic acid sequence; and replicating said first recombinant nucleic acid sequence in said cell, thus producing a pestivirus-like particle having the first replicated recombinant nucleic acid sequence; and preparing a suspension of said multitude of pestivirus-like particles in a suitable diluent; wherein the at least one functional deletion is a cysteine to seine change at position 405 or 381 in E.sup.rns corresponding to a cysteine to serine change at position 144 or 118, respectively, of SEQ ID NO: 22.

21. The non-spreading pestivirus vaccine of claim 20, wherein said pestiviral genomic sequence is derived from a pestivirus vaccine.

22. The non-spreading pestivirus vaccine of claim 20, wherein said pestiviral genomic sequence is from a classical swine fever virus.

23. The non-spreading pestivirus vaccine of claim 20, wherein said pestiviral genomic sequence is from a bovine diarrhea virus.

24. The non-spreading pestivirus vaccine of claim 20, wherein said pestiviral genomic sequence is from a Border disease virus.

25. The non-spreading pestivirus vaccine of claim 20, wherein said pestiviral genomic sequence is from a C-strain swine fever virus.

26. The non-spreading pestivirus vaccine of claim 20, further comprising combining an adjuvant with the suspension.

27. A method for immunizing an animal against a pestivirus infection, said method comprising vaccinating the animal with the non-spreading pestivirus vaccine of claim 1.

28. The method according to claim 27, further comprising testing the animal for the presence of antibodies specific to a wild-type pestivirus.

29. A method for immunizing an animal against a pestivirus infection, said method comprising: vaccinating the animal with the non-spreading pestivirus vaccine of claim 9.

30. The method according to claim 29 further comprising: testing the animal vaccinated with the vaccine for the presence of antibodies specific for a wild-type pestivirus.

31. A method for immunizing an animal against a pestivirus infection, said method comprising: vaccinating the animal with the non-spreading pestivirus vaccine of claim 20.

32. The method according to claim 31 further comprising testing the animal for the presence of antibodies specific to a wild-type pestivirus.

Description

TECHNICAL FIELD

The invention relates to biotechnology, more specifically to vaccines used in the eradication or control of pestivirus infections, particularly those used in pigs or ruminants.

BACKGROUND

The genus Pestivirus of the family Flaviviridae conventionally consists of classical swine fever virus ("CSFV"), Border disease virus ("BDV"), and bovine viral diarrhea virus ("BVDV"). Genomes of several BVDV, BDV and CSFV strains have been sequenced, individual pestiviral proteins have been expressed and viruses derived from (full-length) DNA copies of the RNA genome of BVDV and CSFV have been generated (Renard et al., 1987, EP application 0208672; Collett et al., 1988, Virology 165, 191-199; Mendez et al., J. Virol. 72:4737-4745, 1988; Deng and Brock, 1992, Virology 1991, 865-679; Meyers et al., 1989, Virology 171, 555-567; Moormann et al., 1990, Virology 177, 184-188; Meyers et al., 1989, EP 89104921; Moormann and Wensvoort, 1989, PCT/NL90/00092; Moormann and Van Rijn; 1994, PCT/NL95/00214; Ridpath et al., 1997, Virus Res. 50:237-243; Becker et al., 1998, J. Virol. 72:5165-5173, Meyers et al., J. Virol. 70:1588-1595, 1996).

The pestivirus genome is a positive-stranded RNA molecule of about 12.5 kilobases containing one large open reading frame ("ORF"). The ORF is translated into a hypothetical polyprotein of approximately 4,000 amino acids, which is processed by virus- and cell-encoded proteases. The ORF is flanked by two conserved nontranslated regions, which are probably involved in the replication of the genome. The 5'-noncoding region also plays a role in initiation of translation.

The polyprotein which is co- and post-translationally processed by cellular and viral proteases contains all the viral structural and nonstructural proteins (for review, see, C. M. Rice: In Fields Virology, Third Edition, 1996 Flaviviridae: The Viruses and their Replication: Chapter 30: pp. 931-959). The viral structural proteins, the capsid protein C and the envelope proteins E.sup.rns, E1 and E2, are located in the N-terminal part of the polyprotein. The nonstructural proteins, including the serine protease NS3 and RNA replicase complex NS5A and NS5B, are located in the C-terminal part of the polyprotein.

Pestiviruses are structurally and antigenically closely related. To date, pestiviruses such as BDV, BVDV and CSFV have been isolated from different species, most notably from ruminants and pigs, but infection of humans has also been reported. All pestiviruses have in common the ability to induce congenital infections of fetuses when a pregnant animal is infected. Such fetal infections occur via transplacental infection if the dam undergoes an acute infection during pregnancy or is persistently infected with a pestivirus (Oirschot, J. T. van, Vet. Microbiol. 4:117-132, 1979; Baker J. C., JAVMA 190:1449-1458, 1987; Nettleton P. F. et al., Comp. Immun. Microbiol. Infect. Dis. 15:179-188, 1992; Wensvoort G. and Terpstra C., Res. Vet. Sci. 45:143-148, 1988).

Currently, modified-live, killed and subunit pestivirus vaccines are available. Live-virus vaccines have the advantage over the other types of vaccines of achieving higher levels of immunity without the need of booster vaccination. However, disadvantages include the ability of vaccinal strains to cross the placenta and induce all know consequences of fetal pestivirus infection (Liess B. et al., Zentralblad Veterinarmed. [B] 31:669-681, 1984). Furthermore, modified-live pestivirus vaccines have been reported to cause immunosuppressive effects, probably due to their ability to spread through the vaccinated animal and replicate for several days in lymphocytes and neutrophils, thereby causing leukopenia and horizontal spread (Roth J. A. and Kaeberle M. L., Am. J. Vet. Res. 44:2366-2372, 1983). Furthermore, epizootics of mucosal disease (a consequence of a persistent BVDV infection) and of acute BVDV infections have been reported after vaccination with live-virus vaccines (Lambert G., JAVMA 163:874-876, 1973).

Thus, despite the fact that live vaccines are generally considered as having the best immunological properties, there are distinct downsides to using a live pestiviral vaccine in the control and eradication of pestivirus infections.

These downsides are related to the fact that a conventional live pestiviral vaccine, after inoculation of the animal with the vaccine, undergoes several rounds of replication and spreads through the vaccinated animal. For one thing, this may result in the above-reported shedding of the virus (horizontal spread), which, after all, is a normal result of any viral infection, whereby an animal is infected with a virus after which the virus replicates, spreads through the body, may replicate again, and eventually is shed from the infected animal to spread to and infect a second, contact, animal.

Even more serious, however, are congenital infections with pestiviruses, causing the so-called vertical spread. Fetuses get infected when the virus spreads through the body of a pregnant animal and the virus crosses the transplacental barrier. Depending on the time of gestation and the virulence of the infecting virus, several effects can be noticed. Severe effects include the death of embryos or fetuses, malformations, mummification, stillbirth or perinatal death (Liess B., Vol. 2 Disease Monographs (E. P. J. Gibss, Editor) Academic Press, London. pp 627-650, 1982). Less virulent virus infections, or infections later in gestation, generally result in the birth of congenitally infected offspring (van Oirschot J. T. in: Classical swine fever and related viral infections, B. Liess (ed) Martinus Nijhoff Publishing Boston pp 1-25, 1988), i.e., calves, lambs, or piglets that are commonly, persistently infected for life often do not thrive well, are prone to immunosuppression and (sub)clinical disease (such as mucosal disease with BVDV (Brownlie, J. Arch. Virol. [Suppl. 3]:73-96, 1991)) and, last but not least, are a continuing source of infection for the rest of the population.

DISCLOSURE OF THE INVENTION

The invention provides nucleic acid, pestivirus-like particles and a modified-live pestivirus vaccine comprising the nucleic acid or particle(s) which is capable of eliciting a proper immune response without having the ability to spread throughout the vaccinated animal, thereby avoiding the negative consequences of viral spread. Preferably, the immune response allows for serological discrimination between vaccinated animals and wild-type pestivirus infected animals.

Viral spread or spread of viral nucleic acid in an inoculated animal could, in theory, also be prevented by inoculating an animal with a pestiviral defective interfering particle (DI) as, for example, known from Meyers et al. (J. Virol. 70:1588-1595, 1996) or Kupfermann et al. (J. Virol. 70:8175-8181, 1996) if one could obtain the DI particles free from the helper pestivirus required for their replication, which is, for all practical purposes, near impossible. Inoculating the animal with a DI preparation containing the helper virus as well would defeat all the purposes; the helper virus would spread throughout the animal, thereby subjecting it to the undesired pestivirus infection, allowing horizontal and vertical transmission. Serological discrimination is thus also not possible, since antibodies directed against the helper virus would be detected.

However, even if one succeeded in obtaining the DI particles free of the helper virus, it still would amount to nothing; the pestiviral DI particles contain no nucleic acid that allow it to elicit a proper immune response, since the DI nucleic acid essentially does not contain the nucleic acid encoding for structural proteins or immunodominant parts thereof that are responsible for the proper immune response.

In a first embodiment, the invention provides a recombinant nucleic acid derived from a pestivirus from which nucleic acid, a fragment encoding at least one pestiviral protein or substantial part thereof related to viral spread is functionally deleted, the nucleic acid allowing for RNA replication in a suitable cell and encoding at least one functional structural protein or at least one immunodominant part thereof.

"Functionally deleted" herein comprises any insertion, modification or deletion of the viral genome that results in the production (via transcription and translation of the nucleic acid in a cell, preferably a cell suitable for the transcription and translation of the nucleic acid, preferably a cell in an animal to be vaccinated) of an at least functionally inactivated viral protein or fragment thereof that in its wild-type state is involved in viral spread, or at least in transmission to, or viral infection of, cells. Because of the inactivated protein, even when incorporated into the viral particle comprising the nucleic acid, the functional deletion has disabled the particle to enter or infect a cell which normally, had that protein or functional fragment been functioning properly in the particle, would be infected by the particle. In this way, although the particle may yet still be formed, the particle is no longer infectious for other cells and can, thus, no longer contribute via the route of infection to the spread or transmission of the particle to another cell, notwithstanding the fact that a cell, once infected, may fuse and/or divide, thereby generating multiple cells comprising the particle.

The nucleic acid provided by the invention allows for RNA replication in a suitable cell and encodes at least one functional protective protein or at least one immunodominant part thereof. In a preferred embodiment of the invention, the protective protein is a structural protein, in general, and the immunodominant parts of structural envelope proteins mount the best immune response of the pestiviral proteins. However, some non-structural proteins, such as NS3, are also capable of mounting a sufficient immune response for some purposes and can, therefore, also be included. Thus, although spread-through infection has now been prevented, the fact that RNA replication is possible allows for one or more rounds of transcription and translation in the cell of immunologically dominant proteins against which a vaccinated animal mounts an immune response through which it is at least partly protected against the consequences of infection with a wild-type pestivirus. The translated protein(s) or fragment(s) thereof in themselves (is) are responsible for the immune response and may also become part of a virus-like particle, even comprising the replicate RNA, but the particle is not infectious due to the fact that one essential functional feature of the functionally deleted protein is missing.

Although in one embodiment the nucleic acid as provided by the invention may comprise DNA, as to allow for DNA vaccination, in another embodiment, the invention provides nucleic acid wherein the nucleic acid is RNA to allow for RNA vaccination. Such RNA is, for example, packaged into a virus-like particle in a complementing cell, as provided by the invention, provided with a functional protein or fragment (derived from the complementing cell) responsible for virus-cell interactions allowing the particle to enter or infect a suitable cell, or may be introduced into the animal's cells otherwise, such as via the intradermal route.

In a preferred embodiment, the invention provides a nucleic acid wherein the functional deletion is in a fragment encoding an envelope protein. Essential to infection with pestiviruses is the interaction of viral structural proteins with the surface or a receptor of the susceptible cell. It is through this interaction that the infection takes place. Especially envelope proteins E2 and/or E.sup.rns provide for this interaction, and functionally deleting at least one of these envelope proteins or functional fragments thereof (in particular those fragments involved in receptor or surface interaction) leads to obstruction of infectivity.

Several examples of such functional deletions in a nucleic acid-encoding protein related to viral spread are given in the detailed description of the invention. An example comprises a modification of a cysteine-encoding nucleic acid codon, whereby a conformational change is induced in a fragment of a pestiviral protein, preferably an envelope protein, in such a way that the functionally deleted protein, when incorporated in the particle, has disabled the particle to enter an otherwise accessible cell. One example is the modification of a codon resulting in a cysteine change, for example, at amino acid position 422, or, for that matter, at position 381, of the amino acid sequence of the E.sup.rns protein of CSFV, or at functionally corresponding locations in the E.sup.rns protein of CSFV or other pestiviruses, which, for example, obtained by sequence comparison, are also provided by the invention. Another example comprises deleting larger fragments of a nucleic acid encoding a pestiviral protein, for example, by deleting at least a fragment encoding approximately corresponding positions 170-268 or other functionally related fragments of the capsid proteins C of CSFV or other pestiviruses or by deleting at least a fragment encoding approximately corresponding positions 500-665 or other functionally related fragments of the E1 proteins of CSFV or other pestiviruses, or comprises deleting fragments encoding, etc. Another example comprises deleting larger fragments of a nucleic acid encoding a pestiviral protein, for example, by deleting at least a fragment encoding corresponding positions 381, 422, 381-422, 405-436, 422-436, 422-488 or 273-488 or other functionally related fragments of the E.sup.rns protein of CSFV or other pestiviruses, or comprises deleting fragments encoding corresponding positions 698-1008 or 689-1062 in the E2 protein of CSFV or other functionally related fragments of the E2 protein of CSFV or other pestiviruses.

In a much preferred embodiment, the invention provides a nucleic acid wherein the functional deletion comprises an immunodominant part of the protein. For example, deleting a fragment corresponding to about amino acid positions 422-436 or 422-488 of the E.sup.rns protein, or corresponding to about amino acid positions 693-746, 785-870, 689-870 or 800-864 of the E2 protein or any other fragment related to a discernible immune response against the protein has the additional advantage that a discernible vaccine is provided.

By deleting the serologically discernible fragment, in the end, a marker vaccine is obtained that allows for serological discrimination between vaccinated animals and animals infected with a wild-type pestivirus.

In constructing a vaccine, one has to take into account what (type of) serological test is preferred once the vaccine is employed in the field. For CSFV, it preferably should be genotype specific, which blocks using diagnostic tests based on NS3. However, selecting E2 or E.sup.rns as diagnostic antigens hampers developing a vaccine which uses the protective properties of these proteins. The invention surprisingly provides a pestivirus vaccine in which a protein, preferably an envelope protein comprising a specific immunodominant part, in general, thought responsible for generating protection, has been (functionally) deleted, allowing serological discrimination surprisingly without seriously hampering protective properties.

The protective properties are optimally provided by a nucleic acid according to the invention having a fragment encoding a protective protein that is a functional structural protein, more preferably a functional envelope protein or at least one immunodominant part thereof. Most preferred by the invention is a nucleic acid comprising a fragment encoding a functional deletion in one pestiviral envelope protein, for example, E2 or E.sup.rns, respectively, and further comprising a nucleic acid encoding another protective envelope protein, or immunodominant part thereof, for example, E.sup.rns or E2 or part thereof, respectively.

In a further embodiment, the invention provides a nucleic acid additionally comprising a non-pestivirus nucleic acid fragment, thereby providing a nucleic acid encoding heterologous protein or fragments thereof. Heterologous protein (fragments) may be used as a marker or may be used to elicit a (protective) immune response. Marker sequences are preferably highly antigenic and, in one embodiment of the invention, preferably derived from a (micro)organism not replicating in animals. They may encode known complete gene products (e.g., capsid or envelope proteins or antigenic parts of gene products (e.g., epitopes). Marker sequences may also encode artificial antigens not normally encountered in nature, or histochemical markers like Escherichia coli .beta.-galactosidase, Drosophila alcohol dehydrogenase, human placental alkaline phosphatase, firefly luciferase and chloramphenicol acetyltransferase. Also provided is a nucleic acid wherein the non-pestivirus fragment is derived from a pathogen encoding one or more protein (fragments) inducing protective immunity against disease caused by the pathogen, such as a fragment derived from parvovirus, coronavirus, porcine respiratory and reproductive syndrome virus, herpesvirus, influenza virus, and numerous other pathogens known in the art. Also provided is a nucleic acid wherein the non-pestivirus fragment is derived from a cytokine that induces immuno-regulating or -stimulating signals when expressed. Numerous cytokines are known in the art, such as interleukines, interferons and tumor necrosis or colony-stimulating factors.

The invention further provides a nucleic acid according to the invention wherein the suitable cell comprises a nucleic acid construct encoding at least the pestiviral protein or substantial part thereof related to viral spread. Such a suitable cell, which is also provided by the invention, comprises a cell comprising a recombinant nucleic acid encoding at least one pestiviral protein or substantial part thereof related to viral spread and allows packaging the pestiviral protein or substantial part thereof in a pestivirus-like particle. Such a packaging or complementing cell, according to the invention, allows nucleic acid or replicate nucleic acid according to the invention to be part of a pestivirus-like particle, wherein a substantial part of the protein (fragments) composing the particle is derived from translation and transcription in the cell of nucleic acid according to the invention, being complemented with a protein (fragment) related to viral spread derived from the nucleic acid construct that is also expressed in the complementing cell. Such a protein (fragment) can be transiently expressed from a nucleic acid construct or can be expressed from a helper virus, but preferred is a cell according to the invention wherein the pestiviral protein or substantial part thereof related to viral spread is stably, either inducibly or constitutively, expressed from, for example, a self-replicating nucleic acid or from the cellular genome integrated nucleic acid.

The invention also provides a method for obtaining a pestivirus-like particle comprising transfecting such a cell according to the invention with a nucleic acid according to the invention, further comprising allowing the nucleic acid to replicate in the cell, further comprising allowing replicated nucleic acid to be part of a particle comprising at least the pestiviral protein or part thereof derived from the cell, and further comprising harvesting the particle. Such a use of a nucleic acid according to the invention, or a cell according to the invention, in producing a pestivirus-like particle is provided by the invention. By transfecting the cell with nucleic acid according to the invention and allowing the nucleic acid to replicate, a replicated RNA of the nucleic acid is packaged in the pestivirus-like particle, the particle also comprising a functional protein or set of proteins related to viral spread, at least partly derived from the nucleic acid construct with which the complementing or packaging cell has also been provided. Likewise, the invention provides a pestivirus-like particle obtainable by a method according to the invention; for example, the invention provides a pestivirus-like particle (or a multitude of such particles) comprising nucleic acid derived from a pestivirus, from which nucleic acid, a fragment encoding at least one pestiviral protein, or substantial part thereof related to viral spread, is functionally deleted, the nucleic acid allowing for RNA replication in a suitable cell and encoding at least one functional structural protein or at least one immunodominant part of an immunodominant structural protein. The particle may be derived from one type of pestivirus but can also be a so-called hybrid particle, wherein its genome and part of its constituting protein (fragments) is (are) derived from one type pestivirus, such as BVDV or BDV, but wherein complementing protein (fragments) are derived from another type pestivirus, such as CSFV. The particle, having been produced in the packaging or complementing cell as provided, is itself infectious and, thus, capable of entering a suitable second cell, such as a non-complementing cell capable of being infected with a pestivirus type in general, or such as a susceptible cell in an animal to be vaccinated. When replicating in the second cell (which is non-complementing) a new particle is produced that, however, lacks the possibilities to infect yet another cell and is, thus, unable to spread by infection. Thus, when the particles produced in a complementing cell are used to infect an animal, such as when used in or as a vaccine, the particles will infect suitable cells in the vaccinated animal, from which, however, no new particles that spread by infection to other cells are generated, thereby demonstrating the requirements of a non-spreading (non-transmissible) vaccine.

The invention also provides a method for obtaining a non-spreading pestivirus vaccine comprising obtaining a multitude of particles by a method according to the invention and preparing a suspension of the particles in a suitable diluent. Suitable diluents are known in the art and preferably on a watery basis, such as a (buffered) salt solution or (growth) medium. The invention also provides a method for obtaining a non-spreading pestivirus vaccine comprising obtaining a multitude of particles by a method according to the invention and preparing a suspension of the particles in a method comprising combining the suspension with an adjuvant. Suitable adjuvants are water-oil emulsions, aluminum salts or other adjuvants known in the art, see, for example, Vogel F. R. and Powell M. F, A compendium of adjuvants and excipients. In: Vaccine design. (eds) Powell and Newmann, Pharmaceutical Biotechnology Series, Plenum, New York, 1994. The invention thus provides a non-spreading pestivirus vaccine obtainable by a method according to the invention. The invention provides such a vaccine comprising a nucleic acid according to the invention or a pestivirus-like particle according to the invention, for example, further comprising an adjuvant. Optimal efficacy of the vaccine is achieved if its nucleic acid is targeted to suitable antigen presenting cells. Replication and translation of the viral RNA in these cells will result in processing of viral antigen for optimal presentation to the immune system.

In one embodiment of the invention, the vaccine consists of pestivirus-like particles produced in a complementing packaging cell which, with or without an adjuvant, are applied to the animal via different routes such as, but not limited to, intranasal, intramuscular, intradermal or intravenous vaccination, or a combination of routes. In another embodiment of the invention, the vaccine consists of essentially naked DNA or RNA according to the invention which, with or without an adjuvant, is preferably applied to the animal via the intradermal route. However, alternative routes such as, but not limited to, the intramuscular route are suitable.

Such a vaccine is provided wherein the nucleic acid is derived from any pestivirus (vaccine) strain from which (full-length) cDNA and infectious copies thereof are, or can be, provided, such as C-strain virus or from another pestivirus such as another vaccine-type or wild-type of a classical swine fever virus, a bovine viral diarrhea virus or a Border disease virus, or chimeric virus. For simplicity's sake, the numbering of the C-strain sequence is used herein for all pestivirus sequences. In fact, in other pestivirus sequences, the numbering of the E.sup.rns and the E2 proteins in the (poly)protein may differ slightly due to length differences in the (poly)protein sequences of pestivirus strains. Based on homology, the N and C termini of the E2 or E.sup.rns sequence of any pestivirus strain can, however, easily be determined, such as shown by Rumenapf T. et al., J. Virol., 67:3288-3294, 1993, or Elbers K. et al., J. Virol. 70:4131-4135, 1996.

The invention also provides a method for controlling and/or eradicating a pestivirus infection comprising vaccinating at least one animal with a vaccine according to the invention. The vaccination serves to prevent or mitigate a wild-type pestivirus infection which the animal may have been or may be confronted with due to the presence of wild-type virus in its surroundings. Since no spread or shedding of the vaccine occurs, the vaccinated animal can be safely vaccinated, even when pregnant; no risk of congenital vaccinal infections of its fetus, or shedding of the vaccine from the vaccinated to a non-vaccinated animal, is present due to the non-spreading nature of the vaccine.

Additionally, the invention provides a method for controlling and/or eradicating a pestivirus infection comprising testing an animal vaccinated with a vaccine according to the invention for the presence of antibodies specific for a wild-type pestivirus. In a preferred embodiment, the method for controlling such a vaccine is used as a marker vaccine. The use of such a marker vaccine as provided by the invention allows serological discrimination between vaccinated and field-virus infected animals, and thereby a controlled elimination of the virus. For serological discrimination of pestivirus genotypes, it does not suffice to provide protection with a vaccine comprising the protective proteins E2 or E.sup.rns but not the NS3 protein and detecting infections with tests based on NS3. NS3 is not genotype-specific; at least it does not elicit genotype-specific antibodies to allow discrimination between genotypes. Although no objection can be seen at using the tests to diagnose BVDV or BDV infections, such diagnostic tests can, thus, hardly be used in the aftermath of vaccination campaigns against, for example, CSFV in pigs. Circulating NS3-BDV or NS3-BVDV antibodies will cause a plethora of false-positive results, leading to suspicions of CSFV infections when, in fact, there aren't any in the pig population tested. Preferably, tests are used to detect antibodies against E2, or serologically discernible fragments thereof, when the functional deletion is in the nucleic acid fragment encoding the E2 protein and the protective protein mainly comprises E.sup.rns protective protein or fragments thereof, optionally supplemented with other protective protein (fragments), or vice versa; tests are used to detect antibodies against E.sup.rns, or serologically discernible fragments thereof, when the functional deletion is in the nucleic acid fragment encoding the E.sup.rns protein and the protective protein mainly comprises E2 protein or fragments thereof, optionally supplemented with other protective protein (fragments).

The invention also provides an animal vaccinated with a non-spreading (non-transmissible) pestivirus vaccine according to the invention. Since such an animal bears no risk of spreading the vaccine to contact animals, or to its fetus(es), such an animal has considerable advantages over animals vaccinated with conventional pestivirus vaccines. It can, for example, be traded during that period shortly after vaccination where trade otherwise has to be restricted due to the risk of shedding.

The invention is further explained in detail below without limiting the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic representation of E.sup.rns of CSFV strain C (top) and overview of E.sup.rns plasmids (bottom). Domains of RNase activity are shown by closed bars. Positions of cysteines are indicated with black dots. .sup.a Positions of the deletions or point mutations with respect to the amino acid sequence of the open reading frame (ORF) of CSFV strain C (Moormann et al., J. Virol. 1996, 70:763-770). .sup.b Cysteine-to-serine mutations are depicted with white dots. .sup.c The recombinant E.sup.rns in these plasmids do not harbor a C-terminal HA tag. NA: not available.

FIG. 2 Schematic representation of the construction of the full-length DNA copies pPRKflc23 (A) and pPRKflc22 (B) harboring E.sup.rns deletions: The amino acid sequence numbering is of the open reading frame (ORF) of the CSFV strain C (Moormann et al., 1996, J. Virol., 70:763-770). PCR primers are indicated with solid lines and designated: p(number); N.sup.pro, autoprotease; C, core protein; E.sup.rns, E1 and E2 envelope proteins; 5', 5' non-coding region; 3', 3' non-coding region, Amp, ampicillin resistance gene; CIP, calf intestinal phosphatase; Kan, kanamycin resistance gene; ORF: open reading frame; PNK, polynucleotide kinase; PhCMV, promoter-enhancer sequence of the immediate early gene of human cytomegalovirus; T7, bacteriophage T7 promotor. pPRKflc2 is the wild-type full-length cDNA copy of the CSFV strain C. (B) Plasmid pPRKc129 was the template for the first PCR of E.sup.rns. The NarI site of this PCR product and the ClaI site of the hemagglutinin (HA) epitope have compatible ends. These two PCR fragments were inserted into the BglII/SalI digested vector pPRKc16 via a three-point ligation. See text for detailed information on the construction of the full-length DNA copies and the primer sequences.

FIG. 3 Characterization of recombinant viruses. .sup.a Positions of cysteines are indicated with black dots, cysteine-to-serine mutations are depicted with white dots. .sup.b Positions of the deletions or mutations with respect to the amino acid sequence of CSFV strain C (Moormann et al., J. Virol. 1996, 70:763-770). .sup.c Supernatants from SK6c26-infected cells were used for infection of SK6 and SK6c26 cells. The cells were immunostained with E.sup.rns-specific antibodies (R716 and C5) or an E2-specific Mab (b3) and were scored as positive (+) or negative (-). .sup.d Viruses are considered to be infectious viruses if supernatant of the infected cells can infect SK6 or SK6c26 cells. Spread of virus via cell-to-cell spread and spread of virus due to division of cells is not considered as infectious virus.

FIG. 4 RT-PCR of SK6c26 cells infected with Flc22, Flc23 and Flc2 with primers flanking E.sup.rns and E2. (-) Negative control: mock infected SK6c26 cells; M: 200 bp marker.

FIG. 5 Growth kinetics of the recombinant CSFV viruses Flc22, Flc23 and the wild-type virus Flc2. Subconfluent monolayers of SK6c26 cells were infected at a multiplicity of 0.1. Viruses were adsorbed for 1.5 hr. Virus titers of the cell lysates and supernatant at various time points were determined by end point dilution on SK6c26 cells.

FIG. 6 E.sup.rns amino acid sequence of the recombinant Flc22, Flc23 and the wild-type strain Flc2, |: indicates position of deletion.

FIG. 7 Schematic representation of pPRKflc23 harboring the HA epitope, a non-pestivirus sequence. The depicted sequence (SEQ ID NO:25) shows that the HA epitope is flanked by the 5 utmost N-terminal amino acids and 6 utmost C-terminal amino acids of E.sup.rns.

FIG. 8 Schematic representation of the construction of the full-length DNA copies pPRKflc4 (A) and pPRKflc47 (B). The amino acid sequence numbering is of the open reading frame (ORF) of the CSFV strain C (Moormann et al., 1996, J. Virol.). PCR primers are indicated with solid lines and designated: p(number); N.sup.pro, autoprotease; C, core protein; E.sup.rns, E1 and E2 envelope proteins; p7: p7 protein, NS, nonstructural protein, 5', 5' non-coding region; 3', 3' non-coding region, Amp, ampicillin resistance; Kan, kanamycin resistance. pPRKflc2 is the wild-type full-length DNA copy of the CSFV strain C. (A) The E2 gene of plasmid pPAB16 was inserted in plasmid pPRKc129 by NgoMI/BglII digestion. (B) pPRKflc2 was the template for PCR amplification with primers p1195 and p403. See text for detailed information on the construction of the full-length DNA copies and the primer sequences.

EXAMPLES

Example 1

Construction and Characterization of Recombinant CSFV Strains Flc22, Flc23, Flc30, Flc31, Flc32, and Flc33

Materials and Methods

Cells & Viruses

Swine kidney cells (SK6-M, EP 0 351 901 B1) were grown in Eagle's basal medium containing 5% fetal bovine serum, glutamine (0.3 mg/ml), and the antibiotics penicillin (200 U/ml), streptomycin (0.2 mg/ml), and mycostatin (100 U/ml). Fetal bovine serum was tested for the absence of BVDV and BDV antibodies as described previously (Moormann et al., 1990, Virology 177:184-198).

Recombinant CSFV strain C viruses Flc22, Flc23, FLc30, Flc31, Flc32, and Flc33 were grown and prepared as described earlier (Moormann et al., 1996, J. Virol. 70:763-770) with a slight modification, the growth medium of SK6 cells was changed in supplemented Eagle's basal medium. Virus stocks were prepared by passaging the virus eight to ten times on SK6c26 cells. The obtained virus titers ranged from 5.0 to 5.8 TCID50/ml.

Construction of a Stable SK6 Cell Line Expressing E.sup.rns

Plasmid pPRKc16 contains the E2 gene of CSFV strain C under control of the transcription and translation signals of expression vector pEVhisD12. Plasmid pEVhisD12 is a vector that contains promoter/enhancer sequences of the immediate early gene of the human cytomegalovirus followed by a translation initiation codon and the histidinol dehydrogenase gene (hisD) under control of the SV40 early promoter, which can be used as a selective marker (Peeters et al., 1992, J. Virol. 66:894-905). The E.sup.rns gene of the CSFV strain C was amplified by PCR reaction with primers p974 5' AAG AAA AGA TCT AAA GCC CTA TTG GCA TGG 3' (SEQ ID NO:1) and p976 5' TT GTT ACA GCT GCA TAT GTA CCC TAT TTT GCT TG 3' (SEQ ID NO:2). After BglII digestion, the PCR fragment was ligated into the vector pPRKc16, which was digested with SalI, filled in, and subsequently digested with BglII. The resulting plasmid pPRKc26 contains the E.sup.rns gene of the CSFV strain C.

For transfection of SK6 cells with pPRKc26, lipofectamine (20 .mu.g) (Gibco-BRL) was diluted in 50 .mu.l of Optimem-I (Gibco-BRL) mixed with plasmid DNA (1 .mu.g) diluted in 50 .mu.l Optimem-I (Gibco-BRL) and this mixture was allowed to settle for 15 minutes at room temperature. SK6 cells grown in 10 cm.sup.2 tissue culture plates were washed with Optimem-I. Fresh Optimem-I was added (0.5 ml), followed by the DNA transfection mixture. After 4 hours of incubation at 37.degree. C., the transfection mixture was removed and the wells were supplied with medium containing 5 mM histidinol. After 24 hours of incubation at 37.degree. C., cells were trypsinized and plated on a 90 mm.sup.2 plate. Medium was replaced every 3-4 days. After 15 days, single colonies were picked and plated into 2 cm.sup.2 plates. Expression of E.sup.rns was determined by immunostaining of the cells with Mabs C5 (Wensvoort 1989, Thesis, University of Utrecht) directed against E.sup.rns of CSFV strain C. A second round of cloning was performed by trypsinizing and plating the cells in ten-fold dilution in microtiter plates in medium containing 5 mM histidinol. Wells with individual colonies were trypsinized and expression of E.sup.rns was determined by immunostaining (Wensvoort et al., 1988, Vet. Microbiol. 17, 129-140) the cells with Mab C5. The established SK6 cell line constitutively expressing E.sup.rns was named SK6c26.

Characterization of the Stable Cell Line SK6c26

E.sup.rns expression of the cell line SK6c26 line was tested in an immunoperoxidase staining with E.sup.rns-specific monoclonal antibodies (Mabs) C5, specific for E.sup.rns of strain C (Wensvoort 1989, Thesis, University of Utrecht), 140.1 and 137.5 directed against E.sup.rns of CSFV strains C and Brescia (de Smit et al., unpublished data), and a polyclonal rabbit serum, R716 (Hulst et al., J. Virol. 1998, 72:151-157). The RNase activity of the E.sup.rns expressed in the SK6c26 cell line was measured by a modification of the method of Brown and Ho (Plant Physiol. 1986, 82:801-806) as described by Hulst et al., (J. Virol. 1998, 72:151-157). The amount of E.sup.rns was determined by an indirect ELISA based on Mab C5 as coating antibody and horseradish peroxidase conjugated Mab 140.1 as detection antibody as described by Hulst et al. (J. Virol. 1998, 72:151-157).

Construction of Recombinant CSFV E.sup.rns

pPRKc5 (Hulst et al., Virol. 1998, 72:151-157) is a pEVhisD12 derivative which contains the nucleotide sequence of the autoprotease and structural genes of CSFV strain C, without E.sup.rns (Npro-C and E1-E2, amino acids (a.a.) 5-267 and 495-1063 of the amino acid sequence of CSFV strain C) (Moormann et al., 1996, J. Virol. 70:763-770). A unique StuI site was introduced in pPRKc5 at the position where E.sup.rns was deleted.

Two complementary oligomers, the forward oligomer p1135 (5' CCG AAA ATA TAA CTC AAT GGT TTG GCG CTT ATG 3' (SEQ ID NO:3)) and the reverse oligomer p1136 (5' CAT AAG CGC CAA ACC ATT GAG TTA TAT TTT CGG 3' (SEQ ID NO:4)) were phosphorylated with T4 DNA kinase, hybridized and inserted via ligation in an alkaline phosphatase-treated StuI-digested vector pPRKc5. This construct was named pPRKc48. This construct harbors the five utmost N-terminal amino acids and the six utmost C-terminal amino acids of E.sup.rns (deletion a.a 273-488) (FIG. 1 and FIG. 2A).

A deletion of amino acids 422 to 488 in E.sup.rns of strain C was accomplished by PCR amplification of the E.sup.rns gene using the forward primer p974 and reverse primer p1120 5' GAC GGA TTC GGC ATA GGC GCC AAA CCA TGG GCT CTC TAT AAC TGT AAC 3' (SEQ ID NO:5). The HA epitope, amino acid sequence YPYDVPDYA (SEQ ID NO:6) (Wilson et al., Cell 1984, 37, 767-778), was constructed by annealing the 3' complementary nucleotides of p1124 5' GAC AGA TCT ATC GAT TAC CCA TAC GAT GTT CCA GAT 3' (SEQ ID NO:7) and p1125 5' GAC GTC GAC GGA TCC AGC GTA ATC TGG AAC ATC 3' (SEQ ID NO:8) (underlined is the HA sequence) and filling in the 5' single strand nucleotides in a PCR with Vent polymerase (New England Biolabs). The HA epitope PCR product was digested with ClaI/SalI, and the E.sup.rns PCR product was digested with BglII/NarI. The two digested PCR products were ligated via a three-point ligation into the vector pPRKc16 which was digested with BglII and SalI. This resulted in plasmid pPRKc43 containing a recombinant E.sup.rns with a deletion of amino acids 422 to 488 with C terminally an HA epitope (FIG. 1 and FIG. 2B). After PCR amplification of plasmid pPRKc43 with the forward primer p935 (5' CCG AAA ATA TAA CTC AAT GG 3' (SEQ ID NO:9)) and the reverse primer p925 (5' CAT AAG CGC CAA ACC AGG TT 3' (SEQ ID NO:10)), the PCR product was phosphorylated with T4 DNA kinase and subsequently ligated into the alkaline phosphatase treated StuI digested vector pPRKc5. The resulting construct harboring the nucleotide sequence of the autoprotease, the structural proteins of strain C and the recombinant E.sup.rns lacking amino acids 422 to 488 was named pPRKc50 (FIG. 2B).

A deletion mutant lacking amino acids 436 to 488 of E.sup.rns of strain C was accomplished by