Attenuated forms of bovine viral diarrhea virus

Details Attenuated forms of bovine viral diarrhea virus Attenuated forms of bovine viral diarrhea virus

2000-08-29

2002-06-25

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

Inactivation or attenuation; producing viral subunits

C435S235100, C435S320100, C435S325000, C435S455000, C435S456000, C424S093100, C424S093600, C424S204100, C424S204100, C424S218100, C536S023100

Reexamination Certificate

active

06410299

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to methods of producing an attenuated form of bovine viral diarrhea (BVD) virus by inactivating a specific gene in the viral genome. The attenuated virus, or the mutated viral genome, can be used to produce antibody against BVD virus or in vaccines designed to protect cattle from viral infection.
BACKGROUND OF THE INVENTION
Bovine viral diarrhea (BVD) virus is classified in the pestivirus genus and Flaviviridae family. It is closely related to viruses causing border disease in sheep and classical swine fever. Infected cattle exhibit “mucosal disease” which is characterized by elevated temperature, diarrhea, coughing and ulcerations of the alimentary mucosa (Olafson, et al.,
Cornell Vet
. 36:205-213 (1946); Ramsey, et al.,
North Am. Vet
. 34:629-633 (1953)). The BVD virus is capable of crossing the placenta of pregnant cattle and may result in the birth of persistently infected (PI) calves (Malmquist,
J. Am. Vet. Med. Assoc
. 152:763-768 (1968); Ross, et al.,
J. Am. Vet. Med. Assoc
. 188:618-619 (1986)). These calves are immunotolerant to the virus and persistently viremic for the rest of their lives. They provide a source for outbreaks of mucosal disease (Liess, et al.,
Dtsch. Tieraerztl. Wschr
. 81:481-487 (1974)) and are highly predisposed to infection with microorganisms causing diseases such as pneumonia or enteric disease (Barber, et al.,
Vet. Rec
. 117:459-464 (1985)).
BVD viruses are classified as having one of two different biotypes. Those of the “cp” biotype induce a cytopathic effect in cultured cells, whereas viruses of the “ncp” biotype do not (Gillespie, et al.,
Cornell Vet
. 50:73-79 (1960)). In addition, two major genotypes (type I and II) are recognized, both of which have been shown to cause a variety of clinical syndromes (Pellerin, et al.,
Virology
203:260-268 (1994); Ridpath, et al.,
Virology
205:66-74 (1994)).
The genome of the BVD virus is approximately 12.5 kb in length and contains a single open reading frame located between the 5′ and 3′ non-translated regions (NTRs) (Collett, et al.,
Virology
165:191-199 (1988)). A polyprotein of approximately 438 kD is translated from this open reading frame and is processed into viral structural and nonstructural proteins by cellular and viral proteases (Tautz, et al.,
J. Virol
. 71:5415-5422 (1997); Xu, et al.,
J. Virol
. 71:5312-5322 (1997); Elbers, et al.,
J. Virol
. 70:4131-4135 (1996); and Wiskerchen, et al.,
Virology
184:341-350 (1991)). Among the viral enzymes that participate in this processing are the proteases N
pro
and NS3. N
pro
is the first protein encoded by the viral open reading frame and cleaves itself from the rest of the synthesized polyprotein (Stark, et al.,
J. Virol
. 67:7088-7093 (1993); Wiskerchen, et al.,
Virol
. 65:4508-4514 (1991)).
Among the BVD vaccines that are currently available are those in which virus has been chemically inactivated (McClurkin, et al.,
Arch. Virol
. 58:119 (1978); Fernelius, et al.,
Am. J. Vet. Res
. 33:1421-1431 (1972); and Kolar, et al.,
Am. J. Vet. Res
. 33:1415-1420 (1972)). These vaccines have typically required the administration of multiple doses to achieve primary immunization, provide immunity of short duration and do not protect against fetal transmission (Bolin,
Vet. Clin. North Am. Food Anim. Pract
. 11:615-625 (1995)). In sheep, a subunit vaccine based upon a purified E2 protein has been reported (Bruschke, et al.,
Vaccine
15:1940-1945 (1997)). Unfortunately, only one such vaccine appears to protect fetuses from infection and this protection is limited to one strain of homologous virus. There is no correlation between antibody titers and protection from viral infection.
In addition, modified live virus (MLV) vaccines have been produced using BVD virus that has been attenuated by repeated passage in bovine or porcine cells (Coggins, et al.,
Cornell Vet
. 51:539 (1961); and Phillips, et al.,
Am. J. Vet Res
. 36:135 (1975)) or by chemically induced mutations that confer a temperature-sensitive phenotype on the virus (Lobmann, et al.,
Am. J. Vet. Res
. 45:2498 (1984); and Lobmann, et al.,
Am. J. Vet. Res
. 47:557-561 (1986)). A single dose of MLV vaccine has proven sufficient for immunization and the duration of immunity can extend for years in vaccinated cattle (Coria, et al.,
Can. J. Con. Med
. 42:239 (1978)). In addition, cross-protection has been reported from calves vaccinated with MLV-type vaccines (Martin, et al., In
Proceedings of the Conference Res. Workers' Anim. Dis
., 75:183 (1994)). However, safety considerations, such as possible fetal transmission of the virus, have been a major concern with respect to the use of these vaccines (Bolin,
Vet. Clin. North Am. Food Anim. Pract
. 11:615-625 (1995)).
A clear need exists for new and effective vaccines to control the spread of the BVD virus. Given that the disease caused by this virus is one of the most widespread and economically important diseases of cattle, such vaccines would represent a substantial advance in livestock farming.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that attenuated forms of BVD virus can be produced by deleting or inactivating the N
pro
protease gene. These viruses are much less infectious than their wild-type counterparts in bovine cell lines and are suitable for use in vaccines for cattle. A complete genomic sequence of one such attenuated virus is disclosed herein, and a plasmid encoding this virus, i.e., pBVDdN1, has been deposited with the American Type Culture Collection (ATCC) as ATCC No. 203354.
A. Compositions and Methods Based Upon the BVDdN1 Attenuated Virus
In its first aspect, the present invention is based upon the development of a specific attenuated BVD viral strain. The strain is produced by mutating a wild type viral genome to delete the N
pro
protease gene and its full-length sequence is shown in SEQ ID NO:1 and
FIG. 2
, from nt 39 to nt 12116. Thus, the invention is directed to a virus having a genomic sequence comprising that shown, and preferably consisting essentially of that shown. Ordinarily, the BVD virus has a genome in the form of RNA. When cloned, this will more typically be in the form of DNA. Unless otherwise indicated, the term “nucleic acid” refers to both BVD viral DNA and RNA sequences. For convenience, sequence listing entries only show DNA sequences but the corresponding RNA sequence for each will be readily apparent to those of skill in the art. The term “consisting essentially of” refers to sequences that are substantially the same as those specified both in terms of structure and function. Thus, the invention includes not only the sequences expressly depicted, but also corresponding sequences made by introducing insubstantial additions or substitutions. In particular, the invention includes degenerate nucleic acid sequences that encode the same BVD proteins as SEQ ID NO:1. This particular sequence, i.e., SEQ ID NO:1 from nt 39 to nt 12116, and the corresponding virus it encodes have, for convenience, been designated as the “BVDdN1” genome and virus. Virus can be present either as part of a larger preparation or in substantially purified form, i.e., in a form essentially free from any other viral types.
The invention includes host cells carrying a BVDdN1 nucleic acid molecule of the present invention. The term “host cells” is meant to include any prokaryotic cells carrying a BVDdN1 nucleic acid molecule, and any eukaryotic cells infected with the virus or otherwise carrying a BVDdN1 nucleic acid molecule. For prokaryotic cells, the STBL2 strain of
E. coli
(GibcoBRL) has been found to give the best results for propagating the plasmid, and is generally preferred. For eukaryotic cells, mammalian cells such as MDBK cells (ATCC CCL-22) and RD cells (stable transformed bovine testicular cells) are generally preferred. However, other cultured cells can be used as well. The invention further includes progeny virus produced in such host cells.
The BVDdN1 virus can be used to induce the production of a

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