Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1999-04-30
2003-07-22
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S204100, C536S023720, C435S320100, C435S325000, C435S235100
Reexamination Certificate
active
06596280
ABSTRACT:
BACKGROUND OF THE INVENTION
Infectious bursal disease virus (IBDV), a member of the Bimaviridae family, is the causative agent of a highly immunosuppressive disease in young chickens (Kibenge, F. S. B., et al.,
J. Gen. Virol
., 69, 1757-1775 (1988)). Infectious bursal disease (IBD) or Gumboro disease is characterized by the destruction of lymphoid follicles in the bursa of Fabricius. In a fully susceptible chicken flock of 3-6 weeks of age the clinical disease causes severe immunosuppression, and is responsible for losses due to impaired growth, decreased feed efficiency, and death. Susceptible chickens less than 3 weeks old do not exhibit outward clinical signs of the disease but have a marked infection characterized by gross lesions of the bursa.
The virus associated with the symptoms of the disease is called infectious bursal disease virus (IBDV). IBDV is a pathogen of major economic importance to the nation and world's poultry industries. It causes severe immunodeficiency in young chickens by destruction of precursors of antibody-production B cells in the bursa of Fabricius. Immunosuppression causes increased susceptibility to other diseases, and interferes with effective vaccination against Newcastle disease, Marek's disease and infectious bronchitis disease viruses.
There are two known serotypes of IBDV. Serotype I viruses are pathogenic to chickens whereas serotype II viruses infect chickens and turkeys. The infection of turkeys is presently of unknown clinical significance.
IBDV belongs to a group of viruses called Bimaviridae which includes other bisegmented RNA viruses such as infectious pancreatic necrosis virus (fish), tellina virus and oyster virus (bivalve mollusks) and drosophila X virus (fruit fly). These viruses all contain high molecular weight (MW) double-stranded RNA genomes.
The capsid of the IBDV virion consists of several structural proteins. As many as nine structural proteins have been reported but there is evidence that some of these may have a precursor-product relationship (Kibenge, F. S. B., et al.,
J. Gen. Virol
., 69, 1757-1775 (1988)). The designation and molecular weights of the viral proteins (VP) are as shown below.
Viral Protein
Molecular Weight
VP1
90 kDa
VP2
41 kDa
VP3
32 kDa
VP4
25 kDa
VP5
17 kDa
Two segments of double-stranded RNA were identified in the genome of IBDV. The IBDV genome consists of two segments of double-stranded (ds)RNA that vary between 2827 (segment B) to 3261 (segment A) nucleotide base pairs (Mundt, E. et al.,
Virology
, 209, 10-18 (1995)). The larger segment A encodes a polyprotein which is cleaved by autoproteolysis to form mature viral proteins VP2, VP3 and VP4 (Hudson, P. J. et al.,
Nucleic Acids Res
., 14, 5001-5012 (1986)). VP2 and VP3 are the major structural proteins of the virion. VP2 is the major host-protective immunogen of IBDV, and contains the antigenic regions responsible for the induction of neutralizing antibodies (Azad, et al.,
Virology
, 161, 145-152 (1987)). A second open reading frame (ORF), preceding and partially overlapping the polyprotein gene, encodes a, protein (VP5) of unknown function that is present in IBDV-infected cells (Mundt, E., et al.,
J Gen. Virol
., 76, 437-443, (1995)). The smaller segment B encodes VP1, a 90-kDa multifunctional protein with polymerase and capping enzyme activities (Spies, U., et al.,
Virus Res
., 8, 127-140 (1987); Spies, U., et al.,
J. Gen. Virol
., 71, 977-981 (1990)).
It has been demonstrated that the VP2 protein is the major host protective immunogen of IBDV, and that it contains the antigenic region responsible for the induction of neutralizing antibodies. The region containing the neutralization site has been shown to be highly conformation-dependent. The VP3 protein has been considered to be a group-specific antigen because it is recognized by monoclonal antibodies directed against it from strains of both serotype I and II viruses. The VP4 protein appears to be a virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins.
Although the nucleotide sequences for genome segments A and B of various IBDV strains have been published, it was only recently that the complete 5′- and 3′-noncoding sequences of both segments were determined. The 5′-noncoding region of IBDV segments A and B contain a consensus sequence of 32 nucleotides, whereas the 3′-noncoding terminal sequences of both segments are unrelated, but conserved among IBDV strains of the same serotype (Mundt, E. et al.,
Virology
, 209, 10-18 (1995)). These terminii might contain sequences important in packaging and in the regulation of IBDV gene expression, as demonstrated for other dsRNA containing viruses such as mammalian and plant reoviruses, and rotaviruses (Anzola, et al.,
Proc. Natl. Acad. Sci. USA
, 84, 8301-8305 (1987); Zou, S., et al.,
Virology
, 186, 377-388 (1992); Gorziglia, M. I., et al.,
Proc. Natl. Acad. Sci. USA
, 89, 5784-5788 (1992)).
In recent years, a number of infectious animal RNA viruses have been generated from cloned cDNA using transcripts produced by DNA-dependent RNA polymerase (Boyer, J. C., et al.,
Virology
, 198, 415-426 (1994)). For example poliovirus, a plus-stranded RNA virus; influenza virus, a segmented negative-stranded RNA virus; rabies virus, a non-segmented negative-stranded RNA virus; all were recovered from cloned cDNAs of their respective genomes (van der Werf, S., et al.,
Proc. Natl. Acad. Sci. USA
, 83, 2330-2334 (1986); Enami, M., et al.,
Proc. Natl. Acad. Sci. USA
, 87, 3802-3805 (1990); Schnell, M. J., et al.,
EMBO J
., 13, 41954205 (1994)). For reovirus, it was shown that transfection of cells with a combination of SSRNA, dsRNA and in vitro translated reovirus products generated infectious reovirus when complemented with a helper virus from a different serotype (Roner, M. R., et al.,
Virology
, 179, 845-852 (1990)). However, to date, there has been no report of a recovered infectious virus of segmented dsRNA genome from synthetic RNAs only.
SUMMARY OF THE INVENTION
This invention relates to the infectious bursal disease virus (IBDV) that is associated with Gumboro disease of young chickens. More particularly, this invention relates to a system for the generation of infectious bursal disease virus (IBDV) using synthetic transcripts derived from cloned cDNA. The present invention will facilitate studies of the regulation of viral gene expression, pathogenesis and design of a new generation of live and inactivated vaccines.
DETAILED DESCRIPTION OF THE INVENTION
In an effort to develop a reverse genetics system for IBDV, three independent full-length cDNA clones which contain segment A of serotype I strain D78 or serotype II strain 23/82 and segment B of the serotype I strain P2, respectively, were constructed. Synthetic RNAs of segments A and B were produced by in vitro transcription reaction on linearized plasmids with T7 RNA polymerase. Transcripts of these segments, either untreated or treated with DNase or RNase, were evaluated for the generation of infectious virus by transfection of Vero cells.
The present inventors have demonstrated that synthetic transcripts derived from cloned DNA corresponding to the entire genome of a segmented dsRNA animal virus can give rise to a replicating virus. The recovery of infectious virus after transfecting cells with synthetic plus-sense RNAs derived from cloned cDNA of a virus with a dsRNA genome (IBDV) completes the quest of generating reverse infectious systems for RNA viruses. A number of investigators have generated infectious animal RNA viruses from cloned cDNA (Boyer, J. C., et al.,
Virology
, 198, 415-426 (1994)). Van der Werf et al. were first to generate poliovirus, a plus-stranded RNA virus, using synthetic RNA produced by T7 RNA polymerase on cloned cDNA template (van der Werf, S., et al.,
Proc. Natl. Acad. Sci. USA
, 83, 2330-2334 (1986)). later, Enami et al. rescued influenza virus, a segmented negative-stranded RNA virus (Enami, M., et al.,
Proc. Natl. Acad. Sci. USA
, 87, 3802-3805 (1990)); and
Mundt Egbert
Vakharia Vikram N.
Fuierer Marianne
Hultquist Steven J.
Mosher Mary E.
University of Maryland Biotechnology Institute
Yang Yongzhi
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