Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-01-14
2001-11-20
Ketter, James (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S387100, C530S391300
Reexamination Certificate
active
06320027
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular immunology and virology. More specifically, the present invention relates to nucleotide sequence of the nucleocapsid gene of oropouche virus.
2. Description of the Related Art
Oropouche (ORO) virus, the causative agent of oropouche fever, has emerged during the past 30 years as an increasing health problem in tropical regions of South and Central America. Since 1961 a number of epidemics of oropouche fever have been recorded involving several thousand people. Oropouche fever in humans is characterized by abrupt onset of fever, chills, severe headache, generalized myalgia, arthralgia, anorexia, nausea, vomiting, weakness, dizziness, and photophobia. Because of the non-specific nature of symptoms and paucity of diagnostic virus laboratories in the oropouche-endemic region, the disease often is either unrecognized or misdiagnosed as being an infection caused by dengue or other similar viruses.
The oropouche virus belongs to the genus Bunyavirus of family Bunyaviridae. Based on antigenic characteristics, the bunyavirus genus has been divided into 18 serogroups and oropouche virus is one of 25 viruses that have been placed in Simbu serogroup. The genome of oropouche virus consists of three segments of single stranded, negative sense RNA designated as Large (L), Medium (M) and Small (S) RNAs. The L RNA encodes the L (RNA polymerase) protein; the M RNA encodes two glycoproteins G1 and G2 and a nonstructural protein NSm; and the S RNA encode nucleocapsid (NP) protein and a non-structural (NSs) protein.
To date, the molecular biology of bunyaviruses has focused on a small number of viruses and very little information is available on other viruses, especially the Simbu serogroup viruses. The S RNA has been sequenced for several members of Bunyamwera and California encephalitis serogroups but only Tinaroo, Akabane and Aino viruses have been sequenced for the Simbu serogroup.
Current techniques for the identification and diagnosis of Oropouche infection include neutralization test, hemaglutination inhibition and complement fixation to detect anti-oropouche antibodies. ELISA using antigen produced from infectious virus has also been used but production of antigen is expensive, hazardous and highly variable from batch to batch. This ELISA also gives too many “false positives”.
The prior art is deficient in the lack of knowledge of many aspects of the molecular biological properties of oropouche virus, including the nucleotide sequence of the nucleocapsid gene of oropouche virus. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The S RNA of the Oropouche virus was cloned and sequenced. The sequence analysis of the S RNA revealed that oropouche virus is related to, but genetically distinct from the Simbu serogroup viruses. The total length of the S-RNA was found to be 754 nucleotides and two overlapping open reading frames (ORFs) of 693 and 273 nucleotides were identified which encoded the NP and NSs proteins, respectively.
In addition, the NP protein was also cloned and expressed in an
E.coli
expression system. SDS-PAGE and Western blotting analysis revealed that the molecular weight of the recombinant protein is the same as that of the viral NP and that it was recognized by oropouche-specific antisera. Reactivity pattern of the recombinant protein with sera raised against 15 other Simbu serogroup viruses was indistinguishable from the reactivity pattern of the authentic viral NP. Furthermore, like the viral NP, the recombinant protein failed to react with sera raised against dengue viruses 1 & 2 and 4 bunyaviruses belonging to other serogroups. Thus, based on these data, the recombinant NP protein can be an important diagnostic tool for the identification of Oropouche infection which would be safer, less expensive and easier to produce in large quantities.
It is an object of the present invention to provide the Oropouche NP gene sequence which can be used to design oligonucleotide primers for reverse transcriptase-PCR to amplify Oropouche viral RNA from virus-infected cell cultures and serum samples.
It is also another object of the present invention to express Oropouche virus NP gene in
E. coli.
It is another object of the present invention to provide Oropouche virus NP protein useful in detecting anti-NP antibodies so as to be able to diagnose Oropouche virus infection and seroprevalence.
In one embodiment of the present invention, there is provided DNA encoding a Oropouche NP protein selected from the group consisting of: (a) isolated DNA which encodes a Oropouche NP protein; (b) isolated DNA which hybridizes to isolated viral RNA or complementary DNA of (a) above and which encodes a Oropouche NP protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a Oropouche NP protein.
In another embodiment of the present invention, there is provided a vector capable of expressing the DNA of the present invention adapted for expression in a recombinant cell and regulatory elements necessary for expression of the DNA in the cell.
In yet another embodiment of the present invention, there is provided an isolated and purified Oropouche NP protein encoded by DNA selected from the group consisting of: (a) isolated DNA which encodes a Oropouche NP protein; (b) isolated DNA which hybridizes to isolated DNA of (a) above and which encodes a Oropouche NP protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code, and which encodes a Oropouche NP protein.
In still yet another embodiment of the present invention, there is provided a kit for the immunodetection of Oropouche virus DNA or RNA.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
REFERENCES:
Beaty et al. 1991. Bunyaviridae—Natural History. Current Topics in Microbiology and Immunology 169: 27-78.*
Murphy. 1996. Virus Taxonomy. in Fields, et al., eds. Fundamental Virology, Third Edition. Lippincott-Raven Publishers, Philadelphia. see Table 4 of Chapter 2.
Barrett Alan D. T.
Saeed Mohammad F.
Shope Robert E.
Tesh Robert B.
Wang Heiman
Adler Benjamin Aaron
Gansheroff Lisa
Ketter James
The Board of Regents of the University of Texas
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