THE SEQUENCE, GENOMIC ORGANIZATION, AND EXPRESSION OF THE SMALL GENOMIC SEGMENT OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS FOR THE POTENTIAL USE AS A DIAGNOSTIC ANTIGEN

Abstract

Crimean-Congo hemorrhagic fever (CCHF) virus (Nairovirus, Bunyaviridae) is the causative agent of a serious, often fatal human disease. This disease is present in at least 28 countries and may occur even more widely within the range of its Hyalomma tick vector in Africa and Eurasia. In addition to the naturally acquired disease, the virus is also noted for its ability to cause nosocomial outbreaks, as has occurred in at least seven countries in Asia, the Middle East and Africa. These incidents have resulted in mortality rates as high as 80% among infected health care workers. Current laboratory tests for diagnosis and surveillance of CCHF, lack sensitivity and do not differentiate adequately between the human pathogenic and nonpathogenic members of the Nairovirus genus. The disease syndrome in man is well-described clinically, however, the biochemical properties of the virus are poorly understood. Therefore, a molecular and antigenic analyses of the pathogenic CCHF virus was initiated to define the genes which are relevant to viral diagnosis and immunoprophylaxis. Previous monoclonal antibody studies have indicated that the nucleocapsid (NC) protein was the most type-specific polypeptide. Therefore, subsequent studies to be described, have focused on the determination of the sequence of the CCHF virus S RNA segment. In other Bunyaviridae viruses, the S RNA segment of the viral genome encodes the nucleocapsid (NC) protein. This sequence information will contribute towards the development of polymerase chain reaction (PCR) diagnostic assays, and expression of the NC protein for use as a type-specific diagnostic antigen. An efficient in vitro system to replicate CCHF virus was developed using low multiplicity of infection (MOI) of SW13 cell cultures. Comparative studies of the viral proteins of CCHF virus and other Nairoviruses were initiated using this system. These studies demonstrated a common pattern of polypeptides, consisting of two glycoproteins with relative molecular weights (Mr) of 78 and 37 kDa, and a NC polypeptide with 53.9 kDa Mr, comprising the major structural proteins were present among all the Nairovirus that were evaluated. A pulse-chase experiment indicated that an additional three polypeptides are present in CCHF virus-infected cells, which may be precursors in the formation of the virion glycoproteins. RNA was extracted from viral nucleocapsids purified from infected cells by equilibrium centrifugation, and the L, M, and S RNA segments were separated by gel electrophoresis. Based on the 3' end sequence of the S RNA segment, a single S RNA-specific primer was designed to initiate first strand cDNA synthesis and also to initiate synthesis of a full-length PCR product using CCHF virus RNA as a template. The initial PCR product was found to be specific for the CCHF virus S RNA segment when used as a hybridization probe against CCHF virus and other Nairovirus RNAs in a northern blot analysis. This full-length PCR product was modified for use as a sequencing template and both strands of the DNA product were sequenced using T4 DNA polymerase and in a dideoxy sequencing reaction. The CCHF virus S RNA segment was found to be 1672 nucleotides in length. Computer analysis of this sequence predicted a single open reading frame (ORE'), which could encode a 482 amino acid (53.9 kDa) protein. A PCR product representing this ORF, with exogenous terminal restriction endonuclease sites, was inserted into Autoqrapha californica nuclear polyhedrosis virus (AcNPV), a baculovirus, in lieu of the 5' coding region of the AcNPV polyhedrin gene. Spodoptera fruqiperda (Sf9) cells were infected with the recombinant baculovirus and monitored for NC protein expression. The recombinant expressed protein was characterized using immunofluorescent (IFA) and immunoprecipitation (IP) assays and was found to be antigenically indistinguishable from the authentic CCHF virus NC protein.