Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-07-18
2001-10-02
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C536S024300, C536S024330
Reexamination Certificate
active
06297003
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to a novel nucleotide sequence element identified at or near the 3′ terminus of the hepatitis C virus (HCV) viral genome RNA. This element is highly conserved among HCV genotypes and may be essential for HCV replication.
BACKGROUND OF THE INVENTION
After the development of diagnostic tests for hepatitis A virus and hepatitis B virus, an additional agent, which could be experimentally transmitted to chimpanzees (Alter et al, 1978; Hollinger et al, 1978; Tabor et al, 1978), became recognized as the major cause of transfusion-acquired hepatitis. cDNA clones corresponding to the causative non-A non-B (NANB) hepatitis agent, called hepatitis C virus (HCV), were reported in 1989 (Choo et al, 1989). This breakthrough has led to rapid advances in diagnostics, and in our understanding of the epidemiology, pathogenesis and molecular virology of HCV (see Houghton et al, 1994 for review). Evidence of HCV infection is found throughout the world and the prevalence of anti-HCV antibodies ranges from 0.4-2% in most developed countries to more than 14% in Egypt (Hibbs et al, 1993). Besides transmission via blood or blood products, or less frequently by sexual and congenital routes, sporadic cases, not associated with known risk factors, occur and account for more than 40% of HCV cases (Alter et al, 1990; Mast and Alter, 1993). Infections are usually chronic (Alter et al, 1992) and clinical outcomes range from an inapparent carrier state to acute hepatitis, chronic active hepatitis, and cirrhosis which is strongly associated with the development of hepatocellular carcinoma. Although alpha IFN has been shown to be useful for the treatment of some patients with chronic HCV infections (Davis et al, 1989; DiBisceglie et al, 1989) and subunit vaccines show some promise in the chimpanzee model (Choo et al, 1994), future efforts are needed to develop more effective therapies and vaccines. The considerable diversity observed among different HCV isolates (for review, see Bukh et al, 1995), the emergence of genetic variants in chronically infected individuals (Enomoto et al, 1993; Hijikata et al, 1991; Kato et al, 1992; Kato et al, 1993; Kurosaki et al, 1993; Lesniewski et al, 1993; Ogata et al, 1991; Weiner et al, 1991; Weiner et al, 1992), and the lack of protective immunity elicited after HCV infection (Farci et al, 1992; Prince et al, 1992) present major challenges towards these goals.
Molecular Biology of HCV
Classification
Based on its genome structure and virion properties, HCV has been classified as a separate genus in the flavivirus family, which includes two other genera: the flaviviruses [such as yellow fever virus (YF)] and the animal pestiviruses [bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV)] (Francki et al, 1991). All members of this family have enveloped virions that contain a positive-strand RNA genome encoding all known virus-specific proteins via translation of a single long open reading frame (ORF; see below).
Structure and Physical Properties of the Virion
Little information is available on the structure and replication of HCV. Studies have been hampered by the lack of a cell culture system able to support efficient virus replication and the typically low titers of infectious virus present in serum. The size of infectious virus, based on filtration experiments, is between 30-80 nm (Bradley et al, 1985; He et al, 1987; Yuasa et al, 1991). HCV particles isolated from pooled human plasma (Takahashi et al, 1992), present in hepatocytes from infected chimpanzees, and produced in cell culture (Shimizu et al, 1994a) have been visualized (tentatively) by electron microscopy. Initial measurements of the buoyant density of infectious material in sucrose yielded a range of values, with the majority present in a low density pool of <1.1 g/ml (Bradley et al, 1991). Subsequent studies have used RT/PCR to detect HCV-specific RNA as an indirect measure of potentially infectious virus present in sera from chronically infected humans or experimentally infected chimpanzees. From these studies, it has become increasingly clear that considerable heterogeneity exists between different clinical samples, and that many factors can affect the behavior of particles containing HCV RNA (Hijikata et al, 1993; Thomssen et al, 1992). Such factors include association with immunoglobulins (Hijikata et al, 1993) or low density lipoprotein (Thomssen et al, 1992; Thomssen et al, 1993). In highly infectious acute phase chimpanzee serum, HCV-specific RNA is usually detected in fractions of low buoyant density (1.03-1.1 g/ml) (Carrick et al, 1992; Hijikata et al, 1993). In other samples, the presence of HCV antibodies and formation of immune complexes correlate with particles of higher density and lower infectivity (Hijikata et al, 1993). Treatment of particles with chloroform, which inactivates infectivity (Bradley et al, 1983; Feinstone et al, 1983), or with nonionic detergents, produces RNA containing particles of higher density (1.17-1.25 g/ml) believed to represent HCV nucleocapsids (Hijikata et al, 1993; Kanto et al, 1994; Miyamoto et al, 1992).
There have been many reports of varying levels of negative-sense HCV-specific RNAs in sera and plasma (see Fong et al, 1991). However, it seems unlikely that such RNAs are essential components of infectious particles since some sera with high infectivity can have low or undetectable levels of negative-strand RNA (Shimizu et al, 1993). The virion protein composition has not been rigorously determined, but putative HCV structural proteins include a basic C protein and two membrane glycoproteins, E1 and E2.
HCV Replication
Early events in HCV replication are poorly understood. Cellular receptors for the HCV glycoproteins have not been identified. The association of some HCV particles with beta-lipoprotein and immunoglobulins raises the possibility that these host molecules may modulate virus uptake and tissue tropism. Studies examining HCV replication have been largely restricted to human patients or experimentally inoculated chimpanzees. In the chimpanzee model, HCV RNA is detected in the serum as early as 3 days post-inoculation and persists through the peak of serum alanine aminotransferase (ALT) levels (an indicator of liver damage) (Shimizu et al, 1990). The onset of viremia is followed by the appearance of indirect hallmarks of HCV infection of the liver. These include the appearance of a cytoplasmic antigen (Shimizu et al, 1990) and ultrastructural changes in hepatocytes such as the formation of microtubular aggregates for which HCV previously was referred to as the chloroform-sensitive “tubule forming agent” or “TFA” (reviewed by Bradley, 1990). As shown by the appearance of viral antigens (Blight et al, 1993; Hiramatsu et al, 1992; Krawczynski et al, 1992; Yamada et al, 1993) and the detection of positive and negative sense RNAs (Fong et al, 1991; Gunji et al, 1994; Haruna et al, 1993; Lamas et al, 1992; Nouri Aria et al, 1993; Slierker et al, 1993; Takellara et al, 1992; Tanaka et al, 1993), hepatocytes appear to be a major site of HCV replication, particularly during acute infection (Negro et al, 1992). In later stages of HCV infection the appearance of HCV-specific antibodies, the persistence or resolution of viremia, and the severity of liver disease, vary greatly both in the chimpanzee model and in human patients. Although some liver damage may occur as a direct consequence of HCV infection and cytopathogenicity, the emerging consensus is that host immune responses, in particular virus-specific cytotoxic T lymphocytes, may play a more dominant role in mediating cellular damage (see Rice and Walker, 1995 for review).
It has been speculated that HCV may also replicate in extra hepatic reservoir(s), particularly in chronically infected individuals. In some cases, RT/PCR or in situ hybridization has shown an association of HCV RNA with peripheral blood mononuclear cells including T-cells, B-cells, and monocytes (Blight et al, 1992; Bouffard
Kolykhalov Alexander A.
Rice Charles M.
Howell & Haferkamp LC
Parkin Jeffrey S.
Scheiner Laurie
Washington University
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