Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology.

Abstract

A nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and subsequent amplification with a complex of alkaline phosphatase and anti-alkaline phosphatase antibodies. The intracellular distribution of HCV RNA was monitored in the livers of two chimpanzees experimentally infected with the H strain of HCV and compared with the serum alanine aminotransferase activity, serum HCV RNA, and liver histopathology. Most cells were stained in the cytoplasm as early as 2 days after inoculation, 1 and 2 days, respectively, before the appearance of viral RNA in the serum. The time course of HCV RNA replication was correlated with increases in serum alanine aminotransferase. However, neither one paralleled the appearance of liver cell necrosis nor showed any correlation with the inflammatory response. The NISH signal was not found in liver biopsy specimens taken from these two animals before inoculation with HCV, from chimpanzees with acute hepatitis type A, B, or 6, or from two animals never experimentally infected with any hepatitis agent; moreover, it disappeared when the positive specimens were predigested with RNase and it was not observed after hybridization of positive controls with a labeled oligomer unrelated to HCV RNA. Thus, detection of liver HCV RNA by NISH is a sensitive and specific method for studying HCV replication at the cellular level. Intracellular replication of HCV did not appear to be associated with histopathologic changes in the liver, although the correlation with increases of liver enzyme activity in the serum suggested possible damage to the liver cell membrane.Hepatitis C virus (HCV), the agent responsible for the majority of human non-A, non-B hepatitis cases, has remained elusive for almost two decades. Its discovery and subsequent characterization have indeed followed unconventional routes, such as direct cloning and expression of cDNA prepared from the serum of infected humans (1) or chimpanzees (2). This was necessary because HCV replication in the host is limited (3-6) and the immune response to viral antigens is relatively poor (7-10).Several groups of investigators have reported the partial or complete genome sequence of different isolates of HCV (1,(11)(12)(13)(14)(15)(16)(17)(18). Sequence analyses of the HCV RNA show that HCV is distantly related to human flaviviruses, including yellow fever (YF) virus (another hepatotropic virus), as well as to animal pestiviruses.Sensitive assays have been developed to detect HCV RNA by the polymerase chain reaction (4-6, 9, 19). Infected livers, however, must be processed for nucleic acid extraction, thus hindering morphological evaluation. On...

Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line.

“…4, the PCR titers of plus-and minus-strands were both 100 genomes per cell pellet and, as shown in Table 1, the minus-strand could be detected even when the positive-strand was undetectable. (v) We demonstrated intracellular HCV minus-strand by in situ hybridization with a strand-specific probe, using a modification of a technique recently shown to be specific for HCV sequences in infected hepatocytes (24). (vi) We demonstrated viral core and NS4 antigens by immunofluorescence with monoclonal antibodies from two different sources.…”

“…During the 24 days of culture the cells multiplied with a doubling of 24 hr to attain a 107-fold increase, and on day 14 the culture medium had been diluted -1010-fold by centrifugation/resuspension steps during subcultures. Thus, carryover HCV RNA from the inoculum (103 genomes per 100 p.l by PCR) should have been diluted 104-fold and 107-fold beyond the estimated titers of residual genomes in the cells (day 24) and supernatant (day 14), respectively. (iv) Had the detection of minus-strand in the infected cells been caused by the nonspecific amplification of plus-strand from the inoculum, we should have detected plus-strand in 104-fold excess, based on the experiments described above; this actually was not the case.…”

Associations among clinical, immunological, and viral quasispecies measurements in advanced chronic hepatitis C

“…Serum HCV RNA levels, as a proxy measurement of viral replication, correlate poorly with disease severity, 3,4 whereas conflicting data exist as to whether measurements of intrahepatic HCV replication correlate better with liver injury. [3][4][5][6][7][8][9] Virus-host interactions are also thought to be a significant cause of liver injury. Persistent HCV infection occurs in the context of ongoing HCV-specific cellular and humoral immune responses.…”

Functional B-cell response in intrahepatic lymphoid follicles in chronic hepatitis C

“…3 HCV is known to cause a variety of systemic immunologic reactions or abnormalities such as circulating immune complexes, 4 autoantibodies, 5,6 and autoimmune manifestations. [7][8][9][10] Although the liver is considered the primary site of HCV replication with convincing evidence, 11,12 systemic or extrahepetic immune reactions, as well as intrahepatic responses, can be induced in the course of chronic infection.…”