In situ hybridization for the detection of hepatitis C virus RNA in human liver tissue

Abstract

In situ hybridization (ISH) enables visualization of specific nucleic acid in morphologically preserved cells and tissue sections. Detection of the HCV genomes in clinical specimens is useful for differential diagnosis, particularly between recurrent HCV infection and acute cellular rejection in transplant specimens. We optimized an ISH protocol that demonstrated sensitivity and specificity for detecting genomic and replicative form of HCV RNA in tissue biopsies. Digoxigenin (Dig)-labelled sense and anti-sense riboprobes were synthesized using a plasmid containing a fragment of the highly conserved HCV noncoding region as a template. The efficiency of the Dig-labelled riboprobes in detecting genomic and replicative-intermediate HCV RNA was analysed in 30 liver biopsies from patients infected or uninfected with HCV in a blinded study. A Huh7 cell line that stably replicates genome-length HCV RNA was developed to be used as a positive control. Negative control riboprobes were used in parallel to evaluate and control for background staining. The anti-sense probe detected HCV RNA in 20/21 specimens from HCV-infected liver tissues obtained from patients and in 0/9 samples from patients with non-HCV-related liver diseases, resulting in a sensitivity and specificity of 95% and 100%, respectively. HCV genomic RNA was variably distributed in tissue sections and was located primarily in the perinuclear regions in hepatocytes. Detection of HCV RNA by our optimized ISH protocol appears to be a sensitive and specific method when processing clinical specimens. It may also be revealing when exploring the pathophysiology of HCV infection by verifying the presence of viral genetic material within heptocytes and other cellular elements of diseased liver tissue. This methodology might also evaluate the response to antiviral therapies by demonstrating the absence or alteration of genetic material in clinical specimens from successfully treated patients.

Hepatitis C virus–induced reduction in miR‐181a impairs CD4+ T‐cell responses through overexpression of DUSP6

“…Thus, manipulation of miR‐181a and/or DUSP6 expression may represent a key intervention to strengthen and broaden CD4 + T‐cell responses in HCV‐infected individuals. Based on this study, our previous reports, and other investigators' work, we propose a model, depicted in Fig. , to illustrate the role and pathways involved in the HCV‐induced, miR‐181a‐mediated DUSP6 overexpression in CD4 + T cells to control T‐cell responses in the setting of chronic viral infection.…”

“…After decades of studying this immunomodulatory virus, it has become evident that HCV‐mediated T‐cell dysfunction plays a major role in viral persistence as well as disease progression . We have previously reported that CD4 + T cells play a pivotal role in the host immune responses to pathogenic infection and vaccination . In this study, we further explored the mechanisms of CD4 + T‐cell dysfunction in chronic HCV infection.…”

“…[25][26][27][28][29] We have previously reported that CD4 1 T cells play a pivotal role in the host immune responses to pathogenic infection and vaccination. 11,[25][26][27][28][29][30] In this study, we further explored the mechanisms of CD4 1 T-cell dysfunction in chronic HCV infection. Our studies demonstrate that HCV-induced, miR-181a-mediated DUSP6 overexpression in CD4 1 T cells recalibrates the TCR sensitivity threshold, thus controlling the signaling strength of CD4 1 T cells from HCV-infected individuals, which is evidenced by a more profound dysfunction of CD4 1 T-cell activation and proliferation and IL-2 secretion when compared to HS.…”

Hepatitis C Virus Genotypes and Association With Viral Load in Yazd, Central Province of Iran

“…The previously reported HCV genotypes of 7 through 9 have been re-classified as subtypes of genotype 6 (8). Sequence inconsistency ranges from 31% to 33% among genotypes and from 20% to 25% among subtypes (9). Genotypes 1 to 3 show a worldwide distribution, whereas genotypes 4 to 6 are more geographically restricted (10).…”

Demonstration of Hepatitis C Virus RNA withIn SituHybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

“…In nested RT-PCR negative cases, ISH was negative or partially positive, suggesting that the detection threshold may depend on the virus load. Diffuse HCV expression pattern in the hepatocytes was already reported by Nuriya et al [15], Liang et al [33], and Li et al [34]. …”