Elsevier

The Lancet

Volume 343, Issue 8896, 26 February 1994, Pages 551-552
The Lancet

Letters to the Editor
False detection of negative-strand hepatitis C virus RNA

https://doi.org/10.1016/S0140-6736(94)91509-1Get rights and content

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    Infected cells also cluster bimodally into subpopulations bearing low or high strand counts (Fig. S2). In order to cross-validate our vRNA measurements with an independent assay, we developed a new PCR assay that improves our capacity to discriminate between positive and negative strand vRNAs, since the application of previously published methods yield NPSR values for HCV that range widely from 1:10 to 1:1000, and these readings have been confounded by both interference from the opposite strand, and detection of incomplete vRNAs (McGuinness et al., 1994; Revie and Salahuddin, 2011; Agnello et al., 1998; Komurian-Pradel et al., 2004; Lanford et al., 1995; Lohmann et al., 1999). In this updated assay, total RNA is poly-A tailed, after which a tag oligo-dT anchored primer is used to reverse transcribe from the nascent tail (Fig. S3A); the resulting complementary DNA (cDNA) is then used for qPCR, employing a primer for the tag sequence and an HCV-specific primer for the 3′ end of the target vRNA.

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    Although hepatocytes are the primary site of HCV replication in the body, several studies have identified viral sequences in other anatomic sites, such as blood cells (Lerat et al., 1996), lymphoid tissue (Laskus et al., 2000), and the central nervous system (Fishman et al., 2008; Forton et al., 2004; Laskus et al., 2002; Radkowski et al., 2002), leading to the postulate that HCV may replicate in extrahepatic sites. However, this issue has remained controversial because of the questionable specificity of the tests used to determine the presence of active HCV replication (Farci, 2011), in particular assays for the detection of negative-strand HCV RNA (Lanford et al., 1995; Laskus et al., 1997; McGuinness et al., 1994). Other difficulties are related to the correct preservation of HCV RNA, especially in post-mortem tissues, such as brain tissue.

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    In addition, tissue compartmentalization of the HCV genome has been described during HCV infection7,8and coinfection with HIV.8 Finally, the detection of HCV RNA-negative strand as the theoretical intermediate of replication, although somewhat controversial,9,10has been well documented in PBMCs. Indeed, negative- strand RNA has been reported in mononuclear cells such as B and T lymphocytes and monocytes,11-15in polynuclear lymphocytes,16,17and even in hematopoietic progenitor cells.18

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