Investigation of Putative Multisubtype Hepatitis C Virus Infections In Vivo by Heteroduplex Mobility Analysis of Core/Envelope Subgenomes

Abstract

The frequency that multiple different subtypes of hepatitis C virus (HCV) simultaneously infect a given individual is controversial. To address this question, heteroduplex mobility analysis (HMA) of portions of the HCV core and envelope 1 region was optimized for sensitive and specific detection of mixtures of HCV genomes of different genotype or subtype. Using the standard HCV genotyping approach of 5'-untranslated region (UTR) analysis, 28 of 374 (7.5%) chronic hepatitis C research subjects were classified as having either multiple-subtype HCV infections (n = 21) or switching HCV subtypes over time (n = 7), the latter pattern implying viral superinfection. Upon retesting of specimens by HMA, 25 of 28 multiple-subtype results could not be reproduced. All three patients with positive results were injection drug users with potential multiple HCV exposures. To address the hypothesis of tissue sequestration of multiple-subtype HCV infections, liver (n = 22), peripheral blood mononuclear cell (n = 13), perihepatic lymph node (n = 16), and serum (n = 19) specimens from 23 subjects with end-stage hepatitis C were collected and analyzed by the HMA technique. Whereas 5'-UTR results implicated mixed-subtype HCV infections in 2 subjects, HMA testing revealed no evidence of a second HCV subtype in any tissue compartment (0 of 70 compartments [0%]) or within any given subject (0 of 23 subjects [0%]). In summary, a large proportion of mixed-genotype and switching-genotype patterns generated by 5'-UTR analysis were not reproducible using the HMA approach, emphasizing the need for additional study.

Natural intragenotypic and intergenotypic HCV recombinants are rare in southwest China even among patients with multiple exposures

“…19,20 The wide variation may be explained by the variety of approaches used. 20 Many non-sequencing methods have been applied to detect multitypic HCV infection, which differ in terms of sensitivity to detect minority genotypes and of specificity in the assignment of genotypes. 20 Sequencing a less-conserved subgenomic region, such as envelope 1 (E1) or NS5B, is considered the "gold standard" of genotyping.…”

“…[19][20][21] However, the frequency of multitypic HCV infection is still controversial, with Table 2 The demographic data and virological characteristics of patients detected for multitypic HCV infection with colony analysis of core-envelope 1 regions. (1) a The earliest collected serum sample from each patient was labeled (B), and the latest collected sample was labeled (E).…”

“…20 Many non-sequencing methods have been applied to detect multitypic HCV infection, which differ in terms of sensitivity to detect minority genotypes and of specificity in the assignment of genotypes. 20 Sequencing a less-conserved subgenomic region, such as envelope 1 (E1) or NS5B, is considered the "gold standard" of genotyping. 22 In this study, multitypic HCV infection was identified in 47.4% of the repeatedly exposed individuals by colony analysis of the CE1 region, of which the specificity is assured, because the identity of the minority genotype was characterized by the sequence directly derived from the less-conserved subgenomic region.…”

Genetic diversity of hepatitis C virus predicts recurrent disease after liver transplantation

“…All patients had HCV-related end-stage liver disease at the time of liver transplantation, and all developed recurrent HCV infection as demonstrated by the persistence of post-transplant HCV viremia. HCV genotypes were assigned using the restriction fragment length polymorphism analysis (RFLP) of the 5′-UTR region (Davidson et al, 1995) and confirmed by probe hybridization of the Core/E1 region (Li et al, 2008b). Serum samples and purified peripheral blood mononuclear cells (PBMCs) were obtained immediately prior to liver transplantation and stored at −80°C.…”

Inferring viral population structures using heteroduplex mobility and DNA sequence analyses

“…The ability to subtype different regions of the viral genome made it possible for HMA to identify inter-subtype recombinants (Cham et al, 2000; Heyndrickx et al, 2000; Sarkar et al, 2011; Sawadogo et al, 2003). HMA-based subtyping has also been developed for other viral pathogens, including Hepatitis C (Calvo et al, 1998; Li et al, 2008; White et al, 2000), feline immunodeficiency virus (Bachmann et al, 1997), small-ruminant lentiviruses (Germain, Croise, and Valas, 2008), Influenza (Zou, 1997) and enteric adenoviruses (Soares et al, 2004). The accuracy of HMA subtyping in many of the above studies has been evaluated using DNA sequencing and compared by some to peptide-based serologic methods (Bobkova et al, 2001; Gaywee et al, 1996; Hussein et al, 2000; Wasi et al, 1995a).…”