Molecular Analysis and Ex Vivo Infectivity of Seronegative Occult Hepatitis C Virus: A Study in Single Haemodialysis Centre

Abstract

Background: In occult hepatitis C virus infection (OCI), hepatitis C virus ribonucleic acid (HCV RNA) is detectable in peripheral blood mononuclear cells (PBMCs) but is not evident in serum or plasma. Understanding of OCI in patients with seronegative anti-HCV antibodies is limited.

Methods: In this study, six HCV isolates from haemodialysis (HD) patients with seronegative OCI were identified by molecular assays and phylogenetic analysis. The virus infectivity was assessed ex vivo using a primary naïve PBMC culture system. HCV isolates obtained from the PBMCs of 10 patients with chronic HCV infection (CCI) were characterised concurrently and used as positive controls in the cell culture.

Results: Sequence analysis of the 5' untranslated region (UTR) and non-structural 5B (NS5B) region revealed that HCV genotype 3 was the most prevalent virus type in both the OCI and CCI groups. One of the occult HCV isolates was identified as a mixed type. The mean viral load (log₁₀ RNA copies/106 cells) in the PBMC samples of the OCI group (M = 3.4, SD = 0.7) was lower than that of the CCI group (M = 4.6, SD = 1.7). Upon culture, de novo OCI-HCV replicates were detected in five out of six naïve PBMC cultures. Analysis of the replicates showed a single guanine addition in the domain III of 5'-UTR but the overall molecular structure was retained.

Conclusion: Seronegative OCI is an active form of infection that replicates at a low level in PBMCs. Seronegative OCI may share the same route of transmission as CCI. The retained viral competency may have an implication for its persistence.

Keywords: PBMCs; genotype; hepatitis C virus; patients; sequence analysis.

Figure 1

(A) Infectivity assay. Cells were aliquoted in a triplicate for each sample and incubated with the following inoculums: supernatants from the PBMCs of six occult HCV- infected patients, HCV-infected patients for positive control and healthy individual for negative control. (B) PHA-treated PBMC were observed using Digital Microscope (Dino Lite, Taiwan) in the magnification of 4× (left) and 10× (right)

Figure 1

Detection of genomic and antigenomic HCV RNA in PBMC samples of haemodialysis patients who were also seronegative for anti-HCV antibodies. (A) The genomic HCV RNA was detected in six PBMC samples out of 30 samples. (B) Antigenomic HCV RNA was also detected in the six PMBC samples Notes: M = 100 bp DNA ladder (New England Biolabs, USA); +VE = positive control; NTC = no-template control; lane numbers indicate the sample’s number

Figure 2

Phylogenetic tree of the nucleotide HCV sequences. (A) Phylogenetic analysis based on the 5′UTR sequences shows HCV genotypes 3, 1 and 6 in patients infected with chronic or occult HCV (boxes). (B) Subtyping the HCV infecting PBMCs based on the NS5B phylogenetic analysis reveals subtypes 3a and 1a (boxes). The isolates’ nomenclature: the prefix ‘POS’ of the virus isolates in this study indicates chronic HCV isolates, meanwhile, the prefix ‘HD’ indicates occult HCV. Note that occult HCV isolates are not clustered together suggesting different strains

Figure 3

Molecular detection and nucleotide sequence analysis of the de novo occult HCV isolates. (A) Genomic (right) and antigenomic strands (left) of the HCV RNA were detected in the respective naïve PBMC cultures exposed to the supernatants of PBMC cultures infected with six occult HCV isolates. Supernatant derived from a PBMC culture infected with chronic HCV was run in parallel as the positive control. M = 100 bp ladder (New England Biolabs, USA), +VE = positive control. HCV = hepatitis C virus. (B) Sequence alignment of the HCV isolates shows the addition of guanine (G) nucleotide in the de novo occult HCV sequences (indicated by the black arrow). HCV isolates HD6, HD10, HD15, HD16, HD17 and HD18 with the prefix ‘PRE’ indicates before inoculation, and viral isolates of the post-inoculation are given the prefix ‘POS’. AF217308 is a reference sequence obtained from the NCBI database. (C) Prediction of the domain-III 5’UTR-HCV secondary structure using HD18 sequence reveals no change to the overall 2-D structure despite the addition of G nucleotide. Notes: White arrow indicates the position of G addition; A = adenine; T = thymine; C = cytosine; HCV = hepatitis C virus