Next-generation sequencing analysis of a cluster of hepatitis C virus infections in a haematology and oncology center

Abstract

Molecular characterization of early hepatitis C virus (HCV) infection remains rare. Ten out of 78 patients of a hematology/oncology center were found to be HCV RNA positive two to four months after hospitalization. Only two of the ten patients were anti-HCV positive. HCV hypervariable region 1 (HVR1) was amplified in seven patients (including one anti-HCV positive) and analyzed by next generation sequencing (NGS). Genetic variants were reconstructed by Shorah and an empirically established 0.5% variant frequency cut-off was implemented. These sequences were compared by phylogenetic and diversity analyses. Ten unrelated blood donors with newly acquired HCV infection detected at the time of donation (HCV RNA positive and anti-HCV negative) served as controls. One to seven HVR1 variants were found in each patient. Sequences intermixed phylogenetically with no evidence of clustering in individual patients. These sequences were more similar to each other (similarity 95.4% to 100.0%) than to those of controls (similarity 64.8% to 82.6%). An identical predominant variant was present in four patients, whereas other closely related variants dominated in the remaining three patients. In five patients the HCV population was limited to a single variant or one predominant variant and minor variants of less than 10% frequency. In conclusion, NGS analysis of a cluster of HCV infections acquired in the hospital setting revealed the presence of low diversity, very closely related variants in all patients, suggesting an early-stage infection with the same virus. NGS combined with phylogenetic analysis and classical epidemiological analysis could help in tracking of HCV outbreaks.

Fig 1. Phylogenetic analysis of HVR1 variants in seven hospitalized patients infected with HCV 1b and in ten unrelated controls.

Variant frequencies are expressed as percent values and follow haplotype number. Pt denotes patients from the infection cluster. For clarity, each patient is marked with a different graphical symbol. Controls (C) came from the same geographic area and were infected with the same HCV subtype 1b. Bootstrap values obtained with 1000 replications are shown at the particular bifurcation points. HVR1 genotype 1a sequence (GenBank accession no. EF56024) has been used as an outgroup.

Fig 2. Phylogenetic analysis of HVR1 variants in hospitalized patients 3, 4, 5, 6 and 8.

The trees from patients 3, 4 and 6 are consistent with infection with a single founder (star-like phylogeny). Variant frequencies are expressed as percent values and follow haplotype number. For clarity, each patient is marked with a different graphical symbol (corresponding to Fig 1).

Fig 3. Highlighter plot showing differences (mismatches and gaps) of HVR1 HCV sequence variants in seven hospitalized patients.

Variants are compared to consensus sequence for all sequences present in all patients. A, C, T and G mismatches and gaps are shown in green, blue, orange, red and gray, respectively. Nucleotide numbering follows the reference strain H77 (GenBank accession no. AF009606).

Fig 4. Highlighter plot showing silent and non-silent mutations of HVR1 HCV sequence variants in seven hospitalized patients from the infection cluster.

Variants are compared to consensus sequence for all sequences present in all patients. Silent and non-silent mutations are shown in green and red, respectively. Nucleotide numbering follows the reference strain H77 (GenBank accession no. AF009606).

Fig 5. Highlighter plot showing differences in amino acid composition of HVR1 HCV sequence variants in hospitalized patients from the infection cluster.

Variants are compared to consensus sequence built from all sequences present in all patients. Color coding of amino acid is shown in the legend. Potential N-glycosylation site in the consensus sequence is marked by a purple dot. Amino acid numbering follows the reference strain H77 (GenBank accession no. AF009606). HVR1 spans codons 384–410.