Abstract
Defining genetic diversity of viral infections directly from patient specimens is the ultimate goal of surveillance. Simple tools that can provide full-length sequence information on blood borne viral hepatitis viruses: hepatitis C, hepatitis B and hepatitis D viruses (HCV, HBV and HDV) remain elusive. Here, an unbiased metagenomic next generation sequencing approach (mNGS) was used for molecular characterization of HCV infections (n = 99) from Israel which yielded full-length HCV sequences in 89% of samples, with 7 partial sequences sufficient for classification. HCV genotypes were primarily 1b (68%) and 1a (19%), with minor representation of genotypes 2c (1%) and 3a (8%). HBV/HDV coinfections were characterized by suppressed HBV viral loads, resulting in sparse mNGS coverage. A probe-based enrichment approach (xGen) aiming to increase HBV and HDV coverage was validated on a panel of diverse genotypes, geography and titers. The method extended HBV genome coverage a median 61% (range 8-84%) and provided orders of magnitude boosts in reads and sequence depth for both viruses. When HBV-xGen was applied to Israeli samples, coverage was improved by 28-73% in 4 samples and identified HBV genotype A1, A2, D1 specimens and a dual B/D infection. Abundant HDV reads in mNGS libraries yielded 18/26 (69%) full genomes and 8 partial sequences, with HDV-xGen only providing minimal extension (3-11%) of what were all genotype 1 genomes. Advanced molecular approaches coupled to virus-specific capture probes promise to enhance surveillance of viral infections and aid in monitoring the spread of local subtypes.
Highlights
Viral hepatitis represents a significant global health burden, as many cases lead to cirrhosis and liver cancer, which can be fatal
A total of 99 hepatitis C virus (HCV) RNA positive specimens were collected at the National HIV and Viral Hepatitis Reference Center (NHRL)
A semi-quantitative PCR of libraries indicated that the average viral load was log 5.4 copies/ml, consistent with previously observed trend of most untreated HCV patients having viral loads >4 log IU/ml [26]
Summary
Viral hepatitis represents a significant global health burden, as many cases lead to cirrhosis and liver cancer, which can be fatal. While generation of partial sequences for a given viral genome by Sanger sequencing methods has provided the classical means of surveillance, there are several drawbacks to consider with this approach. Focused sequencing in one region may not adequately inform diagnostic assay development that targets un-sequenced regions of the virus. This method requires the design of primers for amplification that may not work for all genotypes. These issues can be avoided by pursuing complete genome sequencing
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