Near-Complete Genome Sequence of a Hepatitis A Subgenotype IB Virus Isolated from Frozen Raspberries

Hepatitis A virus is one of the most common causes of foodborne viral illness. Here, we report the nearly complete genome sequence of a hepatitis A virus (subgenotype IB) isolated from frozen raspberries using RNA sequencing-based metagenomics.

ABSTRACT

Hepatitis A virus is one of the most common causes of foodborne viral illness. Here, we report the nearly complete genome sequence of a hepatitis A virus (subgenotype IB) isolated from frozen raspberries using RNA sequencing-based metagenomics.

ANNOUNCEMENT

Hepatitis A virus (HAV) is the most frequently identified pathogen of acute viral hepatitis worldwide and is recognized as one of the most common foodborne viruses (1, 2). HAV is a small nonenveloped virus classified into the Hepatovirus genus within the Picornaviridae family. The viral genome is an approximately 7,500-nucleotide (nt) positive-sense, single-stranded RNA. The HAV genome is heterogeneous, enabling classification of the virus into six different genotypes, designated I to VI, where I, II, and III genotypes, which are divided into subgenotypes A and B, have been associated with human disease (3–5). IA is the most prevalent subgenotype in the United States, followed by IB and IIIA (6). HAV is generally acquired through the fecal-oral route, either by direct person-to-person contact or by ingestion of contaminated water or food, particularly shellfish, soft fruit, and raw vegetables (7). Although HAV is considered to be one of the leading foodborne viruses, direct detection of HAV in implicated foods is complicated by the complexity of the food matrix and low contamination levels of the virus. Foodborne HAV infection is not often reported except when triggered by an outbreak. Here, we report the nearly complete genome sequence of a subgenotype IB HAV from frozen raspberries that were collected during an infection event which occurred in New Hampshire in 2013 using a metagenomic technique.

RNA was prepared from frozen raspberries following the U.S. Food and Drug Administration Bacteriological Analytical Manual 26B protocol (8), with the exception of not using murine norovirus 1. The RNA extract was then concentrated with an Amicon ultra 0.5 centrifugal filter (Millipore, MA). RNA amplification, sequencing library preparation, and paired-end sequencing were performed as described previously (9, 10). Briefly, the prepared RNA was reverse transcribed and amplified using the Ovation RNA sequencing (RNA-seq) system v2 (NuGEN, CA), in accordance with the manufacturer’s instructions. Library preparation was conducted with 1 ng of amplified products using the Nextera XT library prep kit (Illumina, CA). The prepared library was loaded in the flow cell of a 500-cycle reagent kit v2, and paired-end sequencing was run on the MiSeq platform (Illumina). The total number of sequence reads was 5,053,188, and the average read length was 194.02 bp. All reads were quality trimmed, and adapter sequences were removed using CLC Genomics Workbench 9.0 (Qiagen, MD). The trimmed reads were assembled de novo using SPAdes 3.8.1 with default parameters (11). We used BLASTn to compare the generated de novo assembled contigs against the NCBI nucleotide sequence database to identify related foodborne viruses. Contigs with sequence similarity to related foodborne viruses were selected for mapping assembly using Burrows-Wheeler aligner (12). A fully assembled genome of HAV was identified as being 7,475 bp long, with a G+C content of 37.86%, and the mean read depth was 60. The genome comprised a 5′ untranslated region (UTR) (734 bp), a single open reading frame (ORF) (6,678 bp), and a 3′ UTR (63 bp). Phylogenetic analysis of the nearly complete genome sequence using Hepatitis A Virus Genotyping Tool version 1.0 (https://www.rivm.nl/mpf/typingtool/hav/) revealed that the HAV associated with the contaminated frozen raspberries belonged to subgenotype IB.

Data availability.

The genome sequence generated in this study was deposited in GenBank under the accession number MK829707. Raw sequence data were deposited in the NCBI Sequence Read Archive under the accession number PRJNA535492.