Genome Sequences of Seven Megrivirus Strains from Chickens in The Netherlands

ANNOUNCEMENT

Viruses in the Picornaviridae family possess a positive-sense single-stranded RNA genome and cause a broad range of diseases in humans and animals (1). Among the 63 genera in this family, at least 15 genera, including Megrivirus, have been identified from avian sources (2). Megriviruses have been detected in both healthy and diseased poultry (3, 4) and are suspected to cause transmissible viral proventriculitis in chickens (5) and hepatitis in turkeys (6). Here, we report 7 nearly complete megrivirus genome sequences identified from 7 out of 8 pooled chicken fecal samples that we sequenced as part of a virome profiling study of farm animals. The samples consisted of pooled fresh fecal droppings from 3 to 4 chicks (Gallus gallus domesticus), collected in the same flock during weeks 4 and 5 of the production cycle from a broiler farm in June 2019 in The Netherlands (Table 1). No clinical signs in the examined flock were reported at the time of sampling.

The genome sequences of the virus were generated using metagenomic sequencing. The fecal suspension (30% [wt/vol] in phosphate-buffered saline) was centrifuged for 10 min at 10,000 × g. The supernatant was collected and treated with TURBO DNase (Invitrogen). Virion-protected nucleic acid was extracted using the QIAamp viral RNA minikit (Qiagen). Reverse transcription was performed using nonribosomal random hexamers (7) and SuperScript III reverse transcriptase (Invitrogen), followed by second-strand cDNA synthesis using Klenow fragment 3′–5′ exo- (New England BioLabs). The resulting DNA was subjected to library preparation using a Nextera XT DNA library preparation kit (Illumina) following the manufacturer’s instructions. The library was then purified and size selected using AMPure XP magnetic beads (Beckman Coulter). The final library was sequenced in paired-end format on the Illumina MiSeq platform using reagent kit v3 (600 cycles; Illumina). The total number of reads generated per sample ranged between 2,020,090 and 4,765,832. The raw reads were analyzed using the automated pipeline Genome Detective Virus Tool v1.126 (8), which utilizes Trimmomatic (9) for adapter and quality trimming, DIAMOND (10) for viral read identification, and metaSPAdes (11) for de novo assembly of the sorted viral reads. De novo-assembled genome sequences were inspected and annotated using Geneious v2020.2.3 (12). The lengths of the 7 nearly complete megrivirus genomes range from 8,993 to 9,592 nucleotides (nt). The depths of coverage range from 34× to 576×. According to BLAST searches, these strains shared 83% to 84% identity at the nucleotide level with chicken picornavirus 5 isolate 27C from Hong Kong (2008; GenBank accession number KF979336), chicken megrivirus strain MG9567 from Brazil (2012; MH806866), and Picornaviridae sp. isolates w3chi090pic1 and w3chi091pic1 from China (2018; MT138368 and MT138369, respectively). These reported genome sequences shared 92% to 93% identity at the amino acid (aa) level when comparing viral polyprotein amino acid sequences. The pairwise nucleotide difference of the complete coding region among the 7 strains ranges from 2 to 18 nt; the pairwise amino acid difference ranges from 0 to 4 aa.

To conclude, we report 7 megrivirus genome sequences identified in The Netherlands. The prevalence of these viruses might have been overlooked in the poultry population thus far. Future studies should investigate the prevalence and diversity of megriviruses and their potential clinical implications.