Genomic Sequence of a Megrivirus Strain Identified in Laying Hens in Brazil

A new strain of chicken megrivirus was identified in fecal samples of layer chickens in a commercial flock in Minas Gerais, Brazil. It is most closely related to the family Picornaviridae, genus Megrivirus, species Melegrivirus A, and has an overall nucleotide identity of up to 85.1% with other megrivirus strains.

T he Picornaviridae family currently consists of 40 genera (1), with at least 13 of these genera identified from avian sources (2). Avian picornaviruses of the genera Megrivirus, Gallivirus, and Avisivirus have been frequently identified in healthy and diseased poultry (3)(4)(5). Recent metagenomics studies focusing on the poultry gut virome have identified an ever-growing number of enteric picornaviruses in fecal samples from broiler chickens (3)(4)(5)(6)(7)(8). In this study, two pooled fecal samples (2 g of fresh fecal material from five sites each in two sheds) were collected from a 102-week-old and a 57-weekold commercial laying flock in Minas Gerais State, Southeast Brazil, in August 2012. No health problems had been reported in the examined flocks.
Total nucleic acid was extracted from a 1:5 dilution of fecal samples using the MagMAX pathogen RNA/DNA kit (Thermo Fisher Scientific, MA) with KingFisher (Thermo Fisher Scientific) (9). Double-stranded cDNA was synthesized using the NEXTflex rapid transcriptome sequencing (RNA-seq) kit (Bioo Scientific Corp., TX). The sequencing library was prepared using the Nextera XT DNA library preparation kit (Illumina, CA) with dual indexing. The pooled libraries were sequenced on an Illumina MiSeq platform at the next-generation sequencing (NGS) lab located in the Veterinary Diagnostic Laboratory at Iowa State University, using the 300-cycle v2 reagent kit (Illumina) to generate 150 base-pair paired-end reads by following standard Illumina protocols. Raw reads of each sample were demultiplexed automatically on the MiSeq platform with the default settings.
Eight contigs of chicken picornaviruses were assembled from the raw data (SRA accession no. SRR8290010) with an N 50 value of 1,268 bp, and specific primers (provided upon request) were then designed to perform reverse transcriptase PCR (RT-PCR) and close the gaps by sequencing of the Nextera XT DNA library of the amplicon on a MiSeq platform (SRA accession no. SRR8290011). Finally, a near-complete sequence of a megrivirus, chicken picornavirus MG/9567, was identified with a genome length of 9,567 nucleotides. The genome had 78.3% nucleotide similarity with the reference Megrivirus C isolate BL 21 (GenBank accession no. KF961186).
The chicken picornavirus MG/9567 genome shared the highest deduced polyprotein amino acid similarity (94.9%) with chicken picornavirus 5 isolate 27 (GenBank accession no. KF979336), which originated from a chicken sample collected in Hong Kong in 2008, and shared 87.7% amino acid (aa) identity with chicken megrivirus strain 3R, collected from a healthy chicken in Brazil in 2018 (GenBank accession no. MG846465). The genome nucleotide similarities were 85.2% and 78.3%, respectively.
Data availability. The genome sequence reported here has been deposited in GenBank under the accession no. MH806866. The sequence data are available under SRA accession no. SRR8290010 and SRR8290011.

ACKNOWLEDGMENTS
We thank Maria Isabel Guedes, Isabela Rehfeld, and Leonardo Lara for providing chicken samples.
Z. I. P. Lobato received a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq). T. Opriessnig was funded by Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grants BB/J004324/1 and BBS/E/D/20241864, awarded to the Roslin Institute. This study received no funding from outside sources.