Comparative complete genome analysis of chicken and Turkey megriviruses (family picornaviridae): long 3' untranslated regions with a potential second open reading frame and evidence for possible recombination

Abstract

Members of the family Picornaviridae consist of small positive-sense single-stranded RNA (+ssRNA) viruses capable of infecting various vertebrate species, including birds. One of the recently identified avian picornaviruses, with a remarkably long (>9,040-nucleotide) but still incompletely sequenced genome, is turkey hepatitis virus 1 (THV-1; species Melegrivirus A, genus Megrivirus), a virus associated with liver necrosis and enteritis in commercial turkeys (Meleagris gallopavo). This report presents the results of the genetic analysis of three complete genomes of megriviruses from fecal samples of chickens (chicken/B21-CHV/2012/HUN, GenBank accession no. KF961186, and chicken/CHK-IV-CHV/2013/HUN, GenBank accession no. KF961187) (Gallus gallus domesticus) and turkey (turkey/B407-THV/2011/HUN, GenBank accession no. KF961188) (Meleagris gallopavo) with the largest picornavirus genome (up to 9,739 nucleotides) so far described. The close phylogenetic relationship to THV-1 in the nonstructural protein-coding genome region and possession of the same internal ribosomal entry site type (IVB-like) suggest that the study strains belong to the genus Megrivirus. However, the genome comparisons revealed numerous unique variations (e.g., different numbers of potential 2A peptides, unusually long 3' genome parts with various lengths of a potential second open reading frame, and multiple repeating sequence motifs in the 3' untranslated region) and heterogeneous sequence relationships between the structural and nonstructural genome regions. These differences suggest the classification of chicken megrivirus-like viruses into a candidate novel species in the genus Megrivirus. Based on the different phylogenetic positions of chicken megrivirus-like viruses at the structural and nonstructural genome regions, the recombinant nature of these viruses is plausible.

Importance: The comparative genome analysis of turkey and novel chicken megriviruses revealed numerous unique genome features, e.g., up to four potential 2A peptides, unusually long 3' genome parts with various lengths containing a potential second open reading frame, multiple repeating sequence motifs, and heterogeneous sequence relationships (possibly due to a recombination event) between the structural and nonstructural genome regions. Our results could help us to better understand the evolution and diversity (in terms of sequence and genome layout) of picornaviruses.

FIG 1

(A) Genome organization with conserved picornaviral motifs and predicted cleavage sites of chicken/B21-CHV/2012/HUN (B21-chicken, GenBank accession no. KF961186) and turkey/B407-THV/2011/HUN (B407-turkey, GenBank accession no. KF961188) megriviruses. Nucleotide (upper numbers) and amino acid (lower numbers) lengths are indicated in each gene box. The pairwise nucleotide (and amino acid) sequence identity values of each genome regions can be found between the two genome maps. The positions of the conserved picornaviral amino acid motifs of chicken/B21-CHV/2012/HUN are indicated with the first amino acid positions of the motif. The locations of the two consensus sequences of chicken/B21-CHV/2012/HUN acquired from pyrosequencing are indicated with gray bars. The steps (reaction no. R1 to R8) of the genome amplification and the locations of the primers are depicted as horizontal lines. (B) Amino acid sequence alignment of VP1-2B genome region of chicken/B21-CHV/2012/HUN (B21) and turkey/B407-THV/2011/HUN (B407). Predicted cleavage sites of the study strains (black arrows) together with the identified Hbox-NC motifs (dotted boxes) are indicated. The locations of the predicted cleavage sites (FQ ↓ AP) are illustrated with a gray background. The presumed cleavage site of genome region “2A0” of turkey/B407-THV/2011/HUN is indicated with a black box. Letters with a black background represent the identical amino acids of the alignment.

FIG 2

Predicted two-dimensional secondary RNA structure of the 5′ UTR IRES of chicken megrivirus (GenBank accession no. KF961186) with the positions of different nucleotides (in dark circles) indicated by black arrows and within frames of dashed lines compared to the IRES of turkey/B407-THV/2011/HUN (GenBank accession no. KF961188). The main domains (II and III), helical segments (III 1 to 4), and hairpins (III a; III c to f) and stems (Stem 1 and Stem 2) similar to those of the similarly labeled structures of hepacivirus/pestivirus-like type IVB IRES are depicted. Gray boxes indicate conserved motifs of IVB IRESes (23). White boxes indicate the conserved unpaired base pairs with respect to DHAVs within the middle loop of domain II. The sequence and location of the apical “8” structure also identified in other avian picornaviruses are indicated with a black frame box (24).

FIG 3

(A) The organization of the 3′ parts of chicken/B21-CHV/2012/HUN (B21, GenBank accession no. KF961186), chicken/CHK-IV-CHV/2013/HUN (CHK-IV, GenBank accession no. KF961187), turkey/B407-THV/2011/HUN (B407, GenBank accession no. KF961188), and pigeon mesiviruses (PiMEVs: GenBank accession no. KC876003 and KC811837). The presumed initiation codons in a weak Kozak context (optimal nucleotides are underlined) of ORF2 are shown in italics after the STOP codon (black star) of the end of the viral polyprotein (3D). Nucleotide (upper numbers) and amino acid (lower numbers) lengths of the presumed ORF2 are indicated in each gene box. Dotted lines corresponding to PiMEVs indicate the deletion of ORF2 region. The identified repeating elements of “unit A” and “unit B” (“B”) together with the location of AUG-rich genome parts are depicted within the gray and white arrows. The pairwise nucleotide (and amino acid) sequence identities between the different regions (indicated by double arrows) of 3′ genome parts are also illustrated. (B) Alignments of the “unit A” and “unit B” sequences of the study strains (B21; CHK-IV and B407), pigeon mesiviruses (PiMEV-1 and -2), and the members of genus Megrivirus (0091.1, 2993D, CHK148, and TRK22). Similar nucleotides are shaded. Asterisks indicate identical nucleotides.

FIG 4

Distance plot analysis based on the comparison of the complete genome of chicken/B21-CHV/2012/HUN (GenBank accession no. KF961186) to those of turkey/B407-THV/2011/HUN (B407-THV; GenBank accession no. KF961188) and pigeon mesivirus 1 (GenBank accession no. KC876003). The genome regions of chicken megrivirus-like picornavirus are drawn to scale. Dashed lines show the borders of 5′ UTR/P1, P1/2A, 2A/2B, and 3D/ORF2.

FIG 5

Phylogenetic positions of the study strains (indicated in bold and by black arrows) among the closest relatives of picornaviruses based on amino acid sequences of the different picornavirus coding regions: P1 (A), VP1 (B), 2C (C), and 3CD (D). Bars indicate 0.2 amino acid substitutions per site. The names of virus species in the different picornavirus genera are underlined.