Molecular detection of novel picornaviruses in chickens and turkeys

Abstract

Fecal specimens, including swabs and litter extracts, collected from chickens, domestic ducks, turkeys, and Canadian geese were tested using degenerate primers targeting regions encoding for conserved amino acid motifs (YGDD and DY(T/S)(R/K/G)WDST) in calicivirus RNA-dependent RNA polymerases. Similar motifs are also present in other RNA viruses. Two fecal specimens and 18 litter extracts collected from chickens and turkeys yielded RT-PCR products. BLAST search and phylogenetic analysis revealed that all amplicons represented picornaviruses that clustered into two major groups. Four chicken and one turkey samples yielded 250 bp amplicons with 84-91% nucleotide identity to the recently described turkey hepatitis viruses, while 280 and 283 bp amplicons obtained from 11 chicken and 4 turkey samples represented novel picornaviruses with the closest nucleotide identity to kobuviruses (54-61%) and turdiviruses (47-54%). Analysis of 2.2-3.2 kb extended genome sequences including the partial P2 (2C) and complete P3 (3A, 3B (VPg), 3C(pro), and 3D(pol)) regions of selected strains indicated that viruses yielding the 280/283 bp amplicons represent a putative new genus of Picornaviridae. The 3'-non-translated region (NTR) of the turkey hepatitis-like viruses described in this study was significantly longer (641-654 nt) than that of any of the other piconaviruses and included a putative short open reading frame (ORF). In summary, we report the molecular detection of novel picornaviruses that appear to be endemic in both chickens and turkeys.

Fig. 1

Phylogenetic analysis of partial 3D^pol protein sequences. The alignment contained 73 amino acids after columns with gaps were removed. The dendrogram was constructed by the Neighbor-Joining clustering method of MEGA version 3.1 with Poisson correction distance calculations and 1,025 bootstrap analyses

Fig. 2

Phylogenetic analysis of complete 3D^pol (a), and 3A–3C^pro (b) protein sequences. The length of the individual proteins are listed in Table 3. The dendrogram was constructed by the UPGMA clustering method of MEGA version 3.1 with Poisson correction distance calculations and 1,025 bootstrap analyses

Fig. 3

ORF maps of CHK148, CHK178, and TRK22 were generated in Omiga v2.0 software (Oxford Molecular Ltd, Oxford, UK). Dots represent start codons (ATG), arrow heads represent stop codons (TAA, TAG, or TGA). Frames are shown in both orientations (+1, +2, +3, −1, −2, −3). The 342 and 294 nt 3′-NTR putative ORFs are boxed (a). Alignment of deduced amino acid sequences of CHK148 and TRK22 3′-NTR putative ORFs were generated in GeneDoc. Identical or similar amino acids are shaded (b)

Fig. 4

RT-PCR products of individual chicken litter samples separated on 2% agarose gels and stained with ethidium bromide. M 1 kb Plus DNA ladder (Invitrogen); −C negative control (water); +C positive control (FT285 RNA)

Fig. 5

Secondary structural analysis of avian picornavirus 3′-NTRs. a Map of the 3′-NTRs and their relative sizes. The gray box indicates the region aligned by secondary structure predictions in b and c. b The secondary structure alignment of CHK 148 and TRK 22 3′-NTRs (terminal 240 nt), the predicted stem-loop structure (below right), and derived thermodynamic and statistical values for the proposed structure (∆g, avg covariation, avg bp probability and canonical bp). The structure shown was predicted by RNAForester using the Webserver for Aligning non-structural RNAs (WAR, http://genome.ku.dk/resources/war/). c The alignment based on structural prediction for the 3′-NTRs of CHK168, TRK 24, Aichi virus, and Turdivirus, the predicted stem-loop structure (bottom right) and values generated in the derivation of this structure (bottom left). Structural alignments were generated using the IUPAC nucleotide ambiguity system. Boxed sequences in alignments b and c correlate with the boxed loops in the secondary structure predications and are provided for reference and orientation

Fig. 6

Secondary structural analysis of the CHK 148 and TRK 22 3′-NTRs. a Map of the region of the CHK 148 and TRK 22 3′-NTRs used for analysis. The gray box indicates the region aligned by secondary structure predictions in b and c. b The secondary structure alignment of the 5′-regions of the CHK 148 and TRK 22 3′-NTRs (250 nt). Structural alignments were generated using the IUPAC nucleotide ambiguity system. As in Fig. 5, the structure shown was predicted by MAFFT-RNAalifold program using the Webserver for Aligning non-structural RNAs (WAR, http://genome.ku.dk/resources/war/). c The predicted stem-loop structure (below right), and derived thermodynamic and statistical values for the proposed structure (∆g, avg covariation, avg bp probability, and canonical bp). Boxed sequences in the alignment in b correlate with the boxed loop in the secondary structure predication in c and is provided for reference and orientation