Screening of Feral and Wood Pigeons for Viruses Harbouring a Conserved Mobile Viral Element: Characterization of Novel Astroviruses and Picornaviruses

A highly conserved RNA-motif of yet unknown function, called stem-loop-2-like motif (s2m), has been identified in the 3′ end of the genomes of viruses belonging to different RNA virus families which infect a broad range of mammal and bird species, including Astroviridae, Picornaviridae, Coronaviridae and Caliciviridae. Since s2m is such an extremely conserved motif, it is an ideal target for screening for viruses harbouring it. In this study, we have detected and characterized novel viruses harbouring this motif in pigeons by using a s2m-specific amplification. 84% and 67% of the samples from feral pigeons and wood pigeons, respectively, were found to contain a virus harbouring s2m. Four novel viruses were identified and characterized. Two of the new viruses belong to the genus Avastrovirus in the Astroviridae family. We propose two novel species to be included in this genus, Feral pigeon astrovirus and Wood pigeon astrovirus. Two other novel viruses, Pigeon picornavirus A and Pigeon picornavirus B, belong to the Picornaviridae family, presumably to the genus Sapelovirus. Both of the novel picornaviruses harboured two adjacent s2m, called (s2m)2, suggesting a possible increased functional effect of s2m when present in two copies

Diagram of the organization of the 3′ part of the avastrovirus genomes.

<p>The nucleotide positions of the start and stop of each open reading frame are shown relative to the beginning of the genome for TAstV-1 and ANV-1. Feral pigeon AstV (594, 6/594, 9) represent two different virus strains.</p

Model of the predicted secondary structure of PiPV-B IRES.

<p>Domains are labelled II and III, stem-loops are labelled IIId, IIIe etc. Stem 1 and stem 2 are elements of the pseudoknot. The underlined AUG triplet in position 429 is the first AUG codon after the pseudoknot. Shaded rectangles indicate bases in domain III that are conserved in the IRES type IV elements. The predicted secondary structure upstream of stem-loop IIId (the sequence between nucleotide 125 and 329) is not shown. The structure was predicted using Mfold.</p

Alignment of the genomic 3′ –end of the two subtypes of the novel pigeon picornavirus, the astrovirus BatAstV-LD77 and the coronavirus ZBCoV.

<p>The two adjacent s2m are shown in red. Stop codon of the polyprotein is underlined (part of the first s2m for PiPV-B). Dots indicate nucleotides identical to the PiPV-A, gaps are shown as dashes.</p

Phylogenetic analysis of A) the complete capsid protein (ORF2) and B) part of the polymerase protein (about 300 amino acids in the C-terminal part of ORF1b) of the astrovirus family.

<p>The trees were constructed by the neighbour-joining method using the Jones-Taylor-Thornton matrix-based model of amino acid substitution. Bootstrap values (>70%) from 1000 replicates are shown. Both trees are plotted to the same scale. The novel isolates are indicated by bold lettering. The viruses marked with ▾ do not harbour s2m.</p

Nucleotide alignment of s2m from various astroviruses (AstV), picornaviruses (PV), coronaviruses (CoV) and noroviruses (NoV) species.

<p>The 24 nucleotides that are absolutely invariant, are marked by asterisks above the sequences.</p

Phylogenetic analysis of A) the complete polymerase protein (3D) and B) the complete P1 capsid region of the picornavirus family.

<p>The trees were constructed by the neighbour-joining method using the Jones-Taylor-Thornton matrix-based model of amino acid substitution. Bootstrap values (>70%) from 1000 replicates are shown. Both trees are plotted to the same scale. The novel isolates are indicated by bold lettering. The viruses marked with ▴ harbour s2m.</p

Sequence of primers used in this study.

<p>*From Jonassen <i>et al.</i> 2005 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025964#pone.0025964-Jonassen2" target="_blank">[9]</a>.</p><p>**From Tang <i>et al.</i> 2005 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025964#pone.0025964-Zuker1" target="_blank">[31]</a>.</p

Virus strains included in the analyses.

<p>*Viruses harbouring s2m.</p><p>**Genus assignment suggested in this paper.</p><p>***The ORF1b sequence was not available.</p