Complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus

Abstract

The complete nucleotide sequence of polyprotein gene 1 and the assembled full-length genome sequence are presented for turkey coronavirus (TCoV) isolates 540 and ATCC. The TCoV polyprotein gene encoded two open reading frames (ORFs), which are translated into two products, pp1a and pp1ab, the latter being produced via -1 frameshift translation. TCoV polyprotein pp1a and pp1ab were predicted to be processed to 15 non-structure proteins (nsp2-nsp16), with nsp1 missing. ClustalW analysis revealed 88.99% identity and 96.99% similarity for pp1ab between TCoV and avian infectious bronchitis virus (IBV) at the amino acid level. The whole genome consists of 27,749 nucleotides for 540 and 27,816 nucleotides for ATCC, excluding the poly(A) tail. A total of 13 ORFs were predicted for TCoV. Five subgenomic RNAs were detected from ATCC-infected turkey small intestines by Northern blotting. The whole genome sequence had 86.9% identity between TCoV and IBV, supporting that TCoV is a group 3 coronavirus.

Fig. 1

(a) Genome organization of TCoV. Diagram shows putative ORFs. UTRs, leader (L), and TRS are not to scale. Above, the genome organization of TCoV are shown the predicted five sgRNA in relative sizes. Genome organization of IBV-Beaudette (NC_001451) is displayed below for comparison with that of TCoV. (b) Mapping of TCoV polyprotein. Predicted non-structure proteins (nsp2–nsp16) for pp1a are shown in relative sizes (bottom panel). Nsp1 is missing from TCoV and nsp11 contains only 23 aa. The sequence is for ATCC isolate (accession number EU022526).

Fig. 2

Phylogenetic relationship between TCoV and other coronaviruses pp1ab. The map was generated by Phylip at http://www.genebee.msu.su/services/phtree_full.html. Sequences of pp1ab of coronaviruses were used for analysis. The sequences accession numbers are listed at the end of the article.

Fig. 3

Northern blotting of total RNA isolated from TCoV infected turkey small intestines. Total RNA (10 μg) was isolated from mock or ATCC infected turkey small intestines 3 days post infection, separated on 1% agarose gel, transferred onto nitrocellulose membrane, and detected with 32P labeled PCR probe corresponding to N gene. The sizes (kb) on the right indicate the predicted size of genomic and subgenomic RNA. X indicates assumed DI RNA.

Fig. 4

Sequences flanking TRS region for TCoV sgRNA. The partial sequences display each sgRNA for ATCC isolate. For each sgRNA, partial genomic leader (gL) and body (gS, gE, gM, g5, and gN) sequences are displayed above and below sgRNA. The star (*) indicates identical nucleotide and the box indicates TRS region where template switch is assumed to occur.