Complete Genome Sequence of a Chinese Virulent Porcine Epidemic Diarrhea Virus Strain

Abstract

CH/S is a virulent porcine epidemic diarrhea virus (PEDV) strain and is used as the virulent strain to evaluate the protection rates of vaccines against PEDV infection in China. Here, we report the complete genome sequence of strain CH/S, which may aid in understanding the molecular characteristics of this strain.

GENOME ANNOUNCEMENT

Porcine epidemic diarrhea virus (PEDV), a member of genus Alphacoronavirus (family Coronaviridae, order Nidovirales), is a single-stranded positive-sense RNA virus that causes an acute and highly contagious enteric disease characterized by severe enteritis, vomiting, and watery diarrhea in swine. Strain CH/S occurred in a swine breeding farm in 1986 in Shanghai and was confirmed as PEDV by electron microscopy, direct immunofluorescence testing, and serum neutralization testing (4). To date, the complete genome sequence of only Belgian strain CV777 has been reported (9). It is thus necessary to analyze the complete genome sequence of CH/S and understand its molecular characteristics.

The 5′ and 3′ ends of the genome of CH/S were confirmed by using a Smarter rapid amplification of cDNA ends (RACE) kit (Clontech, Japan), and the other parts were generated by 12 overlapping cDNA fragments to encompass the entire genome and determined by genome walking sequencing. The coronavirus genome is a single-stranded positive-sense RNA genome of 27 to 32 kb that is infectious (2, 10). The complete genome sequence of CH/S is 28,026 nucleotides (nt) in length [excluding the poly(A) tail]. The 5′ untranslated region (UTR) existing on the extreme 5′ end of the genome has 292 nt and contains a positioned short AUG-initiated open reading frame (ORF), which consists of 39 nt (nt 95 to 133) and encodes a peptide of 12 amino acids. Such small ORFs are present in all coronaviruses sequenced so far (1, 3, 5, 8, 11, 12, 13, 15, 16, 17). The 3′ UTR existing on the extreme 3′ end of the genome is 334 nt in length and possesses an octameric sequence, GGAAGAGC, beginning at base 73 upstream from the poly(A) tail. The genome of CH/S contains six genes, the replicase (Rep), the spike (S), ORF3, envelope (E), membrane (M), and nucleoprotein (N) genes, arranged in the order 5′-Rep-S-ORF3-E-M-N-3′. Two long ORFs (ORF1a and ORF1b) are of 12,351 nt (nt 293 to 12643) and 8,037 nt (nt 12598 to 20634) in length and overlap by 46 nt. At the overlapping region, there is a specific seven-nucleotide “slippery” sequence (UUUAAAC) and a pseudoknot structure (ribosomal frameshifting signal), which are required for the translation of ORF1b (7).

Sequences at the 5′ end of each gene represent signals for the transcription of subgenomic mRNAs of coronavirus (10, 14). These sequences, known as transcription-regulating sequences (TRSs), include a stretch of a highly conserved sequence designated the core sequence (CS), located at sites immediately upstream of most of the genes (7). The CSs in the genome of CH/S are the hexameric motifs 5′-XUA(A/G)AC-3′. The sequence CUAAAC found at the starting sites of gene 1 and the N gene, the GUAAAC sequence found at the starting site of the S gene, the CUAGAC sequence found at the starting sites of gene 3 and the E gene, and the AUAAAC sequence found at the starting site of the M gene are identical to those found in the same sites in the CV777 strain (6).

It is hoped that these data will not only facilitate future investigations of the molecular pathogenesis of PEDV but also contribute to the elucidation of the structure and function of the coronavirus gene-derived products.

Nucleotide sequence accession number.

The complete genome sequence of PEDV strain CH/S was deposited in GenBank under accession no. JN547228.