Natural occurrence and characterization of two internal ribosome entry site elements in a novel virus, canine picodicistrovirus, in the picornavirus-like superfamily

Abstract

Dicistroviridae and Picornaviridae are two phylogenetically related families of positive-sense single-stranded RNA viruses in the picornavirus-like superfamily with similar gene contents but different genome organizations and hosts. In a surveillance study involving 1,472 samples from 368 dogs over a 22-month period, we identified a novel picornavirus-like virus from 47 fecal and urine samples by the use of reverse transcription-PCR (RT-PCR). Sequencing and phylogenetic analysis of three complete genomes revealed that, although it seemed that the virus was most closely related to other picornaviruses, P1, P2, and P3 of the virus possessed very low amino acid identities of <30% to those of all other known picornaviruses and that the amino acid identities between the 3D(pol) and 2C of the virus and the RNA-dependent RNA polymerases and helicases of all other picornaviruses were <35%. Distinct from other picornaviruses, the genomes of the virus contain two putative internal ribosome entry sites (IRESs) and two open reading frames, encoding two polyprotein precursors (844 and 1,406 amino acids), separated by an intergenic region (IGR) of 588 bases. A dual-luciferase activity assay using DNA and RNA transfection revealed that both IRESs were functional. Quantitative RT-PCR showed that numbers of viral RNAs ranged from 7.55 × 10(6) to 1.26 × 10(9) copies/ml of urine and 1.82 × 10(6) to 4.97 × 10(10) copies/ml of fecal sample. This is the first report of the natural occurrence of two functional IRESs in nondicistroviruses. Based on our results, we have proposed a novel species, canine picodicistrovirus (CPDV), to describe this novel member of the picornavirus-like superfamily, which could represent a novel family of viruses.

Fig 1

Schematic representation of the five bicistronic reporter constructs used. pRluc-Fluc, backbone and negative control; pRluc-HCV 5′ IRES-Fluc, positive control; pRluc-EMCV 5′ IRES-Fluc, positive control; pRluc-5′ IRES-Fluc, 5′ IRES of CPDV inserted into pRluc-Fluc backbone; pRluc-IGR IRES-Fluc, intergenic region of CPDV inserted into pRluc-Fluc backbone.

Fig 2

Genome organization of CPDV, dicistrovirus, and picornavirus.

Fig 3

(A) Pairwise alignment between the 5′UTR of CPDV and that of poliovirus. (B) Pairwise alignment between the IGR of CPDV and the 5′UTR of poliovirus.

Fig 4

IRES structures of the 5′ UTR (A) and the IGR (B) in CPDV. The pyrimidine tract and the AUG start codon are underlined.

Fig 5

Maximum-likelihood trees for 3D^pol/RdRp (A) and 2C/S3H (B) of CPDV and representative viruses of the picornavirus-like superfamily. Midpoint rooting was used. aLRT branch support is shown where values are greater than 0.750. The trees are drawn to scale, and scale bars indicate 0.8 (A) and 0.7 (B) amino acid substitutions per site as inferred according to the RtREV+I+G+F model with 4 gamma categories. The three CPDV strains are shown in bold. dsRNA, double-stranded RNA.

Fig 6

Luciferase activities of MDCK cells after transfection of bicistronic construct containing 5′ and IGR IRESs. Vertical bars represent means ± standard deviations of the results of three independent experiments. RF (negative control), pRluc-Fluc; 5′ IRES, pRluc-5′ IRES-Fluc; IGR IRES, pRluc-IGR IRES-Fluc; HCV (positive control), pRluc-HCV 5′ IRES-Fluc.

Fig 7

Luciferase activities of MDCK cells after transfection of in vitro-transcribed capped RNAs. Vertical bars represent means ± standard deviations of the results of three independent experiments. RF, negative control; EMCV, EMCV IRES (positive control).

Fig 8

Luciferase activities of MDCK cells after transfection of pGL3-enhancer experimental constructs with a cotransfection control. Vertical bars represent means ± standard deviations of the results of three independent experiments. Negative control, empty pGL3-enhancer vector; 5′ IRES, pGL3E/5′ IRES; IGR IRES, pGL3E/IGR IRES; positive control, vector containing promoter of the surface gene of hepatitis B virus.