Identification and Genome Characterization of the First Sicinivirus Isolate from Chickens in Mainland China by Using Viral Metagenomics

Abstract

Unlike traditional virus isolation and sequencing approaches, sequence-independent amplification based viral metagenomics technique allows one to discover unexpected or novel viruses efficiently while bypassing culturing step. Here we report the discovery of the first Sicinivirus isolate (designated as strain JSY) of picornaviruses from commercial layer chickens in mainland China by using a viral metagenomics technique. This Sicinivirus isolate, which contains a whole genome of 9,797 nucleotides (nt) excluding the poly(A) tail, possesses one of the largest picornavirus genome so far reported, but only shares 88.83% and 82.78% of amino acid sequence identity to that of ChPV1 100C (KF979332) and Sicinivirus 1 strain UCC001 (NC_023861), respectively. The complete 939 nt 5'UTR of the isolate strain contains at least twelve stem-loop domains (A-L), representing the highest set of loops reported within Sicinivirus genus. The conserved 'barbell-like' structure was also present in the 272 nt 3'UTR of the isolate as that in the 3' UTR of Sicinivirus 1 strain UCC001. The 8,586 nt large open reading frame encodes a 2,862 amino acids polyprotein precursor. Moreover, Sicinivirus infection might be widely present in commercial chicken farms in Yancheng region of the Jiangsu Province as evidenced by all the tested stool samples from three different farms being positive (17/17) for Sicinivirus detection. This is the first report on identification of Sicinivirus in commercial layer chickens with a severe clinical disease in mainland China, however, further studies are needed to evaluate the pathogenic potential of this picornavirus in chickens.

Fig 1. Sequence classification of obtained reads based on BLASTx.

Percentages of reads with similarity to those of eukaryotes, bacteria, phages, viruses, unclassifiable sequences (other, including plasmid vector sequences) and to unknown sequences (Left). Percentages of viral sequence reads are classified by viral types (Right).

Fig 2. Genome organization and the conserved picornaviral motifs.

(A) Predicted genome organization possesses conserved picornaviral motifs of Sicinivirus JSY (KP779642), ChPV1 100C (KF979332), ChPV1 55C (NC_024765), Sicinivirus 1 UCC001 (NC_023861) and Sicinivirus UCC1 (KF366619); the predicted cleavage sites of Sicinivirus JSY are indicated above the junction region. (B) Amino acid sequence alignment of Sicinivirus, Turdivirus 1 (NC_014411), Turkey gallivirus (NC_018400), Feline sakobuvirus A isolate FFUP1 (NC_022802), Aichi virus (NC_001918) and Salivirus A isolate 02394–01 (NC_012986), the identified motifs are indicated with jacinth boxes.

Fig 3. Predicted RNA secondary structure of the Sicinivirus JSY 5' UTR as determined by Mfold and RNAfold.

The complete structure of the 5'UTR (A to L, indicates the type II IRES) has been annotated (inset). Two GNRA motifs and the pyrimidine-rich region are illustrated with gray background boxes.

Fig 4. Predicted RNA secondary structure and the conserved 3' UTR motifs.

(A) Complete structure of the 3'UTR of Sicinivirus JSY (KP779642), the Poly(Y) tract and the 'barbell-like' structure are illustrated with gray background boxes. (B) Nucleotide sequence alignment of Sicinivirus, Turdivirus 1 (NC_014411), Turkey gallivirus (NC_018400), Feline sakobuvirus A isolate FFUP1 (NC_022802), Aichi virus (NC_001918) and Salivirus A isolate 02394–01 (NC_012986), the conserved regions are indicated with jacinth boxes.

Fig 5. Amino acid sequence alignment of VP1 polypeptide of the five Sicinivirus.

Drug-binding pocket sites of Sicinivirus JSY (KP779642), ChPV1 100C (KF979332), ChPV1 55C (NC_024765), Sicinivirus 1 UCC001 (NC_023861) and Sicinivirus UCC1 (KF366619) are illustrated with jacinth frames, I118 and L120 positions are labeled with jacinth frames in bold. rhv_like capsid domain (cd00205) in Sicinivirus JSY (KP779642) is indicated with yellow background.

Fig 6. Phylogenetic analyses of the Sicinivirus strain JSY.

Phylogenetic relationship between Sicinivirus strain JSY and the representative members of the 29 officially recognized genera based upon the complete amino acid sequences of picornavirus P1, 2C, and 3D coding regions. The phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) [17] applying the maximum-likelihood method based on the JTT matrix-based model [18], the robustness of the phylogenetic constructions was evaluated by bootstrapping with 1,000 replicates, initial trees for the heuristic search were obtained automatically by applying neighbour-join and BioNJ algorithms.

Fig 7. Detection of Sicinivirus in clinical stool samples by RT-PCR.

M, Molecular marker D2000; Lanes 1–9, stool samples from farm A; Lanes 10–14, stool samples from farm B; Lanes 15–17, stool samples from farm C; Lanes 18, negative control. The fecal sample that was used to perform the metagenomic analysis is indicated with an orangey-red asterisk (Lane 4).