Identification of a novel picornavirus in healthy piglets and seroepidemiological evidence of its presence in humans

Abstract

In this study, we describe a novel porcine parechovirus-like virus (tentatively named PLV-CHN) from healthy piglets in China using 454 high-throughput sequencing. The complete genome of the virus comprises 6832 bp, encoding a predicted polyprotein of 2132 amino acids that is most similar to Ljungan virus (32% identity). A similar virus that belongs to a novel Picornaviridae genus, named swine pasivirus 1 (SPaV-1), was reported during the preparation of this paper. Sequence analysis revealed that PLV-CHN and SPaV1 shared 82% nucleotide identity and 89% amino acid identity. Further genomic and phylogenetic analyses suggested that both SPaV1 and PLV-CHN shared similar genomic characteristics and belong to the same novel Picornaviridae genus. A total of 36 (20.0%) fecal samples from 180 healthy piglets were positive for PLV-CHN by RT-PCR, while no fecal samples from 100 healthy children and 100 children with diarrhea, and no cerebrospinal fluid samples from 196 children with suspected viral encephalitis, was positive for the virus. However, Western blot and enzyme-linked immunosorbent assays using recombinant PLV-CHN VP1 polypeptide as an antigen showed a high seroprevalence of 63.5% in the healthy population. When grouped by age, the antibody-positivity rates showed that the majority of children under 12 years of age have been infected by the virus. It was suggested that PLV-CHN, SPaV1, or an as-yet-uncharacterized virus can infect humans early in life. Thus, investigation of the role of this novel virus is vital.

Figure 1. List and positions of primers used in this study.

The first and last nucleotide positions (corresponding to the complete nucleotide sequence of PLV-CHN) of the primers are given in the primer names (X-X).

Figure 2. Phylogenetic analysis of amino acid sequences of partial 3D sequences (333 bp) of 36 PLV-CHN strains from porcine fecal samples.

The tree was constructed using the neighbor-joining method. The sequences screened in this study are indicated by “▴”.

Figure 3. Phylogenetic analysis of nucleotide acid sequences of various PLV-CHN regions.

(A) P1 region (B) P2 region (C) P3 region (D) polyprotein. The tree was constructed using the neighbor-joining method by MEGA ver. 4.1 with 1000 bootstrap replicates. The virus in this study is indicated by “•”. Bootstrap values are shown on the branches.

Figure 4. Western blot of purified His-tagged recombinant VP1 polypeptide of PLV-CHN with serum samples from healthy children and adults.

Immmunoreactive protein bands with the expected size of recombinant PLV-CHN VP1 (∼30 kDa) were detected with sera from both an immunized mouse (lane 1) and OD₄₅₀ ≥0.355 (Lane 2, 3, 4, 5, 6 and 7), OD₄₅₀ ≤0.305 serum samples were negative (lanes 8 and 9).

Figure 5. Trends in positivity rates for antibody to PLV-CHN in humans of various age groups.