Virome Analysis for Identification of a Novel Porcine Sapelovirus Isolated in Western China

Abstract

Diarrhea is one of the most important problems associated with the production of piglets, which have a wide range of possible pathogens. This study identified a strain of porcine sapelovirus (PSV) by using next-generation sequencing (NGS) technologies as the pathogen among fecal samples in a pig herd. Phylogenetic analysis showed that the PSV isolates shared a unique polyprotein and clustered with Chinese isolates identified before 2013. The PSV strain was then isolated and named GS01. The in vitro and in vivo biological characteristics of this virus were then described. Our pathogenicity investigation showed that GS01 could cause an inflammatory reaction and induce serious diarrhea in neonatal piglets. To our knowledge, this is the first isolation and characterization of PSV in western China. Our results demonstrate that the PSV GS01 strain is destructive to neonatal piglets and might show an expanded role for sapeloviruses. IMPORTANCE Porcine sapelovirus (PSV) infection leads to severe polioencephalomyelitis with high morbidity and mortality, resulting in significant economic losses. In previous studies, PSV infections were always subclinical or only involved a series of mild symptoms, including spinal cord damage, inappetence, diarrhea, and breathless. However, in our study, we isolated a novel PSV by virome analysis. We also determined the biological characteristics of this virus in vitro and in vivo. Our study showed that this novel PSV could cause an inflammatory response and induce serious diarrhea in neonatal piglets. To our knowledge, this is the first isolation and characterization of PSV in western China. These findings highlight the importance of prevention for the potential threats of PSV.

Keywords: biological characterization; diarrhea; next-generation sequencing; porcine sapelovirus.

FIG 1

Next-generation sequencing identified PSV in diarrhea samples. (A) Workflow of next-generation sequencing for the virome. (B) Statistical results for different RNAs obtained by NGS. (C) The viral annotation showed porcine sapelovirus A (PSV) was the only identified virus. (D) RT-PCR results confirmed that PSV genome RNA existed in diarrhea samples.

FIG 2

Isolation and characterization of PSV/GS01/China/2021. (A) LLC-PK1 cells grown to 90% confluence were inoculated with filtered diarrhea samples. The cells displayed obvious CPE from 48 hpi. IFA results showed there were positive signals distributed among cells. (B) RT-PCR detection of PSV RNA in the supernatant. (C) PSV GS01 strain was purified by plaque assay. (D) LLC-PK1 cells were infected with PSV GS01 at a multiplicity of infection of 0.01. The supertanant was harvested at 12, 24, 36, 48, 60, and 72 hpi to determine the growth kinetics curve for PSV GS01. (E) Electron microscopy of purified PSV GS01 virions.

FIG 3

Phylogenetic analysis of the PSV GS01 strain, comparing the nucleotide sequences of the polyprotein coding regions of PSV strains, conducted with MEGA7.0 software. (A) The phylogenetic tree was constructed using the maximum-likelihood method with 1,000 bootstrap replicates. The PSV strain isolated in this study is indicated by a red dot. All Chinese PSV isolates are indicated by a yellow box and South Korean PSV isolates are indicated by a blue box, while England-USA-India PSV isolates are indicated by a green box. All other members of the genus Sapelovirus are either unshaded or are indicated by a gray box. (B) Differences in the amino acid sequences for the polyproteins of the PSV GS01 strain and reference strains. The yellow bar indicates the Chinese strain. The green bar indicates the England-USA-India strains, while the blue bar indicates the South Korean strains.

FIG 4

Pathogenic analysis of PSV/GS01/China/2021-infected suckling piglets. Twelve-day-old piglets were orally inoculated with PSV GS01 (10⁶ TCID₅₀/pig) or an equal volume of MEM 199. (A to D) Clinical symptoms (A and B), clinical scores (C), and body temperature (D) were observed daily. (E) Fecal swabs were also collected for the detection of shedding virus. (F) At 7 dpi, all piglets were euthanized, and different organs were collected for viral load determinations. (G) Small intestines were also prepared for histopathological analysis.