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

Figures

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  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.

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  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.

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  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.

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  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.