Characterization of porcine sapelovirus isolated from Japanese swine with PLC/PRF/5 cells

Abstract

Porcine sapelovirus (PSV) is a causative agent of neurological disorders, fertility disorders and dermal lesions of swine. In this study, we isolated two PSV strains, Jpsv477 and Jpsv1315, from swine faecal specimens using a PLC/PRF/5 cell culture system. The PSV infection of PLC/PRF/5 cells induced a cytopathic effect (CPE). Two types of virus particles with identical diameter (~35 nm) but different densities (1.300 and 1.285 g/cm ) were observed in the cell culture supernatants. Analysis of the entire genome sequence of Jpsv477 and Jpsv1315 revealed that both strains possess 7,558 nucleotides and the poly (A) tail and have a typical PSV genome organization consisting of a 5' terminal untranslated region (5'UTR), a large open reading frame (ORF), and a 3' terminal untranslated region (3'UTR). The ORF encodes a single polyprotein that is subsequently processed into a leader protein (L), four structural proteins (VP1, VP2, VP3 and VP4) and seven functional proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). The structural proteins VP1, VP2, VP3 and VP4 have molecular masses of ~35, ~26, ~25 and ~6 kDa. The N-terminal amino acid sequence analysis of VP1, VP2, VP3 and VP4 confirmed that the cleavage sites between VP4 and VP2, VP2 and VP3, and VP3 and VP1 are K/A, Q/G and Q/G, respectively. We further confirmed that HepG2/C3A, Vero E6 and primary green monkey kidney cells (PGMKC) were also susceptible to PSV infection. The stability assay demonstrated that PSV was inactivated by heating at 60°C for 10 min or 65°C for 5 min. The virus also lost infectivity by incubation with 62.5 ppm of NaClO for 30 min.

High prevalence, genetic diversity and a potentially novel genotype of Sapelovirus A (Picornaviridae) in enteric and respiratory samples in Hungarian swine farms

“…The viral polyprotein of sapeloviruses is build up from 12 mature peptides located in consecutive order as l-P1(VP4-VP2-VP3-VP1)-P2(2A-2B-2C)-P3(3A-3B-3C-3D) (L=leader peptide) [13,14]. The N-terminal cleavage sites of the P1 capsid proteins of PSVs are experimentally determined [18]. The VP1 capsid protein, which contains major antigenic epitopes, is generally used for sero/genotyping of sapelovirus strains [10,[18][19][20].…”

Porcine sapelovirus enters PK-15 cells via caveolae-dependent endocytosis and requires Rab7 and Rab11

“…PSV can be readily cultivated in pig kidney cells, including PK-15, IBRS-2 and LLC-PK (Lan et al, 2011;Kim et al, 2016). Among cell lines from other species, two PSV strains Jpsv477 and Jpsv1315 are found to replicate in human hepatocarcinoma cell line (PLC/PRF/5 and HepG2/ C3a) and green monkey kidney cell line (Vero E6 and PGMKC) (Bai et al, 2018). Infections by PSV is involved in a wide spectrum of symptoms ranging from asymptomatic infection to clinical symptoms including acute diarrhea, polioencephalomyelitis, pneumonia and reproductive disorders (Lan et al, 2011;Arruda et al, 2017).…”

Sapelovirus

“…PSVs belong to the genus Sapelovirus under the family Picornaviridae. The size of virion particles is small (~35 nm) and they are non-enveloped possessing icosahedral symmetry with a distinct surface morphology visible under the electron microscope (Bai et al 2018). Mature infectious virions are stable in the environment and could resist acidic pH.…”

“…PSV (strain csh) was first isolated from China in 2011 (Lan et al 2011). As on date, 50 complete genomic sequences of PSV strains have been published from China, South Korea, Japan, Germany, France, India and the United Kingdom (Lan et al 2011;Chen et al 2012;Schock et al 2014;Son et al 2014;Ray et al 2018;Piorkowski et al 2018;Bai et al 2018). Different virus strains exhibit affinity towards different target organs, as the PSV-csh strain (China, Shanghai) and PSV-G5 (the United Kingdom) affect mostly nervous system (Lan et al 2011;Schock et al 2014), whereas strains like KS0515, KS04105 and KS0552179 and Chinese PSV strain YC2011 are associated with diarrhoea.…”

“…Additionally, monkey kidney (Vero) and baby hamster kidney fibroblasts are also susceptible to PSVs where a CPE characterised by cell shrinkage, rounding and detachment is commonly seen after 3-5 passages. Recently, a human hepatocarcinoma cell line, PLC/ PRF/5, is normally used to isolate the hepatitis E virus, from swine faecal specimens serendipitously isolated from Japanese PSV strain (Bai et al 2018). Cultured PSVs are generally identified by using virus neutralisation (VN) (Sozzi et al 2010) and immunofluorescence antibody (IFA) assays (Son et al 2014).…”