key: cord-346264-hnwgzt6v
authors: Nagai, Makoto; Wang, Qiuhong; Oka, Tomoichiro; Saif, Linda J.
title: Porcine sapoviruses: Pathogenesis, epidemiology, genetic diversity, and diagnosis
date: 2020-05-26
journal: Virus Res
DOI: 10.1016/j.virusres.2020.198025
sha: 
doc_id: 346264
cord_uid: hnwgzt6v

The first porcine Sapovirus (SaV) Cowden strain was discovered in 1980. To date, eight genogroups (GIII, V-IX) and three genogroups (GIII, GV, and GVI) of porcine SaVs have been detected from domestic pigs worldwide and wild boars in Japan, respectively based on the capsid sequences. Although GIII Cowden strain replicated in the villous epithelial cells and caused intestinal lesions in the proximal small intestines (mainly in duodenal and less in jejunum), leading to mild to severe diarrhea, in the orally inoculated neonatal gnotobiotic pigs, the significance of porcine SaVs in different ages of pigs with diarrhea in the field is still undetermined. This is due to two reasons: 1) similar prevalence of porcine SaVs was detected in diarrheic and non-diarrheic pigs; and 2) co-infection of porcine SaVs with other enteric pathogens is common in pigs. Diagnosis of porcine SaV infection is mainly based on the detection of viral nucleic acids using reverse transcription (RT)-PCR and sequencing. Much is unknown about these genetically diverse viruses to understand their role in pig health and to evaluate whether vaccines are needed to prevent SaV infection.

The first porcine sapovirus (SaV), the Cowden strain was discovered by electron microscopy in the intestinal contents of a 27-day-old diarrheic nursing pig in the United State in 1980 (Saif et al., 1980 . Later it was classified as a genogroup III (GIII) SaV based on the complete genomic sequence analysis (Guo et al., 1999) .

Sapoviruses belong to the Sapovirus genus within the family Caliciviridae. They are non-enveloped viruses that possess a single-stranded, positive-sense RNA genome. Sapovirus particles are small and round with a diameter of 30-40 nm, exhibiting a typical star-of-David structure and cup-shaped surface depressions by electron microscopy (EM) or immune EM (IEM) (Alhatlani et al., 2015; Oka et al., 2015; Saif et al., 1980) . The genome length is 7-8,000 nucleotides (nt) excluding a 3'-end polyadenylated [poly(A)] tail. The 5'-end of the genome covalently links to a small virus-encoded protein (VPg). Sapovirus genomes have two overlapping open reading frames (ORFs): ORF1 and ORF2 (Oka et al., 2015) . ORF1 encodes the nonstructural proteins NS1-NS2-NS3 (putative NTPase)-NS4-NS5 (VPg)-NS6 (protease)-NS7 (RNA-dependent RNA polymerase: RdRp) and the capsid protein, VP1. ORF2 encodes the minor structural protein, VP2. Sapoviruses are genetically highly diverse and have been classified into 19 genogroups based on the VP1 sequences (Farkas et al., 2004; Oka et al., 2016; Scheuer et al., 2013; Yinda et al., 2017) . Among them, eight genogroups (GIII, GV, GVI, GVII, GVIII, GIX, GX, and GXI) and 3 genogroups (GIII, GV, and GVI) of SaVs have been detected from pigs and wild boars, respectively. In this review, we will summarize current knowledge on the pathogenesis of GIII Cowden strain, the epidemiology and genetic diversity of porcine SaVs, and the diagnosis of SaV infection in pigs.

The pathogenesis of most genogroups of porcine SaVs is unknown, except for GIII Cowden strain. The original field sample for the discovery of Cowden strain contained not only SaV particles (33 nm in diameter), but also rotavirus (55 nm and 70 nm in diameter for single-and double-capsid particles, respectively) and 23nm virus-like particles (Saif et al., 1980) . Saif et al. successfully removed rotavirus from the sample using selective membrane ultrafiltration before serial passage in gnotobiotic pigs. The 23-nm virus-like particles failed to replicate in the experimentally inoculated gnotobiotic pigs. At the 12th and above passages, the intestinal contents of the inoculated pigs contained only SaV particles by immune electron microscopy (IEM).

Flynn et al. (Flynn et al., 1988) studied the pathogenesis of porcine SaV Cowden strain in 4-day-old gnotobiotic pigs. They inoculated orally (PO) 18 pigs with the 12th passage of the virus, monitored clinical signs for 14 days, and euthanized pigs at different days post-inoculation (dpi) to examine histopathological changes compared to mock-inoculated pigs at similar ages. They found that SaV Cowden strain caused diarrhea in all the pigs by 3 dpi and persisted for 3-7 days. Most pigs had mild diarrhea during the infection and two pigs (2/18) had severe diarrhea at 4-5 dpi. Porcine SaV replicated in the villous epithelial cells, but not crypt cells, mainly in duodenum, less in jejunum and the least in ileum, but not in the large intestines as determined by immunofluorescent assays (IFA) using pig hyperimmune antisera against porcine SaV Cowden strain. Histologically, porcine SaV-inoculated pigs showed mild to severe villous atrophy in the duodenum with short and flat villi with areas of denudation. Typical SaV particles were detected from the feces and large intestinal contents (LIC) of SaV-inoculated pigs at 1-7 dpi using IEM. Later Guo et al. (Guo et al., 2001) found that infectious porcine SaV entered the blood stream during the acute phase of infection of orally inoculated gnotobiotic pigs. Using more sensitive Taqman real-time RT-PCR assay for the detection of porcine SaV RNA, fecal viral RNA shedding in virus-inoculated pigs started at 1-3 dpi, reached the highest titers [10.8±0.4 log10 genomic copy equivalent (GE)/mL] at 6-10 dpi and lasted for 30±4 days (Lu et al., 2016) . These observations are similar to the pathogenesis of bovine nebovirus, an enteric calicivirus belonging to the Nebovirus genus, that replicated in the proximal portion of the small intestine of calves (Hall et al., 1984; Smiley et al., 2002) .

The 13th passage of porcine SaV Cowden strain from the LIC of a gnotobiotic pig was successfully isolated in primary porcine kidney cells (Flynn and Saif, 1988) . For decades, PoSaV had been the only culturable enteric calicivirus until the successful cultivation of human noroviruses in B cells in 2014 and in intestinal stem cell-derived human enteroids in 2016 (Ettayebi et al., 2016 Jones et al., 2014) . Interestingly, J o u r n a l P r e -p r o o f initial adaptation of PoSaV in primary porcine kidney cells and the subsequent adaptation in LLC-PK, a continuous swine kidney epithelial cell line, required the supplementation of intestinal contents collected from mock-infected gnotobiotic pigs (Flynn and Saif, 1988; Parwani et al., 1991) . Later, the essential components in the intestinal contents for PoSaV replication were identified as bile acids (Chang et al., 2004) . Several human NoVs were grown in enteroids, which occurred exclusively when the culture medium was supplemented with bile or bile acids (Ettayebi et al., 2016) . Bile acids are synthesized in the liver, released with bile into the duodenal lumen, and most of them are recycled back into the liver in the ileum. So, the concentration of bile acids is much higher in the proximal intestine than in other organs and this may be one of the restriction factors for PoSaV replication mainly in duodenum.

Using the LLC-PK cell culture system, α2,3-and α2,6-linked terminal sialic acids on O-linked glycoproteins have been identified as the binding receptor for porcine SaV Cowden strain (Kim et al., 2014) . In the same study, it was also confirmed that these sialic acids are the binding receptor on piglet small intestinal tissues. Recently, the same group found that the tight junction (TJ) protein occludin is a functional receptor for porcine SaV in LLC-PK cells (Alfajaro et al., 2019) . The binding of porcine SaV or virus-like particles or bile acids alone to LLC-PK cells caused the dissociation of TJs and exposed occludin for PoSaV binding.

Then SaV and occludin form a complex and move to late endosomes via Rab5-and Rab7-dependent trafficking to start replication. The fact that more than one receptor is involved in SaV binding and entry is similar to findings for some other caliciviruses. Feline calicivirus (FCV) F9 strain uses a2,6-linked sialic acids on an N-linked glycoprotein as binding factors (Stuart and Brown, 2007) and junctional adhesion molecule 1 (JAM-1) for virus entry into cells (Makino et al., 2006) . Some murine noroviruses use sialic acid linked to ganglioside (CW3 like strains) or protein (CR3 strain) (Taube et al., 2009) for binding and protein receptors CD300lf and/or CD300ld for entry (Haga et al., 2016; Orchard et al., 2016) .

Taken together, cellular receptors (α2,3-and α2,6-linked sialic acids on O-linked glycoproteins and occludin) and bile acids are some of the restriction factors of porcine SaV replication in the proximal small intestine. It may also explain why porcine SaV Cowden strain did not replicate in other organs when piglets were inoculated intravenously (IV) with the virus (Guo et al., 2001) .

To date, porcine SaVs have been detected in the fecal samples of domestic pigs with and without diarrhea worldwide and of wild boars without diarrhea in Japan (Table 1) . Pigs in all growing stages can be infected with porcine SaVs; however, pigs are infected with SaVs early in life and post weaning pigs have higher SaV infection rates than other age groups (Barry et al., 2008; Jeong et al., 2007; Reuter et al., 2010; Valente et al., 2016; Wang, Q.H. et al., 2006a) . This can be explained by lactogenic immunity in nursing pigs and environmental factors (Valente et al., 2016) . Suckling piglets are protected passively by maternal antibodies against SaVs until weaning and post weaning pigs become susceptible to SaV infections when maternal antibodies decline (Alcalá et al., 2010; Barry et al., 2008; Martínez et al., 2006) . On the other hand, nutritional, environmental and social changes during the post-weaning period add significant stress on these animals (Valente et al., 2016) . Although porcine SaVs induced diarrhea and intestinal lesions in experimentally inoculated gnotobiotic piglets (Guo et al., 2001; Flynn et al., 1988; Lu et al., 2016) , there were no significant J o u r n a l P r e -p r o o f differences in the prevalence of SaVs between the same age groups of pigs with diarrhea and without diarrhea in the field (Table 1) . Currently, GIII is the predominant genogroup of porcine SaVs ( SaVs (GIII, GV, and GVIII) in both trees, suggesting that these porcine SaVs possess a common ancestor and are distantly related to other SaVs in the porcine population ( Fig. 1) . Although the end of VP2 of porcine SaVs as well as other SaVs is highly variable (Table 2) , neither deletion nor insertion in the region, like that of the S INDEL strains of porcine epidemic diarrhea virus, is reported.

The Currently, conventional or real-time RT-PCR are the most widely used routine laboratory diagnostic assays for the detection of porcine SaVs from fecal samples, with the advantages of specificity, high sensitivity, broad reactivity, and convenience. Many primers used for the screening of porcine SaVs have been designed (Table 3) . Almost all primers are designed targeting the partial RdRp region, which presents conserved motifs that are useful for molecular diagnosis of genetically highly diverse SaVs ( 

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Salaries and research support for QW and LJS were provided by state and federal funds appropriated to The 

** These primers are universal primers for calicivirus, but not PoSaV-specific. So, their RT-PCR products should be sequenced for confirmation. *** These primers Also detected porcine kobuvirus.J o u r n a l P r e -p r o o f