Porcine Sapovirus Research Articles

Solution structure of the porcine sapovirus VPg core reveals a stable three-helical bundle with a conserved surface patch

Viral protein genome-linked (VPg) proteins play a critical role in the life cycle of vertebrate and plant positive-sense RNA viruses by acting as a protein primer for genome replication and as a protein cap for translation initiation. Here we report the solution structure of the porcine sapovirus VPg core (VPgC) determined by multi-dimensional NMR spectroscopy. The structure of VPgC is composed of three α-helices stabilized by several conserved hydrophobic residues that form a helical bundle core similar to that of feline calicivirus VPg. The putative nucleotide acceptor Tyr956 within the first helix of the core is completely exposed to solvent accessible surface to facilitate nucleotidylation by viral RNA polymerase. Comparison of VPg structures suggests that the surface for nucleotidylation site is highly conserved among the Caliciviridae family, whereas the backbone core structures are different. These structural features suggest that caliciviruses share common mechanisms of VPg-dependent viral replication and translation.

Porcine sapovirus replication is restricted by the type I interferon response in cell culture.

Porcine sapovirus (PSaV) of the family Caliciviridae, is the only member of the genus Sapovirus with cell culture and reverse genetics systems. When combined with the piglet model, these approaches provide a system to understand the molecular basis of sapovirus pathogenesis. The replication of PSaV in cell culture is, however, restricted, displaying an absolute requirement for bile acids and producing lower levels of infectious virus than other caliciviruses. The effect of bile acids has previously been linked to a reduction in the signal transducer and activator of transcription (STAT1)-mediated signalling pathway. In the current study, we observed that even in the presence of bile acids, PSaV replication in cell culture was restricted by soluble factors produced from infected cells. This effect was at least partially due to secreted IFN because treatment of cells with recombinant porcine IFN-β resulted in significantly reduced viral replication. Moreover, IFN-mediated signalling pathways (IFN, STAT1 and the 2′,5′-oligoadenylate synthetase) were activated during PSaV infection. Characterization of PSaV growth in cell lines deficient in their ability to induce or respond to IFN showed a 100–150-fold increase in infectious virus production, indicating that the primary role of bile acids was not the inactivation of the innate immune response. Furthermore, the use of IFN-deficient cell lines enabled more efficient recovery of PSaV from cDNA constructs. Overall, the highly efficient cell culture and reverse genetics system established here for PSaV highlighted the key role of the innate immune response in the restriction of PSaV infection and should greatly facilitate further molecular studies on sapovirus host–cell interactions.

Sapovirus translation requires an interaction between VPg and the cap binding protein eIF4E.

ABSTRACTSapoviruses of the Caliciviridae family of small RNA viruses are emerging pathogens that cause gastroenteritis in humans and animals. Molecular studies on human sapovirus have been hampered due to the lack of a cell culture system. In contrast, porcine sapovirus (PSaV) can be grown in cell culture, making it a suitable model for understanding the infectious cycle of sapoviruses and the related enteric caliciviruses. Caliciviruses are known to use a novel mechanism of protein synthesis that relies on the interaction of cellular translation initiation factors with the virus genome-encoded viral protein genome (VPg) protein, which is covalently linked to the 5′ end of the viral genome. Using PSaV as a representative member of the Sapovirus genus, we characterized the role of the viral VPg protein in sapovirus translation. As observed for other caliciviruses, the PSaV genome was found to be covalently linked to VPg, and this linkage was required for the translation and the infectivity of viral RNA. The PSaV VPg protein was associated with the 4F subunit of the eukaryotic translation initiation factor (eIF4F) complex in infected cells and bound directly to the eIF4E protein. As has been previously demonstrated for feline calicivirus, a member of the Vesivirus genus, PSaV translation required eIF4E and the interaction between eIF4E and eIF4G. Overall, our study provides new insights into the novel mechanism of sapovirus translation, suggesting that sapovirus VPg can hijack the cellular translation initiation mechanism by recruiting the eIF4F complex through a direct eIF4E interaction.IMPORTANCE Sapoviruses, which are members of the Caliciviridae family, are one of the causative agents of viral gastroenteritis in humans. However, human sapovirus remains noncultivable in cell culture, hampering the ability to characterize the virus infectious cycle. Here, we show that the VPg protein from porcine sapovirus, the only cultivatable sapovirus, is essential for viral translation and functions via a direct interaction with the cellular translation initiation factor eIF4E. This work provides new insights into the novel protein-primed mechanism of calicivirus VPg-dependent translation initiation.

Correction: Both α2,3- and α2,6-Linked Sialic Acids on O-Linked Glycoproteins Act as Functional Receptors for Porcine Sapovirus

Correction: Both α2,3- and α2,6-Linked Sialic Acids on O-Linked Glycoproteins Act as Functional Receptors for Porcine Sapovirus

Both α2,3- and α2,6-linked sialic acids on O-linked glycoproteins act as functional receptors for porcine Sapovirus.

Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation.

Complete sequence and phylogenetic analysis of a porcine sapovirus strain isolated from western China.

Sapovirus (SaV) is a type of calicivirus that can cause acute viral gastroenteritis in humans and animals. SaVs have been found in several mammalian species, including humans, pigs, minks, dogs, and bats. Porcine sapovirus (PoSaV) was first identified in 1980 in the United States and has been found to be circulating throughout China in recent years. In this study, the complete genomic characterization of PoSaV CH430, first found in west China, was reported and analyzed. The genome was 7,342 bp excluding the 30 nt poly(A) tail at the 3' terminus and comprised two major open reading frames. Comprehensive evolutionary and phylogenetic analyses indicated that the CH430 strain belongs to genotype III SaVs. However, this particular isolate and DG24 strain occupied an independent branch of the phylogenetic tree we generated, indicating that they could form a separate subgenotype in the near future. We predicted the cleavage sites for the ORF1 polyprotein located at Q56/G57, Q310/A311, E649/A650, E934/A935, E1047/G1048, and E1712/A1713, separately. This is the first PoSaV strain isolated from western China to be fully sequenced and characterized. It provided a reliable experimental basis for studying the genetic nature of emerging PoSaVs.

Detection and characterization of porcine caliciviruses in Italy.

Porcine noroviruses and sapoviruses have been sporadically reported in European countries, and more rarely in Italy. In this study, stools samples were collected from both asymptomatic and diarrheic pigs from northern Italy and were screened for caliciviruses by RT-PCR. Sapoviruses were detected frequently and were genetically related to both the GIII reference strain and the newly described porcine sapovirus genogroups. Porcine norovirus was detected in one asymptomatic pig (0.5%) and was genotyped as GII.11. This is the first detection of porcine norovirus in Italy.

Seroprevalence and molecular detection of porcine sapovirus in symptomatic suckling piglets in Guangdong Province, China

The seroprevalence and genetic identification of sapovirus (SaV) in symptomatic suckling piglets were investigated in Guangdong Province, China, between November 2011 and April 2013. Serum (n = 960) and diarrheic fecal (n = 101) samples collected from symptomatic suckling piglets in Guangdong Province were evaluated for antibodies against SaV using indirect enzyme-linked immunosorbent assay (iELISA). The overall seroprevalence of SaV in symptomatic suckling piglets was 61.9% (594/960). Positive animals were found in all regions with seroprevalence ranging from 52 to 67.8%, but the difference was not statistically significant (P > 0.05). In addition, RNA of SaV was extracted from diarrheic fecal samples, and the partial polymerase gene was amplified by RT-PCR and then sequenced. Seven of 101 (6.9%) samples were found to contain porcine SaV. Phylogenetic analysis showed that all the porcine SaV isolates belong to the porcine SaV genogroup III (GIII). This is the first report of SaV seroprevalence in symptomatic pigs in China.

Internalization of sapovirus, a surrogate for norovirus, in romaine lettuce and the effect of lettuce latex on virus infectivity.

Noroviruses are the leading cause of food-borne outbreaks, including those that involve lettuce. The culturable porcine sapovirus (SaV) was used as a norovirus surrogate to study the persistence and the potential transfer of the virus from roots to leaves and from outer to inner leaves of lettuce plants. Treatment of lettuce with SaV was done through the roots of young plants, the soil, or the outer leaves of mature plants. Sampling of roots, xylem sap, and inner and outer leaves followed by RNA extraction and SaV-specific real-time reverse transcription (RT)-PCR was performed at 2 h and on postinoculation days (PID) 2, 5, 7, 14, and/or 28. When SaV was inoculated through the roots, viral RNA persisted on the roots and in the leaves until PID 28. When the virus was inoculated through the soil, viral RNA was detected on the roots and in the xylem sap until PID 14; viral RNA was detected in the leaves only until PID 2. No infectious virus was detected inside the leaves for either treatment. When SaV was inoculated through the outer leaves, viral RNA persisted on the leaves until PID 14; however, the virus did not transfer to inner leaves. Infectious viral particles on leaves were detected only at 2 h postinoculation. The milky sap (latex) of leaves, but not the roots' xylem sap, significantly decreased virus infectivity when tested in vitro. Collectively, our results showed the transfer of SaV from roots to leaves through the xylem system and the capacity of the sap of lettuce leaves to decrease virus infectivity in leaves.

Expression of Porcine Sapovirus VP1 Gene and VP1 Specific Monoclonal Antibody Production

Sapovirus (SaV) is an agent of human and porcine gastroenteritis and a member of the family Caliciviridae. SaV has been classified based on VP1 full gene nucleotide sequences into five genogroups (GI-GV), among which GIII is known to infect pigs. The VP1 folds into two major domains designated S and P for the shell and protruding domain, respectively. The P domain is divided into two subdomains, P1 and P2. In this study, the VP1 full gene and the S, P, and P2 regions of the VP1 gene of porcine SaV were expressed using a baculovirus expression system. Expressed proteins in the recombinant virus were confirmed by polymerase chain reaction, indirect fluorescence antibody (IFA) testing, and Western blot analysis. Four hybridomas secreting VP1-specific monoclonal antibodies (MAbs) against porcine sapovirus were generated. Four MAbs were characterized according to their IFA and Western blot analysis results. All of the hybridomas produced in this study secreted MAbs binding to S domain of VP1 protein specifically. The MAbs produced in this study can be used as specific diagnostic reagents for detecting porcine SaV.

RT-PCR test for detecting porcine sapovirus in weanling piglets in Hunan Province, China

The prevalence of porcine sapovirus infection in weanling pigs was investigated in Hunan Province, China, between August 2006 and October 2007. A total of 153 diarrheic fecal samples from ten intensive pig farms from ten representative administrative regions in Hunan province were examined for porcine sapoviruses using RT-PCR. Twenty-two of 153 (14.37 %) samples were found to contain porcine sapoviruses. Phylogenetic analysis showed that all the porcine sapovirus isolates in Hunan Province belonged to the porcine sapovirus genogroup III. The results of the present investigation have implications for the control of porcine sapovirus infection in pigs in Hunan Province, China.

Stability of and attachment to lettuce by a culturable porcine sapovirus surrogate for human caliciviruses.

Human noroviruses (HuNoVs) are the leading cause of food-borne illness, accounting for 58% of U.S. cases. Because HuNoVs are unculturable, surrogates are needed to investigate transmission routes and evaluate disinfection methods. However, the current surrogates, feline calicivirus (FCV) and murine NoV (MNV), are less tolerant than HuNoVs to acid and chlorine, respectively. Porcine sapovirus (SaV) is the only culturable enteropathogenic calicivirus. In this study, the resistance of SaV to physicochemical treatments was compared to that of HuNoVs (by reverse transcription-PCR), FCV, and MNV (by infectivity assays). Sapovirus and HuNoV (viral RNA) showed similar resistances to heat (56°C) and to different concentrations of chlorine. However, SaV was more resistant than HuNoVs to ethanol treatment (60% and 70%). Like HuNoVs, SaV was stable at pH 3.0 to 8.0, with a <1.0 log(10) 50% tissue culture infective dose (TCID(50)) reduction at pH 3.0 compared to the value for pH 4.0 to 8.0. SaV and MNV showed similar resistances, and both were more resistant than FCV to heat inactivation (56°C). FCV was more resistant than MNV and SaV to ethanol, and all three viruses showed similar resistances to treatment with low concentrations of chlorine for 1 min. Those results indicate that SaV is a promising surrogate for HuNoVs. Next, we used SaV as a surrogate to examine virus attachment to lettuce at different pHs. Sapovirus attached to lettuce leaves significantly at its capsid isoelectric point (pH 5.0), and the attached viral particles remained infectious on lettuce after 1 week of storage at 4°C. The culturable SaV is a good surrogate for studying HuNoV contamination and transmission in leafy greens and potential disinfectants.

Emerging and Re-emerging Swine Viruses

In the past two decades or so, a number of viruses have emerged in the global swine population. Some, such as porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), cause economically important diseases in pigs, whereas others such as porcine torque teno virus (TTV), now known as Torque teno sus virus (TTSuV), porcine bocavirus (PBoV) and related novel parvoviruses, porcine kobuvirus, porcine toroviruses (PToV) and porcine lymphotropic herpesviruses (PLHV), are mostly subclinical in swine herds. Although some emerging swine viruses such as swine hepatitis E virus (swine HEV), porcine endogenous retrovirus (PERV) and porcine sapovirus (porcine SaV) may have a limited clinical implication in swine health, they do pose a potential public health concern in humans due to zoonotic (swine HEV) or potential zoonotic (porcine SaV) and xenozoonotic (PERV, PLHV) risks. Other emerging viruses such as Nipah virus, Bungowannah virus and Menangle virus not only cause diseases in pigs but some also pose important zoonotic threat to humans. This article focuses on emerging and re-emerging swine viruses that have a limited or uncertain clinical and economic impact on pig health. The transmission, epidemiology and pathogenic potential of these viruses are discussed. In addition, the two economically important emerging viruses, PRRSV and PCV2, are also briefly discussed to identify important knowledge gaps.

Development and application of a SYBR green RT-PCR for first line screening and quantification of porcine sapovirus infection

BackgroundSapoviruses are single stranded positive sense RNA viruses belonging to the family Caliciviridae. The virus is detected in different species including the human and the porcine species as an enteric pathogen causing asymptomatic to symptomatic enteritis. In this study, we report the development of a rapid real time qRT-PCR based on SYBR Green chemistry for the diagnosis of porcine sapovirus infection in swine.ResultsThe method allows the detection of porcine sapoviruses and the quantification of the genomic copies present in stool samples. During its development, the diagnostic tool showed good correlation compared with the gold standard conventional RT-PCR and was ten-fold more sensitive. When the method was applied to field samples, porcine noroviruses from genogroup 2 genotype 11b were also detected. The method was also applied to swine samples from the Netherlands that were positive for PoSaV infection. Phylogenetic results obtained from the samples showed that PoSaV sequences were genetically related to the currently described genogroup III, to the proposed genogroup VII and also to the MI-QW19 sequence (close to the human SaV sequences).ConclusionsA rapid, sensitive, and reliable diagnosis method was developed for porcine sapovirus diagnosis. It correlated with the gold standard conventional RT-PCR. Specificity was good apart for genogroup 2 genotype 11b porcine noroviruses. As a first line screening diagnosis method, it allows a quicker and easier decision on doubtful samples.

Seroprevalence of porcine cytomegalovirus and sapovirus infection in pigs in Hunan province, China

The seroprevalence of porcine cytomegalovirus (PCMV) and sapovirus (SaV) infections in pigs was investigated in Hunan province, China, between May 2005 and October 2010. A total of 500 pig serum samples collected from 10 representative administrative regions in Hunan province were evaluated for antibodies against PCMV and SaV using enzyme-linked immunosorbent assay (ELISA). The overall seroprevalence of porcine cytomegalovirus and sapovirus in pigs was 96.40% (482/500) and 63.40% (317/500), and the seropositivity of 10 herds we surveyed varied, ranging from 94.74% to 98.48% and 56.36% to 72.50%, respectively. The highest prevalence was found in breeding sows (96.67% for PCMV and 83.33% for SaVs). The results of the present survey indicated that infections with porcine cytomegalovirus and sapovirus are highly prevalent in pigs in Hunan province, China.

Molecular characterization of a porcine sapovirus strain isolated from a piglet with diarrhoea: a case report

Porcine sapoviruses, members of the family Caliciviridae, have been considered as an aetiological agent of gastroenteritis in pigs. In this study, we analysed 251 faecal samples obtained from 3 to 90 day-old diarrhoeic pigs in the Czech Republic between January 2005 and June 2010 and tested them by negative staining electron microscopy for the presence of sapoviruses. Only one sample showed the presence of viral particles with characteristic sapovirus morphology. The presence of sapovirus (SaV) was confirmed by an RT-PCR assay with primers specific for the sapoviral RNA polymerase and capsid genes. Phylogenetic analysis based on a partial sequence of the RNA polymerase gene placed the new Czech isolate into the GVII genogroup of porcine sapoviruses; however, analysis of a portion of the capsid gene sequence classified the isolate as GIII of the genus Sapovirus. These contradictory findings indicate that recombinant porcine sapovirus was identified. According to our knowledge this is the first description of porcine sapovirus in domestic pigs in the Czech Republic

Identification of genetic diversity of porcine Norovirus and Sapovirus in Korea

It is well known that Norovirus (NoV) and Sapovirus (SaV) identified in humans and pigs have heterogeneous genome sequences. In this study, a total of three strains of NoV and 37 strains of SaV were detected in 567 porcine fecal samples by RT-PCR, corresponding detection rates of 0.5 and 6.5%, respectively. Phylogenetic analyses were conducted using amino acid sequences of the partial RNA-dependent RNA polymerase (RdRp) and complete capsid proteins of both viruses to determine their genogroups. Analysis with the RdRp sequences indicated that all three NoV strains HW41, DG32, and DO35 detected in this study were classified into genogroup II (GII). A further analysis with the complete capsid sequence demonstrated that the DO35 strain belonged to subgenotype b in GII-21 (GII-21b) along with the SW918 strain. A total of 26 strains out of 27 strains that were selected from the 37 porcine SaVs were classified into genogroup III when they were analyzed with the RdRp sequences. The remaining strain (DO19) was not clustered with any of the previously classified SaV strains, thereby suggesting the advent of a new genogroup virus. Additional analyses with the amino acid sequence of the capsid and the nucleotide sequence of the RdRp and capsid junction region supported the notion that the DO19 strain belonged to a novel genogroup of SaV. To the best of our knowledge, this is the first report to describe a novel porcine SaV belonging to an unknown genogroup in Korea.

Genetic diversity of porcine enteric caliciviruses in pigs raised in Rio de Janeiro State, Brazil

Porcine enteric caliciviruses (PEC) belong to the genera Norovirus and Sapovirus within the family Caliciviridae. They are enteric pathogens and are considered potential zoonotic agents. In this study, the circulation of PEC was evaluated by RT-PCR of stool samples and intestinal contents of pigs raised in Rio de Janeiro State, Brazil. Both porcine norovirus (PoNoV) and porcine sapovirus (PoSaV) were detected. The PoNoV strains were classified as genogroup II, genotypes 11, 18 and 19. The PoSaV strains were classified as genogroups III and VII, though some strains could not be classified into any established genogroup, potentially representing a new one. PEC were detected mainly in animals without clinical signs of gastroenteritis.

Frequent Detection of Noroviruses and Sapoviruses in Swine and High Genetic Diversity of Porcine Sapovirus in Japan during Fiscal Year 2008

A molecular biological survey on porcine norovirus (NoV) and sapovirus (SaV) was conducted in Toyama Prefecture, Japan, during fiscal year 2008. Both NoV and SaV were detected from swine fecal samples throughout the surveillance period, indicating that these viruses were circulating in this region. NoV strains detected in this study belonged to three genotypes that are known as typical swine NoVs. Although human NoVs were occasionally detected, it was unclear whether they replicated in pigs. As for SaV, genogroup VII (GVII) and other divergent genogroups were identified in addition to the dominant genogroup, GIII, which is the prototypic porcine SaV. In addition, 3 strains genetically related to human SaV were detected. Two of these 3 strains were closely related to human SaV GV. Our study showed that genetic diversification of porcine SaV is currently progressing in the swine population.

Molecular characterization and phylogenetic analysis of the complete genome of a porcine sapovirus from Chinese swine

BackgroundPorcine sapovirus was first identified in the United States in 1980, hitherto, several Asian countries have detected this virus. In 2008, the first outbreak of gastroenteritis in piglets caused by porcine sapovirus in China was reported. The complete genome of the identified SaV strain Ch-sw-sav1 was sequenced and analyzed to provide gene profile for this outbreak.MethodsThe whole genome of Ch-sw-sav1 was amplified by RT-PCR and was sequenced. Sequence alignment of the complete genome or RNA dependent RNA polymerase (RdRp) gene was done. 3' end of ORF2 with 21-nt nucleotide insertion was further analyzed using software.ResultsSequence analysis indicated that the genome of Ch-sw-sav1 was 7541 nucleotide long with two ORFs, excluding the 17 nucleotides ploy (A) at the 3' end. Phylogenetic analysis based on part of RdRp gene of this strain showed that it was classified into subgroup GIII. Sequence alignment indicated that there was an inserted 21-nt long nucleotide sequence at the 3' end of ORF2. The insertion showed high antigenicity index comparing to other regions in ORF2.ConclusionCh-sw-sav1 shared similar genetic profile with an American PEC strain except the 21-nt nucleotide at the 3' end of ORF2. The insert sequence shared high identity with part gene of Sus scrofa clone RP44-484M10.