Human Sapovirus Replication in Human Intestinal Enteroids.

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Seroprevalence of sapovirus in dogs using baculovirus-expressed virus-like particles.

Caliciviruses of the Sapovirus genus have been recently detected in dogs. Canine sapoviruses (SaVs) have been identified in the stools of young or juvenile animals with gastro-enteric disease at low prevalence (2.0-2.2%), but whether they may have a role as enteric pathogens and to which extent dogs are exposed to SaVs remains unclear. Here, we report the expression in a baculovirus system of virus like-particles (VLPs) of a canine SaV strain, the prototype virus Bari/4076/2007/ITA. The recombinant antigen was used to develop an enzyme-linked immunosorbent assay (ELISA). By screening an age-stratified collection of serum samples from 516 dogs in Italy, IgG antibodies specific for the canine SaV VLPs were detected in 40.3% (208/516) of the sera. Also, as observed for SaV infection in humans, we observed a positive association between seropositivity and age, with the highest prevalence rates in dogs older than 4 years of age.

Short communication: Immune responses in sows induced by porcine sapovirus virus-like particles reduce viral shedding in suckled piglets.

Porcine sapovirus (PoSaV) is a potential threat to public health owing to its capacity for reassortment with human sapovirus strains. However, there is still no vaccine available for the prevention and control of this infectious disease. In this study, we developed PoSaV virus-like particles (VLPs) using a baculovirus expression system. Immunization with PoSaV VLPs induced high titers of serum antibody specific for VP1 in sows. The results of our challenge study demonstrated that maternally-derived antibodies (MDA) induced by VLP immunization dramatically reduced viral shedding of PoSaV in the feces of next generation piglets. Therefore, the results of this study indicate that the immune responses of sows elicited by PoSaV VLPs can inhibit in vivo viral replication in their offspring and represent a promising strategy for developing vaccines against PoSaV.

Antigenic and Cryo-Electron Microscopy Structure Analysis of a Chimeric Sapovirus Capsid.

UNLABELLED: The capsid protein (VP1) of all caliciviruses forms an icosahedral particle with two principal domains, shell (S) and protruding (P) domains, which are connected via a flexible hinge region. The S domain forms a scaffold surrounding the nucleic acid, while the P domains form a homodimer that interacts with receptors. The P domain is further subdivided into two subdomains, termed P1 and P2. The P2 subdomain is likely an insertion in the P1 subdomain; consequently, the P domain is divided into the P1-1, P2, and P1-2 subdomains. In order to investigate capsid antigenicity, N-terminal (N-term)/S/P1-1 and P2/P1-2 were switched between two sapovirus genotypes GI.1 and GI.5. The chimeric VP1 constructs were expressed in insect cells and were shown to self-assemble into virus-like particles (VLPs) morphologically similar to the parental VLPs. Interestingly, the chimeric VLPs had higher levels of cross-reactivities to heterogeneous antisera than the parental VLPs. In order to better understand the antigenicity from a structural perspective, we determined an intermediate-resolution (8.5-Å) cryo-electron microscopy (cryo-EM) structure of a chimeric VLP and developed a VP1 homology model. The cryo-EM structure revealed that the P domain dimers were raised slightly (∼5 Å) above the S domain. The VP1 homology model allowed us predict the S domain (67-229) and P1-1 (229-280), P2 (281-447), and P1-2 (448-567) subdomains. Our results suggested that the raised P dimers might expose immunoreactive S/P1-1 subdomain epitopes. Consequently, the higher levels of cross-reactivities with the chimeric VLPs resulted from a combination of GI.1 and GI.5 epitopes. IMPORTANCE: We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against heterogeneous VLP antisera than the parental VLPs. The cryo-EM structure of one chimeric VLP (Yokote/Mc114) was solved to 8.5-Å resolution. A homology model of the VP1 indicated for the first time the putative S and P (P1-1, P2, and P1-2) domains. The overall structure of Yokote/Mc114 contained features common among other caliciviruses. We showed that the P2 subdomain was mainly involved in the homodimeric interface, whereas a large gap between the P1 subdomains had fewer interactions.

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-ß 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.

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.

Novel monoclonal antibodies broadly reactive to human recombinant sapovirus-like particles.

Sapovirus (SaV), a member of the family Caliciviridae, is an important cause of acute epidemic gastroenteritis in humans. Human SaV is genetically and antigenically diverse and can be classified into four genogroups (GI, GII, GIV, and GV) and 16 genotypes (7 GI [GI.1-7], 7 GII, [GII.1-7], 1 GIV and 1 GV), based on capsid sequence similarities. Monoclonal antibodies (MAbs) are powerful tools for examining viruses and proteins. PAI myeloma cells were fused with spleen cells from mice immunized with a single type of recombinant human SaV virus-like particles (VLPs) (GI.1, GI.5, GI.6, GII.3, GIV, or GV). Sixty-five hybrid clones producing MAbs were obtained. Twenty-four MAbs were characterized by ELISA, according to their cross-reactivity to each VLP (GI.1, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GIV, and GV). The MAbs were classified by this method into: (i) MAbs broadly cross-reactive to all GI, GII, GIV and GV strains; (ii) those reactive in a genogroup-specific; and (iii) those reactive in a genotype-specific manner. Further analysis of three broadly cross-reactive MAbs with a competitive ELISA demonstrated that at least two different common epitopes are located on the capsid protein of human SaVs in the four genogroups. The MAbs generated and characterized in this study will be useful tools for further study of the antigenic and structural topography of the human SaV virion and for developing new diagnostic assays for human SaV.

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.

Distinct genotype and antigenicity among genogroup II sapoviruses.

SaV, a pathogen of acute gastroenteritis, is divided into five genogroups, GI to GV. However, the relation between SaV antigenicity and genetic clusters is not fully understood. We have recently identified two GII SaV strains, Mc10 and C12, which are grouped into the same cluster based on the polymerase but are grouped into distinct clusters based on the capsid. To evaluate the difference in antigenicity between these two strains, VLP were expressed in mammalian cells. An antigen ELISA demonstrated for the first time that strains in the same GII SaV genogroup, but within different clusters, have distinct antigenicities.

Human sapoviruses: genetic diversity, recombination, and classification.

The family Caliciviridae contains four genera Sapovirus, Norovirus, Lagovirus and Vesivirus, which include Sapporo virus (SaV), Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV) and Feline calicivirus (FCV), respectively. SaV is a causative agent of gastroenteritis in children and adults. SaV can be divided into five genogroups (GI-GV), among which GI, GII, GIV and GV are known to infect humans, whereas SaV GIII infects porcine species. Detection methods include ELISA, RT-PCR and real-time RT-PCR. Since few SaV studies have been conducted, it is difficult to draw correlations between or conclusions about rates of incidence, detection and overall prevalence. Nevertheless, most studies agree that SaV infection is more frequent in young children than adults and that infection in children almost always occurs by 5 years of age. In addition, children at day-care centres and institutions are at greatest risk of SaV-associated infection and transmission. Recently, a number of important findings concerning human SaV were discovered. SaV strains were detected in water samples, which included untreated wastewater specimens, treated wastewater samples and river samples. SaV strains were also detected in shellfish samples destined for human consumption, and recombinant SaV strains were identified in a number of different countries. The purpose of this review was to highlight the current knowledge of human SaV, which appears to be an increasingly important virus causing gastroenteritis in humans.

Development of an enzyme immunoassay for detection of sapovirus-specific antibodies and its application in a study of seroprevalence in children.

Sapoviruses (SVs) are an important cause of acute pediatric gastroenteritis. Due to the lack of appropriate diagnostic methods, the epidemiology of SV-associated illness remains poorly understood. Baculovirus and Escherichia coli expression systems were evaluated for the development of antibody detection enzyme immunoassays (EIA). Age-related antibody prevalence in children was studied using the new EIA. Because of the low yield of the baculovirus system, the E. coli-expressed SV capsid proteins were used to develop the EIA. The antigenic specificities of the E. coli-expressed SV capsid proteins were demonstrated using hyperimmune antisera raised in animals and sera collected from patients. A high prevalence (>90%) of antibodies to both SV (strain Mex340) and norovirus (strain VA387) was observed in children involved in a birth cohort at 20 to 24 months of age; however, at 1 to 3 months of age, <25% of the children possessed anti-SV antibodies versus >90% with anti-NV antibodies. The E. coli-derived SV proteins are an excellent source of antigens for the EIA. SV infection is common in the first 2 years of life. The low prevalence of maternal antibodies detected in Mexican children against SVs in this study is unique and needs to be addressed in future studies.

Genetic variability in the sapovirus capsid protein.

Sapovirus (SV), which causes gastroenteritis in humans, is composed of genetically divergent viruses classified into 5 genogroups. In this study, 2.2-kb nucleotide sequences of the 3' terminus of the genome of 15 SV strains detected in Japan were determined. The 15 SV strains could be classified into four genogroups (GI, GII, GIV and GV), and in two of these, GI and GII, 10 genotypes were identified. The amino acid sequences of the central variable region of the capsid protein showed less than 81% identity when strains belonging to different genotypes were compared. It was therefore supposed that antigenic variety exists between different genotypes. These results will be useful for further genetic and antigenic analyses of SV.

Development of an antigen ELISA to detect sapovirus in clinical stool specimens.

Human sapovirus (SaV) strains are etiological agents of mild and/or acute gastroenteritis in children and adults. In this study, we describe the development of a novel antigen enzyme-linked immunosorbent assay (ELISA) detection system that was based on hyperimmune rabbit and guinea pig antisera raised against SaV genogroup I (GI) virus-like particles. The ELISA had 100% specificity, and sensitivities of 60% and 25% when compared to single-round PCR and nested PCR, respectively. Our results have shown the ELISA was useful in detecting SaV GI antigens in clinical stool specimens collected two days after the onset of illness.

Characterization of polyclonal antibodies raised against sapovirus genogroup five virus-like particles.

Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains were recently divided into five genogroups (GI to GV). We characterized novel polyclonal antibodies raised against SaV GV virus-like particles (VLPs) by Western blot analysis, and both antibody and antigen enzyme-linked immunosorbent assays (ELISAs). Our results have indicated SaV GI and GV VLPs were antigenically distinct by Western blotting and ELISAs. These reagents may be useful for genogroup specific detection of SaV.

Cross-reactivity among several recombinant calicivirus virus-like particles (VLPs) with monoclonal antibodies obtained from mice immunized orally with one type of VLP.

Human caliciviruses (HuCVs) are classified into the Norwalk-like viruses (NLV) and Sapporo-like viruses (SLV) as genera within the family CALICIVIRIDAE: The NLV genus is further classified into genogroups I and II, based on sequence similarities. To study the antigenic determinants on the HuCV capsid protein and develop new diagnostic tools for field samples, we established and characterized monoclonal antibodies (MAbs) against baculovirus-expressed recombinant HuCV virus-like particles (VLPs). Hybrid clones producing MAbs were obtained from cultures of PAI myeloma cells fused with spleen or mesenteric lymph node cells from mice immunized orally with either a single type of recombinant Norwalk virus (rNV), Kashiwa 47 virus (rKAV), Snow Mountain agent (rSMA), or Sapporo virus (rSV) VLP or with mixtures of two types of VLPs from different genogroups. Twenty MAbs, obtained as mouse ascites, were characterized and classified into six groups according to their enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) cross-reactivity patterns to VLPs. Five groups of MAbs reacted by both WB and ELISA and were classified as follows: common cross-reactive MAbs for four genogroup I and six genogroup II VLPs (group A), genogroup I-specific MAbs (group B), genogroup II-specific MAbs (group C), and strain-specific MAbs (groups D and E). One MAb group (group F) reacted only by ELISA. The group A MAbs, which showed broad cross-reactivity with VLPs of both NLV genogroups, were obtained from mice immunized orally with a single type of VLP (either rNV or rKAV). Two MAbs, which were obtained from mice immunized with rSV, reacted with rSV but not with any NLV VLP. These are the first MAbs to be reported for any SLV. These strain-, genogroup-, and genus-reactive MAbs will be useful tools for further study of the antigenic and structural topography of the HuCV virion and for diagnostic assays for HuCVs.