SUMMARY: Sapoviruses cause acute gastroenteritis in humans and animals. They belong to the genusSapoviruswithin the familyCaliciviridae. They infect and cause disease in humans of all ages, in both sporadic cases and outbreaks. The clinical symptoms of sapovirus gastroenteritis are indistinguishable from those caused by noroviruses, so laboratory diagnosis is essential to identify the pathogen. Sapoviruses are highly diverse genetically and antigenically. Currently, reverse transcription-PCR (RT-PCR) assays are widely used for sapovirus detection from clinical specimens due to their high sensitivity and broad reactivity as well as the lack of sensitive assays for antigen detection or cell culture systems for the detection of infectious viruses. Sapoviruses were first discovered in 1976 by electron microscopy in diarrheic samples of humans. To date, sapoviruses have also been detected from several animals: pigs, mink, dogs, sea lions, and bats. In this review, we focus on genomic and antigenic features, molecular typing/classification, detection methods, and clinical and epidemiological profiles of human sapoviruses.

The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in ¹¹⁷¹ Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH ₂ -p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae , especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus .

Sapovirus (SaV), a member of the familyCaliciviridae, is a causative agent of acute gastroenteritis in humans and swine and is currently divided into five genogroups, GI–GV. The proteolytic processing of the SaV open reading frame 1 (ORF1) polyprotein with a human GII SaV Mc10 strain has recently been determined and the products are arranged in the following order: NH₂–p11–p28–p35 (NTPase)–p32–p14 (VPg)–p70 (Pro–Pol)–p60 (VP1)–COOH. The cleavage site between p14 (VPg) and p70 (Pro–Pol) was identified as E¹⁰⁵⁵/A¹⁰⁵⁶by N-terminal amino acid sequencing. To identify other cleavage sites, a series of GII SaV Mc10 full-length clones containing disrupted potential cleavage sites in the ORF1 polyprotein were constructed and used to generate linear DNA templates forin vitrocoupled transcription–translation. The translation products were analysed by SDS-PAGE or by immunoprecipitation with region-specific antibodies. N-terminal amino acid sequencing withEscherichia coli-expressed recombinant proteins was also used to identify the cleavage site between p32 and p14. These approaches enabled identification of the six cleavage sites of the Mc10 ORF1 polyprotein as E⁶⁹/G⁷⁰, Q³²⁵/G³²⁶, Q⁶⁶⁶/G⁶⁶⁷, E⁹⁴⁰/A⁹⁴¹, E¹⁰⁵⁵/A¹⁰⁵⁶and E¹⁷²²/G¹⁷²³. The alignment of the SaV full-length ORF1 amino acid sequences indicated that the dipeptides used for the cleavage sites were either E or Q at the P1 position and A, G or S at the P1′ position, which were conserved in the GI, GII, GIII, GIV and GV SaV ORF1 polyprotein.

Human sapoviruses (HuSaVs) cause acute gastroenteritis similar to human noroviruses. Although HuSaVs were discovered four decades ago, no HuSaV has been grown in vitro, which has significantly impeded the understanding of viral biology and the development of antiviral strategies. In this study, we identified two susceptible human cell lines, that originated from testis and duodenum, that support HuSaV replication and found that replication requires bile acids. HuSaVs replicated more efficiently in the duodenum cell line, and viral RNA levels increased up to ∼6 log₁₀-fold. We also detected double-stranded RNA, viral nonstructural and structural proteins in the cell cultures, and intact HuSaV particles. We confirmed the infectivity of progeny viruses released into the cell culture supernatants by passaging. These results indicate the successful growth of HuSaVs in vitro. Additionally, we determined the minimum infectious dose and tested the sensitivities of HuSaV GI.1 and GII.3 to heat and ultraviolet treatments. This system is inexpensive, scalable, and reproducible in different laboratories, and can be used to investigate mechanisms of HuSaV replication and to evaluate antivirals and/or disinfection methods for HuSaVs. Significance Human sapoviruses (HuSaV) are an important cause of diarrhea and foodborne infections worldwide. Despite the discovery of HuSaVs over 40 y ago, no in vitro cell culture system has been established, limiting research on this important pathogen. In this study, we report successful propagation of HuSaV in a human duodenum cell line in the presence of bile acids. Bile acids are biological components in the intestinal tract, and they recapitulate key physiologic factors present in the viral infection site in the intestine. This inexpensive, reproducible and scalable in vitro cell culture system provides a fundamental scientific tool for HuSaV research and development of infection control strategies in the future.