Date Published: June 5, 2014
Publisher: Public Library of Science
Author(s): Deok-Song Kim, Myra Hosmillo, Mia Madel Alfajaro, Ji-Yun Kim, Jun-Gyu Park, Kyu-Yeol Son, Eun-Hye Ryu, Frederic Sorgeloos, Hyung-Jun Kwon, Su-Jin Park, Woo Song Lee, Duck Cho, Joseph Kwon, Jong-Soon Choi, Mun-Il Kang, Ian Goodfellow, Kyoung-Oh Cho, Timothy L. Tellinghuisen.
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.
Caliciviruses (family Caliciviridae) are small (27–40 nm), non-enveloped, icosahedral viruses that possess a single-strand, plus-sense genomic RNA of 7–8 kb . Caliciviruses are important veterinary and human pathogens which are associated with a broad spectrum of diseases in their respective hosts. A member of the genus Lagovirus, rabbit hemorrhagic disease virus (RHDV), is associated with a fatal liver disease in rabbits . Feline calicivirus (FCV), a member of the genus Vesivirus, causes respiratory disease in cats , . Caliciviruses in the genera Norovirus and Sapovirus are important acute gastroenteritis pathogens in humans and animals , . Each year, human noroviruses cause at least 1.1 million episodes and 218,000 deaths in developing nations as well as approximately 900,000 cases of pediatric gastroenteritis in industrialized nations . Sapoviruses have also been associated with gastroenteritis outbreaks and with disease in pediatric patients . The genus Sapovirus can be divided into five genogroups (GI–GV), among which GI, GII, GIV and GV are known to infect humans, whereas GIII infects porcine species . No fully permissive cell culture system currently exists for the enteric caliciviruses associated with gastroenteritis in humans, hampering the study of viral pathogenesis and immunity of these ubiquitous pathogens .
The lack of an efficient cell culture system of human noroviruses and sapoviruses has hampered the study of virus entry and the molecular mechanisms of virus replication. Among enteric caliciviruses, PSaV Cowden strain is the only cultivable enteric sapovirus and has been shown to replicate in a continuous cell line (LLC-PK), but only in the presence of intestinal contents from gnotobiotic pigs or bile acids as a medium supplement . Although virus-host cell receptor interactions are the first step in the initiation of virus infection, the exact nature of the receptors which are recognized by the Sapovirus genus has not been determined. In the present study, therefore, we used the cell culture adapted PSaV Cowden strain as a model to investigate the entry strategy of an enteric sapovirus in vitro.