Mutational study of sapovirus expression in insect cells

Human sapovirus (SaV), an agent of human gastroenteritis, cannot be grown in cell culture, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the formation of virus-like particles (VLPs). In this study we compared the time-course expression of two different SaV rVP1 constructs. One construct had the native sequence (Wt construct), whereas the other had two nucleotide point mutations in which one mutation caused an amino acid substitution and one was silent (MEG-1076 construct). While both constructs formed VLPs morphologically similar to native SaV, Northern blot analysis indicated that the MEG-1076 rVP1 mRNA had increased steady-state levels. Furthermore, Western blot analysis and an antigen enzyme-linked immunosorbent assay showed that the MEG-1076 construct had increased expression levels of rVP1 and yields of VLPs. Interestingly, the position of the mutated residue was strictly conserved residue among other human SaV strains, suggesting an important role for rVP1 expression

Antigenic Diversity of Human Sapoviruses

Correspondence between virus antigenicity and capsid (VP1) genogrouping and genotyping is likely

Five isoforms of the phosphatidylinositol 3-kinase regulatory subunit exhibit different associations with receptor tyrosine kinases and their tyrosine phosphorylations

AbstractThere are five isoforms of the regulatory subunit for the heterodimeric type of phosphatidylinositol 3-kinase. These five regulatory subunit isoforms were overexpressed using an adenovirus transfection system, and their own tyrosine phosphorylations and associations with various tyrosine kinase receptors were investigated. When overexpressed in CHO-PDGFR cells, the associations of these regulatory subunit isoforms with the platelet-derived growth factor receptor were similar. However, when overexpressed in CHO-IR cells, p55γ exhibited a significantly lower ability to bind with IRS-1 upon insulin stimulation, as compared with other regulatory subunit isoforms. Furthermore, p55α and p55γ were found to be tyrosine-phosphorylated. Finally, interestingly, when overexpressed in CHO-EGFR cells or A431 cells and stimulated with epidermal growth factor (EGF), phosphorylated EGF receptor was detected in p85α, p85β and p50α immunoprecipitates, but not in p55α and p55γ immunoprecipitates. In addition, EGF-induced tyrosine phosphorylation was observed in p85α, p85β, p55α and p55γ, but not in p50α, immunoprecipitates. Thus, each regulatory subunit exhibits specific responses regarding both the association with tyrosine-phosphorylated substrates and its own tyrosine phosphorylation. These results suggest that each isoform possesses specific roles in signal transduction, based on its individual tyrosine kinase receptor

Human Sapovirus in Clams, Japan

Human sapovirus was detected in 4 of 57 clam packages by reverse transcription–PCR and sequence analysis. This represents the first finding of sapovirus contamination in food. Closely matching sequences have been detected in stool specimens from patients with gastroenteritis in Japan, which indicates a possible food-to-human transmission link

Norovirus Infections in Symptomatic and Asymptomatic Food Handlers in Japan▿

Noroviruses are the leading cause of outbreaks of gastroenteritis in the world. At present, norovirus genogroup II, genotype 4 (GII/4), strains are the most prevalent in many countries. In this study we investigated 55 outbreaks and 35 sporadic cases of norovirus-associated gastroenteritis in food handlers in food-catering settings between 10 November 2005 and 9 December 2006 in Japan. Stool specimens were collected from both symptomatic and asymptomatic individuals and were examined for norovirus by real-time reverse transcription-PCR; the results were then confirmed by sequence analysis. Norovirus was detected in 449 of 2,376 (19%) specimens. Four genogroup I (GI) genotypes and 12 GII genotypes, including one new GII genotype, were detected. The GII/4 sequences were predominant, accounting for 19 of 55 (35%) outbreaks and 16 of 35 (46%) sporadic cases. Our results also showed that a large number of asymptomatic food handlers were infected with norovirus GII/4 strains. Norovirus GII had a slightly higher mean viral load (1 log unit higher) than norovirus GI, i.e., 3.81 × 108 versus 2.79 × 107 copies/g of stool. Among norovirus GI strains, GI/4 had the highest mean viral load, whereas among GII strains, GII/4 had the highest mean viral load (2.02 × 108 and 7.96 × 109 copies/g of stool, respectively). Importantly, we found that asymptomatic individuals had mean viral loads similar to those of symptomatic individuals, which may account for the increased number of infections and the predominance of an asymptomatic transmission route

Intertypic reassortment of mammalian orthoreovirus identified in wastewater in Japan

Abstract Mammalian orthoreovirus (MRV), a non-enveloped virus with a ten-segmented double-stranded RNA genome, infects virtually all mammals, including humans. Human infection with MRV seems to be common in early childhood, but is rarely symptomatic. Despite the ubiquitous presence of MRV in mammals as well as in environmental waters, the molecular characterisation of the MRV genome remains to be fully elucidated. In this study, two novel strains, MRV-2 THK0325 and MRV-1 THK0617, were unintentionally isolated from wastewater in Japan via an environmental surveillance of enteric viruses. Homology and phylogenetic analysis demonstrated that all the segments of THK0325 were closely related to the MRV-2 Osaka strains, which were recently proposed to have existed for at least two decades in Japan. Most of the segments in THK0617 also showed a close relationship with the MRV-2 Osaka strains, but the M2, S1, and S3 segments belong to another MRV cluster. According to the S1 sequence, the determinant of serotype THK0617 was classified as MRV-1, and both the M2 and S3 segments were closely related to MRV-1 and -3 from the tree shrew in China. These results suggest that the MRV-2 Osaka-like strain spread widely throughout Japan, accompanied by intertypic reassortment occurring in East Asia