Attempts to grow human noroviruses, a sapovirus, and a bovine norovirus in vitro.

Tomoichiro Oka,Garrett T Stoltzfus,Linda J Saif,Chelsea Zhu,Kwonil Jung,Qiuhong Wang,Christiane E Wobus

doi:10.1371/journal.pone.0178157

Abstract

Noroviruses (NoVs) and Sapoviruses (SaVs) are enteric caliciviruses that have been detected in multiple mammalian species, including humans. Currently, efficient cell culture systems have been established only for murine NoVs and porcine SaV Cowden strain. Establishment of an efficient in vitro cell culture system for other NoVs and SaVs remains challenging; however, human NoV (HuNoV) replication in 3D cultured Caco-2 cells and a clone of Caco-2 cells, C2BBe1, human enteroids and in human B cells has been reported. In this study, we tested various cells and culture conditions to grow HuNoVs and a human SaV (HuSaV) to test the possibility of the propagation in different cells and culture conditions. We also attempted to grow a bovine NoV (BoNoV) in ex vivo organ cultures. We did not observe significant RNA level increases for HuSaV and BoNoV under our test conditions. HuNoV RNA levels increased to a maximum of ~600-fold in long-term Caco-2 cells that were cultured for 1–2 months in multi-well plates and inoculated with HuNoV-positive and bacteria-free human stool suspensions using serum-free medium supplemented with the bile acid, GCDCA. However, this positive result was inconsistent. Our results demonstrated that HuNoVs, BoNoV and HuSaV largely failed to grow in vitro under our test conditions. Our purpose is to share our findings with other researchers with the goal to develop efficient, reproducible simplified and cost-effective culture systems for human and animal NoVs and SaVs in the future.

Highlights

JVM-2 was included as a representative human B cell line
We included a human B cell line JVM-2 based on a recent publication reporting that B cells in the lamina propria of duodenum stained positive for human NoV (HuNoV) antigens in a chimpanzee challenge model [39]
During our study Jones et al reported the propagation of a HuNoV strain in human B cells, BJAB and Raji [11, 17]

Summary

Introduction

Most of these trials failed, we detected increased HuNoV RNA levels once during our study when a sterile mixture of HuNoV GII.4 positive stool specimens was inoculated onto long-term cultured monolayers of Caco-2 cells. RT-qPCR results of both the Caco-2 and Vero cell culture lysates following human GII.4 NoVs or GI.2 SaV infection showed no substantial changes in viral RNA levels compared with the inoculation level (less than 2-fold).

Results

Conclusion