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

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

Fold changes in HuNoV RNA levels at 6 or 9 dpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV GII.4 mixture in long-term cultured HT29-Cl.16E and Caco-2 cells.

<p>Fold changes in HuNoV RNA levels at 6 or 9 dpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV GII.4 mixture in long-term cultured HT29-Cl.16E and Caco-2 cells.</p

Fold changes in HuNoV RNA levels at 96 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV.

<p>Fold changes in HuNoV RNA levels at 96 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV.</p

Fold changes in HuNoV and HuSaV RNA levels at 120 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV in medium supplemented with bacteria or the culture supernatants of co-culturing bacteria and immune cells.

<p>Fold changes in HuNoV and HuSaV RNA levels at 120 hpi relative to 0 hpi in cells (cell lysates) inoculated with HuNoV or HuSaV in medium supplemented with bacteria or the culture supernatants of co-culturing bacteria and immune cells.</p

EFFECTS OF THE S-ADENOSYLHOMOCYSTEINE HYDROLASE INHIBITORS 3-DEAZAADENOSINE AND 3-DEAZAARISTEROMYCIN ON RNA METHYLATION AND SYNTHESIS

The effects of 3-deazaaristeromycin and 3-deazaadenosine on RNA methylation and synthesis were examined in the mouse macrophage cell line, RAW264. S-Adenosylhomocysteine accumulated in cells incubated with 3-deazaaristeromycin while S-3-deazaadenosylhomocysteine was the major product in cells incubated with 3-deazaadenosine and homocysteine thiolactone. RNA methylation was inhibited to a similar extent by the accumulation of either S-adenosylhomocysteine or S-3-deazaadenosylhomocysteine, with S-adenosylhomocysteine being a slightly better inhibitor. In mRNA, the synthesis of N6-methyladenosine and N6-methyladenosine and N6-methyl-2'-O-methyladenosine were inhibited to the greatest extent, while the synthesis of 7-methylguanosine and 2'-O-methyl nucleosides were inhibited to a lesser extent. Incubation of cells with 100 μM 3-deazaaristeromycin or with 10 μM 3-deazaadenosine and 50 μM homocysteine thiolactone produced little inhibition of mRNA synthesis, even though mRNA methylation was inhibited. In contrast, mRNA synthesis was greatly inhibited by treatment of cells with 100 μM 3-deazaadenosine and the inhibition of synthesis was not correlated with an inhibition of methylation