Use of Stem-cell—derived Enteroids to Correlate Nucleic Acid-based Molecular Assays with Human Norovirus Infectivity
Other conference paper


全文

其它資訊
摘要Introduction
Human norovirus accounts for 18% of acute gastroenteritis worldwide. Clinical diagnosis uses highly sensitive, quantitative RT-qPCR assays to detect the presence of norovirus RNA in clinical samples. However, the correlation of these molecular assays to reflect norovirus infectivity remains elusive.

Aim
To establish a threshold for molecular assays to indicate infectious human norovirus.

Methods
We studied the replication kinetics of three strains of pandemic human norovirus GII.Pe-GII.4 Sydney using a stem-cell—derived human jejunal enteroid culture model. Five to seven 3-fold serial dilutions of norovirus-containing stool filtrates mimicking various cycle threshold (Ct) values of a widely-used diagnostic RT-qPCR assay were tested for each virus strain using enteroids. Norovirus RNA levels in the supernatant at 72 hours post-inoculation were measured by the same RT-qPCR assay and a 10-fold or above increase from baseline was used to confirm the presence of infectious virus. The analytical sensitivity of the enteroid model was also compared with that of a FDA-cleared commercial norovirus EIA assay.

Results
The maximum level of norovirus RNA replication achieved in the enteroid line was 45,792 (4.7 log10) folds. The median Ct value of inoculating virus dilution that reflected a transition from a positive-to-negative enteroid culture result was 29.7. No notable virus replication was observed when enteroids were inoculated with filtrate dilutions with Ct values of 30 or higher. The enteroid model was more sensitive than the EIA assay in measuring moderate virus shedding by being able to detect infectious norovirus in filtrate dilutions with Ct values between 25 and 30.

Conclusion
We have experimentally determined a Ct value of less than 30 that can indicate infectious norovirus for a commonly used diagnostic RT-qPCR assay. The biological and clinical relevance of detecting low-level shedding (Ct=30 or above) of presumably non-infectious norovirus in patients and healthy individuals reported in earlier studies deserves a revisit.

Acknowledgements
The project was supported in part by a research grant from the Hong Kong Research Grants Council (reference number 14162217) and a seed fund for gut microbiota research by the Faculty of Medicine of CUHK.
出版社接受日期14.02.2019
著者M Chan, S Cheung, K Mohammad, J Chan, M Estes, P Chan
會議名稱7th Meeting of The European Society for Virology
會議開始日28.04.2019
會議完結日01.05.2019
會議地點Rotterdam
會議國家/地區荷蘭
出版年份2019
語言英式英語
關鍵詞norovirus, enteroid

上次更新時間 2019-06-05 於 17:25