Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment.

Mizuki Fukuta,Taichiro Takemura,Meng Ling Moi,Thi Thu Thuy Nguyen,Futoshi Hasebe,Kouichi Morita,Thi Thanh Ngan Nguyen,Le Khanh Hang Nguyen,Thi Bich Hau Vu,Shingo Inoue,Co Thach Nguyen,Thi Quynh Mai Le

doi:10.3390/ijerph18189845

Abstract

Infectivity and neutralizing antibody titers of flavivirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are frequently measured using the conventional plaque assay. While the assay is useful in the determination of infectivity, conventional plaque assays generally possess lower sensitivity and are time-consuming compared to nucleic acid amplification tests. In this study, a microcrystalline cellulose (MCC), Avicel, was evaluated as an alternative to the conventional virus overlay medium, methylcellulose, for a plaque assay. The plaque assay was performed using dengue and COVID-19 clinical samples and laboratory-established flavivirus and SARS-CoV-2 strains. In virus titration of clinical samples, the plaques were significantly larger, and the virus titers were higher when Avicel MCC-containing overlay medium was used than with conventional methylcellulose overlay medium. In addition, for some clinical samples and laboratory virus strains, infectious particles were detected as plaques in the Avicel MCC-containing medium, but not in the conventional methylcellulose medium. The results suggest that the viremia titer determined using the new overlay medium containing Avicel MCC may better reflect the innate infectious and plaque-forming capabilities of clinical samples and better reflect virus infectivity.

Highlights

Virus shedding during acute viral infection has important implications for the transmission and control of infectious disease [1]
Consistent with the results for flaviviruses, SARS-CoV-2 plaque sizes were up to two times larger when Avicel medium (Av)-containing medium was used compared to Mc medium (Table 3, (p Avicel-methylcellulose medium (AvMc) vs. Mc < 0.05))
One of the challenges encountered in textbook descriptions of flavivirus viremia levels and SARS-CoV-2 virus shedding is that the conventional titration method uses Mc as an overlay medium

Summary

Introduction

Virus shedding during acute viral infection has important implications for the transmission and control of infectious disease [1]. In addition to control measures, high blood viral load levels have been associated with greater disease severity, and an understanding of the detailed patterns of virus shedding and viremia is important for determining disease pathogenesis [2,3,4,5]. The infectivity of a virus is typically determined using permissive cells on which the virus has a cytopathic effect (CPE) or leads to plaque formation on virus infection [9,10]. Measuring the viral titer is further challenged if the clinical virus strain does not have a CPE or lead to plaque formation; measurement of infectivity is limited to the detection of CPE forming viruses. As a result of the reliance on detection of a CPE, the infectivity may not be accurately reflected

Methods

Results

Discussion

Conclusion