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

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.

Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease

“…To our knowledge, this is the first report of the detection of natural DENV infection by an FFA combined to automated analysis of digital imaging. The results presented here are comparable to those rarely reported in the detection of natural infection by plaque-forming assay (PFA) [ 40 ].…”

“…e results presented here are comparable to those rarely reported in the detection of natural infection by plaque-forming assay (PFA) [40].…”

“…The detection of circulating DENV and ZIKV in natural infection using FFAs and plaque-forming assays (PFA) is hardly possible [ 40 ]. FFAs reported for flavivirus infections are generally used in in vitro experiments or to determine the detection limits of molecular tests such as RT-PCR [ 20 ], as well as to measure neutralization potentials in various studies [ 24 , 48 ].…”

Seroprevalence of four endemic human coronaviruses and, reactivity and neutralization capability against SARS-CoV-2 among children in the Philippines

“…(2) For the conventional PRNT, as previously described 40 , VeroE6/TMPRSS2 cells were seeded in 12-well plate with 1 mL of growth medium and incubated until a cell monolayer was formed. The serum samples were diluted in maintenance medium at 1:10 in duplicate, mixed with an equal volume of medium containing 50 plaque forming units of SARS-CoV-2 (SMC-VC-1), and incubated at 37 °C for 60 min.…”

Seroprevalence of four endemic human coronaviruses antibodies among Filipino children and their cross-reactivity and neutralization capacity against SARS-CoV-2

“…(2) For the conventional plaque assay, as previously described 23 , VeroE6/TMPRSS2 cells were seeded in 12-well plates with 1 mL of growth medium and incubated until a cell monolayer was formed. The serum samples were diluted in MM at 1:10 in duplicate, mixed with an equal volume of medium containing 50 plaque forming units of SARS-CoV-2 (SMC-VC-1), and incubated at 37°C for 60 minutes.…”