Flow cytometric detection and serotyping of enterovirus for the clinical laboratory

Abstract

Culture and serotyping of human enteroviruses by fluorescence microscopy are time-consuming and labor-intensive. Flow cytometry has the potential of being more rapid, sensitive, and objective but has not been used for these purposes in a clinical laboratory. Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA). Kinetic studies of coxsackievirus B1 and echovirus 30 infection of PMK cells were performed on days 1-4 after inoculation. Flow cytometry results for echovirus 6, 9, 11, and 30 and coxsackievirus B1 correlated with IFA in all cases. Coxsackievirus B1 and echovirus 30 infections were detected 1 day earlier by flow cytometry than IFA. Flow cytometry can be effectively used for detecting enterovirus-infected cells in a clinical laboratory with the advantages of better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems.

Fig. 1

Uninfected PMK cells and coxsackievirus B1-infected PMK cells were harvested 4 days after virus was inoculated for flow cytometric evaluation. (A) Cellular events were selected for fluorescence analysis based on forward and side scatter as shown (Gate 1). Similar patterns were observed for (B) uninfected PMK cells stained with coxsackievirus B blend antibodies (0.21% positive) and (C) coxsackievirus B1-infected PMK cells stained with isotype control (0.25% positive), (D) echovirus blend (0.24% positive), or (E) coxsackievirus B6 antibody (0.60% positive). (F) The majority (71.52%) of coxsackievirus B1-infected PMK cells stained positively with coxsackievirus B blend antibody above the negative control threshold level.

Fig. 2

Uninfected PMK cells and echovirus 9-infected PMK cells were harvested 1 day after inoculation for flow cytometric evaluation. (A) Uninfected PMK cells stained with echovirus blend antibodies; (B) echovirus 9-infected PMK cells stained with echovirus blend antibody; (C) echovirus 4 specific antibody; (D) echovirus 9 specific antibody. Plots (B) and (D) show positively staining populations (19.0% and 22.3%, respectively) above negative controls (plots A and C).

Fig. 3

Comparison of IFA (left column) and flow cytometry analysis of uninfected PMK cells (center column) and PMK cells days 1–3 after inoculation with coxsackievirus B1 (right column). IFA showed detectable fluorescent cells with apple green cytoplasmic staining at day 2, while flow cytometry was able to detect a small number of cells with bright staining on day 1. Negative cells, counterstained with Evans Blue, are dull red on IFA.