Detection of infectious adenoviruses in environmental waters by fluorescence-activated cell sorting assay

Abstract

Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 10(4) PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r(2) value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r(2) value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.

FIG. 1.

(A) Effects of the initial viral concentration and incubation time on FACS detection of rAd5 GFP on HEK-293A cells. (B) Dose-response curve for rAd5 by FACS-GFP assay after 3-day incubation on HEK-293A cells. The linear correlation is expressed as follows: y = 18.1 log₁₀x + 0.81; r² = 0.97. Each data point represents replication of three or more wells.

FIG. 2.

Optimization of the hexon primary antibody concentration (10 μg/ml, 20 μg/ml, and 30 μg/ml) for FACS-hexon assay using rAd5 incubated on HEK-293A cells for 3 days. In comparison with the FACS-GFP assay, the FACS-hexon assay showed a lower sensitivity. Each data point represents replication of three or more wells.

FIG. 3.

Effects of the initial viral density and incubation time on FACS-hexon detection of rAd5, Ad2, and Ad41 on HEK-293A cells. Each data point represents replication of two or more wells.

FIG. 4.

Comparison of FACS assay of Ad2 based on E1A and hexon protein and FACS-hexon assay using A549 cells and HEK-293A cells after 5-day incubation. Each data point represents replication of two or more wells.

FIG. 5.

The standard curves for quantification of infectious Ads by FACS-hexon assay after 3 days of incubation on HEK-293A cells. The correlations are as follows: y = 9.43 log₁₀x + 2.89 [r² = 0.99]; y = 7.95 log₁₀x + 0.66 [r² = 0.97]; and y = 4.33 log₁₀x + 1.26 [r² = 0.97] for Ad5, Ad41, and Ad2, respectively. Each data point represents replication of two or more wells.

FIG. 6.

Comparison of FACS and qPCR assays of adenovirus after UV irradiation. rAd5 before and after UV exposure was detected by FACS-GFP after 3 days of incubation and qPCR assay. Each data point represents replication of two or more wells.