Detection of Infectious Adenoviruses in Environmental Waters by Fluorescence-activated Cell Sorting Assay

Overview

Journal Appl Environ Microbiol

Specialties Environmental Health
Microbiology

Date 2010 Jan 19

PMID 20080992

Citations 9

Authors

Dan Li

Miao He

Sunny C Jiang

Affiliations

Soon will be listed here.

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.

Citing Articles

Infectious Pepper Mild Mottle Virus and Human Adenoviruses as Viral Indices in Sewage and Water Samples.

Rashed M, El-Senousy W, Sayed E, AlKhazindar M Food Environ Virol. 2022; 14(3):246-257.

PMID: 35713790 PMC: 9458564. DOI: 10.1007/s12560-022-09525-0.

Viruses in wastewater: occurrence, abundance and detection methods.

Corpuz M, Buonerba A, Vigliotta G, Zarra T, Ballesteros Jr F, Campiglia P Sci Total Environ. 2020; 745:140910.

PMID: 32758747 PMC: 7368910. DOI: 10.1016/j.scitotenv.2020.140910.

A Two-Dimensional Affinity Capture and Separation Mini-Platform for the Isolation, Enrichment, and Quantification of Biomarkers and Its Potential Use for Liquid Biopsy.

Guzman N, Guzman D Biomedicines. 2020; 8(8).

PMID: 32751506 PMC: 7459796. DOI: 10.3390/biomedicines8080255.

Detecting Virus-Like Particles from the Umgeni River, South Africa.

Ganesh A, Lin J, Singh M Clean (Weinh). 2020; 42(4):393-407.

PMID: 32313584 PMC: 7159345. DOI: 10.1002/clen.201200564.

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

Morinaga T, Nguyn T, Zhong B, Hanazono M, Shingyoji M, Sekine I Virol J. 2017; 14(1):219.

PMID: 29126418 PMC: 5681831. DOI: 10.1186/s12985-017-0888-0.

References

Quiros C, Herrero M, Garcia L, Diaz M . Application of flow cytometry to segregated kinetic modeling based on the physiological states of microorganisms. Appl Environ Microbiol. 2007; 73(12):3993-4000. PMC: 1932747. DOI: 10.1128/AEM.00171-07. View

Hitt D, Booth J, Dandapani V, Pennington L, Gimble J, Metcalf J . A flow cytometric protocol for titering recombinant adenoviral vectors containing the green fluorescent protein. Mol Biotechnol. 2000; 14(3):197-203. DOI: 10.1385/MB:14:3:197. View

Sirikanchana K, Shisler J, Marinas B . Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication. Appl Environ Microbiol. 2008; 74(12):3774-82. PMC: 2446542. DOI: 10.1128/AEM.02049-07. View

Rodriguez R, Pepper I, Gerba C . Application of PCR-based methods to assess the infectivity of enteric viruses in environmental samples. Appl Environ Microbiol. 2008; 75(2):297-307. PMC: 2620694. DOI: 10.1128/AEM.01150-08. View

Fong T, Lipp E . Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools. Microbiol Mol Biol Rev. 2005; 69(2):357-71. PMC: 1197419. DOI: 10.1128/MMBR.69.2.357-371.2005. View

Graham F, Smiley J, Russell W, Nairn R . Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977; 36(1):59-74. DOI: 10.1099/0022-1317-36-1-59. View

CRUZ J, Caceres P, Cano F, Flores J, Bartlett A, Torun B . Adenovirus types 40 and 41 and rotaviruses associated with diarrhea in children from Guatemala. J Clin Microbiol. 1990; 28(8):1780-4. PMC: 268047. DOI: 10.1128/jcm.28.8.1780-1784.1990. View

KOEPKE J, Landay A . Precision and accuracy of absolute lymphocyte counts. Clin Immunol Immunopathol. 1989; 52(1):19-27. DOI: 10.1016/0090-1229(89)90189-x. View

Sandhu K, Al-Rubeai M . Monitoring of the adenovirus production process by flow cytometry. Biotechnol Prog. 2007; 24(1):250-61. DOI: 10.1021/bp070198s. View

10.

Hammes F, Berney M, Wang Y, Vital M, Koster O, Egli T . Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes. Water Res. 2007; 42(1-2):269-77. DOI: 10.1016/j.watres.2007.07.009. View

11.

Ko G, Jothikumar N, Hill V, Sobsey M . Rapid detection of infectious adenoviruses by mRNA real-time RT-PCR. J Virol Methods. 2005; 127(2):148-53. DOI: 10.1016/j.jviromet.2005.02.017. View

12.

Chang J, Ossoff S, Lobe D, Dorfman M, Dumais C, Qualls R . UV inactivation of pathogenic and indicator microorganisms. Appl Environ Microbiol. 1985; 49(6):1361-5. PMC: 241729. DOI: 10.1128/aem.49.6.1361-1365.1985. View

13.

Cromeans T, Lu X, Erdman D, Humphrey C, Hill V . Development of plaque assays for adenoviruses 40 and 41. J Virol Methods. 2008; 151(1):140-5. DOI: 10.1016/j.jviromet.2008.03.007. View

14.

Zhang L, Mei Y, Wadell G . Human adenovirus serotypes 4 and 11 show higher binding affinity and infectivity for endothelial and carcinoma cell lines than serotype 5. J Gen Virol. 2003; 84(Pt 3):687-695. DOI: 10.1099/vir.0.18666-0. View

15.

Pusch D, Oh D, Wolf S, Dumke R, Schroter-Bobsin U, Hohne M . Detection of enteric viruses and bacterial indicators in German environmental waters. Arch Virol. 2005; 150(5):929-47. DOI: 10.1007/s00705-004-0467-8. View

16.

Avery R, Shelton A, Beards G, Omotade O, Oyejide O, Olaleye D . Viral agents associated with infantile gastroenteritis in Nigeria: relative prevalence of adenovirus serotypes 40 and 41, astrovirus, and rotavirus serotypes 1 to 4. J Diarrhoeal Dis Res. 1992; 10(2):105-8. View

17.

Tiemessen C, Nel M, KIDD A . Adenovirus 41 replication: cell-related differences in viral gene transcription. Mol Cell Probes. 1996; 10(4):279-87. DOI: 10.1006/mcpr.1996.0037. View

18.

Pina S, Puig M, Lucena F, Jofre J, Girones R . Viral pollution in the environment and in shellfish: human adenovirus detection by PCR as an index of human viruses. Appl Environ Microbiol. 1998; 64(9):3376-82. PMC: 106735. DOI: 10.1128/AEM.64.9.3376-3382.1998. View

19.

Ko G, Cromeans T, Sobsey M . UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR. Water Res. 2005; 39(15):3643-9. DOI: 10.1016/j.watres.2005.06.013. View

20.

Svraka S, Duizer E, Vennema H, de Bruin E, van der Veer B, Dorresteijn B . Etiological role of viruses in outbreaks of acute gastroenteritis in The Netherlands from 1994 through 2005. J Clin Microbiol. 2007; 45(5):1389-94. PMC: 1865895. DOI: 10.1128/JCM.02305-06. View