title: An improved infectivity assay combining cell culture with real-time PCR for rapid quantification of human adenoviruses 41 and semi-quantification of human adenovirus in sewage.
abstract: A protocol for the rapid detection and semi-quantification of human enteric adenovirus based on the quantification of viral mRNA during cell culture infectivity assay was developed. Infectivity assays for adenovirus incorporated cell culture and reverse transcription real-time PCR, where viral mRNA detection was used to monitor the progress of adenovirus infection (CC/mRNA qPCR). The cell line used was G293. This specific infectivity assay was calibrated against different initial concentrations of human adenovirus 41. In addition, the expression of the host's housekeeping (HK) gene, GAPDH, served as internal control for the mRNA assays for quality assurance of the mRNA extraction and reverse transcription steps. The concentrations of infectious human adenoviruses in different sewage samples were estimated semi-quantitatively using the CC/mRNA qPCR assay and calibration obtained for adenovirus 41. A linear relationship between concentrations of viral mRNA (hexon gene) and infectious units was observed between 10(7) to 10(1) infectious units per assay (R(2) = 0.97) in samples analyzed 3-5 days post infection. The expressions of host cell GAPDH gene were not significantly affected by infections with different concentrations of human adenovirus 41, and between virus positive and negative cell cultures (p > 0.1). The estimated concentrations of human adenoviruses in sewage samples ranged between 10(2) to 10(3) mRNA-IU/L. Most of the viruses detected in sewage samples were from human adenovirus species F. The CC/mRNA qPCR assay can be used for quantifying infectious human adenovirus 41, estimating the levels of human adenoviruses in sewage samples, and applied to other sample settings. The CC/mRNA qPCR protocol described here represents an improvement in the detection of human enteric adenoviruses by reducing incubation time (5 days); whereas the conventional cell culture method requires longer incubation periods (10-20 days). More importantly, this protocol can be used to more rapidly and semi-quantitatively estimate the levels of infectious human adenoviruses in environmental samples.
authors.affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Roberto.A.Rodriguez@uth.tmc.edu
authors.forename: Roberto A
authors.initials: RA
authors.lastname: Rodríguez
authors.forename: Patsy M
authors.initials: PM
authors.lastname: Polston
authors.forename: Ming Jing
authors.initials: MJ
authors.lastname: Wu
authors.forename: Jianyong
authors.initials: J
authors.lastname: Wu
authors.forename: Mark D
authors.initials: MD
authors.lastname: Sobsey
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics* / Q000472:pathogenicity*; D002138:Calibration; D036022:Capsid Proteins / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D015971:Gene Expression Regulation, Enzymologic; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics / Q000378:metabolism; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012722:Sewage / Q000821:virology*
chemical_list: D036022:Capsid Proteins; D012333:RNA, Messenger; D012722:Sewage; C018315:hexon capsid protein, Adenovirus; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)