Use of cell culture-PCR assay based on combination of A549 and BGMK cell lines and molecular identification as a tool to monitor infectious adenoviruses and enteroviruses in river water

Abstract

Viral contamination in environmental samples can be underestimated because a single cell line might reproduce only some enteric viruses and some enteric viruses do not exhibit apparent cytopathic effects in cell culture. To overcome this problem, we evaluated a cell culture-PCR assay based on a combination of A549 and Buffalo green monkey kidney (BGMK) cell lines as a tool to monitor infectious adenoviruses and enteroviruses in river water. Water samples were collected 10 times at each of four rivers located in Gyeonggi Province, South Korea, and then cultured on group 1 cells (BGMK cells alone) and group 2 cells (BGMK and A549 cells). Reverse transcription and multiplex PCR were performed, followed by phylogenetic analysis of the amplicons. Thirty (75.0%) of the 40 samples were positive for viruses based on cell culture, and the frequency of positive samples grown on group 2 cells (65.0%) was higher than the frequency of positive samples grown on group 1 cells (50.0%). The number of samples positive for adenoviruses was higher with A549 cells (13 samples) than with BGMK cells (one sample); the numbers of samples positive for enteroviruses were similar with both types of cells. By using phylogenetic analysis, adenoviral amplicons were grouped into subgenera A, C, D, and F, and enteroviral amplicons were grouped into coxsackieviruses B3 and B4 and echoviruses 6, 7, and 30, indicating that A549 and BGMK cells were suitable for recovering a wide range of adenoviral and enteroviral types. The cell culture-PCR assay with a combination of A549 and BGMK cells and molecular identification could be a useful tool for monitoring infectious adenoviruses and enteroviruses in aquatic environments.

FIG. 1.

Procedure for the detection of infectious adenoviruses and enteroviruses in river water. PEG, polyethylene glycol.

FIG. 2.

Phylogenetic tree based on nucleotide sequences of 17 adenoviral amplicons. Subgenera A, B, C, D, and F of the adenoviruses are indicated. The reference strains of subgroup E were omitted to simplify the tree. Bovine adenovirus 4 strain THT/62 was used as the outgroup. Nodes supported by bootstrap values of >90% are indicated by solid circles; open circles indicate >70% support, and open squares indicate >50% support. The scale bar corresponds to 0.10 substitution per nucleotide position. Amplicon designations are in boldface type. A, H, I, and S indicate the Ansan River, the Hwajeong River, the Siheung River, and the Sanbon River, respectively, and these letters are followed by sampling dates (01, 12 May 2002; 02, 21 May 2002; 03, 13 June 2002; 04, 28 June 2002; 05, 17 July 2002; 06, 30 July 2002; 07, 15 August 2002; 08, 3 September 2002; 09, 17 September 2002; 10, 1 October 2002); 1, 2A, and 2B indicate the cell line groups and are followed by the subsample number. The numbers in parentheses are accession numbers. See Materials and Methods for details.

FIG. 3.

Phylogenetic tree based on deduced amino acid sequences of 24 enteroviral amplicons. Clusters A, B, and C of the enteroviruses are indicated. The reference strains of cluster D were omitted to simplify the tree. Human rhinovirus 89 was used as the outgroup. The scale bar corresponds to 0.10 substitution per amino acid position. The bootstrap values and amplicon designations are described in the legend to Fig. 2. The numbers in parentheses are accession numbers. See Materials and Methods for details.