Ordering Information:
ORDER NUMBER:  91227
DATE AVAILABLE: Fall 2008

PRINCIPAL INVESTIGATORS:
George D. Di Giovanni, Kristina D. Mena, and Laura Y. Sifuentes

OBJECTIVES:
The main objective of this study was to further develop a method referred to as host cell capture quantitative sequence detection (HCC-QSD) for the rapid detection of potentially infectious viruses in water. Specific objectives were to (1) evaluate different host cell lines and capture conditions for their ability to capture enteric viruses, and (2) evaluate the ability of HCC-QSD to distinguish potentially infectious viruses from those inactivated by free-chlorine and ultraviolet light (UV).

BACKGROUND:
Conventional detection of infectious waterborne viruses involves the use of cell culture, which is time consuming and costly. Many different polymerase chain reaction (PCR) methods for the detection of viruses have been developed, with specificity, speed, and cost advantages over cell culture. However, PCR methods alone do not determine virus infectivity. This project attempted to bridge the gap between the two detection strategies and build on their strengths.

HIGHLIGHTS:
Further development and evaluation of the HCC-QSD method was achieved. Significant progress was made in the following areas of method development:

• Comparison of two different cell lines and viruses for use in HCC-QSD provided evidence for the possible use of a single cell monolayer to detect different viruses in the same sample.
• Comparison of commercially available RNA extraction kits and extraction optimization was performed. The inclusion of DNase was found to significantly improve the PCR quality of the RNA extracts.
• HCC-QSD was found comparable to conventional plaque assay for the detection of infectious and free-chlorine inactivated virus.

APPROACH:

Evaluation of Different Host Cell Lines and Capture Conditions
The Buffalo Green monkey kidney (BGMK) and Vero cell lines were tested for their ability to capture vaccine strain poliovirus and reovirus. Different capture times, inocula volumes, and suspension media were evaluated.

Standard and Advanced Nucleic Acid Extraction for Separating Viral Nucleic Acids From Host Cell Monolayer Nucleic Acids
Two commercial RNA extraction kits were evaluated for recovery of viral RNA from purified virus and virus seeded cell monolayers. The effects of cell lysate homogenization, protein denaturant, carrier RNA, DNase treatment and lysate and purified RNA template volume on HCC-QSD were investigated.

Free-Chlorine and Ultraviolet Light Inactivation Trials
HCC-QSD was compared to conventional plaque assay for its ability to detect and quantify virus inactivation.

RESULTS/FINDINGS:

Evaluation of Different Host Cell Lines and Capture Conditions
Both BGMK and Vero cell lines could be used to capture poliovirus and reovirus, providing evidence for the possible use of a single cell monolayer for the detection of different viruses in the same sample. Monolayers grown in 12-well plates and inoculated with 100 µl samples suspended in serum-free cell culture medium was most effective for virus capture.

Standard and Advanced Nucleic Acid Extraction for Separating Viral Nucleic Acids From Host Cell Monolayer Nucleic Acids
A commercial 96-well plate format RNA extraction method was found to perform best for RNA extraction from virus seeded cell monolayers. The inclusion of DNase in the extraction protocol was found to significantly improve the PCR quality of the RNA extracts and subsequent amplicon detection by both HCC-QSD and conventional gel electrophoresis.

Free-Chlorine and Ultraviolet Light Inactivation Trials
HCC-QSD had poor performance for determining UV-inactivation of virus, likely due to the mechanism of UV-inactivation. In contrast, there was excellent agreement between HCC-QSD and the conventional plaque assay for the detection of infectious and free-chlorine inactivated virus. This suggests that the HCC-QSD assay may be a useful alternative to the plaque assay for the rapid monitoring of chlorine-disinfected water.

IMPACT:
Waterborne viruses present a significant threat to human health, especially for the growing immunocompromised population. Currently, routine water monitoring and risk assessment for infectious viruses in water is hampered by the lack of rapid, specific, and cost-effective methods. In this study, significant progress was made in the further development of HCC-QSD as a rapid method for the detection and quantitation of potentially infectious viruses in water. Further evaluation of HCC-QSD with different enteric viruses and field samples will help us understand its strengths and limitations and allow its use for the routine monitoring of water for infectious viruses.

ISBN 978-1-60573-029-5