Molecular Detection of Waterborne Pathogens
Marylynn V. Yates, Yu-Chen Hwang, Wilfred Chen, and Ashok Mulchandani
University of California, Riverside, California
Current methods to assess the presence of infectious waterborne viruses (e.g., hepatitis A virus, enteroviruses, and rotaviruses) in an environmental sample are based on cell culture techniques, which rely on the production of visible cytopathic effects (CPE), which may take several days or weeks to appear. This limits the use of cell culture for practical applications, such as monitoring drinking water. Molecular methods offer the opportunity to detect viruses much more rapidly (<24 hours), but do not provide information on viral infectivity. Methods that combine cell culture and molecular methods allow rapid, sensitive detection of infective viruses. Two such methods involve the visualization of infected cells using molecular beacons specific for viral RNA and the use of fluorescence resonance energy transfer in engineered cell lines. These methods allow the detection of fewer than 10 infective virus particles in less than 12 hours.
Introduction
Enteric viruses are transmitted predominantly from person to person through the fecal-oral route; poor water quality, sanitation and hygiene are the major origins of infections. Enteric viruses pose a public health threat because they are stable in aquatic environments up to two years and can be easily disseminated or transmitted with a moderate to high morbidity rate. The importance of water as a vehicle for the transmission of enteric viruses, coupled with the very low infectious dose for many of these viruses has resulted in the need for methods that can reliably and rapidly detect small numbers (e.g., 1 plaque-forming unit per 1000 liters of water) of infective virus particles in environmental samples.
Despite significant advances in medicine during the last several decades, infectious disease still accounts for about 26% of deaths worldwide (Kindhause, 2003). Enteric viruses (e.g., enteroviruses, noroviruses, and hepatitis A virus) are among the major groups of viral infectious agents. Enteroviruses, which include coxsackie-, echo-, polio-, and the numbered enteroviruses, are the second most common viral infectious agents in the world (Sawyer, 2002). Coxsackieviruses and echoviruses cause a wide variety of clinical syndromes including aseptic meningitis, myocarditis, encephalitis, and respiratory illness. They have caused a number of epidemics, both worldwide and in the United States (Melnick, 1996). Person-to-person transmission appears to be the primary route of spread in these epidemics; however, the original source of the organisms is rarely, if ever, identified. Because these viruses are shed in the feces, the possibility that sewage-contaminated, inadequately treated drinking water is the source of the viruses exists. These viruses can be isolated from the stools of a relatively high percentage (8.7%) of healthy children. They have been detected in environmental samples including wastewater, surface water, and ground water. However, their role in waterborne disease is not well understood.
Cell culture-based methods have traditionally been used to detect infective viruses and can achieve a detection limit of 1 infective particle per sample. This assay remains the gold standard for virus diagnosis, because it is the only method that detects infective virus particles; however, it is time consuming, taking up to several weeks to produce results. Amplification of viral nucleic acid using the polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR for the detection of mRNA) is extensively used as a research tool for the analysis of environmental samples. PCR-based methods provide the benefit of rapid analysis at relatively low cost, but they are extremely sensitive to contaminants, which become highly concentrated during sample processing, which can lead to false-negative results. A critical disadvantage of molecular approaches, from a pubic health risk viewpoint, is their inability to distinguish between infective and noninfective viruses. Improved methods that can be applied for rapid detection of a single infective virus are essential to provide rapid and quantitative information of their presence for public health risk assessment.
Visualization and quantification of infective coxsackievirus
In the last several years, molecular beacons (MBs)
(Fig. 1) have been used for the construction of probes that are useful for
real-time detection of nucleic acids (Tyagi and Kramer, 1996). These probes are
based on single-stranded nucleic acid molecules that possess a stem-and-loop
structure. A fluorescent moiety is attached to the end of one arm and a
non-fluorescent quenching moiety is attached to the other end. When the probe
encounters a single-strand target, it forms a hybrid with the target,
undergoing a spontaneous conformational change that forces the arm sequences
apart and causes fluorescence to occur. The interaction of MBs with their
targets is extraordinarily specific. No increase in fluorescence is observed
even in the presence of a target strand that contains only a single nucleotide
mismatch (Piatek et al., 1998). MBs have been successfully used to
detect several enteric viruses, including hepatitis A virus (Abd El Galil et
al., 2004) and echovirus (unpublished results).
As described in Wang et al. (2005), to design a MB suitable for targeting viral RNA, a target sequence was selected in a region that was predicted (using MFOLD) to have the least secondary structure and be the most conserved among 22 different enteroviruses. Based on these criteria, MB CB1 targeting an 18-bp region of the 5’non-coding region was selected. To allow the introduction of a sufficient quantity of MB inside the entire population of cells, a simple fixation and permeabilization (with paraformaldehyde and Triton X-100) procedure typically used for in situ RT-PCR was adopted. Uninfected cells produced no detectable fluorescent signal. In contrast, cells highly infected with coxsackievirus B6 (CB6) were brightly fluorescent as visualized by the phase contrast and fluorescence microscopy, confirming the utility of the permeabilization procedure to successfully introduce sufficient quantities of MB to all infected cells (Wang et al., 2005).
Using the 6-h infection window, the efficacy of the assay to quantify the infective dosage of CB6 was tested. Cultures were infected with 1 to 30 PFU and the average number of fluorescent foci was plotted as a function of PFU. A linear correlation was obtained over the entire range of interest (Fig. 2), suggesting that this assay may be useful not only as a detection tool but also for quantification of infective viruses. The infective dosages obtained from the two assays were remarkably similar, with an R2 value of 0.9998, validating the utility of the assay for viral quantification.
Figure 2. The correlation between the average fluorescent foci/grid and the corresponding infective viral dosage (PFU) at 6 h post infection (data from Wang et al., 2005)
Detection of polioviruses by genetically engineered cell line carrying a 2Apro-sensitive FRET pair
In addition to probing intracellular RNA synthesis during viral replication by the use of MBs, other viral replication events inside a host cell can be exploited for non-invasive detection. In particular, different genetically engineered cell lines have been established to probe this process in a non-invasive manner. Several viral-inducible reporter systems have been engineered in the host cell for viral detection based on transcription from viral promoters that are specific for virus-infected cells (e.g., Rider et al., 2003). These transgenic cell lines provide a high level of sensitivity and specificity to facilitate the detection process. Unfortunately, this strategy is not applicable for enteroviruses, which exhibit no defined viral promoter region. Many viruses, such as picornaviruses, retroviruses, and caliciviruses, however, produce a polyprotein that is cleaved into individual proteins by virus-specific proteases (Strauss, 1990). Viral protease is a logical target for the detection of infective viruses because the cleavage event proceeds in a defined manner and is ubiquitous within various viral families. For these viruses, the RNA genome is translated immediately into a single polypeptide upon infection, which is subsequently cleaved by viral proteases to generate mature proteins. This proteolytic process occurs with 100% efficiency and high specificity (Alvey et al., 1991). Furthermore, proteases are diffusible proteins and can act in the cis as well as in the trans form in the infected cells. This proteolytic step serves as a good candidate for viral detection because these proteases are highly expressed at an early stage of infection and the proteolysis is extremely efficient and selective.
A simple way to monitor
this proteolytic event inside a host cell is to engineer a fluorescent protein
pair linked by the target peptide sequence of the protease; proteolysis can be
detected based on changes in the fluorescence resonance energy transfer (FRET)
(Fig. 3). FRET is a phenomenon in which energy is transferred from an excited
fluorophore, the donor, to a light-absorbing molecule, the acceptor, located
within close proximity (Jares-Erijman and Jovin, 2003).
Figure 3. Schematic representation of fluorescent indicator for monitoring viral proteolytic processing in the infected cells.
The feasibility of this method has been demonstrated by genetically engineering a cell line to monitor proteases 2A (2Apro) proteolytic processing in poliovirus-infected cells (Hwang et al., 2006). Specifically, a hybrid fluorescent indicator composed of a linker peptide 1D2A, which is exclusively cleaved by PV 2Apro, flanked with a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) undergoing FRET was constructed. The activity of 2Apro is monitored through the fluorescent probe based on FRET between the donor (CFP) and acceptor (YFP) fluorophore. Both CFP and YFP are highly resistant to various proteases, while the 1D2A sequence is a unique substrate of 2Apro. In the presence of 2Apro, the covalent linkage between the two fluorophores is disrupted, which leads to spatial separation of CFP and YFP and results in a loss of FRET. As few as 10 PFU have been detected within 7 h of infection (Hwang et al., 2006).
Pilot experiments were conducted to evaluate the feasibility of the hypothesis that polioviruses (PV) could be detected in an engineered cell line using the CFP/YFP system. A stable clone of Buffalo Green monkey kidney (BGMK) cells was developed using G418 antibiotic selection to generate a population with positive gene integration. The transfected cells were then exposed to buffer with or without PV. Cell lysate was collected for the measurement of CFP intensity by a fluorometer and Western blotting was conducted to verify the fluorometer result (Fig. 4).
A 440-nm filter was used to generate laser light, which only excites CFP, and a 480-nm filter was selected to measure light emission of CFP. The excitation and emission wavelengths of CFP and YFP exhibit two distinct patterns and there is no cross over between the two fluorophores (Angres and Green, 1999). Cell lysate of the parental BGMK cells exhibited background CFP signal and was negative for CFP/YFP antibody (Fig. 4). However, the transfected cells showed a high expression level of FRET substrate, which was cleaved by PV 2Apro after infection, resulting in the increase in CFP intensity (Fig. 6). Both samples were normalized by the amount of YFP. There was some base level of CFP signal from uninfected cells; this is due to the fact that FRET energy transfer is not 100 % efficient and some fluorescent energy from CFP was released even with conjunction of the donor fluorophore (Fig. 4).
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