Molecular Methods for the Detection of Infectious Enteroviruses in Source or Finished Waters

摘要

Environmental or source water samples and even finished drinking water samples may contain virus particles detectable by molecular methods such as PCR and RT-PCR. These methods in conjunction with confirmatory analyses (e.g., nucleic acid hybridization or nucleotide sequencing) are useful in determining the potential presence or absence of viruses in waters, even at very low levels. However, these tests do not conclusively determine whether the viruses are infectious or non-infectious. The current standard method for detection of infectious viruses in water samples or concentrates is cell culture with two passages for confirmation of cytopathic effect (CPE). Viruses that do not cause CPE or have limited replication are not detected. Furthermore, the procedure requires weeks and is costly. Evidence of replication in cell culture without repeated passage may be obtained by evaluation of infected cells for the presence of viral nucleic acid; however, testing of dilutions is necessary to demonstrate replication. We have developed molecular methods to directly confirm replication or viability of positive-sense, single-stranded RNA viruses in cell culture, regardless of their ability to produce CPE. Key enteric key viral pathogens with such nucleic acid include enteroviruses (coxsackieviruses and echoviruses and polioviruses), caliciviruses, hepatitis A and E viruses and astroviruses. During replication of these viruses, a complimentary negative strand is made that is present primarily paired with the positive strands as replicative intermediate RNAs in infected cells. Detection of the negative strands or the double stranded replicative form RNA complexes containing positive and negative strands is proof of viral replication. We have developed RT-PCR methods to detect the negative strands or double-stranded forms of viral RNA in the infected cultures using Coxsackievirus B3 as a model virus. Specific primer pairs for CVB3 were used for detection of the negative strand. With the use of panenterovirus primers for detection of the double-stranded forms, we have demonstrated that as low as 4 infectious units can be detected within 24 hrs after infection. The methods we have developed offer great promise as rapid, sensitive, and specific approaches to the molecular detection of infectious human enteric viruses in source water and drinking water supplies.