Persistence of Naked Viral RNA Molecules in Groundwater in the presence of Indigenous Bacterial Populations
The Bridges To The Future
Texas A&M Research Center
1380 A&M Circle
El Paso, TX 79927
Year End Report
It is generally assumed that RNA molecules are extremely labile and that naked viral RNA molecules are rapidly degraded in groundwater.
There have been a number of reports documenting that techniques such as RT-PCR are more sensitive than conventional techniques such as tissue culture for the detection of pathogenic viruses in groundwater.
However, questions surrounding the viability and infectivity of such samples still linger. Reports that the detection of enteric viruses in groundwater by RT-PCR based approaches are often not correlated with tissue culture data raises an important question whether these methodologies are detecting naked viral nucleic acids or intact virus particles. Reports suggesting that naked viral RNA molectules are extremely short lived under natural conditions, and thus, RT-PCR results are only indicative of intact capsids containing RNA molecules.
Laboratory studies were performed using groundwater collected from a deep aquifer to determine whether viral RNA molecules extracted from MS2 bacteriophage could resist degradation.
Our objective in these studies was to identify whether naked viral RNA from positive strand viruses can be persistent in groundwater. Given that RNA molecules have a propensity to form secondary structure, we hypothesized that viral RNA molecules could resist rapid degradation in groundwater. The RNA from MS2 bacteriophage was used as the model RNA molecule.
Materials and Methods
Viruses and Viral Genome Extraction: Aliquots of high titer lysates of the MS2 bacteriophage was directly employed in the purification of its nucleic acids. The commercial viral RNA extraction. Absorbance reading were used as the basis to estimate viral RNA concentrations. In experiments where intact viruses were employed, the lysate was cesium chloride purified, dialyzed and resuspended in sterile deionized water prior to use.
Experimental Setups: (200µl, 15ml, and 35ml) samples were inoculated with known amounts of viral RNA in polypropylene tubes. At defined time frames, the samples were concentrated to 11µl, and the presence/absence of specific viral genes were noted using RT-PCR approaches.
Amplification and Detection of Viral Genes: The four bacteriophage genes encompassing the entire genome were employed as the target genes (Figure 1.) Reverse transcription reactions using the 11µl of template were performed as follows: 25°C for 15 min., 42°C for 60 min., and 99°C for 5 min. The PCR amplification conditions were as follows: 94°C for 45 secs., 55°C for 30 secs., and 72°C for 45 secs. A total of 30 amplification cycles were employed. In experiments where intact capsids were involved, the sample was heated to 99ºC for 5 min. prior to the RT step to release the nucleic acids. Agaros gels were employed to detect the amplification products.
Persistence of bacteriophage RNA in groundwater
In the 200ml groundwater experiment, all four genes were present for up to 3 days. On day 10, only three of the four genes were evident, while on day
28, only two of the inner most gene (C and L) were still barely detectable. The presence of two of the four genes even at the end of 28 days suggest that the entire viral genome does not rapidly degrade in groundwater as is commonly believed to occur to RNA molecules. The presence of the two inner most genes suggest that the secondary structure could have played a key role in protecting these two genes from nuclease digestion.
day 3 day 10
In the larger 35 ml microcosm, all four genes were present for only 24 hours, while the inner most genes (C and L) were detectable on day 12. However, none of the four genes were detectable on day 30 (data not included).
· Naked viral RNA can be persistent in groundwater. Previous studies in our laboratory has documented the stability of biral genomes even in the presence of chlorination conditions.
· The persistence of complete genomes in the absence of viable phage particles suggest that viral nucleic acid degradation is not the cause of viral inactiviation in groundwater.
· RT-PCR methodologies for detecting viral particles has the potential of detecting these naked persisting viral genomes, and not necessarily, only intact virus particles.
· The demonstrated structural stability of these RNA genomes forces us to rethink the long standing dogma that RNA molecules are extremely labile.
· Our future studies include similar experiments using Poliovirus. As well as investigating whether these persisting naked RNA molecules retain their biological activities, for example, protein synthesizing ability and infectivity.