The Reactions in Viral Nucleic Acids During Photolysis and Chlorine Disinfection

Abstract

Virus-induced diseases pose risks to public health and cause significant impacts on our economy. People can become infected by waterborne virus pathogens when they come into contact with drinking water and recreational water that was not properly treated and disinfected. Nucleic acids (DNA/RNA) carry the genetic instructions for viruses to replicate in their host cells; therefore, damaging viral nucleic acids is an effective way to inactivate viruses and reduce risks of waterborne infection. UV254 and chlorine are two disinfection methods commonly used in water treatment, and both lead to reactions in viral genomes. Despite the widespread use of disinfection, scientists and engineers still lack a comprehensive understanding of the reactions that take place in viral nucleic acids, the impact of higher order structure on viral genome reactivity during UV254 and chlorine disinfection. With this knowledge, it might become possible to predict the inactivation kinetics of newly emerged viruses and other viruses that are not readily culturable.

To address these knowledge gaps, this dissertation explores the reactions that occur in viral nucleic acids during photolysis and chlorine disinfection. The research spans several levels of nucleic acid reactivity, from the short nucleic acid oligomer level, up to the entire viral genome incorporated in virus particles. In the first portion of this work, the photochemical reactions that take place in viral RNA oligomers were investigated. Specifically, RNA oligomer segments from the genome of bacteriophage MS2 were exposed to UV254, simulated sunlight, and singlet oxygen (1O2), and the oligomer reaction kinetics were analyzed with RT-qPCR and quantitative MALDI-TOF mass spectrometry (MS). One especially important finding of this work was that quantitative MALDI-TOF-MS detected significantly more RNA modifications than RT-qPCR. This suggests that certain chemical modifications in the RNA are not detected by the reverse transcriptase enzyme. High-resolution ESI-Orbitrap MS identified pyrimidine photohydrates as the major UV254 products, which may have contributed to the discrepancy between the MS- and RT-qPCR-based results.

In the second portion, the influence of viral nucleic acid higher order structure on UVC photolysis was examined. We measured the direct UV254 photolysis kinetics of four model viral genomes composed of single-stranded and double-stranded RNA, as well as single-stranded and double-stranded DNA, in ultrapure water, in phosphate buffered saline, and encapsidated in their native virus particles. The photolysis rate constants of naked nucleic acids measured by qPCR (RT-qPCR for RNA) and normalized by the number of bases measured in a particular sequence exhibited the following trend: ssDNA > dsDNA ≈ ssRNA > dsRNA. Interestingly, encapsidation of viral genomes did not affect the photoreactivity of most genome sequences. A large difference in photoreactivity was observed between single and double strands of both RNA and DNA.

In the final portion, the impact of viral genome higher order structure on reactivity with free chlorine was characterized. Chlorine reaction kinetics of the same four model viral genomes were measured when they were naked in solution and when they were incorporated in their native virus particles, respectively. We observed that for most of the nucleic acid regions studied, the naked viral genomes reacted with chlorine significantly faster than encapsidated genomes. The research suggests that dsDNA was the least reactive of the genome types tested. Specifically, the two T3 dsDNA regions were ~72 times more resistant than the ssDNA regions, which was the most reactive genome type tested.