Recent Advancements in Virus Detection and Mechanistic Fate. Krista Wigginton Assistant Professor of Environmental Engineering University of Michigan
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1 Recent Advancements in Virus Detection and Mechanistic Fate Krista Wigginton Assistant Professor of Environmental Engineering University of Michigan
2 Acknowledgments for the graduate students who conducted this work, the funding sources of this work, Project #s: (BRIGE) (CAREER) (PIRE) Sage Chang Yinyin Ye Zhong Qiao and my research team.
3 Outline of today s talk: Overview of virus characteristics that can impact environmental fate and detection Part 1. Review of virus characteristics and detection methods Part 2. Research example of how virus envelopes and proteins influence fate in wastewater Part 3. Development of integrated integrated cell culture-mass spectrometry method for infective virus monitoring
4 Viruses are a major cause of gastrointestinal and respiratory illnesses Gastrointestinal Illnesses Respiratory Illnesses Pathogens in patients Taiwan, N=2647 Source: Chung et al., J.Microbiol.Immunol.Infect., (6) Infant respiratory illness, N = 197 Source: Regamey et al., Peddiatr Infect Dis J., 2008 Feb;27(2) 4
5 Virus characteristics: diameter Norovirus: nm E. coli: 2,000 nm Adenovirus: nm How this might impact fate and detection: too small for most filtration, too small for most microscopy and particle counting techniques
6 Virus characteristics: genome type and size ssrna ssdna dsrna dsdna How this might impact fate and detection: various susceptibilities to UV radiation, no conserved sequences, etc.
7 Virus characteristics: capsid proteins Krishnamani, V., Globisch, C., Peter, C. et al. Eur. Phys. J. Spec. Top. (2016) 225: How this might impact fate and detection: varied reactivity to oxidants, different tendencies to be adsorbed.
8 Virus characteristics: enveloped or nonenveloped How this might impact fate and detection: partitioning, susceptibility to oxidants, susceptibility to surfactants (and solvents), etc.
9 Virus characteristics: tissue tropism
10 Virus detection: culture methods
11 Virus detection: culture methods
12 Virus detection: culture methods
13 Virus detection: culture methods
14 Virus detection: culture methods
15 Virus detection: culture methods When complimentary cells aren t available, virus genes detected by PCR.
16 Virus detection: qpcr and RT-qPCR RNA RT
17 Virus detection: qpcr and RT-qPCR RNA RT cdna Taq Taq
18 Virus detection: qpcr and RT-qPCR DNA Taq
19 Virus detection: qpcr and RT-qPCR DNA Taq
20 Virus detection: qpcr and RT-qPCR Primer design is difficult and often results in false positives/negatives Absolute quantification is complicated by chosen standards and PCR inhibition PCR does not tell you if organism is infective Enzymes that drive PCR do not accurately capture all of the reactions in RNA and DNA OH RT
21 Virus detection: digital droplet PCR Digital PCR is an endpoint assay, meaning we do not care about the signal intensities of our samples and standards (as we do with qpcr). 10,000 to 20,000 mini reactions (nl) Each mini reaction will be + or for target after PCR Use Poisson statistics to calculate concentration: Mean (gene copies per droplet) = λ = -ln(1-p) Quantify total # of mini reactions with target by measuring absolute fluorescence where: p is the probability the droplet is + for the gene target (fraction of droplets with a + fluorescence)
22 Virus detection: concentration requirements Finished water must contain < 2 x10-7 viruses per liter to result in < 1 in 10,000 risk of infection per person per year. < 12 viruses < 1 virus Wigginton, K., Rockey, N., Dodd, M., Kohn, T. and Pecson, B. Bischel, H., Fontaine, N., WRF 14-17: White Paper on Non-Culture-Based Methods for Monitoring in Potable Reuse, in press.
23 Our research aims to provide mechanistic descriptions of virus fate in aquatic environments and to take new approaches to virus detection. To(wastewater( treatment(plant( Wastewater( treatment(plant( Drinking(water( treatment(plant( Wigginton, K. R., Y. Ye, and R. M. Ellenberg. Environmental Science: Water Research & Technology 1.6 (2015):
24 We employ virus models with various characteristics to link particle characteristics with environmental fate and to develop new methods.
25 How does the envelope impact virus fate in water? SARS MERS Fatality rate: 10-35% Avian influenza A (H5N1) Fatality rate: ~50% Zika virus epidemic Fatality rate: ~8% Proteins Lipid membrane Nucleic acids
26 We looked at the inactivation mechanism of an enveloped virus model and compared it with a non-enveloped virus model. Previous study characterized the inactivation mechanism of nonenveloped MS2 with free chlorine. We took the same approach with model enveloped virus Phi6. Wigginton, et al. "Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity." Environmental science & technology (2012): Ye, Yinyin, et al. "Reactivity of Enveloped Virus Genome, Proteins, and Lipids with Free Chlorine and UV254." Environmental science & technology (2018).
27 Part 3: Enveloped Phi6 much more susceptible to chlorine inactivation than MS2. Dose (mgl -1 s) Log inactivation MS2 (non-enveloped) Phi6 (enveloped) k app (mg -1 Ls -1 ) Phi 6 MS2 Ye, Y., et al, in press at Environmental Science & Technology.
28 We then looked at the reactions that take place in the virus components as the virus is inactivated by free chlorine. Free Chlorine Lipid reactions measured with high resolution LC-MS Enveloped virus Phi6 Loss of infectivity measured with plaque assays. UV 254 Genome reactions measured in three 500 bp regions with RT-qPCR Protein reactions measured with heavy isotope labeling and high resolution LC-MS Ye, Y. et al., Environ. Sci. Technol. 2018, 50,
29 Genome reactivity comparison of MS2 and Phi6 genome reactions by FC Does not account for observed inactivation 0.35 k app (mg -1 Ls -1 ) Does account for observed inactivation MS2 Phi6 Ye, Y. et al., Environ. Sci. Technol. 2018, 50,
30 Most reactive proteins in enveloped Phi6 reacted 150x faster than most reactive proteins in nonenveloped MS2 proteins. kapp (L mg -1 s -1 ) k exp (mg -1 L s -1 ) A 3-19 A A A A A Most reactive peptides Phi6 Peptides ~150 x MS2 Reactive methionine residues in Phi6 proteins more exposed than methionine residues in MS2 proteins. kapp (L mg -1 s -1 ) P Free chlorine penetrated lipid membrane to react with inner capsid proteins. P P P Most reactive peptides P P Ye, Y. et al., Environ. Sci. Technol. 2018, 50,
31 Lipids did not degrade significantly over time course of inactivation, but some lipid oxidation products were detected in chlorine experiments. Ye, Y. et al., Environ. Sci. Technol. 2018, 50,
32 We have compared the inactivation and partitioning of model enveloped and nonenveloped viruses in untreated wastewater. MHV Phi 6 MS2 T3 20 nm 100 nm ss RNA 85 nm ds RNA ss RNA 55 nm ds DNA Ye, Yinyin, et al. ES&T, (2016): Survival Adsorption Recovery
33 The enveloped virus surrogates lost infectivity more rapidly than the non-enveloped viruses in wastewater. 10 C, WW 25 C, WW 0 0 Log Inactivation T4 MS2 MHV φ6 T4 MS2 MHV φ T4 MS2 MHV φ6 10 C 25 C T4 MS2 MHV φ Time (h) Time (h) Conditions: Raw wastewater, [virus] 0 = pfu/ml Ye, Yinyin, et al. ES&T, (2016):
34 The enveloped viruses adsorbed slower and to greater extent with WW solids than non-enveloped viruses. MS2 MHV ~40% adsorbed ln (C/C 0 ) -1.0 ln ln (C/C (n f / n fo ) 0 ) Solid-free control Wastewater Pure inactivation Fitting curve Simulation curve Solid-free control Wastewater Pure inactivation Fitting curve Simulation curve Time (hours) Time (hours) Ye, Yinyin, et al. ES&T, (2016):
35 Conclusions for enveloped virus work Model enveloped virus more susceptible to oxidants than model nonenveloped virus Free chlorine inactivation in Phi6 was protein mediated and in MS2 was genome mediated. Free chlorine penetrated lipid bilayer and reacted with inner capsid methionine residues. Two model enveloped viruses partition to greater extent with wastewater solids than nonenveloped viruses. The majority of our four model viruses would remain in the water after primary settling at a WWTP.
36 Part 3: Developing new methods for monitoring environmental viruses. Methods Infectivity Specificity Culture-based methods Yes Low Yes Capability to measure multiple viruses Method optimization High Genome mutations Polymerase chain reaction (PCR), Real-time PCR Integrated cell culture-pcr (ICC-PCR) Integrated cell culturemass spectrometry (ICC-MS) No High No High May fail Yes High No High May fail Yes High Yes Low Lower mutation rates at protein level than genome level 36
37 We hypothesized that an ICC-MS method could detect infectious human viruses in wastewater. Cell culture system Viral protein Database (SwissProt) Liquid chromatography tandem mass spectrometry (LC-MS/MS) Peptide sequences Ye & Wigginton, in prep
38 We first developed the ICC-MS method with a model system including mouse coronavirus (MHV) and its host cells (L2). Murine hepatitis virus (MHV-A59) in media L2 cell lines Viral protein Database (SwissProt) Peptide sequences LC-MS/MS Ye & Wigginton, in prep
39 The ICC-MS method development encompassed the following steps. MHV-A59 2 ~23 cm Wastewater L2 cell lines 11. How long should we culture MHV before detecting with MS? 22. What is the minimum number of viruses that can be detected? 33. What is the impact of wastewater components on the ICC-MS method? Once optimized in mouse model, could the ICC-MS method detect infectious human viruses in wastewater samples? Ye & Wigginton, in prep
40 We next applied the ICC-MS method to try to detect infectious human viruses in wastewater samples. 1.2 ml ~23 cm 2 14 Days 50 concentrated wastewater samples Vero, BSC, BGM Screen/grit Primary treatment Secondary treatment Disinfection Discharge Ye & Wigginton, in prep Sludge treatment
41 Reovirus proteins were detected in primary influent, and effluent pre- UV treatment. Primary influent 1 Accession Sequence Identified viral proteins number coverage (%) P11077 mu-1, Reovirus type 1 32 P11078 mu-1, Reovirus type 3 28 P03527 sigma-3, Reovirus type 3 24 P30211 sigma-3, Reovirus type 2 13 P07939 sigma-3, Reovirus type 1 25 P03525 sigma-2, Reovirus type 3 25 P11314 sigma-2, Reovirus type 1 26 P07940 sigma-ns, Reovirus type 1 19 P03526 sigma-ns, Reovirus type 3 17 P12419 mu-ns, Reovirus type 3 17 Q9PY83 mu-ns, Reovirus type 1 13 Q9WAB2 lambda-1, Reovirus type 1 7 P15024 lambda-1, Reovirus type 3 7 P11079 lambda-2, Reovirus type 3 3 Q00335 mu-2, Reovirus type 1 3 P12418 mu-2, Reovirus type 3 3 Accession number Effluent pre-uv Identified viral proteins Sequence coverage (%) P03527 sigma-3, Reovirus type 3 15 P30211 sigma-3, Reovirus type 2 7 P11077 mu-1, Reovirus type 1 6 Accession number Primary influent 2 Identified viral proteins Sequence coverage (%) P11077 mu-1, Reovirus type 1 40 P11078 mu-1, Reovirus type 3 36 P03527 sigma-3, Reovirus type 3 43 P07939 sigma-3, Reovirus type 1 38 P30211 sigma-3, Reovirus type 2 27 Q9WAB2 lambda-1, Reovirus type 1 29 P12419 mu-ns, Reovirus type 3 24 Q9PY83 mu-ns, Reovirus type 1 19 P12418 mu-2, Reovirus type 3 17 P03526 sigma-ns, Reovirus type 3 22 P07940 sigma-ns, Reovirus type 1 24 P03525 sigma-2, Reovirus type 3 17 P11314 sigma-2, Reovirus type 1 19 Q91RA6 lambda-2, Reovirus type 1 9 P11079 lambda-2, Reovirus type 3 7 P0CK32 lambda-3, Reovirus type 1 3 Accession number Final effluent Identified viral proteins Sequence coverage (%) N.A. N.A. N.A. Ye & Wigginton, in prep
42 We are expanding the method to monitor many types of infectious viruses in wastewater samples using different cell lines. Wastewater 50 concentrated Virus enrichment LC-MS/MS LC-MS/MS LC-MS/MS LC-MS/MS
43 Part 3: Developing new methods for monitoring environmental viruses. Methods Infectivity Specificity Culture-based methods Yes Low Yes Capability to measure multiple viruses Method optimization High Genome mutations Polymerase chain reaction (PCR), Real-time PCR Integrated cell culture-pcr (ICC-PCR) Integrated cell culturemass spectrometry (ICC-MS) No High No High May fail Yes High No High May fail Yes High Yes Low Lower mutation rates at protein level than genome level Ye & Wigginton, in prep
44 Acknowledgments for the graduate students who conducted this work, the funding sources of this work, Project #s: (BRIGE) (CAREER) (INFEWS) (PIRE) Sage Chang Yinyin Ye Zhong Qiao and to my research team.
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