WATER AS A WAY FOR TRANSMISSION OF PLANT VIRUSES Mehle and Ravnikar, Water research, 2012: 4902-4917 PLANT VIRUSES IN WATER
• Some plant viruses are very stable in environment (outside the host) • Pathogenic plant viruses can be transmitted by water (Schwarz, et al. 2010)
The titer of the viruses in environmental waters is extremely low; To monitor viruses in water we need two steps: Efficient concentration + efficient detection Example: survival and water-mediated transmission of potato virus Y, pepino mosaic virus and potato spindle tuber viroid (Mehle et al, 2015, Applied and Environmental Microbiology)
Survival in water at 20 ± 4°C: -PepMV: for up to 3 weeks -PSTVd: for up to 7 weeks -PVY: 1 week Workflow for detection of plant viruses in water
CIM monolithic Influent Eluate chromatography containing RT-qPCR viruses Test for efficiency of concentration
Picor navir idae
Picor navirales
HTS: Illumina Le viviridae
Wastewater Microviridae Tymo virales dsRNA vir uses Effluent unclassified Sipho viridae ssDNA vir uses ssRNA positiv e?str and vir uses , no DNA stage treatment plant dsDNA viruses , no RNA stage ssRNAvir uses
unclassified bacter ial vir uses unclassified RNA vir uses ShiM?2016
Caudovirales Viruses Sipho viridae unclassified RNA vir uses unclassified vir uses
My oviridae
Te ven virinaeSpouna virinae
unclassified P odo viridae P odovir idae
unclassified My ovir idae Pico virinae
N4vir us
A utographivir inae
TEM Phikmvvir us
Virome of the water samples
Infectivity testing CIM® monolithic chromatography
-It is based on electrostatic driven binding of negatively charged viruses to positive monolithic column (CIM QA).
-CIM columns can concentrate different viruses simultaneously from both fresh (Steyer et al 2015) and sea water (Balasubramanian et al, 2016).
- CIM concentration of wastewater samples resulted in an increase of virus concentration of approximately two orders of magnitude (corresponding to approximately 6 units in the Cq values between concentrated and nonconcentrated sample) (Steyer et al. 2015)
(Convective Interaction Media) quaternary amine is a strong anion exchange group that selectively binds molecules with a predominant negative charge Analysis of metagenomic sequencing data Metagenome of wastewater samples
Visualization: METACODER: Foster, Zachary S. L., et al. 2016. bioRxiv, p. 71019, doi:10.1101/071019.
Most commonly detected plant viruses Tomato mosaic virus Cucumber green mottle mosaic virus Cucumber mosaic virus Pepper mild mottle virus Tobacco mild green mosaic virus Melon necrotic spot virus Tobacco mosaic virus Southern bean mosaic virus Turnip vein-clearing virus Plant viruses detected in ww: Cucumber green mottle mosaic virus Paprika mild mottle virus Pepper mild mottle virus Tobacco mild green mosaic virus Tobacco mosaic virus Tomato mosaic virus Turnip vein-clearing virus Hoya chlorotic spot virus Erysimum latent virus Dulcamara mottle virus Grapevine fleck virus Garlic common latent virus Potato virus S Potato virus M Pepino mosaic virus White clover mosaic virus Tulip virus X Garlic virus A Garlic virus B Carnation mottle virus Melon necrotic spot virus Sitke waterborne virus Olive mild mosaic virus Southern bean mosaic virus Ryegrass mottle virus Cocksfoot mottle virus Turnip ringspot virus Watermelon mosaic virus Grapevine fanleaf virus Citrus tristeza virus Cucumber mosaic virus Peanut stunt virus Brome mosaic virus Pepper cryptic virus 2 Infectivity tests
Nicotiana benthamiana Nicotiana occidentalis Cucumis sativus VIRUS DETECTION IN PLANTS After mechanical inoculation with concentrated influent/effluent
Symptoms observation TEM of infected plant material
Tobamovirus morphology RT-qPCR (specific viral detection) Small RNAs for the detection of plant viruses by Illumina sequencing (non-targeted viral detection) Infectivity of plant viruses plants after mechanical inoculation with concentrated influent
MOCK PMMoV (+) MOCK PMMoV (+)
N. benthamiana – PMMoV infection N. occidentalis– PMMoV infection
MOCK ToMV (+) • Tomato Mosaic Virus; ToMV
• Pepper Mild Mottle Virus; PMMoV
N. occidentalis–ToMV infection Infectivity of plant viruses plants after mechanical inoculation with concentrated effluent
PMMoV (+) MOCK PMMoV (+) MOCK TMGMV (+)
N. benthamiana - PMMoV infection N. occidentalis– PMMoV and TMGMV infected
• Pepper Mild Mottle Virus • Tobacco mild green mosaic virus PMMoV TMGMV Conclusions • High diversity of viruses in wastewater was found • We confirmed infectivity of some plant viruses from Tobamovirus genus (family Virgaviridae) even after conventional wastewater treatment • We detected genomes of many viruses that were not reported in Slovenia before • Development of new workflow for metagenomic analysis of environmental water samples and discovery of viruses present in the environmental water
Wastewater treatment plant DOMŽALE KAMNIK ELIMINATION OF VIRUSES FROM WATER HIDRODYNAMIC CAVITATION MS2 inactivation after hydrodynamic cavitation
reduced in more than 4 logs
completely inactivated
Kosel et al., 2017. Efficient inactivation of MS-2 virus in water by hydrodynamic cavitation. Water Res. 124, 465-471. ELIMINATION OF VIRUSES FROM WATER PLASMA TECHNOLOGY ELIMINATION OF VIRUSES FROM WATER PLASMA TECHNOLOGY PVY inactivated after 1 min of treatment
Cold Atmospheric Plasma as a Novel Method for Inactivation of Potato Virus Y in Water Samples Arijana Filipić, Gregor Primc, Rok Zaplotnik, Nataša Mehle, Ion Gutierrez-Aguirre, Maja Ravnikar Miran Mozetič, Jana Žel, David Dobnik Food and Environmental Virology (in press) METHODS FOR CHARACTERIZATION OF VIRUSES
Droplet digital PCR Real-time PCR PCR
from Wikipedia
from Wikipedia Explore mechanisms bio-rad.com Electron of viral inactivation microscopy Nanopore sequencing
Infectivity testing
from Wikipedia nanoporetech.com thermofisher.com