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NERC-EMHH Environmental
Microbiological Risk Symposium
WELCOME
14th March 2018
Education Centre and Main Hall
Royal Geographical Society, London
Environmental Microbiology
and Human Health
Environmental Microbiological
Risk Symposium
Caroline Culshaw
NERC Head of Environment and Health
Welcome and introduction
Research Councils UK
UK Research and Innovation
Our goals
• understand and predict how our
planet works
• manage our environment responsibly
as we pursue new ways of living,
doing business, escaping poverty and
growing economies.
Fund excellent, peer-reviewed environmental
science that helps us:
Our vision
To place
environmental
science at the
heart of
responsible
management of
our planet
Meeting society’s needs
Benefiting from natural resources
Resilience to environmental hazards
Managing environmental change
• Discovery science
Overview of the Environmental
Microbiology and Human Health Programme
Problem
• Environment provides pathways for human exposure to
pathogenic and allergenic microorganisms
• Traditional culture methods are slow and selective
• How to exploit rapid advances in molecular biology?
• Near real-time measurements would greatly enhance exposure
modelling capability and characterise risk to public health
Programme aim
To provide the scientific evidence to support fast and efficient
identification of pathogenic and allergenic microorganisms and
biological material in environmental media, which can be used in
appropriate tools and models for the protection of public health
Programme
Aquatic microbiology
• RESERVOIRS
• VIRAQUA
Bioaerosols
• ENDOTOX II
• RAMBIE
• £5.15 million 2015 - 2019
• Funded four projects in two themes
• Programme integration group
Aquatic microbiology
• RESERVOIRS
Next generation sequencing to reveal human impact on aquatic reservoirs of antibiotic resistant bacteria at the catchment scale
Outcome – informed plans to reduce environmental dissemination, selection and human exposure to ABR bacteria
• VIRAQUA
New approaches for the quantitative detection of human pathogenic viruses within the freshwater-marine continuum
Outcome - new guidelines for assessing infection risk (e.g. in recreational waters, beaches & shellfisheries) and protecting human health
Ambitions for the programme
• Fast, accurate, cheap and reproducible molecular methods to identify
and quantify microorganisms
• Methods to determine viability
• New insights into risk assessment from process studies
• Improved predictive models of pathogenic microorganisms in freshwater
and coastal ecosystems
• Collaboration with industry and policy partners
• Programme-wide coordination and knowledge exchange
Tracing the fate and infectivity of human pathogenic viruses through the environment
Dr Kata Farkas, Bangor University
Caliciviridae40 nmssRNANorovirus Sapovirus
Picornaviridae30 nmssRNAHepatitis A virusAichivirusEnterovirus
Hepeviridae30 nmssRNAHepatitis E virus
Polyomaviridae50 nmssDNAPolyomavirus
Adenoviridae80 nmdsDNAAdenovirus
Enteric viruses
PathogensGastroenteritis, 2-5 daysTransmission: faecal – oralIcosahedral capsid, RNA/DNA genome
IndicatorsAsymptomaticTransmission: faecal/urinal – oralIcosahedral capsid, DNA genome
Environmental transmission
• Contaminated humans are supershedders 105-1011 norovirus particles/g stool
• Released daily in wastewater 102-108 norovirus particles/litre Resistant to treatment
• Contamination Rivers, lakes, sea Groundwater, aquifers Drinking water Fruit and vegetable (irrigation) Shellfish
Viral outbreaks
Objectives
New molecular methods for the quantification of enteric viruses in the environment
Surveillance
Modelling
Risk assessment
Viral ecologyViral
infectivity
Water concentration
Water, 10 L
Tangential flow ultrafiltration, 50 mL
Beef extract elution
PEG precipitation, 2-4 mL
Nucleic acid extraction
q(RT-)PCR
Tangential flow ultrafiltration
Sea Estuarine River
Rec
overy
0
20
40
60
80
100
120NoV GIINoVGII PGMHAVSaVMgVAdV40
pH 8.05 6.91 7.5
T (NTU) 6.37 11.74 3.2
K (mS/m) 40.7 18.88 0.06
• 5000x concentration• 24 hours• £20/sample• 10-100% recovery of enteric viruses• Different water types• Co-concentration of viruses, bacteria, protozoa• Spare viral integrity
Virus concentration in sediment
Sediment, 10-100 g
Beef extract elution
PEG precipitation, 1-2 mL
Nucleic acid extraction
q(RT-)PCR
Elution – concentration
• 10-100x concentration• 24 hours• £1/sample• 80-100% recovery of enteric viruses• Spare viral integrity
Virus concentration in shellfish
Shellfish digestive tissue
Proteinase K tretment
Nucleic acid extraction
q(RT-)PCR
• ISO/TS 15216-1:2013 standard Elution with proteinase K
• Elution with alternative bufferPBS
SM
• Adsorption-twice-elution-extraction (Kittigul et al, 2015)tryptose phosphate broth, pH 9 – arginine, pH 7.5 – chloroform
Virus concentration in shellfish
ISO PBS SM Twice elution
Re
co
ve
ry %
0
20
40
60
80
100
120
140
160MengovirusAdenovirusHepatitis A virusNorovirus GII
Betws-y-Coed WWTP1,200 inhabitants
Llanrwst WWTP4,000 inhabitants
Tal-y-Bont WWTP1,000 inhabitants
Ganol WWTP82,000 inhabitants
Surveillance of enteric viruses in the water environment
CSO Combined Sewer Outflowrainwater + untreated wastewater
Shellfish harvesting area
SW1 – river
SW2 – river
SW3 – tidal limit
SW4 – estuary
Surveillance of enteric viruses in the water environment
Sed1-SF1 – shellfish and sediment
Sed2-SF2 – shellfish and sediment
Sed4 – sediment
GI
BI
BE LI
LE TITE
SW
1SW
2SW
3SW
4
SW
4 Sed
Sed
1
Sed
2SF1
SF2
log
gc
/L o
r lo
g g
c/g
0
1
2
3
4
5
6
7
8Norovirus GINorovirus GIIAdenovirusJC polyomavirus
Surveillance of enteric viruses in the water environment
Enteric pathogens• Norovirus GI/GII and Sapovirus: No diurnal changes in wastewater Strong seasonality in wastewater, surface water,
sediment and shellfish• Hepatitis A/E virus Not detected
Indicator viruses• Adenovirus and JC polyomavirus No diurnal changes in wastewater No seasonal changes Detected at high concentrations in all sample types
Wastewater
Water
Sediment Shellfish
Viral infectivity
qPCR detection
qPCR target region
Viral infectivity
Adenoviruses, polyomaviruses• Infectivity – culturing• 48h assay• Requires skilled staff and equipment
Norovirus?
Norovirus integrity
Porcine Gastric Mucin assay – qPCR detection
Norovirus integrity
Porcine Gastric Mucin assay – qPCR detection
Untreated wastewater: high concentrations
GI 09/1
6
GI 12/1
6
GI 02/1
7
GI 03/1
7
GI 04/1
7
GI 05/1
7
GI 06/1
7
GI 08/1
7
BI 10/1
6
BI 03/1
7
BI 05/1
7
BI 08/1
7
TI 09/1
0
TI 02/1
7
TI 03/1
7
TI 07/1
7
LI 02/1
7
LI 08/1
7
gc/L
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
NoVGI directNoVGI PGM
GI 09/1
6
GI 10/1
6
GI 11/1
6
GI 12/1
6
GI 01/1
7
GI 03/1
7
GI 04/1
7
GI 05/1
7
GI 06/1
7
GI 08/1
7
BI 09/1
6
BI 10/1
6
BI 11/1
6
BI 01/1
7
BI 05/1
7
BI 06/1
7
TI 09/1
6
TI 10/1
6
TI 11/1
6
TI 01/1
7
TI 04/1
7
TI 05/1
7
TI 06/1
7
LI 09/1
6
LI 11/1
6
LI 01/1
7
LI 04/1
7
LI 05/1
7
LI 06/1
7
gc/L
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
NoV GII directNoVGII PGM
Norovirus integrity
Porcine Gastric Mucin assay – qPCR detection
Untreated wastewater: high concentrationsTreated wastewater: 0-4 log reductionWorks better for norovirus GII than for GI
BE
02/1
7
BE
03/1
7
BE
05/1
7
BE
07/1
7
TE
09/1
6
TE
10/1
6
TE
11/1
6
TE
02/1
7
TE
03/1
7
TE
05/1
7
TE
07/1
7
LE
11/1
6
LE
05/1
7
LE
08/1
7
gc/L
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
NoVGI directNoVGI PGM
BE
09/1
6
BE
10/1
6
BE
01/1
7
BE
04/1
7
BE
05/1
7
TE
09/1
6
TE
10/1
6
TE
11/1
6
TE
01/1
7
TE
05/1
7
LE
09/1
6
LE
11/1
6
LE
01/1
7
LE
03/1
7
LE
05/1
7
LE
06/1
7
gc/L
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
NoV GII directNoVGII PGM
Norovirus integrity
Porcine Gastric Mucin assay – qPCR detection
Untreated wastewater: high concentrationsTreated wastewater: 0-1 log reductionWorks better for norovirus GII than for GISurface water: 1-3 log reductionSediment: all negative
SW
1 1
1/1
6
SW
1 0
2/1
7
SW
3 0
2/1
7
SW
3 0
5/1
7
Sed1 1
0/1
6
Sed1 0
2/1
7
Sed1 0
3/1
7
Sed1 0
7/1
7
Sed2 0
2/1
7
Sed4 0
2/1
7
Sed4 0
4/1
7
gc/L
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
NoVGI directNoVGI PGM
SW
1 0
9/1
6
SW
1 1
1/1
6
SW
1 0
5/1
7
SW
3 0
5/1
7
SW
4 0
9/1
6
Sed1 0
9/1
6
Sed1 1
1/1
6
Sed4 1
1/1
6
Sed4 0
4/1
7
gc/L
1e+1
1e+2
1e+3
1e+4
1e+5
1e+6
1e+7
NoV GII directNoVGII PGM
Norovirus integrity
Porcine Gastric Mucin assay – qPCR detection
Semi-degraded viruses?
Illumina HiSeq 4000
viral genomic RNA
wikipedia.org
viral genomes
sapovirus genomes
norovirus genome viral diversity
Metaviromics
Dr Evelien Adriaenssens
Non-targeted sequencing
WW mussels river/est. water sediment
Fam
ily le
vel g
rou
pin
gs
sapo/norovirus
rotavirus
Do the bubbles represent intact/infective viruses?
Intactness inferred from completeness of genome fragments foundCannot give information on infectivity
Example: Two complete sapovirus GII genomes in untreated wastewater only small fragments in mussels degraded genomes
Usefulness:• Identification of novel strains circulating in the environment Pathogens: norovirus GI Indicators: picobirnaviruses
• Trends in viral ecology• Epidemiology
Use the novel human norovirus infectivity assay with environmental samples
• Validation of capsid integrity assays
• Validation of metaviromics results
Dr Myra Hosmillo
Viral infectivity
Summary
• Method validationProtocols on CEFAS website
Two-step concentration of complex water samples for the detection of viruses
Elution and concentration of viruses in sediment*
Quantification of nucleic acids of enteric viruses in concentrated environmental samples**
Capsid integrity assays for the detection and quantification of enteric viruses in environmental samples
Assessment of norovirus infectivity risk in bivalve shellfish using a F-specific coliphage
Viral nucleic acid extraction for metagenomics sequencing
*Farkas K, Hassard F, McDonald JE, Malham SK, Jones DL. (2017) Evaluation of molecular methods for the detection and quantification of
pathogen-derived nucleic acids in sediment. Frontiers in Microbiology 8:53. doi:10.3389/fmicb.2017.00053.
**Farkas K, Peters DE, McDonald JE, De Rougemont A, Malham SK, Jones DL. (2017) Evaluation of two triplex one-step qRT-PCR assays for the
quantification of human enteric viruses in environmental samples. Food and Environmental Virology. 9:342-349. doi:10.1007/s12560-017-9293-5.
Summary
• Method validation
• Identification of potential indicators
• Identification of emerging pathogens
• Seasonality
• Ecology
• Integrity and infectivity
• Modelling
www.viraqua.uk@Viraqua_Project
Viral dispersal in the coastal zone: a method to quantify water quality risk
Peter Robins [email protected] Cooper · Kata Farkas
Shelagh Malham · Davey Jones
Conwy - UK: characteristics andimportance of water quality
Estuary:• Macro-tidal (tidal range 4-6 m) • Embayment-type estuary 20km in length • Strong tidal mixing = vertically mixed• Sensitive to high frequency river flow variability
Catchment:• Size: 380 km2
• Annual rainfall: > 3500 mm• Geology: Impermeable, steep, mountainous• River (1965-2005):20 m3/s (mean)
1.35 m3/s (Q95)45.3 m3/s (Q10)
• Hydrograph: “flashy” (rainfall < 12 hrs to estuary)
Methodology:
Catchment hydrology model
River flows
Estuary model
Tide
Virus flows
Rainfall
CASCADE- Catchment scale water quality model
• 50m spatial grid
• Variable time stepping, 30 min default
• Transport modelled using particle tracking
• Developed under the NERC Macronutrients project to estimate nutrient fluxes, with application to the Conwy catchment
• Used in Viraqua to estimate velocities for virus transport to the estuary
CASCADE – Calculating viral fluxes
• Scenario application with 150 sec time step.
• Assumed virus sources at Llanrwst and Betws-y-Coed waste water treatment works (WwTW), with no other sources.
• River flows estimated from 30 minute simulated flows (low-medium flow conditions).
• 30 min pulse of viral load using typical WwTW flow (.025, .01 m3/s) and high virus (e.g. norovirus) concentrations (200000 gc/L) from WwTW field measurements.
CASCADE - In-river virus transport to the tidal limit
Variability in virus concentration and flux
Fluxes of HAdV virus are probably dominated by flow and not concentration differences between sources.
River Conwy and Llugwy refer to sites upstream of the influence of the two STWs.
Telemac – Estuary and ocean model
• 15 - 500 m spatial grid
• Observational bathymetry
• Tidal and 15-min river boundary conditions
• 2-D depth-averaged
• Used in Viraqua to estimate virus transport in the estuary and surrounding coast
Summary of model simulations.
Run Period River flow Tidal regime Virus
1 Annual 01 Mar ’16 – 01 Apr ‘17 01 Mar ’16 – 01 Apr ‘17Data-assimilated
with/without decay
3 15 d
1 × flash hydrographV1
Mean tide (M2 only)Constant (= 100 ppl) with
decay
2 × flash hydrographs V2
2 × flash hydrographs V1
3 × flash hydrographs V1
1 × slow hydrograph V1
4 15 d 1 × flash hydrograph V1
Spring-to-neap
Constant (= 100 ppl) with
decay
Spring-to-neap (+ 3 hrs)
Spring-to-neap (+ 6 hrs)
Neap-to-spring cycle
5 15 d 30 Jan – 13 Feb 2004 V3 11 – 26 Apr 2016Constant (= 1×105 ppl) with
decay
[Freshwater volume over 7 days: V1 = 37 m3; V2 = 67 m3; V3 = 9.77 m3
Telemac – Baseline simulation
For the majority of the year, the virus did not disperse far away from the estuary.
Worst two-week period:Shellfish/beaches: Prob of exceedance of 1 gc/L > 0.5Offshore (~10 km): Prob of exceedance of 1 gc/L > 0.1Decay: Spatial dispersal reduced.
Total days virus > 1 gc/L (contour lines > 14 d) Estuary: > 200 (50-80 less with decay) Shellfish/beaches: 50-200 (30-50 less with decay) Offshore: < 50 (10-30 less with decay)
Continuous period virus > 1 gc/L Offshore: < 2 weeks (50-80 d less with decay) Shellfish/beaches: 20-30 d (20-50 d less with decay)
Run 3.2 more than doubled
Run 3.3 30% moreRun 3.4 5% more
Run 3.5 10-50% less
3-hour lag between HW and peak flow caused most virus export
20% reduced virus export when occurring at neaps
Extreme scenarioLargest tide + surge
Largest river flow
Highest virus conc.
Future work…
• Point source simulations from CSOs
• Other estuaries
NERC-EMHH Environmental
Microbiological Risk Symposium
DISCUSSION
14th March 2018
Education Centre and Main Hall
Royal Geographical Society, London
NERC-EMHH Environmental
Microbiological Risk Symposium
MORNING COFFEE
14th March 2018
Education Centre and Main Hall
Royal Geographical Society, London