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 p.robins@bangor.ac.ukDavid 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