Tracing the Fate and Infectivity of Human Pathogenic Viruses Through the Environment Enteric Viruses

Tracing the fate and infectivity of human pathogenic viruses through the environment Enteric viruses

Pathogens Indicators Gastroenteritis, 2-5 days Asymptomatic Transmission: faecal – oral Transmission: faecal/urinal – oral Icosahedral capsid, RNA/DNA genome Icosahedral capsid, DNA genome

Polyomaviridae 50 nm Caliciviridae Picornaviridae Hepeviridae Adenoviridae ssDNA 40 nm 30 nm 30 nm 80 nm Polyomavirus ssRNA ssRNA ssRNA dsDNA Norovirus Hepatitis A virus Hepatitis E virus Adenovirus Sapovirus Aichivirus Enterovirus Norovirus: The season not to be jolly Enteric viruses

Viral epidemics are on the rise • 2 million cases of norovirus each year in the UK HPA Norovirus cases in E&W ➢ Costs the NHS £100 million/yr ➢ Costs the UK economy £2 billion/yr • New and emerging viruses (frequent travel) ➢ Hepatitis A/E virus • Acquiring greater virulence ➢ Norovirus GII.4 Sydney 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

Viral Surveillance Viral ecology infectivity

Modelling

Risk assessment Water concentration

Tangential flow ultrafiltration • 5000x concentration Water, 10 L • 24 hours • £20/sample Tangential flow ultrafiltration, 50 mL • 10-100% recovery of enteric viruses Beef extract elution • Concentrate viruses, bacteria, protozoa 120 NoV GII PEG precipitation, 2-4 mL NoVGII PGM 100 HAV SaV MgV Nucleic acid extraction 80 AdV40

60 q(RT-)PCR

Recovery 40

0 Sea Estuarine River pH 8.05 6.91 7.5 T (NTU) 6.37 11.74 3.2 K (mS/m) 40.7 18.88 0.06 Virus concentration in sediment

Elution – concentration

Sediment, 10-100 g • 10-100x concentration • 24 hours Beef extract elution • £1/sample • 80-100% recovery of enteric viruses PEG precipitation, 1-2 mL

Nucleic acid extraction

q(RT-)PCR Virus concentration in shellfish

Shellfish digestive tissue • ISO/TS 15216-1:2013 standard ➢Elution with proteinase K Proteinase K treatment • Elution with alternative buffer ➢PBS Nucleic acid extraction ➢SM • Adsorption-twice-elution-extraction (Kittigul et al., 2015) q(RT-)PCR ➢tryptose phosphate broth, pH 9 – arginine, pH 7.5 – chloroform Virus concentration in shellfish

160 Mengovirus 140 Adenovirus Hepatitis A virus Norovirus GII 120

100

Recovery % Recovery 40

0 ISO PBS SM Twice elution Surveillance of enteric viruses in the water environment: how much?

CSO Combined Sewer Outflow Shellfish harvesting rainwater + untreated wastewater area

Ganol WWTP 82,000 inhabitants

Tal-y-Bont WWTP 1,000 inhabitants

Llanrwst WWTP 4,000 inhabitants

Betws-y-Coed WWTP 1,200 inhabitants Surveillance of enteric viruses in the water environment

Sed1-SF1 – shellfish and sediment Sed2-SF2 – shellfish and sediment SW4 – estuary Sed4 – sediment

SW3 – tidal limit

SW1 – river SW2 – river 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 & HIV ➢ Not routinely detected

Indicator viruses • Adenovirus and JC polyomavirus ➢ No diurnal changes in wastewater ➢ No seasonal changes ➢ Detected at high concentrations in all sample types Viral Infectivity – how much is there?

qPCR detection qPCR target region Norovirus integrity

Porcine Gastric Mucin assay – qPCR detection Norovirus integrity

Porcine Gastric Mucin assay vs. qPCR detection

Untreated wastewater: contains high concentrations of infective virus

1e+7 1e+7 NoVGI direct NoV GII direct NoVGI PGM NoVGII PGM 1e+6 1e+6

1e+5 1e+5

1e+4 1e+4

gc/L

1e+3 1e+3

1e+2 1e+2

1e+1 1e+1

LI 02/17 LI 08/17 LI

TI09/10 TI02/17 TI03/17 TI07/17

BI 10/16 BI 03/17 BI 05/17 BI 08/17 BI

LI 09/16 LI 11/16 LI 01/17 LI 04/17 LI 05/17 LI 06/17 LI

GI 09/16 GI 12/16 GI 02/17 GI 03/17 GI 04/17 GI 05/17 GI 06/17 GI 08/17 GI

TI09/16 TI10/16 TI11/16 TI01/17 TI04/17 TI05/17 TI06/17

BI 09/16 BI 10/16 BI 11/16 BI 01/17 BI 05/17 BI 06/17 BI

GI 09/16 GI 10/16 GI 11/16 GI 12/16 GI 01/17 GI 03/17 GI 04/17 GI 05/17 GI 06/17 GI 08/17 GI Norovirus integrity

Porcine Gastric Mucin assay – qPCR detection

Untreated wastewater: high concentrations Treated wastewater: 0-4 log reduction Works better for norovirus GII than for GI

1e+7 1e+7 NoVGI direct NoV GII direct NoVGI PGM NoVGII PGM 1e+6 1e+6

1e+5 1e+5

1e+4 1e+4

gc/L

1e+3 1e+3

1e+2 1e+2

1e+1 1e+1

LE 09/16 LE 11/16 LE 01/17 LE 03/17 LE 05/17 LE 06/17 LE

LE 11/16 LE 05/17 LE 08/17 LE

TE09/16 TE10/16 TE11/16 TE01/17 TE05/17

TE09/16 TE10/16 TE11/16 TE02/17 TE03/17 TE05/17 TE07/17

BE 09/16 BE 10/16 BE 01/17 BE 04/17 BE 05/17 BE

BE 02/17 BE 03/17 BE 05/17 BE 07/17 BE Viral Infectivity

Adenoviruses, polyomaviruses • Infectivity – culturing • 48h assay • Requires skilled staff and equipment

Norovirus? Norovirus integrity

Porcine Gastric Mucin assay – qPCR detection

Untreated wastewater: high concentrations Treated wastewater: 0-1 log reduction Works better for norovirus GII than for GI Surface water: 1-3 log reduction

Sediment: all negative 1e+7 1e+7 NoVGI direct NoV GII direct NoVGI PGM NoVGII PGM 1e+6 1e+6

1e+5 1e+5

1e+4 1e+4

gc/L

1e+3 1e+3

1e+2 1e+2

1e+1 1e+1

SW109/16 SW111/16 SW105/17 SW305/17 SW409/16

SW111/16 SW102/17 SW302/17 SW305/17

Sed1 09/16 Sed1 11/16 Sed1 11/16 Sed4 04/17 Sed4

Sed1 10/16 Sed1 02/17 Sed1 03/17 Sed1 07/17 Sed1 02/17 Sed2 02/17 Sed4 04/17 Sed4 Norovirus integrity

Porcine Gastric Mucin assay – qPCR detection

Semi-degraded viruses? Norovirus persistency in water

Study site: Betws-y-Coed, North Wales Float-a-Lyzer, Spectrumlabs

Human norovirus GII Murine norovirus Human adenovirus 40 Norovirus persistence

1e+7 NoV GII Direct 1e+6 RNase PGM

1e+5

gc/uL 1e+4

1e+3

1e+2 0 2 4 6 8 10 12 14 Days 1e+7 MNV

1e+6

1e+5

gc/uL

1e+4

1e+3 0 2 4 6 8 10 12 14 Days Metaviromics What viruses are there - surveillance?

viral genomic RNA

Non-targeted sequencing

viral genomes

Illumina HiSeq 4000

norovirus genome viral diversity

sapovirus genomes Dr Evelien Adriaenssens sapo/norovirus

rotavirus Family level groupings level Family

WW mussels river/est. water sediment Do the bubbles represent intact and infective viruses?

Intactness inferred from completeness of genome fragments found Cannot 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 Viral Infectivity

Use the novel human norovirus infectivity assay with environmental samples • Validation of capsid integrity assays • Validation of metaviromics results

Dr Myra Hosmillo Risk modelling Modelling viral dispersal and concentrations through the year

Tide height (m)

River flow (m3/s)

Tidal volume (m3)

Adenovirus at tidal limit (ppl)

Adenovirus at Estuary mouth (ppl) Modelling results

Conservative tracer with viral die off Modelling conclusions

• Enteric viruses can disperse downstream to estuaries within hours whilst active • Coastal waters and shellfisheries are exposed to viruses during ebb tide drainage • Viruses are retained mostly in the estuary (river flow <50 m3 s-1) • Viruses enter the coastal zone during storms (river flow >50 m3 s-1) • Risk is worst after a storm and spring tide • CSO discharges represent a major threat • Human outbreaks are more important than river flow conditions Summary

• Method validation Protocols 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, novel strains • Integrity and infectivity • Modelling

www.viraqua.uk @Viraqua_Project Publications

Robins et al. (2018) Water Res. submitted Farkas et al. (2018) Environ. Sci. Pollut. Res 25, 33391-33401 Farkas et al. (2018) Sci. Tot. Environ. 634, 1174-1183 Adriaenssens et al. (2018) Msystems 3, e00025-18 Farkas et al. (2017) Food Environ. Microbiol. 9, 342-349 Hassard et al. (2016) Front. Microbiol. 7, 1692 Perkins et al. (2016) Sci. Tot. Environ. 572, 1645-1652 Winterbourn et al. (2016) Water Res. 105, 241-250 New NERC project (NE/S004548/1)

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