Rosina Girones

Departament de Genètica, Microbiologia i Estadística,Secció Microbiologia, Virologia i Biotecnologia

Laboratoryof Viruses Contaminants of Water and Food

Aplicació de la metagenòmica en salut pública. reptes, limitacions i utilitats

Viral metagenomicsPCR or q(RT)-PCR methodsCell culture methods

The technology for the analysis of virus in environmental and clinical samples has evolved

Nucleic acid extraction

Concentration of viral particles into

small volumes

Quantification Q(RT)PCR

Nested- PCR and typification

Infectivity Assays

Metagenomics/Amplicon Sequencing

Methodology for detection and quantification of viral pathogens

The InnovaPrep Large Volume Concentration (LVC) Kit, concentration of bacteria and viruses from large liquid volumes (up to a 1000 liters)

• One-pass method• 15 minutes per 10L sample• Based on membrane filtration(REXEED-25A )

• surface area of 2.5 m2

• 30-kDa pore size

• Wet foam elution technology• to rapidly recover

microorganisms into a small final volume (~50 -85 mL)

Dead-end ultrafiltration (DEUF) for large volumes

Concentration of viruses from water

E. coli qPCR 59.6 40.3 - 79 15.6 98.7H. pylori qPCR 30.2 24.4 - 36.1 20.8 41.5

A. castellanii qPCR 20.5 14.9 - 26.1 13 32.1G. lamblia IFA 17.8 15 - 20.7 12.8 21.5C. parvum IFA 12.8 12.5 - 15.2 9.6 17.4

Method% Recovery

Mean % CI 95% of mean min max

Bacteria and protozoa recovery with qPCR and IFA quantification

Virus Method% Recovery

Mean % CI 95% of mean min max

HAdVqPCR 66 53.5 - 78.5 32.2 86.7

IFA 58.7 4.5 - 100 43,5 83,7

MS2q(RT)PCR 23.9 19.6 - 28.1 13.8 36.8

PFU 11.9 9 - 14.7 9.5 13.9

RoVq(RT)PCR 28.2 25.6 - 30.7 16 37.1

TCID50 26.1 17.1 - 35.1 8,1 49,8

BVDVq(RT)PCR 14.7 10.8 - 18.7 12.9 15.8

TCID50 0.7 0.4 -1.1 0.67 0.89

Virus recovery by qPCR and Infectivity quantification

MetawaterNew METAgenomics and molecular based tools for

european scale identification and control of emergent microbial contaminants in irrigation

WATER

Raw Sec. Tertiary

Reservoir RiverDrinking water

Groundwater

1 samples/months6-12 months

Metawater sampling sites

Drinking water from water tanks from the Barcelona’s distribution network.

Reservoir water, collected from the Foixriver basin.

Groundwater from the north-east of Catalonia, a region with intensive farming and agricultural activities.

River water from Fluvià River, 97,2 Km long and a flow of about 10 m³ / s at the mouth of the river.

Sampling sites

Sampling sites: Constructed Wetland

Schematic diagram (plan view and section) of the surface flow constructedwetland 1. Zone planted with Phragmites australis2. Zone planted with Typha latifolia3. Deep zone free of macrophytes4. Island

AA’

41 2

3

A’ A

A single cell with an elongated shape and a surface area of 1 ha, receives a pumped flow of secondary effluent which ranges from 100 to 250 m3/day. Production 120.000 m3 of recycled water every year.

Alfranca O. et al. 20011. Water Sci Technol. 2011;63(5):891-8.

Protocol for the treatment of water samples for metagenomics

10 L

Centrifugation of the sediment

8000 xg

Stirring during 8h

Acidified skimmed milk solution into preconditioned

sample to flocculate viral

particles

Viral concentrate in 4mL

DNAsetreatment

0,45 µm Filtration step

280 μl QIAGEN Nucleic acid Extraction

Eluted DNA/RNA in 60 µl

dsDNAquantification

Nextera XT Illumina Mi-Seq

RT SuperScriptIII using primer A

Second strand synthesis with Sequenase 2.0

25 cycles PCR with primer B

DNA purificationand

concentration

Sampling site Pooled samples UB virusesURV

bacteriaUPV

ProtozoaDrinking water tank 6 months √ √

Sediment 6 months √ √

Raw Sewage

Spring (3 months) √ √ √Summer (3 months) √ √ √Autumn (3 months) √ √Winter (3 months) √ √

Tertiary treatment (Wetland)

Spring (3 months) √ √Summer (3 months) √ √Autumn (3 months) √Winter (3 months) √

Reservoir water 6 months √ √

Groundwater

Spring (3 months) √ √ √Summer (3 months) √ √ √Autumn (3 months) √ √Winter (3 months) √ √

River water

Spring (3 months) √ √ √Summer (3 months) √ √ √Autumn (3 months) √ √Winter (3 months) √ √

Samples tested using metagenomics

Average of 1,2 million reads per sample

Contigs of human viral families detected in raw sewage and irrigation water

Dri

stri

butio

n

Res

ervo

ir

Gro

undw

ater

Riv

er w

ater

Wet

land

(R

ecla

imed

)

Raw

sew

age

Family Genus/Species SP-SU SP-SU SP SU AU WI SP SU AU WI SP SU AU WI SP SU AU WI

Adenoviridae Adenovirus 41 - - - - 1 - - - - - - - - - 1 6 - - Adenovirus 31 - - - - - - - - - - - - - - 1 1 - -

Astroviridae Mamastrovirus 1 - - - - - - - - - - - - - - 8 13 37 32

Caliciviridae Norovirus GI and GII - - - - - - - - 1 - - - - - 5 6 10 13 Sapovirus GI, GII, GIV and GV - - - - - - - - - - - - - - 6 - 4 7

Circoviridae Human cyclovirus - - - - - - - - - - - - - - - 4 - - Hepeviridae Hepatitis E - - - 1 - - - - - - - - - - - 1 - - Papillomaviridae Papillomavirus 104 - - - - - - - - - - - - - - 1 - - - Parvoviridae Human bocavirus - - - - - - - - - - - - - - - - 4 - Human parechovirus - - - - - - - - - - - - - - - 1 - 2

Picornaviridae

Aichivirus - - - - - - - - - - - - - - 2 8 6 1 Enterovirus A (CV-A5, -A10 and -A22, EV-71) - - - - - - - - - - - - - - 4 - - 1

Enterovirus B (ECV-E13, -E18 and -E25) - - - - - - - - - - - - - - 1 2 1 -

Enterovirus C (CV-A22, EV-C99) - - - - - - - - - - - - - - - - 1 1

Klassevirus/Salivirus A - - - - - - - - - - - - - - 3 - 2 6 SP: spring, SU: summer, AU: autumn, WI: Winter, CV: coxsackievirus, ECV: echovirus, EV: enterovirus

Amplicon Sequencing

Methodology

Sampling

• Raw sewage, monthly sampling (April 2016-March 2017). GranollersWWTP

Viral concentration

•Ultracentrifugation (42mL/sample)

•Concentrates (100ul/sample) seasonally pooled

Free DNA elimination

•Turbo DNAseAmbion

Viral lysis and NA extraction

•Qiagen Viral RNA Mini Kit

nPCR

• Specific nPCR withIllumina adaptersincorporation forEV, HPV and HAdV

Amplicons purified and DEEP SEQUENCED

(Illumina Miseq 2x300bp)

Amplicon Sequencing

Enterovirus diversity

CoxsackievrusB5

Echovirus

E18

Echovirus

E30

CoxsackievirusA19, CoxsackievirusA24, CoxsackievirusA5, EchovirusE11, EchovirusE16, EchovirusE20, EchovirusE7, EchovirusE9,EnterovirusA119, EnterovirusA90, EnterovirusA76, EnterovirusA90, EnterovirusC99, RhinovirusA13, RhinovirusA73, RhinovirusB,RhinovirusB72.

Coxsackievirus A16

Enterovirus C99

EnterovirusA71

Urban raw sewage composite sample collected from a Waste Water Treatment Plant

Viral particles from 42ml of water were concentrated by ultracentrifugation.

NA were retrotranscribed to cDNA, tagged and converted to dsDNA. The viral randomly taggeddsDNA was then amplified (25 cycles of amplification).

The libraries were prepared in duplicate following the instructions provided by the manufacturer,one was hybridized with probes designed to capture sequences from vertebrate viral pathogens(VirCapSeq Enrichment Kit, Roche) and the other directly sequenced.

After the capture of the libraries, quality and concentration was checked and the sequencing wasperformed (Illumina Miseq 2x300bp).

Analysis of the sequencing raw data obtained was performed with the Genome Detective.

Target Enrichment vs Direct Metagenomics

Sampling Viral concentration NA extraction NA pre-

amplificationLibrary

constructionViral

sequencescapture

Sequencing

Methodology

Target Enrichment vs Direct Metagenomics

Analysis of the virome in water has shown the presence of high diversity of bacteriophages and human and plant pathogens in urban sewage and with less extension in distribution and superficial and ground water.

A global picture of the viral population is obtained using metagenomics, although a higher sensitivity for human pathogens is observed using target enrichment in complex matrices. The more sensitive method for specific viral groups is observed using amplicon sequencing.

General conclusions

Un repte: La implementació de sistemes de vigilància de la salut pública metagenòmica mitjançant tècniques NGS com a base de dades de la població microbiana present a l’aigua i circulant a la població

Limitacions tècniques: El cost i la rapidesa de resultats estan millorant i ho farà encara més amb les noves technologies de Nanopore com el MinION.

Utilitats: Avaluació del risc global en aigua i aliments de diferent origen; identificar virus nous i emergents circulant a la població; disposar de una base de dades per investigació de brots.

Reptes, limitacions i utilitats

http://www.ub.edu/microbiologia_virology/en

Xavier Fernández CassiEloy Gonzales GustavsonSandra Martínez PucholAyalkibet HundesaMarta RusiñolSílvia Bofill Mas

Computational GenomicsLaboratory, UBJosep Abril

Aknowledgements

VirBaP