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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
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
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
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
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