s urvival and elimination of adenoviruses p ulawy, 12-14 a pril 2010
TRANSCRIPT
• Persistence of infectivity of viruses will be analyzed under selected
conditions relevant to food supply chains.
• Elimination procedures used in the food industry will be studied.
• The efficacy of suggested interventions will be evaluated in the laboratory,
and in pilot and field experiments.
TASK 6.2 SURVIVAL AND ELIMINATION OF VIRUSES
• Human adenoviruses mainly but also murine noroviruses
have been studied.
• The results have been evaluated both considering qPCR
assays and infectivity experiments.
TASK 6.2 SURVIVAL AND ELIMINATION OF VIRUSES
SURVIVAL AND ELIMINATION OF ADENOVIRUSES
• Standard suspensions for qPCR have been produced for human
adenovirus and murine norovirus.
• In the case of murine norovirus comparison between RNA , viral
particles and DNA based standards have been developed. RNA based
standard have been selected.
1. qPCR
DNAse treatment of HAdV2
Enzimatic treatment of samples before molecular detection
RNAse treatment of MNV-1
SURVIVAL AND ELIMINATION OF VIRUSES
• Human adenoviruses 2 and murine noroviruses have been cultured in A549
and RAW 264.7 cell lines respectively.
• Viral stocks obtained have been ultracentrifuged and resuspended in PBS.
• Viral stocks have been quantified both by infectivity assays and qPCR.
• Several infectivity assays have been compared for both viruses.
2. Infectivity assays
Plaque forming units
TCID50
Murine norovirus infectivity assays
Human adenoviruses infectivity assays
TCID50
Plaque forming units
Indirect immunofluorescence
assay
• Time, presence of
citopathic effect and
reproducibility have
been considered.
Elimination of human adenoviruses by chemical disinfection with chlorine
• Chlorine is a low cost chemical
disinfectant commonly used by many
different industries.
• Useful for the treatment of high amounts
of water
• Used in low concentrations
• Easily available
Water samples studied
Temperature (°C) PH Conductivity (µS)
Buffered
demand free
water
23 8 1864
River water 15.1 7.8 1115
Artificial sea
water24 7.75 47400
Sea water 25 7.6 55000
Water sample Chlorine decay analysis
How do we develop chlorine disinfection of HAdV in water?
Spiked viruses
Chlorine
Viral load analysis
Viral infectivity analysis
Water sample
Spiked viruses
Viral load analysis
Viral infectivity analysis
Chlorine decay analysis and considerations
•The free chlorine dose is measured at time
0s, 20 min and 60 min by a colorimetrical
method N,N-dietil-p-Phenilenediamine (DPD).
•The chlorine decay may be
high when organic compounds
are present in the assay.
• Glassware is made chlorine
demand free by overnight
soaking into a solution of 100
mg/l of free chlorine.
Chlorine demand of diluted viral stock
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
0 10 20 30 40 50 60
Time (min)
mg/LArtificial Seawater R1
Natural Seawater R1
Artificial Seawater R2
Natural Seawater R2
mg/
L
Free chlorine decay in sea water
Optimization of the free chlorine initial dose
• The initial free chlorine dose is 2.5 mg/l.
Viral load analysis
•During the assay, aliquots of water are taken at different times from time 0 to 1 h
(0s, 10 min, 20 min, 30 min and 60 min).
• Chlorine is inactivated by adding sodium tiosulphate to each aliquote.
• Nucleic acid extraction is developed by QIAmp viral mini kit (Qiagen, Valencia,
CA, USA).
• The quantification of viral load decay is performed by the qPCR SOP’s.
Viral infectivity analysis
For human adenovirus 2:
• Plaque forming units assay
• Tissue culture infectious dose 50
• Indirect immunofluorescence assay
For murine norovirus 1:
• Plaque forming units assay
• Tissue culture infectious dose 50
Water sample Chlorine decay analysis
How do we develop chlorine disinfection of HAdV in water?
Spiked viruses
Chlorine
Viral load analysis
Viral infectivity analysis
Water sample
Spiked viruses
Viral load analysis
Viral infectivity analysis
Chlorine disinfection of HAdV in sea water
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
0 10 20 30 40 50 60
Time (min)
mg/L
Artificial Seawater R1
Natural Seawater R1
Artificial Seawater R2
Natural Seawater R2
mg/
L
1. Free Chlorine decay during the experiments
Human adenovirus 2 disinfection in natural sea water
HAdV2 con Cloro
HAdV 2 with chlorine disinfection
HAdV 2 without chlorine disinfection
MuNoV without chlorine
MNV 1 con Cloro
Murine norovirus disinfection in natural sea water
HAdV2 con Cloro
MuNoV with chlorine disinfection
Human adenovirus 2 disinfection in artificial sea water
HAdV 2 with chlorine disinfection
HAdV2 without chlorine disinfection
Murine norovirus disinfection in artificial sea water
HAdV2 con Cloro
MuNoV with chlorine disinfection
MuNoV without chlorine disinfection
HAdV 2 without chlorine disinfection
Chlorine disinfection of HAdV in river water
HAdV 2 with chlorine disinfection
Murine norovirus disinfection in natural river water
MuNoV without chlorine disinfection
MuNoV with chlorine disinfection
Human adenovirus 2 disinfection in BDF water
HAdV with chlorine disinfection
HAdV 2 without chlorine disinfection
Murine norovirus disinfection in BDF water
MuNoV without chlorine disinfection
MuNoV with chlorine disinfection
Elimination of human adenoviruses by physical disinfection
• Human adenovirus 2 stocks have been prepared and quantified.
• Two dispersion estrategies for viruses have been tested: chloroform
and glycine buffer treatment. Glycine buffer has provided good
results and has been succesfully applied.
• UV (253,7 nm) dose applied: 100, 200, 300, 400, 600, 800, 1000 and 1400
(J/m2)
OUR NEXT STEPS...
SurfacesFruits and vegetables
Shellfish
Harmonization!
• Develop the statistical analysis for our current data
• Continue working on:
Thank you!
Anna Carratalà
Department of Microbiology
Faculty of Biology
Av. Diagonal 645, 08028 Barcelona
(+34) 93 4039043
A. A. Correa, A. Aregita, A. Carratalà, A. Hundesa, S. Fresno,
J. Rodriguez, M. Rusiñol, L. Guerrero, R. Girones, S. Bofill
MNV-1 en Agua de Mar Natural
Infectivity RT qPCR ET RT qPCR
0,4 1,74 0,51 1,88
10 2,64 0,42 -
20 4,20 1,46 -
30 - 2,04 2,51
45 - 1,84 -
60 - 2,08 4,17
HAdV2 en Agua de Mar Natural
Infectivity qPCR ET qPCR
0,4 0,29 0,73 1,23
10 - 2,51 -
20 - 3,15 -
30 2,25 2,96 2,56
45 - 3,62 -
60 2,27 3,59 3,34
MNV-1 en Agua de Mar Artificial
Infectivity RT qPCR ET RT qPCR
0,4 1,33 1,09 1,80
10 2,42 1,27 -
20 4,37 2,08 -
30 - 1,82 3,47
45 - 3,54 -
60 - 3,84 4,73
HAdV2 en Agua de Mar Artificial
Infectivity qPCR ET qPCR
0,4 0,05 0,75 0,91
10 - 2,28 -
20 - 2,02 -
30 2,12 3,07 2,66
45 - 3,69 -
60 3,08 3,71 2,72
Mean values of 2 replicates
No has acabado