s urvival and elimination of adenoviruses p ulawy, 12-14 a pril 2010

SURVIVAL AND ELIMINATION OF ADENOVIRUSES

PULAWY, 12-14 APRIL 2010

• 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

How do we develop chlorine disinfection of HAdV in water?

Water sample Chlorine decay analysis


Spiked viruses

Chlorine

Viral load analysis

Viral infectivity analysis

Water sample

Spiked viruses

Viral load 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.


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


Spiked viruses

Chlorine

Viral load analysis


Water sample

Spiked viruses

Viral load 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


Natural Seawater R1


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


HAdV2 without chlorine disinfection

Murine norovirus disinfection in artificial sea water

HAdV2 con Cloro


MuNoV without chlorine disinfection


Chlorine disinfection of HAdV in river water


Murine norovirus disinfection in natural river water



Human adenovirus 2 disinfection in BDF water

HAdV with chlorine disinfection


Murine norovirus disinfection in BDF water



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)

Kinetics of inactivation of HAdV2 by infectivity assay and qPCR

Kinetics of inactivation of HAdV2 by infectivity assay and DNAse + qPCR

OUR NEXT STEPS...

SurfacesFruits and vegetables

Shellfish

Harmonization!

• Develop the statistical analysis for our current data

• Continue working on:

Thank you!

Anna Carratalà

[email protected]

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

s urvival and elimination of adenoviruses p ulawy, 12-14 a pril 2010

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