Survival and elimination of adenoviruses Pulawy , 12-14 April 2010

1. Survival and elimination of adenovirusesPulawy, 12-14 April 2010

2. TASK 6.2 SURVIVAL AND ELIMINATION OF VIRUSES • 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.

3. TASK 6.2 SURVIVAL AND ELIMINATION OF VIRUSES • Humanadenovirusesmainlybutalsomurinenoroviruseshavebeenstudied. • The resultshavebeenevaluatedbothconsideringqPCRassays and infectivity experiments.

4. survival and elimination of adenoviruses • StandardsuspensionsforqPCRhavebeenproducedforhuman adenovirus and murinenorovirus. • In the case of murinenoroviruscomparisonbetween RNA , viral particles and DNA basedstandardshavebeendeveloped. RNA basedstandardhavebeenselected. 1. qPCR

5. Enzimatic treatment of samples before molecular detection DNAse treatment of HAdV2 RNAse treatment of MNV-1

6. survival and elimination of viruses • Humanadenoviruses 2 and murinenoroviruseshavebeencultured in A549 and RAW 264.7celllinesrespectively. • Viral stocks obtainedhavebeenultracentrifuged and resuspendedin PBS. • Viral stocks havebeenquantifiedbothbyinfectivityassays and qPCR. • Severalinfectivityassayshavebeencomparedforbothviruses. 2. Infectivity assays

7. Murinenorovirusinfectivityassays Humanadenovirusesinfectivityassays TCID50 TCID50 Plaque forming units Plaque forming units • Time, presence of citopathic effect and reproducibility have been considered. Indirect immunofluorescence assay

9. 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

10. Watersamplesstudied

11. How do wedevelopchlorinedisinfection of HAdV in water?

12. How do wedevelopchlorinedisinfection of HAdV in water? Watersample Chlorinedecayanalysis Spikedviruses Viral load analysis Chlorine Viral infectivityanalysis Viral load analysis Watersample Viral infectivityanalysis Spikedviruses

13. Chlorinedecayanalysis and considerations • The free chlorine dose is measured at time 0s, 20 min and 60 min by a colorimetrical methodN,N-dietil-p-Phenilenediamine (DPD). Chlorine demand of diluted viral stock • 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.

14. 2 1,8 1,6 1,4 Artificial Seawater R1 1,2 Natural Seawater R1 1 mg/L Artificial Seawater R2 0,8 mg/L Natural Seawater R2 0,6 0,4 0,2 0 0 10 20 30 40 50 60 Time (min) Optimization of the free chlorine initial dose Free chlorine decay in sea water • The initial free chlorine dose is 2.5 mg/l.

15. 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.

16. 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

17. How do wedevelopchlorinedisinfection of HAdV in water? Watersample Chlorinedecayanalysis Spikedviruses Viral load analysis Chlorine Viral infectivityanalysis Viral load analysis Watersample Viral infectivityanalysis Spikedviruses

18. 2 1,8 1,6 1,4 Artificial Seawater R1 1,2 Natural Seawater R1 1 mg/L Artificial Seawater R2 0,8 mg/L Natural Seawater R2 0,6 0,4 0,2 0 0 10 20 30 40 50 60 Time (min) Chlorine disinfection of HAdV in sea water 1. Free Chlorine decay during the experiments

19. HAdV 2 with chlorine disinfection HAdV2 con Cloro Human adenovirus 2 disinfection in natural sea water HAdV 2 without chlorine disinfection

20. MuNoV without chlorine MuNoV with chlorine disinfection MNV 1 con Cloro HAdV2 con Cloro Murine norovirus disinfection in natural sea water

21. Human adenovirus 2 disinfection in artificial sea water HAdV2 without chlorine disinfection HAdV 2 with chlorine disinfection

22. MuNoV with chlorine disinfection HAdV2 con Cloro Murine norovirus disinfection in artificial sea water MuNoV without chlorine disinfection

23. Chlorine disinfection of HAdV in river water HAdV 2 without chlorine disinfection HAdV 2 with chlorine disinfection

24. Murine norovirus disinfection in natural river water MuNoV without chlorine disinfection MuNoV with chlorine disinfection

25. Human adenovirus 2 disinfection in BDF water HAdV 2 without chlorine disinfection HAdV with chlorine disinfection

26. Murine norovirus disinfection in BDF water MuNoV without chlorine disinfection MuNoV with chlorine disinfection

27. 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)

28. Kinetics of inactivation of HAdV2 byinfectivityassay and qPCR

29. Kinetics of inactivation of HAdV2 byinfectivityassay and DNAse + qPCR

30. Ournextsteps... • Develop the statistical analysis for our current data • Continue working on: Fruits and vegetables Shellfish Surfaces Harmonization!

31. Thank you! Anna Carratalà acarratala@ub.edu 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

32. No has acabado Mean values of 2 replicates