1. S URVIVAL AND ELIMINATION OF ADENOVIRUSES P ULAWY, 12-14 A PRIL 2010

2. 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

3. 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

4. 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

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

6. 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

7. Plaque forming units TCID 50 Murine norovirus infectivity assays Human adenoviruses infectivity assays TCID 50 Plaque forming units Indirect immunofluorescence assay Time, presence of citopathic effect and reproducibility have been considered.

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. Water samples studied Temperature (°C)PHConductivity (µS) Buffered demand free water 2381864 River water15.17.81115 Artificial sea water 247.7547400 Sea water257.655000

11. How do we develop chlorine disinfection of HAdV in water?

12. 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

13. 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

14. 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 0102030405060 Time (min) mg/L Artificial 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.

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. 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

18. 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 0102030405060 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

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

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

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

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

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

28. Kinetics of inactivation of HAdV2 by infectivity assay and qPCR

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

30. O UR NEXT STEPS... Surfaces Fruits and vegetables Shellfish Harmonization! Develop the statistical analysis for our current data Continue working on:

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. MNV-1 en Agua de Mar Natural InfectivityRT qPCRET RT qPCR 0,41,740,511,88 102,640,42- 204,201,46- 30-2,042,51 45-1,84- 60-2,084,17 HAdV2 en Agua de Mar Natural Infectivity qPCRET qPCR 0,40,290,731,23 10-2,51- 20-3,15- 302,252,962,56 45-3,62- 602,273,593,34 MNV-1 en Agua de Mar Artificial Infectivity RT qPCRET RT qPCR 0,41,331,091,80 102,421,27- 204,372,08- 30-1,823,47 45-3,54- 60-3,844,73 HAdV2 en Agua de Mar Artificial Infectivity qPCRET qPCR 0,40,050,750,91 10-2,28- 20-2,02- 302,123,072,66 45-3,69- 603,083,712,72 Mean values of 2 replicates No has acabado