Shedding some light on inactivation of foodborne viruses. Julie Jean, Ph.D.

Transcription

1 Shedding some light on inactivation of foodborne viruses Julie Jean, Ph.D. MAY 12 TH, 2016

2 Foodborne viruses Virion : non enveloped Capsid : icosahedral Diameter : nm Genome : single-stranded RNA Norovirus (NoV) Hepatitis A virus (HAV) Cannot replicate in food, in very low number Low infective dose (10-100) Difficult to work since no culture system 2

3 Transmission Symptomatic (70 %) Asymptomatic (30 %) Intestinal disease Shedding viruses Infected individual Feces Vomitus Present (40 %) Protected Absent (60 %) Susceptible Previously acquired immunity Transmission vectors Person Food Water Environment Non-shedding (20 %) Resistant Shedding (80 %) Susceptible v5 v Exposed population Hall et coll

4 Inactivation strategies Individual Water Food Environment Chemical and Physical Approaches

5 Inactivation: chemical approach Chlorine Alcohol TOXIQUE ClO 2 gas Stainless steel Quaternary Ammoniums Ozone Lettuce Chlorine dioxide Ozone Peracetic Acid 3-log reduction MNV; 2 ppm, 5 min 3-log reduction MNV; 6 ppm, 30 min Does not seem to be affected by organic matter 1-log reduction MNV; 20 ppm, 1 min Girard et al JPF; Yeap et al., 2016 AEM; Girard et al IJFM; Predmore et al., 2015 FM 5

6 Disinfection: physical approach Heat treatment for complete inactivation of HAV and NoV, more than 180 sec at 90 C in soft shell clams Pulsed light Compared to antibacterial activities, very few antiviral studies Difficult to compare due to differences in equipment (static vs dynamic) Difference in data (UV amount vs fluence) Promising with reduction of several log in short time Reduction of 5-log MNV and HAV in 2-3 sec Sow et al. FPD 2011; Jean et al. FM 2011; Vimont et al. AEM

7 Specific objectives 60% : acid 40% : H 2 O 2 Peracids Pulsed light 1 2 Evaluate the anti-norovirus activity : in suspension on inert surfaces in artificial biofilm Identify the viral targets involved in mechanism of action 7

8 What s a peracid? Organic acid O R OH Hydrogen peroxide peracid O + HO OH R + O OH Peroxide liaison O H 2 FREE RADICALS highly reactive with high antimicrobial activity R O O + HO Sanchez and Myers

9 Inactivation : suspension According to ASTM E MNV : 10 7 PFU ml -1 SOLUTIONS : peracetic / perpropionic / perlactic / percitric CONCENTRATIONS : 50 / 250 / 500 / 1000 mg L -1 CONTACT TIME : 1 / 5 / 10 min 9

10 Viral reduction (log 10 ) Inactivation : suspension PERACETIC ACID Good candidate 5 4 c ab c a a bc a a a a a 3 2 d Peracetic acid concentration (mg L -1 ) 1 min 5 min 10 min 10

11 Viral reduction (log 10 ) Inactivation : suspension PERACETIC ACID PERPROPIONIC ACID H 3 C COOOH H 3 C CH 2 COOOH 5 4 c ab c a a bc a a a a a 5 4 abc ab c a a a a a a ab bc d 2 d [peracide] (mg L -1 ) [peracide] (mg L -1 ) 1 min 5 min 10 min 11

12 Viral reduction (log 10 ) Inactivation : suspension PERACETIC ACID PERLACTIC ACID H 3 C COOOH H 3 C CH COOOH OH 5 4 c ab c a a bc a a a a a 5 4 ab a a a a a 3 2 d 3 2 c c b 1 1 e de d [peracide] (mg L -1 ) [peracide] (mg L -1 ) 1 min 5 min 10 min 12

13 Viral reduction (log 10 ) Inactivation : suspension PERACETIC ACID PERCITRIC ACID H 3 C COOOH 5 4 c ab c a a bc a a a a a d d dc cd d cd bc d bc b d bc a [peracide] (mg L -1 ) [peracide] (mg L -1 ) 1 min 5 min 10 min 13

14 Viral reduction (log 10 ) Inactivation : suspension Reversible reaction O O R + HO OH R + OH O OH Organic acid Hydrogen peroxide Peracid O H Solution d'acide peracétique Acide acétique Peroxde d'hydrogène Peracetic acid Acetic acid Hydrogen peroxide 14

15 BIOFILMS SURFACES ASTM E Inactivation : surfaces and biofilm Peracetic et perpropionic acids : 50 mg L -1 ; 5 min Stainless steel, MNV-1 Treatment Neutralisation PVC 10 4 PFU coupon -1 5 min (clean and soiled) Biofilm + MNV-1 Treatment 10 7 PFU g -1 5 min Neutralisation Dissolution in sodium citrate 15

16 Inactivation : surfaces and biofilm MNV-1 reduction (log 10 ) Stainless steel PVC Biofilm Clean Soiled Clean Soiled Recovery (%) 94,0 ± 1,8 99,9 ± 0,1 97,5 ± 3,0 98,3 ± 0,4 95,2 ± 2,9 Peracetic Perpropionic Total reduction 4 log 10 16

17 Mechanism of action : possible targets Capsid - antigens - structure RNA multiplication Host-cell 17

18 Mechanism of action : samples MNV-1 9,0 log 10 PFU ml -1 Control PBS Moderate treatment 50 mg L -1 ; 5 min Harsh treatment 1000 mg L -1 ; 10 min 8,6 log 10 PFU ml -1 4,9 log 10 PFU ml -1 < 1 log 10 PFU ml -1 18

19 Morphology Control 50 mg L mg L -1 8,6 log 10 PFU ml -1 4,9 log 10 PFU ml -1 < 1 log 10 PFU ml -1 19

20 Viral proteins Control 50 mg L mg L -1 8,6 log 10 PFU ml -1 4,9 log 10 PFU ml -1 < 1 log 10 PFU ml -1 kda VP1 VP1 180 copies of VP SDS-PAGE 20

21 RNA Control 50 mg L mg L -1 8,6 log 10 PFU ml -1 4,9 log 10 PFU ml -1 < 1 log 10 PFU ml -1 Viral RNA Bioanalyzer Internal marker? RNA pico chips 21

22 Proposed mechanism of action R Moderate R treatment O O Fragmentation of RNA O O + HO R O + HO O + HO Compromised replication proteins? Non-recognition by the host-cell 22

23 Conclusion - Peracids Solutions : peracetic and perpropionic acids Regulation (FDA) : 80 mg L -1 Possible applications (reduction of at least 3 log 10 ) : Suspension Clean surfaces Soiled surfaces Biofilm (artificial) 50 mg L -1 ; 5 min Viral targets affected : RNA and proteins 23

24 What is pulsed light? Innovative technology Control panel Non-thermal pasteurization for food preservation X UV IR and µ decontamination of air or packaging material Xenon lamp Treatment chamber 830 J pulse -1 UV-C UV-B UV-A visible n. IR Very short high-intensity pulse nm 8 cm Sample 0,69 J cm -2 0,02 J cm -2 UV (max : 12 J cm -2 ) Approval by FDA ( nm, 2 ms, 12 J cm -2 ) 24

25 Inactivation : suspension MNV : 10 5 PFU ml -1 COMPOSITION Hardness : 0 à 400 mg L -1 Turbidity : 0 à 1000 NTU Wastewater ENERGY 1 to 7 impulsions (0,7 à 4,8 J cm -2 ) Mineral water 25

26 Viral reduction (log 10 ) Inactivation : suspension PBS ,7 2,1 3,5 4,8 Fluence (J cm -2 ) 1 pulse 0.7 sec 3 pulses 1.6 sec 5 pulses 2.7 sec 7 pulses 3.9 sec 26

27 Viral reduction (log 10 ) Inactivation : suspensions PBS HARD WATER P > 0, ,7 2,1 3,5 4,8 0 0,7 2,1 3,5 Fluence (J cm -2 ) Fluence (J cm -2 ) Pulsed light does not seem to be affected by the hardness of water 50 mg L mg L mg L mg L mg L -1 calcium carbonate 27

28 Viral reduction (log 10 ) Inactivation : suspensions PHOSPHATE BUFFER TURBID WATERS P < 0, ,7 2,1 3,5 4,8 0 0,7 2,1 3,5 4,8 Fluence (J cm -2 ) Fluence (J cm -2 ) Pulsed light affected by the water turbidity 50 NTU 100 NTU 200 NTU 500 NTU 1000 NTU bentonite 28

29 Viral reduction (log 10 ) Inactivation : suspensions PBS WASTE AND MINERAL WATER P > 0, ,7 2,1 3,5 4,8 0 0,7 2,1 3,5 4,8 Fluence (J cm -2 ) Fluence (J cm -2 ) Wastewater after grit removal Wastewater after digestion Mineral water 29

30 BIOFILMS SURFACES Inactivation : surfaces and biofilm 1 to 13 pulses (0.7 to 9.0 J cm -2 ) Stainless steel, PVC (clean et soiled) MNV PFU coupon -1 Treatment Biofilm + MNV PFU g -1 Treatment Dissolution 30

31 Viral reduction (log 10 ) Inactivation : surfaces and biofilm 5 STAINLESS STEEL P < ,7 2,1 3,5 4,8 6,2 7,6 9,0 Fluence (J cm -2 ) Soil could protect MNV by pulsed light on surfaces Clean condition Soiled condition 31

32 Viral reduction (log 10 ) Inactivation : surfaces and biofilm STAINLESS STEEL PVC P < P < ,7 2,1 3,5 4,8 6,2 7,6 9,0 0 0,7 2,1 3,5 4,8 6,2 7,6 9,0 Fluence (J cm -2 ) Fluence (J cm -2 ) Clean condition Soiled condition 32

33 Viral reduction (log 10 ) Inactivation : surfaces and biofilm ARTIFICIAL ALGINATE BIOFILM ,7 2,1 3,5 4,8 Fluence (J cm -2 ) Biofilm does not affect the viral inactivation by pulsed light 33

34 Mechanism of action : samples MNV log 10 PFU ml -1 Control 0.0 J cm -2 Moderate treatment 2.1 J cm -2 Harsh treatment 9.0 J cm log 10 PFU ml log 10 PFU ml -1 < 1 log 10 PFU ml -1 34

35 Morphology Control Moderate Harsh 9.0 log 10 PFU ml J cm J cm log < 1 log 10 PFU ml PFU ml -1 35

36 Viral proteins Control 9.0 log 10 PFU ml -1 Moderate 2.1 J cm log 10 PFU ml -1 Harsh 9.0 J cm -2 < 1 log 10 PFU ml -1 kda VP SDS-PAGE 180 copies of VP1 36

37 RNA Control Moderate Harsh 9.0 log 10 PFU ml J cm J cm log < 1 log 10 PFU ml PFU ml -1 Viral RNA Bioanalyzer Internal marker RNA pico chips 37

38 Proposed mechanism of action nm UV-C UV-B UV-A visible IR p. Absorption of photons (UV) especially by nucleic acids = excitation state relaxation Photochemical effect Cleavages of viral RNA H 2 O Photothermical effect Energy in internal content; evaporation of water Increase of internal pressure Structure explosion 38

39 Conclusion Pulsed light Viral inactivation increases with fluence but affected by turbidity and presence of organic matter Regulation (FDA) : 12 J cm -2 Possible applications (reduction of at least 3 log 10 ) : Hard waters : 2,1 J cm -2 (1,6 sec) Turbid waters (200 NTU) : 3,5 J cm -2 (2,7 sec) Waste and mineral water : 2,1 J cm -2 (1,6 sec) Clean surfaces : 3,5 J cm -2 (2,7 sec) Plastic soiled surfaces : 7,6 J cm -2 (6,1 sec) Biofilm (artificial) : 0,7 J cm -2 (0,5 sec) Viral targets achieved : capsid and RNA 39

40 General conclusion Fast No toxic residue Vectors of transmission Person Food Water Environment No heat 60% : acid 40% : H 2 O 2 Peracids Pulsed light 40

41 Acknowledgments Allison Vimont, Ph.D. Maryline Girard, Ph.D. candidate 41

42 Thank you! Julie Jean, Ph.D.

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