Emerging pathogens TUNG - BfR · 2018. 6. 26. · Emerging pathogens - foodborne viruses -...

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Emerging pathogens –

foodborne viruses

Reimar Johne,

Federal Institute for Risk Assessment,

Berlin, Germany

Reimar Johne – Joint international Symposium - 2017 Seite 2

Norovirus

GII.16/II.13

October/November 2012:

10.974 diseased children after consumption

of imported frozen strawberries

Norovirus gastroenteritis outbreak in Germany 2012

Reimar Johne – Joint international Symposium - 2017 Seite 3

Outbreak of hepatitis A in 13 EU countries 2013/2014

Severi et al., 2015

January 2013 – August 2014:

1.589 patients (1.102 hopsitalized)

after ingestion of frozen berry mix

HAV subtype IA

Reimar Johne – Joint international Symposium - 2017 Seite 4

Hepatitis E cases in UK in 2017

(The Telegraph,

22.05.2017)

Reimar Johne – Joint international Symposium - 2017 Seite 5

Important foodborne viruses

virology.net ucdavis.edu dasgesundheitsblog.de pnas.org

Norovirus Rotavirus Hepatitis E-

Virus

Hepatitis A-

Virus

br-online.de

handwashingforlife.com

Vomiting/

Diarrhoa

Virus-

Hepatitis

Reimar Johne – Joint international Symposium - 2017 Seite 6

Transmission of foodborne viruses

HEV

Rota

HEV

NoV

HAV

HEV

Rota - Environmental pollution

- Irrigation water

- Fertilizers

- Food handlers

NoV

HAV

HEV

Rota

NoV

HAV

HEV

Rota

Prevention of

virus contamination!

Reimar Johne – Joint international Symposium - 2017 Seite 7

Transmission of foodborne viruses

HEV

Rota

HEV

NoV

HAV

HEV

Rota - Environmental pollution

- Irrigation water

- Fertilizers

- Food handlers

NoV

HAV

HEV

Rota

NoV

HAV

HEV

Rota

Identification of

virus contamination! Prevention of

virus contamination!

Reimar Johne – Joint international Symposium - 2017 Seite 8

Detection methods

for viruses in food

Reimar Johne – Joint international Symposium - 2017 Seite 9

Detection of pathogens in food

contaminated

food

concentration

of bacteria

(growth in liquid

media)

identification

contaminated

food

identification

concentration

of viruses

(growth in liquid

media)

virus purification

+

concentration

Real time

RT-PCR analysis

bacteria viruses

PCR inhibitors?

Reimar Johne – Joint international Symposium - 2017 Seite 10

ISO 15216 for soft fruits

berry sample

Tris/Glycin/Beef Extract Buffer

and Pectinase

homogenize

adjust to pH 9.5

add PEG/NaCl

RNA extraction

real-time RT-PCR

adjust to pH 9.5

centrifugation 30 min 10.0000 x g

adjust to pH 7.0

stir 1 h at 4°C

centrifugation 30 min 4°C

wash pellet with Chloroform/Butanol

PEG-

Precipitation

Reimar Johne – Joint international Symposium - 2017 Seite 11

Method comparison for NoV detection on strawberries

Artificially NoV-contaminated frozen strawberries

RR Recovery rate | SD Standard deviation

ISO method most reliable

still low recovery rate

Method

RR mean ± SD (%) Reference

ISO/TS 15216 1.71 ± 2.31 ISO (2014)

Ultrafiltration 0.98 ± 0.95 Esseili et al. (2015)

Direct lysis 0.52 ± 0.54 Perrin et al. (2015)

PGM magnetic

Beads

0.04 ± 0.1 Tian et al. (2008)

TriReagent 0.01 ± 0.03 Szabo et al. (2015)

Reimar Johne – Joint international Symposium - 2017 Seite 12

ISO 15216 for soft fruits (expanded)

berry sample

Tris/Glycin/Beef Extract Buffer

and Pectinase

homogenize

adjust to pH 9.5

add PEG/NaCl

RNA extraction

Sephacryl column purification

adjust to pH 9.5

centrifugation 30 min 10.0000 x g

adjust to pH 7.0

stir 1 h at 4°C

centrifugation 30 min 4°C

wash pellet with Chloroform/Butanol

PEG-

Precipitation

real-time RT-PCR

Reimar Johne – Joint international Symposium - 2017 Seite 13

Optimization of the ISO method using Sephacryl columns

Batch

ISO method

NoV RR ± SD (%)

ISO method

+ Sephacryl columns

NoV RR ± SD (%)

3 2.83 ± 2.92 15.28 ± 9.73

4 0.59 ± 0.49 5.60 ± 1.58

NoV Norovirus | RR Recovery rate | SD Standard deviation

Improved detection usiung of Sephacryl columns

Reimar Johne – Joint international Symposium - 2017 Seite 14

Analysis of frozen strawberry samples

involved in the NoV outbreak

in Germany 2012

ISO method

NoV RR ±

SD (%)

ISO method

+ Sephacryl

columns

NoV RR ±

SD (%)

Positive / samples tested

9 / 22 20 / 22

Detection rate in % 40.1 90.9

Higher detection rate using Sephacryl columns

Reimar Johne – Joint international Symposium - 2017 Seite 15

Next Generation Sequencing –

Analysis of frozen strawberry samples

involved in the NoV outbreak

in Germany 2012

~ 29 million reads in total

only 2 human norovirus reads (genotype II.16/II.13 = outbreak strain)

eukaryotes

bacteria

viruses

Reimar Johne – Joint international Symposium - 2017 Seite 16

Hepatitis E Virus

(HEV)

Reimar Johne – Joint international Symposium - 2017 Seite 17

1991

2016

notified

Hepatitis E cases

in Germany

Hepatitis E in Germany

Quelle: survstat @ RKI

- Case/fatality rate <4%: Risk groups are - persons with underlaying liver disease

(- pregnant women)

- immunosuppressed people (transplant patients)

- many subclinical cases (16.8 % seroprevalence in Germany)

Reimar Johne – Joint international Symposium - 2017 Seite 18

Hepatitis E Virus

www.cdc.gov

- Genotypes 1 – 4 with simliar disease course,

but different transmission modes

Genotype 1: human

Genotype 2: human

Genotype 3: human, pig, wild boar, deer

Genotype 4: human, pig

HEV

pnas.org

Reimar Johne – Joint international Symposium - 2017 Seite 19

Transmission pathways of HEV

?

?

Trans-

fusion

Travel to

endemic countries

Reservoir animals Direct contact

Contamination during

irrigation/fertilization

Environmental

contamination

infection

without

symptoms

other animals

Food from

reservoir animals

Reimar Johne – Joint international Symposium - 2017 Seite 20

HEV in reservoir animals in Germany

Wild boar:

29.9% (Adlhoch et al., 2009) to 33.0% (Denzin er al., 2013) antibody-positive

Pig:

42.7% (Dremsek et al., 2013) to 49.8% (Bächlein et al., 2010) antibody-positive

Pig liver:

4 % (Wenzel et al., 2011) HEV RNA-positive

Raw and liver sausage:

20.0% - 22.0% (Szabo et al., 2015) HEV RNA-positive

Reimar Johne – Joint international Symposium - 2017 Seite 21

From:

Pavio, …, Johne:

Vet. Res. 2017, 48:78

Reimar Johne – Joint international Symposium - 2017 Seite 22

From:

Pavio, …, Johne:

Vet. Res. 2017, 48:78

Is the virus still infectious in food?

Reimar Johne – Joint international Symposium - 2017 Seite 23

0

1

2

3

4

5

6

7

8

9

10

d1 d7 d14 d21 d28 d35 d42 d47

passaging

on new cells

log HEV

genome copies

d p.i. of A549 cells

HEV cell culture – isolation of strains

Reimar Johne – Joint international Symposium - 2017 Seite 24

Genome analysis of the isolated HEV strain 47832

genotype 3, related to HEV strains of humans and wild boars from Germany,

contains an unusual insertion in ist ORF 1

0

42.5

5 10 15 20 25 30 35 40

Kernow-C1/human/USA(HQ389544)-3a

LBPR000379/human/USA(JN564006)-3a

100.0

US2/human/USA(AF060669)-3a

100.0

JYO-Hyo03L/human/Japan(AB189075)-3b

100.0

wbGER27/wild boar/Germany(FJ705359)-3i/c

47832/human/Germany(KC618402)-3i/c

100.0

BB02/wild boar/Germany(FJ998008)-3i

100.0

100.0

swX07-E1/pig/Sweden(EU360977)-3

HEV_RKI/human/Germany(FJ956757)-3f

100.0

Osh205/pig/Kyrgyzstan(AF455784)-3g

99.9

T1/human/China(AJ272108)-4

100.0

SAR-55/human/Pakistan(M80581)-1

Mexican/human/Mexico(M74506)-2 99.5

94.9

R63/rat/Germany(GU345042)

ge

no

typ

e 3

genotype 4

genotype 1

genotype 2

ratHEV

Reimar Johne – Joint international Symposium - 2017 Seite 25

Selection of cells for HEV strain 47832

(Schemmerer et al., 2016)

A549

A549/D3

A549/DB3

undil. 1:10 1:100 1:1000 1:10000

1,00E+03

1,00E+04

1,00E+05

1,00E+06

1,00E+07

1,00E+08

8

7

6

5

4

3

Lo

g H

EV

ge

no

me

co

pie

s/m

l

Reimar Johne – Joint international Symposium - 2017 Seite 26

Testing of long-term stability of HEV

(Johne et al., 2016)

4°C

room

temp.

37°C

days

Log

HE

V f

ocus fo

rmin

g u

nits

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

0 5 10 15 20 25 30 35 40 45 50 55

no ffu

4.5

4

3.5

3

2.5

2

1.5

1

0.5

days

Log

HE

V f

ocus fo

rmin

g u

nits

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

0 5 10 15 20 25 30 35 40 45 50 55

no ffu

4.5

4

3.5

3

2.5

2

1.5

1

0.5

days

Log

HE

V f

ocus fo

rmin

g u

nits

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

0 5 10 15 20 25 30 35 40 45 50 55

no ffu

4.5

4

3.5

3

2.5

2

1.5

1

0.5

Reimar Johne – Joint international Symposium - 2017 Seite 27

Testing of short-term heating

(Johne et al., 2016)

Log

HE

V f

ocus fo

rmin

g u

nits heating for 1 min

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

noheat

37°C 50°C 55°C 60°C 65°C 70°C 75°C 80°C 85°C 90°C

no ffu

4.5

4

3.5

3

2.5

2

1.5

1

0.5

little effect strong

effect

< detection

limit

sec

70°C

Log

HE

V f

ocus fo

rmin

g u

nits

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

0 60 120 180 240 300 360 420 480 540 600no ffu

4.5

4

3.5

3

2.5

2

1.5

1

0.5

Reimar Johne – Joint international Symposium - 2017 Seite 28

Summary

- Foodborne viruses have caused large disease outbreaks

during the last years.

- Norovirus and HAV are mainly transmitted by contamination

of food with human excretions.

- Detection of viruses in food is complicated by the absence

of cell culture techniques and the presence of PCR inhibitors

- Hepatitis E poses an increasing problem in industrialized countries

- Pigs and wild boars are the main reservoirs for HEV

- Infectivity of HEV in meat products remains mostly unknown

- A novel cell culture model can be used for estimation of HEV inactivation

under specific physico/chemical conditions

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Acknowledgements

Federal Institute for Risk Assessment

DiedersdorferWeg 1 D-12277 Berlin

Tel. 030 18412 - 1006 Fax 030 18412 - 2064

Reimar.Johne@bfr.bund.de www.bfr.bund.de

Project funding:

Bundesinstitut für

Risikobewertung (BfR):

Kathrin Szabo

Eva Trojnar

Christina Bartsch

Friedrich-Loeffler-Institut (FLI):

Rainer Ulrich

TiHo Hannover:

Günther Klein

ZInstBW Kiel:

Helena Anheyer-Behmenburg

Ulrich Schotte

Alfred Binder

Charité - Virologie:

Jörg Hofmann

Universität Regensburg:

Mathias Schemmerer

Jürgen Wenzel

LAV Sachsen-Anhalt:

Dietrich Mäde