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Emerging pathogens –
foodborne viruses
Reimar Johne,
Federal Institute for Risk Assessment,
Berlin, Germany
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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
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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
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Reimar Johne – Joint international Symposium - 2017 Seite 4
Hepatitis E cases in UK in 2017
(The Telegraph,
22.05.2017)
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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
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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!
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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!
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Reimar Johne – Joint international Symposium - 2017 Seite 8
Detection methods
for viruses in food
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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?
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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
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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)
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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
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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
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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
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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
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Reimar Johne – Joint international Symposium - 2017 Seite 16
Hepatitis E Virus
(HEV)
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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)
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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
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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
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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
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Reimar Johne – Joint international Symposium - 2017 Seite 21
From:
Pavio, …, Johne:
Vet. Res. 2017, 48:78
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Reimar Johne – Joint international Symposium - 2017 Seite 22
From:
Pavio, …, Johne:
Vet. Res. 2017, 48:78
Is the virus still infectious in food?
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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
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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
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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
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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
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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
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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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BU
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Acknowledgements
Federal Institute for Risk Assessment
DiedersdorferWeg 1 D-12277 Berlin
Tel. 030 18412 - 1006 Fax 030 18412 - 2064
[email protected] 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