Download Cell culture and new technologies for in

Cell culture and new technologies for invitro propagation of HEV virus
Alessandra Berto PhD student
Pulawy
14/4/2101
1
HEV
 Is the sole member of the family Hepeviridae
 Is a non enveloped, spherical particle, single stranded
and positive-sense RNA
 Contains three open reading frames (ORF)
 Classified into 4 genotypes
 The disease is that of an acute, icteric hepatitis. The
incubation period ranges from 2 to 9 wk.
 Animals can be infected with the virus
2
Transmission of HEV
 In developing regions is largely waterborne due to
inadequate separation of human sewage from drinking
water supplies
 In developed regions transmission routes are not
clear, but pig appear to play a central role as a
reservoir of infection
3
Vocational exposure:
vets, farmer processing
and retail staff
Sika deer
pig
faeces
Abattoir
effluent
Ingestion
of undercooked
sika deer meat
man
slurry
Human
sewage,
floods
pasture
watercourses
Ingestion
of undercooked
pig meat, liver
corps
Crops via
irrigation
Water
basedreaction
Abstraction
for drinking
water
molluscs
Blood transfusion
Other
mammals
4
man
The exact transmission routes are unclear, largely
because HEV is extremely difficult to propagate in
vitro, but retail pig products have been shown to
contain HEV RNA.
5
General background
•Tanaka et al (Tanaka et al., 2007) have tested 21 cell lines including
PLC/PRF/5 cells using a faecal suspension with high HEV load as
inoculum (Huang et al., 1992, Huang et al., 1995, Huang et al., 1999, Li, 1996). A high load
of HEV was detected in the culture supernatant of cultivated
PLC/PRF/5 cells from day 12 post inoculation.
•At VLA Weybridge laboratory, several attempts were made to
reproduce Tanaka’s work using field swine HEV PCR positive faecal
materials as inoculum, but without success.
6
The lack of an efficient and reliable cell
culture system and a practical animal
model for HEV have hindered studies on
mechanisms of HEV replication,
transmission, pathogenesis and
environmental survival.
7
 There are several reports in the literature
demonstrating the potential of a new 3D culture
system, Rotating Wall Vessel (RWV), for the growth of
fastidious viruses as well as bacteria (Kageyama et al., 2003,
Takahashi et al., 2007, Lorenzo et al., 2008, Goodwin et al., 1993).
 This 3D culture system has been used to grow
fastidious Noroviruses from faecal materials (Kageyama et
al., 2003). The system offers a potential for in vitro
cultivation of HEV.
8
Aim 1

To evaluate the new 3D culture system to assess HEV infectivity. This
is to verify if the HEV virus content detected by PCR in pig and
environmental samples is infectious.

To test the progeny’s infectivity

To compare the efficiency of the 3D system to the conventional 2D
system. In addition, cells grown in the 3D system were transferred to a
2D system and infected. This aims to produce the best tool with which
to examine large numbers of samples and thereby investigate potential
transmission routes.
9
Results
HEV RNA was detected from
15 dpi in the 3D culture infected
with inoculum HEV positive
liver sample obtained from an
experimentally infected pig
Viral Copy Number / ml
1.40E+08
1.20E+08
1.00E+08
8.00E+07
Liver
6.00E+07
Hepatocytes
4.00E+07
2.00E+07
3d
pi
12
dp
i
24
dp
i
32
dp
i
dp
i4 2
58
dp
i
70
dp
12 i
6d
p
17 i
5d
pi
0.00E+00
Two distinct peaks are
observed, the first
between 24 and 42 dpi
and between 30 and 42,
and the second between
62 and 175 dpi for both
10
3 systems developing
3D
2D
3D transferred in 2D
11
•Ct values show a reducing trend in the undiluted and 10^-2 dilution of
inoculum in the period of 40 days
•The Ct values for the 10^-1 dilution of inoculum remained unchanged till
30 dpi
12
A similar trend was observed for the 3D cells transferred to 2D for the neat
inoculum.
13
The Ct values for neat inoculum remained unchanged
14
Discussion
The results have shown detectable Ct values in
quantitative RT-PCR at all dpi. In the parallel 2D system
Ct values were not detectable at any dpi.
The observation of viral replication in PLC/PRF/5 cells in this
system indicates that the 3D system may potentially be used
as a tool to elucidate the pathobiology of HEV, which may, in
turn, facilitate vaccine research, monitoring of HEV
contamination and HEV survival through processing to point
of sale.
15
 In experiment 2, a small titration range was introduced
to give some impression of the relative sensitivity of
3D, 3D transferred to 2D and 2D alone. The virus was
detectable by real time RT- PCR in all three systems
until the end of the experiment.
 This data also demonstrates that importantly, the virus
progeny obtained during experiment 1 was infectious.
16
Aim 2
construction and use of an interferon
knockout cell line to further enhance the
sensitivity of the system to detect viable
virus.
17
PROCEDURE
1)
AMPLIFICATION OF THE PLASMIDS
3 plasmids
pdl’PIV5-V: Expression is controlled by the
constitutive SFFV promoter, which produces a single bicistonic mRNA that
encodes for the PIV5-V together with an eukaryotic resistance marker,
puromycin, for mammalian cell selection.
pCMV R8.91: Plasmid expressing the gag/pol, tat and rev genes of HIV-1.
pMD-G: Plasmid expressing the vesicular stomatitis virus glycoprotein
(VSV-G) gene
The plasmids will be amplified in E. coli
18
2) TRANSFECTION OF 293 T CELLS
to create the virion containing the proteins encoded the
plasmids, these need to be transfect in highly permissive cell
line
3) TRANSDUCTION OF PLC/PRF5
supernatant collected form the 293T cells was transferred in
PLC/PRF/5 cells and subsequentely selected with puromycin
19
Result
 The experiment is failed. Probably explanation:
- the DNA inside the 3 plasmids was degraded
- PLC/PRF/5 are not permissive to the lentivirus
- too much puromycin was added at the last one
- step
20
Before the repeat experiment PLC/PRF/5 were
tested for IFN production through CAT ELISA test
RESULT:
Not infected cells
tested for INF production
result
No IFN activity
HEV positive cells
21
Aim 3
Confocal microscope analysis to compare 3D and
2D cells morphology to proof the major
differentiation of 3D cells
22
2D CD81
3D CD81
23
2D B-Catenin
3D B-Catenin
24
2D E-Caderin
3D E-Caderin
25
Aim 4
to infect cells with not heated liver, liver
heated at 56oC for 1 hour and at 100oC for
15 minutes and detect at which temperature
the virus is inactivated
26
N=5 I.V.
HEV-negative
Not
cooked
4X N=5
HEV- positive
Incubated
At
56°C for
1h
positive
Feagins et al., 2008
Stir-fried
At 191°C
For 5min
negative
Boiled
in water
For 5 min
27
The experiment is still
ongoing………..
28
 Other virus inactivation strategy are in view; such
as inactivate the virus with UV, high pressure
system, detergent like decon or ETOH
 Samples analysis
 EM analysis
29
 It will be tried to implement 3D cell culture for HEV Gt4 propagation in Lelystad.
If this works Gt4 inactivation experiments can be performed
 HEV Gt4 experimental infections in Wild boar and mice
 Establishment of HEV Gt3 and GT4 infectious dose in mice
30
31