Transcript Norovirus

Search for cellular
attachment receptors used
by a GII.4 Norovirus Dijon
Gaurav Dutta Dwivedi
Supervisors: Niklas Arnberg and Carin Årdahl
Introduction
Norovirus are a group of viruses that cause non bacterial gastroenteritis in
humans.
The first outbreak of Norovirus infection was reported in Norwalk, Ohio in
1968.
Human Norovirus infection is popularly known as winter vomiting disease.
Norovirus outbreaks frequently occur in community surroundings.
Noroviruses are predominantly infectious and highly stable in the environment
and immunity following infection generally is short-lived.
Symptoms of Norovirus infection
Nausea and Vomiting
Dehydration
Fever
Abdominal cramping
Diarrhea
Incubation period is 24 to 48 hours, with the symptoms lasting 12 to 60 hours.
Infected hosts can shed virus in stool for up to 2 weeks.
Transmission of Norovirus
Human Noroviruses recognize histo-blood group antigens (HBGAs)
that are expressed on the surface of mucosal epithelial cells.
Noroviruses infect individuals with a functional alpha-1, 2-fucosyltransferase
(FUT2) gene and are designated as secretor-positive and those with defected
FUT2 gene are termed as secretor-negative ,they are resistant to the common
GII.4 strain, however they can still get infected with other Norovirus strains.
Norovirus Classification
The genogroup II, genotype 4 Noroviruses, designated GII.4, are currently
responsible for 70–80% of Norovirus outbreaks worldwide.
Viral Morphology
 Family Caliciviridae.
 Single-stranded, positive-sense RNA genome of
~7.7 kb.
 Non-enveloped and icosahedral.
 27-40 nm.
 The viral genome encodes one major structural
protein of 60 kDa which forms viral capsid.
 Capsid 180 molecules, folds into 90 dimers.
Norovirus genome comprises of three open reading frames (ORFs).
ORF1 encodes the non-structural proteins that are crucial for virus replication, ORF2 and ORF3
encode a major capsid protein VP1 and a minor structural protein VP2, respectively.
VP1 consists of shell domain (S) and the protruding domain (P).
P domain is further divided into two subdomains known as P1 and P2.
Unpublished observations indicate that the presence of specific integrin-binding motifs plays a
role in interactions for binding to integrins and allows virus particles attachment to the host
cells.
Aim
To explore cellular receptors used by the GII.4 Human Norovirus
(NoV) Dijon Virus Like Particles (VLPs) and analyse its binding
characteristics across various integrin expressing cells.
Why we used Norovirus like particles (NoV-LPs) in the
present study ?
The study of NoV has been hampered
by the lack of an efficient cell culture
system, which is still not available for
Human NoV.
Methods
The Dijon NoV-LPs used were produced in Sf9 insect cells.
The VLPs were clarified using ultracentrifugation and sucrose
density gradient methods , finally the purity was confirmed by
using SDS-PAGE and Western blot.
Flow cytometry was used for evaluation of binding characteristics
of NoV-LPs across various integrin expressing cells.
Production of VLPs
Sf9 cell culture
Incubate at 130 rpm,28 ˚C, 5 days
SDS-PAGE
Western blot
Centrifuge at 3000 g,30 min
Ultracentrifuge supernatant at
30000 rpm,2hrs,4˚C
Loading on gel
60%
50%
Ultracentrifuge at
320000 rpm,4hrs,4˚C.
40%
30%
20%
Collect the fractions
1
2
3
4
Collected fractions
Sucrose density gradient
5
Binding Assay
Cell culture
Detach cells and Transfer 200 000 cells/well.
Incubate with
added VLPs
NoV-LPs
FACS BD LSRII
96 well plate
Wash 3 X with 100 µl of PFN and centrifuge every time.
Centrifuge at 1300 rpm,4˚C for 5 mins
Wash 3X with 100 µl of PFN and transfer
Into FACS tubes and analyse the binding.
goat-anti-rabbit Alexa Flour 647
Wash with PFN buffer and
incubate
with the antibodies
Centrifuge
rabbit-anti-NoV
Incubate with primary and secondary antibody
Results
Pooled 3-5
Pooled 6-7
SDS-PAGE showing Fractions1-9 of the
produced VLPs
SDS-PAGE of cell lysate from the sf9 cell culture
Protein gel of pooled concentrated GII.4 Dijon (100K, Amicon filter)
Binding characteristics of NoV-LPs to integrin expressing cells
Figure 1
Figure 2
Figure 1 and Figure 2 represents the binding percentage of various integrin expressing cells to
NoV-LPs .
Western blot for the produced VLPs
Figure 3
Detection of produced NoV-LPs. Figure 3 showing the Western blot results of
Dijon NoV-LPs in various concentrations probed with primary antibody
(rabbit-anti-NoV, serum) and secondary antibody (HRP-conjugated-swineanti-rabbit).
Conclusion
In conclusion, the production and characterization of GII.4 NoV-LPs
in insect cells was validated in this study.
Western blot detected NoV-LPs using NoVs raised antibodies from
rabbit sera.
This receptor binding study indicates approximately 2-6 times
increased binding of NoV-LPs to various integrin expressing cells
signifying the importance of integrins as candidate receptors for
NoV.
Future directions
 Repetition of binding experiments with more integrin
expressing cell lines.
 Immortalization and replication studies using human
cells.
 Blocking studies for analyzing Norovirus binding.
Acknowledgement
 Carin Årdahl thesis supervisor.
 Professor Niklas Arnberg Division
of Virology, Umeå university.
 Division of Medical Microbiology,
Department of Clinical and
Experimental Medicine,
LiU(Linköping University),
Linköping, Sweden.
Reference:
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