Journal of Clinical Virology - Volume 82 - Supplement 1 - September 2016

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Abstracts / Journal of Clinical Virology 82S (2016) S1–S142 S81
ples previously taken from patients presently under investigation
for HEV antibodies.
Materials and methods: Some patients have blood samples
taken earlier in life for other routine diagnostics than HEV, and
therefore it is possible to compare the former and the present
sample, for determination of the HEV status of the patient, and to
conclude if it is a new infection or a reactivation.
We are planning to test approximately 20–30 patients all having
a present HEV sample and a sample taken earlier in life for other
reasons than HEV. All samples will be tested in three different com-
mercial HEV elisa assays, one fromWantai, one fromMikrogen and
one from DSI/Abia, and the results will be compared.
Clinical information is available for some patients and we will
try to retrieve it for the rest of the patients.
Conclusion: The project is ongoing, but the final data will be
ready for the conference.
Reference
[1] P. Grewal, S. Kamili, D. Motamed, Chronic hepatitis E in an immunocompetent
patient: a case report, Hepatology 59 (1) (2014) 347–348.
[2] N. Kamar, J. Izopet, Does chronic hepatitis E virus infection exist in
immunocompetent patients? Hepatology (2013).
Abstract no: 342
Presentation at ESCV 2016: Poster 121
Hepatitis B surface antigen expression: A pilot
study comparing wild type and surface antigen
mutant viruses
A. Jeffery-Smith ∗, J. Poh, S. Ijaz, R. Tedder
Blood Born Virus Unit, Colindale, Public Health
England, UK
Introduction: The immunogenic region of the hepatitis B
surface antigen (HBsAg), the ‘a’ determinant, is formed from a
sequence of amino acids, which through disulphide bonding form
a three dimensional structure. Mutations in this coding region
can lead to amino acid substitutions resulting in a conformational
change in the protein, which may render it unrecognisable to
immunoglobulins.
By creating a construct containing the PreS1/PreS2/S region of
the hepatitis B virus (HBV) genome coding for the three envelope
proteins the effects of mutations in the S gene on the expression of
HBsAg from transfected cells can be investigated
Materials and methods: Five HBV samples were investigated:
a wild type, two G145R HBsAg mutants, and two with T118K,
P120S and M133T, and M133T, P135L, G145R and V168A surface
mutations respectively. Nested PCR was used to obtain a 0.7x HBV
genome (0.7mer) construct containing the PreS1/PreS2/S coding
region.
The TOPO-TA cloning kit (ThermoFisher) was used to clone the
constructs prior to transferring them into a mammalian expression
vector.
Chemical transfection of HepG2 cells was performed with the
FuGENE® HD transfection system (Promega® ) using both 0.5 m
and 2.0 mof DNAof the recombinant expression vector. Cellswere
harvested 72 h post-transfection. Supernatant fluid and cell pellets
were collected. Cells grown on coverslips were fixed and labelled
for microscopy.
Enzyme-linked immunosorbent assay (ELISA) for HBsAg
(Murex) was used to detect HBsAg in cell pellets and supernatant
fluid. Coverslips were labelled with antibodies to HBsAg, golgi and
nuclear proteins for confocal microscopy.
Preliminary results: Using more DNA in the transfection
resulted in higher readouts from the ELISA.
For the 0.5 m DNA transfections, two of the mutant viruses
demonstratedHBsAg secretion deficiency comparedwithwild type
virus. This occurred in one of the G145R HBV mutants and the con-
struct with T118K, P120S and M133T surface mutations, both of
which had higher HBsAg levels in the cell pellet than the super-
natant fluid. The two other viruses appeared to behave like wild
type virus. For the 2.0 mDNA transfections, however, these differ-
ences in secretion were not reflected, with samples demonstrating
higher amounts of HBsAg in the cell pellet than the supernatant
fluid.
Immunofluorescence showed variation in surface antigen
labelling within the HepG2 cells. The cells transfected with one of
the G145R mutant viruses demonstrated greater labelling within
cells than those transfected with wild type and the other mutant
viruses.
Conclusions and further work: The preliminary results of this
pilot work indicate the presence of phenotypic differences between
hepatitis B viruses with surface gene mutations. These mutations
appear to have varying effects on the secretion of HBsAg fromtrans-
fected cells. However, it is difficult to characterise these differences
because of the contradictory results when higher amounts of DNA
are used for transfection. The reasons behind the discrepancy are
not clear and further work to look at the reproducibility of this
phenomenon is needed.
Further phenotyping work using a Luminex® bead based assay
looking at variations in specific epitopes of theHBsAgwill be under-
taken.
Reference
[1] T. Garcia, et al., Drastic reduction in the production of subviral particles does
not impair hepatitis B virus virion secretion, J. Virol. (2009).
Abstract no: 35
Presentation at ESCV 2016: Poster 122
Comparison of immunoassays from three
chemiluminescent automated systems for the
detection of hepatitis B virus serological
markers
J. Lis-Tønder∗, A. Løvig, G.T. Schouborg
Department of Clinical Microbiology, Lillebaelt
Hospital, Vejle, Denmark
Background: HBV infection is a serious global health problem.
More than 350million people suffering fromchronic infectionwhat
results in 500,000 to 1.2million deaths per year n Denmark the
disease is relatively rare – the prevalence in the adult population
estimated per 31st December 2007was 0.24% erological mark-
ers of hepatitis B virus (HBV) are used for laboratory diagnosis and
monitoring of HBV infection or immune status. Hepatitis B surface
antigen (HBsAg) is the hallmark of HBV infection and is the first
serological marker detectable in serum, while antibodies to HBsAg
(anti-HBs) can be formed following a hepatitis B infection or after
hepatitis B vaccination. Hepatitis B core antibodies (IgM and IgG)
are used to follow the progression of the infection from the acute
stage to recovery. Anti-HBc antibodies are sometimes present after
the disappearance of the HBsAg and before appearance of anti-HBs.
In these situations these antibodies serve as the primary marker
for infection. The hepatitis B e antigen is the marker of viral repli-
cation, and anti-HBe is a marker of immune response to HBeAg,