Download Development of RT-PCR and ELISA for detection of Hepatitis virus

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SID 5
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Research Project Final Report
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SID 5 (Rev. 3/06)
Project identification
SE0522
Development of RT-PCR and ELISA for detection of
Hepatitis virus RNA and specific antibody in pigs
Contractor
organisation(s)
Veterinary Laboratories Agency,
Woodham Lane, New Haw,
ADDLESTONE, Surrey
KT15 3NB
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 6
£
56,990
start date ................
01 September 2003
end date .................
01 August 2004
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Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
Hepatitis E caused by the enterically transmitted hepatitis E virus (HEV) is a major public
health problem in many developing regions of the world.
There is now a substantial body of direct and indirect evidence, emerging from
autocthnonously-acquired cases of hepatitis E in industrialised regions, indicating that
hepatitis E virus (HEV) can be transmitted zoonotically. With the exception of pigs and
Sika deer, there is very little evidence for zoonotic transmission from other mammals,
and the ubiquitous nature of HEV in global pig populations has contributed to the
evidence that pigs represent the most likely reservoir for zoonotic transmission.
An ELISA for based on a recombinant antigen was developed and used to detect
antibodies to HEV in pig sera.
An analysis of the HEV genome was made and candidate regions chosen for
assessment in the development of a quantitative RT-PCR.
The RT-PCR was used in collaborative work with Royal Cornwall Hospital Trust (RCHT)
and HPA Infectious diseases centre, Colindale staff and provided more evidence of the
near identity of UK human and pig strains and the increasing number of autochthonous
infections.
For more details of the use of these assays please see attached abstracts of
publications.
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Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
 possible future work; and
 any action resulting from the research (e.g. IP, Knowledge Transfer).
Background
The work completed in a prior project had established the prevalence of HEV in UK pigs but
was not a properly stratified sample of pigs. Furthermore, the testing was done using an
antigen that was a gift from Emerson in the USA, and thus any further testing would require a
new source of antigen. The aim was to express an appropriate HEV antigen in baculovirus
and employ this antigen in an ELISA for HEV antibodies.
The number of UK HEV strains identified prior to this project was 2. This was not a sufficent
base with which to understand the genotype profile of UK pig strains. In addition, it would be
desirable to be able to measure the number of virus particles in pig samples. Acordingly the
PCR was to be further optimised and a quantitative assay developed.
Objectives
 Develop a sensitive and specific ELISA for the detection of specific antibodies to HEV in
pigs
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Optimise and validate RT-PCR and convert to Taqman® format for detection of HEV
nucleic acid in pigs

Use amplicons from selected HEV strains to determine nucleic acid sequence and
construct phylogenetic maps of UK swine strains and contemporary human HEV
strains.

Compile results and conclusions
A recombinant baculovirus carrying a truncated coding region for the HEV capsid (ORF-2)
gene was constructed and checked for recombinant antigen production by polyacryamide gel
electrophoresis (PAGE) and ELISA.
The ELISA results were standardised against the recombinant antigen received from Emerson
in the USA, using a small number of well characterised pig sera. A series of contemporasry
and archived pig sera were tested by the ELISA, establishing that not only had HEV been
present in the pig population in the UK since at least 1980, but that no significant changes in
national or regional prevalence appeared to have occurred between 1980 and the present. A
bulk batch of the antigen was produced using enhanced insect cell culture techniques, this
was aliquoted and stored at the end of the project, since time did not allow full purification and
extraction.
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The RT-PCR was adapted from published methods and used to examine further pig faecal
samples, and in collaboration with the South west Hospital Group and the CFI, HPA,
Colindale, further phylogenetic comparisons were made between human and pig strains of
HEV from the UK. This work further consolidated the view that the pig was a potential
reservoir for hepatitis E virus infection acquired by humans in the UK and in other developed
regions.
The real-time PCR was adapted from collaborators in Sweden. The assay worked reasonably
well in our hands but was not quite as sensitive as the conventional nested PCR. Further
development and optimisation of this assay will be required in order to fulfil it’s full potential in
providing a highly sensitive and quantitative assay for HEV in pig and other environmental
samples.
Future Priorities
The incidence of autochthonous hepatitis E appears to be increasing in the UK. Despite the
evidence, direct and indirect, for zoonotic transmission of HEV, the transmission routes are
still unclear and the possible contribution of other farmed and feral mammals is not known. An
in-vitro cultivation system for HEV is necessary before any meaningful data on environmental
survival, disinfection or significance of PCR-positives in retail meat can be obtained.
Confirmation of the above would enable the preparation of evidence-based risk assessments
and codes of practice aimed at reducing the zoonotic transmission of HEV.
The assays were used for the VLA elements of the work described in the following publications
Banks, M., Bendall, R., Grierson, S., Heath, G., Mitchell, J., & Dalton, H. (2004) Human and porcine
hepatitis E virus strains, United Kingdom. Emerging Infectious Diseases 10, 953-955.
Abstract:
We describe a case of acquired infection of a strain of hepatitis E virus (HEV) with a 100% amino acid identity to the
analogous region in strains of HEV circulating in a United Kingdom pig herd. This case further supports the theory that
autochthonous HEV infection in industrializedcountries is zoonotic.
S Ijaz, E Arnold, M Banks, RP Bendall, ME Cramp, R Cunningham, HR Dalton TJ Harrison, SF Hill, L
MacFarlane, REE Meigh, S Shafi, MJ Sheppard, J Smithson, MP Wilson and CG Teo. (2005) Non travelassociated hepatitis E in the UK: demographic, clinical and molecular epidemiological characteristics. J.
Inf. Dis. 192, 1166-1172.
Abstract
Banks, M. (2004) Hepatitis E: an emerging threat? Pig Journal, 54, 179-184
Abstract
Hepatitis E virus (HEV) has a global distribution in humans and pigs and is present in a range of other mammals.
The virus causes acute hepatitis in humans but is apathogenic in pigs. Most cases in developed regions are
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acquired by travel to one of the many developing regions of the world where the disease is endemic and faecaloral transmission is common. Data from several developed countries suggests a high degree of homology
between porcine and human strains of HEV. Recent evidence related to hepatitis strains isolated from
indigenously-acquired cases in some developed countries indicates that pig meat is a potential source of HEV
and that consumption of raw pigs’ liver may be the main zoonotic transmission route between pigs and humans.
Tolari, F., Chiaro, LD., Card, R., Mazzei, M., Bandecchi, P and Banks, M. (2006) Phylogenetic study of
swine and human hepatitis E virus. Vet Res. Comm. 30: Suppl. 1: 273-276.
Abstract
Hepatitis E is an enterically transmitted viral hepatitis caused by an RNA virus, hepatitis E virus (HEV) first
described in 1984 (Kane et al., 1984). Infection is endemic in many developing countries where seroprevalence is
as high as 25%. Industrialised countries were considered free from this infection and of the few cases that were
reported, almost all related to people who had travelled in endemic areas. However the seroprevalence in human
developed world populations can be high considering the low level of clinical disease. In the last few years there
have been increasing numbers of reports of patients with hepatitis E who had not travelled to endemic regions
(Clemente-Casares et al., 2003; Zanetti et al., 1999). HEV antibodies were detected in several animal species
both in developing and developed countries, suggesting that these animals could be infected by HEV or HEVrelated viruses (Meng., 2000). The presence of hepatitis E antibodies and RNA in swine in Nepal was reported in
1995 (Clayson et al., 1995), and in 1997, sequence data was published indicating that the swine virus was closely
related to the human HEV (Meng et al., 1997). To date, four different HEV genotypes have been identified in man
(I, II, III and IV ) and two in animals (III and IV) and a very high degree of RNA and amino acid sequence similarity
between swine and human isolates from the same geographical area was noticed (Schlauder et al., 1999; Meng
et al,. 2000; Banks et al., 2004), suggesting a possible interspecies transmission. In this paper we report the
results of a study where we compare nucleotide sequences of an ORF-2 fragment from swine and human HEV
isolates.
References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
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The following publications resulted in whole or in part from the work of SE0522
Banks, M., Bendall, R., Grierson, S., Heath, G., Mitchell, J., & Dalton, H. (2004) Human and porcine
hepatitis E virus strains, United Kingdom. Emerging Infectious Diseases 10, 953-955.
S Ijaz, E Arnold, M Banks, RP Bendall, ME Cramp, R Cunningham, HR Dalton TJ Harrison, SF Hill, L
MacFarlane, REE Meigh, S Shafi, MJ Sheppard, J Smithson, MP Wilson and CG Teo. (2005) Non travelassociated hepatitis E in the UK: demographic, clinical and molecular epidemiological characteristics. J.
Inf. Dis. 192, 1166-1172.
Banks, M. (2004) Hepatitis E: an emerging threat? Pig Journal, 54, 179-184
Tolari, F., Chiaro, LD., Card, R., Mazzei, M., Bandecchi, P and Banks, M. (2006) Phylogenetic study of
swine and human hepatitis E virus. Vet Res. Comm. 30: Suppl. 1: 273-276.
Presentations:
Hepatitis E virus in UK pigs – Presented to the South West Gastroenterology Annual Conference,
October, 2003
Hepatitis E: An emerging threat. Presented to the Pig Veterinary Society, May 2004
Hepatitis E Presented at the IAH, Pirbright, September 2004
Hepatitis E – A zoonosis? Presented to the East of England Zoonosis Forum, October 2004
Hepattiis E: The pig perspective – Presented to the South West Zoonosis Group, November 2004
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