SCIENTIFIC REPORT OF THE COST-STSM-929-04468 BETWEEN INIA
(MADRID, SPAIN) AND RIVM (BILTHOVEN, THE NETHERLANDS)
1.-Background
Hepatitis E virus (HEV) is an enterically transmitted RNA virus. It is a common
cause of large waterborne epidemics and sporadic hepatitis in adults in developing
countries with poor sanitary conditions, but evidence increases that also in
industrialized countries locally acquired HEV infections occur.
The HEV is a spherical, non-enveloped viral particle which genome is a single-stranded
positive sense RNA molecule of 7.2 Kb containing 3 open reading frames (ORFs) and a
3´poly A tail.
HEV has been classified in 4 genotypes, all of them infect humans. In general,
when viruses of genotype 3 and 4 have been found in swine and human from the same
geographic region, they are genetically very closely related to each other. Swine HEV
strains in Europe are mainly of genotype 3. Due to the potential risk of zoonotic
transmission, it is important to establish the prevalence of Hepatitis E in the European
swine herds.
Since there is no reliable in vitro (cell culture) infectivity assays, although there
have been recent reports of limited success in these areas, and conventional RT-PCR
assays for HEV are not broadly reactive and are often less sensitive than fluorescence
based real-time RT-PCR assays, this is an efficient method for the detection and
quantification of HEV RNA.
On the other hand, fecal samples are notorious for the presence of substances
that may inhibit PCR, indicating that PCR performance might vary between samples.
To avoid failure of the PCR reaction due to inhibition, an internal quality assurance
system needs to be established, especially when PCR is applied diagnostically to agents
such as HEV that can be found in low concentrations in animal feces. Standardization of
fecal testing of food production animals has recently been included in the program of
the International Organization for Standardization and the European Committee for
Standardization to prepare standardized protocols for the detection of zoonotic agents
by PCR-based methods.
The aim of this COST-STSM, as a collaborative project between the laboratory
of Zoonotic and Environmental Virology (Dpt. Biotechnology, INIA, Madrid, Spain)
and the Laboratory for Zoonoses and Environmental Microbiology (RIVM, The
Netherlands), was to be trained on HEV RNA-detection by real-time RT-PCR, as well
as to be trained on the detection and assessment of RNA internal controls, available at
the host institute.
2.- Activities done at the RIVM
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During my stay at the centre, I got a basic real-time PCR lab training on HEV
using a panel of well characterized samples with the RIVM´s methodology. This
training also allowed me to get familiarized with the procedures and techniques
applied at the RIVM.
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Next, I was trained in the production and assessment of the internal controls,
using the protocols designed at the RIVM, in order to include them in diagnostic
assays to reduce the number of false negative results in the HEV PCR-based
detection technique. These internal controls are going to be used at the INIA
with our own samples according to the RIVM procedures, to compare the results
between standard RT-PCR protocols and the real time methodology applied at
the RIVM.
As expected, to visit the RIVM allowed me to be trained on HEV RNA detection by
real-time RT-PCR, taking advantage of the extensive experience of RIVM, one of the
most important European Institution in the field. COST-STM aims have been
successfully accomplished to a reasonable extent and new tight links have been
established between COST labs. This work has been a great opportunity to develop my
skills at these techniques. It has also been very good to get familiarized with different
laboratory conditions.
All these reasons have made of this short term stay a very good opportunity to
improve in my scientific training.
Ana Belén Blázquez Martín
Laboratory of Zoonotic and Environmental Virology
Dpt. Biotechnology
INIA
Madrid (Spain)