short-term scientific mission - European Network for Environmental

For the attention of the COST Action 929 “A European Network for
Environmental and Food Virology” STSM coordinator
Short Term Scientific Mission, COST 929
Reference code:
Host’s name and Institute: National Institute of Public Health and the
Environment (RIVM), Bilthoven, Netherlands
June 4th – June 8th 2007
First Name: IOANNA
Telephone: 00302551030546
Fax: 00302551030505
Introduction and purpose of the visit
Numerous viruses can be found in the human intestinal tract, but the viruses that
are most commonly transmitted by food are noroviruses and hepatitis A virus.
Various types of food have been implicated in outbreaks of viral disease e.g.
shellfish, vegetables, fruits. Foods served raw can be a transmission risk for
viruses: vegetables destined to the market are harvest and field packed into retail
containers without being washed. Such products are often eaten raw or with
minimal processing and if contaminated with pathogenic enteric viruses may
represent a hazard for the health to the consumers. The control of the presence of
the viruses plays an important role in rending vegetables safe for human
consumption. The importance of foodborne transmission in outbreaks of viral
disease due to consumption of vegetables is increasingly recognized. Moreover in
2003, ECDC notified to the EU Rapid Alert Systems the possible presence of
noroviruses in vegetables harvested around Europe.
To improve microbial monitoring of food quality and to assess the real role of food
in viral transmission, standardized and validated methods need to be developed.
Although significant progress has been made in detection of enteric virus in
shellfish, much needs to be done for other foods before this goal can be reached.
Puprose of the specific STSM was the acquisition of experience with the
techniques used for detection of noroviruses in food samples and also the
disemmination of knowledge of this technology to colleagues to the home Institute.
Description of the work carried out during the visit
The short-term mission of Mrs Ioanna Alexandropoulou (Democritus University of
Thrace) has been performed at the RIVM,in Netherlands. During this visit, Mrs
Alexandropoulou acquired the basic knowledge and technologies for the molecular
detection of Norovirus contamination of vegetables.
The general accomplishment of Mrs Ioanna Alexandropoulou scientific mission
was to define a standardised procedure to detect enteric viruses in vegetables and
improve her knowledge on the automatic molecular biology-based systems for
qualitative and quantitative detection of enteric viruses (Real-Time PCR).
In particular, Mrs Ioanna Alexandropoulou evaluated systems to extract and
concentrate Noroviruses from vegetables, and to eliminate the presence of RTPCR inhibitors in this type of food.
Moreover, she has performed different detection strategies (RT-PCR, Real-Time
RT-PCR) to define the effectiveness of the different techniques to detect
quantitative enteric viruse in vegetables.
During the first three days, Mrs Ioanna Alexandropoulou performed elution and
concentration methods to detect Noroviruses in vegetables. She acquired the
methods for nucleic acid extraction from vegetables and faeces samples. The
methods consisted of removal of virus from vegetables by beef-extract glycine,
then removal of residual food debris by centrifugation, followed by PEG
concentration of virus particles.
The extraction and purification of viral RNA was done by magnetic silica method
(according proposed CEN/TAG4 method), different RNA concentration and
purification methods to remove RT-PCR inhibitors from vegetables and human
The efficiency of the extraction was estimated by qualitative determination using
RT-PCR methods and quantitative determination using Real Time PCR method.
In particular she applied two step Reverse Transcription Polymerase Chain
Reaction (RT-PCR). The amplicons for RT-PCR were visualised by agarose gel
electrophoresis and were confirmed by Southern blot analysis.
During the next two days, she compared different protocols for the developed
quantitative method by Real Time RT-PCR. Primers and probe were used
according to some authors. She has also used one-step RT Real Time PCR. This
is a rapid and simple method which allows detection of RNA viruses in one step in
the same reaction tube. This was facilitated through heat extraction of viral RNA,
and the use of the thermostable enzyme which could perform reverse transcription
and cDNA amplification in the same buffer system (rTth). Genogroup II
Noroviruses fecal suspension was used for contamination of vegetable samples.
Twenty five grams of vegetables were spiked with serial diluitions of fecal
suspension to obtain final concentration of 2x103, 2x102, 2x101 RTPCR /100l.
Genogroup II Noroviruses fecal suspension was used as positive control. Each
contamination was repeated two times.
Samples were extracted by glycine-beef extraction and by PEG concentration.
Each pellet was resuspended in 500μl of phosphate buffer saline (PBS). The final
extracts were used for extraction of viral RNA with magnetic silica column
purification. One step RT Real Time RT-PCR were performed on each dilution.
The amplifications of the DNA target obtained by Real Time RT-PCR were
measured in terms of the increment in the quantity of fluorescence determinated at
the end of each amplification cycle. The resuls were visualized using the software
sequence detector 1.7 provided with tthe ABI Prism 7700 system (Applied
BioSystem), and the standard curve was confirmated by using Microsoft Excel
The results of the tests preformed on vegetables experimentally infected with
concentration of NoV ranging from 103 to 101 RTPCR /100l showed that the
different elution and concentration methods to detect Noroviruses in vegetables
were achieved in each replicate experiment.
In the future this knowledge, transferred to the lab in Greece, will help to study
several aspects of the disease and will be useful to investigate the presence of
enteric virus in vegetables. So far, vegetables are not controlled for viruses in
Greece, so in cooperation with the Ministry of Agriculture, a cooperated project
could start in the future.
Description of the main results obtained
The STSM has improved the skills of I. Alexandropoulou, permitting to improve her
knowledge on molecular biology.
In particular, she developed her competence in detection of NoV in vegetables.
The techniques acquired have been mainly:
1. Application of elution and concentration methods to detect Noroviruses in
2. Application of different RNA concentration and purification methods to detect
Noroviruses in vegetables and human faeces.
3. Qualitative determination of NoV RNA in vegetables and human faeces, using
RT-PCR and Real Time PCR methods.
4. Quantitative determination of NoV RNA in vegetables and human faeces, using
Real Time RT-PCR methods.
Future collaboration with the host institution
The RIVM has been chosen as host institution because it is the most important
public health institution in the Netherlands involved in the problems correlated to
the presence of viruses in vegetables. The host laboratory also has a wide
experience in standardised procedures to detect enteric viruses in vegetables by
using molecular methods.
Thanks to the STMS, a collaboration between the research groups in RIVM and
Democritus University of Thrace has already started, and this relationship will lead
to an exchange of know-how, materials, specimens and techniques.
I would be pleased to provide further information if requested to do so.
Date: 8 June 2007
Signature : Ioanna Alexandropoulou
I enclose the Confirmation by the host institute of the successful execution of the