SHORT-TERM SCIENTIFIC MISSIONS REPORT
For the attention of the STSM coordinator of COST Action 929: «A European Network for
Environmental and Food Virology ENVIRONET»
REFERENCE:
Short Term Scientific Mission, COST 929
(Reference code: COST-STSM-929-02939)
Beneficiary: Tamas Petrovic
Host Institution:
RIJKSINSTITUUT VOOR VOLKSGEZONDHEID
EN MILIEU, BILTHOVEN,
THE NETHERLANDS (RIVM)
Period: from 28/05/2007 to 08/06/2007; place: Bilthoven - The Netherlands
FROM Name: First Names: Petrovic Organisation: Tamas Street: Scientific Veterinary Institute
"Novi Sad" (NIV-NS)
Town and Postcode: :Rumenacki put 20 Country: :21000 Novi Sad Telephone: Telefax:
:SERBIA E-mail Address: : +381 21 518 149
: +381 21 518 544 :
tomy@niv.ns.ac.yu
Final report of the activities performed
Introduction
Hepatitis E virus (HEV) is an enterically transmitted RNA virus from the family hepeviridae and causes liver
inflammation in humans. Hepatitis E virus is currently classified into four genotypes (consecutively named 1
to 4) and 24 subtypes, and the different genotypes have different geographical distributions. Genotype 1 and
genotype 2 strains are found in developing countries as major cause of outbreaks and sporadic cases of
hepatitis E. In Western countries, these strains are exclusively found in patients who have visited a developing
country. Genotype 3 and genotype 4 strains are found predominantly in Western countries as causes of
sporadic cases of locally acquired hepatitis E, which are increasingly observed in industrialized countries. The
origin of HEV in these locally acquired cases is mostly unknown, but a possible role for swine and swine
products has been suggested based on high prevalence of HEV among pigs, and on high similarity between
porcine and human HEV sequences from the same geographical region. Cases of foodborne transmission from
wild boar and deer to humans have been reported for genotype 3 strains in Japan. Hence, genotype 3 strains of
HEV have the potential to be transmitted via foods. Currently, definite evidence for zoonotic transmission
from domestic pigs or products to humans has not been reported. However, pigs were shown to be susceptible
to human HEV strains and primates were susceptible to porcine HEV strains. Furthermore, higher HEV
seroprevalence estimates for groups of veterinarians and swine farm workers compared to control groups were
observed and thus infection seems related to direct contact with pigs. Molecular detection of viral RNA does
not discriminate RNA of infectious viral particles from RNA of defective viral particles. In research focused
on infectious diseases, it is necessary to make that discrimination to estimate implications of detected viral
RNA for public health. Infectivity can be determined by cell culture, but routine cell culture methods are not
available for HEV. To examine whether swine are a source for human hepatitis E, methods such as for
concentration, extraction and detection are needed to be able to determine the presence of HEV in pig samples
such as feces, sera and pig products such as liver. And also the ability to analyse and compare the sequences of
HEV strains in foods enables the assessment of the risk of foodborne HEV transmission.
Objective of Short Term Scientific Mission
To compare methodology for different protocols used in both the Netherlands and Serbia for the extraction of
RNA from feces and liver and detection of HEV in these matrices. Moreover, the application of cell culture
methods for enterovirus and adenovirus in food virology was surveyed to provide information on the
infectivity of the virus which is not accomplished by molecular techniques.
Description of the work carried out during the visit
The short-term mission of Dr. Tamas Petrovic has been performed at the Health related Water and Food
Microbiology group at the RIVM Laboratory for Zoonoses and Environmental Microbiology. During this
visit, Dr. Petrovic acquired the basic knowledge and technologies for the detection of HEV in pig samples
and pig products. The general accomplishment of Dr. Petrovic's scientific mission was to compare
protocols for virus concentration, extraction and detection and improve the knowledge on cell culture
systems for enteric viruses in the environment. Dr. Petrovic evaluated developed two different elution and
concentration methods to detect enteroviruses in environmental samples (surface water). During the first
weak period, he also evaluated cell culture detection of enteric viruses in environmental samples. In
particular, Dr. Petrovic evaluated different systems to concentrate and extract HEV from pig feces and
liver, and to eliminate the presence of RT-PCR inhibitors. Moreover, he has evaluated different detection
strategies (RT-PCR, nestedPCR, Southern blot hibridization) to define the effectiveness of the different
techniques to detect HEV in pig feces and liver. In particular he applied Reverse Transcription
Polymerase Chain Reaction (RT-PCR). This is a rapid and simple method which allows detection of RNA
viruses in two step reaction. The amplicons were visualised by agarose gel electrophoresis. Detection of
specific product was confirmed by Southern blot hybridisation.
List of protocols
Dr Petrovic was familiarised with and has acquired the following protocols:
1. Extraction of RNA from pig feces and liver
2. RT-PCR detection of HEV in pig feces and liver
3. ELISA protocol for HEV antibody detection and specific details of producer
4. Maintaining the BGM cell culture
5. Isolation of enteroviruses on BGM cell culture
6. Maintaining the A549 cell culture
List of reagents
Dr Petrovic was familiarised with and has acquired the following reagents:
1. BGM cells cultures
2. A549 cells cultures
Description of the main results obtained
The STMS has improved the skills of Dr. Petrovic, permitting to improve his knowledge on methodology
for food virology. In particular, he developed competence in detection of HEV in pig samples and
products and also on cell culture techniques for other enteric viruses. The techniques acquired have been
mainly:
1
Application of elution and concentration methods to detect enteroviruses in environmental
samples as surface water.
2
Isolation of enteric viruses on cell culture systems
3
Application of different RNA concentration and purification methods to detect HEV in pig feces
and liver.
4
Determination of HEV RNA in pig feces and liver, using RT-PCR and Southern blot
hybridisation.
Future collaboration with the host institution
The Health related Water and Food Microbiology group at the RIVM Laboratory for Zoonoses and
Environmental Microbiology has been chosen as host institution because it is one of the most important
institution involved in the problems correlated to the detection of HEV and presence of viruses in
environmental samples. The host laboratory also has a wide experience in standardised procedures in
detection of enteric viruses in environment. Thanks to the STMS, collaboration between the research
groups in NIV-NS and RIVM has already started, and this relationship will lead to an exchange of
know-how, materials, specimens and techniques.
Date: 11 June 2007
I enclose the Confirmation by the host institute of the successful execution of the mission