COST STSM – Final Report
Stelian BARAITAREANU
doruvet@yahoo.com
COST Action number: FA0902
STSM Topic: Training in PRRS virus isolation techniques for the Romanian
PRRSV strains study
COST STSM Reference Number: ECOST-STSM-FA0902-060611-008024
Beneficiary’s Name and Institution: Stelian Baraitareanu, University of Agronomical
Science and Veterinary Medicine Bucharest, Romania
Host’s Name and Institution: Jean-Pierre Frossard, AHVLA Weybridge
Period: from June 06, 2011 to August 28, 2011
Place: Animal Health and Veterinary Laboratories Agency, Weybridge, England
(United Kingdom)
COST Office Science Officer:
COST STSM Manager:
• Background:
The identification methods of PRRS agent recommended by the last version of the Manual of Diagnostic
Tests and Vaccines for Terrestrial Animals in May 2010 are split into three groups of techniques: virus
isolation, the detection of nucleic acids, and the detection of viral proteins.
Recently several Romanian laboratories were focused on developing the PCR techniques and,
unfortunately, the viral isolation methods implementation was abandoned. In this context the number of
specialists and laboratories able to perform all viral techniques is decreasing, and few European
laboratories are currently able to perform the PRRS virus isolation technique. The exchange visit is aimed
to learn PRRS diagnostic techniques, including virus isolation.
• Purpose of the STSM:
The aim of this training was to (i) learn and understand the basis of good laboratory practice and biosafety
in virology laboratories; (ii) identify and learn about the equipment, materials and other tools used in
PRRS virus isolation techniques; (iii) participate at the standard operating procedure of alveolar
macrophages harvesting from pig lungs and testing of pulmonary alveolar macrophages batch; (iv)
identify and aliquot the samples used for PRRS virus isolation; and (v) learn and understand the virus
isolation on alveolar macrophages, and molecular diagnostic and characterisation (PCR techniques,
sequencing) of PRRSV isolates.
• Description of the work carried out during the STSM:
The work carried out during the STSM was in concordance with procedures performed by host institution
specialists and it covered the methodology approved for this training.

The basis of good laboratory practice and biosafety in virology laboratories
Theoretical study:
- Working procedures and disposal of waste from the Mammalian Virology Group SAPO cat 0 to
2 labs
Practical works:
- Working with protective wear (laboratory coats, gloves, visors, glasses, face mask.
- Identifying the nominated specialised rooms
- Maintenance and room cleaning activities
- Disposal of non-infectious and infectious waste

Equipment, materials and other tools used in PRRS virus isolation techniques
Theoretical study:
- Working procedures with microbiological safety cabinet, fume hood, incubators, centrifuge,
balance, pipettes, water baths, liquid nitrogen cryostore, x-ray developer
Practical works:
- Working with pipettes, microbiological safety cabinet, centrifuge, incubators.

Harvesting of alveolar macrophages from lungs
Theoretical study:
- Standard operating procedures for preparation of primary lung macrophage cells in CTCS
- Standard operating procedures for general cell culture practice
- Standard operating procedures for cryopreservation of cells in liquid nitrogen
- VLA Health & Safety Policy
Practical works:
- Alveolar macrophages harvesting from lungs of 5 week-old pigs for preparation of primary lung
macrophage cells.
- Alveolar macrophages harvesting from PRRSV infected pig lungs (35 d.p.i.) for pathogenesis
research studies.

Batch testing of alveolar macrophages
Theoretical study:
- Standard operating procedures for counting of cells
- Standard operating procedures for assessment of cell cultures
- Standard operating procedures for cryopreservation of cells in liquid nitrogen
- Standard operating procedures for resuscitation of cells from liquid nitrogen
- VLA Health & Safety Policy
Practical works:
- Test PLM batch for sensitivity to PRRS virus
- Test PLM batch for diagnostic submissions

Samples used for PRRS virus isolation
Theoretical study:
- Standard operating procedures for biosafety in virology laboratories
- Standard operating procedures for PRRSV sampling
- Standard operating procedures for aliquot of oral samples (cotton rope)
- Standard operating procedures for processing and aliquot of oro-pharingeal liquid samples
- Standard operating procedures for processing and aliquot of serum end plasma samples
Practical works:
- Aliquot of oral samples (cotton rope) received from the clinical study experiment of PRRS
(swine experimental infection).
- Processing and aliquot of oro-pharingeal liquid samples (cotton swabs) received from the
clinical study experiment of PRRS (swine experimental infection).
- Processing and aliquot of serum end plasma samples received from the clinical study experiment
of PRRS (swine experimental infection).
- Decontamination of cabinets and disposal of biological contaminated material

Virus isolation on alveolar macrophages
Theoretical study:
- Standard operating procedures for PRRS immunoperoxidase monolayer assay
- Standard operating procedures for PRRS immunoperoxidase monolayer assay plate production
using MARC 145 cells
-
Standard operating procedures for isolation of PRRSV from serum or tissues, using pig lung
macrophage cultures
.1. Seeding macrophages in the microtitre plates
Practical works:
- Defrost the vials, washing the PLM cells with PBS, collecting in RPMI 1640 medium, and
dispensing into microtitre plate.
.2. Preparation of sample
Practical works:
- Dispensing growth medium in microtitre plates wells, adding the samples, and performing
dilutions in dummy plates
.3. Incubation of samples
Practical works:
- Incubate the microtitre plates with samples for 2–5 days and daily observation for CPE.
- Perform the second passage
.4. Reading and interpreting the results
Practical works:
- Examine cultures for cytopathic effects and contamination.
.5. Immunostaining with a PRRSV-positive antiserum or MAb
Practical works:
- Wash, dry, fix and stain the plates infected with different PRRSV strains.

PCR techniques and molecular characterisation (sequencing) of PRRSV isolates
Theoretical study:
- Standard operating procedures for biosafety in virology laboratories
- Laboratory precautions for using PCR in diagnostic testing
- ABI data collection software manual
- ABI Sequence Analysis software manual
- CSU DNA template – Clean un and Quantification
- CSU ABI genetic Analyser Operation – Use and Maintenance
- Samples preparation for PRRSV PCR (ORF5 and ORF7)
- Standard operating procedures for PRRSV detection in serum an tissue using TaqMan PCR
- Standard operating procedures for CSU ABI genetic Analyser – sample preparation
Practical works:
- Handling and storage of samples
- Samples preparation for PRRSV nucleic acid sequencing (ORF7) by CSU ABI genetic Analyser
- Interpretation of RT-PCR, q-PCR and sequencing results.
• Description of the main results obtained:
The training stage has been started with a brief theoretical training course about basis of good
laboratory practice, biosafety in virology laboratories, and description of equipment, materials and other
tools used in PRRS virus isolation techniques. Several exercises with protective wear, pipettes,
microbiological safety cabinets and centrifuges have been daily performed. Skills in safety practice and
appropriate risk assessments for these procedures were improved.
PRRS virus is a fastidious agent which can be propagated on primary cultures of porcine
alveolar macrophages (PAM) [Wensvoort et al., 1991]. Also, PRRSV (North American isolates) was
achieved in the continuous cell lines ATCC VR-2332 and MARC-145 [Bautista et al., 1993; Benfield et
al., 1992; Kim et al.,1993]. However, some isolates could be readily cultured in PAM, and others in
continuous cell lines [Bautista et al., 1993; Kim et al., 1993]. In this context, it has been studied the
procedures of alveolar macrophages harvesting from pig lungs, preparation of primary lung macrophage
cells, and preparation of MARC 145 cells continuous cell lines. Also, it was performed batch testing for
sensitivity and diagnostic purpose of those cell cultures. As a result of training, skills in cell culture
practice were improved.
One of the main problems in PRRSV diagnostics is the quality of sampling. Not all samples are
the entire time positive during the virus infection, and the clinical trials and experimental infections still
test the quality of different samples. In this training stage, different types of biological samples were
processed: oral samples collected by cotton rope, oro-pharingeal liquid samples collected by cotton
swabs, pulmonary lavage, lung and spleen tissue for molecular biology investigations, and serum and
plasma samples received from a clinical study of PRRS experimental infection. Processing and
aliquoting of different types of biological samples were followed by different methods of diagnostis
(immunology, virology and nucleic acid detection). The practical activities improved the skills in PRRS
diagnostic techniques.
Virus isolation on alveolar macrophages and real-time PCR diagnostic methods were used to
correlate the qPCR initial quantity copies and infectious particle values obtained by virus titration on pig
lung macrophage cultures in serum samples collected from pigs experimentally infected with the PRRSVH2 strain. In this context, fifteen serum samples PRRSV-H2 strain positive were selected from the
AHVLA Weybridge cryogenic bank. This selection has been based on previous information about virus
load using a semi-quantitative method (number of positive wells in six replicates) of virus isolation on
PAMs. All samples were selected on the basis of positive results and the variation of semi-quantitative
results (from 1 to 6 positive PAM cell wells). PRRSV Lelystad strain has been used as positive control in
virus titration and to make standard samples (six logarithmic dilutions) for qPCR.
All 15 serum samples collected from pigs experimentally infected with PRRSV-H2 strain were
positive in the microplate cell cultures used for virus titration.
Fig. 1 PRRS virus identification by immunoperoxidase monolayer assay. Pam’s cytoplasm is deeply red
and cytopathic effect is observed.
All 15 serum samples collected from pigs experimentally infected with PRRSV-H2 strain have
yielded positive RT-PCR results. The RNA initial quantities (copies) included in qPCR have been
calculated by MCPro v.4.10 software with equation Ct = -3.821*LOG(copies) + 39.30 (fig. 2). This
equation has been obtained by regression analysis of standard samples. All values have been between
3,700 and 37,000,000 copies/sample.
Fig. 2. Real-Time PCR Quantitation results of 15 serum samples collected from pigs experimentally
infected with PRRSV-H2 strain.
The sample correlation coefficient of RNA copies/ml and Infectious particles/ml has been
calculated to be 0.998. The strong correlation between qPCR and Virus Titration data suggest the
possibility to evaluate the infectious particle load in all remaining samples in the study by using only
qPCR. Also, it validates the use of qPCR to compare infectious particle load between samples, with a
biological relevance.
References
1.
2.
3.
4.
Wensvoort, G., C. Terpstra, J. M. A. Pol, and M. White. 1991. Blue ear disease of pigs. Vet. Rec. 128:574.
Bautista, E. M., S. M. Goyal, I. J. Yoon, H. S. Joo, and J. E. Collins. 1993. Comparison of porcine alveolar
macrophages and CL 2621 for the detection of porcine reproductive and respiratory syndrome (PRRS)
virus and anti-PRRS antibody. J. Vet. Diagn. Invest. 5:163-165.
Benfield, D. A., E. Nelson, J. E. Collins, L. Harris, S. M. Goyal, D. Robinson, W. T. Christianson, R. B.
Morrison, D. Gorcyca, and D. Chladek. 1992. Characterization of swine infertility and respiratory
syndrome (SIRS) virus (isolate ATCC VR-2332). J. Vet. Diagn. Invest. 4:127-133.
Kim, H. S., J. Kwang, I. J. Yoon, H. S. Joo, and M. L. Frey. 1993. Enhanced replication of porcine
reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line.
Arch. Virol. 133:477-483.
• Future collaboration with host institution:
-
Developing the virus bank of PRRSV strains circulating in Europe:
Cooperative Agreement between University of Agronomical Sciences and
Veterinary Medicine Bucharest and AHVLA Weybridge
• Foreseen publications/articles resulting or to result from the STSM
(if applicable):
The data obtained in research of qPCR and Virus Titration on Cell Culture Monolyers
correlation support the study of Dr. Tahar Ait-Ali and Dr Frossard, and will be included in a paper to be
published in the coming year.
• Confirmation by the host institution of the successful execution of the
STSM:
The scientific mission of Dr. Baraitareanu was completed successfully in all respects. His
contribution to the work of the laboratory, particularly in relation to the PoRRSCon EU-funded project,
was considerable. He was enthusiastic throughout his stay, interacting with scientists and veterinarians
from several departments, and making full use of the facilities and resources available at AHVLA. The
links established during this exchange will ensure that future collaboration between the two institutes will
be readily undertaken. Future diagnostic and research efforts in the area of PRRSV in Romania will
therefore benefit from the shared expertise of AHVLA scientists, as intended.