Purpose of Visit
The aim of this work is to define whether there are differences in target cells that
become infected with PRRSV, and to dissect the immunological host responses at
both transcriptional and translational levels. Immunological responses in lungs, blood
and lymphoid tissues of pigs infected with several PRRSV strains with a different
clinical outcome, including subclinical infections with European type PRRSV strain
Lelystad (Netherlands) and a recent Belgian isolate, alongside strains with overt
clinical signs, a European type PRRSV strain LENA (Belarus). With this research it is
aimed to find an explanation for the higher virulence of some strains. The work
completed at the Central Veterinary Institute (CVI), The Netherlands, makes up part
of a larger EU project (PoRRSCon) which is being split between three separate sites,
the CVIthe Veterinary Laboratories Agency, UK, and the Shanghai Veterinary
Research Institute, China. The purpose of the visit to the CVI was not only to help
process the samples and carry out immediate lab work but to observe if any
problems arose that could be addressed before the experiment started at the VLA. It
was also important to observe laboratory methods to ensure that they are kept as
consistent as possible throughout the different sites.
Description of work carried out during visit
Three European strains of PRRSV were selected, Lena, a highly pathogenic Belarus
strain, a recent field isolate from Belgium (07V063) and the European reference
strain Lelystad virus, which will be used across all three sites to act as a control. For
each virus strain, and the uninfected control group, sixteen randomly selected male
pigs were used, four piglets from one sow were spread between each group, to
provide an even genetic background. Each pig was inoculated intranasally with 105
TCID50 of the respective virus, with controls inoculated with PBS. The animals were
clinically scored and had temperatures taken daily. Oropharyngeal samples were
taken at days 0,1,2,3,4,5,6,7,10,14, 21, 26 and 33, the swabs were proceesed and
the samples stored at -80°C for analysis of viral load by qPCR. Blood samples were
taken at days 0, 3, 5, 7, 10, 14, 21, 26 and 33. EDTA blood was used for automated
cell counts, non-treated blood was used to collect serum which was aliquoted and
stored at -80°C for later analysis, blood was taken into paxgene tubes and frozen at 20°C, finally heparin-preserved blood was taken and used for the isolation of
peripheral blood mononuclear cells (PBMCs). PBMCs were phenotyped by flow
cytometry, and used for quantification of PRRSV and aujeszky’s disease virusspecific IFN-γ responses by ELISpot assay. PRRSV Ab were determined in serum by
ELISA. At 3, 7 and 35 dpi, four pigs (eight at day 35, to include four vaccinated at
days 7 and 21) were euthanized and tissues collected for a full histopathological and
virological screening as well as tissues to be examined for host gene expression by
microarray.
Description of main results
PRRSV Antibodies
All animals in the trial were negative for PRRSV Ab before infection and the
control group remained negative throughout. All groups were positive for
PRRSV Ab by day 10 post-infection with levels peaking at day 21.
PRRSV Ab
2.5
Lena
Belgium 07V063
Leystad Virus
Control
S/P Ratio
2.0
1.5
1.0
0.5
33
26
21
14
7
10
-0.5
5
3
0
0.0
d.p.i
Figure 1. Detection of PRRSV-specific Ab by IDEXX ELISA, in serum from
pigs infected with Lena, Belgium 07V063, the Lelystad virus strains of PRRSV
and uninfected controls. An S/P value of >0.4 is considered as a positive for
PRRSV Ab. Mean data for each group is presented and error bars represent
the SEM.
PBMC Phenotyping and IFN-γ Responses.
Results were obtained from flow cytometry with two sets of triple staining with
the
following
monoclonal
antibody
panels;
CD3/CD4/CD8
and
SWC1/SWC3/SWC8. These stains provided data on populations of memory
and naive CD4 T cells, γδ-T cells, CD8+ cytotoxic T cells, natural killer cells,
neutrophils, monocytes and B cells. The IFN-γ ELISpot was performed with
PBMCs plated and stimulated with media, ConA, the PRRSV strain which the
pig was originally infected with and the Aujeszky’s disease virus reference
strain (NIA-3). The data from both the phenotyping of PBMCs and the ELISpot
assays are currently being analysed.
Production of Samples for Later Analysis
The animal experiment has produced numerous samples which have been
stored as it was impossible to perform all analysis during the 2 month visit.
Virus load and distribution is being quantified in tissues, serum and
oropharyngeal swabs by qPCR, those samples which are positive will then be
used to perform virus isolation to determine the amount of infectious virus.
Tissue samples will also be stained for PRRSV antigen.
The humoral
immune response to secondary infection will be determined by looking at
levels of Aujeszky’s disease virus-specific Ab in serum by ELISA. Cytokine
profiles will be determined for pro-inflammatory cytokines (IFN-α, IL-1, IL-6 and TNFα) and other relevant cytokines (including IL-10) by qPCR. Protein levels may be
investigated in those samples which display changes in expression levels. RNA
extracted from lymphoid tissues will be analysed by microarray to assess the
transcription level of different genes during infection. Differences in gene expression
between PRRSV strains may explain different viral replication kinetics related to
virulence. Expression profiling will be performed within the UK Centre for Functional
Genomics in Farm Animals (ARK-Genomics) based at Roslin. With this information
relevant transcripts and pathways will be re-examined in the animal tissues with in
situ hybridisation and immunohistochemistry.
Alongside the data that was produced, or will be produced from analysis of the
samples which were collected during the experiment, the visit has allowed me to
become accustomed to the laboratory work which we will be aiming to replicate in the
experiment at the VLA. Also it has allowed us to identify areas of the original protocol
which may benefit from slight alterations, so that further experiments can run more
smoothly.
Future Collaboration with host institute
Collaboration with the host institute will carry on through the EU PoRRSCon project,
with the possibility of staff members from the CVI coming to the VLA to help with our
experiment. Also the CVI has invited me back to help with analysis of the samples
obtained as well as to perform any ex vivo experiments we may be interested in
performing.
Projected publications
The results of the experiment described above will be published the coming
year..
Confirmation of successful completion of mission
Sophie Morgan assisted during the animal trial at the CVI for the PoRRScon
project. The experiment was very successful and the collaboration with
Sophie was perfect. During the course of the experiment there was an
excellent exchange of knowledge. We would be happy to continue this
collaboration with the VLA.