| dc.description.abstract | Porcine Reproductive and Respiratory Syndrome (PRRS) is the most
economically important disease to the swine industry worldwide. PRRS
causes reproductive failure and abortion in sows and gilts and respiratory
disease, diarrhoea, lethargy and, in some cases, high fever and death. Costs
to producers are a result of piglet losses from still births and abortions, reduced
daily liveweight gain and deaths. The causative agent is the Porcine
Reproductive and Respiratory Syndrome virus (PRRSV) which is a positive
sense, enveloped RNA virus. There are two species of PRRSV: PRRSV-1
and PRRSV-2 both of which display a high level of genetic diversity as a result
of the high rate of mutation in PRRSV. An important consequence is that none
of the available vaccines can control either species or even heterologous
strains within a single subtype.
PRRSV has a tropism for cells of the monocyte/macrophage lineage but there
is a lack of agreement on the mechanisms through which PRRSV enters these
cells. This project focussed on the entry mechanisms of PRRSV. Work began
with an attempt to construct full-length infectious clones of PRRSV. Infectious
clones bring the benefit of knowing the exact sequence of a viral genome at
the beginning, the manipulation of the genome allows a researcher to assess
the effects of deliberate mutations on viral activity. Infectious clones can also
be constructed to include fluorescent proteins to track transfection efficiency
and infections using microscopy or flow cytometry. Two clones were made but
only one appeared to be producing infectious virus and this was at a very low
titre. By the time the successful clone was initially tested, I had to move on
with other areas of the project rather than optimising the protocols. However,
the different approaches demonstrated in this work provides improved
methods to construct PRRSV infectious clones. The lack of an infectious clone
to work with was negated through the use of wild type isolates and the labelling
of anti-PRRSV antibodies which were used in experiments where a secondary
fluorescent antibodies could not be utilised.
The entry pathway was thought to be clathrin-mediated, however, this
conclusion was based on one piece of evidence. The work reported here used
a panel of chemical and pharmacological inhibitors to perturb different
endocytic pathways of primary porcine alveolar macrophages (PAM). Results
of these experiments indicated that the two PRRSV-1 isolates tested were able
to infect cells despite the clathrin pathway being disrupted. In contrast, some
of the inhibitors known to target non-clathrin-mediated pathways significantly
diminished PRRSV infection. This research suggests for the first time that
PRRSV can enter PAM cells independent of clathrin-mediated endocytosis.
Taken together, my data suggest cell entry through macropinocytic/phagocytic
uptake, which may have an impact on both cell receptor use but also potential
antiviral strategies, such as drug treatments or selective breeding.
Several putative cellular receptors/attachment factors have been identified as
being important for PRRSV entry into host cells including; heparin, vimentin,
CD169 and CD151. However, knock-out/knock-down experiments have
demonstrated that they are not solely responsible for PRRSV entry. CD163 is
the only known essential receptor for PRRSV but, not as a surface binding and
entry receptor, its role is in fusion within the endosome, leading to un-coating
and endosomal escape. Thus, the PRRSV entry/binding receptor/attachment
factor is still unknown. As PRRSV initially targets PAM when infecting through
the oronasal route, it was hypothesised that any receptor/attachment factor
utilised by PRRSV must be either specific to M2-type macrophages and/or
highly expressed in PAM. Therefore, a panel of antibodies either raised
against PAM porcine molecules or antibodies predicted to bind to porcine
molecules (due to the sequence similarity) were tested in blocking
experiments. This work identified two antibodies targeting a cell surface
protein able to significantly reduce PRRSV infection. This cell surface protein
receptor has not previously been identified as a PRRSV entry mediator.
Further research is required to characterise the role, if any, of this cell surface
receptor in facilitating PRRSV infection. | en |
