Dissecting and disrupting the interactions between host cells and the porcine reproductive and respiratory syndrome virus

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.