Jean-Pierre Frossard

Increased pathogenicity of European porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine worldwide. Since its first emergence in 1987 the PRRS virus (PRRSV) has become particularly divergent with highly pathogenic strains appearing in both Europe and Asia. However, the underlying mechanisms of PRRSV pathogenesis are still unclear. This study sets out to determine the differences in pathogenesis between subtype 1 and 3 strains of European PRRSV (PRRSV-I), and compare the immune responses mounted against these strains. Piglets were infected with 3 strains of PRRSV-I: Lelystad virus, 215-06 a British field strain and SU1-bel from Belarus. Post-mortem examinations were performed at 3 and 7 days post-infection (dpi), and half of the remaining animals in each group were inoculated with an Aujeszkys disease (ADV) vaccine to investigate possible immune suppression resulting from PRRSV infection. The subtype 3 SU1-bel strain displayed greater clinical signs and lung gross pathology scores compared with the subtype 1 strains. This difference did not appear to be caused by higher virus replication, as viraemia and viral load in broncho-alveolar lavage fluid (BALF) were lower in the SU1-bel group. Infection with SU1-bel induced an enhanced adaptive immune response with greater interferon (IFN)-γ responses and an earlier PRRSV-specific antibody response. Infection with PRRSV did not affect the response to vaccination against ADV. Our results indicate that the increased clinical and pathological effect of the SU1-bel strain is more likely to be caused by an enhanced inflammatory immune response rather than higher levels of virus replication.

Incursion of RHDV2-like variant in Great Britain.

RABBIT haemorrhagic disease virus (RHDV) causes an acute, fulminating and generally fatal disease in the European rabbit ( Oryctolagus cuniculus ). It was first discovered in China in 1984 and then confirmed in the UK in 1992, at …

Porcine reproductive and respiratory syndrome virus: genetic diversity of recent British isolates.

Porcine reproductive and respiratory syndrome (PRRS) continues to be a significant problem for European pig producers, contributing to porcine respiratory disease complex, neonatal piglet mortality, infertility and occasional abortion storms. PRRS virus (PRRSV), a member of the arterivirus family with two defined major genotypes, has been shown to be quite genetically diverse. In the present study, genetic analysis of multiple gene regions of over 100 viruses isolated in Britain between 2003 and 2007 revealed that the diversity of British strains is now far greater than during the early 1990s. All isolates belong to genotype 1 (European). While some recent isolates are still very similar to early isolates, a wide range of more diverse viruses is now also circulating. Interestingly, some isolates were found to be very similar to a modified-live vaccine strain, and it is suggested that use of the vaccine has affected the evolution pattern of PRRS virus strains in Britain. Evidence of deletions in one viral gene, ORF3, and of genome recombination was also seen. A molecular clock model using the ORF7 sequences estimates the rate of substitution as 3.8 × 10(-3) per site per year, thereby dating the most recent common ancestor of all British viruses to 1991, coincident with the first outbreak of disease. Our findings therefore have implications for both the diagnostic and prophylactic methods currently being used, which are discussed.

Porcine reproductive and respiratory syndrome virus: Antigenic and molecular diversity of British isolates and implications for diagnosis

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease of pigs, caused by PRRS virus, a member of the Arteriviridae family. First seen in Britain in 1991, the disease continues to be a significant economic and welfare problem for pig producers. To date, only PRRSV genotype 1 has been found in Britain. At the genetic level, a considerable increase has been reported in the diversity of PRRS viruses isolated in Britain between 2003 and 2007, versus the early 1990 s. In this study, the diversity has been shown to extend to the antigenic level too, with potential consequences for diagnostic methods. Antigenic diversity was assessed using a panel of twelve monoclonal antibodies, only one of which reacted with all isolates tested. Nine diverse viruses were compared as potential antigens in immunoperoxidase monolayer assays, where each one produced quite different results for a common panel of sera. As a single virus is used in each diagnostic assay, results must therefore be interpreted cautiously. For a real-time RT-PCR assay, published oligonucleotide primer and probe sequences were evaluated against available genetic sequences of British and European viruses, and were re-designed where considerable mismatches were found. The multiplex assay incorporating these modified primers to detect genotype 1 and 2 PRRS viruses was then validated for use with diagnostic sera and tissues. As the increasing degree of diversity exhibited by British strains is mirrored in other countries, PRRSV will continue to provide an ongoing challenge to diagnosis at a global, as well as national level.

Proteome-wide screening of the European porcine reproductive and respiratory syndrome virus reveals a broad range of T cell antigen reactivity.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving and diversifying pathogen necessitating the development of improved vaccines. Immunity to PRRSV is not well understood although there are data suggesting that virus-specific T cell IFN-γ responses play an important role. We therefore aimed to better characterise the T cell response to genotype 1 (European) PRRSV by utilising a synthetic peptide library spanning the entire proteome and a small cohort of pigs rendered immune to PRRSV-1 Olot/91 by repeated experimental infection. Using an IFN-γ ELISpot assay as a read-out, we were able to identify 9 antigenic regions on 5 of the viral proteins and determine the corresponding responder T cell phenotype. The diversity of the IFN-γ response to PRRSV proteins suggests that antigenic regions are scattered throughout the proteome and no one single antigen dominates the T cell response. To address the identification of well-conserved T cell antigens, we subsequently screened groups of pigs infected with a closely related avirulent PRRSV-1 strain (Lelystad) and a divergent virulent subtype 3 strain (SU1-Bel). Whilst T cell responses from both groups were observed against many of the antigens identified in the first study, animals infected with the SU1-Bel strain showed the greatest response against peptides representing the non-structural protein 5. The proteome-wide peptide library screening method used here, as well as the antigens identified, warrant further evaluation in the context of next generation vaccine development.

Pathology and Virus Distribution in the Lung and Lymphoid Tissues of Pigs Experimentally Inoculated with Three Distinct Type 1 PRRS Virus Isolates of Varying Pathogenicity

Porcine reproductive and respiratory syndrome (PRRS) continues to be the most economically important disease of swine worldwide. The appearance of highly pathogenic PRRS virus (PRRSV) strains in Europe and Asia has raised concerns about this disease and initiated increased efforts to understand the pathogenesis. In this study, we have compared the pathology and the virus distribution in tissues of pigs experimentally inoculated with three different genotype 1 PRRSV isolates. Sixty 5-week-old pigs were inoculated intranasally with a) the Lelystad virus (LV), b) a field strain from the UK causing respiratory clinical signs (UK) or c) a highly pathogenic strain from Belarus (BE). Sixteen animals were mock-infected and used as controls. The animals were euthanized at 3, 7 and 35 days post-infection (dpi), and lung and lymphoid tissues collected for histopathological examination and PRRSV detection by immunohistochemistry (IHC). Histopathological lesions consisted of interstitial pneumonia with mononuclear cell infiltrates in the lungs, lymphoid depletion, apoptosis and follicular hyperplasia in the spleen, lymph nodes and tonsil and lymphoid depletion in the thymus. Porcine reproductive and respiratory syndrome virus was detected mainly in monocytes-macrophages. BE-infected animals showed the highest pathological scores and the highest presence of virus at 3 and 7 dpi, followed by the UK field strain and then LV. Moderate lesions were observed at 35 dpi with lesser detection of PRRSV by IHC in each infected group. The highly pathogenic BE strain induced more severe pathology in both lungs and lymphoid organs of pigs compared with the classic field isolate and the prototype LV. The increased severity of pathology was in correlation with the presence of a higher number of PRRSV-infected cells in the tissues.

Establishing Porcine Monocyte-Derived Macrophage and Dendritic Cell Systems for Studying the Interaction with PRRSV-1

Monocyte-derived macrophages (MoMØ) and monocyte-derived dendritic cells (MoDC) are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect myeloid cells, such as macrophages (MØ) and dendritic cells (DC). Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated MoMØ were stimulated with activators for classical (M1) or alternative (M2) activation. GM-CSF and IL-4 generated MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype, and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells toward PRRSV-1 infection.

From the field to the lab — An European view on the global spread of PEDV

Abstract Porcine Epidemic Diarrhea Virus (PEDV) is a member of the genus Alphacoronavirus, in the family Coronaviridae, of the Nidovirales order and outbreaks of porcine epidemic diarrhoea (PED) were first recorded in England in the 1970s. Intriguingly the virus has since successfully made its way around the globe, while seemingly becoming extinct in parts of Europe before its recent return from Northern America. In this review we are re-evaluating the spread of PEDV, its biology and are looking at lessons learnt from both failure and success. While a new analysis of PEDV genomes demonstrates a wider heterogeneity of PEDV than previously anticipated with at least five rather than two genotypes, biological features of the virus and its replication also point towards credible control strategies to limit the impact of this re-emerging virus.

Functional analysis of the porcine USP18 and its role during porcine arterivirus replication.

Emerging evidence places deubiquitylation at the core of a multitude of regulatory processes, ranging from cell growth to innate immune response and health, such as cancer, degenerative and infectious diseases. Little is known about deubiquitylation in pig and arterivirus infection. This report provides information on the biochemical and functional role of the porcine USP18 during innate immune response to the porcine respiratory and reproductive syndrome virus (PRRSV). We have shown that UBP gene is the ortholog of the murine USP18 (Ubp43) gene and the human ubiquitin specific protease 18 (USP18) gene and encodes a biochemically functional de-ubiquitin enzyme which inhibits signalling pathways that lead to IFN-stimulating response element (ISRE) promotor regulation. Furthermore we have demonstrated that overexpression of the porcine USP18 leads to reduced replication and/or growth of PRRSV. Our data contrast with the conclusion of numerous reports demonstrating that USP18-deficient mice are highly resistant to viral and bacterial infections and to oncogenic transformation by BCR-ABL, and highlight USP18 as a potential target gene that promotes reduced replication of PRRSV.

A comparative study of the local cytokine response in the lungs of pigs experimentally infected with different PRRSV-1 strains: upregulation of IL-1α in highly pathogenic strain induced lesions.

Porcine reproductive and respiratory syndrome viruses (PRRSV) show high genetic differences both among and within genotypes. Recently, several highly pathogenic PRRSV (HP-PRRSV) strains have been described. This study compares and characterizes the production of cytokines by pulmonary macrophages in pigs experimentally infected with four different PRRSV-1 strains: two low-virulent strains, Lelystad (LV) and a British field strain (215-06); a HP strain (SU1-bel) from Belarus and the attenuated vaccine strain DV (Porcilis(®) PRRS). Animals were clinically monitored and post-mortem examinations were performed at 3, 7 and 35 days post-infection (dpi). Lung samples were processed for histopathological and immunohistochemical studies by using specific antibodies against PRRSV, IL1-α, IL-6, TNF-α, IL-10 and IFN-γ. SU1-bel infected animals presented the highest mean scores for clinical observations, gross and microscopic lesions as well as for PRRSV expression compared with the other infected groups (p≤0.027). These animals displayed the highest expression of IL1-α at 7dpi, together with the highest score for lung pathology, whereas LV, 215-06 and DV inoculated animals only showed a transient enhancement in some of these cytokines. SU1-bel-infected pigs showed a positive correlation between the amount of PRRSV antigen and IL-1α expression (r=0.645, p<0.001). The highest expression of IL-10 was detected in 215-06-infected animals (p≤0.004), with a positive correlation with the numbers of virus-infected cells (r=0.375, p≤0.013). In conclusion, the HP-PRRSV SU1-bel strain replicated more efficiently in the lung of infected animals and induced a higher expression of IL-1α than the other PRRSV-1-infected groups, which may have played a key role in the onset of the clinical signs and interstitial pneumonia.