S. Van Gucht

Correlations between lung proinflammatory cytokine levels, virus replication, and disease after swine influenza virus challenge of vaccination-immune pigs

During experimental infection of pigs with swine influenza virus (SIV), there is a strong temporal correlation between peak virus titers in the lungs, levels of different proinflammatory cytokines in bronchoalveolar lavage (BAL) fluids, and disease. Vaccination against SIV can greatly reduce or prevent virus replication after challenge and the resulting disease. Here, we took advantage of pigs from vaccination-challenge experiments, with different degrees of virological and clinical protection, to further correlate SIV replication with cytokines and disease. Forty-nine pigs were vaccinated twice with a commercial inactivated SIV vaccine or with experimental vaccines, and 35 control pigs were not vaccinated. Between 2 and 4 weeks after the last vaccination, all pigs were challenged intratracheally with SIV. Twenty-four hours after the challenge, we determined body temperatures, respiratory scores, lung virus titers, and neutrophils and cytokines in BAL fluids. Interferon-alpha (IFN-alpha), tumor necrosis factor (TNF-alpha), interleukin-1 (IL-1), and -6 (IL-6) were determined by bioassay, and IL-8 by a commercial ELISA. The results were analyzed for three comparison groups. The unvaccinated control pigs (group 1, n = 35) were positive for all or most parameters examined. Vaccinated pigs with challenge virus replication in the lungs (group 2, n = 28) had slightly lower virus titers than the challenge control pigs, and clear reductions in disease severity and mean titers of all five cytokines, but neutrophil numbers were not affected. Vaccinated pigs without detectable virus replication (group 3, n = 21) were largely protected against clinical signs and neutrophil infiltration. Mean levels of IFN-alpha, TNF-alpha, and IL-6, but not IL-1 or IL-8, were lower than in both other groups. Virus titers in the lungs of individual pigs showed highly significant correlations with IFN-alpha and IL-6, and lower correlations with TNF-alpha and IL-8. Clinical signs were most closely associated with IFN-alpha, IL-6, and TNF-alpha. The relationship between disease and IL-8 or IL-1 was much weaker. Our data provide further evidence for a role of IFN-alpha, TNF-alpha, and IL-6 in the pathogenesis of SIV. The similarities with cytokine profiles during human influenza virus infection are discussed.

Respiratory tract protection upon challenge of pigs vaccinated with attenuated porcine reproductive and respiratory syndrome virus vaccines

In this study, the efficacy of two attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccines was assessed. The virological protection in the lungs of vaccinated pigs upon challenge was studied. Also, challenged pigs were exposed to lipopolysaccharide (LPS) to evaluate clinical protection. Six-week-old pigs were immunized intramuscularly with commercial vaccines based on either an attenuated American or an attenuated European virus strain. Non-immunized pigs and pigs intramuscularly inoculated with the virulent Lelystad strain were included as controls. Six weeks after immunization, pigs were challenged either intratracheally or intranasally with the Lelystad strain, and 3 and 6 days later intratracheally exposed to Escherichia coli LPS. After LPS administration, pigs were monitored for clinical signs. At 4 and 7 days after challenge, pigs were euthanized to determine virus quantities in broncho-alveolar lavage (BAL) fluids and in lungs. Challenge virus was recovered from three out of eight pigs that had been primo-inoculated with the Lelystad strain with titers ranging between 0.3 and 3.1 log(10). Fifteen out of sixteen pigs vaccinated with the attenuated American strain were positive for challenge virus and their mean virus titers were similar to those of non-immunized challenge controls. Eleven out of 16 pigs vaccinated with the attenuated European strain were positive for challenge virus and their mean virus titers were 2.0-2.5 log(10) lower than those of non-immunized challenge controls. Thus, the virological protection in the lungs of vaccinated pigs upon challenge was incomplete, but was more pronounced in the homologous situation. Clinical signs upon LPS exposure in both vaccinated groups were not reproducible in two experiments.

Investigations of the efficacy of European H1N1- and H3N2-based swine influenza vaccines against the novel H1N2 subtype

The efficacy of a commercial swine influenza vaccine based on A/New Jersey/8/76 (Hi N1) and A/Port Chalmers/i /73 (H3N2) strains was tested against challenge with an HI N2 swine influenza virus. Influenza virus-seronegative pigs were vaccinated twice with the vaccine when they were four and eight weeks old, or with the same vaccine supplemented with an Hi N2 component. Control pigs were left unvaccinated. Three weeks after the second vaccination, all the pigs were challenged intratracheally with the swine influenza strain Sw/Gent/7625/99 (Hi N2). The commercial vaccine induced cross-reactive antibodies to Hi N2, as detected by the virus neutralisation (VN) assay, but VN antibody titres were 18 times lower than in the pigs vaccinated with the HIN2-supplemented vaccine. The challenge produced severe respiratory signs in nine of 10 unvaccinated control pigs, which developed high HIN2 virus titres in the lungs 24 and 72 hours after the challenge. Vaccination with the commercial vaccine resulted in milder respiratory signs, but Hi N2 virus replication was not prevented. Mean virus titres in the pigs vaccinated with the commercial vaccine were 1-5 logio lower than in the controls at 24 hours but no different at 72 hours. In contrast, the HI N2-supplemented vaccine prevented respiratory disease in most pigs. There was a 4-5 log10 reduction in the mean virus titre at 24 hours in the pigs vaccinated with this vaccine, and no detectable virus replication at 72 hours. These data indicate that the commercial swine influenza vaccine did not confer adequate protection against the H1 N2 subtype.

Porcine reproductive-respiratory syndrome virus infection predisposes pigs for respiratory signs upon exposure to bacterial lipopolysaccharide

Abstract This study examined whether an infection with porcine reproductive and respiratory syndrome virus (PRRSV) potentiates respiratory signs upon exposure to bacterial lipopolysaccharides (LPS). Five-week-old conventional pigs were inoculated intratracheally with the Lelystad strain of PRRSV and received 5 days later one or two intratracheal LPS administrations. The necessary controls were included. After LPS administration, pigs were intensively monitored for clinical signs. Additionally, some pigs were euthanatized after a second LPS administration for broncho-alveolar cell analysis and virological examinations of the lungs. Broncho-alveolar lavage (BAL) cells were counted and differentiated. Lung suspensions and BAL fluids were titrated for PRRSV. Exposure of pigs to PRRSV only resulted in a fever for time periods ranging from 1 to 5 days and slight respiratory signs. Exposure of pigs to LPS only resulted in general signs, characterized by fever and depression, but respiratory signs were slight or absent. PRRSV–LPS exposed pigs, on the other hand, developed severe respiratory signs upon LPS exposure, characterized by tachypnoea, abdominal breathing and dyspnoea. Besides respiratory signs, these pigs also showed enhanced general signs, such as fever and depression. Lung neutrophil infiltration was similar in non-infected and PRRSV-infected pigs upon LPS exposure. PRRSV quantities were similar in lungs and BAL fluids of pigs infected with PRRSV only and PRRSV–LPS exposed pigs. These data show a clear synergism between PRRSV and LPS in the induction of respiratory signs in conventional pigs. The synergism was observed in 87% of the pigs. So, it can be considered as reproducible and may be used to test the efficacy of preventive and therapeutic measures.

Impact of caspase-1/11, -3, -7, or IL-1β/IL-18 deficiency on rabies virus-induced macrophage cell death and onset of disease

Rabies virus is a highly neurovirulent RNA virus, which causes about 59000 deaths in humans each year. Previously, we described macrophage cytotoxicity upon infection with rabies virus. Here we examined the type of cell death and the role of specific caspases in cell death and disease development upon infection with two laboratory strains of rabies virus: Challenge Virus Standard strain-11 (CVS-11) is highly neurotropic and lethal for mice, while the attenuated Evelyn–Rotnycki–Abelseth (ERA) strain has a broader cell tropism, is non-lethal and has been used as an oral vaccine for animals. Infection of Mf4/4 macrophages with both strains led to caspase-1 activation and IL-1β and IL-18 production, as well as activation of caspases-3, -7, -8, and -9. Moreover, absence of caspase-3, but not of caspase-1 and -11 or -7, partially inhibited virus-induced cell death of bone marrow-derived macrophages. Intranasal inoculation with CVS-11 of mice deficient for either caspase-1 and -11 or -7 or both IL-1β and IL-18 led to general brain infection and lethal disease similar to wild-type mice. Deficiency of caspase-3, on the other hand, significantly delayed the onset of disease, but did not prevent final lethal outcome. Interestingly, deficiency of caspase-1/11, the key executioner of pyroptosis, aggravated disease severity caused by ERA virus, whereas wild-type mice or mice deficient for either caspase-3, -7, or both IL-1β and IL-18 presented the typical mild symptoms associated with ERA virus. In conclusion, rabies virus infection of macrophages induces caspase-1- and caspase-3-dependent cell death. In vivo caspase-1/11 and caspase-3 differently affect disease development in response to infection with the attenuated ERA strain or the virulent CVS-11 strain, respectively. Inflammatory caspases seem to control attenuated rabies virus infection, while caspase-3 aggravates virulent rabies virus infection.

Lung Cell Tropism and Inflammatory Cytokine-Profile of Porcine Respiratory Coronavirus Infection

Knowledge about porcine respiratory coronavirus (PRCV) tropism was limited to morphological identification of target cells and controversial reports on replication in macrophages. This study aimed to clarify the lung cell tropism of a Belgian PRCV strain and to examine the lung profile of inflammatory cytokines for 15 days after intratracheal PRCV inoculation of gnotobiotic piglets. Until 5 days after inoculation, more than 50% of the PRCV-positive cells were type 2 pneumocytes, 30% bronchiolar epithelial cells, and 10% macrophages, as demonstrated by immunofluorescence stainings with specific cell markers. In vitro, PRCV productively infected primary porcine lung epithelial cells, but not porcine al- veolar macrophages. Bronchoalveolar lavage fluids of PRCV-inoculated pigs contained high levels of interferons (IFN-� , IFN-� ) and interleukin-6, moderate interleukin-12 and low tumour necrosis factor-� levels. Our results indicate that PRCV infects primarily type 2 pneumocytes and induces lower lung cytokine levels, compared to swine influenza virus or lipopolysaccharide-complicated PRCV infections.

MALT1 controls attenuated rabies virus by inducing early inflammation and T cell activation in the brain

ABSTRACT MALT1 is involved in the activation of immune responses, as well as in the proliferation and survival of certain cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, further promoting the expression of immunoregulatory genes. Deregulated MALT1 activity has been associated with autoimmunity and cancer, implicating MALT1 as a new therapeutic target. Although MALT1 deficiency has been shown to protect against experimental autoimmune encephalomyelitis, nothing is known about the impact of MALT1 on virus infection in the central nervous system. Here, we studied infection with an attenuated rabies virus, Evelyn-Rotnycki-Abelseth (ERA) virus, and observed increased susceptibility with ERA virus in MALT1−/− mice. Indeed, after intranasal infection with ERA virus, wild-type mice developed mild transient clinical signs with recovery at 35 days postinoculation (dpi). Interestingly, MALT1−/− mice developed severe disease requiring euthanasia at around 17 dpi. A decreased induction of inflammatory gene expression and cell infiltration and activation was observed in MALT1−/− mice at 10 dpi compared to MALT1+/+ infected mice. At 17 dpi, however, the level of inflammatory cell activation was comparable to that observed in MALT1+/+ mice. Moreover, MALT1−/− mice failed to produce virus-neutralizing antibodies. Similar results were obtained with specific inactivation of MALT1 in T cells. Finally, treatment of wild-type mice with mepazine, a MALT1 protease inhibitor, also led to mortality upon ERA virus infection. These data emphasize the importance of early inflammation and activation of T cells through MALT1 for controlling the virulence of an attenuated rabies virus in the brain. IMPORTANCE Rabies virus is a neurotropic virus which can infect any mammal. Annually, 59,000 people die from rabies. Effective therapy is lacking and hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protein involved in innate and adaptive immunity and is an interesting therapeutic target because MALT1-deregulated activity has been associated with autoimmunity and cancers. The role of MALT1 in viral infection is, however, largely unknown. Here, we study the impact of MALT1 on virus infection in the brain, using the attenuated ERA rabies virus in different models of MALT1-deficient mice. We reveal the importance of MALT1-mediated inflammation and T cell activation to control ERA virus, providing new insights in the biology of MALT1 and rabies virus infection.