K. Van Reeth

In Vivo and In Vitro Interferon (IFN) Studies with the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

Some of the interactions between the porcine reproductive and respiratory syndrome virus (PRRSV) and the porcine interferon-alpha (IFN-alpha) system were studied. In a first experiment, it was shown that pretreatment of primary porcine alveolar macrophages (AMs) with recombinant porcine (rPo) IFN-alpha 1 resulted in significant reductions of PRRSV yield and numbers of antigen expressing cells. In a second experiment, sensitivity of PRRSV to IFN-alpha was confirmed in vivo. In pigs inoculated with porcine respiratory coronavirus (PRCV)--a potent inducer of endogenous IFN-alpha in the lungs of pigs--followed 2 days later by PRRSV--lung PRRSV titers were 1.7 to 2.9 log10 TCID50 reduced compared to those in singly PRRSV inoculated pigs. It was concluded therefore that PRRSV has a fairly good sensitivity to the antiviral effects of IFN-alpha. A third experiment documented that in vivo PRRSV infection generally does not affect PRCV-induced IFN-alpha production in the lungs of pigs. In addition, it was shown that the IFN-inducing capacity of PRRSV is at least 159 times lower than that of PRCV. This finding suggests that cells other than AMs may be responsible for IFN production in the lungs of pigs.

Review of influenza A virus in swine worldwide: a call for increased surveillance and research.

Pigs and humans have shared influenza A viruses (IAV) since at least 1918, and many interspecies transmission events have been documented since that time. However, despite this interplay, relatively little is known regarding IAV circulating in swine around the world compared with the avian and human knowledge base. This gap in knowledge impedes our understanding of how viruses adapted to swine or man impacts the ecology and evolution of IAV as a whole and the true impact of swine IAV on human health. The pandemic H1N1 that emerged in 2009 underscored the need for greater surveillance and sharing of data on IAV in swine. In this paper, we review the current state of IAV in swine around the world, highlight the collaboration between international organizations and a network of laboratories engaged in human and animal IAV surveillance and research, and emphasize the need to increase information in high‐priority regions. The need for global integration and rapid sharing of data and resources to fight IAV in swine and other animal species is apparent, but this effort requires grassroots support from governments, practicing veterinarians and the swine industry and, ultimately, requires significant increases in funding and infrastructure.

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.

Virological Surveillance and Preliminary Antigenic Characterization of Influenza Viruses in Pigs in Five European Countries from 2006 to 2008

This study presents the results of the virological surveillance for swine influenza viruses (SIVs) in Belgium, UK, Italy, France and Spain from 2006 to 2008. Our major aims were to clarify the occurrence of the three SIV subtypes – H1N1, H3N2 and H1N2 – at regional levels, to identify novel reassortant viruses and to antigenically compare SIVs with human H1N1 and H3N2 influenza viruses. Lung tissue and/or nasal swabs from outbreaks of acute respiratory disease in pigs were investigated by virus isolation. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined using standard methods. Of the total 169 viruses, 81 were classified as ‘avian‐like’ H1N1, 36 as human‐like H3N2 and 47 as human‐like H1N2. Only five novel reassortant viruses were identified: two H1N1 viruses had a human‐like HA and three H1N2 viruses an avian‐like HA. All three SIV subtypes were detected in Belgium, Italy and Spain, while only H1N1 and H1N2 viruses were found in UK and Northwestern France. Cross‐hemagglutination inhibition (HI) tests with hyperimmune sera against selected older and recent human influenza viruses showed a strong antigenic relationship between human H1N1 and H3N2 viruses from the 1980s and H1N2 and H3N2 human‐like SIVs, confirming their common origin. However, antisera against human viruses isolated during the last decade did not react with currently circulating H1 or H3 SIVs, suggesting that especially young people may be, to some degree, susceptible to SIV infections.

Influenza at the animal-human interface: A review of the literature for virological evidence of human infection with swine or avian influenza viruses other than A(H5N1)

Factors that trigger human infection with animal influenza virus progressing into a pandemic are poorly understood. Within a project developing an evidence-based risk assessment framework for influenza viruses in animals, we conducted a review of the literature for evidence of human infection with animal influenza viruses by diagnostic methods used. The review covering Medline, Embase, SciSearch and CabAbstracts yielded 6,955 articles, of which we retained 89; for influenza A(H5N1) and A(H7N9), the official case counts of t he World Health Organization were used. An additional 30 studies were included by scanning the reference lists. Here, we present the findings for confirmed infections with virological evidence. We found reports of 1,419 naturally infected human cases, of which 648 were associated with avian influenza virus (AIV) A(H5N1), 375 with other AIV subtypes, and 396 with swine influenza virus (SIV). Human cases naturally infected with AIV spanned haemagglutinin subtypes H5, H6, H7, H9 and H10. SIV cases were associated with endemic SIV of H1 and H3 subtype descending from North American and Eurasian SIV lineages and various reassortants thereof. Direct exposure to birds or swine was the most likely source of infection for the cases with available information on exposure.

Acute phase response in porcine reproductive and respiratory syndrome virus infection.

This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10 dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1β was observed at 7 dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection.

Efficacy of commercial swine influenza vaccines against challenge with a recent European H1N1 field isolate.

This study examines the immunogenicity and efficacy of four commercial swine influenza (SI) vaccines against challenge with a recent European H1N1 virus, Sw/Gent/112/07. The vaccines contained different H1N1 strains showing between 77% and 95% genetic homology with the haemagglutinin (HA) of the challenge virus. Four groups of 10 pigs each received a double vaccination, with a 4-week interval, with one of the vaccines; a fifth group served as unvaccinated controls. All pigs were challenged 3 weeks after the second vaccination intratracheally with 10(5.0)EID(50) of Sw/Gent/112/07. Sera were examined in haemagglutination inhibition (HI) tests against the homologous vaccine H1N1 strains, the challenge virus and a panel of five recent H1N1 isolates. Pigs were euthanized at 24 or 72h post-challenge and virus titres were determined in right and left lung halves. Two vaccines, in which the H1N1 strains showed a genetic homology of 93% and 89% to Sw/Gent/112/07, significantly reduced virus replication. The vaccine containing an H1N1 strain with 95% homology to Sw/Gent/112/07, did not offer significant protection, neither did it induce the highest HI titres. In general, pigs with HI antibody titres >or=20 against Sw/Gent/112/07 were virologically protected against challenge. HI titres against other viruses, however, differed compared to the challenge virus and between viruses. Our data clearly show that the genetic homology with the challenge virus is not the ultimate predictor for SI vaccine performance. The true reason for the differences in vaccine potency remains obscure because other factors, such as the antigen dose and/or the adjuvant, also differed between the vaccines.

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.

Clinical Effects of Experimental Dual Infections with Porcine Reproductive and Respiratory Syndrome Virus Followed by Swine Influenza Virus in Conventional and Colostrum‐deprived Pigs

Previous studies demonstrated that experimental dual infections of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) followed by H1N1 influenza virus cause more severe disease and growth retardation than the respective single virus infections. Here three experiments were undertaken to better define the clinical impact of combined PRRSV‐H1N1 infections in conventional and caesarean‐derived colostrum‐deprived (CDCD) pigs. Groups of pigs were inoculated by aerosol with PRRSV followed by H1N1 at 3‐, 7‐ or 14‐day intervals. During the post‐H1N1 period, mean body temperatures, respiratory signs and mean weight gains in the PRRSV‐H1N1 inoculated groups were recorded and compared with those in uninoculated controls (experiments 1 and 2) or in singly virus‐inoculated pigs (experiment 3). In a first experiment with conventional pigs, the PRRSV‐3d‐H1N1 and PRRSV‐7d‐H1N1 infections induced mean body temperatures >40.5°C during 8 days (peaks 41.1 and 41.6°C, respectively) and mean growth reductions of 3.4 and 4.8 kg, respectively, during the 2 weeks after H1N1, along with marked depression and respiratory disease. The PRRSV‐14d‐H1N1 infection, on the contrary, was largely subclinical. In a second experiment with conventional pigs, PRRSV‐3d‐H1N1 and PRRSV‐7d‐H1N1 infections were clinically milder, with smaller increases in mean body temperatures (peak 40.5°C in both groups) and growth reductions (1.4 and 1.6 kg, respectively). In both groups, only one pig showed prominent general and respiratory signs. In a final experiment with CDCD pigs, PRRSV‐7d‐H1N1 infection had minimal effects on mean clinical performances and growth and, except for one pig that was severely affected, differences with the single virus inoculations were negligible. Thus, both the time interval between infections and the sanitary status of pigs can affect the clinical outcome of dual PRRSV‐H1N1 infections. However, factors so far unknown seem to cause large variations in the clinical response between individual pigs.