Eefke Weesendorp

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.

Comparative analysis of immune responses following experimental infection of pigs with European porcine reproductive and respiratory syndrome virus strains of differing virulence

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is difficult to control due to a high mutation rate and the emergence of virulent strains. The objective of this study was to analyze the immunological and pathological responses after infection with the European subtype 3 strain Lena in comparison to subtype 1 strains Belgium A and Lelystad-Ter Huurne (LV). Sixteen pigs were inoculated per strain, and sixteen pigs with PBS. At days 7 and 21 post-inoculation (p.i.), four pigs per group were immunized with an Aujeszky disease vaccine (ADV) to study the immune competence after PRRSV infection. Infection with the Lena strain resulted in fever and clinical signs. This was not observed in the Belgium A or LV-infected pigs. Infection with the Lena strain resulted in high virus titers in serum, low numbers of IFN-γ secreting cells, a change in leukocyte populations and a delayed antibody response to immunization with ADV. Levels of IL-1β, IFN-α, IL-10, IL-12, TNF-α and IFN-γ mRNA of the Lena-infected pigs were increased during the first week of infection. For pigs infected with the Belgium A or LV strain, the effects of infection on these parameters were less pronounced, although for the Belgium A-infected pigs, the level of the analyzed cytokines, except for TNF-α, and leukocyte populations were comparable to the Lena-infected pigs. These results suggest that while the outcome of infection for the three strains was comparable, with mostly clearance of viremia at day 33 p.i, differences in immune responses were observed, perhaps contributing to their virulence.

Dynamics of virus excretion via different routes in pigs experimentally infected with classical swine fever virus strains of high, moderate or low virulence

Classical swine fever virus (CSFV) is transmitted via secretions and excretions of infected pigs. The efficiency and speed of the transmission depends on a multitude of parameters, like quantities of virus excreted by infected pigs. This study provides quantitative data on excretion of CSFV over time from pigs infected with a highly, moderately or low virulent strain. For each strain, five individually housed pigs were infected. Virus excretion was quantified in oropharyngeal fluid, saliva, nasal fluid, lacrimal fluid, faeces, urine and skin scraping by virus titration and quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRRT-PCR). Infectious virus was excreted in all secretions and excretions of pigs infected with the highly and moderately virulent strain, while excretion from pigs infected with the low virulent strain was mostly restricted to the oronasal route. Pigs infected with the highly virulent strain excreted significantly more virus in all their secretions and excretions over the entire infectious period than pigs infected with the moderately or low virulent strains. An exception were the pigs that developed the chronic form of infection after inoculation with the moderately virulent strain. During the entire infectious period, they excreted the largest amounts of virus via most secretions and excretions, as they excreted virus continuously and for a long duration. This study highlights the crucial role chronically infected pigs may play in the transmission of CSFV. Furthermore, it demonstrates the importance of discriminating between strains and the clinical appearance of infection when using excretion data for modelling.

Survival of classical swine fever virus at various temperatures in faeces and urine derived from experimentally infected pigs

Indirect transmission of classical swine fever virus (CSFV) can occur through contact with mechanical vectors, like clothing and footwear or transport vehicles, contaminated with the secretions or excretions of infected pigs. A prerequisite for indirect transmission is survival of the virus on the mechanical vector. Consequently, to obtain more insight into these transmission routes, it is important to know how long the virus remains viable outside the host. In this study we examined the survival of classical swine fever virus in faeces and urine derived from pigs intranasally inoculated with a highly or moderately virulent CSFV strain. Faeces and urine were collected between days 5 and 36 post-inoculation, and stored at 5, 12, 20, and 30 degrees C. Next, the virus titres were determined in the samples by virus titration, and a random selection of these samples was also analyzed by quantitative real-time reverse transcription polymerase chain reaction (qRRT-PCR) to determine the viral RNA decay. Survival curves were generated, and it was shown that the inactivation rate was inversely related to the storage temperature. Average half-life values were between 2 and 4 days at 5 degrees C, and between 1 and 3h at 30 degrees C. Significant differences were observed in survival between virus strains in faeces, however, not in urine. The reduction in viral RNA during the entire study period was limited. This study provided detailed information on survival of CSFV in excretions of infected pigs, which can be used to improve control measures or risk-analysis models.

Lung pathogenicity of European genotype 3 strain porcine reproductive and respiratory syndrome virus (PRRSV) differs from that of subtype 1 strains.

Abstract Porcine reproductive and respiratory syndrome (PRRS) is difficult to control due to a high mutation rate of the PRRS virus (PRRSV) and the emergence of virulent strains. The objective of this study was to analyse early and late pathological responses in the respiratory tract after infection with the European PRRSV subtype 3 strain Lena in comparison to two European PRRSV subtype 1 strains: Belgium A and Lelystad-Ter Huurne (LV). For each virus strain, groups of twelve pigs were inoculated, and four pigs per group were euthanized at days 3, 7 and 35 post-infection (p.i.) for consecutive examination. Infection with strain Lena resulted in a more severe disease than with the subtype 1 strains, an inflammatory response within the first week of infection with expression of IL-1α in the lung and lymph node, and an influx of neutrophils and monocytes in bronchoalveolar lavage fluid (BALF). Infection with strain Belgium A or LV resulted in mild or no pathology within the first week of infection, but inflammatory cell influx in the lung interstititium was increased at the end of the experiment at day 35 p.i. At five weeks p.i., all strains induced a higher percentage of cytotoxic T cells and higher levels of IFN-γ producing cells in BALF. This might have contributed to clearance of virus. In general, subtype 3 strain Lena induced a stronger early inflammatory response which led to more severe clinical disease and pathology. On the other hand, this may have supported an enhanced or faster clearance of virus in tissues, compared to subtype 1 strains.

Quantification of classical swine fever virus in aerosols originating from pigs infected with strains of high, moderate or low virulence

During epidemics of classical swine fever (CSF), the route of virus introduction into a farm is often unclear. One of the suggested routes is via the air. Under experimental conditions, airborne transmission over a short distance seems possible, but analysis of outbreak data is still inconclusive. For a better understanding of the role of airborne transmission, quantitative information is needed on concentrations of virus emitted by infected pigs. This was studied in four groups of 10 pigs in which three pigs were inoculated with either a low virulent strain (Zoelen), a low or high dose of a moderately virulent strain (Paderborn), or a highly virulent strain (Brescia). The other seven pigs in each group served as contact pigs. At several moments after infection, air samples were obtained using gelatine filters. Infectious virus and viral RNA were detected in the air of rooms housing the pigs infected with the moderately and highly virulent strains with titres of 10(1.2) to 10(3.0)TCID(50)/m(3) of infectious virus, and 10(1.6) to 10(3.8)TCID(50)equiv./m(3) of viral RNA. It was observed that the higher the dose or virulence of the virus strain used for inoculation of the pigs, the sooner virus could be detected in the air samples. This is the first study describing the quantification of (infectious) CSFV in air samples of rooms housing infected pigs, enabling to quantify the contribution of individual infected pigs to virus concentrations in aerosols. This can be used as input for quantitative models of airborne spread over large distances.

Phenotypic modulation and cytokine profiles of antigen presenting cells by European subtype 1 and 3 porcine reproductive and respiratory syndrome virus strains in vitro and in vivo.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes continuous problems in the pig industry, due to high costs of outbreaks and reduced welfare of diseased pigs. The severity of infection is, partly, dependent on the virus strain. Recently isolated Eastern-European subtype 3 strains are more pathogenic than the widespread subtype 1 strains. There is, however, almost no information available about the mechanisms involved in the pathogenicity of these subtype 3 strains. The objective of the present study was to characterize the in vitro and in vivo response of two European subtype 1 strains, Belgium A and Lelystad-Ter Huurne (LV), and a virulent subtype 3 strain, Lena, in bone marrow-derived dendritic cells (BM-DC) (in vitro) and alveolar macrophages (in vitro and in vivo). It was shown that infection with the Lena strain resulted in a higher apoptosis of cells in vitro and a higher level of infectivity in vitro and in vivo than the other virus strains. Furthermore, infection with Lena resulted in a small downregulation of the immunologically relevant cell surface molecules SLA-I, SLA-II and CD80/86 in vitro, and SLA-II in vivo. In spite of these differences, in vitro cytokine responses did not differ significantly between strains, except for the absence of IL-10 production by Lena in BM-DC. The higher infectivity, apoptosis and downregulation of the cell surface molecules, may have contributed to the increased pathogenicity of Lena, and have dampened specific immune responses. This could explain the delayed and decreased adaptive immune responses observed after infections with this strain.

Transmission of classical swine fever virus depends on the clinical course of infection which is associated with high and low levels of virus excretion

Infection with moderately virulent strains of classical swine fever virus (CSFV) can lead to different courses of disease: either (sub)acute, resulting in death or recovery, or chronic disease. The virus excretion dynamics between these courses are quite dissimilar, but it is not known if this also results in differences in virus transmission. In this study, the excretion and transmission dynamics of the moderately virulent Paderborn strain were studied in 15 one-to-one experiments. In these experiments, a single inoculated pig was housed with a single susceptible contact pig from day 1 post-inoculation (p.i.). Each contact pig that became infected was removed and replaced by a new contact pig at day 17 p.i. and day 26 p.i. Infection of contact pigs was monitored by reverse transcription quantitative real-time PCR on oropharyngeal fluid samples. Five of the inoculated pigs developed the chronic form or died during the acute phase (high excreting pigs), while 10 pigs recovered from the infection (low excreting pigs). In the first contact period, there was no significant difference in virus excretion between the high and low excreting pigs, while in the second and third contact period, high excreting pigs excreted significantly higher quantities of virus. Over the entire study period, the reproduction ratio differed significantly between the high (143 [56.3-373]) and low excreting pigs (23.1 [11.5-45.0]). This indicates the importance of high excreting pigs in transmission of CSFV. Furthermore, this study showed the rate of CSFV infections from a contaminated environment.

The effect of tissue degradation on detection of infectious virus and viral RNA to diagnose classical swine fever virus

A considerable part of tissue samples that are collected for the monitoring of classical swine fever (CSF) from the wild boar population or from domestic pigs are unsuitable for virus detection using the fluorescent antibody test (FAT) or virus isolation (VI), due to tissue degradation. Reverse transcription polymerase chain reaction (RT-PCR) has a higher sensitivity than the FAT and VI, and is supposed to be less sensitive to sample degradation. Reliable and quantitative information on how long viral RNA and infectious virus can be detected in organs and which organs are most susceptible to degradation is, however, lacking. In the present study we generated survival curves of infectious CSF virus (CSFV) and viral RNA in the tonsil, mesenteric lymph node, spleen and kidney, obtained from 24 pigs infected with a moderately virulent CSFV strain. Tissue samples were stored at room temperature and tested by VI and RT-PCR, directly after storage and 1, 2, 3, 4, 7, 14 and 21 days later. It was shown that the RT-PCR is not only more sensitive than VI on fresh tissue samples, but RT-PCR is also less vulnerable to sample degradation. Average half-life values of viral RNA in the tissues ranged from 0.95 to 2.55 days, while half-life values of infectious virus ranged from 0.21 to 0.31 days. The tonsil and spleen are regarded as the most appropriate organs for the detection of infectious virus and viral RNA, not only in fresh samples, but also in samples that suffer from tissue degradation.

Effect of strain and inoculation dose of classical swine fever virus on within-pen transmission

To improve the understanding of the dynamics and options for control of classical swine fever (CSF), more quantitative knowledge is needed on virus transmission. In this study, virus excretion and within-pen transmission of a strain of low, moderate and high virulence were quantified. Furthermore, the effect of inoculation dose on excretion and transmission were studied. The transmission was quantified using a stochastic susceptible-exposed-infectious-recovered (SEIR) model. Five transmission trials were conducted with ten pigs each. In each trial, three pigs were inoculated with the low virulent strain Zoelen, a low (10(2) TCID(50)), middle (10(3.5) TCID(50)), or high dose (10(5) TCID(50)) of the moderately virulent strain Paderborn, or the highly virulent strain Brescia. The other seven pigs in each trial served as contact pigs. None of the pigs inoculated with the low dose of the Paderborn strain were infected. When it was assumed that the infectiousness of the pigs coincided with virus isolation positive oropharyngeal fluid and/or faeces, no significant differences in transmission rate beta and basic reproduction ratio R(0) between the high inoculation dose of the Paderborn strain (beta= 1.62/day, R(0) = 35.9) and the Brescia strain (beta= 2.07/day, R(0)= 17.5) were observed. When the middle dose of the Paderborn strain was used for inoculation, the beta (5.38/day) was not significantly higher than the Brescia strain or the high inoculation dose of the Paderborn strain, but the R(0) (148) was significantly higher. Infection with the Zoelen strain resulted in a significantly lower beta and R(0) (beta= 0/day, R(0) = 0) than the other strains.