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A retrospect is given on the emergence of porcine epidemic diarrhea (PED) during the early seventies in Europe. While, at first, it appeared as a disease affecting feeder pigs, fattening- and adult swine, it later also became pathogenic for neonatal and suckling pigs hereby drastically increasing its economic impact. Isolation of the causative virus revealed a new porcine coronavirus, the origin of which has never been clarified. Pathogenesis studies with the prototype strain CV777 showed severe villous atrophy in neonatal pigs and the virus-animal interactions showed many similarities with transmissible gastro-enteritis virus (TGEV), another porcine coronavirus. Disease patterns in field outbreaks showed muchvariation but, while farm related factors played a role, possible genetic variations of virus strains in Europe have not been examined and are thus unknown. CV777 in experimental pigs caused diarrheal disease and mortality rates similar to those later encountered in Asia and more recently with the "original" US strains even though genomic typing of the prototype European strain have shown that it belongs to the S-INDEL strains. In Europe, PED has become endemic during the eighties and nineties and subsequently regressed so that, after 2000, swine populations in many countries have largely become seronegative. Sporadic outbreaks have recently reappeared showing a large variety of clinical outcomes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Transmission of white spot syndrome virus (WSSV) in shrimp has been reported to occur by feeding and immersion. In the present study, the impact of the molt process and artificial lesions in the cuticle on shrimp susceptibility to WSSV was examined using intramuscular and immersion routes. For the intramuscular route, Penaeus (Litopenaeus) vannamei shrimp (n=450) were injected with 10(-2.3) up to 10(2.7) shrimp infectious dose 50% end point (SID(50)) of WSSV in early and late post-molt, inter-molt, early and late pre-molt; resp. A-, B-, C-, D1- and D2-stage. The resulting infection titers demonstrated that no difference (p>0.05) in susceptibility existed between different molt stages when virus was injected. For the waterborne route, shrimp in different molt stages were immersed in seawater containing 10(4)SID(50)ml(-1) of WSSV. In a first study, P. vannamei (n=125) incubated in cell culture flasks, became infected with WSSV mostly in post-molt stages. In a second study, 2 groups of P. vannamei (n=100) and P. monodon (n=100) were transferred into plastic bags to prevent damage to the cuticle; and in 1 group a pleopod was cut off prior to incubation. Induction of damage increased infection significantly (p<0.05) in A-stage from 0-40% to 60-100%, in B-stage from 0-20% to 40-60%, in C-stage from 0-20 to 20-60%, while infection was 0% in D-stages with both immersion methods. This study proved that shrimp are more susceptible to WSSV infection via immersion after molting than in the period before molting and wounding facilitates infection.

The concept of polymicrobial disease is well accepted in human and veterinary medicine but has received very little attention in the field of aquaculture. This study was conducted to investigate the synergistic effect of white spot syndrome virus (WSSV) and Vibrio campbellii on development of disease in specific pathogen-free (SPF) shrimp Litopenaeus vannamei. The juvenile shrimp were first injected with WSSV at a dose of 30 SID(50) shrimp(-1) (SID(50) = shrimp infectious dose with 50% endpoint) and 24 h later with 10(6) colony-forming units (cfu) of V. campbellii shrimp(-1). Controls receiving just one of the pathogens or negative inocula were included. In the treatment with WSSV only, shrimp started to die at 48-108 h post injection (hpi) and cumulative mortality reached 100% at 268-336 hpi. In the treatment with only V. campbellii injection (10(6) cfu shrimp(-1)), cumulative mortality reached 16.7%. Shrimp in the dual treatment died very quickly after V. campbellii injection and 100% cumulative mortality was obtained at 72-96 hpi. When WSSV-injected shrimp were given sonicated V. campbellii instead of live V. campbellii, no synergistic effect was observed. Density of V. campbellii in the haemolymph of co-infected moribund shrimp collected 10 h after V. campbellii injection was significantly higher than in shrimp injected with V. campbellii only (P < 0.01). However, there was no difference in WSSV replication between shrimp inoculated with WSSV only compared with dually inoculated ones. This study revealed that prior infection with WSSV enhances the multiplication and disease inducing capacity of V. campbellii in shrimp.

There was an epidemic of diarrhoea affecting pigs of all ages in Italy between May 2005 and June 2006. In 63 herds the cause was confirmed as porcine epidemic diarrhoea virus by electron microscopy, immunoelectron microscopy, pcr and serology. Watery diarrhoea without mucus and blood was usually associated with a reduction of feed consumption. In farrowing-to-weaning herds, diarrhoea affected the sows and suckling piglets, and the mortality in newborn piglets was up to 34 per cent. In growers and fatteners the morbidity ranged from 20 to 80 per cent, but there was either no mortality or it was very low. Depending on the size of the herd and the type of operation, the clinical disease lasted for weeks or months.

Since it first appeared in 1992, white spot syndrome virus (WSSV) has become the most threatening infectious agent in shrimp aquaculture. Within a decade, this pathogen has spread to all the main shrimp farming areas and has caused enormous economic losses amounting to more than seven billion US dollars. At present, biosecurity methods used to exclude pathogens in shrimp farms include disinfecting ponds and water, preventing the entrance of animals that may carry infectious agents and stocking ponds with specific pathogen-free post-larvae. The combination of these practices increases biosecurity in shrimp farming facilities and may contribute to reduce the risk of a WSSV outbreak. Although several control methods have shown some efficacy against WSSV under experimental conditions, no therapeutic products or strategies are available to effectively control WSSV in the field. Furthermore, differences in virulence and clinical outcome of WSSV infections have been reported. The sequencing and characterization of different strains of WSSV has begun to determine aspects of its biology, virulence and pathogenesis. Knowledge on these aspects is critical for developing effective control methods. The aim of this review is to present an update of the knowledge generated so far on different aspects of WSSV organization, morphogenesis, pathology and pathogenesis.

The purpose of this study was to investigate the safety and efficacy of a commercial European porcine reproductive and respiratory syndrome virus (PRRSV)-inactivated vaccine after 18-month use in gilts/sows at a farm with high seroprevalence. In a farrow-to-finish farm with 1100 sows, all sows and gilts were systematically vaccinated with the PRRS-inactivated PROGRESSIS vaccine for a period of 18 months. Farm's reproductive and litter characteristics were longitudinally recorded for this period and historically compared with those of the year prior to vaccination. Serology, employing immunoperoxidase monolayer assay, had confirmed a high prevalence of PRRS-specific antibodies in most age groups within the farm prior to vaccination. Seroprevalence during the experiment ranged between 0% and 100% in weaners and growers, but remained at stable high levels (> 93%) in finishing pigs and gilts throughout all 2-year period of serology measurements. No local or systemic vaccine side effects were noted throughout the trial period. Vaccinations had resulted over time in a significant improvement of sow reproductive performance (e.g. reduction of premature farrowings, abortions and increase of farrowing rate) and litter characteristics (e.g. increase of the number of live born and weaned pigs and decrease of stillborn, mummified, weak and splay-legged piglets). It has also been observed that the higher the degree of immunization of a sow, the better the improvement of her reproductive parameters. Sows after vaccination have shown improved characteristics compared to homoparous sows prior to the application of vaccinations in the farm.

Equine herpesvirus (EHV)-1 is a pathogen of horses, well known for its ability to induce abortion and nervous system disorders. Clinical signs may occur despite the presence of a virus-specific immune response in the horse. The current review will summarize the research, on how, EHV-1-infected cells can hide from recognition by the immune system. Research findings on immune evasion of EHV-1 will be compared with those of other herpesviruses of veterinary importance.

West Nile virus (WNV), an arthropod-borne virus belonging to the family Flaviviridae, had been recognized in Africa, Asia and the south of Europe for many decades. Only recently, it has been associated with an increasing number of outbreaks of encephalitis in humans and equines as well as an increasing number of infections in vertebrates of a wide variety of species. In this article, the data available on the incidence of WNV in vertebrates are reviewed. Moreover, the role of vertebrates in the transmission of WNV, the control of WNV infections in veterinary medicine as well as future perspectives are discussed. A wide variety of vertebrates, including more than 150 bird species and at least 30 other vertebrate species, are susceptible to WNV infection. The outcome of infection depends on the species, the age of the animal, its immune status and the pathogenicity of the WNV isolate. WNV infection of various birds, especially passeriforms, but also of young chickens and domestic geese, results in high-titred viremia that allows arthropod-borne transmission. For other vertebrate species, only lemurs, lake frogs and hamsters develop suitable viremia levels to support arthropod-borne transmission. The role of vertebrates in direct, non-arthropod-borne transmission, such as via virus-contaminated organs, tissues or excretions is less well characterized. Even though direct transmission can occur among vertebrates of several species, data are lacking on the exact amounts of infectious virus needed. Finally, the increased importance of WNV infections has led to the development of killed, live-attenuated, DNA-recombinant and chimeric veterinary vaccines.

In this study, the characteristics of porcine circovirus-2 (PCV2) replication (infectious virus titrations, distribution, and immunophenotyping of infected cells) in lymphoid organs were examined and related to the development of clinical signs and histological lesions in 26 piglets that had been inoculated with PCV2 either in utero or at 1 day of age. Piglets inoculated in utero at 92 or 104 gestational days (n = 12) were collected by Caesarean section at term and either sacrificed immediately or kept in isolators and allowed to live postnatally until 35 days postinoculation (PI). Caesarean-derived piglets inoculated at 1 day of age (n = 14) were sacrificed at 10, 21, 35, 42, and 49 days PI. Spleen and lymph nodes were collected for virologic and histopathological examinations. Clinical signs were not observed in any of the piglets. High virus titers (10(4.5-5.7) TCID50/g [TCID refers to tissue culture infectious dose]) were detected in 6 of the 26 piglets. Three of these 6 piglets were euthanized at 10 days PI, and infected cells of the monocyte-macrophage lineage (SWC3+, CD14+, and sialoadhesin [Sa]+ cells) and infected cells bearing lymphocyte markers (CD4+, CD8+, and immunoglobulin M+ cells) were identified by double-immunofluorescence labeling on serial cryostat sections. The other 3 piglets were euthanized at 21 and 35 days PI, and the majority of infected cells were SWC3+, CD14+, and Sa-. The absence of Sa in these infected cells, together with their localization in lymphocyte-dependent regions, suggests that they were infiltrating monocytic cells. Sialoadhesin is highly expressed in differentiated macrophages and not in peripheral blood mononuclear cells. In all 6 piglets with high virus titers, lymphocyte depletion and infiltration of monocytic cells were observed. In the remaining 20 piglets with virus titers less than 10(4.5) TCID50/g, the majority of infected cells were SWC3+, CD14+, and Sa+. In conclusion, it can be stated that high PCV2 titers in lymphoid organs may lead to the development of histological lesions similar to those observed in pigs with postweaning multisystemic wasting syndrome without causing disease. Furthermore, in lymphoid organs with high virus titers, infection occurs mainly in infiltrating monocytic cells and to a limited extent in cells bearing lymphocyte markers.

This publication reviews some pathogenetic features of the transplacental infection with porcine viruses in sows. Viremia either with virus freely circulating or associated to peripheral blood mononuclear cells (PBMC) is an essential part of such pathogenesis. Virus replication occurs either in fetal tissues only or both in fetal and maternal tissues and the outcome may be different. Since porcine circovirus 2 (PCV2) has been associated with reproductive failure in sows, the question was asked what type of viremia PCV2 causes and what the effect of PCV2 is on the pregnant uterus. Seronegative gilts were oronasally inoculated and plasma and PBMC were monitored for infectious virus and for quantity of viral DNA copies. Infectious virus was found in plasma only at 21 days post-inoculation (DPI). Virus associated to PBMC was detected between 14 and 49 DPI. Viral DNA was found in plasma between 14 and 49 DPI and associated to PBMC between 7 and 63 DPI (end of experiment). Direct intra-fetal inoculation at 57, 75 and 92 days of gestation and collection of fetuses 21 days later showed that the virus replicates highly in fetal tissues, particularly in the heart. Fetal death occurred in the 57 days sows while virus and antibodies were observed in the 75- and 92-day inoculated sows. Inoculation at 57 and 75 days of gestation and collection of the piglets at the end of pregnancy showed that intrauterine spread had occurred to fetuses adjacent to the inoculated ones and that fetal death occurred also in the presence of antibodies. The pregnancy was not interrupted.This study shows that PCV2 causes viremia which is largely cell-associated and that virus replication in fetuses causes fetal death with mummification. Whether such transplacental infection occurs in the immune sow population is questionable.

Blood monocytes infected with pseudorabies virus (PRV), a swine alphaherpesvirus, are not eliminated efficiently by antibody-dependent immunity and may occasionally transport PRV to the pregnant uterus of vaccinated animals. This study examines in vitro the long-term fate of PRV-infected monocytes cultivated in the presence of porcine PRV-specific antibodies. All monocytes were infected and expressed viral late proteins, and 30 % of PRV-infected monocytes cultivated with PRV-specific antibodies survived up to 194 h post-infection (p.i.), the end of the experiment (compared to 0 % for cells cultivated with PRV-negative antibodies). Of these surviving cells, +/-75 % no longer expressed microscopically detectable viral late proteins from 144 h p.i. onwards. Remarkably, monocytes infected with a PRV gB-null virus did not survive in the presence of PRV-specific antibodies. These data suggest that PRV-specific antibodies suppress viral protein levels in infected monocytes, perhaps helping the virus to persist and reach internal organs in vaccinated animals.

Change of porcine circovirus 2 (PCV2) target cells during development from fetal to postnatal life in pigs was examined. PCV2 inoculation was performed in fetuses in utero at either 57, 75 or 92 gestational days and in piglets at 1 day of age. Twenty-one days after virus inoculation, PCV2-infected cells in the heart, lungs, liver, spleen and inguinal lymph nodes were localized and immuno-phenotyped by double-immunofluorescence labeling using different cell markers and PCV2-antibodies. During fetal life, viral antigens were detected in cardiomyocytes, hepatocytes and macrophages and infected cell numbers decreased with increasing fetal age at inoculation. The heart contained the highest number of infected cells and cardiomyocytes were the main target cell. Postnatally, macrophages were the only target cell type in different organs and infected cell numbers were similar to those of fetuses inoculated at 92 days of gestation. One piglet showed exceptionally high number of infected cells in different organs with values 13-513-fold higher compared to littermates. In this piglet, the majority of infected cells in lymphoid tissues could not be typed. This study reveals that PCV2 target cells change from cardiomyocytes, hepatocytes and macrophages during fetal life to only macrophages postnatally.

Pseudorabies virus (PRV)-infected blood monocytes are able to transport virus throughout the body of vaccination-immune pigs. PRV-infected monocytes express viral glycoproteins in their plasma membrane that can be recognized by virus-specific antibodies. Recently, it has been shown that addition of PRV-specific polyclonal immunoglobulins to PRV-infected monocytes at 37 degrees C induces internalization of the majority of plasma membrane-expressed viral glycoproteins. This study investigated whether this process may interfere with efficient antibody-dependent complement-mediated lysis (ADCML) of infected monocytes. Therefore, an ADCML assay was set up in vitro. A significant decrease in the percentage of cells lysed by ADCML was observed when antibody-induced internalization of PRV glycoproteins occurred (P<0.005). Furthermore, it is shown (i) that the PRV gE-gI complex, which, like certain other alpha herpesvirus orthologues, possesses IgG-binding capacity, aids in avoiding efficient ADCML of PRV-infected monocytes and (ii) that the efficiency of PRV gE-gI-mediated evasion of ADCML can be decreased by the presence of gE-gI-specific antibodies.

Pseudorabies virus (PRV) may cause abortion, even in the presence of vaccination-induced immunity. Blood monocytes are essential to transport the virus in these immune animals, including transport to the pregnant uterus. Infected monocytes express viral proteins on their cell surface. Specific antibodies recognize these proteins and should activate antibody-dependent cell lysis. Previous work showed that addition of PRV-specific polyclonal antibodies to PRV-infected monocytes induced internalization of viral cell surface proteins, protecting the cells from efficient antibody-dependent lysis in vitro (immune-masked monocytes). As a first step to reach the pregnant uterus, PRV has to cross the endothelial cell barrier of the maternal blood vessels. The current aim was to investigate in vitro whether immune-masked PRV-infected monocytes can transmit PRV in the presence of virus-neutralizing antibodies via adhesion and fusion of these monocytes with endothelial cells. Porcine blood monocytes, infected with a lacZ-carrying PRV strain, were incubated with PRV-specific antibodies to induce internalization. Then, cells were co-cultivated with endothelial cells for different periods of time. Only PRV-infected monocytes with internalized viral cell surface proteins adhered efficiently to endothelial cells. LacZ transmission to endothelial cells, as a measure for monocyte-endothelial cell fusion, could be detected after co-cultivation from 30 min onwards. Virus transmission was confirmed by the appearance of plaques. Adhesion of immune-masked PRV-infected monocytes to endothelial cells was mediated by cellular adhesion complex CD11b-CD18 and subsequent fusion was mediated by the virus. In conclusion, immune-masked PRV-infected monocytes can adhere and subsequently transmit virus to endothelial cells in the presence of PRV-neutralizing antibodies.

Equine herpesvirus-1 (EHV-1) may cause abortion in vaccination- and infection-immune horses. EHV-1-infected peripheral blood mononuclear cells (PBMCs) play an important role in virus immune evasion. The mechanisms by which infected PBMCs can avoid destruction by EHV-1-specific antibody and equine complement were examined. The majority of EHV-1-infected PBMCs (68.6 %) lacked surface expression of viral antigens and these cells were not susceptible to complement-mediated lysis. In infected PBMCs with surface expression of viral antigens, 63 % showed focal surface expression, whereas 37 % showed general surface expression. General surface expression rendered infected PBMCs susceptible to lysis by antibody and complement (from 5.4 to 31.2 % lysed cells depending on the concentration of antibody and complement). Infected PBMCs with focal surface expression showed significant lysis only in the presence of high concentrations of antibody and complement. Thus, the absence of surface expression protects infected PBMCs against complement-mediated lysis.

The immune system has a variety of tools at its disposal to combat virus infections. These can be subdivided roughly into two categories: 'first line defence', consisting of the non-specific, innate immune system, and 'adaptive immune response', acquired over time following virus infection or vaccination. During evolution, viruses have developed numerous, and often very ingenious, strategies to counteract efficient recognition of virions or virus-infected cells by both innate and adaptive immunity. This review will focus on the different strategies that viruses use to avoid recognition by one of the components of the immune system: the complement system. Complement evasion is of particular importance for viruses, since complement activation is a crucial component of innate immunity (alternative and mannan-binding lectin activation pathway) as well as of adaptive immunity (classical, antibody-dependent complement activation).

Pseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXXPhi motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.

The porcine reproductive and respiratory syndrome virus (PRRSV) has a very restricted tropism for well-differentiated cells of the monocyte-macrophage lineage, which is probably determined by specific receptors on these cells. In this study, the importance of heparinlike molecules on porcine alveolar macrophages (PAM) for PRRSV infection was determined. Heparin interacted with the virus and reduced infection of PAM up to 92 or 88% for the American and European types of PRRSV, respectively. Other glycosaminoglycans, similar to heparin, had no significant effect on infection while heparinase treatment of PAM resulted in a significant reduction of the infection. Analysis of infection kinetics showed that PRRSV attachment to heparan sulfate occurs early in infection. A heparin-sensitive binding step was observed which converted completely into a heparin-resistant binding after 120 min at 4 degrees C. Using heparin-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was observed that the structural matrix (M) and nucleocapsid (N) proteins attached to heparin. Nonreducing SDS-PAGE revealed that M bound to heparin mainly as a complex with glycoprotein GP(5) and that the N protein bound to heparin as a homodimer. GP(3), which was identified as a minor structural protein of European types of PRRSV, did not bind to heparin. Since the N protein is not exposed on the virion surface, it was concluded that the structural M protein and the M-GP(5) complex contribute to PRRSV attachment on a heparinlike receptor on PAM. This is the first report that identifies a PRRSV ligand for a cell surface heparinlike receptor on PAM.

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane anchored viral glycoproteins. This process may interfere with antibody-dependent cell lysis and resembles the well-studied physiological endocytosis process. A confocal study was designed to investigate whether the major cellular components, involved in physiological endocytosis (clathrin, actin, dynein and microtubules), play a role in this virological internalization process. In order to visualize the interaction of endosomes, which contain the internalized viral glycoproteins, with clathrin, actin, dynein and microtubules, a double labeling of viral glycoproteins and different cellular proteins was performed. Porcine monocytes were inoculated with the PrV-strain 89V87 at a multiplicity of infection of 50 for 13h. After the addition of FITC-labeled porcine polyclonal PrV-specific antibodies, cells were fixed with para-formaldehyde at different time points and afterwards permeabilized. The different cellular components were visualized with monoclonal antibodies and a Texas Red-conjugate, with the exception of actin, which was stained with phalloidin-Texas Red. The cells were analyzed by confocal microscopy. A clear co-localization was observed between the viral glycoproteins and clathrin and dynein during the internalization process. The microtubules were in close contact with the internalized vesicles. For actin no co-localization could be observed. It can be stated that clathrin, dynein and microtubules, important components during physiological endocytosis, are also of importance during the antibody-induced internalization of viral glycoproteins.

Equine herpesvirus-1 (EHV-1) is an important pathogen of horses, causing abortion and nervous system disorders, even in vaccinated animals. During the cell-associated viremia, EHV-1 is carried by peripheral blood mononuclear cells (PBMC), mainly lymphocytes. In vitro, monocytes are the most important fraction of PBMC in which EHV-1 replicates, however, mitogen stimulation prior to EHV-1 infection increases the percentage of infected lymphocytes. The role of the cell cycle in viral replication and the role of cluster formation in cell-to-cell transmission of the virus were examined in mitogen-stimulated PBMC. Involvement of the cell cycle was examined by stimulating PBMC with ionomycin/phorbol dibutyrate (IONO/PDB) during 0, 12, 24 and 36 h prior to inoculation. Cell cycle distribution at the moment of inoculation and the percentage of EHV-1 antigen-positive PBMC at 0, 12 and 24 hours post inoculation (hpi) were determined by flow cytometry and immunofluorescence microscopy, respectively. The role of clusters was examined by immunofluorescence staining within clusters of stimulated PBMC using antibodies against EHV-1. Significant correlations were found between the increase of cells in the S- or G2/M-phase after a certain time interval of prestimulation and the increase of EHV-1 antigen-positive cells. The percentage of clusters with adjacent infected cells significantly increased from 3.3% at 8 hpi to 23.7% at 24 hpi and the maximal number of adjacent infected cells increased from 2 to 7. Addition of anti-EHV-1 hyperimmune serum did not significantly alter these percentages. Mitogen stimulation favours EHV-1 infection in PBMC by: (i) initiating cell proliferation and (ii) inducing formation of clusters, thereby facilitating direct cell-associated transmission of virus.

Rounding and loosening of cells is a consequence of infection with pseudorabies virus (PrV), both in vitro and in vivo. These changes in the normal structure of the cell may be the result of cytoskeletal changes. Immunofluorescence staining of actin filaments and microtubule bundles was performed to examine whether PrV induces a reorganization of these cytoskeletal components in infected swine kidney (SK) cells. Every 2h until 12h post-inoculation (p.i.), cells were washed in cytoskeleton stabilizing buffer (CSB), fixed with paraformaldehyde and washed again with CSB. Cells were permeabilized with a 1/1000 dilution of Triton X-100 and actin filaments were stained by incubating cells with phalloidin-Texas Red. Staining of microtubules was done by incubating the cells subsequently with mouse monoclonal anti-alpha-tubulin and goat anti-mouse IgG-FITC. During the course of infection, actin fibers of SK cells were rearranged in the following sequence: (1) disappearance of thick actin stress fibers between 4 and 6h p.i., (2) complete loss of stress fibers between 6 and 8h p.i., and (3) reappearance of thin stress fibers starting from 10h p.i. In contrast to herpes simplex virus 1 (HSV1) or equine herpesvirus 1 (EHV1), PrV infection did not induce changes in the cellular microtubule network. PrV infection induces a temporary disassembly of actin stress fibers.

The ability of porcine circovirus 2 (PCV2) to replicate and cause pathologic abnormalities in foetuses at selected time points of gestation was examined in this study. Two foetuses were inoculated in utero in each of two sows at 57, 75 and 92 days of gestation, respectively, with PCV2 (1121). The remaining foetuses were left uninoculated to assess whether intra-uterine spread occurred. Twenty-one days after inoculation, the foetuses were collected and examined for gross lesions and for virus and infected cells in different organs. Serum samples from all foetuses were tested for PCV2 antibodies. Virus replication was detected in all inoculated foetuses. Spread to non-inoculated foetuses did not occur. Virus replication was significantly higher in foetuses inoculated at 57 days compared to that inoculated at 75 and 92 days. The heart contained the highest virus titre and highest number of viral antigen positive cells. Gross lesions were observed only in foetuses inoculated at 57 days of age. PCV2 antibodies were detected only in foetuses inoculated at 75 and 92 days. This study shows the ability of PCV2 to replicate in foetuses at different stages of gestation and to cause pathologic abnormalities in foetuses inoculated at 57 gestational days.

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and this may interfere with antibody-dependent cell lysis. We investigated the role of actin, microtubules, clathrin, and dynein, the major cellular components involved in physiological endocytosis during this virological internalization. Porcine monocytes were infected in vitro for 13 h and afterward treated with different concentrations of colchicine, cytochalasin D, latrunculin B, and amantadine-HCl, which inhibit polymerization of microtubules, actin/clathrin, actin, and clathrin, respectively. This resulted in a significant reduction of internalization compared to the nontreated control, indicating that these components are involved in the process. A double labeling was performed during the internalization process and a clear colocalization of actin, microtubules, clathrin, and dynein with the viral glycoproteins was observed at different stages during the internalization process. We conclude that these cellular components are used by PrV to generate the antibody-induced internalization of viral glycoproteins., (Copyright 2001 Academic Press.)

In the present study, equine herpesvirus-1 (EHV-1)-infected cells were identified in ionomycin/phorbol dibutyrate (IONO/PDB)-stimulated peripheral blood mononuclear cells (PBMC) and the mechanism by which stimulation increases the percentage of infected cells was examined. In the population of viral antigen-positive PBMC, 38.4+/-4.5% were CD5(+) T-lymphocytes (18.1+/-3.2% CD4(+) 13.6+/-1.8% CD8(+)), 18.1+/-5.4% were B-lymphocytes, 8.5+/-3.9% were monocytes and 35% remained unidentified. The role of the cell cycle in the increased susceptibility to EHV-1 upon stimulation was examined by stimulating PBMC for 0, 12, 24 or 36 h prior to inoculation. A high correlation was found between the increase of cells in the S- (r=0.974) and G(2)/M-phase (r=0.927) at the moment of inoculation and the increase of infected cells at 12 h post-inoculation (p.i.). This suggests that a specific stage of the S-phase or S- and G(2)/M-phase facilitates virus replication. At 24 h p.i. lower correlations were found, suggesting that other effects are involved. From 12 h after addition of IONO/PDB, formation of clusters of PBMC became manifest. We examined whether close intercellular contacts in these clusters facilitated cell-to-cell transmission of EHV-1. Between 8 and 17 h p.i., the percentage of clusters containing adjacent infected cells increased from 1.6 to 13.4% and the maximal number of adjacent infected cells increased from two to four. Confocal microscopy visualized close intercellular contacts between adjacent infected cells. It can be concluded that mitogen stimulation favours EHV-1 infection of PBMC (i) by initiating specific cell cycle events and (ii) by inducing formation of clusters, thereby facilitating transmission of virus between cells.

Twenty-two 4- to 5-week-old gnotobiotic pigs were intranasally inoculated with 10(6.0) TCID(50) of porcine reproductive and respiratory syndrome virus (PRRSV) (Lelystad) and euthanized at different time intervals post-inoculation (p.i.). Bronchoalveolar lavage (BAL) cell populations were characterized, together with the pattern of virus replication and appearance of antibodies in the lungs. Total BAL cell numbers increased from 140x10(6) at 5 days p.i. to 948x10(6) at 25 days p.i. and remained at high levels until the end of the experiment. The number of monocytes/macrophages, as identified by monoclonal antibodies 74-22-15 and 41D3, increased two- to fivefold between 9 and 52 days p.i. with a maximum at 25 days p.i. Flow cytometry showed that the population of differentiated macrophages was reduced between 9 and 20 days p.i. and that between the same time interval, both 74-22-15-positive and 41D3-negative cells, presumably monocytes, and 74-22-15- and 41D3-double negative cells, presumably non-phagocytes, entered the alveolar spaces. Virus replication was highest at 7 to 9 days p.i., decreased slowly thereafter and was detected until 40 days p.i. Anti-PRRSV antibodies were detected starting at 9 days p.i. but neutralizing antibodies were only demonstrated in one pig euthanized at 35 days and another at 52 days p.i. The decrease of virus replication in the lungs from 9 days p.i. can be attributed to (i) shortage of susceptible differentiated macrophages, (ii) lack of susceptibility of the newly infiltrated monocytes and (iii) appearance of anti-PRRSV antibodies in the lungs. Neutralizing antibodies may contribute to the clearance of PRRSV from the lungs.

The addition of porcine pseudorabies virus (PrV)-specific polyclonal IgG antibodies to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and major histocompatibility complex (MHC) class I. Using PrV deletion strains, it was shown that gB and gD are essential for the process to occur. The purpose of the current study was to evaluate whether antibodies directed against single viral glycoproteins are able to induce endocytosis. It was shown that monoclonal antibodies directed against viral glycoprotein gB and gD, but not against gC and gE, are able to induce internalization of their respective ligand. Adding a combination of monoclonal antibodies against gB and gD resulted in endocytosis levels, comparable to the endocytosis levels observed when adding porcine PrV-specific polyclonal antibodies. The addition of genistein and tyrphostin 25, two inhibitors of tyrosine kinase activity, abolished endocytosis induced by monoclonal anti-gB and -gD antibodies in a concentration-dependent manner. The addition of similar concentrations of tyrphostin 1, an inactive tyrphostin, had no effect on endocytosis. It was also shown that a mixture of polyclonal, but not monoclonal, antibodies against gB and gD is able to induce cointernalization of MHC class I. This indicates that MHC class I cointernalization results from a passive catching of the molecules rather than from a specific interaction of the MHC class I molecules with one or more viral glycoproteins. In conclusion, it can be stated that antibody-induced crosslinking of gB and gD induces the activation of a tyrosine phosphorylation-dependent signal transduction pathway, leading to their endocytosis. Cointernalization of other viral glycoproteins and MHC class I is most likely caused by a passive catching of these molecules in the gB and gD aggregates., (Copyright 2000 Academic Press.)

Over the past years, numerous research groups have discovered various strategies that herpesviruses use to hide themselves from recognition by the immune system of their hosts. The current review gives a summary of this research, with emphasis on the mechanisms by which herpesvirus-infected cells escape from elimination by complement, phagocytes, cytotoxic T-lymphocytes and/or natural killer cells.

In the present study, the outcome of an inoculation of equine peripheral blood mononuclear cells (PBMC) with equine herpesvirus type 1 (EHV-1) was studied in vitro. Cytoplasmic and plasma membrane expression of viral antigens, intra- and extracellular virus titres, and plaque formation in co-culture were determined. EHV-1 replicated in monocytes, although in a highly restricted way. Viral antigens were found at maximum levels (8.7% of the monocytes) at 12 h post-infection. The infection was productive in 0.16% of the monocytes. The virus yield was 10(0.7) TCID(50) per productive cell. In a population of resting lymphocytes, 0.9% of cells were infected and less than 0.05% produced infectious virus. After prestimulation with different mitogens, the number of infected lymphocytes increased four to twelve times. The susceptible lymphocytes were T-lymphocytes. In mitogen-stimulated lymphocytes, clear expression of viral antigens was found on the plasma membrane.

In this study, an intranasal immunization strategy was set up in maternally immune pigs in order to protect them not only clinically but also virologically. Two genetically engineered Aujeszky's disease virus (ADV) strains, Kaplan gE-gI- and Kaplan gE-gC-, were used for intranasal immunization. Both strains were safe for 4-week-old pigs. A single intranasal inoculation of 10(6.0) TCID50 of Kaplan gE-gI- and Kaplan gE-gC- at 4 weeks of age in the presence of moderate titres of maternally derived antibodies (SN titres: 12-16) reduced the amount of weight loss, fever and virus excretion upon challenge 6 weeks later. In a second experiment, the effect of an additional intramuscular booster with three different commercial vaccines (containing attenuated Bartha or NIA3-783 or inactivated Phylaxia; all suspended in an oil-in-water emulsion) at 10 weeks of age was evaluated. One month after the last intramuscular booster, between five and seven pigs from each group were selected for challenge. All intranasally/intramuscularly immunized pigs showed a significantly better clinical and virological protection after challenge than the single intranasally immunized pigs. In the double immunized group, the protection was better when Kaplan gE-gC- was used for the intranasal priming (only two of 14 pigs excreted virus with a duration of 4 days) than when Kaplan gE-gI- was used (13 of 18 pigs excreted virus with a duration ranging from 1 to 4 days). The virological protection was not influenced by the type of vaccine used for booster vaccination. Because the intranasal/intramuscular immunization approach is very compatible with current pig movements on farms and pigs with moderate levels of maternally derived antibodies can effectively be immunized, it can be considered as a good alternative to intramuscular/intramuscular vaccinations especially in regions with a high ADV prevalence.

The neural invasion of two virulent Suid Herpesvirus 1 (SHV1) strains was examined in neonatal pigs with or without maternal immunity. One-week-old pigs with comparable levels of maternal immunity (SN-titer = 12-48) were intranasally inoculated with 10(7.0) TCID50 of either of the Ka or E21 strains. The invasion of the strains was examined in the nasal mucosa and in three neuronal levels of the trigeminal nervous pathway as well as in three levels of the olfactory nervous pathway by virus titration and immunohistochemistry (IHC). In control pigs without specific antibodies, both strains invaded up to the end level of each neural pathway. In pigs with maternal immunity, the Ka strain invaded only up to the 2nd level of each pathway with titers being significantly lower (p<0.05) than in the negative controls. However, the E21 strain invaded up to the end levels in both neural pathways of immune pigs with virus titers being similar to those observed in non-immune pigs (p>0.05). IHC revealed that maternal antibodies can protect against a fibroblast-mediated spread of the Ka strain in the lamina propria of the nasal mucosa, as well as against a local spread of the Ka and E21 strains from neurons to their satellite cells in the trigeminal ganglion. In conclusion, the nature of virus strain determines the invasion of SHV1 within the nervous system of maternally-immune neonatal pigs.

Pseudorabies virus (PrV) glycoprotein gE is a nonessential glycoprotein involved in virulence and spread of the virus. It also has an important, yet unknown, function during antibody-induced capping of viral glycoproteins on the plasma membrane of PrV-infected swine kidney cells. In the present study, it was shown, by the use of a PrV strain expressing a truncated gE glycoprotein, that the cytoplasmic tail of gE is of significant importance for viral glycoprotein capping to occur. In addition, using PrV strains carrying point mutations in the cytoplasmic tail of gE, it was demonstrated that two tyrosine-based motifs are very important for correct functioning of gE during viral glycoprotein capping. Furthermore it was shown that genistein and tyrphostin, two tyrosine kinase activity inhibitors, inhibit viral glycoprotein capping in a concentration-dependent manner. In conclusion, it can be stated that efficient antibody-induced viral glycoprotein capping requires the presence of two YxxL sequences in the cytoplasmic tail of glycoprotein gE, as well as the activation of a tyrosine phosphorylation signal transduction pathway., (Copyright 1999 Academic Press.)

In this study, the efficacy of successive intranasal (i.n.)/intramuscular (i.m.) vaccination against Aujeszky's disease virus (ADV) was assessed in experimental pigs. The double deletion-mutant Kaplan gE-gl- was used for i.n. vaccination at 4 weeks of age and the commercially available Bartha strain, suspended in an oil-in-water emulsion, was used for the i.m. booster vaccination at 10 weeks of age. Efficacy was compared with that of the traditional double i.m. vaccination with the commercially available Bartha strain at 10 and 14 weeks of age by challenging the pigs at the end of the fattening period. There were significant differences in clinical signs, mean daily weight gain, and virus excretion between the vaccinated groups and the unvaccinated controls; however, the differences between the vaccinated groups were not statistically significant.

Purified porcine monocytes, the natural carrier cells of pseudorabies virus (PrV) in the pig, were inoculated in vitro with PrV. At different time-points post-inoculation (p.i.) (from 7 to 17 h p.i.), the cells were washed and incubated with fluorescein isothiocyanate-labelled porcine PrV-specific polyclonal antibodies (IgG) at 37 degrees C. At all time-points tested p.i., 1 h of antibody incubation induced passive patching and subsequent internalization of the plasma membrane-anchored viral glycoproteins in approximately 65% of the infected monocytes. This endocytosis process is antibody-dependent, since biotinylated glycoproteins did not undergo spontaneous endocytosis. The process is fast and efficient, since only very low amounts of viral glycoproteins on the plasma membrane (7 h p.i.) and a minimal concentration of antibodies (0.04 mg IgG/ml) were needed to induce endocytosis. Experiments with PrV strains carrying deletions in the genes encoding the 11 different viral glycoproteins showed that viral glycoproteins gB and gD play a very important role in endocytosis (80% reduction with deletion mutants, P < 0.001), while the gE:gI Fc receptor complex, but not gE or gI alone, has a significant but lesser effect (45% reduction, P < 0.05). Double staining of viral glycoproteins and major histocompatibility complex class I (MHC I) showed a clear co-localization and co-endocytosis of MHC I with the viral glycoproteins, suggesting a possible role of the process in immune evasion of the virus.

Porcine alveolar macrophages (AMphi) are the dominant cell type that supports the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in vivo and in vitro. In order to determine the characteristics of the virus-receptor interaction, the attachment of PRRSV to cells was examined by using biotinylated virus in a series of flow cytometric assays. PRRSV bound specifically to AMphi in a dose-dependent manner. Binding of PRRSV to AMphi increased gradually and reached a maximum within 60 min at 4 degrees C. By confocal microscopy, it was shown that different degrees of PRRSV binding exist and that entry is by endocytosis. Virus uptake in vesicles is a clathrin-dependent process, as it was blocked by the addition of cytochalasin D and co-localization of PRRSV and clathrin was found. Furthermore, by the use of two weak bases, NH4Cl and chloroquine, it was demonstrated that PRRSV uses a low pH-dependent entry pathway. In the presence of these reagents, input virions accumulated in large vacuoles, indicating that uncoating was prevented. These results indicate that PRRSV entry into AMphi involves attachment to a specific virus receptor(s) followed by a process of endocytosis, by which virions are taken into the cell within vesicles by a clathrin-dependent pathway. A subsequent drop in pH is required for proper virus replication.

To identify the receptor which may determine the macrophage tropism of porcine reproductive and respiratory syndrome virus (PRRSV), monoclonal antibodies (MAbs) against porcine alveolar macrophages (PAM) were produced. Two MAbs (41D3 and 41D5) which completely blocked PRRSV infection of PAM were further characterized. It was found that they reduce the attachment of PRRSV to PAM and immunoprecipitate a 210-kDa membrane protein from PAM. This protein was detected on the cell membranes of PAM but not of PRRSV-nonpermissive cells. A colocalization was found between the reactive sites of MAb 41D3 and PRRSV on PAM membranes. All PRRSV-infected cells in tissues of experimentally infected pigs reacted with MAb 41D3. Taken together, all these data suggest that the identified 210-kDa membrane protein is a putative receptor for PRRSV on porcine macrophages.

PRRSV has a restricted macrophage tropism. To explore if the difference in susceptibility of porcine alveolar macrophages (PAM) and peripheral blood mononuclear cells (PBMC) to PRRSV is correlated with certain cellular surface antigens which may serve as a virus receptor, polyclonal antibodies against PAM and PBMC were prepared. Anti-PAM but not anti-PBMC antibodies protected PAM from PRRSV infection suggesting that specific receptor(s) may exist on PAM. Furthermore, monoclonal antibodies (MAbs) against putative receptor(s) were produced. Balb/c mice were firstly immune-tolerized with freshly isolated PBMC after which they were immunized with PAM. Two MAbs (41D3 and 41D5) which blocked PRRSV infection of PAM were obtained. MAb 41D3 and 41D5 prevented the attachment of purified PRRSV to PAM. Both MAbs bound to the cellular membrane of PAM but not to that of porcine peritoneal macrophages, PBMC and three porcine cell lines (SK, ST and PK-15) as revealed by flow cytometry. This membrane reactivity correlates well with the susceptibility of these cells to a PRRSV infection. Taken together, these data suggest that MAb 41D3 and 41D5 recognize a potential cellular receptor for PRRSV on PAM.

The frequency with which sows are vaccinated during eradication programmes has been determined rather arbitrarily in the past, without the necessary scientific support. In the present study the efficacy of repeated vaccinations against Aujeszky's disease virus (ADV) with two inactivated vaccines and with one attenuated ADV-strain (Bartha) suspended in either oil-in-water or saline was evaluated in 40 breeding sows originating from five farms by the assessment of the clinical and virological protection upon an experimental challenge. Sows with two different histories of number of booster vaccinations after a base immunization were selected: young sows which had received 1-3 booster vaccinations and old sows which had received 8-10 booster vaccinations. Two seronegative and two gE-positive sows were included as controls. After challenge, clinical signs such as anorexia and fever were observed in the two seronegative sows and only in four out of the 18 sows which had repeatedly been vaccinated with inactivated vaccines. The mean duration of virus excretion significantly differed (P < 0.05) between the experimental groups. In seronegative sows, virus shedding lasted for 12.5 days. This period was reduced to 6.8-7.9 days in the groups of sows vaccinated with inactivated vaccines having no effect by the number of booster vaccinations and type of vaccine. In the groups of sows vaccinated with Bartha vaccine, challenge virus was detected during 3.7-5.3 days when revaccinated 1-3 times. A reduction to 0.7-1.2 days was obtained with a higher number of boosters (8-10) but only when the vaccine virus was suspended in o/w. Considering the mean cumulative values of virus excretion (area under the curve) it can be stated that sows frequently vaccinated with inactivated vaccines excrete significantly (P < 0.05) higher amounts of virus (26.0-31.5) than sows frequently vaccinated with attenuated ones (1.6-23.7). We may conclude from the present data that the attenuated vaccine virus Bartha, especially when suspended in o/w is superior to inactivated vaccines for inducing clinical and virological protection of sows in the field during their whole breeding period.

Fluorescein isothiocyanate-labeled porcine pseudorabies virus (PrV) polyclonal antibodies were added to PrV-infected swine kidney cells in vitro at 37 degrees C. In approximately 47% of the infected cells, the addition induced passive patching and subsequent energy- and microtubule-dependent capping of all viral envelope glycoproteins, expressed on the plasma membranes of the infected cells. Further contraction and extrusion of the capped viral glycoproteins occurred in approximately 30% of the capped cells 2 h after the addition of antibodies and was accompanied by a concentration of F-actin beneath the caps. At that time, about 18% of the extruded caps were shed spontaneously into the surrounding medium. Mechanical force released 85% of the extruded caps, leaving viable cells with no microscopically detectable levels of viral glycoproteins on their plasma membranes. Experiments with PrV deletion mutants showed that viral glycoproteins gE and gI are important in triggering viral glycoprotein redistribution. Since the PrV gE-gI complex exhibits Fc receptor activity which facilitates capping, the importance of gE and gI may be partially explained by antibody bipolar bridging.

Sixteen 6 week old conventional pigs were inoculated by aerosol with a European strain of porcine reproductive and respiratory syndrome virus (PRRSV). Virus replication was followed by virus titration and immunofluorescence in the lungs and in associated and distant lymphoid tissues at 3, 14, 21, 35, 42 and 82 days post-inoculation (DPI). PRRSV replication was detected in alveolar macrophages, lungs, tonsils, spleen, retropharyngeal lymph nodes, bronchial lymph nodes and thoracic aortic lymph nodes at 3 DPI. The same tissues, except retropharyngeal and thoracic aortic lymph nodes, were PRRSV positive at 14 DPI. Lungs and alveolar macrophages were PRRSV positive until 35 DPI. PRRSV was not detected in heart, peripheral blood mononuclear cells and bone marrow cells. Viremia was detected from 3 to 28 DPI. Not more than 2% of alveolar macrophages were PRRSV positive even during the acute stage of infection. 80 to 94% of the PRRSV infected cells in the lungs and in lung lavaged cells were identified as macrophages using a porcine macrophage specific monoclonal antibodies. In the lymph nodes and spleen, 100% of the infected cells were macrophages. Anti-PRRSV antibodies were detected by a blocking ELISA as early as 7 DPI. the antibody titre gradually increased to reach a geometric mean titre (GMT) of 160 at 35 DPI. It remained at that level until the end of the study. These findings clearly demonstrate that PRRSV has a tropism for macrophages. PRRSV mainly replicates in macrophages of the lymphoid tissues and lungs in the acute phase of infection and persists in the lung macrophages.

The degree to which maternally derived antibodies may affect neural invasion of Aujeszky's disease virus (ADV) in neonatal pigs was examined. One-week-old pigs with different levels of maternal immunity were inoculated intranasally with 10(7.0) TCID50 of the Ka strain. The invasion of the virus was studied in both the trigeminal neural pathway (nasal mucosa, trigeminal ganglion = 1st level, pons/medulla = 2nd level and cerebellum/thalamus = 3rd level) and the olfactory neural pathway (olfactory mucosa = 1st level, olfactory bulb = 2nd level and lateral olfactory gyrus = 3rd level) by virus titration and immunohistochemistry (IHC). In control pigs without specific antibodies, virus invaded all neuronal levels in both neural pathways. In pigs with a low concentration of maternal antibodies (SN-titer = 2-3), virus infected all neuronal levels in both neural pathways but, compared to the controls, virus titers were significantly lower (approximately 2 log10) in the trigeminal pathway. In pigs with a high concentration of maternal antibodies (SN-titer = 272-384), virus reached the 2nd neuronal level of the olfactory pathway while no neural tissue had been infected in the trigeminal pathway. Virus titers in the affected neuronal levels of the latter pigs were significantly lower than in the controls. IHC revealed, in non-immune pigs, a fibroblast-mediated spread of the virus in the nasal lamina propria, and a local spread of the virus from neurons to their satellite cells in the trigeminal ganglion. Such a spread of the virus was rarely seen in the nasal mucosa and in the trigeminal ganglion of passively immune pigs. These findings suggest that, in the presence of maternal immunity, defence mechanisms operate at these sites. In conclusion, we can state that a correlation exists between the level of maternal immunity and the protection against invasion of ADV in the nervous system of neonatal pigs.

In this study, the susceptibility of porcine peripheral blood monocytes (BMo), peritoneal macrophages (PM phi) and alveolar macrophages (AM phi) to PRRSV was examined. To test the effect of differentiation and activation on their susceptibility, AM phi and BMo were aged, cultivated in either adhesion or suspension and treated with bacterial lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). It was found that freshly isolated PM phi and BMo were non-permissive to PRRSV. PM phi remained refractory but a few BMo became susceptible after 1 day cultivation. AM phi were permissive with a significant increase of their susceptibility after one day cultivation. In a binding assay, it was demonstrated that the attachment of biotinylated PRRSV to AM phi is much more efficient than to PM phi and BMo. Two monoclonal antibodies (Mabs) 41D3 and 41D5 which block PRRSV infection of AM phi and are directed against a candidate receptor for PRRSV only reacted with the cell membrane of AM phi. PMA treatment of AM phi blocked PRRSV replication in the cells in a dose-dependent manner. The blocking effect of PMA decreased after 9 h continuous pre-treatment and diminished after 24 h continuous pre-treatment. PMA treatment did not affect the binding of PRRSV and MAb 41D3 and 41D5 to AM phi. Direct or indirect treatment of AM phi and BMo with LPS or cultivation in suspension did not significantly affect their susceptibility. These results provide clear evidence that PRRSV has a strongly restricted tropism for only some sub-populations of porcine monocytes/macrophages and that some specific states of differentiation and activation of monocytes/macrophages considerably affect their susceptibility.