Virulence of white spot syndrome virus (WSSV) isolates may be correlated with the degree of replication in gills of Penaeus vannamei juveniles.

A standardized inoculation model was used in 2 separate experiments to gauge the virulence of 3 white spot syndrome virus (WSSV) isolates from Thailand and Vietnam (WSSV Thai-1, WSSV Thai-2, and WSSV Viet) in Penaeus vannamei juveniles. Mortality patterns (Expt 1) were compared and WSSV-positive cells quantified (Expt 2) in tissues following intramuscular inoculation of shrimp with the most (WSSV Thai-1) and least (WSSV Viet) virulent isolates as determined by Expt 1. The results of Expt 1 demonstrated that mortalities began at 36 h post inoculation (hpi) for both Thai isolate groups and at 36 to 60 hpi for the Viet isolate group. Cumulative mortality reached 100% 96 to 240 h later in shrimp challenged with the WSSV Viet isolate compared to shrimp challenged with the Thai isolates. WSSV infection was verified in all groups by indirect immunofluorescence. In Expt 2, WSSV-infected cells were quantified by immunohistochemical analysis of both dead and time-course sampled shrimp. WSSV-positive cells were detected in tissues of Thai-1 inoculated dead and euthanized shrimp from 24 hpi onwards and from 36 hpi onwards in shrimp injected with the Viet isolate. Significantly more infected cells were found in tissues of dead shrimp inoculated with the Thai-1 than in Viet isolate-inoculated shrimp. In these experiments, substantial differences in virulence were demonstrated between the WSSV isolates. The Vietnamese isolate induced a more chronic disease and mortality pattern than was found for the Thai isolates, possibly because it infected fewer cells. This difference was most pronounced in gills.

Increased susceptibility of white spot syndrome virus-infected Litopenaeus vannamei to Vibrio campbellii.

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.

Epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in Italy.

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.

A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus.

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.

Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei.

White spot syndrome virus (WSSV) causes disease and mortality in cultured and wild shrimp. A standardized WSSV oral inoculation procedure was used in specific pathogen-free (SPF) Litopenaeus vannamei (also called Penaeus vannamei) to determine the primary sites of replication (portal of entry), to analyze the viral spread and to propose the cause of death. Shrimp were inoculated orally with a low (10(1.5) shrimp infectious dose 50% endpoint [SID50]) or a high (10(4) SID50) dose. Per dose, 6 shrimp were collected at 0, 6, 12, 18, 24, 36, 48 and 60 h post inoculation (hpi). WSSV-infected cells were located in tissues by immunohistochemistry and in hemolymph by indirect immunofluorescence. Cell-free hemolymph was examined for WSSV DNA using 1-step PCR. Tissues and cell-free hemolymph were first positive at 18 hpi (low dose) or at 12 hpi (high dose). With the 2 doses, primary replication was found in cells of the foregut and gills. The antennal gland was an additional primary replication site at the high dose. WSSV-infected cells were found in the hemolymph starting from 36 hpi. At 60 hpi, the percentage of WSSV-infected cells was 36 for the epithelial cells of the foregut and 27 for the epithelial cells of the integument; the number of WSSV-infected cells per mm2 was 98 for the gills, 26 for the antennal gland, 78 for the hematopoietic tissue and 49 for the lymphoid organ. Areas of necrosis were observed in infected tissues starting from 48 hpi (low dose) or 36 hpi (high dose). Since the foregut, gills, antennal gland and integument are essential for the maintenance of shrimp homeostasis, it is likely that WSSV infection leads to death due to their dysfunction.

Long-term administration of a commercial porcine reproductive and respiratory syndrome virus (PRRSV)-inactivated vaccine in PRRSV-endemically infected sows.

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.

Standardized white spot syndrome virus (WSSV) inoculation procedures for intramuscular or oral routes.

In the past, strategies to control white spot syndrome virus (WSSV) were mostly tested by infectivity trials in vivo using immersion or per os inoculation of undefined WSSV infectious doses, which complicated comparisons between experiments. In this study, the reproducibility of 3 defined doses (10, 30 and 90 shrimp infectious doses 50% endpoint [SID50]) of WSSV was determined in 3 experiments using intramuscular (i.m.) or oral inoculation in specific pathogen-free (SPF) Litopenaeus vannamei. Reproducibility was determined by the time of onset of disease, cumulative mortality, and median lethal time (LT50). By i.m. route, the 3 doses induced disease between 24 and 36 h post inoculation (hpi). Cumulative mortality was 100% at 84 hpi with doses of 30 and 90 SID50 and 108 hpi with a dose of 10 SID50. The LT50 of the doses 10, 30 and 90 SID50 were 52, 51 and 49 hpi and were not significantly different (p > 0.05). Shrimp orally inoculated with 10, 30 or 90 SID50 developed disease between 24 and 36 hpi. Cumulative mortality was 100% at 108 hpi with doses of 30 and 90 SID50 and 120 hpi with a dose of 10 SID50. The LT50 of 10, 30 and 90 SID50 were 65, 57 and 50 hpi; these were significantly different from each other (p < 0.05). A dose of 30 SID50 was selected as the standard for further WSSV challenges by i.m. or oral routes. These standardized inoculation procedures may be applied to other crustacea and WSSV strains in order to achieve comparable results among experiments.

In vivo titration of white spot syndrome virus (WSSV) in specific pathogen-free Litopenaeus vannamei by intramuscular and oral routes.

White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml(-1)) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml(-1)) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 10(6.8) and 10(6.5) SID50 ml(-1), respectively. These titers were not significantly different (p > or = 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection.

West Nile virus in the vertebrate world.

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.

Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages.

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.

Temporary disturbance of actin stress fibers in swine kidney cells during pseudorabies virus infection.

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.

Involvement of cellular cytoskeleton components in antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes.

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.

Porcine circovirus 2 infection in swine foetuses inoculated at different stages of gestation.

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.

Immunological hiding of herpesvirus-infected cells.

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.

Effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs.

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.

Role of anti-gB and -gD antibodies in antibody-induced endocytosis of viral and cellular cell surface glycoproteins expressed on pseudorabies virus-infected monocytes.

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.

Replication of equine herpesvirus type 1 in freshly isolated equine peripheral blood mononuclear cells and changes in susceptibility following mitogen stimulation.

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.