<i>In vivo</i> titration of white spot syndrome virus (WSSV) in specific pathogen-free <i>Litopenaeus vannamei</i> 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 >= 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

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 50 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 experiment

The effect of raising water temperature to 33°C in <i>Penaeus vannamei</i> juveniles at different stages of infection with white spot syndrome virus (WSSV)

This study investigated the effect of high water temperature (33°C) at different stages of infection with a highly virulent and low virulent white spot syndrome virus strain (WSSV Thai-1 and WSSV Viet) in Penaeus vannamei juveniles. Shrimp were inoculated intramuscularly with either a high dose (HD) or low dose (LD). Water temperature was kept either at continuously 27°C or switched from 27°C to 33°C at 0, 12 or 24 h post inoculation (hpi) for both strains and in addition at 48 or 96 hpi for WSSV Viet. The increased temperature 33°C was maintained till the end of the experiments (120–144 hpi with WSSV Thai-1 and 240 hpi with WSSV Viet). To determine the infection status at the moment of temperature increase, five shrimp that were kept continuously at 27 °C were euthanized at 0, 12, 24, 48 and 96 hpi with each dose of two strains. WSSV infections (viral antigen VP28) in dead and euthanized shrimp were demonstrated by indirect immunofluorescence.Shrimp inoculated with HD or LD of WSSV Thai-1 and kept continuously at 27°C till euthanasia were 100% viral antigen positive from 12 (HD) or 24 hpi (LD). Shrimp inoculated with WSSV Viet were 100% positive from 24 (HD) and 48 hpi (LD). Shrimp kept at 27°C, showed clinical signs from 24 (HD) or 24–36 hpi (LD) with both strains. Cumulative mortalities reached 100% with WSSV Thai-1 at 60 (HD) or 84–144 hpi (LD) and with WSSV Viet 100% at 216 hpi (HD) or 90% at 240 hpi (LD). Switch of temperature to 33°C from 0, 12 or 24 hpi was effective in reducing mortality of shrimp inoculated with the LD of both strains and with the HD of WSSV Viet. The switch to 33°C from 24 hpi with the Thai strain (HD) and from 48 and 96 hpi with the Viet strain (LD or HD) had no effect or even accelerated the mortality rate (80–100%). All shrimp were viral antigen positive at death and euthanasia (one shrimp LD WSSV Viet) when kept continuously at 27°C. All dead and euthanized shrimp kept at 33°C from 0 or 12 hpi were viral antigen negative. With 33°C from 24, 48 or 96 hpi, all dead shrimp were viral antigen positive and euthanized shrimp were negative.This study showed that 33°C is effective to prevent disease, reduce mortality and block WSSV replication, but only in the early stages of infection

Antibody-induced endocytosis of viral glycoproteins, expressed on pseudorabies virus-infected monocytes protects these cells from complement-mediated lysis

Pseudorabies virus (PrV) can cause abortion in sows with an immune system activated by vaccination. Virus-carrying blood monocytes are essential for the spread of the virus from the respiratory tract to the pregnant uterus. Two major adaptive immune effector mechanisms should normally be capable of eliminating PrV-infected monocytes. First, newly synthesised viral proteins may be processed and coupled to the major histocompatibility complex class I (MHC I) which then is transported to the plasma membrane. This MHC I-antigen-complex can be recognised by cytotoxic T-lymphocytes (CTLs). Second, specific antibodies are capable of binding to newly synthesised viral envelope glycoproteins, which become expressed in the plasma membrane of the infected cell. Antibodies in association with complement or phagocytes may then result in the lysis of the infected cell. Addition of virus-specific antibodies to PrV-infected swine kidney cells in vitro is known to induce a redistribution of the plasma membrane-anchored viral glycoproteins. This redistribution finally leads to the release of the viral glycoproteins into the surrounding medium, leaving viable cells without visually detectable levels of viral glycoproteins on their plasma membrane. In the present study it was examined whether a similar phenomenon occurs in the natural carrier of the virus, the blood monocyte, in order to evaluate if this process may be significant to the immune evasion of the virus. Blood was collected from the vena jugularis from PrV-negative pigs and blood mononuclear cells were separated on Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). Blood monocytes were purified by plastic adhesion, and were cultivated for 24 h. Afterwards, the cells were inoculated with PrV strain 89V87 or Kaplan and incubated at 37\,^\circC with 5% CO$_2$ for 13 h. After washing of the cells, FITC-labelled virus-specific antibodies were added (0.1 mg IgG/ml), and the cells were incubated at 37\,^\circC for different time periods (0, 5, 10, 30 and 60 min) before fixation with 0.4% formaldehyde and analysis by fluorescence microscopy and/or confocal laser scanning microscopy. Shortly after the addition of antibodies, viral plasma membrane glycoproteins become aggregated (patches). These patches are then internalised by the cell, leaving an infected cell with no visually detectable levels of viral glycoproteins on its plasma membrane. Antibody-induced endocytosis is a fast and efficient process. Endocytosis started at 10 min post-antibody addition, and was completed in 65% of the infected cells at 1 h post-antibody addition. Furthermore, only very few quantities of viral glycoproteins on the plasma membrane (reached after 7 h PI) and very low concentrations of antibodies (0.04 mg IgG/mL) were needed to induce endocytosis. Genistein, a specific inhibitor of tyrosine kinase activity, was found to be a very efficient inhibitor of viral glycoprotein internalisation (100% inhibition at 50 $\mu$g/mL). We also evaluated the effect of viral glycoprotein internalisation on complement-mediated lysis of the infected monocytes. Monocytes were infected for 10 h, and incubated with virus-specific antibodies for 2 h ($\pm 100\%$ of the infected cells displayed internalised viral glycoproteins at this time point). The control cells were incubated with antibodies in the presence of $50\,\mu$g/mL genistein, or were incubated without antibodies. Afterwards, the cells were washed and incubated with different concentrations of guinea pig complement (0-10 IU) for 1 h. Afterwards, 20 $\mu$g/mL of the DNA-staining fluorochrome, propidium iodide, was added for 5 min. Propidium iodide specifically stains dead cells which allows to determine the percentage of dead cells by flow cytometry. Compared relatively to the viability of the cells incubated without either antibodies or the complement, viability of the cells, incubated with the complement for 1 h decreased slightly to 79% $\pm$ 12% for cells incubated without antibodies (no activation of the complement), and to 84% $\pm$ 4% for cells incubated with antibodies (internalised viral glycoproteins and antibodies). The viability dropped to 24% $\pm$ 11% for cells incubated with antibodies and genistein (there was no internalisation of viral glycoproteins and antibodies), which was not caused by toxic effects of genistein. We can therefore state that antibody-induced endocytosis of viral glycoproteins protects PrV-infected cells from complement-mediated lysis. When performing double labelling experiments, we observed that the MHC I co-aggregates and undergoes co-endocytosis with the viral glycoproteins. This may indicate that the addition of virus-specific antibodies to PrV-infected monocytes can hide these cells from both humoral and cellular immune responses. To investigate this hypothesis, we are currently constructing an in vitro assay to evaluate the effect of MHC I co-endocytosis on the capacity of cytotoxic T-lymphocytes to eliminate PrV infected monocytes. Furthermore, we are examining whether the observed processes also occur in vivo. Preliminary experiments, consisting of the injection of colostrum-free piglets with biotinylated PrV-specific antibodies, followed by PrV-inoculation, already showed that endocytosis of antibodies occurs in vivo in infected cells, e.g. in alveolar macrophages

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

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 (101.5 shrimp infectious dose 50% endpoint [SID50]) or a high (104 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

Virulence of white spot syndrome virus (WSSV) isolates may be correlated with the degree of replication in gills of <i>Penaeus vannamei</i> 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