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.

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.

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.

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.

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.

Comparative study of a blocking enzyme-linked immunosorbent assay and the immunoperoxidase monolayer assay for the detection of antibodies to the porcine reproductive and respiratory syndrome virus in pigs.

A blocking enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to the porcine reproductive and respiratory syndrome virus (PRRSV) in pig sera was developed and compared with the immunoperoxidase monolayer assay (IPMA), the most widely used serological diagnostic test in Europe. The blocking ELISA was specific and more sensitive than the IPMA when applied to field sera and to sera which were collected early after an experimental infection with PRRSV. Problems with high background activity as observed in IPMA or indirect ELISA were not encountered.

Cell-free and cell-associated viremia in pigs after oronasal infection with Aujeszky's disease virus.

Nine pigs were examined for the presence of viremia during the first week after oronasal inoculation of 10(8.0) TCID50 Aujeszky's disease virus (ADV). Blood was taken at 1, 2, 3, 5 and 7 days post inoculation (PI) and the presence of cell-free ADV in plasma and of ADV-infected mononuclear cells was examined by titration and by cocultivation with permissive cells, respectively. The mononuclear cells of 6 of the 9 pigs, collected at 3 and 5 days PI were further separated into subpopulations of enriched monocytes and enriched lymphocytes. Both subpopulations were cocultivated. Nasal secretions were collected from 4 of the 9 pigs for the determination of virus titers and interferon concentrations. Both infected mononuclear cells and cell-free ADV were demonstrated in 5 pigs, infected mononuclear cells only were found in 2 pigs, and neither cell-associated or cell-free ADV were detected in 2 pigs. Two of the 7 viremic animals were positive on one single day, 3 on 2 days, 1 on 3 days and 1 on 4 days. The number of infected cells was approximately 5 times higher in monocytes than in lymphocytes. The highest virus titers were present in those nasal fluids with the lowest alpha-interferon concentration. A correlation between the titer of locally produced ADV in the nose and the presence of a viremia was not found. In conclusion, we can state that a viremia regularly occurs under both cell-free and cell-associated form after an oronasal inoculation of ADV and that monocytes are the most susceptible mononuclear cells.

Effect of intratracheal challenge of fattening pigs previously immunised with an inactivated influenza H1N1 vaccine.

Intratracheal inoculation of a field isolate of influenza A H1N1 caused high fever, anorexia and dyspnoea in unvaccinated pigs. In a limited study, it was shown that animals vaccinated once with an inactivated influenza A H1N1 strain showed partial protection at challenge, indicated by mild or absent clinical signs and by the suppression of viral replication. There appeared to be a correlation between the hemagglutination-inhibition titers of the serum of vaccinated pigs and the degree of protection. Animals vaccinated with two spaced injections were completely protected at challenge. Viral replication was inhibited in their respiratory tract since no virus was isolated from animals at slaughter and no increase in antibody titer was observed in challenged vaccinates followed serologically. It was concluded that vaccination of swine against influenza with an inactivated vaccine can result in a protective immunity in the respiratory tract. The New Jersey vaccine strain could protect against swine influenza strains (H1N1) currently prevalent in several European countries.

Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV).

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.

A competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus.

A competitive inhibition ELISA was developed to detect non-neutralizing antibodies to the peplomer protein of transmissible gastroenteritis virus (TGEV) in porcine sera using a monoclonal antibody as an indicator. It was demonstrated that field strains of the TGEV-related porcine respiratory coronavirus (PRCV) did not induce this antibody, whereas the Miller strain and field strains of TGEV did. The sensitivity of the competitive inhibition ELISA appeared to be similar to that of the virus neutralization (VN) test. The test enables differentiation of pigs which were previously infected with TGEV or PRCV and which cannot be distinguished by the classical anti-TGEV neutralization test. The present test is useful for selective serodiagnosis.

Invasion and spread of single glycoprotein deleted mutants of Aujeszky's disease virus (ADV) in the trigeminal nervous pathway of pigs after intranasal inoculation.

The purpose of the study was to evaluate the role which non-essential envelope glycoproteins play in the neuroinvasion and neural spread of ADV. The invasion and spread in the trigeminal nervous pathway with the Ka strain of ADV and its single deletion mutants Ka gI-, Ka gp63- and Ka gIII- were examined after intranasal inoculation in neonatal pigs by virus isolation and immunocytochemistry. Evaluation was performed in the nasal mucosa, trigeminal ganglion (1st neuronal level), ponsmedulla (2nd neuronal level) and thalamus-cerebellum (3rd neuronal level). The Ka gIII- mutant invaded up to the 3rd neuronal level of the trigeminal pathway and spread in a similar way to the parental Ka strain. The Ka gp63- mutant invaded up to the 3rd neuronal level but the spread of this mutant was impaired at all the neuronal levels. The Ka gI- mutant was least neuroinvasive and reached only up to the 2nd neuronal level. The results showed that glycoproteins gI and gp63 play a role in the invasion and spread of ADV in the nervous system. However, the gI glycoprotein appears to be the most important for neuroinvasion and neural spread of ADV in pigs. Therefore, gI deleted vaccines may be considered to be safer with respect to the neuroinvasion than vaccines carrying single deletions of other non-essential envelope glycoproteins.

Extent and duration of virulent virus excretion upon challenge of pigs vaccinated with different glycoprotein-deleted Aujeszky's disease vaccines.

Different deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion optimally upon infection. Groups of pigs were vaccinated once with attenuated deleted Aujeszky's disease vaccine (gI, gX or gp63 negative), suspended in phosphate buffered saline. Two additional groups were vaccinated with a gI deleted vaccine virus suspended in an oil-in-water emulsion. Other groups were vaccinated twice with gI deleted inactivated vaccines. The three control groups included were: pigs immune after infection, unvaccinated pigs and pigs receiving vaccine without known deletion in the envelope. Experimental challenge took place 3 or 4 weeks after the only or the last vaccination. The number of excreting pigs, the duration of excretion and the virus titers excreted, were determined for all the groups. All the pigs vaccinated with glycoprotein deletion vaccines suspended in phosphate buffered saline, excreted virus for 2 to 6 days after challenge. A 100 to 1000 fold reduction in excreted virus titers was obtained in vaccinated pigs compared to unvaccinated ones. Some vaccines suppressed virus excretion better than others, but no correlation could be made between the type of deletion (gI, gX or gp63) and the degree of reduction in virus excretion. Similar results were obtained with two applications of inactivated vaccines. The lowest number of excreting pigs, the lowest duration of excretion and the lowest titers were obtained in groups vaccinated with the attenuated vaccine suspended in an oil-in-water emulsion. No vaccine suppressed virus excretion totally.

Intestinal replication of a porcine respiratory coronavirus closely related antigenically to the enteric transmissible gastroenteritis virus.

One-week-old piglets were inoculated with the porcine respiratory coronavirus (PRCV) either intravenously or directly into the lumen of the gastrointestinal tract. Both inoculation routes resulted in the isolation of virus from the caudal small intestine. Viral replication, however, was only observed upon inoculation into the digestive tract in quantities of greater than or equal to 10(3) TCID50. Replication remained limited to a few unidentified cells located in or underneath the epithelial layer at villus- or crypt-sites. Virus was excreted in the faeces for several days but infection of the respiratory tract occurred rarely in the same pigs. The results of this study indicate that small changes in molecular structure between PRCV and transmissible gastroenteritis virus have resulted in important changes in host cell tropism.

Effect of intratesticular inoculation with Aujeszky's disease virus on genital organs of boars.

Ten 8-10-month-old Belgian Landrace boars were intratesticularly inoculated with 500 TCID50 of a virulent Belgian Aujeszky's disease virus (ADV) isolate (75V19) in 0.1 ml volume. One control boar was similarly inoculated with phosphate-buffered saline solution. The genital organs of six inoculated boars were examined by virus isolation and immunofluorescence. In spite of high virus titers, the fluorescence in the testicles remained limited to a few small foci in the interstitial connective tissue and tunica albuginea at or close to the inoculation site. Neither virus replication, necrosis nor inflammatory lesions could be demonstrated in the epithelium of the seminiferous tubules. However, virus replication was regularly demonstrated in the serosa covering testicles, plexus pampiniformis, ductus deferens and tunica vaginalis. Virus was also isolated from the scrotal fluid. It is suggested that the serosa is the primary target tissue for ADV. The other four boars were inoculated to study the effect of ADV on semen. Severe morphologic alteration and lowered sperm cell concentrations were observed during several weeks after inoculation or until slaughter at 47, 53 and 58 days post inoculation. Virus was isolated from semen of only two out of four boars examined at 9 and 10 days post inoculation.

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.

Effect of the concentration of maternal antibodies on the neural invasion of Aujeszky's disease virus in neonatal pigs.

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.

Neural invasion of two virulent suid herpesvirus 1 strains in neonatal pigs with or without maternal immunity.

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.

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.

Porcine reproductive and respiratory syndrome virus infection of alveolar macrophages can be blocked by monoclonal antibodies against cell surface antigens.

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.