Comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure

OBJECTIVE: To compare the virulence of selected strains of porcine reproductive and respiratory syndrome virus (PRRSV) relative to reproductive performance of pregnant gilts. DESIGN: 16 pregnant gilts (principals) were exposed oronasally to 4 strains (vaccine strain RespPRRS, field strains VR-2385, VR-2431, and NADC-8, 4 gilts/strain) of PRRSV on or about day 90 of gestation, 4 pregnant gilts (controls) were kept under similar conditions, except for exposure to PRRSV. Samples and specimens obtained from gilts, pigs (before ingestion of colostrum), and fetuses were tested for PRRSV and homologous antibody. ANIMALS: 20 pregnant gilts. PROCEDURE: The virulence of each strain of PRRSV was evaluated mainly on the clinical status of the corresponding litters at farrowing. RESULTS: Most gilts remained clinically normal throughout the study and farrowed normally at or near the expected farrowing time. All virus strains crossed the placenta of principal gilts to infect fetuses in utero. The number of late-term dead fetuses (which appeared to be the best measure of relative virulence) ranged from 0 for litters of control gilts and gilts exposed to strain RespPRRS, to 38 for gilts exposed to strain NADC-8. All principal gilts became viremic and developed antibody against PRRSV. All strains persisted in alveolar macrophages of at least some principal gilts for at least 7 weeks after exposure. CONCLUSION: Strains of PRRSV vary in virulence. CLINICAL RELEVANCE: The effects of PRRSV on reproductive performance are strain dependent and this should be considered in making a tentative diagnosis on the basis of clinical observations.     

Isolation of porcine respiratory coronavirus from pigs affected with porcine reproductive and respiratory syndrome

Four cytopathogenic viruses were isolated in CPK cells derived from porcine kidneys from tonsils and lungs of 3 of 15 pigs affected with porcine reproductive and respiratory syndrome virus. Physicochemically and morphologically, the isolates were similar to a coronavirus. The isolates were not distinguished from transmissible gastroenteritis virus (TGEV) by a neutralization test using polyclonal antibodies, but differentiated from TGEV by monoclonal antibodies capable of discriminating between TGEV and porcine respiratory coronavirus (PRCV), indicating that the isolates were PRCV. In a serological survey of 30 serum samples each collected from about 50 days old pigs in the 2 affected farms, 29 (97%) and 15 (50%) sera were positive for neutralizing antibody against the isolate with the titers ranging from 2 to 64, respectively.     

A nonstructural and antigenic glycoprotein is encoded by ORF3 of the IAF-Klop strain of porcine reproductive and respiratory syndrome virus

Open reading frame 3 (ORF3) of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), Quebec strain IAF-Klop, was reverse-transcribed and cloned into the procaryotic expression vector pGEX-4T-1, then subcloned into the eucaryotic expression vector pAdCMV5 which was used as a shuttle vector to generate a replication-defective recombinant adenovirus. The procaryotic GST-ORF3 recombinant fusion protein was used to raise a monospecific antiserum in rabbits. By Western-immunoblotting with PRRSV-infected cell extracts, the ORF3 encoded protein had an estimated molecular mass (M(r)) of 42 kDa, similar to that of the protein expressed by the adenovirus vector. Endoglycosidase F digestion showed that the ORF3 encoded protein occurs in an highly glycosylated form (GP3) in the infected MARC-145 cells. Pulse-chase and radioimmunoprecipitation experiments revealed that the GP3 protein was present in amounts equivalent to those of the N, M, and GP5 proteins in the infected cells, whereas no GP3 could be detected in purified virions. During the first 30 min of chase, the GP3 undergoes a gradual downward shift of its apparent M(r), thought to result from trimming of the mannose-rich glycan structures. Tested convalescent pig sera that were found to be seropositive to PRRSV by indirect immunofluorescence reacted positively with the recombinant GST-ORF3 fusion protein by immunoblotting. Data indicated that the ORF3 protein of the Quebec reference strain of PRRSV is a highly glycosylated and antigenic protein, which is nonstructural.     

Seroprevalence of porcine reproductive and respiratory syndrome virus in Dutch weaning pigs

To determine whether under Dutch field conditions PRRSV infection occurs in weaning pigs before the finishing period, a cross-sectional study was performed on 32 breeding farms to estimate the seroprevalence of antibodies directed against PRRSV in 4- to 5-week-old and 8- to 9-week-old pigs. Farms were visited twice within 5 months, and during each sampling an average of 20 sera were randomly collected from a unit of 4- to 5-week-old and a unit of 8- to 9-week-old pigs. The sera (n = 2568) were tested in the IDEXX-ELISA for the presence of antibodies directed against PRRSV. The seroprevalence of PRRSV in 4- to 5-week-old pigs and 8- to 9-week-old pigs varied between both samplings for each farm. The seroprevalence in the younger pigs was significantly higher than in the older pigs for both samplings (p < 0.05), suggesting the presence of maternal antibodies. In addition, a longitudinal study was performed to evaluate the IDEXX-ELISA in detecting maternal antibodies directed against PRRSV and to determine the rate of decline of these antibodies in field sera. From serological results of eight litters, an average decay function was computed to quantify the maternal immunity to PRRSV. A seroprevalence in 8- to 9-weeks-old pigs of > or = 0.20 was calculated to indicate an active immune response to PRRSV. In the cross-sectional study in the pigs twenty-three percent of the units with 8- to 9-week-old pigs were considered to have an active serological response against PRRSV. We conclude that most Dutch pigs are seronegative for PRRSV at the start of the finishing period, since the results of this study showed that 77% of the units with 8- to 9-week-old pigs had a seroprevalence < 0.20.     

Duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine

The clinical consequences of single or multiple exposure of pregnant gilts to porcine reproductive and respiratory syndrome virus (PRRSV) at various stages of gestation were determined. Thirty-three pregnant gilts were allotted to 6 experimental groups (5 to 7 gilts/group). Gilts of groups 1 to 5 were exposed to strain NADC-8 of PRRSV at the following times: group 1, gestation day (GD) 1; group 2, GDs 1 and 90; group 3, GD 30; group 4, GDs 30 and 90; group 5, GD 90. Virus exposure was by either intrauterine (GD 1) or oronasal (GDs 30 and 90) inoculation. Gilts of group 6 were kept as nonexposed controls. Gilts were either necropsied on or about GD 111 (groups 1 to 5) or were allowed to farrow (group 6). The detection of PRRSV in serum of fetuses and piglets (within 12 hof birth) was considered evidence of transplacental infection. Transplacental infection and virus-induced death were and were not confirmed for groups 3, 4, and 5 and for groups 1, 2, and 6, respectively. Collectively, the results indicated that intrauterine exposure to PRRSV at GD 1 was without clinical effect (groups 1 and 2) and provided protection against subsequent exposure to the same strain of virus at GD 90 (group 2). The highest incidence of transplacental infection and fetal death followed a single exposure to PRRSV at GD 90 (group 5).     

Persistence of porcine reproductive and respiratory syndrome virus in intensive farrow-to-finish pig herds

We analyzed signal-averaged electrocardiograms (ECG) obtained in 50 patients with recent myocardial infarction (RMI: 25 anterior and 25 inferior) and 20 normal subjects to determine the relationship between the initial portion of the signal-averaged QRS complex and cardiac function and infarct size. We examined (1) the root mean square voltage (RMS10-40, microV), (2) the integration (A10-40, microV.msec) at 10-msec intervals over the first 40 msec of the signal-averaged QRS complex, and (3) the intervals (T) of the magnitude of the signal-averaged ECG achieved at 10-microV intervals over the first 40 microV (T10-40, msec). The mean RMS10-40 (p < 0.01) and A10-40 (A10, p < 0.05; A20-40, p < 0.01) were significantly lower and the T10-40 (p < 0.01) was significantly longer in RMI patients than in normal subjects. The RMS10-40 (p < 0.01) and A10-40 (p < 0.05) were significantly lower and the T10-40 (T10.20, p < 0.01; T30.40, p < 0.05) was significantly longer in patients with anterior RMI patients than in patients with inferior RMI. The A30 was correlated with the ejection fraction and total creatine kinase (CK) release in all patients (r = 0.73, and -0.78, respectively, p < 0.001). These results suggest that the A30 may be an important predictor of ventricular dysfunction and infarct size in patients with RMI.     

Monoclonal antibodies against conformationally dependent epitopes on porcine reproductive and respiratory syndrome virus

Monoclonal antibodies (MAbs) against porcine reproductive and respiratory syndrome virus (PRRSV) were prepared and characterized. Four MAbs were developed from the mice immunized with the recombinant GP4 protein expressed in insect cells, and six MAbs were derived from the immunization with recombinant GP5 protein. All of the MAbs showed strong perinuclear fluorescence in PRRSV VR2385 infected cells by immunofluorescence staining. Among the MAbs to GP5 protein, one showed strong reactivity in ELISA and recognized a 26 kDa band of PRRSV in a western blot assay, while another showed neutralizing activity against the VR2385 isolate. Out of the four MAbs to GP4 protein, one showed mild reactivity in ELISA with detergent extracted antigen, but had no reactivity in a western-blot assay. The failure of MAb binding to detergent extracted antigen in ELISA or in western-blot analysis indicated that the MAbs were against conformationally dependent epitopes. Reactivity patterns of the MAbs with PRRSV field isolates tested by fixed-cell ELISA showed that there are antigenic variations in PRRSV GP4 and GP5 proteins. Development of these MAbs will benefit further studies on PRRSV structural proteins as well as in understanding their roles in PRRSV pathogenesis.     

Experimental infection of maternally immune pigs with porcine reproductive and respiratory syndrome (PRRS) virus

To investigate the influence of maternal antibody to porcine reproductive and respiratory syndrome (PRRS) virus infection, the following examination was done using conventional and SPF pigs. Ten 17-day-old conventional pigs with maternal antibody against PRRS virus and 6 44-day-old SPF pigs seronegative were inoculated intranasally with 10(5.0) TCID50 of PRRS virus. Two conventional and 4 SPF pigs were served as non-inoculated control. In conventional pigs, coughing and febrile response were observed after inoculation, and mean rate of weight gain reduced. One of the inoculated conventional pigs died on post-inoculation-day (PID) 28 and Haemophilus parasuis was isolated from the lung. Although febrile response was also observed in the inoculated SPF pigs, reduction in weight gain rate was not recognized. Virus was isolated from all the sera of inoculated conventional and SPF pigs except one conventional pig between PID 7 and 49, and between PID 7 and 28, respectively. Onset of viremia in the several conventional pigs delayed. Virus was isolated from the tissues of the 5 conventional pigs on PID 65 and from the tissues of the dead pig. On the other hand, virus was not isolated from the tissues of non-inoculated conventional pigs, and inoculated and non-inoculated SPF pigs. At the virus inoculation, antibodies by the indirect fluorescent antibody (IFA) assay against PRRS virus were detected in the sera of conventional pigs with antibody titers of 1:20. Antibody titers gradually decreased after inoculation and rose from PID 21 or 28 and were between 1:160 and 1:640 on PID 63. Virus neutralization (VN) antibody titers were 1:2 or 1:4 at the inoculation and gradually decreased. Apparent rise in VN antibody titer was not observed after the inoculation. In the sera of control pigs, both antibody titers gradually decreased and did not rise. In the sera of the SPF pigs, antibodies by the IFA assay were first detected on PID 7 or 14. The titers of antibodies rose and reached their maximum with 1:320 to 1:2,560 on PID 21 to 35. VN antibodies were first detected in PID 42 to 56 and thereafter, the titers ranged between 1:1 to 1:4. Control SPF pigs were free of antibody throughout the examination. Antigenic variability was not recognized between the inoculated and recovered viruses by the VN test. The prolonged duration of viremia and virus isolation from the tissues on PID 65 in conventional pigs with low maternal antibody might support the present of antibody-dependent enhancement activity of PRRS virus infection.     

Transplacental infection following exposure of gilts to porcine reproductive and respiratory syndrome virus at the onset of gestation

The effect of oral amphetamine administration on the Kamin-blocking effect in healthy volunteer subjects was investigated. Against predictions, Kamin blocking was not disrupted by either a high or low oral dose of D-amphetamine under conditions which have, in previous studies, led to disruption of a related learning phenomenon (latent inhibition). This lack of effect of amphetamine administration upon Kamin blocking weakens hypotheses that this cognitive process is mediated by the same changes in dopaminergic activity which affect latent inhibition. Currently, the only data which show strong comparative associations between Kamin blocking and latent inhibition are when they are applied to schizophrenic populations. These results may suggest that Kamin blocking and latent inhibition may be measuring different aspects of schizophrenic cognitive dysfunction.     

Comparison of the antigen distribution of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus

OBJECTIVE: The present study investigates the comorbidity between bulimia nervosa (BN) and the entire range of American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, 3rd rev. ed. (DSM-III-R) personality disorders and controls for the presence of coexisting depression. METHOD: The Personality Disorders Examination (PDE), a structured interview that encompasses all 13 (provisional) DSM-III-R personality disorders, was administered to three groups of subjects: depressed BN patients (n = 15), nondepressed BN patients (n = 15), and nonpsychiatric controls (n = 15). The BN patients were referrals to a dieting disorder unit affiliated with the University of Sydney. They all met DSM-III-R criteria and all had body mass indexes (BMIs) greater than 19. The nonpsychiatric control group were recruited from an undergraduate psychology course. All subjects were given the Bulimic Investigatory Test, Edinburgh (BITE), the Eating Disorders Inventory-2 (EDI-2), the Hamilton Depression Rating Scale (HDRS), and the PDE. RESULTS: 46.7% of depressed BN patients met the criteria for at least one Axis II diagnosis, as assessed by the PDE, and 33.3% of nondepressed BN patients received such a diagnosis, whereas only 6.7% of nonpsychiatric control subjects met this criterion (p < .05). The results of the present study provide support for an increased comorbidity between personality disorders and BN that cannot be attributed to the confounding influence of coexisting depression. DISCUSSION: This finding enables the identification of subgroups of individuals with BN, enabling them to be compared and contrasted. The identification of differences between subgroups may provide information regarding prognosis and differential response to treatment, which could enable more appropriate treatment decisions to be made.     

Apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus

We studied apoptosis caused by porcine reproductive and respiratory syndrome virus (PRRSV) in vivo, focusing on the tissues that constitute the main targets for infection: lung and lymphoid tissues. Previous investigators have shown that the PRRSV glycoprotein p25, encoded by PRRSV open reading frame 5, induces apoptosis when expressed in COS-1 cells. Results of studies conducted in our laboratory indicate the simultaneous occurrence of PRRSV-induced alterations of spermatogenesis and apoptotic death of germinal epithelial cells in the testicle. In this study, the goal was to determine whether virus-induced apoptosis is a direct mechanism of cell death caused by PRRSV in infected pigs. Eight 3-week-old pigs were intranasally inoculated with PRRSV 16244B, a highly virulent field strain. Lung, tonsil, bronchial lymph node, spleen, and heart were assessed histologically at 4 and 7 days postinfection. To characterize PRRSV-infected cells and apoptotic cell death, we used immunohistochemical methods for detection of viral antigen, DNA electrophoresis for detection of DNA fragmentation, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick end labeling method for in situ detection of DNA strand breaks, and electron microscopy for ultrastructural morphologic studies. PRRSV infection resulted in widespread apoptosis in the lungs and lymphoid tissues of infected pigs. Virus infection-induced apoptotic cells were more abundant than PRRSV-infected cells in all tissues. DNA laddering was detected in lung and lymphoid tissues. However, double-labeling experiments demonstrated that the majority of apoptotic cells did not colocalize with PRRSV-infected cells. Our findings suggest the presence of an indirect mechanism in the induction of apoptosis for PRRSV.     

Efficacy of an inactivated vaccine for prevention of reproductive failure induced by porcine reproductive and respiratory syndrome virus

This report describes the results of experiments with an inactivated oily vaccine containing per dose about 10(5.5) median tissue culture infectious dose (TCID50) of the Spanish strain of porcine reproductive and respiratory syndrome (PRRS) virus grown in porcine alveolar macrophages (PAMs). In order to evaluate the efficacy of the vaccine, two experimental infection routes were tested in sows; subsequent intranasal (i.n.) and intravenous (i.v.) (out of a total of 93 piglets born to 7 sows, 16% were mummified, 18.2% were weak and died within 48 h of birth, 37% were stillborn, 5.3% died between 2 and 7 days of age, 22.5% lived for more than one week) and intranasal alone (out of a total of 65 piglets born to 5 sows, 0% were mummified, 22.5% were weak and died within 48 h of birth, 40% were stillborn, 4.6% lived for more than one week). I.N. alone was selected to evaluate the efficacy of the vaccine because this is the natural route of infection. A number of experiments were conducted to test the immunogenicity of the vaccine. In general, after challenge with the homologous strain, protection in vaccinated sows was high (at least 70% of the piglets were born alive and healthy), whereas protection in unvaccinated sows was low (only 10% of the piglets were born alive and healthy). Vaccinated animals devoid of antibodies by immunoperoxidase monolayer assay (IPMA) at the time of challenge were still protected at experimental infection.     

Changes of lymphocyte subpopulations in pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus

Abnormal changes of T-cell subpopulation were observed in the peripheral blood lymphocytes of pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus. Pigs with naturally occurring PRRS revealed increases in CD2+ and CD8+ cells, and decreases in CD4+ cells and the ratios of CD4+/CD8+ cells. Specific-pathogen-free pigs inoculated with PRRS virus showed remarkable decreases in total lymphocytes, CD4+ and CD2+ cells on Postinoculation Day (PID) 3. The decline of CD4+ cells continued for at least 14 days, while CD2+ cells showed a tendency to increase thereafter. On the other hand,CD8+ cells slightly decreased in number on PID 3, and then increased remarkably; their number was significantly larger on PIDs 28 and 35 than on PID 0. The ratios of CD4+/CD8+ cells were significantly low between PIDs 3 and 28 as compared with PID 0. However, there were no differences in thymocyte subpopulations between infected and non-infected pigs, suggesting that the PRRS virus does not modulate intrathymic T-cell differentiation. In an experiment with peripheral blood mononuclear cell cultures, PRRS virus caused neither alteration of T-cell subpopulations nor cell proliferation, suggesting that the virus is not cytotoxic for CD4+ cells and not mitogenic for CD8+ cells.     

Studies of porcine reproductive and respiratory syndrome (PRRS) virus infection in avian species

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently recognized virus of swine. As a newly emerging virus, much of the basic information regarding PRRSV is in the process of discovery. We report three experiments with PRRSV in birds, and a fourth experiment to evaluate the infectivity and transmissibility of avian-derived PRRSV in swine. Experiment 1 compared the susceptibility of Muscovy ducks, Mallard ducks, guinea fowl, and chickens to PRRSV. Birds were exposed to PRRSV (ATCC VR-2402) in drinking water and virus isolation was attempted from feces collected from cages. Based on the duration of fecal shedding of the virus, this experiment showed that Mallard ducks were particularly susceptible to PRRSV. Experiment 2 was done in mallards to corroborate and augment the observations of experiment 1. Virus was isolated from pooled mallard feces up to 25 days post exposure (PE) and from the intestinal contents of 8 of 20 birds euthanized on day 38 PE. No gross or microscopic lesions were observed in ducks collected between 0 and 15 days PE. Experiment 3 evaluated the infectivity and transmissibility of mallard-derived PRRSV in mallards. A cage of mallards orally exposed to PRRSV shed the virus in feces. Exposure of a second cage of mallards to feces from the first cage resulted in fecal shedding of PRRSV by birds in cage two. In turn, exposure to feces from the second cage led to fecal shedding by mallards in a third cage. Experiment 4 assessed the infectivity and transmissibility of mallard-derived virus in swine. Pigs intranasally exposed to PRRSV isolaed from mallard feces in experiment 2 became viremic, seroconverted by ELISA, and transmitted the virus to sentinel swine. Collectively, these studies show that the possibility exists for avian species to be involved in the epidemiology of PRRSV. This is the first report of PRRSV infection in a species other than swine.     

Comparison of four RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus by RT-PCR

We investigated the effect of repeated cold stress (RCS) on the capsaicin-evoked release of glutamate from the primary afferent fibers of the rat, and compared this with the effect of inoculation of complete Freund's adjuvant (adjuvant inoculation). The release of glutamate was measured using a fluorometric on-line continuous monitoring system in which the immobilized glutamate dehydrogenase column was connected to an in vitro superfusion system. In the presence of 0.3 microM tetrodotoxin, the application of 1 microM capsaicin to spinal dorsal horn slices evoked glutamate release (18.6 +/- 1.2 pmol mg(-1) protein, n = 11). In rats subjected to RCS (RCS rats), the release of glutamate evoked by 1 microM capsaicin was markedly increased to 272% (n = 6, P < 0.05) of the value for the control group, although the basal release was not significantly altered (n = 6, P > 0.05). Adjuvant inoculation produced a significant increase in the basal and capsaicin (1 microM) evoked release of glutamate to 141 and 344% (n = 6, P < 0.05) of the value for the control group, respectively. The present results suggest that the facilitated release of glutamate from capsaicin-sensitive primary afferent terminals in the spinal dorsal horn is, at least in part, involved in the hyperalgesia of RCS rats as well as the complete Freund's adjuvant-induced hyperalgesia.     

Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds

Vaccine-like viruses of American type of porcine reproductive and respiratory syndrome virus (PRRSV) were detected in serum samples by RT-PCR. The viruses were analysed by nucleotide sequencing of the genomic region encoding open reading frames 2 to 7. During the ongoing study of Danish isolates of PRRSV by means of nucleotide sequencing, RT-PCR reactions and subsequent nucleotide sequencing showed the presence of American type PRRSV in Danish breeding herds. Most likely, these atypical viruses originated from boars vaccinated with live vaccine of American type (MLV RespPRRS), which were taken to artificial insemination centres and there brought together with unvaccinated boars already at the centres. The nucleotide sequences of three Danish viruses of American type PRRSV were compared to those of known PRRSV isolates. The nucleotide sequence identities of the atypical Danish isolates were between 99.2-99.5% to the vaccine virus RespPRRS and 99.0-99.3% to VR2332 which are the parental virus to the vaccine virus. Phylogenetic analysis including field isolates of American type supports the conclusion that the introduction of American type PRRSV in Denmark was due to spread of vaccine virus.     

Mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia

An experimental model that demonstrates a mycoplasma species acting to potentiate a viral pneumonia was developed. Mycoplasma hyopneumoniae, which produces a chronic, lymphohistiocytic bronchopneumonia in pigs, was found to potentiate the severity and the duration of a virus-induced pneumonia in pigs. Pigs were inoculated with M. hyopneumoniae 21 days prior to, simultaneously with, or 10 days after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV), which induces an acute interstitial pneumonia in pigs. PRRSV-induced clinical respiratory disease and macroscopic and microscopic pneumonic lesions were more severe and persistent in M. hyopneumoniae-infected pigs. At 28 or 38 days after PRRSV inoculation, M. hyopneumoniae-infected pigs still exhibited lesions typical of PRRSV-induced pneumonia, whereas the lungs of pigs which had received only PRRSV were essentially normal. On the basis of macroscopic lung lesions, it appears that PRRSV infection did not influence the severity of M. hyopneumoniae infection, although microscopic lesions typical of M. hyopneumoniae were more severe in PRRSV-infected pigs. These results indicate that M. hyopneumoniae infection potentiates PRRSV-induced disease and lesions. Most importantly, M. hyopneumoniae-infected pigs with minimal to nondetectable mycoplasmal pneumonia lesions manifested significantly increased PRRSV-induced pneumonia lesions compared to pigs infected with PRRSV only. This discovery is important with respect to the control of respiratory disease in pigs and has implications in elucidating the potential contribution of mycoplasmas in the pathogenesis of viral infections of other species, including humans.