A strain of highly pathogenic porcine reproductive and respiratory syndrome virus: genomic characterization, pathogenicity, and construction of an infectious full-length cDNA clone.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which inflicts major economic losses on the global pig farming industry. Based on its similarity to highly pathogenic strains, the GDzj strain isolated in this study was predicted to be highly pathogenic. We therefore analyzed the pathogenicity of this strain experimentally in piglets. All piglets challenged with this virus experienced fever or high fever, loss of appetite, decreased food intake, daily weight loss, shortness of breath, and listlessness, and the necropsy results showed that they had experienced severe interstitial pneumonia. We then used the BAC system to construct a full-length cDNA infectious clone of GDzj, and the rescued virus displayed in vitro proliferation characteristics similar to those of the parental PRRSV strain. In summary, we successfully isolated a highly pathogenic PRRSV strain and constructed a full-length infectious cDNA clone from it, thereby providing an effective reverse genetics platform for further study of viral pathogenesis.

Transcriptional Immune Signatures of Alveolar Macrophages and the Impact of the NLRP3 Inflammasome on Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Replication.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a contagious viral (PRRSV) disease in pigs characterized by poor reproductive health, increased mortality, and reductions in growth rates. PRRSV is known to implement immuno-antagonistic mechanisms to evade detection and mute host responses to infection. To better understand the cellular immunosignature of PRRSV we have undertaken transcriptome and immunomodulatory studies in PRRSV-infected porcine alveolar macrophages (PAMs). We first used genome-wide transcriptome profiling (RNA-seq) to elucidate PRRSV-induced changes in the PAM transcriptome in response to infection. We found a number of cellular networks were altered by PRRSV infection, including many associated with innate immunity, such as, the NLRP3 inflammasome. To further explore the role(s) of innate immune networks in PRRSV-infected PAMs, we used an NLRP3-specific inhibitor, MCC950, to identify the potential functionality of the inflammasome during PRRSV replication. We found that PRRSV does quickly induce expression of inflammasome-associated genes in PAMs. Treatment of PAMs with MCC950 suggests NLRP3 inflammasome activation negatively impacts viral replication. Treatment of PAMs with cell culture supernatants from macrophages subjected to NLRP3 inflammasome activation (via polyinosinic-polycytidylic acid (poly I:C) transfection), prior to PRRSV infection resulted in significantly reduced viral RNA levels compared to PAMs treated with cell culture supernatants from macrophages subjected to NLRP3 inflammasome inhibition (MCC950 treatment/poly I:C transfection). This further supports a role for NLRP3 inflammasome activation in the innate macrophagic anti-PRRSV immune response and suggests that PRRSV is sensitive to the effects of NLRP3 inflammasome activity. Taken together, these transcriptome and immunoregulatory data highlight the complex changes PRRSV infection induces in the molecular immune networks of its cellular host.

Perturbation of Thymocyte Development Underlies the PRRS Pandemic: A Testable Hypothesis.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes immune dysregulation during the Critical Window of Immunological Development. We hypothesize that thymocyte development is altered by infected thymic antigen presenting cells (TAPCs) in the fetal/neonatal thymus that interact with double-positive thymocytes causing an acute deficiency of T cells that produces "holes" in the T cell repertoire allowing for poor recognition of PRRSV and other neonatal pathogens. The deficiency may be the result of random elimination of PRRSV-specific T cells or the generation of T cells that accept PRRSV epitopes as self-antigens. Loss of helper T cells for virus neutralizing (VN) epitopes can result in the failure of selection for B cells in lymph node germinal centers capable of producing high affinity VN antibodies. Generation of cytotoxic and regulatory T cells may also be impaired. Similar to infections with LDV, LCMV, MCMV, HIV-1 and trypanosomes, the host responds to the deficiency of pathogen-specific T cells and perhaps regulatory T cells, by "last ditch" polyclonal B cell activation. In colostrum-deprived PRRSV-infected isolator piglets, this results in hypergammaglobulinemia, which we believe to be a "red herring" that detracts attention from the thymic atrophy story, but leads to our second independent hypothesis. Since hypergammaglobulinemia has not been reported in PRRSV-infected conventionally-reared piglets, we hypothesize that this is due to the down-regulatory effect of passive maternal IgG and cytokines in porcine colostrum, especially TGFß which stimulates development of regulatory T cells (Tregs).

Production diseases in smallholder pig systems in rural Lao PDR.

Pigs in Lao People's Democratic Republic are important for income and food security, particularly in rural households. The majority of pigs are reared in smallholder systems, which may challenge the implementation of any disease control strategies. To investigate risk factors for pig production diseases in such farming systems in the country a serological survey was conducted during 2011. A total of 647 pigs were sampled, accounting for 294 households in Luang Prabang and 353 in Savannakhet province representing upland and lowland, respectively. The results demonstrated that pigs in Lao PDR had antibodies against erysipelas (45.2%), CSF (11.2%), PRRS (8.6%), FMD O (17.2%) and FMD Asia 1, (3.5%). Differences in the housing systems influenced disease risk, for example, penned pigs had reduced odds of FMD and CSF, compared to those in scavenger systems. Pigs owned by farms using a sanaam (a communal area where pigs are kept for some time of the year) had 3.93 (95% confidence interval (CI): 1.09-14.7) times the odds of having pigs seropositive for FMD. Farms on which sudden piglet deaths had been experienced were more likely to have pigs seropositive for FMD O and erysipelas. These diseases constrain the development of village farming and the wider livestock industry due to their impact on productivity and trade. Vaccination coverage for FMD and CSF was low and there was a lack of national funding for livestock disease control at the time of the study. Further investigation into sustainable low-cost control strategies for these pathogens is warranted.

Development and validation of a scoring system to assess the relative vulnerability of swine breeding herds to the introduction of PRRS virus.

Biosecurity is defined as the set of practices carried out to prevent the introduction and spread of infectious agents in a herd. These practices are essential in swine production, especially for highly infectious agents such as porcine reproductive and respiratory syndrome virus (PRRSv). Even with years of research and experience over the last three decades, PRRSv is still causing productivity losses and is the major health problem affecting the global swine industry. Despite knowledge of the various ways in which the virus can be transmitted from one herd to another (e.g. animals, semen, truck, air, and people), determining the most frequent ways in which the virus is transmitted in the field is difficult. A systematic approach to assess vulnerabilities at a herd level related to PRRSv transmission could help producers prioritize biosecurity practices to reduce or avoid the occurrence of outbreaks. The aim of this study was to develop a biosecurity vulnerability score that represents the relative vulnerability of swine breeding herds to the introduction of PRRSv. To create the biosecurity vulnerability score (outcome), a multi-criteria decision analysis methodology was used to rank and quantify biosecurity practices based on expert opinion. To validate the biosecurity vulnerability score, a survey of biosecurity practices and PRRS outbreak histories in 125 breed-to-wean herds in the U.S. swine industry was used. Data on the frequency of PRRS outbreaks was used to test the hypothesis that biosecurity vulnerability scores were different between farms that have a low incidence of PRRS outbreaks, compared to farms that have a high incidence. In the two databases used, the scores consistently showed that farms with higher scores have a higher frequency of PRRS outbreaks. In the first validation, farms that had never had an outbreak investigation before had a significant (p < 0.02) lower score (0.29; 0.21-0.37) when compared to farms that had 2 or more outbreaks (0.43; 0.39-0.46). In the second, the farms of the control group also had significant (p < 0.004) lower scores (0.30; 0.27-0.33) compared to the case group (0.35; 0.33-0.38). Also, the results suggest that events related to swine movements, transmission by air and water, and people movements should be prioritized. The biosecurity vulnerability scores may be useful to assess vulnerabilities on biosecurity protocols in order to reduce the frequency of PRRS outbreaks and may help producers and veterinarians prioritize investments in improving biosecurity practices over time.

Mycoplasma hyorhinis is a potential pathogen of porcine respiratory disease complex that aggravates pneumonia caused by porcine reproductive and respiratory syndrome virus.

The porcine respiratory disease complex (PRDC) caused by numerous bacterial and viral agents has a great impact on pig industry worldwide. Although Mycoplasma hyorhinis (Mhr) has been frequently isolated from lung lesions from pigs with PRDC, the pathological importance of Mhr may have been underestimated. In this study, 383 serum samples obtained from seven herds with a history of PRDC were tested for specific antibodies to Mhr, Mycoplasma hyopneumoniae (Mhp), and porcine reproductive and respiratory syndrome virus (PRRSV). Seropositive rates of PRRSV were significantly correlated with those of Mhr (correlation coefficient, 0.862; P-value, 0.013), but not with those of Mhp (correlation coefficient, -0.555; P-value, 0.196). In vivo experiments demonstrated that pigs co-infected with Mhr and PRRSV induced more severe lung lesions than pigs infected with Mhr or PRRSV alone. These findings suggest that Mhr is closely associated with pneumonia caused by PRRSV and provide important information on Mhr pathogenesis within PRDC. Therefore, effective PRDC control strategies should also consider the potential impact of Mhr in the pathogenesis of PRDC.

Preliminary Study on Prevalence, Risk Factor and Genetic Homogeneity of Porcine Reproductive and Respiratory Syndrome Virus in Registered Pig Farms in Heilongjiang, China.

While porcine reproductive and respiratory syndrome (PRRS) causes great economic losses in southern and central China, systematic studies on the epidemiology of PRRS virus (PRRSV) in Heilongjiang Province had not been performed. Therefore, we conducted a preliminary study to estimate the prevalence and risk factors associated with PRRSV infection, as well as characterize the PRRSV in registered pig farms in Heilongjiang Province, China in 2011. A total of 1237 blood samples were collected from 72 farms and tested by reverse transcription polymerase chain reaction (RT-PCR) for PRRSV. Risk factors associated to PRRSV infection were analysed using logistic regression models. Genes of non-structural protein-2 (Nsp2) and glycoprotein 5 (GP5) from 22 isolates were sequenced for phylogenetic analysis. The results showed that the herd apparent prevalence was 9.7% (95% CI: 6.3, 13.1) in Heilongjiang Province. An increased risk of PRRSV infection on farms was associated with unrestricted movement of external people (OR = 14.1, 95% CI: 1.68, 119.07), close proximity (<1 km) to the nearest house, road or neighbouring farm (OR = 16.2, 95% CI: 1.52, 171.80), and selling farm products at both local and provincial markets (OR = 20.6, 95% CI: 2.02, 210.56). Phylogenetic analysis based on partial amino acid sequences of GP5 and Nsp2 showed that all the 22 PRRSV isolates in Heilongjiang are closely related to the highly pathogenic PRRSV strain JXA1 and belong to the Genotype 2 (American genotype). The prevalence, determination of risk factors and phylogenetic characterization will provide information for future epidemiological studies and a reference for developing surveillance and control strategies in this region.

Pathogenesis of type 1 (European genotype) porcine reproductive and respiratory syndrome virus in male gonads of infected boar.

The objective of this study was to determine the pathogenesis of experimental infection with a type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the sites of viral replication and apoptosis in male gonads from infected boars for a period of 21 days after intranasal inoculation. Microscopically, hypospermatogenesis and abundant germ cell depletion and death were observed in the testes. Such germ cell death occurs by apoptosis, as determined by a characteristic histological patterns and evidence of massive DNA fragment detected in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) reaction. PRRSV was detected in the testicular tissue of infected boars only. Viral nucleic acid was localized in spermatogonia, spermatocytes and spermatids but not in the vesicular and bulbourethral gland. In serial sections, PRRSV-positive cells did not co-localized with apoptotic cells. TUNEL-positive apoptotic cells were more numerous than PRRSV-positive cells in testicular sections. The present study demonstrated that type 1 PRRSV infects the spermatogonia and their progeny, and induces apoptosis in these germ cells.

Effects on boar semen quality after infection with porcine reproductive and respiratory syndrome virus: a case report.

The effect of porcine reproductive and respiratory syndrome virus (PRRSV) on semen quality was examined in a group of 11 spontaneously infected boars in a commercial boar stud. Semen samples were collected 4 weeks prior to 4 weeks post-infection (wpi). Infection with PRRSV of the European genotype subtype 1 (EU-1) was verified by specific quantitative real-time polymerase chain reaction (RT-PCR) in 36% of the serum samples. All boars seroconverted before 4 wpi and remained in normal condition throughout the study. Comparison of the percentage of morphologically intact spermatozoa revealed an increase of acrosome-defective spermatozoa (P = 0.012) between -4 and 4 wpi. Significant deleterious effects on semen quality were detected for membrane integrity when semen had been stored for 2 days after sampling. Analysis of sperm subpopulations in a thermoresistance test on day 7 after sampling revealed alterations in the percentage of circular, progressively motile spermatozoa (P = 0.013), in the percentage of non-linear, progressively motile spermatozoa (P = 0.01), and on the amplitude of lateral sperm head displacement (P = 0.047). There was no difference in the incidence of mitochondrially active spermatozoa (P = 0.075). Investigation of routine production data between pre- and post-infection status showed no differences on ejaculate volume (P = 0.417), sperm concentration (P = 0.788), and percentage of motile spermatozoa (P = 0.321). This case report provides insights into a potential control strategy for PRRSV outbreaks in boar studs.

Epidemiological investigations in regard to porcine reproductive and respiratory syndrome (PRRS) in Quebec, Canada. Part 1: biosecurity practices and their geographical distribution in two areas of different swine density.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a considerable threat to the swine industry and implementing biosecurity measures is essential for the control of its transmission. The aims of this study were: (1) to describe biosecurity practices in production sites located in a moderate density (MD) and a high density (HD) pig area according to production type; (2) to group sites in different patterns according to their biosecurity practices; and (3) to determine the geographical distribution of sites according to biosecurity patterns. Biosecurity practices were selected based on PRRS epidemiology. A questionnaire was completed on 125 breeding sites (MD=54; HD=71) and 120 growing (HD) sites, between 2005 and 2008. Depending on area and production type, the frequency of biosecurity practices used ranged from 0 to 2% for barrier at site entrance, 0 to 19% for use of shower, 25 to 35% for washing truck between loads of pigs, 51 to 57% for absence of rendering or rendering without access to the site, and 26 to 51% for absence of gilt purchase or purchase with quarantine. Better practices pertaining to entrance protocol (i.e. "no-entry" sign, shower, ≥24 h downtime) were reported more frequently on breeding sites in the MD than the HD area (P<0.05). In the HD area, growing sites had in general a lower level of biosecurity than breeding sites. Using a two-step clustering procedure performed separately for breeding and growing sites, two different patterns were obtained for each production type, which corresponded to a high and low level of biosecurity. For breeding sites, a higher biosecurity level was observed at sites located away from other pig sites, set at more than 300 m from the public road, having higher sow inventory, or being part of an integrated production (P<0.05). Spatial clusters of sites for each biosecurity pattern were detected. This study identified some shortcomings regarding biosecurity that should be addressed before implementing any PRRSV regional control. Vicinity of sites with different biosecurity levels also suggests difficulties in planning priorities of intervention based on geographical distribution of sites.

Epidemiological investigations in regard to porcine reproductive and respiratory syndrome (PRRS) in Quebec, Canada. Part 2: prevalence and risk factors in breeding sites.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat for swine industry and understanding factors involved in its epidemiology is undoubtedly essential for disease control. As a part of a larger project, a cross-sectional study was performed on breeding sites in a moderate density area of swine production in Quebec to estimate the prevalence of PRRSV infected sites and to evaluate if characteristics of sites and biosecurity practices, either as specific measures or as a global score, were associated with PRRSV status. A questionnaire and diagnostic procedures were performed on 54 breeding sites between September 2006 and August 2008. A biosecurity score that had been previously computed using two-step clustering procedure was used, classifying breeding sites into two biosecurity patterns (high vs. low) according to 21 specific biosecurity measures. The apparent prevalence of PRRSV infected sites was 74.0% (95% CI, 60.3-85.0). Univariable and multivariable logistic regression models with robust standard errors adjusting for potential clustering of sites due to same ownership were computed. In a first multivariable model evaluating characteristics of sites and specific biosecurity variables, four main effects were significantly associated (P<0.05) with PRRSV positive status: large pig inventory (OR: 10.7), proximity to closest pig site (OR: 7.3), absence of shower (OR: 8.7) and free access to the main entrance of the site by the rendering truck (OR: 7.0). In a second multivariable model including a global biosecurity score as a surrogate for a specific pattern of biosecurity measures, this score was not retained in the final model. The adjusted population attributable fractions were 16% for the proximity to closest pig site variable, 27% for the absence of shower variable, and 10% for the free access to main entrance of the site by the rendering truck. These two latter biosecurity measures, manageable directly on the site, should be prioritized and be part of any intervention strategy designed for PRRSV control.

Direct inoculation of RNA transcripts from an infectious cDNA clone of porcine reproductive and respiratory syndrome virus (PRRSV) into the lymph nodes and tonsils of pigs initiates PRRSV infection in vivo.

The recent construction of PRRSV infectious cDNA clones affords the opportunity for structural and functional studies of PRRSV genes. However, the inherent instability of the PRRSV genome, the requirement of cell culture propagation, and poor virus recovery have limited the usefulness of the PRRSV reverse genetics system for in vivo studies. Here, we report a unique strategy of infecting pigs by bypassing the traditional in vitro cell culture step required for in vivo studies. We demonstrate that inoculation of RNA transcripts of a PRRSV infectious cDNA clone directly into the lymph nodes and tonsils of pigs produces active PRRSV infection. The information from this study will have significant implications for the study of the molecular mechanism of PRRSV pathogenesis using the reverse genetics system.

PMWS: an emerging disease identified in archived porcine tissues.

Postweaning multisystemic wasting syndrome (PMWS) is a disease caused by porcine circovirus type 2 (PCV-2). The disease was present as early as 1986 in Spain, 1989 in Japan and 1993 in Thailand. In view of this, we considered it possible that the disease may also have been present in Switzerland prior to its first description in 2001. A retrospective investigation was performed on paraffin-embedded lymphoid organs and ileum from 496 pigs aged 5-13 weeks collected between 1976 and mid-2001. The sections were investigated immunohistochemically using a monoclonal antibody specific for PCV-2 capsid antigen encoded by ORF2. Virus antigen was detected in tissue samples of 39 pigs from 28 farms. The earliest positive sample originated from 1986. After 1989, positive pigs were found almost every year among the 20-40 cases investigated annually. These results indicate that PCV-2 has been present in Switzerland for some time, and at least since 1986.

A survey of agents associated with neonatal diarrhea in Iowa swine including Clostridium difficile and porcine reproductive and respiratory syndrome virus.

This survey was undertaken to determine the relative frequency of agents that are currently associated with neonatal diarrhea in swine, including Clostridium difficile and porcine reproductive and respiratory syndrome virus (PRRSV). The subjects for this study were the first 100 live 1-7-day-old piglets submitted to the Iowa State University Veterinary Diagnostic Laboratory with a clinical signalment of diarrhea, beginning on January 1, 2000. The evaluation of each pig included bacterial culture of a section of ileum, 2 sections of jejunum, and a single section of colon; a fluorescent antibody test (FAT) or immunohistochemistry (IHC) for transmissible gastroenteritis virus (TGEV); ELISA's for rotavirus and C. difficile toxins; IHC for PRRSV; and microscopic examination of ileum, midjejunum, spiral colon, liver, spleen, and lung. Survey results demonstrate a decline in the relative number of diagnoses of TGEV, Escherichia coli, and Clostridium perfringens type C compared with retrospective data. The combined case frequency rate for these 3 pathogens dropped from 70% in 1988 to 21% in 2000. This survey also demonstrated the emergence of C. difficile as an important pathogen of neonatal swine. Clostridium difficle toxin was detected in the colon contents of 29% of the piglets, and at least 1 toxin-positive animal was identified in 55% of the cases. All 29 C. difficile toxin-positive piglets had mesocolonic edema, and colitis was observed in 21 of 29 toxin-positive animals. PRRSV-positive macrophages were detected in the lamina propria of intestinal villi by IHC in 10 piglets with diarrhea. In 6 of these cases, PRRSV was the only pathogen detected. Gross and microscopic lung lesions were not a reliable indicator of PRRSV infection in these neonatal pigs with diarrhea. The addition of tests for C. difficile and PRRSV to a routine neonatal diarrhea diagnostic protocol resulted in a significant increase in thediagnostic success rate on both individual animal and case bases.

Risk factors for infection of sow herds with porcine reproductive and respiratory syndrome (PRRS) virus.

In 1992, the porcine reproductive and respiratory syndrome virus (PRRSV) of European type (PRRSV-EU) was introduced in Denmark. By 1996, the virus had spread to approximately 25% of the Danish herds. In January 1996, a modified-live vaccine based on the American type of the virus (PRRSV-US) was used in replacement boars for Danish artificial insemination (AI) centres and from July 1996, the vaccine was used in PRRSV-EU infected herds for prevention of disease. Soon after vaccine introduction, PRRSV non-infected herds experienced outbreaks of disease due to infection with PRRSV-US. In this study, we investigated the risk factors (biosecurity level, animals, exposure from PRRSV-US-infected neighbour herds, semen, herd size, pig density and herd density) for infection with PRRSV-US in a cohort of 1071 sow herds; we used a nested case-control study. The retrospective observation period lasted from June 1996 (when they all were non-infected) to October 1997. Seventy-three non-vaccinated, closed sow herds became infected with the vaccine strain during this period. Each case herd was matched with two control herds from the cohort (controls had not been infected at the time of infection in the case herds). The data were analysed using a Cox-regression model. The hazard of infection increased significantly with exposure from PRRSV-US-infected neighbouring herds, purchase of animals from herds incubating PRRSV-US infection, increasing herd size and purchase of semen from boars at PRRSV-US-infected AI centres. The results are consistent with the modified-live vaccine strain spread to other herds by trade with animals and semen and by neighbour (area) transmission. We suggest that virus spread by aerosols was a frequent mode of transmission.

Experimental inoculation of late term pregnant sows with a field isolate of porcine reproductive and respiratory syndrome vaccine-derived virus.

The use of a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in piglets has been associated with reproductive disorders in non-vaccinated sows. Vaccine-derived virus (VDV) has been isolated from foetuses, stillborn pigs, and dead piglets, indicating that the live vaccine spread from vaccinated piglets to non-vaccinated sows, and that the virus might be implicated in the severe reproductive problems observed. In the present study, one such VDV isolate was used to experimentally infect pregnant sows in the last trimester. The chosen isolate, which had more than 99.6% identity to the attenuated vaccine virus, originated from the lungs of a stillborn pig from a swine herd with a sudden high level of stillborn pigs and increased piglet mortality in the nursing period. Intranasal inoculation of sows with the virus isolate resulted in congenital infection, foetal death, and preweaning pig mortality. As such, the present study showed that vaccine-derived PRRSV can cause disease in swine consistent with PRRS.

Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis.

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

Isolation of a cytopathogenic virus from a case of porcine reproductive and respiratory syndrome (PRRS) and its characterization as parainfluenza virus type 2.

From a lung of a fetus of a breeding sow showing PRRS-like symptoms a viral agent could be isolated. It was characterized as an enveloped, hemagglutinating RNA virus. Ultrastructural examination of purified virus revealed paramyxovirus-like pleomorphic virions of approx. 200 nm in diameter. The helical nucleocapsids were about 18 nm in diameter. The virus was found to be antigenically related to simian virus 5 (SV5) a prototype strain of parainfluenza virus type 2, but not to bovine respiratory syncytial virus, parainfluenza virus type 1, parainfluenza virus type 3, and Newcastle disease virus as determined by western blot analysis.

[Boar semen--a possible risk factor in infection occurrence of porcine reproductive and respiratory syndrome]. / Ebersperma--Ein möglicher Risikofaktor im Infektionsgeschehen des Porcinen Reproduktiven und Respiratorischen Syndroms.

Based on an experimental study using two and six PRRS-negative young boars and gilts, respectively, it was proven wether boar semen could be a risk factor in the transmission of the disease. The two boars were inoculated intranasally with the PRRS-virus strain I10 (Intervet) containing 107 TCID50/ml. Using ovulation synchronization six gilts were prepared as bioindicators to be inseminated, two of them with semen of the two boars at day 4, 8 and 12 after infection. Following inoculation of the boars, PRRS virus was shown to be present in blood from the 2nd until the 35th and 40th day p.i., respectively. PRRS virus could also be isolated from nasal swabs at day 6, 9, 12 and 19 and from preputial swabs at day 4, 12 and 27 after infection. PRRS virus could only be detected at day 19 p.i. in semen of one boar. Drastic changes in quality and volume of the ejaculate were observed about day 25 p.i. in both animals. Insemination of the gilts with semen collected at day 4, 8 and 12 p.i., however, did not lead to an infection of the females, because neither clinical signs typical for PRRS nor seroconversion could be observed. Reproductive parameters as well as birth and growing traits of the gilts were in accordance with norm values.

Chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs.

An immunogold-silver immunohistochemical technique was used to determine the chronological distribution and localization of porcine reproductive and respiratory syndrome virus (PRRSV) in experimentally infected gnotobiotic pigs. Thirty-two pigs were randomly allocated to infected (n = 24) or control (n = 8) groups. Pigs in infected groups were inoculated at 3 days of age by nasal instillation of PRRSV isolate ATCC VR-2332 (total dose = 10(2.64) TCID50), and control pigs were exposed in the same manner to uninfected cell culture supernatant. Three infected and one control pigs were euthanatized at 12 hours and at 1, 2, 3, 5, 7, 14, and 21 days postexposure (DPE). Bronchiolar epithelial cells, arteriolar endothelial cells, monocytes, and interstitial, alveolar, and intravascular macrophages stained for PRRSV antigen at 12 hours postexposure. Staining for PRRSV antigen in endothelial cells, monocytes, and alveolar, interstitial, and intravascular macrophages was most intense and widespread in lung sections from 14 and 21 DPE. In the heart, macrophages in the interstitial and subendocardial spaces and endothelial cells in a few arterioles stained for PRRSV antigen at 14 and 21 DPE. Tonsillar macrophages and mucosal epithelium stained for PRRSV antigen at 12 hours postexposure and sporadically with less intensity in subsequent sampling periods. In the nasal turbinate, PRRSV antigen was identified in macrophages within the mucosal epithelium at 12 hours postexposure and again at 14 and 21 DPE. There was focal staining for PRRSV antigen in the choroid plexus in one pig at 14 DPE. Based on the results of this experiment, the pathogenesis of PRRSV infection in gnotobiotic pigs can be described as initial virus entry through nasal epithelial, tonsillar, and pulmonary macrophages, with viremia occurring by 12 hours postexposure followed by the development of pneumonia, myocarditis, encephalitis, rhinitis, vasculitis, and lymphoid necrosis. Although PRRSV can infect macrophages in heart, tonsil, turbinate, and choroid plexus, pulmonary macrophages are predominantly and consistently infected and are the predominantly cells for virus replication in gnotobiotic pigs.