PRRSV-positive Cells Research Articles

Differential innate immune response of endometrial cells to porcine reproductive and respiratory syndrome virus type 1 versus type 2.

Modification of cellular and immunological events due to porcine reproductive and respiratory syndrome virus (PRRSV) infection is associated with pathogenesis in lungs. PRRSV also causes female reproductive dysfunction and persistent infection which can spread to fetus, stillbirth, and offspring. In this study, changes in cellular and innate immune responses to PRRSV type 1 or type 2 infection, including expression of PRRSV mediators, mRNA expression of Toll-like receptors (TLRs) and cytokine, and cytokine secretion, were examined in primary porcine glandular endometrial cells (PGE). Cell infectivity as observed by cytopathic effect (CPE), PRRSV nucleocapsid proteins, and viral nucleic acids was detected as early as two days post-infection (2 dpi) and persisted until 6 dpi. A higher percentage of CPE and PRRSV-positive cells were observed in type 2 infections. PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were upregulated following type 1 and type 2 infection. CD151, CD163 and Sn were upregulated by type 2. In both PRRSV types, mRNA expression of TLR1 and TLR6 was upregulated. However, TLR3 was upregulated by type 1, but TLR4 and TLR8 mRNA and protein were downregulated by type 2 only. Interleukin (IL)-1β, IL-6 and tumor necrotic factor (TNF)-α were upregulated by type 2, but IL-8 was upregulated by type 1. Both PRRSV type 1 and 2 stimulated IL-6 but suppressed TNF-α secretion. In addition, IL-1β secretion was suppressed only by type 2. These findings reveal an important mechanism underlying the strategy of PRRSV infection in the endometrium and associated with the viral persistence.

Role of zinc metalloprotease (ZMPSTE24) in porcine reproductive and respiratory syndrome virus (PRRSV) replication in vitro.

The transmembrane zinc metalloprotease ZMPSTE24 works in cooperation with interferon-induced transmembrane protein 3 (IFITM3) to restrict entry of several enveloped viruses. We investigated the role of ZMPSTE24 in porcine reproductive and respiratory syndrome virus (PRRSV) replication. ZMPSTE24 overexpression significantly reduced PRRSV replication in MARC-145 cells. Interestingly, knockdown of endogenous ZMPSTE24 did not significantly impact virus replication. There was no significant difference in the percentage of PRRSV-positive cells and viral RNA copies at 3 hours postinfection (hpi) between cells transfected with ZMPSTE24-FLAG and the vector control. Our results suggest that ZMPSTE24 overexpression may restrict PRRSV replication at a post-entry step.

Comparison of the Pathogenesis of Single or Dual Infections with Type 1 and Type 2 Porcine Reproductive and Respiratory Syndrome Virus

The aim of this study was to compare the pathogenicity of single or dual infections with type 1 and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) in pigs. Pigs were inoculated intranasally with type 1 or type 2 PRRSV or both viruses together. Pigs infected with type 1 and type 2 PRRSV together had significantly (P <0.05) fewer genomic copies of type 1 PRRSV than did pigs infected with type 1 PRRSV alone. Pigs infected with type 2 PRRSV alone or type 1 and type 2 PRRSV together had significantly (P <0.05) higher gross and microscopical lung lesion scores than did pigs infected with type 1 PRRSV alone. Pigs infected with type 2 PRRSV alone or type 1 and type 2 PRRSV together had significantly (P <0.05) higher scores for PRRSV-positive cells in the lung than did pigs infected with type 1 PRRSV alone. Pigs infected with type 1 PRRSV alone had significantly (P <0.05) higher scores for type 1 PRRSV-positive cells in the lung than did pigs infected with both types of PRRSV together. Pigs infected with both types of PRRSV together developed similar clinical disease and lesions as pigs infected with type 2 PRRSV alone. Significant differences in virulence were not observed between pigs infected with type 2 PRRSV alone and pigs infected with both types of PRRSV together in terms of viraemia, lung lesion score and virus distribution within lung lesions.

Comparative pathogenesis of type 1 (European genotype) and type 2 (North American genotype) porcine reproductive and respiratory syndrome virus in infected boar

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) now has two main genotypes, genotype 1 (European) and genotype 2 (North American). There is a lack of data on the comparison of pathogenicity of the two genotypes in boars. The objectives of the present study were to evaluate the amount of PRRSV present in semen over time and compare the viral distribution and microscopic lesions of type 1 and type 2 PRRSV-infected boars.MethodsTwenty-four 8-month-old PRRSV-naïve Duroc boars were randomly allocated to 3 treatment groups. The boars in groups 1 (n = 9) and 2 (n = 9) were intranasally inoculated with type 1 or type 2 PRRSV, respectively. The boars in groups 1 (n = 6) served as negative controls. Semen and blood samples were collected up to 35 days post-inoculation (dpi), and necropsies were performed on 14, 21, and 35 dpi.ResultsThere were no significant differences in the genomic copy number of PRRSV, microscopic testicular lesion score, number of PRRSV-positive germ cells, or number of apoptotic cells between the type 1 and type 2 PRRSV-infected boars throughout the experiment. Histopathological changes were manifested by the desquamation of spermatocytes and the presence of multinucleated giant cells in seminiferous tubules of both type 1 and type 2 PRRSV-infected boars. The distribution of PRRSV-positive cells was focal; the virus was found in single germ cells or small clusters of germ cells, localized to the spermatogonia, spermatocytes, spermatids, and non-sperm cells in type 1 and type 2 PRRSV-infected boars.ConclusionsThe results of this study demonstrated that two genotypes of PRRSV do not have significantly different virulence toward the male reproductive system of pigs.

Comparative Pathogenicity of Three Korean and One Lelystad Type 1 Porcine Reproductive and Respiratory Syndrome Virus (Pan-European Subtype 1) Isolates in Experimentally Infected Pigs

The aim of this study was to compare the pathogenicity of three type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolates that originated from Korean herds with varying severity of respiratory disease with one Lelystad virus. An experimental infection model was used to study virus distribution, sites of viral replication, viraemia, gross and microscopical lesions and the humoural immune response. Each virus isolate was given intranasally to 3-week-old pigs. Differences were found in the severity of gross and microscopical pulmonary lesions and the distribution of virus-labelled cells in lung and lymph nodes (LNs). The gross and microscopical pulmonary lesion scores were significantly greater in pigs inoculated with the SNUVR100744 isolate. The distribution of PRRSV-labelled cells within tissues and organs was similar for the different virus isolates; however, significantly more PRRSV-positive cells were detected in the lung and LNs of pigs inoculated with the SNUVR100744 isolate than were detected in the same tissues of pigs inoculated with Lelystad virus. The results of the present study demonstrate that type 1 PRRSV isolates differ in their ability to induce viral replication in tissues and induce interstitial pneumonia in pigs.

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 21days 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.

Evaluation of monoclonal antibody–based immunohistochemistry for the detection of European and North AmericanPorcine reproductive and respiratory syndrome virusand a comparison with in situ hybridization and reverse transcription polymerase chain reaction

The objective of the present study was to compare the ability of 2 monoclonal antibodies (mAbs; SDOW17 and SR30) to detect types 1 and 2 Porcine reproductive and respiratory syndrome virus (PRRSV) in formalin-fixed, paraffin-embedded (FFPE) lung tissues by immunohistochemistry (IHC) and to compare the immunohistochemical results with in situ hybridization (ISH) and reverse transcription nested polymerase chain reaction (RT-nPCR) detection techniques. Lungs from 30 experimentally infected pigs (15 pigs with each genotype of PRRSV) and 20 naturally infected pigs (10 pigs with each genotype of PRRSV) with types 1 and 2 PRRSV, respectively, were used for the IHC, ISH, and RT-nPCR analyses. The SR30 mAb-based IHC detected significantly more type 1 PRRSV-positive cells in the accessory and caudal lobes from the experimentally infected pigs at 7 (P = 0.025) and 14 (P = 0.018) days postinoculation, respectively, compared to the SDOW17 mAb-based IHC. The results demonstrated that SR30 mAb-based IHC is useful for detecting both types 1 and 2 PRRSV antigen in FFPE lung tissues.

Type 2 Porcine Reproductive and Respiratory Syndrome Virus Infection Mediated Apoptosis in B- and T-Cell Areas in Lymphoid Organs of Experimentally Infected Pigs

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection is characterized by persisting in lungs and lymphoid tissue, resulting in systemic lymphoid depletion. The aim of this study was to correlate the histological changes, viral antigen expression and apoptosis phenomena in tonsil, medial retropharyngeal and mediastinal lymph nodes of 12 pigs inoculated with a type 2 PRRSV isolate (Chilean strain 2402). Apoptosis phenomena were observed mainly in lymphocytes and secondly in macrophages of lymph nodes and tonsils of inoculated animals, showing a peak of both apoptotic cells and viral antigen expression at the end of the study (21 dpi). However, the number of apoptotic cells was higher than the number of PRRSV-positive cells at the end of the study. This finding together with the location of apoptotic cells and PRRSV-positive cells in different structures of lymphoid organs supports the hypothesis that PRRSV-positive macrophages might modulate the apoptosis phenomena in other cells, mainly lymphocytes, by means of an indirect mechanism. Furthermore, apoptotic cells were detected both in B- and T-cell areas of lymphoid organs, suggesting that apoptosis phenomena may play a role in the impairment of the host immune response during PRRS.

Pathogenesis of Korean Type 1 (European Genotype) Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected Pigs

The aim of this study was to elucidate the pathogenesis of experimental infection with Korean type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the virus distribution, sites of viral replication, viraemia and gross and microscopical lesions in conventional pigs studied for 28 days after intranasal inoculation. Mean rectal temperature was significantly higher in infected pigs than in negative control pigs at 2 days post inoculation (dpi) (P=0.004), 3 dpi (P<0.001), 4 dpi (P=0.003) and 5 dpi (P=0.034). The log(10)TCID(50)/ml of type 1 PRRSV increased significantly at 0-1 dpi (P=0.024) and 5-7 dpi (P=0.029), but decreased at 10-14 dpi (P=0.026) and 14-21 dpi (P=0.012) in infected pigs. Infected pigs developed multifocal, tan-mottled areas of lung tissue with irregular and indistinct borders. Microscopical lesions, when present, were multifocal, mild to moderate, generally most extensive at 5-7 dpi (P=0.036), and were nearly resolved at 28 dpi. Type 1 PRRSV nucleic acid and antigen were detected exclusively within the cytoplasm of macrophages and type I and II pneumocytes. The score for PRRSV-positive cells increased at 3-7 dpi (P<0.05) and decreased at 10-14 dpi (P=0.034) in infected pigs. Thus, respiratory disease was reproduced in conventional pigs by infection with Korean type 1 PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes apoptosis during its replication in fetal implantation sites

Reproductive failure due to porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by late-term abortions, early farrowing and an increase of dead and mummified fetuses and weak-born piglets. The mechanism of PRRSV-induced reproductive failure is poorly understood. Human pregnancies, complicated by some pathogens leading to reproductive disorders exhibit increased apoptosis in the fetal membranes. Because PRRSV-target cells are present in endometrium/fetal placentas from healthy sows and PRRSV-infected macrophages in other organs die by apoptosis, we hypothesized that PRRSV can replicate and induce apoptosis in the fetal implantation sites at the last stage of gestation. In the present study, identification, localization and quantification of the PRRSV-positive and apoptotic cells were performed in the fetal implantation sites. Three dams were inoculated intranasally with 10 5 TCID 50 PRRSV 07V063 at 90 days of gestation and sampled at 10 days post-inoculation. Two non-inoculated dams that were euthanized at 100 days of gestation served as control animals. Inoculation of the dams resulted in a viremia that lasted until the end of the study. Transplacental PRRSV spread was detected in all inoculated dams. Using immunofluorescence staining, single PRRSV-positive cells were found in the endometrial connective tissues adjacent to both PRRSV-positive and PRRSV-negative fetuses. In the fetal placental mesenchyme of the PRRSV-positive fetuses, infected cells were more abundant and spread focally. Double staining showed that all PRRSV-positive cells in the fetal implantation sites were positive for sialoadhesin and CD163. Apoptotic cells (TUNEL+) were detected in endometrium and fetal placentas of both non- and PRRSV-inoculated dams. The number of apoptotic cells was significantly higher in PRRSV-positive endometrium/fetal placentas. PRRSV caused apoptosis in infected cells since 20–61% of PRRSV-positive cells were apoptotic and in surrounding cells since 43–91% of the apoptotic cells were virus-negative. The main conclusion obtained from the present study is that PRRSV replicates in the fetal implantation sites and causes apoptosis in infected macrophages and surrounding cells at the last stage of gestation. The possible mode of PRRSV replication in the fetal implantation sites and the events that might contribute to the reproductive disorders are discussed.

Modulations of phenotype and cytokine expression of porcine bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus

Phenotypic and functional property changes of bone marrow-derived immature dendritic cells (BM-imDCs) after porcine reproductive and respiratory syndrome virus (PRRSV) infection have been detailed in a previous report. A down-regulated expression of MHC I molecules along with an up-regulated expression of CD80/86 were observed in BM-imDCs after the exposure to PRRSV. In this study, we further investigate the expression of surface phenotypes of BM-imDCs in relation to their infection status. Exposure of PRRSV to BM-imDCs resulted in a down-regulated expression of MHC I and an up-regulated expression of CD80/86 in infected cells, as demonstrated by significant alterations in both percentage of expressing cells and mean fluorescence intensity (MFI) in PRRSV-positive cells. A significant suppression in MFI of MHC I and an increase in percentage of cells expressing CD80/86 were observed in noninfected bystander cells. We also demonstrated that exposure of BM-imDCs to PRRSV resulted in a significantly increased secretion of IL-1, IL-6, IL-8, IL-10 and IFN-γ but not IL-12 or TNF-α. In addition, the PRRSV infection modulates cytokine expressions of BM-imDCs through their response to microbial pathogen-associated molecular patterns. These results will prove helpful in clarification of the factors that mediate host defense against PRRSV, as well as the possible interaction mechanisms between PRRSV and other microbes in the pathogenesis of PRRSV infection in pigs.

Evaluation of the Pathogenicity and Transmissibility of a Chilean Isolate of Porcine Reproductive and Respiratory Syndrome Virus

The objective of this study was to determine clinical features, shedding and transmission of a Chilean Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) strain upon experimental inoculation of 4-week-old pigs. Six groups of five animals each were used. The G1 (donor) group was inoculated with PRRSV, maintained in an isolation unit for 35 days, and sampled daily to determine shedding in mucosal secretions and faeces, viraemia and seroconversion. An uninfected control group (G6) was equally maintained and sampled under strict isolation. Four other groups (G2 to G5) were exposed to PRRSV via direct contact with G1 for 5-day periods in a staggered manner, throughout the 35-day period, and were later placed in an independent isolation unit to monitor infection status for 7 days. All the animals in G1 and G6 were killed at 35 days post-inoculation (dpi) and the contact groups at 12 days post-contact (dpc). Samples were obtained from diverse organs for histopathological, immunohistochemical (IHC) and virological analysis. No clinical symptoms were evident in any group, except for a transient fever observed in G1. Histopathologically, all the animals of G1 had interstitial pneumonia, although scarce PRRSV-positive cells were detected in the lung using IHC. PRRSV-positive cells (IHC) were detected in the lymphoid tissue of all animals in infected groups, but especially in G3 and G4. Viraemia was detected in G1 (3-35 dpi) and in the all contact groups (5-12 dpc). Likewise, ranging from 3 to 19 dpi, PRRSV was detected in at least one animal from the tonsils and lungs in all infected groups, in nasal and ocular secretions, saliva or faeces. These results indicate that the donor group excreted infectious PRRSV and was able to transmit the infection to susceptible pigs. The critical shedding period was 7-19 dpi, during which, most likely, transmission took place.

Evidence for the localization of porcine reproductive and respiratory syndrome virus (PRRSV) antigen and RNA in ovarian follicles in gilts.

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) infection in ovary was studied in sexually mature, cycling, nonsynchronized gilts infected with the PRRSV 16244B, a virulent field strain. Previous studies have shown that PRRSV can be isolated from ovaries and is transplacentally passed from gilts to the fetuses. The cause of infertility following PRRSV infection is not known. In this study, we identified the tropism of PRRSV in ovarian tissue from experimentally infected gilts in samples collected between 7 and 21 days postinfection (DPI). Tissues were collected and examined by virus isolation, in situ hybridization (ISH), immunohistochemistry (IHC), and double labeling to identify PRRSV-infected cell types. PRRSV was isolated in ovarian follicles at 7 days DPI. The IHC and ISH indicated that PRRSV-positive cells in ovaries were predominantly macrophages, which were numerous in atretic follicles. No evidence of infection and/or perpetuation of PRRSV in ova was observed, indicating that the female gonad is an unlikely site of persistence. No alteration of the normal ovarian architecture that would support a possible role of PRRSV infection in porcine female infertility was observed.

Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV).

A double in situ hybridization method for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV) genomes in the same tissue section was applied to lung tissues from 9 pigs in which PRRSV and PCV coinfection had been previously demonstrated. Paraffin-embedded tissue sections were simultaneously hybridized with a digoxigenin-labeled antisense RNA probe for PRRSV and a fluorescein-labeled antisense RNA probe for PCV, and hybridization was detected with anti-digoxigenin alkaline phosphatase/fast red and anti-fluorescein peroxidase/diaminobenzidine, respectively. PRRSV and PCV genomes were identified in the same pulmonary cell types as reported previously in all 9 pigs. In all pigs, PCV-positive cells outnumbered PRRSV-positive cells. A small proportion of alveolar macrophages contained both PRRSV and PCV genomes.

In vivo detection of porcine reproductive and respiratory syndrome virus RNA by in situ hybridization at different times postinfection.

We studied the distribution of porcine reproductive and respiratory syndrome virus (PRRSV) RNA in tissues by in situ hybridization at different times postinfection (p.i.). The probe used for in situ hybridization was prepared by reverse transcription of PRRSV RNA, followed by PCR amplification of the cDNA. The sequence amplified corresponded to 433 bp from PRRSV open reading frame 7, which is contained in the nucleocapsid protein gene and which is highly conserved in both European and American strains (H. Mardassi, L. Wilson, S. Mounir, and S. Dea, J. Clin. Microbiol. 32:2197-2203, 1994). An immunohistochemical technique was used to detect PRRSV antigen in tissue from virus-infected animals by using a monoclonal antibody specific for the PRRSV nucleocapsid protein (E.A. Nelson, J. Christopher-Hennings, T. Drew, G. Wensvoort, J.E. Collins, and D.A. Benfield, J. Clin. Microbiol. 31:3184-3189, 1993). The detection of PRRSV RNA was conducted in tissues of 6-week-old pigs that had been infected with one of three different field PRRSV isolates and collected at times ranging from 4 to 42 days p.i. Hybridization signals specific for PRRSV RNA were detected in lung, lymphoid tissues, alveolar macrophages (obtained by lavage at the time of necropsy), Peyer's patches, and kidney. The PRRSV-positive cells in these tissues appeared to be predominantly macrophages. In lung tissue we also obtained evidence suggesting the involvement of type II pneumocytes in the replication of PRRSV. During the acute period of infection there was a close correlation between the detection of RNA and the detection of nucleocapsid protein in individual cells. At later times p.i. (28 and 42 days p.i.), instead, more cells containing only PRRSV RNA than those containing PRRSV RNA and also expressing PRRSV nucleocapsid protein were detected. These results suggest that PRRSV RNA might persist in the tissues of infected animals for a longer time than PRRSV antigen expression.