Porcine Reproductive And Respiratory Syndrome Virus Nucleocapsid Protein Research Articles

Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with N in PRRSV-Infected PAMs.

One of the most significant diseases in the swine business, porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory problems in piglets and reproductive failure in sows. The PRRSV nucleocapsid (N) protein is essential for the virus' assembly, replication, and immune evasion. Stages in the viral replication cycle can be impacted by interactions between the PRRSV nucleocapsid protein and the host protein components. Therefore, it is of great significance to explore the interaction between the PRRSV nucleocapsid protein and the host. Nevertheless, no information has been published on the network of interactions between the nucleocapsid protein and the host proteins in primary porcine alveolar macrophages (PAMs). In this study, 349 host proteins interacting with nucleocapsid protein were screened in the PRRSV-infected PAMs through a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics approach. Bioinformatics analysis, which included gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes database enrichment, and a protein-protein interaction (PPI) network, revealed that the host proteins interacting with PRRSV-N may be involved in protein binding, DNA transcription, metabolism, and innate immune responses. This study confirmed the interaction between the nucleocapsid protein and the natural immune-related proteins. Ultimately, our findings suggest that the nucleocapsid protein plays a pivotal role in facilitating immune evasion during a PRRSV infection. This study contributes to enhancing our understanding of the role played by the nucleocapsid protein in viral pathogenesis and virus-host interaction, thereby offering novel insights for the prevention and control of PRRS as well as the development of vaccines.

Nucleocapsid protein residues 35, 36, and 113 are critical sites in up-regulating the Interleukin-8 production via C/EBPα pathway by highly pathogenic porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious and pathogenic agent that causes considerable economic damage in the swine industry. It regulates the inflammatory response, triggers inflammation-induced tissue damage, suppresses the innate immune response, and leads to persistent infection. Interleukin-8 (IL-8), a pro-inflammatory chemokine, plays a crucial role in inflammatory response during numerous bacteria and virus infections. However, the underlying mechanisms of IL-8 regulation during PRRSV infection are not well understood. In this study, we demonstrate that PRRSV-infected PAMs and Marc-145 cells release higher levels of IL-8. We screened the nucleocapsid protein, non-structural protein (nsp) 9, and nsp11 of PRRSV to enhance IL-8 promoter activity via the C/EBPα pathway. Furthermore, we identified that the amino acids Q35A, S36A, R113A, and I115A of the nucleocapsid protein play a crucial role in the induction of IL-8. Through reverse genetics, we generated two mutant viruses (rQ35-2A and rR113A), which showed lower induction of IL-8 in PAMs during infection. This finding uncovers a previously unrecognized role of the PRRSV nucleocapsid protein in modulating IL-8 production and provides insight into an additional mechanism by which PRRSV modulates immune responses and inflammation.

DExD/H-Box Helicase 36 Signaling via Myeloid Differentiation Primary Response Gene 88 Contributes to NF-κB Activation to Type 2 Porcine Reproductive and Respiratory Syndrome Virus Infection.

DExD/H-box helicase 36 (DHX36) is known to be an ATP-dependent RNA helicase that unwinds the guanine-quadruplexes DNA or RNA, but emerging data suggest that it also functions as pattern recognition receptor in innate immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide. Interstitial pneumonia is considered to be one of the most obvious clinical signs of PRRSV infection, suggesting that the inflammatory response plays an important role in PRRSV pathogenesis. However, whether DHX36 is involved in PRRSV-induced inflammatory cytokine expression remains unclear. In this study, we found that PRRSV infection increased the expression of DHX36. Knockdown of DHX36 and its adaptor myeloid differentiation primary response gene 88 (MyD88) by small-interfering RNA in MARC-145 cells significantly reduced NF-κB activation and pro-inflammatory cytokine expression after PRRSV infection. Further investigation revealed that PRRSV nucleocapsid protein interacted with the N-terminal quadruplex binding domain of DHX36, which in turn augmented nucleocapsid protein-induced NF-κB activation. Taken together, our results suggest that DHX36–MyD88 has a relevant role in the recognition of PRRSV nucleocapsid protein and in the subsequent activation of pro-inflammatory NF-κB pathway.

The integrity of PRRSV nucleocapsid protein is necessary for up-regulation of optimal interleukin-10 through NF-κB and p38 MAPK pathways in porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease, has been constantly causing huge economic losses all over the world. PRRS virus (PRRSV) infection results in immunosuppression and IL-10 up-regulation. The relationship between them is still in dispute. Previous studies demonstrated the protein of PRRSV nucleocapsid (N) protein is able to up-regulate IL-10, yet the underlying molecular mechanisms remain unknown. In this study, the expression kinetics of IL-10 up-regulation induced by PRRSV N protein were analyzed in immortalized porcine alveolar macrophages (PAMs). N protein induced IL-10 expression in a time- and dose-dependent manner. Inhibition experiments of signaling pathways suggested NF-κB and p38 MAPK pathways are both involved in N protein-induced IL-10 up-regulation. Besides, the integrity of N protein is essential for significant IL-10 up-regulation. This research is beneficial for further understanding of the interplay between PRRSV and host immune system.

Development of an Indirect-Elisa to Detect Antibodies against Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein in Gansu China

Porcine reproductive and respiratory syndrome virus (PRRSV), causes reproductive failure in sows and boars, and is an important pig pathogen that is responsible for respiratory symptoms in swine of all ages. PRRS has been a serious problem in swine industry after it was first described in the United States in 1987. PRRSV nucleocapsid protein ( N protein ) is highly conserved among different strains, and it is widely used as a diagnostic antigen for the development of serologic diagnostic tools. In our study, we then developed an indirect enzyme-linked immunosorbent assay (in-ELISA) using the pET-30a (+) expression vector and a Ni-NTA purification kit to purify the viral nucleocapsid protein antigen in order to detect antibodies against the North American porcine reproductive and respiratory syndrome virus (naPRRSV). The in- ELISA was compared with two commercial ELISA kits using 60 infected serum samples , 53 negative serum samples, and 314 serum samples from clinically vaccinated donor pigs, to calculate the specificity and sensitivity. Additional serum samples (979 samples) from different regions of Gansu provinc were used for evaluating the PRRSV antibodies from the different regions. The in-ELISA was more economical, quicker, and easier than the previously described similar methods, and could be suitable for the diagnosis of antibodies and also for epidemiological studies of naPRRSV

Defining the Cellular Target(s) of Porcine Reproductive and Respiratory Syndrome Virus Blocking Monoclonal Antibody 7G10

We produced a monoclonal antibody (MAb) (7G10) that has blocking activity against porcine reproductive and respiratory syndrome virus (PRRSV). In this study, we identified the components of the 7G10 MAb-bound complex as cytoskeletal filaments: vimentin, cytokeratin 8, cytokeratin 18, actin, and hair type II basic keratin. Vimentin bound to PRRSV nucleocapsid protein and anti-vimentin antibodies showed PRRSV-blocking activity. Vimentin was expressed on the surface of MARC-145, a PRRSV-susceptible cell line. Simian vimentin rendered BHK-21 and CRFK, nonsusceptible cell lines, susceptible to PRRSV infection. These results suggest that vimentin is part of the PRRSV receptor complex and that it plays an important role in PRRSV binding with the other cytoskeletal filaments that mediate transportation of the virus in the cytosol.

Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences

The order Nidovirales, which includes the arteriviruses and coronaviruses, incorporate a cytoplasmic replication scheme; however, the nucleocapsid (N) protein of several members of this group localizes to the nucleolus suggesting that viral proteins influence nuclear processes during replication. The relatively small, 123 amino acid, N protein of porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, presents an ideal model system for investigating the properties and mechanism of N protein nucleolar localization. The PRRSV N protein is found in both cytoplasmic and nucleolar compartments during infection and after transfection of gene constructs that express N-enhanced green fluorescent protein (EGFP) fusion proteins. Experiments using oligopeptides, truncated polypeptides and amino acid-substituted proteins have identified several domains within PRRSV N protein that participate in nucleo-cytoplasmic shuttling, including a cryptic nuclear localization signal (NLS) called NLS-1, a functional NLS (NLS-2), a nucleolar localization sequence (NoLS), as well as a possible nuclear export signal (NES). The purpose of this paper is to review our current understanding of PRRSV N protein shuttling and propose a shuttling scheme regulated by RNA binding and post-translational modification.

Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged as an important cause of reproductive disorders and pneumonia in domestic pigs throughout the world. Acute cytocidal replication of PRRSV in alveolar lung macrophages causes the acute pneumonia; however, it remains largely unresolved whether there may also be a predisposition to longer-term local immunodeficiency in the PRRSV-convalescent lung. We applied various flow cytometric techniques to study the interplay between PRRSV replication and macrophage viability/function in pure cultures of porcine alveolar lung macrophages. Monitored by flow cytometric detection of intracellular PRRSV nucleocapsid protein, acute (24 h post infection) PRRSV replication did not impede the ability of alveolar macrophages to ingest fluorescently labelled Escherichia coli. At 48 h post infection, PRRSV-induced cytotoxicity (quantitated by flow analysis of cell size and membrane integrity) led to 40% reduction in the total number of phagocytozing cells. However, viable/uninfected macrophages in PRRSV-infected cultures exhibited normal phagocytic ability at 48 h, indicating that no soluble phagocytosis-suppressive mediators were induced by PRRSV infection in this system. In short, in our minimal system containing only a single cell type, phagocytosis-suppressive effects of PRRSV infection were detected, that acted at the culture level by reducing the total number of alveolar lung macrophages.

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.

Immunohistochemical Identification of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Antigen in the Heart and Lymphoid System of Three-week-old Colostrum-deprived Pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) antigens were detected by a streptavidin-biotin complex technique in tissues of 3-week-old colostrum-deprived pigs that had been inoculated intranasally with PRRSV and had developed moderate respiratory disease. Moderate, multifocal, tan-colored consolidation of the lungs and severe enlargement of the lymph nodes were noted at necropsy. Severe interstitial pneumonia characterized by type 2 pneumocyte proliferation, septal infiltration with mononuclear cells, and accumulation of macrophages and necrotic cells in alveolar spaces was observed at 4 and 9 days postinoculation. Moderate multifocal perivascular lymphohistiocytic myocarditis was observed at 9 days postinoculation. Marked lymphoid follicular hyperplasia and follicular necrosis in the tonsil, spleen, and lymph nodes was observed. A monoclonal antibody that recognizes a conserved epitope of PRRSV nucleocapsid protein was used as primary antibody for immunohistochemistry. Antigen was readily detected in alveolar macrophages in the lung and in endothelial cells and macrophages in the heart. Macrophages and cells resembling dendritic cells in tonsil, lymph nodes, thymus, and spleen also stained intensely positive for viral antigen. PRRSV appears to replicate primarily within macrophages in the respiratory and lymphoid systems of the pig.

Molecular Characterization of Porcine Reproductive and Respiratory Syndrome Virus, a Member of the Arterivirus Group

Porcine reproductive and respiratory syndrome virus (PRRSV)-specific cDNA clones spanning the 3′ terminal 5 kb of the genomic RNA were isolated, sequenced, and used as probes for identification of PRRSV-specific RNAs. The PRRSV genome is a positive-stranded polyadenylated RNA of about 15 kb. In infected cells, a 3′ coterminal nested set of six major subgenomic mRNAs could be demonstrated. Within the 3′ terminal 3.5 kb of the PRRSV genome, six overlapping reading frames (ORFs) were identified, each most likely expressed by one of the subgenomic mRNAs. Amino acid sequence comparisons revealed that the most 3′ terminal ORF (ORF7) encodes the PRRSV nucleocapsid protein with a calculated molecular weight of 14 kDa. It displays 44.8% amino acid identity with the capsid protein of lactate dehydrogenase-elevating virus (LDV) and 23.6% with that of equine arteritis virus (EAV). The product of ORF6, the second 3′ terminal ORF, represents a putative membrane protein and exhibits 53.2 and 27.2% amino acid identity with the corresponding LDV and EAV polypeptides, respectively. Similar to EAV, ORFs 2 through 5 might encode glycosylated viral proteins. The polypeptide deduced from the most 5′ ORF (ORF1b) contains two conserved domains common to EAV and coronavirus polymerases. Genome organization, strategy of gene expression, and the sequence of deduced proteins show that PRRSV belongs to the Arterivirus group of viruses.