Steven Lawson

The Cysteine Protease Domain of Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Possesses Deubiquitinating and Interferon Antagonism Functions

ABSTRACT Porcine reproductive and respiratory syndrome (PRRS) virus nonstructural protein 2 (nsp2) contains a cysteine protease domain at its N terminus, which belongs to the ovarian tumor (OTU) protease family. In this study, we demonstrated that the PRRSV nsp2 OTU domain antagonizes the type I interferon induction by interfering with the NF-κB signaling pathway. Further analysis revealed that the nsp2 OTU domain possesses ubiquitin-deconjugating activity. This domain has the ability to inhibit NF-κB activation by interfering with the polyubiquitination process of IκBα, which subsequently prevents IκBα degradation. To determine whether the nsp2 protein antagonist function can be ablated from the virus, we introduced point mutations into the OTU domain region by use of reverse genetics. The D458A, S462A, and D465A mutations targeting on a B-cell epitope in the OTU domain region generated the viable recombinant viruses, and the S462A and D465A mutants were attenuated for growth in cell culture. The OTU domain mutants were examined to determine whether mutations in the nsp2 OTU domain region altered virus ability to inhibit NF-κB activation. The result showed that certain mutations lethal to virus replication impaired the ability of nsp2 to inhibit NF-κB activation but that the viable recombinant viruses, vSD-S462A and vSD-D465A, were unable to inhibit NF-κB activation as effectively as the wild-type virus. This study represents a fundamental step in elucidating the role of nsp2 in PRRS pathogenesis and provides an important insight in future modified live-virus vaccine development.

Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

Abstract The porcine reproductive and respiratory syndrome virus nsp1 is predicted to be auto-cleaved from the replicase polyprotein into nsp1α and nsp1β subunits. In infected cells, we detected the actual existence of nsp1α and nsp1β. Cleavage sites between nsp1α/nsp1β and nsp1β/nsp2 were identified by protein microsequencing analysis. Time course study showed that nsp1α and nsp1β mainly localize into the cell nucleus after 10 h post infection. Further analysis revealed that both proteins dramatically inhibited IFN-β expression. The nsp1β was observed to significantly inhibit expression from an interferon-stimulated response element promoter after Sendai virus infection or interferon treatment. It was further determined to inhibit nuclear translocation of STAT1 in the JAK–STAT signaling pathway. These results demonstrated that nsp1β has ability to inhibit both interferon synthesis and signaling, while nsp1α alone strongly inhibits interferon synthesis. These findings provide important insights into mechanisms of nsp1 in PRRSV pathogenesis and its impact in vaccine development.

Lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero.

Abstract The ability of porcine reproductive and respiratory syndrome virus (PRRSV) to establish a persistent infection is the principal contributing factor to the world-wide spread of the disease. Several studies have documented the course of viral infection in postnatally infected pigs; however, very little is known regarding sites of virus replication during persistent infection of pigs exposed to PRRSV in utero. In this study, virus replication and PRRSV-specific antibody were followed for several hundred days in a group of pigs derived from three sows infected at 90 days of gestation with PRRSV isolate VR-2332. Eighty-four percent of pigs were born viremic with a mortality of 54% within 21 days after birth. At approximately 60 days sera from pigs were negative for virus by virus isolation. Analysis of virus replication in the tissues of pigs randomly sacrificed between 63 and 132 days showed no evidence of virus in lung and other non-lymphoid organs. However, virus was easily recovered from tonsil and lymph nodes and in situ hybridization identified these tissues as sites of virus replication. Even though replication was at a low level, virus was easily transmitted to sentinel pigs. By 260 days pigs became seronegative and did not transmit virus to sentinel pigs. Sacrifice of remaining pigs after 300 days showed no evidence of virus in blood and tissues. This study shows that congenital PRRSV-infected pigs can support virus replication for an extended period during which virus replication is primarily restricted to tonsil and lymph nodes.

Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response.

Non-structural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the largest protein of this virus. In addition to its crucial role in virus replication, recent studies have indicated its involvement in modulating host immunity. In this study, each of the six identified immunodominant nsp2 B-cell epitopes (ES2-ES7) was deleted using a type I PRRSV cDNA infectious clone. Deletion of ES3, ES4 or ES7 allowed the generation of viable virus. In comparison with the parental virus, the DeltaES3 mutant showed increased cytolytic activity and more vigorous growth kinetics, whilst the DeltaES4 and DeltaES7 mutants displayed decreased cytolytic activity and slower growth kinetics in MARC-145 cells. These nsp2 mutants were characterized further in a nursery pig disease model. The results showed that the DeltaES4 and DeltaES7 mutants exhibited attenuated phenotypes, whereas the DeltaES3 mutant produced a higher peak viral load in pigs. The antibody response reached similar levels, as measured by IDEXX ELISA at 21 days post-infection, and slightly higher levels of mean virus neutralizing titres were observed from pigs infected by the DeltaES4 and DeltaES7 mutants. The expression of innate and T-helper 1 cytokines was measured in peripheral blood mononuclear cells or virus-infected macrophages. The results consistently showed that interleukin-1beta and tumour necrosis factor alpha expression levels were downregulated in cells that were stimulated (or infected) with the DeltaES3 mutant compared with parental virus and the other nsp2 deletion mutants. These results suggest that certain regions in nsp2 are non-essential for PRRSV replication but may play an important role in modulation of host immunity in vivo.

Porcine reproductive and respiratory syndrome virus infection of gnotobiotic pigs: sites of virus replication and co-localization with MAC-387 staining at 21 days post-infection

The organ distribution of PRRSV-infected cells in gnotobiotic piglets at 21 days after infection with PRRSV isolate VR-2332 was examined by in situ hybridization. Cells that expressed PRRSV RNA were identified in all tissues examined, including organs not usually characterized as sites of PRRSV infection. PRRSV-infected cells frequently appeared in clusters and were not always associated with microscopic lesions. The expression of PRRSV RNA co-localized with a macrophage monoclonal antibody, MAC-387, in lymph nodes. Some, but not all infected cells stained with MAC-387. The wide distribution of PRRSV-infected cells and co-localization with MAC-387 staining is consistent with the macrophage-tropism of PRRSV and is similar to observations made during persistent infection with other arteriviruses.

The 2b protein as a minor structural component of PRRSV

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) ORF2 contains an internal ORF that codes for a small non-glycosylated protein known as 2b. Previous work had identified the presence of a 10kDa 2b protein in virus-infected cells and the induction of an anti-2b response in PRRSV-infected pigs, as well as a possible association of 2b with the virion (Wu et al., 2001, Virology 287:183–191). In this study, we utilized two experimental approaches, including the use of a 2b peptide-specific monoclonal antibody, to demonstrate that the PRRSV 2b protein is an integral component of the PRRSV virion. This study suggests that 2b in PRRSV is similar to the E protein in EAV and forms a minor structural component of the virion.

Porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence

Neonatal pigs from three herds of pigs were somnolent and inappetent and had microscopic lesions characterised by severe meningoencephalitis, necrotic interstitial pneumonia and gastric muscular inflammation. Porcine reproductive and respiratory syndrome virus (PRRSV) infection was diagnosed and confirmed by virus isolation, fluorescent antibody examination of frozen lung sections, serology, immunohistochemistry and in situ hybridisation. Each herd had a history of PRRSV infection and was using or had used a modified-live vaccine. The isolates from the affected pigs were genetically distinct from the modified-live vaccine strain of the virus when compared by restriction enzyme analysis and nucleotide sequencing of PRRSV open reading frames 5 and 6. The virus was identified in macrophages or microglia of brain lesions by immunohistochemical staining of brain sections with an anti-PRRsv monoclonal antibody and an anti-macrophage antibody. The replication of the virus in the brain was verified by in situ hybridisation. The meningoencephalitis induced by the virus in pigs from each of the herds was unusually severe and the brain lesions were atypical when compared with other descriptions of encephalitis induced by the virus, which should therefore be considered as a possible diagnosis for neonatal pigs with severe meningoencephalitis. In addition, field isolates of the virus which are capable of causing disease can emerge and coexist with modified-live vaccine virus in some pig herds.

Development of an 8-plex Luminex assay to detect swine cytokines for vaccine development: assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination.

A Luminex (Luminex Corp., Austin, TX) multiplex swine cytokine assay was developed to measure 8 cytokines simultaneously in pig serum for use in assessment of vaccine candidates. The fluorescent microsphere immunoassay (FMIA) was tested on archived sera in a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine/challenge study. This FMIA simultaneously detects innate (IL-1 beta, IL-8, IFN-alpha, TNF-alpha, IL-12), regulatory (IL-10), Th1 (IFN-gamma) and Th2 (IL-4) cytokines. These proteins were measured to evaluate serum cytokine levels associated with vaccination strategies that provided for different levels of protective immunity against PRRSV. Pigs were vaccinated with a modified-live virus (MLV) vaccine and subsequently challenged with a non-identical PRRSV isolate (93% identity in the glycoprotein (GP5) gene). Protection (as defined by no serum viremia) was observed in the MLV vaccinated pigs after PRRSV challenge but not those vaccinated with killed virus vaccine with adjuvant (KV/ADJ) (99% identity in the GP5 gene to the challenge strain) or non-vaccinates. Significantly elevated levels of IL-12 were observed in the KV/ADJ group compared to MLV vaccinated and control groups. However, this significant increase in serum IL-12 did not correlate with protection against PRRSV viremia. Additional studies using this assay to measure the local cytokine tissue responses may help in defining a protective cytokine response and would be useful for the targeted design of efficacious vaccines, not only for PRRSV, but also for other swine pathogens.

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50–500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are “fit for purpose,” how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs.

Development of a fluorescent microsphere immunoassay for detection of antibodies against porcine reproductive and respiratory syndrome virus using oral fluid samples as an alternative to serum-based assays.

ABSTRACT For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies developed in response to porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, we developed a multiplexed fluorescent microsphere immunoassay (FMIA) for detection of PRRSV-specific antibodies in oral fluid and serum samples. Recombinant nucleocapsid protein (N) and nonstructural protein 7 (nsp7) from both PRRSV genotypes (type I and type II) were used as antigens and covalently coupled to Luminex fluorescent microspheres. Based on an evaluation of 488 oral fluid samples with known serostatus, the oral fluid-based FMIAs achieved >92% sensitivity and 91% specificity. For serum samples (n = 1,639), the FMIAs reached >98% sensitivity and 95% specificity. The assay was further employed to investigate the kinetics of the antibody response in infected pigs. In oral fluid, the N protein was more sensitive for the detection of early infection (7 and 14 days postinfection), but nsp7 detected a higher level and longer duration of antibody response (28 days postinfection). In serum, the antibodies specific to nsp7 and N proteins were detected as early as 7 days postinfection, and the responses lasted more than 202 days. This study provides a framework from which a more robust assay could be developed to profile the immune response to multiple PRRSV antigens in a single test. The development of oral fluid-based diagnostic tests will change the way we survey diseases in swine herds and improve our ability to cheaply and efficiently track PRRSV infections in both populations and individual animals.