Antiviral regulation in porcine monocytic cells at different activation states

Monocytic cells, including macrophages and dendritic cells, exist in different activation states that are critical to the regulation of antimicrobial immunity. Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of monocytic cells and interferes with antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages in non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded differently to viral infection. Based on Gene Ontology (GO) analysis, two approaches were used to potentiate antiviral activity: (i) pharmaceutical modulation of cellular lipid metabolism and (ii) in situ PRRSV replication-competent expression of interferon alpha (IFN-α). Both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days postinfection in macrophages. These findings suggest an intricate interaction of viral infection with the activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity

Host genetics of response to porcine reproductive and respiratory syndrome in nursery pigs

PRRS is the most costly disease in the US pig industry. While vaccination, biosecurity and eradication effort have had some success, the variability and infectiousness of PRRS virus strains have hampered the effectiveness of these measures. We propose the use of genetic selection of pigs as an additional and complementary effort. Several studies have shown that host response to PRRS infection has a sizeable genetic component and recent advances in genomics provide opportunities to capitalize on these genetic differences and improve our understanding of host response to PRRS. While work is also ongoing to understand the genetic basis of host response to reproductive PRRS, the focus of this review is on research conducted on host response to PRRS in the nursery and grow-finish phase as part of the PRRS Host Genetics Consortium. Using experimental infection of large numbers of commercial nursery pigs, combined with deep phenotyping and genomics, this research has identified a major gene that is associated with host response to PRRS. Further functional genomics work identified the GBP5 gene as harboring the putative causative mutation. GBP5 is associated with innate immune response. Subsequent work has validated the effect of this genomic region on host response to a second PRRSV strain and to PRRS vaccination and co-infection of nursery pigs with PRRSV and PCV2b. A genetic marker near GBP5 is available to the industry for use in selection. Genetic differences in host response beyond GBP5 appear to be highly polygenic, i.e. controlled by many genes across the genome, each with a small effect. Such effects can by capitalized on in a selection program using genomic prediction on large numbers of genetic markers across the genome. Additional work has also identified the genetic basis of antibody response to PRRS, which could lead to the use of vaccine response as an indicator trait to select for host response to PRRS. Other genomic analyses, including gene expression analyses, have identified genes and modules of genes that are associated with differences in host response to PRRS and can be used to further understand and utilize differences in host response. Together, these results demonstrate that genetic selection can be an additional and complementary tool to combat PRRS in the swine industry

Biogenesis of non-structural protein 1 (nsp1) and nsp1-mediated type I interferon modulation in arteriviruses

AbstractType I interferons (IFNs-α/β) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (PRRSV), has been shown to down-regulate the production of IFNs during infection. Non-structural protein (nsp) 1 of PRRSV has been identified as a viral IFN antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the CREB-binding protein (CBP) and to inhibit the formation of enhanceosome thus resulting in the suppression of IFN production. The study was expanded to other member viruses in the family Arteriviridae: equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). While PRRSV–nsp1 and LDV–nsp1 were auto-cleaved to produce the nsp1α and nsp1β subunits, EAV–nsp1 remained uncleaved. SHFV–nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1β, and nsp1γ), but only two subunits were generated as SHFV–nsp1αβ and SHFV–nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1β motif of nsp1β was functional to generate SHFV–nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV–nsp1β, LDV–nsp1β, EAV–nsp1, and SHFV–nsp1γ were predominantly found in the nucleus. All subunits of arterivirus nsp1 contained the IFN suppressive activity and inhibited both interferon regulatory factor 3 (IRF3) and NF-κB mediated IFN promoter activities. Similar to PRRSV–nsp1α, CBP degradation was evident in cells expressing LDV–nsp1α and SHFV–nsp1γ, but no such degradation was observed for EAV–nsp1. Regardless of CBP degradation, all subunits of arterivirus nsp1 suppressed the IFN-sensitive response element (ISRE)-promoter activities. Our data show that the nsp1-mediated IFN modulation is a common strategy for all arteriviruses but their mechanism of action may differ from each other

A 10-kDa Structural Protein of Porcine Reproductive and Respiratory Syndrome Virus Encoded by ORF2b

AbstractThe major structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) are derived from ORFs 5, 6, and 7. Western blots of sucrose gradient-purified virions and PRRSV-infected MARC-145 cells, probed with immune pig serum, showed the presence of an additional 10-kDa protein. Nucleotide sequence analysis of North American PRRSV isolate SDSU-23983 revealed a small ORF within ORF2, named ORF2b, which, when translated, produced a 73-amino-acid nonglycosylated protein. Recombinant 2b protein expressed by a baculovirus clone, AcVR2, comigrated with the 10-kDa virus-associated protein. The loss of 10-kDa protein immunoreactivity after absorption of immune sera with lysates from AcVR2-infected insect cells demonstrated that the 2b and 10-kDa proteins are immunologically similar. Immunoblots were also used for the detection of anti-2b activity in serum samples from experimentally infected adult pigs. Antibodies against PRRSV were apparent by 14 days postinfection, followed by anti-2b activity and serum neutralizing activity. The putative ORF2b start codon is only 6 nucleotides downstream of the adenine of the ORF2a start codon. The expression of ORF2a and 2b as enhanced green fluorescent fusion proteins showed that both proteins were translated; however, the ORF2b was preferentially expressed. These results suggest that the 2b protein is virion associated and the principal product of ORF2

Comparison of host immune responses to homologous and heterologous type II porcine reproductive and respiratory syndrome virus (PRRSV) challenge in vaccinated and unvaccinated pigs

Porcine reproductive and respiratory syndrome (PRRS) is a high-consequence animal disease with current vaccines providing limited protection from infection due to the high degree of genetic variation of field PRRS virus. Therefore, understanding host immune responses elicited by different PRRSV strains will facilitate the development of more effective vaccines. Using IngelVac modified live PRRSV vaccine (MLV), its parental strain VR-2332, and the heterologous KS-06-72109 strain (a Kansas isolate of PRRSV), we compared immune responses induced by vaccination and/or PRRSV infection. Our results showed that MLV can provide complete protection from homologous virus (VR-2332) and partial protection from heterologous (KS-06) challenge. The protection was associated with the levels of PRRSV neutralizing antibodies at the time of challenge, with vaccinated pigs having higher titers to VR-2332 compared to KS-06 strain. Challenge strain did not alter the cytokine expression profiles in the serum of vaccinated pigs or subpopulations of T cells. However, higher frequencies of IFN-γ-secreting PBMCs were generated from pigs challenged with heterologous PRRSV in a recall response when PBMCs were re-stimulated with PRRSV. Thus, this study indicates that serum neutralizing antibody titers are associated with PRRSV vaccination-induced protection against homologous and heterologous challenge

Molecular characterization and antiviral analyses of porcine type III interferons

Type III interferons (IFNs) are a family of recently identified antiviral cytokines. One to 3 paralogs have been identified in several species; however, little information is available about type III IFNs in pigs. We have identified 2 porcine type III IFNs, Sus scrofa IFN-λ1 (SsIFN-λ1) and SsIFN-λ3, and determined their tissue expression profile and antiviral activities. Open reading frames of SsIFN-λ1 and SsIFN-λ3 are 576 and 588 bp, encoding 191 and 195 amino acid preproteins, respectively. In healthy pigs, SsIFN-λ3 was primarily expressed in mesenteric lymph nodes and intestine, whereas expression of SsIFN-λ1 was found in all tested tissues and was high in mesenteric lymph nodes, intestine, and liver. Porcine cells treated with the viral mimic, dsRNA, robustly increased SsIFN-λ3 expression, with epithelial cells generally displaying the greatest response. Conversely, dsRNA-induced mRNA expressions of SsIFN-λ1, SsIFN-α1, and SsIFN-β were relatively weaker and delayed compared with SsIFN-λ3. SsIFN-λ1 and SsIFN-λ3 peptides exerted similar but lower antiviral potency than SsIFN-α1 and SsIFN-β against a porcine arterivirus and an adenovirus. These findings indicate that pigs have 2 type III IFN paralogs, which have antiviral activity and may serve as targets for modulation of the porcine host–pathogen interaction

Host genetics of response to porcine reproductive and respiratory syndrome in nursery pigs

PRRS is the most costly disease in the US pig industry. While vaccination, biosecurity and eradication effort have had some success, the variability and infectiousness of PRRS virus strains have hampered the effectiveness of these measures. We propose the use of genetic selection of pigs as an additional and complementary effort. Several studies have shown that host response to PRRS infection has a sizeable genetic component and recent advances in genomics provide opportunities to capitalize on these genetic differences and improve our understanding of host response to PRRS. While work is also ongoing to understand the genetic basis of host response to reproductive PRRS, the focus of this review is on research conducted on host response to PRRS in the nursery and grow-finish phase as part of the PRRS Host Genetics Consortium. Using experimental infection of large numbers of commercial nursery pigs, combined with deep phenotyping and genomics, this research has identified a major gene that is associated with host response to PRRS. Further functional genomics work identified the GBP5 gene as harboring the putative causative mutation. GBP5 is associated with innate immune response. Subsequent work has validated the effect of this genomic region on host response to a second PRRSV strain and to PRRS vaccination and co-infection of nursery pigs with PRRSV and PCV2b. A genetic marker near GBP5 is available to the industry for use in selection. Genetic differences in host response beyond GBP5 appear to be highly polygenic, i.e. controlled by many genes across the genome, each with a small effect. Such effects can by capitalized on in a selection program using genomic prediction on large numbers of genetic markers across the genome. Additional work has also identified the genetic basis of antibody response to PRRS, which could lead to the use of vaccine response as an indicator trait to select for host response to PRRS. Other genomic analyses, including gene expression analyses, have identified genes and modules of genes that are associated with differences in host response to PRRS and can be used to further understand and utilize differences in host response. Together, these results demonstrate that genetic selection can be an additional and complementary tool to combat PRRS in the swine industry

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-1) recognition of peptide sequences in CD163 SRCR5

PRRSV is an important swine pathogen that uses macrophages as important target cells for viral replication. CD163, a macrophage specific molecule involved in several homeostatic processes, was also identified as a receptor for PRRSV. Research shows that a complete deletion of CD163 SRCR5 can produce pigs that are entirely resistant to infection with PRRSV. Therefore, our goal is to find the smallest mutation in SRCR5 that will prevent PRRSV-1 infection, but also conserve CD163’s biological functions. The CD163 constructs used in this study were previously generated and tested for permissiveness to PRRSV-2 infection. Briefly, each construct carries an insertion of Proline-Arginine dipeptides (PR) at every 30 bp along the SRCR5 cDNA. All of the CD163 constructs were fused to a green fluorescent protein (GFP) which allowed visualization of the proper expression for each recombinant protein on non-permissive HEK293T cells. In order to test the permissiveness of each CD163 SRCR5 construct to PRRSV-1 infection, the cells expressing each mutant were infected with a PRRSV-1 strain, Lelystad. Infection results were visualized by IFA staining using an antibody recognizing PRRSV-N protein. The results showed a wide range of infection rates, from mutation that showed no or little effect to mutations that almost completely blocked infection. For example, insertion of PR in positions 15, 38, 62 and 78 had no effect on infection, whereas insertion of PR in positions 58 and 100 showed great reduction in PRRSV-1 infection. These results show the possible contact regions between the PRRSV viral proteins and the CD163 receptor

Development of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against African Swine Fever Virus

The incursion of African swine fever virus (ASFV) into Eurasia presents a threat to the world’s swine industry. Highly sensitive and specific diagnostic assays are urgently needed for rapid detection during an outbreak, post-outbreak investigation, and disease surveillance. In this study, a highly specific and repeatable blocking ELISA (bELISA) was developed using a recombinant p30 protein as the antigen combined with biotinylated mAb against p30 as the detection antibody. Initial test validation included sera from 810 uninfected animals and 106 animals experimentally inoculated with ASFV or recombinant alphavirus/adenovirus expressing p30. Receiver operating characteristic (ROC) analysis of the data calculated an optimal percentage of inhibition (PI) cutoff value of 45.92%, giving a diagnostic sensitivity of 98.11% and diagnostic specificity of 99.42%. The coefficient of variation of an internal quality control serum was 6.81% for between runs, 6.71% for within run, and 6.14% for within plate. A time course study of infected pigs showed that bELISA was able to detect seroconversion as early as 7 days post-inoculation. Taken together, these results demonstrate that bELISA can be used as an alternative serological test for detecting ASFV infection.This article is published as Yuan, Fangfeng, Vlad Petrovan, Luis Gabriel Gimenez-Lirola, Jeffrey J. Zimmerman, Raymond RR Rowland, and Ying Fang. "Development of a blocking enzyme-linked immunosorbent assay for detection of antibodies against African swine fever virus." Pathogens 10, no. 6 (2021): 760. DOI: 10.3390/pathogens10060760. Copyright 2021 by the authors. Attribution 4.0 International (CC BY 4.0). Posted with permission

Toll-like receptor 3 activation decreases porcine arterivirus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that initiates infection in pulmonary alveolar macrophages (PAMs), elicits weak immune responses, and establishes a persistent infection. To understand the role of dsRNA intermediates in eliciting host immunity, we sought to determine if toll-like receptor-3 (TLR3), a well-known dsRNA sensor, is involved in the regulation of PRRSV infection. TLR3 gene expression was increased in PAMs of congenitally infected 2-wk-old pigs. Stimulation of PAMs with dsRNA increased gene expression for TLR3 and interferon-β and suppressed PRRSV infectivity. To investigate activation and signaling parameters, expression constructs of wild-type and functional-domain-truncated porcine TLR3 were used in cell transfection studies. When cells that overexpressed porcine TLR3 were stimulated with dsRNA a rapid and robust calcium influx was induced. Moreover, ligand activation of porcine TLR3 expressed in MARC-145 cells elicited an antiviral response to PRRSV. Conversely, transfection of PAMs with small-interfering RNA targeting porcine TLR3 resulted in up to 80% suppression of TLR3 mRNA expression and an increase in PRRSV infectivity. These data provide fundamental genetic and molecular information for porcine TLR3, and implicate its involvement in PRRSV infection, findings that may suggest new strategies to limit this costly pandemic disease