Role of N-linked glycosylation in porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Porcine reproductive and respiratory syndrome (PRRSV) is an enveloped single-stranded positive-sense RNA virus and one of the main pathogens that causes the most significant economical losses in the swine-producing countries. PRRSV is currently divided into two distinct species, PRRSV-1 and PRRSV-2. The PRRSV virion envelope is composed of four glycosylated membrane proteins and three non-glycosylated envelope proteins. Previous work has suggested that PRRSV-linked glycans are critical structural components for virus assembly. In addition, it has been proposed that PRRSV glycans are implicated in the interaction with host cells and critical for virus infection. In contrast, recent findings showed that removal of N-glycans from PRRSV does not influence virus infection of permissive cells. Thus, there are not sufficient evidences to indicate compellingly that N-glycans present in the PRRSV envelope play a direct function in viral infection. To gain insights into the role of N-glycosylation in PRRSV infection, we analysed the specific contribution of the envelope protein-linked N-glycans to infection of permissive cells. For this purpose, we used a novel strategy to modify envelope protein-linked N-glycans that consists of production of monoglycosylated PRRSV and viral glycoproteins with different glycan states. Our results showed that removal or alteration of N-glycans from PRRSV affected virus infection. Specifically, we found that complex N-glycans are required for an efficient infection in cell cultures. Furthermore, we found that presence of high mannose type glycans on PRRSV surface is the minimal requirement for a productive viral infection. Our findings also show that PRRSV-1 and PRRSV-2 have different requirements of N-glycan structure for an optimal infection. In addition, we demonstrated that removal of N-glycans from PRRSV does not affect viral attachment, suggesting that these carbohydrates played a major role in regulating viral entry. In agreement with these findings, by performing immunoprecipitation assays and colocalization experiments, we found that N-glycans present in the viral envelope glycoproteins are not required to bind to the essential viral receptor CD163. Finally, we found that the presence of N-glycans in CD163 is not required for PRRSV infection.

Genetically modified pigs lacking CD163 PSTII-domain-coding exon 13 are completely resistant to PRRSV infection.

CD163 expressed on cell surface of porcine alveolar macrophages (PAMs) serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The extracellular portion of CD163 contains nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. Genomic editing of pigs to remove the entire CD163 or just the SRCR5 domain confers resistance to infection with both PRRSV-1 and PRRSV-2 viruses. By performing a mutational analysis of CD163, previous in vitro infection experiments showed resistance to PRRSV infection following deletion of exon 13 which encodes the first 12 amino acids of the 16 amino acid PSTII domain. These findings predicted that removal of exon 13 can be used as a strategy to produce gene-edited pigs fully resistant to PRRSV infection. In this study, to determine whether the deletion of exon 13 is sufficient to confer resistance of pigs to PRRSV infection, we produced pigs possessing a defined CD163 exon 13 deletion (ΔExon13 pigs) and evaluated their susceptibility to viral infection. Wild type (WT) and CD163 modified pigs, placed in the same room, were infected with PRRSV-2. The modified pigs remained PCR and serologically negative for PRRSV throughout the study; whereas the WT pigs supported PRRSV infection and showed PRRSV related pathology. Importantly, our data also suggested that removal of exon 13 did not affect the main physiological function associated with CD163 in vivo. These results demonstrate that a modification of CD163 through a precise deletion of exon 13 provides a strategy for protection against PRRSV infection.

Identification of CD163 regions that are required for porcine reproductive and respiratory syndrome virus (PRRSV) infection but not for binding to viral envelope glycoproteins.

CD163, a receptor for porcine reproductive and respiratory syndrome virus (PRRSV), possesses nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. To identify CD163 regions involved in PRRSV infection, CD163 mutants were generated. Infection experiments showed resistance to infection following deletion of the SRCR4/5 interdomain or the Exon 13 that encodes a portion of PSTII. The mutation of a pentapeptide domain in SRCR5 and SRCR7 also conferred resistance. Mutant CD163 proteins that resisted infection retained the ability to interact with GP2, GP3, GP4 and GP5 viral glycoproteins. The contribution of multiple domains to infection but not to the binding of viral glycoproteins suggests that the envelope proteins may form multiple interactions with CD163, or that receptor regions important for infection have other cellular binding partners required for PRRSV infection. Finally, we mapped the localization the anti-CD163 2A10 antibody epitope.

Mutations within scavenger receptor cysteine-rich (SRCR) protein domain 5 of porcine CD163 involved in infection with porcine reproductive and respiratory syndrome virus (PRRS).

CD163, a macrophage-specific membrane scavenger receptor, serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The removal of scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR5) of CD163 is sufficient to make transfected cells or genetically modified pigs resistant to PRRSV-1 and PRRSV-2 genotypes, and substitution of SRCR5 with SRCR8 from human CD163-like protein (hCD163L1) confers resistance to PRRSV-1 but not PRRSV-2 isolates. However, the specific regions within the SRCR5 polypeptide involved in PRRSV infection remain largely unknown. In this report, we performed mutational studies in order to identify which regions or amino acid sequences in the SRCR5 domain are critical for PRRSV infection. The approach used in this study was to make proline-arginine (PR) insertions along the SRCR5 polypeptide. Constructs were transfected into HEK293T cells, and then evaluated for infection with PRRSV-2 or PRRSV-1. For PRRSV-2, four PR insertions located after amino acids 8 (PR-9), 47 (PR-48), 54 (PR-55), and 99 (PR-100) had the greatest impact on infection. For PRRSV-1, insertions after amino acids 57 (PR-58) and 99 (PR-100) were critical. Computer simulations based on the crystal structure of SRCR5 showed that the mutations that affected infection localized to a similar region on the surface of the 3-D structure. Specifically, we found two surface patches that are essential for PRRSV infection. PR-58 and PR-55, which were separated by only three amino acids, had reciprocal effects on PRRSV-1 and PRRSV-2. Substitution of Glu-58 with Lys-58 reduced PRRSV-1 infection without affecting PRRSV-2, which partially explains the resistance to PRRSV-1 caused by the SRCR5 replacement with the homolog human SRCR8 previously observed. Finally, resistance to infection was observed following the disruption of any of the four conserved disulfide bonds within SRCR5. In summary, the results confirm that there are distinct differences between PRRSV-1 and PRRSV-2 on recognition of CD163; however, all mutations that affect infection locate on a similar region on the same face of SRCR5.

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.

Epitope mapping of African swine fever virus (ASFV) structural protein, p54.

In the absence of a vaccine for African swine fever virus (ASFV), diagnostic tools are critical for early detection and implementation of control measures. Along with other immunogenic proteins, p54 is a good serological target for conducting ASF detection and surveillance. In this study, a panel of 12 mouse monoclonal antibodies (mAbs) was prepared against a baculovirus-expressed p54(60-178) polypeptide. Further screening showed that five mAbs were positive for reactivity against ASFV-infected cells and recombinant p54 proteins. Mapping studies using five polypeptides and 12 oligopeptides, showed that mAb #154-1 recognized a conserved polypeptide sequence, p54(65-75), and was placed into Group 1. Mabs #143-1 and #7 recognized a region covered by p54(93-113) and were placed into Group 2. Group 3 consisted of mAbs #101 and #117, which recognized p54(118-127). Sera from pigs infected with the low virulent OURT 88/3 strain recognized the same p54 region covered by the Group 3 mAbs. When tested in a neutralization format, only mAb #143-1 showed neutralization activity above background. Together, the results identify important antigenic and immunogenic regions located on p54, which provide new tools for improving ASFV diagnostics.

E2 and Erns isotype-specific antibody responses in serum and oral fluid after infection with classical swine fever virus (CSFV).

Oral fluid sampling for the detection of classical swine fever virus infection provides a relatively inexpensive method for conducting active CSF surveillance. The purpose of this study was to detect CSFV nucleic acid and antibody in serum and oral fluid samples in a group of 10 pigs infected with the moderate CSFV strain, Paderborn. Based on clinical signs, outcome, and other results, pigs were placed into one of three disease outcome groups; Acute, Chronic and Recovered. Oral fluid and serum samples were analyzed for the presence of CSFV nucleic acid along with E2 and Erns surface protein-specific IgM, IgG and IgA responses. The results were summarized into a timeline of detection events beginning with the appearance of E2-IgM in serum (3 DPI) followed by CSFV nucleic acid in serum (6 DPI), CSFV nucleic acid in oral fluid (8 DPI), E2-IgG in serum (20 DPI), and E2-IgG in oral fluid (24 DPI). The results show that a combination of molecular and serological analyses of oral fluid can be incorporated into CSF surveillance.

Effect of bovine leukemia virus on bovine mammary epithelial cells.

Bovine leukemia virus (BLV) is a retrovirus that infects cattle and is associated with an increase in secondary infections. The objective of this study was to analyze the effect of BLV infection on cell viability, apoptosis and morphology of a bovine mammary epithelial cell line (MAC-T), as well as Toll like receptors (TLR) and cytokine mRNA expression. Our findings show that BLV infection causes late syncytium formation, a decrease in cell viability, downregulation of the anti-apoptotic gene Bcl-2, and an increase in TLR9 mRNA expression. Moreover, we analyzed how this stably infected cell line respond to the exposure to Staphylococcus aureus (S. aureus), a pathogen known to cause chronic mastitis. In the presence of S. aureus, MAC-T BLV cells had decreased viability and decreased Bcl-2 and TLR2 mRNA expression. The results suggest that mammary epithelial cells infected with BLV have altered the apoptotic and immune pathways, probably affecting their response to bacteria and favoring the development of mastitis.

Adenovirus-vectored African Swine Fever Virus antigen cocktails are immunogenic but not protective against intranasal challenge with Georgia 2007/1 isolate.

African Swine Fever Virus (ASFV) causes a hemorrhagic disease in swine and wild boars with a fatality rate close to 100%. Less virulent strains cause subchronic or chronic forms of the disease. The virus is endemic in sub-Saharan Africa and an outbreak in Georgia in 2007 spread to Armenia, Russia, Ukraine, Belarus, Poland, Lithuania, and Latvia. In August 2018, there was an outbreak in China and in April 2019, ASFV was reported in Vietnam and Cambodia. Since no vaccine or treatment exists, a vaccine is needed to safeguard the swine industry. Previously, we evaluated immunogenicity of two adenovirus-vectored cocktails containing ASFV antigens and demonstrated induction of unprecedented robust antibody and T cell responses, including cytotoxic T lymphocytes. In the present study, we evaluated protective efficacy of both cocktails by intranasal challenge of pigs with ASFV-Georgia 2007/1. A nine antigen cocktail-(I) formulated in BioMize adjuvant induced strong IgG responses, but when challenged, the vaccinees had more severe reaction relative to the controls. A seven antigen cocktail-(II) was evaluated using two adjuvants: BioMize and ZTS-01. The BioMize formulation induced stronger antibody responses, but 8/10 vaccinees and 4/5 controls succumbed to the disease or reached experimental endpoint at 17 days post-challenge. In contrast, the ZTS-01 formulation induced weaker antibody responses, but 4/9 pigs succumbed to the disease while the 5 survivors exhibited low clinical scores and no viremia at 17 days post-challenge, whereas 4/5 controls succumbed to the disease or reached experimental endpoint. Overall, none of the immunogens conferred statistically significant protection.

Development and characterization of monoclonal antibodies against p30 protein of African swine fever virus.

Among the structural proteins that compose the virion of African swine fever virus (ASFV), p30 is one of the most immunogenic proteins and is produced during early stage of ASFV infection. These two characteristics make p30 a good target for diagnostic assays to detect ASFV infection. In this study, we describe a panel of newly generated p30-specific monoclonal antibodies (mAbs). The reactivity of these mAbs was confirmed by immunoprecipitation and Western blot analysis in Vero cells infected with alphavirus replicon particles that express p30 (RP-p30). Furthermore, this panel of mAbs recognized ASFV strains BA71 V (Genotype I) and Georgia/2007 (Genotype II) in immunofluorescence assays on virus-infected Vero cells and swine macrophages, respectively. These mAbs also detected p30 expression by immunohistochemistry in tissue samples from ASFV-infected pigs. Epitope mapping revealed that a selected mAb from the panel recognized a linear epitope within the 32-amino acid region, 61-93. In contrast, two of the mAbs recognize the C-terminal region of the protein, which is highly hydrophilic, enriched in glutamic acid residues, and predicted to contain an intrinsically disordered protein region (IDPR). This panel of mAbs and mAb-based diagnostic assays potentially represent valuable tools for ASFV detection, surveillance and disease control.

Establishment of Systems to Enable Isolation of Porcine Monoclonal Antibodies Broadly Neutralizing the Porcine Reproductive and Respiratory Syndrome Virus.

The rapid evolution of porcine reproductive and respiratory syndrome viruses (PRRSV) poses a major challenge to effective disease control since available vaccines show variable efficacy against divergent strains. Knowledge of the antigenic targets of virus-neutralizing antibodies that confer protection against heterologous PRRSV strains would be a catalyst for the development of next-generation vaccines. Key to discovering these epitopes is the isolation of neutralizing monoclonal antibodies (mAbs) from immune pigs. To address this need, we sought to establish systems to enable the isolation of PRRSV neutralizing porcine mAbs. We experimentally produced a cohort of immune pigs by sequential challenge infection with four heterologous PRRSV strains spanning PRRSV-1 subtypes and PRRSV species. Whilst priming with PRRSV-1 subtype 1 did not confer full protection against a subsequent infection with a PRRSV-1 subtype 3 strain, animals were protected against a subsequent PRRSV-2 infection. The infection protocol resulted in high serum neutralizing antibody titers against PRRSV-1 Olot/91 and significant neutralization of heterologous PRRSV-1/-2 strains. Enriched memory B cells isolated at the termination of the study were genetically programmed by transduction with a retroviral vector expressing the Bcl-6 transcription factor and the anti-apoptotic Bcl-xL protein, a technology we demonstrated efficiently converts porcine memory B cells into proliferating antibody-secreting cells. Pools of transduced memory B cells were cultured and supernatants containing PRRSV-specific antibodies identified by flow cytometric staining of infected MARC-145 cells and in vitro neutralization of PRRSV-1. Collectively, these data suggest that this experimental system may be further exploited to produce a panel of PRRSV-specific mAbs, which will contribute both to our understanding of the antibody response to PRRSV and allow epitopes to be resolved that may ultimately guide the design of immunogens to induce cross-protective immunity.

T Cell Lymphoma and Leukemia in Severe Combined Immunodeficiency Pigs following Bone Marrow Transplantation: A Case Report.

After the discovery of naturally occurring severe combined immunodeficiency (SCID) within a selection line of pigs at Iowa State University, we found two causative mutations in the Artemis gene: haplotype 12 (ART12) and haplotype 16 (ART16). Bone marrow transplants (BMTs) were performed to create genetically SCID and phenotypically immunocompetent breeding animals to establish a SCID colony for further characterization and research utilization. Of nine original BMT transfer recipients, only four achieved successful engraftment. At approximately 11 months of age, both animals homozygous for the ART16 mutation were diagnosed with T cell lymphoma. One of these ART16/ART16 recipients was a male who received a transplant from a female sibling; the tumors in this recipient consist primarily of Y chromosome-positive cells. The other ART16/ART16 animal also presented with leukemia in addition to T cell lymphoma, while one of the ART12/ART16 compound heterozygote recipients presented with a nephroblastoma at a similar age. Human Artemis SCID patients have reported cases of lymphoma associated with a "leaky" Artemis phenotype. The naturally occurring Artemis SCID pig offers a large animal model more similar to human SCID patients and may offer a naturally occurring cancer model and provides a valuable platform for therapy development.

GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) as a target for homologous and broadly neutralizing antibodies.

Virus neutralization (VN) responses range from narrowly focused antibodies with only homologous neutralizing activity against the virus used for infection, to antibodies that can neutralize both Type 1 and Type 2 viruses, referred to as broadly neutralizing antibody (bnAb). Even though neutralizing epitopes are likely distributed among several structural glycoproteins, this paper focuses on the ectodomain region of GP5 as a model system for investigating the role for neutralizing and non-neutralizing antibodies in protection and disease. Epitope B within GP5 possesses several features common to broadly neutralizing epitopes. In the proposed model, accessibility of antibody to Epitope B is blocked by homologous neutralizing and non-neutralizing antibodies, which bind flanking hypervariable domains. Additional mechanisms for blocking the accessibility of bnAb include conformational alterations within the GP5-M heterodimer and glycan shielding. This model explains how the continuous escape from homologous neutralization provides a mechanism for persistence. The proposed mechanism for immune evasion is not unique to PRRSV, but can be found in other persistent viruses, such as hepatitis C virus (HCV).

Replacement of Porcine CD163 Scavenger Receptor Cysteine-Rich Domain 5 with a CD163-Like Homolog Confers Resistance of Pigs to Genotype 1 but Not Genotype 2 Porcine Reproductive and Respiratory Syndrome Virus.

CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both type 1 and type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knockout (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletion did not express CD163. When placed in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six type 1 and nine type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all type 2 isolates, but no type 1 viruses. Infection of CD163-modified pigs with representative type 1 and type 2 viruses confirmed the in vitro results. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though type 1 and type 2 viruses are considered phenotypically similar at several levels, there is a distinct difference between the viral genotypes in the recognition of CD163. IMPORTANCE: Genetic modification of the CD163 gene creates the opportunity to develop production animals that are resistant to PRRS, the costliest viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important CD163 functions.

Genetically edited pigs lacking CD163 show no resistance following infection with the African swine fever virus isolate, Georgia 2007/1.

African swine fever is a highly contagious, often fatal disease of swine for which there is no vaccine or other curative treatment. The macrophage marker, CD163, is a putative receptor for African swine fever virus (ASFV). Pigs possessing a complete knockout of CD163 on macrophages were inoculated with Georgia 2007/1, a genotype 2 isolate. Knockout and wild type pen mates became infected and showed no differences in clinical signs, mortality, pathology or viremia. There was also no difference following in vitro infection of macrophages. The results do not rule out the possibility that other ASFV strains utilize CD163, but demonstrate that CD163 is not necessary for infection with the Georgia 2007/1 isolate. This work rules out a significant role for CD163 in ASFV infection and creates opportunities to focus on alternative receptors and entry mechanisms.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA.

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

ORF5 of porcine reproductive and respiratory syndrome virus (PRRSV) is a target of diversifying selection as infection progresses from acute infection to virus rebound.

Genetic variation in both structural and nonstructural genes is a key factor in the capacity of porcine reproductive and respiratory syndrome virus (PRRSV) to evade host defenses and maintain within animals, farms and metapopulations. However, the exact mechanisms by which genetic variation contribute to immune evasion remain unclear. In a study to understand the role of host genetics in disease resistance, a population of pigs were experimentally infected with a type 2 PRRSV isolate. Four pigs that showed virus rebound at 42days post-infection (dpi) were analyzed by 454 sequencing to characterize the rebound quasispecies. Deep sequencing of variable regions in nsp1, nsp2, ORF3 and ORF5 showed the largest number of nucleotide substitutions at day 28 compared to days 4 and 42 post-infection. Differences were also found in genetic variations when comparing tonsil versus serum. The results of dN/dS ratios showed that the same regions evolved under negative selection. However, eight amino acid sites were identified as possessing significant levels of positive selection, including A27V and N32S substitutions in the GP5 ectodomain region. These changes may alter GP5 peptide signal sequence processing and N-glycosylation, respectively. The results indicate that the greatest genetic diversity occurs during the transition between acute and rebound stages of infection, and the introduction of mutations that may result in a gain of fitness provides a potential mechanism for persistence.

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

Type I interferons (IFNs-α/ß) 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 nsp1ß subunits, EAV-nsp1 remained uncleaved. SHFV-nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1ß, and nsp1γ), but only two subunits were generated as SHFV-nsp1αß and SHFV-nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1ß motif of nsp1ß was functional to generate SHFV-nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV-nsp1ß, LDV-nsp1ß, 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.

Genome-wide analysis of antiviral signature genes in porcine macrophages at different activation statuses.

Macrophages (MФs) can be polarized to various activation statuses, including classical (M1), alternative (M2), and antiviral states. To study the antiviral activation status of porcine MФs during porcine reproductive and respiratory syndrome virus (PRRSV) infection, we used RNA Sequencing (RNA-Seq) for transcriptomic analysis of differentially expressed genes (DEGs). Sequencing assessment and quality evaluation showed that our RNA-Seq data met the criteria for genome-wide transcriptomic analysis. Comparisons of any two activation statuses revealed more than 20,000 DEGs that were normalized to filter out 153-5,303 significant DEGs [false discovery rate (FDR) ≤0.001, fold change ≥2] in each comparison. The highest 5,303 significant DEGs were found between lipopolysaccharide- (LPS) and interferon (IFN)γ-stimulated M1 cells, whereas only 153 significant DEGs were detected between interleukin (IL)-10-polarized M2 cells and control mock-activated cells. To identify signature genes for antiviral regulation pertaining to each activation status, we identified a set of DEGs that showed significant up-regulation in only one activation state. In addition, pathway analyses defined the top 20-50 significantly regulated pathways at each activation status, and we further analyzed DEGs pertinent to pathways mediated by AMP kinase (AMPK) and epigenetic mechanisms. For the first time in porcine macrophages, our transcriptomic analyses not only compared family-wide differential expression of most known immune genes at different activation statuses, but also revealed transcription evidence of multiple gene families. These findings show that using RNA-Seq transcriptomic analyses in virus-infected and status-synchronized macrophages effectively profiled signature genes and gene response pathways for antiviral regulation, which may provide a framework for optimizing antiviral immunity and immune homeostasis.

Chimeric viruses containing the N-terminal ectodomains of GP5 and M proteins of porcine reproductive and respiratory syndrome virus do not change the cellular tropism of equine arteritis virus.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they are highly species specific and differ significantly in cellular tropism in cultured cells. In this study we examined the role of the two major envelope proteins (GP5 and M) of EAV and PRRSV in determining their cellular tropism. We generated three viable EAV/PRRSV chimeric viruses by swapping the N-terminal ectodomains of these two proteins from PRRSV IA1107 strain into an infectious cDNA clone of EAV (rMLVB4/5 GP5ecto, rMLVB4/5/6 Mecto and rMLVB4/5/6 GP5&Mecto). The three chimeric viruses could only infect EAV susceptible cell lines but not PRRSV susceptible cells in culture. Therefore, these data unequivocally demonstrate that the ectodomains of GP5 and M are not the major determinants of cellular tropism, further supporting the recent findings that the minor envelope proteins are the critical proteins in mediating cellular tropism (Tian et al., 2012).