NRP1 is a receptor for mammalian orthoreovirus engaged by distinct capsid subunits.

Mammalian orthoreovirus (reovirus) is a nonenveloped virus that establishes primary infection in the intestine and disseminates to sites of secondary infection, including the CNS. Reovirus entry involves multiple engagement factors, but how the virus disseminates systemically and targets neurons remains unclear. In this study, we identified murine neuropilin 1 (mNRP1) as a receptor for reovirus. mNRP1 binds reovirus with nanomolar affinity using a unique mechanism of virus-receptor interaction, which is coordinated by multiple interactions between distinct reovirus capsid subunits and multiple NRP1 extracellular domains. By exchanging essential capsid protein-encoding gene segments, we determined that the multivalent interaction is mediated by outer-capsid protein σ3 and capsid turret protein λ2. Using capsid mutants incapable of binding NRP1, we found that NRP1 contributes to reovirus dissemination and neurovirulence in mice. Collectively, our results demonstrate that NRP1 is an entry receptor for reovirus and uncover mechanisms by which NRPs promote viral entry and pathogenesis.

Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus.

Mammalian orthoreovirus (reovirus) infects most mammals and is associated with celiac disease in humans. In mice, reovirus infects the intestine and disseminates systemically to cause serotype-specific patterns of disease in the brain. To identify receptors conferring reovirus serotype-dependent neuropathogenesis, we conducted a genome-wide CRISPRa screen and identified paired immunoglobulin-like receptor B (PirB) as a receptor candidate. Ectopic expression of PirB allowed reovirus binding and infection. PirB extracelluar D3D4 region is required for reovirus attachment and infectivity. Reovirus binds to PirB with nM affinity as determined by single molecule force spectroscopy. Efficient reovirus endocytosis requires PirB signaling motifs. In inoculated mice, PirB is required for maximal replication in the brain and full neuropathogenicity of neurotropic serotype 3 (T3) reovirus. In primary cortical neurons, PirB expression contributes to T3 reovirus infectivity. Thus, PirB is an entry receptor for reovirus and contributes to T3 reovirus replication and pathogenesis in the murine brain.

Neural-Cell-Intrinsic NF-κB Signaling Enhances Reovirus Virulence.

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65-/-) survived. While viral loads in brains of WT and Nsp65-/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65-/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65-/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.

Molecular characterization of emerging variants of PRRSV in the United States: new features of the -2/-1 programmed ribosomal frameshifting signal in the nsp2 region.

In this study, we characterized an emerging porcine reproductive and respiratory syndrome virus (PRRSV) isolate UIL21-0712, which is a lineage 1C variant with ORF5 restriction fragment length polymorphism (RFLP) cutting pattern of 1-4-4. The UIL21-0712 genome sequence has 85.3% nucleotide identity with the prototypic PRRSV-2 strain VR2332. The nsp2 region is the most variable, and the -2/-1 programmed ribosome frameshifting (PRF) signal therein is distinct from historical PRRSV strains. Analysis of PRRSV sequences in GenBank revealed that the majority of the emerging PRRSV variants contain substitutions that disrupt the -1 PRF stop codon to generate a nsp2N protein with a C-terminal extension. Two of the -1 PRF stop codon variant patterns were identified to be predominantly circulating in the field. They demonstrated higher growth kinetics than the other variants, suggesting that the most dominant -1 PRF stop codon variant patterns may provide enhanced growth fitness for the virus.

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression.

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Here we apply ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and to analyse the host response to infection. We calculated programmed ribosomal frameshift (PRF) efficiency at both sites on the viral genome. This revealed the nsp2 PRF site as the second known example where temporally regulated frameshifting occurs, with increasing -2 PRF efficiency likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1ß. Surprisingly, we find that PRF efficiency at the canonical ORF1ab frameshift site also increases over time, in contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a highly expressed 125-codon ORF overlapping nsp12, which is likely translated from novel subgenomic RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporally regulating expression of the 3'-proximal region of ORF1b. We also identified a highly translated, short upstream ORF in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. These findings reveal hidden complexity in the gene expression programmes of these important nidoviruses.

Viruses have tiny genomes. Rather than carry all the genetic information they need, they rely on the cells they infect. This makes the few genes they do have all the more important. Many viruses store their genes not in DNA, but in a related molecule called RNA. When the virus infects cells, it uses the cells' ribosomes ­ the machines in the cells that make proteins ­ to build its own proteins. One of the central ideas in biology is that one molecule of RNA carries the instructions for just one type of protein. But many viruses break this rule. The ribosomes in cells read RNA instructions in blocks of three: three RNA letters correspond to one protein building block. But certain sequences in the RNA of viruses act as hidden signals that affect how ribosomes read these molecules. These signals make the ribosomes skip backward by one or two letters on the viral RNA, restarting part way through a three-letter block. Scientists call this a 'frameshift', and it is a bit like changing the positions of the spaces in a sentence. The virus causes these frameshifts using proteins or by folding its RNA into a knot-like structure. The frameshifts result in the production of different viral proteins over time. The porcine reproductive and respiratory syndrome virus (PRRSV) uses frameshifts to cause devastating disease in pigs. Besides the sequences in its RNA that allow the ribosomes to skip backwards, the viral enzyme that copies the RNA can also skip forward. This results in shortened copies of its genes, which also changes the proteins they produce. To find out exactly how PRRSV uses these frameshifting techniques, Cook et al. examined infected cells in the laboratory. They monitored the RNA made by the virus and looked closely at the way the cells read it using a technique called ribosome profiling. This revealed that frameshifting increases over the course of an infection. This is partly because the viral protein that causes frameshifts builds up as infection progresses, but it also happened with frameshifts caused by RNA knots. The reason for this is less clear. Cook et al. also discovered several new RNAs made later in infection, which could also change the proteins the virus makes. RNA viruses cause disease in humans as well as pigs. Examples include coronaviruses and HIV. Many of these also have frameshift sites in their genomes. A better understanding of how frameshifts change during infection may aid drug development. Future work could help researchers to understand which proteins viruses make at which stage of infection. This could lead to new treatments for viruses like PRRSV.

Monitoring peripheral neutrophil and T-lymphocyte subsets could assist in differentiating the severity and disease progression of coronavirus disease 2019.

Helper T cells (CD3+CD4+ T cells) and cytotoxic T cells (CD3+CD8+ T cells) play direct and indirect antiviral roles. This study retrospectively explored the clinical significance of peripheral lymphocytes, especially the dynamic analysis of T-cell subsets, in determining coronavirus disease 2019 (COVID-19) severity and progression. Seventy-nine patients with COVID-19 in the Public Health Clinical Center of Chengdu from January to February 2020 were included, 59 of which were analyzed for dynamic peripheral T-cell subsets expression. The neutrophil to CD4+ T lymphocyte ratio (N4R) and neutrophil to CD3+ T lymphocyte ratio (N3R) showed clinical significance in differentiating severe or critically-severe COVID-19, with area under receiver operating characteristic curves (AUCs) of 0.933 and 0.900, respectively (P < 0.05). COVID-19 patients with more baseline peripheral lymphocytes or NK cells were prone to test negative to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after therapy (P < 0.05), and the AUC of NK cells for predicting negative results of SARS-CoV-2 RNA detection after therapy was 0.800. When the number of peripheral CD3+CD4+ and CD3+CD8+ T cells in COVID-19 patients continuously increased 6-9 days after baseline, the period of disease exacerbation could be delayed for more than 2 weeks after admission. Baseline N4R and N3R could be potential biomarkers for assisting in differentiating COVID-19 severity, and dynamically monitoring peripheral CD3+CD4+ and CD3+CD8+ T cells 6-9 days after baseline could help clinicians to evaluate disease progression in COVID-19 patients.

A chimeric influenza hemagglutinin delivered by parainfluenza virus 5 vector induces broadly protective immunity against genetically divergent influenza a H1 viruses in swine.

Pigs are an important reservoir for human influenza viruses, and influenza causes significant economic loss to the swine industry. As demonstrated during the 2009 H1N1 pandemic, control of swine influenza virus infection is a critical step toward blocking emergence of human influenza virus. An effective vaccine that can induce broadly protective immunity against heterologous influenza virus strains is critically needed. In our previous studies [McCormick et al., 2015; PLoS One, 10(6):e0127649], we used molecular breeding (DNA shuffling) strategies to increase the breadth of the variable and conserved epitopes expressed within a single influenza A virus chimeric hemagglutinin (HA) protein. Chimeric HAs were constructed using parental HAs from the 2009 pandemic virus and swine influenza viruses that had a history of zoonotic transmission to humans. In the current study, we used parainfluenza virus 5 (PIV-5) as a vector to express one of these chimeric HA antigens, HA-113. Recombinant PIV-5 expressing HA-113 (PIV5-113) were rescued, and immunogenicity and protective efficacy were tested in both mouse and pig models. The results showed that PIV5-113 can protect mice and pigs against challenge with viruses expressing parental HAs. The protective immunity was extended against other genetically diversified influenza H1-expressing viruses. Our work demonstrates that PIV5-based influenza vaccines are efficacious as vaccines for pigs. The PIV5 vaccine vector and chimeric HA-113 antigen are discussed in the context of the development of universal influenza vaccines and the potential contribution of PIV5-113 as a candidate universal vaccine.

Hyper-phosphorylation of nsp2-related proteins of porcine reproductive and respiratory syndrome virus.

Viruses exploit phosphorylation of both viral and host proteins to support viral replication. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus replicase nsp2, and two nsp2-related -2/-1 frameshifting products, nsp2TF and nsp2N, are hyper-phosphorylated. By mapping phosphorylation sites, we subdivide an extended, previously uncharacterized region, located between the papain-like protease-2 (PLP2) domain and frameshifting site, into three distinct domains. These domains include two large hypervariable regions (HVR) with putative intrinsically disordered structures, separated by a conserved and partly structured interval domain that we defined as the inter-HVR conserved domain (IHCD). Abolishing phosphorylation of the inter-species conserved residue serine918, which is located within the IHCD region, abrogates accumulation of viral genomic and subgenomic RNAs and recombinant virus production. Our study reveals the biological significance of phosphorylation events in nsp2-related proteins, emphasizes pleiotropic functions of nsp2-related proteins in the viral life cycle, and presents potential links to pathogenesis.

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.

Double-stranded viral RNA persists in vitro and in vivo during prolonged infection of porcine reproductive and respiratory syndrome virus.

In order to study the mechanism of PRRSV persistence, an in vitro model of persistence was developed by serially passaging PRRSV-infected MARC-145 cells 109 times. Viral persistence was detected to be associated with increased double-stranded (dsRNA) in the infected cells. In PRRSV infected pigs, reduced ratio of plus to minus strands of viral RNA was observed in lymphoid tissues from PRRSV persistent pigs at 52 days post infection. Viral dsRNA was mostly detected in the germinal center during persistent infection compared to the localization of dsRNA in the inter-follicular zones during acute infection. RNA array analysis of antiviral cytokines in persistently infected lymph nodes showed that the presence of dsRNA did not stimulate antiviral immunity. These results suggest that PRRSV dsRNA functions as a mediator for viral persistence. The localization of PRRSV dsRNA in the germinal center of lymphoid tissues reveals a novel mechanism for PRRSV persistence.

Prevalence of Opportunistic Infections and Causes of Death among Hospitalized HIV-Infected Patients in Sichuan, China.

Opportunistic infections (OIs) are the most significant complication of human immunodeficiency virus (HIV) infection. The prevalence of OIs differs among various countries in part due to different climates and socio-economic conditions. We, therefore, carried out the retrospective study at the Public Health Clinical Center of Chengdu, Sichuan to comprehensively investigate the prevalence of OIs, predictors of OIs, and risk factors for in-hospital death among HIV-infected patients. Sichuan in West China is characterized by the largest population living with HIV/Acquired immunodeficiency syndrome (AIDS) across China. In total, we reviewed 954 cases of HIV infection, admitted to the hospital during January 2014 to December 2015, and found that bacterial pneumonia (25.8%) was the most common OIs, followed by candida infection (18.3%), Pneumocystis jiroveci pneumonia (11.9%), tuberculosis (11.5%), infectious diarrhoea (9.3%), cryptococcus infection (7.3%), cytomegalovirus infection (4.9%), toxoplasmosis (4.6%), hepatitis C (4.0%), nontuberculous mycobacteria desease (2.2%) and Penicillium marneffei infection (0.3%). We also found two strongest risk factors for in-hospital mortality: CD4+T cell counts of less than 100 cells/µL and not receiving antiretroviral therapy. Moreover, the study revealed the specific pathogens causing bacterial pneumonia and/or candida infection, the effect of tuberculosis on CD4+T cell counts, and the drug resistance of Mycobacterium tuberculosis among HIV-infected and non-HIV-infected patients. The present findings may aid in the clinical diagnosis and treatment of HIV-infected patients, and could help developing efficient public health strategies in China.

A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase.

Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3Cpro cleavage sites at the 5' and 3' ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-ß expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event.IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order Picornavirales) and torovirus (order Nidovirales). This naturally occurring recombination event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that was inserted into the EVG genome encodes a deubiquitinase/deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights into how a gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity.

A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure.

Porcine circovirus-associated disease (PCVAD) is clinically manifested by postweaning multisystemic wasting syndrome (PMWS), respiratory and enteric disease, reproductive failure, and porcine dermatitis and nephropathy syndrome (PDNS). Porcine circovirus 2 (PCV2) is an essential component of PCVAD, although an etiologic role in PDNS is not well established. Here, a novel circovirus, designated porcine circovirus 3 (PCV3), was identified in sows that died acutely with PDNS-like clinical signs. The capsid and replicase proteins of PCV3 are only 37% and 55% identical to PCV2 and bat circoviruses, respectively. Aborted fetuses from sows with PDNS contained high levels of PCV3 (7.57 × 107 genome copies/ml), and no other viruses were detected by PCR and metagenomic sequencing. Immunohistochemistry (IHC) analysis of sow tissue samples identified PCV3 antigen in skin, kidney, lung, and lymph node samples localized in typical PDNS lesions, including necrotizing vasculitis, glomerulonephritis, granulomatous lymphadenitis, and bronchointerstitial pneumonia. Further study of archived PDNS tissue samples that were negative for PCV2 by IHC analysis identified 45 of 48 that were PCV3 positive by quantitative PCR (qPCR), with 60% of a subset also testing positive for PCV3 by IHC analysis. Analysis by qPCR of 271 porcine respiratory disease diagnostic submission samples identified 34 PCV3-positive cases (12.5%), and enzyme-linked immunosorbent assay detection of anti-PCV3 capsid antibodies in serum samples found that 46 (55%) of 83 samples tested were positive. These results suggest that PCV3 commonly circulates within U.S. swine and may play an etiologic role in reproductive failure and PDNS. Because of the high economic impact of PCV2, this novel circovirus warrants further studies to elucidate its significance and role in PCVAD. IMPORTANCE: While porcine circovirus 2 (PCV2) was first identified in sporadic cases of postweaning multisystemic wasting syndrome in Canada in the early 1990s, an epidemic of severe systemic disease due to PCV2 spread worldwide in the ensuing decade. Despite being effectively controlled by commercial vaccines, PCV2 remains one of the most economically significant viruses of swine. Here, a novel porcine circovirus (PCV3) that is distantly related to known circoviruses was identified in sows with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2, which has previously been associated with these clinical presentations, was not identified. High levels of PCV3 nucleic acid were observed in aborted fetuses by quantitative PCR, and PCV3 antigen was localized in histologic lesions typical of PDNS in sows by immunohistochemistry (IHC) analysis. PCV3 was also identified in archival PDNS diagnostic samples that previously tested negative for PCV2 by IHC analysis. The emergence of PCV3 warrants further investigation.

Construction and characterization of a full-length cDNA infectious clone of emerging porcine Senecavirus A.

A full-length cDNA infectious clone, pKS15-01-Clone, was constructed from an emerging Senecavirus A (SVA; strain KS15-01). To explore the potential use as a viral backbone for expressing marker genes, the enhanced green fluorescent protein (EGFP)-tagged reporter virus (vKS15-01-EGFP) was generated using reverse genetics. Compared to the parental virus, the pKS15-01-Clone derived virus (vKS15-01-Clone) replicated efficiently in vitro and in vivo, and induced similar levels of neutralizing antibody and cytokine responses in infected animals. In contrast, the vKS15-01-EGFP virus showed impaired growth ability and induced lower level of immune response in infected animals. Lesions on the dorsal snout and coronary bands were observed in all pigs infected by parental virus KS15-01, but not in pigs infected with vKS15-01-Clone or vKS15-01-EGFP viruses. These results demonstrated that the infectious clone and EGFP reporter virus could be used as important tools in further elucidating the SVA pathogenesis and development of control measures.

Mutations in a Highly Conserved Motif of nsp1ß Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (nsp1ß) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique -2/-1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif ((123)GKYLQRRLQ(131)) reduced the ability of nsp1ß to suppress interferon beta (IFN-ß) activation and also impaired nsp1ß's function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1ß mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-α expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1ß mutants, IFN-α production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-γ in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1ß function and that modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals. IMPORTANCE: PRRSV infection induces poor antiviral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next-generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing the host immune response. PRRSV nsp1ß was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1ß function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses of infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV modified live-virus (MLV) vaccines and other candidate vaccines.