Shaobo Xiao

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses interferon-β production by interfering with the RIG-I signaling pathway

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of an economically important swine disease that has been devastating the swine industry since the late 1980s. Accumulating evidences have revealed that PRRSV infection fails to induce type I interferon (IFN-α/β), which are normally induced rapidly during virus replication in virus-infected cells. However, the potential mechanisms remain largely unclear. In this study, we showed that PRRSV infection activated the signal transduction components of NF-κB and AP-1, but not of interferon regulatory factor 3 (IRF3), an essential IFN-β transcription factor. Furthermore, PRRSV infection significantly blocked synthetic dsRNA-induced IFN-β production and IRF3 nuclear translocation. To better understand the upstream signaling events that suppress IRF3 activation, we further investigated the roles of individual components of the retinoic acid-inducible gene I (RIG-I)- and Toll-like receptor 3 (TLR3)-mediated signaling pathway for IFN-β production during PRRSV infection. We observed that PRRSV infection significantly inhibited dsRNA-induced IRF3 activation and IFN-β generation by inactivating IFN-β promoter stimulator 1 (IPS-1), an adaptor molecule of RIG-I. In contrast, PRRSV infection only partially reduced the activation of TIR domain-containing adaptor inducing IFN-β (TRIF), an adaptor molecule of TLR3. Our results suggest that PRRSV infection suppresses production of IFN-β primarily by interfering with the IPS-1 activation in the RIG-I signaling pathway.

The Leader Proteinase of Foot-and-Mouth Disease Virus Negatively Regulates the Type I Interferon Pathway by Acting as a Viral Deubiquitinase

ABSTRACT The leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) is a papain-like proteinase that plays an important role in FMDV pathogenesis. Previously, it has been shown that Lpro is involved in the inhibition of the type I interferon (IFN) response by FMDV. However, the underlying mechanisms remain unclear. Here we demonstrate that FMDV Lbpro, a shorter form of Lpro, has deubiquitinating activity. Sequence alignment and structural bioinformatics analyses revealed that the catalytic residues (Cys51 and His148) are highly conserved in FMDV Lbpro of all seven serotypes and that the topology of FMDV Lbpro is remarkably similar to that of ubiquitin-specific protease 14 (USP14), a cellular deubiquitylation enzyme (DUB), and to that of severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a coronaviral DUB. Both purified Lbpro protein and in vivo ectopically expressed Lbpro removed ubiquitin (Ub) moieties from cellular substrates, acting on both lysine-48- and lysine-63-linked polyubiquitin chains. Furthermore, Lbpro significantly inhibited ubiquitination of retinoic acid-inducible gene I (RIG-I), TANK-binding kinase 1 (TBK1), TNF receptor-associated factor 6 (TRAF6), and TRAF3, key signaling molecules in activation of type I IFN response. Mutations in Lbpro that ablate the catalytic activity (C51A or D163N/D164N) or disrupt the SAP (for SAF-A/B, Acinus, and PIAS) domain (I83A/L86A) abrogated the DUB activity of Lbpro as well as its ability to block signaling to the IFN-β promoter. Collectively, these results demonstrate that FMDV Lbpro possesses DUB activity in addition to serving as a viral proteinase and describe a novel mechanism evolved by FMDV to counteract host innate antiviral responses.

miR-365, a Novel Negative Regulator of Interleukin-6 Gene Expression, Is Cooperatively Regulated by Sp1 and NF-κB

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a central role in host defense. IL-6 expression can be regulated at both a transcriptional and a post-transcriptional level. We used a combination of bioinformatics and experimental techniques to demonstrate that the miR-365 is a direct negative regulator of IL-6. Overexpression of miR-365 mimics decreased activity of a luciferase reporter containing the IL-6 3′-UTR and led to repression of IL-6 protein. In contrast, ectopic expression of a miR-365 inhibitor elevated IL-6 expression. The negative regulation of miR-365 was strictly dependent on a microRNA binding element in the 3′-UTR of IL-6 mRNA. Deletion mutant analysis of the miR-365 promoter showed that two transcription factors, Sp1 and NF-κB, are essential for the transcriptional regulation of miR-365. We also demonstrate that the MAPK/ERK pathway contributes to the regulation of miR-365. Furthermore, miR-365 exhibited a greater negative regulatory effect on IL-6 than hsa-let-7a, a previously identified microRNA negatively regulating IL-6. Taken together, our results show that miR-365 is a novel negative regulator of IL-6.

Recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses.

Em2007, a porcine reproductive and respiratory syndrome virus (PRRSV) variant with a unique 68 aa deletion in Nsp2, was recently isolated in China. Phylogenetic and molecular evolutionary analyses indicated that Em2007 is a natural recombinant between a vaccine strain of PRRSV and circulating virus. We also tested its pathogenicity in piglets.

Foot-and-Mouth Disease Virus 3C Protease Cleaves NEMO To Impair Innate Immune Signaling

ABSTRACT Foot-and-mouth disease is a highly contagious viral illness of wild and domestic cloven-hoofed animals. The causative agent, foot-and-mouth disease virus (FMDV), replicates rapidly, efficiently disseminating within the infected host and being passed on to susceptible animals via direct contact or the aerosol route. To survive in the host, FMDV has evolved to block the host interferon (IFN) response. Previously, we and others demonstrated that the leader proteinase (Lpro) of FMDV is an IFN antagonist. Here, we report that another FMDV-encoded proteinase, 3Cpro, also inhibits IFN-α/β response and the expression of IFN-stimulated genes. Acting in a proteasome- and caspase-independent manner, the 3Cpro of FMDV proteolytically cleaved nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), a bridging adaptor protein essential for activating both NF-κB and interferon-regulatory factor signaling pathways. 3Cpro specifically targeted NEMO at the Gln 383 residue, cleaving off the C-terminal zinc finger domain from the protein. This cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of 3Cpro abrogated NEMO cleavage and the inhibition of IFN induction. Collectively, our data identify NEMO as a substrate for FMDV 3Cpro and reveal a novel mechanism evolved by a picornavirus to counteract innate immune signaling.

Epidemiology and Evolutionary Characteristics of the Porcine Reproductive and Respiratory Syndrome Virus in China between 2006 and 2010

ABSTRACT In 2006, an emerging highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV), which causes continuous high fever and a high proportion of deaths in vaccinated pigs of all ages, broke out in mainland China and spread rapidly to neighboring countries. To examine the epidemiology and evolutionary characteristics of Chinese PRRSV after the 2006 outbreak, we tested 2,981 clinical samples collected from 2006 to 2010 in China, determined 153 Nsp2 sequences and 249 ORF5 sequences, and analyzed the epidemiology and genetic diversity of Chinese PRRSV. Our results showed that the percentage of PRRSV-positive specimens collected from sick pigs averaged 60.85% in the past 5 years and that the highly pathogenic PRRSV has become the dominant strain in China. Furthermore, a reemerging strain which apparently evolved from the highly pathogenic PRRSV strain in 2006 appeared to be widely prevalent in China from 2009 onwards. Sequence analyses revealed that the hypervariable region of Nsp2 in most of the isolates contained a discontinuous deletion equivalent to 30 amino acids, along with other types of deletions. Extensive amino acid substitutions in the GP5 sequence translated from ORF5 were found, particularly in the potential neutralization epitope and the N-glycosylation sites. Our results suggest that Chinese PRRSV has undergone rapid evolution and can circumvent immune responses induced by currently used vaccines. Information from this study will help in understanding the evolutionary characteristics of Chinese PRRSV and assist ongoing efforts to develop and use PRRSV vaccines in the future.

Immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus GP5 protein encoded by a synthetic ORF5 gene

Since May 2006, a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV), which causes continuous high fever and a high proportion of deaths in vaccinated pigs of all ages, has emerged and prevailed in Mainland China. Huge efforts should be made towards the development of an efficient vaccine against the highly pathogenic PRRSV. Although the ORF5-encoded GP5 is the most important immunogenic protein, accumulating evidences have demonstrated that incomplete protection conferred by GP5-based vaccines. The inability to induce robust protective immunity has been postulated to be associated with the presence of a non-neutralizing decoy epitope and heavy glycosylation in close to its neutralizing epitope. In this study, a synthetic ORF5 gene (SynORF5) was engineered with the codon usage optimized for mammalian cell expression based on the native ORF5 gene of highly pathogenic PRRSV strain WUH3. Additional modifications, i.e., inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the non-neutralizing decoy epitope, and mutating four potential N-glycosylation sites (N30, N34, N35 and N51) were also included in the synthetic ORF5 gene. The immunogenicity of the SynORF5-encoded GP5 was evaluated by DNA vaccination in mice and piglets. Results showed that significantly enhanced GP5-specific ELISA antibody, PRRSV-specific neutralizing antibody, IFN-gamma level, as well as lymphocyte proliferation response, could be induced in mice and piglets immunized with DNA construct encoding the modified GP5 than those received DNA vaccine expressing the native GP5. The enhanced immunogenicity of the modified GP5 will be useful to facilitate the development of efficient vaccines against the highly pathogenic PRRSV in the future.

Porcine reproductive and respiratory syndrome virus infection activates IL-10 production through NF-κB and p38 MAPK pathways in porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) is an emerging animal virus that has caused high economic losses for the swine industry worldwide. Previous in vitro and in vivo studies demonstrated that PRRSV infection induces significant production of interleukin 10 (IL-10), a pleiotropic cytokine with immuno-modulatory functions involved in host defense. However, the underlying regulatory mechanisms during PRRSV remain largely unknown. In the present study, we analyzed the expression kinetics of IL-10 in PRRSV-infected primary porcine alveolar macrophages (PAMs) and showed that PRRSV infection induced IL-10 mRNA and protein expression in a time- and dose-dependent manner. Inhibition of various molecules of the Toll-like receptor (TLR) or RIG-I-like receptor (RLR) signaling pathways demonstrated that the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) has an important role in IL-10 induction during PRRSV infection. Furthermore, treatment with specific inhibitors or siRNA knockdown assays demonstrated that NF-κB and p38 MAPK (mitogen-activated protein kinase) are required for PRRSV-induced IL-10. Taken together, PRRSV infection significantly induced IL-10 expression and this induction depends on NF-κB activation and p38 MAPK in PAMs.

Genome biology of Actinobacillus pleuropneumoniae JL03, an isolate of serotype 3 prevalent in China.

Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a cause of considerable world wide economic losses in the swine industry. We sequenced the complete genome of A. pleuropneumoniae, JL03, an isolate of serotype 3 prevalent in China. Its genome is a single chromosome of 2,242,062 base pairs containing 2,097 predicted protein-coding sequences, six ribosomal rRNA operons, and 63 tRNA genes. Preliminary analysis of the genomic sequence and the functions of the encoded proteins not only confirmed the present physiological and pathological knowledge but also offered new insights into the metabolic and virulence characteristics of this important pathogen. We identified a full spectrum of genes related to its characteristic chemoheterotrophic catabolism of fermentation and respiration with an incomplete TCA system for anabolism. In addition to confirming the lack of ApxI toxin, identification of a nonsense mutation in apxIVA and a 5′-proximal truncation of the flp operon deleting both its promoter and the flp1flp2tadV genes have provided convincing scenarios for the low virulence property of JL03. Comparative genomic analysis using the available sequences of other serotypes, probable strain (serotype)-specific genomic islands related to capsular polysaccharides and lipopolysaccharide O-antigen biosyntheses were identified in JL03, which provides a foundation for future research into the mechanisms of serotypic diversity of A. pleuropneumoniae.

Complete coding sequences and phylogenetic analysis of porcine bocavirus.

Here we report, for the first time, the nearly full-length genome sequence of porcine bocavirus (PBoV), a recently discovered parvovirus from pigs. Phylogenetic trees based on this genome sequence showed that PBoV belongs to the branch containing the genus Bocavirus, which comprises canine minute virus (CnMV), bovine parvovirus, gorilla bocavirus and human bocavirus (HBoV), and was most closely related to the group containing CnMV. PBoV was predicted to contain three potential ORFs encoding the non-structural protein NS1, the characteristic NP1 protein and the capsid protein VP1/VP2, with an organization similar to that of known bocaviruses. Interestingly, the NS1 gene of PBoV was more similar in length to the homogeneous gene found in HBoV than to those of other known bocaviruses. In addition, highly conserved unique splice-donor and -acceptor sites were identified in the NS1 gene of HBoV and PBoV.