James Zhu

A Conserved Domain in the Leader Proteinase of Foot-and-Mouth Disease Virus Is Required for Proper Subcellular Localization and Function

ABSTRACT The leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV) is involved in antagonizing the innate immune response by blocking the expression of interferon (IFN) and by reducing the immediate-early induction of IFN-β mRNA and IFN-stimulated genes. In addition to its role in shutting off cap-dependent host mRNA translation, Lpro is associated with the degradation of the p65/RelA subunit of nuclear factor κB (NF-κB). Bioinformatics analysis suggests that Lpro contains a SAP (for SAF-A/B, Acinus, and PIAS) domain, a protein structure associated in some cases with the nuclear retention of molecules involved in transcriptional control. We have introduced a single or a double mutation in conserved amino acid residues contained within this domain of Lpro. Although three stable mutant viruses were obtained, only the double mutant displayed an attenuated phenotype in cell culture. Indirect immunofluorescence analysis showed that Lpro subcellular distribution is altered in cells infected with the double mutant virus. Interestingly, nuclear p65/RelA staining disappeared from wild-type (WT) FMDV-infected cells but not from double mutant virus-infected cells. Consistent with these results, NF-κB-dependent transcription was not inhibited in cells infected with double mutant virus in contrast to cells infected with WT virus. However, degradation of the translation initiation factor eIF-4G was very similar for both the WT and the double mutant viruses. Since Lpro catalytic activity was demonstrated to be a requirement for p65/RelA degradation, our results indicate that mutation of the SAP domain reveals a novel separation-of-function activity for FMDV Lpro.

Antiviral Activity of Bovine Type III Interferon Against Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is one of the most serious threats to the livestock industry. Despite the availability of a vaccine, recent outbreaks in disease-free countries have demonstrated that development of novel FMD control strategies is imperative. Here we report the identification and characterization of bovine (bo) interferon lambda 3 (IFN-λ3), a member of the type III IFN family. Expression of boIFN-λ3 using a replication-defective human adenovirus type 5 vector (Ad5-boIFN-λ3) yielded a glycosylated secreted protein with antiviral activity against FMD virus (FMDV) and vesicular stomatitis virus in bovine cell culture. Inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and up-regulation of IFN stimulated gene expression in multiple tissues susceptible to FMDV infection. Our results demonstrate that the type III IFN family is conserved in bovines and boIFN-λ3 has potential for further development as a biotherapeutic candidate to inhibit FMDV or other viruses in cattle.

Differential gene expression in bovine cells infected with wild type and leaderless foot-and-mouth disease virus

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) plays a critical role in viral pathogenesis. Molecular studies have demonstrated that L(pro) inhibits translation of host capped mRNAs and transcription of some genes involved in the innate immune response. We have used microarray technology to study the gene expression profile of bovine cells infected with wild type (WT) or leaderless FMDV. Thirty nine out of approximately 22,000 bovine genes were selectively up-regulated by 2 fold or more in leaderless versus WT virus infected cells. Most of the up-regulated genes corresponded to IFN-inducible genes, chemokines or transcription factors. Comparison of promoter sequences suggested that host factors NF-kappaB, ISGF3G and IRF1 specifically contributed to the differential expression, being NF-kappaB primarily responsible for the observed changes. Our results suggest that L(pro) plays a central role in the FMDV evasion of the innate immune response by inhibiting NF-kappaB dependent gene expression.

Mutations in Classical Swine Fever Virus NS4B Affect Virulence in Swine

ABSTRACT NS4B is one of the nonstructural proteins of classical swine fever virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a putative Toll/interleukin-1 receptor (TIR)-like domain. This TIR-like motif harbors two conserved domains, box 1 and box 2, also observed in other members of the TIR superfamily, including Toll-like receptors (TLRs). Mutations within the BICv NS4B box 2 domain (V2566A, G2567A, I2568A) produced recombinant virus NS4B.VGIv, with an altered phenotype displaying enhanced transcriptional activation of TLR-7-induced genes in swine macrophages, including a significant sustained accumulation of interleukin-6 (IL-6) mRNA. Transfection of swine macrophages with the wild-type NS4B gene partially blocked the TLR-7-activating effect of imiquimod (R837), while transfection with the NS4B gene harboring mutations in either of the putative boxes displayed decreased blocking activity. NS4B.VGIv showed an attenuated phenotype in swine, displaying reduced replication in the oronasal cavity and limited spread from the inoculation site to secondary target organs. Furthermore, the level and duration of IL-6 production in the tonsils of pigs intranasally inoculated with NS4B.VGIv were significantly higher than those for animals infected with BICv. The peak of IL-6 production in infected animals paralleled the ability of animals infected with NS4B.VGIv to resist challenge with virulent BICv. Interestingly, treatment of peripheral blood mononuclear cell cultures with recombinant porcine IL-6 results in a significant decrease in BICv replication.

Patterns of gene expression in swine macrophages infected with classical swine fever virus detected by microarray

Infection of domestic swine with highly virulent, classical swine fever virus (CSFV) strain Brescia, causes lethal disease in all infected animals. However, the molecular mechanisms involved in modulating the host cellular processes and evasion of the immune response have not been clearly established. To gain insight into, the early host response to CSFV, we analyzed the pattern of gene expression in infected swine macrophages, using custom designed swine microarrays. Macrophages, the target cell for CSFV infection, were isolated from primary cultures of peripheral blood mononuclear cells, allowing us to utilize identical uninfected macrophages at the same time points as CSFV-infected macrophages, allowing only genes induced by CSFV to be identified. First, microarray probes were optimized by screening 244,000 probes for hybridization with RNA from infected and uninfected macrophages. Probes that hybridized and passed quality control standards were used to design a 44,000 probe microarray for this study. Changes in expression levels of 79 genes (48 up- and 31 down-regulated) during the first 48h post-infection were observed. As expected many of the genes with an altered pattern of expression are involved in the development of an innate immune response. Several of these genes had differential expression in an attenuated strain NS4B.VGIv, suggesting that some of these differences are responsible for virulence. The observed gene expression profile might help to explain the immunological and pathological changes associated with infection of pigs with CSFV Brescia.

Mechanisms of Foot-and-Mouth Disease Virus Tropism Inferred from Differential Tissue Gene Expression

Foot-and-mouth disease virus (FMDV) targets specific tissues for primary infection, secondary high-titer replication (e.g. foot and mouth where it causes typical vesicular lesions) and long-term persistence at some primary replication sites. Although integrin αVβ6 receptor has been identified as primary FMDV receptors in animals, their tissue distribution alone fails to explain these highly selective tropism-driven events. Thus, other molecular mechanisms must play roles in determining this tissue specificity. We hypothesized that differences in certain biological activities due to differential gene expression determine FMDV tropism and applied whole genome gene expression profiling to identify genes differentially expressed between FMDV-targeted and non-targeted tissues in terms of supporting primary infection, secondary replication including vesicular lesions, and persistence. Using statistical and bioinformatic tools to analyze the differential gene expression, we identified mechanisms that could explain FMDV tissue tropism based on its association with differential expression of integrin αVβ6 heterodimeric receptor (FMDV receptor), fibronectin (ligand of the receptor), IL-1 cytokines, death receptors and the ligands, and multiple genes in the biological pathways involved in extracellular matrix turnover and interferon signaling found in this study. Our results together with reported findings indicate that differences in (1) FMDV receptor availability and accessibility, (2) type I interferon-inducible immune response, and (3) ability to clear virus infected cells via death receptor signaling play roles in determining FMDV tissue tropism and the additional increase of high extracellular matrix turnover induced by FMDV infection, likely via triggering the signaling of highly expressed IL-1 cytokines, play a key role in the pathogenesis of vesicular lesions.

Expression of Porcine Fusion Protein IRF7/3(5D) Efficiently Controls Foot-and-Mouth Disease Virus Replication

ABSTRACT Several studies have demonstrated that the delivery of type I, II, or III interferons (IFNs) by inoculation of a replication-defective human adenovirus 5 (Ad5) vector expressing IFNs can effectively control foot-and-mouth disease (FMD) in cattle and swine during experimental infections. However, relatively high doses are required to achieve protection. In this study, we identified the functional properties of a porcine fusion protein, poIRF7/3(5D), as a biotherapeutic and enhancer of IFN activity against FMD virus (FMDV). We showed that poIRF7/3(5D) is a potent inducer of type I IFNs, including alpha IFN (IFN-α), IFN-β, and IFN-ω but not type III IFN (interleukin-28B), without inducing cytotoxicity. Expression of poIRF7/3(5D) significantly and steadily reduced FMDV titers by up to 6 log10 units in swine and bovine cell lines. Treatment with an IFN receptor inhibitor (B18R) combined with an anti-IFN-α antibody neutralized the antiviral activity in the supernatants of cells transduced with an Ad5 vector expressing poIRF7/3(5D) [Ad5-poIRF7/3(5D)]. However, several transcripts with known antiviral function, including type I IFNs, were still highly upregulated (range of increase, 8-fold to over 500-fold) by poIRF7/3(5D) in the presence of B18R. Furthermore, the sera of mice treated with Ad5-poIRF7/3(5D) showed antiviral activity that was associated with the induction of high levels of IFN-α and resulted in complete protection against FMDV challenge at 6, 24, or 48 h posttreatment. This study highlights for the first time the antiviral potential of Ad5-poIRF7/3(5D) in vitro and in vivo against FMDV. IMPORTANCE FMD remains one of the most devastating diseases that affect livestock worldwide. Effective vaccine formulations are available but are serotype specific and require approximately 7 days before they are able to elicit protective immunity. We have shown that vector-delivered IFN is an option to protect animals against many FMDV serotypes as soon as 24 h and for about 4 days postadministration. Here we demonstrate that delivery of a constitutively active transcription factor that induces the production of endogenous IFNs and potentially other antiviral genes is a viable strategy to protect against FMD.

Treatment with interferon-alpha delays disease in swine infected with a highly virulent CSFV strain

Interferon-alpha (IFNα) can effectively inhibit or abort a viral infection within the host. It has been reported that IFN induction and production is hindered during classical swine fever virus (CSFV) infection. Most of those studies have been performed in vitro, making it difficult to elucidate the actual role of IFNs during CSFV infection in swine. Here, we report the effect of IFNα treatment (delivered by a replication defective recombinant human adenovirus type 5, Ad5) in swine experimentally infected with highly virulent CSFV strain Brescia. Treatment with two different subtypes of IFNα delayed the appearance of CSF-related clinical signs and virus replication although it did not prevent lethal disease. This is the first report describing the effect of IFNα treatment during CSFV infection in swine.

A colorimetric bioassay for high-throughput and cost-effectively assessing anti-foot-and-mouth disease virus activity

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses. This virus is very sensitive to inhibition by type I interferons. Currently, a bioassay based on plaque reduction is used to measure anti-FMDV activity of porcine IFNs. The plaque reduction assay is tedious and difficult to utilize for high-throughput analysis. Using available FMDV susceptible bovine and porcine cells, we developed and tested a colorimetric assay based on cytopathic effect reduction for its ability to quantify FMDV-specific antiviral activity of bovine and porcine type I interferons. Our results show that this new method has significant advantages over other assays in terms of labor intensity, cost, high-throughput capability and/or anti-FMDV specific activity because of simpler procedures and direct measurement of antiviral activity. Several assay conditions were tested to optimize the procedures. The test results show that the assay can be standardized with fixed conditions and a standard or a reference for measuring antiviral activity as units. This is an excellent assay in terms of sensitivity and accuracy based on a statistical evaluation. The results obtained with this assay were highly correlated with a conventional virus titration method.

Clearance of a persistent picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses

Long-term persistent viral infections cause substantial morbidity and associated economic losses in human and veterinary contexts. Yet, the mechanisms associated with establishment of persistent infections are poorly elucidated. We investigated immunomodulatory mechanisms associated with clearance versus persistence of foot-and-mouth disease virus (FMDV) in micro-dissected compartments of the bovine nasopharynx by microarray. The use of laser-capture microdissection allowed elucidation of differential gene regulation within distinct anatomic compartments critical to FMDV infection. Analysis of samples from transitional and persistent phases of infection demonstrated significant differences in transcriptome profiles of animals that cleared infection versus those that became persistently infected carriers. Specifically, it was demonstrated that clearance of FMDV from the nasopharyngeal mucosa was associated with upregulation of targets associated with activation of T cell-mediated immunity. Contrastingly, gene regulation in FMDV carriers suggested inhibition of T cell activation and promotion of Th2 polarization. These findings were corroborated by immunofluorescence microscopy which demonstrated relative abundance of CD8+ T cells in the nasopharyngeal mucosa in association with clearance of FMDV. The findings presented herein emphasize that a critical balance between Th1 and Th2 -mediated immunity is essential for successful clearance of FMDV infection and should be considered for development of next-generation vaccines and antiviral products.