A new immunoassay of serum antibodies against Peste des petits ruminants virus using quantum dots and a lateral-flow test strip.

A fast and ultrasensitive test-strip system combining quantum dots (QDs) with a lateral-flow immunoassay strip (LFIAS) was established for detection of Peste des petits ruminants virus (PPRV) antibody. The highly luminescent water-soluble carboxyl-functionalized QDs were used as the signal output and were conjugated to streptococcal protein G (SPG), which was capable of binding to immunoglobulin G (IgG) from many species through an amide bond to capture the target PPRV IgGs. The PPRV N protein, which was immobilized on the detection zone of the test strip, was expressed by transfecting recombinant Bacmid-PPRV-N with Lipofect into Sf9 insect cells. When exposed to PPRV IgG, QD-SPG bound to PPRV N protein, resulting in the formation of a complex that subsequently produced a bright fluorescent band in response to 365 nm ultraviolet excitation. Sensitivity evaluation showed that the QD-LFIAS limit of detection (LOD) for PPRV antibody was superior to competitive enzyme-linked immunosorbent assay (c-ELISA) and the immunochromatographic strip. No cross reaction was observed when the positive sera of bluetongue virus, canine distemper virus, goat pox virus, and foot-and-mouth disease virus were tested. Further evaluation using field samples indicated that the diagnostic specificity and sensitivity of the QD-LFIAS was 99.47 and 97.67 %, respectively, with excellent agreement between QD-LFIAS and c-ELISA. The simple analysis step and objective results that can be obtained within 15 min indicate that this new method shows great promise for rapid, sensitive detection of PPRV IgG for onsite, point-of-care diagnosis and post vaccination evaluation (PVE). Graphical Abstract Ultrasensitive fluorescent QD immunochromotography in combination with recombinant PPRV N protein could be used to detect PPRV antibody in serum.

Foot-and-mouth disease virus 3C protease cleaves NEMO to impair innate immune signaling.

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 (L(pro)) of FMDV is an IFN antagonist. Here, we report that another FMDV-encoded proteinase, 3C(pro), also inhibits IFN-α/ß response and the expression of IFN-stimulated genes. Acting in a proteasome- and caspase-independent manner, the 3C(pro) 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. 3C(pro) 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 3C(pro) abrogated NEMO cleavage and the inhibition of IFN induction. Collectively, our data identify NEMO as a substrate for FMDV 3C(pro) and reveal a novel mechanism evolved by a picornavirus to counteract innate immune signaling.

The leader proteinase of foot-and-mouth disease virus negatively regulates the type I interferon pathway by acting as a viral deubiquitinase.

The leader proteinase (L(pro)) 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 L(pro) 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 Lb(pro), a shorter form of L(pro), has deubiquitinating activity. Sequence alignment and structural bioinformatics analyses revealed that the catalytic residues (Cys51 and His148) are highly conserved in FMDV Lb(pro) of all seven serotypes and that the topology of FMDV Lb(pro) 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 Lb(pro) protein and in vivo ectopically expressed Lb(pro) removed ubiquitin (Ub) moieties from cellular substrates, acting on both lysine-48- and lysine-63-linked polyubiquitin chains. Furthermore, Lb(pro) 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 Lb(pro) 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 Lb(pro) as well as its ability to block signaling to the IFN-ß promoter. Collectively, these results demonstrate that FMDV Lb(pro) 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.

A DNA vaccine encoding the FMDV capsid precursor polypeptide P1 and the enhancing effect of bovine herpesvirus 1 VP22 protein as molecular adjuvant.

UNLABELLED: DNA vaccines containing the capsid precursor polypeptide P1 gene of foot-and-mouth disease virus (FMDV) alone or combined with the VP22 gene of bovine herpesvirus 1 (BVP22) as molecular adjuvant were constructed and used for immunization of BALB/c mice. The latter were challenged with FMDV and their humoral as well as cell-mediated immune responses and virus clearance capacity were assayed. Both DNA vaccines elicited specific immune responses, however, the DNA vaccine with the BVP22 adjuvant showed stronger responses and more efficient virus clearance. A stronger Th1 response was indicated by the IgG2a/IgG1 ratio. These results indicate that (i) a DNA vaccine based on FMDV P1 can stimulate significant immune responses and virus clearance and (ii) BVP22 is a potentially useful molecular adjuvant for such a vaccine. KEYWORDS: DNA vaccine; foot-and-mouth disease virus; bovine herpesvirus 1.

Development of a novel TaqMan-based real-time PCR assay for the detection of porcine boca-like virus (Pbo-likeV).

The recently discovered porcine boca-like virus (Pbo-likeV) is a member of the Parvoviridae family, genus Bocavirus, and it is potentially associated with swine disease. Several studies have associated Pbo-likeV with postweaning multisystemic wasting syndrome in pigs, but the full spectrum of clinical disease and the epidemiology of Pbo-likeV infection remain unclear. The availability of rapid and reliable molecular diagnostics would aid future studies of this novel virus. Thus, we developed a sensitive and specific TaqMan-based real-time PCR assay to target the Pbo-likeV NP1 gene. The assay reproducibly detected 20 copies of a recombinant DNA plasmid containing the NP1 gene, with a dynamic range of six orders of magnitude (10(2)-10(7) copies). The assay did not cross-react with other animal viruses. Clinical evaluation found that Pbo-likeV was present in Chinese swine herds at a frequency of 44.2% (114/258). Higher infection rates were found in diseased pigs (56.1%, 101/180) compared with healthy pigs (16.7%, 13/78) (P < 0.05). Our assay for the diagnosis and quantification of Pbo-likeV was highly sensitive and specific, and should provide a reliable real-time tool for epidemiological and pathogenetic study of Pbo-likeV infection.

Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced RANTES transcription in PK-15 cells.

The chemokine RANTES (regulated upon activation, normal T-cells expressed and secreted) plays an essential role in inflammation and immune response. Infection with wild-type foot-and-mouth disease virus (FMDV) in PK-15 cells strongly inhibits the expression of RANTES compared to infection with a genetically engineered mutant lacking the leader protein (L(pro)) coding region. This suggests that L(pro) is involved in RANTES regulation. However, the underlying molecular mechanism remains unclear. In this study, we show that transfection of PK-15 cells with a plasmid expressing the L(pro) of FMDV, in the absence of other FMDV proteins, inhibited dsRNA-induced RANTES transcription and promoter activity. Promoter mutagenesis experiments revealed that the interferon-stimulated response element (ISRE) was important for the ability of L(pro) to inhibit dsRNA-induced RANTES promoter activity. Furthermore, over-expression of L(pro) also inhibited IRF-3/7-mediated RANTES activation. Screening L(pro) mutants indicated that catalytic activity and a SAP (for SAF-A/B, Acinus, and PIAS) domain of L(pro) were required to suppress dsRNA-induced RANTES transcription.

Corrigendum: Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms.

[This corrects the article DOI: 10.3389/fimmu.2018.01142.].

Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels.

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-beta (IFN-beta) antagonist that disrupts the integrity of transcription factor nuclear factor kappaB (NF-kappaB). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-alpha1/beta expression caused by L(pro) was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-alpha/beta. Furthermore, overexpression of L(pro) significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L(pro) mutants indicated that the ability to process eIF-4G of L(pro) is not required for suppressing dsRNA-induced activation of the IFN-alpha1/beta promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-kappaB, L(pro) also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms.

Foot-and-mouth disease (FMD) is a highly contagious, severe viral illness notifiable to the World Organization for Animal Health. The causative agent, FMD virus (FMDV), replicates rapidly and efficiently inhibits host translation and the innate immune response for it has developed multiple tactics to evade host defenses and takes over gene expression machinery in the host cell. Here, we report a systemic analysis of the proteome and phosphoproteome of FMDV-infected cells. Bioinformatics analysis suggested that FMDV infection shuts off host cap-dependent translation, but leaves intact internal ribosome entry site (IRES)-mediated translation for viral proteins. Interestingly, several FMDV IRES-transacting factors, including G3BP stress granule assembly factor 1 (G3BP1), were dephosphorylated during FMDV infection. Ectopic expression of G3BP1 inhibited FMDV IRES activity, promoted assembly of stress granules, and activated innate immune responses, collectively suppressing FMDV replication. To counteract these host protective responses, FMDV-induced dephosphorylation of G3BP1, compromising its inhibitory effect on viral IRES. In addition, FMDV also proteolytically cleaved G3BP1 by its 3C protease (3Cpro). G3BP1 was cleaved at glutamic acid-284 (E284) by FMDV 3Cpro, and this cleavage completely lost the abilities of G3BP1 to activate innate immunity and to inhibit FMDV replication. Together, these data provide new insights into the post-translational mechanisms by which FMDV limits host stress and antiviral responses and indicate that G3BP1 dephosphorylation and its proteolysis by viral protease are important factors in the failure of host defense against FMDV infection.

Immunogenicity of foot-and-mouth disease virus structural polyprotein P1 expressed in transgenic rice.

Transgenic plants have become developed as bioreactors for producing heterologous proteins and may even form edible vaccines. In the present study, a transgenic rice expressing the capsid precursor polypeptide (P1) gene of foot-and-mouth disease virus (FMDV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, northern blot, western blot, and ELISA analyses confirmed that the P1 gene was integrated into the transgenic rice and the protein was expressed specifically in the leaves at levels of 0.6-1.3 µg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, FMDV-specific neutralizing antibodies were detected. The immunized mice could clear virus from their sera after FMDV challenge. In addition, FMDV-specific mucosal immune responses were detected in mice after oral immunization with protein extracts from transgenic rice plants. Partial virus clearance was obtained after FMDV challenge. These results indicate the potential of using a transgenic rice-based expression system as an alternative bioreactor for FMDV subunit vaccines.

Foot-and-mouth disease virus (FMDV) leader proteinase negatively regulates the porcine interferon-λ1 pathway.

Foot-and-mouth disease is a highly contagious viral disease caused by foot-and-mouth disease virus (FMDV) of wild and domestic cloven-hoofed animals, and causes an economically important disease in the swine industry. In this study, we found that the replication of FMDV in IBRS-2 cells could be significantly inhibited after treatment with the purified recombinant porcine interferon lambda 1 (IFN-λ1), a newly identified type III interferon. However, FMDV could not activate the IFN-λ1 promoter and IFN-λ1 mRNA expression in infected IBRS-2 cells, suggesting that FMDV has evolved mechanisms to interrupt the antiviral function of IFN-λ1. The cause of this inhibition was determined by screening all structural and non-structural proteins of FMDV, and the leader proteinase (L(pro)) was found to exhibit the highest potential to inhibit poly(I:C)-induced IFN-λ1 promoter activity. Further study revealed that the catalytic activity and a SAP (SAF-A/B, Acinus, and PIAS) domain of L(pro) were required for suppressing poly(I:C)-induced IFN-λ1 production. These data suggest that FMDV replication could be inhibited by porcine IFN-λ1, but that the virus has evolved specific mechanisms to inhibit this action.

Enhanced immunogenicity to food-and-mouth disease virus in mice vaccination with alphaviral replicon-based DNA vaccine expressing the capsid precursor polypeptide (P1).

Recently, alphavirus replicon-based DNA vaccines, also known as suicidal DNA vaccines, have emerged as an important strategy to enhance the potency of DNA vaccines. In this study, two different types of DNA vaccines encoding the capsid precursor polypeptide (P1) of foot-and-mouth disease virus (FMDV) were constructed and the immunogenicity were investigated and compared in mouse model. The first DNA vaccine, pcDP1, is a conventional plasmid DNA vaccine in which P1 was driven directly by a cytomegalovirus promoter. The second DNA vaccine, pSCAP1, is a Semliki Forest virus (SFV) replicon-based DNA vaccine encoding the same antigen. In vitro expression and characterization indicated that two vaccine vectors could correctly produce the P1 antigen. However, pSCAP1 could induce obvious apoptosis of the transfected cells. After immunization in BALB/c mice, the P1-specific ELISA antibodies, neutralizing antibodies, as well as lymphocyte proliferative responses induced by pSCAP1 were significantly higher than those obtained in mice immunized with pcDP1. Notably, mice immunized with the pSCAP1 had the determined ability of clearing virus in their sera after FMDV challenge. These results indicate that the SFV replicon-based DNA vaccine pSCAP1 are more effective than conventional DNA vaccine and it can be considered a promising approach for the development of a safety and efficacious vaccine against FMDV.

[High expression of the foot-and-mouth disease's structural protein P1 in Escherichia coli and analysis of its biology activity].

Foot-and-mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX-KG, resulting in the fusion expression plasmid pKG-P1. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-P1 fusion protein was expressed in high level. The molecular weight of the fusion protein wa 110kD and the expressed products were soluble. Western-blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1-ELISA) based on the purified proteins was developed. Comparison between P1-ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1-ELISA.