Visual and equipment-free reverse transcription recombinase polymerase amplification method for rapid detection of foot-and-mouth disease virus.

BACKGROUND: Foot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages. Accurate, rapid and simple detection of FMDV is critical to containing an FMD outbreak. Recombinase polymerase amplification (RPA) has been explored for detection of diverse pathogens because of its accuracy, rapidness and simplicity. A visible and equipment-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect the FMDV using primers and LF probe specific for the 3D gene. RESULTS: The FMDV LFS RT-RPA assay was performed successfully in a closed fist using body heat for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. The assay could detect FMDV serotypes O, A and Asia1, and there were no cross-reactions with vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2) and pseudorabies virus (PRV). The analytical sensitivity was 1.0 × 102 copies in vitro transcribed FMDV RNA per reaction, which was the same as a real-time RT-PCR. For the 55 samples, FMDV RNA positive rate was 45.5% (25/55) by LFS RT-RPA and 52.7% (29/55) by real-time RT-PCR. For the LFS RT-RPA assay, the positive and negative predicative values were 100% and 80%, respectively. CONCLUSIONS: The performance of the LFS RT-RPA assay was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to be performed. The developed FMDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of FMDV in under-equipped laboratory and at point-of-need facility, which is of great significance in FMD control in low resource settings.

A critical role of interferon-induced protein IFP35 in the type I interferon response in cells induced by foot-and-mouth disease virus (FMDV) protein 2C.

Foot-and-mouth disease virus (FMDV) protein 2C is one of the most highly conserved viral proteins among the serotypes of FMDV. However, its effect on host cell response is not very clear. In our previous report, we showed that FMDV protein 2C interacts with cellular protein N-myc and STAT interactor (Nmi), inducing moderate apoptosis in cells. Here, we show that transfection of HEK293T cells with pEGFP-N1-2C or pEGFP-N1-Nmi induces activation of type I interferon promoters, leading to delayed vesicular stomatitis virus (VSV) growth. Using immunoprecipitation and confocal microscopy assays, we found that interferon-induced protein IFP35 interacts with Nmi. Knockdown of IFP35 expression by siRNA abolished pEGFP-N1-2C and pEGFP-N1-Nmi-induced activation of type I interferon promoters and restored VSV growth, suggesting that IFP35 plays a critical role in the type I interferon response induced by FMDV protein 2C. These findings may help to further understand cell responses to FMDV infection.

Beagle sciatic nerve regeneration across a 30 mm defect bridged by chitosan/PGA artificial nerve grafts.

Longitudinally oriented microstructures are essential for a nerve scaffold to promote the significant regeneration of injured peripheral axons across nerve gaps. In the current study, we present a novel nerve-guiding collagen-chitosan (CCH) scaffold that facilitated the repair of 30 mm-long sciatic nerve defects in beagles. The CCH scaffolds were observed with a scanning electron microscope. Eighteen beagles were equally divided into CCH group, autograft group and non-graft group. The posture and gait of each dog was recorded at 12 and 24 weeks after surgery. Electrophysiological tests, Fluoro-Gold retrograde tracing test, Histological assessment of gastrocnemius and immunofluorescent staining of nerve regeneration were performed. Our investigation of regenerated sciatic nerves indicated that a CCH scaffold strongly supported directed axon regeneration in a manner similar to that achieved by autologous nerve transplantation. In vivo animal experiments showed that the CCH scaffold achieved nerve regeneration and functional recovery equivalent to that achieved by an autograft but without requiring the exogenous delivery of regenerative agents or cell transplantation. We conclude that CCH nerve guides show great promise as a method for repairing peripheral nerve defects.

Engagement of soluble resistance-related calcium binding protein (sorcin) with foot-and-mouth disease virus (FMDV) VP1 inhibits type I interferon response in cells.

Foot-and-mouth disease (FMD) is an acute, highly contagious animal disease caused by FMD virus (FMDV). Although FMDV-induced immunosuppression in host has been well established, the exact molecular mechanism for such induction is not very clear. We report here the identification of FMDV VP1 as an interferon-suppressor by interacting with soluble resistance-related calcium binding protein (sorcin). We found that VP1 suppressed tumor necrosis factor (TNF)-α or Sendai virus (SeV)-induced type I interferon response in HEK293T cells, and that this suppression could be completely abolished by knockdown of sorcin by shRNA. Furthermore, overexpression of sorcin inhibited type I interferon response. Conversely, TNF- or SeV-induced type I interferon response increased when sorcin knocked down, leading to inhibition of vesicular stomatitis virus (VSV) replication. Thus, VP1-induced suppression of type I interferon is mediated by interacting with sorcin, a protein that appears to regulate cell response to viral infections.

A critical role of N-myc and STAT interactor (Nmi) in foot-and-mouth disease virus (FMDV) 2C-induced apoptosis.

Foot-and-mouth disease virus (FMDV) 2C, is one of the most highly-conserved viral proteins among the serotypes of FMDV. However, its effect on host cells is not very clear. Using yeast two-hybrid system and immunoprecipitation approaches, we found that FMDV 2C interacted with the N-myc and STAT interactor (Nmi) protein. When expressed in cells, FMDV 2C is mainly associated with endoplasmic reticulum in the forms of speckles. In the absence of FMDV 2C, Nmi was distributed diffusely in the cytoplasm. However, upon FMDV 2C overexpression Nmi was recruited into FMDV 2C containing speckles where both proteins are co-localized. In addition, FMDV 2C induced apoptosis in BHK-21 cells, which was markedly inhibited by Nmi knockdown, suggesting that Nmi may play a critical role in FMDV 2C-induced apoptosis. These findings may help to further understand the molecular mechanism of pathogenesis of FMDV infection.