Studies on entry events during calicivirus replication

Doctor of PhilosophyDepartment of Diagnostic Medicine and PathobiologyKyeong-Ok ChangCaliciviruses are important pathogens of humans and animals. Noroviruses are major causes of foodborne gastroenteritis cases, but their research is hindered due to the inability to grow human noroviruses in cell culture. Detailed studies on entry events of caliciviruses are lacking and may be crucial for development of cell culture models. We examined the entry events of caliciviruses using porcine enteric calicivirus (PEC), feline calicivirus (FCV) and murine norovirus-1 (MNV-1). PEC replication in LLC-PK cells requires bile acid in the medium, but the mechanism is not well understood. Our studies showed that bile acids are required in the early stage of virus replication, and while internalization of PEC is not dependent of them, they are required for endosomal escape and successful replication. Further examination on virus entry, we demonstrated that endosomal acidification and cathepsin L activity are essential in the replication of PEC, FCV and MNV-1. The results showed that inhibition of endosomal acidification or cathepsin L activity led to retention of viruses in the endosomes. Also we demonstrated that recombinant cathepsin L cleaved structural protein of PEC, FCV or MNV-1, which suggests that the enzyme may facilitate uncoating viruses in endosomes. In addition to bile acids, we found that a cold shock treatment during virus entry supported PEC replication by facilitating the endosomal escape. While PEC alone did not induce ceramide formation, bile acids or cold shock treatment induce ceramide formation on endosomes through activation acid sphingomyelinase (ASM), and this event was crucial for virus replication because inhibition of ASM blocked ceramide formation and significantly reduced PEC replication. Incubation of FCV or MNV-1 with cells led to ceramide formation during virus entry, and inhibition of ASM also significantly reduced their replication. Inhibition of ASM led to endosomal retention of PEC, FCV or MNV-1 during virus entry, which may be the reason for the reduction of viral replication. These studies revealed the important and common events during calicivirus entry for successful replication, including virus endosomal escape, cathepsin L activity and ASM/ceramide formation. This detailed information may provide clues for understanding the replication of fastidious caliciviruses and for potential therapeutic targets

A Study on Traditional and CNN Based Computer Vision Sensors for Detection and Recognition of Road Signs with Realization for ADAS

The aim of this chapter is to provide an overview of how road signs can be detected and recognized to aid the ADAS applications and thus enhance the safety employing digital image processing and neural network based methods. The chapter also provides a comparison of these methods

A Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Confers Full Protection against Rift Valley Fever Challenge in Sheep

Citation: Faburay, B., Wilson, W. C., Gaudreault, N. N., Davis, A. S., Shivanna, V., Bawa, B., . . . Richt, J. A. (2016). A Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Confers Full Protection against Rift Valley Fever Challenge in Sheep. Scientific Reports, 6, 12. doi:10.1038/srep27719Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease outbreaks in Africa and the Arabian Peninsula. The virus has great potential for transboundary spread due to the presence of competent vectors in non-endemic areas. There is currently no fully licensed vaccine suitable for use in livestock or humans outside endemic areas. Here we report the evaluation of the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins. In a previous study, the vaccine elicited strong virus neutralizing antibody responses in sheep and was DIVA (differentiating naturally infected from vaccinated animals) compatible. In the current efficacy study, a group of sheep (n=5) was vaccinated subcutaneously with the glycoprotein-based subunit vaccine candidate and then subjected to heterologous challenge with the virulent Kenya-128B-15 RVFV strain. The vaccine elicited high virus neutralizing antibody titers and conferred complete protection in all vaccinated sheep, as evidenced by prevention of viremia, fever and absence of RVFV-associated histopathological lesions. We conclude that the subunit vaccine platform represents a promising strategy for the prevention and control of RVFV infections in susceptible hosts

Experimental infection of calves by two genetically-distinct strains of rift valley fever virus

Citation: Wilson, W. C., Davis, A. S., Gaudreault, N. N., Faburay, B., Trujillo, J. D., Shivanna, V., . . . Richt, J. A. (2016). Experimental infection of calves by two genetically-distinct strains of rift valley fever virus. Viruses, 8(5). doi:10.3390/v8050145Additional Authors: McVey, D. S.Recent outbreaks of Rift Valley fever in ruminant livestock, characterized by mass abortion and high mortality rates in neonates, have raised international interest in improving vaccine control strategies. Previously, we developed a reliable challenge model for sheep that improves the evaluation of existing and novel vaccines in sheep. This sheep model demonstrated differences in the pathogenesis of Rift Valley fever virus (RVFV) infection between two genetically-distinct wild-type strains of the virus, Saudi Arabia 2001 (SA01) and Kenya 2006 (Ken06). Here, we evaluated the pathogenicity of these two RVFV strains in mixed breed beef calves. There was a transient increase in rectal temperatures with both virus strains, but this clinical sign was less consistent than previously reported with sheep. Three of the five Ken06-infected animals had an early-onset viremia, one day post-infection (dpi), with viremia lasting at least three days. The same number of SA01-infected animals developed viremia at 2 dpi, but it only persisted through 3 dpi in one animal. The average virus titer for the SA01-infected calves was 1.6 logs less than for the Ken06-infected calves. Calves, inoculated with either strain, seroconverted by 5 dpi and showed time-dependent increases in their virus-neutralizing antibody titers. Consistent with the results obtained in the previous sheep study, elevated liver enzyme levels, more severe liver pathology and higher virus titers occurred with the Ken06 strain as compared to the SA01 strain. These results demonstrate the establishment of a virulent challenge model for vaccine evaluation in calves. © 2016 by the authors; licensee MDPI, Basel, Switzerland

360° Map Establishment and Real-Time Simultaneous Localization and Mapping Based on Equirectangular Projection for Autonomous Driving Vehicles

This paper proposes the design of a 360° map establishment and real-time simultaneous localization and mapping (SLAM) algorithm based on equirectangular projection. All equirectangular projection images with an aspect ratio of 2:1 are supported for input image types of the proposed system, allowing an unlimited number and arrangement of cameras. Firstly, the proposed system uses dual back-to-back fisheye cameras to capture 360° images, followed by the adoption of the perspective transformation with any yaw degree given to shrink the feature extraction area in order to reduce the computational time, as well as retain the 360° field of view. Secondly, the oriented fast and rotated brief (ORB) feature points extracted from perspective images with a GPU acceleration are used for tracking, mapping, and camera pose estimation in the system. The 360° binary map supports the functions of saving, loading, and online updating to enhance the flexibility, convenience, and stability of the 360° system. The proposed system is also implemented on an nVidia Jetson TX2 embedded platform with 1% accumulated RMS error of 250 m. The average performance of the proposed system achieves 20 frames per second (FPS) in the case with a single-fisheye camera of resolution 1024 × 768, and the system performs panoramic stitching and blending under 1416 × 708 resolution from a dual-fisheye camera at the same time

Trypsin-independent porcine epidemic diarrhea virus US strain with altered virus entry mechanism

Abstract Background Porcine Epidemic Diarrhea Virus (PEDV) is a coronavirus that infects the intestinal tract and causes diarrhea and vomiting in older pigs or extreme dehydration and death that could reach 100% mortality in neonatal piglets. In the US, the first PEDV outbreaks occurred in 2013 and since then US PEDV strains have quickly spread throughout the US and worldwide, causing significant economic and public health concerns. Currently two conditionally approved vaccines exist in the US, but there is no live attenuated vaccine, which is considered the best option in controlling PEDV by inducing transferrable mucosal immunity to susceptible neonatal piglets. In this study, we passaged an US PEDV isolate under various conditions to generate three strains and characterized their growth and antigenicity in cell culture using various assays including Western blot analysis, serum neutralization assay, sequencing analysis and confocal microscopy. Finally, these strains were evaluated for pathogenicity in nursing piglets (1–4 days old). Results One of the PEDV strains generated in this study (designated as PEDV 8aa) is able to replicate in cells without any protease and grows to a high titer of >8 log10 TCID50/ml in cell culture. Interestingly, replication of PEDV 8aa was severely reduced by trypsin and this correlated with the inhibition of virus attachment and entry into the cells. In neonatal nursing piglets, PEDV 8aa (passage number 70 or 105) was found to be fully attenuated with limited virus shedding. Conclusions These results suggest that applying selective pressure during viral passages can facilitate attainment of viral attenuation and that PEDV 8aa warrants further investigation as an attenuated vaccine