Studies on entry events of porcine epidemic diarrhea virus (PEDV)

Doctor of PhilosophyDepartment of Diagnostic Medicine/PathobiologyKyeong-Ok ChangCoronaviruses are currently the most serious public health concern. The new outbreak of coronavirus disease 2019 (COVID 19) represents a pandemic threat, which led to being declared a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO). The first US outbreak of porcine epidemic diarrhea virus (PEDV) in 2013 and its subsequent spreading to European and Asian countries raised significant economic and public health concerns worldwide. Thus, the development of coronavirus vaccines and therapeutics are urgently needed. Detailed studies on the entry event of coronaviruses may contribute to developing novel therapeutic targets for coronavirus infection. Most cell culture adapted PEDV replication requires the addition of protease in the medium, but the mechanism of protease in PEDV infection is not well demonstrated. Thus, we examined the role of protease during the entry of PEDV using two different proteases-adapted PEDV US strains, PEDV KD and AA. Our study showed that the activity of protease was required at an early stage of PEDV KD replication, particularly after virus binding to cells. The addition of protease facilitated the escape of viruses from the endosome to the cytoplasm leading to a successful replication. The host endosomal protease and endosomal maturation were also shown to be important in the endosomal escape of PEDV by demonstrating endosomal retention of PEDV KD or AA in the presence of inhibitors of cathepsins or endosomal acidification. We also explored the roles of the acid sphingomyelinase (ASM)/ceramide pathway in the entry of PEDV. The infection of PEDV 8aa in Vero cells induced ceramide formation mediated by ASM activation. The inhibition of ASM significantly reduced the replication of 8aa by inhibiting viral endosomal escape. These results demonstrated the importance of interactions among viruses, host cells, and proteases during coronavirus entry for successful replication. During further examination of PEDV and host cell interaction, we observed that protease independent PEDV 8aa infection in Vero cells led to apoptotic cell death. Caspase 6 or 7 cleaved viral nucleocapsid(N) protein at the late stage of the replication while the cells were undergoing the apoptotic process. The caspase-mediated cleavage occurred between D⁴²⁴ and G⁴²⁵ near the C-terminal of N protein. Addition of a pan-caspase inhibitor to prevent the N protein cleavage significantly increased 8aa replication. In conclusion, the achievement of endosomal escape is a crucial step in the PEDV life cycle. The addition of the exogenous protease facilitates the endosomal escape of protease adapted PEDV strains. Activation of ASM/ ceramide pathway led to the efficient replication of protease independent PEDV by facilitating the endosomal escape of the virion. During protease independent PEDV replication, host cells activate caspase-mediated apoptosis as a defense mechanism. These in-depth understandings will provide clues for developing potential PEDV and coronavirus therapeutic targets

Linear Spectral Unmixing Algorithms for Abundance Fraction Estimation in Spectroscopy

Fluorescence spectroscopy is commonly used in modern biological and chemical studies, especially for cellular and molecular analysis. Since the measured fluorescence spectrum is the sum of the spectrum of each fluorophore in a sample, a reliable separation of fluorescent labels is the key to the successful analysis of the sample. A technique known as linear spectral unmixing is often used to linearly decompose the measured fluorescence spectrum into a set of constituent fluorescence spectra with abundance fractions. Various algorithms have been developed for linear spectral unmixing. In this work, we implement the existing linear unmixing algorithms and compare their results to discuss their strengths and drawbacks. Furthermore, we apply optimization methods to the linear unmixing problem and evaluate their performance to demonstrate their capabilities of solving the linear unmixing problem. Finally, we denoise noisy fluorescence emission spectra and examine how noise may affect the performance of the algorithms

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

Comparison of the Oral Microbiomes of Canines and Their Owners Using Next-Generation Sequencing

<div><p>The oral microbiome, which is closely associated with many diseases, and the resident pathogenic oral bacteria, which can be transferred by close physical contact, are important public health considerations. Although the dog is the most common companion animal, the composition of the canine oral microbiome, which may include human pathogenic bacteria, and its relationship with that of their owners are unclear. In this study, 16S rDNA pyrosequencing was used to compare the oral microbiomes of 10 dogs and their owners and to identify zoonotic pathogens. Pyrosequencing revealed 246 operational taxonomic units in the 10 samples, representing 57 genera from eight bacterial phyla. Firmicutes (57.6%), Proteobacteria (21.6%), Bacteroidetes (9.8%), Actinobacteria (7.1%), and Fusobacteria (3.9%) were the predominant phyla in the human oral samples, whereas Proteobacteria (25.7%), Actinobacteria (21%), Bacteroidetes (19.7%), Firmicutes (19.3%), and Fusobacteria (12.3%) were predominant in the canine oral samples. The predominant genera in the human samples were <i>Streptococcus</i> (43.9%), <i>Neisseria</i> (10.3%), <i>Haemophilus</i> (9.6%), <i>Prevotella</i> (8.4%), and <i>Veillonella</i> (8.1%), whereas the predominant genera in the canine samples were <i>Actinomyces</i> (17.2%), Unknown (16.8), <i>Porphyromonas</i> (14.8), <i>Fusobacterium</i> (11.8), and <i>Neisseria</i> (7.2%). The oral microbiomes of dogs and their owners were appreciably different, and similarity in the microbiomes of canines and their owners was not correlated with residing in the same household. Oral-to-oral transfer of <i>Neisseria shayeganii</i>, <i>Porphyromonas canigingivalis</i>, <i>Tannerella forsythia</i>, and <i>Streptococcus minor</i> from dogs to humans was suspected. The finding of potentially zoonotic and periodontopathic bacteria in the canine oral microbiome may be a public health concern.</p></div

Bacteria found in the oral samples from both dogs and owners.

<p>SILVA: Silva ribosomal RNA database, HOMD: Human oral microbiome database, NCBI: NCBI basic local alignment search tool database, Identity: Identity is the percent similarity between the query and subject sequences over the length of the coverage area. The numbers below the sample names were correspond to the relative abundance (sample reads/total reads×100) of the read number of the samples in each OTU.</p><p>Bacteria found in the oral samples from both dogs and owners.</p

Characteristics of the dogs and humans enrolled in this study.

<p>F: female, SF: spayed female, M: male, CM: castrated male, Pom: Pomeranian, Dach: Dachshund, Mal: Maltese, Mix: Mixed breed</p><p>^a Control human who had not raised a dog within the past ten years</p><p>^b Number of times per week teeth were brushed</p><p>^c Closeness score between dog and owner: 0, no contact, 1, nearly no contact with dog kept outdoors, 2, nearly no contact with dog kept indoors, 3, frequent contact without oral contact, 4, frequent contact with oral contact</p><p>Characteristics of the dogs and humans enrolled in this study.</p

Relative distribution of sequences in the OTUs in 10 oral samples.

<p>Distribution at the genus level.</p

Phylogenetic tree showing the similarity in the oral microbiota of canines and their owners based on the neighbor-joining methods.

<p>Phylogenetic tree showing the similarity in the oral microbiota of canines and their owners based on the neighbor-joining methods.</p

Three-dimensional principal coordinate analysis (PCoA) plot of samples using the weighted UniFrac distance metric.

<p>Percentage of the diversity distribution explained by each axis is indicated on the fig.</p

Rottlerin-Liposome Inhibits the Endocytosis of Feline Coronavirus Infection

Rottlerin (R) is a natural extract from Mallotus philippensis with antiviral properties. Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) that is characterized by systemic granulomatous inflammation and high mortality. We investigated the antiviral effect of liposome-loaded R, i.e., rottlerin-liposome (RL), against FCoV. We demonstrated that RL inhibited FCoV replication in a dose-dependent manner, not only in the early endocytosis stage but also in the late stage of replication. RL resolved the low solubility issue of rottlerin and improved its inhibition efficacy at the cellular level. Based on these findings, we suggest that RL is worth further investigation as a potential treatment for FCoV