No description available.

No description available.

Many fish undergo betanodavirus infection. To study the infection process of dragon grouper nervous necrosis virus (DGNNV), native virus and E. coli-produced virus-like particles (VLPs) were used to analyze the binding and internalization in SSN-1 cells. The binding of DGNNV and VLPs to SSN-1 cells was demonstrated using Western blotting and indirect enzyme-linked immunosorbent assay (ELISA). As estimated by ELISA, the DGNNV particles bound SSN-1 cells in a dose-dependent manner up to 8 ¡Ñ 104 particles per cell. The binding of VLPs was sensitive to neuraminidase and tunicamycin, suggesting that cell-surface sialic acid is involved in binding. The recombinant VLPs block attachment of native virus to the surface of cultured fish nerve cells, blocking infection by the native virus. It is suggesting that the outer shell of DGNNV VLPs is structurally indistinguishable from native virus. The penetration of DGNNV into cells, which was monitored by electron microscopy, appeared mainly to occur via the spherical pit and membrane ruffling pathways. Occasionally, a spherical pit was engulfed by membrane ruffling so as to form a large figure 8-shaped vesicle with an open connection. Our observations suggest that DGNNV utilizes both micro- and macro-pinocytosis pathways to enter SSN-1 cells. Both of nucleotide and amino acid sequences of MGNNV protein A were comparison with all of Nodaviridae members, revealed that MGNNV were most closely related to RGNNV. No correlation of sequences of betanodavirus with geographical habitat was detected. All thirteen nodavirus protein A amino acid sequences contained canonical RNA polymerase motifs in their C-terminal halves and conserved elements of predicted secondary structure throughout. By Phyre web server identification, the BVDV RdRp as the best template for fold recognition of the RdRp domain of MGNNV and allowed the construction of a congruent 3D model.

In the U.S., soybean mosaic virus (SMV) is classified into seven strain groups, designated G1 to G7, based on their different responses on resistant soybean [Glycine max (L.) Merr.] cultivars. These responses are: symptomless or resistant (R), necrotic (N), and mosaic or susceptible (S). The gene-for-gene model has been proposed for SMV-soybean interactions. In the majority of cultivars, a single dominant gene, Rsv1, confers both the R and N responses. In the first part of this study, the coat protein (CP) genes of two SMV strains, G1 and G6 were isolated, cloned, and sequenced. Gene isolation was done by reverse transcription-polymerase chain reaction (RT-PCR) on partially purified virus preparation without prior RNA extraction. Amplified products were blunt-end ligated into pNoTA/T7 vector and transformed into competent cells. Sequencing was performed in both directions on heat-denatured double-stranded plasmids. The predicted 265 amino acid sequence of the CP of G1 and G6 strains were 98.9% identical, with only two amino acid differences. Correlating the CP sequences of G1, G2, G6, and G7, with their virulence on resistant soybean cultivars indicated that the CP is not likely to be the R- and/or N-determinant in the SMV-soybean system. The second part of the study involved studying the pathogenesis of G1, G6, and G7 strains on inoculated leaves of R, N, and S soybean cultivars by leaf imprint immunoassay. Results indicated four types of reactions: i) susceptible, showing unrestricted replication and spread; ii) immune, where no virus was detected; iii) systemic spread, showing unrestricted replication but limited spread along the veins; and iv) restricted replication and spread, where infection was restricted to few foci along the veins. Results of this study indicated that Rsv1-mediated resistance is a multicomponent type of resistance that involves both inhibition of virus replication as well as cell-to-cell movement. The third part of the study aimed at investigating Rsv1-mediated resistance at the cellular level. For this purpose, an SMV-soybean protoplast system was developed. Protoplast isolation was based on a combined cellulase-pectolyase Y-23 digestion and metrizamide-sorbitol gradient purification protocol. Virus inoculation of protoplasts was facilitated by either polyethelene glycol (PEG) or poly-L-ornithine (PLO), and method of detection was by Western blotting using antiserum to whole virus. Inoculation by PEG was successful, but results were irreproducible because of the adverse effect of PEG on protoplast viability. Inoculation by PLO was inconclusive because of the high background from residual inoculum. Additional research is needed before a protoplast system can be used to study the mechanism of Rsv1 resistance to SMV at the cellular level. / Ph. D.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (leaves 79-89).

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19192号 / 農博第2131号 / 新制||農||1034(附属図書館) / 学位論文||H27||N4938(農学部図書室) / 32184 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 奥野 哲郎, 教授 佐久間 正幸, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Assembly of virus capsid protein (CP) into icosahedrally symmetric particles is an intriguing and elegant process. In most cases of virus assembly, a large number of identical protein subunits self-assemble to generate a shell that protects the viral genome. Studies on virus assembly have resulted in a new scientific technique that uses these proteinaceous shells as nano-particles for a variety of biological applications. The current thesis deals with understanding the factors that govern the assembly of the Sesbania mosaic virus (SeMV) and a pleomorphic virus, Tobacco streak virus (TSV). CP of SeMV, a T=3 plant virus, consists of a disordered N-terminal R-domain and an ordered S-domain. The importance of the R-domain in the assembly was probed by replacement with polypeptides such as the B-domain of Staphylococcus aureus protein A and polypeptides P10 and P8 of SeMV. These chimera assembled into T=3 or larger virus like particles (VLPs). Addition of divalent cations resulted in the formation of heterogeneous nucleoprotein complexes that disappeared upon treatment with EDTA/RNAse. One of the chimeras (N∆65-B) purified in a dimeric form by affinity chromatography assembled into T=1 VLPs during crystallization. The three dimensional structure of these VLPs showed that they were devoid of divalent ions and the B-domain was disordered. These studies demonstrate the importance of N-terminal residues, metal ions in virus assembly and robustness of the assembly process. Also, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications. Tobacco streak virus (TSV) is a polymorphic virus and a major plant pathogen. TSV capsids encapsidate the tri-partite ss-RNA genome of the virus in three spheroidal particles of diameters 27, 30 and 33 nm, respectively. CPs of ilarviruses are also involved in genome activation. The labile nature of ilarviruses has posed difficulties in their structure determination. This thesis describes the first crystal structure of truncated TSV-CP. The core of TSV CP conforms to the canonical β-barrel jelly roll tertiary structure found in other viral coat proteins. Dimers of CP with swapped C-terminal arms (C-arm) were observed in the two crystal structures determined. The C-arm was found to be flexible and responsible for the polymorphic and pleomorphic nature of TSV capsids. Mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was also found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses.

No description available.

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.

Food poisoning is mainly caused by pathogenic microorganisms and is now a severe problem worldwide. Therefore, rapid and sensitive methods are required to detect foodborne pathogens. A locally isolated bacterium, Bacillus licheniformis B38 was selected for this study. The spores of B. licheniformis B38 were induced by Schaeffer’s sporulation medium containing KCl, MgSO4.7H2O, Ca(NO3)4, MnCl2 and FeSO4. Schaeffer-Fulton endospore staining was used to differentiate spores and vegetative cells, where spores were stained green and vegetative cells were stained red. In order to separate the spores from the cells, a two-phase system was used to obtain pure spore suspension for following experiments. Spore coat proteins were extracted by SDS-8 M urea sample buffer and visualized by two different types of coomassie brilliant blue staining solutions. One of the staining solutions was more suitable for gel elution by diffusion. An ~10 kDa spore coat protein was selected for protein purification. Based on the given results, the protein purification by liquid chromatography was less convincing than using gel elution by diffusion technique. The two hypothetical protein sequences, P06552 and P45693, from the ~10 kDa spore coat protein were identified. In the preliminary study of B. licheniformis B38 spores detection by surface plasmon resonance, several binding parameters were studied. Dot blot was done to verify the reaction between the Bacillus spores polyclonal antibody against the B. licheniformis B38 spore coat protein. The most promising result was the binding of 0.1 mg/mL polyclonal antibody (analyte) to the 0.2 mg/mL spore coat protein at pH 2 (ligand) which showed 5.74 RU. The differences between a dot blot and a SPR detection techniques are described.