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M Niikura

Publications and source records attributed to M Niikura.

At least 19 recordsLinked to original sources
PubMed2006

Stability of Marek's disease virus 132-bp repeats during serial in vitro passages.

The Marek's disease virus (MDV) genome contains 2 sets of 132-bp tandem repeat sequences. An increase in 132-bp repeat units has been associated with attenuation of oncogenicity during in vitro passage. By cloning entire genomes, we demonstrated that the copy number of 132-bp repeats can differ within an individual MDV genome. The stability of the 132-bp repeats during cell passage depended on the initial copy number. When both sets of repeats contained 2 copies, the copy number remained stable, while if even 1 set of repeats contained 6 copies, repeat expansion occurred relatively quickly. This expansion did not affect the in vitro growth curve. However, when MDV clones with low and high copy numbers were passed together, genomes with expanded repeats rapidly predominated, mimicking the behavior of naturally-occurring MDV. These results suggest that the preponderance of high-copy repeats after passage reflects intracellular copy number within individual infected cells rather than an influence on the spread of the virus.

Animals↗
PubMed2005

Direct evidence of host genome acquisition by the alphaherpesvirus Marek's disease virus.

Many herpesviruses including Marek's disease virus (MDV), a poultry alphaherpesvirus, carry homologous host genes presumably acquired during viral evolution. We have characterized one recent acquisition by MDV in considerable detail. The virulent MDV strain Md11 previously was isolated from a commercial chicken and initially propagated on duck cells. In the process of cloning the entire Md11 genome in a bacterial artificial chromosome (BAC), we obtained an infectious clone in which the entire terminal repeat short segment was replaced with a portion of the duck genome that corresponds to chicken chromosome 19. This sequence is not predicted to express any protein even though it contains one exon of the VAMP1 gene. The replacement did not affect MDV replication in vitro, despite the virus having only one copy of ICP4. Furthermore, we have shown that the variant MDV genome containing the duck genome substitution is present in the parental Md11 population and has been maintained through several subsequent propagations of the virus on chicken cells. This finding provides direct evidence that host genome acquisition by MDV actually occurs during virus replication, and that one or more such MDV genomes with host sequences may exist within MDV viral stocks which tend to be polyclonal, due to the cell-associated nature of its infection process.

Animals↗
PubMed2004

Modification of endothelial cell functions by Hantaan virus infection: prolonged hyper-permeability induced by TNF-alpha of hantaan virus-infected endothelial cell monolayers.

Serious vascular leakage is central to the pathogenesis of hantavirus infections. However, there is no evidence suggesting the hantavirus infection of endothelial cells directly causes obvious cell damage or morphological alteration either in vivo or in vitro. In this study, we examined whether Hantaan virus (HTNV) infection modifies the barrier function of endothelial cell monolayers upon the exposure to pro-inflammatory cytokines. Low levels (1 ng/ml) of tumor necrosis factor-alpha initially increased the permeability in both HTNV-infected and uninfected monolayers similarly. Thereafter, however, these monolayers showed significant difference. The HTNV-infected monolayers remained irreversibly hyper-permeable during the experimental period up to 4 days, while the uninfected monolayers completely recovered the barrier function. The prolonged hyper-permeability of HTNV-infected monolayers was not associated with cell death or gap formation in the monolayers, and was independent from their nitric oxide or prostaglandin production. These results are the first evidence that hantavirus infection modifies barrier function of endothelial cell monolayers and suggest that HTNV-infection of endothelial cells may contribute to the increased vascular leakage through the prolonged response to cytokines.

Boron Compounds↗
PubMed2004

DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration.

Delivery of foreign genes to the digestive tract mucosa by oral administration of nonreplicating gene transfer vectors would be a very useful method for vaccination and gene therapy. However, there have been few reports on suitable vectors. In the present study, we found that plasmid DNA can be packaged in vitro into a virus-like particle (VLP) composed of open reading frame 2 of hepatitis E virus, which is an orally transmissible virus, and that these VLPs can deliver this foreign DNA to the intestinal mucosa in vivo. The delivery of plasmid DNA to the mucosa of the small intestine was confirmed by the results of immunohistochemical analyses using an expression plasmid encoding human immunodeficiency virus env (HIV env) gp120. After oral administration of VLPs loaded with HIV env cDNA, significant levels of specific IgG and IgA to HIV env in fecal extracts and sera were found. Moreover, mice used in this study exhibited cytotoxic T-lymphocyte responses specific to HIV env in the spleen, Payer's patches and mesenteric lymph nodes. These findings suggest that VLPs derived from orally transmissible viruses can be used as vectors for delivery of genes to mucosal tissue by oral administration for the purpose of DNA vaccination and gene therapy.

AIDS Vaccines↗
PubMed2004

A comprehensive screen for chicken proteins that interact with proteins unique to virulent strains of Marek's disease virus.

Genetic resistance to Marek's disease (MD) has been proposed as a method to augment current vaccinal control of MD. Although it is possible to identify QTL and candidate genes that are associated with MD resistance, it is necessary to integrate functional screens with linkage analysis to confirm the identity of true MD resistance genes. To help achieve this objective, a comprehensive 2-hybrid screen was conducted using genes unique to virulent Marek's disease virus (MDV) strains. Potential MDV-host protein interactions were tested by an in vitro binding assay to confirm the initial two-hybrid results. As a result, 7 new MDV-chicken protein interactions were identified and included the chicken proteins MHC class II beta (BLB) and invariant (Ii) chain (CD74), growth-related translationally controlled tumor protein (TPT1), complement component Clq-binding protein (C1QBP), retinoblastoma-binding protein 4 (RBBP4), and alpha-enolase (ENO1). Mapping of the encoding chicken genes suggests that BLB, the gene for MHC class II beta chain, is a positional candidate gene. In addition, the known functions of the chicken proteins suggest mechanisms that MDV might use to evade the chicken immune system and alter host gene regulation. Taken together, our results indicate that integrated genomic methods provide a powerful strategy to gain insights on complex biological processes and yield a manageable number of genes and pathways for further characterization.

Animals↗
PubMed2003

Identification of chicken lymphocyte antigen 6 complex, locus E (LY6E, alias SCA2) as a putative Marek's disease resistance gene via a virus-host protein interaction screen.

Marek's disease virus (MDV) is a naturally occurring oncogenic avian herpesvirus that causes neurological disorders and T cell lymphoma disease in domestic chickens. Identification and functional characterization of the individual factors involved in Marek's disease (MD) resistance or pathogenesis will enhance our understanding of MDV pathogenesis and further genetic improvement of chickens. To study the genetic basis for resistance to MD, a strategy that combined protein-protein interaction screens followed by linkage analysis was performed. The MDV protein US10 was used as the bait in an E. COLI two-hybrid screening of a cDNA library derived from activated splenic T cells. The chicken LY6E, also known as SCA2 and TSA1, was found to specifically interact with US10. This interaction was confirmed by an in vitro protein-binding assay. Furthermore, LY6E was found to be significantly associated with MD traits in an MD resource population comprised of commercial chickens. Previously, LY6E was implicated in two independent DNA microarray experiments evaluating differential gene expression following MDV infection. Given that LY6E is involved in T cell differentiation and activation, we suggest that LY6E is a candidate gene for MD resistance and deserves further investigation on its role in MDV pathogenesis, especially with respect to the binding of US10.

Animals↗
PubMed2003

Immunoglobulin G enzyme-linked immunosorbent assay using truncated nucleoproteins of Reston Ebola virus.

We developed an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), using partial recombinant nucleoproteins (rNP) of Reston Ebola virus (EBO-R) and Zaire Ebola virus (EBO-Z). We examined the reaction of 10 sera from cynomolgus macaques naturally infected with EBO-R to each of the partial rNP in the IgG ELISA. All the sera reacted to the C-terminal halves of the rNP of both EBO-R and EBO-Z. Most of the sera reacted to the RdeltaC (amino acid (aa) 360-739), and Rdelta6 (aa 451-551) and/or Rdelta8 (aa 631-739) at a higher dilution than to the corresponding truncated rNPs of EBO-Z. The results indicate that this IgG ELISA is useful for detecting EBO-R specific antibody, and may have a potential to discriminate EBO-R infection from other subtypes.

Animals↗
PubMed2001

Genome structure of Ebola virus subtype Reston: differences among Ebola subtypes. Brief report.

We determined the complete genome sequence of Ebola virus subtype Reston (EBO-R) in the Philippines in 1996. The deduced transcriptional signals were highly conserved among Ebola viruses except for the stop signal of L genes. The intergenic regions were composed of 4 to 7 nucleotides, and of 2 characteristic overlaps and a long intergenic region. The glycoprotein (GP) had several amino acid differences from EBO-R isolated in 1989 and 1992. The variety of GP sequences strongly suggests the independent introduction of EBO-R from unknown natural reservoirs in 1996.

Amino Acid Sequence↗
PubMed2001

Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins.

The full-length nucleoprotein (NP) of Ebola virus (EBO) was expressed as a His-tagged recombinant protein (His-EBO-NP) by a baculovirus system. Carboxy-terminal halves of NPs of EBO and Marburg virus (MBG) were expressed as glutathione S-transferase-tagged recombinant proteins in an Escherichia coli system. The antigenic regions on the NPs of EBO and MBG were determined by both Western blotting and enzyme-linked immunosorbent assay (ELISA) to be located on the C-terminal halves. The C-terminal 110 and 102 amino acids of the NPs of EBO and MBG, respectively, possess strong antigenicity. The full-length NP of EBO was strongly expressed in insect cells upon infection with the recombinant baculovirus, while expression of the full-length NP of MBG was weak. We developed an immunoglobulin G (IgG) ELISA using His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as antigens. We evaluated the IgG ELISA for the ability to detect IgG antibodies to EBO and MBG, using human sera collected from EBO and MBG patients. The IgG ELISA with the recombinant NPs showed high sensitivity and specificity in detecting EBO and MBG antibodies. The results indicate that ELISA systems prepared with the recombinant NPs of EBO and MBG are valuable tools for the diagnosis of EBO and MBG infections and for seroepidemiological field studies.

Animals↗
PubMed2001

Immunofluorescence method for detection of Ebola virus immunoglobulin g, using HeLa cells which express recombinant nucleoprotein.

A novel recombinant baculovirus which expresses Ebola virus (EBO) nucleoprotein (NP) under the control of the cytomegalovirus immediate-early promoter was constructed. HeLa cells abortively infected with the baculovirus expressed EBO NP, and this was used as an immunofluorescent (IF) antigen to detect EBO immunoglobulin G (IgG) antibody. This IF method has high efficacy in detecting EBO IgG antibody in clinical specimens, indicating its usefulness in the diagnosis of EBO infections and seroepidemiological studies.

Antibodies, Viral↗
PubMed2001

Detection of Ebola viral antigen by enzyme-linked immunosorbent assay using a novel monoclonal antibody to nucleoprotein.

With the increase in international traffic, the risk of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all over the world. However, the system for the diagnosis of Ebola virus infection is available in a limited number of countries. In the present study, we developed an Ebola virus antigen-detection enzyme-linked immunosorbent assay (ELISA) system using a novel monoclonal antibody (MAb) to the nucleoprotein (NP). This antibody recognized an epitope defined by a 26-amino-acid stretch near the C terminus of NP. In a sandwich ELISA system with the MAb, as little as 30 ng of purified recombinant NP (rNP) was detected. Although this MAb was prepared by immunization with rNP of subtype Zaire, it also reacted to the corresponding region of NP derived from the Reston and Sudan subtypes. These results suggest that our ELISA system should work with three of four Ebola subtypes. Furthermore, our ELISA system detected the NP in subtype Reston-infected monkey specimens, while the background level in noninfected specimens was very low, suggesting the usefulness of the ELISA for laboratory diagnosis with clinical specimens.

Animals↗
PubMed1999

Nucleotide sequence of thymidine kinase gene of sequential acyclovir-resistant herpes simplex virus type 1 isolates recovered from a child with Wiskott-Aldrich syndrome: evidence for reactivation of acyclovir-resistant herpes simplex virus.

Recurrent acyclovir (ACV)-resistant (ACV-r) herpes simplex virus type 1 (HSV-1) infections occurred in a patient with Wiskott-Aldrich syndrome, an X-linked recessive immunodeficiency syndrome composed of three clinical characteristics of immunodeficiency, thrombocytopenia, and an eczematous dermatitis. The patient had severe and recurrent ACV-r herpes simplex and was treated with vidarabine in a satisfactory manner from 1993 to 1997. During the 4-year observation period, two ACV-sensitive (ACV-s) HSV-1 isolates and five ACV-r HSV-1 isolates were recovered. The nucleotide sequence of the thymidine kinase (TK) gene from these sequential ACV-r isolates was compared with the ACV-s isolates. A single nucleotide deletion of cytosine (C) from homopolymer stretch of four C residues between nucleotide 1061 and 1064 of the open reading frame was found in all ACV-r isolates. No other differences were observed in the TK nucleotide sequence between ACV-s and ACV-r isolates. The TK nucleotide sequences of the two ACV-s isolates were identical to each other and those of the five ACV-r isolates were identical to one another. These results suggest that the ACV-r HSV-1 might have derived from the ACV-s strain in the patient body and that TK-associated ACV-r HSV-1 can reactivate from latency.

Acyclovir↗
PubMed1998

The genetic organization and transcriptional analysis of the short unique region in the genome of nononcogenic Marek's disease virus serotype 2.

Studies on the Marek's disease virus (MDV) serotype 2 (MDV2) genome may be important for understanding the naturally nononcogenic nature of the virus. To determine the complete DNA sequence of MDV2 unique short (Us) region, genomic BamHI fragments F, M1 and R were sequenced. The MDV2 Us region is 12109 bp long and contains 12 potential open reading frames (ORFs) likely to encode for proteins. Seven of them exhibit homologies to herpes simplex virus type 1 (HSV-1) US1 (ICP22), US2, US3 (protein kinase), US6 (gD), US7 (gI), US8 (gE) and US10 genes. These ORFs are conserved in a similar arrangement with those of HSV-1, except for US10 which is transposed in the Us regions of all three MDV serotypes. The predicted amino acid sequence of MDV2 ORF6 is homologous to SORF3 of the other serotypes of MDV serotype 1 (MDV1) and herpesvirus of turkeys (HVT) and to infectious laryngotracheitis virus SR1. In addition, four ORFs, which have been identified around the Us and inverted repeat junction regions, have no apparent relation to any other known herpesvirus genes. The identified ORFs in the MDV2 Us region were more colinear with their previously reported locations of MDV1 than with those of HVT and other alphaherpesviruses. Ten of the 12 ORFs in the MDV2 Us region were expressed and transcribed with 3'-coterminal transcripts and/or a unique transcript in the virus-infected cells. Compared to other MDV serotypes, the MDV2 Us-encoded proteins showed 46-70% and 33-59% identities with equivalent of MDV1 and HVT at the amino acid level, respectively. Our present data will be useful to understand the different pathogenicity among serotypes of MDV and to allow precise manipulation of the genes for a possible use in genetically engineered vaccines.

Amino Acid Sequence↗
PubMed1997

Characterization and expression of the Marek's disease virus serotype 2 glycoprotein E in recombinant baculovirus-infected cells: initial analysis of its DNA sequence and antigenic properties.

In Marek's disease virus (MDV) serotype 2 (MDV2) genome, a gene equivalent to the glycoprotein E (gE) of other alphaherpesviruses was identified and sequenced. The primary translation product comprises 488 amino acids with a M(r) of 54.3 kDa. The predicted amino acid sequence possesses several characteristics typical of membrane glycoproteins, including a N-terminal hydrophobic signal sequence, C-terminal transmembrane and cytoplasmic domains, and extra-cellular region containing four potential N-linked glycosylation sites. Compared with other MDV serotypes, MDV2 gE showed 47.3% identity with MDV1 gE, and 38.9% identity with HVT gE at the amino acid level. In transcriptional analyses, a 2.0 kb mRNA which starts between 65 and 86 bps upstream of the potential translational initiation codon of gE was identified as the gE-specific transcript. By a recombinant baculovirus, this potential gE coding region was expressed as several specific products from 66 to 72 kDa. These products were susceptible to tunicamycin treatment, indicating that they were glycoprotein in nature. Further, the expressed gE reacted with all chicken-antisera raised to each of the three serotypes of MDV (strains GA, SB-1, and FC126), suggesting that gE is expressed by all three serotypes of MDV in infected cells and conserves common antigenic epitope(s) beyond those that are serotype specific.

Amino Acid Sequence↗
PubMed1996

Mechanisms of transcriptional transactivation and restriction of human immunodeficiency virus type I replication in an astrocytic glial cell.

Unique transcriptional transactivation by the human immunodeficiency virus type 1 (HIV-1) Tat protein of long terminal repeat (LTR)-driven RNA expression, in the absence of the transactivator responsive element (TAR), was previously demonstrated in central nervous system (CNS)-derived astrocytic cell-lines, including U87MG. In the present study, RNase protection assays were utilized to reveal the molecular mechanism(s) underlying transactivation of the HIV-1-LTR in these cells. Short transcripts, which represent abortive HIV-1 transcription, could not be detected either in the absence or presence of Tat, and no differences in transcript levels were detected using 5' probes, as compared to 3' probes, in the experiments. Thus, the transactivational effects of Tat, in U87MG cells, were potentially based on the increase of transcriptional initiation, both in TAR-dependent and -independent states. Further, by using newly established stable cellular transformant, containing HIV-1-LTR-reporter gene constructs, TAR-independent transactivation was demonstrated to efficiently function primarily in transiently-transfected U87MG cells. U87MG cells, stably-transfected with the intact HIV-1 proviral genome, produced very low levels of virus after long-term culture, as previously reported in other astrocytic cells. These cells demonstrated profoundly restricted transcription of the HIV-1 genome, with no detectable levels of HIV-1-specific RNA by Northern blotting, indicating that the restriction of viral production in these cells is principally due to the low level of overall transcription from the 5' HIV-1-LTR. Transcription of HIV-1 RNA in this cell could not be significantly up-regulated by various stimulators, such as phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha) and sodium butyrate. These data suggest that the restriction of HIV-1 transcription in these cells may be controlled by different mechanism(s) from those in lymphocytic or monocytic cells.

Astrocytes↗
PubMed1996

Identification of a potential Marek's disease virus serotype 2 glycoprotein D gene with homology to herpes simplex virus glycoprotein D.

The gene of Marek's disease virus (MDV) serotype 2 (MDV2) homologous to glycoprotein D (gD) of herpes simplex virus (HSV) was identified and characterized by its nucleotide and predicted amino acid sequences. The MDV2 gD homologous gene contains an open reading frame capable of specifying a polypeptide of 385 amino acids, which include N- and C-terminal hydrophobic domains consistent with signal and anchor regions, respectively, and two potential N-linked glycosylation sites, one of which was located in a highly conserved region when compared to MDV serotype 1 (MDV1) and herpesvirus of turkeys (HVT). By northern blot analysis using a MDV2 gD-specific DNA probe, two highly abundant polycistronic 6.0 and 4.2 kb transcripts were detected in MDV2-infected cells. The genes encoding MDV2 protein kinase (PK), gD, and glycoprotein I (gI) homologues are transcribed to form 3' coterminal mRNAs of 6.0 kb (encoding PK, gD and gI) and 4.2 kb (encoding gD and gI), respectively. By using rapid amplification cDNA end (RACE) method, several RNA start sites, to be thought those of the 4.2 kb mRNA, were detected in the upstream of MDV2 gD homologue.

Amino Acid Sequence↗
PubMed1996

Expression and properties of feline herpesvirus type 1 gD (hemagglutinin) by a recombinant baculovirus.

We constructed a recombinant baculovirus expressing feline herpesvirus type I (FHV-1) gD in insect cells (Sf9 cells). The expressed product was identified as FHV-1 gD by a panel of monoclonal antibodies specific for the FHV-1 gD, and had an apparent molecular mass of approximately 49 kDa, which was less than that of the authentic FHV-1 gD. When the FHV-1 gD protein were expressed in Sf9 cells and CRFK cells in the presence of tunicamycin, the FHV-1 gD exhibited a molecular mass of 41 kDa. It was shown that the gD protein was transported to the surface of recombinant virus-infected Sf9 cells when examined by membrane-immunofluorescence analysis, and that the gD expressed on the surface of Sf9 cells adsorbed feline erythrocytes. Mice inoculated with a lysate of Sf9 cells expressing FHV-1 gD induced antibodies with virus-neutralizing and hemagglutination-inhibition activities. Therefore, the expressed gD appears to be biologically authentic. These data suggested that recombinant FHV-1 gD produced in Sf9 cells may be a useful immunogen as a feline vaccine.

Animals↗