Minoru Ayata

Wild-Type Measles Virus Infection in Human CD46/CD150-Transgenic Mice: CD11c-Positive Dendritic Cells Establish Systemic Viral Infection

We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFP-expressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/huCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-β was higher in mDC than the profile of IFN-α. Wild-type IC induced markedly high levels of IFN-β compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)−/− mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response.

Prevalence of Norwalk-Like Virus Infections in Cases of Viral Gastroenteritis among Children in Osaka City, Japan

ABSTRACT Surveillance of Norwalk-like virus (NLV) infections in cases of pediatric gastroenteritis between April 1996 and March 2000 showed that NLVs were an important causative agent in viral gastroenteritis cases among children between November and January in those years. The predominant type of NLV was closely related to Lordsdale virus in genogroup 2. During the 1999-2000 season, Arg320-like strains, which may be genetic recombinants, suddenly appeared and spread.

Soluble G protein of respiratory syncytial virus inhibits Toll-like receptor 3/4-mediated IFN-beta induction.

Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR) 3 on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early ( approximately 4 h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) reporter analysis in HEK293 cells, polyI:C- or LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR3/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-I/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV.

Susceptibility of human dendritic cells (DCs) to measles virus (MV) depends on their activation stages in conjunction with the level of CDw150: role of Toll stimulators in DC maturation and MV amplification.

Human CD46 (membrane cofactor protein, or MCP) and CDw150 (signaling lymphocyte activation molecule, or SLAM) serve as receptors for measles virus (MV), which induces marked host immune suppression. Although monocytes express CD46, they are considerably resistant to MV. Once monocytes differentiate into immature myeloid dendritic cells (iDCs) (GM-CSF + IL-4-treated), the cells become susceptible to MV. Therefore, we have identified CD46-adapted and CDw150-adapted strains of MV, and the dynamics of CD46 and CDw150 during monocyte-iDC conversion were examined in conjunction with MV susceptibility. Strikingly, CDw150 was not detected in monocytes and moderately induced in iDCs, while CD46 was constantly expressed in monocyte-to-iDC differentiation. Thus, iDCs were found to become highly permissive to CDw150-adapted MV strains via expression of CDw150. In fact, polyclonal and monoclonal antibodies that specifically blocked the MV receptor function of CD46 or CDw150 cancelled MV replication in iDCs according to the preferential usage of either CD46 or CDw150 in each strain of MV. Next, we showed that DCs that matured via stimulation of their Toll-like receptors (TLRs) 2 and/or 4 exhibited an approximately fivefold increase in CDw150 at the protein level, and concomitantly, higher levels of MV amplification were observed in mixed culture of lymphocytes than in iDCs without TLR2/4 stimuli. Hence, amplification of CDw150-dependent MV strains was augmented in DCs parallel with the levels of CDw150 in the presence of lymphocytes possessing CDw150. TLR-mediated functional potential of DCs may affect the degree of MV amplification through distinct MV strain-specific receptor usage of CDw150 or CD46.

Functional modulation of a peroxygenase cytochrome P450: novel insight into the mechanisms of peroxygenase and peroxidase enzymes

Cytochrome P450BSβ is a peroxygenase that catalyzes the α‐ or β‐hydroxylation of myristic acid by utilizing H2O2. The wild‐type enzyme not only hydroxylated myristic acid, but oxidized 3,5,3′,5′‐tetramethylbenzidine (TMB), a peroxidase substrate, in a myristic acid‐dependent reaction. Study of inhibition of hydroxylation of myristic acid by TMB indicates these two substrates compete for the same highly reactive intermediate during the course of their respective reactions. When deuterated myristic acid was used as a substrate to decrease hydroxylation activity, the rate of TMB oxidation increased. This increased rate of TMB oxidation was greatly enhanced when the R242K mutant enzyme bound with deuterated myristic acid was used. These results suggest that there are critical structural elements at the distal active site which determine whether this enzyme acts as a peroxygenase or a peroxidase.

Genotyping of Norovirus strains detected in outbreaks between April 2002 and March 2003 in Osaka City, Japan.

Noroviruses (NVs) are the major cause of food‐ and waterborne nonbacterial gastroenteritis in Japan. Between April 2002 and March 2003, a total of 111 fecal specimens from 40 outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan were subject to NV detection. Seventy‐two samples (64.9%) from 31 outbreaks (77.5%) were NV positive by a real time reverse transcription (RT)‐PCR assay. To further determine the genotype of individual NV strains, we sequenced the capsid N‐terminal/shell (N/S) domain of some representative strains from each outbreak. The 51 NV strains detected in this study were segregated into 15 genotypes (6 in genogroup I and 9 in genogroup II), and GII/5 genotype NV was a dominant outbreak genotype.

Nucleotide sequences of the matrix protein gene of subacute sclerosing panencephalitis viruses compared with local contemporary isolates from patients with acute measles

Measles viruses isolated from brain cells of patients with subacute sclerosing panencephalitis (SSPE) have numerous mutations, especially in the matrix protein (M) gene. To find whether the M genes of these SSPE viruses were mutated randomly or in a pattern, we sequenced this gene from three strains of defective measles virus isolated in Osaka, Japan. We could deduce the sequence of the possible progenitor measles virus for each patient by comparison of the isolate with measles viruses prevailing at roughly the same time and place as the primary infection. Biased hypermutation affected the M genes of all three SSPE viruses, although the molecular mechanisms for the mutations might be various. Replacements of U with C in the plus strand accounted for 76% of all mutations in two of the strains, but in the other strain, replacements of A with G accounted for 52% of the mutations, and the U residues were unchanged.

Efficient isolation of subacute sclerosing panencephalitis virus from patient brains by reference to magnetic resonance and computed tomographic images

Subacute sclerosing panencephalitis virus has been isolated with difficulty from brains of infected patients. More strains are needed for the study of the pathogenesis of this virus. To make the isolation more efficient, we selected portions to be examined from the brains of three patients with reference to findings of repeated magnetic resonance and computed tomographic imaging. Three cell lines susceptible to measles virus field strains were used. In all three cases viruses were isolated most effectively from recent lesions and with Vero cells. Our results suggested that these imaging methods and Vero cells could be used for improvement in the efficiency of isolation of this virus from patient brains.

The F Gene of the Osaka-2 Strain of Measles Virus Derived from a Case of Subacute Sclerosing Panencephalitis Is a Major Determinant of Neurovirulence

ABSTRACT Measles virus (MV) is the causative agent for acute measles and subacute sclerosing panencephalitis (SSPE). Although numerous mutations have been found in the MV genome of SSPE strains, the mutations responsible for the neurovirulence have not been determined. We previously reported that the SSPE Osaka-2 strain but not the wild-type strains of MV induced acute encephalopathy when they were inoculated intracerebrally into 3-week-old hamsters. The recombinant MV system was adapted for the current study to identify the gene(s) responsible for neurovirulence in our hamster model. Recombinant viruses that contained envelope-associated genes from the Osaka-2 strain were generated on the IC323 wild-type MV background. The recombinant virus containing the M gene alone did not induce neurological disease, whereas the H gene partially contributed to neurovirulence. In sharp contrast, the recombinant virus containing the F gene alone induced lethal encephalopathy. This phenotype was related to the ability of the F protein to induce syncytium formation in Vero cells. Further study indicated that a single T461I substitution in the F protein was sufficient to transform the nonneuropathogenic wild-type MV into a lethal virus for hamsters.

Alterations and diversity in the cytoplasmic tail of the fusion protein of subacute sclerosing panencephalitis virus strains isolated in Osaka, Japan.

We determined the nucleotide sequence of the fusion (F) gene of three strains (Osaka-1, -2, and -3) of nonproductive variants of measles virus (MV). These viral strains were isolated in Osaka, Japan, from brain tissues of patients with subacute sclerosing panencephalitis (SSPE). Phylogenetic analysis revealed a close relationship among the three strains of SSPE virus. The cytoplasmic tail of the F protein, predicted from sequence analysis of the gene, is altered in all three SSPE strains when compared to the MV field strains. However, the extent and mode of alteration are different in each strain. The F protein of the Osaka-1 strain has six nonconservative amino acid substitutions and a 29-residue elongation of its cytoplasmic tail. The F protein of the Osaka-3 strain has two nonconservative substitutions and a 5-residue truncation of its C-terminus. Although the termination codon is not altered in the F protein of the Osaka-2 strain, five or six amino acids are changed in the cytoplasmic tail of the F protein of the two sibling viruses of this strain. The significance of the altered cytoplasmic domain of the SSPE viruses in the SSPE pathogenesis is discussed.