Valosin-containing protein (VCP/p97) plays a role in the replication of West Nile virus

Valosin-containing protein (VCP) is classified as a member of the type II AAA(+) ATPase protein family. VCP functions in several cellular processes, including protein degradation, membrane fusion, vesicular trafficking and disassembly of stress granules. Moreover, VCP is considered to play a role in the replication of several viruses, albeit through different mechanisms. In the present study, we have investigated the role of VCP in West Nile virus (WNV) infection. Endogenous VCP expression was inhibited using either VCP inhibitors or by siRNA knockdown. It could be shown that the inhibition of endogenous VCP expression significantly inhibited WNV infection. The entry assay revealed that silencing of endogenous VCP caused a significant reduction in the expression levels of WNV-RNA compared to control siRNA-treated cells. This indicates that VCP may play a role in early steps either the binding or entry steps of the WNV life cycle. Using WNV virus like particles and WNV-DNA-based replicon, it could be demonstrated that perturbation of VCP expression decreased levels of newly synthesized WNV genomic RNA. These findings suggest that VCP is involved in early steps and during genome replication of the WNV life cycle

A study on Helmholtz resonator tuners for sound transmission control of air-ducts

rights: 社団法人日本建築学

Molecular detection of a novel paramyxovirus in fruit bats from Indonesia

BACKGROUND: Fruit bats are known to harbor zoonotic paramyxoviruses including Nipah, Hendra, and Menangle viruses. The aim of this study was to detect the presence of paramyxovirus RNA in fruit bats from Indonesia. METHODS: RNA samples were obtained from the spleens of 110 fruit bats collected from four locations in Indonesia. All samples were screened by semi-nested broad spectrum reverse transcription PCR targeting the paramyxovirus polymerase (L) genes. RESULTS: Semi-nested reverse transcription PCR detected five previously unidentified paramyxoviruses from six fruit bats. Phylogenetic analysis showed that these virus sequences were related to henipavirus or rubulavirus. CONCLUSIONS: This study indicates the presence of novel paramyxoviruses among fruit bat populations in Indonesia

West Nile Virus in Farmed Crocodiles, Zambia, 2019

We detected West Nile virus (WNV) nucleic acid in crocodiles (Crocodylus niloticus) in Zambia. Phylogenetically, the virus belonged to lineage 1a, which is predominant in the Northern Hemisphere. These data provide evidence that WNV is circulating in crocodiles in Africa and increases the risk for animal and human transmission

MHC class Iを介したウマヘルペスウィルスI型の細胞内侵入機構の解析

Equine herpesvirus 1 (EHV-1) is an endemic virus that affects horse populations worldwide, causing respiratory disease, abortion and a serious neurologic disease known as encephalomyelitis. Outbreaks of EHV-1 encephalomyelitis can have a severe economic impact on farms, racetracks and veterinary hospitals, and currently available vaccines are not effective to protect against EHV-1 encephalomyelitis. Histological analyses of affected horses indicate that viral infection of vascular endothelial cells leads to damage of the central nervous system due to the resulting shortage of blood supply. Although infection of these cells is closely associated with the development of encephalomyelitis, the molecular mechanisms underlying this association remain poorly understood.Alphaherpesviruses enter target cells through sequential multistep processes. Following the initial attachment of the viruses to the cell surface, binding of viral glycoproteins to cell surface receptors triggers fusion of the viral envelope with the cell membrane, resulting in the release of viral capsid (containing viral genome) into the cytoplasm. EHV-1 attaches to cell surface using an interaction between viral glycoprotein C (gC) and cell surface heparan sulfate. Although the role of gC is important for effective infection, it does not trigger viral entry into cells. Glycoprotein D (gD) of EHV-1 is known to be essential for EHV-1 entry into cells. However, functional gD receptor that mediates EHV-1 entry into equine central nervous system (CNS) endothelial cells remains unidentified.In chapter 1, I performed functional cloning using an equine brain microvascular endothelial cell cDNA library and identified that equine major histocompatibility complex (MHC) class I heavy chain conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are resistant to EHV-1 infection. I investigated the role of equine MHC class I in EHV-1 infection and revealed that equine MHC class I acts as an entry receptor for EHV-1 through its binding to EHV-1 gD. I further investigated the role of MHC class I in EHV-1 entry into different types of equine cells.In chapter 2, I constructed equine MHC class I heavy chain specific-RNA probe and performed Northern hybridization to examine the level of equine MHC class I gene expression in the major organs of the adult horse body. The localization of equine MHC class I mRNA in brain tissue was investigated by in situ hybridization.In this thesis, I demonstrated equine MHC class I is a bona fide receptor of EHV-1 entry into equine cells. This study provides new insights into the mechanism of EHV-1 entry into host cells and a potential way to treat and prevent this infectious disease.ウマヘルペスウイルス1型 (EHV-1) はヘルペスウイルス科アルファヘルペスウイルス亜科に属し、馬に脳脊髄炎、流産、鼻肺炎を惹き起こす。罹患馬に重篤な神経症状をもたらす脳脊髄炎では、中枢神経系の血管内皮細胞へのウイルス感染が病態形成に大きな役割を果たすことが明らかになっている。しかしながら、EHV-1の血管内皮向性を規定する宿主因子に関しては不明な点が多い。第1章では、ウマ脳微小血管内皮細胞（EBMECs）より作製したcDNA発現ライブラリーを用いて、EHV-1レセプターのクローニングを試みた。スクリーニングの結果、EHV-1非感受性細胞であるマウス由来細胞株NIH3T3にEHV-1感受性を付与する遺伝子として、ウマ主要組織適合遺伝子複合体（MHC）class I重鎖遺伝子を同定した。ウマMHC class I重鎖遺伝子を安定発現させたNIH3T3細胞へのEHV-1感染は、抗MHC class Iモノクローナル抗体、抗EHV-1 glycoprotein D (gD) ポリクローナル抗体およびgDヒトイムノグロブリン（Ig）融合タンパク（gD-Ig）によって阻害された。また、gD-IgはウマMHC class I分子に特異的に結合した。これらの結果から、ウマMHC class IがEHV-1 gDをリガンドとするエントリーレセプターであることが明らかになった。次に、EHV-1の自然宿主である馬の細胞を用いて、EHV-1感染におけるウマMHC class I分子の関与を解析した。ウマ真皮由来株化細胞E. Derm、EBMECs、ウマ末梢血単核球（PBMC）を抗MHC class Iモノクローナル抗体で前処理すると、EHV-1感染は阻害された。また、β2ミクログロブリン（β2m）分子の発現のノックダウンによってMHC class I分子の細胞表面発現を抑制したE. Derm細胞は、EHV-1感受性が顕著に減少した。以上の結果から、ウマMHC class IはEHV-1のウマ細胞内侵入に重要な役割を果たすことが明らかになった。EHV-1は既知のアルファヘルペスウイルスレセプターを発現していないハムスター由来細胞株CHO-K1に感染し増殖する。そこで、EHV-1のCHO-K1細胞への侵入にハムスターMHC class I分子が関与しているかどうかを検討した。抗MHC class Iモノクローナル抗体の前処理によってEHV-1感染は阻害されなかった。また、β2m分子の発現のノックダウンによってCHO-K1細胞表面におけるMHC class I分子の発現を抑制しても、EHV-1感受性の変化は認められなかった。従って、CHO-K1細胞にはMHC class Iに依存しないEHV-1の細胞内侵入機構が存在することが示唆された。第2章では、ウマ組織におけるMHC class I遺伝子発現の局在を解析した。成馬の全身主要臓器をノーザンハイブリダイゼーションにより解析した結果、検索した全臓器においてウマMHC class I mRNAが検出された。また、成馬脳組織に対してin situハイブリダイゼーション法を施行したところ、ウマMHC class I mRNAの局在を示すシグナルは血管内皮細胞に限局しており、神経細胞を含むその他の細胞にシグナルは認められなかった。以上の結果より、馬の中枢神経系におけるMHC class Iの遺伝子発現はEHV-1の標的細胞である血管内皮細胞に限局しており、MHC class Iの遺伝子発現がEHV-1の脳脊髄炎の病態形成に関与している可能性が示唆された。ウイルスの細胞内侵入は、感染の成立に重要なステップである。本研究によって、ウマMHC class IがEHV-1のエントリーレセプターとして機能することが明らかとなり、これはEHV-1の細胞内侵入機構の解明において重要な知見であると考えられる。また、抗体や組換えタンパクを用いた感染実験の結果より、EHV-1 gDとMHC class Iの結合阻害が、EHV-1感染症の治療法として有用である可能性が示唆された

Mastomys natalensis is a possible natural rodent reservoir for encephalomyocarditis virus

Encephalomyocarditis virus (EMCV) infects a wide range of hosts and can cause encephalitis, myocarditis, reproductive disorders and diabetes mellitus in selected mammalian species. As for humans, EMCV infection seems to occur by the contact with animals and can cause febrile illnesses in some infected patients. Here we isolated EMCV strain ZM12/14 from a natal multimammate mouse (Mastomys natalensis: M. natalensis) in Zambia. Pairwise sequence similarity of the ZM12/14 P1 region consisting of antigenic capsid proteins showed the highest similarity of nucleotide (80.7 %) and amino acid (96.2%) sequence with EMCV serotype 1 (EMCV-1). Phylogenetic analysis revealed that ZM12/14 clustered into EMCV-1 at the P1 and P3 regions but segregated from known EMCV strains at the P2 region, suggesting a unique evolutionary history. Reverse transcription PCR (RT-PCR) screening and neutralizing antibody assays for EMCV were performed using collected tissues and serum from various rodents (n=179) captured in different areas in Zambia. We detected the EMCV genome in 19 M. natalensis (19/179=10.6 %) and neutralizing antibody for EMCV in 33 M. natalensis (33/179=18.4 %). However, we did not detect either the genome or neutralizing antibody in other rodent species. High neutralizing antibody litres (≧320) were observed in both RT-PCR-negative and -positive animals. Inoculation of ZM12/14 caused asymptomatic persistent infection in BALB/c mice with high antibody titres and high viral loads in some organs, consistent with the above epidemiological results. This study is the first report of the isolation of EMCV in Zambia, suggesting that M. natalensis may play a role as a natural reservoir of infection

Multi-reassortant G3P[3] group A rotavirus in a horseshoe bat in Zambia

Group A rotavirus is a major cause of diarrhoea in humans, especially in young children. Bats also harbour group A rotaviruses, but the genetic backgrounds of bat rotavirus strains are usually distinct from those of human rotavirus strains. We identified a new strain of group A rotavirus in the intestinal contents of a horseshoe bat in Zambia. Whole genome sequencing revealed that the identified virus, named RVA/Bat-wt/ZMB/LUS12-14/2012/G3P[3], possessed the genotype constellation G3-P[3]-I3-R2-C2-M3-A9-N2-T3-E2-H3. Several genome segments of LUS12-14 were highly similar to those of group A rotaviruses identified from humans, cows and antelopes, indicating interspecies transmission of rotaviruses between bats and other mammals with possible multiple genomic reassortment events