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日本在来翼手目が保有するウイルスの検索と性状解析

北村(小林), 知也 東京大学 DOI:10.15083/0002002312

2021.10.13

概要

コウモリは哺乳綱翼手目に分類される哺乳類の総称で、約1,300種類が知られている。これは全哺乳類の種数5,416種のうちの約20%を占め、げっ歯目に次いで2番目に大きなグループである。コウモリは重症急性呼吸器症候群(SARS)コロナウイルスやニパウイルスなどのヒトや家畜に対して重篤な病原性を示す様々なウイルスを保有することが知られ、新興・再興感染症の発生母体として注目されている。これまでに海外において、様々なコウモリ種を対象としてそれらが保有するウイルスが調査され、極めて多くの種類のウイルスが検出されている。一方、わが国には35種類のコウモリ在来種が生息しているが、これらのコウモリが保有するウイルスに関してほとんど調べられていない。本研究では、このようにブラックボックスである日本のコウモリが保有するウイルス叢の一端を解明するとともに、その公衆衛生学的な重要性について考察を加えた。

第1章 日本産コウモリからのコウモリコロナウイルスおよびコウモリヘペウイルスの検出と進化系統学的解析
 日本に生息するコウモリがどのようなウイルスを保有しているかを明らかにするため、野生のコウモリを捕獲し、糞検体を採取した。第1章では、ヒトに重篤な感染症を引き起こすSARSや中東呼吸器症候群(MERS)近縁ウイルスの存否を探る目的で、コロナウイルスを標的に調査した。特異的プライマーを用いたRT-PCR法によって、236検体中62検体からコロナウイルスRNAを検出した。それらの塩基配列を決定し進化系統樹解析によって分類したところ、12検体がアルファコロナウイルス属、26検体がベータコロナウイルス属lineage B、24検体がベータコロナウイルス属lineage Cに分類され、日本産コウモリには様々な種類のコウモリコロナウイルスが保有されていることが明らかとなった。Lineage Bおよびlineage Cには、それぞれSARSコロナウイルスやMERSコロナウイルスが属する。しかし、進化系統樹およびスパイクタンパク質のアミノ酸配列の解析によって、今回検出されたウイルスはこれらの病原体とは異なることが示唆された。
 次に、最近になってコウモリからの検出が報告されたヘペウイルスについて調べたところ、81検体中3検体からへぺウイルスRNAが検出された。塩基配列を決定し進化系統樹解析を行ったところ、検出された3検体はヒトのE型肝炎の病原体であるE型肝炎ウイルスとは異なり、既報のコウモリヘペウイルス株と同様に、Orthohepevirus D種に属することが示された。

第2章 日本産コウモリから分離したコウモリアデノウイルスのウイルス学的性状解析
 第1章で用いた236の糞検体のうち163検体を、哺乳動物種由来の11種類の培養細胞に接種し、細胞変性効果を指標にウイルス分離を試みたところ、モモジロコウモリMyotis macrodactylusおよびヒナコウモリVespertilio sinensisの検体からウイルスの分離に成功した。両分離ウイルスは、そのウイルス粒子の特徴やゲノム性状からコウモリアデノウイルス(BtAdV)であることが分かった。DNA polymeraseのアミノ酸配列に基づく進化系統樹解析を行ったところ、両ウイルスはともにMastadenovirus属に属した。モモジロコウモリから分離したBtAdV(BtAdV-Mm32と命名)はコウモリマストアデノウイルスAに分類されたが、ヒナコウモリから分離したBtAdV(BtAdV-Vs9と命名)は他のBtAdVとは進化系統学的に独立し、新種のBtAdVであることが強く示唆された。分離ウイルスは進化系統学的にイヌアデノウイルス(CAdV)に近縁であったが、血清学的抗原性には差異が認められた。複数の哺乳類培養細胞で増殖性を調べたところ、いずれの分離ウイルスも広い細胞指向性を有していた。また、これらの分離ウイルスはBALB/cマウスに経鼻感染させると、弱い病原性を示した。

第3章 日本産コウモリから分離したコウモリアデノウイルスの受容体の同定
 第2章で明らかになったBtAdVの広い細胞指向性のメカニズムを探索するために、第3章では、複数のAdVの受容体として知られるコクサッキー・アデノウイルス受容体(CXADR)のBtAdVの細胞侵入における役割を調べた。まず、分離したBtAdVが効率よく増殖するイヌ由来MDCK細胞からCRISPR-Cas9システムを用いてCXADR遺伝子をノックアウトした細胞を作製したところ、BtAdVの感染性や増殖性が有意に低下することを見出した。一方、組換えレンチウイルスを用いてイヌCXADRを再導入した細胞を作製したところ、BtAdVの感染性や増殖性は野生型細胞と遜色ないレベルまで回復した。これらの結果は、イヌCXADRがMDCK細胞におけるBtAdVの感染性や増殖性を規定していることを示している。さらに、イヌCXADRの代わりにヒトまたはコウモリのCXADRを導入したMDCK細胞においても、BtAdVの感染性や増殖性が、イヌCXADRを再導入した細胞と同様のレベルまで上昇することを見出した。したがって、BtAdVはいくつかの哺乳類由来のCXADRを機能的な受容体として利用できることが明らかとなった。この結果は、BtAdVが他の哺乳類へ感染する潜在性を示すものである。

 以上、本研究によって、日本に生息するコウモリが複数種のコロナウイルス、ヘペウイルス、そしてアデノウイルスを保有していることが明らかとなった。これらの成果は、日本のコウモリが保有するウイルス叢の解明に貢献するものである。また、BtAdVがヒトを含む哺乳動物由来のCXADRを機能的な受容体として利用して、それらの細胞に感染することを初めて明らかにした。この結果は、コウモリのウイルスがヒトを含む哺乳動物に伝播する潜在性を示したものである。今後、対象とするコウモリ種を広げ、それらが保有するウイルスを網羅的に調査することにより、コウモリ種や生息域ごとのウイルス叢や、動物種を超えて感染しうるウイルスの存否が明らかなることを期待する。

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