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Studies on the phylodynamics and pathogenicity of swine and avian influenza viruses

峯, 淳貴 北海道大学

2021.09.24

概要

インフルエンザはオルソミクソウイルス科インフルエンザウイルスを原因とする感染症である。インフルエンザウイルスはその遺伝子、抗原性の違いにより A から D 型に分かれるが、ヒト、ブタ、鳥含む様々な動物に感染し、人獣共通感染症として問題を引き起こすのは A 型インフルエンザウイルス(IAV)である。IAV は 8 分節の遺伝子を有するマイナス鎖の一本鎖 RNA ウイルスであり、一つの細胞に複数のウイルスが感染した際に遺伝子の交換を行うことがある(遺伝子再集合)。IAV は表面蛋白であるヘマグルチニン(HA)およびノイラミニダーゼ(NA)の亜型により、H1 から H18、N1 から N11 に分類され、自然宿主であるカモなどの水禽類はこれらのウイルス(H17、H18、N10、N11 亜型のウイルスを除く)を有している。多様な亜型のウイルスが存在する中で、自然界でニワトリに対して高い病原性を示す鳥インフルエンザウイルス(AIV)は H5亜型と H7 亜型の AIV に限定されている。亜型や遺伝的特徴は宿主により異なるものの、遺伝子再集合により新たな宿主で増殖可能なウイルスが選抜されることがある。豚インフルエンザウイルス(IAV-S)は単独あるいは他の病原体との共感染により養豚場に経済的損失を与える。さらに、2009 年のパンデミックを引き起こしたウイルスのように、他のウイルスとの遺伝子再集合によりパンデミックを起こす能力を獲得する可能性があることから、ウイルスを監視することの重要性が増している。本研究は、国内あるいは世界で分離された IAV-S および AIV について遺伝子情報に加え時期情報、位置情報を加味した系統解析を行うことで、ウイルスの特徴や拡散の背景を詳細に明らかにすることを目的とした。近年の IAV-S の浸潤状況が追究されていなかった日本およびタイの養豚場において分離された IAV-S について時期情報を加味した遺伝子解析を行い、両国で循環している IAV-S の特徴と養豚場内での動態の解析を試みた。さらに、 2017-2018 年冬季に日本で分離された H5N6 亜型高病原性 AIV(HPAIV)の病態を明らかにし、時期情報と位置情報を加えた系統解析により日本でウイルスが拡散する経路の解明を試みた。

日本においては、1970 年以降散発的に IAV-S が分離された報告があるものの、養豚場における IAV-S の循環状況を示すデータは少なく、地域も限られている。そこで第Ⅰ章では、2015 年から 2019 年の間に国内の 21 道県のブタから分離された 424 株の IAV-S について遺伝子の系統学的な解析を試みた。その結果、356 株の IAV-S は 1A.1 classical swine 系統に属する H1 遺伝子を有しており、この遺伝子は他国の IAV-S のものとは遺伝的に大きく異なる独立したクレードを形成した。一方、2015 年から 2019 年に分離された 15 株の H3N2 亜型 IAV-Sは全て近縁であったことから、国内の豚群に定着していることが示唆された。さらに、2009 年にパンデミックを起こした H1N1 ウイルスの HA 遺伝子を有する IAV-S が 53 株分離された。本研究で分離された IAV-S は 1 株を除き全ての IAV-S が 2009 年にパンデミックを起こした H1N1 ウイルス由来の遺伝子を有していたことから、1970 年以降国内で循環していた日本の IAV-S と遺伝子再集合を起こしていることが示唆された。以上の結果は、近年の国内流行株の遺伝的特徴を示しており、有効なワクチンの開発につながる基礎的知見である。

第Ⅱ章では、2011 年から 2017 年にかけてタイの 2 県 4 養豚場において継続的に IAV-S をモニタリングし、合計で 169 株の IAV-S を分離した。このうち 82 株は 2009 年にパンデミックを起こした H1N1 亜型ウイルス由来であった。また、87 株の H3N2 亜型 IAV-S の内部遺伝子は全て 2009 年のパンデミックウイルス由来の遺伝子に置き換わっていたことから、遺伝子再集合が起こったことが示唆された。呼吸器症状を示すブタが頻繁に報告されていた 2 つの養豚場において、H1N1 亜型および H3N2 亜型 IAV-S の抗原性が変化したことが継続的な IAV-S のモニタリングにより明らかになった。系統解析の結果、この農場では抗原性の異なる 2 種類のウイルスが侵入することで、あるいは 1 種類のウイルスが HA 遺伝子への変異の蓄積により抗原性が変化していることがわかった。以上の結果は、IAV-S が実際に野外でどのように多様性を獲得し進化していくかを明らかにするものである。

1996 年に Goose/Guangdong 系統の H5 亜型 HPAIV が中国で発生を起こして以降、このウイルスは世界中に拡散し 20 年以上にわたり循環し続けている。国内での最初の発生は 2004 年に報告され、以降近年まで家禽および野鳥に脅威を与えている。2017-2018 年冬季には、アジアならびにヨーロッパで同時期に H5N6 亜型 HPAIV による発生が記録された。アジアでは 2017 年 11 月に韓国で最初の発生が報告された後、2017-2018 年冬季に家禽の高病原性鳥インフルエンザが香川県で発生し、また島根県、東京都、兵庫県において、死亡野鳥から同じ亜型のウイルスが検出された。第Ⅲ章では、2017-2018 年冬季に家禽ならびに野鳥から分離された H5N6 亜型 HPAIV の遺伝的由来と、ニワトリに対する病原性ならびに伝播能を明らかにした。時期系統解析を行った結果、2017-2018 年冬季に日本で発生を起こした H5N6 亜型 HPAIV(香川株及び日本野鳥株)は、ヨーロッパの H5N8 亜型 HPAIV と野鳥の N6 亜型 AIV が、日本野鳥株については 2016 年夏季に、日本家禽株については 2017 年夏季に遺伝子再集合を起こし出現したと推定された。香川株についてニワトリにおける 50%致死量(EID50)を調べたところ、過去に日本で発生を起こした H5 亜型 HPAIV に比べて 10 倍以上多かった。また、過去の H5 亜型 HPAIV においては、感染した 1 羽のニワトリから同居した 6 羽全てのニワトリへの伝播が成立していたものの、香川株では成立しなかった。以上の結果から、香川株のニワトリに対する病原性は過去に日本で発生を起こした H5 亜型 HPAIV に比べて低く、また伝播能も低いことが示された。2017-2018 年冬季に日本で流行した HPAIV のニワトリに対する病原性が低いことと伝播能の低さは、当該シーズンの流行が大きく広がらなかったことの要因の一つと考えられる。

第 IV 章では、2017-2018 年冬季の H5N6 亜型 HPAIV の N6 遺伝子を含む世界中の AIV の N6 遺伝子を地理的・系統的に解析することで、AIV の拡散動態ならびに渡り鳥の移動との関連性を明らかにした。2018 年 1 月の高病原性鳥インフルエンザ発生原因となった H5N6 亜型 HPAIV の N6 遺伝子を含む 163 株のAIV の NA 遺伝子、2019 年 1 月時点でデータベース(GISAID)に登録されていた 3720 株全ての N6 遺伝子データを収集し地理系統解析を行った。その結果、モンゴルあるいはシベリアの HxN6 亜型 AIV がヨーロッパの AIV と近縁である事例、アジアの AIV と近縁である事例、そしてその両方とも近縁である事例が確認された。このことは、この地域がユーラシア大陸の長距離拡散に重要であることを示唆している。また、近縁な N6 遺伝子を有する AIV が、ユーラシア大陸内だけでなくユーラシア大陸―北アメリカ大陸間、ユーラシア大陸―アフリカ大陸間の長距離を拡散している例が確認された。これらの事例を渡り鳥の飛行経路と照らし合わせると、AIV の広範囲拡散は渡り鳥の飛行経路が関与しており、複数の飛行経路が重なる地点はウイルス拡散の中継点として重要であることが示唆された。

本研究は、IAV-S の養豚場での動態や日本の流行状況、AIV の国間、大陸間の拡散機構の一端を遺伝子情報だけでなく、時期情報、位置情報を加味した系統解析により明らかにしたものである。今後、日本および世界の IAV-S や AIV循環状況を最新の遺伝子解析手法で把握し続けることが、近年流行株に見合ったワクチンの選定や国内に侵入しうる HPAIV の予見といった、現場へ還元できる対策へつながると期待される。

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