資 料 3-1
事
務
連
絡
平成 2 4 年 1 月 2 4 日
日本赤十字社血 液事業本部 御 中
供 血 者 か らの 遡 及 調 査 の 進 捗状況 について
( 目次 )
薬事 ・食 品衛生審議会 血液事業部会事務 局
厚 生 労働省 医薬食 品局血 液対策 課
供血者 か らの遡及調査の進捗状況 について
○
供 血 者か らの遡 及調 査 の進捗状 況 につ いて
(平成 24年1月24日付 け血液対 策課事 務連 絡 )
○
体 血 者 か らの遡 及調 査の進 捗 状 況 につ いて (回答 )
(平成 24年2月7日 付 け 日本赤十字社提 出資 料 )
○
薬 事法 第 77条の4の3に 基 づ く回収報告状況
(平成 23年11月 ∼ 平成 24年1月分 )
標記 につ きま しては、平成 23年 11月 18日 付 け血安第 419号 にて貴社
血液 事業本部長 よ り資料の提 出があ り、 これ を平成 23年 度第 3回 血液事業部
会運営委員会に提出 した ところです。今般 、平成 24年 3月 14日 (水)に 平
成 23年 度 第 4回 血液 事業部 会運営委員会 が 開催 され るこ ととな りま した の
で 、下記 の事項 につ いて改 めて資料 を作成 いただ き、平成 24年 2月 7日 (火)
二￡ に当事務局あて御提出いただきます よ うお願 いいた します。
記
1「 供血者 の供血歴 の確認等 の徹底 につ いて」 (平成 15年 6月 12日 付け医
薬 血発 第 0612001号)に 基 づ く遡及調査 に係 る以下の事項
(1)遡 及調査実施 内容
`
① 調 査の対象 とした献血 件数
② 上 記① の うち、調査 の対象 とした輸 血用血液 製剤 の本数
③ 上 記② の うち、医療機 関に情報提供 を行 つた本数
(2)個 別 NAT関 連情報
の うち、個別 NATの 結果が陽性 となった献血件数
① (1)①
② 上 記①の うち、医療機関 へ供給 され た製剤に関する報告件数
③ 上 記②の うち、受血者情報が判明 した件数
④ 上 記③の うち、医薬品副作用感染症報告を行った件数
2.資 料 の作成に当たつての留意事項
① 本 数又は件数については、病原体別及びその合計を明 らかにす ること。
また、上記 (1)の ③及び (2)の ① ∼③ については、対象期間 ごとに本
数又は件数を記載すること。
② 本 数又は件数については、平成 23年 11月 18日 付け血安第 419号
の提出時において判明 した ものに、その後 の遡及調査の進展状況 を反映 さ
せて記載す ること。
供 血 者から始まる遡 及調 査実施状況
血 安 第 41号
平成 24年 2月 7日
対象期間
平成2 1 年 4 月1 日 ∼
平成2 2 年 3 月3 1 日
平成 2 2 年 4 月 1 日 ∼
平 成2 3 年3 月3 1 日
平成2 3 年 4 月 1 日 ∼
平成2 3 年 1 1 月 3 0 日
HBV
HBV
HBV
HCV
HⅣ
HCV
HIV
HCV
HIV
1)遡及調査実施内容
厚生労働省医薬食品局血液対策課長 様
① 調査の対象とした献血件数(個別NAT実施件数)
1)総 数
1,852
2)イ
回別r卜数
日本赤十字社
血液事業本部長
1.688
1640
1,730
1.588
② 上記①のうち、調査の対象とした輸血用血液製剤の本数
1)総 数
2.014
2 ) 個B l l 本
数
2.072
1,877
1820
1,934
1,760
1.934
1,580
③ 上記②のうち、医療機関に情報提供を行つた本数
供血者か らの遡及調査の進 1/9状
況について (回答)
1)総 数
2.014
2)個 別本数
平成 24年 1月 24日 付事務連絡によりご連絡のあ りま した標記の件 について、別 紙
によ り報告いた します。
1634
1,877
2)個 別NAT関 連情報
)①〕
のうち、個別NATの結果が陽性となつた献血件数
① 遡及調査実施対象〔(￢
1)総 数
2)個 別件数
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
6
0
0
② 上記①のうち、医療機関へ供給された製剤に関する報告件数
1)使 用された本数
2)医 療機関調査中
0
3)院 内で廃棄
6
4)不 明
計
0
6
0
0
152
0
0
106
4
③ 上記②のうちt受血者情報が判明した件数
1)陽 転事例
1
0
0
2)非 陽転事例
0
0
0
3)死 亡
0
0
0
4)退 院 ・
未検査
0
0
0
5)陽 性だが輸 血 前不 明
0
0
計
0
6
1
0
0
④ 上記③のうち、医薬品副作用感染症報告を行つた件数
報告件数
1
4
4
*血液製剤等 に係 る遡 及 調査 ガイドライン(平成20年 12月26日 一 部改正 )に基づ く遊 及調 査 対応 基準 を適 用。
HBV:HB5抗
原CLEIA4確 認試験 (中和試験)又 は個別NAT陽 性の場合は遡及調査を行 う。
:HBctttC口 晨 法陽転の場合は遡及調査を行 う。
Hい ′ :HCVAttCLEIA法 陽転 の血液 及び前回 の血液について個別NATを 実施 し、いずれかが陽性の場合は遡及調査を行 う。
HⅣ :HⅣ 抗体 CLEIA法 で陽転 し、確認試験 (WB法 )又 は個別NAT陽 性の場合 は遡及調査を行 う。
共通 :ス クリーニングNAT陽 転 の場合は遡及調査を行 う。
0
薬事法第 7
0 平 成 2 3 年 1 1 月 ∼平成 2 4 年 1 月
〈
参考〉
供血 者 か ら始まる遡及調 査実施状況
対象期間
平成 1 1 年4 月 1 日∼
平成 1 8 年3 月 3 1 日
HBV
HCV
HA/
平成 1 8 年 4 月 1 日 ∼
平成 1 9 年3 月 3 1 日
平成1 9 年 4 月 1 日 ∼
平成2 0 年 3 月 3 1 日
平成20年 4月 1日 ∼
平成21年 3月 31日
HBV
HBV
H3V
HCV
H:V
HIV
HIV
① 調査の対象とした献血件数
1)遡 及調査の対象件数
23104
2.193
2.694
5,219
② 上記①のうち、個別NAT検 査を実施した本数(検体数)
1)本 数 (検体数)
2,193
23104
2)実 施率
5.219
100%
100%
100%
③ 上記②のうち陽性が判明した本数
本数
3
1
0
1
0
0
0
④ 上記①のうち医療機関に情報提供を行つた件数
1)血 液製剤数 (総数)
曰別本数
33114
/
/
2)情 報提供数
曰別件数
2,408
/
2,062
/
2,062
33114
/
/
2.867
4,034
2.444
2.408
3,552
2.708
2.319
31150
*平 成 11年4月1日∼平成17年3月31日までの情報提供数には、医療機関の廃院等による追跡不能数930件を含む
⑤ 上記③のうち医療機関へ供給された製剤に関する報告件数
1)使 用された本数
2)医療機関調査 中
3
3)院 内で廃棄
卜
)不明
￨
0
7
2
0
0
0
0
1
計
1
2
0
94
0
0
0
0
0
0
53
0
0
0
0
0
0
0
0
⑥ 上記⑤のうち、受血者情報が判明した件数
1)陽 転事例
17
1
1
2)非 陽転事例
4
1
4
0
:1
3)死亡
2
0
)退院 ・
未検査
5)陽性だが輸血前不明
計
0
7
0
3
9
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
⑦ 上記⑥のうち、医薬品副作用感染症報告を行つた件数
積 盲 IT取
0
0
0
Ⅲ個別NATR性 (NArウインドウピリオド)の遡及調査対象血液の輸血により、受血者が陽転じf_3を含む
ウイルス男‖
合_■
3
0
1 6 ・: l 1 1 1 5 1 1 1 0 1 4 1 0 1 0 i n
HBV:28
Hcv:2
HIV:1
′
" β解
同年■
9月
0
0
条の 4
の 3
に基 づ く回収報告 状況
新鮮凍結血渠
H21 11 20
赤血球製剤
血 小板製剤
赤血球濃厚液
45人
3人
者数
H19 2 20
供血
血液製剤
○ 目 爵 泄 当 面コ 叫 夕鶏 叶 冊 満 百Ｏ τ バ
︵
＝ 爵 蛍 楡 Ｎ油 Ч回〓 暉 口＞ 斗 ＋ 囀 洋 渤 鵬 腑 羊 ︶
輸血 された
○ 目 爵 泄 壼 肩コ 叫 夕謝 叶 冊 繊 百Ｏ τ ベ
︵
洲 爵 鍵 や 一コ ミ ロ〓 尋目 館 漱 滸 珊 冊 鱗 醐 譲 ︶ 一一
染が疑われた事例 】
○ 引 爵 器 楡 河 刺 滞 浄 謝 叶 冊 童 Э 叫 針３ 肩Ｏ τべ ω
報告 日
奪灘謝叶 ¨
０ ５べ ︵
針５ ヽ
【HBV感
︿噺 鍛 ﹀ ヽ
・ 蹄 ゆユ 辮 辮 報 Э漸 ざ
〇
○ 罫 ロ コ ロ 爵 泄 壼 ︵鄭 将 吻 瀬 き ざ か冊 菫 編 一
削 覇 ” 圭 マ 熙 器 珊 ︶百
原料血漿 : 2 0 本 中 2 本 確保 。1 8 本 使用済み。
原料血漿 :3本 全て確保。
同 一血液由来の他 製剤等に ついて
供血者 23人
23人 中 21人来訪
HBV関連検査陰性 :21人
赤血球製剤 : 2 2 本 全て供給済み 。
感染が疑われ る輸血時の製剤の 新鮮凍結血漿 : 3 本 全て供給済み 。
保管検体個別 NAT全て陰性
HBV関連検査陰性 :2人
3人 中 2人 来訪
保管検体lE tt NAT全
て陰性
供血者検査結果等
( 平成 2 4 年 2 月 7 日 時点。過去 5 年 間分)
輸 血 用血液製剤で感 染 が 疑われ る事例 につ いて
N
○ 罫 日 ぶ 工ｍく鄭 将 Э 呻 ヨ 準 滸 ︵
副蓼 ・
ｚ＞﹃Э 洋 滞 ︶
ω
平成 2 3 年 6 月 2 7 日 以降 、残 る 2 人 の
来訪な し。
の来訪 な し。
平成 1 9 年 1 0 月 1 9 日 以降 、残 る 1 人
新規報告
目 爵 泄 壼 百コ 斗 夕囲淑藤 囲サ 伊Э 鄭 潜前難 中 冊會 ＝ 肩０ ，バ
平 成 23年 度 感 染 症 報 告 事 例 の ま と め に つ い て
(平 成 23年
11月 ∼ 平 成 23月
12月 報 告 分 )
1 平 成 2 3 年 1 1 月 か ら 2 3 年 1 2 月 ま で に 報 告 ( 新規 及 び 追 加 ) が あ つ た 感 染
症 報 告 ( 疑 い 事 例 を 含 む 。供 血 者 か ら の 情 報 に よ り開 始 した 遡 及 調 査 に よ る
も の を 除 く) は 、 輸 血 用 血 液 製 剤 1 7 件 で あ る 。
輸血 用血 液製剤 の内訳 は、
8件
(1)HBV感
染報 告事 例 :
7件
(2)HCV感
染報 告 事 例 :
0件
(3)HIV感
染報告 事 例 :
(4)そ の他の感 染症 報 告例 : 2件
2 HBV感
染報告 事例
( 1 ) 輸 血 前 後 に 感 染 症 検 査 で H B V ‐D N A 、 H B s 抗 原 等 が 陽 転 した 事 例 は 8 件 。
輸 血 後 NATで 陰 性 又 は輸 血 前 後 で 陽性 は o件 。
(2)使
用 さ れ た 輸 血 用 血 液 製 剤 を 提 供 した 献 血 者 の 保 管 検 体 の 個 別 N A T 陽
性 の事例 は 4件 。
(3)劇
症 化 又 は 輸 血 後 に 死 亡 ( 原 疾 患 又 は 他 の 原 因 に よ る 死 亡 を 除 く) じ
た との 報 告 を 受 け た 事 例 は 0 件 。
3 HCV感
染報告 事例
´
4 HIV感
染報告事 例
の他の感染症報告 事例
(1)B型
肝 炎 及 び C 型 肝 炎 以外 の 肝 障 害 報告 事 例 は 0 件 。
( 2 ) 細 菌 等 感 染 報 告 事 例 に お い て 、使 用 され た 輸 血 用 血 液 製 剤 を 提 供 し た 献
血 者の保 管検 体 の 無菌 試験 陽 性事例 は 0件 。
( 3 ) 輸 血 後 に 死 亡 ( 原 疾 患 又 は 他 の 原 因 に よ る 死 亡 を 除 く) し た と の 報 告 を
受 けた事 例 は 0 件
■ ● “ や尽 ‘ ボ ︶尽 “ 澤
( 1 ) 輸 血 前 後 に 抗 体 検 査 等 が 陽 転 した 事 例 は 0 件 。
( 2 ) 使 用 され た 輸 血 用 血 液 製 剤 を 提 供 した 献 血 者 の 保 管 検 体 の 個 別 N A T 陽
性 事例 は 0件 。
( 3 ) 輸 血 後 に 死 亡 ( 原 疾 患 又 は 他 の 原 因 に よ る 死 亡 を 除 く) し た と の 報 告 を
受 けた事 例 は 0 件 。
5 そ
一３“二‘ 黎躊事 ｏ“”
( 1 ) 輸 血 前 後 に 抗 体 検 査 ( 又 は H C V ―R N A ) 等 が 陽 転 した 事 例 は 7 件 。 輸 血
後 NATで 陰 性 又 は輸 血前 後 で陽性 は 0件 。
( 2 ) 使 用 さ れ た 輸 血 用 血 液 製 剤 を 提 供 した 献 血 者 の 保 管 検 体 の 個 別 N A T 陽
性事例 は 0件 。
( 3 ) 劇 症 化 又 は 輸 血 後 に 死 亡 ( 原 疾 患 又 は他 の 原 因 に よ る 死 亡 を 除 く) し た
との報 告 を受 け た 事 例 は 0 件 。
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の輸Itl用
血液
￨
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担当医より輪 血血液と感楽症の国果 ￨
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平成 24年 3月 14日 開催
薬 事 。食 品衛 生 審議 会
運営 委員 会 提 出資料
別紙
iS蜃ぎ,現踏奮
日本赤十字社
※“
耀根ヨ島
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試行的 H E V 2 0 プ ール N A T 実 施状況について
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( 輸血 後 H E V 感 染 の予防対策)
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北海道赤十字血液センター管内
調査期 間:平成 17年 1月 1日 ∼平成 23年 12月 31日
H17.1∼
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H18.3-H23.12*2
袷)言
汁
341,174
献血者数
HEv―
45
1//7,582
1,590,673
1,931,847
186
RNA陽 性
陽
性率
1//8,552
231
1//8,363
*1北 海道センターにて NAT実 施 (ALT高 値 、検査不合格検体も含 む)
*2血 漿分画センターにて NAT実 施 (ALT高 値 、検査不合格検体は除く)
血 安 第 42号
平成 2 4 年 2 月 7 日
事
務
連
絡
平成 24年 1月 24日
日本赤十字社血液事業本部 御 中
厚生労働省 医薬食品局血液対策課長 様
薬事 ・食品衛生 審議会血液事業部会事務局
厚 生 労 f O J 省医 薬 食 品 局 血 液 対 策 課
日本赤十字社
血液事業本部長
血液 製剤 に関す る報告事項 について
血液製剤 に関す る報告事項について (回答)
血液事 業 の推進 に御努力 いただ き、厚 く御礼 申 し上 げます。
さて、標記 につ きま しては 、平成 23年 11月 18日 付 け血安第 420号 に
て貴社 か ら報告 を頂いた ところですが、平成 24年 3月 14日 (水)、に平成 2
3年 度第 4回 血液事業部会運営委員会が開催 され ますので、下記 の事項につい
て資料 を作成 いただ き、平成 24年 2月 7日 (火)ま でに当事務局あて御提出
いただ きます よ うお願 い します。記 の 3に つい ては 、平成 23年 度第 3回 血液
事業部会運営委員会提 出資料 を更新 の うえ、再度御 提出 くだ さい。
なお、資料 の作成に当た つては 、供血者 、患者及 び医療機 関 の名称並びに こ
れ らの所在地又はこれ らの事項が特定できる情報 を記載 しないよ う、個人情報
及び法人情報 の保護 に特段 の御配慮 をお願 いします 。
平成 24年 1月 24日付事務連絡 によ りご依頼 のあ りま した標記 の件 につ いて、
下記の とお り資料を作成 しま したので報告 いた します。
記
1.平 成 19年 2月 20日 付 けで報告 され た輸血用血液製剤 でHBV(B型
肝炎
ウイル ス)感 染が疑われ る事例 について、残 る 1人 のその後 の来訪な し。(3
名 中 2名 が来所、検査は全て陰性)
記
1.平 成 19年 2月 20日 付 けで報告 され た輸血用 血液 製剤 でHBV(B型
肝
炎 ウイ ル ス)感 染 が疑われ る事例について、残 る 1人 の供血者 のそ の後 の検
査結果。来訪がなければ 、そ の 旨。
炎
(23名
2 平 成 21年 11月 20日 付 けで報告 された輸血用血液製剤で HBV(B型
肝炎 ウイル ス)感 染 が疑われ る事例につ いて 、残 る 2人 の供血者 のその後 の
検査結果。来訪 がなけれ ば,そ の 旨。
は
3 試 行 的HEV20プ
ール NATに
つい て、その後 の調査実施状況。
2.平 成 21年 11月 20日 付 けで報告 された輸血用血液製剤 でHBV(B型
肝
ウイルス)感 染が疑われ る事例 について 、残 る 2人 のその後の来訪 な し。
中 21名 が来所 、検査 は全 て陰性)
3.試 行的HEV20プ
別紙 の とお り。
ール NATに
ついて、その 後 の調査実施状況について
安全 対策業務 の流れ
3-3
1性 別・
年齢区分・
国別
件り
万た
Ю当
件
陽 ＜Ｅ
数
数＞
実
件施
検
血査
献＜
年
¨
嚇
¨
¨
］
体 ・核 酸 増 幅 検 査 陽性 件 数
献血件数 及び HIV抗
件
1987年
( 昭和 6 2 年 )
8,217,340
11(1)
0134
( 昭和 6 3 年 )
7,974,147
9(1)
0113
1989年
( 平成 元 年 )
( 平成 2 年 )
7,876,682
13(1)
0165
1990年
7,743,475
26(6)
0336
1991年
( 平成 3 年 )
8,071,937
29(4)
0359
1992年
( 平成 4 年 )
7,710,693
34(7)
0441
1993年
7,205,514
35(5)
0486
6,610,484
36(5)
0545
1995年
( 平成 5 年 )
・
( 平成 6 年 )
( 平成 7 年 )
6,298,706
46(9)
0730
1996年
( 平成 8 年 )
6,039,394
46(5)
0762
1997年
( 平成 9 年 )
5,998,760
54(5)
0900
1998年
( 平成 1 0 年 )
6,137,378
50(4)
0912
1999年
( 平成 H 年 )
6.139.205
64(6)
1042
2000年
( 平成 1 2 年)
5,877,971
67(4)
[3]
1140
2001年
( 平成 1 3 年)
5,774,269
79(1)
[l]
1368
2002年
( 平成 1 4 年 )
5,784,101
82(5)
[2]
1418
2003年
( 平成 1 5 年)
5,621,096
87(8)
[2]
1548
2004年
( 平成 1 6 年 )
5,473,140
92(4)
[2]
1681
2005年
( 平成 1 7 年 )
5,320,602
78(3)
[2]
1466
2006年
( 平成 1 8 年)
4,987,857
( 平成 1 9 年)
4,939,550
2008年
( 平成 2 0 年 )
5,077,238
2009年
( 平成 2 1 年 )
5,287,101
2010年
( 平成 2 2 年 )
5,318,586
( 平成 2 3 年 )
5,252,182
(速 報 値 )
87(5)
[1]
102(3)
[6]
107(3)
[0]
102(6)
[2]
86(3)
El]
89(8)
[3]
1744
2007年
2011年
(1∼ 12月
)
男
性
女
日本 人
外 国人
人
人
性
日本人 外国人
計
合
計
日本 人 外 国人
計
計
件
1988年
1994年
体 `核 酸 増 幅 検 査 陽 性 献 血 者 数 内 訳
HIV抗
16∼ 19歳
人
人
人
人
人
人
1
メ
l
20∼ 29歳
545
575
628
30∼ 39歳
520
533
561
40∼ 49歳
186
1
187
50∼ 69歳
合
計
1371
1416
198
1
7
0
105
7
7
2
0
1476 i
52
1528
2065
2107
1929
※ 昭和61年 ∼平成23年 12月 (昭和61年 につ いては年途 中から集計)
1617
1695
昭和 6 1 年 は、年 中途 か ら実施 し
ヽ印 “ρ“ 件 、
うち、 陽性件 数 1 1 件 ( 女性 0 ) をぶξぞド を夕
( 注 2 ) ・ 抗 体検 査 及 び核 酸 増 幅 検 査 陽性 の血 液 は廃 棄 され 、製 剤 に は使 用 され ない。
・ 核 醜 雌 査 につ い て は、戦
実施 してい る。
( 注 3 ) ・ 平 成 2 3 年 は、1 月 ∼ 1 2 月 の遡
肇象糟 キ彙 曇 う そ含 冒P に
(注0・
=2-
」
2 都 道 府 県 別 ( 献血 地 別 )
ブ ロ ック別 H I V 抗
体・
核 酸増 幅 検 査 陽性 献 血 者
師 輸
“ 眸
“ 幹
濤 ①
時 ０
箸 出
平成 19年
件
件
1 0 万人
献血者 1陽 性
当たり
人
北海 道
東北
件
東
1.559,391i361
噛
膳
東
海
近
畿
110万
人
1陽性 当たり
件
件
平成23年
(1月∼ 12月)(速報値)
献血者 1陽 性
当たり
件
件
9
件
献血者
性習[´
1陽
件
i5
件
件
1,621,408
36
件
件
件
件
件
件
件
件
件
件
件
件
件
件
件
2お
3
00
轟れ
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帯
11 件
231 2634
件
■ 0925
5
件
件
四 国
件
馴欄
件
件
件
589,760
1
1 件
2:
4
件
-3-
人
平成 22年
545,248t 8t
中 国
合 計
件
献 血者
5
件
関
平成21年
件 一
人
以脚
献血者 レ 性
平成20年
1 130
件
1633
件
件
5,077,2381
件
轟L%
ル
1亀̈
: 件
891
-4-
1 695
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榊 鋼軽 坦 憑 細 郭理 型Ｓ卜 ・
せ 尽 ＞Ｈ
〓展鮨 よ
XMRVに
( 続報 ) ( 平
文献 名
番月
14日 )
血
報告国
液事業部会運営委員会碁昌 岡田 義B召
要約
Hindawi Publishing COrporation
Advancesin Virdogy
Volume 2011,Artide ID 854540,S pages
dot10 1155′
2011/854540
Oakes B, Qiu X, Levine S, Hackett J Jr, Huber BT
Adv Virol. 201 1i2o1 1:854540. Epub 2O11 Jut 27.
米国
Failure to Detect XMRv-Specific Antibodies in the
Plasma of CFS Patients Using Highly Sensitive
Chemiluminescence lmmunoassays.
R“ι
ακtt Ar″
ε
:υ
FailuretO Detect XMRV― Speci■oAntibodies in
the Plas血a ofCFS Patients Using HighlySensitive
Che血 luminescence lnllnlmoassays
Robinson MJ, Tuke PW, Erlwein O, et al.
Adv Virol. 2011:2011:782353.Epub 2011 Jun 9.
英国
No Evidehce of Xl\4RVor l\ruLV Sequences in
Prostate Cancer, Diffuse Large B-Cell Lymphoma,
or the UK Blood Donor Populatioh.
BFendan OakeS,1,2】
缶aodng Qiur susan Levine,4
, O h n H a c k e t, t3 J ■
a n d B F i g Hi ut bt e r■i
Keah€yMF, L6€ K, Bagni RK. et al.
Adv Virol.2011:2011:272'1
93. Epub 201 t Nov 17.
輻
米国
Nucleic Acid, Antibody, and Virus Culture Methods
:o Detect Xenotropic MLv-Reiated Virus in Human
Blood Samples.
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脇響1'‰
:脇
i聯勤務椰惚:Lttχ
CorrcsPondence shoud bc addresscd tO Brlgittc
T Hubeろ
lrigltte
htlbcr@tuns cdu
Zhou Y, Steffen t. MohtalvoL, et al.
rErefusion: 20l 2 Feb:52(2\t33242.
Recelved 8 APH1 201 L AcccPrd lo Junc 2011
Acadcmc Fditor Myra McClure
)evelopment and applicationof a high-throughput
nicroneutElization assayt lack of xenotropic murine
機構 による阻害である。
/irus delection in blood donore.
琴叩警■■■
カナダ
ヽ
o Evidence rOr xMRV Nucleic Acids infectious
Karafin MS. Stramer
Transtusion.
,
柵 :∬
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米国
こ
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[[[:誇
讐勲 捨
蛉L確i[苺
驀墜i:首
SL.
l. Introduction
2012 Feb 152(2):222-5
米国
The scientific
leukemia
chronic
method
al work: xenotropic
murine
virus-related
vhus is neither a cause of
fatigue syndrome nor a threat to the blood
suppry.
漁ι
‖
智[8雪
籍言
凛 1孵
- 1 -
肥
螺l鵠 激 艘 織
In 2006, Urisman et al. identified a new gammaretfovirus
in prostate aner samplesharboring a mutation in a viral
defensegene known m RNASEI Il]. This new virus, xenotropic murine leu-kemiavirus-related retrovirus (XMRV),
was found to be a closerelativeto known murine leukemia
viruses(MLVs) and was the first documented caie of human
infection with a renotropic retrovirus. Although XMRV was
originally associated
with the mutant variant ofthe RNASEI
gene,further researchcould not confrrm this 4sociation but
did find it in about 10oloofprostate cancers[2].
The discoveryof a nry virus that could infect humans
lead Lombardi et al. {31 to test for the viru in patients
sufferingfrom chronic fatigues;mdrome (CFS).CFSIs a diseme of unknown etiology that manifests a neurological,
immunological, and endocrinological dysfunctions.A wide
range of viruseshave been investigatedin the past as caus-
-2-
ative agentsof CFS; howeve5 findings were mixed, and no
onclusive evidene of one virus cauing CFS has been implicated [41. Using a nested polymer-ase
chain reaction
(PCR), Lombardi et al. found that blood samplesof68 out of
l0l (670/o)CFS patients conrained the XMRVgag sequence,
asopposedto only 8 outof2l2 (3.7o/o)samplesfrom healthy
individuais[3]. The 6nding of a viruslinkedro CFSreignited
*citement in the field, leading many laboratories around
the world to iesi for ihis new virus, but the qcitement has
been short lived. Although some support linking XMRV or
MLVs and CFS has been published [3, 5, 6], it has been
overshadowedby reports failing to detect the.virus in CFS
patients [7-20], including a study done by us.
In our original paper I I 7], we failed to find an association
betwen CFS patients and XMRV, using PCR technology.
However, we did detect some XMRV sequencesas well as
other MLV sequencesin some of our smples. Due to the
close relationship between XMRV and MLVs, which are
Advanes in Virology
presentthroughout the mouse genome, we testedall of our
samples for mouse DNA using a thqMm qPCR assayfor
murine mitochondrial cytochrome oxidase,cox2[14], m well
as a single PCR assayfor the highly abundant intracisternal
A-type particle (IAP) long terminal repeat sequence,developed by our group [17]. We fomd rhat every sample rhar
contained an XMRV or MLV sequencewas also positive for
mouse DNA contamination. Although we did not claim that
our findings provided a full *planation of the origin of
XMRV we put fomard a eutionary tale about the risks of
mouse DNA contamination in various ommon laboratory
reaSents.
One ofthe criticisms ofour study [17] was that we only
usedPCR technologyto test fo! the presenc ofXMRV,while
the original paper also included serologiel analyses{31.Specifically, some groups have developednovel serologicaltests
utilizing western blots and ELISAs in the searchfor mtiXMRV antibodies,became the presenceof antibodiesould
not be due to mouse DNA contamination [3,8, 13, 14, 20,
2 I I . Recendy,two prototype direct fomat chemiluminescent
immuno*says (CMIAs) were developed to detect XMRVspecific antibodies [22]. Both CMIAs utilize a direct amy
format in which reombinant pl5E or gp70 protein serves
as both capture and detection antigens.The assaysdemonstrated excellent sensitivity detecting early seroconversion
bleeds in XMRV-infected rhesus maaques [22]. Moreover,
theseassayswere also shown to detect specificmtibodies to
MLVS 1221.In this itudy, we use these two semitive CMIAs
to screenplffma samplesfrom our blinded ohorts for the
presenceof XMRV-specific mtibodies. No samplesftom our
cohort of over 100 CFS patients were positive in either of
these assays,while two samples from the healthy contrcl
cohort tested positive in one of the CMIA assays;howeveq
reactivity of thesesamesampleswas not confirmed by western blot. Thus, thesehighly sensitiveserologicalstudieshave
confirmed our prior concluion that the positiveXMRV PCR
resultswere a result ofmouse DNA contamination, sinceno
antibodies againstXMRV were present.
2. Materials
and Methods
2.1. SampleColledion. All sampleswere collectedacording
to the institutional guidelines of Tufts Uriversity, after receiving informed consent. The 36 healthy individuals (15
femalesand 2l males) were recruited on a voluntary bmis
by the Huber laboratory and were between 18 and 65 years
ofage. The 112 CFS patients (90 females,20 males, and 3
unknown), recruited by Dr. SusanLevine, were between l8
and 65 ),earsof age and resided in the NortheasternUnited
States.All patients were diagnosedfor CFS acording to the
CDC criteria, and the majority ms completely disabled.The
cohort compriseda combination ofthose with an abrupt and
others with a gradual onset ofsymptoms.
2.2. Preparation of Human Blood and Plasma Samples. Approxirirately 30 mL of blood were drawn into three heparinized tubes (Becton Dickinson) and shipped overnight (CFS
patients) or processedimediately (healthy controls). The
-3-
Advancesin Virology
blood collection rubes from each individual were consolidated into one 50 ml tube and diluted with PBS,containing
CaCl2and MgCl2 (sigma) at a I : 1 ratio. 15mL of Ficoll (GE
Healthere) ms added to two nil 50 mL tubes, and 25 mL
of the diluted blood was gently layeredon top of the Ficoll,
followed by a 30min antrifugation in a Sowall RT7 plus
rotor at 2000rpm at room temperature.The PBMCs were
collectedfrom the interfacefollowing the spin and were used
for DNA isolation. Ten mL ofplasma werealsocollectedfrom
eachsampleand storedat -80"C. One m.lof plasmawassent
to Abbott Labson dry ice overnight for further testing.
2.3. XMRV ChemiluminescentImmunoassays(CMIAS). A
detailed procedure can be seen here [22]. Briefly, l00pl of
neatplffma were screenedfor antibodiesto XMRV gp70 ud
p i 5Eproteins using two prototype ARCHITECT chemiluminescentimmunoassays(CMlAs; Abbott Diagnostis, Abbott
Par( Ill) . The CMIAs utilire a direct rosayformat in which E
coliexpressedXMRV pl5E or mammalim-*presed XMRV
gp70 were used as both apture and detection mtigens.
Assay positire controls were derived from XMRV-infected
maaque plasmasat l;1000 (PCl) or 1:4000 (PC2). A
pool of normal hman plasma wa usedas negativecontrol
(NC) and as smple diluents. Cutoff (CO) values of the
ARCHITECT CMIAs were calculatedbaed on the followine
formulas; CO = 0.45 x (Calibrator I Mean RelativeLighi
Units (RLU)) for p15E CMIA and CO = 0.078 x (Calibrator
2 Mean RLU) for gp70 CMIA. Assay results were reported
as the mtio of the sample RLU to the cutotr RLU (S/CO)
for each specimen.Specimenswith S/CO values<1.00 were
considerednonreactive; specimenswith S/CO values >1.00
were considered initially reactive.The S/CO values of the
NC, PCt, and PC2 were 0.16, 12.8, md 3.5 for the gp70
CMIA md O.I3,7.4, and 2.2 for the pl5E CMIA. Initially
reactive specimens were retested in dupliete by either
ARCHITECT pl5E or gp70 CMIAs. Repeatedly reactive
specimenswere mallzed at t : 100dilution by investigational
westernblot assaysusing purified XMRV viral lpate a well
s recombinant gp70 protein.
2.4. WestemBlot Anallsis. Westernblot (WB) analysisusing
purified XMRV viral lysate as well as recombinant gp70
protein w6 performed m described[22]. Briefly, viral lysate
(804g/gel) or recombinant gp70 protein l\\yg/gel) were
separated by electrophoresis on a 4-120/oNuPAGE Bis.Tris
2-dimension gel (lnvitrogen, Carlsbad,Calif) in the presence
of sodium dodecyl sulfate (SDS). The protein bands on the
gei were eiecirophoretically transterred to a poiyvinyiidene
difluoride (PVDF) membrane (invitrogen). After blocking,
the PVDF membrane was cut into 2 lm strips. Strips were
incubated with human samples diluted l:100 or XMRVinfectedmaeque plcma diluted I :200 overnight at 2-8"C.
After removal of unbomd antibodies, strips were incubated
with alkaline phosphataseconiugatedgoat antihuman IgG
(Southern Biotech, Birminghm, Ala) for 30 minutes at
room temperature. The strips were wrohed,and chromogenic substratesolution was added.
Frequency
(a) PISECMIA
Frequency
(b) gpT0CMIA
FrcuRB 1: Distribution of pl5E CMIA (a) md gp70 CMIA (b) log
N of S/CO on 148 smples collected from l12 CFS parients md 36
healthy controls. Nmbers of specimero within each log N of S/CO
value are shown above the olid bars. Asay cutofs were equivalent
to man 16 SD and 12 SD for p15E and gp70 CMIAs, respectiveh
based on blood donor populations [22]. Log N ofs/Cq natural log
trmsfomation
of S/CO.
3. Results
148 blinded plasma smples from our origina.l CFS and
hea.lthy control cohorts were analyzed for the presence of
XMRV-specific antibodies,using the direct format ARCHITECT pi5E md gp70 CMIAS. None of the 148 plasmasamples were reactivein the p15E CMIA (Figure 1(a)). Two of
the 148 samples(ID = 137, 138) were positive in the gp70
CMIA (Figure 1(b)). Both specimenswere weaklyreactivein
the gp70 CMIA with sample/cut-off (S/CO) valuesof 7.77
(log N ofS/CO = 2.05) and 9.02 (log N ofS/CO = 2.20),
respectively. A.lthough the samples were repeat reactive in
the gp70 CMIA, they were not reactive by WB. As shown
in Figure2, both samples showed no visible WB bands
uing either XMRV viral lysate proteins (Figure2(a)) or
recombinant gp70 protein (Figure 2(b)). Unblinding of the
samplo revealedthat the two gp70 reactiyesmples stemmed
fiom two sequential blood collections of a single healthy
control (Thble l).
4. Discussion
In our original study, we found no specific relationship
between the presene of XMRV and CFS {i71. However,
scening the genomic DNA from peripheral blood lymphocyts ofboth healthy control and CFS cohorts, we did detect
PCRproducts that were identiel toXMRV gag sequences,
d
well ro other MLV gag sequencs. Due to the high number
of MLV sequencs in the mouse genomic DNA, we found
(b) Recombinantgp70
FrcuRE2: WB analysisof gp70 CMIA reactivesampleswith (a)
nativeXMRV viral proteinsand (b) recombinantgp70.WB strip
key: I & 2: gp70reactivesmples 137md 138;3: normal blood
donor plama as negativecontrol;4i XMRV-infectedmacaque
ploma aspositiveconrrol.The faintwhirebandin the 65-70kd
regiooin (B, stripsno. l-3) indicatesa lack of specificanti-gp70
antibody.
it prudent to test for mouse DNA contamination in our
samples.Using both a test developed by the Switzer lab at
CDC for mouse mitochondrial DNA [14], as well as a test
developedby the Cof6n lab for the IAP [17], we found that
every sample that was positive for XMRV or other MLVs
PCR products was also positive for mouse DNA. Although
these data provide an explanation for the detection of MLV
sequencesin our samples,they do not rule out the possibility
thatXMRV and mouse DNA contamination could be oresent
in the samesample.To clarifuthis issue,we testedour plasma
samplesfor the presenceofXMRV-specific antibodies.
Recentanimal studiesshowed that XMRV infection elicited a potent humoral immune responsein rhesusmacaques
{221.The infected macaquesdevelopedXMRV-specific antibodies within two weeks of infection and persisted more
than 158days.The predominantresponses
were ro all three
structural proteins of XMRV the envelope protein gp70,
the transmembrane protein p15E, and the epsid protein
p30 [22]. Sensitivity of both pl5E atd gp7\ CMIAs was
validated by the animal model; both CMIAs were able to
detect pl 5E or gp70 specificantibodies asearly as day 9 after
infection [22j. ln contrast,we were unableto deteciXMRV
pl5E or gp70 specific antibodies in the 112 CFS patients
and lhe 36 healthycontrols.AJthough2 samplesfiom rhe
same healthy control had weak reactivity in gp70 CMIA,
the reactivity was not confirmed by recombinmt gp70 WB.
Furthermore, both sampleswere nonreactivein pl5E CMIA
md had no detectable p15E and p30 antibodies by viral
lysate WB. Considered in combination with the negative
PCR data, the observed isolated and weak gp70 reactivity
4
Advancesin Virology
Advances in Virology
５
ｍはＥ
鰤
︲
Ｐ
︲
ａは脚鰤
ｉ
ｔ
壷
︲
懲緻鸞轟襴瀾珊 箸
陽鱗
鮮麟
TH72 1
038
006
TH04 1
TH01 7
016
007
015
006
TH01 8
012
006
TH01 3
TH06 1
015
006
015
0.07
PCR results
XMRV
Unblindcd ID
128
129
132
134
135
GAC
ヽ
lcOx
TH01 1
TH05 1
014
009
136
016
006
THOス 1
a16
Z77
138
7“リス2
α′̀
,の
143
TH10 1
THll l
014
007
014
006
TH02 1
TH01 5
TH21 1
014
007
013
007
015
007
155
TH20 1
016
006
156
TH02 2
017
007
158
013
160
TH08 1
TH03 1
161
TH12 1
011
163
TH19 1
016
072
164
TH16 1
015
007
152
m-ostlikely representsnonspecific reactivity since specificity
of the 8P7OCMlAwas reported as 99.5%o1221.In summary,
the serologic data obtained in this study suggestsa lack of
XMRV infection in our CFS patients and healthy ontrols.
It is theoreticallypossible that XMRV replicatesat very low
levels in humans and fails to induce a humoral immune
response,or, alternatively,that it is sequesteredor latent and
specific antibody titers have declined to mdetectable levels
oy
!iTe. Although these possibilities cannot be formally
excluded, they seem unlikely given responsesobserved to
other human retroviruses,The combination ofnegative molecular and serologicdata do not support an associationbetween CFS and XMRV or other MLVS. Furthermore, the
recent demonstration that XMRV is a recombinant of two
mtrrine MLVs (23) raisesdoubts about the validity (24) of
the original XMRV claims in CFS (3).
5, Conclusion
With the serologicaldata added to our original finding, we
Gn unequivocallyconclude that XMRV is not presentin our
CFS patient or healthy control cohort samples.Although we
have detectedXMRV gag sequencesin three ofour sa;ples,
Repeat
gp70
S′
CO
ス1 るス2 1
&亀 ,77
007
006
075,072
they all tested positive for mouse DNA and testednesatire
for XM RV-specific
antibodies.Laboratorymousestrains,ro
well m wild mice, all erry numerous endogenousMLVs,
md *treme caution must be taken when testins for murin€_
relatedviruses.
References
[1] A. Urisman,R. ]. Molinaro,N. Fischeret a.l.,,,Identifiation
of a novel gmmaretroyirusin prostatetumors of patients
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D. J. Choe,K. R. Brown,H. M. Thaker,md l.
[2] R Schlaberg,
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[3] C. Lombadi, F. W. Ruscetti,J. D. Guptaet al.,,,Detection
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[s] ,. A. Mikovits, V C. Lombardi, M. A. pf;si, K. S. Hagen, and
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in blood ells of patiens wirh chronic fatigue syndrome,,;
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[6] S. C. Lo, N. Pripuava, B. Li et al., ,.Detection of MLVrclated virus gene sequencesin blood ofpatients with chronic
fatigue-syndrome
and healthy blood donors,,' proceedings
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Amnica. vol. 107, no. 36, pp. I 587,+-l 5879, 20 10.
[7] O. Erlwein, S. Kaye, M O. McClure et al., ,,Failure to detecr the
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ONE vol. 5, no. 1, Article lD e8519,2010.
[8] H. C. T. Groom, V C. Boucherit, K Makinson et al., ,,Absence
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[9] F. I. van ltuppweld, A. S. de ]ong, K. H. Ianke et aI., ..prewlence of renotropic murine leukaenia virus_related virus in
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[10] W: M. Switzer, H. lia, O. Hohn et al,, 'Absence of widence of
Xenotrcpic Murine Lukemia Virus-related virus infection in
peroro with Chronic Fatigue Syndrome and healthy controls
in the United States:'R.tuovirology,vol. 7, dticle no, 57, 2010.
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{ I I J P Hong, l. Li, and Y Li, "Failure ro derecrXflotropic murine
leukaemia virus-related virus in Chinese patients wlth chronic
fatigue syndrome," Vrology loumal, rci. 7, artide no. 224,
2010.
[12] T l. Hendch, J. Z, Li, D. Felsensteinet al.,,,Xenotropic murine
leukemia virus-related virus prevalene in patients with
chronic htigue qmdrome or chronic immunomodulatory
conditionsl' I o urna I of lnfec t i ous Di seasa,vol. 202. no. I 0, pp.
1478-1481,2010.
l3j
O. Hohn, K Strchschein, A. U. Brandt er ai., "No evidence for
{
XMRV.in German CFS and MS patients with fatigue despite
the ability of the virus to infect human blood cells in vlno,,,
PloS ONE, vol. 5, no. t2, Anide ID el5612. 2010.
14]
B. C, Sailerfield, R. A. Garcia, H. ,ia et al., ,.serologic and pCR
I
resting ofpersons with chronic fatigue syndrcme in the United
States shows tro Association with xedotropic or polytropic
murine leukenia virus-related viruses,,,Retrovirobgy, vol. g,
article no. 12, 2011.
O.
Erlwein, M. I Robinmn, S. Kaye et ai., ,.lnrestigation into
ll5l
the presence of md serologiol response to XMRV in CFS
patients," PIoS ONE, rcI. 6, no. 3, Article ID e17592, ZOll.
[16] M. r. Robin$oq, O. W. Erlwein, S. IGye et al.,..Mow
DNA
contamination in humil tissue tested for XMRyi Retrovirolo8Z,rcI. 7, article no, 108,2010.
llTl B. Oakes,A.K.Tai,O.Cingozetal,,"Contaminationofhuman
DNA smples with mouse DNA can lead to false detection of
XMRVlike sequences)' Retroirology, vol. 7, article no. 109,
2010.
ll8l E. Sato, R A. Furuta, and T. Miyzawa,'An endogenous murine leukemia viral genome cotrtuinatrt in a commercial
RT-PCR Kit is mpli6ed
sing standild primers for XMRV,"
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[19] S. Hue, E. R Gray, A. Gall a a.l.,,,Disease-osciated XMRV
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Retrovirology,wl.7 , article no. I I t, Z010.
[20] R. A. Furuta, T Milaawa, T. Sugiyama et al., ,,No orcciation of renotropic murine leukemia virus-related virus
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)apan,"
Rerovirobgy, vol. 8, article tro. 20, 201 t.
{211 R. S. Arnold, N. V Makarova, A. O, Osunkoya et al., ..XMRV
inf@tion in patients withprostate cancer: novel serclog.ic asay
and correlation with pCR and FISH," urology, vol. 75, no. 4,
pp.755-761,2010.
I22l X. Qiu, P SMnson, K. C. Luk et al.,',Characteriation of mtibodies elicited by XMRV idfecrion and development of im_
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Hindawi PublishingCorporation
Advancesin Virology
Volume201l, Article ID 7E2353,6pages
dol | 0. | | 55I 20 1| I 782353
2
Research
Article
No Evidence of XMRV or MuLV Sequencesin
Prostate Cancer, Diffuse Large B-Cell Lymphoma,
orthe UK Blood Donor Population
Mark fames Robinson,r Philip William Thke,2 Otto Erlwein,r Kate I. Tettmar,2
Steve Kaye,r Kikkeri N. Naresh,3 Anup Patel,a Marjorie M. Walker,s Takahiro Kimura,6
Ganesh Gopalakrishnan,T Richard S. Tedder,2'8 and Myra 0. McCluret
I Section of Ikfectiow Diserces,
Jeferiss Reseuch Ttust Laboranria, Imperial Cnllege landon, St Mary\ Cahpw,
London W2 lPG, UK
'Transfusion Micrcbiology R6D, Netional
Transfusion Microbiology Labontoies, NHS Blood and Tiansplant,
CoLndalq Iandon NW9 5BG, uK
r Centte
Hospitel Imperial College Health Netuotk NHS Tt6, 115 1st Ftoot L Bb(h
for Pathologt, Hmmersmith
London W12 'HS, UK
a Urolngy Department, St MNy's Hospitul, Iilpaial Colbge Heahhcare
NHS Trwt, Inndot W lNy, LIK
t Histopathology Depaltmeflt, St Mary's Hospital, Impetial CaLlege
Lond.on, London W2 tNy UK
6_Department of Ulology The
j-25-8,
Nkhi-Shinbrehi, Minato-k\
likei Unitersity School of Medicine
Toyko 105-8461, Iapon
7Corculmnt Urclogist, Vedanayagan Hospital, RS Puran, Coimbatore-2
641002, India
3Blood Borne Vituys (Jnit, Viralw Ret'erenceDepanment,
Cenm for Infectiont, Heabh plotdion
Agency,
61 Colindale Avenre, London NW9 SEQ, UK
Correspondence should be addressed to Mak Jama Robinson, mark.robinsonl@imperial,ac.uk
Received l7 March 20ll; Accepted 3l March 20ll
Academic Edior: Yoshinao Kubo
CoPyright @ 20ll Mark lames Robinson et al. This is an opa acss aticle distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, md reproduction in any medium, prcvided the original work is properly
cited.
XenotroPic murine leukaemia virus-related virus (XMRV) is a reently desaibed retrovirus which has been claimed to infect
humus and cause asociated pathology. Initially identified in the US in patients with prostate cancer and subsequently in patients
with chronic fatigue syndrome, doubt now qistJ that XMRV is a hman pathogen. We studied the prevalence ofgenetic saquences
of XMRV and related MuLV sequoces in human prcstate cancer, liom B cell lymphoma patists md frcm Uk blood donors.
Nucleic acid was qtacted from fresh prostate tissue biopsies, formalin-fired paraffinrmbedded (FFPE) prostate tissue and FFPE
B-ell llmphom.
The Presmce of XMRv-sprcific
ITR or MuLV generic gagJike sequences w6 inrestigated by nested PCR. To
cotrtrol for mouse DNA contamination, a PCR that detected intracisternal A-t)'pe particle (IAP) sequenes wo included. In
addition, DNA and RNA were *hacted from whole blood tako ftom UK blood donors and sqemed for XMRV sequences
by real-time PCR )(MRV or MulV-like sequemes were not amplified fiom tissue smplc, Occasionally MuLV gag and iMRVtIR sequenceswere amplified fiom Indian prostate cancer samples,but were alwals detcted in coniunction with codtaminatitrs
murine genomic DNA. We found no wicience ol rMRV or Muty inli:ction in the LIK blood donors.
l. Introduction
In 2006, a new gammaretrovirus,
mia virus-related virus (XMRV),
xenotropic murine leukaewm discovered by the Viro-
chip analysis in prostate cancr tissue from patients homozygous for an RNase L mutation {ll. In these patients, the
innate antiviral defence RNase L pathmy is defective; hence,
thes€ patients are likely to be suseptible to viral infection
and a population
more likely to find a novel virus with
disease association in. When a second US study found that
60/o of all prostate encer patients, independent of RNroe
L mutations, were infected with the virus, thus broadening
the population
at risk [2], interest in XMRV intensified.
However, subsequent studies from the USA [3, 4] and all
Advancesin Virology
European studies [5-7] failed to confirm the presenceof
XMRV in prostatetissue.More recentlyit hasbeen suggested
that XMRV detection in prostate tissue in the US could be
relatedto the specificityand conditions ofthe PCR used [8].
In 2009, Lombardi md colleaguesreported the presence
ofXMRV proviral DNA in peripheral blood leucocytesfrom
3.77oof healthy contrcls and 67% of patients with chronic
fatigue syndrome (CFS) [9]. The detection rate by PCR
amplification ofXMRV proviral DNA subsequendyreduced
the estimated CFS prevalenceto 7o/o,with the explanation
that RNA extraction md cDNA synthesishad been required
to achiwe the 67%0prevalenceoriginally reported 1101.Lo
and colleagues(2010) using predominantly archival material
from patientswith CFS detrcted a high prewlence (86.57o)
of pMuLVs. Theseare similar to, but constitute a different
grcup liom, the xenotropic endogenousMuLVs to which
XMRV belongs IlI]. However,questiom were raised about
how these data were gmerated {12], and a number ofother
studieshave failed to demonstratea link betweenXMRV or
pMuLV infectionmd CFS [13-19].
The causesofB-cell llmphoma are not fully understood
[20], but the clinical and epidemiologiel characteristic
are suggestiveof the involvenent of an infectious agent
i211. Several viruss [22,23] haw been linked to the
risk of B-ell lymphoma, most notably EBtl 124-261, and,
retloviruses are implieted in animal leukaenim. Retroviral
integration could cause somatic DNA changesleading to
clonal upansion of B cellsresulting in leukamia as hasbeen
previouly describedfor adult T-cell leukaemia (ATL) and
HTLV-I 1271.
The geographical discrepancy of XMRV and pMuLV
prevalence remains unexplained. To explore this further, we
have tsted a variety of tissues ftom diverse populations;
(PC) formalin-fixed paraffin-embedded
prostate mcr
(FFPE) tissue from fapm md India, frch prostate tissue
smples received from the Urology Clinic at St Mar/s
Hospital, London, and peripheral blood ftom English blood
donors.
A seriesofrecent papers 128-3Il have demonstratedthe
easewith which specimenscanbe contaminatedwith murine
DNA sequenes.To control for this, all tissuespecimenswere
testedby PCR specificfor intracisternal A particle (IAP), a
retrotansposon presentin multiple copies (- 1000) within.
the mouse genome[32].
2. Methods
and Materials
2.1. Samplesand Nucleic Acid Isolation. Prostate biopsies
were collected from 55 patients admitted to the Urology
Department, St. Mary's Hospital, London, UK to undergo
routine biopsyforprostate cilcer screening.All patientsgave
written informed consentfor theirtissue to be banked for the
purposes of research(ethics number 99/CCC/166, August
1999). The DNA was extracted using the QIAamp DNA
mini kit.(Qiagen,Crawley,UK) following the manufacturert
mstrucnons.
B-cell lymphoma samples were provided by Professor
Kikkiri Naresh, Centre for Pathology, Hammersmith Hospital, London, UK. The DNA from 10 Diffuse Large B-cell
-8-
Lymphoma (DLBLC) patients was extracted from FFPE
tissues of lymph nodal or extranodal diffue large B-cell
lymphoma using the DNeasy Blood & TissueKit (Qiagen).
Briefly, two l5lm sections were cut and transferred to
1.5mL Eppendorftubes. Bladeswere changedbetweensamples to avoid cross-contamination. Sectionswere deparaf6nisedwith xyleneand ethanol,rehydrared,
and incubated
with proteinaseK and lysis buffer in a shakingwater bath at
55'C overnight and the extraction was completedaccording
to the mmufacturer's instructions,
Twenty FFPE prostate specimens including l0 prostate
cancer (PC) and l0 benign prostatic hyperplasia (BPH)
sampleswere supplied by Profesor Gmesh Golpalakrishnan
of VedanayagamHospital, RS Puram, Coimbatore, India
and sixteen specimens from Dr Takahiro Kimura of the
Department of Urology, The fikei University School of
Medicine, Japan.From the Indian blo&s, two 101rMsections
were qtracted with the QIAamp DNA FFPE tissue kit
(Qiagen), accordingto the manufacturert instructions. The
]apanesesampleswere provided preslicedon glassslides.
Rmdom anonymous whole-blood sampleswere obtained Iiom the Donation Testing Department at the National
Health Service Blood and Transplant (NHSBT) Centre at
Colindale, London, UK. Plasma minipools were similarly
obtained from NHSBT. All blood and plasmasampleswere
qtracted on a Qiagen MDx Biorobot and eluted with 804L
ofQiagen buffer AVE.
2.2. XMRU MuLV and Control NestedPCR Sampleswere
tested for the presenceof XMRV and MuLV proviral DNA
using nested PCR, m described previously 1141.Briefly, we
used a set of primers that encompassesthe 24bp deletion
in the XMRV gag leader region, originally describedto distinguish XMRV a a new human virus, along with a second
set ofprimers reflectinga sequenceconservedamongst most
MuLVs. The positive control for the XMRV and MuLV PCRs
wm plasmid \fP62 [t]. ihe PCR method ha been shown to
be sensitiveenough to pick up one copy ofXMRVVP62 plasmid in a background of500ng DNA [28]. tu a conrrol for
sample addition md PCR inhibition, primers to the human
beta-globin (hBG) gene were used. DNA extracted from
LNCaP (humm prostatecancer cells) was usedas a positive
control for humm beta globin. To control for contamination
of sampleswith murine DNA, primers specificto mouse IAP
were used m describedpreviously {28]. The positive control
for IAP was DNA from the Mccoy cell (murine fibroblast
cells,ECAAC 90010305).In all PCRs,at least6 "no templare"
controlswere set up. AJI PCR productswerevisualisedon
Ethidium Bromide-stained 2oloagarosegels.
2.3. XMRV MuLV and Control Reql-Time PCRsfor Blood
Donor Studies. Real-time PCR was performed as detailed in
Table l. For the proviral DNA analysis,l0pL of the nucleic
acid extract were analysed separately in three individual
quantitativePCRS(Q-PCRs).
2.3.1. XMRV Q-PCR and Internal ControL Samples were
testedbyQ-PCR forXMRV asdescribedbyMcCormick et al.
{331 and modified m detailed in Table l. In a Q-PCR to
Advancesin Virology
Advancesin Virology
Tesre l: Details ofPCRs used to tat blood sample.
Sample tested
PCR
Thrget
聰[°
m価dc xM6.q面
gag
:棚
Nヽ
おm ttdc sBttv
Ъ
q MallSBCヽ4V Plasmid
:器
d, PDH Taq Man
PDH himan gene
総2NAsiom価
600 NAs ftom whole
blood
400 NAs ftom plasma
minipools
XN4SIPMu3/RT
Taq Man with BMV
RT Taq Man
gag
BMV
Pdmers od prcbes
Trstt
Cycles(N)
Ragents
Qiagen
QuantiTect Probe kit
ABgene
ABsolute QPCR ROX
mstermix
Numberof cmerous smples
Numberof nonancqous samples(unknownstatu$)
PDH Pobe,RR
ABgene
ABsoluteQPCR ROX
maste!mu
Beta gl●
bin十
XMRV■
MuLV十
P2,F3,R4
BMV Probc,■ R
Qiagen
QuantiTect Probe
RT-PCR kit
メ
トロ
出/Probq R R
SBCWMV℃ PR
,SBCWMVCPR,
VMV237F
SBヽ
The TiqMan assayconditionswere I 5 min at 95"C ( l5 secs95"C, I min 60"C) xN cydes.400 nM concentrationsof primere,and 200nM probeswereused
in all the TaqManssayswith the exceptionofthe CDC MuLv TaqMan wherethe @ncentrationof eachprcbe was 100nM and the PDH TaqManwherethe
primer concentrationswere50 nM.
2
(x) Anplification frcm freshand FFPEti$usbynsted
.
Mean age(range)
PCR
Freshprostate tissue
Japan samples
Indian samples
16155
I 8/55(2l/55)
16
10/20
10720
o
7 2 ( 6 ル8 5 )
43(2783)
unknOwn
55/55
1AP十
mtDNA+
0
unhown
0
0
0
20′
20
2′
20
4′
20
0
nd
0
nd
5′
20
2′
10
16/16
LCBCL samples
1o
10′
lo
o
o
0
nd
(b) SpecificPCRrsults iiom Indian sampl€s
Indian sample number
PCR result using specinc primere
Catrcer status
mtDNA
P-globin
MLV gag
XN/1RV LTR
6489c110
control for the extraction effrciencyand amplification inhibition coextractedsoil-borne cerealmosaic virus (SBCMV)
plasmid DNA was used, (5.4 x 106 copieswere added lo
the 33 mL of Qiagen lysis buffer AL used for qtracting
96 samples on the MDx Biorobot). This reaction was as
described by Ratti et al. [3a]. The primer sequencesfor
this r€action were SBCWMVCPF (5'-CAC TCA GGA CGG
TGA CGA GAT.3'), SBCWMVCPR (5'-GTG ATA CTG TGA
GTC TGG TGA TGA TTT-3') and Wobe SBWMV23TFa(5'
JOE-TTT TGT GAC CTT GGA GGT GAG GCA GTT ATGBHQl-3',).
2.3.2. Q-PCRJor Quantifcatbn of Human DNA. The input
of humari DNA in eachqtract wasmeasuredby a Q-PCR for
the Pltuvate dehydrogenase(PDH) gene.Primers usedPDH
Taq t (s' -TGA AAG TTA TAC A-AA ATT GAG GTC ACT
GTT-3'), PDHTaq 2 (s'- TCCACA GCC CTC GAC TAA CC
-3') with probe PDHP (s'-vlC-CCC CCA GAI ACA CTT
AAG GGA TCA ACT CTT AAI TGT-Tamra-3'). Positive
control for this reaction was a dilution seriesofhuman male
DNA (Applied Biosystems,Warrington, UK, Catalogueno.
4312660).The XMRV Q-PCR raults were validated when
the PDH threshold cycle (Ct) value was greater than the
mean Ct minus 3 SD, and the SBCMV control ws greater
than the mean Ct minus 2 SD. Samples invalid on either
controlwereexcludedfrom the analysis.
2.4. Detection of Gag Sequencesby Nested PCR in Blood
Dozors. Nudease-free water (Severn Biotech, Kidderminslcr, UK) was userj tirrougiroui for tiie cDi.{A anrl PCR
mix preparationsmd as no-template controls. Nucleic acid
extractswere testedby nestedPCR using the gag primers as
describedby Lombardi et al. [9] and Lo et al. I I i ], but using
Applied BiosystemsTaq Gold LD PCR enzyme (Table 1) to
overcomethe problem offalse positivesthat havearisen from
the useoflnvitrogen Taq Polymerase
{301.
2.5. QRT-PCR Amplilicatiofl oJ XMRV/pMuLV in Blood
Donors. An XMRV/pMuLV gag QRT-PCR assaydescribed
by Lo and colleaguesI I 1] but modifred to detectthe pMuLVs
was used to test nucleic acid from whole blood, plasma,and
from plasma minipools. Further details of all QPCR and
QRT-PCR reactionsare listed in Table l. The primers for this
reaction were Fi (''-ACC GTT TGT CTC TCC TAA AC3') and R4 (5'-AGG GTA AAG GGC AGA TCG-3'), with
probe P2 (5'-Fam-CCG ACA GCT CCC GTC CTC CCGTamra-3'), Nuclease-free
water (SevernBiotech)was used
throughout for the RT-PCR mix preparations and m no
template entrols.. RT-PCRwas perfoimed in a total volume
of 501L, containing tx Qiagen QumtiTect M-PCR buffer
and primers, and probes s detailed in Thblel. Synthesis
conditions were 50"C for 30mins, followed by 95'C for
15 mins and 45 cyclesof 95'C for 15sec 60"C for I min.
Twenty pl ofnucleic acid wu anallred in a QRT-PCRwhich
multiplded the XMRV/pMuLV TaqMan with the internal
control TaqMan reaction (Brome mosaicvirus (BMV)) {35].
The BMV RNA was added to the Qiagen AL lysis buffer
md co-extracted with the sample. A smple wm valid if the
BMV Ct value was greater tha the men Ct mins 2 SD.
Samples invalid on the BMV control were qcluded from the
analysis. The sensitivity of this QRT-PCR wu detemined a!
150 RNA copies/ml (75 viral particles/ml) by calculation
fiom the observedllequency of negativesuing the Poisson
disribution.
3. Results
3.1. XMRV Detectbn in Tssue Samplesby N6ted PCR. A
representativestaineci gei foiiowing neste<iPCR is shown
in Figure l. For routine malysis, 0.ll pg of plomid DNA
(representingapproximately 7000copies/PcR) was used as
positive control for XMRV and MuLV All samples were
positive for hBG sequencesby PCR. The sensitivity of the
IAP PCR has been shown previously to detect as little s
0.00llpg DNA in a backgroundof500ng DNA [28]. The
resultsare summarisedin Table2(a). No evidenceof XMRV
or MuLV was found in any of the FFPE prostate tissue
samples fiom lapan or the fresh prostate tissuesfrom the
5383c710
5406a3′
10
2896c′10
BPH
5349c/10
(one prostatecancer,one benign prostatic hyperplasia)were
positive for mtDNA. In both of thae samples,IAP and MuLV
g4g sequencc were amplified. Additionally, one was positive
for XMRV (detailedin Table2(b)).
No evidence of MuLV or XMRV seouenceswas discovered in the DLBCL samplesand none of ihe DCBCL smples
gavean IAP specificproduct.
FrcuaEl: lane l: MWM; luo 24. P-globinPCR on LNCaP
DNA templatelst rcund prcduct, 2nd round product,and notemplatecontlol; lan€s 5-7: XMRV LfR PCR on VP62 pl*mid
DNA tmplate lst omd product,2nd round prcduct, and notemplate
@ntrol;lans 8-10:MulVg4gPCRon VP62plasmidDNA
templatelst round product,2nd rcund product,andno-tcmplate
control;lms 11-12:IAP PCR or McCoyell DNA temDlateaod
no-t@plat€control.
3.2. XMRV Detection in Whole Blood by Real-Time PCR.
XMRV proviral DNA ws not amplified from whole-blood
extracts derived from 540 donors. The averageDNA input
for each amplification wm 93,000 ells (approx 0.5649).
Detection of XMRV/MuLV RNA was undertaken on a
further 600 donon and 400 plasma minipools, derived fiom
19,200individual donations.All samplestestednegativefor
XMRV and MuLV sequences.
4. Discussion
UK. Ofthe 20 Indian samples,four (207o)produed a PCR
signal with the MULV gag primers (three prostate cancer,
one benign prostatic hfperplmmia) md of thce, 2/4 were
positive with XMRV LTR primers (both prostate cmc€r).
The IAP PCR was applied to the same smples to see if
ihe positive signal wm due to mouse DNA @ntamination.
All MuLV/XMRV mplifietion
was concordant with IAP
mplification, qcept for one prostate cancer sample which
was positive for IAP without MuLV/XMRV amplifietion.
Confirmation of murine DNA ontamination wc achieved
uing PCR primers specifc to mous mitochondrial DNA
(mtDNA). Although this PCR hm ben shown to be less
sensitivethan IAP PCR [28), 2120of the Indian smples
Using highly sensitivePCRswith primers that detectXMRV
and primers that detect MulV-like squ€nces, no proviral
DNA was detected in any of the prostate cancer samples
independently of murine DNA contamination. This served
to confirm our previou studies in which FFPE prostate
tisiue was tested and XMRV/MuLV seouencs failed to be
ampli6ed [28]. Here we haveadded further datato show that
no XMRV or MuLVJike sequencescan be detectedin fresh
UK prostate tissue or in prostate cancr samplescollected
from /apan. Samplesfrom India showed evidenceof MuLV
and XMRV sequ€nceswhen viral genomic sequenceswere
amplified bynested PCR Howevel this wasconcordantwith
murine genomicDNAcontamination detectedusingprimers
Advancesin Virology
to IAP. IAPs are retrotransposons present at the level of
Conflict of Interests
around 1000copiesper mouse genome [30]. Thus, IAP PCR
The
authors declareno conflict ofinterests.
representsa highly sensitive detection method for murine
DNA. A.lthoughthe samplesizewassmall (z = l0), we found
Acknowledgments
no evidenceto suggestthatXMRVmight be involved in other
cancers,suchas diffuse large B-ell lynphoma.
The authors would like to thank Dr. R. Silverman for the
It was reported last year that XMRV had been detrcted in
generousdonaiion of the VP62 plmmid and virus used as
greaterthan 6o0/oof 50 samplesfrom English blood donors
positive controls for our PCR and RI-PCR assays.They
In
contrast,
we found no evidene ofXMRV or pJr4uLV would like
[36].
to thank Dr. PG Gnnt for the designof the pDH
in any of 540whole-blood umples liom unselectedNHSBT
TaqMan assay.They would like to thank Dr Giles Budge
donors nor were we able to detect MulVlike sequencesin
for providing the plasmid @ntaining the cloned SBCMV
either the DNA liom whole blood or cDNA prepared frop
PCR product. They Would like to thank Ms Poorvi Patel
the plasma minipools from donors in England. There are
for assistingwith the QRT-PCRsand Ms RenataSzypulska
three possible explanations for this. Firsdy there are no
for msisting with the lobotic extractions and Miss Mahrokh
MuLV infectionsin blood donors in England.Secondly,there
Nohadan for prepaing the FFPE tissue slices. They qr6
are MuLV infections,but that the assaysused failed to detect
grateful for support from the NIHR Biomedical Researlh
them, either due to sensitivity or sequencevuiation. Thirdly,
Centre funding schememd the NHS Blood ad Trmsplmt
thereare MuLV infections,but the prevalenceis too lowto be
for fmding this research.M. l. Robinson and P. W Tuke
detectedin the samplesizestested.
ontributed equally to this study.
Researchinto the presenc of MuLVs in the humm
population is contentious, given disqepant findings [37References
39]. Contamination from sequencescontained in apparently
[t] A. Urisnan, R J. Molin{o, N. Fi$her et al., "ldentification
XMRV-positive samples, amplified products, or plasmids
of a novel gamaretlovirus id prostatetumors of patients
has been suggestedas a reason for the fiading of MuLVs
homozygou for R462Q RNASELvaiant," PIaS Pathogens,
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R. Singh,"XMRV is presentin malignantprostaticepithelium
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and is csociatedwith prostarcener, especially
high-grade
'tumorsl'
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L.
Men€ndez-Arias, "Evidence and controvercies on the role
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-L2-
Hindawi PublishingCorporation
Advancesin Virology
Volume 20I l, Article ID 272193,12 pales
doi:l 0. I l 55I 201| | 272193
2
Research
Article
Nucleic Acid, Antibody, and Virus Culture Methods to Detect
Xenotropic MlV-Related Virus in Human Blood Samples
M. F. Kearnenr K. Lee,r R. K. Bagni,2 A. Wiegand,r f. Spindler,r F. Maldarelli,r p. A. pinto,3
W. M. Linehan,3 C. D. Vocke,3K. A. Delviks-Frankenberry,r R. W. deVereWhite,a
G. Q. Del prete,s 1. W. Mellors,6 f. D. Lifson,5 V. N. KewalRamani,r V. K. pathak,r
I. M. Coffin,7 and S. F. J. Le Gricer
t-HN Drug Resistance
Program, National Cancet Institue at Frederick, Frcdelib MD 21702-l2ll, USA
2Proein Expresion Inboratory,
SAIC-Fredericlg Inc,, NCI-Fredaick, Frcdqick MD 2j702, USA
3 Urohgic Oncology Bran&,
National Canc6 Institute, Bethesdq MD 20gg2, USA
a UC Davis Caner CeflEr, Sacranento,
CA 958i2, USA
5-AIDS ahd Cancer ry';rus Proyafr,
SAlc-Frederick Inc, Natiotal Catcer Institute, Frederick, MD 21702, USA
6Departfrent of Medicine,
Univelsity of p;tttburyh, pittsburgh. pA 1S2@, LISA
7Department of Molecular
Biology and Mictobiotogy, Tufts UniveriE Bosbn, MA 02155, USA
Correspondence should be addressedto M. F. Kearney, kerneym@nail.nih.gov
Received 2l June 2011; R€vised 8 August 20ll; Accepted 27 August 201 I
Academic Editor: Yoshinao Kubo,
Copyiight @ 201 I M. E Keddey et al. This is an open acess article distributed under the Crative Commons Attribution
License,
which permits unrestricted use, distributiotr, and reproduction in my nediun, provided the originalwrk is properlycited.
The Mlv-related
retrovirus, XMRV was recently identified ud reporbd to be ssociated with both prostate cmer ad chronic
fatigue syndrome. At the National caDcer l$titute-Frederick, MD (Ncl-Frederick), we developed ilighly
sensitive methods to
detect XMRV nucleic acids, antibodies,-and replication competent virus. Analysis of ),(MRV_spLd similes md/or
specimens
from mo Piguil maaques experimentally inoculated with 22Rvl cetl-derived I;MRV confumi
the ability of the uai
used to
detect XMRV RNA and DNA, and culture isolatable viru when present, along with XMRV reactive mtiiody
responses. Using
these dsays, we did not detect evidoce of XMRV in blood samples (N = i34) or prostate specimens (N :
is) Ao^ t J
indePendent @horts of Patients with pfostate cdcei Proious studies detected XMRV in prct*e tissues. the pruent
in
study,
we primarily investigated the,levels of xMRvin blood plroma samples collared fiom patients with prostate
cmer These results
demonsttate thatwhile XMRV'related assaysdereloped at the NCt-Frederick can readilymeasure XNiRvnucleic
acidq antibodies,
and replication competent virus, no sidence ofXMRV wa found in the blood ofpatiidtj with prostate 6ncer
l.Introduction
in 2006 in a cohort
of prostate cancer patients
[2]. The
Xenotropic
reukemia
murine
virus-rerar:i6MR]/l': il::i:lff ;lrYlN^X* fi:,'ifi:;f:'J;,"Ti,i:: i;
T,:
arecentlydiscoveredgammaretrovirus reportedlyassociated
with prostate cancerand chronic fatigue syrdrome (cFS) [ l,
2l rhe discoveryof XMRV arosefrom studiesirestigating a
potentialviralcausefordiseasesinpatientswithanRN,4rel
genevariant. This genotlpe, which is observedin a varying
subsetof patients in cohorts with prostatecanar I l, 381,has been associated
with impairment of innate immune
responsesto viral infections [5]. Seeking an etiologically
signifient viral infection msociatedwith impaired RN,4se_Ld€pendmt responses,Urisman et aj. first identified XMRV
a;fi.rg
et al. in 2009 [e]. The prostate cancer stu.ies
*.r. iotto..a by a report from Lombardi et al. presenting
.tid.n." ro. XMRV infection in 67go of individuals with
severeCFS,comparedto 3.7o/oofhalthy individuals {lj.
These high reported frequenci." oi-XllnV infection and
putativelinkageto a debiiitatingillnessprompted concems
iboutth.po.riuitiryoi;;;;i:;;;;;dLroviralepidemic
and stimulated additional ..s."rch towud, deteimining
rhe prevalenceof XMRV infection in Jifferent populationi
*orid*id..
- 1 3-
Several studies supporting high prevalence of XMRV
infection followed. lor mmple, Arnold et al. det@tedantiXMRV antibodiesin 27oloofindividuals with prostatecancer
[10], Schlaberget al. found XMRV nudeic acid.in 230/oof
prostatecancersand 4% ofcontlols I I 11,and Danielsonet al.
detectedXMRV in 22.8y0of extractedDrostatetissuesfrom
individuaiswho had radicalprostatect;miesI t2l. However,
controversyarosewhen other laboratoriescould not demonstrate comparable findings in similar cohorts not only in
the US [13] but in Germany l1al, The Netherlands[15],
and England [16, l7]. Adding to the controversy,Lo et al.
reported the presenceofmouse retroviral sequences,
but not
XMRV, in 86.50loof CFSpatients [18]. Claims weremadethat
such Endings supported the associationofXMRV infection
with CFS,complieting an alreadycontroversialfield.
Severalfactorswere speculativelyproposedto contribute
to the differential detection of XMRV/MLVs by different laboratories. It was suggestedthat inconsistenciesin detection
of XMRV/MLVs in patient samplesould result from varied
prevalenceof infection in differmt populations, differing
critflia for patimt selection,and differing detectionmethodologies utilized [19]. It was also proposed that virus levels
may be chronielly low or episodic in patient plasmaor tissues,making viru detection difficult [19]. Adding to th€
compltrity, detection of XMRV by PCR is highly susceptible
to false positive results due to the very close genetic relationship of XMRV with endogmous MLVs and the high
prevalenceof contaminating mouse genomic DNA in mmy
specimens [20, 2l]. Indeed, studies have suggestedthat
XMRV detection is the result of laboratory contamination
from infected cell lines [22151 or contaminated reagents
[26]. Further suggestionsof laboratory@ntamination came
after publication of a study by Paprotka et al. [25], showing
that XMRV originated in a humm aner cell line generated
by passagingprostatecaner ells through imunocompromised mice. This result indiates that XMRV ould not have
enteredthe humm population until recendy,yet wd aheady
being reported r prevalent in a sireable fraction of prostatic
cancers.Furthermore, it shmed that most "XMRV-specific"
detection assap could, in fact, detect one or the other ofthe
two parentalprovirues (PTeXMRV-i ud 2) that gaverise to
XMRV and are endogenousto some inbred and wild mie.
In asessing this situation, it brcame clear that to rule out
false positive results and reliably detect XMRV infection, one
mut apply several diagn$tic methods used in onjunction
with known positive md negativemntrols.
At the NCl-Frederick, we sought to help clarifr the
XMRV controverey by generating multiple csays, including
rigorous methods to mesure mtibodies to XMRV through
ELISA-basedmethods,to quantifyXMRV proviral DNA and
viral RNA through quantitative PCR ad RT-PCRmethods,
and to measure infectiou virus by viral isolation cultures
using an indicator cell line s)6tem. We characterizedthse
trsays using available positive and negative control smples,
including spiked samples and specimensftom two pigtail
maaques experimentally inoculated with XMRV. We then
applied these methods to specimensfiom two @horts of
prostate cancer patimts to determine the levels of XMRV in
their blood. Overall,we observeda high level ofconcordane
Advancesin Virology
between detection methods and were able to rule out false
positive resultsby applying multiple assayson the same patient samples.Applying this approach,we did not find evidence of XMRV infection in any of the prostatecancer patient-derived specimensstudied.
2. Methods
2.1. Clinical ProstateCaner Samples.The XMRV detection
assays developed at the NCl-Frederick were applied to
samples collected from two cohorts of prostate cancer patients. In total, 134 patients were studied. Plasma samples
from 108 patients were obtained at the UC Davis Cancer
Center.Sampleswere collectedbetween2006and 2010 from
prostate@ncerpatientswho were eithernewlydiagnosed,on
active treatment, or undergoing post-treatmentmonitoring.
Plasma from all 108 patients was tested for XMRV RNA
md antibodies to CA and TM. Institutional Review Board
(IRB) approval uas obtained from the UC Davis Cancs
Center Biorepository and all studysubjectsprovided written
informed consent.
Smples from an additional 26 recentlydiagnosedprostate cancerpatientswere obtained from the Urologic Oncology Branch, NIH Cliniel Center, Bethesda,MD. All 26
blood sampleswere testedfor the presenceofXMRV RNA in
ploma and DNA in whole blood. Testsfor XMRV proviral
DNA were also performed on prostate tissue from 19 of
the 26 individuals in this cohort who had radical prostatectomies.Twenty-two of 26 blood sampleswere tested for
mtibodies to CA and TM. A subsetof 12 sampleswas tested
by virus rescue culture including those that had positive
or indeterminate rsults by X-SCA or ELISA and matched
negativecontrols.The studywas approvedby the IRB ofNCI,
NIH, Bethsda, MD, and all study subjectsprovided written
informed consent.
2.2. XMRV Nucleic Asny Detection with XMRV Single-Copy
Asays (X-SCA). Similar to the single-copyassay(SCA) for
human immunodeficiency virus (HIV) [27], qumtitative
real-time PCR and M-PCR assaysfor detection of XMRV,
alled XMRV single-copy6says (X-SCA),were developedto
qumtiE/ XMRV nucleic acid in plrma, whole blood, and
ell supensions obtained from blood or tissuesamples.The
assayswere designed using amplification primers targeting
a gag leader region consewed between XMRV (as well
as PTe.XMRV-2[25]) and non-XMRV endogenous MLVs
(foryard 5-TGTATCAGTTAACCTACCCGAGT-3', reverse
5-AGACGGGGGCGGGAAGTGTCTC-3'). Consequently,
efEcientamplifietion is achievedfrom both target templates
allowing detection of either XMRV or MLVs prsent in
patient sampls. The Taqmanprobe (5'fam-TGG AGT GGC
TTT GTT GGG GGA CGA- tamra3') used for detection
of amplified products wis designed to span a signature
24 nucleotide deletion in the XMRV (PTeXMRV-2) gag
leader that differentiatesthse from all other MLV sequences
(Figure I(a)). In the event that a positivesampleis identified
by X-SCA, single-pjenomesequencingshould be performed
to confirm that the sourc of amplification ms XMRV and
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Advancesin Virology
Advancesin Virology
13
,n;
t/L\
nFPSE
i iep
--llc
av^^.-;
r^ n nr ,,i
く1 1
0
2833
215356
8242
く1 1
Tesre 2: Immunocsay
ques.
XMRV (fold'dilution from luL 22Rvl supernatant)
645
RcactⅣ
lty witll
CA
TM
8242
0
195
2485
8242
76
12713
544405
14232
0
145
145
14232
76
20108
285277
or absene of plasma,For this reaon, cultures infectedwith
human specimenswere carried out for a minimum of two
week.
j.3. Verifying AssayDetntion Capabilities with Blood Samples
from XMRV-Inoculated Maaques. To nlidate the specificity
of X-SCA and ELISA, we ued specimens from two pigtail
macaquesuperimentally inoculated with XMRV Detailed
results from the macaquestudy will be reported elsewhere
(Del Prete et al., in preparation). In short, sanples tested
by X-SCA revealed that peak viremia was achieved at 5
days after inoculation in one animal and at 13 days in
the sercnd (Table 1). By day 28, levels of XMRV RNA
in plasma had declined to <lcopy/ml in both animals.
PBMC-msociatedXMRV DNA wr alsomerouredbyX-SCA.
DNA levels peaked with similar kinetia as plasma viremia
but persisted with lwels of 23 and 645@pies/106PBMC
in the two animals, respectively, at the end of the followup period, 119 days after inoolation. Antitrody reactivity
to XMRV epsid (CA) and trmsmembrane protein (TM)
measured by ELISA wa undetectableprior to inocularion
but were robustly positive thereafter(Table 2) (Del Preteet
al., in-preparation). Replication competent XMRV cmnot
be cultured from macaque plasma or PBMC samplesdue
to extensive hypermutation of the provirus post-inoculation,
likely due to the effectofAPOBEC proteins (Del Preteer al.,
in preparation).Consequently,XMRv-spiked human plama
w6 used to verity the DERSE.L-iG-Pcells for derection of
XMRV
3.4. TestingProstate Cancer Samplesfor XMRV Nucleic Acid,
Antibodia, and IsolatableViras. Samplesobtained from the
two cohorts of prostate cancer patients were assayedfirst
for XMRV nucleic acid (X-SCA) md antibody reactivify
against XMRV CA and TM protein (Thbles 3 od 4). No
XMRV
22Rvl supernaantalone
22Rvt supernatanttreatedwith CaCl2and heparin
22Rvl supernatantspikedinto human pl6ma
treatedwith CaC!2and heparin
of ploma from XMRV-inoculated maca-
Days after inoculation
︵
ぶτ■●Ｖ■８εヽＬＯ
0
8242
０
8242
５
?/"i
197
０
︲
Fio"'pc
of 22Rvl supernatant were GFP-positive by microscopy
within 4 days of infection (Figure 2) demonstrating the
sensitivity ofthis msayfor detection ofreplication competent
XMRV The sensitivity of this det€ction decreased3-5-fold
in the presenceofEDTA-containing plama samplestreated
as described above. This decreasecould in Dart be due to
the presenceof human comp)ementas hai been recently
reported [24]. Additional days of culture increred the
number ofGFP-positivecellsexposedto virus in the presence
53411
14232
Monkey ID
3.2. Qualifuing XMRV ,4ssay Detection Capabilities with
SpikedHuman Samplcs. Assaysfor detection ofXMRV nucleic acid and replication-competent virus were established
using XMRV-spiked samples* positive control specimens.
To determine the accuracyand sensitivityofX-SCA methods
to detect XMRV in human blood products, we tested a full
panel of plasma md whole blood sanples that were spiked
or not spiked with XMRV derived from 22Rvl cells. The
panel was blinded as to which samples were XMRV positive
and which were XMRV negative and were provided to
us by the XMRV Scientific ResearchWorking Group for
testing by X-SCA 1281.Results from the blinded panel of
spiked sampleswere describedpreviously by Simmons et al.
I28l and demonstrated that we detectedXMRV RNA md
proviral DNA using X-SCA with 1007oaccuracy.The lerel
of sensitivity for detectingXMRV RNA in the spiked plasma
panel was limited by the volume of sampletestedfor XMRV
(270 pL) to 3.3 RNA copies/ml. The level of sensitivity for
detecting XMRV proviral DNA was a singleXMRV-infected
22Rvl all in whole blood samples.A1l unspiked samples
were properly reported o negative for XMRV detection
indicating a very low rate offalse positivity.
The use of DERSE.L-iG-Pcells to detectXMRV was verified using 22Rv1 culture supernatants and XMRV-spiked
human plasma.Figure 2 showsthe resultsfrom virus rescue
experiments performed under the following conditions (i)
22Rvl supernatant alone, (ii) 22Rvl supernatant treated
with CaClz and heparin, (iii) 22Rvl.supernatantspiked into
human plasmatreatedwith CaCl2and heparin. DERSE.LiGP
cells treated with EDTA-containing human plasma alone
are not viable. Proportions of GFP-positive cells detected
by FACS at day 4 and day 8 after infection are shown in
く1 1
0
５
︲
14232
14232
ｍ
網皿 哺舗∝
M∞
kcy lD訛
1:lh
︵Ｓ ︶菫 ８ ●＞■ ８ 、 ヽ餞０
Tesre l: X-SCA Resultson XMRV-inoculated macaoues.
５
２
differentiates these ftom all other MLV sequences (Figure l(a)). Amplifications of XMRV from 22Rvt DNA and
MLV from mouse genomic DNA (extractedftom TA3.CycTl
cells) show that the probe design results in a lower level
of plateau fluoresence from non-XMRV MLV templates
than from XMRV templates (Figure t(b)), likely due to
ineffrcient binding and/or degradation of the probe during
MLV extension compared to XMRV extension. The result
of the probe design is differential amplifietion profiles for
XMRV and MLV, indicating which producr is being detected
in the assayand the proportions ofeach ifboth templatesare
detected,To confirm the result, the products were run on an
agarosegel (Figure I (c)). The XMRV X-SCA product is 86 nt
long and the MLV product 110 nt, easilydistinguishableon a
2o/oagarosegel.
(fold-dilution
22Rvl supernatant alone
El
22Rvl supernatant treatedwith
E
22Rvl supernatant spiked into human plana
treatedwith
(4,
from luL 22Rvl supernatant)
I
CaCl2 and heparin
CaCl2 and heparin
(b)
Frcute 2: Veifying XMRV rcscue by cultwing on DERSE cells wirt 22Rv1 supnnatants ahd wirt XMRv-spiked hunan plasmo, XMRV
culturing under the following onditions: (i) 22Rvl supernatmt alone (black bas), (ii) 22Rvl su;rrnatmt treated with CaCl, + heparin
(white bas), (iii) 22Rvl supem.tant spiked into humm plasma treated with CaCl, + hepain (gray bars). GFP-positive cells were analyad
by FACS at day 4 (a) md day 8 (b).
plsma or prostatetissuesamplesin the NIH prostatecmer
cohort or the UC Davis prostate cancer 6hort were positive
for XMRV nucleic acids or antibodi€s (Tabls 3 and 4).
However, two plasma samples in the NIH @hort (0594,
077I) werc indeteminate for XMRV RNA. One of thse
smples (0594)wm negativeby ELISA, and the other (0771)
had m indeterminateELISAresult.One other patient sample
in the NIH cohort (0781) ws indeterminate for XMRV
mtibody reactivity but negative for XMRV nucleic acid
(Table 3). All three of these smples, along with 9 matched
negative samples,were blinded md t$ted for replicating
viru using the DERSE.L-iG-P assay.Viru could not be
cultured fron any of these plasma sampleswhile it was redily recoveredfrom positive ontrol smples (22Rvl-derived
XMRV spiked into negative human plasma) (Figure 3).
Consequently, by our prospectiwly defined criteria, none of
the 26 patient samplesin the MH ohort were onsidered
to be XMRV infected (positive for nucleic acid, mtibody,
md/orreplication competentvirus) (Table3). All 108plasma
smplc from ?rostate cmcer patients obtained from UC
Davis were assayedfor XMRV RNA and mtibodies (Table4).
All sanples were negative for XMRV nucleic acid except one
(0739), which ws indeterminate. No smple was found to
be mtibody reactive by our ELISA criteria (at least 500/o
reactive relative to the macaque positive control rra) . lwelve
of the 108 sampleswere indeterminate for XMRV reactivity
to either CA or TM (2 standarddeviationsabovethe average
negativehuman sample) but were negativefor nucleic acid
(Table4). No samplewm indeterminateor positive for both
XMRV nucleic acid md antibody, and therefore, all were
determined to be negativefor XMRV infection.
4. Discussion
A.fter publication of the XMRV study by lombardi et al. in
October 2009 suggestinga possiblediseasemsociation with
-18-
CFS and a surprisingly high apparent seroprevalencefor
XMRV even mong healthy ontrol subiects,researchersat
the NCl-Frederick set out to develop rigorous methods to
evaluateth€ prevalenceof XMRV infection, Using control
smples, including spiked specimenswhere appropriate, we
developed assaysto measure plsma XMRV RNA viremia,
cell-associated
XMRV DNA levels,and mtibodis to XMRV
CA and TM. BecauseLombardi et al. reported the presence
of culture rescuablereplication-competentvirus from the
blood ofstudysubjectsusingcoculturewith a human cll line
(LNCap). we created DERSEcells, derivativo of the same
LNCap cells with a fluorescent reporter to detect XMRV
replication. Thee alls broadly ud sensitively detect the
repliation of different MlV-related gamaretrcviruses that
exhibit a tropism for human prostate aner cells. In the
absenceof patient-derived definitive positive and negative
ontrol speciBens, we applied our different assay methods
to samples obtained from two pigtail maaques prior to
and after experimental XMRV inoculation, XMRV plasma
viremia was detectablein both inoculated macaquesfor 23 week after inoculation but then declined to undetectable
levels (Del Prete et al., in prepamtion). However, XMRV
DNA in PBMG and serum antibodies remained at readily
measurablelevels for th€ duration of study follow-up in
both animals (Del Prete et al., in preparation). Evaluation
of samplesfrom the inoculated nracaquesdemonstratedthe
abiliw of our methods to reliablv detectevidence ofXMRV
infection in blood samplesmd showedthat XMRV provirus
and antibodiespersistevenwhen viremia is not detectable.
In the development of diagnostic tools for XMRV infection, it became dear that a single method for XMRV
detection would not be sufhcient for definitive diagnosis due
to a high frequencyof falsepositivesby PCR from contaminating nucleic acids (especiallymouse genomic DNA) and
high background reactivity seen by ELISA, even in samples
， ＰＯ Ｉ
ＩＮ Ｏ Ｉ
計
“
0781 day6
0781 day6
0781 day16
078:day9
0781 daァ
6
0729 day16
0フ
29 day9
0550 daγ
16
0550 da"
0550 day6
0549 day16
0549 day9
2 010
876′
8′6′
2010
く 08
く08
く 06
08
く07
Ⅷ謬
UB10‐0542
8/9/2010
く07
UB10 0547
8′
H ′2 0 1 0
く 08
UB10‐
0548
8ハ 1/2010
く 08
UBIO‐
0549
8711/2010
く 08
UB10 0550
8′
11ノ
2010
く 08
8′19/2010
UB10‐0578
く 08
UB10 0594
82 ′
1/2010
a,
UB10-0643
9/14/2010
く 07
UB10 0665
91 ′
6/2010
く
07
UB10‐0671
9/21/2010
1nvalid test
9/28′ 2010
UB10‐0706
1nvalid test
UB10-0729
9′ 30/2010
く 07
UB10 0771
lo/14′ 2010
1,4
UB10‐0781
10′ 15/2010
く 07
UB10 0738
1o5/2010
1nvand test
UB10-0785
10/18/2010
く
165
UB10-0788
10′ 19/2010
く 165
UB10 0830
1 0 7 2 92 ′
010
く 08
UB10‐0833
11′ 1′
く08
2010
0853
11′ 4′
UB10‐
2010
く
08
UB10-0913
11/19ノ2010
く 08
UB10‐
0539
0 540
UB10‐
8
8
8
GFP
8
ざ
P o s ct ei uてs ( % )
c
Plasma da■
フ
day17
day22
daγ
10
day13
day17
0738
0771
0771
0771
0771
day22
day10
da/11
day17
day22
0671 day22
0738 day10
0738 day13
0594
0594
0671
0671
0671
053,day10
0539 day13
0539 day17
0539 day22
0540d`722
0540 daylo
0540d013
0540 day17
Haslna day22
XM熙 ′十plasma da/5
XMm′ +plasma day7
XN4RV+P:asma daylo
XM熱 ′十Plasma dγ
13
XMR17+plasma day17
XMRV+plaslna day22
Plasma day10
Plasma dγ13
V柳
く
く
く10
く09
く100
く100
く09
く10
く09
く10
く10
く10
く10
く10
く10
く10
く10
く10
く10
く10
く10
く09
く10
く09
く09
10
10
ヽ
0
0
0
0
NA
NA
0
0
0
NA
0
0
0
NA
0
批
174000
し
ヽ
NIA
NA
38
0
器
Nucieic acid testing by X-SCA
澪
8
8
8
Virus Culture
ざ
GFP PoStiК
cens(%)
8
い
TM tt К
tton OttureSult
∬
985
NR
NT
NECAl■
VE
NA
5713
5302
NR
NECATrVE
NECATIVE
NA
2323
2362
NR
NECATIVE
NECATIVE
NA
1505
1864
NR
NECATIVE
NECATrVE
105600
1429
281l
NR
NECATIVE
NECATIVE
307500
1796
1949
NR
ヽ
r
NEGATIVE
81000
2248
4325
NR
ヽ町
NECAnVE
NA
2566
2826
NR
ヽ
r
NECATIVE
59025
5129
6059
NR
NECATn/E
NECATIVE
54825
1412
1414
NR
NECATIVE
NECATIVE
275250
1,412
1,460
NR
NT
NECATIVE
VE
NECATIVE
144075
3,050
3,190
NR
N33畑
32400
5359
5430
NR
NT
NECAnVE
84300
2736
3817
NR
NT
NECバ
HVE
Mヽ
1,490
1,375
NR
NECATIVE
NEG ATIVE
35025
2514
2500
NR
NT
NECATIVE
88950
1,453
1,620
NR
NECATIVE
NECATIVE
NA
10 655
26 030
“
ル
NECATIVE
Indcterminaヒ
吻
NA
8 151
8 451
NECATIVE
物 "'ν NECAttVE
74325
2280
2312
NR
NECATIVE
NECATIVE
27375
3840
3244
NR
NT
NECATIVE
20887
2990
2596
NR
NT
NECATIVE
63225
NT
NT
NT
NT
NECATlVE
4n75
31「
NT
NT
NT
NECATIVE
8535
NT
NT
ヽぼ
NT
NECATIVE
7132
Nr
NT
NT
Nr
NECrIVE
999
Serologic Testing by EUSA
TA8f,B 3: X-SCA, ELISA, and virus culture resulb on prostate cancer sampl$ flom NIH cohort.
ル
0549 day6
0538 day16
0538 day9
0537 day9
U310 0533
8′
5/2010
UB10 0537
8/6/2010
く
UB10‐0538
8/6′ 2010
NA: sample not availabieNT: sanple not tested.
26
22
23
18
19
20
21
15
16
053フday6
XMIⅣ +P16madγ
SamplaD鷺
嶽濶 咬 轟 磯 覇い
10
11
12
13
Ｃ
l
ｒ
誡
ｈ
XM郎 ′+plasma day9
Plasma day9
8
＞ ヽく，●ｏｏ一 一
つ て パヽｏ一ｏ∞ヽ
κ て ，●ｏｏ一 一
ヽ
● く 〓２ ０”ヽ
Advancesin Virology
Adwnces in Virology
Tesre 4: Continued
TABLE4: X-SCA and ELISA results on prostate cdcer samples from UC-Davis cohort.
Plasma RNA
Patiem ID
C.opics′ mL
ル滋 ″r711i"α
″
Patient ID
Pla$ma RNA
Overall result
NECATIVE
Patient lD
P0566
Copies/ml
ELISA result
く1 6 5
NR
Ovcrall result
NECATIVE
Copies/ml
P0388
く 165
P0509
く165
P0511
く165
P0530
く165
NR
NECATIVE
P0572
く165
NR
NECATlVE
く165
NR
NECATIVE
P0592
く165
NR
NECATIVE
P0024
く1 6 5
NR
NEGATIVtt
く165
NR
NECATIVE
P0532
く16:5
P0026
く165
NR
NECATIVT
P0605
く165
NR
NECJATA7E
P0535
く165
P0027
く165
NR
N〕 3ATIVT
P0611
く1 6 5
NR
NECATIVE
P0536
く165
P0031
く165
NR
NEC.ATIVE
く1 6 5
NR
NECATrVE
P0544
く165
P0034
く165
NR
NECArIVE
P0617
く165
NR
NECATIVB
P0562
く165
P0036
く165
NR
NECATrVE
P0632
く165
NR
NECATIVE
P0044
く165
NR
NECATIVT
く165
NR
NEC」ATIVE
P0045
く165
NR
NECATIVE
く165
NR
NEC.ATIVE
P0118
く165
NR
NECATIt/E
く165
NR
NECATIVE
P0133
く165
NR
NEC.ATIVE
P0144
く165
NR
NEGATIVE
NR
NECATIL7E
NR
NEC.ATiVE
P0154
P0593
P0612
P0637
P0641
P0650
P0657
P0659
く165
L′ι
″″
″′
"´″
NECAnVE
NR
NECATIVE
NR
NEQヽ TIVE
く165
NR
NECATIVE
く165
NR
NECATIVE
く165
NR
NECATIVE
く1 6 5
NR
NECATIVE
く1 6 5
NR
NECATA′ E
く165
P0672
P0156
く165
P0162
く1 6 5
P0167
く165
NR
NECATI17E
P0170
く165
NR
NECATIVT
P0172
く165
NR
NECべ TrVE
P0177
く165
NR
NECATIVE
P0721
く1 6 5
NR
NECATIVE
NR
NECATIVE
P0723
く206
NR
NECATIVE
P0185
P0195
く165
P0209
く165
P0219
く1 6 5
14&た rllli4α
“
レ″″ァ
″α″
″′
NR
I12滋κr″“α″
P0673
NEGAT3/E
P0675
P0679
P0685
P0710
NECATIVE
P0726
く165
NECATrVE
P0733
く165
NR
NECATIVE
NECATIVE
P0739
“
NR
NECATIVE
NECATIVE
ル′`セγ
″:れ
α″
NECATIVE
P0232
(165
NR
NECATl1/E
P0766
く206
NR
P0239
く165
NR
NECATIVE
P0778
く165
NR
NEC.ATIVE
P0293
<165
NR
NECATIVE
く165
NR
NECATIVT
P0306
く1 6 5
NR
NECATIVtt
P0792
く165
NR
NECATIVE
P0314
く1 6 5
NR
NECATIVE
P0826
く165
NR
NECATIVE
P0321
く165
NR
NECATIL7E
P0846
く165
NR
NECATIVE
P0322
く165
NR
NEGATIVT
P0848
く165
NR
NECATIVE
P0325
く165
NR
NECATIVE
P0852
く206
P0327
く165
NR
NECヽ T【
ヽT
く165
P0787
NR
NECJATIVE
P0332
く165
ル滋 セrttj"α
″
NECATIVE
P0906
P0916
く165
NR
NECATI1/E
P0340
く1 6 5
ル′αθ
r"レ α″
NECATIVE
P0923
く1 6 5
NR
NECATIVE
P0342
く1 6 5
NECATIVT
P0952
く165
NR
NFCATIVE
P0346
く165
NECATIVE
P0984
く165
NR
NECATIVE
く165
NR
NECム TIVE
P0348
NR
″α″
IItル″r“′
NR
NEGバ TIヽ
電
P0989
P0351
く165
NR
NEGATIVT
P0355
く165
NR
NECATIVE
P0366
く206
NR
NECヽ TIVE
P0380
く165
NR
NECATIVE
P1025
P0382
く165
NR
NECATIVT
P1032
P0384
く1 6 5
NR
NECATIVE
P0996
P0999
P1010
P1063
=21-
あルs、 7″
α″
ヽ■GATR/E
く165
NR
NECATIVE
く165
NR
NECATIVE
く165
NR
NECATIVE
く165
NR
NECATA/E
く165
NR
NEGATⅣ E
く165
NR
NECATIVE
ル
NECATIVE
滋た協":"α
″
Patiert ID
P1076
NECATIVE
Copies/ml
ELISA result
く16 5
P1086
NECATrVE
NR
く165
Pl108
く165
NR
ル
PH 10
く165
NR
NEQヽ TIVE
P1211
く165
NR
く165
NR
く165
NR
P1268
NECATIVE
P1297
NECATIヽ 電
P1304
く165
NECATⅣ 電
P1318
く1 6 5
from healthy control subiects,presumablyreflecting crossreactivity. Therefore,.wesuggesta multiple msay approach
to determinethe XMRV statusof patient smples. We established diagnostic criteria requiring that all replicata ftom
X-SCA analysismut be positive and that serum antibodies
md/or replieting virus nust also be detectable in the
same patient in otd€r to report the patient XMRV positive.
Samplesresulting in discordant resultsfrom PCR replicates
are reported m indeterminate. Despite earlier reports that
evidenceof XMRV infection was detectedin 6 mmy as20olo
of prostatetumors [2, 10-12], usingthe assays
we dryeloped,
we did not 6nd clear evidence for XMRV in the blood of
two independent cohorts of patients with prostate @ncer
(total , -: 134) or in the prostatetissue of a small subset
ofthese indMduals (r = 19). Basedon previomly reported
frequenciesof XMRV detection in prostatecancer patients,
if XMRV is present in the blood of infected indMduals,
we expectedthat approximately 27 of the 134 patients in
our study would be positive for XMRV. One patient from
the NIH ohort (0771) had an indeterminateX-SCA result
(2/3 reactionswere positive for RNA). This samplewas also
positire for reactivity to CA md TM by ELISA.However,no
XMRV DNA wasfound in the wholeblood from this patient,
and replicationcompetentvirus could not be recoveredfrom
the smple. Taken together, these data are considered an
indeterminateresult by our criteria. No other sampleswere
positivebymore than one diagnosticmethod.
The ocaional positive X-SCA reaction is not above
backgroundfor this assay.We regularlyrun 96-well platesof
"no tenplate controls" using both ourX-SCA.primere and
prim€rsttrgetingintracisiernalApaiiicles(IAP) [20,21,33]
that are prBent in high copiesin the mousegenomein order
to monitor the levelsof contaminating mme DNA in the
reagents and in the environment. We have foud that about
5oloof wells are positive with the X-SCA primers and about
2070with the IAP primers. Basedon thesebackgrounds,we
sPect to detectlow l*els of mouse DNA ontamination in
samPlestested,s seenis this study and in others [20, 21, 33].
Therefore,we required that all replicats ofpatient samples
be positiw to obtain a "positive" X-SCA result. We did not
-22-
ヽ
TIVE
NECATIVE
滋 たr″ルαtc・ NECATIVE
NEC.ATR7E
NECATrVE
Overa.ll result
NE(ル
Ｎ
く165
P0015
NR
Overall result
Ｎ
P0013
Plasma RNA
ELISA result
Ｎ Ｎ Ｎ Ｎ Ｎ Ｎ Ｎ
P0005
ELISA rcsulヽ
Plasma RNA
NECATIVE
NECATiVE
NECATIVE
NECATIVE
NEGrrVE
NК ATWE
test the smples directly with IAP primers since we have not
suce$fully found reagentsand an environment that are free
from mouse genomic DNA (on averageabout l/3000 of a
mouse g€nomeper PCR reaction).
Although we had an occasional indeterminate result
for XMRV RNA in the plasma samplc studied, we did
not detect XMRV DNA in any sample tested, despite the
ability of our assayto sensitively detect XMRV DNA in
spiked control samplesand in specimens from inoculated
macaques [28] (Del Prete et al., in preparation). Raults
from the inoculated maeques showed that in experimental
infction, XMRV proviral DNA is readily measurable in
blood alls even when plasma virenia was not detectable
(Del Prete et al., in preparation), further suggestingthat
thesepatients do not @rry XMRV in their blood. Findings
ftom previousstudiesreporting higher prevalencefor XMRV
in similar cohorts [2, 11, 12] typically involved tating of
prostatetumors. None ofthesestudiesreported the detection
of XMRV in blood samplesor the isolation of infectious
virus from clinical specimens,and only one meroured the
presenc ofreactiveantibodiesthrough a virus neutralization
csay [10]. Detection of antibody responsesto specificviral
proteins by ELISA or by reactivity to XMRV immunoblots
Ifwe had usedlessrigorous criteria basing
wro not assessed.
an overall diagnosison a single,nonconfirmed test and not
requiring all replicatesto yield the same result, then our two
ohorts wotrld havegivenriseto m apparent,and in ourview
almost crtainly incorrect, reported XMRV prryalence rate
of approximately l2%o.These considerations may explain
confiicting prior reports for the prevalenceof XMRV and
are consijtent with claims ihai XIVIRV detection is likeiy
the result of laboratory contaminarion [22, 26, ?3, 34). pa;ticularly given the potential for falsepositive results in pCR
and sioiogical assaysfor XMRV our results suggest tbat
appllng multiple diagnosticmethods including measuring
t.u.tr o1 prouiral DIiIA in blood cells provides a mor!
reliable approach for investigatingthe prevalenceof XMRV
Theseresultsalso demonstratethat XMRV nucleic acid, and
antibodies are undetectablein the blood of patients with
prostatecancer.
Advancesin Virology
ll
Acknowledgments
The authors thank Vaierie Boltz for heloful discussionsand
Connie Kinna, Susanlordan, and Susm Toms for administrative support, and JeremySmedley,Rhonda Macallister,
and Mercy Gathuka for expert animal qre. They thank the
XMRV Scientific ResearchWorking Group for providing
samplesused to verify X-SCA methods. IMC wasa Research
Professor of the American Cancer Society, with support
from the FM Kirby Foundation. Funding for this research
was provided by the National Cancer Institute's intramural
Center for CancerResearchand Office forAIDS Researchand
in part with federal funds Ilom the National Cdcer Institute
under contract HHSN2612008000018.This work w* also
supported in part by Bench-to-BedsideAward to V. K.
Pathak.The content of this publietion does not necasarily
reflect the viffs or policiesof the Department of H€lth and
Human Services,nor does mention of trade names, commercial products,or orgmizations imply endorsementby the
US Government.
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{341 J. N. Baraniuk" "Xenotropic murine leukemia virus-related
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2010.
-24-
Br00pp0N0R$
aNpBr00pc0rrEcTr0Nlffia]
Development and application of a high-throughput
microneutralization assay:lack of xenotropic murine leukemia
virus-related virus and/or murine'leukemia virus detection in
blood donors
Yanchen Zhou, Imke steffen, Leilani. Montaluo, Tzong-Hae Lee,ReeueZemel, william M. switzer,
.
shaohuarang'
Hongwei
tia'H:':#:;:::r"!:;#y':#:::,'
chetankumar
s' raitnr,
he short history of xenotropic mudne leukemia
virus (MLV)-related virus (XMRV) is one of controvdrcy and discrepant results. Initial studies
found XMRV nucleic acids and/or Droteins in
prostate cancersr'2and even a low percentage of prostate
tissues ftom individuals with no history of prcstate
cancer.2In contmst, several other studies have failed to
detect )O4RV in prostate cancer.tissue.3,aMuch of this
BACKGROUND:
Xenotropic
murineleukemiavirus
(Mlv)-related virus (XMRV)and other retatedMLVS
have been describedwith chronicfatigue syndromeand
certainlypes ol prostatecancer In addjtion.prevalence
rates as high as 7% have been reportedin blood
donore,raisingthe risk of transfusion-related
lransmission. Severallaboratorieshave utilizedmicroneutralization assays as a surrogatemarkerfor deteclionol
anti-l\4LvserologicresponseFwith up to 2570of Drostate cancer palientsreportedto harbor neutralizinqantiDOOyresponses.
STUDYDESIGNAND METHODS:We devetoped
a
high-throughpulmicroneulralization
assay for research
studieson blood donorsusing retroviralveclors
pseudotypedwiih XMRv-specificenvelopes.Infection
with these pseudotypeswas neutralizedby sera from
both macaquesand mice challengedwith XMRV but
not preimmuneserum.A total of 354 DlasmasamDles
from blood donors in lhe Benorlahoe area were
screenedfor neutralizalion.
RESULTS:A totalol 6.5"/.ot donor samples gave mode€te neulralizationof XIVRV but not controlpseudotypes. However,fudher lesting by Wesiem blot
revealedno evidenceof aniibodiesagainst MLVSin any
ot these samples.Furthermore,no evidenceol inlectious virus or viral nucleicacid was obseryed.
CONCLUSION:A microneutralizalion
assay was developed for detectionof XMRVand can be appliedin a
high-thioughputiomai fo. iarge-scaiesiuciies.Although
a proportionof blood donoredemonslratedthe abilityto
block XMRV envelope-mediated
infeclion,we tound no
evidencethat this inhibitionwas mediatedby specific
antibodieseliciledby exposureto XMRV or MLV.lt is
likelythal this moderateneutralizationis mediated
thrcughanother,nonspecificmechanism.
ABBREVIATIONS: CFS = chronic fatigue $yndrome; DEp = dual
envelope pseudovirus. L6sa-Gp = gly@proteiD oflassa virus;
MLV = murine leukemia virus; MLV-P = pol)4rcpic MLV; qRTPCR = quantitatiye
reverse transqiption-polymerse
chai!
reaction; VW-G = G protein ofvesicular stomatitis virus;
\l{B = Western blot; XMRV = xenotropic murine leukemia virusrelated virus.
From the Blood S]stems Reseach lnstitute, and the DepartMedicine, University of Califomia at San
ment of laboratory
Francisco, San Francisco, California; I2boratory Branch, DiviPrcvention, Centers for Disese Contrcl and
Prevention, Atlanta, Georgia; Creative T6ting Solutions, Tmpe,
6ion of HMAIDS
Aiizona; the Hospita.l for Sick Childret Torontq Ontario,
Cmada; and the Department of Molecular Medicine, Mayo
Clinic, Rochester, Minnesota.
Add.r6s reprint rcquests to: Graham Simmons, Blood
Systems Research Institute, 270 MasonicAvenue, San Fmncisco,
CA 941l8; e-mail; gsimmons@bloodsystem$org.
Thls work @s partially funded by a R2t geit FoF the
National Heart, Lung, and Blood Institute (NHLBI) to cS
(1R21HLl09761). The findings ed coDclusions in rhis paper are
those of the authors and do not necessaily reDresent the views
of their spoNoriug institutions.
Received for publication Octobei 20, 20ll; revision
rcceived November 2I, 2011, ild
ac@pted Nwember 22, 2011.
doi: l0.ll I l/j.1537-2995.201I.03519.x
IRANSFUSION 2012;52.332-342.
332
TRANSFUSION
Volume 52, FebruaN 2012
MICRONEUTRALlzAT10N ASSAY FOR XMRV DETECT:ON
controversy is likely eplained by polyrnerase chain reaction (PCR) and other nucleic acid contmination.sb
Dmpite)Or4RVoriginallybeing isolated from human prostate cancer samples,' it is in all likelihood a laboratory
artifact, created by the passageof human prcstate tissue
through mice.TThis resulted in infection with, and subsequent recombination between, at least two endogenous
MLVs.7Cell lines created from this tissue, and harbodng
)C\4RV were likely distributed to many laboratories
working on prostate cancer
The controversysunounding the associationbetween
)CVIRVand chronic fatigue syndrome (CFS)is, ifanyrhing,
greatel It was reported by Lombardi and colleagues8that
two-thirds ofCFSpatients fromtheUnited Statesharbored
)flURVcompned to 49oofconuols. Irnportantly, this work
was basedon threesepamtelinc ofevidencej l) direct and
indirect nucleic acid detection in peripheral blood mononucleacells (PBMNCS),stimulated PBMNCs, and plasma;
2) cultue of replication-competent )CvtRVfrcm plasma
md PBMNCbycoculture withhumanprostate cells;and 3)
serologic evidence using a flow cytometry assay.In addition to the association with CFS,rhe presence of virus in
plasma and blood cells, coupled with the relatively high
prevalence observed in apparently healthy controls, suggestedthat)(\4RVmay be both blood transfusion transmitted and a real threat to the safety of the US blood supply
However, many other groups failed to detect XMRV in
PBMNC samples ftom CFS or heajthy individua.ls.st3 Ar
letrt two studies tried to fi]lly replicate the initial study
using PCR,culture, and serology,without any convincing
evidence ofXMRV in either CFS patients or healthy controls,ra,rsFurthermore, more recent testing of specimens
from the study by Lombudi and coworkers revealed that
some of the previo$ly reported PCR-positive specimens
were contaminated with XMRv-containing plasmid
sequencesleading to the partial retraction of these pCR
resultstom thepublication byLombardi and colleagus.r6
Additiona4l a recentmultilaboratory blinded study using
I 5 previosly reported XMRV- or MLV-positive subjects as
well as validated negative controls demonstrated that virs
ctr.ltue assays ued in the study by Lombardi md colleagueswere prone to cross-contamination.tTThus, this
Ieavesonlytheserologicresultsaspossibleevidenceforthe
presenceof XMRV or other MLVs in humam. In the same
multilaboratory studyrsthe assaysused by Lombardi and
colleagus detected a serologic response in some specimens; however,this reactivily was not consistent within
replicates of the sme plasma sample and no statistical
association was obseryed in CFS patients compared to
blood donors, while three other highly sensitive assays
in the study failed to detect a serologic response in my
specimen.rT
Microneutralization msays have been used extensively as diagnostic and specificity tests for manyviruses,
including alphavirusesand influema.rs'20Indeed, neutral-
izing antibodies are typically formed as part of a highly
specinc responseto conformational epitopes. Neutfalizarion of )c\4RV in 1l of 40 (27.5%) serum samples was
observed in prostate cancer patients2r suggesting that a
micrcneutralization assayfor )CVIRVwouldbe feasibleand
useftrl. In this study, we generated a microneutralization
assay for studies of blood donors seeking serologic
evidence of XNIRVor MLV infection based on the dual
envelope pseudovirus (DEP) assay system we recently
developed,22which htr been proven to be a rapid, sensitive, and specific high-throughput system for antiviml
drug discovery tageting viral entry This asay system is
composed oftwo viruses.Entry ofthe target virus is driven
bythe XMRV envelopeprotein pseudotyped onto t}le core
of a reporter retrovirus,while infection by a second, internal control pseudovirus is mediated by an unrelated
envelope and is included to reduce the number of false
positives, Using this assay,we screened 354 donors and
identified a smaUnumber with a neutralization signature
warantin g fu rther testing.
MATERIALS
AND METHODS
Sample collection
Anonymized plasma and whole blood aliquots were prepared using residual samples left over from pilot tubs
collected for routine blood donation testing. The samples
selected were ftom 354 different donations from the
United Blood SewicesReno facility. One or tuvoethylenediaminetetnacetate (EDTA)plasma tube(s) were used for
prepffidon of these aliquots depending on the unit collection type. From each EDTA tube two plasma aliquots
were prepaed, the remaining sample was gently inverted
to resuspend,md then three or four whole blood aliquots
were prepared. All aliquots were frozen the day of preparation: Donorsampleswere coded to retainlinlGge only to
the donor's zip code of residence, age, se)6 and racel
etlnicity. Anylin-kageto personal donor information such
as name, address,and telephone nmber wm removed.
AII samples provided were monlmized before shipment
to Blood Systems Research Institute for subsequent
testing. The institutional review board of the University of
California San Franciscoapproved the study protocol.
Cells and reagenls
Human embryonic kidney 293T cells clone 17 (293Tn7)
and hmm prostate LNCaP cells rere obtained from the
AICC and grown in Dulbecco's modified Eagle'smedium
(Invitrogen, Calsbad, CA) supplemented with loyo fetal
bovine serum and penicillin and streptomycin (10 U/mL).
LNCap iGFp cells (DERSE,detectors of exogenousretroviral sequenceelements)were provided byV KewalRamani
(NCI, Frederick, MD).
Volume52, February2012 TRANSFUSTON
333
-25-
ZHOu ET AL
MiCRONEUTRALIZATION ASSAY FOR XMRV DETECT10N
LacZ encoding polytropic MLV (MLV-P;termed LacZ[MCFI3]) viruses were generated by nrst infecting
NIH3T3 cells with replication-competent MCFI3. The
resulting cell line was then infected with replicationdefective lacZ(A-MLV) pseudotyFe virus to introduce the
lacZ gene,?3CHO cells overexpressingmurine ecotropic
MLV receptor mCAT-l (CERD9) have previously been
described.'?a'?51
Sen frord wild mice experimentally or mock-infected
with )ft4RVfor 12weeksz6were used as positive and negative controls. )G4RV-infected rhesus macaque (RIIl0 and
RYHI0) sera were provided by I. Hackett (Abbott, Abbott
Park, IL).'??
Plasmids
)OvlRVenvelope (env) was PCR amplified from 22Rvl
cells with 100% nucleic acid sequence identity to the
)OvlRV22RvI lC\^rR-Rl eny sequence (GenBankAcession
Number FN692043) and cloned into the pCAGGSvector
with Kpnland, Nhel restriction sites.Plasmids encoding G
protein of vesicular stomatitis virs (VSV-G),glycoprotein
ofLassavirus (Iassa-GP),andthe ecotropic MLVenvelope
have been described previously.z3-3'z
'
Pseudotypedviruses with human immunodeficiency
virus (HlV)-based reuwiral backbone were generated
from two plmmids, one encoding eny and the otler
encoding the HIV backbone with a reporter gene.pNl,4-3
Luc-R-E- (pNl--luc) encodes a replication-incompetent
varimt of the HIV-l molecular clone NL4-3; in which the
nef gene has been replaced by a firefly luciferase (luc)
reporter, and the enu and vpr geneswere inaitivated, as
previously described.33Similarly, pNL4-3 Ren-R-E- (pNLren) was constructed by swapping the firefly luciferase
gene for Renilla luciferase.z2 Pseudotyped viruses with
MLV-based retroviral backbone were generated ftom
three plasmids: XMRV en4 Mlv-based firefly luciferase
reporter (MRP-luc),34and MLV gaglpol expression plasmid pHIT60.3s
Virion production
Hlv-based pseudovirions were produced essentially as
previously describedl0 by transfecting 293Tl17 cells with
l0 pg of the corresponding HIV construct (pNl-luc or
pNl-ren vector) and 30 irg of plasmid encoding the viral
er*'elope per iO-cm dish using ihe caicium phosphate
Oansfectionmethod. Similarly, MLV-basedpseudovirions
were produced by transfecting 5 pg of each of the three
plasmid constructs per Lo-cm dish. The next day, expression was induced with sodium butyrate (10 mmol/L) for 6
hous before washing the cells once with phosphatebuffered sa.line md then replacing the medium. Forty
hours after.transfection, the supernatmt was iltered
through a 0.45-pm pore size filter and frozen at -80"C. If
requted, virions were concentrated by ultracentrifuge
concentlation at 141,000x9 through a 2090 sucrose
cushion for 1.5 hours at 4"C.The pellets were resuspended
in Hanks'buffered saline solution and aliquoted for
storageat *80"C. Resultingreporter viruses were classified
according to retmviral backbone, reporter system, and
viral envelope, for enmple, MLV-luc()C\4RVEnv) or HtVren(Lassa-GP).lacz encoding MLV-P was harvested from
3T3LacZMCFl3 cells,filtered through a 0.45-pm pore size
filter, and frozen at -80'C.
Microneutralization assay
Neutralization assayswere performed in 96-well white
tissue culure plates (Nunc, Rochester,I.trY).Donor serum
samples were prepued from plasma by adding thrombin
(King Pharmaceuticals, Bristol, TN) in 0.5 mol/L Mgclr/
CaCl, solution and then removing fibrin clots. The serum
supematant was uansferred to a new tube and heat inactivatedat 56'C for30 minutes. Avolume of 10 pLofserially
diluted test sera or medium alone were transferred to
assaywells, followed by 30 pL of either a single or a tworeporter virus mixture depending on the puryose of the
assay and incubated for I hour at room temperature
before addition of 40uL of 293Tll7 or LNCaP cells
(500,000ceUs/ml) to all wells. Plateswere incubated for 2
days at 37'C and 570COz and fuefly md Rilillaluciferase
reporter expression was determined sequentially as
descdbed in Zhou et al." For the initial high-throughput
micrcneuualization assays,sera samples with final dilutions of80- and 24o-foldwere tested and each emeriment
repeated twice.
Neutralization dose response
For generation of neutralization dose-response curves
with selected donor sera, smples were serially diluted
starting from 40- or 80-fold initial dilutions. Assayswere
performed in tdplicate. lnfection of pseudoviruses MLVluc(XMRV Env) and MLV-luc(VSVG) in 293T/17 cells and
infection of MLVIuC(MLV-E Env) md MLVluc(VSV G) in
CERDgcells were detected using a luciferase assaysystem
(Bright-Glo, Promega, Madison, \M). Infection of LacZ
encoding MLV-P in 293T/17 cells was detected using a
system for chemiluminescent reporter detection of
p-galactosidase (Ga]acto-LightPlus, Applied Biosystems,
Foster City, CA). Additionally, rhe percentage of cells
infected with LacZ encoding MLV-P was measured
with cell fixation and visualization of blue color development under a microscope using a p-gal staining kit
(InvitroBen).
W€stern blot
western blot (\4/B) analysis was performed to detect
)0{RV or MLV antibodies in selected donor sera and
healthy controls as previously desuibed.t116 Briefly,
)0\4Rv-infectedDUl45 prostate cells (C7) were grom in
complete HuMEC serum-free medium supplemented
with l7o HuMEC and 5b pg/ml bovine pituitary extract
(lnvitrogen).Tissueculture supernatantswere clarifiedby
centrifugation and by passage through a 0.45-pm nlter.
XMRVwas purified ffom 150 mLofCT supernatant using a
retrcvirus maxiprep kit (ViraTrap, Bioland Scientific LLC,
Paramount, CA) following the manufacturer's prctocol, A
volume of I50 pL of purified XMRV was denatured with
sodim dodecyl sulfatepolyacrylamide gel electrophoresis sample buffer at g5"C for l0 minutes and viral proteins
were sepilated by gel electrophoresis in a NupAGE
4%-12% Bis-Tris gel (Invitrogen) for WB testing as previously described but modified by using horseradish
peroxidse-conjugated protein G instead of protein
A/G.r'z36Seroreactivity was defiled by rcacdvity to viral
envelope and/or gag proteins of the expectedsize as seen
in the positive control antisera. This \,VBtest accuately
detected XMRV antibodies in three experimentally
infected macaques equivalent to detection using recombinant proteins in recently describedimmunoassays.2T
Quantitative reverse transcription-PGR
RNA was extracted from 100 pL of selected donor whole
blood samples using Qiagen Viral RNA Mini kit. The isolated RNA wtr subjected to revemetransffiption by MLV
reverse tmnscriptase (RT; Roche, Indianapolis, IN). The
resulting CDNA was amplified in a real-time pCR procedure and quantified in a commercially available system
(LightcyclellSo, Roche). Qumtitative reverse trmscripuon (qRT)-PCR w6 performed with Faststart Taq polymerase (Roche) in 45 amplification cycles of 9s and 6O"C
for30 secondseach.T\ryoprimerpairswere used, integrase
(F2 I5'-AACCTGATGGCAGATQMGC.3'], R2. I5'-CCC
AGTTCCCGTAGTCTTTTGAG-3'1, and XMRV probe [S'FAM-AGTTCIAGAAACCTCTACACIC-BHQI-3'J)'3 or gag
(Q445F I5'.GGACTTTTIGGAGTGGCTTIGTT-31,Q52BR
T5aGCGTAAAACCGAAAGCAAAAAT-3'1,
ild )flvIRV probe
F480pRo-BHe
Is'-FAM-AcAcAGAcAcTTcccGcccc
CG-BHQl-3'l;.rz 4 *toff of 40 CIs was ued as evidence
for the presence of XMRV or MLV sequencesin a specimen. Positive conuols represented recombinant plasmid
spiked into whole blood smples in a dilution series from
106to l0{ copies/ml.
Nested RT-PCR amplification ot XMRV sequences
NestedRT-PCRms performed asdescribed.38
Briefly, RNA
was extracted from 0.5 mL of donor plasma using a virus
kit (QlAamp Ultrasens, Qiagen) and subjected to reverse
transcription employing a fust-strand synthesis system
for RT-PCR(Superscript III, Invitrogen). Culture supernatant of the )O4Rv-producing prostate cancer cell line
22Rvl was used at a 10-sdilution as a positive control for
RNA isolation. For amplification ofXMRV gag sequences,
5 pL of the transcribed CDNA was used for the nrst
round of 40-cycle anplificarion with primers 4l9F
(5'-ATCAGTTMCCTACCCGAGTCGGAC-3')and il54R
(5'-GCCGCCTCTTCTTCMTGTTCTC-3')3
and master mix
(Hotstart-ITFideliTaq,USBCorp.,Cleveland,OH). Nested
PCRwas performed for 45-cycleamplificarion wirh 5 pL of
the firsr-round PCR product and two different primer
PAiTS,GAg-I-F (5,.TCTCGAGATCATGGGACAGA-3,)
and
Gag-ln 15|4646GG]AAGGGCAGGGTAA-3') or Npl16
(5'-CATGGGACAGACCGTAACTACC-3')
md NpltT (51
GCAGATCGGGACGGAGGTTG-31.r
To monitor assaysensitiviry, plasmid DNA containing a cloned fragmenr of
)ilr4RVgag'z was included in each PCR run at concentmtions from 1 to I00 copies/pl. PCR and RT-PCRofcApDH
controls with primer pairs, forward (5'-CAIGTTCCAA
TATGATTCAC-3] and reverse (5ICCTGGAAGATGGTG
AIG-3'), were performed to ensure similar levels of DNA
and RNA input in each round of amplification.
Propagation of infectious XMRV in indicator cells
DERSE (detectors of exogenous retroviral sequence elements) indicaror cells were developed at the Narional
Cancer Institute by stable transfection of pBabe.iGFppuro into LNCaP cells. The intron inrerrupted GFp gene
from pBabe.iGFP-puro is only expressedafter mobilization by m infecting gammaretbvirus for a second round
ofinfection.ao To test for the presenceof infectious )C\4RV
in selected donor plasma, DERSE.Li-Gcells were inoculated with donor plasma or control plasma and spin
infection, as described in Steffen and colleagues.3sGFp
expression was monitored every 3 to 4 days for a tota.l
period of3 weelc. As a positive control, cultue supernatmt of the )G4Rv-producing prostate cancer cell line
22Rvl (containing roughly 10, copies/ml) was used as an
inoculm at 10-'?,l0r, and 10{ dilution.
RESULTS
High-throughput
microneutralization
assay development
)G4RV pseudoviruses (MLV-luclXMRV Envl) were generated using a Mlv-based retroviral backbone. These
pseudoviruses infected both 293T and LNCap cells. As
expectedfromprevious studies,arlevels of inlection mediated by the )G4RV envelope were somewhat lower compared to control envelopes.For eremple, on 293T/12 cells,
infecdon of unconcenrrated MLVIuCIXMRV Env) was
about equal ro VSV-G pseudoryped virus stocks diluted
lo-fold (67,714 and 63,742 relative light uirs, respectively). On both cell types MLV-IuCIXMRVEnv) was neutralized by sera from mice (Fig. 1A) and rhesus macaques
Volume52, Fehruary2012 TRANSFUSTON335
334 TRANSFUSIONVdume 52, Februaty2012
- 2 7-
-28-
ZHOU ET AL
MiCRONEUTRALiZAT:ON ASSAY FOR XMRV DETECT:ON
Fig. I . Detectlon of XMRV Bnv neutralizin8
A
Mouse Serum
MousePosL293T
Molse Pre-2937
MousePosfLNCaP
MousePre-LNCaP
)(IIRV whereG no cled neutalization
preimmune
sem or Hryluc(VSV
luc(MLV-E Env) pseudwiruses
ws
observed wlth
G) pseudoviruses.
(C) MLV-
were neutrallzed
b' sda from
rhesus macaquo ch6llenged with )ilvtBv in nCAT-l expressing
CHO e[s (CERDg cells], but no cled neutriliation
oflacz
encodingMLV-P or HMuG(VSV-G)
psddoviruses
observed in 293Tl17 cells, Infection
ofpseudwiruses
w6
121416181
wlth
firefly luciferase reporter w& detected wlth a luciferare ssay
system (Bright-Glo, Promega), whdeas lnfection of IacZ
encoding MLV-P ws measuted uing a slotem fot d€tection of
p-galactosidase (Galacto-Lt8ht Plus, Applled Biosystem$).
B
A.bsolute valus
Primate Serum
for the no sera controls rere tu follows: MLv-
lucoovlRv Env) gave 55,810 relative ltght unlts (RLU) on 2937
cells md 20,213 RLU on LNCaP cellsi HIV-luc(VSV-c) gave
65,961 RUt on 293T @lls dd
ｃｐ ｔ Ｎ一
■ 〓 ，ｏｚ ゞ
51,6?7 RLU on CHO cellsj dd
MLV-P gaw 32,356 RLU on 293T cells dd MLVJUC(MLV:E Env)
gave4l,77l RLU on CHO cells. Resrrls are presented s percentage of neutralization and shoM as mem I SD of triplicate medufemenb.
A replsentative
experiment of at le6t
Mo dperiments
C
p●mate RYH10
Ｃｅ ｔ ● 罵 ● ５０Ｚ ゞ
● VSV‐ CHO
(Fig. 18) challenged with }S4RV wherem no clear neutralization was observedwithpreimmune sera.Similar rsults
were obtained with HlV-luc(XMRV Env) (data not shoM).
Moreover, ecotropic MLV pseudoviruses (MLv-luc[MLV-E
Envl) were also neutializeciby serairom rhesus macaques
challenged with )CvlRV(Fig. lC). However, LacZ encoding
MLV-P or HMuC(VSV G) pseudoviruses (Figs. lB ild IC)
were not neutralized.
To develop a reliable high-throughput assay system
for the sueening of lage nmbers of samples fDr XMnV
infection, we generateda cell-based XMRV microneutralization assay system based on the internally controlled
DEP assay we recently developed to screen for small-
●０ヽ さ ０ヽ３０２ Ｓ
+
*
*
+
in posi-
utibodies
tire controls. MLV-IuG(XMRV Env) IEeudovirus inf@tion of
293Tl17 ud prostate LNCaP cells wd neutralized by sera
from both mice (A) and rh6us macaques (B) chatlenged with
(1-96)
We[Number
- Mean-XMRVMean-L6sa. XMRV-Luo. LassaRenx Control
Fig,2. XMRVEnv nflriallzing mtibody in blood donor em
using a ell-bued XIvIRVmlcroneutEllation dsay system.
Shm ls an qmpl€ scre6 of 80 donor sem samplcs
(8o-fold dilutiono) for )CVIRV
neutalization with viru
combinations of MLV-lucEMRVEnv) dd HIV-ren(I6a-cP)
in a 96-well plate format. Th@ (837, EsB,and BB0)of a rotal
of more thd 80 donor sen showedapproxlmately 5096reduction in XIIRVEnv-,but not Ldsa.GP-,medlatedvlral Infection in 293T,/I 7 cells.
is shom.
nolecule inhibitors,'zzwhich has been proven to be a
rapid, safe,sensitive,and specific high-throughput system
for antiviral drug discovery targeting vira.l en$y. We
adopted a similar approach here for the )G\4RVmicroneutralization asay. The assayswerc performed in 96-well
plate format with the aid of liquid dispensing equipmenl
for high-throughput applications. A-fter preliminary
experiments, a combination of MLVIuc(XMRV Env) and
HIV-ren([assa-GP), which showed no cleil interference
between the two envelopes, was chosen for the sera
screening.This combination proved to give very robust
and reproducible results. A combination of MLV-luc
()iN4RVEnv) md HIV-len(Lassa-GP) from three 96-well
plates indicated that the interplate coefficient ofvuiation
(CV)azwas 8.2 md 5.2% for MLV-luc[XMRVEnv) md HIVren(Lassa-GP),respectivellaA set of20 sera samples indicated that for the intraassays,the CV of sery smple in
triplicate was within 57omd for the interassays, the CV of
every sample from three plates was within i2%, for both
MLV-IuC(XMRVEnv) and Hlv-ren(lassa:cP) (data not
shown)Generally,sera showed relatively higher levels ofneutralization ofXMRVEnv pseudovtuses (approx.30%)than
the Lassa-GPcontrol (approx. 8Vo;Fig.2\. Similar results
were obtained with seraat240-fold dilutions, in indMdual
virus alone, and in LNCaP cells (data not shown). Despite
this higher level of background neutralization, neutraliza,
tion with a number of sera was noticeably morc pronounced. For exmple, in Fig. 2, three (837, B5B,and B80)
of more than 80 donor sera showed approximately s,qo
reduction in )CvlRVEnV but not Lassa-GBmediated vira.l
infection in 293Tl17 cells.
Screening of blood donors
We sed thisassayto screena tota.lof354blood donorsera
collected within the United Blood Seruiceregion ofReno/
Lake Tahoe.The Reno facility ws chosen due to the collection teritory including regions of Nonhern Nevada
and California knom to have clmters ofCFS.alas Patients
from CFS clusters, including the Reno/Lake Tahoe are4
formed the maiority of subjects in the original demonstration ofthe prsence of)O4RV in blood.s T\ryenty-threesera
gave more than 50% reduction in XMRV Env but not
Lassa-GPmediatedviral infection at either 80- or 240-fold
dilutioro.All23 sem smples showed a dose-dependent
neutralization of)O4RV pseudoviruses (apprcx. 6090neutralization at 8o-fold dilution), but unlike the mouse and
macaque mtisera, the blood donor sen demonstrated
very limitcd neutralization for MLV-E pseudoviruses
(<50% at 8o-fold dilution; Fig. 3). No clear neuta.lization
was detected for t'SV pseudorimses (Fig.3) ard IacZ
encoding MLV-P (data not shoM).
Confirmatory testing
The 23 moderately neutralizing sera (>50%) as well as 14
additional poor neutralizers (approx. 3070-50%)and l2
donors with no clear neutralizing ability (<307o) were
further assessedwith a recently developed WB assayr236
336 TRANSFUSION
Volume
52, February
2012
using purified, denatured )0\4RV antigen from XMRVinfected DUl45 prostate cells (C7). All 50 of the tested
blood donor serawere WB-negative (Fig. 4).
To further confirm whether tietewas any evidence of
)0\4RV or other MLV infection in these individuals rhat
would lead to a positive serologic response,we performed
PCR assaysand virus cultures that would detect both specificallyXMRV and more broadlyothu MLVs.Whole blood
samples of the selected donors were tested by qRT-pCR
using primer sets located in either XMRV integraseaoor
gag. No positive signal was seen in any sample with either
primer sel (data not shown). Plasmasamples of the 23
selected donors were also tested and found negative by
nested RT-PCRsing generic MLV primers previously
shown to detect both )OvIRVand the broader family of
xenotropic md polytropic Mlvs3'g(Fig. 5).
To test for the presence of infectious MLVSin donor
plasma,the indicatorcell line DERSEwas used.As a positive control, cultue supernatant of the )fl\4Rv-producing
prostate cancer cell line 22Rv1 (containing roughly RNA
IOe copies/ml) was utilized as an inoculum. Whereas
ceUs inoculated with 22Rvl supernatmts showed a
concentration-dependent GFP expression on Day 7 and
spreadofthe virus on Day2l as prcviouslydescribed,3sno
GFP expiession could be obsered in any of the cells
inoculated with donor plasma from the 23 serorcactive
persons, even when spin infection was used to enhance
the potential infecuon efficiency (data not shown).
DrscussloN
Determining whether serclogic evidence of immune
responsesto gammaretroviruses in humanss2ris an indication of authentic infection or just nompecific crossreactivity is an important final step in the XMRV saga. In
this study,re generateda rcbust, high-throughputmicroneutralization asay for the screening of large numbers
of subjects for serologic evidence of )C\4RVand MLV
infection based on the DEP assay system we recenily
developed.22This assay includes an internal conhol
pseudwirus that is very useful for avoiding nonspecinc
inhibition md a.tsoconuols for cytotoricity. This method
provides a reproducible high-throughput microneutralization researchassayfor large-scaletesting for evidence
of}O4RV and MLV infection.
Cu[entJy, enzyrne immuoassays (ELAS)and \4IB are
the iwo mosr common serologic methods uiilized for virai
diagnosis.aqaT
VVBis limited to the recognition of linear
epitopes and is prone to high-backgromd rates,while EIA
can be restrictedby the quality ofthe antigens,antibodies,
and detection methods. Instead of directly detecting the
eistence of mtiviral antibodies in the sera, the.DEpb6ed microneutralization assayis based on the ability of
a serum to neutralize pseudovirus infection. Comparcd
with standard assayssuch as ElAs, the microneutralizaVOlume 52,February 2012 TRANSFuS:ON 337
-30-
ZHOU ET AL
MiCRONEUTRALiZAT:ON ASSAY FOR XMRV DETECT:ON
XMRV
Fig. 3. Dose-re6ponrccure wlth selectedblood donor sera Neutrallzation of tnfection of HMuG(XMRV Ene) and HIV-luc(VSVG)
pseudovlrses with $etially diluted donor serasuple rercdetected ln 293T/17cells dd HMuc(lrtLV-E Env) in mcAf-l
e{Pre$ed CHOcells (CERD9@ll)' R6ults de presentedaspqcentage ofneuftallzation and shm o meil t SDoftriplicate
meNurements. A reprcoentativeof at leat two experimenb ls shl'w
althoughinthisinstancemanyoftheseseraneutralized
contol viruses in addition to XMRV In contrast, while we
identified 23 of 354 blood donors (6.5%)able to moderately neutralize XMRV Env-mediated infection, control
and other Mlvenvelopes were poorly or not at all neutralized. None of the samples tested showed my evidence of a
serologic response to )0\4RVby WB testing. Furthermore,
べ瑛鵡薦べ漆陵滝銅ド
XMRV oao lStround
← 730 bp 1 419F′
1154R
．
・
一
一
嶽
一一一
・
一
一
一
・・ 一
ｐ
ｂ
０
５
６
100000 1000000
tion assay has fewer steps md can be performed by
automated liquid hudling
equipment, which may
generate less 5D. The disadvantageis a 2-day incubation
period which impacts the clinical usefulness of the
assay.
A recent study identified neutnlizing activily against
)C\4RVin approxim;rcly 147" of blood donor samples,ro
霧鰹
ｐ
ｂ
０
０
３
1000
10000
ution
Serum D‖
‐ Cl
+C2
‐■ C31
- C77
→ ・C78
08
ｐ
ｂ
０
５
６
100
01
C2
C31
077
C78
‐
0‐ 8
ｐ
ｂ
０
０
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一 十 一千一一書
ε Ｏ〓 “Ｎ〓ｃ﹄一ョ ０ 〓 ＾
ド
39
42
76
B37
B58
B80
3
・
・
一一一 一 一 ● 一 一´．
ｉ
VSV
／
3
ｉ
●‐ 380
‐
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ｌ
B37
B58
Ｅ２ ■ Ｎ一
１ ■ ｏ２ ゞ ●２■ 口一
一
１ 一ョ０ア メ
Ｃ， ■ ●■ 〓 ３●〓 ゞ
76
マ
39
42
ニ
MLV‐E
ｙ
MLV‐ E
﹄
・
●‐ 3
い ０ 一 ‘ ● ● 一奪
一 一
337
858
‐
●・ B80
ｌ
ｃρ ¨“Ｎ 竃 〓 ョｏ２ ゞ
ｃｅ ｔ 奎 Ｅ 〓 ●〓 ぶ
76
。¨“詢 ”
獅 呻∞６
‐ Cl
‐ C2
‐■ C31
- C77
‐ C78
‐ 8
39
42
all 23 seroreactivesmples were ncgative forXMRV md MLV sequences using
PCR or virus culture. These PCR and
culture assayswere designed to detect a
Normal Blood Donor Seaa
broad rmge of gammaretroviruses, as
well as XMRV specifically, thus excludinB )G{RVIMLV and other gmmaretroviruses as a source of the nonspeciflc
reactivity The flnding rhat neutralizaり
=83,:`結
蠍 tion by the 23 blood donors was specific
to )O4RV envelopes, but not other
MLV envelopes,was surprising. Pairuise
comparison ofthe amino acid sequence
of the envelope region between XMRV
= 喘 翻 ￨ and MLV-P or MLV-E shows the amino
acid similarity is approximately 89 and
68%, respectively.
ng.4 Absence ofXMRV/MLV antbodies in bicod donor sera by LVB analysis
Given that the true)0\4RV neutralizPurined,denatured XMRV antlgen from xMRV infected DU145 ptOstate cens
ing responses raised in animals were
(C7)was ttsed for WB detecuOn OfxMRV or MLV atubOdles inも el∝ted donor
more broadly ieutralizing (Fig. l), this
sera salllples Results OF positive control antisera tO Purined xMRV andgen
result strongly argues against specific
and 24■ ormal donor sera samPles(B58,B80,E6,E8,E10,D17,D40,C5,C20,C30,
neutralization, but rather suggests the
C33,C35,C45,C47,C49,C50,C5、
C67,3,4,5,6,7,8,from leFt to rightl are
moderate neutra.lization observed was
sllown,locat10ns of reacdwity tO specinc tral prOteins are i五 dicated.Env
mediated by other nonspecific means.
(印69′71)=enVelopc,TM(p15El=tranSmentbFalls Cag(p■
6o,WIA lplつ =mat嶽 ;
This could be cross-reactive antibodies
CA o30)=caPsid.Molectuar weight markers(kDal ale proided On dlele■
of
raised against endogenous retrovira.l
the wB.
elements, completely mrelated proteins, or other nonantibody selum
factors. Human serum potently inhibits
‐
:iiiil!lilii:liiilll
300 bp
650 bp
300 bp
100 bp
100 bp
1聾
霧簿
囃 1議 籐 棗 姜 蝦1難 聰
警
響
`謗 ‐
雑
瘍
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惨
警
臨 繭 顧 耳 FT
GAPDH RNA
‐
壽
CAPDH DNA
‐
轟
Fl8. 5. Absence ofXMRv ga8 *quen@s
in blood donor plcma by nested RT-PCR.A representatire rcsult of lZ donor sampl6 is
containlng I to lo0 @pies,/pl- of a pltrmld hilboring
a cloned fragment of )OiIRv gagu ed negatire wa16r contrcls, Flrst-rcund pcB mp[ncaalon
$ed prlner palr 4l9F and I l54R md secotrd_round pcR amplncation
used
prlmE pairs Ga8-I-F md Ga8-I-R or NPll6 and NPll?. GAPDH RNA and DNA PCR results for ahe same
smples are shown itr
the bottom two pmel$
shoM
with positlve @ntrols
338 TRANSFUSIONVolume52, February2012
Volume52, Februaty2012 TBANSFUSTON339
-31-
-32-
ZHOu ET AL
MiCRONEUTRALiZAT10N ASSAY FOR XMRV DETEC￢
)S4RV;tnhowever, this is lilgely complement driven, md
in our assay serum complement was inactivated by
heating and did not influence our test results. The relatively high level of nonspeciflcity is greater than thar seen
with other microneutralization assays2qa3
and is partly due
to the lack of knom human positive cases that can be
used to accurately set cutoffs for defining specific neutralization. Our results likely a.lso explain other rcported
)O4RV neutralDation results in hman samples.2r
In addition to the initial 6sociation of XMRV to CFS
made by Lombardi and colleagues,sa second publication
by tt md colleagues,3sbased only on PCR analysis, also
yielded a strong dsociation between CFS and MLVlike
viruses.3sThese subsequent viruses demonstmted a far
greater degree ofsequence variation than XMRV with the
majority ofsequencesresembling P-MLV Although Lo and
coworkers3sreported very stringent measuresto minimize
contamination, the most parsimonious explmation,
given the extent of reported contmination of laboratory
reagents, is that their PCR results are false positives
resulting ftom rcagent contamination. lndeed, Lo and
colleaguesused platinum Ta4 (Invitrogen) for PCR amplification, which severalgroups have convincingly demonstrated is contaminated with mouse DNArars,ae
due to the
use of a mouse monoclonal antibody in the enzlme mix,
Furthermore, recent detailed phylogenetic analysis of the
longitudina.l MLV-P sequences reported by Lo and
coworkers showed that these sequences are inconsistent
with retroviral evolution.s0Nonetheless,the nndings of Lo
and colleagues mised the hypothesis that while )C\4RV
itself is clearly a laboratory contminant, the serologic
responses detected in Lombardi and coworkers may be
due to infection by other MLVs or gammaretroviruses.The
serologic assayused by Lombardi md coworkers relies on
antibody binding to the MLV spleen focus-forming virus
(SFFV) Env expressedon the surface of cells. The logic of
this assay is that conformationally dependent crossreactive epitopes shared between this mouse gmmaetrovirus and )0r4RVwould bind )S4RV antibodies, which
would then be detected in a flow cytometry-based assay.
However, it is likely that, as with our microneutralization
assay, mammalian cell culture-based expression of an
unrelated retrovirus Env would be highly prone to nonspecific cross-reactivitythat can confound the testing and
which requires clarincation byWB analysis using purified
antigen.Indeed,when the Lombardi and colleagues'flowbaseci assay was used by two iaboratories on piasma
specimens in a blinded study, high levels of nonspecific
reactivity were observed.rs
ln conclusion, we developed a robust, highthroughput microneutralization dsay to conduct studies
seeking evidence of infection with )O4RV and MLV
AJthough a small proportion of blood donors demonstrated the ability to block )O4Rv-mediated infection, we
found no evidence that this inhibition was mediated bv
specific antibodies elicited by exposue to XMRV or
related MLVS.It is likely that this moderate neutralization
is mediated through another, nonspecific mechanism.
Our ftndings also explainfurther the highly nonreproducible and nonspecific serologic responses detected with
otherassays.srTIn addition, this microneutralization assay
system can be easily adapted to screen donor samples
against other viruses with carefii selection of matching
partner virus envelopes, which will provide importmt
information for neutralizing antibody responses and
infectious diseaseproflles.
Pathak vK. Recombinmt
origin of rhe retrovirus XMRV.
ships of alphaviruses by a simple micro-culture doss,
Science2011;333:97-101.
neutraliation
method. J Gen Virol 1976;32:295-300.
19. Assays for neutralizing antibody to influenza viruses.
Lombardi VC, Ruscetri FW, Das Gupta L Pfost MA, Hagen
KS, Peterson DL, Ruscetri SK Bagni RK, Peuow-Sadoski
C, Gold B, Dem M, Silverman RH, Mikovits JA. Detection
of an infectious retrryirus, XMRV in blood cells ofpatients
with chronic fatigue slndrome, Science 2009;326:585-9.
Report of an informal scieotinc workshop, Dresden, lB-19
March 2003. Wkly Epidemiol Rec 2003;78:290-3.
20. Tempeton Nl, Hoscbler K Major D, Nicolson C, Manvell
R, Hien \/Ivl, Ha do Q, de long M, Zambon M, Takeuchi y,
Erlwein O, Kaye S, Mcclure MO, Weber L Wills G, Collier
D, Wessely S, Cleare A. Failure to detect the novel retrcvi-
Wei$s nA. A sensitive retroviral pseudotype assay for influenza HsNl-neutralizing antibodies. lnfluena Other Re$pi
rus XMRVin chronic fatigue syndrome. PLoS ONE 2010;5:
eB5l9.
Viruses2007;l:105-1221. Arnold RS, MakdoENV,
OsunkoyaAO, Suppiah S, Scott
TA, Johnson NA, Bhosle SM, Liotta D, Hunter E, Marshall
FF, Ly H, Molinarc Rr, Blackwell IL, Petros lA. XMRV infection in patienb with prostate cancer: novel serologic assay
Croom HC, Boucheit VC, Makinson K, Randal E, Baptisra
S, Hage S, Gow JW Mattes FM, Breuer L Kerr lR, Stoye lP,
ACKNOWLEDGMENTS
WethankDrs KyeongEun
(NCI)for
LeeandVineetKewalRamani
providingthe DERSE.Li-G
cellsand Dr Indira Hewletr(FDA)for
adviceon virus culture protocols.We thmk Dr John Hackett
(AbbottDiagnostics)
for macaqueserum.
CONFLICTOF INTEREST
The authors declarethat they have no @nnict of interest.
Bishop IC$.Absence ofxenouopic murine leukaemia
virus-Elated virus in UK patients with chronic fatigue syndrome. Retrwirology 2010;7:I0.
Kustmm
Kt, Bhattachdya T, Flaherty t, phair jp, Wolinsky SM. Absence of xenotopic
mwine
leukemia virus-
related virus in blood cells of men at risk for and infected
with HIV. AIDS 2010;24:1784-5.
Switzer WM, Iia H, Hohn O, Zheng H, Tug S, Shmkar A,
Bannert N Simons
G, Hendry RM, Falkenbeg VR,
United States. Retiovitology
and corelation with PCR and FISH. Urology 2010;25:75561.
22.
Zhou Y, Agidelo J, Lu K coetz DH, Hansell E, Chen yT,
Roush VtrR,McKeEowl, Cnik CS, Amberg SM, Simons c.
Inhibitors of SARS-CoV entry-identificatioo
using d
internally-controlled dual envelope pseudwirion Gsay.
Antivira.l Res 201 l;92:187-94.
23. Tailo! CS, Noud A, lae CG, Koak C, Kabat D. Cloting and
chatacterization ofa cell surface re@pto! for xenotropic
and polyropic murine leulemia viruses. proc NatlAcad
Reev6 WC, Heneine W. Absence of evidence 9f xenotropic
muine leukemiavirus-related vfuus infection in petsons
with chronic fatigue syndrome od
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No Evidencefor XMRVNucleicAcids,InfectiousVirus or
Anti-XMRVAntibodiesin CanadianPatientswith Chronic
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fmke Steffenl'2, D. Lorne Tyrrell3, Eleanor stein4, Leilani Montalvor, Tzong-Hae Leel, yanchen Zhou1,2,
Kai Lul, William M. Switzers, Shaohua Tangs, Hongwei Jias, Darren Hockman3, Deanna M. Santer3,
Michael Logan3, Amir Landi3, John Law3, Michael Houghton3*, Graham Simmonsr,2
I Blood Systems Resarch tn*ltutg san franciscq California, Unted States of Amedca, 2D€pa(ment of laboratory M€dicine, Unive6ity ofCatifornia San FrancL<o, San
F anciscq California,United Stare5ofAm€dca,3 Li & Shing In*itute of Urologtr UniveEty ofAtbeila, Edmonron, Atbeda, Canada,4 Depadmenr of prychiarn Unive6ty
ofCalgau Calgary,Albn., Canada,5Laboratory Branch, Divbion of Hlv/AtOs Prryeniion, Nationat Center for HIV/A|OS.Vilat Heparitts, STD,and TB prcvention, Cenrers
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PCR testing ofpersons with chronic
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H. Reroviral
JP,
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MC, Tedder
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blood ofpatients with chronic fatigue sFdrome and
emptod PLoS ONE 20ll;6:e19953.
healthy blood donors. Proc Natl Acad Sci U S A 2010;107:
Katzourakis A, Hue S, Kellam P, Towers Gt, Phylogenetic
15874-9.
analysis ofmurine
Keamey MF, l€e K Bagni RK, l^/iegmd A, Spindler t,
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Frankenberry KA" devere White RW Del Prete cQ, Mellors
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leukemia virus sequences from longitu-
ctuonic fatigue slndrome
gests PCR contmitration
Cit.iion: St€fienI,TyrrellDl- SteinE,MontaboL,tee T-H,et at.(2Oll) NoEvid€n.efor MRV Nucteic
Acids,Infectious
Virusor AntiXMRV
Antibodiee
in Canadian
PatienBwtrh Chroni.FatigueSyndFm€.PLoSONE6(11):e27870.
doitO.l3Tlljouhat.pone.Ooz787o
Editor: Kl Jeang,Nationalln*ltute of Heahh,UniredSrares
ofAmeric
ReceivedOctober12,2011; AcceptedOdober26,m1lj PublishcdNov€mber17, ZOt1
Thtstsanopen-a.cers
afticle,fie ofall copyright,
andmaybefRly reproduced,
dtseibuted,
r.nsmlned modified,
buih upon,orotheMbeusedby anyonefor
anylawfulpurpse.Thewo* ts madeavailable
underrheCreative
Commons
CCOpublicdom.in dedication.
FqndlngrThisworkwassupponedby the C.nadatucellenc€
Research
Charr(M.H.)andthe Li KaShingInnituleof vrobgy. Thefunde6hadno roreIn *udy
desiqn,datacollection
andanalyris,
dectsion
to publiih,or preparation
of the manusc.ipr.
competing hter6tr Theauthor havedeclaredrhar no compering
interest5
exisl.
| !fr ll mi<hael.houghton@oalbeda.ca
C, Avidity
Lu K, Switzer Vr'M, Tang S, tia H, Hockmm D, Santer DM,
fatigue syndrome. PLoS ONE 20ll;6:e27870.
parients sug-
nther than viral evolution.
t Vilol
Introduction
Chrcnic fatigue syndrome (CFS), also commonly referred to as
myalgic encephalomyelitn (ME), is a complex disorder with an
unknom etiology which is characrerized by disabling physical and
menal fatigue and pain that lasts for at least 6 months and lacks
any obvious cause [,2]. The sudden onset of slmptoms and
underlying activation of inllammatory
pathways suggest an
infectious agent ro the triggering factor. Numerous vinl and
non-viral pathogero have been invstigaled in the cont€xr ofCFS
with 6 yet inconcluive resuls [,2]- The xenotropic murine
leukemia virus (Ml!-related
virus (XMRV)
wc
initially
identified in human prostate cancer cells in 2006 [3]. lt has since
beeu thought to be the only member of the gammaretrovirus
family knom to inGct humas and ib possible rcle in the
development ofprostate cancer has becn widely dircused [4]. ln
2009, Iombardi et al. rcported the detection of XMRV in both
peripheral blood mononuclear cells (PBMC) and plama of67% of
a CFS paticnt cohofr compared n 3.7o/oin healthy controls [5].
This study ha gained a high level ofattention and ws thought to
mak a possible breakthrough in CFS research. Sevenl studies
have since addressed the possible connection bemeen XMRV
infection ad CFS or prostate cece., and rhe reulting evidence is
controversially discussed in the field [4]. While one study reporred
the prLsence of other MLV-like sequencs in CFS patiens [6],
others identilied mouse DNA, human cell lines or commercial
laboratory reagents to be a posible source of MLV conmination
i$j..
342 TRANSFUSIONVolume52, Febtuary2012
-35-
Ailanta, Geoqi4 United 5rats ofAmerica
The gammaretroviruses xenotropic murine leukemia virus (MLv)-related virus {XMRV) and MLV have been reported to be
more prevalent in plasma and peripheral blood mononuclear cells of chronic fatigue syndrome (CFS) patients than rn
healthy controls. Here, we report the complex analysis of whole blood and plasma samples from 58 CFs patients and 57
controls ftom Canada for the presence of XMRV/MLV nucleic acids, infectious virus, and XMRV/fvlLV-sDecificantibodies.
Multiple techniques were employed, including nested and qRT-PCR, cell culture, and immunobloting.
We found no
evidence of XMRV or MLV in humans and conclude that CFS is not associated with these qammaretroviruies.
RS. Chronic fatigue syndrcme in northem
high titer retrovira.l vectors. NucleicAcids Res 1995;23:628-
\^rM. Serologic dd
団
¨
一
⑫
ZHOu ET AL
e,-osote I w.prosone.ors
l7]. Attempts to reproduce the initial findings in different CFS
patient groups world-widc and in pars of the initial cohort have
since failed [4,8,9]. Thus, more research is needed to resolve an
association of MLV-like viruses in humans. In rhis srudy we
performed an extensive analysis of whole blood and plasma
samples from rwo well-characterized Canadian CFS patient
cohorts and healthy controls utilizing muiripie laboratory techniques, including nested and qRT-PCR, cell culture, and
imunoblotting
lor the detection of Xtr4RV/MLV nucleic acids,
infectious viru, and XMRV/MLV-specific
antibodies.
Materials
and
Methods
Ethics statement
All study protocols were reviewed and approved by the Human
Research Erhics Boards of the University of Calgary md the
University of AJbera and all study participants provided written
inlomed
consent. Laboratory testing of the samples was
performed monlmously and blinded.
Cohorts
All patients and controls examined in this study were paft of
cohort from either Calgary or Edmonton, recruited in 2010 and
2011, respectively. All panicipans completed the De Paul
Questionnaire 00] to gather demographic data and to elicit the
Canadian Consensus Criteria (CCC) for ME,/CFS s established
'by Carruthers el al
[]. Moreover, all participanb were screened
November2oll I Volume6 I tssue11 I e27870
No XMRVin CanadianCFSpatients
qRT-PCR
For qRT-PCR analysis, RNA Ms extracted from I00 pl of
either whole blood or plasma using the Qiagen Viral RNA Mini
Kil The isolated RNA was subjected to reverse transcriprion by
murine leukemia virus $4uL!
reverse transcriptae (Roche). The
resulting CDNA wc amplified in a real-time PCR reaction and
quantified in a Roche LightCycler 480. l'wo different primer and
proh" scts wrre used l_orampli!i.-ation o!'wo distinct regrons of the
XMRV genome: primen XMRV-F2
5'-MCCTGATGGCA,
GATCAAGC-3' and XMRV-R2 5''CCCAGTTCCCGTAGTprobe
CTTTTGAG-3'
and
FAM-AGTTCTAGAAACC'I-CTACACTC-BHqI
for ampli6cation of rhe XMRV integrae genc
and WPI primen Q445F 5'-GGACTTTTTGGACTG[l],
GCTTTGT-T'3'
and q528R 5'- GCGTAAAACCGAAAGCAAAAAT'3'
and probe FAM-ACACAGACACTTCCCGCCCCCG-BIIqI
for amplifietion of the XMRV-spaific
g4g
leader equence [2] with FastStart Taq pollmerase (Roche) in 45
.:@,
clos orue I ww.ptosone.org
Virusculture
DERSE (Detectors of Exog€nous Retroviral Sequcnce EIemens) indicator cells were dcreloped at the National Cancer
Institute by stable trdsfecrion of pBabe.iGFP-puro into LNCa?
cells. pBabe.iGFP-puro is an MLV vector encoding puromycin
rcsistece and a CMV promoter drivcn GFP reportq genc which
is interrupted by an inron plaed in sense direction relative of the
vecrcr and tmnscribed antisenscto the vector mRNA. The intron
intenupted GFP gene is only expresed after moblization by an
infecting gammaretrovirus for a second round of infection. After
screening clonal cell populations, the most secitile cloncs ucre
chosen md dcsignated a DERSE.Li-G cells. To test for rhe
presence ofinfectious MLVs in patienr pl6ma, DERSE.Li-G cells
were inoculated with CFS patient pldma or @ntrcl plama. Cells
were sceded 72 houls before infection with 3xl0a cells/ml in 6wcll platq. For spinoolation,
the medium wa removed and
300 pl fresh mcdium and 50 ;rl plama were added per well. The
plabs werc centrifuged at 1,200 rpm for I hour and 0.5 ml fresh
medium wa added. The inoculm ms rmoved the next day and
the cclls were cultured in 2 ml fresh medium and monitored for
GFP expression every 3 to 4 days for a toral period of3 weels. As a
positive conhol, culture supernatmt from the 22Rvl cell line
(containing roughly lOv copies/ml a detemincd from the average
of seven individua.l qPCR asays. dara not shown) wro used m an
inoculum at l0-', and l0-'dilution, respectively.
Serology
Western blot (WB) analysis wa pe rformed ro detecr stiXMRV/MLV
antibodies in CFS patient sen dd healthy
consols. Purified XMRV antigen from XMRV-infened DUl45
prostate cells (C7) wa denaturcd with SDS-PAGE sample buffer
at 95'C for l0 min md analyzcd by immunoblotting as previously
described [9]. Seroreactivity w* defined by reactivity to viral Env
andlor Gag proteins of the expected size a seen in the positive
control antisera (Fig. 2B).
Wherex XMRV gqg sequences were readily detectable in
diluted 22Rvl c€ll supemaranb, XMRV and MLV were not
detected in any of the patient phma samples(Fig. lA and B). The
detection limit of the nesrd PCR assay wx below I copy/ pl
isolated RNA or 5 copies/reaction as determined by the detection
of known amounc of XMRV plamid DNA (Fig. lB). The
sensitivity ofthe qRT-PCR asay was below l0' copies/ml plasma
or whole blood. Regardlessofwhether whole blood or plasma was
tested, all human sepls
were negative for detectable amounb of
XMRV nucleic acid (dah not shown).
DERSE.Li-G cells inmulated with 22Rvl supermhb
showed
a .oncent.a!ion-dependent GFP expression^n day 7 and sprrad of
the virus on day 2 l. GFP expressionwa not obseryedin any ofrhe
DERSE.Li-G cells inoculated with patient plasma (typical example
shom in Fig. 2A).
Seroreactivity was defined by Westem blot reactiviry to v;a]
Env md/or Gag proteins of the expccted size as seen in the
positive control antisera (Fig. 2B). None ofrhe I 15 huma plasma
reacted with the purified XMRV antigen indicating an absence of
antibodies to XMRV/MLV
in the smples (typical example
shou in Fig. 2B). lncreded background noise a obserued for one
―
XMRV WP!p“ mers
51015h25SS40a5
CYclts
Figute 1. Failurc of d€tedlon ot XMRV nudcic .cids in plasma and whole btood of CFs prtlents and healthy aontrots. n, Fifst @und
PCRproduds of a representativenumt€r of RNAsamplesisolatedfrom patient plasmausing primers419F and I I 54R.A t0-5 dilution of 22Rvl cell
cuhureslpehatant and three known concemrationsof XMRVplasmidDNA were included as conuols. 8l Secondround amplificationpioduds of
nestedPCRusingprirers Gagi-F and 6a9l-R of samplesshown in A). ldenticalresultswere obtained with primersNPl 16 and NPI I 7 (seetext, data
not shown).The detection limit was below I copyl Ul isolatedRNA or 5 copies/reaction.C, Resultsof qRT-PCR
for XMRVplasmid (ontiol in serial
dilutionsrangingfiom lO5 to 102copies/mlas well as negative@ntols for both primer pairs used,F2lR2(upper paneDa;d wPl (lower panel).All
patient plasmaand whole blood sampl$ were found to be negativeafter a total of 45 amplifi<ationcycle5(data not shown).
dol1013714ournЫ
pone00278709001
Results
November2oll I Volume6 I lssuetl I e27870
-37-
XMRV F2′R2p“ meE
撻羮 獄一
藍﹁
拳
亜
NEstCd RT-PCR
For detection ofXMRV/\,{!V
sequencesby nested PCR, RNA
was extracted from 0.5 ml plasma using rhe QIAamp Ultrasens
Virus Kit (Qiagen). The isolated RNA was immediatcly subjected
to reversetranscription employirrg the Superscript III FisFshnd
Synthesis System for RT-PCR (Invitrogen). Culture supemabnt
trom the XMRV-producing prostate cancer ceU line 22Rvl wro
usedat a I0-'dilution ro a positiveconrrol for RNA isolarion.For
amplification of XMRV/-MI-V
gag sequences, 5 pl of the
transcribed cDNA were used for the first round of amplification
with primers 4l9F (5'-ATCAGTTAACCTACCCGAGTCGGAC-3') and ll54R
(5'-GCCGCCTCTTCTTCATTGTTCTC-3') [5] and HotSart-IT FideliTaq Mcter Mix (JSB) with
the recommended componen( volumes. The amplfication w6
initiated by incubation for 4 min at 94"C, followed by 40 cycles of
I min at 94"C, I min at 57"C and I min at 72"C, and a final
incubation for l0 min at 72'C. Nested PCR wo perfomed under
the same conditions for 45 amplification cycles with 5 pl ofthe lrsr
rouf,d PCR product and mo di{ferent primer pain, Gag-I-F (5'TCTCGAGATCATGCGACAGA-3)
and Gag-I-R (5'-AGAGGGTAAGCGCAGGGTAA.3')
or NPI I6 (5'-CATGGGACAGACCGTAACTACC-3')
and NPI l7 (5'-GCAGATCGGGACGGAGGTTG-3'), both ofwhich have been shown to detect both
XMRV
and MLV sequenccs [6]. To determile the rosay
scnsitivity, serial dilutions of a cloned fragment of XMRV gag
[9] ranging from I to 100 copics/ pl were included in each PCR.
The resulting PCR amplification products (730 bp for first round
PCR and 413 bp or 380 bp for second round PCR, respectively)
were analyzed by electrophoresis in I.5% agarose gels. Any bands
of approximately the correct size were exci*d and subjected to
sequ€ncing in order to determine homology to MLVs.
amplification cyclcs of 95'C and 60'C for 30 sec each. Seria.l
dilutioro of a cloned fragment of XMRV gag [9] were u*d to
produe standard curus-(Fig. lC). The sensitiviry of the qRTPCR assay wa below lOJ copics/ml plama or whole blood.
¨
”
¨
¨
¨
卿
ゆ
¨
”
”
according to the Fukuda criteria [2]. Two participants did not
meet the CCC af,d one participant did nor meet Fukuda criteria,
but all three were included on clinical grounds. The remainder of
the CFS group met both the CCC and the Fukuda criteria.
Healthy controls who showed more than one syrnptom of ME/
CFS at moderate or grcater severity were excluded. The CFS
group (58 individuals) had a mean age of 48.9110.1 years and
90% were female, compared to the healthy conrrol grcup (57
individuals) with a mean age of47.6t 10-6 years and 89o/ofemale,
reflecting the higher prevalence ofthe diseaseamongst women. A
documented infectious onset could be reponed by 59% ofthe CFS
patients. Of the CFS patients, 93% have been sick for more than 2
years and 3% have been sick lor I 2 years, while 5% showed
symptoms since childhood or adolescence.
鳴 馴'
Figur€ 2. No evlden<e foi infectlous virus or XMRv-speclfic antibodie5 in plasma of CFs patientg and healthy controls. A) GFP
expr€ssionof DERSE.Li-G
cells 7 days (upper panelr)or 21 days (lNs panels)after spinoculationwith two different dilutions of 22Rv1cell cultlre
supematants(l 0-4 and l0-5 dilution) or patient plasma.No GFPexpressioncould be observedin any ofthe cellsinoculatedwith human plasma.8)
lmmunoblotting of C7-purifiedXMRVantigen with patient plasma for detectionof anti-XMRV/MLVantibodies.RepresentativeWB resultsfor CFs
patientsand healthycontrols. Lane l, anti-FdendMuLVwhole virut goat polyclonalantisera;lane 2, anti-Rauscher
fuluLVenvelope,goat polyclonal
antisera;lane 3, XMRVnegative blood donor plasma.Locationsof readivity to specificviral proteins are indicated; Env (gp69l71).envelopelTM
(p15E),transmembrane;MA {pl5), matrix;Gag {pr68);CA (p30),<ap'id.
doi:10.1371ljournal.pone.0027
A7O.gOO2
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oS
ONE
I ―w
pomneOり
November2oll I Volume 6 | lssue1l I e27870
-38-
No XMRVin CanadianCFSPatients
of the CFS patient samples (lane 5, Fig. 28) is most likely due to
the pres€nceof cro$-reactive epitopes.
Discussion
In summary, we were unable to detec( any evidence ofXMRV
or MLV infection in any of the I 15 eemined study pdicipanb,
regardlessofwhether they were suffcring from CFS or represented
healthy controls. The 58 CFS paricnE enrcUed in this srudy werc
carefully selected according to the Canadian Coroensus Criteria
for ME/CI6. Pmitively reeaed parlicipans were onJy included
if they showed slmptoms in at least Mo categories of autonomous,
neuroendocrine, and immune manifeshtions. The sensitivity of
our asays reached copy numben lower thm 120 copies/ml of
plasma lor the detection ofviral nucleic acids, md I0" copies/ml
of plasma for lhe preserce of inGctious paricler
While ir is
posible that XMRV and MLV {e not predominatly bloodborne viruses and as such exist below the detection limit of most
dsays in plasma and whole blood, we believe that the Nays used
in this study are equally sensitive to those rcported in previous
prcsitive studies. Moreover, our broad study desigr and the uE of
degenemte primers with specificity for higNy corueNed sequences
in different MLV-like viruses and XMRV would have allowed us
to identify nucleic acids, infectious particles, and antibodies lor a
number of related murine retroviruscs. However, we could not
dctect any otle. murinc retrcviruses in any of our specimeru,
unlihe the finding ofMlV.like sequencesreported by lo arat [6].
CFS patient cohorc have been tested for the prescnce of
XMRV in the United States,Netherlands, Germmy, China, and
United Kingdom among othcn [4]. Being more awae of the
posible risk of ontmimnb
in commonly used iaboratory
reagens [13], none of these studies were able to reproducc the
initial findings. Moreover, repeated testing of CFS pariens
previouly reported to be infccted with XMRV in the initial study
performed by Lombardi e, al failed to detect my signs of XMRV
infection itr thesepatients [8]. On the contary, it is now becoming
incredingly clear that XMRV found in the prostate cancer cel
Iine 22RvI originated from recombination of mo MLVs present
in the mouse stmins used for pcsaging of the initial pmstate
canccr renograft [4]. The |act that thc viral sequencesinitially
identilied in prcstate and CFS smpies are virtually identical to
those found in 22Rvl cells 05] suggests that rhc Numed
aseiation of XMRV wirh human discases is due ro smradic
laboratory conumination. Moreover. diflerentiai handl.ing of
pati€nt samples compared to controls can intoduce bias and
w6 therefore carefully avoided in this study. Two independent
studies could show that handling ofhuman samplesin laboratory
environmcnt with abundant endogenous MLV prcviruses can
lead o rhe false dctection of XMRV/MLV-like
sequencesdue ro
conhmination 6 proven by PCR detcction ofthe highly abundant
intracisternal A-t ?e pdticle (lAP) Iong reminal repeat in the
same samplcs [6,17]. ln the light of the accumulating evidence
for the artefrctual origin of XMRV and the high burden otMI-Vlike DNA contamination thc inirially Eported comecrion of
XMRV and prosate cdcer is now being ruled out a wel !8].
Thus, although XMRV Ms found to infccr md replicate in a
varie ty of human celb, natural XMRV/MLV
infection of humans
ha not yet been reproduced md is believed to be a false-positive
result lrom mouse DNA and/or Mlv:contaminated
PCR
.eagents 03]. This study eEmines a possible asociation of
XMRV and chroaic fatigue in a Canadian patient cohoft and is
consistst with a number of recendy published reports dedding
no evidence lor the prescnce of MLVlike
viruses in any human
subjece. ln concluion, while this stud md othen fail to support
an osociation beveen XMRV
and CFS, tiey highlight the
urgent nced for fufrher csearch into the root caurcs of CFS.
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Acknowledgments
Wc thsk lae Ann Tyrrcll and Bonnie Bock for thcir inwluable
contributionsin smplc colletion and doomshtion, Drs. K'€ongEun
lec md Vincct KcwalRamani (NCI) for thc kind gift ofDERSE.Li-G clls
and Dr. lndira Hcwi€t (FDA) for advi@ on virus culsrc probcols.
Ulc of tradc mmcs is for idcntification only and d€s nor imply
endorFmcntby thc U.S. Dcparhcnt ofHcalth and Human Scrvic6, the
Public Hcalth Seruicc,or thc Ccntcr for Discac Contrcl and Prevcnrion.
Thc findingsand conclusions
in this reportarc thoscofthe authon md do
noi nccBsarily reresent the vjes oI th€ Cenbs for Disrc Control and
Author Contributions
Concciv€d and ddigncd the *pdim€nts:
DLT ES TLYZ WMS MH cS.
thc dpcrimtrs:
IS LM KL ST I{ DH DMS ML ALJL.
Analyzcd thc daa: lS TL 14/t\4S MH cS. Wrcte the papcr: IS MH GS.
Perfomcd
References
L Caiiu$cB BM, Jd. AK Dildlcir KL, PeieEosDIj Klima NG, .r ,1.
(2003)
Mydsic cnc+halomFlirt/chronic
ratipc sFdrcm.r Cliniel wor[ng
2.
3.
a.
5.
6.
7.
@.
cae d.fdiion, dia$osdc and rcainents proicok.Jolrnal ofChronic Farip€
S y n & o m c I, l : ? l l 5 .
Fuh& K, S!r.u, SE, Hic[c I, Sbrye MC, Dobbins JG, er d. (199a) tc
chrcnic faiip. syndrc6.: a comprchcsivc ryp.@d to is definiior and $udr
hernrtionrl Ckonic Fari$r SFdromc Sody Group. tun Int.rn Md l2l:
953-959.
Udsman A, MohDro RJ, Fische. N, PIlmmcr SJ, Cacy c, d al. (2@6)
Idcntifi@tion of a novel ganmar€rrcd.trs ;r prosbrc tumos of p!ti.d6
hondy$u
for R62Q RNASEL vadanr. PH
Parhos 2: e25. l0.l37ll
jourid.ppar.m2m25 ldoi].
KcnyonJC, Lver AM (2011) XMRV, prostatc cancc. and chronic faripc
(6i1.
rydronc. Br Med 8ol 98i 6t-74. Hfr10 kill;10.1A%/bnh/Hfrt0
bnbardi VC, Rusceni ru, Da cupt.J, tras M
Has.n KS, c! al. (2mg)
Ddccrion of an inGctious rctrodlus, XMRV, in blood c.lh ol padcnc dfi
chronic fadgu. rydrotuc. Scicnce 326: 585-589. 1179052 [pii];t0.1126/
s&nc..l 179052 {doil.
b SC, Pripuzwa N. Li B, Komarcfr AL, Huns OC, d al. (2010) Dciection of
Mlv-r€llt.d
rirs gcnc scqrcnces in blood of pad.nB frth chronic fadflc
sFdrodc and hedthy blood dono6. P.oc Nail Ac.d Si U S A 107:
1 5 8 7 4 - 1 5 8 7 9I.0 0 6 9 0 11 0 7 [ p i i ] ; 1 0 . 1 o ? 3 / p n a s . l 0 0 6 91l 0 7 [ d o ' j .
Sm;th RA (2010) Conramineiion ol.linical 3pccimcns drh Mlvrncoding
nucl.ic acjd!: implicationi lor XMRV
and orhcr candidar€ huBan
rcrrolirurca. RcEovirology 7 : lt2. l?42-+690,1 -t t2 [pii]:10.1 186/1742.
4690-7-l l2 [doi].
rros orr I ww.plosone.org
8. Knoa K, Cdrigd D, Simmors C, Tcquc F, Zlou Y, cr d- 120I l) No Eddercc
ot Mlrinc'Ukc
Gammarcrrcdrus
h CFS Pad.m Prcdoudv ld.ntified *
XMRVJdcctcd Sci6c.. sci.nccl20a963 lpiil;10.1126/$i.nc.l204s3
[doi]
9. swiucr wM,J'a H, Hohn O, Zhcng H, T.ng S,.r al. (2010) Abcnce of
clidencc ofxenoropic murinc lcukcmia dru!-rclaEd drus infedon in peisors
dih chrcnic fadpc syndromc and h.athy @ntrob in dc Unild SEr*.
Rctrcd.olo0 57. | 742'4690.7-57 [pn];t0. I 186/ l7 42.+690-7-57 tdoi).
10. J.!on H, EvaN M, Pone N, B6m
M, BrcM A, €r d, (2010) Th.
d.wlopncnr of a d&d
Caddar nydsic .ncqblodyeh*
chronic Adsc
lyndromc cas dcfinirion. A6ericanjournd of Biodcmisrry and Bior.chnotory
6;120-135.
I l. van Kuppcvcld FJ, d.Josg AS, Lankc KH, v*hacgh cW, Melcl'etr WJ, d d.
(2010)Prev.l.ncc ofxcnotrcpic mudne lokchb
dru,rcl.redvids in pariens
dtfi drcnt
fadpc rytum€
h tfic N.rI.rlailds:
rcffipecriw
an.tFb of
sampler frch ,n cnablihed cohort. BMJ 3€: ct0l8.
12. Donr B, Kim S, Hong S, Das CupraJ, Mabthi K, d d. €m7) h inGcdous
rercvnussusccptibl. ro.n IFN anriiral paihway from human prc'src tumors.
P r o . N a t l & a d S c i U S A l G : 1 6 5 5 - 1 6 6 0 . 0 6 t U 9 1 1 0 4( p i ; 1 i t 0 . t 0 7 3 l
pDa.06l029l le [dd].
13. Silo L Furlh Rd Miyazawa T (2010) An end%do6 nudne l@ldja *,
gcnomc contamhanr itr a rohilqcid
RT-PCR kit t mplfrd
usins 3bndad
primds br XMRV. Rcrrcdrotry
7: ll0. D42^+690-7-lt0 [pit;10.il86/
1742-690-7-ll0 [doi].
14. Pap.orb T, DdvkeFra*.nbery
M, Cinpz O, Martntcz A, Kuns HJ, d at.
(201l) R@om$nlnr O.igin ofrhe RcFodrus XMRV. Sciede. sdcnc.l205292
fliil; I 0. I I 26ls.icncc. I 205292 [doi].
November2011 1 VObme 6 1`sue ll l e27870
-39-
0 1 飩o S O N wE p bl "―nge α
November2oll I Volume 6 | lssue11 | e27870
-40-
00MMENTARV
I董
The scientiflc lnethod at work:xenO● OpiC inurine leukelllia
宙rus―related virus is neither a cause of chromic fatigue syndrome
nor a threat to the b10od supply
Matthew S. Karafin and Susan L. Stramer
ir FrancisBacon (1561-1626),
the tord Chancellor
of England, was thefirstto provide a docmented
philosophical method for investigating a natual
phenomenon. This method later becme lcrown
as "(he scientific method." Unlike many before him, he
suggestedthat our understanding of the world should be
based on data, rather thm faith or dogma. Moreover, his
method required that our unde6tmding of the world be
provisional, with the hope that our current uderstanding
of natuml phenomena would eventually be replaced by
better science. The events that have transDired over the
past 2 yearsregardingthe clinicalrelevmce of xenotropic
murine leukemia virus-relarcd virus (XMRV) haVe shown
that the method of scientific investigation first described
400 yeils ago is very much alive and well today.
The scienti-fic method, as envisioned by Sir Francis
Bacon,starts with observation.Urisman and colleaguesr
first observed that )CVIRVis associated with human
disease in 2006, finding that 4070of men with prostate
cancer and a low activity variant of RNase L, an enzlme
involved in the interferon-induced antiviral response,
were infected with &is virus. Subsequently in 2009 and
2010, two groups of investigators also described finding
)0\4RVorrelated sequencesof polytropic mwine leukemia
viruses (MLVS) in association with cbronic fatigue syndrome (CFS),a diseme characterizedbysevere fatigue and
other related slmptoms lasting more than 6 months.23
X\4RV is currently mderstood to be a retrovim, but
is unrelated to other welldescribed retroviruses such as
From the Deparment
ofPathology, Iohns Hopkins Universitj4
Ba]ti!!!sre,:r4ai],ia!!d;oid the Scie!rtificSuppsri Cmce, /4eri
can Red Cioss, Caithe6burg,
Adix$s
Maryland.
reprint requesrs ,oi Susan Stramel
Scientific
Support Omce, Arnerican Red Cross, 9315 Gaither Road, Gaith.
ersburg, MD 20877; e-mail: strame6@usa-redcross.org.
Received for publication November 28, 20ll; accepted
November 28, 2011.
doi: l0.1lll/i.1537-2995.2011.03518.x
TMNSFUSION 20 l2 ;52t222-225.
222
TRANSFUSION
XMRI TRANSFuS:ON SAFETγ
蔽 61
humm immunodeficiency virus (HIV) and hman T-cell
llmphouopic virus (HTLV). )OvtRV specifically is a
member of the fmily Retroviridae, subfamily Ortho(see AABB
retrovfuinae, and genus Gammrefiouirw
)G4RV Fact Sheet http: / /w.aabb.org/resourcs/bct/
eid/Pages/default.aspx).)O4RVis the first galmiletrovirus to be found in humans, and data indicate that it
originated in mice after the recombination of two muine
proviruses.oVirionsare B0 to I00 nm in diameter, consisting of an envelope, nucleocapsid, md a nucleoid with a
linear dimer of posirive-sense,single-strandedRNA.'
From these initial observations, it was hypothesized
that this virus could be causally related to both prostate
cancerandCFS. Moreovet thefindingofviralsequencesin
the btood of hea.lthy controls in two studiesz'3led to the
concern that this virus cou.ld be transfusion transmitted,
and thus the national blood supply could be at dsk. Specifically, on June 18, 2010,the AABB issued a bulletin to its
membership from its Interorganizational Task Force on
)O4RV that patients diagnosed with CFS be discoumged
from donating blood. Consequently, the Anericm Red
Crossand a number ofotherblood donor centersstartedto
offer edumtional information about CFS md have
requestedvoluntarydeferral of donors who everhave had a
medical diagnosisof this debilitating condition.6
While the risk of trmsmission of XMRV by blood
products was unknown at the time of releaseof this bulletin, the recommeridation of rhe A|I\BB Interorganizational Task Force was reasonable based on the initial
hypothesis that CFS could have m infectious origin. First,
X\4RVis a gammaretrovirus, a genus that contains known
animal pathogens (seeAABB )O4RV Fact Sheet).As other
retrcv'rtuses,sucil as HI\r'and HTLiylare transfusion transmitted, it was plausible that an emerging rekovirus, such
as XMRV could also be transmitted by blood. Second,
studies indicated that )S4RV was physically present in
blood. Arhesus macaque model of)O4RVprevioully demonstrated that the virus can infect lymphoid cells,several
tissues, and organs even though circulation of free virus
was minimal.T Moreover, Lombardi and colleagues,found
)O4RV infection in the llmphocytes of the CFS patients
studied. As lymphocytes are present in transfused cellulu
blood components, such as plarelet products, red blood
cells, and granulocytes,there is a substantial risk that this
virus could be tansmitted by transfusion.
The hlTrothesis that )0\4RVw6 a cause of hman
disease did not, howeve! withstand confirmation by other
researchers who were investigating the same relationship.
One review found that KVIRVhas been detected in 07oto
27,5%ofprostate cancer patients in l2 studies.sMoreover,
while the two original positive CFS studies found that
>C\4RV/MLV
sequenceswere present in up to 86.570ofCFS
patients and up to 7% ofhealthy controls including blood
donors,'z3as of 201l, at least 30 subsequent studies failed
to reproducethese 6ndings in CFSor otherpatient groups
(see AABB )C\4RVFact Sheet table). Additionally, these
studies have failed to demonstnte a relationship beween
)flvlRV/MLV and CFS even with highly sensitive methods
modeled after those described by Lombardi and coworkers,2 including various molecutar, serologic, and culture
procedures, as well as enrolling many of the same CFS
patients for study whose samples were previously ideDtified as )0\4RVor MLV positive.s-rr
Originally, several hlpotheses were posed to explain
the wide variations between study findings, including differencesin cohort selection, testing methods, or sample
source, plepalation, and storage.6r0J2However, recent
data indicate that laboratory, sample,and/or reagent contamination are ttre most likely causes for the results in
those studies with positive findings.aca The Scientific
ResearchWorkingGroup (SRWG)ofthe NHLBI, including
several key authors of the original article associating
)O4RV/MLVSwith CFS,sent samples in a blinded fashion
to nine diferent laboratories, all with research tests for
)0r4RVincluding detection of nucleic acids (l I laboratories), antibody (five labomtories), md virus following
culture (ttuee laboratories). They found that curent
tetting methods do not reproducibly detect)q\4RVevenin
blood samples from patients that were reported to have
)0r4RVinfection previously (including 14 with CFS)or in
XvlRv-negative, healthy controls.rt Of note in this study,
only the two labomtories associatedwjth Lombardi and
colleagues'zobtained positive results for ily samples
(xcluding the spiked positive conffols); howweq these
laboratories fomd positive DNA and antibody results in
healthy controls at the same rate as positive results in CFS
patients with the additional caveat that positive patient
resltts were inconsistent. Perhaps rnost imporiantiy,
Paprotkaand colleaguesaproposed that XMRV originated
asthe result of a labomtory recombination event involving
two mouse proviruses that occurred during the serial
passageofa humm prostate cancerxeno$aft (CWR22)in
nude mice in the 1990s.\A/henaligned, these two proviruses were identical to the sequence of XMRV. Thus, the
authors concluded that )0\4RVis not a real hman pathogen and that positive findings were the result ofcontami
Volume 52, FebruaN 2012
AND HUMAN D:SEASE
nation by a laboratory-dedved virus.{ In a subsequent
related study, it was shom that both provirusesoccurred
in laboratory mice but not in wild strains ofmice, and no
laboratory mouse strain could harbor )OvIRVreplication
due to the lack ofthe required receptor in laboratory mice,
indicating that the xenografted hmm tumor cells were
required for )O4RVpropagation.r3In addition, the genetic
distance among enu and pol sequencesfrom the persistently )O\4Rv-infectedprostate cell line, 22Rvl, derived
ftom the CWR22 xenograft, exceeds that of patientassociated sequences,suggesting laboratory contamination versus human infectious transmission. Thus, )0v1RV
derived from the 22Rvl cell line is the genetic ancestor of
all subsequent isolatesfrom CFS or other patients.ra
XVIRValso does not appear to be a concern for blood
recipient safety.Studies have recently demonstrated that
)0\4RVwould not be able to persist or replicate in human
blood due to cell-mediated antiviral pathways.sA large
recent study further demonstrated that no XMRV antibody could be detected from 17,249 blood donors or
recipients, including 13,399US blood donon from six different regions and 3741 donors linked to I09 recipients of
which 830 samples were tested over a 2-yeu period. A
positive mtibody result required reactivity to three different )S4RV proteins, and the tests used were the same as
those used by the SRWGand representedthose tests that
were automated and could be used for blood donation
screening ifneeded. SinceRNA could alsonot be found in
any recipient or ily donor with isolated antibody reactivity, the study concludes that )O,IRV is not a curent threat
to blood safety.ts
The mounting negative findings failing to associate
)O4RV/MLVwith hman disease,and now documentation
of )C\4RVas a laboratofy iltifact, prompted the Editor of
Scienceto call for a retnction of the 2009 publication by
lombardi and colleaguesin an expressionof concern.r6
Hwwer, the authors of the original study have not agreed
to rebact their original work entirely; a partial retraction
initiated by one author, and signed by the other authors,
has resulted in removat of the polymerase chain reaction
data due to sample contamination with XMRV plasmid
DNA.I?Taken together, the scientific data to date indicate
that XMRV/MLV is neither a human pathogen nor a risk
to the national blood supply (see reviews detailing the
chronology of events and investigations of potetrtial
)G4RVdiseaseassociationssince October 2009r3''z0).
Most
i€cently (December 23, 2O1\\, the Editof-in-Chief of
has
Science
issued il editorial retradion of Lombardi
et al.2due to lhe inabiliryotmultiple laboratoriesto reproduce the study findings, including those of the original
authorS; questions ofquality control related to a number
of specific reported experiments; and m overall loss in
confidence in the validity of the conclusions.zrThis was
followed by a retraction of the Lo et al. mauscript3 by the
authors on December 27,2011. One study exmining the
Volume52. February2012 TRANSFUSION223
-41-
-42-
KARAFIN AND STRAMER
XMRVTRANSFUSION
SAFETYAND HUMANDISEASE
potential link of XMRVwith CFSis stillpending. The study
is sponsoredbythe NIAID and led bylan Lipkin of Colmbia University; this likelywill be the last mator studyinvestigating the diseasepotential of these agents.
Thus, over the past 2 years, XMRV has transformed
from ahagent of potential hmm diseaseassociation,and
a possiblethreat to the national blood supply, to a laboratory contminant without a current thrcat to humans.
This revolution of ideas regarding KVIRV could only have
been made possible by the scientific method. Sir Francis
Bacon aptly described the scientific process with the following metaphor:
of an infectious re[ovirus, XMRV in blood cells of
and distriburion ofthe Mo XMRV parental proviruses.
Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK,
patients with chronic fatigue slmdrome. Science 2009;326:
IVirol 2012;Bti:328-38,
585-9.
Hue S, Clay ER, Gall A, Katzourakis A, Tan CP, Houldcroft
Petrow-Sadowski C, Gold B, Dean M, Mikovits IA. Partial
retnction. Science201t;334:I76.
Lo SC, Prip@ova N, U B, KomiloffAl-,
Hung GC, Wang R,
Alter HF. Detection of MLV-related virus gene sequences in
blood of patients with ch.onic fatigue symdlome and
healthy blood donore. Proc Natl A€d Sci U S A 2010;107:
t5474-9.
Paprotka T, Delviks-Frankenberry
KA, Cingiiz O, Martinez
A, Kung Ht, Tepper CG, Hu WS, Fivash Mi Ir, Comn IM,
Pathak vK, Recombinmt origin of the retrovirus XMRV.
Those who have handled sciences have been either
men of experiment or men of dogmas. The men of
experiment de like the ant; they oDly collect and use:
the reasoners resemble spiders, who make cobwebs
out oftheir own substance.But the bee takes a middle
couse; it gathers its material from the flowers ofthe
garden and the neld, but transforms and digests it by
a power of its own. Not mlike this is the true business
of philosophy; for it neither relies solely or chiefly on
the powers of the mind, nor does it take the matter
which it gathers from natural history and mechmical
experiments and lay it up in the memory whole, as it
finds it; but it lays it up in the unde$tanding altered
and digested. Therefore from a closer and purer
leaguebetween these two faculties, the experimental
and'the rational (such as has never yet been made)
much may be hoped. (Book 1, Aphorism 95)"
CJ, Mclden
S, Pillay D, Futrcal A, carson lA, Pybus OG,
Kellm P, Towers G.J.Disease-associatedXMRV sequences
are consistent with Iaboratory contamination. Retrovirology 2010;7:lI1Dodd RY, Hackett L Linnen tM, Dorsey K Wu Y, Zou S, eiu
X, Swansoo P, Schochetmil c, cao K, Carick rM, Krysztof
DE, Stramer SL. Xenotropic muine leukemia virus-related
virus (n\4RV) does not pose a iisk to blood recipient
Science 201 1;333:97"l0l.
s€fety. Tlarsfusion
Silveman
RH, Nguyen C, Weight CJ, Icein EA The human
retrovirus XMRV in prostate cmcer md chronic fatigue
A.lbertsB. Edilorial expression ofconcern. Science 20I1;
333:335.
slndrome.
Silwmd
Nat Rev Urcl 2010;7:392-4O2.
Klein HG, Dodd RY, Hollinger FB, I(aE LM, I(leiman
Mccleary
tr3(, Silverma
nizational
task force on XMRV xenotropic
2012:52:298-306.
RH, Das Gupta r, Lombardi VC, Ruscerri FW,
lB.
Van Kuppeveld Fl, Vm der Meer lK. XMRV and CFS-the
sad end ofa story. Lancet 2011.doi: l0:1016/50I40-6736(11)60899-4.
19. Robinson ML Brlwein O, Mcclurc MO. Xenottopic murine
leukemia virus,related virus IXMRV) does not cause
chronic fatigue. Trends Microbiol 20ll;19:525-9.
20.
Cohen J, Enserink M. NewsFocus: false positive. Science
201l;333:I 694-701.
21. Alberts B. Retmction. Science 20) l;334j1636.
22. Moore tA. Science as a way of knowing: the foundations
ofmodern biology. London: Harvard University press;
1993. u
S,
RH, Stmmer SL. AABB intelorgamurine leuke-
mia virus-related
vLus QOvIRV)ad blood transfwion:
report of dxe AABB intelorgmizational
)Or{RV task force.
Tfansfusion 20I l;5I:654-61.
Onlamoon N, Das GuptaJ, Sharma P, Rogers K, Suppiah S,
Rhea J, Molinaro RJ, Gaughm C, Dong B, Klein EA, Qiu X,
Dewe S, Schochetme
G, Hackett L Silveman RH, Villinger F, Infection, viral dissemination and etibody
responses ofRhesus macaques eryosed to the human
)O4RV. rVirol 2011;85:4547-57.
Smmuerlovirus
Shin CH, Batemil L, Scttlaberg R, Bunker AM, Leonaid CJ,
Hughetr RW Ught AR, Light KC, Singh IR. Absence of
XMRV ed
orher Mlv-related vinss
in patients with
chronic fatigue slrd rcme. I vircl 201t;85:7 195-202,
Knox K Canigm D, Simmoro c, Teque F, Zhou Y, Hackett
,ust asSir Francis Bacon predicted, the astute combination of a ntional eva.luationof current knowledge with
rigorous experimental obseruation allows the scientific
community to move semlessly from an unproven
working hypothesis to a result based on dre synthesized
accmulation of data disproving the hypothesis. In conclusion, the scientific process remains to this day a powerhrl tool to understand our natural world, and )O4RV
clearly demonstrates the potency of a 40o-yeil-old
method. Sir Fmncis Bacon would be proud.
I Ir, Qiu X, Luk KC, Schochetmm G, Knox A" Kogelnik AM, .
I3vy ,A" No evidence of murine-like gmmdeftoviruses in
CFS patients previously identified 6 )gvlRv-infecred.
Science 20ll;333:94-7,
SimmoN c, clynn SA" Holmberg t Cofnn t, Hewlert I, Lo
SC, Mikovits ,,qq Swi%r \4tM, Umen lM, Bsch MP; for rhe
Blood XMRVScientific ResedchWorking Grcup. The Blood
Xenotropic
Murine I€ukemia Virus-Related Virus Scientific
Research Working Group: mission, progress and plms.
Tlanstusion 20 I 1;51:&3-53.
CONFLICT
OF TNTEREST
ll.
The authorsdeclareno conflictsofinterest.
Kearney MF, Rscetti FW, Pfost MA, Bethel J, Kleinman S,
Holmberg IA" Busch MPj for the Fiood XIdRV Scienrifrc
Reseash Working Group (SRWG). Failure to confirm
REFERENCES
l.
Urismil
A, Molinaro RJ, Fischer N, Plummer St, Casey G,
Klein EA, Malathi K, Magi-Calluzi
C, Tubbs Ri, canem D,
XMRV/MLV in the blood ofpatients with chronic fatigue
Silveman nH, DeRisi tL. Identification of a novel gam'
mdetrovirus in prostate tumoF in patietrts homorygous
syndrome: a multiJaboratorystudy.
for R462Q RNASEL varidt.
Shan H. What is XMRV and should we be worried about ir?
2. tlmbardi
PLoS Pathog 2006;2ie25.
V, Ruscetti FW, Das Gupta ,, Pfost MA, Hagen
C, Gold B, Deil
TRANSFUSION
Science 2011:334:
8t4-7.
Transtusion 20 I I;51 :450-3.
Cingoz O, Paprctka T, Delviks-Frukenberry
KS, Pete6on DL, Ruscetti SK, Bagni RK, Petrow-Sadowski
224
SimmoN G, Glym SA, KomaroffAl,
Mikovits rA, Tobler
l"FI, Hackett I Ir, Tilg N, Switzer WM, Heneine W Hewlett
lK Zh^o J, to SC, Alter HL Umen IM, Gao K, Conn IM,
KA, \4ildt S,
Hu WS, Pathak VK, Coffin tM. Characterization, mapping
M, Silverman RH, Mikovits tA. Detection
Volume 52, February 2012
Volume52, February
2012 TRANSFUSION
225
-i1_
軋
●哀
Press Release
平成2 4 年 2 月2 4 日
医薬食品局血液対策課
( 担当 ・
内線) 課 長補佐 伯 野 ( 2 9 0 5 )
血 液安全係長 松本 ( 2 9 0 8 )
(イ
t 辱長電 言
舌) 0 3 ( 5 2 5 3 ) 1 1 1 1
報道関係者 各位
(直通 電 話 )03(3595)2395
フ ィ ブ リノ ゲ ン製 剤 納 入 先 医 療 機 関 の追 加 調 査 に つ いて
平成 16年12月9日に公表したフィブリノゲン製剤納入先医療機関を対象として、平成 19年
11月7日付で実施した追カロ
調査の結果について、平成 24年2月 10日までに回収した医療機
関からの回答を取りまとめた状況をお知らせいたします。
(参考)C型 肝炎ウイルス検査受診の呼びかけ(下記の厚生労働省ホームページにリンク)
http://www.mhlw.go.ip/houdou/2008/01/h0117-2/index.html
2主 な調査結果
(1)投与 の年月について回答があった医療機 関数と元患者数
9 4 4 施設
医療機関数
元患者数
1 4 , 4 5 4 人 ( 投与年別は別表)
( 2 ) 上記以外 に、過去に投与の事実をお知らせしたという記録 が残されているが、現在で は投与
の年月は特定できないとする回答があった医療機関数と元患者数
医療機関数
元患者数
104施 設
332メ、
(3)(1)と(2)の合計
医療機関数
1,024施設 味 2)
元患者数
14,786人
c型 肝炎ウイルス検査受診 の呼びかけ(フィブリノゲン製剤納 入先
(※2)厚生労働省 ホームペー ジ「
医療機関名の再公表について)Jの公表医療機関等リスト上の該 当医療機関の 「
備考」
ォ
関に、
「
フィブリノゲン製剤を投与されたことが判明した元患者の方がいるとの報告あり。」と記載し
た。
1 回 答状況
( 1 ) 追カロ
調査実施期間
平成 19年 11月7日 ∼ 12月5日 (※1)
(ただし、現在も回収 中)
( 4 ) 元患 者 の 方 へ の 投 与 の 事 実 の お知 らせ の状 況
(※1)(1)の調査以降も毎年度、元患者 の方 へのお知らせ状況等について再度調査を行っており、
(3)回答施設数以降はそれらの結果を反映したものである。
(2)追カロ
調査対象施設数
医療機 関 6,610施 設
(平成 16年公 表施設 のうち、所在地等が不明であつた施 設を除 いた医療機関)
(3)回答施設数
・ 平成 16年公表時に存続 していた5,397施設 のうち、5,291施設 (98%)力も 回答があった。
。 なお、このほか平成 16年 公表時に廃院等していた 1,213施設 のうち、508施 から回答
設
があった。
お知 らせ していない
投与後に原疾患等により死亡
連絡先が不明又は連絡がつかない
肝炎ウイルス検査 の結果が陰性
今後お知らせする予定である
その他 (未記入含む)
8,762人 (59%)(※
6,024メ、 (41%)
3)
1,967メ、 (13%)
2,855人
(19%)
292人 (2%)
442人 (3%)
(※3)元 患者の方に一人でも投与の事実をお知らせ したEXL療
機関は 846施 設であつた。
6り表)
投与の年月について回答があった元 患者数 の投与年別 の内訳
(5)診療録等 の保管状況
平成6年以前の診療録等が次のいずれかにより保管されている施設数
(括弧内は調査対象施設数 に対する割合)
2,045施設 (31%)(※4)
(内訳)(※5)
診療録 (カルテ)
手術記録あるいは分娩記録
製剤使用簿
処方箋
輸液箋あるいは注射指示箋
レセプ トの写し
入 院サ マリー あるいは退 院サ マリー
その他 の 書類
1,498施設
1,578施設
136施設
144施設
(23%)
(24%)
(2%)
(2%)
276｀
施 設 (4%)
83施 設 (1%)
296施 設 (4%)
296施 設 (4%)
(※4)平成 16年 の調査では 「
昭和 63年 6月 30日 以前にフィブリノゲン製斉Jを投与した記録 (診療
投与年
人数
昭和 39年
0 ノヽ
40年
ヘ
7ノ
ヽ
41年
ヘ
9ノ
、
42年
12人
43年
16人
44を
F
19人
45`「
2 0 メ、
46左
F
2 3 ノ、
47年
30人
48を
F
49■
F
48人
人
61ノ
ヽ
50左
F
58人
51年
72人
録、使用簿など)が保管されています か。」との設間であったのに対し、今回の調査では、「
平
52年
93人
成6年 以前のカルテ等 の各種書類が保管されていますか。」との設 間であつたため、保 管して
53年
ヘ
131ノ
、
いると回答した施設の割合が異なったものと思われる。
54年
人
202ノ
ヽ
55年
336ノ、
c型 肝炎ウイルス検査受診 の呼びかけ(フィブリノゲン製剤納入先
(※5)厚生労働省ホームペ ー ジ「
56年
443メ、
の公表医療機関等リスト上の 「
医療機 関名 の再公 表について)」
カルテ等 の有無」
欄 に、平成
6年 以前 のカルテ等の記録が一部 でも保管されている場合、△印を付していたが、さらに保
57年
575メ、
58年
982ノ｀
管されている記録の保管期間、保管状況等を記載した。
59年
人
1,530ノ
、
60年
1,816ン｀
61年
2,495ノヽ
62年
3,037人
63年
1,736人
平成 元 年
244人
2年
181人
3年
人
108ノ
、
4年
63人
5年
53人
6年
44人
計
14,454人
日会 会 料
４議 員
平 薬 血提
時点において低減化処理 に起因する重篤な副作用等の報告は受けていない。
Ｄ御
獅飾聯Ｎ
ｍ
一
Table o cli:lical
cvaluations
日本赤十字社
150S8911
trial - reference group
MIRACLE trial - tvlirasolgroup
Serua
tvtlRACLE
Poland
Spain
血 小 板 製 剤 に対 す る 感 染 性 因 子低 減 化 (不 活 化 )技 術 の
導 入準 備 に つ い て (追 加 報 告 )
MLml
160
175
87
432a
44
evaluation progran
3(scvcrCi)
2 ( s e v e r c)十
12(gradC ll)
368
露鮒鵠 Ⅷ稲1:13∬
冒:織出11℃
よ
糖
島:
酬1母
乱
鰈 腔織選
瞥舞蹴理
雌
靱 魃 踊 脩暮
1 欧 州における導入状況について
①リボフラビン法 (Mirasol)
GrowingAdoptionof Mirasolin Europe& Middle-East
Transfus Med Hemother 2011;38:8-18
Routineuse in over 50
centersin 16 countries
サレン法(Ittercept)
②アモト
INTERCEPT
RoutineCentersper Country
1
■
0
0
5
4
4
FranceDOMfiOM
4
4
1
1
Fr"n."
10
1
10
1
4
Germany
1
0
1
0
5
Greece
1
1
0
0
6
ltaly
14
14
7
7
7
Kazakhstan
11
9
9
7
l(uwait
1
1
0
0
9
Norway
2
2
0
0
l0
Portugal
2
2
0
0
II
Russia
18
12
12
6
t2
Slovenia
1
1
0
0
13
Spain
12
Austria
2
Belgium
3
8
♂
mt
(鴨rumOBCT社 提供資料)
現在、欧州を中心に 16カ 国でリボフラビン法処理血小板がル ーチンで使用されており、そのうち
10カ 国では血漿も処理されている。なお、ベルギー (アモトサレン法が導入されていない北部地方)
では、低減化処理キットの仕様変更のため、現在 医療機関へ の供給は一時中断しているが、本年
夏以降に再開される予定である。
このうちの数力国での低減化処理血小板製剤 の市販後調査が実施 中であるが (次ペ ージ)、現
1
'
5
I
「
2
la
Sweden
7
7
0
0
15
Switzerland
13
13
0
0
Total
103
85
46
28
(Cerus社提供資料)
15カ 国でアモトサ レン法がル ニチンで使用されており、そのうち 14カ 国では血小板も処理されて
いる。これらの国のうち、スイスは 2011年 11月 に全ての血液センターでアモトサレン法で処理した
血小板製剤の製造を開始した。また、フランス本 上では血小板は 1セ ンターのみで変化 はないが、
メチ レンブル ー処理血漿についてヘ モビジランスの結果 、アレルギー反応が多いとの理 由でフラン
ス保健製品衛生安全庁 (AFSSAPS)が 本年度 中の使用 中止を勧告したため、血 漿にアモトサレン
した。一方 、ドイツではアモ トサレン法処理血小板製剤 が早い段階で
法を導入するセンターが増カロ
承認されているが、現時点で血小板製剤にル ーチンで使用しているセンター は無い。
なお、上記表には記載されていないが、本年 2月 1日 にイスラエルがアモトサレン法処理血小板
と血 漿を承認 したとのプレスリリースが発出されている。
3 日 本赤十字社における検討状況等について
①リボフラビン法 (Mirasol)
。
CaridianBCT社 がテルモ社に買収され TerumOBCT社
となつたことにより、テルモ社 の有する
安全性部 門や研究開発部 門の協力を得るこ とができるようになった。現在 、今後 の承認 申請に
必要となるAFSSAPSに 提出された安全性 に関するデ ータの整理を依頼している。
・日本赤十字社 において、製剤としての規格や 品質 にういて決定
するための試 験を実施 している
が、平成 22年 3月 の本委員会に報告した凝集塊 の発生という問題については、未だ解決できて
いない。日本 赤十字社 の検討ではかなりの確率で凝集 塊が発生するのに対 し、他 の 国では類似
した凝集塊 の発生はないとの報告を受けている。現在、TerumOBCT社
2.実 施 中の主な臨床試験 について
Ongoing clinical Studies(2010 to 2013)
・
新興再興感染症対策としてウエストナイル ウイルス(WNV)の 低減化 につ いて検討 したところ、日
本赤十字社のデータとTeru■OBcT社 のデータに乖離 があることが明らかとなつた。乖離 の原 因
を特定するため、種 夕の WniV株 について低減化率を測定した結果、細菌と同様 、使 用した株 に
ａ
ｍ
軸︲
ｓ
ａ
ｐ
″
PREPARES"Tria:―
Sanquin,The
Nethedallds
( 5 ●1 偲■l S 「e h N o m a v 7
+4 sitesin Canada′
2012}￨
と密接 に連絡を取りなが
ら、解決策を探 っているところである。
より低減化率に差が生じることが明らかとなり、ヒトから分離された Ugandai937株 に対 しては 、十
分な低減化効果があることが再確認された。
Study SuFnrnaries
:￨‐
m:燎 :童
ぁ
PL「i,pヽs
(TerumOBCT社 提供資料)
① PREPARES
オランダのサンキン財団を主体としてオランダ、ノル ウェー 、カナダで実施されている、全血採血
ViralStrain
NY‐99F:amingo
Uganda 1937
ATCC#VR-1510
NV,9'〔 4122)
由来 のリボフラビン処理血小 板製剤 と通 常 の血小 板製剤 のランダム試験 (患者登録終 了予定 :
TestSystem
2013年 末)
②IPTAS
96‐we‖ piate
T C : D 5 0 m′L
24‐
we‖ p!ate
TC:D50ノmL
ReporterCells
Vero
Vero
ReductionLevels
3.0‐
4.O Logs
we‖ piate
6‐
PFU/mL
vero
イタリア政府が実施している、添加液 (B墨 )で置換してリボフラビン法またはアモトサ レン法で処
理した血小板製剤と、通常 の血小板製剤との比較試験 (同:2014年 末)
PREPARESは
≧5.l Logs
l.5 Logs
メーカー 主導ではなく症例数が多いこと、また、IPTASは 同じプロトコールで、リ
ボフラビン法及びアモトサ レン法で処理した血小板 の臨床試験が実施 されることから、これ らの試
験 の動向を注 目しているところである。
3
(TerumOBcT社
提供 資料 )
② アモトサ レン法 (Intercept)
・
BloOne社 が精算されたため、現在 、Cerus社 と直接情報交換を行っている。
・
アモトサ レン法 の処理キットには、日本における需要 の 8割 以 上を占める 10単 位製剤の規格に
合うものがなく、また、アモトサレンの吸着除去処理 に長時 間を要することなどから、現状の処理キ
ットを日本 に導入することは困難 であるが、10単 位製剤 に最適化 した処理キットの開発 について
検討 したいとの話があつた。
4 今 後の予定
感染性因子低減 化技術MIRASOL導
平成 23年 度
入 作業計画
平成24年 度
平成25年 度
治 験申童
治験 薬概 要書等 に記載する必 要 が ある
「
品質、毒性 、薬理作用その他 の被 験薬
に関する事項 」に係るデータ等の採取
治験 業務手順書 、
同マニュアルの整備
治験実施
治験薬概要書 、治験 実施計画
書等、治験 の 申請に必要な書
類の作成
機構相談(適宜実施)
candianBcT(TerumoBCr)と の秘密保持 契約 ・
共同開発 契約
EROとの 業務委託契約 ・
秘密保持契約
医療機関とのタ: 約