初探閆麗夢博士第二篇論文參考文獻25中石正麗功能增強實驗內容

作者:Maarago

天使科學家閆麗夢博士在《SARS-CoV-2IsanUnrestrictedBioweapon:ATruthRevealedthroughUncoveringaLarge-Scale,OrganizedScientificFraud(SARS-CoV-2是一種超限生化武器:揭露大規模、有組織的科學騙局的真相)》一文中提到“石正麗課題組常規性地研究該病毒是否能夠感染人體細胞。這種研究活動的模式已經屢見不鮮25,32,36-39。“也就是說石正麗的這幾篇論文就是研究如何可以讓病毒更好感染人體細胞,那麼我們來看一下這幾篇論文第25篇論文的情況。

參考文獻第25:Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus(蝙蝠SARS相關冠狀病毒豐富基因庫的發現為SARS冠狀病毒的起源提供了新見解)

關於殺人女巫石正麗的這篇論文財新是這樣描述的——據財新網2020年01月20日 12:13 發佈的對抗新型冠狀病毒 能從抗擊非典中汲取什麼經驗?

[2013年,她帶領的團隊在《自然》雜誌發表一項研究,在雲南的一個蝙蝠棲息洞中,科學家在菊頭蝠的糞便中分離到一株類似於SARS病毒的活病毒,這種新分離的病毒與已知的SARS病毒具有高度同源性,可以利用ACE2蛋白做為受體感染人的細胞[11]。這項研究清晰地揭示了SARS病毒的來源,石正麗教授的團隊在這個地區的蝙蝠種群中檢測到了組裝SARS病毒所需要的所有基因[17]。他們推斷,SARS病毒很大可能是由感染蝙蝠的各種“類SARS病毒”重組而來,在偶然的情況下,果子狸感染了這種病毒,病毒在果子狸體內進行了複製與進化,通過被人捕獵的方式最終把病毒傳播給了人類[18]。]

在財新這一段文字中列明的參考文獻17就是[17] Hu, B. et al. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. PLoS Pathog 13, e1006698, doi:10.1371/journal.ppat.1006698 (2017).

參考文獻18是[18] 張渺. “蝙蝠女俠”團隊找出SARS病毒源頭. 中國青年報 (2017).

以及財新網2020年02月06日 20:03發佈的從複雜網路小世界、無標度、高聚類特性看新型冠狀病毒肺炎

[回顧2003年造成全球700餘人死亡的SARS傳播[23,24]事件,有研究發現SARS病毒的源頭可能是蝙蝠[25],而不是以前人們認為的果子狸[26,27],果子狸屬於中間宿主,也是SARS病毒的受害者。目前廣為人們接受的關於SARS的傳播途徑的觀點是:蝙蝠——果子狸——人群。2019-nCoV至今仍沒有確定中間宿主是什麼野生動物,雖然已有關於2019-nCoV的很多研究[28,29,30]。更糟糕的是2019-nCoV傳播具有潛伏期,大部分潛伏期1-14天不等,平均7天,潛伏期長增加了疫情控制的難度,使得一些潛在的超級傳播者不能被第一時間發現並隔離。另外,一旦發現感染者,需要向前追溯大量的潛在感染者,增加了工作的難度和複雜度。],這段文字中的參考文獻25就是上文提到的Hu, B. et al. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. PLoS Pathog 13, e1006698, doi:10.1371/journal.ppat.1006698 (2017).這篇論文,根據財新網的兩篇文章的引述,如何看待殺人女巫石正麗的這篇論文呢?從某種意義上說石正麗的這篇偽論文為中共的生化武器罩上了一層迷彩服,在這篇論文的遮蓋下,生化武器的自然宿主隆重登場,如果沒有閆麗夢博士的火眼金睛和一顆天使的心,這套生化武器的迷彩服也許將永遠不會被揭穿,現在每當我們面對數以百萬計的人類感染和數以十萬計的人類死亡的時候,我們如果不能銘記這些與魔鬼共舞的殺人魔頭,我們何以對得起那些正在感染和正在死亡的我們的同類?

這篇論文的作者是(注:作者順序按原論文排序,凡是屬於同一個機構的並列排出):

1、Ben Hu , Lei-Ping Zeng,Xing-Lou Yang(楊興婁) ,Xing-Yi Ge(葛行義), Wei Zhang(張偉), Bei Li, Jia-Zheng Xie, Xu-Rui Shen,

單位:CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases of Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

中國科學院武漢病毒研究所新發傳染病研究中心特殊病原體和生物安全性CAS重點實驗室,武漢

2、Yun-Zhi Zhang(張雲智),

單位:Yunnan Institute of Endemic Diseases Control and Prevention, Dali, China, Dali University, Dali, China

大理大學雲南地方病防治所,大理,中國

3、Ning Wang, Dong-Sheng Luo, Xiao-Shuang Zheng, Mei-Niang Wang,

CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases of Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

中國科學院武漢病毒研究所新發傳染病研究中心特殊病原體和生物安全性CAS重點實驗室,武漢

4、Peter Daszak, EcoHealth Alliance,

New York, New York, United States of America 紐約,紐約,美利堅合眾國

5、Lin-Fa Wang(王林發),

 Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore

新加坡杜克國大醫學院新發傳染病專業

6、Jie Cui(崔傑 , Zheng-Li Shi(石正麗) 

CAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases of Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

中國科學院武漢病毒研究所新發傳染病研究中心特殊病原體和生物安全性CAS重點實驗室,武漢

論文發佈日期:Published: November 30, 2017(2017年11月30日)

總而言之,參與Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus(蝙蝠SARS相關冠狀病毒豐富基因庫的發現為SARS冠狀病毒的起源提供了新見解)這篇造假論文編纂單位有:武漢病毒研究所Ben Hu , Lei-Ping Zeng,Xing-Lou Yang(楊興婁) ,Xing-Yi Ge(葛行義), Wei Zhang(張偉), Bei Li, Jia-Zheng Xie, Xu-Rui Shen, Ning Wang, Dong-Sheng Luo, Xiao-Shuang Zheng, Mei-Niang Wang, Jie Cui(崔傑) , Zheng-Li Shi(石正麗) ,大理大學張雲智,美國Peter Daszak, EcoHealth Alliance兩位學者,新加坡杜克國立大學王林發。

在這篇論文中是如何論述怎麼讓病毒更好感染人體細胞的呢?請注意引用論文及譯文中的黑體字部分:

[Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.

細胞進入研究表明,三個新鑒定出的具有不同S蛋白序列的SARSr-CoV都能夠使用人ACE2作為受體,進一步顯示了該洞穴中的菌株與SARS-CoV之間的密切關係。這項工作為SARS冠狀病毒的起源和演變提供了新的見解,並強調了為SARS樣疾病的未來出現做好準備的必要性。]

we found bat SARSr-CoV strains with different S proteins that can all use the receptor of SARS-CoV in humans (ACE2) for cell entry, suggesting diverse SARSr-CoVs capable of direct transmission to humans are circulating in bats in this cave. Our current study therefore offers a clearer picture on the evolutionary origin of SARS-CoV and highlights the risk of future emergence of SARS-like diseases.

我們發現具有不同S蛋白的蝙蝠SARSr-CoV菌株都可以使用人類SARS-CoV受體(ACE2)進入細胞,這表明能夠直接傳播給人類的各種SARSr-CoV都在蝙蝠中流通。洞穴。因此,我們當前的研究為SARS-CoV的進化起源提供了更清晰的圖景,並突出了SARS樣疾病未來出現的風險。]

Recently we have reported four novel SARSr-CoVs from Chinese horseshoe bats that shared much higher genomic sequence similarity to the epidemic strains, particularly in their S gene, of which two strains (termed WIV1 and WIV16) have been successfully cultured in vitro [17,18]. These newly identified SARSr-CoVs have been demonstrated to use the same cellular receptor (angiotensin converting enzyme-2 [ACE-2]) as SARS-CoV does and replicate efficiently in primary human airway cells [17–19].

最近,我們已經報告了來自中國馬蹄蝙蝠的四種新型SARSr-CoV,它們與該流行病菌株具有更高的基因組序列相似性,特別是在它們的S基因中,其中兩種菌株(稱為WIV1WIV16)已在體外成功培養[17 18] 這些新發現的SARSr-CoV已被證明使用與SARS-CoV相同的細胞受體(血管緊張素轉換酶2 [ACE-2]),並且可以在原代人氣道細胞中有效複製[17-19]。]

[Here we report the latest results of our 5-year longitudinal surveillance of bat SARSr-CoVs in this single location and systematic evolutionary analysis using full-length genome sequences of 15 SARSr-CoV strains (11 novel ones and 4 from previous studies). Efficiency of human ACE2 usage and the functions of accessory genes ORF8 and 8a were also evaluated for some of the newly identified strains.

在這裡,我們報告了我們對蝙蝠SARSr-CoV病毒進行的5年縱向監視的最新結果,該監測結果是在15個SARSr-CoV菌株(11個新菌株和4個來自先前研究的菌株)的全長基因組序列中進行的,是一個單一位置的系統進化分析。 還對一些新鑒定的菌株評估了人類ACE2使用效率以及輔助基因ORF88a的功能。]

注:請重點看下面這一段中英文,我看到這一段我笑了,您呢?因為這裡邊煞有介事地把他們發現的蝙蝠病毒與果子狸病毒進行比較,然後再得出“科學結果”,OMG,原來殺人女巫就是這樣揚名立萬的!

[Based on the diversity of RBD sequences, 11 novel SARSr-CoV strains named by abbreviation of bat species and sample ID (Rs4081, Rs4084, Rs4231, Rs4237, Rs4247, Rs4255, Rs4874, Rs7327, Rs9401, Rf4092 and As6526) were selected for full-length genomic sequencing based on sample abundance, genotype of RBD as well as sampling time. For each RBD genotype and each time of sampling, at least one representative strain was selected. The genome size of these novel SARSr-CoVs ranged from 29694 to 30291 nucleotides (nt). This gave a total of 15 full-length genomes of bat SARSr-CoVs from this single location (13 from R.sinicus, and one each from R. ferrumequinum and A. stoliczkanus), including our previously reported strains, Rs3367, RsSHC014, WIV1 and WIV16 [17,18]. The genomes of all 15 SARSr-CoVs circulating in this single cave shared 92.0% to 99.9% nt sequence identity. The overall nt sequence identity between these SARSr-CoVs and human and civet SARS-CoVs is 93.2% to 96%, significantly higher than that observed for bat SARSr-CoVs reported from other locations in China (88–93%) [9,10,12,14,21,22].

根據RBD序列的多樣性,選擇了11種新的SARSr-CoV菌株,它們以蝙蝠物種的縮寫和樣品ID命名(Rs4081,Rs4084,Rs4231,Rs4237,Rs4247,Rs4255,Rs4874,Rs7327,Rs9401,Rf4092和As6526)樣本的豐度,RBD的基因型以及採樣時間進行全長基因組測序。對於每種RBD基因型和每次採樣,至少選擇一種代表性菌株。這些新穎的SARSr-CoV的基因組大小範圍為29694至30291個核苷酸(nt)。這從該位置總共獲得了蝙蝠SARSr-CoV的15個全長基因組(13個來自R.sinicus,每個來自R. ferrumequinum和A. stoliczkanus),包括我們先前報導的菌株Rs3367,RsSHC014,WIV1和WIV16 [17,18]。在該單個洞穴中迴圈的所有15個SARSr-CoV的基因組都具有92.0%至99.9%的nt序列同一性。這些SARSr-CoV與人類和果子狸SARS-CoV之間的總體nt序列同一性為93.2%至96%,顯著高於從中國其他地區報導的蝙蝠SARSr-CoV88-93%)[9,10 ,, 12,14,21,22]通過Simplot分析檢查了15個SARSr-CoV和SARS-CoV SZ3菌株之間的基因組序列相似性(圖1)。這15個SARSr-CoVs是高度保守的,並且在非結構基因ORF1a(96.6%至97.1%nt序列同一性,98.0%至98.3%aa序列同一性)和ORF1b(96.1%)中與SARS-CoV具有一致高的序列相似性核苷酸序列同一性為96.6%,氨基酸序列同一性為99.0%至99.4%)。相反,在S基因(對應于SZ3基因組位置21477至25244)和ORF8(對應于SZ3基因組位置27764至28132)中顯示出相當大的遺傳多樣性(圖1)。]

注:對於下邊這一段我也是非常好奇,殺人女巫聲稱發現的這些[2003年從蚊子中鑒定出SARS-CoV SZ3。鑒定出SARSr-CoV Rs 672和YN2013 分別來自貴州和雲南省的中華絨螯蟹。 SARSr-CoV Rf1和JL2012分別從湖北和吉林省的鐵銹紅黴菌中鑒定。],對應英文[SARSr-CoV Rs 672 and YN2013 were identified from R. sinicus collected in Guizhou and Yunnan Province, respectively. SARSr-CoV Rf1 and JL2012 were identified from R. ferrumequinum collected in Hubei and Jilin Province, respectively.],這些病毒到底是殺人女巫石正麗在自然中發現的?還是在實驗製造完以後又按同樣的套路“從自然發現的”?

[The key aa residues involved in the interaction with human ACE2 are numbered on top of the aligned sequences. SARS-CoV GZ02, BJ01 and Tor2 were isolated from patients in the early, middle and late phase, respectively, of the SARS outbreak in 2003. SARS-CoV SZ3 was identified from civets in 2003. SARSr-CoV Rs 672 and YN2013 were identified from R. sinicus collected in Guizhou and Yunnan Province, respectively. SARSr-CoV Rf1 and JL2012 were identified from R. ferrumequinum collected in Hubei and Jilin Province, respectively. WIV1, WIV16, RsSHC014, Rs4081, Rs4084, Rs4231, Rs4237, Rs4247, Rs7327 and Rs4874 were identified from R.sinicus, and Rf4092 from R. ferrumequinum in the cave surveyed in this study.

與人ACE2相互作用涉及的關鍵氨基酸殘基在比對序列的頂部編號。 從2003年SARS爆發的早期,中期和晚期分別從患者中分離出SARS-CoV GZ02,BJ01和Tor2。2003年從蚊子中鑒定出SARS-CoV SZ3。鑒定出SARSr-CoV Rs 672和YN2013 分別來自貴州和雲南省的中華絨螯蟹。 SARSr-CoV Rf1和JL2012分別從湖北和吉林省的鐵銹紅黴菌中鑒定。 在本研究調查的洞穴中,從R.sinicus和Rf4092中鑒定出了WIV1,WIV16,RsSHC014,Rs4081,Rs4084,Rs4231,Rs4237,Rs4247,Rs7327和Rs4874,以及Rf4092。]

注:作為非專業人士,對於下邊的這段文字我還是笑了,因為殺人女巫用科學分析在繼續編造從蝙蝠到果子狸再到人類的病毒進化樹。

[Regardless of different host bat species, SARS-CoV and SARSr-CoVs detected in bats from southwestern China (Yunnan, Guizhou and Guangxi province) formed one clade, in which SARSr-CoV strains showing closer relationship to SARS-CoV were all from Yunnan. SARSr-CoVs detected in southeastern, central and northern provinces, such as Hong Kong, Hubei and Shaanxi, formed the other clade which was phylogenetically distant to human and civet SARS-CoVs (Fig 6 and S6 Fig).

無論寄主蝙蝠種類如何,在中國西南(雲南,貴州和廣西)蝙蝠中檢測到的SARS-CoV和SARSr-CoV均形成一個進化枝,其中與SARS-CoV密切相關的SARSr-CoV菌株均來自雲南。在東南,中部和北部省份(例如香港,湖北和陝西)檢測到的SARSr-CoV形成了另一個進化枝,與人類和靈貓SARS-CoV的親緣關係較遠(圖6和S6圖)。]

注:下面這一段又一次說明了殺人女巫石正麗用ACE2做功能增強性實驗,這一次我能記下來英文的說法了,叫做gain of function!

All viruses replicated efficiently in the human ACE2-expressing cells. The results were further confirmed by quantification of viral RNA using real-time RT-PCR (Fig 8).

為了評估這三種新型SARSr-CoV是否可以將人ACE2用作細胞進入受體,我們使用有或沒有人ACE2表達的HeLa細胞進行了病毒感染性研究。所有病毒均在表達人ACE2的細胞中有效複製。通過使用即時RT-PCR定量病毒RNA進一步證實了結果(圖8)。]

接下來,又是gain of function——功能增強實驗:

[Analysis of receptor usage by immunofluorescence assay (A) and real-time PCR (B). Virus infectivity of Rs4874, WIV1-Rs4231S and WIV1-Rs7327S was determined in HeLa cells with and without the expression of human ACE2. ACE2 expression was detected with goat anti-human ACE2 antibody followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG. Virus replication was detected with rabbit antibody against the SARSr-CoV Rp3 nucleocapsid protein followed by cyanine 3 (Cy3)-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI (49,6-diamidino-2-phenylindole).The columns (from left to right) show staining of nuclei (blue), ACE2 expression (green), virus replication (red) and the merged triple-stained images, respectively.

通過免疫螢光分析(A)和即時PCR(B)分析受體使用情況。 在有或沒有人ACE2表達的HeLa細胞中測定Rs4874,WIV1-Rs4231S和WIV1-Rs7327S的病毒感染性。 先用山羊抗人ACE2抗體檢測ACE2表達,再用異硫氰酸螢光素(FITC)偶聯的驢抗山羊IgG檢測。 用抗SARSr-CoV Rp3核衣殼蛋白的兔抗體檢測病毒複製,然後用花青3(Cy3)偶聯的小鼠抗兔IgG檢測。 細胞核用DAPI(49,6-diamidino-2-phenylindole)染色。柱子(從左到右)顯示細胞核染色(藍色),ACE2表達(綠色),病毒複製(紅色)和合併的三重染色 圖片。]

繼續進行功能增強實驗——

[The induction of the ATF6-dependent transcription by the ORF8s of SARS-CoV and bat SARSr-CoVs were investigated using a luciferase reporter, 5×ATF6-GL3. In HeLa cells transiently transfected with the expression plasmids of the ORF8s of bat SARSr-CoV Rf1, Rf4092 and WIV1, the relative luciferase activities of the 5×ATF6-GL3 reporter was enhanced by 5.56 to 9.26 folds compared with cells transfected with the pCAGGS empty vector, while it was increased by 4.42 fold by the SARS-CoV GZ02 ORF8. As a control, the treatment with tunicamyxin (TM) stimulated the transcription by about 11 folds (Fig 9A). The results suggests that various ORF8 proteins of bat SARSr-CoVs can activate ATF6, and those of some strains have a stronger effect than the SARS-CoV ORF8.

使用螢光素酶報導分子5×ATF6-GL3研究了SARS-CoV和蝙蝠SARSr-CoV的ORF8對ATF6依賴性轉錄的誘導。 在暫態轉染了蝙蝠SARSr-CoV Rf1,Rf4092和WIV1的ORF8表達質粒的HeLa細胞中,與空轉染pCAGGS的細胞相比,5×ATF6-GL3報告基因的相對螢光素酶活性提高了5.56至9.26倍 載體,而SARS-CoV GZ02 ORF8將其提高了4.42倍。 作為對照,衣黴素(TM)的處理將轉錄刺激了約11倍(圖9A)。 結果表明,蝙蝠SARSr-CoVs的各種ORF8蛋白都可以啟動ATF6,而某些菌株的蛋白比SARS-CoV ORF8的效果更強。]

繼續功能性增強實驗——

[We conducted transient transfection to examine whether the ORF8a of SARSr-CoV Rs4084 triggered apoptosis. As shown in Fig 9B, 11.76% and 9.40% of the 293T cells transfected with the SARSr-CoV Rs4084-ORF8a and SARS-CoV Tor2-ORF8a expression plasmid underwent apoptosis, respectively. In contrast, transfection with the empty vector resulted in apoptosis in only 2.79% of the cells. The results indicate that Rs4084 ORF8a has an apoptosis induction activity similar to that of SARS-CoV [28].

我們進行了暫態轉染,以檢查SARSr-CoV Rs4084的ORF8a是否觸發了細胞凋亡。 如圖9B中所示,分別用SARSr-CoV Rs4084-ORF8a和SARS-CoV Tor2-ORF8a表達質粒轉染的293T細胞的11.76%和9.40%經歷了細胞凋亡。 相反,用空載體轉染僅導致2.79%的細胞凋亡。 結果表明,Rs4084 ORF8a具有類似於SARS-CoV的凋亡誘導活性[28]。]

接下來繼續胡扯子虛烏有的果子狸——

[The S proteins of RsSHC014, Rs3367, WIV1 and WIV16, which were reported in our previous studies, shared 90% to 97% aa sequence identities to those of human/civet SARS-CoVs [17,18]. Another strain from Rhinolophus affinis in Yunnan termed LYRa11 showed 90% aa sequence identity to SARS-CoV in the S gene [13]. In addition, two studies have described 4 novel SARSr-CoVs (YNLF_31C/34C and GX2013/YN2013) which possessed a full-length ORF8 with substantially higher similarity to that of SARS-CoV [22,30]. These findings provide strong genetic evidence for the bat origin of SARS-CoV with regard to the S gene or ORF8.

我們先前的研究中報導了RsSHC014,Rs3367,WIV1和WIV16的S蛋白與人/果子狸SARS-CoV共有90%至97%的氨基酸序列同一性[17,18]。來自雲南犀牛的另一株名為LYRa11的菌株在S基因中顯示出與SARS-CoV具有90%的氨基酸序列同一性[13]。此外,兩項研究描述了4種新穎的SARSr-CoV(YNLF_31C / 34C和GX2013 / YN2013),它們的全長ORF8與SARS-CoV的同源性高得多[22,30]。這些發現為SARS冠狀病毒蝙蝠起源于S基因或ORF8提供了強有力的遺傳證據。]

接下來,接著再扯果子狸——

[In this cave, we have now obtained full-length genome sequences of additional 11 novel SARSr-CoVs from bats. Our findings suggest the co-circulation of different bat SARSr-CoVs highly similar to SARS-CoV in the most variable S1 (NTD and RBD), ORF8 and ORF3 regions, respectively, in this single location. In the ORF1a, ORF1b, E, M and N genes, the SARSr-CoVs circulating in this cave also shared > 98% aa sequence identities with human/civet SARS-CoVs.

在這個洞穴中,我們現在從蝙蝠中獲得了另外11種新穎的SARSr-CoV的全長基因組序列。 我們的研究結果表明,與蝙蝠SARS-CoV不同的蝙蝠在這個單一位置的可變性最高的S1(NTD和RBD),ORF8和ORF3區域分別與SARS-CoV高度相似。 在ORF1a,ORF1b,E,M和N基因中,在該洞穴中迴圈傳播的SARSr-CoVs與人/果子狸SARS-CoVs也具有> 98%的氨基酸序列同一性。]

接下來,繼續功能增強實驗——

[Our previous studies demonstrated the capacity of both WIV1 and WIV16 to use ACE2 orthologs for cell entry and to efficiently replicate in human cells [17,18]. In this study, we confirmed the use of human ACE2 as receptor of two novel SARSr-CoVs by using chimeric viruses with the WIV1 backbone replaced with the S gene of the newly identified SARSr-CoVs. Rs7327’s S protein varied from that of WIV1 and WIV16 at three aa residues in the receptor-binding motif, including one contact residue (aa 484) with human ACE2. This difference did not seem to affect its entry and replication efficiency in human ACE2-expressing cells. A previous study using the SARS-CoV infectious clone showed that the RsSHC014 S protein could efficiently utilize human ACE2 [33], despite being distinct from SARS-CoV and WIV1 in the RBD (S1 Fig). We examined the infectivity of Rs4231, which shared similar RBD sequence with RsSHC014 but had a distinct NTD sequence, and found the chimeric virus WIV1-Rs4231S also readily replicated in HeLa cells expressing human ACE2 molecule. The novel live SARSr-CoV we isolated in the current study (Rs4874) has an S gene almost identical to that of WIV16. As expected, it is also capable of utilizing human ACE2. These results indicate that diverse variants of SARSr-CoV S protein without deletions in their RBD are able to use human ACE2. In contrast, our previous study revealed that the S protein of a R. sinicus SARSr-CoV with deletions (Rp3) failed to use human, civet and bat ACE2 for cell entry [34]. In this study, in addition to Rs4231 and Rs7327, we also constructed infectious clones with the S gene of Rs4081, Rf4075, Rs4085, Rs4235 and As6526, which all contained the deletions in their RBD. These 7 strains, plus Rs4874 and the previously studied WIV1 and RsSHC014, could represent all types of S variants of SARSr-CoVs in this location (S3A Fig). However, none of the strains with deletions in the RBD could be rescued from Vero E6 cells. Therefore, the two distinct clades of SARSr-CoV S gene may represent the usage of different receptors in their bat hosts。

我們以前的研究表明WIV1和WIV16都具有使用ACE2直系同源物進入細胞並在人類細胞中有效複製的能力[17,18]。在這項研究中,我們通過使用嵌合病毒將WACE1骨架替換為新鑒定的SARSr-CoV的S基因,證實了人ACE2作為兩種新型SARSr-CoV的受體的用途。 Rs7327的S蛋白在受體結合基序的三個氨基酸殘基上不同於WIV1和WIV16,包括一個與人ACE2的接觸殘基(aa 484)。這種差異似乎並未影響其在表達人ACE2的細胞中的進入和複製效率。先前使用SARS-CoV感染性克隆的研究表明,RsSHC014 S蛋白可以有效利用人ACE2 [33],儘管它與RBD中的SARS-CoV和WIV1不同(S1圖)。我們檢查了Rs4231的感染性,該Rs4231與RsSHC014共用相似的RBD序列,但具有獨特的NTD序列,發現嵌合病毒WIV1-Rs4231S也很容易在表達人ACE2分子的HeLa細胞中複製。我們在當前研究中分離出的新穎的即時SARSr-CoV(Rs4874)具有一個與WIV16幾乎相同的S基因。不出所料,它也能夠利用人類ACE2。這些結果表明SARSr-CoV S蛋白的多種變體在其RBD中沒有缺失能夠使用人ACE2。相比之下,我們先前的研究表明帶有缺失(Rp3)的中華R. SARSr-CoV的S蛋白無法使用人,果子狸和蝙蝠ACE2進入細胞[34]。在這項研究中,除了Rs4231和Rs7327外,我們還構建了具有Rs4081,Rf4075,Rs4085,Rs4235和As6526的S基因的感染性克隆,它們均在其RBD中包含缺失。這7個菌株,加上Rs4874和先前研究的WIV1和RsSHC014,可以代表此位置中所有類型的SARSr-CoV的S變異(S3A圖)。但是,RBD中缺失的菌株均無法從Vero E6細胞中拯救出來。因此,SARSr-CoV S基因的兩個不同進化枝可能代表蝙蝠宿主中不同受體的使用。]

繼續進行功能性增強實驗——

[The full-length ORF8 protein of SARS-CoV is a luminal endoplasmic reticulum (ER) membrane-associated protein that induces the activation of ATF6, an ER stress-regulated transcription factor that activates the transcription of ER chaperones involved in protein folding [35]. We amplified the ORF8 genes of Rf1, Rf4092 and WIV1, which represent three different genotypes of bat SARSr-CoV ORF8 (S3C Fig), and constructed the expression plasmids. All of the three ORF8 proteins transiently expressed in HeLa cells can stimulate the ATF6-dependent transcription. Among them, the WIV1 ORF8, which is highly divergent from the SARS-CoV ORF8, exhibited the strongest activation. The results indicate that the variants of bat SARSr-CoV ORF8 proteins may play a role in modulating ER stress by activating the ATF6 pathway. In addition, the ORF8a protein of SARS-CoV from the later phase has been demonstrated to induce apoptosis [28]. In this study, we have found that the ORF8a protein of the newly identified SARSr-CoV Rs4084, which contained an 8-aa insertion compared with the SARS-CoV ORF8a, significantly triggered apoptosis in 293T cells as well.

SARS-CoV的全長ORF8蛋白是一種與腔內質網(ER)膜相關的蛋白,可誘導ATF6的啟動,ATF6是一種ER應激調節的轉錄因數,可啟動參與蛋白折疊的ER伴侶的轉錄[35]。 。我們擴增了代表蝙蝠SARSr-CoV ORF8三種不同基因型的Rf1,Rf4092和WIV1的ORF8基因,並構建了表達質粒。在HeLa細胞中暫態表達的所有三個ORF8蛋白都可以刺激ATF6依賴性轉錄。其中,與SARS-CoV ORF8高度不同的WIV1 ORF8表現出最強的啟動作用。結果表明,蝙蝠SARSr-CoV ORF8蛋白的變異體可能通過啟動ATF6途徑在調節內質網應激中發揮作用。此外,已證明來自後期的SARS-CoV的ORF8a蛋白可誘導細胞凋亡[28]。在這項研究中,我們發現新鑒定出的SARSr-CoV Rs4084的ORF8a蛋白與SARS-CoV ORF8a相比,包含8-aa插入,也顯著觸發了293T細胞的凋亡。]

這一段煞有介事地提出“在該區域的蝙蝠中,各種仍能使用人ACE2的SARSr-CoV仍在流通。因此,有可能溢出到人體內並出現類似SARS的疾病。”,時至今日雲南蝙蝠洞周邊依然安好,殺人女巫恐怕是跑不掉了!

[The data show that frequent recombination events have happened among those SARSr-CoVs in the same cave. While we cannot rule out the possibility that similar gene pools of SARSr-CoVs exist elsewhere, we have provided sufficient evidence to conclude that SARS-CoV most likely originated from horseshoe bats via recombination events among existing SARSr-CoVs. In addition, we have also revealed that various SARSr-CoVs capable of using human ACE2 are still circulating among bats in this region. Thus, the risk of spillover into people and emergence of a disease similar to SARS is possible. This is particularly important given that the nearest village to the bat cave we surveyed is only 1.1 km away, which indicates a potential risk of exposure to bats for the local residents. Thus, we propose that monitoring of SARSr-CoV evolution at this and other sites should continue, as well as examination of human behavioral risk for infection and serological surveys of people, to determine if spillover is already occurring at these sites and to design intervention strategies to avoid future disease emergence.

資料表明,在同一洞穴中的SARSr-CoV之間發生了頻繁的重組事件。雖然我們不能排除在其他地方存在類似的SARSr-CoV基因庫的可能性,但我們提供了足夠的證據來推斷SARS-CoV最有可能是通過現有SARSr-CoV之間的重組事件起源於馬蹄蝠。此外,我們還發現,在該區域的蝙蝠中,各種仍能使用人ACE2的SARSr-CoV仍在流通。因此,有可能溢出到人體內並出現類似SARS的疾病。考慮到距離我們所調查的蝙蝠洞最近的村莊僅1.1公里,這尤其重要,這表明本地居民可能接觸蝙蝠。因此,我們建議應繼續監測該網站和其他網站的SARSr-CoV演變,以及檢查人類感染行為的風險和對人群進行血清學調查,以確定這些網站是否已經發生溢出並設計干預策略避免將來出現疾病。]

對於病毒來自實驗室,殺人女巫石正麗是怎麼回應的呢?——

石正麗回應病毒來源自實驗室言論:是謠言 他的研究方法不對!來源:網易視頻 發佈時間:2020-02-08)

綜述:作為生物知識的小白鼠,我們沒有生物方面的專業素養,但是我想我看懂了閆麗夢博士的報告,那就是——所有關於SAR-COV2來源於自然的科學論斷全是假的!那些與中共沆瀣一氣的科學家還是裝作不懂,其實很簡單對所有中共宣佈在某地發現的病毒再開啟一遍探尋之旅,看看能不能再找到同樣或類似的病毒!對於中共保存的來源於果子狸的類SARS病毒,只要用它來和果子狸做一下親和實驗,看看這個病毒能不能和果子狸的ACE2結合,我想只要一做這個實驗,就能證明管軼聲稱的從果子狸身上發現的類SARS病毒就會像SARS-COV2一樣告訴你——我不喜歡果子狸,我只喜歡人類!我只是借宿一下果子狸,我要過渡到人類!為我辦理到果子狸身上辦理借宿手續的就是中共這幫雜種!為我辦理CHECK-OUT果子裡的也是中共這幫雜種!

(文章內容僅代表作者個人觀點)

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