初探闫丽梦博士第二篇论文参考文献25中石正丽功能增强实验内容

作者:Maarago

天使科学家闫丽梦博士在《SARS-CoV-2IsanUnrestrictedBioweapon:ATruthRevealedthroughUncoveringaLarge-Scale,OrganizedScientificFraud(SARS-CoV-2是一种超限生物武器:揭露大规模、有组织的科学骗局的真相)》一文中提到“

石正丽课题组常规性地研究该病毒是否能够感染人体细胞。这种研究活动的模式已经屡见不鲜25,32,36-39。“也就是说石正丽的这几篇论文就是研究如何可以让病毒更好感染人体细胞,那么我们来看一下这几篇论文第25篇论文的情况。

参考文献第25:Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus(蝙蝠SARS相关冠状病毒丰富基因库的发现为SARS冠状病毒的起源提供了新见解)

关于杀人女巫石正丽的这篇论文财新是这样描述的——据财新网2020年01月20日 12:13 发布的对抗新型冠状病毒 能从抗击非典中汲取什么经验?

[2013年,她带领的团队在《自然》杂志发表一项研究,在云南的一个蝙蝠栖息洞中,科学家在菊头蝠的粪便中分离到一株类似于SARS病毒的活病毒,这种新分离的病毒与已知的SARS病毒具有高度同源性,可以利用ACE2蛋白做为受体感染人的细胞[11]。这项研究清晰地揭示了SARS病毒的来源,石正丽教授的团队在这个地区的蝙蝠种群中检测到了组装SARS病毒所需要的所有基因[17]。他们推断,SARS病毒很大可能是由感染蝙蝠的各种“类SARS病毒”重组而来,在偶然的情况下,果子狸感染了这种病毒,病毒在果子狸体内进行了复制与进化,通过被人捕猎的方式最终把病毒传播给了人类[18]。]

在财新这一段文字中列明的参考文献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).

参考文献18是[18] 张渺. “蝙蝠女侠”团队找出SARS病毒源头. 中国青年报 (2017).

以及财新网2020年02月06日 20:03发布的从复杂网络小世界、无标度、高聚类特性看新型冠状病毒肺炎

[回顾2003年造成全球700余人死亡的SARS传播[23,24]事件,有研究发现SARS病毒的源头可能是蝙蝠[25],而不是以前人们认为的果子狸[26,27],果子狸属于中间宿主,也是SARS病毒的受害者。目前广为人们接受的关于SARS的传播途径的观点是:蝙蝠——果子狸——人群。2019-nCoV至今仍没有确定中间宿主是什么野生动物,虽然已有关于2019-nCoV的很多研究[28,29,30]。更糟糕的是2019-nCoV传播具有潜伏期,大部分潜伏期1-14天不等,平均7天,潜伏期长增加了疫情控制的难度,使得一些潜在的超级传播者不能被第一时间发现并隔离。另外,一旦发现感染者,需要向前追溯大量的潜在感染者,增加了工作的难度和复杂度。],这段文字中的参考文献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).这篇论文,根据财新网的两篇文章的引述,如何看待杀人女巫石正丽的这篇论文呢?从某种意义上说石正丽的这篇伪论文为中共的生化武器罩上了一层迷彩服,在这篇论文的遮盖下,生化武器的自然宿主隆重登场,如果没有闫丽梦博士的火眼金睛和一颗天使的心,这套生化武器的迷彩服也许将永远不会被揭穿,现在每当我们面对数以百万计的人类感染和数以十万计的人类死亡的时候,我们如果不能铭记这些与魔鬼共舞的杀人魔头,我们何以对得起那些正在感染和正在死亡的我们的同类?

这篇论文的作者是(注:作者顺序按原论文排序,凡是属于同一个机构的并列排出):

1、Ben Hu , Lei-Ping Zeng,Xing-Lou Yang(杨兴娄) ,Xing-Yi Ge(葛行义), Wei Zhang(张伟), Bei Li, Jia-Zheng Xie, Xu-Rui Shen,

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

中国科学院武汉病毒研究所新发传染病研究中心特殊病原体和生物安全性CAS重点实验室,武汉

2、Yun-Zhi Zhang(张云智),

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

大理大学云南地方病防治所,大理,中国

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

中国科学院武汉病毒研究所新发传染病研究中心特殊病原体和生物安全性CAS重点实验室,武汉

4、Peter Daszak, EcoHealth Alliance,

New York, New York, United States of America 纽约,纽约,美利坚合众国

5、Lin-Fa Wang(王林发),

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

新加坡杜克国大医学院新发传染病专业

6、Jie Cui(崔杰) , Zheng-Li Shi(石正丽) 

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

中国科学院武汉病毒研究所新发传染病研究中心特殊病原体和生物安全性CAS重点实验室,武汉

论文发布日期:Published: November 30, 2017(2017年11月30日)

总而言之,参与Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus(蝙蝠SARS相关冠状病毒丰富基因库的发现为SARS冠状病毒的起源提供了新见解)这篇造假论文编纂单位有:武汉病毒研究所Ben Hu , Lei-Ping Zeng,Xing-Lou Yang(杨兴娄) ,Xing-Yi Ge(葛行义), Wei Zhang(张伟), Bei Li, Jia-Zheng Xie, Xu-Rui Shen, Ning Wang, Dong-Sheng Luo, Xiao-Shuang Zheng, Mei-Niang Wang, Jie Cui(崔杰) , Zheng-Li Shi(石正丽) ,大理大学张云智,美国Peter Daszak, EcoHealth Alliance两位学者,新加坡杜克国立大学王林发。

在这篇论文中是如何论述怎么让病毒更好感染人体细胞的呢?请注意引用论文及译文中的黑体字部分:

[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.

细胞进入研究表明,三个新鉴定出的具有不同S蛋白序列的SARSr-CoV都能够使用人ACE2作为受体,进一步显示了该洞穴中的菌株与SARS-CoV之间的密切关系。这项工作为SARS冠状病毒的起源和演变提供了新的见解,并强调了为SARS样疾病的未来出现做好准备的必要性。]

[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.

我们发现具有不同S蛋白的蝙蝠SARSr-CoV菌株都可以使用人类SARS-CoV受体(ACE2)进入细胞,这表明能够直接传播给人类的各种SARSr-CoV都在蝙蝠中流通。洞穴。因此,我们当前的研究为SARS-CoV的进化起源提供了更清晰的图景,并突出了SARS样疾病未来出现的风险。]

[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,我们已经报告了来自中国马蹄蝙蝠的四种新型SARSr-CoV,它们与该流行病菌株具有更高的基因组序列相似性,特别是在它们的S基因中,其中两种菌株(称为WIV1和WIV16)已在体外成功培养[17, 18]。 这些新发现的SARSr-CoV已被证明使用与SARS-CoV相同的细胞受体(血管紧张素转换酶2 [ACE-2]),并且可以在原代人气道细胞中有效复制[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.

在这里,我们报告了我们对蝙蝠SARSr-CoV病毒进行的5年纵向监视的最新结果,该监测结果是在15个SARSr-CoV菌株(11个新菌株和4个来自先前研究的菌株)的全长基因组序列中进行的,是一个单一位置的系统进化分析。 还对一些新鉴定的菌株评估了人类ACE2使用效率以及辅助基因ORF8和8a的功能。]

注:请重点看下面这一段中英文,我看到这一段我笑了,您呢?因为这里边煞有介事地把他们发现的蝙蝠病毒与果子狸病毒进行比较,然后再得出“科学结果”,OMG,原来杀人女巫就是这样扬名立万的!

[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].

根据RBD序列的多样性,选择了11种新的SARSr-CoV菌株,它们以蝙蝠物种的缩写和样品ID命名(Rs4081,Rs4084,Rs4231,Rs4237,Rs4247,Rs4255,Rs4874,Rs7327,Rs9401,Rf4092和As6526)样本的丰度,RBD的基因型以及采样时间进行全长基因组测序。对于每种RBD基因型和每次采样,至少选择一种代表性菌株。这些新颖的SARSr-CoV的基因组大小范围为29694至30291个核苷酸(nt)。这从该位置总共获得了蝙蝠SARSr-CoV的15个全长基因组(13个来自R.sinicus,每个来自R. ferrumequinum和A. stoliczkanus),包括我们先前报道的菌株Rs3367,RsSHC014,WIV1和WIV16 [17,18]。在该单个洞穴中循环的所有15个SARSr-CoV的基因组都具有92.0%至99.9%的nt序列同一性。这些SARSr-CoV与人类和果子狸SARS-CoV之间的总体nt序列同一性为93.2%至96%,显着高于从中国其他地区报道的蝙蝠SARSr-CoV(88-93%)[9,10 ,, 12,14,21,22]。通过Simplot分析检查了15个SARSr-CoV和SARS-CoV SZ3菌株之间的基因组序列相似性(图1)。这15个SARSr-CoVs是高度保守的,并且在非结构基因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%)中与SARS-CoV具有一致高的序列相似性核苷酸序列同一性为96.6%,氨基酸序列同一性为99.0%至99.4%)。相反,在S基因(对应于SZ3基因组位置21477至25244)和ORF8(对应于SZ3基因组位置27764至28132)中显示出相当大的遗传多样性(图1)。]

注:对于下边这一段我也是非常好奇,杀人女巫声称发现的这些[2003年从蚊子中鉴定出SARS-CoV SZ3。鉴定出SARSr-CoV Rs 672和YN2013 分别来自贵州和云南省的中华绒螯蟹。 SARSr-CoV Rf1和JL2012分别从湖北和吉林省的铁锈红霉菌中鉴定。],对应英文[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.],这些病毒到底是杀人女巫石正丽在自然中发现的?还是在实验制造完以后又按同样的套路“从自然发现的”?

[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.

与人ACE2相互作用涉及的关键氨基酸残基在比对序列的顶部编号。 从2003年SARS爆发的早期,中期和晚期分别从患者中分离出SARS-CoV GZ02,BJ01和Tor2。2003年从蚊子中鉴定出SARS-CoV SZ3。鉴定出SARSr-CoV Rs 672和YN2013 分别来自贵州和云南省的中华绒螯蟹。 SARSr-CoV Rf1和JL2012分别从湖北和吉林省的铁锈红霉菌中鉴定。 在本研究调查的洞穴中,从R.sinicus和Rf4092中鉴定出了WIV1,WIV16,RsSHC014,Rs4081,Rs4084,Rs4231,Rs4237,Rs4247,Rs7327和Rs4874,以及Rf4092。]

注:作为非专业人士,对于下边的这段文字我还是笑了,因为杀人女巫用科学分析在继续编造从蝙蝠到果子狸再到人类的病毒进化树。

[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).

无论寄主蝙蝠种类如何,在中国西南(云南,贵州和广西)蝙蝠中检测到的SARS-CoV和SARSr-CoV均形成一个进化枝,其中与SARS-CoV密切相关的SARSr-CoV菌株均来自云南。在东南,中部和北部省份(例如香港,湖北和陕西)检测到的SARSr-CoV形成了另一个进化枝,与人类和灵猫SARS-CoV的亲缘关系较远(图6和S6图)。]

注:下面这一段又一次说明了杀人女巫石正丽用ACE2做功能增强性实验,这一次我能记下来英文的说法了,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).

为了评估这三种新型SARSr-CoV是否可以将人ACE2用作细胞进入受体,我们使用有或没有人ACE2表达的HeLa细胞进行了病毒感染性研究。所有病毒均在表达人ACE2的细胞中有效复制。通过使用实时RT-PCR定量病毒RNA进一步证实了结果(图8)。]

接下来,又是gain of function——功能增强实验:

[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.

通过免疫荧光分析(A)和实时PCR(B)分析受体使用情况。 在有或没有人ACE2表达的HeLa细胞中测定Rs4874,WIV1-Rs4231S和WIV1-Rs7327S的病毒感染性。 先用山羊抗人ACE2抗体检测ACE2表达,再用异硫氰酸荧光素(FITC)偶联的驴抗山羊IgG检测。 用抗SARSr-CoV Rp3核衣壳蛋白的兔抗体检测病毒复制,然后用花青3(Cy3)偶联的小鼠抗兔IgG检测。 细胞核用DAPI(49,6-diamidino-2-phenylindole)染色。柱子(从左到右)显示细胞核染色(蓝色),ACE2表达(绿色),病毒复制(红色)和合并的三重染色 图片。]

继续进行功能增强实验——

[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.

使用荧光素酶报道分子5×ATF6-GL3研究了SARS-CoV和蝙蝠SARSr-CoV的ORF8对ATF6依赖性转录的诱导。 在瞬时转染了蝙蝠SARSr-CoV Rf1,Rf4092和WIV1的ORF8表达质粒的HeLa细胞中,与空转染pCAGGS的细胞相比,5×ATF6-GL3报告基因的相对荧光素酶活性提高了5.56至9.26倍 载体,而SARS-CoV GZ02 ORF8将其提高了4.42倍。 作为对照,衣霉素(TM)的处理将转录刺激了约11倍(图9A)。 结果表明,蝙蝠SARSr-CoVs的各种ORF8蛋白都可以激活ATF6,而某些菌株的蛋白比SARS-CoV ORF8的效果更强。]

继续功能性增强实验——

[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].

我们进行了瞬时转染,以检查SARSr-CoV Rs4084的ORF8a是否触发了细胞凋亡。 如图9B中所示,分别用SARSr-CoV Rs4084-ORF8a和SARS-CoV Tor2-ORF8a表达质粒转染的293T细胞的11.76%和9.40%经历了细胞凋亡。 相反,用空载体转染仅导致2.79%的细胞凋亡。 结果表明,Rs4084 ORF8a具有类似于SARS-CoV的凋亡诱导活性[28]。]

接下来继续胡扯子虚乌有的果子狸——

[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.

我们先前的研究中报道了RsSHC014,Rs3367,WIV1和WIV16的S蛋白与人/果子狸SARS-CoV共有90%至97%的氨基酸序列同一性[17,18]。来自云南犀牛的另一株名为LYRa11的菌株在S基因中显示出与SARS-CoV具有90%的氨基酸序列同一性[13]。此外,两项研究描述了4种新颖的SARSr-CoV(YNLF_31C / 34C和GX2013 / YN2013),它们的全长ORF8与SARS-CoV的同源性高得多[22,30]。这些发现为SARS冠状病毒蝙蝠起源于S基因或ORF8提供了强有力的遗传证据。]

接下来,接着再扯果子狸——

[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.

在这个洞穴中,我们现在从蝙蝠中获得了另外11种新颖的SARSr-CoV的全长基因组序列。 我们的研究结果表明,与蝙蝠SARS-CoV不同的蝙蝠在这个单一位置的可变性最高的S1(NTD和RBD),ORF8和ORF3区域分别与SARS-CoV高度相似。 在ORF1a,ORF1b,E,M和N基因中,在该洞穴中循环传播的SARSr-CoVs与人/果子狸SARS-CoVs也具有> 98%的氨基酸序列同一性。]

接下来,继续功能增强实验——

[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。

我们以前的研究表明WIV1和WIV16都具有使用ACE2直系同源物进入细胞并在人类细胞中有效复制的能力[17,18]。在这项研究中,我们通过使用嵌合病毒将WACE1骨架替换为新鉴定的SARSr-CoV的S基因,证实了人ACE2作为两种新型SARSr-CoV的受体的用途。 Rs7327的S蛋白在受体结合基序的三个氨基酸残基上不同于WIV1和WIV16,包括一个与人ACE2的接触残基(aa 484)。这种差异似乎并未影响其在表达人ACE2的细胞中的进入和复制效率。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],尽管它与RBD中的SARS-CoV和WIV1不同(S1图)。我们检查了Rs4231的感染性,该Rs4231与RsSHC014共享相似的RBD序列,但具有独特的NTD序列,发现嵌合病毒WIV1-Rs4231S也很容易在表达人ACE2分子的HeLa细胞中复制。我们在当前研究中分离出的新颖的实时SARSr-CoV(Rs4874)具有一个与WIV16几乎相同的S基因。不出所料,它也能够利用人类ACE2。这些结果表明SARSr-CoV S蛋白的多种变体在其RBD中没有缺失能够使用人ACE2。相比之下,我们先前的研究表明带有缺失(Rp3)的中华R. SARSr-CoV的S蛋白无法使用人,果子狸和蝙蝠ACE2进入细胞[34]。在这项研究中,除了Rs4231和Rs7327外,我们还构建了具有Rs4081,Rf4075,Rs4085,Rs4235和As6526的S基因的感染性克隆,它们均在其RBD中包含缺失。这7个菌株,加上Rs4874和先前研究的WIV1和RsSHC014,可以代表此位置中所有类型的SARSr-CoV的S变异(S3A图)。但是,RBD中缺失的菌株均无法从Vero E6细胞中拯救出来。因此,SARSr-CoV S基因的两个不同进化枝可能代表蝙蝠宿主中不同受体的使用。]

继续进行功能性增强实验——

[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的全长ORF8蛋白是一种与腔内质网(ER)膜相关的蛋白,可诱导ATF6的激活,ATF6是一种ER应激调节的转录因子,可激活参与蛋白折叠的ER伴侣的转录[35]。 。我们扩增了代表蝙蝠SARSr-CoV ORF8三种不同基因型的Rf1,Rf4092和WIV1的ORF8基因,并构建了表达质粒。在HeLa细胞中瞬时表达的所有三个ORF8蛋白都可以刺激ATF6依赖性转录。其中,与SARS-CoV ORF8高度不同的WIV1 ORF8表现出最强的激活作用。结果表明,蝙蝠SARSr-CoV ORF8蛋白的变异体可能通过激活ATF6途径在调节内质网应激中发挥作用。此外,已证明来自后期的SARS-CoV的ORF8a蛋白可诱导细胞凋亡[28]。在这项研究中,我们发现新鉴定出的SARSr-CoV Rs4084的ORF8a蛋白与SARS-CoV ORF8a相比,包含8-aa插入,也显着触发了293T细胞的凋亡。]

这一段煞有介事地提出“在该区域的蝙蝠中,各种仍能使用人ACE2的SARSr-CoV仍在流通。因此,有可能溢出到人体内并出现类似SARS的疾病。”,时至今日云南蝙蝠洞周边依然安好,杀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.

数据表明,在同一洞穴中的SARSr-CoV之间发生了频繁的重组事件。虽然我们不能排除在其他地方存在类似的SARSr-CoV基因库的可能性,但我们提供了足够的证据来推断SARS-CoV最有可能是通过现有SARSr-CoV之间的重组事件起源于马蹄蝠。此外,我们还发现,在该区域的蝙蝠中,各种仍能使用人ACE2的SARSr-CoV仍在流通。因此,有可能溢出到人体内并出现类似SARS的疾病。考虑到距离我们所调查的蝙蝠洞最近的村庄仅1.1公里,这尤其重要,这表明本地居民可能接触蝙蝠。因此,我们建议应继续监测该站点和其他站点的SARSr-CoV演变,以及检查人类感染行为的风险和对人群进行血清学调查,以确定这些站点是否已经发生溢出并设计干预策略避免将来出现疾病。]

对于病毒来自实验室,杀人女巫石正丽是怎么回应的呢?——

石正丽回应病毒来源自实验室言论:是谣言 他的研究方法不对!来源:网易视频 发布时间:2020-02-08)

综述:作为生物知识的小白鼠,我们没有生物方面的专业素养,但是我想我看懂了闫丽梦博士的报告,那就是——所有关于SAR-COV2来源于自然的科学论断全是假的!那些与中共沆瀣一气的科学家还是装作不懂,其实很简单对所有中共宣布在某地发现的病毒再开启一遍探寻之旅,看看能不能再找到同样或类似的病毒!对于中共保存的来源于果子狸的类SARS病毒,只要用它来和果子狸做一下亲和实验,看看这个病毒能不能和果子狸的ACE2结合,我想只要一做这个实验,就能证明管轶声称的从果子狸身上发现的类SARS病毒就会像SARS-COV2一样告诉你——我不喜欢果子狸,我只喜欢人类!我只是借宿一下果子狸,我要过渡到人类!为我办理到果子狸身上办理借宿手续的就是中共这帮杂种!为我办理CHECK-OUT果子里的也是中共这帮杂种!

(文章内容仅代表作者个人观点)

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灭共52165 新中国联邦

take down the ccp, God bless the kind people

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NewFOC

10月 11日