Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis
Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—a new coronavirus that has led to a worldwide pandemic1—has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.
The genetic structure of SARS-CoV-2 does not rule out a laboratory origin
The polybasic furin site in SARS-CoV-2 was created by a 12-nucleotide insert TCCTCGGCGGGC coding for a PRRA amino acid sequence at the S1/S2 junction ( Figure 1). Interestingly, the two joint arginines are coded by two CGGCGG codons, which are rare for these viruses: only 5% of arginines are coded by CGG in SARS-CoV-2 or RaTG13, and CGGCGG in the new insert is the only doubled instance of this codon in SARS-CoV-2. The CGGCGG insert includes a FauI restriction site, of which there are six instances in SARS-CoV-2 and four instances in RaTG13 (and two in MP789). The serendipitous location of the FauI site could allow using restriction fragment length polymorphism ( RFLP) techniques [41] for cloning [42] or screening for mutations, [43] as the new furin site is prone to deletions in vitro.[39, 44]
A study by Zhou et al.[45] reported the discovery of a novel CoV strain RmYN02, which the authors claim exhibits natural PAA amino acid insertions at the S1/S2 cleavage site where SARS-CoV-2 has the PRRA insertion. However, upon close examination of the underlying nucleotide sequence of RmYN02 in comparison with its closest ancestors bat-SL-CoVZC45 and bat-SL-CoVZXC21, no insertions are apparent, just nucleotide mutations (Figure 2).
Therefore, SARS-CoV-2 remains unique among its beta CoV relatives not only due to a polybasic furin site at the S1/S2 junction, but also due to the four amino acid insert PRRA that had created it. The insertion causes a split in the original codon for serine (TCA) in MP789 or RaTG13 to give part of a new codon for serine (TCT) and part of the amino acid alanine (GCA) in SARS-CoV-2 (Figure 3).
30 March 2020 Editors’ note, March 2020: We are aware that this article is being used as the basis for unverified theories that the novel coronavirus causing COVID-19 was engineered. There is no evidence that this is true; scientists believe that an animal is the most likely source of the coronavirus.
我现在也不确定。但是，善意提醒：在下各种结论之前，应该研究一下Patent of Addition和其他 专利法，看看这是不是原因。
隔行如隔山。隔行如隔山。隔行如隔山。
谨慎，谨慎，再谨慎！
Patent of Addition is a cost effective intellectual property weapon used forprotecting the improvements or minor modifications taking place in the existing patent applications.
After arriving at a particular invention, there always remains a window for minor improvements or modifications. The improvements or modification might take place in an ordinary manner. For the minor improvements, it might be difficult to file a new and independent patent application, if the parent application has already been filed, as it will lack the inventive step underSection 2 (1) (j) of the Patents Act, 1970. Therefore, the concept of Patent of Addition came into being. It was first introduced in British Law system.
The concept of “Patent of Addition” came from U.K. patent law system. Thisconcept was first recommended and suggested by Lord Swan Committee.
Lord Swan Committee suggested that “the purpose of patent of addition was to provide reliefs to applicant who has failed to draft their claims in a way to adequately cover their invention, by giving them an opportunity to rectify the claims they have made, in cases where the circumstances permit this.”
While adopting the concept of patent of addition in the Indian patent law, Justice Ayyanger Committee further added that the subject matter of the patent of addition should be some additional or modification disclosure overthe parent patent application and not merely as claimed invention. Other countries such as United States of America, Australia, New Zealand, South Africa and Greece also adopted. In United States of America this concept is called “Continuation-in-Patent Application (CIP).”
另外，Beware of the CIP—Parent Applications Can Be Prior Art 08.25.2014 |
UPDATES Companies file patent applications with the United States Patent and Trademark Office (USPTO) to protect promising innovations. Often, however, improvements, additional uses and refinements surface after filing a patent application. How can a company capture these new, yet related, features?
The options available include filing a new application, a continuation application or a continuation-in-part (CIP) application.
A new application is a straightforward option for protecting new developments, but it cannot take advantage of the priority date of the previously filed application (or “parent application”). The priority date is important because the universe of references that the USPTO can use to reject a patent claim is fixed by an application’s priority date. In general, the earlier the application’s priority date, the higher the chance of obtaining a patent. A continuation application inherits the parent application’s priority date —but it is limited to the parent application’s disclosure. New developments since the original patent filing cannot be described in a continuation application because new matter cannot be added to a continuation application’s disclosure. A CIP application permits a patent applicant to add new subject matter to the existing disclosure of the parent application while retaining the priority date for claims based on the original disclosure. The claims of the CIP can be directed to the new subject matter, the old subject matter or a combination of the two. CIP applications used to be quite common, especially before mid-1995; in that era, the patent term was measured from the date a patent was issued. CIP applications have now fallen out of favor. The CIP application remains an attractive filing choice, but complications that may not be immediately apparent can offset its potential advantages
A CIP application permits a patent applicant to add new subject matter to the existing disclosure of the parent application while retaining the priority date for claims based on the original disclosure. The claims of the CIP can be directed to the new subject matter, the old subject matter or a combination of the two.
【 在 iminosugar (伪糖) 的大作中提到: 】 : 另外，Beware of the CIP—Parent Applications Can Be Prior Art : 08.25.2014 | : UPDATES Companies file patent applications with the United States Patent and : Trademark Office (USPTO) to protect promising innovations. Often, however, : improvements, additional uses and refinements surface after filing a patent : application. How can a company capture these new, yet related, features? : The options available include filing a new application, a continuation : application or a continuation-in-part (CIP) application. : A new application is a straightforward option for protecting new : developments, but it cannot take advantage of the priority date of the : ...................
看来五毛还是花了不少功夫，为什么要下这么大的功夫？
五毛不知道：1. mRNA是有方向的，反方向是没有用的。
2.专利是有方向的，反方向是不受保护的
【 在 atack (小军号) 的大作中提到: 】
: http://www.ncbi.nlm.nih.gov/nuccore/KH664781.1?report=GenBank
: http://www.ncbi.nlm.nih.gov/nuccore/MN908947.3?report=GenBank
: 这个巧合真的古怪。
石正丽的蝙蝠原始病毒有PRRA吗？
【 在 wsn2005 (蓑囝2005) 的大作中提到: 】
: 最早跳出来的美国科学家就说是自然病毒啊。
:
: 2020年春天的时候还说毒性较弱的本版可能已经存在数月甚至数年了。
:
:
: 【 在 simadong (simadong) 的大作中提到: 】
: : 靠，我要是美国，立即叫停，承认新冠病毒来自大自然。
现在可以大胆地猜想了：
2015-2016年，石正丽 提供米蒂原材料 1（BtCoV/4991）
同一时期，米蒂在马来西亚 获得 原材料 2—马来西亚穿山甲冠状病毒毒株（米蒂在马来有生物实验室）。
北卡和MD P4合成了新冠，北卡同时负责瑞德西韦项目（专利），摩德纳则负责新冠
mRNA疫苗（专利，2016年申请）。
他们做过猫实验和ACE2老鼠实验。
2019年病毒被疯子为了贸易战，在春运前夕投毒于中国运输枢纽。
擦
这么说，专利这事是真的？
【 在 ridgway(ridgway) 的大作中提到: 】
: 专利是公开的，石正丽试图改造出了岔子，造出了新冠。
李奇微（柴宁柴大妈）的话，你当放屁就是了。
现在人家头头现在都出来直接否认武汉实验室泄露了。。。
美国国立卫生研究院（隶属卫生与公众服务部）负责人、美国首席遗传学家弗朗西斯•柯林斯称，目前没有证据表明，新冠病毒是从中国的实验室泄漏后开始在世界范
围内传播的。https://sputniknews.cn/science/202106031033823440/
现在福奇邮件曝光，摩德纳在2017年就申请了新冠疫苗专利也引人注目了，你说美国不赶紧出来灭火难道要眼看火烧到自己身上吗？
【 在 I23 (嘿嘿) 的大作中提到: 】
: 擦
: 这么说，专利这事是真的？
:
: 专利是公开的，石正丽试图改造出了岔子，造出了新冠。
:
专利信息放在网上，小军号都能看到，需要什么公开透明的调查？
【在 xwzxjhq(谁的帝)的大作中提到：】
：怪不得时至今日
：
这么说，小军号在造谣？
【在 I23(嘿嘿)的大作中提到：】
：擦
：
没有证据，和否认是两码事。
【在 wsn2005(蓑囝2005)的大作中提到：】
：李奇微（柴宁柴大妈）的话，你当放屁就是了。
：
牛。能发paper吗？
【 在 atack (小军号) 的大作中提到: 】
: http://www.ncbi.nlm.nih.gov/nuccore/KH664781.1?report=GenBank
: http://www.ncbi.nlm.nih.gov/nuccore/MN908947.3?report=GenBank
: 这个巧合真的古怪。
枪铁牛名
我买我菌真的参与书写历史了
制作方法在莫德纳手里。
【 在 ridgwaymj (ridgwaymj) 的大作中提到: 】
: 专利信息放在网上，小军号都能看到，需要什么公开透明的调查？
: ：怪不得时至今日
: ：
人家两张嘴，上下不分。 你就 厚道点儿吧。 LOL
【 在 Timetravler () 的大作中提到: 】
: 李奇微，你真牛逼，说话跟放屁一样
: 你说瞎话张口就来的本事这么高，肯定是骗子岛上的骗子教练吧，肯定挣的是好几个: 1450那么多，太羡慕你了
:
: 专利是公开的，石正丽试图改造出了岔子，造出了新冠。
:
lol
[在 lgw (abcdefg) 的大作中提到：]
:你听他们瞎说。
:摩德纳一直研究mrna，过去做过sars疫苗而已。
:Sars消失了，疫苗没用。
:新冠就是SARS2，稍微改下就能用。
:这帮人一边不让人叫SARS2，一边造谣。非要弄得天下都知道新冠就是SARS2才高兴。
:到时候又该抗议不该叫SARS2了，谁叫谁反华。
:☆ 发自 iPhone 买买提 1.24.11
Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—a new
coronavirus that has led to a worldwide pandemic1—has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B
coronaviruses2. To explore whether the furin cleavage site contributes to
infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2
that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2.
However, the ΔPRRA mutant had reduced replication in a human respiratory
cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA
mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with
coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the
receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant
than against parental SARS-CoV-2, probably owing to an increased ratio of
particles to plaque-forming units in infections with the former. Together,
our results demonstrate a critical role for the furin cleavage site in
infection with SARS-CoV-2 and highlight the importance of this site for
evaluating the neutralization activities of antibodies.
我已经全部截图保存，并复制粘贴保存。
肯定会被删帖。
【 在 atack (小军号) 的大作中提到: 】
: PRRAR是新冠病毒的独特标记：同亚种的相临近的冠状病毒里它是唯一具有PRRAR弗林酶
: 切位点的病毒，另外，两个精氨酸RR是新冠突刺蛋白里唯一用CGG编码的，CGG在整个病
: 毒里也很少用。所以PRRAR是病毒人造论的重要论据。
:
: 另外，去除PRRA的新冠病毒在动物模型中更容易传播，所以从自然进化的角度不大可能
: 会出一个让自己更难传播的插入。
:
: 最后，RRAR是新冠毒性的罪魁祸首。敲除PRRA的新冠病毒的毒性大减。
:
: 【 在 szxpa (military) 的大作中提到: 】
: : 能不能稍微解释一下，门外汉完全不懂啊。
: : : DEFINITION Sequence 11652 from patent US 9587003.
: : : ACCESSION牋 KH664781
: : : VERSION牋牋 KH664781.1
: : : KEYWORDS牋 .
: : : SOURCE牋牋 Unknown.
: :
: :牋 ORGANISM Unknown.
: :
: :牋牋牋牋牋牋 Unclassified.
: : : REFERENCE牋 1 (bases 1 to 3387)
: :
: :牋 AUTHORS牋 Bancel,S., Chakraborty,T., de Fougerolles,A., Elbashir,S
: .M
: : ...................
The genetic structure of SARS-CoV-2 does not rule out a laboratory origin
The polybasic furin site in SARS-CoV-2 was created by a 12-nucleotide insert TCCTCGGCGGGC coding for a PRRA amino acid sequence at the S1/S2 junction (
Figure 1). Interestingly, the two joint arginines are coded by two CGGCGG
codons, which are rare for these viruses: only 5% of arginines are coded by CGG in SARS-CoV-2 or RaTG13, and CGGCGG in the new insert is the only
doubled instance of this codon in SARS-CoV-2. The CGGCGG insert includes a
FauI restriction site, of which there are six instances in SARS-CoV-2 and
four instances in RaTG13 (and two in MP789). The serendipitous location of
the FauI site could allow using restriction fragment length polymorphism (
RFLP) techniques [41] for cloning [42] or screening for mutations, [43] as
the new furin site is prone to deletions in vitro.[39, 44]
A study by Zhou et al.[45] reported the discovery of a novel CoV strain
RmYN02, which the authors claim exhibits natural PAA amino acid insertions
at the S1/S2 cleavage site where SARS-CoV-2 has the PRRA insertion. However, upon close examination of the underlying nucleotide sequence of RmYN02 in
comparison with its closest ancestors bat-SL-CoVZC45 and bat-SL-CoVZXC21, no insertions are apparent, just nucleotide mutations (Figure 2).
Therefore, SARS-CoV-2 remains unique among its beta CoV relatives not only
due to a polybasic furin site at the S1/S2 junction, but also due to the
four amino acid insert PRRA that had created it. The insertion causes a
split in the original codon for serine (TCA) in MP789 or RaTG13 to give part of a new codon for serine (TCT) and part of the amino acid alanine (GCA) in SARS-CoV-2 (Figure 3).
没有。
【 在 noparking (noparking) 的大作中提到: 】
: 石正丽的蝙蝠原始病毒有PRRA吗？
前面不是给图了吗？
【 在 ridgwaymj (ridgwaymj) 的大作中提到: 】
: 没有证据，和否认是两码事。
没有证据，就可以随便造谣吗？
请问你你和王丹的孩子是不是被流产流掉了？
没有证据表明你和你妈乱伦过，但是你也不能否认哦
【 在 ridgwaymj (ridgwaymj) 的大作中提到: 】
: 没有证据，和否认是两码事。
确认
奇怪的是就这一段和cov2匹配
【 在 atack (小军号) 的大作中提到: 】
: http://www.ncbi.nlm.nih.gov/nuccore/KH664781.1?report=GenBank
: http://www.ncbi.nlm.nih.gov/nuccore/MN908947.3?report=GenBank
: 这个巧合真的古怪。
李功友逻辑何在
某宝如何在实验室试验的
对本专利作出了哪些贡献
当然需要调查
【 在 ridgwaymj (ridgwaymj) 的大作中提到: 】
: 专利信息放在网上，小军号都能看到，需要什么公开透明的调查？
: ：怪不得时至今日
: ：
很快就会被下架的
【 在 hurricane1 (飓风) 的大作中提到: 】
: 这个赶紧发推特和油管，还祖国清白
参与历史，见证历史
【 在 Contrarian91 (QuestionEverything) 的大作中提到: 】
: https://www.lens.org/lens/search/patent/list?q=US%209587003&preview=true
: 专利申请2016！！
File日期居然是2016年2月。比想象的早多了。
谁科普一下，中学只学过gtca，这个PRRAR到底是怎么对应到gtca这几个的？
操 搞了半天屁都不懂
一个核苷酸序列
一个是氨基酸序列
【 在 srx(srx) 的大作中提到: 】
<br>: 谁科普一下，中学只学过gtca，这个PRRAR到底是怎么对应到gtca这几个
的？
<br>
我又不是千老
【 在 allienpig (猪 in black) 的大作中提到: 】
: 操 搞了半天屁都不懂
: 一个核苷酸序列
: 一个是氨基酸序列
:
: 谁科普一下，中学只学过gtca，这个PRRAR到底是怎么对应到gtca这几个
: 的？
:
重读 2015年11月的病毒合成雄文，细思极恐。据说当时合成了6种新冠，只选择了一种发表。https://www.nature.com/articles/nm.3985
公开的，能看全文，不需要学校IP的支持。
这篇文章提到了 石争利丽 的经费来源（中国自然科学基金和美国国际开发署）和对合成病毒的贡献（抓蝙蝠提供原材料）。
开篇就是此地无银三百两。
30 March 2020 Editors’ note, March 2020: We are aware that this article is being used as the basis for unverified theories that the novel coronavirus
causing COVID-19 was engineered. There is no evidence that this is true;
scientists believe that an animal is the most likely source of the
coronavirus.
我现在也不确定。但是，善意提醒：在下各种结论之前，应该研究一下Patent of
Addition和其他
专利法，看看这是不是原因。
隔行如隔山。隔行如隔山。隔行如隔山。
谨慎，谨慎，再谨慎！
Patent of Addition is a cost effective intellectual property weapon used forprotecting the improvements or minor modifications taking place in the
existing patent applications.
After arriving at a particular invention, there always remains a window for minor improvements or modifications. The improvements or modification might take place in an ordinary manner. For the minor improvements, it might be
difficult to file a new and independent patent application, if the parent
application has already been filed, as it will lack the inventive step underSection 2 (1) (j) of the Patents Act, 1970. Therefore, the concept of
Patent of Addition came into being. It was first introduced in British Law
system.
The concept of “Patent of Addition” came from U.K. patent law system. Thisconcept was first recommended and suggested by Lord Swan Committee.
Lord Swan Committee suggested that “the purpose of patent of addition was
to provide reliefs to applicant who has failed to draft their claims in a
way to adequately cover their invention, by giving them an opportunity to
rectify the claims they have made, in cases where the circumstances permit
this.”
While adopting the concept of patent of addition in the Indian patent law,
Justice Ayyanger Committee further added that the subject matter of the
patent of addition should be some additional or modification disclosure overthe parent patent application and not merely as claimed invention. Other
countries such as United States of America, Australia, New Zealand, South
Africa and Greece also adopted. In United States of America this concept is called “Continuation-in-Patent Application (CIP).”
另外，Beware of the CIP—Parent Applications Can Be Prior Art
08.25.2014 |
UPDATES Companies file patent applications with the United States Patent and
Trademark Office (USPTO) to protect promising innovations. Often, however, improvements, additional uses and refinements surface after filing a patent application. How can a company capture these new, yet related, features?
The options available include filing a new application, a continuation
application or a continuation-in-part (CIP) application.
A new application is a straightforward option for protecting new
developments, but it cannot take advantage of the priority date of the
previously filed application (or “parent application”). The priority date is important because the universe of references that the USPTO can use to
reject a patent claim is fixed by an application’s priority date. In
general, the earlier the application’s priority date, the higher the chance of obtaining a patent.
A continuation application inherits the parent application’s priority date
—but it is limited to the parent application’s disclosure. New
developments since the original patent filing cannot be described in a
continuation application because new matter cannot be added to a
continuation application’s disclosure.
A CIP application permits a patent applicant to add new subject matter to
the existing disclosure of the parent application while retaining the
priority date for claims based on the original disclosure. The claims of
the CIP can be directed to the new subject matter, the old subject matter or a combination of the two.
CIP applications used to be quite common, especially before mid-1995; in
that era, the patent term was measured from the date a patent was issued.
CIP applications have now fallen out of favor. The CIP application remains an attractive filing choice, but complications that may not be immediately
apparent can offset its potential advantages
密码子，知道吗？
3nt
【 在 allienpig (猪 in black) 的大作中提到: 】
: 操 搞了半天屁都不懂
: 一个核苷酸序列
: 一个是氨基酸序列
:
:
: 【 在 srx(srx) 的大作中提到: 】
: : 谁科普一下，中学只学过gtca，这个PRRAR到底是怎么对应到gtca这几个
: 的？
A CIP application permits a patent applicant to add new subject matter to
the existing disclosure of the parent application while retaining the
priority date for claims based on the original disclosure. The claims of
the CIP can be directed to the new subject matter, the old subject matter or a combination of the two.
所以，如果是CIP，发明者可以加新数据、新claim到原来的专利申请、还保留原来的
priority date。
【 在 iminosugar (伪糖) 的大作中提到: 】
: 另外，Beware of the CIP—Parent Applications Can Be Prior Art
: 08.25.2014 |
: UPDATES Companies file patent applications with the United States Patent
and
: Trademark Office (USPTO) to protect promising innovations. Often, however,
: improvements, additional uses and refinements surface after filing a
patent
: application. How can a company capture these new, yet related, features?
: The options available include filing a new application, a continuation
: application or a continuation-in-part (CIP) application.
: A new application is a straightforward option for protecting new
: developments, but it cannot take advantage of the priority date of the
: ...................
如果更改了，基因库数据要重新上载的。
这份疫苗的基因序列时间是 14-OCT-2017
查了一下，atack网友引用的这个NCBI链接中的内容/信息有好几处错误。我非常怀疑或者基
本上它的准确性。
有时间会仔细查。
现在就仔细查， 立刻，马上。
【 在 iminosugar (伪糖) 的大作中提到: 】
: 查了一下，atack网友引用的这个NCBI链接中的内容/信息有好几处错误。我非常怀疑或
: 者基
: 本上它的准确性。
: 有时间会仔细查。
所以 投毒的侵害了moderna的版权？是不是应该给盖茨专利费？
【 在 busted(busted) 的大作中提到: 】
: 现在就仔细查， 立刻，马上。
因为这个专利的创新之一就是在特定的序列的一些部位加入RRAR之类的酶切位点。专利的Table 8声明了可以插入的各种酶切位点。序列11652不是新冠疫苗，但是使用了
PRRAR插入，所以你只能见到这19个碱基一致。
【 在 krzkrz (krzkrz) 的大作中提到: 】
: 确认
: 奇怪的是就这一段和cov2匹配