सरस चमत्कारी? वुहान कोरोनावायरस की वंशावली

यूएफओ केयर मिनट

महामारी COVID-19, SARS-CoV-2 कोरोनावायरस (2019-nCoV) के कारण होने वाले संभावित गंभीर तीव्र श्वसन संक्रमण की आधिकारिक तौर पर दुनिया में घोषणा की गई है। इस विषय पर हैबे के बारे में बहुत सारी जानकारी है - हमेशा याद रखें कि यह विश्वसनीय / उपयोगी और इसके विपरीत दोनों हो सकता है।

हम आपसे किसी भी प्रकाशित जानकारी के लिए महत्वपूर्ण होने का आग्रह करते हैं।

आधिकारिक स्रोत

• C
• C
• ()

, .

अपने हाथ धोएं, अपने प्रियजनों का ख्याल रखें, जब भी संभव हो घर पर रहें और दूर से काम करें।

के बारे में प्रकाशन पढ़ें: कोरोनावायरस | दूरदराज के काम

शी झेंगली वुहान इंस्टीट्यूट ऑफ वायरोलॉजी में अपनी प्रयोगशाला में

जीवविज्ञान

RBD CoV2, ACE2.
() 2019-nCov. NTD, N- . RBD, - . RBM, - . SD1, 1. SD2, 2. FP, . HR1, 1. HR2, 2. TM, . IC, .
() RBD 2019-nCov. RBM .
() RBD 2019-nCov, ACE2. ACE2 . RBD 2019-nCoV , RBM — . RBD 2019-nCov . N- ACE2, , .

Overall structure of 2019-nCoV RBD bound with ACE2.
(a) Overall topology of 2019-nCoV spike monomer. NTD, N-terminal domain. RBD, receptor-binding domain. RBM, receptor-binding motif. SD1, subdomain 1. SD2, subdomain 2. FP, fusion peptide. HR1, heptad repeat 1. HR2, heptad repeat 2. TM, transmembrane region. IC, intracellular domain.
(b) Sequence and secondary structures of 2019-nCoV RBD. The RBM is colored red.
() Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.

pangolins

, , - 2019 . (Manis pentadactyla) 25 (Manis javanica). ; , , . , , , , , , .

Pangolins used in the study were confiscated by Customs and Department of Forestry of Guangdong Province in March-December 2019. They include four Chinese pangolins (Manis pentadactyla) and 25 Malayan pangolins (Manis javanica). These animals were sent to the wildlife rescue center, and were mostly inactive and sobbing, and eventually died in custody despite exhausting rescue efforts. Tissue samples were taken from the lung, lymph nodes, liver, spleen, muscle, kidney, and other tissues from pangolins that had just died for histopathological and virological examinations.

हमने जमे हुए ऊतकों (फेफड़े, आंत, रक्त) के नमूने प्राप्त किए जो अगस्त 2017 - जनवरी 2018 के दौरान 18 मलय पैंगोलिंस ( मैनिस जावानिका ) से लिए गए थे । ये पैंगोलिन गुआंग्सी सीमा शुल्क द्वारा तस्करी विरोधी अभियानों के दौरान प्राप्त किए गए थे। यह आश्चर्यजनक है कि उनके आरएनए के उच्च-प्रदर्शन अनुक्रमण ने 43 नमूनों में से छह (दो फेफड़े, दो आंत, फेफड़े और आंतों का एक मिश्रण, एक रक्त) में कोरोनाविरस की उपस्थिति का पता लगाया।
...
, , , RaTG13 2019-CoV2 (. 1c, d). , 2019-CoV2 - (RBD; 97,4%; . 1c . 2a), 2019-CoV2 RaTG13. RaTG13 2019-CoV2 89,2% RBD. 2019-CoV2 RBD, RaTG13 2019-CoV2 ( 442, SARS-CoV ).

We received frozen tissue (lungs, intestine, blood) samples that were collected from 18 Malayan pangolins (Manis javanica) during August 2017-January 2018. These pangolins were obtained during the anti-smuggling operations by Guangxi Customs. Strikingly, high-throughput sequencing of their RNA revealed the presence of coronaviruses in six (two lung, two intestine, one lung-intestine mix, one blood) of 43 samples. With the sequence read data, and by filling gaps with amplicon sequencing, we were able to obtain six full or nearly full genome sequences — denoted GX/P1E, GX/P2V, GX/P3B, GX/P4L, GX/P5E and GX/P5L — that fall into the 2019-CoV2 lineage (within the genus Betacoronavirus) in a phylogenetic analysis (Figure 1a).
…
More notable, however, was the observation of putative recombination signals between the pangolins coronaviruses, bat coronaviruses RaTG13, and human 2019-CoV2 (Figure 1c, d). In particular, 2019-CoV2 exhibits very high sequence similarity to the Guangdong pangolin coronaviruses in the receptor-binding domain (RBD; 97.4% amino acid similarity; indicated by red arrow in Figure 1c and Figure 2a), even though it is most closely related to bat coronavirus RaTG13 in the remainder of the viral genome. Bat CoV RaTG and the human 2019-CoV2 have only 89.2% amino acid similarity in RBD. Indeed, the Guangdong pangolin coronaviruses and 2019-CoV2 possess identical amino acids at the five critical residues of the RBD, whereas RaTG13 only shares one amino acid with 2019-CoV2 (residue 442, human SARS-CoV numbering).

दिलचस्प बात यह है कि आरबीडी में केवल पर्यायवाची स्थलों के फेलोजेनेटिक विश्लेषण से पता चला है कि गुआन्गदोंड पैंगोलिन की फाइटोलैनेटिक स्थिति बाकी वायरल जीनोम के अनुरूप है, अर्थात् यह 2019-CoV2 (चित्रा 2 बी) का निकटतम रिश्तेदार नहीं है। इसलिए, यह संभव है कि पैंगोलिन कोरोनाविरस और 2019-सीओवी 2 के आरबीडी में एमिनो एसिड की समानता पुनर्संयोजन के बजाय चुनिंदा मध्यस्थता अभिसरण विकास के कारण है, हालांकि उपलब्ध आंकड़ों के आधार पर इन परिदृश्यों के बीच चयन करना मुश्किल है।

स्रोत इबारत

Interestingly, a phylogenetic analysis of synonymous sites alone in the RBD revealed that the phylogenetic position of the Guangdong pangolin is consistent with that in the remainder of the viral genome, rather than being the closest relative of 2019-CoV2 (Figure 2b). Hence, it is possible that the amino acid similarity between the RBD of the Guangdong pangolin coronaviruses and 2019-CoV2 is due to selectively-mediated convergent evolution rather than recombination, although it is difficult to choose between these scenarios on current data.

क्राउन पर्सन्स की वंशावली

मैं बड़े करीने से मार रहा हूं, लेकिन कठोर हूं

, , , , , SARS-CoV-2. () COVID-19 . SARS-CoV-2 , .

Patients with hypertension, diabetes, coronary heart disease, cerebrovascular illness, chronic obstructive pulmonary disease, and kidney dysfunction have worse clinical outcomes when infected with SARS-CoV-2, for unknown reasons. The purpose of this review is to summarize the evidence for the existence of elevated plasmin(ogen) in COVID-19 patients with these comorbid conditions. Plasmin, and other proteases, may cleave a newly inserted furin site in the S protein of SARS-CoV-2, extracellularly, which increases its infectivity and virulence.

यह पाया गया कि 40% से अधिक SARS-CoV-2 प्रोटीन समरूपता वाले सभी स्पाइक प्रोटीन में बैट-कॉव RaTG13 और SARS-CoV (97.4 के रूप में अनुक्रम पहचान के साथ) में फ़्यूरिन क्लीवेज साइट (चित्र 1, तालिका 1) नहीं थी। % और 78.6%, क्रमशः)। SARS-CoV-2 में RRAR फरिन साइट अपने परिवार में अद्वितीय PRRA डालने के लिए अद्वितीय है। SARS-CoV-2 में यह साइट मुश्किल से MERS, HCoV-HKU1 इत्यादि से विकसित हो सकती है। डेटाबेस में वर्तमान में उपलब्ध अनुक्रमों से, हमारे लिए स्रोत खोजना मुश्किल है। शायद कई और विकासवादी मध्यवर्ती अनुक्रम हैं जो खोज का इंतजार कर रहे हैं।

स्रोत इबारत

It was found that all Spike with a SARS-CoV-2 Spike sequence homology greater than 40% did not have a furin cleavage site (Figure 1, Table 1), including Bat-CoV RaTG13 and SARS-CoV (with sequence identity as 97.4% and 78.6%, respectively). The furin cleavage site “RRAR” in SARS-CoV-2 is unique in its family, rendering by its unique insert of “PRRA”. The furin cleavage site of SARS-CoV-2 is unlikely to have evolved from MERS, HCoV-HKU1, and so on. From the currently available sequences in databases, it is difficult for us to find the source. Perhaps there are still many evolutionary intermediate sequences waiting to be discovered.

यह जांचने के लिए कि मुख्य अमीनो एसिड अवशेषों के अनुक्रम में प्रोटिओलिटिक दरार सेल फ्यूजन गतिविधि को बढ़ावा दे सकता है, हमने दो स्थानों में से एक में फ्यूरिन पहचान साइट (आरआरएसआरआरआर) बनाने के लिए स्पाइक-जैसे एसएआरएस-सीओवी प्रोटीन को उत्परिवर्तित किया।

स्रोत इबारत

To investigate whether proteolytic cleavage at the basic amino acid residues, were it to occur, might facilitate cell–cell fusion activity, we mutated the wild-type SARS-CoV glycoprotein to construct a prototypic furin recognition site (RRSRR) at either position.

एसएआरएस-सीओवी एस 1-एस 2 और एस 2 पदों के संभावित उपयोग की जांच करने के लिए प्रोटिओलिटिक दरार के लिए साइटों के रूप में, हमने पहली बार इन साइटों पर फ्यूरिन क्लीवेज मान्यता साइटों को निम्न म्यूटेशन 664-SLLRSTSQSI - SLL RRSRR SI-671 (S1-S2) से शुरू किया था। 792-LKPTKRSF-LKRTKRSF-799 (S2 ')।

स्रोत इबारत

To examine the potential use of the SARS-CoV S1–S2 and S2? positions as sites for proteolytic cleavage, we first introduced furin cleavage recognition sites at these locations by making the following mutations 664-SLLRSTSQSI — SLLRRSRRSI-671 (S1–S2) and 792-LKPTKRSF — LKRTKRSF-799 (S2?).

बीजिंग 2019

S2' QX , , IBV (WT-IBV). , IBV S2 ( -S2) . IBV .
…
, , FP CoV, , , -, , .

Mutation of the S2' site of QX genotype (QX-type) spike protein (S) in a recombinant virus background results in higher pathogenicity, pronounced neural symptoms and neurotropism when compared with conditions in wild-type IBV (WT-IBV) infected chickens. In this study, we present evidence suggesting that recombinant IBV with a mutant S2' site (furin-S2' site) leads to higher mortality. Infection with mutant IBV induces severe encephalitis and breaks the blood–brain barrier.
…
In summary, our results demonstrate that the furin cleavage site upstream of the FP in S protein is an important site for CoV, modulating entry, cell–virus fusion, adaptation to its host cell, cell tropism and pathogenicity, but not antigenicity.

, HKU4 , HKU4, . , S746R N762A, HKU4.
…
, hPPC hECP HKU4 HKU4 . , HKU4 S1/S2, .

To evaluate the potential genetic changes required for HKU4 to infect human cells, we reengineered HKU4 spike, aiming to build its capacity to mediate viral entry into human cells. To this end, we introduced two single mutations, S746R and N762A, into HKU4 spike. The S746R mutation was expected to restore the hPPC motif in HKU4 spike, whereas the N762A mutation likely disrupted the potential N-linked glycosylation site in the hECP motif in HKU4 spike.
…
Therefore, the reengineered hPPC and hECP motifs enabled HKU4 spike to be activated by human endogenous proteases and thereby allowed HKU4 pseudoviruses to bypass the need for exogenous proteases to enter human cells. These results reveal that HKU4 spike needs only two single mutations at the S1/S2 boundary to gain the full capacity to mediate viral entry into human cells.

संक्षेप में, pseudotyped MERS-CoV- स्पाइक रेट्रोवाइरस, ल्यूसिफरेज रिपोर्टर जीन को व्यक्त करते हुए HEK293T कोशिकाओं के cotransfection द्वारा HIV-1 जीन ले जाने वाले प्लास्मिड के साथ प्राप्त किया गया, Enective दोषपूर्ण व्यक्त luciferase (pNL4-3.luc.RE-) और एक प्लास्मिड एन्कोडिंग दाता। -ओसीवी स्पाइक प्रोटीन।

स्रोत इबारत

Briefly, MERS-CoV-spike-pseudotyped retroviruses expressing a luciferase reporter gene were prepared by cotransfecting HEK293T cells with a plasmid carrying Env-defective, luciferase-expressing HIV-1 genome (pNL4–3.luc.R-E-) and a plasmid encoding MERS-CoV spike protein.

RaTG13 कहां से आया

, - - (RdRp) (BatCoV RaTG13), Rhinolophus affinis , 2019-CoV2. ( GISAID EPI_ISL_402131). Simplot , 2019-CoV2 RaTG13 (Fig. 1c), 96,2%.

We then found that a short region of RNA-dependent RNA polymerase (RdRp) from a bat coronavirus (BatCoV RaTG13) — which was previously detected in Rhinolophus affinis from Yunnan province — showed high sequence identity to 2019-CoV2. We carried out full-length sequencing on this RNA sample (GISAID accession number EPI_ISL_402131). Simplot analysis showed that 2019-CoV2 was highly similar throughout the genome to RaTG13 (Fig. 1c), with an overall genome sequence identity of 96.2%.

मई से जुलाई 2013 तक चीन के चार प्रांतों युन्नान के पांच जिलों में विभिन्न स्थानों पर चमगादड़ पकड़े गए थे।

स्रोत इबारत

Bats were captured from various locations in five counties of four prefectures of Yunnan Province, China, from May to July 2013.

वुहान -1

सबसे महत्वपूर्ण बात, हम वेरो ई 6 कोशिकाओं में बैट मल से पृथक नमूनों से जीवित वायरस एसएल-सीओवी (बैट एसएल-कोव-डब्ल्यूआईवी 1) के पहले अलगाव की रिपोर्ट करते हैं, जिसमें कोरोनोवायरस की एक विशिष्ट आकृति विज्ञान है, 99.9% जीनोम की पहचान Rs3367 और ACE2 लोगों, सिवेट और घोड़े की नाल की हड्डियों का उपयोग सेल में प्रवेश करने के लिए करता है। इन विट्रो परीक्षण में प्रारंभिक से पता चलता है कि WIV1 में व्यापक प्रजाति ट्रॉपिज़्म भी है।

स्रोत इबारत

Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism.

अन्य युन्नान उपभेद

1999: पहला काइमेरिक कोरोनावायरस

निर्देशित आरएनए पुनर्संयोजन का उपयोग करते हुए, हमने कोरोनोवायरस माउस हेपेटाइटिस वायरस (एमएचवी) के एक उत्परिवर्ती का निर्माण किया, जिसमें स्पाइकी प्रोटीन एक्टोडोमैन को उग्र संक्रामक पेरिटोनिटिस वायरस प्रोटीन के अत्यधिक डाइवर्जेंट एक्टोडोमैन द्वारा बदल दिया गया था। परिणामी काइमेरिक वायरस, नामित एफएमएचवी, ने सेलीन कोशिकाओं को संक्रमित करने की क्षमता हासिल कर ली और एक ही समय में संस्कृति में माउस कोशिकाओं को संक्रमित करने की क्षमता खो दी।

स्रोत इबारत

Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture.

अपने स्पाइक-जैसे प्रोटीन में केवल कुछ ही उत्परिवर्तन होते हैं, जो माउस कोरोनावायरस को माउस-सीमित ट्रॉपिज्म से इन विट्रो पैशन में विस्तारित होस्ट रेंज में स्विच करने की अनुमति देता है। ऐसा एक वायरस जिसका हमने अध्ययन किया, दो पुटीय हेपरान सल्फेट बाइंडिंग साइटों का अधिग्रहण किया, जबकि दूसरी जगह फरिन साइट को संरक्षित किया।

स्रोत इबारत

Only a relatively few mutations in its spike protein allow the murine coronavirus to switch from a murine-restricted tropism to an extended host range by being passaged in vitro. One such virus that we studied had acquired two putative heparan sulfate-binding sites while preserving another site in the furin-cleavage motif.

MHV/pi23 — , 23 600 , MHV/BHK, HS- S1 , MHV/BHK, , HS- . MHV/pi23 , , MHV/BHK. HS- , , , S, HR1 (Fig. 1), . S, .

MHV/pi23, a virus obtained after 23 of the 600 passages that resulted in MHV/BHK, also contains a putative HS-binding site in the S1 domain at the same position as in MHV/BHK, albeit as a smaller insertion, while it lacks the putative HS-binding site immediately upstream of the fusion peptide. MHV/pi23 does infect nonmurine cells to some extent but much less efficiently than MHV/BHK. In addition to the multiple HS-binding sites, however, mutations found in other parts of the S protein, such as the HR1 domain and the putative fusion peptide (Fig. 1), might also contribute to the efficient entry into nonmurine cells. We are currently in the process of determining the S protein mutations that are required for the extended host range phenotype.

MERS-CoV प्रजातियों की अनुकूलन क्षमता को बेहतर ढंग से समझने के लिए, हमने वायरल अनुकूलन का अध्ययन करने के लिए सबॉप्टीमल DPP4 संस्करण का उपयोग किया। इस DPP4 संस्करण को व्यक्त करने वाले कोशिकाओं पर वायरस के पारित होने के परिणामस्वरूप वायरल स्पाइक प्रोटीन में उत्परिवर्तन का संचय हुआ, जिससे प्रतिकृति में वृद्धि हुई।

स्रोत इबारत

To better understand the species adaptability of MERS-CoV, we identified a suboptimal species-derived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication.

(एफ) विभिन्न मार्ग में MERS-CoV स्पाइक प्रोटीन में एकल और दोहरे उत्परिवर्तन की उपस्थिति की योजना।
(जी) MERS-CoV स्पाइक प्रोटीन में उत्परिवर्तन का स्थान।

स्रोत इबारत

(F) Schematic of single and double mutation emergence in MERS-CoV spike over different passages.
(G) Location of mutations within MERS-CoV spike.

कैसे बारिक ने सबके लिए रास्ता जलाया

II 59 (MHV-A59). , MHV 31,5 . , MHV-A59 ~ 31,5 ... , , , … , , .

स्रोत इबारत

A novel method was developed to assemble a full-length infectious cDNA of the group II coronavirus mouse hepatitis virus strain A59 (MHV-A59). Seven contiguous cDNA clones that spanned the 31.5-kb MHV genome were isolated. The ends of the cDNAs were engineered with unique junctions and assembled with only the adjacent cDNA subclones, resulting in an intact MHV-A59 cDNA construct of ?31.5 kb in length. The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes… The method has the potential to be used to construct viral, microbial, or eukaryotic genomes approaching several million base pairs in length and used to insert restriction sites at any given nucleotide in a microbial genome.

सार्स 2003

, , SARS-CoV Urbani SARS ( SARS-CoV), , . , , … SARS-CoV , .

Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoV Urbani strain, and have rescued molecularly cloned SARS viruses (infectious clone SARS-CoV) that contained the expected marker mutations inserted into the component clones. Recombinant viruses replicated as efficiently as WT virus and both were inhibited by treatment with the cysteine proteinase inhibitor… Availability of a SARS-CoV full-length cDNA provides a template for manipulation of the viral genome, allowing for the rapid and rational development and testing of candidate vaccines and therapeutics against this important human pathogen.

सार्स-2006

Urbani SARS-CoV (BAC). . , Escherichia coli.
…
SARS-CoV E.coli DH10B 200 , ( ). .

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli.
…
The assembled SARS-CoV infectious cDNA clone was fully stable during its propagation in E. coli DH10B cells for more than 200 generations, considerably facilitating the genetic manipulation of the viral genome (data not shown). The detailed cloning strategy, plasmid maps, and sequences are available upon request.

वुहान 2007

SLS-CoV प्रोटीन की रीढ़ की हड्डी में विभिन्न SARS-CoV स्पाइक प्रोटीन अनुक्रमों को सम्मिलित करके बहुत सारे चिमेरिक स्पाइक प्रोटीन का निर्माण किया गया है।

स्रोत इबारत

A series of S chimeras was constructed by inserting different sequences of the SARS-CoV S into the SL-CoV S backbone.

इन परिणामों से, यह पाया गया कि स्पाइक प्रोटीन BJ01 में 310 से 518 तक का क्षेत्र मानव ACE2 को बांधने के लिए स्पाइक प्रोटीन Rp3 की क्षमता प्राप्त करने के लिए आवश्यक और पर्याप्त था।

स्रोत इबारत

From these results, it was deduced that the region from aa 310 to 518 of BJ01-S was necessary and sufficient to convert Rp3-S into a huACE2-binding molecule.

SARS-CoV से RBM को SL-CoV स्पाइक प्रोटीन में सम्मिलित करने के लिए, स्पाइक प्रोटीन BJ01 में 424 से 494 अमीनो एसिड के कोडिंग क्षेत्र का उपयोग Rp3 प्रोटीन में संबंधित क्षेत्रों को बदलने के लिए किया गया था, जिसके परिणामस्वरूप एक काइमेरिक स्पाइक प्रोटीन जीन (CS), जिसे CS424 के रूप में नामित किया गया था। -494।

स्रोत इबारत

For introduction of the RBM of SARS-CoV S into the SL-CoV S, the coding region from aa 424 to 494 of BJ01-S was used to replace the corresponding regions of Rp3-S, resulting in a chimeric S (CS) gene designated CS424–494.

चिमेरा 2015

SARS-CoV, , SHC014 SARS-CoV. , 2b, SHC014 , SARS (ACE2), in vitro, SARS-CoV. , in vivo . ; , CoV . SHC014 in vitro, in vivo.

स्रोत इबारत

Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo.

(ए) आणविक क्लोन SHC014-CoV की योजना, जिसे छह सन्निहित सीडीएनए सेगमेंट के रूप में संश्लेषित किया गया था (जिसे SHC014A, SHC014B, SHC014C, SHC014D, SHC014E और SHC014F के रूप में निर्दिष्ट) अद्वितीय BglI प्रतिबंध एंजाइम साइटों द्वारा निर्देशित किया गया था 1 ए, 1 बी, स्पाइक, 3, लिफाफा, मैट्रिक्स, 6–8 और न्यूक्लियोकैप्सिड)। रेखांकित न्यूक्लियोटाइड्स प्रतिबंध एंजाइम पाचन के बाद बने हुए अनुक्रम हैं।

स्रोत इबारत

(a) Schematic of the SHC014-CoV molecular clone, which was synthesized as six contiguous cDNAs (designated SHC014A, SHC014B, SHC014C, SHC014D, SHC014E and SHC014F) flanked by unique BglI sites that allowed for directed assembly of the full-length cDNA expressing open reading frames (for 1a, 1b, spike, 3, envelope, matrix, 6–8 and nucleocapsid). Underlined nucleotides represent the overhang sequences formed after restriction enzyme cleavage.

यह उल्लेखनीय है कि SARS-MA15 की तुलना में फेफड़ों में अंतर ट्रोपिज्म और संस्कृतियों में पूर्ण-लंबाई SHC014-CoV का कमजोर होना [मानव उपकला वायुमार्ग] SARS-CoR Urbani के सापेक्ष सुझाव है कि ACE2 के लिए बाध्य करने के अलावा, अन्य कारक - स्पाइक प्रोटीन की प्रक्रिया सहित। , मेजबान की प्रतिरक्षा प्रतिक्रियाओं के रिसेप्टर या प्रतिपक्षी की जैव उपलब्धता - पौरूष में योगदान कर सकती है।

स्रोत इबारत

Notably, differential tropism in the lung as compared to that with SARS-MA15 and attenuation of full-length SHC014-CoV in [human epithelial airway cell] cultures relative to SARS-CoV Urbani suggest that factors beyond ACE2 binding — including spike processivity, receptor bio-availability or antagonism of the host immune responses — may contribute to emergence.

माउस SARS-2007

SARS-CoV ( Urbani) BALB/c. (MA15), . , , , . , . , , 15, , . MA15 , ; SARS-CoV (rMA15), MA15. 15 , SARS .

We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS.

-2008

, 29,7 — (Bat-SCoV), (SARS), SARS-CoV.
…
, RBDs Bat-SCoV SARS-CoV , RBD Bat-SCoV ( 323–505) RBD SARS-CoV ( 319–518) (27, 28) (GenBank FJ211860), , in vivo (Fig. 1B).

Here, we report the design, synthesis, and recovery of the largest synthetic replicating life form, a 29.7-kb bat severe acute respiratory syndrome (SARS)-like coronavirus (Bat-SCoV), a likely progenitor to the SARS-CoV epidemic.
…
To test whether the RBDs of Bat-SCoV and SARS-CoV were interchangeable, we replaced the Bat-SCoV RBD (amino acid 323–505) with the SARS-CoV RBD (amino acid 319–518) (27, 28) (GenBank accession no. FJ211860), simulating a theoretical recombination event that might occur during mixed infection in vivo (Fig. 1B).

(B) , SARS-CoV Bat-SCoV. . Bat-SRBD Bat-SCoV, , RBD Bat-SCoV ( Bat-SCoV 323–505) RBD SARS-CoV ( 319–518) ( GenBank FJ211860 ). Bat-SRBD-MA RBD MA15 SARS-CoV Y436H. Bat-SRBM 13 SARS-CoV, ACE2, RBD 323I-429T Bat-SCoV 426R-518D SARS-CoV. Bat-Hinge Bat-SRBM, Bat-SCoV 392L-397E SARS-CoV 388V-393D. Bat-F 1–24057 SARS-CoV ( 855 ) 3'- Bat-SCoV. 1- (P1). ND , .

स्रोत इबारत

(B) Schematic representation showing organization of the SARS-CoV and Bat-SCoV Spike proteins. The engineered Spike proteins are pictured below with the virus name to the left. Bat-SRBD includes all of the Bat-SCoV Spike sequence except that the Bat-SCoV RBD (Bat-SCoV amino acid 323–505) is replaced with the SARS-CoV RBD (amino acid 319–518) (GenBank accession no. FJ211860). Bat-SRBD-MA includes the MA15 Spike RBD change at SARS-CoV aa Y436H. Bat-SRBM includes the minimal 13 SARS-CoV residues critical for ACE2 contact, resulting in a chimeric RBD of Bat-SCoV amino acid 323I-429T and SARS-CoV amino acid 426R-518D. Bat-Hinge is Bat-SRBM sequence, with Bat-SCoV amino acid 392L-397E replaced with SARS-CoV amino acid 388V-393D. Bat-F includes nt 1–24057 of SARS-CoV (to Spike amino acid 855), with the remaining 3? sequence from Bat-SCoV. To the right of the schematic representations, observation of transcript activity and approximate stock titers at passage 1 (P1) are indicated. ND indicates no infectious virus detected by plaque assay.

बारिक 2016

SARS-CoV (7), WIV1-CoV, , , , (. S1A). WIV1-CoV, SARS WIV1 (WIV1-MA15, . S1B). … Vero SARS-CoV Urbani, WIV1-MA15 WIV1-CoV.

Using the SARS-CoV infectious clone as a template (7), we designed and synthesized a full-length infectious clone of WIV1-CoV consisting of six plasmids that could be enzymatically cut, ligated together, and electroporated into cells to rescue replication competent progeny virions (Fig. S1A). In addition to the full-length clone, we also produced WIV1-CoV chimeric virus that replaced the SARS spike with the WIV1 spike within the mouse-adapted backbone (WIV1-MA15, Fig. S1B). … To confirm growth kinetics and replication, Vero cells were infected with SARS-CoV Urbani, WIV1-MA15, and WIV1-CoV.

बारिक 1990

इस पूरे अध्ययन के दौरान, माउस हेपेटाइटिस वायरस का तनाव A59 (MHV-A59) का उपयोग किया गया था। वायरस को लगातार माउस एस्ट्रोसाइटोमा सेल लाइन (डीबीटी) में तीन बार प्रचारित किया गया और क्लोन किया गया।
...
तापमान-संवेदनशील म्यूटेंट के विभिन्न संयोजनों को 10 के बराबर संक्रमण की बहुलता के साथ कोशिकाओं में मिश्रित और टीका लगाया गया।

स्रोत इबारत

The A59 strain of mouse hepatitis virus (MHV-A59) was used throughout the course of this study. Virus was propagated and cloned three times in the continuous murine astrocytoma cell line (DBT).
…
Various combinations of ts mutants were mixed and inoculated onto cells at a multiplicity of infection of 10 each.

बारिक 2019

साथ में, इन परिणामों से पता चलता है कि प्रोटीज दरार भी HKU5-CoV के साथ वेरो सेल संक्रमण के लिए एक प्रमुख बाधा है। आगे देखते हुए, हमने M1, PDF2180 में S1 / S2, S2 की सीमा और एंडोसोमल सिस्टीन प्रोटीज साइट पर अनुमानित दरार की तुलना की, और HKU5 स्पाइक-जैसे प्रोटीन (चित्र। 6D) (26)। S1 / S2 साइट पर, MERS, युगांडा और HKU5 RXXR दरार साइट को बनाए रखते हैं, हालांकि विभिन्न आंतरिक अमीनो एसिड इसकी प्रभावशीलता को प्रभावित कर सकते हैं। S2 के अनुक्रम के लिए, MERS और HKU5 भी RXXR रूपांकनों को बनाए रखते हैं; हालाँकि, युगांडा स्ट्रेन के स्पाइक्स में पहला आर्गिनिन (SNAR) अनुपस्थित है, जो संभावित रूप से इसके दरार को प्रभावित करता है।

स्रोत इबारत

Together, these results demonstrate that protease cleavage is also the primary barrier to infection of Vero cells with HKU5-CoV. Examining further, we compared the predicted cleavage at S1/S2 border, S2’, and the endosomal cysteine protease site across MERS, PDF2180, and HKU5 spikes (Fig. 6D) (26). For the S1/S2 site, MERS, Uganda, and HKU5 maintain the RXXR cleavage motif, although the different interior amino acids may alter efficiency. For the S2’ sequence, MERS and HKU5 also retain the RXXR motif; however, the Uganda spike lacks the first arginine (SNAR), potentially impacting cleavage.

लाभ का कार्य: "अस्वीकार जारी नहीं रह सकता है"

, (GOF). (Fig. 4a, b) , SHC014-MA15, , . SHC014-MA15 SARS-CoV, , CoV Urbani MA15, , . , Urbani-MA15 CoV, SHC014-MA15 (. 1). , , , . , . GOF; . , , , .

स्रोत इबारत

In addition to offering preparation against future emerging viruses, this approach must be considered in the context of the US government–mandated pause on gain-of-function (GOF) studies. On the basis of previous models of emergence (Fig. 4a,b), the creation of chimeric viruses such as SHC014-MA15 was not expected to increase pathogenicity. Although SHC014-MA15 is attenuated relative to its parental mouse-adapted SARS-CoV, similar studies examining the pathogenicity of CoVs with the wild-type Urbani spike within the MA15 backbone showed no weight loss in mice and reduced viral replication. Thus, relative to the Urbani spike–MA15 CoV, SHC014-MA15 shows a gain in pathogenesis (Fig. 1). On the basis of these findings, scientific review panels may deem similar studies building chimeric viruses based on circulating strains too risky to pursue, as increased pathogenicity in mammalian models cannot be excluded. Coupled with restrictions on mouse-adapted strains and the development of monoclonal antibodies using escape mutants, research into CoV emergence and therapeutic efficacy may be severely limited moving forward. Together, these data and restrictions represent a crossroads of GOF research concerns; the potential to prepare for and mitigate future outbreaks must be weighed against the risk of creating more dangerous pathogens. In developing policies moving forward, it is important to consider the value of the data generated by these studies and whether these types of chimeric virus studies warrant further investigation versus the inherent risks involved.

— , , BSL-4, . ; , . « ».
…
. BSL-4 ; , , , .

, , BSL-4 — , , — . « , , », — .

BSL-4 . , , , , . , , .

« », — . , : « , , ».

त्रेवन का कहना है कि बीएसएल -4 प्रयोगशाला में चीन का निवेश, सबसे ऊपर, दुनिया को साबित करने का एक तरीका हो सकता है कि देश प्रतिस्पर्धी है। "यह जीव विज्ञान में एक बड़ा दर्जा प्रतीक है," वह कहते हैं, "चाहे वे आवश्यक हों या नहीं।"

स्रोत इबारत

Future plans include studying the pathogen that causes SARS, which also doesn’t require a BSL-4 lab, before moving on to Ebola and the West African Lassa virus, which do. Some one million Chinese people work in Africa; the country needs to be ready for any eventuality, says Yuan. “Viruses don’t know borders.”
…
The plan to expand into a network heightens such concerns. One BSL-4 lab in Harbin is already awaiting accreditation; the next two are expected to be in Beijing and Kunming, the latter focused on using monkey models to study disease.
Lina says that China’s size justifies this scale, and that the opportunity to combine BSL-4 research with an abundance of research monkeys — Chinese researchers face less red tape than those in the West when it comes to research on primates — could be powerful. “If you want to test vaccines or antivirals, you need a non-human primate model,” says Lina.
But Ebright is not convinced of the need for more than one BSL-4 lab in mainland China. He suspects that the expansion there is a reaction to the networks in the United States and Europe, which he says are also unwarranted. He adds that governments will assume that such excess capacity is for the potential development of bioweapons.
“These facilities are inherently dual use,” he says. The prospect of ramping up opportunities to inject monkeys with pathogens also worries, rather than excites, him: “They can run, they can scratch, they can bite.”
Trevan says China’s investment in a BSL-4 lab may, above all, be a way to prove to the world that the nation is competitive. “It is a big status symbol in biology,” he says, “whether it’s a need or not.”

हालांकि, इन निष्कर्षों को मनुष्यों में रोगजनक क्षमता में अनुवाद करने के लिए गैर-अमानवीय प्राइमेट पर आगे परीक्षण की आवश्यकता है। यह ध्यान रखना महत्वपूर्ण है कि उपलब्ध चिकित्सीय एजेंटों की कमी उपचार के तरीकों के आगे के अध्ययन और विकास के लिए महत्वपूर्ण आवश्यकता को निर्धारित करती है। इस ज्ञान के साथ, निगरानी कार्यक्रम, नैदानिक ​​अभिकर्मकों, और प्रभावी उपचार विकसित किए जा सकते हैं जो समूह 2 बी-विशिष्ट कोरोनविर्यूज़ जैसे कि SHC014 की उपस्थिति से रक्षा कर सकते हैं, और अन्य कोरोनोवायर शाखाओं पर लागू किए जा सकते हैं जो विषम पूल का समर्थन करते हैं।

स्रोत इबारत

However, further testing in nonhuman primates is required to translate these finding into pathogenic potential in humans. Importantly, the failure of available therapeutics defines a critical need for further study and for the development of treatments. With this knowledge, surveillance programs, diagnostic reagents and effective treatments can be produced that are protective against the emergence of group 2b–specific CoVs, such as SHC014, and these can be applied to other CoV branches that maintain similarly heterogeneous pools.

आत्मज्ञान की भावना कितने चमत्कारिक महामारियों के बारे में तैयार करती है ...

केवल एक वैज्ञानिक ने वायरस के साथ काम किया, और रेयेस ने कहा कि प्रयोगशाला को संदेह है कि वैज्ञानिक ने नवंबर में गलती से एक टेस्ट ट्यूब फेंक दिया था।
...
गैल्वेस्टोन बायोलबोरेटरी में सबसे कड़े सुरक्षा उपाय हैं क्योंकि यह बीएसएल -4 जैव सुरक्षा सामग्रियों, यानी खतरनाक संक्रामक रोगों का अध्ययन कर रहा है जिनमें टीके या ड्रग नहीं हैं। बीएसएल -4 सामग्री में ग्वारनिटो, इबोला और चेचक शामिल हैं।

स्रोत इबारत

Only one scientist worked with the virus, and Reyes said the lab suspects that scientist accidentally threw the vial away in November.
…
Galveston biolab requires the most stringent safety measures because it studies biosafetly level BSL-4 materials, or dangerous infectious diseases that have no vaccines or cures. BSL-4 materials include Guanarito, Ebola and smallpox.

H1N1 1918 , ( 1947 ), 1957 «» H2N2, . H1N1, -, , 20 . 1969 H3N2 H2N2 .

1977 . H1N1 , 1978 . , 1977 . H1N1 - , , . , , , H1N1 1977 H1N1, 1949–1950 , , .
…
2009–2010 . , H1N1 1977 : « — A H1N1, 1977 ».
…
, 1977 . H1N1, , « » (Kung 1978). , , , 1970- ( ) . .

, , 1977–78 . - , , -, H1N1 1978 . H1N1, , . 1976–77 20 H1N1 1957, . 1977 , .

Human influenza H1N1 viruses appeared with the 1918 pandemic, and persisted, slowing accumulating small changes in its genome (with a major change in 1947), until the H2N2 “Asian” flu appeared in 1957, causing a worldwide pandemic. H1N1 influenza virus then apparently became extinct, and was not isolated for 20 years. In 1969 the “Hong Kong” H3N2 virus replaced the H2N2 virus, and is still circulating.
In September 1977 an H1N1 influenza virus was isolated from human infections in the Far East region of the Soviet Union, and in early 1978 the Chinese reported they had isolated H1N1 virus in May of 1977 in northeast China adjacent to the Soviet outbreak. Using the early genetic tools available at the time, the 1977 H1N1 virus was found to be closely related to H1N1 human influenza viruses circulating in 1949–1950, but not to those circulating earlier or later.
…
Only since 2009–2010 did major papers begin to state directly the 1977 emergence of H1N1 influenza was a laboratory related release: “The most famous case of a released laboratory strain is the re-emergent H1N1 influenza A virus which was first observed in China in May of 1977 and in Russia shortly thereafter.”
…
The speculation that the 1977 release may have been related to H1N1 vaccine research is supported by the observation that in the initial outbreaks in China, nine of the ten viral isolates expressed “temperature sensitivity” (Kung 1978). Temperature sensitivity normally an uncommon trait, but one that was in the 1970s (and still is) a fundamental trait for making live attenuated influenza vaccines. Temperature sensitivity generally occurs only after a series of substantial laboratory manipulations and selections.
Interestingly, further investigation indicated the circulating strains in 1977–78 were often comprised of mixed temperature-sensitive and normal components, and that temperature sensitivity apparently disappeared from the post-1978 H1N1 lineage rapidly. Escape of a mid-protocol population of H1N1 virus undergoing laboratory selection for temperature sensitive mutants would provide such a mixed population. In 1976–77 laboratory personnel in their late teens or early 20s would not have been exposed to pre-1957 H1N1 influenza viruses, and been susceptible to laboratory infections. The low severity of the 1977 pandemic might be in part due to the temperature sensitivity of the virus, a trait that limits virus replication in pulmonary tissues.

टेलीग्राम के सूत्रों ने कहा कि उन्हें WIV लैब में गंभीर सुरक्षा मुद्दों के बारे में अलार्म उठाना चाहिए, विशेष रूप से बैट कोरोनवीरस के साथ इसके काम के बारे में। दूतावास के अधिकारियों ने संयुक्त राज्य अमेरिका से इस प्रयोगशाला पर अधिक ध्यान देने और अतिरिक्त सहायता प्रदान करने का आह्वान किया।
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« WIV , , », — 19 2018 , , , WIV. ( .)
WIV के चीनी शोधकर्ताओं ने टेक्सास विश्वविद्यालय के चिकित्सा विभाग और संयुक्त राज्य अमेरिका में अन्य संगठनों के गैल्वेस्टन राष्ट्रीय प्रयोगशाला से मदद प्राप्त की, लेकिन चीनियों ने अतिरिक्त मदद मांगी। टेलीग्राम का तर्क है कि संयुक्त राज्य अमेरिका को वुहान प्रयोगशाला को और सहायता प्रदान करनी चाहिए, मुख्य रूप से क्योंकि उनके बल्ले कोरोनवीरस का अध्ययन महत्वपूर्ण था, लेकिन यह भी खतरनाक था।

स्रोत इबारत

Sources familiar with the cables said they were meant to sound an alarm about the grave safety concerns at the WIV lab, especially regarding its work with bat coronaviruses. The embassy officials were calling for more U.S. attention to this lab and more support for it, to help it fix its problems.
…
“During interactions with scientists at the WIV laboratory, they noted the new lab has a serious shortage of appropriately trained technicians and investigators needed to safely operate this high-containment laboratory,” states the Jan. 19, 2018, cable, which was drafted by two officials from the embassy’s environment, science and health sections who met with the WIV scientists. (The State Department declined to comment on this and other details of the story.)
The Chinese researchers at WIV were receiving assistance from the Galveston National Laboratory at the University of Texas Medical Branch and other U.S. organizations, but the Chinese requested additional help. The cables argued that the United States should give the Wuhan lab further support, mainly because its research on bat coronaviruses was important but also dangerous.

मानव निर्मित के संभावित निशान

SARS-CoV Bat-SCoV.
(A) SARS-CoV Bat-SCoV ( GenBank FJ211859) . () nsp ORF1ab ( , - ; , nsp5 [3C- ]). , , A F. , Bat-SCoV, SARS-CoV, , Bat-SCoV 2 , Bat-E1 Bat-E2, SARS-E.

Schematic representation of SARS-CoV and Bat-SCoV variants.
(A) Schematic representation of SARS-CoV and Bat-SCoV (GenBank accession no. FJ211859) genomes and reverse genetics system. (Top) Arrowheads indicate nsp processing sites within the ORF1ab polyprotein (open arrowheads, papain-like proteinase mediated; filled arrowheads, nsp5 [3C-like proteinase] mediated). Immediately below are the fragments used in the reverse genetics system, labeled A through F. The fragments synthesized to generate Bat-SCoV exactly recapitulate the fragment junctions of SARS-CoV with the exception that the Bat-SCoV has 2 fragments, Bat-E1 and Bat-E2, which correspond to the SARS-E fragment.

, - , SARS-CoV (24, 33, 53) . , Bat-F A-E SARS-CoV F Bat-SCoV, Bgl-NotI. Bat-SCoV Bat-SRBD, Bat-SRBM Bat-Hinge , 7 Bat-SCoV, BglI Bat-A, Bat-B, Bat-C Bat -D, BglI AflII Bat-E1 Bat-E2 BglI NotI Bat-F. , . m7Message mMachine (Ambion), Vero (24, 53).

Viruses containing PCR-generated insertions within the viral coding sequence were produced by using the SARS-CoV assembly strategy (24, 33, 53) with the following modifications. Briefly, for Bat-F virus, full-length cDNA was constructed by ligating restriction products from SARS-CoV fragments A–E and Bat-SCoV fragment F, which required a BglI-NotI digestion. For Bat-SCoV and Bat-SRBD, Bat-SRBM, and Bat-Hinge, plasmids containing the 7 cDNA fragments of the Bat-SCoV genome were digested by using BglI for Bat-A, Bat-B, Bat-C, and Bat-D, BglI and AflII for Bat-E1 and Bat-E2, and BglI and NotI for Bat-F. Digested, gel-purified fragments were simultaneously ligated together. Transcription was driven by using a T7 mMessage mMachine kit (Ambion), and RNA was electroporated into Vero cells (24, 53).

प्रत्येक cDNA के सिरों पर स्थित प्रतिबंध साइटों के जंक्शन साइटों को व्यवस्थित रूप से पूर्ण पूर्ण-आकार के सीडीएनए उत्पाद के संयोजन के दौरान हटा दिया जाता है, जो न्यूक्लियोटाइड्स के संशोधन के बिना पुनर्मूल्यांकन की अनुमति देता है।

स्रोत इबारत

The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes.

सर्वसम्मति क्लोन को जल्दी से इकट्ठा करने के लिए, हमने वर्ग IIS प्रतिबंध एंडोन्यूक्लाइजेस का उपयोग किया, जो असममित क्षेत्रों में कट जाता है और असममित छोरों को छोड़ देता है। ये एंजाइम विशिष्ट विशिष्ट ओवरहैंग उत्पन्न करते हैं जो प्रतिबंध स्थल के बाद के नुकसान के साथ दो सीडीएनए के सहज बंधन प्रदान करते हैं ।

स्रोत इबारत

To rapidly assemble consensus clones, we used class IIS restriction endonucleases that cut at asymmetric sites and leave asymmetric ends. These enzymes generate strand-specific unique overhangs that allow the seamless ligation of two cDNAs with the concomitant loss of the restriction site.

2.2। एक पुनः संयोजक वायरस का निर्माण पुनः
संयोजक विषाणु ryn-S2 / RRKR, जिसमें एक फरिन साइट S2 के साथ एक स्पाइक प्रोटीन होता है, को वैक्सीनिया पुनर्संयोजन द्वारा प्राप्त किया गया था, जैसा कि पहले वर्णित था [20, 28]। संक्षेप में, एक एस in फरिन साइट प्लाज्मिड को सीमलेस असेंबली किट (इनविट्रोजन, कार्ल्सबैड, सीए, यूएसए) का उपयोग करके उत्पन्न किया गया था और सीवी -1 कोशिकाओं में ट्रांसफ़ेक्ट किया गया था जो वाईएन- एस-जीपीटी जीन युक्त वैक्सीनिया वायरस से संक्रमित थे। फ़्यूरिन-एस 2 साइट को एक अस्थायी प्रमुख चयन प्रणाली [25] का उपयोग करके समरूप पुनर्संयोजन द्वारा YN cDNA में पेश किया गया था।

स्रोत इबारत

2.2. Generation of Recombinant Virus
Recombinant rYN-S2/RRKR virus containing an S protein with the furin-S2? site was generated by vaccinia recombination, as described previously [20,28]. Briefly, plasmid with the furin-S2? site was generated using the Seamless Assembly kit (Invitrogen, Carlsbad, CA, USA) and transfected into CV-1 cells infected by vaccinia virus containing the genome of YN-?S-GPT. Furin-S2’ site was introduced into the YN cDNA by homologous recombination using the transient dominant selection system [25].

GeneArt 1 4 , , 30- . E.coli .
• — 4 ( 13 ); ,
• — , , , 90% .
• —
• — - -,
• — , , .

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कोडन प्राथमिकताएं

लेकिन प्रकृति में मानव-पागलपन के खंडन के बारे में क्या?

, SARS-CoV-2 ACE2 , , , RBD SARS-CoV [ SARS — ..] . , SARS-CoV-2 ACE2, , ACE2, . , SARS-CoV-2 .

While the analyses above suggest that SARS-CoV-2 may bind human ACE2 with high affinity, computational analyses predict that the interaction is not ideal and that the RBD sequence is different from those shown in SARS-CoV to be optimal for receptor binding. Thus, the high-affinity binding of the SARS-CoV-2 spike protein to human ACE2 is most likely the result of natural selection on a human or human-like ACE2 that permits another optimal binding solution to arise. This is strong evidence that SARS-CoV-2 is not the product of purposeful manipulation.

, , , , -. , , SARS-CoV-2 - .

Furthermore, if genetic manipulation had been performed, one of the several reverse-genetic systems available for betacoronaviruses would probably have been used. However, the genetic data irrefutably show that SARS-CoV-2 is not derived from any previously used virus backbone.

, O- . in vitro in vivo. , SARS-CoV-2 - , . ACE2, , .

The acquisition of both the polybasic cleavage site and predicted O-linked glycans also argues against culture-based scenarios. New polybasic cleavage sites have been observed only after prolonged passage of low-pathogenicity avian influenza virus in vitro or in vivo. Furthermore, a hypothetical generation of SARS-CoV-2 by cell culture or animal passage would have required prior isolation of a progenitor virus with very high genetic similarity, which has not been described. Subsequent generation of a polybasic cleavage site would have then required repeated passage in cell culture or animals with ACE2 receptors similar to those of humans, but such work has also not previously been described.

शी झेंगली 2020

, SARS-CoV-2 , SARS-CoV, , SARS-CoV-2 .
…
, β- S1/S2, . , SARS-CoV , L . S1/S2 SARS-CoV .

In this study, we have shown that SARS-CoV-2 exhibits much higher capacity of membrane fusion than SARS-CoV, suggesting that the fusion machinery of SARS-CoV-2 is an important target for development of coronavirus fusion inhibitors.
…
Generally, β-B coronaviruses lack the S1/S2 furin-recognition site, and their S proteins are uncleaved in the native state. For example, SARS-CoV enters into the cell mainly via the endosomal membrane fusion pathway where its S protein is cleaved by endosomal cathepsin L and activated. Inducing the S1/S2 furin-recognition site could significantly increase the capacity of SARS-CoV S protein to mediate cellular membrane surface infection.

यह अंत है, सुंदर मित्र

SARS-CoV-2 के प्राणी कौन से प्राणी हैं?
ऐसे जानवर अभी तक नहीं मिले हैं। इस बात के सबूत हैं कि पैंगोलिन संभावित रूप से एक मध्यवर्ती मेजबान हो सकता है, लेकिन पैंगोलिन वायरस SARS-CoV-2 के समान 88-98% हैं। तुलना के लिए, पहले SARS के कीवेट और एक प्रकार का वृक्ष कोरोनवीरस के उपभेद 2003 SARS-CoV उपभेदों के समान 99.8% थे। दूसरे शब्दों में, हम 2003 में सिवनी, एक प्रकार का जानवर और मनुष्यों के उपभेदों के बीच कई उत्परिवर्तन के बारे में बात कर रहे हैं। पैंगोलिन [CoV2 उपभेदों] में 3,000 से अधिक न्यूक्लियोटाइड परिवर्तन होते हैं, इसलिए पैंगोलिन किसी भी तरह से जलाशय की प्रजाति नहीं हो सकते हैं। बिल्कुल मौका नहीं।

स्रोत इबारत

What is the reservoir species of SARS-CoV-2?
They have not identified the actual reservoir species. Reports show that pangolins are potentially the intermediate host, but pangolin viruses are 88–98% identical to SARS-CoV-2. In comparison, civet and racoon dog strains of SARS coronaviruses were 99.8% identical to SARS-CoV from 2003. In other words, we are talking about a handful of mutations between civet strains, racoon dog strains and human strains in 2003. Pangolin [strains of CoV2] have over 3000 nucleotide changes, no way they are the reservoir species. Absolutely no chance.

UPD: RaTG13 और Cov2 के जीनोम के बीच के अंतर का लगभग 4%

. 10-4 , -. , 6 . in vitro , , , .

We obtained an estimate of the spontaneous mutation rate of ca. 10^-4 substitutions per site or lower, a value within the typically accepted range for RNA viruses. A roughly threefold increase in mutation rate and a significant shift in mutation spectrum were observed in samples from patients undergoing 6 months of interferon plus ribavirin treatment. This result is consistent with the known in vitro mutagenic effect of ribavirin and suggests that the antiviral effect of ribavirin plus interferon treatment is at least partly exerted through lethal mutagenesis.

UPD2: RaTG13 - RaBtCoV / 4991 के समान तनाव?

चीन के युन्नान के दक्षिण में पुएर शहर के अधिकार क्षेत्र के तहत मुइज़ियांग स्वायत्त क्षेत्र एक स्वायत्त क्षेत्र है। विकिपीडिया