Sars milagroso? Genealogía del coronavirus de Wuhan

Minuto de atención ovni

La pandemia COVID-19, una infección respiratoria aguda potencialmente grave causada por el coronavirus SARS-CoV-2 (2019-nCoV), se ha anunciado oficialmente en el mundo. Hay mucha información sobre Habré sobre este tema; recuerde siempre que puede ser confiable / útil, y viceversa.

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Shi Zhengli en su laboratorio en el Instituto de Virología Wuhan

Biología

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.

Pangolines

, , - 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.

Recibimos muestras de tejidos congelados (pulmones, intestinos, sangre) que se tomaron de 18 pangolines malaya ( Manis javanica ) durante agosto de 2017 - enero de 2018. Estos pangolines fueron obtenidos durante las operaciones contra el contrabando por la Aduana de Guangxi. Es sorprendente que la secuenciación de alto rendimiento de su ARN revelara la presencia de coronavirus en seis (dos pulmones, dos intestinos, una mezcla de pulmones e intestinos, una sangre) de 43 muestras.
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, , , 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).
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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).

Curiosamente, el análisis filogenético de solo sitios sinónimos en RBD mostró que la posición filogenética del pangolín de guangdong es consistente con la del resto del genoma viral, es decir, que no es el pariente más cercano de 2019-CoV2 (Figura 2b). Por lo tanto, es posible que la similitud de los aminoácidos en la RBD de los coronavirus de pangolín y el 2019-CoV2 se deba a una evolución convergente mediada selectivamente en lugar de la recombinación, aunque es difícil elegir entre estos escenarios en función de los datos disponibles.

Texto de origen

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.

Genealogía de personas coronadas

Estoy golpeando pulcramente, pero fuerte

, , , , , 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.

Se descubrió que todas las proteínas de pico con una homología de la proteína SARS-CoV-2 superior al 40% no tenían un sitio de escisión de furina (Figura 1, Tabla 1), incluidos Bat-CoV RaTG13 y SARS-CoV (con una identidad de secuencia de 97.4 % y 78.6%, respectivamente). El sitio de RRAR furin en SARS-CoV-2 es único en su familia gracias al inserto PRRA único. Este sitio en SARS-CoV-2 difícilmente podría haber evolucionado de MERS, HCoV-HKU1, etc. A partir de las secuencias actualmente disponibles en las bases de datos, nos resulta difícil encontrar la fuente. Quizás haya muchas más secuencias intermedias evolutivas en espera de descubrimiento.

Texto de origen

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.

Para investigar si la escisión proteolítica en la secuencia de los principales residuos de aminoácidos puede promover la actividad de fusión celular, mutamos la proteína SARS-CoV con forma de espiga para crear un sitio de reconocimiento de furina (RRSRR) en uno de dos lugares.

Texto de origen

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.

Para explorar el uso potencial de las posiciones SARS-CoV S1-S2 y S2 'como sitios para la escisión proteolítica, primero introdujimos sitios de reconocimiento de escisión de furina en estos sitios haciendo las siguientes mutaciones 664-SLLRSTSQSI - SLL RRSRR SI-671 (S1-S2) y 792-LKPTKRSF-LKRTKRSF-799 (S2 ').

Texto de origen

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?).

Beijing 2019

S2' QX , , IBV (WT-IBV). , IBV S2 ( -S2) . IBV .
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, , 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.
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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.
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, 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.
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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.

Brevemente, se obtuvieron retrovirus pseudotipados con pico de MERS-CoV que expresan el gen indicador de luciferasa por cotransfección de células HEK293T con un plásmido que porta el gen VIH-1, luciferasa de expresión defectuosa Env (pNL4-3.luc.RE-) y un plásmido que codifica MERS -Cop proteína de pico.

Texto de origen

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.

¿De dónde vino RaTG13?

, - - (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%.

Los murciélagos fueron atrapados en varios lugares en cinco distritos de las cuatro prefecturas de Yunnan, China, de mayo a julio de 2013.

Texto de origen

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

Wuhan-1

Lo más importante es que informamos el primer aislamiento del virus SL-CoV vivo (murciélago SL-CoV-WIV1) a partir de muestras aisladas de heces de murciélago en células Vero E6, que tiene una morfología típica de coronavirus, 99,9% de identidad genómica. Rs3367 y utiliza personas ACE2, huesos de civeta y herradura para ingresar a la célula. Las pruebas preliminares in vitro muestran que WIV1 también tiene un amplio tropismo de especies.

Texto de origen

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.

Otras cepas de Yunnan

1999: primer coronavirus quimérico

Usando la recombinación de ARN dirigida, construimos un mutante del virus de la hepatitis del ratón coronavirus (MHV), en el que el ectodominio en forma de columna fue reemplazado por el ectodominio altamente divergente de la proteína del virus de la peritonitis infecciosa felina. El virus quimérico resultante, designado fMHV, adquirió la capacidad de infectar células felinas y al mismo tiempo perdió la capacidad de infectar células de ratón en cultivo.

Texto de origen

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.

Solo un pequeño número de mutaciones en su proteína con forma de espiga permite que el coronavirus de ratón cambie del tropismo limitado por el ratón a un rango extendido de hospedador por pasivación in vitro. Uno de esos virus que estudiamos adquirió dos supuestos sitios de unión de heparán sulfato, mientras que preservaba el sitio de furina en otros lugares.

Texto de origen

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.

Para comprender mejor la adaptabilidad de las especies MERS-CoV, utilizamos la variante DPP4 subóptima para estudiar la adaptación viral. El paso del virus en las células que expresan esta variante DPP4 dio como resultado la acumulación de mutaciones en la proteína de la punta viral, lo que aumentó la replicación.

Texto de origen

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.

(F) Esquema de la aparición de mutaciones simples y dobles en la proteína espiga MERS-CoV en diferentes pasajes.
(G) Ubicación de mutaciones en la proteína espiga MERS-CoV.

Texto de origen

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

Cómo Barik iluminó el camino para todos

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

Texto de origen

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.

SARS-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.

SARS-2006

Urbani SARS-CoV (BAC). . , Escherichia coli.
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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.
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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.

Wuhan 2007

Se han construido varias proteínas de pico quiméricas insertando varias secuencias de proteína de pico de SARS-CoV en el esqueleto de la proteína SL-CoV.

Texto de origen

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

A partir de estos resultados, se descubrió que la región de 310 a 518 en la proteína de pico BJ01 era necesaria y suficiente para obtener la capacidad de la proteína de pico Rp3 para unirse al ACE2 humano.

Texto de origen

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.

Para insertar RBM de SARS-CoV en la proteína de pico SL-CoV, la región de codificación de 424 a 494 aminoácidos en la proteína de pico BJ01 se usó para reemplazar las regiones correspondientes en la proteína Rp3, dando como resultado un gen de proteína de pico quimérico (CS), designado como CS424 –494.

Texto de origen

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.

Quimera 2015

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

Texto de origen

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.

(a) Esquema del clon molecular SHC014-CoV, que se sintetizó en forma de seis segmentos contiguos de ADNc (designados como SHC014A, SHC014B, SHC014C, SHC014D, SHC014E y SHC014F) flanqueados por sitios únicos de enzimas de restricción BglI que proporcionaron ensamblaje direccional para el ADN 1a, 1b, espiga, 3, envoltura, matriz, 6–8 y nucleocápside). Los nucleótidos subrayados son secuencias sobresalientes formadas después de la digestión con enzimas de restricción.

Texto de origen

(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.

Es de destacar que el tropismo pulmonar diferencial en comparación con el del SARS-MA15 y el debilitamiento de SHC014-CoV de longitud completa en cultivos [vías respiratorias epiteliales humanas] en relación con el SARS-CoV Urbani sugieren que, además de unirse al ACE2, otros factores, incluida la procesividad de la proteína espiga , la biodisponibilidad del receptor o el antagonismo de las respuestas inmunes del huésped pueden contribuir a la virulencia.

Texto de origen

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.

Ratón 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.
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, 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.
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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 , .

Texto de origen

(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.

Barik 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.

Barik 1990

A lo largo de este estudio, se utilizó la cepa del virus de la hepatitis A59 de ratón (MHV-A59). El virus se propagó y clonó tres veces en una línea celular continua de astrocitoma de ratón (DBT).
...
Varias combinaciones de mutantes sensibles a la temperatura se mezclaron e inocularon en células con una multiplicidad de infección igual a 10.

Texto de origen

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.

Barik 2019

Juntos, estos resultados demuestran que la escisión de la proteasa también es una barrera importante para la infección de células Vero con HKU5-CoV. Mirando más allá, comparamos la escisión pronosticada en el borde S1 / S2, S2 'y el sitio de cisteína proteasa endosomal en las proteínas tipo espiga MERS, PDF2180 y HKU5 (Fig. 6D) (26). En el sitio S1 / S2, MERS, Uganda y HKU5 mantienen el sitio de escisión RXXR, aunque varios aminoácidos internos pueden afectar su efectividad. Para la secuencia S2 ', MERS y HKU5 también retienen el motivo RXXR; sin embargo, la primera arginina (SNAR) está ausente en los picos de la cepa de Uganda, lo que potencialmente afecta su escisión.

Texto de origen

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.

Ganancia de función: "Denegar no puede continuar"

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

Texto de origen

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 . , , , , . , , .

« », — . , : « , , ».

Trevan dice que la inversión de China en el laboratorio BSL-4 puede, sobre todo, ser una forma de demostrar al mundo que el país es competitivo. "Este es un gran símbolo de estado en biología", dice, "independientemente de si son necesarios o no".

Texto de origen

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.”

Sin embargo, se requieren más pruebas en primates no humanos para traducir estos hallazgos en potencial patogénico en humanos. Es importante tener en cuenta que la falta de agentes terapéuticos disponibles determina la necesidad crítica de seguir estudiando y desarrollando métodos de tratamiento. Con este conocimiento, se pueden desarrollar programas de vigilancia, reactivos de diagnóstico y tratamientos efectivos que pueden proteger contra la aparición de coronavirus específicos del grupo 2b, como SHC014, y se pueden aplicar a otras ramas de coronavirus que admiten grupos heterogéneos similares.

Texto de origen

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.

Acerca de cuántas epidemias milagrosas prepara el espíritu de la iluminación ...

Solo un científico trabajó con el virus, y Reyes dijo que el laboratorio sospecha que el científico arrojó accidentalmente un tubo de ensayo en noviembre.
...
El Biolaboratorio de Galveston tiene las medidas de seguridad más estrictas porque está estudiando materiales de bioseguridad BSL-4, es decir, enfermedades infecciosas peligrosas que no tienen vacunas ni medicamentos. Los materiales BSL-4 incluyen Guanarito, Ébola y viruela.

Texto de origen

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.

Las fuentes de Telegram dijeron que deberían haber alertado sobre serios problemas de seguridad en el laboratorio de WIV, especialmente con respecto a su trabajo con coronavirus de murciélago. Los funcionarios de la embajada pidieron a los Estados Unidos que presten más atención a este laboratorio y brinden apoyo adicional.
...
« WIV , , », — 19 2018 , , , WIV. ( .)
Los investigadores chinos en WIV recibieron ayuda del Laboratorio Nacional de Galveston en el Departamento Médico de la Universidad de Texas y otras organizaciones en los Estados Unidos, pero los chinos buscaron ayuda adicional. Los telegramas argumentan que Estados Unidos debería brindar más apoyo al laboratorio de Wuhan, principalmente porque su estudio de los coronavirus de murciélago fue importante, pero también peligroso.

Texto de origen

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.

Posibles rastros de los hechos por el hombre.

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).

Los sitios de unión de los sitios de restricción que se encuentran en los extremos de cada ADNc se eliminan sistemáticamente durante el ensamblaje del producto completo de ADNc de tamaño completo, lo que permite el reensamblaje sin modificación de nucleótidos.

Texto de origen

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.

Para ensamblar rápidamente clones de consenso, utilizamos endonucleasas de restricción de clase IIS, que se cortan en regiones asimétricas y dejan extremos asimétricos. Estas enzimas generan voladizos específicos específicos que proporcionan la ligadura perfecta de dos ADNc con la consiguiente pérdida del sitio de restricción .

Texto de origen

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. Creación de un virus recombinante El virus
recombinante rYN-S2 / RRKR, que contiene una proteína espiga con sitio de furina S2 ', se obtuvo por recombinación de vacuna, como se describió anteriormente [20, 28]. Brevemente, se generó un plásmido de sitio de furina S 'usando el kit Seamless Assembly (Invitrogen, Carlsbad, CA, EE. UU.) Y se transfectó en células CV-1 infectadas con el virus vaccinia que contiene el gen YN-? S-GPT. El sitio Furin-S2 se introdujo en el ADNc YN por recombinación homóloga utilizando un sistema de selección dominante temporal [25].

Texto de origen

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% .
• —
• — - -,
• — , , .

The GeneArt Seamless Cloning and Assembly Kit enables the simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute room temperature reaction. The kit contains everything required for the assembly of DNA fragments, and their transformation into E. coli for selection and growth of recombinant vectors.
• Speed and Ease?—?Clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector (up to 13 Kb); no restriction digestion, ligation or recombination sites required
• Precision and Efficiency — Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
• Vector Flexibility — Use our linear vector or a vector of your choice
• Free Tools — Design DNA oligos and more with our free web-based interface that walks you step-by-step through your project
• Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

Preferencias de codones

Pero, ¿qué pasa con la refutación de la creación humana en la naturaleza?

, 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.

Shi Zhengli 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.

Este es el final, hermosa amiga

¿Qué animales son portadores zoonóticos de SARS-CoV-2?
Tales animales aún no se han encontrado. Existe evidencia de que los pangolines podrían ser potencialmente un huésped intermedio, pero los virus de pangolín son solo 88-98% idénticos al SARS-CoV-2. A modo de comparación, las cepas de coronavirus de civeta y mapache del primer SARS fueron 99.8% idénticas a las cepas de SARS-CoV de 2003. En otras palabras, estamos hablando de varias mutaciones entre cepas de civeta, mapache y humanos en 2003. Las pangolinas [cepas CoV2] tienen más de 3.000 cambios de nucleótidos, por lo que las pangolinas no pueden ser una especie de reservorio. Absolutamente no hay posibilidad.

Texto de origen

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: aproximadamente el 4% de la diferencia entre los genomas de RaTG13 y Cov2

. 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: ¿la misma cepa que RaBtCoV / 4991?

La Región Autónoma de Mujiang es una región autónoma bajo la jurisdicción de la ciudad de Puer, en el sur de Yunnan, China. Wikipedia