The accuracy of reverse genetics systems for SARS-CoV-2: Circular polymerase extension reaction versus bacterial artificial chromosome.

Background: Reverse genetics systems to rescue viruses from modified DNA are useful tools to investigate the molecular mechanisms of viruses. The COVID-19 pandemic prompted the development of several reverse genetics systems for SARS-CoV-2. The circular polymerase extension reaction (CPER) method enables the rapid generation of recombinant SARS-CoV-2; however, such PCR-based approaches could introduce unwanted mutations due to PCR errors. Methods: To compare the accuracy of CPER and a classic reverse genetics method using bacterial artificial chromosome (BAC), SARS-CoV-2 Wuhan/Hu-1/2019 was generated five times using BAC and five times using CPER. These 10 independent virus stocks were then deep sequencing, and the number of substitutions for which the frequency was greater than 10% was counted. Results: No nucleotide substitutions with a frequency of greater than 10% were observed in all five independent virus stocks generated by the BAC method. In contrast, three to five unwanted nucleotide substitutions with a frequency of more than 10% were detected in four of the five virus stocks generated by the CPER. Furthermore, four substitutions with frequencies greater than 20% were generated in three virus stocks by using the CPER. Conclusions: We found that the accuracy of the CPER method is lower than that of the BAC method. Our findings suggest care should be used when employing the CPER method.

Reinventing positive-strand RNA virus reverse genetics.

Reverse genetics is the prospective analysis of how genotype determines phenotype. In a typical experiment, a researcher alters a viral genome, then observes the phenotypic outcome. Among RNA viruses, this approach was first applied to positive-strand RNA viruses in the mid-1970s and over nearly 50 years has become a powerful and widely used approach for dissecting the mechanisms of viral replication and pathogenesis. During this time the global health importance of two virus groups, flaviviruses (genus Flavivirus, family Flaviviridae) and betacoronaviruses (genus Betacoronavirus, subfamily Orthocoronavirinae, family Coronaviridae), have dramatically increased, yet these viruses have genomes that are technically challenging to manipulate. As a result, several new techniques have been developed to overcome these challenges. Here I briefly review key historical aspects of positive-strand RNA virus reverse genetics, describe some recent reverse genetic innovations, particularly as applied to flaviviruses and coronaviruses, and discuss their benefits and limitations within the larger context of rigorous genetic analysis.

Versatile SARS-CoV-2 Reverse-Genetics Systems for the Study of Antiviral Resistance and Replication.

The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L-and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12-did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies-providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.

An attenuated TW-like infectious bronchitis virus strain has potential to become a candidate vaccine and S gene is responsible for its attenuation.

TW-like infectious bronchitis virus (IBV) with high pathogenicity is becoming the predominant IBV type circulating in China. To develop vaccines against TW-like IBV strains and investigate the critical genes associated with their virulence, GD strain was attenuated by 140 serial passages in specific-pathogen-free embryonated eggs and the safety and efficacy of the attenuated GD strain (aGD) were examined. The genome sequences of GD and aGD were also compared and the effects of mutations in the S gene were observed. The results revealed that aGD strain showed no obvious pathogenicity with superior protective efficacy against TW-like and QX-like virulent IBV strains. The genomes of strains aGD and GD shared high similarity (99.87 %) and most of the mutations occurred in S gene. Recombinant IBV strain rGDaGD-S, in which the S gene was replaced with the corresponding regions from aGD, showed decreased pathogenicity compared with its parental strain. In conclusion, attenuated TW-like IBV strain aGD is a potential vaccine candidate and the S gene is responsible for its attenuation. Our research has laid the foundation for future exploration of the attenuating molecular mechanism of IBV.

Infectious Clones Produce SARS-CoV-2 That Causes Severe Pulmonary Disease in Infected K18-Human ACE2 Mice.

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has caused extensive mortality and morbidity and wreaked havoc on socioeconomic structures. The urgent need to better understand SARS-CoV-2 biology and enable continued development of effective countermeasures is aided by the production of laboratory tools that facilitate SARS-CoV-2 research. We previously created a directly accessible SARS-CoV-2 toolkit containing user-friendly reverse genetic (RG) infectious clones of SARS-CoV-2. Here, using K18-human ACE2 (hACE2) mice, we confirmed the validity of RG-rescued SARS-CoV-2 viruses to reproduce the infection profile, clinical disease, and pathogenesis already established in mice infected with natural SARS-CoV-2 isolates, often patient derived. RG-rescued SARS-CoV-2-infected K18-hACE2 mice developed substantial clinical disease and weight loss by day 6 postinfection. RG-rescued SARS-CoV-2 was recovered from the lungs and brains of infected K18-hACE2 mice, and infection resulted in viral pneumonia with considerable changes in lung pathology, as seen previously with natural SARS-CoV-2 infection. In mice infected with RG-rescued SARS-CoV-2-mCherry, mCherry was detected in areas of lung consolidation and colocalized with clinically relevant SARS-CoV-2-assocated immunopathology. RG-rescued SARS-CoV-2 viruses successfully recapitulated many of the features of severe COVID-19 associated with the K18-hACE2 model of SARS-CoV-2 infection. With utility in vivo, the RG-rescued SARS-CoV-2 viruses will be valuable resources to advance numerous areas of SARS-CoV-2 basic research and COVID-19 vaccine development.IMPORTANCE To develop COVID-19 countermeasures, powerful research tools are essential. We produced a SARS-COV-2 reverse genetic (RG) infectious clone toolkit that will benefit a variety of investigations. In this study, we further prove the toolkit's value by demonstrating the in vivo utility of RG-rescued SARS-CoV-2 isolates. RG-rescued SARS-CoV-2 isolates reproduce disease signs and pathology characteristic of the K18-hACE2 mouse model of severe COVID-19 in infected mice. Having been validated as a model of severe COVID-19 previously using only natural SARS-CoV-2 isolated from patients, this is the first investigation of RG-rescued SARS-CoV-2 viruses in K18-hACE2 mice. The RG-rescued SARS-CoV-2 viruses will facilitate basic understanding of SARS-CoV-2 and the preclinical development of COVID-19 therapeutics.

Engineering SARS-CoV-2 using a reverse genetic system.

Reverse genetic systems are a critical tool for studying viruses and identifying countermeasures. In response to the ongoing COVID-19 pandemic, we recently developed an infectious complementary DNA (cDNA) clone for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse genetic system can be used to rapidly engineer viruses with desired mutations to study the virus in vitro and in vivo. Viruses can also be designed for live-attenuated vaccine development and engineered with reporter genes to facilitate serodiagnosis, vaccine evaluation and antiviral screening. Thus, the reverse genetic system of SARS-CoV-2 will be widely used for both basic and translational research. However, due to the large size of the coronavirus genome (~30,000 nucleotides long) and several toxic genomic elements, manipulation of the reverse genetic system of SARS-COV-2 is not a trivial task and requires sophisticated methods. Here, we describe the technical details of how to engineer recombinant SARS-CoV-2. Overall, the process includes six steps: (i) prepare seven plasmids containing SARS-CoV-2 cDNA fragment(s), (ii) prepare high-quality DNA fragments through restriction enzyme digestion of the seven plasmids, (iii) assemble the seven cDNA fragments into a genome-length cDNA, (iv) in vitro transcribe RNA from the genome-length cDNA, (iv) electroporate the genome-length RNA into cells to recover recombinant viruses and (vi) characterize the rescued viruses. This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research.

The RNA Architecture of the SARS-CoV-2 3'-Untranslated Region.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3' untranslated region (UTR) of this ß-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3' UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3' UTRs in the infectious virions and the minigene-transfected cells are almost identical.

[A race against the clock: creation of SARS-Cov-2 in the laboratory, a month after its emergence!] / Une course contre la montre - Création du SARS-CoV-2 en laboratoire, un mois après son émergence !

SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2, which emerged in China at the end of 2019, is responsible for a global health crisis resulting in the confinement of more than 3 billion people worldwide and the sharp decline of the world economy. In this context, a race against the clock is launched in order to develop a treatment to stop the pandemic as soon as possible. A study published in Nature by the Volker Thiel team reports the development of reverse genetics for SARS-CoV-2 allowing them to recreate the virus in just a few weeks. The perspectives of this work are very interesting since it will allow the genetic manipulation of the virus and thus the development of precious tools which will be useful to fight the infection. Even though this approach represents a technological leap that will improve our knowledge of the virus, it also carries the germ of possible misuse and the creation of the virus for malicious purposes. The advantages and disadvantages of recreating SARS-CoV-2 in this pandemic period are discussed in this mini-synthesis.

TITLE: Une course contre la montre - Création du SARS-CoV-2 en laboratoire, un mois après son émergence ! ABSTRACT: Le SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2), qui a émergé à la fin de l'année 2019 en République populaire de Chine, est responsable d'une crise sanitaire mondiale qui a entraîné le confinement de plus de 3 milliards d'individus et l'arrêt brutal de l'économie planétaire. Dans ce contexte, une course contre la montre est lancée afin de développer, dans les plus brefs délais, un traitement permettant d'enrayer la pandémie. Une étude de l'équipe de Volker Thiel, parue dans le journal Nature, rapporte la mise au point d'une technique de génétique inverse pour le SARS-CoV-2, leur ayant permis de recréer le virus en seulement quelques semaines. Les perspectives de ces travaux sont très intéressantes puisqu'elles permettent d'envisager la manipulation génétique du virus et ainsi le développement d'outils précieux qui seront utiles pour combattre l'infection. Si la technique représente également un saut technologique qui permettra d'améliorer nos connaissances sur le virus, elle porte aussi en elle le germe d'un possible mésusage et la création d'un virus à des fins malveillantes. Les avantages et inconvénients de recréer le SARS-CoV-2 dans cette période de pandémie sont discutés dans cet article.

Reverse genetic systems: Rational design of coronavirus live attenuated vaccines with immune sequelae.

Since the end of 2019, the global COVID-19 outbreak has once again made coronaviruses a hot topic. Vaccines are hoped to be an effective way to stop the spread of the virus. However, there are no clinically approved vaccines available for coronavirus infections. Reverse genetics technology can realize the operation of RNA virus genomes at the DNA level and provide new ideas and strategies for the development of new vaccines. In this review, we systematically describe the role of reverse genetics technology in studying the effects of coronavirus proteins on viral virulence and innate immunity, cell and tissue tropism and antiviral drug screening. An efficient reverse genetics platform is useful for obtaining the ideal attenuated strain to prepare an attenuated live vaccine.

Insights into SARS-CoV-2, the Coronavirus Underlying COVID-19: Recent Genomic Data and the Development of Reverse Genetics Systems.

The emergence and rapid worldwide spread of a novel pandemic of acute respiratory disease - eventually named coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO) - across the human population has raised great concerns. It prompted a mobilization around the globe to study the underlying pathogen, a close relative of severe acute respiratory syndrome coronavirus (SARS-CoV) called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Numerous genome sequences of SARS-CoV-2 are now available and in-depth analyses are advancing. These will allow detailed characterization of sequence and protein functions, including comparative studies. Care should be taken when inferring function from sequence information alone, and reverse genetics systems can be used to unequivocally identify key features. For example, the molecular markers of virulence, host range and transmissibility of SARS-CoV-2 can be compared to those of related viruses in order to shed light on the biology of this emerging pathogen. Here, we summarize some recent insights from genomic studies and strategies for reverse genetics systems to generate recombinant viruses, which will be useful to investigate viral genome properties and evolution.

SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

Immunoinformatics-guided designing of epitope-based subunit vaccines against the SARS Coronavirus-2 (SARS-CoV-2).

SARS Coronavirus-2 (SARS-CoV-2) pandemic has become a global issue which has raised the concern of scientific community to design and discover a counter-measure against this deadly virus. So far, the pandemic has caused the death of hundreds of thousands of people upon infection and spreading. To date, no effective vaccine is available which can combat the infection caused by this virus. Therefore, this study was conducted to design possible epitope-based subunit vaccines against the SARS-CoV-2 virus using the approaches of reverse vaccinology and immunoinformatics. Upon continual computational experimentation, three possible vaccine constructs were designed and one vaccine construct was selected as the best vaccine based on molecular docking study which is supposed to effectively act against the SARS-CoV-2. Thereafter, the molecular dynamics simulation and in silico codon adaptation experiments were carried out in order to check biological stability and find effective mass production strategy of the selected vaccine. This study should contribute to uphold the present efforts of the researches to secure a definitive preventative measure against this lethal disease.

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform.

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.