Gold Nanoparticles Capped with a Novel Titanium(IV)-Containing Polyoxomolybdate Cluster: Selective and Enhanced Bactericidal Effect Against Escherichia coli.

Bacterial infections are a public health threat of increasing concern in medical care systems; hence, the search for novel strategies to lower the use of antibiotics and their harmful effects becomes imperative. Herein, the antimicrobial performance of four polyoxometalate (POM)-stabilized gold nanoparticles (Au@POM) against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as Gram-negative and Gram-positive bacteria models, respectively, is studied. The bactericidal studies performed, both in planktonic and sessile forms, evidence the antimicrobial potential of these hybrid nanostructures with selectivity toward Gram-negative species. In particular, the Au@GeMoTi composite with the novel [Ti2 (HGeMo7 O28 )2 ]10- POM capping ligand exhibits outstanding bactericidal efficiency with a minimum inhibitory concentration of just 3.12 µm for the E. coli strain, thus outperforming the other three Au@POM counterparts. GeMoTi represents the fourth example of a water-soluble TiIV -containing polyoxomolybdate, and among them, the first sandwich-type structure having heteroatoms in high-oxidation state. The evaluation of the bactericidal mechanisms of action points to the cell membrane hyperpolarization, disruption, and subsequent nucleotide leakage and the low cytotoxicity exerted on five different cell lines at antimicrobial doses demonstrates the antibiotic-like character. These studies highlight the successful design and development of a new POM-based nanomaterial able to eradicate Gram-negative bacteria without damaging mammalian cells.

Spontaneous Interconversion between Different Narrowly Defined Shapes of Rotavirus Double-Layered Particles Studied in Real Time by High-Resolution Mobility Analysis.

Rotavirus double-layered particles (DLPs) are studied in the gas phase with a high-resolution differential mobility analyzer (DMA). DLPs were transferred to 10 mM aqueous ammonium acetate, electrosprayed into the gas phase, converted into primarily singly charged particles, and DMA-analyzed. Up to seven slightly different conformations were resolved, whose apparently random, fast (minutes), and reversible interconversions were followed in real time. They sometimes evolved into just two distinct structures, with periods of one dominating over the other and vice versa. Differences between the DLP structures in solution and in the gas phase are clearly revealed by the smaller DLP diameter found here (60 versus 70 nm). Nevertheless, we argue that the multiple gas-phase conformers observed originate in as many conformations pre-existing in solution. We further hypothesize that these conformers correspond to incomplete DLPs having lost some of the VP6 trimer quintets surrounding each of the 12 5-fold axes. Instances of this peculiar loss have been previously documented by cryoelectron microscopy for the rotavirus Wa strain, as well as via charge detection mass spectrometry for five other rotavirus strains included in the RotaTec vaccine. Evidence of this loss systematically found for all 7 rotavirus types so far studied in aqueous ammonium acetate may be a special feature of this electrolyte.

Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus.

In order to gain insight into the role of the transcription regulatory sequences (TRSs) in the regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus (PRRSV), the enhanced green fluorescent protein (EGFP) gene, under the control of the different structural gene TRSs, was inserted between the N gene and 3'-UTR of the PRRSV genome and EGFP expression was analyzed for each TRS. TRSs of all the studied structural genes of PRRSV positively modulated EGFP expression at different levels. Among the TRSs analyzed, those of GP2, GP5, M, and N genes highly enhanced EGFP expression without altering replication of PRRSV. These data indicated that structural gene TRSs could be an extremely useful tool for foreign gene expression using PRRSV as a vector.

Erratum to: Role of transcription regulatory sequence in regulation of gene expression and replication of porcine reproductive and respiratory syndrome virus.

After publication of the article [1], it has been brought to our attention that an acknowledgement has been omitted from the original article. The authors would like to include the following, The authors also thank Prof. En-Min Zhou (Northwest A&F University) and his laboratory for technical support."

Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform.

Dengue viruses (DENV) have become the most important arthropod-borne viruses, causing dengue and severe dengue fever in at least 50-100 million cases each year, mainly in tropical and subtropical countries. During recent years, important advances in the molecular biology concerning the life cycle of these viruses have allowed the manipulation and generation of recombinant viruses and replicons with multiple applications, mainly in viral biology and the screening of antiviral compounds. In the present study, we describe the construction of an enhanced green fluorescent protein-bearing DENV replicon under the control of the cytomegalovirus immediate early promoter. Following a rational in silico design and cloning by standard molecular biology techniques, a reporter DENV-2 replicon and a replication-deficient mutant were constructed, and characterized by confocal microscopy and real-time RT-PCR. The results showed successful transcription, translation, and autonomous viral RNA replication of the DENV replicon from its DNA clone. This novel DENV replicon will allow the study of viral replication and testing of antiviral candidates without the need for in vitro transcription.

Editorial for SARS-CoV-2 and COVID-19 Topical Collection.

A previously unknown coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was isolated in Wuhan, China in December 2019, from a patient with a respiratory disease linked to potential contact with wild animals [...].

Reverse Genetics of Zika Virus Using a Bacterial Artificial Chromosome.

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has become a global threat to human health. Although ZIKV has been known to circulate for decades causing mild febrile illness, the more recent ZIKV outbreaks in the Americas and the Caribbean have been associated with severe neurological disorders and congenital abnormalities. The development of ZIKV reverse genetics approaches have allowed researchers to address key questions on the biology of ZIKV by genetically engineering infectious recombinant (r)ZIKV. This has resulted in a better understanding of the biology of ZIKV infections, including viral pathogenesis, molecular mechanisms of viral replication and transcription, or the interaction of viral and host factors, among others aspects. In addition, reverse genetics systems have facilitated the identification of anti-ZIKV compounds and the development of new prophylactic approaches to combat ZIKV infections. Different reverse genetics strategies have been implemented for the recovery of rZIKV. All these reverse genetics systems have faced and overcome multiple challenges, including the viral genome size, the toxicity of viral sequences in bacteria, etc. In this chapter we describe the generation of a ZIKV full-length complementary (c)DNA infectious clone based on the use of a bacterial artificial chromosome (BAC) and the experimental procedures for the successful recovery of rZIKV. Importantly, the protocol described in this chapter provides a powerful method for the generation of infectious clones of other flaviviruses with genomes that have stability problems during bacterial propagation.

Bacterial Artificial Chromosome Reverse Genetics Approaches for SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new member of the Coronaviridae family responsible for the coronavirus disease 19 (COVID-19) pandemic. To date, SARS-CoV-2 has been accountable for over 624 million infection cases and more than 6.5 million human deaths. The development and implementation of SARS-CoV-2 reverse genetics approaches have allowed researchers to genetically engineer infectious recombinant (r)SARS-CoV-2 to answer important questions in the biology of SARS-CoV-2 infection. Reverse genetics techniques have also facilitated the generation of rSARS-CoV-2 expressing reporter genes to expedite the identification of compounds with antiviral activity in vivo and in vitro. Likewise, reverse genetics has been used to generate attenuated forms of the virus for their potential implementation as live-attenuated vaccines (LAV) for the prevention of SARS-CoV-2 infection. Here we describe the experimental procedures for the generation of rSARS-CoV-2 using a well-established and robust bacterial artificial chromosome (BAC)-based reverse genetics system. The protocol allows to produce wild-type and mutant rSARS-CoV-2 that can be used to understand the contribution of viral proteins and/or amino acid residues in viral replication and transcription, pathogenesis and transmission, and interaction with cellular host factors.

Transmissible gastroenteritis coronavirus RNA-dependent RNA polymerase and nonstructural proteins 2, 3, and 8 are incorporated into viral particles.

Coronavirus replication and transcription are processes mediated by a protein complex, with the RNA-dependent RNA polymerase (RdRp) as a main component. Proteomic analysis of highly purified transmissible gastroenteritis virus showed the RdRp to be a component of the viral particles. This finding was confirmed by Western blotting, immunofluorescence, and immunoelectron microscopy analyses. Interestingly, the replicase nonstructural proteins 2, 3, and 8 colocalized with the RdRp in the viral factories and were also incorporated into the virions.

Molecular characterization of feline infectious peritonitis virus strain DF-2 and studies of the role of ORF3abc in viral cell tropism.

The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log(10) titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV.

Cytoplasmic RNA viruses as potential vehicles for the delivery of therapeutic small RNAs.

Viral vectors have become the best option for the delivery of therapeutic genes in conventional and RNA interference-based gene therapies. The current viral vectors for the delivery of small regulatory RNAs are based on DNA viruses and retroviruses/lentiviruses. Cytoplasmic RNA viruses have been excluded as viral vectors for RNAi therapy because of the nuclear localization of the microprocessor complex and the potential degradation of the viral RNA genome during the excision of any virus-encoded pre-microRNAs. However, in the last few years, the presence of several species of small RNAs (e.g., virus-derived small interfering RNAs, virus-derived short RNAs, and unusually small RNAs) in animals and cell cultures that are infected with cytoplasmic RNA viruses has suggested the existence of a non-canonical mechanism of microRNA biogenesis. Several studies have been conducted on the tick-borne encephalitis virus and on the Sindbis virus in which microRNA precursors were artificially incorporated and demonstrated the production of mature microRNAs. The ability of these viruses to recruit Drosha to the cytoplasm during infection resulted in the efficient processing of virus-encoded microRNA without the viral genome entering the nucleus. In this review, we discuss the relevance of these findings with an emphasis on the potential use of cytoplasmic RNA viruses as vehicles for the efficient delivery of therapeutic small RNAs.

A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit.

Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3'-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus.

On-chip monitoring of toxic gases: capture and label-free SERS detection with plasmonic mesoporous sorbents.

The detection of the spread of toxic gas molecules in the air at low concentration in the field requires a robust miniaturized system combined with an analytical technique that is portable and able to detect and identify the molecules, as is the case with surface enhanced Raman scattering (SERS). This work aims to address capability gaps faced by first responders in real-time detection, identification and monitoring of neurotoxic gases by developing robust, reliable and reusable SERS microfluidic chips. Thus, the key performance attributes of a portable SERS detection system that must be addressed in detail are its limit of detection, response time and reusability. To this purpose, we integrate a 3D plasmonic architecture based on closely packed mesoporous silica (MCM48) nanospheres decorated with Au nanoparticle arrays, denoted as MCM48@Au, into a Si microfluidic chip designed and used for preconcentration and label-free detection of gases at a trace concentration level. The SERS performance of the plasmonic platform is thoroughly analyzed using DMMP as a model neurotoxic simulant over a 1 cm2 SERS active area and over a range of concentrations from 100 ppbV to 2.5 ppmV. The preconcentration-based SERS signal amplification by the mesoporous silica moieties is evaluated against dense silica counterparts, denoted as Stöber@Au. To assess the potential for applications in the field, the microfluidic SERS chip has been interrogated with a portable Raman spectrometer, evaluated with temporal and spatial resolution and subjected to several gas detection/regeneration cycles. The reusable SERS chip shows exceptional performance for the label-free monitoring of 2.5 ppmV gaseous DMMP.

SARS-CoV-2-encoded small RNAs are able to repress the host expression of SERINC5 to facilitate viral replication.

Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell to restrict the infectivity of certain viruses. Different viruses have developed strategies to antagonize SERINC5 function but, how SERINC5 is controlled during viral infection is poorly understood. Here, we report that SERINC5 levels are reduced in COVID-19 patients during the infection by SARS-CoV-2 and, since no viral protein capable of repressing the expression of SERINC5 has been identified, we hypothesized that SARS-CoV-2 non-coding small viral RNAs (svRNAs) could be responsible for this repression. Two newly identified svRNAs with predicted binding sites in the 3'-untranslated region (3'-UTR) of the SERINC5 gene were characterized and we found that the expression of both svRNAs during the infection was not dependent on the miRNA pathway proteins Dicer and Argonaute-2. By using svRNAs mimic oligonucleotides, we demonstrated that both viral svRNAs can bind the 3'UTR of SERINC5 mRNA, reducing SERINC5 expression in vitro. Moreover, we found that an anti-svRNA treatment to Vero E6 cells before SARS-CoV-2 infection recovered the levels of SERINC5 and reduced the levels of N and S viral proteins. Finally, we showed that SERINC5 positively controls the levels of Mitochondrial Antiviral Signalling (MAVS) protein in Vero E6. These results highlight the therapeutic potential of targeting svRNAs based on their action on key proteins of the innate immune response during SARS-CoV-2 viral infection.

The polypyrimidine tract-binding protein affects coronavirus RNA accumulation levels and relocalizes viral RNAs to novel cytoplasmic domains different from replication-transcription sites.

The coronavirus (CoV) discontinuous transcription mechanism is driven by long-distance RNA-RNA interactions between transcription-regulating sequences (TRSs) located at the 5' terminal leader (TRS-L) and also preceding each mRNA-coding sequence (TRS-B). The contribution of host cell proteins to CoV transcription needs additional information. Polypyrimidine tract-binding protein (PTB) was reproducibly identified in association with positive-sense RNAs of transmissible gastroenteritis coronavirus (TGEV) TRS-L and TRS-B by affinity chromatography and mass spectrometry. A temporal regulation of PTB cytoplasmic levels was observed during infection, with a significant increase from 7 to 16 h postinfection being inversely associated with a decrease in viral replication and transcription. Silencing the expression of PTB with small interfering RNA in two cell lines (Huh7 and HEK 293T) led to a significant increase of up to 4-fold in mRNA levels and virus titer, indicating a negative effect of PTB on CoV RNA accumulation. During CoV infection, PTB relocalized from the nucleus to novel cytoplasmic structures different from replication-transcription sites in which stress granule markers T-cell intracellular antigen-1 (TIA-1) and TIA-1-related protein (TIAR) colocalized. PTB was detected in these modified stress granules in TGEV-infected swine testis cells but not in stress granules induced by oxidative stress. Furthermore, viral genomic and subgenomic RNAs were detected in association with PTB and TIAR. These cytoplasmic ribonucleoprotein complexes might be involved in posttranscriptional regulation of virus gene expression.

RNA-RNA and RNA-protein interactions in coronavirus replication and transcription.

Coronavirus (CoV) RNA synthesis includes the replication of the viral genome, and the transcription of sgRNAs by a discontinuous mechanism. Both processes are regulated by RNA sequences such as the 5' and 3' untranslated regions (UTRs), and the transcription regulating sequences (TRSs) of the leader (TRS-L) and those preceding each gene (TRS-Bs). These distant RNA regulatory sequences interact with each other directly and probably through protein-RNA and protein-protein interactions involving viral and cellular proteins. By analogy to other plus-stranded RNA viruses, such as polioviruses, in which translation and replication switch involves a cellular factor (PCBP) and a viral protein (3CD) it is conceivable that in CoVs the switch between replication and transcription is also associated with the binding of proteins that are specifically recruited by the replication or transcription complexes. Complexes between RNA motifs such as TRS-L and the TRS-Bs located along the CoV genome are probably formed previously to the transcription start, and most likely promote template-switch of the nascent minus RNA to the TRS-L region. Many cellular proteins interacting with regulatory CoV RNA sequences are members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA-binding proteins, involved in mRNA processing and transport, which shuttle between the nucleus and the cytoplasm. In the context of CoV RNA synthesis, these cellular ribonucleoproteins might also participate in RNA-protein complexes to bring into physical proximity TRS-L and distant TRS-B, as proposed for CoV discontinuous transcription. In this review, we summarize RNA-RNA and RNA-protein interactions that represent modest examples of complex quaternary RNA-protein structures required for the fine-tuning of virus replication. Design of chemically defined replication and transcription systems will help to clarify the nature and activity of these structures.

Rapid differentiation of vaccine strain and Chinese field strains of transmissible gastroenteritis virus by restriction fragment length polymorphism of the N gene.

A strain of transmissible gastroenteritis virus (TGEV), designated H16, was isolated in PK-15 cells and passaged serially to level 165. Vaccines based on passages 155-165 in cell cultures are available commercially as vaccines for the prevention and control of infections with TGEV in China. Nucleoprotein (N) sequences of the virus at passages 155 and 165 were aligned and compared using a computer software program. The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiation of the vaccine strain from the other TGEVs was investigated. The RFLP analysis identified a change in the cleavage sites of AclI at passages 155 and 165. This RFLP pattern of the N gene differentiated the Chinese vaccine strain from its parental strain, the 11 TGEVs studied and the other reported TGEVs in the GenBank. Using phylogenetic analysis, the Chinese TGEVs were divided into three groups (G1, G2, and G3). The G3 Chinese TGEVs possessed several specific nucleotides and amino acids that were not found in the G1 and G2 Chinese TGEVs or the other reference TGEVs. Analysis of the phylogenetic trees revealed that the G3 TGEVs represent a separate group that is distinct from the non-Chinese TGEVs and from Chinese TGEVs isolated previously. These findings suggest that Chinese strains of TGEV are evolving continuously.

Molecular characterization of a Chinese vaccine strain of transmissible gastroenteritis virus: mutations that may contribute to attenuation.

Transmissible gastroenteritis virus (TGEV), the etiological agent of transmissible gastroenteritis (TGE), is the major cause of viral enteritis and fetal diarrhea in swine neonates, resulting in significant economic losses to the swine industry. The Chinese vaccine strain H165 of TGEV was derived from a virulent field strain H16 by serial passage in vitro. Strain H165 has been proven to be safe in piglets and pregnant sows and displays efficacy against TGEV infection. In this study, we report the complete genome sequences of strains H165 and H16, obtained by sequencing several overlapping fragments amplified from viral RNA and our findings from sequence and phylogenetic analyses. The genomes were 28,569 nucleotides in length, including the poly (A) tail. No deletions or insertions were detected in the H16 genome sequence after continuous passage in vitro; however, we found 27 nucleotide mutations in strain H165 compared with strain H16, resulting in 16 amino acid changes distributed among the genes 1, S, 3, and sM. An A to G nucleotide mutation was found in the intergenic region between the 3a and 3b genes. Furthermore, six unique nucleotides identified in the genome sequence of H165 could be used as makers to differentiate the H165 vaccine strain from wild-type TGEV strains. Our findings from phylogenetic analysis may enhance our understanding of the evolution of TGEV, as well as the other coronaviruses.

Identification of Inhibitors of ZIKV Replication.

Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently as a global health threat, with recurring outbreaks and its associations with congenital microcephaly through maternal fetal transmission and Guillain-Barré syndrome. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or antivirals to treat ZIKV infections, which underscores an urgent medical need for the development of disease intervention strategies to treat ZIKV infection and associated disease. Drug repurposing offers various advantages over developing an entirely new drug by significantly reducing the timeline and resources required to advance a candidate antiviral into the clinic. Screening the ReFRAME library, we identified ten compounds with antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV). Moreover, we showed the ability of these ten compounds to inhibit influenza A and B virus infections, supporting their broad-spectrum antiviral activity. In this study, we further evaluated the broad-spectrum antiviral activity of the ten identified compounds by testing their activity against ZIKV. Among the ten compounds, Azaribine (SI-MTT = 146.29), AVN-944 (SI-MTT = 278.16), and Brequinar (SI-MTT = 157.42) showed potent anti-ZIKV activity in post-treatment therapeutic conditions. We also observed potent anti-ZIKV activity for Mycophenolate mofetil (SI-MTT = 20.51), Mycophenolic acid (SI-MTT = 36.33), and AVN-944 (SI-MTT = 24.51) in pre-treatment prophylactic conditions and potent co-treatment inhibitory activity for Obatoclax (SI-MTT = 60.58), Azaribine (SI-MTT = 91.51), and Mycophenolate mofetil (SI-MTT = 73.26) in co-treatment conditions. Importantly, the inhibitory effect of these compounds was strain independent, as they similarly inhibited ZIKV strains from both African and Asian/American lineages. Our results support the broad-spectrum antiviral activity of these ten compounds and suggest their use for the development of antiviral treatment options of ZIKV infection.

Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome.

Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.