CPER reactions were then used for transfections without any purification

CPER reactions were then used for transfections without any purification. COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30?kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled Daminozide together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20?kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions. virus family in the order of values are two-sided. Mean values for each treatment are shown??SD. For WT virus, three biological replicates were used, for CPER-generated virus six biological replicates were used, results are from one experiment. f Full lungs from mice infected with WT virus, representative CPER virus, or infected (mock), harvested at 5 days post infection and stained with hematoxylin and eosin (H&E). Scale bar is 5?mm. g Selected H&E stained images of lung sections from mice infected with WT or representative CPER virus showing sloughing of the bronchial JUN epithelium as indicated by the arrowhead, bronchi occluded with edema (O) and red blood cells (R). h Additional features of SARS-CoV-2 infection in lungs of infected mice showing the collapse of alveolar spaces (A) and edema. The scale bar is 100?m. Representative images from fCh are from three independently analyzed samples for each treatment. Source data for d and e are provided in the Source Data file. Full-length SARS-CoV-2 cDNA was assembled from the viral cDNA fragments and the linker fragment into a circular DNA in a single CPER reaction using a high-fidelity DNA polymerase. The process does not require any of the intermediate steps commonly used in other coronavirus reverse genetics systems2,15C17. The CPER reaction mix (Supplementary Fig.?1), without any further purification, was then directly transfected into HEK293T cells. Daminozide To recover the virus, transfected HEK293T cells were cocultured with the highly permissive Vero E6 cells. Two independent transfection experiments were performed (CPER1 and CPER2), with both yielding infectious virus. The CPER viruses were amplified once in Vero E6 cells to generate viral stocks. CPER3 virus was generated by transfecting the CPER reaction mix into ACE2-expressing HEK293T cells18 and amplifying the recovered virus in Vero E6 cells. Nanopore sequencing of the viral cDNA fragments PCR-amplified from P4 viral cDNA showed they had the same swarm/quasi-species sequence variation as the wild-type (WT) P4 virus (Supplementary Table?1). Comparison of the consensus genome sequences between P4 viral cDNA and PCR-amplified fragments used in CPER assembly (deposited to GenBank, ID: “type”:”entrez-nucleotide”,”attrs”:”text”:”MW772455″,”term_id”:”2029247467″,”term_text”:”MW772455″MW772455) did not identify any changes. These data illustrate that the high-fidelity PCR used to generate the cDNA fragments that were subsequently used for the CPER reactions had faithfully amplified the original viral sequence. Culture of SARS-CoV-2 viruses in Vero E6 cells is well known to select rapidly for mutations, often in or around the furin cleavage site19C22. This remains true for a number of reverse genetics systems16,21,23, although deep sequencing of recovered viruses is not always Daminozide provided15,17,24C26. Such selection was also seen for CPER viruses recovered from Vero E6 cells (cocultured with transfected HEK293T) (Supplementary Table?1). The selection of certain amino acid changes was different for CPER1, 2, and 3 viruses (Supplementary Table?1), with such variability in selection also reported for other reverse genetics systems16,21,27. As noted previously21,22, the choice of cell lines can affect these selection processes, with our recovery of CPER3 virus using ACE2-HEK293T cells showed a decrease in the number of affected sites from 4 Daminozide to 1 1 (Supplementary Table?1). Given the SARS-CoV-2 genome encodes 9700 amino acids, the CPER method using ACE2-HEK293T cell thus achieved nearly complete (99.99%) amino acid sequence fidelity. To examine viral properties in cells, a standard plaque assay with crystal violet staining of infected cells at 2 days post infection (dpi) was performed and showed similar plaque sizes for CPER1 and CPER2 viruses and the parental WT QLD02 isolate (Fig.?1c, top row). Immuno-plaque staining (immuno-plaque assay, iPA) with anti-spike protein monoclonal antibody CR3022 showed clear viral foci (Fig.?1c, bottom row). This method was used to determine viral titers (foci forming units, FFU) as it allows detection.