13 include in the recombinant sequence specific markers such as the Bgl1 RSs. The full-length Bat-SCoV infectious clone, generated with the method described by Yount et al. Both the icSARS-CoV and the MA-SARS-CoV have become the most widely used recombinant viruses to study SARS-like viruses and no specific sequences were found in the human SARS-CoV-2Wu. The identity of MA-SARS-CoV compared with the original SARS-CoV-Urbani is 99.97% with only six distinct nucleotides, that cannot be used as markers of this recombinant virus since same mutations are naturally acquired by the WT-SARS-CoV, as demonstrated from the sequences of other isolated SARS-CoV 14. This is expected since the first part of these two genomes show high similarities.Īnother recombinant SARS-CoV was produced in 2007 which derived from fifteen passages of the SARS-CoV-Urbani in BALB/c mouse lungs, therefore it was named Mouse-Adapted (MA)-SARS-CoV 14. All the genomes did not contain these sites (Table S1), in particular, the hSARS-CoV-19-Wuhan and hCoV-19-Italy-Vr.īy analysing the sequences of Bat-CoV-raTG13 and the hCoV-19-Pangolin, we observed that only one sequence from SARS-CoV-Urbani (GCCAGCGTGGT) was found in SARS-CoV-2Wu (Wuhan). We analysed natural sequences isolated from four different SARS-CoV (hCoV-19-Italy-Vr/hSARS-CoV-19-Wuhan/hCoV-19 Pangolin/Bat-Cov-raTG13) to look for Bgl1 RS ‘marker’ (GCCNNNN/NGGC). The newly sequences introduced in the recombinant cDNA of SARS-CoV can be used as markers to follow possible virus laboratory spillage. Also, in this case, the recombinant virus shows several nucleotide mutations which exclude the manipulations performed using modified primers and unique restriction sites. ( D) A specific area of the alignment performed between the mutant SARS-CoV-Urbani MA15 containing the SHC014 spike with the hCoV-19-Italy-VR and the SARS-CoV-19 Wuhan. Despite some small regions are conserved the chimeric spikes show single bp mutation (substitution, deletion, insertions) which support natural evolutions instead of man-made manipulation. ( C) Multiple sequence alignment performed with ClustalW and visualised with JalView show the poor similarities in the RBD between chimeric Spikes generated in the laboratory (line 2 and 4) compared with other SARS-CoV sequences. In the hCoV-19-Italy-VR sequence most of these markers’ sites are not present, while are similar to the wilt type virus HKU3 and RP3. ( B) This alignment shows in violet specific markers used to build a recombinant spike between the Bat-SCoVs genomes HKU3 and RP3. The red boxes show the different nucleotides present in the wilt type SARS-CoV Urbani. The violet box highlight BglI RSs used to build the recombinant icSARS-CoV. ( A) Alignment between the WT SARS-CoV Urbani and the icSARS-CoV. Although other several coronavirus experts have discredited the hypothesis of a man-made coronavirus 8, 9, 10, 11, 12, here we aim to present a different method based on the analysis of restriction site (RS) sequences in the genome of SARS-Cov-2 to reconstruct its origin and follow the new variants.Īnalyses of several RSs sequences in natural and recombinant viruses. In March 2020, Anderson and colleagues published a detailed analysis showing that SARS-CoV-2 does not derive from a laboratory construct 7. Therefore, one of the major discussions around SARS-CoV-2 has been related to its origin with the assumption that SARS-CoV-2 could have been the result of genetic manipulations or spill-over from laboratories studying these viruses. The World Health Organization declared a coronavirus disease 2019 (COVID-19) pandemic in March 2020. Phylogenetic analysis demonstrated the highly similarity between human SARS-CoV-2 and the sequence isolated from the Bat-Cov-raTG13 4 (97.2% identity) and the Pangolin-SARS-CoV 5 (80% identity), particularly in the receptor-binding-domain (RBD) of the S protein, important to mediate binding to human-receptor-angiotensin-converting-enzyme-2 (hACE2) 6.

Although SARS-CoV-2 belongs to the same lineage of CoVs that causes SARS, it is genetically different and it cluster apart exploiting phylogenetic trees 3. In December 2019, a new coronavirus (SARS-CoV-2) started to cause viral pneumonia bringing to severe and fatal infection. They have been associated with two major disease outbreaks, the severe acute respiratory syndrome (SARS-CoV, 2002) and the Middle East respiratory syndrome (MERS-CoV, 2012) 2. Coronaviruses (CoVs) goes into the family Coronaviridae causing symptoms primarily in the upper respiratory tracts which range from common cold to severe to fatal illnesses 1.

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