key: cord-1011488-i4xjk61t authors: Castillo, Andrés E.; Parra, Bárbara; Tapia, Paz; Acevedo, Alejandra; Lagos, Jaime; Andrade, Winston; Arata, Loredana; Leal, Gabriel; Barra, Gisselle; Tambley, Carolina; Tognarelli, Javier; Bustos, Patricia; Ulloa, Soledad; Fasce, Rodrigo; Fernández, Jorge title: Phylogenetic analysis of the first four SARS‐CoV‐2 cases in Chile date: 2020-04-08 journal: J Med Virol DOI: 10.1002/jmv.25797 sha: 66fe185daeec563b540b98ebc26234666ed264f8 doc_id: 1011488 cord_uid: i4xjk61t The current pandemic caused by the new coronavirus is a worldwide public health concern. To aboard this emergency, and like never before, scientific groups around the world have been working in a fast and coordinated way to get the maximum of information about this virus when it has been almost 3 months since the first cases were detected in Wuhan province in China. The complete genome sequences of around 450 isolates are available, and studies about similarities and differences among them and with the close related viruses that caused similar epidemics in this century. In this work, we studied the complete genome of the first four cases of the new coronavirus disease in Chile, from patients who traveled to Europe and Southeast Asia. Our findings reveal at least two different viral variants entries to Chilean territory, coming from Europe and Asia. We also sub‐classified the isolates into variants according to punctual mutations in the genome. Our work contributes to global information about transmission dynamics and the importance to take control measures to stop the spread of the infection. were detected in Wuhan province in China. The complete genome sequences of around 450 isolates are available, and studies about similarities and differences among them and with the close related viruses that caused similar epidemics in this century. In this work, we studied the complete genome of the first four cases of the new coronavirus disease in Chile, from patients who traveled to Europe and Southeast Asia. Our findings reveal at least two different viral variants entries to Chilean territory, coming from Europe and Asia. We also sub-classified the isolates into variants according to punctual mutations in the genome. Our work contributes to global information about transmission dynamics and the importance to take control measures to stop the spread of the infection. reported cases at the time of this report. 1 Near 286 complete genomes of SARS-CoV-2 and related viruses, has been submitted to the GISAID database (www.gisaid.org/ CoV2020) collecting genetic information of the outbreak worldwide. The genomic sequences of all SARS-CoV-2 viruses isolated from patients share a sequence identity about a 99.9%, 7 suggesting a recently zoonotic infection, originated most probably from bats. 5, 6, 8 As the information appears daily, new insights and concepts are being adopted and implemented. Recently Tang et al 9 and GISAD database in the SARS-CoV-2 portal defined three subtypes: S, G, and V, according to nucleotide variants that produce amino acid changes. These changes are located in ORF8 L84S; S (spike protein) D614G and nsp3 G251V, in the nucleotide position 28144, 23403, and 3471, respectively, for S, G, and V, according to the reference sequence NC_045512.2. Chile was the fourth country in South America after Brazil, Ecuador, and Argentina to report COVID-19 in the region. In this report, we present the sequence analysis for the first four complete genomes for SARS-CoV-2 isolates on Chilean patients. Also, a phylogenetic study was performed with worldwide SARS-CoV-2 sequences and the full genomes from Chilean isolates, to identify their genetic similarity. The four cases presented in this report have contracted the infection abroad, either in Southeast Asia or Europe. The first two cases From total RNA extraction we performed the first amplification round using SuperScript III One-Step RT-PCR Platinum Taq DNA Polymerase (Invitrogen) and six pair of specific primers to obtain six complementary DNA fragments around 5 Kbp each, followed by a second amplification round with 24 specific primers (Table S1) to generate two fragments from each first round products, each subfragment (a total of 12) are around 2.3 to 2.7 Kbp. The sequencing quality was analyzed with software Fastqc v0.11.8 and then, the reads were filtered and trimmed using BBDuk software considering a minimum read length of 36 bases and quality more than equal to 10. SARS-CoV-2 assembly was performed with IRMA v0.9.3 using as reference NCBI sequence ID NC_045512.2. Sequence alignment was performed with MAFFT. The phylogenetic tree was built with IQ-TREE v1.6.12 considering a bootstrap of 1000. We consider 218 full complete genome sequences available in the GISAID platform plus the full genome sequences from the first four Chilean cases. where the presence of the virus was confirmed and with an increasing number of cases. We identified the SNPs that generates amino acid changes in all four Chilean genomes (Table 1) A maximum-likelihood phylogeny tree was constructed using 218 complete genome sequences plus the four Chilean cases. Our first two samples, the married couple, mapped together (100% nucleotide identity) and with strains from Wuhan, China and Taiwan. The third sample groups in a well-defined clade with Spanish isolates. The fourth Chilean strain, groups in a European clade with samples from Switzerland, Netherlands, and Germany. In this same clade, we can identify one of the Brazilian and Mexican isolates, representing isolates from Latin America. In addition, the complete genomes were colored according to the variant groups, defined by specific mutations (Figure 1 ). In this early stage of the epidemic, sharing data and information is crucial and the efforts of the scientist worldwide are admirable. After a few weeks since the outbreak started in Wuhan province, the full genome sequence of SARS-CoV-2 was available, and this information paved the way for the development of better detection protocols The authors are thankful to María Ibañez and Jorge Lobos for their valuable technical assistance. The authors declare that there are no conflict of interests. CAE participated in conceptualization, study design, interpreting the data analysis, methodology design, visualization, and wrote the whole manuscript. PB participated in methodology design and experimental assays, TP and TJ participated in data analysis and bioinformatics support. LJ, AL, and BG contributed to genome sequencing. AA, AW, LG, TC, and BP, participate in sample processing and real-time RT-PCR assays. US and FR participated in the critical review of the content. FJ contributed to the conceptualization, study design, supervision, critical review of the content, and approved the final version of the manuscript. ORCID Andrés E. Castillo http://orcid.org/0000-0001-9644-3719 Coronavirus disease Situation Report 53. 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