[Analysis of Nucleotide Changes in RT-PCR Primer/Probe Binding Regions in SARS-CoV-2 Isolates Reported from Turkey]. / Türkiye'den Bildirilen Sars-CoV-2 Izolatlarinda RT-PCR Primer/Prob Baglanma Bölgelerindeki Nükleotit Degisimlerinin Analizi.

The SARS-CoV-2 virus, which caused the COVID-19 epidemic, caused more than 55 million cases and nearly 1.5 million deaths worldwide. For the microbiological diagnosis of the disease, the most valid method is detecting the presence of the viral genome by real-time reverse transcription polymerase chain reaction (rRT-PCR). However, due to the nature of the RNA viruses, frequent mutations may affect the sensitivity of the analyses made on the genetic material of the virus, such as PCR. In this study, we aimed to investigate the mutations in the primer-probe binding regions of the rRT-PCR panels used in COVID-19 diagnosis. SARS-CoV-2 whole genome sequence data (n= 194) isolated from COVID-19 cases in Turkey and uploaded on GISAID database from the centers in Istanbul (n= 78), Ankara (n= 58), Kars (n= 47), Bursa (n= 2), Adiyaman (n= 2), Erciyes (n= 1) and Kocaeli (n= 1) between March 17-September 14, 2020 were analyzed. In order to determine the nucleotide changes, SARS-CoV-2 sequences from Turkey were compared to the reference genome sequence (NC_045512.1) present in "GenBank" website. The constructed data set was aligned using the MAFFT program and was checked manually if the sequences were in the same frame by using the AliView program. Primer-probe binding sites of the thirteen SARS-CoV-2 rRT-PCR panels from seven different institutes (US CDC, China CDC, Charite CDC, Pasteur, HKU, Thailand, NIID) that are being used in COVID-19 diagnosis were evaluated in terms of nucleotide changes within the corresponding regions compared to the reference genome. Sequence diversities in the viral genomes were determined via positional nucleotide numerical calculator and entropy calculator modules and nucleotide and entropy changes in primer-probe binding regions for each rRT-PCR panel were examined. Among thirteen different primer-probe panels, nucleotide changes in the target regions of the seven primer-probe panels were determined. When viral sequences with nucleotide changes in the primer-probe binding regions were examined, the most common changes were observed in the "China CDC" N-forward primer and "US CDC" N3-forward primer binding regions. It is important that the kits to be used as diagnostic tests are designed specific to the regions with less nucleotide changes. Nucleotide changes may not be critical for DNA amplification for most PCR panels, but should be carefully monitored as they may affect the sensitivity of the assay. If the risk of alteration of the designed region is high, the primer - probe binding sites should be checked frequently and updated when necessary.

Analysis of Three Mutations in Italian Strains of SARS-CoV-2: Implications for Pathogenesis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus initially detected in Wuhan in December 2019, responsible for coronavirus disease 2019 (COVID-19), a respiratory syndrome currently affecting >220 countries around the world, with >80 million cases registered and >1.8 million deaths. OBJECTIVE: As several vaccines are still being developed and 2 have been approved, it is particularly important to perform evolutionary surveillance to identify mutations potentially affecting vaccine efficacy. METHODS: DynaMut server has been used to evaluate the impact of the mutation found on SARS-CoV-2 isolates available on GISAID. RESULTS: In this article, we analyze whole genomes sequenced from Italian patients, and we report the characterization of 3 mutations, one of which presents in the spike protein. CONCLUSION: The mutations analyzed in this article can be useful to evaluate the evolution of SARS-CoV-2.

Evidence for mutations in SARS-CoV-2 Italian isolates potentially affecting virus transmission.

Italy is the first western country suffering heavy severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission and disease impact after coronavirus disease-2019 pandemia started in China. Even though the presence of mutations on spike glycoprotein and nucleocapsid in Italian isolates has been reported, the potential impact of these mutations on viral transmission has not been evaluated. We have compared SARS-CoV-2 genome sequences from Italian patients with virus sequences from Chinese patients. We focussed upon three nonsynonymous mutations of genes coding for S(one) and N (two) viral proteins present in Italian isolates and absent in Chinese ones, using various bioinformatics tools. Amino acid analysis and changes in three-dimensional protein structure suggests the mutations reduce protein stability and, particularly for S1 mutation, the enhanced torsional ability of the molecule could favor virus binding to cell receptor(s). This theoretical interpretation awaits experimental and clinical confirmation.

Identification of the nucleotide substitutions in 62 SARS-CoV-2 sequences from Turkey.

A previously unknown coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been shown to cause coronavirus disease 2019 (COVID-19) pandemic. The first case of COVID-19 in Turkey has been declared in March 11th, 2020 and from there on, more than 150,000 people in the country have been diagnosed with the disease. In this study, 62 viral sequences from Turkey, which have been uploaded to GISAID database, were analyzed by means of their nucleotide substitutions in comparison to the reference SARS-CoV-2 genome from Wuhan. Our results indicate that the viral isolates from Turkey harbor some common mutations with the viral strains from Europe, Oceania, North America and Asia. When the mutations were evaluated, C3037T, C14408T and A23403G were found to be the most common nucleotide substitutions among the viral isolates in Turkey, which are mostly seen as linked mutations and are part of a haplotype observed high in Europe.