Comments on 'Development of a PCR-RFLP method for detection of D614G mutation in SARS-CoV-2'.

Dear Editor

It has been widely shown that severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) has a common mutation, known as D614G (A23403G in the whole genome or A1841G in the spike genome) in which the amino acid Aspartate 614 (Nucleotide A23403 or A1841) in the SARS CoV-2 spike protein is substituted with Glycine (Nucleotide G23403 or G1841) as can be found in the Wuhan SARS CoV-2 whole genome sequence (NC_045512.2) as described previously by several studies (Chen et al., 2020; Korber et al., 2020; Plante et al., 2020; Zhang et al., 2020). Thus, A is changed to G.

We wish to comment on an original research paper, recently published by Hashemi et al in the Journal of Infection, Genetics and Evolutions, entitled “Development of a PCR-RFLP method for detection of D614G mutation in SARS-CoV-2” (Hashemi et al., 2020). They aim to develop a novel, rapid, low-cost method for identifying D614G mutation in the SARS CoV-2 genome, using a polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP). The restriction enzyme used in their study was the HpaI enzyme which can cleave only the wild type sequences, GTTAAC.

Interestingly, Hashemi et al have developed a new tool, nonetheless, they have mistakenly identified V615V variant but not D614G. As they have mentioned in their Figure 1 that D614G is caused by a mutation of nucleotide T1845 to G at the position of 1845 of the spike protein. In fact, in the SARS CoV-2 D614G variant, nucleotide A1841 in GAT codon (Aspartate (D)), is changed to G in GGT codon (Glycine (G)). We have shown the highlighted sequences in Fig. 1.

Fig. 1

Short nucleotide sequences of severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome (NCBI Reference Sequence: NC_045512.2). A1841 is the target for D614G mutation. But T1845 is a target for V615.

The (gat) is a codon for Aspartate (D614). The (gtt) is a codon for Valine (V615). When gtt is changed to gtg, which is a codon for Valine too, thus, T1845G mutation leads to V615V, a silent mutation, not D614G.

We think that Hashemi et al have made a mistake that instead of identifying D614G but they have developed a novel method for exploring V615V which is a silent mutation that occurred in Iranian populations when GTT (Valine) is changed to GTG (Valine).

The site of this mutation appears to be rarely targeted in the world as in recent studies, the nucleotide G23405 in the whole genome or G1843 in the spike genome in GTT codon (Valine), is mutated to C23405 or C1843 forming CTT codon (Leucine) and thus the mutation is called V615L (G23405C in the whole genome or G1843C in the spike genome), which has been seldomly reported in the world (Koyama et al., 2020; Li et al., 2020; Yang et al., 2020). Because GTG (Valine), which was mistakenly mentioned as Glycine by Hashemi et al, has been confirmed using DNA sequencing, therefore, we may not envisage the V615L (G23405C or G1843C) variant in the Iranian samples. An in vitro study shows that the V615L play roles in structural impacts and antibody sensitivity of SARS CoV-2 spike protein (Li et al., 2020). However, the roles of silent mutations such as V615V, reported by Hashemi et al, should be elucidated in evolutionary perspectives.

In fact, Hashemi et al have found a novel silent mutation, V615V (T23407G in the whole genome or T1845G in the spike genome) which has been prevalent in 9% of positive samples taken from an intensive care unit of North-eastern Iran within the last six months that may have important roles in increasing the survival fitness of the virus or it could have occurred due to a random mutation. Therefore, we recommend correcting this paper as a corrigendum to be entitled (Development of a PCR-RFLP method for the detection of V615V mutation in SARS-CoV-2).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.