Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant

Abstract

Background: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance.

Methods: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance.

Results: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on February 9th, 2020 while a different mutation in RdRp changing its amino acid composition emerged on February 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001).

Conclusions: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.

Keywords: COVID-19; Drug resistance; Europe; Mutation; Pneumonia; RNA-dependent-RNA-polymerase; RdRp; SARS-CoV-2; Viral mutagenesis.

Fig. 1

SARS-CoV-2 mutation frequency in different geographic areas. Eight novel recurrent hotspots mutations (namely 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881) and 5 hotspots already reported in literature (namely 3036, 8782, 11083, 28144 and 26143) were subdivided into 4 geographic areas: Asia (n = 71), Oceania (n = 15), Europe (n = 101), North America (n = 33). The mutation frequency was estimated for each of them, by normalizing the number of genomes carrying a given mutation in a geographic area, by the overall number of retrieved genomes per geographic area; the graph shows the cumulative mutation frequency of all given mutations present in each geographic area. Mutation locations in viral genes are reported in the legend as well as the proteins (i.e. non-structural protein, nsp) presenting these mutations. The figure shows that genomes from European and North American patients present an increase in mutation frequency compared to Asia. It is also possible to observe that Europe and North America show a differential pattern of mutations: mutation 14408 (red), 23403 (black), 28881 (electric blue) and 26143 (light green) are present mostly in Europe, whereas 18060 (pink), 17857 (purple) and 17746 (light blue) are present mostly in North America

Fig. 2

SARS-CoV-2 Mutation occurrence over time divided per geographic area. Eight novel recurrent hotspots mutations (namely 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881) and 5 hotspots already reported in literature (namely 3036, 8782, 11083, 28144 and 26143) were subdivided first into 5 period subgroups: December 2019 (n = 5), 1st–15th Jan. 2020 (n = 15), 16th–31st Jan 2020 (n = 52), 1st–15th Feb 2020 (n = 13), 16th–29th Feb 2020 (n = 55) and 1st–13th Mar 2020 (n = 80). Next, for each time group, a further subclassification per geographic area (Asia, Oceania, Europe and North America) was performed (number of genomes in each area are reported in the figure inset). The number of mutations in each area was normalized by the number of genomes analyzed for each period of time. This figure shows that mutation frequency increases over time during viral spread out of Asia. No mutations were observed in the Asian genomes analyzed in December 2019. In the time group of February 16th–29th, a defined cluster of mutations emerged in Europe; in March 1st–13th, a different cluster of mutations emerged in North America

Fig. 3

Increment of SARS-CoV-2 mutation frequency after RdRp mutation appearance per geographic area. The increment of mutation frequency before and after February 9th, 2020 across the different geographic areas (Asia, Europe, North America) is shown. The figure shows a diminishment of Asian mutations (i.e. 1397, 8782, 11083, 26143 and 28144) that is simultaneous with the appearance of new mutations such as 2891, 3036, 23403 and 28881, when RdRp novel European mutation located at 14408 (in red) occurred. The upper table shows the increment or decrement for each single mutation, per geographic area

Fig. 4

Number of SARS-CoV-2 mutations associated with the RdRp mutation. Genomes were subdivided into two groups: group 1 contains genomes with mutation in position 14408 (RdRp) (n = 53, 4 North America and 49 European), and group 2 without RdRp mutation (n = 84). We further subdivided group 1 and 2 by the number of mutations present in the genome. Genomes in group 1 (red bars) showed an increased number of mutations compared to group 2 (grey bars). Most genomes of groups 1 (86.8%) have at least 3 or 4 mutations, whereas 76.2% of genomes of group 2 have less than 2 mutations. We found that viral strains with RdRp mutation have a median of 3 point mutations [range: 2–5], whereas viral strains with no RdRp mutation have a median of 1 mutation [range: 0–3] (p value < 0.001, Mann–Whitney test)