Molecular Epidemiology Analysis of SARS-CoV-2 Strains Circulating in Romania during the First Months of the Pandemic

Abstract

Background: The spread of SARS-CoV-2 generated an unprecedented global public health crisis. Soon after Asia, Europe was seriously affected. Many countries, including Romania, adopted lockdown measures to limit the outbreak.

Aim: We performed a molecular epidemiology analysis of SARS-CoV-2 viral strains circulating in Romania during the first two months of the epidemic in order to detect mutation profiles and phylogenetic relatedness.

Methods: Respiratory samples were directly used for shotgun sequencing.

Results: All Romanian sequences belonged to lineage B, with a different subtype distribution between northern and southern regions (subtype B.1.5 and B.1.1). Phylogenetic analysis suggested that the Romanian epidemic started with multiple introduction events from other European countries followed by local transmission. Phylogenetic links between northern Romania and Spain, Austria, Scotland and Russia were observed, as well as between southern Romania and Switzerland, Italy, France and Turkey. One viral strain presented a previously unreported mutation in the Nsp2 gene, namely K489E. Epidemiologically-defined clusters displayed specific mutations, suggesting molecular signatures for strains coming from areas that were isolated during the lockdown.

Conclusions: Romanian epidemic was initiated by multiple introductions from European countries followed by local transmissions. Different subtype distribution between northern and southern Romania was observed after two months of the pandemic.

Keywords: Romania; SARS-CoV-2; WGS; coronavirus; mutations; outbreak; pandemic; phylogenetic analysis.

Figure 1

Phylogenetic analysis of Romanian SARS-CoV-2 sequences. Romanian sequences are represented as red branches, reference sequences are marked in black. SARS-CoV-2 type A cluster is highlighted in orange, type B in yellow and type C in green. Several Romanian clusters were distinctly marked in the tree: Suceava in purple, Bucharest cluster 1 in pink and Bucharest cluster 2 in cyan.

Figure 2

Mutational profile of the forty-nine Romanian SARS-CoV2 sequences. (A) Mutations present in the sequences generated in our study, depicted as either a black (synonymous mutation) or red pin (non-synonymous mutation), aligned using the 045512.2 NCBI reference sequence. Novel mutations were signposted with a black asterisk. Putative mutational signatures of observed clusters were highlighted as follows: pink for Bucharest Cluster 1, blue for Bucharest Cluster 2 and purple for Suceava Cluster. (B) The mutational profile of the other Romanian sequences retrieved from GISAID presented additional nonsense mutations (red pin with a hollowed center).

Figure 3

C-I-Tasser model of Nsp2—local representation of tertiary structure (using Chimera) around the novel K489E mutation. The possible effect of K489E mutation in Nsp2: with cyan lines are represented the H-bonds network in the Nsp2 model. The amino acids in the proximity of K489 are represented as sidechains. Hydrogens are represented on both K489 and K557 Lysins and the amino acids involved in H-bond interactions with K489 and L553 are shown.