Early Diffusion of SARS-CoV-2 Infection in the Inner Area of the Italian Sardinia Island

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the coronavirus disease 2019 (COVID-19) pandemic, which started as a severe pneumonia outbreak in Wuhan, China, in December 2019. Italy has been the first European country affected by the pandemic, registering a total of 300,363 cases and 35,741 deaths until September 24, 2020. The geographical distribution of SARS-CoV-2 in Italy during early 2020 has not been homogeneous, including regions severely affected as well as administrative areas being only slightly interested by the infection. Among the latter, Sardinia represents one of the lowest incidence areas likely due to its insular nature.

Methods: Next-generation sequencing of a small number of complete viral genomes from clinical samples and their virologic and phylogenetic characterization was performed.

Results: We provide a first overview of the SARS-CoV-2 genomic diversity in Sardinia in the early phase of the March-May 2020 pandemic based on viral genomes isolated in the most inner regional hospital of the island. Our analysis revealed a remarkable genetic diversity in local SARS-CoV-2 viral genomes, showing the presence of at least four different clusters that can be distinguished by specific amino acid substitutions. Based on epidemiological information, these sequences can be linked to at least eight different clusters of infection, four of which likely originates from imported cases. In addition, the presence of amino acid substitutions that were not previously reported in Italian patients has been observed, asking for further investigations in a wider population to assess their prevalence and dynamics of emergence during the pandemic.

Conclusion: The present study provides a snapshot of the initial phases of the SARS-CoV-2 infection in inner area of the Sardinia Island, showing an unexpected genomic diversity.

Keywords: COVID-19; SARS-CoV-2; Sardinia Island; epidemiology; genome sequencing; molecular characterization; pandemic; phylogeny.

FIGURE 1

Geographic diffusion of coronavirus disease 2019 (COVID-19) infection in Italy. Autonomous provinces are marked with a *. NW, North-West; NE, North-East; C, Center; S, South; IS, Island. Epidemiological data source: ISS, https://www.iss.it/coronavirus, updated on September 15, 2020.

FIGURE 2

Structural and phylogenetic analysis of the 13 Nuoro severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. (A) Alignment of the 13 viral genomic sequences with respect to SARS-CoV-2 reference genome NC_045512.2 annotated for the position of viral genes (green labels) and their coding DNA sequence (CDS, yellow labels). For the genomic portion encoding ORF1ab polyprotein, the final individual proteins are annotated with white labels. (B) Neighbor-joining tree of the 13 viral genomic sequences and SARS-CoV-2 reference genome (black dot): the percentages of trees in which the associated taxa clustered together (bootstrap test, 100 replicates) are shown above the nodes. The two main clusters—named here 1 and 2—are highlighted with red and blue brackets, respectively, and the correspondent color is also indicated near the aligned sequences. The four nucleotide substitutions shared by all the sequences and the one specifically characterizing cluster 2 members are reported below the alignment in black and blue, respectively. A map of Sardinia showing the geographic distribution of samples is also provided.

FIGURE 3

Phylogenetic analysis of Nuoro severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences and GISAID viral genomes from Italy. Viral genomic sequences identified in Italy have been retrieved from GISAID and analyzed in a neighbor-joining tree. The percentages of trees in which the associated taxa clustered together (bootstrap test, 100 replicates) are shown next to the branches. Samples generated in this work are marked with red and blue dots, depending on their belonging to cluster 1 (clade GR) or cluster 2 (clade G), respectively. SARS-Cov-2 reference genome NC_045512.2 is indicated with a black dot. For the sake of clarity, bootstrap values lower than 30% have been hidden.

FIGURE 4

Phylogenetic analysis of Nuoro severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences and GISAID worldwide viral genomes. About 59,700 viral genomic sequences identified globally have been retrieved from GISAID and analyzed in a maximum likelihood tree. Samples generated in this work are marked with red and blue dots, depending on their belonging to cluster 1 (clade GR) or cluster 2 (clade G), respectively. The corresponding source tree in Newick format is also provided as Supplementary File 1 to allow its detailed inspection in freely available tree viewers.