Genomic epidemiology of SARS-CoV-2 reveals multiple lineages and early spread of SARS-CoV-2 infections in Lombardy, Italy

Abstract

From February to April 2020, Lombardy (Italy) reported the highest numbers of SARS-CoV-2 cases worldwide. By analyzing 346 whole SARS-CoV-2 genomes, we demonstrate the presence of seven viral lineages in Lombardy, frequently sustained by local transmission chains and at least two likely to have originated in Italy. Six single nucleotide polymorphisms (five of them non-synonymous) characterized the SARS-CoV-2 sequences, none of them affecting N-glycosylation sites. The seven lineages, and the presence of local transmission clusters within three of them, revealed that sustained community transmission was underway before the first COVID-19 case had been detected in Lombardy.

Fig. 1. Distribution of SARS-CoV-2 in Lombardy.

a Geographic distribution of COVID-19 confirmed cases and population density among the 12 provinces of Lombardy. b Geographic distribution of SARS-CoV-2 genomes, the lineages detected, and population density among the 12 provinces of Lombardy. Maps were obtained, thanks to the free available software Rgui using the R statistic library tmap.

Fig. 2. Estimated maximum likelihood phylogeny of SARS-CoV-2 genomes from Lombardy (gray taxa with red dots) and genomes from China (black taxa without dots) and other countries (gray taxa without dots), according to lineages.

Local clusters supported by a posterior probability ≥0.98 in the maximum clade credibility tree were highlighted in red (A), in blue (B), in cyan (C), and in orange (D). Gisaid sequences come from Italy (n = 15), East Europe (n = 15), North Europe (n = 10), South America (n = 13), Africa (n = 8), Japan (n = 10), Oceania (n = 25), West Asia (n = 17), South Asia (n = 14), Central Europe (n = 30), East Asia (n = 46), The Netherlands (n = 41), South East Asia (n = 19), North America (n = 52), British Countries (n = 47), China (n = 55). The phylogeny was estimated with Iqtree with 1000 replicates fast bootstrapping.

Fig. 3. Distribution of SARS-CoV-2 genomes against lineages and collection date.

a Cumulative counts of the 346 SARS-Cov-2 genomes circulating in Lombardy against collection date. b Genetic distance for the Sars-Cov-2 genomes circulating in Lombardy against lineages. c Genetic distance for the Sars-Cov-2 genomes against collection date. The Pearson correlation coefficient between genetic distance and collection date is displayed in the top-right corner (r = 0.582). Sequences are colored by sampling location (Lombardy = red, other location = gray). Reference sequence: NC_045512.2. ‘B other’ refers to haplotypes B, B.1.1.1, and B.1.8.

Fig. 4. Prevalence of most representative single nucleotide polymorphisms (SNPs) in SARS-CoV-2 genomes isolated in Lombardy according to geographical locations and their intra-patient prevalence.

a Frequency of single nucleotide polymorphisms (with respect to the Wuhan reference genome NC_045512.2) among SARS-CoV-2 sequences according to different geographical location. Italian sequences include the 6 and 8 sequences from North and Central Italy present in Gisaid at April 10. b Intra-patient prevalence of most representative SNPs in North Italian SARS-CoV-2 sequences according to sample date. Box and whiskers plots indicate the median and interquartile range of intrapatient prevalence. SNP: single nucleotide polymorphism; syn: synonymous; non-syn: non-synonymous; nsp15: non-structural protein 15; S: spike; M: matrix; N: nucleocapsid; RdRp: RNA-dependent RNA polymerase.

Fig. 5. Bayesian phylogeographic reconstruction incorporating location and travel history data of the most informative sequences for virus spread and clustering in Lombardy.

SARS-CoV-2 genomes from Lombardy were shown by red circles. Local clusters of SARS-CoV-2 sequences supported by a posterior probability ≥0.98 by the time-scaled maximum clade credibility tree were highlighted in red (A), in blue (B), in cyan (C), and in orange (D). Two independent chains were run for 25 million states. Parameters and trees were sampled every 1000 states.