Isolation and characterization of an early SARS-CoV-2 isolate from the 2020 epidemic in Medellín, Colombia

Abstract

Introduction: SARS-CoV-2 has been identified as the new coronavirus causing an outbreak of acute respiratory disease in China in December, 2019. This disease, currently named COVID-19, has been declared as a pandemic by the World Health Organization (WHO). The first case of COVID-19 in Colombia was reported on March 6, 2020. Here we characterize an early SARS-CoV-2 isolate from the pandemic recovered in April, 2020. Objective: To describe the isolation and characterization of an early SARS-CoV-2 isolate from the epidemic in Colombia. Materials and methods: A nasopharyngeal specimen from a COVID-19 positive patient was inoculated on different cell lines. To confirm the presence of SARS-CoV-2 on cultures we used qRT-PCR, indirect immunofluorescence assay, transmission and scanning electron microscopy, and next-generation sequencing. Results: We determined the isolation of SARS-CoV-2 in Vero-E6 cells by the appearance of the cytopathic effect three days post-infection and confirmed it by the positive results in the qRT-PCR and the immunofluorescence with convalescent serum. Transmission and scanning electron microscopy images obtained from infected cells showed the presence of structures compatible with SARS-CoV-2. Finally, a complete genome sequence obtained by next-generation sequencing allowed classifying the isolate as B.1.5 lineage. Conclusion: The evidence presented in this article confirms the first isolation of SARSCoV-2 in Colombia. In addition, it shows that this strain behaves in cell culture in a similar way to that reported in the literature for other isolates and that its genetic composition is consistent with the predominant variant in the world. Finally, points out the importance of viral isolation for the detection of neutralizing antibodies, for the genotypic and phenotypic characterization of the strain and for testing compounds with antiviral potential.

Keywords: Coronavirus infections; viral isolation severe acute respiratory syndrome; SARS virus; high-throughput nucleotide sequencing; microscopy; electron; fluorescent antibody technique; indirect.

Figura 1. Identificación de un aislamiento colombiano de SARS-CoV-2. A. y B. Monocapa de células Vero-E6 sin infectar e infectadas; se observa el efecto citopático del SARS-CoV-2 en monocapas de células Vero-E6 a tres días de la inoculación. C., D. y E. Fotografías de placas de IFI preparadas con el suero del paciente en células no infectadas, suero de la etapa aguda en células infectadas y suero de convaleciente en células infectadas

Figura 2. Microscopía electrónica de células Vero-E6 infectadas. A. y B. Fotografías obtenidas por microscopía electrónica de transmisión de partículas virales con tamaño variable entre 87 y 180 nm de diámetro; en A. la flecha señala una estructura compatible con espículas de viriones de SARS-CoV-2. C. y D. Fotografías obtenidas por microscopía electrónica de barrido; se observan estructuras con morfología y tamaño compatibles con de viriones de SARS-CoV-2. En D. también se observa un virión en proceso de gemación (flecha).

Figura 3. Placas con SARS-CoV-2 en monocapa de células Vero-E6. Esta imagen es representativa de la preparación de placas con SARS-CoV-2 en monocapas de células Vero-E6. Se fijaron y se hizo su tinción con una solución de 4 % de formaldehído y 1 % de cristal violeta a los cuatro días de la infección.