In vitro isolation of 23 SARS-CoV-2 strains circulating in Parma (Italy) during the Italian COVID-19 lockdown: cultivation features and viral morphology

Background In Italy, SARS-CoV-2 infection became epidemic on March 3rd, 2020: 219,070 cases with 30,560 deaths were observed until May 10th. This study reports the results of SARS-CoV-2 cultivation from samples of patients attending the tertiary-care University-Hospital of Parma during the Italian lockdown. Methods For 637 respiratory samples analysed in the period 27 February – 10 May 2020, the nucleic acids of the main respiratory viruses, including SARS-CoV-2, were searched for by Real-Time-PCR, and conventional culture was also performed: Intestine 407, Hep-2, Vero, LLC-MK2, MRC5, MDCK-SIAT1 monolayers grown in 24-well plates were inoculated with the sample supernatant. The inoculated cell monolayers were observed daily under an inverted optical microscope in order to check the appearance of a virus-related cytopathic effect. Viral particles from cultures showing a cytopathic effect were identified by electron microscopy and/or Real-Time PCR. Results Among 216 SARS-CoV-2 RNA positive samples, 23 (4 bronchoaspirates, 13 throat swabs, 5 nasopharyngeal aspirates, 1 bronchoalveolar lavage), from as many patients, showed a cytopathic effect (clusters of rounded cells) on VERO (22 cases) and LLC-MK2 (23 cases) cell culture, after 3-10 days from infection. In 2 out of the 23 cases, electron microscopy allowed the detection of 120-nm diameter Coronaviridae, identified as SARS-CoV-2 by Real-Time-PCR, including the first SARS-CoV-2 positive case detected in our area from a nasopharyngeal aspirate, belonging to a 7-week-old baby, sent to our laboratory before 27 February without SARS-CoV-2 suspicion, for whom SARS-CoV-2 RNA on the original sample was retrospectively detected by Real-Time-PCR. For the remaining 18 cases SARS-CoV2 was identified from cell culture only using the specific Real-Time-PCR.Conclusions In this study all samples were submitted to in vitro culture resulting positive for SARS-CoV-2 in 23 cases; all samples negative by PCR were also negative by culture. The results suggest that SARS-CoV-2 diagnosis should not be limited to molecular tools, but it should be performed, were available, together with culture; virus isolation is the only reference laboratory method able to reveal the presence both of infectious agents for diagnostic purposes, or that of emerging viruses, allowing their characterization for epidemiological purposes, such as vaccine and anti-viral drug development.