TITLE: Culture of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; f.2019-nCoV)
AUTHORS: Alyssa Pyke, Ian M. Mackay, Frederick Moore, Andrew Van Den Hurk, Judy Northill, Mitchell Finger, Natalie Simpson, Neelima Nair, Peter Burtonclay, Peter Moore, Sarah Wheatley, Sean Moody, Sonja Hall-Mendelin, Elisabeth Gamez, Amanda De Jong, Ben Huang, Carmel Taylor, David Warrilow, Doris Genge, Glen Hewitson, Inga Sultana, Jamie McMahon, Jean Barcelon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We briefly describe a method to inoculate a susceptible cell line with a human patient sample in order to culture the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease, COVID-19.</div><div class = "text-block">Clinical samples including nasopharyngeal swabs and aspirates were inoculated onto confluent monolayers of African green monkey kidney Vero C1008, clone E6 cells (ATCC®-CRL-1586) grown in Opti-Mem reduced serum growth medium supplemented with 3% foetal bovine serum in polystyrene, flat-sided, screw-cap 3 mL cell culture tubes.</div><div class = "text-block">This work was conducted in Queensland under PC3 laboratory conditions by experienced scientists. </div></div>

[STEPS]
?. [Specimen and uninfected cell preparation]
Cell culture tubes were moved from the 37ºC  incubator to a Class II Biosafety cabinet, within a PC3 laboratory environment.Growth medium from previously prepared Vero E6 tubes was discarded to waste.
?. [Specimen and uninfected cell preparation]
Clinical samples were prepared by diluting in Opti-Mem® reduced serum growth medium without foetal bovine serum (FBS) and filtering through a 0.2 µM, 13 mm Acrodisc® filter (Bio-Strategy Laboratory Products, Australia).
?. [Inoculation of Vero E6 cell monolayers]
150-200 µL of filtered patient material was inoculated onto separate confluent cell monolayers along with a negative control tube (150-200 µL of Opti-Mem alone). NOTE:Fresh specimens are best for succesful viral culture.Samples were absorbed onto cells by incubating tubes for 1 hr at 37ºC before cultures were re-fed with 2 mL of pre-warmed (37ºC) Opti-Mem® reduced serum growth medium.NOTE:It can be informative to collect a 200 µL sample at this point as a Day 0 value to test alongside other culture samples from Day 2 onwards.
?. [Culture incubation and observation]
Cultures were incubated for 2-7 days until signs of cytopathic effect (CPE) was observed.In our hands, cultures that were inoculated with patient sample extracts which, at original testing produced CTs of approximately 20 cycles, developed signs of CPE within 3 days post-inoculation.
?. [Actions to consider in the absence of obvious CPE]
In the event low-levels of the virus were not detected by observation of CPE in the first isolation attempt, negative cell culture supernatants should be further passaged onto fresh Vero E6 monolayers up to 3 times with testing as described in Step 6.
?. [Confirmation of a succesful virus culture]
A cell culture is suspected of hosting virus replication based on the presence of CPE including damage to the monolayer, cell-clearing and morphological changes. Suspected cultures were further confirmed using the Corman et al. E and Northill et al. ORF1ab novel coronavirus reverse-transcription real-time polymerase chain reaction (RT-rPCRs) tests.Samples taken from a suspected culture are expected to contain levels of virus that exceed those present in the original clinical sample so we would expect to see CT values lower than those obtained from testing the original clinical sample extract by RT-rPCRs.SLOW CULTURES: If the CT value from an extract of the first passage of a SARS-CoV-2 culture is the same or higher (suggesting a lower viral load) than the CT value from the RT-rPCR of the original clinical sample, further investigation will be required to ensure the culture has biologically amplified virus. Consideration should be given to whether such a result is due to the detection of viral remnants from the inoculum rather than virus amplification.
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