SARS-CoV-2 virus culture and subgenomic RNA from patients with mild Coronavirus disease

COVID-19: Virus culture and subgenomic RNA for respiratory specimens from patients with mild Coronavirus disease. Jefferson T, Heneghan C.

Published on August 20, 2020

Reference Perera RAPM, Tso E, Tsang OTY, Tsang DNC, Fung K, Leung YWY, et al. SARS-CoV-2 virus culture and subgenomic RNA for respiratory specimens from patients with mild Coronavirus disease. Emerging Infectious Disease journal. 2020;26(11). 1985
Study type
Country China, SAR Hong Kong
Setting Unclear
Funding Details US National Institutes of Health and Natural Science Foundation of China (NSFC) Joint Research Scheme
Transmission mode Respiratory, viral load

Bottom Line

In respiratory specimens of COVID-19 patients mainly with mild disease culturable SARS-CoV-2 and subgenomic RNA (good indicator of replication) was rarely detectable beyond 8 days after onset of illness although virus RNA by RT-PCR remained for up to 70 days.

Evidence Summary

Virus sub-genomic RNA (sgRNA) was detectable in 18/22 (82%) specimens collected eight days or less after symptom onset and in one of 11 specimens (9%) collected nine or more days after onset of disease.

There was a significant association between positivity for sgRNA and virus culture (33 specimens tested for both sgRNA and virus culture were positive for both in 12 cases or 36.3%. 

Both were negative in 36%, sgRNA was positive and culture negative in 7 (21%) while culture was positive and sgRNA was negative in 2 (6%). The median age of the culture positive and negative patients was 39 and 38 years. 

Virus was cultured from 12 of 17 specimens with the virus load ranging from:

  • 7.0 to 9.5 log10 copies per mL of specimen (24 cycle threshold).
  • Three  of 11 specimens with viral load ranging from 6.0 to 6.9 log10 copies per mL (26), 
  • One of seven specimens with viral load ranging from 5.0-5.99 log10 copies per mL (27) and from none of 33 specimens with viral load <5log10 copies per mL (27 ct).

For conversion of logs see:

Log10 copies/mL
8 100,000,000
7 10,000,000
5 10,000
4 1,000
3 100
2 10

What did they do?

The case series reports the findings on a series of 68 respiratory specimens taken from 35 COVID-19 patients in Hong Kong hospitals. Of the 68 specimens, 49 were nasopharyngeal aspirates combined with throat swab, 2 were nasopharyngeal aspirate and then nasopharyngeal swab combined with throat swab (n=3), nasopharyngeal swab (n=2), sputum (n=11) and saliva (n=1).

Patient age ranged from 17 to 75 years (median 38 years, 44 males). 10 had prolonged virus RNA shedding (>30 days) and 6 were re-admitted because of RT-PCR positivity detected after discharge from hospital. 

Nine patients had underlying comorbidities but none was immunocompromised. Three patients were asymptomatic, twenty nine patients had mild clinical illness (mild influenza-like illness symptoms, two patients were in a critical condition and one died. FIve patients received steroids, but not prior to testing.

Specimens were tested for sgRNA with ≥5 log10 N gene copies per mL. The complementary DNA obtained was subjected to PCR (40 cycles). 

Vero E6 cells were seeded and incubated for 24 hours in a CO2 incubator. The culture medium was removed and 125 μL of the clinical specimen in virus transport medium diluted and was inoculated into 2 wells. After 2 hours incubation in a CO2 incubator at 37°C, the plates were incubated at 37°C in a CO2 incubator. 

A sample (100 μL) of supernatant was sampled for a quantitative real-time RT-PCR at 0 and 72 hours post inoculation. At 72 hours, cells were scraped into the supernatant and transferred onto fresh cells in 24-well plates and monitored for an additional 72 hours. A final quota of cells  was collected for quantitative real-time RT-PCR. Cells were observed for cytopathic effect daily and harvested for testing if 25%–50% of cells showed a cytopathic effect.

Study reliability

The patients are well described but their selection is not. Methods appear standard and the number of specimens is reasonable. The text is not clear as to the median age which is reported as slightly different possibly relating to those who were tested for sg RNA and culture.

The explanation for the choice of primer is only reported in the discussion (“Sub-genomic RNA (sgRNA) provides evidence of replicative intermediates of the virus rather than residual viral RNA”).

Clearly defined setting Demographic characteristics described Follow-up length was sufficient Transmission outcomes assessed Main biases are taken into consideration
Unclear Yes Yes Yes Yes

What else should I consider?

This study results are broadly in line with those of similar case series.

About the authors

Carl Heneghan

Carl is Professor of EBM & Director of CEBM at the University of Oxford. He is also a GP and tweets @carlheneghan. He has an active interest in discovering the truth behind health research findings

Jeffrey Aronson

Honorary Consultant Physician & Clinical Pharmacologist