Virological assessment of hospitalized patients with COVID-2019
Wölfel, R., Corman, V.M., Guggemos, W. et al. Virological assessment of hospitalized patients with COVID-2019. Nature (2020). https://doi.org/10.1038/s41586-020-2196-x
Review written by:
Ariel Pourmorady, reviewed by Diogo Teles
Green - published in Nature and peer-reviewed. Although N = 9, they provide sound evidence for their conclusions
SARS-CoV and SARS-CoV-2 are betacoronaviruses with tropism for the ACE2 receptor in the lung. Infection with either virus can cause an ARDS-like syndrome. Unlike SARS-CoV, SARS-CoV-2 is characterized by the additional feature of upper respiratory symptoms in a subset of patients, meaning that site-specific characterization of virus replication, immunity, and infectivity is important. Wolfel et al. recruited 9 COVID-19+ patients all of whom had simultaneous exposure to SARS-CoV-2 from a single index case on January 23rd; were recruited upon first onset of respiratory tract symptoms, generally mild at onset; and were young to middle aged.
Wolfel et al. first sought to identify locations in the body with a detectable viral load as measured by RT-PCR, finding viral RNA in oro/nasopharyngeal swabs, sputum, and stool samples. While viral load varied from person-to-person by orders of magnitude, on average the highest were seen within the first 4-5 days of symptoms. Average sputum viral load was 7 x 10^6 copies/mL and on a pharyngeal swab was 3.4 x 10^6 during the first 5 days of symptoms. Blood and urine were negative for viral RNA at all time points.
Wolfel et al. then aimed to identify the presence and determinants of infectious viral particles. They found that while patients maintained a high viral load in swabs, sputum, and stool for up 2 weeks after symptom onset, infectious viral particles could only be isolated from sputum and pharyngeal swabs in the first 8 days after symptom onset with a background viral load of at least 10^6 copies of viral RNA/mL. Isolation of live virus from stool was never successful.
Since viral RNA and infectious viral particles were present in the pharynx, Wolfel then sought to determine whether there was active infection in the upper respiratory tract. They isolated subgenomic RNA (sgRNA - a measure of intracellularly replicating viral RNA only produced in infected cells) and normalized it to viral genomic RNA (gRNA) to track infection in the pharynx. When measuring sgRNA beginning at day 4-5 after symptom onset, viral replication was observed in sputum until the last measurement on day 11, in the pharynx only on day 4-5, and not at all in stool. These data suggest that active infection begins to wane in the pharynx after day 4-5, but persists in the lungs.
Data from Wolfel et al. support unique populations of infection between the pharynx and lung. They performed full virus genome sequencing in all patients, and in one patient identified a SNP, G6446A, on the pharyngeal swab not seen in the sputum sample. This suggests that there is independent viral replication in the pharynx and the lung, as opposed to simple passive shedding of virus from the lungs via the oropharynx.
Seroconversion occurred by day 7 in 50% of patients and by day 14 in all patients; no live viruses were isolated 7 days after symptom onset, and all patients were able to develop neutralizing antibodies.
Subjects were all young to middle aged, without significant underlying disease and mild clinical course. (This could also be considered a strength, since the majority of patients will only present mild symptoms);
Only 9 patients in the study, small sample size;
All patients were infected with a single genotype SARS-CoV-2 virus; generalizability to other viral subtypes is not addressed;
While sgRNA:gRNA implicitly suggests viral replication at sites of RNA isolation, proving active viral replication requires histopathology of infected tissue samples.