A study team from the US NIH and US NIAID found that SARS-CoV-2 RNA remains active and persists in various anatomical sites, including the brain.
The study findings were published in the peer reviewed journal: Nature.
Here we provide, to our knowledge, the most comprehensive analysis to date of the cellular tropism, quantification and persistence of SARS-CoV-2 across the human body including the brain. Our focus on short postmortem intervals, a comprehensive standardized approach to tissue collection, dissecting the brain before fixation, preserving tissue in RNAlater and flash freezing of fresh tissue allowed us to detect and quantify SARS-CoV-2 RNA levels with high sensitivity by ddPCR and ISH, as well as isolate virus in cell culture from multiple non-respiratory tissues including the brain, which are notable differences compared to other studies.
We show that SARS-CoV-2 disseminates early in infection in some patients, with a significantly higher viral burden in respiratory than non-respiratory tissues. We demonstrated virus replication in multiple non-respiratory sites during the first two weeks following symptom onset and detected subgenomic RNA in at least one tissue in 14 of 27 cases beyond D14, indicating that viral replication may occur in non-respiratory tissues for several months.
Whereas others have proposed that the detection of SARS-CoV-2 in non-respiratory tissues might be due to residual blood within tissues 8,18 or cross-contamination from the lungs during tissue procurement 8, our data indicate otherwise.
Specifically, only 12 of our cases had detectable SARS-CoV-2 RNA in a perimortem plasma sample, only 2 cases had SARS-CoV-2 subgenomic RNA detected in plasma, and negligible, if any, RNA was detected in banked peripheral blood mononuclear cells from representative cases.
Further, we validated detection of SARS-CoV-2 outside the respiratory tract by direct cellular identification of virus in cells through ISH, IHC and IF, isolation of SARS-CoV-2 by cell culture, and detection of distinct SARS-CoV-2 spike sequence variants in non-respiratory sites.
Others have previously reported SARS-CoV-2 RNA within the heart, lymph node, small intestine and adrenal gland6,8,9,10,11,12,18. We replicate these findings and conclusively demonstrate that SARS-CoV-2 is capable of infecting and replicating within these and many other tissues, including brain.
Specifically, we report the recovery of replication-competent SARS-CoV-2 from thalamus of P38 at D13 using a modified Vero E6 cell line that stably expresses ACE2 and TMPRSS2. This along with detection of genomic RNA and subgenomic RNA through PCR, multiple imaging modalities showing SARS-CoV-2 RNA and protein within cells of the CNS, and distinct minor variants detected through sequencing in the CNS prove definitively that SARS-CoV-2 is capable of infecting and replicating within the human brain.
HT-SGS of SARS-CoV-2 spike demonstrates homogeneous virus populations in many tissues, while also revealing informative virus variants in others. Low intra-individual diversity of SARS-CoV-2 sequences has been observed frequently in previous studies 19,20,21, and probably relates to the intrinsic mutation rate of the virus as well as lack of early immune pressure to drive virus evolution.
It is important to note that our HT-SGS approach has both a high accuracy and a high sensitivity for minor variants within each sample, making findings of low virus diversity highly reliable 22. Genetic compartmentalization of SARS-CoV-2 between respiratory and non-respiratory tissues in several individuals supports independent replication of the virus at these sites, although lack of compartmentalization between sites does not rule out independent virus replication.
We note several cases in which brain-derived virus spike sequences showed nonsynonymous changes relative to sequences from other non-CNS tissues. Further studies will be needed to understand whether these cases might represent stochastic seeding of the CNS or differential selective pressure on spike by antiviral antibodies in the CNS, as others have suggested23,24,25.
Our results show that although the highest burden of SARS-CoV-2 is in respiratory tissues, the virus can disseminate throughout the entire body. Whereas others have posited that this viral dissemination occurs through cell trafficking11 due to a reported failure to culture SARS-CoV-2 from blood 3,26, our data support an early viraemic phase, which seeds the virus throughout the body following infection of the respiratory tract.
Recent work 26 in which SARS-CoV-2 virions were pelleted and imaged from plasma of patients with acute COVID-19 supports this mechanism of viral dissemination. Our cohort is predominantly composed of severe and ultimately fatal COVID-19 cases.
However, two cases (P36 and P42) reported only mild or no respiratory symptoms and died with, not from, COVID-19, yet had SARS-CoV-2 RNA widely detected across the body and brain.
Additionally, P36 was a juvenile with an underlying neurological condition, but without evidence of multisystem inflammatory syndrome in children, suggesting that children may develop systemic infection with SARS-CoV-2 without developing a generalized inflammatory response.
Finally, our work begins to elucidate the duration and locations at which SARS-CoV-2 RNA can persist. Although the respiratory tract was the most common location in which SARS-CoV-2 RNA persisted, ≥50% of late cases also had persistent RNA in the myocardium, lymph nodes from the head and neck and from the thorax, sciatic nerve, ocular tissue, and in all sampled regions of the CNS, except the dura mater.
Notably, despite having more than 100 times higher SARS-CoV-2 RNA in respiratory compared to non-respiratory tissues in early cases, this difference greatly diminished in late cases. Less efficient viral clearance in non-respiratory tissues may be related to tissue-specific differences in the ability of SARS-CoV-2 to alter cellular detection of viral mRNA, interfere with interferon signalling, or disrupt viral antigen processing and presentation27,28,29. Understanding mechanisms by which SARS-CoV-2 evades immune detection is essential to guide future therapeutic approaches to facilitate viral clearance.
We detected subgenomic RNA in tissue from more than 60% of the cohort, including in multiple tissues of a case at D99. Although subgenomic RNA is generated during active viral replication, it is less definitive than cell culture at demonstrating replication-competent virus because subgenomic RNA is protected by double-membrane vesicles that contribute to nuclease resistance and longevity beyond immediate viral replication 30,31,32,33.
However, nonhuman primates exposed to γ-irradiated SARS-CoV-2 inoculum with high subgenomic RNA copy numbers through multiple mucosal routes had detectable SARS-CoV-2 genomic RNA but undetectable subgenomic RNA levels in respiratory samples by day 1 post-inoculation 15.
These data suggest that detection of SARS-CoV-2 subgenomic RNA probably reflects recent viral replication.
Prolonged detection of subgenomic RNA in a subset of our cases may, however, represent defective rather than productive viral replication, which has been described in persistent infection with measles virus – another single-strand enveloped RNA virus – in cases of subacute sclerosing panencephalitis 34.
Our study has several important limitations. First, our cohort largely represents older unvaccinated individuals with pre-existing medical conditions who died from severe COVID-19, limiting our ability to extrapolate findings to younger, healthier or vaccinated individuals. Second, our cases occurred during the first year of the pandemic, before widespread circulation of variants of concern, and thus findings might not be generalizable to current and future SARS-CoV-2 variants.
Finally, although it is tempting to attribute clinical findings observed in post-acute sequelae of SARS-CoV-2 to viral persistence, our study was not designed to address this question. Despite these limitations, our findings fundamentally improve the understanding of SARS-CoV-2 cellular distribution and persistence in the human body and brain and provide a strong rationale for pursuing future similar studies to define mechanisms of SARS-CoV-2 persistence and contribution to post-acute sequelae of SARS-CoV-2.