A new study by scientists from the U.S. National Institute of Health (NIH) has found alarming evidence of SARS-CoV-2 viral persistence in the human host body and also in the brain even in those individuals that only had mild symptoms initially during infection and even in the asymptomatic who were deemed as ‘recovered’.
The study findings were published on a preprint server called Research Square and is current under peer review for publication into the journal: Nature.
https://www.researchsquare.com/article/rs-1139035/v1
Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has well described pulmonary and extrapulmonary manifestations1-3, including multiorgan failure and shock among severe and fatal cases.
Some survivors experience Post-Acute Sequelae of SARS-CoV-2 (PASC) – also known as Long COVID – with cardiovascular, pulmonary, and neurological manifestations with or without functional impairment4-5.
These questions include, but are not limited to: What is the burden of infection within versus outside of the respiratory tract? What cell types are infected across extra-pulmonary tissues, and do they support SARS-CoV-2 infection and replication?
To inform these pathogen-focused questions and to evaluate for the presence or absence of associated histopathology in matched tissue specimens, we performed extensive autopsies on a diverse population of 44 individuals who died from or with COVID-19 up to 230 days following initial symptom onset.
Our approach focused on timely, systematic, and comprehensive tissue sampling and preservation of adjacent tissue samples for complementary analyses. We performed droplet digital polymerase chain reaction (ddPCR) for sensitive detection and quantification of SARS-CoV-2 gene targets in all tissue samples collected.
To elucidate SARS-CoV-2 cell-type specificity and validate ddPCR findings, we performed in situ hybridization (ISH) broadly across sampled tissues. Immunohistochemistry (IHC) was used to further validate cell-type specificity in the brain where controversy remains on the regional distribution and cellular tropism of SARS-CoV-2 infection.
In all samples where SARS-CoV-2 RNA was detected by ddPCR, we performed qRT-PCR to detect subgenomic (sg)RNA, an assay suggestive of recent virus replication15. We confirmed the presence of replication-competent SARS-CoV-2 in extrapulmonary tissues by virus isolation in cell culture. Lastly, in six
individuals, we measured the diversity and anatomic distribution of intra-individual SARS-CoV- 2 variants using high-throughput, single-genome amplification and sequencing (HT-SGS).
We categorized autopsy cases of SARS-CoV-2 infection as “early” (n=17), “mid” (n=13), or “late” (n=14) by illness day (D) at the time of death, being ≤D14, D15-D30, or ≥D31, respectively. We defined persistence as presence of SARS-CoV-2 RNA among late cases.
Due to the extensive tissue collection, we analyzed and described the results in terms of grouped tissue categories as the following: respiratory tract; cardiovascular; lymphoid; gastrointestinal; renal and endocrine; reproductive; muscle, skin, adipose, & peripheral nerves; and brain.
COVID-19 histological findings
The histopathology findings from our cohort were similar to those reported in other case series (Extended Data Fig. 4). All but five cases were considered to have died from COVID-19 (Extended Data Table 5), and, of these, 37 (94.5%) had either acute pneumonia or diffuse alveolar damage at the time of death (Supplementary Data 2).
Phases of diffuse alveolar damage showed clear temporal associations, with the exudative phase seen mainly within the first three weeks of infection and the fibrosing phase not seen until after a month of infection (Extended Data Fig. 5). Pulmonary thromboembolic complications, which were also likely related to SARS-CoV-2 infection, with or without infarction, were noted in 10 (23%) cases.
Another finding likely related to SARS-CoV-2 infection included myocardial infiltrates in four cases, including one case of significant myocarditis16 (P3). Some of the cases of microscopic ischemia appeared to be associated with fibrin-platelet microthrombi, and may therefore be related to COVID-19 thrombotic complications. Within the lymph nodes and spleen, we observed lymphodepletion and both follicular and paracortical hyperplasia.
Outside the lungs, histological changes were mainly related to complications of therapy or preexisting co-morbidities: mainly obesity, diabetes, and hypertension. Five cases had old ischemic myocardial scars and three had coronary artery bypass grafts in place.
Given the prevalence of diabetes and obesity in our cohort, it was not surprising to find diabetic nephropathy (10 cases, 23%) or steatohepatitis (5 cases, 12%). One case was known to have chronic hepatitis C with cirrhosis, but the other cases of advanced hepatic fibrosis were likely related to fatty liver disease, even if diagnostic features of steatohepatitis were not present. Hepatic necrosis (13 cases, 30%) and changes consistent with acute kidney injury (17 cases, 39%) were likely related to hypoxic-ischemic injury in these very ill patients.
In the examination of the 11 brains, we found few histopathologic changes, despite the evidence of substantial viral burden. Vascular congestion was an unusual finding that had an unclear etiology and could be related to the hemodynamic changes incurred with infection.
Global hypoxic/ischemic change was seen in two cases, one of which was a juvenile (P36) with a seizure disorder who was found to be SARS-CoV-2 positive on hospital admission, but who likely died of seizure complications unrelated to viral infection.
Discussion
Here we provide the most comprehensive analysis to date of SARS-CoV-2 cellular tropism, quantification, and persistence across the body and brain, in a diverse autopsy cohort collected throughout the first year of the pandemic in the United States.
Our focus on short post- mortem intervals, comprehensive approach to tissue collection, and preservation techniques – RNAlater and flash freezing of fresh tissue – allowed us to detect and quantify viral levels with high sensitivity by ddPCR and ISH, as well as culture virus, which are notable differences compared to other studies.
We show SARS-CoV-2 disseminates across the human body and brain early in infection at high levels, and provide evidence of virus replication at multiple extrapulmonary sites during the first week following symptom onset.
We detected sgRNA in at least one tissue in over half of cases (14/27) beyond D14, suggesting that prolonged viral replication may occur in extra- pulmonary tissues as late as D99.
While others have questioned if extrapulmonary viral presence is due to either residual blood within the tissue8,17 or cross-contamination from the lungs during tissue procurement8, our data rule out both theories. Only 12 cases had detectable SARS-CoV-2 RNA in a perimortem plasma sample, and of these only two early cases also had SARS-CoV-2 sgRNA in the plasma, which occurred at Ct levels higher than nearly all of their tissues with sgRNA.
Therefore, residual blood contamination cannot account for RNA levels within tissues. Furthermore, blood contamination would not account for the SARS-CoV-2 sgRNA or virus isolated from tissues. Contamination of additional tissues during procurement, is likewise ruled out by ISH demonstrating widespread SARS-CoV-2 cellular tropism across the sampled organs, by IHC detecting viral protein in the brain, and by several cases of virus genetic compartmentalization in which spike variant sequences that were abundant in extrapulmonary tissues were rare or undetected in lung samples.
Using both ddPCR and sgRNA analysis to inform our selection of tissue for virus isolation and ISH staining allow us to describe a number of novel findings. Others6,8-12,17 have previously reported SARS-CoV-2 RNA within the heart, lymph node, small intestine, and adrenal gland.
We demonstrate conclusively that SARS-CoV-2 is capable of infecting and replicating within these tissues. Current literature has also reported absent or controversial expression of ACE2 and/or TMPRSS2 in several extrapulmonary tissues, such as the colon, lymphoid tissues, and ocular tissues, calling into question if these tissues can become infected by SARS-CoV-21-3.
However, we observed high levels of SARS-CoV-2 RNA and evidence of replication within these organs, as well as SARS-CoV-2 RNA via ISH in colonic mucosal epithelium and mononuclear leukocytes within the spleen, thoracic cavity lymph nodes, and GI lymphoid aggregates. We believe these ISH positive cells represent either infection or phagocytized virus in resident macrophages. Further, we isolated virus from a mediastinal lymph node and ocular tissue from two early cases (P19, P32).
Our use of a single-copy sequencing approach for the SARS-CoV-2 spike allowed us to demonstrate 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 studies18-20, and likely relates to the intrinsic mutation rate of the virus as well as lack of early immune pressure to drive virus evolution in new infections.
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 reliable21. The virus genetic compartmentalization that we observed between pulmonary and extrapulmonary sites in several individuals supports independent replication of the virus at these sites, rather than spillover from one site to another.
Importantly, lack of compartmentalization between these sites in other individuals does not rule out independent virus replication, as independently replicating populations may share identical sequences if overall diversity is very low. It was also interesting to note several cases where brain-derived virus spike sequences showed non-synonymous differences relative to sequences from other tissues. These differences may indicate differential selective pressure on spike by antiviral antibodies in brain versus other sites, though further studies will be needed to confirm this speculation.
Our results collectively show while that the highest burden of SARS-CoV-2 is in the airways and lung, the virus can disseminate early during infection and infect cells throughout the entire body, including widely throughout the brain. While others have posited this viral dissemination occurs through cell trafficking11 due to a reported failure to culture virus from blood3,22, our data support an early viremic phase, which seeds the virus throughout the body following pulmonary infection. Recent work by Jacobs et al.22 in which SARS-CoV-2 virions were pelleted and imaged from COVID-19 patient plasma, supports this mechanism of viral dissemination.
Although our cohort is primarily made up of severe cases of COVID-19, two early cases had mild respiratory symptoms (P28; fatal pulmonary embolism occurred at home) or no symptoms (P36; diagnosed upon hospitalization for ultimately fatal complications of a comorbidity), yet still had SARS-CoV-2 RNA widely detected across the body, including brain, with detection of sgRNA in multiple compartments.
Our findings, therefore, suggest viremia leading to body-wide dissemination, including across the blood-brain barrier, and viral replication can occur early in COVID-19, even in asymptomatic or mild cases. Further, P36 was a juvenile with no evidence of multisystem inflammatory syndrome in children, suggesting infected children without severe COVID-19 can also experience systemic infection with SARS- CoV-2.
Finally, a major contribution of our work is a greater understanding of the duration and locations at which SARS-CoV-2 can persist. While the respiratory tract was the most common location in which SARS-CoV-2 RNA tends to linger, ≥50% of late cases also had persistence in the myocardium, thoracic cavity lymph nodes, tongue, peripheral nerves, ocular tissue, and in all sampled areas of the brain, except the dura mater.
Interestingly, despite having much lower levels of SARS-CoV-2 in early cases compared to respiratory tissues, we found similar levels between pulmonary and the extrapulmonary tissue categories in late cases. This less efficient viral clearance in extrapulmonary tissues is perhaps related to a less robust innate and adaptive immune response outside the respiratory tract.
We detected sgRNA in tissue of over 60% of the cohort. While less definitive than viral culture23,24, multiple studies have shown that sgRNA levels correlate with acute infection and can be detected in respiratory samples of immunocompromised patients experiencing prolonged infection24.
These data coupled with ISH suggest that SARS-CoV-2 can replicate within tissue for over 3 months after infection in some individuals, with RNA failing to clear from multiple compartments for up to D230. This persistence of viral RNA and sgRNA may represent infection with defective virus, which has been described in persistent infection with measles virus – another single-strand enveloped RNA virus—in cases of subacute sclerosing panencephalitis25.
The mechanisms contributing to PASC are still being investigated; however, ongoing systemic and local inflammatory responses have been proposed to play a role5. Our data provide evidence for delayed viral clearance, but do not support significant inflammation outside of the respiratory tract even among patients who died months after symptom onset.
Understanding the mechanisms by which SARS-CoV-2 persists and the cellular and subcellular host responses to viral persistence promises to improve the understanding and clinical management of PASC.
