In a recent article published in bioRxiv* server, researchers used a preclinical animal model to investigate early pathophysiological mechanisms potentially underlying post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC), especially its neurological symptoms.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

These insights could inform therapeutic interventions needed to reduce the PASC burden.

Background

PASC, a multisystemic condition arising many weeks after acute SARS-CoV-2 infection, affects the brain, leading to impaired neurocognition, psychiatric perturbances, and olfactory dysfunctions. These neurological symptoms affect many individuals and adversely impact their working abilities. 

Non-human primates, such as African green monkeys (AGMs) and Indian Rhesus macaques (RMs), are susceptible to SARS-CoV-2, albeit with species-specific variations in infection proneness and disease outcomes.

Thus, they appear to be appropriate to investigate the PASC pathogenesis following clinical recovery from coronavirus disease 2019 (COVID-19). 

SARS-CoV-2 ribonucleic acid (RNA) has been found in brain tissues of patients who died due to COVID-19. It has been postulated that brain cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are an entry route for SARS-CoV-2 invading the central nervous system (CNS). 

About the study

In the present study, researchers evaluated the persistence of SARS-CoV-2 RNA in the CNS using an RNA in situ hybridization approach in conjunction with a highly specific probe for SARS-CoV-2 messenger RNA (mRNA).

To this end, they first inoculated four African green monkeys (aged 16 years) and four Rhesus macaques (aged 13- 15 years) with the SARS-CoV-2 USA-WA1/2020 isolate by either small particle aerosol or other routes.

The dosage for the former was 1 x 10⁴ plaque-forming units (PFU), and for oral, nasal, intratracheal, and conjunctival inoculations was 4 x 10⁶ PFU. 

The team autopsied the SARS-CoV-2 infected test animals to collect tissue from the pyriform cortex/amygdala and olfactory epithelium for further analysis. The olfactory epithelium may be uniquely prone to SARS-CoV-2 invasion as a result of its neuroanatomical location.

The olfactory epithelium transmits odor-related information to the pyriform cortex for processing, an area of the brain reciprocally connected to regions of the brain such as the amygdala with key roles in emotional processing and cognitive function. Hence, characteristics of the olfactory epithelium and pyriform cortex/amygdala may be important in PASC and its early pathophysiological symptoms.

Further, the team investigated pericytes, brain cells that co-express ACE2 and TMPRSS28, hypothesizing that SARS-CoV-2 enters the CNS through these cells and persists even in the brains of clinically recovered apes.

Specifically, they used RNAscope to investigate the expression of ACE2 and TMPRSS2 in the pyriform cortex/amygdala of clinically recovered and wild-type Rhesus macaques. 

Next, they dual-labeled (with a fluorescent probe) the tissues derived from the pyriform cortex/amygdala for SARS-CoV-2 mRNA and platelet-derived growth factor receptor beta (PDGFRβ). The latter is a biomarker exclusively expressed by pericytes in the adult brain. It helped them directly visualize each SARS-CoV-2 mRNA transcript in the brain, represented by a single-dot staining pattern. 

Finally, the team utilized advanced statistical approaches, including regression analyses, to evaluate whether viral loads and the acute clinical disease phenotype were associated with the total number of SARS-CoV-2 mRNA transcripts, and indicated chronic brain infection.

Results 

The authors detected high levels of SARS-CoV-2 RNA in samples from mucosal swabs and bronchial brush in both African green monkeys and Rhesus macaques. Dose effects or species-specific differences were statistically insignificant.

GM1 and GM2 developed acute respiratory distress syndrome (ARDS), and researchers had to euthanize them. The remaining two African green monkeys and four Rhesus macaques, however, clinically recovered from SARS-CoV-2 infection.

They had undetectable viral loads, and no clinical pathology by the end of the experimentation window, i.e., 21-28 days post-infection. These results confirmed that a non-human primate model is apt for studying early pathophysiological changes occurring during PASC post-acute SARS-CoV-2 infection. 

Wildtype and SARS-CoV-2-inoculated nonhuman primates highly expressed ACE2 and TMPRSS2 in their pyriform cortex/amygdala, as reported in many previous studies. SARS-CoV-2 inoculation, however, markedly downregulated their expression.

ACE2 downregulation resulted in the exacerbation of inflammatory responses. However, the physiological implications of TMPRSS2 downregulation in the CNS require further study.

The authors detected abundant SARS-CoV-2 mRNA in the pyriform cortex/amygdala of clinically recovered non-human primates but not in wild-type non-primates.

Notably, they also found SARS-CoV-2 mRNA in the olfactory epithelium but at much lower levels compared to the pyriform cortex/amygdala. These observations further favor the notion that SARS-CoV-2 persists in the CNS. 

Intriguingly, pericytes harbor SARS-CoV-2 in the CNS even after full clinical recovery, an observation that favors previous findings made using cortical organoids.

Thus, SARS-CoV-2 invasion of pericytes might be one of the causes of neurological manifestations of COVID-19, including blood flow reductions and inflammation. Both SARS-CoV-2 mRNA and PDGFRβ exhibited high co-localization in all African green monkeys and Rhesus macaques. 

Furthermore, the authors noted no significant association between viral loads, clinical assessment scores, lung histopathologic scores, and the SARS-CoV-2 mRNAs in the pyriform cortex, which implies that the acute COVID-19 is not a predictor of the extent of SARS-CoV-2 mRNA invasion in the CNS. 

Conclusions

To summarize, in this study, the researchers identified two pathophysiological mechanisms underlying PASC. First, they noted that SARS-CoV-2 infections downregulated ACE2 and TMPRSS2 mRNA in the pyriform cortex.

Secondly, SARS-CoV-2 mRNA persisted in brain pericytes of primates clinically recovered from acute SARS-CoV-2 infection. 

Thus, interventions aimed at the downregulation of the expression of ACE2/TMPRSS2 and pericytes invasion in the CNS might help effectively reduce the PASC pathology.

However, more importantly, further studies should investigate the long-term effects of SARS-CoV-2 infection on CNS beyond 28 days post-infection.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.