Days after the World Health Organization (WHO) declared COVID-19 to be a pandemic, in March 2020, a study of patients in Italy reported loss of smell and taste as a symptom. The first study to analyze the neurological impact of the disease in hundreds of people was published in April. Since then, research into the consequences of COVID-19 for the brain has discussed the effects observed in the acute stage, and neurological complications reported by some 30% of patients who recover.

“COVID-19 was initially described as a viral infection of the respiratory tract, but we soon learned that the brain is one of several affected organs. Some aspects of the disease remain obscure. The impact on the brain isn’t fully understood. It’s very important to stimulate the sharing of knowledge and experience among researchers around the world,” said Luiz Eugênio Mello, Scientific Director of FAPESP, in his welcoming remarks to the online seminar “What does COVID-19 have to do with the brain?”, held on July 7. The webinar featured speakers from Brazil and Germany and was the latest in the series FAPESP COVID-19 Research Webinars, organized with the support of the Global Research Council (GRC).

Viral entry route 

One of the studies presented to the webinar was conducted at Charité University of Medicine Berlin (Germany) and showed that the novel coronavirus uses the olfactory mucosa as the main route to enter the brain. “This is due to the anatomical proximity between the cells of the mucosa and the blood vessels and nerve cells in the area. Once the virus has entered the olfactory mucosa, it appears to use neuroanatomical connections like the olfactory nerve to reach the brain,” said Helena Radbruch, a researcher in the Department of Neuropathology who analyzed samples from olfactory mucosa and four brain regions of 33 patients who died from severe COVID-19.

Radbruch and her team tracked 180 other patients from the acute stage of the disease until months after they recovered. “The good news, especially for those who have had COVID-19, is that the virus doesn’t stay in the brain. We found it there only in some cases, and it was no longer there a month or two after the acute stage,” she said.

They also found clear evidence of activated immune cells in the brain, medulla, and other parts of the central nervous system. In some cases, there was tissue and neuron damage due to hypercoagulation and acute infarction. 

“The entry of the virus into the central nervous system via the neurons of the olfactory mucosa and other cranial nerves appears to explain neurological symptoms such as loss of smell and taste, which isn’t at all rare among COVID-19 patients,” she said.

In Brazil, researchers at D’Or Institute (IDOR) and the Federal University of Rio de Janeiro (UFRJ) conducted a series of experiments and concluded that the virus enters the brain by other routes besides the olfactory mucosa, including systemic inflammation as the disease progresses and affects different organs.

“Unfortunately, in one autopsy we found severe viral infection of the choroid plexus, a central nervous system structure protected by the blood-brain barrier. This brain region has a large amount of ACE2, the protein to which the virus binds to invade the organism. ACE2 is also abundant in the lungs,” said Marilia Zaluar Guimarães, a researcher at UFRJ and IDOR.

The study arose from the rare case of a one-year-old infant who already suffered from encephalopathy and did not survive COVID-19. The autopsy revealed the presence of viral particles in the lungs, heart, cortex and choroid plexus. “Infection by SARS-CoV-2 caused pneumonia, meningitis and damage to multiple organs owing to thrombosis, including the kidneys, lungs, brain, heart and pancreas,” she explained.

Having confirmed that the virus can breach the blood-brain barrier and infect the brain, the researchers decided to conduct studies with brain organoids, highly simplified genetically engineered models of the brain, and laboratory-cultured neural precursor cells called neurospheres. These were originally developed during the zika epidemic using induced human pluripotent stem cells (hIPSCs), skin or blood cells reprogrammed to return to a stage similar to stem cells. The hIPSCs are stimulated to differentiate into nerve cells such as neuroprogenitors, astrocytes and neurons. 

“The organoids are a very simplified model of the brain, but they contain several cell types that let us study the progression of viral infection. In this manner, we proved that SARS-CoV-2 causes brain damage but isn’t able to replicate in the brain since three days after infection there was no viral presence in the neurospheres, which was puzzling,” Zaluar said. The researchers also found that infection reduced the number of neuroprogenitors but did not prevent these cells from proliferating.

Similar studies conducted previously had detected viral replication in brain organoids and neurospheres, but used much larger quantities of the virus and exposed the cell cultures for 24 hours (versus one hour in her study), she noted, concluding that systemic events could explain most of the neurological damage seen in severe COVID-19 patients, although the virus also causes direct brain damage and neuroinflammation.

The research presented by both Zaluar and Radbruch showed that although the virus is cleared from the brain a short time after infection, the elevated levels of pro-inflammatory cytokines persist well after the acute stage, which probably explains the neurological problems typical of long COVID. 

Glial cells

Scientists affiliated with the University of Campinas (UNICAMP) and the University of São Paulo (USP) conducted a study, with support from FAPESP, in which they analyzed data for 81 subjects who tested positive for COVID-19 but had mild symptoms or none and did not require hospitalization. More than 50 days after they were diagnosed, they were found to have alterations in the structure of the cerebral cortex associated with the olfactory tract. In addition, 28% suffered to some degree from anxiety, 20% from depression, 28% from memory loss, and 34% from cognitive impairment.

The researchers also analyzed brain tissue samples from 26 patients who died after contracting COVID-19. The presence of the virus was confirmed in all samples, and alterations that suggested damage to the central nervous system were detected in five samples. 

“We already knew of such neurological symptoms as loss of smell and taste, but our studies showed for the first time that the virus infects and replicates in astrocytes, which are the most abundant cells in the central nervous system and essential to the maintenance of neurons,” said Marcelo Mori, a professor at UNICAMP’s Institute of Biology (more at: agencia.fapesp.br/34404). 

Researchers who collaborate via the Instituto Pasteur-USP scientific platform highlighted another interesting aspect of the effects of COVID-19 on the brain. Metabolic alterations in infected glial cells (astrocytes and other cell types that support and nourish neurons) may be related not only to the effects of the disease on the brain in the acute stage of the disease but also to long-term neurological complications reported by some patients.

“Animal model studies show that the virus can infect glial cells, where it can replicate, produce new viral copies and cause structural changes that affect cell metabolism,” said Jean Pierre Peron, a researcher at USP’s Biomedical Sciences Institute (ICB) and principal investigator for a project on the topic supported by FAPESP.

The scientists conducted analyses to verify alterations in protein expression by infected cells (proteomics) and in the metabolism (metabolomics). “We found many alterations in protein expression, especially those involved in carbon and glucose metabolism,” Peron said. “Not by chance, these signaling pathways are associated with brain disorders such as Huntington’s, amyotrophic lateral sclerosis and long-term depression.”

The metabolomic analysis showed hyperactivation of glycolytic pathways and mitochondria in infected glial cells. “Overall, we found that during infection by the virus there were changes in protein expression that correlated with the carbon metabolism of these glial cells,” Peron said, noting that the alterations in the expression of the enzyme glutaminase are probably associated with viral replication. “The enzyme is extremely important to glial cells, as 90% of the synapses in the brain are glutamatergic [mediated by the neurotransmitter glutamate]. We believe the virus needs glutamine to replicate in the brain and release fully mature particles. When glutaminase is blocked, the release of pro-inflammatory cytokines is reduced,” he explained. Glutaminase is a mitochondrial enzyme that catalyzes the breakdown of glutamine to form glutamate as a part of the glutaminolysis pathway. The glutamate system is important for information processing in neuronal networks.

A recording of the complete webinar can be watched at: covid19.fapesp.br/o-que-covid-19-tem-a-ver-com-o-cerebro/550.

Featured image: A webinar held by FAPESP featured researchers from Brazil and Germany whose findings offer clues as to how SARS-CoV-2 invades the central nervous system and which cells are most affected (experiments with nerve cells conducted by researchers at D’Or Institute and UFRJ; reproduction)

Provided by FAPESP

In Brazil, researchers at the University of São Paulo’s Medical School (FM-USP) have discovered that SARS-CoV-2 infects and replicates in the salivary glands. 

Analysis of samples from three types of salivary gland obtained during a minimally invasive autopsy procedure performed on patients who died from complications of COVID-19 at Hospital das Clínicas, FM-USP’s hospital complex, showed that tissues specializing in producing and secreting saliva serve as reservoirs for the novel coronavirus. 

The study was supported by FAPESP and reported in an article published in the Journal of Pathology.

The researchers said the discovery helps explain why the virus is so abundant in saliva and has enabled scientists to develop saliva-based diagnostic tests for COVID-19. 

“This is the first report of a respiratory virus’s capacity to infect and replicate in salivary glands. Until now it was thought that only viruses that cause highly prevalent diseases such as herpes used salivary glands as reservoirs. The discovery may help explain why SARS-CoV-2 is so infectious,” Bruno Fernandes Matuck, a PhD candidate at USP’s Dental School and first author of the article, told Agência FAPESP.

A previous study by the same group had already demonstrated the presence of RNA from SARS-CoV-2 in the periodontal tissue of patients who died from COVID-19 (more at: agencia.fapesp.br/35675/). 

Because SARS-CoV-2 is highly infectious compared with other respiratory viruses, they raised the hypothesis that it may replicate in cells of the salivary glands and hence be present in saliva without coming into contact with nasal and lung secretions. Prior research detected ACE2 receptors in salivary gland ducts. The spike protein in SARS-CoV-2 binds to ACE2 in order to invade and infect cells. More recently, other research groups have conducted studies in animals showing that other receptors besides ACE2, such as transmembrane serine protease 2 (TMPRSS2) and furin, both of which are present in salivary glands, are targets of SARS-CoV-2.

To test this hypothesis in humans, ultrasound-guided autopsies were performed on 24 patients who died from COVID-19, with a mean age of 53, to extract tissue samples from the parotid, submandibular and minor salivary glands. 

The tissue samples were submitted to molecular analysis (RT-PCR), which detected the presence of the virus in more than two-thirds. Immunohistochemistry – a form of immunostaining in which antibodies bind to the antigen in the tissue sample, a dye is activated, and the antigen can then be seen under a microscope – also demonstrated the presence of the virus in the tissue. Finally, examination under an electron microscope detected not just the presence of the virus but also its replication in cells and the type of organelle it uses to replicate. 

“We observed several viruses clustering in salivary gland cells, which showed that they were replicating there. They weren’t in these cells passively,” Matuck said.

The mouth as direct point of entry

The researchers now plan to see whether the mouth can be a direct point of entry for SARS-CoV-2, given that ACE2 and TMPRSS2 are found in various parts of the cavity, as well as in gum tissue and oral mucosa. In addition, the mouth has a larger contact area than the nasal cavity, which is widely considered the main way in for the virus.

“We’re going to partner with researchers at the University of North Carolina in the United States to map the distribution of these receptors in the mouth and quantify viral replication in oral tissues,” said Luiz Fernando Ferraz da Silva, a professor at FM-USP and principal investigator for the project.

“The mouth could be a viable medium for the virus to enter the body directly,” Matuck said.

Another idea is to find out whether older people have more ACE2 receptors in their mouths than younger people, given the decrease in salivary secretion with age. Nevertheless, the researchers found a high viral load even in older patients, who have less salivary tissue.

“These patients had almost no salivary tissue, almost only fatty tissue. Even so, viral load was relatively high,” Matuck said.

The article “Salivary glands are a target for SARS-CoV-2: a source for saliva contamination” (doi: 10.1002/path.5679)  by Bruno Fernandes Matuck, Marisa Dolhnikoff, Amaro Nunes Duarte-Neto, Gilvan Maia, Sara Costa Gomes, Daniel Isaac Sendyk, Amanda Zarpellon, Nathalia Paiva de Andrade, Renata Aparecida Monteiro, João Renato Rebello Pinho, Michele Soares Gomes-Gouvêa, Suzana C.O.M. Souza, Cristina Kanamura, Thais Mauad, Paulo Hilário Nascimento Saldiva, Paulo H. Braz-Silva, Elia Garcia Caldini and Luiz Fernando Ferraz da Silva is at: onlinelibrary.wiley.com/doi/10.1002/path.5679.

Featured image: A study conducted at the University of São Paulo suggests that tissues specializing in saliva production and secretion serve as reservoirs for SARS-CoV-2, magnifying its infectious potential (electron microscope image showing novel coronavirus inside salivary glands; credit: Bruno Matuck/USP)

Provided by FAPESP

Scientists have developed a new technology to detect a wider variety of T cells that recognize coronaviruses, including SARS-CoV-2. The technology revealed that killer T cells capable of recognizing epitopes conserved across all coronaviruses are much more abundant in COVID-19 patients with mild disease versus those with more severe illness, suggesting a protective role for these broad-affinity T cells.

The ability to distinguish T cells based on their affinities to SARS-CoV-2 could help scientists elucidate the disparity in COVID-19 outcomes and determine which COVID-19 patients will or will not exhibit a successful immune response against the virus, the authors say.

Their work improves upon the primary tool used to identify T cells – antigen tetramers bound to MHC – by instead attracting the cells to multimerized complexes of antigens bound to MHC called “spheromers.” Because tetramers can harbor a maximum of four MHC-antigen complexes, they tend to miss T cells with a low affinity for certain antigens.

Vamsee Mallajosyula and colleagues tackled these limitations with their spheromers, each of which simultaneously displays 12 copies of an individual peptide-MHC complex. The spheromer is easy to produce and compatible with currently available MHC molecules and tetramer components, allowing for easy adoption of their new protocol, the authors say.

When applied to blood samples from COVID-19 patients and individuals not yet exposed to SARS-CoV-2, the spheromers stained specific T cells more efficiently and captured a more diverse repertoire of TCRs compared with the tetramer.

Using the technology, the authors found that T cells capable of recognizing peptides conserved across all coronaviruses were more abundant and exhibited a “memory” phenotype – a desirable feature among T cells targeted by vaccines – compared with T cells that only recognize SARS-CoV-2.

Indeed, COVID-19 patients with mild disease harbored a greater number of killer T cells with these conserved specificities than those with more severe illness, suggesting the broad-affinity T cells are protective, the authors say. Next steps will require enhancing the spheromer technology to include more MHC proteins, they add.

Reference: Vamsee Mallajosyula, Conner Ganjavi, Saborni Chakraborty, Alana M. McSween, Ana Jimena Pavlovitch-Bedzyk, Julie Wilhelmy, Allison Nau, Monali Manohar, Kari C. Nadeau, Mark M. Davis, “CD8+ T cells specific for conserved coronavirus epitopes correlate with milder disease in COVID-19 patients”, Science Immunology  01 Jul 2021: Vol. 6, Issue 61, eabg5669 DOI: https://doi.org/10.1126/sciimmunol.abg5669

Provided by AAAS

Targeting highly pathogenic coronavirus-induced apoptosis reduces viral pathogenesis and disease severity

A new study using cells, transgenic mouse models, and cultured human lung tissue provides evidence that the ability to trigger programmed cell death (apoptosis) may enable highly pathogenic coronaviruses to spread within their hosts so successfully.

Targeting this process may reduce the severity of coronavirus diseases, the study goes on to show. While scientists have been aware that highly pathogenic coronaviruses leave substantial cell death in their wake as they infiltrate the body, the importance of apoptosis to the internal spread of coronavirus infections – and the underlying mechanisms that account for the virus’ high pathogenicity – have not been clear.

To fill this research gap, Hin Chu and colleagues first observed MERS-CoV in cultured human lung tissue and in the lungs of infected transgenic mice, finding that the virus induced substantial apoptosis. To explore how the virus triggered programmed cell death in its host organ’s cells, the researchers analyzed the mRNA transcripts of human bronchial epithelial cells at 12 and 24 hours after infection with MERS-CoV, finding that genes regulated by the protein kinase R-like endoplasmic reticulum kinase (PERK), which are associated with cell death, were highly expressed.

When Chu et al. inhibited PERK signaling in the infected human lung tissues, they saw substantial reductions in both the cell-to-cell spread of MERS-CoV and in cell death induced by the virus. The researchers also performed tests in which they inhibited PERK signaling in transgenic mice infected with the virus. This treatment down-regulated genes involved in apoptosis and reduced lung damage.

While the authors found that PERK inhibitors did not suppress apoptosis triggered by SARS-CoV or SARS-CoV-2, they did observe that an intrinsic apoptosis inhibitor effectively suppressed the process in mice infected with either SARS-CoV or SARS-CoV-2 and reduced lung damage from SARS-CoV-2.

These findings suggest that while the three highly pathogenic human coronaviruses – SARS-CoV, MERS-CoV, and SARS-CoV-2 – may trigger apoptosis through different mechanisms, inhibiting apoptosis may attenuate the pathogenesis of all three.

The study, “Targeting highly pathogenic coronavirus-induced apoptosis reduces viral pathogenesis and disease severity”, Published in Science Advances on 16 June 2021.

Provided by AAAS