NEW YORK – An international team of researchers has found that the oral cavity is an important site for SARS-CoV-2 infection, implicating saliva in the transmission of the virus.
In a study published on Thursday in Nature Medicine, the researchers noted that the role of the oral cavity in COVID-19 isn't well understood, despite symptoms of infection such as loss of taste, dry mouth, and mucosal lesions. In order to explore this dynamic, they generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva, ultimately identifying 34 unique cell subpopulations between the glands and gingiva.
Importantly, the researchers found that SARS-CoV-2 viral entry factors such as ACE2, TMPRSS2, and TMPRSS4 were broadly enriched in the epithelial cells of the glands and oral mucosae. Further, saliva from people infected with SARS-CoV-2 harbored epithelial cells expressing ACE2 and TMPRSS, and acellular and cellular salivary fractions from asymptomatic individuals were also found to transmit SARS-CoV-2 ex vivo. Indeed, the researchers said, the asymptomatic patients maintained IgG antibodies against SARS-CoV-2 in their saliva upon their recovery from the virus.
"The expression levels of the entry factors are similar to those in regions known to be susceptible to SARS-CoV-2 infection, such as the tissue lining the nasal passages of the upper airway," said study co-corresponding Blake Warner, assistant clinical investigator and chief of the NIH's National Institute of Dental and Craniofacial Research's salivary disorders unit, in a statement.
Kevin Byrd, the study's other senior author and a coordinator of the international Human Cell Atlas' Oral and Craniofacial Biological Network, also noted that the team's findings show the mouth plays a bigger role in SARS-CoV-2 infection than previously thought, through infected oral cells.
"When infected saliva is swallowed or tiny particles of it are inhaled, we think it can potentially transmit SARS-CoV-2 further into our throats, our lungs, or even our guts," he said in the statement.
The researchers began by hypothesizing that the salivary gland and barrier epithelia of the oral cavity and oropharynx could be infected by SARS-CoV-2, which could then contribute to the transmission of the virus. To test this, they generated two human oral single-cell RNA sequencing (scRNA-seq) atlases to predict cell-specific susceptibilities to SARS-CoV-2 infection, and confirmed infection with the virus in autopsy and outpatient samples.
The data revealed that different oral epithelial cells provided entryways for viral infection. To predict the viral tropism of SARS-CoV-2, other coronaviruses (such as SARS-CoV-1 and MERS-CoV), influenza, and rhinovirus C, the researchers performed joint annotation of the salivary glands and gingival scRNA-seq atlases and identified 34 unique cell types, including 12 epithelial, seven mesenchymal, and 15 immune cell clusters. The cell type expression of known viral entry factor genes across cell clusters demonstrated broad potential susceptibilities to viral infection among the epithelial populations, they said.
Similarly to SARS-CoV-1, the SARS-CoV-2 spike glycoprotein binds to ACE2 and is activated by tissue-specific proteases such as TMPRSS2, TMPRSS4, and TMPRSS11D, and endosomal proteases such as CTSB, CTSL, BSG, and FURIN to gain entry for replication. The researchers found that these factors were broadly expressed across many clusters, but that only the epithelia commonly expressed ACE2 and the various TMPRSS members.
Further, an analysis of SARS-CoV-2 entry factor expression revealed that no single oral epithelial subpopulation appeared at singular risk for infection. In fact, ACE2 expression was detected in nine oral epithelial clusters, suggesting that multiple oral epithelial cell subtypes were susceptible to infection.
The researchers then performed droplet digital PCR on a group of autopsy tissues from 18 deceased COVID-19 patients, assessing SARS-CoV-2 transcripts in 28 salivary gland and six mucosal tissue samples. They detected the virus in 57 percent of the salivary glands and found SARS-CoV-2 infection in five of six of the available mucosal sites.
The researchers also found that saliva from asymptomatic individuals with COVID-19 can potentially transmit the virus. In a prospective clinical cohort, they observed a positive correlation between salivary viral load and taste loss, and demonstrated persistent salivary antibody responses to SARS-CoV-2 nucleocapsid and spike proteins.
Notably, they added, these results suggest that expelled oral droplets containing infectious virus and infected cells could be a source of SARS-CoV-2 airborne transmission. Interestingly, when the researchers tested the effectiveness of standard mask wearing to reduce droplet spread in asymptomatic individuals, they found a more than tenfold decrease in expelled salivary droplets, including in some asymptomatic individuals with positive nasopharyngeal or saliva viral load.
"Our findings might partially explain some false-negative tests using only [nasopharyngeal] or saliva testing," the authors concluded. "However, these results raise new questions about COVID-19 pathogenesis," such as whether infection mainly occurs through the nose and then spreads to the mouth or whether the mouth is infected first, and whether the pattern of infection affects disease severity and how the host's immune system responds.
Importantly, the study's findings also raise the question of whether SARS-CoV-2 diagnostics that use saliva as a sample type could eventually be more accurate than those that require nasopharyngeal swabs. Although this study wasn't specifically designed to answer that question, Warner told GenomeWeb, the researchers had initially been curious about the diagnostic importance of saliva.
"I think what our study does support is that looking in the mouth, looking in saliva, is an important place to look for infection," he said, adding that "our study really does support that not only is saliva a good diagnostic fluid and it's easy to collect, but probably you can learn some biology, too, by looking at what's going on in saliva."
One possible advantage saliva tests may eventually have, Warner also noted, is that the use of whole unstimulated saliva (also known as spitting into a cup) would likely be representative of the entire oral cavity at once. So, if there were any variability of infection in the different parts of the mouth, "whole unstimulated saliva is likely to be representative of all of the sites all at once — salivary glands secreting saliva that might contain infectious particles [and] epithelial cells from lots of different sites in the mouth containing either infectious epithelial cells or infectious particles themselves," he said.
So, although the study itself didn't delve into saliva diagnostics, Warner noted that saliva would do "a good job of representing a lot of the oral cavity with one relatively straightforward test."