Enteric viruses found to transmit through the salivary route

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In a recent study published in Nature, researchers reported on the productive and persistent infection of salivary glands (SGs) by enteric viruses, with oral cavity titers similar to intestinal titers.

Study: Enteric viruses replicate in salivary glands and infect through saliva. Image Credit: h2ojs/Shutterstock
Study: Enteric viruses replicate in salivary glands and infect through saliva. Image Credit: h2ojs/Shutterstock

Enteric viruses such as astrovirus, norovirus (NoV), and rotavirus conventionally spread through the fecal-oral route; however, their genomic ribonucleic acid (RNA) has also been detected in saliva. It is essential to assess the salivary route of enteric virus transmission as viruses could rapidly transmit through actions such as coughing, sneezing, kissing, and talking.

About the study

In the present study, researchers reported on saliva as a medium of transmission and the oral cavity as a site of replication for enteric viruses.

Neonatal mouse pups (less than 10 days old) were inoculated orally with murine NoV 1 (MNV-1) or rotavirus [epizootic diarrhea of infant mice (EDIM)] and the viral replication was expressed as median tissue culture infectious dose (TCID50) and quantitative polymerase chain reaction (qPCR) values for MNV-1 and EDIM, respectively. The pups’ small intestinal secretory immunoglobulin A (sIgA) levels were monitored. Mammary dams were immunostained with anti-MNV-1- and anti-EDIM antibodies and non-structural proteins (NSP) 4 and 5, respectively.

Further, the team investigated if viral replication in the mammary glands of dams and the resultant rapid upsurge in milk sIgA were a result of dams infected by their neonatal pups via the conventional fecal-oral route. The levels of EDIM genomic RNA in the dams’ mammary glands, the pups’ small intestines, and the milk sIgA levels were assessed.

For further interrogation of the viral transfer mode, mice pups were inoculated orally with EDIM (A group pups) and were returned to their mother (dam A) for suckling. At one dpi, the mothers were replaced with a foster mother (dam B) from the uninoculated pups (B group pups)-containing cage. Dam A and Dam B suckled the B group pups and A group pups, respectively. At three dpi, animals in both cages died and the replication of viruses in the dams’ mammary glands and the pups’ small intestines was measured.

Furthermore, the team investigated if saliva could be a medium for enteric virus transmission to the dams’ mammary glands during suckling, for which, saliva samples were obtained from mice inoculated orally with MNV-1 or EDIM. In addition, immunoblotting analysis was performed with anti-MNV-1 VP antibodies and anti-EDIM rotavirus VP6. The mice were also inoculated with MNV-3,4, WU23, and CR6 murine NoVs.

The team investigated if murine SG cell spheroids (salispheres) could be used for ex vivo culture studies of murine viruses. Lastly, they explored human NoVs (HuNoVs) replication in SV40-transformed-human SG acinar and ductal cell lines such as NS-SV-TT-AC and NS-SV-TT-DC, respectively, using PCR, immunoblotting analysis using NSP 6,7 and fluorescence in situ hybridization (FISH).


Robust intestinal replication of MNV-1 and EDIM was observed in pup intestines with both viruses peaking between three days post-inoculation (dpi) and five dpi and the viruses cleared after seven to 10 days of inoculation. Similar findings were obtained among adult mice. The team detected a swift surge in pups’ small intestine sIgA titers three dpi onward among pups inculcated with EDIM or MNV-1, which correlated with a swift surge in the dam’s milk sIgA titers.

Isolation of dam mammary glands showed a ~105-fold increase in EDIM and MNV-1 genomic RNA, indicative of replicating mammary enteric viruses, which was confirmed by the immunostaining analysis. In the immunostaining analysis, B lymphocytes and the milk duct-lining epithelial cells were identified as sites of MNV-1 and EDIM replication, respectively.

Contrasting to the dams that suckled virally-infected neonatal pups, no surge in sIgA titers was detected in orally inoculated dams’ milk with the absence of detectable viral genomic RNA in the dams’ mammary glands. On the contrary, 106-fold higher genomic RNA levels of both viruses were detected in the pups’ small intestines at four dpi.

104-fold and 106-fold increases in viral RNA levels in mammary glands (of A and B dams) and intestines (of A and B group pups), respectively, were observed, indicating that both dams acquired infections by suckling A group pups; dam A was initially infected with A group pups and B dam thereafter. B group pups were most likely infected from the feces or mammary glands of dam A by suckling. Taken together, the findings are indicative of the backflow of enteric viruses from the neonatal pups to the mothers via suckling, giving rise to in situ infections of the mother’s mammary glands and a quick upsurge in milk sIgA titers which may have contributed to the clearing of infection among pups.

In the immunoblotting analysis performed with adult mice, both MNV-1 and EBIM demonstrated shedding in saliva within two dpi with 104-fold higher TCID50 values for MNV-1; however, both viruses caused acute infections which cleared in seven to 10 days. On the contrary, MNV-3,4 and WU23 murine NoVs demonstrated persistent infection in the proximal colon with shedding in feces for approximately 21 dpi with a 103-fold increase in titers.

MNV-1,3,4 and WU23 inoculation led to 104-fold increases in the corresponding viral titers in the submandibular SGs (SMGs). Likewise, SMGs of astrovirus- and EDI-inoculated mice showed 103-fold and 105-fold increases in viral genomic RNA levels, respectively. Of note, WU23 and MNV-3,4 viruses demonstrated comparable replication titers in the SMGs and the proximal colon whereas CR6 replication was not observed in the SMGs, indicating differences in replication dynamics among murine NoVs.

EDIM and MNV-1 replicated in epithelial cell adhesion molecule + (EpCAM+) and the cluster of differentiation 45+ (CD45+) SMG cells and the murine NoVs required CD300lf receptors for infecting SMGs. Salispheres demonstrated robust MNV-1, EDIM, and CR6 replication in vivo. Vesicle-cloaked viruses replicated efficiently in the human SG cell lines.


Overall, the study findings highlighted saliva as an alternate route for enteric virus transmission and SG-derived cell lines and spheroids as scalable virus production systems. The salivary route of enteric virus transmission may have diagnostic and therapeutic implications, and appropriate sanitation measures, in addition to those for preventing the traditional fecal-oral transmission, are required to limit the spread of enteric viruses via saliva.   

Journal reference:
Pooja Toshniwal Paharia

Written by

Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.


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