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

Enteric viruses transmitted through the salivary route

In a recent study published in Natureresearchers reported on the prolific and persistent infection of salivary glands (SGs) by enteric viruses, with oral cavity titres comparable to gut titres.

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 by 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 can be rapidly transmitted 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 orally inoculated with murine NoV 1 (MNV-1) or rotavirus [epizootic diarrhea of infant mice (EDIM)] and 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 secretory immunoglobulin A (sIgA) levels of the pups in the small intestine were monitored. Mammary dams were immunostained with anti-MNV-1 and anti-EDIM antibodies and non-structural proteins (NSP) 4 and 5, respectively.

Furthermore, the team examined whether viral replication in the mammary glands of dams and the resulting rapid increase in milk sIgA resulted from dams infected by their neonatal pups through the conventional fecal-oral route. The levels of EDIM genomic RNA in the mammary glands of the dams, the small intestine of the pups and the milk sIgA levels were assessed.

For further questioning of the viral transmission mode, mouse pups were orally inoculated with EDIM (A group pups) and returned to their mother (mother A) for suckling. At one dpi, the mothers were replaced with a foster mother (mother B) from the cage containing uninoculated pups (B group pups). Mother A and Mother B nurse the B-group puppies and A-group puppies, respectively. At three dpi, the animals in both cages died and virus replication was measured in the mammary glands of the dams and the small intestine of the pups.

The team further investigated whether saliva could be a medium for enteric virus transmission to the mammary glands of the dams during lactation, 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 mouse NoVs.

The team investigated whether mouse SG cell spheroids (salispheres) can be used for: ex vivo culture studies of mouse viruses. Finally, they examined 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 the gut of the pups, with both viruses peaking between three days post-inoculation (dpi) and five dpi and the viruses disappearing after seven to ten days of inoculation. Similar findings were obtained in adult mice. The team found a rapid rise in small intestinal sIgA titres in pups of three dpi in pups instilled with EDIM or MNV-1, which correlated with a rapid rise in breast milk sIgA titres.

Isolation of 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 epithelial cells of the milk duct lining were identified as sites of MNV-1 and EDIM replication, respectively.

In contrast to the dams that nursed virally infected neonatal pups, no increase in sIgA titers was detected in orally inoculated breast milk with the absence of detectable viral genomic RNA in the mammary glands of the dams. On the contrary, 106fold higher genomic RNA levels of both viruses were detected in the small intestine of the pups at four dpi.

104-folding and 106-fold increases in viral RNA levels in mammary glands (from A and B dams) and gut (from A and B group pups, respectively) were observed, indicating that both dams contracted infections from nursing A group pups; mother A was initially infected with A group of puppies and B mother afterwards. Group B puppies were most likely infected from mother A’s feces or mammary glands by sucking. Taken together, the findings are indicative of the return of enteric viruses from the neonatal pups to the mothers via lactation, leading to on the spot maternal mammary gland infections and a rapid rise in milk sIgA titers that may have contributed to clearing the infection in puppies.

In the immunoblotting analysis performed on adult mice, both MNV-1 and EBIM showed salivary secretion within two dpi at 104-fold higher TCID50 values ​​for MNV-1; however, both viruses caused acute infections that resolved within seven to 10 days. On the contrary, MNV-3.4 and WU23 mouse NoVs showed persistent infection in the proximal colon with excretion in feces for approximately 21 dpi with a 103-fold increase in titers.

MNV-1,3,4 and WU23 vaccination led to 104-fold increases in the corresponding viral titers in the submandibular SGs (SMGs). Similarly, SMGs from astrovirus- and EDI-inoculated mice showed 103-folding and 105-fold increase in viral genomic RNA levels, respectively. Note that WU23 and MNV-3,4 viruses showed similar replication titers in the SMGs and the proximal colon, while CR6 replication was not observed in the SMGs, indicating differences in replication dynamics between mouse NoVs.

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


Overall, the study results emphasized saliva as an alternative 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 the prevention of traditional faecal-oral transmission, are required to limit the spread of enteric viruses via saliva.

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