In a recent study published in Natureresearchers reported productive and persistent infection of the salivary glands (SG) with enteric viruses, with titers in the oral cavity similar to intestinal titers.
Enteric viruses such as astrovirus, norovirus (NoV), and rotavirus are classically spread by the fecal-oral route; however, their genomic ribonucleic acid (RNA) has also been detected in saliva. Assessing the salivary route of enteric virus transmission is critical because viruses can be transmitted quickly through actions such as coughing, sneezing, kissing, and talking.
About the study
In the present study, researchers reported saliva as a medium of transmission and the oral cavity as a site of replication for enteric viruses.
Neonatal mice (less than 10 days old) were inoculated orally with murine NoV 1 (MNV-1) or rotavirus [epizootic diarrhea of infant mice (EDIM)] and viral replication was expressed as tissue culture median infectious dose (TCID50) and quantitative polymerase chain reaction (qPCR) values for MNV-1 and EDIM, respectively. Levels of secretory immunoglobulin A (sIgA) in the small intestine of the puppies were monitored. Mammary mammaries were immunostained with anti-MNV-1 and anti-EDIM antibodies and non-structural proteins (NSPs) 4 and 5, respectively.
In addition, the team investigated whether the viral replication in the mothers’ mammary glands and the resulting rapid increase in milk sIgAs were the result of mothers infecting their newborn pups through the conventional fecal-oral route. EDIM genomic RNA levels in the mammary glands of the dams, the small intestine of the pups and the levels of IgAs in the milk were assessed.
For further interrogation of the mode of viral transfer, the pups were orally inoculated with EDIM (group A pups) and returned to their mothers (mother A) for nursing. At one day per inch, mothers were replaced with an adoptive mother (mother B) from the cage containing the uninoculated pups (group B pups). Mother A and mother B nursed the pups from group B and pups from group A, respectively. hail of puppies was measured.
In addition, the team investigated whether saliva could be a means of transmission of enteric virus to mammary glands of mothers during lactation, for which saliva samples were obtained from mice orally inoculated with MNV- 1 or EDIM. In addition, immunoblot analysis was performed with anti-MNV-1 VP and anti-EDIM rotavirus VP6 antibodies. The mice were also inoculated with the murine NoVs MNV-3, 4, WU23 and CR6.
The team investigated whether murine SG cell spheroids (salispheres) could be used to ex vivo murine virus culture studies. Finally, they explored the replication of human NoVs (HuNoVs) in SV40-transformed human acinar and ductal SG cell lines such as NS-SV-TT-AC and NS-SV-TT-DC, respectively, using PCR, analysis by immunotransfer using NSP 6.7 and fluorescence in situ hybridization (FISH).
Results
Robust intestinal replication of MNV-1 and EDIM was observed in the intestines of puppies, with both viruses peaking between three days post-inoculation (dpi) and five dpi and the viruses being cleared after seven to 10 days of dpi. ‘inoculation. Similar results were obtained in adult mice. The team detected a rapid increase in pup small intestine sIgA titers from three days per inch among pups instilled with EDIM or MNV-1, which correlated with a rapid increase in sIgA titers of the breastmilk.
Dam mammary gland isolation showed a ~1051-fold increase in EDIM and MNV-1 genomic RNA, indicating replication of enteric mammary viruses, which was confirmed by immunostaining analysis. In immunostaining analysis, B lymphocytes and epithelial cells of the milk duct mucosa were identified as sites of replication of MNV-1 and EDIM, respectively.
Unlike mothers who breastfed virus-infected neonates, no increased sIgA titers were detected in the milk of orally inoculated mothers in the absence of detectable viral genomic RNA in the mammary glands of the infants. mothers. On the contrary, 106Times higher genomic RNA levels of both viruses were detected in the small intestine of puppies at four days per inch.
dix4-fold and 1061-fold increases in viral RNA levels in the mammary glands (of dams A and B) and intestines (of puppies in groups A and B), respectively, were observed, indicating that both dams contracted infections while nursing Group A puppies; mother A was initially infected by group A pups and mother B subsequently. Group B puppies were most likely infected through mother A’s faeces or mammary glands through feeding. Taken together, the results indicate reflux of enteric viruses from small newborns to mothers via breastfeeding, resulting in on the spot infections of the mammary glands of the mother and a rapid increase in IgA titers in the milk which may have contributed to the elimination of the infection in the pups.
In immunoblot analysis performed with adult mice, MNV-1 and EBIM demonstrated excretion in saliva in two dpi with 104– times the upper TCID50 values for MNV-1; however, both viruses caused acute infections that resolved within seven to 10 days. In contrast, mouse NoVs MNV-3,4 and WU23 demonstrated persistent infection in the proximal colon with excretion in the feces for approximately 21 dpi with a 103– times the titer increase.
Inoculation of MNV-1,3,4 and WU23 led to 104-multiplication of the corresponding viral titers in the submandibular SG (SMG). Similarly, SMGs from mice inoculated with astrovirus and EDI showed 103-fold and 105times the viral genomic RNA levels, respectively. Of note, WU23 and MNV-3,4 viruses demonstrated comparable replication titers in SMGs and proximal colon whereas CR6 replication was not observed in SMGs, indicating differences in dynamics of replication among murine NoVs.
EDIM and MNV-1 replicated in epithelial cell adhesion molecule+ (EpCAM+) and the 45+ differentiation cluster (CD45+) SMG cells and murine NoVs required CD300lf receptors to infect SMGs. Salispheres demonstrated robust replication of MNV-1, EDIM and CR6 in vivo. Vesicle-masked viruses replicated efficiently in human SG cell lines.
Conclusion
Overall, the study results highlighted 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 transmission of enteric viruses may have diagnostic and therapeutic implications, and appropriate sanitation measures, in addition to those aimed at preventing traditional fecal-oral transmission, are necessary to limit the spread of enteric viruses via saliva. .
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