Rabies is a zoonotic disease caused by the rabies virus (RABV), which belongs to the genus Lyssavirus of the family Rhabdoviridae (Fooks et al., 2017; Singh et al., 2017). Human rabies is almost invariably fatal once clinical symptoms occur. With a high global death count per year, rabies continues to pose a severe threat to human and animal health (Fooks et al., 2014). Initial symptoms of rabies include generalized symptoms such as fever, pain, and abnormal tingling or burning at the wound. As the virus spreads to the central nervous system (CNS), the disease progresses to fatal encephalitis and encephalomyelitis, leading to the development of the typical clinical signs of furious rabies or paralytic rabies and ultimately death from asphyxia caused by pharyngospasm or respiratory circulatory failure (Mitrabhakdi et al., 2005; Hemachudha et al., 2013; Jackson, 2016).

It has been reported that up to 99% of human rabies cases are caused by a deep bite or scratch from rabid dogs (Fooks et al., 2017). Cases of RABV infection from organ transplantation from infected patients and aerosol transmission are relatively rare (Srinivasan et al., 2005; Maier et al., 2010). Typically, following transmission by a deep bite from an infected animal, RABV replicates in muscle cells and binds to receptors at the neuromuscular junction, initiating early peripheral infection (Lewis et al., 2000). The virus travels through the peripheral nerve bypassing the spinal cord and reaches the CNS by centripetal spread. Once in the brain, RABV replicates on a large scale and spreads centrifugally to the peripheral nervous system, infecting surrounding tissues such as the heart, salivary glands, adrenal glands, gastrointestinal tract and pancreas (Hemachudha et al., 2002; Jogai et al., 2002; Dietzschold et al., 2008; Tobiume et al., 2009). The salivary gland is the primary outlet for the centrifugal spread of RABV and supports viral replication. Previous studies have shown that infected dogs can excrete viral particles in their saliva for up to 14 days before showing obvious clinical signs of rabies (Fekadu and Shaddock, 1984). The salivary gland epithelium contains a high concentration of RABV antigens and can be used as an alternative to brain and cerebrospinal fluid samples for RABV diagnosis (Beauregard and Casey, 1969; Dierks et al., 1969; Charlton et al., 1983).

Saliva is a unique oral bodily fluid. Approximately 90% of saliva is produced by the salivary glands, which mainly include the submandibular gland (SMG), sublingual gland (SLG), and parotid gland (PG) (Zhang et al., 2016a). The secretion of saliva is controlled by the cerebral cortex, and can be influenced by factors such as diet, environment, age, pathogen invasion and salivary gland lesions. Saliva contains various substances, including amylase, lysozyme, peroxidase, mucin, phospholipids, sodium, potassium, calcium, and magnesium, which contribute to its functions of food digestion, antibacterial activity, and protection of the gastric mucosa. Additionally, saliva has the ability to excrete certain pathogens such as RABV (Li et al., 1995) and enteric viruses like murine norovirus (MNV), rotavirus (RV) and astrovirus (AstV) (Kirby et al., 2010; Pisanic et al., 2019; Anfruns-Estrada et al., 2020; Ghosh et al., 2022). Epstein–Barr virus (EBV), herpes simplex virus (HSV), SARS-CoV-2 and human immunodeficiency virus (HIV) can also be excreted or transmitted through the saliva (Corstjens et al., 2000; Lee et al., 2009; Lima et al., 2010; Huang et al., 2021).

In this study, we utilized transcriptomics to characterize the mRNA expression in parotid tissues of mice infected with two RABV strains: the challenge virus standard (CVS)-11 strain and the street rabies virus PB4 strain, and we analyzed the biological functions of the differentially expressed mRNA. The accuracy of the RNA-seq data was confirmed by a quantitative real-time polymerase chain reaction (RT-qPCR). This research represents the first transcriptome analysis of salivary glands in RABV-infected mice and identifies potentially important salivary gland transcripts.

Rabies is a highly fatal zoonotic disease that is primarily transmitted through the bites of infected animals, like dogs in many developing regions of Asia and Africa, and wild animals such as bats and foxes in Western Europe and the Americas (Wilde et al., 2013; Anderson and Shwiff, 2015). Rabies often progresses to a fatal form of encephalomyelitis (Hemachudha et al., 2013). It exhibits neurophilic properties and causes damage to the nervous system (Hemachudha et al., 2013). Salivary excretion of the virus is necessarily influenced by the basic physiology of the salivary gland (Boonsriroj et al., 2016). Transcriptomics has been widely used to screen for potentially functional genes. Previous RABV-related transcriptomics studies have mainly focused on the nervous system, investigating changes in lncRNA, circRNA, and mRNA expression in the mouse brain caused by RABV infection (Zhang et al., 2016b; Sui et al., 2021; Zhao et al., 2021). The spatial and cellular distribution of RABV infection in the mouse brain has also been deeply revealed (Huang and Sabatini, 2020; Zhang et al., 2022). However, studies on the pathogenic mechanism of RABV rarely focus on the salivary glands. To further examine the mechanism of salivary gland function, we infected mice with CVS-11 and PB4 strains and performed RNA sequencing of the PG for the first time. We identified 3,764 DEGs (678 up-regulated and 3,086 down-regulated) in the CVS-11 infected group, and 4,557 DEGs (874 up-regulated and 3,683 down-regulated) in the PB4 infected group.

The salivary glands are mainly composed of SMG, SLG, and PG, along with minor salivary glands (MSG) scattered throughout the mouth. Among these glands, 85–90% of the saliva is produced by the PG and SMG. The PG is located on each side of the face anterior to the ears, and is the main source of stimulated salivary secretion, secreting watery saliva that plays a role in lubricating the oral cavity and maintaining homeostasis. Actions such as biting and chewing are accompanied by contraction of the masseter muscle, which leads to secretion of saliva from the PG into the oral cavity (Brazen and Dyer, 2023). RABV has been found to invade and replicate in the PG of infected hosts (Howard, 1981; Charlton et al., 1984; Silva et al., 2009), and it is possible that a bite from a rabid animal may stimulate the secretory function of the PG. The process of fluid secretion in the salivary glands relies on various ion-transporting proteins expressed in the acinar cells. These proteins are primarily targeted at the apical or basolateral membrane of the acinar cells. In the salivary secretion pathway, several genes that regulate the transport of water and important ion-transporting proteins across membranes were found to be dysregulated in our RABV-infected samples. AQP5, a protein primarily expressed in exocrine glands like salivary and lacrimal glands, plays a crucial role in water transport and is decreased following RABV infection (Agre et al., 1993; Agre, 2004). AQP5 is critical for maintaining the normal physiology of the salivary glands, and its expression in salivary gland tissue could be affected by sialolithiasis and chronic salivary gland inflammation (D'Agostino et al., 2020). Research in AQP5 mutant rats (Murdiastuti et al., 2006), AQP5 knockout mice (Ma et al., 1999), and Sjögren’s syndrome patients (Steinfeld et al., 2001; Ichiyama et al., 2018; Hosoi et al., 2020) has demonstrated that abnormal expression of AQP5 leads to decreased salivary secretion and hypertonic and viscous saliva, which induces thirst or dryness in the oral cavity. Additionally, AQP5 has been found to be associated with viral infections. For instance, PRRSV and adenovirus infection could lead to the downregulation of AQP5, hindering water clearance and edema resolution, and causing abnormal fluid flux during pulmonary inflammation in infected animals (Generous et al., 2014; Zhang et al., 2018). The reduction of AQP5 expression in the infected parotid glands suggests that RABV may inhibit the expression and function of AQP5, thus affecting the intracellular and extracellular fluid balance and the function of water channels. This may be related to the invasion and spread of RABV and the development of the disease. CD38 is a transmembrane glycoprotein that acts as a Ca²⁺ releaser from intracellular Ca²⁺-stores (Lee, 1997; Lee, 2001). Furthermore, the Ca²⁺-activated K⁺ channel of the intermediate single channel conductance encoded by KCNN4 has been identified in exocrine salivary glands (Catalán et al., 2014). In our study, CD38 and KCNN4 were both decreased in mRNA levels, suggesting that RABV may affect the ion transporting system in salivary glands.

There are also dysregulated sequenced genes involved in the salivary secretion signaling pathway. DMBT1, which belongs to the scavenger receptor cysteine-rich (SRCR) superfamily (Generous et al., 2014; Reichhardt et al., 2017) and interacts with various pathogens in saliva (Ligtenberg et al., 2007), was significantly upregulated in both CVS-11 and PB4-infected groups. This may be due to the increased agglutination of virions in the salivary glands. NOS generates nitric oxide (NO) which is thought to contribute to protective and cytotoxic antiviral responses (Wink et al., 1996). Inducible nitric oxide synthase (iNOS) expression and activity have been shown to increase markedly in the brain of rats infected with RABV, indicating its importance in rabies neuropathogenesis (Hooper et al., 1995; Van Dam et al., 1995). Endothelial nitric oxide synthase (eNOS) expression was increased in cerebellar Purkinje cells and brainstem neurons in the brains of rabid cattle. Furthermore, eNOS and RABV were observed to co-localize in Negri bodies, suggesting the involvement of eNOS in the formation of RABV inclusion bodies (Akaike et al., 1995). NOS is widely distributed in various parts of the salivary glands, and is probably activated leading to the generation of cGMP, which opens ion channels to initiate the salivary secretory process (Bi and Reiss, 1995; Lomniczi et al., 1998). In our sequencing results, we observed down-regulation of NOS in the RABV-infected group, indicating that RABV may cause salivary gland damage similar to autoimmune inflammation, resulting in decreased NOS and NO expression levels to facilitate viral infection. Moreover, other signaling pathways are also involved in CVS-11 and PB4 infection, like the PI3K-Akt signaling pathway, the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, and the retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathway. Although it has been reported that RABV could stimulate these immune signaling pathways (Der et al., 1998; Goodbourn et al., 2000; Hornung et al., 2006; Faul et al., 2010), most immunity-related genes in our transcriptome data was restrained, like interferon gamma receptors (Ifngrs), interferon alpha receptors (Ifnars), Janus kinases (Jaks), signal transducers and activators of transcription (Stats), stimulator of interferon response cGAMP interactors (Stings), and TNF receptor associated factor 5 (Traf5). It has been speculated that the host immune system remains in a suppressed state as rabies progresses to late-stage disease, in order to persist the transmission and secretion of virions through saliva. However, the detailed mechanism by which the genes regulating salivary virion production and salivary gland function are dysregulated remains unclear. Further research is needed. Whether these genes could be potential targets for blocking virus transmission through saliva requires further validation. Whether the gene expression map of parotid tissue in the early stage of rabies differs from that in the later stage may merit intensive identification.

In summary, we demonstrated differentially expressed mRNA affected by RABV infection in the parotid tissues by employing the transcriptomic technique, and established GO and KEGG functional enrichment analyses. It is revealed that RABV invasion caused drastic regulation of the saliva secretion process in mice PG along with various host response processes. This study was the first to analyze the gene expression profiles and biological functions of the DEGs derived from RABV-infected mouse salivary tissues, which may have implications for the development of therapeutics, diagnostics and sanitation measures aimed at preventing the spread of rabies through saliva.

RNA-seq was utilized to establish mRNA expression profiles in PG mice infected with CVS-11 and PB4, and the biological functions of DEGs were analyzed. GO and KEGG enrichment analyses showed that most of the dysregulated genes were associated with the salivary secretion pathway, the PI3K-Akt signaling pathway, and the cGMP-PKG signaling pathway. Although some work has been undertaken, the detailed mechanism underlying RABV-induced salivary gland dysfunction remains elusive. These findings will contribute to a deeper understanding of the biology of viral spread.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The animal study was approved by the Institutional Animal Care and Use Committee of Jilin University. The study was conducted in accordance with the local legislation and institutional requirements.

XG: Formal analysis, Investigation, Methodology, Validation, Writing – original draft. MZ: Conceptualization, Funding acquisition, Project administration, Writing – review & editing. YF: Funding acquisition, Investigation, Validation, Writing – review & editing. XL: Formal analysis, Investigation, Writing – review & editing. CW: Formal analysis, Investigation, Writing – review & editing. YZ: Formal analysis, Investigation, Writing – review & editing. ZW: Formal analysis, Investigation, Writing – review & editing. DZ: Investigation, Writing – review & editing. YG: Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing – original draft, Writing – review & editing.