Submandibular Gland Pathogenesis Following SARS-CoV-2 Infection and Implications for Xerostomia.

Although SARS-CoV-2 induces mucin hypersecretion in the respiratory tract, hyposalivation/xerostomia has been reported by COVID-19 patients. We evaluate the submandibular gland (SMGs) pathogenesis in SARS-CoV-2-infected K18-hACE2 mice, focusing on the impact of infection on the mucin production and structural integrity of acini, ductal system, myoepithelial cells (MECs) and telocytes. The spike protein, the nucleocapsid protein, hACE2, actin, EGF, TNF-α and IL-1ß were detected by immunofluorescence, and the Egfr and Muc5b expression was evaluated. In the infected animals, significant acinar hypertrophy was observed in contrast to ductal atrophy. Nucleocapsid proteins and/or viral particles were detected in the SMG cells, mainly in the nuclear membrane-derived vesicles, confirming the nuclear role in the viral formation. The acinar cells showed intense TNF-α and IL-1ß immunoexpression, and the EGF-EGFR signaling increased, together with Muc5b upregulation. This finding explains mucin hypersecretion and acinar hypertrophy, which compress the ducts. Dying MECs and actin reduction were also observed, indicating failure of contraction and acinar support, favoring acinar hypertrophy. Viral assembly was found in the dying telocytes, pointing to these intercommunicating cells as viral transmitters in SMGs. Therefore, EGF-EGFR-induced mucin hypersecretion was triggered by SARS-CoV-2 in acinar cells, likely mediated by cytokines. The damage to telocytes and MECs may have favored the acinar hypertrophy, leading to ductal obstruction, explaining xerostomia in COVID-19 patients. Thus, acinar cells, telocytes and MECs may be viral targets, which favor replication and cell-to-cell viral transmission in the SMG, corroborating the high viral load in saliva of infected individuals.

Murine Cytomegalovirus MCK-2 Facilitates In Vivo Infection Transfer from Dendritic Cells to Salivary Gland Acinar Cells.

The cytomegaloviruses (CMVs) spread systemically via myeloid cells and demonstrate broad tissue tropism. Human CMV (HCMV) UL128 encodes a component of the virion pentameric complex (PC) that is important for entry into epithelial cells and cell-cell spread in vitro. It possesses N-terminal amino acid sequences similar to those of CC chemokines. While the species specificity of HCMV precludes confirmation of UL128 function in vivo, UL128-like counterparts in experimental animals have demonstrated a role in salivary gland infection. How they achieve this has not been defined, although effects on monocyte tropism and immune evasion have been proposed. By tracking infected cells following lung infection, we show that although the UL128-like protein in mouse CMV (MCMV) (designated MCK-2) facilitated entry into lung macrophages, it was dispensable for subsequent viremia mediated by CD11c+ dendritic cells (DCs) and extravasation to the salivary glands. Notably, MCK-2 was important for the transfer of MCMV infection from DCs to salivary gland acinar epithelial cells. Acinar cell infection of MCMVs deleted of MCK-2 was not rescued by T-cell depletion, arguing against an immune evasion mechanism for MCK-2 in the salivary glands. In contrast to lung infection, peritoneal MCMV inoculation yields mixed monocyte/DC viremia. In this setting, MCK-2 again promoted DC-dependent infection of salivary gland acinar cells, but it was not required for monocyte-dependent spread to the lung. Thus, the action of MCK-2 in MCMV spread was specific to DC-acinar cell interactions. IMPORTANCE Cytomegaloviruses (CMVs) establish myeloid cell-associated viremias and persistent shedding from the salivary glands. In vitro studies with human CMV (HCMV) have implicated HCMV UL128 in epithelial tropism, but its role in vivo is unknown. Here, we analyzed how a murine CMV (MCMV) protein with similar physical properties, designated MCK-2, contributes to host colonization. We demonstrate that MCK-2 is dispensable for initial systemic spread from primary infection sites but within the salivary gland facilitates the transfer of infection from dendritic cells (DCs) to epithelial acinar cells. Virus transfer from extravasated monocytes to the lungs did not require MCK-2, indicating a tissue-specific effect. These results provide new information about how persistent viral tropism determinants operate in vivo.

A novel pancreatic cancer model originated from transformation of acinar cells in adult tree shrew, a primate-like animal.

Pancreatic cancer is one of the most lethal common cancers. The cell of origin of pancreatic ductal adenocarcinoma (PDAC) has been controversial, and recent evidence suggested acinar cells as the most probable candidate. However, the genetic alterations driving the transformation of pancreatic acinar cells in fully mature animals remain to be deciphered. In this study, lentivirus was used as a tool to introduce genetic engineering in tree shrew pancreatic acinar cells to explore the driver mutation essential for malignant transformation, establishing a novel tree shrew PDAC model, because we found that lentivirus could selectively infect acinar cells in tree shrew pancreas. Combination of oncogenic KRASG12D expression and inactivation of tumor suppressor genes Tp53, Cdkn2a and Cdkn2b could induce pancreatic cancer with full penetrance. Silencing of Cdkn2b is indispensable for Rb1 phosphorylation and tumor induction. Tree shrew PDAC possesses the main histological and molecular features of human PDAC. The gene expression profile of tree shrew PDAC was more similar to human disease than a mouse model. In conclusion, we established a novel pancreatic cancer model in tree shrew and identified driver mutations indispensable for PDAC induction from acinar cells in mature adults, demonstrating the essential roles of Cdkn2b in the induction of PDAC originating from adult acinar cells. Tree shrew could thus provide a better choice than mouse for a PDAC model derived from acinar cells in fully mature animals.

Psittacid herpesvirus 3 infection in the eclectus parrot (Eclectus roratus) in Australia.

Psittacid herpesvirus 3 (PsHV-3) has recently been implicated as the cause of a severe respiratory disease in Bourke's parrots (Neopsephotus bourkii) in the United States. In this report, the clinical manifestations and gross and microscopic lesions of PsHV-3 infection in 2 eclectus parrots (Eclectus roratus) in Australia are described. The presence of a PsHV-3 infection was confirmed by polymerase chain reaction amplification and sequencing of PsHV-3 DNA using degenerate and PsHV-3 primers. Electron microscopy of infected cells demonstrated the assembly of herpesvirus virions as well as intranuclear tubular structures. The detection of PsHV-3 in Australia in 2 eclectus parrots broadens the list of known affected species and confirms the presence of this virus in Australia.

Gene delivery to pancreatic exocrine cells in vivo and in vitro.

BACKGROUND: Effective gene transfer to the pancreas or to pancreatic cells has remained elusive although it is essential for studies of genetic lineage tracing and modulation of gene expression. Different transduction methods and viral vectors were tested in vitro and in vivo, in rat and mouse pancreas. RESULTS: For in vitro transfection/transduction of rat exocrine cells lipofection reagents, adenoviral vectors, and Mokola- and VSV-G pseudotyped lentiviral vectors were used. For in vivo transduction of mouse and rat pancreas adenoviral vectors and VSV-G lentiviral vectors were injected into the parenchymal tissue. Both lipofection of rat exocrine cell cultures and transduction with Mokola pseudotyped lentiviral vectors were inefficient and resulted in less than 4% EGFP expressing cells. Adenoviral transduction was highly efficient but its usefulness for gene delivery to rat exocrine cells in vitro was hampered by a drastic increase in cell death. In vitro transduction of rat exocrine cells was most optimal with VSV-G pseudotyped lentiviral vectors, with stable transgene expression, no significant effect on cell survival and about 40% transduced cells. In vivo, pancreatic cells could not be transduced by intra-parenchymal administration of lentiviral vectors in mouse and rat pancreas. However, a high efficiency could be obtained by adenoviral vectors, resulting in transient transduction of mainly exocrine acinar cells. Injection in immune-deficient animals diminished leukocyte infiltration and prolonged transgene expression. CONCLUSIONS: In summary, our study remarkably demonstrates that transduction of pancreatic exocrine cells requires lentiviral vectors in vitro but adenoviral vectors in vivo.

Pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo.

Autophagy protects against many infections by inducing the lysosomal-mediated degradation of invading pathogens. However, previous in vitro studies suggest that some enteroviruses not only evade these protective effects but also exploit autophagy to facilitate their replication. We generated Atg5(f/f)/Cre(+) mice, in which the essential autophagy gene Atg5 is specifically deleted in pancreatic acinar cells, and show that coxsackievirus B3 (CVB3) requires autophagy for optimal infection and pathogenesis. Compared to Cre(-) littermates, Atg5(f/f)/Cre(+) mice had an ∼2,000-fold lower CVB3 titer in the pancreas, and pancreatic pathology was greatly diminished. Both in vivo and in vitro, Atg5(f/f)/Cre(+) acinar cells had reduced intracellular viral RNA and proteins. Furthermore, intracellular structural elements induced upon CVB3 infection, such as compound membrane vesicles and highly geometric paracrystalline arrays, which may represent viral replication platforms, were infrequently observed in infected Atg5(f/f)/Cre(+) cells. Thus, CVB3-induced subversion of autophagy not only benefits the virus but also exacerbates pancreatic pathology.

Immunohistochemical and histopathological findings of ovine pulmonary adenocarcinoma (Jaagsiekte) in Egyptian sheep.

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring retrovirus-induced transmissible lung cancer in sheep. Lungs and associated (bronchial and mediastinal) lymph nodes of seven sheep with OPA were examined. Lungs had few multifocal consolidated slightly elevated gray to white masses ranging from 0.5 to 3 cm in diameter. Histopathologically, these masses appeared as well-differentiated acinar adenocarcinoma with little evidence of anaplasia. The acini composed of well-differentiated cuboidal to low columnar epithelium with clear or vacuolated cytoplasm and low mitotic index. No metastases were observed in the bronchial and mediastinal lymph nodes of any animal. The presence of Jaagsiekte sheep retrovirus (JSRV) was demonstrated in the lungs by immunohistochemistry. JSRV protein was detected in all tumor epithelial cells, histologically normal alveolar type II cells, and few bronchiolar epithelial cells, alveolar macrophages, lymphocytes, and plasma cells. This study is the first to confirm the presence of natural OPA in Egypt.