Probes for INS

Abstract

BACKGROUND:

Chromogenic Epstein-Barr virus-encoded RNA (EBER) in situ hybridization (EBER-ISH) is the gold standard to detect Epstein-Barr virus (EBV) but it is difficult to use in conjunction with immunohistochemistry (IHC). In this study, our purpose was to validate the sensitivity and specificity of RNAscope in detection of EBV infection in nasal epithelium and its stroma.

METHODS:

Fluorescence-based RNAscope EBER-ISH, BRLF1-ISH, and lineage marker-IHC were performed on archived formalin-fixed paraffin-embedded tissues from normal nasal cavity (n = 5), nasopharynx (n = 8), and nasopharyngeal carcinoma (NPC) specimens (n = 10).

RESULTS:

The EBERs were detected in 10 of 10 NPC samples but was absent in all normal tissues from the nasal cavity and nasopharynx. The EBERs were exclusively located in pan-cytokeratin (pan-CK)-positive tumor epithelial cells but not in CD45-positive leukocytes and vimentin-positive stromal fibroblasts. The level of EBER expression varied in tumor cells within patient and between patients as well. Additionally, 5 of 10 patients had positive BRLF-ISH.

CONCLUSION:

We developed a simple and reproducible method to simultaneously detect mRNA and protein in formalin-fixed paraffin-embedded tissues of NPC. As a single staining, traditional EBER continues to be useful; however, for interpretation of the phenotype of EBV-infected cells, RNAscope is superior. Significantly, we showed that lytic EBV infection took place in NPC tumors.

Epstein-Barr virus (EBV) is a herpesvirus associated with approximately 1% of tumors worldwide. Although EBV is consistently detected in nasopharyngeal carcinoma(NPC) biopsy, it is hardly detected in normal nasopharyngeal epithelium. The mechanism how virus establishes latent infection in tumor epithelial cells, including the source of virus and the route of entry, has not been fully elucidated largely due to the lack of appropriate in vivo models. We herein aim to investigate the potential route that epithelial cells are infected with EBV. To this end, we established in vivo model system by injection of cell-free EBV or EBV producer line Akata cells together with EBV negative NPC line HONE-1 cells. Akin to in vitro infections, we presented the first in vivo evidence that cell-mediated transfer infection via Akata cells was much more efficient than cell-free virus. These cells then expressed the EBV latency-associated small RNA EBERs, but not lytic antigens, such as BZLF1. However, when cells were inoculated at separate sites, EBV producer line Akata cell failed to demonstrate the ability of migrating from distant location to interact with HONE-1 cell to establish latent infection. In conclusion, cell-cell contact is critical for in vivo EBV infection of nasopharyngeal epithelial cells.

Previously, several studies have been performed to delineate the development and progression of Marburg virus infection in nonhuman primates (NHPs), primarily to clarify the mechanisms of severe (fatal) disease. After the 2013–2016 Ebola virus disease (EVD) epidemic in Western Africa, there has been a reassessment of the available filovirus animal models and the utility of these to faithfully recapitulate human disease. The high lethality of the NHP models has raised doubts as to their ability to provide meaningful data for the full spectrum of disease observed in humans. Of particular interest are the etiologic and pathophysiologic mechanisms underlying postconvalescent sequelae observed in human survivors of EVD and Marburg virus disease (MVD). In the current study, we evaluated the lesions of MVD in NHPs; however, in contrast to previous studies, we focused on the potential for development of sequelae similar to those reported in human survivors of MVD and EVD. We found that during acute MVD in the macaque model, there is frequent inflammation of peripheral nerves, autonomic ganglia, and the iris of the eye. Furthermore, we demonstrate viral infection of the ocular ciliary body and retina, testis, epididymis, ovary, oviduct, uterine endometrium, prostate, and mammary gland. These findings are relevant for both development of postconvalescent sequelae and the natural transmission of virus.

Filoviruses are among the most pathogenic infectious agents known to human, with high destructive potential, as evidenced by the recent Ebola virus epidemic in West Africa. As members of the filovirus family, marburgviruses have caused similar devastating outbreaks, albeit with lower case numbers. In this study we compare the pathogenesis of Ravn virus (RAVV) and Marburg virus (MARV) strains Angola, Musoke, and Ozolin in rhesus and cynomolgus macaques, the 2 nonhuman primate species most commonly used in filovirus research. Our results reveal the most pathogenic MARV strain to be Angola, followed by Musoke, whereas Ozolin is the least pathogenic. We also demonstrate that RAVV is highly pathogenic in cynomolgus macaques but less pathogenic in rhesus macaques. Our results demonstrate a preferential infection of endothelial cells by MARVs; in addition, analysis of tissue samples suggests that lymphocyte and hepatocyte apoptosis might play a role in MARV pathogenicity. This information expands our knowledge about pathogenicity and virulence of marburgviruses.

Epstein-Barr virus (EBV), a common pathogen in humans, is suspected as the cause of multiple pregnancy-related pathologies including depression, preeclampsia, and stillbirth. Moreover, transmission of EBV through the placenta has been reported. However, the focus of EBV infection within the placenta has remained unknown to date. In this study, we proved the expression of latent EBV genes in the endometrial glandular epithelial cells of the placenta and investigated the cytological characteristics of these cells. Sixty-eight placentas were obtained from pregnant women. Tissue microarray was constructed. EBV latent genes including EBV-encoding RNA-1 (EBER1), Epstein-Barr virusnuclear antigen 1 (EBNA1), late membrane antigen (LMP1), and RPMS1 were detected with silver in situ hybridization and/or mRNA in situ hybridization. Nuclear features of EBV-positive cells in EBV-infected placenta were compared with those of EBV-negative cells via image analysis. Sixteen placentas (23.5%) showed positive expression of all 4 EBV latent genes; only the glandular epithelial cells of the decidua showed EBV gene expression. EBV infection status was not significantly correlated with maternal, fetal, or placental factors. The nuclei of EBV-positive cells were significantly larger, longer, and round-shaped than those of EBV-negative cells regardless of EBV-infection status of the placenta. For the first time, evidence of EBV gene expression has been shown in placental tissues. Furthermore, we have characterized its cytological features, allowing screening of EBV infection through microscopic examination.

Sexual transmission of filoviruses was first reported in 1968 after an outbreak of Marburg virus (MARV) disease and recently caused flare-ups of Ebola virus disease in the 2013–2016 outbreak. How filoviruses establish testicular persistence and are shed in semen remain unknown. We discovered that persistent MARV infection of seminiferous tubules, an immune-privileged site that harbors sperm production, is a relatively common event in crab-eating macaques that survived infection after antiviral treatment. Persistence triggers severe testicular damage, including spermatogenic cell depletion and inflammatory cell invasion. MARV mainly persists in Sertoli cells, leading to breakdown of the blood-testis barrier formed by inter-Sertoli cell tight junctions. This disruption is accompanied by local infiltration of immunosuppressive CD4+Foxp3+ regulatory T cells. Our study elucidates cellular events associated with testicular persistence that may promote sexual transmission of filoviruses and suggests that targeting immunosuppression may be warranted to clear filovirus persistence in damaged immune-privileged sites.