Probes for INS

Zika virus (ZIKV) infection can cause fetal developmental abnormalities and Guillain-Barré syndrome in adults. Although progress has been made in understanding the link between ZIKV infection and microcephaly, the pathology of ZIKV, particularly the viral reservoirs in human, remains poorly understood. Several studies have shown that compared to serum samples, patients' urine samples often have a longer duration of ZIKV persistency and higher viral load. This finding suggests that an independent viral reservoir may exist in the human urinary system. Despite the clinical observations, the host cells of ZIKV in the human urinary system are poorly characterized. In this study, we demonstrate that ZIKV can infect renal proximal tubular epithelial cells (RPTEpiCs) in immunodeficient mice in vivo and in both immortalized and primary human renal proximal tubular epithelial cells (hRPTEpiCs) in vitro. Importantly, ZIKV infection in mouse kidneys caused caspase-3-mediated apoptosis of renal cells. Similarly, in vitro infection of immortalized and primary hRPTEpiCs resulted in notable cytopathic effects. Consistent with the clinical observations, we found that ZIKV infection can persist with prolonged duration in hRPTEpiCs. RNA-Seq analyses of infected hRPTEpiCs revealed a large number of transcriptional changes in response to ZIKV infection, including type I interferon signaling genes and anti-viral response genes. Our results suggest that hRPTEpiCs are a potential reservoir of ZIKV in the human urinary system, providing a possible explanation for the prolonged persistency of ZIKV in patients' urine.

Zika virus (ZIKV) has become a global concern because infection of pregnant mothers was linked to congenital birth defects. ZIKV is unique from other flaviviruses, as it is transmitted vertically and sexually in addition to by mosquito vectors. Prior studies in mice, non-human primates, and humans have shown that ZIKV targets the testis in males, resulting in persistent infection and oligospermia. However, its effects on the corresponding female gonads have not been evaluated. Here, we assessed the effects of ZIKV on the ovary in non-pregnant mice. During the acute phase, ZIKV productively infected the ovary causing accumulation of CD4+ and virus-specific CD8+ T cells. T cells protected against ZIKV infection in the ovary, as higher viral burden was measured in CD8-/- and TCRβδ-/- mice. Increased cell death and tissue inflammation in the ovary was observed during the acute phase of infection, but this normalized over time. In contrast to that observed with males, minimal persistence and no long-term consequences of ZIKV infection on ovarian follicular reserve or fertility were demonstrated in this model. Thus, although ZIKV replicates in cells of the ovary and causes acute oophoritis, there is rapid resolution and no long-term effects on fertility, at least in mice.

Zika virus (ZIKV) infection of pregnant women can cause congenital malformations including microcephaly, which has focused global attention on this emerging pathogen1. In addition to transmission by mosquitoes, ZIKV can be detected in the seminal fluid of affected males for extended periods of time and transmitted sexually2. Here, using a mouse-adapted African ZIKV strain (Dakar 41519), we evaluated the consequences of infection in the male reproductive tract of mice. We observed persistence of ZIKV, but not the closely related Dengue virus (DENV), in the testis and epididymis of male mice, and this was associated with tissue injury that caused diminished testosterone and inhibin B levels, and oligospermia. ZIKV preferentially infected spermatogonia, primary spermatocytes, and Sertoli cells in the testis, resulting in cell death and destruction of the seminiferous tubules. Less damage was observed with a contemporary Asian ZIKV strain (H/PF/2013), in part because this virus replicates less efficiently in mice. The extent to which these observations in mice translate to humans remains unclear, but longitudinal studies of sperm function and viability in ZIKV-infected humans seem warranted.

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition, and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high grade prostatic intraepithelial neoplasia or PIN), and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in 8 cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma, and its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target.

Animal models of Zika virus (ZIKV) infection have recently been established in mice, guinea pigs, and nonhuman primates. Tree shrews (Tupaia belangeri) are an emerging experimental animal in biomedical applications, but their susceptibility to ZIKV infection has not been explored. In the present study, we showed that subcutaneous inoculation of ZIKV led to rapid viremia and viral secretion in saliva, as well as to typical dermatological manifestations characterized by massive diffuse skin rash on the trunk. Global transcriptomic sequencing of peripheral blood mononuclear cells isolated from ZIKV-infected animals revealed systematic gene expression changes related to the inflammatory response and dermatological manifestations. Importantly, ZIKV infection readily triggered the production of high-titer neutralizing antibodies, thus preventing secondary homologous infection, in tree shrews. However, neonatal tree shrews succumbed to ZIKV challenge upon intracerebral infection. The tree shrew model described here recapitulates the most common dermatological manifestations observed in ZIKV-infected patients and may greatly facilitate the elucidation of ZIKV pathogenesis and the development of novel vaccines and therapeutics.IMPORTANCEThe reemergence of Zika virus (ZIKV) has caused a global public health crisis since 2016, and there are currently no vaccines or antiviral drugs to prevent or treat ZIKV infection. However, considerable advances have been made in understanding the biology and pathogenesis of ZIKV infection. In particular, various animal models have been successfully established to mimic ZIKV infection and its associated neurological diseases and to evaluate potential countermeasures. However, the clinical symptoms in these mouse and nonhuman primate models are different from the common clinical manifestations seen in human ZIKV patients; in particular, dermatological manifestations are rarely recapitulated in these animal models. Here, we developed a new animal model of ZIKV infection in tree shrews, a rat-sized, primate-related mammal. In vitro and in vivo characterization of ZIKV infection in tree shrews established a direct link between ZIKV infection and the immune responses and dermatological manifestations. The tree shrew model described here, as well as other available animal models, provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccines and therapeutics.

The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses.

Zika virus (ZIKV) has recently been associated with birth defects and pregnancy loss after maternal infection. Because dengue virus (DENV) and ZIKV co-circulate, understanding the role of antibody-dependent enhancement in the context of pregnancy is critical. Here, we showed that the presence of DENV-specific antibodies in ZIKV-infected pregnant mice significantly increased placental damage, fetal growth restriction, and fetal resorption. This was associated with enhanced viral replication in the placenta that coincided with an increased frequency of infected trophoblasts. ZIKV-infected human placental tissues also showed increased replication in the presence of DENV antibodies, which was reversed by FcγR blocking antibodies. Furthermore, ZIKV-mediated fetal pathogenesis was enhanced in mice in the presence of a DENV-reactive monoclonal antibody, but not in the presence of the LALA variant, indicating a dependence on FcγR engagement. Our data suggest a possible mechanism for the recent increase in severe pregnancy outcomes after ZIKV infection in DENV-endemic areas.

Kinase activation by chromosomal translocations is a common mechanism that drives tumorigenesis in spitzoid neoplasms. To explore the landscape of fusion transcripts in these tumors, we performed whole-transcriptome sequencing using formalin-fixed, paraffin-embedded (FFPE) tissues in malignant or biologically indeterminate spitzoid tumors from 7 patients (age 2-14 years). RNA sequence libraries enriched for coding regions were prepared and the sequencing was analyzed by a novel assembly-based algorithm designed for detecting complex fusions. In addition, tumor samples were screened for hotspot TERT promoter mutations, and telomerase expression was assessed by TERT mRNA in situ hybridization (ISH). Two patients had widespread metastasis and subsequently died of disease, and 5 patients had a benign clinical course on limited follow-up (mean: 30 months). RNA sequencing and TERT mRNA ISH were successful in six tumors and unsuccessful in one disseminating tumor because of low RNA quality. RNA sequencing identified a kinase fusion in five of the six sequenced tumors: TPM3-NTRK1 (2 tumors), complex rearrangements involving TPM3, ALK, and IL6R (1 tumor), BAIAP2L1-BRAF (1 tumor), and EML4-BRAF (1 disseminating tumor). All predicted chimeric transcripts were expressed at high levels and contained the intact kinase domain. In addition, two tumors each contained a second fusion gene, ARID1B-SNX9 or PTPRZ1-NFAM1. The detected chimeric genes were validated by home-brew break-apart or fusion fluorescence in situ hybridization (FISH). The two disseminating tumors each harbored the TERT promoter -124C>T (Chr 5:1,295,228 hg19 coordinate) mutation, whereas the remaining five tumors retained the wild-type gene. The presence of the -124C>T mutation correlated with telomerase expression by TERT mRNA ISH. In summary, we demonstrated complex fusion transcripts and novel partner genes for BRAF by RNA sequencing of FFPE samples. The diversity of gene fusions demonstrated by RNA sequencing defines the molecular heterogeneity of spitzoid neoplasms.

Abstract

PURPOSE:

The association of Zika virus (ZIKV) infection and development of neurological sequelae require a better understanding of the pathogenic mechanisms causing severe disease. The purpose of this study was to evaluate the ability and sensitivity of positron emission tomography (PET) imaging using [18F]DPA-714, a translocator protein (TSPO) 18 kDa radioligand, to detect and quantify neuroinflammation in ZIKV-infected mice.

PROCEDURES:

We assessed ZIKV-induced pathogenesis in wild-type C57BL/6 mice administered an antibody to inhibit type I interferon (IFN) signaling. [18F]DPA-714 PET imaging was performed on days 3, 6, and 10 post-infection (PI), and tissues were subsequently processed for histological evaluation, quantification of microgliosis, and detection of viral RNA by in situ hybridization (ISH).

RESULTS:

In susceptible ZIKV-infected mice, viral titers in the brain increased from days 3 to 10 PI. Over this span, these mice showed a two- to sixfold increase in global brain neuroinflammation using [18F]DPA-714 PET imaging despite limited, regional detection of viral RNA. No measurable increase in ionized calcium binding adaptor molecule 1 (Iba-1) expression was noted at day 3 PI; however, there was a modest increase at day 6 PI and an approximately significant fourfold increase in Iba-1 expression at day 10 PI in the susceptible ZIKV-infected group relative to controls.

CONCLUSIONS:

The results of the current study demonstrate that global neuroinflammation plays a significant role in the progression of ZIKV infection and that [18F]DPA-714 PET imaging is a sensitive tool relative to histology for the detection of neuroinflammation. [18F]DPA-714 PET imaging may be useful in dynamically characterizing the pathology associated with neurotropic viruses and the evaluation of therapeutics being developed for treatment of infectious diseases.

Telomerase is reactivated in most cancers and is possibly an early driver event in melanoma. Our aim was to test a novel in situ hybridization technique, RNAscope, for the detection of human telomerase reverse transcriptase (hTERT) mRNA in archival formalin-fixed, paraffin-embedded (FFPE) tissue and to compare the mRNA expression of melanomas and benign naevi. Furthermore, we wanted to see if hTERT mRNA could be a diagnostic or prognostic marker of melanoma. In situ hybridization for the detection of hTERT mRNA was performed on FFPE tissue of 17 melanomas and 13 benign naevi. We found a significant difference in the expression of hTERT mRNA between melanomas and benign naevi (P<0.001) and the expression of hTERT mRNA correlated with Breslow thickness (ρ=0.56, P=0.0205) and the Ki67 proliferation index (ρ=0.72, P=0.001). This study showed that RNAscope was a reliable in situ hybridization method for the detection of hTERT mRNA in FFPE tissue of melanomas and benign naevi. hTERT mRNA was more abundantly expressed in melanomas compared with benign naevi, but cannot be used solely as a diagnostic marker due to an overlap in expression. The hTERT mRNA expression in melanomas correlated with the prognostic markers Breslow thickness and the Ki67 index indicating a prognostic potential of hTERT mRNA.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited.