Probes for INS

The recent coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2. COVID-19 was first reported in China (December 2019) and is now prevalent across the globe. Entry of severe acute respiratory syndrome coronavirus 2 into mammalian cells requires the binding of viral Spike (S) proteins to the angiotensin-converting enzyme 2 receptor. Once entered, the S protein is primed by a specialized serine protease, transmembrane serine protease 2 in the host cell. Importantly, besides the respiratory symptoms that are consistent with other common respiratory virus infections when patients become viremic, a significant number of COVID-19 patients also develop liver comorbidities. We explored whether a specific target cell-type in the mammalian liver could be implicated in disease pathophysiology other than the general deleterious response to cytokine storms. Here, we used single-cell RNA-seq to survey the human liver and identified potentially implicated liver cell-type for viral ingress. We analyzed ~300,000 single cells across five different (i.e., human fetal, healthy, cirrhotic, tumor, and adjacent normal) liver tissue types. This study reports on the co-expression of angiotensin-converting enzyme 2 and transmembrane serine protease 2 in a TROP2+ liver progenitor population. Importantly, we detected enrichment of this cell population in the cirrhotic liver when compared with tumor tissue. These results indicated that in COVID-19-associated liver dysfunction and cell death, a viral infection of TROP2+ progenitors in the liver might significantly impair liver regeneration in patients with liver cirrhosis.

Prenatal exposure to Zika virus (ZIKV) can result in microencephaly and congenital Zika syndrome, though some brain cells and structures are spared by the virus for unknown reasons. Here, a novel murine model of fetal ZIKV infection incorporating intraventricular infection and cell type specific in utero electroporation was used to identify the time course of ZIKV infection and to determine the identity of cells that are initially infected or spared during neocortical neurogenesis. In vivo time course studies revealed the presence of ZIKV in apical radial glial cells (aRGCs) at early time points following virus exposure, while basal intermediate progenitor cells (bIPCs) became maximally (ZIKV+) after 3 days of virus exposure. ZIKV-infected fetal brains exhibited microencephaly as early as one day following infection, regardless of developmental age. This change in brain size was caused in part by apoptosis and reduced proliferation that persisted until birth. While 60% of aRGC basal fibers were perturbed during infection, 40% retained normal morphology, indicating that aRGCs are not uniformly vulnerable to ZIKV infection. To investigate this heterogeneous vulnerability, we performed genetic fate mapping using cell type-specific probes derived from a mouse E15.5 neocortical wall single cell RNA-Seq dataset. The results indicate that one class of aRGCs preferentially express the putative ZIKV entry receptor AXL, and that these cells are more vulnerable to ZIKV infection than other aRGC subtypes with low AXL expression. Together, these data uncover crucial temporal and cellular details of ZIKV fetal brain infection for prevention strategies and for management of congenital Zika syndrome.Significance StatementThe transcriptional signatures of neural precursor cells were utilized for the first time to test Zika virus susceptibility in a direct fetal brain infection model. This novel methodology allowed for elucidation of time point specific differences in neural precursor cell susceptibility that have been debated in the field. Additionally, elucidation of cell morphological features using in utero electroporation revealed substantial but incomplete interruption of basal fibers, a finding that implies interference with neuronal migration. The model presented here, allows for assessment of pre-natal development after exposure to a variety of viruses. The improved specificity of apical radial glial cell labeling afforded by the cell-specific labeling tools uncover functional differences between apical radial glial cell types that will have important implications for children exposed to ZIKV as well as for understanding corticogenesis.

Zika virus is an arthropod-borne virus that has recently emerged as a significant public health emergency due to its association with congenital malformations. Serological and molecular tests are typically used to confirm Zika virus infection. These methods, however, have limitations when the interest is in localizing the virus within the tissue and identifying the specific cell types involved in viral dissemination. Chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) are common histological techniques used for intracellular localization of RNA and protein expression, respectively. The combined use of CISH and IHC is important to obtain information about RNA replication and the location of infected target cells involved in Zika virus neuropathogenesis. There are no reports, however, of detailed procedures for the simultaneous detection of Zika virus RNA and proteins in formalin-fixed paraffin-embedded (FFPE) samples. Furthermore, the chromogenic detection methods for Zika virus RNA published thus far use expensive commercial kits, limiting their widespread use. As an alternative, we describe here a detailed and cost-effective step-by-step procedure for the simultaneous detection of Zika virus RNA and proteins in FFPE samples. First, we describe how to synthesize and purify homemade RNA probes conjugated with digoxygenin. Then, we outline the steps to perform the chromogenic detection of Zika virus RNA using these probes, and how to combine this technique with the immunodetection of viral antigens. To illustrate the entire workflow, we use FFPE samples derived from infected Vero cells as well as from human and mouse brain tissues. These methods are highly adaptable and can be used to study Zika virus or even other viruses of public health relevance, providing an optimal and economical alternative for laboratories with limited resources.

Zika virus (ZIKV), a mosquito-borne flavivirus, can cause severe eye disease and even blindness in newborns. However, ZIKV-induced retinal lesions have not been studied in a comprehensive way, mechanisms of ZIKV-induced retinal abnormalities are unknown, and no therapeutic intervention is available to treat or minimize the degree of vision loss in patients. Here, we developed a novel mouse model of ZIKV infection to evaluate its impact on retinal structure. ZIKV (20 plaque-forming units) was inoculated into neonatal wild type C57BL/6J mice at postnatal day (P) 0 subcutaneously. Retinas of infected mice and age-matched controls were collected at various ages, and retinal structural alterations were analyzed. We found that ZIKV induced progressive neuronal and vascular damage and retinal inflammation starting from P8. ZIKV-infected retina exhibited dramatically decreased thickness with loss of neurons, initial neovascular tufts followed by vessel dilation and degeneration, increased microglia and leukocyte recruitment and activation, degeneration of astrocyte network and gliosis. The above changes may involve inflammation and endoplasmic reticulum stress-mediated cell apoptosis and necroptosis. Moreover, we evaluated the efficacy of preclinical drugs and the safety of ZIKV vaccine candidate in this mouse model. We found that ZIKV-induced retinal abnormalities could be blocked by a selective flavivirus inhibitor NITD008 and a live-attenuated ZIKV vaccine candidate could potentially induce retinal abnormalities. Overall, we established a novel mouse model and provide a direct causative link between ZIKV and retinal lesion in vivo, which warrants further investigation of the underlying mechanisms of ZIKV-induced retinopathy and the development of effective therapeutics.