ika virus (ZIKV) is a neurotropic flavivirus spread to humans by Aedes genus mosquitos. Zika virus was first isolated in the Zika Forest in Uganda in a nonhuman primate in 1947. 1,2 Five years later, Nigeria reported the first cases of ZIKV transmission to humans, and subsequent ZIKV outbreaks have been reported outside Africa. [1][2][3] The first autochthonous case was confirmed in 2015 in Brazil, with a subsequent increase in the number of cases. This event coincided with reports of newborns with microcephaly, congenital malformations, and neurologic syndromes. 1,2 The spread of ZIKV to other parts of Latin America, the Caribbean, and the United States brought epidemiologic concern because of the clinical manifestations, including those associated with congenital Zika syndrome (CZS). [4][5][6] Although microcephaly is considered to be the characteristic feature of CZS, other central nervous system abnormalities and ocular findings have been described as part of this entity. [7][8][9][10] Despite having clinical evidence of the association between CZS and ocular findings, [11][12][13][14][15][16] only a few studies 17,18 have identified the presence of ZIKV in mouse ocular tissues and cultured human cells. Ritter et al 19 described the presence of ZIKV antigen within the neural retina in 1 case of CZS by immunohistochemical analysis, but the histopathologic findings were not described. The only study to date that has provided valuable clinical information regarding the retinal layers affected in CZS in human eyes was performed using optical IMPORTANCE Congenital Zika syndrome (CZS) is known to be associated with severe malformations in newborns. Although microcephaly is the hallmark of this disease, the ocular findings are important given the severe visual impairment that has been observed in these patients. Regardless of the increased number of CZS cases reported, to date, no studies have described the ocular histopathologic findings of this entity.OBJECTIVES To evaluate the presence of Zika virus (ZIKV) antigens and describe the associated ocular histopathologic features of 4 cases of CZS.

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.