Development of a diagnostic kit for Zika virus detection

Abstract

ZIKA virus (ZIKV) is an arbovirus member of the Flaviviridae family and Flavivirus genus, along with Dengue Virus (DENV), Yellow Fever Virus (YFV), Saint Louis Encephalitis Virus (SLEV), West Nile Virus (WNV) and Japanese Encephalitis Virus (JEV). In humans, it was first isolated in 1952 in Uganda and Tanzania. In 2007, it caused an outbreak in Micronesia, South Pacific, in Gabon, Central Africa and in 2013 in French Polynesia. In Brazil, ZIKA infection was primarily reported in 2015 in the Northeast of the country. Recently, an unexpected increase in the diagnosis of fetal and pediatric microcephaly was reported by the Brazilian press. So far, cases were diagnosed in nine Brazilian states, in the same region where the virus was first identified in Brazil. Until November of 2015, 646 cases had been reported only in the state of Pernambuco and despite a direct association has not yet been found, Brazilian Health Ministry confirmed a relation between the infection by ZIKA fever and microcephaly cases. At the present, the confirmation of infection by ZIKA virus is based mainly on PCR (RT-PCR). However, PCR has disadvantages like high cost and high contamination risk, especially when many samples are evaluated at the same time, which is the case in outbreaks. Despite the IgM antibodies against ZIKV can be detected by Elisa, few laboratories are successful using this method. Diagnosis is a challenge due to its low viremia and cross-reactivity of ZIKV antibodies with other Flavivirus (including DENV). The confirmation by neutralization assays is necessary making a rapid diagnosis by serological tests difficult. Considering what has been exposed it justifies the urgency for the development of a simple, rapid and highly specific diagnosis kit based in biotechnology, reducing the possibility of cross diagnosis with other virus or sample contamination, which are currently the major problems of the detection techniques available in the market. The aim of this proposal is the development of a simple and rapid diagnosis system for ZIKA virus, enabling fast and reliable results. A specific viral genomic region will be selected as a target for the diagnosis, taking into account the specificity for ZIKV. Molecular beacon probes will be designed with nucleotide sequences complementary to the target region. These beacons will be carrying a fluorophore that produces an easily detectable signal when it hybridizes. We will also develop magnetic bead probes that can detect the molecular beacons/target RNA hybrids, which will enhance the system specificity and the purity of the reaction. As a result, we expect to obtain a kit for molecular diagnosis of ZIKV, without amplification of the viral genetic material, specific, safe, rapid and with low cost, capable of identifying the infection by ZIKV in large scale and differentiate it from infections caused by other arboviruses. These will allow, in the future, contact with other laboratories in order to stablish a possible negotiation and commercialization of this new and promising tool for viral detection. (AU)