Isolation of infective Zika virus from urine and saliva of patients in Brazil

Abstract

BACKGROUNDZika virus (ZIKV) is an emergent threat provoking a worldwide explosive outbreak. Since January 2015, 41 countries reported autochthonous cases. In Brazil, an increase in Guillain-Barre syndrome and microcephaly cases was linked to ZIKV infections. A recent report describing low experimental transmission efficiency of its main putative vector, Ae. aegypti, in conjunction with apparent sexual transmission notifications prompted the investigation of other potential sources of viral dissemination. Urine and saliva have been previously established as useful tools in ZIKV diagnosis. However, no evidence regarding the infectivity of ZIKV particles present in saliva and urine has been obtained yet.\n\nMETHODOLOGY/PRINCIPAL FINDINGSNine urine and five saliva samples from nine patients from Rio de Janeiro presenting rash and other typical Zika acute phase symptoms were inoculated in Vero cell culture and submitted to specific ZIKV RNA detection and quantification through, respectively, NAT-Zika, RT-PCR and RT-qPCR. Two ZIKV isolates were achieved, one from urine and one from saliva specimens. ZIKV nucleic acid was identified by all methods in four patients. Whenever both urine and saliva samples were available from the same patient, urine viral loads were higher, corroborating the general sense that it is a better source for ZIKV molecular diagnostic. In spite of this, from the two isolated strains, each from one patient, only one derived from urine, suggesting that other factors, like the acidic nature of this fluid, might interfere with virion infectivity. The complete genome of both ZIKV isolates was obtained. Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak.\n\nCONCLUSIONS/SIGNIFICANCEThe detection of infectious ZIKV particles in urine and saliva of patients during the acute phase may represent a critical factor in the spread of virus. The epidemiological relevance of this finding, regarding the contribution of alternative non vectorial ZIKV transmission routes, needs further investigation.\n\nAUTHOR SUMMARYThe American continent has recently been the scene of a devastating epidemic of Zika virus and its severe manifestations, such as microcephaly in newborns and Guillain-Barre Syndrome. Zika virus, first detected in 1947 in Africa, only from 2007 started provoking outbreaks. Zika, dengue and chikungunya viruses are primarily transmitted by Aedes mosquitoes. Dengue is endemic in Brazil for almost 30 years, and the country is largely infested by its main vector, Aedes aegypti. Chikungunya virus entered the country in late 2014 and Zika presence was confirmed eight months later. Nevertheless, Zika notifications multiplied and spread across the country with unprecedented speed, raising the possibility of other transmission routes. This hypothesis was strengthened by some recent reports of Zika sexual transmission in Ae. aegypti-free areas and by the description of a low transmission efficiency to Zika virus in local Ae. aegypti. We found Zika active particles in both urine and saliva of acute phase patients, and a finding that was promptly announced by Fiocruz via Press Conference on February 5, 2016. In this work, we bring up the potential alternative person-to-person infection routes beyond the vectorial transmission, that might have epidemiological relevance.