Isolation of Infective Zika Virus from urine and saliva of patients in Brazil

Submitted by Sandra Infurna ([email protected]) on 2017-04-07T13:08:57Z No. of bitstreams: 1 mirna2_bonaldo_etal_IOC_2016.pdf: 2722947 bytes, checksum: 03f3dfbc2216beffa94b5996752a540d (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-04-07T13:26:32Z (GMT) No. of bitstreams: 1 mirna2_bonaldo_etal_IOC_2016.pdf: 2722947 bytes, checksum: 03f3dfbc2216beffa94b5996752a540d (MD5)Made available in DSpace on 2017-04-07T13:26:32Z (GMT). No. of bitstreams: 1 mirna2_bonaldo_etal_IOC_2016.pdf: 2722947 bytes, checksum: 03f3dfbc2216beffa94b5996752a540d (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Doenças Febris Agudas.Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Mosquitos Transmissores de Hematozoários. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Mosquitos Transmissores de Hematozoários. Rio de Janeiro, RJ, Brasil.Laboratório Nacional de Computação Científica, Petrópolis, RJ, Brasil.Laboratório Nacional de Computação Científica, Petrópolis, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Mosquitos Transmissores de Hematozoários. Rio de Janeiro, RJ, Brasil.Laboratório Nacional de Computação Científica, Petrópolis, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Doenças Febris Agudas.Rio de Janeiro, RJ, Brasil.Zika 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-Barré 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. Here, we described the presence and isolation of infectious ZIKV particles from saliva and urine of acute phase patients in the Rio de Janeiro state, Brazil

ZIKV RNA detection and quantitation.

<p>ZIKV RNA detection and quantitation.</p

Isolation of Zika virus in Vero cell from the saliva of patient 6.

<p>Phase contrast optical microscopy of culture flasks containing (A) Mock-infected Vero cells and (B) saliva-infected Vero cells presenting a clear visible cytopathic effect. Viral plaque detection in saliva (C) and urine (D). The white arrow shows the unique viral plaque detected in the urine sample.</p

Molecular Phylogenetic analysis by Maximum Likelihood method.

<p>The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.9645)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 37.8665% sites). The analysis involved 40 nucleotide sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 10247 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.</p

ZIKV Viral loads from urine and saliva specimens of infected patients measured by RT-qPCR.

<p>Urine specimens are shown in black and saliva specimens are shown in grey. The limit of detection is shown as a dotted line corresponding to 40 viral RNA copies/mL.</p

Differences in amino acid residues in ZIKV polyproteins of Rio-S1 and Rio-U1 isolates.

<p>Differences in amino acid residues in ZIKV polyproteins of Rio-S1 and Rio-U1 isolates.</p

Detection of genomic RNA of Zika virus in urine and saliva samples by RT-PCR analysis.

<p>(A) Shows the profiles obtained from urine samples. The lane numbers indicate the patient code. The lane 1I is the amplicon obtained from the viral isolate from urine of patient 1 (isolate Rio-U1). (B) RT-PCR analysis from patients 5 to 9 where S indicates saliva RNA samples, U, urine RNA samples, and I viral isolate sample. (C) Amplification of Zika virus genome of isolate Rio-U1 (1I) with ZIKV-specific primers that were also employed in the RT-PCR assay of Chikungunya virus RNA (CHIKV), dengue virus RNA (DENV) and Yellow Fever 17DD RNA (YFV). In all of these analyses, a negative control of amplification were included (C). The size marker migration is indicated on the left of the figures.</p