Limits...
Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil.

Bonaldo MC, Ribeiro IP, Lima NS, Dos Santos AA, Menezes LS, da Cruz SO, de Mello IS, Furtado ND, de Moura EE, Damasceno L, da Silva KA, de Castro MG, Gerber AL, de Almeida LG, Lourenço-de-Oliveira R, Vasconcelos AT, Brasil P - PLoS Negl Trop Dis (2016)

Bottom Line: The complete genome of both ZIKV isolates was obtained.Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak.The 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular de Flavivírus, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil.

ABSTRACT

Background: 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.

Methodology/principal findings: Nine 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.

Conclusions/significance: The 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.

No MeSH data available.


Related in: MedlinePlus

Molecular Phylogenetic analysis by Maximum Likelihood method.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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4920388&req=5

pntd.0004816.g004: Molecular Phylogenetic analysis by Maximum Likelihood method.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.

Mentions: The genomic sequences of Vero cell isolates ZIKV Rio-U1 strain (KU926309), isolated from urine and Rio-S1 (KU926310) strain, isolated from saliva, were then determined. The comparison between Rio-U1 and Rio-S1 yielded 99.61% identity, displaying six amino acid variations in the viral proteins (Table 2). For phylogenetic analysis, we used nucleotide sequences coding the complete ZIKV polyprotein. We observed that all sequences sampled in the Americas form a robust monophyletic cluster (bootstrap score = 97%) within the Asian genotype and share a common ancestor with the ZIKV strain that circulated in French Polynesia in November 2013 and remained genetically isolated from African clusters (Fig 4).


Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil.

Bonaldo MC, Ribeiro IP, Lima NS, Dos Santos AA, Menezes LS, da Cruz SO, de Mello IS, Furtado ND, de Moura EE, Damasceno L, da Silva KA, de Castro MG, Gerber AL, de Almeida LG, Lourenço-de-Oliveira R, Vasconcelos AT, Brasil P - PLoS Negl Trop Dis (2016)

Molecular Phylogenetic analysis by Maximum Likelihood method.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920388&req=5

pntd.0004816.g004: Molecular Phylogenetic analysis by Maximum Likelihood method.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.
Mentions: The genomic sequences of Vero cell isolates ZIKV Rio-U1 strain (KU926309), isolated from urine and Rio-S1 (KU926310) strain, isolated from saliva, were then determined. The comparison between Rio-U1 and Rio-S1 yielded 99.61% identity, displaying six amino acid variations in the viral proteins (Table 2). For phylogenetic analysis, we used nucleotide sequences coding the complete ZIKV polyprotein. We observed that all sequences sampled in the Americas form a robust monophyletic cluster (bootstrap score = 97%) within the Asian genotype and share a common ancestor with the ZIKV strain that circulated in French Polynesia in November 2013 and remained genetically isolated from African clusters (Fig 4).

Bottom Line: The complete genome of both ZIKV isolates was obtained.Phylogenetic analysis revealed similarity with strains previously isolated during the South America outbreak.The 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular de Flavivírus, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil.

ABSTRACT

Background: 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.

Methodology/principal findings: Nine 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.

Conclusions/significance: The 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.

No MeSH data available.


Related in: MedlinePlus