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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

ZIKV Viral loads from urine and saliva specimens of infected patients measured by RT-qPCR.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.
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pntd.0004816.g003: ZIKV Viral loads from urine and saliva specimens of infected patients measured by RT-qPCR.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.

Mentions: Viral loads of these samples were then measured by a RT-qPCR assay resulting in data consistent with those obtained by the diagnosis assay kit (Table 1 and Fig 3). Accordingly, the highest viral loads were obtained from those specimens that allowed us to isolate ZIKV by Vero cell infections. The urine of patient 1 exhibited a ZIKV-genomic RNA copies of 2.68 x 103 per ml whereas the patient 6 displayed 2.53 x 105 ZIKV RNA copies per ml in urine and 7.44 x 104 ZIKV RNA copies per ml in saliva. As expected for isolated viral samples, we observed an increase of genomic ZIKV RNA copies in Vero-cell- isolated samples, in which the isolated from patient 1 presented 1.24 x 1010 copies/ml and patient 6, 2.88 x 109 copies/ml (Fig 3). Furthermore, we confirmed positivity of the urine from patient 7 (102 copies/ml) and the positive detection of ZIKV RNA in saliva (40 copies/ml) and urine (431 copies/ml) of patient 9, although this established value is borderline localized in the limit of detection.


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)

ZIKV Viral loads from urine and saliva specimens of infected patients measured by RT-qPCR.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.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004816.g003: ZIKV Viral loads from urine and saliva specimens of infected patients measured by RT-qPCR.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.
Mentions: Viral loads of these samples were then measured by a RT-qPCR assay resulting in data consistent with those obtained by the diagnosis assay kit (Table 1 and Fig 3). Accordingly, the highest viral loads were obtained from those specimens that allowed us to isolate ZIKV by Vero cell infections. The urine of patient 1 exhibited a ZIKV-genomic RNA copies of 2.68 x 103 per ml whereas the patient 6 displayed 2.53 x 105 ZIKV RNA copies per ml in urine and 7.44 x 104 ZIKV RNA copies per ml in saliva. As expected for isolated viral samples, we observed an increase of genomic ZIKV RNA copies in Vero-cell- isolated samples, in which the isolated from patient 1 presented 1.24 x 1010 copies/ml and patient 6, 2.88 x 109 copies/ml (Fig 3). Furthermore, we confirmed positivity of the urine from patient 7 (102 copies/ml) and the positive detection of ZIKV RNA in saliva (40 copies/ml) and urine (431 copies/ml) of patient 9, although this established value is borderline localized in the limit of detection.

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