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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

Isolation of Zika virus in Vero cell from the saliva of patient 6.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.
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pntd.0004816.g001: Isolation of Zika virus in Vero cell from the saliva of patient 6.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.

Mentions: We collected and analyzed urine from patients 1 to 4 and both urine and saliva samples from patients 5 to 9. Vero cells cultures were inoculated at the same date of sample collection and then daily observed through inverted microscopic examination until the appearance of cytopathic effect (CPE). Within one week of incubation, only two samples exhibited CPE (2 out of 14), the urine sample of patient 1 with CPE detected at 4th day of post-inoculation (1 out of 9) and the saliva sample of patient 6 at 5th day post-inoculation (1 out of 5). In this last infection, we recognized small foci of rounded and refractive cells detaching from the monolayer (Fig 1A and 1B). After one-week incubation, we proceeded to split cells from negative cultures by means of trypsinization when monolayer was confluent. This procedure was repeated for three consecutive times. Nevertheless, it was not possible to isolate ZIKV in these samples, neither by detecting CPE in Vero cell monolayers or ZIKV genome by RT-PCR. We also analyzed these samples by plaque forming assay as a way to detect infectious virus particles. Unfortunately, we did not perform this analysis with urine of patient 1, because we received a small aliquot of this specimen. Nevertheless, we detected viral plaques from samples of patient 6 (Fig 1C and 1D), in which the dilution 1:2 of saliva originated in 8 PFU resulting in an original viral concentration of 80 PFU/ml in saliva of patient 6. Interestingly, only one viral plaque was visualized by means of this methodology in urine sample of this patient 6, resulting in a titer of 10 PFU/ml.


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)

Isolation of Zika virus in Vero cell from the saliva of patient 6.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.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004816.g001: Isolation of Zika virus in Vero cell from the saliva of patient 6.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.
Mentions: We collected and analyzed urine from patients 1 to 4 and both urine and saliva samples from patients 5 to 9. Vero cells cultures were inoculated at the same date of sample collection and then daily observed through inverted microscopic examination until the appearance of cytopathic effect (CPE). Within one week of incubation, only two samples exhibited CPE (2 out of 14), the urine sample of patient 1 with CPE detected at 4th day of post-inoculation (1 out of 9) and the saliva sample of patient 6 at 5th day post-inoculation (1 out of 5). In this last infection, we recognized small foci of rounded and refractive cells detaching from the monolayer (Fig 1A and 1B). After one-week incubation, we proceeded to split cells from negative cultures by means of trypsinization when monolayer was confluent. This procedure was repeated for three consecutive times. Nevertheless, it was not possible to isolate ZIKV in these samples, neither by detecting CPE in Vero cell monolayers or ZIKV genome by RT-PCR. We also analyzed these samples by plaque forming assay as a way to detect infectious virus particles. Unfortunately, we did not perform this analysis with urine of patient 1, because we received a small aliquot of this specimen. Nevertheless, we detected viral plaques from samples of patient 6 (Fig 1C and 1D), in which the dilution 1:2 of saliva originated in 8 PFU resulting in an original viral concentration of 80 PFU/ml in saliva of patient 6. Interestingly, only one viral plaque was visualized by means of this methodology in urine sample of this patient 6, resulting in a titer of 10 PFU/ml.

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