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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

Detection of genomic RNA of Zika virus in urine and saliva samples by RT-PCR analysis.(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.
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pntd.0004816.g002: Detection of genomic RNA of Zika virus in urine and saliva samples by RT-PCR analysis.(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.

Mentions: Furthermore, we analyzed all urine and saliva specimens by RT-PCR to confirm the detection of ZIKV (Fig 2A and 2B). In addition, we included RNA samples of ZIKV isolated from patient 1 and 6 in Vero cells. The set of samples of patient 1 and 6 were all positive and an expected-amplicon band of around 300 bp was seen in electrophoretic analyses, demonstrating the presence of ZIKV genome in these samples (Fig 2A and 2B). We also observed a faint band from urine and saliva of patient 9 (Fig 2B). The ZIKV specificity of this approach was confirmed when we tested this protocol in RNA samples of Chikungunya (CHIKV), dengue (DENV) and yellow fever (YFV) viruses (Fig 2C).


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)

Detection of genomic RNA of Zika virus in urine and saliva samples by RT-PCR analysis.(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.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004816.g002: Detection of genomic RNA of Zika virus in urine and saliva samples by RT-PCR analysis.(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.
Mentions: Furthermore, we analyzed all urine and saliva specimens by RT-PCR to confirm the detection of ZIKV (Fig 2A and 2B). In addition, we included RNA samples of ZIKV isolated from patient 1 and 6 in Vero cells. The set of samples of patient 1 and 6 were all positive and an expected-amplicon band of around 300 bp was seen in electrophoretic analyses, demonstrating the presence of ZIKV genome in these samples (Fig 2A and 2B). We also observed a faint band from urine and saliva of patient 9 (Fig 2B). The ZIKV specificity of this approach was confirmed when we tested this protocol in RNA samples of Chikungunya (CHIKV), dengue (DENV) and yellow fever (YFV) viruses (Fig 2C).

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