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Isolation of Infective Zika from Brazil Urine & Saliva - bioRxiv


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Isolation of infective Zika virus from urine and saliva of patients in Brazil

Myrna C BonaldoIeda P RibeiroNoemia S. LimaAlexandre A. C. Santos,Lidiane S.R. MenezesStephanie O.D. CruzIasmim S. MelloNathália D. Furtado,Elaine E. MouraLuana DamascenoKeli A.B. SilvaMarcia G. CastroAlexandra L. GerberLuiz G.P. AlmeidaRicardo Lourenço-de-OliveiraAna Tereza R.VasconcelosPatrícia Brasil
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AUTHOR INFORMATION

  1. Myrna C Bonaldo1 (mbonaldo{at}ioc.fiocruz.br) (http://www.ioc.fiocruz.br),
  2. Ieda P Ribeiro1 (ieda.ribeiro{at}ioc.fiocruz.br) (http://www.ioc.fiocruz.br),
  3. Noemia S. Lima1 (noemiasl{at}gmail.com) (www.ioc.fiocruz.br)
  4. Alexandre A. C. Santos1 (xandeacs{at}ioc.fiocruz.br) (http://www.ioc.fiocruz.br)
  5. Lidiane S.R. Menezes1 (lidiane.raphael{at}ioc.fiocruz.br) (http://www.ioc.fiocruz.br),
  6. Stephanie O.D. Cruz1 (stephanieodc{at}gmail.com) (http://www.ioc.fiocruz.br),
  7. Iasmim S. Mello1 (mimmello123{at}hotmail.com) (http://www.ioc.fiocruz.br),
  8. Nathália D. Furtado1 (nathyfur{at}gmail.com) (http://www.ioc.fiocruz.br),
  9. Elaine E. Moura1 (evangelista.eem{at}gmail.com) (http://www.ioc.fiocruz.br),
  10. Luana Damasceno2 (luanasdamasceno{at}hotmail.com)(http://www.ini.fiocruz.br)
  11. Keli A.B. Silva1 (keli.antunes{at}ioc.fiocruz.br)(http://www.ioc.fiocruz.br)
  12. Marcia G. Castro1 (mcastro{at}ioc.fiocruz.br)(http://www.ioc.fiocruz.br)
  13. Alexandra L. Gerber3 (alegerber{at}lncc.br)(http://www.lncc.br)
  14. Luiz G.P. Almeida3 (lgonzaga{at}lncc.br)(http://www.lncc.br)
  15. Ricardo Lourenço-de-Oliveira1(lourenco{at}ioc.fiocruz.br) (http://www.ioc.fiocruz.br)
  16. Ana Tereza R. Vasconcelos3 (atrv{at}lncc.br) (http://www.lncc.br) and 
  17. Patrícia Brasil2(patricia.brasil{at}ini.fiocruz.br) (http://www.ini.fiocruz.br)
  1. 1 Instituto Oswaldo Cruz, Fiocruz;
  2. 2 Instituto Nacional de Infectologia Evandro Chagas, Fiocruz;
  3. 3 Laboratório Nacional de Computação Científica
  1. * Corresponding author; email: mbonaldo{at}ioc.fiocruz.br
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Abstract

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. However, no evidence regarding the infectivity of ZIKV particles present in saliva and urine has been obtained yet. 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. 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.

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S1 Table

Clinical symptoms

Patient

1

2

3

4

Onset date

01/11/16

01/24/16

01/22/16

01/31/16

Days after symptoms onset *

3

2

5

1

Days after rash onset *

<1**

1

1

1

Rash duration

5 days

6 days

2 days

2 days

Rash type

Macular

Macular and maculo-papular

Maculo-papular

Maculo-papular

Low grade fever

(duration)

-

+

(2 days)

+

(4 days)

-

Headache

-

-

+

-

Retro-orbital pain

-

-

+

-

Photophobia

-

-

NI

-

Fatigue/ malaise

-

+

+

-

Myalgia

-

+

+

-

Arthralgia

(region)

+

(wrist and elbows)

-

+ (large and small joints)

-

Arthritis

-

-

-

-

Anorexia

-

-

+

-

Nausea/ vomiting

-

-

+

-

Diarrhea

-

-

-

-

Abdominal pain

-

-

-

-

Dysuria

-

-

-

-

Bleeding/ petechia

-

-

-

-

Dizziness/ light headedness

-

-

-

-

Pruritus

+

-

+

+

Paresthesias

+

-

-

-

Conjunctivitis

-

-

-

-

Edema

(local/site)

+

(hands)

-

-

-

Lymphadenopathy

(local/ site)

-

-

-

-

Enanthem

NI

-

NI

-

Respiratory symptoms

-

-

-

-

Jaundice

-

-

-

-

Seizures

-

-

-

-

* Regarding the date of sample collection; **the collection date was in the first day of rash manifestation

                                                                                                   

S2 Table

Clinical symptoms

Patient

5

6

7

8

9

Onset date

01/26/16

01/26/16

01/31/16

01/31/16

01/28/16

Days after sypmtons onset *

2

3

1

2

5

Days after rash onset *

2

<1

1

2

2

Rash duration

 

20 days

4 days

5 days

6 days

3 days

Rash type

Macular

Maculo-papular

Maculo-papular

Macular and maculo-papular

Maculo-papular

Low grade fever

(duration)

-

+

(1 day)

-

+

(2 days)

-

Headache

+

-

-

+

+

Retro-orbital pain

+

-

-

+

+

Photophobia

+

-

-

-

+

Fatigue/ malaise

+

+

-

+

-

Myalgia

+

+

-

-

+

Arthralgia

(region)

-

+

(wrists, ankles, knees, hands)

-

+

(large and small joints)

-

Arthritis

-

-

-

-

-

Anorexia

+

+

-

-

-

Nausea/ vomiting

+

-

-

-

-

Diarrhea

+

-

-

-

-

Abdominal pain

-

+

-

-

-

Dysuria

-

-

-

-

-

Bleeding/ petechia

 

-

-

-

-

Dizziness/ light headedness

+

-

-

-

-

Pruritus

+

+

+

+

+

Paresthesias

+

+

-

-

-

Conjunctivitis

+

+

-

-

+

Edema

+

(feet and hands)

 

+

(hands)

-

+

-

Lymphadenopathy

(local/ site)

+

(Cervical and auricular)

-

-

-

+

(cervical)

Enanthem

-

-

-

-

-

Respiratory symptoms

+

-

-

+

-

Jaundice

-

-

-

-

-

Seizures

-

-

-

-

-

* Regarding the date of sample collection; **the collection date was in the first day of rash manifestation

 

 

S3 Table

 

Social-demographic data

Patient

1

2

3

4

Gender

female

female

male

female

Age

36

30

24

42

Gestational age (weeks)

18

33

NA

21

Family members illness

-

-

+

-

Partner illness

-

-

NI

-

Repellent spray use

+

+

NI

+

Previous DENV infection

+

-

NI

-

Domicile in Rio de Janeiro State

Duque de Caxias

Nova Iguaçu

Rio de Janeiro

Duque de Caxias

NA – not applicable; NI- not informed

 

                                                                                   

 

S4 Table

Social demographic data

Patient

5

6

7

8

9

Gender

female

male

female

female

female

Age

30

68

20

27

22

Gestational age (weeks)

NA

NA

17

21

20

Family members illness

-

+

-

-

-

Partner illness

-

-

-

-

-

Repellent spray use

+

-

+

+

+

Previous DENV infection

+

+

-

+

-

Domicile in Rio de Janeiro State

Rio de Janeiro

Rio de Janeiro

Rio de Janeiro

Duque de Caxias

Rio de Janeiro

NA – not applicable; NI- not informed

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