First report of autochthonous transmission of Zika virus in Brazil

Abstract

In the early 2015, several cases of patients presenting symptoms of mild fever, rash,
conjunctivitis and arthralgia were reported in the northeastern Brazil. Although all
patients lived in a dengue endemic area, molecular and serological diagnosis for
dengue resulted negative. Chikungunya virus infection was also discarded.
Subsequently, Zika virus (ZIKV) was detected by reverse transcription-polymerase
chain reaction from the sera of eight patients and the result was confirmed by DNA
sequencing. Phylogenetic analysis suggests that the ZIKV identified belongs to the
Asian clade. This is the first report of ZIKV infection in Brazil.

Full text

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 110(4): 569-572, June 2015 569
online | memorias.ioc.fiocruz.br
First report of autochthonous transmission of Zika virus in Brazil
Camila Zanluca
1
, Vanessa Campos Andrade de Melo
2
, Ana Luiza Pamplona Mosimann
1
,
Glauco Igor Viana dos Santos
2
, Claudia Nunes Duarte dos Santos
1
/
+
, Kleber Luz
3
/
+
1
Laboratório de Virologia Molecular, Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, Brasil
2
Secretaria Estadual de Saúde do Rio Grande do Norte, Natal, RN, Brasil
3
Instituto de Medicina Tropical, Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil
In the early 2015, several cases of patients presenting symptoms of mild fever, rash, conjunctivitis and arthral-
gia were reported in the northeastern Brazil. Although all patients lived in a dengue endemic area, molecular and
serological diagnosis for dengue resulted negative. Chikungunya virus infection was also discarded. Subsequently,
Zika virus (ZIKV) was detected by reverse transcription-polymerase chain reaction from the sera of eight patients
and the result was confirmed by DNA sequencing. Phylogenetic analysis suggests that the ZIKV identified belongs
to the Asian clade. This is the first report of ZIKV infection in Brazil.
Key words: Zika virus - “dengue-like syndrome” - Brazil
doi: 10.1590/0074-02760150192
Financial support: FIOCRUZ, CNPq, CAPES, Fundação Araucária
+ Corresponding authors: clsantos@fiocruz.br, klebergluz@gmail.com
Received 17 May 2015
Accepted 25 May 2015
Zika virus (ZIKV) is an emerging arthropod borne
virus (arbovirus) transmitted by Aedes (Stegomyia)
mosquitoes. The virus belongs to the genus Flavivirus,
family Flaviviridae, and is closely related to other fla-
viviruses of public health relevance including dengue
(DENV), yellow fever and West Nile viruses (Pierson &
Diamond 2013, Faye et al. 2014).
ZIKV was first isolated from a rhesus monkey in the
Zika Forest of Uganda, in 1947 (Dick et al. 1952). For
half a century, the virus was described as causing spo-
radic human infections in Africa and Asia, until 2007
when a Zika fever epidemics took place in Yap Island,
Micronesia (Duffy et al. 2009). In 2013, a large epidemic
was reported in French Polynesia, concomitant with a
dengue epidemic caused by serotypes 1 and 3. ZIKV has
been considered as emergent since 2007 (Cao-Lormeau
et al. 2014, Ioos et al. 2014).
In the early 2015, records of patients presenting a
“dengue-like syndrome” appeared in the public health
service in the city of Natal (05°47’42”S 35°12’32”O), state
of Rio Grande do Norte, Brazil. A physician specialist in
infectious disease evaluated the patients and the clinical
signs and symptoms and laboratory findings indicated
a non-DENV and non-Chikungunya virus (CHIKV) in-
fection. Symptoms included arthralgia, oedema of ex-
tremities, mild fever, maculopapular rashes frequently
pruritic, headaches, retroorbital pain, no purulent con-
junctivitis, vertigo, myalgia and digestive disorder.
By March 2015, the Molecular Virology Laborato-
ry of Carlos Chagas Institute, Oswaldo Cruz Institute,
state of Paraná, Brazil, received 21 acute-phase serum
specimens from those patients exhibiting the “dengue-
like syndrome”. Sera were transported and kept at -80ºC
until use. The protocol was approved by the Brazilian
Ethical Committee (42481115.7.0000.5248).
After RNA extraction, reverse transcription-polymer-
ase chain reaction (RT-PCR) was performed as a rapid
molecular tool to detect viral infection in acute-phase
samples. Viral RNA was extracted from 140 µL of sera
by using QIAamp viral RNA mini kit (Qiagen, USA) ac-
cording to the manufacturer’s instructions.
The sera were first tested for DENV by RT-PCR
(Lanciotti et al. 1992), ELISA for non-structural protein
1 antigen detection (Dengue Early ELISA, PanBio) and
anti-DENV IgM (Dengue IgM Capture ELISA, PanBio),
all yielded negative results. Moreover, all samples ren-
dered negative results in RT-PCR assays for CHIKV in-
fection (Lanciotti et al. 2007).
Since clinical symptoms were compatible with ZIKV
infection, RT-PCR assay to specifically detect ZIKV RNA
was performed, as described by Faye et al. (2008), with the
modification that, instead of a one step, a two step protocol
was used (cDNA synthesis followed by the PCR).
Amplicons of 364 bp encompassing the envelope
protein coding region were obtained for eight out of 21
samples (Fig. 1). Two of the resultant amplicons were
purified with High Pure PCR Purification kit (Roche)
and sequenced in an ABI 3500 Genetic Analyzer (Ap-
plied Biosystems). Sequences were assembled using
the Assembler tool (hpa-bioinformatics.org.uk/cgi-bin/
assembly_tool/seq_assemble.cgi?no=2) (GenBank ac-
cessions KR815989 and KR815990) and their identity
were confirmed by Basic Local Alignment Search Tool
(BLAST) and compared to other ZIKV sequences. Re-
sults demonstrated a high identity with sequences from
the ZIKV Asian lineage. Despite the similarity of the
clinical findings, the remaining samples resulted nega-
tive in RT-PCR, probably due to the short (and low) vi-
raemia period (between the 3rd and 5th day after the
onset of clinical symptoms).

ZIKV transmission in Brazil • Camila Zanluca et al.
570
Phylogenetic analysis of the sequences (Fig. 2) placed
the Brazilian strains in a clade with sequences from the
Asian lineage. The highest identity scores in BLAST
were observed with the H/PF/2013 (99%, GenBank ac-
cession KJ776791), CK-ISL 2014 (99%, GenBank acces-
sion KJ634273) and FSS13025 (99%, GenBank acces-
sion JN860885) isolates. The two generated sequences
were identical between themselves in the region that was
analysed (Fig. 2) and was identified as 15095_BR_2015.
These results corroborate previously published data
about the spread of the ZIKV Asian lineage that was first
reported in the outbreaks of the Pacific Islands (Musso
et al. 2014). To track the introduction of ZIKV in Brazil
a retrospective and more extensive analysis of additional
Fig. 1: agarose gel electrophoresis of reverse transcription-polymerase chain reaction assays for Zika virus (ZIKV) detection. The arrow indicates
the 364 bp amplicon expected for ZIKV. RNA extracted from serum samples of patients in acute-phase of dengue fever and Chikungunya fever
and a negative serum sample were included as negative controls. CHIKV: Chikungunya virus; M: molecular size marker; 1-21: serum samples.
Fig. 2: phylogenetic analysis based on partial E gene nucleic acid sequence (nt 1655-1984 according to AY632535) of a 2015 Brazilian strain of
Zika virus. The tree was inferred using the maximum likelihood algorithm based on the Kimura two-parameter model with invariant sites as
implemented in MEGA 6.05. The numbers shown to the left of the nodes represent bootstrap support values > 70 (1,000 replicates). The tree
was rooted with Spondoweni virus and branch lengths do not represent genetic distance. Strains were labelled according to GenBank accession
/country 2-letter acronym/year of isolation.

571
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 110(4), June 2015
samples from other Brazilian regions is needed, as well
as a complete viral genetic characterisation. However,
one plausible hypothesis is the arrival of the new emer-
gent virus during the soccer World Cup in 2014.
Seven of the eight patients with confirmed ZIKV dis-
ease were female, the median age was 39 years (range, 18-
65), all of them lived in Natal and had relatives with the
same symptoms. The most commonly reported symptoms
were maculopapular rash (100%) (Fig. 3) and pain. The
pain scale was applied for six of them and the pain levels
were high in most patients, with levels of zero (1/7), seven
(2/7), nine (1/7) or 10 (2/7). The pain duration ranged from
two-15 days, except for one patient who had pain for more
than 21 days. Other symptoms included headache (6/8),
retroorbital pain (4/8) and myalgia (6/8). Myalgia was not
intense. Joint pain was reported for seven patients, includ-
ing pain in the hands (5), ankle (4), elbow (4), knee and
wrist (3) and foot (2); one patient reported cervical pain.
During the clinical examination, all the patients were
haemodynamically normal and periarticular swelling
(Fig. 3) was present in six of them, with predominance
in the hands (50%). Fever was reported in six patients,
but high fever (around 39ºC) in only two. The duration
of fever ranged from one-eight days. Submandibular or
cervical lymphadenopathy (Fig. 3) occurred in three pa-
tients. No purulent conjunctivitis was observed. ZIKV
can produce a clinical relevant disease, which lasts for
approximately two weeks. The patients observed in Na-
tal had intense pain resembling CHIKV infection, but
the clinical course was shorter. To date, no death or com-
plications were associated with ZIKV illness in Natal.
The blood cell count revealed that only two patients had
leukopaenia, the others had normal levels of leukocytes and
neutrophils and platelets were normal in all of them. The
tourniquet tests were negative in seven of the patients.
We report here the first identification of ZIKV as the
causative agent of an outbreak in the northeastern Bra-
zil. It represents the first autochthonous transmission of
ZIKV in the country. Although most of the patients had
mild illness, clinicians and public health officials should
be aware of the risk of expansion of this new emerging
virus, especially given the naïve immunological status
of the Brazilian population. Spreading of the disease in
the country might occur by virtue of the large population
mobility and the widespread occurrence of the transmit-
Fig. 3: Zika virus clinical findings in patients from Natal, state of Rio Grande do Norte, Brazil. A: lymphadenopathy; B: maculopapular rash;
C: periarticular swelling.
ting vectors. Furthermore the complex epidemiological
context with the co-circulation of DENV, CHIKV and
ZIKV cannot be neglected. DENV and ZIKV (Dupont-
Rouzeyrol et al. 2015) or DENV and CHIKV (Caron et
al. 2012) co-infections have already been reported.
The effect of the concurrent outbreaks caused by
these three different arboviruses is unknown. ZIKV dis-
ease was associated with neurological and autoimmune
complications in a context of concurrent circulation with
DENV in French Polynesia (Roth et al. 2014). DENV also
circulates in Natal. Further studies are needed to unveil
whether co-infection and subsequent infection by differ-
ent arboviruses can affect the course of the disease, the
occurrence of severe cases and the ways of transmission
(vertical, perinatal, sexual) (Foy et al. 2011, Besnard et
al. 2014, Musso et al. 2015). Of interest, recent paper by
Gourinat et al. (2015) shows evidence of virus secretion
in urine for more than 10 days after onset of disease.
The correct clinical and laboratorial differential di-
agnosis between acute ZIKV, CHIKV and DENV in-
fection will contribute to the patients’ prognosis and to
drive surveillance actions.
ACKNOWLEDGEMENTS
To the Sequencing Platform of the Department of Biochem-
istry and Molecular Biology of the Federal University of Paraná,
for viral genomic sequencing, to Dr Helisson Faoro, for discus-
sion, and to Dra Andreia Suzukawa, for technical assistance.
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