CONTENT

Zika virus
From Wikipedia, the free encyclopedia
This article is about the virus. For the disease, see Zika fever. For the 201516 outbreak, see Zika
virus outbreak (2015present).

Zika virus

Electron micrograph of Zika

virus. Virus particles are
40 nm in diameter, with an
outer envelope and a dense
inner core (source: CDC).

Virus classification

Group:

Group IV

Family:

Flaviviridae

Genus:

Flavivirus

Species:

Zika virus

((+)ssRNA)

[1][2][3][4]

Zika virus /zik, zk/

(ZIKV) is a member of the virus family Flaviviridae and
the genus Flavivirus, transmitted by daytime-activeAedes mosquitoes, such as A. aegypti and A.
albopictus. Its name comes from the Zika Forest of Uganda, where the virus was first isolated in
1947.

[5]

Zika virus is related to dengue, yellow fever, Japanese encephalitis, and West Nile viruses.

[6]

The infection, known as Zika fever, often causes no or only mild symptoms, similar to a mild form
[6]

[7]

of dengue fever. It is treated by rest. Since the 1950s, it has been known to occur within a narrow
equatorial belt from Africa to Asia. In 2014, the virus spread eastward across the Pacific Ocean
to French Polynesia, then to Easter Island and in 2015 to Mexico, Central America, the Caribbean,
and South America, where the Zika outbreak has reached pandemic levels.

[8]

The illness cannot yet

[7]

be prevented by drugs or vaccines. As of February 2016, there is evidence that Zika fever in
pregnant women can cause abnormal brain development in their fetuses by mother-to-child
transmission, which may result in miscarriage
[12]

Zika virus causes microcephaly.

[9]

[10][11]

or microcephaly.

It is not yet known whether

A link has been established with neurologic conditions in infected

[13]

adults, including GuillainBarr syndrome.

In January 2016, the U.S. Centers for Disease Control and Prevention (CDC) issued travel guidance
on affected countries, including the use of enhanced precautions, and guidelines for pregnant
women including considering postponing travel.

[14][15]

Other governments or health agencies soon

[16][17][18]

issued similar travel warnings,

while Colombia, the Dominican Republic, Ecuador, El
Salvador, and Jamaica advised women to postpone getting pregnant until more is known about the
risks.

[17][19]

Contents

[hide]

1Virology

2Transmission
o

2.1Vector

2.2Sexual

2.3During pregnancy

2.4Other, unproven

3Zika fever

4Vaccine development

5History
5.1Virus isolation in monkeys and mosquitoes,

o
1947
o

5.2First evidence of human infection, 1952

5.3Spread in equatorial Africa and to Asia, 1951

1981

5.4Micronesia, 2007

5.5Oceania, 20132014

5.6Americas, 2015present
6See also

7References

8External links
Virology

A video explanation of Zika virus and Zika fever

The Zika virus belongs to Flaviviridae and the genus Flavivirus, and is thus related to
the dengue, yellow fever, Japanese encephalitis, and West Nile viruses. Like other flaviviruses, Zika
virus is enveloped and icosahedral and has a nonsegmented, single-stranded, positivesense RNA genome. It is most closely related to the Spondweni virus and is one of the two viruses
[20][21]

in the Spondweni virus clade.

A positive-sense RNA genome can be directly translated into viral proteins. In other flaviviruses,
such as the similarly sized West Nile virus, the RNA genome genes encode seven nonstructural
proteins and three structural proteins. The structural proteins encapsulate the virus. The replicated
RNA strand is held within a nucleocapsid formed from 12-kDa protein blocks; the capsid is contained
within a host-derived membrane modified with two viral glycoproteins. Replication of the viral
genome would first require creation of an anti-sense nucleotide strand.

[citation needed]

[22]

There are two lineages of the Zika virus: the African lineage, and the Asian lineage.
Phylogenetic
studies indicate that the virus spreading in the Americas is most closely related to the Asian strain,
which circulated in French Polynesia during the 2013 outbreak.
sequence of the Zika virus has been published.

[22][23]

The complete genome

[24]

Transmission
The vertebrate hosts of the virus were primarily monkeys in a so-called enzootic mosquito-monkeymosquito cycle, with only occasional transmission to humans. Before the current pandemic began in
2007, Zika virus "rarely caused recognized 'spillover' infections in humans, even in highly enzootic
areas". Infrequently, other arboviruses have become established as a human disease though, and
spread in a mosquitohumanmosquito cycle, like the yellow fever virus and the dengue fever virus
(both flaviruses), and the chikungunya virus (a togavirus).

[13]

Vector

Global Aedes aegypti predicted distribution. The map depicts

the probability of occurrence (blue=none, red=highest
occurrence).
The Zika virus is transmitted by daytime-active mosquitoes as its vector. It is primarily transmitted by
[25]:2

the female Aedes aegypti in order to lay eggs,

but has been isolated from a number
of arboreal mosquito species in the Aedes genus, such as A. africanus, A. apicoargenteus, A.
furcifer, A. hensilli, A. luteocephalus and A. vittatus with an extrinsic incubation period in mosquitoes
of about 10 days.

[26]

The true extent of the vectors is still unknown. The Zika virus has been detected in many more
species of Aedes, along withAnopheles coustani, Mansonia uniformis, and Culex perfuscus,
[27]

although this alone does not incriminate them as a vector.

Transmission by A. albopictus, the tiger mosquito, was reported from a 2007 urban outbreak in
Gabon where it had newly invaded the country and become the primary vector for the concomitant
[28]

chikungunya and dengue virus outbreaks.

There is concern for autochthonous infections in urban
areas of European countries infested by A. albopictus because the first two cases of laboratory
confirmed Zika virus infections imported into Italy were reported from viremic travelers returning from
French Polynesia.

[29]

The potential societal risk of Zika virus can be delimited by the distribution of the mosquito species
that transmit it. The global distribution of the most cited carrier of Zika virus, A. aegypti, is expanding
[30]

due to global trade and travel.

A. aegypti distribution is now the most extensive ever recorded
across all continents including North America and even the European periphery (Madeira, the
[31]

Netherlands, and the northeastern Black Sea coast).

A mosquito population capable of carrying
the Zika virus has been found in aCapitol Hill neighborhood of Washington, D. C., and genetic
evidence suggests they survived at least four consecutive winters in the region. The study authors
conclude that mosquitos are adapting for persistence in a northern climate.

[32]

Since 2015, news reports have drawn attention to the spread of Zika in Latin America and the
[33]

Caribbean.
The countries and territories that have been identified by the Pan American Health
Organisation as having experienced "local Zika virus transmission" are Barbados, Bolivia, Brazil,
Colombia, the Dominican Republic, Ecuador, El Salvador, French Guiana, Guadeloupe, Guatemala,
Guyana, Haiti, Honduras, Martinique, Mexico, Panama, Paraguay, Puerto Rico, Saint Martin,
Suriname, and Venezuela.

[34][35]

Sexual
As of February 2016, three reported cases indicate that Zika virus could possibly be sexually
transmitted. In 2014, Zika virus capable of reproducing itself was found in the semen of a man at
[36][37]

least two weeks (and possibly up to 10 weeks) after he fell ill with Zika fever.
The second
report is of a United States biologist who had been bitten many times while studying mosquitoes in
Senegal. Six days after returning home in August 2008, he fell ill with symptoms of Zika fever but not
before having unprotected intercourse with his wife, who had not been outside the US in 2008. She
subsequently developed symptoms of Zika fever, and Zika antibodies in both the biologist's and his
[36][38]

wife's blood confirmed the diagnosis.

In the third case, in early February 2016 the Dallas
County Health and Human Services department reported that a person contracted Zika fever after
sexual contact with an ill person who had recently returned from a high risk country. This case is still
under investigation.

[36][39]

It is unknown whether women can transmit Zika virus to their sexual partners. As of February 2016,
the CDC recommends that men "who reside in or have traveled to an area of active Zika virus
transmission who have a pregnant partner should abstain from sexual activity or consistently and
correctly use condoms during sex (i.e., vaginal intercourse, anal intercourse, or fellatio) for the
duration of the pregnancy." Men who reside in or have traveled to an area of active Zika virus
transmission and their non-pregnant sex partners "might consider" abstinence or condom use. The
CDC did not specify how long these practices should be followed with non-pregnant partners
because the "incidence and duration of shedding in the male genitourinary tract is limited to one
case report" and that "testing of men for the purpose of assessing risk for sexual transmission is not
recommended."

[36]

During pregnancy
In 2015, Zika virus RNA was detected in the amniotic fluid of two pregnant women whose fetuses
had microcephaly, indicating that the virus had crossed the placenta and could have caused
[40]

a mother-to-child infection.

Up until February 2016 the link was thought possible but unproven.

[42][43]

[41]

Brain tissue from two newborns with microcephaly who died within 20 hours of birth and
placenta and other tissue of two miscarriages (11 and 13 weeks) from Rio Grande do Norte in Brazil
tested positive for Zika virus by RT-PCR at the CDC.

[9]

According to the WHO on 5 February 2016, a causal link between the Zika virus and microcephaly
was "strongly suspected but not yet scientifically proven" and "Although the microcephaly cases in
Brazil are spatio-temporally associated with the Zika outbreak, more robust investigations and
research is needed to better understand this potential link."

[44]

On 5 February 2016, the United States CDC updated its health care provider guidelines for pregnant
women and women of reproductive age. The new recommendations include offering serologic
testing to pregnant women without Zika fever symptoms who have returned from areas with ongoing
Zika virus transmission in the last 212 weeks; and for pregnant women without Zika symptoms
living in such areas, they recommend testing at the beginning of prenatal care and follow-up testing
[45]

in the fifth month of pregnancy.

Other, unproven
As of February 2016 there are no confirmed cases of Zika virus transmission through blood
[46]

transfusions.
A potential risk is supected based on a study conducted between November 2013
and February 2014 during the Zika outbreak in French Polynesia in which 2.8% (42) of blood
donors tested positive for the Zika virus RNA and were asymptomatic at the time of blood donation.
Eleven of those positive donors reported symptoms of Zika fever after their donation, and only three
of 34 samples grew in culture.

[47]

Since January 2014 nucleic acid testing of blood donors was

implemented in French Polynesia to prevent unintended transmission.

[47]

Zika fever
Main article: Zika fever

Rash on an arm due to Zika virus

Common symptoms of infection with the virus include mild headaches, maculopapular rash, fever,
malaise, conjunctivitis, and joint pains. Three well-documented cases of Zika virus were described in
brief in 1954, whereas a detailed description was published 1964; it began with a mild headache,

and progressed to a maculopapular rash, fever, and back pain. Within two days, the rash started
fading, and within three days, the fever resolved and only the rash remained. Thus far, Zika fever
has been a relatively mild disease of limited scope, with only one in five persons developing
symptoms, with no fatalities, but its true potential as a viral agent of disease is unknown.

[26]

As of 2016, no vaccine or preventative drug is available. Symptoms can be treated with rest, fluids,
and paracetamol (acetaminophen), whileaspirin and other nonsteroidal anti-inflammatory
drugs should be used only when dengue has been ruled out to reduce the risk of bleeding.

[48]

There is a link between Zika fever and neurologic conditions in infected adults, including cases of
[13]

the GuillainBarr syndrome.

Vaccine development
Effective vaccines exist for several flaviviruses. Vaccines for yellow fever virus, Japanese
encephalitis, and tick-borne encephalitis were introduced in the 1930s, while the vaccine for dengue
fever only became available for use in the mid-2010s.

[49][50][51]

Work has begun in the USA towards developing a vaccine for the Zika virus, according to Anthony
[52]

Fauci, director of the National Institute of Allergy and Infectious Diseases.

The researchers at
the Vaccine Research Center have extensive experience from working with vaccines for other
[52]

viruses such as West Nile virus, chikungunya virus, and dengue fever.
Nikos Vasilakis of
the Center for Biodefense and Emerging Infectious Diseases predicted that it may take two years to
develop a vaccine, but 10 to 12 years may be needed before an effective Zika virus vaccine is
approved by regulators for public use.

[53]

An Indian company, Bharat Biotech International, reported in early February 2016 that it was working
[54]

on vaccines for the Zika virus.

The company is working on two approaches to a vaccine:
"recombinant", involving genetic engineering, and "inactivated", where the virus is incapable of
reproducing itself but can still trigger an immune response. The company announced animal trials of
[55]

the inactivated version would commence in late February.

History
See also: Zika fever Epidemiology

Countries that have past or current evidence of Zika virus

transmission (as of January 2016)
[56]

Spread of the Zika virus

[57]

This file is a candidate for speedy deletion. It may be deleted after Wednesday, 17
February 2016.
Virus isolation in monkeys and mosquitoes, 1947
The virus was first isolated in April 1947 from a rhesus macaque monkey that had been placed in a
cage in the Zika Forest of Uganda, near Lake Victoria, by the scientists of the Yellow Fever
[58]

Research Institute.
January 1948.

[59]

A second isolation from the mosquito A. africanus followed at the same site in

When the monkey developed a fever, researchers isolated from

its serum a "filterable transmissible agent" that was named Zika virus in 1948.

First evidence of human infection, 1952

[26][60]

Zika virus had been known to infect humans from the results of serological surveys in Uganda and
Nigeria. A serosurvey of 84 people of all ages showed 50 had antibodies, with all above 40 years of
age being immune.

[61]

It was not until 1954 that the successful isolation of Zika virus from a human was published. This
came as part of a 1952 outbreak investigation of jaundice suspected to be yellow fever. It was found
in the blood of a 10 year old Nigerian female with low grade fever, headache, and evidence of
malaria, but no jaundice, who recovered within three days. Blood was injected into the brain of
laboratory mice, followed by up to 15 mice passages. The virus from mouse brains was then tested
in neutralization tests using rhesusmonkey sera specifically immune to Zika virus. In contrast, no
virus was isolated from the blood of two infected adults with fever, jaundice, cough, diffuse joint
pains in one and fever, headache, pain behind the eyes and in the joints.

[clarification needed]

Infection

[61]

was proven by a rise in Zika virus specific serum antibodies.

A 1952 research study conducted in
India had shown a "significant number" of Indians tested for Zika had exhibited an immune response
to the virus, suggesting it had long been widespread within human populations.

[62]

Spread in equatorial Africa and to Asia, 19511981

From 1951 through 1981, evidence of human infection with Zika virus was reported from other
African countries, such as the Central African Republic, Egypt, Gabon, Sierra Leone, Tanzania, and
Uganda, as well as in parts of Asia including India, Indonesia, Malaysia, the Philippines, Thailand,
and Vietnam.

[26]

From its discovery until 2007, there were only 14 confirmed human cases of Zika

virus infection from Africa and Southeast Asia.

[63]

Micronesia, 2007
Main article: 2007 Yap Islands Zika virus outbreak
In April 2007, the first outbreak outside of Africa and Asia occurred on the island of Yap in the
Federated States of Micronesia, characterized by rash, conjunctivitis, and arthralgia, which was
[64]

initially thought to be dengue, chikungunya, or Ross River disease.

Serum samples from patients
in the acute phase of illness contained RNA of Zika virus. There were 49 confirmed cases, 59
unconfirmed cases, no hospitalizations, and no deaths.

[65]

Oceania, 20132014

This section
requires expansion.(February
2016)

Main article: 20132014 Zika virus outbreaks in Oceania

Between 2013 and 2014, further epidemics occurred in French Polynesia Polynesia, Easter Island,
[4]

the Cook Islands, and New Caledonia.

Americas, 2015present
Main article: Zika virus outbreak (2015present)
Since April 2015, a large, ongoing outbreak of Zika virus that began in Brazil has spread to much
of South and Central America and the Caribbean. In January 2016, the CDC issued a level 2 travel
alert for people traveling to regions and certain countries where Zika virus transmission is ongoing,
[66]

and suggested that women thinking about becoming pregnant should consult with their

physicians before traveling.

[17]

[68]

[67]

Governments or health agencies of the United Kingdom,

[18]

[16]

Ireland,

[18]

New Zealand,
Canada,
and theEuropean Union
soon issued similar travel warnings. In
Colombia, Minister of Health and Social Protection Alejandro Gaviria Uribe recommended avoiding
pregnancy for eight months, while the countries of Ecuador, El Salvador, and Jamaica have issued
similar warnings.

[17][19]

End of January 2016, the authorities in Rio de Janeiro, Brazil, announced plans to try to prevent the
[18]

spread of the Zika virus during the 2016 Summer Olympic Games in that city.

Between October 2015 and January 2016, Brazilian health authorities reported more than 3,500
[69]

microcephaly cases, some with a severe type and some having died.
The worst affected region of
Brazil is its poorest, consisting of the 3 Northeastern states Paraiba, Pernambuco and Bahia, where
about 1 percent of newborns are suspected of being microcephalic.

[70]

As of February 2016 52 travel-associated Zika virus disease cases and no locally acquired vectorborne cases had been reported from the US to the CDC, though there were 9 local cases from US
territories Puerto Rico and the US Virgin Islands.

[71]

RESEARCH ARTICLE
Zika Virus Associated with Microcephaly
Rubin, Eric J., Greene, Michael F., Baden, Lindsey R., . Zika
Virus and Microcephaly. New England Journal of Medicine

Jernej Mlakar, M.D., Misa Korva, Ph.D., Nataa Tul, M.D., Ph.D., Mara Popovi, M.D., Ph.D., Mateja Poljak-Prijatelj, Ph.D., Jerica
Mraz, M.Sc., Marko Kolenc, M.Sc., Katarina Resman Rus, M.Sc., Tina Vesnaver Vipotnik, M.D., Vesna Fabjan Voduek, M.D., Alenka
Vizjak, Ph.D., Joe Piem, M.D., Ph.D., Miroslav Petrovec, M.D., Ph.D., and Tatjana Avi upanc, Ph.D.
February 10, 2016

DOI: 10.1056/NEJMoa1600651

Share:
Abstract
Article
References
Citing Articles (1)

ZIKV, an emerging mosquito-borne flavivirus, was

initially isolated from a rhesus monkey in the Zika
forest in Uganda in 1947.1 It is transmitted by various
species of aedes mosquitoes. After the first human
ZIKV infection, sporadic cases were reported in
Southeast Asia and sub-Saharan Africa.2 ZIKV was
responsible for the outbreak in Yap Island of
Micronesia in 2007 and for major epidemics in French
Polynesia, New Caledonia, the Cook Islands, and
Easter Island in 2013 and 2014.3,4 In 2015, there was
a dramatic increase in reports of ZIKV infection in the
Americas. Brazil is the most affected country, with
preliminary estimates of 440,000 to 1.3 million cases
of autochthonous ZIKV infection reported through
December 2015.5
The classic clinical picture of ZIKV infection
resembles that of dengue fever and chikungunya and
is manifested by fever, headache, arthralgia, myalgia,
and maculopapular rash, a complex of symptoms that
hampers differential diagnosis. Although the disease
is self-limiting, cases of neurologic manifestations and
the GuillainBarr syndrome were described in
French Polynesia and in Brazil during ZIKV

epidemics.5,6 Recent reports from the Ministry of

Health of Brazil suggest that cases of microcephaly
have increased by a factor of approximately 20
among newborns in the northeast region of the
country, which indicates a possible association
between ZIKV infection in pregnancy and fetal
malformations.5
We present a case of vertical transmission of ZIKV in
a woman who was probably infected with ZIKV in
northeastern Brazil at the end of the first trimester of
pregnancy. Our discussion includes details of fetal
imaging and pathological and virologic analyses.

CASE REPORT
In mid-October 2015, a 25-year-old previously healthy
European woman came to the Department of
Perinatology at the University Medical Center in
Ljubljana, Slovenia, because of assumed fetal
anomalies. Since December 2013, she had lived and
worked as a volunteer in Natal, the capital of Rio
Grande do Norte state. She had become pregnant at
the end of February 2015. During the 13th week of
gestation, she had become ill with high fever, which
was followed by severe musculoskeletal and
retroocular pain and an itching, generalized
maculopapular rash. Since there was a ZIKV
epidemic in the community, infection with the virus
was suspected, but no virologic diagnostic testing was
performed. Ultrasonography that was performed at 14
and 20 weeks of gestation showed normal fetal
growth and anatomy.

The patient returned to Europe at 28 weeks of

gestation. Ultrasonographic examination that was
performed at 29 weeks of gestation showed the first
signs of fetal anomalies, and she was referred to the
Department of Perinatology. At that time, she also
noticed reduced fetal movements. Ultrasonography
that was performed at 32 weeks of gestation
confirmed intrauterine growth retardation (estimated
third percentile of fetal weight) with normal amniotic
fluid, a placenta measuring 3.5 cm in thickness
(normal size) with numerous calcifications, a head
circumference below the second percentile for
gestation (microcephaly), moderate ventriculomegaly,
and a transcerebellar diameter below the second
percentile. Brain structures were blurred, and there
were numerous calcifications in various parts of the
brain (Figure 1A and 1BFIGURE 1

Prenatal
Ultrasonographic Images and Photographs of Coronal Slices
of Brain.). There were no other obvious fetal structural

abnormalities. Fetal, umbilical, and uterine blood

flows were normal on Doppler ultrasonography.
The clinical presentation raised suspicion of fetal viral
infection. Because of severe brain disease and
microcephaly, the fetus was given a poor prognosis
for neonatal health. The mother requested that the
pregnancy be terminated, and the procedure was
subsequently approved by national and hospital
ethics committees. Medical termination of the
pregnancy was performed at 32 weeks of gestation.

At the delivery, the only morphologic anomaly was the

prominent microcephaly. Genetic consultation that
included a detailed maternal family history revealed
no suspicion of genetic syndromes or diseases. An
autopsy was performed, as is mandatory in all cases
of termination of pregnancy. The mother provided
written informed consent for the publication of this
case report.

METHODS
Autopsy and Central Nervous System (CNS)
Examination
An autopsy of the fetus and placenta was performed 3
days after termination of the pregnancy, with an
extensive sampling of all organs, placenta, and
umbilical cord. Samples were fixed in 10% buffered
formalin and embedded in paraffin. Fresh tissue
samples were collected for microbiologic
investigations. Brain and spinal cord were fixed in
27% buffered formalin for 3 weeks, after which a
neuropathological examination was performed with
extensive sampling of the brain and spinal cord.
Sections of all tissue samples were stained with
hematoxylin and eosin. Immunostaining for glial
fibrillary acid protein, neurofilament, human leukocyte
antigen DR (HLA-DR), CD3 (to highlight T cells), and
CD20 (to highlight B cells) was performed on
representative CNS samples.
Electron Microscopy

Tissue was collected from formalin-fixed brain and

underwent fixation in 1% osmium tetroxide and
dehydration in increasing concentrations of ethanol.
The sample was then embedded in Epon. Semithin
sections (1.4 m) were made, stained with Azur II,
and analyzed by means of light microscopy. Ultrathin
sections (60 nm) were stained with uranyl acetate and
lead citrate. In addition, a small piece of brain (5 mm3)
was homogenized in buffer. The suspension was then
cleared by low-speed centrifugation, and the obtained
supernatant was ultracentrifuged directly onto an
electron microscopic grid with the use of an Airfuge
(Beckman Coulter). Negative staining was performed
with 1% phosphotungstic acid. Imaging of the ultrathin
sections and brain homogenate was performed with
the use of a 120-kV JEM-1400Plus transmission
electron microscope (JEOL).
Indirect Immunofluorescence
Paraffin-embedded sections of the fetal brain tissue
and brain tissue of an autopsied man as a negative
control were incubated with serum obtained from the
mother of the fetus (dilution, 1:10), followed by
antihuman IgG antibodies labeled with fluorescein
isothiocyanate (FITC) (dilution, 1:50). In addition, fetal
brain tissue was incubated with a serum obtained
from a healthy blood donor, as well as with FITClabeled antihuman IgG antibodies only.
Microbiologic Investigation
RNA was extracted from 10 mg of the placenta, lungs,
heart, skin, spleen, thymus, liver, kidneys, and

cerebral cortex with the use of a TRIzol Plus RNA

purification kit (Thermo Fisher Scientific). Real-time
RT-PCR for the detection of ZIKV RNA (NS5) and
one-step RT-PCR for the detection of the envelopeprotein coding region (360 bp) were performed as
described previously.7,8 In addition, next-generation
sequencing was performed in samples of fetal brain
tissue with the use of Ion Torrent (Thermo Fisher
Scientific) and Geneious software, version 9.0.6.
Reads from both runs were combined and mapped to
the reference sequence (ZIKV MR766; LC002520)
with the use of default measures. For phylogenetic
analysis, complete-genome ZIKV sequences were
used, and multiple sequence alignments (ClustalW)
were performed. A neighbor-joining phylogenetic tree
(GTR+G+I model) was constructed, with the use of
the MEGA6 software system,9 to show the
phylogenetic relationships. The nucleotide sequence
of ZIKV that was obtained in this study has been
deposited in GenBank under accession number
KU527068. A detailed description of the molecular
methods is provided in the Supplementary Appendix,
available with the full text of this article at NEJM.org.
The results of comprehensive serologic analyses of
maternal serum and a description of the molecular
differential diagnostic procedures used with fetal
tissue samples are provided in Tables S1 and S2 in
the Supplementary Appendix. All the authors vouch for
the completeness and accuracy of the data and
analyses presented.

RESULTS
Autopsy and Neuropathological Findings
The fetal body weight was 1470 g (5th percentile), the
length 42 cm (10th percentile), and the head
circumference 26 cm (1st percentile). The only
external anomaly that was noted was microcephaly.
The placenta weighed 200 g, resulting in a placental
fetal weight ratio of 0.136 (<3rd percentile).
Macroscopic examination of the CNS revealed
micrencephaly with a whole-brain weight of 84 g (4
SD below average), widely open sylvian fissures, and
a small cerebellum and brain stem. Almost complete
agyria and internal hydrocephalus of the lateral
ventricles were observed. There were numerous
variable-sized calcifications in the cortex and
subcortical white matter in the frontal, parietal, and
occipital lobes. The subcortical nuclei were quite well
developed (Figure 1C and 1D). In spite of some
autolysis, microscopic examination revealed
appropriate cytoarchitecture of the fetal brain. The
most prominent histopathological features were
multifocal collections of filamentous, granular, and
neuron-shaped calcifications in the cortex and
subcortical white matter with focal involvement of the
whole cortical ribbon, occasionally associated with
cortical displacement (Figure 2A and 2BFIGURE 2

Microscopic Analysis of Brain Tissue.). Diffuse

astrogliosis was present with focal astrocytic outburst

into the subarachnoid space, mostly on the convexity
of the cerebral hemispheres (Figure 2C). Activated
microglial cells and some macrophages expressing
HLA-DR were present throughout most of the cerebral
gray and white matter (Figure 2D). Scattered mild
perivascular infiltrates composed of T cells and some
B cells were present in the subcortical white matter
(Fig. S1 in the Supplementary Appendix). The
cerebellum, brain stem, and spinal cord showed
neither inflammation nor dystrophic calcifications. The
brain stem and spinal cord showed Wallerian
degeneration of the long descending tracts, especially
the lateral corticospinal tract, whereas ascending
dorsal columns were well preserved (Figure 2E).
Indirect immunofluorescence revealed granular
intracytoplasmic reaction in destroyed neuronal
structures, which pointed to a possible location of the
virus in neurons (Figure 2F, and Fig. S1 in
the Supplementary Appendix). Histologic examination
of the placenta confirmed focal calcifications in villi
and decidua, but no inflammation was found. There
were no relevant pathological changes in other fetal
organs or in the umbilical cord or fetal membranes.
Fetal karyotyping with the use of microarray
technology showed a normal 46XY (male) profile.
Electron Microscopy
Although analysis of the ultrathin sections of the brain
showed poorly preserved brain tissue with ruptured
and lysed cells, clusters of dense virus-like particles

of approximately 50 nm in size were found in

damaged cytoplasmic vesicles. Groups of enveloped
structures with a bright interior were also detected. At
the periphery of such groups, the remains of
membranes could be seen. Negative staining of
homogenized brain revealed spherical virus particles
measuring 42 to 54 nm with morphologic
characteristics consistent with viruses of the

Flaviviridae family (Figure 3FIGURE 3

Electron Microscopy of Ultrathin Sections of Fetal Brain and
Staining of a Flavivirus-like Particle.).

Microbiologic Investigation
Positive results for ZIKV were obtained on RT-PCR
assay only in the fetal brain sample, where
6.5107 viral RNA copies per milligram of tissue were
detected. In addition, all autopsy samples were tested
on PCR assay and were found to be negative for
other flaviviruses (dengue virus, yellow fever virus,
West Nile virus, and tick-borne encephalitis virus),
along with chikungunya virus, lymphocytic
choriomeningitis, cytomegalovirus, rubella virus,
varicellazoster virus, herpes simplex virus,
parvovirus B19, enteroviruses, and Toxoplasma
gondii (Table S2 in the Supplementary Appendix).
A complete ZIKV genome sequence (10,808
nucelotides) was recovered from brain tissue.
Phylogenetic analysis showed the highest identity
(99.7%) with the ZIKV strain isolated from a patient

from French Polynesia in 2013 (KJ776791) and ZIKV

detected in Sao Paolo, Brazil, in 2015 (KU321639),
followed by a strain isolated in Cambodia in 2010
(JN860885, with 98.3% identity) and with a strain from
the outbreak in Micronesia in 2007 (EU545988, with
98% identity) (Figure 4FIGURE 4

Phylogenetic

Analysis of the Complete Genome of Zika Virus.). In the

ZIKV polyprotein, 23 polymorphisms were detected in

comparison with the strain from Micronesia and 5
polymorphisms in comparison with the isolate from
French Polynesia; three amino acid changes were
found in the NS1 region (K940E, T1027A, and
M1143V), one in the NS4B region (T2509I), and one
in the FtsJ-like methyltransferase region (M2634V).

DISCUSSION
This case shows severe fetal brain injury associated
with ZIKV infection with vertical transmission.
Recently, ZIKV was found in amniotic fluid of two
fetuses that were found to have microcephaly, which
was consistent with intrauterine transmission of the
virus.10Described cases are similar to the case
presented here and were characterized by severely
affected CNS and gross intrauterine growth
retardation. Calcifications in the placenta and a low
placentalfetal weight ratio,11 which were seen in this
case, indicate potential damage to the placenta by the
virus. Among the few reports of teratogenic effects of
flaviviruses, investigators described the brain and
eyes as the main targets.12,13 No presence of virus

and no pathological changes were detected in any

other fetal organs apart from the brain, which
suggests a strong neurotropism of the virus.
The localization of immunofluorescence signal and
the morphologic appearance of the calcifications,
which resembled destroyed neuronal structures,
indicate a possible location of the virus in neurons.
The consequent damage might cause arrested
development of the cerebral cortex at the embryonic
age of approximately 20 weeks.14 The mechanism
involved in the neurotropism of ZIKV is currently not
clear. The association between ZIKV infection and
fetal brain anomalies was also noted by findings on
electron microscopy that were consistent with ZIKV
detection in the fetal brain. Dense particles consistent
with ZIKV were seen in damaged endoplasmic
reticulum. Groups of enveloped structures with a
bright interior resembling the remains of replication
complexes that are characteristic of
flaviviruses15,16 indicate viral replication in the brain.
The findings on electron microscopy suggest a
possible persistence of ZIKV in the fetal brain,
possibly because of the immunologically secure
milieu for the virus. The number of viral copies that
were detected in the fetal brain were substantially
higher than those reported in the serum obtained from
adult ZIKV-infected patients17 but similar to those
reported in semen samples.18
The complete genome sequence of ZIKV that was
recovered in this study is consistent with the
observation that the present strain in Brazil has

emerged from the Asian lineage.19 The presence of

two major amino acid substitutions positioned in
nonstructural proteins NS1 and NS4B probably
represents an accidental event or indicates a process
of eventual adaptation of the virus to a new
environment. Further research is needed to better
understand the potential implications of these
observations. It is likely that the rapid spread of ZIKV
around the globe will be a strong impetus for
collaborative research on the biologic properties of
the virus, particularly since the risk of neurotropic and
teratogenic virus infections places a high emotional
and economic burden on society.