Review

pubs.acs.org/journal/aidcbc

Zika Virus: Emergence, Phylogenetics, Challenges, and Opportunities

Maaran M. Rajah,† Ryan D. Pardy,† Stephanie A. Condotta,† Martin J. Richer,*,†,‡
and Selena M. Sagan*,†,‡
†
Department of Microbiology and Immunology and ‡Microbiome and Disease Tolerance Centre (MDTC), McGill University,
Montréal, Québec, Canada H3A 2B4

ABSTRACT: Zika virus (ZIKV) is an emerging arthropod-

borne pathogen that has recently gained notoriety due to its
rapid and ongoing geographic expansion and its novel
association with neurological complications. Reports of
ZIKV-associated Guillain−Barré syndrome as well as fetal
microcephaly place emphasis on the need to develop
preventative measures and therapeutics to combat ZIKV
infection. Thus, it is imperative that models to study ZIKV
replication and pathogenesis and the immune response are
developed in conjunction with integrated vector control
strategies to mount an efficient response to the pandemic.
This paper summarizes the current state of knowledge on
ZIKV, including the clinical features, phylogenetic analyses, pathogenesis, and the immune response to infection. Potential
challenges in developing diagnostic tools, treatment, and prevention strategies are also discussed.
KEYWORDS: Zika virus, Guillain−Barré syndrome, fetal microcephaly

■ INTRODUCTION
Zika virus (ZIKV) is an emerging mosquito-borne pathogen
that has recently become a significant global health concern due
to its rapid geographical expansion and pathogenesis associated
with infection. The virus was initially described in 1947 in the
Zika forest region of Uganda, where it was isolated from the
blood of sentinel rhesus macaques.1 Serological surveys
conducted in West Africa demonstrated the presence of
ZIKV antibodies in human populations; however, the first
human infection was not reported until 1964.2,3 For the latter
half of the 20th century the virus remained in relative obscurity
with only isolated human cases reported until the first serious
outbreak in 2007.4 During this outbreak, over 73% of the
population of Yap Island, Federated States of Micronesia,
became infected with ZIKV in a period of 4 months.5,6 This was
followed by a major outbreak in 2013 in French Polynesia;
together, these outbreaks represented the first significant
transmission of ZIKV outside its original endemic regions.6
Since then, ZIKV has been introduced into the Western
Hemisphere, causing an ongoing epidemic in South America,
with localized epidemics in Argentina, Colombia, Brazil, El
Salvador, Guatemala, Paraguay, and Venezuela, as well as recent
outbreaks in the southern United States and Singapore.6−8
Although ZIKV typically causes a mild, self-limiting febrile
illness, its explosive spread across South America and its recent
association with more severe pathogenesis make ZIKV
infection a serious public health concern that must be rapidly
addressed through a concerted collaborative effort between
researchers, clinicians, and public health officials alike.
■ TRANSMISSION AND CLINICAL FEATURES OF
ZIKV INFECTION
ZIKV is an arbovirus that belongs to the Flavivirus genus of the
Flaviviridae family. Viral transmission occurs through both
sylvatic and urban cycles involving a variety of Aedes species
mosquitos and either nonhuman primates or humans as the
amplifying reservoir, respectively. It is not yet clear whether
other mammals or mosquito species can serve as an amplifying
reservoir, but viral replication in cell culture has been
demonstrated in a variety of animal cell lines.9 Recent papers
have also described ZIKV replication in testicular tissue and
excretion in semen, suggesting that sexual transmission is also a
route of ZIKV infection.7,10−12 Although transmission of ZIKV
through breast milk has not been documented, it has been
demonstrated to contain high viral loads, suggesting this may
represent another potential route of transmission.13,14 A deeper
understanding of the amplifying reservoirs for ZIKV and routes
of transmission will be critical to contain the ongoing epidemic
and prevent further spread.
ZIKV replication is first thought to occur in human primary
dermal fibroblasts, epidermal keratinocytes, and immature
dendritic cells at the site of inoculation.15 From the site of
the mosquito bite, ZIKV spreads to the draining lymph node,
where it is amplified and disseminated through the bloodstream
to peripheral tissues and visceral organs. ZIKV is first detectable
in the blood within the first 10 days of infection, with peak viral
Special Issue: Host-Pathogen Interactions
Received: September 14, 2016
Published: October 5, 2016

© 2016 American Chemical Society 763 DOI: 10.1021/acsinfecdis.6b00161

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Figure 1. Phylogenetic analysis of ZIKV. Translated amino acid sequences of 94 ZIKV polyproteins were aligned using ClustalW. Trees were
constructed by neighbor joining of pairwise amino acid distances with the program MEGA7 (according to the distance scale provided). Bootstrap
resampling was used to determine robustness of branches; values of ≥50% (from 1000 replicates) are shown. Human (black circles), monkey (gray
circles), and mosquito (open circles) isolates are indicated. Isolate name, country of origin, and year of isolation, as well as the unique accession
numbers for each sequence, are indicated.

loads coinciding with the onset of symptoms, which typically infection has been reported to cause a self-limiting illness that is
present between 2 and 12 days postinfection.16−18 ZIKV mostly asymptomatic, but can present with mild symptoms in
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up to 20% of cases.19 These symptoms typically include fever,
maculopapular rash, headache, joint and muscle pain, fatigue,
and conjunctivitis.5,6,16 During the Yap Island outbreak, other
symptoms including myalgia, headaches, retro-orbital pain,
edema, and vomiting were also reported.5 The presentation of a
mild febrile illness can be misdiagnosed as Dengue or
Chikungunya virus infection due to their similar clinical
presentation.20 In areas of co-circulation of these pathogens,
multiple infections are likely very common;21,22 however, it
remains to be seen whether there are synergistic effects due to
co-infection, vaccination, or past infections.23−25 In the recent
outbreaks, beginning with the 2013 French Polynesia outbreak,
ZIKV has been linked to an increase in more severe
neurological complications including Guillain−Barré syndrome
and fetal microcephaly. The factors contributing to this sudden
rise in ZIKV-associated neurological symptoms currently
remain unexplained, and research efforts are directed at
identifying host, pathogen, and environmental factors linked
to these complications.
Guillain−Barré Syndrome. Guillain−Barré syndrome is a
severe neurological autoimmune disease causing acute or
subacute flaccid paralysis that is attributable to peripheral
nerve damage.26,27 During the French Polynesia outbreak, there
were 48 reported cases of Guillain−Barré syndrome, and 88%
of the affected patients reported a symptomatic ZIKV infection
prior to the onset of neurological symptoms; this was
comparatively higher than the 5 cases per annum reported
over the previous 4 years.27 A case-controlled study on 42 of
these patients demonstrated that all of them had neutralizing
antibodies directed against ZIKV.27 Although none of these
patients died, 50% of them were still unable to walk without
assistance 3 months after discharge. A similar pattern was
observed in the current epidemic in Latin America and the
Caribbean, where the incidence of Guillain−Barré syndrome in
seven different countries was reported to be 2−9.8-fold higher
than baseline.28 Although fatalities among Guillain−Barré
syndrome patients are rare, the severity and burden of the
disease on the health care system as well as to the affected
patients and their families suggest that research is needed to
better understand the underlying pathology and improve
diagnosis and treatment.26
Fetal Microcephaly. One of the most alarming aspects of
the recent outbreaks has been the association of ZIKV infection
with an increase in congenital malformations including fetal
microcephaly (a neurodevelopmental disorder that results in
malformation of the brain and head), which can result in severe
life-long limitations for the child and family.29 Reports from the
Brazilian Ministry of Health suggest that there is a 20-fold
increase in the incidence of microcephaly that coincides with
the current ZIKV epidemic.30 Recent studies have strongly
linked ZIKV infection during pregnancy to microcephaly,
including the detection of viral RNA in amniotic fluid, ZIKV-
specific IgM antibodies in the cerebrospinal fluid of micro-
cephalic neonates (indicative of active central nervous system
infection), and reports that ZIKV attenuates growth of human
neural progenitor cells.6,31−33 Although questions remain about
the type of exposure and whether symptomatic or asympto-
matic infection poses the greatest risk to the fetus, the first
trimester of pregnancy has been identified as the gestational
period at major risk for microcephaly.34,35 This is further
supported by a recent study that demonstrated ZIKV-induced
apoptosis in first trimester human trophoblasts as well as
detrimental effects on trophoblast differentiation.36 In addition
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to microcephaly, a number of other malformations have been
reported in fetuses and newborns with congenital ZIKV
infection, including intrauterine growth restriction, brain
atrophy, cerebral and placental calcifications, arthrogryposis,
and retinal and optic nerve abnormalities.37−40 Although recent
research has started to decipher some of the mechanisms
through which ZIKV can lead to fetal abnormalities, a
concerted effort is required to further understand all of the
factors associated with ZIKV-induced pathogenesis. Success will
be dependent on a collaborative multidisciplinary research to
assemble epidemiological data along with the establishment of
models to study host−pathogen interactions that ultimately
shape ZIKV pathogenesis.
■ ZIKV GENOME AND PHYLOGENETIC ANALYSES
Similar to other clinically relevant human pathogens belonging
to this family (e.g., yellow fever, West Nile, and Dengue
viruses), ZIKV is a single-stranded, positive-sense RNA virus
with a single open reading frame flanked by highly structured 5′
and 3′ untranslated regions (UTRs). The genome encodes a
single polyprotein that is cleaved by host and viral proteases
into the structural [capsid (C), premembrane protein (prM),
and envelope (E)] and nonstructural proteins (NS1, NS2A,
NS2B, NS3, NS4A, 2K, NS4B, and NS5).19,41,42 The structural
proteins form the virion particle, whereas the nonstructural
proteins participate in polyprotein processing, viral RNA
replication, virion assembly, and evasion of the host immune
response.
Phylogenetic analyses have established that there are two
main ZIKV lineages, African and Asian (Figure 1).41−43
Notably, all of the contemporary human strains have greater
sequence homology to the mosquito strain P6-740 (Malaysia,
1966) than the African strains, suggesting that the currently
circulating strains evolved from the Asian lineage, anchored by
P6-740.42,43 In addition, all of the strains identified in the
2015−2016 epidemic are more closely related to the H/PF/
2013 French Polynesia strain than the FSM/2007 Micronesia
strain, suggesting that these sublineages may have evolved
independently from a common ancestor (Figure 1). Interest-
ingly, the current ZIKV outbreak in Singapore is of the Asian
lineage, but sequence analyses suggest that it evolved
independently from a strain that was already circulating in
Southeast Asia, rather than being derived from an imported
case from the ongoing South American outbreak. Comparative
analyses of historical ZIKV strains in both in vitro and in vivo
models is likely to answer important questions regarding the
emergence and pathogenesis of ZIKV during the current
outbreaks.
Comparative genetic analyses suggest that there are several
amino acid polymorphisms that are common to the Asian
lineage isolates with known clinical outcomes when compared
with the African (MR766, CDC reference strain) and P6-740
strains.42,43 The recent acquisition of these amino acid
polymorphisms could potentially contribute to the increased
pathogenesis and dissemination seen in the current outbreaks.
On the basis of their locations within the ZIKV polyprotein,
these mutations are predicted to contribute to ZIKV infection,
replication, pathogenesis, and the immune response to
infection.42,43 Amino acid polymorphisms in the prM, E, and
NS1 proteins may contribute to infectivity and the immune
response, whereas those in NS2A and NS3 are likely to affect
polyprotein processing and replication. Interestingly, the NS4B
and NS5 proteins contain large clusters of amino acid
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polymorphisms.42,43 This may be of particular interest because
NS4B is implicated in replication, pathogenesis, and antiviral
signaling, whereas the NS5 protein is the viral RNA-dependent
RNA polymerase responsible for viral RNA synthesis.42,43
Although it is clear that ZIKV has undergone significant
evolution since its discovery, little is known about how these
recently acquired nucleotide and amino acid polymorphisms
have contributed to the increased pathogenesis and rapid
dissemination of ZIKV in the current outbreaks.42,43 These
studies have been hampered by the lack of ZIKV reverse
genetics systems and suitable small animal models (discussed in
more detail below).
■ UNDERSTANDING ZIKV PATHOGENESIS AND THE
IMMUNE RESPONSE TO INFECTION
Most of our knowledge of the ZIKV life cycle has been
extrapolated from our knowledge of related flaviviruses. As
such, much remains to be determined regarding ZIKV
replication, cell tropism, transplacental transmission, patho-
genesis and the immune response to infection. Furthermore, it
is unclear what factors have contributed to the explosive spread
and increased virulence of ZIKV in the recent and ongoing
epidemics. The development of genetic tools and experimental
systems, including mosquito transmission models, reverse
genetics systems, and animal models, will be crucial to improve
our understanding of ZIKV transmission, replication and
pathogenesis and the immune response to infection.
Furthermore, these experimental systems will be critical to
facilitate the development of novel vaccine and therapeutic
strategies.
Cell Culture Systems. In vitro and ex vivo models are
useful for dissecting the molecular mechanisms of viral
infection, fitness, replication, and host−virus interactions. To
date, in vitro studies have been focused on determining suitable
cell types for ZIKV infection and replication as well as those
involved in transplacental transmission and neural damage to
investigate ZIKV pathogenesis. The link between ZIKV
infection and fetal microcephaly has been demonstrated by
detection of viral RNA in the placenta, amniotic fluid, and
brain, as well as ZIKV IgM-specific antibodies in the
cerebrospinal fluid of microcephalic neonates.6,29,31−33,44
Recent studies suggest that ZIKV is able to infect and replicate
in primary human placental cells (cytotrophoblasts, endothelial
cells, fibroblasts, and Hofbauer cells) from mid to late gestation
as well as villus explants from first-trimester human placentas,
suggesting two possible routes of ZIKV transmission to the
fetus (placental and paraplacental).45,46 Furthermore, ZIKV
replication in Hofbauer cells (placental macrophages) was
associated with induction of type I interferons (IFN), pro-
inflammatory cytokines, and antiviral gene expression, but
minimal cytopathic effects, suggesting that these cells may allow
ZIKV to gain access to the fetal compartment and could play a
role in viral dissemination.46 In contrast, primary human
trophoblasts (the barrier cells of the placenta) from full-term
placentas are refractory to ZIKV infection and produce type III
IFN that are postulated to protect trophoblast and non-
trophoblast cells from infection during later stages of
pregnancy.47
Several groups have investigated the link between ZIKV
infection and neural pathogenesis in vitro using human neural
progenitor cells (hNPCs), neurons, and cerebral organoids
(three-dimensional, self-organized, stem-cell-derived models
that recapitulate the first trimester of human neurodevelop-
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ment) generated from human induced pluripotent stem
cells.33,48−50 These studies indicate that ZIKV can infect
hNPCs with high efficiency, resulting in increased cell death
and dysregulation of the cell cycle, as well as impaired growth
and morphogenesis of healthy neurospheres.33 Furthermore,
infected hNPCs release infectious viral particles, presenting a
challenge for the development of therapeutics to halt or block
the impact of infection.33,48−50 Importantly, these findings
suggest a potential mechanism of ZIKV-induced microcephaly
as hNPCs are essential for the development of the cortex and
brain. Whereas these cell culture approaches are of great
importance in understanding ZIKV infectivity, viral fitness,
replication, and antiviral responses, these approaches do not
take into account the complexity of host−pathogen interactions
and need to be complemented by animal models of infection.
Animal Models. The establishment of animal models is
extremely important for understanding ZIKV pathogenesis and
the immune response to infection, as well as for the design and
testing of effective vaccine strategies and therapeutics. Nonhu-
man primates are similar to humans in terms of gestation and
fetal development and therefore may provide important
translational insights. To date, the majority of nonhuman
primate ZIKV studies have focused on viral dissemination and
vaccine protection;51,52 however, a recent study demonstrated
that subcutaneous ZIKV infection of a pregnant pigtail
macaque results in arrested fetal brain development and
neuro-invasion.53 Future studies in these models are likely to
further our understanding of ZIKV fetal pathogenesis.
However, nonhuman primates present a number of logistical
challenges that make them less suitable for certain research
avenues than other small animal models.
The use of the wild-type (WT) C57BL/6 or 129 Sv/Ev
mouse strains to create a model of ZIKV pathogenesis has been
hampered by the apparent lack of infectivity of ZIKV in these
mice. Indeed, it has been shown in vitro that although the ZIKV
NS5 protein is capable of impeding IFN signaling in human
cells by binding to the STAT2 protein, it is unable to do so in
murine cells.54 To address this, the majority of mouse models
have used mice lacking the IFN-α/β receptor (IFNAR;
IFNAR−/− or A129 mice), both IFNAR and the IFN-γ receptor
(IFNGR; AG129 mice), or Irf3−/−Irf5−/−Irf7−/− triple-knock-
out mice that produce limited type I IFN (Table 1).10,55−57
The ability of ZIKV to cause severe pathogenesis and even fatal
infection in many of these models emphasizes the importance
of a robust type I IFN response in the innate immune response
to ZIKV.10,55−57 Analyses of viral dissemination and patho-
genesis within these mice have also highlighted ZIKV’s capacity
to infect and replicate within immune privileged sites such as
the reproductive organs, eyes, and brain, similar to reports in
human patients.6,12,58−61 Mice deficient in IFNAR signaling
have also been used to address the effects of ZIKV infection on
fetal development. After subcutaneous infection of pregnant
IFNAR−/− mice, ZIKV was detectable in both the placenta and
fetal brain, which led to intrauterine growth restriction and fetal
demise.58 Similarly, a recent paper demonstrated that intra-
vaginal infection of IFNAR−/− dams mated with WT males led
to total fetal resorption when infection occurred early in
pregnancy.59 Although structural differences between the
human and mouse placenta must be considered, these studies
provide an important first step in understanding the ability of
ZIKV to transverse the placenta, as well as its tropism for neural
progenitor cells in the developing brain.62 Intriguingly,
intravaginal infection of pregnant WT mice still caused
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Table 1. Murine Models of ZIKV Infection: Opportunities
and Challenges
intrauterine growth restriction in the absence of systemic
viremia.59 Together, these models provide excellent tools for
interrogating ZIKV pathogenesis and will be extremely useful
for testing potential therapeutics.
Despite the challenges associated with infection of WT mice,
there is still an urgent need for immunocompetent mouse
models for ZIKV infection. The lack of an intact type I IFN
response in the models described above precludes in-depth
analyses of the immune response to ZIKV infection. In addition
to a role in innate immune responses, type I IFN plays a critical
role in the induction of an optimal adaptive immune response,
including CD8+ T cells, which are required for clearance of
intracellular pathogens.63 To date, only one immunocompetent
mouse model has been proposed, which used WT SJL mice
(Table 1).50 In this strain, pregnant WT mice infected with a
Brazilian ZIKV isolate gave birth to pups with severe
intrauterine growth restriction, ocular deformities, and reduced
cell number and cortical thickness in the brain (symptoms
associated with microcephaly in humans).50 No such defects
were observed in pregnant WT C57BL/6 mice, suggesting
there may be a genetic influence to ZIKV-induced micro-
cephaly.50 Analyses of the ZIKV immune response in both
C57BL/6 and SJL mice will be of interest to determine specific
differences that dictate the apparent increased susceptibility of
SJL mice to ZIKV infection. In addition, immunocompetent
mouse models will be critical to understand whether newly
acquired viral polymorphisms contribute to the capacity of
ZIKV to counter host immune defenses.
Until recently, both in vitro and in vivo studies of ZIKV have
relied on serial passage of ZIKV isolates in suckling mice or cell
culture. However, these systems are not suitable for the
dissection of viral polymorphisms (limited to those mutations
present in viral isolates) and may result in the selection of viral
mutants (e.g., cell culture adaptations). Understanding the
contribution of viral polymorphisms to the explosive trans-
mission and increased pathogenesis of currently circulating
ZIKV strains has been hampered by the lack of ZIKV reverse
genetics models; however, several groups have now reported
the development of full-length infectious cDNA clones.64−67
These ZIKV reverse genetic systems will allow researchers to
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introduce specific mutations as well as generate reporter viruses
that will facilitate antiviral drug discovery efforts. Together,
these advances will significantly accelerate the capacity of the
research community to understand ZIKV molecular biology,
pathogenesis, and the immune response and will aid in the
development of tools to counter the growing threat of ZIKV
infection.
■ CHALLENGES FOR ZIKV RESEARCH
Vector Control. The concept of mosquito vector control
has been suggested for many years as an approach to control
vector-borne infections. However, many challenges arise that
can hamper the implementation of effective control strategies.68
For example, the mosquito life cycle is a major determinant for
executing successful vector control initiatives. The primary
mosquito vector for ZIKV transmission is Aedes aegypti, and
recently Aedes albopictus has also been implicated in trans-
mission.69−72 Although both of these species belong to the
Aedes genus, they have different habitat and behavioral
characteristics. For instance, Aedes aegypti species bite during
the day, typically rest indoors, and lay their eggs in artificial
containers in and around homes,68 whereas Aedes albopictus
species bite in the early morning and late afternoon, rest
outdoors, and prefer to lay their eggs in natural containers such
as tree holes and ground pools.73 These individualistic features
need to be considered when vector control approaches are
employed; as such, entomological surveillance is imperative for
efficacious vector control initiatives.
Prevention targeted at different mosquito life stages is one
control method that can be easily implemented by individuals
at home and through public health community awareness.
Mosquitoes go through four distinct and separate life stages:
egg, larvae, pupae, and adult. Monitoring areas of mosquito
populations and eliminating breeding grounds is the first step.
Individuals at home can remove standing water in receptacles in
and around the home, which will eliminate mosquito eggs,
larvae, and pupae. Spraying with insecticides will kill adult
mosquitoes, and because of their unique resting preferences,
insecticides should be targeted to the appropriate areas to kill
adult Aedes aegypti and Aedes albopictus, thus ultimately
reducing virus transmission. Another aspect to consider is the
possibility of overwintering and vertical transmission.74
Mosquito eggs can survive dry seasons (desiccation) and cold
periods (diapause), facilitating long-term mosquito survival.75
During the change of seasons (shorter days and colder
temperatures), Aedes albopictus is able to lay diapausing eggs,
ensuring survival until the next hatching period.75 A
consequence of this behavior is that there is a possibility that
ZIKV-infected females could vertically transmit ZIKV to
progeny. In fact, vertical transmission of ZIKV has already
been documented in Aedes aegypti, suggesting a potential
mechanism for virus persistence during adverse conditions.76
Thus, although vector control is an attractive option to reduce
ZIKV transmission, there are a large number of variables that
need to be considered to implement effective control protocols.
Biological control and genetic manipulation represent
another set of tools that can be used for vector control. The
bacterium Wolbachia has been demonstrated to reduce the
ability of mosquitoes to transmit viruses to humans.77,78 When
introduced into Aedes aegypti populations, a symbiotic strain of
Wolbachia reduces ZIKV replication within the mosquito,
delaying virus dissemination to the mosquito salivary glands
and therefore reducing vector competence.77,78 Genetic
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modification of mosquitos is another strategy that has been
proposed. The introduction of the OX513A gene into the
mosquito genome results in normal development of larvae;
however, these genetically modified mosquitos die before
adulthood, potentially reducing the risk of virus trans-
mission.79−82 The possibility of environmental damage and a
general public distrust of genetically modified organisms are
two of the current limitations hindering the widespread
implementation of vector control strategies in ZIKV-affected
areas.83 However, the effectiveness of such strategies has been
demonstrated in recent field studies, where the sustained
release of OX513A Aedes aegypti males successfully reduced the
local Aedes aegypti population by 95% in Bahia, Brazil.84 The
future of vector control will thus require an integrated approach
where basic strategies such as eliminating breeding grounds and
larval source management will need to be implemented in
conjunction with more sophisticated strategies, including
biocontrol and genetic manipulation.83
Diagnostics. Definitive diagnosis of ZIKV infection is
challenging due to the similar symptomology and broad cross-
reactivity of Flavivirus antibodies induced during infection. As
such, reliable diagnosis can be achieved only by laboratory
assays. At present, laboratory diagnosis of ZIKV infection relies
on isolation of the virus in culture, detection of viral RNA in
blood or other biological samples, or serologic testing. Thus, a
major challenge in ZIKV research is the development of
diagnostic assays that can rapidly and definitively diagnose
ZIKV infection.
Although the gold standard to diagnose viral infections is
isolation of the virus in culture, this requires sufficient viral load,
appropriate timing of sample collection, and significant
resources to perform. This renders this approach difficult to
implement in the field. Detection of viral RNA using RT-PCR
is the most widespread diagnostic assay used to detect ZIKV in
blood, urine, saliva, or semen specimens.85−87 RT-PCR-based
assays have the best sensitivity during the first week of
symptom onset while patients are still viremic;87 however, viral
RNA can still be detected in samples several months after the
onset of symptoms.11,87−89
Both in-house and commercial assays have been developed
for serologic testing for ZIKV IgM and IgG antibodies in
serum.85 However, many ZIKV immunoassays have significant
cross-reactivity with heterologous flaviviruses from previous
infections or vaccination.18,85 Positive results from enzyme-
linked immunosorbent assays (ELISA) or immunofluorescence
assays should be confirmed by neutralization assays; however,
in patients with secondary Flavivirus infections, neutralization
assays may not be conclusive due to the induction of broadly
neutralizing antibodies from previous infections.18,85 This is a
particular concern in regions of South America, where other
flaviviruses, such as Dengue virus, are endemic and have
infected the large majority of the population. Thus, the
development of more specific antigens is likely required to
improve serologic diagnosis of ZIKV infection in the future.
Therapeutics. To date, there are no clinically approved
antiviral therapeutics for flaviviruses. However, some of the
experience gained from drug discovery efforts for Dengue virus
and related viruses can be applied to ZIKV. Both viral infection
models and viral enzyme assays will be useful in identifying
ZIKV-specific inhibitors from small-molecule compound
libraries. In fact, nucleotide/nucleoside-based inhibitors, such
as 7-deaza-2′-C methyladenosine, T-705 (favipiravir), and 2′-C-
methylated nucleosides, have already shown promise for
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inhibition of several flaviviruses, including ZIKV, suggesting
that these compounds might represent lead candidates for the
development of specific ZIKV antivirals.90−92 An alternative
pharmacologic strategy is to perform drug-repurposing screens
of existing clinical compounds for their potential anti-ZIKV
activity. This approach has been undertaken for other emerging
viruses, and two such screens have now been reported for
ZIKV.93,94 These studies each screened hundreds of clinically
approved drugs, clinical trial drug candidates, and pharmaco-
logically active compounds that exhibited anti-ZIKV activity or
were neuroprotective in nature.93,94 Several active compounds
were identified in both studies that were further validated in
ZIKV-infected human neural stem cells and primary amnion
cells, demonstrating the efficacy of such screening strategies to
identify lead compounds for anti-ZIKV drug development.
As an alternative to small-molecule-based inhibitors,
therapeutic antibodies could also be developed to prevent or
treat ZIKV infections. Neutralizing antibodies have already
been studied in the context of Dengue virus infection and have
demonstrated reduction in viral loads in a variety of
experimental systems.95 In addition to reducing viremia in
the mother, the ability of IgG antibodies to cross the placenta
suggests that ZIKV-neutralizing antibodies could reach the
fetus, protecting against infection and fetal microcephaly.
However, the use of anti-ZIKV antibodies should be carefully
selected to minimize side effects and potential disease
enhancement.96 No matter the strategy, the major challenge
in the development of ZIKV inhibitors will be the time and
caution required to critically evaluate the efficacy and toxicity
for use in pregnant women, who represent the highest risk
population.
Vaccines. A variety of different vaccine platforms are
currently being explored for ZIKV. These include subunit and
DNA-based vaccines as well as the inactivated or live attenuated
vaccines. A recent study reported efficacy of both plasmid
DNA- and rhesus adenovirus vector-based vaccines, both of
which expressed the ZIKV prM and E proteins, which were able
to successfully confer full protection against ZIKV challenge.51
Although the time to development for a subunit or DNA-based
vaccine is likely to be shorter than inactivated or live attenuated
vaccine strategies, this approach is likely to require multiple
doses to achieve protective immunity. In addition, this
approach is likely to induce only an antibody response, thus
making it more susceptible to being countered by viral
evolution. In contrast, live attenuated approaches typically
elicit both antibody and cell-mediated immunity and are thus
likely to provide more robust protection. In line with this, a
recent study demonstrated that a purified inactivated virus
vaccine was indeed able to confer full protection against the
Brazilian and Puerto Rican strains of ZIKV in rhesus
macaques.51 Although each of these approaches has merit,
given that there are already licensed inactivated or live
attenuated vaccines for several flaviviruses, it is likely that one
of these approaches will have the greatest likelihood of
success.97
The major complications surrounding successful vaccine
development arise from distinct features of ZIKV infection and
include the low incidence of clinical presentation, short period
of viremia, the risk of neurological symptoms, and the risk of
sexual transmission.98 Further complications for vaccine
development could result from the close phylogenetic relation-
ship between ZIKV and other arthropod-borne flaviviruses.99
Pre-existing antibodies to these related viruses could attenuate
DOI: 10.1021/acsinfecdis.6b00161
ACS Infect. Dis. 2016, 2, 763−772
ACS Infectious Diseases
optimal adaptive immune responses and hamper the efficacy of
vaccines in Flavivirus endemic populations and may result in
antibody-dependent enhancement (where cross-reactive anti-
bodies from previous Flavivirus infection or vaccination can
enhance subsequent infections).96,99 Two recent studies
suggest that cross-reactive antibodies produced in response to
prior Dengue virus infection can indeed exacerbate ZIKV
infection.96,100 As such, vaccination efforts must carefully
consider the risk of antibody-dependent enhancement and
assess Flavivirus serostatus in test populations.98
■
SUMMARY AND CONCLUSIONS
From its initial isolation in the mid-20th century in Uganda,
ZIKV remained in relative obscurity until recent outbreaks in
Micronesia (2007), French Polynesia (2013), and Latin
America (ongoing) brought it to the forefront of global public
health consciousness. Whereas ZIKV infection typically
presents as a mild febrile illness, recent epidemics have been
associated with novel pathogenesis including Guillain−Barré
syndrome and fetal microcephaly. The rapid mobilization of the
scientific community has already led to several advances in
understanding the pathogenesis of ZIKV and how this may be
linked to these novel neurological complications. Furthermore,
phylogenetic analyses suggest that viral evolution may have
played a role in the rapid emergence and pathogenesis observed
in the recent outbreaks. Comparative genetic analyses and
reverse genetic models, as well as in vitro and in vivo models,
are likely to provide more insight into how host−pathogen
interactions and viral evolution have shaped ZIKV transmission
and the pathogenesis associated with the current outbreaks. In
addition, this outbreak represents a unique opportunity to gain
further insight into the factors that can influence the capacity of
emerging viruses to establish epidemics.
Despite the rapid mobilization of the scientific community,
there remain a number of outstanding questions that need to be
addressed: Did viral evolution lead to changes that resulted in
increased infectivity and pathogenesis of ZIKV? Are there host
genetic factors associated with the increase in neurological symptoms
such as Guillain−Barré syndrome? Has the prevalence of infection
with heterologous f laviviruses in endemic regions contributed to the
sudden rise in pathogenesis? What are the correlates of immune
protection and are these somehow compromised in pregnant
women? The answers to these questions will be critical for the
development of novel therapeutic and vaccine strategies aimed
at preventing ZIKV infection.
Significant research efforts will be required to curb the
growing global threat posed by ZIKV infection. The rapid
response from the research community has already provided
several in vitro and in vivo models that will help provide a
greater understanding of viral transmission, replication, patho-
genesis, and the immune response to infection. Future research
efforts are thus likely to be focused on implementation of
appropriate vector control strategies and development of novel
diagnostic tools and antivirals, as well as design and testing of
ZIKV vaccine strategies.
■
AUTHOR INFORMATION
Corresponding Authors
*(S.M.S.) Mail: McGill University, 3775 University Street,
Room 608, Montréal, QC, Canada H3A 2B4. E-mail: selena.
sagan@mcgill.ca.
769
Review
*(M.J.R.) Mail: McGill University, 3775 University Street,
Room 406A, Montréal, QC, Canada H3A 2B4. E-mail: martin.j.
richer@mcgill.ca.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by start-up funds from McGill
University (S.M.S. and M.J.R.) as well as operating funds from
the Fonds de Recherche du Québec Nature et Technologies
(S.M.S.). S.M.S. is a Tier II Canada Research Chair in RNA
Biology and Viral Infections. M.J.R. received salary support
from the Fonds de Recherche du Québec Santé − Chercheurs-
Boursiers Junior 1. M.M.R. thanks the McGill University
Faculty of Medicine Max E. Binz Fellowship for graduate
training. R.D.P. thanks the Natural Sciences and Engineering
Research Council of Canada (NSERC) Canada Graduate
Scholarship − Masters (CGS-M) for graduate support.
■
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