Zika virus (ZIKV) is a small enveloped positive-sense single-stranded RNA virus belonging
to the genus Flavivirus of the Flaviviridae family that has reemerged in recent years
as a human pathogen with epidemic potential. To date, the ongoing epidemic in the
Americas has led to tens of thousands of confirmed cases in Brazil alone (Pan American
Health Organization Zika-Epidemiological Report, 2 March 2017). More concerning are
the reports of severe neurological disorders such as Guillain-Barré syndrome and congenital
Zika syndrome (microcephaly and other neurodevelopmental defects), which led the World
Health Organization to declare the ZIKV epidemic as a Public Health Emergency of International
Concern in 2016.
Phylogenetic analyses show that ZIKV can be classified into 2 main lineages: the African
lineage and the Asian lineage, the latter of which is responsible for the recent epidemics.
Because recent ZIKV infections were associated with the development of congenital
and neurological disorders, a key question was raised as to whether Asian-lineage
ZIKV strains were phenotypically different from the African lineage strains. It is
well described that mutations acquired during Flavivirus evolution can alter their
virulence and/or tropism [1]. ZIKV is primarily transmitted by the mosquito species
Aedes aegypti and Aedes albopictus. Several studies show that vector transmission
can differ between ZIKV strains, as the overall ZIKV infection prevalence and transmission
rates of African strains may be higher in A. aegypti than Asian strains [2], suggesting
that viral adaptation may have occurred—similar to a single mutation in the chikungunya
virus envelope protein that affected vector specificity and epidemic potential.
During the recent outbreak, it became clear that ZIKV is sometimes able to cause a
prolonged infection. Numerous studies showed that Asian-lineage strains isolated in
South America replicate at low levels in tissues months after the initial infection.
For example, viral genome can be found in stillborn babies who were infected early
during gestation [3], weeks after initial infection in sperm, and in rhesus monkeys,
ZIKV can be found in the cerebral spinal fluid (CSF) 42 days after infection, weeks
after the virus was cleared from the blood [4]. However, to date, no data are available
on the ability of African-lineage strains to cause prolonged infections, but early
studies in 2016 suggested that an African ZIKV strain was highly pathogenic and led
to cell death; when the first reports on a potential link between microcephaly and
ZIKV emerged, laboratories started to study the effect of ZIKV on human neural precursor
cells (hNPCs)/human neural stem cells (hNSCs) with MR766, the most available strain
at the time (Fig 1 and Table 1). These first studies showed strong tropism and deleterious
effects on NSC homeostasis and growth (e.g., [5]). However, some inconsistencies between
this rather strong virulence and the long-term developmental effects associated with
ZIKV infections led researchers to question the biological validity of this strain,
which has been passaged many times in animals and in cells and acquired mutations
and deletions at multiple positions during passaging (Table 1) [6–8]. The relevance
of this strain was also questioned in a study that showed reduced toxicity and long-term
persistence in human neural progenitors of an Asian-lineage ZIKV strain from Puerto
Rico, which seemed more consistent with clinical manifestations [9]. Consequently,
we compared the neurovirulence ex vivo of 2 low-passaged African- and Asian-lineage
ZIKV strains [6]. The African ArB41644 strain was isolated in Central African Republic
in 1989, whereas the Asian H/PF/2013 strain was isolated in 2013 during the French
Polynesian epidemic (Table 1). We showed that the African ZIKV strain displayed a
higher infectivity and replication rate in hNSCs than the Asian ZIKV strain. ArB41644
triggered more defects in proliferation and more apoptosis, which correlated with
a stronger induction of the antiviral response [6]. In a subsequent study, we compared
2 Asian-lineage ZIKV strains (H/PF/2013 and ZIKVNL00013, isolated during the current
epidemic in 2015) with 2 African-lineage ZIKV strains (MR766 and Uganda 927) in human
neural progenitor cells and confirmed the phenotypic differences between ZIKV strains
from the Asian and African lineages [10]. Both African strains infected more cells,
replicated to higher titers, and induced early cell death more frequently than the
Asian-lineage ZIKV strains [10]. The milder virulence of the Asian strains in these
studies could be consistent with a persistent infection and effect of the Asian ZIKV
lineage in developing neural cells. In addition, in dendritic cells, infection by
African (MR766 and Dakar-1984) and Asian (PR-2015) strains led also to a difference
in pathogenicity, as African strains triggered more cellular death [8]. Notably, this
study also showed that despite differences in viral replication rates between the
extensively passaged MR766 strain and the more recently isolated Dakar-1984 strain,
the inductions of cell death were similar, suggesting that the ability to cause cell
death seen in various cellular models with MR766 could still in certain circumstances
be representative for African ZIKV lineage strains [8].
10.1371/journal.pntd.0005821.g001
Fig 1
Phylogenetic analyses of ZIKV strains discussed.
10.1371/journal.pntd.0005821.t001
Table 1
Passage history of ZIKV strains.
Lineage
Strain name
GenBank
Country of origin
Year
Reference
Passage histories
African
MR766
NC_012532; HQ234498.1; AY632535
Uganda
1947
[8]
Unknown + 1x Vero
[11]
Not indicated
[12]
Not indicated
[13]
Not indicated
[10]
Not indicated
[14]
Not indicated
[15]
Not indicated
[16]
Not indicated
[17]
Not indicated
[18]
146x SM, 1x C6/36, 1x Vero
[19]
146x SM, 3x Vero
[20]
Not indicated
[21]
Not indicated
Uganda 976
Uganda
1962
[10]
6x Vero
P6-740
HQ234499.1
Malaysia
1966
[8]
Unknown + 1x Vero
[18]
6x SM, 1x BHK, 1x C6/36, 3x Vero
IbH30656
HQ234500
Nigeria
1968
[16]
Not indicated
Dakar41519
HQ234501
Senegal
1984
[14]
Not indicated
[18]
1x AP61, 1x C6/36, 3x Vero
Dakar 41671
Senegal
1984
[14]
Not indicated
Dakar 41677
Senegal
1984
[14]
Not indicated
DakAr41524
KX198134; KY348860; KX601166
Senegal
1984
[8]
Unknown + 1x Vero
DakAr41525
KU955591
Senegal
1985
[22]
5x Vero, 1x C6/36, 2x Vero
[2]
1x AP61, 1x C6/36
ArB41644
Central African Republic
1989
[10]
5x Vero
[6]
5x Vero
HD78788
KF383039
Senegal
1991
[13]
Not indicated
MP1751
Uganda
1962
[23]
3x SM,4x unknown methods, 1x Vero
Asian
FSS13025
KU955593; JN860885.1
Cambodia
2010
[15]
Not indicated
[22]
1x AP-1, 1x C6/36, 5x Vero
[2]
1x Vero, 1x C6/36
[17]
Not indicated
[18]
5x Vero
[21]
Not indicated
H/PF/2013
KX369547; KJ776791
French Polynesia
2013
[12]
Not indicated
[10]
4x Vero
[14]
Not indicated
[24]
6x C6/36
[6]
5x Vero
[20]
Limited passages in Vero
BeH815744
KU365780
Brazil
2015
[24]
3x C6/36
BeH819015
KU365778.1
Brazil
2015
[20]
Not indicated
FB-GWUH-2016
KU870645
Guatemala
2015
[12]
Not indicated
FLR
KU820897; KX087102
Colombia
2015
[16]
Not indicated
Mex1-7
Mexico
2015
[22]
3x Vero
[2]
4x Vero
PRVABC59
KU501215; KU501215.1
Puerto Rico
2015
[8]
Unknown + 1x Vero
[23]
4x Vero
[15]
Not indicated
[16]
Not indicated
[18]
3x Vero
[19]
ZIKVBR
Brazil
2015
[11]
Not indicated
ZKV2015
KU497555
Brazil
2015
[4]
Not indicated
ZIKVNL00013
KU937936
Surinam
2016
[10]
4x Vero
GZ01
KU820898
Venezuela
2016
[15]
Not indicated
SZ01
KU866423
Samoa
2016
[16]
Not indicated
Abbreviations: BHK, baby hamster kidney, SM suckling mice
Since infection with ZIKV strains from the Asian lineage is associated with microcephaly
in humans, it is likely that ZIKV crosses the placental barrier early in gestation,
when the brain is starting to develop. Some studies showed that cells of the placenta
are susceptible to ZIKV infection, in line with the congenital infections and neurological
symptoms associated. Because infections early in gestation seem to be more linked
to microcephaly [25], it was hypothesized that the placenta vulnerability could differ
depending on the gestational stage. While the placental syncytiotrophoblast, which
is found in mature placenta, appears mostly resistant to ZIKV, a study that compared
an African strain (MR766) to an Asian strain (FSS13025, Cambodia) showed that embryonic
stem cell (ESC)-derived trophoblasts, which recapitulate primitive placenta cells
during implantation, are highly susceptible to ZIKV infection [17]. Interestingly,
infection with the African strain again resulted in higher viral replication and cell
lysis. These data might indicate that infection with African-lineage ZIKV strains—if
they enter the fetus—could result in an early termination of pregnancy, while infection
with an Asian-lineage ZIKV strain would be less destructive and more chronic, thereby
allowing the pregnancy to continue, leading to the development of congenital malformations
[17].
While these ex vivo data suggested that African ZIKV lineage strains are more virulent,
it was important to confirm the difference in (neuro)virulence of these 2 lineages
in vivo. Interferon-α/β receptor (Ifnar)-/- mice injected subcutaneously with African
ZIKV strains (MR766 and 3 strains from Senegal) or with the Asian-lineage H/PF/2013
strain all presented with weight loss and succumbed to the infection after 6 (for
Senegal strains) to 10 (H/PF/2013) days. Interestingly, infection with the African-lineage
strain MR766 resulted in 20% survival at 2 weeks [14]. Another study compared the
symptoms induced by African (Uganda and Senegal) and Asian (Cambodia, Malaysia, and
Puerto Rico) ZIKV strains in signal transducer and activator of transcription (Stat2)-/-
and Ifnar
-/- mice infected subcutaneously [18]. Importantly, infections with African strains
resulted in more weight loss, mortality, and severe prolonged neurological symptoms
compared to Asian strains. Moreover, induction of type 1 and 2 interferon (IFN) were
higher following infection with African strains associated with enhanced levels of
inflammatory cytokines such as interleukin 6 (IL6) or tumor necrosis factor (TNF).
These observations were confirmed in another study showing that an African strain
(MP1751) is pathogenic in A129 mice, contrary to an Asian strain (PRVABC59) that did
not cause signs of illness [26]. These studies confirm the ex vivo results in neural
and nonneural cells regarding the higher virulence of African strains and could suggest
that different ZIKV strains may also display phenotypic differences in human subjects
(Fig 1) (Table 2). Many of the findings from immunocompromised mice have been recapitulated
in nonhuman primates, such as rhesus and cynomolgus macaques. The development of these
different animal models led to new knowledge concerning the pathogenesis of ZIKV.
However, these systems have limitations, in particular, studying viral pathogenesis
using animals lacking a key component of antiviral immunity, which makes it still
difficult to derive definitive conclusions regarding ZIKV virulence in humans. Therefore,
many unanswered questions remain, in particular, regarding the mechanisms of host
restriction, immune evasion, and inflammatory response as well as the long-term neurodevelopmental
implications of congenital infection in humans.
10.1371/journal.pntd.0005821.t002
Table 2
Comparative analyses of ZIKV African and Asian lineages.
Model
Lineage: Strain
Findings
Reference
Ex vivo
Endothelial cells
African: MR766, IbH30656; Asian: PRVABC59, FLR
Asian-lineage strains replicate faster and isolates induced significant cell death
compared to African-lineage strains.
[16]
Astrocytes
African: HD78788, ArB41644; Asian: H/PF/2013
African-lineage strains led to a higher infection rate and viral production infection.
Asian-lineage strain led to the expression of these innate immune response genes early,
while their induction by the African strain was delayed.
[6][13]
Neuronal stem cells
African: DakAr41525, MR766, ArB41644, Uganda 976; Asian: H/FP/2013, Mex1-7, FSS13025,
FB-GWUH-2016, ZIKVNL00013
African-lineage strains led to a higher infection rate and virus production as well
as stronger cell death and antiviral response compared to Asian-lineage strains. Protein
53 (p53) plays an important role in apoptosis induced by Asian-lineage strains but
not by African-lineage strains.
[22][12][6].[21][10]
Cerebral organoids
African: MR766; Asian: ZIKVBR
Asian-lineage strain led to a higher viral production as well as stronger cell death
compared to the African strain.
[11]
Embryonic stem cell–derived trophoblast
African: MR766; Asian: FSS13025
African-lineage strain led to a higher viral production as well as stronger cell death
compared to Asian-lineage strains.
[17]
Monocyte-derived dendritic cells
African: DakAr41524, P6-740, MR766; Asian: PRVABC59
African-lineage strains led to a higher infection rate and viral production as well
as stronger cell death and antiviral response compared to Asian-lineage strains.
[8]
Neuronal cell lines (SK-N-SH, U87 MG)
African: MR766, Uganda 976; Asian: H/FP/2013, ZIKVNL00013
African-lineage strains led to a higher infection rate and virus production.
[10]
In Vivo
Mosquito: Aedes aegypti
African: DAKAR41525, MR766; Asian: MEX1–7, PRVABC59, GZ01, FSS13025, H/PF/2013, BeH815744
Infection prevalence and transmission rate of African-lineage strains is higher compared
to Asian-lineage strains. Infection prevalence and transmission rate of Asian-lineage
strains from the Americas are higher compared to Asian-lineage strains from Asia.
[15][24][2][19]
Mouse: A129
African: MR766, P6-740, DAKAR41519,41667,41671,MP1751; Asian: H/PF/2013, PRVABC59,
FSS13025, BeH819015, PRVABC59
African lineage is pathogenic whereas Asian lineage does not cause sign of illness.
[23]
Ifnar1
-/-
Irf3
-/-
Irf5
-/-
Irf7
-/-
African-lineage strains induced higher mortality and severe neurological symptoms
in a short period as compared to Asian-lineage strains.
[14]
Stat2
-/-
Ifnar1
-/-
Among Asian-lineage strains, the Cambodia strain presented more severe symptoms including
front limb paralysis and lethality, followed by the Malaysia and Puerto Rico strains.
The Brazilian strain is least severe. The Uganda strain showed orders of magnitude
higher viral RNA level compared to all other strains including Senegal, especially
in brain tissue.
[18]
Ifngr1
-/-
Higher levels of interferon type I and type II and inflammatory cytokine induction
by African-lineage strains as compared to the Asian-lineage strains.
[20]
Nonhuman primate
Asian: ZKV2015, PRVABC59
No differences observed
[4]
However, one major challenge in interpreting and comparing the data from these studies
is the lack of harmonization of the virus strains used. In this review alone, in which
we focused on studies that compared multiple ZIKV strains, at least 12 African and
13 Asian strains were found (Fig 1, Tables 1 and 2). While most studies include the
African lineage MR766 strain, this strain cannot be considered as a standard, as it
has been extensively passaged and at least 3 MR766 strains exist with genetic differences,
including a 4–6 codon deletion in the envelope (E) protein [7]. For other strains,
multiple sequences exist, or in some cases, no sequence data are available (Table
1). Therefore, when comparing ZIKV lineages, the strains should be sequenced and experiments
more standardized to allow proper comparison between studies. Ideally, more recent
African ZIKV strains should become available (as the most recent isolate so far is
from 1991) (Table 1).
In conclusion, the data reviewed here indicate that there are intrinsic differences
in the pathogenicity/virulence of African- and Asian-lineage ZIKV strains. Whether
these differences are responsible for differences in clinical presentations should
be confirmed, but one can speculate that the phenotype of Asian ZIKV strains (lower
infection rate, less virus production, and poor induction of early cell death) might
contribute, at least in part, to the ability to cause persistent infections within
the CNS of fetuses, while African-lineage ZIKV strains could result in more acute
infection. However, even though ex vivo and in vivo data point towards a stronger
virulence for African strains, it is still premature to conclude that if an African
epidemic declares itself (due to an African ZIKV strain), neurological symptoms with
similar or worse gravity than those observed in South America will be found. If confirmed,
recent reports of African ZIKV strain infection and potential microcephaly in Guinea
Bissau may be proof of what could be expected in this continent [27]. Determining
the virulence factors for ZIKV using reverse genetic systems that have now been developed
for both African and Asian strains will be crucial to identify the molecular and cellular
mechanisms behind differences in ZIKV pathogenicity and also those of other emerging
arboviruses.