93
ZIKA VIRUS IMPAIRS NEUROBEHAVIORAL DEVELOPMENT AND INDUCES
OXIDATIVE STRESS LINKED TO BLOOD–BRAIN BARRIER DISRUPTION IN A
RAT MODEL OF CONGENITAL INFECTION
Kenjayev Baxtiyor
Assistant lecturer at the Alfraganus University
Email address: ismatovbaxtiyor.67890.@gmail.com
ORCID ID: 0000-0003-3259-8492
https://doi.org/10.5281/zenodo.14783854
Introduction
Zika virus (ZIKV) is an arbovirus belonging to the
Flaviviridae
family and the
Flavivirus
genus, which caused a significant epidemic in the Americas in 2015 (Song et al., 2017).
Gestational ZIKV infection was soon linked to neurodevelopmental complications in fetuses,
leading to various adverse outcomes such as intrauterine growth restriction, fetal death,
miscarriage, stillbirth, and ocular abnormalities (Alvarado & Schwartz, 2017; Coyne & Lazear,
2016). Among these, microcephaly emerged as the most prominent clinical manifestation and
is now well-established as a consequence of ZIKV vertical transmission (Wang & Ling, 2016;
Li et al., 2016). While microcephaly was initially considered the primary neurological outcome
of congenital ZIKV infection, recent studies have demonstrated that children affected by ZIKV
in utero, even without microcephaly, may still exhibit neurodevelopmental delays (Sobral da
Silva et al., 2021). Furthermore, growing evidence suggests that ZIKV-induced pathogenesis
extends beyond the immediate postnatal period, with potential long-term effects on
neurological development.
Several models have been developed to explore the pathophysiology of ZIKV infection
and its impact on the nervous system.
In vitro
studies have been instrumental in elucidating
the cellular and molecular mechanisms underlying ZIKV-induced neurological damage. For
instance, studies using U87-MG (human glioblastoma) and HepG2 (human liver carcinoma)
cell lines have revealed that ZIKV infection leads to increased reactive oxygen species (ROS),
lipid peroxidation, and protein carbonylation, alongside a decrease in antioxidant enzyme
activity, including superoxide dismutase and catalase (Almeida et al., 2020). Similarly, human
neural progenitor cells (hNPCs) infected with ZIKV exhibited cell cycle dysregulation and
increased caspase-3 activation, ultimately leading to cell death (Tang et al., 2016). In radial
glial cells (RGCs), ZIKV infection disrupts mitosis and structural organization and leads to the
sequestration of phosphorylated TBK1 during mitosis (Onorati et al., 2016). Given the crucial
role of RGCs in cortical cell migration, these findings help explain the cortical thinning
observed in ZIKV-infected models.
Another significant consequence of ZIKV infection involves damage to the blood–brain
barrier (BBB), a critical structure that regulates the exchange between systemic circulation
and the central nervous system (Clé et al., 2020). Studies conducted
in vitro
and
in vivo
have
demonstrated that ZIKV infection disrupts BBB integrity (Leda et al., 2019). However, it
remains unclear whether these alterations are transient or contribute to long-term
neuropathological outcomes. Additionally, the full spectrum of BBB-related damage induced
by ZIKV infection has yet to be fully elucidated.
Regarding
in vivo
studies, various rodent models have been employed to investigate
ZIKV pathogenicity, fetal infection, and vertical transmission. Since wild-type (WT) rodents
94
exhibit resistance to ZIKV due to their robust interferon (IFN) response, many studies have
relied on IFN-deficient animal models to assess infection-related outcomes (Kublin &
Whitney, 2018). Immunocompromised mice, such as A129 and AG129 strains, have been
instrumental in understanding the impact of vertical transmission (Vue & Tang, 2021).
Additionally, alternative models have explored different routes of infection, including direct
intrauterine exposure to the developing rodent brain (Li et al., 2016) and postnatal infection
shortly after birth (Lazear et al., 2016; Miner et al., 2016). Given the need to better understand
immune responses, long-term consequences, and potential therapeutic interventions, further
research involving immunocompetent animal models is essential (Morrison & Diamond,
2017).
Methods
Animal Procedures
This study utilized pregnant female Wistar rats, approximately two months old,
obtained from the Animal Reproduction and Experimental Center at the Federal University of
Rio Grande do Sul, Brazil. The animals were housed in individually ventilated cages (IVC)
under a 12-hour light/dark cycle, with a controlled temperature of 21 ± 2°C, and provided
with unrestricted access to food and water. All experimental procedures adhered to the
ethical guidelines established by the National Council for Animal Experimentation of Brazil
and complied with Brazilian legislation for the scientific use of animals (Law 11.794/08).
Additionally, procedures followed the
Guide for the Care and Use of Laboratory Animals
as
outlined by the National Research Council (USA, 2011).
Ethical approval for this study was granted by the Ethics Committee of the Federal
University of Rio Grande do Sul (approval number 33452/2017). All procedures were
conducted in a Biosafety Level 2 (BSL-2) laboratory under Animal Biosafety Level 2 (ABSL-2)
conditions, in accordance with the guidelines of the Centers for Disease Control and
Prevention (CDC) for laboratory work involving Zika virus. Virus handling and animal-related
procedures were performed within a Class II Biological Safety Cabinet (BSC) (Tecniplast® BS
60 class II, Buguggiate, Italy) to ensure biosafety compliance.
On embryonic day 9 (E9), pregnant females were randomly assigned to two groups. Five
females received an intraperitoneal injection of 500 µL containing
1 × 10⁶
plaque-forming
units per milliliter (PFU/mL) of Zika virus isolated in Brazil (
ZIKV_BR
). The control group
consisted of six females that received a 500 µL intraperitoneal injection of sterile diluted
medium.
Neurological Reflexes
Twenty-four hours after birth, litter standardization (5–8 pups per litter) and div
measurements were conducted (Table 1). To assess neurodevelopmental impairments
associated with gestational Zika virus infection, a series of neurological reflex evaluations
were performed. These assessments began on postnatal day (PND) 3 and were conducted
every three days until PND 21. Observations included physical characteristics such as div
weight, eye opening, and incisor tooth eruption (Lubics et al., 2005).
The following reflexes and motor responses were evaluated:
Righting Reflex:
Each pup was placed on its back, and the time taken to return to a
prone position with all four paws in contact with the surface was recorded.
95
Negative Geotaxis:
Pups were positioned on an inclined platform (45°) with their heads
facing downward. The day when they were able to turn their heads upward and climb the
platform was noted, with a maximum time limit of 30 seconds to complete the task.
Limb Placing:
The posterior region of the forepaw and hind paw was gently pressed
against a surface, and the day when the pup successfully placed its paws onto the surface was
recorded.
Limb Grasp:
The back of the forelimb paws was lightly touched against a stem, and the
day when the pup grasped the stem was noted.
Cliff Aversion:
Each pup was placed with its head near the edge of an elevated platform,
and the time taken to turn its head away from the edge was measured. Additionally, the first
day this behavior was observed was recorded.
Gait Assessment:
Pups were placed at the center of a 30 cm-diameter circle, and the
time taken to exit the circle was recorded, along with the first day they exhibited locomotor
movement.
Results
Day of Appearance of Neurological Reflexes
To assess the emergence of neurological reflexes, a
t-test
was conducted, comparing
male ZKV (Zika virus-infected) with male CT (control) and female ZKV with female CT. A
significant delay in the onset of specific neurological reflexes was observed in male ZKV
compared to male CT in the following parameters:
Incisor tooth eruption
(t(28) = 5.82; P < 0.05)
Forelimb placing (right)
(t(28) = 4.58; P < 0.05)
Hind limb placing (right)
(t(28) = 5.15; P < 0.05)
Hind limb placing (left)
(t(28) = 3.35; P < 0.05)
Forelimb grasp (right)
(t(28) = 2.15; P < 0.05)
No significant differences were detected in the timing of
eye opening, negative
geotaxis, forelimb placing (left), forelimb grasp (left), gait, aversion to fall, righting
reflex, or olfactory behavior
.
For females, a similar analysis revealed delayed reflex development in the following:
Hind limb placing (right)
(t(13) = 3.10; P < 0.05)
Hind limb placing (left)
(t(13) = 4.56; P < 0.05)
Righting reflex
(t(28) = 2.28; P < 0.05)
No significant differences were found in other assessed reflexes.
Overall, these findings indicate that gestational Zika virus infection resulted in delays in
the appearance of certain neurological reflexes in both male and female offspring. These
delays may serve as early indicators of potential long-term neurological alterations.
Data are presented as
mean ± SD
.
(ZKV)
refers to offspring from Zika virus-infected
mothers, while
(CT)
represents control animals. Sample sizes: Female CT
(n = 8)
, Female ZKV
(n = 7)
, Male CT
(n = 15)
, Male ZKV
(n = 15)
. Asterisks (*) indicate significant differences
compared to the control group of the same sex (Student’s t-test, p < 0.05).
Discussion
In this study, we expanded the understanding of
congenital Zika virus (ZIKV) syndrome
by developing a rat model of congenital ZIKV infection to investigate
neurodevelopmental
96
impairments and brain tissue disturbances
in Wistar rats shortly after birth. The infection was
induced on
embryonic day 9 (E9)
,
a crucial stage for rodent neurodevelopment (Semple et al.,
2013). Pregnant rats received an
intraperitoneal (i.p.) injection
of ZIKV, and viral presence
was detected in maternal blood as early as
six hours post-infection
.
Additionally, viable virus
was found in the
placenta, spleen, and fetuses
within 24 hours post-inoculation. Notably
,
infected females did not exhibit sickness behavior
despite their offspring showing
neurological impairments, which aligns with previous studies (Sherer et al., 2019). This
finding is particularly relevant, given that
asymptomatic ZIKV circulation has been reported
among pregnant women
in northeastern Brazil (Branco et al., 2021). Furthermore, even
asymptomatic maternal ZIKV infection
has been associated with
neurodevelopmental
impairments in offspring
(Shapiro-Mendoza et al., 2017). Consistently, our study identified
significant neurobehavioral deficiencies
in infected pups, suggesting potential
long-term
cognitive and motor disturbances
.
On
postnatal day 22 (PND 22)
,
blood–brain barrier (BBB)
integrity deficits
in the hippocampus and
altered oxidative status
in the hippocampus and
cortex were observed, highlighting the potential for lasting structural and functional brain
damage
.
Conclusion
This study sought to deepen the understanding of the long-term effects of gestational
Zika virus (ZIKV) infection by developing an immunocompetent rat model and identifying
neurobehavioral markers predictive of future disabilities. Our findings reveal that, even in the
absence of brain morphometric changes or a microcephaly-like phenotype, infected offspring
exhibited significant neurodevelopmental impairments. These deficits were associated with
hippocampal and cortical blood–brain barrier (BBB) disruption
and oxidative stress
imbalance observed 22 days postnatally
.
Together, these results provide strong evidence that the impact of gestational ZIKV
infection extends beyond the neonatal period, with potential long-term consequences for
brain function and development
.
Our study contributes to existing knowledge by highlighting
the need for improved prenatal diagnostics, early interventions, and extended follow-up
studies to further elucidate the mechanisms underlying congenital ZIKV syndrome.
Furthermore, the development of a reliable congenital infection model not only enhances our
understanding of disease pathology but also offers new opportunities for advancing
therapeutic strategies
.
References:
1.
J.B. Alimonti, M. Ribecco-Lutkiewicz, C. Sodja, A. Jezierski, D.B. Stanimirovic, Q. Liu, A.S.
Haqqani, W. Conlan, M. Bani-Yaghoub “
Zika virus crosses an in vitro human blood brain barrier
model
Fluids Barrier” CNS
, 15(1) (2018), 10.1186/S12987-018-0100-Y
2.
L.T. Almeida, A.C. Ferraz, C.C. da Silva Caetano, M.B. da Silva Menegatto, A.C. dos Santos
Pereira Andrade, R.L.S. Lima, F.C. Camini, S.H. Pereira, K.Y. da Silva Pereira, B. de Mello Silva,
L.O. Perucci, A. Talvani, J.C. de Magalhães, C.L. de Brito Magalhães
Zika virus induces oxidative
stress and decreases antioxidant enzyme activities in vitro and in vivo
Virus Res.
, 286 (2020), p.
198084
3.
M.G. Alvarado, D.A. Schwartz “
Zika Virus Infection in Pregnancy, Microcephaly, and
Maternal and Fetal Health: What We Think, What We Know, and What We Think We Know
Arch.
97
Pathol. Lab. Med.
,” 141(1) (2017), pp. 26-32, 10.5858/arpa.2016-0382-RA
4.
M.F.V.V. Aragao, A.C. Holanda, A.M. Brainer-Lima, N.C.L. Petribu, M. Castillo, V. van der
Linden, S.C. Serpa, A.G. Tenório, P.T.C. Travassos, M.T. Cordeiro, C. Sarteschi, M.M. Valenca, A.
Costello
Nonmicrocephalic Infants with Congenital Zika Syndrome Suspected Only after
Neuroimaging Evaluation Compared with Those with Microcephaly at Birth and Postnatally:
How Large Is the Zika Virus “Iceberg”?
AJNR Am J Neuroradiol.
, 38(7) (2017), pp. 1427-1434
5.
G. Barisano, A. Montagne, K. Kisler, J.A. Schneider, J.M. Wardlaw, B.V. Zlokovic
Blood–brain barrier link to human cognitive impairment and Alzheimer’s disease
Nat. Cardiovasc. Res.
https://doi.org/10.1038/s44161-021-00014-4
6.
R.C.C. Branco, P. Brasil, J.M.G. Araújo, F.O. Cardoso, Z.S. Batista, V.M.S. Leitão, M.A.C.N. da
Silva, L.O. de Castro, J.G. Valverde, S.M.B. Jeronimo, J.A. Lima, R. Ribeiro da Silva, M.D.C.L.
Barbosa, L.M.O. Brito, M.A.P. Xavier, M.D.D.S.B. Nascimento
Evidence of Zika virus circulation in
asymptomatic pregnant women in Northeast, Brazil
PLoS Negl. Trop. Dis.
, 15(6) (2021),
https://doi.org/10.1371/journal.pntd.0009412
7.
G.J. Burton, E. Jauniaux “
Oxidative stress
Best Pract. Res. Clin. Obstet. Gynaecol.
”25(3)
(2011), p. 287, 10.1016/J.BPOBGYN.2010.10.016
8.
G. Calvet, R.S. Aguiar, A.S. Melo, S.A. Sampaio, I. de Filippis, A. Fabri, E.S. Araujo, P.C. de
Sequeira, M.C. de Mendonca, L. de Oliveira, D.A. Tschoeke, C.G. Schrago, F.L. Thompson, P.
Brasil,
F.B.
dos
Santos,
R.M.
Nogueira,
A.
Tanuri,
A.M.
de
Filippis
“Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in
Brazil: a case study
Lancet Infect. Dis.
,” 16(6) (2016), 653–660,
9.
Ibroximovna, M. S. (2024). FACTORS OF DEVELOPING OF INTERCULTURAL
COMMUNICATION COMPETENCE IN TEACHING ENGLISH TO CADETS OF MILITARY
UNIVERSITY. Лучшие интеллектуальные исследования, 15(1), 159-163.
10.
Musayeva, S. I. (2024, May). DEVELOPMENT OF INTERCULTURAL COMMUNICATION
COMPETENCE OF CADETS USING INTERACTIVE METHODS. In Proceedings of International
Conference on Scientific Research in Natural and Social Sciences (Vol. 3, No. 5, pp. 276-284).
11.
T.F. Cardoso, R.S. dos Santos, R.M. Corrêa, J.V. Campos, R.D.B. Silva, C.C. Tobias, A. Prata-
Barbosa, A.J.L.A. da Cunha, H.C. Ferreira “
Congenital Zika infection: neurology can occur
without microcephaly Arch. Dis. Child.
, 104(2) (2019), pp. 199-200, 10.1136/ARCHDISCHILD-
2018-314782
12.
M. Clé, C. Desmetz, J. Barthelemy, M.-F. Martin, O. Constant, G. Maarifi, V. Foulongne, K.
Bolloré, Y. Glasson, F. De Bock, M. Blaquiere, L. Dehouck, N. Pirot, E. Tuaillon, S. Nisole, F.
Najioullah, P. Van de Perre, A. Cabié, N. Marchi, F. Gosselet, Y. Simonin, S. Salinas, M.S.
Diamond “
Zika Virus Infection Promotes Local Inflammation, Cell Adhesion Molecule
Upregulation, and Leukocyte Recruitment at the Blood-Brain Barrier
MBio
,” 11(4) (2020),
10.1128/MBIO.01183-20
