(PDF) Aedes aegypti Shows Increased Susceptibility to Zika Virus via Both In Vitro and In Vivo Models of Type II Diabetes
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Aedes aegypti Shows Increased Susceptibility to Zika Virus via Both In Vitro and In Vivo Models of Type II Diabetes
Scott Weaver
2022, Viruses
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Abstract
Chronic conditions like type II diabetes (T2DM) have long been known to exacerbate many infectious diseases. For many arboviruses, including Zika virus (ZIKV), severe outcomes, morbidity and mortality usually only occur in patients with such pre-existing conditions. However, the effects of T2DM and other pre-existing conditions on human blood (e.g., hypo/hyperinsulinemia, hyperglycemia and hyperlipidemia) that may impact infectivity of arboviruses for vectors is largely unexplored. We investigated whether the susceptibility of Aedes aegypti mosquitoes was affected when the mosquitoes fed on “diabetic” bloodmeals, such as bloodmeals composed of artificially glycosylated erythrocytes or those from viremic, diabetic mice (LEPRDB/DB). Increasing glycosylation of erythrocytes from hemoglobin A1c (HgbA1c) values of 5.5–5.9 to 6.2 increased the infection rate of a Galveston, Texas strain of Ae. aegypti to ZIKV strain PRVABC59 at a bloodmeal titer of 4.14 log10 FFU/mL from 0.0 to 40.9 and 4...
Key takeaways
AI
Aedes aegypti exhibits increased susceptibility to Zika virus when fed on blood with diabetic characteristics.
HbA1c levels between 5.5% and 6.2% significantly enhance ZIKV infection rates in Ae. aegypti.
IFNAR blockade in LEPR DB/DB mice resulted in peak ZIKV titers of 4.4 log10 FFU/mL.
Incorporating mammalian factors influenced by chronic conditions is crucial for vector competence studies.
The study highlights the epidemiological implications of chronic conditions on arbovirus transmission dynamics.
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viruses
Article
Aedes aegypti Shows Increased Susceptibility to Zika Virus via
Both In Vitro and In Vivo Models of Type II Diabetes
Sasha R. Azar 1 , Rafael K. Campos 1,2 , Ruimei Yun 2 , Taylor Strange 1 , Shannan L. Rossi 1,3,4,5 ,
Kathryn A. Hanley 6 , Nikos Vasilakis 1,3,4,5,7,8,9, * and Scott C. Weaver 2,4,5,7,8, *
1 Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555-0609, USA;
[email protected]
(S.R.A.);
[email protected]
(R.K.C.);
[email protected]
(T.S.);
[email protected]
(S.L.R.)
2 Department of Microbiology and Immunology, The University of Texas Medical Branch,
Galveston, TX 77555-0610, USA;
[email protected]
3 Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch,
Galveston, TX 77555-0609, USA
4 Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX 77555-0609, USA
5 Institute for Human Infection and Immunity, University of Texas Medical Branch,
Galveston, TX 77555-0610, USA
6 Department of Biology, New Mexico State University, Las Cruces, NM 88003, USA;
[email protected]
7 World Reference Center for Emerging Viruses and Arboviruses, University of Texas Medical Branch,
Galveston, TX 77555-0609, USA
8 Center for Vector-Borne and Zoonotic Diseases, University of Texas Medical Branch,
Galveston, TX 77555-0609, USA
9 Department of Preventive Medicine and Population Health, The University of Texas Medical Branch,
Galveston, TX 77555, USA
* Correspondence:
[email protected]
(N.V.);
[email protected]
(S.C.W.); Tel.: +1-409-747-0650 (N.V.);
+1-409-266-6500 (S.C.W.)
Abstract: Chronic conditions like type II diabetes (T2DM) have long been known to exacerbate
Citation: Azar, S.R.; Campos, R.K.;
many infectious diseases. For many arboviruses, including Zika virus (ZIKV), severe outcomes,
Yun, R.; Strange, T.; Rossi, S.L.;
morbidity and mortality usually only occur in patients with such pre-existing conditions. However,
Hanley, K.A.; Vasilakis, N.; Weaver,
the effects of T2DM and other pre-existing conditions on human blood (e.g., hypo/hyperinsulinemia,
S.C. Aedes aegypti Shows Increased
hyperglycemia and hyperlipidemia) that may impact infectivity of arboviruses for vectors is largely
Susceptibility to Zika Virus via Both
In Vitro and In Vivo Models of Type
unexplored. We investigated whether the susceptibility of Aedes aegypti mosquitoes was affected
II Diabetes. Viruses 2022, 14, 665. when the mosquitoes fed on “diabetic” bloodmeals, such as bloodmeals composed of artificially
tion of erythrocytes from hemoglobin A1c (HgbA1c) values of 5.5–5.9 to 6.2 increased the infection
Academic Editors: Remi N. Charrel
rate of a Galveston, Texas strain of Ae. aegypti to ZIKV strain PRVABC59 at a bloodmeal titer
and Luis Martinez-Sobrido
of 4.14 log10 FFU/mL from 0.0 to 40.9 and 42.9%, respectively. ZIKV was present in the blood of
Received: 25 January 2022 viremic LEPRDB/DB mice at similar levels as isogenic control C57BL/6J mice (3.3 log10 FFU/mL and
Accepted: 18 March 2022 3.6 log10 FFU/mL, respectively. When mice sustained a higher ZIKV viremia of 4.6 log10 FFU/mL,
Published: 23 March 2022
LEPRDB/DB mice infected 36.3% of mosquitoes while control C57BL/6J mice with a viremia of
Publisher’s Note: MDPI stays neutral 4.2 log10 FFU/mL infected only 4.1%. Additionally, when highly susceptible Ae. aegypti Rockefeller
with regard to jurisdictional claims in mosquitoes fed on homozygous LEPRDB/DB, heterozygous LEPRWT/DB, and control C57BL/6J
published maps and institutional affil- mice with viremias of ≈ 4 log10 FFU/mL, 54%, 15%, and 33% were infected, respectively. In total,
iations. these data suggest that the prevalence of T2DM in a population may have a significant impact on
ZIKV transmission and indicates the need for further investigation of the impacts of pre-existing
metabolic conditions on arbovirus transmission.
Copyright: © 2022 by the authors.
Keywords: Aedes aegypti; zika virus; type II diabetes; TGF-β; immunological cross-talk
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
1. Introduction
creativecommons.org/licenses/by/ The last century has seen a marked rise in both the emergence and incidence of arthropod-
4.0/). borne virus (arbovirus) infections [1–4] and the prevalence of chronic medical conditions [5–8]
Viruses 2022, 14, 665. https://doi.org/10.3390/v14040665 https://www.mdpi.com/journal/viruses
Viruses 2022, 14, 665 2 of 15
in the human population. On the arbovirus front, some long-recognized threats, such as
the dengue and yellow fever viruses, have surged in recent decades [3,9–12], while others,
most prominently the Zika (ZIKV) and chikungunya viruses (CHIKV), have emerged into
global prominence only within the last ten years [9–12]. One in three adult patients suffers
from multiple chronic medical conditions simultaneously, a number that is anticipated to
double in developed countries by 2035 [5]. One analysis of data from the National Health
Interview Survey in 2012 found that approximately 117 million US adults (≈50%) had at
least one of ten chronic conditions (hypertension, coronary heart disease, stroke, diabetes,
cancer, arthritis, hepatitis, weak or failing kidneys, current asthma, or chronic obstructive
pulmonary disease), while about one in four suffered from two or more chronic conditions [8].
As many of the chronic conditions listed above are associated with hematologic changes (local
and systemic alterations of blood pH, hyper- and hypoinsulinemia, hyperglycemia, and an
altered basal circulation of inflammatory and anti-inflammatory cytokines), it is reasonable
to hypothesize that they might impact the progression of arboviral replication, disease and
transmission. The observation that infection with many arboviruses [13–21] results in more
severe disease outcomes and mortality in patients with such pre-existing conditions supports
this hypothesis.
In the current study, we focused on Type II diabetes mellitus (T2DM), which causes
hematologic manifestations including hyperglycemia, hyperinsulinemia (and eventual
hypoinsulinemia), metabolic acidosis and hyperlipidemia, as well as chronic inflamma-
tion [22]. Jean-Baptiste et al. reported that the clinical manifestations of CHIKV infection are
more prevalent and longer-lasting in diabetic than non-diabetic patients [23]. However, the
documented hematological effects of T2DM on the arbovirus infection of mosquito vectors
have not been explored. Indeed, comparatively little attention has been afforded to the
impact of blood-borne factors in the modulation of the arbovirus infection of mosquitoes.
This concept, known as “immunological cross-talk”, posits that vertebrate blood factors can
interact with, and signal through, receptors in the mosquito midgut when imbibed. To date,
studies of immunological cross-talk between human blood and vectors has been largely
restricted to investigations of Plasmodium parasites and Anopheline mosquitoes [24–31].
In particular, the cytokine transforming growth factor β (TGF-β) has been extensively
characterized for its ability to regulate innate immune functions in the midguts of Anophe-
line mosquitoes, with direct consequences for their vector competence for Plasmodium
parasites. Specifically, low levels of ingested TGF-β directly decrease malaria parasite
burdens [25,26,30,31]. Whether TGF-β can exert such effects in Aedes spp. mosquitoes and
arboviruses is presently unknown.
Thus, in the present study, we investigated whether the hematologic changes caused
by T2DM could affect the susceptibility of Ae. aegypti mosquitoes to ZIKV. Both artificial
bloodmeals and viremic animals were utilized to produce bloodmeals with a “diabetic
phenotype”. The artificial bloodmeals were produced utilizing human erythrocytes that
underwent in vitro glycosylation to increase the glycosylation of hemoglobin on the cell
surface, often used as a diagnostic tool for glycemic control and diabetes, with a HbA1C
≥6.5% being a diagnostic marker of T2DM [32,33]. To expose mosquitoes to viremic
diabetic animals, we utilized the leptin-receptor mutant LEPRDB/DB model on the C57BL6
mouse background. Following the consumption of ZIKV via artificial bloodmeals or
bloodmeals from viremic animals, the rates of infection and dissemination from the midgut
into the hemocoel were calculated following a set extrinsic incubation period. In both the
in vitro and in vivo experimental paradigms, we observed significant increases in Aedes
aegypti’s susceptibility to ZIKV from phenotypically diabetic bloodmeals compared to
control bloodmeals.
2. Materials and Methods
2.1. Cells and Viruses
African green monkey kidney cells (CCL-81, hereafter referred to as Vero), were
purchased from the American Type Culture Collection (ATCC, Bethesda, MD, USA) and
Viruses 2022, 14, 665 3 of 15
maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, ThermoFisher Scientific,
Waltham, MA, USA) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (FBS,
Atlanta Biologicals, Flowery Branch, GA, USA), 1% (v/v) Penicillin–Streptomycin (P/S,
ThermoFisher Scientific, Waltham, MA, USA; 100 U/mL and 100 µg/mL, respectively) in a
humidified 37 ◦ C incubator with 5% CO2 . The ZIKV strain PRVABC59 (Puerto Rico, 2015)
was received as a lyophilized stock (World Reference Center for Emerging Viruses and
Arboviruses, UTMB) as a passage 4 stock. The virus underwent two additional Vero cell
passages to generate the stocks utilized in these studies.
2.2. Animal Procedures
All the procedures utilizing animals were conducted in full compliance with the
guidelines established by the Animal Welfare Act for the housing and care of laboratory
animals and conducted as laid out in the University of Texas Medical Branch Institutional
Animal Care and Use Committee (UTMB-IACUC)-approved Protocol #1708051A. In exper-
iments conducted with LEPRDB/DB , LEPRWT/DB and C57BL/6J mice, 10-week-old animals
(n = 4 per group per experiment) were pretreated with 1.5–2.0 mg of IFNAR-blockading
antibody (MAR1-5A3, Leinco, St. Louis, MO, USA) to render them permissive for ZIKV
viremia. A 300 µL maximum dosage was allowed; therefore, the functional dose was
contingent on the concentrations provided in the manufacturer vial (concentration range
of 5.0 to 7 mg/mL). The dose range of 1.5 mg or 2.0 mg of anti-IFNAR blockade was
based on previous reports and manufacturer protocols [34]. Animals underwent antibody
treatment one day before and one day after infection with ZIKV. On day 0, mice were
infected via the intraperitoneal route with 5 log10 FFU of ZIKV in a volume of 100 µL of
phosphate buffered saline (PBS). At three or four days after exposure to ZIKV, the mice
were anesthetized via intraperitoneal inoculation with ketamine (100 mg/kg) and xylazine
(10 mg/kg). Following the anesthesia, the mice were placed atop the mesh lid of a 0.5 L
cardboard cup containing sugar-starved Ae. aegypti. Mosquitoes were allowed to feed
for 30 min and then cold-anesthetized, and fully engorged specimens were incubated as
described below. After blood feeding, the mice were humanely euthanized and bled to
quantify the viremia by focus-forming assays.
2.3. Preparation and Administration of Artificial Bloodmeals
All the artificial bloodmeals were created with a calculated titer of 4.3 log10 FFU/mL
of ZIKV. Citrated single-donor human whole blood (gender unspecified, Lampire Biolog-
ical Laboratories, Pipersville, PA, USA) was used as previously described [35], with the
following modifications: The first type of bloodmeal was supplemented with 0.5 ng/mL,
5 ng/mL, or 50 ng/mL recombinant transforming growth factor β1 (rTGFβ-1) (R&D Sys-
tems, Minneapolis, MN, USA) (to act as a 100-fold range that is reported for human health
and disease) [36,37]. The second type of bloodmeal utilized artificially glycosylated human
erythrocytes. Briefly, erythrocytes were washed as previously described and resuspended
in glycation buffer (45 mmol/L glucose in DPBS) to produce a 10% hematocrit suspension
(volume: 30 mL) [38,39], and 300 µL of 100 X P/S was added. These glycation reactions
were incubated for 18 h at 37 ◦ C in a shaking incubator, centrifuged (200 RCF, 15 min)
and resuspended in a 5 mL residual volume. A 20 µL sample was analyzed utilizing an
A1CNow + test kit (PTS Diagnostics, Whitestown, IN, USA) to determine the glycation of
the erythrocytes. A control bloodmeal was mock glycated and processed identically, but re-
suspended and incubated in PBS instead of glycation buffer. The mock-glycosylated blood
was compared against unmodified donor whole blood to confirm identical A1C readouts.
2.4. Mosquitoes
Adult female Aedes aegypti from two colonies, Galveston Texas (F5), or Rockefeller,
unknown generation (ROCK), were used in these studies. The mosquitoes were housed
prior to and following the ingestion of bloodmeals in an incubator with a temperature of
27 ± 1 ◦ C with 80 ± 10% relative humidity and a 16:8 light:dark cycle. The mosquitoes
Viruses 2022, 14, 665 4 of 15
were housed in 0.5 L cardboard cups with mesh lids and provided ad libitum access to
10% sucrose. For vector-competence analyses, female mosquitoes were sorted 3 days
post-eclosion, at which point access to sucrose was removed via replacement with cotton
rounds soaked in water, which were removed 4–6 h prior to blood feeding. After feeding,
sucrose-soaked rounds were provided once again [35,40,41].
2.5. Sampling/Processing Mosquitoes
To determine whether mosquitoes that ingested ZIKV became infected, and whether
the infections disseminated from the midgut into the hemocoel, they were anesthetized
on ice fourteen days post-bloodmeal, and legs of individual mosquitoes were removed
and placed into microfuge tubes containing sterilized steel ball bearings and 500 µL of
mosquito collection medium (MCM) (DMEM, 2% FBS, 1% Pen–Strep, and 2.5 µg/mL of
amphotericin B). The individual carcasses were placed into separate tubes with 500 µL of
MCM and processed by trituration for 5 min at 26 Hz in TissueLyser II (QIAGEN, Hilden
Germany), followed by centrifugation at 200× g for 5 min [35,40–43].
2.6. Detection and Quantification of ZIKV Infection
To determine the presence or absence as well as titers of virus present in the stocks,
mouse sera or bloodmeals, samples either underwent 10-fold serial dilutions in dilution
medium (DMEM, 2% FBS, and 1% Pen–Strep) in 96-well culture plates as previously
described [35,40,41,44,45] or were inoculated directly. Next, 100 µL of samples were added
to 80–95% confluent monolayers of Vero cells on either 12- or 24-well tissue culture plates.
The viral dilutions were allowed to adsorb for one hour in a humidified 37 ◦ C incubator
with 5% CO2 , and then overlayed using a solution of DMEM containing 3% FBS, 1% Pen–
Strep, 1.25 µL/mL amphotericin B, and 0.8% weight/vol methylcellulose. The overlayed
plates were incubated for 5 days in a humidified 37 ◦ C incubator with 5% CO2 ; then,
each well was washed twice with PBS and fixed for a minimum of 30 min in an ice-cold
solution of methanol:acetone, 1:1, vol/vol. Following the fixation, the organic fixative
was removed and the plates were air dried. Following complete air drying, the plates
were washed with PBS and then blocked with 3% FBS in PBS, followed by an overnight
incubation with mouse hyperimmune serum against the ZIKV strain MR-766 (1:2000 in
blocking solution) (WRCEVA, UTMB). The plates were then washed with PBS, followed
by incubation with a goat anti-mouse secondary antibody conjugated to horseradish
peroxidase (KPL, Gaithersburg, MD, USA) diluted 1:2000 in blocking solution. The plates
were washed with PBS, after which an aminoethylcarbazole solution (Enzo Diagnostics,
Farmingdale, NY, USA) prepared according to the manufacturer0 s protocol was added, and
the plates were incubated in the dark. Development was halted by washing in tap water,
and the plates were allowed to air dry at room temperature before scoring.
2.7. Multiplex Cytokine/Chemokine Bead Assay
To determine the levels of cytokines and chemokines in the mice at the time of the
bloodmeal, the serum collected after feeding was subjected to two multiplex bead assays:
Bio-Plex Pro Mouse Cytokine 23-Plex (Bio-Rad, Hercules, CA, USA). Samples were run
in duplicate according to the manufacturer’s instructions, and each value was plotted
alongside the average value per animal cohort.
3. Results
3.1. TGF-β Immunological Cross-Talk in Aedes Aegypti
To establish whether the same types of immunological cross-talk that have been ob-
served in the Plasmodium/Anopheles system [24–29] could potentially occur in the
Ae. aegypti/ZIKV system, a 100-fold range of rTGF-β1 was added to individual bloodmeals,
which were then provided to mosquitoes.
As demonstrated in Figure 1, the addition of rTGF-β1 to bloodmeals significantly
enhanced the percentage of mosquitoes (number positive over number assayed) infected
Viruses 2022, 14, 665 5 of 15
with ZIKV when compared to the control bloodmeal (nominal logistic regression, chi-
squared = 29.0, p < 0.0001). This analysis showed a significant gain in infection between
rTGF-β1 at 0 ng/mL (9.0% of mosquitoes infected) and 0.5 ng/mL (53.1%), with no further
significant increases as the rTGF-β1 concentrations increased. A similar pattern was
observed for the percentage dissemination (the number with positive legs over total number
assayed) (nominal logistic regression, chi-squared = 12.7, p < 0.005), although no significant
differences among adjacent pairs of concentrations were detected.
Figure 1. Effect of TGF-β1 on susceptibility of Ae. aegypti to ZIKV. Strain PRVABC59 at a titer of
4.2 log10 FFU/mL was provided to female mosquitoes (Galveston F5) in artificial bloodmeals that
were supplemented with exogenous human TGF-β1 at indicated doses. Blood-fed mosquitoes were
incubated at 27 ± 1 ◦ C with 80 ± 10% relative humidity and a 16:8 light:dark cycle for fourteen
days, at which point they were collected and analyzed for the presence or absence of ZIKV by Vero
cell assays. Data are presented as percentages: 100 * [number of infected bodies (infection) or legs
(disseminated infections) over total number of mosquitoes assayed in a given condition]. Numbers in
brackets indicate sample sizes. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.
3.2. Infectivity of ZIKV in Artificially Glycosylated Bloodmeals
To investigate whether the infectivity of ZIKV for Ae. aeygpti could be affected by
bloodmeals that had markers of T2DM, an in vitro glycosylation strategy was adopted. In
this methodology, human erythrocytes were treated with a glycation buffer (45 mmol/L
glucose) to increase the level of HbA1c and compared to mock-glycated blood from the
same donor. To facilitate comparisons, the HbA1c values for unmanipulated blood were
also tested (both the unmanipulated whole blood and mock-glycated blood HbA1c values
were found to be 5.5%).
ZIKV in mock-glycated blood failed to infect any of the tested mosquitoes at a titer
of 4.1 log10 FFU/m (Figure 2). Strikingly, glycation to HgbA1c levels of 5.9 or 6.2 yielded
bloodmeals that infected Ae. aegypti at titers of 4.2 (40.9% of the mosquitoes infected)
and 4.1 log10 FFU/m (42.9%), a significant gain relative to the controls (contingency table
analysis with 1 added to every cell to avoid 0 values; chi squared = 24.6, p < 0.0001). The
disseminated infections also increased with increasing glycation of HgbA1c, but the only
significant difference was between the control and the high-glycation bloodmeals (Fisher’s
exact test, p = 0.007).
Viruses 2022, 14, 665 6 of 15
Figure 2. Effect of erythrocyte glycosylation on ZIKV infectivity of mosquitoes. ZIKV strain PRV-
ABC59 was provided to Ae. aegypti (Galveston F5) in artificial bloodmeals at titers of 4.1 log10 FFU/mL
that were made with artificially glycosylated erythrocytes or mock-glycosylated erythrocytes (HbA1c
5.5). Blood-fed mosquitoes were incubated at 27 ± 1 ◦ C with 80 ± 10% relative humidity and a 16:8
light:dark cycle for fourteen days, at which point they were collected and analyzed for the presence
or absence of ZIKV. Data are presented as percentages: 100 * [number of infected bodies (infection)
or legs (dissemination) over total number of mosquitoes assayed in a given condition]. Numbers in
brackets indicate sample sizes. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.
3.3. LEPRDB/DB Mice Require IFNAR Blockade to Become Viremic
While several mouse models of obesity and diabetes exist, the LEPRDB/DB model was
chosen because its obesity and metabolic syndrome do not require researcher manipulation
and the mutation that drives the phenotype is not intrinsically immunological in nature.
LEPRDB/DB mice are produced on a C57BL/6J background, which failed to become viremic
when infected with ZIKV in previous studies [34,46]. To produce and characterize viremia
in LEPRDB/DB mice, animals were treated 1 day prior to and 1 day after ZIKV infection
with the IFNAR-blockading antibody MAR1-5A3.
Concordant with previous experiments in C57BL/6J mice, LEPRDB/DB animals failed
to become viremic in the absence of IFNAR blockade. In the animals treated with MAR1-
5A3, the viremia peaked three days post-infection at an average titer of 4.4 log10 FFU/mL
(Figure 3A), with no apparent difference between males and females (data not shown).
Viruses 2022, 14, 665 7 of 15
Figure 3. Susceptibility to ZIKV ingested from LEPRDB/DB and control animals. (A) LEPRDB/DB were
treated with 1.5–1.8 mg of IFNAR-blockading antibody MAR1-5A3 one day prior to and one day
after infection with 5.00 log10 FFU of ZIKV PRVABC59. Viremia was determined via focus-forming
assay performed on serum from retro-orbitally sampled blood. (B) ZIKV PRVABC59 was provided
to Ae. aegypti (Galveston F5) via infected mice (either LEPRDB/DB or C57BL/6J). Mosquitoes were
exposed to titers of 4.42 ± 4.02 log10 FFU/mL (corresponding to 3 DPI) and 3.56 ± 2.95 log10 FFU/mL
(4 DPI). Blood-fed mosquitoes were incubated at 27 ± 1 ◦ C with 80 ± 10% relative humidity and
a 16:8 light:dark cycle for fourteen days, at which point they were collected and analyzed for the
presence or absence of ZIKV via plaque assays. (C) ZIKV PRVABC59 was provided to Ae. aegypti
(Rockefeller) via infected mice (LEPRDB/DB , LEPRWT/DB or C57BL/6J). Mosquitoes were exposed to
titers corresponding to 3.68 ± 3.01 log10 or 4.32 ± 3.88 log10 FFU/mL. Blood-fed mosquitoes were
incubated at 27 ± 1 ◦ C with 80 ± 10% relative humidity and a 16:8 light:dark cycle for fourteen days,
at which point they were collected and analyzed for the presence or absence of ZIKV via plaque
assays. In B) and C), data are presented as percentages: 100 * [number of infected bodies (infection) or
legs (dissemination) over total number of mosquitoes assayed]. Numbers in brackets indicate sample
sizes. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.
3.4. Infection of Aedes aegypti with ZIKV PRVABC59 via Viremic LEPRDB/DB Mice
Upon establishing that the IFNAR blockade of LEPRDB/DB mice enhanced ZIKV
viremia with titers exceeding 4 log10 FFU/mL, LEPRDB/DB and C57BL/6J mice were
treated with MAR1-5A3 and then presented to Ae. aegypti (Galveston F5) either three or
four days post-infection.
Viremic LEPRDB/DB mice were capable of infecting Ae. aegypti in the relatively low-titer
condition of 3.6 ± 2.9 log10 FFU/mL at a rate of 15.9%, with 2.3% disseminated infections
(Figure 3B), while at this titer, viremic C57Bl/6J only infected 1.2% of mosquitoes, with no
disseminated infections observed. At the higher titer of 4.4 ± 4.0 log10 FFU/mL, viremic
LEPRDB/DB infected 36.3% of the Ae. aegypti, and 12.5% of the mosquitoes demonstrated
disseminated infections. At the same virus titer, the infected control animal (C57BL/6J)
demonstrated substantially lower rates of infection (4.1%) and disseminated infection (1.4%)
in this population of Ae. aegypti. A logistic fit of the data revealed that both the mouse
strain and virus titer had a significant effect on infection (DF = 1 for each parameteter,
p < 0.0001 and p = 0.002, respectively) as well as dissemination (DF = 1 for each parameter,
Viruses 2022, 14, 665 8 of 15
p = 0.0001 and p = 0.004, respectively), with higher infection resulting from higher titers
and LEPRDB/DB mice.
To corroborate and extend these findings, Ae aegypti ROCK mosquitoes, which are
highly permissive to infection, were allowed to feed upon viremic homozygous LEPRDB/DB ,
heterozygous LEPRWT/DB and wild-type C57BL/6J animals with viremia titers of ≈3 or
4 log10 FFU/mL.
In this system, minimal infection and dissemination was observed after the ingestion of
3 log10 FFU/mL (Figure 3C). At titers about 10-fold higher, the infection and dissemination
increased significantly in mosquitoes exposed to all the animals (DF = 1, chi squared = 112.6,
p < 0.0001). With the homozygous mouse strain LEPRDB/DB , infection reached rates of 54%
with 28% dissemination. In this strain of mosquitos, however, titers of ≈4 log10 FFU/mL
in C57BL/6J mice yielded 33% infection and 19% dissemination. Mosquitoes exposed to
heterozygous LEPRWT/DB animals with this viremia titer demonstrated the lowest rates,
of 15% infection and 7% dissemination. A nominal logistic model revealed significant
differences among each pair of genotypes (p < 0.001 for each pairwise comparison).
To determine whether serum cytokines were associated with the differences in mouse
phenotype and, therefore, infection and disseminated infection, mouse sera were collected
immediately after bloodmeals and subjected to multiplex bead analysis for 23 cytokines
(Table 1; data for each individual mouse are provided in Supplementary Table S1). To
reduce the number of comparisons and the possibility of false discoveries, the cytokines
were first grouped into one of four classes (Figure S1) [47]: pro-inflammatory (IL-1 α,
IL-1β, IL-6, IL-13, 1L-17A, G-CSF, IFN-g and TNFa), anti-inflammatory (IL-10, IL-12p40 and
IL-12p70), adaptive immunity (IL-2, IL-3, IL-4, IL-5, IL-9 and GM-CSF) and chemokines
(eotaxin, KC, MCP-1, MIP-1 α, MIP-1 β and RANTES). Each group was subject to principal
component (PC) analysis, and for each group, the first PC explained substantially more
variation in the data than subsequent PCs. Thus, we tested for the variation in the PC-1 of
each group using a Wilcoxon test. Only the chemokines showed a significant difference
among genotypes (DF = 2, chi-squared = 6.04, p = 0.049); while the wild-type was higher
than either the heterozygous or homozygous genotype, a post hoc test revealed a significant
difference only between the wild-type LEPR and the heterozygote (p = 0.03). The PC-1 of
the chemokines was significantly associated with positive values of MCP-1, MIP-1β and
KC and, to a lesser degree, negative values of MIP-1α and RANTES. However, we observe
that one mouse (number 4; see Supplementary Table S1) was flagged as an outlier in most
comparisons. While we chose not to remove this individual, doing so would have changed
the significance of the comparison.
Viruses 2022, 14, 665 9 of 15
Table 1. Cytokine levels in mice varying in the LEPR mutation and infected with ZIKV. Serum
was taken from animals immediately following Aedes aegypti Rockefeller feeding. Serum from each
animal was run in duplicate utilizing a mouse multiplex bead assay to determine circulating levels of
23 cytokines. Data are presented as averages of all animals of a given phenotype, alongside standard
deviations. LOD: limit of detection; N/A: not applicable.
C57BL/6J LEPRWT/DB LEPRDB/DB
Standard Standard Standard
Mean Mean Mean
Cytokine Deviation Deviation Deviation
(pg/mL) (pg/mL) (pg/mL)
(pg/mL) (pg/mL) (pg/mL)
IL-1α 14.82 16.12 4.45 1.57 2.85 2.86
IL-1β
IL-3 3.76 2.36 5.36 1.16 3.58 1.39
IL-4 0.51 0.66 0.31 0.28
IL-9 13.31 6.97 15.96 2.85 13.85 2.09
IL-10 10.60 5.29 11.98 3.67 8.30 3.79
IL-12 (p40) 2158.22 1456.44 1871.29 1452.92 1387.81 754.73
IL-12 (p70) 47.80 32.93 55.44 5.58 48.39 9.13
IL-13 62.10 47.67 44.25 4.58 51.31 8.05
IL-17A 41.86 26.50 52.88 34.07 59.14 24.63
Eotaxin 2548.90 808.52 2747.01 458.67 3717.02 411.93
G-CSF 337.84 74.60 164.98 73.54 159.89 101.84
GM-CSF 5.96 6.29
KC 138.36 53.52 57.20 15.66 102.53 30.01
MCP-1 1068.20 94.62 542.34 105.48 826.28 298.69
MIP-1α 3.55 1.21 3.72 1.04 4.94 0.94
MIP-1β 714.95 1057.70 168.44 19.14 217.29 31.72
RANTES 452.30 216.71 458.00 239.11 472.09 26.57
TNF-α 47.40 14.87 167.51 189.88 48.67 10.34
4. Discussion
To date, there have been limited data on the impacts of T2DM on the arbovirus
infection of vector mosquitoes. Herein, we utilized manipulated bloodmeals and mouse
models of T2DM to assess the impact of “diabetic” blood on ZIKV infectivity for the major
vector, Ae. aegypti.
The incorporation of recombinant mammalian TGF-β1 in artificial bloodmeals in-
creased ZIKV infectivity in Ae. aegypti. Additionally, ZIKV presented in the context of
phenotypically diabetic in vitro and in vivo bloodmeals (artificially glycosylated/viremic
LEPRDB/DB mice) was more infectious. The effect of TGF-β on immunological cross-
talk in Anopheline mosquitoes and Plasmodium parasites has previously been exam-
ined [25,26,28,30,31,48]. Specifically, ingesting mammalian TGF-β at low doses can induce
Plasmodium-parasite killing in Anopheles stephensi mosquitoes via the induction of NO
synthase (AsNOS) [30]. In vitro and ex vivo experiments conducted with dengue virus-1
(DENV1) have previously demonstrated the sensitivity of flaviviruses to NO in the context
Viruses 2022, 14, 665 10 of 15
of isolated human monocytes [49]. Our data appear to differ from these previous studies,
as the rate of ZIKV infection and dissemination was higher in bloodmeals with higher
TGF-β1, regardless of the dosage. This pattern suggests that TGF-β1 does not have a similar
effect on AsNOS in Ae. aegypti mosquitoes to in Anophelines, or that TGF-β1 stimulates
other pathways that favor ZIKV infection and thereby counteract the negative impacts
of NO, and/or that ZIKV is less sensitive to NO than DENV. Therefore, a mechanistic
examination of how mammalian TGF-β1 affects Aedes mosquitoes and ZIKV infection
in vivo is needed.
The examination of the role of HgbA1c in the ZIKV infection of Ae. aegypti revealed
significantly greater infection in the presence of elevated glycosylation. Blood from a
single donor was subjected to artificial glycosylation [38,39], and the resultant erythrocytes
were used to prepare in vitro bloodmeals. While a promising preliminary analysis, this
methodology is highly artificial. Follow-up analyses would ideally be conducted using
whole blood from donors representing a wider range across the HgbA1c spectrum (healthy,
prediabetes and diabetes). The disadvantage of such an approach is the intrinsic variability
in various hematologic parameters among blood donors. Of particular note, the role of
blood glucose is one that is critically important in future analyses. In the latter phases
of T2DM, after β-cell dysfunction and death, plasma insulin falls and plasma glucose
rises [50–53], underscoring the value of using animal models that can exhibit a dynamic
range of both insulin and glucose. One recent analysis demonstrated that mosquitoes
imbibing a bloodmeal supplemented with glucose had significantly increased DENV titers
at both 3 and 7 days after an infectious bloodmeal [54]. This was also accompanied by an
increase in DENV envelope protein levels as a function of the concentration of glucose
and days post-infectious bloodmeal [54]. However, greater concentrations of glucose have
also been shown to inhibit in vitro ZIKV replication in human kidney cells [55], indicating
the potential for acutely hyperglycemic patients to have decreased viremia when infected
with ZIKV. These analyses reinforce the value of in vivo analyses that examine the role of
glucose and insulin in the flavivirus infection of vector mosquitoes.
We also used mouse models of T2DM to investigate the impacts of this condition on
ZIVK infection for mosquitoes. ZIKV in the blood of LEPRDB/DB mice infected Galveston
Ae. aegypti more efficiently than virus from the non-obese genetic background, C57BL/6J.
When the highly susceptible Rockefeller strain of Ae. aegypti was tested using LEPRDB/DB ,
LEPRWT/DB or control mice with a high ZIKV viremia, the DB homozygous mice infected a
significantly higher proportion than the heterozygotes. These results must be considered
in light of other recent studies probing the impact of mammalian insulin on mosquito
immunity and susceptibility to arboviruses. Ahlers et al. showed that mammalian insulin
can trigger Akt and ERK signaling in mosquitoes, leading to the transcription of JAK/STAT-
associated antiviral genes [56]. Moreover, the pre-treatment of cells from both Culex and
Aedes mosquitoes with mammalian insulin suppressed the replication of West Nile virus, a
finding largely recapitulated in Ae. albopictus C6/36 cells with both DENV and ZIKV [56].
We utilized phenotypically T2DM animals, and the serum insulin was not quantified.
Nonetheless, it is well documented that the LEPRDB/DB model becomes hyperinsulinemic
as early as 10–14 days of age, with the peak levels of insulin observed at 3 months of age,
and a drop off in insulin precipitated by β-cell dysfunction and depletion by 6 months of age
occurs [57]. In contrast, LEPRWT/DB animals do not become obese or even hyperinsulinemic
at any age [50,58]. The mice we used were approximately 2.5 months old, and therefore,
the DB homozygotes were in a hyperinsulinemic state, whereas the heterozygotes were
not. At first consideration, the finding that the DB homozygotes infected significantly more
Ae. aegypti with ZIKV than the heterozygotes seems to contradict the finding that insulin
signaling decreases WNV replication in Culex mosquitos [56]. However, it is possible that
the extremely high levels of insulin produced by the DB homozygotes disrupted rather than
enhanced JAK/STAT signaling. Further studies utilizing transcriptomics to investigate this
possibility are warranted. It is worth noting that the LEPRDB/DB mice demonstrate serum
insulin levels of ≈ 1–5 ng/mL, corresponding to 2.25 to 11.23 PM [50,59]. These are levels
Viruses 2022, 14, 665 11 of 15
15.2- to 75.6-fold lower than the 17 PM utilized to produce the phenotype observed in Culex
mosquitoes exposed to WNV [56]. This is particularly important in that, in the current
study, mosquitoes took bloodmeals from viremic animals rather than from an artificial
feeder spiked with non-physiologic levels of insulin, potentially convoluting the infection
outcomes. Regarding the discussion of blood glucose above and blood lipids below, it will
also be important to completely characterize the blood composition in the DB homozygous
and heterozygous mice in future experiments. Finally, the IFNAR blockade may have
obscured or even altered the impact of insulin on JAK–STAT signaling. Unfortunately,
most small animal models of ZIKV rely on immunological defects such as non-functional
interferon α/β receptors, to allow the virus to productively infect [46,60]. The ablation
of mouse STAT2 (which is known to resist ZIKV antagonism) and its replacement with
human STAT2 have been demonstrated to enhance ZIKV replication in immunocompetent
(C57BL/6N) mice [61], raising the possibility of rendering such animals obese via dietary
supplementation for subsequent experiments.
Among a panel of cytokines measured, only the chemokines, particularly MCP-1,
MIP-1β and KC, differed among the three mouse genotypes, with the levels in heterozygote
mice lower than those in both LEPRDB/DB and wild type mice, albeit the difference was only
significant for the heterozygote–wild-type comparison. We have no immediate explanation
for this variation, and it may reflect a complex interaction between the effects of the mouse
genotype per se, ZIKV infection and exposure to mosquito saliva on chemokines. It is
intriguing that the heterozygote mice also infected a smaller percentage of mosquitoes than
either the LEPRDB/DB or wild-type mice. Koerber-Rosso et al. conducted a review of studies
comparing non-human organisms that carried mono-allelic, likely pathogenic, variants at
the LEPR locus (hereafter heterozygotes) to their wild-type counterparts [62]. These studies
showed considerable variation in the effects of the heterozygous state on body weight and
metabolic state. Moreover, to our knowledge, there are relatively few published studies
comparing cytokines in mice heterozygous at the LEPR allele to wild-type mice [63]. Thus,
further studies are needed to characterize the cytokine responses of these heterozygotes
to infection.
Our study has several limitations, including the fact that the methodology utilized
to artificially glycosylate erythrocytes failed to produce glycosylation levels equivalent to
those observed in clinical diabetes; the highest HbA1c value we achieved was 6.2%, which
fails to meet the 6.5% cutoff for a diagnosis of T2DM [32,33]. From an experimental point of
view, artificial glycosylation allows for the erythrocytes of the same donor to be used as both
the test article and the control condition and eliminates the confounding variable of using
multiple donors. However, future studies using human blood from T2DM and control
individuals are needed. Another limitation is that the LEPRDB/DB hyperlipidemia mouse
model does not entirely recapitulate human T2DM and obesity. Human T2DM is gener-
ally characterized by high LDL and VLDL, and low HDL cholesterols, while LEPRDB/DB
animals have highly elevated levels of HDL. Previous studies have demonstrated that
host cholesterols are critical for the flavivirus infection of both mosquitoes and mammals
(reviewed in [64]). In at least one analysis, the administration of LDL to mosquito cell
lines (Aag2 and C6/36) decreased ZIKV infectivity, and the supplementation of an artificial
bloodmeal with 50 mg/dL of LDL reduced the levels of ZIKV RNA compared to that
in mosquitoes fed control bloodmeals. Conversely, however, the treatment of Aag2 and
C6/36 cells with HDL failed to affect ZIKV replication [65]. These results suggest that our
findings may be specific to the LEPRDB/DB model, necessitating corroboration in alternative
models of diabetes, such as via high-fat diets in wild-type animals [66,67].
Given the increasing propensity for arboviral and zoonotic agents to emerge due to
climate change, deforestation and the expansion of the human population into areas associ-
ated with enzootic circulation [10,68,69], as well as the increase in chronic conditions in the
human population [7,8,21,70], it is of paramount importance that vector competence studies
incorporate mammalian factors influenced by chronic conditions. Due to the common find-
ing that pre-existing medical conditions alter the pathogenesis of arboviral pathogens [23],
Viruses 2022, 14, 665 12 of 15
individuals with such conditions are, at the very least, a vulnerable population to consider
epidemiologically when striving to control outbreaks. Furthermore, our data suggest that
that these populations represent a hitherto-unconsidered driver of arbovirus transmission.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/v14040665/s1. Table S1. Serum was taken from animals at the
time point corresponding to the time Aedes aegypti Rockefeller fed upon the animals. Serum from
each animal was run in duplicate utilizing a mouse multiplex bead assay to determine circulating
levels of 23 cytokines at time of feeding. Data shown for each individual animal. Figure S1. Cytokine
outputs from Bioplex analysis were categorized as (IL-1 α, IL-1β, IL-6, IL-13, 1L-17A, G-CSF, IFN-g,
TNFa), anti-inflammatory (IL-10, IL-12p40, IL-12p70), adaptive immunity (IL-2, IL-3, IL-4, IL-5, IL-9
GM-CSF) or chem-okines (eotaxin, KC, MCP-1, MIP-1 α, MIP-1 β, RANTES). PC-1 analyses for each
category is demonstrated in relation to mouse genotype. (A) proinflammatory, (B) chemokines, (C)
anti-inflammatory, and (D) adaptive immunity.
Author Contributions: Conceptualization, S.R.A., N.V., S.L.R., K.A.H. and S.C.W.; methodology,
S.R.A.; formal analysis, S.R.A. and K.A.H.; investigation, S.R.A., R.K.C., R.Y. and S.L.R.; resources,
S.L.R., N.V. and S.C.W.; data curation, S.R.A. and K.A.H.; writing—original draft preparation, S.R.A.;
writing—review and editing, S.R.A., R.K.C., R.Y., T.S., S.L.R., K.A.H., N.V. and S.C.W.; project
administration, S.R.A., S.L.R., N.V. and S.C.W.; funding acquisition, S.L.R., N.V. and S.C.W. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by an Institute for Human Infections and Immunity Pilot
Grant (N.V.) and NIH grant 5R01AI121452-05 (SCW).
Institutional Review Board Statement: All the procedures utilizing animals were conducted in full
compliance with the guidelines established by the Animal Welfare Act for the housing and care of
laboratory animals and conducted as laid out in the University of Texas Medical Branch Institutional
Animal Care and Use Committee (UTMB-IACUC)-approved protocol (protocol code: 1708051A;
approved: August 2020).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or Supplementary Materials. Addi-
tionally, raw data are available from the authors upon request.
Conflicts of Interest: The authors declare no conflict of interest.
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FAQs
AI
What explains Aedes aegypti's increased susceptibility to Zika virus with diabetes?
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The study demonstrates that phenotypically diabetic bloodmeals significantly increase ZIKV infection rates in Aedes aegypti, reaching up to 54% in certain trials.
How do transforming growth factor β levels affect ZIKV infection in mosquitoes?
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Adding recombinant TGF-β1 to bloodmeals increased ZIKV infection rates from 9% to 53% with low doses, establishing a significant correlation.
What methodology was used to assess ZIKV infection in Aedes aegypti?
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Infection was assessed using both artificial bloodmeals modified for diabetic phenotypes and blood from viremic LEPR DB/DB mice.
When did LEPR DB/DB mice exhibit peak ZIKV viremia in the study?
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ZIKV viremia in LEPR DB/DB mice peaked three days post-infection, averaging 4.4 log10 FFU/mL.
What role does glycosylation of erythrocytes play in ZIKV infectivity?
add
Artificial glycosylation of erythrocytes to increase HbA1c levels resulted in significant infection rates in mosquitoes, revealing a connection to ZIKV transmission.
Scott Weaver
University of Texas, Medical Branch at Galveston, Faculty Member
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Zika Virus Growth in Human Kidney Cells Is Restricted by an Elevated Glucose Level
Gilles Gadea
International Journal of Molecular Sciences, 2021
Mosquito-borne Zika virus (ZIKV) became a real threat to human health due to the lack of vaccine and effective antiviral treatment. The virus has recently been responsible for a global outbreak leading to millions of infected cases. ZIKV complications were highlighted in adults with Guillain–Barré syndrome and in newborns with increasing numbers of congenital disorders ranging from mild developmental delays to fatal conditions. The ability of ZIKV to establish a long-term infection in diverse organs including the kidneys has been recently documented but the consequences of such a viral infection are still debated. Our study aimed to determine whether the efficiency of ZIKV growth in kidney cells relates to glucose concentration. Human kidney HK-2 cells were infected with different ZIKV strains in presence of normal and high glucose concentrations. Virological assays showed a decrease in viral replication without modifying entry steps (viral binding, internalization, fusion) under hi...
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Variation in Aedes aegypti Mosquito Competence for Zika Virus Transmission
guilherme ribeiro
Emerging infectious diseases, 2017
To test whether Zika virus has adapted for more efficient transmission by Aedes aegypti mosquitoes, leading to recent urban outbreaks, we fed mosquitoes from Brazil, the Dominican Republic, and the United States artificial blood meals containing 1 of 3 Zika virus strains (Senegal, Cambodia, Mexico) and monitored infection, dissemination, and virus in saliva. Contrary to our hypothesis, Cambodia and Mexica strains were less infectious than the Senegal strain. Only mosquitoes from the Dominican Republic transmitted the Cambodia and Mexica strains. However, blood meals from viremic mice were more infectious than artificial blood meals of comparable doses; the Cambodia strain was not transmitted by mosquitoes from Brazil after artificial blood meals, whereas 61% transmission occurred after a murine blood meal (saliva titers up to 4 log 10 infectious units/collection). Although regional origins of vector populations and virus strain influence transmission efficiency, Ae. aegypti mosquito...
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Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti
Margus Varjak
PLOS Pathogens
As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vectorAedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-in...
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Linking Only Aedes aegypti with Zika Virus Has World-Wide Public Health Implications
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Frontiers in Microbiology
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Distinct Roles of Hemocytes at Different Stages of Infection by Dengue and Zika Viruses in Aedes aegypti Mosquitoes
Alvaro Ferreira
Frontiers in Immunology, 2021
Aedes aegypti mosquitoes are vectors for arboviruses of medical importance such as dengue (DENV) and Zika (ZIKV) viruses. Different innate immune pathways contribute to the control of arboviruses in the mosquito vector including RNA interference, Toll and Jak-STAT pathways. However, the role of cellular responses mediated by circulating macrophage-like cells known as hemocytes remains unclear. Here we show that hemocytes are recruited to the midgut of Ae. aegypti mosquitoes in response to DENV or ZIKV. Blockade of the phagocytic function of hemocytes using latex beads induced increased accumulation of hemocytes in the midgut and a reduction in virus infection levels in this organ. In contrast, inhibition of phagocytosis by hemocytes led to increased systemic dissemination and replication of DENV and ZIKV. Hence, our work reveals a dual role for hemocytes in Ae. aegypti mosquitoes, whereby phagocytosis is not required to control viral infection in the midgut but is essential to restr...
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