PATHOGENESIS AND IMMUNITY

In Vitro and In Vivo Coinfection and Superinfection Dynamics of

Mayaro and Zika Viruses in Mosquito and Vertebrate
Backgrounds
Marco Brustolin,a,b Sujit Pujhari,a,c Gerard Terradas,a Kristine Werling,a Sultan Asad,a Hillery C. Metz,a Cory A. Henderson,a
Donghun Kim,a,d Jason L. Rasgona

a Department of Entomology, the Center for Infectious Disease Dynamics, and the Huck Institutes of the Life Sciences, the Pennsylvania State University, University Park,
Pennsylvania, USA
b Unit of Entomology, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
c Department of Pharmacology Physiology and Neuroscience, University of South Carolina School of Medicine, Columbia, South Carolina, USA
d Department of Vector Entomology, Kyungpook National University, Daegu, South Korea

Marco Brustolin and Sujit Pujhari contributed equally to this article. Author order was determined alphabetically.

ABSTRACT Globalization and climate change have contributed to the simultaneous increase
and spread of arboviral diseases. Cocirculation of several arboviruses in the same geographic
region provides an impetus to study the impacts of multiple concurrent infections within an
individual vector mosquito. Here, we describe coinfection and superinfection with the Mayaro
virus (Togaviridae, Alphavirus) and Zika virus (Flaviviridae, Flavivirus) in vertebrate and mosquito
cells, as well as Aedes aegypti adult mosquitoes, to understand the interaction dynamics of
these pathogens and effects on viral infection, dissemination, and transmission. Aedes aegypti
mosquitoes were able to be infected with and transmit both pathogens simultaneously.
However, whereas Mayaro virus was largely unaffected by coinfection, it had a negative impact
on infection and dissemination rates for Zika virus compared to single infection scenarios.
Superinfection of Mayaro virus atop a previous Zika virus infection resulted in increased
Mayaro virus infection rates. At the cellular level, we found that mosquito and vertebrate cells
were also capable of being simultaneously infected with both pathogens. Similar to our find-

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ings in vivo, Mayaro virus negatively affected Zika virus replication in vertebrate cells, displaying
complete blocking under certain conditions. Viral interference did not occur in mosquito cells.
IMPORTANCE Epidemiological and clinical studies indicate that multiple arboviruses are
cocirculating in human populations, leading to some individuals carrying more than one
arbovirus at the same time. In turn, mosquitoes can become infected with multiple patho-
gens simultaneously (coinfection) or sequentially (superinfection). Coinfection and superin-
fection can have synergistic, neutral, or antagonistic effects on viral infection dynamics and
ultimately have impacts on human health. Here we investigate the interaction between
Zika virus and Mayaro virus, two emerging mosquito-borne pathogens currently circulating
together in Latin America and the Caribbean. We find a major mosquito vector of these
viruses—Aedes aegypti—can carry and transmit both arboviruses at the same time. Our
findings emphasize the importance of considering co- and superinfection dynamics during
vector–pathogen interaction studies, surveillance programs, and risk assessment efforts in
Editor Rebecca Ellis Dutch, University of
epidemic areas. Kentucky College of Medicine
Copyright © 2023 American Society for
KEYWORDS coinfection, Mayaro virus, mosquito, Zika virus, alphavirus, flavivirus,
Microbiology. All Rights Reserved.
vector-borne diseases Address correspondence to Jason L. Rasgon,
jlr54@psu.edu.
The authors declare no conflict of interest.

T he last several decades have seen a dramatic increase in the incidence of arboviral
diseases, a trend attributed to increased international trade and transport, climate
change, and urban crowding. Increasing landscape fragmentation and human activities
Received 17 November 2022
Accepted 19 November 2022

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FIG 1 Geographical range of Mayaro and Zika viruses. Virus distributions are based on reports of human cases,
human serosurvey data, and mosquito surveillance. Countries reporting the presence of ZIKV are shown in red, while
countries reporting the presence of MAYV and ZIKV are shown in blue/red stripes. (No country has reported MAYV
but not ZIKV). The map was generated using mapchart.net/detworld.html and is based on CDC data and previous
publications (17, 60).

are disturbing natural ecosystems, thereby promoting the spillover of new pathogens
with epidemic potential into naive geographic regions (1). More than 150 different
arboviruses have been reported in the neotropical region of the Americas (2), where
they coexist and cocirculate in many areas through sylvatic and/or urban cycles (3, 4).
Despite this diversity and cocirculation of multiple arboviruses in the same geographi-
cal areas, some pathogens often go unnoticed in both vector and host due to lack of
surveillance, overlapping clinical symptoms, and/or the absence of specific diagnostic
tests (5). Clinical studies have identified the cocirculation of multiple arboviruses such
as dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), and Mayaro (MAYV) viruses in epi-
demic areas (6 to 9), but there is only one report of a field-collected mosquito carrying
multiple arboviruses (10). However, this may be an artifact of methodology, as most
surveillance programs analyze pooled mosquito samples to save time and resources,
making it impossible to discern the number of coinfected mosquitoes and their poten-
tial contribution to the transmission cycle of those viruses. In contrast, coinfection and
cotransmission of certain arboviruses have been demonstrated repeatedly under labo-
ratory conditions (11 to 14).

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Mayaro virus (MAYV; Togaviridae, Alphavirus) is an emerging neglected arbovirus thought
to be transmitted by Aedes, Anopheles, and Haemagogus mosquitoes (15). Mayaro virus is
actively circulating in Latin America as well as sporadically in the Caribbean (16, 17) and is
on a trajectory of geographical expansion and increasing incidence, particularly in Brazil and
Peru (17 to 19). For these reasons, MAYV was categorized as an emerging threat to public
health by the Pan American Health Organization (PAHO) in 2019 (20). Zika virus (ZIKV;
Flaviviridae, Flavivirus) is primarily transmitted by Aedes mosquitoes and emerged as a major
arboviral public health concern due to its capacity to cross the placenta in pregnant women
and severely harm the development of human fetuses (21, 22). The overlapping distributions
of these two viruses and the ubiquity of Aedes vectors in Latin America (Fig. 1) both suggest
the potential for coinfection of humans, possibly driven by the bites of coinfected mosqui-
toes. Indeed, there are several reports of MAYV patients coinfected with CHIKV (8, 19), ZIKV
(23), and DENV (23, 24), which could potentially lead to double-infected mosquitoes. Aedes
aegypti is especially worrying as a potential double-infection vector due to its highly anthro-
pophilic behavior (25) and heightened vectorial capacity of ZIKV (26, 27). To date, there are
no empirical data on Ae. aegypti coinfection with MAYV and ZIKV in nature. However, a
recent study identified MAYV and DENV-4 (Flaviviridae, Flavivirus) in pools of Ae. aegypti col-
lected in Mato Grosso (Brazil) (28), possibly indicating the presence of double-infected mos-
quitoes. Considering the vector competence of the species for both ZIKV (26, 27) and MAYV
(15, 29), the overlapping distributions of pathogens and vector in Latin America, and the
field-collected data above, we hypothesize that Ae. aegypti can sustain MAYV and ZIKV trans-
mission cycles simultaneously.

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FIG 2 Double infection routes in mosquito vectors. Schematic depicting how virus-competent
mosquitoes become infected with multiple arboviruses during either a single feeding event (coinfection
[CI]; left) or sequential feeding events 1 and 2 (superinfection [SI]; right).

Simultaneous exposure of a vector to multiple pathogens through a single infection

event is referred to as coinfection (CI), while serial infection of the vector with different
pathogens during sequential feeding events is referred to as superinfection (SI) (30, 31)
(Fig. 2). While both are possible, superinfection may be more likely in nature for mos-
quito species that blood feed multiple times during a single gonotrophic cycle, show
aggressive feeding behavior, and/or seek a second bloodmeal following incomplete
feeding, as is the case for both Ae. aegypti and Ae. albopictus (32). There are three poten-
tial outcomes when multiple arboviruses co- or superinfect the same vector: (i) an
antagonistic interaction that results in partial or total inhibition of one or both viruses,
(ii) a synergistic interaction that enhances one or both viruses, or (iii) a neutral coexis-
tence of both viruses without any alteration of their viral fitness (33). Here, we coinfect
and superinfect Ae. aegypti mosquitoes with MAYV and ZIKV to study their interactions
and consequent effects on vector competence. We also investigate the interaction
kinetics of CI and SI in vertebrate and invertebrate cells, and we demonstrate that coin-
fection occurs at the level of single cells.

RESULTS
Coinfection and superinfection modify infection rate, dissemination rate, and
viral titers in Ae. aegypti. To investigate any effects of CI and SI on vector competence,

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Ae. aegypti females were orally challenged with MAYV and ZIKV via single, simultaneous
(coinfections), or serial (superinfections) spiked blood meals. By quantifying live virus
titers in the bodies, legs, and saliva of Ae. aegypti females following viral challenge, we
calculated infection rates (IR) (mosquitoes with virus-positive bodies/total exposed
mosquitoes), dissemination rates (DIR) (virus-positive legs/virus-positive bodies), and
transmission efficiency (TE) (virus-positive saliva/total exposed mosquitoes).
In the CI experiment, we found differences in viral prevalence for ZIKV but not
MAYV. Specifically, IRs for ZIKV were significantly lower in the coinfected group (CI)
compared to the single-infected group (Z) at both 7 and 14 days postinfection (dpi)
(100% versus 73%, P = 0.0107, and 96% versus 63%, P = 0.0064, respectively; Table 1),
as was the DIR at 7 dpi (72% versus 28%, P = 0.0019; Table 1). In contrast, we did not
find any statistically significant differences in MAYV IR or DIR between groups at either
time point (Table 1).
For both viruses, there were effects of coinfection at the level of titer. Zika virus titers
in the body and legs of coinfected mosquitoes were reduced compared with those of
single infected mosquitoes at 7 dpi (body P , 0.0001; legs P = 0.0019) and 14 dpi (body
P = 0.0002; legs P = 0.0041; Fig. 3B and C). Similarly, compared to single infection, MAYV
titers were lower in body samples of coinfected mosquitoes at 14 dpi (P = 0.0130) but
not 7 dpi (P > 0.05; Fig. 3B). We did not find any statistically significant differences in
the TEs or respective saliva viral titers of MAYV or ZIKV between single and coinfected
groups.

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TABLE 1 Infection rate (IR), dissemination rate (DIR), and transmission efficiency (TE) for MAYV and ZIKV in CI and SI experimentsa
Treatment Groups IR and P value DIR and P value TE and P value
Coinfection
MAYV positivity
7 dpi M vs CI 14/23 (60%) vs 9/19 (47%); ns 13/14 (92%) vs 7/9 (78%); ns 1/23 (4%) vs 0/19; ns
14 dpi M vs CI 17/23 (73%) vs 12/19 (63%); ns 17/17 (100%) vs 11/12 (92%); ns 1/22 (4%) vs 2/19; ns
ZIKV positivity
7 dpi Z vs CI 25/25 (100%) vs 14/19 (73%); P = 0.0107 18/25 (72%) vs 4/14 (28%); P = 0.0019 0/25 vs 0/19; ns
14 dpi Z vs CI 25/26 (96%) vs 12/19 (63%); P = 0.0064 24/25 (96%) vs 11/12 (92%); ns 8/26 (31%) vs 3/19 (16%); ns

Superinfection
MAYV positivity
7 dpi MU vs MZ 12/12 (100%) vs 4/5 (80%); ns 12/12 (100%) vs 3/4 (75%); ns 3/12 (25%) vs 0/5; ns
14 dpi MU vs MZ 12/12 (100%) vs 7/8 (87%); ns 10/12 (83%) vs 6/7 (85%); ns 1/12 (8%) vs 1/8 (12%); ns
7 dpi UM vs ZM 9/14 (64%) vs 20/21 (95%); P = 0.0278 8/9 (88%) vs 18/20 (90%); ns 2/14 (14%) vs 0/21; ns
14 dpi UM vs ZM 13/14 (93%) vs 18/22 (81%); ns 13/13 (100%) vs 17/18 (94%); ns 0/12 vs 1/22 (4%); ns
ZIKV positivity
7 dpi ZU vs ZM 14/14 (100%) vs 21/21 (100%); ns 14/14 (100%) vs 21/21 (100%); ns 9/14 (64%) vs 10/21 (48%); ns
14 dpi ZU vs ZM 11/11 (100%) vs 23/23 (100%); ns 10/11 (90%) vs 20/23 (87%); ns 7/11 (63%) vs 8/23 (35%); ns
7 dpi UZ vs MZ 20/20 (100%) vs 5/0 (100%); ns 17/20 (85%) vs 4/5 (75%); ns 0/20 vs 0/5; ns
14 dpi UZ vs MZ 17/17 (100%) vs 8/8 (100%); ns 17/17 (100%) vs 8/8 (100%); ns 9/17 (53%) vs 5/8 (62%); ns
aInthe coinfection (CI) experiment, we compared single infected groups (MAYV [M] or ZIKV [Z]) to the coinfection group (MAYV 1 ZIKV [CI]). In the superinfection
experiments, superinfected groups (MZ and ZM) have been compared to control groups fed with different combinations of pathogen (M or Z) and uninfected blood (U):
UM, MU, UZ, and ZU, where letter order indicates feeding order. ns, not significant; M, Mayaro virus; Z, Zika virus; U, uninfected blood. In superinfection experiments, two-
letter codes represent two sequential feedings, e.g., MZ = Mayaro virus in the first bloodmeal and Zika virus in the second.

To assess effects of superinfection, mosquitoes were fed two different blood meals
that were either uninfected (U), spiked with ZIKV (Z), or spiked with MAYV (M), totaling
3  2 total treatment groups (UM, UZ, ZU, ZM, MU, and MZ). We tested for effects of
prior infection (e.g., for effects of ZIKV on MAYV by comparing ZM to UM control; Fig.
3E) and subsequent infection (e.g., for effects of ZIKV on MAYV by comparing MZ to
MU control; Fig. 3E), while also controlling for other variables such as the bloodmeal
itself. Thus, we made two statistical comparisons (n = 2 orders of superinfection) per
tissue (n = 3: bodies, legs, saliva)  time point (n = 2: 7 and 14 dpi), totaling 12 compar-
isons per virus (Fig. 3E to J).
Across all the superinfection combinations tested, we observed just one significant

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difference in viral prevalence (i.e., IR, DIR, or TE): an increase in the MAYV IR at 7 dpi fol-
lowing prior infection with ZIKV (i.e., ZM versus UM, 95% versus 64%, P = 0.0278; Table
1). However, this enhancement of MAYV IR did not lead to higher MAYV titers in the
body (Fig. 3E) or to an elevated DIR (Table 1). Conversely, we found that subsequent
ZIKV infection can influence MAYV titer. Specifically, MAYV body and leg titers were
reduced in the MZ group compared to the MU group at 7 dpi (body P = 0.0448, legs
P = 0.0445; Fig. 3E left). The same comparison showed an opposite pattern at 14 dpi
for the body, when a subsequent ZIKV infection intensified the prior MAYV infection
(MZ versus MU, P = 0.0037; Fig. 3E, right).
Likewise, a subsequent MAYV infection slightly intensified prior ZIKV infections in a
time-dependent manner. ZIKV titers in body and legs were greater at 7 but not 14 dpi
following a subsequent MAYV infection (ZM versus ZU, P = 0.0022 and P = 0.0002 for
body and leg samples, respectively; Fig. 3H and I). Salivary ZIKV titers were not statisti-
cally different in either comparison at 7 dpi but showed a reduction in viral titer in the
saliva of the ZM group compared to the ZU group at 14 dpi (P = 0.0221; Fig. 3J).
ZIKV and MAYV double infections in individual Ae. aegypti mosquitoes. We also
determined the percentage of double positive mosquitoes among the CI and SI groups
by assaying the viral titer of both pathogens in different tissues. When challenged with
ZIKV and MAYV simultaneously (CI), 37% (7/19) of mosquitoes became double positive
at both 7 and 14 dpi (Table 2). Additionally, 32% of mosquitoes had double dissemi-
nated infections at 14 dpi (6/19), though none were detected at the earlier time point.

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FIG 3 Infectious Mayaro and Zika viral loads in Ae. aegypti following oral challenge. (A) Schematic timeline for a superinfection experiment. Adult female
mosquitoes were sequentially infected with two arboviruses (or uninfected control), and virus titers quantified in three tissues at 7 and 14 days post
second infection. Single and coinfection designs are not pictured. (B to J) Virus titers are plotted by tissue, treatment group, and time point for (B to D)
both viruses in coinfection experiments, (E to G) MAYV titer in superinfection experiments, and (H to J) ZIKV titer in superinfection experiments. In all
panels, circles depict MAYV titer of individual samples, squares show ZIKV titer of individual samples, and dotted lines depict group medians. CI,
coinfection with MAYV and ZIKV; M, Mayaro virus; Z, Zika virus; U, uninfected blood. Letter order indicates order of infection for superinfection experiments
(i.e., MZ indicates initial infection was with MAYV). Two-tailed Mann-Whitney U tests were used to evaluate significance between groups. Count data (i.e.,
ns for each group) are reported in Table 1. ns, not significant; dpi, days postinfection; FFU, focus forming unit.

Transmission of both pathogens by a single mosquito was not observed at either time
point in the CI group (Table 2).
In the SI experiment, we observed higher IR of double-positive mosquitoes compared
to the CI experiment (Table 2). In addition, double-disseminated infection was recorded

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TABLE 2 Infection rate (IR), dissemination rate (DIR), and transmission efficiency (TE) for
double positive mosquitoes in coinfection and superinfection experimentsa
Treatment Groups IR double positive DIR double positive TE double positive
Coinfection
7 dpi CI 7/19 (37%) 0/19 0/19
14 dpi CI 7/19 (37%) 6/19 (32%) 0/19

Superinfection
7 dpi MZ 4/5 (80%) 3/5 (60%) 0/5
14 dpi MZ 7/8 (86%) 6/8 (75%) 1/8 (12%)
7 dpi ZM 20/21 (95%) 18/21 (86%) 0/21
14 dpi ZM 18/22 (82%) 16/22 (73%) 1/22 (4.5%)
aSignificancewas evaluated using Fisher’s exact tests. Abbreviations are as in Table 1. DIR was calculated as
number of mosquitoes with double positive legs/total number of exposed mosquitoes. In the superinfection
experiment, two-letter codes represent feeding order.

starting from 7 dpi, sooner than in the CI experiment. Saliva samples were double positive
only at 14 dpi. In the ZM group, 1/22 (4.5%) saliva samples tested positive for both viruses,
whereas in the MZ group it was 1/8 (12.5%). Comparisons between IR, DIR, and TE of MZ
and ZM groups did not show any statistically significant differences (data not shown).
There were also no differences between MAYV and ZIKV titers in either CI or SI experiments
(data not shown).
Mayaro virus interferes with ZIKV replication during coinfection and superin-
fection in vertebrate but not mosquito cells. To test whether CI or SI affect viral growth,
we performed growth-curve analyses for MAYV and ZIKV in vitro using cell lines Aag2
(derived from Ae. aegypti; mosquito vector), Vero (derived from African green monkey; non-
human primate), and Huh7.5 cells (derived from human; human host). We again tested sin-
gle, co-, and superinfection, all with a multiplicity of infection (MOI) of 0.1. In the SI experi-
ment, cells were incubated with the inoculum of the first virus for either 2 h or 12 h, after
which it was removed and replaced with either noninfectious media (single infection con-
trols) or an inoculum of the second virus (SI groups; Fig. 4A).
In single-infected Aag2 cells, the titers of MAYV and ZIKV continued to increase after
72 h postinfection, reaching 6.56E 1 05 FFU/mL and 1.31E 1 05 FFU/mL, respectively.
In singly infected vertebrate cells, MAYV titers peaked before decreasing slightly, with
peak titer at 18 h postinfection in Vero (1.97E 1 07 FFU/mL) and 36 h postinfection in
Huh7.5 cells (4.90E 1 07 FFU/mL). ZIKV titers were highest at 60 h postinfection in

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both Vero (7.23E 1 07 FFU/mL) and Huh7.5 (2.20E 1 07 FFU/mL) cells (Fig. 4).
Coinfection and SI both had significant effects on the replication and release of in-
fectious virus particles in vertebrate but not mosquito cells, with ZIKV showing a strong
inhibition. Following CI or initial 12 h infection with MAYV in the SI experiment (MZ),
ZIKV did not replicate effectively in Vero or Huh7.5 cells, indicating a significant out-
competition or inhibition of ZIKV by MAYV (Fig. 4B and C). To further investigate this
phenomenon, we modified the SI experiment in vertebrate cells by adding the second
virus 2 h after the first, rather than after 12 h. With this shortened timeline of superin-
fection, vertebrate cells could support low levels of ZIKV replication and production
(Fig. 4B and C).
MAYV and ZIKV can simultaneously infect and replicate in the same cell. To
determine the capacity of MAYV and ZIKV to infect and replicate in the same cell simul-
taneously, we immunolocalized both viruses in Aag2 and Vero cells (Fig. 5A and B,
respectively) using immunofluorescence microscopy. For vertebrate cells, MAYV, like
other alphaviruses, was predominantly localized to the cell membrane and filopodia
extensions (Fig. 5B, first row; Fig. S1 in the supplemental material). Due to massive and
rapid cytopathic effect caused by MAYV (Fig. S2), most infected vertebrate cells
showed the formation of membrane protrusions. Similar cellular structures and virus
distribution were identified previously in CHIKV-infected cells (13). ZIKV was primarily
localized on the perinucleus and endoplasmic reticulum regions (Fig. 5B, second row),
the proposed replication and assembly sites for flaviviruses (34). Even though they are
found in different cellular compartments, both viruses were able to coinfect the same

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FIG 4 Mayaro and Zika virus in vitro growth curves in vertebrate and invertebrate cells. (A) Schematic timeline for a
superinfection experiment. Cell monolayers (Vero [nonhuman primate], Huh7.5 [human], or Aag2 [mosquito Aedes aegypti])
(Continued on next page)

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FIG 5 Mayaro and Zika viruses can infect and replicate within the same cell. (A) Aag2 and (B) Vero cells were mock, single, or coinfected and viral growth
localized by immunofluorescence. Briefly, at 24 h postinfection (hpi) (Vero) or 48 hpi (Aag2) cells were fixed, permeabilized, and labeled with antibodies for
MAYV, ZIKV, and Hoechst33342 (details in Materials and Methods). Single channels are showed in greyscale. Composite images show MAYV in green (Alexa
Fluor 488), ZIKV in magenta (Alexa Fluor 594), and cell nuclei in blue (Hoechst 33342). Images show some bleed-through. Scale bar = 50 m m. See Fig. S1
for images of coinfection at higher magnification.

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cell. A similar pattern of distribution of MAYV and ZIKV was observed in Aag2 cells,
indicating that both viruses can coinfect the same cell in invertebrate hosts as well.

DISCUSSION
Here, we examine MAYV and ZIKV interactions in a mosquito vector as well as in vitro in
mosquito, primate, and human cells. We find that adult mosquitoes and all tested cell lines
can become infected with MAYV and ZIKV simultaneously. Moreover, the circumstances of
infection—i.e., whether the two pathogens grew as single infections, coinfections, or super-
infections—had significant effects on viral growth in multiple contexts.
Following in vivo coinfection with MAYV, we consistently found reduced ZIKV infec-
tion rates. This finding is similar to previous work, where Rückert et al. (12) demon-
strated a reduction in ZIKV IR when coinfecting with CHIKV, an Alphavirus belonging to
the same antigenic complex as MAYV (Semliki Forest virus complex). Similarly, Muturi

FIG 4 Legend (Continued)

were sequentially infected with two arboviruses (or uninfected control), with either a 2- or 12-h interval between infections.
Viral titers in cell supernatant were then measured at several time points to assay viral growth curves. Single and coinfection
designs are not pictured. All experiments included three technical replicates. (B to D) Viral titers in cell supernatants are plotted
as a function of time for each virus and cell type. Lines depict group means while brackets show 6 SEM. For superinfections
(MZ and ZM), inoculation with each virus was separated by a 12-h interval (solid lines) or 2-h interval (broken lines). All growth
curves are normalized to 0 h postinfection (hpi) with regard to infection with the focal virus. Viral titers were analyzed using
GraphPad Prism 9.

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et al. (31) demonstrated that replication of DENV-4 was suppressed by Sindbis virus
(Togaviridae, Alphavirus) in coinfected Ae. albopictus. In contrast to ZIKV, we did not
observe any variation in the IR of MAYV during coinfection with ZIKV. Our data for
MAYV are consistent with previous studies of Flavivirus/Alphavirus coinfection, which
showed the IR and DIR of CHIKV were not influenced by the presence of ZIKV or DENV-
2 (12, 35). Taken together, these data suggest that in coinfected mosquitoes, the IR of
flaviviruses are negatively affected by the presence of an Alphavirus. However, this
effect is not reciprocal, as the success of an alphavirus was not determined by the pres-
ence of a flavivirus in our study. We did not find any statistically significant differences
in the TEs or respective saliva viral titers of MAYV or ZIKV between single and coin-
fected groups. Consistent with previous studies of Chikungunya/ZIKV coinfection (12,
13), our data suggest that the simultaneous intake of both viruses may negatively
impact their capacity to infect the midgut and subsequently disseminate through the
mosquito body, but the capacity for salivary transmission remains unaffected.
We show that Ae. aegypti can become infected and transmit both pathogens follow-
ing sequential exposure (i.e., superinfection), regardless of the infection order. In gen-
eral, our data suggest that vectors are more permissive to double infection from se-
quential exposure, rather than from simultaneous exposure. Although we analyzed a
relatively small number of animals, our data nevertheless demonstrate the capacity of
the vector to cotransmit both pathogens in its saliva. Transmission of both pathogens
was slow, occurring only 14 days after the second infection event. This implies that,
based on our SI model, the vector may need to survive for approximately 26 to 28 days
in natural conditions to cotransmit MAYV and ZIKV. Field observation and laboratory
experiments indicate Ae. aegypti can survive even longer (36 to 38), but because its
daily survival rate decreases sharply over time, the probability of double transmission
may also drop precipitously (39). Additional experiments—e.g., to assess the mosqui-
to’s extrinsic incubation period or time points between 7 and 14 days after the second
infection (i.e., 9 and 12 dpi)—are needed to understand the minimum time required
for cotransmission.
Interestingly, we observed an increase of MAYV IR in previously ZIKV-infected mos-
quitoes (ZM group). This result indicates a positive effect of a previous ZIKV infection
on MAYV’s ability to stably infect the midgut. The underlying molecular mechanism
remains unclear. However, one possibility is that the previous ZIKV infection could

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have altered the mosquito immune response and thereby created a more suitable
environment for MAYV replication, similar to what has been hypothesized for CHIKV-
ZIKV superinfection (14). Previous studies demonstrated that a subsequent meal with
uninfected blood can increase the DIR in infected mosquitoes and potentially enhance
their TE (40 to 42). This effect may potentially arise from physical expansion of the
midgut following a bloodmeal, which enlarges pores in the basal lamina and allows vi-
ral particles to pass through (40, 43). To investigate whether an increase in DIR in our
experiment could be influenced by the bloodmeal itself and not by the pathogen, we
included control groups fed with uninfected bloodmeals before (U-) or after (-U) viral
challenges (UM, UZ, MU, and ZU). Our results did not show an effect of uninfected
bloodmeals on DIR, even though our assay used a longer interval between the two
bloodmeals (7 days) than the previous study reporting an effect (3 days) (42). This tem-
poral gap was likely sufficient for the basal lamina to revert to normal conformation,
which may limit the window during which a second bloodmeal can affect the DIR.
Further experiments, including different temporal gaps between bloodmeals and a
more in-depth structural analysis of the basal lamina, are needed to investigate this
hypothesis.
Our experimental model assumes that the vector becomes coinfected or superin-
fected with a similar titer of both viruses, a scenario that may not reflect actual field
conditions, where the possible combinations of viruses and their respective titers may
be incredibly variable. Relevant studies have found variable effects when infection pa-
rameters were altered. Muturi et al. (31) showed that varying the virus titer ratio

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Mayaro and Zika Viruses Co- and Superinfection Journal of Virology

between DENV-4 and SINV affected the replication of one or both viruses in an in vitro
SI study. Those data suggest that the relative amounts of different pathogens could
influence the outcome of CI or SI experiments—a parameter we did not test.
Additional studies would be needed to evaluate the impact of different titer combina-
tions in CI and SI double-infection routes.
The cellular and molecular basis of MAYV and ZIKV coinfection and superinfection
dynamics remains unclear. Our in vitro observations demonstrate that dual infection by
these viruses within individual cells of both invertebrates and vertebrates is possible.
However, we noticed several cases of viral interference. Specifically, we observed inhi-
bition of ZIKV replication in mammalian cells during CI and MZ superinfection. At the
same time, partial inhibition was recorded in ZM superinfection. Interestingly, when
the SI experiment in mammalian cells featured a 2-h incubation period between se-
quential infections (instead of 12 h), ZIKV was able to replicate in MAYV-infected cells,
though it could not do so following 12 h of incubation, highlighting that the effect is
time dependent. Interference of arbovirus replication by another arbovirus is influ-
enced by several principal parameters: advantage time of the first virus, order of expo-
sure of the viral pathogens, and difference in MOI (44). Different hypotheses have been
proposed to explain viral interference mechanisms, including competition for replica-
tion sites and cellular substrate resources (44), transacting proteases induced by the
first virus, and superinfection exclusion (45, 46). The latter occurs when a cell infected
by a virus becomes refractory to subsequent infection by the same or a closely related
virus (referred to as a homologous virus) (45). Superinfection exclusion can occur at dif-
ferent steps of the replication cycle, as demonstrated by several studies using Semliki
Forest virus (Togaviridae, Alphavirus) and CHIKV, including binding and internalization,
viral genome transcription, and protein translation (47 to 50). In a recent study,
Boussier et al. (50) showed that CHIKV excludes influenza A virus (Orthomyxoviridae,
Alphainfluenzavirus) replication in vitro, and demonstrated the capacity of alphaviruses
to interfere with the replication cycle of different viral families. Several in vivo studies
also suggest that sequential infection of Alphavirus and Flavivirus results in coinfection,
not in exclusion (13, 51, 52), although none of them have examined MAYV in conjunc-
tion with a flavivirus.
We observed coinfected cells using immunofluorescence microscopy, demonstrat-
ing that a single cell can be simultaneously infected with both viruses (Fig. 5A and B,

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third row). We observed a cytopathic effect in mammalian cells caused by MAYV after
the first 24 to 36 h postinfection (Fig. S2), similar to other alphaviruses (13, 45, 53). The
loss of structural functionality and the partial destruction of cells may partially prevent
infection by ZIKV and/or its subsequent replication, especially when the interval
between infections is longer (e.g., SI with a 12-h initial incubation time). The replication
cycle of alphaviruses is relatively short, and progeny virions can be detected 4 to 6 h
postinfection (54 to 56). Conversely, 8 to 12 h are generally needed to detect progeny
virions of flaviviruses (57). This disparity in viral kinetics could have influenced the
capacity of ZIKV to replicate in the presence of MAYV, which rapidly induces a severe
structural and morphological change to the infected cells. Superinfection with a
shorter incubation time (2 h) likely limited the impact of MAYV-induced cell damage,
which may underlie the smaller effect of MAYV upon ZIKV in the 2-h experiments.
However, across all tested experimental conditions, we found MAYV inhibits ZIKV repli-
cation to some degree, suggesting viral interference at a certain replication stage. We
can therefore speculate that, when introduced in vertebrate cells before or together
with ZIKV, MAYV takes over the cellular transcription and/or translational machinery,
leaving minimal resources for ZIKV replication. Additional studies are required to
understand the molecular dynamics of CI and SI with MAYV and ZIKV in both inverte-
brate and vertebrate models.
Coinfection and superinfection are poorly studied in mosquito vectors. Despite evi-
dence of circulation of multiple pathogens in the same geographical areas, no data are
currently available about prevalence of coinfected vectors in the field. The

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Mayaro and Zika Viruses Co- and Superinfection Journal of Virology

implementation of precise sampling strategies for field-collected mosquitoes would be

fundamental to investigate the prevalence of multi-infected vectors, to understand the
importance of CI and SI in virus transmission cycles and on mosquito fitness (if any), as
well as an in-depth description of the interactions of pathogens at the cellular and mo-
lecular level. Future work could also examine the effects of CI and SI on other vector-
borne pathogens, such as Plasmodium or microfilariae nematodes.

MATERIALS AND METHODS

Mosquitoes. Aedes aegypti (Rockefeller strain) were obtained from Johns Hopkins University and
reared and maintained at the Millennium Sciences Complex insectary (The Pennsylvania State
University, University Park, PA, USA) under the following environmental conditions: 27°C 6 1°C, 12:12 h
light:dark diurnal cycle at 80% relative humidity. The larvae were fed with ground fish flakes (TetraMin,
Melle, Germany). Adult mosquitoes had free access to 10% sucrose solution.
Cells. Vero African green monkey kidney cells (CCL-81; ATCC, Manassas, VA, USA) and Huh7.5 (human
liver cell line) (kind gift from Craig Cameron, University of North Carolina) were cultured in Dulbecco’s
modified Eagle’s medium (DMEM; Life Technologies) containing 10% fetal bovine serum (FBS), penicillin
(100 U/mL), streptomycin (100 m g/mL), 10 mM HEPES, and 200 mM glutamine. Aag2 Ae. aegypti cells (kind
gift from Elizabeth McGraw, The Pennsylvania State University) were grown in Schneider’s insect medium
supplemented with 10% FBS, penicillin (100U/mL), streptomycin (100 m g/mL), and 200 mM glutamine.
Virus. Zika virus strain MR766 (NR-50065; BEI Resources, Manassas, VA, USA) and Mayaro virus strain
BeAr505411 (NR-49910; BEI Resources, Manassas, VA, USA) were propagated on Vero cells. Stock solu-
tions were aliquoted and stored at 270°C until used. Viral stock titers were determined by focus forming
assay (FFA).
In vitro single, co-, and superinfection. Cells were seeded at 80 to 90% confluence in 24-well plates
and infected with ZIKV and/or MAYV with an MOI of 0.1 in FBS-free media the day after seeding. Cells were
incubated independently with either ZIKV or MAYV for single infections, with both viruses at the same time
for coinfection, or sequentially (repeated with two different incubations: 2 h or 12 h apart) for superinfection.
After 1 h of incubation at 37°C, the viral inoculum was removed, cells washed twice to remove unbound vi-
rus, and fresh medium added. At each time point, 200 m L of virus-containing supernatant was collected and
stored at 280°C. Upon sampling, 200 m L of fresh media were added to each well to maintain the same initial
volume. Samples were analyzed by focus forming assay as previously described (15). Briefly, 30 m L of 10-fold
serial dilutions of each sample were used to infect Vero cell monolayers in 96-well plates. After 24 h (for
MAYV) or 36 h (for ZIKV), cells were fixed, permeabilized, and labeled overnight at 4°C using the monoclonal
anti-CHIKV E2 envelope glycoprotein clone CHK-48 (which cross-reacts with MAYV [58]) (BEI Resources,
Manassas, VA, USA) or the monoclonal anti-Flavivirus group antigen (Clone D1-4G2-4-15) (BEI Resources,
Manassas, VA, USA). CI and SI samples were analyzed twice to assess each virus separately using its corre-
sponding primary antibody. We used Alexa-488 goat anti-mouse IgG secondary antibody (Invitrogen, Life
Science, Eugene OR, USA), and resultant green fluorescent foci were viewed and counted using an Olympus
BX41 microscope equipped with an UPlanFI 4 objective and a FITC filter. Three technical replicates were
performed for each condition.

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Vector competence assays. Unfed 5- to 7-day-old female mosquitoes were separated into groups
of approximately 80 individuals and fed with virus-spiked (final titer of 1  107 FFU/mL for each virus)
infected human blood via a glass feeder jacketed with 37°C distilled water for 30 to 45 min. In the CI
experiment, blood contained both MAYV and ZIKV. In the single and SI experiments, blood contained a
single virus (M or Z) (day 0). A control group fed with uninfected blood (U) was included as the negative
control. After feeding, mosquitoes were anesthetized briefly at 4°C, and engorged females were selected
and transferred to a clean cardboard cup with access to 10% sugar solution ad libitum. In the SI experi-
ment, two sequential feeding events were scheduled. During the first feed (day 26), three groups were
exposed to uninfected blood, two groups to ZIKV (Z), and two groups to MAYV (M). During the second
feed (day 0), all groups were provided access to a second blood meal, resulting in seven total treatment
combinations: UM, UZ, MU, ZU, MZ, ZM, and UU. As before, fully engorged females were anesthetized,
selected, and housed as previously described.
At 7 and 14 days postinfection (dpi), living mosquitoes were anesthetized with triethylamine (Sigma,
St. Louis, MO) for approximately 30 s. Legs were dissected from the body, then mosquitoes were forced
to salivate into a glass capillary tube filled with a 1:1 solution of 50% sucrose solution and FBS for
30 min. Bodies and legs were collected in separate 2-mL tubes each containing 1 mL of mosquito dilu-
ent (20% FBS in Dulbecco’s phosphate-buffered saline, 50 m g/mL penicillin/streptomycin, 50 m g/mL
gentamicin, and 2.5 m g/mL fungizone) with a single sterile zinc-plated steel 4.5 mm bead (Daisy, Rogers,
AR, USA). Tissues were homogenized at 30 Hz for 2 min using TissueLyser II (Qiagen GmbH, Hilden,
Germany) then centrifuged for 30 s at 11,000 rpm. Saliva samples were collected in a 2-mL tube contain-
ing 0.1 mL of mosquito diluent. All samples were stored at 270°C until used. Samples were analyzed by
focus forming assay as described above.
All experimental infections were performed at the Eva J. Pell ABSL-3 Laboratory for Advanced
Biological Research (Pennsylvania State University).
Infection rates (IR) (mosquitoes with virus-positive bodies/total exposed mosquitoes), dissemination
rates (DIR) (positive legs/positives bodies), and transmission efficiency (TE) (positive saliva/total exposed
mosquitoes) were calculated for all the tested groups. In the coinfection experiment, we compared M

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Mayaro and Zika Viruses Co- and Superinfection Journal of Virology

versus CI and Z versus CI. In the superinfection experiment, we compared MU versus MZ, ZU versus ZM,
UM versus ZM, and UZ versus MZ.
Viral titers in body, legs, and saliva samples were calculated in units of FFU/mL, and comparisons
were made between the same groups as above.
Immunolocalization of MAYV and ZIKV on Vero and Aag2 cells. Cells were seeded at a density of
1  105 cells/well (Vero cells) or 1  106 cells/well (Aag2 cells) in a 4-well chamber slide and grown at 37°C
with 5% CO2 (Vero cells) or 27°C without CO2 (Aag2). After 24 h, cell monolayers were infected with ZIKV,
MAYV, or both at an MOI of 1 as described above. After 24 (Vero) or 48 (Aag2) h, cells were fixed with 4%
paraformaldehyde (PFA) and permeabilized using 0.02% Tween 20. Cells were then labeled with the mouse
monoclonal anti-CHIKV E2 envelope glycoprotein clone CHK-48 (BEI Resources, Manassas, VA, USA) and the
rabbit monoclonal anti-Flavivirus group antigen (4G2, MAB12411) (The Native Antigen Company, Kildington,
UK). The antibodies used for immunofluorescence included Alexa Fluor 488 conjugated goat anti-mouse IgG
(Invitrogen, Life Science, Eugene OR, USA) and Alexa Fluor 594 conjugated donkey anti-rabbit IgG
(Invitrogen, Life Science, Eugene OR, USA). Hoechst 33342 Fluorescent Stain (Thermo Scientific) was used for
nuclear staining. Upon staining, slides were preserved using ProLong Diamond Antifade mountant (Thermo
Fisher Scientific, Waltham MA, USA). Cells were examined and imaged using an Echo Revolve 2 (Discover
Echo, Inc., San Diego CA, USA) with images processed using ImageJ software (59). For the composite images,
Composite Max mode was used.
Statistical analysis. GraphPad Prism software version 9 was used for all analyses. Differences in IR,
DIR, and TE (i.e., count data) were analyzed by Fisher’s exact test. Two-tailed Mann-Whitney U tests were
used to compare viral titers in body, legs, and saliva samples of different groups. A P value of ,0.05 was
considered statistically significant.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 1.6 MB.

ACKNOWLEDGMENTS
We thank the personnel of Eva J. Pell ABSL-3 Laboratory for Advanced Biological
Research for their help and technical support, Craig Cameron (University of North Carolina)
and Elizabeth McGraw (Pennsylvania State University) for cell lines, and Sage McKeand
(Pennsylvania State University) for Fig. 2 design. The following reagents were obtained
through BEI Resources, NIAID, NIH, as part of the WRCEVA program: Mayaro Virus,
BeAr505411, NR-49910; Zika virus, MR766, NR-50065, Monoclonal Anti-Chikungunya Virus
E2 Envelope Glycoprotein, Clone CHK-48 (produced in vitro), NR-44002, monoclonal anti-
Flavivirus group antigen, Clone D1-4G2-4-15, NR-50327.
This research was funded by NIH grants R21AI128918, R01AI116636, and R01AI150251,
USDA Hatch funds (accession number 1010032; project number PEN04608), and funds from
the Dorothy Foehr Huck and J. Lloyd Huck endowment to J.L.R. C.A.H. was supported in

Downloaded from https://journals.asm.org/journal/jvi on 16 January 2023 by 201.55.48.3.

part by an NSF Graduate Research Fellowship Program award (ID 2018258101). D.K. was
supported in part by a National Research Foundation of Korea (NRF) grant funded by the
Korea government (MSIT; 2019R1G1A1100559).

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