Detection of Wolbachia in Aedes albopictus and Their Effects on Chikungunya Virus
Am. J. Trop. Med. Hyg.
Detection of Wolbachia in Aedes albopictus and Their Effects on Chikungunya Virus
Noor Afizah Ahmad 0 1 2
Indra Vythilingam 0 1
Yvonne A. L. Lim 0 1
Nur Zatil Aqmar M. Zabari 1 2
Han Lim Lee 1 2
0 Department of Parasitology, Faculty of Medicine, University of Malaya , Kuala Lumpur , Malaysia
1 Parasitology, Faculty of Medicine, University of Malaya , Lembah Pantai, 50603 Kuala Lumpur , Malaysia
2 Medical Entomology Unit, World Health Organization Collaborating Centre for Vectors, Institute for Medical Research , Kuala Lumpur , Malaysia
Wolbachia-based vector control strategies have been proposed as a means to augment the currently existing measures for controlling dengue and chikungunya vectors. Prior to utilizing Wolbachia as a novel vector control strategy, it is crucial to understand the Wolbachia-mosquito interactions. In this study, field surveys were conducted to screen for the infection status of Wolbachia in field-collected Aedes albopictus. The effects of Wolbachia in its native host toward the replication and dissemination of chikungunya virus (CHIKV) was also studied. The prevalence of Wolbachia-infected field-collected Ae. albopictus was estimated to be 98.6% (N = 142) for females and 95.1% (N = 102) for males in the population studied. The Ae. albopictus were naturally infected with both wAlbA and wAlbB strains. We also found that the native Wolbachia has no impact on CHIKV infection and minimal effect on CHIKV dissemination to secondary organs.
Aedes aegypti and Aedes albopictus are competent
vectors for dengue virus (DENV) and chikungunya virus
(CHIKV). The latter also known as Asian tiger mosquito
always receive(s) lesser attention as it is considered the
bridge vector to Ae. aegypti-dominated urban epidemics.1
Nonetheless, the Asian tiger mosquitoes can still act as
the principle vector in epidemic areas where Ae. aegypti
is present.1,2 The worldwide expansion of the geographic
range of Ae. albopictus makes this invasive vector of
human pathogenic viruses a major concern in many
locations.1 In 2005, Ae. albopictus was incriminated as a sole
vector responsible for causing chikungunya outbreak of
unprecedented magnitude in the Indian Ocean.3 The
outbreak continued to spread to central Africa,4 India,5 and
then towards Europe,6 Asia,7–10 and North America.11 In
Malaysia, a nationwide outbreak occurred in 2008, starting
in Johor State, which later spread to other states and
federal territories affecting about 10,000 people.9,12,13
Phylogenetic analysis of the viral sequence isolates revealed a
point mutation of alanine to valine at point 226 (A226V) of
the E1 gene of the polyprotein, enhancing the CHIKV
replication and transmission efficacy in Ae. albopictus.14,15
Wolbachia species are obligate intracellular bacteria
that infect a wide range of insects as well as some species
of nematodes, making it the most ubiquitous bacteria
yet described.16,17 Wolbachia infection has also been
detected in mosquitoes including Ae. albopictus but is not
found in Ae. aegypti. Wolbachia are vertically transmitted
from infected females to their progeny. Wolbachia can alter
the reproduction of its host in various ways, one such way is
cytoplasmic incompatibility (CI). CI is a form of sterility in
which if the same and compatible Wolbachia strain is not
present in the egg during embryogenesis, embryonic
development will be disrupted.18,19 CI phenomenon gives a
reproductive advantage to the infected females, at which they can
mate successfully with both infected and uninfected males
and hence enhances Wolbachia invasion in a population.
Wolbachia has drawn much attention as some of the
Wolbachia strains (e.g., wMelPop and wMel+wAlbB) have
shown to reduce mosquito life span and/or induce pathogen
blocking effects on the invertebrate hosts. These effects can
substantially reduce the risk of pathogen transmission.20,21
Nonetheless, the Wolbachia-mediated viral blocking
effect is not ubiquitous. Unlike the case for transinfected
hosts, effect of Wolbachia on virus replication in native
hosts has been reported to be inconsistent. For instance,
the naturally occurring Wolbachia of Aedes notoscriptus
do not induce DENV interference within the native hosts22
contrary to the report on Ae. albopictus that demonstrated
that native Wolbachia can limit transmission of DENV.23
Another study on Drosophila demonstrated that native
Wolbachia render pathogen resistance toward the RNA
viruses in their original hosts.24–26 The Wolbachia-based
vector control strategies have taken the form of either
population replacement or the incompatible insect
technique (IIT) strategy. The population replacement strategy is
highly dependent on the ability of the Wolbachia to invade
and replace the target population with a population that
cannot transmit virus.27,28 On the other hand, the IIT
approach involves a continuous inundated release of males
carrying an incompatible Wolbachia strain with that in the
existing mosquito population, to suppress mosquito numbers
below a threshold that enables continued virus
transmission.29,30 In this study, we aim to determine the Wolbachia
infection in field-collected Ae. albopictus from different
geographical regions. This study is crucial to cater for the
scarcity of information on Wolbachia infection status in
fieldcollected Ae. albopictus population in Malaysia. Furthermore,
we also investigated the effects of the naturally occurring
Wolbachia on the replication of CHIKV in Ae. albopictus.
These findings will help to facilitate the understanding of
the Wolbachia–CHIKV–Ae. albopictus interaction, which will
serve as a platform for the Wolbachia-based vector control
approach to be conducted in Malaysia.
Mosquito collection. Aedes albopictus was collected
from eight collection sites from five states in Malaysia as
shown in Figure 1. A minimum of 50 ovitraps were set in
each location for 5 days and were at least 150 m apart to
minimize the probability of progeny from the same mother.
FIGURE 1. Map of Peninsular Malaysia showing Aedes albopictus collection sites. Samples were collected from eight different collection
sites (indicated by red stars) from five states in Peninsular Malaysia.
Aedes ovitrapping was conducted following the guidelines
of Ministry of Health, Malaysia, on ovitrap deployment.31
Ovitraps were brought back to the insectarium in the
Medical Entomology Unit, Institute for Medical Research (IMR),
Kuala Lumpur. Eggs collected were hatched and larvae
(L3) recovered were individually identified to species level
according to the key by Mahadevan and others.32 The
identified Ae. albopictus larvae from each ovitrap were
placed into a plastic container and were supplied with liver
powder. Once the larvae reached pupal stage, the plastic
containers were placed inside an adult cage (25 × 25 ×
50 cm). Mosquito species were again confirmed. Adult
mosquitoes were supplied with 10% glucose incorporated with
liquid B-complex (Atlantic Laboratories Corp. Ltd, Bangkok,
Thailand) and maintained using standard condition of 28°C
with 70–80% of relative humidity.
DNA extraction. Adults (F0) aged between 7 and 10 days
were subjected to DNA extraction. Briefly, 4–10 mosquitoes
(males and females) recovered from each ovitrap were killed
by placing them in freezer for an hour. The mosquitoes
were individually homogenized in 180 μL cell lysis solution
(ATL Buffer) and incubated in 20 μL proteinase K at 56°C in
water bath for 3 hours. The subsequent procedures were
performed according to the QlAamp® DNA Mini Kit protocol
(Qiagen™, Hilden, Germany).
Detection of Wolbachia. Multiplex polymerase chain
reaction (PCR) was carried out using a temperature profile
of 95°C for 15 seconds, 57°C for 30 seconds, and 72°C for
1 minute for 35 cycles using wsp primers. Primers used
were 328F and 691R for wAlbA strain and 183F and 691R
for wAlbB strain as described by Zhou33 (328F, 5′-CCA
GCA GAT ACT ATT GCG-3′; 183F, 5′-AAG GAA CCG AAG
TTC ATG-3′; 691R, 5′-AAA AAT TAA ACG CTA CTC CA-3′).
The PCR mixture contained 5 μL of extracted DNA, 12.5 μL
of MyTaq™ Mix (Bioline, Taunton, MA), 1 μL of each primer
(10 μM), and 4.5 μL of ddH2O. Negative and positive controls
for the PCR assay were included in each run. The positive
control was obtained by screening the adult Ae. albopictus
(resident strain) using PCR and sequencing of wsp gene to
confirm that the amplified PCR product obtained was
Wolbachia. The quality of the extracted DNA was checked
using the 12S rRNA primer sets (12SA, 5′-AAA CTA GGA
TTA GAT ACC CTA TTA T-3′; 12SB, 5′-AAG AGC GAC
GGG CGA TGT GT-3′) to screen samples that were
negative for wsp primers using the temperature profile of 95°C
for 15 seconds for denaturation, 47°C for 30 seconds for
annealing and 72°C for 1 minute for extension, conducted
for 35 cycles. Samples that were negative for wsp primers
but positive for 12S RNA primers were scored as uninfected.
All the positive PCR products were visualized under 1.5%
agarose gel electrophoresis.
Sequencing of Wolbachia endobacterium. The positive
PCR product was purified using QIAquick® Gel Extraction
Kit (Qiagen™) before DNA sequencing. A minimum of 10
purified DNA extracts from individuals of each locality were
outsourced for sequencing. All sequences were searched
against the GenBank nucleotide database using the Basic
Local Alignment Search Tool (BLAST®) provided by the
National Center for Biotechnology Information (http://blast.
ncbi.nlm.nih.gov/Blast.cgi). Partial wsp gene sequences of
Wolbachia were aligned using the Clustal-W algorithm and
the evolutionary distances of Wolbachia isolates from
Ae. albopictus was constructed using neighbor-joining tree,
utilizing Kimura-2P analysis with 1,000 bootstrap replicates
in MEGA 6.0 software.34 A Wolbachia sequence from Culex
quinquefasciatus was included as an outgroup.
CHIKV production. CHIKV (Asian strain) was provided by
the Virology Unit, IMR, Kuala Lumpur. The virus was isolated
during the outbreak in Bagan Panchor, Perak, in 2006. The
CHIKV was maintained in BHK-21 cell lines. Stock virus
prepared by freeze-thawing the infected cells once, centrifuging
the suspension at 40,000 × g and storing the filtered
supernatants at 80°C. The infected cells were maintained in
Virology Unit, IMR. The titer of the CHIKV stock was
determined using the 50% cell culture infectious dose assay.
Mosquito samples for artificial oral infection. Mosquitoes
from three localities were used for the oral infection
experiment. Each locality was chosen to represent one habitat:
1) Besar Island (tourism island), 2) Tenggol Island (remote
island), and 3) Bandar Rinching (urban residential area).
Females from Besar Island were derived from the same
collections used for Wolbachia screening. Mosquitoes from
Tenggol Island and Bandar Rinching were derived from the
existing colony in insectarium. All mosquitoes used for
artificial oral infection were F6 generation.
Tetracycline treatment to clear Wolbachia. Adult
mosquitoes were provided with a solution of 0.75 mg/mL
tetracycline dissolved in 10% sucrose. After every
treatment, 10 randomly selected treated mosquitoes from
each generation were tested by PCR for Wolbachia
infection. Treatment was continued if any randomly tested
mosquitoes were positive for Wolbachia confirmed via PCR.
This treatment was performed up to four generations of
mosquitoes. Colonies of Wolbachia-free Ae. albopictus were
maintained for a further two generations without
tetracycline before experiments commenced to allow the
reestablishment of beneficial microbiota.
Experimental oral infections. Experimental oral infection
with CHIKV was conducted within an Arthropod
Containment Level 2 insectarium. The artificial membrane feeding
technique was performed using the Hemotek Feeding
Systems (Discovery workshops, Accrington, United Kingdom)
housed in an isolation glove box. Human blood used for
artificial feeding was sent to Virology Unit, IMR, and confirmed
to be negative by neutralization assay for CHIKV antibodies.
Blood suspension containing 1:9 of CHIKV (titer 107
plaqueforming units/mL) in human blood was used for artificial
feeding. Uninfected samples (control group) were obtained
by feeding the mosquitoes with human blood only. A total
of 250 adult mosquitoes from each group Wolbachia
infected (w+) and Wolbachia-free (w−) aged 3–5 days that
have been starved overnight were subjected to artificial
feeding. The blood was presented to the mosquitoes by
placing the cups (containing 50 mosquitoes each) below
the feeder with the surface of the nylon netting of the cup
in contact with the membrane of the feeder. Mosquitoes
were allowed to feed for approximately 20–30 minutes.
The mosquitoes in cups were cold anesthetized by placing
in −20°C freezer for 30 seconds. The mosquitoes were then
sorted. All unfed mosquitoes were discarded. Engorged
mosquitoes were placed in cups (10 per cup) and kept in
incubator at 28°C and humidity of 80% for planned time
points (days 0, 1, 2, 3, 5, 7, and 10) postinoculation (PI)
studies. At each time point, mosquito samples from at least a
single cup were cold anesthetized by placing them in −20°C
for 30 seconds, before dissection to remove midguts,
salivary glands, and ovaries. Individual mosquito was put on a
glass slide. The thorax and head of the mosquito were first
removed, followed by dissection of the salivary glands in a
drop of saline. The abdomen was then dissected to remove
midguts and ovaries in a drop of saline, respectively. Glass
slide was replaced for each individual mosquito, and fresh
drops of saline were used for each organ examined. It was
ensured that the dissecting needles were rinsed in alcohol
between each dissection to prevent contamination. A total
of four engorged mosquitoes fed with clean human blood
at days 0 and 10 PI were kept aside to serve as negative
control. For each experimental time point, the infection rate
is defined as the number of midguts with detectable virus
titer divided by the number of mosquitoes sampled. The
dissemination rate was defined as the number of salivary
glands with detectable virus titer divided by the number of
midguts with detectable virus titer.
Nucleic acid extraction and quantitative PCR. Total
nucleic acid was extracted from the dissected organs
(midguts and salivary glands). Extraction was performed
with innuPREP DNA/RNA Mini Kit (Analytik Jena AG,
Jena, Germany) that enables the isolation of both RNA and
DNA. RNA was used to determine viral load by real-time
reverse transcription PCR (RT-PCR), and DNA to check
for the presence of Wolbachia in ovaries using
conventional PCR. For w+ group, individual mosquito that was
screened negative for Wolbachia (in ovaries) was excluded
from the dataset. A minimum number of nine mosquitoes
were used for each time point. A standard curve was
generated using 10-fold serial dilutions of RNA synthetic
transcript with known copy number.
Determination of limit of detection. RNA template of
known concentration was diluted using six 10-fold serial
dilutions. Each concentration was run in triplicate for a total
of three runs. Dilutions of CHIKV RNA ranged from 2.4 ×
106 RNA copies to 2.4 × 101. The linear range was
established with acceptance criteria of R2 > 0.98 with an
efficiency of > 90%. The limit of detection (LOD) was
defined as the lowest concentration of viral RNA that can
be detected in ≥ 95% of nine replicates.
Statistical analysis. GraphPad Prism version 7.00 for
Windows (GraphPad Software, La Jolla, CA; www.graphpad.
com) was used to construct graphs. All statistical analyses
were conducted using the IBM SPSS Statistics (version 19;
Armonk, NY). CHIKV infection and dissemination rates
were compared with Fisher’s exact test with two-tailed
P values. Non-parametric statistical, Mann–Whitney U tests
was used to assess the statistical differences for CHIKV
titer in w+ and w− groups, and P values < 0.05 were
considered statistically significant. P values were adjusted
for multiple tests using the Kruskal–Wallis test with
Distribution of Wolbachia in Malaysian Ae. albopictus. A
total of 244 Ae. albopictus samples collected from eight
sites in five states (Malacca, Selangor, Terengganu, Perak,
and Pahang) in Malaysia were screened for Wolbachia
infection using wAlbA- and wAlbB-specific wsp gene primers.
Our results showed a high percentage of Wolbachia infection
with 98.6% in females and 95.1% in males. For wsp gene,
phylogenetic analysis revealed that Wolbachia isolates from
the present study were closely related to Wolbachia
isolates from different geographical regions, and the sequences
were grouped into wAlbA and wAlbB clades, respectively
(Figure 2). The size of the wsp fragment for wAlbA was
341 bp and wAlbB was 463 bp.
Table 1 shows the frequency of double and single
infections of Wolbachia in the Ae. albopictus populations
sampled from all the collection sites. For females, 97.2% (138/
142) of the samples were superinfected with both wAlbA
and wAlbB. Only 1.4% (2/142) of samples were singly
infected with either wAlbA or wAlbB. Another 1.4% (2/142)
samples were scored as uninfected. For males, 49.0% (50/
102) was superinfected with wAlbA and wAlbB followed by
46.1% (47/102) infection with wAlbB only. The remaining
4.9% (5/102) samples were uninfected. None of the males
97.2 (138) 0.7 (1) 0.7 (1)
were positive for wAlbA only. The uninfected samples
were confirmed by running the DNA with the 12S RNA
primer set for mitochondrial DNA as a quality check.
Analysis of linearity and LOD determination. The linear
dynamic range for the multiplex RT-PCR assay was 100%
at the range of 100 to 102 RNA copies per reaction but
decreased to 88.8% at 10 copies. The LOD was set at
100 copies, which correspond to the mean Cq of 28.
Samples with Cq value > 28 was scored as uninfected. Only
mosquitoes that score above the LOD was reported to infer
FIGURE 4. The percentage of chikungunya virus (CHIKV) infection in (A) midguts and (B) salivary glands for Wolbachia infected (w+) and
Wolbachia-free (w−) for all localities.
CHIKV infection and dissemination in midguts and salivary
Wolbachia infection status in ovaries of mosquitoes
used in the experimental oral infection study. The
presence of Wolbachia in females used in the experimental oral
infection study was confirmed by screening the ovaries. It
was noticed that ovaries for females were highly infected
with at least 90% infection and above. There was no
significant difference in the percentage of Wolbachia infection
for all the time points for the three localities (P > 0.05,
Fisher’s exact test) (Figure 3).
Laboratory infection of Wolbachia infected (w+) and
uninfected (w−) Ae. albopictus with CHIKV. CHIKV midgut
infections were observed for w+ and w− group for each time
point, the midgut infection rate was consistent with
percentage of at least 60% for w+ and 70% for w− for all localities
(Figure 4A). There was no significant difference in the
infection and dissemination rate between w+ and w− groups for
all the time points tested for the three localities (P > 0.05,
Fisher’s exact test). It was noticed that CHIKV was detected
in salivary glands as early as day 2 PI (Figure 4B).
The number of CHIKV genome copies in midguts was
not significantly different between w+ and w− groups at
any time point tested. CHIKV titer reached the peak as
early as day 2 or day 3. For Besar Island, the peak titer
for w+ and w− groups (median = 106.9 versus 107.9 viral
copies/midgut, P = 0.05) achieved at day 3 PI. For Tenggol
Island, the virus replication reached its peak at day 3 for
w+ (median = 108.9 viral copies/midgut) and day 2 PI
for w− (median = 108.3 viral copies/midgut), respectively.
For mosquitoes sampled from Bandar Rinching, the viral
copies were at the peak of viral infection for w+ and w−
at day 3 PI (median = 107.8 versus 107.2 copies/midgut,
P = 0.143) (Figure 5A).
For salivary glands, the amount of CHIKV copies ranged
from below the LOD (100 CHIKV copies) to 108 CHIKV
copies in both w+ and w− groups. Although there was a
variation in median CHIKV titer among the groups, these
differences were not statistically significant at other time
points, except for day 2 PI (Figure 5B). For Besar Island,
CHIKV copy number of w+ group was almost one log lower
than for w− group for salivary glands (medians = 101.8 versus
102.4, P < 0.05, Mann–Whitney U tests) (Figure 5B). Similarly,
for Bandar Rinching, CHIKV copies in salivary glands for
w+ was lower than w− group (median = 102.1 versus
103.3, P < 0.001, Mann–Whitney U tests) (Figure 5B).
The total CHIKV titer in different organs was compared
between localities as shown in Table 2. The statistical
analysis using Kruskal–Wallis test showed that total
CHIKV titer in midguts were significantly different from at
least one locality (P < 0.05). Subsequent post hoc analysis
using the Bonferroni correction substantiated the
difference. For w+, a significantly lower overall CHIKV titer in
midguts was observed in Besar Island compared with
Bandar Rinching and Tenggol Island. For w− group, a
significantly higher overall CHIKV titer in midguts was observed
in Tenggol Island compared with Bandar Rinching and
Besar Island. No significant difference for CHIKV titer in
salivary glands was observed among the three localities for w+
(P = 0.805) and w− (P = 0.431).
Our study on the distribution of Wolbachia in the
fieldcollected Ae. albopictus showed a high infection rate of
98.6% in females and 95.1% in males. Females showed a
common superinfection of wAlbA and wAlbB, occurred at
a high prevalence of 97.2%. This is in accordance to other
studies, showing superinfection percentage is common in
field-collected Ae. albopictus infecting at least 96% of
mosquitoes.35–39 Wolbachia are expected to rapidly spread
to fixation once a Wolbachia infection enters a
population.40,41 As females that carry both wAlbA and wAlbB strain
can successfully mate with males that are either singly or
superinfected, the superinfection of Wolbachia in females
may explain the high fidelity of maternal transmission of
Wolbachia of the mosquito species in the wild.42 The males
were also most commonly superinfected with wAlbA and
wAlbB at a percentage of 49.0% followed by 46.1% of
wAlbB-only infection. No single infection of wAlbA was
detected in males. Apart from the maternal transmission
efficacy of Wolbachia, the geographical populations are
reported to be strong predictors affecting Wolbachia
infection rate and pattern.43 For example, a single-strain (wAlbA)
infected population has been described in Koh Samui and
Mauritius Islands that are geographically distinct from the
superinfected populations.39,44,45 A study by Tortosa and
others demonstrated that the density of wAlbA significantly
decreased with age in male Ae. albopictus population in
which a complete loss was observed within 5-day period
postemergence.46 Therefore, this finding may serve as a
plausible explanation for the lack of single wAlbA strain
infection in the males despite having females that were
Besar Island, Malacca 55 59 6.07 (5.21, 6.93)a* 2.38 (1.63, 4.63)a 7.02 (4.01, 7.80)a 2.16 (1.22, 3.45)a
Tenggol Island, Terengganu 58 60 7.44 (5.20, 8.09)b 2.70 (1.45, 6.36)a 7.83 (6.10, 8.53)b 2.25 (1.44, 4.34)a
Bandar Rinching, Selangor 60 57 7.01 (6.04, 7.69)b 2.93 (1.73, 5.39)a 6.98 (6.02, 7.50)a 2.81 (1.28, 5.50)a
Data are presented as median CHIKV copy number (IQR) and were compared among localities by Kruskal–Wallis test and Mann–Whitney U test for post hoc pairwise comparisons.
CHIKV = chikungunya virus; IQR = interquartile range.
*Median with different superscripts (i.e., a and b) within the same column indicates significant difference (P < 0.05).
superinfected with both wAlbA and wAlbB strain in the
same population. The small percentage of uninfected
samples may be due to Wolbachia leakage possibly related to
the environmental factors such as high temperature and the
effect of overcrowding during developmental stage of the
larvae, which have been associated with reduced
transmission of Wolbachia.47
The speculative assumption that native Wolbachia might
affect the vectorial competence of the mosquitoes was
further analyzed by investigating the percentage of CHIKV
infection in midguts and dissemination to salivary glands.
The artificial oral infection study was conducted in
mosquitoes sampled from different geographical regions as
genetic susceptibility of Ae. albopictus to CHIKV may vary
by geography.48,49 In this study, removing Wolbachia did
not induce any significant changes of mosquito response
to infection by CHIKV. CHIKV was detected in midguts at a
high rate of at least 60% and 70% for w+ and w−,
respectively, regardless of the population tested. We measured
CHIKV dissemination to secondary organs (salivary glands).
CHIKV was detected as early as day 2 pi for both w+ and
w−, suggesting a short extrinsic CHIKV incubation
regardless the presence of Wolbachia. This finding is in line with
other studies that demonstrated a short CHIKV
incubation period of 2 days.50,51 We also demonstrated that
the total CHIKV titer, but not infection susceptibility, is
statistically affected by the geographical regions as
In this study, CHIKV can massively proliferate in midguts
of w+ and w− groups. No difference in CHIKV titer was
observed between w+ and w− groups for all time points
except for day 2 PI for Besar Island and Bandar Rinching
(salivary glands only). As CHIKV ingested by the
mosquitoes must pass through the epithelium of the mosquito
midgut before infecting salivary gland and other secondary
organs, the occurrence of a midgut escape barrier in w+
group can be suggested, limiting the infection of salivary
glands. However, as CHIKV inhibition effect was only
observed at day 2 PI, it possibly explained a potentially weak
midgut barrier caused by Wolbachia in its native hosts.
Probably, the protection is caused by resource competition
between Wolbachia and CHIKV in tissue in which they
coexists. The presence of Wolbachia might limit the
availability of resources that are important to ensure
achievement of the viral cycle.23,52–54
The higher Wolbachia density confers a better
protection toward viruses.55,56 However, the density of native
Wolbachia in Ae. albopictus showed a high level of
variation, whether it was from field- or laboratory-established
populations.23,35 Additional studies to see correlation between
virus concentration and Wolbachia density may provide a
better insight to explain the inconsistency in the
Wolbachiamediated virus replication in the oral-challenge experiments
in this study. However, the detection of the RNA genome
only did not give definitive evidence of the viability of the
virus. For example, Wong and others in the CHIKV
oralchallenged in Ae. aegypti, wherein a persistent CHIKV RNA
detection was reported in the mosquito eggs and adults
progeny. However, it was not proven to be viable and
infectious virus, omitting the possibility of the vertical
transmission for CHIKV in Aedes sp.57 Nonetheless, we cannot
exclude the limitation for using the quantitative PCR for
virus detection as the RNA viral copy number can give an
overestimation of infectious viral particles. Given the high
sensitivity of real-time PCR in detecting the region encoding
for E1 protein of CHIKV,57 even a slight contamination of
virus nucleic acid on the surface of the organs (from tissue
beside the organs) would have been sufficient to prime the
RT-PCR reaction and hence yielding the overestimation of
the infection percentage in the secondary organs including
Our results suggest a high prevalence of Wolbachia
infection in the wild-caught Ae. albopictus. In accordance to
other studies, the Ae. albopictus are naturally infected with
wAlbA and wAlbB. Our data showed that the presence
of Wolbachia do not pose any significant impact in the
CHIKV infection in the midguts and dissemination to
salivary glands in its native host, Ae. albopictus. The presence
of Wolbachia does not interfere with the extensive CHIKV
replication in midguts. Nonetheless, the native Wolbachia
has a minimal effect on the CHIKV titer in the salivary
glands, explaining why Ae. albopictus is a competent vector
for CHIKV despite naturally infected with Wolbachia.
Acknowledgments: We thank the Director-General of Health,
Malaysia, and the Director, Institute for Medical Research (IMR),
for permission to publish this study. We also thank Apandi Y and his
team from Virology Unit, IMR, for providing and culturing the
chikungunya virus, and Khairul Nizam MK from Molecular Diagnostic
and Protein Unit for assisting us in the molecular work experiment.
We acknowledge Nur Jannah J, Syakinah A, Chandru A, Azahari AH,
Shakirudin N, Mahirah MN, and Khairul Asuad M, from Medical
Entomology Unit, IMR, for their technical assistance in collecting,
identifying, and dissecting the mosquitoes. This work formed a part
of the graduate study (MSc) of the first author at University of
Malaya, Kuala Lumpur, Malaysia.
Financial support: This study was supported by a grant (no.
JPPIMR: 14-011) from the National Institutes of Health, Ministry of
Authors’ addresses: Noor Afizah Ahmad, Medical Entomology
Unit, WHO Collaborating Centre for Vectors, Institute for Medical
Research, Kuala Lumpur, Malaysia, and Department of
Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur,
Malaysia, E-mail: . Indra Vythilingam and Yvonne
A. L. Lim, Department of Parasitology, Faculty of Medicine,
University of Malaya, Kuala Lumpur, Malaysia, E-mails: .
my and . Nur Zatil Aqmar A. Zabari and Han
Lim Lee, Medical Entomology Unit, WHO Collaborating Centre for
Vectors, Institute for Medical Research, Kuala Lumpur, Malaysia,
E-mails: and .
This is an open-access article distributed under the terms of the
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Received June 23, 2016 . Accepted for publication September 27 , 2016 .
Published online December 5 , 2016 .
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