A major mosquito-borne viral disease outbreak caused
by Zika virus (ZIKV) occurred in Bahia, Brazil, in 2015, largely due to
transmission by the mosquito, Aedes aegypti (L.). Detecting
ZIKV in field samples of Ae. aegypti has proven problematic in
some locations, suggesting other mosquito species might be contributing to the
spread of ZIKV. In this study, several (five) adult Aedes albopictus (Skuse)
mosquitoes that emerged from a 2015 field collection of eggs from Camaçari,
Bahia, Brazil, were positive for ZIKV RNA; however, attempts to isolate live
virus were not successful. Results from this study suggest that
field-collected Ae. albopictus eggs may contain ZIKV RNA that
require further tests for infectious ZIKV. There is a need to investigate the
role of Ae. albopictus in the ZIKV infection process in Brazil
and to study the potential presence of vertical and sexual transmission of ZIKV
in this species.
Zika virus, Aedes albopictus, vertical
transmission
Issue Section:
In early 2015, an outbreak of mosquito-borne Zika
virus (ZIKV, Flaviviridae; Flavivirus) occurred in
Bahia, Brazil. Zika virus, a single-stranded RNA arbovirus, can cycle between
mosquitoes and humans in an urban environment (Duffy et al. 2009, Haddow
et al. 2012) and is transmitted to humans primarily by Aedes aegypti (L.)
(Centers for Disease Control and Prevention [CDC], Guerbois et al.
2016, Ferreira-de-Brito et al. 2016). Other Aedes spp.
are thought to be secondary vectors (Diallo et al 2014, Ioos et al.
2014). Aedes albopictus (Skuse) has been shown to be a natural
vector for ZIKV (Grard et al. 2014, Pan American Health Organization/World
Health Organization, [PAHO/WHO] 2016) and is capable of transmitting ZIKV in
the laboratory (Chouin-Carneiro et al. 2016). Multiple Aedes species
and Culex perfuscus Edwards were found infected with live ZIKV
in field studies in Senegal (Diallo et al 2014). During a ZIKV outbreak in
Micronesia, 73% of the human population were infected with ZIKV, and Aedes
hensilli Farner was the most abundant mosquito, suggesting it was the
primary vector (Duffy et al. 2009, Ledermann et al. 2014).
The Zika outbreak in Bahia resulted in ∼110,000 human cases (PAHO/WHO 2016). The first cases
were detected in Camaçari, Bahia, Brazil (Campos et al. 2015), and subsequently,
Zika could be found throughout Brazil (PAHO/WHO). Zika virus has rapidly spread
to most of the southern hemisphere of the Americas and has made inroads into
the southern United States, primarily in local Ae. aegypti populations. Aedes
aegypti in Rio de Janeiro, Brazil, were the primary ZIKV vector while
collections of Ae. albopictus and Culex
quinquefasciatus Say were negative for ZIKV (Ferreira-de-Brito et al.
2016). This study suggested vertical and sexual transmission of ZIKV because
one Ae. aegypti male was positive for ZIKV. Recent studies in
Brazil and China suggested that Cx. quinquefasciatus might
have a role in ZIKV transmission (Ayres 2016, Guo et al. 2016).
Here, we report the detection of ZIKV RNA in
adult Ae. albopictus collected as eggs during 2015 from
Camaçari, Bahia, Brazil.
Materials and Methods
In August 2015, mosquito eggs were collected to
establish a laboratory colony from neighborhoods where Zika cases were
identified (7,391 suspected cases of exanthematous illness like Zika were
reported in Camaçari with 2,626 cases/100,000 inhabitants [Secretaria de Saúde
do Estado da Bahia, 2016]). Standard oviposition cups, containing germination
paper, were placed at three sites in Camaçari (two cups per site; two
collections 7 d apart). Field-collected eggs (≥50) were hatched, aquatic stages
reared, and the adults maintained under standard conditions (Alto et al. 2014).
The field-collected eggs provided 20 female and 19 male adult Ae.
albopictus and one adult Ae. aegypti that was
discarded. The adults were mated with one another; females were blood-fed by
providing chicken blood to produce F1 eggs to establish a colony following
approved IACUC procedures (IACUC number 201507682). The same adult female and
male mosquito bodies were placed individually in 1.5-ml tubes and stored at
−80 °C until processed to extract RNA.
The adult mosquito bodies (not including the F1
mosquitoes) were analyzed to detect ZIKV RNA using reverse transcription and
quantitative PCR (RT-qPCR). The legs from each mosquito were placed in
individual tubes and stored at −80 °C for later processing to detect live
virus. The RNA was extracted from each Ae. albopictus body
using Trizol reagent (Thermo Fisher, Waltham, MA). Primer sequences specific to
ZIKV were designed (IDT, Coralville, IA) to the NS2B gene of a ZIKV isolate
from human blood collected in 2015 in Salvador, Brazil (GenBank
KX520666, Table 1). RNA was treated with DNase (Fisher) and reverse
transcribed using Enhanced Avian Reverse Transcriptase (42 °C for 50 min, Sigma
Aldrich, St. Louis, MO), and qPCR reactions were performed using SsoAdvanced
SYBR green Supermix on a BioRad CFX96 Real-Time PCR Detection System following
standard protocols (BioRad, Hercules, CA). The qPCR conditions were 95 °C for
30 s followed by 39 amplification cycles of 95 °C for 5 s and 60 °C for 30 s. The
positive control used in all qPCR reactions was a ZIKV isolate from French
Polynesia (strain H/PF/2013, GenBank KJ776791.1) provided by the CDC in January
2016. The titer of ZIKV for each positive Ae. albopictus body
was calculated as described elsewhere (Shin et al. 2014), but with ZIKV stock
virus to generate the standard curve. Specifically, ZIKV titration in samples
was performed using the iTaq Universal SYBR Green One-Step Kit (BioRad,
Hercules, CA) on the Bio-Rad CFX96 Real-Time PCR Detection System with the same
ZIKV specific primers as mentioned. The standard curves for ZIKV titer were
obtained by serial dilution of ZIKV stock (7.2 log10 plaque
forming unit). The standard curve was defined as the regression line of the
logarithm of standard copy number versus Cq (quantification cycle) value.
Table 1
Primers and probes used to verify presence of ZIKV in
mosquito samples
|
Primers and probe
name
|
Sequences
|
Position
|
Amplicon (Base pairs)
|
|
ZIKV NS2B set 1 F
|
5ʹ-GTTACGTGGTCTCAGGAAAGAG-3ʹ
|
4274–4295
|
191
|
|
ZIKV NS2B set 1 R
|
5ʹ-CATCAGGACCACCTTGAGTATG-3ʹ
|
4464–4443
|
|
|
ZIKV NS2B F
|
5ʹ-GTTACGTGGTCTCAGGAAAGAG-3ʹ
|
4274–4295
|
135
|
|
ZIKV NS2B R
|
5ʹ-GGGAGAAATCACCACTCTCATC-3ʹ
|
4408–4387
|
|
|
ZIKV NS2B probe
|
FAM/TGCGGAAGT/ZEN/CACTGGAAACAGTCC-3IABkFQ-3ʹ
|
4344–4367
|
|
Quantitative PCR reactions showing ZIKV RNA were
analyzed by gel electrophoresis, and DNA was isolated by following the
protocols using the GenElute Gel Extraction Kit (Sigma Aldrich, Shin et
al. 2014). PCR products (191 nucleotides) were sequenced at Eurofins MWG Operon
LLC (Louisville, KY) in both directions using the same primers from the qPCR
reaction. A mosquito was considered positive for ZIKV RNA if RT-qPCR showed a
Cq ≤ 36 and the PCR product sequence matched ZIKV sequence (≥98% to GenBank
KX520666) by Basic Local Alignment Search Tool analysis (Blastn, GenBank).
Samples with ZIKV RNA were further screened with a nested qRT-PCR reaction
using the iTaq Universal Probes One-Step kit (BioRad). Primers and a probe
specific to ZIKV were designed to the NS2B gene of the same ZIKV isolate
mentioned previously (Table 1). The amplification was performed as follows:
50 °C for 10 min; 95 °C for 2 min; 39 cycles of 95 °C for 15 s and 60 °C for 30
s on the previously mentioned PCR detection system. Quantitative RT-PCR
products were 135 nucleotides and Cq values were ≤35. To rule out contamination
during processing, four replicates were performed for each sample. Standard no
template controls and uninfected mosquito samples were tested in all ZIKV amplification
assays.
Results and Discussion
Adult Ae. albopictus mosquito bodies
from Camaçari were screened for ZIKV. Quantitative PCR found three female
(infection rate [IR] = 15%) and two male (IR= 10%) mosquito bodies with ZIKV
RNA with a Cq ≤ 36. The titer of ZIKV in each body was ∼2log10 ZIKV plaque-forming unit
equivalents (pfue)/ml, consistent with little to no virus replication in
these Ae. albopictus (Table 2). Sequence analysis of five PCR
products revealed matches (≥98%, Blastn GenBank) to several ZIKV isolates,
including ZIKV from human blood collected in 2015 from Salvador, Brazil, and a
human urine sample collected in 2016 from Florida (GenBank KX520666 and
KX922707, respectively). Phylogenetic analysis rooted with Spondweni virus
showed that sequences from the NS2B gene from the Ae. albopictus adults
belonged to ZIKV Asian lineage (Zhu et al. 2016, Supp. Figure S1 [online
only]).
Table 2
Determination of Cq value and associated titer of ZIKV
in adult Ae. albopictus collected in Camaçari
|
Individual mosquito (ID
no.)
|
Sex
|
Avg Cqa
|
Titer (pfue/ml)
|
|
9
|
F
|
35.8
|
154.2
|
|
18
|
F
|
34.9
|
273
|
|
20
|
F
|
33.0
|
793.8
|
|
4
|
M
|
34.9
|
265.5
|
|
11
|
M
|
35.3
|
213.4
|
a
Avg Cq―Average of the Cq values from the technical
replicates for each body.
The detection of ZIKV RNA from five adult Ae.
albopictus reared from eggs collected during the 2015 outbreak in
Camaçari, Bahia, Brazil, is consistent with the potential for vertical or
sexual transmission of ZIKV by Ae. albopictus; however, evidence
supporting this was not conclusive. Vertical transmission has been observed for
dengue, yellow fever, West Nile, Japanese encephalitis, and St. Louis
encephalitis viruses in several species of mosquitoes (Lequime et al. 2016).
Natural vertical transmission of ZIKV was observed in a pool of male Aedes
furcifer (Edwards) collected in Senegal (Diallo et al. 2014). Vertical
and sexual transmission of ZIKV was observed for Ae. aegypti collected
in Rio de Janeiro, Brazil (Ferreira-de-Brito et al. 2016), and vertical
transmission of ZIKV was shown in Ae. aegypti after
intrathoracic inoculation of ZIKV but not for Ae. albopictus (Thangamani
et al. 2016).
Two questions require answers to assess the
significance of this observation. 1) Was the ZIKV RNA owing to contamination
during the processing of these mosquitoes? 2) Does the ZIKV RNA represent
infectious ZIKV? We believe contamination is unlikely. The RNA extractions were
all performed prior to us having ZIKV in our laboratory. Additionally,
sequences from the PCR products of the Ae. albopictus samples
differed sufficiently from our laboratory ZIKV to exhibit separate phylogenetic
clustering. Nonspecific amplification of reverse-transcribed mosquito RNA
during qPCR reactions could be a source of false positives; however, sequence
analysis of positive products did not support this. The low titer of the ZIKV
RNA in the adult samples suggests ZIKV did not replicate in these mosquitoes. Absence
of replication could mean that complete ZIKV genome or live ZIKV was not
introduced into the eggs, or that Ae. albopictus is not a
competent vector for ZIKV. We were unable to isolate live ZIKV from the
Camaçari Ae. albopictus samples using cell culture isolation.
This and the low ZIKV RNA titer is the cause for uncertainty that vertical or
sexual transmission of live ZIKV was responsible for the ZIKV RNA in these
field-collected Ae. albopictus eggs, although this does not
preclude viral replication under different conditions. Future work is needed to
characterize the mechanism responsible for transfer of ZIKV RNA to Ae.
albopictus eggs and whether live ZIKV can accompany this under various
yet unknown conditions.
Finding Ae. albopictus from Brazil
with ZIKV RNA adds further cause for considering Ae. albopictus’
role in ZIKV epidemiology in Bahia, Brazil, in 2015.
Zika virus RNA in field-collected eggs from mosquitoes
where there is current ZIKV transmission is concerning. Samples of mosquitoes,
including those resulting from field-collected eggs that are returned to the
laboratory from regions with ZIKV, must be treated with the potential that
resulting adult mosquitoes or their offspring might be positive for ZIKV RNA.
These mosquitoes must be characterized for live ZIKV to ensure they are
uninfected or they must be treated as if they did contain infectious ZIKV and
maintained under the appropriate required safety and containment practices.
Acknowledgments
We thank J. Day and B. Alto for critical review of
earlier drafts of this manuscript. This research was supported in part by the
Florida Department of Agriculture and Consumer Services Grant 00119227 to
Chelsea T. Smartt.
Subject Editor: William Reisen
© The Authors 2017. Published by Oxford University
Press on behalf of Entomological Society of America. All rights reserved. For
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