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Culex Flavivirus Isolates from Mosquitoes in Guatemala
M. E. MORALES-BETOULLE,1,2 M. L. MONZÓN PINEDA,1 S. M. SOSA,1 N. PANELLA,3
M. R. LÓPEZ B,1 C. CORDÓN-ROSALES,1 N. KOMAR,3 A. POWERS,3 AND B. W. JOHNSON3
J. Med. Entomol. 45(6): 1187Ð1190 (2008)
ABSTRACT A new strain of Culex ßavivirus (family Flaviviridae, genus Flavivirus, CxFV), an insect
virus Þrst described in Japan, was isolated from adult Culex quinquefasciatus Say (Diptera: Culicidae)
collected in 2006 from Izabal Department on the Caribbean coast of Guatemala. Mosquito pools were
assayed for ßavivirus RNA by using ßavivirus group-speciÞc primers that ampliÞed a 720-bp region
of the nonstructural (NS) 5 gene by standard reverse transcriptase-polymerase chain reaction. From
210 pools (1,699 mosquitoes), eight tested positive, and six of these mosquito pools produced virus
isolates in Aedes albopictus Skuse C6/36 cells. Nucleotide sequence comparison of the eight ßavivirus
RNA-positive pools showed that there was 100% identity among them, and phylogenetic analysis of
the NS5 and envelope gene regions indicated that they represent a strain of the recently described
CxFV from Japan. This is the Þrst report of an insect ßavivirus from Central America.
KEY WORDS Flavivirus, Culex quinquefasciatus, insect ßavivirus, Guatemala
Flaviviruses (family Flaviviridae) are single-stranded
RNA viruses made up of ⬇70 known taxa. Most of
these are arthropod-borne, transmitted principally by
mosquitoes or ticks, with 30% having no known vector
(Burke and Monath 2001). Several recently described
ßaviviruses replicate only in arthropods (having been
propagated only in mosquito cell lines) and form a
separate phylogenetic clade (Gaunt et al. 2001,
Hoshino et al. 2007). The Þrst of these ßaviviruses (all
family Flaviviridae, genus Flavivirus) was cell fusing
agent virus (CFAV), isolated originally from a cell line
of the mosquito Aedes aegypti (L.) (Stollar and
Thomas 1975), and later from Þeld-collected Ae. aegypti, Aedes albopictus Skuse, and Culex species in
Puerto Rico (Cook et al. 2006). In central Kenya, a
CFAV-like virus, designated Kamiti River virus
(KRV), was isolated from Þeld-collected Aedes macintoshi mosquitoes (Sang et al. 2003, Crabtree et al.
2003). In Japan, the closely related Culex ßavivirus
(CxFV) was isolated from Þeld-collected Culex pipiens
L. (Hoshino et al. 2007).
We describe a new strain of the insect ßavivirus
CxFV isolated from Culex quinquefasciatus Say
(Diptera: Culicidae) mosquitoes collected from
Puerto Barrios, Department of Izabal, Guatemala. The
characterization of this strain, hereafter termed CxFV
Izabal 2006, was performed by sequencing two portions of the viral genome corresponding to the nonstructural gene NS5, and the complete envelope (E)
1 Centro de Estudios en Salud, CDC-CAP, Universidad del Valle de
Guatemala, 11 calle 15-79 zona 15, Vista Hermosa III, Guatemala,
Guatemala.
2 Corresponding author, e-mail: [email protected].
3 Centers for Disease Control and Prevention, Division of VectorBorne Infectious Diseases, 3150 Rampart Rd., Fort Collins, CO 80521.
gene. Sequences were compared with the corresponding genes of 10 different Flaviviruses to establish the
phylogenetic relationships of CxFV Izabal 2006.
Materials and Methods
Mosquito Collection. Mosquitoes were collected
monthly between March and October 2006, from six
urban and four rural sites within the coastal municipality of Puerto Barrios, Department of Izabal, Guatemala (Fig. 1). One CO2-baited CDC light trap
(John W. Hock Co., Gainesville, FL) and one gravid
trap (Reiter 1983) were placed in each collection
site for one night per month, for a total of 80 collections per trap type. Mosquitoes were killed by
CO2 freezing within their collection bags, transferred to collection tubes, transported frozen on dry
ice to the laboratory, and stored at ⫺70⬚C until
identiÞcation and pooling.
Mosquito Identification and Homogenization.
Adult female mosquitoes were identiÞed by their
morphological characteristics (Clark-Gil and Darsie
1983) and pooled according to species, location,
collection date, and trap type in groups of no ⬎50
mosquitoes in a 2-ml snap-cap polypropylene tube
containing one copper-coated steel BB (Crosman
Corporation, East BloomÞeld, NY). Pools were homogenized by adding 1.75 ml of BA-1 diluent (Medium 199 with HanksÕ balanced salt solution, 2 mM
L-glutamine, 0.05 M Tris-HCl buffer, pH 7.6, 1%
bovine serum albumin, 0.35 g/liter sodium bicarbonate, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 1 mg/ml amphotericin B [Fungizone, Sigma-Aldrich, St. Louis, Mo]), and by shaking on a
mixer mill (model MM-301, Retsch GmbH, Haan,
0022-2585/08/1187Ð1190$04.00/0 䉷 2008 Entomological Society of America
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JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 45, no. 6
Fig. 1. Puerto Barrios municipality, Department of Izabal, Guatemala.
Germany) for 4 min at 25 rps. Homogenates were
clariÞed by centrifugation. A 750-␮l aliquot of the
supernatant was placed in a cryogenic vial for longterm storage.
RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assays. Viral RNA
was extracted from 140 ␮l of mosquito homogenate by
using the QIAamp viral RNA kit (QIAGEN, Valencia,
CA). RNA was eluted from the QIAgen columns in a
Þnal volume of 60 ␮l of elution buffer and stored at
⫺70⬚C.
Flavivirus group-speciÞc RT-PCR was conducted
using the QuantiTect SyBr Green I RT-PCR kit (QIAGEN) with ßavivirus universal primer sets FU2/cFD3
and FU1/cFD2 (Kuno et al. 1998). For RT-PCR by
using FU2/cFD3, the samples were incubated at 50⬚C
for 30 min and 95⬚C 15 min, followed by 45 cycles of
94⬚C for 15 s, 60⬚C for 30 s, and 72⬚C for 30 s. For
RT-PCR by using FU1/cFD2, a similar procedure was
used but the annealing temperature was changed to
55⬚C for 30 s. AmpliÞed cDNA products were analyzed
by electrophoresis on a 2% agarose gel (Promega,
Madison, WI) and visualized by ethidium bromide
staining.
Nucleotide Sequencing. RT-PCR was carried out
with the QIAGEN One-Step RT-PCR kit (QIAGEN)
according to manufacturerÕs instructions (Kuno et al.
1998). The ampliÞed products were puriÞed by agarose gel electrophoresis, followed by extraction using
the QiaQuick gel extraction kit (QIAGEN). PuriÞed
DNA fragments were sequenced in the ABI Prism Big
Dye terminator sequencing ready reaction kit and
analyzed using the ABI 3130 genetic analyzer. Both
strands of the DNA fragments were sequenced, and
they were assembled in the Seqman module of Lasergene version 7 (DNASTAR, Madison, WI). CxFV
Izabal 2006 nucleotide (nt) sequences of 720-bp (nucleotides [nt] 9126 Ð9845) in the nonstructural (NS)
5 gene region were compared with the GenBank database by using the BLAST program.
CxFV Izabal 2006 nt and amino acid (aa) sequences of the complete E gene were compared
with those of 10 ßaviviruses in the GenBank database (all family Flaviviridae, genus Flavivirus):
Culex ßavivirus, CxFV (AB262759 andAB262762);
West Nile virus, WNV (DQ823150); dengue one
virus, DENV1 (EF508204); yellow fever virus, YFV
(X03700); Powassan virus, POWV (AF310922);
Apoi virus, APOIV (AF160193); cell-fusing agent
virus, CFAV (M91671); Kamiti River virus, KRV
(AY149905); Modoc virus, MODV (AJ242984); and
tick-borne encephalitis virus, TBEV (U27495). Sequences were aligned using the Clustal W algorithm in
the MegAlign module of Lasergene. Phylogenetic
analysis was conducted using MEGA version 3.1 (Kumar et al. 2004), with Maximum Parsimony, and 500
bootstrap replicates.
Viral Isolation. One dram shell vials (Thermo
Fisher ScientiÞc, Waltham, MA) containing monolayers of mosquito Aedes albopictus (C6/36) or Vero
cells were used for virus isolation of the RT-PCR
ßavivirus-positive mosquito pools. Cell cultures in
shell vials containing 350 ␮l of maintenance medium
(DulbeccoÕs modiÞed EagleÕs medium with 2% fetal
bovine serum [Atlas, Fort Collins, CO], supplemented with 0.1 mM nonessential amino acids [Invitrogen, Carlsbad, CA], 1 mM sodium pyruvate
[Invitrogen], 100 U/ml penicillin, 100 ␮g/ml streptomycin [Invitrogen], 26 mM sodium bicarbonate,
and 10 mM HEPES) were inoculated with 50 ␮l of
mosquito homogenate. The vials were centrifuged
at 1,000 ⫻ g at 37⬚C for 45 min. The inoculum was
removed and replaced with 1 ml of maintenance
media and incubated at 37⬚C (Vero cells) or 28⬚C
(C6/36 cells) for 7 d. Cells were observed daily for
cytopathic effects (CPE), reported as the percentage of cells affected: 1⫹, ⱕ25% cells; 2⫹, 26 Ð50%
cells; 3⫹, 51Ð75% cells; and 4⫹, ⱖ76% cells. At 4 and
7 d, 200 ␮l of supernatant was removed from the cell
culture, and RNA was extracted. The presence of
viral RNA in the supernatant was identiÞed by RTPCR and sequencing as described above.
November 2008
MORALES-BETOULLE ET AL.: Culex FLAVIVIRUS ISOLATES FROM MOSQUITOES
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Fig. 2. Phylogenetic tree generated by maximum parsimony analysis of Culex ßavivirus strain Izabal 2006, envelope protein
amino acid sequences. The number above the branch is the bootstrap percentage out of 500 replicates and the number below
the branch is the branch length (Kumar et al. 2004). Abbreviations: Culex ßavivirus, CxFV; West Nile virus, WNV; dengue
one virus, DENV1; yellow fever virus, YFV; Powassan virus, POWV; Apoi virus, APOIV; cell-fusing agent virus, CFAV; Kamiti
River virus, KRV; Modoc virus, MODV; tick-borne encephalitis virus, TBEV. The Flavivirus species included in the tree
represent the mosquito/vertebrate group (WNV, DENV1, and YFV), tick/vertebrate group (TBEV and POWV), mosquito
group (CxFV, CFAV, and KRV), and no-known-vector group (APOIV and MODV).
Results and Discussion
In total, 1,699 Cx. quinquefasciatus (Diptera: Culicidae) were identiÞed and grouped into 210 pools, of
which 85% were from urban collection sites. Flavivirus
group-speciÞc primers ampliÞed a product in eight of
these pools. Alignment of the nt sequences from the
eight ßavivirus-positive pools showed that there was
100% sequence identity among them (data not
shown). Results of the BLAST search from the GenBank database of a 720-bp genome segment in the NS5
coding region (CxFV nt 9126 Ð9845) indicated maximum nucleotide sequence homology of 89% with
Culex ßavivirus (CxFV) (Hoshino et al. 2007), 60%
with KRV, and 58% with CFAV. Based on the ßavivirus
taxonomic deÞnition of Kuno et al. (1998), in which
isolates with ⬎84% pairwise nt sequence identity in
the conserved NS5 region were considered to be the
same species, these results indicated that these isolates
would be classiÞed as a strain of CxFV. To further
characterize the isolate, the more variable E gene was
sequenced. Pairwise nt and aa alignment in Megalign
showed sequence identities to CxFV of 89.4 and 90.2%
(AB262759 and AB262762) and 97.4% (AB262762), to
KRV of 48 and 34%, and to CFAV of 67 and 71.9%,
respectively. Phylogenetic analysis was conducted on
alignments of the E aa sequences with 10 other ßavi-
viruses and shows that the isolate clearly clusters with
CxFV (Fig. 2).
Homogenates from the eight ßavivirus RNA-positive mosquito pools were cultured in Vero and C6/36
cells for virus isolation. No CPE was observed in either
C6/36 or Vero cell culture. However, RNA replication
in C6/36 cells was detected by RT-PCR of supernatant
collected from six of the eight C6/36 cell cultures after
4-d incubation.
Based on the sequence data reported herein and
that there are no previous reports of an insect ßavivirus in Central America, we consider CxFV Izabal
2006 to be a novel strain of the Japanese CxFV in the
clade of insect ßaviviruses. The only other insect ßavivirus described to date in North America is CFAV in
Puerto Rico (Cook et al. 2006). In contrast to CxFV
Izabal 2006, other insect ßaviviruses described cause
mild (CxFV) to signiÞcant (CFAV) CPE after cell
culture, whereas no CPE was detected in our viral
isolates.
CxFV Izabal 2006 virus isolates were derived from
mosquito collections in both urban (n ⫽ 5; three different collection sites) and rural collection sites (n ⫽ 1),
during March, April, August, September, and October
2006. These preliminary observations suggest that this
insect ßavivirus is widely distributed in the Cx. quinque-
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JOURNAL OF MEDICAL ENTOMOLOGY
fasciatus population of the Caribbean Coast of Guatemala, with no observable effect by urbanization or season
on the likelihood of mosquito infection.
Insect ßaviviruses circulating in both Aedes and
Culex mosquito species (principal vectors of the mosquito-borne ßaviviruses) may be common in nature
and were not detected previously because of the lack
of (or reduced) CPE in cell culture. Using molecular
techniques to detect ßaviviruses will probably increase the number of strains/isolates corresponding to
insect ßaviviruses described in different geographic
areas. Entire genomic sequencing of novel strains and
in vivo infection studies in mosquitoes will enhance
knowledge of the phylogeny, evolution, and phenotypes of this clade in the genus ßavivirus.
Nucleotide Sequence Accession Numbers. The nucleotide sequences of CxFV Izabal 2006 reported in
this article have been submitted to the DNA Data
Bank of Japan, European Molecular Biology Laboratory, and GenBank data banks under accession numbers EU805806 (NS5 partial coding sequence) and
EU805805 (envelope, complete coding sequence).
Acknowledgments
We thank Danilo Alvarez (Centro de Estudios en SaludUniversidad del Valle de Guatemala [CES-UVG], Centers for
Disease Control and Prevention for Central America and
Panama [CDC-CAP]) for Þeldwork organization; Ramon
Medrano and Bernarda Molina (CES-UVG, CDC-CAP) for
technical assistance; Mary Crabtree (Centers for Disease
Control and Prevention, Division of Vector-Borne Infectious
Diseases [CDC/DVBID]) for assistance with phylogenetic
analysis; Jason Velez (CDC/DVBID) for laboratory assistance; Roger Nasci (CDC/DVBID) for suggesting the name
“Izabal” for this strain of CxFV; and homeowners in Izabal for
granting permission to place mosquito traps. This work was
supported by the Division of Vector-Borne Infectious Diseases, Cooperative Agreement U50/CCU021236 Ð 01 from
Centers for Disease Control and Prevention.
Vol. 45, no. 6
References Cited
Burke, D. S., and T. P. Monath. 2001. Flaviviruses, pp. 1043Ð
1125. In D. M. Knipe and P. M. Howley [eds.], Fields virology. Lippincott Williams & Wilkins, Philadelphia, PA.
Crabtree, M. B., R. C. Sang, V. Stollar, M. D. Dunster, and
B. R. Miller. 2003. Genetic and phenotypic characterization of the newly described insect ßavivirus, Kamiti
River virus. Arch. Virol. 148: 1095Ð1118.
Clark-Gil, S., and R. F. Darsie. 1983. The mosquitoes of
Guatemala. Their identiÞcation, distribution and bionomics, with keys to adult female and larvae in English and
Spanish. Mosq. Syst. 15: 151Ð284.
Cook, S., S. N. Bennett, E. C. Holmes, R. De Chesse, G.
Moureau, and X. Lamballerie. 2006. Isolation of a new
strain of the ßavivirus cell fusing agent virus in a natural
mosquito population from Puerto Rico. J. Gen. Virol. 87:
735Ð748.
Gaunt, M., A. Sall, X. de Lamballerie, A. Falconar, T. Dzhivanian, and E. Gould. 2001. Phylogenetic relationships of
ßaviviruses correlate with their epidemiology, disease
association and biogeography. J. Gen. Virol. 82: 1867Ð
1876.
Hoshino, K., H. Isawa, Y. Tsuda, K. Yano, T. Sasaki, M. Yuda,
T. Takasaki, M. Kobayashi, and K. Sawabe. 2007. Genetic
characterization of a new insect ßavivirus isolated from
Culex pipiens mosquito in Japan. Virology 359: 405Ð 414.
Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: integrated
software for molecular evolutionary genetics analysis and
sequence alignment. Brief. Bioinformatics 5: 150 Ð163.
Kuno, G., G. J. Chang, K. R. Tsuchiya, N. Karabatsos, and C. B.
Cropp. 1998. Phylogeny of the genus Flavivirus. J. Virol.
72: 73Ð 83.
Reiter, P. 1983. A portable, battery-powered trap or collecting
gravid Culex mosquitoes. Mosq. News 43: 496Ð498.
Sang, R. C., A. Gichogo, J. Gachota, M. D. Dunster, V. Ofula,
A. R. Hunt, M. B. Crabtree, B. R. Miller, and L. M.
Dunster. 2003. Isolation of a new ßavivirus related to
Cell fusing agent virus (CFAV) from Þeld-collected
ßood-water Aedes mosquitoes samples from a dambo in
central Kenya. Arch. Virol. 148: 1085Ð1093.
Stollar, V., and V. L. Thomas. 1975. An agent in the Aedes
aegypti cell line (Peleg) which causes fusion of Aedes
albopictus cells. Virology 64: 367Ð377.
Received 13 February 2008; accepted 30 June 2008.