* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Culex Flavivirus Isolates from Mosquitoes in Guatemala
Survey
Document related concepts
Yellow fever wikipedia , lookup
Ebola virus disease wikipedia , lookup
Orthohantavirus wikipedia , lookup
Middle East respiratory syndrome wikipedia , lookup
Influenza A virus wikipedia , lookup
Hepatitis B wikipedia , lookup
Marburg virus disease wikipedia , lookup
2015–16 Zika virus epidemic wikipedia , lookup
Antiviral drug wikipedia , lookup
Herpes simplex virus wikipedia , lookup
Transcript
SHORT COMMUNICATIONS 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 1188 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 1189 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- 1190 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.