Genomic sequence and phylogenetic analysis of Culex flavivirus, an insect-specific flavivirus, isolated from Culex pipiens (Diptera: Culicidae) in Iowa

Abstract

Adult mosquitoes (Diptera: Culicidae) were collected in 2007 and tested for specific viruses, including West Nile virus, as part of the ongoing arbovirus surveillance efforts in the state of Iowa. A subset of these mosquitoes (6,061 individuals in 340 pools) was further tested by reverse transcription-polymerase chain reaction (RT-PCR) using flavivirus universal primers. Of the 211 pools of Culex pipiens (L.) tested, 50 were positive. One of 51 pools of Culex tarsalis Coquillet was also positive. The flavivirus minimum infection rates (expressed as the number of positive mosquito pools per 1,000 mosquitoes tested) for Cx. pipiens and Cx. tarsalis were 10.3 and 1.2, respectively. Flavivirus RNA was not detected in Aedes triseriatus (Say) (52 pools), Culex erraticus (Dyar & Knab) (25 pools), or Culex territans Walker (one pool). Sequence analysis of all RT-PCR products revealed that the mosquitoes had been infected with Culex flavivirus (CxFV), an insect-specific virus previously isolated in Japan, Indonesia, Texas, Mexico, Guatemala and Trinidad. The complete genome of one isolate was sequenced, as were the envelope protein genes of eight other isolates. Phylogenetic analysis revealed that CxFV isolates from the United States (Iowa and Texas) are more closely related to CxFV isolates from Asia than those from Mexico, Guatemala, and Trinidad.

Fig. 1

Geographic locations of the Iowa counties used for mosquito collections, and the CxFV minimal infection rates for each county.

Fig. 2

Phylogenetic analysis of the genomic sequence of a Culex flavivirus isolate from Iowa (CxFV-Iowa07). The displayed phylogeny was estimated by using the program MRBAYES, version 3.1 (Ronquist and Huelsenbeck 2003). The phylogenetic analysis is based on the complete genomic sequence of CxFV-Iowa07 and 24 other flavivirus isolates. An alignment without the Iowa and Texas CxFV sequences had been prepared by aligning the 5′UTR, polyprotein, and 3′UTR separately and then concatenating (Farfan-Ale et al. 2009). For the polyprotein, a protein alignment was back-translated to recover a nucleotide alignment. All alignments were done using the linsi utility of MAFFT (Minin et al. 2005), which was also used to profile align the CxFV isolates to the full genome alignment. The evolutionary model GTR+G+I was assumed based on previous analysis (Farfan-Ale et al. 2009). Phyml was used for the ML tree (Guindon and Gascuel 2003), the Vanilla 1.2 frontend to PAL 1.2 (Drummond and Strimmer 2001) was used for the NJ tree (using the GTR+G model), and Phylip was used for the MP tree (Felsenstein 2004). An initial tree figure was obtained using midpoint rooting in NJplot (Perriere and Gouy 1996). Bootstrap support (out of 100) and posterior support for each branch are indicated. CxFV-Iowa07 is denoted in bold. GenBank accession numbers for sequences used in the phylogenetic analysis are as follows: Cell fusing agent virus (M91671), Culex flavivirus (strain CxFV-Iowa07 from Iowa; FJ663034), Culex flavivirus (strain Tokyo from Japan; AB262759), Culex flavivirus (strain CxFV-Mex07 from Mexico; EU879060), Culex flavivirus (strain TX 24518 from Texas; FJ502995), Dengue type 1 (U88536), Dengue type 2 (U87411), Dengue type 3 (AY099336), Dengue type 4 (AF326825), Entebbe bat (DQ837641), Japanese encephalitis (M18370), Kamiti River (AY149905), Langat (AF253419), Louping ill (Y07863), Modoc (AJ242984), Murray Valley encephalitis (AF161266), Omsk hemorrhagic fever (AY193805), Powassan (L06436), Sepik (DQ837642), St. Louis encephalitis (DQ525916), Tick-borne encephalitis (U27495), Usutu (AY453411), West Nile (DQ211652), Yellow fever (X03700), and Yokose (AB114858) viruses.

Fig. 3

Phylogenetic analysis of the envelope protein genes of nine Culex flavivirus isolates from Iowa. The displayed phylogeny was estimated by using the program MRBAYES, version 3.1 (Ronquist and Huelsenbeck 2003). The phylogenetic analysis is based on the envelope protein genes of 31 flavivirus isolates (nine CxFV isolates from Iowa, 21 CxFV isolates from other geographical locations, and one isolate of Kamiti River virus). All nucleotide sequences were aligned using the MAFFT linsi utility. jModelTest supported the K80+G model under the BIC criterion and GTR+G using Akaike information criterion, but both models gave the same results for all methods, although the methods disagreed with each other (see text). ML, NJ, and MP trees were made as described in the Fig. 1 legend. The displayed tree is rooted by the Kamiti River virus, which is not shown. The MrBayes consensus tree included several multifurcations. All other nodes, except two in the Texas cluster for lack of space, have bootstrap/posterior support indicated. CxFV isolates identified in this study are denoted in bold. GenBank accession numbers for sequences used in the phylogenetic analysis are as follows: CxFV-Iowa07 (FJ663034), IA 377 (FJ663031), IA 318 (FJ663030), IA 383 (FJ663028), IA 657 (FJ663033), IA 599 (FJ663032), IA 635 (FJ663029), IA 380 (FJ663027), IA 1064 (FJ663026), CxFV-Mex07 (EU879060), Hokkaido (AB262762), Izabal (EU805805), Mie (AB262767), Mie pipiens (AB262764), Morioka (AB262765), Narita (AB262760), NIID-21 (AB377213), Osaka (AB262763), Surabaya (AB262766), Tokyo (AB262759), Toyama (AB262761), Trinidad 11264 (FJ503002), Trinidad 11265 (FJ503003), TX-24284 (FJ502997), TX-24471 (FJ502998), TX-24516 (FJ502999), TX-24518 (FJ502995), TX-24519 (FJ502996), TX-24522 (FJ503000), TX-24559 (FJ503001), and Kamiti River virus (AY149905).