Natural Infection by Culex Flavivirus in Culex Quinquefasciatus

Medical and Veterinary Entomology (2019), doi: 10.1111/mve.12374

Natural infection by Culex ﬂavivirus in Culex quinquefasciatus mosquitoes captured in Cuiabá, Mato Grosso Mid-Western Brazil

O. S. MORAES1, B. F.CARDOSO1, T. A. PACHECO1, A. Z. L. P I N T O 1, M. S. CARVALHO1, R. C. HAHN1, T. C. T.BURLAMAQUI2,L.F.OLIVEIRA2,R.S.OLIVEIRA2, J. M. VASCONCELOS2,P.S.LEMOS2, M. R. T.NUNES2 and R. D. SLHESSARENKO1 1Programa de Pós-Graduação em Ciências da Saúde, Faculdade de Medicina, Universidade Federal de Mato Grosso, Cuiabá, Brazil and 2Centro de Inovação Tecnológica, Instituto Evandro Chagas, Ministério da Saúde, Ananindeua, Brazil

Abstract. New species of insect-specific viruses (ISV) have been reported worldwide. In the present study, the complete genome of Culex flavivirus (CxFV) and partial sequences of other ISVs in Culex quinquefasciatus Say 1823 females (n = 3425) sampled in 200 urban areas census tracts of Cuiaba, state of Mato Grosso, were identified via reverse transcriptase-polymerase chain reaction for a NS5 region of flaviviruses, nucleotide and high-throughput sequencing, and viral isolation in C6/36 cells. CxFV was detected in 16 of 403 mosquito pools; sequences found in the study presented a high similarity with isolates from São Paulo, Brazil and other countries in Latin American that belong to genotype II, supporting the geographical influence on CxFV evolution. The monthly maximum likelihood estimation for CxFV ranged from 1.81 to 9.94 per 1000 mosquitoes. In addition to the CxFV complete genome, one pool contained an ORF1 sequence (756 bp) that belongs to a novel Negevirus from the Sandewavirus supergroup most similar to the Santana virus (77.1%) and another pool presented an RNA-dependent RNA polymerase sequence (1081 bp) of a novel Rhabdovirus most similar to Wuhan mosquito virus 9 (44%). After three passages in C6/36 cells, only CxFV was isolated from these co-infected pools. The importance of ISVs relies on their possible ability to interfere with arbovirus replication in competent vectors. Key words. Culex flavivirus, high-throughput sequencing, Negevirus, Rhabdovirus.

Introduction ISVs replicate only in mosquito cells. Their pathogenesis in mosquitoes is largely unknown (Vasilakis et al., 2013; Nunes High-throughput sequencing (HTS) technologies have con- et al., 2015). Collectively, their discovery has opened new views tributed to virus identification and to more accurate taxonomic on the extent of viral diversity and evolution because they appear and evolutionary classifications, leading to the discovery to be ancient viruses compared with arboviruses classified of a wide variety of novel viral species, including several within the same viral families (Vasilakis & Tesh, 2015). Their insect-specific viruses (ISVs) (Bolling et al., 2015; Nouri et al., influence over vector competence to transmit human pathogens 2018). (e.g. arboviruses), as well as their potential development as

Correspondence: Renata Dezengrini Slhessarenko, Programa de Pós-Graduação em Ciências da Saúde – PPGCS, Faculdade de Medicina, Universidade Federal de Mato Grosso, Av. Fernando Corrêa da Costa, n 2367, Centro de Ciências Básicas da Saúde I, Boa Esperança, Cuiabá, Mato Grosso, CEP: 78060-900, Brazil. Tel.: +55 (65) 99213 7333; E-mail: [email protected]

Fig. 1. Culex quinquefasciatus pool distribution in urban regions of Cuiaba, Mato Grosso, and sites of insect-specific virus detection. CxFV, Culex flavivirus. [Colour figure can be viewed at wileyonlinelibrary.com]. biological control agents or novel vaccine platforms, has boosted neighbourhoods distributed into four administrative regions studies involving ISV metagenomics (Bolling et al., 2015). (midwestern, middle-eastern, southern and northern). These A growing number of ISVs have been described, with most of are further subdivided into 804 census tracts, each repre- them being detected in arbovirus surveillance studies (Vasilakis senting 250–350 residents (IBGE, 2011). Characterized by & Tesh, 2015). dry (April to September) and rainy (October to March) sea- Culex flavivirus (CxFV) has been isolated from Culex pipiens sons and mean temperatures ranging from 21.4 to 32.8 ∘C, Linnaeus 1758 complex in several countries (Hoshino et al., the climate is tropical (i.e. hot and humid). For the collec- 2007; Moralles-Betoulle et al., 2008; Cook et al., 2009). In tion period, an average maximum temperature of 32.45% and Brazil, this virus was previously identified in mosquitoes from an average humidity of 77.8% were recorded (IBGE, 2011; the cities of São José do Rio Preto and São Paulo, both in INMET, 2014). the state of São Paulo (Machado et al., 2012; Fernandes et al., Among 804 census tracts, 200 were randomly selected for 2016). adult culicidae capture between January and April 2013 using An increasing number of non-flavivirus ISVs have been Nasci aspirators and hand nets (13.00 to 17.00 hours). For census described in field-collected mosquitoes, indicating that these tract definition, the first sector and fourth sectors were selected, agents are surprisingly more abundant, ancient, diverse and leaving out the middle three sectors (Fig. 1). Three locations widely distributed than previously predicted and implicated as of the central street were randomly selected and sampled for a probable origin of some arboviruses (Vasilakis & Tesh, 2015). at least 30 min in intra- and peri-residential areas of each The present study investigated the presence and phylogenetic census tract. relationships of CxFV and other ISVs in naturally infected adult Specimens (n = 11 090) (Fig. 1) were transported under females of Cx. quinquefasciatus Say 1823 captured in Cuiaba, controlled temperature and humidity conditions, with 10% capital of Mato Grosso (MT), Mid-Western Brazil. sucrose feeding, and were identified alive when in a dor- mant state (4 min at 4 ∘C), with determination of species level using a dichotomous key (Forattini, 2002). Mosquitoes were Materials and methods pooled (1–20 mosquitoes) according to collection date, local, species and sex. Although morphological identification of Study area and Culicidae capture mosquitoes by dichotomous key is a useful method, the identification is inconsistent for Cx. pipiens complex species. Thus, Cuiaba is located in the middle-south region of MT, with an pools initially identified as Cx. pipiens pipiens and Culex sp. estimated population of 575 480 inhabitants living in 173 Linnaeus 1758 (n = 180) were additionally confirmed by an

© 2019 The Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12374 Culex quinquefasciatus insect specific viruses 3 acetylcholinesterase gene region polymerase chain reaction harvested and stored at −80 ∘C. The monolayer of passages 2 (PCR) of Culex quinquefasciatus Say 1823 (Smith & Fonseca, and 3 was subjected to total RNA extraction with Trizol LS 2014). (Invitrogen) and followed by a second round of HTS.

Total RNA extraction and reverse transcriptase (RT)-PCR Sequence analysis and genome assembly for flaviviruses Nucleotide sequences of flavivirus NS5 regionn ( = 16) were In total, 610 pools of culicidae females were obtained, com- analysed, aligned and compared with reference sequences prising 14 species of eight genera (Serra et al., 2016). Among available at blastn and blastp (Nucleotide Basic Local these 610 pools, 403 Cx. quinquefasciatus pools were macer- Alignment Search Tool; NCBI, PubMed) using geneious r10 ated and eluted into 800 𝜇L of RNase-free phosphate-buffered (https://www.geneious.com). saline and centrifuged at 5500 g for 4 min at 4 ∘C; 400 𝜇L Contigs obtained after HTS from total RNA of nine Cx. of the supernatant was subject to total RNA extraction with quinquefasciatus pools and from two isolates (#549 and #1256) Trizol LS reagent (Invitrogen, Carlsbad, CA, U.S.A.). RNA were compared with viral sequences in the NCBI database was immediately subjected to a RT-PCR protocol for a NS5 using the BLASTx algorithm after de novo assembly using gene region (958 bp) of flavivirus genus (Bronzoni et al. 2005; mira 4.0.2 (http://www.chevreux.org/projects_mira.html) and Serra et al., 2016). These products were visualized via 1.5% geneious r10. agarose gel electrophoresis; positive samples were purified Nucleotide sequences obtained in the present study with 20% polyethylene glycol 8000 (Promega, Madison, WI, were deposited in GenBank database (accession numbers: U.S.A.), quantified with specific kits (Quantus Fluorometer; KP764769-KP764781, KY349933, KY401153, KY361743, Promega) and subjected to nucleotide sequencing (3500 Genetic KY435948 and MG682011). The alignment of our sequences and reference sequences available on GenBank (NCBI) was Analyzer; Applied Biosystems, Foster City, CA, U.S.A.). blast generated with MAFFT (Geneious R10). Subsequently, (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (GenBank, PubMed) phylogenetic analysis of the CxFV complete genome, analysis allowed CxFV identification (Cardoso et al., 2015; RNA-dependent RNA polymerase (RdRp) region of Rhab- Serra et al., 2016). dovirus and ORF1 region of Negevirus was inferred via Total RNA of CxFV-positive pools by RT-PCR and nucleotide Bayesian methodology (mrbayes, version 3.2.6; geneious sequencing was also subjected to HTS on an Ion Torrent™ Sys- r10) (Huelsenbeck & Ronquist, 2001). This method is jus- tem (Life Technologies, Carlsbad, CA, U.S.A.). For preparation tified given that the posterior probability of a given nodeon of the library, the samples were reverse transcribed and quanti- inferred topology has an explicit interpretation that is propor- fied using a Qubit™ dsDNA HS Assay kit (Invitrogen) in accor- tional to its presence in the true phylogeny. Phylogenetic trees dance with the manufacturer’s instructions. cDNA (≥ 50 ng) and genomic maps were edited using figtree, version 1.4.3 was fragmented by enzymatic action in 200-bp fragments using (http://tree.bio.ed.ac.uk/software/figtree) and illustrator cc the Ion Xpress Barcode™ Plus Library Kit for AB Library (Adobe Master Collection CS5; Adobe Systems Incorporated Builder with the aid of the Builder System apparatus (Thermo San Jose, CA, U.S.A.). Fisher Scientific, Waltham, MA, U.S.A.) and individually lig- Conserved protein domains, cysteine residues and N potential ated to barcode sequences, which are oligonucleotides of known glycosylation sites were also assessed using the web CD-search sequences, using the Ion Xpress Barcode DNA Adapters 1-32 tool (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb Kit. Prepared libraries were subjected to a 200-bp fragment size .cgi), geneious r10 and netnglyc, version 1.0 (http://www selection using the E-Gel® SizeSelect™ Gels (Thermo Fisher .cbs.dtu.dk/services/NetNGlyc). Scientific) system to remove short sequences. Selected fragments were bound to ion sphere particles and amplified by emul- sion PCR with the Ion PGM Hi-Q View OT2 Kit (Thermo Fisher Data analysis Scientific). Positive ion sphere particles were enriched and prepared for deposition on the Ion 318™ Chip Ion Torrent Kit The digital mesh of the census tracts of Cuiabá provided by the (Thermo Fisher Scientific) and sequenced in accordance with IBGE was plotted using arcmap (ESri ArcGIS; http://www the manufacturer’s instructions. .esri.com/software/arcgis). Data on mosquito distribution and infection were plotted in maps of spatial distribution. Data from mosquito collections were analysed according to census tract, Virus isolation species, sex, number of specimens and ISV positivity. Sizes of the pools varied (1–20 mosquitoes). Supernatant of macerated pools positive for CxFV (n = 16 Maximum likelihood estimation (MLE) was calculated using pools), Rhabdovirus (pool #549) and Negevirus (pool #1256) the PooledInfRate v4 excel (Microsoft Corp., Redmond, WA, were inoculated at a dilution of 1:10 in L-15 medium into U.S.A.) add-in with a confidence interval of 95% (Biggerstaff, C6/36 cells (ATCC CLR-1660). These monolayers, cultivated 2007). The positivity of these pools for ISVs was compared with in L-15 medium supplemented with 5% fetal bovine serum and their positivity for Alphavirus, Flavivirus and Orthobunyavirus ∘ incubated at 28 C with 5% CO2, were monitored daily for as identified in previous studies (Cardoso et al., 2015; Serra 7 days. Three passages were performed and the supernatant was et al., 2016).

Results

Culex flavivirus natural infection in adult Cx. quinquefasciatus females

Among 403 Cx quinquefasciatus pools, 16 (4%) were positive for CxFV (Table 1). Two pools, #549 and #1256, presented the envelope sequence and the complete genome of CxFV, respectively. CxFV was isolated during the second passage in ) coverage Genbank C6/36 cells (pool #1256), which contained two non-engorged e Cx. quinquefasciatus females captured in the middle-eastern region of Cuiaba. A cytopathic effect was not observed in C6/36 cells. CxFV sequences obtained in the present study presented 1081 5.6 KY435948 98.1–99.5% nucleotide identity with those of the virus isolated from Culex spp. in São José do Rio Preto, SP, Brazil d (BR_SJRP_01_CxFV_BR_MT) and in Argentina (Chupali 323), as well as with those from Cx. quinquefasciatus in NS5 838 — KP764769 Guatemala (TR3116) and Trinidad and Tobago (TR3115). c In total, 3465 Cx. quinquefasciatus females were captured and Siriri ORF1 756 3.8 KY361743 grouped in 403 pools out of which 57 (n = 642 females) were Cururu RdRP obtained in January, 86 (n = 405) in February, 127 (n = 1292) in March and 133 (n = 1126) in April. The MLE values and respective confidence intervals of CxFV, expressed as the number of infected mosquitoes per 1000 Cx. quinquefasciatus females along the collection months, were 7.90 (3.00–16.74) No CxFVNo NS5 362 CxFV/ Envelope 244 — 4.4 KP764772 KY401153 in January, 9.94 (3.27–23.13) in February, 3.98 (1.49–8.64) in March and 1.81 (0.32–5.85) in April. As a result of the

large variation in confidence intervals, these results were not b statistically significant. a CxFV complete genomic sequences detected in Cuiaba NoNoNoD4 D4 NoD4 NoNo NoD4OROV No CxFV NoD4 CxFV YesD4 CxFV No NS5D4 NS5D4 CxFVNo CxFV No CxFV NS5 629No No NS5 627OROV CxFV No NS5 No NS5 310 No 359 CxFV No No 593 — CxFV NS5 — 612 CxFV NS5 CxFV NS5 — CxFV KP764770 CxFV NS5 — 544 CxFV KP764771 — NS5 327 Genome NS5 — 417 KP764773 211 10 KP764774 837 493 MG682011 — 425 KP764775 — — — KP764776 — KP764781 — KP764777 KP764778 KY349933 KP764779 KP764780 formed a cluster within genotype II, which includes other Latin

American isolates (Fig. 2B). ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ 30 00 20 40 25 07 20 16 28 20 00 56 05 43 45 10

In addition, eight of 16 (50%) CxFV-positive Cx. quinquefas- ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ 35 35 34 34 33 37 36 36 37 37 36 38 39 38 33 35

ciatus pools, comprising 1–16 mosquitoes, were also positive ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ for Dengue 4 virus (DENV-4) and two of 16 (12.5%) for the ;15 ;15 ;15 ;15 ;15 ;15 ;15 ;15 ;15 ;15 ;15 ;15 ;15 ;15 ;15 ;15 segment S of Oropouche virus (OROV), identified in previous ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ ′′ 22 10 00 40 00 00 02 22 58 00 00 16 05 40 50 20 studies (Table 1) (Cardoso et al., 2015; Serra et al., 2016). ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ 06 57 05 04 06 06 05 04 04 06 02 00 03 02 05 06 ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ ∘ The CxFV complete genome exhibited 97–99% identity at ∘ amino acid and nucleotide levels with other sequences deposited in GenBank and 97–100% identity among those included in the same cluster (Table 2). The CxFV complete genome presents 10 640 nucleotides in length with an open reading frame flanked by a 5’ and a 3’ UTR (29 and 152 amino acids, respectively). The gene CDS spans a region of 10 092 nucleotides and the deduced polyprotein contains 3.363 amino acids, along which the structural and non-structural proteins can be identified (Fig. 2A). The anchored capsid protein C has 137 amino acids, the capsid protein alone has 117 amino acids, the pr protein presents 83 amino acids and the membrane glycoprotein M has 59 amino acids. Protein domains were identified in three non-structural viral proteins of CxFV genome. The NS5 protein presented two conserved domains: the Flavi_NS5 superfamily (PSSM-ID 7 Midwest Quilombo 56 16 South Pedra 90 55 3432 Midwest3 Midwest1 Alvorada Midwest Alvorada Midwest2 Central-East9 Porto Central-East5 Poção 56 Centro Sul2 UFMT 56 6NorthCPA56 Central-East2 South 56 Bela Marina 56 South 56 South 56 Res 56 Coxipó Central-East Res Coxipó Res. São Itamaraty Francisco 56 56 56 56 4 North Paiaguás 56 5 Midwest Duque de Caxias 56

279336), which is the flavivirus RNA dependent RNA poly- Culex quinquefasciatus pools positive for insect specific viruses and arboviruses in Cuiabá, Mato Grosso. merase that possesses a number of short regions and motifs homologous to other RNANA polymerases, and the FtsJ-like 138 806 254 277 329 346 372 549 998 1028 1055 1101 1182 1256 298 methyltransferase (PSSM-ID 307718) in the N terminus of Table 1. sample females105 (n) region neighbourhood location co-infection engorgement virus region sequence(bp

(A) CxFV_BR_SJRP_01_2012 3’UTR 5’UTR

C prM E NS1 NS2a NS2b NS3 NS4a 2k NS4b NSS 10700 nt

5’UTR CxFV_BR/MT CbaAr_1256p2 3’UTR

C prM E NS1 NS2a NS2b NS3 NS4a 2k NS4b NSS 10640 nt Flavi Flavi_NS1 S7 P-loop HELICc FtsJ Flavi_NS5 Glycop (B) (C) NC018615.1NC018615.1 Eilat virus EO329EO329 KU700045.1KU700045.1 Dennstaedtia davallioides MTMTGC2047990GC2047990 NNC034017.1C034017.1 XishuangbannaXishuangbanna aedes fflaviviruslavivirus 100100 KCKC181923.1181923.1 AedesAedes flavivirus AEFV SSPFLDPFLD MMOO 22011011 MPMP66 100 KJKJ741266.1741266.1 AedesAedes flavivirusflavivirus BangkoBangkokk NCNC005064.1005064.1 100 KamitiKamiti RRiveriver vvirusirus 110000 KX669688.1KX669688.1 CalbertadoCalbertado virus 44 1313 KX669689.1KX669689.1 CalbertadoCalbertado virus CALperCALper 100 100100 100 100100 FJ606789.2FJ606789.2 LammiLammi virusvirus Insect Specific NC024806.1NC024806.1 Flaviviruses (ISFV) 100 LammiLammi virusvirus M0719M0719 NC024017.1NC024017.1 NhumirimNhumirim virus BrMSBrMS MMQ10Q10 NC001564.2NC001564.2 CellCell fusing agent virus GalvestonGalveston 100 NC030400.1NC030400.1 NakiwogoNakiwogo virus Uganda08Uganda08 KC505248.1KC505248.1 Palm CreekCreek virus 56 100 HE574573.1HE574573.1 CulexCulex theileri flavivirus RP-2011RP-2011 9797 100100 HE574574.1HE574574.1 CulexCulex theileri flavivirus RP-2011RP-2011 NC021069.1NC021069.1 MosquitoMosquito flavivirus LSFlaviV-A20-09LSFlaviV-A20-09 100100 NC012671.1NC012671.1 QuangQuang Binh virus AB701772.1AB701772.1 Toyama740 2005 FJ663032.1FJ 663032.1 599599 2007 100 100 100 FJ663027.1FJ663027.1 380 20072007 FJ502997.1FJ 502997.1 HOU24284HOU24284 20082008 96 FJ503000.1FJ 503000.1 HOU24522HOU24522 2008 100100 95 FJ503001.1FJ503001. 1 HOU24559HOU24559 2008 KX924633.1KX924633.1 Mos102Mos102 2016 CxFV 100100 AB377213.1AB377213.1 NIID 21-221-2 20032003 Genotype 1 100100 NC008604.2NC008604.2 TokyoTokyo 2003 100100 100 AB262759.2AB262759.2 2003 100 TokyoTokyo 9797 AB262764.1AB262764.1 MieMie 20042004 AB701771.1AB701771.1 Toyama75Toyama75 2004 100100 100 AB701767.1AB701767.1 Toyama734Toyama734 20052005 100 AB262766.1AB262766.1 SurabayaSurabaya 20042004 JQ308189.1JQ308189.1 DG1068DG1068 2010 100 JQ308190.1JQ308190.1 DG1007DG1007 2010 JQ308186.1JQ308186.1HLD102HLD102 20102010 100 JF938690.1JF938690.1 SDDM06-11SDDM06-11 20062006 100 JQ518484.1JQ518484.1 SDDM06-11SDDM06-11 2006 KY401153.1KY401153.1 2013 633 CxFV_BR_MT_CbaAr1256p2CxFV_BR_MT_CbaAr1256p2 100 61 KT726939.1KT726939.1 BR_SJRP_01BR_SJRP_01 2012 KU726615.1KU726615.1 2009 56 Otero_2009Otero_2009 FJ50300FJ503002.12.1 TR3TR3115115 22008008 FJFJ503003.1503003.1 2008 1000 TRTR31163116 KKC700042.1C700042.1 Chupali323 22009009 91 KCKC700041.1700041.1 Chupali20099 2009 CxFV KKC700043.1C700043.1 EbEbCOCO 2009 100100 Genotype 2 100 GUGU289685.1289685.1 MM23132313 2008 82 GGU289700.1U289700.1 MM26652665 2008 GUGU289690.1289690.1 MM26302630 22008008 GGU289694.1U289694.1 MM26442644 20020088 100 GGU289695.1U289695.1 MM26452645 2008 GUGU289686.1289686.1 MM26052605 2008 GGU289687.1U289687.1 2008 100100 MM26142614 GUGU289689.1289689.1 MM26182618 2008 GUGU289696.1289696.1 MM26482648 22008008 GUGU289698.1289698.1 MM26562656 2008

Fig. 2. Genomic map conserved protein domains (A) and phylogenetic tree (B) via Bayesian estimation method for Culex flavivirus (CxFV) complete genome (CxFV_BR_MT_CbaAr1256p2/2013) identified in Culex quinquefasciatus in Cuiaba, Mato Grosso. CxFV sequences obtained from Culex pipiens are indicated in green, Cx. quinquefasciatus in yellow, Culex tritaeniorhynchus in green, Culex restuans in pink, Culex interrogator in red, Culex spp. in grey and Anopheles sinensis in purple. (C) Potential N-glycosylation sites. [Colour figure can be viewed at wileyonlinelibrary.com].

NS5 protein, which is involved in viral RNA capping. The Prediction of GC contents was estimated at 53%. Moreover, P-loop containing nucleoside triphosphate hydrolase (PSSM-ID the presence of TGT/TGC codons predicts the existence of 176 328724) is a member of the P-loop NTPase domain superfamily cysteine residues in CXFV genome. In addition, 12 potential involved in diverse cellular functions, divided into KG (kinase NGlyc sites were also identified (Fig. 2C). GTPase), ASCE ATPases and ATPases associated with a wide variety of activities (AAA) (Fig. 2A). The conserved flavivirus domain flavi_NS1 superfamily Other ISVs partial sequences identified in Cx. quinquefasciatus (PSSM-ID 279316; 360 amino acids) presents 12 cysteine pools residues in the NS1 protein and undergoes glycosylation in a manner similar to other NS proteins. After HTS, pool #1256 presented a region (756 bp) of ORF1 The peptidase_S7 superfamily domain (PSSM-ID 307204; encoding the 3′ end of the viral helicase (VHEL) gene and the 130 amino acids) was found on the NS3 serine protease gene. 5′ end of the RdRp gene of a Negevirus (Fig. 3) 77.1% similar This protein, with NS2B as a cofactor, is responsible for to Santana virus, obtained from Culex sp. in Amapá, Brazil. processing the polyprotein precursor into mature proteins. Phylogenetic analysis demonstrated a relatively low amino The helicase superfamily c-terminal (HELICc) domain acid similarity (75.3–80.72%) between the Negevirus identified (PSSM-ID 238034; 131 amino acids) was found between NS3 in the present study and the other six negeviruses clustering in and NS4A and is characteristic of helicases and helicase related the same branch, supported by high bootstrap values. The Sande- proteins (Fig. 2A). wavirus clade includes the Wallerfield, Goutanap, Dezidougou,

Biratnagar, Tanay and Santana viruses and the negevirus identified in the present study, named Siriri virus. Using blastp, the identity with the closest viruses varied between 52% Tanay virus and 58% with Dezidougou virus, with these data confirming that this sequence belongs to a new negevirus (Fig. 3). HTS also provided a Rhabdovirus RdRp partial sequence (1081 bp) from pool #549, with 32.9% identity and 53.3% amino acid similarity with Wuhan mosquito virus 9, obtained from Cx. tritaeniorhynchus Giles 1901 in China. Phylogenetic analysis demonstrated an evolutionary relationship with other rhabdoviruses isolated from insects not assigned to any of the existent genera. Identity (19–32.9%) and amino acid similarity (48.6–53.3%) with other rhabdoviruses included in the same branch, such as Ive green virus, Drosophila busckii rhabdovirus, jingshan fly vírus, shuangao bebug virus and Wuhan mosquito virus, were relatively low and therefore, support the hypothesis that this also represents a novel virus, named Cururu virus (Fig. 4). After viral isolation, only the CxFV complete genome was recovered by HTS from pool #1256p2, although these pools were co-infected with a new Negevirus. Further attempts to achieve the complete genome sequences of these viruses were unsuccessful.

Discussion

ISVs comprise a large and diverse group of viruses that evolved parallel to their hosts. The present study reports the first identification of the CxFV complete genome in naturally infected Cx. quinquefasciatus females in Mato Grosso, Middle-West Brazil, during the rainy period, when arbovirus outbreaks are common in the area. CxFV-positive pools were also positive for DENV-4 and OROV (Table 1) and two pools presented a putative novel Rhabdovirus and a Negevirus, named Cururu and Siriri viruses, respectively (Table 1). Although the biological importance of dual infections by ISVs is largely unknown, superinfection and

1 2 3 4 5 6 7 8 9 10 11 12co-infection 13 14 15 by 16 arboviruses 17 18 or arboviruses and ISVs is relatively common in their hosts. Culex quinquefasciatus was identified as naturally infected and is a competent vector for arboviruses, especially to Fla- vivirus (Hoch et al., 1987; Segura & Castro, 2007; Heinen et al., 2015; Serra et al., 2016). Mosquito-borne, tick-borne and unknown vector flaviviruses are placed in a phylogenetic sister group to the classical ISVs within the Flavivirus genus, which includes CxFV (Fernan- des et al., 2016). Studies have shown that prior infection by CxFV can suppress the replication of dual-flavivirus infections in mosquito cell lines (Kenney et al., 2014; Bolling et al., 2015). This observation implies that CxFV and other ISVs could poten- tially inhibit the replication of medically important arboviruses in arthropods. It is possible that the coevolution between those insect-specific Chupali323Chupali2009EbCOM2630M2614M2313M2665M2645 KC700042 KC700041M2644M2656M2618M2648 KC700043 99.0 GU289690M2605 GU289687 99.0Otero_2009 GU289685BR_SJRP_01 99.1 99.1 GU289700 99.5 97.2CxFV_BR_MT_CbaAr 1256p2 97.2 99.5 GU289695 97.5 97.5 97.3TR3115 KY401153 97.3 97.7 GU289694 97.7 97.2TR3116 97.2 98.0 97.7 GU289698 97.7 97.2 97.2 98.1 97.7 GU289689 97.7 97.3 98.0 97.3 99.2 97.8 98.1 97.7 GU289696 97.7 98.1 97.3 99.4 98.5 98.0 KU726615 97.3 99.2 KT726939 GU289686 97.9 97.7 97.3 97.7 99.4 99.4 98.1 99.4 98.7 97.3 98.1 97.7 97.5 99.4 99.5 98.1 97.7 99.4 99.4 98.5 97.6 98.1 98.0 97.4 99.7 99.4 99.5 98.1 98.0 97.9 99.5 99.4 98.5 97.5 97.8 97.9 97.5 99.5 98.1 99.4 99.4 98.4 98.0 98.4 99.4 98.5 99.7 FJ503002 97.6 99.5 98.4 97.93 99.6 99.5 99.4 99.4 98.3 98.0 98.5 99.5 FJ503003 98.1 98.0 98.5 99.4 98.3 99.6 99.4 99.4 99.4 99.5 98.4 98.5 98.6 99.6 98.4 98.0 98.1 99.5 99.6 100.0 99.4 99.4 99.4 99.4 99.5 98.5 98.3 98.3 98.2 98.1 98.6 99.6 99.6 97.7 99.5 99.5 99.5 99.4 99.5 98.6 99.4 98.3 98.4 97.7 98.2 99.6 100.0 98.2 97.7 99.6 99.6 99.5 99.6 98.2 98.6 99.4 98.5 99.4 98.5 99.5 97.7 98.3 99.7 99.5 98.0 98.1 99.6 99.7 98.0 98.7 99.5 98.6 99.5 98.5 98.3 99.6 98.2 99.7 99.6 99.5 98.4 98.1 98.8 99.6 98.5 99.6 98.7 98.3 98.3 99.7 99.7 99.6 99.6 98.2 98.5 98.4 98.9 98.3 99.7 99.6 98.4 98.3 98.3 98.5 99.7 99.7 99.7 98.4 98.5 99.0 98.5 98.4 99.6 98.3 98.5 98.2 98.3 99.7 98.2 99.7 99.7 98.5 98.3 98.3 99.3 98.5 98.6 99.7 98.5 98.3 98.3 99.8 98.5 98.3 98.5 98.3 98.5 98.6 99.7 99.7 98.5 98.4 98.4 99.9 98.7 98.5 98.6 98.3 98.6 98.3 99.8 98.5 98.4 98.5 98.5 98.6 98.7 98.4 98.5 98.3 99.9 99.5 98.4 98.5 98.4 99.1 98.5 98.6 98.6 98.4 99.5 98.4 98.5 98.5 98.4 98.5 98.7 98.6 99.1 98.7 98.6 98.5 98.4 98.6 98.8 99.0 98.9 98.7 98.5 98.5 98.6 99.4 99.1 98.7 99.1 98.7 98.5 99.5 98.9 98.7 99.5 98.9 99.3 99.5 99.3 99.5 99.5 99.5 Nucleotide identities of the Culex flavivirus complete genome envelopeand gene sequences of genotype II isolates. flaviviruses, such as CxFV, and their mosquito hosts could favour immune evasion or suppression that would otherwise interfere with the replication of ISV or other

Table 2. number strain1 2 3 4 5 6 7 8 9 accession number 10 11 12 13 14 15 16 17 18 subsequently-infecting viruses (Kent et al., 2010). CxFV

(A)

(B)

YP_009001772YP_009001772 Wallerfield virus YPYP_009094126_009094126 Goutanap virus Sandewavirus AFIAFI2466924669 DDezidougouezidougou vvirusirus YPYP_009351824_009351824 BBiratnagariratnagar vvirusirus 100 BAS69360 NegevNegevirusirus Nona 1 YPYP_009182191_009182191 91 DDaeseongdongaeseongdong vvirusirus 1 100 YPYP_009270625_009270625 BBrejeirarejeira vvirusirus 100 110000 YPYP_009351827_009351827 SSanan Bernardo vvirusirus YPYP_009351830_009351830 PiPiuraura vvirusirus 7766 100 YPYP_009256205_009256205 NNegevegev vvirusirus Nelorpivirus 99 AAQM55322QM55322 Ngewotan vvirusirus 86 100 APAPG77603G77603 HHubeiubei negev-lnegev-likeike vvirusirus 2 YP_YP_009351821009351821 100 BiBigg cypress vvirusirus YPYP_009351835_009351835 110000 LLoretooreto vvirusirus YPYP_009351833_009351833 Cordoba virus APAPG77679G77679 HHubeiubei nenegev-likegev-like vvirusirus 1 110000 NP_NP_056808056808 OOdontoglossumdontoglossum ringspot virvirusus 100 NPNP_597746_597746 Tobaco mosamosaicic vvirusirus Tomovirus NPNP_044577_044577 CCucumberucumber green motile mosaic virus 110000 NPNP_620669_620669 Tobaco rattle vvirusirus 53 NP_NP_620033620033 76 100 PPepperepper rringspotingspot vvirusirus Tobravirus NPNP_049325_049325 PPeaea early-brownearly-browninging vvirusirus 9955 100 NPNP_049335_049335 Soil-borne wwheatheat mosaic viru Argaviridae 110000 NP_NP_059513059513 CChinesehinese whewheatat mosaic virus 110000 Furovirus 100 NPNP_059450_059450 SSoil-borneoil-borne cerecerealal mosaic virus 100 110000 NPNP_059510_059510 OOatat golden strip virvirusus 99 NP_NP_612628612628 BBeeteet soil-bornesoil-borne vivvirusrus 7700 100 NP_NP_620444620444 PPotatootato mop-lop vivvirusrus Pomovirus 100 NP_NP_612605612605 Beet virus Q 99 99 NP_NP_835282835282 IIndianndian ppeanuteanut clumclumpp vvirusirus Peculivirus NP_NP_604481604481 BarleBarleyy strstripip mosamosaicic vvirusirus Hordeivirus 99 AMO03220O Blackford virus YP_009351834 100 Hibiscus Fort Crockett virus 100 AMO03256 Bofa virus AMO03221 Buckhurst virus 63 100 YP_004928118 Hibiscus green spot virus YP_654568 Citrus leprosis virus C 84 YP_004901701_ Blueberryyg necrotic ring blotch virus 89 AFIAFI2467524675 SSantanaantana virus KYKY361743361743 SSiririiriri virus Sandewavirus YPYP_009028558 009028558 TaTanaynay virusvirus

Fig. 3. Genomic map (A) and phylogenetic tree (B) of a Negevirus ORF1 amino acid region generated via the Bayesian estimation method showing Siriri virus_ BR/MT_CbaAr1256/2-13 (2013) identified in Culex quinquefasciatus from Cuiabá, Mato Grosso, Brazil. Outgroup: Virgaviridae family. Viruses obtained from arthropods are marked in yellow and those from plants in green. [Colour figure can be viewed at wileyonlinelibrary.com]. suppress West Nile virus replication in mosquitoes up to 7 days the concept that geographical location plays a role in evolution post-infection and this potential defence mechanism could of CxFV (Machado et al., 2012) because this genotype is pri- include an activated mosquito innate immune response or marily composed of isolates obtained from Cx. quinquefasciatus competition for essential cellular factors (Bolling et al., 2012). in tropical regions (Bittar et al., 2016). Moreover, the cysteine Genetically, CxFV represents a stable group that evolved over residues and potential glycosylation sites found in CxFV many years, in a habitat-dependent manner, with evident trans- sequences in the present study are associated with genomic mission between various mosquito species (Liang et al., 2015). secondary structure maintenance, pathogenicity and viral infec- This virus has been described in several Culex spp. (Newman tivity in their hosts (Murrell et al., 2004; Nunes et al., 2017). et al., 2011; Machado et al., 2012), Anopheles sinensis Chow Negevirus ORF1 (756 bp) phylogenetic analysis revealed a 1950 and Aedes vexans Meigen 1830 (Liang et al., 2015) in major split in two groups, named Nelorpivirus and Sande- several countries, which suggests this virus may infect different wavirus, with a different ancestral origin, corroborating pre- sympatric species and is widely distributed. vious observations indicating that geography may also play a Culex quinquefasciatus MLE values ranged from 1.81 to 9.94. role in Negevirus evolution and sub-speciation (Nunes et al., The month with highest MLE was February and the lowest MLE 2017). Santana and Siriri viruses formed a cluster inside Sande- occurred in April, 2013. Studies have shown a high CxFV annual wavirus subgroup, very close to other negeviruses identified infection rate in Culex spp. in several countries, although, in in mosquitoes worldwide (Vasilakis et al., 2013; Kallies et al., certain areas, CxFV activity is seasonal (Blitvich et al., 2009; 2014). Kim et al., 2009; Farfan-ale et al., 2010). The similarity of the Relatively high infection rates in biting Diptera indicate CxFV genotype II identified in the present study with others that negeviruses are more diverse and abundant than previ- obtained from Culex mosquitoes in Brazil and in other American ously known. These viruses are more related to some plant countries confirms the hypothesis that this virus is disseminated viruses (Fig. 3) and were described in mosquitoes and sand- across North and Latin America. Furthermore, studies support flies, with identification in Aedes larvae demonstrating that these

Fig. 4. Genomic map (A) and phylogenetic tree (B) generated via the maximum-likelihood estimation method for RNA-dependent RNA polymerase amino-acid partial sequences of Cururu virus_BR/MT_CbaAr549/2013, a putative novel rhabdovirus identified in Culex quinquefasciatus females in Cuiabá, Mato Grosso. Viruses from insects are marked in yellow and those from plants in green. [Colour figure can be viewed at wileyonlinelibrary.com]. viruses may be acquired by transovarian route (Nunes et al., of transmission and host species, as observed for other RNA 2017). viruses (Bourhy et al., 2005). Cururu virus clustered with other unclassified Rhabdovirus, Studies have suggested that ISV infections could result in such as Wuhan mosquito virus 9, Shuangao bedbug virus 2, changes in the immune response or in the silencing of mosquito Sanxia water strider virus 5, Jingshan fly virus 2, Drosophila genes involved in the regulation of reproduction, longevity and busckii rhabdovirus and Lye green virus, all isolated from viral pathogenesis, resulting in a selective pressure favouring the insects. These isolates represent a clade sister to the Vari- coevolution of mosquitoes and pathogens (Newman et al., 2011; cosavirus, Cytorhabdovirus and Nucleorhabdovirus clades that Kenney et al., 2014). comprise plant isolates. RdRp or L protein of the Rhabdovirus Thus, these agents might be studied as potential mosquito represent a conserved region useful for distant evolutionary population control strategies that interfere with the replication relationships of this family because few complete genome and dissemination of arboviruses (Bolling et al., 2015). sequences of rhabdoviruses are available to date. These The findings of the present study emphasize the diversity of sequences could be very divergent among rhabdoviruses, ISVs, highlighting the importance of metagenomic studies for although there is sufficient similarity to make a phylogenetic the discovery of new viruses and viral variants, as well as for analysis (Bourhy et al., 2005). Viral sequences from arthropods, obtaining more accurate and complete genomic sequences via vertebrates and plant form distinct clusters in the phylogeny evolution and phylogenetic characterization. (Bourhy et al., 2005; Longdon et al., 2015). Several closely related ISVs belonging to Rhabdoviridae comprise a phylogenetically divergent group and a major split Acknowledgements between those infecting arthropods and other hosts (Bourhy et al., 2005; Vasilakis et al., 2014; Charles et al., 2016). This We thank Fiocruz-AM Instituto Leonidas e Maria Deane major phylogenetic division between Rhabdoviridae genera for nucleotide sequencing; Dr Laura HV Gil (Fiocruz-Recife) indicates that their biology is strongly influenced by mode for providing C6/36 cells; Peter Zeilhofer and Emerson

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