development of a new sindbis virus transducing system and

Insect Molecular Biology (2004)

13

(1), 89–100

© 2004 The Royal Entomological Society

89

Blackwell Publishing, Ltd.

Development of a new Sindbis virus transducing system and its characterization in three Culicine mosquitoes and two Lepidopteran species

B. D. Foy*, K. M. Myles†, D. J. Pierro*, I. Sanchez-Vargas*, M. Uhlí

®

ová‡, M. Jindra§, B. J. Beaty* and K. E. Olson*

*

Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, USA;

†

Center for Disease Control and Prevention, Division of Vector Borne Infectious Diseases, Fort Collins, CO, USA;

‡

University of South Bohemia, Brani

3

ovká 31,

C

eské Bud

e

jovice, Czech Republic; and

§

Institute of Entomology ASCR, Brani

3

ovká 31,

C

eské Bud

e

jovice, Czech Republic

Abstract

Alphavirus transducing systems (ATSs) are alphavirus-based tools for expressing genes in insects. Herewe describe an ATS (5′′′′

dsMRE16ic) based entirely onSindbis MRE16 virus. GFP expression was used tocharacterize alimentary tract infections and dissemi-nation in three Culicine and two Lepidopteran species.Following

per os

infection, 5′′′′

dsMRE16ic-EGFP effi-ciently infected

Aedes aegypti

and

Culex tritaeniorhyn-chus

, but not

Culex pipiens pipiens

.

Ae. aegypti

clearlyshowed accumulation of green fluorescent protein (GFP)in the posterior midgut and foregut/midgut junctionwithin 2–3 days postinfection. Following parenteral infec-tion of larvae,

Bombyx mori

had extensive GFP expressionin larvae and adults, but

Manduca sexta

larvae weremostly resistant. 5′′′′

dsMRE16ic should be a valuable toolfor gene expression in several important insect speciesthat are otherwise difficult to manipulate genetically.

Keywords: Sindbis, alphavirus, mosquito,

Bombyx

,

Manduca

, viral dissemination.

Introduction

Sindbis viruses (family

Togaviridae

, genus

Alphavirus

,species

Sindbis virus

) are single-stranded, enveloped,

positive-sense RNA viruses (Strauss & Strauss, 1994). Thesearboviruses are transmitted naturally between Culicinemosquitoes and vertebrate hosts. Sindbis viruses (SINV)can cause pathology and morbidity in mammalian hosts,but generally produce a noncytopathic, systemic infectionin mosquitoes. Indeed, SINV has been shown to replicatein many different insect tissues, including the midgut,haemolymph, eye, fat body, musculature, neurons and sal-ivary glands (Olson, 2000; Olson

et al

., 2002; Pierro

et al

.,2003). These characteristics make SINV a valuable tool inmosquito and insect biology as a transducing agent. Plasmid-based, infectious DNA clones of these alphaviruses can beconstructed from the viral RNA by reverse-transcriptasepolymerase chain reaction (RT-PCR) and assembly of RT-PCR products (Rice

et al

., 1987; Davis

et al

., 1989; Kuhn

et al

.,1991; Myles

et al

., 2003b). With infectious clones, the viralgenome can be easily manipulated by deleting, rearrangingor adding DNA. Recombinant viruses can be generated

invitro

by first transcribing viral RNA from the linearized plas-mid, and then electroporating the RNA into cultured cells.

The genetic structure and molecular biology of alphavi-ruses has been described elsewhere (Strauss & Strauss,1994). Alphavirus transducing systems (ATSs) are pro-duced by engineering a second subgenomic promotor, fol-lowed by a multiple cloning site (MCS), into the viral genome.When a foreign gene insert is cloned into the MCS, thevirus is forced to transcribe a second subgenomic mRNAcontaining the foreign insert (Olson

et al

., 1992; Pierro

et al

.,2003). With this system, foreign proteins such as green flu-orescent protein (GFP), insect toxins, single-chain anti-bodies and immune peptides can be expressed in

Aedes

mosquito tissues (de Lara Capurro

et al

., 2000; Olson

et al

.,2000; Cheng

et al

., 2001; Pierro

et al

., 2003). Additionally,if only a piece of an endogenous mosquito gene is insertedinto the ATS (in either the sense or the antisense orienta-tion), production of the corresponding subgenomic mRNAcan induce an RNA interference (RNAi) response in themosquito cell, thus silencing expression of the endogenousgene (Johnson

et al

., 1999; Blair

et al

., 2000; Shiao

et al

.,2001; Olson

et al

., 2002). Through foreign gene expressionand endogenous gene silencing, ATSs are powerful toolsfor elucidating gene function in insects. In addition, ATSscould be particularly useful in nonvector insects, such as

Received 28 July 2003; accepted following revision 5 November 2003. Cor-respondence: Dr Brian D. Foy, Arthropod-Borne and Infectious DiseasesLaboratory, Department of Microbiology, Immunology, and Pathology, Colo-rado State University, Fort Collins, CO 80523, USA. Tel.: +1 970 491 4383;fax: +1 970 491 8323; e-mail: [email protected]

90

B. D. Foy

et al.

© 2004 The Royal Entomological Society,

Insect Molecular Biology

,

13

, 89–100

Bombyx mori

or

Manduca sexta

, for which there are limitedmethods for genetic manipulation.

A number of SINV ATSs are currently in use for transduc-tion in mosquitoes and other insects. TE/3

′

2J is an earlygeneration SINV ATS that was constructed from the mouseneurovirulent TE12 SINV strain (Lustig

et al

., 1988; Hahn

et al

., 1992), which was derived from the prototype SINVstrain AR339. In TE/3

′

2J, the duplicated subgenomic pro-motor is located in the 3

′

noncoding region of the viral RNA,immediately after the terminal nucleotides of structuralgenes. The location of this subgenomic promotor is prob-lematic because the resulting virus can be unstable, espe-cially when containing a foreign gene insert. After multiplecell passages, the virus often deletes the insertedsequence (Higgs

et al

., 1999; Pierro

et al

., 2003). To avoidthis complication, some ATSs have been constructed withthe duplicated subgenomic promotor at the 5

′

end of thegenome region encoding virus structural proteins (Raju &Huang, 1991; Pugachev

et al

., 1995; Pierro

et al

., 2003). Inaddition, the TE12 SINV strain was selected to be neuro-virulent in mice, and it poorly infects mosquitoes by the

peros

route (Seabaugh

et al

., 1998). We have previously con-structed infectious clone chimeras using the nonstructuralgenes of TE/3

′

2J and the structural genes of a MalaysianSINV strain MRE16 (Seabaugh

et al

., 1998; Olson

et al

.,2000; Pierro

et al

., 2003). These chimeras are orally infec-tious to mosquitoes, but they do not represent naturallyoccurring alphaviruses. In this study, we have engineeredand characterized a new SINV ATS based entirely on theMalaysian MRE16 SIN strain. We show that this virus,5

′

dsMRE16, is genetically stable, that it is orally infective forseveral mosquito species, and we use it to characterize the

course of alphavirus infection in several Culicine mosqui-toes, in

Manduca sexta

and in

Bombyx mori

.

Results

The ATS SINV infectious clone was engineered to place aninserted gene, 3

′

of the natural viral subgenomic RNA pro-moter and 5

′

of the second subgenomic promoter and viralstructural genes (Fig. 1A). A 5

′

dsMRE16ic virus plasmidmap is shown in Fig. 1B.

In vitro

transcription of genomicRNA and transfection into permissive cells has beendescribed (Olson, 2000).

When mammalian BHK-21 cells were infected at a mul-tiplicity of infection (MOI) of 0.01 with and without enhancegreen fluorescent protein (EGFP) insert, viral titres peak at1

×

10

8

pfu/ml after 24 h (data not shown). In

Aedesabopictus

(C6/36) cells, these viral titres peak at 1

×

10

9

pfu/ml after 72 h (data not shown). We observed that theinsertion of a foreign gene had little effect on viral replica-tion and assembly, and the peak titres and timing of thisvirus in cell culture, with and without an insert, mimic thosefrom wild-type MRE16 virus and the original MRE16infectious clone (MRE16ic) (Myles

et al

., 2003b). The onlyobservable difference in viruses could be seen on viralplaque assays in Vero cells, where 5

′

dsMRE16ic-EGFPtended to produce smaller plaques than those generatedby 5

′

dsMRE16ic or MRE16ic viruses at the same timepostinfection. This observation indicates a delay in cell-to-cell virus dissemination when an insert is present. Wealso observed this phenomenon when examining the rateof dissemination of 5

′

dsMRE16ic-EGFP in mosquitoes(see Table 1).

Table 1. Examination of the oral infectivity and dissemination of 5′dsMRE16ic in three Culicine mosquitoes

Mosquito species: Aedes aegypti – Rexville D White Eye Higgs strain Culex tritaeniorhynchus

Culex pipiens pipiens

Sindbis virus: MRE16ic 5′dsMRE16ic 5′dsMRE16ic-EGFP

Bloodmeal virus titre (pfu/ml): 1 × 108 1 × 108 1 × 108 1 × 107 1 × 106 1 × 105 1 × 104 1 × 103 1 × 108 1 × 108

4 days postinfectionBody 76 ± 10 63 ± 22 44 ± 3 26 ± 14 16 ± 15 7 ± 7 7 ± 7 7 ± 12 15 ± 13 1 ± 2PMG 87 ± 14 87 ± 9 91 ± 8 59 ± 23 21 ± 13 6 ± 6 3 ± 5 – 80 ± 11 –< 33% nd nd 74 (61) 61 (11) 91 (50) 100 (100) 100 (100) – 80 (88) –33–66% nd nd 16 (21) 26 (56) – – – – 6 –> 66% nd nd 10 (18) 13 (33) 9 (50) – – – 12 (13) –

12 days postinfectionBody 89 ± 10 86 ± 4 68 ± 10 53 ± 24 9 ± 5 6 ± 10 5 ± 4 – 16 ± 13 –PMG 96 ± 7 84 ± 2 93 ± 2 72 ± 25 16 ± 6 8 ± 9 2 ± 4 – 20 ± 16 2 ± 3< 33% nd nd 14 (3) 26 (15) 38 (20) 50 (0) – – 100 (100) 10033–66% nd nd 21 (19) 34 (35) 63 (80) – – – – –> 66% nd nd 64 (77) 40 (50) – 50 (100) – – – –

Numbers indicate the percentage infected ± the standard deviation of three replicate feeds (mean n / replicate = 19 mosquitoes).MRE16ic- and 5′dsMRE16ic-infected mosquitoes were examined by IFA of head squashes (Body) or posterior midguts (PMG) using an anti-E2 monoclonal antibody. 5′dsMRE16ic-EGFP-infected mosquitoes were examined by UV fluorescence of whole body (Body) or PMG.The percentage category under PMG refers to the percentage of the PMG epithelium infected; numbers in parentheses refers only to those mosquitoes that were also positive for Body fluorescence.

Alphavirus gene expression and dissemination in insects 91

© 2004 The Royal Entomological Society, Insect Molecular Biology, 13, 89–100

Table 1 summarizes infection and dissemination rates ofMRE16ic, 5′dsMRE16ic and 5′dsMRE16ic-EGFP in threeCulicine mosquitoes that were fed various concentrationsof virus in a bloodmeal. In Aedes aegypti, we observed aclear dose-dependant effect associated with posterior mid-gut (PMG) infection. Higher virus titres in the artificial blood-meal led to higher rates of PMG infection. This dose-effectwas also seen with escape from the alimentary canal andsubsequent viral dissemination to the rest of the body. With-out a foreign gene insert, 5′dsMRE16ic did not differ fromMRE16ic in PMG infection and alimentary canal escape/dissemination, further indicating that the ATS constructiondid not compromise viral replication. Inclusion of EGFP intothe ATS genome did not significantly alter the incidence of

PMG infection (91 ± 8% vs. 87 ± 9% at 4 days postinfection;P = 0.635, Student’s t-test), but it did slow the rate of viralalimentary canal escape and body dissemination (68 ± 10%vs. 86 ± 4% at 12 days postinfection; P = 0.041, Student’st-test). Nevertheless, at high titres in a bloodmeal (= 8 log10

pfu/ml), Ae. aegypti is highly susceptible to this ATS whenit contains a foreign gene insert the size of EGFP (719 bp).

Oral infection of all adult mosquitoes was initiated as asingle focus or multiple foci of cells of the PMG epitheliumwithin the first 24 h after ingestion of a blood meal and a higherdose of virus most often led to multiple foci (Fig. 2). In Ae.aegypti and Culex tritaeniorhynchus, the foci spread out intoneighbouring epithelial cells. The location of the foci appearedto be random in the PMG. In Ae. aegypti, the foci grew over

Figure 1. The strategy for the assembly of p5′dsMRE16ic from pMRE16ic (A) and the final plasmid map of p5′dsMRE16ic (B).

92 B. D. Foy et al.


time and eventually encompassed most of the epithelium(Fig. 2 and Table 1). By contrast, the percentage of Cx. tri-taeniorhynchus infected PMGs was observed to decreaseover time (80 ± 11% at 4 days postinfection vs. 20 ± 16% at12 days postinfection; P = 0.005, Student’s t-test) (Table 1).

Beginning at 2 days postbloodmeal, 5′dsMRE16ic-EGFPvirus began to escape the alimentary canal of Ae. aegyptiand disseminated throughout the haemocoel. The firstinfected cells of the haemocoel were haemocytes and fatbody cells (data not shown). Once virus escaped the ali-mentary tract, it rapidly disseminated throughout the mos-quito and could be seen in whole body EGFP expression(see Fig. 5). Indeed, whole body dissemination is 100%(except to the midgut epithelium) in all mosquitoes if thevirus is parenterally injected (Table 3). However, the ova-ries are one of the few organs resistant to SINV regardlessof the route of infection.

5′dsMRE16ic is not a chimera, unlike our previouslyconstructed orally infectious SINV ATS, ME2/5′2J (Pierroet al., 2003), and therefore its infection and dissemination

properties in mosquitoes should more fully resemble theproperties exhibited by wild-type MRE16. Other than theE2 gene, the genome of ME2/5′2J is derived from the pro-totype AR339 SINV strain, which diverges from the MRE16genome by > 25% in nucleotides and ∼14% in amino acidsand has limited mosquito oral infectivity (Myles et al.,2003a). An examination of the early infection and dissemi-nation patterns of these two viruses in Ae. aegypti revealssome of their differences (Table 2). 5′dsMRE16ic more effi-ciently infects the PMG than ME2/5′2J (cumulative infectedAe. aegypti PMGs over 4 days: 76%, 70/92, from 5′dsMRE16ic-EGFP vs. 50%, 58/116, from ME2/5′2J-EGFP) and usuallyinitiates more infection foci in the PMG. A small percentage(∼12%) of Ae. aegypti orally infected with ME2/5′2J-EGFPexhibit disseminated infections after the first 24 h. However,we have also found that over five comparison feeds of equaltitres, Ae. aegypti mortality within the same 24 h afteringesting a ME2/5′2J-infected bloodmeal can be abnor-mally high (23%, 82/363), when compared with mosquitoesingesting a 5′dsMRE16ic-infected bloodmeal (5%, 15/289).

Figure 2. 5′dsMRE16ic-EGFP efficiently infects midguts of Ae. aegypti when fed at high titre (1 × 108 pfu/ml) in a bloodmeal. Within 24 h of the bloodmeal (A), infection foci of posterior midgut (PMG) epithelial cells are visible in the midgut under UV light (B). The foci spread outward to more PMG epithelial cells over the following days (C–F). PMGs are shown at various stages after a bloodmeal: Day 3 (C, picture taken with UV and visible light simultaneously); Day 7 (D) and Day 12 (E,F). Note that both E and F mosquitoes showed disseminated infections throughout their bodies when they were dissected.



Interestingly, we observed that disseminated 5′dsMRE16ic-EGFP infections of Ae. aegypti were almost always associ-ated with infections of the PMG and the anterior midgut(AMG)/intussuscepted foregut (IF) (Table 2 and Figs 3 and4). Early EGFP expression in the AMG was always limitedto a narrow band just posterior to the IF (Figs 3 and 4). Thisregion of the alimentary canal has been described as apossible site of Rift Valley fever virus dissemination in Culexpipiens pipiens (Romoser et al., 1987).

5′dsMRE16ic-EGFP virus was analysed for genetic sta-bility through multiple passages in vivo (through mosqui-toes) and in vitro (through cell monolayers). We periodicallytested infected Ae. aegypti 12–14 days after ingesting abloodmeal containing 5′dsMRE16ic-EGFP virus and observeda 100% correlation (n = 111 mosquitoes) between SINVE1-positive head tissue as determined by immunofluores-cence analysis (IFA), and EGFP-positive heads as deter-mined by ultraviolet (UV) fluorescence. Thus, most tissuethat contained virus (as determined by IFA) also expressedEGFP (as determined by UV fluorescence), indicating thatlittle to no genetic deletions had occurred. To demonstrateviral stability further, we orally infected Ae. aegypti with5 × 107 pfu/ml of 5′dsMRE16ic-EGFP (after one passageeach in BHK and C6/36 cells) and screened infected adultsfor whole-body EGFP expression 6 days postinfection. Salivawas collected from EGFP-positive individual mosquitoes

and allowed to infect flasks of mammalian Vero cells. Fociof infected cells were visible in all flasks after 48 h and theinfections spread to the whole flask monolayer withinanother 24 h.

The two Culex species that we tested by oral infectionwere more resistant to infection than Ae. aegypti (Table 1).Culex pipiens pipiens was almost completely resistantto oral infection, suggesting that this Culex species hasa midgut infection barrier to 5′dsMRE16ic virus. Cx. tritaen-iorhynchus showed initial PMG infection rates similar to thatof Ae. aegypti (80 ± 11% vs. 91 ± 8% at 4 days postinfection;P = 0.257, Student’s t-test). However, alimentary canalescape and body dissemination was limited to < 20% ofmosquitoes tested regardless of the time point, and thepercentage of infected midguts decreased over time fromthe oral feed. This observation suggests the presence of amidgut escape barrier in this Culex species.

Larval oral infection for all mosquitoes proved to belimited when using previously published techniques(Table 3) (Higgs et al., 1999; Cheng et al., 2001). In a sep-arate experiment designed to achieve greater larval infec-tions, we hatched Ae. aegypti larvae in flasks of infectedC6/36 cells and allowed the developing larvae to feed ontwo consecutive batches of infected C6/36 cells for a totalof 3 days of feeding (Fig. 5D). With these modifications, wewere able to achieve a higher rate of larval infection that

Table 2. Infected alimentary canal tissues of Aedes aegypti immediately following bloodmeals containing either 5′dsMRE16ic-EGFP or ME2/5′2J-EGFP

Day Tissue

5′dsMRE16ic-EGFP ME2/5′2J-EGFP (Pierro et al., 2003)

No dissemination Dissemination No dissemination Dissemination

1 PMG 68 (27/40, avg. 6.4 foci) 50 (26/52, avg. 0.8 foci) 43 (3/7, avg. 1 foci)AMG 18 (7/40) 12 (6/52) 29 (2/7)IFCE 3 (1/40) 4 (2/52)MT 2 (1/52)HG 3 (1/40)

2 PMG 68 (13/19, avg. 5.2 foci) 100 (1/1, 6 foci) 45 (9/20, avg. 3 foci)AMG 5 (1/19) 100 (1/1) 10 (2/20)IFCEMT 5 (1/19) 10 (2/20)HG

3 PMG 92 (12/13, avg. 4.1 foci) 100 (4/4, avg. 2.3 foci) 31 (4/13, avg. 3.8 foci) 50 (2/4, avg. 26 foci)AMG 8 (1/13) 25 (1/4)IF 75 (3/4) 100 (4/4)CEMT 8 (1/13) 50 (2/4)HG 8 (1/13)

4 PMG 83 (10/12, avg. 3.9 foci) 100 (3/3, avg. 5 foci) 67 (12/18, avg. 5.5 foci) 100 (2/2, avg. 1 foci)AMG 8 (2/26) 100 (3/3)IF 4 (1/26) 67 (2/3) 100 (2/2)CEMT 4 (1/26) 6 (1/18)HG 6 (1/18)

Mosquitoes were fed 1 × 107 pfu/ml of virus in each bloodmeal.Numbers refer to the percentage of mosquitoes examined that showed an infection in the respective tissue; dissemination refers to mosquitoes having virus in tissues outside the alimentary canal.PMG, posterior midgut; AMG, anterior midgut; IF, intussuscepted foregut; CE, cardial epithelium; MT, malphigian tubules; HG, hindgut.

94 B. D. Foy et al.


persisted in emerged adults (43% males, 51/121; 16%females, 16/88). Although still rather inefficient, the benefitto this method is that all infected female adults exhibit anear total infection of all their PMG epithelial cells beforethey have taken a single bloodmeal (Fig. 5E). We observed

no undue mortality in pupae or adults that were infectedwith this virus as larvae.

Lastly, we examined viral dissemination in two modelspecies of Lepidopterans. Fourth instar larvae of Manducasexta were injected with approximately 107−8 pfu of

Figure 3. Early alimentary canal infection patterns (4 days postinfection) of 5′dsMRE16ic-EGFP in Aedes aegypti exhibiting a disseminated infection. Virus from an anterior midgut (AMG) infection probably moves forward to infect the intussuscepted foregut (IF) (A, visible light; B, same picture under UV light). (C) Virus in this mosquito had just disseminated from the alimentary tract because only few haemocytes were infected (data not shown). Notice the strong infection of the posterior midgut (PMG), but also the few infected cells in the foregut/midgut junction (arrow). By contrast, other mosquitoes exhibit a relatively poor PMG infection, but have a strong infection of the IF (D, inset is a combination UV/visible light picture of the foregut/midgut junction).

Table 3. Examination of alternative routes of infection with 5′dsMRE16ic-EGFP

Adult mosquitoes examined Aedes aegypti Culex tritaeniorhynchus Culex pipiens pipiens

Parenteral injection (5 × 108 pfu/ml; 1–2 µl) survived 94% (34/36) 24% (8/33) 95% (39/41)infected 100% (34/34) 100% (8/8) 100% (39/39)

Larvae fed on an infected flask of C6/36 female 2% (3/90) 0% (0/33) 5% (5/103)cells (according to Cheng et al., 2001; male 3% (3/109) 0% (0/29) 0% (0/169)Higgs et al., 1999)Larvae fed consecutively on two infected female 16% (16/88) – –flasks of C6/36 cells male 43% (51/121) – –



5′dsMRE16ic-EGFP virus. Even at these high doses ofvirus delivered at multiple sites in the larva, Manduca sextawere highly resistant to widespread infection (data notshown). The earliest signs of infection were visible in about70% of the larvae 3 days after injection. However, expres-sion of the EGFP marker was restricted to only a few cellsof particular tissues and did not spread further. Specificcells of the neural ganglia, such as the ventral unpairedmedian cell (VUM) and dorsal motor neurons, were themost common and often the only targets of viral infection.Sporadically, some animals also showed EGFP-positivemuscle fibres and/or haemocytes (data not shown). Inter-estingly, EGFP signals completely disappeared during thefinal (fifth) larval instar. By contrast, Bombyx mori (silkmoth)fourth instar larvae were highly susceptible to the same orlower titres of infectious SINV. Following injection of virusinto penultimate (fourth) larval instar, we observed efficientEGFP expression throughout the body of 100% of injectedlarvae. Almost all tissues, including the nervous system, fatbody, muscles, silk glands, larval eyes and imaginal primor-dia, were successfully targeted by 5′dsMRE16ic-EGFP(Fig. 6). Viral infection persisted throughout metamorphosisas EGFP expression was observed in pupae and emerged

adults. No EGFP was seen in the silkmoth ovaries and tes-tes, consistent with the observation that SINV does notenter Dipteran reproductive organs.

Discussion

In this paper, we describe the construction of a new ATS-based entirely on the Malaysian SINV strain MRE16 andcharacterize its infection and dissemination in three mos-quito and two Lepidopteran species. This virus representsan advance from previous TE12-based ATSs because it isnot a chimera, it does not cause undue mortality in insects,it is highly orally infective for mosquitoes and it exhibits highstability when containing a foreign gene insert.

We have previously shown that an ATS constructed withthe foreign gene insert 5′ to the structural genes will pro-duce a very stable virus that undergoes minimal rearrange-ment of its genome (Pierro et al., 2003). Here we use asimilar ATS construction technique that was designed tomake a 5′dsATS and still have full-length promotors (+98 to−14) that could most efficiently drive transcription of boththe foreign insert and the viral structural gene 26S RNA. Bymutating a restriction site precisely in the noncoding region

Figure 4. Early alimentary canal infection patterns of 5′dsMRE16ic-EGFP in Aedes aegypti prior to dissemination. Twenty-four hours after a high-titre infectious bloodmeal, between 10 and 20% of mosquitoes exhibit infected anterior midguts (AMG) immediately proximal to the intussuscepted foregut (IF) (A & B). In some mosquitoes, the IF is visibly infected before dissemination outside the alimentary canal occurs (C, mosquito dissected 4 days postinfection).

96 B. D. Foy et al.


between the subgenomic promotor and the capsid startcodon, and then inserting the duplicated subgenomic pro-motor with a 5′ MCS in that site, we could achieve bothgoals. The result is a stable virus that can orally infect mos-quitoes and can be transmitted in the saliva of the samemosquitoes to infect mammalian cells, all the while stillexpressing the foreign gene insert.

In Ae. aegypti, 5′dsMRE16ic infects the PMG in a mannersimilar to that described with MRE16ic using immunofluo-rescence analysis (Myles et al., 2003b). Infection starts asfoci of infected PMG cells, which expand to encompassmuch of the PMG epithelium. However, use of an ATS/EGFP marker enhances our ability to track viral infectionand dissemination and observe, in detail, infected tissues.EGFP is stable in infected cells, it is expressed within 8–12 h postinfection and can be viewed in freshly dissectedtissues under UV light precluding the need to fix infectedtissues with paraformaldehyde or other agents prior toimmunostaining. This advantage has given us insight intothe route of dissemination in Ae. aegypti. Our data point tothe foregut/midgut junction as a possible route of SINVescape from the alimentary canal of Ae. aegypti. Indeed,we have often observed Ae. aegypti with a well-developedPMG epithelium infection, but no sign of disseminatedinfection. We also occasionally observe mosquitoes with

only one or two PMG infection foci, but with disseminatedinfections; these mosquitoes also have infected IF and/orAMG. Romoser et al. (Romoser et al., 1987, 1992) havepreviously described this route of arboviral disseminationby Rift Valley fever virus in Cx. pipiens. It is not clear howrelatively large arboviruses (generally > 40–50 nm in diam-eter) can pass through the mosquito alimentary canal basallamina, which has a pore size of ∼10 nm (Houk et al., 1980,1981). It has been proposed that the foregut/midgut junctionmay facilitate dissemination of some arboviruses becausethe basal lamina may not be fully contiguous at this junction(Lerdthusnee et al., 1995). We are currently studying whetherflaviviruses and other alphaviruses also infect this region ofthe alimentary canal of natural arthropod vectors.

The two Lepidopteran species that we injected with5′dsMRE16ic-EGFP virus vary greatly in their susceptibilityto infection. Compared with Manduca sexta, Bombyx moriis highly susceptible to infection. Of interest is the strongEGFP expression in the silk glands, which may make thisATS a valuable tool for manipulation of silk production (Mori& Tsukada, 2000). The restricted infection and dissemina-tion of 5′dsMRE16ic-EGFP virus in Manduca sexta afterinjecting virus was surprising, because ATS injection usu-ally leads to SINV infection in most tissues of all mosquitospecies tested (Aedes spp., Culex spp., Anopheles spp.), in

Figure 5. Many tissues of Ae. aegypti are infected when 5′dsMRE16ic-EGFP virus disseminates. Many Ae. aegypti exhibit a phenotype that demonstrates whole body infection (A). The hindgut (1), fat body (2), posterior midgut and midgut musculature (3), and various malphigian tubules (4) are all commonly infected in the abdomen (B). The proboscis (1), eyes (2) and salivary glands (3) are commonly infected in disseminated mosquitoes (C). Virus also disseminates throughout the whole body of Ae. aegypti infected as larvae (D). The midguts of adult females that were infected as larvae do not exhibit infection foci, instead they have widespread infections of most of their midgut epithelium (E).



Drosophila melanogaster (unpublished data), and non-Dipteran insects, such as Bombyx mori (shown in this study),Precis coenia and Tribolium castaneum (Lewis et al., 1999).Although it is possible that viral entry is limited by the lackof a receptor, we favour a hypothesis that immune defencemechanisms eliminate the viral infection. Consistent withthis idea is that although some cells become infected (i.e.VUM cells), the infection does not spread and is cleared bythe final instar. A similar phenomenon has been observedin Manduca larvae infected by Autographa californica MNucleopolyhedrovirus (AcMNPV) (Washburn et al., 2000).During early stages of AcMNPV pathogenesis, the virusbehaves as in permissive hosts and enters the primary tar-gets, the midgut and midgut-associated tracheae. How-ever, infection fails to spread and virus is finally cleared.Increased activity of haemocytes around infected tissuessuggested that a cellular immune response was involved.In addition, baculovirus infection in Manduca inducesexpression of some plasma proteins (Finnerty & Granados,1997; Ji et al., 2003) that belong to a complex array of

immune response molecules (Yu et al., 2002), suggestingthat the humoral innate immunity of Manduca may alsocontribute to the suppression of SINV infection. Anotherpossibility is that cells are being infected, but a strong RNAiresponse is quickly suppressing viral replication in cellsbefore the EGFP marker can be expressed.

Others have demonstrated that variations in the E2 glyc-oprotein of alphaviruses can limit infectivity of the mosquitomidgut and thus define the range of mosquito species thatcan transmit the virus (Brault et al., 2002; Myles et al.,2003b). Our results suggest that the midgut epitheliumitself (at least for Cx. tritaeniorhynchus) can modulate viralinfectivity, through an as yet unknown mechanism. In allmosquitoes tested, the tissue tropism of 5′dsMRE16ic iswidespread when parenterally administered. The same istrue of the virus once it escapes from the midgut. In Ae.aegypti midguts, the viral foci greatly expand to encompassmost of the epithelial cells, but in Culex mosquitoes, thesefoci fail to become well established. In Cx. pipiens pipiens,rarely are any midgut cells infected and Cx. tritaeniorhynchus

Figure 6. Dissemination of 5′dsMRE16ic-EGFP virus in Bombyx mori larvae is efficient compared with Manduca sexta. EGFP marker is evenly expressed in the ventral nervous ganglia (A). An example of mosaic EGFP expression in the middle part of the silk gland is shown in B. Green colour, visible on the fourth instar larval body, is caused by intensive EGFP expression in the fat body, one of the first tissues targeted by SINV. Arrow shows viral presence in larval eyes (stemmata) (C). Imaginal discs are also permissive to SINV infection (D).

98 B. D. Foy et al.


clears virus in the PMG by 12 days postinfection overtime. These data highlight the critical nature of mosquitomidgut epithelial cells in mosquito infections (Lehane & Bill-ingsley, 1996, Foy et al., 2003). In the PMG, the peritrophicmatrix and heme-binding proteins are already implicated inprotecting epithelial cells from various pathogens andtoxins (Shao et al., 2001; Beaty et al., 2002; Pascoa et al.,2002). It may be that in Cx. tritaeniorhynchus, infectedmidgut cells are cleared of virus, perhaps through RNAipathways that have been shown to target RNA viruses thatproduce double-stranded RNA intermediates during theirreplication (Li et al., 2002). Another explanation may bethat infected midgut epithelial cells undergo apoptosis andare extruded into the midgut lumen, as has been proposedfor PMG epithelial cells infected with Plasmodium ooki-netes (Han et al., 2000; Zieler & Dvorak, 2000). Manystudies have demonstrated that certain arbovirus/vectorcombinations result in infected PMG epithelium, but dis-semination of the virus is limited by a midgut escape barrier(Whitfield et al., 1973; Turell et al., 1984; Weaver et al.,1984; Kramer et al., 1989). Our studies suggest that someof these barriers could actually be the result of midgutepithelial cells controlling viral replication through mecha-nisms described above that may limit sufficient quantities ofvirus from escaping either the PMG or the foregut/midgutjunction.

In conclusion, the genetic stability and tissue tropism of5′dsMRE16ic should make this ATS an excellent vehicle forexpressing genes in mosquitoes and other insects. Vectorbiologists should find it a very useful tool for studying the invivo effects of expression of various proteins on pathogentransmission, such as single-chain antibodies that bind toarbovirus or parasite surface proteins (de Lara Capurroet al., 2000), or insect immune modulators, such as ser-pins, that may work to limit pathogen infection (Chris-tophides et al., 2002). Alternatively, this ATS can be auseful marker to define virus determinants of vector infec-tion, antiviral pathways in the vector and patterns of vectorgene expression as a consequence of virus infection.

Experimental procedures

Virus construction and propagation

The previously constructed infectious clone plasmid, pMRE16ic(Myles et al., 2003b), was propogated in E. coli and mutated in thenoncoding region after the subgenomic promotor but before thecapsid start codon (+31 to +43, numbering according to Wielgoszet al., 2001) to contain an Sfi I restriction site using Stratagene’sEx-Site mutagenesis kit using primers 5′-GTGTAATATACAGT-AACTGACCAGGCCACATCGGCCGTAAACATGAATCGAGG-3′and 5′-CCTCGATTCATGTTTACGGCCGATGTGGCCTGGTCAGTT-ACTGTATATTACAC-3′. pMRE16ic was used as a PCR template tomake an amplicon (with the primers: 5′-GGCCACATCGGCC-AAGTTTAAACAAAAGCGGCCGCCATCACACCGGTCCTGC-3′and 5′-GGCCGATGTGGCCTGGTCAGTTACTGTATATTACAC-3′)

of the full-length subgenomic promotor (−98 to +43) that also con-tained flanking Sfi I sites and a short multiple cloning site (5′-PmeI–Not I-3′) that preceded the promotor. This amplicon was cloned intothe mutated pMRE16ic following standard cloning protocols. Theresulting plasmid infectious clone was named p5′dsMRE16ic. Thegene encoding EGFP was amplified by PCR from the pEGFPplasmid (Novagen) to contain flanking Not I sites and was clonedinto p5′dsMRE16ic. The plasmids pMRE16ic, p5′dsMRE16ic andp5′dsMRE16ic-EGFP were amplified in bacteria, purified andused in an in vitro transcription reaction to generate infectious viralRNA as previously described (Olson, 2000). The transcribed RNAwas electroporated into BHK-21 cells and virus was amplified bypassage in C6/36 cells, then supernatant containing virus wasaliquoted and frozen at −80 °C. One aliquot for each virus was titreedby plaque assay and the rest were used for all insect infections.

Cells and media

BHK-21, C6/36 and Vero cells were all cultured in 10% MEM (mod-ified Eagle’s medium supplemented with 10% fetal bovine serum,1× nonessential amino acids, 292 µg/ml L-glutamine and 100units/ml penicillin−100 µg/ml streptomycin). BHK-21 and Verocells were maintained at 37 °C with 5% CO2; C6/36 cells weremaintained at 28 °C with 5% CO2.

Insects

Aedes aegypti Rexville D, Higgs White Eye strain, were maintainedat 28 °C, 80% relative humidity, and in a 14 : 10 h light : dark pho-toperiod in our insectaries. Culex pipiens pipiens and Culex tri-taeniorhynchus were reared similarly but at 26 °C. All mosquitoeswere given access to water and sugar ad libitum, except 24 hbefore a bloodmeal to ensure high feeding rates. The Lepidopteranlarvae Manduca sexta and Bombyx mori (Nistari strain) wereraised on artificial diets at 25 °C under a 16 : 8 h light : dark pho-toperiod as previously described (Jindra et al., 1996; Uhlirovaet al., 2002).

Virus titring

Plaque assays were performed as previously described using Verocells (Miller & Mitchell, 1986), except that the first overlay wasadded 24 h after infection (1% agarose/1× MEM − no phenol red,2% FBS, 100 µg/ml gentamycin) and the second overlay (supple-mented with neutral red 50 µg/ml) was added after another 48 h.

Virus infection of insects

All viruses that were fed to or injected into insects had been gen-erated in BHK-21 cells and then passed once in C6/36 cells forvirus amplification and removal of defective interfering viral parti-cles. Per os delivery to mosquitoes was performed with membranefeeders. Viral aliquots were thawed, diluted to an appropriate con-centration with 10% MEM, and mixed with defibrinated sheepblood (Colorado Serum Co., Boulder, CO, USA) to a final volumeof 1 ml. Supernatant : blood never exceded 1 : 1, and was usuallyadjusted to 1 : 3 depending on the viral titre. The bloodmeal wasoffered to mosquitoes in water-jacketed membrane feeders cov-ered with stretched Parafilm and warmed to 37 °C. Mosquitoeswere allowed to feed to repletion (usually 30 min). Mosquitoeswere cold-anaesthetized and fully engorged females wereseparated and returned to the insectary. Larval infections were



performed according to previously described methods (Higgs et al.,1999; Cheng et al., 2001), except when described otherwise.Briefly, L1 larvae were hatched, and placed into large flasks ofC6/36 cells previously infected with virus. The cells were checkedby UV to ensure that the whole monolayer was infected with the5′dsMRE16ic-EGFP virus and, just prior to adding larvae, themonolayer was scraped to suspend the infected cells in the culturesupernatant. Larvae were reared in the flask for 1–2 days in theinsectary, or until they had consumed all of the cells. They werethen returned to normal rearing conditions and allowed to pupateand emerge into adults, when they were analysed for virus infec-tion by GFP expression and IFA analysis. Parenteral infectionswere performed by injecting a small amount of a thawed viralaliquot (1–2 µl) into the adult mosquito thorax via a sterile pulled-glass capillary. Manduca sexta and Bombyx mori larvae werecold-anaesthetized and injected with 15–20 µl of virus betweenthe first and second abdominal segments.

Immunofluorescence and microscopy

When viruses contained the EGFP insert, infection of insects wasusually examined by fluorescence of freshly dissected tissuesunder a wet-mount using UV light. SINV with no EGFP insert wasdetected in the midguts and heads of infected mosquitoes byimmunofluorescence as previously described (Pierro et al., 2003).Microscopy was performed using an Olympus BH2 microscope.Pictures were taken using a Magnafire-SP camera and software,which were connected to the microscope. Composite images(used to visualize tissue or a whole insect) were assembled fromindividual photographs using Adobe Photoshop version 6.0.1.

Acknowledgements

We would like to acknowledge Sarah VanOtterloo andCynthia Meredith for their help in raising and caring for themosquitoes. We also thank Lynn Riddiford for advice anddiscussions on the experiments with Lepidopterans. M.U.and M.J. were supported by the NIH FIRCA grantR03TWO1209-01 and M.U. was also supported by a NATOScience Fellowships Programme. This work was supportedby the National Institute of Allergy and Infectious Diseasesgrants AI46435 and AI46753.

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development of a new sindbis virus transducing system and

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