1 Clinical 1disease 1often 1presents 1as 1excessive 1salivation 1with 1vesicular 1lesions 1of 1the 1gums, tongue, 1 naso-oral 1 mucosa, 1 teats, 1 and 1 coronary 1 bands 1 [1]. 1 Because 1 saliva 1 and 1 vesicular 1 fluid can 1contain 1high 1titers 1of 1virus, 1VSV 1can 1be 1transmi 4 ed 1very 1e ciently 1between 1animals by 1direct 1contact, 1aerosol, 1and 1fomites. 1 Additionally, 1VSV 1is 1vector-borne, 1with 1insects 1acting 1as 1mechanical 1and 1biological 1vectors 1 [2]. 1Typically, 1VS 1occurs 1annually 1in 1enzootic regions 1of 1Central 1and 1South 1America, 1infecting 1a 1large 1percentage 1of 1susceptible 1species [1]. 1In 1contrast, 1in 1the 1U.S., 1it 1appears 1as 1sporadic 1epizootic 1incursions 1of 1viruses 1originating 1from 1enzootic 1regions 1in 1Mexico 1 [3][4][5], 1resulting 1in 1single 1year 1or 1multi-year 1outbreaks with 1overwintering 1genotypes. 1 1 In 1the 1laboratory, 1VSV 1can 1rapidly 1replicate 1to 1high 1titers 1(8-10 1Log10 1 plaque 1forming units 1(PFU)/mL 1in 124-48 1hours) 1in 1a 1variety 1of 1vertebrate 1and 1invertebrate 1host 1cells 1 [6][7][8]. This 1remarkable 1plasticity 1has 1allowed 1the 1study 1of 1continuous 1replication 1in 1specific mammalian 1and 1insect 1cells 1in 1alternating 1environments 1 [9][10][11]. 1In 1vitro, 1VSV 1displays 1high adaptability 1and 1elevated 1variation 1in 1fitness, 1sometimes 1accompanied 1by 1lower 1rates 1of nucleotide 1changes 1 [6,10]. 1During 1replication 1events 1in 1single-host 1cell 1lines, 1VSV 1rapidly increases 1fitness 1to 1the 1cell 1line 1in 1use 1while 1decreasing 1replicative 1fitness 1to 1the 1bypassed host 1 [9,12]. 1In 1nature, 1VSV 1remains 1relatively 1stable, 1with 1evolutionary 1pa 4 erns 1defined by 1similar 1ecological 1conditions 1rather 1than 1geographical 1origin 1or 1immunological 1selection 1 [3,4]. 1Di erences 1in 1post-translational 1glycosylation 1of 1the 1two 1potential 1N-linked 1 glycosylation 1sites 1on 1the 1G 1protein 1in 1mammalian 1cells 1compared 1to 1insect 1cells 1is 1unknown. However, 1host 1cell 1enzymes 1are 1responsible 1for 1the 1addition 1of 1carbohydrates 1 [9,10]; 1thus, the 1composition 1and 1degree 1of 1final 1sialylation 1is 1host-dependent. 1In 1addition 1to 1potential post-translational 1modification 1di erences 1in 1host 1cells, 1the 1error-prone 1nature 1of 1VSV polymerase 1averages 1one 1mutation 1per 1genomic 1replication 1event, 1generating 1a 1wide 1diversity 1of 1genetic 1variants 1and 1quasispecies 1populations 1 [3,6,9,10,13,14]. 1Viruses 1that 1experience 1spatial 1heterogeneity 1with 1lifecycles 1that 1include 1both 1mammalian 1and 1insect hosts 1likely 1have 1even 1greater 1genetic 1diversity 1for 1evolving 1fitness 1 [15]. 1
Culicoides 1biting 1midges 1(Diptera: 1Ceratopogonidae) 1are 1one 1of 1the 1primary 1vectors playing 1a 1key 1role 1in 1VSV 1transmission 1during 1outbreaks 1in 1the 1U.S. 1 [1,3,[16][17][18][19][20][21][22][23]. 1As 1characteristic 1of 1pool-feeders, 1Culicoides 1midges 1use 1their 1mouthparts 1to 1slash 1the 1animal's 1epidermis, 1 often 1 in 1 areas 1 with 1 li 4 le 1 hair 1 such 1 as 1 the 1 naso-oral 1 regions. 1 Blood 1 is 1 ingested 1 along with 1potential 1skin 1surface 1contaminants 1that 1pool 1in 1the 1wound. 1Because 1of 1this 1feeding 1 mechanism, 1and 1because 1demonstrable 1viremia 1in 1infected 1animals 1is 1elusive 1 [2,24], 1acquisition 1of 1VSV 1via 1blood 1feeding 1has 1been 1primarily 1considered 1to 1be 1dependent 1upon the 1availability 1of 1clinically 1infected 1hosts 1exhibiting 1excessive 1salivation 1and 1naso-oral vesicular 1lesions 1as 1sources 1of 1significant 1amounts 1of 1virus 1on 1the 1skin 1 [24][25][26]. 1Once 1VSV is 1ingested 1by 1Culicoides, 1a 1pantropic 1systemic 1infection 1results 1 [18], 1which-despite 1relatively 1low 1whole-body 1virus 1titers 1(3.3-4.6 1Log10 1 PFU/mL 1 [27])-also 1allows 1for 1highly 1efficient 1venereal 1transmission 1between 1female 1and 1male 1midges 1 [28]. 1This 1midge-to-midge VSV 1transmission 1has 1been 1proposed 1as 1a 1maintenance 1mechanism 1by 1which 1epizootic 1 virus 1genotypes 1may 1overwinter, 1resulting 1in 1a 1multi-year 1outbreak 1 [3,29]. 1
To 1understand 1the 1high 1e ciency 1seen 1in 1midge 1venereal 1transmission, 1we 1hypothesized 1that 1VSV 1populations 1propagated 1within 1Culicoides 1have 1increased 1fitness 1for 1subsequent 1midge 1infections. 1Here, 1we 1investigate 1whether 1the 1originating 1host 1cell 1species 1affects 1VSV 1fitness 1for 1Culicoides 1sonorensis 1midge 1infection. 1 1 1
Three 1VSV-New 1Jersey 1(VSV-NJ) 1virus 1strains 1were 1used 1for 1midge 1infections; 1one laboratory-adapted 1positive 1control 1strain 1and 1two 1more 1recent 1outbreak 1isolates 1 [30]. Strain 182-34333 1is 1a 1Colorado 1bovine 1isolate 1from 1the 11982 1epizootic 1outbreak 1 [22] 1that 1has been 1passed 1numerous 1times 1in 1both 1mammalian 1and 1insect 1cells. 1Strain 1rNJ0612NME6 1is a 1highly 1virulent 1synthesized 1equine 1isolate 1from 1a 12012 1epizootic 1outbreak 1in 1New 1Mexico 1 [31]. 1Strain 1rNJ0806VCB 1is 1a 1synthesized 1bovine 1isolate 1from 1a 12006 1enzootic 1outbreak in 1Vera 1Cruz, 1Mexico 1 [31]. 1Both 1synthesized 1strains 1were 1produced 1in 1BHK-21 1cells 1 [30] and 1are 1courtesy 1of 1S. 1Pauszek 1and 1L. 1Rodriguez, 1Plum 1Island 1 Animal 1Disease 1Center, 1NY [31]. 1Virus 1strains 1were 1passaged 1one 1time 1at 1an 1MOI 1of 10.1 1in 1porcine 1skin 1fibroblast 1cells (mammalian-derived 1VSV) 1and 1in 1Culicoides 1cells 1(insect-derived 1VSV) 1to 1produce 1high titer 1viral 1stocks 1that 1were 1stored 1at 1-80 1°C 1for 1subsequent 1midge 1infections. 1
Porcine 1skin 1fibroblast 1cells 1(AG08113; 1Coriell 1Institute, 1Camden, 1NJ) 1were 1maintained 1in 1Eagles 1MEM 1with 1Earle's 1salts 1(Sigma, 1St. 1Louis, 1MO, 1USA) 1containing 12% 1FBS and 1100 1U 1penicillin/streptomycin 1sulfate 1at 137 1°C 1with 15% 1CO2. 1Culicoides 1W8 1cells 1 [32] (USDA, 1Arthropod-Borne 1Animal 1Diseases 1Research 1Unit, 1Manha 4 an, 1KS, 1USA) 1were maintained 1 in 1 serum-free 1 insect 1 media 1 (SFM) 1 (24.5 1 g/L) 1 supplemented 1 with 1 0.4 1 g/L 1 sodium 1 bicarbonate, 10.0585 1g/L 1l-glutamine, 10.006 1g/L 1reduced 1glutathione, 10.03 1g/L 1l-asparagine, 18 1µL 1of 110 1mg/L 1bovine 1insulin, 1and 15% 1FBS 1at 128 1°C 1with 1a 1CO2 1concentration 1of 10.2%. Vero 1MARU 1cells 1(VM; 1Middle 1America 1Research 1Unit, 1Panama 1City, 1Panama) 1grown 1in 1 199E 1media 1containing 12% 1FBS, 1100 1µg/mL 1of 1streptomycin, 1100 1units/mL 1penicillin, 1and 0.25 1ug/mL 1of 1amphotericin 1B 1at 137 1°C 1with 15% 1CO2 1were 1used 1for 1detecting 1and 1titering infectious 1viruses 1from 1midge 1samples 1as 1described 1below. 1
Adult 1Culicoides 1sonorensis 1(Wirth 1and 1Jones) 1midges 1 [33] 1(USDA, 1Arthropod-Borne Animal 1Diseases 1Research 1Unit, 1Manha 4 an, 1KS, 1USA) 1were 1used 1for 1all 1experiments. 1For viral 1exposure 1that 1bypassed 1the 1midge 1midgut 1barriers, 1females 1(1-3 1days 1post-emergence) 1were 1anesthetized 1with 1CO2 1and 1intrathoracically 1injected 1with 160 1nL 1of 1each 1virus as 1 previously 1 described 1 [28]. 1 For 1 all 1 injections, 1 a 1 starting 1 titer 1 of 1 6.4 1 log10 1 PFU/mL 1 was 1 used for 1each 1strain; 1therefore, 1each 1midge 1received 12.1 1log10 1PFU. 1 1 For 1oral 1exposure, 1females 1(1-3 1days 1post-emergence) 1were 1allowed 1to 1feed 1for 11h 1on VSV-blood 1meals 1consisting 1of 1defibrinated 1sheep 1blood 1(Lampire 1Biological 1Products, Pipersville, 1PA, 1USA) 1mixed 1with 1each 1viral 1stock. 1To 1obtain 1the 1highest 1possible 1infection rates 1while 1reflecting 1the 1di erence 1in 1viral 1titers 1observed 1in 1mammalian 1host 1vesicular lesions 1(up 1to 19 1log10 1PFU/mL 1fluid) 1compared 1to 1insect 1vectors 1(up 1to 15 1log10 1GE 1in 1whole 1 bodies 1 [27]), 1we 1used 1the 1highest 1average 1titer 1obtainable 1for 1each 1isolate 1from 1each 1cell source 1(Figure 1S1). 1Infectious 1blood 1meals 1with 1the 1virus 1isolates 1propagated 1in 1mammalian 1cells 1and 1insect 1cells 1had 1titers 1of 18.2 1log10 1PFU/mL 1and 16.4 1log10 1PFU/mL, 1respectively. After 1blood-feeding, 1fully 1engorged 1females 1were 1sorted 1from 1the 1unfed 1and 1partially 1fed 1 and 1placed 1in 1cardboard 1maintenance 1cages. 1Fed 1and 1injected 1midge 1cages 1were 1maintained 1in 1environmental 1chambers 1at 125± 11 1°C 1and 170-80% 1relative 1humidity 1with 1a 113:11 light: 1dark 1cycle 1and 1o ered 110% 1sucrose 1solution 1ad 1libitum. 1
To 1determine 1whole-body 1infection 1rates 1and 1titers 1of 1injected 1midges 1with 1each 1VSV isolate 1propagated 1in 1both 1cell 1types, 1midges 1were 1collected 1at 13-1and 110-days 1post-injection 1(dpi). 1Whole 1midges 1were 1placed 1in 1either 1300 1µL 1of 1TRIzol 1(Invitrogen; 1Thermo Fisher 1Scientific, 1Inc., 1Waltham, 1MA, 1USA) 1for 1RNA 1isolation 1(n=15) 1or 1500 1µL 1of 1antibiotic medium 1(199E 1cell 1culture 1medium 1containing 12% 1FBS 1and 1400 1U/mL 1penicillin, 1400 1 1 µg/mL 1streptomycin, 1200 1µg/mL 1gentamycin, 15 1µg/mL 1ciprofloxacin, 15 1µg/mL 1amphotericin 1 B) 1 for 1 virus 1 isolation 1 (n=15), 1 and 1 stored 1 at 1 -80 1 °C 1 until 1 processed. 1 To 1 determine 1 midgut infection 1and 1dissemination 1rates 1and 1associated 1viral 1titers 1of 1orally 1fed 1midges, 1heads (with 1salivary 1glands) 1were 1separated 1from 1bodies 1of 1midges 1fed 1with 1each 1isolate 1of 1both 1 cell 1types. 1Samples 1were 1collected 1at 13-, 17-, 1and 110-days 1post-feeding 1(dpf) 1and 1placed 1in 1 300 1µL 1of 1TRIzol 1for 1RNA 1isolation 1(n=15) 1or 1500 1µL 1of 1antibiotic 1medium 1for 1virus 1isolation 1(n=15) 1and 1stored 1at 1-80 1°C 1until 1further 1processing. 1In 1addition, 1time-zero 1wholebody 1titer 1samples 1for 1both 1intrathoracically 1injected 1and 1orally 1fed 1midges 1of 1each 1group were 1collected 1in 1300 1µL 1of 1TRIzol 1(n=5) 1or 1500 1µL 1of 1antibiotic 1medium 1(n=5) 1immediately after 1infection. 1
Frozen 1samples 1in 1TRIzol 1were 1thawed 1on 1ice, 1homogenized 1with 1bead-beating, 1and 1 total 1RNA 1was 1extracted 1using 1TRIzol-BCP 1(1-bromo-3chloropropane; 1Thermo 1Fisher 1Life Technologies, 1Waltham, 1MA, 1USA) 1as 1previously 1described 1 [28]. 1RNA 1extracts 1were 1analyzed 1using 1TaqMan 1Fast 1Virus 11-Step 1Master 1Mix 1(Applied 1Biosystems; 1Thermo 1Fisher Scientific, 1Inc.) 1in 1a 1reverse 1transcriptase 1quantitative 1PCR 1(RT-qPCR) 1assay 1detecting 1 VSV-NJ 1L 1(polymerase) 1gene 1as 1previously 1described 1 [28]. 1Standard 1curves 1and 1cycle threshold 1(Ct) 1values 1were 1calculated 1using 17500 1Fast 1Dx 1software 1v1.4 1(Applied 1Biosystems; 1Thermo 1Fisher 1Scientific, 1Inc.). 1RT-qPCR 1reactions 1with 1Ct 1 ´ 1 36.5 1were 1considered positive 1for 1VSV 1RNA 1 [27]. 1Ct 1values 1plo 4 ed 1against 1the 1log 1of 1ssRNA 1VSV 1concentration and 1the 1linear 1regression 1(y= 1-3.30578x 1+ 111.02683) 1were 1used 1to 1determine 1viral 1genomic equivalents 1per 1midge 1[28]. 1
Frozen 1midge 1samples 1stored 1in 1antibiotic 1media 1were 1thawed 1and 1homogenized 1as above. 1Samples 1were 1centrifuged 1at 112,000 1x 1g 1for 16 1min 1to 1pellet 1debris 1and 1200 1µL 1of cleared 1supernatant 1was 1added 1to 1Vero 1monolayers 1with 185-90% 1confluency 1in 124-well plates. 1Additional 1media 1(300 1µL) 1was 1added 1to 1each 1well, 1and 1plates 1were 1incubated 1at 37 1°C 1for 1up 1to 1five 1days. 1Cytopathic 1e ects 1(CPE) 1observed 1after 1two 1passages 1were 1used 1 to 1indicate 1the 1presence 1or 1absence 1of 1infectious 1virus. 1Samples 1with 1positive 1CPE 1at 1the first 1passage 1were 1titered 1by 1plaque 1assay 1using 1200 1µL 1of 1the 1remaining 1original, 1nonpassaged 1homogenate. 1Samples 1that 1showed 1CPE 1only 1after 1a 1second 1passage 1were 1below the 1 limit 1 of 1 detection 1 to 1 quantify 1 by 1 plaque 1 assay. NextSeq 1Illumina 1sequencing 1was 1performed 1by 1the 1Kansas 1State 1University 1Veterinary 1Diagnostic 1Laboratory. 1Consensus 1sequence 1identity 1was 1used 1to 1identify 1nucleotide changes 1between 1the 1original 1BHK 1low-passaged 1rNJ0612NME6 1and 1rNJ0806VCB 1strains and 1these 1strains 1after 1one 1passage 1in 1the 1mammalian 1and 1insect 1cell 1lines. 1Sequences 1for the 182-34333 1positive 1control 1reference 1isolate 1were 1excluded 1from 1the 1sequence 1analysis 1 due 1 to 1 the 1 lab-adapted 1 nature 1 of 1 this 1 isolate, 1 which 1 includes 1 numerous 1 passages 1 in 1 various mammalian 1and 1insect 1cell 1lines. 1
Immediately 1after 1injections 1(time 1zero), 1individual 1midge 1titers 1were 1below 1the 1limit of 1detection 1to 1quantify 1by 1plaque 1assay; 1however, 1all 1homogenates 1were 1positive 1by 1CPE screening 1after 1one 1or 1two 1passages 1(100%; 115/15), 1and 1detectable 1by 1RT-qPCR 1with 1genome 1equivalent 1(GE) 1levels 1ranging 1from 12.1-3.2 1log10 1virus 1particles 1(Figure 1S1), 1indicating 1all 1midges 1received 1statistically 1similar 1doses. 1Viral 1RNA 1was 1detected 1in 1all 1females, independent 1of 1cell 1source 1or 1sampling 1time 1point. 1Mammalian-derived 1VSV 1peaked 1earlier 1than 1insect-derived 1VSV 1with 1significantly 1higher 1virus 1titers 1(GE, 1as 1detected 1by 1RT-qPCR) 1at 13 1days 1post-injection 1(dpi) 1(p= 10.0007; 1Figure 11a). 1Conversely, 1at 110 1dpi, 1RNA 1was significantly 1higher 1in 1midges 1injected 1with 1insect-derived 1VSV 1(p= 10.0002; 1Figure 11a). 1 1
No 1significant 1di erences 1in 1infection 1rates, 1as 1detected 1by 1CPE 1screening 1for 1infectious 1virus, 1were 1found 1in 1midges 1injected 1with 1either 1mammalian-1or 1insect-derived 1VSV at 1either 13 1dpi 1(80% 1and 190%) 1or 110 1dpi 1(100% 1and 1100%) 1(Figure 11b, 1pie 1charts). 1Nor 1were 1 there 1significant 1di erences 1in 1the 1average 1whole-body 1titers 1by 1plaque 1assaying 1the 1original 1homogenate 1from 13 1dpi 1(4.5 1 and 14.6 1 log10 1PFU/mL) 1 or 110 1dpi 1(4.
Immediately 1after 1blood-feeding, 1ingested 1virus 1titers 1were 1measured 1in 1individual fully 1engorged 1females 1by 1plaque 1assay 1and 1RT-qPCR. 1As 1expected, 1mean 1titers 1of 1ingested 1virus, 1as 1detected 1by 1RT-qPCR, 1were 1higher 1in 1midges 1that 1fed 1on 1the 1higher 1titer mammalian-VSV 1blood 1meal 1(Ct 1mean 119.7, 15.5 1log10 1GE) 1than 1in 1midges 1that 1fed 1on 1the lower 1titer 1insect-VSV 1blood 1meal 1(Ct 1mean 126.8, 13.5 1log10 1GE) 1(Figure 1S1). 1Infectious 1virus titers 1in 1single 1whole 1bodies 1immediately 1after 1ingestion, 1as 1detected 1by 1plaque 1assay, 1 were 1achieved 1in 1100% 1(15/15) 1of 1the 1females 1fed 1with 1the 1mammalian-VSV, 1with 1a 1mean 1 titer 1of 15 1log10 1PFU/mL; 1and 1in 166.7% 1(10/15) 1of 1the 1females 1fed 1with 1insect-VSV, 1with 1a 1 mean 1titer 1of 11.9 1log10 1PFU/mL. 1
At 13, 17, 1and 110 1days 1post-feeding 1(dpf), 1individual 1decapitated 1bodies 1and 1their 1associated 1head/gland 1samples 1were 1assayed 1separately 1to 1determine 1VSV 1infection 1and 1dissemination 1rates, 1respectively. 1Although 1the 1highest 1infectious 1dose 1possible 1for 1the 1insect-VSV 1blood 1meals 1(6.4 1log10 1PFU/mL) 1was 1significantly 1lower 1than 1that 1of 1the 1mammalian-VSV 1 blood 1 meals 1 (8.2 1 log10 1 PFU/mL), 1 viral 1 titers 1 (GE) 1 and 1 the 1 proportion 1 of 1 VSV 1 RNApositive 1 bodies 1 detected 1 by 1 RT-qPCR 1 (Figure 1 2a) 1 were 1 significantly 1 higher 1 at 1 all timepoints 1in 1midges 1fed 1with 1insect-derived 1blood 1meals 1(3 1and 17 1dpf 1p< 10.0001, 110 1dpf 1 p= 10.0436). 1Likewise, 1the 1percentages 1of 1VSV-positive 1bodies 1with 1infectious 1virus 1as 1detected 1by 1CPE 1(Figure 12b) 1were 1significantly 1higher 1at 1all 1time 1points 1in 1midges 1fed 1with 1 insect-derived 1blood 1meals 1(3 1dpf 1p= 10.0007, 17 1dpf 1p= 10.0210, 110 1dpf 1p= 10.0029). 1All 1body samples 1were 1below 1the 1detection 1limit 1of 1plaque 1assay 1for 1titration 1purposes. 1
Viral 1titers 1(GE) 1and 1proportions 1of 1positive 1midge 1head/gland 1samples, 1as 1detected by 1RT-qPCR 1(Figure 13a), 1were 1also 1higher 1for 1insect-derived 1VSV 1blood-fed 1midges 1compared 1to 1mammalian-derived 1VSV 1blood-fed 1midges, 1although 1di erences 1were 1only 1significant 1at 17 1dpf 1(p= 10.0356). 1Likewise, 1the 1percentages 1of 1VSV-positive 1head/gland 1samples 1with 1infectious 1virus, 1as 1detected 1by 1CPE 1(Figure 13b), 1were 1higher 1at 1all 1time 1points
The 1field 1isolate 1viral 1stocks 1rNJ0806VCB 1and 1rNJ0612NME6 1(originally 1produced 1in BHK-21 1cells) 1were 1replicated 1once 1in 1each 1of 1our 1test 1host 1cell 1sources 1(porcine 1and 1Culicoides) 1 [30]. 1The 1laboratory 1stock 1isolate, 182-34333, 1had 1undergone 1multiple 1passages 1in multiple 1species 1of 1cell 1lines 1over 130 1years. 1Since 1laboratory 1adaptation 1constitutes 1higher 1 adaptability 1 and 1variation 1 in 1fitness 1 due 1to 1nucleotide 1changes 1 [6,10], 1we 1chose 1to 1compare consensus 1sequences 1of 1the 1more 1recent, 1low 1passage 1field 1isolates, 1rNJ0612NME6 1and rNJ0806VCB, 1before 1and 1after 1the 1single 1passage 1in 1either 1mammalian 1(porcine 1skin 1 1 fibroblast) 1or 1insect 1(Culicoides 1W8) 1cells 1to 1detect 1genome 1changes 1that 1might 1inform 1fitness 1di erences. 1We 1found 1no 1nucleotide 1sequence 1changes. 1The 1sequence 1identity 1for both 1isolates 1in 1both 1cell 1lines 1had 1100% 1identity 1to 1their 1BHK-parental 1strains. 1
VSV 1epidemiology 1is 1very 1complex, 1involving 1a 1broad 1mammalian 1and 1insect 1host range 1with 1direct 1contact 1transmission 1between 1clinical 1mammalian 1hosts 1and 1vectorborne 1transmission 1via 1multiple 1vector 1species 1 [2]. 1VS 1outbreaks 1in 1the 1U.S. 1can 1occur 1as single-year 1events. 1However, 1for 1the 1past 140 1years, 1outbreak 1viruses 1have 1often 1overwintered 1between 1vector 1seasons, 1in 1the 1absence 1of 1infected 1animals, 1with 1the 1same 1viral 1genotype 1re-emerging 1in 1a 1second-year 1outbreak 1 (1982-1983, 11985-1986, 11998-1998, 12004-2006, 12014-2015, 12019-2020) 1 [3,16,34]. 1It 1has 1been 1suggested 1that 1VSV 1is 1being 1inter-epizootically 1 maintained 1 in 1 yet-to-be-identified 1 natural 1 mammalian 1 reservoirs 1 [35,36], 1 or 1 within vector 1populations 1independent 1of 1feeding 1on 1viremic 1animals 1[28, [37][38][39][40]. 1
The 1VSV 1multi-host 1lifecycle 1demonstrates 1the 1flexibility 1of 1VSV 1populations 1and their 1ability 1to 1quickly 1adapt 1to 1infect 1and 1replicate 1in 1very 1di erent 1host 1cell 1environments. 1As 1in 1other 1arbovirus 1systems, 1alternating 1host 1transmission 1cycles 1may 1constrain the 1evolutionary 1rates 1through 1viral 1genetic 1stability 1without 1a ecting 1viral 1fitness 1for 1either 1host 1alone 1 [41][42][43][44]. 1Likewise, 1laboratory 1experiments 1have 1shown 1that 1despite 1VSV mutating 1approximately 1four 1times 1slower 1in 1insect 1cells 1 [45], 1there 1are 1no 1e ects 1in 1the number 1of 1mutations 1accumulated 1or 1any 1fitness 1increase 1or 1cost 1when 1there 1are 1alternating 1 host 1 passages 1 [10,12]. 1 However, 1 host-derived 1 non-genetic 1 di erences 1 such 1 as 1 envelope composition 1and 1protein 1glycosylation 1can 1result 1due 1to 1substantial 1di erences 1in 1posttranslational 1machinery 1and 1the 1cell 1membrane 1constitution 1of 1insect 1and 1mammalian cells 1 [46][47][48]. 1 1
We 1 hypothesized 1 that 1 VSV 1 propagated 1 within 1 Culicoides 1may 1 evolve 1 increased 1 fitness for 1insect 1cells, 1allowing 1persistence 1without 1deleterious 1e ects 1to 1the 1population. 1This adaptability 1would 1further 1enable 1the 1re-emergence 1of 1an 1overwintered 1genotype 1when 1 conditions 1for 1vectored 1bite 1transmission 1are 1once 1again 1ideal. 1For 1some 1arboviruses, 1alternate 1replication 1in 1two 1divergent 1hosts 1may 1reduce 1overall 1virus 1fitness 1in 1both 1hosts compared 1to 1a 1single-host 1virus 1lifecycle 1 [49]. 1However, 1VSV 1populations 1can 1successfully replicate 1in 1multiple 1cellular 1environments 1without 1genetic 1fitness 1constraints 1 [9,10,50]. Non-genetic 1di erences 1accompanied 1by 1di erent 1replicative 1strategies 1in 1invertebrate vectors 1(persistent, 1non-lytic 1replication) 1and 1vertebrate 1hosts 1(acute, 1lytic 1infection) 1may 1 play 1a 1significant 1role 1in 1shaping 1VSV 1evolution 1and 1therefore 1influence 1disease 1transmission 1dynamics. 1To 1evaluate 1the 1potential 1host-specific 1fitness 1increase, 1we 1evaluated 1the infection, 1replication, 1and 1dissemination 1di erences 1of 1insect-1and 1mammalian-derived viruses 1in 1a 1key 1VSV 1vector, 1Culicoides 1sonorensis. 1
First, 1we 1evaluated 1the 1viral 1infection 1dynamics 1after 1intrathoracic 1inoculation 1and after 1oral 1infection 1with 1VSV 1propagated 1in 1a 1mammalian 1host 1(porcine 1epidermal) 1and insect 1vector 1(Culicoides 1W8) 1cell 1lines. 1The 1results 1of 1independent 1infections 1with 1three 1 di erent 1viral 1isolates 1were 1grouped 1by 1cell 1line 1to 1estimate 1the 1e ects 1of 1the 1species-specific 1cell 1environment. 1In 1addition 1to 1highly 1sensitive 1molecular 1methods 1used 1to 1estimate viral 1load, 1infectious 1virus 1isolations 1by 1CPE 1and 1plaque 1assays 1represent 1a 1more 1relevant measurement 1of 1any 1infectiousness 1di erences 1from 1host-derived 1factors. 1Disseminated 1 virus 1in 1heads 1correlate 1with 1salivary 1gland 1infection 1and 1transmission 1potential 1 [18]; therefore, 1the 1dissemination 1rates 1observed 1in 1this 1study 1may 1also 1indicate 1the 1magnitude of 1potential 1bite 1transmission 1competence 1by 1vectors 1infected 1with 1mammalian-sourced VSV 1versus 1insect-sourced 1VSV. 1 1 For 1many 1vector-borne 1diseases, 1most 1predictions 1often 1assume 1that 1vector 1infection rates 1are 1only 1driven 1by 1the 1magnitude 1of 1pathogen 1exposure 1 [27,[51][52][53]. 1However, 1the results 1of 1this 1study 1demonstrate 1that 1infection 1rates 1can 1also 1be 1strongly 1regulated 1by factors 1beyond 1the 1initial 1viral 1load. 1Here 1we 1have 1shown 1that 1both 1mammalian-1and 1insect-derived 1viruses 1can 1e ciently 1infect 1midges 1and 1replicate 1equally 1well 1when 1the 1 1 1 midgut 1barrier 1is 1circumvented 1via 1injection 1(Figure 11). 1However, 1with 1oral 1infection, where 1the 1virus 1was 1required 1to 1infect 1and 1escape 1the 1midgut 1to 1disseminate, 1insect-derived 1VSV 1showed 1significantly 1increased 1infection 1rates 1and 1titers 1in 1bodies, 1as 1well 1as infection 1and 1dissemination 1rates 1in 1heads/glands 1compared 1to 1midges 1infected 1with 1 mammalian-derived 1VSV 1(Figures 12 1and 13) 1at 1all 1time 1points 1tested. 1
In 1midgut 1bypass 1trials, 1although 1all 1midges 1were 1injected 1with 1similar 1titers, 1higher 1 mean 1titers 1of 1viral 1RNA 1were 1detected 1in 1midges 1injected 1with 1mammalian-derived 1VSV at 1the 1early 1time 1point 1(3 1dpi). 1This 1may 1have 1triggered 1an 1enhanced 1immune 1response against 1these 1early 1VSV 1populations, 1resulting 1in 1the 1lower 1titer 1seen 1by 1day 110. 1Similar immune 1responses 1to 1mammalian-derived 1VSV 1may 1have 1influenced 1infection 1outcomes seen 1 in 1 oral 1 exposures 1 due 1 to 1 the 1 higher 1 starting 1 virus 1 titers 1 delivered 1 with 1 the 1 blood 1 meal. However, 1increased 1fitness 1of 1the 1insect-derived 1virus 1at 1the 1midgut 1level 1(measured 1as increased 1genome 1replication) 1may 1also 1be 1explained 1by 1facilitation 1in 1the 1a 4 achment 1and entry 1to 1the 1midgut 1epithelial 1cells 1due 1to 1enhanced 1insect-derived 1viral 1glycoprotein 1fusion 1with 1target 1cells 1 [54][55][56], 1or 1even 1due 1to 1be 4 er 1immune 1evasion 1 [57]. 1 1 As 1similarly 1seen 1in 1field-collected 1insect 1and 1animal 1samples 1during 1outbreaks, 1it 1is 1 important 1to 1note 1that 1during 1propagation 1of 1all 1the 1VSV-NJ 1isolates 1used, 1viral 1production 1(titer) 1was 1lower 1in 1the 1Culicoides 1cells 1than 1in 1the 1porcine 1cells 1(Figure 1S2). 1In 1insect cells, 1where 1some 1receptors 1may 1be 1present 1at 1lower 1densities 1than 1in 1mammalian 1cells, arbovirus 1selection 1may 1favor 1rapid 1entry 1over 1the 1ability 1to 1grow 1to 1high 1titers 1 [44]. 1 1 Within-host 1fitness 1experiments 1have 1been 1frequently 1conducted 1in 1cell 1culture 1experiments 1 [9,10,50,54,[58][59][60]; 1however, 1evaluating 1the 1impact 1of 1within-host 1fitness 1at 1the arthropod 1level 1can 1be 1more 1challenging 1to 1elucidate 1due 1to 1non-genetic 1di erences 1between 1viruses 1derived 1from 1each 1host 1 [46], 1or 1because 1the 1specific 1host 1environment 1may apply 1selection 1pressures 1to 1virus 1populations 1 [61]. 1We 1believe 1that 1host-dependent 1modification 1di erences 1in 1starting 1virus 1populations 1may 1dictate 1infection 1and 1replication success 1and 1subsequently 1influence 1transmission 1rates. 1Such 1di erences 1may 1relate 1to 1the fitness 1and 1robustness 1of 1quasispecies 1populations 1derived 1from 1cells 1of 1the 1di erent 1species. 1As 1is 1indicated 1by 1the 1multi-host, 1multi-vector 1nature 1of 1VSV, 1it 1can 1infect 1multiple host 1cell 1types. 1In 1mammalian 1infections, 1VSV 1is 1restricted 1to 1skin 1lesions 1and 1their 1draining 1lymph 1nodes 1 [62]. 1The 1representative 1mammalian 1cell 1line 1used 1in 1this 1study 1was 1similarly 1restrictive 1in 1that 1it 1was 1a 1clonal 1porcine 1skin 1fibroblast 1line. 1In 1contrast, 1VSV 1infection 1in 1midge 1vectors 1is 1pantropic, 1with 1virus 1detected 1in 1all 1cell 1types 1of 1all 1tissues 1 [18]. Likewise, 1the 1representative 1insect 1cell 1line 1used 1in 1this 1study 1was 1similarly 1diverse 1in 1that it 1 was 1 made 1 from 1 whole 1 midge 1 embryos 1 [32]. 1 The 1 spatial 1 heterogeneity 1 of 1 such 1 a 1 polytypic cell 1line 1may 1result 1in 1a 1larger 1number 1of 1minority 1variants 1 [15] 1which 1have 1a 1greater 1genetic 1diversity 1for 1evolving 1fitness 1more 1rapidly 1 [63]. 1A 1more 1diverse 1virus 1population 1 produced 1in 1midge 1cells 1may 1have 1an 1increased 1ability 1to 1rapidly 1mutate 1toward 1fitness and 1may 1help 1explain 1the 1e ciency 1seen 1in 1venereal 1transmission. 1An 1initial 1analysis 1of minority 1variants 1from 1raw 1genome 1sequence 1reads 1showed 1a 1trend 1toward 1increased 1diversity 1for 1virus 1propagated 1in 1Culicoides 1cells 1compared 1to 1the 1porcine 1cells, 1but 1was 1not conclusive 1as 1deeper 1reads 1are 1needed. 1
With 1dominant 1consensus 1viral 1genome 1sequences 1remaining 1the 1same, 1independent of 1the 1host 1cell 1environment, 1di erences 1in 1observed 1fitness 1cannot 1be 1a 4 ributed 1to 1stabilized 1changes 1in 1the 1genome 1or 1protein-coding 1sequences, 1and 1may 1suggest 1that 1posttranslational 1modifications 1contribute 1to 1fitness. 1In 1the 1case 1of 1enveloped 1viruses, 1hostencoded 1 proteins 1 are 1 embedded 1 in 1 the 1 host-derived 1 lipid 1 bilayer 1 membrane 1 envelope 1 [64]. Alternating 1host 1variation 1in 1the 1lipid 1bilayer 1content 1has 1shown 1to 1have 1subsequent 1effects 1on 1Sindbis 1virus 1stability 1 [59], 1and 1Ross 1River 1and 1Venezuelan 1equine 1encephalitis virus 1capacity 1to 1induce 1immune 1responses 1 [57]. 1Moreover, 1additional 1host 1cytoplasmic 1 factors 1 can 1 be 1 incorporated 1 into 1 viral 1 progeny 1 [64]. 1 Previous 1 VSV 1 proteomic 1 analyses 1 have shown 1host 1cell-type-dependent 1profiles 1influencing 1the 1infectivity 1 of 1 the 1resulting 1virions [64,65]. 1 Additionally, 1the 1envelope 1of 1VSV 1is 1spiked 1with 1glycoprotein 1trimers 1(G 1protein), which 1 are 1 essential 1 for 1 cell 1 receptor 1 binding 1 and 1 entry, 1 thereby 1 playing 1 a 1 role 1 in 1 host 1 range 1 1 determination 1 [66]. 1Host-dependent 1expression 1of 1multiple 1enzymes 1of 1the 1glycosylation 1 pathways 1often 1results 1in 1the 1production 1of 1less 1complex 1glycans 1with 1high 1mannose 1content 1in 1insect 1cells 1 [47,48]. 1Host-derived 1arboviral 1fitness 1due 1to 1glycosylation 1di erences has 1been 1previously 1detected 1in 1Dengue 1 [54], 1La 1Crosse 1 [55], 1West 1Nile, 1and 1Sindbis 1viruses 1 [56]. 1Likewise, 1host-dependent 1modifications 1of 1the 1VSV 1G 1protein 1 [67,68] 1may 1constrain 1virion 1infectivity, 1cell 1tropism, 1and 1levels 1of 1virus 1replication 1in 1di erent 1host 1cell lines 1 [65,66]. 1These 1mechanisms 1considered, 1e ects 1seen 1from 1di erences 1in 1the 1virion 1lipid 1bilayer 1envelope-and/or 1G 1protein 1glycosylation 1with 1mammalian-derived 1VSVwould 1be 1transient, 1 as 1 all 1 progeny 1virions 1would 1have 1insect-derived 1envelopes 1 and 1insect cell-dependent 1G 1protein 1glycosylation 1after 1one 1round 1of 1replication. 1In 1these 1studies, 1the observed 1fitness 1increase 1of 1insect-derived 1VSV 1was 1not 1transient; 1it 1was 1seen 1at 1all 1time points 1(3, 17, 1and 110 1days) 1post-exposure. 1
Di erent 1selective 1pressures 1on 1arboviruses 1primarily 1transmi 4 ed 1through 1bloodfeeding, 1versus 1transovarially 1or 1venereally, 1may 1also 1influence 1the 1degree 1of 1fitness 1observed 1at 1the 1vector 1level 1 [41]. 1For 1viruses 1being 1maintained 1within 1vector 1populations, 1a stronger 1selective 1pressure 1for 1a 4 enuation 1of 1within-host 1virulence 1accompanied 1by 1increased 1vector 1infectivity 1is 1expected 1 [41,69]. 1VSV 1exhibits 1titer 1loss 1during 1persistent 1passages 1in 1insect 1cells 1 [50] 1and 1after 1several 1generations 1of 1solely 1transovarial 1transmission within 1sand 1flies 1 [38,39], 1indicating 1the 1requirement 1of 1clinically 1infected 1mammalian hosts 1to 1perpetuate 1the 1VSV 1lifecycle. 1Nevertheless, 1our 1results 1show 1that 1VSV 1propagation 1in 1Culicoides 1cells 1is 1significantly 1more 1fit 1for 1e cient 1replication 1within 1midges 1and for 1subsequent 1midge-to-midge 1transmission. 1Our 1results 1reinforce 1the 1idea 1that 1host-virus 1interactions 1play 1a 1critical 1role 1in 1VSV 1epidemiology 1and 1suggest 1that 1vector-mediated selection 1may 1play 1a 1role 1in 1virus 1persistence 1during 1interepizootic 1periods. 1