Ci a ion: Jag ap, S.V.; B ink, J.; F ank,
S.C.; Badusche, M.; Leggewie, M.;
S eenu, V.B.; Fuss, J.; Schne le , E.;
Al inli, M. Agua Salud Alpha i us
In ec ion, Dissemina ion and
T ansmission in Aedes aegyp i
Mosqui oes. Vi uses 2023,15, 1113.
h ps://doi.o g/10.3390/ 15051113
Academic Edi o s: Valé ia
Lima Ca alho and Ca los A.
M. Ca alho
Recei ed: 23 Ma ch 2023
Re ised: 25 Ap il 2023
Accep ed: 28 Ap il 2023
Published: 3 May 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
i uses
A icle
Agua Salud Alpha i us In ec ion, Dissemina ion and
T ansmission in Aedes aegyp i Mosqui oes
Swa i V. Jag ap 1,2, Jo n B ink 1, S ea C. F ank 1, Ma lis Badusche 1, Mayke Leggewie 1, Va ipally B. S eenu 3,
Janina Fuss 4, Es he Schne le 1,2,5,* and Mine Al inli 1,2,*
1Be nha d-Noch -Ins i u e o T opical Medicine, 20359 Hambu g, Ge many; [email p o ec ed] (S.V.J.)
2Ge man Cen e o In ec ion Resea ch, Pa ne Si e Hambu g-Lübeck-Bo s el-Riems, 20359 Hambu g, Ge many
3MRC-Uni e si y o Glasgow-Cen e o Vi us Resea ch, Glasgow G61 1QH, UK
4Ins i u e o Clinical Molecula Biology (IKMB), Kiel Uni e si y, 24105 Kiel, Ge many
5Facul y o Ma hema ics, In o ma ics and Na u al Sciences, Uni e si y Hambu g, 20148 Hambu g, Ge many
*Co espondence: [email p o ec ed] (E.S.); [email p o ec ed] (M.A.)
Abs ac :
Mosqui oes a e compe en ec o s o many impo an a h opod-bo ne i uses
(a bo i uses). In addi ion o a bo i uses, insec -speci ic i uses (ISV) ha e also been disco e ed in
mosqui oes. ISVs a e i uses ha eplica e in insec hos s bu a e unable o in ec and eplica e in
e eb a es. They ha e been shown o in e e e wi h a bo i us eplica ion in some cases. Despi e
he inc ease in s udies on ISV–a bo i us in e ac ions, ISV in e ac ions wi h hei hos s and how
hey a e main ained in na u e a e s ill no well unde s ood. In he p esen s udy, we in es iga ed
he in ec ion and dissemina ion o he Agua Salud alpha i us (ASALV) in he impo an mosqui o
ec o Aedes aegyp i h ough di e en in ec ion ou es (pe o al in ec ion, in a ho acic injec ion)
and i s ansmission. We show he e ha ASALV in ec s he emale Ae. aegyp i and eplica es when
mosqui oes a e in ec ed in a ho acically o o ally. ASALV dissemina ed o di e en issues, including
he midgu , sali a y glands and o a ies. Howe e , we obse ed a highe i us load in he b ain han
in he sali a y glands and ca casses, sugges ing a opism owa ds b ain issues. Ou esul s show
ha ASALV is ansmi ed ho izon ally du ing adul and la al s ages, al hough we did no obse e
e ical ansmission. Unde s anding ISV in ec ion and dissemina ion dynamics in Ae. aegyp i and
hei ansmission ou es could help he use o ISVs as an a bo i us con ol s a egy in he u u e.
Keywo ds: Agua Salud alpha i us; insec -speci ic i uses; ho izon al ansmission; Aedes aegyp i
1. In oduc ion
Mosqui oes a e common ec o s o a bo i uses, including medically impo an i uses
such as Zika, chikungunya, dengue, and Yellow e e i uses. In addi ion o he a bo i uses
hey ansmi o e eb a es, an inc easing numbe o insec -speci ic i uses (ISV) ha e been
disco e ed in mosqui oes o e he las decade [
1
–
3
]. ISVs a e a di e se g oup o i uses
ha eplica e in mosqui oes and mosqui o-de i ed cell lines; howe e , hey a e unable o
in ec e eb a es and de i ed cell lines.
ISV s udies ha e gained ac ion du ing he las decade as some ISVs can se e as ac-
cine pla o ms [
4
,
5
], and some can in e e e wi h a bo i us in ec ion [
6
–
10
]. ISV in e e ence
wi h a bo i uses has been in es iga ed mos ly o la i i uses [
3
] and has p esen ed con a-
dic ing esul s anging om in e e ence o he acili a ion o a bo i us in ec ion [
6
,
9
,
11
–
13
].
Howe e , he e a e s ill many open ques ions in e ms o he in e ac ions o ISV wi h hei
hos s, such as how hey a e main ained in na u e and how hey a ec hei mosqui o
hos s [13].
Despi e he high numbe o insec -speci ic la i i uses ha ha e been disco e ed [
14
],
o da e, only i e insec -speci ic alpha i uses ha e been ound in mosqui oes, namely
he Eila i us (EILV) [
15
], Tai Fo es alpha i us (TALV) [
16
], Mwinilunga alpha i us
(MWAV) [
17
], Yada Yada Vi us [
18
] and Agua Salud alpha i us (ASALV) [
19
]. This s a k
Vi uses 2023,15, 1113. h ps://doi.o g/10.3390/ 15051113 h ps://www.mdpi.com/jou nal/ i uses
Vi uses 2023,15, 1113 2 o 13
di e ence in hei p esence in na u al popula ions compa ed o la i i uses could be ela ed
o di e en ansmission ou es and in e ac ions wi h hei mosqui o hos s.
Alpha i uses encompass a la ge g oup o posi i e-sense single-s anded RNA i uses
wi h a di e se hos ange, anging om e eb a es (e.g., humans, monkeys, ho ses, bi ds,
ep iles, and amphibians) o in e eb a es (e.g., mosqui oes and icks). Only wo o he
insec -speci ic alpha i uses ha e been isola ed o da e, namely ASALV and EILV. EILV was
isola ed om a pool o Anopheles cous ani and has been shown o in ec di e en mosqui o
species, including Aedes albopic us,Ae. aegyp i,Anopheles gambiae,Culex quinque ascia us, and
Cx. a salis [
15
,
20
,
21
]. EILV can in e e e wi h he eplica ion o se e al a bo i uses [
22
];
howe e , i is no ye known whe he his is he case o o he insec -speci ic alpha i uses,
including ASALV. ASALV was isola ed om Culex decla a o mosqui oes and sampled
in Panama [
19
], al hough he ASALV hos ange
in i o
has no ye been in es iga ed.
In i o
, ASALV eplica es bo h in Ae. albopic us-de i ed C6/36 and U4.4 cells [
19
] and Ae.
aegyp i-de i ed AF5 cells [
23
]. ASALV has been shown o in e ac wi h he majo an i i al
esponse in mosqui oes wi h RNA in e e ence. RNA in e e ence (RNAi) is a na u al
phenomenon ha egula es gene exp ession and plays an impo an ole in he egula ion
o i al in ec ions in mosqui oes [
24
,
25
]. I can be di ided in o h ee dis inc pa hways
in mosqui oes: he mic oRNA (miRNA), small in e e ing RNA (siRNA), and P-elemen -
induced wimpy es is (PIWI)-in e ac ing RNA (piRNA) pa hway. The siRNA pa hway is
an i i al agains all es ed a bo i uses [
26
] and ASALV, oo [
23
]. The siRNA pa hway is
igge ed by long double-s anded RNAs (dsRNA), o ins ance, eplica ion in e media es,
p oduced du ing i al eplica ion. dsRNAs a e hen cu by Dice 2 in o i us-de i ed
siRNAs ( siRNA) o 21 nucleo ides (n ) in size, and siRNAs a e loaded in o he mul i-
p o ein, RNA-induced silencing complex (RISC). RISC can hen a ge complemen a y i al
RNA o subsequen clea age, esul ing in he inhibi ion o i us eplica ion [
27
,
28
]. In
addi ion o siRNAs, i us-de i ed piRNA ( piRNA) o 25 o 29 n in leng h ha e also been
epo ed in in ec ed mosqui oes and mosqui o-de i ed cells [
29
,
30
]. In Ae.aegyp i-de i ed
cells, piRNA is p oduced h ough a ping-pong ampli ica ion cycle by Ago3 and Piwi5/6.
The esul an piRNAs ha e a bias o ei he u idine a posi ion one o adenine a posi ion
en in he an isense and sense sequences, espec i ely (U1 and A10), and a complemen a y
egion o 10 nucleo ides [29–31].
He e, we in es iga ed ASALV in ec ion and dissemina ion in Ae. aegyp i h ough
di e en in ec ion ou es (o al o in a ho acic injec ion) and i s possible ansmission
ou es. Unde s anding how ISVs in e ac wi h hei mosqui o hos s and ansmission
ou es is impo an o he use o ISVs o a bo i us con ol ools in he u u e.
2. Ma e ials and Me hods
2.1. Mosqui oes
The Ae. aegyp i PAEA s ain (ob ained om P o . Failloux, Ins i u e Pas eu ) was used
in he expe imen s [
32
]. The Cx. quinque ascia us (Malaysia) labo a o y colony was ob ained
om Baye (Baye , Le e kusen, Ge many). Bo h colonies we e ea ed and main ained a
28 ±5◦C and 80% humidi y, wi h a 12 h ligh /da k cycle and 10% uc ose ad libi um.
2.2. Vi us S ocks and Ti a ion
A 70% con luen C6/36 cell lask (T75) was inocula ed wi h ASALV [
30
]. Th ee days
a e inocula ion, he clea ed supe na an was collec ed and s o ed a
−
80
◦
C. ASALV
i a ions we e pe o med using he TCID50 assay wi h C6/36 cells. 4
×
10
4
cells/well
we e seeded in a 96-well pla e wi h a cell medium (Leibo i z’s L15 medium The mo
Fishe Scien i ic, Inc., Wal ham, MA, USA) supplemen ed wi h 10% oe al cal Se um
(The mo Fishe Scien i ic Inc., Wal ham, MA USA) 1x penicillin/s ep omycin (The mo
Fishe Scien i ic Inc., Wal ham, MA, USA) and 10% yp ose phospha e b o h (Gibco Li e
Technologies, Paisley, UK). Fou eplica es we e used o each dilu ion. A e h ee days o
incuba ion a 28
◦
C, he cells we e i s checked o mo phological changes, as low ISV i e s
can cause mo phological changes wi hou causing a clea CPE. Then, he cells we e ixed
Vi uses 2023,15, 1113 3 o 13
wi h 8% o maldehyde and s ained wi h c ys al iole . The i e was calcula ed acco ding
o he Spea man–Kaebe algo i hm [33].
2.3. ASALV T ansmission Expe imen s
2.3.1. Adul In ec ions h ough Feeding and In a ho acic Injec ions
Be ween 7 and 13 days old emale Ae. aegyp i mosqui oes we e ei he in ec ed by
in a ho acic injec ion o o al eeding. Fo in a ho acic injec ion, he mosqui oes we e
immobilised wi h CO
2
, kep on ice, and injec ed wi h ASALV (10
3
PFU/mosqui o, using
Nanojec II (D ummond). Fo in ec ions h ough eeding, mosqui oes we e s a ed o
uc ose o 24 h and hen ed wi h an ASALV-PBS solu ion (10
7
PFU/mL, 10% ATP, and
blue ood dye) o 45 min, using he Hemo ek memb ane eeding sys em wi h syn he ic
chicken skin as he memb ane. Cx. quinque ascia us mosqui oes (Figu e S1) we e in ec ed
using he same i us i e s and injec ion me hod, al hough, o o al in ec ion, co on buds
we e used o deli e he ASALV-PBS solu ion. Mosqui oes we e anaes he ised wi h CO
2
,
and ed mosqui oes we e incuba ed a 28
±
5
◦
C and 80% humidi y, wi h a 12 h ligh /da k
cycle and 10% uc ose ad libi um.
Inocula ed mosqui oes we e ei he ha es ed a 0, 7, o 14 days pos -in ec ion (dpi) o
used in u he expe imen s (sali a ion, dissemina ion, ho izon al ansmission assays).
2.3.2. ASALV Dissemina ion
ASALV- ed o injec ed adul emales we e anaes he ised wi h CO
2
and dissec ed a
7 and 14 dpi. The heads (including sali a y glands), gu s, o a ies, and ca casses o 15
mosqui oes we e pooled pe sample and homogenised in GMEM (The mo Fishe Scien-
i ic, Inc., Wal ham, MA, USA). The o al RNA was isola ed using T izol LS (In i ogen,
Ca lsbad CA, USA), acco ding o he manu ac u e ’s p o ocol. To u he in es iga e he
dissemina ion in he head pa s, heads, sali a y glands and ca casses om ASALV-injec ed
mosqui oes we e dissec ed. Be o e homogenisa ion, he an enna and mou h pa s we e
emo ed om he heads o in es iga e ASALV dissemina ion o he b ain. The body pa s
o 5 mosqui oes we e pooled pe sample, and a o al o 6 samples we e p ocessed.
2.3.3. ASALV Ho izon al T ansmission
To in es iga e ho izon al ansmission du ing he adul li e s age, ASALV-injec ed Ae.
aegyp i emales we e pu in he same cage wi h unin ec ed male mosqui oes. A week la e ,
males and emales we e collec ed and es ed o ASALV in ec ion.
To in es iga e he ho izon al ansmission du ing la al li e s ages, one-day-old
mosqui o la ae we e ans e ed in o a 96-well pla e in a 30
µ
L la ae ood solu ion
(200 mL wa e + 0.2 g ish ood pelle - Se a Wels Tabs XXL). 10
7
PFU ASALV pe well was
added and incuba ed a 28
◦
C
±
5 wi h a 12 h ligh /da k cycle. Two days pos -in ec ion,
he la ae we e ans e ed on o a 24-well pla e in 1 mL ood solu ion. A o al o 300
µ
L
o ood solu ion was added e e y wo days. A e 6 days, he la ae we e ans e ed o
a clean 24-well pla e, gi en esh ood, and le o pupa e. The pupae we e ans e ed o
glass ubes, and eme ging adul s we e collec ed and s o ed a
−
80
◦
C un il analysed. The
expe imen was epea ed wice independen ly.
2.3.4. Sali a ion Assay
A sali a ion assay was pe o med using ASALV-injec ed emale mosqui oes 14 dpi.
Mosqui oes we e anaes he ised wi h CO
2,
and hei legs and wings we e emo ed. They
we e o ced o sali a e in 10
µ
L o PBS in a cu il e ip, as p e iously desc ibed [
32
]. A e
30 min o o ced sali a ion, he ip con aining he PBS-sali a mix u e was emo ed and
placed in o a eac ion ube con aining 10
µ
L o PBS. Finally, 20
µ
L sali a + PBS was added o
C6/36 seeded in 96 well pla es. As no mo phological changes o CPE was obse ed in he
cells, o check whe he i al RNA was p esen , he supe na an was collec ed om he sali a
samples and some nega i e con ol cells. A QIAamp i al isola ion ki (QIAGEN, Hilden,
Ge many) was used o RNA isola ion om he supe na an s. Co esponding emale
Vi uses 2023,15, 1113 4 o 13
mosqui o bodies we e collec ed and homogenised in GMEM (The mo Fishe Scien i ic,
USA) wi hou supplemen s, and RNA was isola ed wi h T izol LS (In i ogen, Ca lsbad
CA, USA). ASALV quan i ica ion was pe o med as desc ibed below.
2.3.5. ASALV Ve ical T ansmission
Be ween 7 and 10 days old emale mosqui oes we e ei he injec ed o ed wi h ASALV
as desc ibed abo e. A e a week, emales we e gi en a blood meal (50% blood, 10% Fe al
Cal Se um, 1% ATP, comple ed wi h F uc ose). Blood- ed emales we e ans e ed o
a cage and le o lay eggs. The eggs we e looded a e a d ying pe iod. L4 la ae and
adul s ( emales and males) we e collec ed, homogenised, and isola ed, while RNA was
es ed o ASALV p esence by i us-speci ic RT-PCR. The expe imen was epea ed wice
independen ly.
2.4. ASALV Quan i ica ion
Whole mosqui o samples o dissec ed issue pools we e homogenised in 500
µ
L o
250
µ
L o GMEM (The mo Fishe Scien i ic, Wal ham, MA, USA) wi hou supplemen s,
espec i ely. The homogena e was cla i ied by cen i uging, and 200
µ
L o he supe -
na an was used o ex ac RNA using TRIzol LS (In i ogen, Ca lsbad CA, USA) (3:1
/TRIzol: homogena e) acco ding o he manu ac u e ’s p o ocol. The RNA concen a ion
was measu ed using a nanod op. RNA was ei he used di ec ly (One-s ep quan i a i e
(q)PCR), o cDNA was syn hesised ( o be used in qPCR) using 1
µ
g o RNA, andom
hexame s, and M-MLV e e se ansc ip ase (P omega, Fi chbu g, WI, USA) acco ding o
he manu ac u e ’s p o ocol.
To quan i y he ASALV load in di e en issues, we pe o med wo-s ep qPCR using
cDNA samples and a Quan iTec SYBR G een PCR ki (QIAGEN, Hilden, Ge many), acco ding
o he manu ac u e ’s p o ocol. The ASALV load was quan i ied ela i e o ibosomal S7 gene
(housekeepe gene, Ae. aegyp i) o GAPDH (housekeepe gene, Cx. quinque ascia us, Figu e S1)
using p e iously published speci ic p ime s (ASALV, F: 5
0
-CCGTACTCGAAACAGACATTGC-
3
0
, R: 5
0
-TCGTCAACGCCTAGATCCTCTA-3
0
; S7, F: 5
0
-CCAGGCTATCCTGGAGTTG-3
0
, R:
5
0
-GACGTGCTTGCCGGAGAAC-3
0
; GAPDH, F:5
0
-TCAAGCAGAAGGTCAAGGAAG-3
0
,
R:5
0
-GTTGTCGTACCAGGAGATGAG-3
0
) [
15
,
34
,
35
]. A one-s ep qRT-PCR (Quan iTec SYBR
G een RT-PCR Ki , QIAGEN, Hilden, Ge many) was pe o med, acco ding o he manu ac-
u e ’s p o ocol, o de ec ASALV in ec ion (in ec ion a e, sali a ion assay, and ho izon al
ansmission assays). Cp > 33.5 was conside ed nega i e based on he s anda d cu e.
All PCR eac ions we e pe o med in h ee echnical eplica es. The da a we e analysed
using a Ligh Cycle 480 II Ins umen (Roche) and he Ligh Cycle 480 so wa e ( e sion
1.5.0 SP4), ei he using absolu e quan i ica ion o ad anced ela i e quan i ica ion. S anda d
cu es o each gene we e c ea ed using dilu ions o he speci ic PCR p oduc s, and genome
copy numbe s we e calcula ed and used o bo h absolu e and ela i e quan i ica ion assays.
2.5. Polyme ase Chain Reac ion (PCR)
Fo ASALV de ec ion, he abo e-men ioned ASALV-speci ic o wa d and e e se
p ime s we e used in combina ion wi h cDNA as a empla e. The ampli ica ion was
pe o med using Go Taq polyme ase (P omega, Fi chbu g, WI, USA) wi h deoxynucleoside
iphospha es (dNTPs) and a 5x G een Go Taq bu e , wi h a inal eac ion olume o 50
µ
L.
The mocycling condi ions o he i s ound o ampli ica ion we e 95
◦
C o 2 min, ollowed
by 32 cycles o dena u a ion (95
◦
C o 30 s), annealing (56
◦
C o 30 s), ex ension (72
◦
C o
45 s), and a inal ex ension a 72
◦
C o 7 min. The ampli ica ion p oduc was isualised in a
2% aga ose-1
×
TAE (T is-ace a e-e hylenediamine e aace ic acid) gel by e hidium b omide
s aining and UV ansillumina ion. The expec ed size o ampli ica ion (PCR p oduc ) was
97 bp.
Vi uses 2023,15, 1113 5 o 13
2.6. Small RNA Sequencing
The midgu s om 15 mosqui oes (ASALV ed, 7 dpi) we e pooled and homogenised in
GMEM (The mo Fishe Scien i ic, Wal ham, MA, USA). The o al RNA was isola ed using
TRIzol LS (In i ogen, Ca lsbad CA, USA) acco ding o he manu ac u e ’s p o ocol, wi h
glycogen as a ca ie . Fo small RNA sequencing, lib a ies we e p epa ed wi h 100 ng o he
o al RNA and he Nex lex small RNA-Seq ki 3 (Pe kinElme Inc., Wal ham, MA, USA),
and lib a ies wi h a size co esponding o (18–35 nucleo ides a e lib a y p epa a ion)
we e sequenced on a No aSeq6000 SP 1.0 pla o m (2
×
50 bp) [
29
]. Sequencing was
pe o med a IKMB (Kiel, Ge many). The da a we e analysed, as desc ibed p e iously [
23
].
As a empla e, he ASALV genome was used (GenBank accession numbe MK959115).
2.7. Da a Analyses
Da a analyses we e conduc ed in R e sion 4.1.2 (1 No embe 2021). To analyse he
ASALV eplica ion in whole mosqui oes a 0, 7 and 14 dpi, we used a non-pa ame ic
K uskal–Wallis es . When he e was a signi ican e ec du ing he day, pai wise compa -
isons we e made using a dunnTes (FSA package) wi h Bon e oni co ec ion. To analyse
he e ec s o issue ype,dpi (7, 14), and ea men (Injec ed, Fed) on he ASALV load in
di e en issues, we i s i ed a gene alised linea model om he Gaussian amily using
log- ans o med da a. Likelihood a io es s o he ull model agains he model wi hou
a gi en e ec we e used o ob ain he deg ees o eedom, de iance and p- alues. The
s a is ical di e ence be ween he sali a y gland, ca casses and b ain issues in e ms o
he ASALV load was es ed using a pai wise- - es wi h Bon e oni co ec ion o mul iple
es ing. A p- alue < 0.05 was conside ed s a is ically signi ican .
3. Resul s
3.1. ASALV In ec s and Replica es in Ae. aegyp i
To de e mine whe he ASALV could in ec and eplica e in Ae. aegyp i mosqui oes,
mosqui oes we e ei he injec ed o ed wi h ASALV and we e collec ed a di e en imes
pos -in ec ion (0, 7, and 14 dpi). ASALV was quan i ied by qRT-PCR. The samples om
0 dpi we e ASALV-posi i e, con i ming ha bo h he eeding and injec ion expe imen s
wo ked well. The injec ed mosqui oes showed a 100% in ec ion a e on 7 and 14 dpi, while
mosqui oes o ally exposed o ASALV showed a lowe in ec ion a e on 14 dpi (Figu e 1A).
A gene al inc ease in he ASALV RNA copy numbe was obse ed a 7 (mean (
±
se),
10
5.17(±0.33)
) and 14 dpi (mean (
±
se), 10
4.89 (±0.37)
) compa ed o ha a 0 dpi (mean (
±
se),
10
3.10(±0.17)
), sugges ing he ac i e in ec ion and eplica ion o ASALV in Ae. aegyp i
mosqui oes o bo h g oups (Figu e 1B). This inc ease was pa icula ly high in he injec ed
g oup wi h a 3 log inc ease om 0 dpi 10
3.26(±0.261)
o 7 dpi 10
6.65(±0.17)
(K uskal–Wallis
χ2
= 44, d = 2, p< 0.001; Funn es : 0–7 dpi p< 0.001; 0–14 dpi p< 0.001). In he ed
g oup, he ASALV load did no di e signi ican ly be ween 0, 7 and 14 dpi (K uskal–Wallis
χ2
= 4, d = 2, p> 0.05), and only some samples collec ed a 7 dpi and 14 dpi exhibi ed
a highe ASALV load compa ed o 0 dpi (Figu e 1B). This sugges ed a s ong bo leneck
passing he midgu ba ie when he mosqui oes we e ed wi h ASALV. Ne e heless, many
mosqui oes emained posi i e a 14 dpi, indica ing ha ASALV could in ec and eplica e
in Ae. aegyp i by eeding.

Vi uses 2023,15, 1113 6 o 13
Vi uses 2023, 15, x FOR PEER REVIEW 6 o 14
compa ed o 0 dpi (Figu e 1B). This sugges ed a s ong bo leneck passing he midgu
ba ie when he mosqui oes we e ed wi h ASALV. Ne e heless, many mosqui oes e-
mained posi i e a 14 dpi, indica ing ha ASALV could in ec and eplica e in Ae. aegyp i
by eeding.
Figu e 1. ASALV in ec ion a e (A) and load (B) in emale Ae aegyp i mosqui oes ollowing in a ho-
acic injec ion and o al in ec ion. Adul emale mosqui oes (7–13 days old) we e ed o injec ed wi h
ASALV and RNA was isola ed om he collec ed mosqui oes a 0, 7 and 14 dpi. ASALV was quan-
i ied using he qRT-PCR in ec ion a e (A), o genome copy numbe s (B) we e calcula ed using
co esponding s anda d cu es. Each da a poin ep esen s indi idual mosqui oes (whole bodies).
E o ba s indica e 95% con idence in e als o he in ec ion a e, n = sample size.
3.2. ASALV In e ac s wi h he RNAi Response in he Midgu
The o al RNA o he dissec ed midgu pool o 15 ASALV-injec ed mosqui oes was
sequenced and analysed o in es iga e ASALV-speci ic small RNA p oduc ion. ASALV-
speci ic 21 n -sized siRNAs we e p oduced in he midgu (Figu e 2A). These siRNAs
we e p oduced along he ASALV genome and an igenome o a simila amoun (Figu e
2B). We obse ed ASALV-speci ic piRNA-sized small RNAs (mos ly mapping o he ge-
nome) in he midgu . Howe e , hese did no show ping-pong ampli ica ion signals (da a
no shown).
Figu e 1.
ASALV in ec ion a e (
A
) and load (
B
) in emale Ae aegyp i mosqui oes ollowing in a ho-
acic injec ion and o al in ec ion. Adul emale mosqui oes (7–13 days old) we e ed o injec ed
wi h ASALV and RNA was isola ed om he collec ed mosqui oes a 0, 7 and 14 dpi. ASALV was
quan i ied using he qRT-PCR in ec ion a e (
A
), o genome copy numbe s (
B
) we e calcula ed using
co esponding s anda d cu es. Each da a poin ep esen s indi idual mosqui oes (whole bodies).
E o ba s indica e 95% con idence in e als o he in ec ion a e, n = sample size.
3.2. ASALV In e ac s wi h he RNAi Response in he Midgu
The o al RNA o he dissec ed midgu pool o 15 ASALV-injec ed mosqui oes was
sequenced and analysed o in es iga e ASALV-speci ic small RNA p oduc ion. ASALV-
speci ic 21 n -sized siRNAs we e p oduced in he midgu (Figu e 2A). These siRNAs we e
p oduced along he ASALV genome and an igenome o a simila amoun (Figu e 2B). We
obse ed ASALV-speci ic piRNA-sized small RNAs (mos ly mapping o he genome) in he
midgu . Howe e , hese did no show ping-pong ampli ica ion signals (da a no shown).
Vi uses 2023, 15, x FOR PEER REVIEW 7 o 14
Figu e 2. ASALV-speci ic small RNA p oduc ion in he Ae. aegyp i emale midgu . Adul emale Ae.
aegyp i mosqui oes we e ed wi h ASALV, and he midgu o 15 emale mosqui oes was pooled a
14 dpi. Size dis ibu ion o small RNAs mapping o he ASALV genome (whi e) o an igenome
(g ey) (A). Dis ibu ion o he 21 n small RNAs along he ASALV genome (whi e) and an igenome
(g ey) (B).
3.3. ASALV Dissemina es o Di e en Tissues and Replica es Mos ly in Heads
The ASALV load was quan i ied using qPCR in pooled issue samples om in a ho-
acically injec ed and o ally in ec ed mosqui oes. In bo h g oups, ASALV was dissemi-
na ed success ully o all dissec ed issues (Figu e 3A). Al hough we pe o med he dissec-
ions a 7 and 14 dpi sepa a ely, he e was no di e ence be ween 7 and 14 dpi samples in
e ms o he ASALV load, so hese da a we e pooled oge he in Figu e 3A (glm, d = 1,
de = 3.27, p = 0.15). A majo di e ence was caused by he in ec ion ou e; injec ed mos-
qui oes gene ally had highe ASALV genomes compa ed o ed mosqui oes (glm, d = 1,
de = 109, p < 0.001; Figu e 3A). The e was no signi ican e ec in he in e ac ion o he
in ec ion ou e and issue ype (glm, d = 3, de = 2.64, p > 0.05), showing ha he injec ion
ou e did no change he issue opism o ASALV. Vi al load di e ed be ween dissec ed
issues (glm, d = 3, de = 18.6, p < 0.001), wi h he highes ASALV load obse ed in head
pools, including he sali a y glands (Figu e 3A). To de e mine whe he he issue opism
o ASALV was speci ic o Ae. aegyp i, o i i exhibi ed he same issue opism in ano he
mosqui o species, we checked he ASALV issue opism in Cx. quinque ascia us using he
same in ec ion p o ocols as Ae. aegyp i. No speci ic opism o he head was obse ed in
Cx. quinque ascia us emale mosqui oes (Figu e S1).
Figu e 2.
ASALV-speci ic small RNA p oduc ion in he Ae. aegyp i emale midgu . Adul emale Ae.
aegyp i mosqui oes we e ed wi h ASALV, and he midgu o 15 emale mosqui oes was pooled a 14
dpi. Size dis ibu ion o small RNAs mapping o he ASALV genome (whi e) o an igenome (g ey) (
A
).
Dis ibu ion o he 21 n small RNAs along he ASALV genome (whi e) and an igenome (g ey) (B).
Vi uses 2023,15, 1113 7 o 13
3.3. ASALV Dissemina es o Di e en Tissues and Replica es Mos ly in Heads
The ASALV load was quan i ied using qPCR in pooled issue samples om in a ho-
acically injec ed and o ally in ec ed mosqui oes. In bo h g oups, ASALV was dissemina ed
success ully o all dissec ed issues (Figu e 3A). Al hough we pe o med he dissec ions a
7 and 14 dpi sepa a ely, he e was no di e ence be ween 7 and 14 dpi samples in e ms o
he ASALV load, so hese da a we e pooled oge he in Figu e 3A (glm, d = 1, de = 3.27,
p= 0.15). A majo di e ence was caused by he in ec ion ou e; injec ed mosqui oes gen-
e ally had highe ASALV genomes compa ed o ed mosqui oes (glm, d = 1, de = 109,
p< 0.001; Figu e 3A). The e was no signi ican e ec in he in e ac ion o he in ec ion
ou e and issue ype (glm, d = 3, de = 2.64, p> 0.05), showing ha he injec ion ou e
did no change he issue opism o ASALV. Vi al load di e ed be ween dissec ed issues
(glm, d = 3, de = 18.6, p< 0.001), wi h he highes ASALV load obse ed in head pools,
including he sali a y glands (Figu e 3A). To de e mine whe he he issue opism o
ASALV was speci ic o Ae. aegyp i, o i i exhibi ed he same issue opism in ano he
mosqui o species, we checked he ASALV issue opism in Cx. quinque ascia us using he
same in ec ion p o ocols as Ae. aegyp i. No speci ic opism o he head was obse ed in
Cx. quinque ascia us emale mosqui oes (Figu e S1).
Vi uses 2023, 15, x FOR PEER REVIEW 8 o 14
Figu e 3. ASALV dissemina ion o di e en issues in ASALV- ed o injec ed adul Ae. aegyp i e-
males. The RNA o di e en issue pools (15 mosqui oes/pool), dissec ed a 7 and 14 dpi, was iso-
la ed and he ASALV load was de e mined by qPCR (A). RNA o b ain issue, sali a y glands (sg)
and ca cass pools (5 mosqui oes/pool) we e isola ed om ASALV-injec ed mosqui oes a 7 dpi (B).
Six pools pe issue we e analysed o ASALV load in he ca cass, sali a y gland and b ain in
ASALV-injec ed Ae. aegyp i emales. ASALV load was de e mined by qPCR and ela i e ASALV
quan i ica ion using he ibosomal S7 gene as a housekeeping gene. ***: p < 0.001. Each da a poin
ep esen s dissec ed issue pools consis ing o 15 (A) o 5 (B) mosqui oes.
To in es iga e which issue was esponsible o he high ASALV load in he heads,
he expe imen was epea ed. This ime, in addi ion o he ca cass, he heads o ASALV-
injec ed mosqui oes (a 7 dpi) we e sepa a ed o c ea e pools o he sali a y glands and
b ains (Figu e 3B). ASALV load was highes in he b ain pool compa ed o he sali a y
glands (pai wise- es wi h Bon e oni co ec ion, p < 0.001) and ca casses (pai wise- es
wi h Bon e oni co ec ion, p < 0.001). ASALV load in he ca cass was no signi ican ly
di e en om sali a y glands.
3.4. ASALV T ansmission Rou es in Ae. aegyp i
To unde s and how ASALV migh be ansmi ed in na u e, we checked ho izon al
ansmission du ing adul and la al s ages. Since ASALV was de ec ed in he sali a y
glands, i s ansmission h ough sali a was checked. Fo his, ASALV-injec ed Ae. aegyp i
was used o pe o m a o ced sali a ion assay a 14 dpi. We did no obse e any in ec ion
signs in he sali a-incuba ed mosqui o cells, no was ASALV RNA de ec ed in he col-
lec ed cell supe na an s. Howe e , all sali a ed emales we e posi i e.
Due o he p esence o ASALV in he o a ies, we in es iga ed he e ical ansmis-
sion o ASALV in F1 o sp ing. The e o e, ASALV’s p esence in F1 La ae (10 L4 es ed
pe g oup) o adul pools (10 pools o 5 males o emales es ed pe g oup) de i ed om
ASALV-injec ed o - ed emales was de e mined by RT-PCR. Expe imen s we e epea ed
wice o each g oup, bu no ASALV was de ec ed in he F1 la ae o adul s.
To in es iga e he ho izon al ansmission be ween adul mosqui oes, we placed 5–7
emales in a cage wi h 15 unin ec ed males in wo independen eplica es (12 emales and
30 males in o al). A week la e , su i ing emales (n = 12) and males (n = 18) we e
Figu e 3.
ASALV dissemina ion o di e en issues in ASALV- ed o injec ed adul Ae. aegyp i emales.
The RNA o di e en issue pools (15 mosqui oes/pool), dissec ed a 7 and 14 dpi, was isola ed and
he ASALV load was de e mined by qPCR (
A
). RNA o b ain issue, sali a y glands (sg) and ca cass
pools (5 mosqui oes/pool) we e isola ed om ASALV-injec ed mosqui oes a 7 dpi (
B
). Six pools pe
issue we e analysed o ASALV load in he ca cass, sali a y gland and b ain in ASALV-injec ed Ae.
aegyp i emales. ASALV load was de e mined by qPCR and ela i e ASALV quan i ica ion using he
ibosomal S7 gene as a housekeeping gene. ***: p< 0.001. Each da a poin ep esen s dissec ed issue
pools consis ing o 15 (A)o 5(B) mosqui oes.
To in es iga e which issue was esponsible o he high ASALV load in he heads, he
expe imen was epea ed. This ime, in addi ion o he ca cass, he heads o ASALV-injec ed
mosqui oes (a 7 dpi) we e sepa a ed o c ea e pools o he sali a y glands and b ains
(Figu e 3B). ASALV load was highes in he b ain pool compa ed o he sali a y glands
(pai wise- es wi h Bon e oni co ec ion, p< 0.001) and ca casses (pai wise- es wi h
Vi uses 2023,15, 1113 8 o 13
Bon e oni co ec ion, p< 0.001). ASALV load in he ca cass was no signi ican ly di e en
om sali a y glands.
3.4. ASALV T ansmission Rou es in Ae. aegyp i
To unde s and how ASALV migh be ansmi ed in na u e, we checked ho izon al
ansmission du ing adul and la al s ages. Since ASALV was de ec ed in he sali a y
glands, i s ansmission h ough sali a was checked. Fo his, ASALV-injec ed Ae. aegyp i
was used o pe o m a o ced sali a ion assay a 14 dpi. We did no obse e any in ec ion
signs in he sali a-incuba ed mosqui o cells, no was ASALV RNA de ec ed in he collec ed
cell supe na an s. Howe e , all sali a ed emales we e posi i e.
Due o he p esence o ASALV in he o a ies, we in es iga ed he e ical ansmission
o ASALV in F1 o sp ing. The e o e, ASALV’s p esence in F1 La ae (10 L4 es ed pe
g oup) o adul pools (10 pools o 5 males o emales es ed pe g oup) de i ed om
ASALV-injec ed o - ed emales was de e mined by RT-PCR. Expe imen s we e epea ed
wice o each g oup, bu no ASALV was de ec ed in he F1 la ae o adul s.
To in es iga e he ho izon al ansmission be ween adul mosqui oes, we placed 5–7
emales in a cage wi h 15 unin ec ed males in wo independen eplica es (12 emales
and 30 males in o al). A week la e , su i ing emales (n = 12) and males (n = 18) we e
collec ed, and he ASALV load was quan i ied using qRT-PCR. All es ed emales we e
highly in ec ed wi h ASALV (Figu e 4). Ou o he 18 males es ed, 9 we e in ec ed,
indica ing a 50% ansmission a e (95% CI: 26-73%). Howe e , ASALV load was lowe
in he males (mean(
±
se):10
3(±0.2)
) compa ed o he emales (mean(
±
se):10
6.62(±0.42)
) ha
we e injec ed.
Vi uses 2023, 15, x FOR PEER REVIEW 9 o 14
collec ed, and he ASALV load was quan i ied using qRT-PCR. All es ed emales we e
highly in ec ed wi h ASALV (Figu e 4). Ou o he 18 males es ed, 9 we e in ec ed, indi-
ca ing a 50% ansmission a e (95% CI: 26-73%). Howe e , ASALV load was lowe in he
males (mean(±se):10
3(±0.2)
) compa ed o he emales (mean(±se):10
6.62(±0.42)
) ha we e in-
jec ed.
Figu e 4. ASALV can be ho izon ally ansmi ed om emale o male Ae. aegyp i mosqui oes.
ASALV-injec ed emales we e kep wi h unin ec ed males o 7 days. ASALV genome copy numbe s
we e quan i ied in emale and male mosqui oes using qRT-PCR. The esul s ep esen he combined
da a o wo independen epea s. Each da a poin ep esen s an indi idual mosqui o (whole body
sample).
To in es iga e he ho izon al ansmission du ing la al s ages, mosqui o la ae we e
ea ed in wa e ha bou ing ASALV. Eme ging adul mosqui oes we e collec ed and es ed
o he p esence o ASALV by PCR. A o al o 10% (95% CI: 2–20%) o he es ed adul
mosqui oes (n = 29, in h ee independen expe imen s) we e ASALV-posi i e, sugges ing
he possibili y o ASALV ho izon al ansmission du ing la al s ages.
4. Discussion
ISVs can only eplica e in insec s and insec -de i ed cell lines and no in e eb a es.
A la ge numbe o ISVs ha e been disco e ed as a esul o ecen ad ances in sequencing
echnology and inc eased ield mosqui o su eillance. Howe e , ou knowledge o ISV
hos ange, hei e ec s on mosqui o i ness, and how hey a e main ained and ansmi ed
in na u e is limi ed. To add ess some o hese open ques ions, we s udied he eplica ion
kine ics, dissemina ion, and ansmission o ASALV in ec o mosqui o species, Ae. ae-
gyp i.
Despi e he inc ease in he de ec ion o many di e se ISVs by sequencing s udies,
ISVs’ hos ange was no always clea ly de ined. In he case whe e he disco e ed ISV was
isola ed, i s eplica ion in mosqui o cells and e eb a e cells was checked. Howe e , he
eplica ion kine ics o ISVs a e a ely checked in o he insec s, wi h only se e al excep-
ions [21,36]. ASALV canno eplica e in cell lines de i ed om ec o he mic hos s o he
Culex mosqui o ( ogs, snakes and ish-de i ed cell lines), sugges ing ha ASALV is an
ISV [19]. P e ious s udies ha e demons a ed ASALV eplica ion in cell lines de i ed
om Aedes species, e en hough i was isola ed om Culex decla a o mosqui oes [19,23].
In his s udy, we showed ha Ae. aegyp i emales a e suscep ible o ASALV when in a ho-
acically and o ally in ec ed. The in ec ion a es in in a ho acically injec ed mosqui oes
we e highe han in o ally in ec ed mosqui oes, and he ASALV load in whole mosqui oes
Figu e 4.
ASALV can be ho izon ally ansmi ed om emale o male Ae. aegyp i mosqui oes. ASALV-
injec ed emales we e kep wi h unin ec ed males o 7 days. ASALV genome copy numbe s we e
quan i ied in emale and male mosqui oes using qRT-PCR. The esul s ep esen he combined da a o
wo independen epea s. Each da a poin ep esen s an indi idual mosqui o (whole body sample).
To in es iga e he ho izon al ansmission du ing la al s ages, mosqui o la ae we e
ea ed in wa e ha bou ing ASALV. Eme ging adul mosqui oes we e collec ed and es ed
o he p esence o ASALV by PCR. A o al o 10% (95% CI: 2–20%) o he es ed adul
mosqui oes (n = 29, in h ee independen expe imen s) we e ASALV-posi i e, sugges ing
he possibili y o ASALV ho izon al ansmission du ing la al s ages.
Vi uses 2023,15, 1113 9 o 13
4. Discussion
ISVs can only eplica e in insec s and insec -de i ed cell lines and no in e eb a es.
A la ge numbe o ISVs ha e been disco e ed as a esul o ecen ad ances in sequencing
echnology and inc eased ield mosqui o su eillance. Howe e , ou knowledge o ISV
hos ange, hei e ec s on mosqui o i ness, and how hey a e main ained and ansmi ed
in na u e is limi ed. To add ess some o hese open ques ions, we s udied he eplica ion
kine ics, dissemina ion, and ansmission o ASALV in ec o mosqui o species, Ae. aegyp i.
Despi e he inc ease in he de ec ion o many di e se ISVs by sequencing s udies,
ISVs’ hos ange was no always clea ly de ined. In he case whe e he disco e ed ISV was
isola ed, i s eplica ion in mosqui o cells and e eb a e cells was checked. Howe e , he
eplica ion kine ics o ISVs a e a ely checked in o he insec s, wi h only se e al excep-
ions [
21
,
36
]. ASALV canno eplica e in cell lines de i ed om ec o he mic hos s o he
Culex mosqui o ( ogs, snakes and ish-de i ed cell lines), sugges ing ha ASALV is an
ISV [
19
]. P e ious s udies ha e demons a ed ASALV eplica ion in cell lines de i ed om
Aedes species, e en hough i was isola ed om Culex decla a o mosqui oes [
19
,
23
]. In his
s udy, we showed ha Ae. aegyp i emales a e suscep ible o ASALV when in a ho acically
and o ally in ec ed. The in ec ion a es in in a ho acically injec ed mosqui oes we e highe
han in o ally in ec ed mosqui oes, and he ASALV load in whole mosqui oes inc eased
signi ican ly in he ASALV-injec ed mosqui oes. On he o he hand, ASALV load did no
signi ican ly inc ease in he whole mosqui oes ollowing o al in ec ion wi h ASALV, bu
mosqui oes s ayed posi i e a 14 dpi, suppo ing i s ac i e eplica ion. In addi ion, he
p esence o ASALV-speci ic 21-n siRNAs in Ae. aegyp i midgu showed ha ASALV
in ec ed and eplica ed in he midgu ollowing o al in ec ion, which igge ed he hos
RNAi esponse. In addi ion o siRNAs, piRNA-sized ASALV-speci ic small RNAs we e
also de ec ed; hese lacked ping-pong ampli ica ion signals, simila o p e ious indings
in Ae. albopic us-de i ed (U4.4) cells [
19
] and Ae. aegyp i-de i ed cells (RNAi-compe en
AF5 cells) [
23
]. O e all, ou esul s show ha ASALV in ec s and eplica es in Ae. aegyp i,
simila o he esul s o p e ious s udies showing success ul EILV in ec ion in Ae. aegyp i
and Cx. a salis. [
20
,
21
]. To da e, all insec -speci ic alpha i uses ha e been disco e ed
in Culex o Anopheles species, al hough he success ul eplica ion o EILV and ASALV
in Ae. aegyp i mosqui oes [
15
–
21
] sugges ha Ae. aegyp i could be a po en ial hos o
insec -speci ic alpha i uses.
When a i us is o ally acqui ed, he midgu is he i s o gan in he mosqui o o become
in ec ed. Following midgu in ec ion, he i us sp eads o seconda y issues, such as he
sali a y glands [
37
,
38
]. P e ious esea ch has shown ha when in ec ed o ally, Ae. aegyp i,
Anopheles gambiae, and Cx. quinque ascia us mosqui oes expe ienced low dissemina ion o
EILV o hei legs and wings. When in ec ed o ally, a high in ec ious i e was necessa y
o dissemina ion in Ae. aegyp i (10
7
PFU/mL), Anopheles gambiae (10
7
PFU/mL), and
Cx. quinque ascia us (10
9
PFU/mL) compa ed o he injec ed ones [
20
]. This sugges s
a s ong midgu ba ie agains insec -speci ic alpha i uses. In his s udy, ASALV was
dissemina ed success ully in Ae. aegyp i wi h a i al i e o 10
7
PFU/mL, simila o EILV.
Howe e , since we did no de e mine he dissemina ion a e (i.e., by es ing indi idual
mosqui oes) in his s udy, ou esul s o ASALV canno be di ec ly compa ed o he
p e iously epo ed dissemina ion a es o EILV wi h he same i e . We also obse ed
a signi ican impac in e ms o he ou e o in ec ion on he ASALV load in dissec ed
issues: ASALV-injec ed mosqui oes had highe i al loads han o ally in ec ed mosqui oes.
Ne e heless, ASALV success ully dissemina ed in di e en issues in bo h ASALV-injec ed
and o ally in ec ed mosqui oes, and he in ec ion ou e did no change he issue opism o
ASALV in Ae. aegyp i.
Ano he open ques ion on ISVs is how hey a ec mosqui os’ i ness. Some ISVs wi h
DNA genomes (e.g., Aedes albopic us denso i uses) exhibi ed isible pa hology and mass
mo ali y du ing he la al s age [
39
], whe eas insec -speci ic RNA i uses did no seem
o cause any easily de ec able pa hology o high mo ali y. Howe e , some RNA i uses
ha e been shown o a ec mosqui o beha iou . Fo ins ance, CxFV in ec ion al e s he