Scale drop disease virus (SDDV) triggering ferroptosis both in vivo and in vitro facilitates virus infection via targeting transferrin receptor 1 (TfR1)

27 S c a l e d r o p d i s e a s e v i r u s ( S D D V ) i s a d i s t i n c t m e m b e r i n g e n u s Megalocytivirus of fam ily Iridoviridae , 28 gar nering in creasing attention d ue to its significant threat to teleost. Ferroptosis is a new type of cell d eath 29 discovered recently and inv olved in v arious viral infections. K nowledges on SDDV induced ferroptosis 30 rem ains u nclear . Here, w e d em onstrated that SDDV infection tri ggers ferr op tosis, as evidenced by 31 ha l lmark fea ture s such as i ron overload, massi ve l ipi d perox i des accu mulatio n, glutathione deple ti on and 32 glutathion e p eroxid ase 4 dow nregulation . SD DV-infected MFF-1 cell s exhibited in creased reacti v e oxygen 33 species produ ction and m itochondrial shrinkage. T reatm ent with Ferrostatin-1, a potent ferroptosis 34 inhibitor , significantly atten u ated SD DV rep l ication in MFF-1 cells and could im p rove th e surviv al of 35 m and arin fish u pon SD DV challenge. T reatm ent with an iron chelator m itigated ferroptosis and red uced 36 the m ortal ity of m and arin fish followin g SDDV in fection, suggesting th at SDD V-indu ced ferroptosi s is 37 iron-dependent. Fin ally , w e d em onstr ated th at SD DV infection could u pregulate the expr ession of 38 tra nsferrin receptor protein 1 (TfR 1 ), a critical iron tra nsp orter , to disrupt cellular iron h o m eostasis, ind u ce 39 ferroptosis, and then facilitate vir al in fection. Collectively , ou r findings pr ovi d e c om p elling evidence that 40 SD DV in fection ind u ces ferroptosis by targeting T fR 1 to facilitate virus in fection. Inhibiting ferroptosis 41 represents a pr om ising im m u n oth erapeu tic strate gy for com bating SDDV in fection in aqu acultur e. 42

Scale dro p disease virus (S DDV); Ferro ptosis; T ransferrin receptor protein 1; Infectivity . 43 44 45 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint 1. Introduction 46 Sca le dr op d is eas e virus (SD D V ) is a sense double-s tranded lar ge D N A v ir us belong ing t o th e 47 Megalo c y t i vir us la tes1 s p e c i e s o f g e n u s M ega locytivirus w i t h i n s u b f a m i l y Al p h a iridov ir id ae . S in ce it w as 48 firs t sc ie nt if i ca l ly id ent ifie d i n 20 15 [1 ] , S D D V h a s b e e n w i d e l y r e p o r t e d i n A s i a n s e a b a s s Lates 49 c a lc a r if er i ndustr y acros s sev eral Sout hea s t Asi an c ountr ies, in clu d in g Sin gap ore, M a la ysi a , a n d 50 Tha i l a n d , a nd h as emer ged as one of th e m o s t co n cern in g v ir a l p atho gens to fa rmed As ia n s e ab ass i n 51 these regi ons [2-4]. S DD V c a n i n f e ct A s i an s e ab a s s o f v a r i o u s s i z es , es p e c i a l l y t h r e a t e n i n g l ar g e- s i z e d 52 o n e s, w i t h ac c um ul a ti v e m o r tali ti e s fr o m 40 % t o 50 % [1 , 4]. The do cu ment a t i o n o f SDD V-a s soc i ate d 53 dise ases, known a s s c a le drop s yndro me ( SD S), c oul d d ate ba ck t o a s ear l y as in the e a r ly- 199 0s in 54 A s i an seab ass farms in P enan g, Ma l a ys ia [4 , 5 ] . D ifferent fr om S D DV in SE c ountr ies jus t infect in g 55 A s i an seabas s, SDD V w as is ol ate d f r om y el lowf in s e abr eam Ac an t h o p a g r u s l a t u s s u f f e r i n g f r o m 56 asc ite s d is eas e s in m a in l and C hin a by our team in 20 20 [ 6 ] . F u rt h e rm o r e , o ur r ec e n t st u d y s h o we d 57 that e v en a lo w d ose of SDD V cou ld c aus e 10 0% mort a l ity in m and ar in f is h Si nip e r ca chuatsi , a h i gh ly 58 su s c ept ible host fish species to bro ad Me ga locytivirus i n f e c t i o n s [7, 8 ] , s u gg e s t i ng tha t ma nd a r i n f i sh 59 m a y s e r v e a s a p r o m i s i n g i n f e c t i o n a n d v a c c i n a t i o n m o d e l f o r t h e s t u d y o f S D D V [9] . Ad di ti o nally , 60 SDD V- clos e vir uses, in c lud in g Eur opean chub iri dov ir us (EC IV) an d a n ov e l ti l ap i a meg a lo cyt iv irus , 61 h a v e a l s o b e en r ep or t e d i n E u r o p e a n c h u b Squa li us cepha lus in UK [1 0 ] a n d t ila pia Or eochr omis s p p . i n 62 the US A [11 ] , r e s p ec ti ve ly . C oll ec ti ve ly , all t h e s e f i ndi n g s hig hlig h t t h e s ig nific a n t t h re a t o f S DDV to 63 g lobal aqu a c u lt ure a nd u nde r score the need for he i ghtene d v ig i l an c e . 64 B e s i de s SD DV , t he g e n u s Megalo c y t i vir use s also in c ludes anoth er species , nam ely M ega locyti virus 65 s p a g ru s1, wh i ch is r e pr e s ente d by the type is ol ate of infect ious s plee n and k idne y necros is virus 66 (ISK N V) [1 2] . N o w a d a y s , Megalo cytivi rus spagrus1 i s f u r t h e r d i v i d e d i n t o t h r e e g e n o t y p e s : I S K N V , 67 red s ea brea m ir id ov irus (R S I V ) and tur bot r e d d ish body ir i d ov irus (TR BIV). These v irus e s hav e 68 c a u s e d l e t h a l s y s t e m i c i n f e c t i o n s i n w i l d a n d f a r m e d f r e s h w a t e r , b r a c k i s h , a n d m a r i n e f i s h s p e c i e s 69 wo rl d wid e [6 , 7, 13 ]. C o m p a r e d t o t h e e x t e n s i v e l y s t u d i e d I S K N V , s t u d i e s o n S D D V r e m a i n a t a 70 r e la ti ve ly p r eli min a ry s ta g e , wi th th e m e c ha ni s m s un d e r l yin g i ts pa t ho g e n e s i s s t ill l a rg ely 71 unex pl o r e d . 72 Ferropt osis is a nov e l non apoptot i c form of pro gra mme d cel l death a nd c aused by l ip i d 73 p e r o x i d a t i o n ( L P O ) d u e t o t h e a c c u m u l a t i o n o f i r o n - d e p e n d e n t r e a c t i v e o x y g e n s p e c i e s ( R O S ) [1 4 ] . It 74 i s c h a r a c t e r i z e d b y t h e d e a c t i v a t i o n o f t h e c e l l u l a r a n t i o x i d a n t s y s t e m , p r i m a r i l y t h r o u g h t h e 75 deple t i o n of g lut ath ione (G SH) and the funct ion a l loss of phos pholip i d peroxid ase g lut ath ione 76 p e r o x i d a s e 4 ( G P x 4 ) [1 5] . N o t a b l y , t h e s e h a l l m a r k s o f f e r r o p t o s i s h a v e b e e n o b s e r v e d d u r i n g v a r i o u s 77 vir a l infe c t ions , suggesti n g a c lose as soci at ion be t w een ferroptos is and v ir a l p atho genesis . For 78 instan ce, ferr o p tosis is l inke d t o sev er e a c ut e resp i rator y syndr o me c o r on avi rus 2 (SAR S - Co V - 2 ) 79 infe c t ion and m a y le a d to mult i-or g a n d am age [1 6] ; New c a s tle d isease v iru s ( ND V ) ha s bee n s hown 80 to in du ce ferr optosis to k i l l tum or cel ls [17 ] ; an d Herpes S imp lex V i r us 1 (HS V-1)-ind u c ed ferr optos is 81 c ontr ibutes to vi ra l en c eph a l it is [18 ] . Add i t i onal l y , a v ar iet y of v ir uses, in c l udin g Inf luen za A V irus 82 (IA V) [ 1 9 ] , Rot av ir us [20] and P or c ine Reproduct i v e a nd Respi r ator y S yndro me V irus (PRR S V) hav e 83 be en report e d to tr ig ge r fe r roptos is to e nhan c e v ira l rep l i cat ion [21] . 84 T rans fe r rin re ce pt or 1 (Tf R1) is a t ype I I tr ansme mbrane g ly c oprote in c ons isted of a 85 d i s ulf id e - b o nd e d h o mo dim e r a nd f u n c ti o n s a s t he p r ima r y c ell ula r ir o n a b s o rp t i o n p r o tei n . T fR1 86 binds t r ansferr in (T F) at the c e l l s urfac e , i mpor t ing iron fr om the e xtr a cel l u l a r e nv ir onme nt into c e l ls 87 v ia r ec e p t o r -m e dia t ed e n d ocy t o si s [ 16 ]. Im ba la nc e in i r o n m e ta b oli s m i s r egar d e d a s a cr u cial fac t o r 88 i n f e r r o p t o t i c c e l l d e a t h . T h u s , T f R 1 i s c o n s i d e r e d t o b e a n i m p o r t a n t r e g u l a t o r o f f e r r o p t o s i s [22 ] . 89 P r e v i o u s s t u d i e s h a v e s h o w n t h a t T f R 1 p l a y s a k e y r o l e i n f er r o p t o s i s i n d u c e d b y v i r a l i n f ec t i o n s . F o r 90 e xamp le , upre gu lat i on of Tf R1 prom ot es fe r roptos is du rin g c ox s a c k iev irus B 3 (C VB 3) infe ct ion [23] . 91 A n d d ys regu l a t ion of T fR1 an d T F c ont ributes to s w i ne inf luenza v irus (SI V)-indu ce d ferr optosis , 92 e nhan c in g v ir a l rep l i c at io n [24 ] . H o w e v e r , r e s e a r c h o n t h e r o l e o f f e r r o p t o s i s i n a q u a t i c v i r a l 93 infe c t ions rem a ins l i m ited, w ith l itt le known about whether aqu at i c v iru ses m e d i ate fer roptosis or 94 how fe r roptos is influen c e s vir a l rep l i ca tion in aqu a c u ltu re spe cies. 95 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint I n t h i s s t u d y , w e d e t e r m i n e d t h a t S D D V i n f e c t i o n i n d u c e s f e r r o p t o s i s a n d T f R 1 i s i d e n t i f i e d a s a 96 ke y r egu l ator of ir on ov er l oa d dur in g SD D V infe ct ion, l ea d ing to ferropt o t i c c el l de ath . The s e 97 find in gs provi d e a nov el persp e ct iv e on S DD V p atho ge nesis a nd offer t he o r e ti c a l s upport for the 98 deve lopment of potent i a l a nt iv ir a l the r ape ut i c s trate g ie s to m it ig ate S D DV inf ec t i o n . 99 2. Materials and methods 100 2.1 . Cell culture a nd v i ral infec t ion 101 Mand ar in fish fr y (MF F-1) cel l l ine w as es ta b l is hed in our l abor atory and u sed for v ir us is ol at io n 102 and infect ion s tud ies [25] . M F F - 1 c e l l s w er e g r o wn i n c o m p l e t e Du l b e c co ' s M o d i f i e d E ag l e ' s M e d i u m 103 c o n tai ni ng 10 % f e tal b o vi n e se ru m (F B S ) , pe nicilli n (1 0 0 IU / ml ) , s tr e p t o myc i n ( 100 μg/ ml ) , a nd 104 amph ote r i cin B ( 0. 25 μ g/ m l; L ife Sc ien c es, US A ) at 2 7 ℃ . 105 S DD V Z H -06 / 20 s t ra in w a s i s o la t e d b y o u r t e a m a nd st o r e d a t −80 ℃ [6 ]. MF F-1 c e l ls w er e 106 in c ub ated w ith Z H-0 6/ 20 at a mu lt ip l i c i ty of infe c t ion (M OI) of 5 . 0 for 1 h in serum -free DME M. Then , 107 the cel ls w e r e w ashed wi th phos phat e-buf fered sal ine ( PBS) and rep len ished w ith fresh DMEM 108 c ont a in in g 5% FBS . Un infected M F F-1 c el ls ser ved as th e m o ck c ontr ol gr oup. Cel ls w ere h a r v e s ted a t 109 6 , 1 2, 2 4 , 3 6 , 4 8 h o u r s p o s t i n f e c t i o n ( h p i ) f o r s u b s e q u e n t e x p e r i m e n t s . A l l e x p e r i m e n t s w e r e 110 c o nd uc t e d in t ri plica t e . 111 2.2 . A r tif i c ial inf e ct ion a nd exp erime nt al gro up s 112 M a n da ri n fi sh ( 50 ±10 g ) w e re o b tai n e d f ro m a q uac ul tu r e fa rme rs i n N a nhai D i st r ic t, F o s ha n , 113 G u a n g d o n g P r o v i n c e , C h i n a . T h e f i s h w e r e t e m p o r a r i l y r e a r e d i n a 5 0 0 L p l a s t i c t a n k . D u r i n g t h e 114 e xperim ent, i nd iv i dua ls w ere r and om l y ass igned to expe r im ental groups . F i sh in the S DDV gr o u p 115 we re i n tr a p e ri to n ea ll y i nj ec te d wi th dilu t e d SD DV Z H -0 6/20 sol u ti o n di s so l ve d i n n o r mal sali n e ( 10 3 116 TCI D 50 / f ish), where a s contr ol group f is h r ece iv e d an equ iv a lent v o lu me of nor m a l s al in e . T iss ue 117 s a m p l e s w e r e c o l l e c t e d a t 2, 4 , 6 , 8 , 1 0 , 1 2 , 1 4 d a y s p o s t - i n f e c t i o n ( d p i ) f r o m e a c h g r o u p f o r 118 morpho lo g i c al, b ioche m i ca l a nd mo le cul ar ana lys e s . Th is s tudy w as approv ed by t he Instit ut ion a l 119 A nima l Ca r e a nd U s e E t hic s Co mmi t t ee o f S u n Y a t - se n U ni ve r s ity un d e r n u mb e r 00 17 09 7003 . 120 2.3 . Q ua ntitat ive r e al t ime PC R (q R T-P CR) 121 Pr ime r s f or qRT -PCR w e r e desi gned u s ing NCBI Pr im er -BLA ST (T able 1 ) . T he β-a c tin g e n e w a s 122 used as the int e r n al referen c e f o r n orm al i z at ion . T ota l R N A w as ex tr a c ted and rev e r se-tr a ns c r ibe d 123 into c omp l ement ary DN A ( c D NA ) us in g Ev o M-M L V RT Pr e m ix f o r qPCR (Ac cur ate B io logy , Hunan , 124 C h i n a ) . T h e q R T - P C R a s s a y s w e r e p e r f o r m e d o n a L i g h t C y c l e r ® 4 8 0 - I I M u l t i w e l l P l a t e 3 8 4 r e a l - t i m e 125 d e t e c t i o n s y s t e m ( R o c h e D i a g n o s t i c s , U S A ) . T h e q R T - P C R r e a c t i o n v o l u m e i n c l u d e d 1 0 μ L o f S Y B R 126 qPCR M aster M i x ( A c c ur ate B i o log y , H un a n, C hin a), 2 μ L of c DNA, 0. 5 μ L o f ea ch p r imer pa i r , an d 7 127 μL d dH 2 O . The a mp l ifi c at ion re act i o n w a s condu cted a t 9 5 ℃ for 1 m i n, 1 cy c le; 4 0 cy cl e s of 128 d e n a tu ra ti o n a t 9 5 ℃ for 10 s a nd a nne a l in g at 60 ℃ for 30 s ; 9 5 ℃ f o r 5 s , 6 0 ℃ f o r 1 m i n a n d 9 5 ℃ , 1 129 cy c le; 50 ℃ for 3 0s, 1 c y cle . A l l ex per i me nts w ere cond u c ted in trip l i c ate. R el at iv e express ion leve ls 130 w e r e an a ly z ed us in g the 2 -ΔΔCt metho d. 131 Ta b l e 1 Pr i mer s us e d i n t hi s s t udy 132 Pri me r Primer sequ enc e (5 ’ -3’) β-a cti n -F AGAGGGAAAT CGTGC G TG β-a cti n -R GAAGGAAGGCT GGAAGAGG SDDV-F AAGAGCGT GAAGCAAT GTC SDDV-R GGGATGAC TAAAT CG CAGA G P X 4- F A C G C A T CC T T G CT TT CC CT T GPX 4 -R TGC T C TT T C AGC CACTTCCA NCOA4-F C CAC TG T C TGTGACCTGT NCOA4-R ACATC TG T T CAGGA GCC T T Tf R1-F TCC C TAAAC AAGCCTGCGAC T C Tf R1-R CGC T ATGTGACC T GCGAAC C IR E B2 -F AGTGCAG T CGTGAGAACG G .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint I R EB 2 - R T G G C TA T A TG G G TG C T CT TT G A FTH1-F CCC GCCACTG AAATT T GAAC C FTH1-R TGTGCAATG C CATA GACAGGT I L -1 β-F AGGGACT GGAC T TG GAGAT TA I L -1 β-R CCG T C CTCCT GA ACTGAAG C I L -8 - F GAAGAGCAGC AGAGTT ATCA T CA I L -8 R ATCT CAG T C TCCT CGC AGT G 2.4 . Imm un ofluoresc ence as sa y ( IF A ) 133 A n I F A w a s p e r f o r m e d t o c o n f i r m S D D V i n f e c t i o n . A m o n o c l o n a l a n t i b o d y ( m A b ) a g a i n s t S D D V 134 ma j or caps id prote in (M CP) (1 :2, 0 0 0) w as used as t he pr im ary ant ibod y . Al e xa F luor 5 5 5 -c onjug ated 135 donkey ant i- mouse Ig G (Inv itr o gen , C ar lsbad, Ca l ifor n i a , US A) w a s used as t he s e cond ar y a nt ibod y . 136 SDD V- infec te d M F F-1 c e l ls serv ed as the posit i ve contro l , wh i l e PB S-tre ate d c e l ls w e r e used as the 137 ne g at iv e contr ol. Nu cleus w ere s ta ined w ith DA PI (Ab c am, Sh angh a i, Ch i na), a nd s ect ions w e r e 138 observ ed under a f luorescen c e m i c r osc ope m i cros c op y . 139 2.5 . Pr us sia n b l ue stainin g s ec t ion o b se rvation 140 Spleen t iss ues fr om m and ar infi sh inf e cte d w it h t he ZH- 0 6 / 20 s tra in w ere co l lecte d an d f ixe d i n 141 al coho l-for m al in - acet i c (AF A) solut ion before embe dd i ng in p a r a f fin . E mbe d d e d t iss ue s w e r e ex cis e d 142 i n t o t h i c k n e s s o f 3 μ m , d e w a x e d i n x y l e n e , r e h y d r a t e d i n g r a d e d e t h a n o l , a n d s t a i n e d w i t h P r u s s i a n 143 b l u e f o r 10–20 mi nu t e s f o ll o win g st a n da r d pr o toc ol s [26] . Observ at ions wer e m ade und e r a 144 mic ro s c o p e ( Ni ko n , T ok yo, Ja p a n ) . 145 2.6 . T ra nsmis sio n e lectron micros co py (TEM ) 146 TEM a n a l ys is of S D D V- infe c ted M FF- 1 cel ls w a s performed at 6 0 hp i , as d e s c r i be d pr e vious ly [23 ] . 147 B r i e f l y , i n f e c t e d c e l l s w e r e f i x e d i n 2. 5 % g l u t a r a l d e h y d e a n d 0 . 1 M P B S c o n t a i n i n g 2 . 0 % o s m i u m 148 t e t r o x i d e . A f t e r d e h y d r a t i n g , p e n e t r a t i n g , e m b e d d i n g a n d p o l y m e r i z a t i o n , t h e s a m p l e s w e r e c u t i n t o 149 ultr ath in sect ions of 6 0 n m. Se c t ions w ere s ta ined w it h uran y l acet ate - lea d citr ate and ex a m ine d 150 u n d e r a P hili p s CM10 el ec tr o n mic ro s co p y ( FE I C om p an y , H ills b o r o , O r eg o n , U SA ) . 151 2.7 . Ir on ass a y 152 The tot a l ir on c on cent rat ion w as deter m ined us in g an iron as say k it (So l arb i o, Be ij in g, Ch in a) in 153 ac cor dan ce wit h the m anuf acturer ’ s ins truct ions . Br i e fl y , appr ox i m ate ly 0. 1 g of t is sue w as 154 homog e n ize d in 1 mL of extr a c t i o n b u ffe r on i ce . In M FF- 1 ce l ls, after r e mov i ng t h e s upernat ant , the 155 c ell s we re dig e st e d wi t h try p s in a n d co lle c t ed . S u b s e q u e n t ly , 1 mL o f e x t rac t i o n b u f f e r w a s a dd e d, 156 a n d t h e c e l l s w e r e s u b j e c t e d t o s o n i c a t i o n o n i c e f o r d i s r u p t i o n . T h e h o m o g e n a t e w a s c e n t r i f u g e d a t 157 4,0 0 0 × g for 1 0 m in at 4 ℃ , an d t he s upernat ant w as co l lecte d. For t he me asur eme nt tube , 60 μL of 158 reagent 1, 120 μL of re age nt 2 , an d 120 μL of the samp l e w e r e adde d. A b l ank t ube w as pr epa r ed w it h 159 120 μL of d is t i l led w ater , an d a stand ar d tube w as prepared w ith 1 20 μ L of gr ad ient- di lute d stand ard 160 solutio n. Subseque nt l y , 6 0 μ L of c h lor ofor m w as a dded to ea c h tube, fo l lo w ed b y i ncub at ion in a 161 b o i l i n g w a t e r b a t h f o r 5 m i n . A f t e r w a r d , t h e s a m p l e s w e r e c e n t r i f u g e d a t 1 0 , 0 0 0 × g f o r 1 0 m i n . T h e 162 opti ca l densit y ( OD ) of ea ch s amp le w as me asur e d at a w av e le ngt h of 5 20 n m. The tot al ir on 163 c on c e n trat io n w a s c a l cu l a t ed b ased on t he prov ided form u l a . 164 2 . 8 F e 2+ c on te n t a s s a y 165 The Fe ²⁺ con c entr at ion in t iss ues or cel ls w as me asur e d us in g a Fe²⁺ con ce ntr at ion as say ki t 166 ac cor d ing to the m anufa c tur e r ’ s ins tru c tions. Approx i m ate ly 0 . 1 g of t is sue w as w e i ghed and 1 m L of 167 r e a g e n t 1 w a s a d d e d . T h e t i s s u e w a s h o m o g e n i z e d a n d t h e n c e n t r i f u g e d a t 1 0 , 0 0 0 × g f o r 1 0 m i n a t 168 4 ° C . F o r M F F - 1 c e l l s , t h e s u p e r n a t a n t w a s r e m o v e d , a n d t h e c e l l s w e r e d i g e s t e d w i t h t r y p s i n , 169 f o l l o w e d b y c o l l e c t i o n o f t h e c e l l s a n d s o n i c a t i o n o n i c e . 1 0 μ L o f a 4 0 m m o l / L s t a n d a r d s o l u t i o n w a s 170 a d d e d t o 9 9 0 μ L o f d i s t i l l e d w a t e r a n d m i x e d t h o r o u g h l y t o p r e p a r e a 4 0 0 μ m o l / L s t a n d a r d s o l u t i o n . 171 The s ta n d a r d s olut ion w as then s eri a l l y d i lut ed w ith r eag ent 1 to obt a in g r ad ie n t con c entr at ions , 172 whi ch w e r e pr e p ared fres h for e a c h ex per im e nt . The b l ank tube r ecei ved 200 μ L o f dd H ₂ O , the 173 standar d tubes w e r e a dde d w it h 200 μ L of the appr o p r i atel y d i lute d stan d ard solut ions, an d the 174 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint s a m p l e t u b e s r e c e i v e d 20 0 μ L o f t h e p r e p a r e d s a m p l e s . T h e n , 1 0 0 μ L o f r e a g e n t 2 w a s a d d e d t o e a c h 175 tube and in cub ated a t 3 7 °C for 10 m in . Afterw ard , 1 00 μ L of ch lorofor m w as a dd e d t o e a c h tube. T he 176 tubes w ere v ort e xed for 5 m in and cent rifuged at 1 2, 0 00 × g f or 10 m in . Th e absor ba n ce at 5 93 n m 177 w a s m e a s u r e d u s i n g a s p e c t r o p h o t o m e t e r . A s t a n d a r d c u r v e w a s p l o t t e d , a n d t h e F e ² ⁺ c o n c e n t r a t i o n 178 w a s c a l cu l a t ed us in g the corr e s pond ing formu l a. 179 2.9 . Glutathione (GS H ) ass a y 180 I n t r a c e l l u l a r G S H l e v e l s w e r e d e t e r m i n e d u s i n g t h e G S H a s s a y k i t ( B e y o t i m e , S h a n g h a i , C h i n a ) 181 fol low in g the m a nuf actur e r ’ s protoc o l. S p le e n tis sues w ere frozen in l i qui d n it r o gen and ground in to 182 i n t o p o w d e r . T h e c e l l s a m p l e w a s d i g e s t e d w i t h t r y p s i n a n d t h e n c e n t r i f u g e d t o r e m o v e t h e 183 supernatant. Then , 3 0 μ L of r e m o v a l r e a g e n t M s o l u t i o n a n d 7 0 μ L o f r e a g en t M s o l u t i o n w e r e a d d e d , 184 and the m ix t ure w a s thorough l y hom o gen i ze d. T he ho mog enate w as centr ifu ge d at 1 0, 00 0 × g for 1 0 185 mi n a t 4 ℃ , a n d t h e s upernat ant w a s u sed for tot a l G S H me asur e ment. Absorban ce w as m easu red a t 186 41 2 nm usi ng a m i crop l ate re ader ( BI O-RA D I nstr uments, C a l ifor ni a, US A ) . 187 2 . 10 . L i p i d pe ro x ida t i o n ( L PO ) a ss ay 188 The L PO c ontent in t is sues w as d e term ine d us in g an LPO c ontent as say k i t (Sol arb io, Be ij in g , 189 C h i n a ) a c c o r d i n g t o t h e m a n u f a c t u r e r ’ s i n s t r u ct i o n s . B r i e f l y , 0 . 1 g o f t i s s u e w a s h o m o g en i z e d i n 1 m L 190 o f e x t r a c t i o n l i q u i d , f o l l o w e d b y c e n t r i f u g e d a t 8 , 0 0 0 × g f o r 1 0 m i n . R e a g e n t s 1 , 2, a n d 3 w e r e a d d e d 191 a cc o rdi n g t o t h e p r o t oc ol . Fo r t h e bla n k t u b e , di s t ille d w a t e r w a s u se d , a n d f o r t h e s t a n da rd t u b e , 192 g r adi e n t - dil u t e d st a n da rd s ol u t i o n w a s pr e p a r ed . T h e sa mpl e s we re i nc u b a te d i n a w a t e r b a t h a t 100 ℃ 193 f o r 6 0 m i n a n d c e n t r i f u g e d a t 8 0 0 0 × g f o r 1 0 m i n . A b s o r b a n c e v a l u e s w e r e m e a s u r e d a t 5 3 2 n m a n d 194 60 0 nm , an d LPO content w a s ca l cu l a t ed us in g the pr ov ided formu l a. 195 2.1 1. 8 - hy drox y-2'-deo xy g ua n os ine ( 8-OH dG ) assay 196 The con c entr at ion o f 8-hy droxy- 2'-de o xygu anosine (8-OH dG) i n t iss ue s w as m e as ured us ing an 197 8-O Hd G a s say k it ( Jon lnb io, Sha ngh ai , Ch i n a ) in ac co r d an c e w it h the ma n u fa ctur e r ’ s gui del i ne s . 198 T h i s k it u t iliz e s a c om pe ti tiv e e nzy m e - li nk e d i mm u n o s o rb e n t a ss a y ( EL IS A ) . T h e 8 - O H dG a nti b o d y 199 w a s p re-coate d onto a m i c r op l ate . S a m p le s or s tan d ards , an d HRP-l abe le d ant igens w ere 200 s e q u e n t i a l l y a d d e d , f o l l o w e d b y i n c u b a t i o n a n d w a s h i n g s t e p s . T h e c o l o r i m e t r i c s u b s t r a t e T M B w a s 201 a d d e d , t r a n s i t i o n i n g f r o m b l u e t o y e l l o w u p o n a c i d i f i c a t i o n . T h e c o l o r i n t e n s i t y , i n v e r s e l y 202 propor tion al to the 8-O HdG con c entr a t i o n , w as meas ured a t 4 5 0 nm us i n g a m i c r op l a t e re ader . 203 Samp l e co n c entr at ions wer e c al cu l ated from the stan dar d cur ve . 204 2.1 2. R eac t ive o xy gen specie s ( R O S) a s s ay 205 ROS lev els in MF F-1 c el ls w e r e d e te cted u sing a ROS assay k it (B ey ot i me, Sh angh a i, C h in a ) , wit h 206 t h e f l u o re s c e n t p ro b e D CF H - DA . T h e p r o b e w a s dilu t e d 1 :1000 a nd a d de d to a d h e re nt MF F -1 c e ll s , 207 then in cubate d for 20 m in. The GFP fl uorescen ce int e ns ity w a s ob s erv ed under a f l uorescen ce 208 mic ro s c o p e . 209 2.1 3. W e stern blot a n alysis 210 P ro te i n s a m ple s we re e x t r a c t e d fr o m MF F - 1 c ell s u s in g RI P A ly si s b u f f e r (Be yo ti m e, S ha ng h ai, 211 C h i n a ) a n d q u a n t i f i e d w i t h t h e P i e r c e B C A P r o t e i n A s s a y K i t ( T h e r m o F i s h e r S c i e n t i f i c , 212 M a s s a c h u s e t t s , U S A ) . P r o t e i n s w e r e s e p a r a t e d v i a 1 0 % s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l 213 e l ec t r o p ho re si s ( S DS - P A G E ) a nd tra n s f e r re d o n t o Im m o bil o n - P PVD F me m b ra n e s (M illi p o r e, M e rck , 214 G erm a ny). The a n t i-SDD V MC P m Ab (1: 2,0 0 0) or rabbit po l y c lon al a nt ibo d ies (pAb s) agains t Tf R 1 215 ( 1 : 2,000 ) w a s us e d a s p ri ma r y a n ti b ody , wi t h β -ac ti n a s t h e i n te r n al c o n t r o l. H R P -c o n j uga te d g oa t 216 ant i-rabb it or ant i- mous e Ig G w as used as second ar y ant ibod y . The bl ot w as vis ua l i z ed us ing t he 217 T a non H i gh-s ig ECL W es te r n B lott in g S ubstr a t e (T anon, Sh a ng ha i , Ch in a). 218 2.1 4 RN A i nte r fe r e nce ( R N A i ) 219 S m a l l i n t e r f e r i n g R N A s ( s i R N A s ) , i n c l u d i n g a n o n - t a r g e t i n g c o n t r o l s i R N A a n d s i R N A t a r g e t i n g 220 TfR1, w ere s ynthes ize d by R i b oB io (G uangzhou , Ch in a). The s iR N As w e r e t ransfected into M FF- 1 221 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint c e l ls us in g the j etPR IME tr ans fe ct io n rea g ent (P lo yp lus ， Strasbour g, F ran ce) fo l low in g th e 222 m a n u f a c t u r e r ’ s p r o t o c o l . B r i e f l y , 2. 5 μ L o f s i R N A w a s d i l u t e d i n 5 0 μ L o f j e t P R I M E b u f f e r a n d m i x e d 223 w i t h 1 μ L o f je t P R I M E t r a n s f e ct i o n r e a g e n t . T h e r e a c t i o n m i x t u r e w a s i n c u b a t ed a t r o om t e m p e r at u r e 224 f o r 10 mi n, a nd t he n a dd e d to t he c ul tu re m e di um in 24 - we ll p la t e s. T h e f i nal s iR NA c o nc e n t r a ti on 225 w a s ad jus te d to 100 nM. For ty-ei gh t hour s post -t ransfection , Tf R 1 mR NA e xpression le v e ls w ere 226 assessed by qR T-PC R, and prote in l ev els w e r e e v a luate d b y w est e r n blot an a lys i s. After 48 hours 227 p o s t - t r a n s f e c t i o n , c e l l s w e r e i n f e c t e d w i t h t h e Z H - 0 6 / 20 a t a M O I o f 5 . 0 . T h e c e l l s w e r e c o l l e c t e d a t 6 , 228 12 , 24, 3 6, 4 8 , 6 0 an d 72 h p i for subs e quent a n a lys e s . The s iRN A sequen c e t ar ge t in g m an d a r infis h 229 TfR1 w as as fo l lows: ACT C TC A AG G CT C TG A TC A . 230 2.1 5. D r u g t re at me nt 231 Ferros tat in-1 ( Fe r -1) w as purch ased fr om GLPBI O ( Ca l iforn i a, U S A) a nd er ast in , ferr i c ci tr ate 232 (F A C ), deferoxa m ine mesy l ate ( DFO ), ne c r ostat in-1 (Nec-1) an d Z-V A D w ere obt a ined fr om 233 M e d C h e m E x p re ss ( N e w J e r se y , US A ) . I n vi tr o, t h e s e c om p o un d s we r e dil u t e d i n dim e t hyl su l f o xid e 234 (DMSO) to prep are w or k ing solut ions at t he de s ired c on centr at io ns a n d ad ded to the ce l l c u ltu re 235 m e d i u m 5 h b e f o r e S D D V i n f e c t i o n . I n v i v o , t h e d r u g s w e r e d i l u t e d w i t h D M S O t o a p p r o p r i a t e 236 c on c e n trat io ns a nd a dm in istered to m a n d ar infish v i a int r amus cu lar in ject ion 5 h pri or t o S D D V 237 infe c t ion. 238 2.1 6. Cell via bility ass a y 239 MFF-1 c el ls w e r e s e eded into 96-w el l p l ates at a dens it y o f 5 × 10 3 ce l ls/w e l l a n d c u lt ure d for 24 h. 240 The te s t drugs w e r e pr e p a r ed at v ar iou s c on c e nt rat i ons, ad ded to the c u ltu re m e d ium , an d in cubated 241 with the ce l ls for 5 h. Subsequentl y , C e l l C ount ing Ki t- 8 ( CC K -8) s o l ut ion (Bey o t i me , Sh angh a i, C hin a ) 242 w a s introdu ce d i nto ea ch we l l an d i nc u bated at 27 ℃ f o r 1 h . C e l l v i a b i l i t y w a s a s s e s s e d b y 243 mea s ur ing the OD at 4 50 nm us ing a m i crop l ate re a de r . V i abi l ity d a t a w ere no rma l i zed r el at iv e to th e 244 c ontr o l gr oup. 245 2.1 7. Statistical 246 The phe n otyp i c c h ar a cter ist i c s of cel l s w ere a s ses s ed us in g a ser ie s of ass a ys , w it h s tat ist i c a l 247 ana lys e s condu cted as fo l lows: Student’ s t -t e s t w a s e m plo y e d to ev alu a t e d iff e r e nces betw e en groups , 248 w h ile P e a r so n ’ s c o rr ela ti o n t e s t w a s u t iliz e d t o e xa mi n e r ela ti o n s h i p s b e t we e n v a ria bl e s . A na ly si s o f 249 v a r i a n c e ( A N O V A ) w a s p e r f o r m e d t o c o m p a r e m u l t i p l e g r o u p s , f o c u s i n g o n b o t h i n t e r - g r o u p a n d 250 intr a-gr o u p v ar i an ces. No t a b l y , the inter- group v ar i an ce r at io w as r e l at i v el y s ma l l . St at ist i c a l 251 sign if i c an c e w a s def ined a s p < 0 . 05. Al l d ata an al yses w ere con du cted using O RIGI N 2021 softw are. 252 3. Results 253 3.1 . S D D V b ut n ot IS KNV an d M RV i n duce s c lassical ferr o pt os is in m an darin fish 254 T o e x p l o r e w h e t h e r S D D V i n d u c e s f e r r o p t o s i s in v i v o , w e appl i ed the S DD V-m and ar infis h 255 m o d e l es t a b l i s h e d b y o u r t e a m an d ex a m i n e d t h e f e a t u r es o f f e r r o p t os i s i n s p l e en t i s s u e, t h e p r i m ar y 256 target of SDD V i nfect ion [9] . A c c u m u l a t i o n o f c e l l u l a r i r o n i s o n e o f t h e m a jo r b i o c h e m i c a l h a l l m a r k s 257 of ferrop tosis . Thus, th e ti ssu e s ec tions of infected m an da r inf is h w e r e s ta ined with Pr uss ian b lue to 258 v i s u ally o b se rve t h e c ha ng e s o f F e 3+ . Add it i ona l l y , the lev els of Fe 2+ a n d t o t a l i r o n w e r e q u a n t i f i e d 259 u s i n g s p e c i f i c a s s a y k i t s . I S K N V , a t y p e s p e c i e s i n M. s pa grus1 a n d M R V , a d i s t i n c t i v e m e m b e r o f 260 ge nu s Ranavirus i n t h e I rido viridae f a m i l y [27, 2 8 ] , w e r e u s e d a s c o n t r o l s . A s s h o w n i n Fig. 1A , 261 h e m o s i d e r i n a c c u m u l a t i o n w a s o b s e r v e d i n t h e s p l e e n t i s s u e o f m a n d a r i n f i s h i n f e c t e d w i t h S D D V , 262 ISKN V , an d M R V . The c ont ents of tot a l ir o n and Fe 2+ i nc r ea s e d s i g ni fic a n tly a ft e r S DDV a nd I SK NV 263 infe c t ion co mp ared to the contr ol group, where as no sign if i c ant in cre a s e w a s observ ed in the M R V 264 g r o u p ( F i g . 1 B ) . F e r r o p t o s i s i s a l s o c h a r a c t e r i z e d b y i n c r e a s e d l i p i d p e r o x i d a t i o n ( L P O ) i n d u c e d b y 265 rea c t iv e ox y gen s pec ies (R O S) and the dys func t ion o f ant iox id ant d efense mechan is ms. C o m p a r e d t o 266 the contro l group, LPO le v e ls w ere s i gn ifi c a nt l y e lev ate d (F i g. 1C ), GS H l ev els w e r e de cre a s ed, 267 rea c h in g the low e s t at 1 2 dp i (F i g. 1C ). G Px4 ex p ressi o n w as als o si gn ifi c ant l y down-regu l ated in the 268 spleen t is sue fol low in g SDD V infec tion ( F ig . 1C). The l e v e ls of 8-hy dr oxy-2-deox ygu a nos in e 269 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint (8-OH d G ), a mar ke r of o x i d at iv e D N A d a m a ge c a u sed by R OS, w ere m ark edl y e lev a t e d fo l low in g 270 ZH-0 6/ 20 infect ion ( Fig. 1C ). I n co nt r a st, a lthou gh 8-O Hd G lev e ls i n c r eased in the I SK NV-infe cted 271 g r o u p , L P O l e v e l s r e m a i n e d u n c h a n g e d a n d e v e n s i g n i f i c a n t l y d e c r e a s e d i n t h e e a r l y s t a g e s o f 272 infe c t ion ( Fig. 1C ) . A ddi ti o nally , t h e c o nt e n t o f G P X4 u n d e rwe n t a s i g ni fica n t u p - r e g u la ti o n a t 3 a nd 273 6 d p i i n t h e I S K N V i n f e c t i o n g r o u p ( Fig. 1C ). Aft e r M R V infe c t ion, the le v els of L PO in cre a s e d 274 s i g n i f i c a n t l y ( F i g . 1 C ) , w h i l e s u b s t a n t i a l c h a n g e s w e r e o b s e r v e d i n t o t a l i r o n c o n t e n t ( Fig. 1B ), a n d 275 8 - O H d G l e v e l s d e c r e a s e d f o l l o w i n g i n f e c t i o n . T a k e n t o g e t h e r , t h e s e r e s u l t s d e m o n s t r a t e d t h a t 276 ferroptos is is present in S DD V-infe c t i on but i s not apparent in IS K N V an d M R V infe ct ions. 277 278 F ig. 1 F erro ptosis o c curs in vivo i n m a n d a r i n f i s h i n f e c t e d w i t h S D D V . A , Repre sen t a t i v e images of Pruss i a n b lue 279 s t a i n i n g i n s p l e e n t i s s u e s f r o m m o c k , S D D V , I S K N V , a n d MR V g r o u p s ( s c a l e b a r = 5 0 μ m ) . B T o t a l i r o n a n d F e ² ⁺ 280 con c entr a tions we re me asured in s ple en tissu es fr o m m ock , SDDV , I SKNV , and MR V gro up s using an i ron assa y k it . C 281 Th e con c ent r a t ions o f G S H, LPO, a nd 8 -OHdG we re qua nti fie d using c omm e rci al assay ki t s fol lowing in fe c ti on s with 282 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint S DDV , I SKNV , a nd MR V . Th e r el ati v e e x pre ssi on of GPX4 w as a n aly ze d by q RT-PCR. n = 3 . *p < 0.0 5. 283 3.2 . S D D V al s o tr igg ers fe r roptosis i n M FF-1 c ell 284 MFF-1 ce l l l ine is h igh l y s usceptib le t o S DD V i nfect io n a nd is w ide l y used for the i sol at ion , 285 i d en ti f ic a ti o n a n d i n f ec ti o n m ec ha ni s m re se a r c h o f i ri do v ir u s e s [25] . T o de term ine w hether S DD V 286 t r i g g e r s f e r r o p t o s i s i n v i t r o , M F F - 1 c e l l s w e r e i n f e c t e d w i t h S D D V . F i r s t l y , w e e v a l u a t e d t h e 287 morpho lo g i c al ch ange s of m ito ch o n dr i a in M FF- 1 c e l ls infecte d w ith SD DV a t a n M O I of 5. 0 for 7 2 h , 288 as m it o chondr i a l shr in k age is a ha l l m a rk of f e r roptos is [29] . Infe c t e d ce l ls exh ibi te d d isru pted cr istae 289 and shri nka ge of m ito c h ond r i a l com pared to un infe cte d ce l ls (Fig. 2A) . N ext, w e tes te d o t her 290 ferroptos is mar kers at d iff e r e nt per iod s after S D D V i nfect ion . C omp are d to the c ontr o l group , the 291 intr a cel lu l ar lev e ls of Fe 2+ in cre ased at 24 hp i a nd peake d at 48 hp i (Fig. 2B) , co in c i d ing w it h 292 e xce s siv e ROS a ccu mu lat i on (Fig. 2C and D) , l e a d i n g t o i n c r e a s e d L P O l e v e l s (Fig. 2E) . In res ponse, 293 intr a cel lu l ar GS H w as ox i d i zed to g l ut a t h ione d is ulf ide (G S SG)to sc a v en g e m as siv e ROS und e r 294 ox i d a t i ve str e s s. The r e f ore, GSH and GPX4 w as d own-r e gu l ated in the m i dd le and l ate sta ges o f 295 infe c t ion, rea ch in g t h e low est v a lue at 6 0 hp i (Fig. 2F and G). In c on clus ion , the abov e r e s ults show e d 296 that SDD V inf ect ion act iv ates ferr optos is in M FF- 1 ce l ls. 297 298 F ig. 2 Ferroptosis c ontrib ut es t o SDD V-in d uce d c e ll de at h i n vitr o . A T r a nsmissi on el ec tr on m icr o s cop y ima ges 299 sho win g mit ochondri a l morpho lo gy a nd vi ra l p a r tic les in c on t r ol a nd SDDV-infe cted MFF-1 ce lls . Black ar rows 300 i n d i c a t e m i t o c h o n d r i a ( s c a l e b a r = 1 μ m ) . B F e ² ⁺ co n ce n tra t i o n s we re q ua n ti fi ed u s i ng a n i ro n a ssa y k it, wit h 301 a bs orb a nce m e a sure d a t 5 93 nm. C, D R O S l e v e l s i n MF F - 1 c e l l s w e r e a s s e s s e d t h r o u g h e x c i t a t i o n w a v e l e n g t h o f 4 8 8 302 n m and an emission w a v e le ngth of 52 5 nm a n d fluore sce nce intensi ty f oll owing S DDV infec ti o n . E L P O 303 con c entr a tions in MFF -1 ce l ls w ere m e a sure d using an L PO ass ay ki t. F GSH le v els w ere qua n tif ied using a GS H ass ay 304 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint k it , wi th a bs orbance me asured a t 4 12 nm. G T h e r e l a t i v e e x p r e s s i o n o f G P X 4 w a s e v a l u a t e d i n c o n t r o l a n d 305 S DDV-infecte d MFF-1 ce l l s by q RT-PCR. n = 3 . *p < 0.0 5. 306 3.3 . F e r roptosis i s the primary for m of cell de at h up on S D DV infec ti on 307 C e l l d e a t h c a u s e d b y v i r a l i n f e c t i o n m a y i n v o l v e a c o m b i n a t i o n o f d i f f e r e n t c e l l d e a t h p a t h w a y s 308 r a t h e r t ha n a si n gl e mo d e [24 ] . Ident ify in g t he pr e do m in ant c e l l de at h mode is ess entia l . I n th is s tudy , 309 MFF-1 c e l ls w e r e treate d w ith v ar iou s con c entr at ions of Fer-1 ( a ferropt osi s inh ibitor ), Er a s tin (a 310 f e r r o p t o s i s i n d u ce r ) , z - V A D ( a p y r o p t o s i s i n h i b i t o r ) a n d N e c 1 ( a n e cr o p t o s i s i n h i b i t o r ) p r i o r t o S D D V 311 infe c t ion, a nd the r el at iv e express ion of the S DD V mcp gene w as meas ured at 6 0 hp i. The r e s ults 312 rev e a led t h a t Fer-1 tr eatm ent e xhib ite d the most si gn if i cant inh ib itory e ff ec t on SDD V rep l i c a t ion 313 ( F i g . 3 A ) , w h i c h w a s f u r t h e r c o n f i r m e d b y i m m u n o f l u o r e s c e n c e a s s a y ( F i g . 3 B ) . T r e a t m e n t w i t h 314 E r a s t i n p r o d u c e d t h e o p p o s i t e e f f e c t ( F i g . 3 A a n d B ) . N o t a b l y , t h e t r e a t m e n t s w i t h z - V A D a n d N e c 1 315 al so si gn ifi c ant l y inh ib ite d S DD V pr o l iferat i on, s ugge s tin g th at S DD V infec t ion indu ce s m u lt ip l e 316 f o r m s o f p r o g r a m m e d c e l l d e a t h i n M F F - 1 c e l l s . H o w e v e r , F e r - 1 h a d t h e m o s t p r o n o u n c e d i n h i b i t o r y 317 e f fe ct, in d i c a t i ng t h a t f e r roptosi s is the dom in ant mod e of c e l l de a th . 318 319 F ig. 3 Ferropt osis is t h e prim a ry mo d e of c el l d e a t h in d uc e d by SDD V i nfe c t io n . A R e lat i ve S D D V ex p r es s i o n l e v el s 320 in MFF -1 cel ls af ter t re atmen t wi th 2 0 μM a nd 5 0 μM er as tin , Fe r -1 , z -V AD, and Ne c-1 fo r 4 h, fol l owed by SDDV 321 in fe ct ion. Th e mock gr o up wa s t r e ate d wi th di lute d DMSO, Differe nt le tte rs ( a , b, c , d) indic a te s ign if ic ant diffe rence s 322 be t w e e n gr o u p s . B Immu no fluore sce n c e anal y s i s o f S DDV at 6 0 hpi in MF F-1 ce ll s pre -tre a t ed with 50 μM Er ast in , 323 F e r-1, z-V AD, o r Nec-1 f o r 5 h pri or t o S DDV challe n ge . n = 3 . 324 3.4 . F e r -1 treatment mitigat e d ferropt os is by SD DV infection 325 T o exam i ne whe t he r th e inh ib it i o n of f er roptosi s c a n pr otect ag ai nst S D DV- ind uced iron ov e r lo ad 326 and l ip id perox id at ion , M FF-1 c e l ls we re tr e ate d w ith Fer-1 , Eras t in, or DMS O, fo l lowe d b y infe ct io n 327 w i t h S D D V a t a n M O I o f 5 . 0 . A s s h o w n i n F i g u r e 4 A , D M S O a n d E r a s t i n t r e a t m e n t s i n d u c e d 328 ac cumu l at ion of Fe 2+ , where as Fer- 1 tr e atment r e m ar k abl y a mel i o r ate d s uch indu c t ions dur in g 329 infe c t ion. W e als o as ses s e d the lev els of R O S and 8 - O H dG in i nfe cte d M FF- 1 ce l ls. After Fe r -1 330 treatment , int ra ce l lul ar RO S ( F i g u r e s 4 B a n d C ) a n d 8 - O H d G (Figure 4D) c o n t e n t sig ni f ica ntly 331 d e c l i n e d c o m p a r e d t o D M S O - t r e a t e d c e l l s , w h i l e E r a s t i n t r e a t m e n t l e d t o e n h a n c e d R O S a n d 332 8 - O H d G p r o d u c t i o n . F u r t h e r m o r e , G S H a n d G P X 4 l e v e l s w e r e r e d u c e d b y E r a s t i n t r e a t m e n t b u t 333 restor e d fol lo win g Fer - 1 t re a tm e nt (Figures 4E and F) . I mp ortant l y , w e observ ed th at S D DV- infected 334 man d ar infis h tre ate d wi th Fe r - 1 e xh ib i ted low er mort a l ity c omp ared to thos e tr e ated w ith Er astin or 335 DM S O (Figure 4G) . The s e results dem onstr a t e th a t Fer- 1 c an suppr es s f e r rop tos is indu ce d by SDD V 336 infe c t ion an d re duc e m or ta l it y i n m a n d a r infish. 337 338 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint 339 F ig. 4 F e r- 1 tre atm ent al l e vi a t e d f erropto s is ind uc ed b y ir o n o v e rlo ad and lip id p e rox idat ion fol lo w ing SD DV 340 infe c tion. Cha n ges in Fe²⁺ concen tr a ti o n (A ), R O S le vel s (B, C) , 8 -OH d G co nten t (D ) , and GS H conte n t (E ) i n MF F - 1 341 cells af ter SDDV infec ti o n . F The rel a t ive e x pr e ssion of GPX4 w a s ana lyzed by qRT-P CR in con tr o l a nd S DDV-infecte d 342 MF F-1 c e lls. G Sur vi v a l c u r ve of ma n d a r in fis h a f te r SDDV infec t ion. Fish we re in tr am usc u la rly injec t ed with DMSO, 343 eras tin , o r F e r-1, f o ll o w ed by S DDV c h a llen ge v i a intr ap eri t on e a l injec ti on a f te r 4 h . T h e m ock gr ou p c o nsis ted o f 344 un tre a t e d man d a rin f is h , whi le the DMSO g r oup was injec te d with d i lute d DMSO 5 h p r ior to SDDV i nfe c ti o n, ser ving 345 a s a c ont ro l f or o ther drug-tre a te d gr ou ps. n = 3 . Diffe re nt le tte r s ( a, b , c , d ) indic a te si gnific a nt di ffere n c es b e twee n 346 gr ou p s . 347 3.5 . S D D V -indu ce d ferropt os is is ir on d e pen dent 348 Fe r roptos is is driv en by iron-d ependen t ROS produ c t io n. T o ev a lu ate the e f fe c t of a lter in g l abi l e 349 iron o n S DD V-in du ce d f e r roptos is in v i v o, m a n dar inf ish w ere in jected intr a mus c ul ar l y w it h D FO (a n 350 iron c hel a tor , 0. 1 g /k g), F AC (a n ir on s upplement , 0 . 1 g/ kg) or DM SO ( contro l, PBS d i l ute d to 20% ) 351 be for e in fect ion w ith SD DV (1 0 3 T C I D 50 / f i s h ) . A s a r e s u l t , D F O t r e a t m e n t r e d u c e d t h e c o n t en t of F e 3 + 352 as observ ed in ir on-st a in ed s pleen t issue sec t ions (Fi g. 5 A). Moreo v er , R OS l evels w e r e s ign if i c a n t l y 353 e lev ated in t he F A C gr oup c omp ared t o the DMS O co ntro l group i n M FF- 1 c e l ls, w h i le the D FO 354 t r e a t m e n t g r o u p m a r k e d l y r e d u c e d t h e s e l ev e l s ( F i g . 5 B ) . I n t h e i n v i v o ex p e r i m e n t s , t h e L P O c o n t e n t 355 w a s hi gher in the F A C group th an in t h e contr o l gr o u p, w h e r eas t he D FO gr oup effectiv el y r e v ers e d 356 t h i s i n c r e a s e ( F i g . 5 C ) . D F O t r e a t m e n t a l s o r e s t o r e d G P x 4 e x p r e s s i o n l e v e l s ( F i g . 5 D ) . I n t e r e s t i n g l y , 357 D F O tr eatme nt pr o t ecte d m and a r in f ish f rom S D D V infect ion. As s hown in F i gur e 5F , F AC tre at me nt 358 sign if i c ant ly in c r eas e d t he tr a ns c r ipt i on of the SD D V m c p g e n e , w h i l e D F O t r e a t m e n t h a d t h e 359 opposite e ff e ct. M ore impor t ant l y , mo rta l ity i n the contr ol an d F AC-tr ea ted groups w as 90% a n d 360 1 0 0 % , r es p ec t i v e l y , wh er e a s D F O t r e a t m en t r e d u c ed m o r t a l i t y t o 5 0 % ( F i g . 5 E ) . Al l to ge ther , these 361 f i n di ng s su g g e st tha t SDD V i n fe c tio n i ndu c e s i r o n o ve r l oa d , R O S acc u m ula tio n, a nd li pi d 362 p e ro x i da ti o n t h r o ug h a n i r o n - de p e n d en t m ec ha ni sm , a n d r e d uc in g ir o n l e v els i m p r o v e s t h e s ur v i v al 363 rate in m a n d a r inf ish infe cted wi th SDD V . 364 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint 365 F ig. 5 R egul at in g l a bi l e ir o n l e v e l in v iv o a ff e ct SD DV inf ectio n. A , Repre se nt at i ve imag e s of Prussi an b lue st a in ing 366 in s p le en t issue s o f mand arinfis h fr om the mo c k , con tr o l , DFO, an d F AC g rou ps (sc ale b ar = 50 μm ). B, C S t a t i s t i c a l 367 a n alysis of lip id ROS a nd LPO le ve ls showe d a si gnif ic a nt dec re ase in the DFO and F AC g r oups compa re d to the 368 mo c k an d D MS O g r ou p . D Rel at ive m RNA express io n of GP X4 w as me asured by qPCR in the DFO and F AC gr ou ps 369 c o m p a r e d t o t h e m o c k a n d D MS O g r o u p . E Mo rta li ty ra t e s o f m a n da ri n fi s h we r e r eco r d ed a ft e r i n t r a p e r i t o n eal 370 in je ct io n wi th F AC a nd DFO, f ol lo w ed b y SDDV c h allenge . F S DDV mR NA lev els wer e qua nt if ied by qP CR a f te r 371 m andar inf ish we re injec te d with F AC and DFO a n d ch alle ng e d with S DDV . The moc k g r oup con s iste d of un tre ate d 372 m andar inf ish, while the DMS O gro up w a s injec ted with d ilu ted DM SO 5 h pri or t o SDDV infe cti o n, se r vin g as a 373 con tr ol f or other drug-tre a te d gr o ups. n = 3 . The l e t t e r s a , b, c , a n d d r e pr e s e nt g r oups t ha t ha v e s i gni f i c a nt di f f e r e nc e s 374 b e twe en th e m ( p < 0 .0 5 ). 375 3.6 . Tf R 1 is in vol ved i n f e rr optosis in S DDV-inf e cted M FF-1 c ells 376 T he regu l a t ion of iron h omeost a s is aff e cts ce l lu lar s e ns itiv it y to f e r roptos is. T o fur ther explor e 377 the me c h an is ms under l y ing S DD V-i n duced ferropt osis, w e focused o n the genes for m a inta i n ing 378 iron ho meostas is, in c lu d ing TfR 1 , ir on respons iv e ele ment bin d ing pr ot e in 2 (I RE B 2), nu c lear 379 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint r e c e p t o r c o a c t i v a t o r 4 ( N C O A 4 ) a n d f e r r i t i n H e a v y C h a i n 1 ( F T H 1 ) . I R E B 2 r e g u l a t e s c e l l u l a r i r o n 380 l e vel s b y m od ula ti ng t he t r a n sla ti o n an d s t a bili t y o f m RNA s t h a t c o n t r ol i r o n me ta b oli s m d u ring 381 i r o n d e p l e t i o n . F T H 1 i s a m a j o r c o m p o n e n t o f f e r r i t i n , a n i r o n s t o r a g e p r o t e i n [30] . N C OA4 m e d ia t e s 382 ferrit i n a ut oph agy and the r ele ase of fr ee iron, wh i ch is ess entia l for in du c ing ferr optosis [3 1 ] . In the 383 s t u d y , t h e r e s u l t s s h o w e d t h a t S D D V i n f e c t i o n s i g n i f i c a n t l y a l t e r e d t h e e x p r e s s i o n o f t h e s e g e n e s i n 384 MFF-1 c e l ls . I n d e t a i l, t he e xpr e s sion le v e ls of NCO A4, TfR 1 and IR E B 2 w as si gn ifi c ant l y upregu l ated 385 (Fig. 6A, C and D) , w h i l e F T H 1 m R N A l e v e l s d e c r e a s e d a t l a t e r s t a g e s o f S D D V i n f e c t i o n (Fig. 6B) . 386 A m ong t he genes an a ly z ed , the most p r onounced c han ge w as obs e r v e d in m a ndar inf ishTf R1 mR N A 387 l e v e l s , w h i c h w e r e s i g n i f i c a n t l y u p r e g u l a t e d b y m o r e t h a n f o u r f o l d a t 6 0 h p i (Fig. 6D) . Cons istentl y , 388 W e s t e r n b l o t a n a l y s i s d e m o n s t r a t e d a t i m e - d e p e n d e n t i n c r e a s e i n T f R 1 p r o t e i n l e v e l s i n r e s p o n s e t o 389 SDD V infe c t ion (Fig. 6E) . Mor e ov e r , o ur previous stu dy id ent ifie d th at TfR 1 i s pa c kaged in t o the 390 S D D V m a t u r e v i r i o n t h r o u g h L C - M S / M S ( U n p u b l i s h e d d a t a ) . B a s e d o n t h i s , w e h y p o t h e s i z e d t h a t 391 TfR1 m ight s e r v e a s a key e ffector gene in SDD V- indu ced ferropt osis. T o t est this hypothes is , w e 392 e v a luate d the effec t of TfR 1 kno c kdo wn. M FF- 1 cel ls were tra ns fe cted w it h e ither an irre lev a nt 393 c ontr o l s iR NA or T fR1-sp e c if i c siR NA . A si gn ifi c ant de c r ease in both Tf R 1 mR NA a nd prote in leve ls 394 w a s observ ed in TfR1 kn o c k d o wn ce l ls com p a r e d wit h scr a mbl e d s iR N A contr ols (Fig. 6F, G, an d H). 395 T w e nty- four hours post tr a ns fec t ion , MFF-1 cel ls w ere inf ected w ith SDD V at an MO I of 5.0 a nd 396 fea tu res of ferr optosis w e r e a s sess e d by mon itor in g the intr ace l lu l ar le v els of Fe 2+ , ROS a n d LPO. As 397 ex p e c t ed , F e 2+ l e v e l w a s l o w e r i n T f R 1 k n o c k d o w n c e l l s c o m p a r e d t o s i R N A c o n t r o l c e l l s ( F i g . 6 I ) . 398 C o r r e s p o n d i n g l y , t h e a c c u m u l a t i o n o f L P O a n d R O S i n d u c e d b y S D D V i n f e c t i o n w a s r e d u c e d i n t h e 399 TfR1 kno c kdo wn cel ls (Fig. 6J a n d K ) . A dd it ion a l l y , w e found that siTfR 1 treatme nt enh an c e d c e l l 400 v ia b ili ty d u ring t h e SD DV i n f ec t i o n pr o c e ss (Fig. 6L) , a s m e a s u r e d b y t h e C C K - 8 a s s a y . T o f u r t h e r 401 e v a l u a t e t h e d i r e c t i m p a c t o f T f R 1 k n o c k d o w n o n S D D V r e p l i c a t i o n , t h e t r a n s c r i p t i o n l e v e l o f t h e 402 SDD V mc p g e n e w a s m ea s u re d a t 24 a n d 48 h pi via RT - q P CR . A s s h ow n i n Fig. 6M , SD DV 403 repl i cat ion w a s s uppressed fol lo w ing siTfR 1 tre atment . In con c lusi o n , TfR1 p l a ys a cr it i c a l r ol e in 404 f e r r o p t o s i s i n d u c e d b y S D D V i n f e c t i o n , a n d d o w n r e g u l a t i o n o f T f R 1 i n h i b i t s f e r r o p t o s i s i n 405 SDD V- infec te d M FF- 1 cel ls . 406 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint 407 F ig. 6 Downreg ula tio n o f TFRC in hib it ed ferropto s is i n SD DV- in f ect ed M FF - 1 c el ls. A -D The m RNA lev e ls o f 408 ferr o pt osis-re l ated gene s we re de tec te d by q P CR. All da t a are p r ese nte d a s me an ± SD. * p < 0. 05 , **p < 0.0 01 . E T h e 409 prote in le ve l o f T fR1 w a s me asure d by wes te rn blot t ing . F- H The effe c ts o f T fR1 knock do wn a t b oth the mRNA le ve l 410 (vi a qP CR) a nd pr ote in lev el ( vi a wes tern b l o t t ing) . I-K S t a t i s t i c a l a n a l y s i s s h o w e d t h a t F e ² ⁺ , L P O , a n d R O S l e v e l s 411 sign if ican tly dec re ased in the s iTfR1 g r o up co m pare d to the s i- NC gro up (*p < 0.0 5, **p < 0.0 01) . L Ce l l vi ab i l i t y i n t he 412 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint siTfR 1 g r oup w as n o t ably impr ov ed foll owi ng S DDV infe ct i o n, as de termined by CCK-8 ass ay s . M T h e r elat i ve 413 express io n o f SDDV mRN A in the siT fR1 g roup was sign if ic ant ly l ower th an th at in t h e s i-NC gro up. Th e mock g r oup 414 refe rs t o un tre ate d ce l ls, whil e th e wide gr o up re p r ese n ts c el ls infec te d wi th SDDV witho ut a ny o ther t re a tmen t. n = 3. 415 Th e l e t ters a , b, c, and d in d ica te gr ou ps with st at ist ica lly signif ican t di ffe r ence s. 416 4. Discussion 417 Fer roptosis r e p resents a new l y re co gni z ed form o f re gu late d cel l de ath, pr i m a r i l y dr iv e n by 418 iron-depen dent l ip i d per oxi d at ion. A l though ferr o p tosis w as on l y re cent ly ident if ied , in c r eas ing 419 e vi d e n ce h i ghl i ghts a st rong assoc iat i o n be t ween v ir a l i nfec t ion s and th is uni que ce l l de a t h p a t hw ay . 420 Fe r roptos is i s ch ar a c t e r i z ed b y elev ated iron le v e ls, wh i c h pro mote the pr o d uct ion of R O S v i a the 421 Fe nt o n r ea ct ion, the r eby a cce le r at ing LPO [3 2, 3 3 ]. Th i s stud y u nc ov ers t he potenti a l ro le a n d 422 u n d e r l y i n g m e c h a n i s m o f f e r r o p t o s i s d u r i n g S D D V i n f e c t i o n , p r o v i d i n g n e w i n s i g h t s i n t o t h e 423 inter a c t ion betw ee n S D D V and its hos t. 424 Fo l low in g S DD V infe c t i o n, t a nn i n sta in in g of splee n t iss ue from m a nd a r inf ish r evea led a 425 pheno m e non of f ree ir on ac c umu l at ion (Fig. 1A) . Add it i ona l l y , the le v e ls of L PO, R OS , an d 8-O HdG 426 in cel ls and t iss ue s esca l ate d , ind i cat in g t he ex isten ce o f oxid a t i ve s tress after infe ct ion (Fig. 1C and 427 Fig. 2C, D, E) . G P x4, a k ey intr a c e l l ul ar a nt iox id ant e n zy me, p l ays a cru c i al ro le in re gu l a t ing 428 ferroptos is and its e xpr e s sion is suppr e ssed dur ing the intr ins i c p athw a y of fer roptos is [23, 29 ]. G P x4 429 mi ti ga t e s li pi d p e r o xid a tio n by u tilizi ng G SH , t h e re b y p ro te c ti ng ce ll s f r o m f e r ro p t o s i s. I n o u r st u d y , 430 w e e xa m ined the a lte r at ions in th e G SH cy c le fo l low i ng S D D V infe c t ion and obser ved a 431 d o w n r e g u l a t i o n o f G P X 4 e x p r e s s i o n , a c c o m p a n i e d b y a s i g n i f i c a n t r e d u c t i o n i n G S H l e v e l s . 432 Furtherm o r e, TEM im a ges of SDD V-i nfec te d c e l ls r e v e a le d m ito ch ond ri a ex h ib it in g ty p i c a l 433 morpho lo g i c al f e atur e s of ferropt osis (Fig. 2A) . Fer-1 , a potent inh ib itor of f e r roptos is, w as s ho wn t o 434 prev e n t the a c c um u l a t i o n of L PO r es ultin g fr om iron ov er l oa d. In th is s tudy , F e r -1 tre at me nt 435 al l e v i ated iron ov er lo ad, r edu c ed LPO and ROS a c cum u l a t i o n , and pr e v e nted the d ec l ine in GSH a nd 436 G P x 4 in S D D V - inf e ct ed MF F-1 cel ls. Add it i ona l l y , F e r -1 tre atment s ign if i c ant l y de cre a s e d the 437 morta l it y of m a nd a r inf ish infected w i th S DD V . In c ontr a s t, tre atme nt w ith Er as tin, a ferr o p tosis 438 indu cer , produ ced th e oppos ite effec t . T he s e fin d ings confir m that the observ ed chan ge s are 439 c h ar a cter ist i c of ferr optosis , in d i cat ing i ts a ct iv at ion dur in g S D DV infe c t ion . 440 Another tw o fis h ir idov ir uses, I SKN V and M R V , w ere intro duc ed for a co mp ar at iv e study . Up on 441 i n f e c t e d w i t h I S K N V , t h e c o n t e n t o f L P O m a r k e d l y r e d u c e d a t 3 d p i (Fig.1C) . A f te r MR V infe ct ion , 442 8-O Hd G l ev els de c r eas e d and G SH le v els in cre ased. These res ults are in c onsis te nt w ith the h al l m a r k 443 c h ar a cter ist i c s of ferroptos is, m ak in g i t ch a l leng in g t o c on c lus iv e ly deter m i ne whe ther ferr o p tosis 444 occurs after I S K N V or MR V i nfect ion . These obs e r v a ti o n s s ugge s t that the p atho ge n i c ity of IS K N V 445 and MR V d iffers fr om that of SD D V . 446 V i r a l infe c t ion- in du c ed cel l death m ay inv o lv e a m ixed pa t te r n of cel l d eath, r ather th an a sin g l e 447 mode [24] . Our s tudy demonst r ate d th a t S D D V infe ct ion in MF F-1 c e l ls w as s uppressed by inh ibit ors 448 of ferr optosis , apoptosis , and ne c r os is , suggest ing th at SD DV tr i gge r s m u lt ip le forms of ce l l de ath . 449 Howev er , the most pr onoun ce d inh ib it ory effect w a s observ ed w ith Fe r - 1 tre atment , ind i cat in g th a t 450 ferroptos is is the p red om in ant for m of c e l l de ath in du ce d by S DDV infect ion. 451 I ron i s an e s sentia l e le ment r e qu ir e d to suppor t basi c ce l lu l ar pr o c ess es, an d it a ls o p l ays a 452 c ruc ial ro le i n th e g ro w t h, vi r ul e nc e a nd pa t h og e nic it y o f vi r u se s, w hic h a c q ui r e i ro n f r o m t h e h o st . 453 Not a bl y , DFO, an iron-ch el a t in g agent t hat redu ce s free iron leve ls , h as b een id entif ied as an e f fect iv e 454 m o d ula t o r o f f e r ro p t o s i s a nd i s well - d oc u me n te d fo r i t s a n ti vi r al ac ti vi ty [3 4] . K a nnan et al . r e por ted 455 that pr e tr eat ment of m ous e pri m ar y m i c r og l ia l ce l ls wi th D FO b lo cke d H I V-1 T at-med i ate d 456 m i c r og l i al act iv at ion in v itr o an d redu ce d the ex p ressi o n an d re le a s e of proi nf lam m a tor y cyt o k i nes 457 [35 ] .Ad d it ion al l y , D FO h as bee n s hown to inh i bit S ARS-C o V -2 infe c t ion an d b lo c k 458 SA R S-Co V-2- indu ced ferr optosis [36 ] . In our s tudy , DF O a d m in istr a t i o n to S D DV-infe cted 459 man d ar infis h l e d to a de c r ease in iron c onte nt , a lon g w ith redu ct io ns in LPO a nd 8-OH d G lev el s, a n d 460 i n c r e a s e s i n G P x 4 a n d G S H . M o r e o v e r , D F O t r e a t m e n t s i g n i f i c a n t l y i n h i b i t e d S D D V r e p l i c a t i o n , 461 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 18, 2025. ; https://doi.org/10.1101/2025.03.18.643978doi: bioRxiv preprint

in g in a 50% r e d u c t ion in mort a l i ty . These find in gs suggest that DF O p r events SDD V-in du ce d 462 ferroptos is and r edu ces v ira l rep l i c at io n by lo w er ing iron c ontent in v iv o.[ 1] 463 T rans fe r rin re ceptor 1 (TfR1) me d i ates c e l lu l ar ir on upt ake an d is ess e nti al for m a int a in ing ir o n 464 home os tasis [ 37] . O u r re su l t s d em o nst r a t e d t ha t T f R 1 e x p re ss i o n w a s s i g n ific a n tl y i nc r ea se d in 465 SDD V- infec te d M FF- 1 cel ls at 7 2 hp i . K no c kdown of T fR 1 a l levi a te d SD D V - i ndu c e d fe r roptos is in 466 MFF-1 cel ls , lea d ing t o re du c ed ir on o v e r lo a d , decre ased LPO lev el s, upr e gu l ated GPX 4 express ion , 467 a n d d e c r e a s e d v i r a l r e p l i c a t i o n . T f R 1 h a s a l s o b e e n s h o w n t o p l a y a v i t a l r o l e i n t h e p r o l i f e r a t i o n o f 468 sev e r al v ir uses. F or exam p le , TfR1 co operates wit h met abotrop i c g lut a mat e r eceptor subtype 2 to 469 med i a t e the intern a l i z at ion of R ab ies vi ru s (R A BV) an d S ARS-CoV- 2 [3 8] ; P o rc i ne e pid e mic d ia rr h ea 470 virus (PED V) S 1 pr ote in int e r acts w ith TfR 1 to prom ote v ir al e nt r y [ 39] . Seve ra l ot he r v irus e s ha v e 471 b e e n s h o w n t o e x p l o i t c e l l u l a r T f R 1 a s a r e c e p t o r , f a c i l i t a t i n g v i r a l i n t e r n a l i z a t i o n t h r o u g h 472 c l a t h r i n - m e d i a t e d e n d o c y t o s i s , i n c l u d i n g N e w W o r l d h e m o r r h a g i c f e v e r a r e n a v i r u s e s [40] , 473 T ransm is sible g a s troenter it is v ir us (TGE V) [ 41] a n d m o u s e m a m m a r y t u m o r v i r u s ( M M T V ) [ 4 2] . 474 F u r t h e r m o r e , o u r p r e v i o u s d a t a s h o w e d t h a t h i g h a b u n d a n c e o f m a n d a r i n f i s h T f R 1 e x i s t e d i n t h e 475 purif ied SD DV v ir us p a r t i cl e s . Therefo re, w e h ypothe s i ze th a t TfR 1 m ay p l ay s a n i mport ant r o le i n 476 t h e p ro c e s s o f SD DV i n f ec ti o n. F u t u re s tu d i e s w ill f u rt h e r i n ve sti ga t e h o w T fR 1 re g u la t e s SD DV 477 infe c t ion an d wh i ch s ta ge s it inf luen c es . 478 5. Conclusion 479 In s umm ar y , S D D V infe c t ion i n du c es fer roptosi s both in v i vo an d in vitr o . F er ro ptosis , a for m of 480 iron-depen dent c e l l de ath , is the pre do m inant m ode of cel l d e at h dur in g S D D V infe ct ion . 481 Mec h an is ti ca l l y , t he ke y iron-r egul at in g gene Tf R1 i s upregu lat ed b y SD D V i nfect ion , lea d ing to the 482 releas e of fr e e iron , wh i c h tr i ggers l ip i d pe r ox i d a t i o n an d ferr optosis . Both the downr egul at ion of 483 TfR1 express ion an d the r edu ct ion of ir on lev els inh ibit ferroptos is, thereb y de c r easi n g v ir al 484 repl i cat ion an d lo w er ing m orta l it y in m and ar inf is h. 485 Acknowledgments 486 This w ork w as fun de d by ke y a r eas R &D Pr ogr am o f G u ang don g Prov in ce u nde r N o. 487 2021B0 202 04 0 002; G ua ng d o ng B a s ic a nd A p p lie d Ba s ic R e s ea r c h F o u n da ti o n u n d e r No . 488 2023A 15 15 1109 3 8; Na tio nal N a t u ral Sc ie n c e F o u nda ti o n o f Chi na u nde r N o . 32 4 03 07 5 ; C hi n a 489 P o s t d o c t o ral Sc ie nc e F o u n da ti o n u n d e r N o. 20 24 M753 721. 490

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