Original article Andes-virus-induced cytokine storm is partially suppressed by ribavirin

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1 Antivirl Therpy 203; 8: (doi: 0.385/IMP2524) Originl rticle Andes-virus-induced cytokine storm is prtilly suppressed by ribvirin Svetln F Khiboullin *, Albert A Rizvnov 2, Vincent C Lombrdi, Sergey P Morzunov 3, Helton J Reis 4, András Plotás 5, Stephen St Jeor 6 Whittemor Peterson Institute, University of Nevd-Reno, Reno, NV, USA 2 Institute of Fundmentl Medicine nd Biology, Kzn (Volg Region) Federl University, Kzn, Russi 3 Deprtment of Pthology nd Nevd Stte Helth Lbortory, University of Nevd-Reno, Reno, NV, USA 4 Lbortório de Neurofrmcologi, Deprtmento de Frmcologi, Instituto de Ciêncis Biológics, Universidde Federl de Mins Geris, Belo Horizonte, Brzil 5 Asklepios-Med (privte medicl prctice nd reserch center), Szeged, Hungry 6 Deprtment of Microbiology nd Immunology, University of Nevd-Reno, Reno, NV, USA *Corresponding uthor e-mil: sv.khiboullin@gmil.com Bckground: Microbe-induced over-ctivtion of cytokines, especilly tumour necrosis fctor (TNF)-, is key to the pthogenesis of hntvirus infection leding to severe inflmmtion with high mortlity rte. Although ribvirin showed promise in inhibiting virl repliction in vitro, its clinicl efficcy remins controversil. Methods: Vrious concentrtions of ribvirin were used to determine its effect on cytokine ctivtion in our infectious model system. Results: Ribvirin decresed the virus lod nd dosedependently inhibited the ccumultion of RANTES messenger RNA in Andes-virus (ANDV)-infected humn endothelil cells, but filed to suppress TNF--induced ctivtion of RANTES nd interleukin-6 in ANDV-inoculted cultures. This report lso shows, for the first time, tht the deleterious over-stimultion by TNF- is medited by nucler fctor-kb, nd describes the effect of ribvirin on cytokine production following ANDV infection. Conclusions: Although highly effective in preventing ANDV repliction nd suppressing ctivtion of select inflmmtory meditors, the therpeutic efficcy of ribvirin is limited due to its inbility to fully inhibit cytokine outburst triggered by hntvirus infection. Introduction Ribvirin (-b-d-ribofurnosyl-,2,4-trizole-3-crboxmide) is synthetic nucleotide nlogue [] tht hs brod spectrum of ntivirl ctivity [2 0]. Vrious mechnisms of ction were suggested to explin ntivirl ctivity, including enzyme suppression [0 5] nd the error ctstrophe effect in which lethl ccumultion of minor muttions leds to stop in virl repliction [6,7]. Although highly effective in vitro, ribvirin s therpeutic efficcy remins controversil nd even conflicting clinicl results re often seen with the sme virl infection, such s with hntvirus [8,9]. For exmple, ribvirin hs been shown to be effective for tretment of ptients with hntvirus hemorrhgic fever with renl syndrome (HFRS), wheres no efficcy ws documented in ptients with hntvirus pulmonry syndrome (HPS) [20]. Although highly effective ginst mny RNA viruses in vitro, cliniclly ribvirin s efficcy hs been proven only in tretment of chronic HCV nd respirtory syncytil virus [2]. It ppers tht direct ntivirl ctivity of ribvirin hs little effect on its overll clinicl efficcy in mny virus infections. Therefore, it hs been suggested tht mechnisms of therpeutic efficcy of ribvirin my involve its immunomodultory properties [22]. A number of studies hve reported cytokine ctivtion in peripherl blood mononucler cells, humn pulmonry epithelil cells nd humn endothelil cells treted with ribvirin in vitro [8,9,23]. Current dt suggests tht ribvirin my fcilitte Th type immune response ctivtion promoting clernce of virus-infected cells [8,9,23]. Clinicl studies hve shown erly cytokine ctivtion in HFRS nd HPS ptients. High serum levels of tumour necrosis fctor (TNF)-, interleukin (IL)-6, nd IL- were found during the cute phse of HFRS [24]. Since 203 Interntionl Medicl Press (print) (online) 575

2 SF Khiboullin et l. hntviruses re not cytopthic, it hs been suggested tht pthogenesis of HFRS is minly ttributed to the ction of virus-ctivted cytokines [24 27]. Therefore, inhibition of hntvirus-induced cytokines my benefit ptients by preventing excessive inflmmtion, which is believed to be the min cuse for crdio-pulmonry shock nd kidney filure in HPS ptients. Although ribvirin hs been shown to be effective for tretment of the subset of HFRS ptients, its effect on cytokine ctivtion during hntvirus infection remins lrgely unknown. We hypothesized tht low efficcy of ribvirin therpy of HPS my be cused by its filure to inhibit cytokine ctivtion cused by hntvirus infection. Endothelil cells re the prime trget for hntvirus infection in vivo [28,29]. Therefore, we pplied n in vitro model using humn endothelil cells to determine the effects of ribvirin on cytokine ctivtion cused by hntvirus infection. Andes virus (ANDV) ws utilized in this study. ANDV hs been shown to be custive gent in severl HPS outbreks in South Americ [24,25]. We hve found tht ribvirin suppresses ANDV repliction in humn umbilicl cord vein endothelil cells (HUVEC) in vitro. Antivirl effects of ribvirin correlted with inhibition of RANTES (chemokine lignd 5 [CCL5]) secretion cused by ANDV repliction in HUVECs. TNF- ctivtes IL-6 nd ugmented RANTES ctivtion in ANDV-infected HUVECs. Ribvirin filed to inhibit TNF--cused RANTES nd IL-6 ctivtion in ANDV-infected HUVECs. We believe tht ctivtion of nucler fctor (NF)-kB by TNF- led to upregultion of IL-6 nd RANTES in ANDV-infected HUVECs. Although ribvirin inhibited ANDV repliction, it did not ffect TNF--triggered NF-kB ctivtion, which sustined trnscriptionl ctivtion of IL-6 nd RANTES. Methods Cell lines nd regents HUVEC nd Vero clone E6 (Vero E6) cells were obtined from ATCC (Mnsss, VA, USA). HUVECs were grown in MCDB 3 medium supplemented with humn vsculr endothelil cell growth fctor, hydrocortisone, 2% fetl bovine serum (FBS), humn fibroblst growth fctor (0.5 ml, mg/ml), scorbic cid, heprin (0.5 ml, mg/ml) nd gentmicin (Clonetics Corportion, Wlkersville, MD, USA). Cells were used between pssges 2 nd 4. Vero E6 cells, isolted from monkey kidney epithelil cells nd generlly used s host cells to grow viruses, were mintined in Dulbecco s modified Egle s medium (DMEM) medium contining 20% FBS nd 50 mg/ml gentmicin. Ribvirin ws obtined from Sigm-Aldrich (St Louis, MO, USA). TNF- ws purchsed from R&D Systems (Minnepolis, MN, USA). Virus ANDV strin 23 used in this study ws kindly provided by T Ksizek (CDC, Atlnt, GA, USA). Virl stock ( plque-forming units [PFU]) ws prepred using Vero E6 cells nd stored t -80 C. In experimentl studies, cells were infected t multiplicity of infection (MOI) of. Virus ws concentrted by ultr-centrifugtion ( h, 22,000 g, 4 C) nd re-suspended in DMEM (0% FBS, 50 mg/ml gentmicin). Virus ws llowed to ttch to cells for h t 37 C in 5% CO 2 tmosphere. Non-ttched virus ws removed, cells were wshed with Hnk s blnced slt solution, nd new medium ws dded. In some experiments new medium ws supplemented with ribvirin nd TNF-. To determine ANDV titre, seril 0-fold dilutions of ANDV ( ml) were dded onto 3 7-dy old Vero E6 cell monolyers nd incubted for h t 37 C in 5% CO 2 tmosphere. Medium ws removed nd 3 ml of overly DMEM (0% FBS, 50 mg/ml gentmicin) supplemented with 0.6% grose (Invitrogen, Crlsbd, CA, USA) ws dded. Monolyers were incubted t 37 C for 7 dys nd then stined by dding 2 ml/well of overly medium contining 5% FBS nd 5% neutrl red solution (Gibco, Rockville, MD, USA). Plques were counted fter 2 dys t 37 C. Rel-time PCR Totl RNA ws extrcted from hntvirus-infected cells t selected time points (, 3, 24 nd 72 h postinfection [PI]) using Trizol regent (Gibco) ccording mnufcturer s recommendtions nd stored t -80 C. TqMn Minor Groove Binder (MGB) fluorogenic probes nd primers were designed using Primer Express softwre (Applied Biosystems, Crlsbd, CA, USA) following the mnufcturer s recommendtions. The sequences of primers nd probes used re summrized in Tble. Primers were obtined from Invitrogen nd probes were synthesized by ABI (Applied Biosystems). Complementry DNA (cdna) synthesis ws performed using rndom primers (Invitrogen) nd stored t -20 C before use. A totl of mg RNA ws used for cdna synthesis. RNA ws first incubted with rndom primers t 70 C for 5 min nd then quickly chilled t 4 C for 5 min. Mixture of reverse trnscriptse buffer, 20 mm nucleoside triphosphtes, RNsin, nd reverse trnscriptse ws dded nd rection ws extended t 42 C for h. At the end of the rection, enzymes were inctivted t 70 C for 5 min. TqMn ws performed using TqMn MGB probes on n ABI Prism 7000 Sequence Detection System (Applied Biosystems). Ech PCR rection (25 ml) consisted of ml of cdna, Pltinum qpcr Supermix-UDG (Invitrogen), 200 nm of ech primer nd 00 nm of probe. 8S ribosoml RNA ws used s n internl control ccording to the mnufcturer s Interntionl Medicl Press

3 Andes-virus nd ribvirin Tble. Primers nd probes for rel-time PCR Gene Forwrd primer Reverse primer TqMn probe RANTES cccgcggggcc gcggtttcccgtctttc ctcgcgggctgc IL-6 ctgcgcgctttgggttc cctgctctttgccgg cgtccgcctgggg ANDV S tccgccggcgtttgg ggctttgcctgtgctgg cttgcttgtggcctt ANDV, Andes-virus; IL, interleukin. instructions. cdna smples were diluted :,000 with nuclese-free wter before use in the 8S ribosoml RNA-specific TqMn rections. Stndrd curves for reltive quntittion of the ANDV S segment RNA, RANTES mrna, IL-6 mrna nd 8S RNA were creted using seril dilutions of cdna purified from infected nd uninfected control smples depending on specifics of experimentl design. In ech experiment, the TqMn vlues obtined for the ANDV S segment RNA, s well s the vlues obtined for the RANTES nd IL-6 mrnas, were normlized to the TqMn vlues of the 8S RNA of the corresponding smple nd presented s reltive units. In ddition, for some experiments these normlized vlues were clculted s the reltive vlues to the sme mrnas produced in the corresponding control group. All TqMn rections were performed in duplicte. Electrophoretic mobility shift ssys Gel shift ssys were performed ccording to the mnufcturer s instructions for the Gel Shift Assy Core System (Promeg, Mdison, WI, USA) using double-strnded NF-kB consensus oligonucleotides. Briefly, 3.5 pmol of consensus oligonucleotides were end-lbelled with mci [γ- 32 ]ATP using 5 units of T4 polynucleotide kinse in 70 mm Tris-HCl (ph 7.6), 0 mm MgCl 2,nd 5 mm DTT t 37 C for 0 min. The rection ws stopped by the ddition of 50 mm EDTA nd diluted with 0 mm Tris HCl (ph 8.0) nd mm EDTA. Unincorported lbel ws removed by size exclusion chromtogrphy with Sephdex G-50 spin columns. DNA binding rections (5 ml) were performed using ml of lbelled consensus oligonucleotides nd 4 mg of nucler extrct in binding buffer contining 4% glycerol, mm MgCl 2, 0.5 mm EDTA, 0.5 mm DTT, 50 mm NCl, 0 mm Tris-HCl (ph 7.5) nd 0.05 mg/ml poly(di-dc). The rections were incubted t room temperture for 20 min nd stopped with n pproprite volume of 0 gel loding buffer contining 250 mm Tris-HCl (ph 7.5), 0.2% bromphenol blue nd 40% glycerol. Smples were loded onto.0 mm, 0 2 cm nondenturing 4% crylmide gels nd electrophoresed t 00 V. Gels were dried overnight t room temperture between cellophne sheets nd exposed to phosphorimger screen. Nucler protein extrction Nucler protein extrctions were performed ccording to stndrd protocols [30]. Briefly, treted cells were rinsed twice nd then scrped in 00 ml of icecold phosphte-buffered sline (PBS; 37 mm NCl, 2.7 mm KCl, 4.3 mm N 2 HPO 4,.4 mm KH 2 PO 4 ) contining mg/ml protinin, mg/ml leupeptin, 0 mg/ml soyben trypsin inhibitor nd mg/ml pepsttin A. The cells were pelleted by centrifugtion t 2,000 g for 5 min t 4 C. The pellet ws wshed twice with ml of buffer A (0 mm HEPES [ph 7.9],.5 mm MgCl 2, 0 mm KCl, 0.5 mm DTT nd 0.5 mm PMSF) nd centrifuged s just described. The superntnt ws discrded, nd the cellulr proteins remining in the pellet were relesed by hypotonic lysis in 00 ml of buffer A contining 0.% Nonidet P-40 nd centrifuging t 0,000 g for 0 min t 4 C. The superntnt contining the cytosolic frction ws trnsferred to new tube, while the nucler proteins remining in the pellet were obtined by extrction in high-slt buffer B (20 mm HEPES [ph 7.9],.5 mm MgCl 2, 0.42 M NCl, 0.2 mm N 2 EDTA, ph 8.0, 25% glycerol, 0.5 mm DTT nd 0.5 mm PMSF) nd centrifugtion t 0,000 g for 0 min t 4 C. The superntnt contining nucler proteins ws then diluted with n equl volume of buffer C (20 mm HEPES [ph 7.9], 50 mm KCl, 25% glycerol, 0.5 mm DTT nd 0.5 mm PMSF). Protein concentrtions for ll extrcts were determined by the bicinchoninic cid method (Sigm-Aldrich) ccording to the mnufcturer s recommendtions. Bovine serum lbumin ws used s the stndrd. TUNEL ssy Terminl deoxynucleotidyl trnsferse dutp nick end lbelling (TUNEL) ssy ws performed using n in situ cell deth detection kit (Roche, Indinpolis, IN, USA) ccording to the mnufcturer s recommendtions. Sttisticl nlyses Sttisticl nlyses were performed using Student s t-test for comprisons between individul experimentl groups (infected nd non-infected). Significnce ws estblished t vlue of P<0.05. Dt re presented s men ±se. Antivirl Therpy

4 SF Khiboullin et l. Figure. Effect of ribvirin on Andes-virus S segment RNA ccumultion in HUVECs Reltive units,000,000 Results 800, , , ,000 0 Untreted infected cells Ribvirin-treted infected cells Error brs represent stndrd error. Humn umbilicl cord vein endothelil cells (HUVECs) were infected with Andes-virus (ANDV) t multiplicity of infection of.0. Ribvirin (50 mg/ml) ws dded into the culture medium h fter infection. TqMn ws performed using TqMn Minor Groove Binder probes on n ABI Prism 7000 Sequence Detection System (Applied Biosystems, Crlsbd, CA, USA). Stndrd curves for reltive quntittion of ANDV S segment RNA nd 8S RNA were creted using seril dilutions of complementry DNA from infected nd uninfected control smples respectively. All TqMn rections were performed in duplicte. P-vlue <0.05 between control nd ribvirin-treted groups. Effect of ribvirin on Andes-virus repliction in HUVECs To determine the in vitro effect of ribvirin on ANDV repliction, HUVECs were infected with ANDV t n MOI of. Mock-infected HUVECs were used s control. At h PI, ribvirin (50 mg/ml) ws dded to the culture medium. Accumultion of the ANDV S segment RNA ws mesured using rel-time PCR. Additionlly, the titre of ANDV in cell culture superntnts ws determined by plque ssy t 72 h PI. Results indicted tht in infected cells without ribvirin, ANDV S segment RNA levels were significntly incresed t 2 h PI, reching mximum t 24 h PI (Figure ). Ribvirin tretment (50 mg/ml) significntly reduced ANDV S segment RNA levels t 2, 24 nd 72 h PI (Figure ). TUNEL ssy ws performed to demonstrte tht ribvirin (50 mg/ml) tretment did not induce poptosis in HUVECs (dt not shown). Likewise, poptosis ws not detected in ANDV-infected HUVECs treted with ribvirin (50 mg/ml; dt not shown). Therefore, we concluded tht ribvirin inhibition of ANDV repliction in HUVECs ws not due to its cytotoxic ctivity. Ribvirin inhibition of ANDV repliction ws lso demonstrted in plque ssy t 72 h PI. ANDV titre in HUVEC culture superntnts ws 0 2 ±20 PFU/ml. ANDV titre in HUVECs treted with ribvirin ws 2.75 ±.9 PFU/ml, which is significntly (P<0.00) lower compred to tht in superntnts of HUVECs without ribvirin tretment. Ribvirin inhibits RANTES expression in Andes-virusinfected HUVECs Previously, we hve shown tht hntvirus infection ctivtes RANTES in HUVECs [3,32]. Therefore, ribvirin s effect on cytokine ctivtion ws evluted using TqMn by ssessing RANTES mrna levels in ANDV-infected HUVECs. RANTES mrna levels were significntly incresed in the ANDV-infected cells t 2, 24, nd 72 h PI when compred to mock-infected cells (Figure 2). Ribvirin tretment did not ffect RANTES mrna levels in the mock-infected cells t ech selected time point (3, 2, 24 nd 72 h PI; Figure 2). However, ribvirin significntly reduced levels of the RANTES mrna in ANDVinfected cells t 2, 24 nd 72 h PI when compred with infected cells without ribvirin tretment (Figure 2). These dt demonstrte tht ribvirin inhibits RANTES ctivtion in ANDV-infected cells. It ppers tht by inhibiting ANDV repliction, ribvirin prevents ANDV-cused ctivtion of cellulr RANTES. Dose-dependent effect of ribvirin on Andesvirus S segment RNA nd RANTES messenger RNA ccumultion in HUVECs HUVECs were infected with ANDV t n MOI of or mock-infected. Ribvirin (, 0 nd 50 mg/ml) ws dded to the culture medium of infected nd mockinfected cells h fter infection. Ribvirin demonstrted dose-dependent effect on ccumultion of the ANDV S segment RNA (Figure 3A). At concentrtions of 0 nd 50 mg/ml, ribvirin significntly inhibited ANDV S segment RNA levels t 2, 24 nd 72 h PI (Figure 3A). By contrst, ribvirin t concentrtion of mg/ml ffected virus RNA level only t 48 h PI (Figure 3A). Similrly, ribvirin hd dose-dependent effect on RANTES gene ctivtion in the infected cells. For instnce, ribvirin tretment (50 nd 0 mg/ml) downregulted RANTES mrna level t 24 nd 72 h PI compred to untreted infected cells (Figure 3B). However, ribvirin pplied t mg/ml hd no effect on RANTES mrna level (Figure 3B). This dt demonstrtes tht inhibitory effects of ribvirin on RANTES ctivtion correlte with its ntivirl ctivity. Ribvirin modultes cellulr responses ctivted by tumour necrosis fctor- TNF- is n importnt meditor of innte inflmmtion nd up-regultes expression of dhesion molecules in vivo nd in vitro [33,34]. Incresed levels of Interntionl Medicl Press

5 Andes-virus nd ribvirin Figure 2. Effect of ribvirin tretment on RANTES messenger RNA levels in Andes-virus-infected HUVECs 0,000,000 Mock-infected control HUVECs HUVECs treted with ribvirin, 50 µg/ml ANDV-infected HUVECs Reltive units 00 0 ANDV-infected HUVECs treted with ribvirin, 50 µg/ml Error brs represent stndrd error. Humn umbilicl cord vein endothelil cells (HUVECs) were infected with Andes-virus (ANDV) t multiplicity of infection of.0. Ribvirin (50 mg/ml) ws dded into the culture medium h fter infection. TqMn ws performed using TqMn Minor Groove Binder probes on n ABI Prism 7000 Sequence Detection System (Applied Biosystems, Crlsbd, CA, USA). Stndrd curves for reltive quntittion of RANTES nd 8S RNA were creted using seril dilutions of complementry DNA from infected nd uninfected control smples respectively. All TqMn rections were performed in duplicte. P-vlue <0.05 between indicted groups. TNF- hve been demonstrted in ser of hntvirusinfected ptients [24,35]. Our dt nd others hve shown tht hntvirus infection does not ctivte TNF- in HUVECs. However, we hve demonstrted ctivtion of TNF- in hntvirus-infected pulmonry mcrophges in vitro [36]. Therefore, we used TNF- supplemented medium to determine effects of ribvirin on cytokine ctivtion in ANDV-infected HUVECs. At h PI with ANDV of HUVECs, TNF- (0 ng/ml) nd ribvirin (50 mg/ml) were dded to the culture medium. Totl RNA ws collected t selected time points, 24 nd 72 h PI. Rel-time PCR ws employed to estimte ccumultion of the ANDV S segment RNA. ANDV S segment RNA ccumultion pttern in HUVECs ws similr to tht observed previously when levels of virl RNA reched its mximum t 24 h PI (Figure 4A). Ribvirin decresed ANDV S segment RNA ccumultion t 24 nd 72 h PI compred to ANDV-infected control cells (Figure 4A). Similrly, ANDV S segment RNA ccumultion in HUVECs ws decresed by TNF- lone nd in combintion with ribvirin t 24 h PI (Figure 4A). Since, levels of ANDV S segment RNA were decresed in ANDV-infected control cells to the sme level s in cells treted with ribvirin nd its combintion with TNF- t 72 h PI (Figure 4A), effects of ribvirin, TNF-, nd their combintion on ANDV repliction were further determined by the virus titre in cell culture superntnts. In plque ssy, ribvirin (50 mg/ml) inhibited ANDV repliction in HUVECs t 72 h PI (Tble 2). TNF- tretment lone did not ffect ANDV repliction. However, combintion of TNF- nd ribvirin suppressed ANDV repliction (Tble 2). Ribvirin (50 mg/ml) did not ffect levels of RANTES mrna in mock-infected HUVECs t 24 h, wheres RANTES mrna levels were significntly lower in cells treted with ribvirin t 72 h (Figure 4B). TNF- (0 ng/ml) significntly incresed RANTES mrna levels in mock-infected HUVECs t 24 nd 72 h compred to tht in control cells (Figure 4B). Similrly, TNF- combined with ribvirin significntly incresed RANTES mrna levels in mock-infected HUVECs t 24 nd 72 h compred to control cells (Figure 4B). Thus ribvirin does not lter TNF- induced RANTES mrna ccumultion in HUVECs. ANDV infection significntly incresed RANTES mrna levels in HUVECs compred to control cells t 24 nd 72 h PI (Figure 4B). As it hd been expected, ribvirin significntly decresed RANTES mrna levels in ANDV-infected cells t 24 nd 72 h PI compred to tht in infected cells without ribvirin (Figure 4B). TNF- significntly up-regulted levels of RANTES mrna in ANDV-infected HUVECs t 24 nd 72 h PI compred to mock-infected cells (Figure 4B). Interestingly, TNF- did not lter ANDVinduced RANTES mrna ccumultion in HUVECs Antivirl Therpy

6 SF Khiboullin et l. t 24 nd 72 h PI. Ribvirin combined with TNF- significntly decresed RANTES mrna levels only t 72 h PI compred to tht in ANDV-infected cells treted with TNF- lone (Figure 4B), lthough verge vlues remined significntly higher compred to tht in infected HUVECs treted with ribvirin lone (Figure 4B). TNF- (0 ng/ml) incresed IL-6 mrna levels in mock-infected HUVECs t 24 nd 72 h (Figure 4C). Ribvirin decresed IL-6 mrna levels only t 72 h PI in mock-infected HUVECs. Ribvirin did not lter IL-6 mrna levels in cells treted with TNF- t 24 nd 72 h (Figure 4C). ANDV infection incresed IL-6 mrna levels in HUVECs t 24 h PI (Figure 4C). However, IL-6 mrna levels were incresed in HUVECs treted with TNF- (0 ng/ml; Figure 4C). Ribvirin did not ffect IL-6 mrna levels in ANDV-infected HUVEC (Figure 4C). Similrly, there ws no effect of ribvirin on IL-6 mrna level in ANDV-infected HUVECs treted with TNF- (Figure 4C). Figure 3. Ribvirin dose-dependent effect on Andes-virus S segment RNA nd RANTES messenger RNA ccumultion in HUVECs A 0,000,000 Reltive units,000,000 ANDV Ribvirin 50 µg/ml Ribvirin 0 µg/ml Ribvirin µg/ml 00,000 B 0, ,000 Reltive units M A M A M A M A M A M A Error brs represent stndrd error. Humn umbilicl cord vein endothelil cells (HUVECs) were infected with Andes-virus (ANDV) t multiplicity of infection of. Ribvirin (, 0 nd 50 mg/ml) ws dded into the culture medium h fter infection. TqMn ws performed using TqMn Minor Groove Binder probes on n ABI Prism 7000 Sequence Detection System (Applied Biosystems, Crlsbd, CA, USA). Stndrd curves for reltive quntittion of ANDV S segment, RANTES nd 8S RNA were creted using seril dilutions of complementry DNA from infected nd uninfected control smples, respectively. All TqMn rections were performed in duplicte. P-vlue <0.05 between control nd ribvirin-treted groups. (A) Dose-dependent effect of ribvirin tretment on ANDV S segment RNA ccumultion. (B) Dose-dependent effect of ribvirin tretment on RANTES mrna ccumultion. A, ANDV-infected cells; M, mock-infected cells Interntionl Medicl Press

7 Andes-virus nd ribvirin These dt demonstrte tht ribvirin fils to inhibit TNF--stimulted RANTES nd IL-6 ctivtion in ANDV-infected HUVECs. Effect of TNF- nd ribvirin on NF-kB ctivtion Our dt reveled tht TNF- increses intrcellulr levels for RANTES nd IL-6 mrna in ANDV-infected HUVECs. NF-kB regultes RANTES nd IL-6 gene ctivtion [37,38]. Therefore, we sought to determine whether NF-kB is ctivted in response to TNF- nd/or ANDV infection. HUVECs were infected with ANDV t MOI of for 72 h nd used for collection of the nucler frctions, which were used in n electrophoretic mobility shift ssy. In mock-infected HUVECs, little binding of the nucler proteins ws seen in the presence of n oligonucleotide encoding the consensus DNA sequence for NF-kB (Figure 5; lne ). This binding is blted in the Figure 4. Ribvirin effect on RANTES nd interleukin-6 ctivtion by tumour necrosis fctor- in Andes-virus-infected HUVECs A 5,000,000 B,000 Reltive units 4,000,000 3,000,000 2,000,000,000,000 Reltive units 00 0 C M A M A Reltive units 00 0 Control Ribvirin TNF-α Ribvirin nd TNF-α 0. M A M A Error brs represent stndrd error. Humn umbilicl cord vein endothelil cells (HUVECs) were infected with Andes-virus (ANDV) t multiplicity of infection of. Ribvirin (50 mg/ml) ws dded into the culture medium h fter infection. Tumour necrosis fctor (TNF)- (0 ng/ml) ws dded into the culture medium together with ribvirin. TqMn ws performed using TqMn Minor Groove Binder probes on n ABI Prism 7000 Sequence Detection System (Applied Biosystems, Crlsbd, CA, USA). Stndrd curves for reltive quntittion of ANDV S segment, RANTES nd 8S RNA were creted using seril dilutions of complementry DNA from infected nd uninfected control smples, respectively. (A) Accumultion of ANDV S segment RNA in HUVECs treted with ribvirin nd TNF- (0 ng/ml). (B) Effect of ribvirin tretment on RANTES messenger RNA ccumultion in TNF--ctivted HUVECs. (C) Effect of ribvirin tretment on interleukin-6 messenger RNA ccumultion in TNF--ctivted HUVECs. All TqMn rections were performed in duplicte. P-vlue <0.05 between control nd experimentl groups. A, ANDV-infected cells; M, mock-infected cells. Tble 2. Andes-virus titre in superntnt of HUVECs treted with tumour necrosis fctor-, ribvirin nd their combintion ANDV ANDV plus ribvirin ANDV plus TNF- ANDV plus ribvirin plus TNF- Titre 72 h PI, PFU ±2 2.5 ± ±3.5 ±0.5 Dt re men ±se. ANDV, Andes-virus; HUVECs, humn umbilicl cord vein endothelil cells; PFU, plque-forming units; PI, post-infection; TNF, tumour necrosis fctor. Antivirl Therpy

8 SF Khiboullin et l. presence of excess unlbelled NF-kB oligonucleotide, indicting tht the increse in binding seen is specific for NF-kB (Figure 5; lne 2). Ribvirin did not ffect nucler protein binding to the NF-kB oligonucleotide (Figure 5; lne 3) while TNF- significntly incresed nucler protein binding to the NF-kB oligonucleotide (Figure 5; lne 4). Nucler protein binding ws slightly decresed in mock-infected HUVECs treted with combintion of TNF- nd ribvirin (Figure 5; lne 5). ANDV infection did not ffect nucler protein binding to the NF-kB oligonucleotide compred to mockinfected cells (Figure 5; lne 6). Similrly, nucler protein binding to the NF-kB oligonucleotide ws not ltered in ANDV-infected HUVECs treted with ribvirin (50 mg/ml; Figure 5; lne 7). TNF- (0 ng/ml) incresed nucler protein binding to the NF-kB oligonucletide in ANDV-infected HUVECs (Figure 5; lne 8). Interestingly, it ppers tht TNF- induced more nucler protein binding to the NF-kB oligonucleotide in ANDV-infected HUVECs compred to tht of mockinfected HUVECs (Figure 5; lne 4 versus 8). Nucler proteins binding to the NF-kB oligonucleotide remined incresed in ANDV-infected HUVECs treted with TNF- nd ribvirin (Figure 5; lne 9). Discussion A number of studies hve reported tht ribvirin exhibits diverse effects on cytokine production by vrious cell types [8,9,23]. Therefore, we sought to determine whether ribvirin tretment ffects RANTES ctivtion in hntvirus-infected cells. Our dt demonstrte tht its dose-dependent inhibition of RANTES in ANDV-infected cells correltes with its inhibition of ANDV repliction. RANTES is known to ttrct mononucler immune effector cells to the site of infection [39 4]. Therefore, ANDV-cused RANTES ctivtion my explin pthogenesis of the interstitil mononucler pneumoni commonly dignosed in HPS ptients [29]. Our dt on ribvirin inhibition of RANTES ctivtion in hntvirus-infected cells suggest potentil therpeutic efficcy of ribvirin in tretment of HPS ptients. However, clinicl studies hve demonstrted tht ribvirin shows no significnt improvement in the clinicl mnifesttion or mortlity rte of HPS ptients [20,42]. It hs been suggested tht virus infection nd cytokine ctivtion ply equl roles in hntvirus pthogenesis. Therefore, we hypothesize tht ribvirin, lthough strong ntivirl gent, hs less effect on virus-cused cytokine ctivtion. Incresed serum concentrtion of TNF- nd IL-6 in hntvirus ptients serum hs been reported [24,35]. We hve shown ctivtion of TNF- in humn lveolr mcrophges infected with Sin Nombre virus Figure 5. Effects of ribvirin on tumour necrosis fctor- ctivtion of nucler fctor-kb in HUVECs TNF-α Ribvirin NF-κB Nonspecific Mock ANDV Humn umbilicl cord vein endothelil cells (HUVECs) were infected with Andesvirus (ANDV) t multiplicity of infection of. Ribvirin nd tumour necrosis fctor (TNF)- were dded into the culture medium h fter infection. Nucler protein frction ws isolted t 72 h post-infection nd used to determine binding of the nucler proteins to the oligonucleotide encoding the DNA consensus sequence (20 nucleotides) for nucler fctor (NF)-kB. Electrophoretic mobility shift ssys were performed using 4 mg of nucler extrcts in the presence of 32 P-lbelled NF-kB consensus oligonucleotides. Excess unlbelled NF-kB oligonucleotide ws dded to the rection in lne 2 s specific binding competitor. A representtive experiment is shown from n=3. [36]. Additionlly, high numbers of TNF--positive cells were described in lung biopsies of HPS ptients [25,26]. These dt suggest tht TNF- my ply significnt role in hntvirus pthogenesis. TNF- is potent ctivtor of n rry of cytokines in endothelil cells including IL-6 [43]. Therefore, we sought to further investigte effects of TNF- on cytokine ctivtion in ANDV-infected HUVECs. Our dt reveled tht TNF- up-regultes IL-6 in ANDV-infected HUVECs. TNF- nd IL-6 shre severl physiologicl effects tht re often synergistic. Both cytokines up-regulte expression of dhesion molecules, promote leukocyte migrtion, nd ctivte cute phse proteins in the liver [44,45]. Thus, it ppers tht TNF- my promote inflmmtory responses in ANDV-infected cells by upregulting IL-6 nd ugmenting RANTES ctivtion. Overll, we hve shown here tht ribvirin filed to vert RANTES nd IL-6 ctivtion in TNF--treted ANDV-infected cells. Thus these dt suggest tht ribvirin hs limited effect on TNF--cused cytokine ctivtion in ANDV-infected cells. RANTES nd IL-6 gene expression is regulted by ctivtion of the trnscription fctor NF-kB [37]. It is lso known tht TNF- stimultes NF-kB signlling [46]. However, there is no dt on NF-kB ctivtion in hntvirus-infected cells. Our dt demonstrtes tht ANDV infection does not ctivte nucler protein binding to NF-kB t 72 h PI. However, TNF- incresed NF-kB Interntionl Medicl Press

9 Andes-virus nd ribvirin binding in mock-infected nd ANDV-infected cells t 72 h. Therefore, it could be suggested tht TNF- cused ctivtion of RANTES nd IL-6 in ANDV-infected cells is regulted by NF-kB. Our dt hs shown tht ribvirin significntly reduces ANDV repliction in HUVECs. Antivirl effect of ribvirin correltes with inhibition of RANTES ctivtion in HUVECs. Although highly effective in inhibiting ANDV repliction when combined with TNF-, ribvirin filed to suppress TNF- induced IL-6 nd RANTES ctivtion in ANDV-infected cells. It is generlly ccepted tht proinflmmtory cytokines such s TNF- nd IL-6 ply significnt role in hntvirus pthogenesis [26,28,29,47]. Therefore, we believe tht, lthough, ribvirin decreses virus lod nd suppresses virus repliction in HPS ptients, it fils to inhibit cytokine ctivtion cusing lung inflmmtion. As result, ribvirin therpeutic efficcy could be limited in HPS ptients, s observed during ribvirin clinicl trils [42]. However, erly initition of ribvirin tretment could be therpeuticlly beneficil due to erly inhibition of hntvirus repliction, which will prevent ctivtion of proinflmmtory cytokines such s TNF-. Recently, combintion of ribvirin with ntibodies ginst proinflmmtory cytokines ws shown to significntly reduce mortlity nd morbidity in virus-infected nimls [48,49]. This pproch combines inhibition of virus repliction nd ongoing inflmmtory responses, s both re involved in development of the morbidity nd mortlity chrcteristic of the HPS. Acknowledgements This study ws supported by the Russin Ministry of Science nd Eduction (FCP ), by the Federl Center for the Collective Use nd Phrmceuticl Reserch nd Eduction, Kzn (Volg Region) Federl University, Kzn, Russi, in prt by NIH grnt AI3648, nd by Asklepios-Med (Szeged, Hungry). Disclosure sttement The uthors declre no competing interests. References. Sidwell RW, Huffmn JH, Khre GP, Allen LB, Witkowski JT, Robins RK. Brod-spectrum ntivirl ctivity of virzole: -bet-d-ribofurnosyl-,2,4-trizole-3- crboxmide. Science 972; 77: Hrusk JF, Bernstein JM, Dougls RG, Jr., Hll CB. Effects of ribvirin on respirtory syncytil virus in vitro. Antimicrob Agents Chemother 980; 7: Povey RC. In vitro ntivirl efficcy of ribvirin ginst feline clicivirus, feline virl rhinotrcheitis virus, nd cnine prinfluenz virus. Am J Vet Res 978; 39: Cssidy LF, Ptterson JL. Mechnism of L Crosse virus inhibition by ribvirin. Antimicrob Agents Chemother 989; 33: de l Torre JC, Alrcon B, Mrtinez-Sls E, Crrsco L, Domingo E. Ribvirin cures cells of persistent infection with foot-nd-mouth disese virus in vitro. J Virol 987; 6: Durr FE, Lindh HF. Efficcy of ribvirin ginst influenz virus in tissue culture nd in mice. Ann N Y Acd Sci 975; 255: Jhrling PB, Peters CJ, Stephen EL. Enhnced tretment of Lss fever by immune plsm combined with ribvirin in cynomolgus monkeys. J Infect Dis 984; 49: Oxford JS. Inhibition of the repliction of influenz A nd B viruses by nucleoside nlogue (ribvirin). J Gen Virol 975; 28: Sidwell RW, Huffmn JH, Brnett BB, Pift DY. In vitro nd in vivo Phlebovirus inhibition by ribvirin. Antimicrob Agents Chemother 988; 32: Whren B, Oberg B. Reversible inhibition of cytomeglovirus repliction by phosphonoformte. Intervirology 980; 4:7 5.. Ben B. Antivirl therpy: current concepts nd prctices. Clin Microbiol Rev 992; 5: Crotty S, Mg D, Arnold JJ, et l. The brod-spectrum ntivirl ribonucleoside ribvirin is n RNA virus mutgen. Nt Med 2000; 6: Goswmi BB, Borek E, Shrm OK, Fujitki J, Smith RA. The brod spectrum ntivirl gent ribvirin inhibits cpping of mrna. Biochem Biophys Res Commun 979; 89: Streeter DG, Witkowski JT, Khre GP, et l. Mechnism of ction of -b-d-ribofurnosyl-,2,4-trizole-3-crboxmide (virzole), new brod-spectrum ntivirl gent. Proc Ntl Acd Sci U S A 973; 70: Stridh S. Determintion of ribonucleoside triphosphte pools in influenz A virus-infected MDCK cells. Arch Virol 983; 77: De Clercq E. Antivirl drugs in current clinicl use. J Clin Virol 2004; 30: Severson WE, Schmljohn CS, Jvdin A, Jonsson CB. Ribvirin cuses error ctstrophe during Hntn virus repliction. J Virol 2003; 77: Meier V, Burger E, Mihm S, Sile B, Rmdori G. Ribvirin inhibits DNA, RNA, nd protein synthesis in PHAstimulted humn peripherl blood mononucler cells: possible explntion for therpeutic efficcy in ptients with chronic HCV infection. J Med Virol 2003; 69: Mrtín J, Nvs S, Quirog JA, Prdo M, Crreno V. Effects of the ribvirin-interferon lph combintion on cultured peripherl blood mononucler cells from chronic heptitis C ptients. Cytokine 998; 0: Huggins JW, Hsing CM, Cosgriff TM, et l. Prospective, double-blind, concurrent, plcebo-controlled clinicl tril of intrvenous ribvirin therpy of hemorrhgic fever with renl syndrome. J Infect Dis 99; 64: Peshuyse J, Dllmeier K, Neyts J. Ribvirin for the tretment of chronic heptitis C virus infection: review of the proposed mechnisms of ction. Curr Opin Virol 20; : Te HS, Rndll G, Jensen DM. Mechnism of ction of ribvirin in the tretment of chronic heptitis C. Gstroenterol Heptol 2007; 3: Jing Z, Kunimoto M, Ptel JA. Autocrine regultion nd experimentl modultion of interleukin-6 expression by humn pulmonry epithelil cells infected with respirtory syncytil virus. J Virol 998; 72: Krkuer T, LeDuc JW, Krkuer H. Serum levels of tumor necrosis fctor-lph, interleukin-, nd interleukin-6 in hemorrhgic fever with renl syndrome. Virl Immunol 995; 8: Mori M, Rothmn AL, Kurne I, et l. High levels of cytokine-producing cells in the lung tissues of ptients with ftl hntvirus pulmonry syndrome. J Infect Dis 999; 79: Antivirl Therpy

10 SF Khiboullin et l. 26. Temonen M, Mustonen J, Helin H, Psternck A, Vheri A, Holthofer H. Cytokines, dhesion molecules, nd cellulr infiltrtion in nephropthi epidemic kidneys: n immunohistochemicl study. Clin Immunol Immunopthol 996; 78: Vplhti O, Lundkvist A, Vheri A. Humn immune response, host genetics, nd severity of disese. Curr Top Microbiol Immunol 200; 256: Knerv M, Mustonen J, Vheri A. Pthogenesis of puuml nd other hntvirus infections. Rev Med Virol 998; 8: Zki SR, Greer PW, Coffield LM, et l. Hntvirus pulmonry syndrome. Pthogenesis of n emerging infectious disese. Am J Pthol 995; 46: Singer CA, Bker KJ, McCffrey A, AuCoin DP, Dechert MA, Gerthoffer WT. p38 MAPK nd NF-kB medite COX-2 expression in humn irwy myocytes. Am J Physiol Lung Cell Mol Physiol 2003; 285:L087 L Khiboullin SF, Rizvnov AA, Deyde VM, St Jeor SC. Andes virus stimultes interferon-inducible MxA protein expression in endothelil cells. J Med Virol 2005; 75: Khiboullin SF, Rizvnov AA, Otteson E, Miyzto A, Mciejewski J, St Jeor S. Regultion of cellulr gene expression in endothelil cells by Sin Nombre nd prospect hill viruses. Virl Immunol 2004; 7: Henninger DD, Pnes J, Eppihimer M, et l. Cytokineinduced VCAM- nd ICAM- expression in different orgns of the mouse. J Immunol 997; 58: Rothlein R, Czjkowski M, O Neill MM, Mrlin SD, Minolfi E, Merluzzi VJ. Induction of intercellulr dhesion molecule on primry nd continuous cell lines by proinflmmtory cytokines. Regultion by phrmcologic gents nd neutrlizing ntibodies. J Immunol 988; 4: Linderholm M, Ahlm C, Settergren B, Wge A, Trnvik A. Elevted plsm levels of tumor necrosis fctor (TNF)-, soluble TNF receptors, interleukin (IL)-6, nd IL-0 in ptients with hemorrhgic fever with renl syndrome. J Infect Dis 996; 73: Khiboullin SF, Netski DM, Krumpe P, St Jeor SC. Effects of tumor necrosis fctor lph on Sin Nombre virus infection in vitro. J Virol 2000; 74: Kim HK, Prk HR, Sul KH, Chung HY, Chung J. Induction of RANTES nd CCR5 through NF-kB ctivtion vi MAPK pthwy in ged rt gingivl tissues. Biotechnol Lett 2006; 28: Xio W, Hodge DR, Wng L, Yng X, Zhng X, Frrr WL. Co-opertive functions between nucler fctors NFkB nd CCAT/enhncer-binding protein-bet (C/EBP-bet) regulte the IL-6 promoter in utocrine humn prostte cncer cells. Prostte 2004; 6: Tub DD, Lloyd AR, Conlon K, et l. Recombinnt humn interferon-inducible protein 0 is chemottrctnt for humn monocytes nd T lymphocytes nd promotes T cell dhesion to endothelil cells. J Exp Med 993; 77: Wiedermnn CJ, Kowld E, Reinisch N, et l. Monocyte hptotxis induced by the RANTES chemokine. Curr Biol 993; 3: Xu LL, Wrren MK, Rose WL, Gong W, Wng JM. Humn recombinnt monocyte chemotctic protein nd other C-C chemokines bind nd induce directionl migrtion of dendritic cells in vitro. J Leukoc Biol 996; 60: Chpmn LE, Mertz GJ, Peters CJ, et l. Intrvenous ribvirin for hntvirus pulmonry syndrome: sfety nd tolernce during yer of open-lbel experience. Antivir Ther 999; 4: Loppnow H, Libby P. Adult humn vsculr endothelil cells express the IL6 gene differentilly in response to LPS or IL. Cell Immunol 989; 22: Hutchins D, Steel CM. Regultion of ICAM- (CD54) expression in humn brest cncer cell lines by interleukin 6 nd fibroblst-derived fctors. Int J Cncer 994; 58: Rmdori G, Christ B. Cytokines nd the heptic cutephse response. Semin Liver Dis 999; 9: Legrnd-Poels S, Schoonbroodt S, Piette J. Regultion of interleukin-6 gene expression by pro-inflmmtory cytokines in colon cncer cell line. Biochem J 2000; 349: Klingström J, Plyusnin A, Vheri A, Lundkvist A. Wild-type Puuml hntvirus infection induces cytokines, C-rective protein, cretinine, nd nitric oxide in cynomolgus mcques. J Virol 2002; 76: Bonville CA, Eston AJ, Rosenberg HF, Domchowske JB. Altered pthogenesis of severe pneumovirus infection in response to combined ntivirl nd specific immunomodultory gents. J Virol 2003; 77: Bonville CA, Lu VK, DeLeon JM, et l. Functionl ntgonism of chemokine receptor CCR reduces mortlity in cute pneumovirus infection in vivo. J Virol 2004; 78: Accepted October 202; published online 8 Jnury Interntionl Medicl Press