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Antiviral Research 90 2011 22 32 Contents lists available at ScienceDirect Antiviral Research journal homepage Inhibition of severe acute respiratory syndrome SARS CoV BALB c mouse model by stinging Yohichi Nathan Donald a Old b CA 94118 c CA d e Department of Microbiology The Chinese University of Hong Kong Shatin Hong Kong China article info Article Received Received Accepted Available online 19 February 2011 Keywords BALB c SARS CoV Urtica abstract 1 the severe Ksiazek originated 0166 3542 doi history 16 August 2010 in revised form 9 February 2011 10 February 2011 mouse dioica agglutinin UDA Urtica dioica agglutinin UDA is a small plant monomeric lectin 8 7 kDa in size with an N acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro In the current study we first examined the efficacy of UDA on the replication of different SARS CoV strains in Vero 76 cells UDA inhib ited virus replication in a dose dependent manner and reduced virus yields of the Urbani strain by 90 at 1 1 0 4H9262g ml in Vero 76 cells Then UDA was tested for efficacy in a lethal SARS CoV infected BALB c mouse model BALB c mice were infected with two LD 50 575 PFU of virus for 4 h before the mice were treated intraperitoneally with UDA at 20 10 5 or 0 mg kg day for 4 days Treatment with UDA at 5 mg kg significantly protected the mice against a lethal infection with mouse adapted SARS CoV p 0 001 but did not significantly reduce virus lung titers All virus infected mice receiving UDA treatments were also significantly protected against weight loss p 0 001 UDA also effectively reduced lung pathology scores At day 6 after virus exposure all groups of mice receiving UDA had much lower lung weights than did the placebo treated mice Thus our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss Furthermore the mode of action of UDA in vitro was further investigated using live SARS CoV Urbani strain virus and retroviral par ticles pseudotyped with SARS CoV spike S UDA specifically inhibited the replication of live SARS CoV or SARS CoV pseudotyped virus when added just before but not after adsorption These data suggested that UDA likely inhibits SARS CoV infection by targeting early stages of the replication cycle namely adsorp tion or penetration In addition we demonstrated that UDA neutralizes the virus infectivity presumably by binding to the SARS CoV spike S glycoprotein Finally the target molecule for the inhibition of virus replication was partially characterized When UDA was exposed to N acetylglucosamine and then UDA was added to cells just prior to adsorption UDA did not inhibit the virus infection These data support the conclusion that UDA might bind to N acetylglucosamine like residues present on the glycosylated envelope glycoproteins thereby preventing virus attachment to cells 2011 Elsevier B V All rights reserved Introduction Severe acute respiratory syndrome coronavirus SARS CoV is causative agent of an emerging human infectious disease acute respiratory syndrome SARS Drosten et al 2003 et al 2003 Peiris et al 2003b Rota et al 2003 SARS in Southern China at the end of 2002 and was character Corresponding author E mail address dale barnard usu edu D L Barnard ized by high mortality and morbidity in infected individuals By July 31 2003 more than 8000 SARS cases and nearly 800 SARS related deaths were reported around the world Studies on the molecu lar evolution of SARS CoV suggest that the virus emerged from non human sources Guan et al 2003 The civet cat coronavirus was shown to have a sequence identity of more than 99 to the SARS CoV with only a limited number of deletions and mutations between both viruses SARS CoV was shown to have a deletion of 29 nucleotides relative to the civet cat virus possibly suggesting that there was direct transmission from civet to humans Guan et al 2003 Virustransmissiontohumansmayhaveoccurredwhencivet see front matter 2011 Elsevier B V All rights reserved 10 1016 j antiviral 2011 02 003 Kumaki a Miles K Wandersee a Aaron J Smith M Nelson a Kevin W Bailey a Zachary G Vest F Smee a Dale L Barnard a Institute for Antiviral Research Department of Animal Dairy and Veterinary Science 5600 Blood Systems Research Institute University of California San Francisco San Francisco Department of Laboratory Medicine University of California San Francisco San Francisco Department of Biology 5305 Old Main Hill Utah State University Logan UT 84322 USA coronavirus replication in a lethal nettle lectin Urtica dioica agglutinin a Yanchen Zhou b c Graham Simmons b c a Joseph K K Li d Paul Kay Sheung Chan e Main Hill Utah State University Logan UT 84322 USA USA 94118 USA Y Kumaki et al Antiviral Research 90 2011 22 32 23 cats probably infected by bats were traded on Chinese wet markets Lau et al 2005 Li et al 2005b Infections caused by SARS CoV pose a serious threat to the human population and represent a challenge for antiviral drug development and administration Groneberg et al 2003 2004 because there are no proven or approved efficacious agents to treat SARS CoV infection Ribavirin in combination with corticosteroids was the most frequently administered antiviral agent during the SARS Tsang nontoxic against ical administration and support In to et is emerge vitro been derived have ence et essential target and interference some Schaffer first we humans in interferon ds shown mice feron epithelial small specificity has selectivity that furt resulting specific ited at against are and was A infected was 2009 mean animals p 100H9262M in an SI 76 9 In addition another lectin the mannose lectin Hippeastrum hybrid agglutinin HHA likely inhib SARS CoV attachment to the cells or acted to inhibit the virus the end of the infectious virus cycle Keyaerts et al 2007 To evaluate the prophylactic potential of antivirals directed SARS CoV infection new lethal animal models for SARS needed to facilitate antiviral research We previously adapted characterized a new strain of SARS CoV strain v2163 that highly lethal in 5 6 week old BALB c mice Day et al 2009 number of compounds were tested for efficacy in SARS CoV BALB c mice including UDA We earlier reported that UDA partially protective in SARS CoV infected mice Day et al UDA at 5 mg kg day was shown to significantly delay the day of death compared to the average onset of death for receiving physiological sterile saline PSS 6 2 1 7 days esis in mice infected with the mouse adapted SARS CoV Further more a higher concentration of UDA was tested to determine its efficacy in reducing lethality and ameliorating the pathogenesis in mice infected with a lethal dose of mouse adapted SARS CoV Our data showed that increasing the concentration of UDA treatment to 15 mg kg day did not increase the efficacy of UDA compared to UDA used at 5 mg kg day Because of the positive results obtained from in vitro assays and the initial studies showing the efficacy of UDA treatmentinsignificantlyreducingmortalityinthelethalSARS CoV mousemodel UDAwasfurtherevaluatedinthelethalmousemodel for SARS CoV to see if efficacy could be improved We optimized the dosage regimen to increase the effectiveness of UDA against SARS CoV in BALB c mouse model in terms of survival We also further investigated the mode of action of UDA in vitro 2 Materials and methods 2 1 Cells Vero 76 cells which were obtained from American Type Culture Collection ATCC Manassas VA were routinely grown in minimal essential medium MEM supplemented with 10 heat inactivated fetalbovineserum FBS ThermoFisherScientificCo Logan UT For invitroantiviralassays theserumwasreducedto2 inVero76cells and gentamicin was added to the medium at a final concentration of 50H9262g ml The human primary embryonic kidney cells 293T which express ACE2 293T ACE2 were obtained from the ATCC and were cultured in Dulbecco s modified Eagle s medium DMEM supplemented with 10 heat inactivated FBS 2 2 Viruses SARS CoV strain Urbani 200300592 was obtained from the Centers for Disease Control CDC Atlanta GA USA The Frankfurt strain was kindly provided by Jindrich Cinatl Klinikum der J W Goethe Universitat Frankfurt Am Main Germany The Toronto 2 strain was supplied by Heinz Feldman National Microbiology Laboratory Winnipeg Manitoba Canada The CHUK W1 strain was received from Paul K S Chan The Chinese University of Hong Kong China All strains were propagated and titrated in Vero 76 cells Personnel entering the facility wore powered air purifying respirators 3M HEPA Air Mate 3M Saint Paul MN and full body protection Tyvek suits 2 3 Development of mouse adapted virus To adapt the human clinical isolate strain Urbani to mice mice were infected intranasally with Urbani strain using a 1 5 dilution of cell cultured amplified virus Three or five days after infection the lungs were removed and homogenized and then a 1 5 dilution of the clarified lung homogenate was used to re infect a subsequent group of mice This infection step continued for 25 times through BALB c mice lungs The virus was then plaque purified 3 times and yielded a virus causing severe lung disease and mortality in infected mice The virus was verified as SARS CoV by ELISA and PCR Day et al 2009 In the mouse model animals infected with three LD 50 of virus die between day 4 and day 8 with 90 100 mortality achieved by day 8 The lungs are severely inflamed and exhibit 24 Y Kumaki et al Antiviral Research 90 2011 22 32 extreme lung histopathology Weight loss is excessive 25 of the total initial body weight The occasional surviving animal may lose 25 or more weight but seems to regain the weight by day 9 or day 10 and lives for at least 21 days or more Virus titers in the lungs often exceed 10 7 ml with the titers peaking at days 3 4 Virus lung titers persist at least until day 7 in mice that survive that long 2 4 Plasmid human 2005 3 of clone luciferase as 2 5 dioica San centration was in interferon were IFN alfacon final was Acetyl Sigma Aldrich 2 6 in replication plates near confluent next to in plates wells Each CPE were per replication control compound defined ing values uninfected and putative show estimated calculated assay NR 2 7 Neutral red NR uptake assay for determination of antiviral efficacy and cytotoxicity of test compound This assay was done for each CPE inhibition test plate described above to verify the inhibitory activity and the cytotoxicity detected by visual observation In our experience the usual correlation between visual and neutral red NR uptake assays in our hands has been greater than 95 The neutral red NR uptake assay was Plasmids encoding spike S glycoprotein from SARS CoV and ACE2 have been described previously Simmons et al 2004 and provided by the Simmon s laboratory Plasmid pNL4 Luc R E pNL luc encodes a replication incompetent variant the human immunodeficiency virus type 1 HIV 1 molecular NL4 3 in which the nef gene has been replaced by a firefly luc reporter andtheenvandvprgeneswereinactivated previously described Connor et al 1995 Compounds The N acetylglucosamine specific stinging nettle lectin U agglutinin UDA was purchased from EY Laboratories Inc Mateo CA Stock solutions of UDA were prepared at a con of 5 mg ml in distilled water and stored at 20 C UDA subsequently diluted in MEM for in vitro experiments For vivo studies UDA was prepared in PSS solution Stock solutions of alfacon 1 IFN alfacon 1 InterMune Inc Brisbane CA provided at a concentration of 30H9262g ml and stored at 20 C 1 was solubilized in MEM for in vitro experiments at a concentration of 3 0 ng ml The interferon inducer poly IC LC obtained from Ribopharm Corporation Bethesda MD N d glucosamine was obtained from the Sigma Aldrich Group St Louis MO Cytopathic effect CPE inhibition assay A modified protocol of Barnard et al 2004a was used for the vitro evaluation of antiviral efficacy of the inhibitors of SARS CoV Vero 76 cells were seeded onto 96 well tissue culture Test compound and virus were added in equal volumes to cell monolayers in 96 well tissue culture plates the day The multiplicity of infection MOI used ranged from 0 01 0 025 in order to produce complete virus cytopathic effects CPE 100 of the cells in the virus control wells within 3 4 days The were incubated at 37 C until the cells in the virus control showed complete viral CPE as observed by light microscopy concentration of drug was assayed for virus inhibition of viral in triplicate and for cytotoxicity in duplicate Six wells per plate set aside as uninfected untreated cell controls and six wells plate received virus only and represented controls for virus IFN alfacon 1 infergen TM was titrated as the positive drug for each set of compounds tested Morphological changes resulting from cytotoxicity of test or virus CPE were graded on a scale of 0 5 with 5 as the appearance of complete cytotoxicity or CPE involv the entire monolayer as observed by light microscopy The obtained were then converted to percentages of untreated controls The 50 cell cytotoxic concentrations CC 50 50 virus inhibitory concentrations IC 50 representing the concentration at which 50 of the monolayers would compound cytotoxicity or virus CPE respectively were by regression analysis A selectivity index SI was using the formula SI CC 50 IC 50 The activity in the CPE was then verified spectrophotometrically by neutral red uptake assay on the same plate performed using a modified method of Cavanaugh et al 1990 as described by Barnard et al 2004b Briefly medium was removed from each well of a plate 0 011 neutral red NR was added to each well of the plate and the plate was incubated for 2 h at 37 Cinthe dark The neutral red NR solution was removed from the wells the wells were rinsed and any remaining dye was extracted using S renson scitratebufferedethanol Absorbancesat540 nm 405 nm were read with a microplate reader Opsys MR TM Dynex Tech nologies Chantilly VA Absorbance values were expressed as percentages of untreated controls and IC 50 CC 50 and SI values were calculated as described above 2 8 Virus yield reduction assay Virus yield reduction assay was used to confirm the results of the CPE inhibition NR uptake assays Infectious virus yield from the CPE inhibition assay was determined on the supernatant from the test well as previously described Barnard et al 2004b After the CPE was scored as described above each plate was frozen at 80 C and then thawed Sample wells at the concentrations of each test compounds were pooled and titered in Vero 76 cells for infectious virus by CPE assay as previously described by Barnard et al 2004a A 90 reduction in virus yield IC 90 was then calculated by linear regression analysis This value represented a one log 10 inhibition in titer when compared to untreated virus controls 2 9 Pseudotype virus production Pseudotyped viruses were generated by cotransfecting 293T cells with 30H9262gofEnv encoding plasmid and 10H9262g of pNL luc reporter backbone per 10 cm dish in the presence of calcium phos phate Forty hours after transfection the supernatant was filtered through a 0 45 H9262m pore size screen and then purified by ultra centrifugation 28 000 rpminanSW28rotor Beckman CoulterInc Brea CA overa20 sucrosecushionandstoredat 80 Casaliquots Zhou et al 2010 The amount of virus was assessed with a p24 antigen capture enzyme linked immunosorbent assay p24 ELISA Aalto Bio Reagents Ltd Dublin Ireland 2 10 Pseudotype virus assay 293T cells 293T ACE2 were seeded to the 96 well white Nun clon surface tissue culture plates Nalgene Nunc Rochester NY at 1 10 4 cells well and grown overnight For pre treatment assay 293T ACE2 cells were pre incubated with UDA at different concen trations at 37 C for 1 h and infected with SARS CoV pseudotyped virus For post treatment assay 293T ACE2 cells were infected with SARS CoV pseudotyped virus for 1 4 or 24 h and treated with UDA at different concentrations In addition SARS CoV pseudotyped virus was pre incubated with UDA at different concentrations at 37 C for 1 or 0 h before addition to 293T ACE2 cells Then plates were incubated for 2 days at 37 C and firefly luciferase reporter expression was determined with reagents from Promega Madison WI and the percentage of infection calculated For cell viabil ity assay 293T ACE2 cells were seeded and treated with UDA The quantity of the ATP present in metabolically active cells was determined with CellTiter Glo Luminescent Cell Viability Assay Systems Promega Madison WI Y Kumaki et al Antiviral Research 90 2011 22 32 25 2 11 Animals Specific pathogen free female 14 18 g BALB c mice were obtained from Charles River Laboratories Wilmington MA for this study They were maintained on standard mouse chow and tap water ad libitum The BALB c mice were quarantined for 24 h prior to use The animal studies were carried out in an approved bio safety level 3 animal facility 2 12 injection tered were days administered after trolling at twice placebo post CoV day every was Animals humanely nization 2 13 mine per change were nence weighed day 2 14 in of with that plum differences followed ance weights tests 2 15 3 was tissue the virus calculated were detected by ANOVA Pairwise comparisons were made by Newman Keuls post tests 2 16 Statistical analysis of death and survival Mice were weighed in groups prior to treatment and then every day thereafter to determine the average weight change for all ani mals in each treatment group Weights were expressed as group Animal experimental design TheBALB cmicewereanesthetizedwitha0 1 mlintraperitoneal of 20 mg kg of Ketamine and SARS CoV was adminis intranasally i n in a volume of 0 05 ml Groups of 20 mice administered UDA intraperitoneally i p twice a day for 4 bid 5 beginning 4 h after virus exposure Poly IC LC was intranasally at 24 h before virus exposure and 8 h exposure to virus and served as a positive control for con the virus infection Thirty mice were treated i p with PSS 24 h prior to virus exposure and 4 h after virus exposure and then a day for three more days Mice in this group represented the controls Animal deaths were recorded for up to 21 days virus exposure Following intranasal administration of SARS 1LD 90 fivemicefromeachgroupweresacrificedonday3and 6 SARS CoV infected and mock infected mice were weighed day and clinical signs of disease were observed Weight loss also determined in all infected and uninfected groups of mice that lost greater than 30 of their initial body weight were euthanized by CO 2 asphyxiation and the day of eutha was designated as the day of death due to infection Compound toxicity determination For UDA a dose range finding experiment was done to deter the maximum tolerated concentration Three mice were used treatment group Toxicity was evaluated in terms of weight and adverse events Adverse events for which observations made included ruffling of fur lethargy paralysis inconti repetitive circular motion and aggression Mice were also every day from 24 h prior to virus infection to day 11 and 15 post virus exposure Lung score lung weight determinations Samples from each mouse lung lobe were weighed and placed a petri dish Lungs were scored based on surface appearance lungs Lungs were then assigned a score ranging from 0 to 4 0 indicating that the lungs looked normal and 4 denoting the entire surface area of the lung was inflamed and showed colored lung discoloration Sidwell et al 1995 Significant in lung scores were determined by Kruskal Wallis test byDunn spairwisecomparisonposttests Analysisofvari ANOVA was used to determine significant differences in lung Pairwise comparisons were made by Newman Keuls post Lung virus titer determination Lung virus titers were analyzed from mice sacrificed on day and day 6 post virus exposure A lobe from each mouse lung homogenized in MEM supplemented with 10 FBS and the fragments were allowed to settle The varying dilutions of supernatant fluids were assayed in triplicate for infectiou