vero e6 cells were infected with 100 focus - forming units of ebov expressing enhanced green fluorescent protein . after a 1-h incubation , the inoculum was removed and replaced with media ( dulbecco s modified eagle 's medium with 2% fetal bovine serum , penn / strep , l - glutamine ) containing chloroquine ( sigma , st .
the supernatant was collected on days 1 , 3 , 5 , 7 , and 9 after infection and media replaced with fresh drug .
viral rna was extracted from the supernatant and quantified by real - time quantitative reverse transcription pcr as previously described ( 10 ) .
concurrently , cell viability was assayed by using cell titer96 aqueous one solution ( promega , madison , wi , usa ) according to the manufacturer s instructions .
ec was determined by using prism6 ( graphpad software , san diego , ca , usa ) . when added 1 h after infection , chloroquine at 5 g / ml and 25 g / ml reduced the viral loads by 0.61 and 1.07 logs , respectively ( peak reduction observed on day 5 ) , without any significant cytotoxicity ( figure 1 ) .
analysis of the data from day 5 resulted in an ec50 of 1.77 g / ml and an ec90 of 23.34 g / ml , concentrations that are comparable with previous data ( 8) ; however , reductions in viral loads at these concentrations at other time points were negligible .
although concentrations of > 50 g / ml of chloroquine reduced viral loads by 24 logs starting on day 3 , this decrease was accompanied by a high level of cytotoxicity ( > 50% ) that was evident both in the cytotoxicity assay and microscopically resulting in poor selectivity of chloroquine .
viral loads from supernatants derived from vero cells infected with ebola virus expressing enhanced green fluorescent protein and treated with chloroquine at the indicated concentrations ( 0 , 5 , 25 , 50 , 100 , and 200 g / ml ) .
six - week - old balb / c mice or syrian hamsters ( both from harlan , indianapolis , in , usa ) were inoculated intraperitoneally with 100 50% lethal dose of ma ebov .
the mouse ( 11 ) and the hamster ( 12 ) are well - established disease models of ebov infection .
treatment groups ( mice and hamsters ) received 90 mg / kg of chloroquine alone ( intraperitoneally ) .
an additional group of hamsters received 50 mg / kg of chloroquine ( intraperitoneally every 24 h ) in combination with 2.5 mg / kg doxycycline ( gavage every 12 h ) and 50 mg / kg azithromycin ( intraperitoneally every 24 h ) .
after inoculation , animals were monitored at least twice daily and euthanized by using a humane endpoint scoring criteria as approved by the animal care and use committee at rocky mountain laboratories ( hamilton , mt , usa ) .
two of 3 mock - challenged mice did not survive because of chloroquine ( 90 mg / kg ) treatment alone ( figure 2 , panel a ) . only 2 of 9 mice infected with ma ebov and treated with
chloroquine survived , and 1 of 9 mice infected with ma ebov and treated with vehicle survived . with median survival of 7 , 8 , and 8 d for mock - challenged / chloroquine - treated mice , ma ebov  infected / chloroquine - treated mice , and ma ebov  infected / vehicle - treated mice , respectively , treatment had no significant effect on survival .
this dose , although previously stated as the maximum tolerated dose in mice ( 8) , was not well tolerated by the animals in this study and clearly did not improve survival in animals challenged with ma ebov .
survival of ma ebov - inoculated mice ( a ) and hamsters ( b ) treated with cq ( 90 mg / kg ) .
c ) survival of ma ebov  infected hamsters treated with a combination of cq ( 50 mg / kg ) , doxycycline ( 2.5 mg / kg ) , and azithromycin ( 50 mg / kg ) .
combo , combination of chloroquine , doxycycline , and azithromycin ; cq ,  chloroquine ; ebov , ebola virus ; ma , mouse - adapted .
when the same dose ( 90 mg / kg ) of chloroquine was given to hamsters challenged with ma ebov , the study had to be terminated on day 2 after treatment .
nearly all the treated animals , in both the ma ebov and the mock - challenged groups , died of acute toxicity after administration of chloroquine intraperitoneally , typically within 30 min after treatment ( figure 2 , panel b ) . in a separate study , hamsters were treated with chloroquine ( 50 mg / kg ) in combination with doxycycline ( 2.5 mg / kg ) and azithromycin ( 50 mg / kg ) to additionally provide broad - spectrum antimicrobial drug coverage .
reperfusion injury of the gut after ebov disease , which would subsequently result in bacterial sepsis , has been suspected as a possible cause of death .
thus , broad - spectrum antimicrobial drugs were proposed to help in this regard . in this study
, no toxicity was observed in the mock - challenged group as a result of the combination treatment .
however , treatment had no effect on survival ; no combination - treated or vehicle - treated groups survived , and median survival times were comparable ( figure 2 , panel c ) .
despite some activity of chloroquine against ebov in vitro , we observed no benefit to its administration in the mouse and hamster models . in the mouse model ,
a dose of 90 mg / kg resulted in toxicity but did not alter survival ; therefore , higher concentrations of chloroquine in the mouse would not be expected to be possible . in the hamsters ,
this dose was already lethal on its own . in the hamster model at a lower dose ( 50 mg / kg ) combined with doxycycline and azithromycin  which together provide broad - spectrum antimicrobial coverage , in addition to doxycycline having a small antiviral effect against ebov 
previous anecdotal reports of the incidental use of chloroquine in patients with filovirus infections also do not support any benefit from its use ( 13,14 ) .
together , these data suggest that chloroquine is unlikely to provide any protection from ebov infection in humans .
given its in vitro activity against many different viruses and its longstanding use in humans , chloroquine has been put into multiple clinical trials . during dengue virus infection ,
viremia did not decrease ( 15 ) , and chloroquine neither prevented influenza virus infection ( 7 ) nor improved outcome of chikungunya virus infection ( 6 ) despite promising in vitro activity against these viruses .
when taken together with previous findings for other less pathogenic viruses , the clinical use of chloroquine seems unlikely to provide any benefit for either prophylaxis or treatment of ebov .
moreover , chloroquine has a small therapeutic window ; dosing for treatment of acute malaria is 15 mg / kg , and lethality starts at 50 mg / kg .
thus , current preclinical data do not support the continued consideration of chloroquine for use against ebov infections in humans .
 the antimalarial drug chloroquine has been suggested as a treatment for ebola virus infection . 
 chloroquine inhibited virus replication in vitro , but only at cytotoxic concentrations . in mouse and hamster models 
 , treatment did not improve survival . 
 chloroquine is not a promising treatment for ebola . 
 efforts should be directed toward other drug classes . 