viruses : the following h5n1 hpaiv strains were used : 
 chicken / yamaguchi/7/04   and 
 chicken / miyazaki / k11/07 , provided by the 
 national institute of animal health ( niah ; tsukuba , japan ) ; chicken / vn - ht/33/2003 , molly 
 duck / vn - hn/77/07 and chicken / vn - ht/30/10 , provided by the national institute of veterinary 
 research ( hanoi , vietnam ) ; and whooper swan / hokkaido/1/08   and whooper swan / hamanaka/11 , isolated in our laboratory .
the following h5 subtype lpaiv strains were used : avian / japan/11og1083/11 
 ( h5n2 ) and avian / japan/9uo0036/09 ( h5n2 ) , isolated in our laboratory ; chicken / ibaraki/8/05 ( h5n2 ) provided by the niah ; duck / hong kong/820/80 ( h5n3 ) , provided by dr . y. 
 sakoda ( hokkaido university , sapporo , japan ) ; and whistling swan / shimane/499/83 ( h5n3 ) 
 , provided by dr . t. ito ( tottori university , 
 tottori , japan ) .
the viruses were propagated in 10-day - old embryonated chicken eggs . cell culture and virus titration : madin  darby canine kidney ( mdck ) cells 
 were cultured as described elsewhere .
viruses 
 were titrated in mdck cells , and virus titers were quantified as the 50% tissue culture 
 infective dose ( tcid50 ) , as described previously .
hemagglutination test : hemagglutination tests were performed according to 
 the who manual on animal influenza diagnosis and surveillance . immunofluorescence assay ( ifa ) : ifa was conducted as previously described 
 . briefly , virus - infected mdck cells were fixed 
 with acetone for 10 min and then reacted with a monoclonal antibody ( mab ) to iav 
 nucleoprotein ( np ) ( clone aa5h , oxford biotechnology , ltd . , london , u.k . ) .
fitc - conjugated 
 rabbit anti - mouse igg ( rockland immunochemicals inc . , gilbertsville , pa , u.s.a . ) was used as 
 a secondary antibody .
fluorescence was observed using the bz-9000 biorevo fluorescence 
 microscope ( keyence corporation , osaka , japan ) .
plant extracts : the following 11 herbal teas were tested for antiviral 
 activities : hibiscus ( althaea officinalis ) , black tea ( camellia 
 sinensis assamica ) , tencha ( rubus suavissimus ) , rosehip tea 
 ( rosa rugosa ) , burdock tea ( arctium lappa ) , green tea 
 ( camellia sinensis sinensis ) , jasmine tea ( jasminum 
 officinale ) , ginger tea ( zingiber officinale ) , lavender tea 
 ( lavandula spica ) , rose tea ( rosa odorata ) and oak tea 
 ( quercus dentata ) .
two grams of each herbal tea were boiled in 
 100-ml ultrapure water for 1 hr ( crude extract ) . in this study , because 
 the hibiscus extract showed high antiviral effects , it was fractionated ( frhibis ) using a 
 vivaspin centrifugal concentrator filter device with an ultrafiltration membrane having a 
 molecular weight cut - off of 3 kda ( sartorius lab instruments gmbh & co. kg , gttingen , 
 germany ) and freeze - dried to estimate the molecular weight of the effective component .
pre - treatment of h5 viruses with the extracts ( pre - treatment test ) : the h5 
 subtype hpaivs and lpaivs used in this test are shown in tables 13table 1.antiviral effects of the crude extracts of 11 teas on chicken / yamaguchi/7/04 
 ( pre - treatment test)tea extractsinfectivity ( log10 
 tcid50/ml)treatment time after mixing the virus with the 
 extract10 min immediatelytreateduntreated ( log10 reduction)treateduntreated(log10 reduction)hibiscus 1.56.1(4.6)<1.56.2(4.7)black 3.05.5(2.5)3.25.5(2.3)tencha4.76.0(1.3)5.06.0(1.0)rosehip 4.06.5(2.0)5.76.0(0.3)burdock ntnt5.56.2(0.7)green 4.06.0(2.0)6.06.3(0.3)jasmine 6.06.0(0.0)6.06.3(0.3)ginger 5.56.2(0.7)ntntrose 3.06.0(3.0)5.56.3(0.8)lavender 6.06.0(0.0)5.86.3(0.5)oak ntnt5.76.2(0.5)a ) the virus was mixed with the extract ( treated ) or medium ( untreated ) and 
 inoculated into the mdck cells .
b ) in effect , 
 a period of 10 sec was required to start the first dilution of the mixture immediately 
 after mixing .
c ) log10 reduction in titers was calculated as follows : 
 ( extract - untreated virus titers)(extract - treated virus titers ) .
d ) bold figures of 
 log reduction > 2.0 indicate effective reduction ; underlined , bold figures > 3.0 
 indicate highly effective reduction .
e ) nt : not tested .. briefly , the virus was mixed with an equal volume of the crude extract or 
 frhibis at room temperature . immediately or 10
min after mixing , the mixture was serially 
 diluted 10-fold with culture medium and titrated in the mdck cells 5 days post inoculation 
 ( dpi ) , as described above .
in effect , a period of 10 sec was required to start the first 
 dilution of the mixture immediately after mixing .
a ) the virus was mixed with the extract ( treated ) or medium ( untreated ) and 
 inoculated into the mdck cells .
b ) in effect , 
 a period of 10 sec was required to start the first dilution of the mixture immediately 
 after mixing .
c ) log10 reduction in titers was calculated as follows : 
 ( extract - untreated virus titers)(extract - treated virus titers ) .
d ) bold figures of 
 log reduction > 2.0 indicate effective reduction ; underlined , bold figures > 3.0 
 indicate highly effective reduction .
antiviral effect of frhibis on viral replication in mdck cells ( post - treatment 
 test ) : mdck cells were inoculated with 200 tcid50 of 
 chicken / yamaguchi/7/04 and adsorbed at 37c for 1 hr . the inocula were then removed , and the 
 cells were washed three times with phosphate - buffered saline ( pbs , ph 7.4 ) .
the cells were 
 then overlaid with medium containing frhibis ( 2.6 and 5.3 mg / ml ) and 
 incubated for 4 days .
the inoculated cells were observed for the presence of a cytopathic 
 effect ( cpe ) , and ha activities in the culture fluids were tested using a hemagglutination 
 test .
rna was 
 extracted from the cells and culture medium at 3 dpi using an rna extraction kit ( nippon 
 gene co. , ltd . ,
treatment of cells with the extract prior to virus inoculation : mdck cells 
 were treated with the crude hibiscus and rosehip extracts or frhibis at 37c for 1 hr and 
 then washed three times with pbs .
the cells were inoculated with chicken / yamaguchi/7/04 ( 200 
 tcid50 ) and observed for 5 days .
reverse transcription - polymerase chain reaction ( rt - pcr ) : cdna was 
 synthesized from rna , which was obtained during the post - treatment tests using random 
 primers and moloney murine leukemia virus reverse transcriptase ( invitrogen corporation , 
 carlsbad , ca , u.s.a . ) under the following conditions : 25c for 10 min , 37c for 60 min and 
 65c for 10 min . to detect viral matrix ( m ) and np genes
, rt - pcr was performed using specific primers for 
 the m   and np genes .
electron microscopy ( em ) and immunogold em : whistling swan / shimane/499/83 
 ( h5n3 ) was purified by ultracentrifugation in 30% and 60% sucrose solution   and mixed with frhibis at room temperature for 10 
 min .
the treated viruses were loaded on a 400-mesh carbon - coated collodion grid and 
 negatively stained with 2% phosphotungstic acid ( pta , ph 6.4 ) . for immunogold em , a grid with frhibis - treated or -untreated viruses , as described above , 
 was blocked for 30 min with 1% bovine serum albumin in pbs and then incubated with anti - h5 
 ha mab against a / vietnam/1203/04 ( clone 15a3 ; rockland immunochemicals , inc . ) for 30 min . 

the grid then was incubated with 10-nm gold - conjugated goat anti - mouse igg 
 + igm ( h&l ) ( bbi solutions , cardiff , u.k . ) for 1 hr , stained with pta and then observed 
 using a hitachi ht-7700 electron microscope ( hitachi high - technologies corporation , tokyo , 
 japan ) .
cytotoxicity of frhibis on the mdck cells : mdck cells cultured in the 
 presence of frhibis ( 5.3 mg / ml ) at 37c for 4 days were microscopically 
 observed daily , and live and dead cells were counted using a luna - fl dual fluorescence cell 
 counter ( logos biosystems , annandale , va , u.s.a . ) .
briefly , the cells were trypsinized and 
 then stained with acridine orange and propidium iodide solution .
live cells were stained 
 green , and dead cells were stained red ( data not shown ) .
pre - treatment of h5 viruses with crude herbal tea extracts and frhibis 
 ( pre - treatment test ) : hibiscus tea , black tea , rosehip tea , green tea and rose 
 tea reduced the chicken / yamaguchi/7/04 titer by 2.0 log10 ( table 1 ) after 10-min pretreatment , and the hibiscus tea extract 
 showed the strongest antiviral effect ( 4.6 log10 reduction in titer ) . no 
 significant reduction in the titers was observed when the mdck cells were inoculated with 
 the virus immediately after mixing with the extracts of rosehip tea , green tea or rose tea , 
 whereas the extracts of hibiscus tea and black tea reduced titers by 4.7 and 2.3 
 log10 , respectively .
five extracts showing antiviral effects against chicken / yamaguchi/7/04 were examined for 
 antiviral effect against h5n2 and h5n3 lpaivs ( table 
 2table 2.antiviral effects of the crude extracts on h5n2 and h5n3 lpaivs ( pre - treatment 
 test)virus10-min treatment after mixing the virus with the 
 extractlog10 reduction in the 
 titerhibiscus teagreen tearosehip teablack tearose teaavian / japan/9uo0036/094.00.71.82.31.0avian / japan/11og1083/114.00.32.23.71.8chicken / ibaraki/8/054.7ntnt4.52.0whistling swan / shimane/499/834.00.82.01.51.0duck / hong kong/820/803.3ntnt2.00.5a ) the virus was mixed with the extract or medium and inoculated into the mdck cells . 

log10 reduction in titers was 
 calculated as indicated in the legend of table 
 1 .
b ) see the description of d ) in the legend of table 1.c ) nt : not tested . ) . a 10-min treatment with the hibiscus tea
the black tea extract reduced 
 the titers of the two h5n2 strains by 3.7 log10 , whereas the green tea , rosehip 
 tea or rose tea extracts did not produce a significant reduction in the titers .
a ) the virus was mixed with the extract or medium and inoculated into the mdck cells . 

log10 reduction in titers was 
 calculated as indicated in the legend of table 
 1 .
b ) see the description of d ) in the legend of table 1.c ) nt : not tested . as the hibiscus extract among the tested extracts highly inactivated the h5n1 virus 
 ( chicken / yamaguchi/7/04 ) in addition to various lpaiv strains , the antiviral effect of 
 frhibis on various h5n1 hpaiv strains
was examined as well . as shown in table 3table 3.antiviral effects of frhibis on various h5n1 hpaivs ( pre - treatment test)virustreatment time after mixing the virus with the 
 extractlog10 reduction in the 
 titer10 minimmediatelychicken / yamaguchi/7/045.05.0whooper swan / hokkaido/1/085.03.0chicken / miyazaki / k11/076.37.0whooper swan / hamanaka/115.03.8chicken / vn - ht/33/20035.04.7molly duck / vn - hn/77/075.22.7chicken / vn - ht/30/105.02.0a ) the virus was mixed with frhibis ( treated ) or medium ( untreated ) and inoculated 
 into the mdck cells .
b ) log10 
 reduction in titers as indicated in the legend of table 1 .
c ) in effect , a period of 10 sec was required to start the first 
 dilution of the mixture immediately after mixing .
d ) see the description of d ) in the 
 legend of table 1 .
, the titers of all strains pretreated with frhibis for 10 min were reduced by 
 5.0 log10 .
reduction in the titers by 3.0 log10 was observed for all 
 h5n1 strains , except the vietnamese strains ( molly duck / vn - hn/77/07 and 
 chicken / vn - ht/30/10 ) , when the mdck cells were inoculated with the virus immediately after 
 mixing with frhibis . however , 3-min pre - treatment reduced the titers of the vietnamese 
 strains by 5.0 log10 ( data not shown ) .
a ) the virus was mixed with frhibis ( treated ) or medium ( untreated ) and inoculated 
 into the mdck cells .
b ) log10 
 reduction in titers as indicated in the legend of table 1 .
c ) in effect , a period of 10 sec was required to start the first 
 dilution of the mixture immediately after mixing .
d ) see the description of d ) in the 
 legend of table 1 .
em and immunogold em : abnormal morphologies , such as swelling and 
 deformity , were observed in the viral particles following 10-min pre - treatment with frhibis 
 ( fig .
purified h5n3 viruses pretreated with frhibis for 10 min 
 were incubated with anti - h5 mab followed by incubation with 10-nm gold - conjugated 
 goat anti - mouse igg + igm .
purified h5n3 viruses pretreated with frhibis for 10 min 
 were incubated with anti - h5 mab followed by incubation with 10-nm gold - conjugated 
 goat anti - mouse igg + igm .
immunogold em revealed gold particles on the surface of the untreated viral particles , 
 indicating that anti - h5 ha mab bound to the ha proteins , whereas no particles were found on 
 the surface of the deformed viral particles pretreated with frhibis ( fig .
inhibition of viral replication in the h5n1 virus - infected cells cultured in the 
 presence of frhibis ( post - treatment test ) : no cpe was observed with 
 5.3-mg / ml frhibis , and the hemagglutination test was negative for the 
 culture fluids of the inoculated cells ( table 
 4table 4.detection of cpe and ha activity in the mdck cells cultured in the presence of 
 frhibis after inoculation with chicken / yamaguchi/7/04 ( post - treatment test)concentration of frhibiscpe and ha titerdays post inoculation12345.3 mg / mlcpeha
titer<1:2nt<1:2<1:22.6 mg / mlcpe++++ha titer<1:2nt1:321:32nonecpe++++ha titer<1:2nt1:321:64a ) ha titers in the culture fluids of the infected cells were determined .
in addition , no viral antigens were detected in the virus - inoculated mdck 
 cells in the presence of 5.3-mg / ml frhibis when examined at 2 dpi ( fig .
2.ifa test to detect viral np antigens in mdck cells infected with h5n1 and cultured in 
 the presence of frhibis .
cells inoculated with chicken / yamaguchi/7/04 were cultured with medium containing 
 5.3-mg / ml frhibis for 48 hr .
3.rt-pcr assay to detect viral np genes in mdck cells inoculated with h5n1 and cultured 
 in the presence of frhibis ( 5.3 mg / ml ) for 72 hr .
m : dna mw standard 
 marker ( phy marker : takara , otsu , japan ) .
ec : cells were 
 cultured with medium containing frhibis . ) and m genes were not amplified in the virus - inoculated cells or the culture fluid in 
 the presence of 5.3-mg / ml frhibis at 3 dpi .
ifa test to detect viral np antigens in mdck cells infected with h5n1 and cultured in 
 the presence of frhibis .
cells inoculated with chicken / yamaguchi/7/04 were cultured with medium containing 
 5.3-mg / ml frhibis for 48 hr .
the inoculated cells were cultured with medium . rt - pcr assay to detect viral np genes in mdck cells inoculated with h5n1 and cultured 
 in the presence of frhibis ( 5.3 mg / ml ) for 72 hr .
m : dna mw standard 
 marker ( phy marker : takara , otsu , japan ) .
treatment of cells with the extract prior to virus inoculation : 
 chicken / yamaguchi/7/04 did not replicate in mdck cells pretreated with the crude hibiscus 
 extract ; however , it replicated in those treated with rosehip crude extract ( table 5table 5.inhibitory effects of the pre - treatment of the mdck cells with crude hibiscus 
 and rosehip extracts and frhibiscell treatment withinfectivity ( log10 
 tcid50/ml)treated cells non - treated cells ( log10 reduction)crude extracthibiscus tea1.56.7(5.2)rosehip tea5.26.7(1.5)frhibis4.56.5(2.0)a ) mdck cells were pretreated with the crude extracts or frhibis for 1 hr and then 
 inoculated with the virus .
b ) the virus titers were measured at 5 dpi.c ) 
 log10 reduction in titers was calculated as indicated in the legend of 
 table 1 .
d ) see the description of d ) 
 in the legend of table 1 . ) .
although the virus titers were reduced by 2.0 log10 
 in cells pretreated with frhibis , no significant reduction was observed , in contrast to that 
 observed in cells pretreated with the crude hibiscus extract .
a ) mdck cells were pretreated with the crude extracts or frhibis for 1 hr and then 
 inoculated with the virus .
b ) the virus titers were measured at 5 dpi.c ) 
 log10 reduction in titers was calculated as indicated in the legend of 
 table 1 .
d ) see the description of d ) 
 in the legend of table 1 .
cytotoxicity of frhibis on the mdck cells : no cytotoxicity was observed 
 when cells were cultured for 4 days in the presence of frhibis ( 5.3 mg / ml ) . 

cell viability was 92% , and the cells were successfully passaged ( data not shown ) .
several studies have been conducted to discover potential anti - influenza agents against 
 h5n1 hpaiv derived from plants .
the extracts of cistus incanus , echinacea purpurea , eugenia jambolana   and pomegranate ( punica granatum l. ) 
  showed only direct antiviral effects against 
 viral particles .
elderberry ( sambucus nigra l. ) extracts reduced the number 
 of foci in infected mdck cells ; however , the effect was limited .
thai herbal plant extracts ( terminalia bellirica , salacia 
 chinensis , zingiber montanum and peltophorum pterocarpum ) 
 moderately reduced viral infectivity in mdck cells simultaneously inoculated with the 
 extract and virus .
thus , not all plants exhibited high antiviral 
 effects against h5n1 hpaiv . however , our study indicated that in addition to the crude 
 hibiscus extract , the fractionated component ( frhibis ) , which has a low molecular weight 
 ( <3 kda ) , also exhibited strong antiviral effects against h5n1 hpaiv strains , 
 irrespective of origin ( table 3 ) .
although 
 frhibis was somewhat less effective against the vietnamese h5n1 strains in a very short 
 treatment time than against the other h5n1 strains , the complete inactivation of the 
 vietnamese strains was achieved within 3 min of exposure to frhibis ( data not shown ) .
this 
 finding suggested that the antiviral effects of frhibis do not significantly differ among 
 the h5n1 strains .
the influenza virus first binds to glycoprotein receptors containing sialic acid on the 
 cell surface via ha and then enters the cell by endocytosis . in the cells treated with frhibis before the viral infection
, the cells 
 allowed the virus to enter ( table 5 ) , indicating 
 no adverse effects of hibiscus on the receptors .
unpublished data indicated that the viruses 
 pretreated with hibiscus lose the ability to agglutinate chicken red blood cells ( data not 
 shown ) .
therefore , the results of the antiviral effects of hibiscus observed in the 
 pre - treatment tests strongly suggested that direct contact between the virus and hibiscus is 
 required to abolish the entry of treated viruses into the cells .
this interpretation was 
 supported by the observations made by em and immunogold em , indicating that the antiviral 
 effect was due to a direct effect against the viral particles as frhibis caused 
 morphological changes to the virus and dysfunction of the viral surface ( ha ) proteins , 
 indicated by the lack of binding of the anti - h5 ha antibody ( fig .
although either the viral envelope or surface proteins seem 
 to be targeted by hibiscus , further studies are needed to clarify the mechanism underlying 
 this antiviral effect in more detail , for example , identifying the target molecules of 
 hibiscus . in the post - treatment test , no viral replication was detected in the mdck cells infected 
 with chicken / yamaguchi/7/04 in the presence of frhibis ( table 4 ) .
in addition , no viral antigens or viral gene expressions were detected 
 ( figs . 2 and 3 ) .
thus , our findings suggest that 
 viral replication may be inhibited at an early stage by the small molecular weight 
 components contained in frhibis , although frhibis did not inhibit viral binding to the cell 
 membrane receptors ( table 
 5 ) . however , the crude hibiscus extract inhibited viral binding to 
 the receptors , which may have been affected by other components contained in the crude 
 extract .
in contrast to our results , hibiscus extract prepared using hexane displayed no antiviral 
 effect against h5n1 hpaiv .
although the reason 
 for this discrepancy between these two studies remains unclear , we believe that the 
 different extraction methods may have influenced the results .
hayashi et al .   reported the anti - human influenza virus activity of a red - fleshed 
 potato anthocyanin .
however , they speculated that its activity was derived from an additive 
 or synergistic effect with other anthocyanin pigments and/or other coexisting pigments . 

therefore , further studies should be conducted to identify the effective components 
 contained in hibiscus and to elucidate potential antiviral mechanisms in more detail .
our preliminary study showed that , in addition to the h5 subtype , hibiscus inactivated 
 seven other subtypes ( data not shown ) , whereas p. sidoides extracts 
 inactivated human influenza viruses ( h1n1 and h3n2 ) but not h5n1 hpaiv .
thus , hibiscus may be a promising candidate as a 
 potent anti - influenza drug , irrespective of subtype .
 viral neuraminidase inhibitors are widely used as synthetic anti - influenza drugs for the 
 prevention and treatment of influenza . however , drug - resistant influenza a virus variants , 
 including h5n1 highly pathogenic avian influenza viruses ( hpaivs ) , have been reported . 
 
 therefore , the discovery of novel and effective antiviral agents is warranted . 
 we screened 
 the antiviral effects of 11 herbal tea extracts ( hibiscus , black tea , tencha , rosehip tea , 
 burdock tea , green tea , jasmine tea , ginger tea , lavender tea , rose tea and oak tea ) 
 against the h5n1 hpaiv in vitro . among the tested extracts , only the 
 hibiscus extract and its fractionated extract ( frhibis ) highly and rapidly reduced the 
 titers of all h5 hpaivs and low pathogenic aivs ( lpaivs ) used in the pre - treatment tests 
 of madin  darby canine kidney ( mdck ) cells that were inoculated with a mixture of the virus 
 and the extract . 
 immunogold electron microscopy showed that anti - h5 monoclonal antibodies 
 could not bind to the deformed h5 virus particles pretreated with frhibis . in 
 post - treatment tests of mdck cells cultured in the presence of frhibis after infection 
 with h5n1 hpaiv , 
 the frhibis inhibited viral replication and the expression of viral 
 antigens and genes . among the plants tested , hibiscus showed the most prominent antiviral 
 effects against both h5 hpaiv and lpaiv 
 . 
MATERIALS AND METHODS
RESULTS
DISCUSSION