preparation of primary goat testis cells : one two - week old lamb ( nubian 
 breed ) was sacrificed , and tissue including his testis and ears was removed . the use and 
 experimental protocols
were approved by the committee on the ethics of animal experiments of 
 national chung hsing university ( approval numbers : 101 - 40 ) .
the testis cells derived from 
 removed testis and fibroblasts from ears were cultured in 1 rpmi 1640 medium with 10% fetal bovine serum ( fbs ) .
when cells grew to 
 full confluency , cells were trypsinized and subcultured in 1:1 or 1:2 ratios . virus inoculation and adaptation to cell culture : samples were collected 
 from orfv affected animals in central taiwan ( hoping ) in 2009 .
half gram of collected scab 
 from mouth of infected goats was soaked and homogenized in 5 ml te buffer 
 ( 50 mm tris - hcl and 10 mm edta , ph 8.0 ) with additional antibiotic .
after centrifugation at 
 3,500 rpm for 10 min , the supernatant was transferred , filtrated through a 0.45 
 m filter and stored as a crude viral stock .
the homogenate was 
 subsequently inoculated into the primary goat testis cells . during viral absorption ,
after 1 hr , the unattached virus 
 was removed by washing the cells twice with pre - warmed 1 pbs , and the cells were maintained 
 in complete medium 1 rpmi 1640 with 2% fbs .
if no cytopathy was found , another run of blind passage and the detection of orf viral 
 dna were conducted .
then , 
 10 microliter of cell lysate was incubated with 4-fold volume of quickextract solution ( dna 
 extraction soln .
1.0 , epicentre , madison , wi , u.s.a . ) at 65c for 6 min and then 98c for 2 
 min , followed by detection of viral dna by pcr ( the method was described in the following 
 section ) .
the pcr - positive cellular lysates were sonicated for three cycles ( 10 sec each 
 cycle at 4c ) .
subsequently , the lysate was added into fresh primary testis cells for 
 further incubation for 7 days , and cytopathy was examined by microscopy . continued passages 
 were repeated until viral cytopathy was obvious .
detection of viral dna by nested pcr : during blind passages , the presence 
 of viral dna was examined by the nested pcr using 2 sets of b2l gene specific primers : outer 
 primer set ovb2lf1 and ovb2lr1 , and inner primer 
 set ovb2lf2 and ovb2lr2 ( sequences are listed in table 1table 1.list of the primersprimersequenceovb2lf15  tccctgaagccctattatttttgtg  3ovb2lr15  gcttgcgggcgttcggaccttc  3ovb2lf25  gcagcttctgctgcaacctgag  3ovb2lr25  aaggcgtggtagcggtagtg  3fp5  gtgttgatcatcgaagactcggtg  3rp15  gtcgcccttgtcgcccttagtctc  3rp25
the expected size of pcr 
 products obtained from the first and second round amplification is 1.2 kbp ( ovb2lf1 and 
 ovb2lr1 ; full length b2l gene ) and 889 bp ( ovb2lf2 and ovb2lr2 ) , respectively . in the first 
 round of nested pcr , the pcr condition started with a denaturation step at 94c for 4 min , 
 followed by 29 cycles of 94c for 1 min ( denaturation ) , 55c for 1 min ( annealing ) and 72c 
 for 1 min ( extension ) , and the reaction was ended by a final extension at 72c for 7 min . 

one microliter of the first round pcr product was used for the next round of pcr which was 
 followed the same conditions , except with 25 amplification cycles .
pcr products were 
 analyzed by 1% agarose gel electrophoresis with health safe nucleic acid stain . for 
 detection of viral gene during the plaque purification , the pcr was conducted using the 
 inner set of b2l - specific primers ( ovb2lf2 and ovb2lr2 ) following the same pcr condition 
 with a 35 amplification cycles .
pcr for strain classification of isolated orfv : after three runs of plaque 
 purification , the picked plaques were selected for identification of viral strains by 
 single - step pcr . viral dna for pcr was prepared by 
 the dna extraction soln .
pcr was conducted with three primers ( fp , rp1 and 
 rp2 ; table 1 ) targeting the c - terminus of the 
 atpase gene .
the pcr program proceeded with an initial denaturation at 94c for 5 min , 35 
 cycles of brief denaturation at 94c for 30 sec , annealing at 63c for 30 sec , extension at 
 72c for 30 sec and a final extension at 72c for 7 min .
restriction enzymes digestion : the primary goat testis cells were seeded 
 on the 10-cm cell culture dish ( approximate 2  10 cells ) and were infected with 
 0.1 moi ( multiplicity of infection ) of orfv .
the infected cells were trypsinized and 
 collected when 80% infected cells showing cytopathy ( ~24 hr ) .
the total dna was obtained by following a 
 method previously described . for each reaction , 
 5 g of extracted dna was digested with 20 units of a restriction enzyme at 
 37c for 2 hr . the mixture was analyzed by electrophoresis at 70 volts in a 0.7% agarose 
 gel .
examination of viral gene expression by rt - pcr : the primary goat testis 
 cells ( 3.5  10 ) were infected with 10 moi of orfv and incubated at 37c with 
 5% co2 .
total cellular rna was extracted from the infected cells following the 
 instruction of the rneasy mini kit ( qiagen , limburg , netherlands ) . after rna quantification , 

one microgram of rna was treatment with rq1 rnase - free dnase ( promega , madison , wi , u.s.a . ) 
 to eliminate dna contamination .
reverse transcription was conducted with 0.5 
 g of rna , and the cdna was synthesized by superscript iii reverse 
 transcriptase ( invitrogen , waltham , ma , u.s.a . ) .
the condition of pcr was a first denaturation at 94c for 4 min , 
 following with 35 cycles of brief denaturation at 94c for 45 sec , annealing at 60c for 45 
 sec and extension at 72c for 45 sec , and a final extension at 72c for 7 min .
western blot analysis : the primary goat testis cells were infected with 1 
 plaque forming unit ( pfu ) of orfv and maintained with 1  rpmi 1640 medium containing 2% fbs 
 at 37c with 5% co2 . the cellular lysate was prepared by rupturing cells with 
 sample buffer after twice washing with pbs . following boiling at 100c for 6 min , proteins 
 were separated by the sds-15% page and transferred onto a pvdf membrane ( amersham , ge 
 healthcare , buckinghamshire , u.k . ) . after blocking with tbst
( 20 mm tris , 150 mm nacl and 
 0.1% tween 20 , ph 7.6 ) containing 5% skim milk , the membrane was incubated with 1 : 2,000 
 diluted mouse polyclonal anti - ov20.0 antibody generated from mice immunized with purified 
 ov20.0 recombinant protein at 4c overnight .
the secondary antibody , the horseradish peroxidase - conjugated goat anti - mouse antibody 
 ( jackson immunoresearch , suffolk , u.k . ) , was added and incubated for 1 hr at room 
 temperature .
the signal was then developed using an enzyme - linked chemiluminescence system 
 ( ecl , amersham , ge healthcare ) .
electron microscopy : electron microscopy was used to examine the 
 morphology of isolated virus particles .
the cells were maintained at 37c with 5% co2 and 
 observed the cpe formation .
when 80% of the infected cells show severe cpe , the cells and 
 medium were collected by scraping off the attached cells with tip .
after brief sonication on 
 ice , the cell debris was removed by centrifugation . the cell supernatant was transferred to 
 a new tube and processed for negative - strain electron microscopy by staining with 2% 
 phosphotungstic acid ( pta ) .
cytokines expression in orfv - infected thp-1 cells : total 1  
 10 thp-1 cells kindly provided by professor s.s .
chiou in graduate institute 
 of microbiology and public health , national chung hsing university were seed in 6 well 
 multiple plates within 1 ml of rpmi 1640 medium containing 10% fbs . 

subsequently , 10 moi of orfv was inoculated into the cells and incubated with the human 
 monocyte thp-1 cells .
forty - eight hr later , the supernatant was collected by brief 
 centrifugation and transferred to a new tube which was then kept at 80c .
the cytokine 
 expression in the culture medium was examined by using the human il-8 and tumor necrosis 
 factor alpha ( tnf- ) elisa kit ( koma biotech inc .
a set of standard cytokines , provided within the kit , with known 
 concentrations were analyzed in parallel for establishment of standard curve .
finally , the 
 od value of each sample was obtained from reading the plate at 450 nm 
 wavelength .
the mean value of the results was obtained from three independent experiments and was 
 then assessed by t - test .
p - value ( p<0.05 ) indicated the 
 significant difference between two groups of samples .
effect of orfv on subsequent influenza virus infection in cells : goat 
 fibroblast primary cells were infected with orfv at moi of 1 , and the cell media were 
 collected at 0 , 6 , 12 and 24 hr post - infection ( hpi ) .
a549 cells were pre - treated with the 
 orfv infected cell medium for 24 hr .
after removal of the cell medium and pbs washing , the 
 a549 cells were infected with 1 moi of influenza virus ( pr8 strain ) .
cellular lysate of infected human lung carcinoma a549 cells obtained 
 from atcc ( ccl-185 ) was harvested at 12 hpi and resolved by sds - page electrophoresis 
 followed by western blot analysis using igy antibody against influenza virus np generated 
 from chickens immunized with purified np recombinant protein .
the overall procedures of 
 western blot analysis followed the description in the previous section .
densitometric 
 quantifications of each band indicating np expression level were carried out using national 
 institutes of health imagej software version 1.43 ( http://rsb.info.nih.gov/ij ) .
effect of orfv on subsequent influenza virus infection in mice : the 
 inhibitory activity on influenza virus infection by orfv was further tested in mice .
six 
 weeks old , female balb / c mice received uv - inactivated 2  10 plaque formation 
 unit ( pfu ) of isolated orfv , hoping strain ( n=4 ) or pbs ( n=4 , as negative control ) by 
 intramuscular ( i m ) or subcutaneous ( sc ) routes .
two days after infection , the mice were 
 intranasally challenged with 1  10 influenza virus ( pr8 strain ) .
seven days 
 post infection , blood was taken from each mouse for determining the concentrations of il-6 
 and tnf- in serum by elisa kits ( biolegend ; san diego , ca , u.s.a . ) performed following the 
 manufacturer s procedure .
subsequently , the mice were scarified , and the lungs of the mice 
 were collected .
after brief centrifugation , the supernatant was used for measurement of the 
 influenza virus titer on mdck cells .
after two to three days post - infection , the infected 
 mdck cells were fixed in methanol and stained with crystal violet .
viral infection and adaptation to the primary testis cells : the crude 
 virus stock was serially diluted and inoculated into the primary goat testis cell .
cells 
 were observed daily for appearance of cpe , and the presence of viral dna was determined by 
 pcr in each run ( 1 week interval ) .
after 3 - 5 rounds of blind passages , 
 stronger bands were amplified in the second round of nested pcr , although no apparent cpe 
 was observed . in subsequent cycles of blind passages , orfv was adapted and enriched in 
 primary goat testis cells ; viral dna can be detected in the first round of the nested 
 pcr .
plaque purification of the orfv : with continued viral passages , the 
 infected primary goat testis cells began to show the cytopathic effect ( cpe ) and form a 
 viral plaque .
the cpe in primary goat testis cells was local and limited on the area of 
 affected cells after four to five days after infection ( fig .
( a ) the infected primary goat testis cells showed rounding , shrinking and 
 detachment , and eventually formed a viral plaque ( 200  magnification ) .
the isolated 
 plaque ( v ) showed the expected sizes ( ~900 bp , as indicated by the arrow ) of 
 amplified products of partial b2l gene .
the positive control ( + ) was 
 the virus - infected cell lysate in pcr ; the negative control (  ) was 
 done without any dna template .
( c ) identification of isolated 
 orfvs by the single - step pcr . as the pcr amplification yielded two dna fragments 
 with characteristic sizes ( 180 and 254 bp , as indicated by arrows ) , it indicated the 
 isolated orfv is the hoping strain ( chan et al . , 2009 ) .
lane 1 : dna treated 
 with ecor i ; lane 2 : treatment with bamh i ; lane 3 : treatment with hind iii ; lane 4 : 
 treatment with kpn i ; lane 5 is the uninfected cell , and the stained dna is smearing 
 after kpn i digestion .
that is consistent with one previous study that parapoxviruses form 
 plaques in primary bovine testis cells .
after 
 three times of plaque purification , the purity of isolated orfv was examined by pcr using 
 primers targeting viral b2l gene .
1b ) , and subsequent dna automated - sequencing 
 confirmed the nucleotide identity of orfv b2l gene ( data not shown ) .
( a ) the infected primary goat testis cells showed rounding , shrinking and 
 detachment , and eventually formed a viral plaque ( 200  magnification ) .
the isolated 
 plaque ( v ) showed the expected sizes ( ~900 bp , as indicated by the arrow ) of 
 amplified products of partial b2l gene .
the positive control ( + ) was 
 the virus - infected cell lysate in pcr ; the negative control (  ) was 
 done without any dna template .
( c ) identification of isolated 
 orfvs by the single - step pcr . as the pcr amplification yielded two dna fragments 
 with characteristic sizes ( 180 and 254 bp , as indicated by arrows ) , it indicated the 
 isolated orfv is the hoping strain ( chan et al . , 2009 ) .
lane 1 : dna treated 
 with ecor i ; lane 2 : treatment with bamh i ; lane 3 : treatment with hind iii ; lane 4 : 
 treatment with kpn i ; lane 5 is the uninfected cell , and the stained dna is smearing 
 after kpn i digestion .
identification of isolated orfv : a single - step pcr developed in our 
 laboratory that shows distinct amplification patterns of three orfv strains in taiwan was 
 used for identifying the isolated viruses .
the 
 nantou and taiping strain could amplify 180 and 217 bp product , respectively , and 2 
 different length fragments ( 180 and 254 bp ) can be produced at the same time in the primary 
 cells from goats .
the nucleotide sequences of the hoping 
 strain were further confirmed by automated dna sequencing ( data not shown ) .
furthermore , the 
 restriction enzyme digestion pattern of viral dna of the hoping strain was also confirmed 
 ( fig .
in comparison with the smearing dna 
 of uninfected sample ( lane 5 in fig . 

1d ) , all the dna of virus - infected samples treated with restriction 
 enzymes showed characteristic cutting patterns . viral gene expression examined by rt - pcr and immunoblotting : to examine 
 viral gene expression in the primary goat testis cell , the viral rna was detected by rt - pcr . 
 despite the weaker expression ,
the transcripts of b2l can be detected at the early stage of 
 infection ( 23 hpi ) , and it was largely synthesized after 12 hpi ( fig . 2afig .
2.detection of the viral b2l gene expression and viral ov20.0 protein in infected 
 primary goat cells .
( a ) rna was extracted from cells infected with orfv at 0 , 1 , 2 , 3 , 
 12 , 20 and 24 hpi ( lanes 17 ) for reverse transcription ( rt )  pcr detection .
the 
 transcription of b2l appeared at 2 and 3 hr post - infection and then was saliently 
 increased at 12 , 20 and 24 hpi .
+ indicates a positive control in which dna template is derived from virus 
 lysate .
( b ) the expression of ov20.0 ( the ortholog of vaccinia virus e3 protein ) was 
 observed at 6 hr , 12 hr and 24 hr after infection ; the expected molecular weight of 
 ov20.0 is 25 kda indicated by the arrowhead .
electron 
 micrograph of orf viruses ( c ) prepared from primary goat testis cells .
the 
 characteristic morphology of orf virus was observed , and viral particles showed 
 ovoid - shape with a spiral crisscross pattern ( bar=100 nm ) . ) .
moreover , the orfv gene expression was verified by western blotting by using the 
 mouse polyclonal anti - ov20.0 antibody .
2b demonstrated the viral ov20.0 protein ( 25 kda ) was produced after 6 , 12 and 24 
 hpi .
these data indicated that genes of isolated orfv can be actively expressed in the 
 primary goat testis cells .
detection of the viral b2l gene expression and viral ov20.0 protein in infected 
 primary goat cells .
( a ) rna was extracted from cells infected with orfv at 0 , 1 , 2 , 3 , 
 12 , 20 and 24 hpi ( lanes 17 ) for reverse transcription ( rt )  pcr detection .
the 
 transcription of b2l appeared at 2 and 3 hr post - infection and then was saliently 
 increased at 12 , 20 and 24 hpi .
+ indicates a positive control in which dna template is derived from virus 
 lysate .
( b ) the expression of ov20.0 ( the ortholog of vaccinia virus e3 protein ) was 
 observed at 6 hr , 12 hr and 24 hr after infection ; the expected molecular weight of 
 ov20.0 is 25 kda indicated by the arrowhead .
electron 
 micrograph of orf viruses ( c ) prepared from primary goat testis cells .
the 
 characteristic morphology of orf virus was observed , and viral particles showed 
 ovoid - shape with a spiral crisscross pattern ( bar=100 nm ) .
electron micrograph observation : morphological confirmation of orf virions 
 in the infected goat testis cells was achieved with electron microscopy .
the electron 
 micrograph results demonstrated the presence of ovoid - shape virions with a spiral crisscross 
 pattern ( fig . 2c ) .
detection of cytokines produced in thp-1 cells with orfv : the orfv 
 infection elicits expression of proinflammatory cytokines , such as several interleukins 
 ( ils ) and tnf- , which contributes to the immune regulation of orfv and has been 
 demonstrated in many studies [ 8 , 9 , 15 , 26 , 37 ] .
it is important to 
 explore whether orfv ( hoping strain ) harbors the immunostimulating activity .
the human 
 monocyte cell line , thp-1 cell , was infected with our local isolate at moi of 10 for 48 hr . 

the cell medium was collected for detection of il-8 and tnf- cytokine production . as the 
 result shown in fig . 3fig .
3.expression of il-8 and tnf- in orfv treated human monocyte thp-1 cells . to examine 

whether our orfv can affect the cellular cytokine levels , thp1 cells were incubated 
 with 10 moi of orfv for 48 hr .
the cell medium was collected for examination of the 
 cytokine il-8 and tnf- expressions by using the elisa kit ( koma biotech inc . )
the 
 results demonstrated that il-8 and tnf- expression level significantly increased in 
 cells infected with orfv , compared with that of pbs control .
the column of each group 
 was the mean ( + /sd ) of three independent experiments .
statistical analysis was 
 performed using unpaired t - test , and p value < 0.05 ( shown with a 
 star symbol ) indicates the statistical significance .
, compared with a mock control , infection of orfv indeed caused the rise of the il-8 
 and tnf- expressions in thp-1 cells .
expression of il-8 and tnf- in orfv treated human monocyte thp-1 cells . to examine 

whether our orfv can affect the cellular cytokine levels , thp1 cells were incubated 
 with 10 moi of orfv for 48 hr .
the cell medium was collected for examination of the 
 cytokine il-8 and tnf- expressions by using the elisa kit ( koma biotech inc . )
the 
 results demonstrated that il-8 and tnf- expression level significantly increased in 
 cells infected with orfv , compared with that of pbs control .
the column of each group 
 was the mean ( + /sd ) of three independent experiments .
statistical analysis was 
 performed using unpaired t - test , and p value < 0.05 ( shown with a 
 star symbol ) indicates the statistical significance .
orfv inhibited influenza virus replication in a549 cells : as an immune 
 modulator , orfv has been shown to act as an inhibitor to prevent other virus infection and 
 also to work as a tumor killer [ 3 , 16 , 19 , 31 ] .
the orfv infected cell medium , of which the infectivity of orfv was under 
 detection , was collected at 6 , 12 and 24 hpi , and overlaid onto a549 cells .
after 24 hr 
 treatment , the a549 cells were then infected with influenza virus pr8 strain for 12 hr . a 
 significant decrease of viral np protein expression was observed in cells pre - treated with 
 orfv infected cell medium ( fig .
4.pre-treatment of orfv - infected cell medium prevents a549 cells from type a influenza 
 virus ( iav ) infection .
the primary goat testis cells were infected with orfv ( hoping 
 strain ) . at two hr post infection ( hpi ) ,
the cells were maintained in complete rpmi 1640 medium with 10% fbs . at 6 , 
 12 and 24
hpi , the medium was collected and used for treatment of human a549 cells . 
 after 24 hr treatment , the a549 cells were infected with 1 moi of iav ( pr8 strain ) . at 
 12 hpi ,
expression of viral nucleoprotein ( np ) of the iav was analyzed by 
 immunoblotting ( panel a ) , and the quantitative analysis of np production was shown in 
 panel b. the column of each group was the mean ( + /sd ) of three independent 
 experiments .
the p value 
 < 0.05 ( shown with a star symbol ) indicates the difference between 2 groups is 
 statistically significant . ) .
noticeably , increase of inhibitory strength coincided with the length of orfv 
 infection ; the longer orfv infection , the stronger inhibitory effect was observed .
it 
 indicates orfv ( hoping strain ) can prevent influenza virus infection in a549 cells .
pre - treatment of orfv - infected cell medium prevents a549 cells from type a influenza 
 virus ( iav ) infection .
the primary goat testis cells were infected with orfv ( hoping 
 strain ) . at two hr post infection ( hpi ) ,
the cells were maintained in complete rpmi 1640 medium with 10% fbs . at 6 , 
 12 and 24
hpi , the medium was collected and used for treatment of human a549 cells . 
 after 24 hr treatment , the a549 cells were infected with 1 moi of iav ( pr8 strain ) . at 
 12 hpi ,
expression of viral nucleoprotein ( np ) of the iav was analyzed by 
 immunoblotting ( panel a ) , and the quantitative analysis of np production was shown in 
 panel b. the column of each group was the mean ( + /sd ) of three independent 
 experiments .
the p value 
 < 0.05 ( shown with a star symbol ) indicates the difference between 2 groups is 
 statistically significant .
inhibition of subsequent influenza virus replication in mice inoculated with 
 uv - inactivated orfv : to further investigate the inhibitory activity on influenza 
 virus infection by uv - inactivated orfv , 6 weeks old female balb / c mice were inoculated with 
 either 2  10 pfu of uv - inactivated orfv ( n=4 ) or pbs ( n=4 , as a negative 
 control ) by i m and sc routes for 2 days and subsequently infected with influenza virus . as 
 shown in fig . 5afig .
orfv was 
 administrated intra - muscularly ( i m ) or subcutaneously ( sc ) to balb / c mice ( 6 weeks 
 old , n=4 ) at dosage of 2  10 pfu before iav infection .
two days later , all groups of mice received a 
 dosage of 1    10 pfu iav via intra - nasal route .
seven days post infection , all the mice were sacrificed , 
 and their lungs were removed for titration of the virus loads ( a ) , and il-6 and tnf- 
 expressions in blood were evaluated by elisa ( biolegend ) ( b ) . values display the means 
 ( + /sd ) of each group .
p value<0.05 , shown with a star symbol , 
 indicates statistical difference between 2 groups .
, in mice groups received the inactivated orfv via both i m and sc routes , the titer of 
 influenza viruses significantly decreased ( fig . 

the cytokine profile was also determined in mice inoculated with orfv . elevated 
 il-6 and tnf- expressions in mice with the inactivated orfv
were noticed ; however , only 
 increased il-6 was statistically significant ( fig . 

orfv was 
 administrated intra - muscularly ( i m ) or subcutaneously ( sc ) to balb / c mice ( 6 weeks 
 old , n=4 ) at dosage of 2  10 pfu before iav infection .
two days later , all groups of mice received a 
 dosage of 1    10 pfu iav via intra - nasal route .
seven days post infection , all the mice were sacrificed , 
 and their lungs were removed for titration of the virus loads ( a ) , and il-6 and tnf- 
 expressions in blood were evaluated by elisa ( biolegend ) ( b ) .
p value<0.05 , shown with a star symbol , 
 indicates statistical difference between 2 groups .
orfv , an epitheliotropic parapoxvirus , causes proliferative dermatitis in goats and sheep , 
 and persistence infection usually occurs in an outbreak farm .
although molecular 
 identification by pcr and phylogenetic analysis of viral dna purified from the animal 
 tissues are common , the isolation of orfv from the field and production of virions in cell 
 culture are still formidable tasks .
the isolation 
 of parapoxviruses from cows , goats and sheep and serows as well as from ruminant animals 
 ( musk ox and sichuan takin ) of a zoo was claimed [ 13 , 
 14 , 18 , 
 22 , 24 , 
 30 , 36 , 
 38 ] ; two of these studies investigated diseases of 
 goats [ 18 , 30 ] .
so far , the most studied orfv strains are nz2 , ia82 , sa00 and d1701 which were 
 isolated from new zealand , north america and germany [ 10 , 25 , 29 ] . in this work ,
a new orfv strain was isolated from current outbreak in taiwan , 
 and the immunoregulation activity of this asian strain was demonstrated for the first 
 time .
the primary cells from goats , sheep or cattle source have been used to isolate 
 parapoxviruses [ 18 , 22 , 24 , 38 ] , and subsequently , some cell lines were found to be susceptible to orfv 
 infection , for examples , madin - darby ovine kidney ( mdok ) cells and madin - darby bovine kidney 
 ( mdbk ) cells [ 13 , 14 , 23 , 30 ] .
the primary testis cells of goat , the original host of orfv , were used to 
 isolate the virus from the field samples .
after several rounds of plaque purification , our 
 local orfv was purified in homogeneity as indicated by pcr amplifications and by electron 
 microscopy ; the typical ovoid - shaped virus particles with particular surface pattern of orfv 
 were demonstrated . on the basis of pcr genotyping
, we confirmed the resulted purified orf 
 viruses were the hoping strain , one of the three strains of orfv identified in taiwan .
reported that the inactivated orfv particles 
 ( iowa strain ) mediated activities against infections of different microbes and transient 
 induction of certain innate immune mechanisms by orfv administration likely contributing to 
 the immunostimulant effects .
weber et 
 al . showed treatment of orfv ( d1701 strain ) protects mice from acquiring and 
 recurrent of herpes simplex virus type 1 infection .
more recently , the effect of orfv particles on inhibition of viral infection 
 was further demonstrated in different models : paulsen et al .
replication of 
 hepatotropic pathogens , i.e. hepatitis b virus and hepatitis c virus , was significantly 
 reduced in a mouse model or in vitro , respectively ; and such an anti - viral activity can be observed in 2 different viral 
 strains ( d1701 and nz2 ) . moreover ,
d1701 strain , a highly attenuated viral strain with 
 reduced pathogenicity , is the main component of 
 the commercially available immunomodulator .
considering the immunoregulatory effect might be 
 variable between strains with different virulence , and hence , in this study , we set out 
 experiments to explore the effect of hoping strain on subsequent influenza virus infection 
 that poses a severe threat to humans and animals and is considered a major worldwide public 
 health problem .
our results indicated that the production of proinflammatory cytokines , such as il-6 and 
 tnf- , was significantly increased in the sera of mice pre - treated with orfv compared with 
 that in the sera of mice receiving the pbs before iav infection ( fig .
5b ) . tnf- and il-8 were also higher in medium of orfv treated 
 human monocyte thp-1 cells ( fig . 

3 ) that indicates an important role for orfv in modulating production 
 of multiple inflammatory cytokines .
this is in line with previous findings ; tnf- 
 contributes to resolving influenza virus infection in the host respiratory tract , and il-6 promotes viral clearance and reduces 
 mortality to influenza virus infection via enhancing neutrophil survival from influenza 
 virus - induced death .
recently , rohde et al . generated 2 orfv recombinants expressing the 
 hemagglutinin ( ha ) or nucleoprotein ( np ) of the highly pathogenic avian influenza virus 
 ( hpaiv , strain h5n1 ) , and via i m route immunization of two doses ( 10 pfu of 
 each ) of ha - expressing orfv protects mice from lethal hpaiv infection .
the present study showed that without expressing any influenza 
 specific antigen , treatment of orfv significantly inhibited expression of np protein in 
 cells , and also , one dose ( 10 pfu ) of orfv immunization is able to reduce the 
 influenza viral load in mouse lung . based on the cytokine expression profile , it appears 
 orfv infection elevates levels of proinflammatory cytokines ( il-6 or il-8 ) .
however , further 
 studies are required to elucidate the mechanisms of suppressing influenza virus infection . 
 in summary ,
 orf virus ( orfv ) , a member of parapoxvirus , is an enveloped virus with genome 
 of double - stranded dna . 
 orfv causes contagious pustular dermatitis or contagious ecthyma 
 in sheep and goats worldwide . in general , detection of viral dna and observing orfv virion 
 in tissues of afflicted animals 
 are two methods commonly used for diagnosis of orf 
 infection ; however , isolation of the orfv in cell culture using virus - containing tissue as 
 inoculum is known to be difficult . in this work , 
 the orfv ( hoping strain ) isolated in 
 central taiwan was successfully grown in cell culture . 
 we further examined the biochemical 
 characteristic of our isolate , including viral genotyping , viral mrna and protein 
 expression . by electron microscopy , one unique form of viral particle from orfv infected 
 cellular lysate 
 was demonstrated in the negative - stained field . moreover , immunomodulating 
 and anti - influenza virus properties of this orfv were investigated . 
 orfv stimulated human 
 monocytes ( thp-1 ) secreting proinflammatory cytokines il-8 and tnf-. and , pre - treatment 
 of orfv - infected cell medium prevents a549 cells from subsequent type a influenza virus 
 ( iav ) infection . 
 similarly , mice infected with orfv via both intramuscular and 
 subcutaneous routes at two days prior to iav infection significantly decreased the 
 replication of iav . in summary , 
 the results of a current study indicated our hoping strain 
 harbors the immune modulator property ; with such a bio - adjuvanticity , we further proved 
 that pre - exposure of orfv protects animals from subsequent iav infection . 