BACKGROUND
japanese encephalitis virus  is the most important cause of epidemic encephalitis in most asian regions, with about  <dig> – <dig>  cases of and  <dig>  deaths from jev infection reported annually  <cit> .
the virus can be found in regions beyond its ecological boundaries, with recent reports of jev having spread as far as northern australia  <cit>  and pakistan  <cit> .
hence, there is concern that jev might become a global threat.
japanese encephalitis  caused by jev is a mosquito-borne zoonotic infectious disease  <cit> .
a variety of animals are susceptible to jev infection, but usually only humans, horses and pigs with infection exhibit symptoms  <cit> .
humans and horses are generally thought to be dead-end jev hosts  <cit> .
pigs are considered the most important amplification and reservoir hosts of jev in endemic regions  <cit> .
jev infection in pregnant sows can cause abortion, stillbirth and other reproductive failure.
in addition, infection of boars can cause acute testicular inflammation and hypospermia  <cit> .
there is no specific treatment available for je, and vaccination is the only effective way to prevent jev infection in humans and domestic animals.
at present, there are three kinds of je vaccines available for humans: the live attenuated virus vaccine sa14-14- <dig>  <cit> , vero cell-derived formalin-inactivated whole-virus sa14-14- <dig> strain vaccine ic <dig> and beijing- <dig> strain vaccine jebikv  <cit> , and recombinant chimeric virus vaccine je-cv  <cit> .
these vaccines have better safety profiles and fewer side effects compared with the mouse brain-derived inactivated vaccine  <cit> .
the attenuated live vaccine is derived from primary hamster kidney cells, is difficult and costly to manufacture, and the infectious agents are always associated with potential biosafety issues.
the use of infectious agents is always a major issue with manufacturing process of the currently used vaccines.
therefore, the development of new types of je vaccines for humans and domestic animals that do not involve the use of infectious jev is imperative.
genetically engineered vaccine is a potential new form of the je vaccine.
previous studies have shown that the pre-membrane  and e protein expressed in mammalian cells could assemble into virus-like particles , and that the expressed jev vlp has the same ability to induce neutralizing antibodies and protective efficiency as the jev virion  <cit> .
thus, studies on second-generation je vaccines have focused on the production of the jev-vlp antigen by genetic engineering of a stable cell line.
different mammalian cell lines including rk <dig>  <cit> , cos- <dig>  <cit>  and cho-k <dig>  <cit>  have been used to construct stable cell lines that continuously express jev-vlp, but do not express efficiently or have additional defects.
in this study, we have generated a new cell line, bj-me, which can stably produce a secreted form of the jev vlp by using bhk- <dig> cells as the parent cell line.
mice immunized with the expressed jev-vlp antigen developed high titres of neutralizing antibodies and complete protection against lethal jev challenges.
RESULTS
establishment of a stable cell clone continuously expressing the prm/m-e antigen
from the cloned colonies of bhk- <dig> cells, 64 g418-resistant and e protein-specific ifa-positive cell colonies were picked and transferred to 24-well plates.
after comparison of e protein expression in the culture medium of the  <dig> cell colonies by western blotting,  <dig> clones with relatively high e protein expression were selected for further comparison.
after ifa testing and further cloning purification, the e protein expression efficiency of all  <dig> cell clones was compared using e protein-specific western blotting.
the clone of most efficiently expression e antigen was selected and designated as bj-me .
the cells were expanded and maintained in g418-containing medium to observe jev antigen production.
the bj-me cell clone had undistinguishable morphology from parental bhk- <dig> cells and induced no polykaryocyte formation.
when the fifth-passage bj-me cells were examined by indirect immunofluorescence and flow cytometry, over 94% of bj-me cells were positively stained with jev e protein-specific mab 5e <dig>  this high percentage of e-expressing cells was maintained till the twentieth passage .western blotting results with the mabs 5e <dig> and 3c <dig> showed that bj-me cells expressed the prm and e protein together, and the majority of prm in the cells did not show cleavage.
when secreted into culture medium, most of the prm protein was cleaved by furin into mature m protein .the morphology of bj-me cells was further examined by transmission electron microscopy.
the results showed that vlp structure was observed in the endoplasmic reticulum of cells , and this jev-vlp structure was further confirmed with purified jev-vlp by negative staining electron microscopy observation .
the appearance of the jev-vlp was in the form of particles with an average diameter of 40–50 nm surrounded by a lipid bilayer.
passage stability and antigen expression of bj-me cells
for bj-me cells, passage  <dig> was defined as the cell culture from the 96-well plate by limiting dilution cloning and with 100% e expression.
after expansion and cryopreservation, the culture medium of bj-me cells cultured in 75 cm <dig> flasks was collected from passage  <dig>  the jev-vlp was purified by ultrafiltration and rate zonal centrifugation.
the purified antigen was used in negative staining electron microscopy observation and used as the antigen standard in an antigen capture elisa quantification assay.
to evaluate the antigen expression dynamics of bj-me cells, the confluent bj-me cells were passaged 1: <dig> into 75 cm <dig> flasks with 20 ml medium containing 10% fbs.
for antigen amounts detection, in next day, the cells started growing in confluence and the medium containing 10% fbs were changed into maintaining medium .
twenty four hours later, 600 μl of the culture medium was replaced with fresh serum-free medium every day.
the amount of jev-vlp was determined by elisa.
as shown in figure 4a, the amount of jev-vlp in the culture medium kept increasing in the first four days.
from the fifth day, it reached a plateau, after which it started showing a slight increase from the fifth to tenth day.
cell culture fluid containing the jev-vlp antigen could be harvested five to seven times continuously at intervals of  <dig> to 6 days during culture, and the concentration of jev-vlp antigen collected at each harvest was over 15 μg/ml .
the stability of jev-vlp antigen expression by bj-me cells was further evaluated using cryopreserved cells.
after thawing, bj-me cells were passaged every 2 days in 75 cm <dig> flasks.
for antigen testing, the culture medium of confluent cells were changed into fresh maintaining medium.
<dig> to 5 days later the culture medium was collected at the 5th, 10th, 15th, and 20th passages and examined for the amount of jev-vlp antigen by elisa .
although the amount of jev-vlp antigen produced by bj-me cells differed little between passages, more than 15 μg/ml of the jev-vlp antigen was detectable throughout the monitoring period.
even at the 20th passage, the frequency of e-expressing cells was over 94% .
immunogenicity and protective efficacy of the jev-vlp
mice administered the jev-vlp with or without adjuvant developed high titers of neutralizing antibody.
even the neutralizing antibody titer in mice that were not administered the adjuvant was somewhat higher than that observed in mice immunized with the live virus vaccine .
all mice immunized with the jev-vlp with or without adjuvant and the live virus vaccine were completely protected against jev.
all mice that were administered pbs died within 10 days after the challenge .
a
atiters are expressed as the maximum serum dilution yielding a 50% plaque reduction.
bjev-vlp antigen.
clive virus vaccine .
nd, not determined.
for the second experiment, the immunogenicity and protective efficacy of different doses of the antigen were evaluated.
as shown in table  <dig>  mice in the groups administered 3 μg and 2 μg of the antigen developed high levels of neutralizing antibody similar to that of the live vaccine group and were completely protected against the jev challenge.
mice in the groups administered  <dig> – <dig>  μg of the antigen developed relatively lower titers of neutralizing antibody, and were partially protected against jev; however, the survival rate in all groups was higher than 70% .
discussion
there is an urgent need for an effective, affordable and safer je vaccine for humans and domestic animals.
biotechnically-engineered cell lines expressing the jev-vlp antigen may meet this requirement.
here, we describe another alternative, a recombinant subunit vaccine candidate, based on a new mammalian cell line.
the bj-me cells described in this study produce extremely high levels of the jev-vlp antigen—15–20 μg/ml—which is higher than that of any other reported cell lines such as j12# <dig>  <cit> , f  <cit>  and je-4b  <cit> .
this value was determined under experimental conditions involving simple changes in the culture medium, and we believe that the titer could be further increased under optimal culture conditions.
moreover, time-course results suggest that 5 days of incubation is sufficient for antigen production.
bj-me cells had similar cell morphology and growth characteristics as the parental bhk- <dig> cells.
in fact, cell clones with different morphogenetic characteristics found during the selection and cloning process were found to release the e antigen inefficiently.
this phenomenon indicates that maintaining similar morphology and growth characteristics with the parental cells may be important in the engineering of cell lines with high efficiency and stable exogenous gene expression.
another factor that may have contributed to the high efficiency of jev-vlp antigen expression in bj-me cells is the genetic codon optimization of the cdna sequence encoding the prm and e proteins.
different species have codon usage bias, and optimizing the genetic codon according to the bias of the expressing host could increase the expression efficiency of the target gene  <cit> .
the bj-me cells showed stable jev-vlp production during cell culture and passage.
even after 20– <dig> passages, the jev-vlp antigen-producing efficiency of bj-me cells was not significantly reduced.
a major obstacle to the stable expression of flavivirus membrane protein in mammalian cells is its cytotoxicity.
in a previous report, attempts to establish cells with high expression of the prm-e protein from vero cells were unsuccessful  <cit> .
this is probably because vero cells are susceptible to jev infection, which makes them sensitive to the cytotoxic effects of the jev-vlp antigen.
although bhk- <dig> cells are susceptible to jev infection, we did not observe any obvious cytotoxic effects of jev vlp.
it was possible to obtain dozens of g418-resistant cell colonies with a single transfection and selection cycle.
the cell line bj-me constructed in this study has the same growth characteristics as the parent cell line bhk- <dig>  so this cell line could easily be cultured in serum-free or low-serum medium.
j12# <dig> cells derived from rk <dig>  a rabbit kidney cell line, was the most stable and highest antigen-producing cell line in all previous reports  <cit> .
however, rk <dig> cells grow slowly and could not reach high cell density, so are not suitable for large-scale culture.
another important point is that rk <dig> cells contain bovine viral diarrhea virus  <cit> , which make them unsuitable for vaccine antigen production.
to overcome the fusogenic cytotoxicity of jev prm-e protein expression in mammalian cell lines, konishi et al.
<cit>  introduced a mutation at the prm/m cleavage site to prevent authentic processing and to reduce the toxic fusion activity of the expressed vlp, and established an f-cell line using cho-k <dig> as the parent cell line.
the elimination of cleavage of the prm protein resulted in the production of immature vlps, which are quite different in their conformational structure from the mature vlps produced by authentic cleavage of prm  <cit> .
these structural differences may eliminate virus conformational neutralizing epitopes.
moreover, reports have shown that most jev-neutralizing epitopes are conformation dependent  <cit> .
the differences in the conformational structure will affect the immunogenicity of the f antigen.
in fact, the protection rate in mice immunized with the f antigen with uncleaved prm protein did not exceed 50%  <cit> .
recently yamaji et al.
further reported expressing the jev prm protein mutated virus-like particles with baculovirus  <cit>  and lepidopteran insect cell lines  <cit> .
obviously the yield of e antigen were increased in both of two expressing systems, especially the e antigen yield reached about 30 μg/ml in lepidopteran insect cell line.
however, these studies did not present immune protection result of these immature jev virus-like particles with uncleaved prm protein.
the differences in structural and immune protective effect between immature and mature jev virus-like particles need further evaluation.
maybe denv-related reports  <cit>  could provide some clues for future survey the relation of immune protective effect and structure of virus-like particles.
as expected be of particulate form, the bj-me antigen was immunogenic.
with or without adjuvant, dose of 2 μg of bj-me induced high titers of neutralizing antibodies and provided complete protection against challenge.
however we observed no enhancement effect of the adjuvant in this experiment which may because the antigen amount of 2 μg is overdosed for mice.
another experiment with different dose of antigen showed that  <dig>  μg of bj-me antigen could induce effective protection neutralizing antibodies titer of 1: <dig> which is higher than surrogate maker for seroprotection of neutralizing antibody titer of 1: <dig>
CONCLUSIONS
the new established cell line bj-me could efficiently and stably produces secreted form of jev-vlp.
bj-me cells expressing the jev-vlp antigen showed good immunogenicity in mice.
an antigen dose of at least  <dig>  μg could induce neutralizing antibody production, and more than 70% of immunized mice survived after lethal challenge with jev.
these results suggest that the recombinant jev virus-like particle antigen produced by the bj-me cell clone is an effective, easy to produce, and safe second-generation subunit je veterinary vaccine candidate.
moreover, the jev-vlp could be used to make diagnostic reagents for je and jev.

 japanese encephalitis virus is the most important cause of epidemic encephalitis in most asian regions, with about – cases of and deaths from jev infection reported annually. the virus can be found in regions beyond its ecological boundaries, with recent reports of jev having spread as far as northern australia and pakistan. hence, there is concern that jev might become a global threat. japanese encephalitis caused by jev is a mosquito-borne zoonotic infectious disease. a variety of animals are susceptible to jev infection, but usually only humans, horses and pigs with infection exhibit symptoms. humans and horses are generally thought to be dead-end jev hosts. pigs are considered the most important amplification and reservoir hosts of jev in endemic regions. jev infection in pregnant sows can cause abortion, stillbirth and other reproductive failure. in addition, infection of boars can cause acute testicular inflammation and hypospermia. there is no specific treatment available for je, and vaccination is the only effective way to prevent jev infection in humans and domestic animals. at present, there are three kinds of je vaccines available for humans: the live attenuated virus vaccine sa14-14-, vero cell-derived formalin-inactivated whole-virus sa14-14- strain vaccine ic and beijing- strain vaccine jebikv, and recombinant chimeric virus vaccine je-cv. these vaccines have better safety profiles and fewer side effects compared with the mouse brain-derived inactivated vaccine. the attenuated live vaccine is derived from primary hamster kidney cells, is difficult and costly to manufacture, and the infectious agents are always associated with potential biosafety issues. the use of infectious agents is always a major issue with manufacturing process of the currently used vaccines. therefore, the development of new types of je vaccines for humans and domestic animals that do not involve the use of infectious jev is imperative. genetically engineered vaccine is a potential new form of the je vaccine. previous studies have shown that the pre-membrane and e protein expressed in mammalian cells could assemble into virus-like particles, and that the expressed jev vlp has the same ability to induce neutralizing antibodies and protective efficiency as the jev virion. thus, studies on second-generation je vaccines have focused on the production of the jev-vlp antigen by genetic engineering of a stable cell line. different mammalian cell lines including rk, cos- and cho-k have been used to construct stable cell lines that continuously express jev-vlp, but do not express efficiently or have additional defects. in this study, we have generated a new cell line, bj-me, which can stably produce a secreted form of the jev vlp by using bhk- cells as the parent cell line. mice immunized with the expressed jev-vlp antigen developed high titres of neutralizing antibodies and complete protection against lethal jev challenges.  establishment of a stable cell clone continuously expressing the prm/m-e antigen from the cloned colonies of bhk- cells, 64 g418-resistant and e protein-specific ifa-positive cell colonies were picked and transferred to 24-well plates. after comparison of e protein expression in the culture medium of the cell colonies by western blotting, clones with relatively high e protein expression were selected for further comparison. after ifa testing and further cloning purification, the e protein expression efficiency of all cell clones was compared using e protein-specific western blotting. the clone of most efficiently expression e antigen was selected and designated as bj-me. the cells were expanded and maintained in g418-containing medium to observe jev antigen production. the bj-me cell clone had undistinguishable morphology from parental bhk- cells and induced no polykaryocyte formation. when the fifth-passage bj-me cells were examined by indirect immunofluorescence and flow cytometry, over 94% of bj-me cells were positively stained with jev e protein-specific mab 5e this high percentage of e-expressing cells was maintained till the twentieth passage.western blotting results with the mabs 5e and 3c showed that bj-me cells expressed the prm and e protein together, and the majority of prm in the cells did not show cleavage. when secreted into culture medium, most of the prm protein was cleaved by furin into mature m protein.the morphology of bj-me cells was further examined by transmission electron microscopy. the results showed that vlp structure was observed in the endoplasmic reticulum of cells, and this jev-vlp structure was further confirmed with purified jev-vlp by negative staining electron microscopy observation. the appearance of the jev-vlp was in the form of particles with an average diameter of 40–50 nm surrounded by a lipid bilayer. passage stability and antigen expression of bj-me cells for bj-me cells, passage was defined as the cell culture from the 96-well plate by limiting dilution cloning and with 100% e expression. after expansion and cryopreservation, the culture medium of bj-me cells cultured in 75 cm flasks was collected from passage the jev-vlp was purified by ultrafiltration and rate zonal centrifugation. the purified antigen was used in negative staining electron microscopy observation and used as the antigen standard in an antigen capture elisa quantification assay. to evaluate the antigen expression dynamics of bj-me cells, the confluent bj-me cells were passaged 1: into 75 cm flasks with 20 ml medium containing 10% fbs. for antigen amounts detection, in next day, the cells started growing in confluence and the medium containing 10% fbs were changed into maintaining medium. twenty four hours later, 600 μl of the culture medium was replaced with fresh serum-free medium every day. the amount of jev-vlp was determined by elisa. as shown in figure 4a, the amount of jev-vlp in the culture medium kept increasing in the first four days. from the fifth day, it reached a plateau, after which it started showing a slight increase from the fifth to tenth day. cell culture fluid containing the jev-vlp antigen could be harvested five to seven times continuously at intervals of to 6 days during culture, and the concentration of jev-vlp antigen collected at each harvest was over 15 μg/ml. the stability of jev-vlp antigen expression by bj-me cells was further evaluated using cryopreserved cells. after thawing, bj-me cells were passaged every 2 days in 75 cm flasks. for antigen testing, the culture medium of confluent cells were changed into fresh maintaining medium. to 5 days later the culture medium was collected at the 5th, 10th, 15th, and 20th passages and examined for the amount of jev-vlp antigen by elisa. although the amount of jev-vlp antigen produced by bj-me cells differed little between passages, more than 15 μg/ml of the jev-vlp antigen was detectable throughout the monitoring period. even at the 20th passage, the frequency of e-expressing cells was over 94%. immunogenicity and protective efficacy of the jev-vlp mice administered the jev-vlp with or without adjuvant developed high titers of neutralizing antibody. even the neutralizing antibody titer in mice that were not administered the adjuvant was somewhat higher than that observed in mice immunized with the live virus vaccine. all mice immunized with the jev-vlp with or without adjuvant and the live virus vaccine were completely protected against jev. all mice that were administered pbs died within 10 days after the challenge. a atiters are expressed as the maximum serum dilution yielding a 50% plaque reduction. bjev-vlp antigen. clive virus vaccine. nd, not determined. for the second experiment, the immunogenicity and protective efficacy of different doses of the antigen were evaluated. as shown in table  mice in the groups administered 3 μg and 2 μg of the antigen developed high levels of neutralizing antibody similar to that of the live vaccine group and were completely protected against the jev challenge. mice in the groups administered –  μg of the antigen developed relatively lower titers of neutralizing antibody, and were partially protected against jev; however, the survival rate in all groups was higher than 70%. discussion there is an urgent need for an effective, affordable and safer je vaccine for humans and domestic animals. biotechnically-engineered cell lines expressing the jev-vlp antigen may meet this requirement. here, we describe another alternative, a recombinant subunit vaccine candidate, based on a new mammalian cell line. the bj-me cells described in this study produce extremely high levels of the jev-vlp antigen—15–20 μg/ml—which is higher than that of any other reported cell lines such as j12#, f and je-4b. this value was determined under experimental conditions involving simple changes in the culture medium, and we believe that the titer could be further increased under optimal culture conditions. moreover, time-course results suggest that 5 days of incubation is sufficient for antigen production. bj-me cells had similar cell morphology and growth characteristics as the parental bhk- cells. in fact, cell clones with different morphogenetic characteristics found during the selection and cloning process were found to release the e antigen inefficiently. this phenomenon indicates that maintaining similar morphology and growth characteristics with the parental cells may be important in the engineering of cell lines with high efficiency and stable exogenous gene expression. another factor that may have contributed to the high efficiency of jev-vlp antigen expression in bj-me cells is the genetic codon optimization of the cdna sequence encoding the prm and e proteins. different species have codon usage bias, and optimizing the genetic codon according to the bias of the expressing host could increase the expression efficiency of the target gene. the bj-me cells showed stable jev-vlp production during cell culture and passage. even after 20– passages, the jev-vlp antigen-producing efficiency of bj-me cells was not significantly reduced. a major obstacle to the stable expression of flavivirus membrane protein in mammalian cells is its cytotoxicity. in a previous report, attempts to establish cells with high expression of the prm-e protein from vero cells were unsuccessful. this is probably because vero cells are susceptible to jev infection, which makes them sensitive to the cytotoxic effects of the jev-vlp antigen. although bhk- cells are susceptible to jev infection, we did not observe any obvious cytotoxic effects of jev vlp. it was possible to obtain dozens of g418-resistant cell colonies with a single transfection and selection cycle. the cell line bj-me constructed in this study has the same growth characteristics as the parent cell line bhk- so this cell line could easily be cultured in serum-free or low-serum medium. j12# cells derived from rk a rabbit kidney cell line, was the most stable and highest antigen-producing cell line in all previous reports. however, rk cells grow slowly and could not reach high cell density, so are not suitable for large-scale culture. another important point is that rk cells contain bovine viral diarrhea virus, which make them unsuitable for vaccine antigen production. to overcome the fusogenic cytotoxicity of jev prm-e protein expression in mammalian cell lines, konishi et al. introduced a mutation at the prm/m cleavage site to prevent authentic processing and to reduce the toxic fusion activity of the expressed vlp, and established an f-cell line using cho-k as the parent cell line. the elimination of cleavage of the prm protein resulted in the production of immature vlps, which are quite different in their conformational structure from the mature vlps produced by authentic cleavage of prm. these structural differences may eliminate virus conformational neutralizing epitopes. moreover, reports have shown that most jev-neutralizing epitopes are conformation dependent. the differences in the conformational structure will affect the immunogenicity of the f antigen. in fact, the protection rate in mice immunized with the f antigen with uncleaved prm protein did not exceed 50%. recently yamaji et al. further reported expressing the jev prm protein mutated virus-like particles with baculovirus and lepidopteran insect cell lines. obviously the yield of e antigen were increased in both of two expressing systems, especially the e antigen yield reached about 30 μg/ml in lepidopteran insect cell line. however, these studies did not present immune protection result of these immature jev virus-like particles with uncleaved prm protein. the differences in structural and immune protective effect between immature and mature jev virus-like particles need further evaluation. maybe denv-related reports could provide some clues for future survey the relation of immune protective effect and structure of virus-like particles. as expected be of particulate form, the bj-me antigen was immunogenic. with or without adjuvant, dose of 2 μg of bj-me induced high titers of neutralizing antibodies and provided complete protection against challenge. however we observed no enhancement effect of the adjuvant in this experiment which may because the antigen amount of 2 μg is overdosed for mice. another experiment with different dose of antigen showed that  μg of bj-me antigen could induce effective protection neutralizing antibodies titer of 1: which is higher than surrogate maker for seroprotection of neutralizing antibody titer of 1:  the new established cell line bj-me could efficiently and stably produces secreted form of jev-vlp. bj-me cells expressing the jev-vlp antigen showed good immunogenicity in mice. an antigen dose of at least  μg could induce neutralizing antibody production, and more than 70% of immunized mice survived after lethal challenge with jev. these results suggest that the recombinant jev virus-like particle antigen produced by the bj-me cell clone is an effective, easy to produce, and safe second-generation subunit je veterinary vaccine candidate. moreover, the jev-vlp could be used to make diagnostic reagents for je and jev. 
there is no specific treatment available for japanese encephalitis, and vaccination is the only effective way to prevent jev infection in humans and domestic animals. cell culture fluid containing the jev-vlp antigen could be harvested five to seven times continuously at intervals of 4– days while maintaining the culture. cell line bj-me can stably produces a secreted form of japanese encephalitis virus virus-like particle which contains the jev envelope glycoprotein and membrane protein. japanese encephalitis virus is the most important cause of epidemic encephalitis in most asian regions. the amount of jev-vlp antigen released into the culture fluid of bj-me cells was as high as 15–20 μg/ml. mice immunized with the jev-vlp antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal jev challenge. these results suggest that the recombinant jev-vlp antigen produced by the bj-me cell line is an effective, safe and affordable subunit japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse. the bj-me cells were engineered by transfecting bhk- cells with a code-optimized cdna encoding jev prm and e protein expression plasmid. we generated a new cell clone that stably produces a secreted form of japanese encephalitis virus virus-like particle. japanese encephalitis virusmammalian cell linevirus-like particlesubunit vaccine the purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of jev for potential use of jev subunit vaccine.
 japanese encephalitis virus is the most important cause of epidemic encephalitis in most asian regions. there is no specific treatment available for japanese encephalitis, and vaccination is the only effective way to prevent jev infection in humans and domestic animals. the purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of jev for potential use of jev subunit vaccine.  we generated a new cell clone that stably produces a secreted form of japanese encephalitis virus virus-like particle. the bj-me cells were engineered by transfecting bhk- cells with a code-optimized cdna encoding jev prm and e protein expression plasmid. cell line bj-me can stably produces a secreted form of japanese encephalitis virus virus-like particle which contains the jev envelope glycoprotein and membrane protein. the amount of jev-vlp antigen released into the culture fluid of bj-me cells was as high as 15–20 μg/ml. jev-vlp production was stable after multiple cell passages and 100% cell expression was maintained without detectable cell fusion or apoptosis. cell culture fluid containing the jev-vlp antigen could be harvested five to seven times continuously at intervals of 4– days while maintaining the culture. mice immunized with the jev-vlp antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal jev challenge.  these results suggest that the recombinant jev-vlp antigen produced by the bj-me cell line is an effective, safe and affordable subunit japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse. japanese encephalitis virusmammalian cell linevirus-like particlesubunit vaccine 