epstein - barr virus ( ebv ) is the etiological agent of infectious mononucleosis and is associated with a variety of human diseases including lymphoproliferative diseases ( lpds ) , malignancies ( e.g. burkitt lymphoma , hodgkin lymphoma , nasopharyngeal carcinoma , and gastric carcinoma ) , and autoimmune diseases ( e.g. multiple sclerosis and rheumatoid arthritis ( ra ) ) ( 1 ) . ebv infection in the majority of hosts is however asymptomatic and the virus persists for life as latent infection . ebv has a unique biologic activity to transform b cells and establish immortalized lymphoblastoid cell lines . in immunocompetent hosts , these transformed cells are readily removed by ebv - specific ctls , because they express highly antigenic viral proteins such as the ebv nuclear antigens 3 ( ebna3s ) and ebna2 ( 2 ) . in immunocompromised hosts , however , ebv - transformed cells may continue to proliferate and cause lpds such as post - transplant lymphoproliferative disease ( ptld ) and aids - associated lymphomas . humans are the only natural host of ebv , while rabbits and limited species of new - world monkeys can be infected with the virus in experimental conditions ( 3 - 6 ) . small animal models of ebv infection and ebv - associated diseases have not been available , rendering studies of pathogenesis and therapies of ebv - associated diseases difficult . recently , the development of severely immunodeficient mouse strains made it possible to transplant human hematopoietic stem cells ( hscs ) and reconstitute human immune system components in mice ( 7 - 10 ) . these ectopic human immune system components express their normal functions and these mice are called humanized mice ( more accurately immune system humanized mice ) . viruses that infect only humans and target cells of the immune system , including ebv , hiv-1 , and human t - lymphotropic virus type 1 have been shown to infect humanized mice and reproduce cardinal features of their infection ( 11,12 ) . in this review , recent studies by the author 's group addressing reproduction of ebv infection and pathogenesis in humanized mice are described . in addition , recent achievements in the field of humanized mouse models of ebv infection are briefly reviewed . the immunodeficient mouse strain nod / shi - scid / il-2r ( nog ) , that lacks b , t , and nk cells completely , was developed by ito and his collaborators ( 7 ) . transplantation of human hscs isolated from cord blood in nog mice resulted in differentiation of human b cells , t cells , nk cells , macrophages , and dendritic cells ( 7,13 - 15 ) . inoculation with ebv of higher doses ( > 10 50% transforming dose ( td50 ) ) resulted in b - cell lpd in the majority of humanized nog mice ( 16 ) . on autopsy , splenomegaly was observed in virtually all mice and tumors in the spleen , kidneys , and/or lymph nodes were seen in a fraction of them . in most cases , this lpd showed the histology of diffuse large b - cell lymphoma with the expression of the b - cell marker cd20 , the b - cell activation marker cd23 , and the germinal center marker mum1 . expression of viral proteins such as ebnas 1 and 2 and the latent membrane proteins ( lmps ) 1 and 2 was detected , being consistent with the latency iii type ebv gene expression . ebv - induced lpd in humanized nog mice was thus remarkably similar to the most common type of ptld and aids - associated lymphomas with respect to histology , marker expression , and ebv gene expression . occasionally , hodgkin - like cells with marked nucleoli and reed - sternberg - like cells with multiple nuclei were seen with hodgkin lymphoma - like histology ( 16 ) . beside the nog mouse - based model described above , models of ebv infection have been produced in other types of humanized mice ( 8 - 10 ) and similar ebv - induced lpd was observed in some of them ( 17 - 20 ) . these humanized mouse models of ebv - associated lpd has been utilized to elucidate the role of individual ebv genes in oncogenesis . ebna3b is one of the nine ebv proteins expressed in the virus - transformed lymphoblastoid cells . ebv recombinants with ebna3b knocked out by homologous recombination , however , did not have any distinct phenotypes in in vitro infection of b cells ( 21 ) , and the role of ebna3b in ebv pathogenesis was not clear . white and others demonstrated that ebna3b knock - out virus , as compared with the wild - type virus , has enhanced ability to generate lymphoma in humanized mice , and suggested a tumor - suppressing role for ebna3b ( 20 ) . bzlf1 is an ebv immediate - early gene and its role in lymphoma genesis was not expected because knocking - out bzlf1 did not affect in vitro transformation of b cells by ebv ( 22 ) . ma and others characterized various ebv recombinants in humanized mice and clearly showed that bzlf1 increases the efficiency of lymphomagenesis in vivo by inducing abortive lytic infection ( 18,19 ) . in similar characterization of ebv recombinants in humanized mice , wahl and others demonstrated that a cluster of micrornas encoded by the ebv bhrf1 gene locus facilitates the development of acute systemic infection but does not substantially enhance lymphomagenesis by the virus ( 23 ) . in contrast to infection at high virus doses , most humanized nog mice inoculated with < 10 td50 ebv survived without any signs of illness ( 16 ) . ebv dna load in the peripheral blood increased only transiently and soon returned to undetectable level in most mice ( 16 ) . however , ebv was found to have persisted in these mice for long after infection ( more than 30 weeks ) , because a few ebv - positive b cells were consistently found on autopsy in the liver and spleen ( 16 ) . this situation might be similar to ebv latency in humans , although further characterization is required . in ebv latency in humans , cocco and others utilized humanized mice to analyze the process by which ebv establishes persistent infection in memory b cells ( 24 ) . ra is a common autoimmune disease associated with progressive disability and systemic complications ; arthritis lesions in ra are characterized by synovial proliferation and destruction of bone and cartilage tissues ( 25 ) . evidence has accumulated suggesting a role for ebv in the pathogenesis of ra ( 26,27 ) . compared with normal controls , patients with ra have higher titers of anti - ebv antibodies and higher levels of ebv dna load in their peripheral blood . they have also high numbers of ebv - specific t cells in their affected joints . furthermore , expression of ebv - encoded small rnas ( ebers ) and lmp1 has been demonstrated in synovial cells of ra lesions ( 28 ) . histological examination of 23 ebv - infected humanized nog mice demonstrated erosive arthritis resembling ra in 15 of them but none in 9 uninfected counterparts ( 29 ) . arthritis of these mice was characterized by massive proliferation of synovial cells and marked infiltration of cd3 t cells of both cd4 and cd8 subsets , cd20 b cells , and cd68 macrophages . most importantly , the pannus , a structure of inflammatory granulation tissue very characteristic to ra , was clearly demonstrated in these mice ( 29 ) . bone marrow tissue adjacent to affected joint showed the histology of edema , another finding reminiscent of ra . these results showed that ebv can trigger erosive arthritis similar to ra in humanized mice . the results obtained so far are however restricted to morphological observations and investigation on the mechanism of this arthritis is required . following infection with ebv , the number of cd3 cells increased dramatically in the peripheral blood of humanized nog mice ( 16 ) . this was mostly due to increase in cd8 t cells and elispot assay detected a large number of ifn--producing cells when cd8 t cells isolated from ebv - infected mice were cultured briefly ( 17h ) with autologous ebv - transformed lymphoblastoid cells . elispot counts suggested that 2~4% of cd8 cells in the peripheral blood of infected mice were ebv - specific . an antibody specific to human mhc class i added to the culture efficiently blocked the production of ifn- , but that specific to murine mhc did not ( 16 ) . no ifn--producing cells were detected when the same cd8 t cell population was cultured with control ebv - transformed cells with mismatch mhc . these results indicated that ebv - specific cd8 t - cell responses restricted by human mhc class i were induced in humanized nog mice . since positive selection of thymic t cells is thought to occur on the surface of epithelial cells and thymic epithelial cells in humanized mice are murine in origin , it is a puzzling question how t - cell responses restricted by human mhc were induced in them . the finding by watanabe and others that differentiation of human t cells is markedly suppressed in nog i - a mice transplanted with human hscs suggests that murine mhc molecules are involved in positive selection of human t cells in humanized nog mice ( 30 ) . differentiation of human t cells was not , however , blocked completely in humanized nog i - a mice , implying a participation of certain human hsc - derived cells , possible b cells or dendritic cells , in the positive selection of t cells in humanized nog mice ( 30 ) . when the possibility of using humanized mice for evaluation of vaccines and immunotherapies is considered , it is an important question whether anti - ebv immune responses mounted in humanized mice have protective effects . a clue to this question was first obtained when the fate of infected mice were compared among those infected at different stages of reconstitution of the immune system ( 31 ) . t - cell development in humanized nog mice occurred substantially later than that of b cells and became evident around six months following transplantation ( 14,15 ) . mice infected at three months after transplantation , when human cells in the peripheral blood were mostly b cells , had shorter lifespan compared with those infected at six months , when sufficient t - cell development was evident . when the okt-3 antibody specific to the t - cell marker cd3 was given intravenously to humanized nog mice that were infected at six months after transplantation of hscs , the number of both cd4 and cd8 subsets of t cells were markedly reduced and ebv dna load in the peripheral blood was significantly higher as compared with control mice . lifespan following infection of these mice were significantly shorter than that of control mice ( 31 ) . importantly , okt-3 antibody did not have any effect on the life span of un - infected mice . administration of antibody specific to the cd8 molecule also shortened the life span of ebv - infected mice ( 31 ) . these results indicated that t - cell responses to ebv , especially those by the cd8 subset , have a protective role in humanized mice . in a regression assay , where ebv - infected b cells were cultured with autologous cd8 t cells isolated from ebv - infected mice , transformation was inhibited in a dose - dependent manner as the number of effector cd8 t cells increased . similar protective roles for ebv - specific t - cell responses in humanized mice have been also reported by strowig and others ( 17 ) . ebv - specific humoral immune responses in humanized nog mice were less efficient than the cell - mediated counterpart . only three out of forty ebv - infected mice produced igm antibodies specific to p18 , a major component of the viral capsid antigen of the virus ( 16 ) . no igg antibodies to ebv proteins were detected . the immunodeficient mouse strain nod / shi - scid / il-2r ( nog ) , that lacks b , t , and nk cells completely , was developed by ito and his collaborators ( 7 ) . transplantation of human hscs isolated from cord blood in nog mice resulted in differentiation of human b cells , t cells , nk cells , macrophages , and dendritic cells ( 7,13 - 15 ) . inoculation with ebv of higher doses ( > 10 50% transforming dose ( td50 ) ) resulted in b - cell lpd in the majority of humanized nog mice ( 16 ) . on autopsy , splenomegaly was observed in virtually all mice and tumors in the spleen , kidneys , and/or lymph nodes were seen in a fraction of them . in most cases , this lpd showed the histology of diffuse large b - cell lymphoma with the expression of the b - cell marker cd20 , the b - cell activation marker cd23 , and the germinal center marker mum1 . expression of viral proteins such as ebnas 1 and 2 and the latent membrane proteins ( lmps ) 1 and 2 was detected , being consistent with the latency iii type ebv gene expression . ebv - induced lpd in humanized nog mice was thus remarkably similar to the most common type of ptld and aids - associated lymphomas with respect to histology , marker expression , and ebv gene expression . occasionally , hodgkin - like cells with marked nucleoli and reed - sternberg - like cells with multiple nuclei were seen with hodgkin lymphoma - like histology ( 16 ) . beside the nog mouse - based model described above , models of ebv infection have been produced in other types of humanized mice ( 8 - 10 ) and similar ebv - induced lpd was observed in some of them ( 17 - 20 ) . these humanized mouse models of ebv - associated lpd has been utilized to elucidate the role of individual ebv genes in oncogenesis . ebna3b is one of the nine ebv proteins expressed in the virus - transformed lymphoblastoid cells . ebv recombinants with ebna3b knocked out by homologous recombination , however , did not have any distinct phenotypes in in vitro infection of b cells ( 21 ) , and the role of ebna3b in ebv pathogenesis was not clear . white and others demonstrated that ebna3b knock - out virus , as compared with the wild - type virus , has enhanced ability to generate lymphoma in humanized mice , and suggested a tumor - suppressing role for ebna3b ( 20 ) . bzlf1 is an ebv immediate - early gene and its role in lymphoma genesis was not expected because knocking - out bzlf1 did not affect in vitro transformation of b cells by ebv ( 22 ) . ma and others characterized various ebv recombinants in humanized mice and clearly showed that bzlf1 increases the efficiency of lymphomagenesis in vivo by inducing abortive lytic infection ( 18,19 ) . in similar characterization of ebv recombinants in humanized mice , wahl and others demonstrated that a cluster of micrornas encoded by the ebv bhrf1 gene locus facilitates the development of acute systemic infection but does not substantially enhance lymphomagenesis by the virus ( 23 ) . in contrast to infection at high virus doses , most humanized nog mice inoculated with < 10 td50 ebv survived without any signs of illness ( 16 ) . ebv dna load in the peripheral blood increased only transiently and soon returned to undetectable level in most mice ( 16 ) . however , ebv was found to have persisted in these mice for long after infection ( more than 30 weeks ) , because a few ebv - positive b cells were consistently found on autopsy in the liver and spleen ( 16 ) . this situation might be similar to ebv latency in humans , although further characterization is required . in ebv latency in humans , cocco and others utilized humanized mice to analyze the process by which ebv establishes persistent infection in memory b cells ( 24 ) . ra is a common autoimmune disease associated with progressive disability and systemic complications ; arthritis lesions in ra are characterized by synovial proliferation and destruction of bone and cartilage tissues ( 25 ) . evidence has accumulated suggesting a role for ebv in the pathogenesis of ra ( 26,27 ) . compared with normal controls , patients with ra have higher titers of anti - ebv antibodies and higher levels of ebv dna load in their peripheral blood . they have also high numbers of ebv - specific t cells in their affected joints . furthermore , expression of ebv - encoded small rnas ( ebers ) and lmp1 has been demonstrated in synovial cells of ra lesions ( 28 ) . histological examination of 23 ebv - infected humanized nog mice demonstrated erosive arthritis resembling ra in 15 of them but none in 9 uninfected counterparts ( 29 ) . arthritis of these mice was characterized by massive proliferation of synovial cells and marked infiltration of cd3 t cells of both cd4 and cd8 subsets , cd20 b cells , and cd68 macrophages . most importantly , the pannus , a structure of inflammatory granulation tissue very characteristic to ra , was clearly demonstrated in these mice ( 29 ) . bone marrow tissue adjacent to affected joint showed the histology of edema , another finding reminiscent of ra . these results showed that ebv can trigger erosive arthritis similar to ra in humanized mice . the results obtained so far are however restricted to morphological observations and investigation on the mechanism of this arthritis is required . following infection with ebv , the number of cd3 cells increased dramatically in the peripheral blood of humanized nog mice ( 16 ) . this was mostly due to increase in cd8 t cells and elispot assay detected a large number of ifn--producing cells when cd8 t cells isolated from ebv - infected mice were cultured briefly ( 17h ) with autologous ebv - transformed lymphoblastoid cells . elispot counts suggested that 2~4% of cd8 cells in the peripheral blood of infected mice were ebv - specific . an antibody specific to human mhc class i added to the culture efficiently blocked the production of ifn- , but that specific to murine mhc did not ( 16 ) . no ifn--producing cells were detected when the same cd8 t cell population was cultured with control ebv - transformed cells with mismatch mhc . these results indicated that ebv - specific cd8 t - cell responses restricted by human mhc class i were induced in humanized nog mice . since positive selection of thymic t cells is thought to occur on the surface of epithelial cells and thymic epithelial cells in humanized mice are murine in origin , it is a puzzling question how t - cell responses restricted by human mhc were induced in them . the finding by watanabe and others that differentiation of human t cells is markedly suppressed in nog i - a mice transplanted with human hscs suggests that murine mhc molecules are involved in positive selection of human t cells in humanized nog mice ( 30 ) . differentiation of human t cells was not , however , blocked completely in humanized nog i - a mice , implying a participation of certain human hsc - derived cells , possible b cells or dendritic cells , in the positive selection of t cells in humanized nog mice ( 30 ) . when the possibility of using humanized mice for evaluation of vaccines and immunotherapies is considered , it is an important question whether anti - ebv immune responses mounted in humanized mice have protective effects . a clue to this question was first obtained when the fate of infected mice were compared among those infected at different stages of reconstitution of the immune system ( 31 ) . t - cell development in humanized nog mice occurred substantially later than that of b cells and became evident around six months following transplantation ( 14,15 ) . mice infected at three months after transplantation , when human cells in the peripheral blood were mostly b cells , had shorter lifespan compared with those infected at six months , when sufficient t - cell development was evident . when the okt-3 antibody specific to the t - cell marker cd3 was given intravenously to humanized nog mice that were infected at six months after transplantation of hscs , the number of both cd4 and cd8 subsets of t cells were markedly reduced and ebv dna load in the peripheral blood was significantly higher as compared with control mice . lifespan following infection of these mice were significantly shorter than that of control mice ( 31 ) . importantly , okt-3 antibody did not have any effect on the life span of un - infected mice . administration of antibody specific to the cd8 molecule also shortened the life span of ebv - infected mice ( 31 ) . these results indicated that t - cell responses to ebv , especially those by the cd8 subset , have a protective role in humanized mice . in a regression assay , where ebv - infected b cells were cultured with autologous cd8 t cells isolated from ebv - infected mice , transformation was inhibited in a dose - dependent manner as the number of effector cd8 t cells increased . similar protective roles for ebv - specific t - cell responses in humanized mice have been also reported by strowig and others ( 17 ) . ebv - specific humoral immune responses in humanized nog mice were less efficient than the cell - mediated counterpart . only three out of forty ebv - infected mice produced igm antibodies specific to p18 , a major component of the viral capsid antigen of the virus ( 16 ) . no igg antibodies to ebv proteins were detected . although b cells and epithelial cells are its major targets , ebv can infect t and nk cells and induces their proliferation in a category of diseases with high morbidity and mortality , called ebv t / nk - cell lymphoproliferative diseases ( ebv t / nk - lpds ) ( 32 - 37 ) . chronic active ebv infection ( caebv ) and ebv - associated hemophagocytic lymphohistiocytosis ( ebv - hlh ) are representatives of this category . since proliferation of ebv - infected t or nk cells has not been observed in the virus - infected humanized nog mice , patient 's pbmcs were transplanted to nog mice to reproduce ebv t / nk - lpds ( 38 ) . one to two months following intravenous transplantation of 1 - 510 pbmcs isolated from patients with caebv , ebv dna load in the peripheral blood of nog mice increased rapidly to the levels of 10 - 10 copies/g dna . the phenotype of ebv - infected cells in recipient mice was identical to that found in the patient from whom the mice received pbmc . tcr repertoire analysis confirmed that an identical ebv - infected t - cell clone was proliferating in patients and their respective recipient mice . ebv - infected t and nk cells proliferating in recipient mice retained the latency ii type ebv gene expression characteristic to caebv . on autopsy obvious tumors were not found , but most mice showed marked splenomegaly . histological analysis exhibited massive infiltration of relatively small ebv - infected lymphoid cells without marked morphological atypia in organs including the spleen , liver , kidneys , and lungs . high - level human cytokine / chemokines , including il-8 , ifn- , and rantes were detected in these mice , as has been reported for patients with caebv ( 38 ) . caebv can be classified into the cd4 , cd8 , t , and nk types , depending on which cells are infected with ebv in the patient , and transplantation with patient 's pbmcs resulted in engraftment of ebv - infected t or nk cells in all four types of caebv . interestingly , however , engraftment of ebv - infected cells was not observed when a cell fraction containing ebv - infected cells was isolated and transplanted to nog mice . the only exception for this was the cd4 fraction isolated from patients with the cd4-type caebv , suggesting that cd4 cells play an important role for the engraftment . consistent with this hypothesis , engraftment was inhibited when cd4 cells are removed from pbmc ; same results were obtained in transplantation from all four types of caebv . furthermore , when cd4 t cells were depleted in vivo by administration of the okt-4 antibody just following transplantation of pbmc , engraftment was completely blocked . these findings taken together indicate that cd4 t cells play an essential role in in vivo proliferation of ebv - infected t and nk cells and suggest that novel therapies targeting cd4 t cells or some factors produced by them might be possible . indeed , okt-4 was effective when administered after engraftment and reduced ebv dna load in the peripheral blood and organs ( unpublished results by imadome and others ) . similar xenotransplantation experiments with pbmcs isolated from patients with ebv - hlh also resulted in systemic proliferation of ebv - infected t cells and splenomegaly . there were however unique finding in these ebv - hlh model mice , including hemorrhagic lesions in thoracic and/or abdominal cavities and extremely high human cytokine levels in the peripheral blood . another unexpected finding in these mice was that although ebv - infected cd8 cells were detected in the peripheral blood , only ebv - infected b cells were found in the spleen and liver . sato and others reported that ebv - infected humanized nog mice can develop hypercytokinemia and hemophagocytosis resembling hlh ( 39 ) . the results described here indicate that humanized mice can reproduce cardinal features of ebv infection including pathogenesis and immune responses . humanized mice are therefore promising tool for elucidation of ebv pathogenesis and evaluation of novel therapies for ebv - associated diseases . although immune functions of current humanized mice are not satisfactory , various trials for their improvement are in progress . for example , introduction of a human mhc class i gene to humanized mice drastically improved their t - cell immune responses to ebv ( 40 )
epstein - barr virus ( ebv ) is etiologically associated with a variety of diseases including lymphoproliferative diseases , lymphomas , carcinomas , and autoimmune diseases . 
 humans are the only natural host of ebv and limited species of new - world monkeys can be infected with the virus in experimental conditions . 
 small animal models of ebv infection , required for evaluation of novel therapies and vaccines for ebv - associated diseases , have not been available . 
 recently the development of severely immunodeficient mouse strains enabled production of humanized mice in which human immune system components are reconstituted and express their normal functions . 
 humanized mice can serve as infection models for human - specific viruses such as ebv that target cells of the immune system . 
 this review summarizes recent studies by the author 's group addressing reproduction of ebv infection and pathogenesis in humanized mice .