the genus enterovirus within family picornaviridae , non - enveloped viruses with a single - stranded rna genome of positive polarity , is comprised of more than 100 serotypes ( pallansch and roos , 2007 ) . human enteroviruses ( hevs ) are presently classified into four species , hev species a , b , c , and d ( hev - a , b , c , and d ) . at present , coxsackievirus a2 ( cva2 ) , cva3 , cva4 , cva5 , cva6 , cva7 , cva8 , cva10 , cva12 , cva14 , cva16 , enterovirus 71 ( ev71 ) , ev76 , ev89 , ev90 , ev91 , and ev92 have been classified as hev - a ( oberste et al . , 2004 , 2005 , 2008 ) . in addition , four types of simian enteroviruses are classified as hev - a ( oberste et al . , 2007 ) . although most enterovirus infections are asymptomatic , particular clinical manifestations are associated with specific types of enteroviruses ( pallansch and roos , 2007 ) . cva2 , cva4 , cva5 , cva6 , and cva10 are commonly associated with herpangina . ev71 and cva16 are major causative agents of hand , foot , and mouth disease ( hfmd ) , a common febrile disease occurring mainly in young children , characterized by skin rash involving palms and soles , and ulcers on oral mucosa . recently , hfmd outbreaks mainly due to cva6 have been reported ( fujimoto et al . , 2012 ) . clinical manifestations of hfmd caused by ev71 , cva6 , and cva16 are usually mild and self - limited . however , ev71 infection causes a diverse range of neurological diseases , such as aseptic meningitis , acute flaccid paralysis , brainstem encephalitis , and neurogenic pulmonary edema , and may result in long - term neurological sequelae , mainly in infants and young children ( alexander et al . recently , two human transmembrane proteins , p - selectin glycoprotein ligand-1 ( psgl-1 ; nishimura et al . , 2009 ) and scavenger receptor class b , member 2 ( scarb2 ; yamayoshi et al . , 2009 ) , were identified as functional receptors for ev71 and cva16 ( patel and bergelson , 2009 ) . in addition , annexin ii ( yang et al . , 2011 ) , sialic acid ( sa ; yang et al . , 2009 ) , and dendritic cell ( dc)-specific icam3-grabbing non - integrin ( dc - sign ; lin et al . , 2009b ) were found to be cellular factors involved in the early stages of ev71 infection . this review summarizes our current understanding of the ev71/cva16 receptors and their role in hev - a infection . patients with severe ev71-associated encephalitis and neurological pulmonary edema showed a significant depletion of t cells and high levels of proinflammatory cytokines ( lin et al . , 2003 ; wang et al . , 2003 ) , suggesting the possible involvement of lymphocytes in ev71 infection and the immunopathogenesis . therefore , we generated a cdna library from jurkat t cells and used it for expression cloning to identify a receptor on lymphocytes that specifically binds to ev71 . finally we identified psgl-1 as a functional ev71 receptor on jurkat t cells ( nishimura et al . , 2009 ) . p - selectin glycoprotein ligand-1 is a sialomucin leukocyte membrane protein expressed as a homodimer of disulfide - linked subunits and it can bind to three different selectins ( p , e , and l ; sako et al . , 1993 ; laszik et al . , 1996 ; somers et al . , 2000 ) the tissue distribution of psgl-1 is restricted to myeloid , lymphoid , and dendritic lineages , and platelets . psgl-1 is also expressed on dcs of lymph nodes and macrophages in the intestinal mucosa ( laszik et al . , 1996 ) , which could be the primary sites of ev71 replication . psgl-1 plays critical roles in the tethering and rolling of leukocytes for the recruitment of cells from blood vessels to the sites of acute inflammation upon stimulation by infection . we found that some representative ev71 strains bind to psgl-1 but other strains did not ( nishimura et al . , 2009 ) . according to their psgl-1 binding capability , we classified the ev71 isolates as psgl-1 binding strains ( ev71-pb ) and psgl-1-non - binding strains ( ev71-non - pb ) . the replication of ev71-pb in jurkat t cells was inhibited by anti - psgl-1 monoclonal antibody ( kpl1 ) , indicating that ev71-pb replicated in jurkat cells in a psgl-1-dependent manner . on the other hand , ev71 replicated in non - leukocyte cells ( such as rd cells ) expressing little or no psgl-1 , and the replication was not affected by kpl1 . therefore we conclude that ev71 does not use psgl-1 as the major cellular receptor on rd cells and other receptor(s ) , including scarb2 or annexin ii , may be responsible for ev71 infection in non - leukocyte cells expressing little or no psgl-1 . post - translational modifications of the n - terminal region of psgl-1 contribute the efficient binding to selectins and chemokines ( pouyani and seed , 1995 ; sako et al . , 1995 ; wilkins et al . , 1995 ; liu et al . , 1998 ; hirata et al . , 2004 ) . in this region , there are a potential o - glycosylation residue ( t57 ) and three potential tyrosine sulfation sites ( y46 , y48 , and y51 ) . we demonstrated that tyrosine sulfation , not o - glycosylation , of the n - terminal region of psgl-1 facilitates its binding to ev71-pb and viral replication in jurkat t cells ( nishimura et al . , 2010 ) . coxsackievirus a16 is genetically and antigenically related to ev71 and is a major causative agent of hfmd as well as ev71 ( oberste et al . , 2004 ) . the inoculation of l - psgl-1.1 cells , mouse l929 cells stably expressing human psgl-1 , with the prototype cva16-g-10 strain induced faint cytopathic effects ( cpe ) ( nishimura et al . , 2009 ) . this result indicated that the prototype cva16 strain may use human psgl-1 and another unidentified receptor(s ) to infect l - psgl-1.1 cells . cva16-g-10 replication in jurkat cells was not apparently inhibited by kpl1 ( nishimura et al . , 2009 ) , but significantly inhibited by a sulfation inhibitor , sodium chlorate ( nishimura et al . , 2010 ) . therefore some sulfated molecules other than psgl-1 might be involved in the replication of cva16 in jurkat cells . cva16-g-10 would use unidentified receptor(s ) to infect jurkat t cells ( nishimura et al . , 2009 ; patel and bergelson , 2009 ) . to investigate the psgl-1-dependent replication phenotype of hev - a , we tested the replication of 10 prototype hev - a strains in l - psgl-1.1 cells in the presence or absence of kpl1 ( figure 1a ) . on day 6 post - inoculation , there was no significant replication of cva4 , cva5 , cva6 , or cva8 in l - bsd cells ( blasticidin - resistant control l929 cells ) or l - psgl-1.1 cells . although higher viral titers were found for cva2 and cva7 in l - psgl-1.1 cells compared with those in l - bsd cells , replication was not affected by kpl1 . these results suggest that cva2 and cva7 may infect to l - psgl-1.1 cells in an alternative pathway via psgl-1 or glycosylated psgl-1 , without the interaction between ev71-pb and the n - terminal region of psgl-1 recognized by kpl1 . we could not demonstrate any psgl-1-dependent replication of the cva3 , cva10 , cva12 , and cva14 strains in l - psgl-1.1 cells , because they replicated even in psgl-1 negative l - bsd cells as previously reported for certain hev - a field isolates ( nadkarni and deshpande , 2003 ; yamayoshi et al . , ( a ) replication of the hev - a strains ( table 1 ) in l - psgl-1.1 cells in the presence or absence of anti - psgl-1 mab ( kpl1 ) or an isotype control . cells were inoculated with viruses at 10 ccid50/cell for 1  h , washed , and incubated in the medium , as described previously ( nishimura et al . , 2009 ) . , the cells were pretreated with 10  g / ml mab for 1  h , washed , and maintained in the medium with 10  g / ml mab . at the indicated time [ just after infection ( 0  h ) and 6  days postinfection ( 6  d ) ] , the infected cells and supernatants were freeze - thawed and viral titers were determined by ccid50 titration using rd cells . the titers are expressed as the mean and error bars indicate sd of triplicate analyses . ( b ) viral replication of hev - b , c , and d in l - psgl-1.1 cells . replication of two hev - b [ cvb3-nancy and echovirus 7 ( e7)-wallace ] and two hev - c strains [ cva21-coe and poliovirus 1 ( pv1)-sabin 1 ] , and one hev - d ( ev70-j670/71 ) strains in l - psgl-1.1 cells in the presence or absence of kpl1 or an isotype control . the titers are expressed as the mean and error bars indicate sd of triplicate analyses . coxsackievirus a7 and cva14 infection induced cpe in l - psgl-1.1 cells , but not in l - bsd cells ( table 1 ) . on the other hand , cva7 and cva14 induced cpe in l - empty cells ( puromycin - resistant control l929 cells ; table 1 ; yamayoshi et al . , 2009 ) . the difference in the cpe induction by some hev - a strains might be due to the maintenance or cultivation conditions of the mouse l929-derived cells regardless of the receptor expression of psgl-1 or scarb2 . some strains of hev - a are able to infect mouse l929 cells regardless of expression of psgl-1 or scarb2 ( nadkarni and deshpande , 2003 ; yamayoshi et al . , 2009 ) . it is therefore impossible to determine receptor usage of hev - a by simply investigating the susceptibility of mouse l929 cells expressing the putative cellular receptor . receptor usage of hev - a should be determined carefully by showing several lines of evidence such as acquisition of susceptibility by expressing a putative receptor in non - susceptible cells , loss of susceptibility by knocking down of the receptor in susceptible cells , and direct binding of the virus to the receptor , etc . blasticidin - resistant l929 cells ( a negative control for l - psgl-1.1 cells ) . puromycin - resistant l929 cells ( a negative control for l - scarb2 cells ) . prototype cva4 strain ( high point ) is unavailable from atcc , therefore we used an in - house reference strain of cva4 , the jr strain . l - psgl-1.1 cells did not support psgl-1-dependent replication of the hev - b and hev - c strains ( figure 1b ) ; however , the prototype ev70 strain ( hev - d ) replicated in l - psgl-1.1 cells more efficiently than in l - bsd cells . although ev70 replication was not affected by kpl1 ( figure 1b ) , we can not exclude the possibility that ev70 utilizes 2,3-linked sa , which could be a receptor for ev70 ( nokhbeh et al . , 2005 ) . recently we reported that that four out of five ev71-pb strains replicated poorly in l - psgl-1.1 cells ( miyamura et al . , 2011 ) . we found that ev71 variants , which were propagated once in l - psgl-1.1 cells , have several possible adaptive mutations , including a putative amino acid determinant of the adaptive phenotype in l - psgl-1.1 cells at vp2 - 149 ( miyamura et al . , 2011 ) . the results suggest that adaptive mutations , along with a pb phenotype , may facilitate efficient psgl-1-dependent replication of the ev71 variants in l - psgl-1.1 cells . it is possible that hev - a strains other than ev71 also require adaptive mutations for efficient replication in l - psgl-1.1 cells . yamayoshi et al . ( 2009 ) identified scarb2 ( also known as lysosomal integral membrane protein ii , or cd36b like-2 ) as an ev71 receptor on rd cells , widely used for isolation of ev71 from clinical specimens . they transfected ev71-non - susceptible mouse l929 cells with the genomic dna of rd cells and selected two cell clones that were susceptible for ev71 infection . by a transcriptome analysis , scavenger receptor class b , member 2 is a heavily n - glycosylated type iii transmembrane protein consists from 478 amino acids and belongs to the cd36 family of scavenger receptor proteins ( fujita et al . , 1992 ; calvo et al . , scarb2 has a n - terminal transmembrane domain , a 400 amino acid lumeral domain , a c - terminal transmembrane domain , and a c - terminal cytoplasmic tail of 20 amino acids ( fujita et al . , scarb2 involves in an enlargement of early endosomes and late endosomes / lysosomes and an impairment of endocytic membrane out of the enlarged compartments ( kuronita et al . , 2002 ) . scarb2 deficiency caused ureteric pelvic junction obstruction , deafness , and peripheral neuropathy in mice ( gamp et al . scarb2 is expressed ubiquitously in human tissues ( eskelinen et al . , 2003 ) ; therefore , it might be involved in systemic ev71 infections ( yamayoshi et al . , 2009 ) . human scarb2 has 10 potential n - glycosylation sites ( fujita et al . , 1992 ) . but the carbohydrate chains of human scarb2 are not essential for the interaction between ev71 and human scarb2 ( yamayoshi and koike , 2011 ) . experiments using a series of chimeric proteins between human and mouse scarb2 identified that the amino acids 142204 of human scarb2 ( encoded by human scarb2 exon 4 ) are responsible for ev71 binding and infection ( yamayoshi and koike , 2011 ) . mouse l929 cells expressing human scarb2 in the presence of puromycin ( l - scarb2 cells ) permitted the replication of all ev71 strains tested , including the non - pb strains ( yamayoshi et al . cva16 induced cpe in l - scarb2 cells , whereas cva2 , cva3 , cva4 , cva5 , cva6 , cva8 , and cva12 did not . cva16 grew efficiently in l - scarb2 , whereas cva2 , cva3 , cva4 , cva5 , cva6 , cva8 , and cva12 did not ( table 1 ) . ( 2009 ) concluded that cva16 also infect l - scarb2 cells in a scarb2-dependent manner and that infection with most other hev - a is not dependent upon scarb2 . cva7 , cva10 , and cva14 induced cpe in both l - empty cells and l - scarb2 cells ( yamayoshi et al . , 2009 ) . they could not determine whether the cpe induced by these viruses were due to human scarb2mediated infection . yang et al . ( 2011 ) identified annexin ii as an ev71 vp1-binding protein on rd cells . using a recombinant vp1 protein of ev71 fused with a calmodulin - binding peptide , they tried to identify vp1-binding proteins from the total cellular proteins of rd cells . a virus - overlay protein - binding assay followed by a mass spectrometry analysis identified annexin ii as a vp1-binding protein . annexin ii is a member of the annexin family  the multifunctional phospholipid - binding proteins . annexin ii on the surface of endothelial cells acts as a profibrinolytic coreceptor for both plasminogen and tissue plasminogen activator facilitating the generation of plasmin ( kim and hajjar , 2002 ) . the interaction to annexin ii was specific to ev71 ; cva16 did not bind to annexin ii in the virus - overlay protein - binding assay ( yang et al . , 2011 ) . sialic acid is usually found as terminal monosaccharides on the glycan chains of glycolipids and glycoproteins ( varki and varki , 2007 ) . coxsackievirus a24 variant ( cva24v ) uses sa - containing glycoconjugates as attachment receptors on corneal cells ( nilsson et al . , 2008 ) . yang et al . ( 2009 ) hypothesized that sa would be important for ev71 infection , as the transmission route of ev71 and cva24v is fecal - oral and/or droplet - aerosol route . ev71 infection to dld-1 intestinal cells was inhibited by an o - glycan synthesis inhibitor , but not by an n - glycan synthesis inhibitor . furthermore , dld-1 cells co - cultured with sa - linked galactose significantly reduced the ev71 infection . recently , neu5ac2,3gal disaccharides on psgl-1 were reported as a candidate receptor of cva24v ( mistry et al . , 2011 ) . it is unknown whether other enteroviruses , including hev - a , recognize sa - containing glycans as the entry receptors . dendritic cells play crucial roles in antiviral immunity by functioning as professional antigen - presenting cells to prime t cells and by secreting cytokines to modulate immune responses . in a mouse model of ev71 infection , dcs from the brains of ev71-infected , but not of uninfected , mice expressed viral antigen and primed t cells efficiently ( lin et al . , 2009a ) . lin et al . ( 2009b ) reported that ev71 infection enhances mouse dcs to elicit protective immune response and also found that ev71 infects human immature dcs and that viral entry is partially inhibited by anti - dc - sign antibody . it is essential to characterize the role of dc - sign and other receptors for ev71 in dcs for understanding the host immunological responses and immunopathogenesis of hev - a including ev71 . identification of psgl-1 and scarb2 as the cellular receptors for ev71 and cva16 has advanced our understanding of the early stages of hev - a infections at the molecular level . however , further experiments using clinical hev - a isolates are necessary to clarify the general role of psgl-1 and scarb2 in hev - a infection and their pathogenesis . most of the prototype ( laboratory - adapted ) hev - a strains other than ev71 and cva16 may use unidentified receptor(s ) to infect susceptible human cells such as rd cells . characterization of the identified and unidentified hev - a receptors is essential to understand the mechanism of hev - a infection and development of a diverse array of the clinical outcomes of hev - a - associated diseases . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
human enterovirus species a ( hev - a ) is one of the four species of hev in the genus enterovirus in the family picornaviridae . among hev 
 - a , coxsackievirus a16 ( cva16 ) and enterovirus 71 ( ev71 ) are the major causative agents of hand , foot , and mouth disease ( hfmd ) . 
 some other types of hev - a are commonly associated with herpangina . although hfmd and herpangina due to hev - a are common febrile diseases among infants and children , ev71 can cause various neurological diseases , such as aseptic meningitis and fatal encephalitis . 
 recently , two human transmembrane proteins , p - selectin glycoprotein ligand-1 ( psgl-1 ) and scavenger receptor class b , member 2 ( scarb2 ) , were identified as functional receptors for ev71 and cva16 . in in vitro infection experiments using the prototype hev - a strains , psgl-1 and scarb2 could be responsible for the specific receptors for ev71 and cva16 . 
 however , the involvement of both receptors in the in vitro and in vivo infections of clinical isolates of hev - a has not been clarified yet . to elucidate a diverse array of the clinical outcome of hev - a - associated diseases 
 , the identification and characterization of hev - a receptors may provide useful information in understanding the hev - a pathogenesis at a molecular level .