Cellular Receptors for Human Enterovirus Species A

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

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a highly sensitive detection of enterovirus in the stool samples of acute flaccid paralysis cases

<p>Abstract</p> <p>Background</p> <p>In the global eradication program for poliomyelitis, the laboratory diagnosis plays a critical role by isolating poliovirus (PV) from the stool samples of acute flaccid paralysis (AFP) cases. In this study, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a rapid and highly sensitive detection of enterovirus including PV to identify stool samples positive for enterovirus including PV.</p> <p>Methods</p> <p>A primer set was designed for RT-LAMP to detect enterovirus preferably those with PV-like 5'NTRs of the viral genome. The sensitivity of RT-LAMP system was evaluated with prototype strains of enterovirus. Detection of enterovirus from stool extracts was examined by using RT-LAMP system.</p> <p>Results</p> <p>We detected at least 400 copies of the viral genomes of PV(Sabin) strains within 90 min by RT-LAMP with the primer set. This RT-LAMP system showed a preference for <it>Human enterovirus species C </it>(HEV-C) strains including PV, but exhibited less sensitivity to the prototype strains of HEV-A and HEV-B (detection limits of 7,400 to 28,000 copies). Stool extracts, from which PV, HEV-C, or HEV-A was isolated in the cell culture system, were mostly positive by RT-LAMP method (positive rates of 15/16 (= 94%), 13/14 (= 93%), and 4/4 (= 100%), respectively). The positive rate of this RT-LAMP system for stool extracts from which HEV-B was isolated was lower than that of HEV-C (positive rate of 11/21 (= 52%)). In the stool samples, which were negative for enterovirus isolation by the cell culture system, we found that two samples were positive for RT-LAMP (positive rates of 2/38 (= 5.3%)). In these samples, enterovirus 96 was identified by sequence analysis utilizing a seminested PCR system.</p> <p>Conclusions</p> <p>RT-LAMP system developed in this study showed a high sensitivity comparable to that of the cell culture system for the detection of PV, HEV-A, and HEV-C, but less sensitivity to HEV-B. This RT-LAMP system would be useful for the direct detection of enterovirus from the stool extracts.</p

Acute Encephalitis Caused by Intrafamilial Transmission of Enterovirus 71 in Adult

Enterovirus 71 (EV71) is a common cause of hand, foot, and mouth disease and sometimes causes severe neurologic complications, mainly in children. We report a case of adult-onset encephalitis caused by intrafamilial transmission of a subgenogroup C4 strain of EV71. This case elucidates the risk for EV71 encephalitis even in adults

Tyrosine Sulfation of the Amino Terminus of PSGL-1 Is Critical for Enterovirus 71 Infection

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease, a common febrile disease in children; however, EV71 has been also associated with various neurological diseases including fatal cases in large EV71 outbreaks particularly in the Asia Pacific region. Recently we identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a cellular receptor for entry and replication of EV71 in leukocytes. PSGL-1 is a sialomucin expressed on the surface of leukocytes, serves as a high affinity counterreceptor for selectins, and mediates leukocyte rolling on the endothelium. The PSGL-1–P-selectin interaction requires sulfation of at least one of three clustered tyrosines and an adjacent O-glycan expressing sialyl Lewis x in an N-terminal region of PSGL-1. To elucidate the molecular basis of the PSGL-1–EV71 interaction, we generated a series of PSGL-1 mutants and identified the post-translational modifications that are critical for binding of PSGL-1 to EV71. We expressed the PSGL-1 mutants in 293T cells and the transfected cells were assayed for their abilities to bind to EV71 by flow cytometry. We found that O-glycosylation on T57, which is critical for PSGL-1–selectin interaction, is not necessary for PSGL-1 binding to EV71. On the other hand, site-directed mutagenesis at one or more potential tyrosine sulfation sites in the N-terminal region of PSGL-1 significantly impaired PSGL-1 binding to EV71. Furthermore, an inhibitor of sulfation, sodium chlorate, blocked the PSGL-1–EV71 interaction and inhibited PSGL-1-mediated viral replication of EV71 in Jurkat T cells in a dose-dependent manner. Thus, the results presented in this study reveal that tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes

Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface.

Enterovirus A71 (EV-A71) infection involves a variety of receptors. Among them, two transmembrane protein receptors have been investigated in detail and shown to be critical for infection: P-selectin glycoprotein ligand-1 (PSGL-1) in lymphocytes (Jurkat cells), and scavenger receptor class B member 2 (SCARB2) in rhabdomyosarcoma (RD) cells. PSGL-1 and SCARB2 have been reported to be expressed on the surface of Jurkat and RD cells, respectively. In the work reported here, we investigated the roles of PSGL-1 and SCARB2 in the process of EV-A71 entry. We first examined the expression of SCARB2 in Jurkat cells, and detected it within the cytoplasm, but not on the cell surface. Further, using PSGL-1 and SCARB2 knockout cells, we found that although both PSGL-1 and SCARB2 are essential for virus infection of Jurkat cells, virus attachment to these cells requires only PSGL-1. These results led us to evaluate the cell surface expression and the roles of SCARB2 in other EV-A71-susceptible cell lines. Surprisingly, in contrast to the results of previous studies, we found that SCARB2 is absent from the surface of RD cells and other susceptible cell lines we examined, and that although SCARB2 is essential for infection of these cells, it is dispensable for virus attachment. These results indicate that a receptor other than SCARB2 is responsible for virus attachment to the cell and probably for internalization of virions, not only in Jurkat cells but also in RD cells and other EV-A71-susceptible cells. SCARB2 is highly concentrated in lysosomes and late endosomes, where it is likely to trigger acid-dependent uncoating of virions, the critical final step of the entry process. Our results suggest that the essential interactions between EV-A71 and SCARB2 occur, not at the cell surface, but within the cell

Experimental Mucosal Infection with Molecularly Cloned Feline Immunodeficiency Viruses

Four of six specific pathogen-free cats were infected after intravaginal exposure to molecularly cloned lymphotropic but non-Crandell feline kidney (CRFK)-tropic feline immunodeficiency virus strain TM2 and its AP-1 deletion mutant. The sequences of the env V3-to-V5 region which defines the CRFK tropism were unchanged in the infected cats through the infection. These data suggest that the strain was transmitted across the mucosal epithelium without a broadening of cell tropism

The Suramin Derivative NF449 Interacts with the 5-fold Vertex of the Enterovirus A71 Capsid to Prevent Virus Attachment to PSGL-1 and Heparan Sulfate.

NF449, a sulfated compound derived from the antiparasitic drug suramin, was previously reported to inhibit infection by enterovirus A71 (EV-A71). In the current work, we found that NF449 inhibits virus attachment to target cells, and specifically blocks virus interaction with two identified receptors--the P-selectin ligand, PSGL-1, and heparan sulfate glycosaminoglycan--with no effect on virus binding to a third receptor, the scavenger receptor SCARB2. We also examined a number of commercially available suramin analogues, and newly synthesized derivatives of NF449; among these, NF110 and NM16, like NF449, inhibited virus attachment at submicromolar concentrations. PSGL-1 and heparan sulfate, but not SCARB2, are both sulfated molecules, and their interaction with EV-A71 is thought to involve positively charged capsid residues, including a conserved lysine at VP1-244, near the icosahedral 5-fold vertex. We found that mutation of VP1-244 resulted in resistance to NF449, suggesting that this residue is involved in NF449 interaction with the virus capsid. Consistent with this idea, NF449 and NF110 prevented virus interaction with monoclonal antibody MA28-7, which specifically recognizes an epitope overlapping VP1-244 at the 5-fold vertex. Based on these observations we propose that NF449 and related compounds compete with sulfated receptor molecules for a binding site at the 5-fold vertex of the EV-A71 capsid