Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus.
(17/171)
Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus of the family Paramyxoviridae and are unique in that they exhibit a broad species tropism and cause fatal disease in both animals and humans. They infect cells through a pH-independent membrane fusion process mediated by their fusion and attachment glycoproteins. Previously, we demonstrated identical cell fusion tropisms for HeV and NiV and the protease-sensitive nature of their unknown cell receptor and identified a human cell line (HeLa-USU) that was nonpermissive for fusion and virus infection. Here, a microarray analysis was performed on the HeLa-USU cells, permissive HeLa-CCL2 cells, and two other permissive human cell lines. From this analysis, we identified a list of genes encoding known and predicted plasma membrane surface-expressed proteins that were highly expressed in all permissive cells and absent from the HeLa-USU cells and rank-ordered them based on their relative levels. Available expression vectors containing the first 10 genes were obtained and individually transfected into HeLa-USU cells. One clone, encoding human ephrin-B2 (EFNB2), was found capable of rendering HeLa-USU cells permissive for HeV- and NiV-mediated cell fusion as well as infection by live virus. A soluble recombinant EFNB2 could potently block fusion and infection and bind soluble recombinant HeV and NiV attachment glycoproteins with high affinity. Together, these data indicate that EFNB2 serves as a functional receptor for both HeV and NiV. The highly conserved nature of EFNB2 in humans and animals is consistent with the broad tropism exhibited by these emerging zoonotic viruses. (+info)
Purification and characterization of Nipah virus nucleocapsid protein produced in insect cells.
(18/171)
The nucleocapsid (N) protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use as a diagnostic reagent, the NiV N gene was cloned into the pFastBacHT vector and the His-tagged fusion protein was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-NiV antibodies produced a band of approximately 62 kDa. A time course study showed that the highest level of expression was achieved after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel-nitrilotriacetic acid resin column revealed different types of structures, including spherical, ring-like, and herringbone-like particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hyrdrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein revealed a high A(260)/A(280) ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay results showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition. (+info)
EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus.
(19/171)
Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70 percent of infected patients, and there is evidence of human-to-human transmission. Endothelial syncytia, comprised of multinucleated giant-endothelial cells, are frequently found in NiV infections, and are mediated by the fusion (F) and attachment (G) envelope glycoproteins. Identification of the receptor for this virus will shed light on the pathobiology of NiV infection, and spur the rational development of effective therapeutics. Here we report that ephrinB2, the membrane-bound ligand for the EphB class of receptor tyrosine kinases (RTKs), specifically binds to the attachment (G) glycoprotein of NiV. Soluble Fc-fusion proteins of ephrinB2, but not ephrinB1, effectively block NiV fusion and entry into permissive cell types. Moreover, transfection of ephrinB2 into non-permissive cells renders them permissive for NiV fusion and entry. EphrinB2 is expressed on endothelial cells and neurons, which is consistent with the known cellular tropism for NiV. Significantly, we find that NiV-envelope-mediated infection of microvascular endothelial cells and primary cortical rat neurons is inhibited by soluble ephrinB2, but not by the related ephrinB1 protein. Cumulatively, our data show that ephrinB2 is a functional receptor for NiV. (+info)
Nipah virus in Lyle's flying foxes, Cambodia.
(20/171)
We conducted a survey in Cambodia in 2000 on henipavirus infection among several bat species, including flying foxes, and persons exposed to these animals. Among 1,072 bat serum samples tested by enzyme-linked immunosorbent assay, antibodies reactive to Nipah virus (NiV) antigen were detected only in Pteropus lylei species; Cynopterus sphinx, Hipposideros larvatus, Scotophilus kuhlii, Chaerephon plicata, Taphozous melanopogon, and T. theobaldi species were negative. Seroneutralization applied on a subset of 156 serum samples confirmed these results. None of the 8 human serum samples was NiV seropositive with the seroneutralization test. One virus isolate exhibiting cytopathic effect with syncytia was obtained from 769 urine samples collected at roosts of P. lylei specimens. Partial molecular characterization of this isolate demonstrated that it was closely related to NiV. These results strengthen the hypothesis that flying foxes could be the natural host of NiV. Surveillance of human cases should be implemented. (+info)
Genetic characterization of Nipah virus, Bangladesh, 2004.
(21/171)
Until 2004, identification of Nipah virus (NV)-like outbreaks in Bangladesh was based on serology. We describe the genetic characterization of a new strain of NV isolated during outbreaks in Bangladesh (NV-B) in 2004, which confirms that NV was the etiologic agent responsible for these outbreaks. (+info)
Potent neutralization of Hendra and Nipah viruses by human monoclonal antibodies.
(22/171)
Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae. Each has a broad species tropism and can cause disease with high mortality in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) envelope glycoproteins (Envs). Seven Fabs, m101 to -7, were selected for their significant binding to a soluble form of Hendra G (sG) which was used as the antigen for panning of a large naive human antibody library. The selected Fabs inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to immunoglobulin G1 (IgG1) significantly increased its cell fusion inhibitory activity: the 50% inhibitory concentration was decreased more than 10-fold to approximately 1 microg/ml. The IgG1 m101 was also exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 microg/ml, and 98% neutralization required only 1.6 microg/ml. The inhibition of fusion and infection correlated with binding of the Fabs to full-length G as measured by immunoprecipitation and less with binding to sG as measured by enzyme-linked immunosorbent assay and Biacore. m101 and m102 competed with the ephrin-B2, which we recently identified as a functional receptor for both HeV and NiV, indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other, suggesting that they bind to overlapping epitopes which are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine-scanning mutants and identified two mutants, P185A and Q191 K192A, which significantly decreased binding to m101 and one, G183, which decreased binding of m102 to G. These results suggest that m101 to -7 are specific for HeV or NiV or both and exhibit various neutralizing activities; they are the first human monoclonal antibodies identified against these viruses and could be used for treatment, prophylaxis, and diagnosis and as research reagents and could aid in the development of vaccines. (+info)
Antibody prophylaxis and therapy against Nipah virus infection in hamsters.
(23/171)
Nipah virus (NiV), a member of the Paramyxoviridae family, causes a zoonotic infection in which the reservoir, the fruit bat, may pass the infection to pigs and eventually to humans. In humans, the infection leads to encephalitis with >40 to 70% mortality. We have previously shown that polyclonal antibody directed to either one of two glycoproteins, G (attachment protein) or F (fusion protein), can protect hamsters from a lethal infection. In the present study, we have developed monoclonal antibodies (MAbs) to both glycoproteins and assessed their ability to protect animals against lethal NiV infection. We show that as little as 1.2 mug of an anti-G MAb protected animals, whereas more than 1.8 mug of anti-F MAb was required to completely protect the hamsters. High levels of either anti-G or anti-F MAbs gave a sterilizing immunity, whereas lower levels could protect against a fatal infection but resulted in an increase in anti-NiV antibodies starting 18 days after the viral challenge. Using reverse transcriptase PCR, the presence of NiV in the different organs could not be observed in MAb-protected animals. When the MAbs were given after infection, partial protection (50%) was observed with the anti-G MAbs when the animals were inoculated up to 24 h after infection, but administration of the anti-F MAbs protected some animals (25 to 50%) inoculated later during the infection. Our studies suggest that immunotherapy could be used for people who are exposed to NiV infections. (+info)
A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L.
(24/171)
The Nipah virus fusion (F) protein is proteolytically processed to F1 + F2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form. (+info)