RESUMO
Hendra (HeV) and Nipah (NiV) viruses are emerging zoonotic pathogens in the Henipavirus genus causing outbreaks of disease with very high case fatality rates. Here, we report the first naturally occurring human monoclonal antibodies (mAbs) against HeV receptor binding protein (RBP). All isolated mAbs neutralized HeV, and some also neutralized NiV. Epitope binning experiments identified five major antigenic sites on HeV-RBP. Animal studies demonstrated that the most potent cross-reactive neutralizing mAbs, HENV-26 and HENV-32, protected ferrets in lethal models of infection with NiV Bangladesh 3 days after exposure. We solved the crystal structures of mAb HENV-26 in complex with both HeV-RBP and NiV-RBP and of mAb HENV-32 in complex with HeV-RBP. The studies reveal diverse sites of vulnerability on RBP recognized by potent human mAbs that inhibit virus by multiple mechanisms. These studies identify promising prophylactic antibodies and define protective epitopes that can be used in rational vaccine design.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus Hendra/imunologia , Henipavirus/imunologia , Testes de Neutralização , Vírus Nipah/imunologia , Receptores Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Antígenos Virais/imunologia , Sítios de Ligação , Ligação Competitiva , Encéfalo/patologia , Quirópteros/virologia , Reações Cruzadas/imunologia , Cristalografia por Raios X , Efrina-B2/metabolismo , Feminino , Furões/virologia , Humanos , Interferometria , Fígado/patologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Receptores Virais/química , Receptores Virais/metabolismoRESUMO
Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse, and hamster target cells using a different, yet unknown, receptor than Nipah virus (NiV) and Hendra virus (HeV) and that NiV- and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryoelectron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing their conformational landscape and distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs.
Assuntos
Vírus Hendra , Infecções por Henipavirus , Henipavirus , Vírus Nipah , Humanos , Animais , Camundongos , Microscopia Crioeletrônica , Glicoproteínas , Internalização do VírusRESUMO
The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.
Assuntos
Vírus Hendra , Lyssavirus , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Ribossômico , Lyssavirus/genética , Lyssavirus/metabolismo , Ribossomos/metabolismo , Vírus Hendra/genética , Vírus Hendra/metabolismo , Fatores de TranscriçãoRESUMO
The henipaviruses, including Nipah virus (NiV) and Hendra virus (HeV), are biosafety level 4 (BSL-4) zoonotic pathogens that cause severe neurological and respiratory disease in humans. To study the replication machinery of these viruses, we developed robust minigenome systems that can be safely used in BSL-2 conditions. The nucleocapsid (N), phosphoprotein (P), and large protein (L) of henipaviruses are critical elements of their replication machinery and thus essential support components of the minigenome systems. Here, we tested the effects of diverse combinations of the replication support proteins on the replication capacity of the NiV and HeV minigenomes by exchanging the helper plasmids coding for these proteins among the two viruses. We demonstrate that all combinations including one or more heterologous proteins were capable of replicating both the NiV and HeV minigenomes. Sequence alignment showed identities of 92% for the N protein, 67% for P, and 87% for L. Notably, variations in amino acid residues were not concentrated in the N-P and P-L interacting regions implying that dissimilarities in amino acid composition among NiV and HeV polymerase complex proteins may not impact their interactions. The observed indiscriminate activity of NiV and HeV polymerase complex proteins is different from related viruses, which can support the replication of heterologous genomes only when the whole polymerase complex belongs to the same virus. This newly observed promiscuous property of the henipavirus polymerase complex proteins likely attributed to their conserved interaction regions could potentially be harnessed to develop universal anti-henipavirus antivirals.IMPORTANCEGiven the severity of disease induced by Hendra and Nipah viruses in humans and the continuous emergence of new henipaviruses as well as henipa-like viruses, it is necessary to conduct a more comprehensive investigation of the biology of henipaviruses and their interaction with the host. The replication of henipaviruses and the development of antiviral agents can be studied in systems that allow experiments to be performed under biosafety level 2 conditions. Here, we developed robust minigenome systems for the Nipah virus (NiV) and Hendra virus (HeV) that provide a convenient alternative for studying NiV and HeV replication. Using these systems, we demonstrate that any combination of the three polymerase complex proteins of NiV and HeV could effectively initiate the replication of both viral minigenomes, which suggests that the interaction regions of the polymerase complex proteins could be effective targets for universal and effective anti-henipavirus interventions.
Assuntos
Genoma Viral , Vírus Nipah , Replicação Viral , Vírus Nipah/genética , Vírus Nipah/fisiologia , Humanos , Proteínas Virais/metabolismo , Proteínas Virais/genética , Vírus Hendra/genética , Vírus Hendra/metabolismo , Vírus Hendra/fisiologia , Animais , Henipavirus/genética , Henipavirus/metabolismo , Infecções por Henipavirus/virologia , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Linhagem CelularRESUMO
Nipah virus (NiV) is a highly pathogenic paramyxovirus capable of causing severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, underscoring the urgent need for the development of countermeasures. The NiV surface-displayed glycoproteins, NiV-G and NiV-F, mediate host cell attachment and fusion, respectively, and are heavily targeted by host antibodies. Here, we describe a vaccination-derived neutralizing monoclonal antibody, mAb92, that targets NiV-F. Structural characterization of the Fab region bound to NiV-F (NiV-F-Fab92) by cryo-electron microscopy analysis reveals an epitope in the DIII domain at the membrane distal apex of NiV-F, an established site of vulnerability on the NiV surface. Further, prophylactic treatment of hamsters with mAb92 offered complete protection from NiV disease, demonstrating beneficial activity of mAb92 in vivo. This work provides support for targeting NiV-F in the development of vaccines and therapeutics against NiV.IMPORTANCENipah virus (NiV) is a highly lethal henipavirus (HNV) that causes severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, highlighting a need to develop countermeasures. The NiV surface displays the receptor binding protein (NiV-G, or RBP) and the fusion protein (NiV-F), which allow the virus to attach and enter cells. These proteins can be targeted by vaccines and antibodies to prevent disease. This work describes a neutralizing antibody (mAb92) that targets NiV-F. Structural characterization by cryo-electron microscopy analysis reveals where the antibody binds to NiV-F to neutralize the virus. This study also shows that prophylactic treatment of hamsters with mAb92 completely protected against developing NiV disease. This work shows how targeting NiV-F can be useful to preventing NiV disease, supporting future studies in the development of vaccines and therapeutics.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Infecções por Henipavirus , Vírus Nipah , Proteínas Virais de Fusão , Vírus Nipah/imunologia , Animais , Infecções por Henipavirus/prevenção & controle , Infecções por Henipavirus/imunologia , Anticorpos Monoclonais/imunologia , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Cricetinae , Humanos , Microscopia Crioeletrônica , Epitopos/imunologia , MesocricetusRESUMO
Batborne henipaviruses, such as Nipah and Hendra viruses, represent a major threat to global health due to their propensity for spillover, severe pathogenicity, and high mortality rate in human hosts. Coupled with the absence of approved vaccines or therapeutics, work with the prototypical species and uncharacterized, emergent species is restricted to high biocontainment facilities. There is a scarcity of such specialized spaces for research, and often, the scope and capacity of research, which can be conducted at BSL-4, is limited. Therefore, there is a pressing need for innovative life-cycle modeling systems to enable comprehensive research within lower biocontainment settings. This work showcases tetracistronic, transcription, and replication-competent minigenomes for the Nipah, Hendra, and Cedar viruses, which encode viral proteins facilitating budding, fusion, and receptor binding. We validate the functionality of all encoded viral proteins and demonstrate a variety of applications to interrogate the viral life cycle. Notably, we found that the Cedar virus replicase exhibits remarkable promiscuity, efficiently driving replication and transcription of minigenomes from all tested henipaviruses. We also apply this technology to Ghana virus (GhV), an emergent species that has so far not been isolated in culture. We demonstrate that the reported sequence of GhV is incomplete, but that this missing sequence can be substituted with analogous sequences from other henipaviruses. The use of our GhV system establishes the functionality of the GhV replicase and identifies two antivirals that are highly efficacious against the GhV polymerase. IMPORTANCE: Henipaviruses are recognized as significant global health threats due to their high mortality rates and lack of effective vaccines or therapeutics. Due to the requirement for high biocontainment facilities, the scope of research which may be conducted on henipaviruses is limited. To address this challenge, we developed innovative tetracistronic, transcription, and replication-competent minigenomes. We demonstrate that these systems replicate key aspects of the viral life cycle, such as budding, fusion, and receptor binding, and are safe for use in lower biocontainment settings. Importantly, the application of this system to the Ghana virus revealed that its known sequence is incomplete; however, substituting the missing sequences with those from other henipaviruses allowed us to overcome this challenge. We demonstrate that the Ghana virus replicative machinery is functional and can identify two orally efficacious antivirals effective against it. Our research offers a versatile system for life-cycle modeling of highly pathogenic henipaviruses at low biocontainment.
Assuntos
Genoma Viral , Henipavirus , Replicação Viral , Humanos , Henipavirus/genética , Regiões Promotoras Genéticas , Animais , Proteínas Virais/genética , Proteínas Virais/metabolismo , Infecções por Henipavirus/virologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Vírus Nipah/genética , Vírus Hendra/genéticaRESUMO
Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.
Assuntos
Infecções por Henipavirus , Henipavirus , Receptores Virais , Humanos , Aminoácidos/genética , Anticorpos Monoclonais/metabolismo , Proteínas de Transporte/metabolismo , Efrina-B3/genética , Efrina-B3/química , Efrina-B3/metabolismo , Epitopos/genética , Epitopos/metabolismo , Gana , Vírus Hendra/metabolismo , Henipavirus/classificação , Henipavirus/genética , Henipavirus/metabolismo , Mutagênese , Vírus Nipah/metabolismo , Proteínas do Envelope Viral/genética , Internalização do Vírus , Receptores Virais/metabolismoRESUMO
First reported in August 2022, the Langya virus (LayV) has emerged as a potential global health threat in the post-COVID-19 era. Preliminary reports show that 35 patients near Shandong and Henan, China experienced a febrile acute LayV infection. We conducted this review following the PRISMA protocol to synthesise current knowledge on LayV's characteristics in terms of molecular, clinical, and public health perspectives. This virus belongs to the Paramyxoviridae family and carries a non-segmented, single-stranded negative-sense RNA genome. Shrews may be the natural reservoir of the virus. Clinical symptoms range from mild flu-like symptoms to severe manifestations involving pneumonia, haematological disorders, and organ dysfunction. Diagnostic methods include PCR and ELISA assays. Despite the absence of established treatments, antiviral drugs such as ribavirin and chloroquine may be useful in some cases. In light of prevention, a comprehensive approach that emphasises multidisciplinary collaboration is crucial for early surveillance and response. Urgent global efforts are needed for vaccine development and preparedness against this potential pandemic threat. As the viral dynamics remain uncertain, a proactive approach is vital to mitigate the impact of not only LayV but also future threats on a large scale in long term.
Assuntos
COVID-19 , Henipavirus , Zoonoses , Animais , Humanos , Zoonoses/epidemiologia , Zoonoses/prevenção & controle , SARS-CoV-2 , Antivirais/uso terapêuticoRESUMO
SignificanceConcern has increased about the pandemic potential of Nipah virus (NiV). Similar to SARS-CoV-2, NiV is an RNA virus that is transmitted by respiratory droplets. There are currently no NiV vaccines licensed for human use. While several preventive vaccines have shown promise in protecting animals against lethal NiV disease, most studies have assessed protection 1 mo after vaccination. However, in order to contain and control outbreaks, vaccines that can rapidly confer protection in days rather than months are needed. Here, we show that a recombinant vesicular stomatitis virus vector expressing the NiV glycoprotein can completely protect monkeys vaccinated 7 d prior to NiV exposure and 67% of animals vaccinated 3 d before NiV challenge.
Assuntos
Infecções por Henipavirus/veterinária , Vírus Nipah/imunologia , Doenças dos Primatas/prevenção & controle , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Biomarcadores , Vetores Genéticos , Estimativa de Kaplan-Meier , Testes de Neutralização , Avaliação de Resultados em Cuidados de Saúde , Doenças dos Primatas/diagnóstico , Doenças dos Primatas/mortalidade , Doenças dos Primatas/virologia , Vacinação , Carga ViralRESUMO
Nipah virus Bangladesh (NiVB) is a bat-borne zoonosis transmitted between people through the respiratory route. The risk posed by related henipaviruses, including Hendra virus (HeV) and Nipah virus Malaysia (NiVM), is less clear. We conducted a broad search of the literature encompassing both human infections and animal models to synthesize evidence about potential for person-to-person spread. More than 600 human infections have been reported in the literature, but information on viral shedding was only available for 40 case-patients. There is substantial evidence demonstrating person-to-person transmission of NiVB, and some evidence for NiVM. Less direct evidence is available about the risk for person-to-person transmission of HeV, but animals infected with HeV shed more virus in the respiratory tract than those infected with NiVM, suggesting potential for transmission. As the group of known henipaviruses continues to grow, shared protocols for conducting and reporting from human investigations and animal experiments are urgently needed.
Assuntos
Vírus Hendra , Infecções por Henipavirus , Vírus Nipah , Animais , Humanos , Infecções por Henipavirus/transmissão , Malásia , Zoonoses/transmissãoRESUMO
IMPORTANCE: Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and regulates multiple cell developmental and signaling processes. It also functions as the cell entry receptor for Nipah virus and Hendra virus, zoonotic viruses that can cause respiratory and/or neurological symptoms in humans with high mortality. Here, we investigate the sequence basis of EFNB2 specificity for binding the Nipah virus attachment G glycoprotein over Eph receptors. We then use this information to engineer EFNB2 as a soluble decoy receptor that specifically binds the attachment glycoproteins of the Nipah virus and other related henipaviruses to neutralize infection. These findings further mechanistic understanding of protein selectivity and may facilitate the development of diagnostics or therapeutics against henipavirus infection.
Assuntos
Efrina-B2 , Vírus Hendra , Infecções por Henipavirus , Vírus Nipah , Proteínas Virais , Humanos , Efrina-B2/genética , Efrina-B2/metabolismo , Glicoproteínas/metabolismo , Ligantes , Proteínas Virais/metabolismoRESUMO
Langya virus (LayV) is a paramyxovirus in the Henipavirus genus, closely related to the deadly Nipah (NiV) and Hendra (HeV) viruses, that was identified in August 2022 through disease surveillance following animal exposure in eastern China. Paramyxoviruses present two glycoproteins on their surface, known as attachment and fusion proteins, that mediate entry into cells and constitute the primary antigenic targets for immune response. Here, we determine cryo-electron microscopy (cryo-EM) structures of the uncleaved LayV fusion protein (F) ectodomain in pre- and postfusion conformations. The LayV-F protein exhibits pre- and postfusion architectures that, despite being highly conserved across paramyxoviruses, show differences in their surface properties, in particular at the apex of the prefusion trimer, that may contribute to antigenic variability. While dramatic conformational changes were visualized between the pre- and postfusion forms of the LayV-F protein, several domains remained invariant, held together by highly conserved disulfides. The LayV-F fusion peptide (FP) is buried within a highly conserved, hydrophobic interprotomer pocket in the prefusion state and is notably less flexible than the rest of the protein, highlighting its "spring-loaded" state and suggesting that the mechanism of pre-to-post transition must involve perturbations to the pocket and release of the fusion peptide. Together, these results offer a structural basis for how the Langya virus fusion protein compares to its Henipavirus relatives and propose a mechanism for the initial step of pre- to postfusion conversion that may apply more broadly to paramyxoviruses. IMPORTANCE The Henipavirus genus is quickly expanding into new animal hosts and geographic locations. This study compares the structure and antigenicity of the Langya virus fusion protein to other henipaviruses, which have important vaccine and therapeutic development implications. Furthermore, the study proposes a new mechanism to explain the early steps of the fusion initiation process that can be more broadly applied to the Paramyxoviridae family.
Assuntos
Henipavirus , Proteínas Virais de Fusão , Animais , Microscopia Crioeletrônica , Henipavirus/metabolismo , Peptídeos , Conformação Proteica , Proteínas Virais de Fusão/metabolismo , Internalização do VírusRESUMO
The genus Henipavirus (family Paramyxoviridae) currently comprises seven viruses, four of which have demonstrated prior evidence of zoonotic capacity. These include the biosafety level 4 agents Hendra (HeV) and Nipah (NiV) viruses, which circulate naturally in pteropodid fruit bats. Here, we describe and characterize Angavokely virus (AngV), a divergent henipavirus identified in urine samples from wild, Madagascar fruit bats. We report the nearly complete 16,740-nucleotide genome of AngV, which encodes the six major henipavirus structural proteins (nucleocapsid, phosphoprotein, matrix, fusion, glycoprotein, and L polymerase). Within the phosphoprotein (P) gene, we identify an alternative start codon encoding the AngV C protein and a putative mRNA editing site where the insertion of one or two guanine residues encodes, respectively, additional V and W proteins. In other paramyxovirus systems, C, V, and W are accessory proteins involved in antagonism of host immune responses during infection. Phylogenetic analysis suggests that AngV is ancestral to all four previously described bat henipaviruses-HeV, NiV, Cedar virus (CedV), and Ghanaian bat virus (GhV)-but evolved more recently than rodent- and shrew-derived henipaviruses, Mojiang (MojV), Gamak (GAKV), and Daeryong (DARV) viruses. Predictive structure-based alignments suggest that AngV is unlikely to bind ephrin receptors, which mediate cell entry for all other known bat henipaviruses. Identification of the AngV receptor is needed to clarify the virus's potential host range. The presence of V and W proteins in the AngV genome suggest that the virus could be pathogenic following zoonotic spillover. IMPORTANCE Henipaviruses include highly pathogenic emerging zoonotic viruses, derived from bat, rodent, and shrew reservoirs. Bat-borne Hendra (HeV) and Nipah (NiV) are the most well-known henipaviruses, for which no effective antivirals or vaccines for humans have been described. Here, we report the discovery and characterization of a novel henipavirus, Angavokely virus (AngV), isolated from wild fruit bats in Madagascar. Genomic characterization of AngV reveals all major features associated with pathogenicity in other henipaviruses, suggesting that AngV could be pathogenic following spillover to human hosts. Our work suggests that AngV is an ancestral bat henipavirus that likely uses viral entry pathways distinct from those previously described for HeV and NiV. In Madagascar, bats are consumed as a source of human food, presenting opportunities for cross-species transmission. Characterization of novel henipaviruses and documentation of their pathogenic and zoonotic potential are essential to predicting and preventing the emergence of future zoonoses that cause pandemics.
Assuntos
Quirópteros , Genoma Viral , Infecções por Henipavirus , Henipavirus , Vírus Nipah , Animais , Quirópteros/genética , Genoma Viral/genética , Glicoproteínas/genética , Henipavirus/classificação , Henipavirus/genética , Infecções por Henipavirus/virologia , Humanos , Madagáscar , Vírus Nipah/genética , Filogenia , Urina/virologia , Zoonoses/genéticaRESUMO
The genus Henipavirus (HNV) includes two virulent infectious viruses, Nipah virus (NiV) and Hendra virus (HeV), which are the focus of considerable public health research efforts and have been classified as priority infectious diseases by the World Health Organization. Both viruses are high risk and should be handled in biosafety level 4 laboratories. Pseudotyped viruses containing the envelope proteins of HNV viruses have the same envelope protein structure as the authentic viruses; thus, they can mimic the receptor-binding and membrane fusion processes of authentic viruses with host cells and can be handled in biosafety level 2 laboratories. These characteristics enable pseudotyped viruses to be widely used in studies of viral infection mechanisms (packaging, budding, virus attachment, membrane fusion, viral entry, and glycosylation), inhibitory drug screening assays, and monoclonal antibody neutralization characteristics. This review will provide an overview of the progress of research concerning pseudotyped virus packaging systems for NiV and HeV.
Assuntos
Vírus Hendra , Vírus Nipah , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Pseudotipagem Viral , Vírus Hendra/genética , Vírus Hendra/metabolismo , Vírus Nipah/genética , Vírus Nipah/metabolismo , Internalização do VírusRESUMO
Nipah virus (NiV) is an emerging bat-borne zoonotic virus that causes near-annual outbreaks of fatal encephalitis in South Asia-one of the most populous regions on Earth. In Bangladesh, infection occurs when people drink date-palm sap contaminated with bat excreta. Outbreaks are sporadic, and the influence of viral dynamics in bats on their temporal and spatial distribution is poorly understood. We analyzed data on host ecology, molecular epidemiology, serological dynamics, and viral genetics to characterize spatiotemporal patterns of NiV dynamics in its wildlife reservoir, Pteropus medius bats, in Bangladesh. We found that NiV transmission occurred throughout the country and throughout the year. Model results indicated that local transmission dynamics were modulated by density-dependent transmission, acquired immunity that is lost over time, and recrudescence. Increased transmission followed multiyear periods of declining seroprevalence due to bat-population turnover and individual loss of humoral immunity. Individual bats had smaller host ranges than other Pteropus species (spp.), although movement data and the discovery of a Malaysia-clade NiV strain in eastern Bangladesh suggest connectivity with bats east of Bangladesh. These data suggest that discrete multiannual local epizootics in bat populations contribute to the sporadic nature of NiV outbreaks in South Asia. At the same time, the broad spatial and temporal extent of NiV transmission, including the recent outbreak in Kerala, India, highlights the continued risk of spillover to humans wherever they may interact with pteropid bats and the importance of limiting opportunities for spillover throughout Pteropus's range.
Assuntos
Quirópteros/virologia , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/transmissão , Infecções por Henipavirus/veterinária , Infecções por Henipavirus/virologia , Vírus Nipah/classificação , Vírus Nipah/genética , Animais , Ásia , Bangladesh/epidemiologia , Surtos de Doenças , Feminino , Especificidade de Hospedeiro , Humanos , Imunidade , Masculino , Modelos Biológicos , Epidemiologia Molecular , Vírus Nipah/imunologia , Filogenia , Zoonoses/epidemiologia , Zoonoses/imunologia , Zoonoses/transmissão , Zoonoses/virologiaRESUMO
A novel Hendra virus variant, genotype 2, was recently discovered in a horse that died after acute illness and in Pteropus flying fox tissues in Australia. We detected the variant in flying fox urine, the pathway relevant for spillover, supporting an expanded geographic range of Hendra virus risk to horses and humans.
Assuntos
Quirópteros , Vírus Hendra , Infecções por Henipavirus , Animais , Austrália/epidemiologia , Vírus Hendra/genética , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/veterinária , CavalosRESUMO
Knowledge of the dynamics and genetic diversity of Nipah virus circulating in bats and at the human-animal interface is limited by current sampling efforts, which produce few detections of viral RNA. We report a series of investigations at Pteropus medius bat roosts identified near the locations of human Nipah cases in Bangladesh during 2012-2019. Pooled bat urine was collected from 23 roosts; 7 roosts (30%) had >1 sample in which Nipah RNA was detected from the first visit. In subsequent visits to these 7 roosts, RNA was detected in bat urine up to 52 days after the presumed exposure of the human case-patient, although the probability of detection declined rapidly with time. These results suggest that rapidly deployed investigations of Nipah virus shedding from bat roosts near human cases could increase the success of viral sequencing compared with background surveillance and could enhance understanding of Nipah virus ecology and evolution.
Assuntos
Quirópteros , Infecções por Henipavirus , Vírus Nipah , Animais , Bangladesh/epidemiologia , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/veterinária , Humanos , Vírus Nipah/genética , RNA Viral/genéticaRESUMO
We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.
Assuntos
Quirópteros , Vírus Hendra , Infecções por Henipavirus , Doenças dos Cavalos , Animais , Austrália/epidemiologia , Vírus Hendra/genética , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/veterinária , Doenças dos Cavalos/epidemiologia , Cavalos , Filogenia , Vigilância de Evento SentinelaRESUMO
Cholesterol has been implicated in various viral life cycle steps for different enveloped viruses, including viral entry into host cells, cell-cell fusion, and viral budding from infected cells. Enveloped viruses acquire their membranes from their host cells. Although cholesterol has been associated with the binding and entry of various enveloped viruses into cells, cholesterol's exact function in the viral-cell membrane fusion process remains largely elusive, particularly for the paramyxoviruses. Furthermore, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytium formation (cell-cell fusion), central to viral pathogenicity. Nipah virus (NiV) is a deadly member of the Paramyxoviridae family, which also includes Hendra, measles, mumps, human parainfluenza, and various veterinary viruses. The zoonotic NiV causes severe encephalitis, vasculopathy, and respiratory symptoms, leading to a high mortality rate in humans. We used NiV as a model to study the role of membrane cholesterol in paramyxoviral membrane fusion. We used a combination of methyl-beta cyclodextrin (MßCD), lovastatin, and cholesterol to deplete or enrich cell membrane cholesterol outside cytotoxic concentrations. We found that the levels of cellular membrane cholesterol directly correlated with the levels of cell-cell fusion induced. These phenotypes were paralleled using NiV/vesicular stomatitis virus (VSV)-pseudotyped viral infection assays. Remarkably, our mechanistic studies revealed that cholesterol reduces an early F-triggering step but enhances a late fusion pore formation step in the NiV membrane fusion cascade. Thus, our results expand our mechanistic understanding of the paramyxoviral/henipaviral entry and cell-cell fusion processes.IMPORTANCE Cholesterol has been implicated in various steps of the viral life cycle for different enveloped viruses. Nipah virus (NiV) is a highly pathogenic enveloped virus in the Henipavirus genus within the Paramyxoviridae family, capable of causing a high mortality rate in humans and high morbidity in domestic and agriculturally important animals. The role of cholesterol for NiV or the henipaviruses is unknown. Here, we show that the levels of cholesterol influence the levels of NiV-induced cell-cell membrane fusion during syncytium formation and virus-cell membrane fusion during viral entry. Furthermore, the specific role of cholesterol in membrane fusion is not well defined for the paramyxoviruses. We show that the levels of cholesterol affect an early F-triggering step and a late fusion pore formation step during the membrane fusion cascade. Thus, our results expand our mechanistic understanding of the viral entry and cell-cell fusion processes, which may aid the development of antivirals.
Assuntos
Colesterol/metabolismo , Fusão de Membrana/fisiologia , Vírus Nipah/fisiologia , Colesterol/deficiência , Células Gigantes/metabolismo , Lipídeos de Membrana/análise , Lipídeos de Membrana/metabolismo , Vírus Nipah/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírion/metabolismo , Internalização do VírusRESUMO
Nipah virus (NiV) is a zoonotic bat henipavirus in the family Paramyxoviridae NiV is deadly to humans, infecting host cells by direct fusion of the viral and host cell plasma membranes. This membrane fusion process is coordinated by the receptor-binding attachment (G) and fusion (F) glycoproteins. Upon G-receptor binding, F fuses membranes via a cascade that sequentially involves F-triggering, fusion pore formation, and viral or genome entry into cells. Using NiV as an important paramyxoviral model, we identified two novel regions in F that modulate the membrane fusion cascade. For paramyxoviruses and other viral families with class I fusion proteins, the heptad repeat 1 (HR1) and HR2 regions in the fusion protein prefusion conformation bind to form a six-helix bundle in the postfusion conformation. Here, structural comparisons between the F prefusion and postfusion conformations revealed that a short loop region (N1) undergoes dramatic spatial reorganization and a short alpha helix (N4) undergoes secondary structural changes. The roles of the N1 and N4 regions during the membrane fusion cascade, however, remain unknown for henipaviruses and paramyxoviruses. By performing alanine scanning mutagenesis and various functional analyses, we report that specific residues within these regions alter various steps in the membrane fusion cascade. While the N1 region affects early F-triggering, the N4 region affects F-triggering, F thermostability, and extensive fusion pore expansion during syncytium formation, also uncovering a link between F-G interactions and F-triggering. These novel mechanistic roles expand our understanding of henipaviral and paramyxoviral F-triggering, viral entry, and cell-cell fusion (syncytia), a pathognomonic feature of paramyxoviral infections.IMPORTANCE Henipaviruses infect bats, agriculturally important animals, and humans, with high mortality rates approaching â¼75% in humans. Known human outbreaks have been concentrated in Southeast Asia and Australia. Furthermore, about 20 new henipaviral species have been recently discovered in bats, with geographical spans in Asia, Africa, and South America. The development of antiviral therapeutics requires a thorough understanding of the mechanism of viral entry into host cells. In this study, we discovered novel roles of two regions within the fusion protein of the deadly henipavirus NiV. Such roles were in allowing viral entry into host cells and cell-cell fusion, a pathological hallmark of this and other paramyxoviruses. These novel roles were in the previously undescribed N1 and N4 regions within the fusion protein, modulating early and late steps of these important processes of viral infection and henipaviral disease. Notably, this knowledge may apply to other henipaviruses and more broadly to other paramyxoviruses.