Neutralization assays for differential henipavirus serology using Bio-Plex protein array systems

Hendra virus (HeV) and Nipah virus (NiV) are related emerging paramyxoviruses classified in the genus Henipavirus. Both cause fatal disease in animals and humans and are classified as biosafety level 4 pathogens. Here we detail two new multiplexed microsphere assays, one for antibody detection and differentiation and another designed as a surrogate for virus neutralization. Both assays utilize recombinant soluble attachment glycoproteins (sG) whereas the latter incorporates the cellular receptor, recombinant ephrin-B2. Spectrally distinct sG(HeV)- and sG(NiV)-coupled microspheres preferentially bound antibodies from HeV- and NiV-seropositive animals, demonstrating a simple procedure to differentiate antibodies to these closely related viruses. Soluble ephrin-B2 bound sG-coupled microspheres in a dose-dependent fashion. Specificity of binding was further evaluated with henipavirus G-specific sera and MAbs. Sera from henipavirus-seropositive animals differentially blocked ephrin-B2 binding, suggesting that detection and differentiation of HeV and NiV neutralizing antibodies can be done simultaneously in the absence of live virus.     

Henipavirus Encephalitis: Recent Developments and Advances

The genus Henipavirus within the family Paramyxoviridae includes the Hendra virus (HeV) and Nipah virus (NiV) which were discovered in the 1990s in Australia and Malaysia, respectively, after emerging to cause severe and often fatal outbreaks in humans and animals. While HeV is confined to Australia, more recent NiV outbreaks have been reported in Bangladesh, India and the Philippines. The clinical manifestations of both henipaviruses in humans appear similar, with a predominance of an acute encephalitic syndrome. Likewise, the pathological features are similar and characterized by disseminated, multi‐organ vasculopathy comprising endothelial infection/ulceration, vasculitis, vasculitis‐induced thrombosis/occlusion, parenchymal ischemia/microinfarction, and parenchymal cell infection in the central nervous system (CNS), lung, kidney and other major organs. This unique dual pathogenetic mechanism of vasculitis‐induced microinfarction and neuronal infection causes severe tissue damage in the CNS. Both viruses can also cause relapsing encephalitis months and years after the acute infection. Many animal models studied to date have largely confirmed the pathology of henipavirus infection, and provided the means to test new therapeutic agents and vaccines. As the bat is the natural host of henipaviruses and has worldwide distribution, spillover events into human populations are expected to occur in the future.     

Monoclonal antibodies against the nucleocapsid proteins of henipaviruses: production, epitope mapping and application in immunohistochemistry

Summary Four monoclonal antibodies (mAbs) were generated by immunizing BALB/C mice with recombinant nucleocapsid protein (N) of Nipah virus (NiV) and Hendra virus (HeV) expressed in E. coli. Two mAbs each were obtained for the HeV N and NiV N, respectively. All four mAbs displayed specific reactivity with the recombinant N proteins of both viruses by western blot, which was further confirmed by immunofluorescent antibody assay using fixed insect cells infected with recombinant baculoviruses expressing either the HeV or NiV N protein. Epitope mapping using a 12-mer random peptide phage display library revealed two linear antigenic sites of the henipavirus N proteins, KLxR (aa 17–20) and FKREM (aa 446–450), which have not been reported previously. Two of the mAbs were able to specifically recognize HeV antigens by immunohistochemical staining of lung tissue sections of a horse experimentally infected with HeV. These reagents will be a useful addition to the collection of tools essential for further research and improvement in diagnosis of henipaviruses. Correspondence: Changchun Tu, Institute of Veterinary Sciences, Academy of Military Medical Sciences, 1068 Qinglong Road, Changchun 130062, China     

重庆地区动物携带Nipah病毒和Hendra病毒的检测

目的 本研究拟建立一步法实时RT-PCR检测Nipah病毒(Nipah virus,NiV)和Hen-dra病毒(Hendra virus,HeV)的方法 ,对采集自中国重庆地区动物外周血样本进行NiV和HeV检测,以获得我国重庆地区的NiV和HeV的病毒流行病学资料.方法 2007年6月起至2008年6月,采集自重庆地区饲养的猪外周血标本580份、奶牛外周血标本250份、山羊外周血标本180份,密度梯度离心法分离外周血淋巴细胞,Trizol法提取细胞总RNA.用已建立的NiV和HeV一步法实时RT-PCR检测方法 ,对RNA样本进行NiV和HeV检测.对阳性样本进行PCR产物序列鉴定及序列分析等研究,在可能的情况下进行病毒分离培养,并提交流行病学调查报告.结果 对采集自中国重庆地区饲养的3种动物的样本进行NiV和HeV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果 .结论 初步流行病学研究提示我国重庆地区饲养的动物猪、牛、羊中NiV和HeV未发现阳性.     

A neutralization test for specific detection of Nipah virus antibodies using pseudotyped vesicular stomatitis virus expressing green fluorescent protein

Nipah virus (NiV) is a new zoonotic paramyxovirus that emerged in 1998 and is now classified in the genus Henipavirus along with the closely related Hendra virus (HeV). NiV is highly pathogenic in several vertebrate species including humans, and the lack of available vaccines or specific treatment restricts it to biosafety level 4 (BSL4) containment. A serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing green fluorescent protein (GFP) and bearing the F and G proteins of NiV (VSV–NiV–GFP). The neutralization titers were obtained by counting GFP-expressing cells or by measuring fluorescence. The performance of this new assay was compared against the conventional test using live NiV with panels of sera from several mammalian species, including sera from NiV outbreaks, experimental infections, as well as HeV-specific sera. The results obtained with the VSV–NiV–GFP based test correlated with those obtained using live NiV. Using a 50% reduction in VSV–NiV–GFP infected cells as the cut-off for neutralization, this new assay demonstrated its potential as an effective tool for detecting NiV neutralizing antibodies under BSL2 containment with greater speed, sensitivity and safety as compared to the conventional NiV serum neutralization test.     

Nipah virus entry can occur by macropinocytosis

Nipah virus (NiV) is a zoonotic biosafety level 4 paramyxovirus that emerged recently in Asia with high mortality in man. NiV is a member, with Hendra virus (HeV), of the Henipavirus genus in the Paramyxoviridae family. Although NiV entry, like that of other paramyxoviruses, is believed to occur via pH-independent fusion with the host cell's plasma membrane we present evidence that entry can occur by an endocytic pathway. The NiV receptor ephrinB2 has receptor kinase activity and we find that ephrinB2's cytoplasmic domain is required for entry but is dispensable for post-entry viral spread. The mutation of a single tyrosine residue (Y304F) in ephrinB2's cytoplasmic tail abrogates NiV entry. Moreover, our results show that NiV entry is inhibited by constructions and drugs specific for the endocytic pathway of macropinocytosis. Our findings could potentially permit the rapid development of novel low-cost antiviral treatments not only for NiV but also HeV.     

Acute Hendra virus infection: Analysis of the pathogenesis and passive antibody protection in the hamster model

Hendra virus (HeV) and Nipah virus (NiV) are recently-emerged, closely related and highly pathogenic paramyxoviruses. We have analysed here the pathogenesis of the acute HeV infection using the new animal model, golden hamster (Mesocricetus auratus), which is highly susceptible to HeV infection. HeV-specific RNA and viral antigens were found in multiple organs and virus was isolated from different tissues. Dual pathogenic mechanism was observed: parenchymal infection in various organs, including the brain, with vasculitis and multinucleated syncytia in many blood vessels. Furthermore, monoclonal antibodies specific for the NiV fusion protein neutralized HeV in vitro and efficiently protected hamsters from HeV if given before infection. These results reveal the similarities between HeV and NiV pathogenesis, particularly in affecting both respiratory and neuronal system. They demonstrate that hamster presents a convenient novel animal model to study HeV infection, opening new perspectives to evaluate vaccine and therapeutic approaches against this emergent infectious disease.     

Emerging trends of Nipah virus: A review

Since emergence of the Nipah virus (NiV) in 1998 from Malaysia, the NiV virus has reappeared on different occasions causing severe infections in human population associated with high rate of mortality. NiV has been placed along with Hendra virus in genus Henipavirus of family Paramyxoviridae. Fruit bats (Genus Pteropus) are known to be natural host and reservoir of NiV. During the outbreaks from Malaysia and Singapore, the roles of pigs as intermediate host were confirmed. The infection transmitted from bats to pigs and subsequently from pigs to humans. Severe encephalitis was reported in NiV infection often associated with neurological disorders. First NiV outbreak in India occurred in Siliguri district of West Bengal in 2001, where direct transmission of the NiV virus from bats‐to‐human and human‐to‐human was reported in contrast to the role of pigs in the Malaysian NiV outbreak. Regular NiV outbreaks have been reported from Bangladesh since 2001 to 2015. The latest outbreak of NiV has been recorded in May, 2018 from Kerala, India which resulted in the death of 17 individuals. Due to lack of vaccines and effective antivirals, Nipah encephalitis poses a great threat to public health. Routine surveillance studies in the infected areas can be useful in detecting early signs of infection and help in containment of these outbreaks.     

Role of endocytosis and cathepsin-mediated activation in Nipah virus entry

The recent discovery that the Nipah virus (NiV) fusion protein (F) is activated by endosomal cathepsin L raised the question if NiV utilize pH- and protease-dependent mechanisms of entry. We show here that the NiV receptor ephrin B2, virus-like particles and infectious NiV are internalized from the cell surface. However, endocytosis, acidic pH and cathepsin-mediated cleavage are not necessary for the initiation of infection of new host cells. Our data clearly demonstrate that proteolytic activation of the NiV F protein is required before incorporation into budding virions but not after virus entry.     

Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase

Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2 μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.     

Nipah virus glycoprotein: production in baculovirus and application in diagnosis

A method for serological diagnosis of Nipah virus (NiV) is described. DNA encoding truncated G protein of NiV was cloned into the pFastBac HT vector, and the fusion protein to His-tag was expressed in insect cells by recombinant baculovirus. The resulting His-G recombinant fusion protein was purified by affinity chromatography and used as the coating antigen for serological testing by indirect enzyme-linked immunosorbant assay (ELISA). When tested against a panel of swine serum samples, the recombinant G protein-based ELISA successfully discriminated all 40 samples previously determined to be serum neutralizing test (SNT) positive from 11 SNT negatives samples. The data show that the recombinant G protein exhibits the antigenic epitopes and conformation necessary for specific antigen-antibody recognition. The main advantage of the recombinant G protein-based NiV ELISA compared to an ELISA using whole virus antigen is the use of a single antigenic protein instead of inactivated whole virus which is required to be prepared under high risk and cost. This test is suitable for routine diagnosis of NiV and also for epidemiological surveys as it allows highly reliable testing of a large number of sera rapidly.     

A hendra virus g glycoprotein subunit vaccine protects african green monkeys from nipah virus challenge

In the 1990s, Hendra virus and Nipah virus (NiV), two closely related and previously unrecognized paramyxoviruses that cause severe disease and death in humans and a variety of animals, were discovered in Australia and Malaysia, respectively. Outbreaks of disease have occurred nearly every year since NiV was first discovered, with case fatality ranging from 10 to 100%. In the African green monkey (AGM), NiV causes a severe lethal respiratory and/or neurological disease that essentially mirrors fatal human disease. Thus, the AGM represents a reliable disease model for vaccine and therapeutic efficacy testing. We show that vaccination of AGMs with a recombinant subunit vaccine based on the henipavirus attachment G glycoprotein affords complete protection against subsequent NiV infection with no evidence of clinical disease, virus replication, or pathology observed in any challenged subjects. Success of the recombinant subunit vaccine in nonhuman primates provides crucial data in supporting its further preclinical development for potential human use.     

Characterization of the antiviral and inflammatory responses against Nipah virus in endothelial cells and neurons

Nipah virus (NiV) is a highly pathogenic paramyxovirus which causes fatal encephalitis in up to 75% of infected humans. Endothelial cells and neurons are important cellular targets in the pathogenesis of this disease. In this study, viral replication and the innate immune responses to NiV in these cell types were measured. NiV infected endothelial cells generated a functionally robust IFN-β response, which correlated with localization of the NiV W protein to the cytoplasm. There was no antiviral response detected in infected neuronal cells. NiV infection of endothelial cells induced a significant increase in secreted inflammatory chemokines, which corresponded with the increased ability of infected cell supernatants to induce monocyte and T-lymphocyte chemotaxis. These results suggest that pro-inflammatory chemokines produced by NiV infected primary endothelial cells in vitro is consistent with the prominent vasculitis observed in infections, and provide initial molecular insights into the pathogenesis of NiV in physiologically relevant cells types.     

Effects of single amino acid substitutions at the E residue in the conserved GDNE motif of the Nipah virus polymerase (L) protein

Summary Nipah virus (NiV) is an emergent zoonotic paramyxovirus. The L proteins of most paramyxoviruses contain a GDNQ motif, thought to be part of the catalytic site for polymerase activity. Conversely, NiV L has GDNE in this position. We substituted the E residue with eight different amino acid residues and examined the effect on L function in an in vitro replication assay. Our results demonstrated that NiV L functioned with similar efficiency with either GDNE or GDNQ, but polymerase activity was severely reduced or abolished when a structurally destabilising residue (such as K, P or G) was introduced at this site.     

Nipah virus fusion protein: Influence of cleavage site mutations on the cleavability by cathepsin L, trypsin and furin

Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae which originated from bats, encodes for a fusion (F) protein which is proteolytically processed within endosomes by cathepsin L. We show here that sequence requirements for NiV F activation differ markedly from other para- or orthomyxoviral fusion proteins. In contrast to other viral fusion proteins with monobasic cleavage sites, processing of NiV F proteins with one single basic amino acid in the cleavage peptide by exogenous trypsin is very inefficient, and introduction of a consensus sequence for furin does not result in cleavage by this ubiquitous protease. In contrast, a multibasic cleavage peptide in the NiV F protein completely impairs proteolytic processing and the generation of biological activity.     

Functional studies of host-specific ephrin-B ligands as Henipavirus receptors

Hendra virus (HeV) and Nipah virus (NiV) are closely related paramyxoviruses that infect and cause disease in a wide range of mammalian hosts. To determine whether host receptor molecules play a role in species-specific and/or virus-specific infection we have cloned and characterized ephrin-B2 and ephrin-B3 ligands from a range of species, including human, horse, pig, cat, dog, bats (Pteropus alecto and Pteropus vampyrus) and mouse. HeV and NiV were both able to infect cells expressing any of the ephrin-B2 and ephrin-B3 molecules. There did not appear to be significant differences in receptor function from different species or receptor usage by HeV and NiV. Soluble ephrin ligands, their receptors and G-specific human monoclonal antibodies differentially blocked henipavirus infections suggesting different receptor affinities, overlapping receptor binding domains of the henipavirus attachment glycoprotein (G) and that the functional domains of the ephrin ligands may be important for henipavirus binding.     

Newcastle disease virus-vectored Nipah encephalitis vaccines induce B and T cell responses in mice and long-lasting neutralizing antibodies in pigs

Nipah virus (NiV), a member of the Paramyxoviridae family, causes deadly encephalitis in humans and huge economic losses to the pig industry. Here, we generated recombinant avirulent Newcastle disease virus (NDV) LaSota strains expressing the NiV G and F proteins respectively (designated as rLa-NiVG and rLa-NiVF), and evaluated their immunogenicity in mice and pigs. Both rLa-NiVG and rLa-NiVF displayed growth properties similar to those of LaSota virus in chicken eggs. Co-infection of rLa-NiVG and rLa-NiVF caused marked syncytia formation, while intracerebral co-inoculation of these viruses in mice showed they were safe in at least one mammalian species. Animal immunization studies showed rLa-NiVG and rLa-NiVF induced NiV neutralizing antibody responses in mice and pigs, and F protein-specific CD8+ T cell responses in mice. Most importantly, rLa-NiVG and rLa-NiVF administered alone or together, induced a long-lasting neutralizing antibody response in pigs. Recombinant rLa-NiVG/F thus appear to be promising NiV vaccine candidates for pigs and potentially humans.     

Sites of phosphorylation of P and V proteins from Hendra and Nipah viruses: newly emerged members of Paramyxoviridae

Hendra (HeV) and Nipah (NiV) viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the subfamily Paramyxoviridae. The polymerase-associated phosphoproteins (P proteins) of paramyxoviruses have been shown, by direct and indirect methods, to be highly phosphorylated. In this study, a comprehensive comparison of in vivo phosphorylation of HeV and NiV P proteins, derived from virus particles, was achieved by a direct approach using electrospray ionization ion trap mass spectrometry (ESI-IT-MS). Phosphorylation sites for the P proteins were determined at Ser-224 and Thr-239 in HeV and at Ser-240 and Ser-472 in NiV. These phosphorylation patterns do not appear to be consistent with those reported for other paramyxoviruses. Protein V, a product of a frame shift in the P protein gene, was identified by specific antibodies in HeV preparations but not in NiV. HeV V protein was found to contain phosphoserine but not phosphothreonine. In addition, P proteins from both viruses were found to be modified by N-terminal acetylation.