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.     

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.     

Novel monoclonal antibodies against Menangle virus nucleocapsid protein

Menangle virus (MenV) is a member of the family Paramyxoviridae isolated in Australia that causes a reproductive disease of pigs. There is a need for specific immunoassays for virus detection to facilitate the diagnosis of MenV infection. Three novel monoclonal antibodies (MAbs) of the IgG1 subtype were generated by immunizing mice with recombinant yeast-expressed MenV nucleocapsid (N) protein self-assembled to nucleocapsid-like structures. One MAb was cross-reactive with recombinant N protein of Tioman virus. The epitopes of MAbs were mapped using a series of truncated MenV N proteins lacking the 29–119 carboxy-terminal amino acid (aa) residues. The epitopes of two MAbs were mapped to aa 430–460 of the MenV N protein, whilst the epitope of one MAb was mapped to residues 460–490. All three MAbs specifically recognized MenV, as indicated by immunohistochemical staining of brain tissue isolated from a field case (a stillborn piglet) of MenV infection. The MAbs against MenV N protein may be a useful tool for immunohistological diagnosis of MenV infection.     

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.     

Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae

Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.     

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.     

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.     

Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus

Nipah virus, a zoonotic paramyxovirus which emerged recently was chemically inactivated using binary ethylenimine (BEI). The inactivated virus was concentrated and purified by sucrose gradient centrifugation. The gradient fractions were examined by electron microscopy and Western immunoblot, and gradient fraction containing mainly Nipah matrix (M) and nucleocapsid (N) proteins was used for immunizing BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones identified five strongly positive clones producing IgG monoclonal antibodies (mAbs) reactive to the Nipah virus antigen. The protein specificity of these mAbs was determined by Western immunoblot using Nipah virus and recombinant Nipah virus proteins expressed in mammalian cells. Four mAbs reacted with Nipah N protein and one reacted with Nipah M protein. None of the mAbs neutralized Nipah virus infectivity in vitro. However, all mAbs recognized Nipah virus in ELISA and immunofluorescence assay. F45G2 mAb was most suitable for immunohistochemistry on long term formalin-fixed Nipah virus infected swine tissues. Three of the anti-nucleocapsid mAbs (F45G2, F45G3 and F45G6) showed cross-reactivity with closely related Hendra virus N protein in both immunofluorescence and Western Immunoblot assays. Two of the mAbs were specific for the Nipah virus only, F45G4 (anti-N) and F45G5 (anti-M), and could be used in the primary identification of Nipah virus. The use of these immunoreagents to develop new diagnostic assays is discussed.     

Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and Erns using phage-displayed random peptide library

Summary. Infection of cells with classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoproteins E2 and E^rns with receptor molecules on the cell surface. These proteins are also the major antigens for eliciting neutralizing antibodies and conferring protective immunity. Here we report the identification of multiple neutralizing epitopes on these proteins by screening a phage-displayed random peptide library with CSFV-specific neutralizing monoclonal antibodies. Two different E2-specific neutralizing mAbs (a18 and 24/10) were found to bind to a common motif SPTxL, which is similar to the sequence SPTTL of the E2 protein (aa 289–293), indicating that this is likely to be an immunodominant epitope. Similarly, an immunodominant epitope corresponding to the sequence DKN of E^rns (aa 117–119) was identified for two independent E^rns-specific neutralizing antibodies, b4-22 and 24/16, respectively. Another binding motif, CxNNxTC, was identified for mAb 24/16, but not for b4-22. Sequencing analysis of the genes coding for the light chain of these mAbs was conducted to ensure that all mAbs were derived from different hybridomas, rather than from different subclones of a common parent line. Inhibition studies using immunofluorescent antibody assay and virus neutralization test demonstrated that the mimotope peptides truly mimicked the antibody binding determinants on the viral proteins. The detailed mapping data for these neutralizing epitopes will be useful for development of improved diagnostic tests and perhaps a peptide-based vaccine for this important swine disease.     

Production and diagnostic application of monoclonal antibodies against influenza virus H5

Nine monoclonal antibodies (mAbs) against avian influenza virus (AI) H5 subtype from mice immunized with inactivated virus H5N1 (A/Turkey/ON/6213/66) were produced. Upon testing, the results indicated that the binding epitopes of eight out of the nine mAbs were conformational, while one mAb (#7) reacted with denatured H5N1 only. Two mAbs #10 and #11 reacted with all of the thirteen H5 strains tested indicating that the binding epitopes of these mAbs were conserved among these H5 subtypes.Possible applications of these mAbs in rapid tests for H5 antigen were explored. Double antibody sandwich (DAS) ELISAs were developed using two selected mAbs #10 and #11. This DAS ELISA detects specific H5 viruses and is able to identify all thirteen H5 strains tested. Three mAbs showed reactivity with AI H5 antigen for both immunofluorescence (IF) and immunohistochemistry. A cELISA used to screen chickens that had been infected with an H5 virus was developed with mAb #9 and recombinant H5 antigen. The sera from chickens that have been infected with an H5N1 virus were examined using the cELISA. 80% of the sera from H5 infected chickens showed a positive H5 specific antibody response at 7 days post-infection (dpi) and remained positive until the end of the experiment on day 30 (>40% inhibition). This panel of the AI H5 specific mAbs is valuable for the development of various immunoassays.     

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.     

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.     

Baculovirus expression and antigenic characterization of the capsid proteins of three Norwalk-like viruses

Summary.  Human caliciviruses (HuCVs) are antigenically diverse. The antigenic relationships among different HuCVs have been difficult to study because HuCVs cannot be passaged in the laboratory. In this study, we describe cloning, sequencing and expression of the viral capsid proteins of three HuCVs that were identified in outbreaks of acute gastroenteritis in Virginia in 1997–1998. Yields of the capsid proteins similar to previously expressed recombinant Norwalk virus were obtained using the baculovirus expression system. Recombinant VA97207 capsid protein (rVA97207) and rVA98387, but not rVA98115, formed virus-like particles (VLPs). All three recombinant capsid antigens detected seroresponses in patients involved in outbreaks of acute gastroenteritis associated with genetically homologous or related HuCVs. The antigenic relationships of the three strains were further characterized using hyperimmune antisera against the three capsid antigens as well as four previously characterized recombinant capsid antigens of Norwalk (rNV), Mexico (rMxV), Hawaii (rHV), and Grimsby viruses (rGrV). VA98387 shared 98% aa identity with GrV; rVA98387 was detected by antisera to GrV. VA98115 shared 87% aa identity with Desert Storm virus and 65% aa identity with prototype Norwalk virus (NV); rVA98115 reacted weakly with NV antisera. VA97207 shared 80% aa identity with Amsterdam and 75% aa identity with Leeds strains and rVA97207 was not detected by any of the heterologous antibodies. In conclusion, VA97207 and VA98115 may belong to CV antigenic types not previously expressed, while VA98387 is a GrV-like virus. Low levels of cross-reactive antibodies were detected between types. Further studies to characterize these antigens and to develop enzyme immune assays (EIAs) for these strains are in progress. Received December 13, 2000 Accepted July 20, 2001     

Mapping of B-cell epitopes of hemagglutinin protein of rinderpest virus

Monoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569-577 and 575-583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527-554 and 588-609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus HN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination.     

Characterization of cross-reactive and serotype-specific epitopes on the nucleocapsid proteins of hantaviruses

The hantavirus nucleocapsid (N) protein fulfills several key roles in virus replication and assembly and is the major antigen in humoral immune responses in humans and mice. Here we report on epitopes involved in serotype-specific and cross-reactive recognition of the N proteins of hantaviruses using monoclonal antibodies (mAbs) against the N proteins of Andes virus (ANDV) and Sin Nombre virus (SNV). The mAbs define at least twelve different epitopic patterns which span eight sequences, including amino acids 17-59, 66-78, 79-91, 157-169, 222-234, 244-263, 274-286 and 326-338 on the SNV and ANDV N proteins. Studies on the cross-reactivity of these mAbs with different hantavirus N proteins indicated that epitopes located within amino acids 244-286 are related to serotype specificity. We analyzed further the location of epitopes with available three-dimensional structure information including the N-terminal coiled-coil and derived exposed and hidden residues of these epitopes. The generated recombinant N proteins and the characterized mAbs are functional tools being now available for hantavirus diagnostics and replication studies.     

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.     

Characterization of monoclonal antibodies against hantavirus nucleocapsid protein and their use for immunohistochemistry on rodent and human samples

Monoclonal antibodies are important tools for various applications in hantavirus diagnostics. Recently, we generated Puumala virus (PUUV)-reactive monoclonal antibodies (mAbs) by immunisation of mice with chimeric polyomavirus-derived virus-like particles (VLPs) harbouring the 120-amino-acid-long amino-terminal region of the PUUV nucleocapsid (N) protein. Here, we describe the generation of two mAbs by co-immunisation of mice with hexahistidine-tagged full-length N proteins of Sin Nombre virus (SNV) and Andes virus (ANDV), their characterization by different immunoassays and comparison with the previously generated mAbs raised against a segment of PUUV N protein inserted into VLPs. All of the mAbs reacted strongly in ELISA and western blot tests with the antigens used for immunization and cross-reacted to varying extents with N proteins of other hantaviruses. All mAbs raised against a segment of the PUUV N protein presented on chimeric VLPs and both mAbs raised against the full-length AND/SNV N protein reacted with Vero cells infected with different hantaviruses. The reactivity of mAbs with native viral nucleocapsids was also confirmed by their reactivity in immunohistochemistry assays with kidney tissue specimens from experimentally SNV-infected rodents and human heart tissue specimens from hantavirus cardiopulmonary syndrome patients. Therefore, the described mAbs represent useful tools for the immunodetection of hantavirus infection.     

A new panel of NS1 antibodies for easy detection and titration of influenza A virus

The non‐structural protein NS1 of the influenza A virus is a good target for the development of diagnostic assays. In this study, three NS1 monoclonal antibodies (mAbs) were generated by using recombinant NS1 protein of H5N1 virus and found to bind both the native and denatured forms of NS1. Two of the mAbs, 6A4 and 2H6, bind NS1 of three different strains of influenza A virus, namely H1N1, H3N2, and H5N1. Epitope mapping revealed that residues 42–53 of H5N1 NS1 are essential for the interaction with both mAbs. Between the three strains, there is only one amino acid difference in this domain, which is consistent with the observed cross‐reactivities. On the other hand, mAb 1G1 binds to residues 206–215 of H5N1 NS1 and does not bind NS1 of H1N1 or H3N2. Furthermore, all three mAbs detected NS1 proteins expressed in virus infected MDCK cells and indirect immunofluorescence staining with mAbs 6A4 and 2H6 provided an alternative method for viral titer determination. Quantifying the numbers of fluorescent foci units yielded viral titers for three different isolates of H5N1 virus that are highly comparable to that obtained by observing cytopathic effect induced by virus infection. Importantly, this alternative method yields results at 1 day post‐infection while the conventional method using cytopathic effect yields results at 3 days post‐infection. The results showed that this new panel of NS1 antibodies can detect NS1 protein expressed during viral infection and can be used for fast and easy titration of influenza A virus. J. Med. Virol. 82:467–475, 2010. © 2010 Wiley‐Liss, Inc.     

小鼠肝炎病毒N蛋白单克隆抗体的制备及鉴定

[目的]制备并鉴定小鼠肝炎病毒(MHV)N蛋白的单克隆抗体(mAb).[方法]以Bac-to-Bac杆状病毒表达系统表达的重组N蛋白为免疫原,免疫BALB/c小鼠,应用常规杂交瘤技术将免疫小鼠脾细胞与Sp2/0骨髓瘤进行融合,用间接ELISA方法筛选分泌N蛋白mAb的杂交瘤细胞系.并用Western blot和IFA方法对所获得的mAb进行鉴定.[结果]共获得4株稳定分泌抗N蛋白mAb的杂交瘤细胞系,其亚类鉴定2株为IgG2a,1株为IgG2b,1株为IgG1,轻链均为k型.经鉴定4株mAb均能与重组N蛋白发生特异性反应.[结论]成功获得了特异性抗MHV N蛋白的mAb,为进一步研究N蛋白的结构功能及建立诊断学方法奠定了基础.