Immunogenicity and performance of an enterovirus 71 virus-like-particle vaccine in nonhuman primates.

A vaccine against human enterovirus 71 (EV-A71) is urgently needed to combat outbreaks of EV-A71 and in particular, the serious neurological complications that manifest during these outbreaks. In this study, an EV-A71 virus-like-particle (VLP) based on a B5 subgenogroup (EV-A71-B5 VLP) was generated using an insect cell/baculovirus platform. Biochemical analysis demonstrated that the purified VLP had a highly native procapsid structure and initial studies in vivo demonstrated that the VLPs were immunogenic in mice. The impact of VLP immunization on infection was examined in non-human primates using a VLP prime-boost strategy prior to EV-A71 challenge. Rhesus macaques were immunized on day 0 and day 21 with VLPs (100 µg/dose) containing adjuvant or with adjuvant alone (controls), and were challenged with EV-A71 on day 42. Complete blood counts, serum chemistry, magnetic resonance imaging (MRI) scans, and histopathology results were mostly normal in vaccinated and control animals after virus challenge demonstrating that the fatal EV-A71-B3 clinical isolate used in this study was not highly virulent in rhesus macaques. Viral genome and/or infectious virus were detected in blood, spleen or brain of two of three control animals, but not in any specimens from the vaccinated animals, indicating that VLP immunization prevented systemic spread of EV-A71 in rhesus macaques. High levels of IgM and IgG were detected in VLP-vaccinated animals and these responses were highly specific for EV-A71 particles and capsid proteins. Serum from vaccinated animals also exhibited similar neutralizing activity against different subgenogroups of EV-A71 demonstrating that the VLPs induced cross-neutralizing antibodies. In conclusion, our EV-A71-B5 VLP is safe, highly immunogenic, and prevents systemic EV-A71-B3 infection in nonhuman primates making it a viable attractive vaccine candidate for EV-A71.

Therapeutic treatment of Nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody.

Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes severe and often fatal disease in pigs and humans. There are currently no vaccines or treatments approved for human use. Studies in small-animal models of NiV infection suggest that antibody therapy may be a promising treatment. However, most studies have assessed treatment at times shortly after virus exposure before animals show signs of disease. We assessed the efficacy of a fully human monoclonal antibody, m102.4, at several time points after virus exposure including at the onset of clinical illness in a uniformly lethal nonhuman primate model of NiV disease. Sixteen African green monkeys (AGMs) were challenged intratracheally with a lethal dose of NiV, and 12 animals were infused twice with m102.4 (15 mg/kg) beginning at either 1, 3, or 5 days after virus challenge and again about 2 days later. The presence of viral RNA, infectious virus, and/or NiV-specific immune responses demonstrated that all subjects were infected after challenge. All 12 AGMs that received m102.4 survived infection, whereas the untreated control subjects succumbed to disease between days 8 and 10 after infection. AGMs in the day 5 treatment group exhibited clinical signs of disease, but all animals recovered by day 16. These results represent the successful therapeutic in vivo efficacy by an investigational drug against NiV in a nonhuman primate and highlight the potential impact that a monoclonal antibody can have on a highly pathogenic zoonotic human disease.

A recombinant Hendra virus G glycoprotein subunit vaccine protects nonhuman primates against Hendra virus challenge.

UNLABELLED: Hendra virus (HeV) is a zoonotic emerging virus belonging to the family Paramyxoviridae. HeV causes severe and often fatal respiratory and/or neurologic disease in both animals and humans. Currently, there are no licensed vaccines or antiviral drugs approved for human use. A number of animal models have been developed for studying HeV infection, with the African green monkey (AGM) appearing to most faithfully reproduce the human disease. Here, we assessed the utility of a newly developed recombinant subunit vaccine based on the HeV attachment (G) glycoprotein in the AGM model. Four AGMs were vaccinated with two doses of the HeV vaccine (sGHeV) containing Alhydrogel, four AGMs received the sGHeV with Alhydrogel and CpG, and four control animals did not receive the sGHeV vaccine. Animals were challenged with a high dose of infectious HeV 21 days after the boost vaccination. None of the eight specifically vaccinated animals showed any evidence of clinical illness and survived the challenge. All four controls became severely ill with symptoms consistent with HeV infection, and three of the four animals succumbed 8 days after exposure. Success of the recombinant subunit vaccine in AGMs provides pivotal data in supporting its further preclinical development for potential human use. IMPORTANCE: A Hendra virus attachment (G) glycoprotein subunit vaccine was tested in nonhuman primates to assess its ability to protect them from a lethal infection with Hendra virus. It was found that all vaccinated African green monkeys were completely protected against subsequent Hendra virus infection and disease. The success of this new subunit vaccine in nonhuman primates provides critical data in support of its further development for future human use.

Serotype-specific host responses in rhesus macaques after primary dengue challenge.

Dengue virus (DENV) is considered to be the most important arthropod-borne viral disease and causes more than 100 million human infections annually. To further characterize primary DENV infection in vivo, rhesus macaques were infected with DENV-1, DENV-2, DENV-3, or DENV-4 and clinical parameters, as well as specificity and longevity of serologic responses, were assessed. Overt clinical symptoms were not present after infection. However, abnormalities in blood biochemical parameters consistent with heart, kidney, and liver damage were observed, and changes in plasma fibrinogen, D-dimers, and protein C indicated systemic activation of the blood coagulation pathway. Significant homotypic and heterotypic serum immunoglobulins were present in all animals, and IgG persisted for at least 390 days. Serum neutralizing antibody responses were highly serotype specific by day 120. However, some heterotypic neutralizing activity was noted in infected animals. Identification of serotype-specific host responses may help elucidate mechanisms that mediate severe DENV disease after reinfection.

Host cell tropism mediated by Australian bat lyssavirus envelope glycoproteins.

Australian bat lyssavirus (ABLV) is a rhabdovirus of the lyssavirus genus capable of causing fatal rabies-like encephalitis in humans. There are two variants of ABLV, one circulating in pteropid fruit bats and another in insectivorous bats. Three fatal human cases of ABLV infection have been reported with the third case in 2013. Importantly, two equine cases also arose in 2013; the first occurrence of ABLV in a species other than bats or humans. We examined the host cell entry of ABLV, characterizing its tropism and exploring its cross-species transmission potential using maxGFP-encoding recombinant vesicular stomatitis viruses that express ABLV G glycoproteins. Results indicate that the ABLV receptor(s) is conserved but not ubiquitous among mammalian cell lines and that the two ABLV variants can utilize alternate receptors for entry. Proposed rabies virus receptors were not sufficient to permit ABLV entry into resistant cells, suggesting that ABLV utilizes an unknown alternative receptor(s).

Paramyxovirus entry.

The family Paramyxoviridae consists of a group of large, enveloped, negative-sense, single-stranded RNA viruses and contains many important human and animal pathogens. Molecular and biochemical characterization over the past decade has revealed an extraordinary breadth of biological diversity among this family of viruses. Like all enveloped viruses, paramyxoviruses must fuse their membrane with that of a receptive host cell as a prerequisite for viral entry and infection. Unlike most other enveloped viruses, the vast majority of paramyxoviruses contain two distinct membrane-anchored glycoproteins to mediate the attachment, membrane fusion and particle entry stages of host cell infection. The attachment glycoprotein is required for virion attachment and the fusion glycoprotein is directly involved in facilitating the merger of the viral and host cell membranes. Here we detail important functional, biochemical and structural features of the attachment and fusion glycoproteins from a variety of family members. Specifically, the three different classes of attachment glycoproteins are discussed, including receptor binding preference, their overall structure and fusion promotion activities. Recently solved atomic structures of certain attachment glycoproteins are summarized, and how they relate to both receptor binding and fusion mechanisms are described. For the fusion glycoprotein, specific structural domains and their proposed role in mediating membrane merger are illustrated, highlighting the important features of protease cleavage and associated tropism and virulence. The crystal structure solutions of both an uncleaved and a cleavage-activated metastable F are also described with emphasis on how small conformational changes can provide the necessary energy to mediate membrane fusion. Finally, the different proposed fusion models are reviewed, featuring recent experimental findings that speculate how the attachment and fusion glycoproteins work in concert to mediate virus entry.

A treatment for and vaccine against the deadly Hendra and Nipah viruses.

Hendra virus and Nipah virus are bat-borne paramyxoviruses that are the prototypic members of the genus Henipavirus. The henipaviruses emerged in the 1990s, spilling over from their natural bat hosts and causing serious disease outbreaks in humans and livestock. Hendra virus emerged in Australia and since 1994 there have been 7 human infections with 4 case fatalities. Nipah virus first appeared in Malaysia and subsequent outbreaks have occurred in Bangladesh and India. In total, there have been an estimated 582 human cases of Nipah virus and of these, 54% were fatal. Their broad species tropism and ability to cause fatal respiratory and/or neurologic disease in humans and animals make them important transboundary biological threats. Recent experimental findings in animals have demonstrated that a human monoclonal antibody targeting the viral G glycoprotein is an effective post-exposure treatment against Hendra and Nipah virus infection. In addition, a subunit vaccine based on the G glycoprotein of Hendra virus affords protection against Hendra and Nipah virus challenge. The vaccine has been developed for use in horses in Australia and is the first vaccine against a Biosafety Level-4 (BSL-4) agent to be licensed and commercially deployed. Together, these advances offer viable approaches to address Hendra and Nipah virus infection of livestock and people.

The distribution of henipaviruses in Southeast Asia and Australasia: is Wallace's line a barrier to Nipah virus?

Nipah virus (NiV) (Genus Henipavirus) is a recently emerged zoonotic virus that causes severe disease in humans and has been found in bats of the genus Pteropus. Whilst NiV has not been detected in Australia, evidence for NiV-infection has been found in pteropid bats in some of Australia's closest neighbours. The aim of this study was to determine the occurrence of henipaviruses in fruit bat (Family Pteropodidae) populations to the north of Australia. In particular we tested the hypothesis that Nipah virus is restricted to west of Wallace's Line. Fruit bats from Australia, Papua New Guinea, East Timor and Indonesia were tested for the presence of antibodies to Hendra virus (HeV) and Nipah virus, and tested for the presence of HeV, NiV or henipavirus RNA by PCR. Evidence was found for the presence of Nipah virus in both Pteropus vampyrus and Rousettus amplexicaudatus populations from East Timor. Serology and PCR also suggested the presence of a henipavirus that was neither HeV nor NiV in Pteropus alecto and Acerodon celebensis. The results demonstrate the presence of NiV in the fruit bat populations on the eastern side of Wallace's Line and within 500 km of Australia. They indicate the presence of non-NiV, non-HeV henipaviruses in fruit bat populations of Sulawesi and Sumba and possibly in Papua New Guinea. It appears that NiV is present where P. vampyrus occurs, such as in the fruit bat populations of Timor, but where this bat species is absent other henipaviruses may be present, as on Sulawesi and Sumba. Evidence was obtained for the presence henipaviruses in the non-Pteropid species R. amplexicaudatus and in A. celebensis. The findings of this work fill some gaps in knowledge in geographical and species distribution of henipaviruses in Australasia which will contribute to planning of risk management and surveillance activities.

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.

Immunization strategies against henipaviruses.

Hendra virus and Nipah virus are recently discovered and closely related emerging viruses that now comprise the genus henipavirus within the sub-family Paramyxoviridae and are distinguished by their broad species tropism and in addition to bats can infect and cause fatal disease in a wide variety of mammalian hosts including humans. The high mortality associated with human and animal henipavirus infections has highlighted the importance and necessity of developing effective immunization strategies. The development of suitable animal models of henipavirus infection and pathogenesis has been critical for testing the efficacy of potential therapeutic approaches. Several henipavirus challenge models have been used and recent successes in both active and passive immunization strategies against henipaviruses have been reported which have all targeted the viral envelope glycoproteins.

Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus.

Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.

A neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge.

Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African green monkeys (AGMs) with HeV was recently demonstrated, and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen, and brain. Here, we demonstrate that m102.4, a potent HeV-neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease after infection with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV, and 12 subjects were infused twice with a 100-mg dose of m102.4 beginning at either 10, 24, or 72 hours after infection and again about 48 hours later. The presence of viral RNA, infectious virus, and HeV-specific immune responses demonstrated that all subjects were infected after challenge. All 12 AGMs that received m102.4 survived infection, whereas the untreated control subjects succumbed to disease on day 8 after infection. Animals in the 72-hour treatment group exhibited neurological signs of disease, but all animals started to recover by day 16 after infection. These results represent successful post-exposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease.

A novel model of lethal Hendra virus infection in African green monkeys and the effectiveness of ribavirin treatment.

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable.

Development of an acute and highly pathogenic nonhuman primate model of Nipah virus infection.

Nipah virus (NiV) is an enigmatic emerging pathogen that causes severe and often fatal neurologic and/or respiratory disease in both animals and humans. Amongst people, case fatality rates range between 40 and 75 percent and there are no vaccines or treatments approved for human use. Guinea pigs, hamsters, cats, ferrets, pigs and most recently squirrel monkeys (New World monkey) have been evaluated as animal models of human NiV infection, and with the exception of the ferret, no model recapitulates all aspects of NiV-mediated disease seen in humans. To identify a more viable nonhuman primate (NHP) model, we examined the pathogenesis of NiV in African green monkeys (AGM). Exposure of eight monkeys to NiV produced a severe systemic infection in all eight animals with seven of the animals succumbing to infection. Viral RNA was detected in the plasma of challenged animals and occurred in two of three subjects as a peak between days 7 and 21, providing the first clear demonstration of plasma-associated viremia in NiV experimentally infected animals and suggested a progressive infection that seeded multiple organs simultaneously from the initial site of virus replication. Unlike the cat, hamster and squirrel monkey models of NiV infection, severe respiratory pathology, neurological disease and generalized vasculitis all manifested in NiV-infected AGMs, providing an accurate reflection of what is observed in NiV-infected humans. Our findings demonstrate the first consistent and highly pathogenic NHP model of NiV infection, providing a new and critical platform in the evaluation and licensure of either passive and active immunization or therapeutic strategies for human use.

A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection.

Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID(50) within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.

A glycosylated peptide in the West Nile virus envelope protein is immunogenic during equine infection.

Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognized the recombinant peptide. Failure of most WNV- and MVEV-positive horse sera to recognize the epitope as a deglycosylated fusion protein confirmed that the N-linked glycan was important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain.

Residues in the stalk domain of the hendra virus g glycoprotein modulate conformational changes associated with receptor binding.

Hendra virus (HeV) is a member of the broadly tropic and highly pathogenic paramyxovirus genus Henipavirus. HeV is enveloped and infects cells by using membrane-anchored attachment (G) and fusion (F) glycoproteins. G possesses an N-terminal cytoplasmic tail, an external membrane-proximal stalk domain, and a C-terminal globular head that binds the recently identified receptors ephrinB2 and ephrinB3. Receptor binding is presumed to induce conformational changes in G that subsequently trigger F-mediated fusion. The stalk domains of other attachment glycoproteins appear important for oligomerization and F interaction and specificity. However, this region of G has not been functionally characterized. Here we performed a mutagenesis analysis of the HeV G stalk, targeting a series of isoleucine residues within a hydrophobic alpha-helical domain that is well conserved across several attachment glycoproteins. Nine of 12 individual HeV G alanine substitution mutants possessed a complete defect in fusion-promotion activity yet were cell surface expressed and recognized by a panel of conformation-dependent monoclonal antibodies (MAbs) and maintained their oligomeric structure. Interestingly, these G mutations also resulted in the appearance of an additional electrophoretic species corresponding to a slightly altered glycosylated form. Analysis revealed that these G mutants appeared to adopt a receptor-bound conformation in the absence of receptor, as measured with a panel of MAbs that preferentially recognize G in a receptor-bound state. Further, this phenotype also correlated with an inability to associate with F and in triggering fusion even after receptor engagement. Together, these data suggest the stalk domain of G plays an important role in the conformational stability and receptor binding-triggered changes leading to productive fusion, such as the dissociation of G and F.

A recombinant subunit vaccine formulation protects against lethal Nipah virus challenge in cats.

Nipah virus (NiV) and Hendra virus (HeV) are closely related deadly zoonotic paramyxoviruses that have emerged and re-emerged over the last 10 years. In this study, a subunit vaccine formulation containing only recombinant, soluble, attachment glycoprotein from HeV (sG(HeV)) and CpG adjuvant was evaluated as a potential NiV vaccine in the cat model. Different amounts of sG(HeV) were employed and sG-induced immunity was examined. Vaccinated animals demonstrated varying levels of NiV-specific Ig systemically and importantly, all vaccinated cats possessed antigen-specific IgA on the mucosa. Upon oronasal challenge with NiV (50,000TCID50), all vaccinated animals were protected from disease although virus was detected on day 21 post-challenge in one animal. The ability to elicit protective systemic and mucosal immunity in this animal model provides significant progress towards the development of a human subunit vaccine against henipaviruses.

Expression of novel genes encoded by the paramyxovirus J virus.

Characterization of the J virus or, in keeping with recent nomenclature recommendations, J paramyxovirus (JPV) genome revealed a unique genome structure, consisting of eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. The small hydrophobic (SH) protein and the transmembrane (TM) protein genes are predicted to encode proteins 69 and 258 aa in size, respectively. The 4401 nt attachment (G) protein gene, much larger than most other paramyxovirus attachment protein genes sequenced to date, encodes a putative 709 aa attachment protein and contains distally a second open reading frame (ORF-X) 2115 nt long. Experiments undertaken in this study were intended to confirm the sequence-based gene allocation of JPV and to determine if proteins encoded by the SH gene, the novel TM gene and ORF-X are expressed. Northern blot analyses carried out on mRNA purified from JPV-infected cells indicated that the putative transcription initiation and termination sequences flanking the SH and TM genes are functional, consistent with their allocation as discrete genes, although a high level of read-through was observed across almost all transcriptional boundaries. Probes specific to the G protein coding region and ORF-X both identified an mRNA species corresponding to the predicted length of the G gene, confirming sequence-based predictions. While the SH and TM proteins were both detected in infected cells, no evidence was found for the expression of ORF-X. Preliminary studies indicate that the novel TM protein is a type II glycosylated integral membrane protein, orientated with its C terminus exposed at the cell surface.

Serotype-independent detection of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) causes a highly contagious vesicular disease affecting cloven hoofed animals and is considered the most economically important disease worldwide. Recent FMD outbreaks in Europe and Taiwan and the associated need for rapid diagnostic turnaround have identified limitations that exist in current diagnostic capabilities. To aid improved diagnosis, a serotype-independent FMDV antigen capture assay was developed using antibodies directed against a highly conserved cross-reactive protein fragment (1AB') located within the structural protein 1AB. Cattle sera raised against all 7 serotypes of FMDV bound purified 1AB' demonstrating its immunogenicity in infected animals. Polyclonal anti-1AB' antiserum was produced in chickens and applied as a universal detector of FMDV antigen. Western blot analysis and ELISA both demonstrated that anti-1AB' serum could recognize FMDV antigens independent of serotype. Two recently characterized anti-FMDV monoclonal antibodies were also evaluated for their ability to capture FMDV antigen independently of serotype. When used in combination with chicken anti-1AB' antibodies in an antigen capture ELISA format, all serotypes of FMDV were detected. These data represent the first demonstration of the use of serotype-independent FMDV antigen capture reagents which may enable the development of rapid laboratory based assays or perhaps more significantly, rapid field-based pen-side or point of entry border control diagnostic tests.