Hendra virus and Nipah virus animal vaccines

Henipaviruses and Fruit Bats, Papua New Guinea

Henipaviruses are emerging zoonotic viruses and causative agents of encephalitis in humans. However, the mechanisms of entry into the central nervous system (CNS) in humans are not known. Here, we evaluated the possible role of olfactory epithelium in virus entry into the CNS. We characterized Hendra virus (HeV) and Nipah virus (NiV) infection of primary human olfactory epithelial cultures. We show that henipaviruses can infect mature olfactory sensory neurons. Henipaviruses replicated efficiently, resulting in cytopathic effect and limited induction of host responses. These results show that human olfactory epithelium is susceptible to infection with henipaviruses, suggesting that this could be a pathway for neuroinvasion in humans. IMPORTANCE Henipaviruses are emerging zoonotic pathogens that can cause acute and severe respiratory and neurological disease in humans. The pathways by which henipaviruses enter the central nervous system (CNS) in humans are still unknown. The observation that human olfactory neurons are highly susceptible to infection with henipaviruses demonstrates that the olfactory epithelium can serve as a site of Henipavirus entry into the CNS.

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.

Nipah virus (NiV) and Hendra virus (HeV) are newly identified members of the family Paramyxoviridae and have been classified in the new genus Henipavirus based on unique genetic characteristics distinct from other paramyxoviruses. Transgenic cell lines were generated that expressed either the attachment protein (G) or the fusion protein (F) of NiV. Functional expression of NiV F and G was verified by complementation with the corresponding glycoprotein, which resulted in the development of syncytia. When exposed to NiV and HeV, expression of NiV G in Crandall feline kidney cells resulted in a qualitative inhibition of both cytopathic effect (CPE) and cell death by both viruses. RT-PCR analysis of surviving exposed cells showed a complete absence of viral positive-sense mRNA and genomic negative-sense viral RNA. Cells expressing NiV G were also unable to fuse with cells co-expressing NiV F and G in a fluorescent fusion inhibition assay. Cell-surface staining for the cellular receptors for NiV and HeV (ephrin-B2 and ephrin-B3) indicated that they were located on the surface of cells, regardless of NiV G expression or infection by NiV. These results indicated that viral interference can be established for henipaviruses and requires only the expression of the attachment protein, G. Furthermore, it was found that this interference probably occurs at the level of virus entry, as fusion was not observed in cells expressing NiV G. Finally, expression of NiV G by either transient transfection or NiV infection did not alter the cell-surface levels of the two known viral receptors.

Nipah virus (NiV) and Hendra virus (HeV), which both belong to the genus henipavirus, are zoonotic pathogens that cause severe systemic, neurological, and/or respiratory disease in humans and a variety of mammals. Therefore, monitoring viral prevalence in natural reservoirs and rapidly diagnosing cases of henipavirus infection are critical to limiting the spread of these viruses. Current laboratory methods for detecting NiV and HeV include virus isolation, reverse transcription quantitative real-time PCR (RT-qPCR), and antigen detection via an enzyme-linked immunosorbent assay (ELISA), all of which require highly trained personnel and specialized equipment. Here, we describe the development of a point-of-care customized immunochromatographic lateral flow (ILF) assay that uses recombinant human ephrin B2 as a capture ligand on the test line and a NiV-specific monoclonal antibody (mAb) on the conjugate pad to detect NiV and HeV. The ILF assay detects NiV and HeV with a diagnostic specificity of 94.4% and has no cross-reactivity with other viruses. This rapid test may be suitable for field testing and in countries with limited laboratory resources.

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections.

Inhibition of Henipavirus infection by RNA interference

The immunohistochemical reactivity of seven clones of mouse monoclonal antibodies raised to Nipah virus antigens were investigated using formalin-fixed, paraffin embedded porcine and equine lung tissues from experimental Nipah and Hendra virus infection, respectively. Either microwave irradiation or enzymatic digestion effectively unmasked the viral antigens in formalin-fixed, paraffin-embedded tissue sections. Four clones showed positive reaction to both Nipah virus-infected porcine lung tissue and Hendra virus-infected equine lung tissue. Two clones (11F6 and 13A5) reacted with Nipah virus-infected porcine lung tissue, but not with Hendra virus-infected equine lung tissue. These Nipah virus-specific monoclonal antibodies may therefore be useful for immunohistological diagnosis of Nipah virus infection and for further research on Nipah virus pathogenesis.

Nipah virus (NIV) belongs to Henipavirus genus in the family of paramaxoviridae, which are negative sense single stranded RNA virus.1 Nipah virus is naturally harboured in fruit eating pteropid bats and is reported to cause neurological and respiratory disease in pigs, horses and humans. NIV infection is prevalent in the south Asian region. NIV infection is associated with high fatality rate, which is reported to be 40-75%. Below is a pictogram (Figure 1) of NIV infection, symptoms, pathology and currently known treatment options,2-4 which will be useful to patients as well as healthcare professionals. Read More. . . .

Nipah virus (NiV) and Hendra virus (HeV) are newly emerging dangerous zoonotic pathogens of the Henipavirus genus of the Paramyxoviridae family. NiV and HeV (HNVs) which are transmitted by bats cause acute respiratory disease and fatal encephalitis in humans. To date, as there is a lack of antiviral drugs or effective antiviral therapies, the development of vaccines against those two viruses is of primary importance, and the immunogen design is crucial to the success of vaccines. In this study, the full-length protein (G), the ectodomain (Ge) and the head domain (Gs) of NiV attachment glycoprotein were delivered by the replication-defective type 5 adenovirus vector (Ad5) respectively, and the recombinant Ad5-NiV vaccine candidates (Ad5-NiVG, Ad5-NiVGe and Ad5-NiVGs) were constructed and their immunogenicity were evaluated in mice. The results showed that all the vaccine candidates stimulated specific humoral and cellular immune responses efficiently and rapidly against both NiV and HeV, and the Ad5-NiVGe elicited the strongest immune responses after a single-dose immunization. Furthermore, the potent conserved T-cell epitope DTLYFPAVGFL shared by NiV and HeV was identified in the study, which may provide valid information on the mechanism of HNVs-specific cellular immunity. In summary, this study demonstrates that the Ad5-NiVGe could be a potent vaccine candidate against HNVs by inducing robust humoral and cellular immune responses.

The Nipah virus (NiV), a highly deadly bat-borne paramyxovirus, poses a substantial threat due to recurrent outbreaks in specific regions, causing severe respiratory and neurological diseases with high morbidity. Two distinct strains, NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B), contribute to outbreaks in different geographical areas. Currently, there are no commercially licensed vaccines or drugs available for prevention or treatment. In response to this urgent need for protection against NiV and related henipaviruses infections, we developed a novel homotypic virus-like nanoparticle (VLP) vaccine co-displaying NiV attachment glycoproteins (G) from both strains, utilizing the self-assembling properties of ferritin protein. In comparison to the NiV G subunit vaccine, our nanoparticle vaccine elicited significantly higher levels of neutralizing antibodies and provided complete protection against a lethal challenge with NiV infection in Syrian hamsters. Remarkably, the nanoparticle vaccine stimulated the production of antibodies that exhibited superior cross-reactivity to homologous or heterologous henipavirus. These findings underscore the potential utility of ferritin-based nanoparticle vaccines in providing both broad-spectrum and long-term protection against NiV and emerging zoonotic henipaviruses challenges.

Functional Properties of the Fusion and Attachment Glycoproteins of Nipah Virus

Nipah virus (NiV) is a highly pathogenic zoonotic virus that causes severe encephalitis and respiratory diseases and has a high mortality rate in humans (>40%). Epidemiological studies on various fruit bat species, which are natural reservoirs of the virus, have shown that NiV is widely distributed throughout Southeast Asia. Therefore, there is an urgent need to develop effective NiV vaccines. In this study, we generated recombinant vaccinia viruses expressing the NiV glycoprotein (G) or fusion (F) protein using the LC16m8 strain, and examined their antigenicity and ability to induce immunity. Neutralizing antibodies against NiV were successfully induced in hamsters inoculated with LC16m8 expressing NiV G or F, and the antibody titers were higher than those induced by other vaccinia virus vectors previously reported to prevent lethal NiV infection. These findings indicate that the LC16m8-based vaccine format has superior features as a proliferative vaccine compared with other poxvirus-based vaccines. Moreover, the data collected over the course of antibody elevation during three rounds of vaccination in hamsters provide an important basis for the clinical use of vaccinia virus-based vaccines against NiV disease. Trial Registration: NCT05398796.

Protection against henipaviruses in swine requires both, cell-mediated and humoral immune response

To study the replication of Nipah virus (NiV), a minigenome replication assay that does not require the use of infectious virus was developed. The minigenome was constructed to encode a NiV vRNA analogue containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative NiV transcription motifs and flanked by the NiV genomic termini. CAT protein was detected only when plasmids encoding the NiV minigenome, nucleocapsid protein (N), phosphoprotein (P) and polymerase protein (L) were transfected into CV1 cells. To determine whether NiV conforms to the rule of six, a series of plasmids encoding minigenomes that differed in length by a single nucleotide was tested in the replication assay. CAT production was detected only with the minigenome whose length was an even multiple of six. The replication assay was also used to show that the N, P and L proteins of NiV recognize cis-acting sequences in the genomic termini of Hendra virus (HeV) but not measles virus. While these results suggest that NiV uses a replication strategy that is similar to those of other paramyxoviruses, they also support the inclusion of NiV and HeV in a separate genus within the subfamily Paramyxovirinae.