Keywords
Nipah virus, innate immunity, adaptive immunity, pathogenesis, animal models, contra-measures
Nipah virus, innate immunity, adaptive immunity, pathogenesis, animal models, contra-measures
Emerging infectious diseases pose a significant threat to human and animal welfare in the world. Nipah virus (NiV) is a recently emerged zoonotic Paramyxovirus, from the Mononegavirales order, capable of causing considerable morbidity and mortality in numerous mammalian species, including humans1–3. Although NiV infection remains rare in humans, this virus has captured the attention of both scientific and public health communities because of its high fatality rate, ranging from 40% in Malaysia to more than 90% in Bangladesh and India, where it was associated with frequent person-to-person transmission4,5. Having the capacity to cause severe zoonosis with serious health and economic problems, without efficient treatment yet available, NiV is considered a possible agent for bioterrorism6, has global pandemic potential7, and is classified as a biosecurity level 4 (BSL4) pathogen. In 2015, the World Health Organization included NiV in the Blueprint list of eight priority pathogens for research and development in a public health emergency context8. Furthermore, the Coalition for Epidemic Preparedness Innovations has targeted NiV as a priority for vaccine development on the basis of its high potential to cause severe outbreaks9.
NiV belongs to Henipavirus genus, along with the highly pathogenic Hendra virus (HeV), which emerged in Australia in 199410, and the non-pathogenic Cedar virus discovered in 201211. Moreover, Henipa-like full-length viral sequences were found in African fruit bats12 and Chinese rats (Moijang virus)13. Two major genotypes of NiV have been identified so far: Malaysia and Bangladesh, which share 92% of nucleotide homology14,15 and present some differences in their pathogenicity16. The NiV genome is composed of a negative-sense, single, non-segmented RNA and contains six transcription units encoding for six viral structural proteins (3′-N-P-M-F-G-L-5′) and three predicted P gene products coding for non-structural proteins, C, V, and W, demonstrated to function as inhibitors of the host innate immune response17–20.
NiV was first identified as the cause of an outbreak of encephalitis in humans during 1998 to 1999 in Malaysia and Singapore21. The virus has been transmitted from infected pigs to humans, and the control of the epidemic necessitated culling over 1 million pigs, presenting a huge economic burden22,23. Although no further outbreaks have occurred in Malaysia since then, annual outbreaks of the new NiV strain have started since 2001 in Bangladesh5. The new NiV cases have been identified in the other parts of Southeast Asia: one in Philippines24 and three in India, with the last one in the state of Kerala, reaching a fatality rate of 91%4, solidifying NiV as a persistent and serious threat in South Asia.
Fruit bats from Pteropus species (flying foxes) have been recognized as the natural host of NiV25. Deforestation in large regions of Southeast Asia damages bat roosting trees and food supplies, leading to the migration of bat colonies toward urban sites, thus increasing the contact with humans26,27. NiV transmission from bats to humans was shown to occur through consumption of raw date palm juice or fruits contaminated with bat saliva or urine28. Alternatively, transmission occurs via close contact with infected domestic animals acting as viral amplifying vectors, such as pigs or horses, and via inter-human transmission in one third of NiV Bangladesh strain infections5,29,30. In addition, NiV and Henipa-like viruses have been molecularly or serologically detected (or both) in Pteropus bats in different countries from Asia and Africa12, and the worldwide distribution of these bat species poses a threat to potential NiV pandemics7.
NiV-caused disease is characterized by the onset of non-specific symptoms, including fever, headache, dizziness, vomiting, and myalgia. Later, patients may develop severe encephalitis and pulmonary disease. Respiratory syndrome is observed more frequently in patients infected with NiV Bangladesh. Recently, the persistence of NiV RNA was described in the semen of a patient surviving NiV infection in India31; this is similar to what has been previously reported for Ebola32 and Zika33 virus. Survivors from NiV infection frequently have long-term neurological sequelae34. Furthermore, another clinical syndrome, late-onset encephalitis, has been observed in some patients following an initial NiV infection that was either mild or asymptomatic. Finally, relapse encephalitis could develop as resurgences of the virus, appearing several months to years after recovering from a symptomatic initial infection35, including a case in which encephalitis occurred 11 years after initial infection36.
Primary human epithelial cells from the respiratory tract were shown to be highly permissive to Henipaviruses and may represent the initial site of infection37. Additionally, the virus shows a high neuro-tropism and the ability to infect muscular cells, suggesting rather ubiquitous expression of its entry receptors in different tissues2. In contrast to some other Paramyxoviridae, NiV is not lymphotropic, and among different blood cell types, NiV could infect dendritic cells only38. Nevertheless, viral dissemination within the host is facilitated by NiV attachment to circulating leukocytes through binding to heparan sulfate without infecting the cells39, using leukocytes as a cargo allowing viral transfer to endothelial vascular cells through a mechanism of transinfection38.
NiV uses Ephrin-B2 and -B3 as entry receptors that are highly conserved among numerous species40–43. Indeed, various mammalian species such as hamsters, ferrets, cats and bigger animals, including horses, pigs, and non-human primates, have been experimentally infected and used to develop potential new therapeutics44,45. Furthermore, Pteropus fruit bats, the natural reservoir of the virus, were experimentally inoculated with NiV in order to study their susceptibility to infection, viral distribution, and pathogenesis46,47. No clinical signs were observed in flying foxes, raising the interest of the scientific community in the study of fruit bat–NiV interactions and understanding their capacity to control NiV infection47–49.
Infection of hamsters with both NiV or HeV induces acute fatal encephalitis with a pathology similar to that of humans50,51 and this small-animal model provides a useful tool in studying both pathogenesis and potential countermeasures. As pigs were the critical amplifying host during the NiV outbreak in Malaysia, they have also been used as a model for NiV infection. Indeed, viral shedding, associated with an invasion of the central nervous system, has been associated with a mortality of 10 to 15% in infected animals52. Interestingly, unlike other species, NiV is able to infect certain populations of swine lymphocytes53. Similarly to what has been observed in hamsters54, the NiV Malaysia strain induces higher virus replication and clinical signs in pigs, compared with the NiV Bangladesh strain55. Remarkably, in the ferret model, the NiV Malaysia and Bangladesh strains showed similar pathogenicity56, although higher amounts of viral RNA were recovered in oral secretions from ferrets infected with NiV Bangladesh57.
Although mice represent a small-animal model convenient to study viral infections providing a well-developed experimental toolbox, NiV induces a subclinical infection in elderly wild-type mice only58. However, it has been demonstrated that NiV infection is highly lethal in interferon receptor type I (IFN-I)-deficient mice59,60.
Development of non-human primate models is particularly important for the advances in anti-viral preventive and therapeutic approaches. Squirrel monkeys61 and African green monkeys (AGMs)62 are susceptible to NiV infection, and the AGM model has been used extensively as its general disease progression and symptomatology are similar to those of NiV-infected humans. NiV infection through the respiratory route in AGM induces a generalized vasculitis and reproduces the clinical symptoms observed in humans, including respiratory distress62,63, a neurological disease64, and a viral persistence in the brain from surviving animals65. Furthermore, concomitant to human infections, NiV Bangladesh is more pathogenic than the NiV Malaysia strain in AGM66. Pathogenesis following NiV infection is observed mainly in the respiratory tract and is characterized by acute respiratory distress syndrome and pneumonia following infection of epithelial cells (Figure 1a). As in other animal models, the virus could be found in a wide range of tissues, including kidneys (Figure 1b), brain, or liver (or a combination of these), suggesting efficient viral dissemination62,67.
Innate immune response plays a critical role in anti-viral host defense and its modulation during NiV infection has been demonstrated in several reports17,68–71. Robust expression of anti-viral genes in lung tissue, including MX1, RSAD2, ISG15, and OAS1, during the early stages of NiV infection in ferrets was not sufficient to contain viral dissemination56. Suppression of IFN-I production is known to promote viral spread by disrupting the first lines of defense, resulting in important tissue damage and leading to death. Several mechanisms have been described and both structural and non-structural NiV proteins were found to be involved in the blocking of IFN-I signaling pathway, using distinct strategies72–78, as summarized in Figure 2.
Inhibition of IFN-I response was observed in different animal models during the course of NiV infection. Indeed, NiV infection of hamsters79 and ferrets56 provides insight into the specific viral signature with a downregulated or delayed IFN-I response during the course of infection. In addition, several in vitro studies allowed the identification of viral proteins involved in immune suppression, providing detailed mechanisms of the modulation of IFN-related pathways. Sanchez-Aparicio et al. reported interactions between non-structural NiV-V protein and both RIG-I and RIG-I regulatory protein TRIM2580. They described the binding of the conserved C-terminal domain of NiV-V to caspase activation and recruitment domains (CARDs) of RIG-I and the SPRY domain of TRIM25, thus preventing ubiquitination of RIG-I and its downstream signaling (Figure 2a). In addition to previously described antagonist effects of NiV-V on MDA5 and STAT1 activation73,81, this recent report highlights the multirole of NiV-V protein in dismantling the IFN-I response. Furthermore, another study described the capacity of NiV matrix protein (M), known to be important in virus assembly and budding, to disrupt IFN-I signaling (Figure 2b). Indeed, NiV-M protein interacts with E3-ubiquitin ligase TRIM6, triggering its degradation and subsequent inhibition of IKKε kinase-mediated IFN-I response83. These results were confirmed by a reduced level of endogenous TRIM6 expression upon NiV infection only when M was expressed. Moreover, the role of NiV nucleoprotein (N) was recently reported in hampering IFN-I signaling by preventing the nuclear transport of both signal transducer and activator of transcription 1 (STAT1) and STAT284, subsequently impairing the expression of IFN-stimulated genes. All together, these recently described routes used by NiV proteins to prevent host anti-viral response provide new insights into viral evasion mechanisms involved in the control of the IFN-I pathway.
NiV causes an important modulation of both humoral and cell-mediated immune responses during the course of infection85,86. The NiV outbreak in Kerala in May 2018 provided the opportunity to study the adaptive immune responses in two surviving patients infected with the NiV Bangladesh strain85. Although absolute number of T-lymphocytes remained normal in blood, the marked elevation of activated CD8 T cells, co-expressing granzyme B and PD-1 was observed, suggesting the increase of lymphocyte population important for the elimination of infected cells. Patients surviving NiV infection also had elevated counts in B-lymphocytes, associated with an important generation of NiV-specific IgM and IgG antibodies. These data support the importance of both humoral and cell-mediated immune responses in the protection against NiV infection. Survivors from NiV infection elicited a stronger, more efficient, and more balanced immune response compared with fatalities.
Three recent studies evaluated immune responses in peripheral blood and tissues in ferrets and monkeys, following infection through the respiratory route56,63,87. Analysis of the gene expression profile in ferrets following the infection with the NiV Bangladesh strain showed a time-dependent increase of macrophage markers and an unchanged level of lymphocyte markers in lungs, while brain infection was characterized by limited immune response56, thus presenting the first global characterization of the host gene expression during Henipavirus infection. Study of the peripheral immune response in NiV-infected AGM highlighted the onset of a cell-mediated immune response through the production of Th1-associated cytokines and an increase in CD8+ T cell activation/proliferation markers in blood, lung, and brain tissues, although neutralizing antibodies were not generated during the 10-day course of infection63. Interestingly, the study of natural killer (NK)-cell response during infection in AGM emphasized an increase in their proliferation, activation, and functional activity during both acute and convalescent phases in surviving animals contrary to succumbing ones87, thus suggesting the implication of NK cells in anti-NiV response.
Several vaccine development strategies have recently been studied in small-animal models, including chimeric rabies-based88, virus-like particle (VLP)-based89, adenovirus-based90, and epitope-based91,92 vaccines. Those approaches induced a protection against NiV by triggering a specific response against its envelope glycoprotein G that will require further development using non-human primates to evaluate their efficiency and safety. An additional study using recombinant vesicular stomatitis virus expressing NiV-G protein, in addition to Ebola virus GP protein (rVSV-EBOV-GP-NiV-G), demonstrated complete protection from a high dose of NiV in the hamster model93. That study was followed by further evaluation of the vaccine vector in the AGM model, where the induction of a robust and rapid protective anti-NiV immune response was observed94,95. Vaccination of animals with rVSV-EBOV-GP-NiV-G vector induced protection against NiV challenge when administered either the day before or at the day of challenge and elicited partial protection when administered up to 1 day post-exposure. A plausible explanation of the mechanism involved in the generation of this fast protection could be the stimulation of the host’s innate immune response, inhibiting viral replication and allowing the development of a virus-specific adaptive immune response. Altogether, this recent work and previous reports highlight the importance of the humoral immune response and the protective role of antibodies directed against viral proteins in the control of NiV infection. Indeed, the human monoclonal antibody specific for Hendra G protein, m102.4, elicited promising results against Henipavirus infection following its passive transfer in infected ferrets96 and AGM97 and is being tested in clinical trials.
Although the development of potential NiV vaccines is ongoing and the scrutiny to get authorized vaccines directed against BSL4 pathogens has been accelerated, the only approved vaccine on the market is an animal vaccine directed against HeV in horses in Australia (Equivac-HeV). The importance of a cell-mediated immune protection against Henipaviruses has been demonstrated in hamsters and pigs86,98,99, indicating that particular attention should be given to this arm of the immune response for the development of new vaccines. Moreover, these reports underline that both well-balanced innate and adaptive immune responses play important roles in the control of NiV infection.
In parallel to vaccines, other therapeutic strategies have been under development. Recent in vitro investigations demonstrated that nucleoside inhibitor 4′-azidocytidine (R1479) and its analogs, previously identified to inhibit flaviviruses, are also capable of inhibiting NiV replication and may present potential broad-spectrum anti-viral candidates for future development100,101. A recent study in hamsters demonstrated the ability of favipiravir (T-705), a viral RNA-dependent RNA polymerase inhibitor that acts as a purine analog, in preventing NiV-induced morbidity and mortality when administered immediately following infection102. Those results indicate that favipiravir, shown previously to protect against Ebola virus infection103, is a potentially good candidate for post-exposure prophylaxis to NiV. A different approach has been developed by specifically inhibiting NiV entry into the cells by acting on the fusion machinery104. Indeed, viral entry is mediated by the viral envelope glycoproteins G and F (fusion protein) and can be targeted by fusion-inhibitory peptides2. Intra-tracheal administration of these peptides conjugated to lipids, shown to increase their efficiency, was protective in both hamsters and AGM against high-dose lethal NiV challenge. Finally, recent promising work has validated the efficiency of remdesivir (GS-5734), a broad-acting anti-viral nucleotide prodrug, against NiV Bangladesh in an AGM model, demonstrating its ability to protect monkeys if given 24 hours post-infection105. Clinical trials of this drug against Ebola virus have recently been started in democratic republic of Congo106 and a similar approach will be required for the evaluation of remdesivir against NiV.
NiV attracts particular attention among members of the Paramyxovirus family, as it possesses high zoonotic potential associated with one of the highest fatality rates observed in infectious diseases. The wide distribution of its natural host, the fruit bats, combined with the possibility of the spread of NiV via the respiratory route, raises the risk that pandemics will be caused by this virus in the future and calls for a better understanding of its pathogenesis and the development of efficient anti-viral approaches. NiV proteins were shown to effectively interact with the immune response and disable the establishment of a protective anti-viral immunity. Understanding the host–pathogen relationship at both molecular and cellular levels in different species and elucidating how bats could efficiently control NiV infection represent exciting challenges for future research and may open new avenues in the development of innovative anti-viral strategies. These studies should lead to novel clinical trials, allowing the generation of drugs efficient in the treatment of NiV infection. Further studies require a multidisciplinary approach, putting together virologists, immunologists, epidemiologists, veterinarians, and physicians within a “one health approach” in the common endeavor to understand and control Henipavirus infections.
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
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