Nipah Virus in Pigs

Overview and Taxonomy of Nipah Virus in Pigs

Nipah virus (NiV) represents one of the most formidable emerging zoonotic pathogens of the 21st century, occupying a unique and dangerous ecological niche at the intersection of wildlife, livestock, and human populations. As a highly pathogenic member of the Henipavirus genus within the family Paramyxoviridae, NiV was first recognized during a devastating outbreak in Malaysia and Singapore between 1998 and 1999, an event that fundamentally altered the understanding of viral spillover dynamics from bats to domestic animals and ultimately to humans [3, 6, 15, 23]. The virus was named after the village of Sungai Nipah in Negeri Sembilan, Malaysia, where it was first isolated from human cases [15, 23]. Critically, domestic pigs (Sus scrofa domesticus) served as the amplifying and intermediate host during this seminal outbreak, facilitating the transmission of NiV from its natural reservoir, Pteropus fruit bats, to humans [1, 2, 8, 11, 17]. This role of pigs as a crucial epidemiological bridge has defined the trajectory of NiV research, vaccine development, and surveillance strategies ever since. Understanding the taxonomy, virological characteristics, and the specific dynamics of NiV infection in pigs is therefore not merely an academic exercise but a fundamental prerequisite for developing effective One Health interventions aimed at preventing future pandemics [6, 10, 20, 27].

Taxonomic Classification and Virological Characteristics

NiV is an enveloped, non-segmented, single-stranded, negative-sense RNA virus belonging to the order Mononegavirales [13, 34]. Within the family Paramyxoviridae, it is classified under the subfamily Paramyxovirinae and the genus Henipavirus, a genus it shares with the closely related Hendra virus (HeV) and the more recently discovered Cedar virus [4, 15, 25, 33]. The genus Henipavirus is distinguished from other paramyxoviruses by its exceptionally large genome size (approximately 18.2 kilobases), its broad host tropism, and its utilization of ephrin-B2 and ephrin-B3 as cellular entry receptors [6, 7, 16]. The NiV genome encodes six major structural proteins: the nucleocapsid (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), attachment glycoprotein (G), and the large RNA-dependent RNA polymerase (L) [17, 25]. Additionally, the P gene encodes accessory proteins (V, W, C) through RNA editing, which are critical for subverting host innate immune responses, particularly interferon signaling [6, 34]. The attachment glycoprotein (G) is the primary determinant of host cell tropism, binding with high affinity to ephrin-B2 and ephrin-B3 receptors that are widely expressed on endothelial cells, neurons, and epithelial cells across mammalian species, including pigs [6, 16, 28]. This receptor conservation explains the remarkable capacity of NiV to infect a wide range of hosts, including humans, pigs, horses, dogs, cats, and ferrets [6, 15, 21, 29].

Genetic and antigenic analyses have led to the classification of NiV into two major genotypes: the Malaysia genotype (NiV-M) and the Bangladesh genotype (NiV-B) [1, 8, 14, 16]. A third proposed genotype from India (NiV-I) has been identified, though some phylogenetic analyses suggest it clusters closely with NiV-B [8]. The NiV-M and NiV-B strains exhibit approximately 92% nucleotide sequence identity and 95% amino acid similarity, with distinct epidemiological and clinical characteristics [8, 14]. The NiV-M strain, responsible for the initial Malaysian outbreak, is associated with high viral replication in the porcine respiratory tract and significant pig-to-human transmission, whereas the NiV-B strain, which has caused recurrent outbreaks in Bangladesh and India, demonstrates enhanced human-to-human transmissibility and a higher case fatality rate in humans [8, 14, 16]. In silico modeling by Hoque et al. (2023) demonstrated that the NiV-B G protein exhibits stronger binding affinity to ephrin-B3 receptors compared to NiV-M, a finding that may correlate with the observed differences in pathogenicity and transmission patterns [16]. This genotypic distinction is not merely academic; it has profound implications for diagnostic assay design, vaccine cross-protection, and epidemiological surveillance. For instance, diagnostic tools such as the one-pot RT-RAA/CRISPR-Cas13a assay developed by Zhang et al. (2026) exploit single nucleotide polymorphisms within the N gene to discriminate between NiV-M and NiV-B genotypes with high sensitivity and specificity [1].

The Role of Pigs as Amplifying and Intermediate Hosts

The epidemiological importance of pigs in NiV transmission cannot be overstated. In the Malaysian outbreak, pigs served as both amplifying hosts, supporting high levels of viral replication and shedding, and as the direct source of infection for nearly 300 human cases [2, 3, 13, 15]. The virus entered pig populations through the consumption of partially eaten fruits contaminated with the saliva or urine of infected Pteropus bats, a process that occurred when pig farms were established within the foraging range of bat colonies [6, 12, 25]. Once introduced into a susceptible pig herd, NiV spread rapidly through direct contact with infected bodily secretions (nasal discharge, saliva, urine, feces), aerosolization within confined swine facilities, and fomite transmission [18, 19, 32]. This explosive spread was facilitated by the high density of pigs in Malaysian commercial farms and the aggressive respiratory transmission of the NiV-M strain [3, 15, 28]. The outbreak ultimately resulted in the culling of over one million pigs, approximately 45% of Malaysia's swine population, to contain the virus, inflicting economic losses exceeding US$500 million [2, 3, 13, 26].

Clinically, NiV infection in pigs presents a spectrum of disease that can range from inapparent or mild respiratory illness to severe, fatal neurological disease, a condition termed "porcine respiratory and encephalitic syndrome" [3, 10, 15]. The hallmark of NiV infection in pigs is a pronounced respiratory component, characterized by a harsh, barking cough (the so-called "barking pig syndrome"), tachypnea, nasal discharge, and in some cases, acute hemorrhagic pulmonary edema [3, 15]. Neurological signs, including tremors, myoclonus, hind limb paresis, ataxia, and seizures, are more common in older animals and often accompany or follow the respiratory phase [15, 18]. The incubation period in pigs is generally 7–14 days, and the case fatality rate is typically low (<5%) under experimental conditions, though secondary bacterial infections such as Streptococcus suis and Enterococcus faecalis can complicate the clinical picture and increase mortality [18, 19]. Importantly, Berhane et al. (2008) demonstrated that oronasally inoculated pigs shed NiV in nasal and oropharyngeal secretions for up to 17 days post-infection, and the virus could be isolated from the serum of animals even in the presence of neutralizing antibodies, suggesting a mechanism of immune evasion and prolonged viremia [18]. The primary sites of viral replication in pigs are the respiratory epithelium (trachea, bronchi, bronchioles), the lymphatic system (tonsils, lymph nodes), and the central nervous system (olfactory bulb, brainstem cerebrum) [18, 28].

Comparative Virology and Host Species Susceptibility

The susceptibility of pigs to NiV infection is mediated by the widespread expression of ephrin-B2 and ephrin-B3 receptors on porcine epithelial and endothelial cells [7, 16]. Zhang and Saito (2025) demonstrated that pig-derived PK-15 cells stably expressing porcine ephrin-B2 exhibited over 1000-fold higher infectivity for NiV pseudovirus compared to wild-type PK-15 cells, confirming that receptor expression is a critical bottleneck for species susceptibility [7]. This finding has direct implications for vaccine development and diagnostic testing, as it provides a more relevant porcine cell culture system for propagating pig-origin NiV isolates [7]. However, not all pig populations are equally susceptible to clinical disease. The NiV-M strain, which was responsible for the Malaysian outbreak, is highly adapted to replication in porcine respiratory tissues, whereas the NiV-B strain is more efficient at human-to-human transmission and has not been associated with large-scale outbreaks in pigs [8, 14]. This difference in host tropism is correlated with the differential binding affinity of the two genotype G proteins to ephrin receptors [16].

Furthermore, pigs are not the only domestic animals susceptible to NiV infection. Serological evidence has documented natural infections in dogs, cats, and horses, although the epidemiological role of these species in NiV transmission remains poorly understood [4, 15, 22]. In the Malaysian outbreak, dogs and cats in close contact with infected pigs developed antibodies to NiV, and in one instance, a horse was found to be seropositive [15]. Hoque et al. (2023) performed in silico docking analyses and predicted that domestic rats (Rattus norvegicus) may exhibit even higher binding affinity for NiV G than pigs, suggesting that peridomestic rodents could serve as unrecognized intermediate hosts in Bangladesh and other endemic regions [16]. This underscores the need for comprehensive serosurveillance across multiple animal species to fully understand the transmission ecology of NiV. Importantly, the World Organisation for Animal Health (WOAH) has classified NiV as a notifiable disease, and Malaysia was granted NiV-free status by WOAH in 2001 after a decade of intensive surveillance [5]. The prolonged serological surveillance program conducted by the Department of Veterinary Services (DVS) Malaysia between 2012 and 2023 tested 26,507 pig sera and 18,248 sera from other domestic animals (cats, dogs, horses) using an in-house indirect ELISA and detected no NiV-specific IgG antibodies, confirming the sustained absence of viral circulation following the eradication efforts [5]. This success story highlights the effectiveness of coordinated One Health strategies that combine pig culling, movement restrictions, enhanced biosecurity, and active surveillance.

Global Distribution and Risk of Re-Emergence

Despite Malaysia's success in eradicating NiV from its pig population, the virus continues to pose a significant threat to the global swine industry due to the widespread distribution of Pteropus bats across South and Southeast Asia, Africa, and Oceania [6, 15, 24, 34]. NiV RNA has been detected in fruit bats across Thailand, Cambodia, Indonesia, and India, and the Bangladesh strain has been found in bat populations in Thailand [9, 24]. In Thailand, surveillance spanning two decades from 2001 to 2021 detected NiV RNA annually in bat urine samples, yet no evidence of infection has been found in pigs or humans, suggesting that the ecological conditions necessary for spillover have not yet converged [9]. However, spatial risk modeling by Thanapongtharm et al. (2015, 2019) identified high-risk zones in the Central Plain of Thailand, where bat colonies, intensive pig farming, and human settlements overlap, indicating that the potential for a future outbreak remains high [24, 31]. Similarly, Wonrak et al. (2020) used multi-criteria decision analysis and network modeling to demonstrate that the risk of NiV dissemination through pig movement networks in Thailand is low but not negligible, with infected subdistricts clustering within 171 km of source subdistricts under baseline transmission parameters [30]. The economic consequences of a re-emergence in a pig-dense region like Thailand or China could be catastrophic, mirroring the devastation wrought on the Malaysian pork industry two decades earlier [2, 3].

Molecular Pathogenesis of Nipah Virus Infection in Pigs

The molecular pathogenesis of Nipah virus (NiV) infection in swine represents a complex, multi-faceted interplay between viral virulence determinants, host receptor tropism, and species-specific innate immune responses that ultimately dictates the clinical trajectory observed in this critical intermediate host. As a biosafety level-4 (BSL-4) pathogen within the Henipavirus genus of the Paramyxoviridae family, NiV orchestrates a precisely choreographed entry, replication, dissemination, and immune evasion strategy within the porcine host that distinguishes it from its pathogenesis in humans or other incidental hosts [6, 15, 37]. Understanding these molecular events at an exhaustive level is paramount, given the pivotal role pigs played in the 1998–1999 Malaysian outbreak, the most economically devastating NiV epizootic on record, and their continued potential as amplifying hosts for spillover into human populations [3, 26, 40].

Receptor Engagement and Cellular Entry: The Ephrin-B2/B3 Axis

The initial molecular event governing NiV tropism in pigs is the high-affinity binding of the viral attachment glycoprotein (G) to the host cell surface receptors ephrin-B2 and ephrin-B3 [6, 16]. These transmembrane ligands are evolutionarily conserved across mammalian species, explaining the remarkably broad host range of henipaviruses. Crucially, the ephrin-B2 receptor is expressed on vascular endothelial cells, smooth muscle cells, and neurons, while ephrin-B3 is predominantly restricted to neurons within the central nervous system [6]. This expression pattern directly dictates the profound vasculotropism and neurotropism that characterize NiV infection in pigs.

In silico protein-protein docking analyses have provided granular insights into the molecular basis for NiV host tropism in swine. Hoque et al. (2023) demonstrated that NiV Bangladesh (NiV-B) strain attachment glycoprotein exhibits significantly stronger binding affinity for both ephrin-B2 and ephrin-B3 receptors compared to the NiV Malaysia (NiV-M) strain, with binding energy (ΔG) scores ranging from -8.0 to -19.1 kcal/mol across 13 domestic and peridomestic mammal species [16]. This differential receptor binding correlates with the higher observed pathogenicity of NiV-B strains in vivo. Notably, among all species examined, the ephrin receptors of domestic rats (Rattus norvegicus) showed the highest binding affinity for NiV G, suggesting these animals may be more susceptible than pigs and could serve as previously unrecognized intermediate hosts [16]. For swine specifically, the expression of porcine ephrin-B2 on target cells is the non-negotiable gateway for productive infection.

The development of a porcine kidney cell line (PK-15) stably expressing swine-derived ephrin-B2 has been a transformative tool for dissecting these early molecular events [7]. Zhang and Saito (2025) reported that NiV pseudovirus infectivity was increased by >1,000-fold in PK-15 cells expressing ephrin-B2 compared to wild-type PK-15 cells, and viral susceptibility in these engineered cells was >30-fold higher than in the standard Vero cell line [7]. This finding underscores a critical species-specific molecular adaptation: pig-derived cells are a far more permissive and physiologically relevant platform for studying NiV pathogenesis in swine than the African green monkey kidney cells traditionally recommended by the World Organisation for Animal Health (WOAH) for virus isolation [7]. Furthermore, the generation of Stat2-knockout PK-15/e phrin-B2 cells, which exhibit stable viral infectivity even in the presence of type I interferon, provides a powerful tool to dissect interferon-mediated antiviral responses at the cellular level [7].

Fusion Machinery and Syncytium Formation

Following receptor attachment by the G glycoprotein, the fusion (F) glycoprotein must be activated to mediate the merger of the viral envelope with the host cell membrane. This process is exquisitely pH-independent and requires direct interaction between the attached G protein and the metastable F protein, which undergoes a dramatic conformational rearrangement from a prefusion to a postfusion state [2, 38]. The fusion protein is synthesized as an inactive precursor (F0) that must be cleaved by host cell proteases into disulfide-linked F1 and F2 subunits to become fusion-competent. In pigs, this proteolytic activation occurs efficiently in the respiratory epithelium, contributing to the pronounced respiratory pathology observed in swine [15, 28].

The molecular clamp-stabilized prefusion F protein (mcsF) has emerged as a key immunogen in vaccine development, as antibodies targeting the prefusion conformation can potently neutralize cell-cell fusion [2, 36]. In pigs vaccinated with the mcsF protein, antibodies capable of neutralizing NiV G and F-mediated syncytia formation were significantly induced, whereas the soluble G protein (sG) vaccine induced superior neutralizing antibody titers against free virus particles [2]. This dichotomy highlights the distinct molecular pathways involved in cell-to-cell spread versus cell-free virus dissemination in the porcine host.

The propensity for NiV to induce syncytium formation, the fusion of infected cells with adjacent uninfected cells, is a hallmark of henipavirus pathogenesis and is particularly pronounced in porcine respiratory epithelium. Vaccinia virus-based vectors expressing both NiV F and G proteins have been shown to generate robust tissue-resident memory CD8α+ T cells in the lungs of vaccinated animals, underscoring the importance of the fusion machinery in shaping the adaptive immune landscape [39]. Molecular studies using bovine herpesvirus-4 (BoHV-4) vectors expressing NiV F or G have further demonstrated that while both glycoproteins are immunogenic, the F protein is particularly effective at eliciting antibodies that block cell-cell fusion, whereas the G protein is superior at inducing neutralizing antibodies against cell-free virus [35].

Early Infection of the Respiratory Epithelium: A Species-Specific Cytokine Milieu

One of the most illuminating findings in NiV molecular pathogenesis is the species-specific difference in the innate immune response mounted by porcine versus human respiratory epithelial cells. Elvert et al. (2020) conducted a landmark comparative study using primary porcine bronchial epithelial cells (PBEpC) and primary human bronchial epithelial cells (HBEpC) [28]. When infected with equivalent doses of NiV, the porcine cells exhibited substantially higher viral RNA loads compared to human cells. Paradoxically, despite this higher viral burden, the porcine cells displayed a markedly blunted interferon response, specifically, reduced expression of type III interferons (IFN-λ) and downstream interferon-stimulated genes [28].

This inherently limited antiviral interferon response in porcine cells appears to be a fundamental species-specific molecular determinant. Even when PBEpC were infected with the same viral RNA copy numbers as HBEpC, the porcine cells consistently showed reduced IFN-λ and IFN-dependent gene expression [28]. Crucially, this suppression of antiviral activity did not extend to proinflammatory cytokines; the expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) was robustly induced in infected PBEpC, comparable to or exceeding that seen in human cells [28]. This molecular configuration, diminished antiviral interferon signaling paired with intact proinflammatory cytokine production, creates a permissive environment for unchecked viral replication while simultaneously driving the acute inflammatory pathology that manifests clinically as severe respiratory disease in pigs. This mechanism elegantly explains why pigs infected with the Malaysian NiV strain consistently develop pronounced respiratory signs, while human infections with the same strain typically present with encephalitis and milder respiratory involvement [15, 28].

Dissemination, Vasculotropism, and Neuroinvasion

Following initial replication in the respiratory epithelium, NiV exploits its ephrin-B2 receptor to target vascular endothelial cells, establishing a systemic vasculitis that is pathognomonic for henipavirus infection [6, 18]. The virus infects endothelial cells throughout the microvasculature, leading to syncytium formation, endothelial sloughing, and fibrinoid necrosis. This vascular pathology compromises the blood-brain barrier, providing a direct conduit for viral neuroinvasion. The widespread endothelial infection also explains the hemorrhagic manifestations and multi-organ involvement occasionally observed in severely affected pigs.

From the respiratory tract, NiV disseminates to the lymphatic system, with virus readily detected in submandibular and bronchial lymph nodes [18]. Viral RNA has been detected in these lymphoid tissues as late as 23 days post-inoculation, suggesting that the lymphatic system may serve as a viral reservoir even after apparent clinical recovery [18]. This persistence in lymphoid tissues raises important questions about potential for recrudescence and prolonged shedding in pigs.

Neuroinvasion occurs via two primary routes: hematogenous spread through infected endothelial cells and direct neuronal transport along the olfactory nerve. The olfactory bulb has been identified as a site of viral persistence in experimentally infected pigs, with RNA detection in this structure at 23 days post-inoculation in some animals [18]. The expression of ephrin-B2 on neurons and ephrin-B3 on specific neuronal subpopulations within the central nervous system facilitates the establishment of non-suppurative meningoencephalitis, characterized by neuronal necrosis, perivascular cuffing, and glial nodule formation [6, 15]. Although neurological signs in pigs are generally less severe than the fatal encephalitis seen in humans, suppurative meningoencephalitis has been documented in experimental infections, particularly in animals that develop secondary bacterial infections [18].

Immune Evasion Strategies at the Molecular Level

NiV has evolved sophisticated molecular countermeasures to subvert the host innate immune response, and these mechanisms are directly relevant to its pathogenesis in pigs. The viral genome encodes accessory proteins, V, W, and C, from the phosphoprotein (P) gene through a process of RNA editing [34]. The W protein, in particular, plays a prominent role in antagonizing interferon signaling by localizing to the nucleus and inhibiting the formation of the interferon-stimulated gene factor 3 (ISGF3) complex [34]. In pigs, the efficiency of these immune evasion strategies appears enhanced relative to human cells, as evidenced by the profoundly suppressed type III interferon response observed in NiV-infected porcine bronchial epithelial cells [28].

This suppression is multifactorial. The NiV matrix (M) protein has been shown to interfere with host cell nuclear transport, while the V protein directly targets the STAT1 and STAT2 proteins for proteasomal degradation, effectively crippling the JAK-STAT signaling pathway downstream of both type I and type II interferon receptors [6]. The net effect in porcine cells is a near-complete ablation of the antiviral transcriptional program, while leaving proinflammatory signaling intact, a molecular environment that simultaneously promotes viral replication and drives immunopathology.

Secondary Infections and Immunosuppression

A notable but underappreciated aspect of NiV molecular pathogenesis in pigs is the evidence for virus-induced immunosuppression, which may predispose infected animals to secondary bacterial infections. Berhane et al. (2008) documented bacterial co-infections in cerebrospinal fluid of five NiV-inoculated pigs, including Streptococcus suis and Enterococcus faecalis [18]. One animal developed exudative epidermitis associated with Staphylococcus hyicus [18]. These bacterial complications occurred in the context of observed lymphoid depletion in lymph nodes of all infected pigs, coupled with the demonstrated ability of NiV to infect porcine peripheral blood mononuclear cells in vitro [18]. The combination of direct lymphocyte infection, STAT-mediated interferon suppression, and depletion of lymphoid follicular architecture creates a window of immunological vulnerability that facilitates opportunistic pathogen invasion. This element of pathogenesis warrants rigorous further investigation, as it may contribute to the variable clinical presentation observed in field outbreaks and complicates the development of accurate diagnostic criteria.

In sum, the molecular pathogenesis of NiV in pigs is defined by a precise sequence: high-affinity engagement of conserved ephrin receptors, efficient F protein-mediated membrane fusion, unchecked replication in bronchial epithelium due to species-specifically blunted interferon responses, systemic vascular and lymphatic dissemination, neuroinvasion facilitated by endothelial infection, and active immune evasion mediated by viral accessory proteins. This molecular architecture, combined with the virus's capacity for direct contact and aerosol transmission in swine populations, solidifies the pig's role as an ideal amplifying host and underscores the critical need for targeted molecular interventions to interrupt this pathogenic cascade.

Epidemiology and Transmission Dynamics of Nipah Virus in Pig Populations

The epidemiology of Nipah virus (NiV) within pig populations represents a critical nexus in the zoonotic spillover pathway, one that has fundamentally shaped our understanding of this highly pathogenic paramyxovirus. The seminal outbreak in Malaysia and Singapore during 1998–1999 remains the most devastating demonstration of pigs serving as amplifying and bridging hosts, a phenomenon that not only resulted in profound human morbidity and mortality but also precipitated an economic and agricultural crisis of unprecedented scale for the regional swine industry [2, 3, 26]. Over one million pigs, approximately 45% of Malaysia's swine population, were culled to contain the outbreak, incurring costs exceeding US$500 million and underscoring the catastrophic intersection of livestock disease and public health [3, 13, 26]. This event established pigs as the archetypal intermediate host for NiV, a role that continues to inform global surveillance and risk assessment frameworks endorsed by the World Organisation for Animal Health (WOAH) and the World Health Organization (WHO) [7, 40]. Understanding the intricate dynamics of NiV transmission within and from pig populations is therefore not merely an exercise in veterinary epidemiology; it is a cornerstone of pandemic preparedness.

Historical Emergence and Geographic Distribution of Swine Infection

The index outbreak was first recognized in September 1998 in the states of Perak, Negeri Sembilan, and Selangor in Malaysia, with subsequent transmission to abattoir workers in Singapore through the importation of infected pigs [3, 13, 23]. This event marked the first identification of NiV as a novel henipavirus, distinct from the related Hendra virus discovered in Australia just a few years prior [23]. Critically, the initial epidemiological investigation revealed that the virus had been circulating asymptomatically or with mild clinical signs in pigs for several weeks prior to human case detection, highlighting the insidious nature of swine infection [3, 15]. In the Malaysian context, transmission was overwhelmingly associated with direct contact with pigs, their respiratory secretions, or bodily fluids, with pig farmers, abattoir workers, and pork traders constituting the highest-risk human populations [32, 43]. The Pteropus fruit bat, now recognized as the definitive natural reservoir, was implicated through spillover events linked to bat foraging in pig farm orchards, where partially eaten fruits contaminated with bat saliva or urine served as the initial source of porcine infection [12, 15, 37]. Since the Malaysian outbreak was contained by 1999, no further swine-associated NiV cases have been reported in Malaysia or Singapore; a comprehensive nationwide surveillance program conducted by the Department of Veterinary Services Malaysia, testing 26,507 pig sera between 2012 and 2023, found no seropositive results, confirming the country's WOAH-acknowledged NiV-free status [5]. However, NiV has continued to cause sporadic outbreaks in other South and Southeast Asian nations, including Bangladesh, India, and the Philippines, though in these regions the primary transmission pathways differ. In Bangladesh and India, human infection is predominantly linked to consumption of bat-contaminated date palm sap, with direct pig-to-human transmission playing a diminished role, although the risk of swine involvement remains a concern due to the presence of Pteropus bats and pig farming in overlapping geographies [8, 20, 25]. The Philippines outbreak in 2014, which involved horse-to-human transmission, further illustrates the epidemiological versatility of NiV across different livestock species [15, 46].

Transmission Dynamics Within Swine Populations: Shedding, Routes, and Viral Kinetics

The transmission dynamics of NiV within pig populations are characterized by efficient horizontal spread, predominantly through the respiratory and oropharyngeal routes. Experimental inoculation studies have provided granular insights into the kinetics of viral shedding and the duration of infectiousness. In a landmark study by Berhane et al. [18], piglets inoculated oronasally with a dose of 2.5 x 10⁵ plaque-forming units (PFU) exhibited nasal and oropharyngeal shedding detectable from as early as 2 days post-inoculation (dpi) and persisting until 17 dpi. This extended shedding period, coinciding with the incubation period of 4 to 14 days, creates a substantial window for within-herd amplification [19, 23]. The virus targets the respiratory epithelium as a primary site of replication, with the porcine bronchial epithelial cells (PBEpC) supporting robust viral replication. Notably, species-specific differences in the host immune response contribute to the unique epidemiology of NiV in pigs compared to humans. Elvert et al. [28] demonstrated that NiV infection in primary PBEpC induces a comparatively diminished type III interferon (IFN-λ) response and reduced IFN-dependent antiviral gene expression relative to human bronchial epithelial cells, despite similar viral RNA loads. Concurrently, however, the expression of proinflammatory cytokines such as IL-6 and IL-8 remains functional in porcine cells. This combination of a blunted antiviral state with preserved inflammatory signaling may be a key factor driving the efficient viral replication and pronounced respiratory pathology observed in pigs infected with the Malaysian NiV strain, facilitating high-titer shedding and subsequent transmission [28]. Direct contact transmission through aerosols, fomites, and contaminated secretions is the principal mode of spread within and between pig herds, a mechanism that was responsible for the rapid dissemination of the virus from Malaysia to Singapore via pig trade chains [2, 3].

Virological clearance in pigs is not uniformly rapid. While infectious virus is typically cleared from tissues by approximately 23 dpi, low levels of viral RNA can persist in the submandibular and bronchial lymph nodes and the olfactory bulb for extended periods [18]. Even more concerning is the observation that neutralizing antibody titers, which peak around 1,280 at 16 dpi, do not guarantee complete viral elimination; infectious virus has been isolated from serum at 24 dpi, and viral RNA was detected in serum at 29 dpi, suggesting a slower clearance from some animals than previously assumed [18]. This prolonged viremia has implications for diagnostic window periods and the potential for recrudescence, although the latter has not been definitively demonstrated in pigs. The role of bacterial co-infections in modulating NiV pathogenesis and transmission is an area of emerging concern. In the same experimental model, Berhane et al. [18] isolated Streptococcus suis and Enterococcus faecalis from the cerebrospinal fluid of five NiV-inoculated pigs, with one animal developing suppurative meningoencephalitis. The observation of lymphoid depletion in lymph nodes of all infected pigs, coupled with the ability of NiV to infect porcine peripheral blood mononuclear cells in vitro, suggests that NiV may induce a transient immunosuppression in swine, predisposing them to secondary bacterial infections that could complicate clinical presentations and potentially enhance viral shedding through increased respiratory secretions [18].

The Role of Pig Trade Networks and Spatial Epidemiology

The 1998-1999 outbreak provided a stark illustration of how pig movement networks can amplify and disseminate NiV over vast geographic distances. The spread from index farms in Malaysia to the Singaporean abattoir sector was a direct consequence of the legal and illegal trade of asymptomatic, incubation-period pigs [3, 26]. Contemporary spatial risk modeling has refined our understanding of the factors that predispose pig populations to NiV incursion. Multi-criteria decision analysis (MCDA) in Thailand, a country where NiV RNA has been detected in resident Pteropus lylei populations but where no swine or human cases have yet been documented, has identified high-suitability areas concentrated around bat colonies in provinces such as Chachoengsao, Chonburi, and Nakhonnayok [9, 31]. Key spatial risk factors include proximity to bat roosts, pig population density, distance to forested areas and orchards, and proximity to water bodies, which serve as congregation points for foraging bats. Critically, these risk maps have been overlaid with nationwide pig movement networks to simulate potential outbreak trajectories. Using a susceptible-exposed-infectious-removed (SEIR) model coupled with network analysis, Wongnak et al. [30] identified 14 index subdistricts in Thailand with a high risk of NiV emergence. Their modeling demonstrated that infected subdistricts tend to cluster within the central plain, with a transmission range of approximately 171 km under baseline conditions, but the virus could be disseminated as far as 528.5 km under higher reproduction number scenarios (R₀ = 5). This spatial dependency underscores that the putative risk of NiV dissemination through pig trade networks in Thailand, while low, is not negligible, and it provides a rational basis for prioritizing surveilance and biosecure regionalization strategies [30].

The case of Thailand is particularly instructive for understanding the pre-emptive One Health surveillance paradigm. Since 2001, integrated surveillance of bats, pigs, and humans in villages near known NiV-infected bat roosts has been conducted under the leadership of the Thai Red Cross Emerging Infectious Diseases Health Science Centre [9]. Despite continuous detection of NiV RNA (predominantly Bangladesh strain) in bat urine and roost samples from 2002 to 2020, with one 2017 genome sharing 99.17% identity to a 2004 Bangladeshi human isolate, no NiV-specific IgG antibodies or RNA have been detected in over 8,000 pig samples or in healthy human volunteers or encephalitis patients from these same villages [9]. This suggests that ecological and behavioral barriers, such as the specific foraging habits of P. lylei and the management practices of Thai pig farms, may currently be sufficient to prevent spillover, but the proximity of high-density pig farms in the agricultural ring around Bangkok to bat habitats represents a persistent vulnerability [24, 31]. The spatial analysis from Thanapongtharm et al. [24] revealed that flying fox colonies are predominantly located on Thailand's Central Plain, often in proximity to Buddhist temples, bodies of water, and orchards, environments that also host intensive pig farming operations. High-risk zones for NiV zoonosis were identified on farms with low biosecurity, those close to water sources, and farms where fruit orchards are concomitantly managed, as these conditions maximize the probability of bat-pig contact [24].

Genotypic Variation and Implications for Swine Epidemiology

A critical dimension of NiV epidemiology in pigs is the existence of two distinct genetic lineages, NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B), which exhibit differences in clinical presentation, transmission efficiency, and host receptor binding. The original Malaysian outbreak was characterized by a predominant swine involvement, with human cases primarily arising from occupational exposure to pigs [8, 17]. In contrast, outbreaks in Bangladesh and India have been more strongly associated with foodborne transmission (e.g., date palm sap) and human-to-human spread, with pigs playing a less prominent role [8, 47]. However, this distinction is not absolute, and the presence of both NiV-M and NiV-B strains in bat populations across Southeast Asia, including Thailand, indicates that the potential for swine-mediated outbreaks exists across a wider geographic range than previously appreciated [9]. In silico protein-protein docking analyses have provided a molecular basis for these epidemiological differences. Hoque et al. [16] demonstrated that the NiV-B attachment glycoprotein (G) exhibits significantly stronger binding affinity to ephrin-B2 and ephrin-B3 receptors, particularly ephrin-B3, compared to the NiV-M G protein. This enhanced receptor engagement may correlate with the higher observed pathogenicity of NiV-B in humans and could theoretically influence transmissibility in intermediate hosts, including pigs. The same study identified that ephrin receptors of the domestic rat (Rattus norvegicus) displayed the highest binding affinity for NiV G among 13 domestic and peridomestic species analyzed, suggesting that rats could serve as previously unrecognized intermediate hosts, potentially bridging the gap between bat reservoirs and pig farms in endemic settings [16]. The development of rapid genotyping tools, such as the one-pot RT-RAA/CRISPR-Cas13a assay described by Zhang et al. [1], which can discriminate between NiV-M and NiV-B within one hour with 100% sensitivity and 94% specificity in simulated pig serum samples, represents a significant advancement for field-based surveillance. The ability to genotype circulating strains in pigs in real-time will be essential for understanding genotype-specific transmission dynamics and tailoring control measures accordingly.

Modeling Transmission Dynamics and Quantifying Outbreak Potential

Mathematical modeling has been instrumental in quantifying the transmission potential of NiV within pig populations and the broader bat-pig-human interface. Compartmental models that account for the bat-to-pig-to-human pathway consistently demonstrate that the basic reproduction number (R₀) for NiV in pigs is highly sensitive to parameters governing bat shedding, pig contact rates, and biosecurity practices [11, 41, 42]. Mabotsa and Munganga [41] formulated a model with three partial basic reproduction numbers corresponding to bat, pig, and human populations; the overall R₀ is the maximum of these three values, and the model's disease-free equilibrium is globally stable only when all three partial R₀ values are ≤1. This framework highlights the importance of controlling NiV in pigs as a means of preventing human spillover, even in the presence of ongoing bat-to-bat transmission [41]. Barua and Dénes [44] extended this analysis to include waning immunity (SIRS dynamics) and seasonality in bat birth rates and transmission rates. Their model, which incorporates periodic environments, revealed that the existence of a disease-free periodic solution and the stability of endemic equilibria depend on thresholds that are modulated by seasonal peaks in pig farming activities and bat foraging behavior. The incorporation of optimal control theory into these models has identified specific intervention strategies with the greatest cost-effectiveness. Loyinmi and Gbodoghe [11] demonstrated that combined strategies, including personal prevention (e.g., farm worker protective equipment), biosecurity measures (e.g., netting to exclude bats from pig feed), and rapid pig testing, were far more effective than any single intervention at reducing the cumulative number of pig and human infections. Similarly, Ozioko et al. [45] found that rapid testing and the prompt burial of infected pigs, when combined with personal protection measures, provided the most robust reduction in R₀. These modeling exercises are not merely theoretical; they provide actionable guidance for veterinary authorities in high-risk regions, such as the Central Plain of Thailand, where a combination of spatial risk mapping, targeted serosurveillance, and movement restrictions on pigs from high-risk zones could prevent the amplification of an incursion into a full-scale epizootic [30, 31].

Diagnostic Approaches for Nipah Virus Detection and Genotyping in Pigs

The accurate and timely diagnosis of Nipah virus (NiV) infection in swine is a cornerstone of effective outbreak control, zoonotic risk mitigation, and global food security. Given the role of pigs as amplifying intermediate hosts, as dramatically demonstrated during the 1998–1999 Malaysian outbreak where over one million pigs were culled [3, 13, 26], diagnostic capacity must be both robust for surveillance and rapid for acute clinical management. The diagnostic landscape for NiV in pigs has evolved considerably, moving from classical virological methods to sophisticated molecular and serological platforms capable of differentiation between the two major genotypes, NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B), which exhibit distinct epidemiological and clinical characteristics [1, 8, 14]. This section provides a comprehensive examination of these diagnostic modalities, emphasizing their biological underpinnings, operational applications, and the critical role of genotyping in understanding viral dynamics within porcine populations.

Serological Surveillance and Antibody Detection

Serological assays remain the primary tool for large-scale epidemiological surveillance, retrospective outbreak investigations, and certification of freedom from infection for international trade, as mandated by the World Organisation for Animal Health (WOAH) guidelines. The detection of anti-NiV antibodies in pig populations has been central to understanding the geographical distribution of henipaviruses and assessing the risk of spillover from bat reservoirs [5, 9, 53].

Enzyme-linked immunosorbent assays (ELISAs) have been developed using recombinant NiV proteins, primarily the attachment glycoprotein (G) and the nucleocapsid (N) protein. The G protein is a critical target due to its surface exposure and role in receptor binding, making it a potent immunogen for generating neutralizing antibodies. Fischer et al. demonstrated that truncated forms of Hendra virus (HeV) and NiV G proteins, alongside full-length NiV N protein, can be used effectively in indirect ELISAs for porcine serum screening [4]. Their work highlighted that the N protein-based ELISA is suitable for broad henipavirus screening, while the G protein-based assays enable differentiation between HeV and NiV infections due to higher homologous reactivity [4]. This is particularly important in regions where both viruses or henipa-like viruses may circulate.

A significant advancement has been the development of a competitive ELISA (cELISA) by Zhu et al., which utilizes a monoclonal antibody (mAb) and recombinant NiV G protein [51]. This format offers enhanced specificity and avoids the need for species-specific secondary antibodies, making it adaptable across multiple animal species, including pigs, mini-pigs, and non-human primates. Using a cutoff of 35% inhibition, the cELISA demonstrated a diagnostic sensitivity of 98.58% and a specificity of 99.92% when evaluated against 1,199 negative and 71 positive porcine sera [51]. Crucially, this assay showed 100% agreement with the gold-standard plaque reduction neutralization test (PRNT) and detected seroconversion as early as 14 days post-infection (dpi), persisting through to 28 dpi [51]. The cELISA represents a simpler, faster, and safer alternative to PRNT, which requires live NiV and high-level biocontainment (BSL-4) facilities [51, 53].

However, the limitations of serology must be acknowledged. The window period between infection and seroconversion can allow for undetected viral shedding. Furthermore, the results of the nationwide surveillance program in Malaysia between 2012 and 2023, where 26,507 porcine sera were tested using an in-house indirect ELISA, yielded no seropositive cases [5]. While this supports Malaysia's NiV-free status, the authors rightly caution that the absence of detectable antibodies does not preclude sporadic low-level infections or the presence of the virus in cryptic wildlife reservoirs [5]. This underscores the need for concurrent molecular surveillance, especially in high-risk zones identified by ecological modeling, such as the Central Plain of Thailand where bat colonies and pig farms overlap [24, 30, 31].

Molecular Diagnostics: Nucleic Acid Amplification and Genotyping

Molecular methods, particularly reverse transcription polymerase chain reaction (RT-PCR), are the gold standard for acute-phase diagnosis due to their high sensitivity and ability to detect viral RNA before an antibody response is mounted [23, 47, 53]. Traditional RT-PCR and quantitative RT-PCR (qRT-PCR) targeting conserved regions of the N gene have been widely adopted. The N gene is highly conserved among NiV strains, making it a robust target for pan-NiV detection [1].

A major recent breakthrough is the integration of isothermal amplification with CRISPR-Cas systems, enabling rapid, point-of-care genotyping without the need for sophisticated thermocyclers. Zhang et al. developed a one-pot reverse transcription recombinase-aided amplification (RT-RAA) combined with CRISPR-Cas13a assay specifically for differentiating NiV-M and NiV-B in pigs [1]. By aligning N gene sequences from 67 NiV strains, conserved regions were identified for RT-RAA primer design, and single nucleotide polymorphisms (SNPs) within these regions were exploited for specific crRNA design. The assay demonstrated remarkable sensitivity, detecting as low as 10⁻¹ IU/mL of NiV pseudovirus in simulated pig sera, outperforming conventional qRT-PCR [1]. Specificity was absolute, with no cross-reactivity against other key porcine pathogens such as PCV2, PEDV, PRRSV, PRV, and SVA [1]. The entire one-pot reaction is completed within one hour, providing a sensitivity of 100% and a specificity of 94% in simulated samples [1]. This technology represents a paradigm shift for field-deployable diagnostics, allowing for early detection and genotyping in resource-limited settings, which is critical for implementing genotype-specific control strategies.

Similarly, Chen et al. described a point-of-care nucleic acid detection (POC-NAD) system integrating one-step RT-PCR with lateral flow immunoassay and microfluidic technology [49]. Targeting conserved G and P gene sequences, this system achieved a limit of detection (LoD) of 199.1 copies/reaction and could simultaneously detect both NiV-M and NiV-B [49]. The use of microfluidic chips provides consistent amplification under variable field conditions, and the lateral flow readout enhances visual interpretation, making it suitable for surveillance in abattoirs and remote pig farming communities [49].

Virus Isolation and Cell Culture Systems

Virus isolation remains the definitive gold standard for confirmatory diagnosis and for generating live virus for research and vaccine development. However, it is restricted to BSL-4 laboratories due to the extreme pathogenicity of NiV [10, 50, 52]. Traditionally, WOAH recommends the use of African green monkey kidney-derived Vero cells for NiV isolation [7]. However, species-specific differences in virus-host interactions can influence isolation efficiency. Zhang and Saito addressed this by developing a porcine-derived PK-15 cell line stably expressing the primary NiV receptor, pig ephrin-B2 (PK-15/Ephrin-B2) [7]. This cell line showed a >1000-fold increase in pseudovirus infectivity compared to wild-type PK-15 cells and was >30-fold more susceptible than Vero cells [7]. Furthermore, by knocking out Stat2 (signal transducer and activator of transcription 2) to create interferon-resistant cells, they established a line that maintains stable infectivity even in the presence of type I interferon, making it exceptionally suitable for processing clinical samples that may contain antiviral cytokines [7]. This platform not only enhances the probability of isolating field strains but also provides a more biologically relevant substrate for neutralization tests and vaccine efficacy studies using porcine sera [7, 16].

Integrated Diagnostic Algorithms and Genotypic Considerations

The clinical presentation of NiV in pigs can be subtle, ranging from subclinical infection to severe respiratory and neurological disease, often complicated by secondary bacterial infections such as Streptococcus suis [15, 18]. Therefore, a multi-modal diagnostic approach is essential. For acute cases, the recommended pathway involves initial screening via RT-PCR or RT-RAA/CRISPR from nasal swabs, oropharyngeal swabs, and serum collected during the febrile phase, which correlates with the period of highest viral shedding (2-17 dpi) [18]. Virus isolation should follow in BSL-4 facilities for confirmation, while serological testing (ELISA followed by PRNT) is applied to convalescent sera or for retrospective surveillance [51, 53].

Genotyping is not merely an academic exercise; it has profound implications for public health and animal management. NiV-M, associated with the Malaysian outbreak, is characterized by efficient pig-to-pig and pig-to-human transmission, presenting primarily as respiratory disease in pigs [14, 25]. In contrast, NiV-B exhibits higher human-to-human transmissibility and a higher case fatality rate in humans (>70%), with pigs potentially serving as an incidental rather than amplifying host in some contexts [14, 43]. The ability to rapidly differentiate these genotypes using the CRISPR-Cas13a platform [1] or by sequencing of the N or G genes allows for the immediate implementation of tailored control measures, such as enhanced biosecurity for pig movements in NiV-M outbreaks or focused human contact tracing in NiV-B scenarios [6, 11, 40]. As mathematical modeling has shown, the potential for NiV to spread through pig trade networks is non-trivial [30], and the early identification of the genotype is a critical variable in model parameterization and the design of optimal control strategies, including vaccination if vaccines such as the ChAdOx1 NiV G or mRNA-vectored candidates become licensed for porcine use [2, 11, 36, 48].

Vaccine Development and Immunoprophylaxis for Nipah Virus in Pigs

The imperative for developing effective vaccines and immunoprophylactic strategies against Nipah virus (NiV) in swine is underscored by the pivotal role pigs played as amplifying hosts during the seminal 1998–1999 outbreak in Malaysia and Singapore. That epizootic resulted in nearly 300 human cases, over 100 fatalities, and necessitated the culling of approximately 45% of Malaysia’s pig population, more than one million animals, at an estimated cost exceeding US$500 million [2, 3, 13, 40]. Despite the catastrophic economic and public health consequences, and the continued potential for pigs to serve as a bridge for zoonotic spillover from pteropid bats to humans, no licensed vaccine is currently available for porcine use. The World Organisation for Animal Health (WOAH) recognizes the significance of this gap, and the World Health Organization (WHO) lists NiV as a priority pathogen for which countermeasure development is urgently needed [2, 7, 21, 26]. The development of a safe, efficacious, and economically viable vaccine for pigs is therefore a cornerstone of a One Health approach to mitigating NiV risk, aiming to protect swine health, safeguard agricultural economies, and prevent the species leap that leads to human outbreaks [2, 3, 6, 20].

Rationale for Porcine Vaccination and Immunological Targets

The rationale for vaccinating pigs against NiV extends beyond direct animal welfare. Pigs infected with the Malaysian strain (NiV-M) typically exhibit a respiratory and neurological syndrome, often with mild or unapparent clinical signs, yet they shed high titers of virus in nasal and oropharyngeal secretions, facilitating rapid intra-species transmission and efficient spillover to humans [3, 15, 18, 28]. Mathematical modeling studies have consistently identified the pig-to-pig and pig-to-human transmission pathways as critical determinants of outbreak magnitude and duration [11, 41, 42, 44]. Vaccination aims to achieve three principal objectives: (1) induce sterilizing immunity or significantly reduce viral replication and shedding, thereby breaking the chain of transmission; (2) prevent clinical disease and reduce the economic burden on producers; and (3) provide a public health buffer in pig-dense regions where bat–pig–human interface zones are established, such as the central plain of Thailand, parts of Malaysia, and potentially other geographies with susceptible populations [9, 24, 30, 31].

The primary immunogens for NiV vaccine development are the two surface glycoproteins essential for viral entry: the attachment glycoprotein (G), which binds to the host cell receptors ephrin-B2 and ephrin-B3, and the fusion glycoprotein (F), which mediates membrane fusion [2, 16, 35, 38]. Both glycoproteins are the principal targets of neutralizing antibodies, and antibodies directed against the G protein are particularly potent at blocking receptor interaction and preventing infection [2, 55, 60]. Additionally, cellular immune responses, particularly CD8+ and CD4+ T-cell responses, are increasingly recognized as important correlates of protection, contributing to viral clearance and long-term immunological memory [2, 35, 60].

Subunit and Protein-Based Vaccine Candidates

Among the most extensively evaluated vaccine platforms in pigs are adjuvanted soluble recombinant protein subunits. McLean et al. (2025) conducted a landmark comparative study assessing three distinct candidate vaccines: (1) an adjuvanted soluble NiV G protein (sG), (2) an adjuvanted pre-fusion stabilized NiV F protein (mcsF), and (3) a replication-deficient adenoviral vector encoding NiV G (ChAdOx1 NiV G) [2]. In this study, the sG subunit vaccine induced the strongest neutralizing antibody responses, as measured by conventional virus neutralization assays. The mcsF formulation, conversely, elicited antibodies with superior capacity to neutralize NiV glycoprotein-mediated cell–cell fusion, a process critical for syncytia formation and viral spread. Prime-boost immunization with all three candidates conferred a high degree of protection against NiV challenge in pigs, despite these divergent immunological profiles. Importantly, follow-up studies demonstrated the longevity of the immune response and confirmed broadly comparable immunogenicity in Bangladeshi pigs maintained under field conditions, supporting the translational potential of these approaches in endemic settings [2].

Gao et al. (2022) independently evaluated a NiV sG subunit vaccine expressed in a mammalian eukaryotic system and adjuvanted with CpG oligodeoxynucleotides and aluminum salt (CpG/Alum) [55]. In pigs, this formulation induced robust humoral and cellular immune responses, including significantly elevated levels of NiV-specific and neutralizing antibodies, along with enhanced proliferation of T helper (Th) cells. The vaccine demonstrated strong protective efficacy in a pseudovirus-based in vivo mouse model, supporting its advancement as a promising candidate for swine [55]. The inclusion of CpG motifs likely skews the response toward a Th1 profile, which is considered advantageous for antiviral immunity.

Viral Vectored Vaccines

Viral vectored platforms offer the advantages of robust antigen expression, intrinsic immunogenicity, and the potential for induction of both humoral and cellular responses without the need for exogenous adjuvant in all formulations. The ChAdOx1 NiV G adenoviral vector, benchmarked in the study by McLean et al. (2025), specifically elicited a prominent CD8+ T-cell response, underscoring its capacity to drive cell-mediated immunity [2]. This property is particularly valuable given the role of T cells in controlling NiV infection and potentially mediating clearance from immunologically privileged sites such as the central nervous system [2, 60].

Pedrera et al. (2020) investigated bovine herpesvirus-4 (BoHV-4) vectors expressing either NiV G or F glycoproteins in pigs [35]. The BoHV-4-A-CMV-NiV-GΔTK vector induced neutralizing antibody titers comparable to those elicited by a canarypox (ALVAC) vector encoding NiV G, which had previously been demonstrated to confer protective immunity. The BoHV-4 vector expressing NiV F induced antibodies uniquely capable of neutralizing cell–cell fusion, while both BoHV-4 vectors elicited robust antigen-specific CD4+ and CD8+ T-cell responses, with the G-expressing vector showing particularly strong T-cell activation. The safety profile and large cloning capacity of BoHV-4 make it an attractive platform for livestock vaccination [35].

Poxvirus-based vectors have also been evaluated, albeit primarily in murine models to date. Modified vaccinia Ankara (MVA) expressing NiV G (MVA-NiV-G) has been shown to activate specific CD8+ and CD4+ T-cell responses in mice, with a soluble version of NiV-G proving advantageous for epitope presentation [60]. Medina-Magües et al. (2023) demonstrated that MVA and raccoon poxvirus (RCN) vectors co-expressing NiV F and G proteins induced high titers of circulating neutralizing antibodies and lung-specific IgG and IgA responses in mice, along with effector and tissue-resident memory CD8α+ T cells in the lungs [39]. These findings suggest that poxvirus-vectored vaccines could be adapted for swine, potentially offering a single-dose regimen capable of inducing durable mucosal immunity.

mRNA-Lipid Nanoparticle Vaccines

The success of mRNA-lipid nanoparticle (LNP) technology during the COVID-19 pandemic has catalyzed its application to NiV. Pedrera et al. (2024) evaluated a nucleoside-modified mRNA vaccine encoding soluble NiV G from the Malaysia strain, formulated in LNPs, in pigs [48]. Following a two-dose regimen, potent antigen-binding and virus-neutralizing antibodies were detected in serum. Notably, the antibody responses effectively neutralized both the Malaysia (NiV-M) and Bangladesh (NiV-B) strains, demonstrating cross-genotype breadth, although neutralization of the related Hendra virus (approximately 80% G amino acid identity) was limited. The vaccine also induced antibodies capable of blocking NiV glycoprotein-mediated cell–cell fusion and elicited measurable T-cell cytokine responses, with evidence for both CD4+ and CD8+ T-cell activation [48]. The speed of production and platform flexibility of mRNA-LNP technology make it ideally suited for rapid response to emerging NiV strains or outbreaks, and this candidate is being pursued for both porcine and human use.

Virus-Like Particle and Novel Delivery Platforms

Virus-like particles (VLPs), which are non-infectious, self-assembling structures displaying native viral glycoproteins, represent another promising avenue. Walpita et al. (2017) produced mammalian cell-derived NiV VLPs composed of G, F, and matrix (M) proteins and evaluated them in a hamster model [59]. Immunization with purified VLPs, either alone or with adjuvant, induced significant neutralizing antibody titers and provided 100% protection against lethal NiV challenge following both single-dose and three-dose regimens. While these studies were conducted in hamsters, the VLP platform is directly translatable to pigs and offers the advantage of presenting the glycoproteins in a conformationally authentic, multimeric array that closely mimics the native virion, potentially maximizing B-cell receptor cross-linking and antibody affinity maturation [59].

Oral vaccination represents a particularly attractive concept for free-roaming pigs or extensive production systems where individual injection is impractical. Shuai et al. (2020) developed recombinant rabies virus (RABV) vectors (rERAG333E/NiVG and rERAG333E/NiVF) expressing NiV G or F, based on a live attenuated RABV strain already used for oral rabies vaccination in dogs [56]. Oral administration of these bivalent vaccines to pigs induced significant NiV-neutralizing antibodies and IgG responses against both NiV G and F, along with robust RABV immunity. The potential for combined NiV and rabies control through oral bait delivery is a compelling example of integrating vaccine strategies within a One Health framework [56]. Similarly, Li et al. (2023) utilized an inactivated recombinant rabies virus (SRV9) displaying NiV F or G to induce Th1-biased humoral and cellular immunity in mice, further supporting the dual-use concept [57].

Immunity, Correlates of Protection, and Serological Monitoring

A critical component of vaccine development is the establishment of validated correlates of protection and the deployment of reliable serological assays for monitoring vaccine-induced immunity and conducting serosurveillance. Fischer et al. (2018) developed indirect ELISA assays based on truncated NiV G and full-length NiV N proteins, enabling sensitive detection of NiV-specific antibodies in porcine sera and differentiation from Hendra virus infection [4]. Zhu et al. (2023) advanced this field further by developing a competitive ELISA (cELISA) using a monoclonal antibody and recombinant NiV G, which demonstrated 98.58% diagnostic sensitivity and 99.92% specificity when validated against a panel of experimental pig sera, and showed strong agreement with the plaque reduction neutralization test (PRNT) [51]. Such assays are indispensable for post-vaccination monitoring, for differentiating infected from vaccinated animals (DIVA), and for large-scale epidemiological surveillance.

The development of low-biocontainment surrogate assays has been essential for characterizing immune responses in pigs without requiring BSL-4 containment. Thakur et al. (2019) established an anti-NiV F/G indirect ELISA, a pseudotype-based microneutralization test, and a microfusion inhibition assay using a quantifiable cell–cell fusion system [36]. These tools enable high-throughput screening of vaccine-induced antibody functionality and have been instrumental in demonstrating that all three lead candidates (sG, mcsF, and ChAdOx1-G) generate robust and functional antibody responses [2, 36]. The availability of porcine cell lines stably expressing the NiV receptor ephrin-B2, such as the PK-15/Ephrin-B2 cells developed by Zhang and Saito (2025), further enhances the capacity to perform neutralization assays in a species-relevant context, reducing reliance on African green monkey Vero cells [7].

For population-level surveillance, the nationwide program conducted by the Department of Veterinary Services Malaysia, which tested over 26,500 pig sera between 2012 and 2023 using an in-house indirect ELISA, found no NiV IgG antibodies, indicating that Malaysia has maintained its NiV-free status since 2001 [5]. However, such surveillance underscores the importance of having validated serological tools ready for rapid deployment should outbreaks recur, and for monitoring vaccine coverage in endemic regions [4, 5, 51].

Regulatory and Deployment Considerations for Porcine Vaccines

The pathway to licensure for a porcine NiV vaccine presents distinct challenges and opportunities compared to human vaccine development. Regulatory agencies, such as the WOAH and national veterinary authorities, require demonstration of safety, potency, and efficacy through controlled challenge studies in the target species, typically using a relevant NiV strain (e.g., NiV-M for pigs) [2, 7, 15]. The economic feasibility of a swine vaccine is heavily dependent on production costs, ease of administration (preferably single-dose, injectable, or oral), and integration into existing swine health management programs. The 1998-1999 outbreak demonstrated that even a relatively mild disease in pigs can trigger massive culling and trade restrictions, creating a strong economic incentive for producers to adopt vaccination [3, 13, 32, 58].

A One Health framework necessitates that porcine vaccination strategies be coordinated with human and bat surveillance. Mathematical models have shown that vaccinating pigs can reduce the infection pressure on human populations and lower the basic reproduction number (R₀) of NiV in regions where pigs serve as the primary amplifying host [11, 41, 45, 54]. The ChAdOx1 NiV G and mRNA vaccine platforms, initially developed with human use in mind, can be evaluated in pigs as a preclinical model and then directly applied to swine as a veterinary product, streamlining the development pipeline [2, 48]. The development of a licensed NiV vaccine for pigs would represent a major victory for the One Health approach, simultaneously protecting animal health, agricultural economies, and global public health [2, 3, 6, 20, 26].

One Health Implications and Control Strategies for Nipah Virus in Pigs

The inextricable linkage between human, animal, and environmental health is starkly exemplified by Nipah virus (NiV), a bat-borne paramyxovirus for which pigs serve as a critical amplifying and bridging host [1, 6, 15]. The emergence of NiV in Malaysia during 1998–1999 was a watershed moment, demonstrating how agricultural intensification, ecological disruption, and the pig production chain could converge to precipitate a devastating zoonotic pandemic [3, 13, 15]. NiV is not merely a veterinary pathogen; it is a quintessential One Health pathogen whose control demands integrated, cross-sectoral strategies spanning wildlife ecology, livestock management, human public health surveillance, and diagnostic innovation [6, 9, 27]. The World Health Organization (WHO) has listed NiV as a priority pathogen for research and development due to its pandemic potential, while the World Organisation for Animal Health (WOAH) recognizes its profound implications for international trade and food security [21, 40, 62]. This section provides an exhaustive analysis of the One Health implications of NiV in pigs and delineates comprehensive control strategies required to mitigate the risk of future spillover events.

The One Health Framework: Ecological, Economic, and Public Health Intersections

The Amplifying Host Paradigm and Zoonotic Bridge

The role of pigs in NiV epidemiology is fundamentally defined by their function as amplifying hosts, animals in which the virus replicates to high titers without necessarily causing severe clinical disease, thereby facilitating efficient transmission to humans [3, 15, 19]. During the Malaysian outbreak, the rapid spread of NiV through intensive swine operations created a massive viral reservoir from which pig farmers, abattoir workers, and veterinarians became infected through direct contact with infected pigs' respiratory secretions, urine, and oropharyngeal fluids [13, 32]. This pathway is mechanistically distinct from the bat-to-human transmission routes observed in Bangladesh and India, where consumption of contaminated date palm sap predominates [8, 12, 23]. The pig-human interface therefore represents a uniquely high-risk spillover conduit, as it involves close occupational contact, high viral loads, and the potential for aerosolization in enclosed swine facilities [15, 18, 28].

Critical to understanding this zoonotic bridge is the species-specific pathogenesis of NiV in pigs. Unlike humans, who frequently develop fatal encephalitis, pigs typically exhibit a mild to moderate respiratory illness characterized by coughing, sneezing, nasal discharge, and neurological signs such as tremors, ataxia, and hindlimb weakness [15, 19, 28]. Importantly, subclinical infections are common, meaning infected pigs can shed virus, detected in nasal and oropharyngeal swabs between 2 and 17 days post-inoculation, without presenting overt clinical signs recognizable to farmers [18]. This stealthy transmission dynamic is compounded by the observation that NiV can induce immunosuppression in swine, as suggested by lymphoid depletion in lymph nodes and secondary bacterial infections like those caused by Streptococcus suis and Enterococcus faecalis [18]. The virus infects porcine bronchial epithelial cells, where it triggers a robust proinflammatory cytokine response (interleukin-6 and interleukin-8) while simultaneously downregulating antiviral interferon responses, creating a microenvironment conducive to efficient viral replication and acute lung pathology [28]. These mechanistic insights underscore why pigs are such effective amplifiers: they can harbor and shed substantial viral loads while remaining sufficiently healthy to continue interacting with conspecifics and human caretakers.

Ecological Drivers of Spillover

The emergence of NiV from its natural reservoir, Pteropus fruit bats, into pig populations is fundamentally driven by ecological perturbation and anthropogenic landscape change [6, 15, 24]. Habitat fragmentation, deforestation, and agricultural expansion have increasingly brought flying fox colonies into proximity with pig farms, particularly in Southeast Asia [6, 24, 27]. Bats are known to forage over large distances, visiting fruit orchards and palm groves, and they can contaminate pig feed or water sources with urine, saliva, and partially eaten fruits [15, 34]. In Thailand, spatial risk assessments using multi-criteria decision analysis have identified high-risk zones for NiV transmission at the bat-pig interface, concentrated around bat colonies in provinces such as Chachoengsao, Chonburi, and Nakhon Nayok, where pig farm densities are also elevated [24, 31]. The resulting "contact zones" represent ecological friction points where spillover is most probable, particularly on farms with low biosecurity, proximity to water bodies, and concomitant orchard cultivation [31].

Climate change further modulates spillover risk by altering bat foraging behavior, migration patterns, and reproductive cycles [6, 15]. Seasonal factors, including the timing of bat births and the availability of fruit resources, have been incorporated into mathematical models that demonstrate periodic recurrence of NiV outbreaks under certain parameter conditions [42, 44]. These models consistently identify the bat-to-pig transmission rate as a critical determinant of outbreak dynamics, with the basic reproduction number (R₀) for the pig population being a function of bat viral prevalence, pig contact rates, and biosecurity measures [11, 41, 42, 44]. Sensitivity analyses reveal that parameters related to pig mortality from culling, pig-to-pig transmission, and the rate of human exposure to pigs are among the most influential in shaping epidemic trajectories [11, 45]. This quantitative framework provides a rational basis for targeting interventions where they will have the greatest impact.

Economic and Food Security Consequences

The economic devastation wrought by NiV outbreaks in pig populations is staggering and represents a powerful argument for proactive One Health investment. The 1998–1999 Malaysian outbreak necessitated the culling of approximately 1.1 million pigs, nearly 45% of the national swine herd, at a cost exceeding US$500 million, with additional losses from trade bans, farm closures, and reduced consumer demand [3, 13, 26, 40]. These losses were not merely financial; they disrupted rural livelihoods, destabilized pork supply chains, and created food insecurity for communities dependent on pig farming [3, 61]. The outbreak also triggered a regional trade embargo, with Singapore prohibiting pig imports from Malaysia, thereby severing a critical economic link [3]. Mathematical modeling of hypothetical NiV introductions into Thailand's pig trade network demonstrates that virus dissemination through movement of infected animals could span distances of up to 528 km (with R₀ = 5), affecting interconnected subdistricts and amplifying economic losses [30]. Such analyses highlight that the cost of controlling NiV through intensive surveillance, biosecurity upgrades, and vaccine development is minuscule compared to the economic catastrophe of an uncontrolled outbreak involving a high-consequence pathogen like NiV.

Surveillance and Diagnostic Strategies for Early Detection

Molecular Diagnostic Platforms and Genotyping

Rapid, sensitive, and field-deployable diagnostic assays are the cornerstone of effective NiV control in pigs, enabling early identification of infected animals and containment before widespread dissemination occurs [1, 49, 50]. Traditional quantitative reverse transcription PCR (qRT-PCR) remains a gold standard, but its reliance on sophisticated laboratory infrastructure limits utility in resource-limited rural settings where NiV is most likely to emerge [50, 53]. Recent innovations in isothermal amplification coupled with CRISPR-Cas-based detection have transformed the diagnostic landscape. Zhang et al. [1] developed a one-pot reverse transcription recombinase-aided amplification (RT-RAA)/CRISPR-Cas13a assay that discriminates between NiV genotypes (NiV-Malaysia and NiV-Bangladesh) within one hour, achieving a detection limit of 10⁻¹ IU/mL in simulated pig serum, superior to conventional qRT-PCR. This platform requires no sophisticated instruments, can be deployed at the point of care, and demonstrated 100% sensitivity (95% CI, 92.87–100%) and 94% specificity (95% CI, 83.78–98.36%) [1]. The ability to genotype is clinically and epidemiologically critical, as the two NiV lineages differ in transmission dynamics, pathogenicity, and geographic distribution, with the Bangladesh strain exhibiting enhanced human-to-human transmissibility and higher case fatality rates [1, 8, 16].

Complementary approaches include microfluidic-integrated point-of-care nucleic acid detection (POC-NAD) systems that combine one-step RT-PCR with lateral flow immunoassay visualization. Chen et al. [49] validated such a system targeting conserved regions of the NiV G and P genes, achieving a limit of detection of 199.1 copies per reaction and 100% concordance with RT-PCR in simulated clinical samples. The use of disposable microfluidic chips ensures consistent amplification across field conditions while minimizing cross-contamination [49]. These technologies, once commercially scaled, could enable frontline veterinary services to conduct real-time NiV surveillance at abattoirs, livestock markets, and farm gates, dramatically shortening the window between viral emergence and response.

Serological Surveillance and Cross-Species Screening

Serological assays are indispensable for retrospective surveillance, outbreak investigation, and monitoring of vaccine-induced immunity. The Veterinary Research Institute of Malaysia's Department of Veterinary Services (DVS) has conducted an ambitious nationwide surveillance program, testing 44,755 serum samples from pigs (26,507), cats, dogs, and horses between 2012 and 2023 using an in-house indirect ELISA [5]. All samples tested negative for NiV IgG antibodies, consistent with Malaysia's WOAH-declared NiV-free status since 2001 [5]. However, the absence of detectable antibodies does not preclude sporadic infections or cryptic circulation in wildlife, emphasizing the need for sustained, risk-based surveillance [5].

Advanced serological tools include competitive ELISA (cELISA) based on recombinant NiV glycoprotein (G) and a virus-specific monoclonal antibody, which demonstrated 98.58% diagnostic sensitivity and 99.92% specificity against a panel of 1,199 negative and 71 NiV-positive porcine sera, with 100% agreement with plaque reduction neutralization tests [51]. This assay detects antibodies as early as 14 days post-infection and is simpler, faster, and safer than live-virus neutralization tests, making it suitable for high-throughput screening in endemic areas [51]. Fischer et al. [4] developed differential ELISAs using truncated Hendra virus (HeV) and NiV G proteins, enabling discrimination between henipavirus infections, a critical capability given the overlapping geographic ranges and serological cross-reactivity between NiV and HeV. The development of porcine cell lines stably expressing the ephrin-B2 receptor (PK-15/ephrin-B2 cells) has further enhanced diagnostic capacity by providing a species-specific platform for virus isolation and neutralization testing that outperforms Vero cells by >30-fold in susceptibility [7]. These tools collectively constitute a comprehensive serodiagnostic arsenal for both active surveillance and retrospective epidemiological investigations.

Integrated Surveillance Systems and Mathematical Modeling

One Health surveillance must transcend species-specific silos and embrace triangulated monitoring across bats, pigs, and humans in high-risk zones. Thailand's two-decade-long proactive surveillance program exemplifies this approach, combining periodic sampling of fruit bats at known roosts with testing of pigs and healthy human volunteers in adjacent communities [9]. NiV RNA (predominantly Bangladesh strain) has been detected annually in Thai bats since 2002, yet no seropositive pigs or humans have been identified, suggesting that current biosecurity and ecological barriers are preventing spillover [9]. This success story highlights the value of pre-emptive cross-sectoral collaboration and community engagement, including training 100 villagers on safe co-existence with bats [9].

Mathematical modeling provides a quantitative framework for optimizing surveillance allocation and predicting outbreak trajectories. Compartmental models that incorporate bat, pig, and human populations with seasonally varying transmission rates have been used to compute partial basic reproduction numbers for each species, with the maximum value determining the overall R₀ [41, 42, 44]. These models demonstrate that the disease-free equilibrium is globally stable when R₀ ≤ 1, whereas endemic equilibria emerge when species-specific thresholds exceed unity [41, 44]. Sensitivity analyses identify the bat-to-pig transmission rate, pig culling rate, and human exposure rate as the most influential parameters for controlling NiV [11, 45]. Optimal control theory, applying Pontryagin's maximum principle, has been used to design cost-effective intervention strategies combining personal protection, rapid testing, infected pig culling, and treatment of human cases [45, 54]. These theoretical frameworks can guide veterinary authorities in prioritizing resources towards farms with the highest spillover risk scores, as identified through spatial multi-criteria decision analysis [30, 31].

Biosecurity, Farm Management, and Quarantine Strategies

Structural and Operational Biosecurity Measures

Preventing NiV incursion into pig herds begins with rigorous biosecurity protocols designed to sever the bat-pig transmission pathway. Given that flying foxes can contaminate pig feed and water with virus-laden urine and saliva, physical exclusion is paramount [15, 34]. Farms in high-risk zones should install bat-proof netting over feed storage areas, pig housing, and water sources, and should avoid planting fruit trees that attract bats in proximity to pig barns [15, 31]. Orchards co-located with pig farms represent a particularly high-risk configuration, as fallen fruit attracts foraging bats that may subsequently roost in barn rafters and defecate into pig feeding troughs [24, 31]. Spatial analyses in Thailand have identified distance to nearest bat colony, distance to nearest forest, and distance to nearest orchard as three of the seven key risk factors for NiV transmission in pigs, with high-suitability areas concentrated within a 50 km radius of major Pteropus lylei roosts [31].

Operational biosecurity also encompasses strict control of pig movements, as animal transportation networks can rapidly disseminate NiV across geographic regions. Network modeling of Thailand's pig trade chain indicates that infected subdistricts cluster within 171 km of source farms, but under worst-case scenarios (R₀ = 5), virus can travel up to 528.5 km through interconnected trading routes [30]. This underscores the need for movement restrictions during outbreaks, pre-movement testing, and traceability systems that allow rapid identification of at-risk farms. The use of multi-criteria decision analysis to generate risk maps, overlaid with pig movement networks, can identify "index" subdistricts where NiV is most likely to emerge, enabling targeted surveillance and pre-emptive movement bans [30].

Culling, Quarantine, and Stamping-Out Protocols

Historically, the most drastic and economically devastating control measure has been mass culling of infected and potentially exposed pig herds. The Malaysian outbreak was ultimately controlled by culling approximately 45% of the national pig population, a measure that, while effective in eliminating the virus, resulted in catastrophic economic losses and raised ethical concerns regarding animal welfare [3, 13, 26]. Mathematical modeling suggests that rapid culling of infected pigs, combined with quarantine of exposed herds, is one of the most effective strategies for reducing R₀ below unity, particularly when implemented within the first week of outbreak detection [11, 45, 54]. Optimal control analyses indicate that combining culling with biosecurity improvements and human personal protection yields the greatest reduction in cumulative infections at the lowest cost [54].

However, the sociopolitical acceptability and feasibility of mass culling vary considerably across cultural and economic contexts. In regions where pigs represent critical household assets or where religious sensitivities surround pig farming, alternative strategies such as ring vaccination, enhanced biosecurity, and controlled marketing may be more appropriate [3, 30]. The development of effective NiV vaccines for pigs (discussed below) could provide a humane alternative to culling, enabling "test-and-vaccinate" approaches that protect herds while maintaining trade continuity.

Vaccination Strategies for Pigs as a One Health Intervention

Overview of Vaccine Candidates and Immunogenicity Profiles

The development of NiV vaccines for pigs represents the single most impactful One Health intervention, as vaccinating the amplifying host would simultaneously protect animal health, prevent pig-to-pig transmission, and eliminate the primary source of human infection [2, 3, 40]. Despite the urgency, no licensed vaccine for NiV exists for either humans or livestock, although several candidates have advanced to preclinical and early clinical evaluation [2, 40, 58]. The most thoroughly evaluated candidates in pigs include: (1) adjuvanted soluble NiV G protein (sG) subunit vaccine; (2) adjuvanted pre-fusion stabilized NiV F protein (mcsF) subunit vaccine; (3) replication-deficient adenoviral vector (ChAdOx1) expressing NiV G; (4) mRNA-vectored NiV G; (5) bovine herpesvirus-4 (BoHV-4) vectors expressing NiV G or F; and (6) rabies virus-vectored oral vaccines [2, 35, 36, 48, 55-57].

McLean et al. [2] conducted a landmark head-to-head comparison of sG, mcsF, and ChAdOx1 NiV G vaccines in pigs, demonstrating that all three confer high protection against NiV challenge despite inducing distinct immunological profiles. The sG subunit vaccine elicited the strongest neutralizing antibody response, with titers persisting for several months; mcsF induced antibodies with superior ability to block NiV glycoprotein-mediated cell-cell fusion; and ChAdOx1 G generated robust CD8+ T-cell responses, which are increasingly recognized as critical for clearing NiV-infected cells [2, 36, 60]. Importantly, these immune responses were comparable in both laboratory-reared pigs and Bangladeshi pigs under field conditions, suggesting the vaccine platform is robust across genetic backgrounds and environmental settings [2]. The mRNA-vectored NiV G vaccine, formulated in lipid nanoparticles, induced potent cross-neutralizing antibodies against both NiV-M and NiV-B strains, though neutralization of the closely related Hendra virus was limited due to ~80% amino acid identity in the G protein [48]. This vaccine also elicited measurable CD4+ and CD8+ T-cell responses, supporting its further evaluation for both pigs and humans [48].

Mechanisms of Protective Immunity and Correlates of Protection

Understanding the immune correlates of protection against NiV in pigs is essential for rational vaccine design and licensure. The NiV attachment glycoprotein (G) and fusion glycoprotein (F) are the primary targets of protective immunity, as antibodies directed against these surface proteins can neutralize virus entry and inhibit cell-to-cell spread [35, 36, 38, 56, 60]. Neutralizing antibodies are considered a major correlate of protection, with high titers strongly associated with survival in animal models [2]. However, T-cell responses, particularly CD8+ cytotoxic T lymphocytes that eliminate infected cells, are increasingly recognized as necessary for complete viral clearance and prevention of persistent infection [2, 35, 39, 60]. The BoHV-4-vectored NiV G vaccine, for instance, induced significantly higher NiV-neutralizing antibodies

References

[1] Zhang H, Cui C, Wang X, Liu S, Wang X, Wang Y, et al.. Development of a one-pot RT-RAA/CRISPR-Cas13a assay for rapid genotyping of Nipah virus in pigs.. Diagnostic microbiology and infectious disease. 2026. DOI: https://doi.org/10.1016/j.diagmicrobio.2026.117316

[2] McLean R, Pedrera M, Thakur N, Elrefaey A, Hodgson S, Lowther S, et al.. Nipah virus vaccines evaluated in pigs as a ‘One Health’ approach to protect public health. npj Vaccines. 2025. DOI: https://doi.org/10.1038/s41541-025-01212-y

[3] McLean R, Graham S. Vaccine Development for Nipah Virus Infection in Pigs. Frontiers in Veterinary Science. 2019. DOI: https://doi.org/10.3389/fvets.2019.00016

[4] Fischer K, Diederich S, Smith GA, Reiche S, Reis VPd, Stroh E, et al.. Indirect ELISA based on Hendra and Nipah virus proteins for the detection of henipavirus specific antibodies in pigs. PLoS ONE. 2018. DOI: https://doi.org/10.1371/journal.pone.0194385

[5] Tulis AN, Ballakrishnan N, Azami MM, Hamid MRM@, Selvia L, Saeid FHM. Serological Detection of Nipah Virus in Domestic Animals in Malaysia Between 2012 and 2023. Journal of Tropical Resources and Sustainable Science (JTRSS). 2026. DOI: https://doi.org/10.47253/jtrss.v14i1.1540

[6] Satapathy T, Sahu P, Satapathy A, Bhardwaj SK, Satapathy A, Yadav N, et al.. Nipah Virus (NiV) at the Human-Animal-Environment Interface: Emerging Insights into Spillover Dynamics, Neurotropism, and Future Pandemic Risk. Journal of Drug Delivery and Therapeutics. 2025. DOI: https://doi.org/10.22270/jddt.v15i11.7457

[7] Zhang H, Saito A. Development of a Porcine Cell Line Stably Expressing Ephrin‐B2 for Nipah Virus Research and Diagnostic Testing. bioRxiv. 2025. DOI: https://doi.org/10.1111/1348-0421.70022

[8] Tan FH, Sukri A, Idris N, Ong K, Schee J, Tan CT, et al.. A systematic review on Nipah virus: global molecular epidemiology and medical countermeasures development. Virus Evolution. 2024. DOI: https://doi.org/10.1093/ve/veae048

[9] Wacharapluesadee S, Ghai S, Duengkae P, Maneeorn P, Thanapongtharm W, Saraya A, et al.. Two decades of one health surveillance of Nipah virus in Thailand. One Health Outlook. 2021. DOI: https://doi.org/10.1186/s42522-021-00044-9

[10] Manna S, Satapathy P, Balasubramanian S, Gupta A. Beyond zoonosis: a one health exploration of nipah virus clinical spectrum. The Evidence. 2024. DOI: https://doi.org/10.61505/evidence.2024.2.1.13

[11] Loyinmi AC, Gbodogbe S. Mathematical Modeling and Control Strategies for Nipah Virus Transmission Incorporating Bat-To-Pig-To-Human Pathway. Educatum. Journal of science, mathematics and technology. 2024. DOI: https://doi.org/10.37134/ejsmt.vol11.1.7.2024

[12] Neha D, Dung ND, Thomas A, John J. Emerging Trends in the Transmission Pathways of Nipah virus: A Comprehensive Review. Jurnal Kebidanan Manna. 2024. DOI: https://doi.org/10.58222/jkm.v3i2.1207

[13] Nair AP. NIPAH VIRUS. International journal of scientific research. 2024. DOI: https://doi.org/10.36106/ijsr/2905763

[14] Mahedi MRA, Rawat A, Rabbi F, Babu K, Tasayco ES, Areche FO, et al.. Understanding the Global Transmission and Demographic Distribution of Nipah Virus (NiV). Research Journal of Pharmacy and Technology. 2023. DOI: https://doi.org/10.52711/0974-360x.2023.00592

[15] Bruno L, Nappo M, Ferrari L, Lecce RD, Guarnieri C, Cantoni A, et al.. Nipah Virus Disease: Epidemiological, Clinical, Diagnostic and Legislative Aspects of This Unpredictable Emerging Zoonosis. Animals. 2022. DOI: https://doi.org/10.3390/ani13010159

[16] Hoque A, Rahman MM, Lamia AS, Islam A, Klena J, Satter S, et al.. In silico prediction of interaction between Nipah virus attachment glycoprotein and host cell receptors Ephrin-B2 and Ephrin-B3 in domestic and peridomestic mammals.. Infection, Genetics and Evolution. 2023. DOI: https://doi.org/10.1016/j.meegid.2023.105516

[17] Askari MRA, Menezes G, Omran HH, Ejaz A, Ejaz H, Hameed SS. Nipah Virus: A Threatening Outbreak. Journal of Clinical and Diagnostic Research. 2023. DOI: https://doi.org/10.7860/jcdr/2023/52734.17504

[18] Berhane Y, Weingartl H, Weingartl H, López J, Neufeld J, Czub S, et al.. Bacterial infections in pigs experimentally infected with Nipah virus.. Transboundary and Emerging Diseases. 2008. DOI: https://doi.org/10.1111/j.1865-1682.2008.01021.x

[19] Nimesh S, Kumar J. An deadly outbreak of Nipah virus in India. Pharmacy & Pharmacology International Journal. 2019. DOI: https://doi.org/10.15406/ppij.2019.07.00232

[20] Singhai M, Jain R, Jain S, Bala M, Singh S, Goyal R. Nipah Virus Disease: Recent Perspective and One Health Approach. Annals of Global Health. 2021. DOI: https://doi.org/10.5334/aogh.3431

[21] Johnson KN, Vu M, Freiberg AN. Recent advances in combating Nipah virus. Faculty Reviews. 2021. DOI: https://doi.org/10.12703/r/10-74

[22] Singh D, Ahuja R, Singh N. EFFECTS OF NIPAH VIRUS IN TODAY’S WORLD. International Journal of Innovative Science & Technology. 2018. DOI: https://doi.org/10.22270/IJIST.V3I2.12

[23] Ang B, Lim TCC, Wang L. Nipah Virus Infection. Journal of Clinical Microbiology. 2018. DOI: https://doi.org/10.1128/JCM.01875-17

[24] Thanapongtharm W, Linard C, Wiriyarat W, Chinsorn P, Kanchanasaka B, Xiao X, et al.. Spatial characterization of colonies of the flying fox bat, a carrier of Nipah Virus in Thailand. BMC Veterinary Research. 2015. DOI: https://doi.org/10.1186/s12917-015-0390-0

[25] Sharma V, Kaushik S, Kumar R, Yadav J, Kaushik S. Emerging trends of Nipah virus: A review. Reviews in Medical Virology. 2018. DOI: https://doi.org/10.1002/rmv.2010

[26] Gurley E, Spiropoulou C, Wit Ed. Twenty Years of Nipah Virus Research: Where Do We Go From Here?. Journal of Infectious Diseases. 2020. DOI: https://doi.org/10.1093/infdis/jiaa078

[27] Chattu VK, Kumar RK, Kumary S, Kajal F, David JK. Nipah virus epidemic in southern India and emphasizing “One Health” approach to ensure global health security. Journal of Family Medicine and Primary Care. 2018. DOI: https://doi.org/10.4103/jfmpc.jfmpc_137_18

[28] Elvert M, Sauerhering L, Maisner A. Cytokine Induction in Nipah Virus-Infected Primary Human and Porcine Bronchial Epithelial Cells.. Journal of Infectious Diseases. 2020. DOI: https://doi.org/10.1093/infdis/jiz455

[29] Seifert SN, Letko M, Bushmaker T, Laing E, Saturday G, Meade-White K, et al.. Rousettus aegyptiacus Bats Do Not Support Productive Nipah Virus Replication. Journal of Infectious Diseases. 2019. DOI: https://doi.org/10.1093/infdis/jiz429

[30] Wongnak P, Thanapongtharm W, Kusakunniran W, Karnjanapreechakorn S, Sutassananon K, Kalpravidh W, et al.. A ‘what-if’ scenario: Nipah virus attacks pig trade chains in Thailand. BMC Veterinary Research. 2020. DOI: https://doi.org/10.1186/s12917-020-02502-4

[31] Thanapongtharm W, Paul M, Wiratsudakul A, Wongphruksasoong V, Kalpravidh W, Wongsathapornchai K, et al.. A spatial assessment of Nipah virus transmission in Thailand pig farms using multi-criteria decision analysis. BMC Veterinary Research. 2019. DOI: https://doi.org/10.1186/s12917-019-1815-y

[32] Saha A, Debnath B. The obscure impact of Nipah virus. Bionatura. 2019. DOI: https://doi.org/10.21931/rb/2019.04.01.13

[33] Sachan D, Verma M, Jain P, Kumar S, Kharya P. Nipah virus outbreak: A comparative study from Southeast Asia. Indian Journal of Community Health. 2019. DOI: https://doi.org/10.47203/ijch.2019.v31i02.002

[34] Zafar S, Fatima Z, Ekram M. Nipah Virus- A Rapidly Emerging Zoonoses: A Mini Review. . 2019. DOI: https://doi.org/10.21089/njhs.44.0158

[35] Pedrera M, Macchi F, McLean R, Franceschi V, Thakur N, Russo L, et al.. Bovine Herpesvirus-4-Vectored Delivery of Nipah Virus Glycoproteins Enhances T Cell Immunogenicity in Pigs. Vaccines. 2020. DOI: https://doi.org/10.3390/vaccines8010115

[36] Thakur N, Barman N, Chang L, Chappell KJ, Gilbert S, Lambe T, et al.. Development of low bio-containment assays to characterise the antibody responses in pigs to Nipah virus vaccine candidates. Access Microbiology. 2019. DOI: https://doi.org/10.1099/ACMI.AC2019.PO0226

[37] Jabeen H, Fatima A, Saeed FU. Insights into Nipah Virus: A Review of Epidemiology, Pathogenesis, and Therapeutic Advances. International Journal of Innovative Science and Research Technology. 2024. DOI: https://doi.org/10.38124/ijisrt/ijisrt24apr1374

[38] Aqsha ZM, Dharmawan MA, Kharisma VD, Ansori A, Sumantri N. Reverse Vaccinology Analysis of B-cell Epitope against Nipah Virus using Fusion Protein. Jordan Journal of Pharmaceutical Sciences. 2023. DOI: https://doi.org/10.35516/jjps.v16i3.1602

[39] Medina-Magües ES, Lopera-Madrid J, Lo MK, Spiropoulou C, Montgomery J, Medina-Magües LG, et al.. Immunogenicity of poxvirus-based vaccines against Nipah virus. Scientific Reports. 2023. DOI: https://doi.org/10.1038/s41598-023-38010-2

[40] Alubaidi GT. Nipah Virus Proposed Vaccines: Are We Prepared For The Expected Pandemic? (Letter to editor). New Armenian Medical Journal. 2026. DOI: https://doi.org/10.56936/18290825-2026.20v.1-119

[41] Mabotsa M, Munganga J. Mathematical modelling of the spread of Nipah virus in bats, humans and pigs. Journal of Interdisciplinary Mathematics. 2025. DOI: https://doi.org/10.47974/jim-2060

[42] Barua S, Ibrahim MA, Dénes A. A compartmental model for the spread of Nipah virus in a periodic environment. AIMS Mathematics. 2023. DOI: https://doi.org/10.3934/math.20231516

[43] Suman N, Khandelwal E, Chiluvuri P, Rami DS, Chansoria S, Jerry A, et al.. NIPAH Virus Encephalitis: Unveiling the Epidemiology, Risk Factors, and Clinical Outcomes – A Systematic Review and Meta-Analysis. Journal of Pharmacy and Bioallied Sciences. 2024. DOI: https://doi.org/10.4103/jpbs.jpbs_935_23

[44] Barua S, Dénes A. Global dynamics of a compartmental model for the spread of Nipah virus. Heliyon. 2023. DOI: https://doi.org/10.1016/j.heliyon.2023.e19682

[45] Ozioko AL, Okeke R, Fadugba SE, Malesela KC, Christopher G, Mbah E. Optimal Nipah virus (NiV) spread control dynamics. Communications in Mathematical Biology and Neuroscience. 2023. DOI: https://doi.org/10.28919/cmbn/8045

[46] Tripathy T, Das S, Singh D, Prusty U, Mishra M, Pattanaik JK, et al.. Homoeopathy in NIPAH Virus. South Asian Research Journal of Applied Medical Sciences. 2023. DOI: https://doi.org/10.36346/sarjams.2023.v05i05.004

[47] Banerjee S, Gupta N, Kodan P, Mittal A, Ray Y, Nischal N, et al.. Nipah virus disease: A rare and intractable disease.. Intractable & Rare Diseases Research. 2019. DOI: https://doi.org/10.5582/irdr.2018.01130

[48] Pedrera M, McLean R, Medfai L, Thakur N, Todd S, Marsh G, et al.. Evaluation of the immunogenicity of an mRNA vectored Nipah virus vaccine candidate in pigs. Frontiers in Immunology. 2024. DOI: https://doi.org/10.3389/fimmu.2024.1384417

[49] Chen W, Cai L, Ye D, Chen J, Ai X, Tang X, et al.. A Stable and Dependable Visual Technique for On-Site Nipah Virus Nucleic Acids Detection. Scientific Reports. 2025. DOI: https://doi.org/10.1038/s41598-025-91593-w

[50] Khutade K, Shah H, Patil S, Patel H. The Era of Molecular Biology: Diagnostics for Nipah Virus. International Journal for Research in Applied Science and Engineering Technology. 2023. DOI: https://doi.org/10.22214/ijraset.2023.56145

[51] Zhu W, Pickering B, Smith GA, Pinette M, Truong T, Babiuk S, et al.. Development and laboratory evaluation of a competitive ELISA for serodiagnosis of Nipah and Hendra virus infection using recombinant Nipah glycoproteins and a monoclonal antibody. Frontiers in Veterinary Science. 2023. DOI: https://doi.org/10.3389/fvets.2023.1120367

[52] Hassan D, Ravindran R, Hossain A. Nipah Virus Mystery: Insight into Transmission and Mechanism of Disease Progression. Journal of Pure and Applied Microbiology. 2022. DOI: https://doi.org/10.22207/jpam.16.1.72

[53] Mazzola LT, Kelly-Cirino C. Diagnostics for Nipah virus: a zoonotic pathogen endemic to Southeast Asia. BMJ Global Health. 2019. DOI: https://doi.org/10.1136/bmjgh-2018-001118

[54] Mohamed Z, Khalil, Rimi M, Mouline J. Modeling Nipah virus dynamics and designing optimal control strategies. Communications in Mathematical Biology and Neuroscience. 2025. DOI: https://doi.org/10.28919/cmbn/9140

[55] Gao Z, Li T, Han J, Feng S, Li L, Jiang Y, et al.. Assessment of the immunogenicity and protection of a Nipah virus soluble G vaccine candidate in mice and pigs. Frontiers in Microbiology. 2022. DOI: https://doi.org/10.3389/fmicb.2022.1031523

[56] Shuai L, Ge J, Wen Z, Wang J, Wang X, Bu Z. Immune responses in mice and pigs after oral vaccination with rabies virus vectored Nipah disease vaccines.. Veterinary Microbiology. 2020. DOI: https://doi.org/10.1016/j.vetmic.2019.108549

[57] Li Z, Zhu Y, Yan F, Jin H, Wang Q, Zhao Y, et al.. Inactivated Recombinant Rabies Virus Displaying the Nipah Virus Envelope Glycoproteins Induces Systemic Immune Responses in Mice. Vaccines. 2023. DOI: https://doi.org/10.3390/vaccines11121758

[58] Afrin S, Mahedi MRA, Radia AA, Devi J. Nipah virus as an emerging threat: mutational dynamics, pathogenesis, and advances in vaccine development- a systematic review. International Journal of Public Health Science (IJPHS). 2026. DOI: https://doi.org/10.11591/ijphs.v15i1.22365

[59] Walpita P, Cong Y, Jahrling P, Rojas O, Postnikova E, Yú S, et al.. A VLP-based vaccine provides complete protection against Nipah virus challenge following multiple-dose or single-dose vaccination schedules in a hamster model. npj Vaccines. 2017. DOI: https://doi.org/10.1038/s41541-017-0023-7

[60] Kalodimou G, Veit S, Jany S, Kalinke U, Broder C, Sutter G, et al.. A Soluble Version of Nipah Virus Glycoprotein G Delivered by Vaccinia Virus MVA Activates Specific CD8 and CD4 T Cells in Mice. Viruses. 2019. DOI: https://doi.org/10.3390/v12010026

[61] Mączyńska A, Zawadzka M, Wojcieszek M, Czarnota M, Fidelis M, Gacka D, et al.. NIPAH VIRUS: CLINICAL PERSPECTIVES ON AN EMERGING PRIORITY PATHOGEN. International Journal of Innovative Technologies in Social Science. 2026. DOI: https://doi.org/10.31435/ijitss.1(49).2026.4936

[62] Gabra M, Ghaith HS, Ebada MA. Nipah Virus: An Updated Review and Emerging Challenges.. Infectious Diseases - Drug Targets. 2022. DOI: https://doi.org/10.2174/1871526522666220117120859