Enzyme-Linked Immunosorbent Assay Using Henipavirus-Receptor EphrinB2 and Monoclonal Antibodies for Detecting Nipah and Hendra Viruses.

The Nipah virus (NiV) and the Hendra virus (HeV) are highly pathogenic zoonotic diseases that can cause fatal infections in humans and animals. Early detection is critical for the control of NiV and HeV infections. We present the development of two antigen-detection ELISAs (AgELISAs) using the henipavirus-receptor EphrinB2 and monoclonal antibodies (mAbs) to detect NiV and HeV. The NiV AgELISA detected only NiV, whereas the NiV/HeV AgELISA detected both NiV and HeV. The diagnostic specificities of the NiV AgELISA and the NiV/HeV AgELISA were 100% and 97.8%, respectively. Both assays were specific for henipaviruses and showed no cross-reactivity with other viruses. The AgELISAs detected NiV antigen in experimental pig nasal wash samples taken at 4 days post-infection. With the combination of both AgELISAs, NiV can be differentiated from HeV. Complementing other henipavirus detection methods, these two newly developed AgELISAs can rapidly detect NiV and HeV in a large number of samples and are suitable for use in remote areas where other tests are not available.

Clinico-epidemiological presentations and management of Nipah virus infection during the outbreak in Kozhikode district, Kerala state, India 2023.

India experienced its sixth Nipah virus (NiV) outbreak in September 2023 in the Kozhikode district of Kerala state. The NiV is primarily transmitted by spillover events from infected bats followed by human-to-human transmission. The clinical specimens were screened using real-time RT-PCR, and positive specimens were further characterized using next-generation sequencing. We describe here an in-depth clinical presentation and management of NiV-confirmed cases and outbreak containment activities. The current outbreak reported a total of six cases with two deaths, with a case fatality ratio of 33.33%. The cases had a mixed presentation of acute respiratory distress syndrome and encephalitis syndrome. Fever was a persistent presentation in all the cases. The Nipah viral RNA was detected in clinical specimens until the post-onset day of illness (POD) 14, with viral load in the range of 1.7-3.3 × 104 viral RNA copies/mL. The genomic analysis showed that the sequences from the current outbreak clustered into the Indian clade similar to the 2018 and 2019 outbreaks. This study highlights the vigilance of the health system to detect and effectively manage the clustering of cases with clinical presentations similar to NiV, which led to early detection and containment activities.

Nipah Virus: An Overview of the Current Status of Diagnostics and Their Role in Preparedness in Endemic Countries.

Nipah virus (NiV) is a paramyxovirus responsible for a high mortality rate zoonosis. As a result, it has been included in the list of Blueprint priority pathogens. Bats are the main reservoirs of the virus, and different clinical courses have been described in humans. The Bangladesh strain (NiV-B) is often associated with severe respiratory disease, whereas the Malaysian strain (NiV-M) is often associated with severe encephalitis. An early diagnosis of NiV infection is crucial to limit the outbreak and to provide appropriate care to the patient. Due to high specificity and sensitivity, qRT-PCR is currently considered to be the optimum method in acute NiV infection assessment. Nasal swabs, cerebrospinal fluid, urine, and blood are used for RT-PCR testing. N gene represents the main target used in molecular assays. Different sensitivities have been observed depending on the platform used: real-time PCR showed a sensitivity of about 103 equivalent copies/reaction, SYBRGREEN technology's sensitivity was about 20 equivalent copies/reaction, and in multiple pathogen card arrays, the lowest limit of detection (LOD) was estimated to be 54 equivalent copies/reaction. An international standard for NiV is yet to be established, making it difficult to compare the sensitivity of the different methods. Serological assays are for the most part used in seroprevalence studies owing to their lower sensitivity in acute infection. Due to the high epidemic and pandemic potential of this virus, the diagnosis of NiV should be included in a more global One Health approach to improve surveillance and preparedness for the benefit of public health. Some steps need to be conducted in the diagnostic field in order to become more efficient in epidemic management, such as development of point-of-care (PoC) assays for the rapid diagnosis of NiV.

A Revised Diagnostic Quantitative RT-PCR for the Detection of Nipah Virus Infection.

From its discovery in Malaysia in the late 1990s, the spillover of the Nipah virus from its pteropid reservoir into the human population has resulted in sporadic outbreaks of fatal encephalitis and respiratory disease. In this chapter, we revise a previously described quantitative reverse transcription polymerase chain reaction method, which now utilizes degenerate nucleotides at certain positions in the probe and the reverse primer to accommodate the sequence heterogeneity observed within the Nipah henipavirus species.

Detection of Nipah and Hendra Viruses Using Recombinant Human Ephrin B2 Capture Virus in Immunoassays.

Nipah virus (NiV) and Hendra virus (HeV) are classified as high-consequence zoonotic viruses characterized by high pathogenicity and high mortality in animals and humans. Rapid diagnosis is essential to containing the outbreak. In this study, the henipavirus receptor ephrin B2 was examined to determine whether it could be used as a universal ligand for henipavirus detection in immunoassays. Enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant ephrin B2 as the capture ligand and two monoclonal antibodies (mAbs) as detection reagents. Using mAb F27NiV-34, which cross-reacts with NiV and HeV, we were able to detect NiV and HeV, while mAb F20NiV-65 was used to detect NiV. Therefore, using these two ELISAs, we were able to differentiate between NiV and HeV. Furthermore, we developed a rapid lateral flow strip test for NiV detection using ephrin B2 as the capture ligand combined with mAb F20NiV-65 as the detector. Taken together, our results show that the combination of ephrin B2 and a specific mAb provides an excellent pairing for NiV and HeV detection.

Development and validation of an IgM antibody capture ELISA for early detection of Hendra virus.

Zoonotic transmission of Hendra virus (HeV) from primary hosts (pteropid bats) to horses, and, occasionally, onward adventitious spread to humans, is associated with high mortality rates in both affected secondary species. The introduction of an effective recombinant G protein vaccine for use in horses has been a major advance for the suppression of disease risk. However, equine HeV vaccination induces neutralising antibody that is indistinguishable from a post infection immune response when using most first line serology assays (eg. VNT and some ELISAs). We have constructed and evaluated an IgM antibody capture (MAC) ELISA which employs yeast expressed HeV nucleoprotein (N). All other serology tests use the G protein which does not detect early infection and is present in the current Hendra virus vaccine and may cause ambiguity in interpretation of results. Thus, this is the first test developed using a N protein which can successfully detect a recent (primarily within the last four weeks) infection of horses with HeV and is not affected by vaccination induced antibody. Testing a limited panel (21 samples) of post infection sera, a normal serum panel (288 samples) and a post vaccination panel (163 samples), we have estimated DSe to be 100 % (95 % CI, 83.9-100.0 %) and DSp to be 98.4 % (95 % CI, 96.8-99.4 %) relative to assigned serology results (VNT, ELISA and Luminex) for the test panels. The HeV IgM MAC ELISA is intended to supplement other molecular and serology test results, with selective use, and is the only serology test which can provide an indication for recent infection which is otherwise not available.

Detection of Nipah virus in Pteropus medius in 2019 outbreak from Ernakulam district, Kerala, India.

BACKGROUND: In June 2019, Nipah virus (NiV) infection was detected in a 21-year-old male (index case) of Ernakulum, Kerala, India. This study was undertaken to determine if NiV was in circulation in Pteropus species (spp) in those areas where the index case had visit history in 1 month. METHODS: Specialized techniques were used to trap the Pteropus medius bats (random sampling) in the vicinity of the index case area. Throat and rectal swabs samples of 141 bats along with visceral organs of 92 bats were collected to detect the presence of NiV by real-time reverse transcriptase-polymerase chain reaction (qRTPCR). Serum samples of 52 bats were tested for anti-NiV Immunoglobulin (Ig) G antibodies by Enzyme-Linked Immunosorbent Assay (ELISA). The complete genome of NiV was sequenced by next-generation sequencing (NGS) from the tissues and swab samples of bats. RESULTS: One rectal swab sample and three bats visceral organs were found positive for the NiV. Interestingly, 20.68% (12/58) of Pteropus were positive for anti-NiV IgG antibodies. NiV sequences of 18,172; 17,200 and 15,100 nucleotide bps could be retrieved from three Pteropus bats. CONCLUSION: A distinct cluster of NiV sequences, with significant net-evolutionary nucleotide divergence, was obtained, suggesting the circulation of new genotype (I-India) in South India. NiV Positivity in Pteropus spp. of bats revealed that NiV is circulating in many districts of Kerala state, and active surveillance of NiV should be immediately set up to know the hotspot area for NiV infection.

Micro-fusion inhibition tests: quantifying antibody neutralization of virus-mediated cell-cell fusion.

Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP-Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.

Nipah@20: Lessons Learned from Another Virus with Pandemic Potential.

Nipah disease is listed as one of the WHO priority diseases that pose the greatest public health risk due to their epidemic potential. More than 200 experts from around the world convened in Singapore last year to mark the 20th anniversary of the first Nipah virus outbreaks in Malaysia and Singapore. Most of these experts are now involved in responding to the coronavirus disease 2019 (COVID-19) pandemic. Here, members of the Organizing Committee of the 2019 Nipah Virus International Conference review highlights from the Nipah@20 Conference and reflect on key lessons learned from Nipah that could be applied to the understanding of the COVID-19 pandemic and to preparedness against future emerging infectious diseases (EIDs) of pandemic potential.

Optimization and diagnostic evaluation of monoclonal antibody-based blocking ELISA formats for detection of neutralizing antibodies to Hendra virus in mammalian sera.

Maintenance of Hendra virus (HeV) in pteropid bat populations has been associated with spillover events in horses, humans and dogs. Experimental studies have demonstrated infections for several other species including guinea pigs, cats and ferrets. The criteria of a sensitive and specific serological test that is effective for a range of species, but which does not require use of live virus, has not been satisfactorily addressed by currently available tests. We have evaluated the use of two HeV neutralizing monoclonal antibodies (mAbs) in a blocking format enzyme-linked immunosorbent assay (bELISA) to detect serum antibody against a recombinant expressed HeV G protein (sol G) in several animal species. The human mAb m102.4 neutralises both HeV and the closely related Nipah virus (NiV); the mouse mAb 1.2 neutralises only HeV. Given these functional differences, we have investigated both antibodies using a bELISA format. Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were optimized using individual thresholds for mAb 1.2 and m102.4. For mAb 1.2 the positive threshold of >33% inhibition yielded DSe and DSp values of 100% (95% CI 95.3-100.0) and 99.5 (95% CI 98.8-99.8) respectively; for mAb m102.4 a positive threshold of >49% inhibition gave DSe and DSp values of 100 (95% CI 95.3-100.0) and 99.8 (95% CI 99.2-100.0) respectively. At these thresholds the DSe was 100% for both tests relative to the virus neutralization test. Importantly, the occurrence of false positive reactions did not overlap across the assays. Therefore, by sequential and selective application of these assays, it is possible to identify false positive reactions and achieve a DSp that approximates 100% in the test population.

Establishment of an immunofluorescence assay to detect IgM antibodies to Nipah virus using HeLa cells expressing recombinant nucleoprotein.

A novel indirect fluorescent antibody test (IFAT) for detection of IgM against Nipah virus (NiV) was developed using HeLa 229 cells expressing recombinant NiV nucleocapsid protein (NiV-N). The NiV IFAT was evaluated using three panels of sera: a) experimentally produced sera from NiV-N-immunized/pre-immunized macaques, b) post-infection human sera associated with a Nipah disease outbreak in the Philippines in 2014, and c) human sera from a non-exposed Malaysian population. Immunized macaque sera showed a characteristic granular staining pattern of the NiV-N expressed antigen in HeLa 229 cells, which was readily distinguished from negative-binding results of the pre-immunized macaque sera. The IgM antibody titers in sequential serum samples (n = 7) obtained from three Nipah patients correlated well with previously published results using conventional IgM capture ELISA and SNT serology. The 90 human serum samples from unexposed persons were unreactive by IFAT. The IFAT utilizing NiV-N-expressing HeLa 229 cells to detect IgM antibody in an early stage of NiV infection is an effective approach, which could be utilized readily in local laboratories to complement other capabilities in NiV-affected countries.

Progressive multifocal leukoencephalopathy in Zambia is caused by JC virus with prototype regulatory region.

There are only few documented cases of progressive multifocal leukoencephalopathy (PML) in Africa. Whether this is caused by a lack of JC virus (JCV) spread or alteration in the JCV genome is unknown. We characterized the clinical presentation, laboratory findings, and JCV regulatory region (RR) pattern of the first documented PML cases in Zambia as well as JCV seroprevalence among HIV+ and HIV- Zambians. We identified PML patients with positive JCV DNA PCR in their cerebrospinal fluid (CSF) among subjects enrolled in an ongoing tuberculous meningitis study from 2014 to 2016 in Lusaka. JCV regulatory region was further characterized by duplex PCR in patients' urine and CSF. Of 440 HIV+ patients, 14 (3%) had detectable JCV DNA in their CSF (age 18-50; CD4+ T cells counts 15-155 × 106/µl) vs 0/60 HIV- patients. The main clinical manifestations included altered mental status and impaired consciousness consistent with advanced PML. While prototype JCV was identified by duplex PCR assay in the CSF samples of all 14 PML patients, only archetype JCV was detected in their urine. All PML Zambian patients tested were seropositive for JCV compared to 46% in a control group of HIV+ and HIV- Zambian patients without PML. PML occurs among HIV-infected individuals in Zambia and is caused by CNS infection with prototype JCV, while archetype JCV strains are present in their urine. JCV seroprevalence is comparable in Zambia and the USA, and PML should be included in the differential diagnosis of immunosuppressed individuals presenting with neurological dysfunction in Zambia.

Henipavirus infection of the central nervous system.

Nipah virus (NiV) and Hendra virus are highly pathogenic zoonotic viruses of the genus Henipavirus, family Paramyxoviridae. These viruses were first identified as the causative agents of severe respiratory and encephalitic disease in the 1990s across Australia and Southern Asia with mortality rates reaching up to 75%. While outbreaks of Nipah and Hendra virus infections remain rare and sporadic, there is concern that NiV has pandemic potential. Despite increased attention, little is understood about the neuropathogenesis of henipavirus infection. Neuropathogenesis appears to arise from dual mechanisms of vascular disease and direct parenchymal brain infection, but the relative contributions remain unknown while respiratory disease arises from vasculitis and respiratory epithelial cell infection. This review will address NiV basic clinical disease, pathology and pathogenesis with a particular focus on central nervous system (CNS) infection and address the necessity of a model of relapsed CNS infection. Additionally, the innate immune responses to NiV infection in vitro and in the CNS are reviewed as it is likely linked to any persistent CNS infection.

Using healthcare-seeking behaviour to estimate the number of Nipah outbreaks missed by hospital-based surveillance in Bangladesh.

BACKGROUND: Understanding the true burden of emergent diseases is critical for assessing public-health impact. However, surveillance often relies on hospital systems that only capture a minority of cases. We use the example of Nipah-virus infection in Bangladesh, which has a high case-fatality ratio and frequent person-to-person transmission, to demonstrate how healthcare-seeking data can estimate true burden. METHODS: We fit logistic-regression models to data from a population-based, healthcare-seeking study of encephalitis cases to characterize the impact of distance and mortality on attending one of three surveillance hospital sites. The resulting estimates of detection probabilities, as a function of distance and outcome, are applied to all observed Nipah outbreaks between 2007 and 2014 to estimate the true burden. RESULTS: The probability of attending a surveillance hospital fell from 82% for people with fatal encephalitis living 10 km away from a surveillance hospital to 54% at 50 km away. The odds of attending a surveillance hospital are 3.2 (95% confidence interval: 1.6, 6.6) times greater for patients who eventually died (i.e. who were more severely ill) compared with those who survived. Using these probabilities, we estimated that 119 Nipah outbreaks (95% confidence interval: 103, 140)-an average of 15 outbreaks per Nipah season-occurred during 2007-14; 62 (52%) were detected. CONCLUSIONS: Our findings suggest hospital-based surveillance missed nearly half of all Nipah outbreaks. This analytical method allowed us to estimate the underlying burden of disease, which is important for emerging diseases where healthcare access may be limited.

Nipah virus infection: A review.

Nipah virus (NiV) is an emerging bat-borne pathogen. It was first identified 20 years ago in Malaysia and has since caused outbreaks in other parts of South and Southeast Asia. It causes severe neurological and respiratory disease which is highly lethal. It is highly infectious and spreads in the community through infected animals or other infected people. Different strains of the virus show differing clinical and epidemiological features. Rapid diagnosis and implementation of infection control measures are essential to contain outbreaks. A number of serological and molecular diagnostic techniques have been developed for diagnosis and surveillance. Difficulties in diagnosis and management arise when a new area is affected. The high mortality associated with infection and the possibility of spread to new areas has underscored the need for effective management and control. However, no effective treatment or prophylaxis is readily available, though several approaches show promise. Given the common chains of transmission from bats to humans, a One Health approach is necessary for the prevention and control of NiV infection.

Advances in diagnostics, vaccines and therapeutics for Nipah virus.

Nipah virus is an emerging zoonotic paramyxovirus that causes severe and often fatal respiratory and neurological disease in humans. The virus was first discovered after an outbreak of encephalitis in pig farmers in Malaysia and Singapore with subsequent outbreaks in Bangladesh or India occurring almost annually. Due to the highly pathogenic nature of NiV, its pandemic potential, and the lack of licensed vaccines or therapeutics, there is a requirement for research and development into highly sensitive and specific diagnostic tools as well as antivirals and vaccines to help prevent and control future outbreak situations.