Over the past few decades, there have been multiple outbreaks of viruses causing severe and severe disease around the world, including known and unknown viral pathogens such as SARS-CoV, Ebola, Lassa fever, Marburg virus, Nipah virus, Hendra virus, Rift Valley fever virus and Middle East respiratory syndrome coronavirus, etc. The diseases caused by the above-mentioned viruses have had a severe impact on human health on a global scale, causing huge economic losses. In late September 1998, the first case of Nipah virus infection was reported near Ipoh, Malaysia, and was successfully isolated for the first time in a patient sample in the Kampungsungai nipah area in March 1999, hence the name Nipah virus (NiV). According to its appearance, NiV is classified as Paramyxovirinae, and because of its strong cross-reaction with Hendra virus (HeV) antiserum, and further sequencing shows that it is a new type of Paramyxovirinae, Therefore, it is classified as Henipavirus together with HeV.
Since the first outbreak of NiV, it has appeared in Malaysia, Singapore, Bangladesh, India and the Philippines in five countries, resulting in severe infection and high mortality in the population. Currently, there are no specific therapeutic drugs and preventive vaccines, and continuous monitoring of animal hosts and environments in high-risk areas of infection can help detect early signs of infection and control disease outbreaks.
NiV Gene Structure
NiV belongs to the paramyxoviridae family Henipavirus genus. It is an enveloped virus with a polymorphic shape and an average diameter of 500 nm (range 180 nm to 1 900 nm), with an envelope and spikes, and an unsegmented single-negative-stranded RNA genome. The genome encodes six structural proteins from the 3′ end, which are nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), glycoprotein (G) and large protein (L). Among them, the P gene also encodes three non-structural proteins C, V and W. The viral nucleocapsid is composed of three proteins, N, L, and P, and the envelope is composed of M, G, and F proteins.
NiV Detection Method
Currently, laboratory diagnosis is the only way to confirm NiV infection, which can be detected by techniques such as virus isolation, histopathology, serology, molecular diagnosis, and immunohistochemistry.
Virus Isolation and Culture
Virus isolation is the gold standard for laboratory diagnosis. NiV virus isolation can help identify new cases or identify new hosts. Infected samples are usually derived from acute-phase patient cerebrospinal fluid, blood, nasal/pharyngeal swabs, urine, or infected animal tissue (lymph nodes, lung, kidney, spleen, etc.). NiV grows well in Vero cells, and cytopathic effect (CPE) can be observed within 3 days of virus infection. CPE is mainly manifested as syncytia composed of 20 or more nuclei, with the nucleocapsid and nuclei usually surrounding the cells, and this phenomenon is more pronounced in the later stages of infection. It is worth noting that the CPE will vary slightly depending on the strain and cell line used.
Molecular Diagnosis
PCR detection Molecular diagnostic techniques such as PCR and genome sequencing play an important role in the detection of viral infections in human and animal diseases due to their high sensitivity and fast detection speed. Before the establishment of real-time quantitative PCR (qPCR) detection methods for NiV, conventional PCR methods have been used for the detection and diagnosis of Henipavirus. The PCR method designed by the US CDC based on the conserved N gene of NiV can be used to detect nucleic acids in different types of specimens, including cerebrospinal fluid, urine, various swabs, and fresh or fixed tissues. Sequencing of PCR products facilitates the rapid identification of virus isolates from tissue supernatants. A nested PCR detection method targeting the N gene with an internal control reduces false negative results due to PCR inhibitors in the sample, thereby improving the detection accuracy and monitoring reliability.
Genome Sequencing
Sanger sequencing plays an important role in the diagnosis of NiV and the identification of potential mutation sites in target regions of the genome, and next-generation sequencing (NGS) has become the first choice for rapid whole-genome sequencing of new Henipavirus isolates. Using sequencing technology, it was found that the NiV strains isolated in Malaysia originated from different hosts such as bats, pigs and humans, but their genome sequences were very similar. The sequences of the NiV strains isolated from Bangladesh and India later were very different from those of the Malaysian strains. Large, genetic evolution analysis indicates that NiV has two main lineages: the Malaysian NiV and the Bangladeshi NiV.
Serological Testing
Enzyme-linked immunosorbent assay Currently, enzyme-linked immunosorbent assay (ELISA) has been widely used to detect NiV antigen and antibody responses. Indirect ELISAs developed using viral antigens for serological detection (IgM and IgG) have been used for sero-antibody transformation in humans and field studies in bats and other animals. Recombinant protein can be used as antigen instead of whole virus for ELISA detection. The expression of recombinant NiV N, F and G proteins in E.coli can react with the convalescent sera of NiV-infected patients, and the recombinant NiV G protein can also react with the sera of late-onset NiV-infected patients.
ELISA detection is fast, safe, low-cost, and high-throughput, but the sensitivity and specificity of the detection are slightly worse than molecular detection, and it is prone to false positives, and positive samples need to be verified by neutralization tests. Nonetheless, ELISA is currently the primary method for serological detection, facilitating NiV epidemiological studies and ongoing surveillance.
Serum Neutralization Test
The Serum neutralization test (SNT) is the reference standard for serological testing and needs to be performed at a BSL-4 facility. Traditional SNT uses Vero cells as host cells, and serum that can reduce or eliminate CPE is considered to have neutralizing activity. In order to reduce the biosafety level of the experimental operation, an improved version of SNT based on the rapid immune plaque test was established. The culture plates obtained in BSL-4, after inactivation by γ-irradiation, can be stained in BSL-2, so that SNT is no longer dependent on the BSL-4 facility, improving experimental operability. In addition, pseudovirus neutralization assays can also replace serum neutralization assays for infectious NiV in areas lacking BSL-4 facilities with high sensitivity and safety. The vesicular stomatitis virus (VSV) vector was used to construct recombinant pseudovirus particles expressing NiV F and G proteins and carrying green fluorescent protein (GFP), which can be used as known viral antigens to detect NiV serum samples . NiV antibody plaque inhibition test can be established by using the pseudovirus expressing NiV F or G protein constructed by this gene recombination technology.
Immunohistochemistry
Anti-NiV antibodies can be used to detect NiV antigens in formalin-fixed tissues. NiV infection in ferrets causes lesions in the respiratory tract, lymphoid tissue, kidney and liver of the body, and viral antigens even exist in neurons and vascular endothelial cells. Therefore, immunohistochemical specimens should include brain, lung, mediastinal lymph nodes, spleen, and kidney. If testing a pregnant animal, the specimen should also include the uterus, placenta, and fetus. The Australian Animal Health Laboratory (AAHL) conducted a comparative study using different antisera and found that the highly immune rabbit serum prepared from NiV recombinant N protein had the best immunohistochemical staining effect on different tissues infected with Henipavirus.Immunohistochemical method for accurate pathogen location is helpful for histopathological research, high safety and traceability is helpful for retrospective pathogen analysis.