Kaikai Jin, Junjie Zhao, Huanxin Chen, Zimo Zhang, Zengguo Cao, Zanheng Huang, Hao Li, Yongsai Liu, Lisi Ai, Yufei Liu, Changqi Fan, Yuanyuan Li, Pei Huang, Hualei Wang, Haili Zhang
Nipah virus (NiV) is a member of the genus Henipavirus of the family Paramyxoviridae, and is an emerging zoonotic virus (Singh et al. 2019). NiV was first observed in 1998 and this outbreak triggered an uncontrollable epidemic lasting until 1999, during which more than a million pigs were destroyed (Ang et al. 2018). Since then, several South and Southeast Asian countries have experienced relatively small sporadic outbreaks almost every year, with mortality rates of up to 40-75 % in humans infected with NiV (Raj Kumar et al. 2019). Therefore, NiV not only does terrible damage to the farming industry but is also a serious threat to human public health and safety. Fruit bats are recognized as natural reservoir hosts for NiV, and other species of bats, as well as pigs, horses, dogs, and humans can also be infected (Soman Pillai et al. 2020). Bats act as asymptomatic carriers of the virus, but they excrete the virus through their saliva, urine, semen, and feces (Soman Pillai et al. 2020). NiV infection in humans and animals primarily occurs through the consumption of date palm juice and fruits that have been contaminated by fruit bats (Bruno et al. 2022). Human-to-human transmission is also possible (Garbuglia et al. 2023). NiV infection in humans and pigs can result in fatal encephalitis and severe respiratory disease (Ma et al. 2019, Ming-Yen et al. 2020). Because of its high pathogenicity and mortality, as well as the lack of effective treatments or vaccines for humans or animals (Gómez Román et al. 2022), NiV is included in the list of epidemiological threats requiring urgent R&D action in the World Health Organization (WHO) R&D Blueprint as one of the pathogens. WHO has therefore prepared a technical brief aimed at guiding countries in their preparedness planning for a NiV event, particularly in countries that have not yet reported a NiV event. The technical brief suggests that early diagnosis of NiV can enhance the survival chances of an infected individual and can also prevent transmission to others.
Currently, the detection methods for NiV are mainly based on traditional serological methods and molecular diagnostic methods. Of the serological detection methods, ELISA, phage plaque assay, and immunofluorescence staining do not require special instruments or equipment, but the NiV-infected samples need to be processed in a Biosafety-Level-4 (BSL-4) laboratory, which is risky for operators and is not available in all regions (Diwakar D et al. 2015, Fischer et al. 2018, Gary et al. 2001). Real-time reverse transcriptase polymerase chain reaction (RT-PCR) tests are the preferred method for viral detection due to their high sensitivity and ability to detect the virus in patients at the earliest stages of infection. However, Real-time RT-PCR requires sophisticated instruments and equipment, as well as specialized operator personnel, and cannot fully satisfy the need for early diagnosis of NiV infection in situ or in poor areas lacking these conditions (Wu et al. 2022). Therefore, the development of a rapid, specific, and sensitive assay that does not require complex instrumentation is critical for the early detection of NiV.
Nucleic acid isothermal amplification technology, characterized by its rapidity, sensitivity, and specificity, is capable of meeting the demands for speed and simplicity detection, and has significant practical application value (Zhao et al. 2015). Of these methods, recombinase-aided amplification (RAA) technology, with its simplicity and ease of use, is ideal for early diagnosis in resource-limited settings (James et al. 2018). Recombinant enzyme-based isothermal amplification tests have been used to develop three rapid assays for NiV that can detect 1,000 copies μL-1 for synthetic NiV RNA in less than 30 min (Pollak et al. 2023). However, these assays require the transfer of the RPA amplification products, exposing the nucleic acid-rich samples to the environment, and leading to potential aerosol contamination. In the present study, a fully enclosed device was employed, combining reverse transcription recombinase-aided amplification technology with lateral flow immunochromatography (RT-RAA-VF), to establish a rapid and sensitive assay for the detection of NiV. This assay avoids false positives caused by aerosols, does not require sophisticated instruments and equipment, does not require specialized personnel to operate, and holds the potential to fulfill the requirements for early diagnosis during the initial stages of a NiV infection outbreak even in poor and remote areas.
To detect all known NiV strains, 55 P gene sequences of different NiV strains, including NiV-B (transmitted in Bangladesh and India), NiV-M (transmitted in Malaysia), and NiV-T (transmitted in Thailand) were retrieved from GenBank (https: //www.ncbi.nlm.nih.gov/) and compared using MAFFT version 7. The highly conserved region of the P gene was selected as the target and was used to design the specific RAA primers and probe by the Prime Primer 5.0 software. The probe contained 55 nucleotides with the tetrahydrofuran (THF) residue replacing the adenine at position 37. The 5' end of the probe was labeled with the fluorescent marker FAM, while the 3' end was labeled with a blocking group comprising a C3 spacer. The 5' end of the reverse primer was labeled with biotin (Table 1). The primers and probe were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The RNA transcript of NiV P (Genebank: NC_002728) and the positive plasmid pcDNA3.1-NiV-P were also synthesized by Sangon Biotech. In the presence of NiV RNA, the reverse transcriptase is activated to reverse transcribe the NiV RNA to cDNA, and the RAA amplification reaction is followed activated by two priming oligonucleotides to produce targets for probe annealing hybridization. The THF site of the probe can then be cleaved by the nfo enzyme to create a new 3'-hydroxyl group, which is used as a start site for polymerase extension, thus converting the probe into a primer that, together with a reverse primer with biotin, produces double-stranded amplification products labeled with two antigens (FAM and biotin). Subsequently, the reaction tube, which contains the amplified products, is positioned within a sealed and disposable nucleic acid visualization paper device. The amplified DNA products are capable of adhering to streptavidin-coated gold nanoparticles, thereby forming a conjugate that is subsequently immobilized by the anti-FAM antibody present on the test line (T line), which manifests as a red stripe. Meanwhile, any streptavidin-labeled gold nanoparticles that are not associated with the amplified DNA are trapped by the anti-streptavidin antibody at the control line (C line) (Fig. 1-A).
To find the optimal RT-RAA reaction time, 10-fold gradient dilutions of the plasmid pcDNA3.1-NiV-P (5×1010 copies μL-1) were used as targets for RT-RAA-VF assay. The reaction was performed at three distinct temperatures—42 °C, 39 °C, and 37 °C—for a consistent period of 20 min. The outcomes indicated that at 42 °C, the emergence of discernible bands on both the C and T lines, indicating of a positive result, was observed at plasmid concentrations of 5 copies μL-1 or greater. At 39 °C and 37 °C, the positive results were only observed at plasmid concentrations of 500 copies μL-1 or more. These results suggested that 42 °C was the optimal reaction temperature for the NiV RT-RAA assay (Fig. 1-B). To determine the most efficient amplification time, the assay was executed for time intervals of 15, 20, 25, and 30 min. Analysis of the data provided in Appendix A confirmed that the optimal amplification duration for the assay was 20 min.
Next, we assessed the sensitivity of the assay using 10-fold gradient dilutions of NiV P RNA transcripts, reacting at 42 °C for 20 min. The results showed that this assay could detect as few as 5 copies μL-1 of RNA transcripts (Fig. 1-C). We further compared the sensitivity of this assay with that of the real-time RT-PCR method currently recommended by the World Organisation for Animal Health (WOAH), using 10-fold serial dilutions of RNA extracted from NiV-infected Vero E6 cells as templates. The results showed that the two methods shared an identical detection limit, and both were capable of detecting NiV RNA from infected cells diluted 10,000 times (Appendix B). To evaluate the specificity of this assay, the nucleic acids of pathogens that can cause neurological symptoms similar to NiV were also tested, including Hendra virus (HeV), herpes simplex virus type 1 (HSV-1), rabies virus (RABV) and Streptococcus suis. The results showed that our established assay only recognized the nucleic acids of NiV, with no cross-reactivity observed with other pathogens (Fig. 1-D).
It has been reported that NiV can be transmitted by droplets, or by contact with throat or nasal secretions, from the respiratory tract of patients or sick pigs. To validate the clinical applicability of this assay, we mixed NiV RNA with RNA extracted from human saliva samples to simulate NiV-infected clinical samples. The simulated NiV clinical samples were evaluated using the RT-RAA-VF assay as well as real-time RT-PCR. Of all 47 samples tested, 25 samples were prepared by 3-, 4-, and 5-fold gradient dilutions of NiV RNA using healthy human RNA as the diluent, and 22 samples were healthy human RNA samples without NiV RNA. The RT-RAA-VF assay was able to distinguish between the 25 positive and 22 negative samples, and the real-time RT-PCR assay also detected 25 positive and 22 negative samples, with Ct values ranging from 25 to 40 for positive samples (Fig. 1-E to G). We also simulated the clinical NiV infected swine samples. Among all 28 samples, 15 samples were obtained by 3-, 4- and 5-fold gradient dilutions of NiV RNA with healthy swine RNA as the diluent, and 13 samples were healthy swine RNA samples without NiV RNA. The results demonstrated that the RT-RAA-VF assay was in accordance with the real-time RT-PCR and was capable of effectively differentiating between 15 positive samples and 13 negative samples (Fig 1-H to J). The assay is therefore expected to be an alternative assay for the clinical detection of NiV, being faster, more specific, and more sensitive than real-time RT-PCR and without the need for complex instrumentation.
As human societies evolve, the combination of urbanization and climate change has resulted in the destruction of bat habitats, leading to increased contact between bats and humans. This has further elevated the risk of NiV infection for both humans and animals (Jonathan A et al. 2004, Kessler et al. 2018). NiV outbreaks have been reported in four countries - Bangladesh, India, Malaysia, and Singapore. However, fruit bats (the natural reservoir host of NiV) have been found in several Southeast Asian countries, suggesting the potential for NiV outbreaks in previously unaffected areas (Mangesh et al. 2022). Given the incubation period associated with NiV, the potential for cross-regional spread due to the movement of infected individuals cannot be dismissed. Consequently, it is imperative to focus on the rapid detection of NiV infections to enhance the survival prospects for those affected and to mitigate the risk of further transmission of the virus to others. In the present study, we developed an RT-RAA-VF assay for the detection of NiV using a sealed disposable nucleic acid visualization test paper device which effectively eliminates false positives caused by aerosol contamination. The assay demonstrated a sensitivity of 5 copies μL-1 for detecting NiV RNA at 42 °C for 20 minutes. The assay exhibits high specificity, with no cross-reactivity observed against other paramyxoviruses or neurological pathogens. Moreover, when evaluated on simulated clinical samples, it demonstrated 100% concordance with the results obtained from the real-time RT-PCR method. With its rapidity, specificity, and sensitivity features, this assay holds promise as an effective tool for early diagnosis of NiV infection.