Development Of A Real-time RT-PCR Assay For The Detection Of Nipah Virus

Nipah virus (NiV) is a newly recognized zoonotic virus. The virus was discovered in 1999. It has caused severe diseases in animals and in humans through contact with infectious animals. The virus is named after the location where it was first detected in Malaysia. NiV is closely related to another newly recognized zoonotic virus, Hendra virus, named after the town where it first appeared in Australia in 1994. Both Nipah and Hendra viruses are members of the virus family Paramyxoviridae. Although members of this group of viruses have only caused a few local outbreaks, the biologic property of these viruses can infect a wide range of hosts and to produce a disease, causing significant mortality (40%-70%) in humans,which has made this emerging viral infection a public heath concern. NiV outbreak in Malaysia in 1998-1999 claimed 105 human lives and resulted in the culling of about 1.1 million pigs, bringing tremendous economic and social impact to the nation. The presence of NiV in bats has also been demonstrated in Cambodia, India and Bangladesh. Because the natural reservoir of NiV, fruit bats distribute widely in South and West China, and China is near to Bangladesh and India, it is of priority for China to develop some methods including RT-PCR to detect NiV for clinic diagnosis or epidemiological surveillance.According to the sequences of N gene of NiV reported in Malaysia, Bangladesh and Indonisia in GenBank,we design twosets priers and probes,which according to two sets of detection system,and then choose a better one with superior sensitivity and specificity.Most RNA positive control currently used for monitoring the quality of RT-PCR assays have some disadvantages, such as instability, inability to monitor the quality of the relevant primers and/or causing indifferentable false positives. To avoid these disadvantages, a simple method to prepare stable and differentiable RNA positive controls is now demonstrated with a real-time RT-PCR assay for the detection of NiV. A DNA sequence which was shorter than its counterpart in the NiV genome and contained the binding sites of the primers of the RT-PCR assay was designed, synthesized and inserted into a vector, and then amplified by PCR with two vector-specific primers both of which contained a T7 promoter at the 5-terminal. The RNA positive control was the dsRNA in vitro transcribed from the PCR amplicons flanked by two T7 promoters. The RNA positive control was stable and able to monitor the quality of the whole concerned RT-PCR assay. False positives caused by contaminations of the RNA positive control or its amplicons could be easily identified because the amplicons of the RNA positive control were obviously shorter than those of real positive samples. Thus, the RNA positive control reported in this study avoided some common disadvantages of current RNA positive controls.The real-time RT-PCR assay established in this study could detect 140 copies of RNA or 17 copies of cDNA in each reaction. Its sensitivity was approximately 100 times of the conventional RT-PCR. Samples (n=127) from E. coli (n=20), Vero cells (n=7) and swine organs (100) were detected using the RT-PCR assay, and all the results were negative, which demonstrated the high specificity of the assay. Among the 100 swine samples, 36 were collected from the regions in Sichuan in the July of 2005, and the detection facilitated the diagnosis of the notorious Streptococcus suis outbreak.Samples including serum, nasopharyngeal swabs, urine and organs from 31 bats were collected in Hainan Province for NiV isolation using Vero cells and NiV antibody detection using an in-house ELSA assay. The samples and the cell culture were detected using the RT-PCR assay established in this study. The results of the virus isolation, RT-PCR and ELISA were all negative, which suggested the little possibility for the bat groups in Hainan from which the samples were collected having been infected with NiV.