The Establishment Of Gene Chip To Detect Major Arbovirus

Arbovirus is a category of viruses that spread between human and livestocks by hematophagus stinging. The viruses could propagate in hematophagus while induce no significant symptoms. So, arboviruse is a category of zoonosis viruses. More than 530 Arboviruses involved in 14 virus families such as Flaviviridae, Togaviridae and Bunyaviridae were found all around the world. 9 viruses among them were isolated in China. However, China is a vast country with diversity climates which is quite suitable for many arboviruses to survive and spread. Therefore, arboviruses spread actually in China may be far more than above listed. In addition, with the rapid increase and development of traveling and international exchanges, the possibility of arboviruses spreading into our country from abroad may also increase significantly. Thus, there is a increase demand of establish rapid and accurate detection methods of arboviruses.Biochip is a new technology which has been developed from 1990's. The significant advantages of biochip are high throughput, high parallel and high automatic. In this study, we established the genechip method to detect 13 main arbovirus, included Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus, Mayaro virus, Dengue fever virus, West Nile virus, Japanese B encephalitis virus, Tick-borne encephalitis virus, Hantaan virus and Bunyamwera virus, from both genus and species levels. The above purpose was accomplished by carefully designing of genus and spesies specific oligonucleotides probes for screening and identification of those arboviruses. The hybridization results of genus and spesies specific oligos could validate each other. This provides a novel method for detection and identification of main arboviruses.Firstly, all known genome sequences of the above arboviruses were downloaded from GenBank. Then genera-specific and species-specific oligonucleotides were designed by Clustal X and BLAST. Oligos of Flavivirus, Alphavirus and Orthounyavirus were designed for genus-spesific screening and each virus-spesific oligos were designed for specific identification. All oligo-probes were 70-mer in length with TM between 75-85℃. The designed speice and genus-specific probes were validated firstly on Internet for their specification by BLAST. And then the optimized oligos were synthesized and spotted on chips. Viruses were cultivated in BSL-2 and BSL-3 labs. Anchoring random PCR was performed to amplify viruses RNA. The PCR products were labeled by fluorochrome incorporation. Hybridization and washing conditions were optimized. The hybridization results were analysed by ScanArray software. The established genechip was verified by detecting of the 13 arboviruses with one or two viruses in one test. The sensibility, specificity and repeatability of the chip were also evaluated.3～10 genera-specific oligonucleotides for Alphavirus, Flavivirus, Orthobunyavirus and 10 virus-specific oligonucleotides were designed. The oligos were all 70-mer in length with TM values between 75-85℃. The total number of oligo was 153 to cover all the strains of these viruses. By using the optimized hybridization conditions, all 13 arboviruses were detected with specific signals both on genus and species levels. Non-specfic signales were not obvious. Specie and genues specific signales were also obtained after mixing RNAs of Japanese encephalitis virus and Mayaro virus. No positive hybridization signals were obtained when using Hepatitis C virus, Rabies virus and Rubella virus as samples, which confirmed the specificity of this chip. Sensitivity of the chip was evaluated by testing Mayaro virus. The result showed that when virus RNA in sample exceed 0.003pmol, the relatively strong positive signales could be observed (lg signales≥2.5).To further validate the specificity of gene chip method, we also carried out specific RT-PCR for each virus by using the same viral RNA specimen. The results showed that gene chip and RT-PCR were coincided with each other. This further indicated that the gene chip method had good specificity.Combined this chip and the human pathogenic viruses chip developed in 2005 by our laboratory, we successfully identified a new isolate as Japanese encephalitis virus. The result was coincided with RT-PCR and sequence analysis which also confirmed the unknown virus was Japanese encephalitis virus.A genechip method was established in this study to detect 13 Arboviruses both on sepecies and genus levels. It could also be used to detect these viruses in mixed infection samples. This provided a new method for detecting of arbovirus.