Dsign And Development Of DNA Microarray For The Detection Of Orthopoxvirus

Poxvirus is the biggest virion in nature, of which the most notorious member is Variola virus that had caused disasters to human being in the past centuries. Variola virus belongs to the genus of orthopoxvirus, and there are some other infectious members, such as Vaccinia virus, cowpox virus, and monkeypox virus. Smallpox disease was considered to be deceased at the end of 1970's, therefore, studies on this topic has been stopped in China. Recently, however, bio-terrorism alarms people that the threat of bio-weapon is still looming large. Variola virus is one of the most possible pathogens to be applied by bio-terrorist, while other species of orthopoxvirus also have the potential to be similarity virulent when modified with genetic engineering. Novel detection method needs to be established urgently at genetic level.DNA microarray can be use to identify the pathogens on high-throughput fashion, and genetic diagnosis could detect the virus infection at the earliest possible time. DNA microarray could be used in pathogen detection with automation and cost-efficiency.As the Variola still is the most potent pathogen, after the WHO declared the victory over small pox diseases, stored pathogen samples had been destroyed. It is now impossible to access pathogen samples. To overcome this difficulty, conservative sequences in the orthopoxvirus were obtained using BLAST analysis from Genebank database. The oligonucleotide probes are designed and the oligonucleotide microarray is prepared. The oligonucleotide microarray was tested by using Vaccinia virus; the later has great homology with that of the Variola virus. DNAs in different infection stages are collected and labeled by Restriction Display (RD) technique, and hybridized to the oLigo-microarray. DNAs from Cowpox virus are also used and experiments found that hybridization signals were strong, which suggested that the oligo-microarray could be used in detection of orthopoxvirus.Vaccinia virus' infection provides us favorable model to explore the infectious mechanism of orthopoxvirus. The best-studied member of thepoxvirus family contains approximately 200 genes that are closely spaced along its 200,000-bp double-stranded DNA genome. Viral replication occurs in the cytoplasm of the host cell, consistent with observations that many proteins necessary for RNA and DNA synthesis are virus encoded. The viral genes can be grouped into two classes, the early genes and the late genes, according to the stage of infection in which they are activated. Early genes are involved in replication and late genes are important in mature of the virions. Soon after entering the cell, a complete transcription system within the virus particle produces early gene mRNAs. The translation products of the early mRNAs include enzymes and factors for DNA replication and late gene expression. The late gene mRNAs encode virion proteins, including the two subunits of the early transcription factors. In this manner, each gene classes expresses during the different infection phrase. In early study, approaches of mutant and revertant were usually used to identify different functions of the genes, which are time consuming and much less effective. In this study, we applied DNA microarray technology to search genes expressed in different infected phase. In the beginning, the library of the Vaccinia virus' probes was constructed, and the probes were printed on the slides to prepare DNA microarray of Vaccinia virus. Then, total cellular RNA was extracted and purified. Total RNA from 3hr and 6hr post-infected cells was treated with acid guanidine thiocyanate phenol chloroform method. cDNA reverse-transcripted from different periods of infection was labeled by RD technique and hybridized to the DNA microarray. Under the same background of cellular gene expression, the distinct pattern of Vaccinia virus gene expression could be detected and these signals could reveal early genes or late genes specifically. This study has identified eighteen genes differently expressed, which include late genes and 7 early...