Development Of A Metagenomics-Based Method For Detection Of Animal Viruses

There exist extreme varieties of viruses in the environment and animals, some of which are infectious agents. However, many unknown viruses are barely detected by means of conventional tests, such as virus isolation and PCR assay, due to the following reasons:most of these viruses could hardly be isolated by cell culture; the amounts of viruses and their nucleic acids are relatively low compared to their host cells; the genomic sequence information of some viruses is absent or insufficient in available databases.Viral metagenomics is a technology used to study a population of viruses in a specific circumstance. This method specially emphasizes on sequence-independent amplification and thus eliminates the process of in vitro viral amplification, which compensates the deficiency of classical methods and shows great advantages in identifying unknown viruses, and it has been extensively utilized in novel virus detection among others. The key to metagenomics-based viral discovery is reducing background nucleic acids.The aim of this study was to develop an unknown virus detection technology platform based on viral metagenomics in combination with molecular diagnosis. Firstly, filtration and enzymatic removal of cellular nucleic acids were performed to reduce background nucleic acids. Next, DNA and RNA were extracted respectively. RNA reverse transcription and double-stranded cDNA/DNA synthesis were performed with a primer with a random hexanucleotide at its3'-end and a defined sequence at the5'-end. Subsequent random PCR was carried out with the defined5'-end of the initial primer. The amplified products were purified and digested with restriction enzyme EcoRV to remove the amplification primer. Products were then separated on an agarose gel and fragments between600and1,500bp in length were excised and extracted. Purified PCR products were ligated to the pSIMPLE18EcoRV/BAP Vector and introduced into chemically competent E coli TOP10cells. Screened positive clones were sequenced to acquire information of possible viruses. We built analysis technology platform for RNA viruses by testing classical swine fever virus (CSFV)-infected cells, and4out of15sequences were highly homologous to CSFV Analysis of a tissue sample infected with porcine circovirus type2(PCV2) showed that12out of20sequences were homologous to PCV2. We amplified16.4%sequences of the simian parainfluenza virus type5genome from a cell culture of unknown virus using the technology platform developed in this study.The results indicated that the developed method was suitable for detection of DNA and RNA viruses, and it also has potential for universal detection of known or unknown viruses. It might play a very important role in diagnosis of emerging infectious diseases.