Metagenomic Analysis Of The Viral Communities In Wild Birds In Ningxia, China

Emerging and re-emerging infectious diseases have posted great threats to animal and human health, so identifying and discovery of new pathogens that may cause emerging infectious diseases is the key to prevent the wide spread of the disease.Wild birds are the natural reservoirs of many important pathogenic viruses, and spread the viruses to a wide range through migration leading to the infections of humans and other animals.Ningxia is located in the Central Asian migratory routes of wild birds bearing large areas of wetlands, which provides good habitants for migratory birds and a suitable place for the transmission of viruses among different birds.To understand the diversity and the natural background of virus community of wild birds in Ningxia using viral metagenomics methods. We firstly used samples of pseudorabies virus cell cultures to explore the best procedure for preprocessing the samples. The cultured pseudorabies cells were differentially centrifuged to remove cell debris and other impurities, then treated with different doses of nuclease and extracted the nucleic acids. At last, the content of the viruses was measured by fluorescence quantitative PCR. The results showed that using 20 uL DNase I in 150 uL sample produces the best results for keeping viral genomes and removing host nucleotide acids at the same time. So this condition was used to prepare samples for viral metagenomeic analysis.Samples from 119 wild birds of 14 species in the cities of Shizuishan, Zhongning, and Qingtongxia of Ningxia were prepared using the previous established sample pretreating method and then the total nucleic acids were extracted and amplified with random primers and the obtained products were sequenced by using Illumina. A total of 32,385,000 useful reads were obtained and assembled into 163,192 contigs using SOAPdenove. Those contigs were then compared with NCBI virus database to find sequences belonging to viral genomes,the annotated avian influenza virus and circovirus are choosed to carry on the specificial PCR detection.Of the total contigs, 0.4%were annotated to belong to the viruses of 19 viral families. There were 63% phages, 33%vertebrate viruses, 3% insect viruses, and 1 % plant viruses in the viral contigs. we found 19 vertebrate viruses and distributed in 11 virus families,including several zoonotic viruses, such as influenza virus and sapporo virus.the result of specificial PCR detection for avian influenza virus and circovirus confirme the viral metagenomics annotation results correctly.The established sample preprocessing method will be a useful protocol for viral metagenomic analysis in the future. The viral community information of the wild birds in Ningxia will enriches the knowledge on wild bird harbored viruses and suggests potential zoonotic disease risks as well.