Screen Viruses Associated With Diarrhea Diseases With Sequence-independent Single Primer Amplification PCR

Background:novel virus and novel subtype virus were discovered with the developing of biotechnology. The novel viruses discovered in the30years are threatening the heath of the world,and result many public healthy difficult problem and serious challenge, such as HIV, hepatitis C virus, hepatitis B virus,SARS,AVI H5N1.About70%pathogeneses of acute upper respiratory tract infection are virus, but some of these pathogeneses are not be discovered up to the present. Gastroenteritis and diarrhea is very common disease caused by virus, but there are20-40%cases whose pathogeneses were not identified. Understanding the pathogeneses is very important to prevent and control the infection diseases. Many methods designed to screen the novel viruses spring up in the last ten years. Some methods were designed to the Morphology of the virus and to Antigen-antibody reaction, another is designed to screen the DNA or RNA,such as sequence-independent single primer amplification (SISPA-PCR) and random PCR,One another is deep sequence.Now,these methods were cooperated to discover the novel pathogeneses, which is more efficiency.Objectives:1.To establish a sequence-independent single primer amplification (DNase-SISPA-PCR) technical platform to screen the virus associated withdia rrhea diseases.2.To discover the novel virus associated with diarrhea diseases.Methods:1. The routine RT-PCR to inspect the familiar viruses associated with diarrhea diseases were used to got the strong positive samples.And the n egative samples to the all familiar viruses, such as rotavirus, calicivirus, bocavi-rus, astrovirus, adenovirus and parechovirus.2. The improvement DNase-SISPA-PCR technical platform was used to screen novel virus associated with diarr hea diseases:The bacteria and cell were removed by the filters of0.45and0. 22um,and the virion were concentrated through ultracentrifuge. The free DNA or RNA of bacteria or the host were removed by DNase Ⅰ and RNAse A. The PCR reaction was work with a primer with a linker. The products more than500bp were purificated and cloned. The plasmids of the random selected clone-s were sequenced. And the bioinformation methods were used to analysis the sequence.3. The genome walking and DNase-SISPA-PCR technical platform were used to get the unknown distraction of the virus sequence. The race PCR was used to get the3’ and5’ sequence of the virus.4. The bioinformation methods were used to analysis the characters of the novel virus and to predict the functions of the novel virus’s motifs and others.Results:1.Four novel viruses (PAstV1, PBoV1, PAstV2, and PPicV) were discovered in the fecal of the pigs younger than90days. One novel virus is a bocavirus-like,two virus are astrovirus-like,and one another is a piconavirus-like.The four viruses have the characters of the virus associated with it’s self. The detection rate of the PAstV1is1.51%, the PAstV2is1.26%, the, PBoV1is12.59%and the PPicV is3.02%in the healthy pig samples.2. The complete sequences of PAstV1and PBoV1were sequenced, and the partial sequences of the PAstV2and PPicV were got.Conclusions:1.The DNase-SISPA-PCR technical platform was established and the platform was efficient that was proved in our study, Genome walking and DNase-SISPA-PCR technical platform are a good methods to get the near complete sequence of an unknown virus.The genome walking cooperates with the DNase-SISPA-PCR technical platform, which is more efficient to discover the novel virus.2. The four virus discoed in the swine fecals were novel virus:astrovirus, bocavirus and piconavirus.3. PBoV1was near to the human bocavirus with the analysis of the phylogenesis, and it has some characters of the parvovirus; PAstV1were near to the human astrovirus in the phylogenies and has the characters of human astrovirus.