Study On The Unkown Pathogens

The study on the unknown pathogens is the hot spot of the biomedical field, though the medical research has been at high level while some unknown pathogens were discoverd. It could promote the understanding of the human body, for competition between the human body and pathogens on the point of evolutional view. On the other hand, it can ease the pain of patients. In theory, it was very difficult to study the unknown pathogens. The one reason is that the instrument or methods were too under-developed to find the unknown pathogens which their size and structure were special. The other reason is some pathogens were conditional pathogenicity. We research the some suspect pathogens below according to existing conditions.1. Identification of a novel strain of EnterovirusA strain of virus(XJ90) with cytopathic effect (CPE) and hemagglutination was isolated from fecal sample of a patient with enterically transmitted non-A, non-B hepatitis (NANBH) by monolayer culture of Hep-G2 cell line in Xinjiang, China in 1986. The immunoelectronmicroscopy (JEM) revealed that the XJ90 strain consisted of spherical, 27-37nm, virus-like empty and complete paticles(VLPs), which could be aggregated by acute phase sera of NANBH patients from Xinjiang, USSR, Burma and India. The results of physics-chemical studies revealed that the RNA virus was acid-fast, ether-resistent and heat-sensitive. Complete gene of XJ90 strain was sequenced, which was classified to Enterovirus of Picornavirus and was higher homogeneity(about 80%). with Coxsackie virus.We developed a new method named random PCR (rPCR)i which was used to identify the unknown pathogeny from cultured cell lines above. Twelve clones were produced by rPCR from the culture of cell line and were sequenced. All of them belong to Enterovirus of Picornavirus and not hepatitis E virus. The rPCR was a very good method of identification of unknown pathogency from cell culture, which was convenient, quick, accurate and specific.We synthesized the some primers on the basis of the 5' and 3' end sequences of twelve clones which were distributed on the most sections of Enterovirus (7.5kb). The complete cDNA of XJ90 was sequenced by overlaped fragment PCR. It was 7443 base pairs and coded a poly protein which was 2189 amino acids. There was 743 bp non-translation region on the 5' end and 130bp(30bp polyA tail) on the 3'end of XJ90.2. Isolation and identification of a strain of suspected L-form bacteriumA microorganism was discoverd from serum of a patient with unknown etiological reason fever. It appeared filament and swimmed quickly. We considered it was L-form bacterium.They were different in length about 3-30um, one piece under the two views of light microscope and one piece under ten views in half a month. The number was no relationship with the state of the illness.In order to show the their configuration more clearly, we observed them by electron microscope and found various shapes, such as like staffs, bent, loop, silk, pseudopod ameba and so on. It is obvious that there are no diaphragm, no cell wall, but division posture and bud.We successfully infected animals with them and explored the cultivation. They didn'tneed the high osmosis and abundance nutrient, but needed blood to survive. This means this L-form bacterium was addicted to the blood. There were no fever and no abnormity viscera of infected animals.3.Study on the unknown hepatitis pathogensThe data from clinic revealed that there were unknown hepatitis pathogens except hepatitis A, B, C, D and E, so we developed a subtractive hybridization mothod which combined the advantages of RDA ( representational difference analysis) and SSH (suppression subtractive hybridization) to search for the novel pathogens from two unknown acute hepatitis patients.The patients were not addicted to smoking and drinking and no liver damage with drug. Ten and eight cDNA sequences were cloned from their serum respectively by our improved method. Seven and two sequence of them were unknown after searching in Genbank database. Further analysis would be done. This study established the foundation to explore the unknown hepatitis pathogens in the future.