Study On The Identification Of Unkown Virus And Viral Hemorrhagic Fever Virus

An infectious disease emerged in 2009 in rural areas of Hubei and Henan provinces. The disease was characterized by fever, gastrointestinal symptoms, thrombocytopenia, and leukocytopenia with an case fatality rate of approximately 10%. This emerging infectious disease was called'Tick bite disease'by local residents. There is an urgent need to confirm pathogen.We isolated an new virus from a patients's serum from Hubei province. Combined techniques of sequence-independent single primer amplification(SISPA) and RNA ligation replication, we completed whole sequencing of 3 segments of this new virus, then, the whole genome of total 11 virus isolated from patients serum collected from 6 provinces were further sequenced. With bioinformatics analysis, we identified this virus is a new member of Phebovirus, Bunyaviridae. But there exist huge difference on the genome sequence between this new virus and other bunyavirus whether for nucleotide level or for amino acid level. This suggested that this new virus might to be the prototype of a new group in the genus of Phlebovirus. The comparison of amino acid sequence similarity provided further support to the separation of SFTS from other phleboviruses. The amino acid sequence similarity for most conserved N protein reached 41%, In contrast, the S segment encoded NSs proteins showed the least amino acid similarity of only 11.2-16.0%. And for RdRp and glycoproteins, is about 21%-36%. Finally, this disease was designated'Severe Fever with Thrombocytopenic Syndrome (SFTS)'and this new virus was termed 'Severe Fever with Thrombocytopenic Syndrome Bunyavirus'(SFTSV).It is urgent to develop sensitive, accurate, and reliable diagnostic tests to meet the needs of disease diangnosis and surveillance, In order to improve the sensitivity and specificity of the test, we choose three segments L, M, S as our target genes simultaneously and developed three sets of primers and probes to detect nucleic acid in serum sample using one-step real-time RT-PCR. We prepared RNA standards produced using in vitro transcription to quantify the viral load of the specimens. In addition, for avoiding false negative results and quality control, an appropriate internal control should be added, we choose the heterologous EGFP gene as the target of internal control. Since naked RNA is susceptible to degradation by RNases and can not reflect efficiency of RNA extraction. We packaged EGFP RNA into pseudoviral particles using the Virapower lentiviral expression system, which renders the RNA protected from RNase-mediated degradation. This internal control can be added before the first step of sample processing, co-extracted and co-amplified with the viral RNA in the same reaction tube, and can monitor the specimen extraction and amplification efficiency. Thus, false-negative results can be avoided with the use of Internal Control. The sensitivity and specificity of this assay was validated by testing confirmed clinical samples. By now,this assay have completed clinical trial and is waiting for commercial kit approval. Up to now, we tested more than 300 susceptible serum sample altogether and the detection rate was about 70%.To further explore the immune mechanism underlying the pathogenesis of SFTS, we studied the expression profile of 27cytokines in the patients'serum using a bead-based multi-assay system. The results showed that the level in serum of IP-10,Eotaxin IL-10,IL-15,IL-1β,G-CSF were markedly elevated during the early stage of SFTS compared with control group, that of IL-6,IL-8,IL-lra,MCP-1,MIP-1 a also increased to varying degrees, on the contrary, the level of PDGFBB, RANTES,VEGF decreased significantly. To our surprise, the interferon was not detected unexpectedly. All the results indicated that SFTSV infection indeed induced immune system disorders and the extent of disorders related with prognosis on a certain degree.When screening for pathogens of diseases, first of all, we must exclude known pathogens at the shortest time, so we try to improve the efficiency of traditional methods. Virus isolation is always considered to be the'gold standard'for confirming viral infection. In the process of virus isolation, we tried to change from observation of cytopathic or immunofluorescence experiments to observation of the reporter protein's expression. In this part of study, the enhanced green florescent protein (EGFP) gene was flanked with the noncoding regions (NCR) of hantavirus(strain 76118 and 8039) L, M and S segments. Then, these constructions were subcloned by anti-sense orientation into the pHH21 vector which have the RNA polymerase I (pol I) promoter and terminator sequences. These different minigenome were transfected into Vero-E6 cells then infected with appropriate virus. The expression level of EGFP was observed 1 or 2 days after infection. The results showed that all NCRs can mediate transcription/replication of a minigenome, but the expression of EGFP is very low, among of which, Minigenome basedon M segment showed the strongest EGFP activity, followed by the S segment and the L segment. Although the system is not up to the original purpose to detect viral infection, it lay the foundations for future studies, Using methods of insertion, deletion, mutation at the DNA level, we can operate on non-coding region to study about the relationship between the viral structures and functions.Due to wide range of pathogens for viral haemorrhagic fever, it is necessary to develop multiple PCR method to save time and sample. Since traditional multiple PCR often come up with amplification bias, we designed a 8-plex PCR based on GeXP system and evaluated initially.