Entire gene sequence of severe fever with thrombocytopenia syndrome virus (SFTSV) and application

A virus genome and syndrome technology, applied in the fields of molecular biology and virology, can solve the problem of unclear etiology of patients, and achieve the effect of huge economic and social benefits

Active Publication Date: 2011-05-25
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the etiology of these patients is not clear

Method used

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  • Entire gene sequence of severe fever with thrombocytopenia syndrome virus (SFTSV) and application
  • Entire gene sequence of severe fever with thrombocytopenia syndrome virus (SFTSV) and application
  • Entire gene sequence of severe fever with thrombocytopenia syndrome virus (SFTSV) and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Virus Isolation

[0054] Since 2004, Zhejiang, Jiangsu, Anhui, Shandong, Henan, Hubei and other provinces have discovered and reported cases of infectious diseases with fever and thrombocytopenia as the main manifestation, and a small number of severe patients developed multiple organ damage and even died. In 2010 Since May 2009, the National CDC Institute of Viral Diseases has collected 197 serum samples from patients with similar symptoms from 6 provinces including Shandong, Hubei, Henan, Jiangsu, Anhui and Liaoning.

[0055] Using conventional tissue culture methods, 197 samples from the above-mentioned 6 provinces were tested for virus isolation. Dilute 100μL of patient serum with DMEM 1:10 to 1ml, inoculate to 25cm 2 In the culture flask vero cells (Greiner Bio-One), adsorb for 1 hour at 37°C, add 4ml DMEM maintenance solution (containing 2% fetal bovine serum and 1000 units / mL penicillin and streptomycin), and place the culture flask at 37°C , 5% CO 2...

Embodiment 2

[0057] Embodiment 2 sequence independent single primer amplification (SISPA)

[0058] Amplification of viral nucleic acid from serum was according to the method of Allander et al. (2001) with some modifications. Firstly, the first acute serum sample HB29 obtained in 2010 was processed: 140 μL of serum was diluted twice with PBS (pH 7.4), centrifuged at 10,000 g for 10 minutes to remove cell debris, and filtered through a filter membrane with a pore size of 0.2 μm Remove cell clumps or bacteria, then add 14U DNase (Ambion), 20U benzonase nuclease (Novagen) and 20U RNase (Promega) to the serum, use 1×DNase buffer (Ambion) as the buffer, and digest at 37°C 2 hours to remove non-viral (no viral particle protection) nucleic acids. Using the Viral RNA Isolation Kit (Qiagen), RNA was extracted from 280 μL of diluted serum according to the instructions and eluted with 40 μL of RNase-free water. Synthesis of first-strand cDNA: Add 2 μL of 10 μM random primers (Promega) to 13 μL of pu...

Embodiment 3

[0065] Embodiment 3 Amplification of the full-length genome of the virus

[0066] Primers were designed according to the sequence originally obtained by SISPA, and forward and reverse primers (Table 2) for RT-PCR were designed from two adjacent fragments to obtain the sequence between the two adjacent fragments. RNA was extracted from patient serum using a viral RNA isolation kit (Qiagen), reverse transcription was performed using a cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA), and amplification was performed using DNA polymerase (Invitrogen), 1.2% agarose gel electrophoresis, DNA gel recovery kit (Qiagen) was used to recover the amplified products, and PCR primers were used for direct sequencing without cloning, and the measured sequences were spliced ​​by DNAStar software.

[0067] In order to determine the sequence of the 3' and 5' ends of the viral genome, rapid amplification of cDNA ends (RACE) was performed, and the operation was carried out according to the kit i...

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Abstract

The invention relates to a severe fever with thrombocytopenia syndrome virus (SFTSV), an entire gene sequence represented by Hubei isolate HB29, amino acid sequences of coding proteins and application. The entire gene sequence of the virus is subjected to homology analysis. The virus belongs to bunyaviridae and comprises three gene segments, namely, L, M and S which represent polymerase and glycoprotein (Gn and Gc), nucleoprotein (NP) and non-structural proteins (NSs) of the virus respectively, and the three segments are all positioned on the branch of phlebovirus but farther from other viruses of phlebovirus. The entire gene sequence and the coding proteins of the virus can be used for developing drugs, vaccines or diagnostic reagents for preventing and treating the epidemic diseases caused by the SFTSV.

Description

technical field [0001] The invention relates to the fields of virology and molecular biology technology, in particular to the whole gene sequence and application of a novel fever with thrombocytopenia syndrome virus. Background technique [0002] Bunyaviruses are spherical, enveloped, and segmented negative-sense RNA viruses. It was named after the representative species of this family, Buniawera virus, was first isolated from Buniawera in western Uganda. Buniavira virus is 90-100 nanometers in diameter, with many glycoprotein protrusions protruding from the envelope, and there are 3 helical and symmetrical nucleocapsids inside, containing 3 large (L), medium (M) and small (S) respectively RNA segments, in which L and M are negative-strand RNAs, which encode viral polymerases (RNA-dependent RNA polymerases, RdRP) and glycoproteins (Gn and Gc), respectively; S segments are ambisense RNAs, which encode nucleoproteins (NP) and Nonstructural proteins (NSs). According to serol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/005C12N15/31C12N15/63A61K38/16A61K38/43A61K48/00A61K35/12A61P31/12C12Q1/68C12Q1/25G01N33/569G01N33/68
Inventor 李德新梁米芳张全福孙玉兰李建东芜为李川曲靖卢静孙丽娜
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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