303 results about "Reverse genetics" patented technology

The instant invention provides stable and novel lineage I WNV reverse genetics systems, and methods for making the reverse genetics systems, specifically, a fully-infectious lineage I WNV cDNA or replicon system engineered with one or more nucleotide sequences each encoding a reporter gene to be used in high throughput cell-based screening assays for the identification of novel antiflaviviral chemotherapeutics and/or vaccines effective to treat and/or immunize against infections by WNV and other emerging flaviviruses, such as, for example, JEV, SLEV, AV, KV, JV, CV, YV, TBEV, DENV-1, DENV-2, DENV-3, DENV-4, YFV and MVEV. The present invention further provides methods of high throughput screening of antiflaviviral compounds or improved derivatives thereof using novel lineage I WNV reverse genetics systems and/or cell lines stably containing the reverse genetics systems. Also, the invention provides novel pharmaceutical compositions comprising an attenuated lineage I WNV that is less virulent but similarly immunogenic as the parent WNV and is capable of providing a protective immune response in a host.

A VII gene type of an attenuated strain of Newcastle disease virus A-NDV-VII and a construction method are disclosed. The invention relates to the application of reverse genetics technique. The invention uses the constructed reverse genetics platform of ZJ1 strain of Newcastle disease virus of goose origin. The invention replaces two envelope glycoprotein gene fragments F and HN of an isolated strain JS-5-05-Go of Newcastle disease virus with high reproductive performance with the corresponding fragments of the ZJ1 strain of Newcastle disease virus of goose origin, so that the recombinant virus NDV-VII is obtained. The VII gene type of Newcastle disease virus A-NDV-VII which is highly attenuated is rescued after the attenuated mutation of the F gene of the recombinant virus. And the virus has a higher reproduction titer on chicken embryo. The invention is suitable for a mass production of vaccine, which can be used for the manufacture of vaccine.

Attenuated influenza virus variants comprising substitutions in NS1 that interfere with viral replication and phosphatidylinositol 3-kinase activation are described. NS1 variant polypeptides, polynucleotides encoding NS1 variant polypeptides, a reverse genetics system for producing attenuated influenza virus NS1 variants, immunogenic compositions comprising live attenuated influenza virus NS1 variants, methods of stimulating an immune response against influenza virus, methods of interfering with influenza virus replication, and methods of treating and preventing influenza virus infection are described.

The invention relates to a method for expanding the antigen spectrum of a foot-and-mouth disease vaccine strain by reverse genetic operation and a preparation method of a vaccine. The amino acid sequence of the VP3 and VP1 structural proteins of the foot-and-mouth disease virus strain of the invention is represented by the amino acid residues from a position 304 to a position 736 in SEQ ID No.4. Experiments show that the vaccine prepared from the mutant virus strain obtained by the invention can resist porcine epidemic viruses of China O/TL/Taiwan/97 lineage, Pan-Asia O/China/99 lineage and Southeast Asia Myanmar O/GS/2010/98 lineage, has a characteristic of wide antigen spectrum, can immunize pigs and obviously improve the rate of protection against foot-and-mouth disease viruses which are of the same type and have antigenicity difference, achieves an immune effect of cross protection, and is expected to play an important role in the prevention and control of foot-and-mouth disease.

The invention discloses a recombinant low-virulent vaccine strain of chicken infectious bursal disease viruses (IBDV) and application thereof. In the invention, a major protective antigen gene VP2 of an epidemic superhigh virulent strain is cloned, the nucleotide of the gene VP2 is modified by mutation and then used for replacing a corresponding segment of a Gt genome of a low-virulent strain of the IBDV, so that the infectious clone of a recombinant genome of the IBDV is constructed, and the recombinant low-virulent vaccine strain is saved and identified by using an IBDV reverse genetic operation system. The microbial collection number of the vaccine strain is CGMCC No.3749. The recombinant low-virulent vaccine strain of the invention has high replicability, genetic stability and safety. The immune effect of the low-virulent vaccine strain of the invention is as good as that of the medium-virulent vaccine strain, but is superior to that of the low-virulent vaccine strain. The biological safety of the low-virulent vaccine strain of the invention is superior to that of the medium-virulent vaccine strain. As the vaccine strain, the recombinant low-virulent vaccine strain of the invention has the characteristics of high efficiency and low toxicity, is a good candidate vaccine strain and can be used for controlling chicken infectious bursal disease.

The invention discloses a genotype VII Newcastle disease virus marker vaccine strain and an application thereof, and belongs to the field of genotype VII Newcastle disease virus marker vaccine strain rescue and application. A built Newcastle disease virus reverse genetic operating platform is utilized for enabling NP protein of a G7 strain to miss 18 amino acids and conducting mutation on F-protein cleavage loci, and the highly-weak virulence and high-virus titer genotype VII Newcastle disease virus marker vaccine strain MG7-NPdelta18+Fmut is rescued through screening. The microbial preservation serial number is CCTCC NO: V201505. The marker vaccine strain has the biological characteristics of high growth titer and low virulence in chick embryos and is genetically stable. The immune protection test result shows that the marker vaccine strain is good in immunogenicity, capable of inducing high-level protective antibodies, and capable of completely protecting immunized chicken, can be used for preventing and controlling a currently-popular genotype VII Newcastle disease virus and lays the foundation of identifying vaccine immunity and wild virus infection.

Live, attenuated recombinant rabies virus vaccines are generated using reverse genetics to combine the antigenic determinants that render the rabies virus non-pathogenic with the determinants that are responsible for the elicitation of an effective anti-rabies immune response. These vaccines do not affect the antigenic, and therefore the immunogenic, properties of the virus. The present invention further relates to recombinant rabies virus vaccines that express a pro-apoptotic protein, such as cytochrome c, to increase the capacity to induce apoptosis, thereby enhancing the protective immunity against rabies. This new generation of live rabies virus vaccines represents a safe and effective approach to the eradication of rabies in wildlife, and subsequently humans and livestock.

The invention provides a preparation method for a triple inactivated vaccine of recombinant Newcastle disease, bird flu and infectious bronchitis. The technical scheme is realized by gene modification of a Newcastle disease virus (DNV) LaSota strain. A recombinant NDV low virulent strain (rLaSota strain) is taken as a vector, firstly an S1 gene of a chicken infectious bronchitis virus (IBV) is inserted between F-HN, a recombinant NDV rLaSota/S1 expressing an S1 protein is constructed by utilizing a reverse genetic technology, and a result shows that rLaSota can serve as a live vector for stably expressing the S1 protein; secondly a recombinant NDV rLaSota/S1-HA expressing an HA gene is constructed, namely, the HA gene is inserted between P-M of the recombinant NDV rLaSota/S1; and thirdly immunogenicity assessment is performed. A result shows that the triple inactivated vaccine of the recombinant Newcastle disease, the bird flu and the infectious bronchitis, prepared through a conventional inactivated vaccine process has relatively good protection effects for the Newcastle disease, the bird flu and the infectious bronchitis.

Rabies Virus compositions and methods are provided. The full-length sequence of Rabies Virus strain Evelyn-Rokitnicki-Abelseth (ERA) is disclosed. A reverse genetics system for producing recombinant ERA virus and derivatives thereof is provided, along with compositions including ERA and/or ERA derivative strain viruses, nucleic acids and/or proteins. In some instances, the compositions are immunogenic compositions useful for the pre- or post-exposure treatment of Rabies Virus.

The present invention describes a reverse genetic system for Phlebovirus such as Rift Valley fever virus. This system comprised of RNA expression plasmids and protein expression plasmids. Additionally, the present invention also discloses the modification of this system to generate a recombinant virus that expresses a non-viral foreign gene. Furthermore, the present invention discloses the use of this system in the development of anti-Rift Valley fever virus vaccines, screening of antivirals testing for anti RVF immune response and developing marker vaccines for Rift Valley fever virus. We also claim the utility of this approach to other phleboviruses.

The invention relates to gene VIb subtype Avian pneumo-encephalitis virus attenuated strain VIbI4 and a construction method thereof. The preserving number of the gene VIb subtype scriber set Avian pneumo-encephalitis virus attenuated strain VIbI4 is CGMCCNo: 6149. The gene VIb subtype Rubulavirus Newcastle disease virus attenuated strain VIbI4 and the construction method thereof relate to a technology for applying reverse genetics; and the method comprises the following steps: carrying out mutation on an F gene by using a transcription carrier Ptvt/071204 containing Avian pneumo-encephalitis virus JS/07/04/Pi full gene genome from pigeons so as to obtain to a transcription carrier ApTVT/071204, and then substituting corresponding parts on the ApTVT/071204 by the NP, P and L genetic fragments of Avian pneumo-encephalitis virus rNDV/I4 so as to obtain recombinant plasmids ApTVT/VIbI4, wherein the plasmids successfully save a recombinant virus VIbI4 after transfecting a BSR-T7/5 cell together with auxiliary plasmids. The virus is relatively high in propagating titre, is suitable for large-scale production of vaccines and can be used for manufacturing vaccines.

The invention discloses an indicator gene used in a TRV-mediated gene silencing system, and the base sequence of the indicator gene is shown in SEQ ID NO:1. Virus induced gene silencing in the invention is a good tool for studying plant gene functions by using reverse genetics; according to the invention, an extremely good indicator gene carrier is provided for a tobacco rattle virus induced gene silencing technology; and the indicator gene carrier is simple in construction, significant in silencing phenotype and lasting in trait expression, and achieves a good indication effect on quick gene function researches.

The invention relates to a Newcastle disease chimeric virus marker vaccine strain as well as a construction method and application thereof, and belongs to the field of rescue and application of Newcastle disease chimeric vaccine strains. A Newcastle disease virus reverse genetic operation platform is utilized to mutate an F protein cleavage site of a Newcastle disease gene GVII type strain into acleavage site of an attenuated strain, F and HN genes of a mutated gene VII type Newcastle disease strain and NP, P, M and L of a gene II type NDV La Sota strain construct a full-length chimeric cDNAsequence, and a 18bp nucleotide marker sequence is inserted into a non-coding region between P and M. A Newcastle disease chimeric virus NDV DC strain is obtained through transfection cell rescue. Theconstructed chimeric virus can reach relatively high culture titer in chick embryos and cells. The chimeric strain contains envelope surface glycoprotein of the gene VII type Newcastle disease strain, contains a skeleton of the gene II type strain, and has immunogenicity of the Newcastle disease gene VII type strain and high reproduction and high safety characteristics of the gene II type La Sotastrain.

The invention discloses a cell line ST/T7 capable of stably expressing T7RNA polymerase, the microorganism preservation number of which is: CGMCC No.24444. In the invention, a T7RNA polymerase gene is inserted into a eukaryotic expression vector pIRES2-EGFP to get the pIRES2-EGFP-T7 to transfect ST cells to obtain the cell line ST/T7 capable of stably expressing T7RNA polymerase. Through RT-PCR detection, expression plasmid containing a red fluorescent protein gene controlled by T7 promotors can be used to transfect the cell line so as to observe the expression of red fluorescent protein in a transcription product amplified form a ST/T7 cell to the T7RNA polymerase. The cell line ST/T7 can provide reverse T7RNA polymerase for use in reverse genetic manipulation of RNA-virus such as hog cholera virus, etc., and can be used as transcription and expression systems in vitro for research on genic structures and functions.

The invention belongs to the technical field of plant gene engineering, particularly relates to application of OsNMCP1 gene in controlling drought tolerance in rice. According to the invention, a candidate gene screening method is adopted to clone a rice drought-tolerant response gene OsNMCP1 by reverse genetics. The nucleotide sequence of the gene is shown in SEQ NO:1, and the protein sequence encoded by the gene is shown in SEQ NO:3. The phenotypic identification of drought stress at seedling stage and heading stage indicates that overexpression of OsNMCP1 gene causes an improvement of the drought resistance of transgenic rice, while the deletion mutation of OsNMCP1 gene results in a significant decrease of rice drought resistance. In the invention, the biological function of the gene and the pathways and methods of application with the same are demonstrated.

The present inventors developed hepatitis C virus 1a/2a and 1b/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and NS2 were replaced by the corresponding genes of the genotype Ia reference strain H77C or TN or the corresponding genes of the genotype Ib reference strain J4. Sequence analysis of recovered 1a/2a and 1b/2a recombinants from 2 serial passages and subsequent reverse genetic studies revealed adaptive mutations in e.g. p7, NS2 and/or NS3. In addition, the inventors demonstrate the possibility of using adaptive mutations identified for one HCV isolate in generating efficient cell culture systems for other isolates by transfer of mutations across isolates, subtypes or major genotypes. Furthermore neutralization studies showed that viruses of e.g. genotype 1 were efficiently neutralized by genotype Ia, 4a and 5a serum, an effect that could be utilized e.g. in vaccine development and immunological prophylaxis. The inventors in addition demonstrate the use of the developed systems for screening of antiviral substances in vitro and functional studies of the virus, e.g. identification of receptors required for HCV entry

The invention provides an influenza B virus Vero cell productive adaptive strain and a preparation method and application thereof. The influenza B virus Vero cell productive adaptive strain is named as B/Yunnan/2/2005va(B) with a preserved number of CGMCC No.2931. The virus strain can be continuously subcultured on the Vero cell, and a clotting titer can be kept over 1: 512. Through detecting, the virus strain is the influenza B virus, belonging to a Yamagata pedigree. The virus strain includes eight genetic fragments of the influenza B virus and has the characteristic of continuously producing the Vero cells. On the one hand, the virus strain is inoculated in the Vero cell, the supernate of a culture is collected to obtain a influenza B virus vaccine which has favorable immunogenicity and proactive effects through an experiment; on the other hand, the virus strain also can be used as a donor virus strain which is reassorted through a natural selective pressure or a reverse genetics to obtain the adaptive strain of an epidemic strain on the Vero cell, thereby an epidemic strain Vero cell influenza vaccine is prepared.

The invention relates to a rabies virus Flury-LEP vaccine strain reverse genetic operating system comprising a recombination vector for coding LEP full length genome cDNA and an auxiliary plasmid system thereof, wherein, the auxiliary plasmid system respectively codes nucleoprotein of LEP, phosphoric acid protein P and polyase protein L. the invention also relates to LEP green fluorescent protein recombination viral vector built by the reverse genetic operating system, concretely, the rabies virus Flury-LEP vaccine strain reverse genetic operating system is pCI-LEP and auxiliary plasmid systems pCAGG-N, pCAGG-P and pCAGG-L, the LEP green fluorescent protein recombination viral vector is pCI_LEP_EGEP. The invention also relates to recombination viruses rescued by the rabies virus Flury-LEP vaccine strain reverse genetic operating system and the LEP green fluorescent protein recombination viral vector, and applications thereof.

The invention relates to recombinant newcastle disease vaccine candidate rAI4-penton to express avian adenovirus penton protein and a construction method thereof. newcastle viral strain rAI4-penton iscollected under CGMCC No: 15492. The construction method includes: using a reconstructed reverse genetics operating platform of a newcastle attenuated strain to penton gene sequence of avian adenovirus into genome full-length transcription vector pNDV/rAI4 of the strain AI4 so as to obtain recombinant newcastle viral genome full-length cDNA clone pNDV/rAI4-penton, and performing transfecting to obtain recombinant viral rAI4-penton that successfully express penton protein. The recombinant virus rAI4-penton has high breeding titer on chicken embryos, can stabilize the penton protein even aftercontinuous passage, is suitable for large-scale production of vaccines, and is suitable for making vaccines.

The present inventors developed hepatitis C virus 6a/2a intergenotypic recombinants in which the JFH1 structural genes (Core, E1 and E2), p7 and the complete NS2 were replaced by the corresponding genes of the genotype 6a reference strain HK6a. Sequence analysis of recovered 6a/2a recombinants from 2 transfection experiments and subsequent reverse genetic studies revealed adaptive mutations in E1 and E2. Conclusion: The developed 6a/2a viruses provide a robust in vitro tool for research in HCV genotype 6, including vaccine studies and functional analyses.

The invention discloses an influenza A virus Vero cell adapted strain and application thereof. The influenza A virus Vero cell adapted strain is named as A/Yunnan/1/2005Va(H3N2), and the preserving number of the strain is CCTCC NO:V200514. The strain comprises an H3 subgroup hemagglutinin gene, an N2 subgroup neuraminidase gene and 6 internal genes for high yield of characteristic influenza viruses on Vero cells. The blood coagulation titer can be maintained at more than 1,024 by continuous subculturing of the strain on the Vero cells. The strain can be taken as a donor strain and subjected to genetic reassortment with an epidemic strain, or reverse genetics technology is adopted to perform genetic reassortment on the 6 internal genes and 2 surface protein genes of the epidemic strain to obtain the Vero cell adapted strain of the epidemic strain, and finally the adapted strain is used for preparing a Vero cell influenza vaccine or a Vero cell pandemic influenza vaccine.