59 results about "Necrovirus" patented technology

The present invention relates to a circular RNA for targeted inhibition of fish neural necrosis virus replication, as well as a preparation method and application thereof. The circular RNA is NNV-circRNA-1, whose nucleotide sequence is shown in SEQ ID NO:1. The circular RNA is prepared by cyclizing linear NNV-circRNA-1 with T4 RNA ligase 1 and then removing residual linear RNA in the reaction system using RNase R enzyme. The circular RNA prepared by the present invention has good stability and high purity. NNV-circRNA-1 has a strong binding ability with NNV-RdRp protein, can effectively inhibit the replication of NNV virus, and can be used in the preparation of vaccines, antiviral drugs and other biological immune products for fish neural necrosis virus in the field of aquaculture.

The invention discloses a detection reagent, a kit and a detection method for pathogens of micropterus salmoides. The micropterus salmoides pathogen detection reagent comprises primers and probes for specifically amplifying an infectious spleen and kidney necrosis virus, a viral nervous necrosis virus, flavobacterium columnar, a perch rhabdovirus, a micropterus salmoides double-RNA virus, edwardsiella tarda, nocardia and a micropterus salmoides iridovirus; specific upstream and downstream primers and probes of the eight pathogens are sequences as shown in SEQ ID NO.1 to SEQ ID NO.24 in sequence. The micropterus salmoides pathogen detection reagent and method can detect eight pathogens at the same time, have the advantages of high sensitivity, high specificity, good repeatability and the like, provide reliable basis for prevention and control of the pathogens, also improve the prevention and control work efficiency, and have good application prospects. The kit is especially suitable for large-scale rapid screening and mixed infection diagnosis of aquaculture pathogens, and has important value and significance for prevention and treatment of micropterus salmoides pathogens.

The present invention relates to the field of biomedicine technology, and specifically discloses an mRNA based on the CP protein gene of fish nervous necrosis virus, a vaccine, and a preparation method and application thereof. The present invention provides an mRNA of fish nervous necrosis virus, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the structure includes a 5' untranslated region, a signal peptide sequence, an NNV virus antigen coding region, a 3' untranslated region and polyA. The mRNA vaccine contains the ORF sequence of the capsid protein CP gene that is resistant to NNV virus infection. The results of animal safety tests on the fish nervous necrosis virus mRNA vaccine prepared by the present invention show that the vaccine is safe and effective, and is convenient and simple to use clinically, and has broad application prospects in the prevention and control of viral nervous necrosis in marine cultured fish.

The invention discloses an application of Rottlerin in preparation of a medicine for preventing and treating nervous necrosis virus. According to the application disclosed by the invention, the fact that the rotinidin has high binding energy to RGNNV capsid protein is found, the rotinidin without toxic concentration is selected to carry out an RGNNV virus infected cell experiment, and the result shows that the rotinidin inhibits the replication of the RGNNV virus in a dose-dependent manner. The safe dosage of Rottlerin is obtained according to the survival rate of zebrafish treated with different doses, the influence of Rottlerin on the survival rate of zebrafish infected with RGNNV is researched, and finally it is proved that Rottlerin has a remarkable treatment effect on zebrafish infected with RGNNV. Rottlerin provides a new natural small molecule drug choice for prevention and treatment of VNN diseases, and has important research and development value and development significance for prevention and treatment of nervous necrosis viruses.

The invention discloses a primer probe combination for detecting various prawn pathogens and application of the primer probe combination. The primer probe combination comprises a first primer probe set for detecting vibrio harveyi, a second primer probe set for detecting infectious subcutaneous and hematopoietic necrosis viruses, a third primer probe set for detecting Taura syndrome viruses and a fourth primer probe set for detecting infectious muscle necrosis viruses. The primer probe combination has the characteristics of high sensitivity, good repeatability and strong specificity, and can be used for specifically detecting vibrio harveyi, infectious subcutaneous and hematopoietic necrosis viruses, Taura syndrome viruses and infectious muscle necrosis viruses.

The application discloses a nerve necrosis virus goliath grouper oral vaccine and a preparation method thereof. The amino acid sequence of the antigen of the nerve necrosis virus goliath grouper oral vaccine is shown as SEQ ID No: 1. The preparation method comprises the following steps: 1) construction of a recombinant plasmid: a fusion gene with a nucleotide sequence shown as SEQ ID No: 1 is synthesized, the fusion gene is inserted into an expression vector to obtain a recombinant vector, the recombinant vector is transformed into a prokaryotic expression bacterium, and the prokaryotic expression bacterium is cultured to select a successfully transformed prokaryotic expression bacterium; 2) the successfully transformed prokaryotic expression bacterium is induced to express; and 3) the induced prokaryotic expression bacterium is used to feed halocyprid larvae, and halocyprid larvae enriched and wrapped with the prokaryotic expression bacterium are obtained to obtain a nerve necrosis virus infection goliath grouper medicine. The nerve necrosis virus goliath grouper oral vaccine has high protection rate and an immune protection efficiency of 100% for goliath groupers. The application can be applied to the prevention and control of nerve necrosis virus diseases in the goliath grouper breeding process.

The invention discloses a method for visually detecting mandarin fish iridovirus and infectious spleen and kidney necrosis virus based on RPA-CRISPR Cas12a / Cas13a, and belongs to the technical field of biological diagnosis. According to the method, a CRISPR / Cas system is combined with RPA amplification, a fluorescent reporter and a test strip are used for naked eye observation, and the method combines the advantages of different isothermal amplification technologies, has the advantages of short detection time, high specificity, no cross reaction, high sensitivity and the like, realizes rapid and accurate diagnosis of the two pathogens, and has a good application prospect. Important technical support is provided for prevention and control of mandarin fish diseases.

The invention provides an application of epigallocatechin gallate in preparation of a medicine for resisting nervous necrosis viruses of fishes. Related experimental results show that the EGCG is used for preparing the medicine for resisting the nervous necrosis viruses of the fishes, infection of the nervous necrosis viruses of the fishes, especially groupers, can be effectively prevented and treated, the economic benefits of cultivation are improved, and a good ecological environment is maintained.

The present invention relates to the technical field of prevention and control of aquatic animal diseases, and specifically relates to a nanocomposite for a nervous necrosis virus, a preparation method therefor, and the use thereof. Provided in the present invention is a nanocomposite consisting of poly(lactic-co-glycolic acid), polyethyleneimine and a siRNA. Also provided in the present invention are a preparation method for the nanocomposite and the use thereof in the preparation of a drug for improving the survival rate of fish eggs infected with a nervous necrosis virus. The present invention constructs a vector for a capsid protein of the nervous necrosis virus by means of simulating the nervous necrosis virus and expresses same in cells, and screens out a siRNA that can effectively inhibit the expression of the viral capsid protein, thus constructing the nanocomposite for the nervous necrosis virus. The nanocomposite can block NNVs at the embryonic phase (fertilized eggs), thereby improving the survival rate of fish fry, and further opening up new possibilities for the treatment of early stage diseases in fish fry. The method is innovative in the field of artificial breeding of aquatic animals and provides new ideas for virus prevention and control in the field of aquaculture.

This invention relates to the field of eDNA enrichment and detection, and discloses a highly efficient method for the enrichment and detection of infectious hematopoietic organ necrosis virus (IHNV) eDNA. The method includes the following steps: preparing and pretreating an IHNV virus sample to obtain a standardized raw solution; subsequently, using a tangential flow enrichment device to efficiently concentrate the IHNV virus particles in the raw solution, achieving the enrichment of viral eDNA; and constructing an RT-qPCR quantitative detection system. By plotting a standard curve and a sample amplification curve, the virus recovery rate is accurately calculated, enabling accurate evaluation of the efficiency of the entire enrichment process. This invention overcomes the shortcomings of traditional methods, such as small processing volume, low efficiency, and cumbersome operation, achieving the goal of efficient and stable recovery of viruses from large-volume water samples.

The invention discloses a primer and probe combination for detecting an infectious spleen and kidney necrosis virus and a rapid detection product, and relates to the technical field of biology. The primer and probe combination comprises an upstream primer of which the nucleotide sequence is shown as SEQ ID NO.6, a downstream primer of which the nucleotide sequence is shown as SEQ ID NO.7 and a probe of which the nucleotide sequence is shown as SEQ ID NO.17. The method has the advantages of high detection sensitivity and strong specificity. The method is adaptive to various basic layer detection scenes such as pond openings and ports, meanwhile, a complex sample pretreatment process is avoided, complex instruments and professional technologies are not needed in operation, integrated diagnosis of on-site sampling, rapid detection and instant interpretation can be achieved, and an efficient, accurate and convenient epidemic disease prevention and control technical support is provided for the aquaculture industry.

The invention discloses a fish nervous necrosis virus resisting medicine which is characterized by comprising baicalin. The invention also discloses a screening method of the drug for resisting the nervous necrosis virus of the fish, which comprises the following steps: constructing a recombinant prokaryotic expression plasmid with the HSC70 gene, transferring the plasmid into a BL21 (DE3) cell, inducing protein expression, and purifying to obtain the HSC70 protein; preparing to-be-detected compound solutions with different concentrations, and then dropwise adding the to-be-detected compound solutions into the solution containing the HSC70 protein for incubation; under the excitation wavelength of 285 nm, the fluorescence signal of the HSC70 protein in the range of 310-350 nm is detected, the compound for targeted inhibition of the HSC70 protein is determined according to the change of the fluorescence signal, and the fish nervous necrosis virus resisting medicine is screened out.

The invention provides a preparation method of a grouper nervous necrosis virus protein antigen and an egg yolk antibody, which comprises the following steps of: obtaining a grouper nervous necrosis virus capsid protein gene sequence, recombining the grouper nervous necrosis virus capsid protein gene sequence into a pET-28a carrier, and introducing into escherichia coli for expression to obtain the grouper nervous necrosis virus protein antigen. The grouper nervous necrosis virus capsid protein antigen obtained after purification, concentration, folding and dialysis is pseudovirus with an icosahedron symmetrical structure, after laying hens are immunized by the pseudovirus, eggs of the laying hens are collected, and egg yolk antibodies are extracted and applied to immune protection of groupers. The obtained antigen is high in immunogenicity, the obtained antibody has excellent titer, has practical value for preventing and treating nervous necrosis virus of grouper, is high in safety and low in cost, and can effectively reduce economic loss of aquaculture.

The invention provides shRNA for interfering nervous necrosis virus B2 protein, a recombinant plasmid as well as a preparation method and application of the shRNA and the recombinant plasmid, and belongs to the technical field of biological medicines. The shRNA (short hairpin ribonucleic acid) interfering with the nervous necrosis virus B2 protein is shRNA1 (short hairpin ribonucleic acid 1) or shRNA2; the positive-sense strand sequence of the shRNA1 is as shown in SEQ ID NO. 2, and the antisense strand sequence of the shRNA1 is as shown in SEQ ID NO. 3; the sequence of a positive-sense strand of the shRNA2 is as shown in SEQ ID NO.5, and the sequence of an antisense strand of the shRNA2 is as shown in SEQ ID NO.6. The shRNA recombinant plasmid is good in stability, has relatively high transfection efficiency in carp epithelial cells, can effectively interfere with the expression of B2 protein in the cells, has a virus replication inhibition rate of 80% or more on nervous necrosis viruses, and can be applied to preparation of drugs for resisting the nervous necrosis viruses in aquaculture so as to realize effective prevention and control on the nervous necrosis viruses in aquaculture.

The application discloses a kind of based on water body virus load and fish body tissue virus load correlation grouper nervous necrosis virus nondestructive testing method.The method is through optimizing the concentration condition of virus in aquaculture water, realize the efficient concentration and quantification of water body virus load, then combine water-water aquaculture animal virus load correlation model, indirectly and nondestructively assess the virus infection state of aquaculture animal.The application detects water environment instead of detecting animals itself, realizes nondestructive, dynamic, early warning monitoring of the health status of aquaculture animal population, with the advantages of low cost, simple operation, frequent implementation, etc., and has important application value in aquatic disease prevention and control, good seed selection and biological safety management.

The present invention relates to a multiplex PCR primer set for simultaneously detecting four pathogens, as well as a detection method and application thereof, belonging to the field of molecular biology. The four pathogens are neural necrosis virus, iridovirus, Streptococcus iniae, and Vibrio harveyi. The primer set comprises SEQ ID NOs. 1-8. Using the primers of the present invention, neural necrosis virus, iridovirus, Streptococcus iniae, and Vibrio harveyi can be simultaneously detected in a short period of time, thereby achieving the purpose of establishing a simple and effective method for simultaneously detecting multiple fish pathogens. Furthermore, the detection primer set and detection method of the present method have good specificity and high sensitivity.

The invention discloses an infectious pancreatic necrosis virus monoclonal antibody and application thereof. The invention provides hybridoma cell strains IPNV-vp2-178-2A7 (CCTCC (China Center For Type Culture Collection) NO: C2024368) and IPNV-vp2-380-1G12 (CCTCC NO: C2024369) as well as two monoclonal antibodies secreted by the two hybridoma cell strains. According to the invention, two monoclonal antibodies are utilized to establish an antigen capture ELISA detection method aiming at the VP2 protein of the IPNV virus, and the effect of the antigen capture ELISA detection method is evaluated to find that the antigen capture ELISA detection method can specifically, rapidly and sensitively detect the infectious pancreatic necrosis virus.

The invention belongs to the technical field of marine aquaculture industry detection, and particularly relates to a primer probe combination for detecting viral nervous necrosis virus, an RPA (recombinase polymerase amplification) visual kit and application thereof. The invention relates to a primer probe combination for detecting viral nervous necrosis virus. The primer probe combination comprises an upstream primer F2, a Biotin downstream primer Biotin-R2 and a fluorescent nfo probe nfo-P2. The kit disclosed by the invention is visual; the specificity is high; the detection sensitivity is high and can reach 100 copies / mu L; the accuracy is high and reliable; the method is convenient and fast to operate, suitable for field detection and wide in application scene.

The present invention discloses a hybridoma cell line, monoclonal antibody and application that secretes anti-infectious pancreatic necrosis virus VP2 monoclonal antibody, which belongs to the field of bioengineering. The hybridoma cell line IPNV9-VP2 was deposited on August 17, 2022 at the China Center for Type Culture Collection, Wuhan University, Wuhan City, Hubei Province, with a deposit number of CCTCC C2022216. It is derived from the recombinant protein of infectious pancreatic necrosis virus VP2. The secreted anti-IPNV-VP2 monoclonal antibody has high purity and can specifically recognize IPNV-VP2 protein. It is used for ELISA to specifically detect the concentration of IPNV-VP2 protein in diseased fish tissues or the distribution of IPNV-VP2 in frozen tissue sections with high sensitivity and accuracy, providing parameter indicators for detecting viral infection. By monitoring the health status of salmon and trout in real time, it provides technical support for the sustainable development of salmon and trout aquaculture.

The invention belongs to the field of prevention and control of aquatic pathogenic microorganisms, and particularly relates to a fish nervous necrosis virus blocking protein as well as a preparation method and application thereof. Due to the special structure of the protein, the protein can be specifically bonded with receptors on the surfaces of fish nervous necrosis virus particles, so that the fish nervous necrosis virus particles lose the infection capability on fish somatic cells, the fish nervous necrosis virus is prevented from infecting fish host cells, and the fish nervous necrosis virus is prevented from being copied and proliferated in a host. The fish nervous necrosis virus blocking protein provided by the invention has an efficient nervous necrosis virus resisting effect, a biological preparation product prepared by taking the fish nervous necrosis virus blocking protein as an active material can remarkably improve the nervous necrosis virus resisting capability of fry in a mixing oral administration manner, is safe and pollution-free, can be subjected to large-scale industrial application, and has a wide application prospect. A new way is provided for preventing and controlling the nervous necrosis virus disease of the fish, so that the economic benefit of fish culture is improved.

The present application belongs to the field of prevention and control of aquatic pathogenic microorganisms, and particularly relates to a fish nervous necrosis virus blocking protein as well as a preparation method and application thereof. Due to the special structure, the protein can specifically bind to the receptors on the surface of fish nervous necrosis virus particles, so that the fish nervous necrosis virus loses the ability to invade fish cells, thereby blocking the fish nervous necrosis virus from infecting the host cells of fish, and avoiding the replication and proliferation of the fish nervous necrosis virus in the host. The fish nervous necrosis virus blocking protein provided by the present application has a high anti-nervous necrosis virus effect. The biological preparation product prepared by using the fish nervous necrosis virus blocking protein as an active material can significantly improve the anti-nervous necrosis virus ability of fish fry by using the oral method, is safe and pollution-free, can be applied on a large scale, provides a new way for the prevention and control of fish nervous necrosis virus disease, and further improves the economic benefits of fish culture.

The application belongs to the technical field of virus vaccine, and particularly relates to a natural gene deletion attenuated virus strain of infectious spleen and kidney necrosis virus and application thereof. In the process of purchasing live experimental animals of mandarin fish, a natural gene deletion attenuated virus strain NH-1398B of ISKNV is separated from the purchased mandarin fish. The genome of the strain is partially deleted in ORF102R and ORF104R, and ORF103R is completely deleted. In the application, whole genome sequencing and virulence determination are carried out on the NH-1398B strain, and the immunoprotective effect of the strain as an ISKNV vaccine is evaluated. The results show that the ISKNV NH-1398B strain has the characteristics of safety, high efficiency and low toxicity, and can be completely used for the immunoprevention of a new infectious spleen and kidney necrosis virus disease.

The invention provides a primer and probe combination for simultaneously detecting white spot virus and infectious subcutaneous and hematopoietic necrosis virus, and relates to the technical field of biological detection. The nucleotide sequences of the primer for detecting the white spot virus are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe for detecting the white spot virus is shown as SEQ ID NO.3; the nucleotide sequence of the primer for detecting the infectious subcutaneous and hematopoietic necrosis virus is shown as SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of the probe for detecting the infectious subcutaneous and hematopoietic necrosis virus is shown as SEQ ID NO.6. The invention further discloses a kit for detecting the infectious subcutaneous and hematopoietic necrosis virus. The method is short in detection time, and detection can be completed within 30 minutes. The method is high in sensitivity, can detect virus nucleic acid as low as 20 copies per reaction, and is suitable for detection of early infection. The specificity is high, the cross reaction with other similar sequences is avoided, and the accuracy of the detection result is ensured.

The present invention discloses the use of bufalin in the preparation of products against IHNV and IPNV. The present invention provides the use of bufalin or its derivatives or its pharmaceutically acceptable salts or substances with bufalin or its derivatives or its pharmaceutically acceptable salts as the active ingredient in the preparation of products for anti-fish viruses; the fish viruses are infectious hematopoietic necrosis virus and / or infectious pancreatic necrosis virus. In order to screen candidate antiviral drugs against IHNV and IPNV, 1,483 traditional Chinese medicine compounds were screened from a traditional Chinese medicine monomer library. The results show that bufalin has antiviral activity against IHNV and IPNV both in vitro and in vivo, and is a potential candidate drug for anti-IHNV and IPNV infections.

The invention relates to the technical field of molecular biological detection, and discloses a composition and a kit for detecting pathogenic microorganisms of fish and application of the composition and the kit. The composition provided by the invention comprises a primer pair aiming at a target sequence of at least one of infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, flavobacterium psychrophilum, aeromonas salmonicida and aeromonas hydrophila; according to the method, multiple fish pathogenic microorganisms in a sample can be detected and confirmed only through one-time PCR, the detection efficiency is greatly improved, the detection cost is reduced, and the method has good specificity, high detection sensitivity, convenience in operation and excellent stability.

The invention discloses an application of MG-132 in preparation of a medicine for resisting nervous necrosis virus. Researches show that the MG-132 can significantly reduce cytopathic effects caused by nervous necrosis virus infection, reduce the number of positive cells expressing RGNNV capsid proteins, and also can significantly reduce the mRNA transcriptional level and protein expression level of RGNNV key genes and the copy number of viral genome RNA; and along with the prolonging of RGNNV infection time, the MG-132 can also significantly inhibit virus gene transcription, protein synthesis and genome replication, and reduce virus titer. Therefore, the virulence of RGNNV on cells can be remarkably reduced through MG-132 treatment, the effect of resisting nervous necrosis virus is achieved, the cytotoxicity is low, and the safety is good. The invention provides a new drug intervention strategy for prevention and treatment of nervous necrosis virus infection, and has important application value for prevention and treatment of NNV related diseases in aquaculture.

Orthotospovirus virions travel through the thrips foregut and enter midgut epithelial cells through the interaction between virus glycoproteins and cellular receptors with several protein motifs thought to be involved in the interaction. Single, double and triple mutant polypeptides in the soybean vein necrosis virus (SVNV) / Neohydatothrips variabilis system are provided herein and several are shown to block viral transmission from the thrips to the soybean plants. Methods for inhibiting viral transmission using these polypeptides or constructs comprising polynucleotides encoding peptides are also provided herein.

The present invention discloses a method for constructing a mixed - infected aquatic animal model. A composition containing 2×10 6 TCID 50 ~3.33×10 6 TCID 50 of nervous necrosis virus and 1×10 6 cfu / mL~1×10 7 cfu / mL of Vibrio is used to infect Perciformes fish with a body length of 4 cm - 9 cm until infection symptoms appear, thus obtaining a mixed - infected aquatic animal model. The method provided by the present invention can quickly establish a multi - pathogen mixed infection of nervous necrosis virus and Vibrio in animals, showing typical mixed - infection symptoms, providing a new technical option for those skilled in the art to solve the problem of frequent multi - pathogen mixed infections in aquaculture, and contributing to the healthy development of the aquaculture industry.

The present application relates to a circRNA vaccine for anti-infectious hematopoietic necrosis virus, the circRNA vaccine is a covalently closed circular structure, comprising an internal ribosome entry site, a G protein sequence of the infectious hematopoietic necrosis virus, and a Kozak sequence located between the internal ribosome entry site and the G protein sequence of the infectious hematopoietic necrosis virus;The circRNA vaccine comprises a cyclization connection sequence formed by a fish tRNA Leu I type intron self-splicing;The internal ribosome entry site is one or more of the following: coxsackievirus 3 IRES, infectious pancreatic necrosis virus IRES, encephalomyocarditis virus IRES, Ythdc2 IRES, and hepatitis C virus IRES;The amino acid sequence of the G protein of the infectious hematopoietic necrosis virus has at least 90% sequence identity with the amino acid sequence encoded by SEQ ID NO. 1.

The invention discloses antisense RNA for targeted inhibition of infectious spleen and kidney necrosis virus, a recombinant vector, a kit and application of the antisense RNA. The antisense RNA is selected from at least one of antisense RNA1 and antisense RNA2; wherein the nucleotide sequences for coding the antisense RNA1 and the antisense RNA2 are respectively as shown in SEQ ID NO.1-2. The invention also discloses a preparation method of the antisense RNA1 and the antisense RNA2. In order to solve the problem that the ISKNV lacks an effective prevention and control means, the long-chain antisense RNA is designed by selecting a virus ORF69 (non-functional protein) and a membrane protein Allo46 as double targets for the first time, the antisense RNA designed by the invention can directly block virus proliferation, the long sequence design tolerates target mutation, and meanwhile, double-target synergistic inhibition can improve the efficiency and delay virus escape. The recombinant vector is introduced into the mandarin fish fries to prepare the anti-ISKNV mandarin fish fries, so that virus outbreak triggered by water temperature can be prevented and controlled from the source, and the economic loss of culture is effectively reduced.