CN102899295B - Duck hemorrhagic ovaritis virus - Google Patents

Abstract

The invention relates to a duck hemorrhagic ovaritis virus (DHOV). Tissue samples of clinical diseased egg ducks are finely separated by SPF (specific pathogen free) chicken embryo fibroblasts (CEF) to obtain a DHOV-JN, and the isolate can proliferate on CEF and produce a typical cytopathoenic effect (CPE), and can also proliferate one MDCKMadin-Darby canine kidney, Vero, BHK-21(baby hamster kidney-21), ST (Swine Testis) and Marc-145 cell monolayer and produce more consistent CPE, and the tilter of the isolate is 105.5TCID5(Tissue Culture Infectious Dose)50/0.1mL; the isolate belongs to a member of flaviviridae, yellow disease and Ntaya virus group; and the strain can develop corresponding RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection/diagnosis methods (reagents) based on the study, and can also develop vaccines and other biological products for clinical prevention of the disease.

Description

One strain duck hemorrhagic ovaritis virus

Technical field

The present invention relates to a strain duck hemorrhagic ovaritis virus.This virus separates from a kind of new duck disease.Belong to animal virology field.

Background technology

Since at the beginning of 2010 4 months, the kind duck in the provinces such as China Zhejiang, Jiangsu, Shandong, Fujian, Hebei, Beijing, egg duck occur a kind ofly to decline rapidly, even stop production and the dead epidemic disease as feature in various degree take laying rate, be commonly called as kind of (egg) duck lay eggs rapid drawdown, ovarian hemorrhage disease, cause the initial called after duck flavivirus of virus (the Duck flavivirus virus of this disease, DFV), now substantially unified called after duck hemorrhagic ovaritis virus (Duck hemorrhagic hvaritis virus, DHOV).Each kind egg duck, kind duck etc. all have generation, but are mainly seen in out laying ducks.After laying ducks morbidity, occur that the laying rate of long-time (40 ~ 60d) is low, after recovering, declining appears in the fertility rate and hatchability of kind of the egg that produces.Morbidity duck food consumption significantly declines, and has loose bowels green just, and spirit is depressed, flocks together, and feather is contrary vertical, and eyeball gets deeply stuck in, One's eyesight is restrained, loses weight.There is astasia, tremble in a small amount of sick duck, difficulty in walking and abnormal posture.Major lesions is hemorrhagic ovaritis, acute nonsuppurative hepatitis and apyetous encephalitis.This disease becomes in the recent period harm China and supports one of important epidemic disease of duck industry.The ill rear 4 ~ 5d of duck group that laying rate is high is dropped rapidly to 20% ~ 30% from lay eggs peak or high laying rate, and serious 7d left and right after morbidity stops production; Just open and produced kind of (egg) duck laying rate and rise slowly or subtract egg, long-time low laying rate or occur without the peak of laying eggs.Different areas, different varieties duck group sickness rate height differs, sickness rate almost 100% in group, and case fatality rate is 0% ~ 12%.

Studying data at home and abroad, flavivirus generally can be bred on the tissues such as mouse brain, C6/36, BHK-21, Vero or cell, and in recent years, also someone inoculates instar chicken embryo on the 9th to 10 or duck embryo allantoic liquid carries out viral Isolation and proliferation.Consider that cell derived is stable, quality controllable, pollute without exogenous virus, our selected SPF source chick embryo fibroblast (CEF) conduct separates unique clone of this virus.As everyone knows, CEF cell derived is in SPF chicken embryo, and its donor background clear (containing any pathogeny), mature preparation process, method are simple, comparatively responsive to fowl source virus.Meanwhile, SPF level duck embryo source is limited, and the CEF propagative viruses of preparing with SPF chicken embryo, its every embryo output is 10 times of left and right of SPF chicken embryo, and it is higher to tire, the tiring on chicken embryo and duck embryo of tiring of chick embryo fibroblast breeding.Therefore, we select the separation and Culture of CEF cell for this virus, and obtain successfully, and strain isolated has been carried out to system identification and the analysis of E gene sequencing.

Owing to being emerging infectious disease, isolation identification, biological characteristics and the major antigen gene expression characteristics research of people to duck hemorrhagic ovaritis virus (Duck hemorrhagic hvaritis virus, DHOV) still has larger blank.

Summary of the invention

The object of the invention is by bacterial isolate in the egg duck body of dying of illness and its isolate is identified and utilized.

The technical solution used in the present invention is: gather the pathological tissues such as duck liver, ovary of dying of illness through grinding, centrifugal, carry out pathogen separation evaluation after filtering, to obtained virus isolated strain carry out cell cultures feature, electron microscopic observation, titration, doubtful cause of disease RT-PCR/PCR detection and order-checking, nucleic acid based evaluation, physicochemical property evaluations, blood clotting mensuration, serum neutralization test, animal experiment is done further evaluation.

1. this research is passed through morbidity sample preparation, the Mammals passage cells such as inoculation primary cell CEF and MDCK, in succession obtain the viral isolates with same CPE performance, through titration, electron microscopic observation, doubtful cause of disease RT-PCR/PCR detects and E gene sequencing, nucleic acid based evaluation, physicochemical property is identified, blood clotting is measured, neutralization test and animal experiment, result shows that this strain isolated is duck hemorrhagic ovaritis virus (Duckhemorrhagic hvaritis virus, DHOV), called after DHOV JN strain, belong to flaviviridae, Flavivirus, ntaya virus group members.

2. cell cultures characteristic DHOV-JN strain isolated has wider cell adapted spectrum, can not only adapt to fowl source cell (CEF), tissue (SPF chicken embryo), also can adapt to the Mammals continuous cell lines such as MDCK, BHK-21, ST, Vero, also this viral host range of side light may be wider, should further investigate this.Consider cell derived controlled, without factors such as external source pollutions, this research selects SPF source CEF as separating, go down to posterity, breed this viral cell, and obtains successfully, virus can adapt to CEF preferably, tires also higher, reaches 10 ^5.5tCID ₅₀/ 0.1mL.This is to carry out the researchs such as cause of disease evaluation, biological characteristics, vaccine to have established fabulous cell platform and Research foundation.

3. Characteristic of pathogenic bacteria has carried out in electron microscopic observation, physicochemical property, suspicious cause of disease detection, gene sequencing and evolutionary analysis, the evaluation of viral nucleic acid type, serum and research, found that: virus particle is greater than 60nm, is hexagon, has cyst membrane; This virus is to thermo-responsive (56 ℃ 15min can deactivation), to chloroform, ether and alkali sensitivity, insensitive to acid, pancreatin; Detect and find with 8 kinds of fowl source virus RT-PCR/PCR such as AIV and flavivirus RT-PCR, only can detect the flavivirus nucleic acid positive, measuring its E mrna length is 1503bp, has built the Phylogenetic tree based on flavivirus E gene, finds that this strain isolated belongs to Flavivirus ntaya virus group's member; IUDR metabolic test result confirms that the nucleic acid based of this strain isolated is RNA; This strain isolated can not be exhaled the positive serum of 8 kinds of common fowl source viruses such as intestines orphan and fowl reticuloendotheliosis to neutralize by bird flu, newcastle disease, egg-decreasing syndrome, duck hepatitis, infectious bursa of Fabricius, infectious bronchitis, kind duck.

4. the pathogenic of virus is pathogenic to bird of checking DHOV-JN strain, and we have carried out the pathogenicity of this strain to SPF chicken embryo, SPF chicken, SPF laying hen, duckling and clinical health egg duck.Found that DHOV-JN strain can make the growth of 9 age in days SPF chicken embryos significantly postpone, dissect find hemorrhage, the moulding chicken embryo of idiosome liver enlargement, present " piebald liver ".The lethal 2 age in days SPF chickens of DHOV-JN strain energy, death in general 3～5 days, mortality ratio is 30%～50%; It is poor that initial stage chicken appetite contrasts, resistance to later without impact; Dead chicken is dissected visible meninx deliquescing, the enlargement a little of kidney and spleen.But this virus does not have significant clinical impact to the SPF chicken of 50 ages in days.But this virus has lethality to squab 2 age in days ducklings, attack 2 age in days ducklings with DHOV-JN strain, experimental group is all dead, dissects and finds that hepatic disease is serious, many organ hemorrhages; Splitting laying ducks has stronger pathogenic, dissect and find, the egg duck laying rate degradation of experimental group, there is the phenomenons such as slow, the hemorrhage and uterine tube atrophy of ovarian follicle liquefaction, follicular development in ovary tissue, liver jaundice, enlargement, quality become fragile, spleen enlargement etc.The above results shows, separate and chicken, duck are all had extremely strong pathogenic from the DHOV-JN of egg duck strain, whether goose is had pathogenic because this research of timing relationship is for carrying out test, but according to Huang Xinmei (Huang Xinmei, Li Yin, Zhao Dongmin, Deng. the separation of novel light yellow viral JS804 strain and evaluation. Jiangsu agricultural journal, 354～360.) etc. 2011,27 (2): research, this virus also has similar pathogenic to goose.These results confirmations, this fowl flavivirus being newly separated to has remarkable harm to aviculture, need to strengthen the researchs such as epidemiology, etiology, immunology.

In sum:

1. this strain virus is duck hemorrhagic ovaritis virus (Duck hemorrhagic hvaritis virus, DHOV) JN strain, this strain virus is preserved in China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center of on March 22nd, 2012 this strain isolated being delivered to No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCCNo.5907.

2. this isolated strain principal feature:

(1) the sick duck that the duck hemorrhagic ovaritis virus CGMCCNo.5907 strain virus separating is called duck hemorrhagic ovaritis from a kind of clinical symptom gathers liver, ovary tissue, separates and obtains with SPF source CEF;

(2) duck hemorrhagic ovaritis virus CGMCC No.5907 strain virus can be bred on CEF, MDCK, Vero, BHK-21, ST and Marc-145 monolayer cell, and can cause these cells and produce more consistent CPE;

(3) recording with SPF source CEF that CGMCC No.5907 strain virus tires is 10 ^5.5tCID ₅₀/ 0.1mL;

(4) CGMCC No.5907 strain virus inoculation chicken/go out typical duck hemorrhagic ovaritis with duck reproducible.

3. duck hemorrhagic ovaritis virus CGMCCNo.5907 strain virus is for the preparation of duck hemorrhagic ovaritis inactivated vaccine.

4. duck hemorrhagic ovaritis virus CGMCC No.5907 strain virus is used as and attacks poison strain in duck hemorrhagic ovaritis inactivated vaccine efficacy test.

Embodiment

1. virus separates

(1) pathological material of disease is processed the inventor by gathering in the sick duck of certain egg duck field egg drop reduction, and every part of pathological material of disease mainly contains the pathological tissues such as part ovary tissue, liver of the egg duck that dies of illness.Aseptic technique clip 5.0g tissue, add a small amount of MEM(containing penicillin, Streptomycin sulphate 1000U/mL), be ground to pasty state, add again the MEM of 4 times of volumes to be diluted to suspension liquid, sample is-80 ℃ and room temperature multigelation 3 times, again through 4 ℃, the centrifugal 10min of 12000r/min, collect the disposable filter Entkeimung of supernatant liquor 0.22 μ m, finally packing-80 are ℃ frozen for subsequent use.

(2) chick embryo fibroblast (CEF) separates with 9 age in days SPF chicken embryo (SPF chicken embryos, SPF laying hen is all purchased from the logical laboratory animal technology of Beijing Cimmeria dimension company limited), under aseptic condition, decaptitate, pawl, internal organ shred with ophthalmology Jian, then adding 0.25% pancreatin digests, after liquid nature muddiness, adding MEM substratum stops, after 6 layers of filtered through gauze with sterilizing, shake up and be distributed into ordinary cells culturing bottle again, put 37 ℃, 5%CO ₂in incubator, cultivate.Amount by 5% after CEF is paved with individual layer accesses the supernatant liquor of organizing through pre-treatment Entkeimung, continue to cultivate and observe to 72h, no matter have or not pathology all to gather in the crops, then continue blind passage to F5 generation, until cytopathy occurs and tend towards stability, results culture-80 ℃ save backup.

Get the pathological material of disease of-80 ℃ of preservations and process supernatant liquor inoculation CEF, passed continuously for 7 generations, reach F4 for starting to occur CPE through observing, the 50h CEF in F5 generation starts to occur CPE, obviously (75% cell has pathology) of pathology when 65h, CPE main manifestations is: the contracting of cell circle, fusion, and draw in the net gradually, disintegration then comes off.See Figure 1,2.Tend towards stability for starting CPE from F5.

(3) propagation of virus on other cells inoculates respectively with-80 ℃ of frozen each 1mL of viral CEF F5 culture thing the MDCK(dog kidney passage cell that has covered with individual layer), Vero(African green monkey kidney cell), BHK-21(breast hamster nephrocyte), ST(pig testis cell) and Marc-145(African green monkey kidney cell clonal cell line) (above passage cell is the preservation of the applicant laboratory), after effect 1～2h, put 37 ℃, 5%CO ₂incubator in carry out cultured continuously observation, record the CPE situation of various cells.Passage fertilizing method is with basic identical by chick embryo fibroblast (CEF) method.

Inoculate respectively MDCK, Vero, BHK-21, ST and Marc-145 monolayer cell for isolate with JN strain CEF F5, all can produce obvious CPE, pathology performance is basically identical with the feature that virus causes CEF.

2. the evaluation of isolate

(1) the CEF cell newly preparing is got in titration, inoculation 96 porocyte culture plates, and every hole inoculating cell suspension 100 μ L, after cell grows up to good individual layer.Get the JN strain CEF F5 that is placed in-80 ℃ of preservations and carry out 10 times of serial dilutions to 10 for isolate ^-7, get each dilution virus liquid, be inoculated in respectively on well-grown CEF cell 96 porocyte culture plates, each titre is inoculated 8 holes, and every hole 100 μ L set up each 8 holes of positive control and negative control simultaneously.Tissue Culture Plate is put to 37 ℃, 5%CO ₂cultivate 3～5d, there is the situation of pathology in observation of cell every day, occurs that CPE person is judged to infection, calculates TCID by Reed-Muench method ₅₀, result is measured the tire (TCID of JN strain CEF F5 for isolate ₅₀) be 10 ^5.5/ 0.1mL.

(2) electron microscopic observation is got JN strain CEF F5 and is done negative staining electron microscopic observation for isolate, found that JN strain CEF F5 is greater than 60nm, is hexagon, has cyst membrane for virus particle, sees Fig. 3.

(3) neutralization test is undertaken by sessile antibody one virus dilution method.Getting the JN strain CEF F5 of-80 ℃ of preservations for isolate, is 100TCID according to viral level measurement result by virus liquid dilution to be checked ₅₀/ 0.1mL, mixes viral dilution liquid respectively with isopyknic AIV, NDV, EDSV, DHV, IBDV, IBV, MDRV and REV standard positive serum (all purchased from China Veterinery Drug Inspection Office) concussion, to 37 ℃ of effect 2h, shake during this time 2～3 times.After effect finishes, neutralizer is inoculated to 96 hole CEF cell plate, every hole 100 μ L set up positive control and normal cell contrast simultaneously.Tissue Culture Plate is put to 37 ℃, 5%CO ₂cultivate 5～7d, there is situation the detailed record of pathology in observation of cell every day.Press Reed-Muench method and calculate TCID ₅₀, result has no any a serum and is neutralized, and illustrates that JN strain does not belong to these common fowl source virus, need to further identify.

(4) nucleic acid based evaluation by 5 '-iodine IUDR that final concentration is 50 μ g/mL processed after DHOV-JN strain CEF F5 culture thing, records its TCID ₅₀be 10 ^5.3/ 0.1mL, untreated control group TCID ₅₀be 10 ^5.5/ 0.1mL, illustrates that 5 '-iodine IUDR can not suppress the propagation of JN strain isolated in CEF, and this virus is RNA viruses.

(5) doubtful cause of disease RT-PCR/PCR detects login GenBank, download 51 representative flavivirus complete genome sequences, after its E gene of DNAstar software analysis, utilized Primer5.0 software design 1 pair detect primer, FLVTF:5'-TTACCATGGACAGGGTCATCA-3', FVTR1:5'-TCCAATTGTGCTCCCACTTCT-3', expection object fragment is 549bp; Increase and electrophoresis detection (Fig. 4) according to preliminary this disease and other the 8 kinds viral RT-PCR detection methods (in table 1) of setting up in the present invention.

Table 1 separates poison and 8 kinds of doubtful fowl source virus PCR testing conditions

(Su Jie, waits .PCR method to detect egg drop syndrome virus for [1] Wang Aihua, Li Quanfu. Jouranl of Agricultural University of Hebei, 2006,29 (4): 88 ~ 91; [2] Cheng Anchun, Wang Mingshu, letter flood one, etc. the foundation of I type duck viral hepatitis virus RT-PCR detection method. Chinese veterinary science, 2007,37 (1): 38 ~ 42; [3] Su Xiaoou, Zhao Deming. the foundation of chicken infectivity bursa of Fabricius virus RT-PCR detection method. animal medicine progress, 2008,29 (9): 5 ~ 8; [4] Cheng Xiaoxia, king encourages, Zhu little Li, etc. the foundation of avian infectious bronchitis virus RT-PCR detection method. Chinese agronomy circular 2009,25 (5): 14 ~ 17; [5] Hu Qilin, Lin Fengqiang, old few warbler, etc. application RT-PCR technology for detection muscovy duck reovirus. Chinese animal doctor's journal, 2004,24 (3): 231 ~ 232; [6] Zhai Xinyan. the foundation of the detection of Reticuloendotheliosis virus and animal infection modal thereof. Changchun: the .2007. of Jilin University)

(6) primer pair of the amplification of DHOV E full length gene and a pair of flavivirus E gene that can increase of the another design of order-checking, FLVEF:

5'-GTTAATAGCCCCAGCGTACAGC-3', FLVER:5'-CTAGCCAAGTCGATTGAGCACC-3', two pairs of primers transfer to Shanghai Sheng Gong biotechnology company limited synthetic.RT-PCR result has amplified band to occur.The recovery object fragment that obtains increasing checks order, and obtains the gene order (seeing sequence 3) of 1554bp through splicing, and comparison finds that the E full length gene of DHOV JN strain is 1503bp.Find on inspection, the JM1 of this fragment and domestic acquisition, JM2, the E mrna length of the local epidemic strain of PD equates, be 1503bp, the E that represents strain with other 51 flaviviruss of now having published is gene constructed Phylogenetic tree (as Fig. 5), result shows that the JN strain that the present invention separates and identify in detail belongs to flaviviridae, Flavivirus, ntaya virus group members, genetic affinity (E gene nucleotide series homology is 87.2%～91.8%) recently with tembusu virus, with Baiyang Lake virus (BYDV, Su J, Li S, Hu X, Yu X, Wang Y, et al. (2011) Duck Egg-Drop Syndrome Caused by BYD Virus, a NewTembusu-Related Flavivirus.PLoS ONE 6 (3): e18106.) homology of E gene is 99.2%～99.6%.

(7) viral physicochemical property identifies that all taking DHOV-JN strain CEF F5 tests, specific as follows.

1) oven test is got the DHOV-JN strain CEF F5 virus liquid that is placed in-80 ℃ of preservations, puts respectively in 50,55,60,65,70,75,80 ℃ of water-baths and acts on after 45min, mensuration heat treated virus liquid and the not TCID of heat treated virus liquid ₅₀.Meanwhile, this virus liquid is placed in to 56 ℃ of effect different times, measures and process virus liquid and the not viral level detection discovery of heat treated virus liquid, this strain virus is at 50 ℃ of water-bath effect 45min, its TCID ₅₀decline nearly 10 times; 55 ℃ of water-bath effect 45min, TCID ₅₀decline more than 100 times; After 60 ℃, 65 ℃, 70 ℃, 75 ℃ and 80 ℃ of water-bath effect 45min, viral infection completely loses (table 2).56 ℃ of effect 15min, virus has been lost infectivity, and there is (table 3) without CPE in inoculation CEF cell, illustrates that this virus has stronger susceptibility to heat.

TCID after table 2 DHOV-JN strain acts on 45min under differing temps ₅₀measurement result

Table 3 DHOV-JN strain is TCID after 56 ℃ of effect different times ₅₀measurement result

2) pH resistance test is got the DHOV-JN strain CEF F5 virus liquid that is placed in-80 ℃ of preservations, adjusts after pH to 3.0,4.0,5.0,6.0,37 ℃ of effect 1h with 0.1mol/L HCl solution, uses 7.5%NaHCO ₃adjust pH to 7.2; With 0.1mol/L NaOH, pH is adjusted to 8.0,9.0,10.0,37 ℃ and acts on after lh, adjust pH to 7.2 with 0.1mol/L HCl solution, measure the TCID that processes virus liquid ₅₀, establish untreated virus liquid simultaneously and compare.Detect and find through soda acid sensitivity test, DHOV-JN strain virus can tolerate sour effect, and to alkali sensitivity, at pH higher than 8.0 o'clock, its infectivity significantly decline (table 4).

TCID after table 4DHOV-JN strain virus acts in acid-base environment ₅₀measurement result

Unit: (TCID ₅₀/ 0.1mL)

3) the DHOV-JN strain CEF F5 virus liquid that is placed in-80 ℃ of preservations is got in the sensitivity test of chloroform, be 5% analytical pure chloroform to adding final concentration in virus liquid, put 4 ℃ of vibration 60min, subsequently with the centrifugal 5min of 3000rpm/min, draw supernatant liquid, measure virus liquid TCID ₅₀, establish untreated virus liquid simultaneously and compare.With the chloroform processing DHOV-JN strain virus 60min of final concentration 5%, after testing, the TCID of chloroform treatment group virus liquid ₅₀/ 0.1mL only reaches l0 ^1.5, and control group is 10 ^5.5, illustrate that this virus is to chloroform sensitivity.

4) the DHOV-JN strain CEF F5 virus liquid that is placed in-80 ℃ of preservations is got in ether sensitization test, be 20% analytical pure ether to adding final concentration in virus liquid, put 4 ℃ of vibration 60min, subsequently with the centrifugal 5min of 3000rpm/min, draw supernatant liquid, measure virus liquid TCID ₅₀, establish untreated virus liquid simultaneously and compare.With the ether processing DHOV-JN strain virus 60min of final concentration 20%, after testing, the TCID of ether treatment group virus liquid ₅₀/ 0.1mL only reaches l0 ^1.25, and control group is 10 ^5.5, illustrate that this virus is to ether sensitivity.

5) pancreatin stability test is got the DHOV-JN strain CEF F5 that is placed in-80 ℃ of preservations for virus liquid, be 0.5% pancreatin solution to adding final concentration in virus liquid, put 37 ℃ of oscillation incubation 10min, 30min, 60min, add subsequently containing the complete cell culture fluid of foetal calf serum and stop, pancreatin is removed in filtration, inoculate the CEF handling well 96 porocytes and cultivate, measure virus liquid TCID ₅₀, establish untreated virus liquid simultaneously and compare.

6) blood clotting measure according to " animal virology (second edition) " (Yin Zhen, Liu Jinghua chief editor. animal virology (second edition). Science Press, Beijing, 1997) described in method carry out, DHOV-JN strain CEF F5 can not aggegation chicken and the red corpuscle of cavy after measured.

3. animal experiment

Be flavivirus feminine gender for examination animal (purchased from Beijing Fu Zhong development in science and technology company limited) through the RT-PCR detected result of setting up.Carry out pathogenicity by CGMCCNo.5907 strain (DHOV-JN strain) virus, specific as follows:

(1) on chick embryo development affect 10 pieces of instar chicken embryos on the 9th, be divided into experiment and control group, 5 pieces every group.Get the DHOV-JN strain CEF F5 of-80 ℃ of preservations for virus liquid, inoculation test group, dosage is 0.1mL/ embryo, the PBS of control group inoculation pH 7.2, dosage is also 0.1mL/ embryo.Two groups of chicken embryos continue hatching and cultivate observation, and the developmental state of observing twice chicken embryo every day, observes 3～5 days, and detailed record.Inoculate after 9 age in days SPF chicken embryos with DHOV-JN strain, compared with the control, this virus has remarkably influenced to the growth of chicken embryo as seen, connects malicious embryo development and seriously lags behind, and after dissecting, visible test group idiosome has the considerable changes such as hemorrhage, liver enlargement, piebald liver.See Fig. 6.

(2) to 20 of the pathogenic use 2 age in days SPF chickens of 2 age in days SPF chickens, be divided at random two groups, 10 every group.Inoculation group is got the DHOV-JN strain CEF F5 of-80 ℃ of preservations for virus liquid, and leg muscle is injected 2 age in days SPF chickens, and 0.2ml/ only.The PBS of control group injection pH7.2,0.2ml/ only.Observe 14 days.The lethal 2 age in days SPF chickens of DHOV-JN strain energy, death in general 3～5 days, mortality ratio is 3/10～5/10, it is poor that initial stage chicken appetite contrasts, resistance to later without impact.Dead chicken is dissected visible meninx deliquescing, the enlargement a little of kidney and spleen.

(3) to the Virulence detection of 50 age in days SPF chickens with 20 of 50 age in days SPF chickens, be divided at random two groups, 10 every group.Inoculation group is got the DHOV-JN strain CEF F5 of-80 ℃ of preservations for virus liquid, leg muscle injection, and 0.5ml/ is only.The PBS of control group injection pH7.2,0.5ml/ only.Observe 14 days.Raise altogether result chicken appetite and spirit and control group no significant difference 14 days.Have no dead, cut open inspection there are no unusual phenomenon.

(4) Virulence detection of SPF laying hen is used to 30 of SPF laying hens, be divided at random two groups, 15 every group.Inoculation group is got the DHOV-JN strain CEF F5 of-80 ℃ of preservations for virus liquid, the injection of SPF laying hen leg muscle, and 2ml/ is only.The PBS of control group injection pH7.2,2ml/ only.In inoculating latter the 3rd, 5,7,9,11 days, each group is cutd open respectively 1 of inspection, observation pathology situation.The results are shown in Table 5.

The pathogenicity test results of table 5DHOV-JN strain to SPF laying hen

(5) to 20 of 2 age in days sheldrakes for the pathogenicity of 2 age in days sheldrakes, be divided at random two groups, 10 every group.Inoculation group is got the DHOV-JN strain CEF F5 of-80 ℃ of preservations for virus liquid, and leg muscle is injected 2 age in days sheldrakes, and 0.2ml/ only.The PBS of control group injection pH7.2,0.2ml/ only.Observe 14 days.Connect after poison the 3rd day duckling and show One's spirits are droopingly, flock together, have slight diarrhoea.Connect poison latter the 4th day, 10 ducks that connect poison group are all dead, and contrast is all good for and is lived.Connect poison group duck body weight obviously light compared with control group.Cut open inspection visible, be atrium hyperemia, kidney enlargement, hyperemia, cerebral tissue has liquefaction phenomenon.

(6) the pathogenic use of splitting laying ducks is opened 20 of laying duckses, is divided at random two groups, 10 every group.Laying ducks is opened in the injection of inoculation group leg muscle.The PBS of control group injection pH7.2,2.0ml/ only.Viewing duration to process of the test in dead egg duck cut open inspection, egg duck dead in process of the test is cutd open inspection after off-test, the pathological material of disease (liver, ovary tissue) of acquisition test egg duck also carries out etiology detection.In table 6.

The pathogenicity test results of table 6 DHOV-JN strain to the negative egg duck of flavivirus

(7) protest test

Inactivated vaccine preparation uses the CGMCC No.5907 cell toxicant of preserving after CEF expansion is numerous, reclaim whole cell cultures, low-speed centrifugal is removed cell debris after the abundant deactivation of formaldehyde, make inactivated vaccine with the ISA206VG adjuvant emulsification according to a certain ratio that match hectogram company produces, save backup through after the assay was approved-4 ℃.

Get 20 of negative clinical health egg ducks, be divided at random 2 groups, 10 every group.Get the inactivated vaccine of above preparation, through leg muscle vaccinate 0.5mL/ only, booster immunization 1 time after 14 days.In two exempt from latter 7 days to every group of total Test duck together with control group, with CGMCC No.5907 strain virus (10 ^6.5tCID ₅₀/ ml) attack every intramuscular injection 1ml, isolated rearing.Attack Continuous Observation 14 days after poison, the spirit of viewing test duck, search for food, drink water and the situation of laying eggs, and within latter 14 days, no matter whether fall ill and all cut open and kill in attacking poison, gather tissue sample and carry out RT-PCR detection.

Attack Continuous Observation 14 days after poison, immune group is attacked spirit, drinking-water and the laying rate no abnormality seen of poison group egg duck, and contrast is attacked the egg duck of poison group and attacked and within the 3rd day, start food consumption after poison and significantly decline, and spirit is depressed, flocks together, and has loose bowels individually green just, and feather is contrary vertical; The laying rate that starts for the 5th day of attacking after poison sharply declines.

Accompanying drawing explanation

Fig. 1 is the normal chick embryo fibroblast (CEF) of virus inoculation not

Fig. 2 inoculates the chick embryo fibroblast (CEF) that rear virus produces cytopathic effect (CPE) afterwards

Fig. 3 viral particle morphology 3-1: single virus particle; Fig. 3-2: multiple virus particle

Fig. 4 specificity experiment M, DNA 2000Marker; 1, positive control; 2, negative control; 3～10, AIV, NDV, EDSV, DHV, IBDV, IBV, MDRV, REV; 11, JN strain isolated

The blue circle of the Phylogenetic tree of Fig. 5 based on flavivirus E gene not of the same race is japanese encephalitis virus group, and red circle is ntaya virus group, and green circle is dengue virus group.

Fig. 6 DHOV-JN strain on the impact of 9 age in days SPF chick embryo developments in the description in table 5.

Microorganism involved in the present invention:

Duck hemorrhagic ovaritis virus DHOV-JN strain, this strain virus is preserved in China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center of on March 22nd, 2012 this strain isolated being delivered to No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCC No.5907.

Positive effect of the present invention

The present invention relates to a strain duck hemorrhagic ovaritis virus (Duckhemorrhagic ovaritis, DHOV).Carefully be separated to a strain DHOV-JN from organizing pathological material of disease of clinical onset egg duck with SPF source chick embryo fibroblast, this strain isolated can be bred and can be caused it and produce typical cytopathy on CEF, also can on MDCK, Vero, BHK-21, ST and Marc-145 monolayer cell, breed and occur more consistent CPE, recording with CEF that strain isolated tires is 10 ^5.5tCID ₅₀/ 0.1mL; This strain isolated belongs to flaviviridae, jaundice genus, ntaya virus group's member; This strain can this research for the corresponding RT-PCR detection/diagnostic method of foundational development (reagent), also can develop for these sick biological products such as vaccine for clinical prevention.

Note: the present invention about testing the main agents relating to is: Trizol LS Regent, DNAzol are purchased from Invitrogen company; MEM, DMEM liquid nutrient medium, import foetal calf serum etc. are all purchased from Gibco; The reagent such as 5-iododeoxyuridine (5-IUDR), chloroform, hydrochloric acid, sodium hydroxide, formaldehyde solution are domestic analytical pure; Complete PCR reaction reagent, the DL 2000DNA Marker such as import ISA206VG adjuvant (match hectogram company) AMV ThermoScript II, Rnase-Inhibitor, rTaq are all purchased from precious biological (Dalian) Engineering Co., Ltd; DNA glue reclaims test kit purchased from Shanghai Sheng Gong biotechnology company limited.

Claims (4)

1. a strain duck hemorrhagic ovaritis virus, it is characterized in that this strain virus is duck hemorrhagic ovaritis virus DHOV-JN strain, this strain virus is preserved in China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center of on March 22nd, 2012 this strain isolated being delivered to No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCCNo.5907; This strain virus has following characteristics:

(1) the sick duck that duck hemorrhagic ovaritis virus CGMCCNo.5907 strain virus is called duck hemorrhagic ovaritis from a kind of clinical symptom gathers liver, ovary tissue, separates and obtains with SPF source CEF;

(2) duck hemorrhagic ovaritis virus CGMCCNo.5907 strain virus can be bred on CEF, MDCK, Vero, BHK-21, ST and Marc-145 monolayer cell, and can cause these cells and produce more consistent CPE;

(3) recording with SPF source CEF that CGMCCNo.5907 strain virus tires is 10 ^5.5tCID ₅₀/ 0.1mL;

(4) CGMCCNo.5907 strain virus inoculation chicken and/or duck reproducible go out typical duck hemorrhagic ovaritis.

2. the application of duck hemorrhagic ovaritis virus as claimed in claim 1 in preparation duck hemorrhagic ovaritis inactivated vaccine.

3. duck hemorrhagic ovaritis virus as claimed in claim 1 is attacked the application of poison strain in preparation duck hemorrhagic ovaritis inactivated vaccine efficacy test.

4. duck hemorrhagic ovaritis virus as claimed in claim 1 is in the application of detect/diagnosis of preparation RT-PCR reagent.

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