KR20120006164A - New attenuated genotype Newcastle disease virus vaccine and Newcastle disease virus vaccine comprising the same - Google Patents

Abstract

The present invention relates to a Newcastle disease virus vaccine that can prevent Newcastle disease (ND), a malignant infectious disease of chickens, and a Newcastle disease virus vaccine comprising the same. More specifically, the nucleic acid sequence of the virus F protein is isolated from domestic poultry ducks. The present invention relates to a Newcastle Disease Virus Vaccine strain belonging to genotype d (Id) of Genotype I of class 1 in Phylogenetic analysis and a Newcastle Disease Virus vaccine comprising the same.

Description

New subtype vaccine strain of lentogenic Newcastle disease virus and the viral vaccine comprising

The present invention relates to a Newcastle disease virus vaccine that can prevent Newcastle disease (ND), a malignant infectious disease of chickens, and a Newcastle disease virus vaccine comprising the same. More specifically, the nucleic acid sequence of the virus F protein is isolated from domestic poultry ducks. The present invention relates to a Newcastle Disease Virus Vaccine strain belonging to genotype d (Id) of Genotype I of class 1 in Phylogenetic analysis and a Newcastle Disease Virus vaccine comprising the same.

Newcastle disease (ND) is a representative malignant infectious disease in poultry that is fast and highly infectious in chickens. Since ND was first reported in Indonesia, the United Kingdom, and Korea in the early 1920s, it continues to occur in many countries, including Asia, Africa, and Europe. The disease causes severe respiratory symptoms, digestive symptoms and high mortality when infected with chickens. In spawning chickens, severe egg production rates are severely reduced and abnormal malformation increases, leading to a significant deterioration in poultry productivity. In particular, if ND occurs in a poultry farm that is not immune, most chickens in the farm can be killed within two weeks.

Newcastle disease virus (NDV), the causative agent of ND, is the Avian paramyxovirus type 1 (APMV-1) in the Avulavirus family belonging to the family Pararamyxoviridae. .

NDV is classified into pathogenic types as velogenic, mesogenic and lentogenic, depending on the degree of damage in chickens. Readily NDV is a virus that causes very severe respiratory symptoms and high mortality in chickens.Nerotropic velogenic, which causes severe gastrointestinal lesions such as severe diarrhea and severe neurological symptoms. Are divided into Medium NDV has low chicken mortality and severely causes respiratory symptoms. Because attenuated NDV has little or no chicken mortality and very low or no disease, some of the attenuated viruses are used as vaccines to prevent Newcastle disease.

NDV has only a single serotype worldwide, but genetically, there are a variety of genotypes or subgenotypes. NDV genotypes are largely classified into type 1 (class 1) and type 2 (class 2), of which the genotype viruses belong to type 2 causing damage in chickens. More specifically, type 2 consists of at least nine genotypes (types I to IX) to date. Genotype I and genotype II are mostly attenuated NDVs, mostly isolated from wild birds or avian species other than chicken and causing no damage to chickens. NDV, which belongs to genotype III to genotype IX, is mainly isolated from chickens and is predominantly caused by severely damaging viruses in chickens. Most of the genotypes of NDV, which have been seriously damaging the poultry industry in Asian countries including Korea, are genotype VII.

The best way to reduce or prevent the damage caused by ND in chickens is to inoculate the chickens with a prophylactic vaccine. Attenuated NDV, which does not cause damage in chickens, is propagated in chicken eggs, and then the virus form that maintains the infectivity of the virus (live poison vaccine) or the virus is removed by formalin or beta propiolactone (sotoxin vaccine). Prophylactic vaccine preparation is a common method.

Zadok vaccine is prepared by mixing the infectious NDV with gel or oil to enhance the immune response. The usual application method is injection into the chicken muscle three to four weeks before the chicken begins laying eggs. To inoculate. Live vaccines are mainly used to prevent ND damage in young chickens. A common application is to add live vaccines to water to feed the chickens (negative) or spray them with a nebulizer to inhale (spray) the sprayed vaccine particles while the chicken is breathing.

NDVs commonly used to make ND vaccines are attenuated NDVs belonging to genotype I or genotype II of type 2. Vaccine viruses corresponding to NDV genotype I are representative of Ulster 2C and V4 strains. Vaccine viruses belonging to NDV genotype II are representative of La Sota, B1 Hitsner and VG / GA.

It is most preferable that these vaccine viruses use a viral strain that causes a strong immune response in chickens and shows no pathogenicity. In most cases of NDV, the stronger the immune response in chickens, the more adverse the pathogenicity of chickens. In particular, La Sota or B1, which is commonly used as a live vaccine, has a strong immune response when inoculated in young chickens within 2 weeks of age, but it has a strong respiratory affinity, resulting in severe respiratory side effects and even death by some individuals. do. Therefore, when vaccinating in young chickens, it is desirable to use NDV as a vaccine virus to form a high immune response while minimizing the pathogenicity in chickens.

In addition, the vaccine virus currently used in domestic poultry is a virus isolated from bird species that are raised or inhabited abroad. Therefore, there is a need to increase the immunity to NDV using a virus isolated from poultry farming in Korea.

Therefore, the present inventors have discovered that there is a new genotype of NDV present in Korea that is genetically different from vaccine viruses currently used in the process of isolating and analyzing NDV from domestically raised poultry ducks. . In addition, the inventors analyzed the isolated NDV, and the new genotype NDV is proliferative in both the respiratory and digestive mucosa without exhibiting pathogenicity in young chickens. The present invention was completed after confirming that the prophylactic effect was exhibited.

Accordingly, it is an object of the present invention to provide a new genotype NDV vaccine strain which is different from the NDV genotypes that are conventionally used as vaccine viruses.

In order to achieve the above object, the present invention provides Newcastle disease virus vaccine strain of Accession No. KCTC 11718BP.

In another aspect, the present invention provides a vaccine for Newcastle disease, including Newcastle disease virus vaccine strain.

The Newcastle disease virus vaccine of the present invention is an attenuated Newcastle disease virus without pathogenicity in chickens, and since the pathogenicity was not reversed, the safety of chickens was confirmed. In addition, the vaccine showed affinity not only in the respiratory tract but also in the digestive tract, and because of these characteristics, the chickens showed excellent immunity when sprayed (respiratory immunity) or inoculated by oral and instillation (digestive immunity). It showed superior protective effect against other vaccines currently on the market.

Figure 1 shows the segment region predicted amino acid sequence of the F protein of NDRL09-01 strain according to the present invention. The boxed part shows the fragment amino acid sequence of the F protein.
Figure 2 is a phylogenetic analysis results based on the F protein gene sequence of NDRL09-01 strain according to the present invention. The content in parentheses is the accession number of the genebank, and the arrow indicates the vaccine strain of the present invention.

The present invention relates to a new attenuated genotype Newcastle disease virus vaccine strain. The present inventors named the new attenuated New Cattle Disease virus isolated from domestic poultry ducks as the New Cattle Disease virus "NDRL09-01", and was deposited on June 25, 2010 at the Korea Institute of Bioscience and Biotechnology, and assigned the accession number KCTC 11718BP. .

The attenuated Newcastle disease virus according to the present invention is a Newcastle disease virus vaccine strain belonging to genotype d (Id) of genotype I of type 1 in phylogenetic analysis based on the nucleic acid sequence of the virus F protein. When inoculated into the chicken's respiratory and digestive mucous membranes to grow locally to form mucosal immunity. In addition, it is excellent for preventing pathogenic NDV without showing pathogenicity in chickens, and when it is inoculated in young chickens, it grows locally in both the respiratory and digestive organs of chickens to form mucosal immunity. desirable.

The present invention also relates to Newcastle disease virus vaccines comprising the new attenuated genotype Newcastle disease virus vaccine strain.

The Newcastle disease virus vaccine according to the present invention can be prepared by a method commonly known in the art, and the vaccine containing the Newcastle disease virus alone or in combination with other disease vaccine strains, the poisoned vaccine inactivated the NDV, and the attenuated Newcastle disease virus Since the gene can be produced in various forms such as NDV subunit vaccine, vector vaccine, chimera vaccine or DNA vaccine produced using the gene, its utilization is very wide. The vaccine may include veterinary acceptable mediators or excipients commonly used in the art, and may further comprise an adjuvant. There is no particular limitation on the method of inoculation of the vaccine of the present invention, but it is preferable to inoculate by the negative, eye drop or spray method.

In one embodiment of the present invention, the attenuated New Cattle Disease virus was inoculated in chicken eggs, and then grown in the form of a live venom vaccine containing the same, but not limited thereto. At this time, the propagation method of the attenuated New Cattle Disease virus according to the present invention, egg inoculation process, culture conditions, vaccine production method and preparation reagents can be used without limitation any method known in the art. In addition, there is no particular limitation on the number of passages of the virus in the egg, but it is preferable that the seed seed virus is passaged up to five times.

In one embodiment of the present invention, the live attenuated vaccine containing the attenuated New Cattle Disease virus caused a sufficient level of immunogenicity to prevent New Cattle disease without adverse side effects in chickens when inoculated in a spray or negative manner to chickens of young age.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

Example 1 Isolation and Identification of Newcastle Disease Virus

In 2009, Newcastle disease virus was isolated from samples collected from ducks on poultry duck farms that were not vaccinated with Newcastle disease. In more detail, first, the feces existing in the total excretory cavity (cloaca) of healthy ducks were collected with a cotton swab, and then thoroughly mixed by adding 1 ml of 0.01 M PBS (phosphate buffered saline, pH 7.4) solution. The fecal mixture was centrifuged at 1,500 g for 30 minutes in a centrifuge, and the centrifugal supernatant was filtered with a disposable 0.45 μm filter to remove bacteria present in the feces and then used as a sample for virus separation.

The specimens were inoculated with 0.1 ml per egg into the intestinal ureum of SPF chicken embryos (embryonating eggs) that developed embryos at 37 [deg.] C. for 9 to 11 days. The inoculation site of the eggshell egg shell was sealed with sterile paraffin, and then put in a 37 ° C. incubator and incubated for 5 days. After that, the inoculated eggs were taken out of the incubator and chilled overnight at 4 ° C., and then egg shells were removed aseptically, and then the chicken blood cells were aggregated aseptically. The virus-proliferating samples of coagulating chicken blood cells (ovulation infiltrating mucosa) were again mixed with Newcastle disease standard antiserum, neutralized for 30 minutes at room temperature, and then examined for hemagglutination inhibition (HI). As a result, it was confirmed that the virus that aggregates chicken blood cells was propagated in the intestinal ureum fluid of the egg inoculated with the poultry sample. In addition, the embryos inoculated with the virus did not die during 5 days of culture, and showed anti-agglutination by Newcastle disease standard antiserum. RT-PCR (reverse transcriptase polymerase chain reaction) test kit for detecting NDV (Intron Biotechnology, Daejeon, Korea) was used to finally identify Newcastle disease virus NDRL09-01, which inhibits chicken blood cell aggregation by standard antiserum among virus samples. .

The isolated Newcastle disease virus NDRL09-01 was purified by cloning three times in SPF chicken eggs by endpoint titration, and then cultured in SPF eggs for large Newcastle disease vaccine strain.

Example 2 Investigation of Proliferative Potency in New Cattle Disease Virus NDRL09-01

The Newcastle disease virus NDRL09-01 of Example 1 was inoculated into SPF eggs to examine the proliferation of the virus. In other words, inoculated with 0.1 ml per egg into the intestinal ureum of SPF egg embryos that developed the fetus for 9 to 11 days in the incubation period, and then incubated again at 37 ° C. incubation period. The inoculated eggs were incubated for 5 days, and then the eggs were removed from the incubator and left to cool overnight at 4 ° C. After removing eggshells from egg eggs, the intestinal urea fluid was aseptically harvested to proliferate and cultured the virus.

Virus HA protein levels were measured using hemagglutination. The virus solution was diluted twice in sterile PBS in 96-well U-type microplate and added 50 μl per well, and then 0.5% (v / v) chicken blood cell solution was added in the same amount and left at room temperature for 40 minutes. . Thereafter, the reciprocal of the maximum dilution factor of the virus fluid in which the chicken blood cells aggregated was determined as the HA titer.

Virus infection amount was measured by the same SPF chicken egg inoculation method as in Example 1. The virus solution was diluted by 10-fold step with sterile PBS, and 5 eggs per dilution were inoculated in the same manner as in Example 1 to SPF eggs. On day 5 of the inoculation, the intestinal serous fluid of the eggs was collected and examined for coagulation of chicken blood cells. When hemagglutination occurred, the egg was considered infected with Newcastle disease virus. The infectivity was measured according to Reed &Muench's method (Reed LJ & Muench), which represents the inverse of the dilution fold of virus fluid that infects 50% of embryonated fetuses (EID ₅₀ : 50% embryo infective dose) (Reed LJ & Muench). H. American J Hyg, 27: 493-497, 1938.

Investigation of virus proliferation in SPF eggs revealed that Newcastle disease virus NDRL09-01 strains had at least 1,000-fold higher virus HA and 10 ^10.0 EID ₅₀ higher viral infection. High infectivity was shown. These results confirmed that the NDRL09-01 strain as a vaccine strain has excellent proliferation in oviposition.

Example 3 Pathogenicity of Chickens with Newcastle Disease Virus NDRL09-01

The pathogenicity of chickens of the Newcastle disease virus NDRL09-01 strain of the present invention is determined by measuring mean death time (MDT) in SPF eggs and intracerebral pathogencity index (ICPI) in 1-day-old SPF chicks. Measurement was made using two methods.

<MDT Measurement>

The Newcastle Disease Virus NDRL09-01 strain of Example 1 was inoculated into 10 SPF chicken eggs. That is, inoculate the virus solution to 5 SPF chicken eggs at 9 am on the day of inoculation and the remaining 5 SPF chicken eggs at 5 pm, and then incubate at 37 ° C. and check each day at 9 am and 5 pm The average lethal time (MDT) was measured by observing the death of the fetus. If the measured MDT was within 60 hours, it was considered to be a strongly pathogenic Newcastle disease virus, from 60 to 90 hours as a pathogenic Newcastle disease virus, and if it was above 90, the pathogenic Newcastle disease virus. The Newcastle disease virus NDRL09-01 strain of the present invention did not cause fetal mortality in any inoculated eggs during 5 days of culture, resulting in MDT of at least 120 hours and belonging to the category of virogenic viruses.

<ICPI Measurement>

The Newcastle Disease Virus NDRL09-01 strain of Example 1 was inoculated into the brains of 10 male 1 day old SPF chickens. That is, the virus was inoculated into 10 brains of 1 day-old SPF chicks by 0.05 ml per chick, and observed for 8 days. During the observation period, the ICPI was finally calculated by dividing the total total score by 80 by cumulative total every day, with a score of 0 per normal chick, 1 per chick indicating disease, and 2 per dead chick. ICPI levels above 1.2 were classified as strongly pathogenic viruses, between 0.6 and 1.2 as pathogenic viruses, and below 0.6 as pathogenic viruses. The Newcastle disease virus NDRL09-01 strain of the present invention belonged to the category of the pathogenic Newcastle disease virus with an ICPI value of 0.2.

ICPI measurement result of Newcastle disease virus NDRL09-01 strain of the present invention Days after inoculation Sum weight sum** One 2 3 4 5 6 7 8 normal 10 * 10 10 10 8 8 8 8 72 0 0 Disease 0 0 0 0 0 0 0 0 0 One 0 Our company 0 0 0 0 2 2 2 2 8 2 16 ICPI = (Sum of Each Total / 80) 16/80 = 0.2

* Number of individuals

** Total = Total * Weight

As a result of MDT and ICPI measurement, which is a conventional method for measuring the pathogenicity of Newcastle disease virus, the Newcastle disease virus NDRL09-01 strain of the present invention was confirmed to be a pathogenic Newcastle disease virus.

Example 4 Genotyping of Newcastle Disease Virus NDRL09-01

Genotype of the Newcastle disease virus NDRL09-01 virus of Example 1 was amplified by the virus F protein gene by RT-PCR method and then subjected to phylogenetic analysis by taking the nucleic acid sequence.

Viral genomic RNA was extracted using a viral RNA-spin kit (viral gene-spin, Intron Biotechnology) according to the manufacturer's recommendations. cDNA synthesis and specific gene amplification were performed using the Accupower RT-PCR premix kit (Bioneer co., Daejeon, Korea) according to the manufacturer's recommended method. In this case, the RT-PCR reaction was performed using the primers shown in Table 2 below, and the reaction conditions were performed as suggested by Lee et al. (Lee et al., Avian Pathol, 2004).

SEQ ID NO: Primer sequence One 5'-GCT GAT CAT GAG GTT ACC TC-3 ' 2 5'-AGT CGG AGG ATG TTG GCA GC-3 '

The primers are designed to amplify a total of 695 bp of nucleic acid from the 1,055th region of the Newcastle Disease Virus M gene to the 508th region of the F gene. The 695 bp nucleic acid material amplified by RT-PCR was extracted purely through agarose gel electrophoresis to determine the base sequence. For sequencing, a cyclic sequencing kit (PRISM ready reaction dye terminator kit, manufactured by Applied Biosystems) and an automatic base sequencer (ABI PRISM version 377 DNA autosequencer, manufactured by Applied Biosystems) were used.

Of the 695 nucleic acid sequences determined through the above procedure, the first 389 nucleic acid sequences of the F gene were taken to analyze the predicted amino acid sequences. As a result, the F1 / F2 segment amino acid of the F0 protein associated with virulence of Newcastle disease virus was ¹¹² Gly (G) -Arg (R) -Gln (Q) -Gly (G) -Arg (R) -Leu (L) ^It had the amino acid sequence of ¹¹⁷ (FIG. 1, SEQ ID NOs: 3 and 4). The non-contiguous basic amino acids in the amino acid constituting the segmental region is a characteristic of attenuated Newcastle disease viruses. This feature is common to conventional vaccine strains such as La Sota strain, Ulster2C strain, B1 strain, and VG / GA strain. Therefore, the amino acid sequence of the F protein segment of the NDRL09-01 strain of Example 1 is only a pathogenic marker indicating that the attenuated New Cattle Disease virus is not characteristic of only the NDRL09-01 strain of the present invention.

To determine the genotype of the NDRL09-01 strain isolated in Example 1, the first 389 nucleic acid sequences of the viral F protein gene were taken and subjected to phylogenetic analysis using a neighboring-joining (NJ) method. The results are shown in FIG. As shown in Figure 2, the NDRL09-01 strain of Example 1 La Sota strain (IIa), B1 strain (IIa), VG / GA strain (IIa), Ulster2C strain (Ic) and V4 strain ( It is classified into a new genotype (Id type) that is completely different from conventional vaccine viruses such as type Ia). Therefore, the present inventors deposited the NDRL09-01 strain of Example 1, a novel genotype Newcastle disease virus, with the KCTC 11718BP, deposited on June 25, 2010, at the Korea Research Institute of Bioscience and Biotechnology.

Next, in order to evaluate the efficacy as a vaccine virus of the NDV NDRL09-01 strain of the present invention, the following experiment was performed.

Test Example 1 Chicken Long-term Affinity Investigation of Newcastle Disease Virus NDRL09-01

In order to investigate the long-term affinity of the NDRL09-01 strain of Example 1, a 6-week-old SPF chick inoculation experiment was performed. That is, eight SPF chickens were inoculated with the eye drop of 10 ⁷ EID ₅₀ Newcastle Disease Virus NDRL09-01 strain. Each experimental group inoculated with La Sota or VG / GA strains, which are currently used as Newcastle disease vaccine strains, was compared. In the control group, 8 SPF chickens were inoculated with sterile PBS solution in the same manner. Trachea, lung, duodenum and cecal tonsil were harvested by euthanizing 4 chickens per experimental group each on day 4 and 7 after inoculation of each virus. The collected tissue sample was emulsified using a mortar and then added sterile PBS solution to 10% (w / v), followed by centrifugation at 1,500 g for 30 minutes in a centrifuge, and the supernatant was filtered with a disposable 0.45 μm filter. Used for virus. Each sample was inoculated into SPF eggs in the same manner as in Example 1 to attempt virus isolation. Inoculation of the intestinal umbilical fluid from the infiltrating oocytes to agglutinate chicken blood cells was considered to be Newcastle disease virus isolated, and finally, whether or not Newcastle disease virus was finally determined by the same RT-PCR test kit (Intron Biotechnology) as in Example 1. Table 3 shows the results of examining the affinity organs of each virus.

Long-term affinity test results in chickens of NDRL09-01 strain of the present invention Inoculation group virus Virus Separation Results by Organs 4th day of inoculation 7th day of inoculation T * L * D * Ct * T L D Ct G1 NDRL09-01 + + + + - - + + G2 VG / GA + + + + - - + + G3 La sota + + - - + + - - Control group PBS - - - - - - - -

T, trachea; L, lung; D, duodenum; Ct, cecal tonsil

In the results of Table 3, the NDRL09-01 strain of the present invention proliferated in both the respiratory (organ and lung) and digestive organs (duodenum, cecal tonsil) on days 4 and 7 after inoculation. La Sota strains, on the other hand, grew only in the respiratory organs (organs and lungs) on days 4 and 7 after inoculation. These results indicate that the NDRL09-01 strain of the present invention is a virus that is affinity not only in the respiratory system but also in the digestive organs. Therefore, the NDRL09-01 strain of the present invention can induce an immune response even by vaccination by various methods such as spray (respiratory induction), eye drop (induced local immune immunity), and oral (induced digestive infection). I could confirm it.

Test Example 2 Investigation of Immune Response in Chickens Spray-Inoculated with NDRL09-01 of the Present Invention

In order to investigate the immune response effect by spray inoculation in chickens, spray inoculation experiments were performed on 1-day-old SPF chicks. That is, 11 No. 1-day-old SPF chicks were treated with the Newcastle disease virus NDRL09-01 strain of Example 1 10 ^6.5 EID _50. Spray inoculation at a concentration (particle size 60-80 μm). In order to compare the immune response of Newcastle disease vaccine, VG / GA and La Sota strains, which are already known vaccines, were used in the same conditions and in the same population. In addition, 6 chicks of the same age were spray-inoculated with sterile PBS solution as a control. The clinical symptoms and mortality were observed and recorded for 14 days after inoculation for all experimental groups. In order to measure the immune response after Newcastle disease vaccination, all individuals were collected before and after 14 days of inoculation to measure the degree of immune response, and the results are shown in Table 4 below.

Experimental results of spray inoculation with NDRL09-01 strain of the present invention in 1-day-old SPF chick Inoculation group Vaccination situation Mortality * Average antibody titer
(HI titer, log2) virus Inoculation Path Inoculation amount Inoculation number Before vaccination 14 days after inoculation G1 NDRL09-01 Spray 10 ^6.5 EID ₅₀ 11 0% 0 2.0 ± 1.0 G2 VG / GA Spray 10 ^6.4 EID ₅₀ 11
0% 0 2.6 ± 1.0 G3 La sota Spray 10 ^6.8 EID ₅₀ 11
27% 0 5.5 ± 2.6 Control group PBS Spray - 6
0% 0 -

* (Number of dead / inoculated) × 100

As can be seen from the results of Table 4, no dead chicks were observed in the experimental group inoculated with the NDRL09-01 strain of Example 1 and the experimental group inoculated with the Newcastle disease VG / GA vaccine strain during the 14-day experimental period. On the other hand, in the experimental group vaccinated with Newcastle disease La Sota vaccine, 27% (3/11) of the inoculated individuals died of respiratory symptoms. The control group did not show any clinical symptoms during the 14-day observation period.

In addition, the results of measuring the degree of immune formation after vaccination are shown in Table 4. The experimental group inoculated with NDRL09-01 strain of the present invention had an average HI antibody value (based on log2) of 2.0 ± 1.0, and the immune response was lower than that of the La Sota vaccine strain (5.5 ± 2.6), which had respiratory side effects in chickens. There was no significant difference from GA vaccination group (2.6 ± 1.0). Based on the above results, La Sota vaccine has the best immune response, but the respiratory side effects are severe enough to kill some chickens. Therefore, La Sota vaccine is not suitable for use as a live venom vaccine at young age. Because it shows similar immune response to VG / GA vaccine strain without side effects such as respiratory symptoms, it was confirmed that it is suitable as a spray vaccine for preventing Newcastle disease.

Test Example 3 Investigation of Immune Response in Chickens Inoculated with NDRL09-01

In order to investigate immunogenicity by negative inoculation or inoculation in chickens of NDRL09-01 strain of Example 1, inoculation experiments were carried out on 10 SPF chicks 1 day old. These SPF chicks were co-inoculated with Newcastle Disease Virus NDRL09-01 at oral and instillation at a concentration of 10 ⁷ EID ₅₀ per allowance. In addition, six 1-day-old SPF chicks were inoculated sterile PBS solution into the mouth and eye drops at the same time as a control. The clinical symptoms and mortality were observed and recorded for 14 days after inoculation for all experimental groups. In order to measure the immune response after Newcastle disease vaccination, each subject was collected before and 7 days and 14 days after inoculation to measure the degree of immune response, and the results are shown in Table 5 below.

Inoculation group virus Average antibody titer (HI titer, log2) Before vaccination 7 days after inoculation 14 days after inoculation G1 NDRL09-01 0 3.7 ± 2.1 5.1 ± 3.3 Control group PBS 0 0 0

In the results of Table 5, the group vaccinated with the NDRL09-01 strain of Example 1 showed no clinical symptoms during the observation period, and HI antibody titer (based on log2) was not detected before the inoculation, but 3.7 ± 2.1 after 7 days. After 14 days, it was 5.1 ± 3.3. The control group did not show any immune response during the observation period.

As described above, the NDRL09-01 strain according to the present invention induces a stronger immune response early in the chicken even with a negative or eye inoculation, it can be seen that the immune formation effect against Newcastle disease in chicken is very excellent. That is, the NDRL09-01 strain according to the present invention exhibited sufficient immune response to the protection against the poisonous New Cattle disease when inoculated by spray, oral and instillation, and the NDRL09-01 strain according to the present invention was sprayed, negative and instilled. When inoculated with tells you that it can exert a protective effect against toxic Newcastle disease. Therefore, the NDRL09-01 strain according to the present invention is suitable as a live vaccine vaccine for preventing the damage caused by high-toxic Newcastle disease.

[Test Example 4] Evaluation of the protective effect against NDRL09-01 strain toxic NDV of the present invention

In order to investigate whether the NDRL09-01 strain of Example 1 has a protective effect against toxic New Cattle disease, all experimental group chickens that completed the above-described experiments were subjected to the corrective strain (10 ^6.3 EID ₅₀ ), a domestic toxic New Cattle disease virus. Inoculated into the muscle, and observed clinical symptoms and mortality for 14 days, the results are shown in Table 6 below.

Experimental group Vaccine Inoculation number Attack vaccination * ERA ** G1 NDRL09-01 11 Calibration 81.8% (9/11) G2 VG / GA 11 Calibration 63.6% (7/11) G3 La sota 8 Calibration 75.0% (6/8) Control group PBS 6 Calibration 0% (0/6)

Intramuscular inoculation of 10 ^6.3 ELD ₅₀ virus per horse

** Survival / Inoculation * 100

In the results of Table 6, the experimental group inoculated with NDRL09-01 strain according to the present invention had an 81.8% (9/11 number) defense rate during the observation period when artificially infected with the highly toxic Newcastle disease virus. The experimental groups inoculated with the other vaccine strains, VG / GA vaccine and La Sota, were 63.6% (7/11) and 75.0% (6/8). The control group died within 5 days after inoculation (defense rate 0%). From the above results, it could be confirmed that the NDRL09-01 strain according to the present invention has the most excellent protective effect against toxic Newcastle disease compared to other vaccine strains.

[Test Example 5] Test for pathogenic recovery in chickens of NDRL09-01 strain according to the present invention

A minimum of 3 days to 5 days old SPF chickens were inoculated simultaneously with oral and instilled 0.1 ml (10 ^7.5 EID ₅₀ ) of NDRL09-01 strain of Example 1 per feed. Trachea was harvested after euthanizing the chicken on day 5 of inoculation. The organs were then aseptically harvested, emulsified to 10% with PBS solution containing antibiotics, and then inoculated again in the same manner as new SPF chickens. In this way, six passages were repeated in chickens, and the final six passages were measured in the brain pathogenicity index (ICPI) in one-day-old SPF chicks in the same manner as in Example 3.

As a result, the six-day-old SPF chicks in chickens had a NDRL09-01 ICI score of 0.15, which corresponds to the attenuation type (less than 0.6). From this, it was confirmed that the NDRL09-01 strain according to the present invention did not recover pathogenicity even after chickens were reversed to chickens.

Korea Research Institute of Bioscience and Biotechnology KCTC11718BP 20100625

<110> REPUBLIC OF KOREA (Management: Ministry for Food, Agriculture, Forestry and Fisheries.National Veterinary Research and Quarantine Service (NVRQS)) <120> New subtype vaccine strain of lentogenic Newcastle disease virus          and the viral vaccine comprising <130> YPD / 201005-0011 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gctgatcatg aggttacctc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agtcggagga tgttggcagc 20 <210> 3 <211> 389 <212> DNA <213> Newcastle disease virus F protein gene <220> <221> CDS (222) (1) .. (387) <400> 3 atg ggc tcc aga tct tct acc agg atc tca gta cct ctg atg cta act 48 Met Gly Ser Arg Ser Ser Thr Arg Ile Ser Val Pro Leu Met Leu Thr   1 5 10 15 gtt cgg gtc gcg ctg gca ctg tgt tgc gtc cgt ctg aca agt tcc ctt 96 Val Arg Val Ala Leu Ala Leu Cys Cys Val Arg Leu Thr Ser Ser Leu              20 25 30 gat ggc agg cct ctt gca gct gca ggg att gtg gta aca gga gat aaa 144 Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys          35 40 45 gca gtc aac ata tac acc tca tcc cag aca ggg tcg atc ata gtc aag 192 Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys      50 55 60 tta ctc ccc aat atg ccc aag gac aaa gag gca tgt gca aaa gcc ccg 240 Leu Leu Pro Asn Met Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro  65 70 75 80 ttg gaa gcg tac aac agg aca ttg act act ctg ctc acc ccc ctt ggt 288 Leu Glu Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly                  85 90 95 gat tct atc cgt agg ata caa gag tcc gtg act aca tct gga gga ggg 336 Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly             100 105 110 aaa cag gga cgc ctt ata ggc gct att atc ggt ggt gcg gct ctc gga 384 Lys Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Ala Ala Leu Gly         115 120 125 gtt gc 389 Val <210> 4 <211> 129 <212> PRT <213> Newcastle disease virus F protein gene <400> 4 Met Gly Ser Arg Ser Ser Thr Arg Ile Ser Val Pro Leu Met Leu Thr   1 5 10 15 Val Arg Val Ala Leu Ala Leu Cys Cys Val Arg Leu Thr Ser Ser Leu              20 25 30 Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys          35 40 45 Ala Val Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys      50 55 60 Leu Leu Pro Asn Met Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro  65 70 75 80 Leu Glu Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly                  85 90 95 Asp Ser Ile Arg Arg Ile Gln Glu Ser Val Thr Thr Ser Gly Gly Gly             100 105 110 Lys Gln Gly Arg Leu Ile Gly Ala Ile Ile Gly Gly Ala Ala Leu Gly         115 120 125 Val    

Claims (9)

Newcastle Disease Virus Vaccine of Accession No. KCTC 11718BP. The Newcastle disease virus vaccine strain according to claim 1, wherein the Newcastle disease virus vaccine belongs to genotype d (Id) of genotype I of type 1 in a phylogenetic analysis based on the nucleic acid sequence of the F protein. According to claim 1, wherein the Newcastle disease virus vaccine is Newcastle disease virus vaccine strain that shows affinity to the respiratory and digestive of chickens. The Newcastle disease virus vaccine strain according to claim 1, wherein the Newcastle disease virus vaccine strain is a virus that locally proliferates in the respiratory and digestive organs of a chicken to form mucosal immunity. A vaccine for preventing Newcastle disease comprising the Newcastle Disease Virus vaccine strain according to any one of claims 1 to 4. 6. The vaccine for preventing Newcastle disease according to claim 5, wherein the vaccine is inoculated into chickens in a negative, eye, or spray manner. The vaccine of claim 5, wherein the vaccine is prepared in the form of a live venom vaccine, a dead venom vaccine, or a subunit vaccine, a vector vaccine, a chimeric vaccine or a DNA vaccine produced using the gene of the New Cattle Disease virus vaccine strain. The vaccine for preventing Newcastle disease according to claim 5, wherein the vaccine comprises a veterinary acceptable medium or excipient. The vaccine for preventing Newcastle disease according to claim 8, wherein the vaccine further comprises an adjuvant.

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