WO2014208822A1

WO2014208822A1 - Novel fowl adenovirus and vaccine thereof

Info

Publication number: WO2014208822A1
Application number: PCT/KR2013/008898
Authority: WO
Inventors: 송창선; 이중복; 최인수; 박승용
Original assignee: 건국대학교기술지주주식회사
Priority date: 2013-06-27
Filing date: 2013-10-04
Publication date: 2014-12-31

Abstract

The present invention relates to a novel fowl adenovirus and a vaccine thereof.

Description

New Poultry Adenovirus and Its Vaccines

The present invention relates to novel poultry adenovirus and vaccines thereof.

This disease, called inclusion body hepatitis or Fowl adenovirus (FAdV), is an infectious disease caused by group 1 poultry adenovirus (FAdV). FAdV belongs to the family Adenoviridae, and gemone consists of double-stranded DNA that is not segmented. There are a total of 12 serotypes in poultry adenovirus belonging to group 1, and the pathogenicity varies greatly among these serotypes. The major serotypes that cause damage to the domestic poultry industry are known to be caused by type 4, type 8 or type 11. Inclusion hepatitis is found in most serotypes, and type 4 is accompanied by pericarditis. It is known to be the strongest. Adenovirus infections are diseases in which liver cells are infected as a main target, forming characteristic inclusion bodies and causing death. Inclusion hepatitis can be transmitted either vertically or horizontally. Vertical propagation usually occurs between three to four weeks of age, and vertical propagation has occurred before seven days of age. The mortality rate of single infection is low around 10%, but mortality is higher when combined with immunosuppressive diseases such as Gamboro and chicken infectious anemia. Infection of serotype 4 with pericardial hydrocephalus is associated with relatively high morbidity and mortality, and mortality rates of up to 80% are known in susceptible broilers, causing significant economic losses to the domestic poultry industry. Situation.

The disease was first isolated from algae in 1949 and is now widely distributed around the world. Since then, in 1987, a serotype 4 adenovirus infection with pericardiomyeloma has been reported in Pakistan and reported worldwide, causing serious damage to the poultry industry. Pericarditis is characterized by high morbidity and mortality due to infection alone, even in the absence of immunosuppressive diseases, unlike conventional inclusion hepatitis. In addition, it is a disease that is highly susceptible to young age, and because it is possible to spread eggplants, it is asymptomatically infected in breeders, and it is a disease that can cause serious disease due to eggplants of later chicks.

According to the report of the Ministry of Agriculture, Food and Rural Affairs and Quarantine, serotype 4 was the highest in 46% of cases of inclusion hepatitis reported from 2007 to 2010, and other types 11 (31%) and 8b (23%). ), Type 4, type 8b and type 11 accounted for a high percentage. In addition, 72% of the total outbreaks were reported in broilers, and laying hens and native chickens were the rest. The damage caused by inclusion hepatitis was found to be greatest in broilers. (Figure 1)

In addition, according to the Agriculture, Forestry and Livestock Quarantine Division, 10 cases out of 39 reported cases of infected chickens from 2 weeks of age due to the vertical spread of the disease continue to report damages from the transmission of inclusion-type hepatitis virus from breeders. It is a situation.

Currently, in Korea, the enveloping hepatitis vaccine is not administered, and the disease is sub-divided, causing problems such as an increase in mortality rate and a decrease in productivity in later chicks. Therefore, there is an urgent need for the introduction of inclusion hepatitis vaccines that can suppress disease infection and maintain high antibody titers and deliver maternal transfer antibodies to breeders.

[Previous Patent Document]

Korean Patent Publication No. 1020110132316

Korean Patent Publication No. 1020110092316

The present invention has been made by the above necessity, and an object of the present invention is to provide a novel poultry adenovirus isolated from Korea.

In order to achieve the above object, the present invention provides a FAdV K4 poultry adenovirus deposited with accession number KCTC 12425BP.

The strain of the present invention was deposited on June 12, 2013 to the Korea Institute of Bioscience and Biotechnology Center (125, Yuseong-gu, Daejeon, Daejeon, Korea, Korea Biotechnology Research Institute, 305-806).

In one embodiment of the present invention, the virus is preferably isolated from domestic chickens, preferably comprising the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The present invention also provides a poultry protection vaccine against diseases caused from poultry adenovirus infection comprising the poultry adenovirus of the present invention and a pharmaceutically acceptable carrier or diluent.

In one embodiment of the invention, the vaccine is preferably effective in preventing or treating inclusion hepatitis, it is preferably in an inactivated form, preferably further comprises an adjuvant, and is infectious to poultry. It is preferred to include one or more vaccine components of other pathogens, but is not limited thereto.

In another aspect, the present invention comprises inoculating a poultry adenovirus defined in the present invention on a substrate susceptible to infection; b) propagating the poultry adenovirus; And c) provides a method for producing poultry adenovirus comprising the step of collecting the poultry adenovirus-containing material.

In one embodiment of the present invention, the substrate is one selected from the group consisting of embryonic liver cells (CEL), chicken embryo fibroblasts (CEF), chicken kidney cells (CK), and Vero cell lines. Preferably, the cells are but are not limited thereto.

In another aspect, the present invention comprises the step of combining the collected poultry adenovirus obtained by the method of the present invention with a pharmaceutically acceptable carrier or diluent, and if necessary, the step is carried out after inactivation of the poultry adenovirus It provides a method for producing a poultry protection vaccine against diseases caused from poultry adenovirus infection.

The present invention also provides a method for controlling a disease caused from poultry adenovirus infection in poultry comprising administering the vaccine of the present invention to birds.

Vaccines of the invention can be prepared according to conventional methods commonly used, for example, in commercial live inactivated poultry adenovirus vaccines. Preparation of veterinary vaccine compositions is particularly described in "Handbunch der Schutzimpfungen in der Tiermedizin" (ed. Mays, A. et al., Verlag Paul Parey, Berlin und Hamburg, Germany, 1984) and "Vaccines or Veterinary Applications" (ed. Peters, AR). Et al., Butterworth-Heinemann Ltd, 1993.

The purpose of inactivating the harvested virus after the propagation step is to prevent regeneration of the virus. In general, this can be done by chemical or physical means. Chemical inactivation can be carried out, for example, by treating the virus with enzymes, formaldehyde, beta-propiolactone, ethylene-imine or derivatives thereof. If necessary, the deactivated compound is later neutralized. The formaldehyde inactivated material may be neutralized with, for example, thiosulfate. Physical inactivation may be preferably carried out by irradiating the virus with sufficient energy, for example UV light. If necessary, the pH may be adjusted to a value of about 7 after treatment.

Vaccines containing inactivated poultry adenovirus may, for example, comprise one or more of the aforementioned pharmaceutically acceptable carriers or diluents suitable for this purpose. Preferably, the inactivated vaccine of the present invention comprises one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include vegetable oils such as aluminum hydroxide, -phosphate, -oxide, mineral oil, vitamin E acetate, and oil-in-water or water-in-oil emulsions based on saponins.

Inactivated vaccines are usually administered parenterally, ie intramuscularly or subcutaneously.

The vaccine of the present invention comprises as an active ingredient an effective amount of poultry adenovirus, i.e., an amount to immunize poultry adenovirus material which will induce immunity in vaccinated birds or their offspring against attack by toxic viruses (antigen administration). . Immunity is defined herein to induce a significantly higher level of protection in avian populations after vaccination compared to the non-vaccinated group.

Typically, the inactivated vaccine comprises an antigen equivalent to 10 ⁴ -10 ¹⁰ TCID50 per bird.

The poultry adenovirus vaccine of the present invention can be used effectively in chickens, but other poultry such as turkey, guinea fowl and quail can also be vaccinated effectively. Chickens include edible chickens, cloned cattle, and laying eggs.

The present invention can effectively protect poultry from poultry adenovirus infection by providing new poultry adenoviruses, vaccines comprising the same and methods for their preparation.

Figure 1 shows the serotype and incidence rate of inclusion hepatitis virus reported in Korea between 2007 and 2010 <Source: Agriculture, Forestry and Livestock Quarantine Headquarters>

2 is another pathogen contamination test

3 is a systematic analysis of the HEXON gene region

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the invention and the scope of the present invention is not to be construed as limited by the following examples.

① Isolation of Fowl Adenovirus FAdV K4 strain and identification of other pathogens

In 2010, livers were collected from broilers from Gyeonggi-based broiler farms, and sterile phosphate buffer solution (pH7.2) was added to make 10 wt% emulsion. Cells and tissues were precipitated by centrifugation at 3,000 g for 15 minutes, and the supernatant was filtered with a 0.45 μm syringe filter. FAdV K4 strains were finally identified by polymerase chain reaction (PCR) by 11-day-old SPF embryonated chorioallantoic membrane (CAM) inoculation.

② Negative test of other pathogens in new FAdV outdoor isolate

Infectious bronchitis virus (IBV), Newcastle disease (NDV), Infectious bursal virus (Infectious bursal virus) disease virus (IBDV), chicken infectious anemia virus (CIAV), mycoplasma, reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR) It was confirmed that no pathogen other than the virus isolate (IBD K7).

③ genome sequence analysis

DNA was extracted by inoculating the 11-day-old SPF embryonated eggs with 150 μl of the urinary membrane solution harvested 5 days later. The DNA thus obtained was mixed with 5 µl of DNA solution, 5 µl of 10 × PCR buffer, 4 µl of 2.5 mM dNTPs, 1 µl of each primer, 0.5 µl of Taq polymerase and 33.5 µl of DW. (G. Meulemans et al., Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses, AVIAN PATHOLOGY 30: 6, 655-660, 2001)

The PCR product obtained using the reaction solution was purified, and the nucleotide sequence was determined using an ABI3100 autobase sequencer. As a result, a genomic nucleotide sequence of FAdV K4 of the present invention represented by HEXON was obtained. (Table 1, Table 2)

Table 1

Table 1 shows the nucleotide sequence and position of primer set used for HEXON base analysis.

TABLE 2

Table 2 shows the HEXON sequences of FAdV K4 virus

④ Molecular Biology of FAdV K4 Virus

The entire FAdV K4 virus genes were compared and analyzed with the inclusion of domestic and foreign inclusion hepatitis viruses on GenBank using the BLAST search (www.ncbi.nlm.nih.gov/blast) and the Bioedit program. As a result, it was confirmed that IBD K4 is serotype 4 and has high homology with domestic epidemic hepatitis. (Figure 3)

⑤ Fabrication and safety test of FAdV K4 inactivated oil vaccine

After culturing FAdV K4 strain in the Chicken Embryo Liver cell (CEL) until the cytopathic effect (CPE), the formalin solution was added to the virus culture at 0.1 ° C. After inactivation for 24 hours, the oil antigen adjuvant ISA70 and antigen, which are widely used in animal poisoning vaccine, were emulsified at a ratio of 30:70. The final virus titer after emulsification was ⁵ × 10 ⁵ tissue culture infective dose (TCID ₅₀ ) / 1 dose (0.5 mL).

To investigate the safety of the prepared inactivated dead venom vaccine, 1 mL (2 dose) was injected into the femoral muscles of 15 SPF chickens. Clinical symptoms, weight gain and mortality were examined for 3 weeks after inoculation, and 5 rats were examined at 3, 4, and 5 weeks, respectively. As a result, FADV K4 strain inactivated dead venom vaccination group showed no clinical symptoms and mortality, and there was no significant difference in the increase rate between the inoculated group and the control group. Table 3.Table 4

TABLE 3

Table 3 shows the clinical symptoms and death of three weeks-old SPF inoculation system after FAdV K4 inactivated oil vaccine.

Table 4

Table 4 shows the changes in body weight and granuloma formation in the three-week-old SPF inoculation system after FAdV K4 inactivated oil vaccine.

⑥ Cottonworking test of SPF chicken of FAdV K4 inactivated dead venom vaccine

To examine the immunogenicity of FAdV K4 inactivated dead venom vaccine, six-week-old SPF chickens were inoculated with FAdV K4 inactivated dead venom vaccine in 15 water SPF chicken thigh muscles for 1 minute each. Three weeks after vaccination, blood was collected and the antibody against poultry adenovirus was confirmed by Agar Gel Precipitation (AGP). As a result of the test, the vaccinated group showed significantly higher antibody positivity than the control group, confirming the immunogenicity of the test vaccine. Table 5

Table 5

Table 5 shows the antibody-forming ability of the 6-week-old SPF inoculation system after FAdV K4 inactivated oil vaccine inoculation.

⑥ Protective effect test against homologous and heterologous serotype virus in SPF chickens inoculated with FAdV K4 inactivated oil vaccine

To test the cross-protective efficacy of FAdV K4 inactivated oil vaccine, 100 two-week-old SPF chickens were divided into 10 groups of 10 animals and then inoculated in the femoral muscles of 5 chickens for 1 minute. Two weeks after inoculation, the inoculation of allogeneic and heterologous serotypes (

serum types

3, 4, 8, 9 and 11) was administered intravenously with 10 ⁶ TCID ₅₀ per dose, followed by observation of clinical symptoms and mortality for 2 weeks. Two weeks later, all groups of chickens were necropsied and virus re-separation was performed using macroscopic lesions (liver edema and necrosis, pericardial myeloma in type 4) and polymerase chain reaction (PCR) caused by adenovirus infection. As a result of the test, the vaccinated group confirmed the cross-protective ability of the test vaccine by confirming that the clinical symptoms and mortality of allogeneic and heterologous viruses were decreased and the attack strain re-separation rate was also reduced compared to the non-vaccinated control group. (Table 6, Table 7)

Table 6

Table 6 shows the clinical symptoms and mortality of allogeneic and heterologous serotype virus challenge after FAdV K4 inactivated oil vaccine.

TABLE 7

Table 7 shows gross lesions and virus re-separation rates for allogeneic and heterologous serotype virus challenge after FAdV K4 inactivated oil vaccine.

⑥ Protective effect test against homologous and heterologous serotype virus in later chicks of breeder inoculated with FAdV K4 inactivated oil vaccine

In order to examine the antibody-forming ability in the breeder of FAdV K4 inactivated oil vaccine and the protection against homologous and heterologous serotype viruses in later chicks, a broiler breeder farm with a negative FAdV antibody was randomly selected. One group of test vaccines were injected into the thigh muscles of 17-week-old breeding dogs. Three weeks after the vaccine, 20 samples were collected from the test group and 10 samples from the control group, and the antibody-forming ability against FAdV was confirmed by the AGP test method. As a result of the test, the vaccinated group confirmed the immunogenicity in broiler breeders by confirming that antibodies to FAdV were formed significantly compared to the vaccinated group.

The 1-day-old posterior chicks of the vaccinated group and the non-vaccinated control group were divided into 10 groups using 100 numbers in the test group and 50 numbers in the control group, and homologous and heterologous serotypes (

serum types

3, 4, 8, 9, 11). Type 2) After challenge inoculation of 10 ⁶ TCID ₅₀ through the muscle, the clinical symptoms and mortality were observed for 2 weeks, and then 2 weeks after the inoculation, all groups of chickens were autopsied to examine the gross lesions caused by adenovirus infection. And histological lesions of necrosis, pericardia in type 4) and liver. The results showed that FAdV significantly reduced clinical manifestations and mortality in the later chicks of the vaccinated group and significantly reduced the gross lesion findings and histologic lesion indices at the time of necropsy. It was confirmed that early infection can be prevented against FAdV. (Table 8, Table 9, Table 10)

Table 8

Table 8 shows antibody-forming ability in 17-week-old broiler breeders after FAdV K4 inactivated oil vaccine

Table 9

Table 9 shows the clinical symptoms and mortality rates of allogeneic and heterologous serotype virus challenge in later chicks inoculated with FAdV K4 inactivated oil vaccine.

Table 10

Table 10 shows gross and tissue lesions in descendants of FAdV K4 inactivated oil vaccine at 2 weeks after allogeneic and heterologous serotype virus challenge.

Claims

FAdV K4 poultry adenovirus deposited with accession number KCTC 12425BP.
The poultry adenovirus according to claim 1, wherein the virus is isolated from domestic chickens.
The poultry adenovirus according to claim 1, wherein the virus has a nucleotide sequence of SEQ ID NO: 1.
A vaccine for poultry protection against diseases caused from poultry adenovirus infection comprising the poultry adenovirus of claim 1 or 2 and a pharmaceutically acceptable carrier or diluent.
The vaccine for protecting poultry according to claim 4, wherein the vaccine is effective in preventing or treating inclusion hepatitis.
5. The poultry protection vaccine according to claim 4, wherein the vaccine is in inactivated form.
The vaccine of claim 4, wherein the vaccine further comprises an adjuvant.
The vaccine of claim 4, wherein the vaccine further comprises one or more vaccine components of other pathogens infectious to poultry.
a) inoculating a poultry adenovirus as defined in claim 1 with a substrate susceptible to infection;

b) propagating the poultry adenovirus; And

c) collecting the poultry adenovirus containing material.
The method of claim 9, wherein the substrate is one cell selected from the group consisting of embryonic liver cells (CEL), chicken embryo fibroblasts (CEF), chicken kidney cells (CK), and Vero cell line. Method for producing poultry adenovirus, characterized in that.
Poultry adenovirus infection comprising combining the collected poultry adenovirus obtained by the method of claim 9 with a pharmaceutically acceptable carrier or diluent, if necessary, after the inactivation of the poultry adenovirus. Method of preparing a vaccine for poultry protection against diseases caused from.
A method of controlling a disease resulting from poultry adenovirus infection in poultry comprising administering the vaccine of claim 4 to birds.