WO2014208821A1

WO2014208821A1 - Novel avian metapneumovirus and vaccine thereof

Info

Publication number: WO2014208821A1
Application number: PCT/KR2013/008897
Authority: WO
Inventors: 최인수; 이중복; 박승용; 송창선
Original assignee: 건국대학교기술지주 주식회사
Priority date: 2013-06-27
Filing date: 2013-10-04
Publication date: 2014-12-31

Abstract

The present invention relates to a novel avian metapneumovirus and a use thereof for a vaccine.

Description

New avian methneumovirus and its vaccines

The present invention relates to novel avian meth pneumoviruses and their use in vaccines.

Avian metapneumovirus (aMPV) has been known to be the major cause of turkey rhinotrachitis (TRT) and Swollen Head Syndrome (SHS) in turkeys and chickens, respectively. The virus was first reported in 1986 and has been distributed worldwide in the United States, Europe, Asia, the Middle East and North Africa. In Korea, clinical and serological cases of head swelling in chickens were confirmed and reported in 1992. In recent years, the causative virus of severe broiler and broiler breeders with severe runny nose and discolored eggs has been reported. It is separated and re-recognized as one of the major diseases causing economic damage to domestic poultry industry.

In chickens, mortality is not very high during aMPV infection, but morbidity is high, especially in respiratory symptoms accompanied by severe runny nose, which lowers the productivity of laying hens. It is the cause.

The causative agent of the disease is aMPV belonging to Genus Metapneumovirus of Family Paramyxoviridae , which is present as a single serotype and reported to have four genotypes (A, B, C and D) based on the G protein gene sequence. It became. Unlike turkeys, where all genotypes develop, only A and B genotypes are known to cause damage in chickens.

In chickens, aMPV is mainly infected in broilers 4-6 weeks of age, which does not cause much mortality, but, like young turkeys, it causes severe respiratory symptoms, leading to a loss of appetite, leading to economic losses in farms. do. In this case, symptoms such as runny nose, sneezing, and tears, which are often expressed as colds, are observed in more than 50% of infectious systems, and head swelling caused by sinusitis, which is known as a representative symptom of aMPV, is rarely observed in less than 10% of infectious systems. . On the other hand, broiler breeders and laying hens can be infected regardless of age, but there are many cases of initiation or spawning peaks (24-52 weeks of age). This is known to be caused by physical stress associated with spawning.

Infection cases in domestic chickens have been reported continuously and vary widely from broilers, laying hens and broiler breeders. AMPV has been detected in farms that show immunosuppression due to severe contraction of thymus in domestic broiler farms. Viruses were also detected in broiler breeders, which had a severe runny nose, decreased feed efficiency and caused persistent mortality with peritonitis. Actually, in case of domestic broiler farms, shipments were delayed to 39 days due to severe increase in weight, and there were cases of serious damage such as 90% of shipments and 190 production index due to the death of secondary bacterial infection. In addition, in the breeder breeder infected with aMPV, up to 10% of white eggs occurred in addition to respiratory symptoms, and also affected egg production rate by 10%. In addition, when aMPV was infected just before the spawning peak, it did not reach the spawning peak, and the mortality increased before and after the spawning peak.

Recently, with the development of new diagnostic technologies such as real-time RT-PCR, the rapid differential diagnosis of aMPV-like respiratory diseases has been increasing. Recently, the number of cases of confirmed aMPV infection damage in domestic broilers, laying hens and broiler farms has increased. . In order to minimize the economic damage caused by aMPV infection, vaccines using live and dead poison vaccines are most effective. Currently, all of the pneumovirus vaccines released in Korea are poisonous oil vaccines, and vaccines against type A viruses are not domestically available.

Currently, in Korea, pneumovirus vaccines are not licensed and therefore are not marketed, so only the vaccines are used to defend against the serotypes. The ability is relatively low, so in order to defend against the two prevalent serotypes in Korea, it is also necessary to use a type A killed vaccine.

[Related Related Patents]

Korean Patent Publication No. 1020100060196

Republic of Korea Patent Publication No. 1020050005412

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a novel avian meth pneumovirus isolated from Korea.

In order to achieve the above object, the present invention provides a Ck / A / Kr / 655/07 avian metapneumovirus deposited with accession number accession number KCTC 12424BP.

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 invention, the virus is preferably isolated from domestic chickens, the virus is preferably of type A, the G protein of the virus preferably comprises a nucleotide sequence of SEQ ID NO: 1 limited to this Not.

In another aspect, the present invention provides a vaccine for poultry protection against diseases caused by avian meth pneumovirus infection comprising the avian meth pneumovirus 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 respiratory symptoms and E. coli peritonitis, the vaccine is preferably in an inactivated form, the vaccine further comprises an adjuvant Preferably, the vaccine preferably further comprises one or more vaccine components of other pathogens infectious to poultry, but is not limited thereto.

In another aspect, the present invention comprises the steps of a) inoculating a bird meth pneumovirus defined in the present invention on a substrate susceptible to infection; b) propagating the avian meth pneumovirus; And c) collecting the avian meth pneumovirus-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.

The present invention also includes the step of combining the collected avian meth pneumovirus obtained by the method of the present invention with a pharmaceutically acceptable carrier or diluent, and if necessary, the step is performed after inactivation of the avian meth pneumovirus Provided is a method of producing a vaccine for poultry protection against diseases caused by avian meth pneumovirus infection.

The present invention also provides a method for controlling a disease caused from avian meth pneumophila 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 avian meth pneumovirus 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 performed, for example, by treating the virus with an enzyme, formaldehyde, β-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 avian meth pneumovirus may comprise one or more of the aforementioned pharmaceutically acceptable carriers or diluents suitable for this purpose, for example. 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 is an active ingredient in an amount effective to immunize avian meth pneumoviruses, ie, avian meth pneumoviral substances that will induce immunity in vaccinated birds or their offspring against attacks by toxic viruses (antigen administration). Include. 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 avian methneumovirus vaccine of the present invention can be used effectively in chickens, but other poultry such as turkeys, guinea fowls and quails can also be vaccinated effectively. Chickens include edible chickens, cloned cattle, and laying eggs.

In the present invention, by providing a novel avian meth pneumovirus, a vaccine comprising the same and a method for producing the same can effectively protect the poultry from avian meth pneumovirus infection.

Figure 1 shows aMPV Ck / A / Kr / 655/07 main G protein gene sequence

2 is a systematic analysis of Ck / A / Kr / 655/07 virus and domestic and foreign aMPV isolates.

Hereinafter, the present invention will be described in more detail through non-limiting examples.

Isolation and identification of avian metapneumovirus aMPV Ck / A / Kr / 655/07 strains in domestic outdoor farms

An oral pharyngeal broiler from a broiler breeder farm in Chungnam, South Korea was resuspended in a MEM medium containing gentamicin. This was centrifuged at 1,000g for 10 minutes to precipitate cells and tissue solution. After extraction of RNA from the supernatant and real-time RT-PCR (Real-time RT PCR), it was diagnosed as aMPV and inoculated into Vero cells (monkey kidney cells) to finally identify aMPV Ck / A / Kr / 655/07 weeks. It was.

Confirmation and subtype classification through real-time RT-PCR

The supernatant after centrifugation of the oral pharynx with a cotton swab and nasal shelf was extracted with RNA using RNeasy kit (Qiagen, USA). Utilizing QuantiTect Probe RT-PCR kit (Qiagen, USA), 10 μl of RNA extract, 12.5 μl of Quantect master mix, 0.25 μl of Quantect RT mix, primer A for primer A for aMPV G protein gene 2 A total of 25 µl was prepared by mixing 0.375 µl (55 uM) of each of the two kinds of primers for type B and 0.375 µl (14 uM) of each of the type A probes and one of the type B probes. (Table 1) The reaction mixture was reacted once at 50 ° C. 30 minutes, 95 ° C. 15 minutes using SmartCycler (Cepheid, USA), and then reacted 45 times at 94 ° C. 15 seconds and 55 ° C. 60 seconds for real-time reverse transcription polymerase chain. The reaction (Real-time RT-PCR) was carried out

Table 1

Table 1 shows the primer and probe sequences used for Real-time RT-PCR.

Genomic sequencing

Positive samples confirmed by real-time RT-PCR were subjected to RT-PCR. Using a primer capable of amplifying each subtype for the aMPV G protein gene, 268 nucleotide sequences for type A and 361 nucleotide sequences for type B were analyzed. Analysis of aMPV Ck / A / Kr / 655/07 virus indicated that it belongs to subtype A. (FIG. 1) (Cavanagh et al ., Subtype B avian metapneumovirus resembles subtype A more closely than subtype C or human metapneumovirus with respect to the phosphoprotein, and second matrix and small hydrophobic proteins, Virus Research 92: 171-178, 2003)

Molecular Biology of aMPV Ck / A / Kr / 655/07 Virus

The aMPV Ck / A / Kr / 655/07 G protein gene sequence was compared and analyzed with domestic and foreign aMPV viruses registered on Genbank using BLAST search (www.ncbi.nlm.nih.gov/blast) and Bioedit program. . As a result, Ck / A / Kr / 655/07 showed higher homology with aMPV from Korea than BUT1 # 8544, a strain of imported aMPV subtype A venom vaccine. (Figure 2)

aMPV virus culture method and virus content test

To culture the virus, Vero cells were dispensed in a cell culture flask at 2 * 10 ⁵ / ml and incubated in a 37 ° C CO2 incubator. After 24 hours, cells formed a monolayer and cell growth culture was removed. The cells attached to the flasks were washed 2-3 times with cell culture medium and sterile PBS and then inoculated with aMPV. (Stock standard 106TCID50 / ml, T75 flask: 0.5 ~ 1ml, T175 flask: 3ml) After inoculation, it was incubated at 37 ° C. for 40 minutes, and then cultured in a 37 ° C. CO 2 incubator with the addition of a cell maintenance medium. After about 5 days of culture, the flask was freeze-thawed three times in a -70 ° C freezer when 80-90% of aMPV-specific cytopathic effect (CPE) was formed. After the last fusion, the cultures in the flask were centrifuged for 3 min at 2000 RPM at 4 ° C. The separated supernatant was used as virus stock solution for passage. Each time five passages were carried out, the vaccine was inoculated with a 10-degree dilution of the monolayer-formed Vero cells within 24 hours of incubation, followed by adsorption, followed by incubation at 37 ° C. Five days after the inoculation, TCID 50 was calculated as positive for the cytopathic effect peculiar to aMPV. Table 2

TABLE 2

Table 2 shows TCID increase according to virus passage

Fresh virus fraud test

When five passages were performed in Vero cells, supernatant of cell culture was completely neutralized with high-immune serum of aMPV and the neutralized supernatant was inoculated into embryonated eggs. And fork formation was not confirmed. Table 3

TABLE 3

Table 3 shows negative virus test results

aMPV inactivated oil vaccine safety test

In order to evaluate the safety of the test vaccine, 14 days after inoculation with 2 minutes of test vaccine, there was no difference in body weight gain compared to the control group, and specific clinical symptoms such as sneezing, head swelling, and sloping were not confirmed. Did. Table 4

Table 4

Table 4 shows the increase rate and clinical symptoms after 2 minutes of inoculation of the test vaccine in 6-week-old SPF chickens.

aMPV inactivated oil vaccine immunogenicity test

In order to evaluate the minimal immunogenicity of the test vaccine, each virus content observed ^{_{(10 3 ~ 6 TCID 50/}} 1 water) of four test groups and one each was inoculated with the test vaccine divided into two non-vaccinated control group Day antibody It was. 10 ⁶ TCID _50/1 antibody demonstrate a positive 100% only in the test group was inoculated water was found to be appropriate for an effective defense aMPV contained a 1 per minute of at least 10 ⁶ TCID ₅₀ Antigen. Table 5

Table 5

Table 5 shows the comparison of antibody levels against aMPV by inoculation concentrations after the test vaccine inoculation in 6-week-old SPF chickens.

aMPV Inactivated oil vaccine Average antibody titer China Index

In order to measure the immunogenicity of the test vaccine, the average antibody titer and neutralization index of each test group were checked three weeks after the 10 ⁶ TCID 50/1 water of each vaccine was inoculated. The average antibody titer was 7050 and the neutralization index was 22. Table 6

Table 6

Table 6 shows the mean antibody titers and neutralization index after 3 weeks of test vaccine in 6-week-old SPF chickens.

aMPV Protective efficacy test of inactivated oil vaccine

In order to confirm the efficacy of the test vaccine, the test group was inoculated with 1 minute of the test vaccine and 3 weeks later, the test group and the control group were inoculated with the inoculation virus 10 ⁴ TCID50 / 0.1ml intranasally. One day later, the challenge virus was re-isolated. The test group and the control group showed no clinical symptoms for 5 days after challenge, and the re-isolation rate of challenge virus was 0%. (Control: 26.7%) (Table 7)

TABLE 7

Table 7 shows the results of defense test after aMPV challenge in 6 weeks-old SPF chickens after 3 weeks of test vaccine.

Claims

Ck / A / Kr / 655/07 avian metapneumovirus deposited with accession number KCTC 12424BP.
The bird meth pneumophila virus according to claim 1, wherein the virus is isolated from domestic chickens.
The avian meth pneumovirus according to claim 1, wherein the virus is type A.
According to claim 1, wherein the G protein of the virus avian meth pneumovirus, characterized in that it has a nucleotide sequence of SEQ ID NO: 1.
A vaccine for poultry protection against diseases caused by avian meth pneumovirus infection comprising the avian meth pneumovirus of claim 1 or 2 and a pharmaceutically acceptable carrier or diluent.
The vaccine for protecting poultry according to claim 5, wherein the vaccine is effective in preventing or treating respiratory symptoms and E. coli peritonitis.
6. The poultry protection vaccine of claim 5, wherein the vaccine is in inactivated form.
The vaccine of claim 5, wherein the vaccine further comprises an adjuvant.
The vaccine of claim 5, wherein the vaccine further comprises one or more vaccine components of other pathogens infectious to poultry.
a) inoculating a susceptible substrate with avian meth pneumovirus as defined in claim 1;

b) propagating the avian methneumovirus; And

c) A method for producing avian meth pneumovirus comprising the step of collecting the avian meth pneumovirus-containing material.
The method of claim 10, 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 avian meth pneumovirus, characterized in that.
A step of combining the collected avian meth pneumovirus obtained by the method of claim 10 with a pharmaceutically acceptable carrier or diluent, if necessary, the step is performed after the inactivation of the avian meth pneumovirus A method for preparing a poultry protection vaccine against diseases caused from pneumovirus infection.
A method of controlling a disease resulting from an avian methneumovirus infection in poultry comprising administering the vaccine of claim 5 to a bird.