TW200540273A - Canine distemper virus isolated in korea and recombinant vaccine using the same - Google Patents

Abstract

Disclosed is a novel Korean canine distemper virus found in Korea and a recombinant vaccine for canine distemper using the same.

Description

200540273 IX. Description of the invention: * [Technical field to which the invention belongs and prior art] The present invention relates to a novel Korean canine lean fever virus found in South Korea and a recombinant vaccine for canine distemper using the virus. Canine fever is a highly contagious and representative acute fever disease in dogs, and it can be seen regardless of region or season. This disease is a viral disease that causes high mortality. It develops into an acute or subacute state in puppies (Qing py) and appears through the gastrointestinal system, the epithelium, or the central nervous system, leading to being Infected young or old dogs with weakened immunity die. The clinical symptoms characteristic of canine distemper are acute inflammation and non-purulent encephalitis in the systemic mucosa, which develop in immunocompromised dogs, such as puppies less than 1 year old (especially 3-6 months) or Old dog. Canine distemper occurs due to oral or respiratory infections of canine distemper virus (hereinafter referred to as "CDV") belonging to the Paramyxovirus () family. It begins when the virus enters the dog's body and continues to show clinical signs With an incubation period of 3.6 days (average 4 days on average), individual viral infections form weak clinical symptoms. However, after CDV infection, these symptoms are often exacerbated by secondary bacterial infections, which are secondary bacterial infections Includes respiratory infections caused by bronchial sepsis (Bordetella palpitations) or Streptococcus pyogenes (stomach / lean), and gastrointestinal infections caused by Salmonella () or E. coli Infection. CD V is most commonly transmitted directly through droplet secretions from dogs infected with CDV, but also due to drip secretions contaminated from the feeding environment contaminated by CDV from nasal or eye excretion and urine. 5 200540273 CDV-transmitted infections eventually infect the entire body's immune system or individuals become impaired and die in the worst cases. Indirect infections are transmitted. Through the small upper respiratory system Into the blood stream and organs. Most of CDV infection 'and therefore susceptible to secondary bacterial infection,

Dou: .CDVs are enveloped RNA viruses that exhibit proteins on the outer envelope that play a role in their pathogenicity and immunogenicity. In particular, alpha has revealed the most antigenic variants in "hemagglutinin (Η) ^, which was involved in the initial viral invasion of host cells, and this protein is widely used as a primary marker for detecting genetic changes in the virus. Therefore In countries around the world: through reverse transcription polymerase chain reaction (hereinafter simply referred to as "RT-PCR"), a gene encoding the hemagglutinin (Η) gene of CDV transmitted in each country, and Restriction fragment length polymorphism (hereafter referred to as RFLP ") assay (in which restriction enzyme digestion is used & = many efforts have been made to prevent dogs with high transmission rates, and thus obtained A worldwide concern from the veterinary clinical field. Initial attempts were made in the United States, European countries, and elsewhere after the contact era to develop inaetivated vaccines using a virus derived from the culture of hamster lung cells. Cells multiply. Recently, live attenuated vaccines have been developed using tissue culture in many countries including South Korea, thus achieving effective prevention of canine disc fever. However, 'many recent reports : After 1 990: generation, in the nearest neighboring country, "there is a new mutant of the CDV isolate in dogs showing signs of canine lean fever syndrome, which has a different world Molecular biological characteristics of the CDV strains present.

6 200540273 η of CDV isolates, many different products were found and separated on an agarose gel to compare RFLP patterns between genes) for analysis of the current CDV vaccine wild CDV strains.

A recent phylogenetic analysis of the amino acid sequence of the plutonium gene in Japan revealed that at least two CDVH genotypes have been transmitted in dogs in Asian regions including Japan;-a species that is transmitted in almost all Japan CDV isolates all belong to its gene $, and another was not previously described in Japan. These two CDV H genotypes contain a plutonium gene that has amino acid sequences that differ from currently available vaccine strains and other lineages or genotypes of CDV strains that are prevalent in the United States, European countries, and elsewhere . In South Korea, good dogs have been immunized with DV vaccines produced in the United States or European countries, and they will gradually re-infect CDV. Based on this phenomenon, it is thought that genetically modified cdv exists especially in Korea. However, there is no precise evidence that a Korean CDv exists. In this regard, the inventors wanted to find a novel Korean CDV strain that has not been reported. SUMMARY OF THE INVENTION Therefore, the present invention aims to provide a novel Korean CDV that causes canine distemper in Korea and a recombinant vaccine for canine distemper using the virus. [Embodiment] The constitution of the present invention The virus of the present invention is a CD V that causes canine distemper, which is characterized in that it is a novel Korean CDV strain that was found especially in South Korea. As described above, phylogenetic differences based on the prion genes encoding prion proteins of CDV have been found, and a variety of variations have been found. A recent Japanese report describes the grouping (M.

Hashimoto et al., Archives of Virology, 2001, 146: 149-155). In the report, RFLp analysis revealed that

KDK-1 strains with different RFLP patterns of the η gene of the vaccine Onderstepoort were classified as Asian / Η1 genotype. In addition, 98-002 isoenzymes obtained from rectal and oral swab samples from individuals infected with CDV showed an RFLp profile different from the Asian / Η1 genotype in which CDV was isolated from swab samples The strain was classified as Asian / H2 genotype. Can be classified as Asian / H2 genotype 98-002, etc. does not refer to isolated viruses, but refers to the plutonium gene itself obtained from rectal and oral swab samples from individuals infected with CDV, that is, the above The report indicates the possibility of CDV of the Asian / Η2 genotype, but such a virus has not been substantially isolated (M. Mochizuki et al., Journal 〇fc 丨 inicai

Microbiology, 1999, 37: 2936-2942). FIG. 2 shows the results of RFLp analysis of a part of each gene of the virus of the present invention (lanes 4-9), mail tank ((ice lane 1), KDK-1 (lane 2), and 98_002 (lane 3), where η was amplified by RT-PCR under the same conditions. The virus of the present invention was found to have a η genotype b that is different from the conventional CDV cancer with the Asian / H1 genotype.人 The human genotype of this genotype genotype is similar to that of Asian / Η2 genotype 98. In addition, as shown in Figure 3, 200540273273, the CDV strain of the present invention is found to be a new genotype virus. The phylogeny is different from the conventional CDV vaccine strain. The CDV strain currently known in Japan. 98.002 is a prion gene obtained from dog rectal and oral swab samples, which can be used as a determinant-species Whether CM is rampant and has Asian / H2 genotype standards, but cannot directly affect the isolation and identification of individual viruses, because it is not a virus. Based on & Discovery 'will be isolated and identified according to the present invention and contains I'NO. I The shown η gene virus is named "Seoul% virus," which Hidden in the Korea Microbial Collection Center (KCCM) of the Korea Culture Collection Alliance on February 25, 2003, and has the number KCCMHM67. In addition, the present invention provides a recombinant vaccine for canine distemper using the present invention Canine distemper virus in whole or in part and contains pharmaceutically acceptable additives. The pharmaceutically acceptable additives may be selected from those commonly used in the art by those skilled in the art. Recombination for canine distemper The vaccine can be prepared using the canine distemper virus of the present invention by any method known in the art. Preferably, the recombinant vaccine is performed using the whole or a part of the Η gene shown in SEQ ID No. i of canine lean fever virus Preparation. The present invention can be better understood through the following examples. The following examples are provided to illustrate the present invention and should not be construed as limiting the present invention. Example 1: Isolation of canine distemper virus (Step 1) The sample was prepared in September 1998, and the body and blood samples of the Beijing Pug puppies 1 month old were submitted through an animal hospital in Seoul, Korea. The CDV immune history of this puppy is unknown, but it is diagnosed with canine distemper. (Step 2) Evaluation of CDV infection Check the CDV infection with the following, that is, the supernatant taken from the organ of the puppy and using the isolated CDV After sensitization, the supernatant of sensitive cells forming cytopathic effect (CPE) and the DNA of Onderstepoortort gene cloned into plastids are obtained.

250 μΐ of each sample was thoroughly mixed with 750 μΐ of RNA-binding salt (trade name; RNaid Kit, BIO10101), and reacted with 10 μΐ of RNAMATRIX (trade name; RNaid Kit, BIO10101) at room temperature for 5 minutes, while often shaking to Prevents RNAMATRIX precipitation. The reaction mixture was centrifuged at 10,000 rpm for 1 minute to precipitate a binding product of RNA and RNAMATRIX. After discarding the supernatant, the centrifugation was repeated again, and the supernatant was completely removed. The pellet was suspended in a 500 μΐ RNA wash solution (RNaid Kit, BIO101) using a micro-pipette, and centrifuged at 10,000 rpm for 1 minute. After discarding the supernatant, the washing step was repeated two more times. After the last wash, centrifuge again and remove the supernatant completely. The pellet was suspended in a 20 μΐ RNase-free distilled water (RNaid Kit, BIO101) using a micro-pipette, and incubated at 50 ° C for 5 minutes to extract RNA. After centrifuging the suspended granular pellets at 1 5,000 rpm for 2 minutes, the supernatant was transferred to a new tube. Reverse Transcription 9 μΐ RNA samples and 1 μΐ random primers (50 pmol / μΐ, 10 200540273

TaKaRa) and allowed to react at 70 ° C for 10 minutes. The sample was immediately placed on ice to stop the reaction. Mix the primary reaction mixture with 4 μΐ 5x RT reaction buffer (RT AMV XL Kit, TaKaRa), 4 μΐ 10 mM dNTP (RT AMV XL Kit, TaKaRa), and 1 μΐ RNasin (20 U / μ Promega) and let It was reacted at 25 ° C for 5 minutes. Then, the resulting reaction mixture was supplemented with 1 μΐ of RT AMV XL (35 U / μΙ, RT AMV XL Kit, TaKaRa), and the conditions were performed at 25 ° C for 10 minutes, 42 ° C for 50 minutes, and 70 ° C for 10 minutes. Reverse transcription to synthesize complementary # DNA (hereinafter referred to as "cDNA"). CDV identification using polymerase chain reaction (PCR) CDV infection is determined by PCR amplification using the obtained cDNA. In a PCR tube, mix 1 μΐ of the cDNA solution with 5 μΐ 10x PCR reaction buffer, 3 μΐ 25 mM MgCl2, 4 μΐ 10 mM dNTP, 1 μΐ Bow I I (CDV H13, CAA / GAC / AAG / GTG / GGT / GCC / TT, lit 33-52 of the H gene of Onderstepoort, 1 μΐ primer II (CDV H18, • CTT / GGT / GAA / ATC / GAA / CTC / CA, nt 207 of the H gene of Onderstepoort -188), 0.5 μΐ Taq polymerase (5 U / μ TaKaRa) and 34.5 μΐ sterile distilled water. Perform PCR with a PCR instrument under certain conditions, including: 1 minute at 94 ° C; 30 seconds at 55 ° C, 30 seconds at 72 ° C, and 30 seconds at 94 ° C, 30 cycles; and an extension at 72 ° C for 5 minutes . The PCR product (175 bp) was then run on a 1.2% agarose gel. The results are given in Figure 1. As shown in Figure 1, the electrophoresis results were consistent in the following samples: 11 200540273 brain (lane 1), bronchi (lane 2), and lung (lane 3) of a dead puppies showing canine distemper symptoms, and Cells sensitized with CDV isolated from it (lane 4) and Onderstepoort strain (lane 5). These results show that the virus whose RNA was isolated from puppies is CDV. Example 2: Evaluation of whether the virus of the present invention has the Asian / H2 genotype (Step 1) Preparation of DNA samples cDNA synthesis

The virus and organ emulsion prepared in Example 1 were subjected to RT-PCR according to the same method as in Example 1 to synthesize cDNA. For KDK-1, 98-002, and Onderstepoort, a plutonium gene previously selected into the plastid was used as a DNA template. (Step 2) RFLP analysis RFLP analysis was performed using the prepared cDNA sample and reference DNA sample (Onderstepoort, KDK-1, and 98-002) to determine the genotype genotype of the virus of the present invention and the conventional CDV strain. Analysis of PCR amplified genes DNA was amplified using the cDNA prepared in Step 1 of Example 2. In a tube, mix 1 μΐ cDNA solution with 5 μΐ 10x PCR reaction buffer, 3 μΐ 25 mM MgCl2, 4 μΐ 10 mM dNTP, 1 μΐ Primer I (CDV F10B, TAT / CAT / GAC / RGY / ART / GGT / TC), 1 μΐ Primer II (CDV R10, CTT / GGT / GAA / ATC / GAA / CTC / CA), 0.5 μΐ Taq polymerase (5 U / μΙ, TaKaRa), and 34.5 μΐ sterile distilled water. Perform PCR with a PCR instrument under certain conditions, including: 12 200540273 94 ° C for 1 minute; 55 ° C for 2 minutes, 72 ° C for 2 minutes, and 94 ° C for 1 minute, 35 cycles; and the final 72 ° C extension 5 minutes. The PCR products were then run on a 1% agarose gel. It was found that the amplified DNA has a size of 87 1 bp, which is equivalent to nt 7,991-8,861 of the Onderstepoort's Η gene. Selecting effective genes from agarose gels Selecting effective genes from agarose gels, purifying them, and The RFLP pattern was evaluated by restriction enzyme digestion. With surgery

# The knife cuts the effective gene band from the agarose gel, and at the same time removes the gel component as much as possible, and weighs it with an electronic weighing machine. Then, the gel pieces were mixed with 200 μΐ of Nal solution (GENECLEAN II Kit, BIO101), incubated at 50 ° C. for 5 minutes to dissolve the gel components, and mixed with 10 μΐ of GLASSMILK (trade name; GENECLEAN II Kit, ΙΟ101). Incubate the reaction mixture at room temperature for 5 minutes to allow DNA to bind to the silica component of GLASSMILK, and often tap the tube with your fingers to suspend the dioxide. The reaction mixture was then centrifuged at 10,000 rpm for 5 seconds to precipitate DNA-bound silica. The granular precipitate was washed with 1 ml of NEW WASH (trade name; GENECLEAN II Kit, BIOl 01) using a micro-dispenser, and the washing was repeated two more times. The tube was placed in vacuum for 5 minutes to completely remove the supernatant, and the pellet was suspended again in 15 μΐ of sterile water and centrifuged at 15,000 rpm for 30 seconds. Transfer the supernatant containing the effective gene to a new sterile tube. Repeat this step once more and combine the secondary and primary supernatants and store at -20 ° C. RFLP analysis of each viral DNA 13 200540273 The effective genes isolated from the agarose gel were digested with restriction enzymes and electrophoresed to determine the RFLP pattern of each viral DNA. Mix 26 μΐ of the effective genes isolated from the agarose gel with 3 μΐ 1 Ox restriction enzyme buffer (NEBuffer 4, NEB) and 1 μΐ Ndel (20 U / μ NEB) and mix in an incubator Incubate at 37 ° C for 8 hours and 30 minutes. The reaction mixture was then at 0.8 ° /. Electrophoresis was performed on an agarose gel. The results are given in Figure 2. As shown in Figure 2, KDK- # 1 strain (lane 2) known to have the Asian / H1 genotype was found to have an RFLP map different from 98-002 DNA (lane 3) known to have the Asian / H2 genotype formula. Organ suspensions (lanes 5-9) and virus-sensitized cells (lanes 4) obtained from puppies infected with the CDV of the present invention were found to have the same RFLP pattern as 98-002 DNA. These results show that, unlike previously isolated CDV strains, the CDV strains of the present invention belong to the Asian / H2 genotype. Example 3: Nucleotide sequence analysis of the tadpole gene

After using the cDNA prepared in Example 1 (Step 2) to PCR-amplify the DNA encoding the Η gene, the nucleotide sequence of the Η gene of the canine distemper virus (Asia / H2 genotype) of the present invention was amplified. Perform analysis. In a PCR tube, mix 1 μΐ cDNA solution with 5 μΐ 10x PCR reaction buffer, 3 μΐ 25 mM MgCl2, 4 μΐ 10 mM dNTP, 1 μΐ Primer I (CDV F8, GTT / GTT / GCT / GAT / TTA / CTG / TT, nt 6800-6819 of the H gene of Onderstepoort), 1 μΐ primer II (CDV R8, CCC / CGT / CTG / TTA / TTT / TGC / TA, nt 9399-93 80 of the H gene of Onderstepoort), 0.5 μΐ EX Taq polymerase (5 U / μΙ, TaKaRa) 14 200540273 and 34.5 μΐ sterile distilled water were mixed. Use a PCR instrument to perform PCR under certain conditions, including 94 ° C for 1 minute and 30 seconds; 52. Cantonese: 1 minute and 30 seconds, 72 ° C for 2 minutes, and 94 ° C for 1 minute and 30 seconds, 35 cycles; and the final 72 ° C extension for 20 minutes. The PCR product was then inserted into a colony load = (PTZ57R, MBI Fermentas). Then, the nucleotide sequence of the plutonium gene of the canine distemper virus (Asia / H2 genotype) of the present invention was determined, and the sequence is shown in SED ID NO · 1. [Advantageous Effects] As mentioned above, the present invention provides a novel Korean canine distemper virus, which is particularly found in South Korea among canine distemper viruses that cause canine distemper, and also provides a virus for dogs using the virus. Spasmic recombinant vaccine. [Brief Description of the Drawings] Fig. 1 shows the results of electrophoretic analysis for determining whether the virus of the present invention is CDV (Canine Distemper Virus). Figure 2 shows the results of an RFLP analysis used to determine whether the virus of the present invention is a CDV belonging to the Asian / h2 genotype. Fig. 3 shows the position of the nucleotide sequence of the plutonium gene of the Korean CDV of the present invention in the phylogenetic tree. [Description of Symbols of Main Components] (None) 15

Claims (1)

200540273 10. Scope of patent application ... 1. An Asian / H2 genotype canine distemper virus, which is used to prepare a vaccine for canine distemper and has the number KCCM 10467. 2. The canine distemper virus according to item 1 of the scope of the patent application, which contains the tadpole gene shown in SEq ID NO. 1. 3. A recombinant vaccine for canine distemper prepared by using whole or part of a canine distemper virus according to item 1 or 2 of the patent application. Eleven, schema: as the next page 16