Title of Invention	ISOLATION OF A KOREAN CANINE DISTEMPER VIRUS OF ASIA/H2 GENOTYPE, AND PREPARATION OF RECOMBINANT VACCINE THEREFROM
Abstract	Disclosed is a novel Korean canine distemper virus found in Korea and a recombinant vaccine for canine distemper using the same.

Full Text

CANINE DISTEMPER VIRUS ISOLATED IN KOREA AND RECOMBINANT VACCINE USING THE SAME Technical Field and Prior Art The present invention relates to a novel Korean canine distemper virus found in Korea and a recombinant vaccine for canine distemper using the same. Canine distemper is a very highly infectious, representative acute febrile disease among dogs, which occurs regardless of regions and seasons. The disease is a viral disease entailing high mortality, which progresses to acute or subacute states in young puppies and occurs through the gastrointestinal, respiratory, epithelial or central nervous systems, leading to death of infected young puppies or old dogs with weak immunity. The characteristic clinical symptoms of canine distemper are acute inflammation and non-suppurative encephalitis in mucous membranes throughout the body, which develop in dogs with weak immunity, such as young puppies lower than one year old (especially between three and six month old) or old dogs. Canine distemper occurs by infection with canine distemper virus (hereinafter, referred to simply as "CDV"), which belongs to the family Paramyxovirus, through the oral or respiratory tract. There is a latent period of 3 to 6 days (average 4 days) from the time the virus enters a dog's body until clinical signs appear, and the single viral infection develops weak clinical symptoms. However, after CDV infection, these symptoms are often exacerbated by secondary bacterial infections, including respiratory infections with Bordetella bronchiseptica or Streptococcus hemolyticus and gastrointestinal infections with Salmonella or E. coll CDV is most often transmitted directly through droplet secretions from CDV-infected dogs, and is also spread by indirect infection through breeding environments contaminated with CDV excreted from nasal or eye discharge and urine. CDV spread via droplet secretions infects the upper respiratory system, enters the blood stream, and eventually infects the systemic immune system or organs. Most individuals infected with CDV come to have weak immunity, are thus susceptible to secondary bacterial infections, and, in the worst cases, die. Many efforts were made to prevent canine distemper, which is highly contagious and thus receiving worldwide interest from the veterinary clinical field. Initial attempts were made in the United States, European countries, and elsewhere, after the 1960s, to develop inactivated vaccines using viruses propagated in cultured cells derived from hamster lung cells. Recently, in many countries including Korea, attenuated live vaccines have been developed using tissue culture, thus realizing effective prevention against canine distemper. However, many recent studies reported that, among CDV isolates fiom dogs displaying clinical signs of canine distemper in the nearest country, 1A Japan, after the 1990s, new mutant strains, which have molecular biological properties different from existing CDV strains common worldwide, are present. CDV is an enveloped RNA virus, which expresses two types of proteins responsible for its pathogenicity and immunogenicity at the external envelope. In particular, the highest antigenic variation has been found in the "hemagglutinin (H) protein", participating in the initial viral invasion into host cells, and this protein is widely used as a major marker to detect genetic changes of the virus. Accordingly, in worldwide countries, when hemagglutinin (H) genes encoding the H protein of CDVs circulating in each country were amplified by Reverse Transcription-Polymerase Chain Reaction (hereinafter, referred to simply as "RT-PCR"), and analyzed by Restriction Fragment Length Polymorphism (hereinafter, referred to simply as "RFLP") assay, in which the RT-PCR products are digested with restriction enzymes and separated on an agarose gel to compare RFLP patterns between the H genes of the CDV isolates, many field CDV strains different from current CDV vaccines have been found. Recently, phylogenetic analysis of the H-gene amino acid sequences in Japan revealed that at least two CDV H-gene genotypes are circulating among dogs in Asia including Japan; one is a genotype to which almost all Japanese CDV isolates, previously circulating in Japan, belong, and the other has not been previously described in Japan. The two CDV H-gene genotypes contain an H gene having an amino acid sequence different from that of the other lineages or genotypes of currently available vaccine strains and CDV strains prevalent in the United States, European countries, and elsewhere. In Korea, even though dogs have been vaccinated with CDV vaccines produced in the United States or European countries, they have been increasingly re-infected with CDVs. Based on this phenomenon, genetically modified CDVs are considered to be present particularly in Korea. However, there is no accurate evidence for the presence of Korean CDVs. In this regard, the present inventors intend to find a novel Korean CDV strain that has not been reported yet. Technical Problem Accordingly, the present invention aims to provide a novel Korean CDV causing canine distemper in Korea and a recombinant vaccine for canine distemper using the same, Brief Description of Drawings FIG. 1 shows the results of electrophoresis analysis for determining whether a virus of the present invention is a CDV (Canine Distemper Virus). 2 FIG. 2 shows the results of RFLP analysis for determining whether a vims of the present invention is a CDV belonging to the Asia/H2 genotype. FIG. 3 shows the location of the nucleotide sequence of the H gene of a Korean CDV of the present invention in a phylogenetic tree. Construction of the Invention A virus of the present invention is a CDV causing canine distemper, which is characterized by being a novel Korean CDV strain found particularly in Korea. As described above, diverse variations have been found based on the phylogenetic difference between H genes encoding the H protein of CDV. A recent Japanese report described the phylogenetic grouping for CDVs found in Asia (M. Hashimoto et al., Archives of Virology, 2001, 146:149-155). In this report, RFLP analysis revealed that the KDK-1 strain, having an RFLP pattern different from the H gene of a conventional CDV vaccine, Onderstepoort, was classified into the Asia/Hi genotype. Also, 98-002 et al., H genes obtained from rectal and oral swab specimens taken from individuals infected with CDV, showed RFLP profiles different from those of the Asia/H1 genotype, wherein CDV isolates in the swab specimens were classified into the Asia/H2 genotype. However, the 98-002 et al., classified into the Asia/H2 genotype, indicates not the isolated viruses but the H gene itself taken from the rectal and oral swab specimens of individuals infected with CDV. That is, the above report indicates the possibility of the presence of CDVs of Asia/H2 genotype, but such viruses have not been substantially isolated (M. Mochizuki et al., Journal of Clinical Microbiology, 1999,37:2936-2942). FIG. 2 shows the results of RFLP analysis of a portion of each of H genes of the virus of the present Invention (lanes 4 to 9), Onderstepoort (lane I), KDK-1 (lane 2) and 98-002 (lane 3), wherein the H genes were amplified by RT-PCR under the same conditions. The viRUs of (he present invention was found to have an H gene genotype, which is different from that of a conventional CDV vaccine, Onderstepoort, and the KDK-1 strain classified into the Asia/H1 genotype, but is similar to that of the 98-002, classified into the Asia/H2 genotype. Also, as shown in FIG, 3, the CDV strain of the present invention was found to be a virus belonging to a new genotype distinct phylogenetically from conventional CDV vaccine strains or CDV strains presently known in Japan. The 98-002, which is the H gene taken from the rectal and oral swab specimens of dogs, is available as the criteria for determining whether a CDV has the Asia/H2 genotype, but does not directly affect the isolation and identification of individual viruses because it is not a virus. Based on this finding, the virus comprising an H gene represented by SEQ ID NO. 1, which was isolated and identified according to the present invention, was designated as "Seoul 98 3 viris", which was deposited at the Korean Culture Center of Microorganisms (KCCM) affiliated with the Korean Federation of Culture Collections on Feb. 25, 2003 and assigned accession number KCCM 10467. In addition, the present invention provides a recombinant vaccine for canine distemper, using the whole or a portion of the canine distemper virus of the present invention and comprising a pharmaceutically acceptable additive. The pharmaceutically acceptable additive may be selected from additives commonly used in the art by those skilled in the art. The recombinant vaccine for canine distemper may be prepared using the canine distemper virus of the present invention by any method known in the art. Preferably, the recombinant vaccine is prepared using the whole or a portion of the H gene, represented by SEQ ID NO. 1, of the canine distemper virus. A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention. EXAMPLE 1: Isolation of canine distemper virus (Step 1) Specimen preparation Dead bodies and blood samples of Pekingese puppies one month old, of which the CDV vaccination history was unknown but which was diagnosed with canine distemper, were submitted by an animal hospital in Seoul, Korea, on September 1998. (Step 2) Evaluation of CDV infection CDV infection was examined in supernatants taken from organs of the puppies, supernatants of sensitive cells developing a cytopathic effect (CPE) after sensitization with an isolated CDV, and Onderstepoort H gene DNA cloned into a plasmid. Isolation of CPVRNA 250 ul of each sample was well mixed with 750 pi of RNA binding salt (trade name; RNaid Kit, BIO101), and reacted with 10 pi of RNAMATRK (trade name; RNaid Kit, BIO101) at room temperature for 5 min while being shaken often to prevent the RNAMATRIX from precipitating. The reaction mixture was centriiliged at 10,000 rpm for 1 min to precipitate the binding products of RNA and RNAMATRIX. After the supernatant was discarded, the centrifugation was repeated again, and the supernatant was completely removed. The pellet was suspended in 500 pi of an RNA washing solution (RNaid Kit, BIO101) with a micropipette, and centrifuged at 10,000 rpm for 1 min. After the supernatant was discarded, this washing step was repeated twice more. After the final washing, centrifugation was carried out once more, and the supernatant was completely removed. 4 The pellet was suspended in 20 µ\ of RNase-free distilled water (RNaid Kit, BIO101) with a micropipette, and incubated at 50°C for 5 min to extract RNA. After the suspended pellet was centrifuged at 15,000 rpm for 2 min, the supernatant was transferred into a new tube. Reverse Transcription 9 uj of a RNA sample was mixed with 1 µ l of random primers (50 pmol/ µ l, TaKaRa) and allowed to react at 70°C for 10 min. Immediately thereafter, the sample was placed on ice to terminate the reaction. The primary reaction mixture was mixed with 4 µ 1 of 5x RT reaction buffer (RT AMV XL Kit, TaKaRa), 4 µ l of 10 mM dNTP (RT AMV XL Kit, TaKaRa) and 1 µ l of RNasin (20 U/ µ l, Promega), and allowed to react at 25°C for 5 min. Then, the resulting reaction mixture was supplemented with 1 µ 1 of RT AMV XL (35 U/ µ l, RT AMV XL Kit, TaKaRa), and reverse transcription was carried out at 25°C for 10 min, 42°C for 50 min and 70°C for 10 min to synthesize complementary DNA (hereinafter, referred to as "cDNA"). Identification of CDV using Polymerase Chain Reaction (PCR) DNA was amplified by PCR using the obtained cDNA to determine CDV infection. In one PCR tube, 1 ui of the obtained cDNA solution was mixed with 5 µ l of 10x PCR reaction buffer, 3 µ l of 25 mM MgCl2, 4 µ l of 10 mM dNTP, 1 µ l of primer I (CDV HO, CAA/GAOAAG/GTG/CKJT/GCOTT, nt 33-52 of the H gene of Onderstepoort), 1 µ l of primer II (CDV HI 8, CTT/GGT/GAA/ ATC/GAA/CTC/CA, nt 207-188 of the H gene of Onderstepoort), 0.5 pi of Taq polyrnerase (5 U/ul, TaKaRa), and 34.5 ul of sterile distilled water. PCR was carried out using a PCR machine under conditions including 94°C for 1 min, 30 cycles of 55°C for 30 sec, 72°C for 30 sec and 94°C for 30 sec, and a final extension of 72°C for 5 min. The PCR products (175 bp) were then electrophoresed on a 1.2% agarose gel. The results are given in FIG. 1. As shown in FIG. 1, electrophoresis results were consistent among the brain (lane 1), the bronchial tube (lane 2) and the lung (lane 3) of the dead puppies displaying canine distemper symptoms, cells (lane 4) sensitized with a CDV isolated therefrom, and the Onderstepoort strain (lane 5). These results indicate that a virus whose RNA was isolated from the puppies is a CDV. EXAMPLE 2: Evaluation of the virus of the present invention for having the Asia/H2 genotype (Step 1) Preparation of DNA samples cDNA synthesis The viruses and the organ emulsions, 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, H genes previously cloned into a plasrnid were used as DNA templates. 5 (Step 2) RFLP analysis RFLP analysis was carried out to determine H gene genotypes of the present virus and the conventional CDV strains using the prepared cDNA samples and reference DNA samples (Onderstepoort, KDK-1 and 98-002). Analysis of gene amplified by PCR DNA was amplified using the cDNA prepared at the step 1 of Example 2. In one tube, 1 ul of the cDNA Solution was mixed with 5 µ 1 of 10x PCR reaction buffer, 3 ul of 25 mM MgCl2,4 µ l of 10 mM dNTPy 1 µ 1 of primer I (CDV Fl OB, TAT/CAT/GAORGY/ART/GGT/TC), 1 µ 1 of primer (CDV RIO'CTT/GGT/GAA/ATaGAA/CTC/CA), 0.5 µ l of Taq polymerase (5 U/ µ l, TaKaRa), and 34.5 ul of sterile distilled waters PCR was carried out using a PCR machine under conditions including 94°C for 1 min, 35 cycles of 55°C for 2 min, 72°C for 2 min and 94°C for 1 min, and a final extension of 72°C for 5 min. The PCR products were then electrophoresed on a 1% agarose gel. The amplified DNA was found to have a size of 871 bp, which was corresponding to nt 7,991-8,861 of the H gene of Onderstepoort. Selection of effective gene from agarose gel An Effective gene was selected from the agarose gel, purified and assessed for RFLP patterns by restriction enzyme digestion. An effective gene band was excised from the agarose gel using a surgical knife while, excluding the gel component as much as possible, and weighed with an electric weighing machine. Then, the gel piece was mixed with 200 ul of Nal solution (GENECLEAN II Kit, BIO101), incubated at 50°C for 5 min to dissolve the ge! component, and mixed with 10 nl of GLASSMILK (trade name: GENECLEAN II Kit, BIO101). The reaction mixture was incubated at room temperature for 5 min to allow the binding of DNA to the silica component of GLASSMILK, while the tube was often tapped with fingers to suspend silica Thereafter, the reaction mixture was centrifuged at 10,000 rpm for 5 sec to precipitate silica bound to DNA. The pellet was washed with 1 ml of NEW WASH (trade name: GENECLEAN H Kit, BIO101) using a micropipette, and this washing was repeated twice more. The tube was placed under vacuum for 5 min to completely remove the supernatant, and the pellet was ^suspended in 15 ul of stenle water and centrifuged at 15,000 rpm for 30 sec. The supernatant containing an effective gene was transferred into a new sterile tube. The step was repeated once more, and the secondary suoernatant was combined with the primary supernatant and stored at -20°C. RFLP analysis of each viral DNA The effective gene isolated from an agarose gel was digested with a restriction enzyme and electrophoresed to determine RFLP patterns of each viral DNA. 26 ul of the effective gene isolated from an agarose gel was mixed with 3 (il of 1 Ox restriction enzyme buffer (NEBuffer 4, NEB) and 1 ul of Ndel (20 U/ul, NEB), and incubated in an incubator at 37°C for 8 hre 30 min. The reaction 6 mixture was then electrophoresed on a 0.8% agarose gel. The results are given in FIG. 2. As shown in FIG. 2, the KDK-1 strain (lane 2), known to have the Asia/Hi genotype, was found to have a RFLP pattern different fan that of the 98-002 DNA (lane 3) known to have the Asia/H2 genotype, The organ suspension (lanes 5 to 9), taken from die puppies infected with the CDV of the present invention, and the virus-sensitized cells (lane 4) were found to have an RFLP pattern identical to that of the 98-002 DNA. These results indicate that, unlike previously isolated CDV strains, the CDV strain of the present invention belongs to the Asia/H2 genotype. EXAMPLE 3: Nucleotide sequence analysis of the H gene After DNA encoding the H gene was amplified by PCR using cDNA prepared in Example 1 (Step 2), the nucleotide sequence of the H gene of the canine distemper virus (Asia/H2 genotype) of the present invention was analyzed. In one PCR tube, 1 µ l of the cDNA solution was mixed with 5 µ \ of 10x PCR reaction buffer, 3 µ l of 25 mM MgC!2,4 µ l of 10 mM dNTP, 1 µ l of primer I (CDV F8, GTT/GTT/GCT/GAT/rTA/CTG/rT, nt 6800-6819 of the H gene of Onderstepoort), 1 µ l of primer II (CDV R8, CCaCGT/CTG/nW TTT/TC3GTA, nt 9399-9380 of the H gene of Onderstepoort), 0.5 uJ of EX Taq polymerase (5 U/ µ l, TaKaRa), and 34.5 µ l of sterile distilled water. PCR was carried out using a PCR machine under conditions including 94°C for 1 min 30 sec, 35 cycles of 52°C for 1 min 30 sec, 72°C for 2 min and 94°C for 1 min 30 sec, and a final extension of 72°C for 20 min. The PCR products were then inserted into a cloning vector (pTZSTR, MBI Fermentas). Then, the nucleotide sequence of the H gene of the canine distemper virus (Asia/H2 genotype) of the present invention, represented by SED ID NO. 1, was determined. Advantageous Effects As described hereinbefore, the present invention provides a novel Korean canine distemper virus found particularly in Korea among canine distemper viruses causing canine distemper, and a recombinant vaccine for canine distemper using the same. ABSTRACT Disclosed is a novel Korean canine distemper virus found in Korea and a recombinant vaccine for canine distemper using the same. / We claim : 1. A canine distemper virus of Asia/H2 genotype, which is used for preparing a vaccine for canine distemper and has accession number KCCM 10467. 2. The canine distemper virus according to claim 1, which comprises an H gene represented by SEQ ID NO. 1. 3. A recombinant vaccine for canine distemper, which is prepared using the whole or a portion of the canine distemper virus of claim 1 or 2. Dated this 8th day of MARCH, 2005. Disclosed is a novel Korean canine distemper virus found in Korea and a recombinant vaccine for canine distemper using the same.

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00145-kol-2005 correspondence-1.1.pdf

00145-kol-2005 correspondence-1.2.pdf

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00145-kol-2005 correspondence.pdf

00145-kol-2005 description (complete).pdf

00145-kol-2005 drawings.pdf

00145-kol-2005 form-1-1.1.pdf

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00145-kol-2005 form-18.pdf

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00145-kol-2005 priority document others.pdf

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145-KOL-2005-FORM 27.pdf

145-KOL-2005-FORM-27.pdf

145-kol-2005-granted-abstract.pdf

145-kol-2005-granted-claims.pdf

145-kol-2005-granted-correspondence.pdf

145-kol-2005-granted-description (complete).pdf

145-kol-2005-granted-drawings.pdf

145-kol-2005-granted-examination report.pdf

145-kol-2005-granted-form 1.pdf

145-kol-2005-granted-form 18.pdf

145-kol-2005-granted-form 2.pdf

145-kol-2005-granted-form 3.pdf

145-kol-2005-granted-form 5.pdf

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