CN113025630A - Infectious cDNA clone of fox-derived canine distemper virus virulent HBF-1 strain and establishment of reverse genetic system - Google Patents

Abstract

The invention discloses infectious cDNA cloning of fox-derived canine distemper virus virulent HBF-1 strain and establishment of a reverse genetic system. The infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain is obtained by reverse genetic operation; the nucleotide sequence of the infectious cDNA clone is shown as SEQ ID No. 1. The reverse genetic operation system consists of a recombinant plasmid containing infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain and three auxiliary plasmids for expressing HBF-1N protein, P protein and L protein. Compared with the parent virus wtHBF-1, the recombinant virus rHBF-1 obtained by rescue through the reverse genetic operation system has similar growth characteristics, and the size and the shape of the recombinant virus are consistent with those of a typical canine distemper virus. The invention provides a basis and a technical means for rescuing fox-derived canine distemper virus virulent HBF-1 strain and deeply exploring a pathogenic attenuated molecular mechanism of the canine distemper virus.

Description

Infectious cDNA clone of fox-derived canine distemper virus virulent HBF-1 strain and establishment of reverse genetic system

Technical Field

The invention relates to the construction of infectious cDNA clone and reverse genetic system of virus, in particular to the construction of infectious cDNA clone and reverse genetic system of fox source canine distemper virus virulent HBF-1 strain. The invention belongs to the field of biotechnology.

Background

The natural infection host of Canine Distemper Virus (CDV) is increasing and poses a life threat to a wide variety of terrestrial and aquatic carnivores worldwide. CDV has different pathogenicity to different susceptible hosts, can cause systemic diseases with different degrees, and has different lethality rates. Wherein, the morbidity and mortality of the domestic cats are 0 percent, the domestic dog is 50 percent, and the ferrets reach 100 percent. Canine distemper causes great economic loss to fur-bearing animal breeding industry and wild animal protection industry. At present, the number of pet dogs and cats owned by all people in China increases year by year, and the influence of canine distemper on the public health of people is not ignored. Furthermore, it has been demonstrated that CDV fusion proteins and hemagglutinin proteins promote osteoclast formation in Paget's disease and humans may become new hosts for CDV.

CDV is a member of the family Paramyxoviridae (Paramyxoviridae) measles virus (Morblivirus) and is a single-stranded negative-strand, non-segmented RNA virus. CDV is divided into 7 genotypes, but only 1 serotype, according to the genetic diversity of the hemagglutinin (H) gene. The CDV genome comprises the following components from the 3 'end to the 5' end: a3 'end non-coding region (3' UTR), a nuclear gene (N), a phosphorus gene (P) (the interior of which contains two non-structural protein genes C and V), a matrix gene (M), a fusion gene (F), a hemagglutinin gene (H), a macropolymerase gene (L) and a 5 'end non-coding region (5' UTR). Under the wrapping of nucleoprotein N, polymerase protein (L) and its accessory factor phosphoprotein (P) act together to form nucleocapsid (RNP), which is the minimum infection unit of CDV, and the sequence following the '6 base principle' is synthesized to cap and start the transcription and translation of virus, so as to generate progeny virus.

The invention of reverse genetic technique provides a powerful tool for in vitro modification of viral RNA genome because the genome of RNA virus is difficult to be arbitrarily operated in vitro. At present, scholars at home and abroad establish a plurality of CDV reverse genetic systems, but mainly aim at CDV attenuated vaccine strains. The CDV attenuated vaccine strain and the CDV virulent strain have larger difference in nucleotide sequence, and the attenuated vaccine strain generally does not cause the morbidity and the death of animals, and is not suitable for explaining the pathogenic mechanism of the canine distemper virus. In addition, the strong toxicity of CDV can cause the morbidity and mortality of animals, but the isolation and culture in vitro are difficult. The CDV virulent virus cannot infect a wild type Vero cell, and only can infect a stable cell line expressing a Signal Lymphocyte Activation Molecule (SLAM), so that the in vitro large-scale proliferation is realized. As adaptation of CDV virulent virus to cells and the number of passages increase, it is likely that the virulence and pathogenicity of CDV are reduced. These characteristics of CDV virulent virus greatly limit the establishment of a reverse genetic system and the rescue and propagation of recombinant viruses related to CDV virulent virus. Therefore, the invention constructs a reverse genetic system of the fox-derived canine distemper virus virulent strain HBF-1. The canine distemper virus virulent strain HBF-1 is a virulent virus separated from an arctic fox disease sample (breeding and pathogenicity research of the canine distemper virus virulent strain HBF-1, high break and the like, China prevention veterinary newspaper, 12 months 2012, volume 32, and phase 12). In order to distinguish the wild-type CDV virus wtHBF-1 strain, a genetic marker locus is artificially added into pcDNA3.2-HBF-1. The in vitro growth characteristics of recombinant virus rHBF-1 and wtHBF-1 were compared. Data results show that: the research successfully rescues the constructed fox-derived CDV virulent HBF-1 reverse genetic system to obtain the recombinant virus rHBF-1, wherein the rHBF-1 has the growth characteristic similar to that of wild type wtHBF-1. The invention provides a powerful platform and a tool for deeply researching the pathogenesis and pathogenesis of CDV, pathogenicity and novel vaccine in the future.

Disclosure of Invention

One of the purposes of the invention is to provide an infectious cDNA clone of fox-derived canine distemper virus (HBF) -1 strain;

the second purpose of the invention is to establish a reverse genetic system of the fox-derived canine distemper virus virulent HBF-1 strain;

the third purpose of the invention is to provide the application of the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain and the reverse genetic system thereof in rescue of the fox-derived canine distemper virus virulent HBF-1 strain and reverse genetic research.

In order to achieve the purpose, the invention adopts the following technical means:

the fox-derived canine distemper virus infectious cDNA clone is constructed by taking a fox-derived canine distemper virus virulent HBF-1 strain adapted to Vero-dSlam cells as a template. After obtaining the whole genome sequence of the virus, the RT-PCR method is used to amplify the whole genome of the canine distemper virus by 4 long segments, and the 4 long segments are sequentially spliced and inserted into the polyclonal enzyme cutting site of eukaryotic expression vector pcDNA3.2 (obtained by transforming the polyclonal site from pcDNA3.1) through the organic combination of enzyme digestion connection and seamless cloning. The full-length cDNA recombinant plasmid (pcDNA3.2-HBF-1) of fox-derived canine distemper virus virulent HBF-1 strain is determined and obtained through enzyme digestion identification, and three auxiliary plasmids (pcDNA3.1-HBF-N, pcDNA3.1-HBF-P and pcDNA3.1-HBF-L) for expressing the canine distemper virus virulent HBF-1 strain N, P, L protein are constructed at the same time. The full-length recombinant plasmid pcDNA3.2-HBF-1 has a sequence consistent with the expectation through enzyme digestion and sequence determination identification. The full-length plasmid pcDNA3.2-HBF-15 mu g, three helper plasmids pcDNA3.1-N1 mu g, pcDNA3.1-P0.8 mu g and pcDNA3.1-L0.5 mu g, a transfection reagent X-treme GENE HP DNA 22 mu L, a BSR cell is CO-transfected, after 3 days, the cell suspension is inoculated on Vero-dSlam cells, and 5% CO is at 37 DEG C₂After 5-10 days of culture, typical syncytial lesions caused by canine distemper virus were observed. The recombinant virus rHBF-1 strain is verified to be rescued through RT-PCR, indirect Immunofluorescence (IFA) and electron microscope observation and identification. Therefore, the invention successfully establishes a reverse genetic system of the fox-derived canine distemper virus strong virus HBF-1 strain, and provides a basis and a technical means for rescuing the fox-derived canine distemper virus strong virus HBF-1 strain and deeply exploring a pathogenic attenuated molecular mechanism of the canine distemper virus.

Therefore, on the basis of the research, firstly, the invention provides an infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain, wherein the infectious cDNA clone is obtained by reverse genetic manipulation of the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain; the nucleotide sequence of the infectious cDNA clone is shown as SEQ ID No. 1.

Secondly, the invention also provides a method for obtaining the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain, which comprises the following steps:

(1) extracting total RNA of HBF-1 virus suspension;

(2) the obtained RNA was subjected to reverse transcription to obtain cDNA.

(3) Utilizing four pairs of primers F1/R1, F2/R2, F3/R3 and F4/R4, taking the reverse transcription product cDNA of HBF-1 genome RNA as a template, amplifying HBF-1 divided into 4 sections by using high-fidelity DNA polymerase, identifying the PCR product by agarose gel electrophoresis, and performing gel recovery after the size is correct;

F1 5’-GCGGCCGCTGTTAAGCGTCTGATGAGTCCGTGAGGACGAAACTATAGGAAAGGAATTCCTATAGTCACCAGACAAAGTTGGCTAAG-3’

R1 5’-GCGTACGTGAAAGCAGTTTTGAGCCT-3’

F2 5’-CTGCTTTCACGTACGCTTAAAAGCAATTATAAAAAACTTAGG-3’

R2 5’-GTTTAAACGCTTTTGAAGGAAATTAGGCGGGACT-3’

F3 5’-TTCCTTCAAAAGCGTTTAAACTGCAACAAATAGTGGCG-3’

R3 5’-GGATTAATTAACACGGTCATCATCCCTCAGTTCAATTGA-3’

F4 5’-GGGATGATGACCGTGTTAATTAATCCCTTACCGATGATTGAATT-3’

R4 5’-CGGATGCCCAGGTCGGACCGCGAGGAGGTGGAGATGCCATGCCGACCCACCAGACAAAGCTGGGTATGATAAC-3’

(4) modification of eukaryotic expression vector pcDNA3.1

Transforming the polyclonal restriction site of eukaryotic expression vector pcDNA3.1, connecting the prepared sequence with Not I, Bsiw I, pmei I, Pac I, Cpo I restriction site and partial hepatitis delta ribozyme to the pcDNA3.1 vector digested by pmei I, and naming the transformed vector as pcDNA3.2;

(5) and sequentially connecting the F1, F2, F3 and F4 fragments to pEASY-Blunt respectively, sequencing, and respectively connecting each gene fragment with correct sequencing to the downstream of a CMV promoter of the modified vector pcDNA3.2 to obtain a plasmid containing the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain.

Wherein, preferably, the nucleotide sequence of the cDNA obtained in step (2) is shown as SEQ ID NO.2, and the sequence of the partial hepatitis delta ribozyme with Not I, Bsiw I, pmeI, PacI and CpoI cleavage sites in step (4) is shown as SEQ ID NO. 3.

The invention further provides application of the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain in rescuing the recombinant fox-derived canine distemper virus virulent rHBF-1 strain.

Furthermore, the invention also provides a reverse genetic operation system of the fox-derived canine distemper virus virulent HBF-1 strain, wherein the operation system consists of a recombinant plasmid containing the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain and three auxiliary plasmids for expressing the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein.

Wherein, preferably, the nucleotide sequences for coding the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein are respectively shown in SEQ ID NO. 4-6.

Preferably, the recombinant plasmid containing the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain is prepared by the following method:

(1) modification of eukaryotic expression vector pcDNA3.1

Transforming a polyclonal restriction site of a eukaryotic expression vector pcDNA3.1, connecting the prepared sequence with Not I, Bsiw I, pmei, PacI and CpoI restriction sites and a part of hepatitis delta ribozyme to a pcDNA3.1 vector digested by pmei, and naming the transformed vector as pcDNA3.2, wherein the sequence with Not I, Bsiw I, pmei, PacI and CpoI restriction sites and a part of hepatitis delta ribozyme is shown as SEQ ID NO. 3;

(2) the infectious cDNA clone of the fox source canine distemper virus virulent HBF-1 strain is connected to the downstream of the CMV promoter of the modified vector pcDNA3.2 to obtain a plasmid containing the whole genome of the CDV HBF-1 strain.

Preferably, the three auxiliary plasmids for expressing the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein are obtained by respectively cloning nucleotide sequences encoding the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein to the downstream of a CMV promoter of a eukaryotic expression vector pcDNA3.1 to obtain the three auxiliary plasmids for expressing the fox-derived canine distemper virus virulent HBF-1N, P, L protein.

Furthermore, the invention also provides application of the reverse genetic operating system of the fox-derived canine distemper virus virulent HBF-1 strain in rescuing the fox-derived canine distemper virus virulent HBF-1 strain and performing reverse genetic research on the fox-derived canine distemper virus virulent HBF-1 strain.

Furthermore, the invention also provides a method for rescuing the fox-derived canine distemper virus virulent HBF-1 strain and a recombinant fox-derived canine distemper virus virulent rHBF-1 strain obtained by the method, wherein the method comprises the following steps:

transfecting a recombinant plasmid containing the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain, three auxiliary plasmids expressing fox-derived canine distemper virus virulent HBF-1N protein, P protein and L protein into BSR cells, preferably transfecting the recombinant plasmid containing the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain and the three auxiliary plasmids expressing fox-derived canine distemper virus virulent HBF-1N protein, P protein and L protein according to the proportion of 5 mu g, 1 mu g, 0.8 mu g and 0.5 mu g; 72h after transfection, cell suspension was taken and added to Vero-dSlam cells at 37 ℃ with 5% CO₂Culturing for 5-10 days, observing typical syncytium lesion (CPE) caused by CDV, collecting cells and supernatant after the CPE appears, continuously carrying out passage in Vero-dSlam cells, culturing for 6-7d, harvesting virus, and obtaining rescued recombinant virus, which is named rHBF-1.

Compared with the prior art, the invention has the beneficial effects that:

the research establishes a reverse genetic operation system of canine polar fox source CDV virulent HBF-1 strain for the first time. CDV virulent HBF-1 can infect canines and cause their death. However, in vitro HBF-1 can not infect ordinary Vero cells, and in order to realize large-scale propagation culture of the Vero cells, Vero-dSlam cells expressing Slam receptor molecules must be utilized, so that the culture and deep research of HBF-1 strains are greatly limited, and a problem is brought to virus rescue work. Therefore, the invention takes the HBF-1 strain adapted to Vero-dSlam cells as a model virus, and constructs a whole genome recombinant plasmid pcDNA3.2-HBF-1 of HBF-1 and three auxiliary plasmids for expressing fox-derived canine distemper virus virulent HBF-1N protein, P protein and L protein. Through optimizing and determining the transfection proportion and the rescue conditions of each plasmid, a reverse genetic operation platform of the canine fox source CDV virulent HBF-1 strain is successfully established. BSR cells were co-transfected with the four-plasmid system, and each of plasmids pCDV3.2-HBF-1, pcCDV3.1-N, pcCDV3.1-P, and pcCDV3.1-L was transfected at a ratio of 5. mu.g, 1. mu.g, 0.8. mu.g, and 0.5. mu.g, with the amount of transfection reagent being 3 times the amount of plasmid. 72h after transfection, the BSR cell suspension was inoculated to Vero-dSlam cells and co-cultured for 6-8d, and syncytium cytopathic effect typical of canine distemper virus was observed.

Considering the strong toxicity of HBF-1, the generation stability and growth characteristics of recombinant virus rHBF-1 are determined. In the invention, rHBF-1 is inoculated to Vero-dSlam cells and is passaged for 9 times, the rHBF-1 gradually adapts to the Vero-dSlam cells, and the virus titer of the rHBF-1 reaches 10 from the 4 th generation⁵TCID₅₀and/mL. The highest titer of rHBF-1 can be stabilized at 10 in the virus passage^5.667TCID₅₀The virus titer of the/mL, wtHBF-1 can be stabilized at 10^5.3TCID₅₀and/mL. rHBF-1 and wtHBF-1 virus suspensions were seeded at MOI ═ 0.1 onto Vero-dSlam cells, supernatants and cells were harvested at different time points, and virus titers were determined separately. As a result, the recombinant virus rHBF-1 has similar growth characteristics compared with the parent virus wtHBF-1. Compared with the parental virus and the recombinant virus, the titer of both the supernatant and the cell-associated virus can reach the highest titer within 72 hours. The size and the shape of rHBF-1 are consistent with those of typical canine distemper virus as can be seen by electron microscope observation。

Drawings

FIG. 1 shows the construction strategy of the full-length infectious cDNA clone of HBF-1 strain;

FIG. 2 shows the restriction enzyme identification result of plasmid pcCDV3.2-HBF-1;

M：DL5000 DNA marker 1:NotⅠ、BsiwⅠ2:PacⅠ、CpoⅠM:DL15000 marker；

FIG. 3 shows the result of helper plasmid identification;

1：NheⅠ2:PmeⅠ3:MluⅠM:DL 15000 DNA marker；

FIG. 4 is an identification of recombinant virus rHBF-1;

(A) cytopathic effect of rHBF-1 on Vero-dSlam cells (. times.40); (B) negative Vero-dSlam cells (. times.40); (C) rHBF-1 immunofluorescent staining (x 40); (D) negative Vero-dSlam cell immunofluorescent staining (x 40);

FIG. 5 shows the restriction enzyme identification of recombinant virus rHBF-1 genetic marker;

(A) RT-PCR and enzyme digestion identification results; (B) sequencing results;

a wtHBF-1 PCR identification product; 2, enzyme digestion result of the product Bsiw I identified by wtHBF-1 PCR; 3.rHBF-1 PCR identification product; 4, identifying the product Bsiw I enzyme digestion result by rHBF-1 PCR; m is DL 2,000 DNA marker;

FIG. 6 shows the virus particle morphology of rHBF-1 strain under an electron microscope;

FIG. 7 shows the growth characteristics of rHBF-1;

(A) passage stability of rHBF-1; (B) one-step growth curves for wtHBF-1 and rHBF-1.

Detailed Description

The present invention is further described with reference to the following specific examples, which are only illustrative and not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 cloning of infectious cDNA of Fox-derived Canine distemper Virus virulent HBF-1 Strain and establishment of reverse genetic System

1 materials and methods

1.1 viruses, cells, vectors

The alopex lagopus source canine distemper virus virulent HBF-1 strain adapted to Vero-dSlam cells, Vero-dSlam cells for expressing Slam receptors (see the literature: establishment and application of Vero cell line for stably expressing racoon dog canine distemper virus cell receptor SLAM, Zhao et al, Microbiol. Proc. in 12 th 2012), and hamster kidney cells (BSR) are stored by special animal epidemic disease prevention and control research laboratories of the institute of specialty of Chinese academy of agricultural sciences; pcDNA3.1 was purchased from invitrogen.

1.2 Primary reagents

Total RNA extraction kit RNeasy mini kit was purchased from QIAGEN; the reverse transcription kit SuperScriptTM III First-Strand Synthesis SuperMix was purchased from Invitrogen; the high fidelity enzyme TransStart FastPfu DNA Polymerase, pEASY-Blunt Cloning Kit, competent cell Trans1-T1 were purchased from Beijing Quanyujin Biotechnology Ltd; DNA gel recovery kits and plasmid miniprep kits were purchased from Axygen; plasmid quantitative extraction kit was purchased from QIAGEN; t4 DNA ligase and other restriction enzymes were purchased from Thermo scientific; 0.25% pancreatin was purchased from all-formula gold; media was purchased from Corning using DMEM containing 10% and 2% fetal bovine serum; x-tremagene HP DNA Transfection Reagent from Roche; CDV N Monoclonal Antibodies (MAB) were purchased from RMRD; secondary green fluorescently labeled goat anti-mouse antibody was purchased from Proteintech.

1.3 primer design and Synthesis

The inventor self-develops a universal specific amplification reaction system for different CDV strains to amplify and sequence determine the whole genome of the fox-derived CDV virulent HBF-1. The single enzyme cutting sites of the whole genome sequence are analyzed by DNAMAN software, four pairs of primers F1/R1, F2/R2, F3/R3 and F4/R4 are designed by Primer Premier 5.0 software, a hammerhead ribozyme sequence (HamRz) is introduced into the upstream of the F1 fragment, a hepatitis delta virus ribozyme sequence (HdvRz) is introduced into the end of the F4 fragment to ensure the accuracy of the end sequence of a transcription product, the corresponding enzyme cutting sites are added into the primers for facilitating the splicing of the whole genome fragment, the specific sequences are shown in Table 1, and upstream and downstream primers for amplifying CDV virulent HBF-1N, P and L genes are designed and synthesized by Shanghai worker biotechnology company.

TABLE 1 primer sequences for amplification

Note: the plus square frame part comprises enzyme cutting sites of Not I, Bsiw I, Pme1, Pac I and Cpo I in sequence, the plus thick part of hammerhead ribozyme (HamRz) sequence and the single underlined part of hepatitis delta virus ribozyme (HdvRz) sequence.

1.4 construction of recombinant plasmid containing Fox-derived CDV virulent HBF-1 strain infectious cDNA clone and three helper plasmids

In order to facilitate the splicing of the whole genome, the selected polyclonal restriction site of eukaryotic expression vector pcDNA3.1 is modified, the prepared sequence (shown in SEQ ID NO. 3) with Not I, Bsiw I, pmeI, PacI, CpoI restriction sites and partial hepatitis delta ribozyme is connected to the pcDNA3.1 vector digested by pmeI, and the modified vector is named as pcDNA3.2. Total RNA from HBF-1 virus suspension was extracted according to the instructions of the QIAGEN Total RNA kit. RNA was reverse transcribed according to the protocol of SuperScriptTM III First-Strand Synthesis Supermix kit to obtain cDNA (SEQ ID NO. 2). Using four pairs of primers F1/R1, F2/R2, F3/R3, and F4/R4 (Table 1) designed by Primer Premier 5.0, HBF-1 was amplified in 4-stage by high fidelity DNA Polymerase (Trans Start Fastpfu DNA Polymerase), and PCR products were identified by 1% agarose gel electrophoresis and recovered in gel after correct size. The F1, F2, F3 and F4 fragments were ligated to pEASY-Blunt, respectively, and sent to the Shanghai Biotechnology company for sequencing. And respectively and sequentially connecting each gene fragment with correct sequencing to the downstream of the CMV promoter of the modified vector pcDNA3.2 to obtain a plasmid pcCDV3.2-HBF-1 (shown in figure 1) containing the CDV HBF-1 strain infectious cDNA clone, wherein the CDV HBF-1 strain infectious cDNA clone nucleotide sequence is shown in SEQ ID NO. 1. In the same manner, using cDNA of reverse transcription product of HBF-1 genome RNA as a template, upstream and downstream primers N-F/N-R, P-F/P-R, L-F/L-R of the CDV HBF-1 strain N, P and L gene amplified in Table 1 and ORF frames of N, P, L of CDV HBF-1 strain respectively are used for amplifying, and the ORF frame of N, P, L of the CDV HBF-1 strain obtained by amplification is cloned to the downstream of CMV promoter of eukaryotic expression vector pcDNA3.1 to obtain three auxiliary plasmids pcCDV3.1-N, pcCDV3.1-P and pcCDV3.1-L for expressing CDV N and P, L proteins, wherein, the nucleotide sequences of N protein, P protein and L protein of the CDV HBF-1 strain are respectively shown in SEQ ID NO. 4-6. After the sequencing identification is correct, high-purity plasmids are prepared by using a QIAGEN plasmid large-scale extraction kit and stored in a refrigerator at the temperature of minus 80 ℃ for later use.

1.5 Virus rescue

After BSR cells in the six-well plate grew to 90%, transfection was started by using 22. mu.L of X-treme GENE HP DNA transfection reagent, and transfection was performed at ratios of 5. mu.g, 1. mu.g, 0.8. mu.g, and 0.5. mu.g of pcCDV3.2-HBF-1, pcCDV3.1-N, pcCDV3.1-P, and pcCDV3.1-L, respectively, according to the instructions. 72h after transfection, approximately 300. mu.L of cell suspension was taken and added to Vero-dSlam cells at a density of approximately 80%, 5% CO at 37 ℃₂After 5-10 days of culture, typical syncytial lesions (CPE) caused by CDV were observed. And after CPE appears, collecting cells and supernatant, continuously carrying out passage on the cells in Vero-dSlam cells, culturing the cells for 6-7d, and harvesting the virus to obtain the rescued recombinant virus, wherein the rescued recombinant virus is named as rHBF-1.

1.6 Indirect Immunofluorescence (IFA) identification of recombinant Virus rHBF-1

Inoculating the harvested rHBF-1 virus liquid to Vero-dSlam cells, fixing the cells for 40min by 4% paraformaldehyde after the cells have CPE, diluting the CDV N protein monoclonal antibody by 1:500 times to serve as a primary antibody, diluting FITC (fluorescein isothiocyanate) labeled goat anti-mouse IgG by 1:100 times to serve as a secondary antibody, carrying out IFA (fluorescence immunoassay), finally, carrying out nuclear staining on DAPI (deoxyribose nucleic acid) and observing green fluorescence in a fluorescence microscope.

1.7 RT-PCR identification of recombinant Virus rHBF-1

And respectively extracting wtHBF-1 and rHBF-1 passaged by Vero-dSlam cells by using an RNA extraction kit, and performing reverse transcription to obtain cDNA. Primers (upstream 5-GGTATCGCCTTGGGTTCA-3 and downstream 5-AGGCAGCGATCCAAATGT-3) are designed at the upper and lower 300bp positions of a Bsiw I enzyme cutting site of rHBF, respective cDNA is used as a template to carry out PCR amplification, one part of a PCR product is sent to a company for sequencing, and the other part of the PCR product is subjected to enzyme cutting by Pme I. The genome of rHBF-1 is artificially added with a Pme I enzyme cutting site, and the wtHBF-1 does not have a recognition sequence of the site.

1.8 viral growth characteristics

The wtHBF-1 and rHBF-1 are respectively inoculated to Vero-dSlam cells according to the MOI of 0.1, and the cells and the supernatant are respectively harvested 12h, 24h, 48h, 72h, 96h, 120h and 144h after infection and are frozen and stored in a refrigerator at the temperature of-80 ℃. 100 μ L of virus suspension harvested at different time points was serially diluted 10-fold. Each dilution in the 96-well plate was repeated 4 times, and 100. mu.L of Vero-dSlam cells (5X 10 cells) were seeded per well⁴One), 6-8d after infection, CPE was observed and the virus Titer (TCID) of supernatant and cell-associated virus was calculated at different time points, respectively₅₀/mL). Virus growth curves were plotted using the software GrapPad Prism 6.

2 results

2.1 determination and analysis of HBF-1 Strain Whole genome sequence

11 gene fragments covering the HBF-1 whole genome are obtained by an RT-PCR method, and are subjected to 1% agarose gel electrophoresis, 11 target bands are obtained, and the size of the 11 target bands is consistent with an expected result. The HBF-1 strain whole genome sequencing result is compared with a Hebei strain sequence in GenBank to find that: there are 13 amino acids which have variation, wherein the P protein mutation rate is 0.39%, and the H protein mutation rate is 0.33%, which are two structural proteins with the highest variation rate.

2.2 HBF-1 full-Length plasmid identification

The recombinant plasmid pcCDV3.2-HBF-1 containing HBF-1 strain infectious cDNA clone is subjected to NotI, Bsiw I, Pac I and Cpo I double enzyme digestion respectively, and the enzyme digestion products are subjected to agarose gel electrophoresis to obtain bands with the sizes of 17300bp, 3500bp, 14100bp and 6700bp respectively, which are consistent with the expected result (figure 2).

2.3 helper plasmid identification

The helper plasmids pcCDV3.1-N, pcCDV3.1-P and pcCDV3.1-L were digested with restriction sites Nhe I, pmei I and Mlu I, respectively, and subjected to 1% agarose gel electrophoresis to obtain 6600bp, 5000bp, 1700bp and 12000bp bands, consistent with the expected results (FIG. 3).

2.4 identification of recombinant Virus rHBF-1

2.4.1 immunofluorescence assay

The recombinant virus rHBF-1 is infected with Vero-dSlam cells, typical cytopathic condition is observed (figure 4A), meanwhile, a blank control has no pathological change (figure 4B), and immunofluorescence is carried out by using a canine distemper N protein monoclonal antibody for further determination. As a result, the diseased cells were found to bind specifically to CDV-NP antibody, giving a positive signal (FIG. 4C), while the blank group was non-fluorescent and negative (FIG. 4D).

2.4.2 RT-PCR test results

Respectively extracting total RNA of wtHBF-1 and recombinant virus rHBF-1, carrying out reverse transcription to obtain cDNA, and carrying out RT-PCR and enzyme digestion identification by using the cDNA as a template. As a result, it was revealed that the wild-type wtHBF-1 was not cleaved by pmeI, and that both the PCR fragment and the cleavage result showed the band of interest of 600bp in size (FIG. 5A). The PCR amplification product of recombinant virus rHBF-1 can be digested by Bsiw I. The size of the PCR product is about 600bp, and after enzyme digestion, the Pme I is cut off into two target bands with the size of about 300 bp. Since the sizes of the 2 target bands are all 300bp, the electrophoresis gel result shows 1 band (FIG. 5A). RT-PCR of rHBF-1 identified the product, and sequencing also verified the presence of the Bsiw I recognition sequence (FIG. 5B).

2.4.3 morphological characterization of rHBF-1

The supernatant of recombinant virus rHBF-1 was used for negative staining and observed under an electron microscope. As a result, the virus particle was enveloped by a membrane, which contains the fiber, and is a morphological feature of paramyxovirus (FIG. 6).

2.4.4 comparison of growth characteristics of recombinant viruses with parental strains on Vero-dSlam cells

Rescued rHBF-1 was passaged 9 times on Vero-dSlam cells and the virus titer contained in each generation of virus suspension was determined. As a result, the virus titer was significantly increased to 10 in the 4 th passage⁵TCID₅₀The titer was closer to that of wild-type wtHBF-1 in/mL (FIG. 7A). wtHBF-1 and rHBF-1 were infected with Vero-dSlam cells at MOI of 0.1, supernatants and cells were collected at different time points, and virus titers were measured at various time periods to plot growth curves. It can be seen that rHBF-1 is present substantially in comparison to wtHBF-1Consistent growth characteristics (fig. 7B).

Sequence listing

<110> institute of specialty products of Chinese academy of agricultural sciences

<120> construction of infectious cDNA clone and reverse genetic system of fox-derived canine distemper virus virulent HBF-1 strain

<130> KLPI190859

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 15690

<212> DNA

<213> Canine Distemper Virus

<400> 1

accagacaaa gttggctaag gatagttaaa ttattggata ttttattaaa aacttagggt 60

caatgatcct accttagaga acaaggtcag ggttcagacc taccagtatg gctagccttc 120

tcaagagcct cacattgttc aagaggactc gggaccaacc cccacttgcc tcgggctccg 180

gaggagcaat aagagggata aagcatgtca ttatagtcct aatcccgggt gattcaagca 240

ttgttacaag gtctcgacta ctggatagac ttgttagatt ggtcggtgat ccggaaatca 300

acggacctaa attaactggg attttaatca gtatcctctc cttgttcgtg gaatcccctg 360

gacagttgat ccagaggatc atagacgacc ctgatgtaag catcaagtta gtagaggtaa 420

tcccaagcat caactctggt tgtggtctta catttgcatc cagaggagca agtttggatt 480

ctgaggcaga tgagttcttc aaaattgtag acgaagggtc gaaagctcaa ggacaattag 540

gctggttgga gaataaggat attgtagaca tagaagttga tgatgctgag caattcaata 600

tattgctagc ttccatcttg gcccaaattt ggatcctgct cgctaaagca gtgactgctc 660

ctgatactgc agccgactcg gaaatgagga ggtggattaa gtatacccaa cagagacgtg 720

tggtcgggga atttagaatg aacaaaatct ggcttgatat tgttagaaac aggattgctg 780

aggacctatc tttgaggcga ttcatggtgg cactcatctt ggacatcaaa cgatccccag 840

ggaacaagcc tagaattgct gaaatgattt gtgatataga taactacatt gtggaagctg 900

gattagctag tttcatctta actatcaaat ttggcattga aactatgtat ccggctcttg 960

ggttgcatga gttttccgga gagttaacaa ctattgaatc ccttatgatg ctatatcaac 1020

agatgggtga aacagcaccg tacatggtta ttctggaaaa ttctgttcag aacaaattta 1080

gtgcaggatc ctacccactg ctctggagtt atgctatggg agttggtgtt gaacttgaaa 1140

actccatggg agggttaaat ttcggtagat cctactttga tccggcctat ttcaggctcg 1200

ggcaagaaat ggtgagaaga tctgccggca aagtaagctc tgcacttgcc gccgagcttg 1260

gcatcaccaa ggaagaggct cagctagtgt cagaaatagc atccaagaca acggaggacc 1320

ggacgattcg cactgctggt cccaagcaat ctcaaatcac ttttctgcac tcagaaagat 1380

ccgaagtcac caatcaacaa cccccaacca tcaacaagag gtccgaaaac caaggaggag 1440

acaaataccc catccacgtc aatgatgaac ggtttccagg gtacacccct gatgtcaaca 1500

gctccgaatg gagtgaatca cgctatgata cccagactat tcaagatgat ggaaacgacg 1560

atgaccggaa atcgatggaa gcaatcgcca agatgagaat gcttactaag atgctcagtc 1620

aacctgggac cagtgaagag agttctcctg tctataatga tagagagcta ctcaattaaa 1680

tattcaagac cagtgttaca tcagtcacca attatccttc taaactcatt ataaaaaact 1740

taggacccag gtccaacaat cccgatcaac cattcatccg accaaccatt ctatctctaa 1800

atggcagaag agcaggccta tcatgtcaat aaagggctgg aatgcctcaa agccctcaga 1860

gagaatcctc ctgacattga ggagattcaa gaggtcagca gtatcagaga tcaaacccgc 1920

gacccaggcc aagagaatgg aaccgcaagc atgcaggaag aagaggtctc tcaggatctc 1980

gatgaatcac acgagccagc aaaaggatca aactatgtcg gccatgtact ccaaaataat 2040

ccgggatgtg gagagagcaa cactgcgctt gtggaggaag agcagcccgc taaagatgat 2100

gtccaaccag gacctgaaat acgatgttat catgtttatg atcacagtgg tgaagaggtt 2160

aagggaatcg aagatgctga cagtctcgtg gtacctgcag gcgctgtcag taatcgagga 2220

ttcgagagag gagaaggaag ccttgatgat agcactgagg attctggcga agattattcc 2280

gagggaaatg cttcatctaa ctggggatat tctttcggcc ttaaaccaga cagagcggct 2340

gatgtgagca tgctgatgga agaggaattg agtactctgc tcaagacaag cagaaatgtg 2400

gggattaaga aaagggatgg gatgactctg cagttcccac acagtcccga aggtaagaca 2460

gaggatccgg agtgtggatc cattaaaaag ggcacaggag agaggtcagc ctcacatgga 2520

atggggatag ttgctggatc gacaagtggt gcaacccaat ctgcactcaa gtcaactggg 2580

ggatcatcag ggccacgtgt gtctgcggag aatgtccgcc aacctgcaat gaatgcaaag 2640

atgacccaga aatgcaaacc cgagtctggt acgcaactcc ctgccaggac ctcaaatgag 2700

gctgaatctg acagtgagta tgacgatgag cttttttctg aaatacaaga aattcgatct 2760

gctatcacta agctaatgga agataatcaa gcaatacttt ctaaactgga taccctatta 2820

ctgcttaaag gagagactga ttcaattaag aaacaaatta gcaagcaaaa tattgctatt 2880

tccacgattg aggggcatct atcaagcatt atgatagcta tacctggttt tgggaaggac 2940

actggagacc ctacggcaaa tgtcgacatt aacccagagc tccgccctat aatagggagg 3000

gattcaggaa gagcactggc ggaagttctc aagcaacccg catcatcccg cggtaatcgg 3060

aaggacagtg gtatcgcctt gggttcaaaa ggtcaactat tgagagacct ccagctgaaa 3120

cctattgaca aagagtctag ttcggcaatc ggatacaaac cgaaggatac cgcaccttcc 3180

aaagctgtac ttgcatcatt gattagatca agcaaaattg atcaaagtca caaacacaac 3240

atgctggccc ttctcaaaaa tattaagggg gatgacaatc taaacgagtt ctaccagatg 3300

atcaagagta tcacacatgc ttaagctgta gcatttacta atctattaac aggctcaaaa 3360

ctgctttcac gtacgcttaa aagcaattat aaaaaactta ggacacaaga gcctaagtcc 3420

tctccacaaa aatgactgag gtgtacgact tcgatcagtc ttcgtgggac accaaaggtt 3480

ccttggcccc cattttgccc accacctacc ccgatggtag gctagtaccc caagtcagag 3540

tgatagatcc aggactcgga gatcgaaaag atgaatgttt catgtatatt tttctactgg 3600

gtataataga agacaacgat ggcctcggac ccccgattgg aagaacattt ggatcgctgc 3660

ctttaggtgt tgggcgcact acagccagac ctgaagaatt attgaaggaa gccaccttgt 3720

tggacattgt ggtaaggcga actgcaggtg tcaaagaaca actggtattt tacaataaca 3780

ccccattgca catcttaact ccgtggaaga aggtccttac gagcgggagt gtattcagtg 3840

ctaatcaagt ctgtaacgcg gtcaatttaa taccattgga catagcacag agatttaggg 3900

tggtatatat gagcatcact cgactatcag acgatggaag ttacagaatt cctcgcggga 3960

tgtttgaatt ccgctccagg aatgctttag catttaatat tttagtcacc attcaagttg 4020

agggagatgt ctgttcaagc cgagggaatt tgagcatgtt caaagatcac caagtgacat 4080

tcatggtgca tatcggcaac ttcagtcgta agaagaacca agcttactct gctgattact 4140

gtaaacttaa aattgaaaag atgggattag tgtttgctct aggagggata ggaggaacca 4200

gtcttcacat acgatgtact ggtaagatga gcaaggcttt gaatgcccag ctaggattca 4260

agaaaatcct gtgttacccg ctcatggaga tcaacgaaga tttgaatcga tttctatgga 4320

gattagagtg caaaatagta agaatccaag cagtcttgca accatcagtc ccacaagatt 4380

tcagaattta taatgatgtt atcatcagtg atgatcaggg tcttttcaaa attctctaaa 4440

tcattagttc atgaactaaa actcaaatgc cttggtggca ttgtccagga tcccttaatc 4500

ctctcaaaca aggattgagg ctacaagtgt caattgtctc ggtgttgctc ctgcatttta 4560

agcaagttct ataggtttct aaacttctca ttcgtgccta ctattctggt gactctgcaa 4620

tacgaagaca gctgaatcaa accaattcat gctcaagagt aggttgatca ttatcggacc 4680

aagaaatgta tggatgcttg gggttttgaa attcacctct aggaatctca cttaaccaat 4740

tatacctcca cgcgcttgcc tgatctcaag ctattactag tagtcctgtt tcacgaagtt 4800

ctgactgtct atctttctat caccaatcgt taataattaa tcaaaactta ggatccagga 4860

cgcagcaagc caacaggcca accaagtcca ccaatccgag gccgggcagg aaccccaaca 4920

aacagacaag ccccatgcac aacaaaatcc ccaaaaaatc caaacccctg ccacacaccc 4980

gacaagatcc cctccaacaa cacagcacca gatccaccga gaccaagacc tcccaaggac 5040

gatatagcat aacatcggct cagcgatcca cgtaccatag tcctcgaaca tcggacaggc 5100

ccgtccacta cataatgaac aggaccaggt cttgcaagca aactagccac agatcggata 5160

acatcccgcc tcacagagac cacgagggta tcatccatca cacaccagag agtgtcaccc 5220

aaggagcgag ttcctggttc aagaggtggc aatccaatgc aaccaatgca ggctctcaat 5280

gcacctggtt agtcctgtgg tgcatcggaa tagccagtct ctttctttgt tccaaggctc 5340

agatacattg gaataatttg tcaacgattg ggattatcgg aactgacagt gtccattata 5400

agatcatgac taggcccagt caccagtact tggtcataaa actaatgcct aatgtttcac 5460

ttatagataa ttgtaccaaa gcagaattag gtgagtatga gaaattatta aattcagtcc 5520

tggagccaat caaccaagct ttgactctaa tgaccaataa tgtgaagccc ctacagtcag 5580

tagggtcagg taggagacaa aggcgttttg caggagtggt gcttgcaggt gcagctttag 5640

gggtggccac agccgcacaa atcactgcag ggatagcttt acatcagtcc aacctcaatg 5700

ctcaagcaat ccaatctctg agaactagcc ttgaacagtc caacaaggct atagaagaaa 5760

ttagggaggc aacccaggaa accgtcattg ctgttcaggg agtccaggat tacgtcaata 5820

atgaactcgt ccctgctatg caacatatgt cgtgtgagtt agttgggcag agattagggt 5880

taaaactgct taggtattac accgagttgt tgtcaatatt tggcccgagt ttacgtgacc 5940

ctatttcagc cgagatatca attcaagcac tgagttatgc tcttggggga gaaattcata 6000

agatacttga gaagttgggg tattctggta atgatatgat tgcaattttg gagagtcggg 6060

ggataaaaac aaaaataact catgtcgatc tccccgggaa actcatcata ttaagtatct 6120

catacccaac tttatcagaa gtcaaggggg ttatagtcca cagactggaa gccgtttctt 6180

ataacatagg gtcacaggag tggtacacca ctgtcccgaa gtatgttgca actaatggtt 6240

acttaatatc aaactttgat gagtcatcct gtgtattcgt ctcagaatca gccatttgta 6300

gccagaactc tctgtacccc atgagcccga ttctacaaca atgcattagg ggcgatactt 6360

catcttgtgc tcggaccttg gtgtctggga ctatgggcaa caagtttatt ctgtcaaaag 6420

gtaatatcgt tgcaaattgt gcttctatac tatgtaagtg ttatagcaca agcacaatta 6480

tcaatcagag tcctgataag ttgctgacat ttattgcctc cgatacctgc ccactggttg 6540

aaatagatgg tgtaactatc caagttggag ggaggcaata ccctgatatg gtatacgaaa 6600

gcaaagttgc cttaggacct gctatatcac ttgagaggtt ggatgtaggt acaaatttag 6660

ggaacgccct taagaaactg gatgatgcta aagtactgat agactcctct aaccagatcc 6720

ttgagacagt taagcgctct tcctttaatt ttggcagtct cctcagcgtt cccatattaa 6780

tctgtacagc tctggctttg ttgttgctaa tttactgctg taaaagacgc taccgacaga 6840

cattcaagca taatactaag gtcgatccga catttaaacc tgatttgact ggaacttcga 6900

aatcatatgt aagatcactc tgaagcactc tgatcacaag tcttacctga ttgttaggct 6960

tgaaatccat aagtcccgcc taatttcctt caaaagcgtt taaactgcaa caaatagtgg 7020

cgaggactga ctccaattat tataattaaa gaaaacttag ggctcaggta gtccaacaat 7080

gctctcttac caagacaagg tgggtgcctt ctacaaggac aatgcaagag ctaattcatc 7140

caagctgtcc ccagtgaccg aagagcaagg gggacggaga ccaccctatt tgctgtttgt 7200

ccttctcatc ctactgattg gaatcctggc cttgcttgcc atcactggag ttcgatttca 7260

ccaagtatca actagcaata tggaatttag cagattgctg aaagaggata tggagaaatc 7320

agaggccgta catcaccaag tcatagatgt cttgacaccg ctcttcaaaa ttattggaga 7380

tgagattggg ttacggttgc cacaaaaact aaacgagatc aaacaattta tccttcaaaa 7440

gacaaacttc ttcaatccga acagggaatt cgacttccgc gatctccact ggtgcattaa 7500

cccacctagc aagatcaagg tgaattttac taattactgt gatacagttg gggtcaaaaa 7560

atctattgca tcggcagcaa atcccatcat tttatcagca ctctccgggg ccagaggtga 7620

catattcccg ccgtacagat gcagtggagc tactacttca gtaggcaggg tattccccct 7680

atccgtatca ttatccatgt ctttgatatc aagaacatca gagataatca atatgctaac 7740

cgctatctca gacggagtgt atggtaaaac ttatttgcta gtgcctgatt atattgaagg 7800

ggagttcgac tcacaaaaga ttcgagtctt tgagataggg tttatcaaac ggtggctgaa 7860

tgacatgcct ttactccaga caaccaacta tatggtcctc ccggaaactt ccaaagccaa 7920

ggtatgtact atagcagtgg gtgagctgac actagcttcc ttgtgtgtag atgagagcac 7980

cgtattgtta tatcatgaca gcaacggttc acaagatggt attctagtag tgacattggg 8040

aatatttggg gcaacaccta tggatcaagt tgaagaggtg atacctaccg ctcacccatc 8100

agtggagaga atacatataa caaatcaccg tgggttcata aaagactcaa tagtaacctg 8160

gatggtgcct gtattggtct ctgagaaaca agaggagcaa aaaaactgtc tggagtctgc 8220

ttgtcacaga aaatcctacc ctatgtgcaa ccaaacgtca tgggaaccct ttggaggagg 8280

acagttgcct tcttatgggc ggttgacatt acctctagat ccaagcattg accttcaact 8340

taacatatca tttacatatg gtccggttat actgaacgga gacggtatgg attattatga 8400

aagcccactt ttggactccg gatggcttac catacctcct aagaacggga cagtccttgg 8460

attgataaac aaagcaagta gaggagacca gttcactgtg accccccatg tgttgacatt 8520

tgcgcccagg gaatcaagtg gaaattgtta tttgcctatt caaacatccc agattatgga 8580

taaagatgtc cttactgagt ccaatttagt ggtgttacct acacagaatt ttagatatgt 8640

catagcaaca tatgatatat cccggggcga tcatgcaatt gtttattatg tttatgaccc 8700

aatccggacg atttcttata catacccatt tagactaact accaagggta gacctgattt 8760

cctaaggatt gaatgttttg tgtgggatga cgatttgtgg tgtcatcaat tttaccgatt 8820

cgaggctaac atcactaact ctacaaccag tgttgagaat ttagtccgta taagattctc 8880

atgtaaccgt tcaaaacctt gatggtatgc tgatacacat ctcaattgaa ctgagggatg 8940

atgaccgtgt taattaatcc cttaccgatg attgaattaa accatcccca gcattataaa 9000

aaaactaagg atccaggatc cttttagtca tggactctgt ttcagtgaac cagattttat 9060

accctgaggt ccatctagat agcccaattg tgaccaataa gctagtggct attttagagt 9120

atgcacgaat tagacataac tatcgactcc ttgacacaac gttagtgcgt aatatcaaag 9180

agagaatttc agaagggtta tcaaaccaga tgatcattaa ctgtatcgaa attgggagca 9240

ttgttaatca gaccttgtta tcttatccca aacacaacca tgtgatatat ccaaattgca 9300

acaaacttct gtttcatgca caggatcgag tcatctctct gaggttgaga aatatattca 9360

aaagaggaaa tagcatctat agtaaaataa cagacggggt caaaaaatgc ttaaacgata 9420

ttaatcttag tattggttta ggaggtgcat tggataagac tattggggcc aaagttgatg 9480

aagcaggcat aattatgcaa agctcacagt ggttcgaacc tttccttctg tggtttacaa 9540

ttaagacaga aatgagatca gtgattaaat cctctactca caactgtcgc aaacgaaggc 9600

agaatcctgt ctttgtaaga ggtgaatcat ttaatgtgtt agtgtctcgg gatcttgtat 9660

gtatcattga cctcaccagt cacaatgttt attacctaac atttgaaatg gtcctgatgt 9720

attgtgatgt gatagaaggg agattaatga ctgataccgc tatggcaatt gatcaccgtt 9780

actcaacttt gcatgtcaga atcaggtatc tttgggatct aattgatgga tttttcccgg 9840

acttaggaaa ttcgacctat caactggtag ctctgctgga gcctctttca ttggcttact 9900

tgcaattaaa agacatcacc ttctctctca ggggtgcttt tttgagtcac tgctttgctg 9960

aaatccagga gattttacag gacaatggct tctatactga agagacattc caaaccttaa 10020

cccaggctct agactttgtt ttcatcacag aggatataca tataacagga gagatctttt 10080

ccttttttag gagtttcggt cacccaagat tagaagcaat aacagcagca gaaaatgtac 10140

ggaaacacat gaatcaaccc aaagttgtct cctatgagac catgatgaag ggacacgcta 10200

ttttctgtgg gataatcatt aacggttatc gggatagaca tggaggaact tggcctccaa 10260

tggatcttcc tgtccatgca tctcctatca tcaggaatgc tcatgcctca ggagagggaa 10320

tcacctatag tcaatgtata gaaaattgga aatcctttgc aggaattcga tttaaatgct 10380

ttatgcccct ctgcctagac agtgatctga ccatgtattt gaaagataag gctttagcag 10440

cccttaaaaa agagtgggat tcagtgtacc caaaagaatt cctcaggtac aacccacctc 10500

gctccactga atctcggaga cttgttaatg tgtttctaga ggactctcag tttgaccctt 10560

ataatatgat tatgtacgtt atctcaggac aatatctaga cgatcctgac ttcaacctat 10620

catacagtct taaagagaaa gagattaaag aagtggggag gttattcgct aaaatgacat 10680

acaaaatgcg ggcctgtcaa gtcatagcag aaaacttaat atctaatgga attgggaagt 10740

acttcaagga caatgggatg gcaaaggatg aacacgatct cactaaagca ttgcacactc 10800

tggctgtgtc cggggttcct aaagacaaga aagactccca tcgcggcctc actaaccagt 10860

gtaagtctaa aaaactgaca ccttatcgag gagcccttca ctccgtctct tctccaagta 10920

gtagatatat ggacccaaac ccaaattttt gcaccagtag aagagaagac aatgacatag 10980

agatctatga gaccgtaagt gcatttataa ctacagatct caaaaagtac tgtctgaatt 11040

ggcgatatga gaccattagt atatttgctc akagattaaa tgaaatctac ggtctcccct 11100

catttttcca atggttgcac agaagattgg aacagtcgat cctatacgta agtgaccccc 11160

actgccctcc agatctcgat cgccatgtgg acttgaacac agtccctaac tctcaaatat 11220

tcatcaaata cccaatggga ggagtagagg gatattgtca aaagttatgg actattagca 11280

caatacctta tctgtacttg gcagcacatg aaagcggtgt caggattgca tcacttgttc 11340

aaggtgataa ccaaaccatt gctgtcacta aaagagttcc aagcacctgg tcatatgcct 11400

tgaagaagtc tgaagccagt cgagtcacca cagaatactt tatagcctta agacagagat 11460

tacatgatgt cggacatcat ttgaaagcaa atgaaacaat aatatcttcc cacttttttg 11520

tatactcaaa aggaatctat tatgatggga tgttaatttc gcaatccttg aaaagtatag 11580

ctaggtgcgt attttggtca gaaacaatag tggatgagac ccgagccgcc tgtagcaaca 11640

tttcaacaac attggcaaaa gccattgaga aagggtttga ccggtattta gcctatgcgc 11700

tgaatatttt aaaaatcatt caacaagtat taatttcatt aggattcact atcaattcag 11760

ctatgacgcg ggatgtgata gaaccccttt tacaagacca ctgtctcttg accaagatgg 11820

caattcttcc tgcacccata ggtggtctta attacctcaa tatgagtagg ctttttgtca 11880

ggaacatcgg ggatcccgtg acatcttcta ttgctgacct caaacgaatg atccgatcag 11940

gccttctcgg agtagagatt ttacatcaag tcatgaccca atacccaggt gactcttcgt 12000

acttagattg ggcaagtgac ccttattctg ctaatctgcc ctgtgtccag agcataaccc 12060

gactccttaa aaatattacg gccaggcatg tccttatcaa cagtccaaat cccatgctga 12120

aaggattgtt ccatgatgaa agtcaggatg aggatgaagc tttagctgct ttcttgatgg 12180

atagaaaaat tattatccca agagctgcac atgaaattct agataacacg atcactggtg 12240

cgagggaggc aattgctgga atgctagata ccacaaaggg gttgataaga gcaagcatga 12300

aaagaggagg gctaacccct agaataataa accgtttgtc aacttatgat tatgagcaat 12360

ttagggcagg tatcagacta ttgtcaggga agggacatga cccactcatc gatcaagact 12420

catgctctgt ccagttagcg agagcattaa ggaaccacat gtgggctaag ttggcgaagg 12480

gtcgtcctat ttatggtcta gaagtcccgg acatccttga atcaatgaag ggctatatga 12540

taagaagaca tgagtcttgt ttgctttgcg catcaggctc tcataactat ggttggtttt 12600

ttgtaccggc aaattgtcaa ttagatagta ttacagaggg aacatctgca ctgagggtac 12660

catacattgg gtccacaaca gaagaaagaa cagacatgaa attagcattc gtcaaatctc 12720

ctagtaggtc tctaaaatca gcagtgagaa tagcaactgt gtactcatgg gcctatggtg 12780

atgatgacga atcttggcaa gaggcttgga ctttggcaaa acagagagcg aacatctcac 12840

ttgaagaatt acggatgatt accccaattt ctacctctac taatctagct caccggctca 12900

gggacaagag tactcaagtc aaatactcag ggacctctct tatcagagta gcacgttatg 12960

caacaatctc taatgataat ctttcttttg tgatagctga caagaaagtg gacacgaact 13020

ttatttatca gcaaggtatg ctcctggggt tggggattct cgagcacttg tttagactgt 13080

cttcaaccac cggcgacacc aacaccgtac tgcatttaca tgttgaaaca gattgttgcg 13140

taatacccat gagcgaccat ccaagggtcc cagggctcag aaaggtcgtt ataccaagaa 13200

atatttgtac aaatcctttg atctatgaca gtaaccctat tattgagaaa gatgcagtca 13260

ggctttataa ccagagtcac aggaagcaca ttgtagagtt tgtcacatgg acaacagggc 13320

agctttatca tgtactagct aaatctactg ctatgtctat ggttgagatg attacaaagt 13380

ttgaaaaaga ccacctaaat gaagtctccg cgttaattgg cgatgacgat atcaatagct 13440

ttatcactga gtttcttctc gttgagccca ggttatttac tgtgtatcta ggtcaatgtg 13500

ctgcaatcaa ctggggcttt gaaattcatt atcaccgacc ttctggaaag tatcaaatgg 13560

gtgaattgtt gttttctttc ttgagtagaa tgagtaaagg agtcttcaaa attttaacca 13620

atgcattgag tcatcccaaa gtatatagac gattttggga cagtgggatg attgaacctg 13680

tacacggacc ctctcttgac tcccagaacc tacacataac tgtatgcaac ctgatctata 13740

actgttacat gatttaccta gaccttctgt taaatgatga gttagatgat ttctcattca 13800

ttttatgcga aagtgatgag gatatcatac ctgaaagatt tgacaacata caagctaggc 13860

acctatgcat cctgtctgac ctttattgta accctcgtga ttgtccccag attcgtgggt 13920

tgacaccaac acagaaatgt gctgtgttat cgaggtactt aaaatcaaaa gctctagagt 13980

cccatgttgg tctgacatgg aatgacaaac ctatcctgat agatcagtat tcatgttccc 14040

tgacatatct gagaagaggc tcaatcaagc agataagatt gagagtggac cccgggttca 14100

tcactgatgc tgttggatgc ttagaaaagc gacctctgag gaaaagtcct atctctaacg 14160

cctcagaatt aaaatcagaa tttgacccac cgaaagatga cctggttaaa ctcctgagtc 14220

agctatcaac aaggacacac aacttaccta ttacaggatt aggagtccga aactatgagg 14280

ttcactcatt cagaagaatt gggatcaatt caacggcatg ttacaaggca gttgaaatag 14340

tctctgttat taagaacgaa ttcacgtctg aagaacatgg attattccta ggagagggtt 14400

caggtgcaat gctgacagta tataaagagc tattgaggtt gtcaagatgt tattataaca 14460

gtggtgtgtc agcagaggct agaactggac aacgagagat ttcaccttac ccttctgagg 14520

tcagccttgt ggaacatcaa ttaggactcg ataaattggt gactgtgctt ttcaatggga 14580

gaccagaggt aacttgggtt gggagtgttg attgttacaa gtacatacta agtcagatat 14640

ctgctagcag tcttggattg attcactcgg atatcgagtc actacctgat aaagacataa 14700

ttgaaaaatt ggaagaactg tctgccatat tatcgatgac tttgatatta gggaaggtag 14760

ggtcagtgtt agtaatcaag atcatgcccg ctagtggcga ctgggttcaa ggatttattc 14820

tgtatgcact cccacatttt cttcgaagtt acataattta cccaagatac agcaattttg 14880

tgtcaacaga ggcctacctc gttttcactg gtcttagagc agggagacta gtcaatccgg 14940

aggggattaa acaacagatt ttgcgagtcg gtattcgaac ttcacccgga ttggtagggc 15000

acatcctttc atcaaagcag acagcatgtg tgcaatcttt gcatggacct ccatttcaag 15060

ctaaatcttt taatccttac ctccagggtt taacaagtat tgagaagatt ttgatcaatt 15120

gtgggcttac aattaacggt cttaaagtat gcaaaaacct gcttcaccat gatatctcgt 15180

caggcgagga agggctgaaa ggatctatca caatccttta tagggaactc gcacggttca 15240

aggataacca ccaattttca catggaatgt tccatgcata cccagtttta atcgcaagtc 15300

aggaaaggga gctcgtatcc accattgcaa ggaagtattg tggttatatt ttgctttact 15360

cgggagactt atacgaaatt accaggatag ttcgagacct gaaagccaac cacataattt 15420

ttgacttaca ccggaactta tttatggata acctatccag atctgaccgg tctctcatcc 15480

tgacgacaat cccaaaaagg aattggctct ttcaacttga gaccaaagag ataaaagagt 15540

ggttcaaact gttggggtat agtgcactga ttagaaacca ctgacgggtt ggtctggttc 15600

ctaaccctct gctattcatt gttattaaat ttaattttac gaaaaaaaac aacgattatt 15660

aataagttat catacccagc tttgtctggt 15690

<210> 2

<211> 15690

<212> DNA

<213> Canine Distemper Virus

<400> 2

accagacaaa gttggctaag gatagttaaa ttattggata ttttattaaa aacttagggt 60

caatgatcct accttagaga acaaggtcag ggttcagacc taccagtatg gctagccttc 120

tcaagagcct cacattgttc aagaggactc gggaccaacc cccacttgcc tcgggctccg 180

gaggagcaat aagagggata aagcatgtca ttatagtcct aatcccgggt gattcaagca 240

ttgttacaag gtctcgacta ctggatagac ttgttagatt ggtcggtgat ccggaaatca 300

acggacctaa attaactggg attttaatca gtatcctctc cttgttcgtg gaatcccctg 360

gacagttgat ccagaggatc atagacgacc ctgatgtaag catcaagtta gtagaggtaa 420

tcccaagcat caactctggt tgtggtctta catttgcatc cagaggagca agtttggatt 480

ctgaggcaga tgagttcttc aaaattgtag acgaagggtc gaaagctcaa ggacaattag 540

gctggttgga gaataaggat attgtagaca tagaagttga tgatgctgag caattcaata 600

tattgctagc ttccatcttg gcccaaattt ggatcctgct cgctaaagca gtgactgctc 660

ctgatactgc agccgactcg gaaatgagga ggtggattaa gtatacccaa cagagacgtg 720

tggtcgggga atttagaatg aacaaaatct ggcttgatat tgttagaaac aggattgctg 780

aggacctatc tttgaggcga ttcatggtgg cactcatctt ggacatcaaa cgatccccag 840

ggaacaagcc tagaattgct gaaatgattt gtgatataga taactacatt gtggaagctg 900

gattagctag tttcatctta actatcaaat ttggcattga aactatgtat ccggctcttg 960

ggttgcatga gttttccgga gagttaacaa ctattgaatc ccttatgatg ctatatcaac 1020

agatgggtga aacagcaccg tacatggtta ttctggaaaa ttctgttcag aacaaattta 1080

gtgcaggatc ctacccactg ctctggagtt atgctatggg agttggtgtt gaacttgaaa 1140

actccatggg agggttaaat ttcggtagat cctactttga tccggcctat ttcaggctcg 1200

ggcaagaaat ggtgagaaga tctgccggca aagtaagctc tgcacttgcc gccgagcttg 1260

gcatcaccaa ggaagaggct cagctagtgt cagaaatagc atccaagaca acggaggacc 1320

ggacgattcg cactgctggt cccaagcaat ctcaaatcac ttttctgcac tcagaaagat 1380

ccgaagtcac caatcaacaa cccccaacca tcaacaagag gtccgaaaac caaggaggag 1440

acaaataccc catccacgtc aatgatgaac ggtttccagg gtacacccct gatgtcaaca 1500

gctccgaatg gagtgaatca cgctatgata cccagactat tcaagatgat ggaaacgacg 1560

atgaccggaa atcgatggaa gcaatcgcca agatgagaat gcttactaag atgctcagtc 1620

aacctgggac cagtgaagag agttctcctg tctataatga tagagagcta ctcaattaaa 1680

tattcaagac cagtgttaca tcagtcacca attatccttc taaactcatt ataaaaaact 1740

taggacccag gtccaacaat cccgatcaac cattcatccg accaaccatt ctatctctaa 1800

atggcagaag agcaggccta tcatgtcaat aaagggctgg aatgcctcaa agccctcaga 1860

gagaatcctc ctgacattga ggagattcaa gaggtcagca gtatcagaga tcaaacccgc 1920

gacccaggcc aagagaatgg aaccgcaagc atgcaggaag aagaggtctc tcaggatctc 1980

gatgaatcac acgagccagc aaaaggatca aactatgtcg gccatgtact ccaaaataat 2040

ccgggatgtg gagagagcaa cactgcgctt gtggaggaag agcagcccgc taaagatgat 2100

gtccaaccag gacctgaaat acgatgttat catgtttatg atcacagtgg tgaagaggtt 2160

aagggaatcg aagatgctga cagtctcgtg gtacctgcag gcgctgtcag taatcgagga 2220

ttcgagagag gagaaggaag ccttgatgat agcactgagg attctggcga agattattcc 2280

gagggaaatg cttcatctaa ctggggatat tctttcggcc ttaaaccaga cagagcggct 2340

gatgtgagca tgctgatgga agaggaattg agtactctgc tcaagacaag cagaaatgtg 2400

gggattaaga aaagggatgg gatgactctg cagttcccac acagtcccga aggtaagaca 2460

gaggatccgg agtgtggatc cattaaaaag ggcacaggag agaggtcagc ctcacatgga 2520

atggggatag ttgctggatc gacaagtggt gcaacccaat ctgcactcaa gtcaactggg 2580

ggatcatcag ggccaagtgt gtctgcggag aatgtccgcc aacctgcaat gaatgcaaag 2640

atgacccaga aatgcaaacc cgagtctggt acgcaactcc ctgccaggac ctcaaatgag 2700

gctgaatctg acagtgagta tgacgatgag cttttttctg aaatacaaga aattcgatct 2760

gctatcacta agctaatgga agataatcaa gcaatacttt ctaaactgga taccctatta 2820

ctgcttaaag gagagactga ttcaattaag aaacaaatta gcaagcaaaa tattgctatt 2880

tccacgattg aggggcatct atcaagcatt atgatagcta tacctggttt tgggaaggac 2940

actggagacc ctacggcaaa tgtcgacatt aacccagagc tccgccctat aatagggagg 3000

gattcaggaa gagcactggc ggaagttctc aagcaacccg catcatcccg cggtaatcgg 3060

aaggacagtg gtatcgcctt gggttcaaaa ggtcaactat tgagagacct ccagctgaaa 3120

cctattgaca aagagtctag ttcggcaatc ggatacaaac cgaaggatac cgcaccttcc 3180

aaagctgtac ttgcatcatt gattagatca agcaaaattg atcaaagtca caaacacaac 3240

atgctggccc ttctcaaaaa tattaagggg gatgacaatc taaacgagtt ctaccagatg 3300

atcaagagta tcacacatgc ttaagctgta gcatttacta atctattaac aggctcaaaa 3360

ctgctttcac tatcgcttaa aagcaattat aaaaaactta ggacacaaga gcctaagtcc 3420

tctccacaaa aatgactgag gtgtacgact tcgatcagtc ttcgtgggac accaaaggtt 3480

ccttggcccc tattttgccc accacctacc ccgatggtag gctagtaccc caagtcagag 3540

tgatagatcc aggactcgga gatcgaaaag atgaatgttt catgtatatt tttctactgg 3600

gtataataga agacaacgat ggcctcggac ccccrattgg aagaacattt ggatcgctgc 3660

ctttaggtgt tgggcgcact acagccagac ctgaagaatt attgaaggaa gccaccttgt 3720

tggacattgt ggtaaggcga actgcaggtg tcaaagaaca actggtattt tacaataaca 3780

ccccattgca catcttaact ccgtggaaga aggtccttac gagcgggagt gtattcagtg 3840

ctaatcaagt ctgtaacgcg gtcaatttaa taccattgga catagcacag agatttaggg 3900

tggtatatat gagcatcact cgactatcag acgatggaag ttacagaatt cctcgcggga 3960

tgtttgaatt ccgctccagg aatgctttag catttaatat tttagtcacc attcaagttg 4020

agggagatgt ctgttcaagc cgagggaatt tgagcatgtt caaagatcac caagtgacat 4080

tcatggtgca tatcggcaac ttcagtcgta agaagaacca agcttactct gctgattact 4140

gtaaacttaa aattgaaaag atgggattag tgtttgctct aggagggata ggaggaacca 4200

gtcttcacat acgatgtact ggtaagatga gcaaggcttt gaatgcccag ctaggattca 4260

agaaaatcct gtgttacccg ctcatggaga tcaacgaaga tttgaatcga tttctatgga 4320

gattagagtg caaaatagta agaatccaag cagtcttgca accatcagtc ccacaagatt 4380

tcagaattta taatgatgtt atcatcagtg atgatcaggg tcttttcaaa attctctaaa 4440

tcattagttc atgaactaaa actcaaatgc cttggtggca ttgtccagga tcccttaatc 4500

ctctcaaaca aggattgagg ctacaagtgt caattgtctc ggtgttgctc ctgcatttta 4560

agcaagttct ataggtttct aaacttctca ttcgtgccta ctattctggt gactctgcaa 4620

tacgaagaca gctgaatcaa accaattcat gctcaagagt aggttgatca ttatcggacc 4680

aagaaatgta tggatgcttg gggttttgaa attcacctct aggaatctca cttaaccaat 4740

tatacctcca cgcgcttgcc tgatctcaag ctattactag tagtcctgtt tcacgaagtt 4800

ctgactgtct atctttctat caccaatcgt taataattaa tcaaaactta ggatccagga 4860

cgcagcaagc caacaggcca accaagtcca ccaatccgag gccgggcagg aaccccaaca 4920

aacagacaag ccccatgcac aacaaaatcc ccaaaaaatc caaacccctg ccacacaccc 4980

gacaagatcc cctccaacaa cacagcacca gatccaccga gaccaagacc tcccaaggac 5040

gatatagcat aacatcggct cagcgatcca cgtaccatag tcctcgaaca tcggacaggc 5100

ccgtccacta cataatgaac aggaccaggt cttgcaagca aactagccac agatcggata 5160

acatcccgcc tcacagagac cacgagggta tcatccatca cacaccagag agtgtcaccc 5220

aaggagcgag ttcctggttc aagaggtggc aatccaatgc aaccaatgca ggctctcaat 5280

gcacctggtt agtcctgtgg tgcatcggaa tagccagtct ctttctttgt tccaaggctc 5340

agatacattg gaataatttg tcaacgattg ggattatcgg aactgacagt gtccattata 5400

agatcatgac taggcccagt caccagtact tggtcataaa actaatgcct aatgtttcac 5460

ttatagataa ttgtaccaaa gcagaattag gtgagtatga gaaattatta aattcagtcc 5520

tggagccaat caaccaagct ttgactctaa tgaccaataa tgtgaagccc ctacagtcag 5580

tagggtcagg taggagacaa aggcgttttg caggagtggt gcttgcaggt gcagctttag 5640

gggtggccac agccgcacaa atcactgcag ggatagcttt acatcagtcc aacctcaatg 5700

ctcaagcaat ccaatctctg agaactagcc ttgaacagtc caacaaggct atagaagaaa 5760

ttagggaggc aacccaggaa accgtcattg ctgttcaggg agtccaggat tacgtcaata 5820

atgaactcgt ccctgctatg caacatatgt cgtgtgagtt agttgggcag agattagggt 5880

taaaactgct taggtattac accgagttgt tgtcaatatt tggcccgagt ttacgtgacc 5940

ctatttcagc cgagatatca attcaagcac tgagttatgc tcttggggga gaaattcata 6000

agatacttga gaagttgggg tattctggta atgatatgat tgcaattttg gagagtcggg 6060

ggataaaaac aaaaataact catgtcgatc tccccgggaa actcatcata ttaagtatct 6120

catacccaac tttatcagaa gtcaaggggg ttatagtcca cagactggaa gccgtttctt 6180

ataacatagg gtcacaggag tggtacacca ctgtcccgaa gtatgttgca actaatggtt 6240

acttaatatc aaactttgat gagtcatcct gtgtattcgt ctcagaatca gccatttgta 6300

gccagaactc tctgtacccc atgagcccga ttctacaaca atgcattagg ggcgatactt 6360

catcttgtgc tcggaccttg gtgtctggga ctatgggcaa caagtttatt ctgtcaaaag 6420

gtaatatcgt tgcaaattgt gcttctatac tatgtaagtg ttatagcaca agcacaatta 6480

tcaatcagag tcctgataag ttgctgacat ttattgcctc cgatacctgc ccactggttg 6540

aaatagatgg tgtaactatc caagttggag ggaggcaata ccctgatatg gtatacgaaa 6600

gcaaagttgc cttaggacct gctatatcac ttgagaggtt ggatgtaggt acaaatttag 6660

ggaacgccct taagaaactg gatgatgcta aagtactgat agactcctct aaccagatcc 6720

ttgagacagt taagcgctct tcctttaatt ttggcagtct cctcagcgtt cccatattaa 6780

tctgtacagc tctggctttg ttgttgctaa tttactgctg taaaagacgc taccgacaga 6840

cattcaagca taatactaag gtcgatccga catttaaacc tgatttgact ggaacttcga 6900

aatcatatgt aagatcactc tgaagcactc tgatcacaag tcttacctga ttgttaggct 6960

tgaaatccat aagtcccgcc taatttcctt caaaagctat caaactgcaa caaatagtgg 7020

cgaggactga ctccaattat tataattaaa gaaaacttag ggctcaggta gtccaacaat 7080

gctctcttac caagacaagg tgggtgcctt ctataaggac aatgcaagag ccaattcatc 7140

caagctgtcc ccagtgaccg aagagcaagg gggacggaga ccaccctatt tgctgtttgt 7200

ccttctcatc ctactgattg gaatcctggc cttgcttgcc atcactggag ttcgatttca 7260

ccaagtatca actagcaata tggaatttag cagattgctg aaagaggata tggagaaatc 7320

agaggccgta catcaccaag tcatagatgt cttgacaccg ctcttcaaaa ttattggaga 7380

tgagattggg ttacggttgc cacaaaaact aaacgagatc aaacaattta tccttcaaaa 7440

gacaaacttc ttcaatccga acagggaatt cgacttccgc gatctccact ggtgcattaa 7500

cccacctagc aagatcaagg tgaattttac taattactgt gatacagttg gggtcaaaaa 7560

atctattgca tcggcagcaa atcccatcat tttatcagca ctctccgggg ccagaggtga 7620

catattcccg ccgtacagat gcagtggagc tactacttca gtaggcaggg tattccccct 7680

atccgtatca ttatccatgt ctttgatatc aagaacatca gagataatca atatgctaac 7740

cgctatctca gacggagtgt atggtaaaac ttatttgcta gtgcctgatt atattgaagg 7800

ggagttcgac tcacaaaaga ttcgagtctt tgagataggg tttatcaaac ggtggctgaa 7860

tgacatgcct ttactccaga caaccaacta tatggtcctc ccggaaactt ccaaagccaa 7920

ggtatgtact atagcagtgg gtgagctgac actagcttcc ttgtgtgtag atgagagcac 7980

cgtattgtta tatcatgaca gcaacggttc acaagatggt attctagtag tgacattggg 8040

aatatttggg gcaacaccta tggatcaagt tgaagaggtg atacctatcg ctcacccatc 8100

agtggagaga atacatataa caaatcaccg tgggttcata aaagactcaa tagtaacctg 8160

gatggtgcct gtattggtct ctgagaaaca agaggagcaa aaaaactgtc tggagtctgc 8220

ttgtcacaga aaatcctacc ctatgtgcaa ccaaacgtca tgggaaccct ttggaggagg 8280

acagttgcct tcttatgggc ggttgacatt acctctagat ccaagcattg accttcaact 8340

taacatatca tttacatatg gtccggttat actgaacgga gacggtatgg attattatga 8400

aagcccactt ttggactccg gatggcttac catacctcct aagaacggga cagtccttgg 8460

attgataaac aaagcaagta gaggagacca gttcactgtg accccccatg tgttgacatt 8520

tgcgcccagg gaatcaagtg gaaattgtta tttgcctatt caaacatccc agattatgga 8580

taaagatgtc cttactgagt ccaatttagt ggtgttacct acacagaatt ttagatatgt 8640

catagcaaca tatgatatat cccggggcga tcatgcaatt gtttattatg tttatgaccc 8700

aatccggacg atttcttata cacacccatt tagactaact accaagggta gacctgattt 8760

cctaaggatt gaatgttttg tgtgggatga cgatttgtgg tgtcatcaat tttaccgatt 8820

cgaggctaac atcactaact ctacaaccag tgttgagaat ttagtccgta taagattctc 8880

atgtaaccgt tcaaaacctt gatggtatgc tgatacacat ctcaattgaa ctgagggatg 8940

atgaccgtgg taagaaatcc cttaccgatg attgaattaa accatcccca gcattataaa 9000

aaaactaagg atccaggatc cttttagtca tggactctgt ttcagtgaac cagattttat 9060

accctgaggt ccatctagat agcccaattg tgaccaataa gctagtggct attttagagt 9120

atgcacgaat tagacataac tatcgactcc ttgacacaac gttagtgcgt aatatcaaag 9180

agagaatttc agaagggtta tcaaaccaga tgatcattaa ctgtatcgaa attgggagca 9240

ttgttaatca gaccttgtta tcttatccca aacacaacca tgtgatatat ccaaattgca 9300

acaaacttct gtttcatgca caggatcgag tcatctctct gaggttgaga aatatattca 9360

aaagaggaaa tagcatctat agtaaaataa cagacggggt caaaaaatgc ttaaacgata 9420

ttaatcttag tattggttta ggaggtgcat tggataagac tattggggcc aaagttgatg 9480

aagcaggcat aattatgcaa agctcacagt ggttcgaacc tttccttctg tggtttacaa 9540

ttaagacaga aatgagatca gtgattaaat cctctactca caactgtcgc aaacgaaggc 9600

agaatcctgt ctttgtaaga ggtgaatcat ttaatgtgtt agtgtctcgg gatcttgtat 9660

gtatcattga cctcaccagt cacaatgttt attacctaac atttgaaatg gtcctgatgt 9720

attgtgatgt gatagaaggg agattaatga ctgataccgc tatggcaatt gatcaccgtt 9780

actcaacttt gcatgtcaga atcaggtatc tttgggatct aattgatgga tttttcccgg 9840

acttaggaaa ttcgacctat caactggtag ctctgctgga gcctctttca ttggcttact 9900

tgcaattaaa agacatcacc ttctctctca ggggtgcttt tttgagtcac tgctttgctg 9960

aaatccagga gattttacag gacaatggct tctatactga agagacattc caaaccttaa 10020

cccaggctct agactttgtt ttcatcacag aggatataca tataacagga gagatctttt 10080

ccttttttag gagtttcggt cacccaagat tagaagcaat aacagcagca gaaaatgtac 10140

ggaaacacat gaatcaaccc aaagttgtct cctatgagac catgatgaag ggacacgcta 10200

ttttctgtgg gataatcatt aacggttatc gggatagaca tggaggaact tggcctccaa 10260

tggatcttcc tgtccatgca tctcctatca tcaggaatgc tcatgcctca ggagagggaa 10320

tcacctatag tcaatgtata gaaaattgga aatcctttgc aggaattcga tttaaatgct 10380

ttatgcccct ctgcctagac agtgatctga ccatgtattt gaaagataag gctttagcag 10440

cccttaaaaa agagtgggat tcagtgtacc caaaagaatt cctcaggtac aacccacctc 10500

gctccactga atctcggaga cttgttaatg tgtttctaga ggactctcag tttgaccctt 10560

ataatatgat tatgtacgtt atctcaggac aatatctaga cgatcctgac ttcaacctat 10620

catacagtct taaagagaaa gagattaaag aagtggggag gttattcgct aaaatgacat 10680

acaaaatgcg ggcctgtcaa gtcatagcag aaaacttaat atctaatgga attgggaagt 10740

acttcaagga caatgggatg gcaaaggatg aacacgatct cactaaagca ttgcacactc 10800

tggctgtgtc cggggttcct aaagacaaga aagactccca tcgcggcctc actaaccagt 10860

gtaagtctaa aaaactgaca ccttatcgag gagcccttca ctccgtctct tctccaagta 10920

gtagatatat ggacccaaac ccaaattttt gcaccagtag aagagaagac aatgacatag 10980

agatctatga gaccgtaagt gcatttataa ctacagatct caaaaagtac tgtctgaatt 11040

ggcgatatga gaccattagt atatttgctc agagattaaa tgaaatctac ggtctcccct 11100

catttttcca atggttgcac agaagattgg aacagtcgat cctatacgta agtgaccccc 11160

actgccctcc agatctcgat cgccatgtgg acttgaacac agtccctaac tctcaaatat 11220

tcatcaaata cccaatggga ggagtagagg gatattgtca aaagttatgg actattagca 11280

caatacctta tctgtacttg gcagcacatg aaagcggtgt caggattgca tcacttgttc 11340

aaggtgataa ccaaaccatt gctgtcacta aaagagttcc aagcacctgg tcatatgcct 11400

tgaagaagtc tgaagccagt cgagtcacca cagaatactt tatagcctta agacagagat 11460

tacatgatgt cggacatcat ttgaaagcaa atgaaacaat aatatcttcc cacttttttg 11520

tatactcaaa aggaatctat tatgatggga tgttaatttc gcaatccttg aaaagtatag 11580

ctaggtgcgt attttggtca gaaacaatag tggatgagac ccgagccgcc tgtagcaaca 11640

tttcaacaac attggcaaaa gccattgaga aagggtttga ccggtattta gcctatgcgc 11700

tgaatatttt aaaaatcatt caacaagtat taatttcatt aggattcact atcaattcag 11760

ctatgacgcg ggatgtgata gaaccccttt tacaagacca ctgtctcttg accaagatgg 11820

caattcttcc tgcacccata ggtggtctta attacctcaa tatgagtagg ctttttgtca 11880

ggaacatcgg ggatcccgtg acatcttcta ttgctgacct caaacgaatg atccgatcag 11940

gccttctcgg agtagagatt ttacatcaag tcatgaccca atacccaggt gactcttcgt 12000

acttagattg ggcaagtgac ccttattctg ctaatctgcc ctgtgtccag agcataaccc 12060

gactccttaa aaatattacg gccaggcatg tccttatcaa cagtccaaat cccatgctga 12120

aaggattgtt ccatgatgaa agtcaggatg aggatgaagc tttagctgct ttcttgatgg 12180

atagaaaaat tattatccca agagctgcac atgaaattct agataacacg atcactggtg 12240

cgagggaggc aattgctgga atgctagata ccacaaaggg gttgataaga gcaagcatga 12300

aaagaggagg gctaacccct agaataataa accgtttgtc aacttatgat tatgagcaat 12360

ttagggcagg tatcagacta ttgtcaggga agggacatga cccactcatc gatcaagact 12420

catgctctgt ccagttagcg agagcattaa ggaaccacat gtgggctaag ttggcgaagg 12480

gtcgtcctat ttatggtcta gaagtcccgg acatccttga atcaatgaag ggctatatga 12540

taagaagaca tgagtcttgt ttgctttgcg catcaggctc tcataactat ggttggtttt 12600

ttgtaccggc aaattgtcaa ttagatagta ttacagaggg aacatctgca ctgagggtac 12660

catacattgg gtccacaaca gaagaaagaa cagacatgaa attagcattc gtcaaatctc 12720

ctagtaggtc tctaaaatca gcagtgagaa tagcaactgt gtactcatgg gcctatggtg 12780

atgatgacga atcttggcaa gaggcttgga ctttggcaaa acagagagcg aacatctcac 12840

ttgaagaatt acggatgatt accccaattt ctacctctac taatctagct caccggctca 12900

gggacaagag tactcaagtc aaatactcag ggacctctct tatcagagta gcacgttatg 12960

caacaatctc taatgataat ctttcttttg tgatagctga caagaaagtg gacacgaact 13020

ttatttatca gcaaggtatg ctcctggggt tggggattct cgagcacttg tttagactgt 13080

cttcaaccac cggcgacacc aacaccgtac tgcatttaca tgttgaaaca gattgttgcg 13140

taatacccat gagcgaccat ccaagggtcc cagggctcag aaaggtcgtt ataccaagaa 13200

atatttgtac aaatcctttg atctatgaca gtaaccctat tattgagaaa gatgcagtca 13260

ggctttataa ccagagtcac aggaagcaca ttgtagagtt tgtcacatgg acaacagggc 13320

agctttatca tgtactagct aaatctactg ctatgtctat ggttgagatg attacaaagt 13380

ttgaaaaaga ccacctaaat gaagtctccg cgttaattgg cgatgacgat atcaatagct 13440

ttatcactga gtttcttctc gttgagccca ggttatttac tgtgtatcta ggtcaatgtg 13500

ctgcaatcaa ctggggcttt gaaattcatt atcaccgacc ttctggaaag tatcaaatgg 13560

gtgaattgtt gttttctttc ttgagtagaa tgagtaaagg agtcttcaaa attttaacca 13620

atgcattgag tcatcccaaa gtatatagac gattttggga cagtgggatg attgaacctg 13680

tacacggacc ctctcttgac tcccagaacc tacacataac tgtatgcaac ctgatctata 13740

actgttacat gatttaccta gaccttctgt taaatgatga gttagatgat ttctcattca 13800

ttttatgcga aagtgatgag gatatcatac ctgaaagatt tgacaacata caagctaggc 13860

acctatgcat cctgtctgac ctttattgta accctcgtga ttgtccccag attcgtgggt 13920

tgacaccaac acagaaatgt gctgtgttat cgaggtactt aaaatcaaaa gctctagagt 13980

cccatgttgg tctgacatgg aatgacaaac ctatcctgat agatcagtat tcatgttccc 14040

tgacatatct gagaagaggc tcaatcaagc agataagatt gagagtggac cccgggttca 14100

tcactgatgc tgttggatgc ttagaaaagc gacctctgag gaaaagtcct atctctaacg 14160

cctcagaatt aaaatcagaa tttgacccac cgaaagatga cctggttaaa ctcctgagtc 14220

agctatcaac aaggacacac aacttaccta ttacaggatt aggagtccga aactatgagg 14280

ttcactcatt cagaagaatt gggatcaatt caacggcatg ttacaaggca gttgaaatag 14340

tctctgttat taagaacgaa ttcacgtctg aagaacatgg attattccta ggagagggtt 14400

caggtgcaat gctgacagta tataaagagc tattgaggtt gtcaagatgt tattataaca 14460

gtggtgtgtc agcagaggct agaactggac aacgagagat ttcaccttac ccttctgagg 14520

tcagccttgt ggaacatcaa ttaggactcg ataaattggt gactgtgctt ttcaatggga 14580

gaccagaggt aacttgggtt gggagtgttg attgttacaa gtacatacta agtcagatat 14640

ctgctagcag tcttggattg attcactcgg atatcgagtc actacctgat aaagacataa 14700

ttgaaaaatt ggaagaactg tctgccatat tatcgatgac tttgatatta gggaaggtag 14760

ggtcagtgtt agtaatcaag atcatgcccg ctagtggcga ctgggttcaa ggatttattc 14820

tgtatgcact cccacatttt cttcgaagtt acataattta cccaagatac agcaattttg 14880

tgtcaacaga ggcctacctc gttttcactg gtcttagagc agggagacta gtcaatccgg 14940

aggggattaa acaacagatt ttgcgagtcg gtattcgaac ttcacccgga ttggtagggc 15000

acatcctttc atcaaagcag acagcatgtg tgcaatcttt gcatggacct ccatttcaag 15060

ctaaatcttt taatccttac ctccagggtt taacaagtat tgagaagatt ttgatcaatt 15120

gtgggcttac aattaacggt cttaaagtat gcaaaaacct gcttcaccat gatatctcgt 15180

caggcgagga agggctgaaa ggatctatca caatccttta tagggaactc gcacggttca 15240

aggataacca ccaattttca catggaatgt tccatgcata cccagtttta atcgcaagtc 15300

aggaaaggga gctcgtatcc accattgcaa ggaagtattg tggttatatt ttgctttact 15360

cgggagactt atacgaaatt accaggatag ttcgagacct gaaagccaac cacataattt 15420

ttgacttaca ccggaactta tttatggata acctatccag atctgaccgg tctctcatcc 15480

tgacgacaat cccaaaaagg aattggctct ttcaacttga gaccaaagag ataaaagagt 15540

ggttcaaact gttggggtat agtgcactga ttagaaacca ctgacgggtt ggtctggttc 15600

ctaaccctct gctattcatt gttattaaat ttaattttac gaaaaaaaac aacgattatt 15660

aataagttat catacccagc tttgtctggt 15690

<210> 3

<211> 94

<212> DNA

<213> artificial sequence

<400> 3

gcggccgccg tacggtttaa acgttaatta agcacggtcc gacctgggca tccgaaggag 60

gacgcacgtc cactcggatg gctaagggag ggcg 94

<210> 4

<211> 1572

<212> DNA

<213> Canine Distemper Virus

<400> 4

atggctagcc ttctcaagag cctcacattg ttcaagagga ctcgggacca acccccactt 60

gcctcgggct ccggaggagc aataagaggg ataaagcatg tcattatagt cctaatcccg 120

ggtgattcaa gcattgttac aaggtctcga ctactggata gacttgttag attggtcggt 180

gatccggaaa tcaacggacc taaattaact gggattttaa tcagtatcct ctccttgttc 240

gtggaatccc ctggacagtt gatccagagg atcatagacg accctgatgt aagcatcaag 300

ttagtagagg taatcccaag catcaactct ggttgtggtc ttacatttgc atccagagga 360

gcaagtttgg attctgaggc agatgagttc ttcaaaattg tagacgaagg gtcgaaagct 420

caaggacaat taggctggtt ggagaataag gatattgtag acatagaagt tgatgatgct 480

gagcaattca atatattgct agcttccatc ttggcccaaa tttggatcct gctcgctaaa 540

gcagtgactg ctcctgatac tgcagccgac tcggaaatga ggaggtggat taagtatacc 600

caacagagac gtgtggtcgg ggaatttaga atgaacaaaa tctggcttga tattgttaga 660

aacaggattg ctgaggacct atctttgagg cgattcatgg tggcactcat cttggacatc 720

aaacgatccc cagggaacaa gcctagaatt gctgaaatga tttgtgatat agataactac 780

attgtggaag ctggattagc tagtttcatc ttaactatca aatttggcat tgaaactatg 840

tatccggctc ttgggttgca tgagttttcc ggagagttaa caactattga atcccttatg 900

atgctatatc aacagatggg tgaaacagca ccgtacatgg ttattctgga aaattctgtt 960

cagaacaaat ttagtgcagg atcctaccca ctgctctgga gttatgctat gggagttggt 1020

gttgaacttg aaaactccat gggagggtta aatttcggta gatcctactt tgatccggcc 1080

tatttcaggc tcgggcaaga aatggtgaga agatctgccg gcaaagtaag ctctgcactt 1140

gccgccgagc ttggcatcac caaggaagag gctcagctag tgtcagaaat agcatccaag 1200

acaacggagg accggacgat tcgcactgct ggtcccaagc aatctcaaat cacttttctg 1260

cactcagaaa gatccgaagt caccaatcaa caacccccaa ccatcaacaa gaggtccgaa 1320

aaccaaggag gagacaaata ccccatccac gtcaatgatg aacggtttcc agggtacacc 1380

cctgatgtca acagctccga atggagtgaa tcacgctatg atacccagac tattcaagat 1440

gatggaaacg acgatgaccg gaaatcgatg gaagcaatcg ccaagatgag aatgcttact 1500

aagatgctca gtcaacctgg gaccagtgaa gagagttctc ctgtctataa tgatagagag 1560

ctactcaatt aa 1572

<210> 5

<211> 1524

<212> DNA

<213> Canine Distemper Virus

<400> 5

atggcagaag agcaggccta tcatgtcaat aaagggctgg aatgcctcaa agccctcaga 60

gagaatcctc ctgacattga ggagattcaa gaggtcagca gtatcagaga tcaaacccgc 120

gacccaggcc aagagaatgg aaccgcaagc atgcaggaag aagaggtctc tcaggatctc 180

gatgaatcac acgagccagc aaaaggatca aactatgtcg gccatgtact ccaaaataat 240

ccgggatgtg gagagagcaa cactgcgctt gtggaggaag agcagcccgc taaagatgat 300

gtccaaccag gacctgaaat acgatgttat catgtttatg atcacagtgg tgaagaggtt 360

aagggaatcg aagatgctga cagtctcgtg gtacctgcag gcgctgtcag taatcgagga 420

ttcgagagag gagaaggaag ccttgatgat agcactgagg attctggcga agattattcc 480

gagggaaatg cttcatctaa ctggggatat tctttcggcc ttaaaccaga cagagcggct 540

gatgtgagca tgctgatgga agaggaattg agtactctgc tcaagacaag cagaaatgtg 600

gggattaaga aaagggatgg gatgactctg cagttcccac acagtcccga aggtaagaca 660

gaggatccgg agtgtggatc cattaaaaag ggcacaggag agaggtcagc ctcacatgga 720

atggggatag ttgctggatc gacaagtggt gcaacccaat ctgcactcaa gtcaactggg 780

ggatcatcag ggccaagtgt gtctgcggag aatgtccgcc aacctgcaat gaatgcaaag 840

atgacccaga aatgcaaacc cgagtctggt acgcaactcc ctgccaggac ctcaaatgag 900

gctgaatctg acagtgagta tgacgatgag cttttttctg aaatacaaga aattcgatct 960

gctatcacta agctaatgga agataatcaa gcaatacttt ctaaactgga taccctatta 1020

ctgcttaaag gagagactga ttcaattaag aaacaaatta gcaagcaaaa tattgctatt 1080

tccacgattg aggggcatct atcaagcatt atgatagcta tacctggttt tgggaaggac 1140

actggagacc ctacggcaaa tgtcgacatt aacccagagc tccgccctat aatagggagg 1200

gattcaggaa gagcactggc ggaagttctc aagcaacccg catcatcccg cggtaatcgg 1260

aaggacagtg gtatcgcctt gggttcaaaa ggtcaactat tgagagacct ccagctgaaa 1320

cctattgaca aagagtctag ttcggcaatc ggatacaaac cgaaggatac cgcaccttcc 1380

aaagctgtac ttgcatcatt gattagatca agcaaaattg atcaaagtca caaacacaac 1440

atgctggccc ttctcaaaaa tattaagggg gatgacaatc taaacgagtt ctaccagatg 1500

atcaagagta tcacacatgc ttaa 1524

<210> 6

<211> 6555

<212> DNA

<213> Canine Distemper Virus

<400> 6

atggactctg tttcagtgaa ccagatttta taccctgagg tccatctaga tagcccaatt 60

gtgaccaata agctagtggc tattttagag tatgcacgaa ttagacataa ctatcgactc 120

cttgacacaa cgttagtgcg taatatcaaa gagagaattt cagaagggtt atcaaaccag 180

atgatcatta actgtatcga aattgggagc attgttaatc agaccttgtt atcttatccc 240

aaacacaacc atgtgatata tccaaattgc aacaaacttc tgtttcatgc acaggatcga 300

gtcatctctc tgaggttgag aaatatattc aaaagaggaa atagcatcta tagtaaaata 360

acagacgggg tcaaaaaatg cttaaacgat attaatctta gtattggttt aggaggtgca 420

ttggataaga ctattggggc caaagttgat gaagcaggca taattatgca aagctcacag 480

tggttcgaac ctttccttct gtggtttaca attaagacag aaatgagatc agtgattaaa 540

tcctctactc acaactgtcg caaacgaagg cagaatcctg tctttgtaag aggtgaatca 600

tttaatgtgt tagtgtctcg ggatcttgta tgtatcattg acctcaccag tcacaatgtt 660

tattacctaa catttgaaat ggtcctgatg tattgtgatg tgatagaagg gagattaatg 720

actgataccg ctatggcaat tgatcaccgt tactcaactt tgcatgtcag aatcaggtat 780

ctttgggatc taattgatgg atttttcccg gacttaggaa attcgaccta tcaactggta 840

gctctgctgg agcctctttc attggcttac ttgcaattaa aagacatcac cttctctctc 900

aggggtgctt ttttgagtca ctgctttgct gaaatccagg agattttaca ggacaatggc 960

ttctatactg aagagacatt ccaaacctta acccaggctc tagactttgt tttcatcaca 1020

gaggatatac atataacagg agagatcttt tcctttttta ggagtttcgg tcacccaaga 1080

ttagaagcaa taacagcagc agaaaatgta cggaaacaca tgaatcaacc caaagttgtc 1140

tcctatgaga ccatgatgaa gggacacgct attttctgtg ggataatcat taacggttat 1200

cgggatagac atggaggaac ttggcctcca atggatcttc ctgtccatgc atctcctatc 1260

atcaggaatg ctcatgcctc aggagaggga atcacctata gtcaatgtat agaaaattgg 1320

aaatcctttg caggaattcg atttaaatgc tttatgcccc tctgcctaga cagtgatctg 1380

accatgtatt tgaaagataa ggctttagca gcccttaaaa aagagtggga ttcagtgtac 1440

ccaaaagaat tcctcaggta caacccacct cgctccactg aatctcggag acttgttaat 1500

gtgtttctag aggactctca gtttgaccct tataatatga ttatgtacgt tatctcagga 1560

caatatctag acgatcctga cttcaaccta tcatacagtc ttaaagagaa agagattaaa 1620

gaagtgggga ggttattcgc taaaatgaca tacaaaatgc gggcctgtca agtcatagca 1680

gaaaacttaa tatctaatgg aattgggaag tacttcaagg acaatgggat ggcaaaggat 1740

gaacacgatc tcactaaagc attgcacact ctggctgtgt ccggggttcc taaagacaag 1800

aaagactccc atcgcggcct cactaaccag tgtaagtcta aaaaactgac accttatcga 1860

ggagcccttc actccgtctc ttctccaagt agtagatata tggacccaaa cccaaatttt 1920

tgcaccagta gaagagaaga caatgacata gagatctatg agaccgtaag tgcatttata 1980

actacagatc tcaaaaagta ctgtctgaat tggcgatatg agaccattag tatatttgct 2040

cagagattaa atgaaatcta cggtctcccc tcatttttcc aatggttgca cagaagattg 2100

gaacagtcga tcctatacgt aagtgacccc cactgccctc cagatctcga tcgccatgtg 2160

gacttgaaca cagtccctaa ctctcaaata ttcatcaaat acccaatggg aggagtagag 2220

ggatattgtc aaaagttatg gactattagc acaatacctt atctgtactt ggcagcacat 2280

gaaagcggtg tcaggattgc atcacttgtt caaggtgata accaaaccat tgctgtcact 2340

aaaagagttc caagcacctg gtcatatgcc ttgaagaagt ctgaagccag tcgagtcacc 2400

acagaatact ttatagcctt aagacagaga ttacatgatg tcggacatca tttgaaagca 2460

aatgaaacaa taatatcttc ccactttttt gtatactcaa aaggaatcta ttatgatggg 2520

atgttaattt cgcaatcctt gaaaagtata gctaggtgcg tattttggtc agaaacaata 2580

gtggatgaga cccgagccgc ctgtagcaac atttcaacaa cattggcaaa agccattgag 2640

aaagggtttg accggtattt agcctatgcg ctgaatattt taaaaatcat tcaacaagta 2700

ttaatttcat taggattcac tatcaattca gctatgacgc gggatgtgat agaacccctt 2760

ttacaagacc actgtctctt gaccaagatg gcaattcttc ctgcacccat aggtggtctt 2820

aattacctca atatgagtag gctttttgtc aggaacatcg gggatcccgt gacatcttct 2880

attgctgacc tcaaacgaat gatccgatca ggccttctcg gagtagagat tttacatcaa 2940

gtcatgaccc aatacccagg tgactcttcg tacttagatt gggcaagtga cccttattct 3000

gctaatctgc cctgtgtcca gagcataacc cgactcctta aaaatattac ggccaggcat 3060

gtccttatca acagtccaaa tcccatgctg aaaggattgt tccatgatga aagtcaggat 3120

gaggatgaag ctttagctgc tttcttgatg gatagaaaaa ttattatccc aagagctgca 3180

catgaaattc tagataacac gatcactggt gcgagggagg caattgctgg aatgctagat 3240

accacaaagg ggttgataag agcaagcatg aaaagaggag ggctaacccc tagaataata 3300

aaccgtttgt caacttatga ttatgagcaa tttagggcag gtatcagact attgtcaggg 3360

aagggacatg acccactcat cgatcaagac tcatgctctg tccagttagc gagagcatta 3420

aggaaccaca tgtgggctaa gttggcgaag ggtcgtccta tttatggtct agaagtcccg 3480

gacatccttg aatcaatgaa gggctatatg ataagaagac atgagtcttg tttgctttgc 3540

gcatcaggct ctcataacta tggttggttt tttgtaccgg caaattgtca attagatagt 3600

attacagagg gaacatctgc actgagggta ccatacattg ggtccacaac agaagaaaga 3660

acagacatga aattagcatt cgtcaaatct cctagtaggt ctctaaaatc agcagtgaga 3720

atagcaactg tgtactcatg ggcctatggt gatgatgacg aatcttggca agaggcttgg 3780

actttggcaa aacagagagc gaacatctca cttgaagaat tacggatgat taccccaatt 3840

tctacctcta ctaatctagc tcaccggctc agggacaaga gtactcaagt caaatactca 3900

gggacctctc ttatcagagt agcacgttat gcaacaatct ctaatgataa tctttctttt 3960

gtgatagctg acaagaaagt ggacacgaac tttatttatc agcaaggtat gctcctgggg 4020

ttggggattc tcgagcactt gtttagactg tcttcaacca ccggcgacac caacaccgta 4080

ctgcatttac atgttgaaac agattgttgc gtaataccca tgagcgacca tccaagggtc 4140

ccagggctca gaaaggtcgt tataccaaga aatatttgta caaatccttt gatctatgac 4200

agtaacccta ttattgagaa agatgcagtc aggctttata accagagtca caggaagcac 4260

attgtagagt ttgtcacatg gacaacaggg cagctttatc atgtactagc taaatctact 4320

gctatgtcta tggttgagat gattacaaag tttgaaaaag accacctaaa tgaagtctcc 4380

gcgttaattg gcgatgacga tatcaatagc tttatcactg agtttcttct cgttgagccc 4440

aggttattta ctgtgtatct aggtcaatgt gctgcaatca actggggctt tgaaattcat 4500

tatcaccgac cttctggaaa gtatcaaatg ggtgaattgt tgttttcttt cttgagtaga 4560

atgagtaaag gagtcttcaa aattttaacc aatgcattga gtcatcccaa agtatataga 4620

cgattttggg acagtgggat gattgaacct gtacacggac cctctcttga ctcccagaac 4680

ctacacataa ctgtatgcaa cctgatctat aactgttaca tgatttacct agaccttctg 4740

ttaaatgatg agttagatga tttctcattc attttatgcg aaagtgatga ggatatcata 4800

cctgaaagat ttgacaacat acaagctagg cacctatgca tcctgtctga cctttattgt 4860

aaccctcgtg attgtcccca gattcgtggg ttgacaccaa cacagaaatg tgctgtgtta 4920

tcgaggtact taaaatcaaa agctctagag tcccatgttg gtctgacatg gaatgacaaa 4980

cctatcctga tagatcagta ttcatgttcc ctgacatatc tgagaagagg ctcaatcaag 5040

cagataagat tgagagtgga ccccgggttc atcactgatg ctgttggatg cttagaaaag 5100

cgacctctga ggaaaagtcc tatctctaac gcctcagaat taaaatcaga atttgaccca 5160

ccgaaagatg acctggttaa actcctgagt cagctatcaa caaggacaca caacttacct 5220

attacaggat taggagtccg aaactatgag gttcactcat tcagaagaat tgggatcaat 5280

tcaacggcat gttacaaggc agttgaaata gtctctgtta ttaagaacga attcacgtct 5340

gaagaacatg gattattcct aggagagggt tcaggtgcaa tgctgacagt atataaagag 5400

ctattgaggt tgtcaagatg ttattataac agtggtgtgt cagcagaggc tagaactgga 5460

caacgagaga tttcacctta cccttctgag gtcagccttg tggaacatca attaggactc 5520

gataaattgg tgactgtgct tttcaatggg agaccagagg taacttgggt tgggagtgtt 5580

gattgttaca agtacatact aagtcagata tctgctagca gtcttggatt gattcactcg 5640

gatatcgagt cactacctga taaagacata attgaaaaat tggaagaact gtctgccata 5700

ttatcgatga ctttgatatt agggaaggta gggtcagtgt tagtaatcaa gatcatgccc 5760

gctagtggcg actgggttca aggatttatt ctgtatgcac tcccacattt tcttcgaagt 5820

tacataattt acccaagata cagcaatttt gtgtcaacag aggcctacct cgttttcact 5880

ggtcttagag cagggagact agtcaatccg gaggggatta aacaacagat tttgcgagtc 5940

ggtattcgaa cttcacccgg attggtaggg cacatccttt catcaaagca gacagcatgt 6000

gtgcaatctt tgcatggacc tccatttcaa gctaaatctt ttaatcctta cctccagggt 6060

ttaacaagta ttgagaagat tttgatcaat tgtgggctta caattaacgg tcttaaagta 6120

tgcaaaaacc tgcttcacca tgatatctcg tcaggcgagg aagggctgaa aggatctatc 6180

acaatccttt atagggaact cgcacggttc aaggataacc accaattttc acatggaatg 6240

ttccatgcat acccagtttt aatcgcaagt caggaaaggg agctcgtatc caccattgca 6300

aggaagtatt gtggttatat tttgctttac tcgggagact tatacgaaat taccaggata 6360

gttcgagacc tgaaagccaa ccacataatt tttgacttac accggaactt atttatggat 6420

aacctatcca gatctgaccg gtctctcatc ctgacgacaa tcccaaaaag gaattggctc 6480

tttcaacttg agaccaaaga gataaaagag tggttcaaac tgttggggta tagtgcactg 6540

attagaaacc actga 6555

Claims (10)

1. The infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain is characterized in that the infectious cDNA clone is an infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain obtained by reverse genetic operation; the nucleotide sequence of the infectious cDNA clone is shown as SEQ ID NO. 1.

2. A method for obtaining an infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain according to claim 1, comprising the steps of:

(1) extracting total RNA of HBF-1 virus suspension;

(2) the obtained RNA was subjected to reverse transcription to obtain cDNA.

(3) Utilizing four pairs of primers F1/R1, F2/R2, F3/R3 and F4/R4, taking the reverse transcription product cDNA of HBF-1 genome RNA as a template, amplifying HBF-1 divided into 4 sections by using high-fidelity DNA polymerase, identifying the PCR product by agarose gel electrophoresis, and performing gel recovery after the size is correct;

F1 5’-GCGGCCGCTGTTAAGCGTCTGATGAGTCCGTGAGGACGAAACTATAGGAAAGGAATTCCTATAGTCACCAGACAAAGTTGGCTAAG-3’

R1 5’-GCGTACGTGAAAGCAGTTTTGAGCCT-3’

F2 5’-CTGCTTTCACGTACGCTTAAAAGCAATTATAAAAAACTTAGG-3’

R2 5’-GTTTAAACGCTTTTGAAGGAAATTAGGCGGGACT-3’

F3 5’-TTCCTTCAAAAGCGTTTAAACTGCAACAAATAGTGGCG-3’

R3 5’-GGATTAATTAACACGGTCATCATCCCTCAGTTCAATTGA-3’

F4 5’-GGGATGATGACCGTGTTAATTAATCCCTTACCGATGATTGAATT-3’R4 5’-CGGATGCCCAGGTCGGACCGCGAGGAGGTGGAGATGCCATGCCGACCCACCAGACAAAGCTGGGTATGATAAC-3’

(4) modification of eukaryotic expression vector pcDNA3.1

Transforming the polyclonal restriction site of eukaryotic expression vector pcDNA3.1, connecting the prepared sequence with Not I, Bsiw I, pmei I, Pac I, Cpo I restriction site and partial hepatitis delta ribozyme to the pcDNA3.1 vector digested by pmei I, and naming the transformed vector as pcDNA3.2;

(5) respectively connecting the F1, F2, F3 and F4 fragments to pEASY-Blunt, sequencing, respectively and sequentially connecting each gene fragment with correct sequencing to the downstream of the CMV promoter of the modified vector pcDNA3.2 to obtain a plasmid containing the infectious cDNA clone of the fox-derived canine distemper virus (HCLV) 1 strain in claim 1.

3. The method of claim 2, wherein the nucleotide sequence of the cDNA obtained in step (2) is shown in SEQ ID NO.2, and the sequence of the partial ribozyme having Not I, BsiwI, pmei, PacI, CpoI cleavage sites and partial hepatitis D in step (4) is shown in SEQ ID NO. 3.

4. The use of the infectious cDNA clone of Fox-derived Canine distemper Virus (HBF) -1 strain according to claim 1 in rescuing recombinant Fox-derived Canine distemper Virus (HBF) -1 strain.

5. A reverse genetic operating system of the fox-derived canine distemper virus virulent HBF-1 strain is characterized in that the operating system consists of a recombinant plasmid containing the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain in claim 1 and three auxiliary plasmids for expressing the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein.

6. The reverse genetic operation system of fox-derived canine distemper virus virulent HBF-1 strain according to claim 5, characterized in that nucleotide sequences encoding the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein are respectively shown in SEQ ID No. 4-6.

7. The reverse genetic manipulation system of the fox-derived canine distemper virus virulent HBF-1 strain according to claim 5, wherein the recombinant plasmid containing the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain according to claim 1 is prepared by the following method:

(1) modification of eukaryotic expression vector pcDNA3.1

Transforming the polyclonal restriction site of eukaryotic expression vector pcDNA3.1, connecting the prepared sequence with Not I, Bsiw I, pmei I, Pac I, Cpo I restriction site and partial hepatitis delta ribozyme to the pcDNA3.1 vector digested by pmei I, and naming the transformed vector as pcDNA3.2; the sequence with Not I, Bsiw I, Pme I, Pac I and Cpo I enzyme cutting sites and part of hepatitis delta ribozyme is shown in SEQ ID NO. 3;

(2) connecting the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain of claim 1 to the downstream of the CMV promoter of the modified vector pcDNA3.2 to obtain a recombinant plasmid containing the whole genome of the CDV HBF-1 strain;

the three auxiliary plasmids for expressing the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein are obtained by respectively cloning nucleotide sequences encoding the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein to the downstream of a CMV promoter of a eukaryotic expression vector pcDNA3.1 to respectively express the fox-derived canine distemper virus virulent HBF-1N, P, L protein.

8. The use of the fox-derived canine distemper virus virulent HBF-1 strain reverse genetic manipulation system according to any one of claims 5-7 in rescue of the fox-derived canine distemper virus virulent HBF-1 strain and reverse genetic study on the fox-derived canine distemper virus virulent HBF-1 strain.

9. A method for rescuing fox-derived virulent HBF-1 canine distemper virus strains is characterized by comprising the following steps:

will contain the rightsObtaining a recombinant plasmid of the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain and three helper plasmids expressing the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein, and transfecting BSR cells, preferably, the recombinant plasmid containing the infectious cDNA clone of the fox-derived canine distemper virus virulent HBF-1 strain of claim 1 and the three helper plasmids expressing the fox-derived canine distemper virus virulent HBF-1N protein, the P protein and the L protein according to the proportion of 5 mu g, 1 mu g, 0.8 mu g and 0.5 mu g; 72h after transfection, cell suspension was taken and added to Vero-dSlam cells at 37 ℃ with 5% CO₂Culturing for 5-10 days, observing typical syncytium lesion (CPE) caused by CDV, collecting cells and supernatant after the CPE appears, continuously carrying out passage in Vero-dSlam cells, culturing for 6-7d, harvesting virus, and obtaining rescued recombinant virus, which is named rHBF-1.

10. A recombinant fox-derived virulent rHBF-1 strain of canine distemper virus, which is rescued by the method according to claim 9.

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