CN101768575B - Construction of recombinant rabies virus of double-expression G gene and biological property analysis thereof - Google Patents

Abstract

The present invention provides a recombinant rabies virus strain in which the G gene is additionally inserted into the genome, as well as a method for constructing the recombinant virus strain and its application. The recombinant virus strain constructed by the present invention has better safety and immunogenicity, and is applicable to In the preparation of inactivated rabies virus vaccine.

Description

Construction of recombinant rabies virus with double expression of G gene and analysis of its biological characteristics

Technical field:

The invention belongs to the field of recombinant vaccines. Specifically, the present invention provides a recombinant rabies virus strain, which additionally inserts a G gene into the genome; the present invention provides a method for constructing the recombinant rabies virus strain and its application for preparing a vaccine.

Background technique

Rabies is an important zoonotic disease, responsible for approximately 60,000 deaths per year ⁽¹⁾ . In many developing countries, dogs are still the main cause of human rabies, while in developed countries, rabies virus is mainly found in wild animal reservoirs. Practice has proved that immunization of animals such as dogs can significantly reduce the incidence of human rabies ⁽²⁾ , so immunization of animals is extremely important for the control of rabies.

Rabies virus belongs to the genus Lyssavirus in Rhabdoviridae. The genome is a single-stranded negative-sense RNA without segments, with a total length of about 12kb, encoding five viral proteins: nucleoprotein N, phosphoprotein P, matrix protein M, and glycoprotein G and RNA polymerase L. Glycoprotein G, located on the capsule, is the main virulence factor and also the main antigen that induces a protective immune response.

At present, the Flury-LEP live virus vaccine is widely used in the immunization of dogs in my country. The virus was isolated from the brain of a girl killed by rabies by Koprowski et al. in 1940. A virulent Flury strain passed through the brain of 1-day-old chicks continuously for 138 generations, and then passed through the yolk sac of chicken embryos for 40 to 50 generations, reducing the It has the property of invading the central nervous system when injected peripherally. The virus is not pathogenic to dogs, but maintains its immunogenicity, but mice are still infected and lethal when injected intracerebrally, and the Flury-LEP vaccine can sometimes cause rabies in 3-month-old puppies. In view of the large safety hazards in the use of attenuated live vaccines, WHO recommends using inactivated rabies vaccines instead of attenuated vaccines.

In order to obtain a rabies vaccine strain with high efficiency and better immunogenicity, a rabies virus rLEP-G containing 2 G genes was constructed by reverse genetic technology in this study. We characterized the biological properties of this recombinant virus strain and compared it with the wild-type strain Flury-LEP. The results showed that the recombinant virus rLEP-G had better safety and immunogenicity than LEP, and was suitable for the development of rabies virus inactivated vaccine.

Contents of the invention

Aiming at the technical problem to be solved by the present invention, the present invention provides in a first aspect a recombinant rabies virus strain with better safety and immunogenicity, which additionally inserts the G gene into the genome.

In one embodiment of the first aspect, the genome comprising two G genes is as shown in Figure 1, and another G gene is inserted at the Pme I restriction site, and the rabies recombinant virus genome after the G gene is inserted is shown in Figure 1A displayed. In one embodiment of the first aspect, the recombinant virus is constructed from the wild-type rabies virus strain Flury-LEP. In one embodiment of the first aspect, the constructed recombinant rabies virus strain is rLEP-G constructed according to the example of the present invention. In one embodiment, the sequence of the inserted G gene is shown in SEQ ID NO: 8.

In a second aspect of the present invention, a method for constructing said recombinant rabies virus strain is provided, said method comprising the following steps;

1) Constructing a transcription plasmid, wherein the G gene sequence is inserted into the genome of the wild-type rabies virus strain to obtain a recombinant genome full-length cDNA clone containing double G genes;

2) Constructing an auxiliary plasmid system, which encodes nucleoprotein N, phosphoprotein P, and polymerase protein L of LEP respectively;

3) using the plasmid vector of the recombinant genome full-length cDNA clone containing the double G gene and the helper plasmid system to co-transfect into a host cell that allows the virus to replicate, and culturing the host cell;

4) Rescue the virus.

In one embodiment of the second aspect, the constructed transcription plasmid is pCI-LEP-G, and the constructed transcription auxiliary plasmids are pCAGG-N, pCAGG-P, and pCAGG-L, respectively. In one embodiment of the second aspect, the host cells used are BHK-21 cells.

In the third aspect of the present invention, a plasmid vector comprising the full-length cDNA clone of the recombinant genome containing the double G gene constructed in the first aspect is provided. In one embodiment of the third aspect, the plasmid vector is pCI.

In the fourth aspect of the present invention, the use of the recombinant virus strain constructed in the first aspect for preparing an inactivated rabies virus vaccine is provided.

Invention effect

The growth kinetics curve of the recombinant rabies virus strain rLEP-G strain comprising two G genes provided by the present invention on nerve cell NA cells and non-neuron cell BHK-21 cells is very similar to that of the parental Flury-LEP strain, and the Beagle dog The immunoassay also shows that inactivated recombinant virus rLEP-G can induce higher neutralizing antibody levels than the wild-type rabies virus strain, indicating that the recombinant rabies virus strain of the present invention can be used to develop safer and more immune Original vaccine.

Description of drawings

Figure 1 (A) Construction of the LEP genome with double G genes

(B) Left panel: Indirect immunofluorescence detection rescued virus rLEP-G from infecting BHK-21 cells,

Right panel: Negative control;

Fig. 2 is the growth kinetic curve of wtLEP and rLEP-G on NA cell (A) and BHK-21 cell (B);

Figure 3 is the Western-blot identification of rLEP-G expressing G protein

1. LEP infected BHK-21 cells; 2. rLEP-G infected BHK-21 cells; 3. BHK-21 cell control; 4. Protein standard molecular weight standard

Detailed ways:

The present invention will be further described by the following examples, but the scope of the present invention is not limited by the examples.

Embodiment 1 Contains the construction and identification of the recombinant virus of two G genes

1 Materials and methods

1.1 Plasmids, cell lines and viruses

Plasmids pCAGG and pCI-RBz were purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and pBluescriptSK(+) was purchased from clonetech. BHK-21 cells (ATCC CC1-10) and 293T cells (CRL-11268) were purchased from ATCC, and the medium was DMEM containing 10% fetal bovine serum (purchased from GIBCO). Mouse brain neuroma cells NA cells come from the Military Veterinary Research Institute of the Academy of Military Medical Sciences, and the culture medium is MEM containing 10% fetal bovine serum (purchased from GIBCO). Rabies virus Flury-LEP and HEP were purchased from the China Veterinary Microbial Culture Collection Management Center of the China Veterinary Drug Administration, amplified on BHK-21 cells, and frozen at -70°C for future use.

1.2 Main reagents and instruments

T4 DNA ligase, restriction endonuclease Sph I, Mlu I, etc. were all purchased from TaKaRa Company. Phusion ^™ Ultra-Fidelity DNA Polymerase. SuperScript ^TM III reverse transcription kit, serum-free medium Opti-MEM, and transfection reagent Lipofectamine ^TM 2000 were all purchased from Invitrogen. Calf serum was purchased from Gibco. LB medium components were purchased from OXOID company. Gel Extraction Mini Kit and Plasmid Mini Kit were purchased from OMEGA Company. Plasmid medium extraction kit QIAfilter Plasmid Midi Kit was purchased from QIAGEN. Mouse anti-rabies virus hyperimmune serum was prepared by our laboratory. FITC-labeled goat anti-mouse fluorescent secondary antibody was purchased from Sigma. Ordinary microscopes were purchased from OLYMPUS Company; fluorescence microscopes were purchased from ZEISS Company. The PCR instrument was purchased from Applied Biosystem. Nucleic acid sequencing adopts BECKMAN CEQ 8000 automatic sequencer, and the reagents used are products of BECKMAN. P2 level biological safety cabinets were purchased from LABCONCO. Carbon dioxide incubators were purchased from Thermo Electron. Different models of centrifuges were purchased from BECKMAN. The -70°C ultra-low temperature refrigerator was purchased from NBS Company. Ordinary refrigerators and freezers were purchased from Haier Company. The fully automatic water purifier MILI-Q was purchased from BIOCEL. Cell culture flasks and cell culture plates of various specifications were purchased from CORNING Company.

1.3 Primers

Referring to the Flury-HEP strain genome sequence (Gene Bank sequence number AB085828) provided in Gene Bank, and the Flury-LEP gene sequence (Gene Bank sequence number DQ099524, the full-length genome cDNA sequence of Flury-LEP is shown in SEQ ID NO: 1), The Flury-LEP genome was divided into three segments (ie, F1, F2 and F3) to design PCR primers respectively. Wherein, fragment F1 is the 1-4063 nucleotide sequence (SEQ ID NO: 2) of Flury-LEP gene sequence, and fragment F2 is the 4013-8254 nucleotide sequence (SEQ ID NO: 3) of Flury-LEP gene sequence, Fragment F3 is the 8187-11925 nucleotide sequence of the Flury-LEP gene sequence (SEQ ID NO: 4). The primers used to construct the full-length genomic cDNA, the primers used to introduce Pme I (Table 1) and the primers used to construct the helper plasmid (Table 2) were all synthesized by Shanghai Handsome Biotechnology Co., Ltd.

Table 1 Primers used for RT-PCR and full-length genomic cDNA construction

Note: The cDNA fragment is consistent with Figure 1. The underlined part is the virus-specific sequence, the restriction site is indicated in bold, and the hammerhead ribozyme (HamRz) and hepatitis D virus ribozyme (HdvRz) are indicated in italics.

The primers used in table 2 auxiliary plasmid construction

Note: The underlined part is the virus-specific sequence, the boldface indicates the restriction site, the mutated base is indicated by a character frame, and the Kozak sequence is indicated by italics.

1.4 Construction of Flury-LEP genome full-length cDNA clone and cDNA fragment clone and auxiliary plasmid system

1.4.1 Extraction, reverse transcription and PCR of viral genome

Take 250 μL of Flury-LEP virus solution, add 750 μL Trizol, and extract genomic RNA by conventional methods. The extracted RNA was reverse-transcribed with the SuperScript ^™ III Reverse Transcription Kit (reverse transcription primers are shown in Table 1) and operated according to the instructions of the kit to obtain 3 viral cDNA fragments F1, F2 and F3 with partially overlapping ends .

1.4.2 Cloning of RT-PCR products

The entire genome was divided into three fragments with partially overlapping ends for RT-PCR amplification, and the PCR products were subjected to 1% agarose gel electrophoresis, and the F1, F2 and F3 fragments were recovered using a gel recovery kit (purchased from OMEGA Company) and cloned separately. To the EcoR v site of pBluscript II KS (+/-) (Clontech), transform competent cells DH5α, select and extract positive plasmids pBlue-F1, pBlue-F2 and pBlue-F3, and sequence to ensure that the cloned genome fragments are consistent with the viral genome The RNA sequences were completely consistent (for specific methods, see "Molecular Cloning Test Guide", edited by J. Sambrook, third edition in 2008, translated by Science Press).

1.4.3 Construction of full-length cDNA clone and cDNA fragment clone of Flury-LEP genome

The restriction enzyme cutting sites existing in the adjacent overlapping parts of the genomic cDNA fragments F1, F2 and F3 were used for ligation and assembly (see Figure 1 for a schematic diagram). Specifically, the F3 segment cDNA was first cloned into pCI-RBz through Nhe I and Rsr II sites, and the resulting plasmid was named pCI-F3, and then the F1 segment was inserted into pCI-F3 through Nhe I and Mlu I, and the resulting plasmid was named pCI-F31. Finally, the F2 segment was cloned into pCI-F31 through Sph I and Mlu I, so far the whole genome cDNA assembly of Flury-LEP was completed, and the constructed viral genome transcription plasmid was named pCI-LEP.

The clone is under the CMV promoter, and the full-length cDNA between the two ribozymes can be transcribed under the action of eukaryotic RNA polymerase, and due to the autocatalytic function of the ribozyme, the 3' end of the transcript can be guaranteed Exactly identical to the viral genome.

1.4.4 Construction of genome full-length cDNA containing double G gene

In order to rescue the recombinant LEP virus expressing the double G gene, on the basis of the viral genome transcription plasmid pCI-LEP, first construct a full-length cDNA clone with a Pme I restriction site between the G and L genes, the specific method is : Using pCI-LEP as a template, using F2-PF and PmeI-PR as primers to amplify the PCR fragment PmeA, using F2-PR and PmeI-PF as primers to amplify the PCR fragment PmeB; and then using the PCR fragment PmeA and PmeB were used as templates, and F2-PF and F2-PR were used as primers to amplify the F2 fragment containing PmeI digestion, which was named F2-P. F2-P was cloned into pCI-F31 via SphI and MluI to construct the recombinant genome transcription vector pCI-LEP-PmeI. Then, with the upstream primer InsertGPF: 5'-ATGC GTTTAAAC AAGTTTATCACTTGTTTACCCTCT-3', the downstream primer InsertGPR: 5'-GCAT GTTTAAAC ACTTGAAGTGTCAAAAAGGATGA-3' (the underline is the restriction site Pme I sequence), and pCI-LEP-Pme I as A 1800bp G gene fragment (SEQ ID NO: 8) with Pme I restriction sites at both ends and a transcription initiation sequence and transcription termination sequence was amplified from the template. After the sequencing was correct, the end of the PCR product was digested with Pme I and inserted into the pCI-LEP-Pme I vector that was also treated with Pme I. The constructed full-length cDNA clone of the recombinant genome containing the double G gene was named pCI-LEP- g.

1.4.5 Construction of transcriptional helper plasmid system

Using pCI-LEP as a template, PCR amplifies the open reading frame (ORF) cDNA sequences of genes encoding nucleoprotein (N), phosphoprotein (P) and polymerase protein (L) (the nucleotide sequences are SEQ ID NOs: 5-7, the primer sequences are shown in Table 2), respectively cloned in the multiple cloning site (MCS) of the pCAGG plasmid. A Kozak sequence (GCCACC) was introduced before the start codon to enhance protein expression; a stop codon was added after the original stop codon to effectively terminate the transcription of the helper plasmid mRNA. The constructed helper plasmids were named pCAGG-N, pCAGG-P and pCAGG-L, respectively.

1.5 Virus Rescue

The day before transfection, transfer BHK-21 cells to a 35mm 6-well plate, and transfection can be carried out when the cell density reaches more than 80%. Add pCI-LEP-G (see 1.3), pCAGG-N, pCAGG-P, and pCAGG-L at the ratio of 4 μg, 2 μg, 1 μg, and 1 μg to 250 μl of serum-free medium OPTI-MEM (purchased from GIBCO) , mix well; take 15μl Lipofectamine ^TM 2000 and dissolve in 250ul OPTI-MEM, mix well and let stand at room temperature ^for 5min; , pCAGG-L (4 μg, 2 μg, 1 μg, 1 μg) were mixed evenly, and incubated at room temperature for 30 minutes. During this period, BHK-21 cells were washed twice with OPTI-MEM, then 1ml of OPTI-MEM was added to each well, and finally Lipofectamine ^TM 2000-plasmid mixture was added to the cell culture wells, and placed in a 37°C _CO2 incubator for 8 hours Afterwards, the transfection solution was aspirated and discarded, and DMEM containing 10% fetal bovine serum was added to continue culturing. After 72 hours, the cells on the culture plate were carefully blown off, and all the culture medium and cell mixture were inoculated with BHK-21 cells, and cultured at 37°C for 3 days. After 3 days, the cell supernatant was diluted 10 ^-1 -10 ^-5 and then inoculated into BHK-21 cells grown in a 24-well plate. After 2 days, the indirect immunofluorescence test was used to detect whether the virus rescue was successful, that is, the dilution was 1:50 The mouse anti-rabies virus hyperimmune serum was used as the primary antibody, mouse negative serum diluted in the same multiple was used as the control, and fluorescein (FITC)-labeled goat anti-mouse IgG (Sigma) was used as the secondary antibody. According to the titer of the rescued virus, it was diluted and passaged in an appropriate proportion to prepare the seed virus. The virus rescued from the full-length plasmid pCI-LEP-G was named rLEP-G.

1.6 RT-PCR identification and sequence determination

Sequence analysis of recombinant virus: take 250 μl of recombinant virus, extract viral RNA with viral RNA (small amount) extraction kit, and use upstream primers

5′-CAATCCTTGGGAGCAATAGA-3′ and downstream primers

5'-CGTTGGTCACTGAAACTGCTA-3', the RT-PCR method was used to amplify the fragment containing two G genes, and the sequence analysis was performed on the amplified PCR product.

1. Preparation and titration of 7 poisons

The above-mentioned rescued recombinant virus strain was amplified on BHK-21 cells and cultured at 37° C. for 3 days. After the virus was harvested, the virus titer was measured, that is, the virus was serially diluted 10 times with sterile PBS to 10 ^-8 , and each dilution was inoculated into 24-well plate monolayer NA cells or BHK-21 cells in three replicate wells. After culturing for 48 hours, IFA was carried out, and the virus titer was expressed as focus forming unit (FFU).

1.8 Determination of multi-step growth curve

To determine the multi-step growth curve of the virus, a monolayer of NA cells or BHK-21 cells was infected with an M.O.I of 0.01, respectively. After 1 hour of sensing, the cells were washed twice with serum-free MEM (purchased from GIBCO) and incubated in MEM with 0.2% FBS or 2% DMEM at 37°C. The cell culture supernatant was collected 24 hours, 72 hours and 120 hours after inoculation, and the titer was measured on NA cells or BHK-21 cells.

1.9 Pathogenicity test of recombinant virus to mice

Dilute the rescued virus and the wild virus L E P with sterilized PBS to a ratio of 10 times to 10 ^-7 , and take 30 μl of each dilution from 10 ^-4 to 10 ^-7 to inoculate five Balbs aged 5 to 6 weeks in the brain. /c female mice. Continuous observation was performed for 21 days, and the average body weight change and death number of mice in each group were recorded. The median lethal dose (LD ₅₀ ) of mice was calculated by Reed-Muench method ⁽⁵⁾ .

10 ⁷ FFU of rLEP and rLEP-G were intramuscularly injected into the hind legs of 4 Balb/c female mice aged 5-6 weeks, observed continuously for 21 days, and the average weight change and death number of mice in each group were recorded.

1.10 Western-blot detection of recombinant virus G gene expression

Recombinant virus rLEP-G and wild-type LEP were used to infect BHK-21 cells at an MOI of 1. After 3 days, the cells were collected for SDS-PAGE electrophoresis, and blank cells were used as control samples. The protein was electrotransferred to the NC membrane, 10% skimmed milk powder was blocked overnight, the mouse anti-G protein hyperimmune serum diluted 1:50 was used as the primary antibody, and IRDye ^TM 700DX labeled goat anti-mouse IgG diluted 1:5000 was used as the secondary antibody . The results were analyzed with an Odyssey infrared fluorescence scanning imaging system.

2. Results

2.1 Construction of LEP full-length genome cDNA clone containing double G gene and rescue of recombinant virus

Through the PCR method, amplify the G gene fragment with Pme I restriction sites at both ends, including the transcription initiation sequence and transcription termination sequence. The end of the PCR product is digested with Pme I and inserted into pCI On -LEP-Pme I, construct the recombinant LEP cDNA cloning plasmid pCI-LEP-G (Fig. 1) containing double G gene.

In order to rescue infectious recombinant virus from cDNA clones, first co-transfect BHK-21 cells with pCI-LEP-G and helper plasmids expressing RVN, P, and L proteins, harvest cells and supernatant after 3 days, and IFA after blind passage for one generation The test result was positive (Fig. 1A), and the virus titer reached 2×10 ⁴ FFU/ml, and the virus was continued to be passaged on BHK-21 cells to prepare seed virus. In order to determine whether the rescued virus was derived from pCI-LEP-G, the recombinant virus was identified by RT-PCR, and a 3732bp fragment was amplified, which contained two G gene sequences. Further sequencing proved that the rescued virus was from pCI-LEP-G.

2.2 Biological characteristics of the recombinant virus

2.2.1 Kinetic curves

The growth kinetics curves of rLEP-G and LEP on neuronal cell NA and non-neuronal cell BHK-21 are shown in the figure (Fig. 2), and the growth performance of rLEP-G on the two cells was not significantly different from that of wtLEP, This indicated that the growth characteristics of rLEP-G rescued from genomic full-length cDNA were consistent with wtLEP.

2.2.2 Neurotropic index

The in vitro neurotropic index of rabies virus refers to the infectivity ratio of the virus on NA cells relative to BHK-21 cells. A neurotropic index of 1 means that the ability of the virus to infect neuronal cells is 10 higher than that on non-neural cells Times ^[6] . It can be seen from Table 1 that the neurotropic index of rLEP-G and wtLEP were 0.85 and 0.84, respectively, and there was no significant difference.

Table 1 In vitro neurotropism of wtLEP and LEP-G

a The titer of the same virus on NA cells and BHK-21 cells

B in vitro neurotropism index=log (the titer on NA)-log (the titer on BHK-21)

2.2.3 Pathogenicity to mice

In order to determine the pathogenicity of rLEP-G and wild-type LEP to mice, each mouse brain was inoculated with virus 10 ⁵ FFU/30μl. Mice infected with rLEP-G and LEP showed clinical symptoms of central nervous system diseases such as paralysis, excitement, and weight loss, and all mice died within 11 days. In addition, we determined the _LD50 of wtLEP and rLEP-G to be 8.8 FFU and 3.4 FFU, respectively. It can be seen that there is no significant difference in the pathogenicity of rLEP-G and wtLEP to mice. However, peripheral infection of mice with 10 ⁷ FFU of LEP can cause disease and death in mice. It can be seen that LEP still has a certain nerve damage ability, while peripheral infection of mice with the same titer of rLEP-G strain can cause weight loss and individual hindlimb paralysis However, it did not cause the death of mice, indicating that rLEP-G is safer than LEP.

2.2.4 Western-blot detection of recombinant virus G gene expression

The expression level of rLEP-G infected BHK-21 cells was analyzed by Western-blot. rLEP-G, wtLEP infected BHK-21 cells with an M.O.I of 1 and collected the cells for lysis. As shown in Figure 3, with β-action as the internal reference, the G expression of rLEP-G was significantly higher than that of wtLEP.

The immunization test of embodiment 2 recombinant virus Beagles

In order to evaluate the immune effect of the recombinant virus on dogs, 8×10 ⁷ FFU/ml of wtLEP and rLEP-G were treated with 1:6000β-propiolactone for 2h to inactivate, and mixed with aluminum phosphate adjuvant (purchased from REHEIS) for 3 : 1 After mixing, immunize 6 each of 3-month-old Beagle dogs (purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army) by intramuscular injection, and set 6 as controls at the same time, and inject 1ml of PBS intramuscularly. Blood was collected 21 days after immunization and serum was separated for detection of neutralizing antibody titers. Neutralization test

Serum samples were first inactivated by treatment in a water bath at 56°C for 30 minutes, and then serially diluted from 20 times to 1280 times. At the same time, negative and positive serum controls were set. The standard immobilized CVS strain was used as the virus. The diluted serum was mixed with equal volumes of virus containing 1000 FFU, incubated at 37°C for 1 hour, and then 100 μl was added to 24-well BHK-21 cells grown overnight. Each sample was repeated 3 times. IFA detection was carried out 48h after infection. The neutralizing antibody titer of the tested serum is the highest multiple of the serum that makes the fluorescence focus inhibition equal to 50%. The initial dilution titer of the positive control serum (purchased from the rabies OIE reference laboratory, France) is 2IU/ml, and the antibody unit to be tested (IU/ml)=the neutralizing titer of the serum to be tested/the neutralizing titer of the standard serum× 2.

Immune Protection of Recombinant Virus rLEP-G in Beagle Dogs

According to the O.I.E standard, the inactivated recombinant virus rLEP-G and wild-type wtLEP induced the level of neutralizing antibodies in Beagle dogs. The results showed that the neutralizing antibodies induced by inactivated rLEP-G immunized Beagle dogs reached 14±4IU/ml, The neutralizing antibody titer induced by immunizing beagle dogs with inactivated wtLEP was 6±2.3IU/ml, and the difference between the two groups was significant (P<0.05). It can be seen that the inactivated recombinant virus rLEP-G can induce a higher neutralizing antibody level than the wild type, and is more suitable for the development of inactivated rabies virus vaccines.

discuss

In this study, the recombinant rabies virus rLEP-G strain was successfully rescued by reverse genetic technology. The recombinant virus was constructed on the basis of the reverse genetic operating system of the parent attenuated vaccine strain Flury-LEP strain. The full-length cDNA of the virus with the same G gene was cloned and rescued. The Flury-LEP strain has excellent immunogenicity and is widely used as a rabies inactivated vaccine strain for humans or animals. It is also used as an attenuated vaccine against canine rabies in many countries including China.

The growth kinetics curves of rLEP-G strain on nerve cell NA cells and non-neuron cell BHK-21 cells are very similar to those of the parental Flury-LEP strain (Figure 2), however, the expression of rLEP-G protein G in BHK-21 cells The amount was significantly higher than that of the parental strain LEP (Figure 3).

Our experimental results also proved that the addition of an additional G gene does not affect the growth performance of the virus, and the expression of G protein in infected cells is significantly increased. The immunization test of Beagle dogs also showed that the inactivated recombinant virus rLEP-G can induce a higher neutralizing antibody level than the wild type, which is more suitable for the development of inactivated rabies virus vaccine.

The envelope glycoprotein G protein is the most important virulence factor of rabies virus ^(6,7) . Mice of LEP strain are still infected and lethal when inoculated in the brain, whether the addition of an additional G protein will enhance their pathogenicity. By measuring the LD50 and observing the state of the mice, we found that the LD ₅₀ of the rLEP-G strain was not significantly different from its parent strain LEP, which indicated that the virulence of the rLEP-G strain was almost the same as that of the parent strain LEP. Peripheral infection of mice with high titer of LEP can cause morbidity and death in mice. It can be seen that LEP still has a certain nerve damage ability. Mice die. One of the factors influencing RV pathogenicity is the ability to evade apoptosis ⁽⁸⁾ . The ability of the recombinant virus with increased G protein content to induce apoptosis is enhanced, and the pathogenicity to mice is reduced during peripheral infection. Correspondingly, the neutralizing antibody titer of the virus is significantly increased, which may be due to the endogenous adjuvant Release stimulates cytotoxic T cell responses and promotes dendritic cell maturation and antigen presentation.

The RNA genome of rabies virus exists in the form of RNP complex, and the RNA genome template and its transcripts are tightly wrapped by N protein, so that it is not degraded by RNase and maintains high activity. Mebatsion et al ^{. (9)} rescued the recombinant virus expressing foreign protein CAT for the first time and could still detect CAT activity after 25 passages. The high genetic stability of the recombinant RV RNA genome after insertion of foreign fragments is one of the advantages of its use as a vector. The rLEP-G strain still retained the extra G gene and maintained a high titer of the virus and high expression of the G protein after multiple passages on BHK-21 cells, indicating the genetic stability of the rLEP-G strain in cultured cells.

references

1. Martinez L. Global infectious disease surveillance. Int J Infect Dis, 2000, 4(4): 222-8.

2. Kammer AR, Ertl HC. Rabies vaccines: from the past to the 21st century. Hybrid Hybridomics, 2002, 21(2): 123-7.

3. Faber M, Pulmanausahakul R, Hodawadekar SS, et al. Overexpression of the rabies virus glycoprotein results in enhancement of apoptosis and antiviralimmune response. J Virol, 2002, 76(7): 3374-81.

4. Hosokawa-Muto J, Ito N, Yamada K, et al. Characterization of recombinantrabies virus carrying double glycoprotein genes. Microbiol Immunol, 2006, 50(3): 187-96.

5. Reed, LJ, Muench, H.A simple method of estimating fifty percent endpoints. Am. J. Hyg, 1938, 27: 493-497.

6. Morimoto K, Hooper DC, Carbaugh H, et al. Rabies virus quasispecies: implications for pathogenesis. Proc Natl Acad Sci USA, 1998, 95(6): 3152-6.

7. Dietzschold B, Wunner WH, Wiktor TJ, et al. Characterization of anantigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proc Naal Acad Sci USA, 1983, 80(1): 70-4.

8. Morimoto K, Hooper DC, Spitsin S, et al. Pathogenicity of different rabies virus variants inversely correlates with apoptosis and rabies virus glycoprotein expression in infected primary neuron cultures. J Virol, 1999, 73-1: 5

9. Mebatsion T, Schnell MJ, Cox JH, et al. Highly stable expression of foreign gene from rabies virus vectors. Proc Natl Acad Sci USA, 1996, 93(14): 7310-4.

sequence listing

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ctatatgggt caagtcagat ctctaaatgc aacggttat gctgcatgtg cccctcatga 1080

gatgtctgtt ctagggggct atttgggaga ggagttcttc ggaaaaggga catttgaaag 1140

aaggttcttc agagacgaga aagaacttca agaatatgag gcggctgaac tgacaaagac 1200

cgacgtggca ctggcagatg acggaaccgt caactccgat gacgaggact acttctccgg 1260

tgaaaccaga agtccagaag ctgtctatac tcgaatcatg atgaatggag gtcgactgaa 1320

gagatcacat atacggagat atgtctcagt cagttccaat catcaagccc gtccaaactc 1380

attcgccgaa tttttaaaca aaacgtattc gagtgactca taaggagttg aatgacaggg 1440

tgccagaaat ccatagattg tgtatatcca tcatgaaaaa aactaacact cctcctttcg 1500

aaccatccca agtatgagca agatctttgt taatccgagt gcaatcagag ccggtctggc 1560

cgatcttgag atggccgaag agactgttga tctgatcaac agaaacatag aagacaatca 1620

ggctcatctc cagggagaac ccatagaagt ggacaatctc cctgaggaca tgaggcaatt 1680

tcacctgggc gatgaaaaat tgtccaacct tggtgagatg gttagggtgg gcgaaggcaa 1740

gtatcgagag gactttcaga tggatgaggg agaggaccccc aacctcctgt tccaatcgta 1800

cctggacaat gttggagtcc aaatagtcag acaaatgagg tcaggagaga gattcctcaa 1860

gatatggtca cagaccgtag aggaaattat atcctatgtc acggtcaact ttcctaaccc 1920

tccaggaagg tcttcggagg ataaatcaac ccaaactact ggccgggagc tcaagaagga 1980

gacaacatcc actctttctc agagaaag ccaaccttca aaagccggaa tggtggctca 2040

agttgcctct ggccctccat cccttgaatg gtctgccacc aatgaagagg atgatctatc 2100

agtagaggct gagatcgctc atcagattgc tgaaagcttt tccaagaagt acaagtttcc 2160

ctctcgatct tcaggaatat tcttgtataa ttttgagcaa ctggagatga accttgatga 2220

catagttaaa gaggcaaaaa atgtaccggg cgtgacccgt ctggcccatg atggatccaa 2280

aatcccccta agatgtgtac tgggatgggt cgctttggcc aactccaaga aattccaatt 2340

gtagtcgag gccgacaagc taagcaaaat catgcaagat gacttggatc gctacacatc 2400

atgctaaccg agtcttcgaa ttcattccct ctagataatg aaaactgaga tgtcatggag 2460

tcgacatgaa aaaaacaggc aacaccacta ataaaatgaa ctttctatgt aagatagtga 2520

aaaactgtag ggatgaggac acccaaaagc cctctcctgt gtcagcccct ccggatggcg 2580

atgacctgtg gcttccacct ccagaatatg tcccgctgaa agaactcaca agcaagaaga 2640

acatgaggaa cttttgtatc aacggggagg ttaaagtgtg tagcccgaac ggttatactcat 2700

tcaggatcct gcggcatatt ctgagatcat tcgacgagat atactctggg aatcatagga 2760

tgattgggtt agtcaaagtt gttattggac tagctttatc aggagctcca gttcctgagg 2820

gcatgaactg ggtatacaaa ttgaggagaa cccttatttt ccagtgggct gattccaggg 2880

gccctcttga agggggaggag ttggaacact ctcaagagat cacttgggac gatgatactg 2940

aattcgtcgg attgcaaatg agagtgagcg caagacaatg tcatattcaa ggcaggatct 3000

ggtgtatcaa catgaactcg agggcatgtc aactatggtc tgacatgtct cttcagacac 3060

aaaggtctga agaggacaaa gactcttctc tgcttctaga ataatcagat tatatcccgc 3120

aagtttatca cttgtttacc tctggaggag agaacatacg ggcttaactc caatccttgg 3180

gagcaataga acaaaaaaac acgccatggt gccattaaac cgctgcattt tatcaaagtc 3240

aagttaatta cctttacatt ttgagcctct tggatgtgaa aaaaactatt aacatccctc 3300

aaaagactta aggaaagatg gttcctcagg ttcttttgtt tgtacccctc ctgggttttt 3360

cattgtgttt cgggaagttc cccatttaca cgataccaga caaacttggt ccctggagcc 3420

ctattgacat aacaccatctc agctgtccaa ataacctggt tgtggaggac gaaggatgta 3480

ccaacctgtc cgagttctct tacatggaac ttaaagtggg atacatctca gccataaaag 3540

tgaacgggtt cacttgcaca ggtgttgtga cagaggcaga aacctacacc aactttgttg 3600

gttatgtcac aaccacattc aagagaaagc atttccgccc caccccagac gcatgtagag 3660

ccgcgtataa ctggaagatg gccggtgacc ccagatatga agagtctcta cacaatccgt 3720

accccgacta ccattggctt cgaactgtaa aaaccaccaa agagtctctc gttatcatat 3780

ccccaagtgt gacagatttg gacccatatg acaaatccct tcactcaagg gtcttccctg 3840

gcggaaattg ctcaggaata acggtgtcct cgacctactg ctcaactaat catgattaca 3900

ccatttggat gcctgagaat ctgagactag ggacatcttg tgacattttt accaatagca 3960

gagggaagag ggcatccaaa ggaggcaaga cttgcggctt tgtggatgaa agaggcctgt 4020

ataagtctct aaagggagca tgcaaactca agttatgtgg agttctcgga cttagactta 4080

tggatggaac atgggtcgcg atgcaaacat cagatgagac caaatggtgc cctccaggtc 4140

agttggtgaa tttgcacgac tttcgctcag acgagattga gcatctcgtt gtggaagagt 4200

tagtcaagaa aagagaggag tgtctggatg cactagagtc catcatgacc accaagtcag 4260

tgagtttcag acgtctcagt cacttgagaa aacttgtccc tgggtttgga aaagcatata 4320

ccatattcaa caaaaccttg atggaggctg atgctcacta caagtctgtc cggacctgga 4380

atgagatcat cccctcaaaa gggtgtttga gagttggggg gaggtgtcat ccccatgtga 4440

acggggtgtt tttcaatggt ataatattag ggtctgacgg ccatgttcta atcccagaga 4500

tgcagtcatc cctcctccag caacatatgg agttgttgga atcttcagtt atccccctga 4560

tgcacccctt ggcagaccct tctacagttt tcaaagacgg tgatgaggtt gaggattttg 4620

ttgaagttca cctccccgat gtgcatgaac aggtctcagg agttgaactg ggtctcccga 4680

actgggggaa gtatgtattg atgattgcag gggccttgat tgccctgatg ttgataattt 4740

tcctgatgac atgttgcaga agagtcaatc gaccagaatc tacgcaaagc agtcttggag 4800

agacaggggag aaatgtgtca gtcacttccc aaagcggaaa agtcatatct tcatgggagt 4860

catataagag tggaggcgag accagactgt gaaggccggt catccttttg acacttcaag 4920

tcccgaggat aacctcctct cggggttggg gggaatcttg ggatccagta gtcctccttg 4980

aactccatcc aacagggtag atttaagagt catgagactt tcattaatca tctcagttga 5040

tcagacatgg tcgtgtagat tctcataaca cgggagatct tctagcagtt tcagtgacca 5100

acggtgcttt cattctccag gaactgatac caaaggttgt ggacaagcca aggggtgctt 5160

cggattactc tgtgcttggg cacagaaaga ggtcgtagtt tgccccttga tagcagattc 5220

aacatgaatt aactaagaaa ggcgatctgc ctcccatgaa ggacataagc aatagttcac 5280

aatcatcttg catctcagtg aagtgtacat aactataaag ggctgggtca tctaagcatt 5340

tcagtcgaga aaaaaactgt agaccaaaag aacaactaac aacacttctc atcccgagac 5400

ccatatcaag atgctggatc cgggagaggt ttatgatgac cctattgatc caattgagtc 5460

agaggctgaa cccagaggaa cccccactgt ccccaacatc ttgaggaact ctgactacaa 5520

tctcaactct cctttgatag aggactctgc caaactaatg ttagaatggt tgaaaacagg 5580

gaacagacct tatcggatga ctttgacaga caattgctcc aggtcttaca aagttttgaa 5640

agattatttc aagaaagtag atttgggttc tctcaaagtg ggaggaactg ctgcacaatc 5700

aatgatttct ctctggttgt atggagccca ctctgagtca aacaggagcc ggagatgtat 5760

aaccgacttg gcccatttct attccaagtc atcccccata gagaagctgt tgaattgtac 5820

gttaggaaac agaggcctga gaatcccacc aggggggtg ttaagttgcc ttgaggggt 5880

cgattatgat aaggcatttg ggaggtatct ggccaacacg tattcctctt atctgttttt 5940

ccatgtaatt accttataca tgaatgccct agactgggaa gaggaaaaaa ccatcctagc 6000

attatggaaa gatctaacct cagtggatac cgggaaggac ttggtcaaat tcaaagatca 6060

aatatgggga ctgctggttg tgacaaagga ctttgtttac tctcagagtt ctaactgtct 6120

ttttgacaga aactatacac tgatgctaaa ggatcttttc ttgtctcgat tcaactcctt 6180

gatgattttg ctttctcccc ctgagccccg atactcagat gacttaatat ctcagctgtg 6240

ccagctatac atcgctgggg atcaagtctt gtccttgtgt gggaactccg gctatgaagt 6300

catcaaaata ttggagccat atgtcgtgaa cagtttggtc cagagggcag agaagtttag 6360

gcctctcatc cactccttgg gagactttcc tatgtttata aaagacaagg tgaatcaact 6420

tgaagggact ttcggtccca gtgcaaaaag gttttttagg gttctagatc aattcgacaa 6480

catacatgat ctagtatttg tgtatggctg ttacagacat tgggggcacc cctatataga 6540

ttatcggaag ggtctgtcga aactatatga tcaagttcac attaagaaag taatagataa 6600

gtcctaccag gagtgtttag caagtgactt ggctagaagg atcctcagat ggggatttga 6660

caagtactcc aagtggtatc tagattcgag attccttgcc ctagaccacc ccttggctcc 6720

ttatatcaag acccaaacat ggccacccaa acatatagta gacttggtgg gggacacatg 6780

gcacaagctc ccgatcacgc agatctttga gattcctgaa tcaatggacc cgtcagagat 6840

actggatgat aaatcacatt ctttcaccag aacaagacta gcttcttggc tgtccgagaa 6900

ccgagggggg cctgttccta gcgagaaggt catttatcacg gccctgtcta agccacctgt 6960

caatccccga gagtttttga aatctatcga cctcggagga ttgccagatg atgacttgat 7020

aattggcctc agaccaaagg aacgggagtt gaagattgag ggccgattct tcgctctaat 7080

gtcatggaat ctaagattat attttgtcat caccgagaag ctcttggcca actacatttt 7140

gccacttttt gacgcactga ctatgacaga caacctgaac aaggtattta aaaagttgat 7200

cgacagggtc accgggcaag ggcttttgga ctattcgagg gtcacatacg catttcacct 7260

ggactatgag aaatggaaca atcatcaaag attggagtca acggaggatg tattttctgt 7320

cctagatcag gtgtttggat tgaagagggt gttttctaga acacacgagt tttttcagaa 7380

gtcctggatc tattattcag acagatcaga cctcattggg ttacgggagg atcagatata 7440

ttgcttggat atgtccaatg gtccaacctg ctggaatggc caagacggcg ggctagaggg 7500

ccttacggcag aagggctgga gtctagtcag cttattgatg atagatagag aatctcaaac 7560

caggaacaca agaaccaaga tactagctca aggagacaac caggttctgt gtccgacata 7620

catgttgtca ccgggattgt ctcaagaggg gcttctctat gagttagaga gcatatcgag 7680

gaatgcactc tcaatatacc gagctatcga ggaaggagca tctaaactgg ggctgatcat 7740

caagaaagaa gagaccatgt gtagttatga ctttctcata tatgggaaga cccccttatt 7800

tcgaggcaac atattagtac ctgaatccaa aagatgggcc cgagtctctt gcatctctaa 7860

cgaccaaata gtcaacctcg ccaatataat gtctacagta tccaccaatg cgctgacagt 7920

ggcacaacac tctcaatctc tgatcaaacc tatgagggat tttctgctca tgtcagtaca 7980

ggcagttttc cactacctgc tgtttagccc aatcttaaaa ggcagggttt ataagattct 8040

gagtgctgaa ggggagagct ttctcctagc catgtcgcgg ataatctacc tagatccttc 8100

tttggggaggg gtgtctggaa tgtctctcgg gaggttccat atacgtcagt tctcagaccc 8160

tgtctctgaa gggttgtcat tctggagaga gatctggtta agctcccatg aatcctggat 8220

tcacgcgttg tgtcaagagg ccgggaaccc cgatcttgga gagagaacac tagagagctt 8280

cactcgcctt ttagaagatc ctactacctt aaatatcaaa ggaggggcca gtcctaccat 8340

tctactcaag gatgctatca gaaaggctct gtacgacgag gtggacagg tggaaaattc 8400

agagtttcga gaggcaatcc tgttgtccaa gacccataga gataacttta tactcttttt 8460

aaaatctgtt gagcctctgt tccctcgatt tctcagtgaa ctcttcagtt cgtccttctt 8520

gggaatacca gagtcaatta ttggactgat acaaaactcc aggacaataa gaaggcagtt 8580

tagaaagagt ctctcaagaa ctttagaaga gtccttctac aactcagaga tccacgggat 8640

taatcggatg accccagacac ctcaaagggt cgggagagtg tggccttgct cttcagagag 8700

ggcagatcta cttagggaga tctcttgggg aaggaaagtg gtaggcacga cagttcctca 8760

cccttccgag atgttggggc tgcttccaaa atcctctatt tcctgcactt gtggagcaac 8820

aggggggaggc aatcctagag tttctgtatc agtactcccg tccttcgatc agtcattttt 8880

ttcacgaggt cccctaaagg gatacttggg ctcgtccacc tccatgtcaa cccagctatt 8940

ccatgcatgg gaaaaagtca ctaatgttca tgtggtgaaa agggctatat cgttaaaaga 9000

atctataaac tggttcatca atagaaattc caatttggct caaactctaa ttagaaacat 9060

catgtctctg acaggccctg atttccctct agaagaggct cctgttttca aacggacagg 9120

gtcagccttg cataggttca agtctgccag atacagcgaa ggagggtatt cttctgtttg 9180

tcctaacctt ctctctcata tctctgtcag tacagacact atgtctgatt tgacccaaga 9240

cgggaagaac tatgatttca tgtttcagcc attgatgctt tatgcgcaaa catggacatc 9300

agagctggta cagagagaca caagactgag agactccacg tttcactggc accttcgatg 9360

caacagatgt gtaaggccca ttgaggatat aacactggaa acttctcaga tcttcgagtt 9420

cccggatgtg tcaaaaagga tatccaggat ggtttctgga gccgtccctc actttcagaa 9480

gcttcctgat atccgtctaa gaccaggtga ttttgaatct ctaagtggta gagaaaagtc 9540

tcgccacata gggtcagctc aggggctctt atactcaatc ttagtagcaa ttcacgattc 9600

aggatacaat gatgggacca tcttccctgt caacatatac ggcaaagttt cccctagaga 9660

ctatttgaga gggctcgcaa gagggatctt gataggatcc tcgatttgct tcttgacacg 9720

aatgacaaat attaatatta aaagacctct tgaattgatc tcaggggtaa tttcctatat 9780

tctcctgagg ctggataatc atccctctct gtatataatg cttagagaac cgtctcttag 9840

aggagagata ttctctatcc ctcagaaaat ccccgccgct tacccaacca ctatgaaaga 9900

aggcaacaga tcgatcttgt gttacctcca acacgtgcta cgctatgagc gagaagtaat 9960

cacggcgtcc ccggagaatg actggctgtg gatcttctca gacttcagaa gtgcgaaaat 10020

gacgtacttg accctcatta cctaccagtc tcacctccta ctccagaggg ttgagcgaaa 10080

cttgtctaag agtatgagag ctactctgcg acaaatgggt tccttaatga ggcaagtgct 10140

gggtgggcac ggagaagaca ccttggagtc agacgacgac attcaacgat tactaaaaga 10200

ctctttgcga aggacaaggt gggtagatca agaggtgcgc catgcagcta gaaccatgag 10260

tggagattac agccccaaca agagagtatc ccgcaaggca ggatgttcag aatgggtctg 10320

ctctgctcaa caggttgccg tctccacctc agccaacccg gcccctgtct cagagcttga 10380

cattagggcc ctatctaaga ggtttcaaaa ccccttgatc tcgggcctaa gagtggttca 10440

gtgggcaacc ggcgcccatt ataagcttaa gcctattcta gatgatctaa atgttttccc 10500

atctctctgt cttgttgttg gagacgggtc agggggaata tcaagggcag ttctcaacat 10560

gtttccagat tctaagcttg tgttcaacag cctattggag gtgaatgatc tgatggcttc 10620

cggaacacat ccactgcctc cttcagcaat catgagtgga ggagatgata tcatctccag 10680

agtgatagac tttgactcaa tctgggaaaa accatccgac ctgaggaact tggccacctg 10740

gagatacttc cagtcagttc aaaagcaggt caacatgtcg tatgacctca ttgtttgtga 10800

tgcagaagtt actgatattg catctatcaa ccggataact ctgttgatgt ctgatttcgc 10860

attgtctata gatggaccac tttatctggt cttcaaaact tacgggacta tgctagtgaa 10920

cccagactat aaagctatcc aacatctgtc aagagcgttc ccttcggtca cagggtttat 10980

aacccaagta acttcgtcct tttcttctga gctatacctc cggttctcca aacgagggaa 11040

gttttttagg gatgctgagt acttgacctc ttccaccctt cgggagatga gccttgtgtt 11100

attcaattgt agcagcccca aaagtgagat gcagagagct cgttccttaa actatcaaga 11160

tcttgtaagg ggatttcctg aagagatcat atcaaatcct tacaatgaaa tgatcataac 11220

tctgattgac agtgatgtag agtccttcct ggtccacaag atggtggatg atcttgagtt 11280

acagagggga actctgtcta aagtggctat catttatcc atcatgattg ttttttccaa 11340

tagagtcttc aacatttcca aacctttgac tgaccccttg ttctatcccc catctgatcc 11400

taaaatcctg aggcacttca acatatgttg cagtactatg atgtatctat ctaccgcttt 11460

aggcgacgtc cctagcttcg caagacttca tgacctatat aatagaccta taacttatta 11520

cttcagaaag caagttatc gagggaatat ttatctatct tggagctggt ccgatgacac 11580

cccagtgttc aagagggtag cctgtaattc tagcttgagt ctgtcatctc actggatcag 11640

gttgattac aagatagtga agactaccag actcgttggc agcatagaag atctatcagg 11700

agaggtagaa cgacacctgc atgggtacaa cagatggatc accctcgagg atatccgatc 11760

tagatcatcc ctactagact acagttgttt gtaagccgga cattaatgaa agcctgtaca 11820

tgctaaaatt cttgtatgat gcatcttgaa aaaaacaaga tcttgaatcc gaacctctgg 11880

ttgtttgatt gtttttttta tctttatgtgt ttatttgtta agcgt 11925

<210>2

<211>4063

<212>DNA

<213> Rabies virus

<400>2

acgcttaaca acaaaaccaa agaagaagca gacagcgtca gttgcaaagc aaaaatgtaa 60

cacccctaca atggatgccg acaagattgt gttcaaagtc aataatcagg tggtctcttt 120

gaagcccgag attatcgtgg atcaatatga gtacaagtac cctgctatca aagatttgaa 180

aaagccttgt ataaccctag ggaaagcccc cgatttaaac aaagcttaca aatcagtttt 240

atcaggcatg aatgccgcca aacttgatcc tgatgatgta tgctcctact tggcagcagc 300

aatgcagttc tttgagggga catgtccgga agactggacc agctatggaa tcctgattgc 360

acgaaaagga gacaagatca ccccagactc tctagtggag ataaagcgta ctgatgtaga 420

agggaattgg gctctgacag gaggcatgga actgacaagg gaccccactg tctccgaaca 480

tgcatcttta gtcggtcttc tcctgagtct gtacagattg agcaaaatat caggacaaaa 540

cactggtaac tataagacaa acattgcgga tagaatagag cagattttcg agacagcccc 600

ttttgttaag atcgtggaac accataccct gatgacaact cacaagatgt gtgctaattg 660

gagtactata ccgaacttca gatttttggc cggaacctac gacatgtttt tctcacggat 720

cgagcatctg tattcggcaa tcagagtggg cacagttgtc accgcttatg aagactgctc 780

aggactggta tcgtttacag ggttcataaa gcagatcaat ctcaccgcaa gagaagcaat 840

actatatttc ttccacaaga actttgaaga agagataagg agaatgtttg agccagggca 900

agagacagct gttcctcact cttatttcat tcacttccgt tcactaggct tgagtgggaa 960

gtctccttat tcatcaaatg ccgtcggtca tgtgttcaat ctcattcact ttgttggatg 1020

ctatatgggt caagtcagat ctctaaatgc aacggttat gctgcatgtg cccctcatga 1080

gatgtctgtt ctagggggct atttgggaga ggagttcttc ggaaaaggga catttgaaag 1140

aaggttcttc agagacgaga aagaacttca agaatatgag gcggctgaac tgacaaagac 1200

cgacgtggca ctggcagatg acggaaccgt caactccgat gacgaggact acttctccgg 1260

tgaaaccaga agtccagaag ctgtctatac tcgaatcatg atgaatggag gtcgactgaa 1320

gagatcacat atacggagat atgtctcagt cagttccaat catcaagccc gtccaaactc 1380

attcgccgaa tttttaaaca aaacgtattc gagtgactca taaggagttg aatgacaggg 1440

tgccagaaat ccatagattg tgtatatcca tcatgaaaaa aactaacact cctcctttcg 1500

aaccatccca agtatgagca agatctttgt taatccgagt gcaatcagag ccggtctggc 1560

cgatcttgag atggccgaag agactgttga tctgatcaac agaaacatag aagacaatca 1620

ggctcatctc cagggagaac ccatagaagt ggacaatctc cctgaggaca tgaggcaatt 1680

tcacctgggc gatgaaaaat tgtccaacct tggtgagatg gttagggtgg gcgaaggcaa 1740

gtatcgagag gactttcaga tggatgaggg agaggaccccc aacctcctgt tccaatcgta 1800

cctggacaat gttggagtcc aaatagtcag acaaatgagg tcaggagaga gattcctcaa 1860

gatatggtca cagaccgtag aggaaattat atcctatgtc acggtcaact ttcctaaccc 1920

tccaggaagg tcttcggagg ataaatcaac ccaaactact ggccgggagc tcaagaagga 1980

gacaacatcc actctttctc agagaaag ccaaccttca aaagccggaa tggtggctca 2040

agttgcctct ggccctccat cccttgaatg gtctgccacc aatgaagagg atgatctatc 2100

agtagaggct gagatcgctc atcagattgc tgaaagcttt tccaagaagt acaagtttcc 2160

ctctcgatct tcaggaatat tcttgtataa ttttgagcaa ctggagatga accttgatga 2220

catagttaaa gaggcaaaaa atgtaccggg cgtgacccgt ctggcccatg atggatccaa 2280

aatcccccta agatgtgtac tgggatgggt cgctttggcc aactccaaga aattccaatt 2340

gtagtcgag gccgacaagc taagcaaaat catgcaagat gacttggatc gctacacatc 2400

atgctaaccg agtcttcgaa ttcattccct ctagataatg aaaactgaga tgtcatggag 2460

tcgacatgaa aaaaacaggc aacaccacta ataaaatgaa ctttctatgt aagatagtga 2520

aaaactgtag ggatgaggac acccaaaagc cctctcctgt gtcagcccct ccggatggcg 2580

atgacctgtg gcttccacct ccagaatatg tcccgctgaa agaactcaca agcaagaaga 2640

acatgaggaa cttttgtatc aacggggagg ttaaagtgtg tagcccgaac ggttatactcat 2700

tcaggatcct gcggcatatt ctgagatcat tcgacgagat atactctggg aatcatagga 2760

tgattgggtt agtcaaagtt gttattggac tagctttatc aggagctcca gttcctgagg 2820

gcatgaactg ggtatacaaa ttgaggagaa cccttatttt ccagtgggct gattccaggg 2880

gccctcttga agggggaggag ttggaacact ctcaagagat cacttgggac gatgatactg 2940

aattcgtcgg attgcaaatg agagtgagcg caagacaatg tcatattcaa ggcaggatct 3000

ggtgtatcaa catgaactcg agggcatgtc aactatggtc tgacatgtct cttcagacac 3060

aaaggtctga agaggacaaa gactcttctc tgcttctaga ataatcagat tatatcccgc 3120

aagtttatca cttgtttacc tctggaggag agaacatacg ggcttaactc caatccttgg 3180

gagcaataga acaaaaaaac acgccatggt gccattaaac cgctgcattt tatcaaagtc 3240

aagttaatta cctttacatt ttgagcctct tggatgtgaa aaaaactatt aacatccctc 3300

aaaagactta aggaaagatg gttcctcagg ttcttttgtt tgtacccctc ctgggttttt 3360

cattgtgttt cgggaagttc cccatttaca cgataccaga caaacttggt ccctggagcc 3420

ctattgacat aacaccatctc agctgtccaa ataacctggt tgtggaggac gaaggatgta 3480

ccaacctgtc cgagttctct tacatggaac ttaaagtggg atacatctca gccataaaag 3540

tgaacgggtt cacttgcaca ggtgttgtga cagaggcaga aacctacacc aactttgttg 3600

gttatgtcac aaccacattc aagagaaagc atttccgccc caccccagac gcatgtagag 3660

ccgcgtataa ctggaagatg gccggtgacc ccagatatga agagtctcta cacaatccgt 3720

accccgacta ccattggctt cgaactgtaa aaaccaccaa agagtctctc gttatcatat 3780

ccccaagtgt gacagatttg gacccatatg acaaatccct tcactcaagg gtcttccctg 3840

gcggaaattg ctcaggaata acggtgtcct cgacctactg ctcaactaat catgattaca 3900

ccatttggat gcctgagaat ctgagactag ggacatcttg tgacattttt accaatagca 3960

gagggaagag ggcatccaaa ggaggcaaga cttgcggctt tgtggatgaa agaggcctgt 4020

ataagtctct aaagggagca tgcaaactca agttatgtgg agt 4063

<210>3

<211>4242

<212>DNA

<213> Rabies virus

<400>3

aggcctgtat aagtctctaa agggagcatg caaactcaag ttatgtggag ttctcggact 60

tagacttatg gatggaacat gggtcgcgat gcaaacatca gatgagacca aatggtgccc 120

tccaggtcag ttggtgaatt tgcacgactt tcgctcagac gagattgagc atctcgttgt 180

ggaagagtta gtcaagaaaa gagaggagtg tctggatgca ctagagtcca tcatgaccac 240

caagtcagtg agtttcagac gtctcagtca cttgagaaaa cttgtccctg ggtttggaaa 300

agcatatacc atattcaaca aaaccttgat ggaggctgat gctcactaca agtctgtccg 360

gacctggaat gagatcatcc cctcaaaagg gtgtttgaga gttgggggga ggtgtcatcc 420

ccatgtgaac ggggtgtttt tcaatggtat aatattaggg tctgacggcc atgttctaat 480

cccagagatg cagtcatccc tcctccagca acatatggag ttgttggaat cttcagttat 540

ccccctgatg cacccccttgg cagacccttc tacagttttc aaagacggtg atgaggttga 600

ggattttgtt gaagttcacc tccccgatgt gcatgaacag gtctcaggag ttgaactggg 660

tctcccgaac tgggggaagt atgtattgat gattgcaggg gccttgattg ccctgatgtt 720

gataattttc ctgatgacat gttgcagaag agtcaatcga ccagaatcta cgcaaagcag 780

tcttggagag acagggagaa atgtgtcagt cacttcccaa agcggaaaag tcatatcttc 840

atgggagtca tataagagtg gaggcgagac cagactgtga aggccggtca tccttttgac 900

acttcaagtc ccgaggataa cctcctctcg gggttggggg gaatcttggg atccagtagt 960

cctccttgaa ctccatccaa cagggtagat ttaagagtca tgagactttc attaatcatc 1020

tcagttgatc agacatggtc gtgtagattc tcataacacg ggagatcttc tagcagtttc 1080

agtgaccaac ggtgctttca ttctccagga actgatacca aaggttgtgg acaagccaag 1140

gggtgcttcg gattactctg tgcttgggca cagaaagagg tcgtagtttg ccccttgata 1200

gcagattcaa catgaattaa ctaagaaagg cgatctgcct cccatgaagg acataagcaa 1260

tagttcacaa tcatcttgca tctcagtgaa gtgtacataa ctataaaggg ctgggtcatc 1320

taagcatttc agtcgagaaa aaaactgtag accaaaagaa caactaacaa cacttctcat 1380

cccgagaccc atatcaagat gctggatccg ggagaggttt atgatgaccc tattgatcca 1440

attgagtcag aggctgaacc cagaggaacc cccactgtcc ccaacatctt gaggaactct 1500

gactacaatc tcaactctcc tttgatagag gactctgcca aactaatgtt agaatggttg 1560

aaaacaggga acagacctta tcggatgact ttgacagaca attgctccag gtcttacaaa 1620

gttttgaaag attatttcaa gaaagtagat ttgggttctc tcaaagtggg aggaactgct 1680

gcacaatcaa tgatttctct ctggttgtat ggagcccact ctgagtcaaa caggagccgg 1740

agatgtataa ccgacttggc ccatttctat tccaagtcat cccccataga gaagctgttg 1800

aattgtacgt taggaaacag aggcctgaga atcccaccag agggggtgtt aagttgcctt 1860

gagagggtcg attatgataa ggcatttggg aggtatctgg ccaacacgta ttcctcttat 1920

ctgtttttcc atgtaattac cttatacatg aatgccctag actgggaaga ggaaaaaacc 1980

atcctagcat tatggaaaga tctaacctca gtggataccg ggaaggactt ggtcaaattc 2040

aaagatcaaa tatggggact gctggttgtg acaaaggact ttgtttactc tcagagttct 2100

aactgtcttt ttgacagaaa ctatacactg atgctaaagg atcttttctt gtctcgattc 2160

aactccttga tgattttgct ttctccccct gagccccgat actcagatga cttaatatct 2220

cagctgtgcc agctatacat cgctggggat caagtcttgt ccttgtgtgg gaactccggc 2280

tatgaagtca tcaaaatatt ggagccatat gtcgtgaaca gtttggtcca gagggcagag 2340

aagtttaggc ctctcatcca ctccttggga gactttccta tgtttataaa aagacaaggtg 2400

aatcaacttg aagggacttt cggtcccagt gcaaaaaggt tttttagggt tctagatcaa 2460

ttcgacaaca tacatgatct agtatttgtg tatggctgtt acagacattg ggggcacccc 2520

tatatagatt atcggaaggg tctgtcgaaa ctatatgatc aagttcacat taagaaagta 2580

atagataagt cctaccagga gtgtttagca agtgacttgg ctagaaggat cctcagatgg 2640

ggatttgaca agtactccaa gtggtatcta gattcgagat tccttgccct agaccacccc 2700

ttggctcctt atatcaagac ccaaacatgg ccacccaaac atatagtaga cttggtgggg 2760

gacacatggc acaagctccc gatcacgcag atctttgaga ttcctgaatc aatggacccg 2820

tcagagatac tggatgataa atcacattct ttcaccagaa caagactagc ttcttggctg 2880

tccgagaacc gaggggggcc tgttcctagc gagaaggtca ttatcacggc cctgtctaag 2940

ccacctgtca atccccgaga gtttttgaaa tctatcgacc tcggaggatt gccagatgat 3000

gacttgataa ttggcctcag accaaaggaa cgggagttga agattgaggg ccgattcttc 3060

gctctaatgt catggaatct aagattatat tttgtcatca ccgagaagct cttggccaac 3120

tacattttgc cactttttga cgcactgact atgacagaca acctgaacaa ggtatttaaa 3180

aagttgatcg acagggtcac cgggcaaggg cttttggact attcgagggt cacatacgca 3240

tttcacctgg actatgagaa atggaacaat catcaaagat tggagtcaac ggaggatgta 3300

ttttctgtcc tagatcaggt gtttggattg aagagggtgt tttctagaac acacgagttt 3360

tttcagaagt cctggatcta ttattcagac agatcagacc tcattgggtt acgggaggat 3420

cagatatatt gcttggatat gtccaatggt ccaacctgct ggaatggcca agacggcggg 3480

ctagaggggct tacggcagaa gggctggagt ctagtcagct tattgatgat agatagagaa 3540

tctcaaacca ggaacacaag aaccaagata ctagctcaag gagacaacca ggttctgtgt 3600

ccgacataca tgttgtcacc gggattgtct caagaggggc ttctctatga gttagagagc 3660

atatcgagga atgcactctc aatataccga gctatcgagg aaggagcatc taaactgggg 3720

ctgatcatca agaaagaaga gaccatgtgt agttatgact ttctcatata tgggaagacc 3780

cccttatttc gaggcaacat attagtacct gaatccaaaa gatgggcccg agtctcttgc 3840

atctctaacg accaaatagt caacctcgcc aatataatgt ctacagtatc caccaatgcg 3900

ctgacagtgg cacaacactc tcaatctctg atcaaaccta tgagggatt tctgctcatg 3960

tcagtacagg cagttttcca ctacctgctg tttagcccaa tcttaaaagg cagggtttat 4020

aagattctga gtgctgaagg ggagagcttt ctcctagcca tgtcgcggat aatctaccta 4080

gatccttctt tgggaggggt gtctggaatg tctctcggga ggttccatat acgtcagttc 4140

tcagaccctg tctctgaagg gttgtcattc tggagagaga tctggttaag ctcccatgaa 4200

tcctggattc acgcgttgtg tcaagaggcc gggaaccccg at 4242

<210>4

<211>3739

<212>DNA

<213> Rabies virus

<400>4

gagagatctg gttaagctcc catgaatcct ggattcacgc gttgtgtcaa gaggccggga 60

accccgatct tggagagaga acactagaga gcttcactcg ccttttagaa gatcctacta 120

ccttaaatat caaaggaggg gccagtccta ccattctact caaggatgct atcagaaagg 180

ctctgtacga cgaggtggac aaggtggaaa attcagagtt tcgagaggca atcctgttgt 240

ccaagaccca tagagataac tttatactct ttttaaaatc tgttgagcct ctgttccctc 300

gatttctcag tgaactcttc agttcgtcct tcttgggaat accagagtca attattggac 360

tgatacaaaa ctccaggaca ataagaaggc agtttagaaa gagtctctca agaactttag 420

aagagtcctt ctacaactca gagatccacg ggattaatcg gatgacccag acacctcaaa 480

gggtcgggag agtgtggcct tgctcttcag agagggcaga tctacttagg gagatctctt 540

ggggaaggaa agtggtaggc acgacagttc ctcacccttc cgagatgttg gggctgcttc 600

caaaatcctc tatttcctgc acttgtggag caacaggggg aggcaatcct agagtttctg 660

tatcagtact cccgtccttc gatcagtcat ttttttcacg aggtccccta aagggatact 720

tgggctcgtc cacctccatg tcaacccagc tattccatgc atgggaaaaa gtcactaatg 780

ttcatgtggt gaaaagggct atatcgttaa aagaatctat aaactggttc atcaatagaa 840

attccaattt ggctcaaact ctaattagaa acatcatgtc tctgacaggc cctgatttcc 900

ctctagaaga ggctcctgtt ttcaaacgga cagggtcagc cttgcatagg ttcaagtctg 960

ccagatacag cgaaggaggg tattcttctg tttgtcctaa ccttctctct catatctctg 1020

tcagtacaga cactatgtct gatttgaccc aagacgggaa gaactatgat ttcatgtttc 1080

agccattgat gctttatgcg caaacatgga catcagagct ggtacagaga gacacaagac 1140

tgagagactc cacgtttcac tggcaccttc gatgcaacag atgtgtaagg cccattgagg 1200

atataacact ggaaacttct cagatcttcg agttcccgga tgtgtcaaaa aggatatcca 1260

ggatggtttc tggagccgtc cctcactttc agaagcttcc tgatatccgt ctaagaccag 1320

gtgattttga atctctaagt ggtagagaaa agtctcgcca catagggtca gctcaggggc 1380

tcttatactc aatcttagta gcaattcacg attcaggata caatgatggg accatcttcc 1440

ctgtcaacat atacggcaaa gtttccccta gagactattt gagagggctc gcaagaggga 1500

tcttgatagg atcctcgatt tgcttcttga cacgaatgac aaatattaat attaaaagac 1560

ctcttgaatt gatctcaggg gtaatttcct atattctcct gaggctggat aatcatccct 1620

ctctgtatat aatgcttaga gaaccgtctc ttagaggaga gatattctct atccctcaga 1680

aaatccccgc cgcttaccca accactatga aagaaggcaa cagatcgatc ttgtgttacc 1740

tccaacacgt gctacgctat gagcgagaag taatcacggc gtccccggag aatgactggc 1800

tgtggatctt ctcagacttc agaagtgcga aaatgacgta cttgaccctc attacctacc 1860

agtctcacct cctactccag agggttgagc gaaacttgtc taagagtatg agagctactc 1920

tgcgacaaat gggttcctta atgaggcaag tgctgggtgg gcacggagaa gacaccttgg 1980

agtcagacga cgacattcaa cgattactaa aagactcttt gcgaaggaca aggtgggtag 2040

atcaagaggt gcgccatgca gctagaacca tgagtggaga ttacagcccc aacaagagag 2100

tatcccgcaa ggcaggatgt tcagaatggg tctgctctgc tcaacaggtt gccgtctcca 2160

cctcagccaa cccggcccct gtctcagagc ttgacattag ggccctatct aagaggtttc 2220

aaaacccctt gatctcgggc ctaagagtgg ttcagtgggc aaccggcgcc cattataagc 2280

ttaagcctat tctagatgat ctaaatgttt tcccatctct ctgtcttgtt gttggagacg 2340

ggtcaggggg aatatcaagg gcagttctca acatgtttcc agattctaag cttgtgttca 2400

acagcctatt ggaggtgaat gatctgatgg cttccggaac acatccactg cctccttcag 2460

caatcatgag tggaggagat gatatcatct ccagagtgat agactttgac tcaatctggg 2520

aaaaaccatc cgacctgagg aacttggcca cctggagata cttccagtca gttcaaaagc 2580

aggtcaacat gtcgtatgac ctcattgttt gtgatgcaga agttactgat attgcatcta 2640

tcaaccggat aactctgttg atgtctgatt tcgcattgtc tatagatgga ccactttatc 2700

tggtcttcaa aacttacggg actatgctag tgaacccaga ctataaagct atccaacatc 2760

tgtcaagagc gttcccttcg gtcacagggt ttataaccca agtaacttcg tccttttctt 2820

ctgagctata cctccggttc tccaaacgag ggaagttttt tagggatgct gagtacttga 2880

cctcttccac ccttcggggag atgagccttg tgttattcaa ttgtagcagc cccaaaagtg 2940

agatgcagag agctcgttcc ttaaactatc aagatcttgt aaggggattt cctgaagaga 3000

tcatatcaaa tccttacaat gaaatgatca taactctgat tgacagtgat gtagagtcct 3060

tcctggtcca caagatggtg gatgatcttg agttacagag gggaactctg tctaaagtgg 3120

ctatcattat atccatcatg attgtttttt ccaatagagt cttcaacatt tccaaacctt 3180

tgactgaccc cttgttctat cccccatctg atcctaaaat cctgaggcac ttcaacatat 3240

gttgcagtac tatgatgtat ctatctaccg ctttaggcga cgtccctagc ttcgcaagac 3300

ttcatgacct atataataga cctataactt attacktcag aaagcaagtt attcgaggga 3360

atatttatct atcttggagc tggtccgatg acaccccagt gttcaagagg gtagcctgta 3420

attctagctt gagtctgtca tctcactgga tcaggttgat ttacaagata gtgaagacta 3480

ccagactcgt tggcagcata gaagatctat caggagaggt agaacgacac ctgcatgggt 3540

acaacagatg gatcaccctc gaggatatcc gatctagatc atccctacta gactacagtt 3600

gtttgtaagc cggacattaa tgaaagcctg tacatgctaa aattcttgta tgatgcatct 3660

tgaaaaaaac aagatcttga atccgaacct ctggttgttt gattgttttt tttatcttta 3720

ttgtttattt gttaagcgt 3739

<210>5

<211>1353

<212>DNA

<213> Rabies virus

<400>5

atggatgccg acaagattgt gttcaaagtc aataatcagg tggtctcttt gaagcccgag 60

attatcgtgg atcaatatga gtacaagtac cctgctatca aagatttgaa aaagccttgt 120

ataaccctag ggaaagcccc cgatttaaac aaagcttaca aatcagtttt atcaggcatg 180

aatgccgcca aacttgatcc tgatgatgta tgctcctact tggcagcagc aatgcagttc 240

tttgagggga catgtccgga agactggacc agctatggaa tcctgattgc acgaaaagga 300

gacaagatca ccccagactc tctagtggag ataaagcgta ctgatgtaga agggaattgg 360

gctctgacag gaggcatgga actgacaagg gaccccactg tctccgaaca tgcatcttta 420

gtcggtcttc tcctgagtct gtacagattg agcaaaatat caggacaaaa cactggtaac 480

tataagacaa acattgcgga tagaatagag cagattttcg agacagcccc ttttgttaag 540

atcgtggaac accataccct gatgacaact cacaagatgt gtgctaattg gagtactata 600

ccgaacttca gatttttggc cggaacctac gacatgtttt tctcacggat cgagcatctg 660

tattcggcaa tcagagtggg cacagttgtc accgcttatg aagactgctc aggactggta 720

tcgtttacag ggttcataaa gcagatcaat ctcaccgcaa gagaagcaat actatatttc 780

ttccacaaga actttgaaga agagataagg agaatgtttg agccagggca agagacagct 840

gttcctcact cttatttcat tcacttccgt tcactaggct tgagtgggaa gtctccttat 900

tcatcaaatg ccgtcggtca tgtgttcaat ctcattcact ttgttggatg ctatatgggt 960

caagtcagat ctctaaatgc aacggttat gctgcatgtg cccctcatga gatgtctgtt 1020

ctagggggct atttgggaga ggagttcttc ggaaaaggga catttgaaag aaggttcttc 1080

agagacgaga aagaacttca agaatatgag gcggctgaac tgacaaagac cgacgtggca 1140

ctggcagatg acggaaccgt caactccgat gacgaggact acttctccgg tgaaaccaga 1200

agtccagaag ctgtctatac tcgaatcatg atgaatggag gtcgactgaa gagatcacat 1260

atacggagat atgtctcagt cagttccaat catcaagccc gtccaaactc attcgccgaa 1320

tttttaaaca aaacgtattc gagtgactca taa 1353

<210>6

<211>894

<212>DNA

<213> Rabies virus

<400>6

atgagcaaga tctttgttaa tccgagtgca atcagagccg gtctggccga tcttgagatg 60

gccgaagaga ctgttgatct gatcaacaga aacatagaag acaatcaggc tcatctccag 120

ggagaaccca tagaagtgga caatctccct gaggacatga ggcaatttca cctgggcgat 180

gaaaaattgt ccaaccttgg tgagatggtt agggtgggcg aaggcaagta tcgagaggac 240

tttcagatgg atgagggaga ggaccccaac ctcctgttcc aatcgtacct ggacaatgtt 300

ggagtccaaa tagtcagaca aatgaggtca ggagagagat tcctcaagat atggtcacag 360

accgtagagg aaattatatc ctatgtcacg gtcaactttc ctaaccctcc aggaaggtct 420

tcggaggata aatcaaccca aactactggc cgggagctca agaaggagac aacatccact 480

ctttctcaga gagaaagcca accttcaaaa gccggaatgg tggctcaagt tgcctctggc 540

cctccatccc ttgaatggtc tgccaccaat gaagaggatg atctatcagt agaggctgag 600

atcgctcatc agattgctga aagcttttcc aagaagtaca agtttccctc tcgatcttca 660

ggaatattct tgtataattt tgagcaactg gagatgaacc ttgatgacat agttaaagag 720

gcaaaaaatg taccgggcgt gacccgtctg gcccatgatg gatccaaaat ccccctaaga 780

tgtgtactgg gatgggtcgc tttggccaac tccaagaaat tccaattgat agtcgaggcc 840

gacaagctaa gcaaaatcat gcaagatgac ttggatcgct acacatcatg ctaa 894

<210>7

<211>6384

<212>DNA

<213> Rabies virus

<400>7

atgctggatc cgggagaggt ttatgatgac cctattgatc caattgagtc agaggctgaa 60

cccagaggaa cccccactgt ccccaacatc ttgaggaact ctgactacaa tctcaactct 120

cctttgatag aggactctgc caaactaatg ttagaatggt tgaaaacagg gaacagacct 180

tatcggatga ctttgacaga caattgctcc aggtcttaca aagttttgaa agattatttc 240

aagaaagtag atttgggttc tctcaaagtg ggaggaactg ctgcacaatc aatgatttct 300

ctctggttgt atggagccca ctctgagtca aacaggagcc ggagatgtat aaccgacttg 360

gcccatttct attccaagtc atcccccata gagaagctgt tgaattgtac gttaggaaac 420

agaggcctga gaatcccacc agaggggtg ttaagttgcc ttgagagggt cgattatgat 480

aaggcatttg ggaggtatct ggccaacacg tattcctctt atctgttttt ccatgtaatt 540

accttataca tgaatgccct agactgggaa gaggaaaaaa ccatcctagc attatggaaa 600

gatctaacct cagtggatac cgggaaggac ttggtcaaat tcaaagatca aatatgggga 660

ctgctggttg tgacaaagga ctttgtttac tctcagagtt ctaactgtct ttttgacaga 720

aactatacac tgatgctaaa ggatcttttc ttgtctcgat tcaactcctt gatgattttg 780

ctttctcccc ctgagccccg atactcagat gacttaatat ctcagctgtg ccagctatac 840

atcgctgggg atcaagtctt gtccttgtgt gggaactccg gctatgaagt catcaaaata 900

ttggagccat atgtcgtgaa cagtttggtc cagagggcag agaagtttag gcctctcatc 960

cactccttgg gagactttcc tatgtttata aaagacaagg tgaatcaact tgaagggact 1020

ttcggtccca gtgcaaaaag gttttttagg gttctagatc aattcgacaa catacatgat 1080

ctagtatttg tgtatggctg ttacagacat tgggggcacc cctatataga ttatcggaag 1140

ggtctgtcga aactatatga tcaagttcac attaagaaag taatagataa gtcctaccag 1200

gagtgtttag caagtgactt ggctagaagg atcctcagat ggggatttga caagtactcc 1260

aagtggtatc tagattcgag attccttgcc ctagaccacc ccttggctcc ttatatcaag 1320

acccaaacat ggccacccaa acatatagta gacttggtgg gggacacatg gcacaagctc 1380

ccgatcacgc agatctttga gattcctgaa tcaatggacc cgtcagagat actggatgat 1440

aaatcacatt ctttcaccag aacaagacta gcttcttggc tgtccgagaa ccgagggggg 1500

cctgttccta gcgagaaggt cattatcacg gccctgtcta agccacctgt caatccccga 1560

gagtttttga aatctatcga cctcggagga ttgccagatg atgacttgat aattggcctc 1620

agaccaaagg aacgggagtt gaagattgag ggccgattct tcgctctaat gtcatggaat 1680

ctaagattat attttgtcat caccgagaag ctcttggcca actacatttt gccacttttt 1740

gacgcactga ctatgacaga caacctgaac aaggtattta aaaagttgat cgacagggtc 1800

accgggcaag ggcttttgga ctattcgagg gtcacatacg catttcacct ggactatgag 1860

aaatggaaca atcatcaaag attggagtca acggaggatg tattttctgt cctagatcag 1920

gtgtttggat tgaagagggt gttttctaga acacacgagt tttttcagaa gtcctggatc 1980

tattattcag acagatcaga cctcattggg ttacgggagg atcagatata ttgcttggat 2040

atgtccaatg gtccaacctg ctggaatggc caagacggcg ggctagaggg cttacggcag 2100

aagggctgga gtctagtcag cttattgatg atagatagag aatctcaaac caggaacaca 2160

agaaccaaga tactagctca aggagacaac caggttctgt gtccgacata catgttgtca 2220

ccgggattgt ctcaagaggg gcttctctat gagttagaga gcatatcgag gaatgcactc 2280

tcaatatacc gagctatcga ggaaggagca tctaaactgg ggctgatcat caagaaagaa 2340

gagaccatgt gtagttatga ctttctcata tatgggaaga cccccttatt tcgaggcaac 2400

atattagtac ctgaatccaa aagatgggcc cgagtctctt gcatctctaa cgaccaaata 2460

gtcaacctcg ccaatataat gtctacagta tccaccaatg cgctgacagt ggcacaacac 2520

tctcaatctc tgatcaaacc tatgagggat tttctgctca tgtcagtaca ggcagttttc 2580

cactacctgc tgtttagccc aatcttaaaa ggcagggttt ataagattct gagtgctgaa 2640

ggggagagct ttctcctagc catgtcgcgg ataatctacc tagatccttc tttgggaggg 2700

gtgtctggaa tgtctctcgg gaggttccat atacgtcagt tctcagaccc tgtctctgaa 2760

gggttgtcat tctggagaga gatctggtta agctcccatg aatcctggat tcacgcgttg 2820

tgtcaagagg ccgggaaccc cgatcttgga gagagaacac tagagagctt cactcgcctt 2880

ttagaagatc ctactacctt aaatatcaaa ggaggggcca gtcctaccat tctactcaag 2940

gatgctatca gaaaggctct gtacgacgag gtggacagg tggaaaattc agagtttcga 3000

gaggcaatcc tgttgtccaa gacccataga gataacttta tactcttttt aaaatctgtt 3060

gagcctctgt tccctcgatt tctcagtgaa ctcttcagtt cgtccttctt gggaatacca 3120

gagtcaatta ttggactgat acaaaactcc aggacaataa gaaggcagtt tagaaagagt 3180

ctctcaagaa ctttagaaga gtccttctac aactcagaga tccacgggat taatcggatg 3240

accccagacac ctcaaagggt cgggagagtg tggccttgct cttcagagag ggcagatcta 3300

cttagggaga tctcttgggg aaggaaagtg gtaggcacga cagttcctca cccttccgag 3360

atgttggggc tgcttccaaa atcctctatt tcctgcactt gtggagcaac aggggggaggc 3420

aatcctagag tttctgtatc agtactcccg tccttcgatc agtcattttt ttcacgaggt 3480

cccctaaagg gatacttggg ctcgtccacc tccatgtcaa cccagctatt ccatgcatgg 3540

gaaaaagtca ctaatgttca tgtggtgaaa agggctatat cgttaaaaga atctataaac 3600

tggttcatca atagaaattc caatttggct caaactctaa ttagaaacat catgtctctg 3660

acaggccctg atttccctct agaagaggct cctgttttca aacggacagg gtcagccttg 3720

cataggttca agtctgccag atacagcgaa ggagggtatt cttctgtttg tcctaacctt 3780

ctctctcata tctctgtcag tacagacact atgtctgatt tgacccaaga cgggaagaac 3840

tatgatttca tgtttcagcc attgatgctt tatgcgcaaa catggacatc agagctggta 3900

cagagagaca caagactgag agactccacg tttcactggc accttcgatg caacagatgt 3960

gtaaggccca ttgaggatat aacactggaa acttctcaga tcttcgagtt cccggatgtg 4020

tcaaaaagga tatccaggat ggtttctgga gccgtccctc actttcagaa gcttcctgat 4080

atccgtctaa gaccaggtga ttttgaatct ctaagtggta gagaaaagtc tcgccacata 4140

gggtcagctc aggggctctt atactcaatc ttagtagcaa ttcacgattc aggatacaat 4200

gatgggacca tcttccctgt caacatatac ggcaaagttt cccctagaga ctatttgaga 4260

gggctcgcaa gagggatctt gataggatcc tcgatttgct tcttgacacg aatgacaaat 4320

attaatatta aaagacctct tgaattgatc tcaggggtaa tttcctatat tctcctgagg 4380

ctggataatc atccctctct gtatataatg cttagagaac cgtctcttag aggagagata 4440

ttctctatcc ctcagaaaat ccccgccgct taccaacca ctatgaaaga aggcaacaga 4500

tcgatcttgt gttacctcca acacgtgcta cgctatgagc gagaagtaat cacggcgtcc 4560

ccggagaatg actggctgtg gatcttctca gacttcagaa gtgcgaaaat gacgtacttg 4620

accctcatta cctaccagtc tcacctccta ctccagaggg ttgagcgaaa cttgtctaag 4680

agtatgagag ctactctgcg acaaatgggt tccttaatga ggcaagtgct gggtgggcac 4740

ggagaagaca ccttggagtc agacgacgac attcaacgat tactaaaaga ctctttgcga 4800

aggacaaggt gggtagatca agaggtgcgc catgcagcta gaaccatgag tggagattac 4860

agccccaaca agagagtatc ccgcaaggca ggatgttcag aatgggtctg ctctgctcaa 4920

caggttgccg tctccacctc agccaacccg gcccctgtct cagagcttga cattagggcc 4980

ctatctaaga ggtttcaaaa ccccttgatc tcgggcctaa gagtggttca gtgggcaacc 5040

ggcgcccatt ataagcttaa gcctattcta gatgatctaa atgttttccc atctctctgt 5100

cttgttgttg gagacgggtc agggggaata tcaagggcag ttctcaacat gtttccagat 5160

tctaagcttg tgttcaacag cctattggag gtgaatgatc tgatggcttc cggaacacat 5220

ccactgcctc cttcagcaat catgagtgga ggagatgata tcatctccag agtgatagac 5280

tttgactcaa tctgggaaaa accatccgac ctgaggaact tggccacctg gagatacttc 5340

cagtcagttc aaaagcaggt caacatgtcg tatgacctca ttgtttgtga tgcagaagtt 5400

actgatattg catctatcaa ccggataact ctgttgatgt ctgatttcgc attgtctata 5460

gatggaccac tttatctggt cttcaaaact tacgggacta tgctagtgaa cccagactat 5520

aaagctatcc aacatctgtc aagagcgttc ccttcggtca cagggtttat aacccaagta 5580

acttcgtcct tttcttctga gctatacctc cggttctcca aacgagggaa gttttttagg 5640

gatgctgagt acttgacctc ttccaccctt cgggagatga gccttgtgtt attcaattgt 5700

agcagcccca aaagtgagat gcagagagct cgttccttaa actatcaaga tcttgtaagg 5760

ggatttcctg aagagatcat atcaaatcct tacaatgaaa tgatcataac tctgattgac 5820

agtgatgtag agtccttcct ggtccacaag atggtggatg atcttgagtt acagagggga 5880

actctgtcta aagtggctat catttatcc atcatgattg ttttttccaa tagagtcttc 5940

aacatttcca aacctttgac tgaccccttg ttctatcccc catctgatcc taaaatcctg 6000

aggcacttca acatatgttg cagtactatg atgtatctat ctaccgcttt aggcgacgtc 6060

cctagcttcg caagacttca tgacctatat aatagaccta taacttatta cttcagaaag 6120

caagttatc gagggaatat ttatctatct tggagctggt ccgatgacac cccagtgttc 6180

aagagggtag cctgtaattc tagcttgagt ctgtcatctc actggatcag gttgattac 6240

aagatagtga agactaccag actcgttggc agcatagaag atctatcagg agaggtagaa 6300

cgacacctgc atgggtacaa cagatggatc accctcgagg atatccgatc tagatcatcc 6360

ctactagact acagttgttt gtaa 6384

<210>8

<211>1801

<212>DNA

<213> Rabies virus

<400>8

aagtttatca cttgtttacc tctggaggag agaacatacg ggcttaactc caatccttgg 60

gagcaataga acaaaaaaac acgccatggt gccattaaac cgctgcattt tatcaaagtc 120

aagttaatta cctttacatt ttgagcctct tggatgtgaa aaaaactatt aacatccctc 180

aaaagactta aggaaagatg gttcctcagg ttcttttgtt tgtacccctc ctgggttttt 240

cattgtgttt cgggaagttc cccatttaca cgataccaga caaacttggt ccctggagcc 300

ctattgacat aacaccatctc agctgtccaa ataacctggt tgtggaggac gaaggatgta 360

ccaacctgtc cgagttctct tacatggaac ttaaagtggg atacatctca gccataaaag 420

tgaacgggtt cacttgcaca ggtgttgtga cagaggcaga aacctacacc aactttgttg 480

gttatgtcac aaccacattc aagagaaagc atttccgccc caccccagac gcatgtagag 540

ccgcgtataa ctggaagatg gccggtgacc ccagatatga agagtctcta cacaatccgt 600

accccgacta ccattggctt cgaactgtaa aaaccaccaa agagtctctc gttatcatat 660

ccccaagtgt gacagatttg gacccatatg acaaatccct tcactcaagg gtcttccctg 720

gcggaaattg ctcaggaata acggtgtcct cgacctactg ctcaactaat catgattaca 780

ccatttggat gcctgagaat ctgagactag ggacatcttg tgacattttt accaatagca 840

gagggaagag ggcatccaaa ggaggcaaga cttgcggctt tgtggatgaa agaggcctgt 900

ataagtctct aaagggagca tgcaaactca agttatgtgg agttctcgga cttagactta 960

tggatggaac atgggtcgcg atgcaaacat cagatgagac caaatggtgc cctccaggtc 1020

agttggtgaa tttgcacgac tttcgctcag acgagattga gcatctcgtt gtggaagagt 1080

tagtcaagaa aagagaggag tgtctggatg cactagagtc catcatgacc accaagtcag 1140

tgagtttcag acgtctcagt cacttgagaa aacttgtccc tgggtttgga aaagcatata 1200

ccatattcaa caaaaccttg atggaggctg atgctcacta caagtctgtc cggacctgga 1260

atgagatcat cccctcaaaa gggtgtttga gagttggggg gaggtgtcat ccccatgtga 1320

acggggtgtt tttcaatggt ataatattag ggtctgacgg ccatgttcta atcccagaga 1380

tgcagtcatc cctcctccag caacatatgg agttgttgga atcttcagtt atccccctga 1440

tgcacccctt ggcagaccct tctacagttt tcaaagacgg tgatgaggtt gaggattttg 1500

ttgaagttca cctccccgat gtgcatgaac aggtctcagg agttgaactg ggtctcccga 1560

actgggggaa gtatgtattg atgattgcag gggccttgat tgccctgatg ttgataattt 1620

tcctgatgac atgttgcaga agagtcaatc gaccagaatc tacgcaaagc agtcttggag 1680

agacaggggag aaatgtgtca gtcacttccc aaagcggaaa agtcatatct tcatgggagt 1740

catataagag tggaggcgag accagactgt gaaggccggt catccttttg acacttcaag 1800

t 1801

Claims (6)

1. one kind has better security and immunogenic recombinant rabies virus strain, described virus strain is characterised in that inserts extra G gene between G gene in its genome and L gene, it comprises two G genes in genome, described recombinant virus is to be built by the wild-type Flury-LEP of rabies virus strain, and the sequence of the G gene of described insertion is as shown in SEQ ID NO:8.

2. method that builds the recombinant rabies virus strain of claim 1, described method comprises the following steps;

1) structure is transcribed plasmid, wherein with between G gene and L gene in the genome of G gene order insertion wild-type rabies virus strain Flury-LEP, obtains containing the recombination group full length cDNA clone of two G genes;

2) build the helper plasmid system, described helper plasmid system is nucleoprotein N, phosphoric acid albumen P and the polymerase protein L of the encoding wild type LEP of rabies virus strain respectively;

3) use the plasmid vector of the recombination group full length cDNA clone contain two G genes and helper plasmid cotransfection to the host cell of allowing described virus replication, cultivate described host cell;

4) rescue virus.

3. the method for claim 2, wherein host cell used is the BHK-21 cell.

4. a plasmid vector, wherein contain the recombination group full length cDNA clone that contain two G genes constructed just like claim 1.

5. the plasmid vector of claim 4, it builds from plasmid pCI-RBz.

6. the recombinant virus that builds as claim 1 is for the preparation of the application of rabies virus inactivated vaccine.

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