# Patent

## Title
Attenuated vaccinia virus KVAC103 strain

## Abstract

The attenuated vaccinia virus strain KVAC103 was obtained by subculturing a vaccinia virus strain 103 times in VERO cells. The attenuated vaccinia virus strain KVAC103 has significantly low toxicity, shows reduced skin lesions, and, at the same time, induces effectively immune responses to poxvirus. Thus, it can be used as a poxvirus vaccine while causing less side effects.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims priority to Korean Patent Application No. 10-2014-0140150, filed Oct. 16, 2014, the contents of such application being incorporated by reference herein.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the novel attenuated vaccinia virus strain KVAC103 having reduced toxicity and side effects.
Description of the Prior Art
Vaccinia virus is an enveloped DNA virus which has an about 120-180 kb double-stranded linear DNA genome encoding about 200 different genes (Hendrickson et al., 2010). Each of the genes is composed of a short 5′-promoter, a single ORF encoding a protein without intron, a short 3′-polyadenylation site. These proteins are expressed in a promoter-dependent manner in the intermediate-early (IE), early (E) or late (L) stage of viral infection. The sequences of the early and late promoters have been well characterized through functional experiments (Rosel et al., 1986; Yuen et al., 1987; Davision et al., 1989a and 1989b; Chakrabarti et al., 1997).
Since vaccinia virus was first used as a vaccine for smallpox by Edward Jenner in the 18th century, it has become a general term for immunomodulators such that it would give the etymology of the word “vaccine”. Smallpox is an acute contagious disease caused by the variola virus. In smallpox caused by the variola major virus, maculopapular exanthema appears after an incubation of 7-17 days (12 days on average), and then progresses to blisters, pus blisters, etc. Smallpox has a mortality rate of 30% or higher, and the survivors are left with scars on the face. The variola virus that causes smallpox belongs to the Orthopoxvirus genus together with monkeypox, cowpox and vaccinia virus. Orthopoxviruses have the characteristic of inducing strong cross-immunity therebetween, and thus a vaccine produced from the vaccinia virus, which is less pathogenic than variola virus, has been used in a global program for the eradication of smallpox. The effect of the vaccine was demonstrated by the 1980's World Health Organization (WHO) declaration of eradication of smallpox (WHO Declaration of global eradication of smallpox, WkIy Epideminol Rec 1980:55:148). The variola virus that causes smallpox is characterized in that: 1) it is stable in an aerosol state; 2) it is easy to mass-produce; 3) it is contagious in a small amount; 4) it is highly contagious among humans; 5) it has a long incubation period of 7-17 days; and 6) it has high mortality rate. Due to such characteristics, there have been concerns over the potential for the variola virus to be developed as biological weapons. Due to the uncertainty over the eradication of smallpox and the danger of developing the variola virus into biological terror weapons, countries have retained smallpox vaccines for use in case of emergency. As is known, in the case of the USA, the first-generation smallpox vaccine produced from cows has been used for some risk groups. Some developed countries including Japan have also taken their own measures, and for example, developed smallpox vaccine by themselves in order to meet the demand for smallpox vaccines in their countries, or purchased the first-generation smallpox vaccine.
Vaccinia virus used in the early years had a good immunogenic efficacy and greatly contributed to the eradication of smallpox, but people vaccinated with the vaccinia virus sometimes showed serious side effects such as systemic infection or progressive infection. To reduce such side effects, Virulence-attenuated vaccinia virus strains including MVA, NYVAC, and LC16m8 were developed. Among them, modified vaccinia virus Ankara (MVA) as disclosed in Korean Patent No. 1009102970000 is an attenuated virus strain obtained by Subculturing the vaccinia virus CVA strain 500 times or more in chick embryo fibroblasts (CEFs), and it does not proliferate in most mammalian cells and has a 30 kb deleted region in six regions in the genome. This virus showed excellent safety in animal models, and thus have been developed as a smallpox vaccine and a vaccine delivery vehicle by Bavarian Nordic (Denmark). LC16m8, an attenuated virus developed by the Chiba Serum Institute of Japan, forms small plaques and shows reduced virulence. It was found to have a mutation in the B5R gene in the genome sequence. NYVAC is a virus strain obtained by deleting 18 ORFs from five regions containing 18 ORFs in the genome of the Copenhagen strain by a genetic engineering technique, and has been developed as various recombinant viral delivery vehicles.
SUMMARY OF THE INVENTION
An aspect of the present invention provides a novel attenuated vaccinia virus strain in order to develop a highly pure, safe poxvirus vaccine which can be produced by cell culture.
The present inventors subcultured vaccinia virus 103 times in Vero cells, thereby isolating and identifying the novel attenuated vaccinia virus strain KVAC103 which has excellent immunogenicity while having reduced toxicity due to virulence attenuation.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with the color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 shows the survival rates of 5-day-old suckling mice after selected vaccinia virus clones were inoculated into the brains of the mice in order to compare the safety of the clones.
FIG. 2 shows the results of evaluating humoral immunogenicity by a plaque reduction neutralization test (PRNT50) after mouse immunization with each of selected vaccinia virus clones.
FIG. 3 shows the results of evaluating humoral immunogenicity by a plaque reduction neutralization test (PRNT50) after rabbit immunization with each of selected vaccinia virus clones.
FIG. 4 shows the results of performing ICS (intracellular cytokine staining) to evaluate cell-mediated immunogenicity in mice after immunization with viruses.
FIGS. 5A and 5B shows the results of an ELISPOT immunoassay to evaluate cell-mediated immunogenicity in mice after immunization with viruses.
FIG. 6 shows the results of analyzing the skin toxicity of the attenuated vaccinia virus strain KVAC103 in rabbits.
FIG. 7 shows a comparison between the nucleotide sequence of a deletion region in the genome of the novel attenuated virus strain KVAC103 (corresponding to nucleotide residues 14992-15054 and 120327-120391 of SEQ ID NO: 1) and the entire nucleotide sequence of reference strain VACV107 (corresponding to nucleotide residues 15163-15224, 34601-34662, 139946-140008 and 142453-142516 of SEQ ID NO: 7).
FIG. 8 is a gene map showing the deletion region of the genome of KVAC103.



DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail. However, the present invention can be embodied in different forms and is not limited to the embodiments described herein.
In one aspect, the present invention relates to the attenuated vaccinia virus strain KVAC103 (accession No. KCCM11574P).
In an embodiment of the present invention, the attenuated vaccinia virus strain KVAC103 may be one wherein a gene having a nucleotide sequence represented by SEQ ID NO: 1 is deleted or damaged.
In one aspect, the present invention relates to an immunogenic composition for preventing or treating poxvirus infection, the composition containing the attenuated vaccinia virus strain KVAC103 (accession No. KCCM11574P) as an active ingredient.
In an embodiment of the present invention, the attenuated vaccinia virus strain KVAC103 may be attenuated by subculture.
In an embodiment, the composition may contain at least one pharmaceutically acceptable carrier or excipient.
For preparation of the preventive composition (i.e., vaccine) of the present invention, the attenuated vaccinia virus according to the present invention is converted into a physiologically acceptable form. This can be done based on the experience in the preparation of poxvirus vaccines used for vaccination against smallpox (as described by Stickl, H. et al. Dtsch. med. Wschr. 99, 2386-2392 [1974]). For the preparation of vaccine shots, for example, virus particles are lyophilized in 100 ml of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule. Alternatively, the vaccine shots are produced by stepwise freeze-drying of the virus in a formulation. This formulation can contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other aids, such as antioxidants or inert gas, stabilizers or recombinant proteins (for example, human serum albumin) suitable for in vivo administration. The glass ampoule is then sealed and can be stored between 4° C. and room temperature for several months. However, as long as no immediate need exists, the ampoule can preferably be stored at temperatures below −20° C.
The therapeutically effective dose of the therapeutic composition of the present invention can vary depending on various factors, for example, an administration method, a target area, the subject's conditions, etc. Thus, when the composition is to be used in the human body, the dose of the composition should be suitably determined by taking into consideration both safety and efficiency. It is also possible to estimate the dose for human administration from the effective dose determined through an animal test. Such considerations to be taken in the determination of the effective dose are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E. W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
The composition of the present invention may also comprise a carrier, a diluent, an excipient, or a combination of two or more thereof, which are commonly used in biological formulations. The pharmaceutically acceptable carrier for use in the present invention is not specifically limited, as long as it is suitable for in vivo delivery of the composition. Examples of a pharmaceutically acceptable carrier that may be used in the present invention include the compounds described in Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures containing one or more of these components. If necessary, the composition may contain other conventional additives such as antioxidants, buffers, bacteriostatic agents and the like. In addition, the composition can be prepared into injectable formulations, such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets, by adding diluents, dispersing agents, surfactants, binders and lubricants thereto. Furthermore, the pharmaceutical composition may preferably be formulated according to each disease or component by a suitable method known in the art or by using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton Pa., 18th, 1990).
The composition of the present invention may further comprise pharmaceutically acceptable additives. Examples of pharmaceutically acceptable additives that may be used in the present invention include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, Arabia rubber, pregelatinized starch, corn starch, cellulose powder, hydroxypropyl cellulose, Opadry, starch sodium glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol, talc, etc. The pharmaceutically acceptable additives that are used in the present invention are preferably contained in an amount of 0.1-90 parts by weight based on the total weight of the composition, but are not limited thereto.
The therapeutic composition of the present invention may be administered orally or parenterally (for example, intravenous, subcutaneous, intraperitoneal or topical application) according to the intended method. The dose of the composition of the present invention can vary depending on various factors, including the subject's weight, age, sex and health conditions, diet, administration time, administration method, excretion rate and the severity of the disease. The daily dose of the composition according to the present invention is 0.0001-10 mg/ml, preferably 0.0001-5 mg/ml, and is preferably administered once or several times a day.
In one aspect, the present invention relates to a method for preventing or treating poxvirus infection in mammals excluding humans, the method comprising a step of administering the composition of the present invention to the mammals.
In an embodiment of the present invention, the poxvirus may be Orthopoxvirus.
For vaccination or therapy, the lyophilisate of the composition can be dissolved in 0.1-0.5 ml of an aqueous solution, preferably physiological saline or Tris buffer, and administered either systemically or locally, i.e. parenterally, subcutaneously, intramuscularly, or any other administration routes known to those skilled in the art. The mode of administration, the dose and the number of administrations can be optimized by those skilled in the art in a known manner. However, most commonly, a patient is vaccinated with a second shot about one month to six weeks after the first vaccination shot.
Hereinafter, the present invention will be described in further detail with reference to the following examples. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
EXAMPLES
Example 1: Establishment of Attenuated Vaccinia Virus Strain
1-1: Subculture of Vaccinia Virus
To make an attenuated vaccinia virus, a Korean smallpox vaccine obtained from the Korea National Institute of Health was used as a parent strain, and subcultured in monkey kidney-derived Vero cells (ATCC CCL-81). Specifically, Vero cells cultured as a monolayer in a T75 flask were infected with 2 ml of the virus for 2 hours, after which the supernatant was removed, and the cells were cultured in 1% FBS-containing DMEM medium for 2-3 days while they were observed. A cytopathic effect (CPE) in the entire cells was observed by a phase contrast microscope, and the virus was harvested. To harvest the virus, the T-flask containing the infected cells was sealed, and freezing at −80° C. and thawing at room temperature was repeated twice to lyse the cells, after which the lysed cells were centrifuged at 1800 rpm for 5 minutes to remove the precipitate, and the supernatant containing the virus was collected. Infection of Vero cells with the virus-containing supernatant and subculture of the cells was repeated 103 times. As a result, viral stock containing various genetic variants was obtained.
1-2: Separation of Attenuated Viral Plaques
Each of the viral stocks of Example 1-1 was serially diluted 10-fold, and Vero cells cultured as a monolayer in a 6-well plate were infected with the diluted viral stock for 2 hours. Then, a 1:3 mixture of 4% agarose gel (Gibco-BRL, Cat#18300-012) and 1% FBS-containing DMEM was overlaid on the cells. Thereafter, the cells were cultured for 3 days, and then stained with 0.06% neutral red dye solution (Sigma, N2889) diluted in DMEM. Based on the results of the staining, plaques thought to have a suitable shape and size were selected, and previously prepared Vero cells were infected with the selected plaques and cultured for 3 days, thereby obtaining clones showing a cytopathic effect (CPE).
Example 2: Selection of Attenuated Virus Strain
2-1: Examination of Cerebral Toxicity
To compare the safety of isolated vaccinia virus clones selected after subculture, the clones were inoculated into the brains of suckling mice, and then the survival rate of the mice was evaluated. In the experiment, 5-day-old Balb/C suckling mice purchased from Orient Co., Ltd. (Korea) were used. The mice were stabilized for at least two days after purchase but before use in the experiment. Each mouse of each mouse group consisting of 12-15 suckling mice (5 days old) was inoculated with 10 PFU of the virus by an intracerebral route and observed for 14 days. Clones having low toxicity were selected (FIG. 1).
2-2: Analysis of Humoral Immune Response
Mice were immunized by injection with each of the selected vaccinia virus clones. 1, 2, 3, 4 and 5 weeks after immunization, immunogenicity was analyzed by ELISA. Specifically, each mouse group consisting of 5 female Balb/C mice of 4-weeks was inoculated with the virus. Inoculation was performed by a scarification method in which the tail was picked up 3-4 times with a bifurcated needle. Thereafter, clones showing excellent humoral immune activity were selected (FIG. 2).
2-3: Measurement of Neutralizing Antibody Titer
Blood was collected at one-week intervals from the eyeball of vaccinia clone-immunized mice for 6 weeks and from the ear vein of vaccinia clone-rabbits for 5 weeks. Serum was separated from the blood and inactivated at 56° C. for 30 minutes. One day before the experiment, BSC-40 cells were seeded in a 12-well plate. The antibody-containing mouse serums was serially diluted 2-fold and were mixed with 50 PFU of the vaccinia virus Lister strain at a ratio of 1:1, and the mixture was incubated at 37° C. for 1 hours. Then, the cells were infected with the incubated mixture for 2 hours. After the supernatant was removed, the cells were cultured in DMEM+1% FES medium containing carboxymethyl cellulose (CMC) for 2 days, and then stained with 0.06% crystal violet solution. Based on the results of the staining, the number of the plaques was measured to determine a serum concentration corresponding to a 50% or more reduction in the plaque number (FIG. 3).
2-4: Analysis of Cell-Mediated Immune Response
To analyze cell-mediated immunogenicity, 5 mice/group were inoculated with 6.5×106 PFU of the isolated virus strain. 4 weeks after immunization, the spleen was taken from the mice, and cells were extracted from the spleen. Using the extracted cells, cell-mediated immunogenicity was evaluated by ELISPOT and ICS (intracellular cytokine staining). Specifically, 5×105 splenocytes/well were cultured and stimulated with 0.1 MOI of the vaccinia Lister strain in vitro, and the number of splenocytes secreting IFN-γ was measured by ELISPOT and ICS (intracellular cytokine staining) (FIGS. 4, 5A and 5B).
As a result, clone 7 (KVAC) showed a decrease in the mouse cerebral toxicity compared to the conventional vaccine strain, had a constant plaque size and shape, and would be highly useful as a vaccine as proven by the immunogenicity test. Thus, this clone was named “KVAC103”, and deposited with the Korean Culture Center of Microorganisms (KCCM), in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms, on Oct. 1, 2014 under the accession number KCCM11574P, that all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of a patent.
2-5: Skin Toxicity Test
To compare safety between the conventional vaccine and the novel clones, each of the selected KVAC103 vaccinia virus and the vaccinia virus Lister strain was inoculated into the skin of rabbits, and then the size of lesions generated after inoculation was compared. As the rabbits, about 2-month-old female New Zealand white rabbits (weight: about 2 kg) were used. Inoculation of the rabbits was performed similar to inoculation of humans. Specifically, an area having a diameter of 5 cm or more on both sides of the back was shaved, and the shaved skin was inoculated with 105 PFU of the virus by a scarification method in which the skin was lightly picked 10 times or more with a smallpox vaccine inoculation device (bifurcated needle). Then, the skin was observed for 2 weeks. As a result, it could be seen that the skin toxicity of the attenuated vaccinia virus strain KVAC103 of the present invention was very low (FIG. 6).
Example 3: Characterization of Attenuated Vaccinia Virus Strain KVAC103
3-1: Analysis of Entire Genome Sequence of KVAC103
To analyze the nucleotide sequence of KVAC103, PCR amplification was performed. A vaccinia virus reference sequence for primer design was used based on the genome sequence (Genbank: DQ121394; 189,421 bp) of the VACV107 strain. The primers used were designed such that PCR products of the entire nucleotide sequence would be produced with a size of about 1,000-1,500 bp and the calculated melting temperature (Tm) would be between 53° C. and 55° C. (Table 1). For PCR amplification, the genomic DNA (20 ng/μl) of the virus was used as a template, and 10 pmole of each primer, 2.5 U of Taq polymerase (Cosmogenetech, Korea), 1× buffer and dNTP (each 2.5 mM) were mixed with distilled water to a total volume of 50 μl and used in PCR amplification. The PCR reaction was performed under the following conditions: 95° C. for 3 min; and then 35 cycles, each consisting of 95° C. for 1 min, 50° C. for 30 sec, and 72° C. for 80 sec; and then 72° C. for 3 min. Each of the PCR products was electrophoresed on agarose gel, and then the electrophoresis product bands were excised. Each PCR product was purified using a gel elution kit (Cosmogenetech, Korea), and the entire nucleotide sequence of KVAC103 was determined using both end primers. As a result, the entire nucleotide sequence (167122 bp) of KVAC103 could be determined, and the determined nucleotide sequence is shown in SEQ ID NO: 1.










TABLE 1





PCR





product


Tm


No.
Primer name
Sequence (5′->3′)
(° C.)







  1
     1-23F
GGT ACA CTT TTT TAA TTC GTG GT
53.1



  1223-1200R
AAG ATT CTT CCT CCA AAC AGT TAA
53.6


 


  2
  1102-1125F
GTA GTC TTG AGT ATT GGT ATT ACT
53.6



  2328-2305R
ATA AGA TAG ATT CCA TCA TCG TGA
53.6


 


  3
  2217-2237F
TGT CTG TCT GAA TGT ATG GCT
53.9



  3446-3423R
TTC TGT CAA CAA TGT CTG TCA TAT
53.6


 


  4
  3327-3347R
TGT AAT GGA GAG TTA CCT CGT
53.9



  4563-4544R
TAT CCT CTG CAC GAC TAC TT
53.4


 


  5
  4454-4474F
GCC ATT GTT CGA TAC GTG ATT
53.9



  5679-5656R
AGA ATA GCT ATG GAT TAT TGT GGT
53.6


 


  6
  5565-5588F
TTG ATA AGT TGT GAC ACG TTT CAA
53.6



  6700-6677R
CCT ATT AAA GGA GTA TGT CAT GAA
53.6


 


  7
  6584-6607F
GCT AAA GCT ATC TAT ACT ATC AGA
53.6



  7823-7803R
ATG GCT TAT TAT CGA GGA GAC
53.9


 


  8
  7697-7717F
CAT TAC GAT AGT ATG CAG GCA
53.9



  8906-8886R
TCG GAG GAT TCT CTA TTA TCG
53.9


 


  9
  8759-8783F
TTA GTA GAT AAC ATA CCA CAC CAT T
54.0



  9995-9976R
TCG ATA GCG TTA CCA CTA TC
53.4


 


 10
  9878-9898F
ACA CAC ACT GAG AAA CAG CAT
53.9



 11041-11020R
CAA TTG CCG TTC TAG ACA TAT C
54.4


 


 11
 10892-10915F
CAA CCA AGT AAT GAT CAT CTA TTG
53.6



 12106-12083R
GGA TAA TGC ATA CTG TTA GTC TTA
53.6


 


 12
 11993-12012F
TCG CGG ATA TGG AAT TCG AT
53.4



 13227-13204R
CAA GAT GAG AAT CCT ATT TCT CAT
53.6


 


 13
 13115-13138F
ATT CTC CAG AAG ATG TTA CAA TGT
53.6



 14336-14313R
AAT GTG TAG TAT TGT ACC ACT ATG
53.6


 


 14
 14223-14246F
CCG AAT GTC ATA TAC TCA ATT AGT
53.6



 15466-15443R
CAT GAT GGT AGT AAT AAA GGA TCT
53.6


 


 15
 15365-15385F
ACC TCC GTT AAT ACC TCC ATT
53.9



 16607-16584R
GAT GAA GGA GCT ACA TTA TAT AGA
53.6


 


 16
 16472-16496F
GGT TAT CAT CAT TGT CAT TAT CTA C
54.0



 17714-17691R
ACT AAT AGT ACT GAC ACT ACA GTA
53.6


 


 17
 17577-17600F
TAT CTT ATC GTT AAC CAT GAT TGG
53.6



 18818-18798R
GTT GGC TTA TTC CAT AGT AGC
53.9


 


 18
 18696-18716F
CTG AAT GGA TGA ACG AAT ACC
53.9



 19937-19917R
GAG AGA ATG GAT TAT GTG TGC
53.9


 


 19
 19793-19816F
ATT GGT CTG TGT TAC ATA TCT CTT
53.6



 21023-21000R
CGA CAC AAT AGA TAT ATC TGA TTG
53.6


 


 20
 20882-20905F
CAC ATT ATC ATC TGT TAG AGT AGT
53.6



 22126-22106R
GTA ACT CAT AAG TCA CTG CCA
53.9


 


 21
 22019-22039F
ATG TTA TCC TGG ACA TCG TAC
53.9



 23257-23236R
CTA TAT AAT GCG TCG ATG TCA G
54.4


 


 22
 23133-23153F
TTT ACG TTA ACG TCT TCG TGG
53.9



 24366-24342R
GAT CTA ATG ATT GAT CTA TAT GGT G
54.0


 


 23
 24298-24318F
GTA CTC ATT ATC ATT CGG CAG
53.9



 25496-25479R
CTT TGT ACT TAT GCA TCC GGA
53.9


 


 24
 25392-25415F
AAT ACT TCG TAA GAT ACT CCT TCA
53.6



 26638-26618R
ATG CTA CAA GCT ATC GAA CCT
53.9


 


 25
 26522-26542F
TGG AGA GTA TCC TCT ATG AAG
53.9



 27770-27745R
CGA CAT TAA TAT CTA TTC TGC TAA TC
54.4


 


 26
 27661-27684F
TTG TAT AGG AGT CAG ACT TGT ATT
53.6



 28899-28876R
ATG ATG GCT CCT AGT ATG TTT AAT
53.6


 


 27
 28785-28805F
CTT GTT CGT CGA CAT CTA TCT
53.9



 30018-29998R
GGC AGA GTA TAC GAG AAT GAT
53.9


 


 28
 29926-29946F
ATG CAT CTT AAC ACT CTC TGC
53.9



 31182-31163R
AGG AGT ACG AGT TAG AGT AG
53.4


 


 29
 31055-31075F
TAT GAA GAA CTC CTC CTA GGA
53.9



 32309-32289R
CGT CTC CTT CCT CTA TAT TGA
53.9


 


 30
 32188-32211F
GCA CTC ATG ATT CAC GTT ATA TAA
53.6



 33442-33419R
TAG CAA TAA TGT TGA TCA CGA CTA
53.6


 


 31
 33346-33369F
CGT TGA TAG ATA ATC GAG TAT GTT
53.6



 34553-34533R
GAT TCC AAT TCC TCC GAT GAA
53.9


 


 32
 34440-34459F
AAC CAA CGC TCA ACA GAT GT
53.4



 35657-35637R
ATA GAA TTC GTC GCG GAT AGA
53.9


 


 33
 35576-35596F
CCA TGA AAT CAA ACG GAT TGG
53.9



 36865-36846R
ATA TCC AAC AGA GTC GCT AG
53.4


 


 34
 36712-36732F
GGA TTC ACA AAT GTT ACG CAC
53.9



 37933-37913R
TTA ATA TGA CGC TCG TCA TGG
53.9


 


 35
 37832-37852F
TGT AAC ATC CTT CTC TTC CCT
53.9



 39090-39067R
GCA TTA GAA GAA TTC ATC ATG TGT
53.6


 


 36
 39014-39034F
ATT GGG TGA AGA ATG GAA ACC
53.9



 40269-40249R
ATG GGT GTT GCC AAT GAT TCA
53.9


 


 37
 40117-40137F
TAA CAA TCT AAC TGA CGG AGC
53.9



 41346-41326R
GCA TTC CAT CGA GAT CAA AGA
53.9


 


 38
 41165-41185F
GGA TAC GAA GAT GCT ATC CAT
53.9



 42423-42403R
TAT ACT GGC CCA ATA ACT GTG
53.9


 


 39
 42336-42356F
TAC GCA TCC ATC CAA ATA ACC
53.9



 43516-43496R
AGT CGA CGA CGA ATA TGT TCA
53.9


 


 40
 43372-43391F
GCT AGA TAC CCA ATC TCT CT
53.4



 44611-44591R
TAG ACG AAG ATG ACA TCA TGG
53.9


 


 41
 44505-44525F
CGA TTC TAG AAT ATC ATC GGC
53.9



 45678-45655R
GAT AGT TAA TTC CAA TGT TAC GTG
53.6


 


 42
 45506-45526F
GAT TAT CCA ATT GAG AAG CGC
53.9



 46835-46816R
GAC ACA TCG TGT GAT TTC GA
53.4


 


 43
 46697-46717F
CAC ATA AAC CTC TGG CAC TTA
53.9



 47961-47933R
AAT AGG AAT CCT GAT CAG AAT ACT T
54.0


 


 44
 47863-47886F
ATA TGA TAT TCG TTC ACA CTA GGT
53.6



 49107-49084R
CTA TAC ATT CTA CGT TAG TAA TGG
53.6


 


 45
 49021-49041F
TAT CGA TGT CGA TCT CGT CTA
53.9



 50317-50294R
TAC GTC ATC ATT AGA TTC TGA TTC
53.6


 


 46
 50182-50205F
ATA CTA GTC GCG TTA ATA GTA CTA
53.6



 51395-51372R
ATG TGA TAT GAT TAA GGG TAC TAG
53.6


 


 47
 51220-51240F
AGT TAG GAC ACG GTG TAT TGT
53.9



 52462-52442R
GAG GGT ATC TTC TTC AGA TTC
53.9


 


 48
 52214-52237F
ATT CGT TAT AGA ATG CAT CCA ATG
53.6



 53487-53467R
CTA TGC CTG TAT CTT TCT TGC
53.9


 


 49
 53302-53325F
GAG AAC TTA TAG GCG TAA ATT ATG
53.6



 54575-54555R
ATC CGT CTT GGT TAG ATG GAT
53.9


 


 50
 54432-54452F
CCT AGT ATT CTT CCA TCG CTA
53.9



 55722-55699R
ATT ATA GAT TGG AAT GGA AGT CAG
53.6


 


 51
 55623-55643F
GTT CTC TGG TAG ATA CGT ATC
53.9



 56898-56878R
TCC GTG TTT ACA GAG ATA GAC
53.9


 


 52
 56764-56784F
CAG ATT CTT ATA TAC CGC CTC
53.9



 57989-57969R
CCA CAG GTA ATT ATG TGA CTG
53.9


 


 53
 57873-57896F
ATG TAT CAT TAG GTA AAG TAG GAC
53.6



 59155-59135R
GGA TGT TCG GTG CAT TAA TTG
53.9


 


 54
 59075-59094F
TCA CCA TTT CGA CAT CTG GA
53.4



 60342-60322R
CTT AAT CGG TCA ACG ATG TTG
53.9


 


 55
 60225-60245F
TCC GTA TAC CAA CAT GTC TGA
53.9



 61479-61459R
TGA GAA TGT AGT CAA GGC TAC
53.9


 


 56
 61360-61380F
GTA GCT CCT ACA GGA ATA TCT
53.9



 62639-62618R
GAC GAA TCG TCA ATC TAT GTG
53.9


 


 57
 62523-62543F
AGC GTT CGT CAA CAT ACT ATG
53.9



 63785-63765R
TTG GCC AAG GCA ATT ATC ACA
53.9


 


 58
 63656-63676F
TAT CTC CAG AGA GTC CGA TAA
53.9



 64896-64876R
GTG GAA CCG TAT ACC AGT AAT
53.9


 


 59
 64772-64795F
CTA TTC TTG ATC TCA TCA TTC CAT
53.6



 66069-66049R
GAT GAT AGC ATA GAG GGT ATC
53.9


 


 60
 65949-65968F
TAC CGG AGA TAT AGC TTC CA
53.4



 67260-67240R
GCG TCT AAA CAC AAT CTT GGT
53.9


 


 61
 67125-67148F
AAC TGG AAA GCT AGA CTT GAT TAT
53.6



 68325-68302R
CTA TTT AAC TTG CAA GAT CTA TCC
53.6


 


 62
 68164-68188F
AGT TAT TCA TCG TCG TCT ACT ATT
53.6



 69504-69481R
TGT AAG AGA ATA CAT TAA CGC AGT
53.6


 


 63
     1-24F
CAT CTA TAT TGC TAC ATA ATC CAG
53.6



  1224-1200R
CAT TCT GGA AGA TGG ATT TAT CTT A
54.0


 


 64
  1102-1120F
GAT GAC AGG GAA CGG CTA A
54.9



  2321-2299R
CCA ATC TAT TGA GCA AGA ATA CC
54.9


 


 65
  2199-2222F
CTT TTG TAG ACA CGA CTA AAC ATT
53.6



  3438-3415R
ATA TCG GGA TCG AAT ATC GTA ATT
53.6


 


 66
  3302-3324F
TTA CCG TTT GTA CTT ACT GGA AT
53.1



  4530-4510R
TTA TAA CAC GGC CGT ATA CAC
53.9


 


 67
  4418-4440F
GAA CAT GTA GAT TTA TCG GAG GA
54.9



  5613-5591R
CTT CTT CCA TAA ATC CGG TAA CA
54.9


 


 68
  5499-5519F
CAA GAC GTT AGA GAC AAG AGA
53.9



  6732-6710R
TGT TAA GTT ACG TTG AGG TTC TA
53.1


 


 69
  6571-6589F
GTG TTC CAA CTC GTG TGC T
54.9



  7801-7781R
GGT GAG CCT ATT ATA GTA GCT
53.9


 


 70
  7684-7707F
CAT ATG TGG TAT TCA CTA TAT CAG
53.6



  8924-8904R
CAG TAT TAC CAG GAG TCT TCA
53.9


 


 71
  8811-8830F
CCG TCA TTC ATC TAG AAT GG
53.4



 10043-10021R
ACA TGA CAA TCT TAA TGA GGA AG
53.1


 


 72
  9909-9929F
GAG TGT TCG AAT GCC AAT GTT
53.9



 11093-11075R
GGT CGA AGT ATA GCA GGA C
54.9


 


 73
 10991-11012F
CTG ATC GTC TAG ACG ATA TAG T
54.4



 12230-12208R
ACG AGG TGA TAG AAA TAT ACC AG
54.9


 


 74
 12113-12134F
ATC TTG TTT CGG TGG CTG ATT A
54.4



 13350-13329R
GAT GTT TGA GTT GTC ATC CAT G
54.4


 


 75
 13227-13250F
ATA TGT TCT TCA TGC CTA AAC GAA
53.6



 14404-14382R
CTA CAC ACC GAT TGA TAC ATA TC
54.9


 


 76
 14292-14314F
CTA ACT GCT GTG TGT ATG AAA TG
54.9



 15530-15505R
ATA CTC TAT ATC GGT AGT ATA TCT C
54.0


 


 77
 15401-15423F
GTT AGT CAT GAA CCA ATA CAA CG
54.9



 16632-16607R
CAT GAC TAA TGA TAA TAT CTG TAG CA
54.4


 


 78
 16504-16529F
GAA ATT TGA TTG TAT ACT TTC GGT TC
54.4



 17742-17721R
TAG CTC TGA TAG ATG AAG CGA T
54.4


 


 79
 17628-17648F
CAT TTA TTG CCT GGT GAT TGG
53.9



 18818-18801R
CAG TGT TCC GGT ACG TGA
54.3


 


 80
 18672-18690F
CCA GAC TCT AAG GAT CCA G
54.9



 19900-19880R
TGA TTG AAG TCG TCA TAT CCC
53.9


 


 81
 19779-19801F
GAA CCG GAA GAA TTG AAT CTT AG
54.9



 21014-20995R
: TTG TAC GCC TAC TAT AGC CT
53.4


 


 82
 20879-20900F
TCC AGT TGA ACG TAG TAG TAA G
54.4



 22110-22086R
ACG ATA ACG TTA TTG AGG ATA TTA C
54.0


 


 83
 22075-22096F
GAC GAA GAA ACG TAA TAT CCT C
54.4



 23297-23275R
CAT CTC CGT GGT TAT ATA CGA TT
54.9


 


 84
 23180-23201F
ATC CAA TTC CTC CGG CAT TAT A
54.4



 24401-24380R
ACC AGA GAT TAA GAA GAT ACC G
54.4


 


 85
 24273-24294F
GGA ATA CCG ATG TGT CTA CAT A
54.4



 25490-25469R
TTA ACG ATA TTG TCG GTA GCC A
54.4


 


 86
 25371-25389F
AGA GCT CGA TGA TAG CGA C
54.9



 26593-26572R
CGT GAT CTA CGA CTA TAG ATA G
54.4


 


 87
 26485-26506F
AGA GCT TAT ATG GCA ACG ACT A
54.4



 27709-27689R
CAG AGA TGT ATT AGG CCT TGA
53.9


 


 88
 27560-27584F
CGA CTA GAA TTA GTC AAT CTT ATT C
54.0



 28769-28748R
CAT TAT TGG CGT ATT GAT GTC C
54.4


 


 89
 28658-28682F
GGA AAT CAA CAT AAT ATC ATA GTC G
54.0



 29866-29846R
ATG AAC AGG CCA GAT GTT ATC
53.9


 


 90
 29751-29769F
GGA CCT TCC AAC TGT GGA T
54.9



 30981-30963R
GGA TCT ATA CCG CAC ACA C
54.9


 


 91
 30871-30889F
AGT AGC CAG TTG GCT GCT A
54.9



 32100-32079R
CCA CGT AAG TGA ATA GGT AAT C
54.4


 


 92
 31999-32017F
CTC TTC GGG TTG TAG GTA C
54.9



 33213-33192R
CAA TGA CAC AAG GTT CTG TCA A
54.4


 


 93
 33088-33110F
CAT TTA TCG CTA ACA TGC ATT TG
53.1



 34315-34295R
ATC ACG GTC ATA AGT TCT CCA
53.9


 


 94
 34205-34228F
CGA TAA CGT CAT TAA TTA TAA CGG
53.6



 35439-35416R
GAC ATC TCT TGA AGA ATA GAG TAT
53.6


 


 95
 35321-35345F
AAC GAT TAC ACA CAG GAT GAA TTA A
54.0



 36556-36536R
TTG TCG TCG TCT AAT CAT GAG
53.9


 


 96
 35321-35345F
AAC GAT TAC ACA CAG GAT GAA TTA A
54.0



 36556-36536R
TTG TCG TCG TCT AAT CAT GAG
53.9


 


 97
 36433-36453F
AGG TAG TTG CTG CTC GTA TAA
53.9



 37613-37591R
ACT GTT ATA CGT TCA TCA CTT TC
53.1


 


 98
 37498-37519F
ATC GAT CCT CAC TTT GAA GAG T
54.4



 38693-38675R
GGC GTA GAT GGT CGA GTA T
54.9


 


 99
 38538-38561F
CAT AGA GGA TGT TAT TAC GAA TCA
53.6



 39797-39774R
TGT TCG TAC ATA GTT AAT AAC GAG
53.6


 


100
 39605-39626F
TAG TAG CCA TAC GTC TCA GAA T
54.4



 40834-40816R
CGT AAG CGA TAG CTG GTT G
54.9


 


101
 40703-40723F
ACA CTG TTC ACA TCC TTC CAA
53.9



 41935-41912R
AAT CTT TGC TCA CAT ATC ACA TAG
53.6


 


102
 41825-41846F
GAT TCG GCT GAT CTA TTA TCT C
54.4



 43036-43015R
GGA TGA CTC TAT TAA ACG GTC T
54.4


 


103
 42916-42934F
AGC CTG ATT GTC TGG ACC A
54.9



 44083-44062R
GTA TTG CGG AGA TTA TTG TCT C
54.4


 


104
 43984-44005F
CAT CTA CAT CAT CCG TGG ATA T
54.4



 45218-45197R
CCG ATT TCT ATC AAT TCC AAG G
54.4


 


105
 45112-45132F
ACT CCA GAA CAT CTT CCA TAG
53.9



 46317-46294R
TGG TAG ATG AAG ATC ATA TTC ACA
53.6


 


106
 46197-46217F
TTG GTA CGT TGA TTT CTA GCC
53.9



 47428-47408R
CTA GTG ACT CTC CAT CTT CTT
53.9


 


107
 47320-47343F
CGA TTA TAA GAT TAA ATG GCA GAC
53.6



 48562-48541R
CCG AAG ATA TTG TAT CCG ATT C
54.4


 


108
 48465-48485F
TTG TTG GAG AAC TTG ATA CGC
53.9



 49697-49673R
GAT AGT AAG TCC GTA TAT CCT TAA T
54.0


 


109
 49582-49603F
GTT CCA TAT TAG CAG TCA TTC C
54.4



 50804-50783R
TAA TCC CTT CGT ATA CAC TCA G
54.4


 


110
 50681-50701F
AAT AGA TGT ATC GCA CGC TCT
53.9



 51890-51869R
GAT TCC ACA GCC AAT GAA CAT
53.9


 


111
 51774-51794F
TAG TAC GGG CGC TGT AAT TAA
53.9



 53001-52981R
GTG GAG ATA TTA CAG GAG AAG
53.9


 


112
 52881-52905F
GTT GTA ATG TTA TCC AAC ATA TCA C
54.0



 54132-54108R
GAT ATC AAT CTC TTA TTC CTA GAC T
54.0


 


113
 54023-54043F
GGT TAC ATT CAC TGC AGC ATT
53.9



 55266-55247R
GAT AAT AGT GGC CGG TGA AT
53.4


 


114
 55154-55174F
ACA ACG TGG TAG ATA GAG AAC
53.9



 56339-56319R
CTC GTC TTC TTC TAC ATC AAC
53.9


 


115
 56225-56245F
TTG GAC AAT CTG ACC ATC CAT
53.9



 57355-57335R
AAG TAA TTA CGA GCC GTT GCT
53.9


 


116
 57208-57232F
CGA ATG AAC AAA GTG GAA TAT AAA C
54.0



 58446-58426R
TTA TCA GAA CGA GGT AAC TCC
53.9


 


117
 58319-58338F
TTG CTA CGC TAT CAC TAT CG
53.4



 59497-59478R
ATG ATC CCG GTT CAA CTT CT
53.4


 


118
 59394-59416F
GAT GGA GTA TAA TCT TTA TGC CG
54.9



 60640-60618R
ATA GTT CTG TTT CTC GAC ATA CC
54.9


 


119
 60527-60547F
CCG GTA CTG GTT TAG ATA TTC
53.9



 61770-61750R
TAC ACG CAC TTC GCA TAT CAT
53.9


 


120
 61634-61657F
TAA TAT TGA TCC GGG CTA TTA CAT
53.6



 62875-62851R
GAA GAA CTG TAG TGT ATT CAT ATT G
54.0


 


121
 62759-62779F
TAG AAG TCA AGG ATA ACT CCG
53.9



 63980-63959R
GGA TGA TAT CTT CGA ACA ACA G
54.4


 


122
 63869-63892F
ACT ATG CAC ATT ATT CTA TCC AAG
53.6



 65111-65088R
CTA ATG AAT TCT AGA CTC ACT CTA
53.6


 


123
 65003-65026F
ATA TAT CAC AAT TGG AAG CGT TGA
53.6



 66221-66201R
ATG TCA GAG AAT GTC ATG TCG
53.9


 


124
 66118-66138F
GGA AGA TTA GTC AGA CCA TTC
53.9



 67375-67355R
: AAG GCT TAT CCG TTT CAG GAT
53.9


 


125
 67249-67272F
ATC GAT ACA TAT ATG CAA TTC GCT
53.6



 68474-68455R
TTA TGG AGT TCG ATC GCC TT
53.4


 


126
 68333-68354F
CTA CGT TCA GAT TCC AAT TCA C
54.4



 69556-69538R
GAC AAC TGA GTG AAT GCC G
54.9


 


127
138802-138825F
ATT ATT ACG TCT ACA GTC GTT CAA
53.6



140039-140019R
TTC TTC TAC TAA CTC CCG AAG
53.9


 


128
139916-139936F
GTA ATA GGC TTA GGC AAA TGC
53.9



141149-141126R
AGT TGA TAG GTT AGA ACA TCA CAT
53.6


 


129
141032-141052F
ATG TGG ATG GAC AGT AGG TAA
53.9



142302-142322R
GGA CTT TAT GAA AGC CTA TCC
53.9


 


130
142202-142222F
CGC TAG CAT GGT CTT ATG ATA
53.9



143438-143415R
GGA TGT AAT TCT AGG TTA GAA TCA
53.6


 


131
143347-143367F
TTA CAC GCA TCA GAC AAT GCT
53.9



144769-144746R
AAT CAT CAT GAT ATA TAC CTC CTC
53.6


 


132
144492-144515F
TAG TAA AGC TGC AAT TAC ACA CTT
53.6



145817-145797R
TCC TCC CTA CTA ACA ACC TTA
53.9


 


133
145664-145684F
ATC CTC CCG TTA AAA CCA TTC
53.9



146930-146907R
TAA CAC ATA GTA CAG ATT GAG TAC
53.6


 


134
146831-146851F
CAA CTA AAC AGT ACG ACG GAT
53.9



148074-148051R
AAG GAT TGG ATG AAT AGT TAG GAT
53.6


 


135
147963-147989F
CGT AAT GAA CAG ACT ATT TAT CAG
53.6



149211-149191R
GAG AAT GAA TCC ATT CCG TAC
53.9


 


136
149084-149107F
GAG CAT TAG TAT TTC TGT GGA TTA
53.6



150345-150322R
GTG TAT AAT ACG TCG TCT AAT AAG
53.6


 


137
150192-150215F
TCA ACG ATA TTC TAA CTC TTG ACA
53.6



151465-151445R
GTG TGG TGT ACT CGA TTA AGA
53.9


 


138
151358-151378F
GTA ATG GTA ACT TCG AAA CCG
53.9



152592-152572R
TAC GTA GAA GCA ACA CTA GAC
53.9


 


139
152425-152445F
TAC GGT ATC GCG ATT AGT GTA
53.9



153660-153640R
GAC ACG ATA CAT TTA CTG AGC
53.9


 


140
153505-153524F
GCA CGT TAA CCG TAG ATG AA
53.4



154795-154772R
GCC AAT AAT TCC GTA ATC ATG AAT
53.6


 


141
154671-154691F
TTA CCG TGT TGC TTA CAT TGC
53.9



155921-155901R
GTA TTA ACC GCG CAA CCA ATA
53.9


 


142
155792-155813F
CAC ACT ACA CTG TCG AAT TTG
53.9



157100-157081R
CGT GCA ACC ATC CAA AGA TT
53.4


 


143
156973-156993F
TCC ACA TCT ATA GAC GAC GAT
53.9



158221-158201R
CAA CTC AGT CTG ATA GTT CTC
53.9


 


144
158129-158152F
ATC ATG TGT AGA TCA AAC TTG GAT
53.6



159376-159353R
GTA CAT TAT GTT CGT CTA CAA GAA
53.6


 


145
159264-159287F
AGT ACT CTC TCA TAA AGT GGA TTA
53.6



160493-160473R
GAG TCT TGT CAT CGT CAT CTA
53.9


 


146
160385-160405F
TAT CTG GAT AAT GCG GTA TCC
53.9



161696-161673R
TCA TAG ATA TGC AAT CGT ATA CAG
53.6


 


147
161562-161585F
AAT TCG GTA CTA TAG AAG AAC TCA
53.6



162801-162778R
TGA ATA ATA CAC TAA CCA AGT AGC
53.6


 


148
162697-162717F
GAC TTG ATC CAT TTC CTC CAT
53.9



163964-163941R
GAA TTT GTA CAT GTA TTG TAC GCT
53.6


 


149
163835-163858F
TAT CTT CAC GTA GAT ATA GGT GTA
53.6



165082-165062R
ATC AGT GTC ATT TGT AGG CGA
53.9


 


150
164962-164982F
ATC TTA CGA CTC TCC ATA CGA
53.9



166199-166179R
TAA TAG TGT CGA ATA GAC CGG
53.9


 


151
166091-166111F
AAG CTT CGT CGT AGA AAC ACT
53.9



167414-167394R
CCA TGA TTT CGG TTG TAC ACA
53.9


 


152
167292-167312F
ATT ACG ACA AGT TTC GGC ACA
53.9



168654-168634R
ATT CTC TAC CAG TCT GAG GTA
53.9


 


153
168505-168525F
ACT AAG AAC ACG TAT ACG GCA
53.9



169812-169792R
TCC ACG TTG TTG ATA TCG TGT
53.9


 


154
169631-169650F
GAA GAC AGT TAC GGT TGT AC
53.4



170994-170974R
TCA TAG TGG GTA CAG TAC ATG
53.9


 


155
170872-170892F
TAG TGC TCG ACA GTG TAT ACT
53.9



172171-172151R
CCA TTA AGT GTA TCC ATC ACC
53.9


 


156
172069-172089F
ACA GGC TAT TTA CAA GAT GCG
53.9



173320-173300R
ATG CGG ATA TGT CGT ATG TTC
53.9


 


157
173078-173101F
ACT TCA GTG ACA GTA GTC AAA TAA
53.6



174421-174402R
CGT GAT ATA CCC TAG CCA TA
53.4


 


158
174323-174343F
CTA ATA GCA CGA TCG TGG TTA
53.9



175665-175645R
GTC TTG AAA CTG TTG CTC CAA
53.9


 


159
175559-175582F
ATA CAA GAG TGG AAA CTC ACA TAT
53.6



176896-176877R
CCA CTA GTA CAG AAG TTG CT
53.4


 


160
176800-176820F
AAT TCA GAT GTG AGT GTC GAC
53.9



178144-178124R
CTG CGT TTA CGT TAC TAG CAA
53.9


 


161
178021-178041F
ATC GTG TCT GTC TCA GAA TCA
53.9



179428-179405R
TAA GGT AGG TGA TTC AGT TCT ATA
53.6


 


162
179288-179308F
ATT ACA ATA GCA TGA TCC GCG
53.9



180639-180616R
AGT ATA GGC GTT AAT CCA TGA TTA
53.6


 


163
180502-180525F
GTT AGA TAA TTG TGG TAA TAC ACC
53.6



181757-181737R
CAG TAG ATG CGA GTA AGT CTT
53.9


 


164
181579-181599F
TGG TAA CTG TGT TAC ATG TGC
53.9



182827-182807R
GAG GAA TGA GAG TGT CTT ATC
53.9


 


165
182676-182699F
ACC AAG GTA GTT AGT TAA TAC ACA
53.6



183996-183973R
TAC ATA CAG GTA CGA AAT ACG TAA
53.6


 


166
183869-183889F
TAT GCA ATC AAT GGT CTC GGA
53.9



185155-185135R
ACG CCT GAT ATG TAG ACA TTC
53.9


 


167
185027-185047F
CAG ATA CGC CTT AAT CCT AGA
53.9



186289-186269R
TGT TGC ACA ATC GTT CCA TGA
53.9


 


168
186151-186171F
TAT GGA GCA AAC ATT AAC GCG
53.9



187445-187425R
CTA TTG TGA GTC GTG TTA CAC
53.9


 


169
187318-187338F
ATG CGA TAG CAA GAC TAA CAC
53.9



188444-188464R
ATC TGA CTC GGA CTC TGT AAT
53.9


 


170
188303-188323F
AGT CTT CAG CAA TCA TCC TCA
53.9



189118-189099R
GGA ATA TAG TGT CCG GTA CA
53.4









3-2: Characterization of KVAC103 by Comparison of Nucleotide Sequences
The novel attenuated virus strain KVAC103 was characterized by comparing the entire nucleotide sequence of KVAC103 with the entire nucleotide of VACV107 (Genbank: DQ121394). As a result, it was shown that the attenuated virus strain KVAC103 had a 19.5 kb [C9L-F3L] deletion region on the left and a 2.5 kb [A25L-A26L] deletion region on the right (FIGS. 7 and 8). Thus, the identity of the attenuated strain KVAC103 could be identified by amplifying the deletion regions in the genome by PCR and determining the nucleotide sequences of the PCR products. The 19.5 kb deletion region of KVAC103 was amplified using a primer pair of 15F (SEQ ID NO: 2) and 35R (SEQ ID NO: 3), and the 2.5 kb deletion region was amplified using a primer pair of 140F (SEQ ID NO: 4) and 143R (SEQ ID NO: 5). The amplification products were separated on agarose gel, and then sequenced, thereby identifying deleted sequence of KVAC103. In addition, the wild-type vaccinia virus DNA was identified by PCR amplification using a primer pair of 15F (SEQ ID NO: 2) and 16R (SEQ ID NO: 6).












TABLE 2










SEQ



Region
Primer
Primer sequence
ID



amplified
name
(5′->3′)
NO.









19.5 kb  
15F
AGG AAC AGG ATC 
2



deletion

ATT GTC ATT ACA




region






 




35R
TGA ACT AAA TGT 
3





TCC AGA TGA GGA




 



2.5 kb  
140F
AGT TCT GCA TTC 
4



deletion

AAT TCG GTG AGT




region






 




143R
AAT TGT ACC AAC 
5





GGT TCA AGA TGT




 



Wild-type  
15F
AGG AAC AGG ATC 
2



vaccinia

ATT GTC ATT ACA




virus






 




16R
TCG TTT ATC AAC 
6





ACT ACC GTT AGA










3-3: Optimization of KVAC103 Culture Conditions
The proliferation levels of the virus in Vero cells (ATCC CCL-81), in various culture media and at various serum concentrations, were analyzed to determine the optimum culture conditions. Specifically, Vero cells were seeded in a 12-well plate, and then cultured with 1% FBS (Gibco #16000)-containing DMEM (Gendepot #CM0027050), Advanced-DMEM (Gibco #12491), OptiMEMI (Gibco #31985), VP-SFM (Gibco #11681) or OptiPro-SFM (Gibco #2309) medium for 24 hours. Then, the cells were infected with KVAC103 at a titer of 100 PFU/well for 2 hours. Thereafter, the medium in each well was replaced with the same medium containing 0%, 1%, 2% or 5% FBS, followed by culture for 3 days. The medium was removed, and the virus plaques were observed by staining with crystal violet solution. As a result, it was shown that the plaques were the clearest and largest in the OptiMEMI medium containing 1% FBS. Under such culture conditions, the proliferation of the virus was smooth, indicating that these conditions are suitable for the replication of KVAC103 and the production of a recombinant virus.
As described above, the attenuated vaccinia virus strain KVAC103 according to the present invention has reduced toxicity, shows reduced skin lesions, and effectively induces humoral and cell-mediated immune responses. Thus, it can be expected that the attenuated vaccinia virus strain KVAC103 can be effectively used as a vaccine composition which has excellent immunogenic effect together with reduced toxicity.
Accession Number
Depository authority: Korean Culture Center of Microorganisms;
Accession number: KCCM11574P;
Deposit date: Oct. 1, 2014.

## Claims

1. The attenuated vaccinia virus strain KVAC103 (Korean Culture Center of Microorganisms [KCCM] accession number KCCM11574P) which has a nucleotide sequence represented by SEQ ID NO: 1.

2. An immunogenic composition for inhibiting or treating symptoms associated with a poxvirus infection, the composition containing the attenuated vaccinia virus strain KVAC103 (accession number KCCM11574P) of claim 1 as an active ingredient.

3. The composition of claim 2, which contains at least one pharmaceutically acceptable carrier or excipient.

4. The composition of claim 2, wherein the attenuated vaccinia virus strain KVAC103 is one attenuated by subculture.

5. A method for inhibiting or treating symptoms associated with a poxvirus infection in mammals excluding humans, the method comprising a step of administering the composition of claim 2 to the mammals.

6. An immunogenic composition for inhibiting or treating symptoms associated with a poxvirus infection, the composition containing the attenuated vaccinia virus strain KVAC103 (accession number KCCM11574P) of claim 1 as an active ingredient and at least one pharmaceutically acceptable carrier or excipient.

7. The composition of claim 6, wherein the attenuated vaccinia virus strain KVAC103 is one attenuated by subculture.

8. A method for inhibiting or treating symptoms associated with a poxvirus infection in mammals excluding humans, the method comprising a step of administering the composition of claim 6 to the mammals.

9. An attenuated vaccinia virus, wherein the genome of said attenuated vaccinia virus comprises a nucleotide deletion from 15,195 to 34,632 and from 139,978 to 142,484 as compared to SEQ ID NO: 7.