1. INTRODUCTION

The invention is directed to a novel attenuated canine parvovirus (CPV) strain which may be used as a veterinary vaccine against CPV disease. The invention is further directed to a virus stock generated from a genomic DNA clone of such attenuated CPV virus for use as a veterinary vaccine, and methods for its production.

2. BACKGROUND OF THE INVENTION

During 1978 and 1979, outbreaks of previously unrecognized disease were observed in dogs in a number of countries. Identification of the causative agent, canine parvovirus (CPV) , was made when comparisons between the disease seen in dogs and those caused in cats by feline panleukopenia virus (FPV) or in minks by mink enteritis virus (MEV) , both parvoviruses, were noted. CPV has become endemic in domestic and wild dog populations around the world. Rapid global spread of the virus was most likely due to the high viral titers found in feces of infected dogs (Parrish, C.R., Adv. Virus Res. 38:403-450, 1990).

Two clinical conditions are recognized - enteric disease in dogs older than 4 to 5 months, and yocardial disease in pups 3 to 16 weeks old. CPV is responsible for serious illness and mortality in dogs, and pups less than 6 months old are particularly susceptible (Carmichael et al., Cornell Vet. 73:13-29, 1983) .

Canine parvovirus (CPV) is an autonomous parvovirus with a DNA genome of about 5,000 bases of single-stranded DNA. Two structural genes are characterized (VP-1 and VP-2) , as well as one or two non-structural (NS) genes (Parrish et al. J. Virol, 65:6544-6552, 1991).

The original strain of CPV (CPV-2) , first identified in 1978, was almost completely replaced between 1979 and 1982 by an antigenic and genetic variant, CPV-2a. A later antigenic variant, CPV-2b, emerged around 1984 and became the

- l -

predominant virus type by 1988. It has largely replaced the previous strains in the United States, such that over 90% of infected dogs now carry this strain. DNA sequence analysis shows that sequence variation in the VP1/VP2 genes gave rise to successive antigenic virus types, such that the CPV-2a strain differed in only 5 or 6 amino acids from CPV-2, while CPV-2b differs in only 2 amino acids from CPV-2a (Parrish et al. J. Virol 65:6544-6552, 1991).

Vaccines designed to elicit protection against previous strains of CPV have been developed, including live (U.S. Patent No. 4,303,645 dated Dec. 1, 1981; U.S. Patent No. 4,810,494 dated Mar. 7,1989) inactivated (U.S. Patent No. 4,193,991 dated Mar. 18, 1980), heterotypic (U.S. Eatent No. 4,193,990 dated Mar. 18, 1980) as well as reco binant subunit vaccines (U.S. Patent No. 4,971,793 dated Nov. 20, 1990; Lopez de Turiso et al., J. Virol. 66:2748-2753, 1992). Baculovirus expression of the CPV capsid genes generated empty parvoviral capsids which could be used to immunize dogs against challenge with CPV-2b (Mazzara et al., Vaccines ' 87, Cold Spring Harbor Laboratory Press, 1987, p. 419-424; Saliki et al., J. Gen. Virol. 73:369-374, 1992).

The advantage of a vaccine derived from an attenuated virus over a heterotypic, inactivated, or recombinant vaccine is that the virus is able to reproduce in the host system such that an immune response is maintained over time.

No attenuated vaccines have yet been developed which are derived from the most recent CPV-2b strain that is the most prevalent form of the virus found in infected dogs.

3. SUMMARY OF THE INVENTION

It is an object of the present invention to provide attenuated canine parvoviruses derived from serial passaging of a virulent CPV-2b isolate. Such viruses are provided herein, the DNA of which differs in nucleotide sequence from that of wild-type CPV-2b. The DNA from the attenuated strains is used for the production of infectious molecular DNA clones, which, in turn, can be transfected into cells to generate

stable master stocks of the virus. The attenuated viruses can be used in dogs as a vaccine for the prevention of CPV disease. In a preferred embodiment, the attenuated virus

VBI440 (ATCC Deposit ) is used as the vaccine.

5

4. DESCRIPTION OF THE FIGURES

FIG. 1 represents a schematic diagram of the full-length CPV genome and the subcloned fragments of the genome which were ligated to form a continuous fragment that was cloned 10 into plasmid pGEM5Z.

FIG. 2 represents a schematic diagram of the positions of the sequence changes detected in pBI440 relative to the control genome of the #5 passage, showing the 5' and 3' changes relative to coding regions and secondary structures in 15 the CPV genome.

FIG. 3 represents the DNA sequence of CPV-39 passage #60 which was cloned into pBI440.

FIG. 4 represents the DNA sequence of CPV-39 passage #5.

20 5. DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the invention, a novel attenuated strain of canine parvovirus useful as a veterinary vaccine against CPV disease has been isolated. An infectious molecular DNA clone based on the genome of the attenuated

25 strain has been produced and it may be used to generate a stable master stock of the attenuated CPV strain.

5.1. Isolation of Attenuated Canine Parvovirus

This embodiment of the present invention is directed to

30 the isolation of an attenuated strain of canine parvovirus as a vaccine to protect animals, such as wild or domestic dogs, against CPV. Attenuation of a virulent isolate of CPV is achieved by serial passaging of such virus in a suitable host cell line (see e.g., Carmichael et al., Cornell Vet. 71:408-

35 471, 1981) over time so that mutations accumulate that confer attenuation on the isolate. Serial passaging refers to the infection of a cell line with a virus isolate, the recovery of

the viral progeny from the host cells, and the subsequent infection of host cells with the viral progeny to generate the next passage. Cell lines for the passaging of CPV include Norden Laboratory feline kidney (NLFK) , mink lung cells, Madin-Darby canine kidney cells, canine A72 cells, and Crandell feline kidney (CRFK) cells.

Virulent CPV may be recovered from the feces of infected dogs and subsequently grown in tissue culture cells (Appel et al.. Vet. Rec. 105:156-159, 1979). Isolation of CPV may be performed by disruption of infected cells with sonication or cycles of freezing and thawing followed by virus purification (Parrish, Virology 183:195-205, 1991).

CPV isolates which may be used to develop an attenuated strain that is protective against CPV-2b include, but is not limited to, CPV-39 as well as other isolates known to those skilled in the art (Parrish et al., J. Virol. 65:6544-6552, 1991) .

Serial passaging of a virulent (disease-causing) strain of CPV results in the isolation of variants which are attenuated, i.e., infectious, yet not capable of causing disease. These attenuated variants are identified through testing of a passaged isolate on a suitable subject population, i.e., dogs, so that the clinical profile of the infected subjects can be ascertained. When a passaged virus is identified that infects dogs, yet is incapable of causing disease, it is characterized as attenuated. This passaged virus is then further characterized for its ability to serve as a vaccine against CPV disease, i.e., to confer protective immunity against challenge with virulent CPV. In one embodiment of the invention, a virulent CPV-2b isolate was serially passaged in NLFK cells to derive the attenuated strain. Serial passaging was performed by infecting NLFK cells with the virulent strain, incubating the infected cells for several days, collecting and then freezing and thawing the infected cells to release virus. An inoculum from the previous passage was then applied to fresh, thinly seeded NLFK cells to generate the next passage. Each passage

was similarly performed and collected. He agglutination (HA) assay of selected passages was used to identify the endpoint dilution of virus, and this dilution was used to generate the next passage. In an embodiment of the invention described, infra, various passages in the series were tested for clinical effect. Dogs from 8-35 weeks of age were inoculated with passaged virus, either by oro-nasal or subcutaneous routes. The virulence of a passaged virus, i.e., the ability to cause disease, was assessed by daily monitoring of reduced appetite, malaise, elevated temperature, vomiting/diarrhea, lymphopenia, weight loss and fecal shed in the infected dogs. Virulence was retained up to the 15th passage, while attenuation was observed at subsequent passages. The 60th passage was attenuated, as judged by the inability of this virus to elicit clinical signs of CPV disease (Table 1, infra ) (Abstract presented at the Fifth Parvovirus Workshop, Crystal River, Florida, November 10-14, 1993.) Serial backpassages of this strain in dogs did not cause reversion to virulence.

5.2. Construction of an Infectious CPV Clone from an Attenuated Strain and the Generation of an Attenuated Stock

The DNA genome of autonomous parvoviruses is able to initiate a productive infection when introduced into host cells by transfection. The infectious DNA genome of an attenuated virus is engineered into a vector for introduction into host cells for the production of progeny virus which are genetically identical to the parent attenuated isolate. The vector may be engineered to carry the viral genome using standard in vitro recombinant DNA techniques known to those skilled in the art (Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, New York, 1989) . Vectors which may be used to deliver the genome into host cells include pGEM3Z, pGEM5Z (Promega Corporation, Madison, WI) , and other similar plas id

vectors. Stable master stocks of a virus with desirable characteristics, i.e., attenuation, may be generated.

The ability to generate viral progeny through plasmid- mediated introduction of a viral genome can also be used to produce viruses with defined molecular changes. In this embodiment of the invention, stable virus stocks can be produced that contain altered DNA sequences that confer desired properties on the virus, for example, reduced virulence. This approach can also be used to assess the effect of molecular changes on various properties of the virus, i.e., antigenic type, virulence, or attenuation by introducing desired sequence changes into the viral genome, producing virus progeny from the genome, and recovering the virus progeny for characterization. In addition, this approach can be used to construct a virus with heterologous sequences inserted into the viral genome that are concurrently delivered by the virus to generate an immune response against other diseases. Such diseases include, but are not limited to, canine adenovirus, canine distemper virus, canine corona virus, and Leptospira.

Construction of viral genomes with defined molecular changes can be accomplished using standard techniques such as oligonucleotide-directed, linker-scanning or polymerase chain reaction-based mutagenesis techniques known to those skilled in the art (Zoller and Smith DNA 3:479-488, 1984; Botstein and Shortle Science 229:1193, 1985; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, New York, 1989, Chapter 8) . Ligation of the genome into a suitable vector for transfer may be accomplished through standard techniques known to those skilled in the art. Transfection of the vector into host cells for the production of viral progeny may be done using any of the standard techniques such as calcium-phosphate or DEAE- dextran mediated transfection, electroporation, protoplast fusion, and other techniques known to those skilled in the art (Sambrook et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) .

In one embodiment of the present invention, viral DNA was prepared from the attenuated strain derived from the 60th passage of CPV-2b and cloned into a plasmid for transfer into host cells by transfection. Progeny virus was produced and recovered from the transfected cells. The resulting virus stock (vBI440) was confirmed as an attenuated strain. This virus was used as a vaccine against virulent CPV-2b.

5.3. Sequence of Attenuated Canine Parvovirus The isolation of an attenuated virus may be followed by a sequence analysis of its qenome to determine the basis for the attenuated phenotype. This is accomplished by sequencing the viral DNA and identifying nucleotide changes in. the attenuated isolate relative to the genomic sequence of a control virus. Therefore, the molecular changes that confer attenuation on a virulent strain can be characterized.

In an embodiment of the invention, the sequence of the DNA genome isolated from the attenuated virus (60th passage) was determined and compared to a control genome (5th passage) . Nucleotide sequence variations between the virulent strain and the attenuated strain were identified. Four nucleotide alterations were found in the attenuated virus genome, in the 5' and 3' nontranslated regions (Table 2) .

The invention provides for attenuated CPV-2b viruses which have one or more of the following sequence alterations relative to the sequence of the control (5th passage) wild- type CPV-2b (SEQ. ID NO. 2): nucleotide at position 59 A or C or T nucleotide at position 97 A or G or T nucleotide at position 4745 A or G or C nucleotide at position 4881 A or G or T In one embodiment of the invention provided herein, the viral genome with alterations at all 4 positions (SEQ. ID. NO. 1) relative to the wild-type sequence was used to produce an attenuated virus stock. Other embodiments include the introduction of sequence changes at 1, 2 or 3 of the sites noted above in order to generate attenuated virus progeny.

Viral genomes with such alterations can be produced by any of the techniques described in Section 5.2, supra. for the introduction of nucleotide changes into cloned DNA. A genome may then be ligated into an appropriate vector for transfection into host cells (Section 5.2, infra) for the production of viral progeny.

5.4. Attenuated Canine Parvovirus as a Vaccine

The invention may be used as a vaccine to protect dogs from disease resulting from challenge with all extant strains of CPV, types 2, 2a and 2b. The vaccine may be an attenuated virus isolate, or, alternatively, the vaccine may be comprised of virus which has been generated from an infectious genomic clone of an attenuated virus. Preparation of the vaccine may be accomplished by growing large-scale stocks of the attenuated virus in tissue culture. Alternatively, the plasmid which contains the genome of the attenuated virus may be transfected into host cells to generate large-scale virus stock. The viruses may be recovered from host cells by disruption by, for example, sonication or cycles of freezing and thawing (Parrish, Virology 183:195-205, 1991).

Determination of virus yield may be performed with the use of a hemagglutination assay (Carmichael et al., Am. J. Vet. Res. 41: 784-791, 1980) or plaque assay (Chang et al., J. Virol. 66:6858-6867, 1988).

The vaccine may be comprised of an inoculum which is an aliquot from an attenuated virus stock. The vaccine may be prepared as a suspension or, alternatively, the vaccine may be lyophilized. The attenuated virus may be suspended in a pharmaceutically acceptable carrier, including but not limited to, phosphate-buffered saline.

The attenuated virus may be combined with other ingredients and antigens for the production of a vaccine. The attenuated virus may be given in combination with other antigens, including, but not limited to, canine adenovirus, canine distemper virus, canine coronavirus, or Leptospira

antigens to form a combination vaccine. Stabilizers may be added to such a vaccine, including but not limited to, gelatin, sorbitol, mannitol, glucose, sucrose, dextran, albumin, SPGA (Bovarnick, J. Bact. 59:509, 1950) or others known to those skilled in the art.

Subject populations for evaluation of a candidate vaccine include wild and domestic dogs.

Dosage of the vaccine may range from about 10 ² to about 10 ⁷ tissue culture infectious dose ₅₀ (TCID _J0 ) . A dosage greater than 10 ⁷ TCID _JO ^ma y -° ^e given. In a preferred embodiment, the dosage is 10 ⁵ TCID ₅₀ . A minimal immunizing dose (MID) may be determined by vaccinating a subject population with graded 10- fold dilutions of an attenuated virus stock, and assaying the animals for HI titer (Carmichael et al., Cornell Vet. 73:13- 29, 1983) .

The vaccine may be administered by several parenteral routes, including subcutaneously, intramuscularly, or intravenously. Repeated administration may be given, including but not limited to, yearly booster shots. The safety of the vaccine can be determined by the absence of adverse effects, i.e., evidence of illness generated by the administration of the vaccine in a test population prior to challenge with virulent virus.

The immune response generated by the vaccine can be assayed by testing sera from inoculated dogs using hemagglutination-inhibition (HI) titers (Carmichael et al.. Am. J. Vet. Res. 41: 784-791, 1980) . The development of antibodies to the attenuated virus correlates with an increase in HI titer, and the generation of this protective response is defined as vaccination. Protection can be correlated with the development of an HI titer that exceeds 1:40.

The efficacy of the vaccine can be determined by the ability of the vaccine to confer resistance on a subject population when challenged with virulent CPV. Challenge may performed when an interval has elapsed after vaccination, for example, from 14-20 days. Duration of immunity afforded by the vaccine may be determined by periodic assessment of HI

titers in vaccinated animals or by challenge with virulent CPV and observation of clinical signs of CPV disease.

In one embodiment of the invention, virus derived from the infectious molecular clone of the attenuated strain (vBI440) was used to vaccinate pups (Section 9, infra) . After challenge with virulent CPV, the vaccinated dogs did not evidence signs of disease. Hemagglutination-inhibition (HI) titers of the vaccinated animals showed the development of a serological response to the attenuated virus vaccine.

6. EXAMPLE: ISOLATION OF ATTENUATED CANINE PARVOVIRUS STRAIN 6.1. Methods

To produce a virus strain capable of eliciting, protection against virulent CPV-2b, a CPV-2b isolate (CPV-39) was obtained from the Texas Veterinary Medical Diagnostic

Laboratory (accession number C84176071, No. 2) . This isolate was confirmed as a CPV-2b isolate by typing against a panel of monoclonal antibodies (Parrish et al., J. Virol. 65:6544-6552, 1991) . The virus was originally passaged five times in NLFK cells to establish a stock virus preparation.

The NLFK host cells used in these experiments were grown in a mixture of 50% McCoy's 5a and 50% Leibovitz L15 media with 5% fetal bovine serum at 37°C.

CPV-39 was serially passaged in NLFK cells. Serial passaging was performed by infecting NLFK cells with CPV-39, incubating the infected cells for between 5-7 days, and then freezing and thawing the infected cells to release viral progeny. Virus from the cell lysate (1-2 ml) was then applied to fresh, thinly seeded NLFK cells to generate the subsequent passage in the series. Each passage was similarly performed and collected.

At various passages (pass 3, 15, 20, 25, 35, 44, 50 and 60) the virus-containing materials were diluted in a 10-fold series in tissue culture medium, and the dilutions inoculated onto thinly seeded NLFK cells. After incubation for 4-5 days, the cultures were frozen and thawed, and the medium tested for virus hemagglutination of rhesus macaque erythrocytes (HA

assay) . The subsequent passage in each case was made using the culture inoculated with the endpoint dilution of the virus as determined in the HA assay.

The HA assay was performed in a icrotiter plate by incubating 0.025 ml of a virus dilution per well with 0.025 ml of barbitol-buffered saline (BBS) to which is added 0.050 ml of a 0.5% v/v suspension of rhesus erythrocytes in BBS/bovine serum albumin (BSA) . The plate was shaken to mix and placed at 4°C for the cells to settle. The HA titer is read as the last well containing >50% agglutinated cells.

Various passages in the series were tested for the ability to induce disease in dogs. In each test between 2 and 5 specific-pathogen-free (SPF) beagle dogs, aged between 8-35 weeks, were inoculated with 10 ⁵⁵ to 10 ⁶⁵ tissue culture infectious dose-50 (TCID ₅₀ ) units of passaged virus, either by oro-nasal (ON) or subcutaneous (SC) routes. Virulence was judged by reduced appetite, malaise, elevated temperature, vomiting/diarrhea and weight loss, while attenuation was judged by the reduction or absence of these symptoms within 10 days post-infection.

6.2. Results

Virulence was retained up to the 15th passage (#15) , was greatly reduced by the 20th passage (#20) , while the 60th passage (#60) caused no clinical symptoms in susceptible puppies (Table 1) , and was characterized as attenuated. Non- inoculated control dogs remained clinically normal.

7. EXAMPLE: PRODUCTION OF VIRUS FROM

MOLECULAR CLONE OF ATTENUATED STRAIN

7.1. Methods

Stock virus was prepared from the 60th passage by infecting thinly seeded NLFK cells. After culturing the virus-infected cells for 36 hours, the cells were lysed and the viral replicative form (RF) DNA (double-stranded) was recovered using a modification of the Hirt procedure for the isolation of low molecular weight DNA (Parrish et al., Amer.

J. Vet. Res. 45:2591-2599, 1984). The recovered DNA was purified by preparative agarose gel electrophoresis (Ausubel et al.. Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, New York, 1989) , and the viral RF DNA was recovered from the gel by electroelution (Ausubel et al., supra) .

The viral DNA genome was subcloned as shown in FIG. 1 by restriction and assembled into a single continuous sequence, then ligated into plasmid pGEM5Z to derive plasmid pBI440. 10 μg of this plasmid was electroporated into NLFK cells (at 220V/330uF) to initiate a virus infection from the genome. Virus generated by the transfection of the infectious genomic clone was recovered from the NLFK cells by .freezing and thawing. The resulting virus (vBI440) was tested against a panel of type-specific monoclonal antibodies by hemagglutination-inhibition (HI) assay (see Section 9, infra) and the derivation of this virus from the original CPV-39 was confirmed (Parrish et al., J. Virol. 65: 6544-6552, 1991). Eight 9 week-old beagle pups were inoculated subcutaneously with 10 ^s5 to 10 ⁶⁵ tissue culture infectious dose- 50 (TCID _JO ) units of the VBI440 strain to determine if the attenuated phenotype of the parent virus (#60 passage) was retained.

7.2. Results

Clinical analysis of the pups inoculated with VBI440 (#60-cloned strain) revealed that this virus did not cause any signs of CPV disease (Table 1) , as judged by the absence of CPV-like symptoms, and therefore could be characterized as attenuated. Serial backpassages of this strain in dogs did not cause illness.

The virus derived from passage #60 in the series was passaged for another 15 passages in NLFK cells, at which time a non-hemagglutinating variant emerged. In contrast, 15 passages of vBI440 in NLFK cells showed no apparent phenotypic variation, illustrating the stability of the virus derived from the genome of the attenuated strain.

Table 1

Clinical Responses of Pups Inoculated by the Oral-Nasal (On) or Subcutaneous (Sc) Route with Different Passage Levels of CPV-2b and Infectious Clone of Passage #60

O to

—I

σ>

I

ft?

Number of dogs/total inoculated at this passage with temperature > 103°F.

Lymphopenia - lymphocyte counts <50% of pre-inoculation value. Total WBC counts done by Coulter counter. No dog had a panleukopenia.

Weight loss = failure to gain normal weight or actual weight loss between tine of infection and 2 weeks later.

Fecal hemagglutination titer >2048 for at least 2 days.

8. EXAMPLE: SEQUENCE ANALYSIS OF CLONED ATTENUATED VIRUS

8.1. Methods

Plasmid pBI440, containing the viral DNA of passage #60 cloned into pGEM5Z, was subcloned into M13 phage vectors mpl8 and mpl9 and the DNA was sequenced by the dideoxy method (Ausubel et al.. Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, New York, 1989) using primers as described previously (Parrish et al., 1991, Virology 183:195-205) . DNA prepared from the CPV-39 5th passage (#5) was also cloned into pGEM5Z and pGEM3Z and sequenced as a comparative control.

8.2. Results

The sequence of the attenuated virus genome derived from passage #60 is given in FIG. 3 (SEQ. ID. NO. 1) . The sequence of the control virus used for sequence comparison (passage #5) is given in FIG. 4 (SEQ. ID. NO. 2). Comparative differences between these genomes are shown in Table 2. Nucleotide changes at 4 positions in pBI440 (passage #60) relative to that observed in the control plasmid (passage #5) were identified. Two changes at the 5' end, at positions 4745 and 4881, occurred in the non-coding region. At the 3' end, nucleotide changes at positions 59 and 97 were also in the non-coding region, and within the hairpin potentially formed by the palindromic sequence at that terminus (FIG. 2) .

9. EXAMPLE: VACCINATION WITH ATTENUATED VBI440

9.1. Methods

Five SPF 12.5 week-old beagle pups were vaccinated by the subcutaneous route with 1 dose (10 ^s TCID _J0 /ml) of VBI440. Two control pups were kept in isolation until challenge with virulent CPV-39. Challenge was performed at 20 days after vaccination and consisted of inoculation with 10 ⁶² TCID _S0 units in 3 ml of inoculum.

Pre-inoculation blood samples were taken for leukocyte counts and at intervals for 8 days post-challenge. Dogs were observed twice daily for signs of illness, including rectal temperatures. Fecal samples were also collected during the same time period and pooled samples from each isolation unit were prepared as a 10% volume suspension in phosphate-buffered saline and then tested for virulent viral shed using an HA assay.

Blood for serological testing was obtained on post- vaccination days 0, 7 and 21 and 10 days after challenge with virulent CPV-2b. He agglutination-inhibition (HI) titers of the vaccinated animals were performed by collecting animal sera and diluting 1:5 in BBS, then heat inactivating at 56°C for 30 minutes. To the sera, 0.010 ml 50% packed red blood cells were added, and the mixture was allowed to stand for 1 hour at 4°C. In a microtiter well, 0.025 ml BBS, and serial dilutions of heat-inactivated sera, beginning at 0.025 ml, were added, followed by 0.025 ml of diluted antigen. Incubation of the plates for 1 hour at room temperature was followed by the addition of 0.050 ml 0.5% RBC suspension. An antigen control was set up by setting up a parallel plate without sera. All plates were shook and placed at 4°C for 4-

16 hours. The HI titer is the reciprocal of the highest serum dilution that completely prevents hemagglutination.

9.2. Results The vaccine, vBI440, did not cause symptoms of illness, as indicated by the normal temperature observed in vaccinated animals 1411-1415 (Table 3A, column days post-inoculation

(DPI) : -1, 0) as well as normal leukocyte values (Table 3A, column DPI: 0) .

For 9 days following challenge with virulent CPV-39, all vaccinated animals (1411-1415) maintained normal temperature and normal blood values (WBC and Ly/PMN) (Table 3A, column DPI: 1-9). Fecal HA titers indicated no shed of virulent virus in the vaccinated animals. The vaccination with VBI440 offered protection against challenge with CPV-39, while non- vaccinated animals were susceptible to challenge. In contrast, the non-vaccinated animals (1418, 1419) showed elevated temperatures by day 5 in the course of a 9-day period post-challenge, as well as a marked lymphopenia on days 4-7 (Table 3B, column Ly/PMN: 4-7) and leukopenia OQ day 6 (column WBC: 6) . Fecal shed of virulent virus was observed on days 4 and 5, as determined from an HA assay.

The vaccine VBI440 elicited serological protection for the 5 vaccinated pups challenged with virulent CPV-39, as evidenced by the HI antibody titers in Table 4A. For vaccinated animals 1411-1415, HI titers had increased by 1 week post-vaccination relative to pre-inoculation levels (Table 4A, 1 wk. PI) , further increasing by 3 weeks post- vaccination (Table 4A, 3 wk. PI) , indicating that the antibodies to VBI440 had developed which afforded protection against the subsequent challenge with CPV-39. In contrast, in non-vaccinated animals 1418 and 1419, no significant HI titers were detected prior to challenge (Table 4B, prechallenge) . Following challenge, non-vaccinated animals evidenced the development of antibodies to CPV after the course of disease had abated, as indicated by the increased HI titers observed at 10 and 21 days post-challenge (Table 4B) .

Table 3

CLINICAL RESPONSES TO VACCINATION WITH VBI 40 AND CHALLENGE WITH VIRULENT CPV-2b

FECAL HA: Titers were all less than 1:32 (generally 1:8-1:16) before and after challenge, indicating no shed of virulent (challenge) virus.

SPF controls challenged with CPV-2b (passage 5 ₎ ;

Post- 1418-C 1419- _C challenge day

Table 4

SEROLOGICAL RESPONSES TO VACCINATION WITH VBI440

A. HI Antibody Titers (CPV-2b antigen) :

1411 1415 Pre-inoc. <10 <10

CD

1 wk. PI 2560 2560

3 wk. PI (pre-challenge) 5120 10,240 5120 10,240 10,240 m I 10 days post-challenge 5120 10,240 2560 2560 2560

B. Serology (HI antibody titer)

1418-C 1419-C

Prechallenge: <10 <10

10 days 5120 5120

21 days 10,240 5120

10. DEPOSIT OF MICROORGANISMS

The following microorganisms were deposited with the American Type Culture Collection (ATCC) , Rockville, MD, on October 28, 1994, and have been assigned the following accession numbers:

Virus Accession Numbers

CPV-39 passage #60 ATCC Vft Z- ^

VBI440 ATCC \ ( Z. <iϋ

CPV-39 passage #5 ATCC yfl 27<7/

Plasmid pBI440 ATCC *738

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosure of which are incorporated by reference in their entireties.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Parrish, Colin R. Gruenberg, Allen Carmichael, Leland E.

(ii) TITLE OF INVENTION: Attenuated Canine Parvovirus Vaccine

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pennie & Edmonds

(B) STREET: 1155 Avenue of the Americas

(C) CITY: New York

(D) STATE: NY

(F) ZIP: 10036-2711

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Gordon, Jennifer

(B) REGISTRATION NUMBER: 30753

(C) REFERENCE/DOCKET NUMBER: 7937-006

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 790-9090

(B) TELEFAX: (212) 869-8864/9741

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5049 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Parvovirus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATCATTCTTT AGAACCAACT GACCAAGTTC ACGTACGTAT GACGTGATGA CGCGCGCTAC 60

GCGCGCTGCC TACGGCAGTC ACACGTCATA CGTACGTTCC TTGGTCAGTT GGTTCTAAAG 120

AATGATAGGC GGTTTGTGTG TTTAAACTTG GGCGGGAAAA GGTGGCGGGC TAATTGTGGG 180

CGTGGTTAAA GGTATAAAAG ACAAACCATA GACCGTTACT GACATTCGCT TCTTGTCTTT 240 GACAGAGTGA ACCTCTCTTA CTTTGACTAA CCATGTCTGG CAACCAGTAT ACTGAGGAAG 300 TTATGGAGGG AGTAAATTGG TTAAAGAAAC ATGCAGAAAA TGAAGCATTT TCGTTTGTTT 360 TTAAATGTGA CAACGTCCAA CTAAATGGAA AGGATGTTCG CTGGAACAAC TATACCAAAC 420 CAATTCAAAA TGAAGAGCTA ACATCTTTAA TTAGAGGAGC ACAAACAGCA ATGGATCAAA 480 CCGAAGAAGA AGAAATGGAC TGGGAATCGG AAGTTGATAG TCTCGCCAAA AAGCAAGTAC 540 AAACTTTTGA TGCATTAATT AAAAAATGTC TTTTTGAAGT CTTTGTTTCT AAAAATATAG 600 AACCAAATGA ATGTGTTTGG TTTATTCAAC ATGAATGGGG AAAAGATCAA GGCTGGCATT 660 GTCATGTTTT ACTTCATAGT AAGAACTTAC AACAAGCAAC TGGTAAATGG CTACGCAGAC 720 AAATGAATAT GTATTGGAGT AGATGGTTGG TGACTCTTTG TTCGGTAAAC TTAACACCAA 780 CTGAAAAGAT TAAGCTCAGA GAAATTGCAG AAGATAGTGA ATGGGTGACT ATATTAACAT 840 ACAGACATAA GCAAACAAAA AAAGACTATG TTAAAATGGT TCATTTTGGA AATATGATAG 900 CATATTACTT TTTAACAAAG AAAAAAATTG TCCACATGAC AAAAGAAAGT GGCTATTTTT 960 TAAGTACTGA TTCTGGTTGG AAATTTAACT TTATGAAGTA TCAAGACAGA CAAATTGTCA 1020 GCACACTTTA CACTGAACAA ATGAAACCAG AAACCGTTGA AACCACAGTG ACGACAGCAC 1080 AGGAAACAAA GCGCGGGAGA ATTCAAACTA AAAAGGAAGT GTCAATCAAA TGTACTTTGC 1140 GGGACTTGGT TAGTAAAAGA GTAACATCAC CTGAAGACTG GATGATGTTA CAACCAGATA 1200 GTTATATTGA AATGATGGCA CAACCAGGAG GTGAAAATCT TTTAAAAAAT ACACTTGAAA 1260 TTTGTACTTT GACTTTAGCA AGAACAAAAA CAGCATTTGA ATTAATACTT GAAAAAGCAG 1320 ATAATACTAA ACTAACTAAC TTTGATCTTG CAAATTCTAG AACATGTCAA ATTTTTAGAA 1380 TGCACGGATG GAATTGGATT AAAGTTTGTC ACGCTATAGC ATGTGTTTTA AATAGACAAG 1440 GTGGTAAAAG AAATACAGTT CTTTTTCATG GACCAGCAAG TACAGGAAAA TCTATCATTG 1500 CTCAAGCCAT AGCACAAGCT GTGGGTAATG TTGGTTGTTA TAATGCAGCA AATGTAAATT 1560 TTCCATTTAA TGACTGTACC AATAAAAATT TAATTTGGAT TGAAGAAGCT GGTAACTTTG 1620 GTCAACAAGT TAATCAATTT AAAGCAATCT GTTCTGGACA AACAATTAGA ATTGATCAAA 1680 AAGGTAAAGG AAGTAAGCAA ATTGAACCAA CTCCAGTAAT TATGACAACT AATGAAAATA 1740 TAACAATTGT GAGAATTGGA TGTGAAGAAA GACCTGAACA TACACAACCA ATAAGAGACA 1800 GAATGTTGAA CATTAAGTTA GTATGTAAGC TTCCAGGAGA CTTTGGTTTG GTTGATAAAG 1860 AAGAATGGCC TTTAATATGT GCATGGTTAG TTAAACATGG TTTTGAATCA ACCATGGCTA 1920 ACTATACACA TCATTGGGGA AAAGTACCAG AATGGGATGA AAACTGGGCG GAGCCTAAAA 1980 TACAAGAAGG TATAAATTCA CCAGGTTGCA AAGACTTAGA GACACAAGCG GCAAGCAATC 2040 CTCAGAGTCA AGACCAAGTT CTAACTCCTC TGACTCCGGA CGTAGTGGAC CTTGCACTGG 2100 AACCGTGGAG TACTCCAGAT ACGCCTATTG CAGAAACTGC AAATCAACAA TCAAACCAAC 2160 TTGGCGTTAC TCACAAAGAC GTGCAAGCGA GTCCGACGTG GTCCGAAATA GAGGCAGACC 2220

TGλGAGCCAT CTTTACTTCT GAACAATTGG AAGAAGATTT TCGAGACGAC TTGGATTAAG 2280

GTACGATGGC λCCTCCGGCA AAGAGAGCCA GGAGAGGTAA GGGTGTGTTA GTAAAGTGGG 2340

GGGAGGGGAA AGλTTTAλTλ λCTTλλCTλλ GTλTGTGTTT TTTTλTλGGA CTTGTGCCTC 2400

CAGGTTATAA λTλTCTTGGG CCTGGGλλCλ GTCTTGACCA λGGλGAACCA ACTAACCCTT 2460

CTGACGCCGC TGCAAAAGAA CACGACGAAG CTTACGCTGC TTλTCTTCGC TCTGGTλλAA 2520

ACCCATACTT ATATTTCTCG CCAGCAGATC AACGCTTTAT AGATCAAACT AAGGACGCTA 2580

AAGATTGGGG GGGGAAAATA GGACATTATT TTTTTAGAGC TAλλAAGGCA ATTGCTCCAG 2640

TATTAACTGA TACACCAGAT CATCCATCAA CλTCAAGACC AACAAAACCA ACTAAAAGAA 2700

GTAAACCACC ACCTCATATT TTCATCAATC TTGCAAAAAA AAAAAAAGCC GGTGCAGGAC 2760

AAGTAAAAAG AGACAATCTT GCACCAATGA GTGATGGAGC AGTTCAACCA GACGGTGGTC 2820

AACCTGCTGT CAGAAATGAA AGAGCTACAG GATCTGGGAλ CGGGTCTGGA GGCGGGGGTG 2880

GTGGTGGTTC TGGGGGTGTG GGGATTTCTλ CGGGTλCTTT CAATAATCAG ACGGAATTTA 2940

AλTTTTTGGλ AAACGGATGG GTGGAAATCA CAGCAAACTC AAGCAGACTT GTACATTTAA 3000

ATATGCCAGA AAGTGAAAAT TATAGAAGAG TGGTTGTAAA TAATTTGGAT AAAACTGCAG 3060

TTAACGGAAA CATGGCTTTA GATGATACTC ATGCACAAAT TGTAACACCT TGGTCATTGG 3120

TTGATGCAAA TGCTTGGGGA GTTTGGTTTA ATCCAGGAGA TTGGCAACTA ATTGTTAATA 3180

CTATGAGTGA GTTGCATTTA GTTAGTTTTG AACAAGAAAT TTTTAATGTT GTTTTAAAGA 3240

CTGTTTCAGA ATCTGCTACT CAGCCACCAA CTAAAGTTTA TAATAATGAT TTAACTGCAT 3300

CATTGATGGT TGCATTAGAT AGTAATAATA CTATGCCATT TACTCCAGCA GCTATGAGAT 3360

CTGAGACATT GGGTTTTTAT CCATGGAAAC CAACCATACC AACTCCATGG AGATATTATT 3420

TTCAATGGGA TAGAACATTA ATACCATCTC ATACTGGAAC TAGTGGCACA CCAACAAATA 3480

TATACCATGG TACAGATCCA GATGATGTTC AATTTTATAC TATTGAAAAT TCTGTGCCAG 3540

TACACTTACT AAGAACAGGT GATGAATTTG CTACAGGAAC ATTTTTTTTT GATTGTAAAC 3600

CATGTAGACT AACACATACA TGGCAAACAA ATAGAGCATT GGGCTTACCA CCATTTCTAA 3660

ATTCTTTGCC TCAATCTGAA GGAGGTACTA ACTTTGGTTA TATAGGAGTT CAACAAGATA 3720

AAAGACGTGG TGTAACTCAA ATGGGAAATA CAAACTATAT TACTGAAGCT ACTATTATGA 3780

GACCAGCTGA GGTTGGTTAT AGTGCACCAT ATTATTCTTT TGAGGCGTCT ACACAAGGGC 3840

CATTTAAAAC ACCTATTGCA GCAGGACGGG GGGGAGCGCA AACAGATGAA AATCAAGCAG 3900

CAGATGGTGA TCCAAGATAT GCATTTGGTA GACAACATGG TCAAAAAACT ACCACAACAG 3960

GAGAAACACC TGAGAGATTT ACATATATAG CACATCAAGA TACAGGAAGA TATCCAGAAG 4020

GAGATTGGAT TCAAAATATT AACTTTAACC TTCCTGTAAC AGATGATAAT GTATTGCTAC 4080

CAACAGATCC AATTGGAGGT AAAACAGGAA TTAACTATAC TAATATATTT AATACTTATG 4140

GTCCTTTAAC TGCATTAAAT AATGTACCAC CAGTTTATCC AAATGGTCAA ATTTGGGATA 4200

AAGAATTTGA TACTGACTTA AAACCAAGAC TTCATGTAAA TGCACCATTT GTTTGTCAAA 4260

ATAATTGTCC TGGTCAATTA TTTGTAAAAG TTGCGCCTAA TTTAACAAAT GAATATGATC 4320

CTGATGCATC TGCTAATATG TCAAGAATTG TAACTTACTC AGATTTTTGG TGGAAAGGTA 4380

AATTAGTATT TAAAGCTAAA CTAAGAGCCT CTCATACTTG GAATCCAATT CAACAAATGA 4440

GTATTAATGT AGATAACCAA TTTAACTATG TACCAAGTAA TATTGGΛGGT ATGAAAATTG 4500

TATATGAAAA ATCTCAACTA GCACCTAGAA AATTATATTA ACATACTTAC TATGTTTTTA 4560

TGTTTATTAC ATATCAACTA GCACCTAGAA AATTATATTA ATATACTTAC TATGTTTTTA 4620

TGTTTATTAC ATATTATTTT AAGATTAATT AAATTACAGC ATAGAAATAT TGTACTTGTA 4680

TTTGATATAG GATTTAGAAG GTTTGTTATA TGGTATACAA TAACTGTAAG AAATAGAAGA 4740

ACATCTAGAT CATAGTTAGT AGTTTGTTTT ATAAAATGTA TTGTAAACCA TTAATGTATG 4800

TTGTTATGGT GTGGGTGGTT GGTTGGTTTG CCCTTAGAAT ATGTTAAGGA CCAAAAAAAA 4860

TCAATAAAAG ACATTTAAAA TTAAATGGCC TCGTATACTG TCTATAAGGT GAACTAACCT 4920

TACCATAAGT ATCAATCTGT CTTTAAGGGG GGGGTGGGTG GGAGATGCAC AACATCAGTA 4980

GACTGACTGG CCTGGTTGGT TGCTCTGCTT AATCAACCAG ACCGCGTAGC GGTCTGGTTG 5040

ATTAAGCGC 5049 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5049 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Parvovirus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

ATCATTCTTT AGAACCAACT GACCAAGTTC ACGTACGTAT GACGTGATGA CGCGCGCTGC 60

GCGCGCTGCC TACGGCAGTC ACACGTCATA CGTACGCTCC TTGGTCAGTT GGTTCTAAAG 120

AATGATAGGC GGTTTGTGTG TTTAAACTTG GGCGGGAAAA GGTGGCGGGC TAATTGTGGG 180

CGTGGTTAAA GGTATAAAAG ACAAACCATA GACCGTTACT GACATTCGCT TCTTGTCTTT 240

GACAGAGTGA ACCTCTCTTA CTTTGACTAA CCATGTCTGG CAACCAGTAT ACTGAGGAAG 300

TTATGGAGGG AGTAAATTGG TTAAAGAAAC ATGCAGAAAA TGAAGCATTT TCGTTTGTTT 360

TTAAATGTGA CAACGTCCAA CTAAATGGAA AGGATGTTCG CTGGAACAAC TATACCAAAC 420

CAATTCAAAA TGAAGAGCTA ACATCTTTAA TTAGAGGAGC ACAAACAGCA ATGGATCAAA 480

CCGAAGAAGA AGAAATGGAC TGGGAATCGG AAGTTGATAG TCTCGCCAAA AAGCAAGTAC 540

AAACTTTTGA TGCATTAATT AAAAAATGTC TTTTTGAAGT CTTTGTTTCT AAAAATATAG 600

AACCAAATGA ATGTGTTTGG TTTATTCAAC ATGAATGGGG AAAAGATCAA GGCTGGCATT 660

GTCATGTTTT ACTTCATAGT AAGAACTTAC AACAAGCAAC TGGTAAATGG CTACGCAGAC 720

AAATGAATAT GTATTGGAGT AGATGGTTGG TGACTCTTTG TTCGGTAAAC TTAACACCAA 780

CTGAAAAGAT TAAGCTCAGA GAAATTGCAG AAGATAGTGA ATGGGTGACT ATATTAACAT 840

ACAGACATAA GCAAACAAAA AAAGACTATG TTAAAATGGT TCATTTTGGA AATATGATAG 900

CATATTACTT TTTAACAAAG AAAAAAATTG TCCACATGAC AAAAGAAAGT GGCTATTTTT 960

TAAGTACTGA TTCTGGTTGG AAATTTAACT TTATGAAGTA TCAAGACAGA CAAATTGTCA 1020

GCACACTTTA CACTGAACAA ATGAAACCAG AAACCGTTGA AACCACAGTG ACGACAGCAC 1080

AGGAAACAAA GCGCGGGAGA ATTCAAACTA AAAAGGAAGT GTCAATCAAA TGTACTTTGC 1140

GGGACTTGGT TAGTAAAAGA GTAACATCAC CTGAAGACTG GATGATGTTA CAACCAGATA 1200

GTTATATTGA AATGATGGCA CAACCAGGAG GTGAAAATCT TTTAAAAAAT ACACTTGAAA 1260

TTTGTACTTT GACTTTAGCA AGAACAAAAA CAGCATTTGA ATTAATACTT GAAAAAGCAG 1320

ATAATACTAA ACTAACTAAC TTTGATCTTG CAAATTCTAG AACATGTCAA ATTTTTAGAA 1380

TGCACGGATG GAATTGGATT AAAGTTTGTC ACGCTATAGC ATGTGTTTTA AATAGACAAG 1440

GTGGTAAAAG AAATACAGTT CTTTTTCATG GACCAGCAAG TACAGGAAAA TCTATCATTG 1500

CTCAAGCCAT AGCACAAGCT GTGGGTAATG TTGGTTGTTA TAATGCAGCA AATGTAAATT 1560

TTCCATTTAA TGACTGTACC AATAAAAATT TAATTTGGAT TGAAGAAGCT GGTAACTTTG 1620

GTCAACAAGT TAATCAATTT AAAGCAATCT GTTCTGGACA AACAATTAGA ATTGATCAAA 1680

AAGGTAAAGG AAGTAAGCAA ATTGAACCAA CTCCAGTAAT TATGACAACT AATGAAAATA 1740

TAACAATTGT GAGAATTGGA TGTGAAGAAA GACCTGAACA TACACAACCA ATAAGAGACA 1800

GAATGTTGAA CATTAAGTTA GTATGTAAGC TTCCAGGAGA CTTTGGTTTG GTTGATAAAG 1860

AAGAATGGCC TTTAATATGT GCATGGTTAG TTAAACATGG TTTTGAATCA ACCATGGCTA 1920

ACTATACACA TCATTGGGGA AAAGTACCAG AATGGGATGA AAACTGGGCG GAGCCTAAAA 1980

TACAAGAAGG TATAAATTCA CCAGGTTGCA AAGACTTAGA GACACAAGCG GCAAGCAATC 2040

CTCAGAGTCA AGACCAAGTT CTAACTCCTC TGACTCCGGA CGTAGTGGAC CTTGCACTGG 2100

AACCGTGGAG TACTCCAGAT ACGCCTATTG CAGAAACTGC AAATCAACAA TCAAACCAAC 2160

TTGGCGTTAC TCACAAAGAC GTGCAAGCGA GTCCGACGTG GTCCGAAATA GAGGCAGACC 2220

TGAGAGCCAT CTTTACTTCT GAACAATTGG AAGAAGATTT TCGAGACGAC TTGGATTAAG 2280

GTACGATGGC ACCTCCGGCA AAGAGAGCCA GGAGAGGTAA GGGTGTGTTA GTAAAGTGGG 2340

GGGAGGGGAA AGATTTAATA ACTTAACTAA GTATGTGTTT TTTTATAGGA CTTGTGCCTC 2400

CAGGTTATAA ATATCTTGGG CCTGGGAACA GTCTTGACCA AGGAGAACCA ACTAACCCTT 2460

CTGACGCCGC TGCAAAAGAA CACGACGAAG CTTACGCTGC TTATCTTCGC TCTGGTAAAA 2520

ACCCATACTT ATATTTCTCG CCAGCAGATC AACGCTTTAT AGATCAAACT AAGGACGCTA 2580

AAGATTGGGG GGGGAAAATA GGACATTATT TTTTTAGAGC TAAAAAGGCA ATTGCTCCAG 2640

-25-

TATTAACTGλ TACACCAGAT CATCCATCAA CATCAAGACC AACAAAACCA ACTAAAAGAA 2700

GTAAACCACC ACCTCATATT TTCATCAATC TTGCAAAAAA AAλλAAAGCC GGTGCAGGλC 2760

AAGTAAAAAG AGACAATCTT GCACCAATGA GTGATGGAGC AGTTCAACCA GACGGTGGTC 2820 λλCCTGCTGT CAGAAATGAA AGAGCTACAG GATCTGGGλλ CGGGTCTGGA GGCGGGGGTG 2880

GTGGTGGTTC TGGGGGTGTG GGGATTTCTλ CGGGTACTTT CAATAATCAG ACGGAATTTA 2940

AATTTTTGGA AAACGGATGG GTGGAAATCA CAGCAAACTC AAGCAGACTT GTACATTTAA 3000

ATATGCCAGA AAGTGAAAAT TATAGAAGAG TGGTTGTAAA TAATTTGGAT AλAACTGCAG 3060

TTAλCGGλAA CλTGGCTTTA GATGATACTC ATGCACAAAT TGTλλCACCT TGGTCATTGG 3120

TTGATGCAAA TGCTTGGGGλ GTTTGGTTTA ATCCAGGAGA TTGGCAACTA ATTGTTAATλ 3180

CTλTGAGTGA GTTGCλTTTλ GTTAGTTTTG AλCAAGAAAT TTTTAATGTT GTTTTAAAGA 3240

CTGTTTCAGλ ATCTGCTACT CAGCCACCAA CTAAAGTTTA TAATAATGAT TTAACTGCAT 3300

CATTGATGGT TGCλTTAGAT AGTAATAATA CTATGCCATT TACTCCAGCA GCTATGAGAT 3360

CTGAGACATT GGGTTTTTAT CCATGGλAAC CAACCATACC AACTCCATGG AGATATTATT 3420

TTCλATGGGA TλGAACλTTA ATACCATCTC ATACTGGAAC TAGTGGCACA CCAACAAATA 3480

TATACCλTGG TACλGATCCA GλTGATGTTC AATTTTATAC TATTGAAAAT TCTGTGCCAG 3540

TλCACTTλCT AAGAACAGGT GATGAATTTG CTACAGGAAC ATTTTTTTTT GATTGTAAAC 3600

CATGTAGACT AACACATACA TGGCAAACAA ATAGAGCATT GGGCTTACCA CCATTTCTAA 3660

ATTCTTTGCC TCAλTCTGAA GGAGGTACTA ACTTTGGTTA TATAGGAGTT CAACAAGATA 3720

AAAGACGTGG TGTAACTCAA ATGGGAAATA CAAACTATAT TACTGAAGCT ACTATTATGA 3780

GACCAGCTGA GGTTGGTTAT AGTGCACCAT ATTATTCTTT TGAGGCGTCT ACACAAGGGC 3840

CATTTAAAAC ACCTATTGCA GCAGGACGGG GGGGAGCGCA AACAGATGAA AATCAAGCAG 3900

CAGATGGTGλ TCCAAGATAT GCATTTGGTA GACAACATGG TCAAAAAACT ACCACAACAG 3960

GAGAAACACC TGAGAGATTT ACATATATAG CACATCAAGA TACAGGAAGA TATCCAGAAG 4020

GAGATTGGAT TCAAAATATT AACTTTAACC TTCCTGTAAC AGATGATAAT GTATTGCTAC 4080

CAACAGλTCC AATTGGAGGT AAAACAGGAA TTAACTATAC TAATATATTT AATACTTATG 4140

GTCCTTTAλC TGCATTAAAT AATGTACCAC CAGTTTATCC AAATGGTCAA ATTTGGGATA 4200

AAGAATTTGA TACTGACTTA AAACCAAGAC TTCATGTAAA TGCACCATTT GTTTGTCAAA 4260

ATAATTGTCC TGGTCAATTA TTTGTAAAAG TTGCGCCTAA TTTAACAAAT GAATATGATC 4320

CTGATGCATC TGCTAATATG TCAAGAATTG TAACTTACTC AGATTTTTGG TGGAAAGGTA 4380

AATTAGTATT TAAAGCTAAA CTAAGAGCCT CTCATACTTG GAATCCAATT CAACAAATGA 4440

GTATTAATGT AGλTAACCAA TTTAACTATG TACCAAGTAA TATTGGAGGT ATGAAAATTG 4500

TATλTGAAAA ATCTCAACTA GCACCTAGAA AATTATATTA ACATACTTAC TATGTTTTTA 4560

TGTTTATTAC ATλTCAACTA GCACCTAGAA AATTATATTA ATATACTTAC TATGTTTTTA 4620

TGTTTATTAC ATATTATTTT AAGATTAATT AAATTACAGC ATAGAAATAT TGTACTTGTA 4680

TTTGATλTλG GATTTAGAAG GTTTGTTATA TGGTATACAA TAACTGTAAG AAATAGAAGA 4740

ACλTTTAGλT CλTλGTTλGT λGTTTGTTTT λTλλAATGTλ TTGTλλACCA TTλλTGTλTG 4800

TTGTTλTGGT GTGGGTGGTT GGTTGGTTTG CCCTTAGAAT ATGTTAAGGA CCAAAAλλλA 4860

TCAATAAAAG ACATTTAAAA CTλλλTGGCC TCGTATλCTG TCTATλλGGT GAACTAACCT 4920

TACCATAAGT ATCAATCTGT CTTTAAGGGG GGGGTGGGTG GGAGATGCAC AACATCAGTA 4980

GACTGACTGG CCTGGTTGGT TGCTCTGCTT AATCAACCAG λCCGCGTλGC GGTCTGGTTG 5040

ATTAAGCGC 5049

International Application No: PCT/ /

MICROORGANISMS

Optional Sheet in connection with the microorganism referred to on page 20, lines 1-2 ₂ of the description '

A. IDENTIFICATION OF DEPOSIT '

Further deposits are identified on an additional sheet

Name of depositary institution ' American Type Culture Collection

Address of depositary institution (including postal code and country) '

12301 Par lawn Drive Rockville. MD 20852 US

Date of deposi ' October 28. 1994 Accession Number ' VR 2489

B. ADDITIONAL INDICATIONS ^• (leave blank if not applicable). This informal ion u continual on a ιe-nraιe attached sheet

C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ' a

D. SEPARATE FURNISHING OF INDICATIONS ' (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later ' (Specify the general nature of the indications e g , "Accession Number of Deposit")

E. E This sheet was received with the International application when filed (to be checked by the receiving Office)

(Authori£zed O&fficer)**_»-* ^■

D The date of receipt (from the applicant) by the International Bureau "

(Authorized Officer) Form PCT/RO/134 (January 1981 )

-27/1-

International Application No: PCT/

Form PCT/RO/134 (cont.)

American Type Culture Collection

12301 Parklawn Drive Rockville. MD 20852 US

Accession No. Date of Deposit

VR 2490 October 28, 1994

VR 2491 October 28, 1994

75938 October 28, 1994

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