AU7219800A - Recombinant poxvirus-feline infectious peritonitis virus, compositions thereof and methods for making and using them - Google Patents

Description

S&F Ref: 421704D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Virogenetics Corporation Rensselaer Technology Park 465 Jordan Road Troy New York 12180 United States of America Enzo Paoletti, Russell Gettig Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Recombinant Poxvirus-feline Infectious Peritonitis Virus, Compositions Thereof and Methods for Making and Using Them The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c RECOMBINANT POXVIRUS FELINE INFECTIOUS PERITONITIS VIRUS, COMPOSITIONS THEREOF AND METHODS FOR MAKING AND USING THEM Related Applications Reference is made to US Patent No 5,494,807 and US Patent No 5,364,773, the contents of which are hereby incorporated herein by reference.

Field of the Invention The present invention relates to modified recombinant poxviruses, compositions thereof and to methods of making and using the same; for instance, a vaccinia virus or o0 avipox canarypox or fowlpox) virus. For example, the invention relates to modified poxvirus-feline infectious peritonitis virus (FIPV) recombinants, compositions thereof, and methods for making and using the recombinants and compositions. The invention further relates to such recombinants which are attenuated recombinants, especially NYVAC- or ALVAC-FIPV recombinants, compositions thereof and methods for making and using the recombinants and compositions. Thus, the invention relates to a recombinant poxvirus-FIPV, such recombinants which express(es) gene product(s) of FIPV, compositions containing such recombinants and/or gene product(s), and methods for making and using the recombinants or compositions. The gene product can be FIPV N, M, and three versions of S (S1-complete spike; S2-spike minus the signal sequence; and S3-spike C- .i 11 \)ayLib\LIBFF]25810spec.doc:gcc 2 terminal section) or combinations thereof such as M and N. The recombinants or compositions containing them can induce an immunological response against FIPV infection, when administered to a host. The host is preferably a feline, a cat or kitten. The response can be protective. Thus, the composition can be immunological, or antigenic, or a vaccine.

The invention additionally relates to the products of expression of the poxvirus which by themselves are useful for eliciting an immune response raising antibodies or stimulating cell-mediated responses, which antibodies or responses are useful against FIPV infection, or which expression products or antibodies elicited thereby, isolated from a cell culture or from an animal, are useful for preparing a diagnostic kit, test or assay for the detection of FIPV, or of the recombinant virus, or of infected cells, or, of the expression of the antigens or products in other systems. The isolated expression products and antibodies elicited by the recombinant virus are especially useful in kits, tests or assays for detection of antibodies or antigens in a system, host, serum or sample; and the expression products are useful for generation of antibodies.

Several publications are referenced in this 25 application. Full citation to these references is found at the end of the specification immediately preceding the claims or where the publication is mentioned; and each of these publications is hereby incorporated herein by ~reference.

30 BACKGROUND OF THE INVENTION Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequenceS'flanking a S"foreign genetic element in a donor plasmid and homologous oo e sequences present in the rescuing poxvirus (Piccini et al., 1987).

Specifically, the recombinant poxviruses are constructed in two steps known in the art which are analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Patent Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587, and 5,179,993, the disclosures of which are incorporated herein by reference.

First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Maniatis et al., 1982).

Second, the isolated plasmid containing the DNA gene 25 sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential 30 region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.

35 Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA.

In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.

Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.

However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and S. the products of that recombination are then detectable by the presence of that genetic marker or gene in the 25 recombinant viral genome. Additional strategies have recently been reported for generating recombinant vaccinia virus.

Successful expression of the inserted DNP genetic sequence by the modified infectious virus requires two 30 conditions. First, the insertion must be into a oooo nonessential region of the virus in order that the modified virus remain viable. The second condition for go expression of inserted DNA is the presence of a promoter e• in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream S"from the DNA sequence to be expressed.

eeo:e Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. In the course of its history, many strains of vaccinia have arisen. These different strains demonstrate varying immunogenicity and are implicated to varying degrees with potential complications, the most serious of which are postvaccinial encephalitis and generalized vaccinia (Behbehani, 1983) With the eradication of smallpox, a new role for vaccinia became important, that of a genetically engineered vector for the expression of foreign genes.

Genes encoding a vast number of heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990a, 1990b) The genetic background of the vaccinia vector has been shown to affect the protective efficacy of the expressed foreign immunogen. For example, expression of Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine Sstrain of vaccinia virus did not protect cottontop tamarins against EBV virus induced lymphoma, while expression of the same gene in the WR laboratory strain S* 25 of vaccinia virus was protective (Morgan et al., 1988).

A fine balance between the efficacy and the safety of a vaccinia virus-based recombinant vaccine candidate is extremely important. The recombinant virus must present the immunogen(s) in a manner that elicits,a 30 protective immune response in the vaccinated animal but lacks any significant pathogenic properties. Therefore attenuation of the vector strain would be a highly desirable advance over the current state of technology.

A number of vaccinia genes have been identified 35 which are non-essential for growth of the virus in tissue culture and whose deletion or inactivation reduces virulence in a variety of animal systems.

The gene encoding the vaccinia virus thymidine kinase (TK) has been mapped (Hruby et al., 1982) and sequenced (Hruby et al., 1983; Weir et al., 1983).

Inactivation or complete deletion of the thymidine kinase gene does not prevent growth of vaccinia virus in a wide variety of cells in tissue culture. TK- vaccinia virus is also capable of replication in vivo at the site of inoculation in a variety of hosts and administered by a variety of routes.

It has been shown for herpes simplex virus type 2 that intravaginal inoculation of guinea pigs with TKvirus resulted in significantly lower virus titers in the spinal cord than did inoculation with TK virus (Stanberry et al., 1985). It has been demonstrated that herpesvirus encoded TK activity in vitro was not important for virus growth in actively metabolizing cells, but was required for virus growth in quiescent cells (Jamieson et al., 1974) Attenuation of TK- vaccinia has been shown in mice inoculated by the intracerebral and intraperitoneal routes (Buller et al., 1985). Attenuation was observed both for the WR neurovirulent laboratory strain and for the Wyeth vaccine strain. In mice inoculated by the intradermal route, TK- recombinant vaccinia generated 25 equivalent anti-vaccinia neutralizing antibodies as compared with the parental TK vaccinia virus, indicating that in this test system the loss of TK function does not significantly decrease immunogenicity of the vaccinia virus vector. Following intranasal inoculation of mice 30 with TK- and TK recombinant vaccinia virus (WR strain) significantly less dissemination of virus to other locations, including the brain, has been found (Taylor et Sal., 1991a) Another enzyme involved with nucleotide metabolism is ribonucleotide reductase. Loss of virally encoded ribonucleotide reductase activity in herpes simplex virus (HSV) by deletion of the gene encoding the large subunit was shown to have no effect on viral growth and DNA synthesis in dividing cells in vitro, but severely compromised the ability of the virus to grow on serum starved cells (Goldstein et al., 1988). Using a mouse model for acute HSV infection of the eye and reactivatable latent infection in the trigeminal ganglia, reduced virulence was demonstrated for HSV deleted of the large subunit of ribonucleotide reductase, compared to the virulence exhibited by wild type HSV (Jacobson et al., 1989).

Both the small (Slabaugh et al., 1988) and large (Schmidtt et al., 1988) subunits of ribonucleotide reductase have been identified in vaccinia virus.

Insertional inactivation of the large subunit of ribonucleotide reductase in the WR strain of vaccinia virus leads to attenuation of the virus as measured by intracranial inoculation of mice (Child et al., 1990).

The vaccinia virus hemagglutinin gene (HA) has been mapped and sequenced (Shida, 1986). The HA gene of vaccinia virus is nonessential for growth in tissue culture (Ichihashi et al., 1971). Inactivation of the HA gene of vaccinia virus results in reduced neurovirulence in rabbits inoculated by the intracranial route and smaller lesions in rabbits at the site of intradermal 25 inoculation (Shida et al., 1988). The HA locus was used for the insertion of foreign genes in the WR strain (Shida et al., 1987), derivatives of the Lister strain (Shida et al., 1988) and the Copenhagen strain (Guo et al., 1989) of vaccinia virus. Recombinant HA- vaccinia 30 virus expressing foreign genes have been shown to be immunogenic (Guo et al., 1989; Itamura et al., 1990; Shida et al., 1988; Shida et al., 1987) and protective against challenge by the relevant pathogen (Guo et al., 1989; Shida et al., 1987) Cowpox virus (Brighton red strain) produces red "(hemorrhagic) pocks on the chorioallantoic membrane of chicken eggs. Spontaneous deletions within the cowpox i genome generate mutants which produce white pocks (Pickup et al., 1984). The hemorrhagic function maps to a 38 kDa protein encoded by an early gene (Pickup et al., 1986). This gene, which has homology to serine protease inhibitors, has been shown to inhibit the host inflammatory response to cowpox virus (Palumbo et al., 1989) and is an inhibitor of blood coagulation.

The u gene is present in WR strain of vaccinia virus (Kotwal et al., 1989b). Mice inoculated with a WR vaccinia virus recombinant in which the u region has been inactivated by insertion of a foreign gene produce higher antibody levels to the foreign gene product compared to mice inoculated with a similar recombinant vaccinia virus in which the u gene is intact (Zhou et al., 1990). The u region is present in a defective nonfunctional form in Copenhagen strain of vaccinia virus (open reading frames B13 and B14 by the terminology reported in Goebel et al., 1990a,b) Cowpox virus is localized in infected cells in cytoplasmic A type inclusion bodies (ATI) (Kato et al., 1959). The function of ATI is thought to be the protection of cowpox virus virions during dissemination from animal to animal (Bergoin et al., 1971). The ATI region of the cowpox genome encodes a 160 kDa protein which forms the matrix of the ATI bodies (Funahashi et al., 1988; Patel et al., 1987). Vaccinia virus, though containing a homologous region in its genome, generally does not produce ATI. In WR strain of vaccinia, the ATI region of the genome is translated as a 94 kDa protein (Patel et al., 1988). In Copenhagen strain of vaccinia virus, most of the DNA sequences corresponding to the ATI region are deleted, with the remaining 3' end of the S*region fused with sequences upstream from the ATI region to form open reading frame (ORF) A26L (Goebel et al., 35 1990a,b) A variety of spontaneous (Altenburger et al., 1989; Drillien et al., 1981; Lai et al., 1989; Moss et al., 1981; Paez et al., 1985; Panicali et al., 1981) and engineered (Perkus et al., 1991; Perkus et al., 1989; Perkus et al., 1986) deletions have been reported near the left end of the vaccinia virus genome. A WR strain of vaccinia virus with a 10 kb spontaneous deletion (Moss et al., 1981; Panicali et al., 1981) was shown to be attenuated by intracranial inoculation in mice (Buller et al., 1985). This deletion was later shown to include 17 potential ORFs (Kotwal et al., 1988b). Specific genes within the deleted region include the virokine N1L and a kDa protein (C3L, by the terminology reported in Goebel et al., 1990a,b). Insertional inactivation of NIL reduces virulence by intracranial inoculation for both normal and nude mice (Kotwal et al., 1989a). The 35 kDa protein is secreted like N1L into the medium of vaccinia virus infected cells. The protein contains homology to the family of complement control proteins, particularly the complement 4B binding protein (C4bp) (Kotwal et al., 1988a). Like the cellular C4bp, the vaccinia 35 kDa protein binds the fourth component of complement and inhibits the classical complement cascade (Kotwal et al., 1990). Thus the vaccinia 35 kDa protein appears to be involved in aiding the virus in evading host defense mechanisms.

25 The left end of the vaccinia genome includes two genes which have been identified as host range genes, K1L (Gillard et al., 1986) and C7L (Perkus et al., 1990).

Deletion of both of these genes reduces the ability of vaccinia virus to grow on a variety of human cell Xines (Perkus et al., 1990) Two additional vaccine vector systems involve the use of naturally host-restricted poxviruses, avipox S"viruses. Both fowlpoxvirus (FPV) and canarypoxvirus S(CPV) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920's by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982) and there are no reports in the literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and human applications an attractive proposition.

FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988a). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor et al., 1988a). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor et al., 1990; Edbauer et al., 1990).

Despite the host-restriction for replication of FPV and CPV to avian systems, recombinants derived from these viruses were found to express extrinsic proteins in cells 25 of nonavian origin. Further, such recombinant viruses were shown to elicit immunological responses directed towards the foreign gene product and where appropriate were shown to afford protection from challenge against the corresponding pathogen (Tartaglia et al., 1993a,b; 30 Taylor et al., 1992; 1991b; 1988b) Feline infectious peritonitis virus (FIPV) produces a chronic, progressive, immunologically-mediated disease in felines such as domestic and exotic cats. The route of FIPV infection is thought to occur primarily through 35 the oral cavity and pharynx. Clinically apparent FIP occurs after the virus crosses the mucosal barrier and a primary viremia takes FIPV to its many target organs (liver, spleen, intestine and lungs). Two forms of the disease have been described as effusive (wet) and noneffusive (dry). The effusive form results in the classic fluid accumulation seen in infected cats which is caused by an Arthus-type vasculitis in the target organs mediated by complement activation and an intense inflammatory response. The non-effusive form is characterized by little or no ascitic fluid accumulation but internal organs may be infiltrated with granular fibrinous deposits. Thus, antibodies formed in response to FIPV infection (primarily to the spike protein) tend to enhance the pathogenesis of the disease and are obviously unwanted in a vaccine or immunological composition (Olsen and Scott, 1991). (However, expression of such proteins by a recombinant and the recombinants themselves are useful if one desires antigens or antibodies therefrom for a kit, test or assay or the like).

FIPV is a member of the Coronaviridae family.

Coronaviruses are large, positive stranded RNA viruses with genomic lengths of 27-30 kb. The virion is enveloped and is studded with peplomeric structures called spikes. The left half of the FIPV genome encodes a large polyprotein which is cleaved into smaller 25 fragments, some of which are involved in RNA replication.

The right half of the FIPV genome encodes 3 major structural proteins designated nucleocapsid matrix and spike The FIPV S gene product mediates attachment of the virus to the cell receptor, triggers 30 membrane fusion, and elicits virus-neutralizing antibodies. The N protein is necessary for encapsidating genomic RNA and directing its incorporation into the capsid, and is thought to be involved in RNA replication.

The FIPV M glycoprotein appears to be important for FIP viral maturation and for the determination of the site at which virus particles are assembled (Spann et al., 1988) Because of the antibody-dependent enhancement (ADE) of FIP in cats, attempts to produce a safe and efficacious vaccine or immunological composition against FIPV have been largely unsuccessful. Inactivated FIPV vaccines and heterologous live coronavirus vaccines did not afford any protection against FIPV infection and vaccination usually resulted in increased sensitization to the disease. A modified live virus vaccine, Primucell, is the first and only commercially marketed FIPV vaccine. Primucell is a temperature sensitive strain of FIPV that can replicate at the cooler temperatures of the nasal cavity, but not at systemic body temperatures (Gerber et al., 1990). Thus, intranasally administered Primucell is thought to produce a localized immunity to FIPV. However, serious questions remain concerning the efficacy and enhancement potential of this vaccine (Olsen and Scott, 1991) Vaccinia virus has been used as a vector for generating recombinant viruses expressing FIPV structural genes. A recombinant expressing the FIP M gene was shown to increase the survival time of cats after challenge with FIPV (Vennema et al., 1990).

Vennema, et al. (1991) relates to primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and to certain recombinant vaccinia viruses thereof introduced into kittens. The Vennema et al. FIPV matrix gene was cloned from a Spathogenic strain (79-1146) and its sequence appears S" identical to the matrix gene (discussed herein). The Vennema et al. recombinant, vFM, contains the coding region of matrix coupled to the vaccinia 7.5K early/late 30 promoter inserted at the thymidine kinase (tk) locus.

en.

Note that the promotor was not linked precisely to the matrix ATG initiation codon, but rather to a position 48 bp upstream from the ATC. Also, a vaccinia T5NT early transcriptional termination signal (Yuen et al., 1987) located in the coding region of the matrix gene was not removed.

Moreover, the vaccinia strain in Vennema et al. is the WR strain (Vennema et al. at page 328, left column, first 2 lines; see also, the donor plasmids and control viruses as mentioned on the same page in the section "Construction of Recombinant Vaccinia Viruses expressing the FIPV M and N proteins" beginning at mid-left column clearly indicate via literature citations that the WR strain is used). The choice of strain is important because the WR strain is a laboratory virus not a vaccine strain and the virulence characteristics of the WR strain do not make it a presently acceptable vector for a recombinant that may contact humans, let alone a recombinant in a composition such as a vaccine or antigenic or immunological composition targeted to felines, such as kittens, or other animals in contact with humans, especially young children or immunosuppressed individuals, due to recent concerns of contact transmission (such "other animals" could be laboratory cell cultures or animals for antigen expression or for antibody production for making kits, tests or assays) Thus, the Vennema, et al. articles fail to teach or suggest the recombinants, compositions and methods of the present invention.

More particularly, recombinants in the present invention preferably employ NYVAC or vectors (NYVAC and ALVAC are highly attenuated vectors having a BSL1 containment level) Further, in constructs of the present invention, preferably the coding region is coupled to the promotor in a precise coupling to the ATG codon with no 30 intervening sequence. (Any T5NT sequence can be inactivated by a base substitution which does not change the amino acid sequence but will prevent early transcriptional termination in a poxvirus vector) In eeee addition, multiple, two, copies of the coding 35 region directly coupled to the promotor can be present in each recombinant viral genome in the present invention.

The Vennema et al. efficacy study used SPF kittens (13-14 weeks old) which were vaccinated subcutaneously at day 0 and day 21 with 1 x 10B and 5 x 108 pfu respectively. On day 35 the cats were challenged orally with FIP strain 79-1146.

The herein protocol was similar, with the major difference being a lower vaccination dose (1 x The Vennema protection results were based on mortality with 3 of 8 cats vaccinated with vFM surviving Vennema et al. deemed their challenge sufficient in that 7 of 8 unvaccinated cats succumbed to the challenge exposure and died. Upon necropsy, all challenged cats, in Vennema et al. including the three surviving vFM vaccinated cats, had pathological signs of FIP infection including peritoneal effusions and granulomatous lesions on the viscera.

By contrast, the trials herein were more stringent.

Herein applicants scored protection as surviving and being free from FIP pathology upon necropsy. Using this criteria, Applicants had 3 out of 5 cats vaccinated with vCP262 protected with 0% of the unvaccinated cats protected. If the Vennema et al. results were scored using Applicants' criteria, Vennema would have had no protection; and ergo no recombinant suitable for vaccine use. In addition, the Vennema et al. observed fever and weight loss in all challenged cats. In Applicants' trials, (see trial 3 in particular) Applicants' observed even no weight loss and a lower febrile response after challenge.

Thus, the recombinants of the present invention employ an acceptable vector for all uses and a surprisingly higher protection level at a lower dose than S* the Vennema et al. vaccinia recombinant.

Recent studies using monoclonal antibodies directed against the S gene (Olsen et al., 1992) have shown also that mABs which neutralize the virus also cause ADE. No 35 enhancement is observed with mABs against matix or nucleocapsid proteins.

Thus, prior to the present invention, there has been a need for poxvirus-FIPV recombinants,.especially such 4 o recombinants using an acceptable vector and such recombinants having expression at low doses which indeed affords protection; and, there has been a need for compositions containing such recombinants, as well as a need for methods for making and using them. And, moreover, it would be especially surprising and unexpected if this poxvirus-FIPV recombinant was modified so as to be attenuated, an attenuated vaccinia virus-FIPV recombinant or an attenuated avipox-FIPV recombinant, such as a NYVAC-FIPV or ALVAC-FIPV recombinant; because, for instance, from attenuation and, diminished or lack of productive replication of the poxvirus in the host, one skilled in the art would have not expected and would be surprised by the usefulness of the attenuated recombinant, especially in a composition for felines and other hosts, and more especially in such a composition which provides a response including protection in felines.

Attenuated poxvirus vectors would also be especially advantageous for antigenic or vaccine compositions, particularly in view of attenuated vectors providing diminished or little or no pathogenic properties with regard to the intended host or, to unintended, possibly accidental hosts, such as those who work with the vector in formulating or administering the vector or antigen, or who may otherwise come into contact with it. That is, attenuated poxvirus vectors provide diminished or little or no pathogenic properties to intended hosts such as cats, kittens and the like and to unintended, possibly 30 accidental hosts, such as humans engaged in formulating S""the vector into a composition for administration or in administering the composition veterinarians, technicians, other workers) or, who may otherwise come into contact with the vector pet owners) 35 It can thus be appreciated that provision of a FIPV recombinant poxvirus, and of compositions and products therefrom, particularly NYVAC or ALVAC based FIPV recombinants and compositions and products therefrom, 16 would be a highly desirable advance over the current state of technology.

Objects and Summary of the Invention It is therefore an object of this invention to provide modified recombinant viruses, which viruses have enhanced safety, and to provide a method of making such recombinant viruses.

Additional objects of this invention include: to provide a recombinant poxvirus- FIPV, compositions containing the recombinant, antigen(s) from the recombinant or from the composition, methods for making the recombinant and composition, methods of using the compositions or the recombinant, in vivo and in vitro uses for expression by administering or infecting. Preferably the poxvirus-FIPV recombinant composition is an antigenic, or vaccine or immunological composition a composition containing a recombinant which expresses antigen, or the product from expression of the antigen).

It is a further object of this invention to provide a modified vector for expressing a gene product in a host, wherein the vector is modified so that it has attenuated virulence in the host.

It is another object of this invention to provide a method for expressing a gene product in a cell cultured in vitro using a modified recombinant virus or modified vector having an increased level of safety and to provide the use of such product in compositions.

According to a first embodiment of the invention, there is provided a recombinant poxvirus containing therein DNA from feline infectious peritonitis virus in a non-essential region of the poxvirus genome wherein the poxvirus is a vaccinia virus wherein J2R, B13R B14R, A26L, A56R, C7L-KIL and I4L are deleted from the virus, or a thymidine kinase gene, a hemorrhagic region, an A type 25 inclusion body region, a hemagglutinin gene, a host range region, and a large subunit, ribonucleotide reductase are deleted from the virus; or, the poxvirus is (ii) canarypox which was attenuated through more than 200 serial passages on chick embryo fibroblasts, a master seed therefrom was subjected to four successive plaque purifications under agar, from which a plaque clone was 30 amplified through five additional passages.

According to a second embodiment of the invention, there is provided an immunological composition comprising a recombinant poxvirus as defined in accordance with the first embodiment of the invention, and a carrier.

I :\I)ayLib\LIBFF]25810spec.doc:gcc 16a According to a third embodiment of the invention, there is provided a method for inducing an immunological response in a host comprising administering a recombinant poxvirus as defined in accordance with the first embodiment of the invention, or a composition as defined in accordance with the second embodiment of the invention.

According to a fourth embodiment of the invention, there is provided the recombinant poxvirus as defined in accordance with the first embodiment of the invention, or a composition as defined in accordance with the second embodiment of the invention, when used in inducing an immunological response in a host.

According to a fifth embodiment of the invention, there is provided the use of the I0 recombinant poxvirus as defined in accordance with the first embodiment of the invention, or a composition as defined in accordance with the second embodiment of the invention, in the preparation of a vaccine for inducing an immunological response in a host.

According to a sixth embodiment of the invention, there is provided a method for expressing a gene product in vitro comprising introducing into a cell a virus poxvirus as defined in accordance with the first embodiment of the invention, and continuing a cell culture in vitro under appropriate conditions for expression of the gene product.

In one aspect, the present invention relates to a modified recombinant virus having inactivated virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The functions can be non-essential, or associated with virulence. The virus is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. The modified recombinant virus can include, within a non-essential e *i II \DayLib\IIBFF]258iOspec.doc:gcc region of the virus genome, a heterologous DNA sequence which encodes an antigen or epitope derived from FIPV.

In another aspect, the present invention relates to an antigenic, immunological or vaccine composition or a therapeutic composition for inducing an antigenic or immunological or protective response in a host animal inoculated with the composition, said composition including a carrier and a modified recombinant virus having inactivated nonessential virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The virus used in the composition according to the present invention is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. The modified recombinant virus can include, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein, derived from FIPV. The composition can contain a recombinant poxvirus which contains coding for and expresses FIPV antigen(s) or the isolated antigen(s) In yet another aspect, the present invention relates to methods employing the aforementioned recombinant or composition; for instance, for obtaining an in vivo response to FIPV antigen(s). The method can comprise administering the recombinant or composition either to felines or other hosts, laboratory animals such as rodents such as rats, mice, gerbils or the like for antibody production for kits, assays and the like.

In a further aspect, the present invention relates oeooo to a method for expressing a gene product in a cell in vitro by introducing into the cell a modified recombinant Svirus having attenuated virulence and enhanced safety.

The modified recombinant virus can include, within a nonessential region of the virus genome, a heterologous 35 DNA sequence which encodes an antigenic protein, e.g.

derived from FIPV virus. The product can then be administered to individuals, felines or mice to stimulate an immune response. The antibodies raised can e o °e eeeoe be useful in individuals for the prevention or treatment of FIPV or and, the antibodies from individuals or animals or the isolated in vitro expression products can be used in diagnostic kits, assays or tests to determine the presence or absence in a sample such as sera of rabies or other maladies or antigens therefrom or antibodies thereto (and therefore the absence or.presence of the virus or of the products, or of an immune response to the virus or antigens) In a still further aspect, the present invention relates to a modified recombinant virus and compositions containing such. The virus can have nonessential virusencoded genetic functions inactivated therein so that the virus has attenuated virulence, and the modified recombinant virus further contains DNA from a heterologous source in a nonessential region of the virus genome. The DNA can code for FIPV antigen(s). In particular, the genetic functions are inactivated by deleting an open reading frame encoding a virulence factor or by utilizing naturally host restricted viruses.

The virus used according to the present invention is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. Advantageously, the open reading frame is selected from the group consisting of J2R, B13R B14R, A26L, A56R, C7L K1L, and I4L (by the terminology reported in Goebel et al., 1990a,b); and, the combination thereof. In this respect, the open reading frame comprises genomic regions which comprise a thymidine 30 kinase gene, a hemorrhagic region, an A type inclusion body region, a hemagglutinin gene, a host range gene region or a large subunit, ribonucleotide reductase; or, the combination thereof. A suitable modified Copenhagen strain of vaccinia virus is identified as NYVAC 35 (Tartaglia et al., 1992), or a vaccinia virus from which has been deleted J2R, B13R+B14R, A26L, A56R, C7L-K11 and I4L or a thymidine kinase gene, a hemorrhagic region, an A type inclusion body region, a hemagglutinin gene, a host range region, and a large subunit, ribonucleotide reductase (See also U.S. Patent No. 5,364,773).

Alternatively, a suitable poxvirus is an ALVAC or, a canarypox virus (Rentschler vaccine strain) which was attenuated, for instance, through more than 200 serial passages on chick embryo fibroblasts, a master seed therefrom was subjected to four successive plaque purifications under agar from which a plaque clone was amplified through five additional passages.

The invention in yet a further aspect relates to the product of expression of the inventive poxvirus-FIPV recombinant and uses therefor, such as to form antigenic, immunological or vaccine compositions, for administration to a host, animals, such as felines, or for administration for protection or response or for treatment, prevention, diagnosis or testing, and, to methods employing such compositions. The FIPV antigen(s), or the DNA encoding FIPV antigen(s) can code for M, N, and the three versions of S; Sl, S2, S3, or combinations thereof, M+N.

The present invention (recombinants, compositions and methods and uses) finds a basis in the discoveries that NYVAC and ALVAC recombinants, particularly NYVACand ALVAC-FIPV recombinants, surprisingly have expression despite attenuation, and expression which can confer a truly protective response in a susceptible host.

These and other embodiments are disclosed or are obvious from and encompassed by the follow detailed description.

S" 30 BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, 35 in which: Figure 1 shows the DNA sequence of FIPV matrix gene open reading frame (strain 79-1146); t Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 shows the DNA sequence of the FIPV matrix gene donor plasmid (The modified matrix gene coding region is initiated at 2408 and terminates at 1620; the entomopox 42K promoter starts at 2474; the C5 left arm is from 1 to 1549 and the C5 right arm is from 2580 to 2989); shows the DNA sequence of FIPV nucleocapsid gene open reading frame (strain 79-1146); shows the DNA sequence of the FIPV nucleocapsid gene donor plasmid (the nucleocapsid gene coding region initiates at 2101 and terminates at 968; the vaccinia I3L promoter starts at 2160; the C3 left arm is from 1 to 939 and the C3 right arm is from 2285 to 4857); shows the DNA sequence of FIPV spike gene open reading frame (strain 79-1146); shows the DNA sequence of the FIPV spike gene donor plasmid (the modified spike gene coding region is initiated at 591 and terminates at 4976; the vaccinia H6 promoter starts at 471; the C6 left arm is from 1 to 387 and the C6 right arm is from 4983 to 6144); shows the DNA sequence of the FIPV spike gene minus signal sequence donor plasmid (the modified spike gene coding region is initiated at 591 and terminates at 4922; the vaccinia H6 promoter starts at 471; the C6 left arm is from 1 to 387 and the C6 right arm is from 4929 to 6090); shows the DNA sequence of the FIPV spike gene C-terminal fragment donor plasmid (the modified spike gene coding region initiates at 591 and terminates at 2369; the vaccinia H6 promoter starts at 471; Figure 7 Figure 8 the C6 left arm is from 1 to 387 and the C6 right arm is from 2376 to 3537); Figure 9 shows the DNA sequence of a 7351 bp fragment of canarypox DNA containing the C3 open reading frame (the C3 ORF is initiated at position 1458 and terminates at position 2897); Figure 10 shows the DNA sequence of a 3208 bp fragment of canarypox DNA containing the C5 open reading frame (the C5 ORF is initiated at position 1537 and terminates at position 1857); and, Figure 11 shows the DNA sequence of a 3706 bp fragment of canarypox DNA containing the C6 open reading frame (the C6 ORF is initiated at position 377 and terminates at position 2254).

DETAILED DESCRIPTION OF THE INVENTION To develop a new vaccinia vaccine strain, NYVAC (vP866), the Copenhagen vaccine strain of vaccinia virus was modified by the deletion of six nonessential regions of the genome encoding known or potential virulence factors. The sequential deletions are detailed below (See U.S. Patent No. 5,364,773). All designations of vaccinia restriction fragments, open reading frames and nucleotide positions are based on the terminology reported in Goebel et al., 1990a,b.

The deletion loci were also engineered as recipient loci for the insertion of foreign genes.

The regions deleted in NYVAC are listed below. Also listed are the abbreviations and open reading frame designations for the deleted regions (Goebel et al., 1990a,b) and the designation of the vaccinia recombinant (vP) containing all deletions through the deletion specified: thymidine kinase gene (TK; J2R) vP410; hemorrhagic region B13R B14R) vP553; A type inclusion body region (ATI; A26L) vP618; hemagglutinin gene (HA; A56R) vP723; host range gene region (C7L K1L) vP804; and large subunit, ribonucleotide reductase (I4L) vP866 (NYVAC) NYVAC is a genetically engineered vaccinia virus strain that was generated by the specific deletion of eighteen open reading frames encoding gene products associated with virulence and host range. NYVAC is highly attenuated by a number of criteria including i) decreased virulence after intracerebral inoculation in newborn mice, ii) inocuity in genetically or chemically (cyclophosphamide) immunocompromised mice, iii) failure to cause disseminated infection in immunocompromised mice, iv) lack of significant induration and ulceration on rabbit skin, v) rapid clearance from the site of inoculation, and vi) greatly reduced replication competency on a number of tissue culture cell lines including those of human origin.

Nevertheless, NYVAC based vectors induce excellent responses to extrinsic immunogens and provided protective immunity.

TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolate derived from the FP-1 vaccine strain of fowlpoxvirus which is licensed for vaccination of chicks. ALVAC is an attenuated canarypox virus-based vector that was a plaque-cloned derivative of the licensed canarypox vaccine, Kanapox (Tartaglia et al., 1992). ALVAC has some general properties which are the same as some general properties of Kanapox. ALVAC-based 30 recombinant viruses expressing extrinsic immunogens have also been demonstrated efficacious as vaccine vectors (Tartaglia et al., 1993a,b). This avipox vector is restricted to avian species for productive replication.

On human cell cultures, canarypox virus replication is 35 aborted early in the viral replication cycle prior to viral DNA synthesis. Nevertheless, when engineered to :express extrinsic immunogens, authentic expression and processing is observed in vitro in mammalian cells and inoculation into numerous mammalian species induces antibody and cellular immune responses to the extrinsic immunogen and provides protection against challenge with the cognate pathogen (Taylor et al., 1992; Taylor et al., 1991b). Recent Phase I clinical trials in both Europe and the United States of a canarypox/rabies glycoprotein recombinant (ALVAC-RG) demonstrated that the experimental vaccine was well tolerated and induced protective levels of rabiesvirus neutralizing antibody titers (Cadoz et al., 1992; Fries et al., 1992). Additionally, peripheral blood mononuclear cells (PBMCs) derived from the ALVAC-RG vaccinates demonstrated significant levels of lymphocyte proliferation when stimulated with purified FIPV (Fries et al., 1992).

NYVAC, ALVAC and TROVAC have also been recognized as unique among all poxviruses in that the National Institutes of Health Public Health Service), Recombinant DNA Advisory Committee, which issues guidelines for the physical containment of genetic material such as viruses and vectors, guidelines for safety procedures for the use of such viruses and vectors which are based upon the pathogenicity of the particular virus or vector, granted a reduction in physical containment level: from BSL2 to BSL1. No other poxvirus has a BSL1 physical containment level. Even the Copenhagen strain of vaccinia virus the common smallpox vaccine has a higher physical containment level; namely, BSL2. Accordingly, the art has recognized that NYVAC, ALVAC and TROVAC have a lower pathogenicity than 30 any other poxvirus.

Clearly based on the attenuation profiles of the NYVAC, ALVAC, and TROVAC vectors and their demonstrated ability to elicit both humoral and cellular immunological responses to extrinsic immunogens (Tartaglia et al., 35 1993a,b; Taylor et al., 1992; Konishi et al., 1992) such recombinant viruses offer a distinct advantage over previously described vaccinia-based recombinant viruses.

e* oeooo The invention provides poxvirus-FIPV recombinants, preferably NYVAC- and ALVAC-FIPV recombinants which contain exogenous DNA coding for any or all of FIPV, M, N, and the three versions of S; SI, S2, S3, or combinations thereof, M+N.

The administration procedure for recombinant poxvirus-FIPV or expression product thereof, compositions of the invention such as immunological, antigenic or vaccine compositions or therapeutic compositions, can be via a parenteral route (intradermal, intramuscular or subcutaneous). Such an administration enables a systemic immune response, or humoral or cell-mediated responses.

More generally, the inventive poxvirus-FIPV recombinants, antigenic, immunological or vaccine poxvirus-FIPV compositions or therapeutic compositions can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical or veterinary art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts taking into consideration such factors as the age, sex, weight, species and condition of the particular patient, and the route of administration. The compositions can be administered alone, or can be co-administered or sequentially administered with compositions, with "other" immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or "cocktail" or combination compositions of the invention and methods employing them. Again, the ingredients and manner (sequential or co-administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular patient, and, the route of administration. In this regard, reference is "35 made to U.S. Serial No. 08/486,969, filed June 7, 1995, incorporated herein by reference, and directed to rabies .compositions and combination compositions and uses thereof.

0* e 0 Examples of compositions of the invention include liquid preparations for orifice, oral, nasal, anal, vaginal, peroral, intragastric, etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration injectable administration) such as sterile suspensions or emulsions.

In such compositions the recombinant poxvirus or antigens may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. Suitable dosages can also be based upon the examples below.

Further, the products of expression of the inventive recombinant poxviruses and compositions comprising them can be used directly to stimulate an immune response in individuals or in animals. Thus, the expression products can be used in compositions of the invention instead or ~in addition to the inventive recombinant poxvirus in the aforementioned compositions •o 30 Additionally, the inventive recombinant poxvirus and the expression products therefrom and compositions of the S"invention stimulate an immune or antibody response in animals; and therefore, those products are antigens.

From those antibodies or antigens, by techniques well known in the art, monoclonal antibodies can be prepared and, those monoclonal antibodies or the antigens, can be employed in well known antibody binding assays, diagnostic kits or tests to determine the presence or absence of particular FIPV antigen(s); and therefore, the presence or absence of the virus or of the antigen(s) or to determine whether an immune response to the virus or antigen(s) has simply been stimulated. Those monoclonal antibodies or the antigens can also be employed in immunoadsorption chromatography to recover or isolate FIPV antigen(s) or expression products of the inventive recombinant poxvirus or compositions of the invention.

Methods for producing monoclonal antibodies and for uses of monoclonal antibodies, and, of uses and methods for FIPV antigens the expression products of the inventive poxvirus and compositions are well known to those of ordinary skill in the art. They can be used in diagnostic methods, kits, tests or assays, as well as to recover materials by immunoadsorption chromatography or by immunoprecipitation.

Monoclonal antibodies are immunoglobulins produced by hybridoma cells. A monoclonal antibody reacts with a single antigenic determinant and provides greater specificity than a conventional, serum-derived antibody.

Furthermore, screening a large number of monoclonal antibodies makes it possible to select an individual antibody with desired specificity, avidity and isotype.

Hybridoma cell lines provide a constant, inexpensive 25 source of chemically identical antibodies and preparations of such antibodies can be easily Sstandardized. Methods for producing monoclonal antibodies are well known to those of ordinary skill in Sthe art, Koprowski, H. et al., U.S. Patent No.

30 4,196,265, issued April 1, 1989, incorporated herein by reference.

Uses of monoclonal antibodies are known. One such use is in diagnostic methods, David, G. and Greene, H. U.S. Patent No. 4,376,110, issued March 8, 1983; i 35 incorporated herein by reference. Monoclonal antibodies have also been used to recover materials by immunoadsorption chromatography, Milstein, C. 1980, 27 Scientific American 243:66, 70, incorporated herein by reference.

Accordingly, the inventive recombinant poxvirus and compositions have several herein stated utilities. Other utilities also exist for embodiments of the invention.

A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.

EXAMPLES

DNA Cloning and Synthesis. Plasmids were constructed, screened and grown by standard procedures (Maniatis et al., 1982; Perkus et al., 1985; Piccini et al., 1987). Restriction endonucleases were obtained from Bethesda Research Laboratories, Gaithersburg, MD, New England Biolabs, Beverly, MA; and Boehringer Mannheim Biochemicals, Indianapolis, IN. Klenow fragment of E.

coli polymerase was obtained from Boehringer Mannheim Biochemicals. BAL-31 exonuclease and phage T4 DNA ligase were obtained from New England Biolabs. The reagents were used as specified by the various suppliers.

Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as previously described (Perkus et al., 1989). DNA sequencing was performed by the dideoxy-chain 25 termination method (Sanger et al., 1977) using Sequenase (Tabor et al., 1987) as previously described (Guo et al., 1989). DNA amplification by polymerase chain reaction (PCR) for sequence verification (Engelke et al., 1988) ~was performed using custom synthesized oligonucleotide primers and GeneAmp DNA amplification Reagent Kit (Perkin Elmer Cetus, Norwalk, CT) in an automated Perkin Elmer Cetus DNA Thermal Cycler. Excess DNA sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by BAL-31 S* 35 exonuclease and mutagenesis (Mandecki, 1986) using synthetic oligonucleotides.

Cells, Virus, and Transfection. The origins and conditions of cultivation of the Copenhagen strain of vaccinia virus has been previously described (Guo et al., 1989). Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filters and screening for B-galactosidase activity are as previously described (Piccini et al., 1987).

The origins and conditions of cultivation of the Copenhagen strain of vaccinia virus and NYVAC has been previously described (Guo et al., 1989; Tartaglia et al., 1992). Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filters and screening for B-galactosidase activity are as previously described (Panicali et al., 1982; Perkus et al., 1989).

NYVAC is prepared by reference to U.S. Patent No.

5,364,773 and allowed U.S. application Serial No.

105,483, incorporated herein by reference.

The parental canarypox virus (Rentschler strain) is a vaccinal strain for canaries. The vaccine strain was obtained from a wild type isolate and attenuated through more than 200 serial passages on chick embryo fibroblasts. A master viral seed was subjected to four successive plaque purifications under agar and one plaque clone was amplified through five additional passages after which the stock virus was used as the parental virus in in vitro recombination tests. The plaque 25 purified canarypox isolate is designated ALVAC.

The strain of fowlpox virus (FPV) designated FP-1 has been described previously (Taylor et al., 1988a). It is an attenuated vaccine strain useful in vaccination of day old chickens. The parental virus strain Duvette was 30 obtained in France as a fowlpox scab from a chicken. The virus was attenuated by approximately 50 serial passages in chicken embryonated eggs followed by 25 passages on •chicken embryo fibroblast cells. The virus was subjected to four successive plaque purifications. One plaque 35 isolate was further amplified in primary CEF cells and a stock virus, designated as TROVAC, established.

NYVAC, ALVAC and TROVAC viral vectors and their derivatives were propagated as described previously (Piccini et al., 1987; Taylor et al., 1988a,b). Vero cells and chick embryo fibroblasts (CEF) were propagated as described previously (Taylor et al., 1988a,b) EXAMPLE 1 GENERATION OF ALVAC-BASED FIPV

RECOMBINANTS

1. Generation of an ALVAC Recombinant Expressing the Feline Infectious Peritonitis Virus (FIPV) Matrix Glycoprotein Gene Open Reading Frame (vCP262) The 79-1146 FIPV strain was obtained from Dr. F.

Scott (Cornell University, Ithaca, NY). Total RNA was isolated from FIPV infected CRFK cells using the quanidium isothiocyanate-cesium chloride procedure of Chirgwin, et al., (1979). First strand cDNA was synthesized using AMV reverse transcriptase and random oligonucleotide primers (6 mers) by the procedure of Watson and Jackson (1985), yielding single-stranded cDNA complementary to the FIPV positive strand mRNA.

The matrix gene was amplified by PCR from the first strand cDNA using oligonucleotide primers RG739 (SEQ ID NO:1) (5'-TAAGAGCTCATGAAGTACATTTTGCT-3') and RG740 (SEQ ID NO:2) (5'-ATTGGTACCGTTTAGTTACACCATATG-3') These primers were derived from Genbank sequence COFIPVMN 25 (Accession X56496) (Vennema et al., 1991). This 800 bp PCR fragment was digested with Asp718/SacI, gel purified, and ligated into pBluescript SK+ digested with Asp718/SacI to yield pBSFIPM. The M gene ORF was sequenced and is presented in Figure 1 (SEQ ID NO:3) 30 pBSFIPM was transformed into GM48 (dam-) cells, and plasmid DNA isolated which was demethylated (pBSFIPMdemeth). A 330 bp PCR fragment was amplified from pBSFIPM using oligonucleotides RG751 (SEQ ID NO:4)

TCTGAGCTCTTTATTGGGAAGAATATGATATATTTT-

GGGATTTCAAAATTGAAAATATATAATTACAATATAAAATGAAGTACATTTTGCT-

and RG752 (SEQ ID TTGTGGTCTGCCATATTG TAACACTGTTATAAATACAATC-3') and digested with SacI/BclI. This fragment was gel purified and ligated into pBSFIPM (demeth) digested with BclI to yield pFIPM42K. An 85 bp fragment was generated as a PCR primer-dimer from oligonucleotides RG749 (SEQ ID NO:6) 3')and RG750 (SEQ ID NO:7)

TACGAGCTCAAGCTTCCCGGGTTAATTAATTAGTCATCAGGCAGGGCGAGAACG-

This fragment was digested with SacI and ligated into pFIPM42K digested with SacI to yield pFIPM42KVQ.

This plasmid construct contains an expression cassette consisting of the complete FIPV matrix ORF (with a mutated T5NT early transcriptional stop signal) coupled to the entomopox 42K promoter (SEQ ID NO:8) ATTTCAAAATTGAAAATATATAATTACAATATAAA-3'). The sequence is modified such that it no longer functions as an early transcription stop signal and no amino acids are changed. This cassette was excised by digesting pFIPM42KVQ with Asp718/HindIII and isolated as a 950bp fragment. The ends of this fragment were blunted using Klenow polymerase and ligated into the ALVAC C5 locus insertion plasmid pNC5LSP-5, digested with SmaI. The resulting donor plasmid, pC5FIPM42K, was confirmed by DNA sequence analysis. It consists of the entomopox 42K promoter coupled to the FIPV matrix ORF at the ATG flanked by the left and right arms of the ALVAC insertion locus (Figure 2 (SEQ ID NO:9)).

SThis donor plasmid, pC5FIPM42K, was used in in vivo recombination (Piccini et al., 1987) with the ALVAC virus vector to generate the recombinant virus vCP262.

Immunoprecipitation analysis from a radiolabeled 30 lysate of VERO cells infected with vCP262 using a FIP matrix specific monoclonal antibody designated 15A9.9 (Olsen et al., 1992) showed expression of a 30 kDa polypeptide band. This was consistent with the expected size of the M gene product. In addition, the band 35 comigrated with an immunoprecipitated band from FIPV infected cells. Fluorescent activated cell sorting (FACS) analysis using the same monoclonal antibody showed 31 this expressed protein from vCP262 was localized in the cytoplasm of the infected cell.

2. Generation of an ALVAC Recombinant Expressing the FIPV Nucleocapsid Gene Open Reading Frame (vCP261A) The FIPV nucleocapsid gene was amplified by PCR using the first strand cDNA (described in 1 above.) as template and oligonucleotide primers RG741 (SEQ ID (5'-TAAGAGCTCATG-GCCACACAGGGACAA-3') and RG742 (SEQ ID NO:11) (5'-TATGGTACCTTA-GTTCGTAACCTCATC-3'). These primers were derived from Genbank sequence COFIPVMN (Accession X56496)(Vennema et al., 1991). The resulting 1150 bp fragment was digested with Asp718/SacI and ligated into pBluescript SK+ digested with Asp718/SacI resulting in pBSFIPN. The N gene ORF was sequenced and is presented in Figure 3 (SEQ ID NO:12) The vaccinia I3L promoter (SEQ ID NO:13)

TGAGATAAAGTGAAAATATATATCATTATATTACAAAGTACAATTATTTAGGTTTAA

TC-3')(Schmitt and Stunnenberg, 1988) was coupled to the ATG of the N ORF as follows. A 370 bp fragment was amplified by PCR using pBSFIPN as template and oligonucleotide primers RG747 (SEQ ID NO:14) CATCAGCATGAGGTCCTGTACC-3') and RG748 (SEQ ID 25

TCATTATATTACAAAGTACAATTATTTAGGTTTAATCATGGCCACACAGGGACAA-

This fragment was digested with SacI/PPuMI and ligated into pBSFIPN digested with SacI/PPuMI resulting in pFIPNI3L. An 85 bp fragment was generated as a PCR 30 primer-dimer from oligonucleotides RG749 (SEQ ID NO:6) and RG750 (SEQ ID

TACGAGCTCAAGCTTCCCGGGTTAATTAATTAGTCA

TCAGGCAGGGCGAGAACG-3'). This fragment was digested with SacI and ligated into pFIPNI3L digested with SacI to yield pFIPNI3LVQ. The N gene expression cassette (I3L promoted N) was excised as a 1300 bp fragment by digesting pFIPNI3LVQ with Asp718/HindIII. The ends of this fragment were blunted using Klenow polymerase and ligated into the C3 insertion plasmid, pSPCP3LSA (see below), digested with SmaI. The resulting donor plasmid, pC3FIPNI3L, was confirmed by DNA sequence analysis. It consists of the vaccinia I3L promoter coupled to the FIPV N gene ORF flanked by the left and right arms of the ALVAC C3 insertion locus (Figure 4 (SEQ ID NO:16)).

This donor plasmid, pC3FIPNI3L, was used in in vivo recombination (Piccini et al., 1987) with the ALVAC virus vector to generate the recombinant virus vCP261A.

Immunoprecipitation analysis from a radiolabeled lysate of VERO cells infected with vCP261A using a FIP nucleocapsid specific monoclonal antibody designated 17B7.1 (Olsen et al., 1992) showed expression of a 45 kDa polypeptide band. This was consistent with the expected size of the N gene product. In addition, the band comigrated with an immunoprecipitated band from FIPV infected cells. FACS analysis using the same monoclonal antibody showed this expressed protein from vCP261A was localized in the cytoplasm of the infected cell.

3. Generation of an ALVAC Recombinant Expressing both the FIPV Matrix and Nucleocapsid Open Reading Frames (vCP282) Plasmid pC5FIPM42K (Figure 2, SEQ ID NO:9) 25 containing the FIPV matrix gene ORF coupled to the entomopox 42K promoter was used in in vivo recombination (Piccini et al., 1987) with the ALVAC-FIP-N recombinant (vCP261A) (described in 2 above)to generate the double recombinant vCP282. This recombinant contains the FIPV M 30 gene ORF (42K promoter) inserted into the C5 locus and the FIPV N gene ORF (I3L promoter) inserted into the C3 locus.

Immunoprecipitation analysis from a radiolabeled lysate of VERO cells infected with vCP282 using a FIP matrix specific monoclonal antibody designated 15A9.9 (Olsen et al., 1992) showed expression of a 30 kDa polypeptide band while using a nucleocapsid specific monoclonal antibody designated 17B7.1 showed expression of a 45 kDa polypeptide band. This was consistent with 33 the expected size of the M and N gene products respectively. In addition, both bands comigrated with an immunoprecipitated bands from FIPV infected cells.

Fluorescent activated cell sorting (FACS) analysis using the same monoclonal antibodies showed these expressed proteins from vCP282 were localized in the cytoplasm of the infected cell.

4. Generation of an ALVAC Recombinant Expressing the Complete FIPV Spike Glycoprotein Gene ORF (vCP281) The FIPV spike gene was obtained by PCR amplification from first strand cDNA template (described in 1 above) in three sections. PCR primers were synthesized based on Genbank sequence COFIPE2 (Accession #X06170) (De Groot et al., 1987). The 5' end was amplified by PCR using oligonucleotide primers JP53 (SEQ ID NO:17) (5'-CATCATGAGCTCATGATTGTGCTCGTAAC-3') and JP77 (SEQ ID NO:18) (5'-AACAGCCGCTTGTGCGC-3'). The isolated 1630 bp fragment was digested with SacI/HindIII and ligated into pBluescript SK+ digested with SacI/HindIII to yield pBSFIP-SA, which was confirmed by DNA sequence analysis.

The middle section of S was amplified by PCR using oligonucleotide primers JP84 (SEQ ID NO:19) CTTGGTATGAAGCTTAG-3') and JP85 (SEQ ID NO:20) o GGTGACTTAAAGCTTGC-3'). The isolated 1715 bp fragment was digested with HindIII and ligated into pBluescript SK+ digested with HindIII. Two clones, pKR5 and pKW13 were 30 sequenced and found to have errors (based on Genbank sequence COFIPE2) but in different locations. To correct these PCR errors, a section of pKW13 was replaced with a subfragment from pKR5 as follows. PKR5 was digested with Clal, blunted with Klenow polymerase, digested with BstEII and a 750 bp fragment isolated and cloned into pKR13 digested with SmaI/BstEII. The resulting plasmid, pBSFIPS-MII, was confirmed by DNA sequence analysis.

The 3' section of S was amplified by PCR using oligonucleotide primers JP71 (SEQ ID NO:21) i TAATGATGCTATACATC-3') and JP90 (SEQ ID NO:22) CATCATGGTACCTTAGTGGACATGCACTTT-3'). The isolated 1020 bp fragment was digested with HinDIII/Asp718 and ligated into pBluescript SK+ digested with HinDIII/Asp718 to yield pBSFIPS-C, which was confirmed by DNA sequence analysis.

The complete DNA sequence of the FIPV Spike gene as derived from the 79-1146 strain cDNA is presented in Figure 5 (SEQ ID NO:23) The spike ORF contains three T5NT early transcriptional stop signals. Two were eliminated from the middle section by introducing mutations via PCR. A 330 bp PCR fragment was amplified from pBSFIPS-MII using oligonucleotide primers RG757B (SEQ ID NO:24)

CATTAGACTCTGTGACGCCATGTGATGTAA-

GCGCACAAGCGGCTGTTATCGATGGTGCCATAGTTGGAGCTATGACTTCCATTAACA

GT- GAACTGTTAGGCCTAACACATTGGACAACGACACCTAATTTCTATTAC- 3')and RG758B (SEQ ID NO:25)

CATTAGACTGTAAACCTGCATGTATTCAACTTG-

CACAGATATTGTAAAATTTGTAGGTATCGTGACATTACCAGTGCTAATTGGTTGCAC

GT-CTCCGTCAGAATGTGTGACGTTAATAAATACCAAAG-3'), digested with HgaI/BspMI and cloned into HgaI/BspMI digested pBSFIPS-MII to yield pMJ5. Sequence analysis of revealed a 33 bp deletion which was corrected by 25 replacing the 250 bp StuI/BspMI fragment with a PCR fragment amplified from pBSFIPS-MII using oligonucleotide primers RG758B (SEQ ID NO:25) and JP162 (SEQ ID NO:26) The isolated fragment was digested with StuI/BspMI 30 and ligated into pMJ5 digested with StuI/BspMI to yield pNR3. This plasmid had a base change at position 2384 which was corrected using the U.S.E. mutagenesis kit (Pharmacia) to yield pBSFIPS-MIIDII. This plasmid contains the middle section of the S gene with changed 35 T5NT sequences and the introduction of new ClaI and StuI sites while maintaining the correct amino acid sequence.

In order to couple the vaccinia H6 promoter (SEQ ID NO:27)

TTCTTTATTCTATACTTAAAAAGTGAAAATAAATACAAAGGTTCTTGA-

GGGTTGTGTTAAATTGAAAGCGAGAAAAAAAATAATCATAAATTATTTCATTATCGC

G-ATATCCGTTAAGTTTGTATCGTA-3') (Perkus et al., 1989) to the ATG of the S gene the following was performed. The 3' end of the H6 promoter coupled to the S gene amplified as a PCR fragment from pBSFIPS-A section of S gene) using oligonucleotide primers RG755 (SEQ ID NO:28) CTTGTATGCATTCATTATTTG-3') and RG756 (SEQ ID NO:29) TCCGAGCTCGATATCCGTTAAGTTTGTATCGTAATGATTGTGCTCGTAAC-3').

The 100 bp fragment was digested with SacI/NsiI and ligated to pBSFIPS-A digested with SacI/NsiI to yield pBSFIPS-AH6.

To remove the T5NT sequence in the 5' section of the spike gene without altering the amino acid sequence, a 350 bp PCR fragment was amplified from pBSFIPS-AH6 using oligonucleotide primers RG753 (SEQ ID NO:30) TCACTGCAGATGTACAATCTG-3') and RG754 (SEQ ID NO:31)

CAGTATACGATGTGTAAGCAATTGTCCAAAAA-

GCTCCACTAACACCAGTGGTTAAAT-

TAAAAGATATACAACCAATAGGAAATGTGCTAAAGAAATTGTAACCATTAATATAGA

AATGG-3'). The fragment was digested with PstI/AccI and ligated into pBSFIPS-AH6 digested with PstI/AccI to yield pNJ1.

The middle and 3' ends of the S gene were coupled together to form the complete ORF as follows.

First, the 3' section was excised as a 1000 bp fragment by digesting pBSFIPS-C with Asp718/HinDIII and ligating into pNJI section) digested with Asp718/HinDIII yielding pBSFIPS-A/CHG. The middle section was added by 30 excising a 1700 bp fragment from pBSFIPSMIIDII by digesting with HinDIII and ligating into pBSFIPS-A/CH6 digested with HinDIII and screened for orientation. The resulting plasmid, pBSFIPSH6II, contains the complete S ORF coupled to the 3' end of the H6 promoter with all .35 three T5NT sequences eliminated.

To insert the complete S ORF into a C6 donor plasmid, a 4.4 kb cassette was excised from pBSFIPSH6II by digesting with EcoRV/EcoRI and filling in the ends i with Klenow polymerase. This cassette was ligated into pJCA070 digested with EcoRV/EcoRI and filled in with Klenow polymerase. The resulting plasmid, pOG9, was found by DNA sequence analysis to have a 110 bp insert in the H6 promoter between the NruI and EcoRV sites. To remove these sequences, pOG9 was digested with NruI/EcoRV and religated to yield the donor plasmid pC6FIPSH6II which has the complete H6 promoter minus four base pairs between the NruI and EcoRI sites which is not required for early and late transcription. This plasmid consists of the left arm of the C6 locus, the H6 promoter, complete S gene ORF and the right arm of the C6 locus (Figure 6 (SEQ ID NO:32)). A mutation in the stop codon adds an additional nine amino acids to the C-terminus of spike (Figure 7) This donor plasmid, pC6FIPSH6II, was used in in vivo recombination (Piccini et al., 1987) with the ALVAC virus vector to generate the recombinant virus vCP281.

Immunoprecipitation analysis from a radiolabeled lysate of CRFK cells infected with vCP281 using a FIP spike specific monoclonal antibody designated 23F4.5 (Olsen et al., 1992) showed expression of a 220 kDa polypeptide band. This was consistent with the expected size of the S gene product. In addition, the band comigrated with an immunoprecipitated band from FIPV infected cells, consistent with proper glycosylation.

FACS analysis using the same monoclonal antibody showed this expressed protein from vCP281 was localized in the .cytoplasm of the infected cell. However, inoculation of 30 monolayers of CRFK cells with vCP281 showed strong fusigenic activity, indicating the protein was also on the surface of these cells. No fusigenic activity was observed in CRFK cells infected with the ALVAC parental virus (control) 35 5. Generation of an ALVAC Recombinant Expressing the FIPV Spike Glycoprotein Gene ORF Minus the Signal Sequence (vCP283B) The 57 bp signal sequence was removed from the Nterminus of the S gene and replaced by an ATG by inserting a 270 bp PCR fragment into pOG9 as follows.

The PCR fragment was amplified from pBSFIPS-A using oligonucleotide primers RG759 (SEQ ID NO:33) GCTATTTTCCATGGCTTCC-3') and RG760 (SEQ ID NO:34)

TCCGAGCTCGATATCCGTTAAGTTTGTATCGTAATGA-CAACAAATAATGAATGC-

The fragment was digested with EcoRV/NcoI and ligated into pOG9 digested with EcoRV/NcoI to yield pOM12. pOM12 was digested with EcoRV/NruI and religated to remove the 110 bp insert in the H6 promoter. The resulting donor plasmid, pC6FIPSH6-SS, was confirmed by DNA sequence analysis (Figure 7 (SEQ ID This donor plasmid, pC6FIPSH6-SS, was used in in vivo recombination (Piccini et al., 1987) with the ALVAC virus vector to generate the recombinant virus vCP283B.

Immunoprecipitation analysis from a radiolabeled lysate of CRFK cells infected with vCP283B using a cat FIP-immune serum (#511) showed expression of a polypeptide band of about 145±10 kDa. This was consistent with the predicted size of a non-glycosylated S gene product. Immunofluorescence analysis using the same polyclonal serum showed this expressed protein was localized in the cytoplasm of vCP283B infected CEF cells.

25 No fusigenic activity was observed in CRFK cells.

6. Generation of an ALVAC Recombinant Expressing the C-terminal Section of the FIPV Spike Glycoprotein Gene ORF (vCP315) 30 The C-terminal 1749 bp of the S gene (terminal 582 aa out of 1452 aa total) was linked to the H6 promoter as follows. pOG9 was digested with NruI/BstEII and a-6.2 kb fragment isolated. This fragment contains the 1749 bp Cterminal portion of the S gene. A fragment containing the 3' end of the H6 promoter coupled to an ATG codon flanked by a BstEII site was generated by annealing oligonucleotides JP226 (SEQ ID NO:36)

CATTAGCATGATATCCGTTAAGTTTGTATCGT-AATGGGTAACCCTGAGTAGCAT-

and JP227 (SEQ ID NO:37)

ATGCTACTCAGGGTTACCCATTACGATACAAACTTAACGGATATCATGCTAATG-

and digesting with NruI/BstEII. This fragment was ligated into the 6.2 kb pOG9 fragment (see 4 above) to yield the donor plasmid pC6FIPSH6-C, which was confirmed by DNA sequence analysis (Figure 8 (SEQ ID NO:38)) This donor plasmid, pC6FIPSH6-C, was used in in vivo recombination (Piccini et al., 1987) with the ALVAC virus vector to generate the recombinant virus vCP315.

Western blot analysis from a lysate of CRFK cells infected with vCP315 using a cat FIP-immune serum (#511) showed expression of a 56 kDa polypeptide band. This was slightly smaller than the predicted size of the truncated, non-glycosylated S gene product (64 kDa) Immunofluorescence analysis using the same polyclonal serum showed a weak detection of the protein localized in the cytoplasm of vCP315 infected CEF cells. No fusigenic activity was observed in CRFK cells.

EXAMPLE 2 GENERATION OF C3, C5 AND C6 INSERTION

PLASMIDS

Generation of C3 insertion plasmid pSPCP3LA.

An 8.5 kb canarypox BglII fragment was cloned into the BamI site of pBluescript SK+ (Stratagene, La Jolla, CA) to yield pWW5. Nucleotide sequence analysis of this 25 fragment revealed an open reading frame designated C3 initiated at position 1458 and terminated at position .2897 in the sequence presented in Figure 9 (SEQ ID NO:39). In order to delete the entire C3 open reading frame (ORF), PCR primers were designed to amplify a 30 and a 3' fragment relative to the C3 ORF. Oligonucleotide primers RG277 (SEQ ID NO:40) GTACCACTGGTATTTTATTTCAG-3') and RG278 (SEQ ID NO:41)

TATCTGAATTCCTGCAGCCCGGGTTTTTATAGCTAATTAGTCAAATG-

TGAGTTAATATTAG-3') were used to amplify the 5' fragment from pWW5 and oligonucleotide primers RG279 (SEQ ID NO:42) AA-GCATACAAGC-3') were used to amplify the 3' fragment from pWW5. The 5' fragment was digested with 39 Asp718/EcoRI and the 3' fragment digested with EcoRI/SacI. The 5' and 3' arms were then ligated into pBluescript SK+ digested with Asp718/SacI to yield pC3I.

This plasmid contains the C3 insertion locus with the C3 ORF deleted and replaced with a multiple cloning site flanked by vaccinia early transcriptional and translational termination signal. pC3I was confirmed by DNA sequence analysis.

The flanking arms of pC3I were lengthened as follows. A 908 bp fragment upstream of the C3 locus was obtained by digestion of pWW5 with NsiI and SspI. A 604 bp PCR fragment was amplified from pWW5 using oligonucleotide primers CP16 (SEQ ID NO:43)(5'- TCCGGTACCGCGGCCGCAGATATTTGTTAGCTTCTGC-3') and CP17 (SEQ ID NO:44) (5'-TCGCTCGAGTAGGATACCTACCTACTACCTA-CG-3'), digested with Asp718/XhoI and ligated into (International Biotechnologies, Inc., New haven, CT) to yield pSPC3LA. pSPC3LA was digested within pIBI25 with EcORV and within the insert (canarypox DNA) with NsiI and ligated to the 908 bp Nsi/SspI fragment generating pSPCPLAX which contains 1444 bp of canarypox DNA upstream of the C3 locus. A 2178 bp BglII/StyI fragment of canarypox DNA was isolated from pXX4 (which contains a 6.5 kb NsiI fragment of canarypox DNA cloned into the S25 PstI site of pBluescript A 279 bp PCR fragment was amplified from pXX4 using oligonucleotide primers CP19 (SEQ ID NO:45) (5'-TCGCTCGAGCTTTCTTGACAATAACATAG-3') and (SEQ ID NO:46) digested with XhoI/SacI and ligated into digested with SacI/XhoI to yield pSPC3RA.

To add additional unique sites to the multiple cloning site (MCS) in pC3I, pC3I was digested with EcoRI/ClaI (in the MCS) and ligated to kinased and annealed oligonucleotides CP12 (SEQ ID NO:47) S. 35 AATTCCTCGAGGGATCC-3') and (SEQ ID NO:48) CGGGATCCCTCG-AGG-3') (containing an EcoRI sticky end, XhoI site, BamHI site and a sticky end compatible with Clal) to yield pSPCP3S. pSPCP3S was digested within the canarypox sequences downstream of the C3 locus with StyI and SacI (from pBluescript SK+)and ligated to a 261 bp BglII/SacI fragment from pSPC3RA and the 2178 bp BglII/StyI fragment from pXX4 generating pCPRAL containing 2572 bp of canarypox sequences downstream of the C3 locus. pSPCP3S was digested within the canarypox sequences upstream of the C3 locus with Asp718 (in pBluescript SK+) and AccI and ligated to a 1436 bp Asp718/AccI fragment from pSPCPLAX generating pCPLAI containing 1457 bp of canarypox DNA upstream of the C3 locus. pCPLAI was digested within the canarypox sequences downstream of the C3 locus with StyI and SacI (in pBluescript SK+) and ligated to a 2438 bp StyI/SacI fragment from pCPRAL generating plasmid pSPCP3LA. The left arm of pSPCP3LA was shortened by about 500 bp as follows. pSPCP3LA was digested with NotI/NsiI and a 6433 bp fragment was isolated. Oligonucleotides CP34 (SEQ ID NO:49) (5'-GGCCGCGTCGACATGCA-3') and CP35 (SEQ ID (5'-TGTCGACGC-3') were annealed and ligated into this fragment to yield pSPCP3LSA. This is the C3 insertion plasmid which consists of 939 bp of canarypox DNA upstream of the C3 locus, stop codons in six reading frames, early transcriptional termination signal, an MCS, early transcriptional termination signal, stop codons in 0 25 six reading frames and 2572 bp of canarypox DNA downstream of the C3 locus.

Generation of C5 insertion plasmid A genomic library of canarypox DNA was constructed in the cosmid vector pVK102 (Knauf and Nester, 1982) 30 probed with pRW764.5 (a pUC9 based plasmid containing an 880 bp canarypox PvuII fragment which includes the ORF) and a cosmid clone containing a 29 kb insert was identified (pHCOS1). A 3.3 kb Clal fragment from pHCOS1 containing the C5 region was identified. The C5 ORF is 35 initiated at position 1537 and terminated at position 1857 in the sequence shown in Figure 10 (SEQ ID NO:51) The C5 insertion vector was constructed in two steps. The 1535 bp upstream sequence was generated by PCR amplification from purified genomic canarypox DNA using oligonucleotide primers C5A (SEQ ID NO:52) ATCATCGAATTCTGAATGTTAAATGTTATACTTTG-3') and C5B (SEQ ID NO:53) (5'-GGGGGTACCTTTGAGAGTACCACTTCAG-3'). This fragment was digested with EcoRI and ligated into pUC8 digested with EcoRI/Smal to yield pC5LAB. The 404 bp arm was generated by PCR amplification using oligonucleotides (SEQ ID NO:54) GGGTCTAGAGCGGCCGCTTATAAAGATCTAAAATGCATAATTTC-3') and (SEQ ID NO:55) This fragment was digested with PstI and cloned into SmaI/PstI digested pC5LAB to yield pC5L. pC5L was digested within the MCS with Asp718/NotI and ligated to kinased and annealed oligonucleotides CP26 (SEQ ID NO:56)

GTACGTGACTAATTAGCTATAAAAAGGATCCGGTACCCTCGAGTCTAGAATCGATCC-

CGGGTTTTTATGACTAGTTAATCAC-3') and CP27 (SEQ ID NO:57)

GGCCGTGATTAACTAGTCATAAAAACCCGGGATCGATTCTAGACTCGAGGGTACCGG

ATCCTTTTTATAGCTAATTAGTCAC-3') to yield pC5LSP. This plasmid was digested with EcoRI, ligated with kinased and self-annealed oligonucleotide CP29 (SEQ ID NO:58) (5'-AATTGCGGCCGC-3') and digested with NotI. The linearized plasmid was purified and self-ligated to generate pNC5LSP-5. This C5 insertion plasmid contains 1535 bp of canarypox DNA upstream of the Cs ORF, translation stop codons in six reading frames, vaccinia early transcription termination signal, an MCS with BamHI, KpnI, XhoI, Clal and SmaI restriction sits, 30 vaccinia early termination signal, translation stop codons in six reading frames and 404 bp of downstream canarypox sequence (31 bp of C5 coding sequence and 373 bp of downstream canarypox sequence) Generation of C6 insertion plasmid PC6L.

Figure 11 (SEQ ID NO:59) is the sequence of a 3.7 kb *segment of canarypox DNA. Analysis of the sequence revealed an ORF designated C6L initiated at position 377 and terminated at position 2254. The following describes a C6 insertion plasmid constructed by deleting the C6 ORF and replacing it with an MCS flanked by transcriptional and translational termination signals. A 380 bp PCR fragment was amplified from genomic canarypox DNA using oligonucleotide primers C6A1 (SEQ ID NO:60) ATCATCGAG-CTCGCGGCCGCCTATCAAAAGTCTTAATGAGTT-3') and C6B1 (SEQ ID NO:61) TTCGTAAGTAAGTATTTTTATTTAA-3'). A 1155 bp PCR fragment was amplified from genomic canarypox DNA using oligonucleotide primers C6C1 (SEQ ID NO:62)

CCCGGGCTGCAGCTCGAGGAATTCTT-

TTTATTGATTAACTAGTCAAATGAGTATATATAATTGAAAAAGTAA-3') and C6D1 (SEQ ID NO:63) GATGATGGTACCTTCATAAATACAAGTTTGATTAAACTT-AAGTTG-3'). The 380 bp and 1155 bp fragments were fused together by adding them together as template and amplifying a 1613 bp PCR fragment using oligonucleotide primers C6A1 (SEQ ID NO:49) and C6D1 (SEQ ID NO:52). This fragment was digested with SacI/KpnI and ligated into pBluescript SK+ digested with SacI/KpnI. The resulting plasmid, pC6L was confirmed by DNA sequence analysis. It consists of 370 bp of canarypox DNA upstream of C6, vaccinia early termination signal, translation stop codons in six reading frames, an MCS containing SmaI, PstI, XhoI and SEcoRI sites, vaccinia early termination signal, translation stop codons in six reading frames and 1156 bp of downstream canary pox sequence.

pJCA070 was derived from pC6L by ligating a cassette 30 containing the vaccinia H6 promoter coupled to another foreign gene into the SmaI/EcoRI sites of pC6L. Cutting pJCA070 with EcoRV/EcoRI excises the foreign gene and the 5' end of the H6 promoter.

EXAMPLE 3 EFFICACY TRIALS WITH ALVAC-BASED FELINE INFECTIOUS PERITONITIS VIRUS RECOMBINANTS Trial 1 Safety, antigenicity and efficacy trial Swith vCP261A(N), vCP262 and vCP282 (M+N) 43 Twenty five specific pathogen-free (SPF) 10-12 week old cats from Harlan Sprague Dawley, Inc. were randomly divided into five groups (5 cats/group). Groups were vaccinated subcutaneously (neck area) twice (day 0 and day 21) with 107 TCID 50 /dose with either vCP261, vCP262, vCP282 or vCP261A vCP262. Five cats in one group were not vaccinated and served as challenge controls. At day all cats were challenged orally with 10 3 5 TCIDO per cat with a virulent FIP virus (strain 1146). The cats were observed daily for 33 days post challenge to monitor mortality and visible manifestations of FIP virus infection. At day 33, all surviving cats were necropsied and examined for FIP pathology. The non-effusive form was detected by isolation of FIP virus from the intestinal tract and identification by virus-neutralization tests.

Cats with the effusive form had a thick yellow fluid in the peritoneal cavity, white edematous fluid in the pleural cavity and lesions on the intestine, spleen and liver. Some infected cats showed ocular involvement with conjunctivitis, blepharospasm and opalesent retina.

None of the vaccinated cats showed any adverse local or systemic postvaccination reactions. All five nonvaccinated cats either died with FIP signs or when necropsied had FIP signs, thus validating the challenge 25 dose. Dead and dying cats displayed signs of both effusive and non-effusive forms of FIP. The results from the ALVAC-FIP recombinant vaccinated cats is presented in Table 1. None of these cats developed virus neutralizing antibody prior to challenge on day 35. All cats had a 30 febrile response following challenge. All vaccinated groups showed partial protection with the best protection in the vCP262 and vCP282 vaccinated groups, each having 3/5 cats with no FIP mortality or signs. Thus, it appears from this study that the ALVAC-FIP matrix recombinants provided the best overall protection.

Trial 2 Safety, antigenicity and efficacy trial with vCP262 in comparison with

PRIMUCELL.

Twenty three SPF cats aged 10-12 weeks from Hill Grove, Great Britain were used in this trial. Ten cats were vaccinated subcutaneously with vCP262 at a dose of 108 pfu on days 0 and 21. Five cats received a commercially available FIP vaccine (PRIMUCELL, Smithkline Beecham) which was given as recommended by the manufacturer (2 doses, 21 days apart, intranasal, 104.8 TCIDO per dose). Eight cats were non-vaccinated and served as challenge controls. On day 35, all cats were challenged with a virulent FIP virus (strain 79-1146) at a dose of 320 DECP 0 given intranasally. Surviving cats were rechallenged on day 84 and those surviving were necropsied on day 104 and examined for FIP pathology.

None of the vaccinated cats showed any adverse local or systemic postvaccination reactions. Within the control group, four of the cats either died or had FIP pathology when necropsied. The remaining four controls (housed in a separate unit from the other controls) survived both challenges and appeared to be protected.

They all showed significant increase in serum neutralizing antibodies to FIP following challenge, thus indicating exposure to the virus. Whether this indicates technical problems with the challenge protocol or a natural protection is unknown.

Serological analysis showed no significant viral neutralizing antibody titers to FIP in cats receiving two inoculations of vCP262. In contrast, significant titers were observed after one inoculation of PRIMUCELL and these titers were boosted after the second inoculation.

30 Cats in both groups showed high titers following challenge.

The mortality data results for the vaccinated cats is presented in Table 2. In the vCP262 group, 8/10 cats survived the first challenge, while 6/10 survived both challenges In contrast, in the PRIMUCELL group, only 1/5 cats survived the first challenge. The surviving cat also survived the second challenge. It is important to note that 3 of the 4 dead PRIMUCELL vaccinated cats died on or before day 11 which indicates an enhancement of the normal progression of the disease. No enhancement was observed with vCP262 vaccinated cats. Thus, compared to PRIMUCELL, vCP262 provides greater protection with no enhancement of the disease.

Trial 3 Safety, antigenicity and efficacy trial with vCP262 in combination with the spike recombinants (vCP281(Sl), vCP283B(S2) and vCP315(S3)) Thirty six 9 week old SPF cats were received from Harlan Sprague Dawley, Inc. and randomly divided into six groups (6 cats/group). Groups received two subcutaneous inoculations (dose of about 10 7 TCIDS for each recombinant at day 0 and day 21,) with the following recombinants: 1) vCP262 (matrix), 2) vCP262 plus vCP281 (Si spike complete), 3) vCP262 plus vCP283B (S2 spike minus signal sequence) and 4) vCP262 plus vCP315 (S3 spike C-terminal section). One group was vaccinated intranasally with a commercially available FIP vaccine (PRIMUCELL, Pfizer Animal Health) as recommended by the manufacturer (2 doses, day 0 and day 21). One group was not vaccinated and served as challenge controls. Fifteen 25 days following the second vaccination (day 36), all cats were challenged orally with 1 0 3S TCIDS 0 per cat with a virulent FIP virus (NVSL FIP-1146, 89-5-1). The cats were monitored for weight, temperature, serologic response and mortality for 35 days post challenge.

Necropsy was performed on the majority of dead cats to look for FIP signs and FIPV virus was isolated from two cats to confirm infection.

None of the cats vaccinated with ALVAC recombinants showed any adverse local or systemic postvaccination reactions. All cats vaccinated with PRIMUCELL had virus neutralizing titers. In the recombinant groups, only S. cats in the group receiving matrix plus complete spike had virus neutralizing titers (3/6 after the second vaccination) 46 The mortality data is presented in table 3.

Necropsied cats showed signs of both the effusive (majority) and non-effusive forms of the disease. One cat had FIP induced encephalitis (control group). The lowest mortality was observed in the group vaccinated with vCP262 (matrix) alone. Groups receiving vCP262 plus any of the spike recombinants showed little, if any protection. The PRIMUCELL vaccinated group showed a mortality of 66.7%. Antibody induced enhancement (early death) was observed in both the PRIMUCELL and vCP281 (Sl complete spike) groups. Five out of six of the control nonvaccinated cats died from FIP infection which validated the challenge.

Fever and weight loss are indicators of FIP disease.

There was relative postchallenge weight loss in all the groups. However the vCP262 vaccinated group showed only a slight weight loss as compared to PRIMUCELL and the control groups. Chronic fever was observed in all cats, however the group that was vaccinated with vCP262 exhibited consistently lower temperatures that the other groups.

From this study it was concluded that vCP262 provided protection against a severe FIP challenge. In addition, cats vaccinated with this recombinant showed a lower febrile response and less weight loss following challenge. The other FIP recombinants (vCP281, vCP283B, and vCP315) as well as PRIMUCELL provided poor protection and even enhancement of mortality (PRIMUCELL, vCP281) ftft f ft ftf ft ft 47 TABLE 1 Results of FTP Efficacy Trial with ALVAC Matrix Nucleocapsid Recombinants Groups Virus Mortality Neutralizing Protection 3 Antibody Titer (GMAT)l Day 35 Day 63 Alive 2 Dead Control <2 >14,190 2(2FIP+) 3 0/5 (0%6) vCP26lA <2 446 2(IFIP+) 3 1/4

(N)

vCP262 <2 >11,585 4(1FIP+) 1 3/5 vCP282 <2 >16,384 4(lFIP+) 1 3/5 vCP261A <2 >16, 384 3(lFIP+) 2 2/5 (400%) vCP262 (M) 1. Titers expressed as reciprocal of final serum dilution.

2. Numbers in parenthesis represent cats with FTP signs at necropsy.

3. No mortality or FTP signs.

S.

C 48 TABLE 2 Results of Efficacy Trial Comparing ALVAC Matrix Recombinant with PRIMUCELL Groups Number of Mortality Protection Cats 1st 2nd Challenge Challenge' Day 35 Day 84 Control 8 3 1 4/8 vCP262 10 2 2 6/10 PRIMUCELL 5 42 0 1/5 1.

104.

Includes cats necropsied with FIP pathology at day 2. Three of these cats died on or before day 11 indicating -enhancement.

u 49 TABLE 3 Mortality Data Comparing ALVAC-based Matrix and Spike Recombinants with PRIMUCELL.

Group Mortality Enhancement' vCP262 2/6 (33%1) NO vCP262 vCP281 (Si) 6/6 (100-1) YES vCP262 vCP283 (S2) 5/6 (83 NO vCP262 vCP315 (S3) 5/6 (83.30-) NO PRIMLICELL 4/6 YES Control 5/6 NO 1. Death on or prior to day 15 post challenge.

EXAMPLE 4 GENERATION OF NYVAC-BASED FIPV

RECOMBINANTS

Using insertion loci and promoters as in USSN 105,483, incorporated herein by reference, such as by modifying plasmid pRW842 for insertion of rabies glycoprotein G gene into TK deletion locus (used for generation of vP879), by excising out of pRW842 the rabies DNA and inserting therefor the herein disclosed FIPV DNA coding for M, N, and the three versions of S; S1, S2, S3, or combinations thereof (for instance M and N) and by then employing the resultant plasmids in recombination with NYVAC, vP866, NYVAC-FIPV(M), and the three versions of and (M N) recombinants are generated; and analysis confirms expression.

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope thereof.

0 e *o* *o e

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Virol. 63:3829-3836 (1989).

55. Piccini, Perkus, Paoletti, Methods in Enzymology 153:545-563 (1987).

56. Piccini, M.E. Perkus, and E. Paoletti, Methods in Enzymology 153:545-563 (1987).

57. Pickup, B.S. Ink, W. Hu, C.A. Ray and W.K.

Joklik, Proc. Natl. Acad. Sci. USA 83:7698-7702 (1986).

58. Pickup, B.S. Ink, B.L. Parsons, W. Hu and W.K.

Joklik, Proc. Natl. Acad. Sci. USA 81:6817-6821 (1984).

59. Sanger, Nickel, S. Coulson, Proc. Natl.

Acad. Sci. 74:5463-5467 (1977).

Schmidtt, J.F.C. and H.G. Stunnenberg, J. Virol.

62:1889-1897 (1988).

61. Schmitt, J.F.C. and Stunnenberg, J. Virology 62:1889-1897 (1988).

62. Shida, Hinuma, Hatanaka, Morita, M., Kidokoro, Suzuki, Maruyzam, Takahashi- Nishimaki,.F., Sugimoto, Kitamura, Miyazawa, and Hayami, J. Virol. 62:4474-4480 (1988).

63. Shida, Virology 150:451-462 (1986).

64. Shida, T. Tochikura, T. Sato, T. Konno, K.

Hirayoshi, M. Seki, Y. Ito, M. Hatanaka, Y. Hinuma, M. Sugimoto, F. Takahashi-Nishimaki, T. Maruyama, K.

Miki, K. Suzuki, M. Morita, H. Sashiyama and M.

Hayami, EMBO 6:3379-3384 (1987).

Slabaugh, N. Roseman, R. Davis and C. Mat'hews, J. Virol. 62:519-527 (1988).

66. Spann, Cavanagh, and Horzinek, J. Gen.

*50 Virol. 69:2939-2952 (1988).

67. Stanberry, Kit, Myers, J. Virol.

:3 22 -328 (19 68. Tabor, S. and C. C. Richardson, Proc. Natl. Acad.

Sci. USA 84:4767-4771 (1987).

69. Tartaglia, Pincus, Paoletti, Critical Reviews in Immunology 10:13-30 (1990a).

Tartaglia, J. and Paoletti, In Immunochemistry of Viruses, II, eds. M.H.V. van Regenmortel A.R.

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71. Tartaglia, J. Taylor, W.I. Cox, Audonnet, M.E. Perkus, A. Radaelli, C. de Giuli Morghen, B.

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THE FOLLOWING PAGE(S) APPEAR AFTER THE DESCRIPTION AND BEFORE THE CLAIMS.

Figure 1 1 ATGAAGTACA TTTTGCTAAT ACTCGCGTGC ATAATTGCAT GCGTTTATGG TGAACGCTAC

TGTGCCATGC

TGCTT7TGAAC

ATATTGATTG

TATGGCATTA

TTTALATGCAT

CGTGCAGTTG

AGAAGAAOOA

GCATTGGGTA

CTACTTTCAG

CATTTGCCTA

GGAAAACAAT

GGTGATTACT

ATGGTGTAA

AAGACAGTGG

GTGGTGATCT

TTTTTATAAC

AAATGCTGAT

ACTCTGAGTA

TAACGTTTGC

AATCATGGTG

GAAGTTATG'T

GAAATCTATA

AATAOGTCAT

TAAAAGCAAC

CAACAGAAGC

CTTGCAGTGT

TATTTGGCAT

AGTGTTACAA

CATGTGGCTA

CCAAGTTTCC

ACTTTGGATG

GT=TTTT

GCTTC,-CTTA

T7GCTGAAGGT GATTO OTACA

TAOTGCCACA

ACGTACTGAC

ATTAATGGCA

CTTGCTAACT

TATGGCAGAC

TTATGGCCTA

AGATATGTAA

ATGTATTTTG

OCTGAGACTA

GATGGTACTC

TTCAAAATFGG

CCTAGTAGAA

GGATGGGCTT

AATTTGAGTG

CAAAf]7CAAG

GGAACTTCAG

CACAATTTAG

TTGTTCTAGC

TGTTCGG OTT

TGAGATCTGT

ATGCAAT

C'TGGTGGTTT

CCATCTTTA

ACTACGTAAA

AACATO-AAAA

ATGTCAAACO

CTGGTCTGTA

CTGGCTCGTT

GOTTAOGATT

TAGTGTTGOA

TOAGOTATAT

TTGTGTTAAT

TACO OTTACT

AAOOATCGAC

TAOATTAGTT

ATOTAAAGOT

ATTATTACAT

1/30 Figure 2 121 181 2 11 .301 3 6 1 7891 84 1 901 961 7081 1201 9261 1081 1201 1261 1321 1681 1741 GAATTGCOG C

AAAAATAATC

ACTAAOCTTA

CTAACAAATA

'FCAOOAATGG

TACAATTACT

CATGATAATT

TGGAAAGATG

CTATCGGAAG

TCTGCAATAT

CCATTTATCT

ATGAGATTAC

AAATGAAAAA

CAAGATGGCT

TAGGTTTTTG

TGAAATGGCT

TAAACCTGTA

CTACAAAATA

AGOCTTTACT

ATTGGCTCAT

AGCOGTATCA

TOACTTGCTG

TOAAATATGT

TTGCCAGCTG

GATGTAAACT

GAAAGTTACT

AATTAGCTAT

TACACCATAT

TAATCACCAG

TGTTTTCCAA

COCTGAATGT

CATTAAAGA

TTCTTAACGA

ACTAAAACAT

GGTTAAATAT

ATTACGAATA

GGOTACGACA

GATTTGACAG

ATAGGATACC

TCGTAAAAGA

CAACGACATC

TATIAAACTTT

GTATAGAAGC

TACATATACG

GACAATGGAT

GTAATGrI'CA

GTTACTGAAT

GTGAAAGATC

CCTTTGTOTT

TCGOCGGATG

AATAAAAATT

GATAACATGG

AGCACACTAC

TAATTCATGG

ACATCTTTGA

CTGAGACACA

AAAAAGGATC

GTAATAATTT

CTTTAGATTT

CTAATGTATA

TAAATGTTAT

AAGGATTCAA

CGCTTTAALAT

AAAAATAATA

TTATATCACG

TGCAAGAOAT

TAGTGATAAA

ATGTAACTTA

AGTTATATTA

TGAAGATTAC

GTC-TAATTCT

TTGTATACTT

TGTTCACGAG

TCTGTOAGGC

TCGACCCTAA

AGAATACCOA

GCACAACTTC

TGTTGAAZGAA

TOG CAGCTTA

TAOATATTTC

TAACAATCOT

GACGTACTCC

~TTAAAAAAAA

TAGAAAAGAA

AAGAAATGGA

AAAGAOOTAG

COGTACCCTC

TTCATGTTCA

TACGTAGTAAi

AACGATGGTT

ACTTTGGATG

ATACTACAA-A

ATACACAAAT

AAAGGAAATG

TGTATATCTA

AATAAGATTA

TOCTATTTCG

ATAGGTGCA--.

TACA.PAAAATC

TGCGAAITTTC

TCCATGTTTT

ATATTCCOTA

CGGTTGTTGA

TATCATGGAT

CACGGAATAT

GGCTATAAAA

TTOTCTG CAT

TAACTATGTA

CCTTAACAAA

AAACACGGAT

TAAACTTCTA

TTTAATGATC

TAAAATOTCC

GTGCTCAGGC

AAATCATATA

CTGAAGTGGT

GAGTCTAGAA

CTCAAATTOT

GCCCATCCTG

CTACTAGGTG

AAGCTATAAA

ACCTAAGCGA

AAACATAATT

TAATATCGTA

TACTOTTATC

CGTATTTAAG

CATCGTTACA

AAATCOTTAAA

ACTOOTTOUGA

TAAACTATGA

ATGTATOTGT

AACTATATTA

AAACAACAAA

AATOACAATG

GGTACTCTAC

ATCTTGATGA

GATGCGGTGT

AACAA-GTTC

GTTAATTTGO

CGGTTAACTC

TTGAACAAAG

GCTGTACAAT

AGAACTGGGA

TACTTTTCAA

CTGTTTTOGA

ACTCTCAAAG

TCGATCCCGT

CAOTACOTGC

TGGCAGTAGT

TAG CAATCAT

TATGCATTOG

TAATATGTTA

TTTGTATAAC

ATTATTTTAC

GTATACTCTT

AOAATCTTGT

TAAAGTCAGT

TAACAGCATT

TAAA ACAGATI

CAATAAAAAG-

TTCAGATA77

ATCATGAAGA

ATTATACATT

CATCTCTAAA

A.ATCTCCTCT

GOTATGGAGC

TGAGAGACGA

TPTTACAC C

TTAAACTTCT

CTCTACATAT

GTG OTOATAC

CTGOAAATAT

AAAATTGATC

CAAAGGAGCA

ATTGATTAAA

GTACGTGACT

ACCGTTTAGT

TTCTGTTGAG

TGCTTTTAAT

GACGTATTTA

0 000.

0 2/30 Figure 2 (cont'd.) 1801 1861 1921 1981 20,11 2101 2161 2281 23,i 1 2401 2461 2521 2 58 1 2641 2701 2761 2821 21881 2941

GGCAAATGCT

GAAAGTAGAG

CCCAATGCAT

GTTCTTCTAT

ACTGCACCTG

GCA77AAAAA

ATGCCATAAA

ATCAATATTA

TCAAAGCACGG

ATGGCACAGT

TACTTCATTT

TTCTTCCCAA

TGATGACTAA

TATAAAGATC

GTTAGTATAT

TGATGTTTTA

CGATGATTAT

GGTTACAAAG

GAAAATGTTG

ATCTCCTTTG

CGATGGTTAA

TAAGGGTAAC

TAACACAAAG

ATAGCTGAAC

CAACACTAAA

TCGTAAGCGC

CGAGCCAGCT

CAGACCAGCT

TTTGACATCT

AG CCTT CAC C

TATATTGTAA

TAAAGAGCTC

TTA-ATTAACC

TAAAATG CAT

TTTACAATGG

GAAAAGAAAG

TGTTGTAAAT

TATAAGTCTA

TTAGATTATG

CACATAATTT

ACCACCAGCC

ACCTGTAGGA

AATTGCATTA

AGATCTCACA

GCCGAACATT

TAGAACAATA

AAATTGTGGT

GAAGTTCCAG

TGAAT7TGTG ATAAACG CAT

TTATATATTT

TAATTAATTA

CCC GAAG CTG

AATTTCTAAA

AGATTAACG C

TTATTGAATA

CTGTTTTAGA

TACTACTA-AT

ATTATGAAALA

CATCTATTCC

ATTTGAAAC

GTACCATCTA

GTCTCAGGAT

AAATACATCA

ACATATCTGG

GGCCATAATA

CTGCCATATT

TTAGCAAGAT

CC CATT ,ATAC

GCAATTATGC

TCAATTTTGA

ACGAGCAGAT

GGTTTTTATG

TAATGAAAAA

TCTATACCGT

TGAAAACTTT

TGAAGAAGAT

GGCGACTTGT

ACCAAATAAA

TAGTTTAGAA

CTTCAGCATA

AGGGAAGCAC

TAAAAGACCA

TCCAAAGTGC

AAACTTGGTA

GCCACATGAT

GTAACACTGT

GCCAAATAAG

AC TGCAAG CC

ACGCCAGTAT

AATCCCAAAA

AGTCTCGTTC

ACTAGTTAAT

AAGTACATCA

TCTATGTTTA

AATGAAGATG

GACGCGCTAA

GCAAGAAGGT

TCAGATCCAT

TACCTGCAG

TAGATTTCCT

ATAACTTCTA

CCATGATTTG

AAACGTTACA

CTCAGAGTAT

CAGCATTTA

TATAAATACT

ATCACCACGT

ACTGTCTTGC

TAGCAAAATG

TATTATCATA

TCGCCCTGCC

CACGGCCGCT

TGAGCAACGC

TTGATTCAGA

AAGATGACGA

AGTATACTAT

ATAGTATACT

ATCTAAAGGT

*0 3/30 Figure 3 1 61 121 181 1 301 336 1 '121 6 1 721 781 81; 1 90C)1 961 1021 1081

ATGGCCACAC

TCTAACTCTC

CTCGAACAAG

GGTAATAAGG

GGCCAGCGTA

GCTGATGCTA

ATGAACAAGC

TT17GATGGTA

AGGTCTGGTT

CATTCCAATA

GCGT'TACTG

AGGGACACAA

GATC-TGACAA

GTTG CCAATG

TCTAGCATAA

ACGCTCACTC

CAGATTGACG

CGTTCTAAGT

ACAGATGTGT

AGGGACAACG

GTGGTCGGAA

GATCTAAATT

ATCAACAAAT

AGGAACTCGC

AATTCAAAGA

CCACAACGCT

AGATACCGCC

CTCAGTCTAG

ACCAGAATAA

ACAAACAAAG

CACCTAAGAA

CTTTCTATGG

GTAACGCTGC

TCTTTGGCAG

ACACCTACTA

CTTACAAGCG

CTGCTGATAA

TTGATGACAC

CGTCAACTGG

GAATAATGAT

TTGGAATTTA

TGGTTA FFGG

TGAGAGGTGG

CAAGA FrGAT

TGGCACTCGT

ACAGTTTCAG

ATCTGTTTCA

TAATGTTGAG

GTCACGTTCT

TG CCAACAAA

TGCTAGAAGT

CAAATGCTAC

TCAATGGTCT

CCTGOCCAAAG

ACCTTCTGAA

GAAGCCTGAG

ACAGGTTGAG

GGAGATGAAC

ATACCTTTGT

TGTCCGAGAG

AATAGACAGA

TTCTTTTACT

r2GAGTCTTCT

GGAACCAATA

CTTGAAGTGA

AGAAACAGAT

GATACAJJTTG

AAACCTAGAG

CACACCTGGA

AGTTCAGCTA

CCTCAGATAG

GCTGAAGAAG

GATGATGCCA

GTGGCTAAGG

GAGTTGTCTG

ATGATTGAT G

CTTCCAAAAG

CArTCTACAA

ACCTTGTTCC

TTCGTTATCG

TCTTAGGTAC

GGGTTGCAAG

ACGAATCCAA

ACCGTTCTAG

CCAA-CTAG

FL-JCGTAG]TGA

,ACAAAACTGC

ACTTTGGTGA

CTGAATGTGT

CTGGTGATCA

AAACTAGTCA

ATCAGAGGCA

TAACTCTTGT

AG'STTAC GAA

ACGTGGTCGT

CCCCATTACC

CAAAGGAATA

TA FrGTAAAA

AGGACCTCAT

GGATGGTGCC

ACCACTGAGA

GAACAATTCA

AGGAAGACAC'

TTC CAAA Ct

AGGCAAGGGIA

TAGTGATCTC

TCCATCAGTG

AGTGAAAGTC

ATTCCTAGAA

AAGAAGATCC

GOAGO CATAC

CTAA

4/30 Figure 4 1 GCGGCCGCGT CGACATGCAT TGTTAGTTCT GTAGATCAGT AACGTATAGC ATACGAGTAT 61 121 181 24 1 301 3 61 4 2 1 411 541i 601 661 721 781 811 901 961 1021 1081 114 1 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741

AATTATCGTA

CAATTCAGCA

TGTTACGATA

TATGAAAAAT

AAGGGTAATT

CATGTTAGAA

GAAATTACTT

'1ATTCCCAAG

AAGCTTGACG

CATACGAGAG

ACTCATCTTT

TTATATACTA

GAAAGTAAAA

ATTCAGTTAT

ATAAATTAGT

CCGTACCTTA

TGTATGCCTC

GGGATCTTCT

GTTCTAGGAA

TGACTTTCAC

ACACTGATGG

CGAGATCACT

CTCCCTTGCC

TAGGTTTGGA

CTAATTTTTC

GGTGTCTTCC

TTGAATTGTT

ATCTCAGTGG

TGGCACCATC

GGTAGTAGGT

ATTTCTCTAT

GTATTTCTAA

ATAGTAATGT

TTTACATATC

GTAAAAAAGA

AGTATGTATA

TTGAGAAACG

TTTCCTATAA

TAACTACTCA

GATGTGGTAT

TATAGTATTA

TACTATAAAT

ATTGTTTTAT

AATGTAGTAT

GTTCGTAACC

CACAAGAGTT

TTGCCTCTGA

TTGACTAGTT

TTGATCACCA

AACACATTCA

ATCACCAAAG

TGCAGTTTC

ATCACTACGT

AAGCACGGCT

TCTAGATTGA

CCTAGAACGG

TTTGGATTCG

CCTTGCAACC

ATCCTAAAAT

TATCATGATA

AGTAAAGAGC

ACATATTTCT

TATATACGCT

AAGAACTAAT

TAATGTATAA

GTATALATAGA

TG CCTACTAA

TCGTATAACT

AAATGTATAA

AAAATTATAT

ATGTATCTCT

AAAAGCTAAA

ACTAATATTA

TCATCAATCA

ACAGACAACT

TCCTTAGCCA

TTGGCATCAT

GCTTCTTCAG

GCTATCTGAG

TTAGCTGAAC

TTCCAGGTGT

TCTCTAGGTT

ACAATTGTAT

GATCTGTTTC

TTCACTTCAA

TTATTGGTTC

CAGAAGACTC

AAATCTGATA

ATGATTAATA

AGGAATCCCT

AATGTTAACA

TATTACAGYJ'

TTTACAAAGT

AGGTATGAAT

TATATTT-ICTA

GAAA-ACTAGA

ACTGTTGCTA

TAACTATATT

TTGTATAATT

TATTTATAAC

TGCTACTAGA

ACTCACATTT

TCTCAACCTG

CCTCAGGCTT

CTTCAGAAGG

CCTTTGGCAG

CAGACCATTG

GGTAGCAT

TACTTCTAGC

GTTTGTTGc3C

TAGAACGTGA

CCTCAACATT

TTGAAACAGA

GCTGAAACTG

CACGAGTGCC

CATCAATCTT

CAGATAATAA

CACAGCGTGT

AGTATAATAG

TATTTATACG

ATTAAAAATA

GCTTTACCPA

ATCAC.AAACA

SATACCATTA

AGATACATAC

ACAGTGACAC

ACACTGGTAT

ATATTATTAT

TTATTAGTAA

TTGATATAA-A

GACTAATTAG

TGTGTCATCA

CTTATCAGCA

TCGCTTGTAA

GTAGTAGGTG

ACTGCCAAAG

GGCAGCGTTA

ACCATAGAAA

ATTCTTAGGT

CCTTTGTTTG

ATTATTCTGG

TCTAGACTGA

TGGCGGTATC

AAGCGTTGTG

GTCTTTGAAT

CTTTGTAAATl

CGTTATTT

AAATAATCCA

TAAATCCAGG

TACTTGCAAAJi

AATGCCAATO

ATACTAXCG U TGATGTTATtA-

TTTATTTCACJ

AT'TCAGTGTA

AGTATGTACT

TGAATATGTA

CTATAAAAAC

AACACATCTC

GACTTAGAAC

GCGTCAATCT

TGACTGAGCC-

ATTATGCTAG

CCATTGGCA-P

GTTGTCACAT

C-TTGTGTCCC

TCAGTALACAC

T TATTGGAAT

GAACCAGACC

TTACCATCAA

GGCTTGTTCA

TTAGCATCAC

5/30 Figure 4 (cont'd.) 1801 1861 1921 1981 2041 2101 2 16 1 24 1 24 6 1 21 258i 2641 2701 2761 2 82 1 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3 54 1

CATGAGGTCC

CTTTTACAAT

CTATTCCTTT

GGGTAATGGG

AACGACCACG

TGATTAAACC

GAG CTCTAAT

TTAACCCGGG

AATCAAATAA

GTGTAAACTA

GGGATTAGAT

CGTTAGGTTA

ATGTGATATT

TGAAAATTTC

AGAAGAGATG

TGTAGTTATG

TGCGGTACCC

TCACATAACA

GTGCATGTCC

TAAGTATTTG

ACGGTTTTTA

AGAAATGAGT

AAATTTAGTA

GTCTTTGTAT

CGGTGCCGAT

TACAGAAATT

AAATAGTCCG

AAAAAAAGGT

GATATCTGAT

TAGAAAT

TGTACCTAAG

ACGATAACGA

GGGAACAAGG

GTTGTAGAAT

TCTTTTGGAA

TAAATAATTG

TAATTAACGA

AAGCTGGGCT

AAAGCATACA

AGCCACATAG

GTTAAGGTTC

GATACTGATG

TTTCCTCATA

AAAAAGCAAA

TGTTTTCCTC

AAACTGGAGG

TGTTCGAAGG

GTAGGATATG

AAGTTTAGGG

GTATAATTTA

TTAGAATAAA

AATGGAAGAC

AGGTATATAC

TTAGCCGTAA

ATTTTAAAAT

GCTAAACTAC

TTATATATTT

GTTAATTGTA

GATATGTATA

GAAACTCCGT

AAGTAAAAGA

ATCTGTCTAT

TCTCTCGGAC

GACAAAGGTA

GGTTCATCTC

TACTTTGTAA

GCAGATAGTC

G CAGGAATTC AG CTATTG CT 7TIGCCAATGA

CTTGGGATTA

TTACAGATTA

TAACTCTTGG

TALACTGATCA

AGAGTAACGC

TATCTGATGA

ACGTGTTTGG

TTA-AGGAGGA

GGCAAGAAAT

TTAAATAGTA

ATAGAGATAA

TTATAAATGA

TTAAAAAATG

GTATTTCTGA

GTAAAAATCC

TAATAGATTC

CTGTATATAG

ATAGATTCTT

AAATATTTAT

TACATTACGC

ACCACCTCTC

TCCAATAACC

ATAAATTCCA

TATCArI'ATT

CCCAGTTGAC

TATAATGATA

TCGTTCTCGC

CTCGAGGGAT

TCGCTATCGT

AAAAAATAGT

TAGTAACTGG

TAATAATGTT

ALATAGCAAAT

AGATTTACAG

CTCTAAACAG

ACTTAGAGCC

TGATATCACA

CGATGTCGAA

ACAAGTTCTA

TAATTATAAC

TATCATAATG

ACTGCATAAA

CAAATACAAT

TATAGAAATG

TCCTCTTCAT

TGGCGCTGAC

AAACAATAAG

TCTAAATTAT

AGATTTTAAT

TATAAAGTAT

AGCGAGTTCC

AATTTGTTGA

AAATTTAGAT

CTTCCGACCA

GCG'FFGTCCC

TATATTTTCA

CCTGCCTGAT

CCCGA=TT

TACAAAATGG

AGAAAGGATA

GCATCTGTTA

ACAATAAAAT

ATGGATCAAT

ACTATTTCTA

TTGGGAGCGA

CTAAGAAATC

GTAGATAATC

AACAAGAAAC

GGATGGTATT

AAATAATAAA

ATATATAATA

GCTATAAGGT

AACGTAAATA

GTAAAATTAT

AAAGCTGCTA

ATAGAACAGA

TCATTAACTA

TACGATGTAC

ATTGATCTTA

AAGAATATAG

TTACGCTGGC

TCCTTATTAC

CCTTGTTCGA

CGAGAGTTAG

TGTGTGGCCA

CTTTATCTCA

GACTAATTAA

ATGACTAGTT

CAGGAATTTTT

CTATTTTAAj-,

ACTTTTACGA

ACATGACAGG

GTGATAGATT

TAGTCTGTAA

AAGGATGCGC

TTCTGCTGAJ.4

CGTGGAATCC

GCCTAATGGA

AATALAG TAT C

TAACATGATA

CTTCATTACC

ATAGAGATAT

TACTATCAAC

TACTAGAACA

GTTTAGATAA

TACATTCTOG

GATATTTATT

TGTATGATAAk

ATATACAAAC

ATTTAATTAG

6/30 Pigure 4 tconr'd.) 3601 3661 3721 3781 3841 3901 3961 4 02 1 4 3 21 4381 44 11 4501 4561 41621 4 68 1 4741 4801

GATATTGTTA

CATAAAGGCA

AGTGCCTATA

TAGAAGAAAA

TGACTGTATG

GACACTTTTA

AAATATAGCT

TGATACAAAG

TATTAATATA

AGGTTTCACT

ACTTGACCAC

TAAAGCTATG

CTATAATTCT

CAAGATAGCT

AGC'TAATTCA

ATCTATAAAA

TATATATTCT

TAATCCTAGA

AAATAAATCA

GAATCTAGGA

AACTAATAAT

GATAATAGTA

CTTAAAAATA

AACGAACAAG

GATGTAACAG

GGCAGTCCCT

GAAAGAGGAT

GTTGCATCTA

TTGGTAGGAT

CTGAP.TGCGA

CCTCTATACA

GGTGCTTACG

TTATCTAATA

CTAAATAATC

ATTATGATAA

GAAGGTTTTA

GAATCATGCG

TTTAATATCT

GTTAATAAGA

TTAGCTTTTC

ATAATAGGTA

GATTTACATT

TTAAAATAGA

ATTGTAGTTA

ATGATTTAGG

CACTTCTGTT

TACATTACGC

CTAATGTTAA

AAAACAAALAC

TAGATAAACA

TCTTATTATA

TGGCAGTTAG

TAAATGCTAA

GTTTAATAA

ACGGTAATAC

TATCTAAAAT

TAGTAAACAT

AAAAAGAACT

TTCTTGACAA

TACCTGCATG

ATAGACATCA

GGTTACCTA'r

CTGTTATCAC

TAAAAGTTTA

CGATATAATA

TAAAACCCCA

AAATCTAGGA

TGTT'TCACGT

TGTGGTTAAT

TATAGTAAAC

'I'TT-ATTCAC

TrGTTCCTA'r

T-TCTATGAAA

AG CTAAGTTA

TATAAAATTA

GCCTCTAACT

GATGTTAGAA

GGAACATATA

AGATGTTATA

TAACATAGAT

TATACGTATA

GCTAATAGTT

AGATATCAAA

CAGCTGTTGT

TTTTTGCATA

GCGTTACTTA

TTACATCATT

GCTGATATAA

AACGATATCG

AATCATATAG

TTAT7ACTFGA

ATAGCTATAG

G TAA CCT C

ACAGAATTTG

TCTGGAAATA

CTTTTATCTT

TGTGTTAGCT

ATATCTAAAA

AACAGTAATA

ACACATATAA

CTTATGGTAA

TATAGGGAAT

AAAG CTGTAA

CATATAATAA

AACCCAGTAG

AACAGTATCT

TAAATCACGG

CGGTAATTAA

ACGTAATAGA

AAACAACAA-A

ATACCGTTCT-

AGTACGGTAC

AAATIGALAAGIA-

A T A T i--A T 1h 1TI~AATCTT

CTCCTTTACIA

ATAACGCCGA

TTTTAGATGA

ATCCTGAALAT

AAAC-ACTACT

AGTTAAATTC

AGTTCGTAAC

TAATACGGAJ.

AAGAGAGTAA

TGGAACTATT

TATAAAG

7/30 Figure 1 61 121 181 241 301 361 421 481 54 1 601 661 721 781 841 901 961 1011 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741

ATGATTGTGC

ACAAATAATG

AGAGA'PTTTC

CCTACAGAGG

AATAATATAC

CGTGGTAAAC

TATAGGGATG

IkATCGCCATA C CTGACAGAA

CTTGAGTGGA

ATCAATACTA

TGGGAATACA

AATAACACCA

TATGCTACCA

AACAATTGGT

CAACCATTAT

TF1'TGTTTTG

GATGTTATTA

GTATTTTCAC

GTGAGTGAGT

CGATACTGTT

AGTGTAAAGG

TTTAGCACAT

TTTTGGACAA

ATTAAAAATG

GCTAATTTGA

AGTGTTGTGT

GGTATGAAGC

CCAATGCAGG

GTTCATTCCA

TCGTAACTTG

AATGCATACA

TGTTTAGTAA

TGTGGTACAA

ATGCCTTTA

CATTATTATT

ATGTGCAACA

TTAACTATGA

AAATTCCTTT

ATGATGACTT

ATTGGTTTAA

GTGCTGCATA

ATGGTCTAAA

ATGTATTTC

TCTTGCTTAC

TGATTAATTG

AAGGTGCACA

GATTCAACCT

TGAATACAAC

CTAGTTCTTA

ATGTACTTTA

AAATCGCTAT

TTCC7ATTGG

TTGCTTACAC

TGACGTATTG

ATAATGGATT

TATTACCTAG

TTAGTGGTTA

ATAACAATAC

CTTGCAAAAG

CCTCTTGTTG

AGTTAACGTA

CTTTAAAGAA

CTGCTCTAGA

TTTTGTTATG

TCATGTGCAT

PLAGGCCCCTT

ACAATTCACC

CTCTGTCATA

TGTTACAGCT

CAATGTCACA

TGCTTACCAA

AACCTATGAA

TCCGACATCA

AAATAGTTCC

CTTGTGGCCA

GTTTAGCCAA

TAATTTCACT

AGGTGGTGTC

CAGTTATGGT

CAATGGCACA

TAGTAAGTGG

TTGTATATCT

ATCGTATACT

TAACAGTCAC

TTATCCTGTT

CTTTTTCACA

TGGTCAACCC

TGATGTGTAC

TTCTTTATGG

TTATGTTCAT

ACACAATTGG

GAAGGAAGTG

ACAGCTCGAA

GAAGCCATGG

GGTGAGCCTG

TTAAAACATG

TCCAACCAGT

CCCACGGACA

TATATTAGTG

CTTTTGTATT

GGTc3TTTCTA

TTATGTGAAG

GGTGGTTACA

ACTTTTGTTA

GTGCCCAGTT

TGTAATGGTG

GCAGATGTAC

ATTCTTGAAA

GAAATCCCGT

GCTCTTAAAT

GGCCATTTTT

TTTAATTTAA

GAAGCATTAG

ATTAATAACA

GCTTCAAGTG

TACACCGCTG

ATAGCCTCGA

TGTATTCGTT

GACAATA=T

ACCACACAGT

CTGGCAATGA

TAGTTGTTGG

CTACTGCCTT

AAAATAGCAC

TTAGTGTTA'T

GGTTAGTC-TG

GGAATTCCAC

ATGC-AACIVAJ\

GTCGTTCTTA

CACCTCAAC

ACTTCACTTA

ATTATGA.ACA

TACCTGATCG

GTGGCAGGTT

TTGGTGTAC

TGTCTTTAAA

ALAT CTGG TAT

TTTCATGTTA

TCGGCATAA4C

ATTTAGGAAC

ATATTAATCG

CCACTGGTOT

TACAAC-TTGA

TTAAATGTTC-

AAGTAGGTTT

TCAATATAAC

CACTAAGTAA

CTAACCAATT

TTAATCAAGA

TTTGAGTACA

AAACCTTATC

TGGTTATTAC

TCAGTATTTT

TGGTAATGCA

TATATCGGCT

CATAACTAAA

ATGTACGGGT

AAT'1'CTTGC-'T

TCACTTGAAC

CACTGCTACC

TTACA.AGTTA

TTGCACTGGC

ATTTAGTTTT

TGTAACAAAT

AC CACAAGAA

TAACACACTG

GGGTGCTACA

TACTCACACA

TGACGGACCA

ATTACCACCC

TTACAATTTC

TACTCCAGCT

AAACACAGCT

-AACTTACT

CGTTAATAAC

CATTGATCTT

CATCACACTA

CTCACTTTAT

CTGCACGGAT

8/30 Figure 5 (cont'd.) 1801 GTTTTAGAGG 1861 AACAATTACT 1921 AAGT'=GATC 1981 ATATATGAAG 2041 TTGTCTGTGC 2101 GGTATTATTA 2161 GGTGATr1'GT "221 GATGTAAGCG 2281 AACAGTGAAC 234 1 ATATATPATT 2; 01 TGTGAACCTG '461 ATTAACGTCA 2521 CCTACAAATT 2581 TCAATAGATT 2641 CAATATGTGT 2701 AACATGGAGG 2761 GAGGCGTTCA 2821 GGTTCTTGGC 2881 GGTTCTGCTA 294 1 GATGAAGATT 3001 TATTACAATG 3061 ACAGCATCAC 3121 CCTTTTGCAG 3181 AATAAAAACC 3241 GCTTTTGGTA 3301 AAAGCGTTGG 3361 ACAGTACAAT 3421 AGGCTTGACG 3481 GCACTTAATG

CTACAGCTGT

TGACTTTTAA

TTGCTGCACG

AAGGAGACAA

TACACCTAGA

GACGAACTAA

TAGGCTTTAA

CACAAGCGGC

TGTTAGGTCT

ACACAAGTGA

TCATAACCTA

CACATTCTGA

TTACCATATC

GTGCAAGATA

CTGCATGTCA

TTGATTCCAT

ATAGTACAGA

TAGGAGGTCT

TAGAAGATTT

ATAAACGTTG

GCATCATGGT

TTGCAGGTGG

TAGCAGTACA

AACAGATCCT

AGGTTAATGA

CAAAAGTGCA

TGCAAAATAA

AACTGAGTGC

CA=TGTGTC

TATAAAAACT

CAAGTTCTGT

TACAAGAACC

CATAGTGGGT

CTCCTGTACA

CAGTACGCTA

AAATGTTAGT

TGTTATTGAT

AACACATTGG

GAGGACTCG'r

TTCTAATATA

CGGAGACGTG

TGTGCAAGTT

CGTTTGTAAT

AACTATTGAA

GTTGTTTGTC

AAATTTAGAT

AAAAGATATA

GCTTTTTAT

TACTGGTGGT

TCTACCAGGT

TATAACATTA

GGCTAGACTT

GGCTAATGCT

TGCTATACAT

AGATGTTGTC

TTTTCAAGCC

TGATGCACAP

TCAGACT=T

GGTACTTGTC

TTGTCGTTGA

AATGAGCAGG

GTACCGTCTG

GATTACAATA

CTTAGTGGCT

GATGGTGTCA

GGTGCCATAG

ACAACGACAC

GGCACTGCAA

GGTGTTTGTA

CAACCAATTA

GAATACATGC

GGTAACCCTA

CAAGCACTTG

TCGGAAALATG

CCTATTTACA

CTACCGTCCC

AAAGTTGTAA

TACGACATAG

GTAGCTAATG

GGTGCACTTG

AATTATGTTG

TTCAATCAAG

CAAACATCAC

AACACACAAG

ATTAGTAG FI

GTTGATAGGC

ACCAGACAAC

CTTTCTCATT T GTCCTGTTGG TI TTGTTAGAAG TI ATAATAGCGG T TATATGt3TAG TATATTACAC I TTTATTCTGT G TTGGAGCTAT C r2TAATTT-TA 7TGACAGTAA

AAAATGGTGC

GCACTGGTAA

AGGTTTACAC

GATGTAACAA

CAATGGGTGC

CCCTTAAATT

AAGAATGGCC

ATAATAGCAA

CATCTGGTTT

CAGACTTGGT

CTGACAAGAT

GTGGTGGCGC

CTCTACAAAC

CTATTGGTA.A

AAGGTCTTGC

GGCAAGCTTT

CTATTAGTGA

TGATTACAGG

CAGAGGTTAG

'GATAAATTG

'GCTAATTGC

CTATATGTA

CTGCACGAT

ACTGGTGTT

TCACTATCA

;ACGCCATGT

ACTTCCATT

['ACTAC7 C-'

CGATGTTGAT

TTGGTTTT-

IGTCACGATA

TACACCAGTA

ATTGTTAACA

CAGACTTGAA

GGCATCTGTT

TAG CATAGGT

ACGTAAGTAT

AGGTACAGTT

GTGTGCTCAA

GACTATGTAC

CGTGGCTATA

TGATGTATTG

CATTACACAG

CACTGTTGCT

AAGTCACCTT

TATTTATAAC

TAGACTTACA

GGCTAGTAGA

.999 .9 9 3541 CAACTTGCCA AAGACAAGGT TAATGAATGT GTTAGGTCTC AGTCTCAGAG ATTCGGATTC 9/30 Figure 5 (cont'd.) 3601 3661 3721 3781 3 84,1 3901 3961 4C021 4 08 1 414 1 4 20 1 4 2 61

TGTGGTAATG

TTTCATACAG

GCTTCAGATG

CGTAATCTAG

ACTAGTTCTG

ATTGATTTGC

TTAGAAAATT

ACCTATTTAA

AACAC'-AC.Zk

GAATGGCTCA

GGTTTAGTAG

TGTGGATGCA

GTACACATTT

TACTATTACC

GCGATCGCAC

ATGACAAGTT

ATTTTGTTCA

CTAGTATTAT

ACAGACCAAA

ATCTGAC-TGG

TAGA.ACTTGC'

ATrAGAATTGA

TAGTATTTTG

TAGGTTGTTT

GTTTTCACTA

AACAGCTTAT

TTCGGACTT

CTATTTGACC

AATTGAAGGG

ACCTGACTAT

CTGGACTGTA

TGAAATTGAT

CATTCTCATT

AACTTATGTA

CATACCATTA

AGGAAGTTGT

GCAAATGCAG

GAAACTGTAA

GTCGTTAAAG

CCCAGAACTA

TGTGATGTGT

ATTGACATTA

CCTGAATTTA

GACTTAGAGT

GATAACAT7 IA

AAATGGCCTT

CTC CTATTTT

TGTCACTCTA

CACCAAATGG

CAGCTTGGTC

ATGTGCAGTT

TGTATCAGCC

TGTTTGTCAA

ATCAAACTGT

CACTTGATAT

TTACCTCAGA

A-AATACATT

2OTArC'1GTG

GCTGTTTTAG

TATGTAGTAG

CATGATTTTC

AGGTATTTGT

GACGTTGTTT

TAGAGTTGCA

CGCGACTGTA

TCA-AGACATA

TTTCAACGCA

AAAGCTACAT

AGTCAA-C-:-

GCTACTGATA

CACAC-CTTG'I

AAGACAATTT

4321 GAAAATTATG AACCAATTGA AAAAGTGCAT GTCCACTAA 10/30 Figure 6 1 61 121 181 241 301 361 42 1 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1 501 1561 1621 1681 1741 GAG CTCGCGG

TACAAAGGTA

GATGATAGTA

TAATCATCAC

CGATGTATTT

TrACATAATGG

ATAAAAATAC

TTATTAGACA

TATACTTAAA~

AGAAATA-ATC

TCGTAACTTG

AATGCATACA

TGTT'PAGTAA

TGTGGTACAA

ATGCCTTTTA

CATTATTATr

ATGTGCAACA

TTAACTATGA

AAATTCCTTT

ATGATGACTT

ATTGGTTTAA

GTGCTGCATA

ATGGTCTAAA

ATGTATTTGC

TCTTGCTTAC

TGATTAATTG

AAGGTGCACA

GATTCAACCT

TGAATACAAC

CTAGTTCTTA

CCGCCTATCA

TTCATATTTC

GATAATAGAT

GCGTTCATAA

GAGAGAGATT

AT=]TGTTAT

TTACTTACGA

AGGTGAAAAC

AAGTGAAAAT

ATAAATTATT

CCTCTTGTTG

AGTTAACGTA

CTTAAAGAA

CTGCTCTAGA

'FFTGTTATG

TCATGTGCAT

AAGG CCC CTT

ACAATTCACC

CTCTGTCATA

TGTTACAGCT

CAATGTCACA

TGCTTACCAA

AACCTATGAA

TCCGACATCA

AAATAGTTCC

CTTGTGGCCA

GTTTAGCCAA

TAATTTCACT

AGGTGGTGTC

CAGTTATGGT

AAAGTCTTAA

CTATCAATTC

ACGCTCATAT

GTTTCAACTG

GGACATCTAA

CATCAGTTAT

AAAAATGACT

GAAACTATr

AAATACAAAG

TCATTATCGA

TTATGTTCAT

ACACAATTGG

GAAGGAAGTG

ACAGCTCGAA

GAAGCCATGG

GGTGAGCCTG

TTAAAACATG

TCCAACCAGT

CCCACGGACA

TATATTAGTG

CTTTTGTATT

GGTGTTTCTA

TTATGTGAAG

GGTGGTTACA

ACTTTTGTTA

GTGCCCAGTT

TGTAATGGTG

GCAGATGTAC

ATTCTTGAAA

GAAATCCCGT

TGAGTTAGGT

TAAAGTAGAT

AATGACTGCA

CATAGATCAA

CTACGCTAAA

ATTTAACATA

AATTAGCTAT

GTAGCTTAAT

GTTCTTGAG]

I'CCGTTAAG'T

ACCACACAGT

CTGGCAATGA

TAGTTGTTGG

CTACTGCCTT

AAAATAGCAC

TTAGTGTTAT

GGTTAGTGTG--

GGAATTCCA(C

ATGGAACAAA

GTCGTTCTTA

CACGCTCAAG

ACTTCACTTA

ATTATGAACA

TACCTGATGG

GTGGCAGGTT

TTGGTGTAGC

TGTCTTTAAA~

AATCTGGTAT

TTTCATGTTA

TCGGCATAAC

GTAGATAGTA

GATATTAATA

AATTTGGACG

AATCTCACTA

GAAATTACAG

AGTACAATAA

AAAAACCCTT

TATAGAGC

'T7CTGTTAA

TTGTATCGTA

TTTGAGTACA

AAACCTTATC

TGGTTATTAC

TCAGTATTTT

TGGTAATGCA

TATATCGGCT

CATAACTAAA

A'7GTACGGGT

AATCTATGGT

TCA CTTGAA C

CACTGCTACC

TTACAAGTTA

TTGCACTGGC

ATTTAGTTTT

TGTAACAAAT

AGCACAAGAA

TAACACAGTG

GGGTGCTACA

TAGTGACACA

TGACGGACCA

TAGATATTAC

ACTCAAAGAT

GTTCACATTT

AAAAGATAGC

TTATAAATAA

AAAGTATTAA

AATTAATTAG

TTCTTATTC

ATTGAAAC(-'

ATGATTGTGC

ACAAATAATG

AGAGATTTTt' CCTACAGAG1

AATAATATAC

CGTGGTAAAC

TATAGGGATG

AATCGCCATA

GCTGACAGAA

CTTGAGTGGA

ATCAATACTA

TCGGAATACA

AATAACACCA

TATGCTACCA

AACAATTGGT

CAACCATTAT

TTTTGTTTTG

GATGTTATTA

GTATTTTCAC

GTGAGTGAGT

CGATACTGTT

'9 11/30 Figure 6 (cont'd.) 1801 1861 1921 1981 2 04 1 2 1 01 2 4 61 2 59 1 2641 2701 27 61 282 1 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541

ATGTACTTTA

AAATCGCTAT

TTCCTATTGG

TTGCTTACAC

TGACGTATTG

ATAATGGATT

TATTACCTAG

T'TAGTGGTiTA

ATAACAATAC

CTTGCAAAAG

CTACAGCITGT

TGACTTTTAA

TTGCTGCACG

AAGGAGACAA

TACACCTAGA

GACGAACTAA

TAGGCTTTAA

CACAAGCGGC

TGTTAGGCCT

ACACAAGTGA

TCATAACCTA

CACATTCTGA

TTACCATATC

GTGCAAGATA

CTGCATGTCA

TTGATTCCAT

ATAGTACAGA

TAGGAGGTCT

TAGAAGATTT

ATAAACGTTG

CAATGGCACA GCTCTTAAAT ATTTAGGAAC ATTACCACCC AGTGTAAAGG

TAGTAAGTGG

TTGTATATCT

ATCGTATACT

TAACAGTCAC

TTATCCTGTT

CTTTTTCACA

TGGTCAACCC

TGATGTCTAC

TTCTTTATGG

TATAAAAACT

CAAGTTCTGT

TACAAGAACC

CATAGTGGGT

CTCCTGTACA

CAGTACGCTA

AAATGTTAGT

TGTTATCGAT

AACACATTGG

GAGGACTCGT

TTCTAATATA

CGGAGACGTG

TGTGCAAGTT

CGTTTGTAAT

AACTATTGAA

GTTGTTTGTC

AAATTTAGAT

AAAAGATATA

GCTTTTTGAT

TACTGGTGG7

GGCCATTTCT

TTTAATTTAA

GAAGCATTAG

ATTAATAACA

GCTTCAAGTG

TACACCGCTG

ATAGCCTCGA

TGTATTCGTT

GACAATATTT

GGTACTTGTC

TTGTCGTTGA

AATGAGCAGG

GTACCGTCTG

GATTACAATA

CTTAGTGGCT

GATGGTGTCA

GGTGCCATAG

ACAACGACAC

GGCACTGCAA

GG3TGTTTGTA

CAACCAATTA

GAATACATC

GGTAACCCTA

CAAGCACTTG

TCGGAAAATG

CCTATTTACA

CTACCGTCCC

AAAG FTGTAP

TACGACATAC

ATATTAATGG

CCACTGGTGT

TACAAGTTGA

TTAAATGTTC

AAGTAGGTTT

TCAATATAAC

CACTAAGTAA

CTAACCAATT.'

TTAATCAAGA.

CTTTCTCATT

GTCCTGTTGG

TTGTTAGAAG

ATAATAGCGG

TATATGGTAG

TATATTACAC

TTTATTCTGT

TTGGAGCTAT

CTAATTTCTA

TTGACAGTAA

AAAATGGTGC

GCACTGGTAA

AGGTTTACAC

GATGTAACAA

CAATGGGTGC

CCCTTAAATT

AAGAATGGCC

ATAATAG CAA CATCTGGrT CAGACTTGGTl

TTACAATTTC

TAGTGGAGCT

AAACACAGCT

TCAACTTACT

CGTTAATAAG

CATTGATCTT

CATCACACTA

CTCAGTTTAT~

CTGCACGGAT

TGATAAATTG

TGCTAATTGC

TCTATATGTA

TCTGCACGAT

AACTGGTGTT

ATCACTATCA

GACGCCATGT

C-ACTTCCATT

TTACTACTCT

CGATGTTGAT

TTTGGTATTT

TGTCACGATA

TACACCAGTA

ATTGTTAACA

CAGACTTGAA

GGCATCTGTT

TAGCATAGGT

ACGTAAGTAT

AGGTACAGTT

GTGTGCTCAA

TTTAGCACAT

TTTTGGACAA

TTTAAAAATG

GCTAATTTGA

AGTGTTGTGT

GGTATGAAGC

CCAATGCAGC

GTTCATFTC C.

GTTTT'AGAGC]

AACAATTACE

AAGTTTGAT2-

ATATATGAAC

TTGTCTGTC-

GGTATTATTA

GGTGATTTGT

GATGTAAGCG

AACAGTGAAC

ATATATAATT

TGTGAACCTO

ATTAACGTCA

CCTACAAATT

TCAATAGATT

CAATATGTGT

AACATGGAGG

6AGGCGTTCA

GGTTCTTGGC

GGTTCTGCTA

GATGAAGATT

TATTACAATG

12/30 Figure 6 (cont'd.) 3601 3661 3721 3781 38,;1 3 91 4 2?? ,49 1 4501 46z 1 48 Cl 4 8621 4981 50411 101, 5161 5221 5281 534 1

GCATCATGGT

TTGCAGGTGG

TAGCAGTACA

AACAGATCCT

AGGTTAATGA

CAAAAGTGCA

TOCAAAATAA

.PACTGAGTGC

'ATTTCTGTC

.!A-GACAAGGT

I-ACACATTT

TACTATTACC

C "CGATCGCAC

ATGACAAGTT

ATTTTGTTCA

CTAGTATTAT

ACAGACCAAA

ATCTGACTGG

TAGAACTTGC

ATAGAATTGA

TAGTATTTTG

TAGGTTGTTT

AACCAATTGA

ATGAGTATAT

GTAATAGACA

AATTAGATAA

TCAGACACAC

ACTTATGGGT

AGACTTATTC

ATTAAAATAG

TCTACCAGGT

TATAACATTA

GGCTAGACTT

GGCTAATGCT

TGCTATACAT

AGATGTTGTC

TTTTCAACCC

TGATGCACAA

TCAGACTCTA

TAATGAATGT

GTTTTCACTA

AACAGCTTAT

TTTCGGACTT

CTATTTGACC

AATTGAAGGG

ACCTGACTAT

CTGGACTGTA

TGAAATTGAT

CATTCTCATT

AACTTATGTA

CATACCATTA

AGGAAGTTGT

AAAAGTGCAT

ATALATTGAAA

GGAACTGGCA

AAATGATACA

C'PrATTACAA

ATAATATAAT

CATCAACCCC

ATTGTTTAAG

GTAGCTAATG

GGTGCACTTG

AATTATGTTG

TTCAATCAAG

CAAACATCAC

AACACACAAG

ATTAGTAGTT

GTTGATAGGC

AC CAGACALAG

GTTAGGTCTC

CAAATGCAG

GAAACTGTAA

GTCGTTAAAG

CCCAGAACTA

TGTGATGTGT

ATTGACATTA

C CTGAATTTA

GACTTAGAGT

GATAACATTA

AAATGGCCTT

CTGCTATTTT

TGTCACTCTA

GTCCACAAGG

AAGTAAAATA

GATTCTTCTT

GCAAATACAG

ACTAACTAAG

AAAGATTCAT

TTCAAACCTT

AGATGTAAAT

CTGACAAGAT

GTGGTGGCGC

CTCTACAAAC

CTATTGGTAA

AAGGTCTTGC

GCCAAGCTTT

CTATTAGTGA

TGATTACAGG

CAGAGCT'IAG

AGTCTCAGAG

CACCAAATGG

CAGCTTGOTC

ATGTGCAGTT

TGTATCAGCC

TGTTTGTCAA

ATCAAACTGT

CACTTGATAT

TTAGGTCAGA

ATAATACATT

GGTATGTGTG

GCTGTTTTAG

TATGTAGTAG

TACAATTCTT

TAAATCATAT

CTAATGAAGT

CTTCATTCAA

TCAGATGATG

GATATTAATA

TCTGGATATT

AATTATTTGG

GACTATGTAC

CGTGGCTATA

TGATGTATTG

CATTACACAG

CACTGTTGCT

AAGTCACCTTr

TATTTATAAC

TAGACTTACA

GOCTAGTAGA

ATTCGGATTC

CATGATTTTC

AGGTATTTGT

GACCTTGTTT

TAGAGTTGCA

CO CGAC TOTA

TCAAGACATA

TTTCAACGCA

AAAGCTACAT

AGTCAATCTT

GCTACTGATA

CACAGGTTGT

AAGACAATTT

TTTATTGATT

AATAATGAAA

AAGTACTGCT

CGAATTACCT

AGAAAGTAAA

ATTTACTTAA

ATAAAATACC

AGGTAAAGGA

ACAGCATCAC

CCTTTTGCAG

AATAAAAACC

GCTTTTGGTA

AAAGCGTTGG

ACAGTACAAT

AGGCTTGACC

3 CACTTAATG

CAAC'TIC;CCA!.

TGTGGTAA-D]

T-TCATACAG

GCTTCAGATG

CGTAATCTAG

ACTAGTTCTG

ATTGATTTGC

TTAGAAAATT

ACCTATTTAA

AACACTACAG

GAATGGCTCA

GGTTTAGTAG

TGTGGATGCA

GAAAATTATG

AACTAGTCAA

CGAAATATCA

AAATCTCCAA

TTTAATTTTT

TATAAATTTA

CGATGTTAAT

AGTTAATGAT

TATAAAATTA

a. a a a a.

13/30 Figure 6 (cont'd.) 5401 GTCTATCTTT CACATGGAAA TG3AATTACCT AATATTAATA ATTATGATAG GAATFTTTTTA 5461 5521 5581 5641 5701 5761 5821 8 81 5941 6001 6061 6121

GGATTTACAG

TGTAACGGGA

TCTATAAACA

ACAACAAAAA

AGTCTATCTT

GAAAGAAATG

TTAAAATCTA

CATAACAG TA

GGAATAAGCA

AATACTAAAA

GAAGAAATAC

CAAACTTGTA

CTGTTATATG

AGCAGCATTC

TTTTACCACA

AATTTAACGA

ATCTACAAGA

TATACAAAAA

GACTTAGTAT

GTACATTAAT

ATATGCCAAT

ATAGGATACG

TTTGTTCTAT

TTTATGALAGG

TATCAACAAT

TATGGTAACT

AATAATAGGA

TGTATGGCCA

TATGAAAGAA

CGTGGAAGCT

AACAAAACAC

CAG TGATATA

TATCTCTAAT

TGATAGGCTC

ACCTTCGGAG

TAC'-

ACAGGCAGAT

GGCCTATGTT

iCCTCTAGAT

GAAGTATTTT

GATAATCATT

TTTATATTAA

TTAAAT GCCA CT01GJAACGAT

ATTTTAACT

-TAAAAJGCTG

GAAAGAACTT

CTATGGTTAT

TAATAGCCAG

ATTTAATATT

CTACTA-ATAA

TAGTAGTAGC

ATAGCATATT

ATATCGATTC

C lACAGA C TC

TAGAACTAAA

CAATAAATAG

TAGAACAACT

GGTAAAACAC

ATCAT=rAC

ATATCTAACA

AGATAAAGA'I

TACTAATATG

ACTAGAAGAT

TATATTT CAT

AACT-A'IGCAA-,

ACGTTCTACC

TAAGGATGTA

TAAGTTTAAT,

14/30 igure 7 841 901 961 1021 1081 1 14 1 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 GAG CTCGCGG

TACAAAGGTA

GATGATAGTA

TAATCATCAC

CGATGTATTT

TACATAATGG

ATAAAAATAC

TTATTAGACA

TATACTTAAA

AGAAATAATC

ATAATGAATG

ATTTTCTGTT

CAGAGGTGTG

ATATACATGC

GTAAACCATT

GGGATGATGT

GCCATATTAA

ACAGAAAAAT

AGTGGAATGA

ATACTAATTG

AATACAGTGC

ACACCAATGG

CTACCAATGT

ATTGGTTCTT

CATTATTGAT

GTTTTGAAGG

TTATTAGATT

TTTCACTGAA

GTGAGTCTAG

ACTGTTATGT

CCGCCTATCA

TTCATATTC

GATAATAGAT

GCGTTCATAA

GAGAGAGATT

ATTTTGTTAT

TTACTTACGA

AGGTGAAAAC

AAGTGAAAAT

ATAAATTATT

CATACAAGTT

TAGTAACTTT

GTACAACTGC

CTTTTATTTT

ATTATTTCAT

GCAACAAAGG

CTATGAACAA

TCCTTTCTCT

TGACTTTGTT

GTTTAACAAT

TGCATATGCT

TCTAAAAACC

ATTGCTCCG

GCTTACAAAT

TAATTGCTTG

TGCACAGTTT

CAACCTTAAT

TACAACAGGT

TTCTTACAGT

ACTTTACAAT

AAAGTCTTAA

CTATCAATTC

ACGCTCATAT

GTTTCAACTG

GGACATCTAA

CATCAGTTAT

P.AAAATGACT

GAAACTATTT

AAATACAA-AG

TCATTATCGA

AACGTAACAC

AAAGAAGAAG

TCTAGAACAG

GTTATGGAAG

GTGCATGGTG

CCCCTTTTAA

TTCACCTCCA

GTCATACCCA

ACAG CTTATA

GTCACACTTT

TACCAAGGTG

TATGAATTAT

ACATCAGGTG

AGTTCCACTT

TGGCCAGTGC

AGCCAATGTA

TTCACTGCAG

GGTGTCATTC

TATGGTGAAA

GGCACAGCTC

TGAGTTAGGT

TAAAGTAGAT

AATGACTGCA

CATAGATCAA

CTACGCTAAA

ATTTAACATA

AATTAGCTAT

GTAGCTTAAT

G-TTCTTGAGG

TCCGTTAAGT

AATTGGCTGG

GAAGTGTAGT

CTCGAACTAC

CCATGGAAA-A

AG CCTGTTAG

AACATGGGTT

ACCAGTGGAA

CGGACAATGG

TTAGTGGTCG

TGTATTCACG

TTTCTAACTT

GTGAAGATTA

GTTACATACC

T ]GTTAGTGG

CCAGTTTTGG

ATGGTGTGTC

ATGTACAATC

TTGAAATTTC

TCCCGTTCGG

TTAAATATTT

GTAGATACTA

GATATTAATA

AATTTGGACG

AATCTCACTA

GAAATTACAG

AGTACAATAA

AAAAACCCTT

TAATTAGAGC

GTGTGTTAA

TTGTATCGTA

CAATGAAAAC

TGTTGGTGGT

TGCCTTTCAG

TAG CACTGGT

TGTTATTATA

AGTGTGCATA

TTCCACATGT

PA4CAAAAATC

TTCTTATCAC

CTCAAGCACT

CACTTATTAC

TGAACATTGC

TGATGGATTT

CAGGTTTGTA

TGTAGCAGCA

TTTAAATAAC

TGGTATGGGT

ATGTTATAGT

CATAACTGAC

AGGAACATTA

TAGATATTAC

ACTCAAAGAT

GTTCACA'l

AAAAGATAGC

TTATAAATAA

AAAGTATTAA

ALATTAATTAG

'1T7CTTTATTC

A'-TCGAAAGCG-

ATGACAACAA

CTT ATCAGArG

TATTACCCTA

TATTTTAATA

AATGCACGTG

TCGGCTTATA

ACTAAAAATC

ACGGGTGCTG

TATGGTCTTG

TTGAACATCA

GCTACCTGGG

A.AGTTAAATA

ACTGGCTATG

AGTTTTALACA

ACAAATCAAC

CAAGAATTTT

ACAGTGGATG

GCTACAGTAT

GACACAGTGA

GGACCACGAT

CCACCCAGTG

15/30 Figure (cont'd.) 1801 TAAAGGAAAT 1861 GCACATTCC 1921 GGACAATTGC 1981 AAAATGTGAC 204 1 ATTTGAATAA 21f;1 TTG'FGTTATT 21(1 TGAAGCTTAG 221TGCAGGATAA

"JATTCCAC-TTC

231 AGAGGCTAC 2461! TTGATGTTGC 2521 ATGAAGALAGG 251CTGTGCTACA 2641. TTATTAGACG 2701 ATTTGTTAGG 2761 TAAGCGCACA 2E21 GTGAACTGTT 2881 ATAATTACAC 2 ci: ACCTGTCAT 3001 ACGTCACACA 3061 CAAATTTTAC 3121 TAGATTGTGC 3181 ATGTGTCTGC 3241 TGGAGCTTGA 3301 CGTTCAATAG 3361 CTTGGCTAGG 3421 CTGCTATAGA 3481 AAGATTATAA 3541 ACAATGGCAT

CGCTATTAGT

TATTGGTTGT

TTACACATCG

GTATTGTAAC

TGGATTTTAT

ACCTAGCTTT

TGGTTATGGTF

CAATACTGAT

CAAAGTTC*-

AGCTGTTATA

TTTThACAAG

TGCACGTACA

AGACALACATA

CCTAGACTCC

AACTAACAGT

CTTTAAAAAT

AGCGGCTGTT

AGGCCTAACA

AAGTGAGAGG

AACCTATTCT

TTCTGACGGA

CATATCTGTG

AAGATACGTT

ATGTCAAACT

TTCCATGTTG

TACAGAAAAT

AGGTCTAAAA

AGATTTGCTT

ACGTTGTACT

CATGGTTCTA

AAGTGGGGCC

ATATCTTTTA

TATACTGAAG

AGTCACATTA

CCTGTTGCTT

TTCACATACA

CAACCCATAG

GTGTACTGTFA

7TATC-GGACA

AAAACTGGTA

T-CTGTTTGT

AGAACCAATG

GTGGGTGTAC

TGTACAGATT

ACGCTACTTA

GTTAGTGATG

ATCGATGGTG

CATTGGACAA

ACTCGTGGCA

AATATAGGTG

GACGTGCAAC

CAAGTTGAAT

TGTAATGGTA

ATTGAACAAG

TTTGTCTCGG

TTAGATCCTA

GATATACTAC

TTTGATAAAG

GGTGGTTACG

CCAGGTGTAG

ATTTCTATAT

ATTTAACCAC

CATTAGTACA

ATAACATTAA

CAAGTGAAGT

CCGCTGTCAA

CCTCGACACT

TTCGTTCTAA

ATrATTTTTAA

CTTCTCCTTT

CCTTGAGTCC

AGCAGGTTGT

CGTCTGATAA

ACAATATATA

GTGGCTTATA

GTGTCATTTA

CCATAGTTGG

CGACACCTAA

CTGCAATTGA

TTTGTAAAAA

CAATTAGCAC

ACATGCAGGT

ACCCTAGATG

CACTTGCAAT

AAAATGCCCT

TTTACAAAGA

CGTCCCATAA

TTGTAACATC

ACATAGOAGA

CTAATGCTGA

TAATGGTTAC

TGGTGTTAGT

AGTTGAAAAC

ATGTTCTCAA

AGGTTTCGTT

TATAACCATT

AAGTAACA'rC '2CAA-TTCTCA

T:A.AGACTFGC

CTICATTTGAT

T!GTTGGTGCT

TAGAAGTCTA

TAG CGGTCTG

TGGTAGAACT

TTACACATCA

TTCTGTGACG

AGCTATGACT

TTTCTATTAC

CAGTAACGAT

TGGTGCTTTG

TGGTAATGTC

TTACACTACA

TAACAAATTG

GGGTGCCAGA

TAAATTGGCA'-

ATGGCCTAGC

TAGCAAACGT

TGGTTTAGGT

CTTGGTGTGT

CAAGATGACT

AATTTCTTTA

GGAGCTTTTT

ACAGCTATTA

CTTACTGCTA

AATALAGAGTG

GATCTTGGTA

ACACTACCAA

GTTTATCTTC

ACGGAI'GTTT-

AAATTGAACA

AATTGCA.AG"T

TATGTAATAT

CACGATTTGT

GGTGTTGGTA

CTATCAGGTG

CCATGTGATG

TCCATTAACA

TACTCTATAT

GTTGATTGTG

GTATTTATTA

ACGATACCTA

CCAGTATCA

TTAACACAAT

CTTGAAAACA

-CTGTTGAGG

ATAGGTGGTT

AAGTATGGTT

ACAGTTGATG

G CTCAATATT

ATGTACACAG

16/30 Figure 7 (cont'd.) 3601 CATCACTTGC AGGTGGTATA ACATTAGGTG CACTTGGTGG TGGCGCCGTG C CTATACC 3661 TTGCAGTAGC AGTACAGGCT AGACTTAATI ATGTTGCTCT ACAAACTGAT GTATTGAATA 3721 AAAACCAACA GATCCTGGCT AATGCTTTCA ATCAAGCTAT TGGTAACATT ACACAGGCTT 3781 38411 3901 3961 41 0 i 201 41.*, ,201 4261 4321 41381 4441 4501 4561 41621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341

TTGGTAAGGT

CGTTGGCAA-A

TACAATTGCA

'FrGACGAACT

TTAATGCATT

TTGCCAAAGA

GTAATGGTAC

ATACAGTACT

CAGATGGCGA

ATCTAGATGA

GTTCTGATTT

ATTTGCCTAG

AAAATTACAG

ATTTAAATCT

CTACAGTAGA

GGCTCAATAG

TAGTAGTAGT

GATGCATAGG

ATTATGAACC

AGTCAAATGA

ATATCAGTAA

CTCCAAAATT

ATTTTTTCAG

AATTTAACTT

GTTAATAGAC

AATGATATTA

AAATTAGTCT

TAATGATGCT

AGTGCAAGAT

AAATAATTTT

GAGTGCTGAT

TGTGTCTCAG

CAAGGCTTAAT

ACATTTG'TTT

ATTACCAACA

TCGCACTTTC

CAAGTTCTAT

TGTTCAAATT

TATTATACOT

ACCAAACTGG

GACTGGTGAA

ACTTGCCATT

AATTGAAACT

ATTTTGCATA

TTGTTTAGGA

AATTGAAAAA

GTATATATAA

TAGACAGGAA

AGATAAAAAT

ACACACCTTA

ATGGGTATAA

TTATTCCATC

AAATAGATTG

ATCTTTCACA

ATACATCAAA

GTTGTCAACA

CAAC CCATTA

GCACAAC-'TTG

ACTCTAAC CA

GAA"TCTGC!T':

TCACTAGCAA4

GCTTATGAAA

GGACTTCTCG

TTGACCCCCA

GAAGGGTGTG

GACTATATTG

ACTGTACCTG

ATTGATGACT

CTCATTGATA

TATGTAAAAT

CCATTACTGC

AGTTGTTGTC

GTGCATGTCC

TTGAAAAAGT

CTGGCAGATT

GATACAGCAA

TTACAAACTA

TATAATAAAG

AACCCCTTCA

TTTAAGAGAT

TGGAAATGAA

CATCACAACG

CACAAGGGCA

GTAGTTCTAT

ATAGGCTGAT

GACAAGCAGA

CGITCTCAC'7,-

ATGCAGCACC-

CTGTAACAGC

TTAAAGATGT

GAACTATGTA

ATGTGTTGTT

ACATTAATCA

AATTTACACT

TAGAGTTTAG

ACATTAATAA

GGCCTTG-TA

TATTTTGCTG

ACTCTATATG

ACAAGGTACA

AA-AATATAAA

CTTCTTCTAA

ATACAGCTTC

ACTAAGTCAG

ATTCATGATA

AACCTTTCTG

GTAAATAATT

TTACCTAATA

TCTTGCCACT

AGCTTTAAGT

TAGTGATATT

TACAGGTAGA

GGTTAGGGCT

TCAGAGATTC

AAATGGCATG

TTGGTCAGGT

GCAGTTGACG

TCAGCCTAGA

TGTCAACGCG

AACTGTTCAA

TGATATTTTC

GTCAGAAAAG

TACATTAGTC

TGTGTGGCTA

TTTTAGCACA

TAGTAGAAGA

ATTCTTTTTA

TCATATAATA

TGAAGTAAGT

ATTCAACGAA

ATGATGAGAA

TTAATAATTT

GATATTATAA

ATTTGGAGGT

TTAATAATTA

GTTGCTAAAG

CACCTTACAG

TATAACACGG CTTACAGCACf AGTAGACNAA I: G GAT TrC

ATTTGTGCTT

TTGTTTCGA

GTTGCAACTA

ACTGTAATT.2

GACATATTAG

AACGCAACCT

CTACATAACA

AATCTTGAAT

CTGATAGGTT

GGTTC-TTGTG

CAATTTGAAA

TTGATTAACT

ATGAAACGAA

ACTGCTAAAT

TTACCTTTTA

AGTAAATATA

ACTTAACGAT

AATACCAGTT

AALAGGATATA

TGATAGGAAT

a

S

S

S. Sa

S.

SS*SS

17/30 Pigure V cont'd.) 5401 5461 5521 5581 5641 5701 576 1 586i 6061 TTTTTAGGAT TTACAGCTGT TATATGTATC AACAATACAG C-CAGATCTAT GGTTATGGTA AAACACTGTA ACGGGAAGCA GCATTCTATG GTAACTGGCC TATGTTTAAT AGCCAGATCA TTTTACTCTA TAAACATTTT ACCACAAATA ATAGGATOCT CTAGATATTT AATATTATAT CTAACAACAA CAAAAAAATT TAACGATGTA TGGCCAGAAG TATTTTCTAC TAATAAAGAT AAAGATAGTC TATCTTATCT ACAAGATATG AAAGAAGATA ATCATTTAG'T AGTAGCTACT AATATGGAAA GAAATGTATA CAAAAACGTG GAACCTTTTA TATTAAATAG CATATTACTA GAAGATTTAA AATCTAGACT TAGTATAACA AAACAGTTAA ATGCCAATAT CGATTCTATA TTTCATCATA ACAGTAGTAC ATTAATCAGT GATATACTGA AACGATCTAC AGACTCAACT ALTGCAAGG.AA TAG.ATAT GCCAATTATG TCTAATAT7T? TACTTC A-AAAC 'rCTACCAATA CTAAAAATAG GATACGTGAT AGGCTGTTAA PAACZT'IGCAAx'1 AJVVTAGTAA3 GATGTAGAAG AAATACTTTG TTCTATACCT TCC-GAGGAAA SPAACTTTAGA ACAACTTAAG TTTAATCAAA CTTGTATTTA TGAAGGTACC 4. S S 5S5S5*

S

S. S

S.

S.

S

*SS*SS

55 S S *5

*.SSSS

S

18/30 Figure 8 6 12 18 24 36 48 66 72 78 84 96 102 108 114 120 126 132 138 144 150 156 162 168 174 1 GAGCTCGCGG 1 TACAAAGGTA 1 GATGATAGTA I TAATCATCAC I CGATGTATTT 1 TACATAATGG 1 ATAAAAATAC 1 TTATTAGACA 2. TATACTTAAA I AGAAATAATC 1 fCTAGATGTAA 1 TTGCAATGCG I ATGCCCTTAA 1 ACAAAGAATO 1 CCCATAATAG 1 TAACATCTCG

CCGCCTATCA

TTCATATTTC

GATAATAGAT

GCGTTCATAA

GAGAGAGATT

ATTTTGTTAT

TTACTTACGA

AGGTGAAAAC

AAGTGAAAAT

ATAAATTATT

CAAATTGTTA

TGCCAGACTT

ATTGGCATCT

GCCTAGCATA

CAAACGTAAG

TTTAGGTACA

GGTGTGTGCT

GATGACTATG

CGCCGTGGCT

AACTGATGTA

TAACATTACA

TGCCACTGTT

TTTAAGTCAC

TGATATTTAT

AGGTAGACTT

TAGGGCTAGT

GAGATTOGGA

TGGCATGATT

GTCAGGTATT

GTTGACGTTG

AAAGTCTTAA

CTATCAATTC

ACGCTCATAT

GTTTCAACTG

GGACATCTAA

CATCAGTTAT

AAAAATGACT

GAAACTAT-77 AAA7ACAAAG

TCATTATCGA

ACACAATATG

GAAAACATGG

GTTGAGC C T

GGTGGTTCT

TATGGTTCTG

GTTGATGAAG

CAATATTACA

TACACAG CAT

ATACCTTTTG

TTGAATAAAA

CAGGCTTTTG

GCTAAAGCGT

CTTACAGTAC

AACAGGCTTG

ACAGCACTTA

AGACAACTTG

TTCTGTGGTA

TTCTTTCATA

TGTGCTTCAG

TTTCGTAATC

TGAGTTAGGT

TAAAGTAGAT

AATGACTGCA

CATAGATCAA

CTACGCTAA-A

ATTTAACATA

AATTAGCTAT

GTAGCTTAAT

GTT'1CTTGAGI

TCCGTTAAGI'

TGTrCTGCAT,3

AGGTTGATT(C

TCAATAGTAC

GGCTAGGAGG

CTATAGAAGA

ATTATAAACG

ATGGCATCAT

CACTTGCAGG

CAGTAGCAGT

ACCAACAGAT

GTAAGGTTAA

TGGCAAAAGT

AATTGCAAAA

ACGAACTGAG

ATGCATTTGT

CCAAAGACAA

ATGGTACACA

CAGTACTATT

ATGGCGATCG

TAGATGACAA

GTAGATAGTA

GATATTAATA

AATTTGGACG

AATCTCACTA

GAAATTACAG

AGTACAATAA

AAAACCCTT

TAATTAGAGC

C?-TGTGTTAA

7TGTATCGTA

.,-,.AL-TATT

CATGTTGTTT

AGAAAATTTA

T2TA-AAGAT

TTTGCTTTTT

T7GTACTGGT

GGTTCTACCA

TGG-ATAACA

ACAGGCTAGA

CCTGGCTAAT

TGATGCTATA

GCAAGATGTT

TAATTTTCAA

TGCT7GATGCA

GTCTCAGACT

GGTTAATGAA

TTTGTTTTCA

ACCAACAGCT

CACTTTCGGA

GTTCTATTTG

TAGATATTAC

ACTCAAAGAT

GTTCACATTT

AAAAGATAGC

TTATAAATAA

AAAGTATrAA

AATTAATTAG

TTCTTTATTC

A TTGAAAGCG

ATIGGGTAACC:

GAACAAGCAC

C-TCTCGGAALA

GATCCTATTT

ATACTACCGT

GATAAAGTTG

GGTTACGACA

GGTGTAGCTA

TTAGGTGCAC

CTTA.ATTATG

GCTTTCAATC

CATCAAACAT

GTCAACACAC

GCCATTAGTA

CAAGTTGATA

CTAACCAGAC

TGTGTTAGGT

CTAGCAAATG

TATGAAACTG

CTTGTCGTTA

ACCCCCAGAA

go S.

S S: of SS of 1 1 1 1 '1 .1 :1 .1 .1 1 1

K

K

TAGCAGACTT

ATGCTGACAA

TTGGTGGTGG

TTGCTCTACA

AAGCTATTGG

CACAAGGTCT

AAGGGCAAGC

GTTCTATTAG

GGCTGATTAC

AAGCAGAGGT

CTCAGTCTCA

CAGCACCAAA

TAACAGCTTG

AAGATGTGCA

19/30 Figure 8 (cont'd.) 1801 CTATGTATCA GCCTAGAGTT GCAACTAGTT CTGATTTTGT TCAAATTGAA GGGTGTGATG 1861 TGTTGTTTGT CAACGCGACT GTAATTGATT TGCCTAGTAT TATACCTGAC TATATTGACA 1921 TTAATCAAAC 1982 TTACACTTGA '041 AGTTTAGGTC 2101 TTPATATAC 1 16. CTTGGTATGTr .221 TTTGCTGTTT- 221CTATATGTAG 23'!11 AGGTACAATT 2 :ATATAAATCA :2461 CTTCTAATGA 25-'1 CAGCTTCATT 2581 AAGTCAGATG 26411 CATGATATTA 2701 CTTTCTGGAT 2761 AATAATTATT 2821 CCTAATATTA 2881 ATACAGGCA 2941 ACTGGCCTAT 3001 GGATCCTCTA 3061 CCAGAAGTAT 3121 GAAGATAATC 3181 GCTTTTATAT 3241 CAGTTAAATG 3301 ATACTGAAAC 3361 AATATTTTAP 3421 CTGTTAAAAC 3481 GAGGAAAGAP

TGTTCAAGAC

TATTITCAAC

AGAAAAGCTA

ATTAGTCAAT

GTGGCTACTG

TAG CACAGGT TAG AAC-ACAA

CTTTTTATTG

TATAATAATG

AGTAAGTACT

CAACGAATTA

ATGAGAAAGT

ATAATTTACT

ATTATAAAAT

TGGAGGTAAA

ATAATTATGA

GATCTATGGT

GTTTAATAGC

GATATTTAAT

TTTCTACTAA

ATTTAGTAGT

TAAATAGCAT

CCAATATCGA

GATCTACAGA

C=TAGAACT

CTGCAATAAA1

CTTTAGAACPA

ATATTAGAPA

GCAACCTATT

CATAACACTA

CTTGAATGGC

ATAGGTTTAG

TGTTGTG CAT

TTTGAAAP'T"

ATTAACTAGT

AAACGAAATA

GCTAAATCTC

CCTTTTA-ATT

AAATATAAAT

TAACGATGTT

ACCAGTTAAT

GGATATAAAA

TAGGAATTTT

TATGGTAAA

CAGATCATTT

ATTATATCTA

TAAAGATAAA

AGCTACTAAT

ATTACTAGAA

TTCTATATTT

CTCAACTATG

AAAACGTTCT

'ACTTAAGTTT

ATTACAGACC

TAAATCTGAC

CAGTAGAACT

TCAATAGAAT

TAGTAGTATT

GCATAGGTTG

ATGAACCAA'T'

CAAATGAGT:,A

TCAGTAATAG

CAAAATTAGA

TT.TTCAGACA

TTAACTTATG

AATAGACTTA

GATATTAAAA

TTAGTCTATC

TTAGGATTTA

CACTGTAACG

TACTCTATAA

ACAACAACAA

GATAGTCTAT

ATGGAAAA

GATTTAAAAI

CATCATAACI

CAAGGAATA?

ACCAATACTI

GTAGAAGAA7

AATCA-AACT'

AAACTGGACT GTACCTGAAT TGGTGAAATT GATGACTTAG TGCCATTCTC ATTGATAACA TGAAACTTAT GTAAAATGGC TTGCATACCA TTACTGCTAT TTTAGGAAGT TGTTGTCACT- TGAAAAAGTG CATGTCrCAC.L TATATAATTG AAAAAGTAAA- ACAGGAACTG GCAGATTCTT- TAAAAATGAT ACAGCAAATA CACCTTATTA CAAACTAACT GGTATAATAT AATAALAGATT TTCCATCAAC CCCTTCAAAC TAGATTGTTT AAGAGATGTA TTTCACATGG AAATGAATTAZ' CAGCTGTTAT ATGTATCA-AC GGAAGCAGCA TTCTATGGTA ACATTTTACC ACAAATAATA AAAAATTTAA CGATGTATGG CTTATCTACA AGATATGAAA ATGTATACAA AAACGTGGAA CTAGACTTAG TATAACAAAA GTAGTACATT AATCAGTGAT GCAATATGCC AATTATGTCT1 \AAAATAGGAT ACGTGATAGG STACTTTGTTC TATACCTTCG F GTATTTATGA AGGTACC 0O 6@ S

S@

'SOS

55 S

S

S

*5 5 0@

S.

S.

*5.S

S

55 0e 55

S

OSSOSS

S

OS S

S.

0e

S

S S 20/30 Figure 93 1 AGATATTTGT 61 GGTCTTATCT 121 TTGCATGAAG 181 TGTACGAGTC 2411 ACTGAYPCAT 301 CGATCTAGAA 361 CCAAACCTA -i 1 CTATTACTAC 4'81 AAGACTAAG 5-1TTAGTTCTGT 601 CCTAAAATAA .661 TCATGATAAT 721 TAAGAGCAG 781 ATATTTCTA 841 TATACGCTTA 901. GAACTAATTT 961 ATGTATAAAG 1021 ATAATAGATA 1081 CCTACTAAGA 1141 GTATAACTAC 1201 ATGTATAATA 1261 AATTATATTT 1321 GTATCTCTTA 1381 AAGCTAAATG 1441 TAATATTAAC 1501 CAAACATGTT 1561 TACTTAAAAA 1621 AACATTTAGA 1681 TAGATAAGAA 1741 TTAAACTCCT

TAGCTTCTGC

ATTCTGTCAG

GTCACTGTGT

CTTCTAACAC

TTTCATCTGT

GTCTAATAAC

CAAATATAGG

TCAAAkAGAGA

CTGTAGAAC

AGATCAGTAA

ATCTGATACA

GATTAATACA

GAATCCCTAG

TGTTAACATA

TTACAGTTAT

TACAAAGTGC

GTATGAATAT

TATTTCTAGA

AAACTAGAAG

TGTTGCTAAC

ACTATATTAC

GTATAATTAT

TTTATAACTT

CTACTAGATT

TCACATTATG

AACAGTTTA

AGATATTAAT

CATTACATTP

CAATCGTACP

ATTAAAAAA3l CGGAGATACC G CAGAGTAGGT I AGAGTTCAAA I TGTGGTTTAT2

CAACGTTTCT

TGCTAAGTAT

AGAAGCTTCT

TATTACATTA

TGTTATGAAG

CGTATAG CAT

GATAATAACT

CAGCGTGTCG

TATALATAGAA

TTTATAGGTA

TAAAAATATA

TTTACCAAAA

CACAAACAGC

TACCATTAAT

ATACATACAT

AGTGACACTG

ACTGGTATTT

ATTATTATAT

ATTAGTAAAG

GATATAAATG

AATACTACTA

AAAGCCATTA

G'FrAATAGAT

TGTA-ATATAC

CCGTTGTTTT

GGCGCGA-ATG

~TGAAAATCT A CCTCTAATG A \CTGATCATC A

PGGCTGGAAT

'TAAGAGATT C %TTATTGGAT T CTTATGAAAC J1 ATTATGTGAT C

AATATCTTAT

ACGAGTATAA

TTGTAAATCA

TTATTTTTTG

ATAATCCATA

AATCCAGGAA

CTTGCAAACA

TGCCAATGGA

AALATCGGCTA

AACCTTATAA

ACTAACGCCA

ATGTTATAAC

TATTTCAGTT

TCAGTGTAGA

TATGTACTAT

AATATGTAAT

ATCACGAAGA

GTAATAAACA

TATTAACTAG

TTATAGAACG

ATGCGGTAAA

TAAATTTACA

TTTTCTGGA A ~CGAAGACAA I

~GTGTTTGAT

LkAAGGATAA I :ATAGGTATT I 'TAACGCGCT I

;ACGCATCCA

'AGATATATT

FTATCGTAGC

%TTCAGCAAT

TTACGATAGT

TGAAAAATAT

GGGTAATTTT

TGTTAGAAGT

AATTACTTAG

TTCCCAAGTT

GCTTGACGTT

TACGAGAGTA

TCATCTTTGA

ATATACTATA

AAGTAAAATA

TCAGTTATAT

AAATTAGTAA

ATGCAGTAAA

GTACAATATA

TTATTCTAAC

TOCACCAGAC

GAATAATGAT

AGATAGTATA

~GGAAAGGGA

'AGTGAATAC

ACTCTAGCG

\GACACCTAT

TTATTACAT

~TAAACG CAT

-FT-TACTCTTA

\CATATAAAG

AGATGCATT;

T AGTAGGTAT

FTCTCTATTA

1TTTCTAAAG

AGTAATGTAC

TACATATOTA

AAAAAAGAAA

TATGTATATA

GAGAAACGGT

TCCTATAATG

ACTACTCATC

TGTGGTATAA

TAGTATTAAA

CTATAAATAT

TGTTTTATAA

TGTAGTATAC

ALI,' ITGATA

ATTAAGTCTT

GAAATATATA

ATAAACATTA

TATGATATGG

GGATATTCA'I

21/30 F~igure 9 (cont'd.) 1801 GTCTTCACAT CGCAGGTATA CATAATAGTA ACATAGAAAT AGTAGATGCA TTGATATCAT 1861 ACAAACCAGA TTTAAACTCC CGCGATTGGG TAGGTAGAAC ACCGCTACAT, ATCTTCGTGA 1921 TAGAATCTAA CTTTGAAGCT GTGAAATTAT TATTAAAGTC AGGTGCATAT GTAGGTTTGA 1981 AAGACAAATG TAAGCATTTT CCTATACACC ATTCTGTAAT GAAATTAGAT CACTTAATA'r 2041 CAGGATTGTT ATTAAAATAT GGAGCAAATC CAAATACAAT TACGGCAA7 GG~AACAT 211TATTAAGCAT TGCTGTAACA TCTATAATrA CACTACTGGT AGAACAGCTG CTGTTATATG 161 GAGCAGAAGT TIAATAATGGT CGTTATGATG 71'CCAGCTCC "'ATl'ATA'PCC GCTGTCAc-TC 221 TTAACAATTA TGATATTGTT AAGATACTGA TACATAATGO TGCGAATATA AATGTATCCA 28 1 CGGAAGATGC TAGAACGTCT TTACATACA(] -TATGTTT-,G CiA]*JAACG-7 AAAATAATAG .TGA-TTGOCT TAAC-.7TGGAi AGTO C7xTJ ACAGCGTAG.' C3 AGAACTC-Gr I TATCTTGTTA TCGTAGCTTA AGTTATGATIA1 TC:GCTACTAA AC-,APTAT2(-A CGTATrCATTrA 2461 TACAGATGT CTATCGTGAA GCACCAGTk4 ATATCAGCGG A2TTITA7XTT AATTT~AA 2521 CTATAGAAAA TATGATATA TTCAATTAJ'. TTAAGATGA TTGTATTAAP. GAGATAAACA 258 TACTTAAG TATAACCCTT ATAAA.TTTC ATTCATCTGA CATATTTATA CGATATAATA 26411 CTGATATATG TTTATTAACG AGATTTATTC AACATCCAA GATAATAGAA CTAGACAAA.

2701 AACTCTACGC TTATAAATCT ATACTCAACG AGAGAAAAAT CAAGCTACT TACACGTATT 2761 ATCAAATAAA AAAGTATTA ACTGTACTAC CTTTTTCAGG ATATTTCT.2T- ATATTGCCGT, 2821 TTGATGTGTT AGTATATATA CTTGAATTCA TCTATGATkAx TAA~TATGTTG GTACTTATGA *2681 GAGCGTTATC ATTAAAATGA AATAAAAAGC ATACAAGCTA 'FTGCTTCGCT ATCGTTACAA *2941 AATGGCAGGA ATTTTGTGTA AACTAAGCCA CATACTT=C2 AATGAAAAAA ATAGTAOAAA *3001 GGATACTATT TTAATGGGAT TAGATGTTAA GGTTCCTTGG GATTATAGTA ACTGGGCATC *3061 TGTTAACTTT TACGACGTTA GGTTAGATAC TGATGTTACA GATTATAATA ATGTTACAAT 3121 AAAATACATG ACAGGATGTG ATATTTTTCC TCATATAACT CTTGGAATAG CAAATATGGA 3181 TCAATGTGAT AGATTTGAAA ATTTCAAAAA GCAAATAACT GATCAAGA7T TACAGACTAT 3241 TTCTATAGTC TGTAAAGAAG AGATGTGTTT TCCTCAGAGT AACGCCTCTA AACAGTTGGG 3301 AGCGAAAGGA TGCGCTGTAG TTATGAAACT GGAGGTATCT GATGAACTTA GAGCCCTAAG 3361 AAATGTTCTG CTGAATGCGG TACCCTGTTC GAAGGACGTO TTTGGTGATA TCACAGTAGA 3421 TAATCCGTGG AATCCTCACA TAACAGTAGG ATATGTTAAG GAGGACGATG TCGAAAACAJ\ 3481 GAAACGCCTA ATGGAGTGCA TGTCCAAGTT TAGGGGGCAA GAAATACAAG TTCTAGGATG 354'1 GTATTAATAA GTATCTAAGT ATTTGGTATA ATTTATTAAA TAGTATAATT ATAACAAATA 22/30 Figure 9 (cont'd.) 3601 ATAAATAACA 3661 TAATACTTCA 3721 AAGGTATAGA 3781 AAATATACTA 3841 ATTATTACTA 3901 TGCTAGTTTA 3561 ACAGATACAT

AACTACATAT

fl C8 I TGTACTOTAT I TCTTAA'IATA -021 TATAGATTTA -1261 GCATAAACAG 4321 ACTTATAAAT 41381 TCATTCGGTA 4441 TATAACGTA 4501 7ATCG.ACA -1561 TATAGATACC 41621 ACTGAAGTAC -681 TATAAATG 4741 CGTCTATAAT 4801 ATTTGTTA 4861 AAATACTCCT 4921 ATCTTATAAC 4981 TAOCTTTTTA 5041 TAAAAATCCT 5101 TAATAAAAGA 5161 TATAAAGTTA 5221 GGTAAAGTTC 5281 GGAATTAATA 5341 TGTAAAAGAG

TGATAACGGT

TTACCAGAAA

GATATAAATT

TCAACGTCTT

GAACACGGTG

GATAATACAG

TCTGGAAATA

TTATTAAAAA4

GATAAGATAT

CAAACTIAGAA

ATTAGGATAT

TATCTCATAA

CACGGAGTOC

ATTAATAGAA

ATAGATGACT

ACAAAGACAC

OTTCTAAATA

GGTACTGATA

AAAGATATTA

CATAAAGGTT

CTCTTACTTG

TTACATAAAG

GCCGACTATA

GATGACAAGA

GAAATAGCTA

CTACTATCTA

AATTCTATAT

GTAACTAATC

CGGAAAAATA

AGTAAGAATC

TTTATTAGA

TGAGTAATGG

TAGTAAGGTA

TGTATTTAGC

CCGATATTTT

AAATTGCTAA

GTCCGTTATA

AAGGTGTTAA

CTGATGATAT

ATTTTGAAAC

TGTTAOATAA

AGO CACTTAA

CTATAAACGA

GAAAAGATGT

GTATGGGCAG

TTTTAGAAAG

TAO CTGTTO C

CAAAOTTGGT

ATATACTGAA

TCACTCCTCT

ACCACGGTGC

CTATGTTATC

ATTCTCTAA.A

TAGCTATTAT

ATTCAGAAGG

TAAAAGAATC

ATTCTTTTAA

CTAGAGTTAA

AATCATTAGC

TAGGAATAAT

ATAAAATAGA

AAGACTTATA

TATACTTAAA

CGTAAGTATT

AAAATGTAAA

ACTACTAATA

TATTTCTGTA

TTGTAATAGA

GTATAAAATA

TCCGTTACAT

TAGTATTAAA

AAATAATTGT

ACAAGATGAT

AACAGCACTT

TCCCTTACAT

AGGATCTAAT

ATCTAAAAAC

AGGATTAGAT

TGCGATCTTA

ATACATGGCA

TTACGTAAAT

TAATAGTTTT

TAATCACGGT

GATAATATCT

TTTTATAGTA

ATGCGAAAAA

TATCTTTCTT

TAAGATACCT

TTTTCATAGA

AGGTAGGTTA

GATAATATCA

AATGAAC'FGC

AAATGCAAAT

TCTGATATAG

AAT CCT C CTC

GATTCTGGCG

TATAGAAACA

?T]CTTTICTAA

TTTATAGATT

TACO C1ATAA

ATAC-ATAAAA

AGTTACGATA

TTAGGTAAAA

CTGTTAAATC

TACO CTGTTT

GTTAATGTOG

AkAACTATAG

AAACATGTTA

TTATATGGTT

GTTAGTTCTA

GCTAA.AOCTA

AATAATATAA

AATACOCCTC

AAAATOATOT

AACATGGAAC

GAACTAGATO

GACAATAACA

OCATGTATAC

CATCAGCTAA

CCTATAGATA

TAATGATATA

ATAAAGCTAT

ACAATAACGT

AAATGGTAAA

TTCATAAAGC

CTrGACATAGA Af'AAGTCATT

ATTATTACO;.

TTAATATTGA

AGTATAACPJ.

OTTTATTTTT

TAATAGCGTT

CCC CATTACA

TAOGAOCTOA

CACGTAACGA

TTAATA-ATCA

TAAACTTATT

TTCACATAGC

GCTATGTAAA

TOAAAACAOA

AGTTATCTGG

AATTACTTTT

TAACTTGTGT

TAGAAATATC

ATATAAACAG

TTATAACACA

TAOATCTTAT

OTATATATAG

TAGTTAAAOC

TCAAACATAT

23/30 Figure 9 (cont'd.) 5401 5461 5521 5581 5641 5701 5761 5821 588 1 6001 6061 6 12'1 6181 6241 6301 6361 6421 6481 6541 6601 6661 6721 6781 6841 6901 6961 7021 7081 7141

AATAATGGAA

AGTAGTATAA

GAGTTATGAG

AGAAAATTAT

ATATAAGGCT

TGAATATGTT

AAATTTTTCT

TCAAkAAATCT

ATATGGTAAT

TAI\CGAAGAG

AGACGATTTA

AGCTAAAAAG

ACCACTAGCG

TGATGCGAAT

CGTAGGTACC

TGTAGGAGAT

TCTGTTAATG

TTTTACTCCT

CGGGGCTGAT

TTTTGATAAT

ATATAACGAT

AAACGATACT

TAAAGACATA

AATAGACAGA

TTAACTTACA

TTCATACGTT

GATGTTTGAT

AAGGACTTTA

TACCTTCTTT

AATACGGGGC

CTATTAAGTA

AGTGATTTTA

TATTTAACTA

CTTGAGTCTT

GTTGAGTTTA

AAAGTAGATA

CTCATAGACG

AAAGATAGTA

GATAGTGATA

ATAGATATTA

GGAAACACA-

TTACTAGATT

TGTGCCGTTA

CCTGATTCAT

GGTAATATAG

AAATCTGGAG

GAGATGCTAC

TTGTTTAATG

ATCAATATTA

AAATATGTAA

GAAAGATTGT

CTTAAAGTTA

GATGCAGATA

TGGCATACTA

AGii~TGATCGA

AGTTAAATCT

AAATAAAAGA

TGAAATGACT

AATTATAGCA

AAAATTAAAT

ATAATGATTT

TTCAATTACG

AAGTTACTTT

CATTCCATC

GAAATGTAAA

GTCATGGTGT

CTGACATGTA

ACGAATTTCA

,TA.A.TAAC-Tr.

TAGATCTTT'7 CTTTGCAT7A

GTGGAGCAGA

ATACTTGCA

CTTCCTCATA

ATATTGTAAG

TTACTCCTTT

TAGATAGCGG

CAGTATATGA

CTGACTCTTA

ATTCAATAAT

TTCCACCTGG

TAGCTAAAAA

ACGTATTATT

CATGTAAAAT

GGTCACTTAT

AACGATGTAA

AATAGAAGAA

GAAATGTTAT

GCTAAAAATA

GATATTTATT

ACATTCTGTT

AAGATAAACA

AGGTACAAAT

ACCGTCTAAA

TGCTGTAAAA

CTCGCCTTTA

TTCAGAATTTI

ACGAG-TAG'?

TCTAACATT7A C ATAAA 7A-A; CTCGoTGTGAT-

'CCTAACATA

CGAGATACTA

TTTTTTAGG7

ATCTTTACTT

GCACGTTGCT

GGCAGATCCA

TCATAACCGT

CGGAAATACT

TATCTTACAA

TATGATAAAA

GTGTAATTCG

GGAGCTTTTA

ATCTTAAATA

TATACTCTTT

TACGTGTTCG

ATCAGTCACC

TGCTAAATAA

ATGACTTACC

TAAGGGACAC

ATCACCAGCT

TTAAATTTGT,

AAAATATTAT

TTTATTATTA

AAAATATTAC

CATATTATAG

AATGAAATTA

13.,AATAGA7O -ATG CTAAG2

GTTAATGATT

rGTAGATATT"

ACTAATGTGT

ACGGCTGGTG

SCAGCTGATA

Nk4TATAAAA-

ATAAAGTTAT

2-CTCTTACTT

ATATATCTAC

AATTTAAACT

TTAATACGCT

GAGGAAGAGG

GTAATTAAAT

AATAATGGGT

TGAATTAATA

TGTATATATA

TGCAAGTCTA

TATGATAAAA

AGCATTAATG

GTTGTAACCC

TAACAGATAT

GTAATATAAT

AAACCTTATT

AGAATGATAT

CTATGCCTTC

GTAATAGACT

CAATTA'rAGA

ATATFACAGAG

TAkAATATTAA

GATCAAAGAT

TAGGTGTTAC

TGTTAAATAA

TACATACAGC

CCAATCCTAA

AAGACAGTTA

GCGCAAACGG

TATTTCTTTA

ATATGACTAA

TTAAAAAAGA

TTATAGAATC

ATAAGAAAAA

AAGAAGATGA

CATTGAAATA

ACAAAGAGTA

AAGGA TGATA

GCTATATACA

GATACTAAAA

TTATTGATAC

ATAGCTCTCA

S

24/30 Figure 9 (cont 0.) 7201 GAAATGGTTA CCTAGATATA GCTGAATATT TACTTTCATT AGGAGCAGAA TTTGTTAAA'r 7261 ACAGACATAA GGTAATATAT AAATATCTAT CAAAAGATGC GTATGAATTA CTTTTTAGAT 7321 TTAATTATGA CGTTAATATA ATAGATTGAG A 25/30 Figure 1 TGAATGTTAA ATGTTATACT TTGGATGAAG CTATAAATAT GCATTGGAAA AATAATCCAT 71 16 13 1 1381 150i 1321 174 1

TTAAAGAAAG

TTAACGACGC

AAAACATAAA

TAAATATTTA

ACGAATATGC

TACGACATAG

TTGACAGATG

GGATACCAGT

TAAAAGATGA

CGACATCGC-G

AAACTTTTTG

TAGAAGCTGT

ATATACGTCT

AATGGATTCG

ATGTTCAAGA

ACTGAATGCA

AAAGATCTGT

TTGTGTTTGG

GCGGATGTAG

AAAAATTTAA

AACATGGGAC

ACACTACTTA

TTCATGGTAG

TCTTTGAAAG

AGACACAAAA

AGAAGTAGAG

ATGGTCATAA

AGGATAGTTA

TTAAAATTTA

GATTCAAATA

TTTAAATATA

AATAATAAAA

TATCACGTGT

AAGAGATAAT

TGATAAATGC

TAACTTAATA

TATATTATAC

AGATTACTO C

TAATTCTTCC

TATACTTATA

TCACGA-CG

GTGAGC-CTAT

ACC CTAACAC

ATACCGAGGC

CAACTTCTTG

TGAAGAATAA

CAGCTTACCT

ATATTTCAAA

CAATGGTTAA

GTACTCCTTT

AAAAAAATAA

AAAAGAAGTG

AAATGGAAAA

GAGGTAGCTG

AAATAACACT

ATAACTCTGA

AAAATAGAAA

TAAATACGCA

CTACAAAACC

CACAAATAAA

GGAAATGTAA

ATATCTATAC

AAGATTACGT

TATTTCG CAT

GTOCAAAAA.

AAAAATCACTl GAATTTG'I'AAi

ATGTTTTATG

TTCCGTAAAC

TTGTTGAAAA

CATGGATAAT

GGAATATGGT

TATAAAAATC

TCTGCATGAT

CTATGTAAAC

TAACAAAGTT

CACGGATCGG

ACTTCTATTG

AATGATCGCT

AATGTC CAGA CTCAGC CTAC

TCATATACTG

AAGTGGTACT

TTATGACTTT

TATTGCAAGT

AAAAGAGTTA

TAATAATAAA

TAAGCGATA

CATAATTTTT

TATCGTAATT

TGTTATCG7A

ATTTAAGAGA

CGTTACATAA

TGTTAA\T 4

GGTTGGATAA.

A T-'A"GAA

TATGTGTTTC'

TATATTAATC

CAACAAAATT

GACAATG CAT-

ACTCTACAAT

TTGATGAGGT

GCGGTGTTGA'

AATGTTCTTT

AATTTGGT-A

TTAACTCCTC-

A-ACAA-AGGTG

GTACAATCTG

ACTGGGAAAA

TTTTCAACAA

TTTTGGAATT

CTCAAkAATGC

CTTAGTTGTA

AAATGCAATA

ATTTGTAGGG

AATAGATTAT

TATGTTALACT

GTATAACCTA

ATTTTACTCA

TACTCTTTAC

ATCTTGTCAT

AGTCAGTTGG

CAGC.)ATTCT:A

/AAAT7'Y,-CT "'AAAYAJ\,G C C ,A

.ACATATTATG

ATGAAGAA.AA

ATACATTCAA

CTCTAAATAG

CTCCTCTTGA

ATGGAGCTAA

GAGACGACTA

ACAGCC-GAGG

AACTCCTATT

TACATATAGC

CTGATACTGA

GAAATATTGA

ATTC-ATCTTG

AGGAGCAGAT

GATTAAAGAA

AGAACGATGA

GAAAAGATAG

ATAAGTTAGA

TTAAAATAAT

ACTTATTACC

AAGCTTATTC

ACAAATAACT

GGAATGGGGT

AATTACTATT

GATAATTGGG

AAAGATGGA7 TCGC-A4AATA'-

GCAATA'-TCC;

AGATTACTAT

TGAAAAAGTA

GATGGCTTAC

GTTTTTGGAC

AATGG CTGTA

ACCTGTAGTT

CAAAATAGT7G

CTTTACTCCT

GGCTCATTCG-

CGTATCAAAT

CTTGCTGGAT

AATATGTAGC

CCAGCTGTAA

GTAAACTACA

AGTTACTCTC

CTGCGAAGCA

AGATATAATG

TTTATTTAAA

ACATAAGATC

TTCAGAGATA

26/30 Figure 10 (cont'd.) 1801 1861 1921 1981 201 -211 23K 1 'A401 2461 21521 2581 2641 270: 2761 2821 2881 2941 3001 3061 3121 3181

AAATTAAGA

TGAAAAAAAG

ATACCGTTCT

AAACTTTAAT

AGAAGATGAC

GACTTGTGCA

AAATAAATCA

TTTAGAATAC

AGAAGATTAT

AATCAGAGAA

TGATGAAATC

AGTAAATTAC

ACAGGATATG

TAAAGATAAT

AACTCTATTA

CAAAATCTTA

AAAATAGGAT

AGGGCTATAA

CTCATAGAAA

AATTCTTTTC

AATCTTAATA

ATCATCTGTT

TTATTTTTAA

AATCCACTTA

TATTTACTTA

TACATCATGA

ATGTTTATTG

GAAGATGAAG

GCGCTAAAGT

AGAAGGTATA

GATCCATATC

TTTTCATTAT

GTTAACTCTG

GTTCTAAAAG

AACAAGGAGG

AAGGATGTTT

ATAAAG CTGT

CTTATTAAAA

CTAATAACTC

TTGACATCA-A

GTAAGAACTT

ACAATGAAAC

AATTCATTTC

AAGGATTCAA

ACCATGAACT

ATTATAAGCA

CTACATATTT

GAATTTCTAG

TTTAACTTAT

GCAACGCGTT

ATTCAGATGA

ATGACGACGA

ATACTATGGT

GTATAGTGAA

TAAAGGTATC

ATFTTGTTTAC

CTAATAAGAT

GA-VATAAAAAA

AACTGAATAT

ACGGTTCTTC

TAATCGATCA

AAAAATAATA

CAGTGGATAT

GTTAGuATTGT

ACTAGAATGT

GATTAAAAAT

TGAAAGTATA

TAATAAAT-TG

AAAAAAAATT

AAGATGCTTG

GATGTTCATT

TTATCTAG

AAAGATCTAA

AGTATATTTT

TGTTTTAGALA

TGATTATTGT

TACAAAGTAT

AATGTTGTTA

TCCTTTGCAC

AGCTGAAGAC

TCTAACTGAT

AG CTPACTA

AGCTCTCCAT

TGGAGCTGAT

TCACGTTTAG

GAACATAATA

GAAAATGAGA

TTTATCAATA

TATAAAAATC

CTAAGACACG

C CTTACGAGA

TTAGATAATA

TTGCCAATAA

CTCTTTATAT

AATGCATAAT

ACAATGGAGA

AAGAAAGTTA

TGTAAATCTG

AAGTCTATAC

GATTATGATT

ATAATTTCAT

GAAAkAAAATA GAGTCTG'FGik

CAGGATATAA

TTGTTAGATA

AGAGCTGCTA

CTAAACTCTT

TAATATTAAA

CGAAGTTTAT

TTATGAAATT

ATGATATGAA

ATTTCCCTAT

AATTATTGGA

TTCAGTACAT

TACATTAAAA

TATACAACAG

AGTATACACA

TTCTAAATAA

TTAACGCTCT

TTGAATATGA

TTTTAGATGA

TACTAATGGC

ATGAAAAAC C

CTATTCCTAG

TAT CC A'AA-T

TAATAGCTAT

AAACATTGGUK

GAG GGGC CkLA TTGG TAG GAA

TAACTATTGC

ATATATTAAT

ACATTC-TCAT

AAGGAATACA

TACAGCTATCT

ATATAATACG

TGGAGTTATA

TATACTGGAG

AGGTAAATAG

GTATTTGTTT

GAAAATTCAT

27/30 F'igure 11 1 AAGCrFTCTArT 61 TATTCATATT 121 TAGATAATAG 181 ACGCGTTCAT 2-11 TTGAGAGAGA 3o1 GQGATTTTGTT 361 ACTTACTTAC iAGAGTCC:TTT' ~1 ATTTGATAAA

I'TAGTOATACA

61GTTAGATATT 72'1 AATAGGAGAC 781 TACTAAAGGA 8411 ACAGTATAAT 901l CATATGGGTT 961 TCTAAGCC 1021 TGCGGATAAA 101ACGAGTAGGT 111TATACTTACT 1201 ATATTGGCC 1261 TTCATCTTCT 1321 AGCTATACTC 1381 ATATTGGACA 1411 AAAATATATA 1501 TGTATATAAT 1561 ATTCTCCGGA 1621 ATTTTATCCG 16 31 TTACTTTATA 1741 TATACTCGCA

CAAAAGTCTT

TCCTATCAAT

ATACGCTCAT

AAGTTTCAAC

TTGGACATCT

ATCATCAGTT

GAAAAAATGT

AAGAGTTATA

GTTGTAGTAA

ATTCTGTTTT

ATATTAC-ATA

CTAAAAAAAA

AATAGTAACT

TTATTAGATT

AAAAAGAATA

AAAAATGTTA

GCTGCTGAAG

TTATACACTT

ACAATAATTG

GATTTTCTAT

GGATATTTTT

GAATGGGTAA

TTTGATGCTA

AAGAAAACTT

ATTAAGAAAG

CACGTAACTT

ATGATAGATA

AAACGTGTTT

TCAGATGTT7A

GAAAACTACT

AATGAGTTAG

TCTAAAGTAG

ATAATGACTG

TGCATAGATC

AACTACGCTA

ATATTTAACA

CATTATTACA

ATTTAAAAGA

'1AAATGAAGA

TTIAACTCCTT

TAGATACTCA

GAG CGTGTGA

TATATTATAA

ACAAGTTTGT

CTATAATAGA

TAGAATACTG

ATGATTGGTT

TCGAGTTCAT

TACATCCAA.A

CTTACGTAGA

TAGAATTCTT

TGAATAGTAA

GTTTATTTTT

TGCAGTATAA

ATAGTCAGGT

ACTTAATGGA

TACTACTGTT

CTGTAACTAA

ATAAATTCAG

ATCCAAAAGG

GTGTAGATAG

ATGATATTAA

CAAATTTGGA

AAAATCTCAC

AAGAAATTAC

TAAGTACAAT

AAAACTATATi TAkACCATAAT!

'FAATGTACT'G

TAATAAC'G C-

TAATTATTAT

TTTAGAATTA

AGATATGACT

ATTATTG CC TATAATACA7

TTCTCCTGGC

AAGATACGAT

AGTTATATTA

CAAGATAATA

AGAACTAATA

TGAGACCACT

CTGTTTAGTA

CCAACTCTCT

GAACTTTTTT

GATAGATAGA

TACGTTTAAA

TGGAATATTG

TATAATATCA

TAAAAAGATA

AAGGCCCTAT

TATAGATATT

I'AACTCAAAG

CGGTTCACAT

TAAAAAGATA

AGTTATAAAT

AAAAAGTATT

TTTACAGAAC

GAA-A'TTA

TTATCTACA.A

'FTATC!AA-AAT

ATACCTAGTT

GAAGATCTAA

TACATGAATA

GATGTAGATA

CGCGATAACA

TATATATTAT

AACCGTATAA

GAAAATAATA

GCTAAGGTA

TATCATCATA

ATTTTATCAG

CACCTGAAAA

ACTAAAAGCA

AAAGACGGTA

GTATGTTATT

ATTCCTGGTT

CATAAGGATA

GAATCTATCT

GAATATAAAA

TTTACACATA

k.CTACAAAGG

%TGATGATAG

['TTAATCATC

3CCGATGTArl

)LATACATAAT

1,AAT-AAAAATI A AT C-1AT AG 7 C. CCA GA 7.01-CXk4' TA7: A7JTATGCGTT

ATTOGTTATT

AAT7GTTACAA

GACAGTATAA

GGTACATGA

ACGAATTATC

TAAAACATTT

CA? CTAATAA

ATTCATCTAT

AATTTATTTC

CAGGGTATGA

ATTATGTAAA

AACAGTTAGC

TACACGCAGC

TTGATTTTAA

ATGAGAATAT

ATGCAGATTT

CTATGTTTCC

CATCTAACGA

28/30 Figure 11 lcont'd.) 1801 AGATCTTCTG TCTATCTGTT TATGCGAAGT AACAGTTTGT AAAGATATAA AAAATCCATT 1861 ATTATATTCT AAAAAGGATA TATCAGCAAA ACGATTCATA GGTTTATTTA CATCTGTCGA 1921 TATAAATACG 1981 GCCTGAAAAG 20411 AAGACGTrTA 21(11 TACCAGAGAT 2161 ATCGTATACT 2 2? 1 TAA4ATATAAT

ATAAATCATA

I TCTAATGAAC 4 G CTTCATTCA 2411 GTCAGATGAT 2521 TGATATTAAT 2581 TTCTGGATAT 2641 TAATTATTTG 2701 TAATATTAAT 276:1 TACAGGCAGA 2821 TGGCCTATGT 280'1 ATCCTOTAGA 2941 AGAAGTATTT 3001 AGATAATCAT 3061 TTTTATATTA 3121 GTTAAATGCC 3181 ACTGAAACGA 3241 TATTTTAACT 3301 TGTTAAAAGC 3361 AGGAAAGAAC 3421 AAATTATGGA 3481 ATACGGCTAA 3541 CTCTCATA-AG

GCTGTTGAGT

ATAAAAATAT

GATATTCTAC

GGAGTCAGAA

TATAAATTAC

CAG GTAATAT

'AATAATGAA

TA.AGTACTC

ACGAATTACC

GAGAAAGTAA

AATTTACTTA

TATAAAATAC

GAGGTAAAGG

AATTATGATA

TCTATGGTTA

TTAATAGCCA

TATTTAATAT

TCTACTAATA

TTAGTAGTAG

AATAGCATAT

AATATCGATT

TCTACAGACT

TTAGAACTAA

TGCAATAAAT

TTTAGAACAA

AGATACAAGT

CTTATATAAA

TAGGATTAAT

TAAGAGGATA

TTAATTCTAA

ATTCCTATCT

ATAATTTACC

TAAATTG CCA

ATAATCCTAT

ACGAAATATC

TAAATCTCCA

TTTTAATTTT

ATATAAATTT

ACGATGTTAA

CAGTTAATGA

ATATAAAATT

GGAATTTTTT

TGGTAAAACA

GATCATTTTA

TATATCTAAC

AAGATAAAGA

CTACTAATAT

TACTAGAAGA

CTATATTTCA

CAACTATGCA

AACGATTCTA

AGTAAGGATG

CTTAAGTTTA

AAAAGAATGG

GTGTACGGAC

AATATTATAG

TAAAATAAGA

TCCTACATAC

GCTTAAATTT

TATAATTTCTI

TATGTACAAT

ATAGGAGTAT

AGTAATAGAC

AAATTAGAT-A.

TTCAGACACA

AACTTATGGG

TAGACTTATT

TATTAAAATA

AGTCTATCTT

AGGATTTACA

CTGTAACGGG

CTCTATAAAC

AACAACAAAA

TAGTCTATCT

GGAAAGAAAT

TTTAAAATCT

TCATAACAGT

AGGAATAAGC

CCAATACTAA

TAGAAGAAAT

ATCAAACTTC-

ATGTTGAATG

AAAACGAATA

AAACTTTAAA

GTAATAGGAT

A.TTAGATTAT1

A-ATATAACAG

7TTATCGTCA

TCGTGTAAGA

ATATAATTGA

AGGAACTGGC

;AAA-GATAC

!OTTATTACA

TATA.4TATAA

C'CATCAACCC

GATTGTTTAA

T'CACATGGAA

C CTGTTATAT

AAGCAGCATT

ATTTTACCAC

AAA TTTAACG

TATCTACAAG

GTATACAAAA

AGACTTAGTA

AC-TACATTAA

AATATGCCAA

AAATAGGA'-A

ACTTTGTTCT

TATTTATGAA

TCGTAGTTTA

TATGATTACT

ATATAATCTG

GTTTAGAATG

TACTAACAGA

AG GATATAG C

GTTATTGTAG

TAACAAAGTG

.AAAAGTAAA-A

AGATTCTT C- AG CAAATAC.

AACTAACTAA.

TAAAGATTCA

CTTCAAACCT

GAGATGTAALA

ATGAATTACC

GTATCAACAA

CTATGGTAAC

AAATAATAGG

ATGTATGGCC

ATATGAAAGA

ACGTGGAAGC

TAACAAAACA

TCAGTGATAT

TTATGTCTAA

CGTGATAGGC

ATACCTTCGG

CACTATAAALA

GAACATAACT

TATATACTAG

GTGGGGCTAG

29/30 Figure 11 (cont'd.) 3601 ACGAATCTAC AATACGTAAT ATAAAT'TATA TAATTTCACA AAGAACAAAA AAAAATCAGT 3661 TTCTAATACC TTATAGATAA ACTATATTTT TTACCACTGA CAACAC .00, fee*@ 30/30

Claims (22)

1. A recombinant poxvirus containing therein DNA from feline infectious peritonitis virus in a non-essential region of the poxvirus genome wherein the poxvirus is a vaccinia virus wherein J2R, B13R B14R, A26L, A56R, C7L-K1L and I4L are deleted from the virus, or a thymidine kinase gene, a hemorrhagic region, an A type inclusion body region, a hemagglutinin gene, a host range region, and a large subunit, ribonucleotide reductase are deleted from the virus; or, the poxvirus is (ii) canarypox which was attenuated through more than 200 serial passages on chick embryo fibroblasts, a master seed therefrom was subjected to four successive plaque purifications under agar, from which a plaque clone was amplified through five additional passages.

2. The recombinant poxvirus of claim 1 wherein the poxvirus is the canarypox virus.

3. The recombinant poxvirus of claim 2 which is vCP262, vCP261A, vCP282, is vCP281, vCP283B, vCP315.

4. The recombinant poxvirus of claim 1 wherein the feline infectious peritonitis virus DNA encodes M, N, and the three versions of S; S1, S2, S3, or combinations thereof.

5. The recombinant poxvirus of claim 4 wherein the DNA encodes M. 20

6. The recombinant poxvirus of claim 4 wherein the DNA encodes N.

7. The recombinant poxvirus of claim 4 wherein the DNA encodes S.

8. The recombinant poxvirus of claim 4 wherein the DNA encodes S1.

9. The recombinant poxvrus of claim 4 wherein the DNA encodes 2. 9. The recombinant poxvirus of claim 4 wherein the DNA encodes S2.

10. The recombinant poxvirus of claim 4 wherein the DNA encodes S3. 25

11. The recombinant poxvirus of claim 4 wherein the DNA encodes M+N.

12. The recombinant poxvirus of any one of claims 1-11, wherein the poxvirus is the vaccinia virus. .54S*

13. The recombinant poxvirus of claim 3 which is vCP262.

14. An immunological composition comprising a recombinant poxvirus as 30 claimed in any one of claims 1-13, and a carrier.

A method for inducing an immunological response in a host comprising administering an immunologically effective amount of a recombinant poxvirus as claimed in any one of claims 1-13, or the composition of claim 14.

16. The recombinant poxvirus of any one of claims 1-13, or the composition of claim 14, when used in inducing an immunological response in a host. [I:\DayLib\LIBFF]25810spec.doc:gcc

17. Use of the recombinant poxvirus as claimed in any one of claims 1-13, or the composition of claim 14 in the preparation of a vaccine for inducing an immunological response in a host.

18. A method for expressing a gene product in vitro comprising introducing into a cell a virus as claimed in any one of claims 1-13, and continuing a cell culture in vitro under appropriate conditions for expression of the gene product.

19. A recombinant poxvirus containing therein DNA from feline infectious peritonitis virus in a non-essential region of the poxvirus genome substantially as hereinbefore described with reference to any one of the examples.

20. A method for expressing a gene product in vitro, substantially as hereinbefore described with reference to any one of the examples.

21. A gene product expressed in accordance with the method of claim 18 or S S. S. o Do [I:\DayLib\LIBFF]25810spec.doc:gcc 59 1. A recombinant poxvirus containing therein exogenous DNA from feline infectious peritonitis virus in a non-essential region of the poxvirus genome, wherein the poxvirus is an NYVAC virus, or a virus having the characteristics of NYVAC. 2. The recombinant poxvirus of claim 1, wherein said virus is selected from the group consisting of: vP1126, vP1128, vP1145, vP992, vP1184, vP1196, vP1210, vP1214, vP1216. vP1251, vP1262, vP1302, vP1173, vP1183, vP1205B, vP893, vPll61. vP1160, vPl186. vP1201, vP1238, vP1247, vP1312, vP1302B, vP1001 or vP1399. 3. The recombinant poxvirus of claim 1 or 2 wherein the feline infectious peritonitis virus DNA encodes M, N, and the three versions of S; S1, S2, S3, or i0 combinations thereof. 4. The recombinant poxvirus of claim 3 wherein the DNA encodes M. The recombinant poxvirus of claim 3 wherein the DNA encodes N. 6. The recombinant poxvirus of claim 3 wherein the DNA encodes S. 7. The recombinant poxvirus of claim 3 wherein the DNA encodes S 1. Is 8. The recombinant poxvirus of claim 3 wherein the DNA encodes S2. 9. The recombinant poxvirus of claim 3 wherein the DNA encodes S3. The recombinant poxvirus of claim 3 wherein the DNA encodes M+N. 11. The recombinant poxvirus of any one of claims 1-10, wherein the poxvirus is the vaccinia virus. S 20 12. An immunological composition comprising a recombinant poxvirus as 0 claimed in any one of claims 1-11, and an immunologically acceptable carrier. 13. A method for inducing an immunological response in a host comprising administering an immunologically effective amount of a recombinant poxvirus as claimed in any one of claims 1-11, or the composition of claim 12. 25 14. The recombinant poxvirus of any one of claims 1-11, or the composition of claim 12, when used in inducing an immunological response in a host. Use of the recombinant poxvirus as claimed in any one of claims 1-11, or the composition of claim 12 in the preparation of a vaccine for inducing an immunological response in a host. 30 16. A method for expressing a gene product in vitro comprising introducing into a cell a virus as claimed in any one of claims 1-11, and continuing a cell culture in vitro under appropriate conditions for expression of the gene product. 17. An antibody elicited by in vivo expression of an antigen from a virus as claimed in any one of claims 1-11, or by administration of a feline infectious peritonitis [I:\DayLib\LIBFF2581 Ospec.doc:gcc virus associated antigen obtained by in vitro expression of the virus of any one of claims 1-11. 18. A feline infectious peritonitis antigen prepared from in vitro expression of the virus of any one of claims 1-11. 19. A recombinant NYVAC poxvirus containing therein DNA from feline infectious peritonitis virus in a non-essential region of the poxvirus genome substantially as hereinbefore described with reference to any one of the examples. A method for expressing a gene product in vitro, substantially as hereinbefore described with reference to any one of the examples. j0 21. A gene product expressed in accordance with the method of claim 16 or 18.

22. A vaccine prepared in accordance with the use of claim Dated 12 December, 2000 Virogenetics Corporation i1 Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON S.. S.* 0 S S S S. [I \DayLib\LIBFF]258IOspec.doc:gcc