WO2014074927A1

WO2014074927A1 - Methods of identifying, preventing, and treating virulent aleutian mink disease virus using replikin sequences

Info

Publication number: WO2014074927A1
Application number: PCT/US2013/069310
Authority: WO
Inventors: Samuel Bogoch; Elenore S. Bogoch; Samuel Winston Bogoch; Anne-Elenore Bogoch Borsanyi
Original assignee: Samuel Bogoch; Bogoch Elenore S; Samuel Winston Bogoch; Anne-Elenore Bogoch Borsanyi
Priority date: 2012-11-09
Filing date: 2013-11-08
Publication date: 2014-05-15

Abstract

The present invention provides methods of predicting the virulence of Aleutian mink disease virus and outbreaks of said virus comprising comparing Replikin concentrations in different isolates of virus and identifying relatively virulent isolates as those having relatively higher Replikin concentrations. The present invention further provides diagnostic, preventive, and therapeutic applications for Replikin peptides identified in Aleutian mink disease virus and for Replikin Peak Genes identified in Aleutian mink disease virus.

Description

METHODS OF IDENTIFYING, PREVENTING, AND TREATING VIRULENT ALEUTIAN MINK DISEASE VIRUS USING REPLIKIN

SEQUENCES

[0001] This application claims priority to U.S. Provisional Appln. Ser. No. 61/724,538, filed November 9, 2012, which is incorporated herein by reference.

[0002] This application incorporates by reference in its entirety each of the following applications: U.S. Provisional Appln. Ser. No. 61/891,677, filed October 16, 2013, PCT/US 13/3911 1, filed May 1, 2013, U.S. Provisional Appln. Ser. No. 61/813,889, filed April 19, 2013, U.S. Appln. Ser. No. 13/791,609, filed March 8, 2013, PCT/2013/ 30013, filed March 8, 2013, U.S. Provisional Appln. Ser. No. 61/765, 106, filed February 15, 2013, U.S. Appln. Ser. No. 13/553, 137, filed July 19, 2012, PCT/US2012/047451, filed July 19, 2012, U.S. Provisional Appln. Ser. No. 61/687,818, filed May 2, 2012, U.S. Provisional Appln. Ser. No. 61/609,074, filed March 9, 2012, U.S. Provisional Appln. Ser. No. 61/509,896, filed July 20, 201 1, U.S. Appln. Ser. No. 12/581, 1 12, filed October 16, 2009, U.S. Provisional Appln. Ser. No. 61/246,006, filed September 25, 2009, U.S. Appln. Ser. No. 12/538,027, filed August 7, 2009, U.S. Provisional Appln. Ser. No. 61/185, 160, filed June 8, 2009, U.S. Provisional Appln. Ser. No.

61/179,686, filed May 19, 2009, U.S. Provisional Appln. Ser. No. 61/172, 115, filed April 23, 2009, U.S. Appln. Ser. No. 12/429,044, filed April 23, 2009, PCT/US09/41565, filed April 23, 2009, U.S. Provisional Appln. Ser. No.

61/143,618, filed January 9, 2009, U.S. Provisional Appln. Ser. No. 61/087,354, filed August 8, 2008, U.S. Provisional Appln. Ser. No. 61/054,010, filed May 16, 2008, U.S. Appln. Ser. No. 12/108,458, filed April 23, 2008,

PCT/US2008/61336, filed April 23, 2008, U.S. Appln. Ser. No. 12/010,027, filed January 18, 2008, U.S. Provisional Appln. Ser. No. 60/991,676, filed November 30, 2007, U.S. Appln. Ser. No. 11/923,559, filed October 24, 2007, now U.S. Patent No. 8,050,871, U.S. Provisional Appln. Ser. No. 60/982,336, filed October 24, 2007, U.S. Provisional Appln. Ser. No. 60/982,333, filed October 24, 2007, U.S. Provisional Appln. Ser. No. 60/982,338, filed October 24, 2007, U.S. Provisional Appln. Ser. No. 60/935,816, filed August 31, 2007, U.S. Provisional Appln. Ser. No. 60/935,499, filed August 16, 2007, U.S.

Provisional Appln. Ser. No. 60/954,743, filed August 8, 2007, U.S. Appln. Ser. No. 1 1/755,597, filed May 30, 2007, U.S. Provisional Appln. Ser. No. 60/898,097, filed January 30, 2007, U.S. Provisional Appln. Ser. No.

60/880,966, filed January 18, 2007, U.S. Provisional Appln. Ser. No.

60/853,744, filed October 24, 2006, U.S. Appln. Ser. No. 11/355, 120, filed February 16, 2006, now U.S. Patent No. 7,894,999, U.S. Appln. Ser. No.

1 1/1 16,203, filed April 28, 2005, now U.S. Patent No. 7,774,144, U.S. Appln. Ser. No. 10/660,050, filed June 4, 2004, now U.S. Patent No. 7,442,761, U.S. Appln. Ser. No. 10/189,437, filed July 8, 2002, now U.S. Patent No. 7,452,963, U.S. Appln. Ser. No. 10/105,232, filed March 26, 2002, now U.S. Patent No. 7, 189,800, U.S. Appln. Ser. No. 09/984,057, filed October 26, 2001, now U.S. Patent No. 7,420,028, and U.S. Appln. Ser. No. 09/984,056, filed October 26, 2001, now U.S. Patent No. 7, 176,275.

SEQUENCE LISTING

[0002.1] The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 8, 2013, is named 13795-48476_SL.txt and is 7,238 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Aleutian mink disease is a disease of mink caused by a virus in the Parvoviridae family presently known as the Aleutian mink disease virus or AMDV. The disease is also sometimes referred to as Mink Plasmacytosis. Symptoms of viral infection in mink include lethargy, loss of appetite, tarry diarrhea, a rough coat, and weight loss. Mink infected with AMDV have been observed to carry the virus for several weeks or months after infection prior to obvious symptoms. Once symptoms arise, death of the infected mink is generally considered certain. Commercial operations infected with AMDV often find extensive death in their population following extreme environmental temperatures. Deaths also tend to follow from secondary infections including pneumonia in the young. A commercial operation that is extensively affected by AMDV often also experiences significant reductions in reproductive capacity. Infected mink generally are observed to have enlarged liver, lymph nodes, and spleen with mottled kidneys and general dehydration. Kidney failure is a common cause of death. [0004] Aleutian mink disease was apparently first described in 1956 when the symptoms of the disease were identified in domesticated Aleutian-type colored mink. The disease is now known to be present in minks of all types but differences in severity have been noted based on type. Aleutian mink disease has been observed globally in the mink-raising industry and is considered the most significant pathogen affecting commercial mink operations. The virus is also known to infect ferrets and it is thought that, on very rare occasions, the virus may play a role in human disease.

[0005] There currently exists no known treatment for Aleutian mink disease. Diagnosis and culling is a method of disease control presently practiced in the mink industry. Nevertheless, differences in virulence have been observed between strains of AMDV. As a result, methods of differentiating the severity of strains of virus infecting a commercial operation would provide information to help in determining whether culling or isolation or some other disease-control approach is most appropriate.

[0006] Before the current invention, attempts at designing vaccines against AMDV have all resulted in practical failure. As a result, a vaccine or method of designing a vaccine that provides an effective therapy against AMDV provides a surprising result. In addition, methods of predicting outbreaks of AMDV and of comparing relative virulence of AMDV using genomic methods have not before been available in the art and are very useful in the practice of raising AMDV. These methods provide surprising and practical results for the mink industry.

[0007] In view of the previous absence of therapies against AMDV, there is a need in the art for methods of preventing and treating outbreaks of AMDV. There is also a need in the art for methods of identifying and differentiating virulent forms of AMDV. Such identification and differentiation allows for development of vaccines and other therapies against expanding outbreaks of AMDV and for other measures of disease control. A need also exists for economical means of diagnosing AMDV.

[0008] The inventors have found a family of conserved small protein sequences that relate to rapid replication in influenza and other viruses. The family of protein sequences is known as Replikins. Rapid replication is characteristic of virulence in viruses where Replikin sequences are identified or where concentrations of Replikin sequences are present. Rapid replication has been associated with the presence of Replikin sequence structures in protein sequences. Replikin sequences have been further associated with viral outbreaks, epidemics and increased rates of host mortality. The inventors provide herein methods of predicting, preventing, treating, and diagnosing Aleutian mink disease virus.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods of determining the relative virulence, replication rate, and/or lethality of an isolate, strain, or population of Aleutian mink disease virus (AMDV). The present invention further provides compounds and compositions for prevention and treatment of AMDV, including immunogenic compositions and vaccines against AMDV.

[00010] A first non-limiting aspect of the present invention provides an isolated or chemically-synthesized protein, protein fragment, polypeptide, or peptide comprising a Replikin peptide sequence, functional fragment of a Replikin peptide sequence, or homologue of a Replikin peptide sequence identified in or derived from an isolate of Aleutian Mink Disease Virus

(AMDV). In a non-limiting embodiment, the isolated or chemically-synthesized protein, protein fragment, polypeptide, or peptide is identified in or derived from an isolate of AMDV. In a non-limiting embodiment, a homologue of a Replikin peptide sequence in a protein, protein fragment, polypeptide, or peptide is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% homologous with a Replikin peptide sequence.

[00011] In a non-limiting embodiment, the protein, protein fragment, polypeptide, or peptide comprises 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, or 400 or more amino acid residues longer than the Replikin peptide sequence, functional fragment of the Replikin peptide sequence, or homologue of the Replikin peptide sequence identified or derived from an isolate of AMDV. In another non-limiting embodiment, the amino acid residues that make the protein, protein fragment, polypeptide, or peptide longer than the Replikin peptide sequence, functional fragment of Replikin peptide sequence, or homologue of Replikin peptide sequence identified or derived from the isolate of AMDV begin at the C-terminus and/or N-terminus of the Replikin peptide sequence, functional fragment of Replikin peptide sequence, or homologue of Replikin peptide sequence.

[00012] In a non-limiting embodiment, the protein, protein fragment, polypeptide, or peptide is an AMDV protein or protein fragment comprising the Replikin peptide sequence, the functional fragment of the Replikin peptide sequence, or the homologue of the Replikin peptide sequence. In another non- limiting embodiment, the protein, protein fragment, polypeptide, or peptide comprises a functional fragment of a Replikin peptide sequence identified in an isolate of AMDV. In a non-limiting embodiment, a protein fragment, polypeptide, or peptide may consist essentially of an AMDV Replikin peptide sequence or may consist of an AMDV Replikin peptide sequence.

[00013] In a non-limiting embodiment, an isolated or chemically- synthesized protein, protein fragment, polypeptide, or peptide may comprise at least one peptide sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% or more homologous with any one of SEQ ID NO(s): 1-35. A protein, protein fragment, polypeptide, or peptide may comprise at least one peptide sequence of any one of SEQ ID NO(s): 1-35, may comprise at least one functional fragment of any one of SEQ ID NO(s): 1-35, may consist essentially of any one of SEQ ID NO(s): 1-35, or may consist of any one of SEQ ID O(s): 1-35.

[00014] A non-limiting embodiment of the first aspect of the invention provides an isolated or chemically-synthesized Replikin peptide sequence of AMDV where the peptide sequence consists of 7 to about 50 amino acid residues and comprises (1) at least one lysine residue located 6 to 10 residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues. In a non-limiting embodiment, the isolated or chemically- synthesized Replikin peptide sequence has at least one lysine residue on one end of the peptide and at least one lysine residue or at least one histidine residue on the other end of the peptide. In a non-limiting embodiment, the isolated or chemically-synthesized Replikin peptide sequence is the Replikin peptide sequence reflecting the shortest amino acid sequence of 7 to 50 amino acid residues comprising (1) a first lysine residue located six to ten residues from a second lysine residue, (2) at least one histidine residue; and (3) at least 6% lysine residues. [00015] A second non-limiting aspect of the invention provides a composition comprising at least one protein, protein fragment, polypeptide, or peptide comprising a Replikin peptide sequence, a functional fragment of a Replikin peptide sequence, or a homologue of a Replikin peptide sequence. Any of the proteins, protein fragments, polypeptides, or peptides described herein may be comprised in such a composition. In a non-limiting embodiment, the composition may be an immunogenic composition or a blocking composition comprising any of the proteins, polypeptides, protein fragments, or peptides described above or herein.

[00016] In a non-limiting embodiment, an immunogenic composition or blocking composition may comprise at least two proteins, protein fragments, polypeptides, and/or peptides described herein wherein each protein, protein fragment, polypeptide, or peptide comprises a different Replikin peptide sequence, a functional fragment of a Replikin peptide sequence, and/or a homologue of a Replikin peptide sequence. In a non-limiting embodiment, a composition, immunogenic composition, or blocking composition may comprise a mixture of proteins, protein fragments, polypeptides, or peptides. In a non- limiting embodiment the mixture comprises at least one protein fragment or peptide comprising SEQ ID NO: 1, at least one protein fragment or peptide comprising SEQ ID NO: 2, at least one protein fragment or peptide comprising SEQ ID NO: 3, at least one protein fragment or peptide comprising SEQ ID NO: 4, at least one protein fragment or peptide comprising SEQ ID NO: 5, at least one protein fragment or peptide comprising SEQ ID NO: 6, at least one protein fragment or peptide comprising SEQ ID NO: 7, at least one protein fragment or peptide comprising SEQ ID NO: 8, at least one protein fragment or peptide comprising SEQ ID NO: 9, at least one protein fragment or peptide comprising SEQ ID NO: 10, at least one protein fragment or peptide comprising SEQ ID NO: 1 1, at least one protein fragment or peptide comprising SEQ ID NO: 12, at least one protein fragment or peptide comprising SEQ ID NO: 13, at least one protein fragment or peptide comprising SEQ ID NO: 14, at least one protein fragment or peptide comprising SEQ ID NO: 15, and at least one protein fragment or peptide comprising SEQ ID NO: 16. As such, a composition comprises a mixture of isolated or chemically-synthesized protein fragments and/or peptides, including, respectively, at least one isolated or chemically- synthesized peptide consisting essentially of each of SEQ ID NO(s): 1-16.

[00017] In a non-limiting embodiment the mixture comprises at least one protein fragment or peptide comprising SEQ ID NO: 3, at least one protein fragment or peptide comprising SEQ ID NO: 4, at least one protein fragment or peptide comprising SEQ ID NO: 5, at least one protein fragment or peptide comprising SEQ ID NO: 6, at least one protein fragment or peptide comprising SEQ ID NO: 7, at least one protein fragment or peptide comprising SEQ ID NO: 1 1, at least one protein fragment or peptide comprising SEQ ID NO: 17, at least one protein fragment or peptide comprising SEQ ID NO: 18, at least one protein fragment or peptide comprising SEQ ID NO: 19, at least one protein fragment or peptide comprising SEQ ID NO: 20, at least one protein fragment or peptide comprising SEQ ID NO: 21, at least one protein fragment or peptide comprising SEQ ID NO: 22, at least one protein fragment or peptide comprising SEQ ID NO: 23, at least one protein fragment or peptide comprising SEQ ID NO: 24, and at least one protein fragment or peptide comprising SEQ ID NO: 25. As such, a composition comprises a mixture of isolated or chemically-synthesized protein fragments and/or peptides, including, respectively, at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID NO(s): 3-7, 11, 17-25.

[00018] In a non-limiting embodiment, the mixture comprises at least one protein fragment or peptide comprising SEQ ID NO: 26, at least one protein fragment or peptide comprising SEQ ID NO: 27, at least one protein fragment or peptide comprising SEQ ID NO: 28, at least one protein fragment or peptide comprising SEQ ID NO: 29, at least one protein fragment or peptide comprising SEQ ID NO: 30, and at least one protein fragment or peptide comprising SEQ ID NO: 31. As such, a composition comprises a mixture of isolated or chemically- synthesized protein fragments and/or peptides, including, respectively, at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID NO(s): 26-31.

[00019] In a non-limiting embodiment, the mixture comprises at least one protein fragment or peptide comprising SEQ ID NO(s): 28, at least one protein fragment or peptide comprising SEQ ID NO: 30, at least one protein fragment or peptide comprising SEQ ID NO: 32, at least one protein fragment or peptide comprising SEQ ID NO: 33, at least one protein fragment or peptide comprising SEQ ID NO: 34, and at least one protein fragment or peptide comprising SEQ ID NO: 35. That is, a composition comprises a mixture of isolated or chemically- synthesized protein fragments and/or peptides, including, respectively, at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID NO(s): 28 and 30-35, respectively. The mixture may further comprise at least one polypeptide comprising SEQ ID NO: 36.

[00020] In a non-limiting embodiment, a composition, immunogenic composition, or blocking composition comprises at least one isolated or chemically-synthesized protein fragment or peptide consisting essentially of SEQ ID NO: 13.

[00021] In another non-limiting embodiment, a composition, immunogenic composition, or blocking composition may comprise at least one isolated or chemically-synthesized peptide consisting essentially of any one of SEQ ID NO(s): 1-36.

[00022] A composition, immunogenic composition, or blocking

composition may comprise a pharmaceutically-acceptable carrier, excipient, and/or adjuvant.

[00023] A third non-limiting aspect of the present invention provides a vaccine or therapeutic against AMDV. A non-limiting vaccine or therapy may comprise at least one of any one of the proteins, protein fragments, polypeptides, or peptides discussed above and herein. A non-limiting embodiment may comprise a protein, protein fragment, polypeptide, or peptide comprising a Replikin peptide sequence, functional fragment of a Replikin peptide sequence, or homologue of a Replikin peptide sequence identified in or derived from an isolate of AMDV. A non-limiting embodiment may comprise an isolated or chemically-synthesized protein, protein fragment, polypeptide, or peptide identified in or derived from an isolate of AMDV. In a non-limiting

embodiment, a homologue of a Replikin peptide sequence in a protein, protein fragment, polypeptide, or peptide may be at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more homologous with a Replikin peptide sequence. In a non-limiting embodiment, a protein fragment, polypeptide, or peptide may comprise 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, or 400 or more amino acid residues longer than the Replikin peptide sequence, functional fragment of the Replikin peptide sequence, or homologue of the Replikin peptide sequence identified or derived from an isolate of AMDV. In another non-limiting embodiment, the amino acid residues that make the protein, protein fragment, polypeptide, or peptide longer than the Replikin peptide sequence, functional fragment of Replikin peptide sequence, or homologue of Replikin peptide sequence identified or derived from the isolate of AMDV may begin at the C-terminus and/or N-terminus of the Replikin peptide sequence, functional fragment of Replikin peptide sequence, or homologue of Replikin peptide sequence.

[00024] In a non-limiting embodiment, a vaccine may comprise at least two proteins, protein fragments, polypeptides, and/or peptides described above or herein, wherein said two proteins, protein fragments, polypeptides, and/or peptides may comprise different Replikin peptide sequences, functional fragments of Replikin peptide sequences, and/or homologues of Replikin peptide sequences. A vaccine may comprise a mixture of proteins, protein fragments, polypeptides, and/or peptides. In a non-limiting embodiment, a mixture of proteins, protein fragments, polypeptides, and/or peptides may comprise proteins, protein fragments, polypeptides, or peptides comprising each of SEQ ID NO(s): 1-16, respectively. In a non-limiting embodiment, a vaccine may comprise at least one isolated or chemically-synthesized peptide consisting essentially of any one of SEQ ID NO(s): 1-35.

[00025] In a non-limiting embodiment, a vaccine may comprise a mixture of isolated or chemically-synthesized protein, protein fragments, polypeptides, and/or peptides, including at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID NO(s): 1-16, respectively, including at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID NO(s): 3-7, 11, 17-25, respectively, at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID NO(s): 26-31, respectively, and/or at least one isolated or chemically-synthesized peptide consisting essentially of SEQ ID NO(s): 28 and 30-35, respectively. A vaccine may comprise at least one isolated or chemically-synthesized peptide consisting essentially of SEQ ID NO: 13. A vaccine may comprise a pharmaceutically-acceptable carrier, excipient, and/or adjuvant. [00026] A fourth non-limiting aspect of the present invention provides a method of determining the relative virulence of an isolate A of AMDV comprising determining a Replikin concentration of at least one amino acid sequence A isolated from an isolate A of AMDV and comparing the Replikin concentration of the at least one amino acid sequence A to the Replikin concentration of at least one amino acid sequence B isolated from an isolate B of AMDV and determining that isolate A is relatively more virulent than isolate B if the Replikin concentration of said a least one amino acid sequence A is greater than the Replikin concentration of said at least one amino acid sequence B or determining that isolate A is relatively less virulent than isolate B if the Replikin concentration of said at least one amino acid sequence A is less than the Replikin concentration of said at least one amino acid sequence B.

[00027] In a non-limiting embodiment, at least one amino acid sequence A is encoded in the same region of the genome of isolate A of AMDV as at least one amino acid sequence B encoded in the genome of isolate B. In a non- limiting embodiment, sequence A is the amino acid sequence of a protein and sequence B is the amino acid sequence of the same protein as sequence A. In another non-limiting embodiment, sequence A is a Replikin Peak Gene of isolate A and sequence B is a Replikin Peak Gene of isolate B.

[00028] In another non-limiting embodiment, at least one sequence A is a plurality of sequences A isolated from a plurality of isolates of a population A of AMDV and at least one sequence B is a plurality of sequences B isolated from a plurality of isolates of a population B of AMDV where a mean Replikin concentration of the plurality of sequences A is compared to a mean Replikin concentration of the plurality of sequences B and virus population A is determined to be relatively more virulent than virus population B if the mean Replikin concentration of the plurality of sequences A is greater than the mean Replikin concentration of the plurality of sequences B or the virus population A is determined to be relatively less virulent than virus population B if the Replikin concentration of the plurality of sequences A is less than the Replikin concentration of the plurality of sequences B.

[00029] In a non-limiting embodiment, the population A of AMDV is a population of a particular strain of AMDV isolated within a time period A and the population B of AMDV is a population of the same strain of AMDV isolated within a time period B. In a non-limiting embodiment, the time period A may be a particular calendar year and the time period B may be a different calendar year.

[00030] In a non-limiting embodiment, at least one step of the method is processed on a computer. In another non-limiting embodiment, all steps of the method of determining the relative virulence of an isolate A of AMDV are processed on a computer.

[00031] In a non-limiting embodiment, determination of higher relative virulence in a population in mink is followed by the steps of culling infected subjects in the population or isolating infected subjects in a population or isolating populations infected with relatively more virulent AMDV from other populations.

[00032] A fifth non-limiting aspect of the present invention provides a method of making a preventive or therapeutic AMDV vaccine comprising identifying at least one Replikin sequence, at least one functional fragment of the at least one Replikin sequence, or at least one homologue of a Replikin sequence in at least one protein, protein fragment, polypeptide, or peptide of AMDV wherein a Replikin sequence consists of 7 to 50 amino acid residues and comprises (1) at least one lysine residue six to ten amino acid residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues, and making the virus vaccine comprising the at least one protein, protein fragment, polypeptide, or peptide of AMDV.

[00033] In a non-limiting embodiment, the at least one protein, protein fragment, polypeptide, or peptide is isolated or chemically-synthesized and combined with a pharmaceutically-acceptable carrier, excipient, and/or adjuvant to make the virus vaccine. In a non-limiting embodiment, the at least one Replikin sequence is conserved for at least two consecutive years in a plurality of isolates of AMDV. In a non-limiting embodiment, the at least one Replikin sequence has at least one lysine residue on one end of the sequence and at least one lysine residue or at least one histidine residue on the other end of the sequence. In a non-limiting embodiment, the vaccine is made with the shortest Replikin sequences identified in a virulent strain of AMDV. In a non-limiting embodiment, the vaccine is made with the longest Replikin sequences identified in a virulent strain of AMDV. [00034] A sixth non-limiting aspect of the invention provides a method of preventing, mitigating, or treating AMDV infection or an outbreak of AMDV infection comprising administering at least one compound comprising an AMDV Replikin peptide sequence, a functional fragment of an AMDV Replikin peptide sequence, or a homologue of a Replikin peptide sequence, to a subject. In a non-limiting embodiment, a compound comprises a protein, protein fragment, polypeptide, or peptide comprising an AMDV Replikin peptide sequence, a functional fragment of an AMDV Replikin peptide sequence, or a homologue of an AMDV Replikin peptide sequence.

[00035] A seventh non-limiting aspect of the present invention provides a method of stimulating the immune system of a subject comprising administering at least one AMDV Replikin peptide sequence, at least one functional fragment of at least one AMDV Replikin peptide sequence, or at least one homologue of a AMDV Replikin peptide sequence or at least one protein, protein fragment, polypeptide, or peptide comprising said at least one AMDV Replikin peptide sequence, said functional fragment of said at least one AMDV Replikin peptide sequence, or said at least one homologue of said AMDV Replikin peptide sequence to a subject to stimulate the immune system.

[00036] An eighth non-limiting aspect of the present invention provides a binding agent that specifically binds at least a portion of an AMDV Replikin peptide sequence. In a non-limiting embodiment, the binding agent is an isolated antibody or antibody fragment. In another non-limiting embodiment, the binding agent is an antagonist of the replication of AMDV and interacts with at least a portion of an AMDV Replikin peptide sequence or functional fragment thereof. In a non-limiting embodiment, an antagonist may interact with at least a portion of a Replikin peptide sequence of AMDV and not fully bind the portion.

[00037] A ninth non-limiting aspect of the present invention provides an isolated or chemically-synthesized Replikin Peak Gene of AMDV wherein the Replikin Peak Gene is identified as the portion of the genome, protein or protein fragment of a virion of the virus consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to other portions of the genome, protein or protein fragment of the virion of the virus. A Replikin Peak Gene may be identified as the portion of a protein or protein fragment consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to all other proteins or protein fragments in the virion of the virus. In a non-limiting embodiment, an isolated or chemically-synthesized peptide or polypeptide may consist essentially of an AMDV Replikin Peak Gene. In a non- limiting embodiment, an isolated or chemically-synthesized peptide or polypeptide may consist of an AMDV Replikin Peak Gene. In a non-limiting embodiment, an isolated or chemically-synthesized Replikin sequence or functional fragment thereof may be identified in a Replikin Peak Gene of AMDV. In a non-limiting embodiment, at least one isolated or chemically- synthesized Replikin Peak Gene may be comprised in a composition, an immunogenic composition, or a blocking composition. A composition, immunogenic composition, or blocking composition may comprise at least one Replikin sequence or functional fragment thereof identified in a Replikin Peak Gene.

[00038] A non-limiting embodiment comprises a chemically-synthesized AMDV peptide or polypeptide comprising an AMDV Replikin Peak Gene, where the Replikin Peak Gene is identified as the portion of the genome, protein or protein fragment of a virion of the virus consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to other portions of the genome, protein or protein fragment of the virion of the virus. An isolated or chemically-synthesized peptide or polypeptide may be an AMDV protein or protein fragment comprising a Replikin Peak Gene.

[00039] A non-limiting embodiment provides a method of stimulating the immune system of a subject comprising administering at least one AMDV Replikin Peak Gene or at least one polypeptide comprising at least one AMDV Replikin Peak Gene to a subject to stimulate the immune system. Another non- limiting embodiment provides a vaccine against AMDV comprising at least one isolated or chemically-synthesized AMDV polypeptide comprising an AMDV Replikin Peak Gene. In a non-limiting embodiment, a vaccine may comprise an isolated or synthesized AMDV polypeptide comprising an AMDV Replikin Peak Gene. The isolated or chemically-synthesized polypeptide may be an AMDV protein or protein fragment comprising a Replikin Peak Gene. An isolated or chemically-synthesized polypeptide may consist essentially of an AMDV Replikin Peak Gene. An isolated or chemically-synthesized polypeptide may consist of an AMDV Replikin Peak Gene. [00040] A non-limiting embodiment provides a binding agent that specifically binds at least a portion of an AMDV Replikin Peak Gene. In a non- limiting embodiment, a binding agent may be an antibody or antibody fragment. In a non-limiting embodiment, a binding agent may be an antagonist of the replication of AMDV that interacts with at least a portion of an AMDV Replikin Peak Gene. In a non-limiting embodiment, an antagonist may bind at least a portion of an AMDV Replikin Peak Gene. In a non-limiting embodiment, an antagonist may be an antibody.

[00041] A tenth non-limiting embodiment provides a method of preventing, mitigating, or treating an outbreak of AMDV predicted to have an expansion of population comprising predicting an expansion of the population of a first AMDV pathogen comprising

i. identifying at least one cycle of Replikin concentration in isolates of AMDV and predicting that an expansion of the population of the AMDV will take place after the occurrence of a rising portion of the at least one cycle of Replikin concentration, and

ii. administering to an animal or patient a compound comprising an isolated or chemically-synthesized portion of the structure or genome of the pathogen to mitigate, prevent, or treat the predicted outbreak of the pathogen.

[00042] A non-limiting embodiment provides a method comprising mitigating an outbreak of AMDV comprising

i. predicting an expansion of the population of a first strain of AMDV as compared to another isolate or plurality of isolates of the same or a related strain of AMDV comprising: (1) identifying a first cycle in the Replikin concentration of a plurality of isolates of said first strain of AMDV, (2) identifying a first peak in the Replikin concentration within the identified first cycle at a first time point or time period, and (3) predicting an increase in the virulence of an isolate of the same or related strain of pathogen isolated at a second time point or time period subsequent to the first time point or time period; and

ii. administering to an animal or a patient a compound comprising an isolated or synthesized portion of the structure or genome of the at least one isolate of the AMDV to prevent, mitigate, or treat the outbreak of AMDV.

[00043] In a non-limiting embodiment, an isolated or chemically- synthesized portion of the structure or genome of the at least one isolate of AMDV is a protein or protein fragment comprising a Replikin peptide and/or a Replikin Peak Gene, a Replikin peptide identified within a Replikin Peak Gene, or any structure or portion of the structure of said AMDV.

[00044] In a non-limiting embodiment, an isolated or chemically- synthesized portion of a structure or genome may consists essentially of a Replikin peptide or a Replikin Peak Gene or may consist of a Replikin peptide or a Replikin Peak Gene. In a non-limiting embodiment, a second time point or time period is up to three years after a first time point or time period, a second time point or time period is about one year after a first time point or time period, or a second time point or time period is about six months after a first time point or time period, or more or less.

[00045] An eleventh non-limiting aspect of the present invention provides a method of predicting an expansion of the population of AMDV comprising identifying at least one cycle of Replikin concentration in isolates of AMDV and predicting that an expansion of the population of AMDV will take place after the occurrence of a rising portion of the at least one cycle of Replikin concentration. In a non-limiting embodiment, a rising portion comprises a peak where the expansion of the population of AMDV is predicted after the occurrence of the peak. In a non-limiting embodiment, a cycle comprises at least a first rising portion and a second rising portion where a first rising portion occurs prior in time to a second rising portion. In a non-limiting embodiment, a cycle comprises at least three rising portions where the at least three rising portions are at least rising portion A', rising portion B' and rising portion C In a non- limiting embodiment, the rising portion B' comprises a peak and the rising portion A' comprises a peak, and the peak of rising portion B' has a greater Replikin concentration than the peak of rising portion A'.

[00046] In a non-limiting embodiment, at least one step of the method is processed on a computer. In another non-limiting embodiment, each step of the method is processed on a computer. [00047] In a non-limiting embodiment, a cycle comprises more than one cycle including from peak to trough to peak to trough or from trough to peak to trough to peak. In a non-limiting embodiment, a cycle comprises three peaks or three troughs or more.

[00048] A non-limiting embodiment provides a method of preventing, mitigating, or treating an outbreak of AMDV comprising

i. predicting an expansion of a strain of AMDV comprising (1) determining a mean Replikin Count and a standard deviation of said mean Replikin Count for a plurality of isolates of a strain of AMDV for a first time period in a first geographic region, (2) determining a Replikin Count of at least one isolate of the same or a related strain of AMDV from a second time period and/or second geographic region wherein said second time period is different from said first time period and/or said second geographic region is different from said first geographic region, and (3) predicting an expansion of said strain of pathogen isolated in said second time period and/or second geographic region if the Replikin Count of said at least one isolate is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said first time period and in said first geographic region; and

ii. administering to an animal or a patient a compound comprising an isolated or synthesized portion of the structure or genome of the at least one isolate of AMDV virus to prevent or treat the outbreak of AMDV virus.

[00049] In a non-limiting embodiment, a first period is one year and a first geographic region is a country. In a non-limiting embodiment, a second time period is one year. In a non-limiting embodiment, a second geographic region is a country. In a non-limiting embodiment, the isolated or chemically-synthesized portion of the structure or genome of the at least one isolate of pathogen is a protein or protein fragment comprising a Replikin peptide. In a non-limiting embodiment, a protein or protein fragment is a Replikin peptide. In a non- limiting embodiment, a protein or protein fragment consists essentially of a Replikin peptide or a Replikin Peak Gene. In a non-limiting embodiment, a protein or protein fragment comprises a Replikin Peak Gene. In a non-limiting embodiment, a protein or protein fragment consists of a Replikin Peak Gene. In a non-limiting embodiment, a protein or protein fragment is a Replikin peptide identified within a Replikin Peak Gene.

[00050] In a non-limiting embodiment, an isolated or chemically- synthesized portion of the structure or genome is a nucleic acid encoding a Replikin Peak Gene, a nucleic acid encoding a Replikin peptide or plurality of Replikin peptides within a Replikin Peak Gene, or a nucleic acid encoding a Replikin peptide.

[00051] Another non-limiting embodiment provides a method of predicting an expansion of a strain of AMDV comprising

i. determining a mean Replikin Count and a standard deviation of said mean Replikin Count for a plurality of isolates of said strain of AMDV for a first time period in a first geographic region;

ii. determining a Replikin Count of at least one isolate of the same or a related strain of AMDV from a second time period and/or second geographic region wherein said second time period is different from said first time period and/or said second geographic region is different from said first geographic region; and

iii. predicting an expansion of said strain of AMDV isolated in said second time period and/or second geographic region if the Replikin Count of said at least one isolate from a second time period and/or second geographic region is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said first time period and in said first geographic region.

[00052] In a non-limiting embodiment, a least one step of the method is processed on a computer. In another non-limiting embodiment, all steps of the method are processed on a computer.

BRIEF DESCRIPTION OF THE DRAWINGS

[00053] Figure 1 illustrates annual mean Replikin concentration (Replikin Count) of all protein sequences (available to applicants) of isolates of Aleutian mink disease virus from Vendsyssel, Denmark from 2000 through 2009. In Figure 1, gray columns represent annual mean Replikin Count and black-capped lines represent standard deviation from the annual mean. Table 1 in Example 1 contains the data reflected in Figure 1. A rise in Replikin Count may be seen beginning between 2001 and 2002. This rise in 2002 was followed by an increase in percentage of incidence of Aleutian mink disease virus in

Vendsyssel, Denmark beginning in 2003. The rise in incidence of Aleutian mink disease is illustrated in Figure 2.

[00054] Figure 2 illustrates annual percent incidence of Aleutian mink disease virus in Vendsyssel, Denmark from 2000 through 2008. Table 2 in Example 1 contains the data reflected in Figure 2. A rise in incidence is observed beginning in 2003. This rise follows a rise in Replikin Count in isolates from the Vendsyssel mink population beginning in 2002 (as seen in Figure 1).

[00055] Figure 3 illustrates annual mean Replikin concentration (Replikin Count) of all protein sequences of isolates of Aleutian mink disease virus reported at www.pubmed.com between 1983 and 2011. In Figure 3, gray columns represent annual mean Replikin Count and black-capped lines represent standard deviation from the annual mean. Table 4 in Example 8 contains the data reflected in Figure 3.

[00056] Figure 4 illustrates a high-level block diagram of a computer system incorporating a system and method for identifying Replikin patterns in amino acid sequences.

[00057] Figure 5 illustrates a simple flow chart illustrating a general method for locating a Replikin pattern in a sequence of amino acids.

[00058] Figure 6 illustrates a flow chart illustrating a generalized method for locating a plurality of Replikin-like patterns in a sequence of amino acids.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[00059] As used herein, a "Replikin sequence" is an amino acid sequence of 7 to about 50 amino acids comprising (1) a first lysine residue located six to ten residues from a second lysine residue, (2) at least one histidine residue; and (3) at least 6% lysine residues. A Replikin sequence may have a lysine residue on one end of the sequence and a lysine residue or histidine residue on the other end of the sequence. For the purpose of determining Replikin concentration, a Replikin sequence is the shortest amino acid sequence of 7 to 50 amino acid residues comprising (1) a first lysine residue located six to ten residues from a second lysine residue, (2) at least one histidine residue; and (3) at least 6% lysine residues. For purposes of determining Replikin concentration, the Replikin sequence may comprise any number of lysine residues and any number of histidine residues so long as any two lysine residues and any one histidine residue reflect the requirements of the Replikin sequence. As a result, a Replikin sequence counted as part of the Replikin concentration of a sequence of amino acid residues may comprise overlapping Replikin sequences.

[00060] The term "Replikin sequence" can also refer to a nucleic acid sequence encoding an amino acid sequence having 7 to about 50 amino acids comprising:

(1) at least one lysine residue located six to ten amino acid residues from a second lysine residue;

(2) at least one histidine residue; and

(3) at least 6% lysine residues,

wherein the amino acid sequence may have a lysine residue on one end of the sequence and a lysine residue or histidine residue on the other end of the sequence or may be the shortest amino acid sequence having any two lysine residues and any one histidine residue reflecting the requirements of the Replikin sequence.

[00061] As used herein, "Replikin Count" or "Replikin concentration" refers to the number of Replikin sequences per 100 amino acids in a protein, protein fragment, virus, or organism. A higher Replikin concentration in a first strain of a virus or organism has been found to correlate with more rapid replication of the first virus or organism as compared to a second, earlier-arising or later-arising strain of the virus or organism having a lower Replikin concentration. Replikin concentration is determined by counting the number of Replikin sequences in a given amino acid sequence or in a nucleic acid that encodes at least one Replikin peptide sequence.

[00062] As used herein, the term "peptide" or "protein" refers to a compound of two or more amino acids in which the carboxyl group of one amino acid is attached to an amino group of another amino acid via a peptide bond. As used herein, "isolated" or "synthesized" peptide or biologically active portion thereof refers to a peptide that is, after purification, substantially free of cellular material or other contaminating proteins or peptides from the cell or tissue source from which the peptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized by any method, or substantially free from contaminating peptides when synthesized by recombinant gene techniques. A protein or peptide may be isolated in silico from nucleic acid or amino acid sequences that are available through public or private databases or sequence collections.

[00063] An "encoded" or "expressed" protein, protein sequence, protein fragment sequence, or peptide sequence is a sequence encoded by a nucleic acid sequence that encodes the amino acids of the protein or peptide sequence with any codon known to one of skill in the art now or hereafter. It should be noted that it is well-known in the art that, due to redundancy in the genetic code, individual nucleotides can be readily exchanged in a codon and still result in an identical amino acid sequence. As will be understood by one of skill in the art, a method of identifying a Replikin amino acid sequence also encompasses a method of identifying a nucleic acid sequence that encodes a Replikin amino acid sequence wherein the Replikin amino acid sequence is encoded by the identified nucleic acid sequence.

[00064] As used herein, a "protein fragment" is a polypeptide that has less than the total number of amino acid residues of an expressed whole protein. A "whole protein" or an "expressed whole protein" as used in this specification reflects a protein that is expressable from an intact gene of a virus from a start codon to a stop codon. A whole protein or expressed whole protein may also reflect a whole protein or expressed whole protein that has been subject to cellular processing to create a protein that is capable of functioning within the virus replication system in a proper manner for virus replication.

[00065] As used herein, "conserved" or "conservation" refers to conservation of particular amino acids due to lack of substitution.

[00066] As used herein, "outbreak" is an increase in virulence, morbidity, and/or mortality in a pathogenic disease or an expansion in the population of pathogen as compared to a baseline of an earlier occurring epidemiological pattern of infection in the same disease. One of ordinary skill in the art will know how to determine an epidemiological baseline.

[00067] As used herein, an "isolate" is any virus or organism isolated from a natural source wherein a natural source includes, but is not limited to, a reservoir of an organism or virus, a vector of an organism or virus, or a host of an organism or virus. "Obtaining," "isolating," or "identifying" an isolate is any action by which an amino acid or nucleic acid sequence within an isolate is obtained including, but not limited to, isolating an isolate and sequencing any portion of the genome or protein sequences of the isolate, obtaining any nucleic acid sequence or amino acid sequence of an isolate, wherein the nucleic acid sequence or amino acid sequence may be analyzed for Replikin concentration, or any other means of obtaining the Replikin concentration of a virus isolated from a natural source at a time point or within a time period. Isolated or related words may also mean: identified within such a Replikin sequence identified within a larger polypeptide. A Replikin sequence may be analyzed in silico from any medium, including from a database such as the databases at www.pubmed.com. Nevertheless, isolated sequences in this specification refers to sequences that have been physically isolated.

[00068] "Functional derivatives" of the Replikin sequences as described herein are fragments, variants, analogs, or chemical derivatives of Replikin sequences that retain at least a portion of the immunological cross reactivity with an antibody specific for the Replikin sequence. A fragment of the Replikin peptide refers to any subset of the molecule. A "functional fragment" of the Replikin peptide or a Replikin Peak Gene is any subset of the molecule that retains at least a portion of immunological cross reactivity with an antibody specific for the Replikin peptide or Replikin Peak Gene. Variant peptides may be made by direct chemical synthesis, for example, using methods well known in the art. An analog of a Replikin to a non-natural protein substantially similar to either the entire protein or a fragment thereof. Chemical derivatives of a Replikin contain additional chemical moieties not normally a part of the peptide or peptide fragment. KLNRVIKTTH (SEQ ID NO: 29) is an example of a functional fragment of KLNRVIKTTHEKFH (SEQ ID NO: 30). Likewise, RVIKTTH (SEQ ID NO: 36) is a functional fragment of KLNRVIKTTHEKFH (SEQ ID NO: 30) and a functional fragment of KLNRVIKTTH (SEQ ID NO: 29). SEQ ID NO: 29 is likewise 79.4% homologous with SEQ ID NO: 30. SEQ ID NO: 36 is 70% homologous with SEQ ID NO: 29 and 50% homologous with SEQ ID NO: 30. [00069] As used herein, "homologous" or "homology" or "sequence identity" are used to indicate that an amino acid sequence or nucleic acid sequence exhibits substantial structural or functional equivalence with another sequence. Any structural or functional differences between sequences having sequence identity or homology will be de minimus; that is, they will not affect the ability of the sequence to function as indicated in the desired application. Structural differences are considered de minimus if there is a significant amount of sequence overlap or similarity between two or more different sequences or if the different sequences exhibit similar physical characteristics even if the sequences differ in length or structure. Such characteristics include, for example, the ability to hybridize under defined conditions, or in the case of proteins, immunological cross-reactivity, similar enzymatic activity, etc. The ordinary skilled practitioner can readily determine each of these characteristics by art-known methods. Because of the crucial nature of the lysine and histidine residues in a Replikin peptide sequence related to the function of rapid replication and antigenicity, functional characteristics of Replikin sequences are conserved even when larger than expected sequence differences are noted.

[00070] To determine the percent identity or percent homology of two sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences as compared to the total length of the sequence identified as a reference sequence. [00071] As used herein, an amino acid sequence of a protein fragment, polypeptide, or peptide is "derived from" an identified protein or gene area of an AMDV isolate (such as the VP 1, V2, or VP3 capsid proteins) if one of ordinary skill in the art would understand from the structure, history, or other relevant information of the amino acid sequence that it originated from an amino acid sequence of the identified protein or gene area. Among other methods, one of ordinary skill may employ analysis of the homology of the amino acid sequence with the identified protein or gene area. One of ordinary skill may also employ the history of research used in developing the amino acid sequence to determine that the amino acid sequence is derived from an original sequence of the identified protein or gene area. One of ordinary skill would understand that a protein fragment, polypeptide, or peptide is derived from an identified protein, polypeptide, or peptide if it is traceable to the identified protein, polypeptide, or peptide, if it is deducible or inferable from the identified protein, polypeptide, or peptide, if the identified protein, polypeptide, or peptide is the source of the peptide, or if the protein fragment, polypeptide, or peptide is derived from the identified protein, polypeptide, or peptide as understood by one of skill in the art. One of ordinary skill may employ any method known now or hereafter for determining whether an amino acid sequence is derived from an identified protein or gene area of an AMDV.

[00072] As used herein, a "Replikin cycle" or "a cycle of Replikin

concentration" or "a cycle of Replikin Count" means Replikin concentrations of a plurality of isolates of a species of virus or organism wherein at least four of said plurality of isolates are isolated at successive time points or in successive time periods, wherein a Replikin concentration of a second individual isolate or second mean of a plurality of isolates at a second time point or time period is higher than a Replikin concentration of a first individual isolate or a first mean of a plurality of isolates at a first time point or time period, a Replikin concentration of a third individual isolate or a third mean of a plurality of isolates at a third time point or time period is lower than the Replikin

concentration at a second time point or time period, and a Replikin concentration of a fourth individual isolate or fourth mean of a plurality of isolates at a fourth time point or time period is higher than the Replikin concentration at a third time point or time period; or wherein a Replikin concentration of a second individual isolate or second mean of a plurality of isolates at a second time point or time period is lower than a Replikin concentration of a first individual isolate or a first mean of a plurality of isolates at a first time point or time period, a Replikin concentration of a third individual isolate or a third mean of a plurality of isolates at a third time point or time period is higher than the Replikin concentration at a second time point or time period, and a Replikin concentration of a fourth individual isolate or fourth mean of a plurality of isolates at a fourth time point or time period is lower than the Replikin concentration at a third time point or time period. Within the Replikin cycle, cycle of Replikin concentration, or cycle of Replikin Count, the second time point or time period must be later in time than the first time point or time period, the third time point or time period must be later in time than the second time point or time period, and the fourth time point or time period must be later in time than the third time point or time period. Within a Replikin cycle, any rising portion is predictive of an expansion in population or an increase in virulence, morbidity, and/or mortality of a pathogen in hosts and any decreasing portion is predictive of a contracting population or a decrease in virulence, morbidity, and/or mortality of pathogen in hosts. A cycle need not be complete to be predictive; a decreasing portion followed by a rising portion is predictive of an expanding population or an increase in virulence, morbidity, and/or mortality. Likewise, a rising portion followed by a decreasing portion followed by a rising portion is predictive of an expanding population or an increase in virulence, morbidity, and/or mortality. As such, cycles need not be complete cycles to provide predictive capacity concerning an expansion or contraction (or change in virulence, morbidity, and/or mortality) of a pathogen in hosts.

[00073] As used herein, a "step-wise" cycle is any set of cycles wherein a first Replikin cycle peak in time is lower than a second Replikin cycle peak in time or a first Replikin cycle peak in time is higher than a second Replikin cycle peak in time. A step-wise cycle also occurs when successive peaks are observed to move lower. A step-wise cycle may also be observed if successive troughs move higher or lower. Step-wise cycles provide additional predictive capacity for predictions of expansion or contraction of a population.

[00074] As used herein, a "rising portion" of a Replikin cycle means the Replikin concentration of an isolate or the mean Replikin concentration of a plurality of isolates, wherein the isolate or isolates were isolated at a time point or time period of the Replikin cycle where the trend of Replikin concentration in the Replikin cycle is increasing from at least a first time point or time period to at least a second time point or time period. Additionally, the rising portion may include a peak.

[00075] As used herein, a "decreasing portion" of a Replikin cycles means the opposite of a rising portion, wherein a decreasing portion may include a trough.

[00076] As used herein, a "peak" in a Replikin cycle means a second time point or time period within a Replikin cycle, wherein the Replikin concentration at a first time point or time period sequentially preceding the second time point or time period is lower than the Replikin concentration at the second time point or time period, and the Replikin concentration at a third time point or time period sequentially following the second time point or time period is lower than the Replikin concentration at the second time point or time period. One of skill in the art will understand that because of the variability of biological systems, a peak may include a general region of a cycle that is generally higher than a sequentially preceding region and generally higher than a sequentially following region rather than an exact time point or time period.

[00077] As used herein, a "trough" in a Replikin cycle means the opposite of a peak in a Replikin cycle.

[00078] As used herein, a "Replikin Count Virus Expansion Index" or "RCVE Index" or a "Replikin Count Expansion Index" or "RCE Index" is the number of Replikin Counts of a plurality of isolates from a first time period and/or first geographic region that are greater than one standard deviation of the mean of the Replikin Count of a plurality of isolates isolated in a second time period and in a second geographic region, divided by the number of Replikin Counts of said plurality of isolates from said first time period and/or said first geographic region that are less than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said second time period in said second geographic region. An RCE or RCVE Index predicts the expansion of a pathogen in a particular region and/or time period if the ratio of the RCE or RCVE Index is greater than one. An RCE or RCVE Index predicts the contraction, retraction, reduction, or failure of a pathogen in a particular region and/or time period if the ratio of the RCE or RCVE Index is less than one. An RCE or RCVE Index predicts equilibrium between expansion and contraction in the pathogen population if the ratio of the RCVE Index is equal to one.

[00079] As used herein, different "time periods" or different "time points" are any two time periods or time points that may be differentiated from each other. For example, an isolate of an organism or virus isolated during the year 2004 may be considered to be isolated in a different time period than an isolate of the same organism or virus isolated during the year 2005. Likewise, an isolate of an organism or virus isolated in May 2004 may be considered to be isolated in a different time period than an isolate of the same organism or virus isolated in June 2004. When comparing Replikin concentrations of different isolates, one may use comparable time periods. For example, an isolate from 2004 may be compared to at least one other isolate from some other year such as 2002 or 2005. Likewise, an isolate from May 2004 may be compared to at least one isolate from some other month of some year, for example, an isolate from December 2003 or from June 2004.

[00080] As used herein, "an earlier-arising" virus or organism or a virus or organism isolated at "an earlier time point" or during "an earlier time period" is a specimen of a virus or organism collected from a natural source of the virus or organism on a date prior to the date on which another specimen of the virus or organism was collected from a natural source. A "later-arising" virus or organism or a virus or organism isolated at a "later time point" or during a "later time period" is a specimen of a virus or organism collected from a natural source of the virus (including, but not limited to, a reservoir, a vector, or a host) or a natural source of the organism on a date subsequent to the date on which another specimen of the virus or organism was collected from a natural source.

[00081] As used herein, a "Replikin Peak Gene (RPG)" (or sometimes a Replikin Peak Gene Area-RPGA) means a segment of a genome, protein, segment of protein, or protein fragment in which an expressed gene or gene segment has a highest concentration of continuous, non-interrupted and overlapping Replikin sequences (number of Replikin sequences per 100 amino acids) when compared to other segments or named genes of the genome. More than one RPG may be identified within a gene, gene segment, protein, or protein fragment. An RPG may have a terminal lysine or a terminal histidine, two terminal lysines, or a terminal lysine and a terminal histidine. For diagnostic, therapeutic and preventive purposes, an RPG may have a terminal lysine or a terminal histidine, two terminal lysines, or a terminal lysine and a terminal histidine or may likewise have neither a terminal lysine nor a terminal histidine so long as the terminal portion of the RPG contains a Replikin sequence or Replikin sequences defined by the definition of a Replikin sequence, namely, an amino acid sequence having 7 to about 50 amino acids comprising:

(2) at least one histidine residue; and

(3) at least 6% lysine residues.

Further, for diagnostic, therapeutic, preventive and predictive purposes, an RPG may include the protein or protein fragment that contains an identified RPG. For predictive purposes, a Replikin Count in the RPG may be used to track changes in virulence and lethality. Likewise the RPG may be used as an immunogenic compound or as a vaccine. Whole proteins or protein fragments containing RPGs are likewise useful for diagnostic, therapeutic and preventive purposes, such as, for example, to be included in immunogenic compounds, vaccines and for production of therapeutic or diagnostic antibodies.

[00082] As used herein, "expansion" of a pathogen or a population of pathogen and "expanding" pathogen or population of pathogen means an increase in virulence, morbidity, and/or lethality of a pathogen (e.g., a strain of AMDV) and/or an expansion of the population of a pathogen (e.g., a strain of AMDV) wherein said expansion includes an increase in the occurrence of the pathogen in a given geographic region or in a given time period or both, or a spreading of the occurrence of the pathogen to another geographic region.

[00083] As used herein, an increase or decrease in "virulence" includes an increase or decrease in virulence, morbidity, lethality, host mortality, rate of replication, rate of distribution, and/or expansion of a pathogen, such as an AMDV.

[00084] As used herein, "geographic region" or similar term is an area differentiated from another area by space including spaces on mink ranches. For example, China is a geographic region that may be differentiated from the geographic region of Denmark. Likewise a geographic region may be a town, or city, or continent or any area differentiable from another area. A geographic region may encompass the entire earth if an isolate or plurality of isolates from a given time period is compared to isolates from another time period over the entire earth and no geographic differentiation is undertaken for the comparison. A geographic region may denote a space on a mink ranch that is separated from another space such as one building separated from another building, one room separated from another room, or one pen separated from another pen.

[00085] As used herein, the term "continuous Replikin sequences" means a series of two or more Replikin sequences that are overlapped and/or are directly covalently linked.

Replikin Counts from AMDV Isolates in Vendsyssel, Denmark Correlate with Increases in Percent Incidence of AMDV Disease

[00086] Replikin Counts were determined for all genomic sequences available to applicants for isolates of AMDV from mink ranches in Vendsyssel,

Denmark from 2000 through 2009. Annual mean Replikin Count with standard deviation from the mean was determined for each year. See Figure 1. Percent incidence of AMDV in mink ranches in Vendsyssel was determined. See Figure

2. It was observed that annual mean Replikin Count began to rise between 2001 and 2002. It was further observed that percent incidence began to rise in 2003 following the 2002 rise in Replikin Count. Applicants determined that Replikin

Count correlated with and predicted the 2003 rise in disease incidence. One non- limiting aspect of the present invention is, therefore, a method of forecasting rise in incidence, virulence, and morbidity in AMDV in mink.

Vaccine against outbreaks of AMDV in Vendsyssel, Denmark, Canada, China, and Bornholm

[00087] Applicants have designed a vaccine that targets AMDV outbreaks by targeting Replikin sequences in AMDV in isolates from Vendsyssel and Saeby Denmark. The vaccine comprises at least one sequence of SEQ ID NO(s): 1-16. The vaccine may further comprise more than one sequence of SEQ ID NO(s): 1-16 and may comprise each sequence of SEQ ID NO(s): 1-16. The vaccine may further comprise a functional fragment of any of SEQ ID NO(s): 1- 16. The vaccine may further comprise a homologue of any of SEQ ID NO(s): 1- 16. The vaccine may further comprise a polypeptide, protein fragment, or protein comprising at least one of SEQ ID NO(s): 1-16, a functional fragment of SEQ ID O(s): 1-16, or a homologue of SEQ ID NO(s): 1-16. The vaccine may comprise a pharmaceutically-acceptable carrier and/or adjuvant including sterile water or other carrier and may be administered by any method known to one of skill in the art including spray to the eyes or nose or parenteral administration. The vaccine may comprise equal parts by weight of each of SEQ ID NO(s): 1-16 and may be combined with any of SEQ ID NO(s): 17-36.

[00088] Applicants have further designed a vaccine that targets AMDV outbreaks by targeting Replikin sequences in AMDV in isolates from all parts of Denmark. The vaccine comprises at least one sequence of SEQ ID NO(s): 3-7, 1 1, and 17-25 or any target of SEQ ID NO(s): 3-7, 11, and 17-25 including a functional fragment of SEQ ID NO(s): 3-7, 11, and 17-25 and/or a homologue of SEQ ID NO(s): 3-7, 11, and 17-25 and may comprise any combination of peptides or polypeptides, proteins, or protein fragments comprising any one or more of SEQ ID NO(s): 3-7, 1 1, and 17-25. The vaccine may further comprise any one or more of SEQ ID NO(s): 1, 2, 8-10, 12-16, and 26-36.

[00089] Applicants have further designed a vaccine that targets AMDV outbreaks by targeting Replikin sequences in AMDV in isolates from Canada. The vaccine comprises at least one sequence of SEQ ID NO(s): 26-31 or any target of SEQ ID NO(s): 26-31 including a functional fragment of SEQ ID NO(s): 26-31 and a homologue of SEQ ID NO(s): 26-31 and may comprise any combination of peptides or polypeptides, proteins, or protein fragments comprising any one or more of SEQ ID NO(s): 26-31. The vaccine may further comprise any one or more of SEQ ID NO(s): 1-25 and 32-36.

[00090] Applicants have further designed a vaccine that targets AMDV outbreaks by targeting Replikin sequences in AMDV in isolates from China. The vaccine comprises at least one sequence of SEQ ID NO(s): 28 and 30-35 or any target of SEQ ID NO(s): 28 and 30-35 including a functional fragment of SEQ ID NO(s): 28 and 30-35 or a homologue of SEQ ID NO(s): 28 and 30-35 and may comprise any combination of peptides or polypeptides, proteins, or protein fragments comprising any one or more of SEQ ID NO(s): 28 and 30-35. The vaccine may further comprise any one or more of SEQ ID NO(s): 1-27, 29, and 36.

[00091] Applicants have further designed a vaccine that targets AMDV outbreaks by targeting Replikin sequences in AMDV in isolates from Bornholm, Denmark. The vaccine comprises SEQ ID NO: 13 or any target of SEQ ID NO(s): 13 including a functional fragment of SEQ ID NO(s): 13 or a homologue of SEQ ID NO: 13 and may comprise any combination of peptides or polypeptides, proteins, or protein fragments comprising SEQ ID NO: 13. The vaccine may further comprise any one or more of SEQ ID NO(s): 1-12 and 14- 36.

[00092] A vaccine comprising any one of SEQ ID NO(s): 1-36 or any combination thereof or any functional fragment thereof or any homologue thereof and may be directed against outbreaks of AMDV since these sequences have been identified as conserved across regions and times. A vaccine comprising a homologue of any one of SEQ ID NO(s): 1-36 may likewise be directed at outbreaks of AMDV.

[00093] The vaccines provided in this application are directed against conserved mechanisms of rapid replication in AMDV using antigenic sequences and sequences capable of blocking rapid replication. As a result, previous failures of vaccine attempts are overcome by direct targeting of rapid replication.

[00094] Vaccines, immunogenic compositions, blocking compositions, and other compositions of the invention are useful because they stimulate the immune system against Replikin sequence targets or initiate blocking of Replikin sequence targets in the replication mechanism of AMDV. Replikin sequences, homologues of Replikin sequences, and functional fragments of Replikin sequences may be targeted within a protein or within the replication mechanism of AMDV to control replication, infectivity, and lethality of the virus. A polypeptide comprising a Replikin sequence, a homologue of a Replikin sequence, or a functional fragment of a Replikin sequence provides the Replikin sequence, homologue, or fragment as an antigenic or blocking structure that stimulates antagonism of replication, infectivity, and lethality of the virus. As a result, a protein comprising a Replikin sequence, homologue, or fragment, or a polypeptide or protein fragment comprising a Replikin sequence, homologue, or fragment may be 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, or 400 or more amino acid residues longer than the Replikin peptide sequence, functional fragment of the Replikin peptide sequence, or homologue of the Replikin peptide sequence identified or derived from an isolate of AMDV. The additional residues may begin at the C-terminus and/or N-terminus of the Replikin peptide sequence, homologue, or fragment. Nevertheless, the Replikin peptide sequence, homologue, or fragment remains a target to stimulate an immune response or initiate a blocking response in a subject to which the protein, protein fragment, or polypeptide has been administered.

Determination of Relative Virulence of AMDV

[00095] An aspect of the present invention provides methods of determining the relative virulence of an isolate, plurality of isolates, or population of AMDV. Relative virulence in AMDV includes increased or decreased morbidity, lethality, host mortality or viral lethality, rate of replication, rate of distribution, expansion of a pathogen and/or any metric known to one of skill in the art concerning the virulence of a virus. One non-limiting embodiment of the present invention provides a method of determining the relative virulence of an isolate A of AMDV by determining the Replikin concentration of at least one amino acid sequence isolated from isolate A of AMDV and comparing the Replikin concentration to the Replikin concentration of at least one amino acid isolated from an isolate B of AMDV. If the Replikin concentration of the at least one amino acid sequence A is greater than the Replikin concentration of said at least one amino acid sequence B then isolate A is determined to be relatively more virulent than isolate B. If the Replikin concentration of the at least one amino acid sequence A is less than the Replikin concentration of the at least one amino acid sequence B then isolate A is determined to be relatively less virulent than isolate B.

[00096] The at least one amino acid sequence A may be encoded in the same region of the genome of the virus as the at least one amino acid sequence B. Amino acid sequence A and B may be isolated from the same protein of AMDV or from the same region of the same protein. Sequence A and B may also be the amino acid sequence of the same protein in isolate A and isolate B, respectively. Sequences A and B may also be respective Replikin Peak Genes of isolate A and B.

[00097] Mean Replikin concentration of plurality of sequences A from a plurality of isolates of AMDV may be compared to the Replikin concentration of at least one amino acid sequence B. Mean Replikin concentration of a plurality of sequences B isolated from a plurality of isolates of AMDV may likewise be compared to the Replikin concentration of the at least one amino acid sequence A or to a mean Replikin concentration of a plurality of sequences A. [00098] A plurality of sequences A may also be isolated from a plurality of isolates of virus population A and a plurality of sequences B may be isolated from a plurality of isolates of virus population B where virus population A is determined to be relatively more virulent than virus population B if the mean Replikin concentration of the plurality of sequences A is greater than the mean Replikin concentration of the plurality of sequences B or virus population A may be determined to be relatively less virulent than virus population B if the Replikin concentration of the plurality of sequences A is less than the Replikin concentration of the plurality of sequences B.

[00099] Virus population A may be a population of a strain A of AMDV and virus population B may be a population of a strain B of AMDV where strain A is any particular strain of AMDV and strain B is any particular strain of AMDV that is different from strain A. Strain A may differ from strain B based on physical or genetic differences between strain A and strain B or may differ based on the time or region in which strain A was isolated as compared to the time or region in which strain B was isolated. Strain A may also differ from strain B based on any difference understood by one of skill in the art.

[000100] For example, virus population A may be a population of a particular strain of AMDV isolated within a time period A and virus population B may be a population of the same strain of AMDV isolated within a time period B. Time period A may be a particular month and time period B a different month. Time period A may be a particular year and time period B a different year. Time periods A and B may also represent different calendar years.

[000101] Another non-limiting embodiment provides a method of determining an increased probability of an outbreak of AMDV within about six months to about three years following an increase in Replikin concentration in at least one isolate of AMDV. The method comprises identifying an increase in the concentration of Replikin sequences in at least one first isolate of AMDV as compared to at least one other isolate of AMDV where the at least one first isolate is isolated at a later time point than the at least one other isolate is isolated, and where the increase in the concentration of Replikin sequences signifies an increased probability of outbreak of AMDV within about six months to about three years following the increase in the concentration of Replikin sequences. The at least one first isolate may be isolated at least one month later than the at least one other isolate. The at least one first isolate may be isolated at least six months later than the at least one other isolate. The at least one first isolate may be isolated at least one year or more later than the at least one other isolate.

[000102] One non-limiting aspect also provides a method of determining an increased probability of an outbreak of AMDV comprising:

(1) obtaining a plurality of isolates of AMDV wherein at least one of said isolates is isolated about six months to about 3 years later than at least one other of said isolates;

(2) analyzing the amino acid sequence of at least one protein or protein

fragment in each isolate of the plurality of isolates for the presence and concentration of Replikin sequences;

(3) comparing the concentrations of Replikin sequences in the at least one protein or protein fragment in each isolate of the plurality of isolates one to another;

(4) identifying an increase in the concentration of Replikin sequences in the plurality of isolates over at least one time period of about six months or greater; and

(5) predicting an outbreak of AMDV within about six months to about three or more years following said the identified increase in the concentration of Replikin sequences.

The outbreak of AMDV may be predicted within about six months to about five years. The outbreak likewise may be predicted within about one year to about three years or within about one year.

Emergence of AMDV using scout viruses

[000103] A non-limiting aspect of the present invention provides methods of determining emergence of AMDV. One non-limiting embodiment provides a method of determining emergence of AMDV in regions and times by analyzing the concentration and structure of the genomic Replikin sequences in scout viruses isolated from initial cases up to one year or longer before an outbreak. Knowledge of this emergence provides advance warning and the ability to respond to the coming specific outbreaks with vaccines and therapies that are a non-limiting aspect of the invention. [000104] Analysis of a the Replikin concentration of a plurality of isolates of AMDV in a population allows for the determination of a mean Replikin concentration with standard deviation of the mean (SD). See, e.g., Figures 1 and 3. The standard deviation of the mean indicates the population distribution of viruses with different Replikin concentrations. At relatively low virus replication rates, the standard deviation is small. However, standard deviation is seen to increase with virus rapid replication. An increase in mean Replikin concentration is then seen to follow in time. See, e.g., 2002-2004 of Figure 1.

[000105] This advance warning affords the time to produce specific solid- phase synthetic vaccines based on identified Replikin sequences in seven days, and to test them thoroughly. Bulk production also takes days rather than months. Storage does not require refrigeration.

[000106] Identification of specific Replikin sequences in the AMDV genome, both conserved and novel, provide for vaccines against populations with currently-increasing Replikin concentrations. Increasing Replikin concentrations provides knowledge of increased risk of large outbreaks.

[000107] When the Replikin sequences change in a population, vaccines and therapies against AMDV can be changed in 7 days. Examples of successful vaccines using this design have been disclosed in shrimp in taura syndrome virus and in chickens in H5N1 influenza virus. See, e.g., Jackwood MW, Bogoch, S., Bogoch ES, Hilt, D and Williams SW. Efficacy of a Replikin peptide Vaccine Against Low Pathogenicity Avian Influenza H5 Virus. Avian Diseases

53(4):613-617, 2009 and Examples 2 and 3 of WO 2008/156914.

AMDV Replikin sequences and their utilities

[000108] An isolated or chemically-synthesized AMDV Replikin peptide sequence consists of 7 to about 50 amino acid residues and comprises (1) at least one lysine residue located 6 to 10 residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues. An isolated or synthesized Replikin peptide sequence may also have at least one lysine residue on one end of the peptide and at least one lysine residue or at least one histidine residue on the other end of the peptide. An isolated or synthesized Replikin peptide may have at least one lysine residue on one end of the peptide and at least one lysine residue on the other end of the peptide or may have at least one lysine residue on one end of the peptide and at least one histidine residue on the other end of the peptide. An isolated or synthesized Replikin peptide may contain as few as two lysine residues and may contain all lysines with the exception of one histidine. The isolated or synthesized Replikin peptide may contain as few as one histidine residue and may contain many histidine residues so long as the peptide contains at least 6% lysines with two lysines in the requisite positions.

[000109] An isolated AMDV polypeptide may comprise an AMDV Replikin peptide or a synthesized polypeptide comprising an AMDV Replikin peptide. The synthesized polypeptide may be derived from a polypeptide of AMDV as understood by one of skill in the art. The synthesized polypeptide may be homologous with an AMDV polypeptide. The synthesized or isolated polypeptide may be an AMDV protein or protein fragment comprising a Replikin peptide. A synthesized or isolated peptide or polypeptide may consist essentially of an AMDV Replikin peptide.

[000110] In another non-limiting embodiment an isolated or synthesized peptide or polypeptide may comprise a plurality of Replikin peptides or a mixture of at least one Replikin peptide and at least one Replikin Peak Gene. An isolated or synthesized peptide or polypeptide may likewise consist essentially of a plurality of Replikin peptides or a mixture of at least one Replikin peptide and at least one Replikin Peak Gene. An isolated or synthesized peptide or polypeptide may also consist of a plurality of Replikin peptides or a mixture of at least one Replikin peptide and at least one Replikin Peak Gene.

[000111] A composition may comprise at least one AMDV Replikin peptide. In a non-limiting embodiment, the composition may comprise a

pharmaceutically-acceptable carrier and/or adjuvant. An immunogenic composition may comprise at least one AMDV Replikin peptide. The immunogenic composition may comprise a pharmaceutically-acceptable carrier and/or adjuvant and/or excipient. A blocking composition may comprise at least one AMDV Replikin peptide. The blocking composition may comprise a pharmaceutically-acceptable carrier and/or adjuvant and/or excipient.

[000112] A non-limiting vaccine may comprise at least one AMDV Replikin sequence and/or may comprise an isolated or synthesized AMDV polypeptide comprising an AMDV Replikin peptide. The isolated or synthesized

polypeptide may be an AMDV protein or protein fragment comprising a Replikin peptide. A vaccine may comprise a peptide or polypeptide consisting essentially of an AMDV Replikin peptide or plurality of AMDV Replikin peptides or may comprise a mixture of at least one Replikin peptide and at least one Replikin Peak Gene. A non-limiting vaccine may comprise at least one protein fragment, polypeptide, or peptide comprising any one of SEQ ID NO(s): 1-36, at least one polypeptide or peptide consisting essentially of any one of SEQ ID NO(s): 1-36, or at least one polypeptide or peptide consisting of any one of SEQ ID O(s): 1-36.

Homologues of AMDV Replikin sequences and their utilities

[000113] A non-limiting aspect of the present invention provides an isolated or chemically-synthesized polypeptide comprising at least one Replikin peptide sequence, a functional fragment of at least one Replikin peptide sequence, or at least one homologue of a Replikin peptide sequence. A homologue of a Replikin peptide sequence is an amino acid sequence exhibiting substantial structural or functional equivalence with the Replikin peptide sequence. To determine the percent identity or percent homology of two sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).

[000114] Applicants have identified many homologues of Replikin sequences in expressed proteins of AMDV. Many examples are provided in Table 3. One example of homologues in Table 3 is HHCDIQNIKDPEDRAKNLK (SEQ ID NO: 3) and HHCDOODIKDPEAKADNLK (SEQ ID NO: 26) where residues in SEQ ID NO: 26 that are not identical to residues in SEQ ID NO: 3 are bolded and underlined. SEQ ID NO: 26 has six amino acid residues that are not identical to residues at the same position in SEQ ID NO: 26. These two sequences are both 19 residues long. SEQ ID NO: 26 has 13 residues out of 19 that match the residues of SEQ ID NO: 3. As a result, SEQ ID NO: 26 is 68.4% homologous with SEQ ID NO: 3. SEQ ID NO: 3 has been identified in isolates of AMDV in Denmark from 1999, 2003, 2004, 2005, 2007, 2008, and 2009. See Table 3. SEQ ID NO: 26 has been identified in isolates of AMDV in Canada from 2005. See Table 3. These homologues are useful in vaccines against AMDV in either country. Further, because these homologues are conserved in various regions and across time, the sequences are useful as targets for controlling AMDV across regions and time.

[000115] As another example, HCDOQDIKDPEAKADNLK (SEQ ID NO: 17) is 94.7% homologous with HHCDOQDIKDPEAKADNLK (SEQ ID NO: 26) because SEQ ID NO: 17 matches SEQ ID NO: 26 in all nineteen residues except the first residue (a histidine) in SEQ ID NO: 26, which is not present in SEQ ID NO: 17. SEQ ID NO: 17 has been identified in Denmark in 2005. SEQ ID NO: 26 has been identified in Canada in 2005. See Table 3. As a result, these homologues are useful as targets for controlling AMDV across regions and time.

[000116] Another example of homologues is HKQTKQDYNK (SEQ ID NO: 4) and KQTKKDYVKMVH (SEQ ID NO: 34). SEQ ID NO: 4 is 58.3% homologous with SEQ ID NO: 34 because SEQ ID NO: 4 matches SEQ ID NO: 34 at seven out of twelve residues. SEQ ID NO: 4 has been identified in AMDV isolates in Denmark in 1999, 2003, 2004, 2005, 2007, 2008, and 2009. SEQW ID NO: 34 has been identified in AMDV isolates in China in 1992, 1993, 1994, 2009, and 2010. As a result, these homologues are useful as targets for controlling AMDV across regions and time.

[000117] Table 3 provides extensive examples of homologues identified in various isolates of AMDV from different regions and in different years. A vaccine comprising a sequence that is homologous with sequences in other isolates from other regions and is conserved or has homologues that are conserved over time is a vaccine that may be used against AMDV across regions and time. Table 3 provides many such examples.

Replikin peptides in AMDV vaccines

[000118] A non-limiting embodiment of the aspect of the present invention provides a method of making a preventive or therapeutic AMDV vaccine comprising identifying at least one Replikin sequence or at least one functional derivative fragment of the at least one Replikin sequence or at least one homologue of at least one Replikin peptide in at least one protein, protein fragment, polypeptide, or peptide of AMDV where the at least one Replikin sequence comprises 7 to 50 amino acid residues and comprises (1) at least one lysine residue six to ten amino acid residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues, and making the virus vaccine comprising the at least one protein, protein fragment, polypeptide, or peptide of AMDV. The at least one protein, protein fragment polypeptide, or peptide may be isolated or synthesized and further combined with a pharmaceutically acceptable carrier and/or adjuvant to make the AMDV vaccine. The Replikin sequence may be conserved in AMDV and further may be conserved for at least two consecutive years. In a non-limiting embodiment, the at least one Replikin sequence may consist of 7 to 50 amino acid residues. In a further non-limiting embodiment, the at least one Replikin sequence may have at least one lysine residue on one end of the sequence and at least one lysine residue or at least one histidine residue on the other end of the sequence.

[000119] Another non-limiting embodiment of the aspect of the present invention provides a method of making an AMDV vaccine comprising identifying at least one Replikin sequence, at least one functional derivative fragment of at least one Replikin sequence, or at least one homologue of at least one Replikin sequence in an amino acid sequence of AMDV where the at least one Replikin sequence comprises 7 to 50 amino acid residues and comprises (1) at least one lysine residue six to ten amino acid residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues, and making the AMDV vaccine comprising the at least one Replikin sequence. The Replikin sequence may consists of 7 to 50 amino acids and my further have at least one lysine residue on one end of the sequence and at least one lysine residue or at least one histidine residue on the other end of the sequence.

[000120] A vaccine may comprise a protein or protein fragment comprising a Replikin sequence. A protein fragment of up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acid residues on either or both termini of at least one Replikin sequence may be comprised in a vaccine.

[000121] Another non-limiting embodiment of the aspect of the present invention provides a method of preventing, mitigating, or treating AMDV infection or an outbreak of AMDV infection comprising administering at least one compound comprising an AMDV Replikin peptide to a subject. A compound may comprise a protein, protein fragment, other polypeptide, or peptide comprising an AMDV Replikin peptide. A compound may further comprise a protein, protein fragment, other polypeptide, or peptide consisting essentially of an AMDV Replikin peptide. [000122] A non-limiting embodiment of the aspect of the present invention provides a method of stimulating the immune system of a subject by

administering at least one AMDV Replikin peptide to stimulate the immune system. The immune system may be stimulated against AMDV. The immune system may be stimulated to provide protection of the subject against infection of AMDV. The subject may be a mink, a ferret, or a human or any animal infected by AMDV.

[000123] Another non-limiting aspect of the present invention provides a binding agent that binds at least a portion of an AMDV Replikin peptide. The binding agent may be an antibody or antibody fragment or other binding agent. In a non-limiting embodiment, an antibody specifically binds at least a portion of an AMDV Replikin peptide. An antibody may specifically bind to a portion of an AMDV Replikin peptide with at least two-times more affinity than it binds to another AMDV epitope or with at least 10-times more affinity than it binds to another AMDV epitope. In a non-limiting embodiment, the antibody is an isolated antibody. In a further non-limiting embodiment, the antibody is a single chain antibody, a chimeric antibody, or an antibody fragment.

[000124] A non-limiting embodiment is an antagonist of the replication of AMDV where the antagonist interacts with at least a portion of an AMDV Replikin peptide. An antagonist may bind at least a portion of an AMDV Replikin peptide.

[000125] A non-limiting embodiment of an aspect of the present invention provides a method of antagonizing the replication of AMDV comprising administering to a subject an agent capable of interacting with and/or binding at least a portion of an AMDV Replikin peptide. The agent may bind at least a portion of an AMDV Replikin peptide comprised within a protein, protein fragment, other polypeptide or peptide. The agent may bind a portion of the AMDV Replikin peptide and a portion of the protein, protein fragment, other polypeptide, or peptide comprising the AMDV Replikin peptide. The agent may be an antibody, which may include a chimerized antibody, a single chain antibody, or an antibody fragment.

Immunogenic fragments of AMDV Replikin sequences and their utilities

[000126] Another non-limiting aspect of the present invention provides an isolated or synthesized peptide that is an immunogenic fragment (functional fragment) of an AMDV Replikin sequence. Immunogenic fragments of an AMDV Replikin sequence are fragments that provide at least a portion of cross- reactivity with an antibody or antibody fragment against the AMDV Replikin sequence. One embodiment of the present invention provides an isolated or synthesized immunogenic fragment of at least one AMDV Replikin sequence as a target for control of replication of the virus. One embodiment provides the isolated or synthesized immunogenic fragment as an antigen for stimulating the immune system of a subject, including but not limited to a mink, to produce antibodies against the fragment. Another embodiment of the invention provides the isolated or synthesized immunogenic fragment as a vaccine or as a component of a vaccine in combination with an acceptable pharmaceutical carrier or in combination with other proteins, peptides, immunogenic substances, and/or adjuvant(s).

AMDV Replikin Peak Gene sequences and their utilities

[000127] Another non-limiting aspect of the present invention provides an isolated or synthesized Replikin Peak Gene of AMDV where the Replikin Peak Gene is identified as the portion of the genome, protein or protein fragment of a virion of the virus consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to other portions of the genome, protein or protein fragment of the virion of the virus. The portion of a protein or protein fragment consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to all other proteins or protein fragments in the virion of the virus may likewise be a Replikin Peak Gene.

[000128] An isolated or synthesized Replikin Peak Gene is useful for diagnosis, prevention or treatment of an outbreak of AMDV. One example of a Replikin Peak Gene may be prepared by the method comprising:

( 1 ) obtaining a plurality of isolates of AMDV;

(2) analyzing the protein sequences or protein sequence fragments of each isolate of the plurality of isolates for the presence and concentration of Replikin sequences;

(3) identifying the protein sequence or the protein sequence fragment having the highest concentration of Replikin sequences in each isolate;

(4) comparing the protein sequence or protein sequence fragment having the highest concentration of Replikin sequences in each isolate to the protein sequence or protein sequence fragment having the highest concentration of Replikin sequences in each of the other isolates of the plurality of isolates;

(5) selecting the protein sequence or protein sequence fragment having the highest concentration of Replikin sequences;

(6) identifying the amino acid sequence of the selected protein sequence or protein sequence fragment as the Replikin Peak Gene of the plurality of isolates of AMDV; and

(7) isolating or synthesizing the identified Replikin Peak Gene of at least one of the isolates of the plurality of isolates for diagnosis, prevention or treatment of an outbreak of AMDV.

[000129] A Replikin sequence from the Replikin Peak Gene or a peptide consisting essentially of the Replikin sequence from the Replikin Peak Gene or a fragment of the Replikin Peak Gene may be isolated or synthesized for diagnostic, preventive, or therapeutic purposes.

[000130] An isolated or synthesized Replikin Peak Gene or an isolated or synthesized Replikin sequence identified within a Replikin Peak Gene is useful when comprised in an immunogenic or blocking composition because the sequences are antigenic and related to mechanisms of rapid replication. A non- limiting embodiment of this aspect of the present invention also provides an isolated or synthesized AMDV polypeptide comprising an AMDV Replikin Peak Gene. The isolated or synthesized polypeptide may be an AMDV protein or protein fragment comprising a Replikin Peak Gene. The peptide or polypeptide may consist essentially of an AMDV Replikin Peak Gene.

[000131] A composition may comprise at least one AMDV Replikin Peak Gene and may further comprise a pharmaceutically-acceptable carrier, excipient, and/or adjuvant. The compositions may be an immunogenic composition or blocking composition.

[000132] Administering at least one AMDV Replikin Peak Gene to a subject stimulates the immune system. Likewise, administering a Replikin sequence identified within the Replikin Peak Gene stimulates the immune system against AMDV to provide protection to the subject against infection of AMDV, including protection in mink. [000133] As a result, the technology provides a vaccine against AMDV where a vaccine may comprise at least one isolated or synthesized AMDV Replikin Peak Gene or may comprise an isolated or synthesized AMDV polypeptide comprising an AMDV Replikin Peak Gene. The isolated or synthesized polypeptide may be an AMDV protein or protein fragment comprising a Replikin Peak Gene or may consist essentially of an AMDV Replikin Peak Gene or plurality of AMDV Replikin Peak Genes.

[000134] The technology, therefore, provides a method of making a preventive or therapeutic AMDV vaccine by identifying at least one Replikin Peak Gene sequence or at least one functional derivative fragment of the at least one Replikin Peak Gene sequence in at least one protein, polypeptide, or peptide of AMDV and making the virus vaccine comprising the at least one protein, polypeptide, or peptide of AMDV. The protein, polypeptide, or peptide in which said at least one Replikin Peak Gene sequence or said at least one functional derivative fragment of said at least one Replikin Peak Gene sequence may be isolated or synthesized and combined with a pharmaceutically-acceptable carrier, excipient, and/or adjuvant to make the AMDV vaccine. A Replikin Peak Gene sequence may be conserved in AMDV and may be conserved for at least two consecutive years. The Replikin Peak Gene sequence may have at least one lysine residue on one end of the sequence and at least one lysine residue or at least one histidine residue on the other end of the sequence. The vaccine may also comprise an isolated or chemically-synthesized Replikin Peak Gene sequence.

[000135] Another non-limiting embodiment provides a method of preventing, mitigating, or treating AMDV infection or an outbreak of AMDV infection comprising administering to a subject at least one compound comprising an AMDV Replikin peptide. The compound may comprise a protein, protein fragment, other polypeptide, or peptide comprising an AMDV Replikin peptide or may comprise a protein, protein fragment, other polypeptide, or peptide consisting essentially of an AMDV Replikin peptide.

[000136] An antibody that binds at least a portion of an AMDV Replikin Peak Gene is provided. The antibody may be isolated from other biological or chemical contaminants and may be isolated from a subject such as a mink. The antibody may specifically bind at least a portion of an AMDV Replikin Peak Gene and may bind with at least two-times more affinity than it binds another AMDV epitope or may bind with at least 10-times more affinity than it binds to another AMDV epitope. An antibody may be a single chain antibody, a chimeric antibody, an antibody fragment, or any other portion or chimera of the antibody or any agent that behaves like an antibody.

[000137] An antagonist of the replication of AMDV is provided where the antagonist may interact with at least a portion of an AMDV Replikin Peak Gene. The antagonist may bind at least a portion of an AMDV Replikin Peak Gene. The antagonist may be an antibody.

[000138] As a result, a method of antagonizing the replication of AMDV comprises administering to a subject an agent capable of binding at least a portion of an AMDV Replikin Peak Gene. The Replikin Peak Gene may be comprised within a protein, protein fragment, other polypeptide or peptide. The agent may bind a portion of the AMDV Replikin Peak Gene and a portion of the protein, protein fragment, other polypeptide, or peptide comprising the AMDV Replikin peptide.

Methods of preventing, mitigating, and treating expanding AMDV

[000139] Another non-limiting aspect of the present invention provides a method of preventing, mitigating, or treating an outbreak of AMDV predicted to have an expansion of population. The method comprises predicting an expansion of a population of AMDV and administering a compound comprising an isolated or synthesized portion of the structure or genome of the AMDV predicted to expand.

[000140] For example, the method comprises:

predicting an expansion of the population of a first AMDV population comprising identifying at least one cycle of Replikin concentration in isolates of AMDV and predicting that an expansion of the population of the AMDV will take place after the occurrence of a rising portion of the at least one cycle of Replikin concentration, and

administering to an animal or patient a compound comprising an isolated or synthesized portion of the structure or genome of the AMDV predicted to expand to mitigate, prevent, or treat the predicted outbreak of the pathogen. [000141] The technology is also useful as a method of preventing, mitigating, or treating an outbreak of AMDV comprising

predicting an expansion of the population or an increase in virulence, morbidity, and/or mortality of an isolate or plurality of isolates of a first strain of AMDV as compared to another isolate or plurality of isolates of the same or a related strain of AMDV comprising: (1) identifying a first cycle in the Replikin concentration of a plurality of isolates of said first strain of AMDV, (2) identifying a first peak in the Replikin concentration within the identified first cycle at a first time point or time period, and (3) predicting an increase in the virulence of an isolate of the same or related strain of pathogen isolated at a second time point or time period subsequent to the first time point or time period; and

administering to an animal or a patient a compound comprising an isolated or synthesized portion of the structure or genome of the at least one isolate of the AMDV to prevent, mitigate, or treat the outbreak of AMDV. A second time point or time period may be up to three years after the first time point or time period or may be about one year after the first time point or time period. The second time point may be about six months after the first time point or time period.

[000142] An identified peak in the cycle of Replikin concentration may also have a higher Replikin concentration than a chronologically earlier peak in the cycle of Replikin concentration. An identified peak in the cycle of Replikin concentration may be significantly higher than the earlier peak. For example, a p value may be less than 0.01 or may be less than 0.001.

[000143] The administered isolated or synthesized portion of the structure or genome of the at least one isolate of AMDV may be a protein or protein fragment comprising a Replikin peptide and/or a Replikin Peak Gene, a Replikin peptide identified within a Replikin Peak Gene, or any structure or portion of the structure of said AMDV or may be a nucleic acid encoding a Replikin Peak Gene, a Replikin peptide or a plurality of Replikin peptides within a Replikin Peak Gene, or a Replikin peptide or plurality of Replikin peptides. The isolated or synthesized portion may consist essentially of a Replikin peptide or a Replikin Peak Gene. Methods of predicting expanding cyclic populations of AMDV

[000144] Another non-limiting aspect of the present invention provides a method of predicting an expansion of the population of AMDV comprising identifying at least one cycle of Replikin concentration in isolates of AMDV and predicting that an expansion of the population of AMDV will take place after the occurrence of a rising portion of the at least one cycle of Replikin concentration, where the at least one cycle is cycle A.

[000145] The rising portion may comprise a peak wherein the expansion of the population of AMDV is predicted after the occurrence of the peak. The cycle may include at least a first rising portion and a second rising portion, where the first rising portion occurs prior in time to the second rising portion or may include at least three rising portions, where the at least three rising portions are at least rising portion A', rising portion B' and rising portion C Rising portion B' may comprise a peak and rising portion A' may comprise a peak, and the peak of rising portion B' may have a greater Replikin concentration than the peak of rising portion A'. The method may be processed on a computer. A cycle may incorporate more than one cycle including, for example, from peak to trough to peak to trough or from trough to peak to trough to peak. The cycle may include three peaks or three troughs or more.

[000146] A method of prediction may comprise identifying at least one other cycle of Replikin concentration in isolates of at least one other strain of AMDV, where the at least one other cycle is cycle B, and where cycle B shares synchrony with cycle A; and predicting that an expansion of the population of the AMDV will occur after the occurrence of a rising portion in cycle A that corresponds to a rising portion in cycle B. Expansion of the population of the first pathogen may be predicted within three years after the peak and may be predicted within one year after the peak.

[000147] A further non-limiting embodiment provides a method of predicting an expansion of a population of AMDV or an increase in the virulence, morbidity, and/or mortality of AMDV relative to the population or the virulence, morbidity, and/or mortality of another strain of AMDV comprising: (1) identifying a cycle in the Replikin concentration of isolates of a plurality of a first strain of AMDV, (2) identifying a first peak in the Replikin concentration of isolates of a plurality of said first strain of AMDV within the identified cycle at a first time point or time period, and (3) predicting an expansion of the population of the first strain of AMDV or an increase in the virulence, morbidity, and/or mortality of a pathogen of the same or a related strain of AMDV isolated at a second time point or time period subsequent to the first time point or time period.

[000148] In another non-limiting embodiment, the second time point or time period is up to three years after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about one year after the first time point or time period. In a further non-limiting embodiment, the second time point or time period is about six months after the first time point or time period.

[000149] In another non-limiting embodiment, the identified peak in the cycle of Replikin concentration has a higher Replikin concentration than a chronologically earlier peak in the cycle of Replikin concentration. In a further non-limiting embodiment of the invention, the identified peak in the cycle of Replikin concentration is significantly higher than the earlier peak. In a further non-limiting embodiment the identified peak is significantly higher than the earlier peak with a p value less than 0.01. In a further non-limiting embodiment the identified peak is significantly higher than the earlier peak with a p value less than 0.001.

[000150] A further non-limiting embodiment provides a method of predicting an expanding population of AMDV or an increase in virulence, morbidity, and/or mortality in AMDV comprising: (1) determining the mean Replikin Count in a plurality of isolates of at least two strains of AMDV at a plurality of successive time points; (2) comparing the mean Replikin Count at at least four successive time points for each strain and identifying at least one cycle of increasing mean Replikin Counts over the at least four time points for each of the at least two strains; (3) identifying at least partial synchrony between the at least one cycle of increasing mean Replikin Counts for each of the at least two strains; and (4) predicting an increase in virulence following in time the increase in mean Replikin Count in the at least one cycle in said at least two strains wherein said at least one cycle in said at least two strains occurs at a

corresponding time period. In a further non-limiting embodiment, step-wise cycles are identified between successive time points. In a further non-limiting embodiment, specific conserved Replikin sequences are identified within the step-wise cycles. In a further non-limiting embodiment, Replikin sequences are identified at the peak of a stepwise cycle. The Replikin sequences identified at the peak of a stepwise cycle are useful for developing a vaccine or therapeutic composition of an isolated or synthesized Replikin peptide for use in preventing or treating outbreaks of malaria with relatively higher mortality.

[000151] Another non-limiting embodiment provides a method of predicting a contraction or failure of a population of a strain of AMDV, wherein an isolate of said pathogen is isolated at a time point or time period subsequent to a decreasing portion of a Replikin cycle.

[000152] A further non-limiting embodiment provides a method for making a vaccine comprising predicting an expanding population of AMDV or related strain of AMDV or an increase in virulence, morbidity, and/or mortality of AMDV or a related strain of AMDV and identifying a portion of the structure or genome of said isolated influenza virus to be comprised in a vaccine.

[000153] A further non-limiting embodiment provides an isolated or synthesized portion of the structure or genome of AMDV wherein said AMDV is predicted to have an expansion of the population of the pathogen. In a further embodiment, the isolated or synthesized portion is a protein, protein fragment, or peptide comprising a Replikin peptide or a Replikin Peak Gene. In a further non- limiting embodiment, the isolated or synthesized portion of the structure or genome of AMDV consists of one or more Replikin peptides and/or one or more Replikin Peak Genes. In a further non-limiting embodiment, the one or more Replikin peptides are conserved during a cycle in Replikin concentration at at least two successive time points or time periods in the cycle.

[000154] Another non-limiting embodiment provides Replikin peptides for diagnostic, therapeutic, and/or preventive purposes identified as conserved in an isolate of AMDV from among a plurality of isolates of AMDV, wherein said isolates are isolated during a cycle in Replikin concentration at at least two successive time points or time periods, and the cycle preferably includes at least two peaks or two troughs.

[000155] Any of the above-listed or herein identified Replikin peptides may be comprised in an immunogenic compound of the invention. [000156] A further non-limiting embodiment provides a computer readable medium having stored thereon instructions which, when executed, cause a processor to perform a method of predicting an expansion of a strain of AMDV or an increase in virulence, morbidity, and/or mortality of AMDV. In a further embodiment, the processor reports a prediction to a display, user, researcher, or other machine or person. In a further embodiment, the processor identifies to a display, user, researcher, or other machine or person, a portion of a pathogen predicted to be an expanding AMDV or predicted to increase in virulence, morbidity, and/or mortality, wherein said portion may be employed as a therapeutic or diagnostic compound. Said portion may be a Replikin peptide or plurality of Replikin peptides or any other structure or portion of said genome of said pathogen including a Replikin Peak Gene.

[000157] Figure 3 illustrates a cycle in the annual mean Replikin

concentration of protein sequences of isolates of Aleutian mink disease virus between 2001 and 2011. In Figure 3, gray bars represent annual mean Replikin Count and black-capped lines represent standard deviation from the annual mean. As may be seen in Figure 3, analysis of the annual mean Replikin Count of protein sequence of AMDV available at www.pubmed.com for isolates isolated between 2001 and 2011 revealed a cycle of Replikin Count. A rising portion of the cycle is seen between 2001 and 2005. A trough is seen in 2006 and 2007. A new rising portion is seen between 2007 and 2009 followed by a trough in 201 1. The data is graphically illustrated in Figure 3 and summarized in Table 4 above.

[000158] The data for Figure 3 are seen in Table 4 in Example 8 below. Mean annual Replikin Count, standard deviation, significance of annual mean Replikin Count to the lowest annual mean Replikin Count and to the previous annual mean Replikin Count, and number of accession numbers analyzed per annum is provided.

[000159] Changes in AMDV virulence and mortality may be predicted by identifying a peak within an identified cycle in the Replikin concentration of isolates of a plurality of AMDV and predicting an increase in the virulence, morbidity, and/or mortality of AMDV isolated at a time point or time period subsequent to the time point or time period of the identified peak in the cycle of Replikin concentration. [000160] Cyclic increases in Replikin concentration in the genome can be a mechanism of expansion of a virus. The Replikin concentration in each Replikin cycle apparently builds on the previous one. Timely, repeated analyses of cyclic changes in AMDV's Replikin structure is useful to bring current the targets for the chemical synthesis of Replikin vaccines having a best fit for emerging pathogens having increased virulence, morbidity, and/or mortality. These strain- specific vaccines may be manufactured in seven days as has been demonstrate with a 91% protection of shrimp against the lethal taura syndrome virus. See, e.g., U.S. Appln. Ser. No. 12/108,458, filed April 23, 2008 (incorporated herein in its entirety by reference) and with 100% blocking of excretion in low- pathogenic H5N1 in chickens. See, e.g., Example 10 U.S. Appln. Ser. No. 12/429,044, filed April 23, 2009

Methods of identifying emerging strains of AMDV

[000161] Another non-limiting aspect of the present invention provides the identification of an emerging strain of AMDV comprising:

(1) obtaining a plurality of isolates of AMDV from different time periods wherein at least one of said isolates is isolated about six months to about 3 years later than at least one other of said isolates;

(2) identifying the Replikin Peak Gene or Replikin Peak Gene Area in the plurality of isolates of AMDV;

(3) analyzing the amino acid sequence of a protein or protein fragment of the Replikin Peak Gene or Replikin Peak Gene Area of each isolate of the plurality of isolates for the presence and concentration of Replikin sequences;

(4) comparing the concentrations of Replikin sequences in each of the

proteins or protein fragments of the Replikin Peak Gene or Replikin Peak Gene Area of each isolate of the plurality of isolates to the concentration of Replikin sequences in each of the proteins or protein fragments of the Replikin Peak Gene or Replikin Peak Gene Area of each of the other isolates of the plurality of isolates; and

(5) identifying the isolate having the highest concentration of Replikin

sequences in the protein or protein fragment of the Replikin Peak Gene or Replikin Peak Gene Area as an emerging strain of AMDV. [000162] Another embodiment provides the identification of an emerging strain of AMDV comprising:

(1) obtaining a plurality of isolates of AMDV from different time periods;

(2) identifying a Replikin concentration in at least one protein or protein fragment in the plurality of isolates of AMDV;

(3) analyzing the amino acid sequence of the at least one protein or protein fragment of each isolate of the plurality of isolates for the presence and concentration of Replikin sequences;

(4) comparing the concentrations of Replikin sequences in each of the

proteins or protein fragments of each isolate of the plurality of isolates to the concentration of Replikin sequences in each of the proteins or protein fragments of each of the other isolates of the plurality of isolates; and

(5) identifying the isolate having the highest concentration of Replikin sequences in the protein or protein fragment as an emerging strain of AMDV.

[000163] Another embodiment provides a method of selecting an AMDV peptide for inclusion in a preventive or therapeutic virus vaccine comprising:

(1) obtaining at least one isolate of AMDV;

(2) analyzing the amino acid sequence of at least one protein or protein fragment in the at least one isolate for the presence and concentration of Replikin sequences;

(3) comparing the concentration of Replikin sequences in the amino acid sequence of the at least one isolate to the concentration of Replikin sequences observed in the amino acid sequence in at least one other isolate isolated at at least about six months to about three years earlier to provide the concentrations of Replikin sequences for at least two time points;

(4) identifying the isolate having the higher increase in the concentration of Replikin sequences between the at least two time points; and

(5) selecting at least one Replikin sequence present in the isolate

identified in step (4) as a peptide for inclusion in the virus vaccine. An Expansion Index for Populations of Pathogens

[000164] The present invention also provides a method of predicting an expansion of a strain of pathogen such as AMDV by (1) determining a mean Replikin Count and a standard deviation of the mean Replikin Count for a plurality of isolates of a strain of pathogen for a first time period in a first geographic region; (2) determining a Replikin Count of at least one isolate of the same or a related strain of pathogen from a second time period and/or second geographic region wherein the second time period is different from the first time period and/or the second geographic region is different from the first geographic region; and (3) predicting an expansion of the strain of pathogen isolated in the second time period and/or second geographic region, if the Replikin Count of the at least one isolate is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in the first time period and in the first geographic region.

[000165] In the above-described method, at least one isolate of the same or related strain of pathogen from a second time period and/or second geographic region may be a plurality of isolates from the second time period and/or second geographic region. In this case, the Replikin Count of each isolate of the plurality of isolates from the second time period and/or second geographic region is compared separately to one standard deviation of the mean. An expansion of pathogen isolated in the second time period and/or second geographic region may also be predicted if the number of Replikin Counts of a plurality of isolates from the second period and/or second geographic region that is greater than one standard deviation of the mean is greater than the number of Replikin Counts of said plurality of isolates from the second period and/or second geographic region that is less than one standard deviation of the mean.

[000166] The method may also employ a ratio of the number of Replikin Counts that are greater than one standard deviation of the mean divided by the number of Replikin Counts that are less than one standard deviation of the mean. The ratio is called a Replikin Count Expansion Index (RCE Index). Another way to determine the RCE Index is to divide the percent of Replikin Counts in a plurality of isolates of influenza virus grouped by time and/or region that are higher than one standard deviation of the mean by the percent of Replikin Counts that are lower than one standard deviation of the mean. An RCE Index may be used to quantify the future risk of an outbreak of pathogen by tracking Replikin Counts in strains of pathogen over time.

[000167] In determining a RCE Index, the mean Replikin Count of the plurality of isolates from the first time period and geographic region may be considered a control. A control population preferably has a relatively large number of isolates with a relatively small variability in the Replikin Count of the isolates but any population may be deemed a control when a comparison between the control and a related isolate or plurality of isolates is desired. A control may be related to the population that is being studied. For example, if an infection in a bird species, such as swans, is being studied, the control may be something closely related, such as chickens, wherein isolates from chickens may be relatively numerous (if available) and relatively stable (if possible) wherein stability in Replikin Count through the population demonstrates a level of equilibrium between the expansion and contraction of the strain or a related strain of influenza virus in chickens. A control may reflect a highest number of isolates reported in a year or in several years in a geographic area.

[000168] Any Replikin sequence, Replikin Peak Gene, or protein fragment containing a Replikin sequence or Replikin Peak Gene identified in a strain of pathogen that is predicted to have an increase in virulence, morbidity, or mortality may be isolated and/or synthesized as a diagnostic, therapeutic, or prophylactic agent to mitigate the predicted outbreak of the pathogen.

Computer Methods for Determining Virulence in AMDV

[000169] A prediction of expansion or retraction or a determination of relative virulence of an AMDV isolate or population may be performed by a processor. A prediction or determination of relative virulence may be output to a user or display. Likewise, a particular Replikin peptide or Replikin Peak Gene within an isolate or population of isolates of AMDV predicted to be expanding or retracting in replication or lethality or virulence and this prediction or relative virulence may be output to a user or display. A machine-readable storage medium may contain executable instructions that, when executed by a processor, cause the processor to provide sufficient data to a user, a printout, or a display such that the user or a user of the printout or display may predict expansion or retraction or relative virulence of a population of AMDV. [000170] A computer system may include a processor coupled to a network, and a memory coupled to a processor, wherein the memory contains a plurality of instruction to perform the methods of prediction discussed herein.

[000171] A user of outputted data from a processor, storage medium, machine-readable medium, or computer system may include any person or any machine that records or analyzes the outputted data. A display or printout may include any mechanism by which data is outputted so that any person or any machine may record or analyze the outputted data, including a printed document, a visual impulse, an aural impulse, or any other perceivable impulse, a computer monitor, a set of numbers, or any other display or printout of data including a digital recording medium.

[000172] Concerning a non-limiting computer system that may implement methods disclosed in the application, Figure 4 is a high-level block diagram of a computer system incorporating a system and method for identifying Replikin patterns in amino acid sequences. As shown in Figure 4, computer workstation 410 may be a computer having a processor and a memory configured to permit a researcher to search protein databases and to scan protein descriptions for selected amino acid patterns. To accomplish these functions, computer workstation 410 may include protein and amino acid research system 430, which may receive instructions from a user/researcher to conduct protein searching and amino acid scanning operations. According to an embodiment, protein and amino acid research system 430 may further include amino acid sequence scanner 440 that scans and searches retrieved protein and amino acid sequences for specific patterns of amino acids, including Replikin patterns. Protein and amino acid research system 430 may communicate with network interface 420 to obtain protein sequences and amino acid sequences from resources on network 460, which may include the Internet. Alternatively, protein and amino acid research system 430 may obtain protein sequences and amino acid sequences from a local protein database 450. In addition, protein and amino acid research system 430 may obtain protein sequences and amino acid sequences directly from other input means, such as keyboard input. Protein and amino acid research system 430 may also communicate with network interface 420 to transmit results to other computers on network 460. [000173] Figure 5 is a simple flow chart illustrating a general method for locating a Replikin pattern in a sequence of amino acids, according to an embodiment of the present invention. The method 500 may begin after a sequence of amino acids has been obtained. Typically, the sequence of amino acids may be represented by three alphabetic characters. However, other encodings are envisioned by the present invention as well. Referring to Figure 5, once a sequence of amino acids has been obtained, the sequence is searched for a Replikin pattern (510), which comprises a subsequence (or string) of amino acids that includes the following characteristics:

(1) the string contains from 7 to about 50 amino acids;

(2) the string contains at least one lysine residue located 6 to 10 positions from a second lysine residue;

(3) the string contains at least one histidine residue; and

(4) the string contains at least 6% lysine residues.

[000174] Once a string of amino acids is found to match the Replikin pattern, the string may be identified or marked (520) accordingly.

[000175] A given sequence of amino acids may contain many subsequences or strings that match the Replikin pattern. Additionally, Replikin patterns may overlap each other. Thus, to locate and identify all possible Replikin patterns in a sequence of amino acids, method 700 may be invoked iteratively for each subsequence of amino acids contained within the original sequence of amino acids.

[000176] When method 500 is invoked iteratively to identify and locate all possible Replikin patterns in an amino acid sequence, the number of resulting Replikin patterns may be counted. A Replikin count may be reported as an absolute number. Additionally, a ratio of the number of Replikins per N amino acids in the sequence may be calculated. For example, it may be determined that a given protein contains a ratio of 6 Replikin sequences for every 100 amino acid residues. Replikin ratios have been shown by laboratory experiment and by epidemiological evidence to correlate directly to the rate that a given protein replicates. Rapid replication of proteins in pathogens is an indication of disease. For example, the presence of relatively high ratios of Replikin patterns has been correlated to epidemics of influenza. Similarly, an increase in the count of Replikin patterns observed in a protein over time indicates a future disease caused by the organism from which the protein was obtained.

[000177] Figure 6 is a flow chart illustrating a generalized method 600 for locating a plurality of Replikin patterns in a given sequence of amino acids, according to an embodiment of the present invention. The method 600 begins by locating a first lysine residue in the given sequence (610). Then, the method 600 may determine whether a second lysine residue resides within kmin to kmax positions of the first lysine residue (620). As indicated in Figure 6, kmin and kmax define the limits on the distance between the first and second lysine residues. For a typical Replikin pattern, kmin will equal 6 and kmax will equal 10. However, these values may be varied by a researcher interested in discovering other similar patterns.

[000178] Once method 600 has identified two lysine residues that are close enough to each other (620), the method 600 may examine every histidine residue that resides within rmax positions of both the first and second lysine residues (630). When method 600 is employed to identify and locate typical Replikin patterns, rmax will usually be set to equal 50. For every histidine residue that resides within rmax positions of the two lysine residues identified in steps (610) and (620), method 600 will construct the shortest string of amino acid residues that includes the first lysine residue, the second lysine residue, and the identified histidine residue (640). Then, method 600 will determine whether the length of that shortest string is within the desired range - that is, whether it contains at least rmin amino acid residues and no more than rmax amino acid residues (650). Finally, if the identified string of amino acids also contains at least kpercent of lysine residues (660), the string will be identified as matching the desired Replikin-like pattern (670).

[000179] Still referring to Figure 6, it is apparent that method 600 may identify several Replikin-like patterns from a single given amino acid sequence. This may happen because method 600 may examine more than one histidine residue that resides within rmax positions of the two identified lysine residues. Each identified histidine residue may, in combination with the two lysine residues, match the desired Replikin-like pattern. Methods of diagnosing AMDV using Replikin sequences

[000180] A method of diagnosing AMDV is also provided. The method comprises identifying an AMDV Replikin sequence or AMDV Replikin Peak Gene sequence in a mink or other animal, including human, infected by AMDV. In a non-limiting embodiment, the blood or tissue of mink or other animal may be screened for an AMDV Replikin sequence or an AMDV Replikin Peak Gene sequence. The AMDV Replikin sequence or the AMDV Replikin Peak Gene sequence may be identified in a Replikin cycle in AMDV. In a further non- limiting embodiment, an antibody or other binding agent may be used to screen for an AMDV Replikin sequence or AMDV Replikin Peak Gene sequence. In a further non-limiting embodiment, the blood or tissue of mink or other animal may be screened for DNA or RNA encoding an AMDV Replikin sequence or an AMDV Replikin Peak Gene sequence. In another non-limiting embodiment, clothing, tools, or other equipment in a commercial mink operation may be screened for DNA or RNA encoding an AMDV Replikin sequence or an AMDV Replikin Peak Gene sequence.

[000181] In a further non-limiting embodiment, Replikin or Replikin Peak Gene nucleotide sequences may be used in hybridization assays of biopsied tissue or blood, e.g., Southern or Northern analysis, including in situ

hybridization assays, to diagnose the presence of AMDV in a tissue sample or an environmental sample. A further non-limiting embodiment contemplates kits containing antibodies specific for particular Replikin sequences that are present in a particular pathogen of interest, or containing nucleic acid molecules (sense or antisense) that hybridize specifically to a particular Replikin sequence, and, optionally, various buffers and/or reagents needed for diagnosis.

Diagnostics and Therapies Using Replikin Peptides Identified in Replikin Cycles

[000182] The present invention further provides the opportunity to identify Replikin sequences (including nucleic acid sequences and peptide sequences) for diagnostic, therapeutic, or preventive purposes (such as the construction of vaccines and other pharmaceuticals). The present invention contemplates, for example, Replikin peptides identified within a pathogen where the pathogen is predicted to have an expanding population or a higher virulence, morbidity, and/or mortality than another pathogen of the same or a related species based on the predictive methods of the invention. Replikin peptides identified in an isolate of a pathogen, wherein said isolate is isolated during a rising portion of a cycle in Replikin concentration among a plurality of isolates of the pathogen or is isolated at a peak in a cycle in Replikin concentration among a plurality of isolates of the pathogen, are useful for diagnostic, therapeutic, and preventive purposes. For example, a Replikin peptide identified in the genome of an isolate identified in a rising portion of a cycle in Replikin concentration or identified at a peak in a cycle in Replikin concentration is useful as a peptide to stimulate the immune system of a human or animal to produce an immune response against infection by the pathogen or to produce antibodies against a pathogen predicted to have higher virulence, morbidity, and/or mortality. One of ordinary skill in the art will recognize that antibodies against these pathogens are useful for diagnosing the more highly virulent or mortal disease in a subject or useful as therapies against the infection either as a prophylactic or after onset of the infection.

[000183] Additionally, Replikin peptides identified during a rising portion in Replikin concentration in a Replikin cycle or identified at or near a peak in Replikin concentration in a Replikin cycle that are conserved during the rising portion of the Replikin cycle are useful as compounds for diagnostic, therapeutic, and preventive purposes. Conservation of the Replikin peptides during a rise in virulence, morbidity, and/or mortality provides targets that are more constant and likely more involved in the mechanisms of rapid replication that provide the predicted increase in virulence, morbidity, and/or mortality. As such, these conserved Replikin peptides are of use as compounds or in compositions for stimulating the immune system of a subject to produce an immune response, an antibody response, and/or a protective effect in the subject.

[000184] Replikin peptides identified and isolated using the methods of the invention include and are not limited to SEQ ID NO(s): 1-36.

[000185] Identification of portions of a pathogen (such as Replikin Peak Genes or Replikin peptides) predicted to expand in population provide unique compounds for diagnostics and treatment of expanding pathogens, wherein the unique compounds would otherwise not be identifiable but for the methods of the invention and the compounds disclosed herein. [000186] A non-limiting embodiment of the invention further contemplates use of the Replikin peptide as immunogenic compositions and contemplates the immunogenic compositions as vaccines, including vaccines that provide an immune response, vaccines that provide a humoral immune response, vaccines that provide an innate immune response, vaccines that provide an antigenic immune response, and vaccines that provide a protective effect. The invention additionally contemplates an antibody to the Replikin peptides of the invention.

[000187] High Replikin Counts and RPGs have been shown to be related to rapid replication, viral outbreaks, epidemics, morbidity and host mortality, for example, in influenza virus strains, including H5N1, in SARS coronavirus, in shrimp taura syndrome virus, and in foot and mouth disease virus. Replikin sequences identified at or near the peak of the Replikin cycle or during a rising portion of the Replikin cycle in a pathogen are appropriate peptides for diagnostics, vaccines, and other treatments.

[000188] Because Replikin sequences are chemically defined, the sequences may be synthesized by organic chemistry rather than biological techniques, and thus are potentially more specific, more reproducible and more reliable. The chemically defined Replikin sequences identified by Applicants are likewise potentially freer from adverse reactions that are characteristic of biologically derived vaccines and antibodies.

Structure and function in Replikin peptide sequences and homologues

[000189] Because the peptides disclosed herein are peptides (and homologues) that are conserved over time and across regions, one of skill in the art expects such peptides (and peptides that are similar in structure and function) to also be useful in immunogenic compounds and blocking compounds for AMDV infections of various strains. This expectation is based on, for example, the function of the peptide sequences identified herein and the commonality of structure and position of the peptides and their homologues as described herein as well as the functionality of the peptides and the homologues in AMDV. This expectation is also based in part on the conservation of Replikin peptides generally and the commonality of function of Replikin peptides across different viruses and organisms. See, e.g., Tables 7a, 8, 9, and 10 with descriptions and Examples 6 and 7 in U.S. Appln. Ser. No. 1 1/355,120, filed February 16, 2006 and Table 8 with description in U.S. Patent No. 7,442,761, and Figures 1-21 in U.S. Appln. Ser. No. 12/010,027, filed January 18, 2008. For example, Replikin peptides have been shown to be broadly antigenic, to be conserved, and to be related to rapid replication and outbreaks across many different viruses. See, e.g., WO 2008/143717, Figures 1-21. Additionally, the crucial lysine and histidine residues defining the structure of Replikin peptides have been demonstrated to be related to rapid replication and to be conserved in fixed positions within functional proteins across viruses and organisms. This conservation with fixed positions is even found in highly mutable viruses such as HIV. See, e.g., Table 8 with description in columns 62 and 63 in U.S. Patent No. 7,442,761. As a result, the peptides and their homologues described herein are, among other things, antigenic, common to various strains of AMDV in both position and function, conserved in various strains over time, conserved in specific positions in gene areas over time, conserved in their lysine and histidine residues within the Replikin structure, and associated with mechanisms of virulence and rapid replication. As a result, one of ordinary skill in the art expects the Replikin peptides and their homologues described herein to be useful in immunogenic compounds for therapies against AMDV within strains, across strains, and across time.

[000190] One non-limiting aspect of the present invention provides an isolated or synthesized polypeptide or peptide comprising a peptide A that has about the same number of amino acid residues as a peptide B, where peptide B is a Replikin peptide identified in AMDV, and where the lysine residues and histidine residues in peptide A are conserved as compared to the lysine residues and histidine residues in peptide B, wherein said isolated or synthesized polypeptide or peptide may further comprise up to 100 more amino acid residues than does peptide A, and where the up to 100 more amino acid residues of the isolated or synthesized polypeptide or peptide are positioned to the amino- terminus and/or carboxy-terminus of the lysine or histidine termini of peptide A. The up to 100 more amino acid residues may be up to one, two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, or fifty more amino acid residues. The isolated or synthesized polypeptide or peptide may consist essentially of or consist of peptide A.

[000191] The lysine and histidine residues of the Replikin peptide sequence of AMDV are structural components related to rapid replication and virulence. Upon administration to a subject, the sequences are antigenic and are also capable of blocking mechanisms of replication and virulence through humoral or non-humoral responses. This structure/function relationship is herein surprisingly further identified in exemplary vaccines in AMDV. Vaccines in the art had previously been unsuccessful and not useful in the mink-raising industry. Vaccines, Treatments and Therapeutics

[000192] The observations of specific Replikin sequences and their concentration in AMDV proteins provide specific quantitative early chemical correlates of outbreaks and increases in mortality and provide for production and timely administration of vaccines tailored specifically to treat the prevalent emerging or re-emerging strain virus in a particular region of the world, a country, a province, a town, a farm, or of any size mink operation. Synthesis of these vaccines may be accomplished in seven days or less, which allows for administration of vaccines that are a best fit for a particular virulent strain of virus.

[000193] By analyzing the protein sequences of isolates of an AMDV for the presence, concentration and/or conservation of Replikins, pandemics, epidemics, and other changes in virulence and mortality can be predicted and treatments developed. Furthermore, the severity of such outbreaks can be significantly lessened by administering a peptide vaccine based on the Replikin sequences found to be most abundant or shown to be on the rise in virus isolates over a given time period, such as about one to about three years.

[000194] A peptide vaccine of the invention may include a single Replikin peptide sequence or may include a plurality of Replikin sequences observed in particular virus strains. A peptide vaccine may comprise a Replikin peptide or plurality of Replikin peptides, it may consist essentially of a Replikin peptide or a plurality of Replikin peptides, or may consist of a Replikin peptide or plurality of Replikin peptides. A vaccine may include a conserved Replikin peptide(s) in combination with a new Replikin(s) peptide or may be based on new Replikin peptide sequences. A vaccine may likewise comprise a protein or protein fragment that comprises a Replikin peptide. The protein or protein fragment may be identified by identifying the Replikin peptide.

[000195] Replikin peptides can be synthesized by any method, including chemical synthesis or recombinant gene technology, and may include non- Replikin sequences. Vaccine composition may comprise a pharmaceutically acceptable carrier and/or adjuvant. Among the Replikin peptides for use in a virus or pathogen vaccine are those Replikins observed to "re- emerge" after an absence from the amino acid sequence for one or more years.

[000196] The vaccines of the present invention can be administered alone or in combination with antiviral drugs, such as gancyclovir; interferon; interleukin; M2 inhibitors, such as, amantadine, rimantadine; neuraminidase inhibitors, such as zanamivir and oseltamivir; and the like, as well as with combinations of antiviral drugs.

[000197] The vaccine of the present invention may be administered to any animal capable of producing antibodies in a humoral immune response or to an animal capable of an innate immune response. For example, the vaccine of the present invention may be administered to a mink, a rabbit, a chicken, a shrimp, a pig, or a human. Because of the universal nature of Replikin sequences, a vaccine of the invention may be directed at a range of strains of a virus or a particular strain of virus.

[000198] The Replikin peptides of the invention, alone or in various combinations are administered to a subject, in a non-limited embodiment, by i.v., intramuscular injection, by mouth, or by spray inhalation, intranasal

administration, or intraocular administration. The peptides are administered in order to stimulate the immune system of the subject to produce antibodies to the peptide. Generally the dosage of peptides is in the range of from about 0.01 μg to about 500 mg, about 0.05 μg to about 200 mg, or about 0.075 μg to about 30 mg, from about 0.09 μg to about 20 mg, from about 0.1 μg to about 10 mg, from 10 μg to about 1 mg, and from about 50 μg to about 500 μg. The skilled practitioner can readily determine the dosage and number of dosages needed to produce an effective immune response.

[000199] In another aspect of the invention, isolated Replikin peptides may be used to generate antibodies, which may be used, for example to provide passive immunity in an individual or for diagnostics. See, e.g., U.S. Appln. Ser. No. 1 1/355, 120, filed February 16, 2006 and U.S. Appln. Ser. No. 12/010,027, filed January 18, 2008 (each incorporated herein by reference in their entirety). Antibodies and antibody derivatives and passive immunity

[000200] In another aspect of the invention, isolated Replikin peptides may be used to generate antibodies, which may be used, for example for diagnostic purposes, to identify protein or protein fragments of interest for development of vaccines and other therapies, or, for example, to provide passive immunity in an subject. Various procedures known in the art may be used for the production of antibodies to Replikin sequences. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by a Fab expression library. Antibodies that are linked to a cytotoxic agent may also be generated. Antibodies may also be administered in combination with an antiviral agent. Furthermore, combinations of antibodies to different Replikin sequences, fragments, or homologues may be administered as an antibody cocktail.

[000201] An antibody of the invention may bind to a Replikin peptide or a Replikin Peak Gene or a fragment or homologue. It may bind to a protein or protein fragment comprising a Replikin peptide or a Replikin Peak Gene. It may also bind to a portion of a Replikin peptide or a portion of a Replikin Peak Gene. It may bind to a portion of a Replikin peptide or a portion of a Replikin Peak Gene and a portion of a protein, protein fragment, polypeptide, or peptide comprising a Replikin peptide or Replikin Peak Gene or fragment or homologue.

[000202] An antibody that specifically binds to a portion of a Replikin peptide or a portion of a Replikin Peak Gene generally binds to an epitope on the Replikin peptide or an epitope that is at least partially on the Replikin peptide or to an epitope on the Replikin Peak Gene or an epitope that is at least partially on the Replikin Peak Gene when the antibody or fragment of the antibody binds to the epitope more readily than it would bind to a random, unrelated epitope.

[000203] Monoclonal antibodies to Replikins may be prepared by using any technique that provides for the production of antibody molecules. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al, 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of chimeric antibodies (Morrison et al., 1984, Proc. Nat. Acad. Sci USA, 81 :6851-6855) or other techniques may be used. Alternatively, techniques described for the production of single chain antibodies (U.S. 4,946,778) can be adapted to produce Replikin-specific single chain antibodies.

[000204] Antibodies to any peptides observed to be present in an emerging or re-emerging strain of virus and combinations of such antibodies are useful in the treatment and/or prevention of viral infection, including Replikin peptides, Replikin Peak Gene peptides, and Replikin sequences isolated within Replikin Peak Gene peptides.

[000205] Antibody fragments that contain binding sites for a Replikin may be generated by known techniques. For example, such fragments include but are not limited to F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecules and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be generated (Huse et al, 1989, Science, 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

[000206] In another aspect of the invention, immune serum containing antibodies to one or more Replikin sequences obtained from an individual exposed to one or more Replikin sequences may be used to induce passive immunity in another individual or animal. Immune serum may be administered via i.v. to a subject in need of treatment. Passive immunity also can be achieved by injecting a recipient with preformed antibodies to one or more Replikin sequences. Passive immunization may be used to provide immediate protection to individuals who have been exposed to an infectious organism. Administration of immune serum or preformed antibodies is routine and the skilled practitioner can readily ascertain the amount of serum or antibodies needed to achieve the desired effect. One of the reasons that vaccines directed towards a particular protein antigen of a disease causing agent have not been fully effective in providing protection against the disease (such as foot and mouth vaccine which has been developed against the VP1 protein or large segments of the VP1 protein) is that the best antibodies have not been produced, that is - it is likely that the antibodies to the Replikin sequences have not been produced.

[000207] A vaccine or immunogenic composition of the invention can be generated using any Replikin peptide. A vaccine or immunogenic composition may comprise at least one Replikin sequence isolated in a RPG. A vaccine may also comprise a RPG peptide. A vaccine or immunogenic composition or blocking composition may comprise conserved Replikin sequences observed to "re-emerge" after an absence from the amino acid sequence for one or more years.

Vaccine formulations

[000208] A vaccine may be formulated with a pharmaceutically acceptable excipient, carrier, or adjuvant. One pharmaceutically acceptable carrier or excipient is water. Excipients, carriers, or adjuvants may include, but are not limited to, excipients, carriers and adjuvants known to those of skill in the art now or hereafter.

[000209] The compositions of the invention may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic, transdermal (topical), transmucosal or any other routes. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

Supplementary active compounds can also be incorporated into the

compositions.

[000210] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for intranasal, intraocular, spray inhalation, parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water (for dermal, nasal, or ocular application, spraying, or injection), saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as

ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [000211] Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include

physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. Preferred pharmaceutical formulations are stable under the conditions of manufacture and storage and must be preserved against the contaminating action of

microorganisms such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.

[000212] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[000213] Administration of the vaccine via any method may produce an immune response in the animal or human, it may further produce an antibody response in the animal or human, or a blocking response. In a further non- limiting embodiment, the vaccine may produce a protective effect in the animal or human. For example, the vaccine of the present invention may be administered to a rabbit, a chicken, a shrimp, a pig, a ferret, a human, or any animal capable of an immune response. Because of the universal nature of Replikin sequences, a vaccine of the invention may be directed at a range of strains of influenza.

Replikin Sequences in AMDV

[000214] The present invention provides methods of determining the relative virulence, replication rate, and/or lethality of an isolate, strain, or population of Aleutian mink disease virus (AMDV). The present invention also provides compounds for preventing, treating, and diagnosing AMDV. The present invention further provides methods of identifying Replikin sequences and Replikin Peak Gene sequences to be comprised in therapies and diagnostics for AMDV. The present invention also provides methods of predicting outbreaks of AMDV. The examples herein are illustrative of the uses of Replikin sequences and Replikin Peak Gene sequences in the invention and do not limit the scope of the invention described herein. One of ordinary skill in the art will understand the full scope of utilities of the Replikin sequences and Replikin Peak Gene sequences described herein based on the description and examples provided herein.

Example 1

Analysis of Replikin Count in Aleutian Mink disease virus in Vendsyssel, Denmark Correlates with Increased Incidence of Disease

[000215] Replikin Counts were determined for all genomic sequences available to applicants at www.pubmed.com for isolates of AMDV from mink ranches in Vendsyssel, Denmark from 2000 through 2009. Annual mean

Replikin Counts with standard deviation from the mean were determined for each year. See Figure 1. Percent incidence of AMDV in mink ranches in

Vendsyssel was determined. See Figure 2. It was observed that annual mean

Replikin Count began to rise between 2001 and 2002. It was further observed that percent incidence began to rise in 2003 following the 2002 rise in Replikin

Count. Surprisingly, Applicants determined that Replikin Count correlated with and predicted the 2003 rise in disease incidence. One non-limiting aspect of the present invention is, therefore, a method of forecasting rise in incidence, virulence, and morbidity in AMDV in mink.

[000216] The mean Replikin Count for each year for isolates of virus from mink ranches in Vendsyssel, Denmark along with standard deviation from the mean is provided in Table 1 below. Number of isolates per year is also provided. A graphical representation of the data is provided in Figure 1.

Table 1 - AMDV Annual Mean Replikin Count Vendsyssel, Denmark

2007 1 20.2 0

2008 17 11.3 9.1

2009 15 9.4 6.8

[000217] The percent incidence of AMDV disease for mink ranches in Vendsyssel, Denmark is provided in Table 2. A graphical representation of the data is provided in Figure 2.

Table 2 - AMDV Percent Incidence Vendsyssel, Denmark

Example 2

Vaccine Against AMDV in Vendsyssel, Denmark

[000218] Replikin sequences identified as conserved in isolates from mink ranches in Vendsyssel and Saeby, Denmark were analyzed and prepared for synthesis. A vaccine was designed using the conserved synthetic peptide sequences.

[000219] The following conserved sequences were identified for use in a vaccine. The sequences are noted along with the years in which AMDV isolates were analyzed having the conserved sequence.

KSLGWFIKRLNKDLAVIYSNH (SEQ ID NO: 9) 2003, 2004, 2005, 2007, 2008,

2009

KVWVEDGPTKPYKYYH (SEQ ID NO: 10) 2003, 2004, 2005, 2007, 2008,

2009

KYYHKQTK (SEQ ID NO: 11) 2003, 2004, 2005, 2007, 2008,

2009

KYFHKQTK (SEQ ID NO: 12) 1999, 2009

KNEDPKDIHK (SEQ ID NO: 13) 1999, 2005, 2008

KPYKYFHK (SEQ ID NO: 14) 1999, 2009

KSLGWFIKKLNKDLAVVYSNH (SEQ ID NO: 15) 1999, 2002, 2009

KQTKQDYNKPVH (SEQ ID NO: 16) 1994, 1999

[000220] A vaccine was designed comprising peptides of each of the

conserved sequences SEQ ID NO(s): 1-16 in equal parts by weight in sterile water. The vaccine is administered in mink via parenteral injection. The mink are inoculated with AMDV. The vaccine provides protection against AMDV.

Example 3

Homologues of Replikin sequences in AMDV shared in isolates from

Vendsyssel, Denmark, all of Denmark, Canada, China, and Bornholm,

Denmark

[000221] Replikin sequences identified as conserved in isolates of AMDV from Saeby and Vendsyssel, Denmark were compared with Replikin sequences identified as conserved in isolates of AMDV from all of Denmark, as well as

Canada, China, and Bornholm, Denmark. As may be seen Table 3 below,

specific Replikin sequences share homology across the different geographic regions. Table 3 lists the accession numbers in which the conserved sequences were initially identified, the years in which each sequence was identified in one or more isolates, and sequences sharing homology across geographical regions.

Certain differences in specific amino acid residues among listed homologues are bolded and underlined.

[000222] The conserved and shared sequences listed in Table 3 are

particularly appropriate for use in vaccines against AMDV in all geographic regions. Each sequence may be targeted to control replication in AMDV. Each sequence likewise may be comprised in a vaccine against AMDV either

individually or in combination with other conserved sequences or sequences identified in a particular region. Immunogenic compositions may comprise the Replikin sequences or a functional fragment or a homologue thereof individually or comprised in a larger peptide, in a polypeptide, or in an isolated protein. Table 3 - Conserved AMDV Sequences Across Various Geographic Regions

[000223] One embodiment of the present invention provides one or more of the peptides listed in Table 3. Another embodiment provides functional fragments of one or more of the peptides listed in Table 3 as well as peptides sharing percent sequence identity or homology with one or more of the peptides listed in Table 3. Percent sequence identity or homology may be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more. Peptides sharing percent sequence identity share functional characteristics.

[000224] Another embodiment provides proteins, protein fragments, polypeptides, or other compounds comprising one or more of the peptides listed in Table 3, functional fragments of one or more of the peptides listed in Table 3, or peptides sharing percent identity or homology with one or more peptides or functional fragments of the peptides listed in Table 3.

[000225] As may be seen in Table 3, significant numbers of sequences are shared among the isolates from various regions listed in the table. Any sequence that is shared among regions is useful for targeting rapid replication in AMDV across regions and is useful for diagnostic and therapeutic purposes across regions. Any homologue of a sequence in Table 3 is likewise useful for diagnostic and therapeutic purposes. Example 4

Vaccine Against AMDV in Denmark

[000226] Replikin sequences identified as conserved in isolates from mink ranches across all of Denmark were analyzed in Accession Nos. ABQ82130, ABQ82127, and ABQ2123 and prepared for synthesis. A vaccine was designed using the conserved synthetic peptide sequences.

[000227] The following conserved sequences were identified for use in a vaccine. The sequences are noted along with the years in which AMDV isolates were analyzed having the conserved sequence.

Example 5

Vaccine Against AMDV in Canada

[000228] Replikin sequences identified as conserved in isolates from mink ranches across Canada were analyzed in Accession No. ACF04392 and prepared for synthesis. A vaccine was designed using the conserved synthetic peptide sequences.

[000229] The following conserved sequences were identified for use in a vaccine. The sequences are noted along with the years in which AMDV isolates were analyzed having the conserved sequence.

Example 6

Vaccine Against AMDV in China

[000230] Replikin sequences identified as conserved in isolates from mink ranches across China were analyzed and prepared for synthesis. A vaccine was designed using the conserved synthetic peptide sequences.

[000231] The following conserved sequences were identified for use in a vaccine. The sequences are noted along with the years in which AMDV isolates were analyzed having the conserved sequence.

Example 7

Vaccine Against AMDV in Bornholm

[000232] A Replikin sequence identified as conserved in isolates from mink ranches across Bornholm, Denmark was analyzed in Accession No. ADU60608 and prepared for synthesis. A vaccine was designed using the conserved synthetic peptide sequence.

[000233] The following conserved sequence was identified for use in a vaccine. The sequence is noted along with the year in which AMDV isolates were analyzed having the conserved sequence.

KNEDPKDIHK (SEQ ID NO: 13) [ 08

Example 8

Analysis of Replikin Count in Global Aleutian Mink disease virus

[000234] Publicly available sequences of isolates of Aleutian mink disease virus at www.pubmed.com were analyzed using proprietary search tool software (ReplikinsForecast^®, a service available in the United States through

REPLIKINS LLC, Boston, MA) for years 2000 to 2011 to determine the mean Replikin Count of all reported protein sequences of all isolates available in each of those years. [000235] The mean Replikin Count for each year is reported in Table 4. Standard deviation and significance as compared to the mean Replikin Count of the previous year and of the lowest mean Replikin Count within the data set are also provided along with the mean Replikin Count for each year.

Table 4 - AMDV Annual Mean Replikin Count

[000236] Analysis of the annual mean Replikin Count of protein sequence of AMDV available at www.pubmed.com for isolates isolated between 2000 and 201 1 revealed a cycle of Replikin Count. A rising portion of the cycle is seen between 2001 and 2005. A trough is seen in 2006 and 2007. A new rising portion is seen between 2007 and 2009 followed by a trough in 201 1. The data is graphically illustrated in Figure 3 and summarized in Table 4 above.

Example 9

Analysis of Replikin Count in Aleutian Mink disease virus in Canada

[000237] Publicly available sequences at www.pubmed.com of isolates of Aleutian mink disease virus isolated in Canada were analyzed using proprietary search tool software (ReplikinForecast^®, a service available in the United States through REPLIKXNS LLC, Boston, MA) for years having available isolates between 2000 and 2010 to determine the mean Replikin Count for the entire genome of all isolates available in each of those years.

[000238] The mean Replikin Count for each year is reported in Table 5. Standard deviation and significance as compared to the mean Replikin Count of the previous year and of the lowest mean Replikin Count within the data set are also provided along with the mean Replikin Count for each year.

Table 5 - AMDV Annual Mean Replikin Count in Canada

Example 10

Analysis of Replikin Count in Aleutian Mink disease virus in China

[000239] Publicly available sequences at www.pubmed.com of isolates of Aleutian mink disease virus isolated in China were analyzed using proprietary search tool software (ReplikinForecast^®, a service available in the United States through REPLIKI S LLC, Boston, MA) for years having available isolates between 2005 and 2010 to determine the mean Replikin Count for the entire genome of all isolates available in each of those years.

[000240] The mean Replikin Count for each year is reported in Table 6. Standard deviation and significance as compared to the mean Replikin Count of the previous year and of the lowest mean Replikin Count within the data set are also provided along with the mean Replikin Count for each year.

Table 6 - AMDV Annual Mean Replikin Count in China

Claims

CLAIMS What is claimed is:

1. An isolated or chemically-synthesized protein fragment or peptide comprising a Replikin peptide sequence, functional fragment of a Replikin peptide sequence, or homologue of a Replikin peptide sequence identified in an isolate of Aleutian Mink Disease Virus (AMDV).

2. The isolated or chemically-synthesized protein fragment or peptide of claim 1 identified in an isolate of AMDV.

3. The isolated or chemically-synthesized protein fragment or peptide of claim 1 wherein said protein fragment or peptide comprises said homologue of a Replikin peptide sequence that is at least 50% homologous with said Replikin peptide sequence.

4. The isolated or chemically-synthesized protein fragment or peptide of claim 1 wherein said protein fragment or peptide comprises said homologue of a Replikin peptide sequence that is at least 75% homologous with said Replikin peptide sequence.

5. The isolated or chemically-synthesized protein fragment or peptide of claim 1 wherein said protein fragment or peptide comprises said homologue of a Replikin peptide sequence that is at least 90% homologous with said Replikin peptide sequence.

6. The isolated or chemically-synthesized protein fragment or peptide of claim 1 wherein said protein fragment or peptide comprises said homologue of a Replikin peptide sequence that is at least 95% homologous with said Replikin peptide sequence.

7. The isolated or chemically-synthesized protein fragment or peptide of claim 1 , wherein said protein fragment or peptide is an AMDV protein fragment or peptide comprising said Replikin peptide sequence.

8. The isolated or chemically-synthesized protein fragment or peptide of claim 1, wherein said protein fragment or peptide comprises said functional fragment of said Replikin peptide sequence identified in said isolate of AMDV.

9. The isolated or chemically-synthesized protein fragment or peptide of any of claims 1-8, wherein the protein fragment or peptide is up to 100 amino acid residues longer than the Replikin peptide sequence, the homologue of the Replikin peptide sequence, or the functional fragment of a Replikin peptide sequence.

10. The isolated or chemically-synthesized protein fragment or peptide of any of claims 1-8, wherein the protein fragment or peptide is up to 200 amino acid residues longer than the Replikin peptide sequence, the homologue of the Replikin peptide sequence, or the functional fragment of a Replikin peptide sequence.

1 1. The isolated or chemically-synthesized protein fragment or peptide of any of claims 1-8, wherein the protein fragment or peptide is up to 50 amino acid residues longer than the Replikin peptide sequence, the homologue of the Replikin peptide sequence, or the functional fragment of a Replikin peptide sequence.

12. The isolated or chemically-synthesized protein fragment or peptide of claim 1 consisting essentially of an AMDV Replikin peptide.

13. The isolated or chemically-synthesized protein fragment or peptide of claim 1, wherein said protein fragment or peptide consists of an AMDV Replikin peptide sequence.

14. The isolated or chemically synthesized protein fragment or peptide of claim 1, wherein said protein fragment or peptide comprises at least one peptide sequence at least 50% homologous, at least 75% homologous, at least 90% homologous, or at least 95% homologous with any one of SEQ ID NO(s): 1-35.

15. The isolated or chemically synthesized protein fragment or peptide of claim 1, wherein said protein fragment or peptide comprises at least one peptide sequence of any one of SEQ ID NO(s): 1-35.

16. The isolated or chemically synthesized protein fragment or peptide of claim 1, wherein said protein fragment or peptide comprises at least one functional fragment of any one of SEQ ID NO(s): 1-35.

17. The isolated or chemically synthesized protein fragment or peptide of claim 1, wherein said protein fragment or peptide is a peptide consisting essentially of any one of SEQ ID NO(s): 1-35.

18. The isolated or chemically synthesized protein fragment or peptide of claim 1, wherein said protein fragment or peptide is a peptide consisting of any one of SEQ ID O(s): 1-35.

19. An immunogenic composition comprising at least one protein fragment or peptide of any one of claims 1-18.

20. The immunogenic composition of claim 19 further comprising at least two protein fragments and/or peptides of any one of claims 1-18.

21. The immunogenic composition of claim 19 further comprising a mixture of protein fragments and/or peptides of claim 1, wherein said mixture respectively comprises at least one protein fragment and/or peptide comprising each of SEQ ID O(s): 1-16.

22. The immunogenic composition of claim 19 comprising at least one isolated or chemically-synthesized peptide consisting essentially of any one of SEQ ID NO(s): 1-35.

23. The immunogenic composition of claim 19 comprising a pharmaceutically - acceptable carrier, excipient, and/or adjuvant.

24. A vaccine comprising at least one protein fragment or peptide of any one of claims 1-18.

25. The vaccine of claim 24 comprising at least two protein fragments or peptides of any one of claims 1-18.

26. The vaccine of claim 24 further comprising a mixture of protein fragments and/or peptides of claim 1, wherein said mixture respectively comprises at least one protein fragment or peptide comprising each of SEQ ID NO(s): 1-16.

27. The vaccine of claim 24 comprising at least one isolated or chemically- synthesized peptide consisting essentially of any one of SEQ ID NO(s): 1-35.

28. The vaccine of claim 24 comprising a mixture of isolated or chemically- synthesized protein fragments and/or peptides, including, respectively, at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID O(s): 1-16.

29. The vaccine of claim 24 comprising a mixture of isolated or chemically- synthesized protein fragments and/or peptides, including, respectively, at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID O(s): 3-7, 11, 17-25.

30. The vaccine of claim 24 comprising a mixture of isolated or chemically- synthesized protein fragments and/or peptides, including, respectively, at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID NO(s): 26-31.

31. The vaccine of claim 24 comprising a mixture of isolated or chemically synthesized protein fragments and/or peptides, including, respectively, at least one isolated or chemically-synthesized peptide consisting essentially of each of SEQ ID NO(s): 28 and 30-35.

32. The vaccine of claim 24 comprising at least one isolated or chemically- synthesized peptide consisting essentially of SEQ ID NO: 13.

33. The vaccine of any one of claims 24-32 further comprising a

pharmaceutically-acceptable carrier, excipient, and/or adjuvant.

34. A method of determining the relative virulence of an isolate A of AMDV comprising determining a Replikin concentration of at least one amino acid sequence A isolated from said isolate A of AMDV and comparing the Replikin concentration of said at least one amino acid sequence A to the Replikin concentration of at least one amino acid sequence B isolated from an isolate B of AMDV and determining that isolate A is relatively more virulent than isolate B if the Replikin concentration of said a least one amino acid sequence A is greater than the Replikin concentration of said at least one amino acid sequence B or determining that isolate A is relatively less virulent than isolate B if the Replikin concentration of said at least one amino acid sequence A is less than the Replikin concentration of said at least one amino acid sequence B.

35. The method of claim 34, wherein said at least one amino acid sequence A is encoded in the same region of the genome of isolate A of AMDV as said at least one amino acid sequence B is encoded in the genome of isolate B.

36. The method of claim 35, wherein said sequence A is the amino acid sequence of a protein and said sequence B is the amino acid sequence.

37. The method of claim 34, wherein said sequence A is a Replikin Peak Gene of isolate A and sequence B is a Replikin Peak Gene of isolate B.

38. The method of claim 34, wherein said at least one sequence A is a plurality of sequences A isolated from a plurality of isolates of a population A of AMDV and said at least one sequence B is a plurality of sequences B isolated from a plurality of isolates of a population B of AMDV and wherein a mean Replikin concentration of said plurality of sequences A is compared to a mean Replikin concentration of said plurality of sequences B and virus population A is determined to be relatively more virulent than virus population B if the mean Replikin concentration of said plurality of sequences A is greater than the mean Replikin concentration of said plurality of sequences B or said virus population A is determined to be relatively less virulent than virus population B if the Replikin concentration of said plurality of sequences A is less than the Replikin concentration of said plurality of sequences B.

39. The method of claim 38 further comprising, determining the standard deviation of the mean Replikin concentration of said plurality of sequences A and the standard deviation of the mean Replikin concentration of said plurality of sequence B and predicting an outbreak of population a virulent version of population A if the standard deviation of the mean Replikin concentration of said plurality of sequences A is larger than the standard deviation of the mean Replikin concentration of said plurality of sequences B.

40. The method of claim 38, wherein said population A of AMDV is a population of a particular strain of AMDV isolated within a time period A and said population B of AMDV is a population of the same strain of AMDV isolated within a time period B.

41. The method of claim 40, wherein said time period A is a particular calendar year and said time period B is a different calendar year.

42. A method of making a preventive or therapeutic AMDV vaccine comprising identifying at least one Replikin sequence, at least one functional fragment of a Replikin sequence, or at least one homologue of a Replikin sequence in at least one protein, protein fragment, polypeptide, or peptide of AMDV, wherein a Replikin sequence consists of 7 to 50 amino acid residues and comprises (1) at least one lysine residue six to ten amino acid residues from at least one other lysine residue, (2) at least one histidine residue, and (3) at least 6% lysine residues, and making the virus vaccine comprising the at least one protein, protein fragment, polypeptide, or peptide of AMDV.

43. The method of claim 42, wherein said at least one protein, protein fragment, polypeptide, or peptide is isolated or chemically-synthesized and combined with a pharmaceutically-acceptable carrier, excipient, and/or adjuvant to make the virus vaccine.

44. The method of claim 42, wherein the at least one Replikin sequence is conserved for at least two consecutive years in a plurality of isolates of AMDV.

45. The method of claim 42, wherein the at least one Replikin sequence has at least one lysine residue on one end of the sequence and at least one lysine residue or at least one histidine residue on the other end of the sequence.

46. A method of preventing, mitigating, or treating AMDV infection or an outbreak of AMDV infection comprising administering at least one compound comprising an AMDV Replikin peptide sequence, a functional fragment of an AMDV Replikin peptide sequence, or a homologue of an AMDV Replikin peptide sequence to a subject.

47. The method of claim 46, wherein said compound comprises a protein, protein fragment, polypeptide, or peptide comprising an AMDV Replikin peptide sequence, functional fragment of an AMDV Replikin peptide sequence, or a homologue of a Replikin peptide sequence.

48. A method of stimulating the immune system of a subject comprising administering at least one AMDV Replikin peptide sequence, at least one functional fragment of an AMDV Replikin peptide sequence, or at least one homologue of an AMDV Replikin peptide sequence or at least one protein, protein fragment, polypeptide, or peptide comprising said at least one AMDV Replikin peptide sequence, said functional fragment of said AMDV Replikin peptide sequence, or said at least one homologue of said AMDV Replikin peptide sequence to a subject to stimulate the immune system.

49. A binding agent that specifically binds at least a portion of an AMDV Replikin peptide sequence.

50. The binding agent of claim 49, wherein said binding agent is an isolated antibody or antibody fragment.

51. The binding agent of claim 49, wherein said binding agent is an antagonist of the replication of AMDV and interacts with at least a portion of an AMDV Replikin peptide sequence or functional fragment thereof.

52. An isolated or chemically-synthesized Replikin Peak Gene of AMDV wherein said Replikin Peak Gene is identified as the portion of the genome, protein or protein fragment of a virion of the virus consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to other portions of the genome, protein, or protein fragment of the virion of the virus.

53. The isolated or chemically-synthesized Replikin Peak Gene of claim 52, wherein said Replikin Peak Gene is the portion of a protein or protein fragment consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to all other proteins or protein fragments in the virion of the virus.

54. An isolated or chemically-synthesized Replikin sequence, functional fragment, or homologue thereof identified in a Replikin Peak Gene of AMDV, wherein said Replikin Peak Gene is identified as the portion of the genome, protein or protein fragment of a virion of the virus consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to other portions of the genome, protein, or protein fragment of the virion of the virus.

55. An immunogenic composition comprising at least one isolated or chemically-synthesized Replikin Peak Gene of claim 52.

56. An immunogenic composition comprising at least one Replikin sequence, functional fragment, or homologue thereof of claim 54.

57. An isolated or chemically-synthesized AMDV peptide or polypeptide comprising an AMDV Replikin Peak Gene, wherein said Replikin Peak Gene is identified as the portion of the genome, protein or protein fragment of a virion of the virus consisting of the highest number of continuous Replikin sequences per 100 amino acids as compared to other portions of the genome, protein or protein fragment of the virion of the virus.

58. The isolated or chemically-synthesized peptide or polypeptide of claim 57 that is an AMDV protein or protein fragment comprising a Replikin Peak Gene.

59. A method of stimulating the immune system of a subject comprising administering at least one AMDV Replikin Peak Gene or at least one polypeptide comprising said AMDV Replikin Peak Gene to the subject to stimulate the immune system.

60. A vaccine against AMDV comprising at least one isolated or chemically- synthesized polypeptide comprising an AMDV Replikin Peak Gene.

61. The vaccine of claim 60 comprising at least one isolated or chemically- synthesized AMDV polypeptide comprising an AMDV Replikin Peak Gene.

62. The vaccine of claim 60, wherein said at least one isolated or chemically- synthesized polypeptide is an AMDV protein or protein fragment comprising a Replikin Peak Gene.

63. The vaccine of claim 60, wherein said at least one isolated or chemically- synthesized polypeptide consists essentially of an AMDV Replikin Peak Gene.

64. The vaccine of claim 63 comprising a plurality of isolated or chemically- synthesized polypeptides each consisting of an AMDV Replikin Peak Gene.

65. A binding agent that specifically binds at least a portion of an AMDV Replikin Peak Gene.

66. The binding agent of claim 65, wherein said binding agent is an isolated antibody or antibody fragment.

67. The binding agent of claim 65 that is an antagonist of the replication of AMDV that interacts with at least a portion of an AMDV Replikin Peak Gene.

68. The binding agent of claim 67, wherein said antagonist binds at least a portion of an AMDV Replikin Peak Gene.

69. The binding agent of claim 68, wherein said antagonist is an antibody.

70. A method of preventing, mitigating, or treating an outbreak of AMDV predicted to have an expansion of population comprising predicting an expansion of the population of a first AMDV pathogen comprising:

71. The method of claim 70, comprising mitigating an outbreak of AMDV comprising

72. The method of claim 71, wherein said isolated or chemically-synthesized portion of the structure or genome of the at least one isolate of AMDV is a protein or protein fragment comprising a Replikin peptide and/or a Replikin Peak Gene, functional fragment, or homologue thereof, a Replikin peptide identified within a Replikin Peak Gene, functional fragment, or homologue thereof, or any structure or portion of the structure of said AMDV.

73. The method of claim 71, wherein said isolated or chemically-synthesized portion of the structure or genome consists essentially of a Replikin peptide or a Replikin Peak Gene.

74. The method of claim 71, wherein said isolated or chemically-synthesized portion of the structure or genome consists of a Replikin peptide or a Replikin Peak Gene.

75. The method of claim 71, wherein the second time point or time period is up to three years after the first time point or time period.

76. The method of claim 71, wherein the second time point or time period is about one year after the first time point or time period.

77. The method of claim 71 , wherein the second time point or time period is about six months after the first time point or time period.

78. A method of predicting an expansion of the population of AMDV comprising identifying at least one cycle of Replikin concentration in isolates of AMDV and predicting that an expansion of the population of AMDV will take place after the occurrence of a rising portion of the at least one cycle of Replikin concentration.

79. A method of preventing, mitigating, or treating an outbreak of AMDV comprising

80. The method of claim 79, wherein said first period is one year and said first geographic region is a country.

81. The method of claim 79, wherein said second time period is one year.

82. The method of claim 79, wherein second geographic region is a country.

83. The method of claim 79, wherein said isolated or chemically-synthesized portion of the structure or genome of the at least one isolate of pathogen is a protein or protein fragment comprising a Replikin peptide.

84. The method of claim 83, wherein said protein or protein fragment is a Replikin peptide, functional fragment, or homologue thereof.

85. The method of claim 84, wherein said protein or protein fragment is a Replikin peptide identified within a Replikin Peak Gene.

86. A method of predicting an expansion of a strain of AMDV comprising (1) determining a mean Replikin Count and a standard deviation of said mean Replikin Count for a plurality of isolates of said strain of AMDV for a first time period in a first geographic region; (2) determining a Replikin Count of at least one isolate of the same or a related strain of AMDV from a second time period and/or second geographic region wherein said second time period is different from said first time period and/or said second geographic region is different from said first geographic region; and

(3) predicting an expansion of said strain of AMDV isolated in said second time period and/or second geographic region if the Replikin Count of said at least one isolate from a second time period and/or second geographic region is greater than one standard deviation of the mean of the Replikin Count of the plurality of isolates isolated in said first time period and in said first geographic region.

87. The method of claim 86, wherein at least one step of the method is processed on a computer.

88. The method of claim 87, wherein all steps of the method are processed on a computer.