WO2020236741A2

WO2020236741A2 - Synthetic bacteriophage and compositions and methods of selection and use

Info

Publication number: WO2020236741A2
Application number: PCT/US2020/033445
Authority: WO
Inventors: Jorge BARDALES; Andrew Harper FAVOR; Carlos Daniel LLANOS
Original assignee: Nextbiotics, Inc.
Priority date: 2019-05-17
Filing date: 2020-05-18
Publication date: 2020-11-26
Also published as: WO2020236741A3

Abstract

Described herein are novel engineered bacteriophages and bacteriophage compositions, methods of production thereof, and therapeutic uses thereof. The engineered bacteriophages may have properties conferring enhanced stability when subjected to conditions of elevated heat, extreme pH, or other medically relevant conditions. These properties may provide advantages as alternatives to the current reliance upon antimicrobials.

Description

SYNTHETIC BACTERIOPHAGE AND COMPOSITIONS AND METHODS OF

SELECTION AND USE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/849,831, filed on May 17, 2019, entitled“SYNTHETIC BACTERIOPHAGE AND COMPOSITIONS AND METHODS OF SELECTION AND USE”, which disclosure is herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference for all purposes.

FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to the field of microbiology. Specifically, it includes methods to identify and engineer genes within a bacteriophage genome of importance for bacteriophage stability, efficacy and specificity and related properties. Moreover, the disclosure herein provides methods that allow the generation of engineered bacteriophage having an improved structural and/or functional properties as compared to wild-type bacteriophage. The engineered bacteriophage described herein may be used to treat or prevent disease, including for antimicrobial uses and microbiome modulation, among other biotechnological uses.

BACKGROUND

[0004] The recent increase in drug-resistant bacterial infections has created a need to develop alternative therapies. Bacteriophages are viruses that can replicate inside bacteria, thereby suppressing bacterial growth and/or infectivity. The use of bacterial viruses as therapeutic agents was explored as early as 1919, but the emergence of small molecule and other commercially produced antibiotics limited the successful development and commercialization of phage therapy products, at least in the U.S. There has been a recent resurgence of interest in phage therapy due to the emergence of antibiotic resistant bacteria.

[0005] There are challenges to using phase therapy in commercial settings. For example, many phages are not stable under one or more conditions, such as increases in temperature and variations in pH, and many phages are lacking adequate efficacy. In addition, phage may be slow to replicate. To be reliably used in therapeutic applications, phages with increased stability and/or efficacy are needed. Additionally, more efficient methods are needed to determine how to modify phages to cause an increase in at least one of stability and/or efficacy.

SUMMARY OF THE DISCLOSURE

[0006] Described herein are engineered bacteriophage proteins (and nucleotide sequences encoding them), bacteriophage expressing them, methods of using the proteins and/or bacteriophage expressing them to treat, prevent or ameliorate pathogen infection, compositions including such proteins and/or synthetic bacteriophage expressing them, and/or methods of identifying modifications in proteins and/or genes in a bacteriophage genome that, once modified, can be used create a synthetic bacteriophage with one or more enhanced properties or characteristics.

[0007] Examples of characteristics of the synthetic bacteriophage described herein having one or more enhanced properties and characteristics include, without limitation, an increase in heat stability, an increase in pH stability, an increase in blood exposure stability, an increase in bile acid stability, an increase in stability in one or more internal and/or external bodily fluids, an increase in efficacy against one or more types of bacteria, a change in specificity to one or more bacteria, and a change in the host-range. This list is intended to provide examples of enhanced properties, but other phage properties and characteristics can be modified using methods herein.

[0008] The present disclosure also includes synthetic peptide (amino acid) and/or nucleotide sequences that provide a synthetic bacteriophage with one or more of the enhanced properties or characteristics. One or more of the synthetic nucleotide sequences may be in one or more of structural, functional and regulatory genes.

[0009] In a first aspect, a bacteriophage is provided having a nucleotide sequence including an engineered nucleotide sequence in at least one of: a region that encodes for an endonuclease, a region that encodes for a head-to-tail joining protein, a region that encodes for an internal core protein, a region that encodes for a tail fiber protein gene, and a direct repeat region, where the engineered nucleotide sequence results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, internal and/or external bodily fluid stability, and host range of the bacteriophage compared to a respective wild-type bacteriophage. The nucleotide sequence including the engineered nucleotide sequence of the bacteriophage may have more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99% identity with a nucleotide sequence of the wild-type bacteriophage. In some variations, the bacteriophage may be a synthetic bacteriophage. The synthetic

bacteriophage may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

[00010] Also described herein are methods of treating an animal with a bacteriophage composition that include administering to the animal the bacteriophage composition, wherein the bacteriophage composition comprises a synthetic bacteriophage comprising mutations to two or more of: a head-to-tail joining protein , an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D, wherein the two or more mutations results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, and internal and/or external bodily fluid stability compared to a respective wild-type bacteriophage, and wherein the mutations comprise point mutations such that a synthetic bacteriophage has more than 99% identity with a nucleotide sequence of the wild-type bacteriophage.

[00011] For example, the synthetic bacteriophage may include a mutation in the head-to-tail protein and a mutation in the internal core protein, relative to the nucleotide sequence of the wild-type bacteriophage. In some variations, the synthetic bacteriophage includes a mutation in the head-to-tail protein and a mutation in the internal core protein and a mutation in the tail fiber protein (e.g., point mutations), relative to the nucleotide sequence of the wild-type

bacteriophage. For example, the synthetic bacteriophage may comprise mutations to three or more of: a head-to-tail joining protein, an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D.

[00012] The synthetic bacteriophage may include a mutation in two or more of: a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, a T3p48 gene, T3p38 gene, T3p39 gene, T3p46 gene, T7p43 gene, T7p51 gene, or a homologous gene of a different bacteriophage species thereof. In some variations, the synthetic bacteriophage comprises a mutation in two or more of: a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, a T3p48 gene, T3p38 gene, T3p39 gene, T3p46 gene, or a homologous gene of a different bacteriophage species thereof. The synthetic bacteriophage may comprise a mutation in two or more of: T7p43 gene, T7p51 gene, or a homologous gene of a different bacteriophage species thereof. The synthetic bacteriophage may comprise two or more mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO: 5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO: 73. For example, the synthetic bacteriophage may comprise a nucleotide sequence of two or more of SEQ ID NOS: 3, 9, 14, 16, 18, 24, 26, 28, 38, 40, 46, 48, 56, 58, 63, 67, 69, and 75. The synthetic bacteriophage may comprise a nucleotide sequence of two or more of SEQ ID NOS: 4, 10, 15, 17, 19, 25, 27, 29, 39, 41, 47, 49, 57, 59, 64, 68, 70, and 76. In some variations, the synthetic bacteriophage comprises an amino acid sequence comprising a sequence two or more of SEQ ID NOS: 5, 11, 20, 30, 32, 34, 42, 50, 52, 60, 65, 71, and 73. The synthetic bacteriophage may comprise an amino acid sequence comprising a sequence two or more of SEQ ID NOS: 6, 12, 21, 31, 33, 35, 43, 51, 53, 61, 66, 72, 74, 77, and 78. The synthetic bacteriophage comprising the two or more mutations may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera

Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage

[00013] The wild-type bacteriophage may be, e.g., a bacteriophage of a Podoviridae genus, Caudoviridae genus, or Siphoviridae genus. For example, if the wild-type bacteriophage is a Podoviradae bacteriophage, then the wild-type bacteriophage may be a member of a T7 family of bacteriophages. The wild-type bacteriophage may be a T3 or a T7 phage.

[00014] The mutations may provide an increase in heat stability at 65°C in the bacteriophage, compared with a heat stability of the wild-type bacteriophage. Alternatively or additionally, the mutations may provide an increase in at least one of pH stability at pH 3 or pH stability at pH 12 in the bacteriophage relative to a respective pH stability of the wild-type bacteriophage.

[00015] The mutation may produce an amino acid sequence comprising at least one amino acid that is more hydrophobic or more bulky than an amino acid sequence produced by the nucleotide sequence of the wild-type bacteriophage. The bacteriophage composition may be formulated as a liquid, solid or a gel.

[00016] In any of these methods, the animal may be a mammal, shellfish, fish, or bird. For example, the animal may be a livestock animal. In some variations, the animal is a human.

[00017] Administering may include administering an effective amount of the bacteriophage composition to maintain a healthy microbiome of the animal. The bacteriophage composition may be administered to a subject in the form of a feed additive, a drinking water additive, or a disinfectant. In some variations, administering comprises delivering the bacteriophage composition in a drinking water composition. Administering may comprise administering to an animal to treat or prevent an Escherichia coli infection.

[00018] For example, described herein are methods of treating an animal with a bacteriophage composition, comprising administering to the animal the bacteriophage composition, wherein the bacteriophage composition comprises a synthetic bacteriophage having two or more stabilizing mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO: 5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO:

73 in a pharmaceutically acceptable carrier. The bacteriophage composition may be formulated as a liquid, solid or a gel. The synthetic bacteriophage having two or more stabilizing mutations may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to an activity of the wild-type bacteriophage.

[00019] For example, a method of treating a subject with a bacteriophage composition may include administering to the subject the bacteriophage composition, wherein the bacteriophage composition comprises a synthetic bacteriophage having three or more stabilizing mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO:

5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO:

73 in a pharmaceutically acceptable carrier, wherein the three or more stabilizing mutations results in an increase in one or more of a heat stability, a pH stability, a virulence, a blood stability, a bile acid stability, a bodily fluid stability, and a host range of the bacteriophage compared to a wild-type bacteriophage. The synthetic bacteriophage having three or more stabilizing mutations may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter,

Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to an activity of the wild-type bacteriophage.

[00020] Also described herein are methods of preparing an animal feed composition using any of the bacteriophage (engineered, selected or synthetic bacteriophage) described herein. For example, the method may include: providing a bacteriophage composition comprising bacteriophage as an active ingredient; and mixing the composition with an animal feed base to provide the animal feed composition, wherein the bacteriophage composition comprises a synthetic bacteriophage having two or more stabilizing mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO: 5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO: 73 in a pharmaceutically acceptable carrier. The synthetic bacteriophage having two or more stabilizing mutations may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to an activity of the wild-type bacteriophage

[00021] A method of preparing an animal feed composition may include: providing a bacteriophage composition comprising bacteriophage as an active ingredient; and mixing the composition with an animal feed base to provide the animal feed composition, wherein the bacteriophage comprising mutations to two or more of: a head-to-tail joining protein , an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D, wherein the two or more mutations results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, and internal and/or external bodily fluid stability compared to a respective wild-type bacteriophage, and wherein the mutations comprise point mutations such that a synthetic bacteriophage has more than 99% identity with a nucleotide sequence of the wild-type bacteriophage. The synthetic bacteriophage comprising the two or more mutations may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

[00022] A synthetic bacteriophage as described herein may include mutations to two or more of: a head-to-tail joining protein , an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D, wherein the two or more mutations results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, and internal and/or external bodily fluid stability compared to a respective wild-type bacteriophage, and wherein the mutations comprise point mutations such that a synthetic bacteriophage has more than 99% identity with a nucleotide sequence of the wild-type bacteriophage. As mentioned, the synthetic bacteriophage may comprise a mutation in the head-to-tail protein and a mutation in the internal core protein, relative to the nucleotide sequence of the wild-type bacteriophage. The synthetic bacteriophage may include mutations to three or more of: a head-to-tail joining protein, an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D. The synthetic bacteriophage may include a mutation in two or more of: a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, a T3p48 gene, T3p38 gene, T3p39 gene, T3p46 gene, T7p43 gene, T7p51 gene, or a homologous gene of a different bacteriophage species thereof. The synthetic bacteriophage may include a mutation in two or more of: a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, a T3p48 gene, T3p38 gene, T3p39 gene, T3p46 gene, or a homologous gene of a different bacteriophage species thereof.

[00023] The synthetic bacteriophage may include a mutation in two or more of: T7p43 gene, T7p51 gene, or a homologous gene of a different bacteriophage species thereof. The synthetic bacteriophage may include two or more mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO: 5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO: 73. The synthetic bacteriophage may include a nucleotide sequence of two or more of SEQ ID NOS: 3, 9, 14, 16, 18, 24, 26, 28, 38, 40, 46, 48, 56, 58, 63, 67, 69, and 75. The synthetic bacteriophage may include a nucleotide sequence of two or more of SEQ ID NOS: 4, 10, 15, 17, 19, 25, 27, 29, 39, 41, 47, 49, 57, 59, 64, 68, 70, and 76. The synthetic bacteriophage may include an amino acid sequence comprising a sequence two or more of SEQ ID NOS: 5, 11, 20, 30, 32, 34, 42, 50, 52, 60, 65, 71, and 73. The synthetic bacteriophage may include an amino acid sequence comprising a sequence two or more of SEQ ID NOS: 6, 12, 21, 31, 33, 35, 43, 51, 53, 61, 66, 72, 74, 77, and 78. The synthetic

bacteriophage comprising the two or more mutations may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage

[00024] In some variations, the engineered nucleotide sequence may include a mutation in a region that encodes for a head-to-tail protein and a mutation in a region that encodes for an internal core protein, relative to the nucleotide sequence of the wild-type bacteriophage. In some embodiments, the mutation in the region encoding for an internal core protein may include internal virion protein B. In some variations, the engineered nucleotide sequence may further include a mutation in a region that encodes for a tail fiber protein, relative to the nucleotide sequence of the wild-type bacteriophage.

[00025] In some variations, the engineered nucleotide sequence may include a mutation in a region that encodes for an endonuclease, relative to the nucleotide sequence of the wild-type bacteriophage. In some embodiments, the engineered nucleotide sequence may include a mutation in a 5’UTR capsid assembly protein non-coding region, relative to the nucleotide sequence of the wild-type bacteriophage. In some other embodiments, the engineered nucleotide sequence may include a mutation in a 5’UTR internal core protein non-coding region, relative to the nucleotide sequence of the wild-type bacteriophage.

[00026] In some other variations, the engineered nucleotide sequence may include a mutation in a direct repeat region, relative to the nucleotide sequence of the wild-type bacteriophage. In some embodiments, the engineered nucleotide sequence may include a mutation in a plurality of direct repeat regions, relative to the nucleotide sequence of the wild-type bacteriophage. In some embodiments, the engineered nucleotide sequence may include a mutation in a 5’UTR capsid assembly protein non-coding region, relative to the nucleotide sequence of the wild-type bacteriophage. In some other embodiments, the engineered nucleotide sequence may include a mutation in a 5’UTR internal core protein non-coding region, relative to the nucleotide sequence of the wild-type bacteriophage.

[00027] In some other variations, the mutation of the engineered nucleotide sequence may be in at least one of a T3p37 gene, a T3p38 gene, a T3p39 gene a T3p45 gene, or a T3p48 gene, or a homologous region of a different bacteriophage species thereof. In some further variations, the engineered nucleotide sequence may include a mutation at more than one of the T3p37 gene, the T3p38 gene, the T3p39 gene, the T3p45 gene, or the T3p48 gene, or the homologous gene of a different bacteriophage species thereof. In yet other variations, the mutation of the engineered nucleotide sequence may be in at least one of a T7p43 gene, a T7p51 gene, or a homologous gene of a different bacteriophage species thereof.

[00028] In some variations, the bacteriophage may include a nucleotide sequence of at least one of SEQ ID NOS 3, 4, 9, 10, 14, 15, 16, 17, 18, 19, 24, 25, 26, 27, 28, 29, 38, 39, 40, 41, 46, 47, 48, 49, 56, 57, 58, 59, 63, 64, 67, 68, 69, 70, 75, or 76. In some variations, the engineered nucleotide sequence may produce an amino acid sequence including a sequence of at least one of SEQ ID NOS 5, 6, 12, 20, 21, 30, 31, 32, 33, 34, 35, 42, 43, 50, 51, 52, 53, 60, 61, 65, 66, 71, 72, 73, 74, 77, or 78.

[00029] In some variations, the wild-type bacteriophage may be a bacteriophage of a

Podoviridae genus, Caudoviridae genus, or Siphoviridae genus. In some embodiments, when the wild-type bacteriophage is a Podoviradae bacteriophage, then the wild-type bacteriophage may be of a T7 family of bacteriophages. In some embodiments, the wild-type bacteriophage may be a T3 or a T7 phage.

[00030] In some variations, the engineered nucleotide sequence may provide an increase in heat stability at 65 °C in the bacteriophage, compared with a heat stability of the wild-type bacteriophage. In some embodiments, the engineered nucleotide sequence may further provide an increase in at least one of pH stability at pH 3 or pH stability at pH 12 in the bacteriophage relative to a respective pH stability of the wild-type bacteriophage.

[00031] In some variations, the engineered nucleotide sequence of the bacteriophage may provide an increase in at least one of pH stability at pH 3 or pH stability at pH 12 in the bacteriophage relative to a respective pH stability of the wild-type bacteriophage.

[00032] In some variations, the engineered nucleotide sequence of the bacteriophage may provide an increase in stability of 10% or more in the bacteriophage, relative to the wild-type bacteriophage.

[00033] In some variations, the engineered nucleotide sequence may produce an amino acid sequence including at least one amino acid that is more hydrophobic or more bulky than an amino acid sequence produced by the nucleotide sequence of the wild-type bacteriophage. In some variations, the engineered nucleotide sequence may provide an amino acid sequence including at least 15%, at least 20%, at least 25%, or at least 30% more hydrophobic and/or hydrophilic amino acids.

[00034] In another aspect, an engineered bacteriophage is provided having an enhanced stability compared to a wild-type T3 phage, the engineered bacteriophage including an engineered nucleotide sequence having at least 95% sequence identity relative to a nucleotide sequence of a wild-type T3 phage, the engineered nucleotide sequence having at least one mutation relative to the wild-type nucleotide sequence in one or more of a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, or a T3p48 gene, where the increased stability includes one or more of: a heat stability, pH stability, virulence, blood stability, bile acid stability, internal and/or external bodily fluid stability, infectivity, or efficacy, including any combination thereof, compared to the wild-type phage. In some variations, the engineered nucleotide sequence may include more than one mutation in at least one of the T3p37 gene, the T3p38 gene, the T3p39 gene, the T3p45 gene, or the T3p48 gene. In some variations, the engineered bacteriophage may be a synthetic bacteriophage. The engineered bacteriophage may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage. [00035] In yet another aspect, a method is provided for creating an engineered bacteriophage having an engineered nucleotide sequence, including creating at least one mutation in a wild-type bacteriophage. In some variations, creating at least one mutation in a wild-type bacteriophage may include creating at least one mutation in a wild type bacteriophage of the genera

Podoviridae. In some variations, creating at least one mutation may include creating at least one mutation in a bacteriophage of a T7 family of bacteriophages. In some embodiments, creating at least one mutation may include creating at least one mutation in a T3 or T7 bacteriophage.

[00036] In some variations, creating the mutation may include creating a mutation one or more of a T3p37 gene, T3p45 gene, a T3p48 gene, a T3p38 gene, a T3p39 gene, a T3p46 gene, a or a homologous gene of a different bacteriophage species. In some other variations, creating the mutation may also or alternatively include creating a mutation in at least one of a T7p43 gene, a T7p51 gene or a homologous gene of a different bacteriophage species.

[00037] In another aspect, a composition is provided, including a bacteriophage having a nucleotide sequence including an engineered nucleotide sequence in at least one of: a region that encodes for an endonuclease, a region that encodes for a head-to-tail joining protein, a 5’UTR capsid assembly protein non-coding region, a 5’ UTR internal core protein non-coding region, a region that encodes for an internal core protein, a region that encodes for an tail fiber protein gene, and a direct repeat region, where the engineered nucleotide sequence results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, internal and/or external bodily fluid stability, and host range of the bacteriophage compared to a respective wild-type bacteriophage; and a pharmaceutically acceptable carrier. The

bacteriophage having a nucleotide sequence including an engineered nucleotide sequence may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage. The nucleotide sequence including the engineered nucleotide sequence may have more than more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99% identity with a nucleotide sequence of the wild-type bacteriophage.

[00038] In some variations, the composition may be formulated as a liquid. In some other variations, the composition may be formulated as a solid. In yet other variations, the composition may be formulated as a gel, an ointment, a paste, a suspension, a semi-solid, spreadable, or dispersible.

[00039] In some variations, the bacteriophage may be any bacteriophage having an engineered nucleotide as described herein. The bacteriophage having a nucleotide sequence including an engineered nucleotide sequence may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella,

Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

[00040] In another aspect, a method is provided for administering a composition to an animal, including: administering an effective amount of a composition to the animal, where the composition includes a bacteriophage having a nucleotide sequence including an engineered nucleotide sequence in at least one of: a region that encodes for an endonuclease, a region that encodes for a head-to-tail joining protein, a 5’UTR capsid assembly protein non-coding region, a 5’ UTR internal core protein non-coding region, a region that encodes for an internal core protein, a region that encodes for an tail fiber protein gene, and a direct repeat region, where the engineered nucleotide sequence results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, internal and/or external bodily fluid stability, and host range of the bacteriophage compared to a respective wild-type bacteriophage, in a pharmaceutically acceptable carrier. The nucleotide sequence including the engineered nucleotide sequence may have more than more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99% identity with a nucleotide sequence of the wild-type bacteriophage. The bacteriophage may be any bacteriophage having a nucleotide sequence including an engineered nucleotide sequence as described herein. The bacteriophage having a nucleotide sequence including an engineered nucleotide sequence may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera

Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

[00041] In some variations, the composition may be formulated as a liquid. In some other variations, the composition may be formulated as a solid. In yet other variations, the composition may be formulated as a gel, an ointment, a paste, a suspension, a semi-solid, spreadable, or dispersible. In some variations, the effective amount of the composition may maintain a healthy microbiome of the animal.

[00042] In some variations, the animal may be a mammal, shellfish, fish, or bird. In some embodiments, the animal may be a livestock animal configured to enter a food supply chain. In some embodiments, the animal may be a human.

[00043] In yet another aspect, a method is provided for treating a bacterial infection, including: administering an effective amount of a composition to an animal in need thereof, where the composition includes a bacteriophage having a nucleotide sequence including an engineered nucleotide sequence in at least one of: a region that encodes for an endonuclease, a region that encodes for a head-to-tail joining protein, a 5’UTR capsid assembly protein non coding region, a 5’ UTR internal core protein non-coding region, a region that encodes for an internal core protein, a region that encodes for an tail fiber protein gene, and a direct repeat region, where the engineered nucleotide sequence results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, internal and/or external bodily fluid stability, and host range of the bacteriophage compared to a respective wild-type bacteriophage in a pharmaceutically acceptable carrier. The nucleotide sequence including the engineered nucleotide sequence may have more than more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99% identity with a nucleotide sequence of the wild-type bacteriophage. The bacteriophage may be any

bacteriophage having a nucleotide sequence including an engineered nucleotide sequence as described herein. The bacteriophage having a nucleotide sequence including an engineered nucleotide sequence may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter,

Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

[00044] In some variations, the composition may be formulated as a liquid. In some other variations, the composition may be formulated as a solid. In yet other variations, the composition may be formulated as a gel, an ointment, a paste, a suspension, a semi-solid, spreadable, or dispersible.

[00045] In some variations, the animal may be a mammal, shellfish, fish, or bird. In some embodiments, the animal may be a livestock animal configured to enter a food supply chain. In some embodiments, the animal may be a human.

[00046] In another aspect, a method is provided for mutating a nucleotide sequence of a first bacteriophage to enhance at least one phenotype of the first bacteriophage, including: creating a plurality of bacteriophage mutants by mutating at least one gene or sequence of a nucleotide sequence of the first bacteriophage; exposing the plurality of mutants to a test environment; testing the plurality of mutants thereby determining whether each of the plurality of mutants exhibits a desired characteristic in response to exposure to the test environment; and selecting at least one mutant bacteriophage exhibiting the desired characteristic in response to the test environment. In some variations, the first bacteriophage may be a wild-type bacteriophage. In some variations, the first bacteriophage may be a bacteriophage of a Podoviridae genus, Caudoviridae genus, or Siphoviridae genus. In some embodiments, when the first bacteriophage is a Podoviradae bacteriophage, then the wild-type bacteriophage may be of a T7 family of bacteriophages.

[00047] In some variations, the at least one mutant bacteriophage may be configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

[00048] In some variations, the desired characteristic may be an increase of stability to the test environment or is an increase of infectivity against one or more bacterial species, relative to a stability or infectivity of the first bacteriophage. In some embodiments, the stability or the infectivity may be increased by 50% relative to the stability or infectivity of the first

bacteriophage.

[00049] In some variations, testing may include performing a test on each of the plurality of mutants to determine if an indicator of mutant survival (e.g., % survival) includes at least a survival threshold.

[00050] In some variations, selecting may include selecting a mutant bacteriophage having the at least survival threshold indicator of mutant survival. In some variations, selecting at least one mutant bacteriophage exhibiting the desired characteristic in response to the test environment may further include selecting a plurality of mutant bacteriophages, where each of the plurality of mutant bacteriophages exhibit the desired characteristic.

[00051] In some variations, exposing the plurality of mutants to a test environment may include exposing the plurality of mutants to an environment including at least one of a temperature at or above a threshold temperature, a pH at or above a threshold pH, a pH at or below a threshold pH, blood, bile acid, an internal bodily fluid, or an external bodily fluid.

[00052] In some embodiments, the test environment may include a temperature of at least 65°C. In some other embodiments, the test environment may include a pH of pH 3 or pH 12.

[00053] In some variations, the method may further include repeating: creating the plurality of bacteriophage mutants; exposing the plurality of mutants to a test environment, subsequently testing the plurality of mutants thereby determining whether each of the plurality of mutants exhibits a desired characteristic, and selecting the at least one mutant bacteriophage exhibiting the desired characteristic, where repeating is performed from 5 to 50 times. In some variations, repeating may include creating the plurality of bacteriophage mutants; exposing the plurality of mutants to a test environment, subsequently testing the plurality of mutants thereby determining whether each of the plurality of mutants exhibits a desired characteristic, and selecting the at least one mutant exhibiting the desired characteristic, wherein repeating may be performed from 9 to 29 times.

[00054] In some variations, when selecting the at least one mutant exhibiting the desired characteristic, the indicator of mutant survival may be progressively increased in each repetition.

[00055] In some variations, where exposing the plurality of mutants to a test environment may further includes changing the test environment each time the method is repeated.

[00056] In some embodiments, when the desired characteristic includes enhanced stability to heat, repeating may include incubating the plurality of mutant bacteriophages in a test environment including incubating at progressively higher temperatures. In some embodiments, a temperature of the test environment may be increased in 5°C increments. In some embodiments, a temperature of the test environment may initially be a temperature at which bacterial host grows optimally. In some embodiments, a temperature of the test environment may be increased to at least 65 °C.

[00057] In some embodiments, when the desired characteristic includes enhanced stability at a pH lower than neutral pH, repeating may include incubating the plurality of mutant

bacteriophages in a test environment including incubating at progressively lower pH. In some embodiments, the test environment may include an initial pH of 5. In some embodiments, a pH of the test environment may be decreased in 0.5 pH unit increments. In some embodiments, the pH of the test environment may be decreased to a pH of 3, thereby selecting a mutant having the at least survival threshold indicator at pH 3.

[00058] In some embodiments, when the desired characteristic includes enhanced stability at a pH higher than neutral pH, and where repeating includes incubating the plurality of mutant bacteriophages in a test environment including incubating at progressively higher pH. In some embodiments, the test environment may include an initial pH of 9. In some embodiments, the pH of the test environment may be increased in 0.5 pH unit increments. In some embodiments, a pH of the test environment may be increased to a pH of 12, thereby selecting a mutant having the at least survival threshold indicator at pH 12.

[00059] In some embodiments, when the desired characteristic includes enhanced stability relative to a stability of the first bacteriophage to at least one of blood, bile acids, internal bodily fluids, or external bodily fluids, and where the test environment includes incubating the plurality of mutant bacteriophages in at least one of blood, bile acids, internal bodily fluids, or external bodily fluids in progressively longer periods of time. In some embodiments, the test period may include incubating for a period of 30 min. In some embodiments, a time period of incubation of the test environment may be increased by at least 30 minutes to at least 24 hours, thereby selecting a mutant having the at least survival threshold indicator of stability for 24 hours to at least one of blood, bile acids, internal bodily fluids, or external bodily fluids. In some embodiments, the time period of the test environment may be not longer than 72 hours.

[00060] In some embodiments, when the desired characteristic includes an enhanced host range, exposing the plurality of mutants to a test environment may include incubating the plurality of mutant bacteriophages in a mixture of first bacterial cells and second bacterial cells, where the first bacterial cells include bacterial cells for which the first bacteriophage is infective and where the second bacterial cells include bacterial cells for which the first bacteriophage is not infective. In some embodiments, the test environment may include equal amounts

(PFU/PFU) of the first bacterial cells and the second bacterial cells. In some embodiments, the test environment may include progressively increasing greater percentages of the second bacterial cells relative to the first bacterial cells, thereby selecting a mutant having the at least survival threshold indicator for infectivity towards the second bacterial cells.

[00061] In some embodiments, when the desired characteristic includes an increase in the efficiency of lytic activity, testing the plurality of mutants may include performing an assay identifying mutants configured to lyse bacterial cells at an earlier time point than a lysing timepoint of the wild-type bacteriophage. In some embodiments, mutating at least one gene or sequence of a nucleotide sequence of a bacteriophage may include at least one of biochemical, chemical, or physical mutagenesis. In some embodiments, mutating at least one gene or sequence of a nucleotide sequence of the first bacteriophage may include modifying nucleobases of the at least one gene or the nucleotide sequence resulting in base mispairing. In some embodiments, modifying nucleobases of the at least one gene or the nucleotide sequence may include administering ethyl methanesulfonate or nitrosoguanidine. In some other embodiments, mutating at least one gene or sequence of a nucleotide sequence of a bacteriophage may include exposing the first bacteriophage to at least one of X-ray radiation or UV wavelength radiation. In yet other embodiments, mutating at least one gene or sequence of a nucleotide sequence of a bacteriophage may include exposing the first bacteriophage to an enzyme with a defective enzymatic activity involved in DNA repair and/or proofreading. In some embodiments, the enzyme with a defective enzymatic activity involved in DNA repair and/or proofreading may include an error-prone DNA polymerase.

[00062] In some variations, the method may further include identifying a location of at least one mutated base in the at least one mutated gene or sequence of the nucleotide sequence of the mutant bacteriophage relative to the nucleotide sequence of the first bacteriophage.

[00063] In some variations, the method may further include identifying a frequency of at least one mutated base in the at least one mutated gene or sequence of the nucleotide sequence across the at least one selected mutant bacteriophage exhibiting the desired characteristic. In some embodiments, identifying the frequency of the at least one mutated base may further include identifying a frequency of the at least one mutated base above a pre-selected threshold.

[00064] In some variations, the method may further include predicting the at least one mutated base conferring the desired characteristic.

[00065] In some variations, the method may further include identifying a location of each of a plurality of mutated bases of the at least one mutated gene or sequence of the nucleotide sequence of the mutant bacteriophage relative to the nucleotide sequence of the first

bacteriophage. In some variations, the method may further include identifying a frequency of each of the plurality of mutated bases in the at least one mutated gene or sequence of the nucleotide sequence of the mutant bacteriophage relative to the nucleotide sequence of the first bacteriophage.

[00066] In some variations, identifying may include identifying a DNA sequence.

[00067] In some variations, the method may further include creating an engineered bacteriophage including the at least one mutated base at the identified location within a nucleotide sequence of the engineered bacteriophage. In some variations, the engineered bacteriophage may be any bacteriophage having an engineered nucleotide sequence as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[00068] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[00069] The novel features of the invention are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[00070] FIG. 1 schematically illustrates a method of directed evolution to engineer bacteriophages according to some embodiments of the disclosure. As illustrated in FIG. 1, a lytic phage is taken through iterative cycles of mutagenesis and phenotype selection to enhance a desired trait/phenotype. As illustrated in FIG. 1, this procedure consists of multiple cycles of the following steps, (1) bacteriophage genomes are randomly mutagenized. (2) Upon replication within a host, a mismatch mutation is introduced into the genome and expressed. (3) The mutant library then is subjected to phenotypic selection to enrich for phages with the desired trait (e.g. thermal stabilization, enhanced lytic activity, bacterial host-range); (4) phages are amplified and carried over to subsequent cycles. Next-generation sequencing may be used to map location and frequency of mutations across phage genomes. Mutations may be manually modified, combined and used to engineer one or more bacteriophage which may then be further engineered or selected.

[00071] FIG. 2 is a graph illustrating the improved thermal stability of the engineered bacteriophage T3 (after 10, 20 and 30 generations of selection) compared to wild-type bacteriophage according to some embodiments of the disclosure.

[00072] FIG. 3A is a graph illustrating the thermal stability and half-life of the engineered T3 bacteriophage after 30 generations of directed evolution (G30) according to some embodiments of the disclosure. FIG. 3B is a graph illustratingthe phage survival rate of the G30 bacteriophage and the wild-type phage at room temperature. FIG. 3C is a graph illustrating the half-life of the engineered G30 bacteriophage.

[00073] FIG. 4 illustrates increased pH stability of the engineered bacteriophage G30 compared to a wild-type bacteriophage at reduced pH according to some embodiments of the disclosure.

[00074] FIG. 5 graphically illustrates lytic dynamics for both wild type T3 and G10, G20, and G30 T3 mutated bacteriophages according to some embodiments of the disclosure..

[00075] FIG. 6 illustrates an example of next-generation sequencing mapping of engineering changes (engineered mutations) across the genome of the T3 bacteriophage according to some embodiments of the disclosure. Sequencing data shows the genomic locations where high- frequency mutations occur after various rounds of directed evolution.

[00076] FIG. 7A illustrates graphically the mutation frequency enrichment for selected commonly observed mutations for T3 bacteriophage according to some embodiments of the disclosure.

[00077] FIG. 7B graphically illustrates the relationship of survival fraction vs. the normalized mutation frequency with respect to the thermal tolerance induced according to some

embodiments of the disclosure.

[00078] FIGS. 8 A and 8B graphically illustrate the ratios of types of amino acid substitution between wild-type and mutated phages according to some embodiments of the disclosure.

[00079] FIG. 9A is a schematic representation of an in vitro gene assembly and phage reboot according to some embodiments of the disclosure.

[00080] FIG. 9B is a graphical representation of a comparison of survival at room temperature and elevated temperature for wild-type, G30 phage produced by the methods herein, and five single bacteriophage mutants according to some embodiments of the disclosure. [00081] FIG. 10A schematically illustrates an example of next-generation sequencing mapping of engineering changes (engineered mutations) across the genome of the T7 bacteriophage according to some embodiments of the disclosure.

[00082] FIG. 10B graphically illustrates the comparison of survival for wild-type T7 and G15 mutagenized phage at room temperature and at elevated temperature according to some embodiments of the disclosure.

[00083] FIG. 11 is a table illustrating mutation locations, protein affected, and specific modifications made to nucleotide sequence and protein sequence for both T3 and T7

bacteriophages, according to some embodiments of the disclosure.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE FISTING

[00084] SEQ ID NO: 1 is a polynucleotide listing of a wild-type T3 head to tail joining protein (e.g., T3p37).

[00085] SEQ ID NO: 2 is an amino acid sequence listing of the wild-type T3 head to tail joining protein (e.g., T3p37).

[00086] SEQ IDs NO: 3-6 are sequences of a stabilizing mutation in T3 head to tail joining protein (e.g., T3p37).

[00087] SEQ ID NO: 7 is a polynucleotide listing of a wild-type T3 internal core

protein/intemal virion protein B (T3p45).

[00088] SEQ ID NO: 8 is an amino acid sequence listing of the T3 internal core

protein/intemal virion protein B (T3p45).

[00089] SEQ IDs NO: 9-12 are sequences of a stabilizing mutation in T3 internal core protein/intemal virion protein B (T3p45).

[00090] SEQ ID NO: 13 is a polynucleotide listing of a wild-type T3 tail fiber protein (e.g., T3p48).

[00091] SEQ ID NO: 79 is an amino acid sequence listing of the native T3 tail fiber protein (e.g., T3p48).

[00092] SEQ IDs NO: 14-21 are sequences of a stabilizing mutation in T3 tail fiber protein (e.g., T3p48).

[00093] SEQ ID NO: 22 is a polynucleotide listing of a wild-type T3 capsid assembly protein (e.g., T3p38).

[00094] SEQ ID NO: 23 is an amino acid sequence listing of the wild-type T3 capsid assembly protein (e.g., T3p38).

[00095] SEQ IDs NO: 24-35 are sequences of stabilizing mutations in T3 capsid assembly protein (e.g., T3p38). [00096] SEQ ID NO: 36 is a polynucleotide listing of a wild-type T3 minor capsid protein (e.g., T3p39).

[00097] SEQ ID NO: 37 is an amino acid sequence listing of the wild-type T3 minor capsid protein (e.g., T3p39).

[00098] SEQ IDs NO: 38-43 are sequences of a stabilizing mutation in T3 minor capsid protein (e.g., T3p39).

[00099] SEQ ID NO: 44 is a polynucleotide listing of a wild-type T3 in internal virion protein C (e.g., T3p46).

[000100] SEQ ID NO: 45 is an amino acid sequence listing of the wild-type T3 in internal virion protein C (e.g., T3p46).

[000101] SEQ IDs NO: 46-53 are sequences of stabilizing mutations in T3 in internal virion protein C (e.g., T3p46).

[000102] SEQ ID NO: 54 is a polynucleotide listing of a wild-type T7 capsid assembly protein (e.g., T7p43).

[000103] SEQ ID NO: 55 is an amino acid sequence listing of the wild-type T7 capsid assembly protein (e.g., T7p43).

[000104] SEQ IDs NO: 56-60 are sequences of a stabilizing mutation in T7 capsid assembly protein (e.g., T7p43).

[000105] SEQ ID NO: 61 is a polynucleotide listing of a wild-type T7 internal virion protein D (e.g·, T7p51).

[000106] SEQ ID NO: 62 is an amino acid sequence listing of the wild-type T7 internal virion protein D (e.g., T7p51).

[000107] SEQ IDs NO: 63-78 are sequences of stabilizing mutations in T7 internal virion protein D (e.g., T7p51).

DETAILED DESCRIPTION

[000108] The disclosure herein relates to engineered bacteriophage having enhanced properties or characteristics, which may be referred to herein as a phenotype. This methodology can be used successive times to favor the accumulation of beneficial mutations, in a manner similar to directed evolution. Any suitable sequencing method including Sanger sequencing or massively parallel sequencing method (e.g., next-generation sequencing (NGS) such as emulsion PCR, solid phase PCR, DNA nanoball sequencing, pyrosequencing, semiconductor sequencing, and the like, available from Illumina, Ion Torrent, Oxford Nanopore, Complete Genomics, Pacific Biosciences and others) can then be used to map the exact genomic location of the mutations (i.e. for a mutation in a coding region or non-coding region: the gene, domain, and amino acid substitution), which furthers the understanding of the structural and functional effects of these mutations on phage biology. The detailed mapping of the beneficial mutations can then be used to generate de-novo mutations in a rational and precise manner in other phages. In some variations, detection of a nucleotide sequence may be made by technique other than massively parallel sequencing. A nucleotide sequence maybe be detected by any suitable nucleotide hybridization techniques and may be quantified using hybridization techniques such as quantitative PCR (e.g., qPCR).

[000109] By using this methodology, one or more of the following phage properties can be enhanced: thermal stability, pH stability, blood stability, bile acid stability, other internal bodily fluid stability, external bodily fluids, and modulation of the lytic activity, specificity or host- range of phage infection.

[000110] The rapid escalation of drug resistant bacterial infections and decreased investment in antibiotic research make it imperative to develop novel antimicrobial therapies. An approach regaining significant interest is phage therapy (PT) whereby bacteria targeting viruses

(bacteriophages or phages for short) may be used as antimicrobials or for delivery of genetic circuits (e.g., intracellular components responsible for encoding RNA and/or protein, where the genetic circuits may be synthetically derived, such as by employing CRISPR synthetic modification techniques) with antimicrobial or physiological activities. Phages are exquisitely selective of their host, which makes phage therapy less destructive of the normal and beneficial microflora of the patient compared to conventional chemical antibiotics. Bacteriophages are also functionally orthogonal to antibiotics, as they are generally unaffected by acquisition of antibiotic resistance. These properties make bacteriophages particularly adapted to the treatment of Anti-Microbial Resistant (AMR) infections. A further advantage of phages is their self-dosing capacity, they can replicate to the extent of the infection, and, once there are no viable target bacteria, no further replication may follow. However, these attributes also makes traditional pharmacodynamics methods inadequate for Phage Therapy (PT).

[000111] Although independent of antibiotic resistance mechanisms, bacteria have evolved various resistance solutions against phage predation. Bacteriophage initiate infection through the specific recognition of a surface exposed receptor molecule, protein, lipopolysaccharide (LPS) or capsule component. However, if these biomolecules become mutated or masked, the virus can be deprived of its entry port. Resistance to phages may also arise from acquisition of dedicated phage defense mechanisms such as CRISPR or abortive infection systems. Finally, phages may recognize a specific receptor and therefore, for many naturally occurring phages, there is a relatively narrow host range. This in turn, means that no single phage may be active against all (or a medically relevant fraction of) bacteria involved in any given disease. [000112] These issues are may be alleviated by assembling and regularly updating

bacteriophage cocktails of un-related bacteriophages that are collectively able to eliminate the variety of bacteria causing affliction.

[000113] Various approaches have been undertaken to expand the host range of phages to combat resistance, including hybridization between already characterized bacteriophages with known and desired host ranges.

[000114] Some studies have relied on traditional phage mutant selection procedures which utilize natural evolution. This process typically proceeds through single mutations at a time and some of these mutations may be deleterious initially though required towards the evolutionary goal set. Thus, the natural evolution procedure often reaches bottlenecks where too many concomitant mutations are necessary to both obtain the selected phenotype and have a viable organism.

[000115] Previous studies have demonstrated that the T7-family of phages is particularly amenable to phage host range engineering, T7-family phages have an extremely host independent life cycle so that DNA entry into the host range is the most significant barrier to generating progeny. The experiments described here have focused on phage T3 and T7. The two phages are extremely similar and share an extremely similar developmental cycle. However, the methods described herein are not so limited to T3 and T7 bacteriophages but may be extended to other bacteriophage species, families, and genera.

[000116] Studies of bacterial resistance to T3 bacteriophages and T7 bacteriophages have revealed that phages routinely adapt to resistance through mutations within genes 11, 12, and/or 17 for T7 and within 17 exclusively for T3. Both T3 and T7 rely on binding to the outer core LPS for absorption; however, they bind to different LPS moieties which leads to slightly different host ranges. The T7 gpl7 tip was crystallized and its structure resolved. It is 75% identical to the corresponding region of T3 gpl7, and the structure of the T3 tail fiber tip can therefore be modelled with high accuracy using homology modelling tools. The distal 106 aa of gpl7 form an intertwined globular domain shaped by an eight stranded beta barrel (labelled B to I) connected by random coils. Four of those coils, BC, DE, FG and HI, are pointed towards the exterior side of the tail fiber and are therefore uniquely positioned to contact the host and recognize the receptor moiety.

[000117] Disclosed herein are strategies and methods for engineering synthetic bacteriophages with expanded resistance to many of the environmental factors that inhibit phage activity and commercial use. The mutations described herein are compatible with other forms of phage engineering (synthetic genome reconstruction or Gibson assembly of full phage genomes), selection of other phenotypes (e.g., selection of faster or slower replication rates or altered immunogenicity of the phage, including modulation of the release of endotoxin

(lipopolysaccharide, LPS) after the end of the replication cycle within the bacterial cell)), and are also compatible with random mutagenesis to enrich mutations outside of the immediately targeted region.

[000118] The engineered bacteriophage described herein may also be part of a phage library or used to form a phage library. A combinatorial-based approach may be used in which the various modifications described herein may be combined in combinations of two, three or more.

[000119] Thus, disclosed herein are bacteriophages (e.g., engineered bacteriophages, including synthetic bacteriophages and also including de-novo engineered bacteriophages) having one or more mutations (e.g., an engineered nucleotide sequence)in one or more of a non-coding and/or coding region, such as: a coding region of the phage head-to-tail joining protein, a coding region of a phage capsid assembly protein, a coding region of the phage internal core protein, an internal virion protein (e.g. virion protein D), a coding region for an endonuclease, a direct repeat region and/or a coding region of a phage tail fiber protein. The bacteriophage having an engineered nucleotide sequence may be a synthetic bacteriophage. In some variations, the engineered nucleotide sequence of the bacteriophage may be in a non-coding region such as, for example, a 5’UTR capsid assembly protein non-coding region or a 5’ UTR internal core protein non-coding region, The engineered nucleotide sequences may be mutations of specific base pairs, insertions, deletions or combination of these. The engineered nucleotide sequences (e.g., mutated nucleotide sequences) may provide an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, internal and/or external bodily fluid stability and host range of the bacteriophage having the engineered nucleotide sequence compared to a respective wild-type bacteriophage. The nucleotide sequence of the bacteriophage, including the engineered nucleotide sequence, may have more than about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity with a nucleotide sequence of the wild type bacteriophage

[000120] In some variations, the bacteriophage may have an engineered nucleotide sequence (e.g., mutation) in more than one region, including in a coding region of the phage head-to-tail joining protein and a coding region of an internal core protein. In some variations, the coding region of the internal core protein may further include a region of the internal virion protein B. In some other variations, the bacteriophage may have an engineered nucleotide sequence in a coding region of a phage fiber protein. In yet other variations, the bacteriophage may have an engineered nucleotide sequence in a coding region of the phage head-to-tail joining protein, a coding region of an internal core protein (which may include a region of the internal virion protein B), and a coding region of a phage fiber protein. In yet other variations, the bacteriophage may have an engineered nucleotide sequence (e.g., mutation) in a region that encodes for an endonuclease.

[000121] In some variations, the bacteriophage may have an engineered nucleotide sequence (e.g., mutation) in a direct repeat region. In other variations, the bacteriophage may have an engineered nucleotide sequence (e.g., mutation) in a plurality of direct repeat regions.

[000122] In yet other variations, the engineered nucleotide sequence comprises a mutation in a 5’UTR capsid assembly protein non-coding region. In some other variations, the engineered nucleotide sequence comprises a mutation in a 5’UTR internal core protein non-coding region.

In some variations, the bacteriophage may have an engineered nucleotide sequence (e.g., mutation) in a region that encodes for an endonuclease, and in a 5’UTR capsid assembly protein non-coding region. In other variations, the bacteriophage may have an engineered nucleotide sequence (e.g., mutation) in a region that encodes for an endonuclease, and in a 5’UTR internal core protein non-coding region. In some other variations, the bacteriophage may have an engineered nucleotide sequence (e.g., mutation) in a direct repeat region or in a plurality of direct repeat regions and in a 5’UTR capsid assembly protein non-coding region. In yet other variations, the bacteriophage may have an engineered nucleotide sequence (e.g., mutation) in a direct repeat region or in a plurality of direct repeat regions and in a 5’UTR internal core protein non-coding region.

[000123] The engineered mutations (e.g., engineered nucleotide sequence) identified herein may be generated by non-natural methods such as synthesis of sequences (or replacement of sequences) or removal (deletion) of sequence, to introduce mutations relative to the wild-type sequence. Accordingly, the bacteriophage may be a synthetic bacteriophage.

[000124] Thus, mutations introduced to produce the bacteriophages (e.g., engineered bacteriophage, including a synthetic bacteriophage) can be substitution mutations, deletions, or insertions/additions. It also is possible to add amino acids or delete amino acids. The types of mutations can be mixed such that a substitution mutation of one or more amino acids and an addition and/or deletion mutation may be present in the same phage. The types of mutations also can be mixed such that, for example, one phage contains both a substitution mutation of one or more amino acids, and an addition and/or deletion mutation.

[000125] In some embodiments, the engineered mutations are the only mutations in the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage). The bacteriophage may contain multiple mutations in multiple proteins (and/or in non-coding regions), such as for providing the bacteriophage with one or more additional functional features.

[000126] As shown herein, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) can be a T3 bacteriophage. Other similar bacteriophage can likewise be generated to have any, all, or some of the mutations described herein, such as a T7 bacteriophage or a bacteriophage having about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to a T3 bacteriophage mutated sequence region.

[000127] As phages generally have homologous nucleotide sequence regions for the limited functional repertoire of a phage, the specific mutations described herein for a T3 or a T7 may be extrapolated to the homologous region of the homologous gene and type of mutation in other bacteriophage species and genera. In some other variations, the wild-type bacteriophage which is mutated by the methods described herein may be a bacteriophage of a Podoviridae genus. In some other variations, the wild-type bacteriophage which is mutated by the methods described herein may be a bacteriophage of a Caudoviridae genus. In some other variations, the wild-type bacteriophage which is mutated by the methods described herein may be a bacteriophage of a Siphoviridae genus. In some variations, when the wild-type bacteriophage is a Podoviradae bacteriophage, then the wild-type bacteriophage may be of a T7 family of bacteriophages. In some other variations, the wild-type bacteriophage may be a T3 or a T7 phage.

[000128] Accordingly, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) may have an engineered nucleotide sequence (e.g., mutation) in at least one of a T3pl7 gene, a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, a T3p48 gene, or a homologous gene of a different bacteriophage species thereof. In some other variations, the bacteriophage may have an engineered nucleotide sequence at more than one of the T3p37 gene, the T3p38 gene, the T3p39 gene, the T3p45 gene, or the T3p48 gene or the homologous gene(s) of a different bacteriophage species thereof. In yet other variations, the bacteriophage may have an engineered nucleotide sequence in at least one of a T7p43 gene, a T7p51 gene, or a homologous gene of a different bacteriophage species thereof.

[000129] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) may have an engineered nucleotide sequence (e.g., mutation) of at least one of SEQ ID NOS 3, 4, 9, 10, 14, 15, 16, 17, 18, 19, 24, 25, 26, 27, 28, 29, 38, 39, 40, 41, 46, 47, 48, 49, 56, 57, 58, 59, 63, 64, 67, 68, 69, 70, 75, or 76. In some embodiments, the engineered nucleotide sequence may be a homolog of at least one of SEQ ID NOS 3, 4, 9, 10, 14, 15, 16, 17, 18, 19, 24, 25, 26, 27, 28, 29, 38, 39, 40, 41, 46, 47, 48, 49, 56, 57, 58, 59, 63, 64, 67, 68, 69, 70, 75, or 76, in a different bacteriophage species In some other variations, t the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) may have an engineered nucleotide sequence (e.g., mutation) may produce an amino acid sequence of at least one of SEQ ID NOS 5, 6, 12, 20, 21, 30, 31, 32, 33, 34, 35, 42, 43, 50, 51, 52, 53, 60, 61, 65, 66, 71, 72, 73, 74, 77, or 78. [000130] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have an increase in heat stability at about 55 C, about 57°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C or about 65°C, compared with a heat stability of the wild-type bacteriophage. In some variations, the bacteriophage having an engineered nucleotide sequence may have an increase in heat stability at about 60°C or about 62°C, compared with a heat stability of the wild- type bacteriophage. The increase in heat stability, compared to the wild-type bacteriophage, may be at least a 10% increase, a 20% increase, a 30 % increase, a 40% increase, a 50% increase, a 60% increase, a 70% increase, a 80% increase, a 90% increase, or a 100% increase.

[000131] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have an increase in at least one of pH stability at pH 3 or pH stability at pH 12 in the bacteriophage relative to a respective pH stability of the wild-type bacteriophage. The increase in pH stability at pH 3 or pH stability at pH 12 compared to the wild-type bacteriophage, may be at least a 10% increase, a 20% increase, a 30 % increase, a 40% increase, a 50% increase, a 60% increase, a 70% increase, a 80% increase, a 90% increase, or a 100% increase. In some variations, the increase in heat stability may provide an increase in a survival rate of the bacteriophage relative to a survival rate of the wild-type bacteriophage.

[000132] In some variations, the bacteriophage having an engineered nucleotide sequence may have an increase in heat stability, and an increase in at least one of pH stability at pH 3 or pH stability at pH 12 in the bacteriophage relative to a respective pH stability of the wild-type bacteriophage. In some variations, the increase in pH stability may provide an increase in a survival rate of the bacteriophage relative to a survival rate of the wild-type bacteriophage.

[000133] In some variations, the bacteriophage having an engineered nucleotide sequence having an increase in heat stability, and/or an increase in at least one of pH stability at pH 3 or pH stability at pH 12, may produce an amino acid sequence comprising at least one amino acid, at least two amino acids, at least 4 amino acids, at least 5 amino acids, or more that are more hydrophobic or more bulky than an amino acid sequence produced by the nucleotide sequence of the wild-type bacteriophage. In some embodiments, the bacteriophage having an engineered nucleotide sequence having an increase in heat stability, and/or an increase in at least one of pH stability at pH 3 or pH stability at pH 12, may produce an amino acid sequence comprising at least about 5% more, about 10% more, about 15% more, about 20% more, about 25% more, about 30% more or about 35% more hydrophobic amino acids within the amino acid sequence compared to the amino acid sequence of the wild-type bacteriophage. In some other variations, the bacteriophage having an engineered nucleotide sequence having an increase in heat stability, and/or an increase in at least one of pH stability at pH 3 or pH stability at pH 12, may produce an amino acid sequence comprising at least about 5% more, about 10% more, about 15% more, about 20% more, about 25% more, about 30% more or about 35% more bulky (e.g. having more residue volume) amino acids within the amino acid sequence compared to the amino acid sequence of the wild-type bacteriophage.

[000134] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have an increase in virulence, compared with a virulence of the wild-type bacteriophage. The virulence of the bacteriophage having an engineered nucleotide sequence may be increased about 5%. about 10%, about 20%, about 30%, about 50%, about 75%, about 100% or more compared to the virulence of the wild-type bacteriophage.

[000135] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have an increase in stability in blood, compared with a stability in blood of the wild-type bacteriophage. The stability in blood of the bacteriophage having an engineered nucleotide sequence may be increased about 5%. about 10%, about 20%, about 30%, about 50%, about 75%, about 100%, about 200% or more compared to the stability in blood of the wild-type bacteriophage.

[000136] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have an increase in stability to bile acid, compared with a stability to bile acid of the wild-type bacteriophage. The stability to bile acid of the bacteriophage having an engineered nucleotide sequence may be increased about 5%. about 10%, about 20%, about 30%, about 50%, about 75%, about 100%, about 200% or more compared to the stability to bile acid of the wild-type bacteriophage.

[000137] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have an increase in stability to internal and/or external bodily fluid, compared with a stability to internal and/or external bodily fluid of the wild-type bacteriophage. The stability to internal and/or external bodily fluid of the bacteriophage having an engineered nucleotide sequence may be increased about 5%. about 10%, about 20%, about 30%, about 50%, about 75%, about 100%, about 200% or more compared to the stability to internal and/or external bodily fluid of the wild-type bacteriophage.

[000138] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have an increase in infectivity, compared with an infectivity of the wild-type bacteriophage. The infectivity of the bacteriophage having an engineered nucleotide sequence may be increased about 5%. about 10%, about 20%, about 30%, about 50%, about 75%, about 100%, about 200% or more compared to the infectivity of the wild-type bacteriophage.

[000139] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have an increase in efficacy, compared with an efficacy of the wild-type bacteriophage. The efficacy of the bacteriophage having an engineered nucleotide sequence may be increased about 5%. about 10%, about 20%, about 30%, about 50%, about 75%, about 100%, about 200% or more compared to the efficacy of the wild-type bacteriophage.

[000140] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have a shift in host range compared with the host range of the wild-type bacteriophage, permitting the bacteriophage to infect and eliminate/reduce other bacterial species beyond the typical target bacteria of the wild-type bacteriophage.

[000141] In some variations, the bacteriophage (e.g., engineered bacteriophage, including a synthetic bacteriophage) having an engineered nucleotide sequence may have an increased stability in more than one of heat stability, pH stability, virulence, blood stability, bile acid stability, internal and/or external bodily fluid stability, infectivity, efficacy, or shift in host range, in any combination thereof,

[000142] Compositions. Compositions of the bacteriophages (e.g., engineered bacteriophages, including synthetic bacteriophages and also including de-novo engineered bacteriophages) having one or more mutations (e.g., an engineered nucleotide sequence)in one or more of a non coding and/or coding region, also are provided. The bacteriophage having an engineered nucleotide sequence may be any bacteriophage having an engineered nucleotide sequence as described herein. Such compositions can include a pharmaceutically-acceptable carrier.

Generally, for pharmaceutical use, the synthetic bacteriophages may be formulated as a pharmaceutical preparation or compositions comprising at least one synthetic bacteriophage and at least one pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active compounds. Such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such administration forms may be solid, semi-solid or liquid, depending on the manner and route of administration. An administration form may be a gel, an ointment, a paste, a suspension, a semi-solid, spreadable, or dispersible. For example, formulations for oral administration may be provided with an enteric coating that will allow the bacteriophages in the formulation to resist the gastric environment and pass into the intestines. More generally, bacteriophage formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract. In addition, suitable suppositories may be used for delivery into the gastrointestinal tract. Various pharmaceutically acceptable carriers, diluents and excipients useful in bacteriophage

compositions are known to the skilled person.

[000143] The bacteriophage(s) (e.g., engineered bacteriophage, including synthetic

bacteriophage and also including de-novo engineered bacteriophage) having one or more mutations (e.g., an engineered nucleotide sequence) in one or more of a non-coding and/or coding region may be present in the formulation at a concentration of about 10³PFU/ml; about 10⁴PFU/ml; about 10⁵PFU/ml; about 10⁶PFU/ml; about 10⁷PFU/ml; about 10⁸PFU/ml; about 10⁹PFU/ml; about 10¹⁰PFU/ml; or about 10^uPFU/ml. In other embodiments, the

bacteriophage(s) may be present at a concentration of about 10³PFU/g; about 10⁴PFU/g; about 10⁵PFU/g; about 10⁶PFU/g; about 10⁷PFU/g; about 10⁸PFU/g; about 10⁹PFU/g; about

10¹⁰PFU/g; or about 10^uPFU/g.

[000144] The bacteriophage compositions have, in some embodiments, a single type of synthetic bacteriophage. More typically, however, the bacteriophage compositions include two or more variants or types of bacteriophages that have different mutations in the tail fiber tip protein, head-to-tail joining protein, and/or internal core protein such as, but not limited to internal virion protein B, i.e., a“cocktail” of bacteriophages. In some embodiments, the two or more types of bacteriophages advantageously have different host ranges.

[000145] Also provided are collections (also referred to as“libraries” or“banks”) of bacteriophages, which include a plurality of bacteriophages having different mutations engineered therein. As noted above, such mutations may be substitutions, additions, or deletions.

[000146] Methods of Administration. Also provided are methods for administering a composition as described herein to an animal. The method may include administering an effective amount of a composition to the animal, where the composition includes a bacteriophage (e.g. an engineered bacteriophage, including a synthetic and/or de-novo engineered

bacteriophage) having a nucleotide sequence including an engineered nucleotide sequence (e.g. at least one mutation) in at least one of: a region that encodes for an endonuclease, a region that encodes for a head-to-tail joining protein, a 5’UTR capsid assembly protein non-coding region, a 5’ UTR internal core protein non-coding region, a region that encodes for an internal core protein, a region that encodes for an tail fiber protein gene, and a direct repeat region, wherein the engineered nucleotide sequence results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, internal and/or external bodily fluid stability, and host range of the bacteriophage compared to a respective wild-type bacteriophage, and wherein the nucleotide sequence including the engineered nucleotide sequence has more than 95% identity with a nucleotide sequence of the wild-type bacteriophage in a

pharmaceutically acceptable carrier. The nucleotide sequence of the bacteriophage, including the engineered nucleotide sequence, may have more than about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity with a nucleotide sequence of the wild type bacteriophage. The bacteriophage having an engineered nucleotide sequence may be any bacteriophage having an engineered nucleotide sequence as described herein. The pharmaceutically acceptable carrier may be any suitable carrier as described herein or know in the art. The animal may be a mammal, shellfish, fish, or bird. In some embodiments, the animal may be a livestock animal. In some embodiments, a livestock animal may include a cow, pig, goat, elk, deer, turkey, chicken, duck, pigeon, shrimp, bass, oyster, clam, mussel, salmon, or tilapia. The livestock animal may be configured to enter a food chain. In other embodiments, the animal may be a human.

[000147] In some variations, the effective amount of the composition may include about 10³PFU/kg; about 10⁴PFU/kg; about 10⁵PFU/kg; about 10⁶PFU/kg; about 10⁷PFU/kg; about 10⁸PFU/kg about 10⁹PFU/kg; about 10¹⁰PFU/kg; about 10^uPFU/kg; about 10¹²PFU/kg; about 10¹³PFU/kg; about 10¹⁴PFU/kg; or about 10¹⁵PFU/kg of animal weight.

[000148] In some variations, the effective amount of the composition may be administered to the animal to maintain a healthy microbiome. That is, the animal may not have pathological symptoms of a bacterial infection. The composition may be administered as a probiotic composition. When administering to a livestock animal, administration may be made throughout the lifetime of the animal or may be made at near the end of the lifetime of the animal. The compositions may be administered to prevent or reduce the likelihood that the animal develops a bacterial infection or pathological symptoms thereof. A healthy microbiome may include a variety of flora, both bacterial and fungal, and administration of the composition may maintain desirable flora in balance, particularly commensal bacteria of the gut.

[000149] Also provided are methods for treating a bacterial infection using the

bacteriophage(s) having an engineered nucleotide sequence, as disclosed herein. The methods include administering an effective amount of a composition to an animal in need thereof, wherein the composition comprises a bacteriophage having a nucleotide sequence comprising an engineered nucleotide sequence in at least one of: a region that encodes for an endonuclease, a region that encodes for a head-to-tail joining protein, a 5’UTR capsid assembly protein non coding region, a 5’ UTR internal core protein non-coding region, a region that encodes for an internal core protein, a region that encodes for an tail fiber protein gene, and a direct repeat region, wherein the engineered nucleotide sequence results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, internal and/or external bodily fluid stability, and host range of the bacteriophage compared to a respective wild-type bacteriophage, and wherein the nucleotide sequence comprising the engineered nucleotide sequence has more than 95% identity with a nucleotide sequence of the wild-type bacteriophage in a pharmaceutically acceptable carrier. The nucleotide sequence of the bacteriophage, including the engineered nucleotide sequence, may have more than about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity with a nucleotide sequence of the wild type bacteriophage. The bacteriophage having an engineered nucleotide sequence may be any bacteriophage having an engineered nucleotide sequence as described herein. The pharmaceutically acceptable carrier may be any suitable carrier as described herein or know in the art. The animal may be a mammal, shellfish, fish, or bird. In some embodiments, the animal may be a livestock animal. In some embodiments, a livestock animal may include a cow, pig, goat, elk, deer, turkey, chicken, duck, pigeon, shrimp, bass, oyster, clam, mussel, salmon, or tilapia. The livestock animal may be configured to enter a food chain. In other embodiments, the animal may be a human.

[000150] In some variations, the effective amount of the composition may include about 10³PFU/kg; about 10⁴PFU/kg; about 10⁵PFU/kg; about 10⁶PFU/kg; about 10⁷PFU/kg; about 10⁸PFU/kg or about 10⁹PFU/kg of animal weight.

[000151] Bacterial targets. Bacteriophage typically target selected bacteria. For example, T3 and/or T7 bacteriophage inactivate varieties of Escherichia coli ( E . coli ) by lytic growth, e.g., the life cycle of the bacteriophage requires lysis (and subsequent death) of the infected bacterial cell. However, naturally occurring T3 and/or T7 phage may infect other selected bacteria as well. One goal of the methods described herein is to identify mutant bacteriophages having modified capacity for infecting different species of E. coli or different genera of bacteria. For example, an engineered T3 or T7 bacteriophage may selectively infect, and reduce populations of the genera of Pseudomonas (including P. aeruginosa); Staphylococcus spp. (including Methicillin resistant S. aureous MRSA)); virulent E. coli spp.; Salmonella (including antibiotic resistant Salmonella DT104); Escherichia; Shigella; Acinetobacter; Klebsiella; Campylobacter; Pasteurella;

Aeromonas; Vibrio; or Yersinia (including Y. enterocolitica).

[000152] In some variations, methods are provided for using one or more engineered bacteriophages (including one or more synthetic bacteriophages) or a composition including such bacteriophage(s), as described herein for manufacturing a medicament for treating a bacterial infection in an animal, e.g., treating a population of undesired bacteria in an animal to reduce or eliminate the population of undesired bacteria, which may in particular be an Escherichia coli {e. coli ) bacteria, that may cause infections in animals. In another variations, methods are provided for using one or more engineered bacteriophages (including one or more synthetic bacteriophages) or a composition including such bacteriophage(s), as described herein for treating an animal or for manufacturing a medicament for maintaining a healthy microbiome in an animal, that is, the animal may not have a bacterial infection (e.g., an overpopulation of a bacteria causing pathological symptoms. The medicament may prevent or reduce the likelihood that a bacterial infection may occur in the animal so treated.

[000153] Examples of infections caused by e. coli that may be treated as described herein include, but are not limited to: infections, including cholecystitis, bacteremia, cholangitis, urinary tract infection (UTI), enteric infection, and traveler's diarrhea, and other clinical infections such as neonatal meningitis and pneumonia.

[000154] The methods may include any combination of bacteriophages and features of the bacteriophages or compositions as described herein.

[000155] Methods of producing a bacteriophage having an engineered nucleotide sequence. Methods of producing one or more bacteriophages (e.g., engineered bacteriophages, including synthetic bacteriophages and also including de-novo engineered bacteriophages) having one or more mutations (e.g., an engineered nucleotide sequence)in one or more of a non coding and/or coding region, also are provided. In such methods, one or more sequences of a bacteriophage DNA is mutated to produce a bacteriophage having the corresponding target mutation(s) described herein. The engineered bacteriophage described herein may be generated by one or more techniques.

[000156] In one variation, mutations can be introduced by synthesizing portions of the target protein using degenerate primers that vary the nucleotide sequence, and thereby introduce substitutions of amino acids (or additions or deletions) in the target sequences.

[000157] In some variations, a method as illustrated in FIG. 1 schematically illustrates one exemplary method of generating bacteriophage having directed mutations described. This method generally modifies nucleotides of the at least one gene or the nucleotide sequence resulting in base mispairing. This method allows the accumulation of beneficial mutations by iterating though a cycle that consists of 4 successive processes. In in the first process of the method, a plurality of mutations (e.g., tens of thousands) can be generated across the genome of a wild-type bacteriophage 105 producing a mixture of mutated phages 115, the mutations (e.g., alkylated bases in this example) represented by different asterisked arrows at different locations within the genome. The mutations may be introduced by a variety of agents. Typically, a mutagenizing agent that produces mutations across the genome may be used, such as a chemical agent (e.g., nitrosoguanidine, ethyl methanesulfonate, or the like); irradiation by X-ray or UV wavelength irradiation; contact with a defective enzymatic activity involved in DNA repair and/or proofreading, such as an error-prone polymerase, amongst others. Mutagenesis increases the genetic diversity to accelerate the speed of evolution.

[000158] The method as described in this exemplar uses a chemical mutagenesis process (e.g. ethyl methanesulfonate (EMS)), but the methods of the disclosure are not so limited. In this exemplar, EMS induces random alkylation, typically at guanine bases throughout the nucleotide sequence of the bacteriophage At process 120, the phage mutants 115 are amplified in the host bacteria 107 to allow the expression of mutated protein and the formation of the mutated bacteriophage particles 125, incorporating the mutations at starred arrows as shown. In process 130, mutant bacteriophages are selected for one or more desired properties or characteristics by exposure to a test environment in which evolution/selection of the mutant bacteriophages may be challenged, which refine the gene pools towards variants more successful at surviving the test environment. Surviving mutant phages 135 are selected, and in process 140, are amplified in a suitable host bacteria 117, which may be the same or different from host bacteria 107. The amplified mutant phages 135 may be returned through the cycle of processes 110, 120, 130, and 140 again until mutants with desired properties or characteristic are obtained. In process 150, mutant phages 135 may be subjected to analysis including qPCR and/or sequencing, including NGS sequencing to map location and the frequency of mutations across phage genomes. The cycle, including processes 110, 120, 130, 140, and optionally 150 may be repeated n times where n is from 1 to about 200; from 1 to about 100, from 1 to about 50, from 1 to about 30; from 1 to about 20 from 1 to about 15; from 1 to about 10; or from 1 to about 10.

[000159] The mapped locations and the frequency of mutations across phage genome can then be used to help understand the structural and functional effects of these mutations on phage biology. De-novo mutations can then be made in a rational and precise manner in other phages, which can be used in therapeutic applications.

[000160] The method illustrated in FIG. 1 can be the basis for a method of mutating a bacteriophage genome to enhance at least one phenotype of the bacteriophage, where the method includes creating a plurality of bacteriophage mutants by mutating at least one gene or sequence of a genome of a wild type bacteriophage; exposing the plurality of mutants to a test

environment; after exposure to the test environment, performing a test on the plurality of mutants to determine if each of the plurality of mutants exhibits a desired characteristic in response to exposure to the test environment; and selecting at least one mutant from the plurality of mutants if the mutant exhibits the desired characteristic when exposed to the test environment. In some variations, the method may include selecting a plurality of mutant bacteriophages, where each of the plurality of mutant bacteriophages exhibit the desired characteristic. The plurality of mutant bacteriophages may have different mutations within the engineered nucleotide sequence of the mutant bacteriophage.

[000161] The methodology described herein may be used on wide variety of different phages to enhance one or more phage properties. For example, without limitation, bacteriophage that may be used in the methodology herein may be of the genera Podoviridae, Caudoviridae, or

Siphoviridae. Exemplary bacteriophage of the genera Podoviridae include the T3 and T7 bacteriophage, which are similar in structure.

[000162] The methods may permit identification of at least one mutant bacteriophage (e.g. an engineered bacteriophage, including a synthetic and/or de-novo engineered bacteriophage) having a nucleotide sequence including an engineered nucleotide sequence (e.g. at least one mutation) configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage

[000163] The method may provide bacteriophages (e.g., engineered bacteriophages, including synthetic bacteriophages and also including de-novo engineered bacteriophages) having one or more mutations (e.g., an engineered nucleotide sequence) in one or more of a non-coding and/or coding region with improved properties for one or more of stability to heat, pH ranges outside of physiological, blood stability, bile acid stability, internal and/or external bodily fluid stability; improved virulence, improved infectivity, improved efficacy and/or shifted host range. The bacteriophage having an engineered nucleotide sequence may have an improved stability, property or differentiated host range that may be increased about 5%. about 10%, about 20%, about 30%, about 50%, about 75%, about 100%, about 200% or more compared to the stability, property or host range of the wild-type bacteriophage. In an embodiment where it is desirable to shift the host range, the bacteriophage having an engineered nucleotide sequence may be capable of infecting a bacterial species that the wild-type bacteriophage cannot infect at all or that the wild-type bacteriophage can infect only minimally.

[000164] Test environment. The test environment (e.g., challenge conditions) may be chosen to select for desirable mutated bacteriophages that can survive or preferentially function under the chosen environment. This can direct evolution over the course of several challenge incubations to select for more optimally functioning engineered bacteriophages.

[000165] For example, one test environment may be an elevated temperature such as about 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees Celsius, driving isolation of bacteriophages which are more heat stable than the wild type bacteriophage. This can offer advantages in both manufacture of the compositions and medicaments of the disclosure, e.g., heat treatment to neutralize byproducts of the manufacturing process, such as LPS as well as extending the in-vivo lifetime of the engineered bacteriophage relative to that of the wild type organism. The unnatural temperature chosen (60 degrees C) is higher than a temperature in which bacteriophage may be typically found (e.g., soils or bodies of water) or a body temperature of any species in which bacteriophage may be found or administered to. For example, humans, cattle, and equine normal body temperatures may range from about 36 degrees C to about 39 degrees C; leporid, porcine, caprinaeid normal body temperature may range from about 38 to about 40 degrees C; and poultry normal body temperature may range from about 40 degrees C to about 43 degrees C.

[000166] Another test environment that may be used to direct evolution of a wild type bacteriophage to provide advantageously engineered bacteriophages is altered pH. For example, exposure to repeated incubations at reduced pH, e.g., at about a pH of about 2.0, 2.5, 3.0, 3.5 or about 4.0 may provide mutant bacteriophages having nucleotide and/or protein sequence alterations providing increased stability at reduced pHs, which may be advantageous for potential oral administration. Orally administered low pH tolerant engineered bacteriophages may survive passage through the stomach environment and may further may be more stable to bile acids present within the small intestine. The repeated incubations at such lowered pHs provide an unnatural test environment to drive the desired mutations.

[000167] Alternatively, a test environment may be selected to direct evolution of a wild type bacteriophage at an elevated pH. An unnatural test environment of a pH above about 9.0, 9.5. 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, or about 13, over repeated incubations, can direct evolution of an engineered bacteriophage that is advantageously more stable to basic conditions found in the intestines.

[000168] In other variations, a test environment may be selected to direct evolution of a wild type bacteriophage to enhance the stability of an evolved bacteriophage to exposure in blood, bile acid, an internal bodily fluid (e.g., a fluid such as plasma, serum, lymphatic fluid, stomach acid, vagina fluid, prostatic fluid and the like), or an external bodily fluid (e.g., tear fluid). Any of these fluids may have proteases, nucleases, and acidic components which can degrade bacteriophages, and may do so synergistically. Additionally, these substances may have other components which may assist in destabilizing a wild-type bacteriophage including but not limited to macrophages, and other active scavenger species. A test condition environment may provide an artificially high concentration of one or more components of the challenging blood, bile acid, an internal bodily fluid, or an external bodily fluid; may include an artificially warmer temperature than body temperature when contacting the mutated phages to the test environment; may use a synthetic composition to approximate or control the ratios of components making up the challenging blood, bile acid, an internal bodily fluid, or an external bodily fluid; or may contact the mutated phages to the test environment for a longer test period than typically found in-vivo. A test condition may be any of these, singly or in combination, and may be altered as described herein in order to further drive the selection of mutant bacteriophages with greater stability.

[000169] In other variations, a test environment may be selected to direct evolution of a wild type bacteriophage to enhance the infectivity and/or efficacy of an evolved bacteriophage.

Selection cycles may be made more rapidly to select bacteriophages with shorter life cycle and/or more effective entry/lysis of the host bacteria.

[000170] In yet further variations, a test environment may be selected to direct evolution of a wild-type bacteriophage to shift or expand bacterial host range. The test environment may initially contain both the host bacteria species for which the wild-type bacteria typically target, as well as a different bacteria species to which the expanded or shifted host range is desired.

[000171] Testing the plurality of mutants to determine whether each of the plurality of mutants exhibits a desired characteristic in response to exposure to the test environment may include performing a test on each of the mutants to determine if an indicator of mutant survival (e.g., % survival) comprises at least a survival threshold. For example, when the mutants may be tested to determine what percentage of the initial inoculant population survives the test environment, the survival threshold may be set at the same percentage as the survival percentage of the wild-type bacteriophage (e.g., cannot be less stable). In other embodiments, the survival threshold may be set at a higher level than that of the wild-type bacteriophage. The mutant may be selected after testing when the bacteriophage having the engineered nucleotide sequence has a percentage survival that is about 5%; about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 70%, about 100% or more, than that of the wild-type bacteriophage.

[000172] In some variations, the method may further include repeating the processes of:

creating the plurality of bacteriophage mutants; exposing the plurality of mutants to a test environment, testing the plurality of mutants thereby determining whether each of the plurality of mutants exhibits a desired characteristic, and selecting the at least one mutant bacteriophage exhibiting the desired characteristic, where repeating is performed from 4 or 5 time to 50 times. In some other variations, the processes may be repeated about 2 to about 29 time. In some variations, the processes may be repeated at least 5 times, up to about 200 times.

[000173] Repeating the cycle. When the method includes repeating the cycle of processes, selecting the at least one mutant exhibiting the desired characteristic may include progressively increasing the indicator of mutant survival in each repetition. That is, for example, where in the first cycle the survival threshold was set at 5% survival as the selection criteria, the second cycle (repeat of the processes) may increase the survival threshold to 10% or 15% for selection, and each cycle thereafter ,the survival threshold may be further increased either a same incremental amount or an increasing incremental amount.

[000174] When the method includes repeating the cycle of processes, exposing the plurality of mutants to a test environment may include changing the test environment each time the method is repeated. The test condition may become increasingly challenging in each successive repeat of the cycle. For example, when the desired characteristic includes enhanced stability to heat, repeating the cycle of processes may include incubating the plurality of mutant bacteriophages (e.g., bacteriophages having an engineered nucleotide sequence) in a test environment comprising incubating at progressively higher temperatures. In some variations, the temperature of the test environment may be increased in 5°C increments. The temperature may be set in the initial cycle at a temperature at which the host bacteria grows optimally, for example 25 degrees C or up to about 38 degrees C. As the number of cycles increase, the temperature of the test condition may be increased to about 65 degrees C or about 68 degrees C.

[000175] In another example, the desired characteristic may include enhanced stability at a pH lower than neutral pH, and in the repeating cycles, incubating the plurality of mutant

bacteriophages in a test environment may be performed by incubating at progressively lower pH. For example, an initial test environment may have a pH of 5. In succeeding cycles, the pH of the test environment may be decreased in about 0.5 pH, 04 pH, 0.3 pH, 0.2 pH, or about 0.1 pH unit increments. The cycles maybe repeated until a pH of the test environment is decreased to a pH of 3, thereby permitting the selection of a mutant (e.g., a bacteriophage having an engineered nucleotide sequence) having the at least survival threshold indicator at pH 3. In yet another example, the desired characteristic may include enhanced stability at a pH higher than neutral pH, and in repeating cycles, incubating the plurality of mutant bacteriophages in a test environment may include incubating at progressively higher pH. An initial test condition may set an initial pH of about 9, and the pH of the test environment may be set about 0.1, about 0.2, about 0.3, about 0.4, or about 0.5 pH units higher in each succeeding repeated cycle. In some embodiments, the pH of the test environment may be increased to a pH of 12, permitting selection of a mutant having the at least survival threshold indicator at pH 12.

[000176] In yet other variations, the desired characteristic may be an enhanced stability relative to a stability of the wild-type bacteriophage to at least one of blood, bile acids, internal bodily fluids, or external bodily fluids, and wherein the test environment comprises incubating the plurality of mutant bacteriophages in at least one of blood, bile acids, internal bodily fluids, or external bodily fluids in progressively longer periods of time. In some variations, the test period may include incubating for a period of 30 min. In some variations, as the cycle is repeated, a time period of incubation of the test environment may be increased by at least 30 minutes to at least 24 hours, and may permit selection of a mutant (e.g., a bacteriophage having an engineered nucleotide sequence) having the at least survival threshold indicator of a stability of 24 hours to at least one of blood, bile acids, internal bodily fluids, or external bodily fluids. In some variations, the time period of the test environment may not be longer than 72 hours.

[000177] In some other variations, the desired characteristic may include an enhanced host range, and exposing the plurality of mutants (e.g., a bacteriophage having an engineered nucleotide sequence) to a test environment may include incubating the plurality of mutant bacteriophages in a mixture of first bacterial cells and second bacterial cells, wherein the first bacterial cells are bacterial cells for which the first bacteriophage is infective (typical host bacterial cells) and wherein the second bacterial cells comprise bacterial cells for which the wild- type bacteriophage is not infective, or is minimally infective. In some variations, the test environment may include equal amounts (PFU/PFU) of the first bacterial cells and the second bacterial cells. In some variations, the test environment may include progressively increasing greater percentages of the second bacterial cells relative to the first bacterial cells, thereby selecting a mutant having the at least survival threshold indicator for infectivity towards the second bacterial cells.

[000178] In some other variations, the desired characteristic may include an increase in the efficiency of lytic activity, and testing the plurality of mutants may include performing an assay identifying mutants (e.g., a bacteriophage having an engineered nucleotide sequence) configured to lyse bacterial cells at an earlier time point than a lysing timepoint of the wild-type

bacteriophage. The mutant phages that are capable of lysing bacterial cells early, below the calculate latent period, may be identified based on one-step assays relative to that of the wild- type bacteriophage.

[000179] Identifying the location of the mutation. The method may include identifying a location of at least one mutated base in the at least one mutated gene or sequence of the engineered nucleotide sequence of the mutant bacteriophage. In some variations, identifying may include identifying locations of a plurality of mutated bases in the engineered nucleotide of the mutant bacteriophage. Identifying may include identifying a DNA sequence. Identification may be performed by PCR, for example using hybridization probe techniques such as Taqman, Scorpion or other FRET based assays and further may be quantitated using qPCR. Identifying a DNA sequence may include sequencing via Sanger or any massively parallel sequencing technique. The sequencing may be targeted sequencing focused at a selected region of the gene or nucleotide sequence of the bacteriophage or may be whole genome sequencing performed by massively parallel sequencing, e.g., NGS. Identifying may further include identifying a frequency of at least one mutated base in the at least one mutated gene or sequence of the engineered nucleotide sequence across the at least one selected mutant bacteriophage exhibiting the desired characteristic. Identifying the frequency of the at least one mutated base may further include identifying a frequency of the at least one mutated base above a pre-selected threshold. The pre-selected threshold may be above about 1%; about 5%; about 10%; about 15%; about 20%; about 25%; about 30%; about 50%; about 75%; about 80%; about 90%; or more. The method may further include identifying a frequency of each of the plurality of mutated bases in the at least one mutated gene or sequence of the engineered nucleotide sequence of the mutant bacteriophage. The method may further include predicting the at least one mutated base conferring the desired characteristic.

EXAMPLES

[000180] Experiment 1. Directed evolution of T3 bacteriophage to enhance thermal stability. T3 bacteriophage (Obtained from D'Herelle phage center, Laval University), having a nucleic acid sequence as shown in Appendix A (NC 003298.1), were subjected to the method of directed evolution as shown in FIG. 1, in this example, to drive to increased thermal stability.

The wild-type T3 population was treated with Ethyl methanesulfonate (EMS) sourced from Sigma Aldrich. Before incubation, EMS was diluted in Dulbecco's phosphate-buffered saline to generate a 180mM EMS solution. 100 uL of phage stock (10⁹ PFU/ml) was mixed with 50 mL EMS solution. The phages were then incubated with the mutagen at 37C for 1 hour, with rapid shaking. 20 mL of mutagenized phage was then mixed into another microplate well with a 180 mL solution of Escherichia coli BL21 host bacteria (0.5 OD600), then allowed to reach complete lysis. 100 uL lysate was then removed, and heated in a thermocycler at a temperature of pronounced denaturation. 100 uL of heat- selected phages were then added to another well with 100 uL fresh bacteria host (0.5 OD600), and lysis was allowed to complete. This process of mutagenesis and selection was repeated up to 30 times, with a gradual increase of the selection temperature from 50 degrees C to 68 degrees C. Aliquots of the surviving phages were retained, particularly after cycle 10, cycle 20 and the final 30^th cycle.

[000181] The thermal stability of the evolved phages was tested by quantifying the number of infective page particles with Escherichia coli BL21 after incubation at ambient temperature (25 degrees C) and at a high temperature (60 degrees C) for 1 hour, for each of wild-type, generation 10 phage (G10); generation 20 phage (G20) and generation 30 phage (G30). Inoculation was at 10⁹ PFU (100%). FIG. 2 shows images of the successive dilutions of the surviving phage stock 10-1, 10-2, 10-3, 10-4, and 10-5, in two horizontal rows for each bacteriophage. In the top panel, wild-type bacteriophage demonstrated a 100% survival when incubated at 25 degrees C, but only 6.6% survival after incubation at 60 degrees C. The second panel shows G10 phage, which again showed a 100% survival after incubation at 25 degrees, and an improved survival of 36.0% after incubation at 60 degrees C. G20 phages demonstrated further improvement in survival after incubation at 60%, with 59.1% survival (and 100% survival at 25 degrees C). G30 phages improved further, with 69.9% survival after incubation at 60 degrees (100% survival at 25 degrees incubation). This showed that application of the directed evolution cycle was capable of significantly improving the heat stability of G30 phages. The improvement in heat stability appears to have been introduced rapidly during initial rounds of directed evolution, while further mutations conferring further improvement occurred less frequently.

[000182] To better understand the thermal stability of the evolved phages, time-dependent survival assays were performed for both wild-type and G30 phages. After incubation at three elevated temperatures of 60 degrees C; 62 degrees C; and 64 degrees C, phages were introduced to Escherichia coli BL21 and survivability evaluated. As shown in FIG. 3 A, wild-type phages demonstrated highly impaired survivability within even short periods of time: at 62 or 64 degrees C, only a few percent of the original inoculated population were still viable even after 10 min.

No detectable amounts of wild-type phages survived for 50 minutes at any of the elevated temperatures. In contrast, the G30 phage population showed significantly improved survival at 60 degrees C, with about 10% surviving the 1 hour incubation. Survival at 62 or 64 degrees C was significantly affected, but the G30 phages were still viable at shorter periods of incubation (10 or 20 min), providing at least a usable population. FIG. 3B shows the survival rate of each group (wild-type and G30, here labelled as“Mutant”) at room temperature (25 degrees C) in Dulbecco’s phosphate-buffered saline for 38 days, using a 10⁸ PFU/ml starting concentration of phages. The ability to form plaques was determined at the time points shown (survivability assay). The half-life for each of the wild-type and G30 phage population were calculated from these experiments and are shown in FIG. 3B. As expected, the G30 phage population, produced by directed evolution, has significantly increased half-lives at 25 degrees C and at 60, 62, and 64 degrees C. These results appear to indicate that the directed evolution process produced the G30 population having significantly modulated kinetics of denaturation relative to the wild type phage population.

[000183] Interestingly, the phage evolution process, while directed to improving thermal stability, also provided enhanced stability in other types of physical behavior, likely due to structural stabilization_· Survival assays were performed using Escherichia coli BL21 as host bacterial species, incubating at 25 degrees C, for both wild-type and G30 populations, testing pH stability at pH 7.0; pH 3.7; and pH 3.5 for a 1 hour period. The two acidic pHs were selected to probe the extent of increased stabilization around pH levels where degradation of phages are known to accelerate significantly. As shown in FIG. 4, the two graphs show the stability at pH 7 and a test pH of either 3.7 or 3.5 for each of the wild-type (WT) and G30 phage populations. While both populations were stable at pH 7.0, the G30 population showed significantly greater percentages of phage survival, 67.8% at pH 3.7 and 30.4% at pH 3.5 compared to survival of wild-type at 49.6% (pH 3.7) and only 10% (pH 3.5). Therefore, the structural changes induced by the directed evolution process to enhance thermal stability, also enhanced tolerance to acidic conditions. These paired effects may permit development of phages having better oral dosing profiles (AUC, and the like) for orally administered therapeutics.

[000184] Additional investigation was made to determine whether other unrelated traits had been modified by the directed evolution process for thermal stability. Wild-type, G10, G20 and G30 bacteriophage populations were assayed for lytic ability by inoculation into host strain bacteria, Rosetta cells, at multiplicity of infection (MOI) of 10 ¹ (phage: host cell) and 10⁵ (phage: host cell). As shown in FIG. 5, the resultant curves for each of the wild-type and respective mutated phage generation populations are coincident, and shows an expected increase of time to the inflection point as the concentration of phages were decreased from the left hand panel (MOI 10 ¹) to right hand panel (MOI 10⁵), which is also the same for wild-type and mutated populations. Therefore, the infection dynamics are not affected by the structural changes wrought by the directed evolution of this experiment. Further, the host range of the mutated populations were the same as that of the wild-type phage parent (data not shown).

[000185] Mutation analysis. Pools of T3 mutated phages from each of the G10, G20 and G30 were lysed and prepared for sequencing, generally using massively parallel sequencing approaches. Detection of variants and the frequency of mutations relative to the wild-type was analyzed. FIG. 6 shows the pattern of mutation location for the most prevalent mutations seen in G10, G20 and G30, and was similar between all three generations. The engineered bacteriophage shares greater than 99% identity with the wild-type. The majority of the mutations were found within the region of the nucleotide sequence of the genome for structural genes, including the head-to-tail joining protein (T3p37); tail-tubular proteins (T3p42, 43) and internal virion proteins (T3p44-47). These genes either encode protein sub-units of the tail complex or are involved in the mechanical activities of structure- assembly and DNA injection. A limited number of mutations were observed across the phage genome during early rounds of mutagenesis and these mutations became enriched during the later rounds of mutagenesis. Some of these mutations reached saturation levels as the rounds of mutagenesis proceeded, as shown in FIG. 7A. One such early mutation which reached saturation by G10 is found at base location 33152, modifying the wild-type base from T to C, which is the T3 tail fiber protein (T3p48), which was propagated throughout in greater than 80% of the mutated phages by G10 and 100% of the mutated phages by G20. Another early mutation is at 19559 (19660) within theT3 head to tail joining protein (e.g., T3p37) mutating from A to T, also represented in nearly 80% of the G10 phages, and in 100% of the G20 phages. Importantly, the rate of mutation enrichment correlated with the rate of enhancement of thermal stability seen in the temperature stability assays. As shown in FIG. 7B, survival fraction vs. the normalized mutation frequency (R2 = 0.968), where the normalized mutation frequency is calculated as the sum of all mutation frequencies across a genome at a given round of mutagenesis/test environment, divided by the total net mutation frequency achieved at the final round of evolution, in this case, 30 rounds. The linear correlation shown indicates that the thermal tolerance induced is a function of mutation enrichment.

[000186] Analysis of the type of substitution made in the mutants was also analyzed. As shown in FIG. 8A, the residue volume of the mutated amino acids (which included the G30 mutations seen in this experiment and also including the respective G30 mutation data from Experiment 2, describing below) were compared against the residue volume of the original amino acids of the wild-type phage (T3 for Experiment 1, and the closely related T7 for Experiment 2). What is seen is that when amino acids having small side chain substituents, (e.g., glycine and the like) are replaced in the mutant, the resulting mutated amino acid has a much higher proportion of large residue volume side chains such as phenylalanine, valine, leucine and the like, increasing from about 20% to over 40%. In FIG. 8B, the degree of hydrophobicity in the amino acids that are mutated, of the wild type bacteriophage (T3 and T7), is compared to the hydrophobicity of amino acids replacing them in the G30 mutated phages(from both Experiment 1 and Experiment 2). In the wild-type phages, about 30% of the amino acids that become replaced in the G30 populations were hydrophilic amino acids, (such as serine or aspartic acid) about 60% were amphipathic, and a little over 10% were hydrophobic. In the resulting G30 populations, mutagenized amino acids now comprise about 50% hydrophobic amino acids such as phenylalanine, leucine, and the like, while the proportion of hydrophilic amino acids declines to less than 10% of the mutagenized amino acids, with the remainder being amphipathic amino acids, such as tyrosine, tryptophan, and methionine. The shift in amino acid types point to a more tightly held molecular structure for the G30 mutants as well as increased hydrophobic contact points for initial association with target bacteria perhaps assisting with initial entry.

[000187] Confirmatory experiments. Despite the random nature of the mutagenizing chemical agent (which is also true for radiation induced mutagenesis and use of poor fidelity amplification), applying the experimental steps employed above in a second attempt to enhance thermal stability in T3 bacteriophage led to similar patterns of mutagenesis, in the same specific genes, again, mostly structural proteins. Across both sets of mutagenesis protocols, two of the most mutagenized regions were the T3p37 and T3p45 genes. The high mutation rate at the T3p37 gene (coding for the head-to-tail joining protein, which bridges the capsid and tail of the bacteriophage) further suggested that modification of this structure plays a critical role in conferring the improvement in structural stability observed over the course of directed evolution. The T3p45 gene codes for an internal virion protein, which assembles at the capsid interior, and participates in the DNA-injection process. These results show that the primary sites of enriched mutations under a thermal selection criteria were genes involved in structural integrity, and that the mutations observed enhance the ability of these proteins to perform this function.

[000188] To further validate the role of these observed mutations, each of five high frequency mutations was introduced singly into T3 phage variants, in order to assess the influence each mutation has upon survivability under high temperature conditions. Three of the variants were located in the structural genes T3p37, T3p45 and Tp348, while two were at non-coding sites (upstream of 44, 38). This was performed, as shown schematically in FIG. 9A, by identification of the mutated sequence (910) that arose from wild-type 905 and gene assembly (920, 930)>

This was followed by phage rebooting by introduction to a suitable bacterial host species (940); reproduction (950); lytic release (960) to produce the single mutant 915. (See Kilcher et al.

PNAS, vol. 115, no. 3, 567-572, 2018, incorporated herein by reference in its entirety.) Single variants Mut37 (incorporating the T3p37 mutation), Mut38 (non-coding), Mut44 (non coding), Mut45, and Mut48 (incorporating the T3p48 mutation) were produced by this method. FIG. 9B shows the results of survival assays performed as described above for each of the single mutants. The five mutant phages, wild-type phage and the G30 phage population which contains all three of the mutations in T3p37, T3p45 and T3p48 were evaluated, both at 25 degrees C and 60 degrees C, demonstrating that the two mutants Mut37 and Mut45 do contribute to the thermal stability seen in G30 phage population.

[000189] Experiment 2. Directed evolution of T7 bacteriophage to enhance thermal stability. The highly related T7 bacteriophage was subjected to the same mutagenesis protocol as described above in Experiment 1, to enhance thermal stability. The protocol was continued for 30 rounds as in Experiment 1. It was observed that the mutagenesis protocol enhanced T7 phage stability. As shown in FIG. 10B, 46.5% of the T7 G15 phage population survived a survival assay performed as above at 60 degrees C, while only 3.2% of the wild-type survived the 1 hour 60 degree incubation. Additionally, in a similar manner to T3, a series of mutations enriched across the phage genome occur predominantly in structural genes, as shown in FIG. 10A.

[000190] Mutations within several common genes were found to be enriched in both T3 and T7 phages over the course of directed evolution (compare FIG. 6 with FIG. 10A). The region coding for T7’s head-to-tail joining protein (T7p42 gene) was again a significant site of mutagenesis. Several other genes were prevalent sites of mutagenesis in both phages, as shown in FIG. 11, including structural genes such as those encoding capsid assembly proteins, and internal virion proteins.

[000191] The improvements seen in both species indicate that this approach is an effective way to induce phenotypic changes in different species of phages. Additionally, the convergent effects of mutagenesis seen in the genes shared by the T3 and T7 phages suggest that, despite the random nature of this protocol’s mutagenesis step, the steps involved in applying a given selection criteria guide the genomic changes within the experimental population towards semi- directional patterns of regional mutagenesis within structural genes. Analysis of the crystal structures of highly mutated proteins (data not shown) indicates that many mutations occur at the interfacial regions between subunits of the assembled phage. Because these three proteins are nearly identical in both T3 and T7, mutations sites (represented at magenta residues) could be mapped from both species to the structures (and corresponding gene-IDs): head-to-tail joining protein (T3p37, T7p42), tail-tubular protein A (T3p42, T7p46), and tail-tubular protein B (T3p43, T7p47).

[000192] Wild-type T3 and T7 bacteriophage sequence listings

[000193] As compared to wild-type T3 bacteriophage sequence listing FIG. 11 illustrates some of the functional mutations (point mutations) identified as described herein.

[000194] The genomic sequence for T3 bacteriophage is known in the art, and may be found, e.g., at: https://www.ncbi.nlm.nih.gov/nuccore/NC_003298. l?report=fasta.

[000195] The genomic sequence for T7 bacteriophage is known in the art, and may be found, e.g., at: https://www.ncbi.nlm.nih.gov/nuccore/NC_003298. l?report=fasta.

[000196] As mentioned above, other bacteriophage having similar corresponding regions (e.g., 60% or greater identity, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, etc.) in the identified modification region may also be modified as shown.

[000197] Specific and frequent mutations in T3 or T7 bacteriophages identified through the processes described in Experiments 1 and 2, which confer enhanced stability to the resultant bacteriophage, as follows:

[000198] A. Mutation in the T3 head to tail joining protein (e.g., T3p37)

SEQ ID NO: 1

TYPE: DNA

// WT DNA sequence: T3 head to tail joining protein (e.g., T3p37) ATGGCTGATTCAAAACGTACAGGATTGGGCGAAGACGGTGCTAAAGCTACCTATGA

CCGCCTAACAAACGACCGTAGAGCCTATGAGACTCGTGCGGAGAACTGTGCGCAAT

ACACCATTCCGTCCTTGTTCCCGAAGGAGTCCGATAACGAATCTACCGACTACACGA

CTCCGTGGCAGGCTGTAGGTGCGCGGGGTCTCAACAATCTAGCCTCTAAGTTAATGC

TTGCGTTATTCCCGATGCAGTCGTGGATGAAGCTGACCATTAGCGAATATGAGGCGA

AGCAGCTTGTTGGAGACCCTGATGGACTCGCTAAGGTGGACGAAGGTCTGTCAATG

GTTGAGCGCATAATCATGAACTATATCGAATCCAACAGTTACCGCGTAACACTCTTT

GAGTGCCTCAAGCAGTTGATCGTGGCTGGTAACGCCCTGCTTTACTTACCGGAACCA

GAAGGTAGCTACAATCCGATGAAGCTGTACCGATTGTCTTCTTATGTTGTCCAAAGA

GACGCATACGGCAATGTGTTACAGATTGTCACTCGTGACCAGATAGCCTTTGGTGCT

CTCCCGGAAGACGTTAGGTCTGCGGTAGAGAAATCTGGTGGTGAGAAGAAGATGGA

CGAAATGGTCGATGTGTACACCCATGTGTATCTCGATGAAGAGTCCGGCGATTACCT

CAAGTACGAGGAAGTAGAGGACGTTGAGATTGATGGTTCCGATGCCACCTATCCGA

CTGACGCGATGCCCTACATTCCGGTTCGCATGGTTCGCATTGATGGCGAGTCTTACG

GTCGCTCCTACTGTGAAGAATACTTAGGTGACTTAAGGTCGCTTGAGAATCTCCAAG

AGGCTATCGTTAAGATGAGTATGATTAGCGCGAAGGTCATTGGTCTGGTGAACCCG

GCTGGCATTACGCAGCCCCGTAGATTAACCAAAGCTCAGACTGGTGACTTCGTTCCA

GGCCGTCGAGAAGATATTGACTTCCTGCAACTGGAGAAGCAAGCTGACTTTACCGT

AGCGAAAGCTGTGAGTGACCAGATAGAAGCACGCTTATCGTATGCCTTTATGTTGAA

CTCTGCGGTACAGCGCACAGGCGAACGTGTGACCGCCGAAGAGATTCGATACGTTG

CGTCAGAACTGGAAGATACGCTTGGTGGCGTCTACTCGATTCTGTCTCAAGAATTGC

AATTGCCTCTGGTACGTGTGCTCTTGAAGCAACTCCAAGCAACCTCGCAGATTCCTG

AGCTACCGAAAGAAGCCGTTGAGCCTACTATCAGTACAGGTCTGGAAGCAATTGGT

CGTGGTCAAGACCTCGATAAGCTGGAGCGTTGCATCTCAGCGTGGGCGGCTCTTGCC

CCTATGCAGGGAGACCCGGACATTAATCTTGCTGTCATTAAGCTACGCATTGCTAAC

GCTATAGGTATTGATACTTCTGGTATCCTACTGACGGATGAACAGAAGCAAGCCCTT

ATGATGCAGGATGCGGCACAAACAGGCGTCGAGAATGCTGCGGCTGCTGGTGGTGC

TGGTGTTGGTGCTTTGGCTACCTCAAGTCCAGAAGCCATGCAAGGTGCTGCTGCACA

GGCTGGCCTCAACGCCACCTAA

SEQ ID NO: 2

TYPE: DNA

//WT AA sequence: T3 head to tail joining protein (e.g., T3p37) MADSKRTGLGEDGAKATYDRLTNDRRAYETRAENCAQYTIPSLFPKESDNESTDYTTP

WQAVGARGLNNLASKLMLALFPMQSWMKLTISEYEAKQLVGDPDGLAKVDEGLSMV

ERIIMN YIES N S YRVTLFECLKQLIV AGN ALLYLPEPEGS YNPMKL YRLS S YV V QRD A Y G

NVLQIVTRDQIAFGALPEDVRSAVEKSGGEKKMDEMVDVYTHVYLDEESGDYLKYEEV

EDVEIDGSDATYPTDAMPYIPVRMVRIDGESYGRSYCEEYLGDLRSLENLQEAIVKMSM

ISAKVIGLVNPAGITQPRRLTKAQTGDFVPGRREDIDFLQLEKQADFTVAKAVSDQIEAR

LS Y AFMLN S A V QRTGER VT AEEIRY V AS ELEDTLGG V Y S ILS QELQLPLVR VLLKQLQ A

TSQIPELPKEAVEPTISTGLEAIGRGQDLDKLERCISAWAALAPMQGDPDINLAVIKLRIA

NAIGIDTSGILLTDEQKQALMMQDAAQTGVENAAAAGGAGVGALATSSPEAMQGAAA

QAGLNAT

[000199] The nucleotide in position 19660 may be modified from A to a T. This region may be recognized by looking for the sequence of the regions before and after the modification (e.g., CAGAAGCC before and TGCAAGGT after, etc.). 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs before and/or 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs after may be identified as 100% identical, 95% or more identical, 90% or more or more identical, 85% or more identical, etc. For example:

SEQ ID NO: 3

Type: DNA

Organism: Artificial sequence: stabilizing mutation in T3 head to tail joining protein (e.g., T3p37)

CAGAAGCCTTGCAAGGT

SEQ ID NO: 4

Type: DNA

// Mut DNA sequence: stabilizing mutation in T3 head to tail joining protein (e.g., T3p37)

ATGGCTGATTCAAAACGTACAGGATTGGGCGAAGACGGTGCTAAAGCTACCTATGA

CCGCCTAACAAACGACCGTAGAGCCTATGAGACTCGTGCGGAGAACTGTGCGCAAT

ACACCATTCCGTCCTTGTTCCCGAAGGAGTCCGATAACGAATCTACCGACTACACGA CTCCGTGGCAGGCTGTAGGTGCGCGGGGTCTCAACAATCTAGCCTCTAAGTTAATGC

TTGCGTTATTCCCGATGCAGTCGTGGATGAAGCTGACCATTAGCGAATATGAGGCGA

AGCAGCTTGTTGGAGACCCTGATGGACTCGCTAAGGTGGACGAAGGTCTGTCAATG

GTTGAGCGCATAATCATGAACTATATCGAATCCAACAGTTACCGCGTAACACTCTTT

GAGTGCCTCAAGCAGTTGATCGTGGCTGGTAACGCCCTGCTTTACTTACCGGAACCA

GAAGGTAGCTACAATCCGATGAAGCTGTACCGATTGTCTTCTTATGTTGTCCAAAGA

GACGCATACGGCAATGTGTTACAGATTGTCACTCGTGACCAGATAGCCTTTGGTGCT

CTCCCGGAAGACGTTAGGTCTGCGGTAGAGAAATCTGGTGGTGAGAAGAAGATGGA

CGAAATGGTCGATGTGTACACCCATGTGTATCTCGATGAAGAGTCCGGCGATTACCT

CAAGTACGAGGAAGTAGAGGACGTTGAGATTGATGGTTCCGATGCCACCTATCCGA

CTGACGCGATGCCCTACATTCCGGTTCGCATGGTTCGCATTGATGGCGAGTCTTACG

GTCGCTCCTACTGTGAAGAATACTTAGGTGACTTAAGGTCGCTTGAGAATCTCCAAG

AGGCTATCGTTAAGATGAGTATGATTAGCGCGAAGGTCATTGGTCTGGTGAACCCG

GCTGGCATTACGCAGCCCCGTAGATTAACCAAAGCTCAGACTGGTGACTTCGTTCCA

GGCCGTCGAGAAGATATTGACTTCCTGCAACTGGAGAAGCAAGCTGACTTTACCGT

AGCGAAAGCTGTGAGTGACCAGATAGAAGCACGCTTATCGTATGCCTTTATGTTGAA

CTCTGCGGTACAGCGCACAGGCGAACGTGTGACCGCCGAAGAGATTCGATACGTTG

CGTCAGAACTGGAAGATACGCTTGGTGGCGTCTACTCGATTCTGTCTCAAGAATTGC

AATTGCCTCTGGTACGTGTGCTCTTGAAGCAACTCCAAGCAACCTCGCAGATTCCTG

AGCTACCGAAAGAAGCCGTTGAGCCTACTATCAGTACAGGTCTGGAAGCAATTGGT

CGTGGTCAAGACCTCGATAAGCTGGAGCGTTGCATCTCAGCGTGGGCGGCTCTTGCC

CCTATGCAGGGAGACCCGGACATTAATCTTGCTGTCATTAAGCTACGCATTGCTAAC

GCTATAGGTATTGATACTTCTGGTATCCTACTGACGGATGAACAGAAGCAAGCCCTT

ATGATGCAGGATGCGGCACAAACAGGCGTCGAGAATGCTGCGGCTGCTGGTGGTGC

TGGTGTTGGTGCTTTGGCTACCTCAAGTCCAGAAGCCTTGCAAGGTGCTGCTGCACA

GGCTGGCCTCAACGCCACCTAA

SEQ ID NO: 5

Type: Protein

SPEALQGAA SEQ ID NO: 6:

Type: PROTEIN

// Mut A A Sequence:

MADSKRTGLGEDGAKATYDRLTNDRRAYETRAENCAQYTIPSLFPKESDNESTDYTTP

WQAVGARGLNNLASKLMLALFPMQSWMKLTISEYEAKQLVGDPDGLAKVDEGLSMV

ERIIMN YIES N S YRVTLFECLKQLIV AGN ALLYLPEPEGS YNPMKL YRLS S YV V QRD A Y G

NVLQIVTRDQIAFGALPEDVRSAVEKSGGEKKMDEMVDVYTHVYLDEESGDYLKYEEV

EDVEIDGSDATYPTDAMPYIPVRMVRIDGESYGRSYCEEYLGDLRSLENLQEAIVKMSM

ISAKVIGLVNPAGITQPRRLTKAQTGDFVPGRREDIDFLQLEKQADFTVAKAVSDQIEAR

LS Y AFMLN S A V QRTGER VT AEEIRY V AS ELEDTLGG V Y S ILS QELQLPLVR VLLKQLQ A

TSQIPELPKEAVEPTISTGLEAIGRGQDLDKLERCISAWAALAPMQGDPDINLAVIKLRIA

NAIGIDTSGILLTDEQKQALMMQDAAQTGVENAAAAGGAGVGALATSSPEALQGAAA

QAGLNAT

[000200] B. Mutation in the T3 internal core protein/internal virion protein B (T3p45)

SEQ ID NO: 7

// WT DNA sequence: T3 internal core protein/internal virion protein B (T3p45)

ATGTGCTGGATGGCAGCGATTCCTATTGCTATGGCGGGTGCCCAAGCTCTAAGTAGC

CAGAACAGTGCTGACAAGGCGCGCGTGGCACAGACAGAAGCTGGACGCCGACAGG

CAATTGAGATGGTAAAAGAGATGAATATCCAAAATGCCAACGCCTCATTGGAACAA

CGGGACGCCCTTGAAGCTGCATCCTCTGAGTTGACTTCACGTAACATGCAGAAGGTA

CAGGCTATGGGAACCATCCGTGCAGCGATTGGTGAGGGTATGCTCGAAGGTGAATC

CATGAAGCGCATCAAGCGTATCGAAGAAGGCAACTACATTCGGGAGGCAAATAGTG

TCACCGAGAATTACCGCCGAGACTACGCGAGTATCTTTGCGCAACAGTTGGGACGC

ACTCAGTCCACAGCAAGTCAAGTCGATGCAATGTACAAGAGCGAGGCCAAAGGTAA

GTCTGGTCTGATGCGTGTACTAGACCCTCTGTCCATTATGGGTCAGGAAGCTGCAAG TCAATATGCGGCTGGTGGATTTGACAAGAAAGGTGGCAACCAAGCAGCACCTATCA

GTGCCGCCAAAGGAACTAAGACCGGGAGGTAA

SEQ ID NO: 8

// WT AA sequence: T3 internal core protein/internal virion protein B (T3p45)

MC WM A AIPIAM AG AQ ALS S QN S ADKAR V AQTE AGRRQ AIEM VKEMNIQN AN AS LEQR DALEAASSELTSRNMQKVQAMGTIRAAIGEGMLEGESMKRIKRIEEGNYIREANSVTEN YRRD Y AS IFAQQLGRTQS T AS Q VD AM YKS E AKGKS GLMRVLDPLS IMGQE A AS Q Y A A GGFDKKGGN QAAPIS AAKGTKTGR

[000201] The nucleotide in position 26962 may be modified from C to T. This region may be recognized by looking for the sequence of the regions before and after the modification (e.g., TCCTATTG before and TATGGCG after, etc.). 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs before and/or 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs after may be identified as 100% identical, 95% or more identical, 90% or more or more identical, 85% or more identical, etc. For example:

SEQ ID NO: 9

Type: DNA

Organism: Artificial sequence derived from T3 internal core protein/intemal virion protein B (T3p45)

TCCTATTGTTATGGCGG

SEQ ID NO: 10

Type: DNA

//Mut DNA Sequence derived from T3 internal core protein/internal virion protein B (T3p45):

ATGTGCTGGATGGCAGCGATTCCTATTGTTATGGCGGGTGCCCAAGCTCTAAGTAGC

CAGAACAGTGCTGACAAGGCGCGCGTGGCACAGACAGAAGCTGGACGCCGACAGG

CAATTGAGATGGTAAAAGAGATGAATATCCAAAATGCCAACGCCTCATTGGAACAA

CGGGACGCCCTTGAAGCTGCATCCTCTGAGTTGACTTCACGTAACATGCAGAAGGTA CAGGCTATGGGAACCATCCGTGCAGCGATTGGTGAGGGTATGCTCGAAGGTGAATC

CATGAAGCGCATCAAGCGTATCGAAGAAGGCAACTACATTCGGGAGGCAAATAGTG

TCACCGAGAATTACCGCCGAGACTACGCGAGTATCTTTGCGCAACAGTTGGGACGC

ACTCAGTCCACAGCAAGTCAAGTCGATGCAATGTACAAGAGCGAGGCCAAAGGTAA

GTCTGGTCTGATGCGTGTACTAGACCCTCTGTCCATTATGGGTCAGGAAGCTGCAAG

TCAATATGCGGCTGGTGGATTTGACAAGAAAGGTGGCAACCAAGCAGCACCTATCA

GTGCCGCCAAAGGAACTAAGACCGGGAGGTAA

SEQ ID NO: 11

Type: PROTEIN

Organism: Artificial sequence derived from T3 internal core protein/intemal virion protein B (T3p45):

AIPIVMAGA

SEQ ID NO: 12

Type: PROTEIN

// Mut AA sequence derived from T3 internal core protein/internal virion protein B (T3p45):

MC WM A AIPIVM AG AQ ALS S QN S ADKAR V AQTE AGRRQ AIEM VKEMNIQN AN AS LEQR DALEAASSELTSRNMQKVQAMGTIRAAIGEGMLEGESMKRIKRIEEGNYIREANSVTEN YRRD Y AS IFAQQLGRTQS T AS Q VD AM YKS E AKGKS GLMRVLDPLS IMGQE A AS Q Y A A GGFDKKGGN QAAPIS AAKGTKTGR

[000202] C. Mutations in the T3 tail fiber protein (e.g., T3p48)

SEQ ID NO: 13

WT DNA sequence: T3 tail fiber protein (e.g., T3p48)

ATGGCTAACGTAATTAAAACCGTTTTGACTTACCAGTTAGATGGCTCCAATCGTGAT

TTTAATATCCCGTTTGAGTATCTAGCCCGTAAGTTCGTAGTAGTAACCCTTATTGGCG

TAGACCGCAAGGTCCTTACGATTAATGCAGACTACCGTTTTGCTACGCGTACTACCA TCTCACTTACCAAGGCTTGGGGTCCAGCGGATGGATACACTACCATCGAGTTACGCC

GAGTAACCTCCACAACCGACCGATTGGTTGACTTTACGGATGGTTCAATCCTCCGTG

CGTATGACCTTAACGTCGCTCAGATTCAAACGATTCACGTAGCGGAAGAGGCCCGT

GACCTCACTACTGATACCATAGGTGTCAATAATGATGGTCATTTGGATGCTCGTGGT

CGTCGAATTGTTAACCTAGCGAACGCTGTGGATGACCGCGACGCTGTTCCGTTTGGT

CAACTTAAGACCATGAACCAGAACTCGTGGCAGGCGCGTAATGAGGCACTACAGTT

CCGTAATGAGGCTGAGACTTTCAGAAATCAAACGGAGGTTTTTAAGAATGAGTCCG

GTACTAACGCTACGAACACAAAGCAGTGGCGAGATGAGGCTAATGGGTCCCGAGAT

GAAGCCGAGCAGTTCAAGAATACGGCTGGTCAATACGCTACATCTGCTGGGAACTC

TGCTACTGCTGCGCATCAATCTGAGGTAAACGCTGAGAACTCCGCTACAGCAGCAG

CGAACTCTGCGAATTTGGCAGAACAACACGCAGACCGTGCGGAACGTGAAGCAGAC

AAGCTGGGGAATTTTAATGGACTGGCTGGTGCAATTGACAGGGTGGATGGAACCAA

TGTGTACTGGAAAGGAGGTATCCATGCGAACGGACGCCTTTACCTTACCTCAGATGG

TTTCGACTGTGGTCAGTATCAACAGTTCTTTGGTGGTTCTGCTGGTCGTTACTCTGTC

ATGGAGTGGGGTGATGAGAACGGATGGCTGATGCATGTTCAACGTAGAGAGTGGAC

AACAGCGATAGGTGATAACATCCAGCTAGTAGTAAACGGACATATCATCGCCCAAG

GTGGAGACATGACTGGTCCGCTGAAATTGCAGAATGGACATGCCCTTTACTTAGAGT

CCGCATCCGACAAGGCGCAATATATTCTATCTAAAGATGGTAACAGAAACAACTGG

TACATTGGTAGAGGATCAGATAACAACAATGACTGTACCTTCCACTCCTATGTGTAT

GGTACGAACTTAACACTCAAGCCGGACTATGCAGTAGTTAACAAACGCTTCCACGT

AGGTCAGGCAGTTGTAGCCACTGATGGTAATATTCAAGGTACTAAGTGGGGAGGTA

AGTGGCTTGATGCTTACCTAAACGATACTTACGTTAAGAAGACAATGGCCTGGACTC

AAGTATGGGCTGCTGCTAGTGGTAGTCACATGGGAGGAGGTTCTCAGACTGATACTC

TCCCACAGGACTTGCGATTCCGCAACATATGGATTAAGACCAGAAACAACTATTGG

AACTTCTTCCGAACTGGTCCTGACGGTATCTACTTCCTTTCAGCCGAGGGCGGTTGG

CTAAAATTCCAGATACACTCTAATGGCAGGGTATTTAAGAACATAGCGGATAGAGA

TGCGCCTCCAACAGCAATAGCCGTAGAGGACGTGTAA

[000203] The nucleotide in position 33152 may be modified from T to G (or A or C). This region may be recognized by looking for the sequence of the regions before and after the modification (e.g., CAAACGAT before and CACGTAG after, etc.). 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs before and/or 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs after may be identified as 100% identical, 95% or more identical, 90% or more or more identical, 85% or more identical, etc. For example: SEQ ID NO: 14

Type: DNA

Organism: Artificial sequence derived from T3 tail fiber protein (e.g., T3p48)

C A A ACG AT GC AC GT AG

SEQ ID NO: 15

Type: DNA

//Mut DNA sequence derived from T3 tail fiber protein (e.g., T3p48)

ATGGCTAACGTAATTAAAACCGTTTTGACTTACCAGTTAGATGGCTCCAATCGTGAT

TTTAATATCCCGTTTGAGTATCTAGCCCGTAAGTTCGTAGTAGTAACCCTTATTGGCG

TAGACCGCAAGGTCCTTACGATTAATGCAGACTACCGTTTTGCTACGCGTACTACCA

TCTCACTTACCAAGGCTTGGGGTCCAGCGGATGGATACACTACCATCGAGTTACGCC

GAGTAACCTCCACAACCGACCGATTGGTTGACTTTACGGATGGTTCAATCCTCCGTG

CGTATGACCTTAACGTCGCTCAGATTCAAACGATGCACGTAGCGGAAGAGGCCCGT

GACCTCACTACTGATACCATAGGTGTCAATAATGATGGTCATTTGGATGCTCGTGGT

CGTCGAATTGTTAACCTAGCGAACGCTGTGGATGACCGCGACGCTGTTCCGTTTGGT

CAACTTAAGACCATGAACCAGAACTCGTGGCAGGCGCGTAATGAGGCACTACAGTT

CCGTAATGAGGCTGAGACTTTCAGAAATCAAACGGAGGTTTTTAAGAATGAGTCCG

GTACTAACGCTACGAACACAAAGCAGTGGCGAGATGAGGCTAATGGGTCCCGAGAT

GAAGCCGAGCAGTTCAAGAATACGGCTGGTCAATACGCTACATCTGCTGGGAACTC

TGCTACTGCTGCGCATCAATCTGAGGTAAACGCTGAGAACTCCGCTACAGCAGCAG

CGAACTCTGCGAATTTGGCAGAACAACACGCAGACCGTGCGGAACGTGAAGCAGAC

AAGCTGGGGAATTTTAATGGACTGGCTGGTGCAATTGACAGGGTGGATGGAACCAA

TGTGTACTGGAAAGGAGGTATCCATGCGAACGGACGCCTTTACCTTACCTCAGATGG

TTTCGACTGTGGTCAGTATCAACAGTTCTTTGGTGGTTCTGCTGGTCGTTACTCTGTC

ATGGAGTGGGGTGATGAGAACGGATGGCTGATGCATGTTCAACGTAGAGAGTGGAC

AACAGCGATAGGTGATAACATCCAGCTAGTAGTAAACGGACATATCATCGCCCAAG

GTGGAGACATGACTGGTCCGCTGAAATTGCAGAATGGACATGCCCTTTACTTAGAGT

CCGCATCCGACAAGGCGCAATATATTCTATCTAAAGATGGTAACAGAAACAACTGG

TACATTGGTAGAGGATCAGATAACAACAATGACTGTACCTTCCACTCCTATGTGTAT

GGTACGAACTTAACACTCAAGCCGGACTATGCAGTAGTTAACAAACGCTTCCACGT AGGTCAGGCAGTTGTAGCCACTGATGGTAATATTCAAGGTACTAAGTGGGGAGGTA

AGTGGCTTGATGCTTACCTAAACGATACTTACGTTAAGAAGACAATGGCCTGGACTC

AAGTATGGGCTGCTGCTAGTGGTAGTCACATGGGAGGAGGTTCTCAGACTGATACTC

TCCCACAGGACTTGCGATTCCGCAACATATGGATTAAGACCAGAAACAACTATTGG

AACTTCTTCCGAACTGGTCCTGACGGTATCTACTTCCTTTCAGCCGAGGGCGGTTGG

CTAAAATTCCAGATACACTCTAATGGCAGGGTATTTAAGAACATAGCGGATAGAGA

TGCGCCTCCAACAGCAATAGCCGTAGAGGACGTGTAA

SEQ ID NO: 16

Type: DNA

Organism: Artificial sequence derived from T3 tail fiber protein (e.g., T3p48)

C A A ACG AT AC AC GT AG

SEQ ID NO: 17

Type: DNA

//Mut DNA sequence derived from T3 tail fiber protein (e.g., T3p48)

ATGGCTAACGTAATTAAAACCGTTTTGACTTACCAGTTAGATGGCTCCAATCGTGAT

TTTAATATCCCGTTTGAGTATCTAGCCCGTAAGTTCGTAGTAGTAACCCTTATTGGCG

TAGACCGCAAGGTCCTTACGATTAATGCAGACTACCGTTTTGCTACGCGTACTACCA

TCTCACTTACCAAGGCTTGGGGTCCAGCGGATGGATACACTACCATCGAGTTACGCC

GAGTAACCTCCACAACCGACCGATTGGTTGACTTTACGGATGGTTCAATCCTCCGTG

CGTATGACCTTAACGTCGCTCAGATTCAAACGATACACGTAGCGGAAGAGGCCCGT

GACCTCACTACTGATACCATAGGTGTCAATAATGATGGTCATTTGGATGCTCGTGGT

CGTCGAATTGTTAACCTAGCGAACGCTGTGGATGACCGCGACGCTGTTCCGTTTGGT

CAACTTAAGACCATGAACCAGAACTCGTGGCAGGCGCGTAATGAGGCACTACAGTT

CCGTAATGAGGCTGAGACTTTCAGAAATCAAACGGAGGTTTTTAAGAATGAGTCCG

GTACTAACGCTACGAACACAAAGCAGTGGCGAGATGAGGCTAATGGGTCCCGAGAT

GAAGCCGAGCAGTTCAAGAATACGGCTGGTCAATACGCTACATCTGCTGGGAACTC

TGCTACTGCTGCGCATCAATCTGAGGTAAACGCTGAGAACTCCGCTACAGCAGCAG

CGAACTCTGCGAATTTGGCAGAACAACACGCAGACCGTGCGGAACGTGAAGCAGAC

AAGCTGGGGAATTTTAATGGACTGGCTGGTGCAATTGACAGGGTGGATGGAACCAA TGTGTACTGGAAAGGAGGTATCCATGCGAACGGACGCCTTTACCTTACCTCAGATGG

TTTCGACTGTGGTCAGTATCAACAGTTCTTTGGTGGTTCTGCTGGTCGTTACTCTGTC

ATGGAGTGGGGTGATGAGAACGGATGGCTGATGCATGTTCAACGTAGAGAGTGGAC

AACAGCGATAGGTGATAACATCCAGCTAGTAGTAAACGGACATATCATCGCCCAAG

GTGGAGACATGACTGGTCCGCTGAAATTGCAGAATGGACATGCCCTTTACTTAGAGT

CCGCATCCGACAAGGCGCAATATATTCTATCTAAAGATGGTAACAGAAACAACTGG

TACATTGGTAGAGGATCAGATAACAACAATGACTGTACCTTCCACTCCTATGTGTAT

GGTACGAACTTAACACTCAAGCCGGACTATGCAGTAGTTAACAAACGCTTCCACGT

AGGTCAGGCAGTTGTAGCCACTGATGGTAATATTCAAGGTACTAAGTGGGGAGGTA

AGTGGCTTGATGCTTACCTAAACGATACTTACGTTAAGAAGACAATGGCCTGGACTC

AAGTATGGGCTGCTGCTAGTGGTAGTCACATGGGAGGAGGTTCTCAGACTGATACTC

TCCCACAGGACTTGCGATTCCGCAACATATGGATTAAGACCAGAAACAACTATTGG

AACTTCTTCCGAACTGGTCCTGACGGTATCTACTTCCTTTCAGCCGAGGGCGGTTGG

CTAAAATTCCAGATACACTCTAATGGCAGGGTATTTAAGAACATAGCGGATAGAGA

TGCGCCTCCAACAGCAATAGCCGTAGAGGACGTGTAA

SEQ ID NO: 18

Type: DNA

Organism: Artificial sequence derived from T3 tail fiber protein (e.g., T3p48)

CAAACGATCCACGTAG

SEQ ID NO: 19

Type: DNA

//Mut DNA Artificial sequence derived from T3 tail fiber protein (e.g., T3p48)

ATGGCTAACGTAATTAAAACCGTTTTGACTTACCAGTTAGATGGCTCCAATCGTGAT

TTTAATATCCCGTTTGAGTATCTAGCCCGTAAGTTCGTAGTAGTAACCCTTATTGGCG

TAGACCGCAAGGTCCTTACGATTAATGCAGACTACCGTTTTGCTACGCGTACTACCA

TCTCACTTACCAAGGCTTGGGGTCCAGCGGATGGATACACTACCATCGAGTTACGCC

GAGTAACCTCCACAACCGACCGATTGGTTGACTTTACGGATGGTTCAATCCTCCGTG

CGTATGACCTTAACGTCGCTCAGATTCAAACGATCCACGTAGCGGAAGAGGCCCGT

GACCTCACTACTGATACCATAGGTGTCAATAATGATGGTCATTTGGATGCTCGTGGT CGTCGAATTGTTAACCTAGCGAACGCTGTGGATGACCGCGACGCTGTTCCGTTTGGT

CAACTTAAGACCATGAACCAGAACTCGTGGCAGGCGCGTAATGAGGCACTACAGTT

CCGTAATGAGGCTGAGACTTTCAGAAATCAAACGGAGGTTTTTAAGAATGAGTCCG

GTACTAACGCTACGAACACAAAGCAGTGGCGAGATGAGGCTAATGGGTCCCGAGAT

GAAGCCGAGCAGTTCAAGAATACGGCTGGTCAATACGCTACATCTGCTGGGAACTC

TGCTACTGCTGCGCATCAATCTGAGGTAAACGCTGAGAACTCCGCTACAGCAGCAG

CGAACTCTGCGAATTTGGCAGAACAACACGCAGACCGTGCGGAACGTGAAGCAGAC

AAGCTGGGGAATTTTAATGGACTGGCTGGTGCAATTGACAGGGTGGATGGAACCAA

TGTGTACTGGAAAGGAGGTATCCATGCGAACGGACGCCTTTACCTTACCTCAGATGG

TTTCGACTGTGGTCAGTATCAACAGTTCTTTGGTGGTTCTGCTGGTCGTTACTCTGTC

ATGGAGTGGGGTGATGAGAACGGATGGCTGATGCATGTTCAACGTAGAGAGTGGAC

AACAGCGATAGGTGATAACATCCAGCTAGTAGTAAACGGACATATCATCGCCCAAG

GTGGAGACATGACTGGTCCGCTGAAATTGCAGAATGGACATGCCCTTTACTTAGAGT

CCGCATCCGACAAGGCGCAATATATTCTATCTAAAGATGGTAACAGAAACAACTGG

TACATTGGTAGAGGATCAGATAACAACAATGACTGTACCTTCCACTCCTATGTGTAT

GGTACGAACTTAACACTCAAGCCGGACTATGCAGTAGTTAACAAACGCTTCCACGT

AGGTCAGGCAGTTGTAGCCACTGATGGTAATATTCAAGGTACTAAGTGGGGAGGTA

AGTGGCTTGATGCTTACCTAAACGATACTTACGTTAAGAAGACAATGGCCTGGACTC

AAGTATGGGCTGCTGCTAGTGGTAGTCACATGGGAGGAGGTTCTCAGACTGATACTC

TCCCACAGGACTTGCGATTCCGCAACATATGGATTAAGACCAGAAACAACTATTGG

AACTTCTTCCGAACTGGTCCTGACGGTATCTACTTCCTTTCAGCCGAGGGCGGTTGG

CTAAAATTCCAGATACACTCTAATGGCAGGGTATTTAAGAACATAGCGGATAGAGA

TGCGCCTCCAACAGCAATAGCCGTAGAGGACGTGTAA

SEQ ID NO: 20

Type: PROTEIN

Organism: Artificial sequence derived from T3 tail fiber protein (e.g., T3p48)

QIQTMHVAE

SEQ ID NO: 21

Type: PROTEIN

// Mut AA sequence: Artificial sequence derived from T3 tail fiber protein (e.g., T3p48) MANVIKTVLTYQLDGSNRDFNIPFEYLARKFVVVTLIGVDRKVLTINADYRFATRTTISL TKAWGPADGYTTIELRRVTSTTDRLVDFTDGSILRAYDLNVAQIQTMHVAEEARDLTTD TIGVNNDGHLDARGRRIVNLANAVDDRDAVPFGQLKTMNQNSWQARNEALQFRNEAE TFRN QTE VFKNES GTN ATNTKQWRDE AN GS RDE AEQFKNT AGQ Y ATS AGN S AT A AHQ SEVNAENSATAAANSANLAEQHADRAEREADKLGNFNGLAGAIDRVDGTNVYWKGGI HAN GRLYLTS DGFDC GQ Y QQFFGGS AGR Y S VME W GDEN GWLMH V QRRE WTT AIGDN IQLVVNGHIIAQGGDMTGPLKLQNGHALYLESASDKAQYILSKDGNRNNWYIGRGSDN NNDCTFHSYVYGTNLTLKPDYAVVNKRFHVGQAVVATDGNIQGTKWGGKWLDAYLN DT Y VKKTM A WTQ VW A A AS GS HMGGGS QTDTLPQDLRFRNIWIKTRNN YWNFFRTGP DGIYFLS AEGGWLKF QIHS N GR VFKNIADRD APPT AIA VED V

[000204] D. Mutations in the T3 capsid assembly protein (e.g., T3p38)

SEQ ID NO: 22

// WT DNA sequence T3 capsid assembly protein (e.g., T3p38)

ATGGCTGAATCTAATGCAGACGTTTATGCGTCCTTCGGTGTGAACAACGCGGTAATG

ACCGGAAGCACACCTACTGAACACGAACAGAATATGCTGAGTCTCGACGTTGCTGC

CCGTGATGGCGATGATGCAATCGTGCTTAGCGATGAACCGACTTCCCATAACGATGA

CCCCTATGCGGCAGGTGTAGACCCGTTCGCTGATGGTGAAGATGATGAGGGCCGCA

TTCAGGTTCGTATCAGTGAAGATGGCAATGAAGCCGGGTTCGACACTGATGGCGAT

AACTCTGAGGTGGAGACCGAAGGTGAGGACGTTGAGTTTGAACCGCTGGGTGACAC

CCCAGAAGAACTAAGCCAAGTGACTGAGCAATTAGGTCAGCACGAAGAAGGCTTTC

AGGCGATGGTCGAGCAGGCAGTTGAGCGTGGACTGAGCGCAGACTCTGTGAGTCGA

ATCTACGAAGAGTATGAAGCCGATGGCATCTCCGAGAAATCCTATGCTGAACTAGA

AGCTGCTGGCTATAGTCGTGCCTTTGTGGACTCATACATCTCAGGTCAGGAAGCTCT

GGTAGACCAGTACGTCAATCAGGTAGTTGCCTTTGCTGGTGGTCAGGAGCGCTTTAG

TGCAATCCATACTCACCTCGAAGCGACAAACCCTGCTGCTGCTGAGTCCCTTGAGTC

TGCCATGATGAACCGAGACTTGGCGACCGTCAAAGCGATTATCAATCTGGCTGGTG

AGAGCTACACGAAGAAATTCGGTAAGCCTGCCAACCGTAGTGTTACCAAGCGTGCT

ACTCCGGTTAAACCCGTAGCTCGTCAGAAAGAGGGCTTTACGAATCAGGCTGAGAT

GATTAAAGCTATGAGCGACCCGCGCTATCGTAGCGATTCTGCCTACCGCCAAATGGT

AGAACAGAAGGTTATCGACTCTAGTTTCTAA SEQ ID NO: 23

// WT AA sequence: T3 capsid assembly protein (e.g., T3p38)

MAES N AD V Y ASF G VNN A VMT GS TPTEHEQNMLS LD V A ARDGDD AIVLS DEPTS HNDD

PYAAGVDPFADGEDDEGRIQVRISEDGNEAGFDTDGDNSEVETEGEDVEFEPLGDTPEE

LSQVTEQLGQHEEGFQAMVEQAVERGLSADSVSRIYEEYEADGISEKSYAELEAAGYSR

AFVDSYISGQEALVDQYVNQVVAFAGGQERFSAIHTHLEATNPAAAESLESAMMNRDL

ATVKAIINLAGESYTKKFGKPANRSVTKRATPVKPVARQKEGFTNQAEMIKAMSDPRY

RSDSAYRQMVEQKVIDSSF

[000205] The nucleotide in position 20469 may be modified from C to T (or A or G). This region may be recognized by looking for the sequence of the regions before and after the modification (e.g., TGCTGAGT before and CCTTGAGT after, etc.). 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs before and/or 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs after may be identified as 100% identical, 95% or more identical, 90% or more or more identical, 85% or more identical, etc. For example:

SEQ ID NO: 24

Type: DNA

Organism: Artificial sequence derived from T3 capsid assembly protein (e.g., T3p38)

TGCTGAGTTCCTTGAGT

SEQ ID NO: 25

Type: DNA

//Mut DNA sequence derived from T3 capsid assembly protein (e.g., T3p38)

ATGGCTGAATCTAATGCAGACGTTTATGCGTCCTTCGGTGTGAACAACGCGGTAATG

ACCGGAAGCACACCTACTGAACACGAACAGAATATGCTGAGTCTCGACGTTGCTGC

CCGTGATGGCGATGATGCAATCGTGCTTAGCGATGAACCGACTTCCCATAACGATGA

CCCCTATGCGGCAGGTGTAGACCCGTTCGCTGATGGTGAAGATGATGAGGGCCGCA

TTCAGGTTCGTATCAGTGAAGATGGCAATGAAGCCGGGTTCGACACTGATGGCGAT AACTCTGAGGTGGAGACCGAAGGTGAGGACGTTGAGTTTGAACCGCTGGGTGACAC

CCCAGAAGAACTAAGCCAAGTGACTGAGCAATTAGGTCAGCACGAAGAAGGCTTTC

AGGCGATGGTCGAGCAGGCAGTTGAGCGTGGACTGAGCGCAGACTCTGTGAGTCGA

ATCTACGAAGAGTATGAAGCCGATGGCATCTCCGAGAAATCCTATGCTGAACTAGA

AGCTGCTGGCTATAGTCGTGCCTTTGTGGACTCATACATCTCAGGTCAGGAAGCTCT

GGTAGACCAGTACGTCAATCAGGTAGTTGCCTTTGCTGGTGGTCAGGAGCGCTTTAG

TGCAATCCATACTCACCTCGAAGCGACAAACCCTGCTGCTGCTGAGTTCCTTGAGTC

TGCCATGATGAACCGAGACTTGGCGACCGTCAAAGCGATTATCAATCTGGCTGGTG

AGAGCTACACGAAGAAATTCGGTAAGCCTGCCAACCGTAGTGTTACCAAGCGTGCT

ACTCCGGTTAAACCCGTAGCTCGTCAGAAAGAGGGCTTTACGAATCAGGCTGAGAT

GATTAAAGCTATGAGCGACCCGCGCTATCGTAGCGATTCTGCCTACCGCCAAATGGT

AGAACAGAAGGTTATCGACTCTAGTTTCTAA

SEQ ID NO: 26

Type: DNA

TGCTGAGTACCTTGAGT

SEQ ID NO: 27

Type: DNA

//Mut DNA sequence derived from T3 capsid assembly protein (e.g., T3p38)

ATGGCTGAATCTAATGCAGACGTTTATGCGTCCTTCGGTGTGAACAACGCGGTAATG

ACCGGAAGCACACCTACTGAACACGAACAGAATATGCTGAGTCTCGACGTTGCTGC

CCGTGATGGCGATGATGCAATCGTGCTTAGCGATGAACCGACTTCCCATAACGATGA

CCCCTATGCGGCAGGTGTAGACCCGTTCGCTGATGGTGAAGATGATGAGGGCCGCA

TTCAGGTTCGTATCAGTGAAGATGGCAATGAAGCCGGGTTCGACACTGATGGCGAT

AACTCTGAGGTGGAGACCGAAGGTGAGGACGTTGAGTTTGAACCGCTGGGTGACAC

CCCAGAAGAACTAAGCCAAGTGACTGAGCAATTAGGTCAGCACGAAGAAGGCTTTC

AGGCGATGGTCGAGCAGGCAGTTGAGCGTGGACTGAGCGCAGACTCTGTGAGTCGA

ATCTACGAAGAGTATGAAGCCGATGGCATCTCCGAGAAATCCTATGCTGAACTAGA

AGCTGCTGGCTATAGTCGTGCCTTTGTGGACTCATACATCTCAGGTCAGGAAGCTCT GGTAGACCAGTACGTCAATCAGGTAGTTGCCTTTGCTGGTGGTCAGGAGCGCTTTAG

TGCAATCCATACTCACCTCGAAGCGACAAACCCTGCTGCTGCTGAGTACCTTGAGTC

TGCCATGATGAACCGAGACTTGGCGACCGTCAAAGCGATTATCAATCTGGCTGGTG

AGAGCTACACGAAGAAATTCGGTAAGCCTGCCAACCGTAGTGTTACCAAGCGTGCT

ACTCCGGTTAAACCCGTAGCTCGTCAGAAAGAGGGCTTTACGAATCAGGCTGAGAT

GATTAAAGCTATGAGCGACCCGCGCTATCGTAGCGATTCTGCCTACCGCCAAATGGT

AGAACAGAAGGTTATCGACTCTAGTTTCTAA

SEQ ID NO: 28

Type: DNA

TGCTGAGTGCCTTGAGT

SEQ ID NO: 29

Type: DNA

//Mut DNA sequence derived from T3 capsid assembly protein (e.g., T3p38):

ATGGCTGAATCTAATGCAGACGTTTATGCGTCCTTCGGTGTGAACAACGCGGTAATG

ACCGGAAGCACACCTACTGAACACGAACAGAATATGCTGAGTCTCGACGTTGCTGC

CCGTGATGGCGATGATGCAATCGTGCTTAGCGATGAACCGACTTCCCATAACGATGA

CCCCTATGCGGCAGGTGTAGACCCGTTCGCTGATGGTGAAGATGATGAGGGCCGCA

TTCAGGTTCGTATCAGTGAAGATGGCAATGAAGCCGGGTTCGACACTGATGGCGAT

AACTCTGAGGTGGAGACCGAAGGTGAGGACGTTGAGTTTGAACCGCTGGGTGACAC

CCCAGAAGAACTAAGCCAAGTGACTGAGCAATTAGGTCAGCACGAAGAAGGCTTTC

AGGCGATGGTCGAGCAGGCAGTTGAGCGTGGACTGAGCGCAGACTCTGTGAGTCGA

ATCTACGAAGAGTATGAAGCCGATGGCATCTCCGAGAAATCCTATGCTGAACTAGA

AGCTGCTGGCTATAGTCGTGCCTTTGTGGACTCATACATCTCAGGTCAGGAAGCTCT

GGTAGACCAGTACGTCAATCAGGTAGTTGCCTTTGCTGGTGGTCAGGAGCGCTTTAG

TGCAATCCATACTCACCTCGAAGCGACAAACCCTGCTGCTGCTGAGTGCCTTGAGTC

TGCCATGATGAACCGAGACTTGGCGACCGTCAAAGCGATTATCAATCTGGCTGGTG

AGAGCTACACGAAGAAATTCGGTAAGCCTGCCAACCGTAGTGTTACCAAGCGTGCT

ACTCCGGTTAAACCCGTAGCTCGTCAGAAAGAGGGCTTTACGAATCAGGCTGAGAT GATTAAAGCTATGAGCGACCCGCGCTATCGTAGCGATTCTGCCTACCGCCAAATGGT

AGAACAGAAGGTTATCGACTCTAGTTTCTAA

SEQ ID NO: 30

Type: PROTEIN

AAAEFLESA

SEQ ID NO: 31

Type: PROTEIN

//Mut AA sequence derived from T3 capsid assembly protein (e.g., T3p38)

M AES N AD V Y ASF G VNN A VMT GS TPTEHEQNMLS LD V A ARDGDD AIVLS DEPTS HNDD

PYAAGVDPFADGEDDEGRIQVRISEDGNEAGFDTDGDNSEVETEGEDVEFEPLGDTPEE

LSQVTEQLGQHEEGFQAMVEQAVERGLSADSVSRIYEEYEADGISEKSYAELEAAGYSR

AFVDSYISGQEALVDQYVNQVVAFAGGQERFSAIHTHLEATNPAAAEFLESAMMNRDL

ATVKAIINLAGESYTKKFGKPANRSVTKRATPVKPVARQKEGFTNQAEMIKAMSDPRY

RSDSAYRQMVEQKVIDSSF

SEQ ID NO: 32

Type: PROTEIN

AAAEYLESA

SEQ ID NO: 33

Type: PROTEIN

// Mut AA sequence derived from T3 capsid assembly protein (e.g., T3p38) MAES N AD V Y ASF G VNN A VMT GS TPTEHEQNMLS LD V A ARDGDD AIVLS DEPTS HNDD

PYAAGVDPFADGEDDEGRIQVRISEDGNEAGFDTDGDNSEVETEGEDVEFEPLGDTPEE

LSQVTEQLGQHEEGFQAMVEQAVERGLSADSVSRIYEEYEADGISEKSYAELEAAGYSR

AFVDSYISGQEALVDQYVNQVVAFAGGQERFSAIHTHLEATNPAAAEYLESAMMNRDL

ATVKAIINLAGESYTKKFGKPANRSVTKRATPVKPVARQKEGFTNQAEMIKAMSDPRY

RSDSAYRQMVEQKVIDSSF

SEQ ID NO: 34

Type: PROTEIN

AAAECLESA

SEQ ID NO: 35

Type:PRT

//Mut AA sequence derived from T3 capsid assembly protein (e.g., T3p38)

MAES N AD V Y ASF G VNN A VMT GS TPTEHEQNMLS LD V A ARDGDD AIVLS DEPTS HNDD

PYAAGVDPFADGEDDEGRIQVRISEDGNEAGFDTDGDNSEVETEGEDVEFEPLGDTPEE

LSQVTEQLGQHEEGFQAMVEQAVERGLSADSVSRIYEEYEADGISEKSYAELEAAGYSR

AFVDSYISGQEALVDQYVNQVVAFAGGQERFSAIHTHLEATNPAAAECLESAMMNRDL

ATVKAIINLAGESYTKKFGKPANRSVTKRATPVKPVARQKEGFTNQAEMIKAMSDPRY

RSDSAYRQMVEQKVIDSSF

[000206] E. Mutations in the T3 minor capsid protein (e.g., T3p39)

SEQ ID NO: 36

// WT DNA sequence T3 minor capsid protein (e.g., T3p39)

ATGGCTAACATTCAAGGCGGACAGCAAATTGGTACTAATCAGGGTAAGGGTCAGTC

CGCAGCGGACAAATTGGCGCTGTTCCTGAAAGTGTTCGGCGGTGAAGTCCTGACGG

CTTTCGCTCGCACCTCCGTGACCATGCCTCGTCACATGCTGCGCTCTATTGCTTCTGG TAAGTCCGCACAGTTCCCTGTGATTGGTCGCACCAAAGCTGCTTACCTGAAACCGGG

TGAGAACCTCGATGACAAACGTAAAGATATCAAACACACCGAGAAGGTAATCCACA

TTGATGGCCTGCTGACTGCGGATGTGCTGATTTACGACATTGAGGACGCGATGAACC

ACTATGACGTTCGCGCTGAGTACACCGCCCAGTTGGGTGAATCTCTGGCGATGGCGG

CTGACGGTGCTGTACTGGCAGAACTGGCTGGTCTTGTTAATCTGCCGGACGGCTCTA

ACGAGAACATTGAGGGTCTCGGTAAGCCAACCGTACTGACTCTGGTTAAGCCTACC

ACTGGCAGCCTGACTGACCCGGTTGAGTTGGGTAAAGCGATTATTGCTCAGTTGACT

ATCGCTCGTGCATCCCTGACCAAGAACTACGTTCCGGCTGCTGATCGCACCTTCTAC

ACCACTCCTGACAACTACTCTGCGATTCTGGCTGCTCTGATGCCGAACGCAGCAAAC

TATCAGGCACTGCTCGACCCTGAGCGCGGTACTATCCGTAACGTGATGGGCTTCGAG

GTGGTTGAGGTTCCGCACCTGACCGCTGGTGGTGCAGGCGATACCCGTGAGGATGC

CCCGGCTGACCAGAAGCACGCTTTCCCGGCTACTTCCAGCACTACCGTTAAGGTTGC

TCTGGATAACGTTGTGGGCCTGTTCCAGCACCGCTCTGCGGTTGGTACGGTCAAACT

GAAAGACTTGGCTCTGGAGCGTGCTCGTCGTGCGAACTATCAGGCTGACCAGATTAT

CGCTAAATATGCGATGGGTCACGGCGGTCTGCGTCCAGAAGCTGCTGGCGCTATCGT

GCTCCCAAAGGTGTCGGAG

SEQ ID NO: 37

// WT AA sequence T3 minor capsid protein (e.g., T3p39)

MANIQGGQQIGTNQGKGQSAADKLALFLKVFGGEVLTAFARTSVTMPRHMLRSIASGK SAQFPVIGRTKAAYLKPGENLDDKRKDIKHTEKVIHIDGLLTADVLIYDIEDAMNHYDV RAEYTAQLGESLAMAADGAVLAELAGLVNLPDGSNENIEGLGKPTVLTLVKPTTGSLT DPVELGKAIIAQLTIARASLTKNYVPAADRTFYTTPDNYS AILAALMPNAANY QALLDPE RGTIRN VMGFE V VE VPHLT AGG AGDTRED AP ADQKH AFP AT S S TT VKV ALDN V V GLFQ HRSAVGTVKLKDLALERARRANYQADQIIAKYAMGHGGLRPEAAGAIVLPKVSE

[000207] The nucleotide in position 21835 may be modified from C to A (or G). This region may be recognized by looking for the sequence of the regions before and after the modification (e.g., CGTGCGAA before and TATCAGGC after, etc.). 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs before and/or 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs after may be identified as 100% identical, 95% or more identical, 90% or more or more identical, 85% or more identical, etc. For example: SEQ ID NO: 38

Type: DNA

Organism: Artificial sequence derived from T3 minor capsid protein (e.g., T3p39)

CGTGCGAAATATCAGGC

SEQ ID NO: 39

Type:DNA

//Mut DNA sequence derived from T3 minor capsid protein (e.g., T3p39)

ATGGCTAACATTCAAGGCGGACAGCAAATTGGTACTAATCAGGGTAAGGGTCAGTC

CGCAGCGGACAAATTGGCGCTGTTCCTGAAAGTGTTCGGCGGTGAAGTCCTGACGG

CTTTCGCTCGCACCTCCGTGACCATGCCTCGTCACATGCTGCGCTCTATTGCTTCTGG

TAAGTCCGCACAGTTCCCTGTGATTGGTCGCACCAAAGCTGCTTACCTGAAACCGGG

TGAGAACCTCGATGACAAACGTAAAGATATCAAACACACCGAGAAGGTAATCCACA

TTGATGGCCTGCTGACTGCGGATGTGCTGATTTACGACATTGAGGACGCGATGAACC

ACTATGACGTTCGCGCTGAGTACACCGCCCAGTTGGGTGAATCTCTGGCGATGGCGG

CTGACGGTGCTGTACTGGCAGAACTGGCTGGTCTTGTTAATCTGCCGGACGGCTCTA

ACGAGAACATTGAGGGTCTCGGTAAGCCAACCGTACTGACTCTGGTTAAGCCTACC

ACTGGCAGCCTGACTGACCCGGTTGAGTTGGGTAAAGCGATTATTGCTCAGTTGACT

ATCGCTCGTGCATCCCTGACCAAGAACTACGTTCCGGCTGCTGATCGCACCTTCTAC

ACCACTCCTGACAACTACTCTGCGATTCTGGCTGCTCTGATGCCGAACGCAGCAAAC

TATCAGGCACTGCTCGACCCTGAGCGCGGTACTATCCGTAACGTGATGGGCTTCGAG

GTGGTTGAGGTTCCGCACCTGACCGCTGGTGGTGCAGGCGATACCCGTGAGGATGC

CCCGGCTGACCAGAAGCACGCTTTCCCGGCTACTTCCAGCACTACCGTTAAGGTTGC

TCTGGATAACGTTGTGGGCCTGTTCCAGCACCGCTCTGCGGTTGGTACGGTCAAACT

GAAAGACTTGGCTCTGGAGCGTGCTCGTCGTGCGAAATATCAGGCTGACCAGATTAT

CGCTAAATATGCGATGGGTCACGGCGGTCTGCGTCCAGAAGCTGCTGGCGCTATCGT

GCTCCCAAAGGTGTCGGAG

SEQ ID NO: 40

Type: DNA

Organism: Artificial sequence derived from T3 minor capsid protein (e.g., T3p39) CGTGCGAAGTATCAGGC

SEQ ID NO: 41

Type:DNA

//Mut DNA sequence derived from T3 minor capsid protein (e.g., T3p39)

ATGGCTAACATTCAAGGCGGACAGCAAATTGGTACTAATCAGGGTAAGGGTCAGTC

CGCAGCGGACAAATTGGCGCTGTTCCTGAAAGTGTTCGGCGGTGAAGTCCTGACGG

CTTTCGCTCGCACCTCCGTGACCATGCCTCGTCACATGCTGCGCTCTATTGCTTCTGG

TAAGTCCGCACAGTTCCCTGTGATTGGTCGCACCAAAGCTGCTTACCTGAAACCGGG

TGAGAACCTCGATGACAAACGTAAAGATATCAAACACACCGAGAAGGTAATCCACA

TTGATGGCCTGCTGACTGCGGATGTGCTGATTTACGACATTGAGGACGCGATGAACC

ACTATGACGTTCGCGCTGAGTACACCGCCCAGTTGGGTGAATCTCTGGCGATGGCGG

CTGACGGTGCTGTACTGGCAGAACTGGCTGGTCTTGTTAATCTGCCGGACGGCTCTA

ACGAGAACATTGAGGGTCTCGGTAAGCCAACCGTACTGACTCTGGTTAAGCCTACC

ACTGGCAGCCTGACTGACCCGGTTGAGTTGGGTAAAGCGATTATTGCTCAGTTGACT

ATCGCTCGTGCATCCCTGACCAAGAACTACGTTCCGGCTGCTGATCGCACCTTCTAC

ACCACTCCTGACAACTACTCTGCGATTCTGGCTGCTCTGATGCCGAACGCAGCAAAC

TATCAGGCACTGCTCGACCCTGAGCGCGGTACTATCCGTAACGTGATGGGCTTCGAG

GTGGTTGAGGTTCCGCACCTGACCGCTGGTGGTGCAGGCGATACCCGTGAGGATGC

CCCGGCTGACCAGAAGCACGCTTTCCCGGCTACTTCCAGCACTACCGTTAAGGTTGC

TCTGGATAACGTTGTGGGCCTGTTCCAGCACCGCTCTGCGGTTGGTACGGTCAAACT

GAAAGACTTGGCTCTGGAGCGTGCTCGTCGTGCGAAGTATCAGGCTGACCAGATTAT

CGCTAAATATGCGATGGGTCACGGCGGTCTGCGTCCAGAAGCTGCTGGCGCTATCGT

GCTCCCAAAGGTGTCGGAG

SEQ ID NO: 42

Type: PROTEIN

ARRAKYQAD SEQ ID NO: 43

Type:PRT

//Mut A A sequence:

MANIQGGQQIGTNQGKGQSAADKLALFLKVFGGEVLTAFARTSVTMPRHMLRSIASGK SAQFPVIGRTKAAYLKPGENLDDKRKDIKHTEKVIHIDGLLTADVLIYDIEDAMNHYDV RAEYTAQLGESLAMAADGAVLAELAGLVNLPDGSNENIEGLGKPTVLTLVKPTTGSLT DPVELGKAIIAQLTIARASLTKNYVPAADRTFYTTPDNYS AILAALMPNAANY QALLDPE RGTIRN VMGFE V VE VPHLT AGG AGDTRED AP ADQKH AFP AT S S TT VKV ALDN V V GLFQ HRSAVGTVKLKDLALERARRAKYQADQIIAKYAMGHGGLRPEAAGAIVLPKVSE

[000208] F. Mutations in the T3 in internal virion protein C (e.g., T3p46)

SEQ ID NO: 44

// WT DNA sequence T3 in internal virion protein C (e.g., T3p46)

ATGGCTAGTAAACTAAATAGTGTTTTAGGCAACATGGCGACTCCCGGTATGGAACG

ACTCAGGGGAGTCCGGGGGATGGACTACAGGGCAGCAACCATTCAGGCAGAGCAA

CCT AG AGCG AGT CTTCT GG ACT CC ATT GGTCG ATTC GCT A AGGCT GGT GCC GAT AT G

TATATCGCTAAGGAACAACGAGCACGCGACCTAGCTGACGAACGCTCTAACGAGAT

TATCCGTAAGCTGACACCTGAGCAACGTCGAGAGGCTCTCAACAATGGGACCCTTCT

GTATCAGGATGACCCATACGCTATGGAAGCACTGCGAGTCAAGACTGGTCGTAACG

CTGCGTACCTTGTGGACGACGACGTTATGCAGAAGGTCAAAGAAGGTGTCTTCCGTA

CTCGTGAGGAGATGGAACAGTATCGCCATAGTCGTCTTCAAGAGGGCGCTAAGGCA

TACGCTGAGCAGTTCGGTATTGACCCTGAGGACGTTGATTATCAGCGTGGTTTCAAT

GGGGACATTACCGAGCGTAACATCTCACTGTATGGCGCACACGATAACTTCTTGAGC

CAGCAAGCTCAGAAGGGTGCCATCATGAACAGCCGAGTGGAACTCAACGGTGTCCT

TCAAGACCCTGATATGCTTCGTCGCCCAGACTCTGCGGACTTCTTTGAGAAGTACAT

CGACAACGGTCTGGTTACCGGAGCAATCCCGTCTGACGCTCAGGCTACACAGCTTAT

AAGCCAAGCGCTCAGTGACGCTTCTAGCCGTGCTGGTGGTGCTGACTTCCTGATGCG

AGTCGGTGACAAGAAGGTAACACTTAATGGAGCCACTACGACTTACCGAGAGTTGA

TGGGTGAAGAACAGTGGAATGCTCTCATGGTCACAGCACAACGTTCTCAGTTTGAG

AATGACGCTAAGCTGAACGAGCAGTACCGCTTGAAGATTAACTCTGCGCTGAACCA

AGAGGACCCTCGTACTGCGTGGGAGATGCTTCAAGGTATCAAGGCTGAACTCGATA

AGGTTCAACCTGATGAGCAGATGACACCGCAACGTGAGTGGCTAATCTCGGCACAG GAACAAGTTCAGAATCAGATGAACGCATGGACGAAGGCTCAAGCCAAAGCTCTGGA

TGACTCAATGAAGTCTATGAACAAACTTGACGTAATCGACAAGCAGTTCCAGAAGC

GAATCAACGGTGAGTGGGTCTCAACGGACTTCAAGGATATGCCAACAAACGAGAAC

ACTGGTGAGTTCAAACATAGTGATATGGTTAACTACGCCAATAAGAAGCTCGCGGA

GATTGACCGCATGGACATCCCAGATAGCGCCAAGGACATGATGAAGTTGAAGTACC

TTCAAGCGGACTCTAAGGACGGGGCATTCCGTACAGCAATCGGAACTATGGTGACT

GACGCTGGTCAAGAGTGGTCTGCCGCTGTGATTAACGGTAAGTTGCCAGAACGAAC

CCCAGCTCTGGATGCTCTACGTAGAATCCGTAATGCCGACCCCCAGTTGATTGCTGC

GCTATACCCAGACCAAGCTGAGCTATTCCTGACGATGGACATGATGGACAAGCAGG

GTATTGACCCTCAGGTTATTCTTGACGCTGACCGACTGACTGCCAAGCGTTCCAAAG

AGCAACGATTCGAGGACGATAAAGCATTCGAGTCTGCATTGAATAGCTCTACGGCC

CCTGAGATTGCCCGTATGCCAGCGTCACTTCGTGAATCTGCACGTAAGATTTATGAC

TCAGTTAAGTACCGCTCTGGGAACGAAAGCATGGCTATGGAGCAGATGACCAAGTT

CCTTAAGGAATCTACCTACACGTTCACTGGTGACGATGTTGACGGTGATACCATCGG

TGTGATTCCTAAGAACATGATGCAAGTCAACTCTGACCCGAAATCATGGGAGCAAG

GTCGCGATATTCTGGAGGAAGCACGTAAGGGAATCATTGCGAGCAACCAGTGGATA

ACCAACAAGCAACTGACCATGTATTCTCAAGGTGACTCCATTTACCTCATGGACACC

ACTGGTCAAGTCCGCGTCCGTTATGATAAAGAGTTACTCTCGAAGGTCTGGAGTGAG

AACCAGAAGAAACTCGAAGAGAAGGCTCGTGAGAAGGCTCTGGCTGATGTGAACA

AGC GGGC ACCT ATC GTT GCC GCA AC G A AGGCCC GTG A AT CT GCT GCT A A AC GAGT C

CGAGAGAAACGTAAACAGACTCCGAAGTTCATCTATGGACGCAAGGAGTAA

SEQ ID NO: 45

// WT AA sequence T3 in internal virion protein C (e.g., T3p46)

MASKLNSVLGNMATPGMERLRGVRGMDYRAATIQAEQPRASLLDSIGRFAKAGADMY IAKEQRARDLADERSNEIIRKLTPEQRREALNNGTLLYQDDPYAMEALRVKTGRNAAYL VDDDVMQKVKEGVFRTREEMEQYRHSRLQEGAKAYAEQFGIDPEDVDYQRGFNGDIT ERNIS FY G AHDNFFS QQ AQKG AIMN S RVEFN G VFQDPDMFRRPDS ADFFEK YIDN GFVT GAIPSDAQATQFISQAFSDASSRAGGADFFMRVGDKKVTFNGATTTYREFMGEEQWNA EM VT AQRS QFEND AKFNEQYRFKINS AFN QEDPRT AWEMFQGIKAEFDKV QPDEQMT PQREWLIS AQEQ V QN QMN A WTKAQ AKALDDS MKS MNKLD VIDKQF QKRIN GEW V S T DFKDMPTNENTGEFKHSDMVNYANKKLAEIDRMDIPDSAKDMMKLKYLQADSKDGAF RTAIGTMVTDAGQEWSAAVINGKLPERTPALDALRRIRNADPQLIAALYPDQAELFLTM DMMDKQGIDPQVILDADRLTAKRSKEQRFEDDKAFESALNSSTAPEIARMPASLRESAR KIYDSVKYRSGNESMAMEQMTKFLKESTYTFTGDDVDGDTIGVIPKNMMQVNSDPKS WEQGRDILEE ARKGIIASNQWITNKQLTM Y S QGDS IYLMDTTGQVRVR YD KELLS KVW SENQKKLEEKAREKALADVNKRAPIVAATKARESAAKRVREKRKQTPKFIYGRKE

[000209] The nucleotide in position 28066 may be modified from G to A (or T). This region may be recognized by looking for the sequence of the regions before and after the modification (e.g., CAGACCAA before and CTGAGCTA after, etc.). 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs before and/or 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs after may be identified as 100% identical, 95% or more identical, 90% or more or more identical, 85% or more identical, etc. For example:

SEQ ID NO: 46

Type: DNA

Organism: Artificial sequence derived from T3 in internal virion protein C (e.g., T3p46)

CAGACCAAACTGAGCTA

SEQ ID NO: 47

Type: DNA

// Mut DNA sequence derived from T3 in internal virion protein C (e.g., T3p46)

ATGGCTAGTAAACTAAATAGTGTTTTAGGCAACATGGCGACTCCCGGTATGGAACG

ACTCAGGGGAGTCCGGGGGATGGACTACAGGGCAGCAACCATTCAGGCAGAGCAA

CCT AG AGCG AGT CTTCT GG ACT CC ATT GGTCG ATTC GCT A AGGCT GGT GCC GAT AT G

TATATCGCTAAGGAACAACGAGCACGCGACCTAGCTGACGAACGCTCTAACGAGAT

TATCCGTAAGCTGACACCTGAGCAACGTCGAGAGGCTCTCAACAATGGGACCCTTCT

GTATCAGGATGACCCATACGCTATGGAAGCACTGCGAGTCAAGACTGGTCGTAACG

CTGCGTACCTTGTGGACGACGACGTTATGCAGAAGGTCAAAGAAGGTGTCTTCCGTA

CTCGTGAGGAGATGGAACAGTATCGCCATAGTCGTCTTCAAGAGGGCGCTAAGGCA

TACGCTGAGCAGTTCGGTATTGACCCTGAGGACGTTGATTATCAGCGTGGTTTCAAT

GGGGACATTACCGAGCGTAACATCTCACTGTATGGCGCACACGATAACTTCTTGAGC

CAGCAAGCTCAGAAGGGTGCCATCATGAACAGCCGAGTGGAACTCAACGGTGTCCT TCAAGACCCTGATATGCTTCGTCGCCCAGACTCTGCGGACTTCTTTGAGAAGTACAT

CGACAACGGTCTGGTTACCGGAGCAATCCCGTCTGACGCTCAGGCTACACAGCTTAT

AAGCCAAGCGCTCAGTGACGCTTCTAGCCGTGCTGGTGGTGCTGACTTCCTGATGCG

AGTCGGTGACAAGAAGGTAACACTTAATGGAGCCACTACGACTTACCGAGAGTTGA

TGGGTGAAGAACAGTGGAATGCTCTCATGGTCACAGCACAACGTTCTCAGTTTGAG

AATGACGCTAAGCTGAACGAGCAGTACCGCTTGAAGATTAACTCTGCGCTGAACCA

AGAGGACCCTCGTACTGCGTGGGAGATGCTTCAAGGTATCAAGGCTGAACTCGATA

AGGTTCAACCTGATGAGCAGATGACACCGCAACGTGAGTGGCTAATCTCGGCACAG

GAACAAGTTCAGAATCAGATGAACGCATGGACGAAGGCTCAAGCCAAAGCTCTGGA

TGACTCAATGAAGTCTATGAACAAACTTGACGTAATCGACAAGCAGTTCCAGAAGC

GAATCAACGGTGAGTGGGTCTCAACGGACTTCAAGGATATGCCAACAAACGAGAAC

ACTGGTGAGTTCAAACATAGTGATATGGTTAACTACGCCAATAAGAAGCTCGCGGA

GATTGACCGCATGGACATCCCAGATAGCGCCAAGGACATGATGAAGTTGAAGTACC

TTCAAGCGGACTCTAAGGACGGGGCATTCCGTACAGCAATCGGAACTATGGTGACT

GACGCTGGTCAAGAGTGGTCTGCCGCTGTGATTAACGGTAAGTTGCCAGAACGAAC

CCCAGCTCTGGATGCTCTACGTAGAATCCGTAATGCCGACCCCCAGTTGATTGCTGC

GCTATACCCAGACCAAACTGAGCTATTCCTGACGATGGACATGATGGACAAGCAGG

GTATTGACCCTCAGGTTATTCTTGACGCTGACCGACTGACTGCCAAGCGTTCCAAAG

AGCAACGATTCGAGGACGATAAAGCATTCGAGTCTGCATTGAATAGCTCTACGGCC

CCTGAGATTGCCCGTATGCCAGCGTCACTTCGTGAATCTGCACGTAAGATTTATGAC

TCAGTTAAGTACCGCTCTGGGAACGAAAGCATGGCTATGGAGCAGATGACCAAGTT

CCTTAAGGAATCTACCTACACGTTCACTGGTGACGATGTTGACGGTGATACCATCGG

TGTGATTCCTAAGAACATGATGCAAGTCAACTCTGACCCGAAATCATGGGAGCAAG

GTCGCGATATTCTGGAGGAAGCACGTAAGGGAATCATTGCGAGCAACCAGTGGATA

ACCAACAAGCAACTGACCATGTATTCTCAAGGTGACTCCATTTACCTCATGGACACC

ACTGGTCAAGTCCGCGTCCGTTATGATAAAGAGTTACTCTCGAAGGTCTGGAGTGAG

AACCAGAAGAAACTCGAAGAGAAGGCTCGTGAGAAGGCTCTGGCTGATGTGAACA

AGC GGGC ACCT ATC GTT GCC GCA AC G A AGGCCC GTG A AT CT GCT GCT A A AC GAGT C

CGAGAGAAACGTAAACAGACTCCGAAGTTCATCTATGGACGCAAGGAGTAA

SEQ ID NO: 48

Type: DNA

Organism: Artificial sequence derived from T3 in internal virion protein C (e.g., T3p46) C AGACC AATCTGAGCT A

SEQ ID NO: 49

Type: DNA

//Mut DNA sequence derived from T3 in internal virion protein C (e.g., T3p46)

ATGGCTAGTAAACTAAATAGTGTTTTAGGCAACATGGCGACTCCCGGTATGGAACG

ACTCAGGGGAGTCCGGGGGATGGACTACAGGGCAGCAACCATTCAGGCAGAGCAA

CCT AG AGCG AGT CTTCT GG ACT CC ATT GGTCG ATTC GCT A AGGCT GGT GCC GAT AT G

TATATCGCTAAGGAACAACGAGCACGCGACCTAGCTGACGAACGCTCTAACGAGAT

TATCCGTAAGCTGACACCTGAGCAACGTCGAGAGGCTCTCAACAATGGGACCCTTCT

GTATCAGGATGACCCATACGCTATGGAAGCACTGCGAGTCAAGACTGGTCGTAACG

CTGCGTACCTTGTGGACGACGACGTTATGCAGAAGGTCAAAGAAGGTGTCTTCCGTA

CTCGTGAGGAGATGGAACAGTATCGCCATAGTCGTCTTCAAGAGGGCGCTAAGGCA

TACGCTGAGCAGTTCGGTATTGACCCTGAGGACGTTGATTATCAGCGTGGTTTCAAT

GGGGACATTACCGAGCGTAACATCTCACTGTATGGCGCACACGATAACTTCTTGAGC

CAGCAAGCTCAGAAGGGTGCCATCATGAACAGCCGAGTGGAACTCAACGGTGTCCT

TCAAGACCCTGATATGCTTCGTCGCCCAGACTCTGCGGACTTCTTTGAGAAGTACAT

CGACAACGGTCTGGTTACCGGAGCAATCCCGTCTGACGCTCAGGCTACACAGCTTAT

AAGCCAAGCGCTCAGTGACGCTTCTAGCCGTGCTGGTGGTGCTGACTTCCTGATGCG

AGTCGGTGACAAGAAGGTAACACTTAATGGAGCCACTACGACTTACCGAGAGTTGA

TGGGTGAAGAACAGTGGAATGCTCTCATGGTCACAGCACAACGTTCTCAGTTTGAG

AATGACGCTAAGCTGAACGAGCAGTACCGCTTGAAGATTAACTCTGCGCTGAACCA

AGAGGACCCTCGTACTGCGTGGGAGATGCTTCAAGGTATCAAGGCTGAACTCGATA

AGGTTCAACCTGATGAGCAGATGACACCGCAACGTGAGTGGCTAATCTCGGCACAG

GAACAAGTTCAGAATCAGATGAACGCATGGACGAAGGCTCAAGCCAAAGCTCTGGA

TGACTCAATGAAGTCTATGAACAAACTTGACGTAATCGACAAGCAGTTCCAGAAGC

GAATCAACGGTGAGTGGGTCTCAACGGACTTCAAGGATATGCCAACAAACGAGAAC

ACTGGTGAGTTCAAACATAGTGATATGGTTAACTACGCCAATAAGAAGCTCGCGGA

GATTGACCGCATGGACATCCCAGATAGCGCCAAGGACATGATGAAGTTGAAGTACC

TTCAAGCGGACTCTAAGGACGGGGCATTCCGTACAGCAATCGGAACTATGGTGACT

GACGCTGGTCAAGAGTGGTCTGCCGCTGTGATTAACGGTAAGTTGCCAGAACGAAC

CCCAGCTCTGGATGCTCTACGTAGAATCCGTAATGCCGACCCCCAGTTGATTGCTGC

GCTATACCCAGACCAATCTGAGCTATTCCTGACGATGGACATGATGGACAAGCAGG GTATTGACCCTCAGGTTATTCTTGACGCTGACCGACTGACTGCCAAGCGTTCCAAAG

AGCAACGATTCGAGGACGATAAAGCATTCGAGTCTGCATTGAATAGCTCTACGGCC

CCTGAGATTGCCCGTATGCCAGCGTCACTTCGTGAATCTGCACGTAAGATTTATGAC

TCAGTTAAGTACCGCTCTGGGAACGAAAGCATGGCTATGGAGCAGATGACCAAGTT

CCTTAAGGAATCTACCTACACGTTCACTGGTGACGATGTTGACGGTGATACCATCGG

TGTGATTCCTAAGAACATGATGCAAGTCAACTCTGACCCGAAATCATGGGAGCAAG

GTCGCGATATTCTGGAGGAAGCACGTAAGGGAATCATTGCGAGCAACCAGTGGATA

ACCAACAAGCAACTGACCATGTATTCTCAAGGTGACTCCATTTACCTCATGGACACC

ACTGGTCAAGTCCGCGTCCGTTATGATAAAGAGTTACTCTCGAAGGTCTGGAGTGAG

AACCAGAAGAAACTCGAAGAGAAGGCTCGTGAGAAGGCTCTGGCTGATGTGAACA

AGC GGGC ACCT ATC GTT GCC GCA AC G A AGGCCC GTG A AT CT GCT GCT A A AC GAGT C

CGAGAGAAACGTAAACAGACTCCGAAGTTCATCTATGGACGCAAGGAGTAA

SEQ ID NO: 50

Type: PROTEIN

YPDQTELFL

SEQ ID NO: 51

Type: PROTEIN

//Mut AA sequence derived from T3 in internal virion protein C (e.g., T3p46)

MASKLNSVLGNMATPGMERLRGVRGMDYRAATIQAEQPRASLLDSIGRFAKAGADMY

IAKEQRARDLADERSNEIIRKLTPEQRREALNNGTLLYQDDPYAMEALRVKTGRNAAYL

VDDDVMQKVKEGVFRTREEMEQYRHSRLQEGAKAYAEQFGIDPEDVDYQRGFNGDIT

ERNIS LY G AHDNFLS QQ AQKG AIMN S RVELN G VLQDPDMLRRPDS ADFFEK YIDN GLVT

GAIPSDAQATQLISQALSDASSRAGGADFLMRVGDKKVTLNGATTTYRELMGEEQWNA

LM VT AQRS QFEND AKLNEQYRLKINS ALN QEDPRT AWEMLQGIKAELDKV QPDEQMT

PQREWLIS AQEQ V QN QMN A WTKAQ AKALDDS MKS MNKLD VIDKQF QKRIN GEW V S T

DFKDMPTNENTGEFKHSDMVNYANKKLAEIDRMDIPDSAKDMMKLKYLQADSKDGAF

RTAIGTMVTDAGQEWSAAVINGKLPERTPALDALRRIRNADPQLIAALYPDQTELFLTM

DMMDKQGIDPQVILDADRLTAKRSKEQRFEDDKAFESALNSSTAPEIARMPASLRESAR KIYDSVKYRSGNESMAMEQMTKFLKESTYTFTGDDVDGDTIGVIPKNMMQVNSDPKS WEQGRDIFEE ARKGIIASNQWITNKQFTM Y S QGDS IYFMDTTGQVRVRYDKEFFS KVW SENQKKLEEKAREKALADVNKRAPIVAATKARESAAKRVREKRKQTPKFIYGRKE

SEQ ID NO: 52

Type: PROTEIN

YPDQSELFL

SEQ ID NO: 53

Type: PROTEIN

//Mut AA sequence derived from T3 in internal virion protein C (e.g., T3p46)

MASKLNSVLGNMATPGMERLRGVRGMDYRAATIQAEQPRASLLDSIGRFAKAGADMY

IAKEQRARDLADERSNEIIRKLTPEQRREALNNGTLLYQDDPYAMEALRVKTGRNAAYL

VDDDVMQKVKEGVFRTREEMEQYRHSRLQEGAKAYAEQFGIDPEDVDYQRGFNGDIT

ERNIS LY G AHDNFLS QQ AQKG AIMN S RVELN G VLQDPDMLRRPDS ADFFEK YIDN GLVT

GAIPSDAQATQLISQALSDASSRAGGADFLMRVGDKKVTLNGATTTYRELMGEEQWNA

LM VT AQRS QFEND AKLNEQYRLKINS ALN QEDPRT AWEMLQGIKAELDKV QPDEQMT

PQREWLIS AQEQ V QN QMN A WTKAQ AKALDDS MKS MNKLD VIDKQF QKRIN GEW V S T

DFKDMPTNENTGEFKHSDMVNYANKKLAEIDRMDIPDSAKDMMKLKYLQADSKDGAF

RTAIGTMVTDAGQEWSAAVINGKLPERTPALDALRRIRNADPQLIAALYPDQSELFLTM

DMMDKQGIDPQVILDADRLTAKRSKEQRFEDDKAFESALNSSTAPEIARMPASLRESAR

KIYDSVKYRSGNESMAMEQMTKFLKESTYTFTGDDVDGDTIGVIPKNMMQVNSDPKS

WEQGRDILEE ARKGIIASNQWITNKQLTM Y S QGDS IYLMDTTGQVRVR YD KELLS KVW

SENQKKLEEKAREKALADVNKRAPIVAATKARESAAKRVREKRKQTPKFIYGRKE

[000210] G. Mutations in the T7 capsid assembly protein (e.g., T7p43)

SEQ ID NO: 54

// WT DNA sequence of T7 capsid assembly protein (e.g., T7p43) ATGGCTGAATCTAATGCAGACGTATATGCATCTTTTGGCGTGAACTCCGCTGTGATG

TCTGGTGGTTCCGTTGAGGAACATGAGCAGAACATGCTGGCTCTTGATGTTGCTGCC

CGTGATGGCGATGATGCAATCGAGTTAGCGTCAGACGAAGTGGAAACAGAACGTGA

CCTGTATGACAACTCTGACCCGTTCGGTCAAGAGGATGACGAAGGCCGCATTCAGG

TTCGTATCGGTGATGGCTCTGAGCCGACCGATGTGGACACTGGAGAAGAAGGCGTT

GAGGGCACCGAAGGTTCCGAAGAGTTTACCCCACTGGGCGAGACTCCAGAAGAACT

GGTAGCTGCCTCTGAGCAACTTGGTGAGCACGAAGAGGGCTTCCAAGAGATGATTA

ACATTGCTGCTGAGCGTGGCATGAGTGTCGAGACCATTGAGGCTATCCAGCGTGAGT

AC G AGG AG A AC G A AG AGTTGT CC GCC G AGT CCT AC GCT A AGCT GGCT G A A ATT GGC

TACACGAAGGCTTTCATTGACTCGTATATCCGTGGTCAAGAAGCTCTGGTGGAGCAG

TACGTAAACAGTGTCATTGAGTACGCTGGTGGTCGTGAACGTTTTGATGCACTGTAT

AACCACCTTGAGACGCACAACCCTGAGGCTGCACAGTCGCTGGATAATGCGTTGAC

CAATCGTGACTTAGCGACCGTTAAGGCTATCATCAACTTGGCTGGTGAGTCTCGCGC

TAAGGCGTTCGGTCGTAAGCCAACTCGTAGTGTGACTAATCGTGCTATTCCGGCTAA

ACCTCAGGCTACCAAGCGTGAAGGCTTTGCGGACCGTAGCGAGATGATTAAAGCTA

TGAGTGACCCTCGGTATCGCACAGATGCCAACTATCGTCGTCAAGTCGAACAGAAA

GTAATCGATTCGAACTTCTGA

SEQ ID NO: 55

// WT AA sequence T7 capsid assembly protein (e.g., T7p43)

MAES N AD V Y ASF G VN S A VMS GGS VEEHEQNMLALD V A ARD GDD AIELAS DE VETERD LYDNSDPFGQEDDEGRIQVRIGDGSEPTDVDTGEEGVEGTEGSEEFTPLGETPEELVAAS EQLGEHEEGF QEMINI A AERGMS VETIE AIQRE YEENEELS AES Y AKLAEIGYTKAFIDS Y IRGQEALVEQYVNSVIEYAGGRERFDALYNHLETHNPEAAQSLDNALTNRDLATVKAII NLAGES R AKAF GRKPTRS VTNRAIP AKPQ ATKREGF ADRS EMIKAMS DPRYRTD AN YR RQVEQKVIDSNF

[000211] The nucleotide in position 22828 may be modified from C to A (or G). This region may be recognized by looking for the sequence of the regions before and after the modification (e.g., GATGCCAA before and TATCGTCG after, etc.). 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs before and/or 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs after may be identified as 100% identical, 95% or more identical, 90% or more or more identical, 85% or more identical, etc. For example: SEQ ID NO: 56

Type: DNA

Organism: Artificial sequence derived from T7 capsid assembly protein (e.g., T7p43)

GATGCCAAATATCGTCG

SEQ ID NO: 57

Type: DNA

// Mut DNA sequence derived from T7 capsid assembly protein (e.g., T7p43)

ATGGCTGAATCTAATGCAGACGTATATGCATCTTTTGGCGTGAACTCCGCTGTGATG

TCTGGTGGTTCCGTTGAGGAACATGAGCAGAACATGCTGGCTCTTGATGTTGCTGCC

CGTGATGGCGATGATGCAATCGAGTTAGCGTCAGACGAAGTGGAAACAGAACGTGA

CCTGTATGACAACTCTGACCCGTTCGGTCAAGAGGATGACGAAGGCCGCATTCAGG

TTCGTATCGGTGATGGCTCTGAGCCGACCGATGTGGACACTGGAGAAGAAGGCGTT

GAGGGCACCGAAGGTTCCGAAGAGTTTACCCCACTGGGCGAGACTCCAGAAGAACT

GGTAGCTGCCTCTGAGCAACTTGGTGAGCACGAAGAGGGCTTCCAAGAGATGATTA

ACATTGCTGCTGAGCGTGGCATGAGTGTCGAGACCATTGAGGCTATCCAGCGTGAGT

TACACGAAGGCTTTCATTGACTCGTATATCCGTGGTCAAGAAGCTCTGGTGGAGCAG

TACGTAAACAGTGTCATTGAGTACGCTGGTGGTCGTGAACGTTTTGATGCACTGTAT

AACCACCTTGAGACGCACAACCCTGAGGCTGCACAGTCGCTGGATAATGCGTTGAC

CAATCGTGACTTAGCGACCGTTAAGGCTATCATCAACTTGGCTGGTGAGTCTCGCGC

TAAGGCGTTCGGTCGTAAGCCAACTCGTAGTGTGACTAATCGTGCTATTCCGGCTAA

ACCTCAGGCTACCAAGCGTGAAGGCTTTGCGGACCGTAGCGAGATGATTAAAGCTA

TGAGTGACCCTCGGTATCGCACAGATGCCAAATATCGTCGTCAAGTCGAACAGAAA

GTAATCGATTCGAACTTCTGA

SEQ ID NO: 58

Type: DNA

Organism: Artificial sequence derived from T7 capsid assembly protein (e.g., T7p43) GATGCCAAGTATCGTCG

SEQ ID NO: 59

Type: DNA

// Mut DNA sequence derived from T7 capsid assembly protein (e.g., T7p43)

ATGGCTGAATCTAATGCAGACGTATATGCATCTTTTGGCGTGAACTCCGCTGTGATG

TCTGGTGGTTCCGTTGAGGAACATGAGCAGAACATGCTGGCTCTTGATGTTGCTGCC

CGTGATGGCGATGATGCAATCGAGTTAGCGTCAGACGAAGTGGAAACAGAACGTGA

CCTGTATGACAACTCTGACCCGTTCGGTCAAGAGGATGACGAAGGCCGCATTCAGG

TTCGTATCGGTGATGGCTCTGAGCCGACCGATGTGGACACTGGAGAAGAAGGCGTT

GAGGGCACCGAAGGTTCCGAAGAGTTTACCCCACTGGGCGAGACTCCAGAAGAACT

GGTAGCTGCCTCTGAGCAACTTGGTGAGCACGAAGAGGGCTTCCAAGAGATGATTA

ACATTGCTGCTGAGCGTGGCATGAGTGTCGAGACCATTGAGGCTATCCAGCGTGAGT

TACACGAAGGCTTTCATTGACTCGTATATCCGTGGTCAAGAAGCTCTGGTGGAGCAG

TACGTAAACAGTGTCATTGAGTACGCTGGTGGTCGTGAACGTTTTGATGCACTGTAT

AACCACCTTGAGACGCACAACCCTGAGGCTGCACAGTCGCTGGATAATGCGTTGAC

CAATCGTGACTTAGCGACCGTTAAGGCTATCATCAACTTGGCTGGTGAGTCTCGCGC

TAAGGCGTTCGGTCGTAAGCCAACTCGTAGTGTGACTAATCGTGCTATTCCGGCTAA

ACCTCAGGCTACCAAGCGTGAAGGCTTTGCGGACCGTAGCGAGATGATTAAAGCTA

TGAGTGACCCTCGGTATCGCACAGATGCCAAGTATCGTCGTCAAGTCGAACAGAAA

GTAATCGATTCGAACTTCTGA

SEQ ID NO: 60

Type: PROTEIN

RTDAKYRRQ

SEQ ID NO: 61

Type: PROTEIN // Mut AA sequence derived from T7 capsid assembly protein (e.g., T7p43)

MAES N AD V Y ASF G VN S A VMS GGS VEEHEQNMLALD V A ARD GDD AIELAS DE VETERD LYDNSDPFGQEDDEGRIQVRIGDGSEPTDVDTGEEGVEGTEGSEEFTPLGETPEELVAAS EQLGEHEEGF QEMINI A AERGMS VETIE AIQRE YEENEELS AES Y AKLAEIGYTKAFIDS Y IRGQEALVEQYVNSVIEYAGGRERFDALYNHLETHNPEAAQSLDNALTNRDLATVKAII NLAGES R AKAF GRKPTRS VTNRAIP AKPQ ATKREGF ADRS EMIKAMS DPRYRTD AKYR RQVEQKVIDSNF

[000212] H. Mutations in the T7 internal virion protein D (e.g., T7p51)

SEQ ID NO: 61

// WT DNA sequence T7 internal virion protein D (e.g., T7p51)

ATGGATAAGTACGATAAGAACGTACCAAGTGATTATGATGGTCTGTTCCAAAAGGC

TGCTGATGCCAACGGGGTCTCTTATGACCTTTTACGTAAAGTCGCTTGGACAGAATC

ACGATTTGTGCCTACAGCAAAATCTAAGACTGGACCATTAGGCATGATGCAATTTAC

CAAGGCAACCGCTAAGGCCCTCGGTCTGCGAGTTACCGATGGTCCAGACGACGACC

GACTGAACCCTGAGTTAGCTATTAATGCTGCCGCTAAGCAACTTGCAGGTCTGGTAG

GGAAGTTTGATGGCGATGAACTCAAAGCTGCCCTTGCGTACAACCAAGGCGAGGGA

CGCTTGGGTAATCCACAACTTGAGGCGTACTCTAAGGGAGACTTCGCATCAATCTCT

GAGGAGGGACGTAACTACATGCGTAACCTTCTGGATGTTGCTAAGTCACCTATGGCT

GGACAGTTGGAAACTTTTGGTGGCATAACCCCAAAGGGTAAAGGCATTCCGGCTGA

GGTAGGATTGGCTGGAATTGGTCACAAGCAGAAAGTAACACAGGAACTTCCTGAGT

CCACAAGTTTTGACGTTAAGGGTATCGAACAGGAGGCTACGGCGAAACCATTCGCC

AAGGACTTTTGGGAGACCCACGGAGAAACACTTGACGAGTACAACAGTCGTTCAAC

CTTCTTCGGATTCAAAAATGCTGCCGAAGCTGAACTCTCCAACTCAGTCGCTGGGAT

GGCTTTCCGTGCTGGTCGTCTCGATAATGGTTTTGATGTGTTTAAAGACACCATTACG

CCGACTCGCTGGAACTCTCACATCTGGACTCCAGAGGAGTTAGAGAAGATTCGAAC

AGAGGTTAAGAACCCTGCGTACATCAACGTTGTAACTGGTGGTTCCCCTGAGAACCT

CGATGACCTCATTAAATTGGCTAACGAGAACTTTGAGAATGACTCCCGCGCTGCCGA

GGCTGGCCTAGGTGCCAAACTGAGTGCTGGTATTATTGGTGCTGGTGTGGACCCGCT

TAGCTATGTTCCTATGGTCGGTGTCACTGGTAAGGGCTTTAAGTTAATCAATAAGGC

TCTTGTAGTTGGTGCCGAAAGTGCTGCTCTGAACGTTGCATCCGAAGGTCTCCGTAC CTCCGTAGCTGGTGGTGACGCAGACTATGCGGGTGCTGCCTTAGGTGGCTTTGTGTT

TGGCGCAGGCATGTCTGCAATCAGTGACGCTGTAGCTGCTGGACTGAAACGCAGTA

AACCAGAAGCTGAGTTCGACAATGAGTTCATCGGTCCTATGATGCGATTGGAAGCC

CGTGAGACAGCACGAAACGCCAACTCTGCGGACCTCTCTCGGATGAACACTGAGAA

CATGAAGTTTGAAGGTGAACATAATGGTGTCCCTTATGAGGACTTACCAACAGAGA

GAGGTGCCGTGGTGTTACATGATGGCTCCGTTCTAAGTGCAAGCAACCCAATCAACC

CTAAGACTCTAAAAGAGTTCTCCGAGGTTGACCCTGAGAAGGCTGCGCGAGGAATC

AAACTGGCTGGGTTCACCGAGATTGGCTTGAAGACCTTGGGGTCTGACGATGCTGAC

ATCCGTAGAGTGGCTATCGACCTCGTTCGCTCTCCTACTGGTATGCAGTCTGGTGCCT

CAGGTAAGTTCGGTGCAACAGCTTCTGACATCCATGAGAGACTTCATGGTACTGACC

AGCGTACTTATAATGACTTGTACAAAGCAATGTCTGACGCTATGAAAGACCCTGAGT

TCTCTACTGGCGGCGCTAAGATGTCCCGTGAAGAAACTCGATACACTATCTACCGTA

GAGCGGCACTAGCTATTGAGCGTCCAGAACTACAGAAGGCACTCACTCCGTCTGAG

AGAATCGTTATGGACATCATTAAGCGTCACTTTGACACCAAGCGTGAACTTATGGAA

AACCCAGCAATATTCGGTAACACAAAGGCTGTGAGTATCTTCCCTGAGAGTCGCCAC

AAAGGTACTTACGTTCCTCACGTATATGACCGTCATGCCAAGGCGCTGATGATTCAA

CGCTACGGTGCCGAAGGTTTGCAGGAAGGGATTGCCCGCTCATGGATGAACAGCTA

CGTCTCCAGACCTGAGGTCAAGGCCAGAGTCGATGAGATGCTTAAGGAATTACACG

GGGTGAAGGAAGTAACACCAGAGATGGTAGAGAAGTACGCTATGGATAAGGCTTAT

GGTATCTCCCACTCAGACCAGTTCACCAACAGTTCCATAATAGAAGAGAACATTGA

GGGCTTAGTAGGTATCGAGAATAACTCATTCCTTGAGGCACGTAACTTGTTTGATTC

GGACCTATCCATCACTATGCCAGACGGACAGCAATTCTCAGTGAATGACCTAAGGG

ACTTCGATATGTTCCGCATCATGCCAGCGTATGACCGCCGTGTCAATGGTGACATCG

CCATCATGGGGTCTACTGGTAAAACCACTAAGGAACTTAAGGATGAGATTTTGGCTC

TCAAAGCGAAAGCTGAGGGAGACGGTAAGAAGACTGGCGAGGTACATGCTTTAATG

GATACCGTTAAGATTCTTACTGGTCGTGCTAGACGCAATCAGGACACTGTGTGGGAA

ACCTCACTGCGTGCCATCAATGACCTAGGGTTCTTCGCTAAGAACGCCTACATGGGT

GCTCAGAACATTACGGAGATTGCTGGGATGATTGTCACTGGTAACGTTCGTGCTCTA

GGGCATGGTATCCCAATTCTGCGTGATACACTCTACAAGTCTAAACCAGTTTCAGCT

AAGGAACTCAAGGAACTCCATGCGTCTCTGTTCGGGAAGGAGGTGGACCAGTTGAT

TCGGCCTAAACGTGCTGACATTGTGCAGCGCCTAAGGGAAGCAACTGATACCGGAC

CTGCCGTGGCGAACATCGTAGGGACCTTGAAGTATTCAACACAGGAACTGGCTGCT

CGCTCTCCGTGGACTAAGCTACTGAACGGAACCACTAACTACCTTCTGGATGCTGCG

CGTCAAGGTATGCTTGGGGATGTTATTAGTGCCACCCTAACAGGTAAGACTACCCGC

TGGGAGAAAGAAGGCTTCCTTCGTGGTGCCTCCGTAACTCCTGAGCAGATGGCTGGC ATCAAGTCTCTCATCAAGGAACATATGGTACGCGGTGAGGACGGGAAGTTTACCGT

TAAGGACAAGCAAGCGTTCTCTATGGACCCACGGGCTATGGACTTATGGAGACTGG

CTGACAAGGTAGCTGATGAGGCAATGCTGCGTCCACATAAGGTGTCCTTACAGGATT

CCCATGCGTTCGGAGCACTAGGTAAGATGGTTATGCAGTTTAAGTCTTTCACTATCA

AGTCCCTTAACTCTAAGTTCCTGCGAACCTTCTATGATGGATACAAGAACAACCGAG

CGATTGACGCTGCGCTGAGCATCATCACCTCTATGGGTCTCGCTGGTGGTTTCTATG

CT AT GGCT GC AC ACGT C A A AGC AT AC GCT CT GCCT A AGG AG A A AC GT A AGG AGT AC

TTGGAGCGTGCACTGGACCCAACCATGATTGCCCACGCTGCGTTATCTCGTAGTTCT

CAATTGGGTGCTCCTTTGGCTATGGTTGACCTAGTTGGTGGTGTTTTAGGGTTCGAGT

CCTCCAAGATGGCTCGCTCTACGATTCTACCTAAGGACACCGTGAAGGAACGTGACC

CAAACAAACCGTACACCTCTAGAGAGGTAATGGGCGCTATGGGTTCAAACCTTCTG

GAACAGATGCCTTCGGCTGGCTTTGTGGCTAACGTAGGGGCTACCTTAATGAATGCT

GCTGGCGTGGTCAACTCACCTAATAAAGCAACCGAGCAGGACTTCATGACTGGTCTT

ATGAACTCCACAAAAGAGTTAGTACCGAACGACCCATTGACTCAACAGCTTGTGTTG

AAG ATTT AT G AGGC G A ACGGT GTT A ACTT G AGGG AGCGT AGG A A AT A A

SEQ ID NO: 62

// WT AA sequence T7 internal virion protein D (e.g., T7p51)

MDKYDKNVPSDYDGLFQKAADANGVSYDLLRKVAWTESRFVPTAKSKTGPLGMMQF TKATAKALGLRVTDGPDDDRLNPELAINAAAKQLAGLVGKFDGDELKAALAYNQGEG RLGNPQLEAYSKGDFASISEEGRNYMRNLLDVAKSPMAGQLETFGGITPKGKGIPAEVG LAGIGHKQKVTQELPESTSFDVKGIEQEATAKPFAKDFWETHGETLDEYNSRSTFFGFKN AAE AELS NS V AGM AFRAGRLDN GFD VFKDTITPTRWN S HIWTPEELEKIRTE VKNP A YI NV VT GGS PENLDDLIKLANENFENDS R A AE AGLG AKLS AGIIG AG VDPLS Y VPM V G VT G KGFKLINKALV V G AES A ALN V AS EGLRT S V AGGD AD Y AG A ALGGFVF G AGMS AIS DA VAAGLKRSKPEAEFDNEFIGPMMRLEARETARNANSADLSRMNTENMKFEGEHNGVPY EDLPTERG A V VLHD GS VLS AS NPINPKTLKEFS E VDPEKA ARGIKL AGFTEIGLKTLGS D DADIRRVAIDLVRSPTGMQSGASGKFGATASDIHERLHGTDQRTYNDLYKAMSDAMKD PEFS T GG ARMS REETR YTIYRR A ALAIERPELQKALTPS ERIVMDIIKRHFDTKRELMENP AIFGNTKAVSIFPESRHKGTYVPHVYDRHAKALMIQRYGAEGLQEGIARSWMNSYVSRP EVKARVDEMLKELHGVKEVTPEMVEKY AMDKAY GISHSDQFTNSSIIEENIEGLVGIEN N S FLE ARNLFDS DLS ITMPDGQQF S VNDLRDFDMFRIMP A YDRR VN GDIAIMGS T GKTT KELKDEILALKAKAEGDGKKTGEVHALMDTVKILTGRARRNQDTVWETSLRAINDLGF FAKNAYMGAQNITEIAGMIVTGNVRALGHGIPILRDTLYKSKPVSAKELKELHASLFGK E VDQLIRPKR ADIV QRLRE ATDT GP A V ANIV GTLKY S T QELA ARS PWTKLLN GTTN YLL DAARQGMLGDVISATLTGKTTRWEKEGFLRGASVTPEQMAGIKSLIKEHMVRGEDGKF TVKDKQAFSMDPRAMDLWRLADKVADEAMLRPHKVSLQDSHAFGALGKMVMQFKSF TIKS LN S KFLRTFYDGYKNNRAID AALS IITSMGLAGGFY AM AAHVKA Y ALPKEKRKE Y LERALDPTMIAH A ALS RS S QLG APLAM VDLV GG VLGFES S KM ARS TILPKDT VKERDPN KP YT S RE VMG AMGS NLLEQMPS AGFV AN V G ATLMN A AG V VN S PNKATEQDFMTGLM NSTKELVPNDPLTQQLVLKIYEANGVNLRERRK

[000213] The nucleotide in position 32893 may be modified from G to C. This region may be recognized by looking for the sequence of the regions before and after the modification (e.g., CATTCCTT before and AGGCACGT after, etc.). 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs before and/or 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs after may be identified as 100% identical, 95% or more identical, 90% or more or more identical, 85% or more identical, etc. For example:

SEQ ID NO: 63

Type: DNA

Organism: Artificial sequence derived from T7 internal virion protein D (e.g., T7p51)

CATTCCTTCAGGCACGT

SEQ ID NO: 64

Type: DNA

// Mut DNA sequence derived from T7 internal virion protein D (e.g., T7p51)

ATGGATAAGTACGATAAGAACGTACCAAGTGATTATGATGGTCTGTTCCAAAAGGC

TGCTGATGCCAACGGGGTCTCTTATGACCTTTTACGTAAAGTCGCTTGGACAGAATC

ACGATTTGTGCCTACAGCAAAATCTAAGACTGGACCATTAGGCATGATGCAATTTAC

CAAGGCAACCGCTAAGGCCCTCGGTCTGCGAGTTACCGATGGTCCAGACGACGACC

GACTGAACCCTGAGTTAGCTATTAATGCTGCCGCTAAGCAACTTGCAGGTCTGGTAG

GGAAGTTTGATGGCGATGAACTCAAAGCTGCCCTTGCGTACAACCAAGGCGAGGGA

CGCTTGGGTAATCCACAACTTGAGGCGTACTCTAAGGGAGACTTCGCATCAATCTCT

GAGGAGGGACGTAACTACATGCGTAACCTTCTGGATGTTGCTAAGTCACCTATGGCT GGACAGTTGGAAACTTTTGGTGGCATAACCCCAAAGGGTAAAGGCATTCCGGCTGA

GGTAGGATTGGCTGGAATTGGTCACAAGCAGAAAGTAACACAGGAACTTCCTGAGT

CCACAAGTTTTGACGTTAAGGGTATCGAACAGGAGGCTACGGCGAAACCATTCGCC

AAGGACTTTTGGGAGACCCACGGAGAAACACTTGACGAGTACAACAGTCGTTCAAC

CTTCTTCGGATTCAAAAATGCTGCCGAAGCTGAACTCTCCAACTCAGTCGCTGGGAT

GGCTTTCCGTGCTGGTCGTCTCGATAATGGTTTTGATGTGTTTAAAGACACCATTACG

CCGACTCGCTGGAACTCTCACATCTGGACTCCAGAGGAGTTAGAGAAGATTCGAAC

AGAGGTTAAGAACCCTGCGTACATCAACGTTGTAACTGGTGGTTCCCCTGAGAACCT

CGATGACCTCATTAAATTGGCTAACGAGAACTTTGAGAATGACTCCCGCGCTGCCGA

GGCTGGCCTAGGTGCCAAACTGAGTGCTGGTATTATTGGTGCTGGTGTGGACCCGCT

TAGCTATGTTCCTATGGTCGGTGTCACTGGTAAGGGCTTTAAGTTAATCAATAAGGC

TCTTGTAGTTGGTGCCGAAAGTGCTGCTCTGAACGTTGCATCCGAAGGTCTCCGTAC

CTCCGTAGCTGGTGGTGACGCAGACTATGCGGGTGCTGCCTTAGGTGGCTTTGTGTT

TGGCGCAGGCATGTCTGCAATCAGTGACGCTGTAGCTGCTGGACTGAAACGCAGTA

AACCAGAAGCTGAGTTCGACAATGAGTTCATCGGTCCTATGATGCGATTGGAAGCC

CGTGAGACAGCACGAAACGCCAACTCTGCGGACCTCTCTCGGATGAACACTGAGAA

CATGAAGTTTGAAGGTGAACATAATGGTGTCCCTTATGAGGACTTACCAACAGAGA

GAGGTGCCGTGGTGTTACATGATGGCTCCGTTCTAAGTGCAAGCAACCCAATCAACC

CTAAGACTCTAAAAGAGTTCTCCGAGGTTGACCCTGAGAAGGCTGCGCGAGGAATC

AAACTGGCTGGGTTCACCGAGATTGGCTTGAAGACCTTGGGGTCTGACGATGCTGAC

ATCCGTAGAGTGGCTATCGACCTCGTTCGCTCTCCTACTGGTATGCAGTCTGGTGCCT

CAGGTAAGTTCGGTGCAACAGCTTCTGACATCCATGAGAGACTTCATGGTACTGACC

AGCGTACTTATAATGACTTGTACAAAGCAATGTCTGACGCTATGAAAGACCCTGAGT

TCTCTACTGGCGGCGCTAAGATGTCCCGTGAAGAAACTCGATACACTATCTACCGTA

GAGCGGCACTAGCTATTGAGCGTCCAGAACTACAGAAGGCACTCACTCCGTCTGAG

AGAATCGTTATGGACATCATTAAGCGTCACTTTGACACCAAGCGTGAACTTATGGAA

AACCCAGCAATATTCGGTAACACAAAGGCTGTGAGTATCTTCCCTGAGAGTCGCCAC

AAAGGTACTTACGTTCCTCACGTATATGACCGTCATGCCAAGGCGCTGATGATTCAA

CGCTACGGTGCCGAAGGTTTGCAGGAAGGGATTGCCCGCTCATGGATGAACAGCTA

CGTCTCCAGACCTGAGGTCAAGGCCAGAGTCGATGAGATGCTTAAGGAATTACACG

GGGTGAAGGAAGTAACACCAGAGATGGTAGAGAAGTACGCTATGGATAAGGCTTAT

GGTATCTCCCACTCAGACCAGTTCACCAACAGTTCCATAATAGAAGAGAACATTGA

GGGCTTAGTAGGTATCGAGAATAACTCATTCCTTCAGGCACGTAACTTGTTTGATTC

GGACCTATCCATCACTATGCCAGACGGACAGCAATTCTCAGTGAATGACCTAAGGG

ACTTCGATATGTTCCGCATCATGCCAGCGTATGACCGCCGTGTCAATGGTGACATCG CCATCATGGGGTCTACTGGTAAAACCACTAAGGAACTTAAGGATGAGATTTTGGCTC

TCAAAGCGAAAGCTGAGGGAGACGGTAAGAAGACTGGCGAGGTACATGCTTTAATG

GATACCGTTAAGATTCTTACTGGTCGTGCTAGACGCAATCAGGACACTGTGTGGGAA

ACCTCACTGCGTGCCATCAATGACCTAGGGTTCTTCGCTAAGAACGCCTACATGGGT

GCTCAGAACATTACGGAGATTGCTGGGATGATTGTCACTGGTAACGTTCGTGCTCTA

GGGCATGGTATCCCAATTCTGCGTGATACACTCTACAAGTCTAAACCAGTTTCAGCT

AAGGAACTCAAGGAACTCCATGCGTCTCTGTTCGGGAAGGAGGTGGACCAGTTGAT

TCGGCCTAAACGTGCTGACATTGTGCAGCGCCTAAGGGAAGCAACTGATACCGGAC

CTGCCGTGGCGAACATCGTAGGGACCTTGAAGTATTCAACACAGGAACTGGCTGCT

CGCTCTCCGTGGACTAAGCTACTGAACGGAACCACTAACTACCTTCTGGATGCTGCG

CGTCAAGGTATGCTTGGGGATGTTATTAGTGCCACCCTAACAGGTAAGACTACCCGC

TGGGAGAAAGAAGGCTTCCTTCGTGGTGCCTCCGTAACTCCTGAGCAGATGGCTGGC

ATCAAGTCTCTCATCAAGGAACATATGGTACGCGGTGAGGACGGGAAGTTTACCGT

TAAGGACAAGCAAGCGTTCTCTATGGACCCACGGGCTATGGACTTATGGAGACTGG

CTGACAAGGTAGCTGATGAGGCAATGCTGCGTCCACATAAGGTGTCCTTACAGGATT

CCCATGCGTTCGGAGCACTAGGTAAGATGGTTATGCAGTTTAAGTCTTTCACTATCA

AGTCCCTTAACTCTAAGTTCCTGCGAACCTTCTATGATGGATACAAGAACAACCGAG

CGATTGACGCTGCGCTGAGCATCATCACCTCTATGGGTCTCGCTGGTGGTTTCTATG

TTGGAGCGTGCACTGGACCCAACCATGATTGCCCACGCTGCGTTATCTCGTAGTTCT

CAATTGGGTGCTCCTTTGGCTATGGTTGACCTAGTTGGTGGTGTTTTAGGGTTCGAGT

CCTCCAAGATGGCTCGCTCTACGATTCTACCTAAGGACACCGTGAAGGAACGTGACC

CAAACAAACCGTACACCTCTAGAGAGGTAATGGGCGCTATGGGTTCAAACCTTCTG

GAACAGATGCCTTCGGCTGGCTTTGTGGCTAACGTAGGGGCTACCTTAATGAATGCT

GCTGGCGTGGTCAACTCACCTAATAAAGCAACCGAGCAGGACTTCATGACTGGTCTT

ATGAACTCCACAAAAGAGTTAGTACCGAACGACCCATTGACTCAACAGCTTGTGTTG

AAG ATTT AT G AGGC G A ACGGT GTT A ACTT G AGGG AGCGT AGG A A AT A A

SEQ ID NO: 65

Type: PROTEIN

NSFLQARNL SEQ ID NO: 66

Type:PRT

//Mut AA sequence derived from T7 internal virion protein D (e.g., T7p51)

MDKYDKNVPSDYDGLFQKAADANGVSYDLLRKVAWTESRFVPTAKSKTGPLGMMQF

TKATAKALGLRVTDGPDDDRLNPELAINAAAKQLAGLVGKFDGDELKAALAYNQGEG

RLGNPQLEAYSKGDFASISEEGRNYMRNLLDVAKSPMAGQLETFGGITPKGKGIPAEVG

LAGIGHKQKVTQELPESTSFDVKGIEQEATAKPFAKDFWETHGETLDEYNSRSTFFGFKN

AAE AELS NS V AGM AFRAGRLDN GFD VFKDTITPTRWN S HIWTPEELEKIRTE VKNP A YI

NV VT GGS PENLDDLIKLANENFENDS R A AE AGLG AKLS AGIIG AG VDPLS Y VPM V G VT G

KGFKLINKALV V G AES A ALN V AS EGLRT S V AGGD AD Y AG A ALGGFVF G AGMS AIS DA

VAAGLKRSKPEAEFDNEFIGPMMRLEARETARNANSADLSRMNTENMKFEGEHNGVPY

EDLPTERG A V VLHD GS VLS AS NPINPKTLKEFS E VDPEKA ARGIKL AGFTEIGLKTLGS D

DADIRRVAIDLVRSPTGMQSGASGKFGATASDIHERLHGTDQRTYNDLYKAMSDAMKD

PEFS T GG ARMS REETR YTIYRR A ALAIERPELQKALTPS ERIVMDIIKRHFDTKRELMENP

AIFGNTKAVSIFPESRHKGTYVPHVYDRHAKALMIQRYGAEGLQEGIARSWMNSYVSRP

EVKARVDEMLKELHGVKEVTPEMVEKY AMDKAY GISHSDQFTNSSIIEENIEGLVGIEN

NSFLQARNLFDSDLSITMPDGQQFSVNDLRDFDMFRIMPAYDRRVNGDIAIMGSTGKTT

KELKDEILALKAKAEGDGKKTGEVHALMDTVKILTGRARRNQDTVWETSLRAINDLGF

FAKNAYMGAQNITEIAGMIVTGNVRALGHGIPILRDTLYKSKPVSAKELKELHASLFGK

E VDQLIRPKR ADIV QRLRE ATDT GP A V ANIV GTLKY S T QELA ARS PWTKLLN GTTN YLL

DAARQGMLGDVISATLTGKTTRWEKEGFLRGASVTPEQMAGIKSLIKEHMVRGEDGKF

TVKDKQAFSMDPRAMDLWRLADKVADEAMLRPHKVSLQDSHAFGALGKMVMQFKSF

TIKS LN S KFLRTFYDGYKNNRAID AALS IITSMGLAGGFY AM AAHVKA Y ALPKEKRKE Y

LERALDPTMIAH A ALS RS S QLG APLAM VDLV GG VLGFES S KM ARS TILPKDT VKERDPN

KP YT S RE VMG AMGS NLLEQMPS AGFV AN V G ATLMN A AG V VN S PNKATEQDFMTGLM

NSTKELVPNDPLTQQLVLKIYEANGVNLRERRK

[000214] The nucleotide in position 33479 may be modified from G to T (or C). This region may be recognized by looking for the sequence of the regions before and after the modification (e.g., TGATACCG before and ACCTGCCG after, etc.). 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs before and/or 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. base pairs after may be identified as 100% identical, 95% or more identical, 90% or more or more identical, 85% or more identical, etc. For example:

SEQ ID NO: 67

Type: DNA

TGATACCGTACCTGCCG

SEQ ID NO: 68

Type: DNA

//Mut DNA sequence derived from T7 internal virion protein D (e.g., T7p51)

ATGGATAAGTACGATAAGAACGTACCAAGTGATTATGATGGTCTGTTCCAAAAGGC

TGCTGATGCCAACGGGGTCTCTTATGACCTTTTACGTAAAGTCGCTTGGACAGAATC

ACGATTTGTGCCTACAGCAAAATCTAAGACTGGACCATTAGGCATGATGCAATTTAC

CAAGGCAACCGCTAAGGCCCTCGGTCTGCGAGTTACCGATGGTCCAGACGACGACC

GACTGAACCCTGAGTTAGCTATTAATGCTGCCGCTAAGCAACTTGCAGGTCTGGTAG

GGAAGTTTGATGGCGATGAACTCAAAGCTGCCCTTGCGTACAACCAAGGCGAGGGA

CGCTTGGGTAATCCACAACTTGAGGCGTACTCTAAGGGAGACTTCGCATCAATCTCT

GAGGAGGGACGTAACTACATGCGTAACCTTCTGGATGTTGCTAAGTCACCTATGGCT

GGACAGTTGGAAACTTTTGGTGGCATAACCCCAAAGGGTAAAGGCATTCCGGCTGA

GGTAGGATTGGCTGGAATTGGTCACAAGCAGAAAGTAACACAGGAACTTCCTGAGT

CCACAAGTTTTGACGTTAAGGGTATCGAACAGGAGGCTACGGCGAAACCATTCGCC

AAGGACTTTTGGGAGACCCACGGAGAAACACTTGACGAGTACAACAGTCGTTCAAC

CTTCTTCGGATTCAAAAATGCTGCCGAAGCTGAACTCTCCAACTCAGTCGCTGGGAT

GGCTTTCCGTGCTGGTCGTCTCGATAATGGTTTTGATGTGTTTAAAGACACCATTACG

CCGACTCGCTGGAACTCTCACATCTGGACTCCAGAGGAGTTAGAGAAGATTCGAAC

AGAGGTTAAGAACCCTGCGTACATCAACGTTGTAACTGGTGGTTCCCCTGAGAACCT

CGATGACCTCATTAAATTGGCTAACGAGAACTTTGAGAATGACTCCCGCGCTGCCGA

GGCTGGCCTAGGTGCCAAACTGAGTGCTGGTATTATTGGTGCTGGTGTGGACCCGCT

TAGCTATGTTCCTATGGTCGGTGTCACTGGTAAGGGCTTTAAGTTAATCAATAAGGC

TCTTGTAGTTGGTGCCGAAAGTGCTGCTCTGAACGTTGCATCCGAAGGTCTCCGTAC

CTCCGTAGCTGGTGGTGACGCAGACTATGCGGGTGCTGCCTTAGGTGGCTTTGTGTT TGGCGCAGGCATGTCTGCAATCAGTGACGCTGTAGCTGCTGGACTGAAACGCAGTA

AACCAGAAGCTGAGTTCGACAATGAGTTCATCGGTCCTATGATGCGATTGGAAGCC

CGTGAGACAGCACGAAACGCCAACTCTGCGGACCTCTCTCGGATGAACACTGAGAA

CATGAAGTTTGAAGGTGAACATAATGGTGTCCCTTATGAGGACTTACCAACAGAGA

GAGGTGCCGTGGTGTTACATGATGGCTCCGTTCTAAGTGCAAGCAACCCAATCAACC

CTAAGACTCTAAAAGAGTTCTCCGAGGTTGACCCTGAGAAGGCTGCGCGAGGAATC

AAACTGGCTGGGTTCACCGAGATTGGCTTGAAGACCTTGGGGTCTGACGATGCTGAC

ATCCGTAGAGTGGCTATCGACCTCGTTCGCTCTCCTACTGGTATGCAGTCTGGTGCCT

CAGGTAAGTTCGGTGCAACAGCTTCTGACATCCATGAGAGACTTCATGGTACTGACC

AGCGTACTTATAATGACTTGTACAAAGCAATGTCTGACGCTATGAAAGACCCTGAGT

TCTCTACTGGCGGCGCTAAGATGTCCCGTGAAGAAACTCGATACACTATCTACCGTA

GAGCGGCACTAGCTATTGAGCGTCCAGAACTACAGAAGGCACTCACTCCGTCTGAG

AGAATCGTTATGGACATCATTAAGCGTCACTTTGACACCAAGCGTGAACTTATGGAA

AACCCAGCAATATTCGGTAACACAAAGGCTGTGAGTATCTTCCCTGAGAGTCGCCAC

AAAGGTACTTACGTTCCTCACGTATATGACCGTCATGCCAAGGCGCTGATGATTCAA

CGCTACGGTGCCGAAGGTTTGCAGGAAGGGATTGCCCGCTCATGGATGAACAGCTA

CGTCTCCAGACCTGAGGTCAAGGCCAGAGTCGATGAGATGCTTAAGGAATTACACG

GGGTGAAGGAAGTAACACCAGAGATGGTAGAGAAGTACGCTATGGATAAGGCTTAT

GGTATCTCCCACTCAGACCAGTTCACCAACAGTTCCATAATAGAAGAGAACATTGA

GGGCTTAGTAGGTATCGAGAATAACTCATTCCTTGAGGCACGTAACTTGTTTGATTC

GGACCTATCCATCACTATGCCAGACGGACAGCAATTCTCAGTGAATGACCTAAGGG

ACTTCGATATGTTCCGCATCATGCCAGCGTATGACCGCCGTGTCAATGGTGACATCG

CCATCATGGGGTCTACTGGTAAAACCACTAAGGAACTTAAGGATGAGATTTTGGCTC

TCAAAGCGAAAGCTGAGGGAGACGGTAAGAAGACTGGCGAGGTACATGCTTTAATG

GATACCGTTAAGATTCTTACTGGTCGTGCTAGACGCAATCAGGACACTGTGTGGGAA

ACCTCACTGCGTGCCATCAATGACCTAGGGTTCTTCGCTAAGAACGCCTACATGGGT

GCTCAGAACATTACGGAGATTGCTGGGATGATTGTCACTGGTAACGTTCGTGCTCTA

GGGCATGGTATCCCAATTCTGCGTGATACACTCTACAAGTCTAAACCAGTTTCAGCT

AAGGAACTCAAGGAACTCCATGCGTCTCTGTTCGGGAAGGAGGTGGACCAGTTGAT

TCGGCCTAAACGTGCTGACATTGTGCAGCGCCTAAGGGAAGCAACTGATACCG!AC

CTGCCGTGGCGAACATCGTAGGGACCTTGAAGTATTCAACACAGGAACTGGCTGCT

CGCTCTCCGTGGACTAAGCTACTGAACGGAACCACTAACTACCTTCTGGATGCTGCG

CGTCAAGGTATGCTTGGGGATGTTATTAGTGCCACCCTAACAGGTAAGACTACCCGC

TGGGAGAAAGAAGGCTTCCTTCGTGGTGCCTCCGTAACTCCTGAGCAGATGGCTGGC

ATCAAGTCTCTCATCAAGGAACATATGGTACGCGGTGAGGACGGGAAGTTTACCGT TAAGGACAAGCAAGCGTTCTCTATGGACCCACGGGCTATGGACTTATGGAGACTGG

CTGACAAGGTAGCTGATGAGGCAATGCTGCGTCCACATAAGGTGTCCTTACAGGATT

CCCATGCGTTCGGAGCACTAGGTAAGATGGTTATGCAGTTTAAGTCTTTCACTATCA

AGTCCCTTAACTCTAAGTTCCTGCGAACCTTCTATGATGGATACAAGAACAACCGAG

CGATTGACGCTGCGCTGAGCATCATCACCTCTATGGGTCTCGCTGGTGGTTTCTATG

TTGGAGCGTGCACTGGACCCAACCATGATTGCCCACGCTGCGTTATCTCGTAGTTCT

CAATTGGGTGCTCCTTTGGCTATGGTTGACCTAGTTGGTGGTGTTTTAGGGTTCGAGT

CCTCCAAGATGGCTCGCTCTACGATTCTACCTAAGGACACCGTGAAGGAACGTGACC

CAAACAAACCGTACACCTCTAGAGAGGTAATGGGCGCTATGGGTTCAAACCTTCTG

GAACAGATGCCTTCGGCTGGCTTTGTGGCTAACGTAGGGGCTACCTTAATGAATGCT

GCTGGCGTGGTCAACTCACCTAATAAAGCAACCGAGCAGGACTTCATGACTGGTCTT

ATGAACTCCACAAAAGAGTTAGTACCGAACGACCCATTGACTCAACAGCTTGTGTTG

AAG ATTT AT G AGGC G A ACGGT GTT A ACTT G AGGG AGCGT AGG A A AT A A

SEQ ID NO: 69

Type: DNA

TGATACCGCACCTGCCG

SEQ ID NO: 70

Type: DNA

// Mut DNA sequence derived from T7 internal virion protein D (e.g., T7p51)

ATGGATAAGTACGATAAGAACGTACCAAGTGATTATGATGGTCTGTTCCAAAAGGC

TGCTGATGCCAACGGGGTCTCTTATGACCTTTTACGTAAAGTCGCTTGGACAGAATC

ACGATTTGTGCCTACAGCAAAATCTAAGACTGGACCATTAGGCATGATGCAATTTAC

CAAGGCAACCGCTAAGGCCCTCGGTCTGCGAGTTACCGATGGTCCAGACGACGACC

GACTGAACCCTGAGTTAGCTATTAATGCTGCCGCTAAGCAACTTGCAGGTCTGGTAG

GGAAGTTTGATGGCGATGAACTCAAAGCTGCCCTTGCGTACAACCAAGGCGAGGGA

CGCTTGGGTAATCCACAACTTGAGGCGTACTCTAAGGGAGACTTCGCATCAATCTCT

GGTAGGATTGGCTGGAATTGGTCACAAGCAGAAAGTAACACAGGAACTTCCTGAGT

CCACAAGTTTTGACGTTAAGGGTATCGAACAGGAGGCTACGGCGAAACCATTCGCC

AAGGACTTTTGGGAGACCCACGGAGAAACACTTGACGAGTACAACAGTCGTTCAAC

CTTCTTCGGATTCAAAAATGCTGCCGAAGCTGAACTCTCCAACTCAGTCGCTGGGAT

GGCTTTCCGTGCTGGTCGTCTCGATAATGGTTTTGATGTGTTTAAAGACACCATTACG

CCGACTCGCTGGAACTCTCACATCTGGACTCCAGAGGAGTTAGAGAAGATTCGAAC

AGAGGTTAAGAACCCTGCGTACATCAACGTTGTAACTGGTGGTTCCCCTGAGAACCT

CGATGACCTCATTAAATTGGCTAACGAGAACTTTGAGAATGACTCCCGCGCTGCCGA

GGCTGGCCTAGGTGCCAAACTGAGTGCTGGTATTATTGGTGCTGGTGTGGACCCGCT

TAGCTATGTTCCTATGGTCGGTGTCACTGGTAAGGGCTTTAAGTTAATCAATAAGGC

TCTTGTAGTTGGTGCCGAAAGTGCTGCTCTGAACGTTGCATCCGAAGGTCTCCGTAC

CTCCGTAGCTGGTGGTGACGCAGACTATGCGGGTGCTGCCTTAGGTGGCTTTGTGTT

TGGCGCAGGCATGTCTGCAATCAGTGACGCTGTAGCTGCTGGACTGAAACGCAGTA

AACCAGAAGCTGAGTTCGACAATGAGTTCATCGGTCCTATGATGCGATTGGAAGCC

CGTGAGACAGCACGAAACGCCAACTCTGCGGACCTCTCTCGGATGAACACTGAGAA

CATGAAGTTTGAAGGTGAACATAATGGTGTCCCTTATGAGGACTTACCAACAGAGA

GAGGTGCCGTGGTGTTACATGATGGCTCCGTTCTAAGTGCAAGCAACCCAATCAACC

CTAAGACTCTAAAAGAGTTCTCCGAGGTTGACCCTGAGAAGGCTGCGCGAGGAATC

AAACTGGCTGGGTTCACCGAGATTGGCTTGAAGACCTTGGGGTCTGACGATGCTGAC

ATCCGTAGAGTGGCTATCGACCTCGTTCGCTCTCCTACTGGTATGCAGTCTGGTGCCT

CAGGTAAGTTCGGTGCAACAGCTTCTGACATCCATGAGAGACTTCATGGTACTGACC

AGCGTACTTATAATGACTTGTACAAAGCAATGTCTGACGCTATGAAAGACCCTGAGT

TCTCTACTGGCGGCGCTAAGATGTCCCGTGAAGAAACTCGATACACTATCTACCGTA

GAGCGGCACTAGCTATTGAGCGTCCAGAACTACAGAAGGCACTCACTCCGTCTGAG

AGAATCGTTATGGACATCATTAAGCGTCACTTTGACACCAAGCGTGAACTTATGGAA

AACCCAGCAATATTCGGTAACACAAAGGCTGTGAGTATCTTCCCTGAGAGTCGCCAC

AAAGGTACTTACGTTCCTCACGTATATGACCGTCATGCCAAGGCGCTGATGATTCAA

CGCTACGGTGCCGAAGGTTTGCAGGAAGGGATTGCCCGCTCATGGATGAACAGCTA

CGTCTCCAGACCTGAGGTCAAGGCCAGAGTCGATGAGATGCTTAAGGAATTACACG

GGGTGAAGGAAGTAACACCAGAGATGGTAGAGAAGTACGCTATGGATAAGGCTTAT

GGTATCTCCCACTCAGACCAGTTCACCAACAGTTCCATAATAGAAGAGAACATTGA

GGGCTTAGTAGGTATCGAGAATAACTCATTCCTTGAGGCACGTAACTTGTTTGATTC

GGACCTATCCATCACTATGCCAGACGGACAGCAATTCTCAGTGAATGACCTAAGGG

TCAAAGCGAAAGCTGAGGGAGACGGTAAGAAGACTGGCGAGGTACATGCTTTAATG

GATACCGTTAAGATTCTTACTGGTCGTGCTAGACGCAATCAGGACACTGTGTGGGAA

ACCTCACTGCGTGCCATCAATGACCTAGGGTTCTTCGCTAAGAACGCCTACATGGGT

GCTCAGAACATTACGGAGATTGCTGGGATGATTGTCACTGGTAACGTTCGTGCTCTA

GGGCATGGTATCCCAATTCTGCGTGATACACTCTACAAGTCTAAACCAGTTTCAGCT

AAGGAACTCAAGGAACTCCATGCGTCTCTGTTCGGGAAGGAGGTGGACCAGTTGAT

TCGGCCTAAACGTGCTGACATTGTGCAGCGCCTAAGGGAAGCAACTGATACCGCAC

CTGCCGTGGCGAACATCGTAGGGACCTTGAAGTATTCAACACAGGAACTGGCTGCT

CGCTCTCCGTGGACTAAGCTACTGAACGGAACCACTAACTACCTTCTGGATGCTGCG

CGTCAAGGTATGCTTGGGGATGTTATTAGTGCCACCCTAACAGGTAAGACTACCCGC

TGGGAGAAAGAAGGCTTCCTTCGTGGTGCCTCCGTAACTCCTGAGCAGATGGCTGGC

ATCAAGTCTCTCATCAAGGAACATATGGTACGCGGTGAGGACGGGAAGTTTACCGT

TAAGGACAAGCAAGCGTTCTCTATGGACCCACGGGCTATGGACTTATGGAGACTGG

CTGACAAGGTAGCTGATGAGGCAATGCTGCGTCCACATAAGGTGTCCTTACAGGATT

CCCATGCGTTCGGAGCACTAGGTAAGATGGTTATGCAGTTTAAGTCTTTCACTATCA

AGTCCCTTAACTCTAAGTTCCTGCGAACCTTCTATGATGGATACAAGAACAACCGAG

CGATTGACGCTGCGCTGAGCATCATCACCTCTATGGGTCTCGCTGGTGGTTTCTATG

TTGGAGCGTGCACTGGACCCAACCATGATTGCCCACGCTGCGTTATCTCGTAGTTCT

CAATTGGGTGCTCCTTTGGCTATGGTTGACCTAGTTGGTGGTGTTTTAGGGTTCGAGT

CCTCCAAGATGGCTCGCTCTACGATTCTACCTAAGGACACCGTGAAGGAACGTGACC

CAAACAAACCGTACACCTCTAGAGAGGTAATGGGCGCTATGGGTTCAAACCTTCTG

GAACAGATGCCTTCGGCTGGCTTTGTGGCTAACGTAGGGGCTACCTTAATGAATGCT

GCTGGCGTGGTCAACTCACCTAATAAAGCAACCGAGCAGGACTTCATGACTGGTCTT

ATGAACTCCACAAAAGAGTTAGTACCGAACGACCCATTGACTCAACAGCTTGTGTTG

AAG ATTT AT G AGGC G A ACGGT GTT A ACTT G AGGG AGCGT AGG A A AT A A

SEQ ID NO: 71

Type: PROTEIN

ATDTVPAVA SEQ ID NO: 72

Type: PROTEIN

//Mut AA sequence derived from T7 internal virion protein D (e.g., T7p51)

MDKYDKNVPSDYDGLFQKAADANGVSYDLLRKVAWTESRFVPTAKSKTGPLGMMQF TKATAKALGLRVTDGPDDDRLNPELAINAAAKQLAGLVGKFDGDELKAALAYNQGEG RLGNPQLEAYSKGDFASISEEGRNYMRNLLDVAKSPMAGQLETFGGITPKGKGIPAEVG LAGIGHKQKVTQELPESTSFDVKGIEQEATAKPFAKDFWETHGETLDEYNSRSTFFGFKN AAE AELS NS V AGM AFRAGRLDN GFD VFKDTITPTRWN S HIWTPEELEKIRTE VKNP A YI NV VT GGS PENLDDLIKLANENFENDS R A AE AGLG AKLS AGIIG AG VDPLS Y VPM V G VT G KGFKLINKALV V G AES A ALN V AS EGLRT S V AGGD AD Y AG A ALGGFVF G AGMS AIS DA VAAGLKRSKPEAEFDNEFIGPMMRLEARETARNANSADLSRMNTENMKFEGEHNGVPY EDLPTERG A V VLHD GS VLS AS NPINPKTLKEFS E VDPEKA ARGIKL AGFTEIGLKTLGS D DADIRRVAIDLVRSPTGMQSGASGKFGATASDIHERLHGTDQRTYNDLYKAMSDAMKD PEFS T GG ARMS REETR YTIYRR A ALAIERPELQKALTPS ERIVMDIIKRHFDTKRELMENP AIFGNTKAVSIFPESRHKGTYVPHVYDRHAKALMIQRYGAEGLQEGIARSWMNSYVSRP EVKARVDEMLKELHGVKEVTPEMVEKY AMDKAY GISHSDQFTNSSIIEENIEGLVGIEN N S FLE ARNLFDS DLS ITMPDGQQF S VNDLRDFDMFRIMP A YDRR VN GDIAIMGS T GKTT KELKDEILALKAKAEGDGKKTGEVHALMDTVKILTGRARRNQDTVWETSLRAINDLGF FAKNAYMGAQNITEIAGMIVTGNVRALGHGIPILRDTLYKSKPVSAKELKELHASLFGK E VDQLIRPKR ADIV QRLRE ATDT VP A V ANIV GTLKY S T QELA ARS PWTKLLN GTTN YLL DAARQGMLGDVISATLTGKTTRWEKEGFLRGASVTPEQMAGIKSLIKEHMVRGEDGKF TVKDKQAFSMDPRAMDLWRLADKVADEAMLRPHKVSLQDSHAFGALGKMVMQFKSF TIKS LN S KFLRTFYDGYKNNRAID AALS IITSMGLAGGFY AM AAHVKA Y ALPKEKRKE Y LERALDPTMIAH A ALS RS S QLG APLAM VDLV GG VLGFES S KM ARS TILPKDT VKERDPN KP YT S RE VMG AMGS NLLEQMPS AGFV AN V G ATLMN A AG V VN S PNKATEQDFMTGLM NSTKELVPNDPLTQQLVLKIYEANGVNLRERRK

SEQ ID NO: 73

Type: PROTEIN

ATDTAPAVA SEQ ID NO: 74

Type:PRT

//Mut AA sequence derived from T7 internal virion protein D (e.g., T7p51)

MDKYDKNVPSDYDGLFQKAADANGVSYDLLRKVAWTESRFVPTAKSKTGPLGMMQF TKATAKALGLRVTDGPDDDRLNPELAINAAAKQLAGLVGKFDGDELKAALAYNQGEG RLGNPQLEAYSKGDFASISEEGRNYMRNLLDVAKSPMAGQLETFGGITPKGKGIPAEVG LAGIGHKQKVTQELPESTSFDVKGIEQEATAKPFAKDFWETHGETLDEYNSRSTFFGFKN AAE AELS NS V AGM AFRAGRLDN GFD VFKDTITPTRWN S HIWTPEELEKIRTE VKNP A YI NV VT GGS PENLDDLIKLANENFENDS R A AE AGLG AKLS AGIIG AG VDPLS Y VPM V G VT G KGFKLINKALV V G AES A ALN V AS EGLRT S V AGGD AD Y AG A ALGGFVF G AGMS AIS DA VAAGLKRSKPEAEFDNEFIGPMMRLEARETARNANSADLSRMNTENMKFEGEHNGVPY EDLPTERG A V VLHD GS VLS AS NPINPKTLKEFS E VDPEKA ARGIKL AGFTEIGLKTLGS D DADIRRVAIDLVRSPTGMQSGASGKFGATASDIHERLHGTDQRTYNDLYKAMSDAMKD PEFS T GG ARMS REETR YTIYRR A ALAIERPELQKALTPS ERIVMDIIKRHFDTKRELMENP AIFGNTKAVSIFPESRHKGTYVPHVYDRHAKALMIQRYGAEGLQEGIARSWMNSYVSRP EVKARVDEMLKELHGVKEVTPEMVEKY AMDKAY GISHSDQFTNSSIIEENIEGLVGIEN N S FLE ARNLFDS DLS ITMPDGQQF S VNDLRDFDMFRIMP A YDRR VN GDIAIMGS T GKTT KELKDEILALKAKAEGDGKKTGEVHALMDTVKILTGRARRNQDTVWETSLRAINDLGF FAKNAYMGAQNITEIAGMIVTGNVRALGHGIPILRDTLYKSKPVSAKELKELHASLFGK E VDQLIRPKR ADIV QRLRE ATDT AP A V ANIV GTLKY S T QELA ARS PWTKLLN GTTN YLL DAARQGMLGDVISATLTGKTTRWEKEGFLRGASVTPEQMAGIKSLIKEHMVRGEDGKF TVKDKQAFSMDPRAMDLWRLADKVADEAMLRPHKVSLQDSHAFGALGKMVMQFKSF TIKS LN S KFLRTFYDGYKNNRAID AALS IITSMGLAGGFY AM AAHVKA Y ALPKEKRKE Y LERALDPTMIAH A ALS RS S QLG APLAM VDLV GG VLGFES S KM ARS TILPKDT VKERDPN KP YT S RE VMG AMGS NLLEQMPS AGFV AN V G ATLMN A AG V VN S PNKATEQDFMTGLM NSTKELVPNDPLTQQLVLKIYEANGVNLRERRK

[000215] Further, both positions 32893 and 33479 may be mutated at the same time, providing:

SEQ ID NO: 75

// Mut DNA sequence derived from T7 internal virion protein D (e.g., T7p51) ATGGATAAGTACGATAAGAACGTACCAAGTGATTATGATGGTCTGTTCCAAAAGGC

TGCTGATGCCAACGGGGTCTCTTATGACCTTTTACGTAAAGTCGCTTGGACAGAATC

ACGATTTGTGCCTACAGCAAAATCTAAGACTGGACCATTAGGCATGATGCAATTTAC

CAAGGCAACCGCTAAGGCCCTCGGTCTGCGAGTTACCGATGGTCCAGACGACGACC

GACTGAACCCTGAGTTAGCTATTAATGCTGCCGCTAAGCAACTTGCAGGTCTGGTAG

GGAAGTTTGATGGCGATGAACTCAAAGCTGCCCTTGCGTACAACCAAGGCGAGGGA

CGCTTGGGTAATCCACAACTTGAGGCGTACTCTAAGGGAGACTTCGCATCAATCTCT

GAGGAGGGACGTAACTACATGCGTAACCTTCTGGATGTTGCTAAGTCACCTATGGCT

GGACAGTTGGAAACTTTTGGTGGCATAACCCCAAAGGGTAAAGGCATTCCGGCTGA

GGTAGGATTGGCTGGAATTGGTCACAAGCAGAAAGTAACACAGGAACTTCCTGAGT

CCACAAGTTTTGACGTTAAGGGTATCGAACAGGAGGCTACGGCGAAACCATTCGCC

AAGGACTTTTGGGAGACCCACGGAGAAACACTTGACGAGTACAACAGTCGTTCAAC

CTTCTTCGGATTCAAAAATGCTGCCGAAGCTGAACTCTCCAACTCAGTCGCTGGGAT

GGCTTTCCGTGCTGGTCGTCTCGATAATGGTTTTGATGTGTTTAAAGACACCATTACG

CCGACTCGCTGGAACTCTCACATCTGGACTCCAGAGGAGTTAGAGAAGATTCGAAC

AGAGGTTAAGAACCCTGCGTACATCAACGTTGTAACTGGTGGTTCCCCTGAGAACCT

CGATGACCTCATTAAATTGGCTAACGAGAACTTTGAGAATGACTCCCGCGCTGCCGA

GGCTGGCCTAGGTGCCAAACTGAGTGCTGGTATTATTGGTGCTGGTGTGGACCCGCT

TAGCTATGTTCCTATGGTCGGTGTCACTGGTAAGGGCTTTAAGTTAATCAATAAGGC

TCTTGTAGTTGGTGCCGAAAGTGCTGCTCTGAACGTTGCATCCGAAGGTCTCCGTAC

CTCCGTAGCTGGTGGTGACGCAGACTATGCGGGTGCTGCCTTAGGTGGCTTTGTGTT

TGGCGCAGGCATGTCTGCAATCAGTGACGCTGTAGCTGCTGGACTGAAACGCAGTA

AACCAGAAGCTGAGTTCGACAATGAGTTCATCGGTCCTATGATGCGATTGGAAGCC

CGTGAGACAGCACGAAACGCCAACTCTGCGGACCTCTCTCGGATGAACACTGAGAA

CATGAAGTTTGAAGGTGAACATAATGGTGTCCCTTATGAGGACTTACCAACAGAGA

GAGGTGCCGTGGTGTTACATGATGGCTCCGTTCTAAGTGCAAGCAACCCAATCAACC

CTAAGACTCTAAAAGAGTTCTCCGAGGTTGACCCTGAGAAGGCTGCGCGAGGAATC

AAACTGGCTGGGTTCACCGAGATTGGCTTGAAGACCTTGGGGTCTGACGATGCTGAC

ATCCGTAGAGTGGCTATCGACCTCGTTCGCTCTCCTACTGGTATGCAGTCTGGTGCCT

CAGGTAAGTTCGGTGCAACAGCTTCTGACATCCATGAGAGACTTCATGGTACTGACC

AGCGTACTTATAATGACTTGTACAAAGCAATGTCTGACGCTATGAAAGACCCTGAGT

TCTCTACTGGCGGCGCTAAGATGTCCCGTGAAGAAACTCGATACACTATCTACCGTA

GAGCGGCACTAGCTATTGAGCGTCCAGAACTACAGAAGGCACTCACTCCGTCTGAG

AGAATCGTTATGGACATCATTAAGCGTCACTTTGACACCAAGCGTGAACTTATGGAA

AACCCAGCAATATTCGGTAACACAAAGGCTGTGAGTATCTTCCCTGAGAGTCGCCAC AAAGGTACTTACGTTCCTCACGTATATGACCGTCATGCCAAGGCGCTGATGATTCAA

CGCTACGGTGCCGAAGGTTTGCAGGAAGGGATTGCCCGCTCATGGATGAACAGCTA

CGTCTCCAGACCTGAGGTCAAGGCCAGAGTCGATGAGATGCTTAAGGAATTACACG

GGGTGAAGGAAGTAACACCAGAGATGGTAGAGAAGTACGCTATGGATAAGGCTTAT

GGTATCTCCCACTCAGACCAGTTCACCAACAGTTCCATAATAGAAGAGAACATTGA

GGGCTTAGTAGGTATCGAGAATAACTCATTCCTTCAGGCACGTAACTTGTTTGATTC

GGACCTATCCATCACTATGCCAGACGGACAGCAATTCTCAGTGAATGACCTAAGGG

ACTTCGATATGTTCCGCATCATGCCAGCGTATGACCGCCGTGTCAATGGTGACATCG

CCATCATGGGGTCTACTGGTAAAACCACTAAGGAACTTAAGGATGAGATTTTGGCTC

TCAAAGCGAAAGCTGAGGGAGACGGTAAGAAGACTGGCGAGGTACATGCTTTAATG

GATACCGTTAAGATTCTTACTGGTCGTGCTAGACGCAATCAGGACACTGTGTGGGAA

ACCTCACTGCGTGCCATCAATGACCTAGGGTTCTTCGCTAAGAACGCCTACATGGGT

GCTCAGAACATTACGGAGATTGCTGGGATGATTGTCACTGGTAACGTTCGTGCTCTA

GGGCATGGTATCCCAATTCTGCGTGATACACTCTACAAGTCTAAACCAGTTTCAGCT

AAGGAACTCAAGGAACTCCATGCGTCTCTGTTCGGGAAGGAGGTGGACCAGTTGAT

TCGGCCTAAACGTGCTGACATTGTGCAGCGCCTAAGGGAAGCAACTGATACCGTAC

CTGCCGTGGCGAACATCGTAGGGACCTTGAAGTATTCAACACAGGAACTGGCTGCT

CGCTCTCCGTGGACTAAGCTACTGAACGGAACCACTAACTACCTTCTGGATGCTGCG

CGTCAAGGTATGCTTGGGGATGTTATTAGTGCCACCCTAACAGGTAAGACTACCCGC

TGGGAGAAAGAAGGCTTCCTTCGTGGTGCCTCCGTAACTCCTGAGCAGATGGCTGGC

ATCAAGTCTCTCATCAAGGAACATATGGTACGCGGTGAGGACGGGAAGTTTACCGT

TAAGGACAAGCAAGCGTTCTCTATGGACCCACGGGCTATGGACTTATGGAGACTGG

CTGACAAGGTAGCTGATGAGGCAATGCTGCGTCCACATAAGGTGTCCTTACAGGATT

CCCATGCGTTCGGAGCACTAGGTAAGATGGTTATGCAGTTTAAGTCTTTCACTATCA

AGTCCCTTAACTCTAAGTTCCTGCGAACCTTCTATGATGGATACAAGAACAACCGAG

CGATTGACGCTGCGCTGAGCATCATCACCTCTATGGGTCTCGCTGGTGGTTTCTATG

TTGGAGCGTGCACTGGACCCAACCATGATTGCCCACGCTGCGTTATCTCGTAGTTCT

CAATTGGGTGCTCCTTTGGCTATGGTTGACCTAGTTGGTGGTGTTTTAGGGTTCGAGT

CCTCCAAGATGGCTCGCTCTACGATTCTACCTAAGGACACCGTGAAGGAACGTGACC

CAAACAAACCGTACACCTCTAGAGAGGTAATGGGCGCTATGGGTTCAAACCTTCTG

GAACAGATGCCTTCGGCTGGCTTTGTGGCTAACGTAGGGGCTACCTTAATGAATGCT

GCTGGCGTGGTCAACTCACCTAATAAAGCAACCGAGCAGGACTTCATGACTGGTCTT

ATGAACTCCACAAAAGAGTTAGTACCGAACGACCCATTGACTCAACAGCTTGTGTTG

AAG ATTT AT G AGGC G A ACGGT GTT A ACTT G AGGG AGCGT AGG A A AT A A SEQ ID NO: 76

// Mut DNA sequence derived from T7 internal virion protein D (e.g., T7p51)

ATGGATAAGTACGATAAGAACGTACCAAGTGATTATGATGGTCTGTTCCAAAAGGC

TGCTGATGCCAACGGGGTCTCTTATGACCTTTTACGTAAAGTCGCTTGGACAGAATC

ACGATTTGTGCCTACAGCAAAATCTAAGACTGGACCATTAGGCATGATGCAATTTAC

CAAGGCAACCGCTAAGGCCCTCGGTCTGCGAGTTACCGATGGTCCAGACGACGACC

GACTGAACCCTGAGTTAGCTATTAATGCTGCCGCTAAGCAACTTGCAGGTCTGGTAG

GGAAGTTTGATGGCGATGAACTCAAAGCTGCCCTTGCGTACAACCAAGGCGAGGGA

CGCTTGGGTAATCCACAACTTGAGGCGTACTCTAAGGGAGACTTCGCATCAATCTCT

GAGGAGGGACGTAACTACATGCGTAACCTTCTGGATGTTGCTAAGTCACCTATGGCT

GGACAGTTGGAAACTTTTGGTGGCATAACCCCAAAGGGTAAAGGCATTCCGGCTGA

GGTAGGATTGGCTGGAATTGGTCACAAGCAGAAAGTAACACAGGAACTTCCTGAGT

CCACAAGTTTTGACGTTAAGGGTATCGAACAGGAGGCTACGGCGAAACCATTCGCC

AAGGACTTTTGGGAGACCCACGGAGAAACACTTGACGAGTACAACAGTCGTTCAAC

CTTCTTCGGATTCAAAAATGCTGCCGAAGCTGAACTCTCCAACTCAGTCGCTGGGAT

GGCTTTCCGTGCTGGTCGTCTCGATAATGGTTTTGATGTGTTTAAAGACACCATTACG

CCGACTCGCTGGAACTCTCACATCTGGACTCCAGAGGAGTTAGAGAAGATTCGAAC

AGAGGTTAAGAACCCTGCGTACATCAACGTTGTAACTGGTGGTTCCCCTGAGAACCT

CGATGACCTCATTAAATTGGCTAACGAGAACTTTGAGAATGACTCCCGCGCTGCCGA

GGCTGGCCTAGGTGCCAAACTGAGTGCTGGTATTATTGGTGCTGGTGTGGACCCGCT

TAGCTATGTTCCTATGGTCGGTGTCACTGGTAAGGGCTTTAAGTTAATCAATAAGGC

TCTTGTAGTTGGTGCCGAAAGTGCTGCTCTGAACGTTGCATCCGAAGGTCTCCGTAC

CTCCGTAGCTGGTGGTGACGCAGACTATGCGGGTGCTGCCTTAGGTGGCTTTGTGTT

TGGCGCAGGCATGTCTGCAATCAGTGACGCTGTAGCTGCTGGACTGAAACGCAGTA

AACCAGAAGCTGAGTTCGACAATGAGTTCATCGGTCCTATGATGCGATTGGAAGCC

CGTGAGACAGCACGAAACGCCAACTCTGCGGACCTCTCTCGGATGAACACTGAGAA

CATGAAGTTTGAAGGTGAACATAATGGTGTCCCTTATGAGGACTTACCAACAGAGA

GAGGTGCCGTGGTGTTACATGATGGCTCCGTTCTAAGTGCAAGCAACCCAATCAACC

CTAAGACTCTAAAAGAGTTCTCCGAGGTTGACCCTGAGAAGGCTGCGCGAGGAATC

AAACTGGCTGGGTTCACCGAGATTGGCTTGAAGACCTTGGGGTCTGACGATGCTGAC

ATCCGTAGAGTGGCTATCGACCTCGTTCGCTCTCCTACTGGTATGCAGTCTGGTGCCT

CAGGTAAGTTCGGTGCAACAGCTTCTGACATCCATGAGAGACTTCATGGTACTGACC AGCGTACTTATAATGACTTGTACAAAGCAATGTCTGACGCTATGAAAGACCCTGAGT

TCTCTACTGGCGGCGCTAAGATGTCCCGTGAAGAAACTCGATACACTATCTACCGTA

GAGCGGCACTAGCTATTGAGCGTCCAGAACTACAGAAGGCACTCACTCCGTCTGAG

AGAATCGTTATGGACATCATTAAGCGTCACTTTGACACCAAGCGTGAACTTATGGAA

AACCCAGCAATATTCGGTAACACAAAGGCTGTGAGTATCTTCCCTGAGAGTCGCCAC

AAAGGTACTTACGTTCCTCACGTATATGACCGTCATGCCAAGGCGCTGATGATTCAA

CGCTACGGTGCCGAAGGTTTGCAGGAAGGGATTGCCCGCTCATGGATGAACAGCTA

CGTCTCCAGACCTGAGGTCAAGGCCAGAGTCGATGAGATGCTTAAGGAATTACACG

GGGTGAAGGAAGTAACACCAGAGATGGTAGAGAAGTACGCTATGGATAAGGCTTAT

GGTATCTCCCACTCAGACCAGTTCACCAACAGTTCCATAATAGAAGAGAACATTGA

GGGCTTAGTAGGTATCGAGAATAACTCATTCCTTCAGGCACGTAACTTGTTTGATTC

GGACCTATCCATCACTATGCCAGACGGACAGCAATTCTCAGTGAATGACCTAAGGG

ACTTCGATATGTTCCGCATCATGCCAGCGTATGACCGCCGTGTCAATGGTGACATCG

CCATCATGGGGTCTACTGGTAAAACCACTAAGGAACTTAAGGATGAGATTTTGGCTC

TCAAAGCGAAAGCTGAGGGAGACGGTAAGAAGACTGGCGAGGTACATGCTTTAATG

GATACCGTTAAGATTCTTACTGGTCGTGCTAGACGCAATCAGGACACTGTGTGGGAA

ACCTCACTGCGTGCCATCAATGACCTAGGGTTCTTCGCTAAGAACGCCTACATGGGT

GCTCAGAACATTACGGAGATTGCTGGGATGATTGTCACTGGTAACGTTCGTGCTCTA

GGGCATGGTATCCCAATTCTGCGTGATACACTCTACAAGTCTAAACCAGTTTCAGCT

AAGGAACTCAAGGAACTCCATGCGTCTCTGTTCGGGAAGGAGGTGGACCAGTTGAT

TCGGCCTAAACGTGCTGACATTGTGCAGCGCCTAAGGGAAGCAACTGATACCGCAC

CTGCCGTGGCGAACATCGTAGGGACCTTGAAGTATTCAACACAGGAACTGGCTGCT

CGCTCTCCGTGGACTAAGCTACTGAACGGAACCACTAACTACCTTCTGGATGCTGCG

CGTCAAGGTATGCTTGGGGATGTTATTAGTGCCACCCTAACAGGTAAGACTACCCGC

TGGGAGAAAGAAGGCTTCCTTCGTGGTGCCTCCGTAACTCCTGAGCAGATGGCTGGC

ATCAAGTCTCTCATCAAGGAACATATGGTACGCGGTGAGGACGGGAAGTTTACCGT

TAAGGACAAGCAAGCGTTCTCTATGGACCCACGGGCTATGGACTTATGGAGACTGG

CTGACAAGGTAGCTGATGAGGCAATGCTGCGTCCACATAAGGTGTCCTTACAGGATT

CCCATGCGTTCGGAGCACTAGGTAAGATGGTTATGCAGTTTAAGTCTTTCACTATCA

AGTCCCTTAACTCTAAGTTCCTGCGAACCTTCTATGATGGATACAAGAACAACCGAG

CGATTGACGCTGCGCTGAGCATCATCACCTCTATGGGTCTCGCTGGTGGTTTCTATG

TTGGAGCGTGCACTGGACCCAACCATGATTGCCCACGCTGCGTTATCTCGTAGTTCT

CAATTGGGTGCTCCTTTGGCTATGGTTGACCTAGTTGGTGGTGTTTTAGGGTTCGAGT

CCTCCAAGATGGCTCGCTCTACGATTCTACCTAAGGACACCGTGAAGGAACGTGACC CAAACAAACCGTACACCTCTAGAGAGGTAATGGGCGCTATGGGTTCAAACCTTCTG GAACAGATGCCTTCGGCTGGCTTTGTGGCTAACGTAGGGGCTACCTTAATGAATGCT GCTGGCGTGGTCAACTCACCTAATAAAGCAACCGAGCAGGACTTCATGACTGGTCTT ATGAACTCCACAAAAGAGTTAGTACCGAACGACCCATTGACTCAACAGCTTGTGTTG AAG ATTT AT G AGGC G A ACGGT GTT A ACTT G AGGG AGCGT AGG A A AT A A

SEQ ID NO: 77

Type:PRT

//Mut AA sequence derived from T7 internal virion protein D (e.g., T7p51)

MDKYDKNVPSDYDGLFQKAADANGVSYDLLRKVAWTESRFVPTAKSKTGPLGMMQF

TKATAKALGLRVTDGPDDDRLNPELAINAAAKQLAGLVGKFDGDELKAALAYNQGEG

RLGNPQLEAYSKGDFASISEEGRNYMRNLLDVAKSPMAGQLETFGGITPKGKGIPAEVG

LAGIGHKQKVTQELPESTSFDVKGIEQEATAKPFAKDFWETHGETLDEYNSRSTFFGFKN

AAE AELS NS V AGM AFRAGRLDN GFD VFKDTITPTRWN S HIWTPEELEKIRTE VKNP A YI

NV VT GGS PENLDDLIKLANENFENDS R A AE AGLG AKLS AGIIG AG VDPLS Y VPM V G VT G

KGFKLINKALV V G AES A ALN V AS EGLRT S V AGGD AD Y AG A ALGGFVF G AGMS AIS DA

VAAGLKRSKPEAEFDNEFIGPMMRLEARETARNANSADLSRMNTENMKFEGEHNGVPY

EDLPTERG A V VLHD GS VLS AS NPINPKTLKEFS E VDPEKA ARGIKL AGFTEIGLKTLGS D

DADIRRVAIDLVRSPTGMQSGASGKFGATASDIHERLHGTDQRTYNDLYKAMSDAMKD

PEFS T GG ARMS REETR YTIYRR A ALAIERPELQKALTPS ERIVMDIIKRHFDTKRELMENP

AIFGNTKAVSIFPESRHKGTYVPHVYDRHAKALMIQRYGAEGLQEGIARSWMNSYVSRP

EVKARVDEMLKELHGVKEVTPEMVEKY AMDKAY GISHSDQFTNSSIIEENIEGLVGIEN

NSFLQARNLFDSDLSITMPDGQQFSVNDLRDFDMFRIMPAYDRRVNGDIAIMGSTGKTT

KELKDEILALKAKAEGDGKKTGEVHALMDTVKILTGRARRNQDTVWETSLRAINDLGF

FAKNAYMGAQNITEIAGMIVTGNVRALGHGIPILRDTLYKSKPVSAKELKELHASLFGK

E VDQLIRPKR ADIV QRLRE ATDT VP A V ANIV GTLKY S T QELA ARS PWTKLLN GTTN YLL

DAARQGMLGDVISATLTGKTTRWEKEGFLRGASVTPEQMAGIKSLIKEHMVRGEDGKF

TVKDKQAFSMDPRAMDLWRLADKVADEAMLRPHKVSLQDSHAFGALGKMVMQFKSF

TIKS LN S KFLRTFYDGYKNNRAID AALS IITSMGLAGGFY AM AAHVKA Y ALPKEKRKE Y

LERALDPTMIAH A ALS RS S QLG APLAM VDLV GG VLGFES S KM ARS TILPKDT VKERDPN

KP YT S RE VMG AMGS NLLEQMPS AGFV AN V G ATLMN A AG V VN S PNKATEQDFMTGLM

NSTKELVPNDPLTQQLVLKIYEANGVNLRERRK SEQ ID NO: 78

Type:PRT

//Mut AA sequence derived from T7 internal virion protein D (e.g., T7p51)

MDKYDKNVPSDYDGLFQKAADANGVSYDLLRKVAWTESRFVPTAKSKTGPLGMMQF

TKATAKALGLRVTDGPDDDRLNPELAINAAAKQLAGLVGKFDGDELKAALAYNQGEG

RLGNPQLEAYSKGDFASISEEGRNYMRNLLDVAKSPMAGQLETFGGITPKGKGIPAEVG

LAGIGHKQKVTQELPESTSFDVKGIEQEATAKPFAKDFWETHGETLDEYNSRSTFFGFKN

AAE AELS NS V AGM AFRAGRLDN GFD VFKDTITPTRWN S HIWTPEELEKIRTE VKNP A YI

NV VT GGS PENLDDLIKLANENFENDS R A AE AGLG AKLS AGIIG AG VDPLS Y VPM V G VT G

KGFKLINKALV V G AES A ALN V AS EGLRT S V AGGD AD Y AG A ALGGFVF G AGMS AIS DA

VAAGLKRSKPEAEFDNEFIGPMMRLEARETARNANSADLSRMNTENMKFEGEHNGVPY

EDLPTERG A V VLHD GS VLS AS NPINPKTLKEFS E VDPEKA ARGIKL AGFTEIGLKTLGS D

DADIRRVAIDLVRSPTGMQSGASGKFGATASDIHERLHGTDQRTYNDLYKAMSDAMKD

PEFS T GG ARMS REETR YTIYRR A ALAIERPELQKALTPS ERIVMDIIKRHFDTKRELMENP

AIFGNTKAVSIFPESRHKGTYVPHVYDRHAKALMIQRYGAEGLQEGIARSWMNSYVSRP

EVKARVDEMLKELHGVKEVTPEMVEKY AMDKAY GISHSDQFTNSSIIEENIEGLVGIEN

NSFLQARNLFDSDLSITMPDGQQFSVNDLRDFDMFRIMPAYDRRVNGDIAIMGSTGKTT

KELKDEILALKAKAEGDGKKTGEVHALMDTVKILTGRARRNQDTVWETSLRAINDLGF

FAKNAYMGAQNITEIAGMIVTGNVRALGHGIPILRDTLYKSKPVSAKELKELHASLFGK

E VDQLIRPKR ADIV QRLRE ATDT AP A V ANIV GTLKY S T QELA ARS PWTKLLN GTTN YLL

DAARQGMLGDVISATLTGKTTRWEKEGFLRGASVTPEQMAGIKSLIKEHMVRGEDGKF

TVKDKQAFSMDPRAMDLWRLADKVADEAMLRPHKVSLQDSHAFGALGKMVMQFKSF

TIKS LN S KFLRTFYDGYKNNRAID AALS IITSMGLAGGFY AM AAHVKA Y ALPKEKRKE Y

LERALDPTMIAH A ALS RS S QLG APLAM VDLV GG VLGFES S KM ARS TILPKDT VKERDPN

KP YT S RE VMG AMGS NLLEQMPS AGFV AN V G ATLMN A AG V VN S PNKATEQDFMTGLM

NSTKELVPNDPLTQQLVLKIYEANGVNLRERRK

SEQ ID NO: 79

// WT AA sequence: T3 tail fiber protein (e.g., T3p48)

MANVIKTVLTYQLDGSNRDFNIPFEYLARKFVVVTLIGVDRKVLTINADYRFATRTTISL

TKAWGPADGYTTIELRRVTSTTDRLVDFTDGSILRAYDLNVAQIQTIHVAEEARDLTTDT

IGVNNDGHLDARGRRIVNLANAVDDRDAVPFGQLKTMNQNSWQARNEALQFRNEAET FRN QTE VFKNES GTN ATNTKQWRDE AN GS RDE AEQFKNT AGQ Y ATS AGN S AT A AHQS EVNAENSATAAANSANLAEQHADRAEREADKLGNFNGLAGAIDRVDGTNVYWKGGIH AN GRLYLTS DGFDC GQ Y QQFFGGS AGR Y S VME W GDEN GWLMH V QRRE WTT AIGDNI QLVVNGHIIAQGGDMTGPLKLQNGHALYLESASDKAQYILSKDGNRNNWYIGRGSDNN NDCTFHSYVYGTNLTLKPDYAVVNKRFHVGQAVVATDGNIQGTKWGGKWLDAYLND TYVKKTM AWTQVW AAAS GSHMGGGS QTDTLPQDLRFRNIWIKTRNNYWNFFRTGPD GIYFLS AEGGWLKFQIHS N GRVFKNIADRD APPT AIA VED V

[000216] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/".

[000217] A“coding sequence” is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.

[000218] To“treat” means to inhibit, through a toxic effect, the ability of a pathogen (e.g., e. coli ) to survive, grow, feed, and/or reproduce, or to limit damage or harm to the animal .

[000219] “Corresponding to” in the context of the present invention means that when amino acid sequences (or polynucleotide sequences, depending on the context) are aligned with each other, the amino acids (or polynucleotides) that“correspond to” certain enumerated positions are those that align with these positions in the native (e.g., wild type) protein (or gene), but that are not necessarily in these exact numerical positions relative to the particular amino acid sequence.

[000220] To“deliver” a composition to the animal means that the composition comes in contact with the animal. The composition can be delivered in many recognized ways, e.g., orally by ingestion or by contact, formulated as a feed material, sprayable protein composition(s), injected, inhaled, or any other art-recognized delivery system.

[000221] A synthetic or engineered bacteriophage refers to a bacteriophage having at least one mutation or more specifically, a combination of mutations, which is/are not known to naturally occur in a wild type protein. Bacteriophage that have been engineered (synthesized) according to the invention have substantially improved properties compared to native bacteriophage. Particularly, these bacteriophage have improved heat stability, a pH stability, a virulence, a blood stability, a bile acid stability, a bodily fluid stability, and a host range of the bacteriophage compared to a wild-type bacteriophage.

[000222] A“gene” is a defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion. A gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example introns.

[000223] A“heterologous” nucleic acid sequence is a nucleic acid sequence not naturally associated with a bacteriophage into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence.

[000224] A“homologous” nucleic acid sequence is a nucleic acid sequence naturally associated with a bacteriophage into which it is introduced.

[000225] An“isolated” nucleic acid molecule or protein is a nucleic acid molecule or protein that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or protein may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.

[000226] A“nucleic acid molecule” or“nucleic acid sequence” is a linear segment of single- or double- stranded DNA or RNA that can be isolated from any source. The nucleic acid molecule or nucleic acid sequence may be a segment of DNA.

[000227] “Transformed/transgenic/recombinant” refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A“non- transformed”,“non-transgenic”, or“non-recombinant” host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.

[000228] Nucleic acids are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise indicated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Len; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[000229] Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[000230] Although the terms“first” and“second” may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one

feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[000231] Throughout this specification and the claims which follow, unless the context requires otherwise, the word“comprise”, and variations such as“comprises” and“comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term

“comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[000232] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and/or steps may alternatively be exclusive, and may be expressed as“consisting of’ or alternatively“consisting essentially of’ the various components, steps, sub-components or sub-steps.

[000233] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "X" is disclosed the "less than or equal to X" as well as "greater than or equal to X" (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point“10” and a particular data point“15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[000234] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative

embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others.

Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical

substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

CLAIMS What is claimed is:

1. A method of treating an animal with a bacteriophage composition, the method

comprising administering to the animal the bacteriophage composition, wherein the bacteriophage composition comprises a synthetic bacteriophage having two or more stabilizing mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO: 5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO: 73 in a

pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the bacteriophage composition is formulated as a liquid, solid or a gel.

3. The method of claim 1, wherein the animal is a mammal, shellfish, fish, or bird.

4. The method of claim 3, wherein the animal is a livestock animal.

5. The method of claim 1, wherein the animal is a human.

6. The method of claim 1, wherein administering comprises administering an effective amount of the bacteriophage composition to maintain a healthy microbiome of the animal.

7. The method of claim 1, wherein the bacteriophage composition is administered to a subject in the form of a feed additive, a drinking water additive, or a disinfectant.

8. The method of claim 1, wherein administering comprises delivering the bacteriophage composition in a drinking water composition.

9. The method of claim 1, wherein administering comprises administering to an animal to treat or prevent an Escherichia coli infection.

10. The method of claim 1, wherein the two or more stabilizing mutations comprise point mutations such that a synthetic bacteriophage has more than 99% identity with a nucleotide sequence of the wild-type bacteriophage.

11. The method of claim 1, wherein the synthetic bacteriophage having two or more

stabilizing mutations is configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio,

Staphyloccocus, or Yersinia relative to an activity of the wild-type bacteriophage.

12. A method of treating a subject with a bacteriophage composition, the method comprising administering to the subject the bacteriophage composition, wherein the bacteriophage composition comprises a synthetic bacteriophage having three or more stabilizing mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO: 5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO: 73 in a pharmaceutically acceptable carrier, wherein the three or more stabilizing mutations results in an increase in one or more of a heat stability, a pH stability, a virulence, a blood stability, a bile acid stability, a bodily fluid stability, and a host range of the bacteriophage compared to a wild-type bacteriophage.

13. A method of treating an animal with a bacteriophage composition, the method

comprising administering to the animal the bacteriophage composition, wherein the bacteriophage composition comprises a synthetic bacteriophage comprising mutations to two or more of: a head-to-tail joining protein , an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D, wherein the two or more mutations results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, and internal and/or external bodily fluid stability compared to a respective wild-type bacteriophage, and wherein the mutations comprise point mutations such that a synthetic bacteriophage has more than 99% identity with a nucleotide sequence of the wild-type bacteriophage.

14. The method of claim 13, where the synthetic bacteriophage comprises a mutation in the head-to-tail protein and a mutation in the internal core protein, relative to the nucleotide sequence of the wild-type bacteriophage.

15. The method of claim 13, where the synthetic bacteriophage comprises mutations to three or more of: a head-to-tail joining protein , an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D.

16. The method of claim 13, wherein the synthetic bacteriophage comprises a mutation in two or more of: a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, a T3p48 gene, T3p38 gene, T3p39 gene, T3p46 gene, T7p43 gene, T7p51 gene, or a homologous gene of a different bacteriophage species thereof.

17. The method of claim 13, wherein the synthetic bacteriophage comprises a mutation in two or more of: a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, a T3p48 gene, T3p38 gene, T3p39 gene, T3p46 gene, or a homologous gene of a different bacteriophage species thereof.

18. The method of claim 13, wherein the synthetic bacteriophage comprises a mutation in two or more of: T7p43 gene, T7p51 gene, or a homologous gene of a different bacteriophage species thereof.

19. The method of claim 13, wherein the synthetic bacteriophage comprises two or more mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO: 5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO: 73.

20. The method of claim 13, wherein the synthetic bacteriophage comprises a nucleotide sequence of two or more of SEQ ID NOS: 3, 9, 14, 16, 18, 24, 26, 28, 38, 40, 46, 48, 56, 58, 63, 67, 69, and 75.

21. The method of claim 13, wherein the synthetic bacteriophage comprises a nucleotide sequence of two or more of SEQ ID NOS: 4, 10, 15, 17, 19, 25, 27, 29, 39, 41, 47, 49, 57, 59, 64, 68, 70, and 76.

22. The method of claim 13, wherein the synthetic bacteriophage comprises an amino acid sequence comprising a sequence two or more of SEQ ID NOS: 5, 11, 20, 30, 32, 34, 42, 50, 52, 60, 65, 71, and 73.

23. The method of claim 13, wherein the synthetic bacteriophage comprises an amino acid sequence comprising a sequence two or more of SEQ ID NOS: 6, 12, 21, 31, 33, 35, 43, 51, 53, 61, 66, 72, 74, 77, and 78.

24. The method of claim 13, wherein the wild-type bacteriophage is a bacteriophage of a Podoviridae genus, Caudoviridae genus, or Siphoviridae genus.

25. The method of claim 24, wherein when the wild-type bacteriophage is a Podoviradae bacteriophage, then the wild-type bacteriophage is of a T7 family of bacteriophages.

26. The method of claim 13, wherein the wild-type bacteriophage is a T3 or a T7 phage.

27. The method of claim 13, wherein the mutations provide an increase in heat stability at 65°C in the bacteriophage, compared with a heat stability of the wild-type bacteriophage.

28. The method of claim 13, wherein the mutations provide an increase in at least one of pH stability at pH 3 or pH stability at pH 12 in the bacteriophage relative to a respective pH stability of the wild-type bacteriophage.

29. The method of claim 13, wherein the mutation produces an amino acid sequence

comprising at least one amino acid that is more hydrophobic or more bulky than an amino acid sequence produced by the nucleotide sequence of the wild-type

bacteriophage.

30. The method of claim 13, wherein the bacteriophage composition is formulated as a

liquid, solid or a gel.

31. The method of claim 13, wherein the animal is a mammal, shellfish, fish, or bird.

32. The method of claim 31, wherein the animal is a livestock animal.

33. The method of claim 13, wherein the animal is a human.

34. The method of claim 13, wherein administering comprises administering an effective amount of the bacteriophage composition to maintain a healthy microbiome of the animal.

35. The method of claim 13, wherein the bacteriophage composition is administered to a subject in the form of a feed additive, a drinking water additive, or a disinfectant.

36. The method of claim 13, wherein administering comprises delivering the bacteriophage composition in a drinking water composition.

37. The method of claim 13, wherein administering comprises administering to an animal to treat or prevent an Escherichia coli infection.

38. The method of claim 13, wherein the synthetic bacteriophage comprising the two or more mutations is configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio,

Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

39. A method of preparing an animal feed composition, the method comprising:

providing a bacteriophage composition comprising bacteriophage as an active

ingredient; and

mixing the composition with an animal feed base to provide the animal feed

composition,

wherein the bacteriophage composition comprises a synthetic bacteriophage having two or more stabilizing mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO: 5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO: 73 in a pharmaceutically acceptable carrier.

40. The method of claim 39, wherein the synthetic bacteriophage comprising the two or more stabilizing mutations is configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio,

41. A method of preparing an animal feed composition, the method comprising:

providing a bacteriophage composition comprising bacteriophage as an active

ingredient; and

mixing the composition with an animal feed base to provide the animal feed

composition,

wherein the bacteriophage comprising mutations to two or more of: a head-to-tail joining protein , an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D, wherein the two or more mutations results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, and internal and/or external bodily fluid stability compared to a respective wild- type bacteriophage, and wherein the mutations comprise point mutations such that a synthetic bacteriophage has more than 99% identity with a nucleotide sequence of the wild-type bacteriophage.

42. The method of claim 41, wherein the bacteriophage comprising the two or more

mutations is configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

43. A synthetic bacteriophage comprising mutations to two or more of: a head-to-tail joining protein , an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D, wherein the two or more mutations results in an increase in one or more of heat stability, pH stability, virulence, blood stability, bile acid stability, and internal and/or external bodily fluid stability compared to a respective wild-type bacteriophage, and wherein the mutations comprise point mutations such that a synthetic bacteriophage has more than 99% identity with a nucleotide sequence of the wild-type bacteriophage.

44. The synthetic bacteriophage of claim 43, where the synthetic bacteriophage comprises a mutation in the head-to-tail protein and a mutation in the internal core protein, relative to the nucleotide sequence of the wild-type bacteriophage.

45. The synthetic bacteriophage of claim 43, comprising mutations to three or more of: a head-to-tail joining protein , an internal core protein, a tail fiber protein, a capsid assembly protein, a minor capsid protein, an internal virion protein C, and an internal virion protein D.

46. The synthetic bacteriophage of claim 43, wherein the synthetic bacteriophage comprises a mutation in two or more of: a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, a T3p48 gene, T3p38 gene, T3p39 gene, T3p46 gene, T7p43 gene, T7p51 gene, or a homologous gene of a different bacteriophage species thereof.

47. The synthetic bacteriophage of claim 43, wherein the synthetic bacteriophage comprises a mutation in two or more of: a T3p37 gene, a T3p38 gene, a T3p39 gene, a T3p45 gene, a T3p48 gene, T3p38 gene, T3p39 gene, T3p46 gene, or a homologous gene of a different bacteriophage species thereof.

48. The synthetic bacteriophage of claim 43, wherein the synthetic bacteriophage comprises a mutation in two or more of: T7p43 gene, T7p51 gene, or a homologous gene of a different bacteriophage species thereof.

49. The synthetic bacteriophage of claim 43, wherein the synthetic bacteriophage comprises two or more mutations selected from: a head-to-tail joining protein including the amino acid sequence of SEQ ID NO: 5; an internal core protein including the amino acid sequence of SEQ ID NO: 11; a tail fiber protein including the amino acid sequence of SEQ ID NO: 20; a capsid assembly protein including the amino acid sequence of one of: SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34; a minor capsid protein including the amino acid sequence of SEQ ID NO: 42; and an internal virion protein C including the amino acid sequence of one of: SEQ ID NO: 71 or SEQ ID NO: 73.

50. The synthetic bacteriophage of claim 43, wherein the synthetic bacteriophage comprises a nucleotide sequence of two or more of SEQ ID NOS: 3, 9, 14, 16, 18, 24, 26, 28, 38, 40,

46, 48, 56, 58, 63, 67, 69, and 75.

51. The synthetic bacteriophage of claim 43, wherein the synthetic bacteriophage comprises a nucleotide sequence of two or more of SEQ ID NOS: 4, 10, 15, 17, 19, 25, 27, 29, 39, 41,

47, 49, 57, 59, 64, 68, 70, and 76.

52. The synthetic bacteriophage of claim 43, wherein the synthetic bacteriophage comprises an amino acid sequence comprising a sequence two or more of SEQ ID NOS: 5, 11, 20,

30, 32, 34, 42, 50, 52, 60, 65, 71, and 73.

53. The synthetic bacteriophage of claim 43, wherein the synthetic bacteriophage comprises an amino acid sequence comprising a sequence two or more of SEQ ID NOS: 6, 12, 21,

31, 33, 35, 43, 51, 53, 61, 66, 72, 74, 77, and 78.

54. The synthetic bacteriophage of claim 43, wherein the wild-type bacteriophage is a

bacteriophage of a Podoviridae genus, Caudoviridae genus, or Siphoviridae genus.

55. The synthetic bacteriophage of claim 43, wherein when the wild-type bacteriophage is a Podoviradae bacteriophage, then the wild-type bacteriophage is of a T7 family of bacteriophages.

56. The synthetic bacteriophage of claim 43, wherein the wild-type bacteriophage is a T3 or a T7 phage.

57. The synthetic bacteriophage of claim 43, wherein the mutations provide an increase in heat stability at 65 °C in the bacteriophage, compared with a heat stability of the wild-type bacteriophage.

58. The synthetic bacteriophage of claim 43, wherein the mutations provide an increase in at least one of pH stability at pH 3 or pH stability at pH 12 in the bacteriophage relative to a respective pH stability of the wild-type bacteriophage.

59. The synthetic bacteriophage of claim 43, wherein the mutation produces an amino acid sequence comprising at least one amino acid that is more hydrophobic or more bulky than an amino acid sequence produced by the nucleotide sequence of the wild-type bacteriophage.

60. The synthetic bacteriophage of claim 43, wherein the synthetic bacteriophage is

configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella,

Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

61. A method of mutating a nucleotide sequence of a first bacteriophage to enhance at least one phenotype of the first bacteriophage, comprising: creating a plurality of bacteriophage mutants by mutating at least one gene or sequence of a nucleotide sequence of the first bacteriophage;

exposing the plurality of mutants to a test environment;

testing the plurality of mutants thereby determining whether each of the plurality of mutants exhibits a desired characteristic in response to exposure to the test environment; and

selecting at least one mutant bacteriophage exhibiting the desired characteristic in response to the test environment.

62. The method of claim 61, wherein the first bacteriophage is a wild-type bacteriophage.

63. The method of claim 61, wherein the desired characteristic is an increase of stability to the test environment or is an increase of infectivity against one or more bacterial species, relative to a stability or infectivity of the first bacteriophage.

64. The method of claim 61, wherein the stability or the infectivity is increased by 50%

relative to the stability or infectivity of the first bacteriophage.

65. The method of claim 61, wherein testing comprises performing a test on each of the

plurality of mutants to determine if an indicator of mutant survival comprises at least a survival threshold.

66. The method of claim 65, wherein selecting comprises selecting a mutant bacteriophage comprising the at least survival threshold indicator of mutant survival.

67. The method of claim 61, wherein exposing the plurality of mutants to a test environment comprises exposing the plurality of mutants to an environment comprising at least one of a temperature at or above a threshold temperature, a pH at or above a threshold pH, a pH at or below a threshold pH, blood, bile acid, an internal bodily fluid, or an external bodily fluid.

68. The method of claim 67, wherein the test environment comprises a temperature of at least 65°C.

69. The method of claim 61, wherein the test environment comprises a pH of pH 3 or pH 12.

70. The method of claim 61, wherein the method further comprises repeating: creating the plurality of bacteriophage mutants; exposing the plurality of mutants to a test

environment, subsequently testing the plurality of mutants thereby determining whether each of the plurality of mutants exhibits a desired characteristic, and selecting the at least one mutant bacteriophage exhibiting the desired characteristic, wherein repeating is performed from 5 to 50 times.

71. The method of claim 70, wherein repeating comprising creating the plurality of

bacteriophage mutants; exposing the plurality of mutants to a test environment, subsequently testing the plurality of mutants thereby determining whether each of the plurality of mutants exhibits a desired characteristic, and selecting the at least one mutant exhibiting the desired characteristic, is performed from 9 to 29 times.

72. The method of any one of claims 70-71, when selecting the at least one mutant exhibiting the desired characteristic, the indicator of mutant survival is progressively increased in each repetition.

73. The method of any one of claims 70-71, wherein exposing the plurality of mutants to a test environment further comprises changing the test environment each time the method is repeated.

74. The method of any one of claims 70-71, wherein the desired characteristic comprises enhanced stability to heat, and wherein repeating comprises incubating the plurality of mutant bacteriophages in a test environment comprising incubating at progressively higher temperatures.

75. The method of claim 74, wherein a temperature of the test environment is increased in 5°C increments.

76. The method of claim 74, wherein a temperature of the test environment is initially a temperature at which bacterial host grows optimally.

77. The method of claim 74, wherein a temperature of the test environment is increased to at least 65°C.

78. The method of any one of claims 70-71, wherein the desired characteristic comprises enhanced stability at a pH lower than neutral pH, and wherein repeating comprises incubating the plurality of mutant bacteriophages in a test environment comprising incubating at progressively lower pH.

79. The method of claim 78, wherein the test environment comprises a pH of 5.

80. The method of claim 78, wherein a pH of the test environment is decreased in 0.5 pH unit increments.

81. The method of claim 78, wherein a pH of the test environment is decreased to a pH of 3, thereby selecting a mutant having the at least survival threshold indicator at pH 3.

82. The method of any one of claims 70-71, wherein the desired characteristic comprises enhanced stability at a pH higher than neutral pH, and wherein repeating comprises incubating the plurality of mutant bacteriophages in a test environment comprising incubating at progressively higher pH.

83. The method of claim 82, wherein the test environment comprises a pH of 9.

84. The method of claim 82, wherein a pH of the test environment is increased in 0.5 pH unit increments.

85. The method of claim 82, wherein a pH of the test environment is increased to a pH of 12, thereby selecting a mutant having the at least survival threshold indicator at pH 12.

86. The method of any one of claims 70-71, wherein the desired characteristic comprises enhanced stability relative to a stability of the first bacteriophage to at least one of blood, bile acids, internal bodily fluids, or external bodily fluids, and wherein the test environment comprises incubating the plurality of mutant bacteriophages in at least one of blood, bile acids, internal bodily fluids, or external bodily fluids in progressively longer periods of time.

87. The method of claim 86, wherein the test period comprises incubating for a period of 30 min.

88. The method of claim 86, wherein a time period of incubation of the test environment is increased by at least 30 minutes to at least 24 hours, thereby selecting a mutant having the at least survival threshold indicator of stability for 24 hours to at least one of blood, bile acids, internal bodily fluids, or external bodily fluids.

89. The method of claim 88, wherein the time period of the test environment is not longer than 72 hours.

90. The method of any one of claims 70-71, wherein the desired characteristic comprises an enhanced host range, and wherein exposing the plurality of mutants to a test environment comprises incubating the plurality of mutant bacteriophages in a mixture of first bacterial cells and second bacterial cells, wherein the first bacterial cells comprise bacterial cells for which the first bacteriophage is infective and wherein the second bacterial cells comprise bacterial cells for which the first bacteriophage is not infective.

91. The method of claim 90, wherein the test environment comprises equal amounts

(PFU/PFU) of the first bacterial cells and the second bacterial cells.

92. The method of claim 90, wherein the test environment comprises progressively

increasing greater percentages of the second bacterial cells relative to the first bacterial cells, thereby selecting a mutant having the at least survival threshold indicator for infectivity towards the second bacterial cells.

93. The method of any one of claims 70-71, wherein the desired characteristic comprises an increase in the efficiency of lytic activity, and wherein testing the plurality of mutants comprises performing an assay identifying mutants configured to lyse bacterial cells at an earlier time point than a lysing timepoint of the wild-type bacteriophage.

94. The method of claim 56, wherein mutating at least one gene or sequence of a nucleotide sequence of a bacteriophage comprises at least one of biochemical, chemical, or physical mutagenesis.

95. The method of claim 56, wherein mutating at least one gene or sequence of a nucleotide sequence of the first bacteriophage comprises modifying nucleobases of the at least one gene or the nucleotide sequence resulting in base mispairing.

96. The method of claim 90, wherein modifying nucleobases of the at least one gene or the nucleotide sequence comprises administering ethyl methanesulfonate or

nitro soguanidine .

97. The method of claim 56, wherein mutating at least one gene or sequence of a nucleotide sequence of a bacteriophage comprises exposing the first bacteriophage to at least one of X-ray radiation or UV wavelength radiation.

98. The method of claim 56, wherein mutating at least one gene or sequence of a nucleotide sequence of a bacteriophage comprises exposing the first bacteriophage to an enzyme with a defective enzymatic activity involved in DNA repair and/or proofreading.

99. The method of claim 93, wherein the enzyme with a defective enzymatic activity involved in DNA repair and/or proofreading comprises an error-prone DNA polymerase.

100. The method of claim 61, further comprising identifying a location of at least one mutated base in the at least one mutated gene or sequence of the nucleotide sequence of the mutant bacteriophage relative to the nucleotide sequence of the first bacteriophage.

101. The method of claim 61, further comprising identifying a frequency of at least one mutated base in the at least one mutated gene or sequence of the nucleotide sequence across the at least one selected mutant bacteriophage exhibiting the desired characteristic.

102. The method of claim 101, wherein identifying the frequency of the at least one mutated base further comprises identifying a frequency of the at least one mutated base above a pre-selected threshold.

103. The method of claim 101, further comprising predicting the at least one mutated base conferring the desired characteristic.

104. The method of claim 100, further comprising identifying a location of each of a plurality of mutated bases of the at least one mutated gene or sequence of the nucleotide sequence of the mutant bacteriophage relative to the nucleotide sequence of the first bacteriophage.

105. The method of claim 104, further comprising identifying a frequency of each of the plurality of mutated bases in the at least one mutated gene or sequence of the nucleotide sequence of the mutant bacteriophage relative to the nucleotide sequence of the first bacteriophage.

106. The method of any one of claims 100-102 or 104-105, wherein identifying comprises identifying a DNA sequence.

107. The method of claim 100, further comprising creating an engineered bacteriophage comprising the at least one mutated base at the identified location within a nucleotide sequence of the engineered bacteriophage.

108. The method of claim 107, wherein the engineered bacteriophage is the bacteriophage of any one of claims 43-60.

109. The method of claim 61, wherein the first bacteriophage is a bacteriophage of a Podoviridae genus, Caudoviridae genus, or Siphoviridae genus.

110. The method of claim 61, wherein when the first bacteriophage is a Podoviradae

bacteriophage, then the wild-type bacteriophage is of a T7 family of bacteriophages.

111. The method of claim 61 , wherein the at least one mutant bacteriophage is configured to more efficiently decrease or inhibit the growth of a bacteria in at least one of the genera Salmonella, Escherichia, Shigella, Acinetobacter, Klebsiella, Campylobacter, Pasteurella, Aeromonas, Pseudomonas, Vibrio, Staphyloccocus, or Yersinia relative to the activity of the wild-type bacteriophage.

112. The method of claim 61, wherein selecting at least one mutant bacteriophage exhibiting the desired characteristic in response to the test environment further comprises selecting a plurality of mutant bacteriophages, wherein each of the plurality of mutant

bacteriophages exhibit the desired characteristic.