TABLE 3.
Oligonucleotide primersa
| Analysis | Primer | Sequence (5′–3′)b | Coordinates or accession no. |
|---|---|---|---|
| Synthesis of inserts | |||
| CyHV-3 ORF25 | ORF25outF | CCTCCGACTCTGAAGACGAT | 45389–45408 |
| ORF25outR | GGTACGTGATGCTGTAGAAG | 47569–47588 | |
| CyHV-3 ORF32 | ORF32outF | CCAGGTCCAGGCAGTCTC | 54216–54233 |
| ORF32outR | ATGCGCGTCTACAAGATCG | 55619–55601 | |
| CyHV-3 ORF59 | ORF59outF | GACGCCTGTCTCTGGTAGA | 101541–101559 |
| ORF59outR | GCGTCTCCAAAAAGAGCGG | 102941–102923 | |
| CyHV-3 ORF64 | ORF64outF | GTCGAGATATCCGAGGCAGA | 119716–119735 |
| ORF64outR | TTCATAGCCACAACCGGAGT | 122203–122184 | |
| CyHV-3 ORF65 | ORF65outF | TGGAGAACATCAAGCAGCAC | 121920–121939 |
| ORF65outR | GAAGTCTGAAACTGTCTGAATG | 124106–124127 | |
| CyHV-3 ORF81 | ORF81outF | GGTTTAGTCCAAATCCGACCT | 150519–150539 |
| ORF81outR | GCAGCTTCTGCTTCAGAGTG | 152023–152004 | |
| CyHV-3 ORF83 | ORF83outF | ACACGCTGATGGTCACGAG | 152549–152567 |
| ORF83outR | ACACCAACCAGTTCGTGCAG | 153638–153619 | |
| CyHV-3 ORF99 | ORF99intF | GCTTAGCCTGTTCGGCAC | 184445–184462 |
| ORF99intR | AAGATCTGGGACACGGACTG | 185515–185496 | |
| CyHV-3 ORF106 | ORF106outF | GCTGACACCTGTCACAACCA | 195629–195648 |
| ORF106outR | TCGTGAGGACAAACCGTCTC | 196189–196170 | |
| CyHV-3 ORF108 | ORF108outF | GATGATGAAGGGTGTTCATG | 201067–201086 |
| ORF108outR | CATCTACAAGTCGGACAACC | 201937–201956 | |
| CyHV-3 ORF115 | ORF115outF | CACGTAAACGAAGCCCCATA | 207951–207970 |
| ORF115outR | ATTCGTGGCGGCTGTTATAC | 210734–210715 | |
| CyHV-3 ORF131 | ORF131outF | GCCCTGGTCCTCGTACTTTT | 224971–224990 |
| ORF131outR | CTGATCAGATTCTCAGGAGCAG | 227271–227250 | |
| CyHV-3 ORF132 | ORF132outF | GCTCCTGAGAATCTGATCAG | 227252–227271 |
| ORF132outR | GTTAATGGTCACAGAAGCGC | 228155–228174 | |
| CyHV-3 ORF136 | ORF136outF | GTGTCAAGTACGTGGAGCGT | 231157–231176 |
| ORF136outR | GGCCTCTGAATGTGTTATTGC | 231991–232011 | |
| CyHV-3 ORF148 | ORF148outF | CATGGTTCGAGGTTGTGAAG | 253808–253827 |
| ORF148outR | CAGCATTCGTCTCTTAGTGC | 255933–255952 | |
| CyHV-3 ORF149 | ORF149outF | ACCCTATCATGATTGACGGC | 255770–255789 |
| ORF149outR | CTTTCGTTCTACTGTTCCTC | 258172–258191 | |
| Synthesis of galK recombination cassettes | |||
| ORF 25 Del galK | ORF25 galK F | GTCATTTTTCCTCGTGGTGTGCAGCTTTTTCTAACGTGTGGTAGGACGCTAACCATTACCAAGTAAACCATTACGCCTGTTGACAATTAATCATCGGCA | 45495–45569 |
| ORF25 galK R | TGACCAGGGTAGAATGATAGACCCGCCGCTAAGAAAAATGACTCTCCGTCCGCTACCGTCGCCCGCTATAGTGTTTCAGCACTGTCCTGCTCCTT | 47376–47450 | |
| ORF 32 Del galK | ORF32 galK F | ATAATACGCACCCTGGTCACTTTCTTTAACGTGTCCGTGTCGGGCTCCCTACACGTTTCCTCCGAGTCGCCGATCCCTGTTGACAATTAATCATCGGCA | 54417–54491 |
| ORF32 galK R | CTGTATCCCATCAGGGGCTGATCTGCTGACGGCCTCGACGTGGTGGTGATGGTGGTGGTGGTGGTGATGATGATGTCAGCACTGTCCTGCTCCTT | 54491–54417 | |
| ORF 59 Del galK | ORF59 galK F | CCCGCACGACGTCGGACGCTCCCTACCGAGTGAGCCGTCTCTCTGACCCGGGTCGTGCGGGCGCCACCAGCCCTTCCTGTTGACAATTAATCATCGGCA | 101961–102035 |
| ORF59 galK R | AGCAGTGGTAGTCCTCTCACCCCCCCCCCCCCCCCCTTGACCTTCACCCTTCACCCCTAACCCCAACAACCAACCTCAGCACTGTCCTGCTCCTT | 102521–102447 | |
| ORF 64 Del galK | ORF64 galK F | TGGTCCTCCCATGCTTTTTATTCAATCCTGCTCCTCTTGAAAAGCTTAAGCCTGTTGACAATTAATCATCGGCA | 119814–119863 |
| ORF64 galK R | GTACACTATATTGCGTTTATTTGTTTTTCATAAAAAGCACATACATAATATCAGCACTGTCCTGCTCCTT | 122082–122033 | |
| ORF 65 Del galK | ORF65 galK F | CAATATAGTGTACACCGTGGTCTTTATTAATTTGATAACATAATAATGTACCTGTTGACAATTAATCATCGGCA | 122070–122119 |
| ORF65 galK R | CAAGACGCCTCACACGACGGAAACCCGCAACACCACCCCACAAACAAACGTCAGCACTGTCCTGCTCCTT | 123911–123960 | |
| ORF 81 Del galK | ORF81 galK F | GCACTTGAGAGCAGCGGAGGAGAAGACCGGTGCATCCATCTCGACGCTCGTCAGGTTGTGACTGTCCTCGTCCTCCCTGTTGACAATTAATCATCGGCA | 150722–150796 |
| ORF81 galK R | CGGCTGCTGTGTTTTGTGGTGCTGGCCTCGACTGTGGGGTGGTGGTGGCGGCGACTGCGAGAGGCTTCGGAATCAGCACTGTCCTGCTCCTT | 150796–150722 | |
| ORF 83 Del galK | ORF83 galK F | CACAATAAAAAGCTCCCCTCGGGGATCACCAAGACAACCTTTCAGTAGCCCCTGTTGACAATTAATCATCGGCA | 153481–153530 |
| ORF83 galK R | TCTTCGCCCTCGCCCTCGTCCCTCAACCTGTTGTACCTATACGCGCTCATTCAGCACTGTCCTGCTCCTT | 152758–152807 | |
| ORF 99 Del galK | ORF99 galK F | TGCAGGCCACCGCCGCCAACGCCGAGATGTCCAACAACCTCCAGACGCAGCCTGTTGACAATTAATCATCGGCA | 184714–184763 |
| ORF99 galK R | AGGGCTTGCTCTTGAGGAAGGGCTGGTTGACGGGGCAGCCCTGCAGGTAATCAGCACTGTCCTGCTCCTT | 185214–185165 | |
| ORF 106 Del galK | ORF106 galK F | ACTACTCGCCCACCGCCTTATACAACTTCACCGCGTATCCGCCGTACATCCCTGTTGACAATTAATCATCGGCA | 195727–195776 |
| ORF106 galK R | GGGTTTGGTAGAGGGCGCTGCGGGGGATGAGCCCGACGATACCGCTGCGGTCAGCACTGTCCTGCTCCTT | 196063–196014 | |
| ORF 108 Del galK | ORF108 galK F | CACTTAGTCTACCACTCACTTTACCTTACAACCACCCAGACACCAACACACCACACCACCCACACTACGAGCGAGCCTGTTGACAATTAATCATCGGCA | 201156–201230 |
| ORF108 galK R | ACCAAGCAACACGTTCATGTGATTTTTTTTTATTGTTGATGTTGTTTGATGTATTACAGTTACAATAAACGTAATTCAGCACTGTCCTGCTCCTT | 201819–201893 | |
| ORF 115 Del galK | ORF115 galK F | ATCGTGTCCTCCGTCTCGGTCATCTCTGTGGTTTTTTCCAGCTTTGCGCCCCTGTTGACAATTAATCATCGGCA | 208152–208226 |
| ORF115 galK R | GCTCAAAATAAAAAAAACATGTCTGGACCTTTACATCTTGTTTCAATAATTCAGCACTGTCCTGCTCCTT | 210641–210567 | |
| ORF 131 Del galK | ORF131 galK F | GTGAGGGAGTGATATGGAGTGAACGTAAATGGAGGGGCGCTGCGGAGGTTCCTGTTGACAATTAATCATCGGCA | 225410–225484 |
| ORf131 galK R | TCGAGACGCCCGAACTGGTCGAGGCCTACGTGAACGACGTCAAGGTCCGCTCAGCACTGTCCTGCTCCTT | 226426–226352 | |
| ORF 132 Del galK | ORF132 galK F | TCAAGTCTTTATTATTACTTTATTTAATGGGGTTCGGTTGGAGAAGAGCAGTCAAGTGAGCAGAAGAGAAGATCGCCTGTTGACAATTAATCATCGGCA | 227369–227443 |
| ORF132 galK R | AACTGAGGCCTCTCCGGCTATCCGCACCGAGTGATATACCGCCCGCCACTACCCCAGAACCCCCACACAGCCGCATCAGCACTGTCCTGCTCCTT | 227957–228031 | |
| ORF 136 Del galK | ORF136 galK F | AAGCTTGCCTTTGAAACATGGAAAAGAGCTCTCGTGTGTTTCTTTAGAATAAGACGACGTCGAGCGACGACCATCCCTGTTGACAATTAATCATCGGCA | 231264–231337 |
| ORF136 galK R | TGTTGGTTTGCTTTGGCTTGATAGCACACAAAAATCACATTTGTAAGAGTTTTATTGTTTGACAATGATACATCATCAGCACTGTCCTGCTCCTT | 231801–231875 | |
| ORF 148 Del galK | ORF148 galK F | GAGGGTAGAGAGGGCTGGAGAGTAGGAGAGGAGAGAGGGGAAAGTTTTGAAGTTCTTGCACATGTTGAGGGGAAGCCTGTTGACAATTAATCATCGGCA | 253881–253955 |
| ORF148 galK R | AAAACCAAAATTTTTCTGTTATCGTATCGTGATGAGCGCGACCGAGGCGACGGTCCAGTAACTCCGCCGTCAATCTCAGCACTGTCCTGCTCCTT | 255780–255854 | |
| ORF 149 Del galK | ORF149 galK F | TTTGCTTTTTTTATTATGTGATGCTGTAGTGTACACACGTATTAGAAACAATAGTAGAGCAAGCAAGCAAAAGCCCTGTTGACAATTAATCATCGGCA | 255861–255935 |
| ORF149 galK R | TTTTCTTTTGACGCGCAATGACCGACGGCTCAAGTGACGGCTACCTAGACGATACGTCACAGACGGCTGAGGACCTCAGCACTGTCCTGCTCCTT | 257997–258071 | |
| Nonsense mutagenesis of essential genesc | |||
| ORF32 | ORF32.T237G-sense | GACAACGGCTGGGGCTAGACCTTTCTGATCCAATC | 54711–54745 |
| ORF32.A237C-antisense | GATTGGATCAGAAAGGTCTAGCCCCAGCCGTTGTC | 54745–54711 | |
| ORF59 | ORF59.C144G-sense | CATGCAGCGCTAGCAACTGTGCGAG | 102315–102291 |
| ORF59.G144C-antisense | CTCGCACAGTTGCTAGCGCTGCATG | 102291–102315 | |
| ORF81 | ORF81.C207G-sense | TTGCGCACGCCATGTAGTCCAACGATCCC | 150987–151015 |
| ORF81.G207C-antisense | GGGATCGTTGGACTACATGGCGTGCGCAA | 151015–150987 | |
| ORF83 | ORF83.G225A-sense | GTTCATGTACTGAGTCACCCTACGC | 153268–153244 |
| ORF83.C225T-antisense | GCGTAGGGTGACTCAGTACATGAAC | 153244–153268 | |
| ORF99 | ORF99.C3039G-sense | CAACGACAGGTTCTCGTAGGTGGCGAGCC | 184946–184974 |
| ORF99.G3039C-antisense | GGCTCGCCACCTACGAGAACCTGTCGTTG | 184974–184946 | |
| ORF106 | ORF106.CA108AT-sense | CTTCGTCGTCTAATAGGCCCAGCAC | 195872–195896 |
| ORF106GT108TA-antisense | GTGCTGGGCCTATTAGACGACGAAG | 195896–195872 | |
| ORF115 | ORF115.C744A-sense | GGACCTGGTCCTGTAATCCCAGACGAACGTG | 208955–208985 |
| ORF115.G744T-antisense | CACGTTCGTCTGGGATTACAGGACCAGGTCC | 208985–208955 | |
| ORF131 | ORF131.C735G-sense | TCCAGGGCCGTGTAGGAGGCCGC | 226054–226032 |
| SDM131.G735C-antisense | GCGGCCTCCTACACGGCCCTGGA | 226032–226054 | |
| Synthesis of probes for Southern analysis (probe name) | |||
| GalK | GalK.Int2-F | AGGTGAGGAACTAAACCCAG | |
| GalK.Int2-R | CGTATTGCAGCAGCTTTATC | ||
| ORF32 | ORF32.SB-F | CAGATGTGGCTGGACATGA | 54567–54585 |
| ORF32.SB-R | GCTACCCAGGTGGTGTTGTT | 55006–54987 | |
| ORF59 | ORF59.SB-F | GTTCTGCGTCGAGGATGATG | 102042–102061 |
| ORF59.SB-R | CGTTCAGCACCTCGGTCTAC | 102427–102408 | |
| ORF81 | ORF81.SB-F | AAAGCTCAACTGGCCAAGAG | 150809–150828 |
| ORF81.SB-R | ACACCGCCGTCTTCGAGTAG | 151259–151240 | |
| ORF83 | ORF83.SB-F | TGCTCTCCACGTCCTTGTA | 152838–152856 |
| ORF83.SB-R | ATGTCTCCTTTGTGCGGTC | 153480–153462 | |
| ORF99 | ORF99.SB-F | CCTCCAGACGCAGATCAACT | 184751–184770 |
| ORF99.SB-R | CTTGAGGAAGGGCTGGTTG | 185204–185186 | |
| ORF106 | ORF106.SB-F | GCAGCAGCAGCAGAAGTG | 195783–195800 |
| ORF106.SB-R | TCAGTTGGTCTTGGGGCC | 196013–195996 | |
| ORF115 | ORF115.SB-F | CTACGCCAACGACGAACC | 209426–209443 |
| ORF115.SB-R | AGCACCACGAACCACACTC | 209776–209758 | |
| ORF131 | ORF131.SB-F | ACTGGAGCGGGTGATAGTTG | 225487–225506 |
| ORF131.SB-R | GGACTCGTCGTGCCTCTC | 225937–225920 | |
| PCR | |||
| ORF25 | ORF25int_F | ATCAAGCGCTACGACGACTT | 45948–45967 |
| ORF25int_R | TGTTGCAGGAGGTGTAGACG | 46442–46461 | |
| ORF64 | ORF64int_F | CCATAGTCCAGGACGACGAT | 121431–121450 |
| ORF64int_R | TGCTGCTTGATGTTCTCCAC | 121919–121938 | |
| ORF65 | ORF65int_F | GATGGTCATGTTGGTGTTGC | 122996–123015 |
| ORF65int_R | CCAAGAACGAGCTCTTCACC | 123494–123513 | |
| ORF108 | ORF108int_F | ACCAACTACACGACCGTCTC | 201246–201265 |
| ORF108int_R | CCGGGTTGGTGTAGGTAGAA | 201677–201696 | |
| ORF132 | ORF132int_F | TTGGTTTTTGTTGGTGACGA | 227468–227487 |
| ORF132int_R | TGACGGGTTCCAAGATTAGC | 227936–227955 | |
| ORF136 | ORF136int_F | CTGGTTACATGGGTGGCTTT | 231360–231379 |
| ORF136int_R | TAGCCCCTGTTGTAGGATGC | 231738–231757 | |
| ORF148 | Pr148fw2 | GGTTGTTGGAGTAGTGGTGC | 254196–254215 |
| Pr148rev2 | CTCATTCAGGCTCGGAGAC | 254592–254610 | |
| ORF149 | Pr149fw1 | GTAGTCGCTGGATGTGACG | 256662–256680 |
| Pr149rev1 | GTCAACACGGACTGCTCCG | 257093–257111 | |
| ORF112 | ORF112intFW | CCAGTGTCATCACCACAAGC | 205336–205355 |
| ORF112intREV | ACGGACATCCTGGGTATCAA | 205571–205590 | |
| qPCR | |||
| CyHV-3 ORF89 | KHV-86F | GACGCCGGAGACCTTGTG | AF411803 |
| KHV-163R | CGGGTTCTTATTTTTGTCCTTGTT | ||
| KHV-109P | (6FAM) CTTCCTCTGCTCGGCGAGCACG (BHQ1) | ||
| Carp glucokinase | CgGluc-162F | ACTGCGAGTGGAGACACATGAT | AF053332 |
| CgGluc-230R | TCAGGTGTGGAGCGGACAT | ||
| CgGluc-185P | (6FAM) AAGCCAGTGTCAAAATGCTGCCCACT (BHQ1) |
a
Coordinates are listed in relation to GenBank accession number NC_009127.1.
b
Underlined: segments correspond to the CyHV-3 sequence; italicization indicates sequences corresponding to galK sequence. Mutated nucleotides are both underlined and italicized.