Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.2 (PDK1)
 2,238 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To identify the molecular determinants for attenuation of wild-type Japanese encephalitis (JE) virus strain SA14, the RNA genome of wild-type strain SA14 and its attenuated vaccine virus SA14-2-8 were reverse transcribed, amplified by PCR and sequenced. Comparison of the nucleotide sequence of SA14-2-8 vaccine virus with virulent parent SA14 virus and with two other attenuated vaccine viruses derived from SA14 virus (SA14-14-2/PHK and SA14-14-2/PDK) revealed only seven amino acids in the virulent parent SA14 had been substituted in all three attenuated vaccines. Four were in the envelope (E) protein (E-138, E-176, E-315 and E-439), one in non-structural protein 2B (NS2B-63), one in NS3 (NS3-105), and one in NS4B (NS4B-106). The substitutions at E-315 and E-439 arose due to correction of the SA14/CDC sequence published previously by Nitayaphan et al. (Virology 177, 541-552, 1990). The mutations in NS2B and NS3 are in functional domains of the trypsin-like serine protease. Attenuation of SA14 virus may therefore, in part, be due to alterations in viral protease activity, which could affect replication of the virus.
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PMID:Molecular basis of attenuation of neurovirulence of wild-type Japanese encephalitis virus strain SA14. 784 60

We identified nine nucleotide differences between the genomes of dengue-2 (DEN-2) 16681 virus and its vaccine derivative, strain PDK-53. These included a C-to-T (16681-to-PDK-53) mutation at nucleotide position 57 of the 5'-untranslated region, three silent mutations, and substitutions prM-29 Asp to Val, NS1-53 Gly to Asp, NS2A-181 Leu to Phe, NS3-250 Glu to Val, and NS4A-75 Gly to Ala. Unpassaged PDK-53 vaccine contained two genetic variants as a result of partial mutation at NS3-250. We constructed infectious cDNA clones for 16681 virus and each of the two PDK-53 variants. DEN-2 16681 clone-derived viruses were identical to the 16681 virus in plaque size and replication in LLC-MK2 cells, replication in C6/36 cells, E and prM epitopes, and neurovirulence for suckling mice. PDK-53 virus and both clone-derived PDK-53 variants were attenuated in mice. However, the variant containing NS3-250-Glu was less temperature sensitive and replicated better in C6/36 cells than did PDK-53 virus. The variant containing NS3-250-Val had smaller, more diffuse plaques, decreased replication, and increased temperature sensitivity in LLC-MK2 cells relative to PDK-53 virus. Both PDK-53 virus and the NS3-250-Val variant replicated poorly in C6/36 cells relative to 16681 virus. Unpassaged PDK-53 vaccine virus and the virus passaged once in LLC-MK2 cells had genomes of identical sequence, including the mixed NS3-250-Glu/Val locus. Although the NS3-250-Val mutation clearly affected virus replication in vitro, it was not a major determinant of attenuation for PDK-53 virus in suckling mice.
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PMID:Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53. 914 86

The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK(2) cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 10(7.3) PFU/ml or greater in LLC-MK(2) cells. The crippled replication of PDK-53 virus in C6/36 cells and its attenuation for mice were determined primarily by the 5'NC-57-T and NS1-53-Asp mutations. The temperature sensitivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mutations. The 5'NC-57, NS1-53, and NS3-250 loci all contributed to the small-plaque phenotype of PDK-53 virus. Reversions at two or three of these loci in PDK-53 virus were required to reconstitute the phenotypic characteristics of the parental 16681 virus. The prM-29 locus had little or no effect on viral phenotype. Sequence analyses showed that PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.
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PMID:Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5' noncoding region and nonstructural proteins 1 and 3. 1070 15

The genetic stabilities of the three attenuation loci of the candidate dengue 2 (D2) PDK-53 vaccine virus were evaluated for the PDK-53 virus and PDK-53-vectored chimeric D2/1, D2/3, and D2/4 viruses following 10 sequential passages in Vero cells. Sequencing revealed that the dominant NS1-53-Asp and the NS3-250-Val attenuation loci were extremely stable, whereas reversion occurred at the 5'NCR-57-U locus in 10 of the 18 viral lineages tested. A more sensitive and quantitative assay, the TaqMan mismatch amplification mutation assay (TaqMAMA), was employed to more finely discriminate the level of reversion at the 5'NCR-57 locus. This rapid genetic assay permitted detection of <or=1% reversion of 5'NCR-57 U-to-C in viral populations. By TaqMAMA, various levels of reversion at 5'NCR-57 were detected in all 18 of the PDK-53-based viral lineages tested at Vero passage 10, but only 3 lineages had reversion levels>80% in the viral population. Chimeric viruses based on the PDK-53-V (all three mutations present) genetic background were more stable than those developed in the PDK-53-E (5'NCR and NS1 mutations present) background. The TaqMAMA can be applied in quality control analyses to ensure that attenuated vaccine seeds contain undetectable or minimal levels of reversion at a given attenuation locus.
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PMID:Determining genetic stabilities of chimeric dengue vaccine candidates based on dengue 2 PDK-53 virus by sequencing and quantitative TaqMAMA. 1608 48