Epigallocatechin gallate (EGCG) modification of structural and functional properties of whey protein isolate.

The objective of this paper was to investigate the interactions between (-)-Epigallocatechin-3-gallate (EGCG) and whey protein isolate (WPI) by covalent and non-covalent combinations and the effects of the interactions on the conformational and functional changes of whey protein. Conformational changes in the secondary structure of whey protein with various concentrations of EGCG were studied using FTIR spectra. EGCG was more likely to form covalent bonds than non-covalent bonds when it interacted with whey proteins. The addition of EGCG altered the conformation of whey protein. The content of ß-sheet decreased, while that of ß-turn increased, however, the random coil remained unchanged. An reduction in surface hydrophobicity was observed in all the WPI-EGCG complexes, suggesting that modification in secondary structure of WPI were induced by EGCG. Additionally, the emulsifying and foaming attributes of WPI were enhanced after interaction with EGCG. This study confirms that EGCG can enhance the functional properties of WPI. It is also a pointer to the possible application of WPI-EGCG complexes in the dairy industry.

Amino acid mutations in the env gp90 protein that modify N-linked glycosylation of the Chinese EIAV vaccine strain enhance resistance to neutralizing antibodies.

The Chinese EIAV vaccine is an attenuated live virus vaccine obtained by serial passage of a virulent horse isolate (EIAVL) in donkeys (EIAVD) and, subsequently, in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAVL) with attenuated strains serially passaged in donkey MDM (EIAVDLV) and donkey dermal cells (EIAVFDDV). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g10. To investigate the biological significance of these changes, reverse-mutated viruses were constructed in the backbone of the EIAVFDDV infectious molecular clone (pLGFD3). The resulting virus stocks were characterized for replication efficiency in donkey dermal cells and donkey MDM, and were tested for sensitivity to neutralization using sera from two ponies experimentally infected with EIAVFDDV. Results clearly show that these mutations generated by site-directed mutagenesis resulted in cloned viruses with enhanced resistance to serum neutralizing antibodies that were also able to recognize parental viruses. This study indicates that these mutations played an important role in the attenuation of the EIAV vaccine strains.

Reverse mutation of the virulence-associated S2 gene does not cause an attenuated equine infectious anemia virus strain to revert to pathogenicity.

The contribution of S2 accessory gene of equine infectious anemia virus (EIAV) to the virulence of pathogenic strains was investigated in the present study by reverse mutation of all four consensus S2 mutation sites in an attenuated EIAV proviral strain, FDDV3-8, to the corresponding sequences of a highly pathogenic strain DV117. The S2 reverse-mutated recombinant strain FDDVS2r1-2-3-4 replicated with similar kinetics to FDDV3-8 in cultivated target cells. In contrast to the results of other studies of EIAV with dysfunctional S2, reverse mutation of S2 only transiently and moderately increased the plasma viral load of inoculated horses, and induction of transient immunosuppression did not boost viral pathogenicity. In addition, inoculation of FDDVS2r1-2-3-4 induced partial protection to a challenge pathogenic virus. These results suggest that the attenuated EIAV vaccine strain with multiple mutations in multiple genes will not easily revert to a virulent phenotype.

Amino acid mutations in the env gp90 protein that modify N-linked glycosylation of the Chinese EIAV vaccine strain enhance resistance to neutralizing antibodies.

The Chinese EIAV vaccine is an attenuated live-virus vaccine obtained by serial passage of a virulent horse isolate (EIAV(L)) in donkeys (EIAV(D)), and subsequently in donkey cells in vitro. In this study, we compare the env gene of the original horse virulent virus (EIAV(L)) with attenuated strains serially passaged in donkey MDM (EIAV(DLV)), and donkey dermal cells (EIAV(FDDV)). Genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substitutions/deletions in gp90 that resulted in a loss of three potential N-linked glycosylation sites, designated g5, g9, and g10. To investigate the biological significance of these changes, reverse-mutated viruses were constructed in the backbone of the EIAV(FDDV) infectious molecular clone (pLGFD3). The resulting virus stocks were characterized for replication efficiency in donkey dermal cells and donkey MDM, and were tested for sensitivity to neutralization using sera from two ponies experimentally infected with EIAV(FDDV). The results clearly show that these mutations generated by site-directed mutagenesis resulted in cloned viruses with enhanced resistance to serum-neutralizing antibodies that were also able to recognize parental viruses. The results of this study indicate that these mutations play an important role in the attenuation of the EIAV vaccine strains.

[Construction and in vitro evaluation of an infectious clone of the equine infectious anemia virus vaccine strain EIAV(FDDV) with four reverse-mutated vaccine-specific sites in the S2 gene].

To elucidate the function of the S2 gene in equine infectious anemia virus (EIAV) and its role in the attenuation of the Chinese attenuated EIAV vaccine strains, the S2 in the EIAV vaccine strain EIAV (FDDV) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. Based on the sequence variation of the S2 gene between the EIAV virulent strains and vaccine strains, all the four vaccine-specific sites in the S2 of an EIAV(FDDV) infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAV(DV115). The reverse-mutated molecular clone pFDDVS2r1-3-4-5 was used to transfect fetal donkey dermal (FDD) cells for rescuing the derived virus vpFDDVS2r1-3-4-5. The production and replication of vpFDDVS2r1-3-4-5 in FDD cells were proved by RT-PCR, immune fluorescence assay and reverse transcriptase activity assay. Typical virons of EIAV were clearly observed under the electron microscopy. The parallel analysis of the dynamic replication of the reverse-mutated viral clone vpFDDVS2r1-3-4-5 and its parental virus vpFDDV3-8 showed that the virus with four reverse mutations in the S2 replicated only slightly slower than its parental vaccine strain in FDD cells. This result implicates that the mutations in the S2 of the EIAV vaccine strains did not significantly alter the viral replication in vitro. Further studies on the in vivo replication of the reverse-mutated viral clone are required for understanding the relationship between the S2 and the attenuated pathogenesis of EIAV attenuated vaccines.

[Construction of an infectious clone of equine infectious anemia virus by N-glycosylation reverse-mutations].

To elucidate the role of N-glycosylation in fetal donkey dermal cell (FDD)-attenuated equine infectious anemia virus (EIAV), we constructed an N-glycosylation reverse-mutation molecular clone, pLGN191N236N246. This viral molecular clone was derived from the infectious clone pLGFD3-8 by site-directed mutagenesis. This clone was used to transfect fetal donkey dermal (FDD) cells. Infectious characteristics of transfectants were monitored by RT-PCR, indirect immune fluorescence and reverse transcriptase activity assay. After three passages in FDD cells, viral replications in the supernatant of cell cultures were detected by all the above three methods and viral particles were clearly observed by electron microscopy. The construction of the N-glycosylation reverse-mutation infectious clone provides a solid basis for further study of the mechanism of attenuated pathogenesis and increased immune protection of EIAV attenuated vaccines.