| Changes (mutation or deletion) in nonstructure protein 3A and copies of nonstructure protein 3B of FMDV have been associated with altered host range. However, knowledge regarding the molecular mechanism of host range of FMDV is poorly understood.In this study, whole-genome sequencing and the construction of full-length infectious cDNA clone of FMDV O/SEA/Mya-98 strain O/GZSB/2011 and O/GD/2/CHN/2010 isolated from cattle and swine, respectively, were carried out. Based on the established full-length infectious cDNA clone, we generated 3A-truncated mutants (rvSBA93-102, rvSBΔ133-143, rvGDA93-102 and rvGDΔ133-143),3A chimeric viruses (rvSB70, rvOZ70 and rvSBqO),3A-tagged FMDVs(rvSBm, rvSBh and rvSBf) and 3B mutant viruses (rvB1323, rv2B3, rvB13 and rv1B3). Evaluation of the biology characteristics of the recombinant viruses and its parental viruses in different cell cultures, our results indicated, the following results were obtained:1. All of the 3A-truncated mutants grew well and displayed growth properties and plaque phenotypes similar to those of the parental virus in baby hamster kidney (BHK-21) cells, porcine kidney (PK-15) cells, and primary fetal porcine kidney (FPK) cells. While the 3A-truncated mutants rvSBΔ133-143, and rvGDΔ133-143 exhibited similar growth properties and plaque phenotypes with their parental virus in primary fetal bovine kidney (FBK) cells, the 3A-truncated mutants rvSBA93-102 and rvGDA93-102 grew at a slower rate and had a smaller plaque size phenotype in FBK cells than that of the parental virus. Therefore, the results suggest that the deletion at positions 93-102 of 3A protein does not affect FMDV replication ability in BHK-21, PK-15, FPK and FBK cells, but affects virus replication efficiency in FBK cells, although, cannot alone account for the inability to replicate in bovine cells.2. All the chimeric viruses and their parental viruses had similar growth characterizations and plaque phenotypes in BHK-21 and FPK cells. While rvSBqO and rvSB70 displayed lower growth rate and smaller plaque size phenotypes than those of the parental virus in FBK cells, rvOZ70 and O/HN/CHA/93 were completely cannot unable to produce any visible plaques in FBK cells. Therefore, the 3A sequences outside the residues 93-102 can affect the host range of FMDV, and sequences in the FMDV genome except the 3 A region also can affect the host range of FMDV.3. Foreign epitopes can stably maintained and expressed during serial passages of the 3A-tagged viruses in BHK-21 cells. The 3A-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the 3A-tagged viruses had similar growth properties and plaque phenotypes in FBK cells, but exhibited lower growth rate and smaller plaque size phenotypes than those of the parental virus in FBK cells. These results demonstrate that the amino acid substitution at positions 93-102 in 3 A protein of FMDV affect the growth rate of FMDV in FBK cells, and that the 93-102 residues that play key roles in decreased ability to replicate in FBK cells could be reduced to 8 aa residues at positions 94-101 in 3A protein.4.3B mutant viruses rvB1323, rv2B3 and rvB13 displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 and FBK cells. However, rv1B3 which only contained 3B3 exhibited lower growth rate and smaller plaque size phenotypes than those of the parental virus in BHK-21 and FBK cells. When BHK-21 and FBK cells were infected with the same amount of 3B mutant virus particles, respectively, the viruses titres obtained in BHK-21 cells were similar to those in FBK cells for each of the 3B mutant virus, and the order of the viruses titre in BHK-21 and FBK cells was rvGZSB>rvB1323>rv2B3>rvB13>rv1B3. These results show that the copies of 3B protein in FMDV affect the replication efficiency of FMDV, but do not affect the host range of FMDV.Taken together, these studies provided a foundation for further study the gene function, host range and pathogenesis of FMDV. |
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