Liu Xiangtao

Alteration of BHK-21 cells proteome after foot-and-mouth disease virus infection

Foot-and-mouth disease is a highly contagious viral illness of wild and domestic cloven-hoofed animals. The complex relationship of FMDV and with the host cells leads to its replication and spread. BHK-21 cell line is an in vitro model for FMDV infection and is commonly used for viral seed preparation. In order to better understand the molecular basis of this relationship, a proteomics study on baby hamster kidney cells infected with FMDV was performed. The differential proteomes of BHK-21 cells, with and without BHK-21 infection, were analyzed with two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF identification. Mass spectrometry identified 30 altered protein spots (19 up-regulated, 9 down-regulated and 2 viral protein spots), which included metabolic processes proteins, cytoskeletal proteins, microfilament-associated proteins, stress response proteins and FMD viral proteins. Western blot analysis further confirmed the differential expression of protein NME-2 in the proteomic profiles. Subcellular location demonstrated NME2 protein was distributed in BHK-21 cell cytoplasm and nucleolus. Thus, this work provides useful proteinrelated informations to further understand the underlying pathogenesis of FMDV infection.

Variation analysis of the VP1 and 3ABC genes from yellow cattle isolates persistently infected by Foot-and-mouth disease virus.

The potential relationship between the establishment of Foot-and-mouth disease virus (FMDV) persistent infection and gene variation was identified by investigating the variation of VP1 and 3ABC genes from yellow cattle persistent infection isolates. Five yellow cattle were inoculated on their tongue with 1.0×10 4 ID 50 /ml of FMDV O/Akesu/58 strain. After displaying clinical or subclinical signs, they probably became asymptomatic carriers. Oesophageal–pharyngeal fluids were collected monthly from the carriers with a probang and inoculated into a baby hamster kidney cell line (BHK-21); 12 FMDV isolates were obtained. The VP1 and 3ABC genes of the 12 isolates were then amplified by reverse-transcriptase polymerase chain reaction (RT-PCR). Cloning and sequencing revealed that the homology of the VP1 nucleotide and amino-acid sequence of all the isolates was above 98%, with no base deletion or insertion. When compared with the O/Akesu/58 FMDV strain, the homology of the VP1 nucleotide sequence of the isolates was only 85%, and that of the deduced amino-acid sequence only 90%.There were several nucleotide mutations in the VP1 gene of the isolates, including 16 consistent nucleotide mutations, with only two of them leading to a change in amino acid (I 56 →T, A 210 →E). Moreover, it was found that four nucleotide points and three amino-acid points had transversions among all isolates. The 3ABC gene had only 13 nucleotide transversions and five amino-acid mutations. It was presumed that persistent FMDV infection might have little connection with variation in the VP1 and 3ABC genes, and was probably related to other structural protein gene and key factors.