Cell And Molecular Biology Research On Borna Disease Virus Infection In Ningxia

Objective To investigate the natural infection of borna disease virus in sheep in Ningxia, we sequenced the nucleotide sequences of positive samples to gain detailed insights into the genetic characters of the virus, and to establish phylogenetic trees of the virus so as to clarify the natural infection of humans and animals in Ningxia and its potential source of the infection. Using deeply research on transfected oligodendroglial cell to gain the location of virus nucleoprotein, furthermore, we make a preliminary study on the infection and pathogenesis.Methods Borna disease virus p24 and p40 gene segment were detected by fluorescence quantitative nested RT-PCR from brain tissue and peripheral blood mononuclear cells of sheep in Ningxia. Only when both p24 and p40 gene fragments were positive specimens, p24 gene fragment was connected, cloned, and sequenced. Then, applying EMBL, DNASP4.0 and MEGA4.0 software to analyze the homology similarity of nucleotide and amino acid sequence, reconstruct genes phylogenetic tree and analyze the possible sources of virus infection. For transfected oligodendroglial cell we used laser confocal microscopy and Western blot method to detect nucleoprotein and its intracellular location in the cell.Results The detecting discovered borna disease virus p24 and p40 were positive examples in 163 sheep brain and among 710 sheep blood in Ningxia, positive detecting efficiency is 3.68%(6/163) and 1.27%(9/710) respectively. Data from 29 collections of positive test samples of gene sequence analysis proved that the homologous similarity sequences of both the nucleotide and the amino acid was between 95.3% and 100% and highly homophylic with He/80 that detected from ill horses in Germany. The reconstructed genes phylogenetic tree showed one part of nucleotide sequences formed the'Ningxia independent branch'while the other one belonged to the'Germany-Ningxia of China-Japan mixed branch'. There was a high identity within the branch. We found that the nucleoprotein can be positioned in the cytoplasm and cell membrane by laser scanning confocal microscope and Western blot results appeared that there was nucleoprotein in the constitutive protein, but was not detected in the cytoplasm.Conclusions There may exist borna disease virus infection in sheep in Ningxia. A Ningxia independent borna disease virus strain from gegraphical origin might exist while the epidemic strains were imported with multiple sources. It has indicated that the transfected oligodendroglial cells stablely express of borna disease virus nucleoprotein, and its location is in the cell structure of proteins, particularly in the cytoplasm and more richer in cell membrane, which indicated their may play a key role in cell signal transduction or into the cell-mediated viral infection.