Swine diseases could be caused by unrecognized or minor pathogens. In this study, two unknown cytopathogenic agents were isolated from swine, through cell culture. In order to identify these two cytopathogenic agent (designated CP129 and #2045-7), a particle associated nucleic acids PCR (PAN-PCR) from previous paper was used with simple modification. The cloning procedure was more specified in this study by adding cell control system. According to the modified PAN-PCR, two and four agents-specific DNA sequences were obtained from CP129 and #2045-7, respectively, and they were identified as Mycoplasma (M.) hyorhinis and Mammalian orthoreovirus by nucleotide BLAST. Since M. hyorhinis (CP129) was filterable and non-visible by microscope, this unusual virus-like nature of M. hyorhinis (CP129) was discussed. Especially, the reovirus (#2045-7) was a serotype 3 and a triple reassortant among three serotypes of reoviruses. It was grouped with recently reported reoviruses from disease cases (swine, human and feline), based on the genetic analysis of L1 and S1 partial sequences. In conclusion, two unknown cytopathogenic agents were successfully identified using modified PAN-PCR with cell control system and they were characterized in this study.
Base Sequence ; Cell Culture Techniques ; Clone Cells ; Cloning, Organism ; Humans ; Mammalian orthoreovirus 3 ; Mycoplasma ; Mycoplasma hyorhinis ; Nucleic Acids ; Orthoreovirus, Mammalian ; Polymerase Chain Reaction ; Swine ; Swine Diseases

Adult ; Antibodies, Viral ; blood ; Female ; Humans ; Male ; Middle Aged ; Neutralization Tests ; Orthoreovirus, Mammalian ; immunology ; isolation & purification ; Severe Acute Respiratory Syndrome ; virology

OBJECTIVETo study a newly isolated domestic mammalian reovirus, BYD1, its ability to induce apoptosis analyze the three-dimensional structure of its major membrane penetration protein to predict its function in inducing apoptosis.

METHODSHeLa cells infected with BYD1 reovirus were metered with flow cytometer (FCM) to quantify the ratio of apoptotic cells. The data were analyzed with Student's t-test to judge the ability of BYD1 strain to induce apoptosis. The primary sequence ranged from 582 to 675 per microliter protein of BYD1, T1L, T2J and T3D were aligned and compared. The three-dimensional comparative protein structure model of microliter protein was generated by homology-modeling pipeline SWISS MODEL was applied to annotate its secondary and tertiary structure.

RESULTSBYD1 strain was verified with the ability to induce the apoptosis of HeLa cells. The 643-675 segment composing an alpha-helix showed major difference compared with prototype T2J.

CONCLUSIONThe newly isolated reovirus BYD1 is an apoptosis inducing strain. The alpha-helix (residues 643 to 675) of microliter protein of BYD1 may play a key role to induce the proapoptotic activity of infected cells.

Apoptosis ; Apoptosis Regulatory Proteins ; chemistry ; genetics ; physiology ; Cell Differentiation ; Female ; Flow Cytometry ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; Models, Molecular ; Orthoreovirus, Mammalian ; genetics ; metabolism ; physiology ; Protein Conformation ; Protein Structure, Tertiary ; Reoviridae ; genetics ; metabolism ; physiology ; Uterine Cervical Neoplasms ; pathology ; virology ; Viral Regulatory and Accessory Proteins ; chemistry ; genetics ; physiology