Detection of PERV by polymerase chain reaction and its safety in bioartificial liver support system.
OBJECTIVE
To establish a method detecting porcine endogenous retrovirus (PERV) in China experimental minipigs and to evaluate the safety of PERV in three individuals treated with bioartificial liver support systems based on porcine hepatocytes.


METHODS
Porcine hepatocytes were isolated with two-stage perfusion method, then cultured in the bioreactor, which is separated by a semipermeable membrane (0.2 microm) from the lumen through which the patients' blood plasma was circulated. After post-hemoperfusion, patients' blood was obtained for screening. Additionally, samples of medium collected from both intraluminal and extraluminal compartments of the laboratory bioreactor and culture supernate in vitro was analyzed. The presence of viral sequences was estimated by polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, the infection of virus in the supernate of common culture was ascertained by exposure to the fetal liver cells.


RESULTS
PERV-specific gag sequences were found in the porcine hepatocytes using RT-PCR. and were detected in all samples from the intraluminal, extraluminal samples and culture supernate. However, culture supernatant from primary porcine hepatocytes (cleared of cellular debris) failed to infect human fetal liver cells. Finally, RT-PCR detected no PERV infection was found in the blood samples obtained from three patients at various times post-hemoperfusion.


CONCLUSIONS
The assays used are specific and sensitive, identified by second PCR. PERVs could be released from hepatocytes cultured in bioreactor without the stimulation of mitogen and could not be prevented by the hollow fiber semipermeable membrane, indicating the existence of PERV safety in extracorporeal bioartificial liver support system (EBLSS).
Department of Endocrine Diseases, Southwestern Hospital, Third Military Medical University, Chongqing 400038, China.
Hai-Hui
HH
Wang
Ying-Jie
YJ
Wang
Hong-Ling
HL
Liu
Jun
J
Liu
Yan-Ping
YP
Huang
Hai-Tao
HT
Guo
Yu-Ming
YM
Wang
D000818:Animals; D019149:Bioreactors; D002478:Cells, Cultured; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D020077:Endogenous Retroviruses / Q000235:genetics / Q000302:isolation & purification*; D015750:Genes, gag; D022781:Hepatocytes / Q000737:chemistry / Q000166:cytology / Q000821:virology*; D006801:Humans; D017093:Liver Failure / Q000628:therapy; D019164:Liver, Artificial / Q000821:virology*; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013552:Swine; D013556:Swine, Miniature / Q000821:virology*
D004279:DNA, Viral