Prevalence of porcine endogenous retrovirus in Chinese pig breeds and in patients treated with a porcine liver cell-based bioreactor

AIM： To determine the prevalence of porcine endogenous retrovirus （PERV） in various pig breeds raised in China including Chinese experimental mini-pigs by PERV-reverse transcriptase （PERV-RT enzyme）. Moreover, the potential for infection of PERV was investigated in patients treated with a bioreactor based on porcine liver cells （n = 3）. METHODS： Pig serum, liver and muscle cell-free supernatants were collected from various Chinese pig breeds. Porcine hepatocytes were isolated with a two-step perfusion method. Three patients with acute or chronic liver failure were treated with a bioartificial liver support system （BALSS） for 8-12 h and serum samples were collected from the patients before, immediately after and 30 d after treatment. RESULTS： The activities of PERV-RT enzyme in pig liver and muscle cell-free supernatants were higher than in normal human controls. PERV-TR enzyme activity did not increase in patients before and after 1 mo of treatment. PERV-RT activities were not significantly different when compared with pre-treatment group （1.544±0.155576）, the post-treatment groups （1.501±0.053507, 1.461±0.033808 and 1.6006667±0.01963 for 0, 14 and 30 d post-treatment, respectively, P〉0.05）, and normal control group （1.440±1.0641, P〉0.05）. RT enzyme activity in Chinese experimental mini-pigs was higher than in normal human control group （1.440±1.0641 U/mL, P〈0.05）, and not significantly different （P〉0.05） when compared with the pig breeds except in the muscle supernatants. All the samples including muscle and liver cell supernatants from the Chinese mini-experimental pigs and the four domestic Chinese pig breeds contained PERVs. CONCLUSION： These results suggest that the risk of PERV infection through BALSS containing porcine liver cells without immunosuppressants may be quite low. Although there were PERVs in Chinese experimental mini-pigs and porcine liver cell culture suspensions, we did not find any evidence of persistent PERV infection in patients treated with this porcine hepatocyte-based bioartificial liver.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes

BACKGROUND： A novel hybrid bioartificial liver（HBAL） was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells（MSCs）. This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses（PERVs） into canines with acute liver failure（ALF） undergoing HBAL.METHODS： Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity.RESULTS： PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative.CONCLUSION： No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

Ancient origin and complex evolution of porcine endogenous retroviruses

Porcine endogenous retroviruses(PERVs)are proviruses that have medical value in xenotransplantation.However,the evolutionary process leading to the generation of modern PERVs is not well understood.In this study,we in silico mined 14 pig genomes and other available 304 mammalian genomes,which led to the documentation of 185 full-length PERVs or PERV-like sequences.Notably,we found that lesser Egyptian jerboa(Jaculus jaculus),rock hyrax(Procavia capensis)and eight Muridae species all harbored ancestral PERV-like sequences,indicative of ancient cross-species transmission events from none-porcine species to pigs.We also revealed that 11 genomic rearrangement events were mediated via PERVs,during the process of porcine evolution.In conclusion,these new findings help us to understand the complex evolution of the modern PERVs.

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes

AIM: To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. METHODS: Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assaykits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. RESULTS: The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. CONCLUSION: Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.