All xenografts from pigs impose infection risk by porcine endogenous retrovirus (PERV). The purpose of this study was to investigate the env constructs with the comparison of the ratio of the competent form to the defective one of env in subtypes, PERV-A, PERV-B and PERV-C in different pig breeds. The results of PCR amplification of env represented that all env subtypes had more than two defective forms which cannot bind to host cells due to the absence of binding regions of env in miniature pigs, SNU and PWG, and farm pig breeds, Duroc, Yorkshire and Landrace. In addition, comparing the full sequences with the defective ones in three subtypes demonstrated that the present percentages of env sequences in defective PERV-A, PERV-B and PERV-C were approximately 50%, 38~45% and 4~11%, respectively, in SNU and PWG pigs whereas PERV-A and PERV-B occupied around 40 to 60% but PERV-C was not detected in farm pigs. Quantitative real-time PCR assays with primers and probes targeted to proline-rich region (PRR) of each env subtype were done to measure the copy numbers of each env subtype. When the reference was set with copy number of PERV-A, the ratio of those of PERV-B and PERV-C to the reference were 1.5 to 6.0 folds high in SNU and PWG pigs while 1.0 or less in farm pigs. These contradictory results of PERV-C constructs and copy numbers in SNU pigs suggests that many truncated or short defective sequences of PERV-C might be present in them.
Endogenous Retroviruses* ; Heterografts ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Swine* ; Transplantation, Heterologous

This study was performed to investigate the expression of two porcine endogenous retrovirus (PERV) elements, PERV gag and full-length conserved PERV, in blood cells collected periodically from organ-recipient monkeys that underwent pig to non-human primate xenotransplantation. The heart and kidney-respectively acquired from alpha-1,3-galactosyltransferase knockout (GT-KO) pigs that survived for24 and 25 days-were xenografted into cynomolgus monkeys. The two PERV elements expressed in the xenografted GT-KO pig organs were not present in the blood cells of the recipient monkeys. In the present study, we deduced that PERVs are not transmitted during GT-KO pig to monkey xenotransplantation.
Blood Cells ; Endogenous Retroviruses* ; Haplorhini* ; Heart ; Heterografts ; Macaca fascicularis ; Primates ; Swine ; Transplantation, Heterologous*

BACKGROUND: Xenotransplantation is thought to be one of the alternative methods to overcome the shortage of human organs for transplantation. Recipients should be immunosuppressed for graft survival, and thus, there is a need for developing diagnostic modality that can detect diverse infections originating from animals and recipients rapidly, in the early stage, and with high sensitivity using small volume of samples. This study was carried out to develop a fast, simple, and robust technique for the preparation of HCMV DNA and PERV RNA using small volume of samples. MATERIALS AND METHODS: Nucleic acids were extracted from serially diluted samples with one step extraction method as well as with Qiagen kit. The presence of genomic DNA of human cytomegalovirus (HCMV) and porcine endogenous retrovirus (PERV) was detected by PCR and specific primer set, respectively. RNA of HCMV and PERV was extracted and then detected by RT-PCR and specific primer set, respectively. For absolute quantification of HCMV, standard curve was established by real time PCR. RESULTS: HCMV DNA and PERV RNA were prepared from culture supernatant and cells for PCR or RT-PCR with one step extraction method. It was possible to extract both the DNA and RNA from the samples in about 20 minutes with one step extraction method in a single tube. HCMV and PERV could also be detected by PCR and one step extraction method, respectively. It was also good with small quantity samples. CONCLUSIONS: One step extraction method is simpler and faster method than other extraction methods when there are two types of DNA and RNA viruses in one sample. From these results, we could see that the one step extraction method could be very useful in detecting HCMV and PERV rapidly from the pig cells or organ transplanted recipients with a small amount of sample.
Animals ; Cytomegalovirus ; DNA ; Endogenous Retroviruses ; Graft Survival ; Humans ; Nucleic Acids ; Polymerase Chain Reaction ; RNA ; RNA Viruses ; Transplantation, Heterologous ; Transplants

Quantitative RT(reverse transcriptase) assay was established to detect the reverse transcriptase in plasma of thirty-four Chinese Banna minipig inbred in this work. The protocol was given in the RT kit (Roche), using HIV-1 as the positive control of the kit and supernatant of PK-15 as the PERV positive control respectively. The results show that positive reverse transcriptase reaction can be detected in the plasma of the pigs, but the levels are much lower than that of HIV-1 and lower than that of PERV in supernatant of PK-15.
Animals ; Animals, Inbred Strains ; Endogenous Retroviruses ; enzymology ; RNA-Directed DNA Polymerase ; blood ; Swine ; blood ; virology ; Swine, Miniature ; blood ; virology

OBJECTIVE: To detect porcine endogenous retrovirus (PERV) in Daweizi pigs and to provide basic parameters of evaluating the biological safety for xenotransplantation from pigs to humans. METHODS: Ear tissues from 42 individuals were randomly collected from a Daweizi pig population. PCR and RT-PCR were performed to detect PERV proviral DNA and mRNA respectively. Finally, env-A, env-B, and env-C were amplified, sequenced, and analyzed using the BLAST software in National Center for Biotechnology Information. RESULTS: PERV proviral DNA and mRNA could be detected in the 42 individuals by PCR and RT-PCR, respectively. env-A, env-B and env-C were detected in all the individuals. Compared with other pig species (AY288779, DQ011794 and AY534304), there was 1 and 8 bp differences in the sequences of env-A and env-C, while no difference in env-B. CONCLUSION PERV exists and has transcriptive activity in Daweizi pigs. The predominate subtype is PERV-ABC. Env genes are firstly cloned and sequenced in Daweizi pigs and there are polymorphism in the breed. As to the biological safety, the breed was not suitable as a donor in xenotransplantation.
Animals ; DNA, Viral ; isolation & purification ; Endogenous Retroviruses ; isolation & purification ; Humans ; Polymerase Chain Reaction ; methods ; Swine ; virology ; Transplantation, Heterologous

Xenotransplantation using porcine organs could potentially associate with the risk of pathogenic infections, because human tropic porcine endogenous retrovirus (PERV) particles could be released from pig cells or organs. While there is no evidence of PERV transmission to human, safety issues become a paramount concern. For the prevention of this transmission, specific immunological tools must be provided for PERV transmission detection. In this study we described the expression of PERV envelope proteins and the production of a specific antibody against PERV envelope (Env) glycoprotein. The nucleotide sequence harboring the partial region of glycoprotein 70 was cloned into the pET vector and envelope protein was expressed in E. coli. Approximately 42 kDa recombinant Env protein (PERV Env-aa357) was purified by the Ni-affinity column. For antibody production, mice were immunized with the recombinant PERV Env-aa357. The generated anti-serum was tested using Western blot and immunocytochemical assay. We found that anti-PERV Env serum displayed the specificity against the PERV Envs (PERV-A and PERV-B) expressed not only in E. coli but also in mammalian cells, and PERV particles within the porcine cell lines (PK 15 and PK-1). Taken together, PERV antibody could be useful for detecting PERV infection or xenotransplantation transmission.
Animals ; Antibody Formation ; Base Sequence ; Blotting, Western ; Cell Line ; Clone Cells ; Endogenous Retroviruses ; Gene Products, env ; Glycoproteins ; Humans ; Mice ; Proteins ; Sensitivity and Specificity ; Transplantation, Heterologous

Porcine endogenous retroviruses (PERVs) are members of family Retroviridae, genus Gamma retrovirus, and transmitted by both horizontally and vertically like other endogenous retroviruses (ERVs). PERV was initially described in the 1970s having inserted its gene in the host genome of different pig breeds, and three classes, PERV-A, PERV-B, and PERV-C are known. The therapeutic use of living cells, tissues, and organs from animals called xenotransplantation might relieve the limited supply of allografts in the treatment of organ dysfunction. Because of ethical considerations, compatible organ sizes, and physiology, the pig has been regarded as an alternative source for xenotransplantation. Sensitive duplex reverse transcription-polymerase chain reaction protocols for simultaneously detecting PERV gag mRNA and porcine glyceraldehydes 3-phosphate dehydrogenase mRNA in one tube was established. To compare the age-related PERV expression patterns of the lung, liver, spleen, kidney, heart, and pancreas in commercial pigs, 20 pigs from four age groups (5 heads each in 10 days-, 40 days-, 70 days-, and 110 days-old, respectively) were used in this study. The expression patterns of PERV were statistically different among age groups in lung, liver, and kidney (ANOVA, p<0.05). These data may support in the selection of appropriate donor pigs expressing low levels of PERV mRNA.
Animals ; Endogenous Retroviruses/*metabolism ; Gene Expression Regulation, Viral/*physiology ; RNA, Messenger/genetics/metabolism ; RNA, Viral/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/methods/*veterinary ; Sensitivity and Specificity ; Swine/*virology

Human contains large number of human endogenous retroviruses (HERVs) in its genome. One of the HERV families, HERV-K, entered human genome most recently and includes many members with full-length intact proviruses. Normally, these proviruses do not express but infrequently they seem to express in cancers or autoimmune disease patients. To investigate expression mechanisms of these endogenous retroviruses, a DNA copy of HERV-K was cloned and its expression was studied. The transfection of the full-length clone into human cell lines did not produce any detectable viral capsid protein, Gag, and the transcription from its own promoter in LTR was extremely poor. The transcription was less than 10 percent compare to the exogenous retrovirus. However, when the Gag coding region was cloned under CMV promoter, Gag could be expressed efficiently and secreted as particles, probably virus like particles. The efficient expression also required a nuclear export signal. The expressed Gag could also package its own genomic RNA. These results indicate that the LTR of HERV-K is normally not active but its genes have a potential to express and possibly produce infectious particles.
Autoimmune Diseases ; Capsid Proteins ; Cell Line ; Clinical Coding ; Clone Cells* ; DNA* ; Endogenous Retroviruses ; Genome ; Genome, Human ; Humans* ; Nuclear Export Signals ; Product Packaging* ; Proviruses ; Retroviridae ; RNA ; Transfection

Most of the endogenous retroviral genes integrated into the primate genome after the split of New World monkeys in the Oligocene era, approximately 33 million years ago. Because they can change the structure of adjacent genes and move between and within chromosomes they may play important roles in evolutionas well as in many kinds of disease and the creation of genetic polymorphism. Comparative analysis of HERVs (human endogenous retroviruses) and their LTR (long terminal repeat) elements in the primate genomes will help us to understand the possible impact of HERV elements in the evolution and phylogeny of primates. For example, HERV-K LTR and SINE-R elements have been identified that have been subject to recent change in the course of primate evolution. They are specific elements to the human genome and could be related to biological function. The HERV-M element is related to the superfamily of HERV-K and is integrated into the periphilin gene as the truncated form, 5'LTR-gag-pol-3'LTR. PCR and RT-PCR approaches indicated that the insertion of various retrotransposable elements in a common ancestor genome may make different transcript variants in different primate species. Examination of the HERV-W elementrevealed that env fragments were detected on human chromosomes 1, 3-7, 12, 14, 17, 20, and X, whilst the pol fragments were detected on human chromosomes 2-8, 10-15, 20, 21, X, and Y. Bioinformatic blast search showed that almost full-length of the HERV-W family was identified on human chromosomes 1-8, 11-15, 17, 18, 21, and X. Expression analysis of HERV-W genes (gag, pol, and env) in human tissues by RT-PCR indicated that gag and pol were expressed in specific tissues, whilst env was constituitively expressed in all tissues examined. DNA sequence based phylogenetic analysis indicated that the gag, pol and env genes have evolved independently during primate evolution. It will thus be of considerable interest to expand the current HERV gene information of various primates and disease tissues.
Base Sequence ; Chromosomes, Human ; Endogenous Retroviruses ; Genes, env ; Genome ; Genome, Human ; Humans* ; Phylogeny ; Platyrrhini ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Primates ; Retroelements* ; Zidovudine

OBJECTIVETo investigate the potential transmissibility of porcine endogenous retrovirus (PERV) from a newly-developed porcine hepatocyte bioartificial liver (BAL) system prior to human clinical trial by using a live canine model.

METHODSFive normal beagles were treated with the new BAL support system for six hours. Samples of plasma from the BAL system and whole blood from the beagles were collected at regular intervals over the six month study period. DNA and RNA were isolated from both the peripheral blood mononuclear cells (PBMCs) and plasma for evaluation by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR, respectively, to detect PERV and the Sus scrofa cytochrome B normalization standard. In addition, RT activity and the in vitro infectivity of the plasma were detected in HEK293 cells.

RESULTSAll five beagles remained in stable physical health throughout the treatment and survived until the end of the study. PERV RNA-positivity and RT activity were only detected in the plasma samples from the 3rd BAL treatment cycle. All other samples, including PBMCs and plasma, were negative for PERV RNA, PERV DNA, and RT activity. In addition, none of the sera samples showed in vitro infectivity.

CONCLUSIONApplication of our BAL system does not lead to PERV transmission.

Animals ; Cell Line ; Dogs ; Endogenous Retroviruses ; HEK293 Cells ; Hepatocytes ; virology ; Humans ; Leukocytes, Mononuclear ; virology ; Liver, Artificial ; adverse effects ; Models, Animal ; Swine