Silencioso e negligenciado: o que se sabe após quatro décadas do descobrimento do HTLV-1? / Silent and neglected: what is known after four decades of the discovery of the HTLV-1? / Silencioso y descuidado: ¿qué se sabe después de cuatro décadas del descubrimiento del HTLV-1?

O vírus linfotrópico T humano tipo 1 (HTLV-1) foi o primeiro retrovírus humano descoberto, descrito pela primeira vez há 41 anos. Esse retrovírus está associado ao desenvolvimento de duas doenças graves: a leucemia/linfoma de células T do adulto (ATLL) e a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). Este trabalho teve como objetivo analisar as atualizações sobre o HTLV-1, destacando os aspectos clínicos, os avanços e as limitações no tratamento e na prevenção da infecção pelo HTLV-1. Para isso, foi realizada uma revisão integrativa, por meio de coleta de dados nas plataformas PubMed, LILACS e SciELO, entre março e abril de 2021. Foram incluídos 61 artigos de diferentes países. O Brasil foi o país com maior número de publicações na área: 12. Os resultados obtidos mostram que existem avanços importantes no que diz respeito ao tratamento e à prevenção da infecção pelo HTLV-1. No entanto, a falta de estudos específicos sobre o vírus, que abordem os aspectos clínicos da infecção, foi um fator limitante para este estudo, o que reforça a necessidade de investimento em novas pesquisas sobre o tema.

The Human T-lymphotropic Virus 1 (HTLV-1) was the first human retrovirus discovered, described for the first time 41 years ago. This retrovirus is associated with the development of two serious diseases: adult T-cell leukemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (HAM/TSP). This study aimed to analyze the updates about HTLV-1, highlighting the clinical aspects, advances, and limitations in the treatment and prevention of HTVL-1 infection. To this end, an integrative review was carried out, with data collection on PubMed, LILACS, and SciELO platforms, between March and April 2021. A total of 61 articles from different countries were included. Brazil was the country with the largest number of publications in the area: 12. The results showed effective advances regarding treating and preventing HTLV-1 infection. However, the lack of specific studies about the virus, which address the clinical aspects of the infection, was a limiting factor for this study, which reinforces the need for investment in new research about this topic.

El virus linfotrópico T tipo 1 humano (HTLV-1) fue el primer retrovirus humano descubierto y se describió por primera vez hace 41 años. Este retrovirus está asociado con el desarrollo de dos enfermedades graves: leucemia/linfoma de células T del adulto (ATLL) e mielopatía asociada a HTLV-1/paraparesia espástica tropical (HAM/TSP). Este estudio tuvo como objetivo analizar las actualizaciones sobre HTLV-1, destacando los aspectos clínicos, los avances y limitaciones en el tratamiento y prevención de la infección por HTLV-1. Para ello, se realizó una revisión integradora, a través de la recolección de datos en las plataformas PubMed, LILACS y SciELO entre marzo y abril de 2021. Se incluyeron 61 artículos de diferentes países. Brasil fue el país con mayor número de publicaciones en el área: 12. Los resultados obtenidos muestran que existen avances efectivos en cuanto al tratamiento y prevención de la infección por HTLV-1. Sin embargo, la falta de estudios específicos sobre el virus que aborden los aspectos clínicos de la infección fue un factor limitante para el presente estudio, lo que refuerza la necesidad de invertir en nuevas investigaciones sobre este virus.

Doença do neurônio motor no contexto da infecção pelo HIV: perfil clínico, pesquisa de HERV-K e impacto da terapia antirretroviral (estudo preliminar) / Doença do neurônio motor no contexto da infecção pelo HIV: perfil clínico, pesquisa de HERV-K e impacto da terapia antirretroviral (estudo preliminar)

OBJETIVO: Descrever a forma clínica e evolução de pacientes com doença do neurônio motor (DNM) no contexto da infecção pelo HIV, avaliando fatores clínicos, imunológicos, virológicos e expressão de Herv-K (retrovírus endógeno humano da família k) na casuística estudada. Avaliar a mudança no curso clínico da doença do neurônio motor nos casos com necessidade de intervenção padrão, buscando uma TARV (terapia antirretroviral) com alta penetrância em sistema nervoso central e baixa toxicidade. MÉTODOS: Estudo de série de casos, numa coorte de indivíduos soropositivos, em acompanhamento no Instituto de Infectologia Emílio Ribas (IIER) - São Paulo/SP, no período de julho de 2018 a julho de 2019. Identificou-se seis pessoas portadoras do HIV e diagnosticadas com doença do neurônio motor, com idade de dezoito anos ou mais, em acompanhamento ambulatorial. As variáveis sociodemográficas, clínicas e resultados de exames laboratoriais e complementares foram obtidos por meio de questionários próprios padronizados preenchidos com informações do prontuário do paciente. A pesquisa de Herv-K no soro foi realizada após extração do RNA com Trizol. A avaliação cognitiva foi realizada através da bateria de avaliação neuropsicológica padronizada do serviço para investigação de HAND (Distúrbios neurocognitivos associados ao HIV) e a avaliação da funcionalidade foi realizado através da Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS). RESULTADOS: Foram identificados inicialmente seis pacientes. Três evoluíram a óbito em menos de três meses de acompanhamento. Três pacientes conseguiram realizar o protocolo de um ano de seguimento completo. Dentre os pacientes que completaram o protocolo, um deles apresentava a forma clássica da ELA (Esclerose Lateral Amiotrófica) e dois apresentavam a forma localizada (Flail arm e Flail Leg). Em todos os casos pesquisados, a carga viral plasmática do Herv-K foi indetectável. Em relação aos outros três casos que evoluíram a óbito rapidamente, todos apresentavam a forma clássica da ELA. Nos dois pacientes com a forma localizada houve estabilização do quadro neurológico após modificação da TARV. No caso com Flail arm, foi possível a intervenção terapêutica, com otimização do esquema da TARV para melhor penetração no SNC, e neste caso observou-se uma estabilização da doença neurológica desde a modificação da terapia. No caso com Flail leg, houve estabilização do quadro neurológico após a boa adesão à terapia antirretroviral e a terapia guiada pela genotipagem viral. Ambos os pacientes com as formas localizadas apresentavam a forma leve da demência do HIV e depressão menor. CONCLUSÕES: Observou-se na amostra estudada que completou o protocolo de seguimento que houve uma maior prevalência de casos com a forma localizada, pesquisa do Herv-k negativa, com mudança na evolução clínica com o uso da TARV, e com avaliação neuropsicológica evidenciando sintomas cognitivos de demência pelo HIV e depressão menor, além da não adesão ao tratamento logo após o diagnóstico da infecção pelo HIV.

Regulation of porcine endogenous retrovirus by dual LTR1+2 (Long Terminal Region) miRNA in primary porcine kidney cells

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.

Analysis of swine leukocyte antigen class I gene profiles and porcine endogenous retrovirus viremia level in a transgenic porcine herd inbred for xenotransplantation research

Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.

Triptolide induces apoptosis of human acute T lymphocytic leukemia Jurkat cells via inhibiting transcription of human endogenous retrovirus HERV-K Np9 gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the molecular?mechanisms by which triptolide induces apoptosis of human acute T lymphocytic leukemia Jurkat cells.</p><p><b>METHODS</b>MTT assay was employed to detect the proliferation inhibition of Jurkat cells by triptolide, and the IC50 was calculated by OriginPro8. Flow cytometry was used to analyze apoptosis of Jurkat cells. Np9 mRNA levels were detected by RT-PCR and analyzed quantitatively by Kodak 1D 3.6 software. Correlation between the inhibition of Np9 transcription and the cell apoptosis was analyzed by SPSS 19.0.Western blotting was employed to determine Np9 downstream signaling molecules c-myc, β-catenin, ERK, AKT and Notch1 protein level in Jurkat cells after exposure to different concentrations of triptolide for 48 h.</p><p><b>RESULTS</b>Triptolide treatment resulted in dose-dependent inhibition of Jurkat cells proliferation and its IC50 was 12.7 nmol/L. Triptolide induced apoptosis of Jurkat cells in dose- dependent manner. Furthermore, triptolide inhibited Np9 mRNA transcription level in Jurakt cells in a dose-dependent manner. There was a correlation between the triptolide-mediated the apoptosis and the inhibition of Np9 transcription of Jurkat cells (R(2)=0.907). Western blotting results displayed that triptolide inhibited transcription levels of Np9 mRNA with a concomitant decrease of its downstream signaling molecules c-myc, β-catenin, ERK, AKT and Notch1 at protein levels.</p><p><b>CONCLUSION</b>Inhibition of HERV-K Np9 mRNA and its downstream signaling molecules c-myc, β-catenin, ERK, Akt and Notch1 protein might be one of important molecular?mechanisms by which triptolide induces apoptosis of human acute T lymphocytic leukemia Jurkat cells.</p>

Analysis of env Subtypes of Porcine Endogenous Retrovirus in SNU Miniature Pigs

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.

No expression of porcine endogenous retrovirus after pig to monkey xenotransplantation / 한국실험동물학회지

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.

Los retrovirus endógenos y su rol en el melanoma cutáneo / The role of endogenous retroviruses in cutaneous melanoma

Los retrovirus endógenos son elementos virales ancestrales insertos en el genoma humano desde hace miles de años. Aunque permanecen quiescentes a lo largo de la vida, ciertos estímulos y mutaciones genéticas pueden reactivar estos elementos genéticos. El másestudiado de los retrovirus endógenos, el HERV-K, aporta diferentes proteínas que tienen acción inmunosupresora y alteran la migración y el fenotipo celular, lo que conduce finalmente a una génesis y progresión de diferentes neoplasias, como el melanoma cutáneo. A través dediferentes métodos, pueden detectarse anticuerpos dirigidos contra las proteínas retrovirales, las cuales correlacionan con un pronóstico desfavorable en pacientes con melanoma en diferentes estadios. La terapéutica dirigida contra HERV-K es una de las futuras herramientasen estudio para reducir la morbimortalidad de esta neoplasia cutánea...

Transposable Elements: No More 'Junk DNA'

Since the advent of whole-genome sequencing, transposable elements (TEs), just thought to be 'junk' DNA, have been noticed because of their numerous copies in various eukaryotic genomes. Many studies about TEs have been conducted to discover their functions in their host genomes. Based on the results of those studies, it has been generally accepted that they have a function to cause genomic and genetic variations. However, their infinite functions are not fully elucidated. Through various mechanisms, including de novo TE insertions, TE insertion-mediated deletions, and recombination events, they manipulate their host genomes. In this review, we focus on Alu, L1, human endogenous retrovirus, and short interspersed element/variable number of tandem repeats/Alu (SVA) elements and discuss how they have affected primate genomes, especially the human and chimpanzee genomes, since their divergence.

New evidence of porcine endogenous retrovirus transmission with new bio-artificial liver system: a experimental study / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Five 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.</p><p><b>RESULTS</b>All 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.</p><p><b>CONCLUSION</b>Application of our BAL system does not lead to PERV transmission.</p>

No Evidence of the Productive Replication of Porcine Endogenous Retrovirus (PERV) from SNU Miniature Pigs in Human Cell Line / 감염과화학요법

BACKGROUND: The presence of porcine endogenous retrovirus (PERV) has been considered as one of the main hurdles to transplant pig's organs or tissues to human beings. There has been no report that PERV infection is associated with human diseases. Because pigs have their own characteristics of PERV according to pig strain, it is necessary to analyze the infectivity of PERV from SNU miniature pig to human cells for future utilization as a transplantation donor. MATERIALS AND METHODS: Human cell lines were infected with culture supernatant from porcine cell line or immunomodulator-stimulated peripheral blood mononuclear cells (PBMC) of SNU miniature pigs. They were also co-cultured with PBMC or islet cells of SNU miniature pigs. The presence of PERV genes and general pig marker gene in cells was determined by nested PCR with primer set for PERV pol and pig mitochondrial cytochrome oxidase II (COII), respectively. RESULTS: Infection test with the culture supernatant from PBMC of SNU miniature pigs showed that PERV pol but not COII was detected only in a few cases, but there was no uniform infection pattern in scope of stimulators and cell types. PERV pol was not demonstrated in co-cultures of human cell line with PBMC or islet cells from SNU miniature pigs after 80 days of co-cultures. CONCLUSIONS: In vitro infectivity test suggests that PERV from SNU miniature pig might not replicate productively in human cell lines although it could infect human cells and integrate into chromosome.

Detection of Human Cytomegalovirus (HCMV) and Porcine Endogenous Retrovirus (PERV) with One Step Extraction Method / 감염과화학요법

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.

Characterization of Clones of Human Cell Line Infected with Porcine Endogenous Retrovirus (PERV) from Porcine Cell Line, PK-15 / 감염과화학요법

BACKGROUND: Porcine endogenous retroviruses (PERVs) form part of the chromosomes of all pigs. Since they can be produced as infectious virion and infect human cells, safety issues on PERVs infection to human are still controversial and is one of main hurdles of xenotransplantation using pig cells or organs. It has been reported that the established porcine cell line, PK-15, produces PERVs and can infect the human cell lines. Therefore, clonal analysis on human cell line infected with PERV is a prerequisite to characterize the infectivity to human cells and to investigate the harmfulness of PERVs to human. MATERIALS AND METHODS: For the characterization of PERV that originates from porcine cell line, PK-15, full length PERV cloning from genomic DNA of PK-15 was performed and partial sequences of both ends were achieved. Cell clones from human cell line, 293, persistently infected with PERVs from PK-15 were established by the method of limiting dilution. Nested PCR and direct sequencing of PCR products in each clone were carried out so as to confirm the PERV genomes in each clone. The growth rate of each clone was checked using cell counting and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, the infectivity by reverse transcriptase (RT) assay, and genetic analysis by karyotyping. RESULTS: A total of 12 genomic PERV clones could be retrieved; 1 with full length, 4 with defective forms, and others with irrelevant sequences. Intact PERV was thought to be able to infect 293 and the PERV-infected cell clones were selected by limiting dilution. PCR results confirmed that nine cell clones were infected with PERV, and sequence alignment data on PCR products of pol region from PK-15 and human cell clones with PERV showed very similar results. Cell counting and MTT assay for growth kinetics of each clone indicated that two clones showed reduced growth rate. However, it was difficult to verify the effect of PERV infection on the cell growth because of the presence of many genetic alterations in 293 parental cells. No RT activities were detected in the culture supernatant from PERV-infected 293 cell clones. CONCLUSION: The sequences of PERVs were detected in human cell clones after PERV infection, but PERV virions could not be detected from the culture supernatant by RT assay.

Characterization of Clones of Human Cell Line Infected with Porcine Endogenous Retrovirus (PERV) from Porcine Cell Line, PK-15 / 감염과화학요법

BACKGROUND: Porcine endogenous retroviruses (PERVs) form part of the chromosomes of all pigs. Since they can be produced as infectious virion and infect human cells, safety issues on PERVs infection to human are still controversial and is one of main hurdles of xenotransplantation using pig cells or organs. It has been reported that the established porcine cell line, PK-15, produces PERVs and can infect the human cell lines. Therefore, clonal analysis on human cell line infected with PERV is a prerequisite to characterize the infectivity to human cells and to investigate the harmfulness of PERVs to human. MATERIALS AND METHODS: For the characterization of PERV that originates from porcine cell line, PK-15, full length PERV cloning from genomic DNA of PK-15 was performed and partial sequences of both ends were achieved. Cell clones from human cell line, 293, persistently infected with PERVs from PK-15 were established by the method of limiting dilution. Nested PCR and direct sequencing of PCR products in each clone were carried out so as to confirm the PERV genomes in each clone. The growth rate of each clone was checked using cell counting and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, the infectivity by reverse transcriptase (RT) assay, and genetic analysis by karyotyping. RESULTS: A total of 12 genomic PERV clones could be retrieved; 1 with full length, 4 with defective forms, and others with irrelevant sequences. Intact PERV was thought to be able to infect 293 and the PERV-infected cell clones were selected by limiting dilution. PCR results confirmed that nine cell clones were infected with PERV, and sequence alignment data on PCR products of pol region from PK-15 and human cell clones with PERV showed very similar results. Cell counting and MTT assay for growth kinetics of each clone indicated that two clones showed reduced growth rate. However, it was difficult to verify the effect of PERV infection on the cell growth because of the presence of many genetic alterations in 293 parental cells. No RT activities were detected in the culture supernatant from PERV-infected 293 cell clones. CONCLUSION: The sequences of PERVs were detected in human cell clones after PERV infection, but PERV virions could not be detected from the culture supernatant by RT assay.

Current Status and Future Perspectives of Xenotransplantation / 대한이식학회지

Xenotransplantation using pigs as the transplant source holds great promise to resolve the severe shortage of human organ donors. Although stem-cell-derived organ and tissue regeneration have a potential to solve this as well for the future, it still remains as very early experimental phase. Likewise, artificial organs and mechanical devices have been simply used for bridge therapy to transplant. Therefore, xenotransplantation might provide the most imminent solution to the scarcity of human organ donors. In the last two decades, major progress has been made in understanding the mechanisms of xenografts rejection, zoonotic infections including porcine endogenous retrovirus (PERV) and production of genetically engineered pigs including alpha1,3-galactosyltransferase-deficient pigs. With these elaborations, it is now on the threshold of first clinical application. Particularly promising first target is porcine pancreatic islet xenotransplantation. Graft survival has been prolonged to almost one year in the non-human primate study and is waiting for the development of relatively non-toxic or clinically applicable immunosuppressive or tolerance-inducing regimens. This review highlights the currently known obstacles to translate xenotransplantation into clinical therapies and the possible strategies to overcome these hurdles, as well as current status and future perspective for clinical xenotransplantaion.

Construction of the Porcine Endogenous Retrovirus Envelope Glycoprotein A and B Specific Antibody

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.

Comparison of the age-related porcine endogenous retrovirus (PERV) expression using duplex RT-PCR

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.

A specific PCR assay and a nested PCR assay for the screening of ovine pulmonary adenomatosis / 病毒学报

Ovine pulmonary adenomatosis (OPA) is a naturally occurring contagious lung tumor of sheep which was caused by an exogenous retrovirus of sheep, jaagsiekte retrovirus (JSRV). Although no specific circulating antibodies against the virus coud be detected in infected sheep, exogenous JSRV proviral DNA sequences (exJSRV) and JSRV RNA transcripts could be detected in lung tumors, lymphoreticular system and peripheral blood mononuclear cells (PBMC) from sheep affected by OPA. The sheep genome carried 15 to 20 copies of endogenous retrovirus loci (enJSRV) that were similar to JSRV in structural genes but the divergene in U3. Therefore, primers specific for the U3 sequences of exJSRV were designed for the specific PCR and nested PCR (n-PCR). Sensitivity between specific PCR assay and n-PCR assay was compared by using serial dilutions of positive plasmid pJSRV-LTR in a background of 700ng sheep genome DNA. Sensitivity of n-PCR was ten-fold higher than specific PCR. The n-PCR was only available in blood test for detection of JSRV infected sheep and might be useful in epidemiological studies and disease control of OPA.

Existence of proviral porcine endogenous retrovirus in fresh and decellularised porcine tissues.

PURPOSE: Swine are expected to be utilized as xenograft donors for both whole organ and cellular transplantation. A major concern in using porcine organs for transplantation is the potential of transmission of porcine endogenous retrovirus (PERV). Tissue-engineered or decellularised heart valves have already been implanted in humans and have been marketed by certain companies after Food and Drug Administration (FDA) approval. The aim of this study was to examine the existence of porcine endogenous retrovirus (PERV) in fresh and decellularised porcine tissues. METHODS: Porcine tissues (both fresh and decellularised) were analysed using validated assays specific for PERV: polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: PERV specific GAG sequences were found in the porcine heart tissue samples using PCR for DNA and RT- PCR for RNA. All tissue samples (both fresh and treated tissues) like aortic valve, pulmonary valve and heart muscle showed the presence of PERV DNA. RT PCR for PERV was positive in all fresh tissues and was found to be negative in decellularised treated tissues. CONCLUSIONS: PCR is a rapid, specific test for the detection of PERV virus in xenografts. These findings have demonstrated that the presence of proviral DNA form of PERV in porcine tissues needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.