Diseases in animals or humans which are induced by or associated with horizontally-transmitted exogenous retroviruses, include feline leukemias or sarcomas, chicken leukemias or sarcomas, mouse leukemias or sarcomas, equine infectious anemia, bovine leukemia, caprine arthritis-encephalitis, human adult T-cell leukemia, human tropic spastic paraparesis, and AIDS.
Spumavirus human foamy virus has not yet been definitely associated with disease in humans. Infection with human T-cell leukemia virus type 1 HTLV-l , an exogenous C-type oncovirus, results in a spectrum of clinical manifestations ranging from asymptomatic infection to lymphoproliferative and neurologic disorders Fig.
Most HTLV-l-infected individuals are asymptomatic, with normal white blood cell and differential counts. However, some asymptomatic individuals may present with moderate lymphocytosis and aberrant lymphocytes. Approximately half of these latter individuals will progress to a chronic form of adult T-cell leukemia ATL , characterized by small numbers of leukemic cells in the peripheral blood, by skin lesions, and by a lack of involvement of other organ systems.
Approximately 15 to 20 percent of adult T-cell leukemia cases follow a chronic course. T cell lymphomas occur and may involve a variety of organ systems. The most common malignant sequela of HTLV-l infection is acute adult T-cell leukemia, which follows an aggressive course characterized by polylobular malignant T cells, hypercalcemia, leukemic cell infiltrates of the dermis and epidermis, and immunosuppression leading to opportunistic infections.
The predominant clinical manifestation is progressive weakness and partial paralysis of the lower extremities. The clinical features may mimic those of multiple sclerosis.
In fact, there is some speculation that an HTLV-related virus is involved in multiple sclerosis. There have also been reports that HTLV-l is associated with large granular lymphocytic leukemia and malignant hypereosinophilic syndrome. However, these claims have not been substantiated. The relation of these viruses to human disease is unclear.
HTLV-2 has been associated with two cases of hairy-cell leukemia and has been reported in one case of T-cell prolymphocytic leukemia and one of T-cell chronic lymphocytic leukemia. HTLV-5 has been isolated from a patient with Tac-antigen-negative cutaneous T cell lymphoma-leukemia mycosis fungoides.
HTLVs are members of the oncovirus family of retroviruses, which are distinguished from other viruses by the presence of reverse transcriptase, an enzyme that transcribes RNA into DNA. Most retroviruses are spherical particles, approximately nm in diameter, consisting of an internal protein core surrounded by an envelope of glycoproteins embedded in a lipid bilayer. The core contains several copies of reverse transcriptase bound to two identical single-stranded RNA molecules.
In the viruses that cause chronic leukemias, the viral genes encode the core proteins gag , envelope proteins env , replication enzymes pol , and regulatory proteins tax and rex.
Some animal retroviruses, in contrast, are acutely transforming. The env gene of these viruses is truncated and replaced with a cellular gene onc that causes acute transformation of the target cells. The envelope of HTLV consists of two glycoproteins of molecular weights approximately 20, and 46, daltons.
Three gag proteins molecular weights of 9,, 15,, 24, to 26, daltons constitute the viral core. Unlike most animal retroviruses, the genome of HTLV codes for two nonstructural proteins, tax and rex, which are involved in the regulation of HTLV expression. Tax may also be indirectly involved in HTLV transformation of lymphocytes. A virus is classified as an HTLV by the presence of reverse transcriptase and by its isolation from and infection of mature T cells.
The genomes of HTLV hybridize only weakly with the genomes of other animal retroviruses. The HTLVs share cross-reacting internal core proteins which differ slightly in molecular weight. Although the long terminal repeat sequences of these two viruses are quite dissimilar, specific regulatory regions in the long terminal repeat are highly conserved. The genome of HTLV-5 is less well characterized. Retroviruses have a common mode of viral replication that is based on the function of the reverse transcriptase molecules Fig.
The initial event in retroviral infection is the attachment of the virus to the cell membrane. The specific cellular receptor s for HTLV has not yet been identified, but recent experiments have shown that a receptor is encoded by a gene on human chromosome After binding to the cell membrane, the virion enters the cell and is uncoated to release the viral RNA.
The virion-associated integrase then enables the viral DNA to integrate into the host cell genome. The integrated viral DNA, now called a provirus, can either remain inactive or be transcribed into viral RNA and into messenger RNA that is translated into viral structural and regulatory proteins.
Progeny viral RNA and structural proteins assemble at the cell surface and bud from the cell membrane. Since the provirus is duplicated along with the cellular DNA during the replication cycle of the cell, infection of the cell will persist throughout the lifespan of the clone. The HTLVs appear to induce disease in a fundamentally different way from other oncogenic animal retroviruses. The acutely transforming animal retroviruses cause malignant transformation by their onc genes which were originally derived from cellular growth genes, but are no longer under cellular control and rather are under the control of the viral promoter.
The chronic oncogenic animal retroviruses induce leukemia after a long latent period. Often the provirus inserts near a cellular onc gene. As a result, the cellular gene comes under the control of the viral promoter, thereby enhancing its expression. Therefore, the virally transformed animal leukemic cells have proviral copies integrated at specific sites in the cellular DNA. In contrast, the HTLVs do not possess an onc gene, nor do they integrate in the same site in different tumors i.
The rex gene encodes a protein of molecular weight 26, to 27, daltons that is localized in the nucleus and is required for the accumulation of unspliced gag mRNA and for efficient expression of the gag gene products.
The tax gene product is a nuclear protein of molecular weight 37, to 40, daltons that transactivates viral gene expression. The exact mechanism of tax-induced transactivation is unknown, but it has been suggested that the tax gene product may activate a constitutively expressed cellular transcription factor that binds to the viral promoter and indirectly enhances viral gene expression. Although expression of the tax gene can lead to cancer in some strains of transgenic mice, the resulting tumors are neurofibromas and do not demonstrate a direct effect of tax on leukemogenesis.
One mechanism by which the HTLV tax gene product may play a role in leukemogenesis is by activating cellular genes that are involved in T-cell growth Fig. Mitogen- or antigen-stimulated T cells produce a factor, interleukin-2 IL-2 , that binds to a receptor on the surface of activated T cells and induces T-cell multiplication. Therefore, it has been hypothesized that the tax gene product is responsible for stimulating the production of IL-2 receptors through the induction of host transcription factors that bind to the IL-2 receptor gene.
Research has identified in the IL-2 receptor gene a region that is essential for tax-mediated regulation of gene expression. It has also been suggested that the tax protein can transactivate the IL-2 gene itself, although less strongly than the IL-2 receptor gene. HTLV can also stimulate T-cell growth without actually infecting the cell.
Exposure of T cells to inactivated HTLV-1 or to partially purified viral proteins can result in mitogenic stimulation and proliferation of the T cells in the absence of exogenous IL Two features of infection indicate that HTLV-induced leukemogenesis must involve additional pathogenic events. The first is the long incubation period between infection with HTLV and the appearance of leukemic cells: it is thought that leukemia may take several decades to develop.
The second feature is the monoclonal nature of the malignant cells in adult T-cell leukemia patients. Therefore, although HTLV may polyclonally stimulate the growth of T cells via either tax-mediated mechanisms or mitogenic stimulation, the outgrowth of leukemic cells presumably depends on as yet unidentified secondary events. Infection with HTLV-1 also results in immunosuppression.
In vitro, HTLV-1 infection not only alters helper T-cell function by causing increased proliferation, but also induces nonspecific polyclonal immunoglobulin production by B cells regardless of the type of antigen-presenting cell. Infection of cytotoxic T-cell clones leads to reduction or loss of cytotoxic function.
Cells expressing HTLV-1 have abnormal expression of major histocompatibility complex MHC antigens cell surface proteins essential for the functional interaction of immune cells.
Inappropriate expression of these antigens on infected T-cells would impede or abolish their normal function. In addition, the HTLV-1 envelope and major histocompatibility complex proteins share certain antigenic determinants, so the immune system may be tricked into thinking that the HTLV-infected cell is self and need not be eliminated by the cytotoxic T-cell response.
The pathogenesis of the second major clinical manifestation of HTLV-1 infection, tropical spastic paraparesis, is unknown. Several animal retroviruses, particularly members of the lentivirus family, can infect the central nervous system and cause chronic neurologic disease in animals. The damage to the central nervous system is generally considered to result not from direct infection of neuronal cells but rather from the release of toxic factors by cells infected with HTLV Another hypothesis is that neural tissue is damaged by the host immune system via an autoimmune mechanism.
Even rarer is the occurrence of both diseases in the same individual. Possible explanations for these observations include the existence of distinct adult T-cell leukemia-inducing strains and tropical spastic paraparesis-inducing strains of HTLV-1 or genetic determinants or host cell factors that contribute to variable disease expression.
A hallmark of HTLV infection is its persistence. Once an individual is infected with HTLV, the virus remains in that individual for life. Specific antibodies are produced against a variety of HTLV proteins, including env, gag, and tax. Cytotoxic lymphocytes that recognize viral proteins have also been identified. In patients with tropical spastic paraparesis, antibody against HTLV-1 is synthesized intrathecally. The role of HTLV-specific antibodies and T-cell cytotoxicity in the prevention of new infections or in disease progression is unclear.
HTLV-1 is not lysed by human serum, although human complement can lyse other animal retroviruses. The persistence of HTLV infection in vivo indicates that completely effective immune responses do not occur naturally. HTLV-l infection occurs, with varying degrees of prevalence, in several regions of the world Table The highest prevalence is in southwestern Japan, where six endemic areas have been identified.
The prevalence of anti-HTLV-1 antibodies in inhabitants of these areas ranges from 6 to 37 percent. The Caribbean is another region where HTLV-1 is endemic, with an overall seropositivity rate of 4 percent. Between 19 and 48 percent of family members of HTLV-1 -infected individuals in these two endemic areas are seropositive.
In both regions, the prevalence of anti-HTLV-1 antibodies increases with age, from 2 percent in young children to 30 percent in adults in their mid-forties. Although fewer data are available for Africa, HTLV- 1 infection is also endemic there, with seropositivity rates ranging from 4 to 7 percent. HTLV infection is also found in defined populations e. The predominant modes of transmission of HTLV infection are by sexual contact, via contaminated blood or blood products, and from mother to child via breast milk.
Studies in Japan have shown that between 48 and 82 percent of recipients of seropositive blood seroconverted. One unique characteristic of HTLV-1 infection is the extremely long incubation period between initial infection and the occurrence of disease. The incubation period for adult T-cell leukemia can range from years to decades and may be as long as 40 years; the incubation period for tropical spastic paraparesis is thought to be shorter but is still several years.
Antibodies to HTLV-1 are found in the vast majority of patients with adult T-cell leukemia and tropical spastic paraparesis. However, the incidence of T-cell cancers or tropical spastic paraparesis in HTLVinfected individuals is extremely low. Studies on the incidence of adult T-cell leukemia in Japan estimate that fewer than 0. Adult T-cell leukemia is usually found in individuals older than 40 years, and mostly in individuals who were infected as infants.
A positive assay is confirmed by direct radioimmunoprecipitation assays or Western immunoblots. The polymerase chain reaction assay, which is based on the amplification of viral DNA segments, is used to distinguish between these two viruses.
Since the interval between HTLV infection and the appearance of antibodies is not known, the polymerase chain reaction can also be used to diagnose HTLV infection in antibody-negative individuals. The presence of modest leukocytosis with abnormal-appearing lymphocytes in an asymptomatic, HTLV-infected individual is indicative of pre-adult T-cell leukemia.
Acute adult T-cell leukemia-lymphoma is characterized by the presence of pleomorphic neoplastic cells with mature T lymphocyte markers. Since adult T-cell leukemia cells express high levels of Tac antigen a protein chain that is part of the IL-2 receptor , diagnosis should include staining for the Tac antigen.
Clinical features may include dermal or epidermal infiltrates, hypercalcemia, osteolytic bone lesions, hepatosplenomegaly, and increased susceptibility to opportunistic infections. Chronic smoldering adult T-cell leukemia is diagnosed on the basis of skin lesions, low levels of circulating leukemic cells, and an absence of visceral involvement.
Tropical spastic paraparesis is characterized by a meningeal inflammatory process largely limited to the spinal cord, with progressive weakness in the lower extremities and sensory abnormalities. HTLV-1 can be isolated from the blood and cerebrospinal fluid of many patients with tropical spastic paraparesis. Control of HTLV infection and disease involves three approaches: therapy, education and public health, and vaccination Table Treatment of HTLV-1 infection and its sequelae is extremely difficult.
Current treatment regimens for adult T cell leukemia involve combination chemotherapy protocols which have not significantly increased survival. The elimination of Tac-positive cells by anti-Tac antibodies coupled to toxin is being considered as an experimental therapy. Glucocorticoids have been used in treating tropical spastic paraparesis, but with only limited success. The effect of these agents on HTLV in vivo is unknown. Although there is no treatment for HTLV infection, asymptomatic HTLV-infected individuals should be routinely screened for evidence of disease progression and counseled on ways to avoid spreading the infection.
There is no vaccine against HTLV infection or disease. The development of vaccines for retrovirus infections in general has proven extremely difficult. Several experimental HTLV-1 vaccines have been designed that use the envelope portion of the virus as the immunogen.
One HTLV-1 recombinant hybrid envelope product has been tested in cynomolgus monkeys. The receptive partner is at greatest risk There is an increased risk of transmission if partners have other sexually transmitted diseases and during primary HIV infection.
This is the second most common route of transmission world wide. Infection may occur in utero during birth commonest post-natally, through breast feeding. Exposure to blood: Intra-venous drug abusers - sharing of needles Needle-stick injuries - risk approximately 0. Most individuals experience a febrile illness about weeks after exposure.
This illness co-incides with sero-conversion development of antibodies and so is often referred to as the sero-conversion illness. The symptoms are similar to those of glandular fever, namely fever, sore throat, night sweats, lymphadenopathy, diarrhoea.
The illness is self limiting. Asymptomatic phase Following the primary infection, the patient enters a stage of clinical latency.
During this time the patient feels fine, but they are infectious as they have on-going viral replication. This healthy state may last many years. Prodromal phase As the CD4 counts drop, there is a gradual onset of a variety of prodromal disorders, such as weight loss, fever, persistant lymphadenopathy, oral candidiasis and diarrhoea. These symptoms precede the progression to AIDS. Acquired Immunodeficiency Syndrome AIDS Syndrome with the following features: 1 Constitutional disease: fever, diarrhoea, weight loss, skin rashes 2 Neuro-cognitave defects : dementia, myelopathy, peripheral neuropathy 3 Immunodeficiency: Increased susceptibility to opportunistic infections: 4 Rare malignancies : Kaposi sarcoma, oral hairy leukoplakia, lymphomas.
When a new infection is established, the first cells to be exposed are the dendritic cells. These cells are resident in the skin and genital mucosa. It is their job to take up antigen in the tissues and to transport it to regional lymph nodes where they present it to T cells.
Cycles of infection are set up in the CD4 cells in the lymphoid tissue. Helper T cells are the primary target of HIV. They are cytokine secreting cells that provide the signals to control the immune response. Without them the immune response cannot function. In the early days after infection, HIV is able to replicate to very high levels while the immune system learns to deal with it. The CD4 cell population in the gut is particularly severely affected early on. However, an immune response to the virus does develop after a while and virus levels in the blood fall to a steady state level.
Unfortunately, the immune response is not able to control the infection completely and virus replication continues in the lymphoid tissue. As time passes, the antiviral immunity begins to fail and virus levels begin to rise again and the person succumbs to the infection. Productive infection of the cell by the virus causes cell death with the release of new progeny virions.
Lysis of infected cells by the host's CTLs. Apoptosis activation induced cell death of uninfected cells. It is in the interests of the patient that the CTL killing of infected cells is efficient. If the immune system can kill the infected cells before they release new progeny viruses, virus production is less efficient and levels of virus are lower.
Thus patients with a strong CTL response have lower viral loads and survive for longer. Patients with a weak CTL response have higher viral loads and survive for a shorter time.
Dying T cells are replaced by de novo synthesis of new T cells in the thymus or by cell division of mature cells in the lymphoid organs. Only when the ability of the immune system to replace dead T cells fails, do T cell numbers begin to fall. Immune activation fuels disease progression. This is caused by ongoing virus replication and immune attack in the lymphoid tissue which damages the delicate network of immune cells.
The integrity of this network is crucial for the immune system to function effectively. Once this starts to fail, cells receive incorrect signals leading to: Inappropriate activation and death of un-infected cells by apoptosis Impairment of the function of the remaining cells Failure to regenerate new cells.
Immune activation is made worse by the fact that the lymphoid tissue of the gut is depleted early during the clinical course of infection and the mucosal barrier to the entry of bacterial products from the gut is compromised.
These products can reach the systemic lymphoid organs such as lymph nodes and spleen and induce local inflammatory responses. The source of infection is usually the mother.
About one third of babies born to HIV positive mothers will be infected unless antiretroviral prophylaxis is given to mother and baby.
The most risky time for transmission is during delivery, but in utero transmissions can also occur as well as post natal transmission during breast feeding. Because of their immature immune responses, about half of the infected infants do not have a phase of clinical latency, but instead develop a progressive illness and die in the first year of life.
The others will experience a latent period and may survive for years or longer. Symptoms of HIV infection in children include: Failue to thrive, Lymphadenopathy, diarrhoeal disease, opportunistic infections, interstitial pneumonia, parotitis etc. Tuberculosis, Pneumocystis jiroveci and CMV are very common opportunistic infections that cause the death of HIV infected children in the first year of life. Concerning liver transplantation, in , growth continued in the number of new waiting list registrations 12, in the United States; however, only transplants were performed, including living-donor transplants [ 8 ].
The situation is worse with other organs. In the United States, heart transplants were performed in , but almost the same number of patients were waiting for a heart [ 9 ]. In the Eurotransplant region, hearts were transplanted in , but the active waiting list at the end of was [ 10 ], indicating that the demand for heart transplants far exceeds the number of donated human organs.
In addition, Eurotransplant reported a decrease of transplanted organs from On this background, xenotransplantation offers several advantages over allotransplantation, among them nearly unlimited availability and increased microbiological safety. Since the pigs are generated under specified or designated pathogen-free conditions, the donor animals will be free of exogenous infectious microorganisms. In contrast, several viruses have been transmitted with solid organ allotransplantations, among them the human immunodeficiency virus HIV , the rabies virus, the human cytomegalovirus HCMV , Epstein-Barr virus, and others [ 11 ].
As a matter of fact, there are excellent achievements in the field of xenotransplantation, especially remarkable survival times of pig organ transplants in nonhuman primate recipients Table 1. It is important to note that no PERV transmission was observed in these trials.
In addition, the first clinical trials transplanting encapsulated pig islet cells into diabetic patients in New Zealand and Argentina and the preclinical trials with pig islet cells in nonhuman primates were successful [ 2 , 12 , 13 ]. The excellent survival times were achieved due to numerous genetic modifications of the pigs, improved immunosuppression regimens, and removal of pathogenic viruses from the donor pigs.
The first patient who received a human heart survived only 18 days [ 15 ]; the first patient in Germany, 27 h [ 16 ]. Retroviruses are enveloped RNA viruses. They encode an enzyme called reverse transcriptase, which is able to transcribe their single-stranded RNA genome into a double-stranded DNA copy.
Using another enzyme, the integrase, this DNA copy is integrated into the genome of the infected cell. No HIV-1 proviruses can be found, for example, in liver cells. HIV-1 is an exogenous retrovirus. When, however, a retrovirus infects and integrates into a sperm cell or an oocyte or into their precursor cells, after the fertilization of the oocyte by the sperm, the integrated retroviral provirus will be present in each cell of the developing embryo, and later of the whole organism.
These integrated retroviruses in all cells of an organism are called endogenous retroviruses. Endogenous retroviruses are found in all reptiles, birds, and mammals, including humans. Most of the human endogenous retroviruses HERVs are defective due to mutations and deletions, only some; e. Antibodies against HERV-K have been found in tumor patients and pregnant women, indicating that virus proteins are expressed [ 25 , 26 ].
It is well known now that the envelope proteins of endogenous retroviruses of numerous species are functioning as syncytins in the placenta development for review, see [ 27 , 28 ]. The genes and open reading frames are typical for gammaretrovirus and have been described in detail [ 3 ] Figure 1. The RNA genome encodes the core proteins Gag, group-specific antigen , a polymerase Pol and other enzymes, and the envelope proteins Env.
Schematic presentation of the genome of PERV. LTR, long terminal repeat; gag, group specific antigen; pol, polymerase; env, envelope. The env gene codes for the surface SU envelope protein and the transmembrane TM envelope protein.
The envelope proteins are responsible for binding to the cellular receptor and inducing membrane fusion. In the SU envelope protein, a receptor-binding domain RBD is located, binding to the receptor molecule. Purified viruses, recombinant TM proteins, and synthetic peptides corresponding the ISU domain have been shown to inhibit lymphocyte stimulation and to modulate the cytokine release of lymphocytes for review, see [ 32 , 33 ].
An immunosuppressive activity has also been shown for PERV [ 34 ]. The LTRs contain binding sites for transcription factors, and viruses with LTRs containing more enhancer repeats are characterized by higher expression and replication [ 35 ].
A productive infection characterized by replication of PERV was observed for some immortalized human cell lines such as the kidney cell line , and cat cells for review, see [ 3 , 36 ].
An infection without replication was observed for cells of minks, rhesus monkeys, baboons, gorillas, and chimpanzees. No infection was observed in the case of mouse, rat, rabbit, cotton rat, horse, pig-tailed macaque, African green monkey, and cynomolgus monkey cells. In contrast to human cells, which allow production of PERV because they lost intracellular restriction factors [ 37 ], other human cell lines such as THP-1 and C cells could be infected, but did not support PERV replication.
Of interest are the infection of primary human cells. Endothelial cells, vascular fibroblast, and mesangial cells could be infected with PERV [ 38 ]. However, it remains unclear in both cases whether the virus was produced.
Based on these results, infection experiments in vivo were performed. Neither small animals nor nonhuman primates could be infected, even when pharmaceutical immunosuppression was applied for review, see [ 3 ]. Only in the case of guinea pigs was a limited infection without evidence of replication observed in inoculated animals [ 40 ].
There are no new achievements in the field of viral receptors. They are members of the riboflavin transporter, also known as human riboflavin transporter 3 hRFT3 , and human riboflavin transporter 1 hRFT1 , respectively.
The PERV receptor on baboon and other nonhuman primate cells was functional, but deficient by a mutation, explaining the low replication in these cells [ 43 ]. In mice, the receptor is mutated [ 44 ], explaining that mouse cells could not be infected, and infection experiments with high doses in vivo also failed [ 45 ]. Transgenic mice had been generated carrying the HuPAR-2, and it was reported that they could be infected with PERV [ 46 ], but no further investigation followed.
Rats had only a low expression of the functional receptor, explaining that rat cells could not be infected; however, transfection with human or rat PAR-1 conferred susceptibility [ 44 ]. In summary, no animal models of PERV infection were found that would allow testing of antiretroviral drugs and vaccines.
PERV is the result of a trans-species transmission of a retrovirus or retroviruses from other species to the pig [ 47 , 48 ]. Trans-species transmission of retroviruses was and is a common mechanism of retrovirus distribution. The best investigated example is HIV [ 49 , 50 , 51 ] Figure 2. PERVs are the result of a trans-species transmission of precursor retroviruses from different animals and further evolution in the pig genome. Ancestral PERV-like sequences were found in lesser Egyptian jerboas Jaculus jaculus , in rock hyraxes Procavia capensis , and in eight murid species, indicating an ancient trans-species transmission from non-porcine species to pigs [ 47 , 48 ] Figure 2.
Examples of trans-species transmission of retroviruses. A Transmission of simian immunodeficiency viruses SIV from chimpanzee cpz or sooty mangabey sm , which are apathogenic in their natural hosts, resulting in highly pathogenic human immunodeficiency viruses HIV-1 and HIV B Example of a trans-species transmission of a gammaretrovirus, the koala retrovirus KoRV , which is closely related to PERV, which induces lymphoma and immunodeficiency in koalas, and which was possibly derived from bats or rodents [ 52 , 53 ].
C Trans-species transmission from different species resulted in integrated PERVs in the pig genome [ 47 , 48 ]. Numerous methods have been developed to detect PERV, both in the porcine donor and in the transplant recipient. These methods are methods that either directly detect viral RNA, proviral DNA, viral proteins, viral reverse transcriptase enzymatic activity, or infectious virus particles, or indirectly detect PERV-specific antibodies as sign of a viral infection.
The detection methods, or better, the detection systems, which are defined as the complex of sample generation, sample preparation, sample origin, time of sampling, and the necessary negative and positive controls, along with the specific detection methods either PCR-based, cell-based, or immunological methods , are well described in several reviews [ 3 , 54 , 55 , 56 ].
Of great importance for the evaluation of the safety of xenotransplantation is an assay detecting infectious viruses. At present, the most favored assay is based on infection of highly susceptible human cells [ 57 ]; however, this assay is very insensitive, and more sensitive tests should be developed [ 58 ].
Further improvement of the detection systems and their application in virus elimination programs will lead to clean donor animals and a safe xenotransplantation. The detection of PERV is usually one part of strategies to screen for a broad spectrum of porcine microorganisms that may be zoonotic. Such comprehensive strategies and the tested microorganisms were described in detail [ 59 , 60 , 61 , 62 , 63 , 64 ]. New methods were added to the plethora of already described ones [ 65 ].
One of the new methods is droplet digital PCR ddPCR , a method allowing to the correct measurement of the number of integrated proviruses. The copy number of PERVs in the genome of pigs; e. Since retroviral DNA molecules are not able to replicate autonomously like episomes, they depend on integration for stable maintenance in cells [ 17 ].
The analysis of the copy number revealed that PERV is still active, and that the copy number increases during fetal development and after birth. The copy number of PERV proviruses was much lower in expanded potential stem cells EPSCs than in young and older pigs, confirming the increase in copy number during their lifetime [ 69 ].
The replication of a virus in host cells significantly depends on the presence or absence of cellular restriction factors. The biology of the restriction factors inhibiting PERV is well described [ 3 , 36 , 70 ]. The authors concluded that transgenic overexpression of tetherin may reduce the risk of PERV transmission in xenotransplantation. These recombinants were integrated only in the genome of somatic cells, but not in the germ line [ 57 ].
The recombinants have a higher replication rate compared with the paternal PERV-A [ 75 ], and there are several genetic elements responsible for their high infectivity [ 76 ]. On the other hand, miniature swine that do not produce replication-competent PERV-C have been identified [ 78 ]. De novo infections and recombinations take place mainly in proliferating immune cells, because gammaretroviruses integrate only in proliferating cells. In diseased animals, which are setting up an effective immune response, the immune cells should proliferate massively.
This assumption agrees with our finding that mitogen-stimulation of pig lymphocytes of some kind simulating the immune stimulation led to an increased expression of PERV [ 83 , 84 , 85 ]. Budding viruses red arrow , maturating viruses green arrow , and mature viruses lilac arrow can be seen. Bar— nm. Courtesy of L. Laue, Robert Koch Institute, Berlin.
Endogenous retroviruses have been found highly expressed in embryonic stem cells ESCs and induced pluripotent stem cells iPSCs of humans and mice, and they were used as markers for pluripotency [ 87 , 88 , 89 , 90 ].
These cells were shown to express key pluripotency genes, to be genetically stable, and to differentiate to derivatives of the three germ layers, and additionally to trophoblast [ 92 ]. Therefore, EPSCs represent a unique state of cellular potency. Endogenous retroviruses were often found highly expressed in murine and human tumors; for example, the human endogenous retrovirus-K HERV-K was found expressed in human melanomas [ 93 ], prostate cancer [ 94 ], and other human tumors for review, see [ 95 , 96 ].
It remains unclear whether the endogenous retrovirus contributes to the tumor development itself, or whether it is expressed due to transcriptional activation in the tumor cells.
PERV particles were released from transformed pig kidney cells and lymphoma cells for review, see [ 3 ]. On the other hand, no PERV expression was found in two newly established pig lymphoma cell lines and L23 pig lymphoma cells [ 98 ]. Since in all three lymphoma cell lines the expression of PERV was very low, it seems unlikely that PERVs were involved in the pathogenesis of these lymphomas. However, all three lines were infected with the porcine lymphotropic herpesvirus-3 PLHV-3 , which may have been involved in lymphoma development.
In all preclinical and clinical trials performed until now, no PERV has been transmitted to the recipients. In the past, more than humans have received a xenotransplantation product comprising pig cells, or tissues including ex vivo perfusion of pig organs or pig cell-based bioreactors for review, see [ 3 ] and [ 99 ].
In the best documented human trials, encapsulated islet cells from Auckland Island pigs were transplanted to diabetic patients, and no PERV transmission was observed using both PCR-based and immunological methods [ , , ]. Concerning the preclinical trials, in recent studies transplanting islet cell in marmosets [ ] and cynomolgus monkeys [ ], no PERV transmission was observed Table 2. No PERV transmission was observed in a preclinical trial transplanting pig hearts from genetically modified pigs to baboons, with survival times of and days [ 26 , ] Table 2.
These long survival times were achieved because in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion, and control of post-transplantation growth of the transplant, the transmission of the porcine cytomegalovirus PCMV was prevented [ ]. In cases where PCMV was transmitted to the baboon recipient, and the survival time of the transplant was significantly reduced, PERV transmission also was not observed [ ].
When analyzing streptozotocin-induced diabetic cynomolgus macaques that received porcine islet macrobeads implanted intraperitoneally, no PERV transmission was observed when their PBMCs were screened by PCR [ 63 ] Table 2. Regarding the cornea, transmission of PERV was not evident in both in vitro [ ] and in vivo corneal transplantation studies [ , ]. In patients, PERV was not detectable up to 3.
Many other preclinical trials transplanting pig hearts, kidneys, islet cells, and cornea have been performed using effective immunosuppression regimens [ 27 , 63 , , , , , ]. Unfortunately, in these studies, PERV transmission was not analyzed, but at least no clinical signs of a retrovirus infection were observed. In order to prevent transmission of PERV to the recipient, a range of different strategies have been developed. The expression of PERV differs significantly between animals of one breed and between different breeds [ 83 , 84 , 85 ].
The antiretroviral drugs can be used in case an infection of the recipient has taken place. However, the experience with the treatment of acquired immunodeficiency syndrome AIDS demonstrated that a monotherapy with a single antiviral may soon lead to resistance [ ]. In this case, a combination therapy should be developed. Whereas there is no vaccine against the retrovirus HIV-1, there are effective vaccines against different gammaretroviruses.
Commercial vaccines against the FeLV, closely related to PERV, are on the market [ ], and experimental vaccines against the murine leukemia virus, also closely related to PERV, have been developed [ , ]. Using the recombinant surface envelope and transmembrane envelope proteins of PERV, neutralizing antibodies were induced in several animal species, suggesting that such antibodies could also be induced in humans [ , , , , ].
The combination of both proteins as ingredients in one vaccine resulted in higher titers of neutralizing antibodies compared with each envelope protein in a single application [ ]. Because there is no animal model to test such vaccines against PERV, the corresponding transmembrane and surface envelope protein of the related FeLV were used to induce neutralizing antibodies against FeLV for review, see [ ].
Using this vaccination strategy, strong neutralizing antibodies binding to similar epitopes, as in the case of PERV, were induced, and cats could be protected from FeLV disease [ ]. Why the animals produce antibodies against the core protein p27GAG at the same time remains unclear [ ].
Genome editing is a powerful tool to inactivate single genes in cells and animals [ ]. The situation with PERV is more complicated, as it is integrated 50—70 times in the genome of a cell. A highly conserved target sequence in the polymerase of all known proviruses was selected that should inactivate all PERVs in the genome. Unfortunately, the high expression of the ZFN was toxic to the transfected cells, most likely due to the specific cutting of the high copy number of the PERV proviruses [ ].
The altered morphology was possibly an off-target effect on the Gag protein or protease. The nuclei of these treated cells were transferred into pig oocytes, giving rise to embryos that were then transferred to surrogate sows.
This process resulted in the birth of healthy piglets with inactivated PERVs [ , ]. As demonstrated above, until now in all clinical trials, among them transplantations of pig islet cells from Auckland Island pigs in diabetic patients in New Zealand and Argentina, no transmission of PERV was observed [ 3 , 99 , , , ]. Furthermore, in all preclinical trials in nonhuman primates, no transmission of PERVs was observed [ , , , ]. However, nonhuman primates are not an ideal animal model to assess the risk of PERV transmission in xenotransplantation [ ].
In infection experiments in small animals and nonhuman primates with or without pharmaceutical immunosuppression, PERV transmission also was not observed: The mouse receptor was mutated and not effective, and the rat receptor was expressed only at low concentrations on the cell surface [ 44 ], showing that mouse and rat cells could not be infected [ 45 , 46 , ].
Xenotransplantation using pig cells, tissues, or organs is regarded as the next great medical revolution [ ]. PERVs are integrated in the genome of all pigs; they can be released as infectious particles, and some of them can infect human cells, therefore posing a risk for xenotransplantation. PERVs are typical gammaretroviruses, closely related to viruses inducing leukemia and immunodeficiencies in their hosts.
They are, like many other endogenous and exogenous retroviruses, the result of a trans-species transmission. Accumulated knowledge of the biology, replication, release, and mutation of PERV, as well as numerous preclinical and clinical trials, allow for a better risk evaluation; however, there are no more experimental approaches to evaluate the full risk until we move to the clinic. To prevent PERV transmission, numerous strategies have been developed, including selection of PERV-C-free animals, RNA interference, antiviral drugs, vaccination, and genome editing, all of which can be applied in clinical trials.
The author declares no conflict of interest.
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