Suppressing active replication of a live attenuated simian immunodeficiency virus vaccine does not abrogate protection from challenge.

Although safety concerns preclude the use of live attenuated HIV vaccines in humans, they provide a useful system for identifying the elusive correlates of protective immunity in the SIV/macaque animal model. However, a number of pieces of evidence suggest that protection may result from prior occupancy of susceptible target cells by the vaccine virus rather than the immune response. To address this, we developed a Nef-deletion variant of an RT-SHIV whose active replication could be shut off by treatment with RT-inhibitors. Groups of macaques were inoculated with the ∆Nef-RT-SHIV and immune responses allowed to develop before antiretroviral treatment and subsequent challenge with wild-type SIVmac239. Vaccinated animals either resisted infection fully or significantly controlled the subsequent viremia. However, there was no difference between animals undergoing replication of the vaccine virus and those without. This strongly suggests that competition for available target cells does not play a role in protection.

Induction of neutralizing antibodies specific for the envelope proteins of the koala retrovirus by immunization with recombinant proteins or with DNA.

BACKGROUND: The koala retrovirus (KoRV) is the result of a transspecies transmission of a gammaretrovirus with fatal consequences for the new host. Like many retroviruses, KoRV induces lymphoma, leukemia and an immunodeficiency that is associated with opportunistic infections in the virus-infected animals. We recently reported the induction of neutralizing antibodies by immunization with the recombinant ectodomain of the transmembrane envelope protein p15E of KoRV. Since the neutralization titers of the p15E-specific sera were only moderate, we investigated the use of the surface envelope protein gp70 to induce neutralizing antibodies. FINDINGS: We immunized rats and goats with the recombinant gp70 protein of the KoRV, an unglycosylated protein of 52kD (rgp70/p52) or with the corresponding DNA. In parallel we immunized with recombinant rp15E or with a combination of rp15E and rgp70/p52. In all cases binding and neutralizing antibodies were induced. The gp70-specific sera had titers of neutralizing antibodies that were 15-fold higher than the p15E-specific sera. Combining rp15E and rgp70/p52 did not significantly increase neutralizing titers compared to rgp70/p52 alone. High titers of neutralizing antibodies specific for gp70 were also induced by immunization with DNA. Since KoRV and PERV are closely related, we investigated cross-neutralization of the antisera. The antisera against p15E and gp70 of PERV and KoRV inhibited infection by both viruses. CONCLUSION: The envelope proteins of the KoRV may therefore form the basis of an effective preventive vaccine to protect uninfected koalas from infection and possibly an immunotherapeutic treatment for those already infected.

A novel small animal model to study the replication of simian foamy virus in vivo.

Preclinical evaluation in a small animal model would help the development of gene therapies and vaccines based on foamy virus vectors. The establishment of persistent, non-pathogenic infection with the prototype foamy virus in mice and rabbits has been described previously. To extend this spectrum of available animal models, hamsters were inoculated with infectious cell supernatant or bioballistically with a foamy virus plasmid. In addition, a novel foamy virus from a rhesus macaque was isolated and characterised genetically. Hamsters and mice were infected with this new SFVmac isolate to evaluate whether hamsters are also susceptible to infection. Both hamsters and mice developed humoral responses to either virus subtype. Virus integration and replication in different animal tissues were analysed by PCR and co-cultivation. The results strongly indicate establishment of a persistent infection in hamsters. These studies provide a further small animal model for studying FV-based vectors in addition to the established models.

Staufen-1 interacts with the human endogenous retrovirus family HERV-K(HML-2) rec and gag proteins and increases virion production.

The human endogenous retrovirus family HERV-K(HML-2) Rec protein is an RNA transport factor that enhances nuclear export of intron-containing retroviral transcripts. Using the yeast two-hybrid approach, we have newly identified human Staufen-1 as a Rec-interacting protein. The interaction was confirmed by coimmunoprecipitation experiments, and the relevant site in Staufen-1 has been mapped to double-stranded RNA binding domain 4 (RBD4). Staufen-1 is in several aspects functionally related to retroviral RNA transport proteins. It binds mRNAs and targets its ribonuclear cargo to polysomes for efficient translation. We observed an accumulation of Staufen-1 in the nucleus of Rec-expressing cells and colocalization in the nucleoli as well as in the cytoplasm. Overexpression of Staufen-1 resulted in a 5-fold enhancement in nuclear export and/or translation of unspliced HERV-K(HML-2) viral RNAs in the presence of Rec and its Rec-responsive element (RcRE) binding site together with a clear increase in virus production. Staufen-1 was previously shown to interact with the Gag protein of HIV-1, promoting Gag oligomerization and RNA encapsidation. We demonstrate here that Staufen-1 also binds to the Gag protein of HERV-K(HML-2). Under stress conditions, Rec colocalizes with Staufen-1 in stress granules in cells that express viral RNA but not in mRNA-decay-related processing bodies. Our results suggest a new role for Staufen-1 as a cellular Rec and HERV-K(HML-2) Gag cofactor.

The Rec protein of HERV-K(HML-2) upregulates androgen receptor activity by binding to the human small glutamine-rich tetratricopeptide repeat protein (hSGT).

The expression of endogenous retroviruses of the HERV-K(HML-2) family is strongly upregulated in germ cell tumors and several other cancers. Although the accessory Rec protein of HERV-K(HML-2) has been shown to induce carcinoma in situ in transgenic mice, to increase the activity of c-myc and to interact with the androgen receptor (AR), whether or not Rec expression is indeed implicated causally in the initiation or progression of any human malignancies remains unclear. We used the yeast two-hybrid system involving the Rec protein of a recently integrated HERV-K(HML-2) element in an effort to identify potential Rec-related oncogenic mechanisms. This revealed the human small glutamine-rich tetratricopeptide repeat (TPR)-containing protein (hSGT) to be a cellular binding partner. The interaction of Rec with this known negative regulator of the AR was confirmed by coimmunoprecipitation, pull-down assays and colocalization studies. The interaction involves the TPR motif of hSGT and takes place in the cytoplasm and in the nucleoli. Using an AR-responsive promoter and gene we could demonstrate that Rec interference with hSGT resulted in an up to five-fold increase in the activity of AR. Furthermore, in AR positive cells, Rec was shown to act as transactivator by enhancing AR-mediated activation of the HERV-K(HML-2) LTR promoter. This is in line with previous observations of elevated HERV-K(HML-2) expression in steroid-regulated tissues. On the basis of our findings we propose a "vicious cycle" model of Rec-driven hyperactivation of the AR leading to increased cell proliferation, inhibition of apoptosis and eventually to tumor induction or promotion.

Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element.

BACKGROUND: The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. RESULTS: By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and proline-rich peptides, QP1 and QP2, spanning 23 and 19 amino acids, respectively. CONCLUSIONS: Expression of reconstituted sequences of original HERV elements is an important tool for studying fundamental aspects of the biology of these ancient viruses. The analysis of HERV-K(HML-2) Gag processing and the nature of the mature Gag proteins presented here will facilitate further studies of the discrete functions of these proteins and of their potential impact on the human host.

Joint production of prime/boost pairs of Fowlpox Virus and Modified Vaccinia Ankara recombinants carrying the same transgene.

Pairs of recombinant MVA (Modified Vaccinia Ankara) and FPV (Fowlpox Virus) expressing the same transgene are reasonable candidates for prime/boost regimens, because cross-reacting immune responses between the two vectors, both non-replicative in mammalian hosts, are very limited. The acceptor virus FPD-Red, a derivative of FPV, carrying a red fluorescent protein gene flanked by the homology regions of MVA deletion III, was constructed. The same MVA Transfer Plasmid Green, designed to insert transgenes into the MVA deletion III locus, can therefore be used to transfer transgenes into both acceptor viruses MVA-Red and FPD-Red with the described recently Red-to-Green gene swapping method. Cells infected by either recombinant virus can be sorted differentially by a simple and reliable FACS-based purification protocol. The procedure is carried out in primary chick embryo fibroblasts grown in serum-free media and was applied to the production of three rMVA/rFPV pairs expressing the H5N1 avian influenza antigens M1, M2 and NP. The viral genes were human codon-optimized and expressed at high levels in both chick and mammalian cells. Both single-step and multiple-step growth analyses showed no significant differences in growth due to the transgenes in either rMVA or rFPV derivatives.

Vaccine protection against lethal homologous and heterologous challenge using recombinant AAV vectors expressing codon-optimized genes from pandemic swine origin influenza virus (SOIV).

The recent H1N1 influenza pandemic and the inevitable delay between identification of the virus and production of the specific vaccine have highlighted the urgent need for new generation influenza vaccines that can preemptively induce broad immunity to different strains of the virus. In this study we have produced AAV-based vectors expressing the A/Mexico/4603/2009 (H1N1) hemagglutinin (HA), nucleocapsid (NP) and the matrix protein M1 and have evaluated their ability to induce specific immune response and protect mice against homologous and heterologous challenge. Each of the vaccine vectors elicited potent cellular and humoral immune responses in mice. Although immunization with AAV-M1 did not improve survival after challenge with the homologous strain, immunization with the AAV-H1 and AAV-NP vectors resulted in survival of all mice, as did inoculation with a combination of all three vectors. Furthermore, trivalent vaccination also conferred partial protection against challenge with the highly heterologous and virulent A/PR/8/34 strain of H1N1 influenza.

Generation of neutralising antibodies against porcine endogenous retroviruses (PERVs).

Antibodies neutralising porcine endogenous retroviruses (PERVs) were induced in different animal species by immunisation with the transmembrane envelope protein p15E. These antibodies recognised epitopes, designated E1, in the fusion peptide proximal region (FPPR) of p15E, and E2 in the membrane proximal external region (MPER). E2 is localised in a position similar to that of an epitope in the transmembrane envelope protein gp41 of the human immunodeficiency virus-1 (HIV-1), recognised by the monoclonal antibody 4E10 that is broadly neutralising. To detect neutralising antibodies specific for PERV, a novel assay was developed, which is based on quantification of provirus integration by real-time PCR. In addition, for the first time, highly effective neutralising antibodies were obtained by immunisation with the surface envelope protein of PERV. These data indicate that neutralising antibodies can be induced by immunisation with both envelope proteins.

Increased neutralizing antibody response after simultaneous immunization with leucogen and the feline leukemia virus transmembrane protein.

To develop improved vaccination strategies against feline leukemia virus (FeLV), rats were immunized with the transmembrane envelope protein p15E of FeLV alone or in combination with the commercial vaccine Leucogen® comprising the nonglycosylated FeLV surface envelope protein. Binding and neutralizing antibodies were induced in both groups and in the group immunized with Leucogen alone. Higher titers of antibodies neutralizing FeLV were induced by simultaneous immunization with Leucogen and p15E compared to the responses using Leucogen or p15E alone, suggesting that combination vaccines should be used in the future. Epitope mapping of p15E-specific antibodies induced by simultaneous immunization with Leucogen and p15E revealed the same pattern of response as obtained after immunization with p15E alone: one epitope was localized in the membrane-proximal external region (MPER) and the other in the fusion peptide-proximal region, and they are related to the epitopes detected after immunization with p15E of the porcine endogenous retrovirus and the koala retrovirus. The data indicate that these epitopes in the MPER are an effective target for neutralization and that antigens containing them may therefore prove to be a useful component of vaccines against retroviruses, including HIV-1.

Immunization with the transmembrane protein of a retrovirus, feline leukemia virus: absence of antigenemia following challenge.

A major challenge in the development of vaccines against retroviruses is the induction of neutralizing antibodies since they prevent infection of the cells where the virus may persist. The transmembrane envelope (TM) protein contains highly conserved domains and seems to be a suitable target. To study whether vaccinating with a TM protein of a retrovirus could protect from infection in vivo, cats were immunized with the TM protein p15E of feline leukemia virus (FeLV) and subsequently challenged. For the first time we show that immunization with a retroviral TM protein prevented antigenemia. The level of neutralizing antibodies after the boost immunization correlated with the outcome of FeLV infection.

Characterisation of a human cell-adapted porcine endogenous retrovirus PERV-A/C.

BACKGROUND: Porcine endogenous retroviruses (PERVs) pose a potential risk for xenotransplantation using pig cells, tissues or organs. A special threat comes from viruses generated by recombination between human-tropic PERV-A and ecotropic PERV-C. Serial passages of a recombinant PERV-A/C on human 293 cells resulted in increased infectious titers and a multimerization of transcription factor binding sites in the viral long terminal repeat (LTR). In contrast to the LTR, the sequence of the env gene did not change, indicating that the LTR represents the determinant of high infectivity. MATERIAL/METHODS: The virus was further propagated on human cells and characterized by different methods (titration, sequencing, infection experiments, electron microscopy). RESULTS: Further propagation on human 293 cells resulted in deletions and mutations in the LTR. In contrast to low-titer viruses, the high-titer virus was infectious for cells from non-human primates including chimpanzees. Scanning electron microscopy revealed clustering of budding virions at the cell surface of infected human cells and transmission electron microscopy indicated that the virus infects them via receptor-mediated endocytosis. CONCLUSIONS: After propagation of PERV on human cells without selection pressure, viruses with different LTR were generated. High titer PERV was shown to infect cells from non-human primates. The experiments performed here simulate the situation in vivo and give an extended characterization of human cell-adapted PERVs.

Regulation of human endogenous retrovirus-K expression in melanomas by CpG methylation.

The overall prognosis of patients with advanced melanoma is poor due to the lack of effective treatment. A key factor for successful therapy is an early detection of disease. Therefore, reliably detection methods and meaningful tumor markers are required. Expression of the human endogenous retrovirus (HERV)-K(HML-2) was found elevated in melanomas and it was shown that HERV-K supports the in vitro transition of melanoma cells from adherent to a more malignant, nonadherent phenotype. Furthermore, the detection of HERV-K-specific antibodies in melanoma patients was found to correlate with reduced survival. However, the reason for HERV-K expression in melanomas still remains unclear and its use as a tumor marker needs further investigation. Therefore, the tumor-specific transcriptional regulation of HERV-K expression in melanoma was studied in detail. Human melanoma cell lines were investigated for HERV-K expression using real-time PCR. Five cell lines showed very high levels of HERV-K mRNA as a result of increased promoter activity. This promoter activity was directly silenced by DNA methylation in reporter gene experiments. Higher levels of long terminal repeat (LTR) methylation in cells not expressing HERV-K compared with cells expressing HERV-K were found using methylation-sensitive PCR and bisulfite sequencing. Treatment of cell lines with the demethylating agent 5-aza-2'-deoxycytidine resulted in increased levels of HERV-K expression in cells previously not expressing HERV-K and it was shown that this increase is not the result of transcription factor activation. These results demonstrate that increased HERV-K expression in melanomas may be due to increased promoter activity and demethylation of the 5'LTR.

No evidence for XMRV in German CFS and MS patients with fatigue despite the ability of the virus to infect human blood cells in vitro.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV), a novel human retrovirus originally identified in prostate cancer tissues, has recently been associated with chronic fatigue syndrome (CFS), a disabling disease of unknown etiology affecting millions of people worldwide. However, several subsequent studies failed to detect the virus in patients suffering from these illnesses or in healthy subjects. Here we report the results of efforts to detect antibody responses and viral sequences in samples from a cohort of German CFS and relapsing remitting multiple sclerosis (MS) patients with fatigue symptoms. METHODOLOGY: Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors. Fatigue severity in MS patients was assessed using the Fatigue Severity Scale (FSS). Validated Gag- and Env-ELISA assays were used to screen sera for XMRV antibodies. PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene. In addition, PBMC cultures were exposed to 22Rv1-derived XMRV to assess infectivity and virus production. CONCLUSION: None of the screened sera from CFS and MS patients or healthy blood donors tested positive for XMRV specific antibodies and all PBMC (and PBMC plus LNCaP) cultures remained negative for XMRV sequences by nested PCR. These results argue against an association between XMRV infection and CFS and MS in Germany. However, we could confirm that PBMC cultures from healthy donors and from CFS patients can be experimentally infected by XMRV, resulting in the release of low levels of transmittable virus.

Beneficial and detrimental effects of human endogenous retroviruses.

In this mini review, we aim to evaluate the structure and function of Human Endogenous Retroviruses (HERVs) with respect to the benefit they may have for humans or the damage they may cause. Emphasis is laid on their putative roles, if any, in pregnancy, in gene regulation and in cancer. As a basis for this discussion it will first be necessary to briefly describe the structure and function of retroelements, including HERVs, before addressing their positive or negative effects at the cellular and organismal level. Finally, we will give an outlook in which we will attempt to define priorities for future research.

Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients.

BACKGROUND: A novel gammaretrovirus named xenotropic murine leukemia virus-related virus (XMRV) has been recently identified and found to have a prevalence of 40% in prostate tumor samples from American patients carrying a homozygous R462Q mutation in the RNaseL gene. This mutation impairs the function of the innate antiviral type I interferon pathway and is a known susceptibility factor for prostate cancer. Here, we attempt to measure the prevalence of XMRV in prostate cancer cases in Germany and determine whether an analogous association with the R462Q polymorphism exists. RESULTS: 589 prostate tumor samples were genotyped by real-time PCR with regard to the RNaseL mutation. DNA and RNA samples from these patients were screened for the presence of XMRV-specific gag sequences using a highly sensitive nested PCR and RT-PCR approach. Furthermore, 146 sera samples from prostate tumor patients were tested for XMRV Gag and Env antibodies using a newly developed ELISA assay. In agreement with earlier data, 12.9% (76 samples) were shown to be of the QQ genotype. However, XMRV specific sequences were detected at neither the DNA nor the RNA level. Consistent with this result, none of the sera analyzed from prostate cancer patients contained XMRV-specific antibodies. CONCLUSION: Our results indicate a much lower prevalence (or even complete absence) of XMRV in prostate tumor patients in Germany. One possible reason for this could be a geographically restricted incidence of XMRV infections.

Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain.

Endogenous retroviruses present in the human genome provide a rich record of ancient infections. All presently recognized elements, including the youngest and most intact proviruses of the human endogenous retrovirus K(HML-2) [HERV-K(HML-2)] family, have suffered postinsertional mutations during their time of chromosomal residence, and genes encoding the envelope glycoprotein (Env) have not been spared these mutations. In this study, we have, for the first time, reconstituted an authentic Env of a HERV-K(HML-2) provirus by back mutation of putative postinsertional amino acid changes of the protein encoded by HERV-K113. Aided by codon-optimized expression, we demonstrate that the reconstituted Env regained its ability to be incorporated into retroviral particles and to mediate entry. The original ancient HERV-K113 Env was synthesized as a moderately glycosylated gp95 precursor protein cleaved into surface and transmembrane (TM) subunits. Of the nine N-linked oligosaccharides, four are part of the TM subunit, contributing 15 kDa to its apparent molecular mass of 41 kDa. The carbohydrates, as well as the cytoplasmic tail, are critical for efficient intracellular trafficking, processing, stability, and particle incorporation. Whereas deletions of the carboxy-terminal 6 residues completely abrogated cleavage and virion association, more extensive truncations slightly enhanced incorporation but dramatically increased the ability to mediate entry of pseudotyped lentiviruses. Although the first HERV-K(HML-2) elements infected human ancestors about 30 million years ago, our findings indicate that their glycoproteins are in most respects remarkably similar to those of classical contemporary retroviruses and can still mediate efficient entry into mammalian cells.

Enhanced T- and B-cell responses to simian immunodeficiency virus (SIV)agm, SIVmac and human immunodeficiency virus type 1 Gag DNA immunization and identification of novel T-cell epitopes in mice via codon optimization.

As a prelude to primate studies, the immunogenicity of wild-type and codon-optimized versions of simian immunodeficiency virus (SIV)agm Gag DNA, with and without co-administered granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA, was directly compared in two strains of mice. Gag-specific T cells in the splenocytes of BALB/c and C57BL/6 mice immunized by gene gun were quantified by ELISpot using panels of overlapping synthetic peptides (15mers) spanning the entire capsid proteins of SIVagm, SIVmac and human immunodeficiency virus type 1. Specific antibodies were measured by ELISA. Codon optimization was shown to significantly increase the immune response to the DNA immunogens, reducing the amount of DNA necessary to induce cellular and antibody responses by one and two orders of magnitude, respectively. Co-administration of murine GM-CSF DNA was necessary for the induction of high level T- and B-cell responses. Finally, it was possible to identify both known and novel T-cell epitopes in the Gag proteins of the three viruses.

Mode of interaction between the HIV-1-neutralizing monoclonal antibody 2F5 and its epitope.

OBJECTIVE: To determine the mechanism of interaction between the HIV-1 gp41-specific broadly neutralizing monoclonal antibody (mAb) 2F5, its epitope in the membrane proximal external region and a domain located in the fusion peptide proximal region in the N-terminal region of gp41. Knowledge of these interactions would be useful for the design of antigens used to induce 2F5-like antibodies. METHODS: The binding and avidity of the mAb 2F5 were analyzed using enzyme-linked immunosorbent assays, epitope mapping and surface plasmon resonance analysis. Inhibition of virus neutralization by 2F5 was analyzed using peptides corresponding to the gp41 sequence. RESULTS: Using transmembrane envelope proteins of gammaretroviruses, we had previously induced neutralizing antibodies that recognize two epitopes, one located in the N-terminal part of the transmembrane protein (designated E1) and the other in the C-terminal membrane proximal external region (E2). The E2 epitope corresponds to the mAb 2F5/4E10 epitope in the gp41 of HIV and we have now identified a corresponding E1 domain in gp41. Although 2F5 did not bind directly to E1, the presence of E1 peptides increased the binding of 2F5 to peptides carrying its epitope. Neutralization of HIV-1 by 2F5 was inhibited more effectively by both gp41-derived peptides E1 and E2 together than with the peptide E2 alone. CONCLUSION: The interaction between the E1 and E2 domains of gp41 increased the efficacy of mAb 2F5 binding to its E2 epitope. Such an interaction may occur after gp41 folding into a six-helix bundle. Antigens containing both domains might be used to induce broadly neutralizing 2F5-like antibodies.

Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation.

BACKGROUND: Multi-transgenic pigs produced for use in xenotransplantation have to be screened for the presence and expression of porcine endogenous retroviruses (PERV) to select animals with low PERV load. The production of transgenic pigs may also be associated with the integration of the transgene adjacent to or into the locus of a PERV provirus, potentially leading to an enhanced virus expression. METHODS: Non-transgenic animals, single-transgenic, and multi-transgenic pigs were screened for the presence of PERV-A, -B, and -C and recombinant PERV-A/C using polymerase chain reaction (PCR). PERV expression was determined by real time reverse transcriptase-PCR. An assay based on the activation of PERV in peripheral blood mononuclear cells by mitogens was used to discriminate between low and high PERV producer animals. RESULTS: All animals carried PERV-A and -B. A total of 176 from 181 (97.2%) animals carried PERV-C in the germ line and 18 from 64 animals carried PERV-A/C in the genome of lymphoid cells but not in the germ line. The expression of PERV was very low in all animals and not different between transgenic pigs and non-transgenic animals. PERV expression differed between various pig lines. The highest expression was found in mini-pigs and crossing other pig lines with mini-pigs resulted in increased PERV expression in the progeny. However, expression of viral proteins and particle release were not observed in all transgenic animals. CONCLUSIONS: No evidence for elevated PERV expression in (multi-) transgenic pigs was observed. Differences in PERV expression correlated with the genetic background of the animals, not with the specific transgene. Mini-pigs consistently had the highest level of PERV expression and animals with a mini-pig background had a higher level of expression compared with animals without mini-pig background.