Improved pig donor screening including newly identified variants of porcine endogenous retrovirus-C (PERV-C).

Porcine endogenous retroviruses (PERV) are widely distributed in the genomes of pigs. PERV-A and PERV-B are present in all pigs. They infect human cells in vitro and therefore represent a risk for xenotransplantation when pig cells, tissues or organs are used. PERV-C infects only pig cells and is not present in the genomes of all pigs. However, PERV-A/C recombinants infecting human cells and characterized by high replication titers were found in pigs. To select PERV-C-free animals that cannot generate such recombinants, PCR-based assays were developed (Kaulitz et al., J Virol Methods, 175:60, 2011). When screening for PERV-C in German wild boars (Sus scrofa scrofa), applying these methods, a new variant of PERV-C was identified. Whereas in all 125 wild boar only the new variant of PERV-C was found, different variants were present in some landrace pigs, and most importantly, some pigs were totally free of PERV-C.

Increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (PERVs).

Despite enormous difficulties to induce antibodies neutralizing HIV-1, especially broadly neutralizing antibodies directed against the conserved membrane proximal external region (MPER) of the transmembrane envelope protein, such antibodies can be easily induced in the case of gammaretroviruses, among them the porcine endogenous retroviruses (PERVs). In addition to neutralizing antibodies directed against the transmembrane envelope protein p15E, neutralizing antibodies were also induced by immunization with the surface envelope protein gp70. PERVs represent a special risk for xenotransplantation using pig tissues or organs since they are integrated in the genome of all pigs and infect human cells and a vaccine may protect from transmission to the recipient. To investigate the effect of simultaneous immunization with both proteins in detail, a study was performed in hamsters. Gp70 and p15E of PERV were produced in E. coli, purified and used for immunization. All animals developed binding antibodies against the antigens used for immunization. Sera from animals immunized with p15E recognized epitopes in the MPER and the fusion peptide proximal region (FPPR) of p15E. One MPER epitope showed a sequence homology to an epitope in the MPER of gp41 of HIV-1 recognized by broadly neutralizing antibodies found in HIV infected individuals. Neutralizing antibodies were detected in all sera. Most importantly, sera from animals immunized with gp70 had a higher neutralizing activity when compared with the sera from animals immunized with p15E and sera from animals immunized with gp70 together with p15E had a higher neutralizing activity compared with sera from animals immunized with each antigen alone. These immunization studies are important for the development of vaccines against other retroviruses including the human immunodeficiency virus HIV-1.

Expression of porcine endogenous retroviruses (PERV) in different organs of a pig.

Porcine endogenous retroviruses (PERVs) represent a particular risk for xenotransplantation using pig cells, tissues or organs. PERVs are integrated in the genome of all pig strains and can be released as particles that infect human cells. We performed for the first time a systematic analysis of PERV expression in different organs of a miniature pig using in parallel quantitative real-time RT-PCR, Western blot analysis, and immunohistochemistry. All three types of PERV, PERV-A, PERV-B and PERV-C were present in the germ line of the animal. In addition, recombinant PERV-A/C were detected in some tissues, but not in the germ line. Expression of the viral full-length and spliced mRNA and proteins was found in many organs, but at different levels. A high expression was found in lymphoid organs.

Development of sensitive methods for detection of porcine endogenous retrovirus-C (PERV-C) in the genome of pigs.

Porcine endogenous retroviruses (PERV) represent a risk for xenotransplantation using pig cells, tissues or organs. PERV-A and PERV-B are present in the genome of all pigs and both infect human cells in vitro. PERV-C infects only pig cells and it is integrated in the genome of most, but not all pigs. Recombinants between PERV-A and PERV-C were described that infect human cells and replicate at high titres. To avoid such recombinations, PERV-C positive animals should not be used for breeding animals suited for xenotransplantation. In order to detect PERV-C positive pigs, different methods were developed such as specific PCRs using different primers, a highly sensitive nested PCR and a real-time PCR allowing measurement of proviral copy numbers. The real-time PCR was found to be useful to discriminate between contamination and actual provirus copies. The PCRs were optimized and their sensitivity was determined. Screening can be started with PCR1, if the result is negative, PCR2 to PCR5 or the nested PCR should be used, if the result is positive, the real-time PCR should be used to exclude contaminations. All methods were used to evaluate the prevalence of PERV-C and to identify PERV-C free animals. Due to the risk of contamination with cells from other animals testing should be performed with blood cells, not with ear biopsies.