Study of full-length porcine endogenous retrovirus genomes with envelope gene polymorphism in a specific-pathogen-free Large White swine herd

Abstract

Specific-pathogen-free (SPF) swine appear to be the most appropriate candidate for pig to human xenotransplantation. Still, the risk of endogenous retrovirus transmission represents a major obstacle, since two human-tropic porcine endogenous retroviruses (PERVs) had been characterized in vitro (P. Le Tissier, J. P. Stoye, Y. Takeuchi, C. Patience, and R. A. Weiss, Nature 389:681-682, 1997). Here we addressed the question of PERV distribution in a French Large White SPF pig herd in vivo. First, PCR screening for previously described PERV envelope genes envA, envB, and envC (D. E. Akiyoshi, M. Denaro, H. Zhu, J. L. Greenstein, P. Banerjee, and J. A. Fishman, J. Virol. 72:4503-4507, 1998; Le Tissier et al., op. cit.). demonstrated ubiquity of envA and envB sequences, whereas envC genes were absent in some animals. On this basis, selective out-breeding of pigs of remote origin might be a means to reduce proviral load in organ donors. Second, we investigated PERV genome carriage in envC negative swine. Eleven distinct full-length PERV transcripts were isolated. The sequence of the complete envelope open reading frame was determined. The deduced amino acid sequences revealed the existence of four clones with functional and five clones with defective PERV PK-15 A- and B-like envelope sequences. The occurrence of easily detectable levels of PERV variants in different pig tissues in vivo heightens the need to assess PERV transmission in xenotransplantation animal models.

FIG. 1

Distribution of proviral elements in SPF pigs in Ploufragan. PCR with primer pairs specific for envA, envB, and envC resulted in amplification products of 359 bp, 263 bp, and 281 bp, respectively. Here, a pig family was defined as the descendants of one boar. S₁ to S₃, mother sows; O₁ to O₄, piglets sired by boars 1 to 4, respectively.

FIG. 2

Characterization of full-length PERV RNA and DNA. Long PCR performed on (A) plasmid Tsukuba-1 DNA, (B) porcine DNA extracted from whole blood of one offspring in each pig family, and (C) cDNA derived from major pig organs (lanes +) and negative controls without reverse transcriptase (lanes −). A specific amplification product was obtained for cloned PERV sequence, whereas porcine DNA revealed multiple products of viral origin. RT-PCR demonstrated the existence of full-length and spliced viral RNA in all pig tissues studied.

FIG. 3

Amino acid sequences of untruncated envelope ORFs deduced from PERV transcripts. Dashes indicate gaps, and dots indicate identical amino acids.