PCR-based cloning and immunocytological titration of infectious porcine endogenous retrovirus subgroup A and B.
Two pig endogenous retroviruses (PERV), PERV-A and -B, productively infect human cells and are therefore considered to constitute a potential risk in pig-to-human xenotransplantation. A PCR-based cloning technique to isolate infectious PERV proviruses was established. Overlapping 3' half and 5' halves of PERV proviral genomes were amplified using DNA extracted from human 293 cells infected with PERV-A or -B. These clones were fused at a unique restriction site in the overlapping region and tested for their infectivity. Representative constructs possessed the same infectious properties as their parent isolates. We also developed a polyclonal anti-PERV serum by using recombinant PERV capsid protein derived from one of the infectious constructs as immunogen and established an immunocytological method for detection and titration of PERV infection. This detection method proved to be more sensitive than the current method of choice (transfer of MLV-lacZ vectors) for infectivity assessment of PERV. These findings should be considered for future characterization of PERV isolates.
Wohl Virion Centre, The Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK1.
Birke
B
Bartosch
Wohl Virion Centre, The Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK1.
Robin A
RA
Weiss
Wohl Virion Centre, The Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK1.
Yasuhiro
Y
Takeuchi
D000818:Animals; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis / Q000096:biosynthesis; D020077:Endogenous Retroviruses / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity; D005814:Genes, Viral; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D011533:Proviruses / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity; D011994:Recombinant Proteins / Q000276:immunology; D011995:Recombination, Genetic; D013552:Swine / Q000821:virology*; D020079:Terminal Repeat Sequences
D004279:DNA, Viral; D011994:Recombinant Proteins