Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue

Science

Volumn 285, Issue 5431, 1999, Pages 1236-1241

  Paradis, Khazal ^a   Langford, Gillian ^a   Long, Zhifeng ^a   Heneine, Walid ^a   Sandstrom, Paul ^a   Switzer, William M ^a   Chapman, Louisa E ^a   Lockey, Chris ^a   Onions, David ^a   Otto, Edward ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

DNA;

ANIMAL TISSUE; ARTICLE; DNA DETERMINATION; HUMAN; IMMUNOBLOTTING; MICROCHIMERISM; NONHUMAN; ORGAN DONOR; POLYMERASE CHAIN REACTION; PRIORITY JOURNAL; RETROVIRUS; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; SWINE; VIREMIA; VIRUS TRANSMISSION; XENOTRANSPLANTATION;

ADOLESCENT; ADULT; AGED; ANIMALS; ANTIBODIES, VIRAL; CHILD; CHILD, PRESCHOOL; CHIMERA; DNA, VIRAL; EXTRACORPOREAL CIRCULATION; FEMALE; GAMMARETROVIRUS; HUMANS; IMMUNOBLOTTING; ISLETS OF LANGERHANS TRANSPLANTATION; MALE; MIDDLE AGED; RETROSPECTIVE STUDIES; RETROVIRIDAE INFECTIONS; REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION; RNA, VIRAL; SKIN TRANSPLANTATION; SWINE; TRANSPLANTATION, HETEROLOGOUS; TUMOR VIRUS INFECTIONS; VIREMIA; ZOONOSES;

EID: 0033588002     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5431.1236     Document Type: Article

References (36)

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28. RNA was reverse-transcribed with a primer containing a 19-base sequence at the 3′-end specific for PERV pol and a 30-base tag sequence at the 5′-end, 5′-GAACATCGATGACAAGCTTAGGTATCGATAACAGCCGTTGGTGTGGTCA-3′, for 30 min at 42°C using 50 U of MuLV reverse transcriptase (RT) followed by 10 min at 90°C Control reactions with no RT were also performed as a control for contamination with PERV genomic DNA. After reverse transcription, the cDNA was amplified with primers specific for PERV pol (5′-AGCTCCGGGAGGCCTACTC-3′) and the cDNA specific 30-base tag primer (5′-GAACATCGATGACAAGCTTAGGTATCGATA-3′) and probe 5′-FAMCCACCGTGCAGGAAACCTCGAGACT-TAMRA-3′. Amplification was detected with the 7700 sequence detection system (ABI/Perkin-Elmer). The cycling parameters were 2 min at 50°C, 10 min at 95°C, and 42 cycles of 15 s at 95°C and 2 min at 65°C.
29. At Q-One, RNA was reverse-transcribed for 50 min at 42°C by means of 5 U of Moloney murine leukemia virus (MuLV) reverse transcriptase (RT) with random hexanucleotides. After transcription, ribonuclease H was added to the reaction mix, which was then heated to 37°C for 15 min. Control reactions with no RT were also performed as a control for contamination with PERV genomic DNA. After reverse transcription, 5 μl of the sample was amplified using primers from within the PERV gag gene, 5′-GCGACCCACGCAGTTGCATA-3′ and 5′-CAGTTCCTTGCCCAGTGTCCTT-3′. A second nested PCR assay was carried out using the primers 5′-TGATCTAGTGAGAGAGGCAGAG-3′ and 5′-CGCACACTGGTCCTTGTCG-3′. RT-PCR products were analyzed by agarose gel electrophoresis.
30. Purified recombinant PERV protein or virions were treated with SDS/β-mercaptoethanol and separated by electrophoresis through a tris-glycine acrylamide gel (12%) before immunoelectrotransfer onto PVDF membrane (Imobilon) (200 mA, 1 to 2 hours). The membrane was washed in phosphate-buffered saline (PBS)/ Tween before being treated with milk protein. Test sera were diluted to 1:200 in PBS/Tween containing 5% milk protein and incubated with membranes for 1 to 2 hours at room temperature. Blots were washed in PBS/ Tween and incubated with the secondary antibody, an alkaline phosphatase-labeted antiserum diluted at 1:1000, for 1 hour at room temperature. After incubation, the membrane was washed repeatedly in PBS/ Tween and developed with 5-bromo-4-chloro-2-indoyl phosphate/nitro blue tetrazolium, which gives a dark blue precipitate when labeled antibody has bound. Negative controls were performed by incubating membranes with serum from individuals who had not been exposed to porcine tissues. The protein immunoblot assay was validated with serum samples from a variety of sources (200 healthy humans, 58 HIV-1-positive humans, 18 HIV-2-positive humans, 13 HTLV-positive humans, four butchers with lymphoma, 20 transplant patients, and 10 CMV-positive humans with transplants).
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