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Kim, Jung, Park, Kim, and Hwang: No Evidence of the Productive Replication of Porcine Endogenous Retrovirus (PERV) from SNU Miniature Pigs in Human Cell Line
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Infect Chemother 2010;42(3):175-180.

Published online: 30 June 2010

DOI: https://doi.org/10.3947/ic.2010.42.3.175

No Evidence of the Productive Replication of Porcine Endogenous Retrovirus (PERV) from SNU Miniature Pigs in Human Cell Line

Jung Heon Kim1, 3, Eun-Suk Jung1, 3, Chung-Gyu Park1, 4, 5, 6, 7, Sang Joon Kim2, 7, Eung Soo Hwang1, 3, 8

1Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Korea

2Department of Surgery, Seoul National University College of Medicine, Seoul, Korea

3Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul, Korea

4Transplantation Research Institute, Seoul National University Medical Research Center, Seoul, Korea

5Seoul National University Cancer Research Institute, Seoul, Korea

6Center for Animal Resource Development, Seoul National University College of Medicine, Seoul,, Korea

7Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, Korea

8BK21 Division of Human Life Science, Seoul National University College of Medicine, Seoul, Korea

Corresponding author: Eung Soo Hwang, M.D., Ph.D. Department of Microbiology and Immunology, Seoul National University College of Medicine, 28 Yeongeon-dong, Jongro-gu, Seoul 110-799, Korea Tel: +82-2-740-8307, Fax: +82-2-743-0881 E-mail: hesss@snu.ac.kr

Received 20 April 2010       Revised 26 May 2010       Accepted 31 May 2010

Copyright © 2010 The Korean Society of Infectious Diseases; Korean Society for Chemotherapy

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The presence of porcine endogenous retrovirus (PERV) has been considered as one of the main hurdles to transplant pig’s organs or tissues to human beings. There has been no report that PERV infection is associated with human diseases. Because pigs have their own characteristics of PERV according to pig strain, it is necessary to analyze the infectivity of PERV from SNU miniature pig to human cells for future utilization as a transplantation donor.

Materials and Methods

Human cell lines were infected with culture supernatant from porcine cell line or immunomodulator-stimulated peripheral blood mononuclear cells (PBMC) of SNU miniature pigs. They were also co-cultured with PBMC or islet cells of SNU miniature pigs. The presence of PERV genes and general pig marker gene in cells was determined by nested PCR with primer set for PERV pol and pig mitochondrial cytochrome oxidase II (COII), respectively.

Results

Infection test with the culture supernatant from PBMC of SNU miniature pigs showed that PERV pol but not COII was detected only in a few cases, but there was no uniform infection pattern in scope of stimulators and cell types. PERV pol was not demonstrated in co-cultures of human cell line with PBMC or islet cells from SNU miniature pigs after 80 days of co-cultures.

Conclusions

In vitro infectivity test suggests that PERV from SNU miniature pig might not replicate productively in human cell lines although it could infect human cells and integrate into chromosome.

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Keywords: Porcine endogenous retrovirus, Infectivity, Human cell line, Co-culture, miniature pig, Xenotransplantation

Table 1.
Primer sets used for nested PCR in this study
primary nested
pol 5‘ GCA TTC AGT GCT GCT ACA AC 3’ 5’ GCT ACA ACC ATT AGG AAA ACT AAA AG 3’
5’ ATT GGA CAG GAA CTA GGA TG 3’ 5’ ACC CAG GAC TGT ATA TCT TGA TCA G 3’
COII 5’ CTT ACC CTT TCC AAC TAG GCT TC 3’ 5’ CAC ACA CTA GCA CAA TGG ATG CC 3’
5’ TTC GAA GTA CTT TAA TGG GAC AAG 3’ 5’ GAG GAT ACT AAT ATT CGG ATT GTT AT 3’

COII, cytochrome oxidase II

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ic-42-175-g001.tif
Figure 1.
Detection of PERV RNA in plasma (A) and culture supernatant of PBMC (B) from SNU miniature pig by RT-PCR. (A) M, molecular marker; 1, PK-15; 2, plasma; 3, distilled water control (B) M, molecular marker; 1, PK-15; 2, culture supernatant of PK-15; 3-8, culture supernatant of PBMC treated with PMA, PHA, PMA plus PHA, LPS, PGE2, and media; 9, distilled water control.

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Table 2.
Detection of PERV in Human Cell Lines Infected with Culture Supernatant of Stimulator-activated PBMC from SNU Miniature Pig*
PMA PHA LPS PGE2 PMA+PHA media
Exp-1
Day 3 ECV304 +/+ +/+ +/+ +/- +/+ +/+
HEK-293 +/- +/- +/+ +/+ +/+ +/-
THP-1 -/- +/- +/- +/- +/- +/-
Raji +/+ +/+ +/+ +/+ +/+ +/+
Jurkat +/+ +/+ +/+ +/- +/+ +/-
Day 6 THP-1 +/- +/+ +/- +/+ +/+ +/+
Raji +/+ +/+ +/+ +/+ +/+ +/+
Jurkat +/- +/+ +/- +/+ +/+ +/-
Day 9 THP-1 -/- +/- -/- -/- +/+ -/-
Raji -/- -/- -/- -/- +/+ +/-
Jurkat +/+ -/- -/- -/- +/- -/-
Exp-2
Day 1 THP-1 -/- -/- -/- -/- -/- -/-
Raji +/+ -/- -/- -/- +/- -/-
Jurkat +/+ -/- -/- -/- -/- -/-
HEK-293 -/- -/- -/- -/- -/- -/-
ECV304 -/- -/- -/- -/- -/- -/-
Day 7 THP-1 +/+ -/- +/+ +/- +/+ +/+
Raji -/- -/- +/- -/- +/+ -/-
Jurkat -/- +/- -/- +/+ +/- -/-
HEK-293 -/- -/- +/- -/- -/- -/-
ECV304 +/+ +/+ +/- +/- +/- +/+
Exp-3
Day 2 hPBMC -/- +/- +/- +/- +/+ +/-
THP-1 +/+ +/- +/- +/- +/+ +/-
Jurkat +/+ +/- +/- +/- +/+ +/-
Day 4 THP-1 +/+ +/- +/+ +/+ +/+ +/+
Raji +/+ +/- +/+ +/+ +/+ +/+
Jurkat +/+ +/- +/- +/- +/+ +/+
HEK-293 +/+ +/+ +/+ +/- +/+ +/+
Day 6 THP-1 +/+ +/- +/+ +/- +/- +/-
Raji +/- +/- +/- +/- +/- +/-
Jurkat +/+ +/- +/- +/- +/+ +/-

* +/+, pol(+)/COII(+); +/-, pol(+)/COII(-); -/-, pol(-)/COII(-)

PMA, phorbol myristate acetate; PHA, phytohemagglutinin; LPS, lipopolysaccharide; PGE2, prostaglandin E2

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Table 3.
Detection of PERV in Human Cell Lines Co-cultured with Peripheral Blood Mononuclear Cells from SNU Miniature Pig*
Exp-1 Day 2 Day 30 Day 87 Day 98 Day 127 Day 147 Day 162
Jurkat +/+ ND ND ND -/- ND -/-
T47D +/- ND -/- -/- -/- -/- -/-
ECV304 +/+ -/+ -/- -/- -/- -/- -/+
HEL299 +/+ +/+ -/- ND ND -/- -/+
U373MG +/- ND ND -/- -/- -/- -/-
Exp-2 D-2 D-7 D-21 D-38 D-48 D-63 D80 D91 D-105
THP-1 +/+ ND ND ND +/- -/- -/- -/-
Raji +/+ ND ND -/+ ND +/- ND -/-
Jurkat +/+ ND ND ND -/- -/- -/- -/-
T47D ND +/+ +/+ ND ND ND ND ND -/-
ECV304 +/+ ND +/+ ND -/- -/- -/- -/-

* +/+, pol(+)/COII(+); -/+, pol(-)/COII(+); -/-, pol(-)/COII(-); ND, not done

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Table 4.
Detection of PERV in HEK-293 Cells Co-cultured with Stimulator-activated Islet Cells from SNU Miniature Pig*
Exp-1 Day 5 Day 13 Day 22 Day 41 Day 62 Day 79 Day 90
Media +/+ +/+ +/+ -/- -/- -/- -/-
PMA +/+ +/+ +/+ -/- -/- -/- -/-
PHA +/+ +/+ +/+ -/- -/- -/+ -/-
LPS +/+ +/+ +/+ -/- -/- -/- -/-
PGE2 +/+ +/+ +/+ -/- -/- +/+ -/-
Exp-2 Day 30 Day 40 Day 58 Day 69 Day 90
Media +/+ -/+ -/- -/- -/-
PMA ND +/+ -/- -/+ -/-
PHA +/+ -/+ -/- -/- -/-
LPS -/+ -/+ -/- -/- -/-
PGE2 +/+ -/+ -/- -/- -/-

* +/+, pol(+)/COII(+); -/+, pol(-)/COII(+); -/-, pol(-)/COII(-); ND, not done.

PMA, phorbol myristate acetate; PHA, phytohemagglutinin; LPS, lipopolysaccharide; PGE2, prostaglandin E2

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