• 미생물학회지
• /
• 제46권1호
• /
• pp.15-20
• /
• 2010

돼지를 이용한 이종간 장기이식은 인간 세포주를 감염시킬 수 있는 것으로 알려진 돼지 내인성 레트로바이러스의 존재로 인해 실제 적용에 어려움이 있다. PERV (Porcine Endogenous Retrovirus: PERV)-A와 PERV-B는 in vitro 상에서 인간 세포주와 돼지 세포주를 동시에 감염시킬 수 있으나 PERV-C는 단지 돼지 세포주만 감염시킬 수 있다. 또한 PERV-A와 PERV-B 또는 PERV-A와 PERV-C사이에 재조합이 일어나 새로운 위험한 바이러스가 출현할 가능성이 있다. 최초의 재조합 바이러스인 PERV-A14/220은 대부분 PERV-C의 유전자로 구성 되어있으며 수용체와 결합하는 부위만 PERV-A의 외막 유전자로 재조합이 되어 있는데 PERV-A보다 500배 이상 높은 감염가를 가진다. PERV-A14/220의 경우 PERV-A 외막 단백질 140 번째 아미노산과 PERV-C 외막 PRR (proline rich region) 부위가 높은 감염가에 관여하는 것으로 알려져 있다. 본 연구에서는 막 융합에 관여하는 PERV-C의 세포질쪽 C-말단 부위 또한 재조합 바이러스의 높은 감염가에 관여하는지 알아보기 위해 PERV-A/C의 재조합 외막 당단백질을 가진 pseudotype 바이러스를 만들어 사람 세포주에서 감염가를 측정하였는데 PERV-A 보다 재조합 바이러스가 10배 이상 높은 감염가를 나타내었다. 이러한 연구 결과는 PERV-C의 C-말단 막당단백질에 존재하는 양친매성 부위가 재조합 바이러스의 높은 감염가에 관여한 것으로 판단된다.

• Journal of Microbiology and Biotechnology
• /
• 제18권10호
• /
• pp.1735-1740
• /
• 2008

Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.

• Asian-Australasian Journal of Animal Sciences
• /
• 제25권10호
• /
• pp.1357-1363
• /
• 2012

Pigs may need to be exploited as xenotransplantation donors due to the shortage of human organs, tissues and cells. Porcine endogenous retroviruses (PERVs) are a significant obstacle to xenotransplantation because they can infect human cells in vitro and have the potential for transmission of unexpected pathogens to humans. In this research, 101 pigs, including four commercial breeds (23 Berkshire, 13 Duroc, 22 Landrace and 14 Yorkshire pigs), one native breed (19 Korean native pigs) and one miniature breed (10 NIH miniature pigs) were used to investigate insertional variations for 11 PERV loci (three PERV-A, six PERV-B and two PERV-C). Over 60% of the pigs harbored one PERV-A (907F8) integration and five PERV-B (B3-3G, B3-7G, 742H1, 1155D9 and 465D1) integrations. However, two PERV-A loci (A1-6C and 1347C1) and one PERV-B locus (B3-7F) were absent in Duroc pigs. Moreover, two PERV-C loci (C2-6C and C4-2G) only existed in Korean native pigs and NIH miniature pigs. The results suggest that PERV insertional variations differ among pig breeds as well as among individuals within a breed. Also, the results presented here can be used for the selection of animals that do not have specific PERV integration for xenotransplantation research.

• Journal of Animal Science and Technology
• /
• 제49권3호
• /
• pp.405-414
• /
• 2007

돼지의 내인성 레트로 바이러스인 PERV의 존재는 돼지의 무균 사육을 통해서도 제거할 수 없으며, 장기 이식 시 인수 공통 감염의 위험성을 내포하고 있으므로, 이종 간 장기 이식 시 면역 거부 반응과 더불어 해결해야 할 가장 큰 문제점 중의 하나이다. 본 연구에서는 국내에서 사육되고 있는 Berkshire, Duroc, Landrace, Yorkshire 종을 비롯하여 국내 미니 돼지 및 제주도 토종 흑돼지에서 PERV의 존재 유무 및 종류에 대하여 확인 및 검출하는 중합효소 연쇄반응(PCR) 기반 기법을 확립하였다. 사용된 모든 공시 돼지의 genomic DNA로부터 PERV- A 및 PERV-B가 모두 검출되었으며, PERV-C의 경우 음성대조구인 PK15 세포에서의 결과와 비교했을 때, Duroc종에서 매우 낮은 양의 PCR 산물이, 국내 미니돼지와 제주 토종 흑돼지에서는 상대적으로 높은 양의 PCR 산물이 검출되었다. 또한 PERV-A와 PERV-B의 경우 특이적 primer와 제한효소 BamHI을 이용한 PCR- RFLP를 통하여 확인할 수 있었다. 본 연구의 PERV 검출법은 이종 간 장기 이식 시 PERV에 대한 안전 검정 및 PERV의 분류 방법으로서 활용가능할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
• /
• 제21권4호
• /
• pp.387-390
• /
• 2011

Nude mice (BALB/c) were grafted with human 293 cells and PERV (porcine endogenous retrovirus)-IRES-EGFP (a packageable retroviral vector plasmid containing an internal ribosome entry site-enhanced green fluorescent protein)-producing pig PK15 cells in order to determine whether the pig cells could transmit PERV-IRES-EGFP to mice and human 293 cells in vivo. None of the transplanted human 293 cell lines were infected by PERV, but PCR analysis identified PERV-B provirus integration into both the heart and salivary gland of the inoculated nude mice. Our data indicate that hearts and salivary glands can be used to identify PERV-B receptors.

• Journal of Animal Science and Technology
• /
• 제46권3호
• /
• pp.307-314
• /
• 2004

돼지를 이용한 이종간 장기 이식은 차세대 장기 공급 부족 문제를 해결해 줄 수 있는 가능성을 제공해준다. 그러나 돼지 장기를 이용한 이종간 장기 이식은 면역학적인 문제 외에도 가축 유래 전염성 병원균에 대한 안전성 문제가 심각하게 고려되어지고 있다. 대부분의 외인성 병원균은 무균 사육환경을 통해 제어되는 반면 내인성 레트로 바이러스(Endogenous Retrovirus, PERVs)는 germ line을 통해 전파됨으로 사육 조건으로 해결할 수는 없다. 본 연구는 이종간 장기 이식용 무균 돼지 생산시 가장 문제가 되는 PERVs에 대한 분포를 알아보고자 국내 돼지(Landrace, Berkshire, Yorkshire, Duroc)의 genome 내 PERVs 분포와 유전자 염기 서열을 비교하였다. 모든 공시 돼지(20두)의 genomic DNA로부터 PCR을 이용하여 PERV A/B/C와 PERV-E의 존재를 확인하였다. pol 유전자 일부 염기 서열 분석 결과 같은 품종내 개체간 또는 품종간 상동성이 99.1과 98.8%로 매우 높게 나왔다. 본 연구의 결과 모든 국내 돼지 유전자내에 PERV A/B/C와 PERV E가 provirus 형태로 많이 존재하며 바이러스간의 높은 상동성은 바이러스 제어에 많은 잇점으로 작용하리라 사료되며 아직 밝혀지지 않은 내인성바이러스에 대한 연구가 요구되어진다. 그러므로 본 연구의 자료는 이종간 장기 이식용 무균돼지 생산 시 PERVs 를 제어하기 위한 기초 자료로 활용될 수 있을 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제20권4호
• /
• pp.461-465
• /
• 2007

Porcine Endogenous Retroviruses (PERVs) are a major concern in relation to xenotransplantation. Previous research indicated that PERVs are present at about 50 copies in the pig genome and their chromosomal insertion sites are different among pig breeds. We examined nine Korean native pigs and seven Asian Wild Boars for the presence of a PERV-A at SSC 1q2.4 and a PERV-B at SSC 7p1.1-2 previously reported in a Large White pig. The PERV-B at locus 7p1.1-2 displayed insertional variability in Korean native pigs and Asian Wild Boars. Using the primers for the PERV-A at 1q2.4 from Large White pig, we only can amplify an unclassified 798 bp sequence, which showed insertional variability only in Korean native pigs. This study indicates that there are differences within and between Asian and European pigs in PERV insertions and suggests that selection could generate PERV-free lines of pigs more suitable for xenotransplantation.

• Journal of Microbiology and Biotechnology
• /
• 제18권3호
• /
• pp.585-590
• /
• 2008

Human tropic Porcine Endogenous Retroviruses (PERVs) are the major concern in zoonosis for xenotransplantation because PERVs cannot be eliminated by specific pathogen-free breeding. Recently, a PERV A/C recombinant with PERV-C bearing PERV-A gp70 showed a higher infectivity (approximately 500-fold) to human cells than PERV-A. Additionally, the chance of recombination between PERVs and HERVs is frequently stated as another risk of xenografting. Overcoming zoonotic barriers in xenotransplantation is more complicated by recombination. To achieve successful xenotransplantation, studies on the recombination in PERVs are important. Here, we cloned and sequenced proviral PERV env sequences from pig gDNAs to analyze natural recombination. The envelope is the most important element in retroviruses as a pivotal determinant of host tropisms. As a result, a total of 164 PERV envelope genes were cloned from pigs (four conventional pigs and two miniature pigs). Distribution analysis and recombination analysis of PERVs were performed. Among them, five A/B recombinant clones were identified. Based on our analysis, we determined the minimum natural recombination frequency among PERVs to be 3%. Although a functional recombinant envelope clone was not found, our data evidently show that the recombination event among PERVs may occur naturally in pigs with a rather high possibility.

• Asian-Australasian Journal of Animal Sciences
• /
• 제21권11호
• /
• pp.1572-1575
• /
• 2008

The xenotransplantation of pig organs and cells can be related with a risk of transmission of infectious diseases to human. Previous findings indicate that the regulatory region of PERV for retroviral transcription, replication and integration into the cellular DNA is located on the 5' Long Terminal Repeat (LTR). The objective of this study is the investigation of methylation and deletion status of the PERV 5' LTR region which can be used for regulating PERV expression. We compared the sequences of genomic DNA and bisulfite-treated genomic DNA from PK-15 cells expressing PERV to observe the methylation status of the 5' LTR. Our results showed that the CpG sites of U3 were methylated and methylation was inconsistent in the R and U5 regions. Also, variable numbers of 18 bp repeats and 21 bp repeats were detected on 5' LTR by sequencing analysis. The consistent U3 methylation might be indicative of host suppression of expression of the retroviruses.

• Journal of Animal Science and Technology
• /
• 제47권2호
• /
• pp.177-186
• /
• 2005

Xenotransplantation may help to overcome the critical shortage of human tissues and organs for human transplantation, Swine represents an ideal source of such organs owing to their anatomical and physiological similarities to human besides their plentiful supply, However, the use of organs across the species barrier may be associated with the risk of transmission of pathogens, specially porcine endogenous retroviruses (PERVs).• Although most of these potential pathogens could be eliminated by pathogen-free breeding, PERVs are not eliminated by this treatment. PERVs are integrated into the genome of all pigs and produced by normal pig cells and infect human cells. They belong to gamma retroviruses and are of three classes viruses: A, B and C. In the present study, PCR based cloning was performed with chromosomal DNA extracted from pigs from domestic pigs in Korea. Amplified PCR fragments of about 1.5 Kb, covering the partial env gene, were cloned into pCR2.l-TOPO vectors and sequenced. A total of 91 env clones were obtained from domestic pigs, Berkshire, Duroc, Landrace and Yorkshire in Korea. Phylogenetic analysis of these genes revealed the presence of only PERV class A and B in the proportion of 58 % and 42 %, respectively. Among these, 28 clones had the correct open reading frame: 18 clones in class A and 10 clones in class B. Since both these PERV classes are polytropic and have the capacity to infect human cells, our data suggest that proviral PERVs have the potential to generate infectious viruses during or after xenotransplantation in human.