Type C Retrovirus Released from Porcine Primary Peripheral Blood Mononuclear Cells Infects Human Cells

Wilson, Carolyn A.; Wong, Susan; Müller, Jacqueline; Davidson, Cynthia E.; Rose, Timothy M.; Burd, Parris R.

doi:10.1128/jvi.72.4.3082-3087.1998

Cited by 354 publications

(160 citation statements)

References 24 publications

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“…Microarrays provide a powerful tool for viral discovery provided the new viruses are sufficiently related to those already known to permit specific hybridisation. Degenerate PCR primers are based on conserved sequences within viral groups and have an impressive track record having identified numerous macaque herpesviruses [1][2][3][4][5], GBV-C [10,11], HCoV-NH [18], animal retroviruses [6,7] and picornaviruses from seawater [9]. This approach is limited by each viral group requiring the use of different degenerate primer sets and the use of highly degenerate primers for highly variable viral groups.…”

Section: Methods Based On Specific Nucleic Acid Hybridisation and Antmentioning

confidence: 99%

“…Cell culture combined with visual observation for cytopathic effects, followed by testing for immunological cross-reactivity using large panels of sera, is a powerful and relatively rapid method when the unknown viral agents replicate in the particular cell lines used and cross-reactive reagents are available. Tentative identification of the virus then allows the use of more specific reagents, particularly degenerate PCR primers, targeting the likely viral group for definitive genetic characterisation [1][2][3][4][5][6][7][8][9][10][11]. Strictly molecular methods that do not require in vitro replication and scarce serological or antigenic reagents have also been developed and successfully used to allow the characterisation of numerous novel animal viruses.…”

Section: Introductionmentioning

confidence: 99%

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Viral metagenomics

Delwart

2007

Reviews in Medical Virology

250

206

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Characterisation of new viruses is often hindered by difficulties in amplifying them in cell culture, limited antigenic/serological cross-reactivity or the lack of nucleic acid hybridisation to known viral sequences. Numerous molecular methods have been used to genetically characterise new viruses without prior in vitro replication or the use of virus-specific reagents. In the recent metagenomic studies viral particles from uncultured environmental and clinical samples have been purified and their nucleic acids randomly amplified prior to subcloning and sequencing. Already known and novel viruses were then identified by comparing their translated sequence to those of viral proteins in public sequence databases. Metagenomic approaches to viral characterisation have been applied to seawater, near shore sediments, faeces, serum, plasma and respiratory secretions and have broadened the range of known viral diversity. Selection of samples with high viral loads, purification of viral particles, removal of cellular nucleic acids, efficient sequence-independent amplification of viral RNA and DNA, recognisable sequence similarities to known viral sequences and deep sampling of the nucleic acid populations through large scale sequencing can all improve the yield of new viruses. This review lists some of the animal viruses recently identified using sequence-independent methods, current laboratory and bioinformatics methods, together with their limitations and potential improvements. Viral metagenomic approaches provide novel opportunities to generate an unbiased characterisation of the viral populations in various organisms and environments.

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Section: Methods Based On Specific Nucleic Acid Hybridisation and Antmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Viral metagenomics

Delwart

2007

Reviews in Medical Virology

250

206

View full text Add to dashboard Cite

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“…DNA and RNA were therefore not extracted from the same tissue fragment; the different methods used permitted better purity and yield for each nucleic acid preparation. 1-2 mg of total RNA were reverse transcribed in a reaction volume of 30-60 ml containing 0.5 mM (each) dNTP, 10 mM DTT, 12.5 U SUPERase-In (Ambion, Austin, TX, USA), 0.5 mg Oligo dT [12][13][14][15][16][17][18] , 200U Superscript II (Life Technologies, Carlsbad, CA, USA) and reaction buffer (supplied with the enzyme) at 421C for 62 min. DNA and cDNA were stored at À201C before PCR analysis.…”

Section: Dna and Rna Extractionmentioning

confidence: 99%

Transient transmission of porcine endogenous retrovirus to fetal lambs after pig islet tissue xenotransplantation

et al. 2007

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Evidence for the in vivo transmission of porcine endogenous retrovirus (PERV) from porcine xenografts to various recipient animals has been inconsistent. To characterize the contribution of the host immune system to the potential for PERV transmission from pig islet tissue xenografts to host tissues, we examined two immunoincompetent animal models, thymectomizsed fetal lambs and NODscid mice. Pig proislets were grafted into fetal lambs or adult NODscid mice. Conventional, nested and real-time PCR/RT-PCR tests were used to search for PERV and pig cell-specific sequences (porcine mitochondrial cytochrome oxidase II (COII) or mitochondrial ribosomal 12S) in pig proislets, host liver and spleen at 5-84 days (lambs) or 96 days (mice) after transplantation. Xenografts were harvested at the same time points. The copy number of PERV sequences and host cell-specific nuclear (palmitoylcarnitine transferase) sequences was assessed by real-time PCR to estimate the proportion of PERV-infected host cells. Pig proislets were shown to be PERV+ve by PCR and immunohistochemistry (PERV B env protein p15E). PERV transmission (PERV A, B or C DNA in the absence of porcine COII or 12S sequences) was detected by nested PCR and real-time PCR in 4/12 fetal lamb liver samples 5-23 days after transplantation; the maximum copy number of PERV B env sequences was found at day 5 (700 copies/1Â10 6 lamb cells). A total of 4/12 fetal lambs demonstrated both PERV and 12S porcine sequences in liver samples (days 5-84) by real-time PCR, suggesting that pig cells had migrated to those tissues and established microchimerism; nested PCR showed evidence for microchimerism (porcine COII sequences alone) in 2/12 lambs (day 5). The incidence of PERV transmission and frequency of microchimerism was similar in host spleen analysed by real-time PCR. Histological examination showed complete xenograft rejection by 23 days after transplantation to fetal lambs. In contrast, pig proislet xenografts survived long term (Xday 96) in NODscid mice but no PERV transmission was found. Both nested and real-time PCR assays revealed that 2/3 mice had become microchimeric. Long-term expression of PERV A, B and C as well as porcine 12S or COII RNAs was found at the graft site (day 96) only, indicating that PERV transcription and possibly replication, continued in the donor pig islet tissue after transplantation. Overall, detection of PERV transmission and microchimerism was limited by the sensitivity of the PCR assay and the primers chosen. The absence of stable PERV transmission and microchimerism in fetal lambs and the rejection of pig proislet xenografts correlated in time with the establishment of host immunocompetence. We therefore suggest that the frequent failure to identify PERV transmission late after transplantation could be due to the immunological destruction of PERV-infected host cells. Recipient NODscid mice demonstrated long-term microchimerism and intragraft PERV expression, which was consistent with their stable immunoincompetence.

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“…[5][6][7][8][9][10] Two main concerns for porcine primary hepatocytes to become an ideal cell source of BALs for clinical application are their immunogenicity and risk of transmitting porcine endogenous retrovirus (PERVs). Although there is no report of PERVs transmission from pig to human in clinical trials to date, [11][12][13][14] it is already known that PERVs can infect human cells in vitro, 15,16 and retroviral vector pseudotypes bearing PERV-A and PERV-C (PERV-A/C recombinants) have a much higher chance to infect humans. 17,18 Immunogenic reactions are not taken the top priority to be settled when patients with acute liver failure are urgent for BALs, because immunosuppressive treatment will be undertaken during the surgery process.…”

Section: Introductionmentioning

confidence: 99%

Establishment of gene‐edited pigs expressing human blood‐coagulation factor VII and albumin for bioartificial liver use

Meng

Zou

et al. 2019

J of Gastro and Hepatol

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Background and Aim Bioartificial livers (BALs) are considered as a solution to bridge patients with acute liver failure to liver transplantation or to assist in spontaneous recovery for patients with end‐stage liver disease. Pig is the best donor of hepatocytes for BALs in clinical trials, because metabolic and detoxification function of its liver are close to human. However, using pig hepatocytes for BALs remains controversial for safety concern owing to nonhuman proteins secretion. Herein, we attempt to establish modified pigs expressing humanized liver proteins, blood‐coagulation factor VII (F7), and albumin (ALB). These pigs should also be porcine endogenous retrovirus subtype C (PERV‐C) free so that their ability of transmitting PERV to human could be diminished seriously. Methods We devised both homology‐dependent and independent knock‐in approaches to insert a fusion of hF7 and hALB gene downstream the site of pig endogenous F7 promoter in pig fetal fibroblasts negative for PERV‐C. The modified pigs were then generated through somatic cell nuclear transfer. Results We obtained 14 and 10 cloned pigs by homology‐dependent and independent approaches, respectively. Among them, 19 cloned pigs were with expected gene modification and 13 are alive to date. These modified pigs can successfully express hF7 and hALB in the liver and serum, and the expressed hF7 exhibits normal coagulation activity. Conclusions The gene‐edited pigs expressing hF7 and hALB in the liver were generated successfully. We anticipate that our pigs could provide an alternative cell source for BALs as a promising treatment for patients with acute liver failure.

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Type C Retrovirus Released from Porcine Primary Peripheral Blood Mononuclear Cells Infects Human Cells

Cited by 354 publications

References 24 publications

Viral metagenomics

Viral metagenomics

Transient transmission of porcine endogenous retrovirus to fetal lambs after pig islet tissue xenotransplantation

Establishment of gene‐edited pigs expressing human blood‐coagulation factor VII and albumin for bioartificial liver use

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