Optimizing lentiviral vector formulation conditions for efficient ex vivo transduction of primary human T cells in chimeric antigen receptor T-cell manufacturing.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing. METHODS: To identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVOTM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 h post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized. RESULTS: Post-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVOTM 15, and 10% trehalose-20 mM MgCl2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 h at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality. CONCLUSION: A buffer consisting of 10% trehalose-20 mM MgCl2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 h post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing.

Cytokines impact natural killer cell phenotype and functionality against glioblastoma in vitro.

Objective: Natural killer (NK) cells are a part of the innate immune system and first-line defense against cancer. Since they possess natural mechanisms to recognize and kill tumor cells, NK cells are considered as a potential option for an off-the-shelf allogeneic cell-based immunotherapy. Here, our objective was to identify the optimal cytokine-based, feeder-free, activation and expansion protocol for cytotoxic NK cells against glioblastoma in vitro. Methods: NK cells were enriched from human peripheral blood and expanded for 16 days with different activation and cytokine combinations. The expansion conditions were evaluated based on NK cell viability, functionality, expansion rate and purity. The cytotoxicity and degranulation of the expanded NK cells were measured in vitro from co­cultures with the glioma cell lines U­87 MG, U­87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell­selection steps (Depletion of CD3+ cells, enrichment of CD56+ cells, and depletion of CD3+ cells followed by enrichment of CD56+ cells); four activation protocols (continuous, pre-activation, re-activation, and boost); and four cytokine combinations (IL-2/15, IL­21/15, IL­27/18/15 and IL-12/18/15). Results: The expansion rates varied between 2-50-fold, depending on the donor and the expansion conditions. The best expansion rate and purity were gained with sequential selection (Depletion of CD3+ cells and enrichment of CD56+ cells) from the starting material and pre-activation with IL­12/18/15 cytokines, which are known to produce cytokine-induced memory-like NK cells. The cytotoxicity of these memory-like NK cells was enhanced with re-activation, diminishing the donor variation. The most cytotoxic NK cells were produced when cells were boosted at the end of the expansion with IL-12/18/15 or IL-21/15. Conclusion: According to our findings the ex vivo proliferation capacity and functionality of NK cells is affected by multiple factors, such as the donor, composition of starting material, cytokine combination and the activation protocol. The cytokines modified the NK cells' phenotype and functionality, which was evident in their reactivity against the glioma cell lines. To our knowledge, this is the first comprehensive comparative study performed to this extent, and these findings could be used for upscaling clinical NK cell manufacturing.

PeptiCHIP: A Microfluidic Platform for Tumor Antigen Landscape Identification.

Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology reduces the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs.

Porcine circovirus 2 (PCV2) population study in experimentally infected pigs developing PCV2-systemic disease or a subclinical infection.

Porcine circovirus 2 (PCV2) is a single stranded DNA virus with one of the highest mutation rates among DNA viruses. This ability allows it to generate a cloud of mutants constantly providing new opportunities to adapt and evade the immune system. This pig pathogen is associated to many diseases, globally called porcine circovirus diseases (PCVD) and has been a threat to pig industry since its discovery in the early 90's. Although 11 ORFs have been predicted from its genome, only two main proteins have been deeply characterized, i.e. Rep and Cap. The structural Cap protein possesses the majority of the epitopic determinants of this non-enveloped virus. The evolution of PCV2 is affected by both natural and vaccine-induced immune responses, which enhances the genetic variability, especially in the most immunogenic Cap region. Intra-host variability has been also demonstrated in infected animals where long-lasting infections can take place. However, the association between this intra-host variability and pathogenesis has never been studied for this virus. Here, the within-host PCV2 variability was monitored over time by next generation sequencing during an experimental infection, demonstrating the presence of large heterogeneity. Remarkably, the level of quasispecies diversity, affecting particularly the Cap coding region, was statistically different depending on viremia levels and clinical signs detected after infection. Moreover, we proved the existence of hyper mutant subjects harboring a remarkably higher number of genetic variants. Altogether, these results suggest an interaction between genetic diversity, host immune system and disease severity.

Transcriptomics uncovers substantial variability associated with alterations in manufacturing processes of macrophage cell therapy products.

Gene expression plasticity is central for macrophages' timely responses to cues from the microenvironment permitting phenotypic adaptation from pro-inflammatory (M1) to wound healing and tissue-regenerative (M2, with several subclasses). Regulatory macrophages are a distinct macrophage type, possessing immunoregulatory, anti-inflammatory, and angiogenic properties. Due to these features, regulatory macrophages are considered as a potential cell therapy product to treat clinical conditions, e.g., non-healing diabetic foot ulcers. In this study we characterized two differently manufactured clinically relevant regulatory macrophages, programmable cells of monocytic origin and comparator macrophages (M1, M2a and M0) using flow-cytometry, RT-qPCR, phagocytosis and secretome measurements, and RNA-Seq. We demonstrate that conventional phenotyping had a limited potential to discriminate different types of macrophages which was ameliorated when global transcriptome characterization by RNA-Seq was employed. Using this approach we confirmed that macrophage manufacturing processes can result in a highly reproducible cell phenotype. At the same time, minor changes introduced in manufacturing resulted in phenotypically and functionally distinct regulatory macrophage types. Additionally, we have identified a novel constellation of process specific biomarkers, which will support further clinical product development.

Exploratory metagenomic analyses of periweaning failure-to-thrive syndrome-affected pigs.

Modern pig farming is characterised by the emergence of several syndromes whose aetiology is unclear or has a multifactorial origin, including periweaning failure-to-thrive syndrome (PFTS). In fact, its specific aetiology remains elusive, although several causes have been investigated over time. The present study aimed to investigate the potential role of viral agents in PFTS-affected and healthy animals by evaluating the virome composition of different organs using a metagenomic approach. This analysis allowed demonstrating a higher abundance of Porcine parvovirus 6 (PPV6) in healthy subjects while Ungulate bocaparvovirus 2 (BoPV2), Ungulate protoparvovirus 1 (PPV) and Porcine circovirus 3 (PCV-3) were increased in pigs with PFTS. No differential abundance of RNA viruses was found between PFTS-affected and control pigs. Remarkably, this is the first molecular characterisation of PPV6 and BoPV2 in Spain and one of the few all around the world, supporting their apparent widespread circulation. Interestingly, PCV-3 has been recently identified in several clinical-pathological conditions as well as in healthy pigs, while BoPV2 pathogenic potential is unknown. Although obtained results must be taken as preliminary, they open the door for further studies on the potential role of these viruses or their combination as predisposing factor/s for PFTS occurrence.

Porcine circovirus 2 (PCV-2) genetic variability under natural infection scenario reveals a complex network of viral quasispecies.

Porcine circovirus 2 (PCV-2) is a virus characterized by a high evolutionary rate, promoting the potential emergence of different genotypes and strains. Despite the likely relevance in the emergence of new PCV-2 variants, the subtle evolutionary patterns of PCV-2 at the individual-host level or over short transmission chains are still largely unknown. This study aimed to analyze the within-host genetic variability of PCV-2 subpopulations to unravel the forces driving PCV-2 evolution. A longitudinal weekly sampling was conducted on individual animals located in three farms after the first PCV-2 detection. The analysis of polymorphisms evaluated throughout the full PCV-2 genome demonstrated the presence of several single nucleotide polymorphisms (SNPs) especially in the genome region encoding for the capsid gene. The global haplotype reconstruction allowed inferring the virus transmission network over time, suggesting a relevant within-farm circulation. Evidences of co-infection and recombination involving multiple PCV-2 genotypes were found after mixing with pigs originating from other sources. The present study demonstrates the remarkable within-host genetic variability of PCV-2 quasispecies, suggesting the role of the natural selection induced by the host immune response in driving PCV-2 evolution. Moreover, the effect of pig management in multiple genotype coinfections occurrence and recombination likelihood was demonstrated.

Temporal Dynamics of Gene Expression During Endothelial Cell Differentiation From Human iPS Cells: A Comparison Study of Signalling Factors and Small Molecules.

Endothelial cell (EC) therapy may promote vascular growth or reendothelization in a variety of disease conditions. However, the production of a cell therapy preparation containing differentiated, dividing cells presenting typical EC phenotype, functional properties and chemokine profile is challenging. We focused on comparative analysis of seven small molecule-mediated differentiation protocols of ECs from human induced pluripotent stem cells. Differentiated cells showed a typical surface antigen pattern of ECs as characterized with flow cytometry analysis, functional properties, such as tube formation and ability to uptake acetylated LDL. Gene expression analysis by RNA sequencing revealed an efficient silencing of pluripotency genes and upregulation of genes related to cellular adhesion during differentiation. In addition, distinct patterns of transcription factor expression were identified during cellular reprogramming providing targets for more effective differentiation protocols in the future. Altogether, our results suggest that the most optimal EC differentiation protocol includes early inhibition of Rho-associated coiled-coil kinase and activation of cyclic AMP signaling, and inhibition of transforming growth factor beta signaling after mesodermal stage. These findings provide the first systematic characterization of the most potent signalling factors and small molecules used to generate ECs from human induced pluripotent stem cells and, consequently, this work improves the existing EC differentiation protocols and opens up new avenues for controlling cell fate for regenerative EC therapy.

DHRS9 Is a Stable Marker of Human Regulatory Macrophages.

BACKGROUND: The human regulatory macrophage (Mreg) has emerged as a promising cell type for use as a cell-based adjunct immunosuppressive therapy in solid organ transplant recipients. In this brief report, dehydrogenase/reductase 9 (DHRS9) is identified as a robust marker of human Mregs. METHODS: The cognate antigen of a mouse monoclonal antibody raised against human Mregs was identified as DHRS9 by immunoprecipitation and MALDI-MS sequencing. Expression of DHRS9 within a panel of monocyte-derived macrophages was investigated by quantitative PCR, immunoblotting and flow cytometry. RESULTS: DHRS9 expression discriminated human Mregs from a panel of in vitro derived macrophages in other polarisation states. Likewise, DHRS9 expression distinguished Mregs from a variety of human monocyte-derived tolerogenic antigen-presenting cells in current development as cell-based immunotherapies, including Tol-DC, Rapa-DC, DC-10, and PGE2-induced myeloid-derived suppressor cells. A subpopulation of DHRS9-expressing human splenic macrophages was identified by immunohistochemistry. Expression of DHRS9 was acquired gradually during in vitro development of human Mregs from CD14 monocytes and was further enhanced by IFN-γ treatment on day 6 of culture. Stimulating Mregs with 100 ng/mL lipopolysaccharide for 24 hours did not extinguish DHRS9 expression. Dhrs9 was not an informative marker of mouse Mregs. CONCLUSION: DHRS9 is a specific and stable marker of human Mregs.

Development of an indirect ELISA assay for the detection of IgG antibodies against the ORF1 of Torque teno sus viruses 1 and 2 in conventional pigs.

Torque teno sus viruses (TTSuV, family Anelloviridae) cause long lasting and persistent infection in pigs under subclinical scenarios, and are potentially linked to several economically important swine diseases. Currently, little is known about swine immune response against TTSuV infections. In this study, an ELISA assay was developed based on the ORF1-A recombinant protein of two known TTSuVs, namely TTSuV1 (genus Iotatorquevirus) and TTSuV2 (genus Kappatorquevirus). The assay was used to study the development of the humoral immune response against TTSuV1 and TTSuV2 in longitudinally sampled clinically healthy pigs and their dams. Anti ORF1-A IgG was found in serum of pigs and sows for both TTSuVs. From 15 sows, 15 (100%) and 13 (83%) had anti ORF1-A IgG against TTSuV1 and TTSuV2, respectively. Pig sero-prevalences at the first sampling (4 weeks of age) were 65% (24/37) and 5% (2/37) for TTSuV1 and TTSuV2, respectively. For TTSuV1, the highest anti ORF1-A IgG prevalence was observed at weeks 21 and 25, with 68% (25/37) sero-positive pigs. Quantitative PCR (qPCR) results at week 21 revealed that 26 out of 32 (81%) pigs were positive for TTSuV1. In the case of TTSuV2, the highest anti ORF1-A IgG prevalence was observed at week 21, with 84% (31/37) pigs being sero-positive. At the same week, 92% (34/37) of pigs were qPCR positive. In summary, anti ORF1-A IgGs were detected in both sows and piglets at different ages, indicating that these animals could mount a humoral immune response against both TTSuVs. However, the high percentage of viremic pigs in presence of anti ORF1-A IgG suggests that these antibodies are not able to remove TTSuVs from circulation.

Vaccination of pigs reduces Torque teno sus virus viremia during natural infection.

Anelloviruses are a group of single-stranded circular DNA viruses infecting several vertebrate species. Four species have been found to infect swine, namely Torque teno sus virus (TTSuV) 1a and 1b (TTSuV1a, TTSuV1b; genus Iotatorquevirus), TTSuVk2a and TTSuVk2b (genus Kappatorquevirus). TTSuV infection in pigs is distributed worldwide, and is characterized by a persistent viremia. However, the real impact, if any, on the pig health is still under debate. In the present study, the impact of pig immunization on TTSuVk2a loads was evaluated. For this, three-week old conventional pigs were primed with DNA vaccines encoding the ORF2 gene and the ORF1-A, ORF1-B, and ORF1-C splicing variants and boosted with purified ORF1-A and ORF2 Escherichia coli proteins, while another group served as unvaccinated control animals, and the viral load dynamics during natural infection was observed. Immunization led to delayed onset of TTSuVk2a infection and at the end of the study when the animals were 15 weeks of age, a number of animals in the immunized group had cleared the TTSuVk2a viremia, which was not the case in the control group. This study demonstrated for the first time that TTSuV viremia can be controlled by a combined DNA and protein immunization, especially apparent two weeks after the first DNA immunization before seroconversion was observed. Further studies are needed to understand the mechanisms behind this and its impact for pig producers.

Porcine circovirus 2 immunology and viral evolution.

Porcine circovirus 2 (PCV2) has and is still causing important economic losses to pig industry. This is due to PCV2-systemic disease (PCV2-SD), formerly known as postweaning multi-systemic wasting syndrome (PMWS), which increases mortality rates and slows down the growth of the animals, as well as other conditions collectively included within the so-called porcine circovirus diseases (PCVD). PCV2-SD affected pigs are considered to be immunosuppressed, with severe lymphocyte depletion and evidence of secondary infections. However, PCV2-infected pigs not developing the disease are able to mount humoral and cellular immune responses and clear the virus or limit the infection. On the contrary, insufficient amounts of neutralizing antibodies have been linked to increased PCV2 replication, severe lymphoid lesions and development of PCV2-SD. Central role in controlling PCV2 infection are played by the antigen specific memory T cells. These cells persist long term post-infection or vaccination and are able to expand rapidly after recall antigen recognition. Most farms in the main pig producing countries are applying vaccination against PCV2 to prevent the disease and improve the farm performance. Vaccines do not induce sterilizing immunity and PCV2 keeps on circulating even in farms applying vaccination. This, together with the high mutation rate of PCV2, world-wide fluctuations in the genotype dominance and emergence of novel genetic variants, warrant close molecular survey of the virus in the field.

Genetic variability of porcine circovirus 2 in vaccinating and non-vaccinating commercial farms.

Vaccines against porcine circovirus 2 (PCV2) are now widely used to control the diseases caused by the virus. Although the vaccines protect pigs against the disease, they do not lead to sterilizing immunity and therefore infections with PCV2 continue in farms. It is expected that, due to its high evolutionary rate, PCV2 can adapt quickly to environmental pressures such as vaccination. The goal of this study was to elucidate the molecular variation of PCV2 in relation to vaccination. PCV2 variability was investigated from samples of infected pigs from five farms where vaccination had never been applied and two farms where pigs had been vaccinated for at least 2 years. For the genetic analysis, full PCV2 genomes were amplified and subsequently pooled by vaccination status from serum of eight vaccinated, infected pigs and 16 non-vaccinated, infected pigs. Variability of viral populations was quantified using next-generation sequencing and subsequent bioinformatics analysis. The number of segregating sites was similar in the non-vaccinated (n=109) and vaccinated pools (n=96), but the distribution of these sites in the genome differed. Most notably, in the capsid gene, the number of segregating sites was observed only in the non-vaccinated population. Based on the structural analysis, it is expected that some low-frequency amino acids result in biologically low-fit viruses. On the contrary, D294 in replicase represents a novel amino acid which was dominant and unique in the vaccinated pool. This work showed that variable PCV2 populations were circulating in commercial farms, and that this variability was different in samples obtained from vaccinating and non-vaccinating farms.

Detection of porcine anelloviruses in pork meat and human faeces.

Torque teno viruses (TTV) are icosahedral, single-stranded circular DNA viruses infecting several vertebrate species. Currently, these viruses are considered non-pathogenic although they are suggested to be co-factors in several diseases. Recently single-stranded circular DNA viruses have been found in human faeces. Considering the consumption of pork meat products and the ubiquitous nature of swine TTV (Torque tenosus virus, TTSuV), the human population is frequently exposed to these viruses. To determine if TTSuVs could be delivered through food, human faecal samples were analysed for their presence. Indeed, the results of this study show that up to 25% of faecal samples were positive for known TTSuVs by PCR and sequencing. Additionally, all commercially available pork products purchased in Spanish supermarkets contained DNA of TTSuV.

Torque teno sus virus 1 and 2 distribution in tissues of porcine circovirus type 2-systemic disease affected and age-matched healthy pigs.

The present work was aimed to investigate the torque teno sus virus 1 and 2 (TTSuV1 and 2) loads of different tissues of pigs affected by porcine circovirus type 2 (PCV2)-systemic disease (PCV2-SD) and healthy age-matched ones. A total of 20 pigs (10 healthy and 10 diagnosed as PCV2-SD) were chosen for the study. From each pig, a total of 7 tissues were analyzed, including lung, kidney, liver, ileum, bone marrow, and mesenteric and mediastinal lymph nodes. TTSuV 1 and 2 were quantified by means of species-specific real time quantitative PCR methods. Both TTSuVs were distributed systemically. Results showed the highest loads for TTSuV2 in tissues from diseased pigs. The highest TTSuV1 DNA load was found in bone marrow, lung, and liver, while it was in bone marrow, mediastinal lymph node and liver for TTSuV2. Bone marrow was the tissue with the highest viral DNA burden for both TTSuV species. Overall, statistically significant differences were observed in viral DNA load, with TTSuV2 showing higher loads than TTSuV1 (p<0.05). Also, tissues from PCVD-SD affected pigs showed higher TTSuV2 loads than tissues of the healthy group (p<0.05). It was concluded that both TTSuVs had a wide tissue distribution all over the body, with differential load comparing diseased versus healthy pigs. It is likely that other tissues not included in this study would also be infected by TTSuVs.

The natural history of porcine circovirus type 2: from an inoffensive virus to a devastating swine disease?

The present review summarizes the current knowledge on the natural history of porcine circovirus type 2 (PCV2) infection and its related diseases. The perception about PCV2 as a significant pathogen has markedly changed in the last 15 years. The ubiquitous nature of the virus, the retrospective evidence of this infection long before disease association, the multifactorial aetio-pathogenesis of PCV2-systemic disease (SD) and the lack of consistent demonstration of Koch's postulates caused great controversy about the real causal capabilities of this virus. The advent of vaccines against PCV2 radically changed such perception and this virus is nowadays regarded as a very important pig pathogen. Moreover, the current PCV2 vaccines are ones of the most widely used in pig producing countries. On the other hand, how the virus causes disease is still a not fully solved complex scientific question, but host, infection timing and the virus itself are pivotal factors to consider explaining disease presentation at an individual level. The appearance of PCV2-SD as an epidemic problem at the end of 1990 s or early-mid 2000s might be related with a number of known and unknown variables. Based on available data, the international trade of pigs may have played a major role in the dissemination of more susceptible swine genetic lines as well as in the global PCV2 genotype replacement (PCV2b over PCV2a) during such period.

Discovery of a novel Torque teno sus virus species: genetic characterization, epidemiological assessment and disease association.

The study describes a novel Torque teno sus virus (TTSuV) species, provisionally named Torque teno sus virus k2b (TTSuVk2b), originally found in commercial pig sera by applying the rolling-circle amplification technique. Full-length sequences of TTSuVk2b were obtained, annotated and used in the phylogenetic analyses, which revealed that TTSuVk2b is a novel Anellovirus species within the genus Kappatorquevirus of the family Anelloviridae. Quantitative PCR techniques were developed to determine total TTSuV DNA quantities as well as the prevalence and viral DNA quantities of TTSuV1, TTSuVk2a and TTSuVk2b. The mean total TTSuV load in seven commercial sera was determined at 6.3 log(10) DNA copies ml(-1) of serum, with TTSuVk2b loads being the lowest at 4.5 log(10) DNA copies ml(-1) of serum. Subsequently, prevalence and loads of TTSuVs were determined in pig sera from 17 countries. TTSuVk2b prevalence ranged from 0 to 100 % with viral loads from 3.3 to 4.6 log(10) copies ml(-1) of sera. TTSuVk2a, so far the only species in the genus Kappatorquevirus, has been linked to an economically important swine disease, namely post-weaning multisystemic wasting syndrome (PMWS). Considering the grouping of TTSuVk2b in the same genus as TTSuVk2a, TTSuVk2b prevalence and viral DNA load were determined in PMWS-affected animals and healthy counterparts. This revealed that TTSuVk2a and TTSuVk2b are not only genetically related, but also that their viral loads in serum are elevated in PMWS animals compared with those of healthy pen mates. In summary, the present work describes a novel TTSuV species including its genetic characterization, epidemiological assessment and potential disease association.

Globalisation and global trade influence molecular viral population genetics of Torque Teno Sus Viruses 1 and 2 in pigs.

Globalisation, in terms of the rapid and free movement of people, animals and food, has created a new paradigm, increasing the range and rate of distribution of many pathogens. In the present study, Torque teno sus viruses (TTSuVs) have been used as a model to evaluate the effects of global trade on viral heterogeneity, and how the movement of live pigs can affect the distribution and composition of virus populations. Seventeen countries from different parts of the world have been screened for TTSuV1 and TTSuvV2. High levels of genetic diversity have been found as well as two new TTSuV subtypes. A small fraction of this diversity (<5%) was related with spatial structure; however the majority (>50%) was best explained by the exchange of live pigs among countries, pointing to the direct relationship between the movement of hosts and the diversity of their accompanying viruses. Taking TTSuVs as sentinels, this study revealed that the distribution and diversity of comensal microflora in live animals subjected to global trade is shaped by the commercial movements among countries. In the case of TTSuVs, it appears that commercial movements of animals are eroding the genetic composition of the virus populations that may have been present in pig herds since their domestication.

Expression profile and subcellular localization of Torque teno sus virus proteins.

In the present study, the expression, generation and subcellular localization of Torque teno sus virus (TTSuV) proteins were characterized into two genetically distinct TTSuV species (TTSuV1 and TTSuV2). Following transfection of three TTSuV1 and TTSuV2 full-length ORF (ORF1, ORF2 and ORF3) expression constructs into porcine kidney cells, alternative splice variants encoding new TTSuV protein isoforms were identified for the first time. Proteins encoded from ORF1 and ORF3 were localized in the nucleoli of porcine kidney cells and that of ORF2 in the cytoplasm and nucleus excluding the nucleoli. The subcellular localization of the different protein isoforms was not only similar between distinct TTSuV species but also to the ones described in human Torque teno virus (TTV). Results of the present in vitro study were not based on full-length viral clones but suggested that alternative splicing strategy to generate TTSuV protein isoforms probably occurs in vivo. Obtained data provide new information on molecular biology of TTSuV and anelloviruses, which until now has been solely based on results obtained from human TTV.