Direct in vivo CAR T cell engineering.

T cells modified to express intelligently designed chimeric antigen receptors (CARs) are exceptionally powerful therapeutic agents for relapsed and refractory blood cancers and have the potential to revolutionize therapy for many other diseases. To circumvent the complexity and cost associated with broad-scale implementation of ex vivo manufactured adoptive cell therapy products, alternative strategies to generate CAR T cells in vivo by direct infusion of nanoparticle-formulated nucleic acids or engineered viral vectors under development have received a great deal of attention in the past few years. Here, we outline the ex vivo manufacturing process as a motivating framework for direct in vivo strategies and discuss emerging data from preclinical models to highlight the potency of the in vivo approach, the applicability for new disease indications, and the remaining challenges associated with clinical readiness, including delivery specificity, long term efficacy, and safety.

A Combined Approach for the Characterization of Small Ruminant Lentivirus Strains Circulating in the Islands and Mainland of Greece.

Small ruminant lentiviruses are a group of viruses infecting goat and sheep worldwide. These viruses exhibit an extraordinary degree of genetic and antigenic variability that severely influence in vivo and in vitro features, as well as diagnostic test results. Small ruminant farming is the most important animal farming business in Greece, with a high impact on the Greek primary economy. Although SRLV infection and its impact on animal production are well established in the country, little is known about the circulating SRLV strains and their prevalence. The aim of this study was to characterize SRLVs circulating in Greece with a combined serological and molecular approach, using the bulk milk matrix collected from 60 farms in different municipalities. This study allowed us to estimate a seroprevalence of around 52% at the herd level. The B1, B2 and A3 subtypes and a novel A viral cluster were identified. Moreover, the amplicon sequencing method allowed us to identify more than one viral subtype in a sample. These results again confirm the high variability of these viruses and highlight the importance of the constant monitoring of viral evolution, in particular in antigens of diagnostic interest.

Capsid-dependent lentiviral restrictions.

Host antiviral proteins inhibit primate lentiviruses and other retroviruses by targeting many features of the viral life cycle. The lentiviral capsid protein and the assembled viral core are known to be inhibited through multiple, directly acting antiviral proteins. Several phenotypes, including those known as Lv1 through Lv5, have been described as cell type-specific blocks to infection against some but not all primate lentiviruses. Here we review important features of known capsid-targeting blocks to infection together with several blocks to infection for which the genes responsible for the inhibition still remain to be identified. We outline the features of these blocks as well as how current methodologies are now well suited to find these antiviral genes and solve these long-standing mysteries in the HIV and retrovirology fields.

Pro-inflammatory feedback loops define immune responses to pathogenic Lentivirus infection.

BACKGROUND: The Lentivirus human immunodeficiency virus (HIV) causes chronic inflammation and AIDS in humans, with variable rates of disease progression between individuals driven by both host and viral factors. Similarly, simian lentiviruses vary in their pathogenicity based on characteristics of both the host species and the virus strain, yet the immune underpinnings that drive differential Lentivirus pathogenicity remain incompletely understood. METHODS: We profile immune responses in a unique model of differential lentiviral pathogenicity where pig-tailed macaques are infected with highly genetically similar variants of SIV that differ in virulence. We apply longitudinal single-cell transcriptomics to this cohort, along with single-cell resolution cell-cell communication techniques, to understand the immune mechanisms underlying lentiviral pathogenicity. RESULTS: Compared to a minimally pathogenic lentiviral variant, infection with a highly pathogenic variant results in a more delayed, broad, and sustained activation of inflammatory pathways, including an extensive global interferon signature. Conversely, individual cells infected with highly pathogenic Lentivirus upregulated fewer interferon-stimulated genes at a lower magnitude, indicating that highly pathogenic Lentivirus has evolved to partially escape from interferon responses. Further, we identify CXCL10 and CXCL16 as important molecular drivers of inflammatory pathways specifically in response to highly pathogenic Lentivirus infection. Immune responses to highly pathogenic Lentivirus infection are characterized by amplifying regulatory circuits of pro-inflammatory cytokines with dense longitudinal connectivity. CONCLUSIONS: Our work presents a model of lentiviral pathogenicity where failures in early viral control mechanisms lead to delayed, sustained, and amplifying pro-inflammatory circuits, which in turn drives disease progression.

Serological and Molecular Characterization of Small Ruminant Lentiviruses in Morocco.

Recent studies that investigated the origins of SRLV strains offered new insights into their distribution among domestic ruminants. The aim of the study was to investigate SRLV circulation in Morocco. A total of 51 farms were selected in different geographical locations and tested by screening and genotyping ELISA. Whole blood was used for DNA extraction and nested gag PCR. The sample size allowed for an estimation of prevalence lower than 20% (CI 95%). Surprisingly, a large proportion of screening-positive samples were not correctly serotyped. Sanger and NGS amplicon sequencing approaches allowed us to obtain new sequences even from difficult-to-amplify samples. The serological data support the evidence of an intrinsic difficulty of SRLV to spread, likely due to management practices. The low rate of success by genotyping ELISA led us to suppose that divergent strains might have escaped from diagnostic tools, as partially confirmed by the evidence of an A subtype carrying a mismatch in serotyping epitope. The sequence analysis revealed the circulation of novel B and recombinant A/B subtypes. This study highlights the importance of monitoring viral sequences and their evolution to develop specific diagnostic tests, particularly in countries where control measures are in place.

Acellular scaffold-based approach for in situ genetic engineering of host T-cells in solid tumor immunotherapy.

BACKGROUND: Targeted T-cell therapy has emerged as a promising strategy for the treatment of hematological malignancies. However, its application to solid tumors presents significant challenges due to the limited accessibility and heterogeneity. Localized delivery of tumor-specific T-cells using biomaterials has shown promise, however, procedures required for genetic modification and generation of a sufficient number of tumor-specific T-cells ex vivo remain major obstacles due to cost and time constraints. METHODS: Polyethylene glycol (PEG)-based three-dimensional (3D) scaffolds were developed and conjugated with positively charged poly-L-lysine (PLL) using carbamide chemistry for efficient loading of lentiviruses (LVs) carrying tumor antigen-specific T-cell receptors (TCRs). The physical and biological properties of the scaffold were extensively characterized. Further, the scaffold loaded with OVA-TCR LVs was implanted in B16F10 cells expressing ovalbumin (B16-OVA) tumor model to evaluate the anti-tumor response and the presence of transduced T-cells. RESULTS: Our findings demonstrate that the scaffolds do not induce any systemic inflammation upon subcutaneous implantation and effectively recruit T-cells to the site. In B16-OVA melanoma tumor-bearing mice, the scaffolds efficiently transduce host T-cells with OVA-specific TCRs. These genetically modified T-cells exhibit homing capability towards the tumor and secondary lymphoid organs, resulting in a significant reduction of tumor size and systemic increase in anti-tumor cytokines. Immune cell profiling revealed a significantly high percentage of transduced T-cells and a notable reduction in suppressor immune cells within the tumors of mice implanted with these scaffolds. CONCLUSION: Our scaffold-based T-cell therapy presents an innovative in situ localized approach for programming T-cells to target solid tumors. This approach offers a viable alternative to in vitro manipulation of T-cells, circumventing the need for large-scale in vitro generation and culture of tumor-specific T-cells. It offers an off-the-shelf alternative that facilitates the use of host cells instead of allogeneic cells, thereby, overcoming a major hurdle.

Replicative fitness and pathogenicity of primate lentiviruses in lymphoid tissue, primary human and chimpanzee cells: relation to possible jumps to humans.

BACKGROUND: Simian immunodeficiency viruses (SIV) have been jumping between non-human primates in West/Central Africa for thousands of years and yet, the HIV-1 epidemic only originated from a primate lentivirus over 100 years ago. METHODS: This study examined the replicative fitness, transmission, restriction, and cytopathogenicity of 22 primate lentiviruses in primary human lymphoid tissue and both primary human and chimpanzee peripheral blood mononuclear cells. FINDINGS: Pairwise competitions revealed that SIV from chimpanzees (cpz) had the highest replicative fitness in human or chimpanzee peripheral blood mononuclear cells, even higher fitness than HIV-1 group M strains responsible for worldwide epidemic. The SIV strains belonging to the "HIV-2 lineage" (including SIVsmm, SIVmac, SIVagm) had the lowest replicative fitness. SIVcpz strains were less inhibited by human restriction factors than the "HIV-2 lineage" strains. SIVcpz efficiently replicated in human tonsillar tissue but did not deplete CD4+ T-cells, consistent with the slow or nonpathogenic disease observed in most chimpanzees. In contrast, HIV-1 isolates and SIV of the HIV-2 lineage were pathogenic to the human tonsillar tissue, almost independent of the level of virus replication. INTERPRETATION: Of all primate lentiviruses, SIV from chimpanzees appears most capable of infecting and replicating in humans, establishing HIV-1. SIV from other Old World monkeys, e.g. the progenitor of HIV-2, replicate slowly in humans due in part to restriction factors. Nonetheless, many of these SIV strains were more pathogenic than SIVcpz. Either SIVcpz evolved into a more pathogenic virus while in humans or a rare SIVcpz, possibly extinct in chimpanzees, was pathogenic immediately following the jump into human. FUNDING: Support for this study to E.J.A. was provided by the NIH/NIAID R01 AI49170 and CIHR project grant 385787. Infrastructure support was provided by the NIH CFAR AI36219 and Canadian CFI/Ontario ORF 36287. Efforts of J.A.B. and N.J.H. was provided by NIH AI099473 and for D.H.C., by VA and NIH AI AI080313.

STIM2 regulates NMDA receptor endocytosis that is induced by short-term NMDA receptor overactivation in cortical neurons.

Recent findings suggest an important role for the dysregulation of stromal interaction molecule (STIM) proteins, activators of store-operated Ca2+ channels, and the prolonged activation of N-methyl-D-aspartate receptors (NMDARs) in the development of neurodegenerative diseases. We previously demonstrated that STIM silencing increases Ca2+ influx through NMDAR and STIM-NMDAR2 complexes are present in neurons. However, the interplay between NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM1/STIM2 with regard to intracellular trafficking remains unknown. Here, we found that the activation of NMDAR endocytosis led to an increase in STIM2-GluN2A and STIM2-GluN2B interactions in primary cortical neurons. STIM1 appeared to migrate from synaptic to extrasynaptic sites. STIM2 silencing inhibited post-activation NMDAR translocation from the plasma membrane and synaptic spines and increased NMDAR currents. Our findings reveal a novel molecular mechanism by which STIM2 regulates NMDAR synaptic trafficking by promoting NMDAR endocytosis after receptor overactivation, which may suggest protection against excessive uncontrolled Ca2+ influx through NMDARs.

A Longitudinal Cohort Study of Risk Factors Associated with Small Ruminant Lentivirus Seropositivity in Intensively Reared Dairy Ewes in Greece.

A two-year longitudinal cohort study was conducted on a total of 407 purebred Chios and Lacaune ewes from four intensive dairy sheep farms to assess potential risk factors for small ruminant lentiviruses (SRLVs) seropositivity. Ewes were serologically tested semiannually at pre-mating and pre-lambing, and their age, breed, and body condition score (BCS) were recorded. Εwes were categorized as constantly seronegative, constantly seropositive, seroconverted, seroreverted, or animals with an intermittent presence of antibodies. Mixed binary logistic regression models were used to estimate the adjusted relative risks of the studied risk factors for (i) the individual ewes' seropositivity, (ii) the manifestation of specific serological patterns, and (iii) the occurrence of seroconversion and seroreversion incidents. Increased age was associated with seropositivity and constantly seropositive status (p < 0.001 in both cases). On the other hand, age was negatively associated with constantly seronegative pattern, seroconversion incident, and the intermittent presence of antibodies (p < 0.05 in all cases). Moreover, breed was recognized as a risk factor: Lacaune ewes demonstrated increased seropositivity, whereas Chios ewes were more likely to demonstrate an intermittent presence of antibodies (p < 0.01 in both cases). Seropositive status (p < 0.001), seropositivity in animals with an intermittent presence of antibodies (p = 0.001), and seroconversion incidents (p < 0.001) were significantly increased at pre-lambing compared to pre-mating. The risk factors recognized in our study contribute to a better understanding of SRLVs epidemiology and the evidence-based designation of SRLVs' control programs in intensive dairy sheep farms in Greece.

Exploiting a rodent cell block for intrinsic resistance to HIV-1 gene expression in human T cells.

IMPORTANCE: Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a "mousified" version of one such host protein, cyclin T1 (CCNT1), in human T cells. CCNT1 is essential for efficient HIV-1 transcription, making it an intriguing target for gene-based inactivation of virus replication. We show that isogenic cell lines engineered to encode CCNT1 bearing a single mouse-informed amino acid change (tyrosine in place of cysteine at position 261) exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cell biology. These results provide proof of concept for targeting CCNT1 in the context of one or more functional HIV-1 cure strategies.

Different Patterns of Codon Usage and Amino Acid Composition across Primate Lentiviruses.

A common feature of the mammalian Lentiviruses (family Retroviridae) is an RNA genome that contains an extremely high frequency of adenine (31.7-38.2%) while being extremely poor in cytosine (13.9-21.2%). Such a biased nucleotide composition has implications for codon usage, causing a striking difference between the frequency of synonymous codons in Lentiviruses and that in their hosts. To test whether primate Lentiviruses present differences in codon and amino acid composition, we assembled a dataset of genome sequences that includes SIV species infecting Old-World monkeys and African apes, HIV-2, and the four groups of HIV-1. Using principal component analysis, we found that HIV-1 shows a significant enrichment in adenine plus thymine in the third synonymous codon position and in adenine and guanine in the first and second nonsynonymous codon positions. Similarly, we observed an enrichment in adenine and in guanine in nonsynonymous first and second codon positions, which affects the amino acid composition of the proteins Gag, Pol, Vif, Vpr, Tat, Rev, Env, and Nef. This result suggests an effect of natural selection in shaping codon usage. Under the hypothesis that the use of synonyms in HIV-1 could reflect adaptation to that of genes expressed in specific cell types, we found a highly significant correlation between codon usage in HIV-1 and monocytes, which was remarkably higher than that with B and T lymphocytes. This finding is in line with the notion that monocytes represent an HIV-1 reservoir in infected patients, and it could help understand how this reservoir is established and maintained.

Small Ruminant Lentivirus Infection in Sheep and Goats in North Portugal: Seroprevalence and Risk Factors.

Small ruminant lentiviruses (SRLVs) are transmitted among ovine and caprine species. This disease is a severe problem for small ruminant production, not only for animals' well-being but also for flocks' efficiency. The main aim of this research was to quantify the seroprevalence and associated risk factors for SRLV infection in the northern region of Portugal. Samples were collected from a total of 150 flocks, of which 129 (86.0%; 95% CI: 80.67%-91.33%) had at least one seropositive animal. Out of 2607 individual blood samples, 1074 (41.2%) were positive for SRLVs. Risk factors associated with SRLV infection were species (caprine), age (>2 years old), flock size (>100 animals), production system (intensive), food production system (milk), type of activity (professional), participation in livestock competitions (yes), replacement young ewe bought (yes), and natural feeding management (yes). This knowledge empowers the implementation of effective preventive measures. Overall, biosecurity measures should be promoted and implemented with the main aim of reducing viral transmission and reducing the prevalence of this disease. We recognise that government authorities should promote and audit voluntary control and eradication programs in small ruminant flocks in the region studied.

FAM46C Is an Interferon-Stimulated Gene That Inhibits Lentiviral Particle Production by Modulating Autophagy.

FAM46C is a multiple myeloma (MM) tumor suppressor whose function is only starting to be elucidated. We recently showed that in MM cells FAM46C triggers apoptosis by inhibiting autophagy and altering intracellular trafficking and protein secretion. To date, both a physiological characterization of FAM46C role and an assessment of FAM46C-induced phenotypes outside of MM are lacking. Preliminary reports suggested an involvement of FAM46C with regulation of viral replication, but this was never confirmed. Here, we show that FAM46C is an interferon-stimulated gene and that the expression of wild-type FAM46C in HEK-293T cells, but not of its most frequently found mutant variants, inhibits the production of both HIV-1-derived and HIV-1 lentiviruses. We demonstrate that this effect does not require transcriptional regulation and does not depend on inhibition of either global or virus-specific translation but rather mostly relies on FAM46C-induced deregulation of autophagy, a pathway that we show to be required for efficient lentiviral particle production. These studies not only provide new insights on the physiological role of the FAM46C protein but also could help in implementing more efficient antiviral strategies on one side and lentiviral particle production approaches on the other. IMPORTANCE FAM46C role has been thoroughly investigated in MM, but studies characterizing its role outside of the tumoral environment are still lacking. Despite the success of antiretroviral therapy in suppressing HIV load to undetectable levels, there is currently no HIV cure, and treatment is lifelong. Indeed, HIV continues to be a major global public health issue. Here, we show that FAM46C expression in HEK-293T cells inhibits the production of both HIV and HIV-derived lentiviruses. We also demonstrate that such inhibitory effect relies, at least in part, on the well-established regulatory role that FAM46C exerts on autophagy. Deciphering the molecular mechanism underlying this regulation will not only facilitate the understanding of FAM46C physiological role but also give new insights on the interplay between HIV and the cellular environment.

Viral Vector-Based Gene Therapy.

Gene therapy is a technique involving the modification of an individual's genes for treating a particular disease. The key to effective gene therapy is an efficient carrier delivery system. Viral vectors that have been artificially modified to lose their pathogenicity are used widely as a delivery system, with the key advantages of their natural high transduction efficiency and stable expression. With decades of development, viral vector-based gene therapies have achieved promising clinical outcomes. Currently, the three key vector strategies are based on adeno-associated viruses, adenoviruses, and lentiviruses. However, certain challenges, such as immunotoxicity and "off-target", continue to exist. In the present review, the above three viral vectors are discussed along with their respective therapeutic applications. In addition, the major translational challenges encountered in viral vector-based gene therapies are summarized, and the possible strategies to address these challenges are also discussed.

Protocol Optimization for Direct Reprogramming of Primary Human Fibroblast into Induced Striatal Neurons.

The modeling of neuropathology on induced neurons obtained by cell reprogramming technologies can fill a gap between clinical trials and studies on model organisms for the development of treatment strategies for neurodegenerative diseases. Patient-specific models based on patients' cells play an important role in such studies. There are two ways to obtain induced neuronal cells. One is based on induced pluripotent stem cells. The other is based on direct reprogramming, which allows us to obtain mature neuronal cells from adult somatic cells, such as dermal fibroblasts. Moreover, the latter method makes it possible to better preserve the age-related aspects of neuropathology, which is valuable for diseases that occur with age. However, direct methods of reprogramming have a significant drawback associated with low cell viability during procedures. Furthermore, the number of reprogrammable neurons available for morphological and functional studies is limited by the initial number of somatic cells. In this article, we propose modifications of a previously developed direct reprogramming method, based on the combination of microRNA and transcription factors, which allowed us to obtain a population of functionally active induced striatal neurons (iSNs) with a high efficiency. We also overcame the problem of the presence of multinucleated neurons associated with the cellular division of starting fibroblasts. Synchronization cells in the G1 phase increased the homogeneity of the fibroblast population, increased the survival rate of induced neurons, and eliminated the presence of multinucleated cells at the end of the reprogramming procedure. We have demonstrated that iSNs are functionally active and able to form synaptic connections in co-cultures with mouse cortical neurons. The proposed modifications can also be used to obtain a population of other induced neuronal types, such as motor and dopaminergic ones, by selecting transcription factors that determine differentiation into a region-specific neuron.

Genetic Diversity of the LTR Region of Polish SRLVs and Its Impact on the Transcriptional Activity of Viral Promoters.

A long terminal repeat (LTR) plays an indispensable role in small ruminant lentivirus (SRLV) gene expression. In this study, we present the LTR sequence of Polish SRLVs representing different subtypes, and analyzed their impact on SRLV promoter activity, as measured in transient transfection assays. Although certain nucleotide motifs (AML(vis), TATA box and the polyadenylation site (AATAAA)) were conserved across sequences, numerous mutations within the LTR sequences have been identified. Single nucleotide polymorphisms (SNPs) were detected in both regulatory (AP-1, AP-4, Stat and Gas) and non-regulatory sequences, and subtype-specific genetic diversity in the LTR region of Polish SRLVs was observed. In vitro assays demonstrated subtype-specific functional differences between the LTR regions of distinct SRLV subtypes. Our results revealed that the promoter activity of Polish strains was lower (1.64-10.8-fold) than that noted for the K1514 reference strain; however, the differences in most cases were not statistically significant. The lowest promoter activity was observed for strains representing subtype A5 (mean 69.067) while the highest promoter activity was observed for strain K1514 representing subtype A1 (mean 373.48). The mean LTR activities of strains representing subtypes A12, A17, A23, A18 and A24 were 91.22, 137.21, 178.41, 187.05 and 236.836, respectively. The results of the inter-subtype difference analysis showed that the promoter activity of strains belonging to subtype A5 was significantly lower than that for subtype A12 strains (1.32-fold; p < 0.00). The promoter activities of the A5 strain were 1.98-fold and 2.58-fold less active than that of the A17 and A23 strains, and the promoter activities of A12 strains were 1.955 and 1.5 times lower than the promoter activity of A23 and A17 strains, respectively. Furthermore, the promoter activity of A17 strains was 1.3 lower than the promoter activity of A23 strains. Our findings suggest that subtype-specific genetic diversity, mainly in the transcription factor's binding sites, has an impact on their transcriptional activity, producing a distinct activity pattern for the subtypes. This study provides new information that is important for better understanding the function of the SRLV LTR. However, further research including more strains and subtypes as well as other cell lines is needed to confirm these findings.

Identification of a Novel Post-transcriptional Transactivator from the Equine Infectious Anemia Virus.

All lentiviruses encode a post-transcriptional transactivator, Rev, which mediates the export of viral mRNA from the nucleus to the cytoplasm and which is required for viral gene expression and viral replication. In the current study, we demonstrate that equine infectious anemia virus (EIAV), an equine lentivirus, encodes a second post-transcriptional transactivator that we designate Grev. Grev is encoded by a novel transcript with a single splicing event that was identified using reverse transcription-PCR (RT-PCR) and RNA-seq in EIAV-infected horse tissues and cells. Grev is about 18 kDa in size, comprises the first 18 amino acids (aa) of Gag protein together with the last 82 aa of Rev, and was detected in EIAV-infected cells. Similar to Rev, Grev is localized to the nucleus, and both are able to mediate the expression of Mat (a recently identified viral protein of unknown function from EIAV), but Rev can mediate the expression of EIAV Gag/Pol, while Grev cannot. We also demonstrate that Grev, similar to Rev, specifically binds to rev-responsive element 2 (RRE-2, located in the first exon of mat mRNAs) to promote nuclear export of mat mRNA via the chromosome region maintenance 1 (CRM1) pathway. However, unlike Rev, whose function depends on its multimerization, we could not detect multimerization of Grev using coimmunoprecipitation (co-IP) or bimolecular fluorescence complementation (BiFC) assays. Together, these data suggest that EIAV encodes two post-transcriptional transactivators, Rev and Grev, with similar, but not identical, functions. IMPORTANCE Nuclear export of viral transcripts is a crucial step for viral gene expression and viral replication in lentiviruses, and this export is regulated by a post-transcriptional transactivator, Rev, that is shared by all lentiviruses. Here, we report that the equine infectious anemia virus (EIAV) encodes a novel viral protein, Grev, and demonstrated that Grev, like Rev, mediates the expression of the viral protein Mat by binding to the first exon of mat mRNAs via the chromosome region maintenance 1 (CRM1) pathway. Grev is encoded by a single-spliced transcript containing two exons, whereas Rev is encoded by a multiple-spliced transcript containing four exons. Moreover, Rev is able to mediate EIAV Gag/Pol expression by binding to rev-responsive element (RRE) located within the Env-coding region, while Grev cannot. Therefore, the present study demonstrates that EIAV encodes two post-transcriptional regulators, Grev and Rev, suggesting that post-transcriptional regulation patterns in lentivirus are diverse and complex.

A Novel, Fully Spliced, Accessory Gene in Equine Lentivirus with Distinct Rev-Responsive Element.

All lentiviruses encode the accessory protein Rev, whose main biological function is to mediate the nuclear export of unspliced and incompletely spliced viral transcripts by binding to a viral cis-acting element (termed the Rev-responsive element, RRE) within the env-encoding region. Equine infectious anemia virus (EIAV) is a member of the lentivirus genus in the Retroviridae family and is considered an important model for the study of lentivirus pathogenesis. Here, we identified a novel transcript from the EIAV genome that encoded a viral protein, named Mat, with an unknown function. The transcript mat was fully spliced and comprised parts of the coding regions of MA and TM. Interestingly, the expression of Mat depended on Rev and the chromosome region maintenance 1 (CRM1) pathway. Rev could specifically bind to Mat mRNA to promote its nuclear export. We further identified that the first exon of Mat mRNA, which was located within the Gag-encoding region, acted as an unreported RRE. Altogether, we identified a novel fully spliced transcript mat with an unusual RRE, which interacted with Rev for nuclear export through the CRM1 pathway. These findings updated the EIAV genome structure, highlighted the diversification of posttranscriptional regulation patterns in EIAV, and may help to expand the understanding of gene transcription and expression of lentivirus. IMPORTANCE In lentiviruses, the nuclear export of viral transcripts is an important step in controlling viral gene expression. Generally, the unspliced and incompletely spliced transcripts are exported via the CRM1-dependent export pathway in a process mediated by the viral Rev protein by binding to the Rev-responsive element (RRE) located within the Env-coding region. However, the completely spliced transcripts are exported via an endogenous cellular pathway, which was Rev independent. Here, we identified a novel fully spliced transcript from EIAV and demonstrated that it encoded a viral protein, termed Mat. Interestingly, we determined that the expression of Mat depended on Rev and identified that the first exon of Mat mRNA could specifically bind to Rev and be exported to the cytoplasm, which suggested that the first exon of Mat mRNA was a second RRE of EIAV. These findings provided important insights into the Rev-dependent nuclear export of completely spliced transcripts in lentiviruses.

Pathological, molecular, and serological study of small ruminant lentiviruses in Jordan.

Background and Aim: Maedi-visna is a chronic viral disease of sheep with worldwide distribution causing substantial economic losses to the small ruminant industry. Pneumonia and mastitis are the main manifestations of the disease. This study aimed to investigate the occurrence of maedi-visna virus (MVV) in sheep using histopathology and nested polymerase chain reaction (PCR) techniques and also to estimate the seroprevalence of small ruminant lentiviruses (SRLVs) in sheep and goats using commercially available enzyme-linked immunosorbent assay (ELISA). Materials and Methods: Lung tissue samples from 380 sheep were collected and fixed in 10% formalin for histopathology and molecular diagnosis of MVV. Separately, 806 serum samples were randomly collected from 633 sheep and 173 goats to detect the seroprevalence of SRLVs using ELISA. Results: The results showed that 4.7% of lung samples (n=190) were positive by both histopathology and nested PCR, 5.8% (n = 380) were positive by histopathology only (have lymphoid follicular hyperplasia), and 7.4% (n = 190) were positive by nested PCR only. Statistical analysis revealed a moderate agreement between the two tests (Kappa=0.451, n = 190). Serology results revealed that sheep and/or goats herd prevalence was 59.8% (n = 87), while individual seroprevalence in sheep (40.1%, n = 633) was significantly higher than that in the other six countries and also significantly higher than that in goats (18.5%, n = 173) (at p < 0.05). Conclusion: The moderate statistical agreement between nested PCR and histopathological diagnosis of MVV in formalin-fixed paraffin-embedded sheep lung tissue samples (Kappa=0.451, n = 190) suggests combining both tests for more sensitive MVV detection in sheep lung samples. SRLVs seropositivity in sheep was significantly higher than in goats, thus, it is of high concern and urges the inquiry into the economic impact of the disease and the financial benefit of adopting eradication measures.

A Novel Mouse Model of TGFß2-Induced Ocular Hypertension Using Lentiviral Gene Delivery.

Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) ß2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFß2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFß2 in the TM. We developed an LV vector-encoding active hTGFß2C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFß2C226,228S. We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFß2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFß2C226,228S exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFß2C226,228S that induces TM dysfunction and outflow resistance.