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Hepatitis C Virus belongs to the Flaviviridae family. One proposed mechanism of HCV persistence in the ability to infect hematopoietic cells, including Dendritic cells (DCs). HCV infection of DCs could impair their functions that represent one of the mechanisms, thus hampering viral clearance by the host immune system. Among HCV-encoded proteins, the highly conserved Core protein has been suggested to be responsible for the immunomodulatory properties of this Hepacivirus. Recombinant viral vectors expressing the HCV Core protein and allowing its transduction and therefore the expression of the protein into DCs could be useful tools for the analysis of the properties of the Core protein. Vaccinia Virus and retrovirus have been used to transduce human DCs. Likewise, gene transfer into DCs using Semliki Forest Virus has been reported. This study aimed to express the HCV Core protein in human monocyte-derived DCs using an SFV vector, in which the subgenomic RNA encoding the structural proteins was replaced by the HCV Core sequence and then analyze the effects of its expression on DCs functions.

• A production approach for viral particles as a delivery system of RNA encoding the rabies virus Glycoprotein was set up. • The vectored particles were able to enter different mammalian cell lines and promote the expression of the target protein. • The vectored particles were able to RNA delivery to 5 mammalian cell lines (BHK-21, HuH-7, Vero, L929, and HEK-293T). The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 μg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 μg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 μg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 μg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique. [ABSTRACT FROM AUTHOR]

Hepatitis C Virus belongs to the Flaviviridae family. One proposed mechanism of HCV persistence in the ability to infect hematopoietic cells, including Dendritic cells (DCs). HCV infection of DCs could impair their functions that represent one of the mechanisms, thus hampering viral clearance by the host immune system. Among HCV-encoded proteins, the highly conserved Core protein has been suggested to be responsible for the immunomodulatory properties of this Hepacivirus. Recombinant viral vectors expressing the HCV Core protein and allowing its transduction and therefore the expression of the protein into DCs could be useful tools for the analysis of the properties of the Core protein. Vaccinia Virus and retrovirus have been used to transduce human DCs. Likewise, gene transfer into DCs using Semliki Forest Virus has been reported. This study aimed to express the HCV Core protein in human monocyte-derived DCs using an SFV vector, in which the subgenomic RNA encoding the structural proteins was replaced by the HCV Core sequence and then analyze the effects of its expression on DCs functions. [ABSTRACT FROM AUTHOR]

Abstract Viruses are obligate intracellular parasites of the host cells. A commonly accepted view is the requirement of internal membranous structures for various aspects of viral life cycle. Organelles enable favourable intracellular environment for several viruses. However, studies reporting organelle dynamics upon viral infections are scant. In this review, we aim to summarize and highlight modulations caused to various organelles upon viral infection or expression of its proteins. [ABSTRACT FROM AUTHOR]

Simian foamy viruses (SFVs) infect most nonhuman primate species and appears to co-evolve with its hosts. This co-evolutionary signal is particularly strong among great apes, including orangutans (genus Pongo ). Previous studies have identified three distinct orangutan SFV clades. The first of these three clades is composed of SFV from P . abelii from Sumatra, the second consists of SFV from P . pygmaeus from Borneo, while the third clade is mixed, comprising an SFV strain found in both species of orangutan. The existence of the mixed clade has been attributed to an expansion of P . pygmaeus into Sumatra following the Mount Toba super-volcanic eruption about 73,000 years ago. Divergence dating, however, has yet to be performed to establish a temporal association with the Toba eruption. Here, we use a Bayesian framework and a relaxed molecular clock model with fossil calibrations to test the Toba hypothesis and to gain a more complete understanding of the evolutionary history of orangutan SFV. As with previous studies, our results show a similar three-clade orangutan SFV phylogeny, along with strong statistical support for SFV-host co-evolution in orangutans. Using Bayesian inference, we date the origin of orangutan SFV to > 4.7 million years ago (mya), while the mixed species clade dates to approximately 1.7 mya, > 1.6 million years older than the Toba super-eruption. These results, combined with fossil and paleogeographic evidence, suggest that the origin of SFV in Sumatran and Bornean orangutans, including the mixed species clade, likely occurred on the mainland of Indo-China during the Late Pliocene and Calabrian stage of the Pleistocene, respectively. [ABSTRACT FROM AUTHOR]

Semliki Forest virus (SFV) is an alphavirus that uses the very-low-density lipoprotein receptor (VLDLR) as a receptor during infection of its vertebrate hosts and insect vectors. Herein, we used cryoelectron microscopy to study the structure of SFV in complex with VLDLR. We found that VLDLR binds multiple E1-DIII sites of SFV through its membrane-distal LDLR class A (LA) repeats. Among the LA repeats of the VLDLR, LA3 has the best binding affinity to SFV. The high-resolution structure shows that LA3 binds SFV E1-DIII through a small surface area of 378 Å2, with the main interactions at the interface involving salt bridges. Compared with the binding of single LA3s, consecutive LA repeats around LA3 promote synergistic binding to SFV, during which the LAs undergo a rotation, allowing simultaneous key interactions at multiple E1-DIII sites on the virion and enabling the binding of VLDLRs from divergent host species to SFV. [Display omitted] • The receptor VLDLR binds multiple E1-DIII sites of SFV through its LA repeats • Single LA binds E1-DIII with a small contact surface and a low binding affinity • Multiple LAs promote synergistic binding and a high binding affinity to SFV Semliki Forest virus (SFV) enters host cells by binding to repeat regions of the host cell's very-low-density lipoprotein receptors. The repeat regions enable synergic, multimodal binding with SFV, which is highly tolerant of mutations, allowing SFV to bind efficiently to the receptors of highly divergent species. [ABSTRACT FROM AUTHOR]

Despite the success of immune checkpoint blockade for cancer therapy, many patients do not respond adequately. We aimed to improve this therapy by optimizing both the antibodies and their delivery route, using small monodomain antibodies (nanobodies) delivered locally with a self-amplifying RNA (saRNA) vector based on Semliki Forest virus (SFV). We generated nanobodies against PD-1 and PD-L1 able to inhibit both human and mouse interactions. Incorporation of a dimerization domain reduced PD-1/PD-L1 IC50 by 8- and 40-fold for anti-PD-L1 and anti-PD-1 nanobodies, respectively. SFV viral particles expressing dimeric nanobodies showed a potent antitumor response in the MC38 model, resulting in >50% complete regressions, and showed better therapeutic efficacy compared to vectors expressing conventional antibodies. These effects were also observed in the B16 melanoma model. Although a short-term expression of nanobodies was observed due to the cytopathic nature of the saRNA vector, it was enough to generate a strong proinflammatory response in tumors, increasing infiltration of NK and CD8+ T cells. Delivery of the SFV vector expressing dimeric nanobodies by local plasmid electroporation, which could be more easily translated to the clinic, also showed a potent antitumor effect. • Nanobodies able to block human and mouse PD-1/PD-L1 interactions have been developed. • Dimerization of nanobodies considerably decreased IC50 for PD-1/PD-L1 interactions. • SFV viral particles expressing dimeric nanobodies led to potent antitumor effects. • Delivery of SFV saRNA by plasmid electroporation showed a potent antitumor effect. [ABSTRACT FROM AUTHOR]

The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.

It is now known that all human retroviruses have a non-human primate counterpart. It has been reported that the presence of these retroviruses in humans is the result of interspecies transmission. Several authors have described the passage of a simian retrovirus, simian foamy virus (SFV), from primates to humans. To better understand this retroviral “zoonosis” in natural settings, we evaluated the presence of SFV in both captive and wild non-human primates and in humans at high risk, such as hunters and people bitten by a non-human primate, in Gabon, central Africa. A high prevalence of SFV was found in blood samples from non-human primates and in bush meat collected across the country. Mandrills were found to be highly infected with two distinct strains of SFV, depending on their geographical location. Furthermore, samples collected from hunters and non-human primate laboratory workers showed clear, extensive cross-species transmission of SFV. People who had been bitten by mandrills, gorillas and chimpanzees had persistent SFV infection with low genetic drift. Thus, SFV is presumed to be transmitted from non-human primates mainly through severe bites, involving contact between infected saliva and blood. In this review, we summarize and discuss our five-year observations on the prevalence and dissemination of SFV in humans and non-human primates in Gabon.

Interferon inducible transmembrane proteins ( IFITMs) are broad-spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo-, flavi-, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus ( SFV), which both use endocytosis and acid-induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH-induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant ( Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV. [ABSTRACT FROM AUTHOR]

The role of long noncoding RNAs (lncRNAs) in viral infection is poorly studied. We have identified hepatitis C virus (HCV)-Stimulated lncRNAs (CSRs) by transcriptome analysis. Interestingly, two of these CSRs (PVT1 and UCA1) play relevant roles in tumorigenesis, providing a novel link between HCV infection and development of liver tumors. Expression of some CSRs seems induced directly by HCV, while others are upregulated by the antiviral response against the virus. In fact, activation of pathogen sensors induces the expression of CSR32/EGOT. RIG-I and the RNA-activated kinase PKR sense HCV RNA, activate NF-κB and upregulate EGOT. EGOT is increased in the liver of patients infected with HCV and after infection with influenza or Semliki Forest virus (SFV). Genome-wide guilt-by-association studies predict that EGOT may function as a negative regulator of the antiviral pathway. Accordingly, EGOT depletion increases the expression of several interferon-stimulated genes and leads to decreased replication of HCV and SFV. Our results suggest that EGOT is a lncRNA induced after infection that increases viral replication by antagonizing the antiviral response. [ABSTRACT FROM AUTHOR]

Highlights: [•] We tested 118 samples from 6 different primates species from Cambodia for retroviruses. [•] We showed no evidence of SIV infection. [•] We observed high prevalence of SFV infection complying with host/virus co-divergence. [•] We identified a well-supported clade of Cambodian STLV-1 from 3 different species. [ABSTRACT FROM AUTHOR]

It is now known that all human retroviruses have a non-human primate counterpart. It has been reported that the presence of these retroviruses in humans is the result of interspecies transmission. Several authors have described the passage of a simian retrovirus, simian foamy virus (SFV), from primates to humans. To better understand this retroviral "zoonosis" in natural settings, we evaluated the presence of SFV in both captive and wild non-human primates and in humans at high risk, such as hunters and people bitten by a non-human primate, in Gabon, central Africa. A high prevalence of SFV was found in blood samples from non-human primates and in bush meat collected across the country. Mandrills were found to be highly infected with two distinct strains of SFV, depending on their geographical location. Furthermore, samples collected from hunters and non-human primate laboratory workers showed clear, extensive cross-species transmission of SFV. People who had been bitten by mandrills, gorillas and chimpanzees had persistent SFV infection with low genetic drift. Thus, SFV is presumed to be transmitted from non-human primates mainly through severe bites, involving contact between infected saliva and blood. In this review, we summarize and discuss our five-year observations on the prevalence and dissemination of SFV in humans and non-human primates in Gabon. [ABSTRACT FROM AUTHOR]

Clin Microbiol Infect 2012; 18: 514-520 Abstract It is now well established that simian immunodeficiency viruses (SIVs) from chimpanzees (SIVcpz) and gorillas (SIVgor) from west Central Africa are at the origin of HIV-1/AIDS. Apes are also infected with other retroviruses, notably simian T-cell lymphotropic viruses (STLVs) and simian foamy viruses (SFVs), that can be transmitted to humans. We discuss the actual knowledge on SIV, STLV and SFV infections in chimpanzees, gorillas, and bonobos. We especially elaborate on how the recent development of non-invasive methods has allowed us to identify the reservoirs of the HIV-1 ancestors in chimpanzees and gorillas, and increased our knowledge of the natural history of SIV infections in chimpanzees. Multiple cross-species events with retroviruses from apes to humans have occurred, but only one transmission of SIVcpz from chimpanzees in south-eastern Cameroon spread worldwide, and is responsible for the actual HIV pandemic. Frequent SFV transmissions have been recently reported, but no human-to-human transmission has been documented yet. Because humans are still in contact with apes, identification of pathogens in wild ape populations can signal which pathogens may be cause risk for humans, and allow the development of serological and molecular assays with which to detect transmissions to humans. Finally, non-invasive sampling also allows the study of the impact of retroviruses and other pathogens on the health and survival of endangered species such as chimpanzees, gorillas, and bonobos. [ABSTRACT FROM AUTHOR]

Abstract: Semliki Forest virus (SFV), an alphavirus, replicates in vertebrate host and mosquito vector cells. The virus-specific part of the replicase complex constitutes nonstructural proteins 1–4 (nsP1–nsP4) and is bound to cytoplasmic membranes by an amphipathic helix inside of nsP1 and through the palmitoylation of cysteine residues in nsP1. In mammalian cells, defects in these viral functions result in a nonviable phenotype or the emergence of second-site compensatory mutations that have a positive impact on SFV infection. In most cases, these second-site compensatory mutations were found to compensate for the defect caused by the absence of palmitoylation in mosquito cells (C6/36). In C6/36 cells, however, all palmitoylation-defective viruses had severely reduced synthesis of subgenomic RNA; at the same time, several of them had very efficient formation of defective interfering genomes. Analysis of C6/36 cells that individually expressed either wild type (wt) or palmitoylation-deficient nsP1 forms revealed that similar to mammalian cells, the wt nsP1 localized predominantly to the plasma membrane, whereas its mutant forms localized to the cytoplasm. In contrast to transfected mammalian cells, all forms of nsP1 induced the formation of filopodia-like structures on some, but not all, transfected mosquito cells. These findings indicate that the plasma membrane and associated host factors may have different roles in alphavirus replicase complex formation in mammalian and mosquito cells. In general, the lack of nsP1 palmitoylation had a less severe effect on the function of the replication complex in mammalian cells when compared with that in mosquito cells. [ABSTRACT FROM AUTHOR]

Background Vaccine combining structural and regulatory proteins is an emerging approach to develop an HIV/AIDS vaccine and therefore, the immunogenicity and efficacy of two regimens of immunization combining structural (Gag/Pol, Env) and regulatory (Rev, Tat, Nef) Simian immunodeficiency virus (SIV) proteins were compared in cynomolgus monkeys. Methods Monkeys were immunized with Modified Vaccine Ankara vector (MVA-J5) (protocol 1) or with DNA, Semliki forest virus and MVA vectors (DNA/SFV/MVA) (protocol 2). At week 32, all monkeys were challenge intravenously (protocol 1) or intrarectally (protocol 2) with 50 MID50 of SIVmac251. Humoral, proliferative responses and in particular in protocol 2, the frequency of IFN-γ producing cells, were measured in all monkeys before and after the challenge. Results Both vaccine regimens elicited humoral and proliferative responses but failed to induce neutralizing antibodies. Upon intravenous challenge, two out of three MVA-J5 vaccinated monkeys exhibited a long-term control of the viral replication whereas DNA/SFV/MVA vaccine abrogated the virus replication up to undetectable level in three out of four vaccinated monkeys. A major contribution to this vaccine effect appeared to be the IFN-γ/ELISPOT responses to vaccine antigens (Gag, Rev Tat and Nef). Conclusions These results, indicate that multiprotein heterologous prime-boost vaccination can induce a robust vaccine-induced immunity able to abrogate virus replication. [ABSTRACT FROM AUTHOR]

Abstract: Background: Vectors derived from alphaviruses are gaining interest for their high transfection potency and strong immunogenicity. Objectives: After a brief introduction on alphaviruses and their vectors, an overview is given on current preclinical immunotherapy studies using vector systems based on alphaviruses. The efficacy of alphavirus vectors in inducing immune responses will be illustrated by a more detailed description of immunization studies using recombinant Semliki Forest virus for the treatment of human papilloma virus-induced cervical cancer. Results: Immunization with recombinant alphavirus results in the induction of humoral and cellular immune responses against microbes, infected cells and cancer cells. Preclinical studies demonstrate that infectious diseases and cancer can be treated prophylactically as well as therapeutically. Conclusions: Alphavirus-based genetic immunization strategies are highly effective in animal model systems, comparing quite favourably with any other approach. Therefore, we hope and expect to see an efficient induction of tumour-or microbial immunity and a positive outcome in future clinical efficacy studies. [Copyright &y& Elsevier]

This article presents results obtained from the performance monitoring of the inverter, part of the first Colombian interconnected photovoltaic system (PVS), which was installed and in operation since June 2003, at the Physics Department of the Universidad National de Colombia, Bogotá Campus; the PVS is constituted of a 980 Wp photovoltaic generator and a 1000 VA inverter. Data acquisition and analysis equipment was developed from virtual instrumentation capable of permanent monitoring parameters yielding information on the performance of the inverter (current and d.c voltage generated by the photovoltaic array, current and a.c voltage at inverter output, inverter efficiency, irradiance and ambient temperature measurements) and on the PVS efficiency. This equipment was developed by using hardware made up of Field Point modules and a NI-6024E data acquisition board, along with LabVIEW 7.1 graphic programming software. The results obtained, from a three-month monitoring, indicate the viability of the installation and operation of the interconnected to the local electric grid photovoltaic systems, as embedded systems capable of supporting the power supplied by the distribution grid. [ABSTRACT FROM AUTHOR]

Despite promising preclinical results of various therapeutic anticancer immunization strategies, these approaches may not be effective enough to eradicate tumors in cancer patients. While most animal models are based on fast-growing transplantable tumors, malignancies in, for example, cervical cancer patients in general develop much more slowly, which may lead to immune suppression and/or immune tolerance. As a consequence, the immunomodulating signal of any therapeutic immunization regimen should be sufficiently potent to overcome this immunocompromised condition. In previous studies, we demonstrated that an experimental vaccine against human papillomavirus (HPV)-induced cervical cancer, based on Semliki Forest virus (SFV), induces robust HPV-specific cellular immune responses in mice. Now we studied whether this strategy is potent enough to also prime a cellular immune response in immune-tolerant HPV transgenic mice, in which CTL activity cannot be induced using protein or DNA vaccines. We demonstrate that, depending on the route of immunization, SFV-expressing HPV16 E6 and E7 indeed has the capacity to induce HPV16 E7-specific cytotoxic T cells in HPV-transgenic mice.Gene Therapy (2005) 12, 1410–1414. doi:10.1038/sj.gt.3302536; published online 21 April 2005 [ABSTRACT FROM AUTHOR]

We have recently shown that the NS3-based genetic immunogens should contain also hepatitis C virus (HCV) nonstructural (NS) 4A to utilize fully the immunogenicity of NS3. The next step was to try to enhance immunogenicity by modifying translation or mRNA synthesis. To enhance translation efficiency, a synthetic NS3/4A-based DNA (coNS3/4A-DNA) vaccine was generated in which the codon usage was optimized (co) for human cells. In a second approach, expression of the wild-type (wt) NS3/4A gene was enhanced by mRNA amplification using the Semliki forest virus (SFV) replicon (wtNS3/4A-SFV). Transient tranfections of human HepG2 cells showed that the coNS3/4A gene gave 11-fold higher levels of NS3 as compared to the wtNS3/4A gene when using the CMV promoter. We have previously shown that the presence of NS4A enhances the expression by SFV. Both codon optimization and mRNA amplification resulted in an improved immunogenicity as evidenced by higher levels of NS3-specific antibodies. This improved immunogenicity also resulted in a more rapid priming of cytotoxic T lymphocytes (CTLs). Since HCV is a noncytolytic virus, the functionality of the primed CTL responses was evaluated by an in vivo challenge with NS3/4A-expressing syngeneic tumor cells. The priming of a tumor protective immunity required an endogenous production of the immunogen and CD8+ CTLs, but was independent of B and CD4+ T cells. This model confirmed the more rapid in vivo activation of an NS3/4A-specific tumor-inhibiting immunity by codon optimization and mRNA amplification. Finally, therapeutic vaccination with the coNS3/4A gene using gene gun 6-12 days after injection of tumors significantly reduced the tumor growth in vivo. Codon optimization and mRNA amplification effectively enhances the overall immunogenicity of NS3/4A. Thus, either, or both, of these approaches should be utilized in an NS3/4A-based HCV genetic vaccine.Gene Therapy (2004) 11, 522-533. doi:10.1038/sj.gt.3302184 [ABSTRACT FROM AUTHOR]

Huntington''s disease (HD) is a progressive, hereditary, neurodegenerative disorder caused by an expanded polyglutamine tract in huntingtin protein, leading to misfolding and abnormal protein–protein interactions. Reducing the initial misfolding should lead to decreased pathogenesis. We show that malonate stress increases the number of dead or dying cells when organotypic slice cultures are transduced to express pathological-length huntingtin fragments. Co-transfected anti-HD single-chain Fv (sFv) intrabodies can reverse this HD-specific increase in malonate-induced morbidity. [Copyright &y& Elsevier]

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses . [ABSTRACT FROM AUTHOR]

This article provides a review of the pathogenesis of Semliki Forest virus (SFV) encephalitis. In mice, outcome of infection varies according to age of the mouse and strain of the virus and can include acute encephalitis, subacute demyelinating meningoencephalomyelitis, and persistent subclinical central nervous system (CNS) infection. All strains of virus are virulent in mice infected < 12 days of age. The L10 strain is also virulent in mice > 14 days age, whereas the A7(74) strain is avirulent. The genetic difference between these strains maps to the nsp3 gene. For A7(74) virus, age-related virulence correlates with ability of CNS neurons to replicate virus and undergo apoptotic cell death. Immature developing neurons support complete virus replication but as neuronal populations and circuits mature in the postnatal brain, virus infection becomes progressively restricted and nonproductive. This restricted replication can be overcome by gold I compounds, which may function by inducing neuronal dedifferentiation to a state permissive for virus replication. Biochemical pathways associated with membrane biogenesis may be an important determinant of this effect. Infection of some developing neuronal populations results in apoptosis, whereas infection of mature neurons results in persistent infection. An active type-I interferon system prevents virus spread in extraneural tissues. An initial high-titer plasma viremia is controlled by immunoglobulin M (IgM) antibodies. Virus enters the brain across cerebral endothelial cells and initiates scattered foci of perivascular infection. The blood-brain barrier is disrupted. Neurons and oligodendrocytes are the cell types most frequently infected. Infectivity in the brain can be eliminated by IgG antibodies, though an active T-cell response is required for virus elimination. Lesions of inflammatory demyelination require the presence of CD8[sup +] T lymphocytes and probably result from destruction by these cells of virally infected oligodendrocytes. [ABSTRACT FROM AUTHOR]

Immunotoxins are the rapeutic agents with a high degree of specificity and unique mechanism of action. An immunotoxin is achimeric protein consisting of a targeting moiety linked to a toxin. The targeting moiety selectively binds to a tumor cell and targets it for death via the attached toxin. Generally, immunotoxins are specifically potent against cancer cells in vitro and in animal models of human malignancies. However, immunotoxins can be limited clinically by immunogenicity, toxicity, and instability. In this review, weofferwaysto overcome these limitations to create “ideal immunotoxins” for cancer therapy. These include producing single chain targeting/toxin fusion proteins of fully human origin that are extracellularly stable but once internalized, can be cleaved by intracellular proteases to free the toxin and facilitate its translocation to the cytosol. [ABSTRACT FROM AUTHOR]

The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined. Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen. However, immediately after induction at either 30°C or 37°C there was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant. The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic \-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis. Bacteria incubated at 30°C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/ A at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded. Osmotic-shock procedures did not release L6 sFv. However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location. French press disruption resulted in the release of correctly but not incorrectly folded material. Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane. These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis. It is also suggested that membrane association in the periplasmic space may impede proper folding. [ABSTRACT FROM AUTHOR]

Old World monkeys (OWM), simians inhabiting Africa and Asia, are currently affected by at least four infectious retroviruses, namely, simian foamy virus (SFV), simian immunodeficiency virus (SIV), simian T-lymphotropic virus (STLV), and simian type D retrovirus (SRV). OWM also show chromosomal evidence of having been infected in the past with four more retroviral species, baboon endogenous virus (BaEV), Papio cynocephalus endogenous virus (PcEV), simian endogenous retrovirus (SERV), and Rhesus endogenous retrovirus-K (RhERV-K/SERV-K1). For some of the viruses, transmission to other primates still occurs, resulting, for instance, in the HIV pandemic. Retroviruses are intimately connected with their host as they are normally spread by close contact. In this review, an attempt to reconstruct the distribution and history of OWM retroviruses will be made. A literature overview of the species infected by any of the eight retroviruses as well as an age estimation of the pathogens will be given. In addition, primate genomes from databases have been re-analyzed for the presence of endogenous retrovirus integrations. Results suggest that some of the oldest retroviruses, SERV and PcEV, have travelled with their hosts to Asia during the Miocene, when a higher global temperature allowed simian expansions. In contrast, younger viruses, such as SIV and SRV, probably due to the lack of a primate continuum between the continents in later times, have been restricted to Africa and Asia, respectively.

Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)