Chandipura viral glycoprotein (CNV-G) promotes Gectosome generation and enables delivery of intracellular therapeutics.

Overexpression of vesicular stomatitis virus G protein (VSV-G) elevates the secretion of EVs known as gectosomes, which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (viral glycoprotein from the Chandipura virus [CNV-G]) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison with VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity toward specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor, which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli in vitro and in the brain of mice in vivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly.

Shear-reversible clusters of HIV-1 in solution: stabilized by antibodies, dispersed by mucin.

IMPORTANCE: The phenomenon of reversible clustering is expected to further nuance HIV immune stealth because virus surfaces can escape interaction with antibodies (Abs) by hiding temporarily within clusters. It is well known that mucin reduces HIV virulence, and the current perspective is that mucin aggregates HIV-1 to reduce infections. Our findings, however, suggest that mucin is dispersing HIV clusters. The study proposes a new paradigm for how HIV-1 may broadly evade Ab recognition with reversible clustering and why mucin effectively neutralizes HIV-1.

Whole genome CRISPR screening strategy to identify genes contributing to SARS-CoV-2 spike and VSV-G mediated entry.

Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole-genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using live viruses and cellular lethality for selection, these screens can identify an overwhelming number of genes without specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole-genome screen and counter-screen strategy based on a pseudoviral platform that allowed identification of genes specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and vesicular stomatitis virus glycoprotein (VSV-G) mediated entry. Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed the identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein packaged into the pseudovirus was used to bypass retro-transcription and integration by directly activating a floxed fluorescent protein reporter upon entry  reduced the number of gene hits and increase specificity for viral entry. Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and low-density lipoprotein receptors, respectively, and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry-associated genes. Validation of a few hits demonstrates that this approach distinguishes envelope-specific host factors from genes affecting reporter expression. Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication.

Niosomal virosome derived by vesicular stomatitis virus glycoprotein as a new gene carrier.

Virosomes as membranous vesicles with viral fusion protein in their membrane are versatile vehicles for cargo delivery. The vesicular stomatitis virus glycoprotein (VSV-G) is a common fusogenic protein used in virosome preparation. This glycoprotein has been used in liposomal systems so far, but in this study, we have tried to use the niosomal form instead of liposome for. Niosomes are vesicular systems composed of non-ionic surfactants. Niosomes were constructed by the thin-film hydration method. VSV-G gene in pMD2.G plasmid was expressed in the HEK293T cell line and then was reconstituted in the niosome bilayer. The formation of niosomal virosomes was confirmed with different methods such as SDS-PAGE gel, western blotting, and transmission electron microscopy (TEM). The efficiency of niosomal virosome was investigated with the pmCherry reporter gene. SDS-PAGE and western blotting proved the expression and successful insertion of protein into the bilayer. The TEM images showed the spike projection of VSV-G on the surface of niosomes. The transfection results showed high efficiency of niosomal virosomes as a novel carrier. This report has verified that niosome could be used as an efficient bilayer instead of liposome to construct virosomes.

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction.

BACKGROUND AIMS: Viral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct. METHODS: Assay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations. RESULTS: The limit of detection was 10 copies/µL, whereas negative samples showed <1.3 copies/µL. Within and between assay imprecision coefficients of variation across the reportable range (10-10 000 copies/µL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R2 >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations). CONCLUSIONS: DDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors.

BACKGROUND AIMS: Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs. METHODS: The authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro. RESULTS: Blocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFPexpression of 40-50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality. CONCLUSIONS: The authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors' protocol can be readily adapted to generate clinical-grade NK cells and thus has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future.

Comparative analysis of lentiviral gene transfer approaches designed to promote fetal hemoglobin production for the treatment of ß-hemoglobinopathies.

ß-Hemoglobinopathies are among the most common single-gene disorders and are caused by different mutations in the ß-globin gene. Recent curative therapeutic approaches for these disorders utilize lentiviral vectors (LVs) to introduce a functional copy of the ß-globin gene into the patient's hematopoietic stem cells. Alternatively, fetal hemoglobin (HbF) can reduce or even prevent the symptoms of disease when expressed in adults. Thus, induction of HbF by means of LVs and other molecular approaches has become an alternative treatment of ß-hemoglobinopathies. Here, we performed a head-to-head comparative analysis of HbF-inducing LVs encoding for: 1) IGF2BP1, 2) miRNA-embedded shRNA (shmiR) sequences specific for the γ-globin repressor protein BCL11A, and 3) γ-globin gene. Furthermore, two novel baboon envelope proteins (BaEV)-LVs were compared to the commonly used vesicular-stomatitis-virus glycoprotein (VSV-G)-LVs. Therapeutic levels of HbF were achieved for all VSV-G-LV approaches, from a therapeutic level of 20% using γ-globin LVs to 50% for both IGF2BP1 and BCL11A-shmiR LVs. Contrarily, BaEV-LVs conferred lower HbF expression with a peak level of 13%, however, this could still ameliorate symptoms of disease. From this thorough comparative analysis of independent HbF-inducing LV strategies, we conclude that HbF-inducing VSV-G-LVs represent a promising alternative to ß-globin gene addition for patients with ß-hemoglobinopathies.

A CRISPR screen for factors regulating SAMHD1 degradation identifies IFITMs as potent inhibitors of lentiviral particle delivery.

The InterFeron Induced TransMembrane (IFITM) proteins are interferon stimulated genes that restrict many viruses, including HIV-1. SAMHD1 is another restriction factor blocking replication of HIV-1 and other viruses. Some lentiviruses evolved Vpx/Vpr proteins to degrade SAMHD1. However, this viral antagonism can be perturbed by host mechanisms: a recent study showed that in interferon (IFN) treated THP1 cells, Vpx is unable to degrade SAMHD1. In the present work, we designed an Interferon Stimulated Genes (ISGs)-targeted CRISPR knockout screen in order to identify ISGs regulating this phenotype. We found that IFITM proteins contribute to the IFNα-mediated protection of SAMHD1 by blocking VSV-G-mediated entry of the lentiviral particles delivering Vpx. Consistent with this, IFNα treatment and IFITM expression had no effect when the A-MLV envelope was used for pseudotyping. Using an assay measuring viral entry, we show that IFNα and IFITMs directly block the delivery of Vpx into cells by inhibiting VSV-G viral fusion. Strikingly, the VSV-G envelope was significantly more sensitive to this IFNα entry block and to IFITMs than HIV-1's natural envelope. This highlights important differences between VSV-G pseudotyped and wild-type HIV-1, in particular relative to the pathways they use for viral entry, suggesting that HIV-1 may have evolved to escape restriction factors blocking entry.

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli.

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b5 at the N-terminal of interferon (b5-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b5 at the C-terminal of interferon (Met-IFN-b5-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b5-chimera), resolved this problem and gave enhanced biological activity.

Flexible pseudotyping of retrovirus using recombinase-mediated cassette exchange.

OBJECTIVE: Develop an engineered cell line containing two flexible gene expression systems enabling the continuous production of tailor-made recombinant gammaretrovirus with predictable productivities through targeted integration. RESULTS: Dual-FLEX cells (dFLEX) contain two independent recombinase-mediated cassette exchange (RMCE) systems which confer flexibility to the expression of different transgene and envelope combinations. The flexible envelope expression in dFLEX cells was validated by pseudotyping retrovirus particles with three different viral envelope proteins-GaLV, 4070A and VSV-G. Our results show that dFLEX cells are able to provide high titers of infectious retroviral particles with a single-copy integration of the envelope constructs after RMCE. The integrated CRE/Lox tagging cassette was amenable to express envelope proteins both using constitutive (i.e. CMV) and inducible (i.e. Tet-on) promoters. CONCLUSIONS: dFLEX cell line provides predictable productivities of recombinant retrovirus pseudotyped with different envelope proteins broadening the tropism of particles that can be generated and thus accelerating the research and development of retrovirus-based products.

Kinetically distinct sorting pathways through the Golgi exhibit different requirements for Arf1.

To investigate the role of cytoplasmic sequences in directing transmembrane protein trafficking through the Golgi, we analyzed the sorting of VSV tsO45 G fusions with either the native G cytoplasmic domain (G) or an alternative cytoplasmic tail derived from the chicken AE1-4 anion exchanger (G(AE) ). At restrictive temperature G(AE) and G accumulated in the ER, and upon shifting the cells to permissive temperature both proteins folded and underwent transport through the Golgi. However, G(AE) and G did not form hetero-oligomers upon the shift to permissive temperature and they progressed through the Golgi with distinct kinetics. In addition, the transport of G through the proximal Golgi was Arf1 and COPI-dependent, while G(AE) progression through the proximal Golgi was Arf1 and COPI-independent. Although Arf1 did not regulate the sorting of G(AE) in the cis-Golgi, Arf1 did regulate the exit of G(AE) from the TGN. The trafficking of G(AE) through the Golgi was similar to that of the native AE1-4 anion exchanger, in that the progression of both proteins through the proximal Golgi was Arf1-independent, while both required Arf1 to exit the TGN. We propose that the differential recognition of cytosolic signals in membrane-spanning proteins by the Arf1-dependent sorting machinery may influence the rate at which cargo progresses through the Golgi.

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells.

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24 h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.

Rab6 dependent post-Golgi trafficking of HSV1 envelope proteins to sites of virus envelopment.

Herpes simplex virus 1 (HSV1) is an enveloped virus that uses undefined transport carriers for trafficking of its glycoproteins to envelopment sites. Screening of an siRNA library against 60 Rab GTPases revealed Rab6 as the principal Rab involved in HSV1 infection, with its depletion preventing Golgi-to-plasma membrane transport of HSV1 glycoproteins in a pathway used by several integral membrane proteins but not the luminal secreted protein Gaussia luciferase. Knockdown of Rab6 reduced virus yield to 1% and inhibited capsid envelopment, revealing glycoprotein exocytosis as a prerequisite for morphogenesis. Rab6-dependent virus production did not require the effectors myosin-II, bicaudal-D, dynactin-1 or rabkinesin-6, but was facilitated by ERC1, a factor involved in linking microtubules to the cell cortex. Tubulation and exocytosis of Rab6-positive, glycoprotein-containing membranes from the Golgi was substantially augmented by infection, resulting in enhanced and targeted delivery to cell tips. This reveals HSV1 morphogenesis as one of the first biological processes shown to be dependent on the exocytic activity of Rab6.

Lentiviral HSV-Tk.007-mediated suicide gene therapy is not toxic for normal brain cells.

BACKGROUND: Gene therapeutic strategies with suicide genes are currently investigated in clinical trials for brain tumors. Previously, we have shown that lentiviral vectors delivering the suicide gene HSV-Tk to experimental brain tumors promote a highly significant treatment effect and thus are promising vectors for clinical translation. METHODS: In the present study, we tested lentiviral vectors delivering the suicide gene HSV-Tk.007, a highly active mutant of HSV-Tk, to rat brains as a preclinical toxicity study. We injected 10(6) vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped functional lentiviral particles harboring the suicide gene HSV-Tk.007 into the brain of healthy, immunocompetent rats. During prodrug treatment with ganciclovir (GCV), we measured weight and assessed the behavior of the rats in an open field test. After 14 days of GCV treatment, we analyzed HSV-Tk.007 expression in different brain cell populations, as well as inflammatory responses and apoptosis. RESULTS: During prodrug treatment with GCV, behavior experiments did not reveal differences between the treated rats and the control groups. Analysis of HSV-Tk expression in different brain cell populations showed that transduced normal brain cells survived GCV treatment. There were no statistically significant differences in the number of transduced cells between treatment and control groups. Furthermore, inflammatory responses and apoptosis of brain cells were not observed. CONCLUSIONS: We show that HSV-Tk.007-mediated suicide gene therapy is not toxic to normal brain cells. This observation is of high relevance for the translation of lentivirus-mediated suicide gene therapies into the clinic for the treatment of brain tumor patients. Copyright © 2016 John Wiley & Sons, Ltd.

Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria.

The objective of this study was to determine if plasma membrane vesicles (PMVs) could be exploited for efficient transfer of macro-biomolecules and mitochondria. PMVs were derived from mechanical extrusion, and made fusogenic (fPMVs) by incorporating the glycoprotein G of vesicular stomatitis virus (VSV-G). Confocal microscopy examination revealed that cytoplasmic proteins and mitochondria were enclosed in PMVs as evidenced by tracing with cytoplasmically localized and mitochondria-targeted EGFP, respectively. However, no fluorescence signal was detected in PMVs from cells whose nucleus was labeled with an EGFP-tagged histone H2B. Consistently, qRT-PCR measurement showed that mRNA, miRNA and mitochondrial DNA decreased slightly; while nuclear DNA was not measureable. Further, Western blot analysis revealed that cytoplasmic and membrane-bound proteins fell inconspicuously while nuclear proteins were barely detecsle. In addition, fPMVs carrying cytoplamic DsRed proteins transduced about ~40 % of recipient cells. The transfer of protein was further confirmed by using the inducible Cre/loxP system. Mitochondria transfer was found in about 20 % recipient cells after incubation with fPMVs for 5 h. To verify the functionalities of transferred mitochondria, mitochodria-deficient HeLa cells (Rho0) were generated and cultivated with fPMVs. Cell enumeration demonstrated that adding fPMVs into culture media stimulated Rho0 cell growth by 100 % as compared to the control. Lastly, MitoTracker and JC-1 staining showed that transferred mitochondria maintained normal shape and membrane potential in Rho0 cells. This study established a time-saving and efficient approach to delivering proteins and mitochondria by using fPMVs, which would be helpful for finding a cure to mitochondria-associated diseases. Graphical abstract Schematic of the delivery of macro-biomolecules and organelles by fPMVs. VSV-G-expressing cells were extruded through a 3 µm polycarbonate membrane filter to generate fusogenic plasma membrane vesicles (fPMVs), which contain bioactive molecules and organelles but not the nucleus. fPMVs can be endocytosed by target cells, while the cargo is released due to low-pH induced membrane fusion. These nucleus-free fPMVs are efficient at delivery of cytoplasmic proteins and mitochondria, leading to recovery of mitochondrial biogenesis and proliferative ability in mitochondria-deficient cells.

RhoD is a Golgi component with a role in anterograde protein transport from the ER to the plasma membrane.

RhoD is a member of the Rho GTPase family and it coordinates actin dynamics and membrane trafficking. Activation of RhoD results in formation of filopodia, dissolution of stress fibers, and the subsequent formation of short actin bundles. In addition, RhoD localizes to early endosomes and recycling endosomes, and has a regulatory role in endosome trafficking. In this study, we report on a function of RhoD in the regulation of Golgi homeostasis. We show that manipulation of protein and activation levels of RhoD, as well as of its binding partner WHAMM, result in derailed localization of Golgi stacks. Moreover, vesicle trafficking from the endoplasmic reticulum to the plasma membrane via the Golgi apparatus measured by the VSV-G protein is severely hampered by manipulation of RhoD or WHAMM. In summary, our studies demonstrate a novel role for this member of the Rho GTPases in the regulation of Golgi function.

Further Characterization of the Antiviral Transmembrane Protein MARCH8.

The cellular transmembrane protein MARCH8 impedes the incorporation of various viral envelope glycoproteins, such as the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G-glycoprotein (VSV-G), into virions by downregulating them from the surface of virus-producing cells. This downregulation significantly reduces the efficiency of virus infection. In this study, we aimed to further characterize this host protein by investigating its species specificity and the domains responsible for its antiviral activity, as well as its ability to inhibit cell-to-cell HIV-1 infection. We found that the antiviral function of MARCH8 is well conserved in the rhesus macaque, mouse, and bovine versions. The RING-CH domains of these versions are functionally important for inhibiting HIV-1 Env and VSV-G-pseudovirus infection, whereas tyrosine motifs are crucial for the former only, consistent with findings in human MARCH8. Through analysis of chimeric proteins between MARCH8 and non-antiviral MARCH3, we determined that both the N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, of MARCH8 are critical for its antiviral activity. Notably, we found that MARCH8 is unable to block cell-to-cell HIV-1 infection, likely due to its insufficient downregulation of Env. These findings offer further insights into understanding the biology of this antiviral transmembrane protein.

A straightforward microfluidic-based approach toward optimizing transduction efficiency of HIV-1-derived lentiviral vectors in BCP-ALL cells.

Background: HIV-1-derived lentiviral vectors (LVs) are capable of transducing human cells by integrating the transgene into the host genome. In order to do that, LVs should have enough time and space to interact with the surface of the target cells. Herein, we used a microfluidic system to facilitate the transduction of BCP-ALL cells. Methods and Results: We used a SU-8 mold to fabricate a PDMS microfluidic chip containing three channels with a 50 µm height and a surface matching 96-well plates. In order to produce LVs, we used HEK293T cells to package the second generation of LVs. First, we evaluated the cell recovery from the microfluidic chip. Cell recovery assessment showcased that 3 h and 6 h of incubation in microfluidic channels containing 100,000 NALM-6 (BCP-ALL) cells with 2µL of culture media yielded 87±7.2% and 80.6 ± 10% of cell recovery, respectively. Afterward, the effects of LV-induced toxicity were evaluated using 10-30% LV concentrations in time frames ranging from 3 h to 24 h. In 96-well plates, it took 12-24 h for the viruses with 20% and 30% concentrations to affect the cell survival significantly. These effects were intensified in the microfluidic system implying that microfluidic is capable of enhancing LV transduction. Based on the evidence of cell recovery and cell survival we chose 6 h of incubation with 20% LV. Conclusion: The results from EGFP expression showcased that a microfluidic system could increase the LV transduction in BCP-ALL cells by almost 9-folds. All in all, the microfluidic system seems to be a great armamentarium in optimizing LV-based transduction.

Synergistic surface-enhanced Raman scattering effect to distinguish live SARS-CoV-2 S pseudovirus.

The COVID-19 pandemic negatively affected the economy and health security on a global scale, causing a drastic change on lifestyle, calling a need to mitigate further transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. Surface-enhanced Raman spectroscopy (SERS) has shown great potential in the sensitive and rapid detection of various molecules including viruses, through the identification of characteristic peaks of their outer membrane proteins. Accurate detection can be developed through the synergistic integration effect among SERS-active substrate, the appropriate laser wavelength, and the target analyte. In this study, gold nanocavities (Au NC) and Au nanoparticles upon ZrO2 nano-bowls (Au NPs/pZrO2) were tested and used as SERS-active substrates in detecting SARS-CoV-2 pseudovirus containing S protein as a surface capsid glycoprotein (SARS-CoV-2 S pseudovirus) and vesicular stomatitis virus G (VSV-G) pseudo-type lentivirus (VSV-G pseudovirus) to demonstrate their virus detection capability. The optimized Au NCs and Au NPs/pZrO2 substrates were then verified by examining the repetition of measurement, reproducibility, and detection limit. Due to the difference in geometry and composition of the substrates, the characteristic peak-positions of live SARS-CoV-2 S and VSV-G pseudoviruses in the obtained Raman spectra vary, which were also compared with those of inactivated ones. Based on the experimental results, SERS mechanism of each substrate to detect virus is proposed. The formation of hot spots brought by the synergistic integration effect among substrate, analyte, and laser induction may result differences in the obtained SERS spectra.

Fusogenic Viral Protein-Based Near-Infrared Active Nanocarriers for Biomedical Imaging.

An effective drug delivery system (DDS) relies on an efficient cellular uptake and faster intracellular delivery of theranostic agents, bypassing the endosomal mediated degradation of the payload. The use of viral nanoparticles (VNPs) permits such advancement, as the viruses are naturally evolved to infiltrate the host cells to deliver their genetic material. As a proof of concept, we bioengineered the vesicular stomatitis virus glycoprotein (VSV-G)-based near-infrared (NIR) active viral nanoconstructs (NAVNs) encapsulating indocyanine green dye (ICG) for NIR bioimaging. NAVNs are spherical in size and have the intrinsic cellular-fusogenic properties of VSV-G. Further, the NIR imaging displaying higher fluorescence intensity in NAVNs treated cells suggests enhanced cellular uptake and delivery of ICG by NAVNs compared to the free form of ICG. The overall study highlights the effectiveness of VSV-G-based VNPs as an efficient delivery system for NIR fluorescence imaging.