VSV Pseudotype Research Articles

Identification of Novel Antiviral Compounds Targeting Entry of Hantaviruses.

Hemorrhagic fever viruses, among them orthohantaviruses, arenaviruses and filoviruses, are responsible for some of the most severe human diseases and represent a serious challenge for public health. The current limited therapeutic options and available vaccines make the development of novel efficacious antiviral agents an urgent need. Inhibiting viral attachment and entry is a promising strategy for the development of new treatments and to prevent all subsequent steps in virus infection. Here, we developed a fluorescence-based screening assay for the identification of new antivirals against hemorrhagic fever virus entry. We screened a phytochemical library containing 320 natural compounds using a validated VSV pseudotype platform bearing the glycoprotein of the virus of interest and encoding enhanced green fluorescent protein (EGFP). EGFP expression allows the quantitative detection of infection and the identification of compounds affecting viral entry. We identified several hits against four pseudoviruses for the orthohantaviruses Hantaan (HTNV) and Andes (ANDV), the filovirus Ebola (EBOV) and the arenavirus Lassa (LASV). Two selected inhibitors, emetine dihydrochloride and tetrandrine, were validated with infectious pathogenic HTNV in a BSL-3 laboratory. This study provides potential therapeutics against emerging virus infection, and highlights the importance of drug repurposing.

Analysis of VSV pseudotype virus infection mediated by rubella virus envelope proteins

Rubella virus (RV) generally causes a systemic infection in humans. Viral cell tropism is a key determinant of viral pathogenesis, but the tropism of RV is currently poorly understood. We analyzed various human cell lines and determined that RV only establishes an infection efficiently in particular non-immune cell lines. To establish an infection the host cells must be susceptible and permissible. To assess the susceptibility of individual cell lines, we generated a pseudotype vesicular stomatitis virus bearing RV envelope proteins (VSV-RV/CE2E1). VSV-RV/CE2E1 entered cells in an RV envelope protein-dependent manner, and thus the infection was neutralized completely by an RV-specific antibody. The infection was Ca2+-dependent and inhibited by endosomal acidification inhibitors, further confirming the dependency on RV envelope proteins for the VSV-RV/CE2E1 infection. Human non-immune cell lines were mostly susceptible to VSV-RV/CE2E1, while immune cell lines were much less susceptible than non-immune cell lines. However, susceptibility of immune cells to VSV-RV/CE2E1 was increased upon stimulation of these cells. Our data therefore suggest that immune cells are generally less susceptible to RV infection than non-immune cells, but the susceptibility of immune cells is enhanced upon stimulation.

Comparison of platform technologies for assaying antibody to Ebola virus

BackgroundThe recent Ebola outbreak in West Africa led to the use of a variety of different platform technologies for assaying antibodies because of the difficulties of handling the live virus. The same types of method could be applied rapidly to other infections when they emerge. There is a need to compare quantitative results of different assays, which means that the assays must measure similar parameters and give comparable results. MethodsA collaborative study was carried out to establish an International Reference Reagent through WHO. Nine samples were sent to 16 laboratories and the results from 22 different assays compared to those obtained by neutralisation assays using the wild type virus. FindingsQuantitative correlation with the wild type neutralisation assays was very variable but generally poor, with only five of the twenty-two assays giving a correlation coefficient of 0.7 or greater; the five best assays included methods based on wild type and VSV pseudotype neutralisation and ELISA. They could be applicable to other rapidly emerging diseases. The remaining assays including neutralisation of lentiviral pseudotypes need further development. InterpretationThe assay platform should be chosen with care to ensure that it is fit for purpose. Many of the assays were not suitable for quantitation of antibody levels, a finding that is not surprising given the urgency with which they had to be implemented but some may be of generic value. Antibody titres in samples from a vaccine trial were comparable to those from convalescent patients or lower. FundingFunding was from the UK DoH and the Wellcome Tust.

Inability of rat DPP4 to allow MERS-CoV infection revealed by using a VSV pseudotype bearing truncated MERS-CoV spike protein.

Middle East respiratory syndrome (MERS) coronavirus (Co-V) contains a single spike (S) protein, which binds to a receptor molecule, dipeptidyl peptidase 4 (DPP4; also known as CD26), and serves as a neutralizing antigen. Pseudotyped viruses are useful for measuring neutralization titers against highly infectious viruses as well as for studying their mechanism of entry. In this study, we constructed a series of cytoplasmic deletion mutants of MERS-CoV S and compared the efficiency with which they formed pseudotypes with vesicular stomatitis virus. A pseudotype bearing an S protein with the C-terminal 16 amino acids deleted (MERSpv-St16) reached a maximum titer that was approximately tenfold higher than that of a pseudotype bearing a non-truncated full-length S protein. Using MERSpv-St16, we demonstrated the inability of rat DPP4 to serve as a functional receptor for MERS-CoV, suggesting that rats are not susceptible to MERS-CoV infection. This study provides novel information that enhances our understanding of the host range of MERS-CoV.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-015-2506-z) contains supplementary material, which is available to authorized users.

Digital Detection of Nanoparticles: Viral Diagnostics and Multiplexed Protein and Nucleic Acid Assays

ABSTRACTSynthetic nanoparticles have made significant impact across a broad range of technological applications including optical nanoantennas, ultra-sensitive imaging and sensing, and diagnostics and therapeutics. Natural nanoparticles such as viruses and pollutants are major concerns for human health. High-throughput characterization of nanoparticles in terms of their size and shape is crucial for practical applications of synthetic nanoparticles and highly sensitive pathogen identification. Recently, we have demonstrated Interferometric Reflectance Imaging Sensor (IRIS) with the ability to detect single nanoscale particles [1,2].In single-particle modality of IRIS (SP-IRIS), the interference of light reflected from the sensor surface is modified by the presence of particles producing a distinct signal that reveals the size of the particle. In our approach, the dielectric layered structure acts as an optical antenna optimizing the elastic scattering characteristics of nanoparticles for sensitive detection and analysis. We have demonstrated identification of virus articles in complex samples for various viruses in multiplexed format. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of non-specifically bound particles to achieve a limit-of-detection (LOD) of 5x103 pfu/mL for the Ebola and Marburg VSV pseudotypes. We have demonstrated the simultaneous detection of multiple viruses in serum or whole blood as well as in samples contaminated with high levels of bacteria [3]. Single nanoparticle detection with IRIS has shown promising results for protein [4] and DNA arrays with attomolar detection sensitivity.

Cotton rat (Sigmodon hispidus) signaling lymphocyte activation molecule (CD150) is an entry receptor for measles virus.

Cotton rats (Sigmodon hispidus) replicate measles virus (MV) after intranasal infection in the respiratory tract and lymphoid tissue. We have cloned the cotton rat signaling lymphocytic activation molecule (CD150, SLAM) in order to investigate its role as a potential receptor for MV. Cotton rat CD150 displays 58% and 78% amino acid homology with human and mouse CD150, respectively. By staining with a newly generated cotton rat CD150 specific monoclonal antibody expression of CD150 was confirmed in cotton rat lymphoid cells and in tissues with a pattern of expression similar to mouse and humans. Previously, binding of MV hemagglutinin has been shown to be dependent on amino acids 60, 61 and 63 in the V region of CD150. The human molecule contains isoleucine, histidine and valine at these positions and binds to MV-H whereas the mouse molecule contains valine, arginine and leucine and does not function as a receptor for MV. In the cotton rat molecule, amino acids 61 and 63 are identical with the mouse molecule and amino acid 60 with the human molecule. After transfection with cotton rat CD150 HEK 293 T cells became susceptible to infection with single cycle VSV pseudotype virus expressing wild type MV glycoproteins and with a MV wildtype virus. After infection, cells expressing cotton rat CD150 replicated virus to lower levels than cells expressing the human molecule and formed smaller plaques. These data might explain why the cotton rat is a semipermissive model for measles virus infection.

SGP Serves as a Structural Protein in Ebola Virus Infection

sGP, which is perceived as nonstructural, secretory glycoprotein, shares 295 amino acids at its N-terminal with GP(1,2), which include the specific residue necessary to interact with GP(2). In the present study, we tested whether the sGP protein of Zaire ebolavirus (ZEBOV) could substitute for GP(1) and form a complex with GP(2), thus serving as a structural protein. We expressed ZEBOV GP(1,2), VP40, and NP proteins, together with sGP protein, from expression plasmids and examined the resultant virus-like particles by using Western blot. Cells expressing GP(2) in combination with either GP(1) or sGP were analyzed by using flow cytometry with the KZ52 antibody, which recognizes a GP(1,2) conformational epitope. A VSV pseudotype, VSVΔG*, which expresses a GFP reporter gene instead of the G protein, was used to produce pseudotyped viruses encoding sGP and variants of GP to test the contribution of sGP to infectivity. Western blot and flow cytometric analyses suggested the existence of a covalently linked sGP-GP(2) molecule. VSVΔG*(sGP + GP(2)) and VSVΔG*(GP(1,2)) infected Vero E6 cells and were neutralized by the KZ52 antibody. Overexpression of sGP reduced the titer of VSVΔG*(GP(1,2)). ZEBOV sGP can substitute for GP(1), forming a sGP-GP(2) complex and conferring infectivity. Our studies suggest a novel role for sGP as a structural protein.

Development of a neutralization assay for Nipah virus using pseudotype particles

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes several days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.

Tandem Repeats of Lactoferrin‐Derived Anti‐Hepatitis C Virus Peptide Enhance Antiviral Activity in Cultured Human Hepatocytes

Previously, we found that bovine and human lactoferrin (LF) specifically inhibited hepatitis C virus (HCV) infection in cultured non-neoplastic human hepatocyte-derived PH5CH8 cells, and we identified 33 amino acid residues (termed C-s3-33; amino acid 600-632) from human LF that were primarily responsible for the binding activity to the HCV E2 envelope protein and for the inhibiting activity against HCV infection. Since the anti-HCV activity of C-s3-33 was weaker than that of human LF, we speculated that an increase of E2 protein-binding activity might contribute to the enhancement of anti-HCV activity. To test this possibility, we made two repeats [(C-s3-33)(2)] and three repeats [(C-s3-33)(3)] of C-s3-33 and characterized them. Far-Western blot analysis revealed that the E2 protein-binding activities of (C-s3-33)(2) and (C-s3-33)(3) became stronger than that of the C-s3-33, and that the binding activity of (C-s3-33)(3) was stronger than that of (C-s3-33)(2). Using an HCV infection system in PH5CH8 cells, we demonstrated that the anti-HCV activities of (C-s3-33)(2) and (C-s3-33)(3) became stronger than that of the C-s3-33. Furthermore, using a recently developed infection system with a VSV pseudotype harboring the green fluorescent protein gene and the native E1 and E2 genes, we demonstrated that the antiviral activities of (C-s3-33)(2) and (C-s3-33)(3) were stronger than that of C-s3-33. These results suggest that tandem repeats of LF-derived anti-HCV peptide are useful as anti-HCV reagents.

Coexpression of hepatitis C virus E1 and E2 chimeric envelope glycoproteins displays separable ligand sensitivity and increases pseudotype infectious titer.

We have previously reported that a pseudotype virus generated by reconstitution of hepatitis C virus (HCV) chimeric envelope glycoprotein E1-G or E2-G on the surface of a temperature-sensitive mutant of vesicular stomatitis virus (VSVts045) interacts independently with mammalian cells to initiate infection. Here, we examined whether coexpression of both of the envelope glycoproteins on pseudotype particles would augment virus infectivity and/or alter the functional properties of the individual subunits. Stable transfectants of baby hamster kidney (BHK) epithelial cells expressing either one or both of the chimeric envelope glycoproteins of HCV on the cell surface were generated. The infectious titer of the VSV pseudotype, derived from a stable cell line incorporating both of the chimeric glycoproteins of HCV, was approximately 4- to 5-fold higher than that of a pseudotype bearing E1-G alone or approximately 25- to 30-fold higher than that of E2-G alone when assayed with a number of mammalian cell lines. Further studies suggested that that the E1-G/E2-G or E2-G pseudotype was more sensitive to the inhibitory effect of heparin than the E1-G pseudotype. Treatment of the E1-G/E2-G pseudotype with a negatively charged sulfated sialyl lipid (NMSO3) displayed a approximately 4-fold-higher sensitivity to neutralization than pseudotypes with either of the two individual glycoproteins. In contrast, VSVts045, used as a backbone for the generation of pseudotypes, displayed at least 20-fold-higher sensitivity to NMSO3-mediated inhibition of virus plaque formation. The effect of low-density lipoprotein on the E1-G pseudotype was greater than that apparent for the E1-G/E2-G pseudotype. The treatment of cells with monoclonal antibodies to CD81 displayed an inhibitory effect upon the pseudotype with E1-G/E2-G or with E2-G alone. Taken together, our results indicate that the HCV E1 and E2 glycoproteins have separable functional properties and that the presence of these two envelope glycoproteins on VSV/HCV pseudotype particles increases infectious titer.

International Collaborative Study to Compare Assays for Antibodies that Neutralize Human Immunodeficiency Virus

A variety of techniques are currently in use to measure antibodies that neutralize human immunodeficiency virus (HIV), and standardization of these assays is needed. Eleven laboratories participated in this comparison study of 14 methods for detection of HIV neutralizing antibodies (NA). A panel of 10 coded sera and a positive and a negative control serum were tested in each assay. The 10 coded samples included aliquots of the same sera that were distributed as positive and negative control sera, an aliquot of the WHO reference human anti-HIV-1 serum, and seven other sera from people with HIV-1 infection. Each laboratory reported features of its test methods and results. The results demonstrated excellent within laboratory and between laboratory consistency. The most important variable appeared to be the virus strain used. Cell line, conditions of neutralization or culture, method of endpoint determination, or use of VSV pseudotypes did not appear to be important variables. The results indicate that standardization of HIV-1 NA assays should be readily achievable.

Synthetic peptides approach to identification of epitopes on bovine leukemia virus envelope glycoprotein gp51

Peptides corresponding to residues 21–28, 39–48, 57–67, 59–69, 78–92, 144–155, 144–157, 195–205, 255–268, and 260–268 of envelope glycoprotein gp51 of bovine leukemia virus (BLV) were chemically synthesized and coupled to keyhole limpet hemocyanin or tetanus toxoid. All peptides were immunogenic in rabbits and induced production of antipeptide antibodies. Enzyme-linked immunosorbent assays using Tween 80-purified gp51 or BLV particles showed that antibodies against peptides 78–92, 255–268, and to a lesser extent 39–48 and 144–157 were able to react with the parent glycoprotein, purified or as an integer part of BLV particles. Antisera against peptides 39–48, 78–92, and 144–157 neutralized VSV (BLV) pseudotypes in vitro, the highest neutralization titer being obtained with the 78–92 cyclized peptide. These observations confirm that the NH 2 moiety of gp51 carries epitopes involved in virus infectivity.

Rescued human virus (RHV): association with melanoma.

RHV, which is presumably a defective human retrovirus, has been recovered from the human melanoma cell line HMB2. In the presence of Moloney mouse leukemia virus (MLV), used as a helper, RHV is serially transmissible in mouse NIH-3T3 cells and can provide envelope antigens for vesicular stomatitis virus pseudotype--VSV(RHV). This pseudotype is neutralizable with an inhibitor, present in all human sera tested; the inhibitory activity is resistant to heating at 100 degrees C. To detect whether any specific response to RHV is connected with the disease, we isolated IgG from 140 human sera (70 melanoma patients and 70 control group donors) and tested it for neutralization of VSV(RHV) pseudotype in a randomized, blind experiment. Sixteen samples of IgG from the sera of melanoma patients, but only one control IgG, neutralized the pseudotype; this difference was significant (p less than 0.001). RHV thus appears to be in some way associated with melanoma.

Neutralization of diverse HIV-1 strains by monoclonal antibodies raised against a gp41 synthetic peptide

Three IgM monoclonal antibodies raised against synthetic peptide analogs of a hydrophilic region of the gp41 transmembrane env protein of HIV-1 neutralize different HIV-1 isolates but not HIV-2 isolates, as determined by HIV titration and by syncytial inhibition assays. VSV (HIV-1) pseudotypes, however, were not neutralized, indicating that gp41 was not accessible to these antibodies on the pseudotype particles. The antibodies affect early steps in adsorption and penetration of HIV-1.

Human immunodeficiency virus infection of CD4-bearing cells occurs by a pH-independent mechanism.

The effect of weak bases (NH4Cl and amantadine) and carboxylic ionophores (monensin) on the infection of CD4 (T4) positive human cell lines by HIV-1 is examined. These reagents, which raise the pH of acidic intracellular organelles, fail to inhibit HIV-1 entry and the events leading to viral protein synthesis at concentrations inhibitory for low pH-dependent fusogenic enveloped viruses. The infectivity of VSV (HIV-1) pseudotypes is unaffected by weak bases at concentrations causing 95% plaque reduction of VSV in its own envelope. HIV-1 dependent cell--cell fusion (syncytium formation) occurs in medium maintained at pH 7.4-7.6, and virions are not irreversibly inactivated by incubation in acid medium. Our results show that HIV-1 entry and membrane fusion do not require exposure to low pH. The production of infectious HIV-1 particles, however, is inhibited in cells treated with NH4Cl.

Detection of VSV(MuLV) pseudotypes by an immunobiochemical technique

Vesicular stomatitis virus (murine leukemia virus) (VSV(MuLV)) pseudotypes containing a [ 3H]uridine-labeled VSV RNA genome and MuLV envelope glycoproteins (gp70) were produced by phenotypic mixing of the two viruses. In order to better detect such pseudotypes, an immunobiochemical (IB) technique was developed. [ 3H]Uridine-labeled virus progeny of the dual virus infection was immunoprecipitated by monospecific MuLV gp70 antibodies complexed with fixed Staphylococcus aureus. The immunoprecipitated 3H-labeled genomic RNA was identified as that of VSV by its sedimentation coefficient, by the lack of polyadenylate, and by molecular hybridization with complementary VSV RNA. By the IB technique, approximately 11% of the progeny of the dual virus infection were found to be VSV(MuLV). By neutralization and other biological assays, however, only 0.1% of the progeny were found to be VSV(MuLV) pseudotypes. Apparently, the IB technique is capable of detecting VSV pseudotypes encapsidated with only a few molecules of MuLV gp70. The IB technique, therefore, offers a quantitative and molecular technique for the detection of VSV(MuLV) pseudotypes and can be modified to detect other viral pseudotypes when other assays are lacking. In spite of its sensitivity however, the IB technique did not detect the formation of MuLV (VSV) pseudovirions among the virus progeny of the dual virus infection. These results confirmed a similar observation made previously using immunoelectron microscopy.

The use of vesicular stomatitis virus pseudotype production in the study of a temperature-sensitive murine leukemia virus

A mutant of Moloney murine leukemia virus (MoLV), designated ts 3 was recently shown to have a temperature-sensitive defect associated with the release of mature virus particles budding from the cell membrane [Wong, P. K. Y., and McCarter, J. A., Virology 58, 396–408 (1974)] . In an attempt to determine whether the defective function resides in an envelope component of the virion, the formation of pseudotypes between VSV and ts 3 was studied under nonpermissive and permissive conditions of ts 3 infection. Whereas similar levels of phenotypic mixing were observed between VSV and wild-type MoLV at both 39 and 34°, the level of pseudotypes formed between VSV and ts 3 was found to be considerably lower at 39° (nonpermissive temperature) than at 34° (permissive temperature). The results of temperature-shift experiments indicate that two separate blocks to VSV ts 3 pseudotype production may occur depending on the length of time ts 3-infected cells are incubated at the nonpermissive temperature. Preincubation of ts 3-infected cells for 24 hr at 39°, prior to superinfection with VSV at 39°, prevents pseudotype formation. In contrast, brief incubation at 39°C, coincident with VSV infection, introduces a reversible block on the release of VSV (ts 3) pseudotypes from the cell membrane. Complementation of ts 3 through ts 3 (VSV) pseudotype production was not detected at the nonpermissive temperature.

VSV Pseudotype Produced in Cell Line derived from Human Mammary Carcinoma

RECENT findings suggest that a virus related to the mouse mammary tumour virus (MMTV) could be involved in human breast cancer. B type particles have been detected in human mammary tumours1 and in human milk2 by electron microscopy, reverse transcriptase enzyme was found in preparations from human milk3,4 and RNA isolated from human breast tumours hybridized with DNA primed by MMTV-RNA5.