Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm), while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm). Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.

Multi-virion infectious units arise from free viral particles in an enveloped virus.

Many animal viruses are enveloped in a lipid bilayer taken up from cellular membranes. Because viral surface proteins bind to these membranes to initiate infection, we hypothesized that free virions may also be capable of interacting with the envelopes of other virions extracellularly. Here, we demonstrate this hypothesis in the vesicular stomatitis virus (VSV), a prototypic negative-strand RNA virus composed of an internal ribonucleocapsid, a matrix protein and an external envelope1. Using microscopy, dynamic light scattering, differential centrifugation and flow cytometry, we show that free viral particles can spontaneously aggregate into multi-virion infectious units. We also show that, following establishment of these contacts, different viral genetic variants are co-transmitted to the same target cell. Furthermore, virion-virion binding can determine key aspects of viral fitness such as antibody escape. In purified virions, this process is driven by protein-lipid interactions probably involving the VSV surface glycoprotein and phosphatidylserine. Whereas we found that multi-virion complexes occurred unfrequently in standard cell cultures, they were abundant in other fluids such as saliva, a natural VSV shedding route2. Our findings contrast with the commonly accepted perception of virions as passive propagules and show the ability of enveloped viruses to establish collective infectious units, which could in turn facilitate the evolution of virus-virus interactions and of social-like traits3.

Real-Time Capture and Visualization of Individual Viruses in Complex Media.

Label-free imaging of individual viruses and nanoparticles directly in complex solutions is important for virology research and biosensing applications. A successful visualization technique should be rapid, sensitive, and inexpensive, while needing minimal sample preparation or user expertise. Current approaches typically require fluorescent labeling or the use of an electron microscope, which are expensive and time-consuming to use. We have developed an imaging technique for real-time, sensitive, and label-free visualization of viruses and nanoparticles directly in complex solutions such as serum. By combining the advantages of a single-particle reflectance imaging sensor, with microfluidics, we perform real-time digital detection of individual 100 nm vesicular stomatitis viruses as they bind to an antibody microarray. Using this approach, we have shown capture and visualization of a recombinant vesicular stomatitis virus Ebola model (rVSV-ZEBOV) at 100 PFU/mL in undiluted fetal bovine serum in less than 30 min.

Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients.

Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4(+) T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM-100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4(+) T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4(+) T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4(+) T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients.

Virus inhibition induced by polyvalent nanoparticles of different sizes.

The development of antiviral agents is one of the major challenges in medical science. So far, small monovalent molecular drugs that inhibit the late steps in the viral replication cycle, i.e., virus budding, have not worked well which emphasizes the need for alternative approaches. Polyvalently presented viral receptors, however, show potential as good inhibitors of virus-cell binding, which is the first step in the viral infection cycle. By gradually increasing the size of ligand functionalized gold nanoparticles, up to virus-like dimensions, we are now able to quantify the polyvalent enhancement of virus-cell binding inhibition and to identify varying mechanisms of virus inhibition with different efficacies: by employing a new binding assay we found that surface area-normalized polysulfated gold nanoparticles of diameters equal to and larger than the virus diameter (>50 nm) more efficiently inhibit the binding of vesicular stomatitis virus (VSV) to cells than smaller particles. On a per particle basis, larger sized gold nanoparticles were surprisingly shown to inhibit the viral infection up to two orders of magnitude more efficiently than smaller particles, which suggests different mechanisms of virus inhibition. Based on complementary electron microscopic data, we noticed that larger gold nanoparticles act as efficient cross-linkers between virions, whereas smaller gold nanoparticles decorate the surface of individual virus particles. Our systematic study accentuates the need for the design of biodegradable, virus-sized inhibitors capitalizing on polyvalent binding.

Virion-associated complement regulator CD55 is more potent than CD46 in mediating resistance of mumps virus and vesicular stomatitis virus to neutralization.

Enveloped viruses can incorporate host cell membrane proteins during the budding process. Here we demonstrate that mumps virus (MuV) and vesicular stomatitis virus (VSV) assemble to include CD46 and CD55, two host cell regulators which inhibit propagation of complement pathways through distinct mechanisms. Using viruses which incorporated CD46 alone, CD55 alone, or both CD46 and CD55, we have tested the relative contribution of these regulators in resistance to complement-mediated neutralization. Virion-associated CD46 and CD55 were biologically active, with VSV showing higher levels of activity of both cofactors, which promoted factor I-mediated cleavage of C3b into iC3b as well as decay-accelerating factor (DAF) activity against the C3 convertase, than MuV. Time courses of in vitro neutralization with normal human serum (NHS) showed that both regulators could delay neutralization, but viruses containing CD46 alone were neutralized faster and more completely than viruses containing CD55 alone. A dominant inhibitory role for CD55 was most evident for VSV, where virus containing CD55 alone was not substantially different in neutralization kinetics from virus harboring both regulators. Electron microscopy showed that VSV neutralization proceeded through virion aggregation followed by lysis, with virion-associated CD55 providing a delay in both aggregation and lysis more substantial than that conferred by CD46. Our results demonstrate the functional significance of incorporation of host cell factors during virion envelope assembly. They also define pathways of virus complement-mediated neutralization and suggest the design of more effective viral vectors.

Mast cells elicit proinflammatory but not type I interferon responses upon activation of TLRs by bacteria.

Balanced induction of proinflammatory and type I IFN responses upon activation of Toll-like receptors (TLRs) determines the outcome of microbial infections and the pathogenesis of autoimmune and other inflammatory diseases. Mast cells, key components of the innate immune system, are known for their debilitating role in allergy and autoimmunity. However, their role in antimicrobial host defenses is being acknowledged increasingly. How mast cells interact with microbes and the nature of responses triggered thereby is not well characterized. Here we show that in response to TLR activation by Gram-positive and -negative bacteria or their components, mast cells elicit proinflammatory but not type I IFN responses. We demonstrate that in mast cells, bound bacteria and TLR ligands remain trapped at the cell surface and do not undergo internalization, a prerequisite for type I IFN induction. Such cells, however, can elicit type I IFNs in response to vesicular stomatitis virus which accesses the cytosolic retinoic acid-inducible gene I receptor. Although important for antiviral immunity, a strong I IFN response is known to contribute to pathogenesis of several bacterial pathogens such as Listeria monocytogenes. Interestingly, we observed that the mast cell-dependent neutrophil mobilization upon L. monocytogenes infection is highly impaired by IFN-beta. Thus, the fact that mast cells, although endowed with the capacity to elicit type I IFNs in response to viral infection, elicit only proinflammatory responses upon bacterial infection shows that mast cells, key effector cells of the innate immune system, are well adjusted for optimal antibacterial and antiviral responses.

Cryo-EM model of the bullet-shaped vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a bullet-shaped rhabdovirus and a model system of negative-strand RNA viruses. Through direct visualization by means of cryo-electron microscopy, we show that each virion contains two nested, left-handed helices: an outer helix of matrix protein M and an inner helix of nucleoprotein N and RNA. M has a hub domain with four contact sites that link to neighboring M and N subunits, providing rigidity by clamping adjacent turns of the nucleocapsid. Side-by-side interactions between neighboring N subunits are critical for the nucleocapsid to form a bullet shape, and structure-based mutagenesis results support this description. Together, our data suggest a mechanism of VSV assembly in which the nucleocapsid spirals from the tip to become the helical trunk, both subsequently framed and rigidified by the M layer.

[Comparison of characteristics of SVCV strains isolated in China and in Europe].

In the nationwide epidemiological investigation, SVCV-741 was for the first time isolated in Beijing region, China in 2003, and designated as SVCV Asian strain. In this paper, we compared SVCV-741 (Asian strains isolated in China) with SVCV-10/3 (Europe reference strain) on their physico-chemical, biological and morphological characteristics. The results indicated that there were no distinct differences between two SVCV strains on phycico-chemical and morphological characteristics. The main existing differences were: (1) The stability of SVCV-741 to temperature in cell culture was higher than that of SVCV-10/3, which might have some evolutionary and biological implication of SVCV; (2) No SVC outbreak ever occurred caused by SVCV-741;Furthermore we found that both SVCV-741 and SVCV-10/3 grew faster and produced higher virus titer in CO cells than other cell lines. It indicated that CO cell lines might be useful tool for SVCV research.

Site-specific labeling of enveloped viruses with quantum dots for single virus tracking.

This study reports a general method of labeling enveloped viruses with semiconductor quantum dots (QDs) for use in single virus trafficking studies. Retroviruses, including human immunodeficiency virus (HIV), could be successfully tagged with QDs through the membrane incorporation of a short acceptor peptide (AP) that is susceptible to site-specific biotinylation and attachment of streptavidin-conjugated QDs. It was found that this AP tag-based QD labeling had little effect on the viral infectivity and allowed for the study of the kinetics of the internalization of the recombinant lentivirus enveloped with vesicular stomatitis virus glycoprotein (VSVG) into the early endosomes. It also allows for the live cell imaging of the trafficking of labeled virus to the Rab5(+) endosomal compartments. This study further demonstrated by direct visualization of QD-labeled virus that VSVG-pseudotyped lentivirus enters cells independent of clatherin- and caveolin-pathways, while the entry of VSVG-pseudotyped retrovirus occurs via the clathrin pathway. The studies monitoring HIV particles using QD-labeling showed that we could detect single virions on the surface of target cells expressing either CD4/CCR5 or DC-SIGN. Further internalization studies of QD-HIV evidently showed that the clathrin pathway is the major route for DC-SIGN-mediated uptake of viruses. Taken together, our data demonstrate the potential of this QD-labeling for visualizing the dynamic interactions between viruses and target cell structures.

The ESCRT-I subunit TSG101 controls endosome-to-cytosol release of viral RNA.

Like other enveloped viruses, vesicular stomatitis virus infects cells through endosomes. There, the viral envelope undergoes fusion with endosomal membranes, thereby releasing the nucleocapsid into the cytoplasm and allowing infection to proceed. Previously, we reported that the viral envelope fuses preferentially with the membrane of vesicles present within multivesicular endosomes. Then, these intra-endosomal vesicles (containing nucleocapsids) are transported to late endosomes, where back-fusion with the endosome limiting membrane delivers the nucleocapsid into the cytoplasm. In this study, we show that the tumor susceptibility gene 101 (Tsg101) subunit of the endosomal sorting complexes required for transport (ESCRT)-I complex, which mediates receptor sorting into multivesicular endosomes, is dispensable for viral envelope fusion with endosomal membranes and viral RNA transport to late endosomes but is necessary for infection. Our data indicate that Tsg101, in contrast to the ESCRT-0 component Hrs, plays a direct role in nucleocapsid release from within multivesicular endosomes to the cytoplasm, presumably by controlling the back-fusion process. We conclude that Tsg101, through selective interactions with its partners including Hrs and Alix, may link receptor sorting and lysosome targeting to the back-fusion process involved in viral capsid release.

First detection and confirmation of spring viraemia of carp virus in common carp, Cyprinus carpio L., from Hamilton Harbour, Lake Ontario, Canada.

In June 2006, 150 wild common carp were sampled from Hamilton Harbour, Lake Ontario, Canada. Tissue pools consisting of kidney, spleen and encephalon were screened for viruses as a condition facilitating the export of live carp to France. Cytopathic effect (CPE), indicative of a viral infection, became evident after 8 days of incubation at 15 degrees C. Eighteen of 30 tissue pools (five fish per pool) eventually demonstrated viral CPE. The viral pathogen was initially cultured and isolated on the epithelioma papulosum cyprini cell line and subsequently shown to produce CPE in the fathead minnow and bluegill fin cell lines. Electron microscopy demonstrated the virus to be a rhabdovirus. Reverse transcriptase-polymerase chain reaction assay and nucleotide sequence analysis identified the isolate as spring viraemia of carp virus (SVCV). Phylogenetic analysis of a 533 bp region of the glycoprotein gene grouped the Canadian isolate in SVCV genogroup Ia together with isolates from Asia and the USA. Sequence comparisons revealed the Hamilton Harbour, Lake Ontario isolate to be most similar to an isolate obtained from common carp in the Calumet Sag Channel in Illinois in 2003 (98.9% nucleotide identity). This is the first report of the detection of SVCV in Canada.

Vesicular stomatitis New Jersey virus (VSNJV) infects keratinocytes and is restricted to lesion sites and local lymph nodes in the bovine, a natural host.

Inoculation of vesicular stomatitis New Jersey virus (VSNJV) by skin scarification of the coronary-band in cattle, a natural host of VSNJV, resulted in vesicular lesions and 6-8 log(10) TCID(50) increase in skin virus titers over a 72 h period. Virus infection was restricted to the lesion sites and lymph nodes draining those areas but no virus or viral RNA was found in the blood or in 20 other organs and tissues sampled at necropsy. Scarification of flank skin did not result in lesions or a significant increase in viral titer indicating that viral clinical infection is restricted to skin inoculation at sites where lesions naturally occur. Viral antigens co-localized primarily with keratinocytes in the coronary band, suggesting these cells are the primary site of viral replication. Viral antigen also co-localized with few MHC-II positive cells, but no co-localization was observed in cells positive for macrophage markers. Although granulocyte infiltration was observed in lesions, little viral antigen co-localized with these cells. This is the first detailed description of VSNJV tissue distribution and infected cell characterization in a natural host. The pathogenesis model shown herein could be useful for in-vivo tracking of virus infection and local immune responses.

P-protein of Chandipura virus is an N-protein-specific chaperone that acts at the nucleation stage.

The nucleocapsid protein N of Chandipura virus is prone to aggregation in vitro. We have shown that this aggregation occurs in two phases in a nucleation-dependent manner. Electron microscopy suggests that the aggregated state may have a ring-like structure. Using a GFP fusion, we have shown that the N-protein also aggregates in vivo. The P-protein suppresses the N-protein aggregation efficiently, both in vitro and in vivo. Increased lag phase in the presence of the P-protein suggests that chaperone-like action of the P-protein occurs before the nucleation event. The P-protein, however, does not exert any chaperone-like action against other proteins, suggesting that it binds to the N-protein specifically. Surface plasmon resonance and fluorescence enhancement indeed suggest that the P-protein binds tightly to the native N-protein. The P-protein is thus an N-protein-specific chaperone which inhibits the nucleation phase of N-protein aggregation, thus keeping a pool of encapsidation-competent N-protein for viral maturation.

Isolation of a vesicular virus belonging to the family rhabdoviridae from the aqueous humor of a patient with bilateral corneal endotheliitis.

PURPOSE: To report bilateral corneal endotheliitis caused by a vesicular virus (family Rhabdoviridae). METHODS: Case report of a 49-year-old man with a complaint of sudden onset of decreased vision in both eyes had diffuse corneal stromal edema with extensive folds in Descemet's membrane and was diagnosed as having bilateral viral endotheliitis. Virologic investigations were performed using aqueous humor from the right eye. RESULTS: An ether- and chloroform-sensitive cytopathic agent was isolated in Vero and BHK-21 cell lines from the aqueous humor. It was identified as a vesicular virus belonging to the family Rhabdoviridae by electron microscopy. Neutralizing antibody was demonstrated at a titer greater than 1 in 4,096 dilutions in the convalescent serum. Neurologic complications included loss of hearing and postinfectious polyradiculopathy affecting both lower limbs. Best-corrected visual acuity was 20/120 OD and 20/20 OS. Six months later, he developed glaucoma in the right eye. Trabeculectomy with intraoperative application of 5-fluorouracil was performed. CONCLUSION: This is the first report of bilateral endotheliitis caused by a vesicular virus and confirmed by virus isolation from the aqueous humor of the affected eye.

Ultrastructural aspects of replication of the New Jersey serotype of vesicular stomatitis virus in a suspected sand fly vector, Lutzomyia shannoni (Diptera: Psychodidae).

Transmission electron microscopy was used to examine replication of the New Jersey serotype of vesicular stomatitis virus (VSNJ) (Rhabdoviridae: Vesiculovirus) in Lutzomyia shannoni (Diptera: Psychodidae), a recently implicated sand fly vector. Following ingestion of an infectious blood meal, female sand flies were fixed and examined at approximately 12-hr intervals for six days. The New Jersey serotype of vesicular stomatitis virus was first detected in the abdominal midgut after 34 hr of incubation. Virus next appeared in fat body and the thoracic midgut at 48 hr, while salivary glands first contained visible virus in apical cavities 5-6 days after infection. Flight muscles and nervous tissue occasionally contained small numbers of VSNJ virions, while virus was never detected in the ovaries or malphigian tubules. The midgut and fat body appeared to be major sites of VSNJ virus replication. In all tissues examined, virus matured primarily by budding from the plasma membrane. Virions were occasionally observed within vacuoles, along with nucleocapsids. In the midgut, budding occurred exclusively from the basolateral plasma membrane, while maturation in salivary gland cells involved apical budding. Accumulation of virions adjacent to basal laminae surrounding several tissues suggested that this structure physically impedes virus dissemination within the sandfly. The paucity of virus budding 120-144 hr after infection suggested that the VSNJ virus infection was modulated in Lu. shannoni.

Physical properties of New Jersey serotype of vesicular stomatitis virus and its defective particles.

The wild-type New Jersey serotype of vesicular stomatitis virus generated two types of defective interfering T-particles. The physical properties of these particles and the wild-type virion were determined by laser light scattering spectroscopy, sedimentation measurements, and electron microscopy.