Incomplete Particles Research Articles

Inhibitory effects of typical inert gases on the flame propagation and structures in TiH2 dust clouds

The inhibitory effects of CO2, N2 and Ar on TiH2 dust explosion had been experimentally studied. The results showed that the inhibitory effects of the three inert gases on the flame morphology, propagation velocity and temperature of TiH2 dust explosion were in the order of CO2 > N2 > Ar under the same oxygen concentration. CO2 accelerated the cooling and extinguishing processes of the incomplete burning particles to inhibit the further dehydrogenation and combustion reactions of TiH2 particles. N2 reacted with TiH2 particles and form ternary Ti-O-N solutions on its surface to hinder the binding progress of TiH2 and O2. In the process of Ar dilution, OH radical was adsorbed on the surface of TiH2-x to enhance its surface reactivity. N2 and CO2 mainly inhibited the explosion of TiH2 dust cloud through dilution of O2, cooling and chemical reactions, whereas Ar mainly played the roles of diluting O2 and cooling.

Incomplete bunyavirus particles can cooperatively support virus infection and spread.

Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented arthropod-borne Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations unable to spread autonomously are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that complementing incomplete virus particles can co-infect mosquitoes, resulting in the reconstitution of infectious virus that is able to disseminate to the mosquito salivary glands. Computational models of infection dynamics predict that incomplete virus particles can positively impact virus spread over a wide range of conditions, with the strongest effect at intermediate multiplicities of infection. Our findings suggest that incomplete particles may play a significant role in within-host spread and between-host transmission, reminiscent of the infection cycle of multipartite viruses.

The role of hepatitis B virus surface proteins in regulating the maturation and secretion of complete and incomplete virions.

The expression of various forms of hepatitis B virus (HBV) surface proteins regulates the release of mature virion, but whether they affect the release of other incomplete viral particles, such as naked capsid, is not clear. Here, by stable overexpression of large or middle/small hepatitis B surface proteins (LHBs, M/SHBs) in HepAD38 cells, we evaluated their effects on the release of complete and incomplete viral particles. Overproduction of LHBs inhibited the release of all surface proteins, which increased the ratio of naked capsids/virions. This effect was accompanied by the elevated extracellular HBV RNA. On the other hand, overexpression of M/SHBs greatly improved the secretion of enveloped viral and subviral particles. In situ visualization of viral DNA and LHBs revealed intracellular retention of mature virions when LHBs were overexpressed. These results indicate that the molecular decision on secretion of enveloped or unenveloped viral particles is modulated by the intracellular ratio of large, middle and small surface antigens. This mechanism may be relevant in the progression and resolution of HBV-induced chronic liver disease.

Antiviral activity of chitosan nanoparticles for controlling plant-infecting viruses

Chitosan nanoparticles (ChiNPs) are a potentially effective means for controlling numerous plant diseases. This study firstly describes the antiviral capabilities of ChiNPs to control plant viral diseases compared to its bulk form. Bean yellow mosaic virus (BYMV) was used as a model plant virus affecting faba bean plants and many other legumes. The antiviral effectiveness of ChiNPs and chitosan were evaluated as a curative application method, using six dosage rates (50, 100, 200, 250, 300 and 400 mg/L). Results indicated that ChiNPs curatively applied 48 h post virus inoculation entirely inhibit the disease infectivity and viral accumulation content at 300 mg/L and 400 mg/L. The virus titre was greatly alleviated within the plant tissues by 7.71% up to100% depending on ChiNP dosage rates. However, chitosan used in its bulk-based material form revealed a relatively low to an intermediate reduction in virus infectivity by 6.67% up to 48.86%. Interestingly, ChiNPs affect the virus particle’s integrity by producing defective and incomplete BYMV viral particles, defeating their replication and accumulation content within the plant tissues. Simultaneously, ChiNP applications were appreciably shown to promote the pathogenesis-related (PR-1) gene and other defence-related factors. The mRNA of the PR-1 gene was markedly accumulated in treated plants, reaching its maximum at 400 mg/L with 16.22-fold relative expression change over the untreated control. Further, the total phenol dynamic curve was remarkably promoted for 30 days in response to ChiNP application, as compared to the untreated control. Our results provide the first report that chitosan-based nanomaterials have a superior effect in controlling plant viruses as an antiviral curing agent, suggesting that they may feasibly be involved in viral disease management strategies under field conditions without serious health concerns and environmental costs.Significance: Our findings show that chitosan nanoparticles have a powerful curing antiviral activity against BYMV disease. These findings open the door for the use of eco-friendly nano-based tools in controlling numerous plant viruses. The use of eco-friendly nano-based materials could result in a successful integrative control strategy for plant viruses under field conditions, negating the need for the conventional measure used to control most of the insect-transmitted plant viruses, that is insecticide application against vector insects.

Analysis of Recombinant Adenovirus Vectors by Ion Trap Charge Detection Mass Spectrometry: Accurate Molecular Weight Measurements beyond 150 MDa.

Adenovirus is one of the largest nonenveloped, double-stranded DNA viruses. It is widely used as a gene therapy vector and has recently received a lot of attention as a novel vaccine platform for SARS-CoV-2. Human adenovirus 5 (HAdV5) contains over 2500 protein molecules and has a 36 kbp genome. Adenovirus is well beyond the range of conventional mass spectrometry, and it was unclear how well such a large complex could be desolvated. Here, we report molecular weight (MW) distributions measured for HAdV5 and for 11 recombinant AdV vectors with genomes of varying lengths. The MW distributions were recorded using ion trap charge detection mass spectrometry (CDMS), a single-particle technique where m/z and charge are measured for individual ions. The results show that ions as large as 150 MDa can be effectively desolvated and accurate MW distributions obtained. The MW distribution for HAdV5 contains a narrow peak at 156.1 MDa, assigned to the infectious virus. A smaller peak at 129.6 MDa is attributed to incomplete particles that have not packaged a genome. The ions in the 129.6 MDa peak have a much lower average charge than those in the peak at 156.1 MDa. This is attributed to the empty particles missing some or all of the fibers that decorate the surface of the virion. The MW measured for the mature virus (156.1 MDa) is much larger than that predicted from sequence masses and copy numbers of the constituents (142.5 MDa). Measurements performed for recombinant AdV as a function of genome length show that for every 1 MDa increase in the genome MW, the MW of the mature virus increases by around 2.3 MDa. The additional 1.3 MDa is attributed to core proteins that are copackaged with the DNA. This observation suggests that the discrepancy between the measured and expected MWs for mature HAdV5 is due to an underestimate in the copy numbers of the core proteins.

Effects of coal particle size on the two-phase flow and slagging performance in a swirl burner

Swirl burner is widely used in coal-fired boilers. Coal particle size is an important factor affecting the two-phase flow as well as the combustion performances of a swirl burner. A bench-scale experiment with CFD simulation was conducted with full burner complexity. The results show that asymmetry of the particle phase is gradually weakened as the particle size increases, opposite to the variation trend of the continuous flow field in a hot state. The asymmetry of the particle phase is much more affected by the asymmetry of the secondary air jet flow than that of the primary air jet flow. As the particle size increases within a certain range, the combustibility of pulverized coal decreases, which enhances the asymmetry of the continuous flow field. When the particle size increases more than 50 μm, an increase in the concentration of incomplete combustion particles in the side recirculation zone around the burner occurs, intensifying the tendency of slagging. Thus, controlling the pulverized coal particle size below 50 μm is a considerable choice to improve combustion efficiency and avoid slagging.

The skin as a critical window in unveiling the pathophysiologic principles of COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), is a single-stranded RNA virus whose sequence is known. COVID-19 is associated with a heterogeneous clinical phenotype ranging from asymptomatic to fatal disease. It appears that access to nasopharyngeal respiratory epithelia expressing angiotensin-converting enzyme (ACE) 2, the receptor for SARS-CoV-2, is followed by viral replication in the pulmonary alveolar septal capillary bed. We have demonstrated in earlier studies that incomplete viral particles, termed pseudovirions, dock to deep subcutaneous and other vascular beds, potentially contributing to the prothrombotic state and systemic complement activation that characterizes severe and critical COVID-19. A variety of skin eruptions have been described in the setting of SARS-CoV-2 infection and more recently, after COVID-19 vaccination. The vaccines deliver a laboratory-synthesized mRNA that encodes a protein that is identical to the spike glycoprotein of SARS-CoV-2, allowing the production of immunogenic spike glycoprotein that will then elicit T cell and B cell adaptive immune responses. In this contribution, we review an array of cutaneous manifestations of COVID-19 that provide an opportunity to study critical pathophysiologic mechanisms that underlie all clinical facets of COVID-19, ranging from asymptomatic/mild to severe and critical COVID-19. We classify cutaneous COVID-19 according to underlying pathophysiologic principles. In this regard we propose three main pathways: (1) complement mediated thrombotic vascular injury syndromes deploying the alternative and mannan binding lectin pathways and resulting in the elaboration of cytokines like interleukin 6 from endothelium in the setting of severe and critical COVID-19 and (2) the robust T cell and type I interferon-driven inflammatory and (3) humoral-driven immune complex mediated vasculitic cutaneous reactions observed with mild and moderate COVID-19. Presented are novel data on cutaneous vaccine reactions that manifest a clinical and morphologic parallel with similar eruptions observed in patients with mild and moderate COVID-19 and in some cases represent systemic eczematoid hypersensitivity reactions to a putative vaccine-based antigen versus unmasking subclinical hypersensitivity due to immune enhancing effects of the vaccine. Finally, we demonstrate for the first time the localization of human synthesized spike glycoprotein after the COVID-19 vaccine to the cutaneous and subcutaneous vasculature confirming the ability of SARS-CoV-2 spike glycoprotein to bind endothelium in the absence of intact virus.

Stable Display of Artificially Long Foreign Antigens on Chimeric Bamboo mosaic virus Particles.

Plant viruses can be genetically modified to generate chimeric virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo mosaic virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines.

Visualizing the ribonucleoprotein content of single bunyavirus virions reveals more efficient genome packaging in the arthropod host

Bunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.

Outbreak of Encephalitis Caused by Human Herpesvirus Type 1 (HSV-1) in NonHuman Captive Primates in São Paulo, SP, Brazil

Human herpesvirus type 1 (HSV-1), a Alphaherpesvirinae, has a broad range of cross-species infectivity with considerable variation in expression and disease severity among different hosts. Infection in humans, results in mild disease characterized by mucocutaneous lesions. In some species of nonhuman primates, however, the infection can be lethal. Transmission occurs through food contaminated by humans with labial herpes, offered to monkeys, constituting an important anthropozoonosis. In this study, we describe the occurrence of two outbreaks of encephalitis caused by HSV-1, occurring in the State of São Paulo, Brazil. The first outbreak affected a Brown howler monkey and three marmosets. The Brown howler monkey had tongue ulcers and enteritis and the marmosets were found dead without presenting clinical symptomatology. The second outbreak affected ten marmosets that were found dead suddenly in the enclosure without presenting symptoms or clinical signs. Organ fragments were processed for transmission electron microscopy, histopathology and inoculation in cell culture techniques. Ballooning degeneration, foci of monolymphocytic inflammatory reaction, corpuscle of eosinophilic inclusion, monolymphocytic meningitis and desquamative monolymphocytic enterocolitis, were the main lesions observed by histopathology. By the negative staining technique, enveloped and non-enveloped, herpesvirus particles were found, measuring 120-200 nm in diameter in all samples of organ fragments. The presence of aggregates formed by antigenantibody complex, characterized the positive result obtained in the immunoelectron microscopy technique for HSV-1. Using the immunocytochemistry technique, the antigen-antibody interaction was strongly enhanced by the colloidal gold particles over the herpesvirus, confirming the presence of HSV-1. Intranuclear incomplete particles measuring 80-100 nm in diameter and complete or enveloped scattered in the cytoplasm, were visualized in ultrathin sections of liver and cell culture measuring 140 nm of diameter. Immature particles budding from cell membranes were also observed. In viral isolation in VERO cells, typical cytopathic effect was observed in brain samples.

Droplet-vaporization behavior during plasma-assisted mist chemical vapor deposition of zinc oxide films

Droplet vaporization behavior during plasma-assisted mist chemical vapor deposition of zinc oxide films was investigated. The time required for a droplet to fully evaporate is simulated as a function of the initial droplet temperature, droplet-vapor interactions, and initial droplet size. Droplets injected into the plasma are vaporized by the heat flux from the plasma. It was found that micrometric size droplets evaporate completely within several microseconds due to heat flux from the plasma. Moreover, owing to differences in vaporization rates, solid particles were formed from some of the droplets during their passage through the plasma, but other droplets formed incomplete particles or remained as droplets due to incomplete evaporation. Particles formed in the plasma and subsequently deposited on a substrate maintained their shape.

Evolution of Intermediates during Capsid Assembly of Hepatitis B Virus with Phenylpropenamide-Based Antivirals.

Self-assembly of virus capsids is a potential target for antivirals due to its importance in the virus lifecycle. Here, we investigate the effect of phenylpropenamide derivatives B-21 and AT-130 on the assembly of hepatitis B virus (HBV) core protein. Phenylpropenamides are widely believed to yield assembly of spherical particles resembling native, empty HBV capsids. Because the details of assembly can be overlooked with ensemble measurements, we performed resistive-pulse sensing on nanofluidic devices with four pores in series to characterize the size distributions of the products in real time. With its single particle sensitivity and compatibility with typical assembly buffers, resistive-pulse sensing is well-suited for analyzing virus assembly in vitro. We observed that assembly with B-21 and AT-130 produced a large fraction of partially complete virus particles that may be on-path, off-path, or trapped. For both B-21 and AT-130, capsid assembly was more sensitive to disruption under conditions where the interprotein association energy was low at lower salt concentrations. Dilution of the reaction solutions led to the rearrangement of the incomplete particles and demonstrated that these large intermediates may be on-path, but are labile, and exist in a frustrated dynamic equilibrium. During capsid assembly, phenylpropenamide molecules modestly increase the association energy of dimers, prevent intermediates from dissociating, and lead to kinetic trapping where the formation of too many capsids has been initiated, which results in both empty and incomplete particles.

The immunological function of extracellular vesicles in hepatitis B virus-infected hepatocytes.

Hepatitis B virus (HBV) generates large amounts of complete and incomplete viral particles. Except for the virion, which acts as infectious particles, the function of those particles remains elusive. Extracellular vesicles (EVs) have been revealed to have biological functions. The EVs which size are less than 100 nm in diameter, were collected from HBV infected-patients. These vesicles contain, complete and incomplete virions, and exosomes, which have been recently shown to be critical as intercellular communicators. Here, the effects of the exosome, the complete, and the incomplete particles on the target cells were investigated. These particles are endocytosed by monocyte/macrophages and function primarily to upregulate PD-L1. The functions and composition of the EVs were affected by nucleotide reverse transcriptase inhibitors (NRTIs), suggesting that the EVs are involved in the pathogenesis of HBV hepatitis and clinical course of those patients treated by NRTIs.

Detection of size of manufactured sand particles based on digital image processing

The size distribution of manufactured sand particles has a significant influence on the quality of concrete. To overcome the shortcomings of the traditional vibration-sieving method, a manufactured sand casting/dispersing system was developed, based on the characteristics of the sand particle contours (as determined by backlit image acquisition) and an extraction mechanism. Algorithms for eliminating particles from the image that had be repeatedly captured, as well as for identifying incomplete particles at the boundaries of the image, granular contour segmentation, and the determination of an equivalent particle size, are studied. The hardware and software for the image-based detection device were developed. A particle size repeatability experiment was carried out on the single-grade sands, grading the size fractions of the manufactured sand over a range of 0.6–4.75 mm. A method of particle-size correction is proposed to compensate for the difference in the results obtained by the image-based method and those obtained by the sieving method. The experimental results show that the maximum repeatability error of single-grade fractions is 3.46% and the grading size fraction is 0.51%. After the correction of the image method, the error between the grading size fractions obtained by the two methods was reduced from 7.22%, 6.10% and 5% to 1.47%, 1.65%, and 3.23%, respectively. The accuracy of the particle-size detection can thus satisfy real-world measuring requirements.

Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients.

Extracellular HBV RNA has been detected in both HBV-replicating cell culture media and sera from chronic hepatitis B (CHB) patients, but its exact origin and composition remain controversial. Here, we demonstrated that extracellular HBV RNA species were of heterogeneous lengths, ranging from the length of pregenomic RNA to a few hundred nucleotides. In cell models, these RNAs were predominantly associated with naked capsids, although virions also harbored a minority of them. Moreover, HBV RNAs in hepatitis B patients' blood circulation were localized in unenveloped capsids in the form of capsid-antibody complexes (CACs) and in virions. Furthermore, we showed that extracellular HBV RNAs could serve as the template for viral DNA synthesis. In conclusion, extracellular HBV RNAs mainly consist of pgRNA or the pgRNA species degraded by the RNase H domain of the polymerase in the process of viral DNA synthesis and circulate as CACs and virions. Their presence in blood circulation of CHB patients may be exploited to develop novel biomarkers for HBV persistence.IMPORTANCE Although increasing evidence suggests the presence of extracellular HBV RNA species, their origin and molecular forms are still under debate. In addition to the infectious virions, HBV is known to secrete several species of incomplete viral particles, including hepatitis B surface antigen (HBsAg) particles, naked capsids, and empty virions, during its replication cycle. Here, we demonstrated that extracellular HBV RNAs were associated with naked capsids and virions in HepAD38 cells. Interestingly, we found that unenveloped capsids circulate in the blood of hepatitis B patients in the form of CACs and, together with virions, serve as vehicles carrying these RNA molecules. Moreover, extracellular HBV RNAs are heterogeneous in length and represent either pregenomic RNA (pgRNA) or products of incomplete reverse transcription during viral replication. These findings provide a conceptual basis for further application of extracellular RNA species as novel biomarkers for HBV persistence.

Fabrication of Hierarchical Co/MgO Catalyst for Enhanced CO2 Reforming of CH4 in a Fluidized‐Bed Reactor

This work reports facile fabrication of fluidizable hierarchical Co–MgO particles with high dispersion of Co nanoparticles (NPs) and high surface oxygen vacancies for matching with the fluidized‐bed reactor. The hierarchical Co–MgO catalyst exhibits higher activity and thermal stability for CH4–CO2 reforming than the conventional impregnation Co/MgO catalyst. The enhanced prevention of Co NPs sintering of the hierarchical Co–MgO catalyst is attributed to the multilayer structure and the exposed MgO (111) face, which creates high surface oxygen vacancies, induces high Co dispersion to restrain Co NPs sintering, and supplies high resistance to graphite carbon deposition. A formation mechanism for the hierarchical MgO structure is also proposed. This formation of incomplete rhombohedron MgO particles with an exposed octahedral MgO (111) face is attributed to the electrostatic interaction of OH−. The proposed approach to improving catalytic activity by creation of hierarchical MgO particles can be expanded to other metal oxide catalysts. © 2018 American Institute of Chemical Engineers AIChE J, 65: 120–131, 2019

Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-Free Hepatitis B Virions.

During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (i) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (ii) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required but could stimulate empty virion secretion. Also, substitutions in L that eliminated secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that blocked secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids and mature nucleocapsids interact with the S and L proteins during the formation of complete and empty virions.IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases, including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV-infected cells also secrete noninfectious, incomplete viral particles in large excess over the number of complete virions. In particular, the empty (or genome-free) virion shares with the complete virion the outer envelope and interior capsid but contains no genome. We have carried out a comparative study on the capsid and envelope requirements for the secretion of these two types of virion particles and uncovered both shared and distinct determinants on the capsid and envelope for their secretion. These results provide new information on HBV morphogenesis and have implications for efforts to develop empty HBV virions as novel biomarkers and a new generation of HBV vaccine.

The 3.3\u2009\xc5 structure of a plant geminivirus using cryo-EM

Geminiviruses are major plant pathogens that threaten food security globally. They have a unique architecture built from two incomplete icosahedral particles, fused to form a geminate capsid. However, despite their importance to agricultural economies and fundamental biological interest, the details of how this is realized in 3D remain unknown. Here we report the structure of Ageratum yellow vein virus at 3.3 Å resolution, using single-particle cryo-electron microscopy, together with an atomic model that shows that the N-terminus of the single capsid protein (CP) adopts three different conformations essential for building the interface between geminate halves. Our map also contains density for ~7 bases of single-stranded DNA bound to each CP, and we show that the interactions between the genome and CPs are different at the interface than in the rest of the capsid. With additional mutagenesis data, this suggests a central role for DNA binding-induced conformational change in directing the assembly of geminate capsids.

Human Embryonic Stem Cell-Derived Neurons Are Highly Permissive for Varicella-Zoster Virus Lytic Infection.

Varicella-zoster virus (VZV) is highly cell associated when grown in culture and has a much higher (4,000- to 20,000-fold increased) particle-to-PFU ratio in vitro than herpes simplex virus (HSV). In contrast, VZV is highly infectious in vivo by airborne transmission. Neurons are major targets for VZV in vivo; in neurons, the virus can establish latency and reactivate to produce infectious virus. Using neurons derived from human embryonic stem cells (hESC) and cell-free wild-type (WT) VZV, we demonstrated that neurons are nearly 100 times more permissive for WT VZV infection than very-early-passage human embryonic lung cells or MRC-5 diploid human fibroblasts, the cells used for vaccine production or virus isolation. The peak titers achieved after infection were ∼10-fold higher in human neurons than in MRC-5 cells, and the viral genome copy number-to-PFU ratio for VZV in human neurons was 500, compared with 50,000 for MRC-5 cells. Thus, VZV may not necessarily have a higher particle-to-PFU ratio than other herpesviruses; instead, the cells previously used to propagate virus in vitro may have been suboptimal. Furthermore, based on electron microscopy, neurons infected with VZV produced fewer defective or incomplete viral particles than MRC-5 cells. Our data suggest that neurons derived from hESC may have advantages compared to other cells for studies of VZV pathogenesis, for obtaining stocks of virus with high titers, and for isolating VZV from clinical specimens.IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox and shingles. Cell-free VZV has been difficult to obtain, both for in vitro studies and for vaccine production. While numerous cells lines have been tested for their ability to produce high titers of VZV, the number of total virus particles relative to the number of viral particles that can form plaques in culture has been reported to be extremely high relative to that in other viruses. We show that VZV grows to much higher titers in human neurons than in other cell types in vitro and that the number of total virus genomes relative to the number of viral particles that can form plaques in culture is much lower in human neurons than other cultured cells. These findings indicate that human neurons may be useful for studying VZV in vitro, for growing preparations of virus with high titers, and for isolating the virus from human samples.

A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid.

Hepatitis B virus (HBV) core protein is a model system for studying assembly and disassembly of icosahedral structures. Controlling disassembly will allow re-engineering the 120 subunit HBV capsid, making it a molecular breadboard. We examined removal of subunits from partially crosslinked capsids to form stable incomplete particles. To characterize incomplete capsids, we used two single molecule techniques, resistive-pulse sensing and charge detection mass spectrometry. We expected to find a binomial distribution of capsid fragments. Instead, we found a preponderance of 3 MDa complexes (90 subunits) and no fragments smaller than 3 MDa. We also found 90-mers in the disassembly of uncrosslinked HBV capsids. 90-mers seem to be a common pause point in disassembly reactions. Partly explaining this result, graph theory simulations have showed a threshold for capsid stability between 80 and 90 subunits. To test a molecular breadboard concept, we showed that missing subunits could be refilled resulting in chimeric, 120 subunit particles. This result may be a means of assembling unique capsids with functional decorations.