Uncovering the Rab5‐Independent Autophagic Trafficking of Influenza A Virus by Quantum‐Dot‐Based Single‐Virus Tracking

Autophagy is closely related to virus‐induced disease and a comprehensive understanding of the autophagy‐associated infection process of virus will be significant for developing more effective antiviral strategies. However, many critical issues and the underlying mechanism of autophagy in virus entry still need further investigation. Here, this study unveils the involvement of autophagy in influenza A virus entry. The quantum‐dot‐based single‐virus tracking technique assists in real‐time, prolonged, and multicolor visualization of the transport process of individual viruses and provides unambiguous dissection of the autophagic trafficking of viruses. These results reveal that roughly one‐fifth of viruses are ferried into cells for infection by autophagic machineries, while the remaining are not. A comprehensive overview of the endocytic‐ and autophagic‐trafficking process indicates two distinct trafficking pathway of viruses, either dependent on Rab5‐positive endosomes or autophagosomes, with striking similarities. Expressing dominant‐negative mutant of Rab5 suggests that the autophagic trafficking of viruses is independent on Rab5. The present study provides dynamic, precise, and mechanistic insights into the involvement of autophagy in virus entry, which contributes to a better understanding of the relationship between autophagy and virus entry. The quantum‐dot‐based single‐virus tracking is proven to hold promise for autophagy‐related fundamental research.     

Single‐Particle Tracking Reveals the Sequential Entry Process of the Bunyavirus Severe Fever with Thrombocytopenia Syndrome Virus

The Bunyavirales is one of the largest groups of RNA viruses, which encompasses many strains that are highly pathogenic to animals and humans. Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick‐borne bunyavirus that causes severe disease in humans, with a high fatality rate of up to 30%. To date, the entry process of bunyavirus infection remains obscure. Here, using quantum dot (QD)‐based single‐particle tracking and multicolor imaging, the dynamic molecular process of SFTSV entry and penetration is systematically dissected. The results show that internalization of SFTSV into host cells is initiated by recruiting clathrin onto the cell membrane for the formation of clathrin‐coated pits and further pinching off from the plasma membrane to form discrete vesicles. These vesicular carriers further deliver virions to Rab5+ early endosomes, and then to Rab7+ late endosomes. The intracellular transport of virion‐carrying endocytic vesicles is dependent first on actin filaments at the cell periphery, and then on microtubules toward the cell interior. The final fusion events occur at ≈15–60 min post‐entry, and are triggered by the acidic environment at ≈pH5.6 within the late endosomes. These results reveal the multistep SFTSV entry process and the dynamic virus–host interactions involved.     

Exploring Sialic Acid Receptors‐Related Infection Behavior of Avian Influenza Virus in Human Bronchial Epithelial Cells by Single‐Particle Tracking

Human respiratory tract epithelial cells are the portals of human infection with influenza viruses. However, the infection pathway of individual avian influenza viruses in human respiratory cells remains poorly reported so far. The single‐particle tracking technique (SPT) is a powerful tool for studying the transport mechanism of biomolecules in live cells. In this work, we use quantum dots to label avian influenza H9N2 virus and elaborate on the infection mechanism of the virus in human bronchial epithelial (HBE) cells using a three‐dimensional SPT technique. We have found that the H9N2 virus can infect HBE cells directly and the virus infection follows an actin filament‐ and microtubule‐dependent process with a three‐stage pattern. The transport behaviors show a high degree of consistency between the sialic acid receptors and the influenza virus. Real‐time SPT provides dynamic evidence of the sialic acid receptors‐related infection behavior of the avian influenza virus in live cells. The study of the influence of sialic acid receptors on virus infection may contribute to a better understanding of the cross‐species transmission of the avian influenza virus.     

Self-organization: making complex infectious viral particles from purified precursors

Viruses have served as excellent model systems in which to study biological self-organization. Purified virion structural constituents have been shown to self-assemble into particles that can initiate a productive infection in the host cell resulting in the release of progeny virions. Accumulating information on virus structures and assembly principles has revealed unexpected similarities between viruses that infect hosts as diverse as bacteria and humans, suggesting that these viruses had an early common ancestor. I will describe, in more detail, the assembly pathway of a complex double-stranded RNA bacterial virus. In this system, infectious viral particles are produced starting from purified protein and nucleic acid constituents through an elaborate self-assembly, RNA-packaging and synthesis pathway.     

Anchoring of nematic liquid crystals on viruses with different envelope structures

The ordering of synthetic liquid crystals near surfaces is known to be dependent on the nanoscopic structure and chemical functionality of surfaces. In this letter, we report that the orientational ordering of synthetic liquid crystals on surfaces decorated with viruses is also dependent on the structures of the viruses. Each of the four virions investigated had diameters of approximately 100 nm, but three of the viruses (influenza virus, La Crosse virus, and vesicular stomatitis virus) were enveloped in a lipid bilayer, whereas one virus (adenovirus) was not. We observed that lipid bilayer-enveloped viruses induce homeotropic (perpendicular) ordering of a nematic liquid crystal upon contact with the liquid crystal. In contrast, nonenveloped virus (adenovirus)-treated surfaces caused a near-planar orientation of the liquid crystal. We conclude that the homeotropic ordering of liquid crystals is a signature of the presence of enveloped viruses present on surfaces. These results suggest new approaches to the design of nanostructured materials that incorporate viruses as well as suggest methods that can be used to amplify the presence of nanoscopic virions into micrometer-sized domains of liquid crystal that can be optically probed.     

Importance of Viral Disease in Dairy Cow Fertility

Many viral diseases are endemic in cattle populations worldwide. The ability of many viruses to cross the placenta and cause abortions and fetal malformations is well understood. There is also significant evidence that viral infections have additional actions in dairy cows, which are reflected in reduced conception rates. These effects are, however, highly dependent on the time at which an individual animal first contracts the disease and are less easy to quantify. This paper reviews the evidence relating to five viruses that can affect fertility, together with their potential mechanisms of action. Acute infection with non-cytopathic bovine viral diarrhea virus (BVDV) in mid-gestation increases abortion rates or causes the birth of persistently infected calves. BVDV infections closer to the time of breeding can have direct effects on the ovaries and uterine endometrium, which cause estrous cycle irregularities and early embryo mortality. Fertility may also be reduced by BVDV-induced immunosuppression, which increases the susceptibility to bacterial infections. Bovine herpesvirus (BHV)-1 is most common in pre-pubertal heifers, and can slow their growth, delay breeding, and increase the age at first calving. Previously infected animals subsequently show reduced fertility. Although this may be associated with lung damage, ovarian lesions have also been reported. Both BHV-1 and BHV-4 remain latent in the host following initial infection and may be reactivated later by stress, for example associated with calving and early lactation. While BHV-4 infection alone may not reduce fertility, it appears to act as a co-factor with established bacterial pathogens such as Escherichia coli and Trueperella pyogenes to promote the development of endometritis and delay uterine repair mechanisms after calving. Both Schmallenberg virus (SBV) and bluetongue virus (BTV) are transmitted by insect vectors and lead to increased abortion rates and congenital malformations. BTV-8 also impairs the development of hatched blastocysts; furthermore, infection around the time of breeding with either virus appears to reduce conception rates. Although the reductions in conception rates are often difficult to quantify, they are nevertheless sufficient to cause economic losses, which help to justify the benefits of vaccination and eradication schemes.     

How sticky should a virus be? The impact of virus binding and release on transmission fitness using influenza as an example

Budding viruses face a trade-off: virions need to efficiently attach to and enter uninfected cells while newly generated virions need to efficiently detach from infected cells. The right balance between attachment and detachment—the right amount of stickiness—is needed for maximum fitness. Here, we design and analyse a mathematical model to study in detail the impact of attachment and detachment rates on virus fitness. We apply our model to influenza, where stickiness is determined by a balance of the haemagglutinin (HA) and neuraminidase (NA) proteins. We investigate how drugs, the adaptive immune response and vaccines impact influenza stickiness and fitness. Our model suggests that the location in the ‘stickiness landscape’ of the virus determines how well interventions such as drugs or vaccines are expected to work. We discuss why hypothetical NA enhancer drugs might occasionally perform better than the currently available NA inhibitors in reducing virus fitness. We show that an increased antibody or T-cell-mediated immune response leads to maximum fitness at higher stickiness. We further show that antibody-based vaccines targeting mainly HA or NA, which leads to a shift in stickiness, might reduce virus fitness above what can be achieved by the direct immunological action of the vaccine. Overall, our findings provide potentially useful conceptual insights for future vaccine and drug development and can be applied to other budding viruses beyond influenza.     

Imaging Viral Behavior in Mammalian Cells with Self‐Assembled Capsid–Quantum‐Dot Hybrid Particles

Unique spectral properties of quantum dots (QDs) enable ultrasensitive and long‐term biolabeling. Aiming to trace the infection, movement, and localization of viruses in living cells, QD‐containing virus‐like particles (VLPs) of simian virus 40 (SV40), termed SVLP‐QDs, are constructed by in vitro self‐assembly of the major capsid protein of SV40. SVLP‐QDs show homogeneity in size (≈24 nm), similarity in spectral properties to unencapsidated QDs, and considerable stability. When incubated with living cells, SVLP‐QDs are shown to enter the cells by caveolar endocytosis, travel along the microtubules, and accumulate in the endoplasmic reticulum. This process mimics the early infection steps of SV40. This is the first paradigm of imaging viral behaviors with encapsidated QDs in living cells. The method may provide a new alternative for various purposes, such as tracing viruses or viral components, targeted nanoparticle delivery, and probing of drug delivery.     

Electrochemical differentiation of epitope-specific aptamers

DNA aptamers are promising immunoshielding agents that could protect oncolytic viruses (OVs) from neutralizing antibodies (nAbs) and increase the efficiency of cancer treatment. In the present Article, we introduce a novel technology for electrochemical differentiation of epitope-specific aptamers (eDEA) without selecting aptamers against individual antigenic determinants. For this purpose, we selected DNA aptamers that can bind noncovalently to an intact oncolytic virus, vaccinia virus (VACV), which can selectively replicate in and kill only tumor cells. The aptamers were integrated as a recognition element into a multifunctional electrochemical aptasensor. The developed aptasensor was used for the linear quantification of the virus in the range of 500-3000 virus particles with a detection limit of 330 virions. Also, the aptasensor was employed to compare the binding affinities of aptamers to VACV and to estimate the degree of protection of VACV using the anti-L1R neutralizing antibody in a displacement assay fashion. Three anti-VACV aptamer clones, vac2, vac4, and vac6, showed the best immunoprotection results and can be applied for enhanced delivery of VACV. Another two sequences, vac5 and vac46, exhibited high affinities to VACV without shielding it from nAb and can be further utilized in sandwich bioassays.     

Spectroscopic evaluation of the effect of a red microalgal polysaccharide on herpes-infected Vero cells

The sulfated polysaccharide obtained from a species of red microalga has proved to be a potent antiviral agent against various members of the herpes family. In the present study, we used microscopic Fourier transform infrared spectroscopy (FT-IR) to investigate differences between normal cells, those infected with herpes viruses, and infected cells treated with red microalgal polysaccharide. FT-IR enables the characterization of cell or tissue pathology based on characteristic molecular vibrational spectra of the cells. The advantage of microscopic FT-IR spectroscopy over conventional FT-IR spectroscopy is that it facilitates inspection of restricted regions of cell cultures or tissue. Our results showed significant spectral differences at early stages of infection between infected and noninfected cells, and between infected cells treated with the polysaccharide and those not treated. In infected cells, there was an impressive decrease in sugar content and a considerable increase in phosphate levels in conjunction with the infection progress. Our results also proved that sugars penetrated and accumulated inside cells treated with the red microalgal polysaccharide. These could have been sugar fragments of low molecular weight present in the polysaccharide solution, despite purification by dialysis. Such sugar accumulation might be responsible for a breakdown in the internal steps of the viral replication cycle.     

Modelling dynamics of the type I interferon response to in vitro viral infection.

Innate immunity is crucial in the early stages of resistance to novel viral infection. The family of cytokines known as the interferons (IFNs) forms an essential component of this system: they are responsible for signalling that an infection is underway and for promoting an antiviral response in susceptible cells. We construct a spatial stochastic model, parameterized by experimental data and informed by analytic approximation, to capture the dynamics of virus-IFN interaction during in vitro infection of Madin-Darby bovine kidney cell monolayers by Herpes simplex virus 1. The dose dependence of infection progression, subsequent monolayer destruction and IFN-beta production are investigated. Implications for in vivo infections, in particular the priming of susceptible cells by IFN-beta during infection, are considered.     

Patterned assembly of genetically modified viral nanotemplates via nucleic acid hybridization

The patterning of nanoparticles represents a significant obstacle in the assembly of nanoscale materials and devices. In this report, cysteine residues were genetically engineered onto the virion surface of tobacco mosaic virus (TMV), providing attachment sites for fluorescent markers. To pattern these viruses, labeled virions were partially disassembled to expose 5' end RNA sequences and hybridized to virus-specific probe DNA linked to electrodeposited chitosan. Electron microscopy and RNAase treatments confirmed the patterned assembly of the virus templates onto the chitosan surface. These findings demonstrate that TMV nanotemplates can be dimensionally assembled via nucleic acid hybridization.     

Effect of different modes of viral spread on the dynamics of multiply infected cells in human immunodeficiency virus infection

Infection of individual cells with more than one HIV particle is an important feature of HIV replication, which may contribute to HIV pathogenesis via the occurrence of recombination, viral complementation and other outcomes that influence HIV replication and evolutionary dynamics. A previous mathematical model of co-infection has shown that the number of cells infected with i viruses correlates with the ith power of the singly infected cell population, and this has partly been observed in experiments. This model, however, assumed that virus spread from cell to cell occurs only via free virus particles, and that viruses and cells mix perfectly. Here, we introduce a cellular automaton model that takes into account different modes of virus spread among cells, including cell to cell transmission via the virological synapse, and spatially constrained virus spread. In these scenarios, it is found that the number of multiply infected cells correlates linearly with the number of singly infected cells, meaning that co-infection plays a greater role at lower virus loads. The model further indicates that current experimental systems that are used to study co-infection dynamics fail to reflect the true dynamics of multiply infected cells under these specific assumptions, and that new experimental techniques need to be designed to distinguish between the different assumptions.     

Hep-Pred: Hepatitis C Staging Prediction Using Fine Gaussian SVM

Hepatitis C is a contagious blood-borne infection, and it is mostly asymptomatic during the initial stages. Therefore, it is difficult to diagnose and treat patients in the early stages of infection. The disease’s progression to its last stages makes diagnosis and treatment more difficult. In this study, an AI system based on machine learning algorithms is presented to help healthcare professionals with an early diagnosis of hepatitis C. The dataset used for our Hep-Pred model is based on a literature study, and includes the records of 1385 patients infected with the hepatitis C virus. Patients in this dataset received treatment dosages for the hepatitis C virus for about 18 months. A former study divided the disease into four main stages. These stages have proven helpful for doctors to analyze the liver’s condition. The traditional way to check the staging is the biopsy, which is a painful and time-consuming process. This article aims to provide an effective and efficient approach to predict hepatitis C staging. For this purpose, the proposed technique uses a fine Gaussian SVM learning algorithm, providing 97.9% accurate results.     

Aptamer-based viability impedimetric sensor for viruses

The development of aptamer-based viability impedimetric sensor for viruses (AptaVISens-V) is presented. Highly specific DNA aptamers to intact vaccinia virus were selected using cell-SELEX technique and integrated into impedimetric sensors via self-assembly onto a gold microelectrode. Remarkably, this aptasensor is highly selective and can successfully detect viable vaccinia virus particles (down to 60 virions in a microliter) and distinguish them from nonviable viruses in a label-free electrochemical assay format. It also opens a new venue for the development of a variety of viability sensors for detection of many microorganisms and spores.     

Functionalized Fullerene for Inhibition of SARS-CoV-2 Variants

As virus outbreaks continue to pose a challenge, a nonspecific viral inhibitor can provide significant benefits, especially against respiratory viruses. Polyglycerol sulfates recently emerge as promising agents that mediate interactions between cells and viruses through electrostatics, leading to virus inhibition. Similarly, hydrophobic C₆₀ fullerene can prevent virus infection via interactions with hydrophobic cavities of surface proteins. Here, two strategies are combined to inhibit infection of SARS-CoV-2 variants in vitro. Effective inhibitory concentrations in the millimolar range highlight the significance of bare fullerene's hydrophobic moiety and electrostatic interactions of polysulfates with surface proteins of SARS-CoV-2. Furthermore, microscale thermophoresis measurements support that fullerene linear polyglycerol sulfates interact with the SARS-CoV-2 virus via its spike protein, and highlight importance of electrostatic interactions within it. All-atom molecular dynamics simulations reveal that the fullerene binding site is situated close to the receptor binding domain, within 4 nm of polyglycerol sulfate binding sites, feasibly allowing both portions of the material to interact simultaneously.     

Robust adaptive Lyapunov‐based control of hepatitis B infection

A new robust adaptive controller is developed for the control of the hepatitis B virus (HBV) infection inside the body. The non‐linear HBV model has three state variables: uninfected cells, infected cells and free viruses. A control law is designed for the antiviral therapy such that the volume of infected cells and the volume of free viruses are decreased to their desired values which are zero. One control input represents the efficiency of drug therapy in inhibiting viral production and the other control input represents the efficiency of drug therapy in blocking new infection. The proposed controller ensures the stability and robust performance in the presence of parametric and non‐parametric uncertainties (and/or bounded disturbances). The global stability and tracking convergence of the process are investigated by employing the Lyapunov theorem. The performance of the proposed controller is evaluated using simulations by considering different levels of uncertainties. Based on the obtained results, the proposed strategy can achieve its desired objectives with different cases of uncertainties.Inspec keywords: medical control systems, drugs, drug delivery systems, cellular biophysics, microorganisms, diseases, Lyapunov methods, adaptive controlOther keywords: robust adaptive Lyapunov‐based control, robust adaptive controller, hepatitis B virus infection, uninfected cells, free viruses, antiviral therapy, drug therapy, viral production, global stability, tracking convergence     

Development of methodology based on commercialized SERS-active substrates for rapid discrimination of Poxviridae virions

Surface-enhanced Raman spectroscopy (SERS) can be made an attractive approach for identification of Raman-active compounds and biological materials (i.e., toxins, viruses, or intact bacterial cells/spores) through development of reproducible, spatially uniform SERS-active substrates. Recently, reproducible (from substrate-to-substrate), spatially homogeneous (over large areas) SERS-active substrates have been commercialized and are now available in the marketplace. We have utilized these patterned surfaces to acquire SERS spectral signatures of intact bovine papular stomatitis, pseudocowpox, and Yaba monkey tumor viruses. Salient spectral signature features make it possible to discriminate among these genetically distinct Poxviridae-Chordopoxvirinae virions. In addition, partial least-squares, a multivariate calibration method, has been used to develop personal computer-borne algorithms useful for classification of unknown Parapoxvirus (e.g., bovine papular stomatitis virus and pseudocowpox virus) samples based solely on SERS spectral signatures. To our knowledge, this is the first report detailing application of these commercial-off-the-shelf (COTS) SERS-active substrates to identification of intact poxviruses.     

High-throughput detection and sizing of individual low-index nanoparticles and viruses for pathogen identification

Rapid, chip-scale, and cost-effective single particle detection of biological agents is of great importance to human health and national security. We report real-time, high-throughput detection and sizing of individual, low-index polystyrene nanoparticles and H1N1 virus. Our widefield, common path interferometer detects nanoparticles and viruses over a very large sensing area, orders of magnitude larger than competing techniques. We demonstrate nanoparticle detection and sizing down to 70 nm in diameter. We clearly size discriminate nanoparticles with diameters of 70, 100, 150, and 200 nm. We also demonstrate detection and size characterization of hundreds of individual H1N1 viruses in a single experiment.     

High-speed mass measurement of nanoparticle and virus

Until now, there have been no relatively easy methods to measure the mass and mass distributions of nanoparticles/viruses. In this work, we report the first set of measurements of mass and mass distributions for nanoparticles/viruses using a novel mass spectrometry technology. In the past, mass spectrometry was typically used to measure the mass of a particle or molecule with a mass less than 1,000,000 Da. We developed cell mass spectrometry that can measure the mass of a cell or a microparticle. Nevertheless, there is a gap for mass measurement methods in the mass region of a nanoparticle or virus (1 MDa to 1 GDa). Here, we developed a nanoparticle/virus mass spectrometry technique to make rapid and accurate mass and mass distribution measurements of nanoparticles/viruses. This technique should be valuable for the quality control of nanoparticle production and the identification of various viruses. In the future, this method can also serve to monitor drug delivery when nanoparticles are used as carriers. Furthermore, it may be possible to measure the degree of infection by measuring the number of viruses in specific cells or in plasma.