Single Virus Research Articles

Trapping and Detection of Single Viruses in an Optofluidic Chip.

Accurate single virus detection is critical for disease diagnosis and early prevention, especially in view of current pandemics. Numerous detection methods have been proposed with the single virus sensitivity, including the optical approaches and immunoassays. However, few of them hitherto have the capability of both trapping and detection of single viruses in the microchannel. Here, we report an optofluidic potential well array to trap nanoparticles stably in the flow stream. The nanoparticle is bound with single viruses and fluorescence quantum dots through an immunolabeling protocol. Single viruses can be swiftly captured in the microchannel by optical forces and imaged by a camera. The number of viruses in solution and on each particle can be quantified via image processing. Our method can trap and detect single viruses in the 1 mL serum or water in 2 h, paving an avenue for the advanced, fast, and accurate clinical diagnosis, as well as the study of virus infectivity, mutation, drug inhibition, etc.

1774P Exploration of torque teno virus (TTV) levels as marker for immune suppression during systemic therapy in advanced solid cancer patients

Torque teno virus (TTV) is an apathogenic non-enveloped, circular single strand DNA virus and is used as a marker for the immune suppression status after solid organ transplantation. We investigated the role of TTV as possible marker for monitoring the immune function and changes in metastatic solid cancer patients during systemic therapies. TTV level quantification was performed by polymerase chain reaction of full blood samples from newly-diagnosed therapy-naïve metastatic solid cancer patients before the first application of systemic therapy and at the following re-staging after 1-4 months. Systemic inflammation markers (neutrophil-to-lymphocyte ratio, leucocyte-to-lymphocyte ratio, platelet-to-lymphocyte ratio, monocyte-to-lymphocyte ratio) were obtained. 71 patients (31/71, 43.7% females; 40/71, 56.3% males) with a median age of 64 years (range 27-89) with longitudinal TTV levels at baseline (BL) and the following re-staging were included in the analyses. The most common tumor entities included lung cancer (27/7; 38.0%), pancreatic cancer (9/71; 12.7%), sarcomas (7/71; 9.9%) and renal cell carcinoma (5/71; 7.0%). TTV levels at BL were not associated with age or cancer type (p>0.05). BL TTV levels were numerically higher in patients achieving a therapy response (median TTV level 3.5E+5) compared to patients showing disease progression at the following re-staging (median TTV level 1.2E+5; Mann-Whitney U test, p=0.071). There was no association of BL TTV levels with BL systemic inflammation markers (p>0.05). Thirty-one/71 (43.7%) patients showed a significant change (±1 log level) of TTV levels during systemic therapy (6/31, 19.4% patients with significant increase; 25/31, 80.6% patients with significant decrease). Increase or decrease of TTV levels was not associated with type of systemic therapy, therapy response nor with change of systemic inflammation markers (p>0.05). Significant changes of TTV levels could be detected in a high portion of our cohort of advanced solid cancer patients under systemic therapy. Further longitudinal observation is warranted in order to specify the role of TTV levels as potential new immunological marker in cancer patients.

Jamestown Canyon and snowshoe hare virus seroprevalence in New Brunswick.

Jamestown Canyon virus (JCV) and snowshoe hare virus (SSHV) are wide-ranging mosquito-borne arboviruses in the California serogroup viruses (CSGV) that are known to circulate in New Brunswick. Despite potential for debilitating central nervous system manifestations, the prevalence of human exposure to these viruses in New Brunswick is unknown. The goal of this study was to quantify rates of human exposure in New Brunswick to these neglected arboviruses. A retrospective, anonymized provincial serosurvey was performed using a stratified random sample of residual sera submitted between May 2015 and August 2016. To determine the seroprevalence of JCV and SSHV, competitive enzyme-linked immunosorbent assay-positive samples were confirmed positive using plaque-reduction neutralization testing (PRNT). A total of 452 serum samples were screened. The seroprevalence of antibodies against CSGV was estimated to be 31.6% (95% CI 27.4% to 36.1%) with 143 positive samples. PRNT results indicated that most single virus exposures were due to JCV (38 of 143; 26.6%) rather than SSHV (3 of 143; 2.1%). The species of CSGV, to which the remaining 102 seropositive people were exposed, could not be precisely determined. The prevalence of human exposure to CSGV is high but comparable to rates observed in other Atlantic Canadian jurisdictions. Studies such as this provide important baseline epidemiological data regarding the risk of exposure to these neglected arboviruses. SSHV and JCV should be considered in the differential diagnosis for undiagnosed febrile and neuroinvasive illness during mosquito season, particularly when testing for common aetiologies is negative or inconclusive.

Compact RNA editors with small Cas13 proteins.

CRISPR-Cas13 systems have been developed for precise RNA editing, and can potentially be used therapeutically when temporary changes are desirable or when DNA editing is challenging. We have identified and characterized an ultrasmall family of Cas13b proteins-Cas13bt-that can mediate mammalian transcript knockdown. We have engineered compact variants of REPAIR and RESCUE RNA editors by functionalizing Cas13bt with adenosine and cytosine deaminase domains, and demonstrated packaging of the editors within a single adeno-associated virus.

Rapid detection of neutralizing antibodies to SARS-CoV-2 variants in post-vaccination sera.

The uncontrolled spread of the COVID-19 pandemic has led to the emergence of different SARS-CoV-2 variants across the globe. The ongoing global vaccination strategy to curtail the COVID-19 juggernaut, is threatened by the rapidly spreading Variants of Concern (VOC) and other regional mutants, which are less responsive to neutralization by infection or vaccine derived antibodies. We have previously developed the hiVNT system which detects SARS-CoV-2 neutralizing antibodies in sera in less than three hours. In this study, we modify the hiVNT for rapid qualitative screening of neutralizing antibodies (nAb) to multiple VOC of SARS-CoV-2, and assess the neutralizing efficacy of the BNT162b2 mRNA vaccine on seven epidemiologically relevant SARS-CoV-2 variants. Here we show that the BNT162b2 mRNA vaccine can activate humoral immunity against the major SARS-CoV-2 mutants that are currently in circulation. Albeit a small sample size, we observed that one dose of vaccine was sufficient to elicit a protective humoral response in previously infected people. Using a panel of seven SARS-CoV-2 variants and a single prototype virus, our modified hiVNT would be useful for large-scale community wide testing to detect protective immunity that may confer vaccine/immune passport in the ongoing COVID-19 pandemic.

Nanoscale Wetting of Single Viruses.

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ~1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

In-situ quantitation of genome release of Japanese encephalitis viruses by quantum dot-based single-virus tracking

The fusion of the viral membrane and genome release are hallmark events of enveloped virus infections. However, the related dynamic mechanisms of most viruses remain poorly understood. In this study, we combine single-virus tracking with the in-situ pH quantification of the dual-labeled Japanese encephalitis virus (JEV) to label its viral envelope and RNA by using quantum dots and pH-sensitive dyes, respectively. This robust technique allows us to visualize and quantify the process of genome release at the level of a single virus in real time, and to thus collect more information on the dynamics of genome release. The results of our experiments show that the infection pathway of the JEV is dependent on actin and microtubules, and viral fusion events predominantly occur in maturing endosomes that represent the intermediate stage between early and late endosomes. The kinetic insights gained in this study address long-standing questions regarding where and how the JEV harnesses endosomal compartments and releases its genome for replication, and are expected to facilitate a comprehensive understanding of the infection mechanism of JEV.

Mirtron-mediated RNA knockdown/replacement therapy for the treatment of dominant retinitis pigmentosa

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.

2020 Hindsight: Should evolutionary virologists have expected the unexpected during a pandemic?

2020 Hindsight: Should evolutionary virologists have expected the unexpected during a pandemic?

Implementation Of Health Protocol In Preventing The Spread Of The Covid 19 Virus

Covid-19 is a positive single strain RNA virus that infects the respiratory tract. This virus spreads quickly, the only way is to prevent it. As time progressed, the conditions of transmission became more widespread and took many victims, ranging from adults, teenagers to children. This virus can attack anyone, this virus can also attack children with special needs. Children with special needs are children with special characteristics that are different from children in general without always showing mental, emotional, or physical disabilities. One of them is mental retardation. The more widespread the spread of the COVID-19 virus, the application and knowledge of health protocols must also touch the mentally retarded. This obstacle will affect them to adjust to the implementation of health protocols every day and homework for SLB C Muzdalifah to provide a safe place for students. Prepare students and teaching units in the implementation of New Normal Education. And Educate students so that they apply health protocols at all times. The approach method used in this community service is socialization through posters of health protocols and placement of hand sanitizers. Information on preventing the spread of covid 19 through posters and placing hand sanitisers in the SLB C Muzdalifah area as a reminder and teaching regularity in implementing health protocols. This information is expected to become a habit in the New Normal.

Rotavirus Binding to Cell Surface Receptors Directly Recruiting α2 Integrin

Rotavirus interactions with endogenous cell surface receptors are of fundamental interest in virology and medicine; however, the evidence of rotavirus directly binding to the receptors and the consequent dynamic behaviors are still elusive. Force–distance curve‐based atomic force microscopy allows for the extraction of biophysical properties underlying binding of single virions to receptors and clarification of the dynamics of rotavirus–receptor interactions. Unfortunately, this method is time‐consuming due to the lack of automation when analyzing large data sets. Herein, rotavirus–receptor interactions and early endocytosis behaviors using automated high‐throughput analysis are examined. It is demonstrated that rotavirus binds to α‐linked sialic acid and α2β1 integrin. The effect of trypsinization is investigated on the capsid protein VP4 binding to the receptors. Using fluidic force microscopy, it is demonstrated that the interaction leads to α2 integrin recruitment to the cell‐bound rotavirus on the plasma membrane. Further, it is illustrated that an integrin‐derived peptide can impede binding and alter downstream dynamics. Taken together, these results open a new understanding of the infection mechanism of rotavirus and suggest a novel inhibitory peptide against rotavirus binding.

Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress.

The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.

African Horse Sickness Virus Serotype 1 on Horse Farm, Thailand, 2020.

To investigate an outbreak of African horse sickness (AHS) on a horse farm in northeastern Thailand, we used whole-genome sequencing to detect and characterize the virus. The viruses belonged to serotype 1 and contained unique amino acids (95V,166S, 660I in virus capsid protein 2), suggesting a single virus introduction to Thailand.

Self-Assembled Chromogenic Polymeric Nanoparticle-Laden Nanocarrier as a Signal Carrier for Derivative Binary Responsive Virus Detection

In the current biosensor, the signal generation is limited to single virus detection in the reaction chamber. An adaptive strategy is required to enable the recognition of multiple viruses for diagnostics and surveillance. In this work, a nanocarrier is deployed to bring specific signal amplification into the biosensor, depending on the target viruses. The nanocarrier is designed using pH-sensitive polymeric nanoparticle-laden nanocarriers (PNLNs) prepared by sequential nanoprecipitation. The nanoprecipitation of two chromogens, phenolphthalein (PP) and thymolphthalein (TP), is investigated in three different solvent systems in which PNLNs demonstrate a high loading of the chromogen up to 59.75% in dimethylformamide (DMF)/dimethyl sulfoxide (DMSO)/ethanol attributing to the coprecipitation degree of the chromogens and the polymer. The PP-encapsulated PNLNs (PP@PNLNs) and TP-encapsulated PNLNs (TP@PNLNs) are conjugated to antibodies specific to target viruses, influenza virus A subtype H1N1 (IV/A/H1N1) and H3N2 (IV/A/H3N2), respectively. After the addition of anti-IV/A antibody-conjugated magnetic nanoparticles (MNPs) and magnetic separation, the enriched PNLNs/virus/MNPs sandwich structure is treated in an alkaline solution. It demonstrates a synergy reaction in which the degradation of the polymeric boundary and the pH-induced colorimetric development of the chromogen occurred. The derivative binary biosensor shows feasible detection on IV/A with excellent specificities of PP@PNLNs on IV/A/H1N1 and TP@PNLNs on IV/A/H3N2 with LODs of 27.56 and 28.38 fg mL-1, respectively. It intrigues the distinguished analytical signal in human serum with a variance coefficient of 25.8% and a recovery of 93.6-110.6% for one-step subtype influenza virus detection.

Label-free detection of virus-like particles employing rotationally symmetric nanowire array based whispering gallery and quasi-whispering gallery resonant modes onto a silicon platform.

In spite of tremendous advancements in modern diagnostics, there is a dire need for reliable, label-free detection of highly contagious pathogens like viruses. In view of the limitations of existing diagnostic techniques, the present theoretical study proposes a novel scheme of detecting virus-like particles employing whispering gallery and quasi-whispering gallery resonant modes of a composite optical system. Whereas whispering gallery mode (WGM) resonators are conventionally realized using micro-disk, -ring, -toroid or spherical structures, the present study utilizes a rotationally symmetric array of silicon nanowires which offers higher sensitivity compared to the conventional WGM resonator while detecting virus-like particles. Notwithstanding the relatively low quality factor of the system, the underlying multiple-scattering mediated photon entrapment, coupled with peripheral total-internal reflection, results in high fidelity of the system against low signal-to-noise ratio. Finite difference time domain based numerical analysis has been performed to correlate resonant modes of the array with spatial location of the virus. The correlation has been subsequently utilized for statistical analysis of simulated test cases. Assuming detection to be limited by resolution of the measurement system, results of the analysis suggest that for only about 5% of the simulate test cases the resonant wavelength shift lies within the minimum detection range of 0.001-0.01 nm. For a single virus of 160 nm diameter, more than 8 nm shift of the resonant mode and nearly 100% change of quality factor are attained with the proposed nanowire array based photonic structure.

The spatial-temporal dynamics of respiratory syncytial virus infections across the east-west coasts of Australia during 2016-17.

Respiratory syncytial virus (RSV) is an important human respiratory pathogen. In temperate regions, a distinct seasonality is observed, where peaks of infections typically occur in early winter, often preceding the annual influenza season. Infections are associated with high rates of morbidity and mortality and in some populations exceed that of influenza. Two subtypes, RSV-A and RSV-B, have been described, and molecular epidemiological studies have shown that both viruses mostly co-circulate. This trend also appears to be the case for Australia; however, previous genomic studies have been limited to cases from one Eastern state—New South Wales. As such, the broader spatial patterns and viral traffic networks across the continent are not known. Here, we conducted a whole-genome study of RSV comparing strains across eastern and Western Australia during the period January 2016 to June 2017. In total, 96 new RSV genomes were sequenced, compiled with previously generated data, and examined using a phylodynamic approach. This analysis revealed that both RSV-A and RSV-B strains were circulating, and each subtype was dominated by a single genotype, RSV-A ON1-like and RSV-B BA10-like viruses. Some geographical clustering was evident in strains from both states with multiple distinct sub-lineages observed and relatively low mixing across jurisdictions, suggesting that endemic transmission was likely seeded from imported, unsampled locations. Overall, the RSV phylogenies reflected a complex pattern of interactions across multiple epidemiological scales from fluid virus traffic across global and regional networks to fine-scale local transmission events.

A New Infectious Unit: Extracellular Vesicles Carrying Virus Populations.

Viral egress and transmission have long been described to take place through single free virus particles. However, viruses can also shed into the environment and transmit as populations clustered inside extracellular vesicles (EVs), a process we had first called vesicle-mediated en bloc transmission. These membrane-cloaked virus clusters can originate from a variety of cellular organelles including autophagosomes, plasma membrane, and multivesicular bodies. Their viral cargo can be multiples of nonenveloped or enveloped virus particles or even naked infectious genomes, but egress is always nonlytic, with the cell remaining intact. Here we put forth the thesis that EV-cloaked viral clusters are a distinct form of infectious unit as compared to free single viruses (nonenveloped or enveloped) or even free virus aggregates. We discuss how efficient and prevalent these infectious EVs are in the context of virus-associated diseases and highlight the importance of their proper detection and disinfection for public health.

Burden of Respiratory Syncytial Virus Associated Severe Pneumonia in Hospitalized Children.

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory infections. We examined the burden of RSV-associated severe community-acquired pneumonia among hospitalized children and factors that predict RSV etiology. A hospital-based prospective study examined children below five years of age admitted with radiologically confirmed severe or very severe pneumonia in two tertiary care centers in Sri Lanka. Nasopharyngeal secretions (NPS) were tested for 19 viruses by multiplex RT-PCR. Univariate and multivariate analysis was performed to determine whether RSV etiology could be predicted based on clinical, sociodemographic, environmental, radiological, and laboratory parameters. A total of 108 children with severe or very severe were included in the study. At least one virus was found in NPS in 92.5% of children. Forty-six children had RSV (+) pneumonia. Mean RSV proportion was 42.6% (95% CI: 33.1-52.5%, p value = 0.149). RSV as a single virus was found in 41.3% (19/46). The children with RSV (+) pneumonia were younger (p = 0.026) and had lower C-reactive protein (p = 0.003) and household crowding (p = 0.012) than the RSV (-) group, after controlling for confounding covariates. In conclusion, the present study demonstrated that respiratory syncytial virus was the commonest virus associated with CAP in children under five years. Younger age, crowded housing, and lower C-reactive protein levels were predictors of severe RSV-associated pneumonia.

Robust molecular detection of the new Tomato brown rugose fruit virus in infected tomato and pepper plants from Turkey

Tomato brown rugose fruit virus (ToBRFV) causes severe fruit loss in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants. It is an emerging Tobamovirus that is spreading globally. The major challenge is to develop a reliable method for the detection of the virus, and to better characterize the symptoms it causes. The aims of this study, therefore, were to characterize the symptom development on tomato and pepper plants, and to establish a reliable detection method for the virus. Following infection of the tomato and pepper plants with ToBRFV, the leaves turned chlorotic, mosaic or mottled, while the fruit became rugose, necrotic and marbled, and showed discoloration with yellow or brown spots. Transmission electron microscopy (TEM) revealed single rod-like virus particles characteristic of the Tobamoviruses. Classical reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR) with specific primers and probes confirmed that the virus is ToBRFV. We found that the resistance genes from tomato, Tm-22, and pepper, L1, L2, L3 and L4, did not confer resistance to ToBRFV. Here, we present a PCR-based method as a diagnostic test for detecting ToBRFV in infected seeds. This method will help to prevent further spread of the virus in commercial seeds.