Small Virus Particles Research Articles

Non-Detection of HCMV Total Genomic DNA in Human Glioma Cells Genome.

To demonstrate if the human cytomegalovirus (HCMV) genome, that is involved in the pathogenesis of gliomas, is part of the genomic DNA of glioma cells or not. The study included U87MG glioblastoma cell culture and tumor samples from glioma patients. The genomic DNA of tumor samples and U87MG cells were extracted and real-time quantitative PCR was used to assess the presence of the human cytomegalovirus genomic DNA. Consequently, HCMV positivity was not detected in the tumor and cell line genomic DNA under the aforementioned experimental conditions. We found that the genomic DNA of all the samples was negative for HCMV genomic DNA. Thus, HCMV could not be detected in human glioma tumors and we put forward that HCMV genomic DNA was not incorporated into the genomic DNA of glioma cells. Thus, total viral DNA is not involved in the pathogenesis of glioma; however, small viral particles or specific genes might be incorporated into the genomic DNA of glioma cells, leading to cancer development. This prompts further studies for verification.

Development of a Specific Anti-capsid Antibody- and Magnetic Bead-Based Immunoassay to Detect Human Norovirus Particles in Stool Samples and Spiked Mussels via Flow Cytometry.

Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 105gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.

Social Intervention by the Numbers: Evidence Behind the Specific Public Health Guidelines in the COVID-19 Pandemic.

Social Intervention by the Numbers: Evidence Behind the Specific Public Health Guidelines in the COVID-19 Pandemic.

Cost-Effective Filtrating Suction to Evacuate Surgical Smoke in Laparoscopic and Robotic Surgery During the COVID-19 Pandemic.

The wide and fast spread of COVID-19 around the world has led to a dramatic increase in the need for protection products both for carers and for populations. Surgical team protection includes a systematic screening of patients, wearing protection devices by all the operating staff, and adequate management of aerosols. The risk of aerosol dispersal is particularly high during laparoscopic and robotic surgeries due to the interaction between circulating CO2 and surgical smoke that may contain small viral particles. To decrease the risk of virus transmission, many recommendations have been implemented including the use of integrated insufflation devices comprising smoke evacuation and filtration mode. Such devices are lacking in many centers around the world and to overcome this urgent unmet need, we designed a cost-effective filtrating suction as a more readily available alternative.

An Overview on the Role of Relative Humidity in Airborne Transmission of SARS-CoV-2 in Indoor Environments

COVID-19 disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, China and spread with an astonishing rate across the world. The transmission routes of SARS-CoV-2 are still debated, but recent evidence strongly suggests that COVID-19 could be transmitted via air in poorly ventilated places. Some studies also suggest the higher surface stability of SARS-CoV-2 as compared to SARS-CoV-1. It is also possible that small viral particles may enter into indoor environments from the various emission sources aided by environmental factors such as relative humidity, wind speed, temperature, thus representing a type of an aerosol transmission. Here, we explore the role of relative humidity in airborne transmission of SARS-CoV-2 virus in indoor environments based on recent studies around the world. Humidity affects both the evaporation kinematics and particle growth. In dry indoor places i.e., less humidity (< 40% RH), the chances of airborne transmission of SARS-CoV-2 are higher than that of humid places (i.e., > 90% RH). Based on earlier studies, a relative humidity of 40–60% was found to be optimal for human health in indoor places. Thus, it is extremely important to set a minimum relative humidity standard for indoor environments such as hospitals, offices and public transports for minimization of airborne spread of SARS-CoV-2.

Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles.

The nature and concentration of lipids and proteins at the surface of viruses are essential parameters for determining particle infectiveness. Historically, averaged bulk analysis of viral particles has been the primary method to quantitatively investigate these parameters, though this neglects heterogeneity within populations. Here we analyze the properties of Junin virus particles using a sensitive flow virometry assay and further sort virions, while conserving their infectivness. This method allows us to characterize the relationship between infectivity, virus size, and RNA content and to compare particles secreted by Vero cells with those from physiologically relevant human primary macrophages. Our study highlights significant differences in particle infectivity according to its nature, the type of producer cells and the lipid membrane composition at the budding site. Together, our results present the flow virometry assay as a powerful and versatile tool to define virus particle profiles.

Exposure to Influenza Virus Aerosols During Routine Patient Care

Defining dispersal of influenza virus via aerosol is essential for the development of prevention measures. During the 2010-2011 influenza season, subjects with influenza-like illness were enrolled in an emergency department and throughout a tertiary care hospital, nasopharyngeal swab specimens were obtained, and symptom severity, treatment, and medical history were recorded. Quantitative impaction air samples were taken not ≤0.305 m (1 foot), 0.914 m (3 feet), and 1.829 m (6 feet) from the patient's head during routine care. Influenza virus was detected by rapid test and polymerase chain reaction. Sixty-one of 94 subjects (65%) tested positive for influenza virus. Twenty-six patients (43%) released influenza virus into room air, with 5 (19%) emitting up to 32 times more virus than others. Emitters surpassed the airborne 50% human infectious dose of influenza virus at all sample locations. Healthcare professionals (HCPs) were exposed to mainly small influenza virus particles (diameter, <4.7 µm), with concentrations decreasing with increasing distance from the patient's head (P < .05). Influenza virus release was associated with high viral loads in nasopharyngeal samples (shedding), coughing, and sneezing (P < .05). Patients who reported severe illness and major interference with daily life also emitted more influenza virus (P < .05). HCPs within 1.829 m of patients with influenza could be exposed to infectious doses of influenza virus, primarily in small-particle aerosols. This finding questions the current paradigm of localized droplet transmission during non-aerosol-generating procedures.

Isolation of two Chinese bovine enteroviruses and sequence analysis of their complete genomes

In this study, RNA corresponding to bovine enterovirus (BEV) was detected in 24.6 % of faecal samples (17/69) from diarrheic and healthy cattle in six different areas in China by an RT-PCR screening method. Furthermore, two cytopathic agents, designated as BHM26 and BJ50, were isolated from the bovine diarrheic fecal samples. During passage in MA104 cells, ultrathin sections of virus-infected monolayers were examined using a transmission electron microscope, and a large number of symmetrical virus crystals were seen in the cytoplasm, with monomorphic small viral particles of 27-30 nm in diameter. The full-length RNA genomes were 7433 and 7416 nucleotides long, respectively, with a genome organization analogous to that of picornaviruses. Phylogenetic analysis of the VP1 and VP3 capsid protein coding sequences suggested that the viruses BHM26 and BJ50 belong to genotype 2 of the BEV cluster B (BEV-B). In addition, sequence comparisons of the 5′ and 3′ UTRs and P1, P2 and P3 subgenomic regions of the two isolates suggested that there were intergenotypic recombination events occurring during evolution of the BHM26 and BJ50 isolates.

Evidence for an enterovirus as the cause of encephalitis lethargica.

BackgroundThe epidemic of encephalitis lethargica (EL), called classical EL, was rampant throughout the world during 1917–1926, affecting half a million persons. The acute phase was lethal for many victims. Post-encephalitic parkinsonism (PEP) affected patients for decades. Our purpose was to investigate the cause of classical EL by studying the few available brain specimens. Cases of PEP and modern EL were also studied. Transmission electron microscopy (TEM) and immunohistochemistry were employed to examine brain from four classical EL cases, two modern EL cases and one PEP case.MethodsStandard methods for TEM, immunohistochemistry and RTPCR were applied.Results27 nm virus-like particles (VLP) were observed in the cytoplasm and nuclei of midbrain neurons in all classical EL cases studied. Large (50 nm) VLP and 27 nm intranuclear VLP were observed in the modern EL cases and the PEP case. Influenza virus particles were not found. VLP were not observed in the control cases. TEM of cell cultures inoculated with coxsackievirus B4 and poliovirus revealed both small and large intranuclear virus particles and small cytoplasmic particles, similar to the VLP in EL neurons. In the EL brains, nascent VLP were embedded in putative virus factories and on endoplasmic reticulum (ER). The VLP in the cases of classical EL survived, whereas ribosomes underwent autolysis due to the lack of refrigeration and slow formaldehyde fixation of whole brain. The VLP were larger than ribosomes from well preserved brain. Immunohistochemistry of classical EL cases using anti-poliovirus and anti-coxsackievirus B polyclonal antibodies showed significant staining of cytoplasm and nuclei of neurons as well as microglia and neuropil. Purkinje cells were strongly stained.A 97-bp RNA fragment of a unique virus was isolated from brain tissue from acute EL case #91558. Sequence analysis revealed up to 95% identity to multiple human Enteroviruses. Additional cases had Enterovirus positive reactions by real time PCR.ConclusionsThe data presented here support the hypothesis that the VLP observed in EL tissue is an Enterovirus.

Resolving structure and mechanical properties at the nanoscale of viruses with frequency modulation atomic force microscopy.

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25–50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called “dissipation channel” in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.

Tangential flow filtration for virus purification

Purification of virus particles and viral vectors for viral vaccines and gene therapy applications is a major large-scale separations challenge. Purification of parvovirus particles such as adeno-associated virus, the leading candidate for gene therapy applications, is particularly challenging given their small size, typically 18–26 nm. We have investigated the use of ultrafiltration for purification of Aedes aegypti densonucleosisvirus, a mosquito parvovirus. Four ultrafiltration membranes with molecular weight cut offs of 30, 50, 100 and 300 kDa have been tested in tangential flow mode. Experiments have also been conducted where part of the permeate is pumped co-current to the feed. This results in a more constant transmembrane pressure drop along the entire membrane. Maintaining a more constant transmembrane pressure results in improved passage of host cell proteins and smaller virus particles. Currently chromatographic separations such as ion exchange are used to remove DNA and host cell proteins. Our results indicate that by carefully selecting the molecular weight cut off of the membrane and maintaining a constant transmembrane pressure along the membrane, virus particles may be purified and concentrated by ultrafiltration. Reducing the impurity load to subsequent chromatographic steps could result in the use of smaller columns leading to cost savings.

Small round viruses: classification and role in food-borne infections.

Since the first observation of Norwalk virus in the electron microscope in 1972, many different small virus particles in the size range 20-40 nm have been described world-wide in association with outbreaks of gastroenteritis. Progress characterizing these agents has been hampered by the relatively small numbers of particles present in clinical material and the lack of success in culturing them. Although the relationship between some of these viruses remains confusing, a number of distinct groups has emerged, based on morphological features and limited physical data. Immuno-electron microscopy has proved valuable in detecting viruses but the addition of antibody can mask surface morphological features. Examination of viruses in negatively stained preparations without added antibody has revealed distinct morphological differences and viruses previously thought to be simply antigenic variants within the Norwalk group of viruses clearly belong to other groups. Preliminary evidence suggests that one human virus unrelated to Norwalk has a single-stranded DNA genome and is a parvovirus. Some groups have been implicated in outbreaks of food-borne gastroenteritis, particularly after the consumption of shellfish, and their role in other food-borne and water-borne outbreaks is being increasingly recognized.

Structure of Virioplankton in the Charente Estuary (France): Transmission Electron Microscopy versus Pulsed Field Gel Electrophoresis

Changes in the composition of viral communities were investigated along a salinity gradient and at different times by means of transmission electron microscopy (TEM) and pulsed field gel electrophoresis (PFGE). Samples were collected in fresh (Charente River), estuarine (Charente Estuary), and coastal (Pertuis d'Antioche, French Atlantic coast) waters. Both methods revealed similar patterns in viral community structure with a dominance of small viral particles (capsid and genome size). Viruses with a head size below 65 nm made up 71 +/- 5% of total virus-like particles, and virus-like genomes (VLG) below 100 kb accounted for 89 +/- 9% of total VLG. Despite this apparent stability of virioplankton composition over spatial scale (salinity gradient), the occurrence of large viruses (capsid and genome size) in estuarine and seawater samples indicated the presence of viral populations specific to a geographical location. Temporal changes in the structure (capsid and genome size) of viral communities were more pronounced than those reported at the spatial scale. From January to May 2003, seasonal changes in viral abundance and bacterial production occurred concomitantly with an increase in viral genomic diversity (richness), suggesting that virioplankton composition was strongly linked to changes in microbial activity and/or in the structure of the host communities. Although PFGE and TEM yielded complementary results in the description of virioplankton structures, it seems that the use of PFGE alone should be enough for the monitoring of community changes.

Ultrafiltration Membrane for Electrophoretic Capturing of Pathogens for AFM Imaging

This communication describes a simple and rapid technique for electrophoretically assisted capture of phages, viruses, and other pathogens on the surface of an ultrafiltration membrane that can be considered smooth at the nanoscale. The surface was prepared by coating commercial dialysis membrane with a micrometer-thick layer of cross-linked dextran or globular proteins. To ensure strong adherence of the coating, the surface of the dialysis membrane was activated in cold plasma. It was shown that the root-mean-square roughness of the coating was well below 1 nm when the polymer solution used for coating was allowed to slowly dry through a dialysis membrane left in direct contact with mica. Relatively small viral particles (e.g., fd phages 0.7 microm long and only 3.5 nm high in the dry state) are readily visible by AFM following electrophoretic capture from suspensions containing as few as 1 x 10(6) particles/mL onto membranes prepared as described.

Alteration of Sendai Virus Morphogenesis and Nucleocapsid Incorporation due to Mutation of Cysteine Residues of the Matrix Protein

The matrix (M) protein of Sendai virus (SeV) has five cysteine residues, at positions 83, 106, 158, 251, and 295. To determine the roles of the cysteine residues in viral assembly, we generated mutant M cDNA possessing a substitution to serine at one of the cysteine residues or at all of the cysteine residues. Some mutant M proteins were unstable when expressed in cultured cells, suggesting that cysteine residues affect protein stability, probably by disrupting the proper conformation. In an attempt to generate virus from cDNA, SeV M-C(83)S, SeV M-C(106)S, and SeV M-C(295)S were successfully recovered from cDNA, while recombinant SeVs possessing other mutations were not. SeV M-C(83)S and SeV M-C(106)S had smaller virus particles than did the wild-type SeV, whereas SeV M-C(295)S had larger and heterogeneously sized particles. Furthermore, SeV M-C(106)S had a significant amount of empty particles lacking nucleocapsids. These results indicate that a single-point mutation at a cysteine residue of the M protein affects virus morphology and nucleocapsid incorporation, showing direct involvement of the M protein in SeV assembly. Cysteine-dependent conformation of the M protein was not due to disulfide bond formation, since the cysteines were shown to be free throughout the viral life cycle.

Structural maturation of the transmissible gastroenteritis coronavirus.

During the life cycle of the transmissible gastroenteritis coronavirus (TGEV), two types of virus-related particles are detected in infected swine testis cells: large annular viruses and small dense viruses. We have studied the relationships between these two types of particles. Immunoelectron microscopy showed that they are closely related, since both large and small particles reacted equally with polyclonal and monoclonal antibodies specific for TGEV proteins. Monensin, a drug that selectively affects the Golgi complex, caused an accumulation of large annular viral particles in perinuclear elements of the endoplasmic reticulum-Golgi intermediate compartment. A partial reversion of the monensin blockade was obtained in both the absence and presence of cycloheximide, a drug that prevented the formation of new viral particles. After removal of monensin, the Golgi complex recovered its perinuclear location, and a decrease in the number of perinuclear large viral particles was observed. The release of small dense viral particles into secretory vesicles and the extracellular medium was also observed, as was a partial recovery of infectivity in culture supernatants. Small viral particles started to be seen between the third and the fourth Golgi cisternae of normally infected cells. All of these data strongly indicate that the large annular particles are the immature precursors of the small dense viruses, which are the infectious TGEV virions. The immature viral particles need to reach a particular location at the trans side of the Golgi stack to complete their morphological maturation.

The role of environmental contamination with small round structured viruses in a hospital outbreak investigated by reverse-transcriptase polymerase chain reaction assay

In May 1994 an outbreak of vomiting and diarrhoea occurred in a 28-bed long-stay ward for the mentally infirm. The predominant symptoms were vomiting, diarrhoea, malaise and abdominal pain lasting for approximately 12 h in most cases. The attack rate was 62% ( 13 21 ) for patients and 46% ( 16 35 ) for staff members. Infection control measures were implemented (containment of infectious individuals, hand hygiene among staff and environmental decontamination) and the ward was closed to admissions. Affected staff were excluded from contact with patients and their food until asymptomatic for 72 h. The outbreak lasted for 17 days. Faecal samples from nine symptomatic persons were negative for bacterial enteric pathogens, Giardia, Cryptosporidium and group A rotavirus. Electron microscopy of 12 faecal samples and one sample of vomitus revealed small round structured virus (SRSV) particles in one faecal sample. A further 30 faecal samples and seven vomitus samples were tested by reverse transcription polymerase chain reaction (RT-PCR) for SRSV of which 12 (40%) and 1 (14%) were positive respectively. Twenty-eight throat swabs from symptomatic and asymptomatic patients were collected, three (9·5%) of which were positive for SRSV by RT-PCR. Thirty-six environmental swabs were collected on the affected ward, and 11 (30%) were positive by RT-PCR. Positive swabs were from lockers, curtains and commodes and confined to the immediate environment of symptomatic patients. The distribution of contamination supports the rationale of cohorting sick patients.

Two types of virus-related particles are found during transmissible gastroenteritis virus morphogenesis.

The intracellular assembly of the transmissible gastroenteritis coronavirus (TGEV) was studied in infected swine testis (ST) cells at different postinfection times by using ultrathin sections of conventionally embedded infected cells, freeze-substitution, and methods for detecting viral proteins and RNA at the electron microscopy level. This ultrastructural analysis was focused on the identification of the different viral components that assemble in infected cells, in particular the spherical, potentially icosahedral internal core, a new structural element of the extracellular infectious coronavirus recently characterized by our group. Typical budding profiles and two types of virion-related particles were detected in TGEV-infected cells. While large virions with an electron-dense internal periphery and a clear central area are abundant at perinuclear regions, smaller viral particles, with the characteristic morphology of extracellular virions (exhibiting compact internal cores with polygonal contours) accumulate inside secretory vesicles that reach the plasma membrane. The two types of virions coexist in the Golgi complex of infected ST cells. In nocodazole-treated infected cells, the two types of virions coexist in altered Golgi stacks, while the large secretory vesicles filled with virions found in normal infections are not detected in this case. Treatment of infected cells with the Golgi complex-disrupting agent brefeldin A induced the accumulation of large virions in the cisternae that form by fusion of different membranous compartments. These data, together with the distribution of both types of virions in different cellular compartments, strongly suggest that the large virions are the precursors of the small viral particles and that their transport through a functional Golgi complex is necessary for viral maturation.

Outbreaks of gastroenteritis in elderly nursing homes and retirement facilities associated with human caliciviruses.

Eleven outbreaks of acute gastroenteritis, eight of which were in nursing homes or retirement facilities, were reported in virginia during the winter of 1993-1994. Serum samples (four outbreaks) and stool samples (two outbreaks) from involved people were tested for human calicivirus (HuCV) infection by enzyme immune assays (EIAs) using recombinant Norwalk virus (rNV) and Mexico virus (rMX) capsid antigens and reverse transcription-polymerase chain reaction (RT-PCR). Of the 31 pairs of acute and convalescent serum specimens tested, 24 had a fourfold or more titer increase to rMX and 4 responded to rNV. In all four outbreaks, the geometric mean titers (GMTs) against rMX were significantly higher than those against rNV in the convalescent, but not in the acute phase of illness. The antibody response to rMX among these patients was also higher than to rNV (summary mean 32-fold increase vs. 0.7-fold increase, respectively, P < .001). Antigen was detected in 5 of 21 stool specimens tested by the rMX EIA, RNA in 12 of 17 stool specimens tested by RT-PCR, and small round structured virus (SRSV) particles in 12 of 21 by electron microscopy (EM); none were positive by the rNV EIA. Sequence analysis of the RT-PCR-amplified products from the viral RNA polymerase region revealed 92-93% amino acid identity with Snow Mountain agent (SMA), 86% with MX, 58-59% with NV, and 31-32% with Sapporo HuCV, suggesting that these viruses belong to the SMA HuCV genogroup.

The removal of phages T1 and PP7, and poliovirus from fluids with hollow-fiber ultrafilters with molecular weight cut-offs of 50,000, 13,000, and 6000.

We tested the ability of hollow-fiber ultrafilters with molecular weight cut-offs (MWCOs) of 50,000, 13,000, and 6000 to remove and detect viral agents (phage T1, 50-150 nm; phage PP7, poliovirus, 28-30 nm) from ultrapure water, 0.85% saline with 1% trypticase soy broth, and Dulbecco's modified Eagle minimum essential medium with 10% fetal bovine serum (DMEM-10). Virus diluted in saline and DMEM-10 were tested to evaluate filter performance under conditions that minimize the adsorption of viral particles to the filter matrix. During filtration, the retentate was returned to the input reservoir, and the permeate was removed to a separate vessel. Thus, the virus concentration in the feed increased over the course of filtration. Filter performance was evaluated by comparing the concentration of infectious virus in the initial virus suspension with the virus concentration in the permeate and retentate. Very efficient removal of phages T1 and PP7 was observed with the filters with MWCOs of 13,000 and 6000 (titer reduction > 7 logs) for all three fluids tested. No poliovirus was detected in the permeate of the ultrafilters with MWCOs of 13,000 or 6000 (titer reduction > 6 logs). These results indicate that the ultrafilters with MWCOs of 13,000 and 6000 were very effective in removing small viral particles (25-30 nm) by size exclusion. The recovery efficiency of the virus in the retentate varied by fluid type. However, filtration with virus diluted in DMEM-10 resulted in consistent recovery of the viruses tested. The results suggest that these ultrafilters may have the dual potential of removing viral contaminants from fluids and concentrating virus in the retentate.