Non-enveloped Viruses Research Articles

Simultaneous extraction and detection of DNA and RNA from viruses, prokaryotes, and eukaryotes in wastewater using a modified COPMAN

Wastewater surveillance can offer a comprehensive grasp of infectious disease prevalence and human health because wastewater contains various human-derived microbial pathogens, including viruses, bacteria, and fungi. However, methods capable of simultaneous detection of multiple groups of targets in the automated systems and large-scale surveillance are still under development. Here, we demonstrated the modification, involving the addition of bead-beating, to the existing COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater) enabled enhanced detection of various microorganisms, including SARS-CoV-2. The modified method, termed bead-beating COPMAN (BB-COPMAN), was evaluated through spike-and-recovery experiments and comparative analysis against three previously reported methods for simultaneous DNA/RNA detection. Our study targeted a range of microorganisms, including enveloped and non-enveloped RNA viruses (SARS-CoV-2, PMMoV), a DNA virus (crAssphage), archaea, gram-negative and gram-positive bacteria (E. coli, Lachnospiraceae), antibiotic resistance gene (ampC), and fungi (Candida albicans). The recovery rates of BB-COPMAN for gram-negative and gram-positive bacteria were 17 and 2.1-fold higher, respectively, compared to the method for DNA/RNA detection. Additionally, BB-COPMAN exhibited the highest extraction efficiency among the tested methods, achieving 1.2–5.7 times more DNA and 1.1–69 times more RNA yield on average. BB-COPMAN allowed the detection of SARS-CoV-2 from all nine samples and PMMoV at concentrations 39–97 times higher than other methods. Moreover, BB-COPMAN detected larger amounts of DNA for four out of six DNA targets than the previously reported DNA/RNA detection method. These results demonstrated that BB-COPMAN enables enhanced detection of multiple targets in a single flow of nucleic acid extraction, making the method well-suited for automated systems. In conclusion, BB-COPMAN is a promising method in wastewater surveillance for assessing the prevalence of wide range of pathogenic microorganisms.

Effect of the Functional VP1 Unique Region of Human Parvovirus B19 in Causing Skin Fibrosis of Systemic Sclerosis.

Human parvovirus B19 (B19V) is a single-stranded non-enveloped DNA virus of the family Parvoviridae that has been associated with various autoimmune disorders. Systemic sclerosis (SSc) is an autoimmune connective tissue disorder with high mortality and has been linked to B19V infection. However, the precise mechanism underlying the B19V contribution to the development of SSc remains uncertain. This study investigated the impacts of the functional B19V-VP1 unique region (VP1u) in macrophages and bleomycin (BLE)-induced SSc mice. Cell experimental data showed that significantly decreased viability and migration of both B19V-VP1u-treated U937 and THP-1 macrophages are detected in the presence of celastrol. Significantly increased MMP9 activity and elevated NF-kB, MMP9, IL-6, TNF-α, and IL-1β expressions were detected in both B19V-VP1u-treated U937 and THP-1 macrophages. Conversely, celastrol revealed an inhibitory effect on these molecules. Notably, celastrol intervened in this pathogenic process by suppressing the sPLA2 activity of B19V-VP1u and subsequently reducing the inflammatory response. Notably, the administration of B19V-VP1u exacerbated BLE-induced skin fibrosis in mice, with augmented expressions of TGF-β, IL-6, IL-17A, IL-18, and TNF-α, ultimately leading to α-SMA and collagen I deposits in the dermal regions of BLE-induced SSc mice. Altogether, this study sheds light on parvovirus B19 VP1u linked to scleroderma and aggravated dermal fibrosis.

Household dishwashing detergents efficiently inactivate HSV-1, but not the non-enveloped viruses HAV and MNV, while all viruses were removed from glass by manual scrubbing

This study investigated the stability of herpes simplex virus 1 (HSV-1), hepatitis A virus (HAV) and murine norovirus (MNV) on glass, their inactivation by three household dishwashing detergents and during a glass rinsing process. After drying on glass and storage in darkness, virus titers decreased by 4.7, 5.5 and 4.5 log10 plaque forming units (PFU) in 21, 28 and 14 days for HSV-1, HAV and MNV, respectively. Daylight storage resulted in shorter survival times. While HAV and MNV titers were not significantly affected by 60 s incubation in dishwashing detergents at up to 43 °C, HSV-1 titer decreased significantly by > 4 log10 PFU/mL at the same conditions with each detergent used. After cleaning artificially contaminated drinking glasses with a manual glass scrubbing device, HSV-1, HAV and MNV titers decreased significantly by 4.4 ± 0.1 to 5.3 ± 0.0 log10 PFU. Our study shows, that all tested viruses can remain infectious after drying on glass up to several weeks. Household dishwashing detergents efficiently inactivate the enveloped virus HSV-1, but not the non-enveloped viruses HAV and MNV. The applied manual glass rinsing procedure efficiently removed all three viruses from drinking glasses, probably due to the combination of chemical and mechanical treatments.

Electron probe microanalysis and gene expression analysis of melanization caused by summer atrophy virus in the Akoya pearl oyster (Pinctada fucata)

The Akoya pearl oyster, Pinctada fucata, is the major species used for pearl culture in Japan. However, a massive mortality of juvenile and mantle atrophy of adult P. fucata have occurred during summer in Japan's major pearl production areas since 2019, and the symptoms were named “summer atrophy,” where a brown substance was deposited on the inner surface of their shell and mainly at the edge of the mantle. Summer atrophy is caused by a non-enveloped virus with a diameter of <100 nm. This study aimed to reveal whether the deposits are melanin and determine the gene expression in the mantle with the deposition through an infectious experiment. The body fluid of diseased P.fucata containing the causative virus was inoculated into the adductor muscle of three-year-old P. fucata. P. fucata were sampled at 0 (before injection), 1, 3, 6, 10, 14, 21, and 35 days post-injection. First, an electron probe microanalyzer was used to observe the detailed structure and material composition of the deposits of experimentally and spontaneously diseased P. fucata. Protuberances and wrinkled structures differed from a normal inner surface shell. Cluster analysis of these elemental composition ratios revealed that the brown deposits formed a cluster with the melanin standard, a prismatic layer of Mytilus galloprovincialis. Therefore, the brown deposits were considered to be melanin. The gene expression of infected mantles was analyzed by quantitative PCR. RNA was extracted from the mantle and body fluids, and the gene expression of shell matrix protein genes (msi31 and msi60), tyrosinase genes involved in melanin formation (OT47 and pfty1), and superoxide dismutase (SOD), which is an indicator of reactive oxygen species, was analyzed. A general linear model was created using the presence of deposits as an objective variable, with virus gene copy numbers in the body fluid and mantle and expression levels of the above five genes as explanatory variables. Then, an optimal model was selected. As a result, OT47, msi60, and SOD were selected as explanatory variables. The results suggest that virus infection induced the up-regulation of SOD and tyrosinase, inducing the deposits on the inner surface of shells in summer atrophy. Melanization is known in many animals and plays a major role in immunity, particularly in invertebrates. In bivalves, melanization caused by bacterial diseases has been reported, but this is the first report of melanization caused by viral diseases.

Comprehensive profiling of neutralizing polyclonal sera targeting coxsackievirus B3

Despite their fundamental role in resolving viral infections, our understanding of how polyclonal neutralizing antibody responses target non-enveloped viruses remains limited. To define these responses, we obtained the full antigenic profile of multiple human and mouse polyclonal sera targeting the capsid of a prototypical picornavirus, coxsackievirus B3. Our results uncover significant variation in the breadth and strength of neutralization sites targeted by individual human polyclonal responses, which contrasted with homogenous responses observed in experimentally infected mice. We further use these comprehensive antigenic profiles to define key structural and evolutionary parameters that are predictive of escape, assess epitope dominance at the population level, and reveal a need for at least two mutations to achieve significant escape from multiple sera. Overall, our data provide a comprehensive analysis of how polyclonal sera target a non-enveloped viral capsid and help define both immune dominance and escape at the population level.

Isolation and characterization of a novel parvovirus from a red-crowned crane, China, 2021

BackgroundParvoviruses are icosahedral, nonenveloped viruses with single-stranded DNA genomes of approximately 5 kb in length. In recent years, parvoviruses have frequently mutated and expanded their host range to cause disease in many wild animals by altering their tissue tropism. Animal infection mainly results in acute enteritis and inflammation of other organs. In this study, we used a viral metagenomic method to detect a novel parvovirus species in a red-crowned crane that died due to severe diarrhea in China.ResultsThe presence of the viral genome in the kidney, lung, heart, liver, and intestine were confirmed by PCR. Histopathological examination of the intestine showed a large number of infiltrated inflammatory cells. The JL21/10 strain of the red-crowned crane parvovirus was first isolated from the intestine. Whole-genome sequence analysis showed that JL21/10 shared high identity with the red-crowned crane Parvovirinae strains yc-8 at the nucleotide level (96.61%). Phylogenetic analysis of the complete genome and NS1 gene revealed that the JL21/10 strain clustered with strains in chicken and revealed a close genetic relationship with the red-crowned crane parvovirus strains.The complete of VP2 gene analysis showed that JL21/10 shared identity with the red-crowned crane yc-8 strains (97.7%), chicken (55.4%),ducks(31.0%) and geese(30.1%) at the amino acid level. The result showed that red-crowned crane parvovirus may be cross-species transmission to chicken. However, There is little possibility of transmission to ducks and geese.ConclusionThis is the first isolation and identification of a parvovirus in red-crowned crane that was associated with severe diarrhea.

A novel in vitro system of supported planar endosomal membranes (SPEMs) reveals an enhancing role for cathepsin B in the final stage of Ebola virus fusion and entry.

Ebola virus (EBOV) causes a hemorrhagic fever with fatality rates up to 90%. The EBOV entry process is complex and incompletely understood. Following attachment to host cells, EBOV is trafficked to late endosomes/lysosomes where its glycoprotein (GP) is processed to a 19-kDa form, which binds to the EBOV intracellular receptor Niemann-Pick type C1. We previously showed that the cathepsin protease inhibitor, E-64d, blocks infection by pseudovirus particles bearing 19-kDa GP, suggesting that further cathepsin action is needed to trigger fusion. This, however, has not been demonstrated directly. Since 19-kDa Ebola GP fusion occurs in late endosomes, we devised a system in which enriched late endosomes are used to prepare supported planar endosomal membranes (SPEMs), and fusion of fluorescent (pseudo)virus particles is monitored by total internal reflection fluorescence microscopy. We validated the system by demonstrating the pH dependencies of influenza virus hemagglutinin (HA)-mediated and Lassa virus (LASV) GP-mediated fusion. Using SPEMs, we showed that fusion mediated by 19-kDa Ebola GP is dependent on low pH, enhanced by Ca2+, and augmented by the addition of cathepsins. Subsequently, we found that E-64d inhibits full fusion, but not lipid mixing, mediated by 19-kDa GP, which we corroborated with the reversible cathepsin inhibitor VBY-825. Hence, we provide both gain- and loss-of-function evidence that further cathepsin action enhances the fusion activity of 19-kDa Ebola GP. In addition to providing new insights into how Ebola GP mediates fusion, the approach we developed employing SPEMs can now be broadly used for studies of virus and toxin entry through endosomes. IMPORTANCE Ebola virus is the causative agent of Ebola virus disease, which is severe and frequently lethal. EBOV gains entry into cells via late endosomes/lysosomes. The events immediately preceding fusion of the viral and endosomal membranes are incompletely understood. In this study, we report a novel in vitro system for studying virus fusion with endosomal membranes. We validated the system by demonstrating the low pH dependencies of influenza and Lassa virus fusion. Moreover, we show that further cathepsin B action enhances the fusion activity of the primed Ebola virus glycoprotein. Finally, this model endosomal membrane system should be useful in studying the mechanisms of bilayer breaching by other enveloped viruses, by non-enveloped viruses, and by acid-activated bacterial toxins.

Biocompatible Alginate Hydrogel Film Containing Acetic Acid Manifests Broad-Spectrum Antiviral and Anticancer Activities.

Acetic acid, a colourless liquid organic acid with a characteristic acrid smell, is obtained naturally and has applications in both the food and pharmaceutical industries. It has been reported to have beneficial uses for lifestyle-related diseases, and its efficient disinfectant properties are well known. In this study, an alginate crosslinked with Ca2+ hydrogel film was treated with acetic acid to explore its biological properties for biomedicine. The results showed that the novel calcium alginate/acetic acid film was biocompatible in vitro using human keratinocyte cells and in vivo with Caenorhabditis elegans. It also had antiviral properties against enveloped and non-enveloped viruses and anticancer properties against melanoma and colon cancer cells. This novel film thus showed promise for the biomedical and pharmaceutical industries, with applications for fabricating broad-spectrum antiviral and anticancer materials.

Development of a simple and low-cost method using Moringa seeds for efficient virus concentration in wastewater

Effective virus concentration methods are essential for detecting pathogenic viruses in environmental waters and play a crucial role in wastewater-based epidemiology. However, the current methods are often expensive, complicated, and time-consuming, which limits their practical application. In this study, a simple and low-cost method was developed using the extract of Moringa oleifera (MO) seeds (MO method) to recover both enveloped and non-enveloped viruses, including pepper mild mottle virus (PMMoV), murine norovirus (MNV), Aichivirus (AiV), murine hepatitis virus (MHV), and influenza A virus subtype H1N1[H1N1] in wastewater. The optimal conditions for the MO method were determined to be a concentration of MO extract at the UV280 value of 0.308 cm−1 and an elution buffer (0.05 M KH2PO4, 1 M NaCl, 0.1 % Tween80 [v/v]) for recovering the tested viruses in wastewater. Compared to other commonly used virus concentration methods such as InnovaPrep, HA, PEG, and Centricon, the MO method was found to be more efficient and cost-effective in recovering the tested viruses. Moreover, the MO method was successfully applied to detect various types of viruses (PMMoV, AiV, norovirus of genotype II [NoV II], enterovirus [EV], influenza A virus [matrix gene] [IAV], and SARS-CoV-2) in raw wastewater. Thus, the developed MO method could offer a simple, low-cost, and efficient tool to concentrate viruses in wastewater.

Bubble manipulates the release of viral aerosols in aeration

Bubble bursting is a common phenomenon in many industrial and natural processes, plays an important role in mediating mass transfer across the water-air interface. But the interplay between bubbles and pathogens remains unclear and the mechanisms of virus aerosolization by the bubble properties have not been well studied. The main objective of this study was to evaluate the water-to-air transfer of viruses by bubbles of different sizes. Unlike the dominant view of smaller bubbles less bioaerosols, it was found that the smaller bubbles could generate significantly more viral aerosols regardless of the virus species (Phi6, MS2, PhiX174, and T7), when the Sauter mean bubble diameters were between 0.56 and 1.65 mm under constant aeration flow rate. The mechanism studies denied the possibilities of more aerosols or better dispersion of viruses in the aerosols generated by the smaller bubbles. However, deeper bubbling could transfer more viruses to the air for MS2, PhiX174, and T7. Their concentrations in aerosols were linearly related to the bubbling depth for these non-enveloped viruses, which demonstrates the bubble-scavenging effect as a main mechanism except for the enveloped virus Phi6. Whereas, unlike these three non-enveloped viruses, Phi6 could survive relatively better in the aerosols generated from the smaller bubbles, though the enhancement of aerosolization by the smaller bubbles was much larger than the improvement of survival. Other mechanisms still remain unknown for this enveloped virus. This study suggests that the attempt of decreasing the bubble size in aeration tank of the wastewater treatment plant might significantly increase the solubility of oxygen as well as the risk of viral aerosols.

Broad-Spectrum Antiviral Effect of Cannabidiol Against Enveloped and Nonenveloped Viruses.

Introduction: Cannabidiol (CBD), the main non-psychoactive cannabinoid of the Cannabis sativa plant, is a powerful antioxidant compound that in recent years has increased interest due to causes effects in a wide range of biological functions. Zika virus (ZIKV) is a virus transmitted mainly by the Aedes aegypti mosquitoes, which causes neurological diseases, such as microcephaly and Guillain-Barre syndrome. Although the frequency of viral outbreaks has increased recently, no vaccinations or particular chemotherapeutic treatments are available for ZIKV infection. Objectives: The major aim of this study was to explore the in vitro antiviral activity of CBD against ZIKV, expanding also to other dissimilar viruses. Materials and Methods: Cell cultures were infected with enveloped and nonenveloped viruses and treated with non-cytotoxic concentrations of CBD and then, viral titers were determined. Additionally, the mechanism of action of the compound during ZIKV in vitro infections was studied. To study the possible immunomodulatory role of CBD, infected and uninfected Huh-7 cells were exposed to 10 μM CBD during 48 h and levels of interleukins 6 and 8 and interferon-beta (IFN-β) expression levels were measured. On the other hand, the effect of CBD on cellular membranes was studied. For this, an immunofluorescence assay was performed, in which cell membranes were labeled with wheat germ agglutinin. Finally, intracellular cholesterol levels were measured. Results: CBD exhibited a potent antiviral activity against all the tested viruses in different cell lines with half maximal effective concentration values (CE50) ranging from 0.87 to 8.55 μM. Regarding the immunomodulatory effect of CBD during ZIKV in vitro infections, CBD-treated cells exhibited significantly IFN-β increased levels, meanwhile, interleukins 6 and 8 were not induced. Furthermore, it was determined that CBD affects cellular membranes due to the higher fluorescence intensity that was observed in CBD-treated cells and lowers intracellular cholesterol levels, thus affecting the multiplication of ZIKV and other viruses. Conclusions: It was demonstrated that CBD inhibits structurally dissimilar viruses, suggesting that this phytochemical has broad-spectrum antiviral effect, representing a valuable alternative in emergency situations during viral outbreaks, like the one caused by severe acute respiratory syndrome coronavirus 2 in 2020.

Canine bufavirus (Carnivore protoparvovirus-3) infection in dogs with respiratory disease.

Canine bufavirus (CBuV) or Carnivore protoparvovirus-3, a nonenveloped DNA virus belonging to the genus Protoparvovirus, family Parvoviridae, has been identified in dogs with respiratory and enteric diseases. Although CBuV detection has been reported in multiple countries, descriptions of pathologic findings associated with infection have not yet been provided. In this study, the authors necropsied 14 dogs (12 puppies and 2 adult dogs) from a breeding colony that died during multiple outbreaks of respiratory diseases. Postmortem investigations revealed extensive bronchointerstitial pneumonia with segmental type II pneumocyte hyperplasia in all necropsied puppies but less severe lesions in adults. With negative results of common pathogen detection by ancillary testing, CBuV DNA was identified in all investigated dogs using a polymerase chain reaction (PCR). Quantitative PCR demonstrated CBuV DNA in several tissues, and in situ hybridization (ISH) indicated CBuV tissue localization in the lung, tracheobronchial lymph node, and spinal cord, suggesting hematogenous spread. Dual CBuV ISH and cellular-specific immunohistochemistry were used to determine the cellular tropism of the virus in the lung and tracheobronchial lymph node, demonstrating viral localization in various cell types, including B-cells, macrophages, and type II pneumocytes, but not T-cells. Three complete CBuV sequences were successfully characterized and revealed that they clustered with the CBuV sequences obtained from dogs with respiratory disease in Hungary. No additional cases were identified in small numbers of healthy dogs. Although association of the bufavirus with enteric disease remains to be determined, a contributory role of CBuV in canine respiratory disease is possible.

Low-density hepatitis C virus infectious particles are protected from oxidation by secreted cellular proteins.

Assessments of viral stability on surfaces or in body fluids under different environmental conditions and/or temperatures are often performed, as they are key to understanding the routes and parameters of viral transmission and to providing clues on the epidemiology of infections. However, for most viruses, the mechanisms of inactivation vs stability of viral particles remain poorly defined. Although they are structurally diverse, with different compositions, sizes, and shapes, enveloped viruses are generally less stable than non-enveloped viruses, pointing out the role of envelopes themselves in virus lability. In this report, we investigated the properties of hepatitis C virus (HCV) particles with regards to their stability. We found that, compared to alternative enveloped viruses such as Dengue virus (DENV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), hepatitis delta virus (HDV), and Crimean-Congo hemorrhagic fever virus (CCHFV) that infect the liver, HCV particles are intrinsically labile. We determined the mechanisms that drastically alter their specific infectivity through oxidation of their lipids, and we highlighted that they are protected from lipid oxidation by secreted cellular proteins, which can protect their membrane fusion capacity and overall infectivity.

A Coxsackievirus B1-mediated nonlytic Extracellular Vesicle-to-cell mechanism of virus transmission and its possible control through modulation of EV release.

Like most non-enveloped viruses, CVB1 mainly uses cell lysis to spread. Details of a nonlytic virus transmission remain unclear. Extracellular Vesicles (EVs) transfer biomolecules between cells. We show that CVB1 entry into HeLa cells results in apoptosis and release of CVB1-induced 'medium-sized' EVs (CVB1i-mEVs). These mEVs (100-300 nm) harbour CVB1 as shown by immunoblotting with anti-CVB1-antibody; viral capsids were detected by transmission electron microscopy and RT-PCR revealed CVB1 RNA. The percentage of mEVs released from CVB1-infected HeLa cells harbouring virus was estimated from TEM at 34 %. Inhibition of CVB1i-mEV production, with calpeptin or siRNA knockdown of CAPNS1 in HeLa cells limited spread of CVB1 suggesting these vesicles disseminate CVB1 virions to new host cells by a nonlytic EV-to-cell mechanism. This was confirmed by detecting CVB1 virions inside HeLa cells after co-culture with CVB1i-mEVs; EV release may also prevent apoptosis of infected cells whilst spreading apoptosis to secondary sites of infection.

Possibilities of using mussels (Mytilus galloprovincialis) to predict rotavirus contamination in Albania.

Rotaviruses are non-enveloped viruses that each consist of 11 double-stranded RNA molecules. These viruses are able to persist in the environment, and therefore play a fundamental role in the epidemiology of gastroenteritis and severe diarrhoea in children worldwide. While mussels have been primarily used as indicators of chemical pollution, they can also be used to monitor viral contamination. The purpose of this study was to demonstrate that the Mytilus galloprovincialis mussel can also be used to detect microbial contamination, owing to its tendency to naturally concentrate viruses and other pathogens. A total of 102 Mytilus galloprovincialis mussel samples from Albania were collected over a three-year period: 37 samples off the Cape of Stillo in 2015, 39 samples from Butrinti Lake in 2019 and 26 samples from Butrinti Lake in 2021. The presence of rotavirus in the Cape of Stillo samples in 2015 was noted in 47% of samples from site 1, 33% from site 2, and 52% from site 3. In Butrinti Lake the percentage of infected individuals in 2019 was 33% from site 1, 41% from site 2, and 33% from site 3, whereas in 2021, it was 50% from site 1, 19% from site 2, and 0% from site 3. In total the percentage of infected individuals off the Cape of Stillo in 2015 was 44%, in Butrinti Lake in 2019 it was 36%, and in Butrinti Lake in 2021 it was 23 %. These results indicate the presence of rotavirus in the shellfish specimens tested, and further analysis is needed to assess the potential health risks associated with consuming these shellfish. This study also indicates that mussels can be used in marine virological surveillance programmes.

The Protective Effect of Virus Capsids on RNA and DNA Virus Genomes in Wastewater.

Virus concentrations measured in municipal wastewater help inform both the water treatment necessary to protect human health and wastewater-based epidemiology. Wastewater measurements are typically PCR-based, and interpreting gene copy concentrations requires an understanding of the form and stability of the nucleic acids. Here, we study the persistence of model virus genomes in wastewater, the protective effects provided by the virus capsids, and the relative decay rates of the genome and infectious viruses. In benchtop batch experiments in wastewater influent at 25 °C, extraviral (+)ssRNA and dsDNA amplicons degraded by 90% within 15-19 min and 1.6-1.9 h, respectively. When encapsidated, the T90 for MS2 (+)ssRNA increased by 424× and the T90 for T4 dsDNA increased by 52×. The (+)ssRNA decay rates were similar for a range of amplicon sizes. For our model phages MS2 and T4, the nucleic acid signal in untreated wastewater disappeared shortly after the viruses lost infectivity. Combined, these results suggest that most viral genome copies measured in wastewater are encapsidated, that measured concentrations are independent of assay amplicon sizes, and that the virus genome decay rates of nonenveloped (i.e., naked) viruses are similar to inactivation rates. These findings are valuable for the interpretation of wastewater virus measurements.

Human Papillomavirus-Associated Tumor Extracellular Vesicles in HPV+ Tumor Microenvironments.

Most infections with human papillomaviruses (HPVs) are self-resolving and asymptomatic. However, some infections can lead to the development of cancer at different mucosal sites, such as the cervix and the head and neck. Head and neck cancers (HNCs) are dichotomized into HPV-positive (HPV+) or HPV-negative (HPV-) based on their respective etiologies. Notably, the tumor microenvironment (TME) of the HPV+ subtype has an immune landscape characterized with increased immune infiltration, higher levels of T cell activation, and higher levels of immunoregulatory stimuli compared to their HPV- counterparts. Both enveloped and nonenveloped viruses hijack the extracellular vesicle (EV) biogenesis pathway to deploy a "trojan horse" strategy with a pseudoviral envelope to enhance infectivity and evade inflammation. EVs derived from HPV-infected tumor cells could allow for the stealth transport of viral cargo to neighboring nonmalignant cellular populations or infiltrating immune cells within the TME. Furthermore, viral cargo or altered cellular cargo from HPV-associated tumor EVs (HPV-TEVs) could alter the functional state or biological responses of the recipient cellular populations, which could shape the distinctive HPV+ TME. This review will cover the impact of EVs released from HPV-infected cells on HPV-induced carcinogenesis, their role in shaping the distinctive HPV+ tumor microenvironment, and current efforts to develop a painless EV-based liquid biopsy for HPV+ cancers.

Rotavirus Particle Disassembly and Assembly In Vivo and In Vitro.

Rotaviruses (RVs) are non-enveloped multilayered dsRNA viruses that are major etiologic agents of diarrheal disease in humans and in the young in a large number of animal species. The viral particle is composed of three different protein layers that enclose the segmented dsRNA genome and the transcriptional complexes. Each layer defines a unique subparticle that is associated with a different phase of the replication cycle. Thus, while single- and double-layered particles are associated with the intracellular processes of selective packaging, genome replication, and transcription, the viral machinery necessary for entry is located in the third layer. This modular nature of its particle allows rotaviruses to control its replication cycle by the disassembly and assembly of its structural proteins. In this review, we examine the significant advances in structural, molecular, and cellular RV biology that have contributed during the last few years to illuminating the intricate details of the RV particle disassembly and assembly processes.

The atlastin ER morphogenic proteins promote formation of a membrane penetration site during non-enveloped virus entry.

During entry, non-enveloped viruses penetrate a host membrane to cause infection, although how this is accomplished remains enigmatic. Polyomaviruses (PyVs) are non-enveloped DNA viruses that penetrate the endoplasmic reticulum (ER) membrane to reach the cytosol en route to the nucleus for infection. To penetrate the ER membrane, the prototype PyV simian virus 40 (SV40) induces formation of ER-escape sites, called foci, composed of repeating units of multi-tubular ER junctions where the virus is thought to exit. How SV40 triggers formation of the ER-foci harboring these multi-tubular ER junctions is unclear. Here, we show that the ER morphogenic atlastin 2 (ATL2) and ATL3 membrane proteins play critical roles in SV40 infection. Mechanistically, ATL3 mobilizes to the ER-foci where it deploys its GTPase-dependent membrane fusion activity to promote formation of multi-tubular ER junctions within the ER-foci. ATL3 also engages an SV40-containing membrane penetration complex. By contrast, ATL2 does not reorganize to the ER-foci. Instead, it supports the reticular ER morphology critical for the integrity of the ATL3-dependent membrane complex. Our findings illuminate how two host factors play distinct roles in the formation of an essential membrane penetration site for a non-enveloped virus. IMPORTANCE Membrane penetration by non-enveloped viruses, a critical infection step, remains enigmatic. The non-enveloped PyV simian virus 40 (SV40) penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol en route for infection. During ER-to-cytosol membrane penetration, SV40 triggers formation of ER-associated structures (called ER-foci) that function as the membrane penetration sites. Here, we discover a role of the ATL ER membrane proteins-known to shape the ER morphology-during SV40-induced ER-foci formation. These findings illuminate how a non-enveloped virus hijacks host components to construct a membrane penetration structure.

Instantaneous Inactivation of Herpes Simplex Virus by Silicon Nitride Bioceramics.

Hydrolytic reactions taking place at the surface of a silicon nitride (Si3N4) bioceramic were found to induce instantaneous inactivation of Human herpesvirus 1 (HHV-1, also known as Herpes simplex virus 1 or HSV-1). Si3N4 is a non-oxide ceramic compound with strong antibacterial and antiviral properties that has been proven safe for human cells. HSV-1 is a double-stranded DNA virus that infects a variety of host tissues through a lytic and latent cycle. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) tests of HSV-1 DNA after instantaneous contact with Si3N4 showed that ammonia and its nitrogen radical byproducts, produced upon Si3N4 hydrolysis, directly reacted with viral proteins and fragmented the virus DNA, irreversibly damaging its structure. A comparison carried out upon testing HSV-1 against ZrO2 particles under identical experimental conditions showed a significantly weaker (but not null) antiviral effect, which was attributed to oxygen radical influence. The results of this study extend the effectiveness of Si3N4's antiviral properties beyond their previously proven efficacy against a large variety of single-stranded enveloped and non-enveloped RNA viruses. Possible applications include the development of antiviral creams or gels and oral rinses to exploit an extremely efficient, localized, and instantaneous viral reduction by means of a safe and more effective alternative to conventional antiviral creams. Upon incorporating a minor fraction of micrometric Si3N4 particles into polymeric matrices, antiherpetic devices could be fabricated, which would effectively impede viral reactivation and enable high local effectiveness for extended periods of time.