Structural and Proteomic Characterization of the Initiation of Giant Virus Infection.

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four infection intermediates in Samba virus (Mimivirus genus, lineage A) as visualized by cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-ET), and scanning electron microscopy (SEM). Each of these four intermediates reflects similar morphology to a stage that occurs in vivo. We show that these genome release stages are conserved in other mimiviruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved among disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.

Persistence of Influenza H5N1 and H1N1 Viruses in Unpasteurized Milk on Milking Unit Surfaces.

Examining the persistence of highly pathogenic avian influenza A(H5N1) from cattle and human influenza A(H1N1)pdm09 pandemic viruses in unpasteurized milk revealed that both remain infectious on milking equipment materials for several hours. Those findings highlight the risk for H5N1 virus transmission to humans from contaminated surfaces during the milking process.

Stability of Monkeypox Virus in Body Fluids and Wastewater.

An outbreak of human mpox infection in nonendemic countries appears to have been driven largely by transmission through body fluids or skin-to-skin contact during sexual activity. We evaluated the stability of monkeypox virus (MPXV) in different environments and specific body fluids and tested the effectiveness of decontamination methodologies. MPXV decayed faster at higher temperatures, and rates varied considerably depending on the medium in which virus was suspended, both in solution and on surfaces. More proteinaceous fluids supported greater persistence. Chlorination was an effective decontamination technique, but only at higher concentrations. Wastewater was more difficult to decontaminate than plain deionized water; testing for infectious MPXV could be a helpful addition to PCR-based wastewater surveillance when high levels of viral DNA are detected. Our findings suggest that, because virus stability is sufficient to support environmental MPXV transmission in healthcare settings, exposure and dose-response will be limiting factors for those transmission routes.

Inherent Heterogeneity of Influenza A Virus Stability following Aerosolization.

Efficient human-to-human transmission represents a necessary adaptation for a zoonotic influenza A virus (IAV) to cause a pandemic. As such, many emerging IAVs are characterized for transmissibility phenotypes in mammalian models, with an emphasis on elucidating viral determinants of transmission and the role host immune responses contribute to mammalian adaptation. Investigations of virus infectivity and stability in aerosols concurrent with transmission assessments have increased in recent years, enhancing our understanding of this dynamic process. Here, we employed a diverse panel of 17 human and zoonotic IAVs, inclusive of seasonally circulating H1N1 and H3N2 viruses, as well as avian and swine viruses associated with human infection, to evaluate differences in spray factor (a value that assesses efficiency of the aerosolization process), stability, and infectivity following aerosolization. While most seasonal influenza viruses did not exhibit substantial variability within these parameters, there was more heterogeneity among zoonotic influenza viruses, which possess a diverse range of transmission phenotypes. Aging of aerosols at different relative humidities identified strain-specific levels of stability with different profiles identified between zoonotic H3, H5, and H7 subtype viruses associated with human infection. As studies continue to elucidate the complex components governing virus transmissibility, notably aerosol matrices and environmental parameters, considering the relative role of subtype- and strain-specific factors to modulate these parameters will improve our understanding of the pandemic potential of zoonotic influenza A viruses. IMPORTANCE Transmission of respiratory pathogens through the air can facilitate the rapid and expansive spread of infection and disease through a susceptible population. While seasonal influenza viruses are quite capable of airborne spread, there is a lack of knowledge regarding how well influenza viruses remain viable after aerosolization and whether influenza viruses capable of jumping species barriers to cause human infection differ in this property from seasonal strains. We evaluated a diverse panel of influenza viruses associated with human infection (originating from human, avian, and swine reservoirs) for their ability to remain viable after aerosolization in the laboratory under a range of conditions. We found greater diversity among avian and swine-origin viruses compared to seasonal influenza viruses; strain-specific stability was also noted. Although influenza virus stability in aerosols is an underreported property, if molecular markers associated with enhanced stability are identified, we will be able to quickly recognize emerging strains of influenza that present the greatest pandemic threat.

Dynamical Differences in Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a leading viral cause of pediatric respiratory infections and early infant mortality. Despite extensive development efforts currently underway, there remain no vaccines available for the prevention of RSV. RSV is an enveloped, negative-strand RNA virus that utilizes two different proteins (G and F) to mediate attachment and entry into host cells. These G and F proteins are the primary determinants of viral strain-specific differences and elicit protective neutralizing antibodies during natural infection in humans. Earlier studies have demonstrated that these proteins play an additional role in regulating the stability of RSV particles in response to temperature and pH. However, it remains unclear how much variability exists in the stability of RSV strains and what contribution changes in temperature and pH make to the clearance of virus during an active infection. In this study, we evaluated the impacts of changes in temperature and pH on the inactivation of four different chimeric recombinant RSV strains that differ exclusively in G and F protein expression. Using these data, we developed predictive mathematical models to examine the specific contributions and variations in susceptibility that exist between viral strains. Our data provide strain-specific clearance rates and temperature-pH landscapes that shed light on the optimal contributions of temperature and pH to viral clearance. These provide new insight into how much variation exists in the clearance of a major respiratory pathogen and may offer new guidance on optimization of viral strains for development of live-attenuated vaccine preparations.

Role of Phage Capsid in the Resistance to UV-C Radiations.

The conformational variation of the viral capsid structure plays an essential role both for the environmental resistance and acid nuclear release during cellular infection. The aim of this study was to evaluate how capsid rearrangement in engineered phages of M13 protects viral DNA and peptide bonds from damage induced by UV-C radiation. From in silico 3D modelling analysis, two M13 engineered phage clones, namely P9b and 12III1, were chosen for (i) chemical features of amino acids sequences, (ii) rearrangements in the secondary structure of their pVIII proteins and (iii) in turn the interactions involved in phage capsid. Then, their resistance to UV-C radiation and hydrogen peroxide (H2O2) was compared to M13 wild-type vector (pC89) without peptide insert. Results showed that both the phage clones acquired an advantage against direct radiation damage, due to a reorganization of interactions in the capsid for an increase of H-bond and steric interactions. However, only P9b had an increase in resistance against H2O2. These results could help to understand the molecular mechanisms involved in the stability of new virus variants, also providing quick and necessary information to develop effective protocols in the virus inactivation for human activities, such as safety foods and animal-derived materials.

Effective inactivation of Nipah virus in serum samples for safe processing in low-containment laboratories.

BACKGROUND: Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes severe encephalitis and respiratory disease with a high mortality rate in humans. During large outbreaks of the viral disease, serological testing of serum samples could be a useful diagnostic tool, which could provide information on not only the diagnosis of NiV disease but also the history of an individual with previous exposure to the virus, thereby supporting disease control. Therefore, an efficient method for the inactivation of NiV in serum samples is required for serological diagnosis. METHODS: We determined the optimal conditions for the inactivation of NiV infectivity in human serum using heating and UV treatment. The inactivation method comprised UV irradiation with a cover of aluminum foil for 30 min and heating at 56 °C for 30 min. RESULTS: With an optimized protocol for virus inactivation, NiV infectivity in serum samples (containing 6.0 × 105 TCID50) was completely inactivated. CONCLUSIONS: We developed a recommended protocol for the effective inactivation of NiV. This protocol would enable a regional or local laboratory to safely transport or process samples, including NiV, for serological testing in its biosafety level-2 facility.

Half-Life of African Swine Fever Virus in Shipped Feed.

African swine fever virus is transmissible through animal consumption of contaminated feed. To determine virus survival during transoceanic shipping, we calculated the half-life of the virus in 9 feed ingredients exposed to 30-day shipment conditions. Half-lives ranged from 9.6 to 14.2 days, indicating that the feed matrix environment promotes virus stability.

Evaluation of the role of respiratory syncytial virus surface glycoproteins F and G on viral stability and replication: implications for future vaccine design.

Respiratory syncytial virus (RSV) remains a leading cause of infant mortality worldwide and exhaustive international efforts are underway to develop a vaccine. However, vaccine development has been hindered by a legacy of vaccine-enhanced disease, poor viral immunogenicity in infants, and genetic and physical instabilities. Natural infection with RSV does not prime for enhanced disease encouraging development of live-attenuated RSV vaccines for infants; however, physical instabilities of RSV may limit vaccine development. The role of RSV strain-specific differences on viral physical stability remains unclear. We have previously demonstrated that the RSV fusion (F) surface glycoprotein is responsible for mediating significant differences in thermostability between strains A2 and A2-line19F. In this study, we performed a more comprehensive analysis to characterize the replication and physical stability of recombinant RSV A and B strains that differed only in viral attachment (G) and/or F surface glycoprotein expression. We observed significant differences in thermal stability, syncytia size, pre-fusion F incorporation and viral growth kinetics in vitro, but limited variations to pH and freeze-thaw inactivation among several tested strains. Consistent with earlier studies, A2-line19F showed significantly enhanced thermal stability over A2, but also restricted growth kinetics in both HEp2 and Vero cells. As expected, no significant differences in susceptibility to UV inactivation were observed. These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability.

Ebola Virus Stability Under Hospital and Environmental Conditions.

The West African outbreak of Ebola virus (EBOV) is largely contained, but sporadic new cases continue to emerge. To assess the potential contribution of fomites to human infections with EBOV, we tested EBOV stability in human blood spotted onto Sierra Leonean banknotes and in syringe needles under hospital and environmental conditions. Under some of these conditions, EBOV remained infectious for >30 days, indicating that EBOV-contaminated items may pose a serious risk to humans.

Spectrum of Newcastle disease virus stability in gradients of temperature and pH.

Newcastle disease (ND) is one of the highly pathogenic viral diseases of avian species. The disease is endemic in many developing countries where agriculture serves as the primary source of national income. Newcastle disease virus (NDV) belongs to the family Paramyxoviridae and is well characterized member among the avian paramyxovirus serotypes. The failure of vaccination is one of the major causes of NDV outbreaks in field condition. The present study gives a brief picture about the biology of NDV genome and its proteins under different conditions of temperature and pH. Our results indicate that the NDV is non-infective above 42 °C and unstable above 72 °C. The study will be useful in defining an optimum storage condition for NDV without causing any deterioration in its viability.

Survival of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in the Environment.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine, with losses due to poor reproductive performance and high piglet and growing pig mortality. Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) may occur by both direct and indirect routes; the latter includes exposure to PRRSV-contaminated fomites, aerosols, and arthropod vectors. This review has collected available data on the ex-vivo environmental stability and persistence of PRRSV in an effort to highlight important sources of the virus and to determine the role of environmental conditions on the stability of the virus, especially temperature. The ex-vivo settings include fomites (solid, porous, and liquid fomites), insects, people, and pork meat, as well as the role of environmental conditions on the stability of the virus, especially temperature.

Antiviral activity of salt-coated materials against SARS-CoV-2.

The SARS-CoV-2 pandemic demonstrated the importance of human coronaviruses and the need to develop materials to prevent the spread of emergent respiratory viruses. Coating of surfaces with antiviral materials is a major interest in controlling spread of viruses, especially in high-risk or high-traffic areas. A number of different coatings for surfaces have been proposed, each with their own advantages and disadvantages. Here we show that simple salt coating on a range of surfaces, including a novel biomass aerogel can reduce the infectivity of SARS-CoV-2 placed onto the surface. This suggests that a simple to apply coating could be applied to a range of materials and have an antiviral effect against SARS-CoV-2, as well as other potential emerging viruses.

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

IMPORTANCE: 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.

SARS-CoV-2 Survival on Surfaces. Measurements Optimisation for an Enthalpy-Based Assessment of the Risk.

The present work, based on the results found in the literature, yields a consistent model of SARS-CoV-2 survival on surfaces as environmental conditions, such as temperature and relative humidity, change simultaneously. The Enthalpy method, which has recently been successfully proposed to investigate the viability of airborne viruses using a holistic approach, is found to allow us to take a reasoned reading of the data available on surfaces in the literature. This leads us to identify the domain of conditions of lowest SARS-CoV-2 viability, in a specific enthalpy range between 50 and 60 kJ/Kgdry-air. This range appears well-superimposed with the results we previously obtained from analyses of coronaviruses' behaviour in aerosols, and may be helpful in dealing with the spread of infections. To steer future investigations, shortcomings and weaknesses emerging from the assessment of viral measurement usually carried out on surfaces are also discussed in detail. Once demonstrated that current laboratory procedures suffer from both high variability and poor standardisation, targeted implementations of standards and improvement of protocols for future investigations are then proposed.

A comparative study on the persistence and viability of SARS-CoV-2 wild-type and omicron variant on artificially contaminated surfaces: the role of fomites.

Indirect transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been investigated but it is still not completely understood. The present study aimed to compare the persistence and viability of the lineage B.1 and omicron BA.1 subvariant in five daily-use materials to evaluate the role of fomites as a possible source of infection. Artificial contamination was performed in the first set of materials, ethylene vinyl acetate (EVA), cardboard, polystyrene, aluminium, and plastic. Further surfaces using BA.1 (glass, plexiglass, cotton, polyester, and tetrapak) were conducted. The persistence, viability of Vero E6 cell cultures and the residual infectivity of the two lineages were evaluated over 5 days. The results showed different stabilities between the tested matrices. In cotton and polyester, the RNA was undetectable in 24 and 48h post-contamination (p.c.), respectively, and the virus was not viable within 30 min, while in the other surfaces, both lineages, RNA was detectable until 120h p.c. A rapid decay of the viral load was revealed on cardboard, mostly for the omicron variant. Furthermore, on all the materials, longer stability of BA.1 was demonstrated, but showing a less intense CPE than the wild-type. EVA was the material that was able to better sustain virus stability as the virus developed CPE up to 72h p.c. In conclusion, the potential spread of SARS-CoV-2 through fomites is conceivable, albeit it is difficult to establish the real capacity to infect people. Nevertheless, thise information is fundamental to adopting the appropriate measures to mitigate the spread of SARS-CoV-2 and its variants.

Stability of African swine fever virus genome under different environmental conditions.

Background and Aim: African swine fever (ASF), a globally transmitted viral disease caused by ASF virus (ASFV), can severely damage the global trade economy. Laboratory diagnostic methods, including pathogen and serological detection techniques, are currently used to monitor and control ASF. Because the large double-stranded DNA genome of the mature virus particle is wrapped in a membrane, the stability of ASFV and its genome is maintained in most natural environments. This study aimed to investigate the stability of ASFV under different environmental conditions from both genomic and antibody perspectives, and to provide a theoretical basis for the prevention and elimination of ASFV. Materials and Methods: In this study, we used quantitative real-time polymerase chain reaction for pathogen assays and enzyme-linked immunosorbent assay for serological assays to examine the stability of the ASFV genome and antibody, respectively, under different environmental conditions. Results: The stability of the ASFV genome and antibody under high-temperature conditions depended on the treatment time. In the pH test, the ASFV genome and antibody remained stable in both acidic and alkaline environments. Disinfection tests revealed that the ASFV genome and antibody were susceptible to standard disinfection methods. Conclusion: Collectively, the results demonstrated that the ASFV genome is highly stable in favorable environments but are also susceptible to standard disinfection methods. This study focuses on the stability of the ASFV genome under different conditions and provides various standard disinfection methods for the prevention and control of ASF.

Environmental Stability of Enveloped Viruses Is Impacted by Initial Volume and Evaporation Kinetics of Droplets.

Efficient spread of respiratory viruses requires the virus to maintain infectivity in the environment. Environmental stability of viruses can be influenced by many factors, including temperature and humidity. Our study measured the impact of initial droplet volume (50, 5, and 1 µL) and relative humidity (RH; 40%, 65%, and 85%) on the stability of influenza A virus, bacteriophage Phi6 (a common surrogate for enveloped viruses), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) under a limited set of conditions. Our data suggest that the drying time required for the droplets to reach quasi-equilibrium (i.e., a plateau in mass) varied with RH and initial droplet volume. The macroscale physical characteristics of the droplets at quasi-equilibrium varied with RH but not with the initial droplet volume. Virus decay rates differed between the wet phase, while the droplets were still evaporating, and the dry phase. For Phi6, decay was faster in the wet phase than in the dry phase under most conditions. For H1N1pdm09, decay rates between the two phases were distinct and initial droplet volume had an effect on virus viability within 2 h. Importantly, we observed differences in virus decay characteristics by droplet size and virus. In general, influenza virus and SARS-CoV-2 decayed similarly, whereas Phi6 decayed more rapidly under certain conditions. Overall, this study suggests that virus decay in media is related to the extent of droplet evaporation, which is controlled by RH. Importantly, accurate assessment of transmission risk requires the use of physiologically relevant droplet volumes and careful consideration of the use of surrogates. IMPORTANCE During the COVID-19 pandemic, policy decisions were being driven by virus stability experiments with SARS-CoV-2 in different droplet volumes under various humidity conditions. Our study, the first of its kind, provides a model for the decay of multiple enveloped RNA viruses in cell culture medium deposited in 50-, 5-, and 1-µL droplets at 40%, 65%, and 85% RH over time. The results of our study indicate that determination of half-lives for emerging pathogens in large droplets may overestimate transmission risk for contaminated surfaces, as observed during the COVID-19 pandemic. Our study implicates the need for the use of physiologically relevant droplet sizes with use of relevant surrogates in addition to what is already known about the importance of physiologically relevant media for risk assessment of future emerging pathogens.

Prediction of frozen virus stability based on degradation mechanisms, real-time data and modeling.

Aim: Critical virus reagents in regulated bioanalytical assays require stability monitoring. Although stability at ultra-low frozen temperatures is generally assumed, published data are limited and real-time studies are time consuming. Materials & methods: The authors reviewed literature data, typical mechanisms of molecular degradation, glass transition temperatures of commonly used buffers and available real-time storage data to model frozen virus reagent stability. Results: Storage at ultra-low temperatures below the glass transition temperature was critical for virus stability. Modeling of real-time data suggested that virus potency remained within 0.5 log10 of its starting potency at a probability of >99, 90 and 73% after 10, 20 and 30 years, respectively. Conclusion: The study supports the practice of virus storage at -70°C or below for 20-30 years.

Stability of African swine fever virus in feed during environmental storage.

African swine fever virus (ASFV) causes high case fatality in pigs and a trade-limiting disease resulting in significant economic losses to pork production. ASFV is resistant to environmental degradation and maintains infectivity in feed ingredients exposed to transoceanic shipment conditions. As ASFV is transmissible through consumption of contaminated feed, the objective of this study was to evaluate the stability of ASFV Georgia 2007 in three feed matrices (complete feed, soybean meal, ground corncobs) exposed to three environmental storage temperatures (40°F, 68°F, 95°F) for up to 365 days. ASFV DNA was highly stable and detectable by qPCR in almost all feed matrices through the conclusion of each study. Infectious ASFV was most stable in soybean meal, maintaining infectivity for at least 112 days at 40°F, at least 21 days at 68°F and at least 7 days at 95°F. These data help define risk of ASFV introduction and transmission through feed ingredients.