Viable Virus Research Articles

Survivability of H5N1 avian influenza virus in homemade yogurt, cheese and whey

ABSTRACT Influenza virus is typically associated with respiratory infections, but H5N1 in US dairy cows raises public health concerns about milk by-products. We show that simple home recipes can inactivate H5N1 in cheese, yogurt, and whey. While viral RNA was present, no viable virus was found, ensuring food safety.

Fish Snx27 promotes viral products by modulating the innate immune response and exosomal machinery.

Viral nervous necrosis caused by the nervous necrosis virus (NNV) poses a significant threat to the global aquaculture industry. Developing preventive methods to minimize economic losses due to NNV infections is crucial. This study explored the role of the sorting nexin 27 (Snx27) gene, encoded by the orange-spotted grouper (Epinephelus coioides) and referred to as EcSnx27, as an immune regulator affecting red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro. Our findings revealed that EcSnx27 negatively regulates interferon (IFN)-related cytokines and the promoter activities of fish ISRE and NF-κB. Furthermore, we identified the SNX-FERM and SNX-FERM-like domains as responsible for the interaction between EcSnx27 and RGNNV coat protein. Through the detection of viable virions associated with EcSnx27-containing exosomes, we propose that EcSnx27 may contribute to the release process of RGNNV by influencing the apoptosis-linked gene 2-interacting protein X (ALIX)-associated exosomal pathway. Consequently, our study suggests that EcSnx27 promotes RGNNV replication by inhibiting the IFN immune response and facilitating virus production and release through ALIX-mediated exosomal machinery.IMPORTANCERed grouper nervous necrosis virus (RGNNV), a member of the Nodaviridae family, has emerged as a significant cause of fish diseases worldwide, leading to high morbidity and mortality rates. This study investigated the sorting nexin 27 (Snx27) gene encoded by the orange-spotted grouper (Epinephelus coioides) on RGNNV infection in grouper kidney cells. Our findings revealed that EcSnx27 negatively regulated the interferon pathway, resulting in the promotion of RGNNV replication. Additionally, we observed that EcSnx27 could interact with apoptosis-linked gene 2-interacting protein X (ALIX) and the RGNNV coat protein, suggesting its potential involvement in viral release processes through modulation of the exosomal pathway. Our study identified EcSnx27 as a key target that RGNNV exploits to enhance viral production. This finding offers valuable insights into the immune evasion and viral release mechanisms of non-enveloped RNA viruses.

Phi6 virus transmission in Arctic outdoor air and the effects of solar UV radiation on virus and host viability

Virus seasonality is affected by many environmental factors. UV radiation is a less studied but potentially important factor in virus outdoor aerosol transmission. Virus aerosol transmission in an Arctic environment was simulated by nebulizing Phi6 outdoors to study longer range (0-75 m) transmission. Aerosolized viruses were collected using host-containing agar plates, Biosamplers and filter collectors. Additionally, the effects of long-term exposure to solar UV radiation were studied on Phi6 and its host bacteria Pseudomonas phaseolicola. Phi6 remained infectious in aerosols for at least 50 m outdoors, however, potential for longer transmission distance still exists. Exposure to solar UV radiation did not significantly affect Phi6 viability, however, a decreasing trend was seen over an 8 h exposure time. Potential UV radiation related damages were found in the host bacteria's genome, but not in Phi6. This study shows that some viruses can remain infectious and travel for relatively long distances outdoors. Solar UV radiation was found to have minimal or no effect on Phi6 aerosol transmission at these distances, however, Phi6 is known to be resilient against UV radiation. Further studies are required with longer distance collection to understand the full potential of virus aerosol transmission outdoors. Additionally, other model viruses should be experimented with to understand how different viruses remain infectious in aerosols.

Influencing Factors Analysis of Infectious SARS-CoV-2 Aerosols Sampling

Assessing the potential infectivity of airborne viruses is critical for evaluating the risk of their transmission through air. This study investigated the factors influencing the collection of infectious SARS-CoV-2 aerosols using three common aerosol samplers: the impactor sampler AGI-30, the SKC Biosampler, and a cyclone sampler WA-400III. It was found that the sampling process over time significantly impacted the infectivity of SARS-CoV-2 captured in the sampler, and the infectivity loss of different SARS-CoV-2 variants in the process varied. Additionally, adding newborn calf serum in the collection suspension could effectively preserve viral infectivity in the sampler. Further tests conducted under various ventilation occasions indicated that ventilation can reduce the virus concentration in the environment and rapidly clear viral particles after aerosol generation. Although the flow rate of WA-400III is higher, it only could collect higher concentrations of viral RNA instead of live viruses than AGI-30 or SKC Biosampler when aerosol was generated constantly in the environment. After aerosol generation stopped, the WA-400III collected more infectious viruses and viral RNA. This study highlights the impact of the sampling process on captured virus should be assessed and protective agent is needed to preserve viral viability. And it is crucial to select appropriate sampler based on environmental characteristics and research objectives for obtaining optimal results. The AGI-30 and SKC Biosampler are preferable for collecting infectious viruses in environments with high aerosol concentrations, while the cyclone sampler WA-400III is more effective for collecting viral nucleic acids and enriching virus samples in confined spaces with lower viral loads.

Immunogenicity and Efficacy of Combined mRNA Vaccine Against Influenza and SARS-CoV-2 in Mice Animal Models

Background. The combined or multivalent vaccines are actively used in pediatric practice and offer a series of advantages, including a reduced number of injections and visits to the doctor, simplicity of the vaccination schedule and minimization of side effects, easier vaccine monitoring and storage, and lower vaccination costs. The practice of widespread use of the combined vaccines has shown the potential to increase vaccination coverage against single infections. The mRNA platform has been shown to be effective against the COVID-19 pandemic and enables the development of combined vaccines. There are currently no mRNA-based combined vaccines approved for use in humans. Some studies have shown that different mRNA components in a vaccine can interact to increase or decrease the immunogenicity and efficacy of the combined vaccine. Objectives. In the present study, we investigated the possibility of combining the mRNA vaccines, encoding seasonal influenza and SARS-CoV-2 antigens. In our previous works, both vaccine candidates have shown excellent immunogenicity and efficacy profiles in mice. Methods. The mRNA-LNPs were prepared by microfluidic mixing, immunogenicity in mice was assessed by hemagglutination inhibition assay, enzyme-linked immunoassay and virus neutralization assay. Immunological efficacy was assessed in a mouse viral challenge model. Results. In this work, we demonstrated that the individual mRNA components of the combined vaccine did not affect the immunogenicity level of each other. The combined vaccine demonstrated excellent protective efficacy, providing a 100% survival rate when mice were infected with the H1N1 influenza virus and reducing the viral load in the lungs. Four days after the challenge with SARS-CoV-2 EG.5.1.1., no viable virus and low levels of detectable viral RNA were observed in the lungs of vaccinated mice. Conclusions. The combination does not lead to mutual interference between the individual vaccines. We believe that such a combined mRNA-based vaccine could be a good alternative to separated human vaccinations for the prevention of COVID-19 and influenza.

Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications.

RT-qPCR is commonly used for virological endpoints during clinical trials for antiviral therapy to determine the quantity and presence of virus in a sample. However, RT-qPCR identifies viral RNA and cannot determine if viable virus is present. Existing culture-based techniques for SARS-CoV-2 are insensitive and not sufficiently standardized to be employed as clinical study endpoints. The use of a culture system to monitor replicating viruses could mitigate the possibility of molecular techniques identifying viral RNA from inactive or lysed viral particles. The methodology optimized in this study for detecting infectious viruses may have application as a secondary virological endpoint in clinical trials of therapeutics for SARS-CoV-2 in addition to numerous research processes.

Two neglected contributors to water biosafety risks: The impacts of microbial co-existence and light on viral inactivation

Virus-induced risks to water quality persist, with the viability of viruses in complex aquatic environments impacted by various factors, including coexisting bacteria and exposure to light. This study investigated the disinfection efficacy of UV254 irradiation and free chlorine (FC) on Bacteriophage phiX174 in the presence of coexisting bacteria, specifically Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). To elucidate the underlying mechanisms, this study examined the effects of bacterial compounds (i.e., E. coli lipopolysaccharide (LPS) and B. subtilis peptidoglycan (PGN)) on viral inactivation, thermal stability, and environmental persistence. The research results demonstrated that bacterial presence and their associated compounds significantly impaired viral inactivation, with greater concentrations of coexisting bacteria and their compounds exacerbating this effect. Additionally, the photoreactivation of phiX174 was explored following UV254 irradiation, indicating that high-dose UV254, coupled with low nutrient availability and minimal host bacteria levels, effectively suppressed viral photoreactivation. This study is the first to investigate the multifactorial effects on viral photoreactivation. The results offer novel insights into the challenges associated with virus-related pressures in water treatment, with potential implications for optimizing disinfection processes.

Microcosm experiment investigating climate-induced thermal effects on human virus viability in seawater: qPCR vs capsid integrity for enhanced risk management

Climate change is intensifying extreme weather events in coastal areas, leading to more frequent discharge of untreated wastewater containing human viruses into coastal waters. This poses a health risk, especially during heatwaves when bathing activity increases. A study examined the survival and viability of seven common wastewater viruses in seawater at different temperatures. Viral genomes were quantified using direct qPCR, whilst viability was assessed using Capsid Integrity qPCR. Results showed that T90 values from direct qPCR were much higher than those from CI-qPCR, suggesting that risk mitigation should be based on viral integrity tests. All viruses remained potentially viable for at least 72 h in environmental seawater and longer in sterile artificial seawater, highlighting the importance of biotic processes in viral inactivation. Viral persistence decreased with increasing temperature. Whilst heatwaves may partially reduce risks from human viral pathogens in coastal waters, they do not eliminate them entirely.

Aerodynamic Size-Dependent Collection and Inactivation of Virus-Laden Aerosol Particles in an Electrostatic Precipitator.

Electrostatic precipitators (ESPs) may enable high particle collection efficiency with minimal pressure drop in HVAC systems. However, studies of pathogen collection and inactivation in ESPs at medium to higher flow rates are limited. Here, a single-stage, wire-plate ESP operated at flow rates of 51 and 85 m3 h-1 was used to study the removal of virus-laden aerosol particles for three different airborne viruses: (1) bovine coronavirus (BCoV), (2) influenza A virus (IAV), and (3) porcine reproductive and respiratory virus (PRRSV). Size-resolved measurements of collection efficiency were obtained using Andersen cascade impactors (ACI) sampling upstream and downstream of the ESP. All measurements were analyzed based on three distinctive but complementary methods: (1) fluorimetry to assess physical collection, (2) RT-qPCR to assess viral RNA concentrations and (3) virus titration to assess virus viability. In general, log reductions by virus titration were highest followed by those from RT-qPCR, and last fluorimetry, suggesting that a portion of virus may be potentially inactivated in flight in the ESP. An effective migration (deposition) velocity ranging from 3.10 to 10.05 cm s-1 was also determined using the spatially resolved measurements of virus collection on the ESP plates.

Association between FTO polymorphism and COVID-19 mortality among older adults: A population-based cohort study

ObjectivesCOVID-19 caused a global pandemic with millions of deaths. Fat mass and obesity-associated gene (FTO) (alias m6A RNA demethylase) and its functional rs17817449 polymorphism are candidates to influence COVID-19-associated mortality since methylation status of viral nucleic acids is an important factor influencing viral viability. MethodsWe tested a population-based cohort of 5233 subjects (aged 63-87 years in 2020) where 70 persons died from COVID-19 and 394 from other causes during the pandemic period. ResultsThe frequency of GG homozygotes was higher among those who died from COVID-19 (34%) than among survivors (19%) or deaths from other causes (20%), P <0.005. After multiple adjustments, GG homozygotes had a higher risk of death from COVID-19 with odds ratio = 2.01 (95% confidence interval; 1.19-3.41, P <0.01) compared with carriers of at least one T allele. The FTO polymorphism was not associated with mortality from other causes. ConclusionsOur results suggest that FTO variability is a significant predictor of COVID-19-associated mortality in Caucasians.

ANTIVIRAL TEXTILES AND ANTIVIRAL ACTIVITY TESTING - THE USE OF BACTERIOPHAGE SURROGATE FOR ANTIVIRAL ACTIVITY TESTING

The risk of dissemination of highly contagious viral diseases (as COVID-19, Ebola) led in the increasing need to develop functional textiles and surfaces with antiviral effect. Antiviral textiles are designed to reduce the viability and infectivity of viruses on their surfaces and by this way to reduce the cases of infection (including re-infection or cross-infection with contaminated textiles). Different antiviral agents and diverse techniques of their application are used for functionalized textiles manufacturing. The most often used antivirals are metallic and ionic silver and copper, iron oxide, quaternary ammonium salts. The aim of the process is to prepare textiles with long-term durable finishing effective in viral activity inhibition. The basic step of functionalized antiviral textiles development is antiviral effectivity testing. The safe method of testing with the use of Phi6 bacteriophage, SARS-CoV-2 and Ebola virus surrogate, was modified for antiviral textiles testing. The samples of textiles with antiviral finishing were tested by the bacteriophage-based method and excellent antiviral activity was detected for all tested materials. The woven cotton was used as reference untreated material, the different textile cotton structures with similar square weight were compared and no statistically significant difference was found between the resulting antiviral efficacy values. A simple and quickly feasible screening method for determining the antiviral properties of textiles, especially with leaching-type of treatment, was also designed and tested.

Minimal influenza virus transmission from touching contaminated face masks: a laboratory study

The risk of virus transmission via the touching of contaminated masks has long been assumed by infection control teams. Yet, robust evidence to support this belief has been lacking. This risk was investigated in a laboratory setting by measuring the amount of viable influenza virus successfully transferred from artificially contaminated medical (surgical) mask surfaces to a human finger used to swipe their outer surface under various experimental conditions. Despite being exposed to high levels of virus contamination on the masks, very little or no viable virus was successfully transferred from the mask to the finger in these experiments.

No evidence of spread of Linda pestivirus in the wild boar population in Southern Germany

Lateral-shaking inducing neuro-degenerative agent virus (LindaV) is a novel member of the highly diverse genus Pestivirus within the family Flaviviridae. LindaV was first detected in Austria in 2015 and was associated with congenital tremor in piglets. Since then, the virus or specific antibodies have been found in a few further pig farms in Austria. However, the actual spatial distribution and the existence of reservoir hosts is largely unknown. Since other pestiviruses of pigs such as classical swine fever virus or atypical porcine pestivirus can also infect wild boar, the question arises whether LindaV is likewise present in the wild boar population. Therefore, we investigated the presence of neutralizing antibodies against LindaV in 200 wild boar samples collected in Southern Germany, which borders Austria. To establish a serological test system, we made use of the interchangeability of the surface glycoproteins and created a chimeric pestivirus using Bungowannah virus (species Pestivirus australiaense) as synthetic backbone. The E1 and E2 glycoproteins were replaced by the heterologous E1 and E2 of LindaV resulting in the chimera BV_E1E2_LV. Viable virus could be rescued and was subsequently applied in a neutralization test. A specific positive control serum generated against the E2 protein of LindaV gave a strong positive result, thereby confirming the functionality of the test system. All wild boar samples, however, tested negative. Hence, there is no evidence that LindaV has become highly prevalent in the wild boar population in Southern Germany.

Genetic trans-complementation of L-protease fails to rescue the infectious foot-and-mouth disease virus from the Lbpro defective genome

Outbreaks of the foot-and-mouth disease (FMD) have major economic impact on the global livestock industry by affecting the animal health and product safety. L-protease, a non-structural protein of FMDV, is a papain-like cysteine proteinase involved in viral protein processing as well as cleavage of host proteins for promoting the virus growth. FMDV synthesizes two forms of leader proteinase, Lpro (Labpro and Lbpro), where the deletion of Labpro is lethal and Lbpro deletion is reported to be attenuated. Defective replicons have been used by trans-complementing the deleted gene to produce one time replicating virus; thus, the bio-safety procedure can be compromised in the production units. Attempts are made to rescue of ΔLbproFMDV Asia1 virus by co-expressing the Lbpro protein carried in pcDNA plasmid. Mutant FMDV cDNA, pAsia-ΔLbpro, was constructed by PCR mediated mutagenesis using inverse primers. Transfection of BHK-21 cells with in-vitro transcribed RNA from the constructs failed to produce an infective mutant FMDV. Genetic trans-complementation of the Lbpro, which was done by co-transfecting the pcDNALbpro plasmid DNA along with the pAsia-ΔLbpro RNA in BHK-21 cells also failed to produce viable virus. Expression experiments of reporter genes and indirect immune-fluorescence confirmed the production of the viral proteins in wild type FMDV pAsiaWT; however, it was absent in the pAsia-ΔLbpro indicating that the leaderless virus was unable to produce infectious progeny and infect the cells. Failure to produce virus either by Lbpro deleted mutant clone or by genetic complementation suggests little chance of reversion of the disabled virus with large deletions of FMDV genome.

Inactivation Validation of Ebola, Marburg, and Lassa Viruses in AVL and Ethanol-Treated Viral Cultures.

High-consequence pathogens such as the Ebola, Marburg, and Lassa viruses are handled in maximum-containment biosafety level 4 (BSL-4) laboratories. Genetic material is often isolated from such viruses and subsequently removed from BSL-4 laboratories for a multitude of downstream analyses using readily accessible technologies and equipment available at lower-biosafety level laboratories. However, it is essential to ensure that these materials are free of viable viruses before removal from BSL-4 laboratories to guarantee sample safety. This study details the in-house procedure used for validating the inactivation of Ebola, Marburg, and Lassa virus cultures after incubation with AVL lysis buffer (Qiagen) and ethanol. This study's findings show that no viable virus was detectable when high-titer cultures of Ebola, Marburg, and Lassa viruses were incubated with AVL lysis buffer for 10 min, followed by an equal volume of 95% ethanol for 3 min, using a method with a sensitivity of ≤0.8 log10 TCID50 as the limit of detection.

Inactivation of Highly Pathogenic Avian Influenza Virus with High-temperature Short Time Continuous Flow Pasteurization and Virus Detection in Bulk Milk Tanks

Infections of dairy cattle with clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) were reported in March 2024 in the U.S. and viable virus was detected at high levels in raw milk from infected cows. This study aimed to determine the potential quantities of infectious HPAIV in raw milk in affected states where herds were confirmed positive by USDA for HPAIV (and therefore were not representative of the entire population), and to confirm that the commonly used continuous flow pasteurization using the FDA approved 72 °C (161°F) for 15 s conditions for high−temperature short time (HTST) processing, will inactivate the virus. Double-blinded raw milk samples from bulk storage tanks from farms (n = 275) were collected in four affected states. Samples were screened for influenza A using quantitative real-time RT-PCR (qrRT-PCR) of which 158 (57.5%) were positive and were subsequently quantified in embryonating chicken eggs. Thirty-nine qrRT-PCR positive samples (24.8%) were positive for infectious virus with a median titer of 3.5 log10 50% egg infectious doses (EID50) per mL. To closely simulate commercial milk pasteurization processing systems, a pilot-scale continuous flow pasteurizer was used to evaluate HPAIV inactivation in artificially contaminated raw milk using the most common legal conditions in the US: 72 °C (161°F) for 15 s. Among all replicates at two flow rates (n = 5 at 0.5 L/min; n = 4 at 1 L/min), no viable virus was detected. A mean reduction of ≥5.8 ± 0.2 log10 EID50/mL occurred during the heating phase where the milk is brought to 72.5 °C before the holding tube. Estimates from heat-transfer analysis support that standard U.S. continuous flow HTST pasteurization parameters will inactivate >12 log10 EID50/mL of HPAIV, which is ∼9 log10 EID50/mL greater than the median quantity of infectious virus detected in raw milk from bulk storage tank samples. These findings demonstrate that the US milk supply is safe when pasteurized.

Evaluation of coating materials on the characterization of size and viability of virus-laden particles collected with an Andersen cascade impactor

Airborne pathogens are typically associated with particles, and the transport behavior of these particles is largely driven by their size. To better understand airborne transmission of viral diseases and develop effective control measures, proper size characterization of virus-laden particles is essential. The Andersen cascade impactor (ACI) is an 8-stage air sampler that separates aerosol particles into 9 aerodynamic size fractions. During sampling with an ACI under certain conditions, particles may bounce upon impact with the collection plates of the ACI, leading to eventual deposition on a stage further downstream than their target stage. Coating collection plates with adhesive materials may help decrease particle bounce; however, it may also affect the viability of collected pathogens. In this study, we evaluated different materials for their ability to minimize particle bounce while conserving virus viability during the collection of viral aerosol particles with an ACI. We evaluated nine materials - Tween® 80, silicone oil, Span® 85, Brij® 35, glycerol, mineral oil, gelatin, bovine serum albumin, and virus growth media - on their effect to inactivate H1N1 influenza virus and bovine coronavirus, a surrogate of SARS-CoV-2. Plates coated with gelatin, silicone oil, and mineral oil resulted in the least reduction of viability for both viruses. These materials were then used to sample viral aerosol particles in a wind tunnel. Results of physical particle collection, viral load and viral viability from the various ACI stages revealed no significant differences in aerodynamic size distribution between coated and uncoated plates, and the size distribution was similar to that reported by an optical particle sizer. Overall, our results did not support the need to coat ACI collection plates when characterizing viral aerosol particles under the conditions of this study. However, we did identify potential coating materials which could conserve virus viability maximally, if particle bounce is of concern.

Optimal environmental sampling conditions for electrostatic aerosol-to-hydrosol collection of airborne viruses

Due to adverse effects of viral outbreaks on human health, accurate detection of airborne pathogens is essential. Among many methods available for bioaerosol sampling, electrostatic precipitation (ESP) has been used to directly collect bioaerosols as hydrosols. The performance of an ESP sampler depends on its design, operational and environmental parameters such as air relative humidity (RH), air temperature, sampling liquid type and liquid temperature. Thus, it is essential to identify and maintain optimal conditions throughout sampling process to operate the sampler at its highest capacity. This study provides crucial insights into parameters that affect the collection efficiency of the aerosol-to-hydrosol ESP sampler and its virus recovery. The results indicate that air temperature does not affect collection efficiency, meanwhile, air RH, sampling liquid temperature, and salt concentration are the main parameters that significantly affect collection efficiency. Likewise, when deionized water is used as sampling liquid, hydrogen peroxide concentration increases proportionally with increasing air RH, resulting in significant decrease of virus viability. Consequently, for ESP samplers similar to our study, the following conditions are recommended: air RH of 55–65%, air and sampling liquid temperature of 37 °C, and a mixture of 10–20 mM ascorbic acid in PBS as sampling liquid.

SARS-CoV-2 genome detection and viral viability in breast milk samples of unvaccinated postpartum women.

SARS-CoV-2 genome detection and viral viability in breast milk samples of unvaccinated postpartum women.

Laser wavelength and sample conditioning effects on biochemical monitoring of SARS-CoV-2 VLP production upstream stage by Raman spectroscopy

This work assessed the impact of laser wavelength and sample conditioning on offline monitoring (viable cell density, cell viability, virus titer, glucose, lactate, glutamine, glutamate, and ammonium) of SARS-CoV-2 virus-like particles production upstream stage by Raman spectroscopy. The evaluated chemometrics techniques were Partial Least Squares (PLS) and Artificial Neural Networks (ANN), and different spectral filtering approaches were also considered. ANN showed better prediction capacity for most of the parameters, but ammonium and lactate, better predicted by PLS, and glutamine, no difference between modeling techniques was detected. For cell growth parameters and virus titer, samples without cells measured at 785 nm Raman laser wavelength originated better-adjusted models. This laser wavelength was also more appropriate for the remaining monitored experimental parameters except for glucose, in which the best model came from the spectral database acquired at 1064 nm wavelength. Cell remotion significantly increased the accuracy of viable cell density, cell viability, glutamate, and virus titer models. However, glucose, lactate, and ammonium models showed better prediction capacity for samples containing cells. Thus, it was demonstrated that laser wavelength, sample conditioning, spectral preprocessing, and chemometric modeling techniques need to be tailored for each experimental parameter to improve accuracy.