Viable Virus Research Articles

Exploiting Hydrophobic Amino Acid Scanning to Develop Cyclic Peptide Inhibitors of the SARS-CoV-2 Main Protease with Antiviral Activity.

The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (Mpro) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 Mpro to enhance its cell permeability and in vitro antiviral activity. By identifying mutation-tolerant amino acid residues within the peptide sequence, we describe the development of a second-generation Mpro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS-CoV-2 Mpro.

Duration of Highly Pathogenic Avian Influenza Virus and Newcastle Disease Virus Infectivity in Dried Ornithologic Study Skins.

Ornithologic study skins are specimens of avian skins that have been preserved by drying after removing the viscera and muscle. Because of the high value of study skins for scientific studies, specimens are shared among researchers. There is concern that study skins might be contaminated with high-consequence diseases such as highly pathogenic avian influenza virus (HPAIV) or Newcastle disease virus (NDV). To mitigate risk, thermal or chemical treatment of study skins may be required before transfer; however, such treatments might damage the specimens. Therefore, a study was conducted to evaluate the duration of infectivity of HPAIV and NDV in study skins prepared from infected chickens (Gallus gallus). Study skins were prepared from 10 chickens infected with each virus. Skin and feather pulp samples were taken at the time of study skin preparation to establish starting titers. Mean starting titers in the skin was 4.2 log10 and 5.1 log10 50% egg infectious doses (EID50) for HPAIV and NDV groups respectively, and were 6.7 log10 EID50 for HPAIV, and 6.4 log10 EID50 for NDV in feather pulp. Samples were collected at 2 and 4 wk of drying to quantify viable virus. At 2 wk, fewer samples had detectable virus and mean titers were 1.8 log10 (skin) and 2.1 log10 (feathers) EID50 for HPAIV, and 1.7 log10 (skin) and 3.5 log10 (feathers) EID50 for NDV. At 4 wk viable virus could not be detected in either tissue type.

Neuropathogenesis of SARS-CoV-2 in human neuronal, microglial and glial cells.

Neurological complications, both acute and chronic, are reported commonly in COVID-19 affected individuals. In this context, the understanding of pathogenesis of SARS-CoV-2 in specific cells of central nervous system (CNS) origin is relevant. The present study explores infection biology of a clinical isolate of SARS-CoV-2 in human cell lines of neural origin such as the glioblastoma (U87-MG), neuroblastoma (SHSY5Y) and microglia (C20). Despite showing clear evidence of infection by immunofluorescence with an anti-spike protein antibody, all the three neural cell lines were observed to be highly restrictive to the replication of the infecting virus. While the U87-MG glioblastoma cells demonstrated no cytopathic effects and a low viral titre with no signs of replication, the SHSY5Y neuroblastoma cells exhibited cytopathic effects with bleb formation but no evidence of viable virus. The C20 microglial cells showed neither signs of cytopathic effects nor viable virus. Ultrastructural studies demonstrated intracellular virions in infected neural cells. The presence of lipid droplets in infected SHSY5Y cells suggested an impact on host cell metabolism. The decrease in viral RNA levels over time in all the neural cell lines suggested restricted viral replication. In conclusion, this study highlights the limited susceptibility of neural cells to SARS-CoV-2 infection. This reduced permissibility of neural cell lines to SARS-CoV-2 may point to their inherent lower expression of receptors that support viral entry in addition to the intracellular factors that potently inhibit viral replication. The study findings prompt further investigation into the mechanisms of SARS-CoV-2 infection of neural cells.

How I treat: Coronavirus disease 2019 in leukemic patients and hematopoietic cell transplant recipients.

Among immunocompromised hosts, leukemia patients, and hematopoietic cell transplant recipients are particularly vulnerable, facing challenges in balancing coronavirus disease 2019 (COVID-19) management with their underlying conditions. In this How I Treat article, we discuss how we approach severe acute respiratory syndrome coronavirus 2 infections in daily clinical practice, considering the existing body of literature and for topics where the available data are not sufficient to provide adequate guidance, we provide our opinion based on our clinical expertise and experience. Diagnostic approaches include nasopharyngeal swabs for polymerase chain reaction testing and chest computed tomography scans for symptomatic patients at risk of disease progression. Preventive measures involve strict infection control protocols and prioritizing vaccination for both patients and their families. Decisions regarding chemotherapy or hematopoietic cell transplantation in leukemia patients with COVID-19 require careful consideration of factors such as COVID-19 severity and treatment urgency. Treatment protocols include early initiation of antiviral therapy, with nirmatrelvir/ritonavir or remdesivir. For cases of prolonged viral shedding, distinguishing between viable and non-viable viruses remains challenging but is crucial for determining contagiousness and guiding management decisions. Overall, individualized approaches considering immune status, clinical presentation, and viral kinetics are essential for effectively managing COVID-19 in leukemia patients.

Investigating the effectiveness of commercially available mouthwash on SARS-CoV-2 in vivo using viable virus titre as the primary outcome. A randomised controlled trial.

This multi-arm, parallel group, single-blinded randomised controlled trial aimed to assess three commercially available mouthwashes effectiveness against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This manuscript has been written in accordance with the CONSORT statement. Methods. Eligible participants were SARS-CoV-2 positive with a positive test in the last 72 h. All participants had mild to moderate symptoms and could provide five saliva samples over a 60 min period. Participants delivered a baseline saliva sample and then used a mouthwash as per manufacturer's instructions. They provided further saliva samples at minute 1, 10, 30 and 60. Participants were randomised to one of four groups; OraWize+, Total Care Listerine, Cool Mint Listerine and water (control). The lab-based research team were blind to the intervention. The research question was: can SARS-CoV-2 be rendered inactive in saliva by using a mouthwash and how long does this effect last? The primary outcome was the amount of viable infectious SARS-CoV-2 virus in the sample, compared to the baseline sample. The secondary outcome measure was the amount of genetic material from the SARS-CoV-2 virus in the sample, measured via PCR testing. Results. In total 100 participants were recruited (25 per group). Eight participants did not receive the allocated intervention and did not have saliva samples collected. There were no adverse events. In total 42 of the 92 participants had viable virus which could be cultured at baseline. Statistical analysis of the primary outcome was not advised due to the reduced level of viable virus at baseline and the positive skewness present in the distribution of log10(titre) data. Observational data of the primary outcome measure is presented. Analysis of the secondary outcome PCR measure showed that there was strong evidence for a decrease in SARS-CoV-2 RNA levels compared to water for all mouthwashes after 1 min, OraWize+ -0.49 (-0.92, -0.05), p-value 0.029, Cool Mint Listerine -0.81 (-1.25, -0.38), p-value<0.001, Total Care Listerine -1.05 (-1.48, -0.62), p-value<0.001. For the remaining timepoints there was generally no evidence of virus level reduction compared to water although there is weak evidence for a decrease at ten minutes using Total Care Listerine -0.44 (-0.88, 0.01), p-value 0.053. Conclusion. The three mouthwashes included in this trial observationally demonstrated a reduction in virus titre level 1 min after use, with virus levels normalising up to 60 min compared to the control. Although an interesting observation, this result could not be statistically analysed. Using the secondary outcome PCR measure all three included mouthwashes reduced virus levels compared to water at 1 min and these results were statistically significant. Clinically this result does not support the use of the included mouthwashes to reduce SARS-CoV-2 levels in saliva.

Phenotypes of cytomegalovirus genetic variants encountered in a letermovir clinical trial illustrate the importance of genotyping validation

Emergence of drug resistance is rare after use of letermovir (LMV) as prophylaxis for post-transplant cytomegalovirus (CMV) infection. In a recent study involving renal transplant recipients, no known LMV resistance mutations were detected in those receiving LMV prophylaxis. However, uncharacterized viral amino acid substitutions were detected in LMV recipients by deep sequencing in viral subpopulations of 5%–7%, at codons previously associated with drug resistance: UL56 S229Y (n = 1), UL56 M329I (n = 9) and UL89 D344Y (n = 5). Phenotypic analysis of these mutations in a cloned laboratory CMV strain showed that S229Y conferred a 2-fold increase in LMV EC50, M329I conferred no LMV resistance, and D344Y knocked out viral viability that was restored after the nonviable clone was reverted to wild type D344. As in previous CMV antiviral trials, the detection of nonviable mutations, even in multiple study subjects, raises strong suspicion of genotyping artifacts and encourages the use of replicate testing for authentication of atypical mutation readouts. The non-viability of UL89 D344Y also confirms the biologically important locus of the D344E substitution that confers resistance to benzimidazole CMV terminase complex inhibitors, but does not feature prominently in LMV resistance.

Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.

Deformed wing virus in bee bread: infectivity and thermal inactivation

Bee-collected pollen is stored in the colony and mixed with bee saliva and microorganisms, resulting in pollen’s fermentation and acidification. Pathogens infecting the colony, including the deformed wing virus (DWV), may be transferred into the bee bread (BB). In this study, we used an in vivo infection assay to determine whether DWV remains infective in BB. We also used Lactobacillus kunkeei to ferment UV-treated fresh pollen. This fermented pollen in vitro (FPV) was used as a virus-free reference for the initial viral loads in BB and to evaluate the effect of controlled bacterial fermentation on the infectivity of DWV. Finally, we investigated the thermal inactivation of DWV in BB and FPV. To accomplish these goals, we added DWV inoculum to BB and FPV and heated them at 70 °C or 60 °C for 1 h. Suspensions of the virus recovered from the treated pollen samples were injected into pupae to evaluate the degree of viral viability and the efficacy of thermal inactivation. We found that the virus recovered from non-thermally treated BB and FPV samples remained highly infectious in the pupal injection assay. Overall, BB and FPV treated at 60 °C had an average reduction of 98.69% of viable DWV copies, while treatments at 70 °C inactivated DWV almost entirely (99.99%). This study demonstrates that DWV remains infective in BB and that thermal inactivation is an effective strategy for significantly reducing levels of viable DWV in contaminated pollen.

PSI-5 Evaluation of EhV (surrogate for African swine fever virus) inactivation in corn- and soybean-based feed ingredients and diets at various times and temperatures during storage

Abstract African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA viruses (NCLDVs) and remains stable in a variety of environments, including animal feed ingredients as shown in previous laboratory experiments and simulations. Emiliania huxleyi virus (EhV) is another member of NCLDV, which has a restricted host range limited to a species of marine algae called Emiliania huxleyi. This algal NCLDV has many similar morphological and physical characteristics to ASFV which makes it a safe and reliable surrogate for studying ASFV survival and inactivation in feed ingredient matrices in-situ under various time and temperature storage conditions. The objective of the present study was to investigate the inactivation of ASFV in various feedstuffs under a wide range of time and temperature conditions during storage using EhV as a surrogate. Corn- and soybean-based feed ingredients including corn, corn fermented protein, distiller dried grains with solubles, extruded soybean meal, high protein distiller’s grains, solvent-extracted soybean meal, soybean hulls, complete feed, and a positive control (no matrix) were inoculated with EhV (1 × 108 viruses/mL). Negative controls were inoculated with medium containing no EhV for each matrix. To assess the effects of different times and temperatures of storage, all samples were stored at temperatures of 4, 24, and 34 °C for 1, 5, 60, and 120 d. For each timepoint and temperature combination, samples were evaluated for virus presence (qPCR) and viability using a previously validated viability qPCR (v-qPCR) method. On average, we observed a 0.2 log reduction in viable viral DNA content over the 120-d period, with a 0.1 log reduction across varying temperatures. No reductions in EhV viability exceeding 1 log were detected for any matrix and the positive control at any time and temperature combination evaluated. No EhV was recovered from the negative control samples. Given that the experimental error was calculated to be within the range of 1 log, we concluded that storing EhV at 4, 24, or 34 °C for up to 120 d did not result in the inactivation of EhV in any of the ingredients, complete feed, or the control with no matrix. These results demonstrate that ASFV-like NCLDVs is extremely resilient and can maintain viability in various feed matrices during long-term storage and suggest that extending storage time alone may not be an effective mitigation practice against African swine fever virus.

Human coronavirus 229E inactivation on porous and nonporous materials using dielectric barrier discharge (DBD) plasma.

Viruses can spread through contaminated aerosols and contaminated surface materials, and effective disinfection techniques are essential for virus inactivation. Nonthermal plasma-generated reactive oxygen and nitrogen species can effectively inactivate the coronavirus. We aim to interpret the coronavirus inactivation level and mechanism of surface interaction with materials with and without dielectric barrier discharge (DBD) plasma treatment. Nonthermal plasma, particularly surface-type DBD plasma, can inactivate human coronavirus 229E (HCoV-229E) on porous (paper, wood, mask) and nonporous (plastic, stainless steel, glass, Cu) materials. Virus inactivation was analyzed using a 50% tissue culture infectivity dose (TCID50) using cell line, flow cytometry, and immunofluorescence. Surfaces contaminated with HCoV-229E were treated at different time intervals (0-5 h) with and without plasma exposure (natural decay in ambient air conditions). HCoV-229E persistence conformed to the following order: plastic > cover glass > stainless steel > mask > wood > paper > Cu with and without plasma exposure. HCoV-229E was more stable in plastic, cover glass, and stainless steel in 5 h, and the viable virus titer gradually decreased from its initial log10 order of 6.892 to 1.72, 1.53, and 1.32 TCID50/mL, respectively, under plasma exposure. No virus was observed in Cu after treatment for 5 h. The use of airflow, ambient nitrogen, and argon did not promote virus inactivation. Flow cytometry and immunofluorescence analysis demonstrated a low expression level of spike protein (fluorescence intensity) during plasma treatment and in E and M genes expression compared with the virus control.

Safety, tolerability, viral kinetics, and immune correlates of protection in healthy, seropositive UK adults inoculated with SARS-CoV-2: a single-centre, open-label, phase 1 controlled human infection study

A SARS-CoV-2 controlled human infection model (CHIM) has been successfully established in seronegative individuals using a dose of 1×101 50% tissue culture infectious dose (TCID50) pre-alpha SARS-CoV-2 virus. Given the increasing prevalence of seropositivity to SARS-CoV-2, a CHIM that could be used for vaccine development will need to induce infection in those with pre-existing immunity. Our aim was to find a dose of pre-alpha SARS-CoV-2 virus that induced infection in previously infected individuals. Healthy, UK volunteers aged 18-30 years, with proven (quantitative RT-PCR or lateral flow antigen test) previous SARS-CoV-2 infection (with or without vaccination) were inoculated intranasally in a stepwise dose escalation CHIM with either 1×101, 1×102, 1×10³, 1×104, or 1×105 TCID50 SARS-CoV-2/human/GBR/484861/2020, the same virus used in the seronegative CHIM. Post-inoculation, volunteers were quarantined in functionally negative pressure rooms (Oxford, UK) for 14 days and until 12-hourly combined oropharyngeal-nasal swabs were negative for viable virus by focus-forming assay. Outpatient follow-up continued for 12 months post-enrolment, with additional visits for those who developed community-acquired SARS-CoV-2 infection. The primary objective was to identify a safe, well tolerated dose that induced infection (defined as two consecutive SARS-CoV-2 positive PCRs starting 24 h after inoculation) in 50% of seropositive volunteers. This study is registered with ClinicalTrials.gov (NCT04864548); enrolment and follow-up to 12 months post-enrolment are complete. Recruitment commenced on May 6, 2021, with the last volunteer enrolled into the dose escalation cohort on Nov 24, 2022. 36 volunteers were enrolled, with four to eight volunteers inoculated in each dosing group from 1×101 to 1×105 TCID50 SARS-CoV-2. All volunteers have completed quarantine, with follow-up to 12 months complete. Despite dose escalation to 1×105 TCID50, we were unable to induce sustained infection in any volunteers. Five (14%) of 36volunteers were considered to have transient infection, based on the kinetic of their PCR-positive swabs. Transiently infected volunteers had significantly lower baseline mucosal and systemic SARS-CoV-2-specific antibody titres and significantly lower peripheral IFNγ responses against a CD8+ T-cell SARS-CoV-2 peptide pool than uninfected volunteers. 14 (39%) of 36 volunteers subsequently developed breakthrough infection with the omicron variant after discharge from quarantine. Most adverse events reported by volunteers in quarantine were mild, with fatigue (16 [44%]) and stuffy nose (16 [44%]) being the most common. There were no serious adverse events. Our study demonstrates potent protective immunity induced by homologous vaccination and homologous or heterologous previous SARS-CoV-2 infection. The community breakthrough infections seen with the omicron variant supports the use of newer variants to establish a model with sufficient rate of infection for use in vaccine and therapeutic development. Wellcome Trust and Department for Health and Social Care.

Oral mouthwashes for asymptomatic to mildly symptomatic adults with COVID-19 and salivary viral load: a randomized, placebo-controlled, open-label clinical trial

BackgroundRecent randomized clinical trials suggest that the effect of using cetylpyridinium chloride (CPC) mouthwashes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in COVID-19 patients has been inconsistent. Additionally, no clinical study has investigated the effectiveness of on-demand aqueous chlorine dioxide mouthwash against COVID-19.MethodsWe performed a randomized, placebo-controlled, open-label clinical trial to assess for any effects of using mouthwash on the salivary SARS-CoV-2 viral load among asymptomatic to mildly symptomatic adult COVID-19-positive patients. Patients were randomized to receive either 20 mL of 0.05% CPC, 10 mL of 0.01% on-demand aqueous chlorine dioxide, or 20 mL of placebo mouthwash (purified water) in a 1:1:1 ratio. The primary endpoint was the cycle threshold (Ct) values employed for SARS-CoV-2 salivary viral load estimation. We used linear mixed-effects models to assess for any effect of the mouthwashes on SARS-CoV-2 salivary viral load.ResultsOf a total of 96 eligible participants enrolled from November 7, 2022, to January 19, 2023, 90 were accepted for the primary analysis. The use of 0.05% CPC mouthwash was not shown to be superior to placebo in change from baseline salivary Ct value at 30 min (difference vs. placebo, 0.640; 95% confidence interval [CI], -1.425 to 2.706; P = 0.543); 2 h (difference vs. placebo, 1.158; 95% CI, -0.797 to 3.112; P = 0.246); 4 h (difference vs. placebo, 1.283; 95% CI, -0.719 to 3.285; P = 0.209); 10 h (difference vs. placebo, 0.304; 95% CI, -1.777 to 2.385; P = 0.775); or 24 h (difference vs. placebo, 0.782; 95% CI, -1.195 to 2.759; P = 0.438). The use of 0.01% on-demand aqueous chlorine dioxide mouthwash was also not shown to be superior to placebo in change from baseline salivary Ct value at 30 min (difference vs. placebo, 0.905; 95% CI, -1.079 to 2.888; P = 0.371); 2 h (difference vs. placebo, 0.709; 95% CI, -1.275 to 2.693; P = 0.483); 4 h (difference vs. placebo, 0.220; 95% CI, -1.787 to 2.226; P = 0.830); 10 h (difference vs. placebo, 0.198; 95% CI, -1.901 to 2.296; P = 0.854); or 24 h (difference vs. placebo, 0.784; 95% CI, -1.236 to 2.804; P = 0.447).ConclusionsIn asymptomatic to mildly symptomatic adults with COVID-19, compared to placebo, the use of 0.05% CPC and 0.01% on-demand aqueous chlorine dioxide mouthwash did not lead to a significant reduction in SARS-CoV-2 salivary viral load. Future studies of the efficacy of CPC and on-demand aqueous chlorine dioxide mouthwash on the viral viability of SARS-CoV-2 should be conducted using different specimen types and in multiple populations and settings.

Environmental health of water bodies from a Brazilian Amazon Metropolis based on a conventional and metagenomic approach.

The present study aimed to use a conventional and metagenomic approach to investigate the microbiological diversity of water bodies in a network of drainage channels and rivers located in the central area of the city of Belém, northern Brazil, which is considered one of the largest cities in the Brazilian Amazon. In eight of the analyzed points, both bacterial and viral microbiological indicators of environmental contamination-physical-chemical and metals-were assessed. The bacterial resistance genes, drug resistance mechanisms, and viral viability in the environment were also assessed. A total of 473 families of bacteria and 83 families of viruses were identified. Based on the analysis of metals, the levels of three metals (Cd, Fe, and Mn) were found to be above the recommended acceptable level by local legislation. The levels of the following three physicochemical parameters were also higher than recommended: biochemical oxygen demand, dissolved oxygen, and turbidity. Sixty-three bacterial resistance genes that conferred resistance to 13 different classes of antimicrobials were identified. Further, five mechanisms of antimicrobial resistance were identified and viral viability in the environment was confirmed. Intense human actions combined with a lack of public policies and poor environmental education of the population cause environmental degradation, especially in water bodies. Thus, urgent interventions are warranted to restore the quality of this precious and scarce asset worldwide.

Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A: Impacts on rescue of sequence-modifying viruses

Senecavirus A (SVA) has a positive-sense, single-stranded RNA genome. Its 5´ untranslated region harbors an internal ribosome entry site (IRES), comprising 10 larger or smaller stem-loop structures (including a pseudoknot) that have been demonstrated to be well conserved. However, it is still unclear whether each stem-loop subdomain, such as a single stem or loop, is also highly conserved. To clarify this issue in the present study, a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis (SDM) on the IRES. The SDM-modified scenarios included: (1) stem-formed complementary sequences exchanging with each other; (2) loop transversion; (3) loop transition; and (4) point mutations. All cDNA clones were separately transfected into cells for rescuing viable viruses, whereas only four SVAs of interest could be recovered, and were genetically stable during 20 passages. One progeny grew significantly slower than the other three did. The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity. Our previous study indicated that a single motif from any of the ten stem structures, if completely mutated, would cause the failure of virus recovery. Interestingly, our present study revealed three stem structures, whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs. Moreover, one apical loop was demonstrated to have the ability to tolerate its own full-length transition, also having no impact on the recovery of sequence-modifying SVA. The present study suggested that not every stem-loop structure was strictly conserved in its conformation, while the full-length IRES itself was well conserved. This provides a new research direction on interaction between the IRES and many factors.

Relative Humidity-Dependent Phase Transitions in Submicron Respiratory Aerosols.

Respiratory viruses, such as influenza and severe acute respiratory syndrome coronavirus 2, represent a substantial public health burden and are largely transmitted through respiratory droplets and aerosols. Environmental factors such as relative humidity (RH) and temperature impact virus transmission rates, and a precise mechanistic understanding of the connection between these environmental factors and virus transmission would improve efforts to mitigate respiratory disease transmission. Previous studies on supermicrometer particles observed RH-dependent phase transitions and linked particle phase state to virus viability. Phase transitions in atmospheric aerosols are dependent on size in the submicrometer range, and actual respiratory particles are expelled over a large size range, including submicrometer aerosols that can transmit diseases over long distances. Here, we directly investigated the phase transitions of submicrometer model respiratory aerosols. A probe molecule, Nile red, was added to particle systems including multiple mucin/salt mixtures, a growth medium, and simulated lung fluid. For each system, the polarity-dependent fluorescence emission was measured following RH conditioning. Notably, the fluorescence measurements of mucin/NaCl and Dulbecco's modified Eagle's medium particles indicated that liquid-liquid phase separation (LLPS) also occurs in submicron particles, suggesting that LLPS can also impact the viability of viruses in submicron particles and thus affect aerosol virus transmission. Furthermore, the utility of fluorescence-based measurements to study submicrometer respiratory particle physicochemical properties in situ is demonstrated.

Quantification and Potential Viability of Human Noroviruses in Final Effluent from Wastewater Treatment Works in Pretoria, South Africa

Growing global concerns over water scarcity, worsened by climate change, drive wastewater reclamation efforts. Inadequately treated wastewater presents significant public health risks. Previous studies in South Africa (SA) have reported high norovirus levels in final effluent and sewage-polluted surface water, indicating pathogen removal inefficiency. However, the viability of these virions was not explored. This study assessed human norovirus viability in final effluent from wastewater treatment works (WWTWs) in Pretoria, SA. Between June 2018 and August 2020, 200 samples were collected from two WWTWs, including raw sewage and final effluent. Norovirus concentrations were determined using in-house RNA standards. Viability of noroviruses in final effluent was assessed using viability RT-qPCR (vPCR) with PMAxx™-Triton X-100. There was no significant difference in GI concentrations between raw sewage (p = 0.5663) and final effluent (p = 0.4035) samples at WWTW1 and WWTW2. WWTW1 had significantly higher GII concentrations in raw sewage (p < 0.001) compared to WWTW2. No clear seasonal pattern was observed in norovirus concentrations. At WWTW1, 50% (7/14) of GI- and 64.9% (24/37) of GII-positive final effluent samples had no quantifiable RNA after vPCR. At WWTW2, the majority (92.6%, 25/27) of GII-positive final effluent samples showed a 100% RNA reduction post vPCR. PMAxx™-Triton X-100 vPCR provides a more accurate reflection of discharge of potentially viable noroviruses in the environment than standard RT-qPCR. Despite significant reductions in potentially viable noroviruses after wastewater treatment, the levels of potentially viable viruses in final effluent are still of concern due to the high initial load and low infectious dose of noroviruses.

Phase II Trial of the Impact 0.5% Povidone-Iodine Nasal Spray (Nasodine®) on Shedding of SARS-CoV-2.

A Phase II trial was conducted to determine if nasal disinfection with a commercial Good Manufacturing Practice-manufactured 0.5% povidone-iodine nasal spray (Nasodine®) may be a useful adjunct in the management of COVID-19 by reducing viral shedding and prevention of transmission of SARS-CoV-2. The aim was to confirm the results from a human single-dose pilot study by assessing repeated and frequent doses on nasal shedding of SARS-CoV-2 from adult subjects with confirmed COVID-19. A multicenter, randomized, double-blinded, placebo-controlled Phase II clinical trial involving adults with early COVID-19 symptoms. Baseline nasal swabs were collected to quantify pretreatment SARS-CoV-2 nasal viral load, followed by Nasodine treatment eight times daily over 3 calendar days. Daily nasal swabs were collected post-dose to assess the impact of treatment on nasal viral load, measured by log10 TCID50 in quantitative culture. Nasodine subjects exhibited significantly improved reduction in viral load (log10 TCID50) on Days 2-4 compared to placebo recipients (p = 0.028), rate of nasal clearance of viable virus (p = 0.032), and complete (100%) nasal and throat clearance of the virus by Day 5. No difference was seen in antigen shedding as measured by time transition from Rapid Antigen Test (RAT) positivity to RAT negativity. A total of 20 doses of Nasodine® nasal spray administered over 2.5 days significantly reduced the titers of viable SARS-CoV-2 virus in the nasal passages of COVID-19 subjects. This is the first study demonstrating the efficacy of a tolerable intranasal formulation of povidone-iodine on viral shedding in COVID-19 subjects. Nasal disinfection may diminish viral transmission to others. 2 Laryngoscope, 134:3947-3952, 2024.

SARS-CoV-2 live virus culture and sample freeze-thaw stability

The effect of freeze-thaw on SARS-CoV-2 viral viability is not well established. We isolated virus from 31 split clinical samples cultured fresh or after a 7- or 17/18-day freeze. We found that freeze-thaw did not significantly affect viral culture isolation. Therefore, frozen samples may be used to assess SARS-CoV-2 infectiousness.

SARS-CoV-2 Contamination on Healthy Individuals' Hands in Community Settings During the COVID-19 Pandemic.

Introduction Hand hygiene is an infection control measure for COVID-19 in our daily lives; however, the contamination levels of SARS-CoV-2 in the hands of healthy individuals remain unclear. Thus, we aimed to evaluate SARS-CoV-2 contamination levels by detecting viral RNA and viable viruses in samples obtained from the hands of 925 healthy individuals. Methods Swab samples were collected from the palms and fingers of healthy participants, including office workers, public officers, university students, university faculty and staff, and hospital staff between December 2022 and March 2023. The collected swab samples were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for SARS-CoV-2 RNA detection. Viral RNA-positive samples were subjected to plaque assay to detect viable viruses. Results We collected 1,022 swab samples from the hands of healthy participants. According to the criteria for data collection, 97 samples were excluded, and 925 samples were analyzed using RT-qPCR. SARS-CoV-2 RNA was detected in three of the 925 samples. The viral RNA detection rate was 0.32% (3/925), and the viral RNA copy numbers ranged from 5.0×103to 1.7×105copies/mL. The RT-qPCR-positive samples did not contain viable viruses, as confirmed by the plaque assay results. Conclusions The detection rate of SARS-CoV-2 RNA from the hands of healthy individuals was extremely low, and no viable viruses were detected. These results suggest that the risk of contact transmission via hands in a community setting is extremely rare.

Randomized controlled trial of molnupiravir SARS-CoV-2 viral and antibody response in at-risk adult outpatients

Viral clearance, antibody response and the mutagenic effect of molnupiravir has not been elucidated in at-risk populations. Non-hospitalised participants within 5 days of SARS-CoV-2 symptoms randomised to receive molnupiravir (n = 253) or Usual Care (n = 324) were recruited to study viral and antibody dynamics and the effect of molnupiravir on viral whole genome sequence from 1437 viral genomes. Molnupiravir accelerates viral load decline, but virus is detectable by Day 5 in most cases. At Day 14 (9 days post-treatment), molnupiravir is associated with significantly higher viral persistence and significantly lower anti-SARS-CoV-2 spike antibody titres compared to Usual Care. Serial sequencing reveals increased mutagenesis with molnupiravir treatment. Persistence of detectable viral RNA at Day 14 in the molnupiravir group is associated with higher transition mutations following treatment cessation. Viral viability at Day 14 is similar in both groups with post-molnupiravir treated samples cultured up to 9 days post cessation of treatment. The current 5-day molnupiravir course is too short. Longer courses should be tested to reduce the risk of potentially transmissible molnupiravir-mutated variants being generated. Trial registration: ISRCTN30448031