Reduction In Viral Load Research Articles

Immunogenicity and protective efficacy of a rhesus adenoviral vaccine targeting conserved COVID-19 replication transcription complex

The COVID-19 pandemic marks the third coronavirus pandemic this century (SARS-CoV-1, MERS, SARS-CoV-2), emphasizing the need to identify and evaluate conserved immunogens for a pan-sarbecovirus vaccine. Here we investigate the potential utility of a T-cell vaccine strategy targeting conserved regions of the sarbecovirus proteome. We identified the most conserved regions of the sarbecovirus proteome as portions of the RNA-dependent RNA polymerase (RdRp) and Helicase proteins, both of which are part of the coronavirus replication transcription complex (RTC). Fitness constraints suggest that as SARS-CoV-2 continues to evolve these regions may better preserve cross-reactive potential of T-cell responses than Spike, Nucleocapsid, or Membrane proteins. We sought to determine if vaccine-elicited T-cell responses to the highly conserved regions of the RTC would reduce viral loads following challenge with SARS-CoV-2 in mice using a rhesus adenovirus serotype 52 (RhAd52) vector. The RhAd52.CoV.Consv vaccine generated robust cellular immunity in mice and led to significant reductions in viral loads in the nasal turbinates following challenge with a mouse-adapted SARS-CoV-2. These data suggest the potential utility of T-cell targeting of conserved regions for a pan-sarbecovirus vaccine.

Reduction of SARS-CoV-2 viral load in exhaled air by antiseptic chewing gum: a pilot trial

PurposeThe dominant route of transmission of SARS-CoV-2 is airborne, through respiratory transmission by aerosols or droplets which can be measured by viral load in exhaled air. Several natural substances have shown antiviral activity. The aim of this pilot study was to investigate the effect of a chewing gum containing natural antiseptic ingredients (cinnamon-, peppermint- and lemon-oil, quercetin, spermidine, ginger and ginseng) on viral load in exhalative air in patients infected with SARS-CoV-2.MethodsNine patients infected with SARS-CoV-2 were enrolled and exhaled forcefully into a special mouthpiece at different time points before and after chewing the antiseptic gum. The mouthpiece contained a filter paper serving for extraction of coronaviruses following real-time PCR to quantify the viral load.Results and conclusionCycle threshold (Ct) values of all patients increased after chewing the gum. The mean difference between the Ct values at baseline (before chewing the antiseptic gum) and time point 30 min (15 min after chewing) was 3.8 ± 2.6; (93% viral load reduction; p = 0.002). Time point 15 min (2.7 ± 1.7 (83% viral load reduction; p = 0.003)), 60 min (3.0 ± 3.4 (88% viral load reduction; p = 0.028)), 90 min (3.7 ± 1.8 (92% viral load reduction; p = 0.004)) and 120 min (3.0 ± 3.7 (91% viral load reduction; p = 0.05)) showed similar results. The antiseptic chewing gum demonstrated a significant potential to reduce SARS-CoV-2 viral load in exhalative air and, in this way, reduce further spread and infection risk. Larger placebo-controlled clinical trials are required to confirm these findings further.

Superior antiviral activity of IFNβ in genital HSV-1 infection.

Type I interferons (IFNs) present the first line of defense against viral infections, providing antiviral, immunomodulatory and antiproliferative effects. The type I IFN family contains 12 IFNα subtypes and IFNβ, and although they share the same receptor, they are classified as non-redundant, capable to induce a variety of different IFN-stimulated genes. However, the biological impact of individual subtypes remains controversial. Recent data propose a subtype-specificity of type I IFNs revealing unique effector functions for different viruses and thus expanding the implications for IFNα-based antiviral immunotherapies. Despite extensive research, drug-resistant infections with herpes simplex virus type 1 (HSV-1), which is the common agent of recurrent orogenital lesions, are still lacking a protective or curing therapeutic. However, due to the risk of generalized infections in immunocompromised hosts as well as the increasing incidence of resistance to conventional antiherpetic agents, HSV infections raise major health concerns. Based on their pleiotropic effector functions, the application of type I IFNs represents a promising approach to inhibit HSV-1 replication, to improve host immunity and to further elucidate their qualitative differences. Here, selective IFNα subtypes and IFNβ were evaluated for their therapeutic potential in genital HSV-1 infections. Respective in vivo studies in mice revealed subtype-specific differences in the reduction of local viral loads. IFNβ had the strongest antiviral efficacy against genital HSV-1 infection in mice, whereas IFNα1, IFNα4, and IFNα11 had no impact on viral loads. Based on flow cytometric analyses of underlying immune responses at local and peripheral sites, these differences could be further assigned to specific modulations of the antiviral immunity early during HSV-1 infection. IFNβ led to enhanced systemic cytokine secretion and elevated cytotoxic responses, which negatively correlated with viral loads in the vaginal tract. These data provide further insights into the diversity of type I IFN effector functions and their impact on the immunological control of HSV-1 infections.

Artificial Cell Membrane Polymersome-Based Intranasal Beta Spike Formulation as a Second Generation Covid-19 Vaccine.

Current parenteral coronavirus disease 2019 (Covid-19) vaccines inadequately protect against infection of the upper respiratory tract. Additionally, antibodies generated by wild type (WT) spike-based vaccines poorly neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. To address the need for a second-generation vaccine, we have initiated a preclinical program to produce and evaluate a potential candidate. Our vaccine consists of recombinant Beta spike protein coadministered with synthetic CpG adjuvant. Both components are encapsulated within artificial cell membrane (ACM) polymersomes, synthetic nanovesicles efficiently internalized by antigen presenting cells, including dendritic cells, enabling targeted delivery of cargo for enhanced immune responses. ACM vaccine is immunogenic in C57BL/6 mice and Golden Syrian hamsters, evoking high serum IgG and neutralizing responses. Compared to an ACM-WT spike vaccine that generates predominantly WT-neutralizing antibodies, the ACM-Beta spike vaccine induces antibodies that neutralize WT and Beta viruses equally. Intramuscular (IM)-immunized hamsters are strongly protected from weight loss and other clinical symptoms after the Beta challenge but show delayed viral clearance in the upper airway. With intranasal (IN) immunization, however, neutralizing antibodies are generated in the upper airway concomitant with rapid and potent reduction of viral load. Moreover, antibodies are cross-neutralizing and show good activity against Omicron. Safety is evaluated in New Zealand white rabbits in a repeated dose toxicological study under Good Laboratory Practice (GLP) conditions. Three doses, IM or IN, at two-week intervals do not induce an adverse effect or systemic toxicity. Cumulatively, these results support the application for a Phase 1 clinical trial of ACM-polymersome-based Covid-19 vaccine (ClinicalTrials.gov identifier: NCT05385991).

Airway-delivered short-chain fatty acid acetate boosts antiviral immunity during rhinovirus infection

Microbiota are recognized to play a major role in regulation of immunity through release of immunomodulatory metabolites such as short-chain fatty acids (SCFAs). Rhinoviruses (RVs) induce upper respiratory tract illnesses and precipitate exacerbations of asthma and chronic obstructive pulmonary disease through poorly understood mechanisms. Local interactions between SCFAs and antiviral immune responses in the respiratory tract have not been previously investigated. We sought to investigate whether pulmonary metabolite manipulation through lung-delivered administration of SCFAs can modulate antiviral immunity to RV infection. We studied the effects of intranasal administration of the SCFAs acetate, butyrate, and propionate on basal expression of antiviral signatures, and of acetate in a mouse model of RV infection and in RV-infected lung epithelial cell lines. We additionally assessed the effects of acetate, butyrate, and propionate on RV infection in differentiated human primary bronchial epithelial cells. Intranasal acetate administration induced basal upregulation of IFN-β, an effect not observed with other SCFAs. Butyrate induced RIG-I expression. Intranasal acetate treatment of mice increased interferon-stimulated gene and IFN-λ expression during RV infection and reduced lung virus loads at 8 hours postinfection. Acetate ameliorated virus-induced proinflammatory responses with attenuated pulmonary mucin and IL-6 expression observed at day 4 and 6 postinfection. This interferon-enhancing effect of acetate was confirmed in human bronchial and alveolar epithelial cell lines. In differentiated primary bronchial epithelial cells, butyrate treatment better modulated IFN-β and IFN-λ gene expression during RV infection. SCFAs augment antiviral immunity and reduce virus load and proinflammatory responses during RV infection.

Prefusion-specific antibody-derived peptides trivalently presented on DNA-nanoscaffolds as an innovative strategy against RSV entry

Human respiratory syncytial virus (RSV) is the primary cause of acute lower respiratory tract infections in children and the elderly worldwide, for which neither a vaccine nor an effective therapy is approved. The entry of RSV into the host cell is mediated by stepwise structural changes in the surface RSV fusion (RSV-F) glycoprotein. Recent progress in structural and functional studies of RSV-F glycoprotein revealed conformation-dependent neutralizing epitopes which have become attractive targets for vaccine and therapeutic development. As RSV-F is present on viral surface in a trimeric form, a trivalent binding interaction between a candidate fusion inhibitor and the respective epitopes on each of the three monomers is expected to prevent viral infection at higher potency than a monovalent or bivalent inhibitor. Here we demonstrate a novel RSV entry inhibitory approach by implementing a trimeric DNA nanostructure as a template to display up to three linear peptide moieties that simultaneously target an epitope on the surface of the prefusion RSV-F protein. In order to design synthetic binding peptides that can be coupled to the DNA nanostructure, the prefusion RSV-F-specific monoclonal antibody (D25) was selected. Complementarity-determining region 3 (CDR3) derived peptides underwent truncation and alanine-scanning mutagenesis analysis, followed by systematic sequence modifications using non-canonical amino acids. The most effective peptide candidate was used as a binding moiety to functionalize the DNA nanostructure. The designed DNA-peptide construct was able to block RSV infection on cells more efficiently than the monomeric peptides, however a more moderate reduction of viral load was observed in the lungs of infected mice upon intranasal application, likely due to dissociation or absorption of the underlying DNA structure by cells in the lungs. Taken together, our results point towards the inhibitory potential of a novel trimeric DNA-peptide based approach against RSV and open the possibility to apply this platform to target other viral infections.

Synthesis and Evaluation of a Silver Nanoparticle/Polyurethane Composite That Exhibits Antiviral Activity against SARS-CoV-2.

In this proof-of-concept study, we aim to produce a polyurethane (PU)-based composite that can reduce the amount of viable SARS-CoV-2 virus in contact with the surface of the polymeric film without further interventions such as manual cleaning. Current protocols for maintaining the hygiene of commonly used touchpoints (door handles, light switches, shop counters) typically rely on repeated washing with antimicrobial products. Since the start of the SARS-CoV-2 pandemic, frequent and costly surface sanitization by workers has become standard procedure in many public areas. Therefore, materials that can be retrofitted to touchpoints, yet inhibit pathogen growth for extended time periods are an important target. Herein, we design and synthesise the PU using a one-pot synthetic procedure on a multigram scale from commercial starting materials. The PU forms a robust composite thin film when loaded with 10 wt% silver nanoparticles (AgNPs). The addition of AgNPs increases the ultimate tensile strength, modules of toughness and modulus of elasticity at the cost of a reduced elongation at break when compared to the pristine PU. Comparative biological testing was carried out by the addition of pseudotyped virus (PV) bearing the SARS-CoV-2 beta (B.1.351) VOC spike protein onto the film surfaces of either the pristine PU or the PU nanocomposite. After 24 h without further human intervention the nanocomposite reduced the amount of viable virus by 67% (p = 0.0012) compared to the pristine PU treated under the same conditions. The significance of this reduction in viable virus load caused by our nanocomposite is that PUs form the basis of many commercial paints and coatings. Therefore, we envisage that this work will provide the basis for further progress towards producing a retrofittable surface that can be applied to a wide variety of common touchpoints.

Self-amplifying mRNA bicistronic influenza vaccines raise cross-reactive immune responses in mice and prevent infection in ferrets

Vaccines are the primary intervention against influenza. Currently licensed inactivated vaccines focus immunity on viral hemagglutinin (HA). Self-amplifying mRNA (sa-mRNA) vaccines offer an opportunity to generate immunity to multiple viral proteins, including additional neuraminidase (NA). This evaluation of a bicistronic approach for sa-mRNA vaccine development compared subgenomic promoter and internal ribosome entry site strategies and found consistent and balanced expression of both HA and NA proteins in transfected cells. In mice, sa-mRNA bicistronic A/H5N1 vaccines raised potent anti-HA and anti-NA neutralizing antibody responses and HA- or NA-specific CD4+ and CD8+ Tcell responses. The addition of NA also boosted the cross-neutralizing response to heterologous A/H1N1. Similar immunogenicity results were obtained for bicistronic seasonal A/H3N2 and B/Yamagata vaccines. In ferrets, sa-mRNA bicistronic A/H1N1 vaccine fully protected lung from infection by homologous virus and showed significant reduction of viral load in upper respiratory tract, warranting further evaluation of sa-mRNA bicistronic vaccine in humans.

S2489 Differentiating CMV Colitis From GVHD in Post-Transplant Patients: A Case Study

Introduction: Cytomegalovirus (CMV) infection can complicate the clinical course of immunocompromised patients including allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, where the infection can be serious. CMV can involve different organs including lungs, brain, eyes, and the GI tract. Signs and symptoms of CMV infection may overlap with other conditions including graft-versus-host disease (GVHD), a multisystem disorder commonly seen in allo-HSCT recipients. Case Description/Methods: A 57-year-old female presented with abdominal pain and diarrhea. Past medical history included relapsed acute myeloid leukemia, treated with azacitidine and venetoclax followed by allo-HSCT. She was recently hospitalized for suspected GVHD with skin, liver, and gastrointestinal involvement. She was also found to have a CMV viral load of 920 IU/mL. Endoscopy showed diffuse inflammation across the GI tract, with biopsies revealing apoptotic bodies consistent with acute GVHD. She was treated with ruxolitinib and prednisone for GVHD, and valganciclovir prophylactically for CMV viremia. At this presentation, labs were notable for lymphocytes of 39 x103/uL and platelets of 22 x 103/uL, and CMV viral load of 1,822,242 IU/mL. CT imaging revealed thickened distal small bowel loops, cecum, and right colon, and edematous gastric walls with submucosal enhancement. Infectious work-up, including stool culture, ova/parasite, and clostridium, was negative. She underwent endoscopic evaluation; EGD showed white discoloration that washed off in the esophagus and diffuse erythematous mucosa in the stomach (Figure). Biopsies from gastric mucosa and right colon mucosa revealed focal nuclear changes. Immunohistochemical stains were positive for CMV. She was diagnosed with refractory CMV colitis and treated with maribavir with improvement of symptoms and reduction of viral load, and prior course of steroids was tapered. Discussion: Immunosuppression for GVHD has been identified as an important risk factor for CMV reactivation. Differentiating CMV colitis and GI-GVHD poses a diagnostic challenge, and endoscopic evaluation is needed to address this. Endoscopy can identify cecal ulcers, a reliable finding for CMV colitis in patients with GVHD after allo-HSCT.1 It is important to timely identify CMV colitis to prevent post-transplant morbidity and mortality. This case also demonstrates effectiveness of maribavir, a novel antiviral, for refractory post-transplant CMV colitis.Figure 1.: EGD images showing white discoloration in the entire esophagus and diffuse erythematous mucosa in the entire examined stomach.

Shortening Duration of Swine Exhibitions to Reduce Risk for Zoonotic Transmission of Influenza A Virus.

Reducing zoonotic influenza A virus (IAV) risk in the United States necessitates mitigation of IAV in exhibition swine. We evaluated the effectiveness of shortening swine exhibitions to <72 hours to reduce IAV risk. We longitudinally sampled every pig daily for the full duration of 16 county fairs during 2014–2015 (39,768 nasal wipes from 6,768 pigs). In addition, we estimated IAV prevalence at 195 fairs during 2018–2019 to test the hypothesis that <72-hour swine exhibitions would have lower IAV prevalence. In both studies, we found that shortening duration drastically reduces IAV prevalence in exhibition swine at county fairs. Reduction of viral load in the barn within a county fair is critical to reduce the risk for interspecies IAV transmission and pandemic potential. Therefore, we encourage fair organizers to shorten swine shows to protect the health of both animals and humans.

Efficacy and safety of polyherbal formulation as an add-on to standard-of-care in mild-to-moderate COVID-19: A randomized, double-blind, placebo-controlled trial

BackgroundNovel corona virus disease-2019 (COVID-19) pandemic is a significant contributor to morbidity and mortality in affected individuals. Modulating the immune response in COVID-19 is now an established treatment approach. Polyherbal formulations have long been assessed for their potential immune modulating effects and are expected to be beneficial on COVID-19. MethodsThis study aims at assessing the efficacy and safety of polyherbal formulation (referred as IP) in comparison to placebo, as add on to the standard of care (SOC), in patients with mild to moderate COVID-19 patients. Hospitalized RT-PCR positive patients were randomized to either SOC + IP or SOC + Placebo arm. The viral load (VL) was assessed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Immunological parameters were also assessed. The clinical improvement was assessed using a numeric rating scale (NRS) and WHO ordinal scale, and follow-up period was 30 days. ResultsSeventy-two patients were randomized to SOC + IP (n = 39) and SOC + Placebo (n = 33) arms. There was significant reduction in VL in SOC + IP arm from day 0–4 (p = 0.002), compared to SOC + Placebo arm (p = 0.106). Change in the NRS score and WHO score was significant in both arms, however, the difference between the two arms was statistically significant in favour of IP arm. The increase in Th1 response was significant in SOC + IP arm (p = 0.023), but not in SOC + Placebo arm. COVID-19 specific antibodies were numerically higher in the SOC + IP arm. ConclusionThe study finds that polyherbal formulation significantly reduces VL and contributes to immunomodulation and improvement in clinical conditions without side effects.

Atazanavir-Concentrate Loaded Soft Gelatin Capsule for Enhanced Concentration in Plasma, Brain, Spleen, and Lymphatics.

This study investigates the development of atazanavir-concentrate loaded soft gelatin capsule for achieving enhanced atazanavir (ATV) concentration in plasma, brain, spleen, and lymphatics beneficial in the significant reduction of viral load in HIV infection. For this purpose, ATV-concentrate in the presence and absence of Soluplus with corn oil, oleic acid, tween 80, and propylene glycol was developed. The developed ATV-concentrate was found to have enhanced dispersibility with no signs of precipitation after dilution with simulated G.I fluid as evident from particle size (16.49±0.32 nm) and PDI (0.217±0.02) analysis. The rheological and molecular docking studies explainedthe reduction of viscosity of SuATV-C due to the intermolecular H-bond between ATV and Soluplus that helps to retard crystallization. The shell of the soft gelatin capsule retains its integrity when subjected to a folding endurance test on a texture analyzer depicting that the concentrate did not affect the integrity of the soft gelatin capsule shell. An ex vivo and in vivo pharmacokinetic study in rats revealed that the SuATV-C soft gelatin capsule (SuATV-C SGC) indicated 2.9 fold improvement in rate and extent of permeation and absorption than that of ATV-suspension. The tissue distribution study also exhibited higher drug concentration in the brain (2.5 fold), lymph nodes (2.7 fold), and spleen (1.2 fold) administered with SuATV-C SGC, revealing the overwhelming influence of Soluplus and corn oil. In a nutshell, these studies demonstrated that SuATV-C SGC seems to have the potential to deliver an anti-retroviral drug to the viral sanctuaries for the better management of HIV.

Mineral oil interferes with potato virus Y in aphid stylets

AbstractMineral oils have been used for many decades to control the transmission of plant viruses and their ensuing epidemics. Although it is not clearly defined, the mode of action of these oils has been extensively studied, and effects on the feeding behaviour and orientation of the vector or the transmission of the virus by its vector have often been put forward as possible explanations. This study examines the effect of mineral oil on the interaction between potato virus Y (PVY) and its vector, the green peach aphid Myzus persicae, with particular focus on the transmission organ. A lipophilic staining technique was used to visualize the oil inside the insect's stylets for the first time. Virus binding and localization were also examined in aphid stylets following PVY acquisition. The results indicate that the presence of mineral oil on leaves reduces the likelihood of acquiring the virus. This study verified that this reduced acquisition could be accompanied by a reduction in viral load, as measured by reverse transcription‐quantitative PCR on the stylets. The effect of the oil on virus retention inside the stylets was also measured. The mechanism by which the oil impacts virus acquisition seems to take place in the very early stages of acquisition, well before the virus attaches to the stylets.

Latent HIV-1 provirus in vitro suppression using combinatorial CRISPR/Cas9 strategy

Antiretroviral therapy (ART) effectively controls HIV-1 replication. However, it is unable to eliminate latent proviruses and the occurrence of viral resistance. In this study, CRISPR/Cas9 was used to knockdown Pol, Gag, Rev, Tat, and Vif of HIV-1. The CRISPR construct (combined_nuc) was investigated against different subtypes of HIV-1 positive ART naïve PBMCs, and the frequency of mutation, viral load reduction, and changes to immune markers were determined. Combined_nuc knocked down targeted HIV-1 genes and caused the most significant decrease in Tat mRNA expression. Combined_nuc suppressed up to 87.54 % of HIV gene expression and improved CD4+ T cell expression by up to 94.72 %. The treatment facilitated an efficient viral suppression and positive immune response in HIV-1 patient samples in vitro, with the highest efficacy in the young male patient sample with subtype B. This pilot study suggests that combinatorial CRISPR/Cas9 could be an alternative therapy to control HIV-1 infection.

Cross protective efficacy of the Non-Neurotropic live attenuated herpes simplex virus type 1 vaccine VC-2 is enhanced by intradermal vaccination and deletion of glycoprotein G

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2 respectively) cause life-long latent infections resulting in recurrent orofacial and genital blisters or sores. Ensued disease can be painful and may lead to significant mental anguish of infected individuals. Currently, there are no FDA-approved vaccines for either prophylactic or therapeutic use, and recent clinical trials of subunit vaccines failed to achieve endpoints goals. Development of a safe live-attenuated herpes simplex vaccine may provide the antigenic breadth to ultimately protect individuals from acquiring HSV disease. We have previously shown that prophylactic use of the non-neurotropic live attenuated HSV-1 vaccine, VC-2, provides potent and durable protection from genital HSV-2 disease in the guinea pig model. Here, we investigated the effects of intradermal administration as well as the deletion of the viral glycoprotein G (gG) on the efficacy of prophylactic vaccination. Vaccination with either VC-2, VC-2 gG null, or gD2 MPL/Alum offered robust protection from acute disease regardless of route of vaccination. However, both the VC-2 gG-null and the ID vaccination route were more effective compared to the parent VC2 administered by the IM route. Specifically, the VC-2 gG-null administered ID, reduced HSV-2 vaginal replication on day 2 and day 4 as well as mean recurrent lesion scores more effectively than VC2 administered IM. Most importantly, only VC-2 gG null IM and VC-2 ID significantly reduced the frequency of recurrent shedding, the most likely source for virus transmission. Similarly, while all vaccinated groups demonstrated a significant reduction in the number of animals testing PCR-positive for HSV-2 in their dorsal root ganglia following challenge only VC2 ID vaccinated animals demonstrated a significant reduction in DRG viral load. All vaccinations induced neutralizing antibodies to HSV-2 MS when compared to unvaccinated guinea pigs. Therefore, further investigation of VC-2 gG null delivered ID is warranted.

Efficacy of washing produce in removing human coronavirus OC43 and murine norovirus.

AimsFresh produce is often a vehicle for the transmission of foodborne pathogens such as human norovirus. Thus, it is recommended to wash the surface of produce before consumption, and one of the most common ways to wash produce is by rinsing under running tap water. This study determined the effectiveness of removal of human coronavirus‐OC43 (HCoV‐OC43), as a surrogate for severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and murine norovirus‐1 (MNV‐1), as a surrogate for human norovirus, from contaminated lettuce, apples and cucumbers.Methods and ResultsThe produce surfaces were artificially inoculated in conjunction with faecal material to represent natural contamination. Rinsing under tap water for 10 s at 40 ml/s removed 1.94 ± 0.44, 1.42 ± 0.00 and 1.42 ± 0.42 log of HCoV‐OC43 from apple, cucumber and lettuce respectively. The same washing technique removed 1.77 ± 0.17, 1.42 ± 0.07 and 1.79 ± 0.14 log of MNV‐1 from apple, cucumber and lettuce respectively. This washing technique was effective at reducing a significant amount of viral contamination, however, it was not enough to eliminate the entire contamination. There was no significant difference in the reduction of viral load between the two viruses, nor between the three surfaces tested in this study.ConclusionsOur data suggest that washing under tap water would be an efficient way of reducing the risk of foodborne viral transmission only if the level of contamination is less than 2 log PFU.Significance and Impact of StudyThis study demonstrates that running tap water was effective at reducing the amount of infectious HCoV‐OC43 and MNV on produce surfaces, and washing produce continues to be an important task to perform prior to consumption to avoid infection by foodborne viruses, particularly for foods which are eaten raw.

415.6: Exploring Cell-Specific Mechanisms of Ischemia/Reperfusion Injury Mediated Reactivation of Cytomegalovirus From Latency in the Orthotopic Mouse Lung Transplantation

Purpose: Cytomegalovirus (CMV) is the most common opportunistic infection in lung transplant recipients, with the highest risk seen in CMV seronegative recipients (R-) of seropositive organs (D+). This study aims to explore key molecular pathways responsible for transplant-mediated CMV reactivation by using single-cell RNA sequencing (scRNA-seq) in a mouse model of syngeneic D+/R- lung transplantation. Methods: Left lungs from naïve (D-) mice or MCMV latently infected (D+) mice were transplanted to syngeneic R- recipients, while the contralateral right lungs served as controls. Lung tissues from the transplants at day 2 post-transplant (POD) or controls were harvested for viral RNA analysis and single cell isolation. CD31+ and CD45+ enriched single cell suspensions were processed using 10X Genomics, followed by sequencing. The sequenced data were processed using Cell Ranger pipeline and Seurat. Additional transplants were treated with small molecule Myd88 inhibitor to determine whether innate immunity plays a role in transplant induced CMV reactivation. Results: MCMV IE-1 mRNA transcripts were significantly upregulated at POD 2, but were not detectable in the controls, indicating that transcriptional activation is induced in the early phase of transplant and ischemia/reperfusion (I/R) injury associated with the transplant procedures is an important contributing factor. In addition, viral DNA amplification was detected in the salivary glands of D+/R- recipients at POD14, confirming that transplant of a D+ graft results in viral reactivation and dissemination. These data support the mouse lung transplant model as a viable approach to study mechanisms of CMV reactivation. Data from scRNAseq analysis showed distinct lung and immune cell clusters in lung transplants and controls. There were minimal differences in transcriptome landscapes between D- and D+ lungs prior to transplant (controls). In contrast, the differences were prominent between lungs from D-/R- transplant vs D+/R- lung grafts in the myeloid populations [e.g. neutrophils, macrophages (Macs)] and endothelial cells. Gene expression of Macs showed more differences between transplant lungs and control lungs than any endothelial cells or neutrophils. Pathway analysis of differentially expressed genes for myeloid cell types showed several significant immune response pathways including MIF regulation of innate immunity and communication between innate and adaptive immune cells. Expression of Myd88 and c-fos were increased in the lung grafts regardless of latent MCMV infection. Furthermore, our preliminary study using a Myd88 inhibitor showed a trend of CMV viral load reduction in lung grafts. Conclusions: Early I/R injury following lung transplant resulted significant alterations of transcriptome landscape unique to different cell types. Myd88/AP-1 plays a critical role in CMV reactivation; thus targeting Myd88 has a great therapeutic potential to prevent I/R injury-mediated CMV reactivation. NIH NIAID R21AI163876. NIH/NIAID P01AI112522.

The Discovery of GSK3640254, a Next-Generation Inhibitor of HIV-1 Maturation.

GSK3640254 is an HIV-1 maturation inhibitor (MI) that exhibits significantly improved antiviral activity toward a range of clinically relevant polymorphic variants with reduced sensitivity toward the second-generation MI GSK3532795 (BMS-955176). The key structural difference between GSK3640254 and its predecessor is the replacement of the para-substituted benzoic acid moiety attached at the C-3 position of the triterpenoid core with a cyclohex-3-ene-1-carboxylic acid substituted with a CH2F moiety at the carbon atom α- to the pharmacophoric carboxylic acid. This structural element provided a new vector with which to explore structure-activity relationships (SARs) and led to compounds with improved polymorphic coverage while preserving pharmacokinetic (PK) properties. The approach to the design of GSK3640254, the development of a synthetic route and its preclinical profile are discussed. GSK3640254 is currently in phase IIb clinical trials after demonstrating a dose-related reduction in HIV-1 viral load over 7-10 days of dosing to HIV-1-infected subjects.

Antiviral efficacy of favipiravir against Zika and SARS-CoV-2 viruses in non-human primates

The COVID-19 pandemic has exemplified that rigorous evaluation in large animal models is key for translation from promising in vitro results to successful clinical implementation. Among the drugs that have been largely tested in clinical trials but failed so far to bring clear evidence of clinical efficacy is favipiravir, a nucleoside analogue with large spectrum activity against several RNA viruses in vitro and in small animal models. Here, we evaluate the antiviral activity of favipiravir against Zika or SARS-CoV-2 virus in cynomolgus macaques. In both models, high doses of favipiravir are initiated before infection and viral kinetics are evaluated during 7 to 15 days after infection. Favipiravir leads to a statistically significant reduction in plasma Zika viral load compared to untreated animals. However, favipiravir has no effects on SARS-CoV-2 viral kinetics, and 4 treated animals have to be euthanized due to rapid clinical deterioration, suggesting a potential role of favipiravir in disease worsening in SARS-CoV-2 infected animals. To summarize, favipiravir has an antiviral activity against Zika virus but not against SARS-CoV-2 infection in the cynomolgus macaque model. Our results support the clinical evaluation of favipiravir against Zika virus but they advocate against its use against SARS-CoV-2 infection.

Vemurafenib Inhibits Enterovirus A71 Genome Replication and Virus Assembly.

Enterovirus A71 (EV-A71) infection is a major cause of hand, foot, and mouth disease (HFMD), which may be occasionally associated with severe neurological complications. There is currently a lack of treatment options for EV-A71 infection. The Raf-MEK-ERK signaling pathway, in addition to its critical importance in the regulation of cell growth, differentiation, and survival, has been shown to be essential for virus replication. In this study, we investigated the anti-EV-A71 activity of vemurafenib, a clinically approved B-Raf inhibitor used in the treatment of late-stage melanoma. Vemurafenib exhibits potent anti-EV-A71 effect in cytopathic effect inhibition and viral load reduction assays, with half maximal effective concentration (EC50) at nanomolar concentrations. Mechanistically, vemurafenib interrupts both EV-A71 genome replication and assembly. These findings expand the list of potential antiviral candidates of anti-EV-A71 therapeutics.