An automated assay platform for the evaluation of antiviral compounds against polioviruses.

High-throughput screening requires assays that have flexibility to test large numbers of specimens while being accurate to ensure reproducibility across all specimens and variables tested. Previously, we used a low-throughput, cell-based assay to identify compounds with antiviral activity against polioviruses. In this report, we report the development and implementation of a high-throughput automation platform for the identification of compounds with antiviral activity against polioviruses. The platform uses off-the-shelf automated equipment combined with a modified assay, with minimal changes to existing laboratory space. We evaluated automation systems from Hudson Robotics Inc., Agilent Technologies, and a microplate reader from PerkinElmer during the platform design. Optimization for high throughput was focused on bulk reagent additions, serial dilutions, microplate washing and measuring results from the tens-to-hundreds of microplates. We evaluated the automated cell-based assay for selectivity, sensitivity, accuracy, precision, and reproducibility. This platform can be applied to screen novel antivirals against polioviruses and non-polio enteroviruses.

Antiviral Activity of Ag5IO6, a Unique Silver Compound.

Pentasilver hexaoxoiodate (Ag5IO6) has broad-spectrum antimicrobial efficacy, including the long-term prevention of microbial adherence, the rapid killing of planktonic microorganisms, and the elimination of mature biofilms. This study's goal was to determine whether it may also have antiviral activity against structurally distinct viruses. Ag5IO6 was tested following ASTM E1052-20, Standard Practice to Assess the Activity of Microbicides Against Viruses in Suspension, against adenovirus type 5, murine norovirus, poliovirus type 1, SARS-CoV-2 (original), and SARS-CoV-2 (omicron) (host cells: H1HeLa, RAW 264.7, LLC-MK2, Vero E6, and Vero E6, respectively). A 0.1 g/mL Ag5IO6 suspension was prepared and the viruses were exposed for 30 min, 4 h, or 24 h. Exposure to Ag5IO6 resulted in complete kill of SARS-CoV-2 (omicron) within 30 min, as well as complete kill of both SARS-CoV-2 (original) and the murine norovirus within 4 h. Ag5IO6 showed increasing activity over time against the adenovirus, but did not achieve a 3-log reduction within 24 h, and showed no antiviral activity against the poliovirus. These results demonstrate that Ag5IO6 has antiviral activity against medically important viruses, in addition to its well-characterized antimicrobial activity, suggesting that it may be valuable in situations where the prevention or simultaneous treatment of microbes and viruses are necessary.

Effective of different industrial disinfection in subzero cold-chain environment.

Effective disinfection methods are crucial in the cold chain transportation process of food due to the specificity of temperature and the diversity of contaminated flora. The objective of this study was to investigate the sanitizing effect of different disinfectants on various fungi at - 20 °C to achieve accurate disinfection of diverse bacterial populations. Peracetic acid, hydrogen peroxide, and potassium bisulfate were selected as low-temperature disinfectants and were combined with antifreeze. The sanitizing effect of these cryogenic disinfectants on pathogens such as Bacillus subtilis black variant spores (ATCC9372), Staphylococcus aureus (ATCC 6538), Candida albicans (ATCC 10231), Escherichia coli (8099), and poliovirus (PV-1) was sequentially verified by bactericidal and virus inactivation experiments. After a specified time of disinfection, a neutralizing agent was used to halt the sanitizing process. The study demonstrates that different disinfectants exhibit selective effects during the low-temperature disinfection process. Peracetic acid, hydrogen peroxide, and potassium monopersulfate are suitable for the low-temperature environmental disinfection of bacterial propagules, viruses, and fungal contaminants. However, for microorganisms with strong resistance to spores, a low-temperature disinfectant based on peracetic acid should be chosen for effective disinfection treatment. Our results provide a valuable reference for selecting appropriate disinfectants to sanitize various potential pathogens in the future.

Immunization Against Poliomyelitis and the Challenges to Worldwide Poliomyelitis Eradication.

Both inactivated poliovirus vaccine (IPV) and oral poliovirus vaccine (OPV) have contributed to the rapid disappearance of paralytic poliomyelitis from developed countries despite possessing different vaccine properties. Due to cost, ease of use, and other properties, the Expanded Programme on Immunization added OPV to the routine infant immunization schedule for low-income countries in 1974, but variable vaccine uptake and impaired immune responses due to poor sanitation limited the impact. Following launch of the Global Polio Eradication Initiative in 1988, poliomyelitis incidence has been reduced by >99% and types 2 and 3 wild polioviruses are now eradicated, but progress against type 1 polioviruses which are now confined to Afghanistan and Pakistan has slowed due to insecurity, poor access, and other problems. A strategic, globally coordinated replacement of trivalent OPV with bivalent 1, 3 OPV in 2016 reduced the incidence of vaccine-associated paralytic poliomyelitis (VAPP) but allowed the escape of type 2 vaccine-derived polioviruses (VDPV2) in areas with low immunization rates and use of monovalent OPV2 in response seeded new VDPV2 outbreaks and reestablishment of type 2 endemicity. A novel, more genetically stable type 2 OPV vaccine is undergoing clinical evaluation and may soon be deployed prevent or reduce VDPV2 emergences.

Hypothetical emergence of poliovirus in 2020: part 2. exploration of the potential role of vaccines in control and eradication.

OBJECTIVES: The emergence of human pathogens with pandemic potential motivates rapid vaccine development. We explore the role of vaccines in control and eradication of a novel emerging pathogen. METHODS: We hypothetically simulate emergence of a novel wild poliovirus (nWPV) in 2020 assuming an immunologically naïve population. Assuming different nonpharmaceutical interventions (NPIs), we explore the impacts of vaccines resembling serotype-specific oral poliovirus vaccine (OPV), novel OPV (nOPV), or inactivated poliovirus vaccine (IPV). RESULTS: Vaccines most effectively change the trajectory of an emerging disease when disseminated early, rapidly, and widely in the background of ongoing strict NPIs, unless the NPIs successfully eradicate the emerging pathogen before it establishes endemic transmission. Without strict NPIs, vaccines primarily reduce the burden of disease in the remaining susceptible individuals and in new birth cohorts. Live virus vaccines that effectively compete with the nWPVs can reduce disease burdens more than other vaccines. When relaxation of existing NPIs occurs at the time of vaccine introduction, nWPV transmission can counterintuitively increase in the short term. CONCLUSIONS: Vaccines can increase the probability of disease eradication in the context of strict NPIs. However, successful eradication will depend on specific immunization strategies used and a global commitment to eradication.

Anti-poliovirus activity of Nerium oleander aqueous extract.

Nerium oleander (NO), a member of the Apocynaceae family, is an ornamental plant. In this study, we evaluated the antiviral activity of hot and cold extract of NO against six different viruses such as herpes simplex virus type 1 (HSV-1), polio virus type 1 (Sb-1), vesicular stomatitis virus (VSV), reovirus type-1 (Reo-1), human immunodeficiency virus type-1 (HIV-1), and yellow fever virus (YFV). Interestingly the results of plaque reduction assay demonstrated that both, hot extract and cold extract (breastin) of NO inhibited Sb-1 viral infection.In order to identify the mechanism by which NO exerts its antiviral activity, the virucidal effect, the time of addition and the adsorption assay were carried out. Results demonstrated that NO exerts its effect after infection period, particularly during the first two hours post infection.

Decontamination efficacy of sodium hypochlorite solutions for poliovirus.

Effective decontamination procedures are critical to the successful manufacture and control of poliovirus vaccines to minimize the risk to personnel and the environment. Polio viruses have been reported to be more resistant to disinfectants than many other viruses. We assessed the efficacy of sodium hypochlorite-containing disinfectants for decontamination for three poliovirus serotypes to implement decontamination procedures that are fully compliant with the WHO GAP III and Health authorities' requirements. A 10.4 log reduction was observed with a 0.63% sodium hypochlorite solution in a suspension with high protein and high poliovirus concentrations diluted 10-fold compared with a 6 log reduction in an undiluted sample. Treatment efficacy increased with sodium hypochlorite content and decreased with sample protein content. The surface tests showed that two 1-min treatments, 5-min apart, with a 0.63% Chl sodium hypochlorite solution effectively reduced the concentration of all poliovirus serotypes by 10 log10, irrespective of the protein and virus concentration in the sample. Sodium hypochlorite solutions lower than 0.52% were less effective for complete inactivation of poliovirus. In conclusion, we demonstrated that a high level of virus reduction (>10 log10) can be achieved with sodium hypochlorite solutions with poliovirus in suspension and dried on surfaces.

Ozone disinfection kinetics of poliovirus 1 determined by cell culture assay, RT-qPCR and ethidium monoazide qPCR reduction in a continuous quench-flow reactor.

AIMS: A continuous quench-flow (CQF) reactor was developed to collect samples at the reaction times of less than one second. The reactor is applied to determine ozone disinfection kinetics of poliovirus and to study whether EMA-qPCR can assess the viral infectivity after ozone disinfection. METHODS: Ozone disinfection of poliovirus was conducted in the developed CQF, and the disinfection kinetics were tested in the range of 0·7-5·0 s at ozone concentration of 0·08 and 0·25 mg l-1 . Inactivation, damage on viral genome and damage on capsid integrity were determined by plaque assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and ethidium monoazide treatment coupled with RT-qPCR (EMA-qPCR), respectively. RESULTS: By using CQF, 2·18 and 2·76 log10 reductions were observed at the reaction time of 0·7 s and ozone concentration of 0·08 and 0·25 mg l-1 , respectively, followed by tailing. Ozone disinfection kinetics of poliovirus 1 were better fit by the efficiency factor Hom model than by the Chick-Watson model, or the modified Chick-Watson model. Kinetics observed were similar between RT-qPCR and EMA-qPCR assays at the reaction times of <2·0 s and ozone concentrations of 0·08 and 0·25 mg l-1 . At reaction times > 5 s, viral concentration evaluated by EMA-qPCR was reduced in comparison to stable RT-qPCR results. Both assays still underestimated the virus inactivation. CONCLUSION: The simple developed reactor can be used to investigate viral ozone disinfection kinetics and to elucidate inactivation characteristics or mechanisms at very short exposure times. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed CQF reactor is beneficial for better understanding of virus inactivation by ozone, and the reactor can be used to better elucidate disinfection kinetics and mechanisms for future research. This work constitutes an important contribution to the existing knowledge of the application and limitation of the EMA/PMA-qPCR to assess virus infectivity after ozone disinfection.

Microbiota-independent antiviral effects of antibiotics on poliovirus and coxsackievirus.

Coxsackieviruses primarily infect the gastrointestinal tract of humans, but they can disseminate systemically and cause severe disease. Using antibiotic treatment regimens to deplete intestinal microbes in mice, several groups have shown that bacteria promote oral infection with a variety of enteric viruses. However, it is unknown whether antibiotics have microbiota-independent antiviral effects for enteric viruses or whether antibiotics influence extra-intestinal, systemic infection. Here, we examined the effects of antibiotics on systemic enteric virus infection by performing intraperitoneal injections of either coxsackievirus B3 (CVB3) or poliovirus followed by quantification of viral titers. We found that antibiotic treatment reduced systemic infection for both viruses. Interestingly, antibiotics reduced CVB3 titers in germ-free mice, suggesting that antibiotic treatment alters CVB3 infection through a microbiota-independent mechanism. Overall, these data provide further evidence that antibiotics can have noncanonical effects on viral infection.

Bio-redox potential of Hyphaene thebaica in bio-fabrication of ultrafine maghemite phase iron oxide nanoparticles (Fe2O3 NPs) for therapeutic applications.

Maghemite (Fe2O3-NPs) nanoparticles were synthesized by a convenient, green and cost effective method using aqueous fruit extracts of Hyphaene thebaica. Different techniques like FTIR, XRD, UV-Vis, Raman, HR-TEM, EDS. SAED, Zeta potential were used to establish the nature of Fe2O3-NPs, while the therapeutic properties were studied using different biological assays including antiviral, antibacterial, antifungal, antioxidant and enzyme inhibition assays. XRD pattern revealed sharp peaks and a crystalline nature of Fe2O3-NPs. HR-TEM revealed quasi-spherical and cuboidal morphologies, while the particle size in ~10 nm. FTIR indicated a sharp peak centered at ~444 cm-1 which is the characteristic FeO band vibration. SAED pattern indicated the crystalline nature while EDS also confirmed the synthesis of Fe2O3 NPs. Zeta potential was obtained in different solvents and physiological buffers indicating highest value in water (-26.5 mV) and lowest in DMSO (-15.8 mV). Tested bacterial strains, Bacillus subtilis was found to be inhibited significantly. Aspergillus flavus appeared to be susceptible to all of the tested concentration of Fe2O3 NPs. Maximum 40.78% FRSA was obtained at 400 µg/mL. Cell culture based studies on RD cells and L20B cells indicated reduction in viability of cells with increase concentration of Fe2O3 NPs. Moderate inhibition of polio virus-1 and polio virus-2 was observed, after culturing the virus in the L20B cells. Excellent Protein Kinase (PK) inhibition was revealed. Hemolytic potential and cytotoxic potential was indicated to be dose dependent. In conclusion, the present report for the first time reports the synthesis of Fe2O3 NPs from H. thebaica fruits and reveals their biomedical potential including antiviral potential.

Clearance of Immunodeficiency-associated Vaccine-derived Poliovirus Infection With Pocapavir.

Pocapavir exhibits antiviral activity against both polio and nonpolio enteroviruses. There is limited experience of the use of this investigational drug in young children with enteroviral infection. We describe the successful clearance of prolonged immunodeficiency-associated vaccine-derived type 3 poliovirus infection by pocapavir in an infant with underlying X-linked agammaglobulinemia.

Preliminary Anti-Coxsackie Activity of Novel 1-[4-(5,6-dimethyl(H)- 1H(2H)-benzotriazol-1(2)-yl)phenyl]-3-alkyl(aryl)ureas.

BACKGROUND: Coxsackievirus infections are associated with cases of aseptic meningitis, encephalitis, myocarditis, and some chronic disease. METHODS: A series of benzo[d][1,2,3]triazol-1(2)-yl derivatives (here named benzotriazol-1(2)-yl) (4a-i, 5a-h, 6a-e, g, i, j and 7a-f, h-j) were designed, synthesized and in vitro evaluated for cytotoxicity and antiviral activity against two important human enteroviruses (HEVs) members of the Picornaviridae family [Coxsackievirus B 5 (CVB-5) and Poliovirus 1 (Sb-1)]. RESULTS: Compounds 4c (CC50 >100 µM; EC50 = 9 µM), 5g (CC50 >100 µM; EC50 = 8 µM), and 6a (CC50 >100 µM; EC50 = 10 µM) were found active against CVB-5. With the aim of evaluating the selectivity of action of this class of compounds, a wide spectrum of RNA (positive- and negativesense), double-stranded (dsRNA) or DNA viruses were also assayed. For none of them, significant antiviral activity was determined. CONCLUSION: These results point towards a selective activity against CVB-5, an important human pathogen that causes both acute and chronic diseases in infants, young children, and immunocompromised patients.

Related Enteric Viruses Have Different Requirements for Host Microbiota in Mice.

Accumulating evidence suggests that intestinal bacteria promote enteric virus infection in mice. For example, previous work demonstrated that antibiotic treatment of mice prior to oral infection with poliovirus reduced viral replication and pathogenesis. Here, we examined the effect of antibiotic treatment on infection with coxsackievirus B3 (CVB3), a picornavirus closely related to poliovirus. We treated mice with a mixture of five antibiotics to deplete host microbiota and examined CVB3 replication and pathogenesis following oral inoculation. We found that, as seen with poliovirus, CVB3 shedding and pathogenesis were reduced in antibiotic-treated mice. While treatment with just two antibiotics, vancomycin and ampicillin, was sufficient to reduce CVB3 replication and pathogenesis, this treatment had no effect on poliovirus. The quantity and composition of bacterial communities were altered by treatment with the five-antibiotic cocktail and by treatment with vancomycin and ampicillin. To determine whether more-subtle changes in bacterial populations impact viral replication, we examined viral infection in mice treated with milder antibiotic regimens. Mice treated with one-tenth the standard concentration of the normal antibiotic cocktail supported replication of poliovirus but not CVB3. Importantly, a single dose of one antibiotic, streptomycin, was sufficient to reduce CVB3 shedding and pathogenesis while having no effect on poliovirus shedding and pathogenesis. Overall, replication and pathogenesis of CVB3 are more sensitive to antibiotic treatment than poliovirus, indicating that closely related viruses may differ with respect to their reliance on microbiota.IMPORTANCE Recent data indicate that intestinal bacteria promote intestinal infection of several enteric viruses. Here, we show that coxsackievirus, an enteric virus in the picornavirus family, also relies on microbiota for intestinal replication and pathogenesis. Relatively minor depletion of the microbiota was sufficient to decrease coxsackievirus infection, while poliovirus infection was unaffected. Surprisingly, a single dose of one antibiotic was sufficient to reduce coxsackievirus infection. Therefore, these data indicate that closely related viruses may differ with respect to their reliance on microbiota.

Structural characterization and antiviral activity of pectin isolated from Inga spp.

Several studies have been conducted on polysaccharides derived from natural sources, and their different biological properties have been reported. Their low toxicity and antiviral effects i.e., their action on several steps of viral replication, have been extensively examined. In this work, pectin isolated from Inga spp. fruit pulp was first characterized and evaluated using HEp-2 cells against the herpes simplex virus type 1 (HSV-1) and the poliovirus (PV). The isolated pectin (denoted as PDTS) was characterized by infrared spectroscopy, NMR and Gel permeation chromatography. The cytotoxicity was analyzed by the MTT method and antiviral activity by plaque reduction assay, immunofluorescence assay (IF) and polymerase chain reaction (PCR). The cytotoxic concentration (CC50) of PDTS was 870 µg.mL-1 and the inhibitory concentrations (IC50) were 179 µg.mL-1 and 58 µg.mL-1 for HSV-1 and PV, respectively. Greater inhibitory effect was observed when the cells were simultaneously treated with PDTS and infected, suggesting that PDTS inhibited the initial viral replication stages, revealing its antiviral potential.

Tungsten carbide nanoparticles show a broad spectrum virucidal activity against enveloped and nonenveloped model viruses using a guideline-standardized in vitro test.

Five tungsten carbide nanoparticle preparations (denoted WC1-WC5) were investigated for broad spectrum virucidal activity against four recommended model viruses. These are modified vaccinia virus Ankara (MVA), human adenovirus type 5 (HAdV-5), poliovirus type 1 (PV-1) and murine norovirus (MNV). All virucidal tests were performed two to five times using the quantitative suspension test, which is a highly standardized test method to evaluate the virucidal efficacy of disinfectants in accordance with the European norm EN 14476+A1 and the German DVV/RKI guidelines. Quantitative detection of viruses was conducted by endpoint titration and quantitative real-time PCR. Results showed that three of the five tested compounds (WC1-WC3) were able to reduce the infectivity of all model viruses by at least four log10 of tissue culture infective dose 50% per ml after 15 min, whereas the other two compounds exhibited only limited efficacy (WC4) or showed cytotoxicity (WC5). Virucidal activity of nanoparticles increased with incubation time and a dose-effect curve showed dependence of virucidal activity with particle concentration. Whereas WC1-WC4 showed little cytotoxicity, WC5 which was doped with copper exhibited a significant cytotoxic effect. These findings propose tungsten carbide nanoparticles to be very promising in terms of new disinfection techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study investigates the virucidal activity of tungsten carbide nanoparticles using the quantitative suspension test in accordance with the European norm EN 14476+A1 and the German DVV/RKI guidelines. Due to highly standardized assay conditions, results of this test are considered very reliable for evaluation of the virucidal activity of disinfectants. Broad-spectrum activity and high efficacy of three different tungsten carbide nanoparticles preparations is concluded.

Botryosphaeran and sulfonated derivatives as novel antiviral agents for herpes simplex and dengue fever.

Sulfated polysaccharides are known to display activity against enveloped viruses, such as herpes and dengue. The aim of this work was to assess the antiviral activity of botryosphaeran, a fungal exocellular (1 → 3)(1 → 6)-ß-d-glucan devoid of sulfate groups, and its chemically sulfonated derivatives, against herpes simplex virus (HSV), dengue virus (DENV) and poliovirus (PV). The natural parent polysaccharide inhibited acyclovir-sensitive HSV (HSV-KOS) infection in Vero cells (IC50 of 39.3 µg mL-1), while the IC50 against acyclovir-resistant HSV (HSV-AR) was 47.5 µg mL-1. Botryosphaeran was derivatized by sulfonylation with chlorosulfonic acid to prepare two sulfonated derivatives, S1 and S2, with degrees of substitution (DS) of 0.4 and 1.1, respectively. Antiviral evaluation of S1 and S2 gave the IC50 of 3.0 and 2.4 µg mL-1 against HSV-KOS, and 7.3 and 2.7 µg mL-1 against HSV-AR, respectively. This study demonstrated for the first time that native botryosphaeran inhibited HSV infection, albeit moderately, while its sulfonated derivatives developed high activity against viral infection. DENV inhibition was weak for botryosphaeran, but remarkably stronger for S1 and S2. All compounds were inactive against PV, as it lacked a viral envelope. The presence of sulfate groups and the DS were confirmed to be important features for antiviral activity.

Inactivation of Poliovirus by Ozone and the Impact of Ozone on the Viral Genome.

OBJECTIVE: To investigate the mechanisms underlying ozone-induced inactivation of poliovirus type 1 (PV1). METHODS: We used cell culture, long-overlapping RT-PCR, and spot hybridization assays to verify and accurately locate the sites of action of ozone that cause PV1 inactivation. We also employed recombinant viral genome RNA infection models to confirm our observations. RESULTS: Our results indicated that ozone inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ozone specifically damaged the 80-124 nucleotide (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 lacking this region was non-infectious. CONCLUSION: In this study, we not only elucidated the mechanisms by which ozone induces PV1 inactivation but also determined that the 80-124 nt region in the 5'-NCR is targeted by ozone to achieve this inactivation.

Effect of HIV-exposure and timing of anti-retroviral treatment on immunogenicity of trivalent live-attenuated polio vaccine in infants.

INTRODUCTION: The prevalence of HIV infection in South African pregnant women has been approximately 30% over the past decade; however, there has been a steady decline in mother-to-child transmission of HIV from 8% in 2008 to <2% in 2015. We evaluated the immunogenicity of live-attenuated trivalent oral polio vaccine (OPV) following the primary vaccination series (doses at birth, 6, 10 and 14 weeks of age) in HIV-exposed uninfected (HEU), HIV-infected infants initiated on early anti-retroviral treatment (HIV+/ART+), HIV-infected infants on deferred ART (HIV+/ART-) and HIV-unexposed infants (HU) as the referent group. METHODS: Serum polio neutralization antibody titres were evaluated to serotype-1, serotype-2 and serotype-3 at 6, 10 and 18 weeks of age. Antibody titres ≥8 were considered seropositive and sero-protective. RESULTS: At 18 weeks of age, following the complete primary series of four OPV doses, no differences in GMTs, percentage of infants with sero-protective titres and median fold change in antibody titre (18 weeks vs 6 weeks) were observed in HEU infants (n = 114) and HIV+/ART+ infants (n = 162) compared to HU infants (n = 104) for the three polio serotypes. However, comparing HIV+/ART- infants (n = 70) to HU infants at 18 weeks of age, we observed significantly lower GMTs for serotype-1 (p = 0.022), serotype-2 (p<0.001) and serotype-3 (p<0.001), significantly lower percentages of infants with sero-protective titres for the three serotypes (p<0.001), and significantly lower median fold change in antibody titre for serotype-1 (p = 0.048), serotype-2 (p = 0.003) and serotype-3 (p = 0.008). CONCLUSION: Delaying initiation of ART in HIV-infected infants was associated with an attenuated immune response to OPV following a four-dose primary series of vaccines, whereas immune responses to OPV in HIV-infected children initiated on ART early in infancy and HEU children were similar to HU infants.