Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

Cryptic bacterial pathogens of diatoms peak during senescence of a winter diatom bloom.

Diatoms are globally abundant microalgae that form extensive blooms in aquatic ecosystems. Certain bacteria behave antagonistically towards diatoms, killing or inhibiting their growth. Despite their crucial implications to diatom blooms and population health, knowledge of diatom antagonists in the environment is fundamentally lacking. We report systematic characterisation of the diversity and seasonal dynamics of bacterial antagonists of diatoms via plaque assay sampling in the Western English Channel (WEC), where diatoms frequently bloom. Unexpectedly, peaks in detection did not occur during characteristic spring diatom blooms, but coincided with a winter bloom of Coscinodiscus, suggesting that these bacteria likely influence distinct diatom host populations. We isolated multiple bacterial antagonists, spanning 4 classes and 10 bacterial orders. Notably, a diatom attaching Roseobacter Ponticoccus alexandrii was isolated multiple times, indicative of a persistent environmental presence. Moreover, many isolates had no prior reports of antagonistic activity towards diatoms. We verified diatom growth inhibitory effects of eight isolates. In all cases tested, these effects were activated by pre-exposure to diatom organic matter. Discovery of widespread 'cryptic' antagonistic activity indicates that bacterial pathogenicity towards diatoms is more prevalent than previously recognised. Finally, examination of the global biogeography of WEC antagonists revealed co-occurrence patterns with diatom host populations in marine waters globally.

Serological immune biomarker for disease severity in dengue-infected pediatric hospitalized patients.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using an enzyme-linked immunoassay assay system which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources.

The Virus-Induced Cytopathic Effect.

The cytopathic effect comprises the set of cellular alterations produced by a viral infection. It is of great relevance since it constitutes a direct marker of infection. Likewise, these alterations are often virus-specific which makes them a phenotypic marker for many viral species. All these characteristics have been used to complement the study of the dynamics of virus-cell interactions through the kinetic study of the progression of damage produced by the infection. Various approaches have been used to monitor the cytopathic effect, ranging from light microscopy, immunofluorescence assays, and direct labeling with fluorescent dyes, to plaque assay for the characterization of the infection over time. Here we address the relevance of the study of cytopathic effect and describe different experimental alternatives for its application.

Interlaboratory comparison of Pseudomonas aeruginosa phage susceptibility testing.

Standardized approaches to phage susceptibility testing (PST) are essential to inform selection of phages for study in patients with bacterial infections. There is no reference standard for assessing bacterial susceptibility to phage. We compared agreement between PST performed at three centers: two centers using a liquid assay standardized between the sites with the third, a plaque assay. Four Pseudomonas aeruginosa phages: PaWRA01ø11 (EPa11), PaWRA01ø39 (EPa39), PaWRA02ø83 (EPa83), PaWRA02ø87 (EPa87), and a cocktail of all four phages were tested against 145 P. aeruginosa isolates. Comparisons were made within measurements at the two sites performing the liquid assay and between these two sites. Agreement was assessed based on coverage probability (CP8), total deviation index, concordance correlation coefficient (CCC), measurement accuracy, and precision. For the liquid assay, there was satisfactory agreement among triplicate measurements made on different days at site 1, and high agreement based on accuracy and precision between duplicate measurements made on the same run at site 2. There was fair accuracy between measurements of the two sites performing the liquid assay, with CCCs below 0.6 for all phages tested. When compared to the plaque assay (performed once at site 3), there was less agreement between results of the liquid and plaque assays than between the two sites performing the liquid assay. Similar findings to the larger group were noted in the subset of 46 P. aeruginosa isolates from cystic fibrosis. Results of this study suggest that reproducibility of PST methods needs further development.

Enhancement of SARS-CoV-2 infection and growth by an ACE2-specific monoclonal antibody.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) pandemic and continues to pose a threat to global public health through genetic mutation. In this study, we have found that an angiotensin-converting enzyme 2-specific monoclonal antibody at low concentration was able to enhance SARS-CoV-2 infection and growth in cell culture. Strikingly, it promotes SARS-CoV-2 plaque formation, resulting in accurate titration of different SARS-CoV-2 variants, particularly the newly emerged Omicron variants, which otherwise cannot be determined by standard plaque assays. Quantification of infectious titers of the newly emerged variants will facilitate the development and evaluation of vaccines and antiviral drugs against SARS-CoV-2.

Comparative characterization of Crimean-Congo hemorrhagic fever virus cell culture systems with application to propagation and titration methods.

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is a biosafety level 4 and World Health Organization top priority pathogen. Infection leads to an often fatal hemorrhagic fever disease in humans. The tick-borne virus is endemic in countries across Asia, Europe and Africa, with signs of spreading into new regions. Despite the severity of disease and the potential of CCHFV geographic expansion to cause widespread outbreaks, no approved vaccine or treatment is currently available. Critical for basic research and the development of diagnostics or medical countermeasures, CCHFV viral stocks are commonly produced in Vero E6 and SW-13 cell lines. While a variety of in-house methods are being used across different laboratories, there has been no clear, specific consensus on a standard, optimal system for CCHFV growth and titration. In this study, we perform a systematic, side-by-side characterization of Vero E6 and SW-13 cell lines concerning the replication kinetics of CCHFV under different culture conditions. SW-13 cells are typically cultured in a CO2-free condition (SW-13 CO2-) according to the American Type Culture Collection. However, we identify a CO2-compatible culture condition (SW-13 CO2+) that demonstrates the highest viral load (RNA concentration) and titer (infectious virus concentration) in the culture supernatants, in comparison to SW-13 CO2- and Vero E6 cultures. This optimal viral propagation system also leads to the development of two titration methods: an immunostaining-based plaque assay using a commercial CCHFV antibody and a colorimetric readout, and an antibody staining-free, cytopathic effect-based median tissue culture infectious dose assay using a simple excel calculator. These are anticipated to serve as a basis for a reproducible, standardized and user-friendly platform for CCHFV propagation and titration.

Invasion of HeLa Cells by Shigella Species Clinical Isolates Recovered from Pediatric Diarrhea.

Shigella is considered a major public health concern, especially for children younger than 5 years of age in developing countries. The pathogenicity of Shigella is a complex process that involves the interplay of multiple genes located on a large, unstable virulence plasmid as well as chromosomal pathogenicity islands. Since various factors (including virulence and antibiotic resistance genes) are associated with the severity and duration of shigellosis, in this article, we aim to evaluate whether the invasion of HeLa cells is affected by Shigella spp. isolates with different characteristics (including serogroups, virulence gene profiles, and antibiotic resistance patterns) recovered from pediatric patients in Tehran, Iran. Cell invasion ability of 10 Shigella isolates with different serogroups (Shigella flexneri and Shigella sonnei), gene profiling (virA, sen, ipgD, ipaD, ipaC, ipaB, and ipaH), and antibiotic resistance phenotyping (ampicillin, azithromycin, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, cefixime, cefotaxime, minocycline, and levofloxacin) were measured by plaque-forming assay in HeLa cell lines. The results show that all the selected Shigella spp. isolates recovered from pediatric patients were able to invade HeLa cells, but the total number and average size of plaques were different between the isolates. The higher invasion ability of S. flexneri isolates in HeLa cells compared to S. sonnei isolates was attributed to the presence of particular virulence genes; however, the role of each of these virulence factors remains to be determined.

Mechanically Ventilated Patients Shed High-Titer Live Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) for Extended Periods From Both the Upper and Lower Respiratory Tract.

BACKGROUND: SARS-CoV-2 infection can lead to severe acute respiratory distress syndrome needing intensive care admission and may lead to death. As a virus that transmits by respiratory droplets and aerosols, determining the duration of viable virus shedding from the respiratory tract is critical for patient prognosis, and informs infection-control measures both within healthcare settings and the public domain. METHODS: We prospectively examined upper and lower airway respiratory secretions for both viral RNA and infectious virions in mechanically ventilated patients admitted to the intensive care unit (ICU) of the University Hospital of Wales. Samples were taken from the oral cavity (saliva), oropharynx (subglottic aspirate), or lower respiratory tract (nondirected bronchoalveolar lavage [NBAL] or bronchoalveolar lavage [BAL]) and analyzed by both quantitative PCR (qPCR) and plaque assay. RESULTS: 117 samples were obtained from 25 patients. qPCR showed extremely high rates of positivity across all sample types; however, live virus was far more common in saliva (68%) than in BAL/NBAL (32%). Average titers of live virus were higher in subglottic aspirates (4.5 × 107) than in saliva (2.2 × 106) or BAL/NBAL (8.5 × 106) and reached >108 PFU/mL in some samples. The longest duration of shedding was 98 days, while most patients (14/25) shed live virus for ≥20 days. CONCLUSIONS: ICU patients infected with SARS-CoV-2 can shed high titers of virus both in the upper and lower respiratory tract and tend to be prolonged shedders. This information is important for decision making around cohorting patients, de-escalation of personal protective equipment, and undertaking potential aerosol-generating procedures.

Inactivation mechanisms of human adenovirus by e-beam irradiation in water environments.

This study aims to study the kinetics and mechanisms of human adenovirus inactivation by electron beam. Human adenovirus type 5 (HAdV-5) was inoculated in two types of aqueous substrates (phosphate-buffered saline - PBS, domestic wastewater - WW) treated by electron beam at a dose range between 3 and 21 kGy. Samples were evaluated for virus infectivity, PCR amplification of fragments of HAdV-5 genome and abundance and antigenicity of the virion structural proteins. The maximum reduction in viral titre, in plaque-forming units (PFU) per millilitre, was about 7 and 5 log PFU/mL for e-beam irradiation at 20 kGy in PBS and 19 kGy in wastewater, respectively. Among the virion structural proteins detected, the hexon protein showed the higher radioresistance. Long (10.1 kbp) genomic DNA fragments were differently PCR amplified, denoting a substrate effect on HAdV-5 genome degradation by e-beam. The differences observed between the two substrates can be explained by the protective effect that the organic matter present in the substrate may have on viral irradiation. According to the obtained results, the decrease in viral viability/infectivity may be due to DNA damage and to protein alterations. In summary, electron beam irradiation at a dose of 13 kGy is capable of reducing HAdV-5 viral titres by more than 99.99% (4 log PFU/mL) in both substrates assayed, indicating that this type of technology is effective for viral wastewater disinfection and may be used as a tertiary treatment in water treatment plants. KEY POINTS: • The substrate in which the virus is suspended has an impact on its sensitivity to e-beam treatment. • E-beam irradiation at 13 kGy is capable of reducing by 4 Log PFU/mL the HAdV-5 viral titre. • The decrease in viral viability/infectivity may be due to DNA damage and to protein alterations.

Hit-to-Lead Short Peptides against Dengue Type 2 Envelope Protein: Computational and Experimental Investigations.

Data from the World Health Organisation show that the global incidence of dengue infection has risen drastically, with an estimated 400 million cases of dengue infection occurring annually. Despite this worrying trend, there is still no therapeutic treatment available. Herein, we investigated short peptide fragments with a varying total number of amino acid residues (peptide fragments) from previously reported dengue virus type 2 (DENV2) peptide-based inhibitors, DN58wt (GDSYIIIGVEPGQLKENWFKKGSSIGQMF), DN58opt (TWWCFYFCRRHHPFWFFYRHN), DS36wt (LITVNPIVTEKDSPVNIEAE), and DS36opt (RHWEQFYFRRRERKFWLFFW), aided by in silico approaches: peptide-protein molecular docking and 100 ns of molecular dynamics (MD) simulation via molecular mechanics using Poisson-Boltzmann surface area (MMPBSA) and molecular mechanics generalised Born surface area (MMGBSA) methods. A library of 11,699 peptide fragments was generated, subjected to in silico calculation, and the candidates with the excellent binding affinity and shown to be stable in the DI-DIII binding pocket of DENV2 envelope (E) protein were determined. Selected peptides were synthesised using conventional Fmoc solid-phase peptide chemistry, purified by RP-HPLC, and characterised using LCMS. In vitro studies followed, to test for the peptides' toxicity and efficacy in inhibiting the DENV2 growth cycle. Our studies identified the electrostatic interaction (from free energy calculation) to be the driving stabilising force for the E protein-peptide interactions. Five key E protein residues were also identified that had the most interactions with the peptides: (polar) LYS36, ASN37, and ARG350, and (nonpolar) LEU351 and VAL354; these residues might play crucial roles in the effective binding interactions. One of the peptide fragments, DN58opt_8-13 (PFWFFYRH), showed the best inhibitory activity, at about 63% DENV2 plague reduction, compared with no treatment. This correlates well with the in silico studies in which the peptide possessed the lowest binding energy (-9.0 kcal/mol) and was maintained steadily within the binding pocket of DENV2 E protein during the MD simulations. This study demonstrates the use of computational studies to expand research on lead optimisation of antiviral peptides, thus explaining the inhibitory potential of the designed peptides.

A fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity enables live imaging of infection in single cells and viral plaques.

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

Isolation and genetic characterization of SARS-CoV-2 from Indian patients in a single family without H/O travel abroad.

In view of the rapidly progressing COVID-19 pandemic, our aim was to isolate and characterize SARS-CoV-2 from Indian patients. SARS-CoV-2 was isolated from nasopharyngeal swabs collected from the two members of a family without any history of (H/O) travel abroad. Both the virus isolates (8003 and 8004) showed CPE on day 3 post-inoculation, viral antigens by immunofluorescence assay and produced distinct, clear and uniform plaques. Infectious virus titers were 5 × 106 and 4 × 106 Pfu/ml by plaque assay and 107.5 and 107 by CPE-based TCID50/ml, respectively. Phylogenetic analysis grouped our isolates with the Italian strains. On comparison with Wuhan strain, 3 unique mutations were identified in nsp3 (A1812D), exonuclease (P1821S) of Orf1ab and spike protein (Q677H) regions, respectively. Both the viruses grouped with Italian strains of SARS-CoV-2 suggesting possible source being the virus imported from Italy. These fully characterized virus isolates will be useful in developing neutralization/virological assays for the evaluation of vaccines/antivirals.

Examining the persistence of human Coronavirus 229E on fresh produce.

Human coronaviruses (HCoVs) are mainly associated with respiratory infections. However, there is evidence that highly pathogenic HCoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome (MERS-CoV), infect the gastrointestinal (GI) tract and are shed in the fecal matter of the infected individuals. These observations have raised questions regarding the possibility of fecal-oral route as well as foodborne transmission of SARS-CoV-2 and MERS-CoV. Studies regarding the survival of HCoVs on inanimate surfaces demonstrate that these viruses can remain infectious for hours to days, however, there is limited data regarding the viral survival on fresh produce, which is usually consumed raw or with minimal heat processing. To address this knowledge gap, we examined the persistence of HCoV-229E, as a surrogate for highly pathogenic HCoVs, on the surface of commonly consumed fresh produce, including: apples, tomatoes, cucumbers and lettuce. Herein, we demonstrated that viral infectivity declines within a few hours post-inoculation (p.i) on apples and tomatoes, and no infectious virus was detected at 24h p.i, while the virus persists in infectious form for 72h p.i on cucumbers and lettuce. The stability of viral RNA was examined by droplet-digital RT-PCR (ddRT-PCR), and it was observed that there is no considerable reduction in viral RNA within 72h p.i.

The FDA-Approved Drug Nelfinavir Inhibits Lytic Cell-Free but Not Cell-Associated Nonlytic Transmission of Human Adenovirus.

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-ß-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multiround infection, nelfinavir mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of human immunodeficiency virus (HIV) aspartyl protease that is used to treat AIDS. It is not effective against single-round HAdV infections. Here, we show that nelfinavir inhibits lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighboring uninfected cells. HAdV lacking ADP was insensitive to nelfinavir but gave rise to comet-shaped foci, indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and -B14p1 lacking ADP were highly sensitive to nelfinavir, although HAdV-A31, -B3, -B7, -B11, -B16, -B21, -D8, -D30, and -D37 were less sensitive. Conspicuously, nelfinavir uncovered slow-growing round HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of nonlytic cell-to-cell transmission. Our study demonstrates the repurposing potential of nelfinavir with postexposure efficacy against different HAdVs and describes an alternative nonlytic cell-to-cell transmission mode of HAdV.

Cross-Protection of Dengue Virus Infection against Congenital Zika Syndrome, Northeastern Brazil.

The Zika virus outbreak in Latin America resulted in congenital malformations, called congenital Zika syndrome (CZS). For unknown reasons, CZS incidence was highest in northeastern Brazil; one potential explanation is that dengue virus (DENV)-mediated immune enhancement may promote CZS development. In contrast, our analyses of historical DENV genomic data refuted the hypothesis that unique genome signatures for northeastern Brazil explain the uneven dispersion of CZS cases. To confirm our findings, we performed serotype-specific DENV neutralization tests in a case-control framework in northeastern Brazil among 29 Zika virus-seropositive mothers of neonates with CZS and 108 Zika virus-seropositive control mothers. Neutralization titers did not differ significantly between groups. In contrast, DENV seroprevalence and median number of neutralized serotypes were significantly lower among the mothers of neonates with CZS. Supported by model analyses, our results suggest that multitypic DENV infection may protect from, rather than enhance, development of CZS.

An antiviral drug screening system for enterovirus 71 based on an improved plaque assay: A potential high-throughput method.

Plaque assay plays an irreplaceable role in a variety of virological studies, including determining titers of viruses. Our previous study showed that a simple and highly repeatable plaque assay could be used for enterovirus 71 (EV-A71). Now, we show that using a subclone of a clinical EV-A71 isolate and a rhabdomyosarcoma cell line (RD), a plaque assay based on an EV-A71/RD model could exhibit the most rapid formation of plaques (<2 days), with much higher repeatability and consistency. Inspired by a plaque inhibitory test for testing ribavirin and interferon, as well as a plaque reduction neutralization test, this modified method has been used to establish a convenient system by using 96-well plates for screening anti-EV-A71 drugs from a 130-compound library containing multiple types of inhibitors. Nine candidate effective compounds for EV-A71 have been screened out, and among them, nobiletin (flavonoid) was found to be a novel effective compound at the concentration of 10 µM. Our findings imply that this improved method based on an EV-A71/RD model proved to be a potential high-throughput method in screening novel antiviral drugs for EV-A71. Undoubtedly, this method can also be applied to other viruses that can produce an obvious cytopathic effect.

Optimization of 1,3-disubstituted urea-based inhibitors of Zika virus infection.

Zika virus (ZIKV) has become a public health concern worldwide due to its association with congenital abnormalities and neurological diseases. To date, no effective vaccines or antiviral drugs have been approved for the treatment of ZIKV infection, and new inexpensive therapeutic options are urgently needed. In this study, we have used an in vitro plaque assay to assess an antiviral activity of the second generation of anti-ZIKV compounds, based on 1,3-disubstituted (thio)urea scaffold. Several compounds in the library were found to possess excellent activity against Zika virus with IC50 values <200 pM. The most active analog, A5 exhibited an exceptional IC50 = 85.1 ±â€¯1.7 pM. Further analysis delineated structural requirements necessary for potent antiviral effects of this class of compounds. Collectively, our findings suggest that 1,3-disubstituted (thio)urea derivatives are excellent preclinical candidates for the development of anti-ZIKV therapeutics.

Inactivation of airborne viruses using a packed bed non-thermal plasma reactor.

Outbreaks of airborne infectious diseases such as measles or severe acute respiratory syndrome can cause significant public alarm. Where ventilation systems facilitate disease transmission to humans or animals, there exists a need for control measures that provide effective protection while imposing minimal pressure differential. In the present study, viral aerosols in an airstream were subjected to non-thermal plasma (NTP) exposure within a packed-bed dielectric barrier discharge reactor. Comparisons of plaque assays before and after NTP treatment found exponentially increasing inactivation of aerosolized MS2 phage with increasing applied voltage. At 30 kV and an air flow rate of 170 standard liters per minute, a greater than 2.3 log reduction of infective virus was achieved across the reactor. This reduction represented ~2 log of the MS2 inactivated and ~0.35 log physically removed in the packed bed. Increasing the air flow rate from 170 to 330 liters per minute did not significantly impact virus inactivation effectiveness. Activated carbon-based ozone filters greatly reduced residual ozone, in some cases down to background levels, while adding less than 20 Pa pressure differential to the 45 Pa differential pressure across the packed bed at the flow rate of 170 standard liters per minute.

Identification of novel small-molecule inhibitors of Zika virus infection.

The recent re-emergence of Zika virus (ZIKV), a member of the Flaviviridae family, has become a global emergency and a serious public health threat worldwide. ZIKV infection causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women causing various developmental abnormalities. Currently, there are no effective methods of preventing or treating ZIKV infection, and new treatment options are urgently needed. Therefore, we have used an in vitro plaque assay to screen a limited proprietary library of small organic compounds and identified highly bioactive leads, with the most active analogs showing activity in low picomolar range. Identified "hits" possess certain common structural features that can be used in the design of the next generation(s) of ZIKV inhibitors. Collectively, our findings suggest that identified compounds represent excellent template(s) for the development of inexpensive and orally available anti-Zika drugs.