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Retail foodservice establishments (FSE) frequently utilize washes with sanitizing agents during fresh produce preparation. This study evaluated the efficacy of ozonated water on the inactivation of viruses, bacteria, and viruses in association with bacteria on produce surfaces. Boston bibb lettuce (BB) and cherry tomatoes were spot inoculated with viruses (murine norovirus (MNV) and MS2 bacteriophage), bacteria (Enterobacter cloacae and Bacillus cereus), or MNV associated with E. cloacae or B. cereus. Following inoculation, produce was held at 4 °C for 90 min (virus, virus + bacteria) or 24 h (virus, bacteria) prior to treatment. A batch wash ozone sanitation system (BWOSS) was prepared with ice (3–5 °C) and 0.5 ppm initial ozone concentration or no ozone. Produce samples were treated for 40 min with an ozonated water (0.86–0.99 ppm) or water-only wash with samples taken every 10 min. Samples were processed for microbial recovery, and plaque forming units (PFU) and colony forming units remaining on the produce were determined. Although microbial reductions of 99 to 99.99% were achieved during ozone treatments, few statistically significant differences (P > 0.1) were detected when comparing the ozonated water to water-only wash. Notably, a significant difference (P = 0.009) in log reduction of MNV + bacteria and MNV alone on BB was observed after 40 min ozonated water wash. Specifically, MNV with B. cereus achieved a 1−log greater reduction (2.60 log PFU/ml) compared to MNV alone (1.63 log PFU/ml). Overall, washing produce in ozonated water did not significantly increase microbial inactivation compared to water alone under the conditions presented here. Variables impacting ozone wash effectiveness should be considered when implementing produce wash sanitation systems within FSE.

Objective This study determined the passage time and phage propagation time of a salmonella specific phage, Felix O1, in bearded dragons, based on reisolation from cloacal swabs and faecal samples following oral administration, as a possible tool for reducing the zoonotic risk of salmonella from pet reptiles. An application scheme for this phage in bearded dragons was developed. Material and methods Ten healthy bearded dragons (Pogona vitticeps) were used in the study. The pH tolerance of the phage was tested and drugs were used to evaluate their influence on the gastric pH of the reptiles. After pH adjustment, the phage was administered orally for 12 consecutive days. Over 60 days, swabs were taken from the cloaca and examined for the presence of phages using culture and PCR. Furthermore, faecal samples were collected for phage quantification. Results Felix O1 displayed no activity at pH below 2.8. A calcium- and magnesium carbonate buffer induced an appropriate gastric pH increase for 30 minutes. Phages were reisolated for up to 24 days (mean shedding: 19 days) after last administration. Titres between 105 and 107 plaque forming units/g faeces were detected. The animals did not show any clinical signs related to phage application. Conclusion and clinical relevance The study provides first results on oral administration, passage time, and reisolation of a phage in reptiles. It could be shown that the phage was able to replicate in the intestine, and was shed for a prolonged period and therefore could potentially contribute to a reduction of salmonella shedding.

Purpose To evaluate the antiviral potential of five multipurpose disinfecting solutions against coronavirus (mouse hepatitis virus, a surrogate for SARS-CoV-2 human corona virus). Methods Test solutions (Biotrue, renu Advanced [Bausch and Lomb], ACUVUE RevitaLens [Johnson and Johnson Vision], cleadew [Ophtecs corp.] or AOSept Plus [Alcon]) were mixed with the coronavirus mouse hepatitis virus at 104 plaque forming units (PFU)/mL as the final concentration and incubated at room temperature for the specified disinfection time. Surviving virus from each sample was then quantified by standard plaque forming unit assay and the reduction of PFU for each disinfectant was compared to the phosphate buffer saline (PBS) treated negative control. A regimen test was also conducted using Biotrue. Results The three multipurpose disinfecting solutions Biotrue (containing PHMB and polyquaternium-1), renu Advanced (PHMB, polyquaternium-1 and alexidine) and ACUVUE RevitaLens (polyquaternium-1 and alexidine) did not kill the coronavirus at the manufacturers recommended disinfection time in the stand alone test. After treatment, the virus’s titer (3.8 ± 0.2 log10 for Biotrue, 3.7 ± 0.1 log10 for renu and 3.7 ± 0.2 log10 for RevitaLens) was similar to the negative control (3.7 ± 0.1 log10; p ≥ 0.996). AOSept Plus (hydrogen peroxide) and cleadew (povidone iodine) significantly (p

The consequences of SARS-CoV-2 infection on the musculoskeletal system represent a dangerous knowledge gap. Aging patients are at added risk for SARS-CoV-2 infection; therefore, a greater understanding of the resulting musculoskeletal sequelae of SARS-CoV-2 infection may help guide clinical strategies. This study examined fundamental bone parameters among mice treated with escalating viral loads. Male C57BL/6J (WT, n = 17) and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (K18-hACE2 transgenic mice, n = 21) expressing human ACE2 (TG) were divided into eight groups (n = 4–6/group) and subjected to intranasal dosing of 0, 1 × 103, 1 × 104, and 1 × 105 PFU (plaque forming units) of human SARS-CoV-2. Animal health was assessed daily by veterinary staff using established and validated scoring criteria (activity, posture, body condition scores and body weight). We report here that mock and WT infected mice were healthy and completed the study, surviving until 12–14 days post infection (dpi). In contrast, the TG mice infected with 1 × 105 PFU all experienced severe health declines that necessitated early euthanasia (6–7 dpi). For TG mice infected with 1 × 104 PFU, 2 mice were also euthanized after 7 dpi, while 3 mice showed signs of moderate disease at day 6 dpi, but recovered fully by day 11 dpi. Four of the 5 TG mice that were infected with 1 × 103 PFU remained healthy throughout the study. This suggests that our study mimics what is seen during human disease, where some patients develop severe disease resulting in death, while others have moderate to severe disease but recover, and others are asymptomatic. At necropsy, femurs were extracted and analyzed by μCT. No difference was found in μCT determined bone parameters among the WT groups. There was, however, a significant 24.4% decrease in trabecular bone volume fraction (p = 0.0009), 19.0% decrease in trabecular number (p = 0.004), 6.2% decrease in trabecular thickness (p = 0.04), and a 9.8% increase in trabecular separation (p = 0.04) among surviving TG mice receiving any viral load compared to non-infected controls. No differences in cortical bone parameters were detected. TRAP staining revealed surviving infected mice had a significant 64% increase in osteoclast number, a 27% increase in osteoclast surface, and a 38% increase in osteoclasts per bone surface. While more studies are needed to investigate the long-term consequences of SARS-CoV-2 infection on skeletal health, this study demonstrates a significant reduction in several bone parameters and corresponding robust increases in osteoclast number observed within 2 weeks post-infection in surviving asymptomatic and moderately affected mice., Graphical abstract Unlabelled Image

Virus destabilization and inactivation are critical considerations in providing safe drinking water. We demonstrate that iron electrocoagulation simultaneously removed (via sweep flocculation) and inactivated a non-enveloped virus surrogate (MS2 bacteriophage) under slightly acidic conditions, resulting in highly effective virus control (e.g., 5-logs at 20 mg Fe/L and pH 6.4 in 30 min). Electrocoagulation simultaneously generated H2O2 and Fe(II) that can potentially trigger electro-Fenton reactions to produce reactive oxygen species such as •OH and high valent oxoiron(IV) that are capable of inactivating viruses. To date, viral attenuation during water treatment has been largely probed by evaluating infective virions (as plaque forming units) or genomic damage (via the quantitative polymerase chain reaction). In addition to these existing means of assessing virus attenuation, a novel technique of correlating transmission electron micrographs of electrocoagulated MS2 with their computationally altered three-dimensional electron density maps was developed to provide direct visual evidence of capsid morphological damages during electrocoagulation. The majority of coliphages lost at least 10-60% of the capsid protein missing a minimum of one of the 5-fold and two of 3- and 2-fold regions upon electrocoagulation, revealing substantial localized capsid deformation. Attenuated total reflectance-Fourier transform infrared spectroscopy revealed potential oxidation of viral coat proteins and modification of their secondary structures that were attributed to reactive oxygen species. Iron electrocoagulation simultaneously disinfects and coagulates non-enveloped viruses (unlike conventional coagulation), adding to the robustness of multiple barriers necessary for public health protection and appears to be a promising technology for small-scale distributed water treatment.

We report the results of a laboratory sensitivity and specificity evaluation of the Rapid Analyte Measurement Platform (RAMP®) Dengue Virus (DENV) antigen detection assay, which is designed to detect all serotypes of DENV in mosquito pools. The RAMP DENV assay was able to detect geographically distinct strains of all 4 DENV serotypes in virus-spiked mosquito pools that contained at least 4.3 log10 plaque forming units/ml, although discrete sensitivity limits varied slightly for each serotype. The RAMP DENV assay also detected DENV 1–4 in mosquito pools containing a single infected mosquito and 24 laboratory-reared uninfected mosquitoes. No false positives were detected in negative control mosquito pools or in samples containing high titers of nontarget arboviruses. We found that while the kit-supplied RAMP buffer reduced the infectious titer of DENV, it did not completely inactivate all serotypes. We recommend adding a detergent, Triton X-100, to the buffer to ensure complete inactivation of DENV if the assay is to be conducted at a lower biosafety level than required for DENV handling.

Susceptibility to SARS-CoV-2 and the outcome of COVID-19 have been linked to underlying health conditions and the age of affected individuals. Here we assessed the effect of age on SARS-CoV-2 infection using a ferret model. For this, young (6-month-old) and aged (18-to-39-month-old) ferrets were inoculated intranasally with various doses of SARS-CoV-2. By using infectious virus shedding in respiratory secretions and seroconversion, we estimated that the infectious dose of SARS-CoV-2 in aged animals is ∼32 plaque forming units (PFU) per animal while in young animals it was estimated to be ∼100 PFU. We showed that viral replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets when compared to young animals. Similar to observations in humans, this was associated with higher transcription levels of two key viral entry factors - ACE2 and TMPRSS2 - in the upper respiratory tract of aged ferrets. Importance In humans, ACE2 and TMPRSS2 are expressed in various cells and tissues, and a differential expression have been described in young and old people, with a higher level of expressing cells being detected in the nasal brushing of older people when compared to young individuals. We described the same pattern occurring in ferrets and we demonstrated that age affects susceptibility of ferrets to SARS-CoV-2. Aged animals were more likely to get infected when exposed to lower infectious dose of the virus when compared to young animals and the viral replication in the URT and shedding is enhanced in aged ferrets. Together these results suggest that the higher infectivity and enhanced ability of SARS-CoV-2 to replicate in aged individuals is associated - at least in part - with transcription levels of ACE2 and TMPRSS2 at the sites of virus entry. The young and aged ferret model developed here may represent a great platform to assess age-related differences in SARS-CoV-2 infection dynamics and replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of ∼107 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of ∼106.5 TCID50/ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts.

To understand susceptibility of California sea lions and Northern elephant seals to influenza A virus (IAV), we developed an ex vivo respiratory explant model and used it to compare infection kinetics for multiple IAV subtypes. We used a similar approach with explants from rhesus macaques to establish the system and to compare infection kinetics with marine mammals. Trachea, bronchi, and lungs from 11 California sea lions, 2 Northern elephant seals and 10 rhesus macaques were inoculated within 24 hours post-mortem with 6 strains representing 4 IAV subtypes. Explants from all 3 species showed similar IAV infection kinetics with peak viral titers 48-72 hours post-inoculation (hpi) that increased by 2-4 log10 PFU/explant relative to the inoculum. Immunohistochemistry localized IAV infection to apical epithelial cells. These results demonstrate that respiratory tissue explants from marine mammals support IAV infection. In the absence of the ability to perform experimental infections of marine mammals, this ex vivo culture of respiratory tissues mirrors the in vivo environment and serves as a tool to study IAV susceptibility, host-range, and tissue tropism. We adapted the explant approach and used it to inoculate tissues from 2 rhesus macaques with severe acute respiratory syndrome virus 2 (SARS-CoV-2). SARS-CoV-2 titers increased by 2-4 log10 PFU/explant relative to the inoculum and peaked 48 or 72 hpi in trachea, bronchi, and kidney of both macaques and in the lung of 1 animal. These results demonstrate that this ex vivo model can define infection dynamics for 2 respiratory viruses of significant public health importance. Importance Although influenza A virus can infect marine mammals, a dearth of marine mammal cell lines and ethical and logistical challenges prohibiting experimental infections of living marine mammals means that little is known about IAV infection kinetics in these species. We circumvented these limitations by adapting a respiratory tract explant model first to establish the approach with rhesus macaques and then for use with marine mammals. We observed that multiple strains representing 4 IAV subtypes infected trachea, bronchi, and lungs of macaques and marine mammals with variable peak titers and kinetics. We adapted the approach for SARS-CoV-2 and observed that the infection kinetics in inoculated rhesus macaque explants parallel observations from ex vivo human lungs. This ex vivo model can define infection dynamics for 2 respiratory viruses of significant public health importance and use of explants from animals euthanized for other reasons reduces use of animals in research.

Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act “universally” against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10-times higher than for a previously reported batch cultivation were observed. Further, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing.Key pointsAn automated perfusion process for production of IAV DIPs was establishedDIP titers of 7.40E+9 plaque forming units per mL were reachedA novel tubular cell retention device enabled continuous virus harvesting

Influenza remains a world-wide health concern, causing 290,000–600,000 deaths and up to 5 million cases of severe illnesses annually. Noticing the host factors that control biological responses, such as inflammatory cytokine secretion, to influenza virus infection is important for the development of novel drugs. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and has essential biological functions in inflammation. However, the kinetic effects of influenza virus infection on physiological S1P levels and their signaling in multiple tissues remain unknown. In this study, we utilized a mouse model intranasally infected with 50 or 500 plaque forming units (PFU) of A/Puerto Rico/8/34 (H1N1; PR8) virus to investigate how S1P levels and expression of its regulating factors are affected by influenza virus infection by the liquid-chromatography/mass spectrometry and real-time PCR, respectively. The S1P level was significantly high in the plasma of mice infected with 500 PFU of the virus than that in control mice at 6 day-post-infection (dpi). Elevated gene expression of sphingosine kinase-1 (Sphk1), an S1P synthase, was observed in the liver, lung, white adipose tissue, heart, and aorta of infected mice. This could be responsible for the increased plasma S1P levels as well as the decrease in the hepatic S1P lyase (Sgpl1) gene in the infected mice. These results indicate modulation of S1P-signaling by influenza virus infection. Since S1P regulates inflammation and leukocyte migration, it must be worth trying to target this signaling to control influenza-associated symptoms.

H9N2 avian influenza virus has been continuously circulating among poultry and could infect mammals, indicating that this virus is a potential pandemic strain. During influenza pandemics, secondary bacterial (particularly pneumococcal) pneumonia usually contributes to excess mortality. In the present study, we observed the dynamic effect of H9N2 virus infection on host defense against secondary pneumococcal infection in mice. BALB/c mice were intranasally inoculated with 1.2 × 105 plaque forming units (PFU) of H9N2 virus followed by 1 × 106 colony forming units of Streptococcus pneumoniae on 7, 14 or 28 days post-H9N2 infection (D.P.I.). The bacterial load, histopathology, body weight and survival were assessed after pneumococcal infection. Our results showed that H9N2 virus infection had no significant impact on host resistance to secondary pneumococcal infection on 7 D.P.I. However, H9N2 virus infection increased pulmonary pneumococcal clearance and reduced pneumococcal pneumonia-induced morbidity after secondary pneumococcal infection on 14 or 28 D.P.I., as reflected by significantly decreased bacterial loads, markedly alleviated pulmonary histopathological changes and significantly reduced weight loss in mice infected with H9N2 virus followed by S. pneumoniae compared with mice infected only with S. pneumoniae. Further, the significantly decreased bacterial loads were observed when mice were previously infected with a higher dose (1.2 × 106 PFU) of H9N2 virus. Besides, similar to the results obtained in BALB/c mice, improvement in pulmonary pneumococcal clearance was also observed in C57BL/6 mice. Overall, our results showed that pulmonary pneumococcal clearance is improved after resolution of H9N2 virus infection in mice.

Although antivirals are important tools to control the SARS-CoV-2 infection, effective vaccines are essential to control the current pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here we report the immunogenicity and protection induced in macaques by intramuscular injections of VLP bearing SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytosine phosphoguanine (CpG) 1018. Although a single dose of unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after prime) and adjuvants significantly improved both responses with a higher immunogenicity and protection provided by AS03 adjuvanted CoVLP. Fifteen microgram CoVLP adjuvanted with AS03 induced a balanced IL-2 driven response along with IL-4 expression in CD4 T cells and mobilization of CD4 follicular helper cells (Tfh). Animals were challenged by multiple routes (i.e. intratracheal, intranasal and ocular) with a total viral dose of 106 plaque forming units of SARS-CoV-2. Lower viral replication in nasal swabs and broncho-alveolar lavage (BAL) as well as fewer SARS-CoV-2 infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of pro-inflammatory cytokines and chemotactic factors in BAL were observed in the animals immunized with CoVLP adjuvanted with AS03. No clinical, pathologic or virologic evidences of vaccine associated enhanced disease (VAED) were observed in vaccinated animals. CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.

Rationale: Immune stimulation with immune checkpoint inhibitors (ICIs) has emerged as a highly effective treatment for several cancer types. Research also suggests these agents may be therapeutic for viral infections. However, by interrupting inhibitory signaling pathways, ICIs can cause immune-related adverse events including pneumonitis. A critical question during the present SARS-CoV-2 pandemic has been whether prior ICI treatment aggravates or improves virus-associated lung injury. Methods: To address this question, we first developed a lethal coronavirus acute lung injury model in A/J mice by infecting them intratracheally (IT) with mouse hepatitis virus-1 (MHV-1), a betacoronavirus that can be studied at Biosafety Level-2 (Study-1). We then investigated the effects of anti-PD-L1 monoclonal antibody (anti-PD-L1mAb;clone 10F.9G2, Bio X Cell) pretreatment on outcomes with MHV-1 lung challenge (Study-2). Results: In Study 1 testing 8 increasing doses of virus [5 to 2000 plaque forming units (PFU)/mouse], IT administration of 12.5 (n=8), 25 (n=8), or 50 (n=16) PFU/mouse produced lethality rates closest to 50% (Figure A). At 14d, surviving mice receiving any of these three doses had decreased circulating lymphocyte and increased lavage lymphocyte percentages and protein concentrations compared to diluent-challenged control animals (p≤0.04 averaged across the three MHV doses). Experiments in noninfected animals showed that compared to isotype-mAb (control) treatment, 4 doses of anti-PD-L1mAb (300μg/mouse) administered intraperitoneally every 3d significantly reduced lung immune cell PD-L1 expression (normalized mean fluorescence intensity, p=0.04;Figure B) and produced anti-PD-L1mAb levels at 14d consistent with those measured in ICI-treated cancer patients ( 283.2 ± 112.4 ug/mL). Therefore, in Study 2, mice were treated with either isotype-mAb or anti-PD-L1mAb (300ug/mouse, every 3 days) starting 12d before and continuing until 3d after IT challenge with 25 or 50PFU/mouse of MHV-1 (Experiments 1 and 2 respectively). Compared to control animals [12 survivors of 12 total animals in Experiment 1 (100%), and 4 of 12 in Experiment-2 (33%)], survival was decreased with PD-L1-mAb in both experiments [11 of 12, (91%) and 2 of 12 (17%), respectively] but these survival differences were not significant (p>0.05) (Figure C). Conclusions: Intratracheal MHV-1 challenge in A/J mice produced lethality and late changes in circulating lymphocytes and lung lavage lymphocytes and protein that appear consistent with changes observed clinically with SARS-CoV2 infection. Prior treatment with anti-PD-L1mAb in this model did not improve and potentially aggravated the lethal effects of MHV-1, but requires further study.

The current global COVID-19 pandemic led to an unprecedented effort to develop effective vaccines against SARS-CoV-2. mRNA vaccines were developed very rapidly during the last year, and became the leading immunization platform against the virus, with highly promising phase-3 results and remarkable efficacy data. Since most animal models are not susceptible to SARS CoV-2 infection, pre-clinical studies are often limited to infection-prone animals such as hamsters and non-human primates. In these animal models, SARS-CoV-2 infection results in viral replication and a mild disease disease. Therefore, the protective efficacy of the vaccine in these animals is commonly evaluated by its ability to elicit immunologic responses, diminish viral replication and prevent weight loss. Our lab recently reported the design of a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). These experiments demonstrated the development of a robust and specific immunologic response in RBD-hFc mRNA-vaccinated BALB/c mice. In the current study, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18-hACE2 mouse model. We report that administration of RBD-hFc mRNA vaccine to K18-hACE2 mice led to a robust humoral response comprised of both binding and neutralizing antibodies. In accordance with the recorded immunologic immune response, 70% of vaccinated mice were protected against a lethal dose (3000 plaque forming units) of SARS-CoV-2, while all control animals succumbed to infection. To the best of our knowledge, this is the first non-replicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.

BACKGROUND: Avian influenza A (H7N9) virus has caused more than 1,500 cases of human infection since its emergence in early 2013. Displaying little or no pathogenicity in poultry, but a 40% case-fatality rate in humans, five waves of H7N9 human infections occurred in China during 2013–2017, caused solely by a low pathogenicity strain. However, avian isolates possessing a polybasic connecting peptide in the hemagglutinin (HA) protein were detected in mid-2016, indicating that a highly pathogenic virus had emerged and was co-circulating with the low pathogenicity strains. METHODS: Here we characterize the pathogenicity of a newly emerged human H7N9 variant with a PEVPKRKRTAR/GLF insertion motif at the cleavage site of the HA protein in vitro and in vivo. RESULTS: This variant replicates in MDCK cells independently of TPCK-trypsin, which is indicative of high pathogenicity in chickens. The 50% mouse lethal dose (MLD(50)) of this novel isolate was less than 10 plaque forming units (PFU), compared with 3.16×10(4) for an identical virus lacking the polybasic insertion, indicating a high virulence phenotype. CONCLUSIONS: Our results demonstrate that the multiple basic amino acid insertion in the HA protein of the H7N9 variant confers high virulence in mammals, highlighting a potential risk to humans. Continuous viral surveillance is therefore necessary in the China region to improve pandemic preparedness.

Background The purpose of the study was to evaluate the in vitro antibacterial effect of experimental eye drops with bacteriophages in elimination of Staphylococcus spp. isolated from dogs with bacterial conjunctivitis.. The bacterial material was collected from dogs with independent clinical signs of bacterial conjunctivitis. Staphylococcus spp. were identified by phenotypic and genotypic methods (MALDI-TOF MS mass spectrometry). Antibiotic resistance was determined by the disc-diffusion method. Phage activity (Plaque forming units, PFU) was determined on double-layer agar plates. Phages with lytic titres > 108 PFU were used to prepare eye drops. The stability of the antibacterial titre was evaluated for preparations stored in sealed bottles as well as after opening and reclosing. Results The tests confirmed the occurrence of Staphylococcus spp. strains as etiological agents of bacterial conjunctivitis in dogs. A high percentage of strains were resistant to more than three antibiotics. The experimental phage eye drops used in the study exhibited 100% efficacy in vitro against the tested Staphylococcus isolates. Particularly noteworthy is the long duration of activity and constant antibacterial lytic titre of ≥108 PFU/mL of two eye drop solutions, nos. 7 and 12, after the bottle had been opened (21 days) and after hermetically sealed packaging (28 days) at 4–8 °C. Conclusions The results represent the first stage of research and require continuation in vivo. If positive effects are obtained in animals, the results can be used in applied research in humans and animals.

IntroductionNosocomial transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a major feature of the COVID-19 pandemic. Evidence suggests patients can auto-emit aerosols containing viable viruses; these aerosols could be further propagated when patients undergo certain treatments, including continuous positive airway pressure (PAP) therapy. Our aim was to assess 1) the degree of viable virus propagated from PAP circuit mask leak and 2) the efficacy of a ventilated plastic canopy to mitigate virus propagation.MethodsBacteriophage phiX174 (108 copies·mL−1) was nebulised into a custom PAP circuit. Mask leak was systematically varied at the mask interface. Plates containing Escherichia coli host quantified viable virus (via plaque forming unit) settling on surfaces around the room. The efficacy of a low-cost ventilated headboard created from a tarpaulin hood and a high-efficiency particulate air (HEPA) filter was tested.ResultsMask leak was associated with virus contamination in a dose-dependent manner (χ2=58.24, df=4, p−1) was associated with virus counts equivalent to using PAP with a vented mask. The highest frequency of viruses was detected on surfaces 3·h−1 eradicated all evidence of virus contamination.ConclusionsMask leak from PAP may be a major source of environmental contamination and nosocomial spread of infectious respiratory diseases. Subclinical mask leak levels should be treated as an infectious risk. Low-cost patient hoods with HEPA filtration are an effective countermeasure.

Several techniques are currently available to quickly and accurately quantify the number of virus particles in a sample, taking advantage of advanced technologies improving old techniques or generating new ones, generally relying on partial detection methods or structural analysis. Therefore, characterization of virus infectivity in a sample is often essential, and classical virological methods are extremely powerful in providing accurate results even in an old-fashioned way. In this chapter, we describe in detail the techniques routinely used to estimate the number of viable infectious coronavirus particles in a given sample. All these techniques are serial dilution assays, also known as titrations or end-point dilution assays (EPDA).

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), is transmitted person-to-person via respiratory droplets and, likely, via smaller droplet nuclei light enough to remain suspended in the air for hours and contaminate surfaces particularly in indoor conditions. Thus, effective measures are needed to prevent SARS-CoV-2 transmission in indoor environments. In this regard, we have investigated whether a system based on a filter combining Tungsten Trioxide-Based (WO3) photocatalysis and an antiviral fabric treated-copper nanocluster could inactivate SARS-CoV-2. To this purpose, an infectious SARS-CoV-2 suspension was introduced in the upper opening of a closed cylinder containing a WO3 filter and a lightbased system that activates WO3 and the antiviral fabric. From the bottom exit, aliquots of fluid were collected every 10 min (up to 60 min) and tested for their infectivity by means of a viral plaque assay in Vero cells whereas, in parallel, the viral RNA content was measured by quantitative PCR (qPCR). As we have previously shown for SARS-CoV, a 1:1,000 ratio of plaque forming units (PFU) vs. viral RNA copies was observed also for SARS-CoV-2. After 10 min, the infectious viral content was already decreased by 98.2% reaching 100% inactivation after 30 min whereas the SARS-CoV-2 RNA load was decreased of 1.5 log10 after 30 min. Thus, in spite of only a partial decrease of viral RNA, SARS-CoV-2 infectivity was completely abolished by the WO3 photocatalysis system by 30 min. These results support the hypothesis that this system could be exploited to achieve SARS-CoV-2 inactivation in indoor environments.

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2)(1-3). Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in both the lung and brain leading to lethality. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the lung, and no clinical signs of infection with a challenge dose of 104 plaque forming units. The K18-hACE2 model provides a stringent model for testing the ability of vaccines and antivirals to protect against disease, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.

Post Influenza bacterial pneumonia is associated with significant mortality and morbidity. Dendritic cells (DCs) play a crucial role in host defense against bacterial pneumonia, but their contribution to post influenza-susceptibility to secondary bacterial pneumonia is incompletely understood. WT and CCR2−/− mice were infected with 100 plaque forming units (pfu) H1N1 intranasally alone or were challenged on day 5 with 7×107 colony forming units (cfu) methicillin-resistant Staphylococcus aureus intratracheally. WT mice express abundant CCL2 mRNA and protein post-H1N1 alone or dual infection. CCR2−/− mice had significantly higher survival as compared to WT mice, associated with significantly improved bacterial clearance at 24 and 48 hours (10 fold and 14 fold, respectively) post-bacterial challenge. There was robust upregulation of IL-23 and IL-17 as well as down-regulation of IL-27 expression in CCR2−/− mice following sequential infection as compared to WT mice, which was also associated with significantly greater accumulation of CD103+ DC. Finally, WT mice treated with a CCR2 inhibitor showed improved bacterial clearance in association with similar cytokine profiles as CCR2−/− mice. Thus, CCR2 significantly contributes to increased susceptibility to bacterial infection after influenza pneumonia likely via altered dendritic cell responses and thus, CCR2 antagonism represents a potential therapeutic strategy.

BACKGROUND:The arenavirus Junin virus (JUNV), causative agent of the argentine hemorrhagic fever, is able to modulate several signaling pathways involved in cell survival and multiplication.OBJECTIVES:We aimed to characterize the infection of rat osteoblasts (OBCs) with JUNV and its consequence on the modulation of osteogenic genes expression, thus studying the ability of this virus to induce cell differentiation. In addition, we evaluated the effect of purinergic agonists on viral replication.METHOD:Quantification of infectivity by plaque forming unit (PFU) assay, synthesis of viral proteins by western blot and immunofluorescence, and expression of osteogenic differentiation markers (ODM) by quantitative real-time polymerase chain reaction were employed.RESULTS:Infection of OBCs with JUNV (MOI 0.01 PFU/cell) showed a peak of infectivity, reaching 1.5 × 105 PFU/mL at the second day post-infection (p.i.). A marked restriction in multiplication was detected at day 7 p.i. that did not impair the establishment of a persistent stage of infection in OBCs. Analysis of mRNAs corresponding to ODM such as alkaline phosphatase, bone sialo-protein, and bone morphogenetic proteins (BMPs) 4 and 6 revealed that only the levels of BMP-6 were significantly higher in infected cells. Treatment with the purinergic agonists ATPγS, UTP, ADP, or UDP diminished viral titer and reduced the expression of the viral nucleoprotein. Also, treatment with 10 μM ATPγS reduced the stimulation of BMP-6 expression induced by the infection.CONCLUSIONS:These data demonstrate that JUNV is capable of infecting OBCs and point out BMP-6 as a key factor during this process. Fil: Ayala Peña, Victoria Belen. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Armiento, María Nieves. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernandez Bell Fano, Pablo Maria. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Santillan, Graciela Edith. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina Fil: Scolaro, Luis Alberto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

COVID-19 has become a pandemic and is spreading fast worldwide. The COVID-19 virus is transmitted mainly through respiratory droplets and close contact. However, the fecal-oral transmission of the virus has not been ruled out and it is important to ascertain how acidic condition in the stomach affects the infectivity of the virus. Besides, it is unclear how stable the COVID-19 virus is under dry and wet conditions. In the present study, we have shown that the COVID-19 virus is extremely infectious as manifested by the infection of Vero-E6 cells by one PFU (Plaque Forming Unit) of the virus. We then investigated the stability of the COVID-19 virus in wet, dry and acidic (pH2.2) environments at room temperature. Results showed that the COVID-19 virus could survive for three days in wet and dry environments, but the dry condition is less favorable for the survival of the virus. Our study also demonstrated that the COVID-19 virus at a relative high titer (1.2 x 103 PFU) exhibits a certain degree of tolerance to acidic environment at least for 60 minutes. When the virus titer was ≤1.0 x 103 PFU, acid treatment (pH2.2) for 30 or 60 minute resulted in virus inactivation. It suggests that the virus at a high concentration may survive in the acidic environment of the stomach. The finding of the present study will contribute to the control of the spread of the COVID-19 virus.

The three encephalitic alphaviruses, western, eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV) are potential biothreat agents due to high infectivity through aerosol exposure, ease of production in large amounts, and relative stability in the environment. Currently, there is no licensed vaccine for human use to these three encephalitic alphaviruses, and efforts to move vaccine candidates forward into clinical trials have not been successful. In this study, the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform was used to construct and produce three monovalent recombinant MVA-BN-based encephalitic alphavirus vaccines, MVA-BN-W, MVA-BN-E, and MVA-BN-V. Additionally, a MVA-BN-based construct was designed to produce antigens against all three alphaviruses, the trivalent vaccine MVA-BN-WEV. The protective efficacy of these vaccines was evaluated in vivo. Female BALB/c mice were immunized with two doses of each monovalent MVA-BN-based alphavirus vaccine, a mixture of the three monovalent vaccines, MVA-BN-W + E + V, or the trivalent vaccine MVA-BN-WEV at a four-week interval. Two weeks after the booster immunization, the mice were instilled intranasally with 5 × 103 to 1 × 104 plaque forming units of WEEV, EEEV, or VEEV. All mice immunized with monovalent vaccines survived the respective virus challenge without any signs of illness or weight loss, while all the control mice died. The triple mixture of vaccines or the trivalent vaccine also provided 90 to 100% protection to the mice against WEEV and VEEV challenges, and 60% to 90% protection against EEEV challenge. These data suggest that each monovalent MVA-BN-W, MVA-BN-E, and MVA-BN-V is a potential vaccine candidate against respective encephalitic alphavirus and the three monovalent vaccines can be given in a mixture (MVA-BN-W + E + V) or the trivalent vaccine MVA-BN-WEV can serve as a true multivalent vaccine without significantly reducing efficacy against WEEV and VEEV despite slightly reduced efficacy against EEEV challenge.

Herein, we introduced a molecular imprinting based capacitive biosensor for real-time and highly sensitive bacteriophage detection. The sensing mechanism was based on the binding of target phage into the specific cavities on the electrode surface which resulted in a measurable change in the total capacitance of the system. Phage detection was investigated in the concentration range of 1.0 × 101–1.0 × 105 plaque forming units (pfu)/mL and the limit of detection (LOD) was measured as 10 pfu/mL which shows the high sensitivity of the system compared to results reported for previous studies. The system also allowed the detection of phages in river water samples which is very important for the usability of the system as in-field analysis for different applications e.g. investigating the contamination of drinking water via wastewater or reservoir water in the future. Recently, due to their high specificity towards their host bacteria, being cost-effective and also stable in harsh environments, bacteriophages have been used as biorecognition elements in many studies. Due to this reason, the applicability of the phage imprinted biosensor was also investigated for host bacteria detection. E. coli detection has been performed in the concentration range of 1.0 × 102–1.0 × 107 colony forming units (cfu)/mL with a LOD value of 100 cfu/mL. This system offers direct, real-time, very sensitive and rapid detection of bacteriophage and its host bacteria.

This study investigated the susceptibility of American singer canaries ( Serinus canaria) to West Nile virus (WNV) infection. Adult canaries were inoculated with 105, 102, and 101 plaque forming units (PFU) of WNV. All birds became infected and mortality occurred by 5 days postinoculation. The load of viral RNA as determined by RT-qPCR was dose dependent, and was higher at all doses than the level of viral RNA detected in American crows ( Corvus brachyrhynchos) challenged with 105 PFU of WNV. In a subset of birds, viremia was detected by virus isolation; canaries inoculated with 101 PFU of WNV developed viremia exceeding 1010 PFU/mL serum, a log higher than American crows inoculated with 105 PFU of virus. In canaries euthanized at 3 days postinoculation, WNV was isolated at >107 PFU of virus/100 mg of lung, liver, heart, spleen, and kidney tissues. Pallor of the liver and splenomegaly were the most common macroscopic observations and histologic lesions were most severe in liver, spleen, and kidney, particularly in canaries challenged with 102 and 101 PFU. Immunoreactivity to WNV was pronounced in the liver and spleen. IgG antibodies to WNV were detected in serum by enzyme immunoassay in 11 of 21 (52%) challenged canaries and, in 4 of 5 (20%) of these sera, neutralization antibodies were detected at a titer ≥ 1:20. American singer canaries provide a useful model as this bird species is highly susceptible to WNV infection.

The influenza A virus (IAV)-cytokine-trypsin/matrix metalloproteinase-9 (MMP-9) cycle is one of the important mechanisms of multiple organ failure in severe influenza. Clarithromycin, a macrolide antibiotic, has immune modulatory and anti-inflammatory effects. We analyzed the effects of clarithromycin on the induction of chemokines, cytokines, MMP-9, trypsin, vascular hyper-permeability and inflammatory aggravation in mice with IAV infection. IAV/Puerto Rico/8/34(H1N1) infection increased the levels of monocyte chemoattractant protein-1 (MCP-1) and cytokines in serum, and MMP-9 and trypsin in serum and/or the lungs and heart. Clarithromycin significantly suppressed the induction of serum MCP-1 and MMP-9 and vascular hyperpermeability in these organs in the early phase of infection, but did not suppress the induction of trypsin, IL-6 or IFN-γ. Histopathological examination showed that clarithromycin tended to reduce inflammatory cell accumulation in the lungs and heart. These results suggest that clarithromycin suppresses infection-related inflammation and reduces vascular hyperpermeability by suppressing the induction of MCP-1 and MMP-9.

The family Filoviridae, which includes the genera Marburgvirus and Ebolavirus, contains some of the most pathogenic viruses in humans and non-human primates (NHPs), causing severe hemorrhagic fevers with high fatality rates. Small animal models against filoviruses using mice, guinea pigs, hamsters, and ferrets have been developed with the goal of screening candidate vaccines and antivirals, before testing in the gold standard NHP models. In this review, we summarize the different animal models used to understand filovirus pathogenesis, and discuss the advantages and disadvantages of each model with respect to filovirus disease research.

The present study aimed to establish the kinetics of the appearance of coliphage plaques using the double agar layer titration technique to evaluate the feasibility of using traditional coliphage plaque forming unit (PFU) enumeration as a rapid quantification method. Repeated measurements of the appearance of plaques of coliphages titrated according to ISO 10705-2 at different times were analysed using non-linear mixed-effects regression to determine the most suitable model of their appearance kinetics. Although this model is adequate, to simplify its applicability two linear models were developed to predict the numbers of coliphages reliably, using the PFU counts as determined by the ISO after only 3 hours of incubation. One linear model, when the number of plaques detected was between 4 and 26 PFU after 3 hours, had a linear fit of: (1.48 × Counts3 h + 1.97); and the other, values >26 PFU, had a fit of (1.18 × Counts3 h + 2.95). If the number of plaques detected was

BACKGROUND Concerned over the risk of Zika virus (ZIKV) transfusion transmission, public health agencies recommended the implementation of mitigation strategies for its prevention. Those strategies included the use of pathogen inactivation for the treatment of plasma and platelets. The efficacy of amotosalen/ultraviolet A to inactivate ZIKV in plasma had been previously demonstrated, and the efficacy of inactivation in platelets with the same technology was assumed. These studies quantify ZIKV inactivation in platelet components using amotosalen/ultraviolet A. STUDY DESIGN AND METHODS Platelet components were spiked with ZIKV, and ZIKV infectious titers and RNA loads were measured by cell culture-based assays and real-time polymerase chain reaction in spiked platelet components before and after photochemical treatment using amotosalen/ultraviolet A. RESULTS The mean ZIKV infectivity titers and RNA loads in platelet components before inactivation were either 4.9 log10 plaque forming units per milliliter, or 4.4 log10 50% tissue culture infective dose per milliliter and 7.5 log10 genome equivalents per milliliter, respectively. No infectivity was detected immediately after amotosalen/ultraviolet A treatment. No replicative virus remained after treatment, as demonstrated by multiple passages on Vero cell cultures; and ZIKV RNA was not detected from the first passage after inactivation. Additional experiments in this study demonstrated efficient inactivation to the limit of detection in platelets manufactured in 65% platelet additive solution, 35% plasma, or 100% plasma. CONCLUSION As previously demonstrated for plasma, robust levels of ZIKV inactivation were achieved in platelet components. With inactivation of higher levels of ZIKV than those reported in asymptomatic, RNA-reactive blood donors, the pathogen-inactivation system using amotosalen/ultraviolet A offers the potential to mitigate the risk of ZIKV transmission by plasma and platelet transfusion.

The infectivity of equine herpesvirus (EHV)-9 has been studied in different animal models including immunocompromised animals. The current study focused on the infectivity of EHV-9 in different mouse strains (C3H, C57BL, DBA, BALB/c-nu/nu, BALB/c and ICR) by intranasal inoculation of 2 × 106 plaque forming units (PFU). Various organs, including head and lungs, were collected 7 days post infection (dpi) to investigate microscopical lesions and the distribution of EHV-9 antigen. Immunopositivity of tissue sections was scored using ImageJ software. Open reading frame (ORF) 30 expression in lung tissues was quantified using quantitative reverse transcriptase polymerase chain reaction. Pathological examination revealed different degrees of rhinitis in the different mouse strains. Severe rhinitis was detected in C3H and BALB/c-nu/nu strains, moderate rhinitis was observed in C57BL and DBA strains and no lesions were detected in BALB/c mice. Immunopositivity for EHV-9 antigens was detected in the olfactory epithelium of C3H and BALB/c-nu/nu strains. Compared with C57BL, DBA, BALB/c-nu/nu, ICR and BALB/c strains, the C3H strain showed greater expression of EHV-9 antigens in the brain. The proportion of areas with high positive to positive immunoreactivity for EHV-9 were 7.57, 3.42, 3.12, 2.51, 1.79 and 0.03% for C3H, C57BL, DBA, BALB/c-nu/nu, ICR and BALB/c strains, respectively. The proportions of areas with low positive to negative immunoreactivity were 92.42, 96.70, 96.87, 97.48, 98.16 and 99.96%, respectively. The highest relative expression levels for EHV-9 ORF30 in the lungs were in C3H mice. No significant differences in the expression of ORF30 were observed in other strains. In conclusion, of the strains examined, C3H, C57BL, DBA, BALB/c-nu/nu and ICR were the most susceptible to EHV-9 infection, and the BALB/c strain was less susceptible.

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Tick borne encephalitis (TBE) is a dangerous neurological disease that is transmitted to humans through the bite of Ixodid ticks. The disease exhibits an estimated 16 000 cases recorded annually over 30 European and Asian countries. The agent of TBE is the tick borne encephalitis virus (TBEV), belonging to the family Flaviviridae, genus Flavivirus. In spite the significant impact of TBE on human health, there is a serious lack of specific treatment against this disease. The only specific drug available is the human anti-TBEV immunoglobulin from vaccinated blood donors. The drug is produced and used in Russia only, both to prevent and to cure the TBE. In this work, we evaluated the ability of TBEV-specific DNA-aptamers and extracts of traditional medicine plants to neutralize the TBEV Selection of aptamers was performed using SELEX approach. Extracts of the seeds of Momordica cochinchinensis and Terminalia chebula were produced by boiling the ground seeds in water, clarified by centrifugation and filtration steps and filter sterilized. The SELEX had produced two aptamers - My13 and My38. Neither of two was capable to neutralize TBEV in vitro. The ability to shield the antibody binding sites on the surface of TBEV virions was also absent. The extract of M. cochinchinensis exhibited no neutralizing activity as well. Surprisingly, the T. chebula extract completely neutralized the TBEV after 30 min of incubation at 37 °C. The possible explanations and further development of the project are discussed.

Virotherapy is emerging as an alternative treatment of cancer. Among the candidate oncolytic viruses (OVs), Newcastle disease virus (NDV) has emerged as a promising non-engineered OV. In the present communication, we explored the oncolytic potential of R2B Mukteshwar strain of NDV using SW-620 colon cancer cells. SW-620 cells were xenografted in nude mice and after evaluation of the safety profile, 1 x 107 plaque forming units (PFU) of NDV were inoculated as virotherapeutic agent via the intratumoral (I/T) and intravenous (I/V) route. Tumor growth inhibition was compared with their respective control groups by gross volume and histopathological evaluation. Antibody titer and virus survival were measured by hemagglutination inhibition (HI)/serum neutralization test (SNT) and real-time PCR, respectively. During the safety trial, the test strain did not produce any abnormal symptoms nor weight loss in BALB/c mice. Significant tumor lytic activity was evident when viruses were injected via the I/T route. There was a 43 and 57% tumor growth inhibition on absolute and relative tumor volume basis, respectively, compared with mock control. On the same basis, the I/V route treatment resulted in 40 and 16% of inhibition, respectively. Histopathological examination revealed that the virus caused apoptosis, followed by necrosis, but immune cell infiltration was not remarkable. The virus survived in 2/2 mice until day 10 and in 3/6 mice by day 19, with both routes of administration. Anti-NDV antibodies were generated at moderate level and the titer reached a maximum of 1:32 and 1:64 via the I/T and I/V routes, respectively. In conclusion, the test NDV strain was found to be safe and showed oncolytic activity against the SW-620 cell line in mice.

In 2008, viral hemorrhagic septicemia virus (VHSV) genotype IVb, the causative agent of viral hemorrhagic septicemia (VHS) in a wide range of fish, was isolated from sea lamprey (Petromyzon marinus) in the Great Lakes Basin. To better understand the implications of the reported isolation, we utilized four virus detection methods to follow the pathogenesis on an experimental infection of VHSV IVb in 60 wild, adult lamprey. Thirty lamprey were infected by intracoelomic cavity injections with 106 plaque forming units (pfu) of the VHSV IVb MI03 isolate, and 30 control lamprey were injected with sterile culture media. All media-injected controls tested negative by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and cell culture. Twenty-six of the 30 infected lamprey were positive for VHSV by qRT-PCR, and nine were positive by cell culture. In-vivo replication was demonstrated by a significant temporal increase in the mean concentration of VHS viral RNA copy number equivalents in pooled viscera measured by qRT-PCR from day 7 to day 28 and an increase in cell culture positive pools at day 28. Microscopically, infected lamprey did not develop lesions typical of virulent VHSV. Despite this, the virus was identified by immunohistochemistry in multiple organs, most readily in the kidney, where localization progressed from the interstitium to the tubular lumens. The combined results from qRT-PCR, cell culture, histopathology, and immunohistochemistry suggest that VHSV causes a sublethal infection in lamprey characterized by depletion of renal interstitial cells and in-vivo viral replication with maintenance in the brain at later stages of infection.

Background: Nosocomial transmission of SARS-CoV-2 has been a major cause of morbidity and mortality in the COVID-19 pandemic. Emerging evidence suggests patients auto-emit aerosols containing viable respiratory viruses. These aerosols could be further propagated when patients undergo certain treatments including continuous positive airway pressure (PAP) therapy. This study aimed to assess the degree of viable virus propagated from mask leak in a PAP circuit and the mitigation of virus propagation by an air filter combined with a plastic canopy. Methods: Bacteriophage PhiX174 (108 copies/mL) was nebulised into a custom PAP circuit within a non-vented clinical room. Mask leak was systematically varied to allow 0, 7, 21, 28 and 42 L/min at the mask interface. Plates containing Escherichia coli host assessed the degree of viable virus (via plaque forming unit) settling on surfaces around the room. In order to contain virus spread, the efficacy of a simple, low-cost ventilated headboard, created from a plastic tarpaulin hood and a high efficiency particulate air (HEPA) filter was tested. Findings: Increasing mask leak was associated with virus contamination in a dose response manner (χ2 = 58.24, df=4, p

The U.S. FDA Food Safety Modernization Act Preventive Controls for Human Food Rule emphasizes the importance of an effective environmental monitoring (EM) program. This study aims to characterize polyurethane foam (PUF) EM tools—currently used in the food industry for the recovery of bacteria from food contact surfaces—for their efficacy in the release and recovery of human enteric viruses. Two viruses (human norovirus [hNoV] and Tulane virus [TV]) were compared at varying inoculum levels, with two EM tools (PUF swab and sponge), two delayed processing times (24 h and 72 h), and one surface type (stainless steel [SS]). Specifically, the objectives were to (1) determine the ability of PUF devices to release viruses for detection and (2) assess the ability of PUF devices to recover viruses from SS surfaces. For TV release from the sponge, there was a significant difference (p = 0.0064) when compared across inoculum level (105 plaque forming unit [PFU]/sponge vs. 102 PFU/sponge). Release of hNoV at a single inoculum level by PUF sponge and swab was compared resulting in a significant difference (p

Numerous animal models of systemic orthopoxvirus disease have been developed to evaluate therapeutics against variola virus (VARV), the causative agent of smallpox. These animal models do not resemble the disease presentation in human smallpox and most used surrogate Orthopoxviruses. A rodent model using VARV has a multitude of advantages, and previous investigations identified the CAST/EiJ mouse as highly susceptible to monkeypox virus infection, making it of interest to determine if these rodents are also susceptible to VARV infection. In this study, we inoculated CAST/EiJ mice with a range of VARV doses (102-106 plaque forming units). Some animals had detectable viable VARV from the oropharynx between days 3 and 12 post inoculation. Despite evidence of disease, the CAST/EiJ mouse does not provide a model for clinical smallpox due to mild signs of morbidity and limited skin lesions. However, in contrast to previous rodent models using VARV challenge (i.e. prairie dogs and SCID mice), a robust immune response was observed in the CAST/EiJ mice (measured by Immunoglobulin G enzyme-linked immunosorbent assay). This is an advantage of this model for the study of VARV and presents a unique potential for the study of the immunomodulatory pathways following VARV infection.

Oncolytic viruses have shown excellent safety profiles in preclinical and clinical studies; however, in most cases therapeutic benefits have been modest. We have previously reported the generation of a chimeric poxvirus (CF33), with significantly improved oncolytic characteristics, through chimerization among different poxviruses. Here we report the sequence analysis of CF33 and oncolytic potential of a GFP-encoding CF33 virus (CF33-GFP) with a J2R deletion in lung cancer models. Replication of CF33-GFP and the resulting cytotoxicity were higher in cancer cell lines compared to a normal cell line, in vitro. After infection with virus, cancer cells expressed markers for immunogenic cell death in vitro. Furthermore, CF33-GFP was safe and exerted potent anti-tumor effects at a dose as low as 1000 plaque forming units in both virus-injected and un-injected distant tumors in A549 tumor xenograft model in mice. Likewise, in a syngeneic model of lung cancer in mice, the virus showed significant anti-tumor effect and was found to increase tumor infiltration by CD8+ T cells. Collectively, these data warrant further investigation of this novel chimeric poxvirus for its potential use as a cancer bio-therapeutic.

Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic diversity among its serotypes, requiring several anti-FMDV antibodies for its laboratory diagnosis, which complicated the used techniques. To conquer this cumbersome, we developed a new panel of different single-chain fragment variable (scFv) for serotype-independent detection of FMDV. The recombinant VP2 capsid protein, as a relatively conserved protein among FMDV serotypes, was expressed in E. Coli, and injected in mice. Spleen's RNA was extracted for isolating the coding sequences of IgG variable domains that were assembled into repertoires of scFv. Phage library displaying scFv was constructed with ∼1.9 × 108 plaque forming units. Characterization of the library showed eight of unique scFvs, which were expressed as bacterial periplasmic proteins with apparent molecular weight of ∼27 kDa. Our data revealed the broad-spectrum binding affinity of the eight scFvs as both coating and tracing antibodies to FMDV serotypes A, O, and SAT 2.

The production of phages for therapeutic purposes demands fast, efficient and scalable purification procedures. Phage lysates have a wide range of impurities, of which endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species are harmful to humans. The highest allowed endotoxin concentration for parenterally applied medicines is 5 EU/kg/h. The aim of this study was to evaluate the feasibility of different purification methods in endotoxin and protein toxin removal in the production of phage preparations for clinical use. In the purification assays, we utilized three phages: Escherichia phage vB_EcoM_fHoEco02, Acinetobacter phage vB_ApiMiHyAci03, and Staphylococcus phage vB_SauMiRuSau02. The purification methods tested in the study were precipitation with polyethylene glycol, ultracentrifugation, ultrafiltration, anion exchange chromatography, octanol extraction, two different endotoxin removal columns, and different combinations thereof. The efficiency of the applied purification protocols was evaluated by measuring phage titer and either endotoxins or staphylococcal enterotoxins A and C (SEA and SEC, respectively) from samples taken from different purification steps. The most efficient procedure in endotoxin removal was the combination of ultrafiltration and EndoTrap HD affinity column, which was able to reduce the endotoxin-to-phage ratio of vB_EcoM_HoEco02 lysate from 3.5 x 10(4) Endotoxin Units (EU)/10(9) plaque forming units (PFU) to 0.09 EU/10 9 PFU. The combination of ultrafiltration and anion exchange chromatography resulted in ratio 96 EU/10(9) PFU, and the addition of octanol extraction step into this procedure still reduced this ratio threefold. The other methods tested either resulted to less efficient endotoxin removal or required the use of harmful chemicals that should be avoided when producing phage preparations for medical use. Ultrafiltration with 100,000 MWCO efficiently removed enterotoxins from vB_SauM_fRuSau02 lysate (from 1.3 to 0.06 ng SEA/10(9) PFU), and anion exchange chromatography reduced the enterotoxin concentration below 0.25 ng/ml, the detection limit of the assay.

The host E. coli and its phage were isolated from sewage water. The phage infected host, E coli cells were encapsulated in alginate at specific time interval of co-cultivation, beads were stored at different temperatures. Beads were solublised to release phages and stability of released phages was evaluated by performing plaque assay at monthly interval. The number of plaque forming units increased with time of co-cultivation and by 45mins of phage-host co-cultivation, count raised from 106 to 108 pfu/ml. The infected host cells were entrapped in calcium alginate at a stage when multiple copies of mature phage are ready within host cell, and well before commencement of cell lysis.

Biofouling is a pervasive challenge in industrial and medical settings with enormous economic and health impacts. Use of quorum-quenching phages is a potential solution in either combating the biofilm or inhibiting biofilm formation when an engineered phage is designed for a specific bacterial system. In the event of a real-life application of synthetic phage, it is necessary to consider the effect of environmental conditions on the synthetic phage. This study focused on both (1) stability of phage lysate under different types of carbon sources and (2) competition of the engineered phage in comparison to a wild-type (wt) phage. Optimal results were found (in plaque forming units [PFUs]) with glucose as a carbon source and were kept stable for almost 30 days. Results suggested that competition happens before 4 h. After that time, polymerase chain reaction results from PFU samples showed an increase in T7aiiA, which indicates that these conditions favored the phage replication of the engineered pha...

In cell culture infections with vaccinia virus the number of counted virus particles is substantially higher than the number of plaques obtained by titration. We found that standard vaccine preparations of recombinant Modified Vaccinia virus Ankara produce only about 20–30% plaque-forming virions in fully permissive cell cultures. To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant viruses expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and automated counting by fluorescent microscopy indicated that virtually all virus particles can enter cells and switch on viral gene expression. Although most of the non-plaque-forming infections are arrested at the level of viral early gene expression, we detected activation of late viral transcription in 10–20% of single infected cells. Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of vaccinia virus vaccines., Highlights • Recombinant vaccinia virus MVA preparations contain >70% non-plaque-forming virions. • Non-plaque-forming particles can enter cells and switch on viral gene expression. • Non-plaque-forming virions are likely to contribute to vaccine immunogenicity.

The most severe manifestation of Herpes Simplex Type 1 virus (HSV-1) infection is encephalitis characterized by arousal impairment and seizures that can evolve to coma and death. Previous studies reported the involvement of suppressor of cytokine signaling (SOCS) proteins, specifically SOCS1 and SOCS3, in HSV-1 infection, suggesting that other members of this family could be involved in the immune response against HSV-1. No previous study has reported the role of SOCS2 in HSV-1 infection. In the current study, C57BL/6 wild-type mice (WT) and mice deficient in SOCS2 gene (SOCS2−/−) were subjected to intracranial inoculation with 102 plaque forming units (PFU) of HSV-1. Survival curve, neuroinflammatory parameters and neuropathology were evaluated. Infected SOCS2−/− mice had increased survival in comparison with infected WT animals. This better outcome was associated with reduced leukocyte infiltration, concentration of cytokines, and structural changes in the brain. SOCS2 seems to play a detrimental role in HSV-1 encephalitis. Moreover, the control of neuroinflammatory response in HSV-1 infection was of paramount importance to clinical outcome.

A modified enzyme-linked immunosorbent assay (ELISA) with nanomaterials is an effective and powerful method to amplify the signal and reduce the cost of detecting and measuring trace biomarkers or proteins. In this study, an ultra-sensitive colorimetric immunoassay was designed, and its ability to detect influenza viruses using positively charged gold nanoparticles ((+)Au NPs) was assessed as a possible role for peroxidase-mimic inorganic enzymes. This method detected influenza virus A (H1N1) with a linear range up to 10 pg mL(-1) and clinically isolated influenza virus A (H3N2) up to 10 plaque forming units (PFU) mL(-1) , where its sensitivity improved to 500-fold higher than that of commercial virus kits. The sensitivity of this proposed method was not declined even though in complex biological media in compared to conventional ELISA. These results revealed that the (+)AuNP-based colorimetric immunoassay could be suitable for lab-on-a-chip device and open new opportunities for clinical protein diagnostics. Biotechnol. Bioeng. 2016;113: 2298-2303. © 2016 Wiley Periodicals, Inc.

There is currently no animal model to evaluate the preventive efficacy of antiviral drugs for the treatment of smallpox in human patients with a weak immune system. Therefore, the possibility of using outbred severe combined immunodeficiency (SCID) mice as an animal model was examined by electron microscopy and virological, histological, and statistical methods. There were no clinical signs of disease after the intranasal infection of mice with the variola virus (VARV) at a dose of 5.2 log10 PFU (plaque forming unit). At the same time, the median infective dose (ID50) of VARV estimated by registering the presence of the virus in the lungs of animals four days after infection was 3.5 log10 PFU, and thus, was close to that theoretically determined in human patients by identifying the clinical picture of the disease. The reproduction of the virus was detected only in the respiratory organs of mice infected with VARV at a dose of 5.2 log10 PFU (50 ID50). The values of the VARV concentration in the lungs and nose were similar to those observed in human patients and well-known animal models such as Macaca cynomolgus and ICR mice respiratorily infected with almost the same doses of VARV. The known animal models were not shown to be significantly different from SCID mice in terms of the duration of the presence of the virus in the lungs. Moreover, the inflammatory and necrotic pathomorphological changes observed in the respiratory organs of SCID mice were similar to those occurring in other animal models and human patients. The use of SCID mice for evaluating the preventive efficacy of the NIOCH-14 and ST-246 antiviral drugs led to the results agreeing well with the previously reported data. Thus, SCID mice may be used as an experimental animal model for simulating smallpox to develop antiviral drugs for the treatment of human patients with a severe immunosuppressive state.

The in vitro replication of viral haemorrhagic septicaemia virus (VHSV) isolates from each VHSV genotype and the associated cellular host Mx gene expression were analysed. All the isolates were able to infect RTG-2 cells and induce increased Mx gene expression (generic assay detecting isoforms 1 and 3 [Mx1/3]). A trout pathogenic, genotype Ia isolate (J167), showing high replication in RTG-2 cells (by infective titre and N gene expression) induced lower Mx1/3 gene expression than observed in VHSV isolates known to be non-pathogenic to rainbow trout: 96-43/8, 96-43/10 (Ib); 1p49, 1p53 (II); and MI03 (IVb). Paired co-inoculation assays were analysed using equal number of plaque forming units per ml (PFU) of J167 (Ia genotype) with other less pathogenic VHSV genotypes. In these co-inoculations, the Mx1/3 gene expression was significantly lower than for the non-pathogenic isolate alone. Of the three rainbow trout Mx isoforms, J167 did not induce Mx1 up-regulation in RTG-2 or RTgill-W1 cells. Co-inoculating isolates resulted in greater inhibition of Mx in both rainbow trout cell lines studied. Up-regulation of sea bream Mx in SAF-1 cells induced by 96-43/8 was also lower in co-inoculation assays with J167. The RTG-P1 cell line, expressing luciferase under the control of the interferon-induced Mx rainbow trout gene promoter, showed low luciferase activity when inoculated with pathogenic strains: J167, DK-5131 (Ic), NO-A-163/68 (Id), TR-206239-1, TR-22207111 (Ie), 99-292 (IVa), and CA-NB00-01 (IVc). Co-inoculation assays showed a J167-dose dependent inhibition of the luciferase activity. The data suggest that virulent VHSV isolates may interfere in the interferon pathways, potentially determining higher pathogenicity.