Median Tissue Culture Infective Dose Research Articles

Multi-sulfonated ligands on gold nanoparticles as virucidal antiviral for Dengue virus

Dengue virus (DENV) causes 390 million infections per year. Infections can be asymptomatic or range from mild fever to severe haemorrhagic fever and shock syndrome. Currently, no effective antivirals or safe universal vaccine is available. In the present work we tested different gold nanoparticles (AuNP) coated with ligands ω-terminated with sugars bearing multiple sulfonate groups. We aimed to identify compounds with antiviral properties due to irreversible (virucidal) rather than reversible (virustatic) inhibition. The ligands varied in length, in number of sulfonated groups as well as their spatial orientation induced by the sugar head groups. We identified two candidates, a glucose- and a lactose-based ligand showing a low EC50 (effective concentration that inhibit 50% of the viral activity) for DENV-2 inhibition, moderate toxicity and a virucidal effect in hepatocytes with titre reduction of Median Tissue Culture Infectious Dose log10TCID50 2.5 and 3.1. Molecular docking simulations complemented the experimental findings suggesting a molecular rationale behind the binding between sulfonated head groups and DENV-2 envelope protein.

Oncolytic virus pelareorep plus carfilzomib phase I trial in carfilzomib-refractory patients (NCI 9603): Responses with cytokine storm.

8535 Background: Viral oncolytic therapy with intravenous Pelareorep, the infusible form of reovirus, is supported by preclinical data indicating that its antitumor activity results from direct cytolysis and an immune response against infected MM cells. Our preclinical data has shown that monocytes serve as carriers of reovirus from the peripheral blood to the bone marrow (Dona et al, ASH, 2019). In this abstract we present patients treated to date that were Carfilzomib refractory at enrollment. Methods: Pelareorep (P), Carfilzomib (K), and dexamethasone (d) were all infused on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle. Patients were pretreated with dexamethasone 20 mg intravenously, then Carfilzomib 20 mg/m2 on days 1 and 2 of cycle 1, and 56 mg/m2 thereafter. Pelareorep dose levels were at 3x1010, 4.5*1010, and planned 9*1010 median tissue culture infectious dose (TCID50). Results: All 6 evaluable patients showed reovirus infection in the post-treatment marrow aspirates cycle 1 day 9. Despite all patients having Carfilzomib-refractory disease, 2 patients achieved partial responses, two patients with short duration stable disease but few side effects, and 2 patients with PD. Of the two responders, one patient developed fever and grade 4 thrombocytopenia during cycle 1 and withdrew mid cycle. The other patient with a PR developed a cytokine storm - this patient presented cycle 1 day 3 hypoxemic with pneumonia, systolic failure (LVEF 69%-- > 26%), with biochemical evidence of hemophagocytic syndrome with elevated IL-2R, IL-6, and ferritin > 25K. This patient responded to tocilizumab, but ultimately died two weeks later with sepsis. Autopsy revealed pneumonia, no areas of reovirus infection other than in myeloma cells, and reovirus productive infection in 5-35% of myeloma cells (i.e. capsid protein). Conclusions: While infection of myeloma cells was seen in all evaluable patients on cycle 1 day 9, those with a clinical response demonstrated concomitant CD8 & NK cell recruitment, PD L1 upregulation, activated caspase-3 expression, increased viral protein production within the myeloma cells, and these patients demonstrated mild to severe signs and symptoms consistent with secondary HLH. This is the first report of cytokine storm after oncolytic virus in a patient with a blood cancer, a syndrome thought to be related to T-cell activation from the combination treatment. This trial was supported by the NCI Division of Cancer Treatment & Diagnosis Cancer Therapy Evaluation Program (CTEP). Clinical trial information: NCT02101944 .

Alterations of fecal microbiome characteristics by dietary soy isoflavone ingestion in growing pigs infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important disease, and the ingestion of soy isoflavones (ISF) may benefit PRRSV-infected pigs due to demonstrated anti-inflammatory and antiviral properties. The objective of this study was to quantify the effects of ISF consumption on fecal microbiome characteristics at different timepoints across a disease challenge and determine whether any changes, if present, elude to potential biological mechanisms for previously observed performance benefits. In total, 96 weaned barrows were group-housed in a Biosafety Level-2 containment facility and allotted to one of three experimental treatments that were maintained throughout the study: noninfected pigs receiving an ISF-devoid control diet (NEG, n = 24) and infected pigs receiving either the control diet (POS, n = 36) or that supplemented with total ISF in excess of 1,600 mg/kg (ISF, n = 36). Following a 7-d adaptation, pigs were inoculated intranasally with either a sham-control (phosphate-buffered saline) or live PRRSV (1 × 105 median tissue culture infectious dose[TCID]50/mL, strain NADC20). Fecal samples were collected from 48 individual pigs at pre-infection (-2 d post-inoculation [DPI]), peak-infection (10 DPI), and post-infection (144 DPI) timepoints. Extracted DNA was used to quantify fecal microbiota profiles via 16S bacterial rRNA sequencing. Differences in bacterial communities among diet groups were evaluated with principal coordinate analysis and permutational multivariate analysis of variance using UniFrac distance matrices based on both unweighted and weighted UniFrac distances using QIIME 2. All other data were analyzed by one-way ANOVA performed on square root transformations using R. Across all timepoints, only a few differences were observed due to ISF alone mainly in lowly abundant genera. The most notable differences observed were decreased relative abundance of Actinobacteria at 144 DPI between noninfected and infected treatments (P < 0.05), which is consistent with various dysbioses observed in other disease models. Our findings indicate that the differences present were mainly due to PRRSV-infection alone and not strongly influenced by diet, which implies that previously observed performance benefits conferred by dietary ISF are not likely due to the changes in microbiome composition.

Anti-viral activity of Zingiber officinale (Ginger) ingredients against the Chikungunya virus

Chikungunya is one of the highly infectious viral disease without vaccine and anti-viral. Aim of present study is to check the anti-chikungunya activities of Zingiber officinale (Ginger) in the animal cell culture model. The medicinal plant extract was prepared from Z. officinale rhizome. Median tissue culture infective dose (TCID50) of Chikungunya virus (CHIKV) and Maximum non-toxic dose (MNTD) of Z. officinale extract was determined in Vero cell-line on the basis of cell viability followed by MTT assay. In vitro anti-chikungunya activity was performed in Vero cell-line with MNTD and half of MNTD of Z. officinale medicinal plant extract. The anti-viral effect of Z. officinale was studied by observing the cytopathic effects and cell viability measured by MTT assay. Maximum non-toxic dose of Z. officinale plant extract was found 62.5 μg/ml. During anti-chikungunya experimentation, cell viability increased to 51.05% and 35.10%, when Vero cells were pre-treated with MNTD and half of MNTD of Z. officinale extract respectively. Similarly, in co-treatment, when MNTD, half of MNTD of Z. officinale and Median tissue culture infective dose CHIKV were inoculated simultaneously, then the viability of Vero cell-line was increases by 52.90% and 49.02% respectively. The rhizome extracts of Z. officinale have high potential to treat CHIKV. Medicinal plants and their metabolites are most important sources of antimicrobial and can be utilized for the development of new drugs. In view of the rapid expansion of CHIKV at the global level, there is an urgent need to develop newer anti-chikungunya drugs.

In vitro screening antiviral activity of Thai medicinal plants against porcine reproductive and respiratory syndrome virus

BackgroundPorcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) results in economic losses in the swine industry globally. Several studies have investigated the use of plant extracts in the prevention and control of PRRS outbreaks. Thai medicinal plants may be useful for treating PRRSV infection in pigs. Therefore, we investigated the in vitro anti-PRRSV and antioxidant properties of seven Thai medicinal plants: Caesalpinia sappan Linn., Garcinia mangostana Linn., Houttuynia cordata, Perilla frutescens, Clinacanthus nutans, Phyllanthus emblica, and Tiliacora triandra.ResultsUsing antiviral screening, we observed that T. triandra extract strongly inhibited PRRSV infectivity in MARC-145 cells [virus titer 3.5 median tissue culture infective dose (TCID50)/ml (log10)] at 24 h post-infection, whereas C. sappan extract strongly inhibited PRRSV replication [virus titer 2.5 TCID50/ml (log10)] at 72 h post-infection. C. sappan extract had the highest total phenolic content [220.52 mM gallic acid equivalent/g] and lowest half-maximal inhibitory concentration [1.17 mg/ml in 2,2-diphenyl-1-picrylhydrazyl and 2.58 mg/ml in 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulfonic acid) diammonium salt].ConclusionT. triandra extract could inhibit PRRSV infectivity, whereas C. sappan extract was the most effective in inhibiting PRRSV replication in MARC-145 cells. This study elucidates the antiviral activities of Thai medicinal plant extracts in vivo. The results promise that Thai medicinal plant extracts, particularly T. triandra and C. sappan extracts, can be developed into pharmaceutical drugs for the prevention of PRRS in pigs.

Virus isolation and full-length genome sequencing of a representative canine distemper virus wild type strain of the South America 2 clade

Canine Distemper Virus (CDV) is a highly contagious pathogen of dogs that causes severe respiratory, gastrointestinal and nervous signs. Although vaccines have been used to prevent infections, CDV has been reported worldwide, even in vaccinated animals.In the present study, a representative wild type CDV strain (Arg24) was isolated from a sick vaccinated dog and its genome was completely sequenced using Illumina technology. This strain produced a strong cytopathic effect in Vero SLAM (Signaling Lymphocyte Activation Molecule) cells with a higher titer of 1.1 × 105 Median Tissue Culture Infectious Dose (TCID50/mL) at 32 h post infection, in cell-associated virus. The Arg24 strain genome, showed values of 97.1, 90.3, 96.7, 90.6, 89.8 and 97.3 % of amino acid identity with respect to the Onderstepoort vaccine strain (Nucleoprotein, Phosphoprotein, Matrix, Fusion, Hemagglutinin and Large polymerase, respectively).Focusing on the Hemagglutinin gene, which is the target for genetic characterization, Arg24 showed four additional potential glycosylation sites, with respect to the Onderstepoort.The availability of Arg24 strain, which can be easily grown in Vero SLAM cells, is an important tool to perform immunological and antigenic comparative studies, between wild type and vaccine CDV strains.

Inactivation by osmotic dehydration and air drying of Salmonella, Shiga toxin-producing Escherichia coli, Listeria monocytogenes, hepatitis A virus and selected surrogates on blueberries

Osmotically dehydrated and air dried berry fruits are used as ingredients for the production of yoghurts, chocolates, cereal bars and mixes, ice creams and cakes and these fruits are often subjected to mild thermal treatments only, posing questions around their microbiological safety. As osmotic dehydration methods and parameters vary considerably within the industry and minimally processed high quality fruits are increasingly sought, the scope of this study was to determine which temperatures are required for the inactivation of relevant bacteria and viruses during osmotic dehydration of berries, using blueberries as a model berry in a thawed state to mimic common industrial practices. Additionally, we studied the inactivation of osmotic dehydration at 23 °C, sometimes referred to “cold infusion” followed by air drying at 100 °C to determine the microbiological safety achieved by this combined treatment. Four pathogens (Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes and hepatitis A virus (HAV)) and five surrogates (Enterococcus faecium, Escherichia coli P1, Listeria innocua, murine Norovirus (MNV) and bacteriophage MS2) were inoculated on blueberries and reductions were measured after different treatment combinations. After osmotic dehydration of bacterial strains at 40 °C no survivors were detected on blueberries, with the exception of E. faecium. Inactivation of the viruses at 45 °C showed no survivors for MS2 and mean reductions of 1.5 and 3.4 log10 median tissue culture infectious dose (TCID50)/g for HAV and MNV, respectively. Similarly, in the sugar solution at 40 °C, no survivors were observed, with the exception of E. faecium and the three viruses. The combined process (osmotic dehydration at 23 °C followed by air-drying at 100 °C) achieved an >6 log reduction of all tested bacterial strains and MS2. For HAV and MNV, 2.6 and >3.4 log10 TCID50/g were measured. In summary, the present study shows that osmotic dehydration appears an efficient control measure for the control of L. monocytogenes, S. enterica and E. coli O157:H7 if carried out at 40 °C or at 23 °C and followed by air-drying at 100 °C. Based on the results generated with MNV, the combined treatment is also expected to reduce human Norovirus (NoV) but does not appear to be sufficient to fully control HAV. The results contribute to a better management of the microbial safety of osmotically dehydrated and dried berries and especially the results generated for the viruses emphasize that within a robust food safety management system, safety must be assured through the entire food supply chain and therefore must start at primary production with the implementation of Good Agricultural Practices (GAP).

Titration methods for rVSV-based vaccine manufacturing

The recombinant Vesicular Stomatitis Virus (rVSV) is an emerging platform for viral vector-based vaccines. Promising results have been reported in clinical trials for the rVSV-ZEBOV vaccine for Ebola virus disease prevention. In this study, we describe the titration tools elaborated to assess the titre of rVSV-ZEBOV productions.• A streamlined Median Tissue Culture Infectious Dose (TCID50) assay to determine the infectious titer of this vaccine was established.• A digital polymerase chain reaction (dPCR) assay to assess the total number of viral particles present in cell-free culture supernatants of rVSV productions was developed.• These assays are used to titre rVSV-ZEBOV samples and characterize the ratio of total particles to infectious units for monitoring process robustness and product quality attributes and can be used to titre samples generated in the production of further rVSV vectors.

Povidone-iodine demonstrates rapid in vitro virucidal activity against SARS-CoV-2, the virus causing COVID-19 disease

Introduction. As of 22 June 2020, Severe Acute Respiratory Syndrome (SARS)-coronavirus (CoV)-2 has infected more than 8.95 million people worldwide, causing >468,000 deaths. The virus is transmitted through respiratory droplets and physical contact from contaminated surfaces to the mucosa. Hand hygiene and oral decontamination among other measures are key to preventing the spread of the virus. We report the in vitro virucidal activity of topical and oral povidone-iodine (PVP-I) products against SARS-CoV-2. Method. Suspension assays were used to assess the virucidal activity of PVP-I against SARS-CoV-2. Products were tested at a contact time of 30 s for virucidal activity. Viral titres were calculated using the Spearman‑Kärber method and reported as median tissue culture infectious dose (TCID50)/mL. Results. All four products [antiseptic solution (PVP-I 10%), skin cleanser (PVP-I 7.5%), gargle and mouth wash (PVP-I 1%) and throat spray (PVP-I 0.45%)] achieved ≥ 99.99% virucidal activity against SARS-CoV-2, corresponding to ≥ 4 log10 reduction of virus titre, within 30 s of contact. Conclusions. This study provides evidence of rapid and effective virucidal activity of PVP-I against SARS-CoV-2. PVP-I-based products are widely available for medical and personal use for hand hygiene and oral decontamination, and could be readily integrated into the coronavirus disease (COVID-19) infection control measures in hospital and community settings.

Povidone-iodine demonstrates rapid in vitro virucidal activity against SARS-CoV-2, the virus causing COVID-19 disease

Introduction. As of 22 June 2020, Severe Acute Respiratory Syndrome (SARS)-coronavirus (CoV)-2 has infected more than 8.95 million people worldwide, causing >468,000 deaths. The virus is transmitted through respiratory droplets and physical contact from contaminated surfaces to the mucosa. Hand hygiene and oral decontamination among other measures are key to preventing the spread of the virus. We report the in vitro virucidal activity of topical and oral povidone-iodine (PVP-I) products against SARS-CoV-2. Method. Suspension assays were used to assess the virucidal activity of PVP-I against SARS-CoV-2. Products were tested at a contact time of 30 s for virucidal activity. Viral titres were calculated using the Spearman‑Kärber method and reported as median tissue culture infectious dose (TCID50)/mL. Results. All four products [antiseptic solution (PVP-I 10%), skin cleanser (PVP-I 7.5%), gargle and mouth wash (PVP-I 1%) and throat spray (PVP-I 0.45%)] achieved ≥ 99.99% virucidal activity against SARS-CoV-2, corresponding to ≥ 4 log10 reduction of virus titre, within 30 s of contact. Conclusions. This study provides evidence of rapid and effective virucidal activity of PVP-I against SARS-CoV-2. PVP-I-based products are widely available for medical and personal use for hand hygiene and oral decontamination, and could be readily integrated into the coronavirus disease (COVID-19) infection control measures in hospital and community settings.

Isolation and characterization of a variant subgroup GII-a porcine epidemic diarrhea virus strain in China

BackgroundHighly virulent variants of porcine epidemic diarrhea virus (PEDV) have been closely associated with recent outbreaks of porcine epidemic diarrhea (PED) in China, which have resulted in severe economic losses to the pork industry. MethodsIn the current study, the variant PEDV strain HM2017 was isolated and purified and a viral growth curve was constructed according to the median tissue culture infective dose (TCID50). HM2017 were amplify with RT-PCR and analyzed by phylogeny analysis. Animal pathogenicity experiment was carried to evaluate the HM2017 clinical assessment. ResultsGenome-based phylogenetic analysis revealed that PEDV strain HM2017 was clustered into the variant subgroup GII-a that is currently circulating in pig populations in China. The highest median tissue culture infectious dose of strain HM2017 after 15 passages in Vero cells was 1.33 × 107 viral particles/mL. Strain HM2017 was highly virulent to suckling piglets, which exhibited clinical symptoms at 12 h post-infection (hpi) (i.e., weight loss at 12–84 hpi, increased body temperatures at 24–48 hpi, high viral loads in the jejunum and ileum, and 100% mortality by 84 hpi). ConclusionThe present study reports a variant subgroup GII-a PEDV HM2017 strain in China and characterize its pathogenicity. PEDV strain HM2017 of subgroup GII-a presents a promising vaccine candidate for the control of PED outbreaks in China.

Downregulation of microRNA-30a-5p contributes to the replication of duck enteritis virus by regulating Beclin-1-mediated autophagy

BackgroundMicroRNAs (miRNAs) is increasingly recognized as an important element in regulating virus-host interactions. Our previous results showed that cellular miR-30a-5p was significantly downregulated after duck enteritis virus (DEV) infection cell. However, whehter or not the miR-30a-5p is involved in DEV infection has not been known.MethodsQuantitative reverse-transcription PCR (qRT-PCR) was used to measure the expression levels of miRNAs(miR-30a-5p) and Beclin-1 mRNA. The miR-30a-5p - Beclin-1 target interactions were determined by Dual luciferase reporter assay (DLRA). Western blotting was utilized to analyze Beclin-1-mediated duck embryo fibroblast (DEF) cells autophagy activity. DEV titers were estimated by the median tissue culture infective dose (TCID50).ResultsThe miR-30a-5p was significantly downregulated and the Beclin-1 mRNA was significantly upregulated in DEV-infected DEF cells. DLRA confirmed that miR-30a-5p directly targeted the 3′- UTR of the Beclin-1 gene. Overexpression of miR-30a-5p significantly reduced the expression level of Beclin-1protein (p < 0.05), leading to the decrease of Beclin-1-mediated autophagy activity, which ultimately suppressed DEV replication (P < 0.05). Whereas transfection of miR-30a-5p inhibitor increased Beclin-1-mediated autophagy and triggered DEV replication during the whole process of DEV infection (P < 0.01).ConclusionsThis study shows that miR-30a-5p can inhibit DEV replication through reducing autophagy by targeting Beclin-1. These findings suggest a new insight into virus-host interaction during DEV infection and provide a potential new antiviral therapeutic strategy against DEV infection.

Porcine circovirus type 2 induces a strong cytopathic effect in the serum-free culture cell line CPK-NS

When the adherent stable serum-free porcine kidney cell line CPK-NS were inoculated with porcine circovirus type 2 (PCV2) and passaged, viral titre concentration-dependent cell detachment was observed. The copy number of viral genes in supernatants of the infected CPK-NS cells decreased as cell detachment progressed. Furthermore, cell detachment was completely inhibited via neutralisation of the virus using antisera collected from PCV2-infected specific pathogen-free pigs. These results indicated that detachment of CPK-NS cells is a cytopathic effect (CPE) caused via infection with PCV2. Only a single round of cell passaging was required to observe clear a CPE when the inoculated viral titre was significantly high [≥104.5 median tissue culture infectious dose (TCID50)/mL]. Our study confirms that PCV2, which is normally non-cytopathogenic, is capable of inducing a distinct CPE in CPK-NS cells. Application of CPK-NS cells for detection of viruses may contribute towards the diagnosis and control of PCV2-mediated infectious diseases.

Enterovirus A71 Containing Codon-Deoptimized VP1 and High-Fidelity Polymerase as Next-Generation Vaccine Candidate.

Enterovirus A71 (EV-A71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD), which occasionally results in severe neurological complications. In this study, we developed four EV-A71 (rgEV-A71) strains by reverse genetics procedures as possible vaccine candidates. The four rgEV-A71 viruses contained various codon-deoptimized VP1 capsid proteins (VP1-CD) and showed replication rates and antigenicity similar to that of the wild-type virus, while a fifth virus, rg4643C4VP-CD, was unable to form plaques but was still able to be examined by median tissue culture infectious dose (TCID50) titers, which were similar to those of the others, indicating the effect of CD on plaque formation. However, the genome stability showed that there were some mutations which appeared during just one passage of the VP1-CD viruses. Thus, we further constructed VP1-CD rgEV-A71 containing high-fidelity determinants in 3D polymerase (CD-HF), and the number of mutations in CD-HF rgEV-A71 was shown to have decreased. The CD-HF viruses showed less virulence than the parental strain in a mouse infection model. After 14 days postimmunization, antibody titers had increased in mice infected with CD-HF viruses. The mouse antisera showed similar neutralizing antibody titers against various CD-HF viruses and different genotypes of EV-A71. The study demonstrates the proof of concept that VP1 codon deoptimization combined with high-fidelity 3D polymerase decreased EV-A71 mutations and virulence in mice but retained their antigenicity, indicating it is a good candidate for next-generation EV-A71 vaccine development.IMPORTANCE EV-A71 can cause severe neurological diseases with fatality in infants and young children, but there are still no effective drugs to date. Here, we developed a novel vaccine strategy with the combination of CD and HF substitutions to generate the genetically stable reverse genetics virus. We found that CD combined with HF polymerase decreased the virulence but maintained the antigenicity of the virus. This work demonstrated the simultaneous introduction of CD genome sequences and HF substitutions as a potential new strategy to develop attenuated vaccine seed virus. Our work provides insight into the development of a low-virulence candidate vaccine virus through a series of genetic editing of virus sequences while maintaining its antigenicity and genome stability, which will provide an additional strategy for next-generation vaccine development of EV-A71.

Application of Concanavalin A–Linked Magnetic Beads for the Detection of Hepatitis A Virus

Prompt and inexpensive detection of hepatitis A virus (HAV) is essential to control acute hepatitis outbreaks associated with the consumption of contaminated raw or minimally processed food. In this study, various carbohydrate-binding lectins, including concanavalin A (Con A), wheat germ agglutinin, and soybean agglutinin, were compared for their binding affinity to HAV. Con A, which showed significantly higher binding affinity than other lectins, was used to develop an alternative and affordable method to conventional antibody-linked immunomagnetic separation prior to detection of HAV using reverse transcriptase PCR. This method, Con A-linked immunomagnetic separation combined with reverse transcriptase PCR, can detect HAV at a dilution concentration of 10-4 of the virus stock (titer: 104 median tissue culture infective dose per mL), indicating that Con A could be a promising candidate for concentrating HAV.

Key role of singlet oxygen and peroxynitrite in viral RNA damage during virucidal effect of plasma torch on feline calicivirus

A dielectric barrier discharge (DBD) plasma torch has been used to evaluate the mechanism underlying inactivation of feline calicivirus (FCV) by plasma treatment. Plasma treatment of cell lysate infected with FCV F9 strain reduced the viral titer of the median tissue culture infectious dose (TCID50). The D value (treatment time required to lower the viral titer to 1/10) was 0.450 min, while the viral titer dropped below the detection limit within 2 min. FCV was not significantly inactivated by heat or UV applied at levels corresponding to those generated from the DBD plasma torch after 2 min (38.4 °C and 46.79 mJ/cm2 UV, respectively). However, TCID50 was reduced by 2.47 log after exposure to 4.62 mM ONOO−, corresponding to the concentration generated after 2 min of plasma treatment. Radical scavengers, including superoxide dismutase, dimethyl sulfoxide, and catalase, did not significantly affect viral titers; however, sodium azide, uric acid, and ascorbic acid, which are scavengers of 1O2 radicals, ONOO−, and peroxynitrous acid (ONOOH; produced from ONOO− under acidic conditions), respectively, significantly increased TCID50 and intact viral RNA. These findings suggest that ONOO− and 1O2 play an important role in FCV inactivation by attacking viral RNA during DBD plasma torch treatment.

Development of a Rhinovirus Inoculum Using a Reverse Genetics Approach

BackgroundExperimental inoculation is an important tool for common cold and asthma research. Producing rhinovirus (RV) inocula from nasal secretions has required prolonged observation of the virus donor to exclude extraneous pathogens. We produced a RV-A16 inoculum using reverse genetics and determined the dose necessary to cause moderate colds in seronegative volunteers.MethodsThe consensus sequence of RV-A16 from a previous inoculum was cloned, and inoculum virus was produced using reverse genetics techniques. After safety testing, volunteers were inoculated with either RV-A16 (n = 26) or placebo (n = 10), Jackson cold scores were recorded, and nasal secretions were tested for shedding of RV-A16 ribonucleic acid.ResultsThe reverse genetics process produced infectious virus that was neutralized by specific antisera and had a mutation rate similar to conventional virus growth techniques. The 1000 median tissue culture infectious dose (TCID50) dose produced moderate colds in most individuals with effects similar to that of a previously tested conventional RV-A16 inoculum.ConclusionsReverse genetics techniques produced a RV-A16 inoculum that can cause clinical colds in seronegative volunteers, and they also serve as a stable source of virus for laboratory use. The recombinant production procedures eliminate the need to derive seed virus from nasal secretions, thus precluding introduction of extraneous pathogens through this route.

PATHOGENICITY OF TAIWAN FERRET BADGER RABIES VIRUS IN RODENTS

The outbreaks of Taiwan ferret badger rabies reported in 2013 terminated the “rabies-free” status of Taiwan. Subsequent phylogenetic and divergence analyses have demonstrated that Taiwan ferret badger rabies virus (RABV-TWFB) might have emerged 100 years previously; however, most rabies cases were restricted to the Formosan ferret badger. In this study, pathogenic characteristics of mouse intracranial median lethal dose (MICLD[Formula: see text], median tissue culture infectious dose (TCID[Formula: see text], mortality development patterns, and peripheral infection of RABV-TWFB were evaluated in experimental rodents. The results revealed that RABV-TWFB had low MICLD[Formula: see text] titers, whereas TCID[Formula: see text] titers could not be determined. Compared with the typical street rabies virus, the overall mortality development patterns were later onset and slower progression. RABV-TWFB was unable to produce peripheral infection in the experimental rodents. Taken together, RABV-TWFB was less virulent to experimental rodents than other more typical RABV strains. To provide more appropriate strategies for epidemics management, the pathogenic properties of RABV-TWFB should be further investigated using ferret badgers and sympatric animals as models.

Ultraviolet germicidal irradiation of influenza-contaminated N95 filtering facepiece respirators

Safe and effective decontamination and reuse of N95 filtering facepiece respirators (FFRs) has the potential to significantly extend FFR holdings, mitigating a potential shortage due to an influenza pandemic or other pandemic events. Ultraviolet germicidal irradiation (UVGI) has been shown to be effective for decontaminating influenza-contaminated FFRs. This study aims to build on past research by evaluating the UVGI decontamination efficiency of influenza-contaminated FFRs in the presence of soiling agents using an optimized UVGI dose. Twelve samples each of 15 N95 FFR models were contaminated with H1N1 influenza (facepiece and strap), then covered with a soiling agent-artificial saliva or artificial skin oil. For each soiling agent, 3 contaminated FFRs were treated with 1 J/cm2 UVGI for approximately 1 minute, whereas 3 other contaminated FFRs remained untreated. All contaminated surfaces were cut out and virus extracted. Viable influenza was quantified using a median tissue culture infectious dose assay. Significant reductions (≥3 log) in influenza viability for both soiling conditions were observed on facepieces from 12 of 15 FFR models and straps from 7 of 15 FFR models. These data suggest that FFR decontamination and reuse using UVGI can be effective. Implementation of a UVGI method will require careful consideration of FFR model, material type, and design.

Development and characterization of a guinea pig model for Marburg virus.

The Angolan strain of Marburg virus (MARV/Ang) can cause lethal disease in humans with a case fatality rate of up to 90%, but infection of immunocompetent rodents do not result in any observable symptoms. Our previous work includes the development and characterization of a MARV/Ang variant that can cause lethal disease in mice (MARV/Ang-MA), with the aim of using this tool to screen for promising prophylactic and therapeutic candidates. An intermediate animal model is needed to confirm any findings from mice studies before testing in the gold-standard non-human primate (NHP) model. In this study, we serially passaged the clinical isolate of MARV/Ang in the livers and spleens of guinea pigs until a variant emerged that causes 100% lethality in guinea pigs (MARV/Ang-GA). Animals infected with MARV/Ang-GA showed signs of filovirus infection including lymphocytopenia, thrombocytopenia, and high viremia leading to spread to major organs, including the liver, spleen, lungs, and kidneys. The MARV/Ang-GA guinea pigs died between 7–9 days after infection, and the LD50 was calculated to be 1.1×10–1 TCID50 (median tissue culture infective dose). Mutations in MARV/Ang-GA were identified and compared to sequences of known rodent-adapted MARV/Ang variants, which may benefit future studies characterizing important host adaptation sites in the MARV/Ang viral genome.