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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.

Objective. Optimization of vaccine virus cultivation in the suspended cell culture BHK-21 for infectious activity increment of virus-containing suspension as the base material for smallpox vaccine preparations. Materials and methods. We used suspended culture line of the cells BHK-21 of 72-hour age and nutrient medium of the MEM type in accordance with the guidelines on preparation in our studies. For challenging of the cells, vaccine virus (strain B-51) was used. The virus was adapted through three consequent passages on horion-allantois shell of developing chicken embryos of commercial dermovaccine series 449а at the premises of the Federal State Budgetary Institution “the 48th Central Research Institute” of the Ministry of Defense of the Russian Federation. Information on its genetic features is absent. Cultivation and precipitation of infected cells BHK-21 was carried out in bioreactor with priming volume of 1 liter at (36.5±0.5) °C and aeration with air mixture with varying content of CO2. Results and conclusions. Gas massexchange intensity was enhanced alongside simultaneous maintaining of sparing hydrodynamic conditions for mixing suspended cell cultures in bioreactor. Two-fold increase (up to (4.48±0.63)·109 cell/l) in suspended BHK-21 cell culture concentration at the end of reproduction cycle was achieved. Concentration of the vaccine virus was 3–5 times raised, from (8.1±0.3) lg PFU (plaque forming unit)/ml up to the level of infectious activity – (8.8±0.3) lg PFU/ml. Specific multiplicity of cell infection in recalculation per a cell was 1–5 PFU/cell and by virus yield – 20–100 PFU/cell. Enhanced infectious activity of the virus in concentrated suspension of infected BHK-21 cells substantiates the perspectives of the proposed method for improvement of vaccine virus accumulation phase in the development of anti-smallpox preparations based on cell cultures.

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

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.

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.

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.

Staphylococcus aureus phage Henu2 was isolated from a sewage sample collected in Kaifeng, China, in 2017. In this study, Henu2, a linear double-stranded DNA virus, was sequenced and found to be 43513bp long with 35% G + C content and 63 putative open reading frames (ORFs). Phage Henu2 belongs to the family Siphoviridae and possesses an isometric head (63 nm in diameter). The latent time and burst size of Henu2 were approximately 20 min and 7.8 plaque forming unit (PFU)/infected cells. The Henu2 maintained infectivity over a wide range of temperature (10–60 °C) and pH values (4–12). Phylogenetic and comparative genomic analyses indicate that Staphylococcus aureus phage Henu2 should be a new member of the family of Siphoviridae class-II. In this paper, Phage Henu2 alone exhibited weak inhibitory activity on the growth of S. aureus. However, the combination of phage Henu2 and some antibiotics or oxides could effectively inhibit the growth of S. aureus, with a decrease of more than three logs within 24 h in vitro. These results provide useful information that phage Henu2 can be combined with antibiotics to increase the production of phage Henu2 and thus enhance the efficacy of bacterial killing.

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.

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.

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 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.

Houttuynia cordata is an herbal plant rich in polysaccharides and with several pharmacological activities. Human noroviruses (HuNoVs) are the most common cause of foodborne viral gastroenteritis throughout the world. In this study, H. cordata polysaccharide (HP), with a molecular weight of ~43 kDa, was purified from H. cordata water extract (HWE). The polysaccharide HP was composed predominantly of galacturonic acid, galactose, glucose, and xylose in a molar ratio of 1.56:1.49:1.26:1.11. Methylation and NMR analyses revealed that HP was a pectin-like acidic polysaccharide mainly consisting of &alpha, 1,4-linked GalpA, &beta, 1,4-linked Galp, &beta, 1,4-linked Glcp, and &beta, 1,4-linked Xylp residues. To evaluate the antiviral activity of H. cordata extracts, we compared the anti-norovirus potential of HP with HWE and ethanol extract (HEE) from H. cordata by plaque assay (plaque forming units (PFU)/mL) for murine norovirus-1 (MNV-1), a surrogate of HuNoVs. Viruses at high (8.09 log10 PFU/mL) or low (4.38 log10 PFU/mL) counts were mixed with 100, 250, and 500 &mu, g/mL of HP, HWE or HEE and incubated for 30 min at room temperature. H. cordata polysaccharide (HP) was more effective than HEE in reducing MNV-1 plaque formation, but less effective than HWE. When MNV-1 was treated with 500 &mu, g/mL HP, the infectivity of MNV-1 decreased to an undetectable level. The selectivity indexes of each sample were 1.95 for HEE, 5.74 for HP, and 16.14 for HWE. The results of decimal reduction time and transmission electron microscopic revealed that HP has anti-viral effects by deforming and inflating virus particles, thereby inhibiting the penetration of viruses in target cells. These findings suggest that HP might have potential as an antiviral agent in the treatment of viral diseases.

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...

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.

The genus Orthobunyavirus are a group of viruses within arbovirus, with a zoonotic cycle, some of which could lead to human infection. A characteristic of these viruses is their lack of antiviral treatment or vaccine for its prevention. The objective of this work was to study the in vitro antiviral activity of nordihydroguaiaretic acid (NDGA), the most important active compound of Larrea divaricata Cav. (Zigophyllaceae), against Fort Sherman virus (FSV) as a model of Orthobunyavirus genus. At the same time, the effect of NDGA as a lipolytic agent on the cell cycle of this viral model was assessed. The method of reducing plaque forming units on LLC-MK2 cells was used to detect the action of NDGA on CbaAr426 and SFCrEq231 isolates of FSV. NDGA did not show virucidal effect, but it had antiviral activity with a similar inhibition in both isolates, which was dose dependent. It was established that the NDGA has a better inhibition 1-h post-internalization (p.i.), showing a different behavior in each isolate, which was dependent upon the time p.i. Since virus multiplication is dependent on host cell lipid metabolism, the antiviral effect of NDGA has been previously related to its ability to disturb the lipid metabolism, probably by interfering with the 5-lipoxigenase (5-LOX) and the sterol regulatory element-binding proteins (SREBP) pathway. We determined by using caffeic acid, a 5-LOX inhibitor, that the inhibition of this enzyme negatively affected the FSV replication; and by means of resveratrol, a SREBP1 inhibitor, it was showed that the negative regulation of this pathway only had action on the SFCrEq231 reduction. In addition, it was proved that the NDGA acts intracellularly, since it showed the ability to incorporate into LLC-MK2 cells. The information provided in this work converts the NDGA into a compound with antiviral activity in vitro against FSV (Orthobunyavirus), which can be subjected to structural modifications in the future to improve the activity.

The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) causes Coronavirus disease-2019 (COVID-19), which is an ongoing pandemic that has significantly affected the health, economy, and socio-economic status of individuals worldwide. Laboratory research using in vitro, ex vivo and in vivo models has been accelerated to understand the pathogenesis of SARS-CoV-2 infection. However, such experimental research involving SARS-CoV-2 is restricted to biocontainment/safety level-3 (BSL-3) settings, due to the high pathogenicity of this virus. Since many of the downstream analyses of SARS-CoV-2-infected biological samples need to be conducted in a non-BSL3 setting, it is important to ensure that the samples are fully decontaminated and safe for subsequent analysis. Here, we report the effectiveness of standard procedures used to fix cells and tissues for pathological analysis, including 2% or 4% paraformaldehyde, 50%–70% ethanol, 10% neutral buffered formalin and ultrafiltration using membranes with a molecular weight cut-off (MWCO) ranging from 3 to 30 kDa, for inactivating or eliminating SARS-CoV-2. We validated these methods in experimental laboratory samples, such as viral inoculum in cell culture media, SARS-CoV-2 infected host cells and animal tissue lysates. We found that 15 minutes’ treatment of viral inoculum (105 plaque-forming units; PFU) or SARS-CoV-2 infected cells with paraformaldehyde or 70% ethanol resulted in complete inactivation of the virus. The treatment of infected hamster lung tissues with 10% neutral buffered formalin also fully inactivated the virus. However, only 3 kDa ultracentrifuge filter was effective in eliminating the virus to an undetectable limit in the filtrate. Our validated methods are useful for decontaminating biological samples to reduce infection risk and safe handling in BSL2 facilities.

Zika virus (ZIKV) has emerged since 2013 as a significant global human health threat following outbreaks in the Pacific Islands and rapid spread throughout South and Central America. Severe congenital and neurological sequelae have been linked to ZIKV infections. Assessing the ability of common mosquito species to transmit ZIKV and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We compared infection, dissemination, and transmission rates by measuring ZIKV RNA levels in cohorts of mosquitoes that ingested blood meals from type I interferon-deficient mice infected with either a Puerto Rican ZIKV strain from 2015 (PR15), a Brazilian ZIKV strain from 2015 (BR15), or an ancestral Asian-lineage Malaysian ZIKV strain from 1966 (MA66). With PR15, Cx. quinquefasciatus was refractory to infection (0%, N = 42) and Cx. tarsalis was infected at 4% (N = 46). No ZIKV RNA was detected in saliva from either Culex species 14 or 21 days post feeding (dpf). In contrast, Ae. aegypti developed infection rates of 85% (PR15; N = 46), 90% (BR15; N = 20), and 81% (MA66; N = 85) 14 or 15 dpf. Although MA66-infected Ae. aegypti showed higher levels of ZIKV RNA in mosquito bodies and legs, transmission rates were not significantly different across virus strains (P = 0.13, Fisher’s exact test). To confirm infectivity and measure the transmitted ZIKV dose, we enumerated infectious ZIKV in Ae. aegypti saliva using Vero cell plaque assays. The expectorated plaque forming units PFU varied by viral strain: MA66-infected expectorated 13±4 PFU (mean±SE, N = 13) compared to 29±6 PFU for PR15-infected (N = 13) and 35±8 PFU for BR15-infected (N = 6; ANOVA, df = 2, F = 3.8, P = 0.035). These laboratory vector competence results support an emerging consensus that Cx. tarsalis and Cx. quinquefasciatus are not vectors of ZIKV. These results also indicate that Ae. aegypti from California are efficient laboratory vectors of ancestral and contemporary Asian lineage ZIKV., Author summary Assessing the ability of common mosquito species to transmit Zika virus (ZIKV) and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We observed variation in infection loads between ZIKV strains in Ae. aegypti, but transmission rates were not different. In addition, there was a positive relationship between ZIKV RNA levels in infected mosquitoes ascertained from bodies and ZIKV RNA transmission rates. Our data add to the growing body of evidence supporting the role of Aedes aegypti as a ZIKV vector and refute Cx. quinquefasciatus and Cx. tarsalis as vectors.

Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r=0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations., This work was supported by the Portuguese Science Foundation [project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124- FEDER-027462)], [project IF/01413/2013]; and DNA mimics [ref. PIC/IC/82815/ 2007] from Fundação para a Ciência e Tecnologia (FCT) and Ministério da Ciência, Tecnologia e Ensino Superior (MCTES).

Aims Human norovirus (HuNoV) is estimated to cause 19–21 million illnesses each year in the US. A major limitation in HuNoV research is the lack of an in vitro culture system; therefore, surrogate viruses including murine norovirus (MNV) and feline calicivirus (FCV) are used to study HuNoV. Here, we aim to establish the physiochemical properties of Tulane virus (TV)—a newer HuNoV surrogate. Methods and Results For thermal inactivation, TV was exposed to 37°C for 2 h, and 56, 63 and 72°C for 30 min. For ethanol tolerance, TV was treated with 60, 70 and 90% ethanol at room temperature (RT) for 5 min. Tulane virus pH stability at pH 2, 3, 7, 9 and 10 was performed at RT for 90 min. At 37°C, there was no significant reduction in TV after 2 h. However, at 56, 63 and 72°C, D-values of 4·03, 1·18, and 0·24 min, were calculated respectively. The D-values obtained for TV ethanol tolerance were 1·46, 1·93, and 0·35 min at 60, 70 and 90% respectively. Less than 1 log10 plaque forming units (PFU) reduction was observed for TV at all pH levels except pH 10 where about a 2-log10 PFU reduction was observed. Tulane virus was also tolerant to chlorine disinfection on a solid surface with D-values of 15·82 and 5·42 min at 200 and 1000 ppm respectively. Conclusions Tulane virus is likely a suitable surrogate to study HuNoV thermal stability as well as ethanol tolerance below 90%. Tulane virus also is a promising surrogate to study HuNoV pH stability and chlorine tolerance. Significance and Impact of the Study Based on current work, in vitro studies demonstrate that TV is an overall more conservative and suitable surrogate for the study of HuNoV physicochemical properties.

West Nile virus (WNV) and Rift Valley fever virus (RVFV) are two emerging arboviruses transmitted by Culex pipiens species that includes two biotypes: pipiens and molestus. In Lebanon, human cases caused by WNV and RVFV have never been reported. However, the introduction of these viruses in the country is likely to occur through the migratory birds and animal trades. In this study, we evaluated the ability of Cx. pipiens, a predominant mosquito species in urban and rural regions in Lebanon, to transmit WNV and RVFV. Culex egg rafts were collected in the West Bekaa district, east of Lebanon and adult females of Cx. pipiens were experimentally infected with WNV and RVFV Clone 13 strain at titers of 1.6×108 and 1.33×107 plaque forming units (PFU)/mL, respectively. We estimated viral infection, dissemination and transmission at 3, 7, 14 and 19 days post infection (dpi). Results showed that infection was higher for WNV than for RVFV from 3 dpi to 19 dpi. Viral dissemination and transmission started from 3 dpi for WNV; and only from 19 dpi for RVFV. Moreover, Cx. pipiens were able to excrete in saliva a higher number of viral particles of WNV (1028 ± 405 PFU/saliva at 19 dpi) than RVFV (42 PFU/saliva at 19 dpi). Cx. pipiens from Lebanon are efficient experimental vectors of WNV and to a lower extent, RVFV. These findings should stimulate local authorities to establish an active entomological surveillance in addition to animal surveys for both viruses in the country., Author summary West Nile virus (WNV) and Rift Valley fever virus (RVFV) are two emerging mosquito-borne arboviruses mainly transmitted by Culex mosquitoes. WNV considered one of the most important causative agent of viral encephalitis has a wide distribution in many tropical and temperate countries including the Middle East. RVFV is mainly distributed in Sub-Saharan Africa but epizootics were also reported in Egypt, Saudi Arabia and Yemen. The mosquito vector belongs to the Culex pipiens species which includes two biotypes: pipiens and molestus. Both biotypes are the most widely distributed mosquitoes in Lebanon. Using experimental infections of mosquitoes, our study showed that Cx. pipiens populations collected in West Bekaa were susceptible to infection by these two viruses and ensured efficient transmission of WNV and to a lesser extent, RVFV. Our findings may help to prepare a control strategy more adapted to these mosquito vectors.

Rabies is an ancient neglected tropical disease that causes tens of thousands of human deaths and millions of cattle deaths annually. In order to develop a new vaccine for potential use in bats, a reservoir of rabies infection for humans and animals alike, an in silico antigen designer tool was used to create a mosaic glycoprotein (MoG) gene using available sequences from the rabies Phylogroup I glycoprotein. This sequence, which represents strains more likely to occur in bats, was cloned into raccoonpox virus (RCN) and the efficacy of this novel RCN-MoG vaccine was compared to RCN-G that expresses the glycoprotein gene from CVS-11 rabies or luciferase (RCN-luc, negative control) in mice and big brown bats (Eptesicus fuscus). Mice vaccinated and boosted intradermally with 1 x 107 plaque forming units (PFU) of each RCN-rabies vaccine construct developed neutralizing antibodies and survived at significantly higher rates than controls. No significant difference in antibody titers or survival was noted between rabies-vaccinated groups. Bats were vaccinated either oronasally (RCN-G, RCN-MoG) with 5x107 PFU or by topical application in glycerin jelly (RCN-MoG, dose 2x108 PFU), boosted (same dose and route) at 46 days post vaccination (dpv), and then challenged with wild-type big brown variant RABV at 65 dpv. Prior to challenge, 90% of RCN-G and 75% of RCN-MoG oronasally vaccinated bats had detectable levels of serum rabies neutralizing antibodies. Bats from the RCN-luc and topically vaccinated RCN-MoG groups did not have measurable antibody responses. The RCN-rabies constructs were highly protective and not significantly different from each other. RCN-MoG provided 100% protection (n = 9) when delivered oronasally and 83% protection (n = 6) when delivered topically; protection provided by the RCN-G construct was 70% (n = 10). All rabies-vaccinated bats survived at a significantly (P ≤ 0.02) higher rate than control bats (12%; n = 8). We have demonstrated the efficacy of a novel, in silico designed rabies MoG antigen that conferred protection from rabies challenge in mice and big brown bats in laboratory studies. With further development, topical or oronasal administration of the RCN-MoG vaccine could potentially mitigate rabies in wild bat populations, reducing spillover of this deadly disease into humans, domestic mammals, and other wildlife., Author summary Rabies remains a significant and costly zoonotic disease worldwide. While control of canine rabies can significantly diminish the threat to human health, spillover of rabies and related lyssaviruses from bats into terrestrial animals and humans continues to be an important issue. Here we describe the development of a novel rabies vaccine, using raccoonpox virus (RCN) as a viral vector, and a computer designed rabies virus mosaic antigen. We demonstrate that this new vaccine leads to protection against experimental challenge in wild caught big brown bats when administered oronasally or topically. This technology could be adapted to target other bat species and also be directly applicable toward control of vampire-bat associated rabies in Mexico and Central and South America.

Biofilm formation, one of the most important virulence factors of pathogenic bacteria, protects bacteria against desiccation, antibiotics, phages and host immune responses. However, phage-derived depolymerases show antibiofilm activity and demonstrate great potential to treat infections caused by biofilm-forming bacteria. In this study, the Escherichia coli phage vB_EcoM_ECOO78 was isolated and characterised, and we observed its ability to lyse five out of 34 tested E. coli clinical isolates. The highest phage titre was observed at a multiplicity of infection of 10-5 and a burst size of approximately 74 plaque forming units (PFU)/infection. Electron micrographs indicated that vB_EcoM_ECOO78 belongs to the family Myoviridae. The presence of increasing halos surrounding the lysis plaques formed by vB_EcoM_ECOO78 indicated that this phage may encode a depolymerase. Based on a sequencing analysis, the complete genome of vB_EcoM_ECOO78 was found to be 41,289 bp in size, with a GC content of 53.07%. Additionally, vB_EcoM_ECOO78 has 56 predicted open reading frames, 51 (91.07%) of which are assumed to be functional. A BLAST analysis indicated that ORF42 of vB_EcoM_ECOO78 (Dpo42) has low identity with other reported phage-associated depolymerases. Dpo42 was expressed and purified as a soluble protein using E. coli BL21. The biofilm formation ability of E. coli isolates and the antibiofilm activity of Dpo42 were tested by performing spot assays and using a 96-well micro-titre plate method. Dpo42 degraded the capsular polysaccharides surrounding E. coli and exhibited dose-dependent biofilm-formation prevention activity. Based on these results, Dpo42 appears to be a novel phage-derived depolymerase that represents a new potential strategy for preventing E. coli biofilm formation.

Rotavirus is a major cause of acute gastroenteritis in young children in developed and developing countries. The use of probiotics for the treatment of gastrointestinal diseases is both safe and easily accessible. In this study, we evaluated the anti-rotaviral activities of probiotic mixtures in a Sprague-Dawley rat. 24 litters with their dams were randomly assigned to four groups; placebo, phosphate buffered saline (PBS), and two probiotic mixture (PRO-1 and PRO-2) groups. All rats were inoculated with rotavirus at dose of 8 log plaque forming units per rat at 5 days old. Animals in the PRO-1 and PRO-2 groups were orally administered probiotic mixtures 1 or 2, respectively, at a dose of 8 log colony forming units daily during 4 days. For control purposes, placebo and PBS groups were orally administered the same amount of placebo (containing maltose and polydextrose) or PBS once daily for 4 days, respectively. Antiviral analysis was performed by real-time quantitative PCR (RT-qPCR) and observing intestinal villi. As a result, weights of small intestines were greater in the PRO-1, PRO-2 groups than in control groups. Villi were short and villous epithelial necrosis was exhibited in control groups, but these morphological changes were not observed in PRO-1, PRO-2 treated rats. RT-qPCR analysis showed that VP7 gene level of rotavirus in fecal samples and small intestinal epithelial cells were lower in the PRO-1 and PRO-2 groups. These findings suggest that probiotic mixtures may be useful probiotics for the treatment of or as alternative therapies for rotaviral gastroenteritis.

A previously described phage infecting Escherichia coli O157:H7 was added to raw and cooked beef pieces at concentrations ranging from 101–108 plaque forming units/cm2 to either low (2) or high (104 CFU/cm2) concentrations of host bacterial cells. Incubation for up to 24 h was performed at 5℃ and 24℃ to simulate refrigerated and room temperature storage/temperature abuse. Surviving bacteria were enumerated during the incubation period, with phages being counted at the first and last sampling times. Significant reductions of E. coli O157:H7 of the order of >4 log10 CFU/cm2 at both temperatures could be achieved compared to phage-free controls. There was a trend for greater inactivation to occur with increasing phage concentration. While re-growth of surviving cells occurred in nearly all samples incubated for 24 h at 24℃, these conditions are not typical of those experienced by perishable foods. It was concluded that phages can be used to reduce the concentration of a bacterial pathogen on meat, but the concentration of phages needs to be high (>4–5 log10 plaque forming units/cm2) for reductions to occur. A concentration of the order 8 log10 plaque forming units/cm2 was needed to achieve a 4 log10 CFU/cm2 reduction.

Virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrosis virus were quantified in penultimate instar Pseudoplusia includens (soybean looper) larvae using a recombinant encoding the lacZ reporter gene ( Ac MNPV- hsp70/lacZ ). Larvae inoculated orally with Ac MNPV -hsp70/lacZ occlusion bodies (OB) or intrahemocoelically with budded virus (BV) were susceptible to fatal infection (LD 50 = 40.8 OB; 13.8 BV plaque forming units). Pseudoplusia includens displayed increased developmental resistance as larvae were orally inoculated with OB at later times during the penultimate instar. The optical brightener M2R, an inhibitor of the apoptotic processes that drive developmental resistance, did not significantly affect the virulence of Ac MNPV- hsp70/lacZ OB. To study pathogenesis, newly molted penultimate instar P. includens were orally inoculated with 60 OB and examined from 0.5 to 96 h post inoculation (h.p.i). Infection in the midgut was first detected at 4 h.p.i in 32% of the larvae and was apparent in cells of the tracheal system at 8 h.p.i. LacZ-positive (LacZ + ) hemocytes were first observed 2 h later. At 18 h.p.i, a low proportion of the hemocytes were LacZ + (5.6%). However, flow cytometry analysis of cell surface expression of the viral protein GP64 showed that 84.5% of the hemocytes collected at 18 h.p.i were infected with Ac MNPV, suggesting that flow cytometry may be a more sensitive method for identifying Ac MNPV-infected cells. Because P. includens display no physiological barriers to Ac MNPV OB and is permissive to fatal infection, this species could be controlled in organic cropping systems using naturally occurring strains of Ac MNPV.

Laban jameed is a dried salty dairy product obtained by fermentation of milk using a complex population of lactic acid bacteria. Jameed is considered a traditional food product in most eastern Mediterranean countries and is usually made from sheep or cow milk. The aim of this study was to assess phage contamination of jameed dairy product. Two phages were isolated: one from sheep milk jameed (PPUDV) and the other from cow milk jameed (PPURV). Each of the two bacteriophages was partially characterized. The PPUDV phage was identified as a single stranded DNA virus with an approximately 20 kb genome that was resistant to RNase, whereas PPURV phage possessed a double stranded RNA genome of approximately 20 kb and was resistant to DNase. The phage bacterial strain hosts were identified as Lactobacillus helveticus and Bacillus amyloliquefaciens for PPUDV and PPURV, respectively. One step growth curve using a double layer plaque assay test was carried out to monitor the phage life cycle after host infection. PPUDV showed a latent period of about 36 h, burst period of 70 h and a burst size of about 600 Plaque Forming Units (PFU) per infected cell. PPURV phage showed a latent period of about 24 h, burst period of 47 h and a burst size of about 700 PFU per infected cell. SDS-PAGE analysis of total viral proteins showed at least three major bands (27, 40, and 45 kDa) for PPUDV. This is the first study to report the isolation of both DNA and RNA bacteriophages from laban jameed. This study adds new insights into the complexity of dairy contamination and fermentation microbiology of the jameed revealing the existence of two viral genomes in this highly dried and salty dairy product.

The increase of foodborne viral outbreaks highlights the need for a rapid and sensitive method for the prediction of viral infectivity in food samples. This study assesses the use of propidium monoazide (PMA) coupled with real-time PCR methods (RT-qPCR or qPCR for RNA or DNA viruses, respectively) in the determination of viral infectivity in complex animal-related food matrices. Clam and Spanish fermented sausage (“chorizo”) samples were spiked with infectious and heat-inactivated human adenovirus-2 (HAdV-2) and mengovirus (vMC0). PMA-qPCR/RT-qPCR discriminated infective virus particles, with significant reductions (>2.7 log10 or 99.7%). Additionally, infectious HAdV-2 and vMC0 were quantified by plaque assay (in plaque forming units, PFU), and compared with those in virus genomes copies (GCs) quantified by PMA-qPCR/RT-qPCR. A consistent correlation (R2 > 0.92) was showed between PFU and GCs along serial 10-fold dilutions in both DNA and RNA virus and in both food matrices. This study shows the use of PMA coupled to qPCR/RT-qPCR as a promising alternative for prediction of viral infectivity in food samples in comparison to more expensive and time-consuming methods and for those viruses that are not able to grow under available cell culture techniques., ThisstudywasfinanciallysupportedbytheRTA2014-00024- C04-01 fromtheSpanishMinistryofEconomyandInnovation and theBrazilianCNPqProjectnumber472804/2013-8,andby CAPES/PNPD andCAPES/PDSE.

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections.

No microbial source tracking tool satisfies all the characteristics of an ideal indicator of human fecal pollution. For this reason, the potential of Enterococcus faecalis phages (enterophages) as markers of this type of contamination was tested by using eight Enterococcus type strains as the possible hosts. The prevalence of enterophages in animal feces and domestic sewage were determined, as were the inactivation rates in raw sewage at 4 °C and surface and tap waters at 22 °C. Enterophages were exclusively detected in raw sewage (up to 66.0 plaque forming units (PFU)/100 mL), suggesting a strictly human origin; and exhibited inactivation rates of approximately 0.002 to 0.05, 0.3 to 0.5 and 0.4 to 1.4 log day−1 in raw sewage and surface and tap waters, respectively, similar to those of previous reports on human enteric viruses under similar conditions. Interestingly, phages infecting other Enterococcus type strains were detected in both animal feces and domestic sewage in concentrations of up to 335.8 PFU/g and 96.0 PFU/100 mL, and certain phage isolates infected several of the strains tested. This clearly indicates the possible promiscuous nature of some Enterococcus phages and thus opens up the opportunity to further characterize these as indices of specific fecal sources.

Single and combined effects of three GRAS (generally recognized as safe) antimicrobials including, bacteriophage P100 (phage P100), lauric arginate (LAE), and potassium lactate-sodium diacetate mixture (PL-SD) were evaluated against Listeria monocytogenes cold growth in queso fresco cheese (QFC). The fate of phage P100 when exposed to LAE (200 ppm) or PL-SD (2.8% PL and 0.2% SD) was determined at 4°C and 30°C in a broth model. Phage P100 was found to be stable in the presence of these antimicrobial agents as plaque forming units (PFU) did not vary between control, LAE or PL-SD treatments. When 9 log CFU/ml of stationary phase cells of L. monocytogenes was exposed to these antimicrobials in tryptic soy broth, there was a 3 to 5 log CFU/ml reduction with phage P100 and a complete 9 log CFU/ml reduction with LAE but no measurable reduction with PL-SD after 24h at 4°C or 30°C. In QFC, the L. monocytogenes populations increased from the initial 3.5 log CFU/cm(2) to 7.7 log CFU/cm(2) in 28 days at 4°C. Treatment with 7.8 log PFU/cm(2) of phage P100 or 200 ppm of LAE showed strong listericidal effect initially by reducing L. monocytogenes counts by 2 to 3.5-4 log CFU/cm(2) while there was a subsequent regrowth of L. monocytogenes at 4°C. Treatment with PL-SD showed strong listeriostatic effect without decreasing L. monocytogenes counts but growth was prevented for 28 days at 4°C. Only the combined treatment of listericidal phage P100 or LAE with listeriostatic PL-SD reduced the initial L. monocytogenes counts by 2-4 log CFU/cm(2) and also kept the L. monocytogenes counts at that reduced level in QFC for 28 days at 4°C.

Quantification of bacteriophages by real-time quantitative PCR (qPCR) is an interesting alternative to the traditional plaque assay. Importantly, the method should in principle be able to discriminate between closely related phages that are indistinguishable by most other means. Here, a method is presented that employs qPCR to discriminate and quantify ten closely related lambdoid phages of Escherichia coli str. K-12. It is shown that (1) treatment of samples with DNase efficiently removes non-encapsidated DNA, while the titer of plaque forming units is not affected, (2) individual phage types can be accurately quantified in mixed lysates, and (3) the detection limit corresponds to that of a plaque assay. The method is used to quantify individual phage types that are released from lysogens that carry up to three different prophages.

Graphical abstract, Sinupret®, a herbal medicinal product made from Gentian root, Primula flower, Elder flower, Sorrel herb, and Verbena herb is frequently used in the treatment of acute and chronic rhinosinusitis and respiratory viral infections such as common cold. To date little is known about its potential antiviral activity. Therefore experiments have been performed to measure the antiviral activity of Sinupret® oral drops (hereinafter referred to as “oral drops”) and Sinupret® dry extract (hereinafter referred to as “dry extract”), in vitro against a broad panel of both enveloped and non-enveloped human pathogenic RNA and DNA viruses known to cause infections of the upper respiratory tract: influenza A, Chile 1/83 (H1N1) virus (FluA), Porcine Influenza A/California/07/2009 (H1N1) virus (pFluA), parainfluenza type 3 virus (Para 3), respiratory syncytial virus, strain Long (RSV), human rhinovirus B subtype 14 (HRV 14), coxsackievirus subtype A9 (CA9), and adenovirus C subtype 5 (Adeno 5). Concentration-dependent antiviral activity (EC50 between 13.8 and 124.8 μg/ml) of Sinupret® was observed against RNA as well as DNA viruses independent of a viral envelope. Remarkable antiviral activity was shown against Adeno 5, HRV 14 and RSV in which dry extract was significantly superior to oral drops. This could be ascertained with different assays as plaque-reduction assays in plaque forming units (PFU), the analyses of a cytopathogenic effect (CPE) and with enzyme immunoassays (ELISA) to determine the amount of newly synthesised virus. Our results demonstrate that Sinupret® shows a broad spectrum of antiviral activity in vitro against viruses commonly known to cause respiratory infections.

Salmonella is a foodborne pathogenic bacterium that causes human illnesses and morbidity and mortality in swine. Bacteriophages are viruses that prey on bacteria and are naturally found in many microbial environments, including the gut of food animals, and have been suggested as a potential intervention strategy to reduce Salmonella levels in the live animal. The present study was designed to determine if anti-Salmonella phages isolated from the feces of commercial finishing swine could reduce gastrointestinal populations of the foodborne pathogen Salmonella Typhimurium in artificially inoculated swine. Weaned pigs (n = 48) were randomly assigned to two treatment groups (control or phage-treated). Each pig was inoculated with Salmonella Typhimurium (2 × 10(10) colony forming units/pig) via oral gavage at 0 h and fecal samples were collected every 24 h. Swine were inoculated with a phage cocktail via oral gavage (3 × 10(9) plaque forming units) at 24 and 48 h. Pigs were humanely killed at 96 h, and cecal and rectal intestinal contents were collected for quantitative and qualitative analysis. Fecal Salmonella populations in phage-treated pigs were lower (p 0.09) than controls after 48 h. Phage treatment reduced intestinal populations of inoculated Salmonella Typhimurium in pigs compared to controls at necropsy. Cecal populations were reduced (p = 0.07) by phage treatment1.4 log(10) colony forming units/g digesta, and rectal populations were numerically reduced. The number of pigs that contained inoculated Salmonella Typhimurium was reduced by phage treatment, but a significant (p 0.05) reduction was only observed in the rectum. We conclude that phages can be a viable tool to reduce Salmonella in swine. Further research needs to be performed to determine the most efficacious dosing regimens and the most effective combinations of phages targeting the diverse Salmonella population found in swine before they can enter the food supply.

Infectious laryngotracheitis (ILT) is an upper respiratory tract disease of chickens that is caused by infectious laryngotracheitis virus (ILTV), an alphaherpesvirus. This disease causes significant economic loses in poultry industries worldwide. Despite widespread use of commercial live attenuated vaccines, many poultry industries continue to experience outbreaks of disease caused by ILTV. Efforts to improve the control of this disease have resulted in the generation of new vaccine candidates, including ILTV mutants deficient in virulence factors. A glycoprotein G deletion mutant vaccine strain of ILTV (ΔgG ILTV), recently licenced as Vaxsafe ILT (Bioproperties Pty Ltd), has been extensively characterised in vitro and in vivo, but the minimum effective dose required to protect inoculated animals has not been determined. This study performed a vaccination and challenge experiment to determine the minimum dose of ΔgG ILTV that, when delivered by eye-drop to seven-day-old specific pathogen-free chickens, would protect the birds from a robust challenge with a virulent field strain of virus (class 9 ILTV). A dose of 10(3.8) plaque forming units was the lowest dose capable of providing a high level of protection against challenge, as measured by clinical signs of disease, tracheal pathology and virus replication after challenge. This study has shown that the ΔgG ILTV vaccine strain is capable of inducing a high level of protection against a virulent field virus at a commercially feasible dose. These results lay the foundations upon which a commercial vaccine can be developed, thereby offering the potential to provide producers with another important tool to help control ILTV.

In an evaluation of a bacteriophage treatment for infection, ten dogs were included with chronic Pseudomonas aeruginosa otitis. Each received, directly into the auditory canal of one ear, a single dose of a topical preparation containing approximately 1 × 10(5) plaque forming units (PFU) of each of 6 bacteriophage strains, active against P. aeruginosa. At the time of bacteriophage administration and 48 h later each dog's core temperature was taken, its ear was assigned a clinical score (higher=worse condition) and aural swabs were taken for bacteriophage and P. aeruginosa counts. Forty eight hours after treatment the clinical score and P. aeruginosa count of all ears had fallen (mean score fall: 30.1%, range 7.7-56.3%, p

Bacteriophage Listex P100 (phage P100) was approved by the U.S. Food and Drug Administration and U.S. Department of Agriculture's Food Safety and Inspection Service for Listeria monocytogenes control on both raw and ready-to-eat food products. In this article, we present the proof of concept on the influence of phage dose, phage contact time, and storage temperature on the listericidal activity of phage P100 in reducing the L. monocytogenes loads on the surface of fresh channel catfish fillet. The fresh catfish fillet samples were surface inoculated with approximately 4.3 log(10) colony forming units (CFU)/g of a two serotype mix (1/2a and 4b) of L. monocytogenes cells and then surface treated with phage P100. L. monocytogenes reduction was influenced by phage contact time and phage dose regardless of higher or lower temperature regimes tested on catfish fillet. The reduction in L. monocytogenes loads (p0.05) with the phage P100 dose of 2 x 10(7) plaque forming units (PFU)/g (7.3 log(10) PFU/g) was 1.4-2.0 log(10) CFU/g at 4 degrees C, 1.7-2.1 log(10) CFU/g at 10 degrees C, and 1.6-2.3 log(10) CFU/g at room temperature (22 degrees C) on raw catfish fillet. The phage contact time of 30 min was adequate to yield greater than 1 log(10) CFU/g reduction in L. monocytogenes, whereas 15 min contact time with phage yielded less than 1 log(10) CFU/g reduction in L. monocytogenes loads on catfish fillet. Phage P100 titer was stable on catfish fillet samples, and overall reductions in L. monocytogenes counts were still maintained over a 10-day shelf life at 4 degrees C or 10 degrees C by phage P100 treatment. These findings illustrate the effectiveness of an alternative generally recognized as safe antimicrobial such as bacteriophage Listex P100 in quantitatively reducing L. monocytogenes from fresh catfish fillet surfaces.

Peste des Petits Ruminants (PPR) is a highly contagious animal disease caused by the Peste des Petits Ruminants virus (PPRV) belonging to the genus morbillivirus and family Paramyxoviridae. The disease results in high morbidity and mortality in goats, sheep and in some small wild ruminants. The presence of large number of small ruminants reared in endemic areas makes PPR a notorious disease threatening the livelihood of poor farmers. Conventional vaccination using a live, attenuated vaccine gives adequate protection but cannot be used in case of eradication of the disease due to difficulty in differentiation of infected animals from the vaccinated ones.In the present study, we constructed two recombinant viruses using attenuated Modified Vaccinia virus Ankara virus (MVA) namely MVA-F and MVA-H expressing the full length PPRV fusion (F) and hemagglutinin (H) glycoproteins, respectively. Goats were vaccinated intramuscularly with 105 plaque forming units (PFU) each of the recombinant viruses and a live attenuated vaccine (RAKSHA PPR) and challenged 4 months later with PPRV challenge virus (10(3) goat LD(50)). All goats were completely protected from the clinical disease. This study gave an indication that mass vaccination of small ruminants with either of the above or both recombinant inexpensive virus vaccines could help in possible eradication of PPRV from endemic countries like India and subsequent seromonitoring of the disease for differentiation of infected animals from vaccinated ones.

The continuing outbreaks of avian influenza A H5N1 virus infection in Asia and Africa have caused worldwide concern because of the high mortality rates in poultry, suggesting its potential to become a pandemic influenza virus in humans. The transmission route of the virus among either the same species or different species is not yet clear. Broilers and BABL/c mice were inoculated with the H5N1 strain of influenza A virus isolated from birds. The animals were inoculated with 0.1 mL 10(6.83) TCID(50) of H5N1 virus oronasally, intraperitoneally and using eye drops. The viruses were examined by virological and pathological assays. In addition, to detect horizontal transmission, in each group, healthy chicks and mice were mixed with those infected. Viruses were detected in homogenates of the heart, liver, spleen, kidney and blood of the infected mice and chickens. Virus antigen was not detected in the spleen, kidney or gastrointestinal tract, but detected by Plaque Forming Unit (PFU) assay in the brain, liver and lung without degenerative change in these organs (in the group inoculated using eye drops. The detection results for mice inoculated using eye drops suggest that this virus might have a different tissue tropism from other influenza viruses mainly restricted to the respiratory tract in mice. All chicken samples tested positive for the virus, regardless of the method of inoculation. Avian influenza A H5N1 viruses are highly pathogenic to chickens, but its virulence in other animals is not yet known. To sum up, the results suggest that the virus replicates not only in different animal species but also through different routes of infection. In addition, the virus was detection not only in the respiratory tract but also in multiple extra-respiratory tissues. This study demonstrates that H5N1 virus infection in mice can cause systemic disease and spread through potentially novel routes within and between mammalian hosts.

Forty-eight strains of Mycobacterium ranae (Bergey et al.) have been phage typed with various dilutions of a suspension of mycobacteriophage BK4. A close correlation between lysis by 5 times 102 or less Plaque Forming Units (P.F.U.) of the phage and inositol-utilising activity has been found. Strains that utilise inositol are lysed by 5 times 102 or less P.F.U., whereas strains that are unable to utilise inositol require 5 times 104 or more P.F.U. for lysis. In one group of strains of M. ranae the inositol-utilisation is greatly reduced and only detectable by a sensitive technique. This is considered to be due to permeability factors rather than a defect in the inositol locus.

The effects of pH (3-7), NaCl (0-6%), and temperature on pressure inactivation of hepatitis A virus (HAV) were determined. The HAV samples were treated at 400 MPa for 1 min at 5, 20, and 50 degrees C. Decreasing solution pH enhanced pressure inactivation of HAV. This enhanced inactivation effect was most evident at 20 degrees C. A baroprotective effect was observed for NaCl concentrations from 1 to 6%. For example, a treatment of 400 MegaPascals (MPa) for 1 min at 50 degrees C reduced the HAV titers by 4.0, 4.1, 1.3 and 0.4 log plaque forming units (PFU)/ml for NaCl concentrations of 0, 1, 3, and 6%, respectively. Overall, increasing the treatment temperature enhanced pressure inactivation of HAV in the solutions. The pressure resistance of HAV in oysters was also examined. Temperature in the range of 5 to 50 degrees C did not significantly affect the pressure inactivation of HAV within oyster homogenates. It is concluded that HPP treatment of oysters at temperatures above room temperature would not provide any additional benefit for inactivation of HAV. However, the observation that HAV inactivation is enhanced in acidic matrices is information that may be useful for designing product formulations and processing parameters for high pressure processing of products such as low pH fruit juices and salsa.

There are increasing concerns of zoonotic transmission of some animal enteric viruses, such as calicivirus, hepatitis E virus, and rotavirus, which are closely related to human pathogenic strains. Most enteric viruses are detected by molecular techniques because they cannot be cultured. Surrogates such as F-RNA coliphages are cultivable but few molecular methods exist. Individual real-time TaqMan RT-PCR assays for the replicase gene of F-RNA coliphage genogroups I and IV were developed and multiplexed with a real-time TaqMan RT-PCR assay for feline calicivirus as a sample process control for the simultaneous detection and enumeration of genogroup I and IV F-RNA coliphages. Genogroup IV were successfully detected with the multiplexed assay in 80% of fecal samples that contained F-RNA coliphage levels ≥3.2 log plaque forming units (pfu). F-RNA coliphage were at or below the limit of detection in most fecal samples when levels were ≤4 log pfu/g.