Ruxolitinib accelerates influenza A virus adaptation in the Madin-Darby canine kidney (MDCK) cell line.

AIM: To investigate the effect of ruxolitinib medium supplement, separately and in combination with trypsin, on influenza A virus (IAV) adaptation and propagation in the Madin-Darby canine kidney (MDCK) cell line. METHODS AND RESULTS: Two consecutive passages of three egg-based IAV strains were performed in the MDCK cell line with medium (a) without additives; (b) with a combination of ruxolitinib and trypsin; (c) with ruxolitinib; and (d) trypsin. Adaptation without a medium additive failed in both passages. After a single passage, the probability of the IAV adaptation was highly significantly influenced by the type of additive (binomial generalized linear model, χ22 = 23.84, P < 0.00001). The highest probability of adaptation was achieved with the combination of ruxolitinib and trypsin, followed by ruxolitinib alone and trypsin. After the two consecutive passages, the influence of the type of medium additive on the probability of virus adaptation was no longer significant. In two of three IAV MDCK-adapted strains, the type of medium additive had no significant influence on virus yields. CONCLUSION: Ruxolitinib accelerates the adaptation of IAV in the MDCK cell line both individually and together with trypsin.

Coronavirus Persistence on a Plastic Carrier Under Refrigeration Conditions and Its Reduction Using Wet Wiping Technique, with Respect to Food Safety.

The global SARS-CoV-2 pandemic dictates that anti-contagion strategies should become matters of essential routine in everyday life. Fomite transference is one of the routes of transmission that has been considered for this virus. However, the risks associated with contaminated surfaces of food packaging kept in refrigerators have not yet been adequately assessed. In this study, a surrogate virus, Alphacoronavirus 1, was used to investigate the persistence of coronavirus dried on a plastic carrier at 4 °C. Techniques of wet wiping, with or without disinfectant saturation, were employed to evaluate their effectiveness in the elimination of the virus. If not wiped, the loss of infectivity of the virus on plastic surfaces was, on average, 0.93 log10 (i.e. 83%) per day of storage at 4 °C. Wiping with water-saturated material reduced the initial virus titre on the plastic carrier by 2.4 log10 (99.6%); the same results were achieved through wiping with bactericidal wipes containing ethanol. Wipes saturated with a combination of disinfectant agents (didecyl-dimethyl-ammonium chloride, hydrogen peroxide) decreased the virus titre still more efficiently, by 3.8 log10 (99.98%) and also significantly prevented further transfer of the virus to a secondary surface through wiping. Thus SARS-CoV-2 transmission potential via contaminated plastic packaging and food may be efficiently eliminated by wet-wiping, especially when wipes saturated with specific disinfectants are used.

Virucides in apiculture: persistence of surrogate enterovirus under simulated field conditions.

BACKGROUND: Honeybee viruses have been recognized as being among the most important factors leading to colony losses worldwide. Colony food and faeces are regarded as possible sources of infectious viruses able to contaminate the environment and equipment of apiaries. Thus, methods for elimination of viruses are required. No cell culture assay for testing the effect of disinfectants on honeybee viruses is yet available. Therefore, surrogate virus was employed for testing of the efficacy of iodophor- and peracetic acid-based disinfectants in combination with six organic contaminants at +6 °C and +22 °C. Moreover, we evaluated the persistence of the surrogate in honey at +6 °C, +22 °C, and +50 °C. RESULTS: Iodophor-based disinfectant showed a maximum reduction of virus titre of 3.4 log10 . Peracetic acid reduced the titre (≥4 log10 ) only at 22 °C and without yeast extract/bovine serum albumin. After 25 days of incubation of the virus - honey mix, no decrease of virus titre was observed at +6 °C, whereas a significant reduction (3.5 log10 ) was found at +50 °C already after 1 day. CONCLUSIONS: Both tested disinfectants can serve as appropriate virucides in apiaries. The effect of peracetic acid significantly depended on temperature and organic contaminants. The iodophor-based disinfectant showed a stable antiviral effect at different temperatures and with different contaminants. © 2017 Society of Chemical Industry.

Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices.

The detection and quantification of enteric RNA viruses is based on isolation of viral RNA from the sample followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). To control the whole process of analysis and in order to guarantee the validity and reliability of results, process control viruses (PCV) are used. The present article describes the process of preparation and use of such PCV- MS2 phage-like particles (MS2 PLP) - in RT-qPCR detection and quantification of enteric RNA viruses. The MS2 PLP were derived from bacteriophage MS2 carrying a unique and specific de novo-constructed RNA target sequence originating from the DNA of two extinct species. The amount of prepared MS2 particles was quantified using four independent methods - UV spectrophotometry, fluorimetry, transmission electron microscopy and a specifically developed duplex RT-qPCR. To evaluate the usefulness of MS2 PLP in routine diagnostics different matrices known to harbor enteric RNA viruses (swab samples, liver tissue, serum, feces, and vegetables) were artificially contaminated with specific amounts of MS2 PLP. The extraction efficiencies were calculated for each individual matrix. The prepared particles fulfill all requirements for PCV - they are very stable, non-infectious, and are genetically distinct from the target RNA viruses. Due to these properties they represent a good morphological and physiochemical model. The use of MS2 PLP as a PCV in detection and quantification of enteric RNA viruses was evaluated in different types of matrices.

3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses.

Virus quantitation by transmission electron microscopy, TCID50, and the role of timing virus harvesting: a case study of three animal viruses.

Quantitation of viruses is practised widely in both basic and applied virology. Infectious titration in cell cultures, the most common approach to it, is quite labour-intensive and alternative protocols are therefore sought. One of the alternatives is transmission electron microscope (TEM) quantitation using latex particles at a known concentration as a reference for counting virus particles. If virus TCID50 is determined in parallel, the ratio of infectious to non-infectious virus particles may be established. This study employs such an approach to compute the number of virus particles and TCID50, and establish their correlation for three viruses: Canine adenovirus 1 (CAdV-1), Feline calicivirus (FCV) and Bovine herpesvirus 1 (BoHV-1). Each of the viruses was grown in five replicates until complete cytopathology was recorded (time 0), then frozen. They were thawed, filter-sterilised and left for additional periods of 16, 32 and 48 h at 37°C. At each time point, the infectious ability of the virus was characterised by TCID50 and the number of virions quantified by TEM, in order to evaluate the influence of timing on virus harvest. The virus particle count determined by TEM did not change for any of the viruses throughout the experiment. The relationship between virus particle counts with TCID50 at time 0 showed good linearity response; their ratio was almost constant. The virus particle-to-TCID50 ratio varied between 146 and 426 (mean±SD: 282±103) for CAdV-1, between 36 and 79 (57±18) for FCV and between 110 and 249 (167±53) for BoHV-1. The proportion of non-infectious particles did not change throughout the experiment for either CAdV-1 or BoHV-1. However, a decrease in virus infectious ability disclosed by TCID50 indicated that the fraction of non-infectious particles in FCV increased 300,000 times when time 0 and 48 h were compared. The quantitation of viruses with TEM is a simple and rapid protocol for virus quantitation but account must be taken of the type of virus and harvesting time as virus counts need not necessarily correlate with virus infectious ability.

Genetic diversity of porcine sapoviruses, kobuviruses, and astroviruses in asymptomatic pigs: an emerging new sapovirus GIII genotype.

Small, non-enveloped RNA viruses belonging to the genera Sapovirus, Kobuvirus, and Mamastrovirus are usually associated with gastroenteritis in humans and animals. These enteric pathogens are considered potential zoonotic agents. In this study, the prevalence and genetic diversity of sapoviruses (SaVs), kobuviruses (KoVs), and astroviruses (AstVs) in asymptomatic pigs were investigated using a PCR approach. KoV was found to be the most prevalent virus (87.3 %), followed by AstV (34.2 %) and SaV (10.2 %). Interestingly, the intra- and inter-cluster distances between porcine SaV capsid sequences revealed one strain (P38/11/CZ) that formed a new genotype within genogroup III of porcine SaVs, and it is tentatively called "P38/11-like" genotype. Moreover, this is the first report of porcine kobuvirus detection on Czech pig farms. The high prevalence rate of gastroenteritis-producing viruses in clinically healthy pigs represents a continuous source of infection of pigs, and possibly to humans.