Virus Suspension Research Articles

Stimulated Low-Frequency Raman Scattering in Brome Mosaic Virus

We experimentally register stimulated low-frequency Raman scattering (SLFRS) in the suspension of brome mosaic virus (BMV) in phosphate buffer with very high conversion efficiency. We identify two components of the SLFRS spectrum as the breathing and quadrupole modes of BMV and determine damping characteristics and gain factors for these modes. We show that, using the core–shell model for BMV and taking into account the influence of the environment, the acoustic properties of individual components of such a composite nanosystem can be determined. Thus, we define the sound velocity in the RNA core of BMV, in view of spectral characteristics of SLFRS.

Anticoronavirus Activity of Water-Soluble Pristine C60 Fullerenes: In Vitro and In Silico Screenings.

The emergence of a new member of the Coronaviridae family, which caused the 2020 pandemic, requires detailed research on the evolution of coronaviruses, their structure and properties, and interaction with cells. Modern nanobiotechnologies can address the many clinical challenges posed by the COVID-19 pandemic. In particular, they offer new therapeutic approaches using biocompatible nanostructures with "specific" antiviral activity. Therefore, the nanosized spherical-like molecule (0.72 nm in diameter) composed of 60 carbon atoms, C60 fullerene, is of interest in terms of fighting coronaviruses due to its high biological activity. In here, we aim to evaluate the effectiveness of anticoronavirus action of water-soluble pristine C60 fullerene in the model and in vitro systems. As a model, apathogenic for human coronavirus, we used transmissible gastroenteritis virus of swine (TGEV), which we adapted to the BHK-21 cell culture (kidney cells of a newborn Syrian hamster). The shape and size of the particles present in C60 fullerene aqueous colloidal solution (C60FAS) of given concentration, as well as C60FAS stability (value of zeta potential) were studied using microscopic (STM, scanning tunneling microscopy, and AFM, atomic force microscopy) and spectroscopic (DLS, dynamic light scattering) methods. The cytopathic effect of TGEV was determined with the help of a Leica DM 750 microscope and the degree of monolayer changes in cells was assessed. The microscopy of the viral suspension was performed using a high resolution transmission electron microscope (HRTEM; JEM-1230, Japan). Finally, the search for and design of optimal possible complexes between C60 fullerene and target proteins in the structure of SARS-CoV-2 coronavirus, evaluation of their stability in the simulated cellular environment were performed using molecular dynamics and docking methods. It was found that the maximum allowable cytotoxic concentration of C60 fullerene is 37.5±3.0μg/ml. The investigated C60FAS reduces the titer of coronavirus infectious activity by the value of 2.00±0.08TCID50/ml. It was shown that C60 fullerene interacts directly with SARS-CoV-2 proteins, such as RdRp (RNA-dependent RNA polymerase) and 3CLpro (3-chymotrypsin-like protease), which is critical for the life cycle of the coronavirus and, thus, inhibits its functional activity. In both cases, C60 fullerene fills the binding pocket and gets stuck there through stacking and steric interactions. Pioneer in vitro study to identify the anticoronavirus activity of water-soluble pristine C60 fullerenes indicates that they are highly promising for further preclinical studies, since a significant inhibition of the infectious activity of swine coronavirus of transmissible gastroenteritis in BHK-21 cell culture was found. According to molecular modeling results, it was shown that C60 fullerene can create the stable complexes with 3CLpro and RdRp proteins of SARS-CoV-2 coronavirus and, thus, suppress its functional activity.

Attenuation of a Turkeypoxvirus field strain as an alternative to heterologous vaccination in turkeys

Avian pox can severely impact turkey production systems. Vaccination programs in Mexico use commercially available Fowlpoxvirus vaccines, that are used across different bird species. Nonetheless, there are reports of sporadic disease outbreaks among vaccinated turkeys, which suggest that heterologous vaccines may provide limited immunity, presenting the need to develop homologous vaccines that can better protect turkeys. This study compared the protection granted to turkey chicks by a commercial Fowlpoxvirus vaccine and by a live attenuated Turkeypoxvirus vaccine after a challenge with a field isolated Turkeypoxvirus virus. Histopathology, polymerase chain reaction, and sequencing of DNA were used for viral identification. A Turkeypoxvirus strain was first isolated in chicken embryo lesions, and subsequently adapted through serial passes in chorioallantoic membrane to produce the homologous vaccine. The attenuated virus was used as a vaccine when a 104.4 embryo ID50/mL titre was reached. Three groups of three-week-old turkey chicks were used for challenge experiments. Subjects in Group 1 were immunized with the attenuated Turkeypoxvirus vaccine (homologous vaccine). Chicks in Group 2 were vaccinated with the commercially available heterologous vaccine (Fowlpoxvirus). Subjects in Group 3 were not vaccinated and received only saline solution (control group). Two weeks after vaccination, animals from Group 1 reached a 97.7 ND50 seroneutralization titre, while levels reached in Group 2 birds and in control chicks were 11.7 ND50 (Group 2) and zero, respectively. At this time, all groups were challenged with a suspension of a field-isolated Turkeypox virus. The homologous vaccine afforded 100% protection in Group 1 (10/10 individuals), while only 10% (1/10) of individuals in Group 2 were protected by the commercial heterologous Fowlpoxvirus vaccine. None of the non-immunized birds in Group 3 were protected (0/10). These results show that the homologous vaccine afforded a greater protection against a Turkeypox virus infection than that observed for the heterologous vaccine, and that a homologous vaccine can be efficiently produced by isolating and attenuating the virus from turkeypox lesions, through chorioallantoic membrane serial passes.

Radiosensitivity of Feline Calicivirus F9 on Iceberg Lettuce Surface after Combined Treatments with γ-Radiation

The surface of iceberg lettuce (Lactuca sativa L.) is favorable to the survival of pathogens such as bacteria, parasites, and viruses such as norovirus. The present study was conducted to investigate the antiviral properties of treatment with cranberry juice (CJ), ozone (O3), and γ-radiation alone or in combination against feline calicivirus (FCV) F9 present on the surface of iceberg lettuce. The lettuce leaves were inoculated with virus suspensions at ∼6 log TCID50 (50% tissue culture infective dose)/mL and treated with CJ, O3, and γ-radiation alone and in combination during storage at 4°C. The D10-values of 1.21 kGy, 2.23% CJ, and 14.93 ppm of O3 were obtained when samples were treated with various radiation doses, CJ, and O3, respectively. Relative radiosensitization of FCV-F9 virus on lettuce was 1.20, 1.50, 1.09, and 1.00 after combined CJ treatments of 0.1, 0.25, 0.50, and 1.50%, respectively. Optimum treatments were 5 ppm for 7.5 min for O3, 0.25% CJ, and γ-radiation at 1.5 kGy when each treatment was used alone. The combination of the three treatments produced the highest reduction of 2.15 log TCID50/mL (from initial inoculation of ∼7 log TCID50/mL) during 10 days of storage at 4°C. Antibacterial properties of treatments and physicochemical quality of lettuce were investigated during 13 days of storage at 4°C. The treatment of lettuce with γ-radiation alone (1.5 kGy) reduced the total flora by 3 log CFU/g; however, the combination of CJ (0.25%) with irradiation (1.5 kGy) reduced it by ∼5 log CFU/g after 13 days of storage at 4°C. The texture and color of the lettuce treated with the combined treatments changed slightly during storage, and chlorophyll increased by 3.81 μg/mL after 10 days of storage at 4°C. Significant differences in taste and color were observed in lettuce without treatments after 5 days of storage, whereas no difference was observed after the 0.25% CJ or the combined treatments.

Trails of use of nano-particles as adjuvant for inactivated bovine viral diarrheoa vaccine

Bovine viral diarrhea virus (BVDV) with different genotypes considered as one of the most important viruses in genus Pestivirus at animal level is consider an ideal research model for hepatitis C virus of the same genus of Flavivirus. Many traditional inactivated BVDV vaccine have been used adjuvanted by Alhydragel or oil. The current study focused on formulation anew inactivated BVDV vaccine using innovated nano material (meso porous nano silica) prepared with as 9 parts of viral suspension: 1- Part of dissolved adjuvant. Sterility and safety of the adjuvant alone and with the prepared vaccine containing two genotypes of BVDV (1and 2) and potency test on in vaccinated calves revealed that such adjuvant and vaccine are free from foreign contaminants and the vaccine is potent provided calves with protection against BVDV coverage a period of 9 month for both genotypes using two doses of 1.5mL of both inactivated virus vaccine. This study recommended other complementary study to clarify the detail keeping quality issues and field study.

Antiviral photodynamic therapy: Inactivation and inhibition of SARS-CoV-2 in vitro using methylene blue and Radachlorin.

IntroductionRecently, the COVID-19 pandemic has spread globally, necessitating the development of new methods for its prevention and treatment. The purpose of this study was to evaluate the antiviral activity of photodynamic therapy (PDT) against SARS-CoV-2 in vitro.MethodsVero E6 cells and SARS-CoV-2 isolated in Russia were used for PDT with methylene blue (MB) and Radachlorin. A continuous laser with wavelength λ = 662 nm in doses of 16 J/cm2 and 40 J/cm2 laser irradiation was used for PDT of a viral suspension and SARS-CoV-2-infected cells. The direct cytopathogenic effect of SARS-CoV-2 was evaluated via light microscopy to calculate the TCID50 in the samples and perform statistical analysis.ResultsViral suspensions of SARS-CoV-2 that had a TCID50 greater than 103 were inactivated by PDT in the presence of MB and Radachlorin. Vero E6 cells were protected from 104 TCID50 of SARS-CoV-2 by PDT post infection. The range of protective concentrations was 1.0–10.0 μg/ml and 0.5–5.0 μg/ml for MB and Radachlorin, respectively. Additionally, it was found that MB and Radachlorin also possess significant antiviral activity even without PDT. The 50 % inhibitory concentration (IC50) against 102 TCID50 of SARS-CoV-2 was found to be 0.22 and 0.33 μg/mL with the addition of MB and Radachlorin, respectively, to cells concomitantly with virus, whereas in the case of applying the photosensitizers at 3.5 h post infection, the IC50 was 0.6 and 2.0 μg/mL for MB and Radachlorin, respectively.ConclusionPDT shows high antiviral activity against SARS-CoV-2 when combined with MB and Radachlorin in vitro.

Electrostatic wipes as simple and reliable methods for influenza virus airborne detection

The performance of an in-house protocol for virus detection on commercialized electrostatic wipes (EWs) was assessed experimentally by impregnating them with suspensions of cytomegalovirus, adenovirus, and influenza virus, and by determining the recovery efficiency, repeatability, and detection limit of the protocol. The protocol was sensitive enough to detect 4 log10 gene copies of virus. At room temperature, influenza RNA was stable on EWs for at least four days. When EWs were placed high in 32 influenza-infected patients' rooms, influenza RNA was detectable in 75% (N= 24) of EWs, suggesting that EWs are simple and reliable methods for influenza virus airborne detection.

Using Algal Virus Paramecium bursaria Chlorella Virus as a Human Adenovirus Surrogate for Validation of UV Treatment Systems.

Adenovirus is among the most UV-resistant waterborne human pathogens. There is a need to identify nonpathogenic surrogates for adenovirus for the water treatment industry. In this study, the feasibility of using the algal virus Paramecium bursaria chlorella virus (PBCV-1) as an adenovirus surrogate for validation of UV reactors was evaluated. The UV dose-response behavior of PBCV-1 to monochromatic UV radiation at 254 nm and action spectrum for wavelengths ranging from 214 to 289 nm were measured. A culture-based infectivity assay was used to evaluate viral inactivation, and a quantitative PCR assay was used to quantify DNA damage. A UV254 dose of 150 mJ/cm2 resulted in roughly 5-log10 units of reduction of PBCV-1, which is similar to that of adenovirus. Furthermore, the inactivation action spectrum of PBCV-1 was similar to that of adenovirus between 214 and 289 nm. A simplified and inexpensive prepurification method was also developed to prepare PBCV-1 viral suspensions with similar inactivation behavior to purified PBCV-1. Overall, PBCV-1 appears to represent an appropriate adenovirus surrogate for UV system performance evaluation and illustrates the potential of using algal viruses as nonpathogenic, easy to culture, and readily available surrogates for human pathogens.

Coronavirus rotational diffusivity.

Just 11 weeks after the confirmation of first infection, one team had already discovered and published [D. Wrapp et al., “Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation,” Science 367(6483), 1260–1263 (2020)] in exquisite detail about the new coronavirus, along with how it differs from previous viruses. We call the virus particle causing the COVID-19 disease SARS-CoV-2, a spherical capsid covered with spikes termed peplomers. Since the virus is not motile, it relies on its own random thermal motion, specifically the rotational component of this thermal motion, to align its peplomers with targets. The governing transport property for the virus to attack successfully is thus the rotational diffusivity. Too little rotational diffusivity and too few alignments are produced to properly infect. Too much, and the alignment intervals will be too short to properly infect, and the peplomer is wasted. In this paper, we calculate the rotational diffusivity along with the complex viscosity of four classes of virus particles of ascending geometric complexity: tobacco mosaic, gemini, adeno, and corona. The gemini and adeno viruses share icosahedral bead arrangements, and for the corona virus, we use polyhedral solutions to the Thomson problem to arrange its peplomers. We employ general rigid bead–rod theory to calculate complex viscosities and rotational diffusivities, from first principles, of the virus suspensions. We find that our ab initio calculations agree with the observed complex viscosity of the tobacco mosaic virus suspension. From our analysis of the gemini virus suspension, we learn that the fine detail of the virus structure governs its rotational diffusivity. We find the characteristic time for the adenovirus from general rigid bead–rod theory. Finally, from our analysis of the coronavirus suspension, we learn that its rotational diffusivity descends monotonically with its number of peplomers.

Rapid Selective Detection of Potentially Infectious Porcine Epidemic Diarrhea Coronavirus Exposed to Heat Treatments Using Viability RT-qPCR.

Coronaviruses (CoVs) cause severe respiratory, enteric, and systemic infections in a wide range of hosts, including humans and animals. Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of porcine epidemic diarrhea (PED), a highly contagious intestinal disease affecting pigs of all ages. In this study, we optimized a viability real-time reverse transcriptase polymerase chain reaction (RT-qPCR) for the selective detection of infectious and heat-inactivated PEDV. PEMAX™, EMA™, and PMAxx™ photoactivable dyes along with PtCl4 and CDDP platinum compounds were screened as viability markers using two RT-qPCR assays: firstly, on PEDV purified RNA, and secondly on infectious and thermally inactivated virus suspensions. Furthermore, PMAxx™ pretreatment matched the thermal inactivation pattern obtained by cell culture better than other viability markers. Finally, we further optimized the pretreatment by coupling viability markers with Triton X-100 in inoculated serum resulting in a better estimation of PEDV infectivity than RT-qPCR alone. Our study has provided a rapid analytical tool based on viability RT-qPCR to infer PEDV infectivity with potential application for feed and feed ingredients monitoring in swine industry. This development would allow for greater accuracy in epidemiological surveys and outbreak investigations.

Effects and mechanism of mitochondrial transcription factor A and cytochrome c oxidase pathway in the energy production of hypoxic cardiomyocytes of rats regulated by tumor necrosis factor receptor associated protein 1

Objective: To investigate the effects and mechanism of mitochondrial transcription factor A (TFAM) and cytochrome c oxidase (COX) pathway in the energy production of hypoxic cardiomyocytes of rats regulated by tumor necrosis factor receptor associated protein 1 (TRAP1). Methods: The cardiomyocytes were isolated from 135 neonatal Sprague-Dawley rats (aged 1-3 d) and cultured for the following experiments. (1) Cells were collected and divided into normoxia blank control (NBC) group, hypoxia blank control (HBC) group, hypoxia+ TRAP1 over-expression control (HTOC) group, and hypoxia+ TRAP1 over-expression (HTO) group according to the random number table (the same grouping method below), with 1 bottle in each group. Cells in NBC group were cultured routinely, cells in HBC group were cultured in hypoxic condition for 6 hours after routine culture, cells in HTOC and HTO groups were respectively added with TRAP1 over-expression empty virus vector and TRAP1 over-expression adenovirus vector virus suspension for transfection for 48 hours after routine culture and then cultured in hypoxic condition for 6 hours. The protein expression of TFAM of cells in each group was detected by Western blotting. (2) Cells were collected and divided into NBC, HBC, HTOC, HTO, HTO+ TFAM interference control (HTOTIC), and HTO+ TFAM interference (HTOTI) groups, with 1 well in each group. Cells in the former 4 groups were dealt with the same methods as the corresponding groups in experiment (1). Cells in HTOTIC and HTOTI groups were respectively added with TFAM interference empty virus vector and TFAM interference adenovirus vector virus suspension for transfection for 48 hours, and the other processing methods were the same as those in HTO group. The content of ATP of cells in each group was determined by ATP determination kit and microplate reader, and the COX activity of cells in each group was determined by COX activity assay kit and microplate reader. (3) Cells were collected and divided into NBC group, normoxia+ sodium azide (NSA) group, HBC group, and hypoxia+ sodium azide (HSA) group, with 1 well in each group. Cells in NBC and HBC groups were respectively dealt with the same methods as the corresponding groups in experiment (1). Cells in NSA and HSA groups were respectively added with 32 nmol sodium azide at 30 min before experiment or hypoxia, and then cells in HSA group were cultured in hypoxic condition for 6 hours. The content of ATP was determined by the same method as above. The above three experiments were repeated for three times. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: (1) Compared with that in NBC group, the protein expression of TFAM of cells in HBC group was significantly decreased (P<0.01). Compared with that in HBC group or HTOC group, the protein expression of TFAM of cells in HTO group was significantly increased (P<0.01). (2) Compared with 0.552±0.041 and 1.99±0.15 in NBC group, the COX activity (0.270±0.044) and ATP content (1.09±0.11) of cells in HBC group were significantly decreased (P<0.01). Compared with 0.269±0.042 and 1.17±0.12 in HBC group and those in HTOC group, the COX activity (0.412±0.032 and 0.404±0.016) and ATP content (1.75±0.06 and 1.69±0.07) of cells in HTO and HTOTIC groups were significantly increased (P<0.01). Compared with those in HTO and HTOTIC groups, the COX activity (0.261±0.036) and ATP content (1.23±0.07) of cells in HTOTI group were significantly decreased (P<0.01). (3) Compared with that in NBC group, the ATP content of cells in NSA and NBC groups was significantly decreased (P<0.01). Compared with that in HBC group, the ATP content of cells in HSA group was significantly decreased (P<0.01). Conclusions: TRAP1 can increase the COX activity of cardiomyocytes by raising the expression of TFAM, and finally alleviate the impairment in energy production of cardiomyocytes caused by hypoxia.

Water-soluble tetra-cationic porphyrins display virucidal activity against Bovine adenovirus and Bovine alphaherpesvirus 1.

Porphyrins are photoactive compounds that can absorb the energy of light and transfer it to oxygen molecules, producing reactive oxygen species (ROS). Once produced, ROS may alter biological molecules and cellular mechanisms, leading to cell apoptosis or inactivation of microorganisms, such as bacteria, fungi, and viruses. Therefore, the objective of this study was to evaluate the in vitro virucidal activity of six tetra-cationic porphyrins against two bovine viruses (Bovine alphaherpesvirus 1, BoHV-1, enveloped; and Bovine adenovirus, BAV, non-enveloped). For this, viral suspensions were incubated with each porphyrin (H2TMeP, ZnTMeP, and CuTMeP at 4.0 μM, NiTMeP at 5.0 μM, and CoClTMeP and MnClTMeP at 1.0 μM) and exposed to white-light irradiation for 0, 60, 120, and 180 min (BAV) or 0, 30, 60, 90, and 120 min (BoHV-1). Then, the remaining viral titers were determined by limiting dilution and compared with the control (virus + porphyrins without light exposition). The porphyrins H2TMeP and ZnTMeP showed the highest virucidal activity against both viruses. ZnTMeP inactivated BoHV-1 after 30 min of photoactivation and H2TMeP after 60 min. The BAV was photo-inactivated by both porphyrins at 180 min of white-light exposition. CuTMeP, NiTMeP, and CoClTMeP porphyrins had weak virucidal activity against BoHV-1 and MnClTMeP showed no virucidal activity against both viruses. These results indicated that free-base H2TMeP and ZnTMeP porphyrins present virucidal activity against non-enveloped and enveloped viruses, opening the possibility for their use to inactivate viruses on surfaces, biological substrates, and solutions.

Antiviral Components from Cleome droserifolia and Lotus creticus

Objectives: Searching for new antiviral compounds is important due to continuous appearance of new viral strains that resist the commonly used drugs and due to toxic side effects of some of these drugs. This current study was designed to investigate some herbs to discover new natural antiviral agents. Methods: Eight herbal extracts were screened for antiviral activities against Hepatitis A, Herpes Simplex-1 and Coxsackie viruses. Bio guided fractionation of the active extracts using different solvents and further investigation of the bioactive one using various chromatographic techniques including normal phase column chromatography, poly amide column and preparative TLC led to isolation and identification of twelve compounds. Structural elucidation of these compounds was done using different spectroscopic techniques including IR, HR-MS, 1D and 2D-NMR. The antiviral activities of the isolated compounds were assessed by MTT assay that depends on mixed treatment assay which test inactivation of virus particles in the virus suspension by samples. The concentration of samples which inactivate virus particles in virus suspension by 50% compared with untreated control was determined for each sample and was compared with IC50 of positive control, Acyclovir. Findings: Twelve known compounds were isolated from the bioactive fractions of Cleome droserifolia and Lotus creticus. Nine compounds (1-9) from methylene chloride and ethyl acetate fractions of Cleome droserifolia and four compounds (10-13) from ethyl acetate and butanol fractions of Lotus creticus. Teuclatriol (4) was the most potent, with IC50 value of 4.89 0.061mM against HSV-1 and a selective index (SI) value of 4.8. Moreover, the pentamethoxyflavone (3) was the most active against HAV with IC50 value of 9.520.329 mM and SI of 3.4. The potent activity against Cox. B4 virus with IC50 of 13.054 0.348 mM and SI of 3.4 exhibited by b -sitosterol 3-O-b -D-glucoside(6, 12). Novelty: The results highlight that these compounds are candidates for the development of new lead antiviral compounds. This is the first report for proving antiviral activities of these compounds. Keywords: Antiviral activity; MTT; pentamethoxyflavone; teuclatriol

Antimicrobial Activity of Chitosan-Based Films Enriched with Green Tea Extracts on Murine Norovirus, Escherichia coli, and Listeria innocua.

Edible films can be designed to serve as carriers of antimicrobial agents and be used to control pathogenic foodborne viruses and bacteria. This research tested this concept by dissolving green tea extract (GTE) in chitosan film-forming solutions (FFS) and using it to prepare dried chitosan edible films. As a control, the GTE was also dissolved in deionized water (DW). The FFS and the dried chitosan films with the GTE and the DW without chitosan were all evaluated against murine norovirus (MNV-1), Escherichia coli K12, and Listeria innocua. Both the FFS and the DW with GTE were incubated with ~107 PFU/ml of the virus suspensions for 3 h. The chitosan films with GTE were incubated for 4 and 24 h at 23 ± 1°C. The results showed that the DW containing 1, 1.5, and 2.5% aqueous GTE, significantly (p < 0.05) reduced MNV-1 plaques by 1.7, 2.5, and 3.3 logs after 3 h exposure, respectively. Similarly, FFS containing 2.5 and 5.0% GTE reduced MNV-1 counts by 2.5 and 4.0 logs, respectively, after 3 h exposure. The dried chitosan films with 5, 10, and 15% GTE were also effective against MNV-1 infectivity. After 24 h incubation, the 5 and 10% chitosan GTE films produced significant (p < 0.05) titer reductions of 1.6 and 4.5 logs, respectively. Chitosan films containing 15% GTE reduced MNV-1 plaques to undetectable levels in 24 h. All chitosan GTE films reduced E. coli K12 and L. innocua populations to undetectable levels in tryptic soy broth after 24 h exposure. The results of this study showed that edible films enriched with GTE have potential to reduce both foodborne viruses and bacteria.

Determination of the effects of Enrofloxacin, Linco-Spectin and Florphenicol antibiotics on BHK-21 cell culture and FMD 146S Virus particles-infective titers

Abstract: Enrofloxacin, linko-spectin and florphenicol antibiotics were intended to be used in the BHK-21 An30 cells and the foot and mouth disease virus (FMDV) culture during the vaccine production process. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and cell cultures assays were performed in the BHK-21 An30 cells treated with the antibiotics that took place in this study to determine the concentration that inhibits cell proliferation and adverse effects on cell morphology. Virus cultures were performed by inoculating of FMDV serotypes (A/TUR/11, O/TUR/07, Asia-1/TUR/15) to the treated cell cultures with the antibiotics. 146S and infective tites of the obtained virus suspensions were determined. The non-toxic upper limit was determined to be 15μg /ml for enrofloxacin and 300μg/ml for linco-spectin in terms of cell morphology and cell numbers versus positive control (gentamycin, penicillin-streptomycin) and negative control (antibiotic-free medium) as a result of MTT and cell culture tests on BHK cells. It was observed that Florfenicol also inhibited cell proliferation and induced cell degeneration, even at a concentration of 5μg/ml. The mean 146S values of FMD virus cultures containing enrofloxacin and linco-spectin were determined to be 0.49, 0.46, ; 0.53, 0.47 and 0.30, 0.28µg/ml for serotype A/TUR/11, O/TUR/07 and Asia-1/TUR/15 respectively. The mean values of the infective titres were 107,04, 107,25, 106,04, 106,59 and 107,26, 107,6 pfu/ml. for serotype A/TUR/11, O/TUR/07, and Asia-1/TUR/15 respectively. In the control group used gentamycin, penicillin-streptomycin and antibiotic-free medium, the mean 146S FMD virus particles were obtained as 0.51, 0.50, 0.50; 0.52, 0.55, 0.52 and 0.36, 0.33, 0.30 µg/ml for A/TUR/11, O/TUR/07 and Asia-1/TUR/15 respectively. The mean values of the FMD infective virus titres were 107,35, 107,40, 107,11; 106,24, 106,46, 106,62, and 107,70, 107,75, 107,77 pfu/ml for A/TUR/11, O/TUR/07 and Asia-1/TUR/15 respectively. As a result, the infective FMDV titer and 146S results obtained in the control group (gentamicin, penicillin-streptomycin) and FMD virus cultures using Enrofloxacin (15µg / ml) and linco-spectin (300µg / ml) were very close to each other. According to these data, it was concluded that enrofloxacin and linco-spectin can be used up to the upper limit in the BHK-21 An30 cell and FMD virus cultures. However, florfenicol should not be used in cell and virus cultures.

RAAV8 and rAAV9-Mediated Long-Term Muscle Transduction with Tacrolimus (FK506) in Non-Human Primates

To establish an efficient, safe immunosuppressive regimen of adeno-associated vector (AAV)-mediated gene therapy for Duchenne muscular dystrophy (DMD), we evaluated the effect of tacrolimus (FK506) on skeletal muscle transduction with AAV8 and AAV9 vectors expressing the LacZ and microdystrophin (M3) genes labeled by FLAG. We utilized 3- to 4-year-old Macaca fascicularis, screened for neutralizing antibodies against AAV. 3 days before AAV injection and throughout the experiment, 0.06 mg/kg tacrolimus was intravenously administered. A viral suspension of 1 × 1013 viral genomes/muscle was intramuscularly injected bilaterally at the tibialis anterior and biceps brachii muscles, which were biopsied at 8, 16, 24, and 42 weeks after injection. Without tacrolimus, AAV8- and AAV9-mediated LacZ expression disappeared 8 and 16 weeks after transduction, respectively. With tacrolimus, AAV8/9-mediated LacZ expression persisted for at least 42 weeks after injection. At 42 weeks after AAV8CMVLacZ and AAV9CMVLacZ injection, nearly 50% and 17% of muscle fibers were positive for β-galactosidase, respectively. AAV8/9-mediated M3-FLAG expression lasted for up to 42 weeks using tacrolimus. No significant generalized toxicity was observed in any monkey. These results indicate that tacrolimus administration regulated the immune response to transgenes and truncated microdystrophin in normal primates and may enhance the benefits of AAV-mediated gene therapy for DMD.

Human adenovirus 36 improves insulin sensitivity and lipid profiles and increases inflammatory markers in Wistar rats

Human adenovirus 36 (Ad-36) causes obesity with increased adiposity, in contrast, Ad-36 infection reduces glucose and lipid metabolism; the results, however, are not consistent. In the current study, the effects...

Inactivated rabies vaccines: Standardization of an in vitroassay for residual viable virus detection.

Human rabies, a neglected viral zoonosis, is preventable through domestic animals vaccination and post-exposure prophylaxis using inactivated rabies vaccines. During vaccine production, several mandatory in vivo quality control trials, such as potency, live virus, and safety, are responsible for the use of large numbers of laboratory animals. Over the years, global organizations encouraged the development of alternative methods to reduce, replace and refine the use of animals in the pharmaceutical industry. In this study we standardized an in vitro assay for determination of residual live virus combining viral isolation techniques with direct immunofluorescence detection and viral quantification by a molecular method. Standardization of viral recovery steps and quantification by RT-qPCR were performed and the combined method was shown to be 3 fold more sensitive than the in vivo assay. It was possible to identify viral suspensions cultures, which still had residual viable rabies virus particles, evidencing the importance to implement this method in quality control schemes of rabies vaccine production. In addition, this developed assay is more practical, inexpensive and less time consuming, producing results in just 4 days, which may allow greater agility in the internal quality control of the vaccine. The in vitro method may reduce 2/3rd of laboratory animals numbers used for this purpose, since it can be applied in the intermediate quality control of inactivated rabies vaccine production.

Progress in the use of an electro-optical sensor for virus detection

This paper describes, for the first time, the real-time virus detection with an electro-optical (EO) sensor, using transmissible gastroenteritis virus (TGEV) as an example. The possibility of detecting an intact but inactivated TGEV by using specific antibodies is shown. The antibody-aided detection of the TGEV is possible in the presence of extraneous virus particles. The changes in the frequency dependence of the EO signal of virus suspensions before and after the addition of specific antibodies is an optimally informative parameter. The limit for reliable virus detection is 104 virus particles ml−1, and the time of analysis is 10–15 min. The EO sensor is promising for the recording of virus-antibody interactions and for rapid virus detection.

The efficacy of vacuum-ultraviolet light disinfection of some common environmental pathogens

BackgroundThis study is to elucidate the disinfection effect of ozone producing low-pressure Hg vapor lamps against human pathogens. Ozone producing low-pressure Hg vapor lamps emit mainly 254 nm ultraviolet light C (UVC) with about 10% power of Vacuum-ultraviolet (VUV) light at 185 nm. The combination of UVC and VUV can inactivate airborne pathogens by disrupting the genetic materials or generation of reactive oxygen species, respectively. In this study, inactivation of common bacteria including Escherichia coli ATCC25922 (E. coli), Extended Spectrum Beta-Lactamase-producing E. coli (ESBL), Methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis (MTB), and that of influenza A viruses H1N1 and H3N2 under the radiation from ozone producing low-pressure Hg vapor lamps was examined. Log reduction values at different treatment durations were determined.MethodsIn vitro tests were carried out. Various bacterium and virus suspensions were added onto nitrocellulose filter papers and subjected to the illumination from ozone producing low-pressure Hg vapor lamps. The extents of pathogen inactivation at different illumination times were investigated by conducting a series of experiments with increasing duration of illumination. log10 reduction in CFU/ml and reduction at log10(TCID50) were respectively measured for bacteria and viruses. The disinfection effectiveness of this type of lamps against the pathogens under the environment with a moderate barrier to light was therefore evaluated.ResultsOzone producing low-pressure Hg vapor lamp successfully inactivated these human pathogens. Nevertheless, among these pathogens, disinfection of MTB required more intense treatment. In the best tested situation, 3-log10 inactivation of pathogens can be achieved with ≤10 min of VUV treatment except MTB which needed about 20 min. This demonstrated the high resistance against UV disinfection of MTB.ConclusionsFollowing the criteria that valid germicidal results can be reflected with 3-log10 inactivation for bacteria, 4-log10 inactivation for viruses and 5-log10 inactivation for MTB, most of the bacteria required ≤10 min of VUV treatment, 20 min for the influenza viruses while MTB needed about 30 min VUV treatment. This indicated that VUV light is an effective approach against different environmental microorganisms.