Genomic and In Vitro Phenotypic Comparisons of Epidemic and Non-Epidemic Getah Virus Strains

2021 ◽  
Author(s):  
Noor-Adila Mohamed-Romai-Noor ◽  
Sing-Sin Sam ◽  
Boon-Teong Teoh ◽  
Zur-Raiha Hamim ◽  
Sazaly AbuBakar
Keyword(s):  
Virus Titer ◽  
Vero Cells ◽  
Phylogenetic Groups ◽  
Cytopathic Effects ◽  
Group I ◽  
Getah Virus ◽  
High Virus ◽  
Disease Epidemics ◽  
Kinetics Of

Abstract Getah virus is an emerging mosquito-borne animal pathogen. Four phylogenetic groups of GETV, Group I (GI), GII, GIII and GIV, were identified. However, only the GETV GIII was associated with disease epidemics suggesting possible virulence difference in this virus group. Here, we compared the genetic and in vitro phenotypic characteristics between the epidemic and non-epidemic GETV. Our complete coding genome sequence analyses revealed several amino acid substitutions unique to the GETV GIII and GIV groups, which were found mainly in the hypervariable domain of nsP3 and E2 proteins. Replication kinetics of the epidemic (GIII MI-110 and GIII 14-I-605) and non-epidemic GETV strains (prototype GI MM2021 and GIV B254) were compared in mammalian Vero cells and mosquito C6/36 and U4.4 cells. In all cells used, both epidemic GETV GIII MI-110 and GIII 14-I-605 strains showed replication rates and mean maximum titers at least 2.7-fold and 2.3-fold higher than those of GIV B254, respectively (Bonferroni posttest, P<0.01). In Vero cells, the epidemic GETV strains caused more pronounced cytopathic effects in comparison to the GIV B254. Our findings suggest that higher virus replication competency to produce high virus titer during infection may be the main determinant of virulence and epidemic potential of GETV.

scholarly journals Evidences for lipid involvement in SARS-CoV-2 cytopathogenesis

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Roberta Nardacci ◽  
Francesca Colavita ◽  
Concetta Castilletti ◽  
Daniele Lapa ◽  
Giulia Matusali ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Vero Cells ◽  
Blood Cholesterol ◽  
Ultrastructural Changes ◽  
Type Ii ◽  
Cytopathic Effects ◽  
Type Ii Pneumocytes ◽  
Infected Cells ◽  
Accumulation Of Lipids

AbstractThe pathogenesis of SARS-CoV-2 remains to be completely understood, and detailed SARS-CoV-2 cellular cytopathic effects requires definition. We performed a comparative ultrastructural study of SARS-CoV-1 and SARS-CoV-2 infection in Vero E6 cells and in lungs from deceased COVID-19 patients. SARS-CoV-2 induces rapid death associated with profound ultrastructural changes in Vero cells. Type II pneumocytes in lung tissue showed prominent altered features with numerous vacuoles and swollen mitochondria with presence of abundant lipid droplets. The accumulation of lipids was the most striking finding we observed in SARS-CoV-2 infected cells, both in vitro and in the lungs of patients, suggesting that lipids can be involved in SARS-CoV-2 pathogenesis. Considering that in most cases, COVID-19 patients show alteration of blood cholesterol and lipoprotein homeostasis, our findings highlight a peculiar important topic that can suggest new approaches for pharmacological treatment to contrast the pathogenicity of SARS-CoV-2.

scholarly journals Dengue Virus Non-structural (NS) 1 Gene as A Molecular Marker for Early Detection of in vitro Dengue Virus Infection

2021 ◽  
Vol 50 (10) ◽  
pp. 3035-3043
Author(s):  
Nur Azizah A Rahman ◽  
Fadhilah Moh Djamil ◽  
Vinod RMT Balasubramaniam ◽  
Sharifah Syed Hassan ◽  
Wei Boon Yap
Keyword(s):  
Early Detection ◽  
Dengue Virus ◽  
Early Phase ◽  
Disease Onset ◽  
Vero Cells ◽  
Specific Primers ◽  
Cytopathic Effects ◽  
Ns1 Gene ◽  
Infected Cells

Serology-based dengue assays at times produce inaccurate results especially in the early phase of disease onset. A more precise diagnostic approach detecting dengue infections in the early phase enables better management of the disease. This helps reduce dengue-associated morbidity and mortality. Besides, an early diagnosis of dengue is also very beneficial in a dengue outbreak and in endemic regions. In this light, this study aimed to determine the potential of the dengue virus (DENV) non-structural 1 (NS1) gene as an early detection biomarker. The cytopathic effects (CPE) were monitored and the cell death of DENV serotype-2 (DENV2)-infected Vero cells was evaluated for fourteen consecutive days. Only Lemos and in-house NS1-specific primer pairs showed positive amplifications in the preliminary primer validation. Thus, both of the primer pairs were then used to amplify the NS1 gene from the infected cells. The NS1 gene was detected as early as day-2 post-infection using the in-house primers. There was no amplicon produced using the Lemos primers. This is speculated to be attributable to the relatively lower complementarity of the primer sequences with that of the template and low amount of viral mRNA in the DENV2-infected cells. Conclusively, the DENV NS1 gene is a potential early detection marker, however, the NS1-specific primers should be pre-validated to ensure a reliable dengue diagnosis.

scholarly journals Bisbenzylisoquinoline Alkaloids of Cissampelos Sympodialis With in Vitro Antiviral Activity Against Zika Virus

2021 ◽  
Vol 12 ◽  
Author(s):  
Poliena Gomes da Silva ◽  
Aventino H. Fonseca ◽  
Malu P. Ribeiro ◽  
Taizia D. Silva ◽  
Cristiane F. F. Grael ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Zika Virus ◽  
High Performance ◽  
Antiviral Agents ◽  
Virus Titer ◽  
Vero Cells ◽  
Positive Control ◽  
Bisbenzylisoquinoline Alkaloids ◽  
Bioassay Guided Fractionation

In search of new antiviral compounds against Zika virus we conducted a bioassay-guided fractionation of bisbenzyilisoquinoline alkaloids isolated from Cissampelos sympodialis (Menispermaceae), a medicinal plant species endemic to Brazil. Six subfractions were obtained from a tertiary alkaloidal fraction of the rhizomes (TAFrz) using preparative high-performance liquid chromatography. All the subfractions were tested against Zika virus-infected Vero cells as the cellular model to evaluate cytotoxicity and antiviral effective concentrations. The results showed that three of the six TAFrz subfractions tested were active. The most active ones were the subfraction 6 (that consisted of the alkaloids methylwarifteine and warifteine present as a mixture at a ratio of 8.8:1.2 respectively) and the subfraction 5, that was later identified as warifteine, the major tertiary alkaloid of this species. Warifteine was able to significantly reduce virus titer in Zika virus-infected Vero cells with an IC50 of 2.2 μg/ml and this effect was selective (selectivity index, SI = 68.3). Subfraction 6 had an IC50 = 3.5 μg/ml and was more cytotoxic than pure warifteine, with SI = 6.14. Fraction 5 and fraction 6 were more potent in decreasing the viral titer of Zika virus-infected Vero cells than 6-methylmercaptopurine riboside (IC50 = 24.5 μg/ml and SI = 11.9), a mercaptopurine riboside with ZIKV antiviral activity used as a positive control. Our data demonstrate that alkaloids of the bisbenzylisoquinoline type may be explored as new antiviral agents or as an useful pharmacophore for investigating ZIKV antiviral activity.

scholarly journals Evaluation of Residual Infectivity after SARS-CoV-2 Aerosol Transmission in a Controlled Laboratory Setting

2021 ◽  
Vol 18 (21) ◽  
pp. 11172
Author(s):  
Luisa Zupin ◽  
Sabina Licen ◽  
Margherita Milani ◽  
Libera Clemente ◽  
Lorenzo Martello ◽  
...  
Keyword(s):  
Viral Load ◽  
Virus Titer ◽  
Clinical Samples ◽  
Laboratory Model ◽  
Post Inoculation ◽  
Experimental Laboratory ◽  
High Virus ◽  
Respiratory Droplets

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is mainly transmitted through respiratory droplets, aerosols, or direct contact with fomites from an infected subject. It has been reported that SARS-CoV-2 is stable and viable in aerosol up to 16 h in controlled laboratory conditions. However, the aerosolization conditions varied a lot between the studies. In this work, an experimental laboratory model of SARS-CoV-2 aerosolization was established, employing an impinger nebulizer, a cylindrical chamber for aerosol travel, and a SKC biosampler for the collection of particles. The efficiency of the system was assessed based on the molecular determination of the viral load in the nebulizer after the aerosolization and in the aerosol collected at the end of the travel. Moreover, the residual infectivity was tested in vitro on the Vero E6 cell line, through the observation of the cytopathic effect (CPE), and the quantification of the viral load in the supernatants at 7 days post inoculation (dpi). A high RNA viral load was found in the SKC biosampler after aerosolization, indicating that it was possible to transport a high virus titer through the 30-cm chamber with all the dilutions (initial 105, 104, 103 plaque forming unit—PFU/mL). At the 7 dpi, an increment of the RNA viral load was determined for the dilutions 105 and 104 PFU/mL tested, while only the initial 105 PFU/mL resulted in visible CPE. Our findings allowed us to achieve the resilience of SARS-CoV-2 in aerosol form, at a concentration comparable to those reported for clinical samples. This mode of transmission should be considered for the mitigation and preventive measures to counteract SARS-CoV-2 spreading.

scholarly journals Synergistic anti-influenza virus A (H1N1) activities of PM-523 (polyoxometalate) and ribavirin in vitro and in vivo.

1997 ◽  
Vol 41 (7) ◽  
pp. 1423-1427 ◽  
Author(s):  
S Shigeta ◽  
S Mori ◽  
J Watanabe ◽  
S Soeda ◽  
K Takahashi ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Mdck Cells ◽  
Lethal Dose ◽  
Virus Titer ◽  
Cytopathic Effects ◽  
Aerosol Exposure ◽  
Wide Range ◽  
Darby Canine Kidney

A Kegin-type polyoxometalate, PM-523, in combination with ribavirin, was tested for its therapeutic effectiveness against influenza virus (FluV) A (H1N1) infection in tissue culture and in mice. PM-523 [(PriNH3)6H [PTi2W10O38(O2)2] x H2O, where Pri is isopropanol] and ribavirin individually inhibited FluV A-induced cytopathic effects in Madin-Darby canine kidney (MDCK) cells at median effective concentrations (EC50s) of 30 and 34 microM, respectively, and at 70% effective concentrations (EC70s) of 48 and 72 microM, respectively. On the other hand, a combination of PM-523 and ribavirin at a ratio of 1:16 exhibited lower EC50s and EC70s than each compound used singly, and combination indices were less than 1. A wide range of combinations of PM-523 and ribavirin at ratios of from 1:128 to 1:1 exhibited additive or synergistic anti-FluV effects in MDCK cells. When these compounds were tested for their anti-FluV A activities in vivo by aerosol exposure of mice which had been infected with a lethal dose of FluV A by an intranasal route, a 1:16 combination of PM-523 and ribavirin was found to have a significantly better therapeutic effect than a single dose of either compound used singly with respect to both the survival rate of the mice and the virus titer in the lungs of the infected mice. PM-523 was effective for the treatment of experimental FluV infection, and in combination with ribavirin, PM-523 exhibited enhanced anti-FluV effects in vitro and in vivo compared with the effect of PM-523 alone.

scholarly journals Deep Transfer Learning Approach for Automatic Recognition of Drug Toxicity and Inhibition of SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 610
Author(s):  
Julia Werner ◽  
Raphael M. Kronberg ◽  
Pawel Stachura ◽  
Philipp N. Ostermann ◽  
Lisa Müller ◽  
...  
Keyword(s):  
Transfer Learning ◽  
Drug Toxicity ◽  
Experimental Approach ◽  
Virus Titer ◽  
Inhibitory Effects ◽  
Simple Method ◽  
Cytopathic Effects ◽  
Toxic Concentrations ◽  
Fine Tune

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 and is responsible for the ongoing pandemic. Screening of potential antiviral drugs against SARS-CoV-2 depend on in vitro experiments, which are based on the quantification of the virus titer. Here, we used virus-induced cytopathic effects (CPE) in brightfield microscopy of SARS-CoV-2-infected monolayers to quantify the virus titer. Images were classified using deep transfer learning (DTL) that fine-tune the last layers of a pre-trained Resnet18 (ImageNet). To exclude toxic concentrations of potential drugs, the network was expanded to include a toxic score (TOX) that detected cell death (CPETOXnet). With this analytic tool, the inhibitory effects of chloroquine, hydroxychloroquine, remdesivir, and emetine were validated. Taken together we developed a simple method and provided open access implementation to quantify SARS-CoV-2 titers and drug toxicity in experimental settings, which may be adaptable to assays with other viruses. The quantification of virus titers from brightfield images could accelerate the experimental approach for antiviral testing.

scholarly journals An in vitro study regarding an animal model for the attenuation of SARS-CoV-2: Are cats possible candidates?

2021 ◽  
Author(s):  
Tuba Cigdem Oguzoglu ◽  
Alireza Hanifehnezhad ◽  
Aykut Özkul
Keyword(s):  
Animal Model ◽  
Virus Titer ◽  
In Vitro Study ◽  
Vero Cells ◽  
Vitro Study ◽  
Source Of Infection ◽  
Host Status ◽  
Strain Virus ◽  
Pcr Techniques

Abstract Although the origin of the SARS-CoV-2 pandemic is not definitively known, this virus is seen that adapted to human. It is known yet, several animal species are naturally susceptible for SARS-CoV-2, including pets. In this in vitro study, it was investigated whether cats could have a host status in this infection. It was also following questioned, whether cats could be an animal model for vaccine attenuation. Felis catus whole fetus 4 (FCWF-4) and Vero cells were used in this study for propagation of SARS-CoV-2-Ank1 strain. Virus replication was controlled by immunohistochemistry and real time pcr techniques. While an increase in virus titer was detected in the serious passages made in Vero cells, no increase was observed in the FCWF-4 cell. The results were confirmed by immunohistochemistry. Due to the fact that SARS CoV 2 is a zoonotic potential, its presence in domestic animals frequently investigated in current studies. Cats can get this virus from infected owners with whom they share the same habitat. The results of this study also showed that cats are not candidates to be a natural source of infection for SARS-CoV-2.

scholarly journals Local antiviral activity of the drug «Thymogen®», nasal dosed spray, against SARS-CoV-2 coronavirus in vitro

2021 ◽  
Vol 66 (5-6) ◽  
pp. 11-16
Author(s):  
I. A. Leneva ◽  
V. S. Smirnov ◽  
T. A. Kudryavtseva ◽  
E. B. Fayzuloev ◽  
A. V. Gracheva ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Virus Titer ◽  
Vero Cells ◽  
Urgent Problem ◽  
Route Of Transmission ◽  
Series Of Experiments ◽  
Coronavirus Infection ◽  
Poor Ventilation ◽  
Restrictive Measures

On account of the COVID-19 pandemic, the global pharmaceutical industry has achieved impressive results in the development and introduction of various types of vaccines causing the formation of acquired immunity against the SARS-CoV-2 coronavirus into clinical practice. However, none of them currently show the declared one hundred percent guarantee of protection. In the case of the COVID-19 disease, patients with concomitant pathologies are the most vulnerable to the occurrence of severe complications. The aerosol route of transmission of SARS-CoV-2 contributes to the emergence of outbreaks of the new coronavirus infection in crowded places and closed rooms with poor ventilation. In this regard, an urgent problem is the search for drugs with local antiviral activity, which, together with restrictive measures and mask wearing policy, can potentially reduce the likelihood of contracting coronavirus. In this experimental in vitro study on Vero CCL81 cell culture (ATCC), the local antiviral activity of the drug Thymogen® spray against the SARS-CoV-2 virus was studied in comparison with the antiseptic Miramistin® solution. As a result of the experiment, no toxic effects on Vero cells were detected in the drugs in the studied concentrations. In a series of experiments, Thymogen® spray showed local antiviral activity against SARS-CoV-2 when the virus titer was 5,2 lg TCID50. Therefore, the drug Thymogen® dosed nasal spray has high potential as a topical drug for prevention and treatment of COVID-19 disease, which requires additional confirmation in relevant clinical studies.

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