scholarly journals Establishment of an antiviral assay system and identification of severe fever with thrombocytopenia syndrome virus inhibitors

2017 ◽  
Vol 25 (3) ◽  
pp. 83-89 ◽  
Author(s):  
Masanori Baba ◽  
Masaaki Toyama ◽  
Norikazu Sakakibara ◽  
Mika Okamoto ◽  
Naomichi Arima ◽  
...  
Keyword(s):  
Antiviral Agents ◽  
Vero Cells ◽  
Assay System ◽  
Viral Rna ◽  
University Hospital ◽  
Selective Inhibitors ◽  
Reverse Transcription Pcr ◽  
The Family ◽  
Severe Fever

Aims Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease. SFTS is epidemic in Asia, and its fatality rate is around 30% in Japan. The causative virus severe fever with thrombocytopenia syndrome virus (SFTSV) is a phlebovirus of the family Phenuiviridae (the order Bunyavirales). Although effective treatments are required, there are no antiviral agents currently approved for clinical use. Ribavirin and favipiravir were examined for their anti-SFTSV activity and found to be selective inhibitors of SFTSV replication in vitro. However, their activity was not sufficient. Therefore, it is mandatory to identify novel compounds active against SFTSV. To this end, we have established a safe and rapid assay system for screening selective inhibitors of SFTSV. Methods The virus was isolated from SFTS patients treated in Kagoshima University Hospital. Vero cells were infected with SFTSV and incubated in the presence of various concentrations of test compounds. After three days, the cells were examined for their intracellular viral RNA levels by real-time reverse transcription-PCR without extracting viral RNA. The cytotoxicity of test compounds was determined by a tetrazolium dye method. Results Among the test compounds, the antimalarial agent amodiaquine was identified as a selective inhibitor of SFTSV replication. Its 50% effective concentration (EC50) and cytotoxic concentration (CC50) were 19.1 ± 5.1 and >100 µM, respectively. The EC50 value of amodiaquine was comparable to those of ribavirin and favipiravir. Conclusion Amodiaquine is considered to be a promising lead of novel anti-SFTSV agents, and evaluating the anti-SFTSV activity of its derivatives is in progress.

scholarly journals Infection of SCID mice with Montana Myotis leukoencephalitis virus as a model for flavivirus encephalitis

2002 ◽  
Vol 83 (8) ◽  
pp. 1887-1896 ◽  
Author(s):  
Nathalie Charlier ◽  
Pieter Leyssen ◽  
Jan Paeshuyse ◽  
Christian Drosten ◽  
Herbert Schmitz ◽  
...  
Keyword(s):  
Yellow Fever Virus ◽  
Antiviral Agents ◽  
Antiviral Drug ◽  
Vero Cells ◽  
Scid Mice ◽  
Fever Virus ◽  
Viral Rna ◽  
Interferon Α ◽  
Immunocompetent Mice ◽  
Antiviral Targets

We have established a convenient animal model for flavivirus encephalitis using Montana Myotis leukoencephalitis virus (MMLV), a bat flavivirus. This virus has the same genomic organization, and contains the same conserved motifs in genes that encode potential antiviral targets, as flaviviruses that cause disease in man (N. Charlier et al., accompanying paper), and has a similar particle size (approximately 40 nm). MMLV replicates well in Vero cells and appears to be equally as sensitive as yellow fever virus and dengue fever virus to a selection of experimental antiviral agents. Cells infected with MMLV show dilation of the endoplasmic reticulum, a characteristic of flavivirus infection. Intraperitoneal, intranasal or direct intracerebral inoculation of SCID mice with MMLV resulted in encephalitis ultimately leading to death, whereas immunocompetent mice were refractory to either intranasal or intraperitoneal infection with MMLV. Viral RNA and/or antigens were detected in the brain and serum of MMLV-infected SCID mice, but not in any other organ examined: MMLV was detected in the olfactory lobes, the cerebral cortex, the limbic structures, the midbrain, cerebellum and medulla oblongata. Infection was confined to neurons. Treatment with the interferon-α/β inducer poly(I)·poly(C) protected SCID mice against MMLV-induced morbidity and mortality, and this protection correlated with a reduction in infectious virus titre and viral RNA load. This validates the MMLV model for use in antiviral drug studies. The MMLV SCID model may, therefore, be attractive for the study of chemoprophylactic or chemotherapeutic strategies against flavivirus infections causing encephalitis.

scholarly journals Novel Antiviral Agent DTriP-22 Targets RNA-Dependent RNA Polymerase of Enterovirus 71

2009 ◽  
Vol 53 (7) ◽  
pp. 2740-2747 ◽  
Author(s):  
Tzu-Chun Chen ◽  
Hwan-You Chang ◽  
Pei-Fen Lin ◽  
Jyh-Haur Chern ◽  
John Tsu-An Hsu ◽  
...  
Keyword(s):  
Potent Inhibitor ◽  
Antiviral Agents ◽  
Enterovirus 71 ◽  
Antiviral Agent ◽  
Viral Rna ◽  
Drug Resistant ◽  
Rna Dependent Rna Polymerase ◽  
Rna Accumulation ◽  
Elongation Activity

ABSTRACT Enterovirus 71 (EV71) has emerged as an important virulent neurotropic enterovirus in young children. DTriP-22 (4{4-[(2-bromo-phenyl)-(3-methyl-thiophen-2-yl)-methyl]-piperazin-1-yl}-1-pheny-1H-pyrazolo[3,4-d]pyrimidine) was found to be a novel and potent inhibitor of EV71. The molecular target of this compound was identified by analyzing DTriP-22-resistant viruses. A substitution of lysine for Arg163 in EV71 3D polymerase rendered the virus drug resistant. DTriP-22 exhibited the ability to inhibit viral replication by reducing viral RNA accumulation. The compound suppressed the accumulated levels of both positive- and negative-stranded viral RNA during virus infection. An in vitro polymerase assay indicated that DTriP-22 inhibited the poly(U) elongation activity, but not the VPg uridylylation activity, of EV71 polymerase. These findings demonstrate that the nonnucleoside analogue DTriP-22 acts as a novel inhibitor of EV71 polymerase. DTriP-22 also exhibited a broad spectrum of antiviral activity against other picornaviruses, which highlights its potential in the development of antiviral agents.

scholarly journals In vitro Assessment of Antiviral Effect of Natural Compounds on Porcine Epidemic Diarrhea Coronavirus

2021 ◽  
Vol 8 ◽  
Author(s):  
Manuel Gómez-García ◽  
Héctor Puente ◽  
Héctor Argüello ◽  
Óscar Mencía-Ares ◽  
Pedro Rubio ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Formic Acid ◽  
Antiviral Agents ◽  
Antiviral Effect ◽  
Vero Cells ◽  
Optical Microscope ◽  
Dependent Manner ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Organic acid and essential oils (EOs), well-known antimicrobials, could also possess antiviral activity, a characteristic which has not been completely addressed up to now. In this study, the effect of two organic acids (formic acid and sodium salt of coconut fatty acid distillates) and two single EO compounds (thymol and cinnamaldehye) was evaluated against porcine epidemic diarrhea virus (PEDV). The concentration used for each compound was established by cytotoxicity assays in Vero cells. The antiviral activity was then evaluated at three multiplicities of infection (MOIs) through visual cytopathic effect (CPE) evaluation and an alamarBlue assay as well as real-time reverse-transcription PCR (RT-qPCR) and viral titration of cell supernatants. Formic acid at at a dose of 1,200 ppm was the only compound which showed antiviral activity, with a weak reduction of CPE caused by PEDV. Through the alamarBlue fluorescence assay, we showed a significant anti-CPE effect of formic acid which could not be observed by using an inverted optical microscope. RT-qPCR and infectivity analysis also showed that formic acid significantly reduced viral RNA and viral titers in a PEDV MOI-dependent manner. Our results suggest that the antiviral activity of formic acid could be associated to its inhibitory effect on viral replication. Further studies are required to explore the anti-PEDV activity of formic acid under field conditions alone or together with other antiviral agents.

scholarly journals Hantavirus N Protein Exhibits Genus-Specific Recognition of the Viral RNA Panhandle

2006 ◽  
Vol 80 (22) ◽  
pp. 11283-11292 ◽  
Author(s):  
M. A. Mir ◽  
B. Brown ◽  
B. Hjelle ◽  
W. A. Duran ◽  
A. T. Panganiban
Keyword(s):  
Viral Rna ◽  
Genomic Rna ◽  
N Protein ◽  
High Affinity ◽  
The Andes ◽  
The Family ◽  
Viral Genomic Rna ◽  
Negative Sense

ABSTRACT A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into “panhandle” hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5′ and 3′ ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.

scholarly journals Development of a Novel Marker Gene Based Assay System for Detection and Evaluation of Antiviral Agents with Activity against Papillomaviruses

1991 ◽  
Vol 2 (6) ◽  
pp. 363-370 ◽  
Author(s):  
K. May ◽  
D. N. Planterose ◽  
M. J. Browne ◽  
R. M. Perkins
Keyword(s):  
Retinoic Acid ◽  
Human Papillomavirus ◽  
Marker Gene ◽  
Antiviral Agents ◽  
Assay System ◽  
Cell Phenotype ◽  
Bovine Papillomavirus ◽  
Tissue Plasminogen

A novel assay system has been developed in which expression of a human tissue-plasminogen activator (t-PA) gene, carried on a recombinant papillomavirus vector, is used as a marker for the presence of bovine papillomavirus type 1 (BPV-1) within transformed mouse C127 cells. This provides a relatively quick and simple means of identifying and evaluating agents with anti-papillomavirus activity. Using this system the antiviral activity and cytotoxicity of interferon and retinoic acid, have been investigated. After seven subcultures in the presence of 200 Units ml−1 mouse α and β interferon, t-PA expression was completely inhibited, with a concurrent alteration in cellular morphology, and restoration of contact inhibition. In accordance with the problems encountered with interferon therapy of human papillomavirus infections, these effects were dependent on the continued presence of interferon, its removal leading to a rapid return of t-PA expression, and reversion of cells to the transformed phenotype. In comparison, 2.0 μg ml−1 retinoic acid partially reduced t-PA expression (this effect was largely maintained even after removal of the inhibiting compound) but did not affect the transformed cell phenotype. These results are discussed in relation to other in vitro studies and also to the clinical treatment of human papillomavirus (HPV) disease.

scholarly journals In Vivo Expression of the Mannose-Resistant Fimbriae of Photorhabdus temperata K122 during Insect Infection

2004 ◽  
Vol 186 (3) ◽  
pp. 611-622 ◽  
Author(s):  
L. M. Meslet-Cladiere ◽  
A. Pimenta ◽  
E. Duchaud ◽  
I. B. Holland ◽  
M. A. Blight
Keyword(s):  
Pathogenic Bacteria ◽  
Galleria Mellonella ◽  
Liquid Cultures ◽  
Reverse Transcription Pcr ◽  
Greater Wax Moth ◽  
The Family ◽  
Complex Regulation ◽  
Photorhabdus Temperata

ABSTRACT Photorhabdus temperata K122 is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae. Surface fimbriae are important for the colonization of many pathogenic bacteria, and here we report the nucleotide sequence and analysis of the expression of a 12-kbp fragment encoding the mannose-resistant fimbriae of P. temperata (mrf). The mrf gene cluster contains 11 genes with an organization similar to that of the mrp locus from Proteus mirabilis. mrfI (encoding a putative recombinase) and mrfA (encoding pilin), the first gene in an apparent operon of nine other genes, are expressed from divergent promoters. The mrfI-mrfA intergenic region contains inverted repeats flanking the mrfA promoter. This region was shown to be capable of inversion, consistent with an ON/OFF regulation of the operon. In in vitro liquid cultures, both orientations were detected. Nevertheless, when we analyzed the expression of all of the genes in the mrf locus by semiquantitative reverse transcription-PCR during infection of Galleria mellonella (greater wax moth) larvae, expression of mrfA was not detected until 25 h postinfection, preceding the death of the larvae at 32 h. In contrast, mrfJ (a putative inhibitor of flagellar synthesis) was expressed throughout infection. Expression of mrfI was also detected only late in infection (25 to 30 h), indicating a possible increase in inversion frequency at this stage. In both in vitro liquid cultures and in vivo larval infections, the distal genes of the operon were expressed at substantially lower levels than mrfA. These results indicate the complex regulation of the mrf cluster during infection.

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 Rescue of Two Recombinant Canine Distemper Viruses That Separately Express Dabie Bandavirus Gn and Gc in Vitro

2021 ◽  
Author(s):  
Fuxiao Liu ◽  
Jiahui Lin ◽  
Qianqian Wang ◽  
Hu Shan
Keyword(s):  
Reverse Genetics ◽  
Open Reading Frames ◽  
Canine Distemper ◽  
Significant Difference ◽  
The Family ◽  
Potential Vaccine ◽  
Severe Fever ◽  
Reading Frames

Abstract Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonosis with a high mortality rate in humans. Additionally, dogs are frequently reported to be infected with this disease. There has been no commercially available vaccine for humans and animals as yet. The SFTS is caused by Dabie bandavirus (DBV), formerly known as SFTS virus. The DBV is now classified into the genus Bandavirus in the family Phenuiviridae. DBV Gn and Gc can induce specific immune responses in vivo. In this study, we used reverse genetics to construct two recombinant canine distemper viruses (rCDV), rCDV-Gn and -Gc, which could express Dabie bandavirus Gn and Gc in vitro, respectively. Two foreign sequences, Gn and Gc open reading frames, were genetically stable during twenty serial viral passages in cells. Growth curve of the rCDV-Gc basically coincided with that of a wild-type CDV, but showed a significant difference from that of the rCDV-Gn. The rCDV-Gn and -Gc were derived from a common parental CDV, the virulence-attenuating QN strain. Therefore, if proven to be efficient in resisting both canine distemper and SFTS in dogs, either or both recombinant CDVs would be potential vaccine candidates.

scholarly journals Antiviral activity of silymarin and baicalein against dengue virus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhao Xuan Low ◽  
Brian Ming OuYong ◽  
Pouya Hassandarvish ◽  
Chit Laa Poh ◽  
Babu Ramanathan
Keyword(s):  
Antiviral Activity ◽  
Dengue Virus ◽  
Viral Entry ◽  
Dengue Infection ◽  
Antiviral Agents ◽  
Antiviral Drug ◽  
Vero Cells ◽  
Viral Envelope ◽  
E Protein

AbstractDengue is an arthropod-borne viral disease that has become endemic and a global threat in many countries with no effective antiviral drug available currently. This study showed that flavonoids: silymarin and baicalein could inhibit the dengue virus in vitro and were well tolerated in Vero cells with a half-maximum cytotoxic concentration (CC50) of 749.70 µg/mL and 271.03 µg/mL, respectively. Silymarin and baicalein exerted virucidal effects against DENV-3, with a selective index (SI) of 10.87 and 21.34, respectively. Baicalein showed a better inhibition of intracellular DENV-3 progeny with a SI of 7.82 compared to silymarin. Baicalein effectively blocked DENV-3 attachment (95.59%) to the Vero cells, while silymarin prevented the viral entry (72.46%) into the cells, thus reducing viral infectivity. Both flavonoids showed promising antiviral activity against all four dengue serotypes. The in silico molecular docking showed that silymarin could bind to the viral envelope (E) protein with a binding affinity of − 8.5 kcal/mol and form hydrogen bonds with the amino acids GLN120, TRP229, ASN89, and THR223 of the E protein. Overall, this study showed that silymarin and baicalein exhibited potential anti-DENV activity and could serve as promising antiviral agents for further development against dengue infection.

scholarly journals Potential antiviral agents of Rosmarinus officinalis extract against herpes viruses 1 and 2

2020 ◽  
Vol 40 (6) ◽  
Author(s):  
Wafa A. AL-Megrin ◽  
Norah A. AlSadhan ◽  
Dina M. Metwally ◽  
Razan A. Al-Talhi ◽  
Manal F. El-Khadragy ◽  
...  
Keyword(s):  
Superoxide Anion ◽  
Viral Infections ◽  
Antiviral Agents ◽  
Vero Cells ◽  
Rosmarinus Officinalis ◽  
Rosemary Extract ◽  
Scavenging Activity ◽  
Herpes Simplex Viruses ◽  
Hsv 1

Abstract Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) belong to the herpesviridae family and cause neurological disorders by infecting the nervous system. The present study aimed to investigate the effects of Rosmarinus officinalis L. (rosemary) extract against HSV-1 and HSV-2 in vitro. The antioxidant activity of this extract was investigated by superoxide anion and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical assays. Rosemary extract was evaluated by an HSV-1 antiviral assay, in which viral replication in Vero cells was determined and quantified using a cytopathic effect assay. The present study showed that rosemary extract at 30 µg/ml caused 55% inhibition of HSV-1 plaques, whereas 40 µg/ml rosemary extract caused 65% inhibition of HSV-2 plaques. The extracts completely inhibited HSV-1 and HSV-2 plaque formation at 50 µg/ml. Scavenging activity of the superoxide anion radical was observed at 65.74 mg/ml, whereas 50% scavenging activity of the DPPH radical was observed at 67.34 mg/ml. These data suggest that rosemary extract may be suitable as a topical prophylactic or therapeutic agent for herpes viral infections. However, further research is required to elucidate the plant’s active constituents, which may be useful in drug development.

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