Characterization of #Usutu virus NS5 protein. #Polymerase activity, protein-protein interaction and cellular localization (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Characterization of Usutu virus NS5 protein. Polymerase activity, protein-protein interaction and cellular localization.

L. Albentosa-González, P. Clemente-Casares, R. Sabariegos, A. Mas

DOI: 10.1128/AAC.01573-19

 

ABSTRACT

Usutu virus (USUV) has become increasingly relevant in recent years with large outbreaks that sporadically have affected humans, being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive single-stranded RNA genome which is replicated by the activity of the non-structural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyl-transferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we have cloned and expressed the entire NS5 and the RdRpD in a heterologous system and have used purified preparations for protein characterizations. We have determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA, and that the RdRp activity is not inhibited by Sofosbuvir. To further examine the characteristics of USUV polymerase in a more biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed its subcellular location. NS5 is predominantly found in the cytoplasm, a significant proportion is directed to the nucleus and this translocation involves nuclear location signals (NLS) located, at least, between the MTase and RdRpD domains.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Flavivirus; Usutu Virus; Sofosbuvir.

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#Antinflammatory compound shows #therapeutic safety and efficacy against #flavivirus infection (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Anti-inflammatory compound shows therapeutic safety and efficacy against flavivirus infection

Fu-Kai Chuang, Shih-Ming Huang, Ching-Len Liao, An-Rong Lee, Shu-Pei Lien, Yu-Lung Chiu, Tsung-Hsien Chang, Pei-Ling Tsai, Ren-Jye Lin, Chih-Chin Shih, Yi-Jing Tsai, Gu-Jiun Lin, Li-Chen Yen

DOI: 10.1128/AAC.00941-19

 

ABSTRACT

Flaviviruses comprise several medically important viruses, including Japanese encephalitis virus, West Nile virus, dengue virus (DENV), yellow fever virus and Zika virus (ZIKV). A large outbreak of DENV and ZIKV occurred recently, leading to many cases of illness and death. However, despite decades of efforts, we have no clinically specific therapeutic drugs against DENV and ZIKV. Previous studies showed that inflammatory responses play a critical role in dengue and Zika pathogenesis. Thus, in this study, we examined a series of novel anti-inflammatory compounds and found that treatment with compound 2d could dose-dependently reduce viral protein expression and viral progeny production in HEK-293 and Raw264.7 cells with four serotypes of DENV and ZIKV infection. As well, considering medication safety, compound 2d could not suppress cyclooxygenase-1 (COX-1) enzymatic activities and thus could prevent bleeding side effect. Moreover, compound 2d significantly inhibited COX-2 enzymatic activities and prostaglandin E2 levels, associated with viral replication, as compared with a selective COX-2 inhibitor, celecoxib. Furthermore, administering 5 mg/kg compound 2d to DENV-2–infected AG129 mice prolonged survival and reduced viremia and serum cytokine levels. Overall, compound 2d showed therapeutic safety and efficacy in vitro and in vivo and could be further developed as a potential therapeutic agent for flavivirus infection.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Flavivirus; Antivirals.

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#NS5 from #Dengue virus serotype 2 can adopt a conformation analogous to its #ZIKV and #JEV homologues (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

NS5 from Dengue virus serotype 2 can adopt a conformation analogous to its ZIKV and JEV homologues.

Abbas El Sahili, Tingjin Sherryl Soh, Jonas Schiltz, Aïcha Gharbi-Ayachi, Cheah Chen Seh, Pei-Yong Shi, Siew Pheng Lim, Julien Lescar

DOI: 10.1128/JVI.01294-19

 

ABSTRACT

Flavivirus non-structural protein 5 (NS5) contains a N-terminal methyltransferase domain (MTase) and a C-terminal polymerase domain (RdRp) fused through a nine-amino acid linker. While the individual NS5 domains are structurally conserved, in the full-length protein, their relative orientations fall into two classes: NS5 from JEV and ZIKV adopt one conformation, while NS5 from DENV3 another. Here, we report a crystallographic structure of NS5 from DENV2 in a conformation similar to the extended one seen in JEV and ZIKV NS5 crystal structures, albeit looser. Substituting DENV2 NS5 linker with DENV1, -3, -4, JEV and ZIKV NS5 linkers had modest or minimal effects on in vitro DENV2 MTase and RdRp activities. Heterotypic DENV NS5 linkers attenuated DENV2 replicon growth in cells, whilst JEV and ZIKV NS5 linkers abolished replication. Thus, JEV and ZIKV linkers likely hindered essential DENV2 NS5 interactions with other viral or host proteins within the virus replicative complex. Overall, this work sheds light on the dynamics of the multifunctional flavivirus NS5 protein and its interdomain linker. Targeting the NS5 linker is a possible strategy for producing attenuated flavivirus strains for vaccine design.

 

IMPORTANCE

Flaviviruses include important human pathogens such as dengue or Zika virus. NS5 is a non-structural protein essential for flavivirus RNA replication, with dual MTase and RdRp enzyme activity and thus constitutes a major drug target. Insights into NS5 structure, dynamics and evolution should inform the development of antiviral inhibitors and vaccine design. We found that NS5 from DENV2 can adopt a conformation resembling NS5 from JEV and ZIKV. Substitution of DENV2 NS5 linker with JEV and ZIKV NS5 linkers abolished DENV2 replication in cells, without significantly impacting in vitro DENV2 NS5 enzymatic activities. We propose that heterotypic Flavivirus NS5 linkers impeded DENV2 NS5 protein-protein interactions that are essential for virus replication.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Flavivirus; Dengue fever; Zika Virus; Japanese Encephalitis virus.

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Infection of Western Gray #Kangaroos (Macropus fuliginosus) with #Australian #Arboviruses Associated with #Human #Infection (Vector Borne Zoo Dis., abstract)

[Source: Vector Borne and Zoonotic Diseases, full page: (LINK). Abstract, edited.]

Infection of Western Gray Kangaroos (Macropus fuliginosus) with Australian Arboviruses Associated with Human Infection

Narayan Gyawali, Andrew W. Taylor-Robinson, Richard S. Bradbury, Abbey Potter, and John G. Aaskov

Published Online: 26 Sep 2019

 

Abstract

More than 75 arboviruses (arthropod-borne viruses) have been identified in Australia. While Alfuy virus (ALFV), Barmah Forest virus (BFV), Edge Hill virus (EHV), Kokobera virus (KOKV), Murray Valley encephalitis virus (MVEV), Sindbis virus (SINV), Ross River virus (RRV), Stratford virus (STRV), and West Nile virus strain Kunjin (KUNV) have been associated with human infection, there remains a paucity of data regarding their respective transmission cycles and any potential nonhuman vertebrate hosts. It is likely that these viruses are maintained in zoonotic cycles involving native animals rather than solely by human-to-human transmission. A serosurvey (n = 100) was undertaken to determine the prevalence of neutralizing antibodies against a panel of Australian arboviruses in western gray kangaroos (Macropus fuliginosus) obtained from 11 locations in the midwest to southwest of Western Australia. Neutralizing antibodies against RRV were detected in 25%, against BFV in 14%, and antibodies to both viruses in 34% of serum samples. The prevalence of antibodies against these two viruses was the same in males and females, but higher in adult than in subadult kangaroos (p < 0.05). Twenty-one percent of samples had neutralizing antibodies against any one or more of the flaviviruses ALFV, EHV, KOKV, MVEV, and STRV. No neutralizing antibodies against SINV and KUNV were detected. If this sample of kangaroo sera was representative of the broader Australian population of macropods, it suggests that they are common hosts for RRV and BFV. The absence or low seroprevalence of antibodies against the remaining arboviruses suggests that they are not prevalent in the region or that kangaroos are not commonly infected with them. The detection of neutralizing antibodies to MVEV requires further investigation as this virus has not been identified previously so far south in Western Australia.

Keywords: Arbovirus; Flavivirus; Kangaroos; Wildlife; Human; Australia.

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#Vector #Competence: What Has #Zika Virus Taught Us? (Viruses, abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Viruses. 2019 Sep 17;11(9). pii: E867. doi: 10.3390/v11090867.

Vector Competence: What Has Zika Virus Taught Us?

Azar SR1,2,3, Weaver SC4,5,6.

Author information: 1 Department of Microbiology and Immunology, University of Texas Medical Branch, 300 University Blvd, Galveston, TX 77555, USA. srazar@utmb.edu. 2 Institute for Translational Sciences, University of Texas Medical Branch, 300 University Blvd, Galveston, TX 77555, USA. srazar@utmb.edu. 3 Institute for Human Infections and Immunity, University of Texas Medical Branch, 300 University Blvd, Galveston, TX 77555, USA. srazar@utmb.edu. 4 Department of Microbiology and Immunology, University of Texas Medical Branch, 300 University Blvd, Galveston, TX 77555, USA. sweaver@utmb.edu. 5 Institute for Translational Sciences, University of Texas Medical Branch, 300 University Blvd, Galveston, TX 77555, USA. sweaver@utmb.edu. 6 Institute for Human Infections and Immunity, University of Texas Medical Branch, 300 University Blvd, Galveston, TX 77555, USA. sweaver@utmb.edu.

 

Abstract

The unprecedented outbreak of Zika virus (ZIKV) infection in the Americas from 2015 to 2017 prompted the publication of a large body of vector competence data in a relatively short period of time. Although differences in vector competence as a result of disparities in mosquito populations and viral strains are to be expected, the limited competence of many populations of the urban mosquito vector, Aedes aegypti, from the Americas (when its susceptibility is viewed relative to other circulating/reemerging mosquito-borne viruses such as dengue (DENV), yellow fever (YFV), and chikungunya viruses (CHIKV)) has proven a paradox for the field. This has been further complicated by the lack of standardization in the methodologies utilized in laboratory vector competence experiments, precluding meta-analyses of this large data set. As the calls for the standardization of such studies continue to grow in number, it is critical to examine the elements of vector competence experimental design. Herein, we review the various techniques and considerations intrinsic to vector competence studies, with respect to contemporary findings for ZIKV, as well as historical findings for other arboviruses, and discuss potential avenues of standardization going forward.

KEYWORDS: Aedes aegypti; Flaviviruses; Zika virus; arbovirus; mosquitoes; vector competence

PMID: 31533267 DOI: 10.3390/v11090867

Keywords: Mosquitoes; Aedes aegypti; Flavivirus; Zika Virus.

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Insights into the #ZIKV NS1 #Virology from Different #Strains through a Fine Analysis of #Physicochemical Properties (ACS Omega, abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

ACS Omega. 2018 Nov 29;3(11):16212-16229. doi: 10.1021/acsomega.8b02081. eCollection 2018 Nov 30.

Insights into the ZIKV NS1 Virology from Different Strains through a Fine Analysis of Physicochemical Properties.

Poveda-Cuevas SA1,2,3, Etchebest C4,5,6,3, Barroso da Silva FL1,2,3,7.

Author information: 1 Programa Interunidades em Bioinformática, Universidade de São Paulo, São Paulo 05508-090, Brazil. 2 Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo 14040-903, Brazil. 3 University of São Paulo and Université Sorbonne Paris Cité Joint International Laboratory in Structural Bioinformatics. 4 Institut National de la Transfusion Sanguine, Paris 75015, France. 5 Biologie Intégrée du Globule Rouge, Equipe 2, Dynamique des Structures et des Interactions Moléculaires, Institut National de la Santé et de la Recherche Médicale, UMR_S 1134, Paris 75015, France. 6 Université Sorbonne Paris Cité and Université Paris Diderot, 75013 Paris, France. 7 Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27606, United States.

 

Abstract

The flavivirus genus has several organisms responsible for generating various diseases in humans. Recently, especially in tropical regions, Zika virus (ZIKV) has raised great health concerns due to the high number of cases affecting the area during the last years that has been accompanied by a rise in the cases of the Guillain-Barré syndrome and fetal and neonatal microcephaly. Diagnosis is still difficult since the clinical symptoms between ZIKV and other flaviviruses (e.g., dengue and yellow fever) are highly similar. The understanding of their common physicochemical properties that are pH-dependent and biomolecular interaction features and their differences sheds light on the relation strain-virulence and might suggest alternative strategies toward differential serological diagnostics and therapeutic intervention. Due to their immunogenicity, the primary focus of this study was on the ZIKV nonstructural proteins 1 (NS1). By means of computational studies and semiquantitative theoretical analyses, we calculated the main physicochemical properties of this protein from different strains that are directly responsible for the biomolecular interactions and, therefore, can be related to the differential infectivity of the strains. We also mapped the electrostatic differences at both the sequence and structural levels for the strains from Uganda to Brazil, which could suggest possible molecular mechanisms for the increase of the virulence of ZIKV in Brazil. Exploring the interfaces used by NS1 to self-associate in some different oligomeric states and interact with membranes and the antibody, we could map the strategy used by the ZIKV during its evolutionary process. This indicates possible molecular mechanisms that can be correlated with the different immunological responses. By comparing with the known antibody structure available for the West Nile virus, we demonstrated that this antibody would have difficulties to neutralize the NS1 from the Brazilian strain. The present study also opens up perspectives to computationally design high-specificity antibodies.

PMID: 31458257 PMCID: PMC6643396 DOI: 10.1021/acsomega.8b02081

Keywords: Flavivirus; Zika Virus; Brazil.

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Previous #dengue or #Zika virus #exposure can drive to #infection #enhancement or neutralisation of other #flaviviruses (Mem Inst Oswaldo Cruz, abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Mem Inst Oswaldo Cruz. 2019;114:e190098. doi: 10.1590/0074-02760190098. Epub 2019 Aug 12.

Previous dengue or Zika virus exposure can drive to infection enhancement or neutralisation of other flaviviruses.

Oliveira RA1,2, de Oliveira-Filho EF1,3, Fernandes AI4,5, Brito CA6, Marques ET1,7, Tenório MC1, Gil LH1.

Author information: 1 Fundação Oswaldo Cruz, Instituto Aggeu Magalhães, Departamento de Virologia, Recife, PE, Brasil. 2 Universidade Federal da Paraíba, Departamento de Fisiologia e Patologia, João Pessoa, PB, Brasil. 3 Charité-Universitätsmedizin Berlin, Berlin, Germany. 4 Universidade Federal da Paraíba, Hospital Universitário Lauro Wanderley, Serviço de Doenças Infecciosas e Parasitárias, João Pessoa, PB, Brasil. 5 Universidade Federal da Paraíba, Escola Técnica de Saúde, Grupo de Estudos e Pesquisas em Imunologia Humana, João Pessoa, PB, Brasil. 6 Universidade Federal de Pernambuco, Departamento de Medicina Clínica, Recife, PE, Brasil. 7 University of Pittsburgh, Center for Vaccine Research, Department of Infectious Diseases and Microbiology, Pittsburgh, PA, USA.

 

Abstract

BACKGROUND:

Dengue virus (DENV) has circulated in Brazil for over 30 years. During this time, one serotype has cyclically replaced the other, until recently, when all four distinct serotypes began to circulate together. Persistent circulation of DENV for long time periods makes sequential infections throughout a person’s life possible. After primary DENV infection, life-long immunity is developed for the infecting serotype. Since DENV and Zika virus (ZIKV) are antigenically similar, the possibility of cross-reactions has attracted attention and has been demonstrated in vitro.

OBJECTIVE:

The aim of this study was to investigate whether immune-sera from DENV and ZIKV infected patients would cross-react in vitro with other Flaviviridae family members.

METHODS:

Cross-reaction of the studied samples with yellow fever virus (YFV), West Nile virus (WNV), Rocio virus (ROCV), Saint Louis virus (SLEV) and Ilheus virus (ILHV) has been investigated by plaque reduction neutralisation test (PRNT) and the antibody-dependent enhancement (ADE) by flow-cytometry.

FINDINGS:

Antibodies against ZIKV and DENV virus cross-reacted with other flaviviruses either neutralising or enhancing the infection. Thus, viral entrance into FcRFcɣRII-expressing cells were influenced by the cross-reactive antibodies. ZIKV or DENV immune sera enhanced cellular infection by WNV, ILHV, ROCV and SLEV. Finally, DENV immune sera presented higher neutralising activity for YFV and SLEV. While ZIKV immune sera neutralised WNV, ILHV and ROCV with high frequencies of positivity.

MAIN CONCLUSIONS:

The co-circulation of those viruses in the same area represents a risk for the development of severe infections if they spread throughout the country. Successive flavivirus infections may have an impact on disease pathogenesis, as well as on the development of safe vaccine strategies.

PMID: 31411310 DOI: 10.1590/0074-02760190098

Keywords: Flavivirus; Dengue fever; Zika Virus; WNV; Rocio Virus; St Louis Virus; Ilheus virus; Yellow Fever; Serology; ADE; Brazil.

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