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 Albentosa-González et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Keywords: Arbovirus; Flavivirus; Usutu virus; Viral pathogenesis.

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#Clinical and #virological #findings in patients with #Usutu virus #infection, northern #Italy, 2018 (Euro Surveill., abstract)

[Source: Eurosurveillance, full page: (LINK). Abstract, edited.]

Clinical and virological findings in patients with Usutu virus infection, northern Italy, 2018

Monia Pacenti 1,2, Alessandro Sinigaglia 2,3, Thomas Martello 2, Elena De Rui 3, Elisa Franchin 2,3, Silvana Pagni 2,3, Elektra Peta 3, Silvia Riccetti 3, Adelaide Milani 4, Fabrizio Montarsi 4, Gioia Capelli 4, Carlo Giovanni Doroldi 5, Francesco Bigolin 5, Luca Santelli 6, Lucia Nardetto 6, Marco Zoccarato 6, Luisa Barzon 2,3

Affiliations: 1 Microbiology and Virology Unit, Padua University Hospital, Padova, Italy; 2 These authors contributed equally as first authors; 3 Department of Molecular Medicine, University of Padova, Padova, Italy; 4 Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro PD, Italy; 5 Medicine Unit, Camposampiero Hospital, Azienda ULSS 6 Euganea, Padova, Italy; 6 Neurology Department, Ospedale S. Antonio, Azienda ULSS 6 Euganea, Padova, Italy

Correspondence:  Luisa Barzon

Citation style for this article: Pacenti Monia, Sinigaglia Alessandro, Martello Thomas, De Rui Elena, Franchin Elisa, Pagni Silvana, Peta Elektra, Riccetti Silvia, Milani Adelaide, Montarsi Fabrizio, Capelli Gioia, Doroldi Carlo Giovanni, Bigolin Francesco, Santelli Luca, Nardetto Lucia, Zoccarato Marco, Barzon Luisa. Clinical and virological findings in patients with Usutu virus infection, northern Italy, 2018. Euro Surveill. 2019;24(47):pii=1900180. https://doi.org/10.2807/1560-7917.ES.2019.24.47.1900180

Received: 12 Mar 2019;   Accepted: 20 May 2019

 

Abstract

Background

Usutu virus (USUV) is a mosquito-borne flavivirus, which shares its transmission cycle with the phylogenetically related West Nile virus (WNV). USUV circulates in several European countries and its activity has increased over the last 5 years.

Aim

To describe human cases of USUV infection identified by surveillance for WNV and USUV infection in the Veneto Region of northern Italy in 2018.

Methods

From 1 June to 30 November 2018, all cases of suspected autochthonous arbovirus infection and blood donors who had a reactive WNV nucleic acid test were investigated for both WNV and USUV infection by in-house molecular methods. Anti-WNV and anti-USUV IgM and IgG antibodies were detected by ELISA and in-house immunofluorescence assay, respectively; positive serum samples were further tested by WNV and USUV neutralisation assays run in parallel.

Results

Eight cases of USUV infection (one with neuroinvasive disease, six with fever and one viraemic blood donor who developed arthralgia and myalgia) and 427 cases of WNV infection were identified. A remarkable finding of this study was the persistence of USUV RNA in the blood and urine of three patients during follow-up. USUV genome sequences from two patients shared over 99% nt identity with USUV sequences detected in mosquito pools from the same area and clustered within lineage Europe 2.

Conclusions

Clinical presentation and laboratory findings in patients with USUV infection were similar to those found in patients with WNV infection. Cross-reactivity of serology and molecular tests challenged the differential diagnosis.

©  This work is licensed under a Creative Commons Attribution 4.0 International License.

Keywords: Arbovirus; Flavivirus; Usutu virus; Italy.

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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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#Usutu Virus #Epizootic in #Belgium in 2017 and 2018: Evidence of Virus Endemization and Ongoing Introduction Events (Vector Borne Zoo Dis., abstract)

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

Usutu Virus Epizootic in Belgium in 2017 and 2018: Evidence of Virus Endemization and Ongoing Introduction Events

Emna Benzarti, Michaël Sarlet, Mathieu Franssen, Daniel Cadar, Jonas Schmidt-Chanasit, Jose Felipe Rivas, Annick Linden, Daniel Desmecht, and Mutien Garigliany

Published Online: 3 Sep 2019 / DOI: https://doi.org/10.1089/vbz.2019.2469

 

Abstract

Wildlife surveillance allowed the monitoring of the zoonotic mosquito-borne Usutu virus (USUV) in birds and bats (Pipistrellus pipistrellus) in southern Belgium in 2017 and 2018. USUV-RNA was detected in 69 birds (of 253) from 15 species, among which 7 species had not previously been reported to be susceptible to the infection. Similarly, 2 bats (of 10) were detected positive by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). USUV-associated lesions were mainly found in Eurasian Blackbirds (Turdus merula), in which USUV antigens were demonstrated by immunohistochemistry in the brain, heart, liver, kidney, intestine, and lung. Partial nonstructural protein 5 gene-based phylogenetic analysis showed several identical or closely related strains from 2016, 2017, and 2018 clustering together within Europe 3 or Africa 3 lineages. Further, one USUV strain detected in a common chaffinch (Fringilla coelebs) manifested a close genetic relationship with the European 1 strains circulating in Hungary and Austria. Our data provide evidence of USUV endemization in southern Belgium in local birds and bats, extension of the host range of the virus and ongoing virus introduction from abroad, likely by migratory birds. Our results highlight the need for vigilance in the forthcoming years toward new virus-associated outbreaks in birds and possible human infections in Belgium.

Keywords: Usutu virus; Bats; Wild Birds; Belgium.

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#Extraordinary increase in #WNV cases and first confirmed #human #Usutu virus #infection in #Hungary, 2018 (Euro Surveill., abstract)

[Source: Eurosurveillance, full page: (LINK). Abstract, edited.]

Extraordinary increase in West Nile virus cases and first confirmed human Usutu virus infection in Hungary, 2018

Anna Nagy1,2, Eszter Mezei2,3, Orsolya Nagy1,4, Tamás Bakonyi5,6, Nikolett Csonka1, Magdolna Kaposi1, Anita Koroknai1, Katalin Szomor7, Zita Rigó7, Zsuzsanna Molnár3, Ágnes Dánielisz3, Mária Takács1,4

Affiliations: 1 National Reference Laboratory for Viral Zoonoses; National Public Health Center, Budapest, Hungary; 2 These authors contributed equally to this work; 3 Department of Communicable Diseases Epidemiology and Infection Control; National Public Health Center, Budapest, Hungary; 4 Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary; 5 Department of Microbiology and Infectious Diseases, University of Veterinary Medicine, Budapest, Hungary; 6 Viral Zoonoses, Emerging and Vector-borne Infections Group, Institute of Virology, University of Veterinary Medicine, Vienna, Austria; 7 National Reference Laboratory for Viral Exanthematous Diseases; National Public Health Center, Budapest, Hungary

Correspondence: Anna Nagynagy.annannk.gov.hu

Citation style for this article: Nagy Anna, Mezei Eszter, Nagy Orsolya, Bakonyi Tamás, Csonka Nikolett, Kaposi Magdolna, Koroknai Anita, Szomor Katalin, Rigó Zita, Molnár Zsuzsanna, Dánielisz Ágnes, Takács Mária. Extraordinary increase in West Nile virus cases and first confirmed human Usutu virus infection in Hungary, 2018. Euro Surveill. 2019;24(28):pii=1900038. https://doi.org/10.2807/1560-7917.ES.2019.24.28.1900038

Received: 07 Jan 2019;   Accepted: 02 Apr 2019

 

Abstract

Background

During the 2018 WNV transmission season, similarly to other endemic areas in Europe, a large number of human West Nile virus (WNV) infections were reported in Hungary.

Aims

We summarise the epidemiological and laboratory findings of the 2018 transmission season and expand experiences in flavivirus differential diagnostics.

Methods

Every patient with clinical suspicion of acute WNV infection was in parallel tested for WNV, tick-borne encephalitis virus and Usutu virus (USUV) by serological methods. Sera, whole blood and urine samples were also tested for the presence of viral nucleic acid.

Results

Until the end of December 2018, 215 locally acquired and 10 imported human WNV infections were notified in Hungary. All reported cases were symptomatic; most of them exhibited neurological symptoms. In a large proportion of tested individuals, whole blood was the most appropriate sample type for viral nucleic acid detection, but because whole blood samples were not always available, testing of urine samples also extended diagnostic possibilities. In addition, the first human USUV infection was confirmed in 2018 in a patient with aseptic meningitis. Serological cross-reactions with WNV in different serological assays were experienced, but subsequent molecular biological testing and sequence analysis identified Europe lineage 2 USUV infection.

Conclusion

Careful interpretation and simultaneous application of different laboratory methods are necessary to avoid misdiagnosis of human USUV cases. Expansion of the laboratory-confirmed case definition criteria for detection of viral RNA in any clinical specimens to include urine samples could increase diagnostic sensitivity.

© This work is licensed under a Creative Commons Attribution 4.0 International License.

Keywords: WNV; Usutu virus; Hungary.

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Establishment of a #Cell #Culture Model of Persistent #Flaviviral #Infection: #Usutu Virus Shows Sustained Replication during Passages and Resistance to Extinction by Antiviral Nucleosides (Viruses, abstract)

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

Viruses. 2019 Jun 17;11(6). pii: E560. doi: 10.3390/v11060560.

Establishment of a Cell Culture Model of Persistent Flaviviral Infection: Usutu Virus Shows Sustained Replication during Passages and Resistance to Extinction by Antiviral Nucleosides.

Sempere RN1,2, Arias A3.

Author information: 1 Life Science & Bioengineering Building, Technical University of Denmark, 2800 Kongens Lyngby, Denmark. rnsempere@abiopep.com. 2 Abiopep Sociedad Limitada, Parque Científico de Murcia, 30100 Murcia, Spain. rnsempere@abiopep.com. 3 Life Science & Bioengineering Building, Technical University of Denmark, 2800 Kongens Lyngby, Denmark. aaesteban2@gmail.com.

 

Abstract

Chronic viral disease constitutes a major global health problem, with several hundred million people affected and an associated elevated number of deaths. An increasing number of disorders caused by human flaviviruses are related to their capacity to establish a persistent infection. Here we show that Usutu virus (USUV), an emerging zoonotic flavivirus linked to sporadic neurologic disease in humans, can establish a persistent infection in cell culture. Two independent lineages of Vero cells surviving USUV lytic infection were cultured over 82 days (41 cell transfers) without any apparent cytopathology crisis associated. We found elevated titers in the supernatant of these cells, with modest fluctuations during passages but no overall tendency towards increased or decreased infectivity. In addition to full-length genomes, viral RNA isolated from these cells at passage 40 revealed the presence of defective genomes, containing different deletions at the 5′ end. These truncated transcripts were all predicted to encode shorter polyprotein products lacking membrane and envelope structural proteins, and most of non-structural protein 1. Treatment with different broad-range antiviral nucleosides revealed that USUV is sensitive to these compounds in the context of a persistent infection, in agreement with previous observations during lytic infections. The exposure of infected cells to prolonged treatment (10 days) with favipiravir and/or ribavirin resulted in the complete clearance of infectivity in the cellular supernatants (decrease of ~5 log10 in virus titers and RNA levels), although modest changes in intracellular viral RNA levels were recorded (<2 log10 decrease). Drug withdrawal after treatment day 10 resulted in a relapse in virus titers. These results encourage the use of persistently-infected cultures as a surrogate system in the identification of improved antivirals against flaviviral chronic disease.

KEYWORDS: antiviral therapies; chronic viral infection; defective viral genomes; emerging arboviruses; lethal mutagenesis

PMID: 31212939 DOI: 10.3390/v11060560

Keywords: Flavivirus; Usutu virus; Antivirals; Favipiravir; Ribavirin.

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First #Evidence of #Fatal #Usutu Virus Natural #Infections in an #Anatidae, the Common Scoter (Melanitta nigra) (Vector Borne Zoo Dis., abstract)

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

First Evidence of Fatal Usutu Virus Natural Infections in an Anatidae, the Common Scoter (Melanitta nigra)

Emna Benzarti, Mutien Garigliany, Dany Hauman, Julien Paternostre, Annick Linden, Mathieu Franssen, Michael Sarlet, Dominique Cassart, and Daniel Desmecht

Published Online: 28 May 2019 / DOI: https://doi.org/10.1089/vbz.2019.2460

 

Abstract

While fatal infections caused by the Usutu virus appeared to concern only passerines (especially the blackbird) and Strigiformes (especially the great gray owl), we report herein that the virus also naturally causes a fatal disease in an anseriforme species, the common scoter (Melanitta nigra).

Keywords: Usutu virus; Wild birds.

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