Abstract

Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUGMet→GCCAla mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.

Highlights

  • Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 virus species [1]

  • Sequencing of segment 10 (Seg-10) of this passage showed a point deletion resulting in repair of the open reading frame (ORF) of NS3/NS3a thereby inserting an Alanine at position 38 of NS3 (StyI-rev1, Fig. 2B)

  • The mechanisms of virus release from the infected cell of arthropod borne viruses is very intriguing, since it might be linked to pathogenesis and viremia as well as to transmission of virus between host and insect vector

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Summary

Introduction

Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 virus species [1]. Three of these orbivirus species, bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV), and African horsesickness virus (AHSV) cause a ‘notifiable disease’ as listed by the Office International des Epizooties (OIE) [2]. Virus transmission between ruminants (BTV and EHDV) or equids (AHSV) occurs in majority by bites of specific species of Culicoides. BTV is causing severe haemorrhagic disease in ruminants with fever, lameness, coronitis, swelling of the head ( the lips and tongue) and death. BTV has been the most extensively studied orbivirus and serves as representative of arthropod-borne orbiviruses. Twenty-four serotypes of BTV have been recognized as defined by crossneutralization assays, and recent BTV isolates are considered as serotype 25 and 26 which was partially based on sequence data [3,4]

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