Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis

Femke Feenstra, B.S. Drolet, Jan Boonstra, P.A. Van Rijn*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

11 Citations (Scopus)

Abstract

Background: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. However, deletion of NS3/NS3a leads to delayed virus release from mammalian cells and largely reduces virus release from insect cells. NS3/NS3a knockout BTV in sheep causes no viremia, but induces sterile immunity and is therefore proposed to be a Disabled Infectious Single Animal (DISA) vaccine candidate. In the absence of viremia, uptake of this vaccine strain by blood-feeding midges would be highly unlikely. Nevertheless, unintended replication of vaccine strains within vectors, and subsequent recombination or re-assortment resulting in virulent phenotypes and transmission is a safety concern of modified-live vaccines. Methods: The role of NS3/NS3a in replication and dissemination of BTV1, expressing VP2 of serotype 2 within colonized Culicoides sonorensis midges was investigated. Virus strains were generated using reverse genetics and their growth was examined in vitro. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV with or without expressing NS3/NS3a and replication in the midge was examined using RT PCR. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. Results: Although the parental NS3/NS3a expressing strain was not able to replicate and disseminate within C. sonorensis after oral feeding, this virus was able to replicate efficiently when the midgut infection barrier was bypassed by intrathoracic injection, whereas the NS3/NS3a knockout mutant was unable to replicate. This demonstrates that NS3/NS3a is required for viral replication within Culicoides. Conclusion: The lack of viremia and the inability to replicate within the vector, clearly demonstrate the inability of NS3/NS3a knockout DISA vaccine strains to be transmitted by midges.

Original languageEnglish
Article number476
Number of pages9
JournalParasites & Vectors
Volume8
Issue number1
DOIs
Publication statusPublished - 2015

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Ceratopogonidae
Bluetongue virus
Virus Release
Vaccines
Viremia
Proteins
Viruses
Reverse Genetics
Ruminants
Virus Replication
Infection
Genetic Recombination
Insects
Immunity
Sheep
Head
Phenotype
Safety
Polymerase Chain Reaction
Injections

Keywords

  • Arbovirus
  • Bluetongue virus
  • Culicoides
  • DISA vaccine
  • Midge
  • NS3/NS3a

Cite this

@article{703fd3ce90824b0cb05ca709a39a993f,
title = "Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis",
abstract = "Background: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. However, deletion of NS3/NS3a leads to delayed virus release from mammalian cells and largely reduces virus release from insect cells. NS3/NS3a knockout BTV in sheep causes no viremia, but induces sterile immunity and is therefore proposed to be a Disabled Infectious Single Animal (DISA) vaccine candidate. In the absence of viremia, uptake of this vaccine strain by blood-feeding midges would be highly unlikely. Nevertheless, unintended replication of vaccine strains within vectors, and subsequent recombination or re-assortment resulting in virulent phenotypes and transmission is a safety concern of modified-live vaccines. Methods: The role of NS3/NS3a in replication and dissemination of BTV1, expressing VP2 of serotype 2 within colonized Culicoides sonorensis midges was investigated. Virus strains were generated using reverse genetics and their growth was examined in vitro. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV with or without expressing NS3/NS3a and replication in the midge was examined using RT PCR. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. Results: Although the parental NS3/NS3a expressing strain was not able to replicate and disseminate within C. sonorensis after oral feeding, this virus was able to replicate efficiently when the midgut infection barrier was bypassed by intrathoracic injection, whereas the NS3/NS3a knockout mutant was unable to replicate. This demonstrates that NS3/NS3a is required for viral replication within Culicoides. Conclusion: The lack of viremia and the inability to replicate within the vector, clearly demonstrate the inability of NS3/NS3a knockout DISA vaccine strains to be transmitted by midges.",
keywords = "Arbovirus, Bluetongue virus, Culicoides, DISA vaccine, Midge, NS3/NS3a",
author = "Femke Feenstra and B.S. Drolet and Jan Boonstra and {Van Rijn}, P.A.",
year = "2015",
doi = "10.1186/s13071-015-1063-3",
language = "English",
volume = "8",
journal = "Parasites & Vectors",
issn = "1756-3305",
publisher = "Springer Verlag",
number = "1",

}

Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis. / Feenstra, Femke; Drolet, B.S.; Boonstra, Jan; Van Rijn, P.A.

In: Parasites & Vectors, Vol. 8, No. 1, 476, 2015.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis

AU - Feenstra, Femke

AU - Drolet, B.S.

AU - Boonstra, Jan

AU - Van Rijn, P.A.

PY - 2015

Y1 - 2015

N2 - Background: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. However, deletion of NS3/NS3a leads to delayed virus release from mammalian cells and largely reduces virus release from insect cells. NS3/NS3a knockout BTV in sheep causes no viremia, but induces sterile immunity and is therefore proposed to be a Disabled Infectious Single Animal (DISA) vaccine candidate. In the absence of viremia, uptake of this vaccine strain by blood-feeding midges would be highly unlikely. Nevertheless, unintended replication of vaccine strains within vectors, and subsequent recombination or re-assortment resulting in virulent phenotypes and transmission is a safety concern of modified-live vaccines. Methods: The role of NS3/NS3a in replication and dissemination of BTV1, expressing VP2 of serotype 2 within colonized Culicoides sonorensis midges was investigated. Virus strains were generated using reverse genetics and their growth was examined in vitro. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV with or without expressing NS3/NS3a and replication in the midge was examined using RT PCR. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. Results: Although the parental NS3/NS3a expressing strain was not able to replicate and disseminate within C. sonorensis after oral feeding, this virus was able to replicate efficiently when the midgut infection barrier was bypassed by intrathoracic injection, whereas the NS3/NS3a knockout mutant was unable to replicate. This demonstrates that NS3/NS3a is required for viral replication within Culicoides. Conclusion: The lack of viremia and the inability to replicate within the vector, clearly demonstrate the inability of NS3/NS3a knockout DISA vaccine strains to be transmitted by midges.

AB - Background: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. However, deletion of NS3/NS3a leads to delayed virus release from mammalian cells and largely reduces virus release from insect cells. NS3/NS3a knockout BTV in sheep causes no viremia, but induces sterile immunity and is therefore proposed to be a Disabled Infectious Single Animal (DISA) vaccine candidate. In the absence of viremia, uptake of this vaccine strain by blood-feeding midges would be highly unlikely. Nevertheless, unintended replication of vaccine strains within vectors, and subsequent recombination or re-assortment resulting in virulent phenotypes and transmission is a safety concern of modified-live vaccines. Methods: The role of NS3/NS3a in replication and dissemination of BTV1, expressing VP2 of serotype 2 within colonized Culicoides sonorensis midges was investigated. Virus strains were generated using reverse genetics and their growth was examined in vitro. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV with or without expressing NS3/NS3a and replication in the midge was examined using RT PCR. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. Results: Although the parental NS3/NS3a expressing strain was not able to replicate and disseminate within C. sonorensis after oral feeding, this virus was able to replicate efficiently when the midgut infection barrier was bypassed by intrathoracic injection, whereas the NS3/NS3a knockout mutant was unable to replicate. This demonstrates that NS3/NS3a is required for viral replication within Culicoides. Conclusion: The lack of viremia and the inability to replicate within the vector, clearly demonstrate the inability of NS3/NS3a knockout DISA vaccine strains to be transmitted by midges.

KW - Arbovirus

KW - Bluetongue virus

KW - Culicoides

KW - DISA vaccine

KW - Midge

KW - NS3/NS3a

U2 - 10.1186/s13071-015-1063-3

DO - 10.1186/s13071-015-1063-3

M3 - Article

VL - 8

JO - Parasites & Vectors

JF - Parasites & Vectors

SN - 1756-3305

IS - 1

M1 - 476

ER -