INFECTIOUS SCHMALLENBERG VIRUS FROM CLONED CDNAS AND USES THEREOF

Abstract
The present invention belongs to the field of animal health and relates to a nucleic acid sequence which comprises the complete genome of an infectious Schmallenberg virus (SBV) useful for studying viremia and diseases caused by SBV in ruminants, and in the development of vaccines, therapeutics and diagnostics for the prophylaxis, treatment and diagnosis of viremia and diseases caused by SBV.
Description
SEQUENCE LISTING

This application contains a sequence listing in accordance with 37 C.F.R. 1.821-1.825. The sequence listing accompanying this application is hereby incorporated by reference in its entirety.


BACKGROUND OF THE INVENTION

1. Technical Field


The present invention belongs to the field of animal health and relates to nucleic acid sequences comprising the complete genome sequences of the genome segments of an infectious Schmallenberg virus. The invention also relates to the use of the nucleic acid sequences for producing infectious Schmallenberg virus to study the viremia and clinical symptoms induced by Schmallenberg virus in ruminants, and in the development of vaccines, therapeutics and diagnostics for the prophylaxis, treatment and diagnosis of a Schmallenberg virus infection.


2. Background Information


A novel orthobunyavirus, the Schmallenberg virus (SBV), was discovered in Europe in November 2011. After the first detection, the reported cases of SBV in sheep, cattle, and goats dramatically accumulated in several European countries to several thousand cases of PCR-positive malformed lambs and calves (1, 2). The virus was detected by metagenomics at the Friedrich-Loeffler-Institut (ELI) in samples of cattle with milk drop and fever. The investigated samples were collected in a farm near the city of Schmallenberg (North Rhine-Westphalia, Germany), and consequently the virus was named Schmallenberg virus (SBV). SBV is a member of the genus Orthobunyavirus within the family Bunyaviridae. It is related to the so-called Simbu serogroup viruses (1). SBV is like Akabane virus (AKAV) able to cross the placental barrier in pregnant cows and sheep, infect the fetus and cause fatal congenital defects during a susceptible stage in pregnancy (2). Therefore, SBV is a serious threat to ruminant livestock in Europe since vaccines are currently not available.


Orthobunyaviruses have a segmented, negative stranded RNA genome and are mainly transmitted by insect vectors like midges and mosquitis. The three segments (S, M and L) of the Orthobunyavirus genome allow genetic reassortment, which naturally occurs resulting in the emergence of viruses with new biological properties (3). The largest segment L encodes the RNA-dependent RNA polymerase. The M-segments encodes the viral surface glycoproteins Gn and Gc which are responsible for cell fusion, viral attachment and the induction of neutralizing antibodies. The small S-segment encodes the nucleocapsid N which is also involved in complement fixation (4). The relationship between Orthobunyaviruses were often only determined by serological cross-reactivity (5). In the era of DNA sequencing, phylogenetics has additionally been assessed by comparison of partial genome sequences (full N and partial Gc gene) (6). Therefore, available and published genome sequence information of full-length genomes is sparse. As a consequence, in-depth phylogenetic analyses are difficult. In conclusion, a detailed and reliable taxonomic classification of SBV could not be made. Preliminary investigations showed similarities of the M- and L-segment sequences to partial AKAV and Aino virus (AINOV) sequences. The N gene was most closely related to Shamonda virus (SHAV) (1).


SBV was the first orthobunyavirus of the Simbu serogroup detected in Europe. The virus is apparently transmitted by arthropod vectors. Biting midges probably play an important role in transmission. According to the current state of knowledge, ruminants are susceptible to infection with SBV. Adult animals may develop mild disease, if any. However, transplacental infection occurs frequently and can lead to severe congenital malformation of the vertebral column (Kyphosis, lordosis, scoliosis, torticollis) and of the scull (macrocephaly, brachygnathia inferior) as well as variable malformations of the brain (hydrancenphaly, porencephaly, cerebellar hypoplasia, hypoplasia of the brain stem) and of the spinal cord in lambs, kids and calves. The infection spread rapidly over large parts of North Western Europe. Belgium, Germany, France, Italy, Luxembourg, the Netherlands, Spain and the United Kingdom have been affected so far.


The Simbu serogroup, named according to the prototype virus, is the largest serogroup of Orthobunyavirus and contains at least 25 viruses, among them medically important viruses such as Akabane virus, Oropouche virus, Sathuperi virus or Douglas virus, most of which can cause malformations in new born ruminants, but also human beings can be affected. Akabane virus, for instance, causes congenital defects in ruminants and circulates in Asia, Oceania and Africa, whereas Oropouche virus is responsible for large epidemics of Oropouche fever, a zoonosis similar to dengue fever, in human populations in South Africa. Sathuperi virus has lent his name to the Sathuperi serogroup, to which belong also Douglas virus and SBV.


Reverse genetic systems for Bunya viruses are technically challenging, which is reflected by a small number of publicated systems. For Orthobunyaviruses a minigenome system (7), a transcription and replication competent trVLP (virus like particle) system (8) and full-length clone systems (9, 10) have been described. However, although the rescue system to recover infectious Bunyamvera virus of the Group C serogroup (genus Orthobunyavirus) entirely from cloned cDNA, that uses T7 RNA Polymerase has already been described in 1996 (9, 10), and comparable system exists for a Simbu serogroupe virus. One rescue system, which is based on cloned cDNAs but utilizes RNA polymerase I for the production of viral transcripts, had been described for Akabane virus, so far. However, there is a strong need for reverse genetic systems, particularly with regard to T7 RNA polymerase-driven systems allowing to produce infectious Schmallenberg viruses, for a better understanding of the diseases induced by said virus, for reproducing said disease in its different forms, for comparative tests, and as platform for the development of new vaccines, medications and diagnostics for the prophylaxis, treatment and diagnosis of viremia and diseases caused by SBV.







DESCRIPTION OF THE INVENTION

The solution to the above technical problem is achieved by the description and the embodiments characterized in the claims.


Thus, the invention in its different aspects is implemented according to the claims.


In one aspect, the invention provides a nucleic acid molecule, in particular a cDNA molecule, comprising the genomic sequence of a Schmallenberg virus (SBV) genome segment, in particular comprising the complete genomic sequence of a genome segment of an infectious Schmallenberg virus (SBV), wherein said molecule comprises a nucleic acid sequence selected from the group consisting of:

    • a nucleic acid sequence having at least 97.8% sequence identity with the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:7,
    • a nucleic acid sequence having at least 82.2% sequence identity with the nucleic acid sequence of SEQ ID NO:2, and
    • a nucleic acid sequence having at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO:3.


Preferably, the nucleic acid molecule of the invention comprises the genomic sequence of the S segment of Schmallenberg virus, wherein said molecule comprises a nucleic acid sequence having at least 97.8%, preferably at least 98%, more preferably at least 99%, still more preferably at least 99.5%, and in particular preferably 100% sequence identity with the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:7, and wherein this nucleic acid molecule is also termed “nucleic acid molecule (S)” or “DNA molecule (S)” hereinafter.


In another aspect, the nucleic acid molecule of the invention comprises the genomic sequence of the M segment of Schmallenberg virus, wherein said molecule comprises a nucleic acid sequence having at least 82.2%, in particular at least 85%, more particular at least 90% or at least 95%, preferably at least 98%, more preferably at least 99%, still more preferably at least 99.5%, and in particular preferably 100% sequence identity with the nucleic acid sequence of SEQ ID NO:2, and wherein this nucleic acid molecule is also termed “nucleic acid molecule (M)” or “DNA molecule (M)” hereinafter.


In a further aspect, the nucleic acid molecule of the invention comprises the genomic sequence of the L segment of Schmallenberg virus, wherein said molecule comprises a nucleic acid sequence having at least 93%, in particular at least 95%, more particular at least 97%, preferably at least 98%, more preferably at least 99%, still more preferably at least 99.5% or at least 99.8%, and in particular preferably 100% sequence identity with the nucleic acid sequence of SEQ ID NO:3, and wherein this nucleic acid molecule is also termed “nucleic acid molecule (L)” or “DNA molecule (L)” hereinafter.


Sequence identity in the context of the invention is understood as being based on pairwise sequence alignments. For purposes of the present invention, pairwise sequence alignments are done with ClustalW as implemented in Mega5 (K. Tamura et. al., MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 28, 2731-2739 (2011)), using the default settings (gap opening penalty of 15 and gap extension penalty of 6.66; DNA weight matrix: ClustalW 1.6; Transition weight of 0.5). Sequence identities of the aligned sequences are preferably calculated using BioEdit version 7.0.9.0.


The term “having 100% sequence identity”, as used herein, is understood to be equivalent to the term “being identical”.


As used herein, it is in particular understood that the term “sequence identity with the nucleic acid sequence of SEQ ID NO:X” is equivalent to the term “sequence identity with the nucleic acid sequence of SEQ ID NO:X over the length of SEQ ID NO: X” or to the term “sequence identity with the nucleic acid sequence of SEQ ID NO:X over the whole length of SEQ ID NO: X”, respectively. In this context, “X” is any integer selected from 1 to 10 so that “SEQ ID NO: X” represents any of the SEQ ID NOs mentioned herein.


In another aspect, the invention comprises a combination of at least two, preferably two, nucleic acid molecules selected from the group consisting of:

    • the nucleic acid molecule (S), i.e., as defined herein, a nucleic acid molecule comprising the genomic sequence of the S segment of Schmallenberg virus, wherein said molecule comprises a nucleic acid sequence having at least 97.8%, preferably at least 98%, more preferably at least 99%, still more preferably at least 99.5%, and in particular preferably 100% sequence identity with the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:7,
    • the nucleic acid molecule (M), i.e., as defined herein, a nucleic acid molecule comprising the genomic sequence of the M segment of Schmallenberg virus, wherein said molecule comprises a nucleic acid sequence having at least 82.2%, in particular at least 85%, more particular at least 90% or at least 95%, preferably at least 98%, more preferably at least 99%, still more preferably at least 99.5%, and in particular preferably 100% sequence identity with the nucleic acid sequence of SEQ ID NO:2,
    • and
    • the nucleic acid molecule (L), i.e., as defined herein, a nucleic acid molecule comprising the genomic sequence of the L segment of Schmallenberg virus, wherein said molecule comprises a nucleic acid sequence having at least 93%, in particular at least 95%, more particular at least 97%, preferably at least 98%, more preferably at least 99%, still more preferably at least 99.5% or at least 99.8%, and in particular preferably 100% sequence identity with the nucleic acid sequence of SEQ ID NO:3,


      and wherein in particular the combination of the nucleic acid molecule (S) and the nucleic acid molecule (M), preferably each having at least 98% or at least 99% sequence identity with SEQ ID NO:1 and SEQ ID NO:2, respectively, is preferred, or wherein in particular the combination of the nucleic acid molecule (S) and the nucleic acid molecule (M), preferably each having at least 98% or at least 99% sequence identity with SEQ ID NO:7 and SEQ ID NO:2, respectively, is preferred.


Preferably, the nucleic acid molecules described herein are isolated nucleic acid molecules. According to the invention, the combination of the nucleic acid molecule (S), the nucleic acid molecule (M), and the nucleic acid molecule (L) is most preferred, in particular a combination of the nucleic acid molecule (S), the nucleic acid molecule (M) and the nucleic acid molecule (L), each having at least 98% or at least 99% sequence identity with SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, or in particular a combination of the nucleic acid molecule (S), the nucleic acid molecule (M) and the nucleic acid molecule (L), each having at least 98% or at least 99% sequence identity with SEQ ID NO:7, SEQ ID NO:2, and SEQ ID NO:3, respectively.


The term “combination”, as used herein, in particular refers to any bringing together or admixture of the nucleic acid molecules, of the DNA constructs, preferably the cDNA constructs or of the RNA transcripts to be combined according to the invention, or preferably refers to a composition containing the nucleic acid molecules, the DNA constructs, preferably the cDNA constructs or the RNA transcripts of the combination.


Preferably, the combination of the nucleic acid molecule (S), the nucleic acid molecule (M), and the nucleic acid molecule (L), is capable of producing infectious Schmallenberg virus when transfected into cells. Since Schmallenberg virus has a negative stranded RNA genome, the presence of an RNA polymerase, preferably of T7 RNA polymerase or the RNA polymerase encoded by the Schmallenberg virus, in the transfected cells is required. Most preferred is the use of the T7 RNA polymerase. The presence of the RNA polymerase in the transfected cells can be provided, for instance, by co-transfection of a plasmid coding for and expressing the RNA polymerase or by penetrating the cells with RNA polymerase protein. According to the invention, in this regard, the use of transgenic cells producing RNA polymerase is particularly preferred, such as the transfection of the combination of the nucleic acid molecule (S), the nucleic acid molecule (M), and the nucleic acid molecule (L) into BSR-T7/5 cells. Alternatively, the cells can also be transfected with the mRNA that codes for the RNA polymerase and which is translated into the RNA polymerase when transfected into the host cells.


In two exemplary embodiments, the transfection may be performed with or without the co-transfection of at least one, preferably two or three, helper plasmid(s).


The term “infectious Schmallenberg virus” according to the invention is in particular understood as a Schmallenberg virus which infects mammals and/or insects and causes viremia in the infected mammal and/or insect.


As used herein, the term “viremia” is particularly understood as a condition in which Schmallenberg virus particles reproduce and circulate in the bloodstream of an animal, in particular of a mammal or of an insect.


Said infection of a mammal and/or insect by the Schmallenberg virus being produced by the nucleic acid molecules of the present invention in particular includes attachment of the virus to a host cell, entry of the virus into the cell, uncoating of the virion in the cytoplasm, replication and transcription of the viral genome, expression of viral proteins and assembly and release of new infectious viral particles.


Preferably, the mammal as mentioned herein is a ruminant, in particular selected from the group consisting of cattle, sheep, goats, deer, elk, giraffes, bison, moose, yaks, water buffalo, camels, alpacas, llamas, antelope, pronghorn, and nilgai. More preferably, the mammal as mentioned herein is a ruminant selected from the group consisting of cattle, sheep and goats.


The insect, as mentioned herein, is preferably selected from the group consisting of midges, in particular Culicoides spp., biting flies and mosquitoes.


The term “helper plasmids” as mentioned herein, is in particular directed to plasmids that contain one or more SBV coding sequence(s), e.g. under the control of a T7 promotor, to express the protein(s) of SBV.


The present invention further provides a DNA construct, preferably a cDNA construct, comprising the cDNA molecule according to the invention, wherein said DNA construct is in particular a cDNA vector such as a plasmid.


Herein, the DNA construct. preferably the cDNA construct, of the present invention which comprises the cDNA molecule (S) is also termed “DNA construct (S)”, the DNA construct of the present invention which comprises the DNA molecule (M) is also termed “DNA construct (M)”, and the DNA construct of the present invention which comprises the DNA molecule (L) is also termed “DNA construct (L)”.


According to the invention, preferred DNA vectors or plasmids into which the nucleotide molecule of the present invention can be inserted are pGEM-T Easy, pUC18, pcDNA, pX8δT or pT7riboSM2. The cDNA construct, as described herein, is preferably an isolated cDNA construct.


Exemplary cDNA constructs of the invention are provided with the sequences set forth in SEQ ID NOs: 4-6, wherein SEQ ID No: 4 shows an example of the sequence of a DNA construct (S), SEQ ID No: 5 shows an example of the sequence of a DNA construct (M) and SEQ ID No: 6 shows an example of the sequence of a DNA construct (L). Further exemplary cDNA constructs of the invention are provided with the sequences set forth in SEQ ID NOs: 8-10, wherein SEQ ID No: 8 shows an example of the sequence of a DNA construct (S), SEQ ID No: 9 shows an example of the sequence of a DNA construct (M) and SEQ ID No: 10 shows an example of the sequence of a DNA construct (L).


The invention also provides a combination of at least two, preferably two, different DNA constructs selected from the group consisting of:

    • the DNA construct (S), i.e., as defined herein, a cDNA construct which comprises the DNA molecule (S),
    • the DNA construct (M), i.e., as defined herein, a cDNA construct which comprises the DNA molecule (M),
    • and
    • the DNA construct (L), i.e., as defined herein, a cDNA construct which comprises the DNA molecule (L),


      wherein the at least two different cDNA constructs are preferably isolated cDNA constructs, and wherein in particular the combination of the DNA construct (S) and the DNA construct (M) is preferred, preferably each comprising the nucleic acid molecule (S) or the nucleic acid molecule (M), respectively, having at least 98% or at least 99% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, respectively, or preferably each comprising the nucleic acid molecule (S) or the nucleic acid molecule (M), respectively, having at least 98% or at least 99% sequence identity with SEQ ID NO:7 or SEQ ID NO:2, respectively.


According to the invention, the combination of the DNA construct (S), the nucleic acid molecule (M), and the nucleic acid molecule (L), is most preferred, in particular each comprising the nucleic acid molecule (S), the nucleic acid molecule (M) or the nucleic acid molecule (L), respectively, having at least 98% or at least 99% sequence identity with SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, respectively, or in particular each comprising the nucleic acid molecule (S), the nucleic acid molecule (M) or the nucleic acid molecule (L), respectively, having at least 98% or at least 99% sequence identity with SEQ ID NO:7, SEQ ID NO:2, or SEQ ID NO:3, respectively.


Further, the invention provides a preferably isolated RNA transcript of the cDNA construct of the invention.


In the following, the RNA transcript of the DNA construct (S) of the present invention is also termed “RNA transcript (S)”, the RNA transcript of the DNA construct (M) of the present invention is also termed “RNA transcript (M)”, and the RNA transcript of the DNA construct (L) is also termed “RNA transcript (L)”.


The invention also provides a combination of at least two, preferably two, different RNA transcripts, preferably isolated RNA transcripts, selected from the group consisting of:

    • the RNA transcript (S), i.e., as defined herein, the RNA transcript of the DNA construct (S),
    • the RNA transcript (M), i.e., as defined herein, the RNA transcript of the DNA construct (M),
    • and
    • the RNA transcript (L), i.e., as defined herein, the RNA transcript of the DNA construct (L),


      wherein in particular the combination of the RNA transcript (S) and the RNA transcript is preferred, preferably transcribed from the DNA construct (S) and the DNA construct (M), respectively, each comprising the nucleic acid molecule (S) or the nucleic acid molecule (M), respectively, having at least 98% or at least 99% sequence identity with SEQ ID NO:1 or SEQ ID NO:2, respectively, or preferably transcribed from the DNA construct (S) and the DNA construct (M), respectively, each comprising the nucleic acid molecule (S) or the nucleic acid molecule (M), respectively, having at least 98% or at least 99% sequence identity with SEQ ID NO:7 or SEQ ID NO:2, respectively.


According to the invention, the combination of the RNA transcript (S), the RNA transcript (L), and the RNA transcript (M), is most preferred, in particularly transcribed from the DNA construct (S), the DNA construct (M) and the DNA construct (L), respectively, each comprising the nucleic acid molecule (S), the nucleic acid molecule (M) or the nucleic acid molecule (L), respectively, having at least 98% or at least 99% sequence identity with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, respectively, or in particularly transcribed from the DNA construct (S), the DNA construct (M) and the DNA construct (L), respectively, each comprising the nucleic acid molecule (S), the nucleic acid molecule (M) or the nucleic acid molecule (L), respectively, having at least 98% or at least 99% sequence identity with SEQ ID NO:7, SEQ ID NO:2 or SEQ ID NO:3, respectively.


The present invention also provides a cell transfected with the DNA construct described herein or with the combination of DNA constructs described herein, wherein said cell is preferably an isolated cell.


Thus, the present invention also provides Schmallenberg virus produced by the aforementioned cell, wherein said Schmallenberg virus is preferably an isolated Schmallenberg virus. Furthermore, the present invention also provides a cell, preferably a cultured host cell which comprises the Schmallenberg virus produced by or in the presence of one or more of the nucleic acid constructs provided herein.


Further, the present invention provides a cell transfected with the RNA transcript mentioned herein or with the combination of RNA transcripts mentioned herein, wherein said cell is preferably an isolated cell.


Hence, the present invention also provides Schmallenberg virus produced by the aforementioned cell, wherein said Schmallenberg virus is preferably an isolated Schmallenberg virus.


The present invention further provides a Schmallenberg virus whose genome comprises the nucleic acid molecule of the present invention or the combination of nucleic acid molecules of the present invention, wherein said Schmallenberg virus is preferably an isolated Schmallenberg virus


In another aspect, the present invention provides a method for producing a Schmallenberg virus, said method comprising transfecting a cell with the DNA construct or with the combination of DNA constructs described herein.


Moreover, the present invention provides a method for producing a Schmallenberg virus, said method comprising transfecting a cell with the RNA transcript or with the combination of RNA transcripts mentioned herein.


Since Schmallenberg virus has a negative stranded RNA genome, preferably the method of producing the Schmallenberg virus is done in the presence of an RNA polymerase, preferably of T7 RNA polymerase or the RNA polymerase encoded by the Schmallenberg virus. Most preferred is the use of the T7 RNA polymerase. The presence of the RNA polymerase in the transfected cells can be provided, for instance, by co-transfection of a plasmid coding for and expressing the RNA polymerase. According to the invention, in this regard, the use of transgenic cells producing RNA polymerase is particularly preferred, such as the transfection of the combination of the nucleic acid molecule (S), the nucleic acid molecule (M), and the nucleic acid molecule (L) into BSR-T7/5 cells. Alternatively, the cells can also be transfected with the mRNA that codes for the RNA polymerase and which is translated into the RNA polymerase when transfected into the host cells.


In yet another aspect, the present invention provides a composition, said composition comprising the nucleic acid molecule according to the invention or the combination of nucleic acids according to the invention, suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient.


Production of the nucleic acid molecules described herein is within the skill in the art and can be carried out according to recombinant techniques described, among other places, in Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel, et al., 2003, Current Protocols In Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY; Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed), 1994, PCR Technology, Oxford University Press, New York, all of which are incorporated herein by reference.


Example 1

Establishment of a reverse genetics system for the generation of recombinant SBV, which allows further investigation on the molecular biology of Orthobunyaviruses as well as the generation of save and efficient vaccines.


SBV was isolated from infected cattle and passaged on KC cells and BHK-21 cells. RNA was extracted from infected cells and transcribed into cDNA. PCR fragments of the three RNA segments were amplified by using gene specific primers and were inserted into the plasmid pX8δT (11) by restrictions-free cloning (13). The resulting plasmids pX8δT_SBV_S, pX8δT_SBV_M and pX8δT_SBV_L contain the full-length antigenome of SBV.


Transfection experiments are done by using BSR T7/5 cells, stably expressing the phage T7 polymerase (12) and plasmid DNA of all of the three constructs pX8δT_SBV_S, pX8δT_SBV_M and pX8δT_SBV_L. Supernatants of the cells are harvested after various times following transfection and transferred to susceptible cell lines. The cell monolayers are investigated for expression of SBV proteins by indirect immunofluorescence staining.


Results


Three cDNA clones spanning the complete genomic sequence of the segments S, M and L were generated from viral RNA by fusion PCR. RNA transcripts were produced by bacteriophage T7 polymerase in BSR T7/5 cells. The exact 3′ end of the RNA is specified by self-cleavage of the RNA by the hepatitis delta virus antigenome ribozyme sequence. Rescue of infectious SBV, growth characteristics of recombinant viruses and manipulation of the full-length genome like the deletion of relevant domains can be demonstrated.


Conclusions


A reverse genetic system for the recovery of SBV, the first European Simbu serogroup virus, can be established. The new system can be used for the generation of recombinant SBV, by transfection of cells stably expressing phage T7 RNA polymerase with the plasmids pX8δT_SBV_S, pX8δT_SBV_M pX8δT_SBV_L allowing expression of antigenomic SBV RNA and the viral proteins. By using SBV reverse genetics, defined mutants can be designed enabling the mechanistic investigation of virus-host interactions as well as the molecular basis of SBV pathogenesis. Furthermore, the approach will be useful for the design of next generation vaccines like packaged replicons and defective in second cycle virions, chimera or modified deletion mutants.


In the following, the construction of the plasmids pX8δT_SBV_S, pX8δT_SBV_M and pX8δT_SBV_L and the transfection and recovery of recombinant SBV, as mentioned above, is described in closer detail.


The construction of the plasmids pX8δT_SBV_S, pX8δT_SBV_M and pX8δT_SBV_L was done by using the plasmid vector X8δT (11). cDNA of the Schmallenberg Virus (SBV) RNA segments was inserted into this plasmid by restrictions-free cloning (fusion PCR) (13), respectively. The construction of the cDNA clones is shown in FIG. 1A-1C. The plasmids contain a bacteriophage T7 promotor (T7) before 5′ SBV cDNA to enable in vitro transcription of cDNA into RNA, the Hepatitis delta virus ribozyme sequence (Hep custom-character for the generation of the exact 3′ end by self-cleavage of the nascent RNA by the Hepatitis delta virus antigenome ribozyme and the T7 transcription termination sequence (T7 term) downstream the 3′ end of the SBV cDNA. Location of the used primers and nucleotide positions corresponding to the Schmallenberg antigenome are indicated by arrows.


RNA of Schmallenberg virus (BH80/11-4) infected BHK 21 cells was isolated by using QIAmp viral RNA Mini Kit (Qiagen) and transcribed by using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Plasmids were amplified in Escherichia coli DH10B™ cells (Invitrogen). For Megaprimer-PCR and fusion PCR the Phusion High Fidelity PCR Master Mix with HF Buffer (New England Biolabs) and Phusion High-Fidelity Master Mix (Finnzymes) were used. Plasmid DNA was purified by using Qiagen Plasmid Mini or Midi Kit (Qiagen). Sequencing was carried out using the Big Dye® Terminator v1.1 Cycle sequencing Kit (Applied Biosystems). Nucleotide sequences were read with an automatic sequencer (3130 Genetic Analyzer, Applied Biosystems) and analyzed using the Genetics Computer Group software version 11.1 (Accelrys Inc., San Diego, USA). Primers were synthesized by biomers.net GmbH and are listed in table 1.









TABLE 1







Nucleotide sequence of primers used for Megaprimer-PCR and fusion


PCR









Primer
Sequenz 5′ → 3′a
Genomic region





P_Ph_S1F

CTTGTAATACGACTCACTATA
GGGAGTAGTG

   1-22a (+sense)



AGCTCCACTATTAAC






P_Ph_S1

GTGGAGATGCCATGCCGACCCAGTAGTGTTC

 830-808a (−sense)



TCCACTTATTAAC






P_Ph_M1F

CTTGTAATACGACTCACTATA
GGGAGTAGTG

   1-20b (+sense)



AACTACCACAATC






P_Ph_M1R

GTGGAGATGCCATGCCGACCCGCACTTGGAG

1686-1665b (−sense)



AGGGCACAACTG






P_M2F
CTCAGCTTACAATAGAGCACC
1453-1473b (+sense)





P_Ph_M2R

GTGGAGATGCCATGCCGACCCGTGACCCAAC

3080-3063b (−sense)



CATCTTGATG






P_M3F
TCGAGTCGCACATCCCTGC
2854-2872b (+sense)





P_Ph_M3R

GTGGAGATGCCATGCCGACCCGTCAGTCTCC

4105-4082b (−sense)



AATaGAAAGATAGG






P_M4F
CCTATCTTTCTATTGGAGACTGAC
4082-4105b (+sense)





P_Ph_MR

GTGGAGATGCCATGCCGACCCAGTAGTGTTCTAC

4373-4355b (−sense)



CACATG






P_Ph_L1F

CTTGTAATACGACTCACTATA
GGGAGTAGTG

   1-24c (+sense)



TACCCCTAATTACAATC






P_Ph_L1R

GTGGAGATGCCATGCCGACCCGTTTGCACAA

1626-1606c (−sense)



CACACTACACG






P_L2F
GTTCAAAGGATACATGGGATCAG
1478-1500c (+sense)





P_Ph_L2R

GTGGAGATGCCATGCCGACCCGTCATCAGAA

3543-3524c (−sense)



TGAACCATAG






P_L3F
CTGCAGGGGAATCTCAATTACAC
3409-343c (+sense)





P_Ph_L3R

GTGGAGATGCCATGCCGACCCGATTGATAGA

5570-55494c (−sense)



TCAATTGGACCAGTAG






P_L4F
GCAGAAGAGCAGATCACATGG
5500-5520c (+sense)





P_Ph_L4R

AGGTGGAGATGCCATGCCGACCCCAAACTTT

6781-6762c (−sense)



GATCTGCCACCC






P_L5F
GAGCCATGGGTGTCTATACTG
6637-6657c (+sense)





P_Ph_LR

GTGGAGATGCCATGCCGACCCAGTAGTGTGC

6882-6862c (−sense)



CCCTAATTACATG






anucleotide position corresponding to SBV segment S sequence (unpublished)




bnucleotide position corresponding to SBV segment M sequence (unpublished)




cnucleotide position corresponding to SBV segment L sequence (unpublished)







Sequences derived from plasmid X8δT are underlined, and three additional G residues are in italics.


Construction of pX8δT_SBV_S (FIG. 1A): In a first step segment S cDNA was synthesized with primer P_Ph_S1F and used as template for the generation of a megaprimer PCR fragment. As primers P_Ph_S1F and P_Ph_S1R were utilized. By fusion PCR, SBV segment S sequences were introduced into the plasmid pX8δT.


Construction of pX8δT_SBV_M (FIG. 1B): In a multi-step cloning procedure the cDNA clone pX8δT_SBV_M was constituted from four megaprimer PCR fragments which were assembled into plasmid vector pX8δT by fusion PCR. In a first step segment M cDNA was synthesized with primer P_Ph_M1F and P_M3F and used as template for the generation of the megaprimers 1, 2, 3 and 4, respectively. As primers for the generation of megaprimer 1 primers P_Ph_M1F and P_Ph_M1R, for the generation of megaprimer 2 the primers P_M2F and P_Ph_M2R, for the generation of megaprimer 3 the primers P_M3F and P_Ph_M3R and for the generation of megaprimer 4 the primers P_M4F and P_Ph_MR were used. By fusion PCR the megaprimers were introduced into the plasmid pX8δT, successively.


Construction of pX8δT_SBV_L (FIG. 1C): In a multi-step cloning procedure the cDNA clone pX8δT_SBV_L was constituted from five megaprimer PCR fragments which were assembled into plasmid vector pX8δT by fusion PCR. In a first step segment L cDNA was synthesized with primer P_Ph_L1F and P_L3F and used as template for the generation of the megaprimers 1, 2, 3, 4 and 5, respectively. As primers for the generation of megaprimer 1 primers P_Ph_L1F and P_Ph_L1R, for the generation of megaprimer 2 the primers P_L2F and P_Ph_L2R, for the generation of megaprimer 3 the primers P_L3F and P_Ph_L3R, for the generation of megaprimer 4 the primers P_L4F and P_Ph_L4R and for the generation of megaprimer 5 the primers P_L5F and P_Ph_LR were used. By fusion PCR the megaprimers were introduced into the plasmid pX8δT, successively.


Transfection and Recovery of Recombinant SBV


Transfection experiments are done using BHK 21 cells, clone BSR T7/5, stably expressing the phage T7 RNA polymerase (12), according to Lowen et al. (10). About 6×105 cells grown to 80% confluency are transfected with various amounts of plasmid DNA e.g. 0.25 μg pX8δT_SBV_L, 0.1 μg pX8δT_SBV_S, 1 μg pX8δT_SBV_M using a transfection reagent e.g., Lipofectin (Invitrogen), Lipofectamin (Invitrogen), Superfect (Qiagen) and DAC-30 (Eurogentec) according to suppliers protocols. Transfected cells are incubated for various times (e.g. 4-5 days) at 37° C. The supernatant fluid is collected, clarified by low speed centrifugation and various volumes (e.g 200 μl) are inoculated into highly susceptible cells (KC, BHK 21). Detection of infectious SBV can be done by indirect IF-staining using SBV-specific monoclonal and polyclonal antibodies.


Example 2

Establishment of a reverse genetics system using the plasmid pT/riboSM2 for the generation of recombinant SBV, which allows further investigation on the molecular biology of Orthobunyaviruses as well as the generation of save and efficient vaccines.


SBV was isolated from infected cattle and passaged on KC cells and BHK-21 cells. RNA was extracted from infected cells and transcribed into cDNA. PCR fragments of the three RNA segments were amplified by using gene specific primers and were subcloned into the plasmid pX8δT (11) by restrictions-free cloning (13). The resulting plasmids pX8δT_SBV_S, pX8δT_SBV_M and pX8δT_SBV_L contain the full-length antigenome of SBV. Afterwards, by using segment-specific primers and the full-length plasmids as template DNA, full-length PCR fragments were amplified and inserted into plasmid pT/riboSM2 (14) either by restrictions-free cloning or by digestion with appropriate restriction enzymes (e.g. Esp3I, BsmBI) and ligation. The resulting plasmids pT7ribo_SBV_S, pT7ribo_SBV_M and pT7ribo_SBV_L contain the full-length antigenome of SBV.


Transfection experiments are done by using BSR T7/5 cells, stably expressing the phage T7 polymerase (12) and plasmid DNA of all of the three constructs pT7ribo_SBV_S, pT7ribo_SBV_M and pX8δT_SBV_L or pT7ribo_SBV_L. Supernatants of the cells are harvested after various times following transfection and transferred to susceptible cell lines. The cell monolayers are investigated for expression of SBV proteins by indirect immunofluorescence staining.


Results


Three cDNA clones spanning the complete genomic sequence of the segments S, M and L were generated from viral RNA. RNA transcripts were produced by bacteriophage T7 polymerase in BSR T7/5 cells. The exact 3′ end of the RNA is specified by self-cleavage of the RNA by the hepatitis delta virus antigenome ribozyme sequence. Rescue of infectious SBV, growth characteristics of recombinant viruses and manipulation of the full-length genome like the deletion of relevant domains can be demonstrated. The virus rescue is more efficient, compared to example 1.


Conclusions


A reverse genetic system for the recovery of SBV, the first European Simbu serogroup virus, can be established. The new system can be used for the generation of recombinant SBV, by transfection of cells stably expressing phage T7 RNA polymerase with the plasmids pT7ribo_SBV_S, pT7ribo_SBV_M and pX8δT_SBV_L or pT7—SBV_L allowing expression of antigenomic SBV RNA and the viral proteins. By using SBV reverse genetics, defined mutants can be designed enabling the mechanistic investigation of virus-host interactions as well as the molecular basis of SBV pathogenesis. Furthermore, the approach will be useful for the design of next generation vaccines like packaged replicons and defective in second cycle virions, chimera or modified deletion mutants.


In the following, the construction of the plasmids pT7ribo_SBV_S, pT7ribo_SBV_M, pX8δT_SBV_L and pT7ribo_SBV_L and the transfection and recovery of recombinant SBV, as mentioned above, is described in closer detail.


The construction of the plasmids pT7ribo_SBV_S, pT7ribo_SBV_M pX8δT_SBV_L and pT7ribo_SBV_L was done by using the plasmid vectors X8δT (11) and pT7riboSM2 (14). cDNA of the Schmallenberg Virus (SBV) RNA segments was inserted into this plasmid by standard cloning methods using restriction enzyme BsmB or by restriction-free cloning (fusion PCR) (13), respectively. The construction of the cDNA clones is shown in FIG. 2A-2C. The plasmids contain a bacteriophage T7 promotor (T7) before 5′ SBV cDNA to enable in vitro transcription of cDNA into RNA, the Hepatitis delta virus ribozyme sequence (Hep custom-character for the generation of the exact 3′ end by self-cleavage of the nascent RNA by the Hepatitis delta virus antigenome ribozyme and the T7 transcription termination sequence (T7 term) downstream the 3′ end of the SBV cDNA. Location of the used primers and nucleotide positions corresponding to the Schmallenberg antigenome are indicated by arrows.


RNA of Schmallenberg virus (BH80/11-4) infected BHK 21 cells was isolated by using QIAmp viral RNA Mini Kit (Qiagen) and transcribed by using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche). Plasmids were amplified in Escherichia coli DH10B™ cells (Invitrogen). For Megaprimer-PCR and fusion PCR the Phusion High Fidelity PCR Master Mix with HF Buffer (New England Biolabs) and Phusion High-Fidelity Master Mix (Finnzymes) were used. Digestion of the plasmids and DNA fragments was done with The restriction enzymes BsmBI (New England Biolabs) and Esp3I (Fisher Scientific). For ligation of plasmid DNA and DNA fragments, T4 DNA ligase (Promega) was used. Plasmid DNA was purified by using Qiagen Plasmid Mini or Midi Kit (Qiagen). Sequencing was carried out using the Big Dye® Terminator v1.1 Cycle sequencing Kit (Applied Biosystems). Nucleotide sequences were read with an automatic sequencer (3130 Genetic Analyzer, Applied Biosystems) and analyzed using the Genetics Computer Group software version 11.1 (Accelrys Inc., San Diego, USA). Primers were synthesized by biomers.net GmbH and are listed in table 2.









TABLE 2







Nucleotide sequence of PCR primers









Primer
Sequenz 5′ → 3′a
Genomic region





P_Ph_S1F

CTTGTAATACGACTCACTATA
GGGAGTAG

   1-22a (+sense)



TGAGCTCCACTATTAAC






P_Ph_S1R

GTGGAGATGCCATGCCGACCCAGTAGTGT

 830-808a (−sense)



TCTCCACTTATTAAC






P_Ph_S2F

CTTGTAATACGACTCACTATA
GAGTAGTG

   1-22a (+sense)



AACTCCACTATTAAC






P_Ph_M1F

CTTGTAATACGACTCACTATA
GGGAGTAG

   1-20b (+sense)



TGAACTACCACAATC






P_Ph_M1R

GTGGAGATGCCATGCCGACCCGCACTTGG

1686-1665b (−sense)



AGAGGGCACAACTG






P_M2F
CTCAGCTTACAATAGAGCACC
1453-1473b (+sense)





P_Ph_M2R

GTGGAGATGCCATGCCGACCCGTGACCC

3080-3063b (−sense)



AACCATCTTGATG






P_M3F
TCGAGTCGCACATCCCTGC
2854-2872b (+sense)





P_Ph_M3R

GTGGAGATGCCATGCCGACCCGTCAGTCT

4105-4082b (−sense)



CCAATaGAAAGATAGG






P_M4F
CCTATCTTTCTATTGGAGACTGAC
4082-4105b (+sense)





P_Ph_MR

GTGGAGATGCCATGCCGACCCAGTAGTGTTC

4373-4355b (−sense)



TACCACATG






P_M_BsmBI_F

CTAC

CGTCTCCTATA

GAGTAGTGAACTACCA





CAATC






P_M_BsmBI_R

GTACCGTCTCCACCCAGTAGTGTTCTACCACA





TG






P_Ph_L1F

CTTGTAATACGACTCACTATA
GGGAGTAG

   1-24c (+sense)



TGTACCCCTAATTACAATC






P_Ph_L1R

GTGGAGATGCCATGCCGACCCGTTTGCAC

1626-1606c (−sense)



AACACACTACACG






P_L2F
GTTCAAAGGATACATGGGATCAG
1478-1500c (+sense)





P_Ph_L2R

GTGGAGATGCCATGCCGACCCGTCATCAG

3543-3524c (−sense)



AATGAACCATAG






P_L3F
CTGCAGGGGAATCTCAATTACAC
3409-343c (+sense)





P_Ph_L3R

GTGGAGATGCCATGCCGACCCGATTGATA

5570-55494c (−sense)



GATCAATTGGACCAGTAG






P_L4F
GCAGAAGAGCAGATCACATGG
5500-5520c (+sense)





P_Ph_L4R

AGGTGGAGATGCCATGCCGACCCCAAAC

6781-6762c (−sense)



TTTGATCTGCCACCC






P_L5F
GAGCCATGGGTGTCTATACTG
6637-6657c (+sense)





P_Ph_LR

GTGGAGATGCCATGCCGACCCAGTAGTGT

6882-6862c (−sense)



GCCCCTAATTACATG






P_Mut
GAAAAGTACACCCAGATCTTTGGtGAtGCATT
 340-388c (+sense)


L_BsmBIF
GTCAGAATTGCCGTTTG






P_L_BsmBI_F
CTACCGTCTCCTATAGAGTAGTGTACCCC
   1-20c (+sense)



TAATTAC






P_L_BsmBI_R
CTACCGTCTCCACCCAGTAGTGTGCCCCT
6882-6859c (−sense)



AATTAC






anucleotide position corresponding to SBV segment S sequence (unpublished)




bnucleotide position corresponding to SBV segment M sequence (unpublished)




cnucleotide position corresponding to SBV segment L sequence (unpublished)







Sequences derived from plasmids X8δT and pT7riboSM2 are underlined, mutated nucleotides are in lower case and additional G residues and restriction sites are in italics.


Construction of pT7—SBV_S (FIG. 2A): In a first step segment S cDNA was synthesized with primer P_Ph_S1F and used as template for the generation of a megaprimer PCR fragment. As primers P_Ph_S1F and P_Ph_S1R were utilized. By fusion PCR, SBV segment S sequences were introduced into the plasmid pX8δT. Plasmid pX8δT_SBV_S was used as template, to amplify a full-length megaprimer PCR fragment by using primers P_Ph_S2F and P_Ph_S1R. By fusion PCR, SBV segment S sequences were introduced into the plasmid pT7ribo_SM2, resulting in plasmid pT7ribo_SBV_S.


Construction of pT7ribo_SBV_M (FIG. 2B): In a multi-step cloning procedure the cDNA clone pX8δT_SBV_M was constituted from four megaprimer PCR fragments which were assembled into plasmid vector pX8δT by fusion PCR. In a first step segment M cDNA was synthesized with primer P_Ph_M1F and P_M3F and used as template for the generation of the megaprimers 1, 2, 3 and 4, respectively. As primers for the generation of megaprimer 1 primers P_Ph_M1F and P_Ph_M1R, for the generation of megaprimer 2 the primers P_M2F and P_Ph_M2R, for the generation of megaprimer 3 the primers P_M3F and P_Ph_M3R and for the generation of megaprimer 4 the primers P_M4F and P_Ph_MR were used. By fusion PCR the megaprimers were introduced into the plasmid pX8δT, successively. Plasmid pX8δT_SBV_M was used as template, to amplify a full-length PCR fragment by using primers P_M_BsmBI_F and P_M_BsmBI_R. This PCR fragment was digested with BsmBI and ligated into BsmBI-digested plasmid pT7ribo_SM2, resulting in plasmid pT7ribo_SBV_M.


Construction of pX8δT_SBV_L (FIG. 2C): In a multi-step cloning procedure the cDNA clone pX8δT_SBV_L was constituted from five megaprimer PCR fragments which were assembled into plasmid vector pX8δT by fusion PCR. In a first step segment L cDNA was synthesized with primer P_Ph_L1F and P_L3F and used as template for the generation of the megaprimers 1, 2, 3, 4 and 5, respectively. As primers for the generation of megaprimer 1 primers P_Ph_L1F and P_Ph_L1R, for the generation of megaprimer 2 the primers P_L2F and P_Ph_L2R, for the generation of megaprimer 3 the primers P_L3F and P_Ph_L3R, for the generation of megaprimer 4 the primers P_L4F and P_Ph_L4Rand for the generation of megaprimer 5 the primers P_L5F and P_Ph_LR were used. By fusion PCR the megaprimers were introduced into the plasmid pX8δT, successively. In order to generate pT7ribo_SBV_L, the BsmBI-site within pX8δT_SBV_L had to be deleted by site-directed mutagenesis. A PCR fragment (megaprimer) was amplified by using primers P_Mut L_BsmBIF, P_Mut L_BsmBIR and plasmid pX8δT_SBV_L as template DNA. By fusion PCR the megaprimer was introduced into the plasmid pX8δT_SBV_L, resulting in the plasmid pX8δT_Mut_L_BsmBI. Plasmid pX8δT_Mut_L was used as template, to amplify a full-length PCR fragment by using primers P_L_BsmBI_F and P_L_BsmBI_R. This PCR fragment was digested with BsmBI and ligated into BsmBI-digested plasmid pT7ribo_SM2, resulting in plasmid pT7ribo_SBV_L.


Transfection and Recovery of Recombinant SBV


Transfection experiments are done using BHK 21 cells, clone BSR T7/5, stably expressing the phage T7 RNA polymerase (12), according to Lowen et al. (10). About 6×105 cells grown to 80% confluency are transfected with various amounts of plasmid DNA e.g., 3 μg pT7robo_SBV_S, 3 μg pT7ribo_SBV_M, 3 μg pX8δT_SBV_L or 3 μg pT7ribo_SBV_L using a transfection reagent e.g. Lipofectin (Invitrogen), Lipofectamin (Invitrogen) and Superfect (Qiagen) according to suppliers protocols. Transfected cells are incubated for various times (e.g. 4-5 days) at 37° C. The supernatant fluid is collected, clarified by low speed centrifugation and various volumes (e.g 0.1-1.0 ml) are inoculated into highly susceptible cells (KC, BHK 21). Detection of infectious SBV can be done by indirect IF-staining using SBV-specific monoclonal and polyclonal antibodies.


LIST OF FIGURES


FIG. 1A: Construction of the plasmid pX8dT_SBV_S.



FIG. 1B: Construction of the plasmid pX8dT_SBV_M.



FIG. 1C: Construction of the plasmid pX8dT_SBV_L.



FIG. 2A: Construction of the plasmid pT7ribo_SBV_S.



FIG. 2B: Construction of the plasmid pT7ribo_SBV_M.



FIG. 2C: Construction of the plasmid pT7ribo_SBV_L.


IN THE SEQUENCE LISTING

SEQ ID NO:1 corresponds to the complete genome sequence of a S segment of an infectious Schmallenberg virus (BH80/11-4),


SEQ ID NO:2 corresponds to the complete genome sequence of a M segment of an infectious Schmallenberg virus (BH80/11-4),


SEQ ID NO:3 corresponds to the complete genome sequence of a L segment of an infectious Schmallenberg virus (BH80/11-4),


SEQ ID NO:4 corresponds to the sequence of plasmid pX8δT_SBV_S,


SEQ ID NO:5 corresponds to the sequence of plasmid pX8δT_SBV_M,


SEQ ID NO:6 corresponds to the sequence of plasmid pX8δT_SBV_L,


SEQ ID NO: 7 corresponds to SEQ ID NO:1, wherein the nucleotide at position 9 is “a” instead of “g”,


SEQ ID NO:8 corresponds to the sequence of plasmid pT7ribo_SBV_S,


SEQ ID NO:9 corresponds to the sequence of plasmid pT7ribo_SBV_M,


SEQ ID NO:10 corresponds to the sequence of plasmid pT7ribo_SBV_L.


REFERENCES

All references cited herein are entirely incorporated by reference.

  • 1. B. Hoffmann, M. Scheuch, D. Hoper, R. Jungblut, M. Holsteg, H. Schirrmeier, M. Eschbaumer, K. V. Goller, K. Wernike, M. Fischer, A. Breithaupt, T. C. Mettenleiter, M. Beer, Novel orthobunyavirus in Cattle, Europe, 2011. Emerg. Infect. Dis. 18, 469-472 (2012).
  • 2. M.-M. Gariglinany et al., Schmallenberg virus in calf born at term with porencephaly, Belgium. Emerg. Infect. Dis. 18 (2012), doi: 10.3201/eid1806.120104.
  • 3. M. D. Bowen et al., A reassortant bunyavirus isolated from acute hemorrhagic fever cases in Kenya and Somalia. Virology. 291, 185-190 (2001).
  • 4. A. M. Q. King, M. J. Adams, E. B. Carstens, E. J. Lefkowitz, Eds., Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. (Elsevier, San Diego, USA, 2011), pp 725-731.
  • 5. R. M. Kinney, C. H. Calisher, Antigenic relationships among Simbu serogroup (Bunyaviridae) viruses. Am. J. Trop. Med. Hyg. 30, 1307-1318 (1981).
  • 6. M. F. Saeed, L. L1, H. Wang, S. C. Weaver, A. D. Barrett, Phylogeny of the Simbu serogroup of the genus Bunyavirus. J. Gen. Virol. 82, 2173-2181 (2001).
  • 7. E. F. Dunn, D. C. Pritlove, H. Jin, R. M. Elliott, Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins. Virology 211 133-143 (1995).
  • 8. X. Shi, A. Kohl, V. H., Leonard, P. Li, A. McLees, R. M. Elliott, Requirement of the N-terminal region of orthobunyavirus nonstructural protein NSm for virus assembly and morphogenesis. J. Virol. 80, 8089-8099 (2006).
  • 9. A. Bridgen, R. M. Elliot, Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proc. Natl. Acad. Sci. U.S.A. 93, 15400-15404 (1996).
  • 10. A. C. Lowen, C. Noonan, A. McLees, R. M. Elliotts, Efficient bunyavirus rescue from cloned cDNA. Virology 330, 493-500 (2004).
  • 11. M. J. Schnell, T. Mebatsion, K. K. Conzelmann, Infectious rabies viruses from cloned cDNA. EMBO Journal 13, 4195-4203 (1994).
  • 12. U. J. Buchholz, S. Finke, K. K. Conzelmann, Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV promotor. J. Virol. 73, 251-259 (1999).
  • 13. M. Geiser, R. Cebe, D. Drewello, R. Schmitz, Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase. Biotechniques 31, 88-90, 92 (2001).
  • 14. M. Habjan, N. Penski, M. Spiegel, F. Weber, T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus. J Gen Virol 89, 2157-2166 (2008).

Claims
  • 1. An isolated nucleic acid molecule comprising the complete genomic sequence of a Schmallenberg virus (SBV) genome segment, wherein said molecule comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence having at least 97.8% sequence identity with the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:7,a nucleic acid sequence having at least 82.2% sequence identity with the nucleic acid sequence of SEQ ID NO:2,a nucleic acid sequence having at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO:3, andany combinations thereof.
  • 2. The nucleic acid molecule of claim 1 comprising the complete genomic sequence of the S segment of Schmallenberg virus, wherein said molecule comprises a nucleic acid sequence having at least 97.8% sequence identity with the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:7.
  • 3. The nucleic acid molecule of claim 1 comprising the complete genomic sequence of the M segment of Schmallenberg virus, wherein said molecule comprises a nucleic acid sequence having at least 82.2% sequence identity with the nucleic acid sequence of SEQ ID NO:2.
  • 4. The nucleic acid molecule of claim 1 comprising the complete genomic sequence of the L segment of Schmallenberg virus, wherein said molecule comprises a nucleic acid sequence having at least 93% sequence identity with the nucleic acid sequence of SEQ ID NO:3.
  • 5. A combination of two nucleic acid molecules selected from the group consisting of the nucleic acid molecule of claim 2, the nucleic acid molecule of claim 3, and the nucleic acid molecule of claim 4.
  • 6. A combination of the nucleic acid molecule of claim 2, the nucleic acid molecule of claim 3, and the nucleic acid molecule of claim 4.
  • 7. The combination of claim 6, wherein the nucleic acid molecules contained therein are capable of producing infectious Schmallenberg virus when transfected into cells.
  • 8. The combination of claim 7, wherein the virus is able to induce SBV viremia in mammals and/or insects.
  • 9. A cDNA construct comprising a cDNA molecule according to claim 1.
  • 10. A combination of two different cDNA constructs selected from the group consisting of a cDNA construct comprising a cDNA molecule according to claim 2,a cDNA construct comprising a cDNA molecule according to claim 3, anda cDNA construct comprising a cDNA molecule according to claim 4.
  • 11. A combination of the cDNA constructs comprising: a cDNA construct comprising a cDNA molecule according to claim 2,a cDNA construct comprising a cDNA molecule according to claim 3, anda cDNA construct comprising a cDNA molecule according to claim 4.
  • 12. An RNA transcript of the cDNA construct of claim 9.
  • 13. A combination of two RNA transcripts selected from the group consisting of: an RNA transcript of a cDNA construct comprising a cDNA molecule according to claim 2,an RNA transcript of a cDNA construct comprising a cDNA molecule according to claim 3, andan RNA transcript of a cDNA construct comprising a cDNA molecule according to claim 4.
  • 14. A combination of the RNA transcripts comprising: an RNA transcript of a cDNA construct comprising a DNA molecule according to claim 2,an RNA transcript of a cDNA construct comprising a cDNA molecule according to claim 3, andan RNA transcript of a cDNA construct comprising the cDNA molecule according to claim 4.
  • 15. A cell transfected with the cDNA construct of claim 9.
  • 16. A cell transfected with a combination of cDNA constructs according to claim 10.
  • 17. A cell transfected with a combination of cDNA constructs according to claim 11.
  • 18. A cell transfected with the RNA transcript of claim 12.
  • 19. A cell transfected with a combination of RNA transcripts according to claim 13.
  • 20. A cell transfected with a combination of RNA transcripts according to claim 14.
  • 21. A Schmallenberg virus produced by the cell of claim 15.
  • 22. A Schmallenberg virus produced by the cell of claim 16.
  • 23. A Schmallenberg virus produced by the cell of claim 17.
  • 24. A Schmallenberg virus produced by the cell of claim 18.
  • 25. A Schmallenberg virus produced by the cell of claim 19.
  • 26. A Schmallenberg virus produced by the cell of claim 20.
  • 27. A Schmallenberg virus whose genome comprises a nucleic acid molecule according to claim 1.
  • 28. A Schmallenberg virus whose genome comprises a nucleic acid molecule according to a combination of nucleic acid molecules according to claim 5.
  • 29. A method for producing a Schmallenberg virus comprising transfecting a cell with the cDNA construct of claim 9.
  • 30. A method for producing a Schmallenberg viruses comprising transfecting a cell with the cDNA construct of the combination of cDNA constructs according to claim 10.
  • 31. A method for producing a Schmallenberg viruses comprising transfecting a cell with the cDNA construct of the combination of cDNA constructs according to claimll.
  • 32. A method for producing a Schmallenberg virus comprising transfecting a host cell with the RNA transcript of claim 12.
  • 33. A method for producing a Schmallenberg virus comprising transfecting a host cell with the RNA transcript of the combination of RNA transcripts according to claim 13.
  • 34. A method for producing a Schmallenberg virus comprising transfecting a host cell with the RNA transcript of the combination of RNA transcripts according to claim 14.
  • 35. A composition comprising a nucleic acid molecule of claim 1 suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient.
  • 36. A composition comprising a combination of nucleic acid molecules according to claim 5, suspended in a suitable amount of a pharmaceutically acceptable diluent or excipient.
  • 37. The cells according to claim 15, wherein the cell contains an RNA polymerase.
  • 38. The cells according to claim 37, wherein the cell expresses the RNA polymerase.
Priority Claims (1)
Number Date Country Kind
12170630.3 Jun 2012 EP regional