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Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus
Virus Research 77 (2001) 19 – 23 www.elsevier.com/locate/virusres
Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus Alan M. Betts *, David M. Stone The Centre for the En6ironment, Fisheries and Aquaculture Science, Weymouth Laboratory, Barrack Road, Weymouth, Dorset DT4 8UB, UK Received 20 July 2000; received in revised form 16 October 2000; accepted 16 October 2000
Abstract A mammalian expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used to demonstrate that CAT could be successfully used as a reporter system in fish cells growing at low temperatures. We then constructed a viral haemorrhagic septicaemia virus (VHSV) minigenome by cloning the CAT reporter gene between the viral leader and trailer sequences. This construct was used in transfection experiments with helper VHSV to demonstrate that the minigenome can be encapsidated and transcribed by helper virus proteins. In addition, passaging of viruses collected from cells expressing the minigenome showed that the minigenome was being packaged and replicated in the presence of helper virus. These experiments provide the initiating steps for a reverse genetics system for VHSV. © 2001 Elsevier Science B.V. All rights reserved. Keywords: VHSV; Minigenome; Helper virus; CAT
Viral haemorrhagic septicaemia virus (VHSV) is a fish rhabdovirus and the causative agent of viral haemorrhagic septicaemia, a disease causing considerable losses in farmed trout throughout Europe. The virus usually causes skin haemorrhages and haemorrhaging of the kidney and liver, with mortality rates as high as 90%. Initial indications are that the Pacific and Atlantic marine strains of VHSV are considerably less virulent for rainbow trout than those isolated from freshwater species. However, the mutation * Corresponding author. Tel.: + 44-130-5206647; fax: + 44130-5206601. E-mail address: [email protected] (A.M. Betts).
rate of RNA viruses is known to be high and it is possible therefore, that intensive farming conditions could provide the selective pressures that favour the virulent strains of VHSV that would subsequently lead to the development of a VHS outbreak. VHSV has a non-segmented negative-sense RNA genome of 11,161 nucleotides coding for five structural proteins: the nucleocapsid (N), the RNA polymerase associated protein (P), the matrix protein (M), the glycoprotein (G) and an RNA polymerase (L). In addition, a non-structural protein termed the Nv protein is encoded between the G and L genes. The complete nucleotide sequence of VHSV has been determined
0168-1702/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1702(01)00261-1
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A.M. Betts, D.M. Stone / Virus Research 77 (2001) 19–23
(Schutze et al., 1999) and several strains of VHSV have recently been sequenced (Betts and Stone, 2000). However, coding regions involved in the determination of virulence have not yet been identified and discriminating between virulent and avirulent strains other than by experimental challenge remains problematic. There have been significant advances recently in the recovery of non-segmented negative-strand RNA viruses entirely from cDNA (reviewed by Conzelmann, 1998; Peosz et al., 1999). Such systems allow for manipulation of viral RNA genomes and can provide an invaluable tool for
the investigation of viral pathogenicity. Therefore we have carried out the initial stages in the development of a reverse genetics system for VHSV by constructing a VHSV minigenome and demonstrating replication and rescue of this construct by helper virus at the low temperatures required for VHSV replication. In this study we have used VHSV strain 96-43, isolated from an Atlantic herring caught in the English Channel that has been shown to be avirulent for rainbow trout under experimental conditions (Dixon et al., 1997). This virus strain shares \ 98.6% amino acid sequence identity with the
Fig. 1. (A) Schematic representation of the VHSV minigenome construct. Genetic element abbreviations are: T7 promoter (T7-P), VHSV 3% leader (Leader), chloramphenicol acetyltransferase (CAT), VHSV 5% trailer (Trailer), hepatitis delta virus ribozyme (HDV) and T7 terminator (T7-T). (B) Nucleotide sequence of the T7 promoter, 3% and 5% terminal ends of VHSV and the start of the hepatitis delta ribozyme sequence are shown. Arrows indicate the transcription start site of T7 RNA polymerase and the ribozyme cleavage site.
Fig. 2. The effect of temperature on CAT production. EPC cells were transfected with plasmid pcDNA3CAT at various temperatures and incubated for 48 h before CAT production was analysed.
A.M. Betts, D.M. Stone / Virus Research 77 (2001) 19–23
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Fig. 3. Replication and transcription of a VHSV minigenome by helper virus. EPC cells were transfected with pVHSVCAT or infected with VHSV as negative controls. Cells were also transfected with pcDNA3CAT as a positive control. Cells were then infected with VHSV and subsequently transfected with in vitro transcribed pVHSVCAT and CAT production was assayed. Virus was harvested from minigenome transfected plates and passaged onto fresh EPC cells which were then assayed for CAT production.
virulent isolates 14-58 and Hededam (Betts and Stone, 2000) making it an ideal candidate for determining which amino acids are involved in the determination of virulence for rainbow trout. Viruses were grown in EPC cells and genomic RNA extracted as described previously (Stone et al., 1997). A minigenome was then constructed. Primers were designed for RT-PCR of nucleotides 1-168 of the 3%-leader sequence and nucleotides 11010– 11161 of the 5%-trailer sequence of the VHSV genome (Schutze et al., 1999; Betts and Stone, 2000). First strand cDNA synthesis and PCR were carried out in duplicate using standard protocols (Sambrook et al., 1989). Products were cloned into the pGEM-T vector to create pGEMT-LEADER and pGEMT-TRAILER according to the manufacturer’s protocol (Promega) and several clones were sequenced in both directions using an Applied Biosystems 310 automated sequencer. A primer containing the T7 promoter and two additional G residues and the first 15 nucleotides of the leader sequence and a primer containing the final 15 nucleotides of the leader sequence and the first 15 nucleotides of the chloramphenicol acetyltransferase (CAT) gene were designed. These primers were used in a PCR reaction with pfu DNA polymerase (Promega) and plasmid pGEMT-LEADER to amplify a T7leader PCR product containing 15 nucleotides of
the CAT gene. The full open reading frame of CAT was then PCR amplified from plasmid pcDNA3CAT and joined to the T7-leader product by overlapping PCR. Primers were then designed to amplify the trailer region of VHSV from pGEMT-TRAILER containing the final 15 nucleotides of the CAT gene and the first 15 nucleotides of the hepatitis delta ribozyme (HDV) sequence. This product was joined to the T7leader-CAT construct using overlapping PCR to produce a T7-leader-CAT-trailer product. DNA fragments corresponding to the HDV ribozyme and the T7 terminator were amplified from plasmid pOLTV5 (Peeters et al., 1999) and joined to the T7-leader-CAT-trailer construct as described previously. The resulting construct was ligated into the mammalian expression vector pCI (Promega) creating pVHSVCAT (Fig. 1). Constructs were sequenced in duplicate to confirm that errors had not been introduced during PCR. Minigenome constructs were linearised with Sma1 and 1 mg was transcribed in vitro using the Ribomax T7 transcription kit (Promega). EPC cell monolayers grown in six well plates were infected at a m.o.i of 1 with VHSV and incubated at 18°C for 1 h. The cells were then washed with PBS and transfected with 5 mg of in vitro transcribed RNA using Transfast transfection reagent (Promega). The cells were incubated at 18°C for 48 h and
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A.M. Betts, D.M. Stone / Virus Research 77 (2001) 19–23
then assayed for CAT production using a CAT ELISA (Roche Biochemicals). As a positive control for transfections, EPC cells were transfected with 1 mg of the mammalian expression vector pcDNA3CAT (Invitrogen) which contains the CAT gene under control of the CMV promoter. Initial experiments were carried out using pcDNA3CAT to optimise the transfection conditions. In addition, a range of temperatures from 17 to 28°C were tested to check that a functional CAT protein could be expressed in fish cells at the low temperatures required for VHSV replication. The CAT activity increased from 90 pg/well at 17°C to 1590 pg/well at 28°C (Fig. 2) and probably reflects an increase in the efficiency of the CMV promoter in pcDNA3CAT at higher temperatures. Although CAT production was 18-fold lower at 17°C than at 28°C the results demonstrate that the CAT reporter system can be used in fish cells grown at low temperatures. As VHSV strain 96–43 replicates most efficiently between 14 and 18°C and does not produce a productive infection at temperatures \ 20°C (Betts, unpublished observations), subsequent VHSV minigenome experiments were carried out at 18°C. To confirm that the leader and trailer sequences of the VHSV minigenome would allow replication and packaging by helper virus, EPC cells were infected with VHSV and then transfected with in vitro transcribed minigenome RNA. A similar approach has been used for other single-stranded negative-sense RNA viruses (Baron and Barrett, 1997; Sidhu et al., 1995; Peeters et al., 1999; Yunus et al., 1999) and this strategy relies upon the transfected minigenome RNA becoming encapsidated and then transcribed and replicated by helper virus proteins. The results are illustrated in Fig. 3. There was no background production of CAT in EPC cells or in cells infected with VHSV alone. Cells infected with helper VHSV and then transfected with in vitro transcribed pVHSVCAT expressed CAT, albeit at relatively low levels (Fig. 3, ‘pVHSVCAT +VHSV’). Identical experiments were carried out with a minigenome construct that lacked the two additional G residues from the T7 promoter, however this construct produced very low levels of CAT activity (data not shown).
This suggests that the two extra G residues are tolerated by the virus and suggests both transcription and encapsidation of the model genome by helper virus. Viruses were then harvested from the medium of infected and transfected cells, passaged onto fresh cells and assayed for CAT activity (Fig. 3, ‘passaged pVHSVCAT’). These cells showed an increased CAT production compared to minigenome plus helper virus and demonstrates that the minigenome is being replicated. Plasmids encoding the VHSV N, P and L genes have been constructed in the expression vector pcDNA3.1 under the control of the T7 promoter and we are currently constructing a vector containing a cDNA copy of the entire VHSV genome. These constructs will be used in a vaccinia virus driven rescue system similar to that used for recovery of recombinant snakehead rhabdovirus (Johnson et al., 2000). However, the low temperature required by fish viruses to replicate efficiently may be problematic as the T7 vaccinia virus expression system is less efficient at low temperatures. We are currently optimising our minigenome recovery system using the viral N, P and L constructs prior to the recovery of infectious VHSV entirely from cDNA. Acknowledgements We would like to thank Dr Ben Peeters and Dr Michael Baron for their kind gifts of plasmids pOLTV5 and pMDB1, respectively. This work was funded by MAFF contract number F1118. References Baron, M.D., Barrett, T., 1997. Rescue of rinderpest virus from cloned cDNA. J. Virol. 71, 1265 – 1271. Betts, A.M., Stone, D.M., 2000. Nucleotide sequence analysis of the entire coding regions of virulent and avirulent strains of viral haemorrhagic septicaemia virus. Virus Genes 20, 259 – 262. Conzelmann, K.K., 1998. Nonsegmented negative-strand viruses: genetics and manipulation of viral genomes. Ann. Rev. Genet. 132, 123 – 162. Dixon, P.F., Feist, S., Kehoe, E., Parry, L., Stone, D.M., Way, K., 1997. Isolation of viral haemorrhagic septicaemia virus from Atlantic herring, Clupea harengus from the English channel. Dis. Aquat. Org. 30, 81 – 89.
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Sidhu, M.S., Chan, J., Kaelin, K., Speilhofer, P., Schneider, H., Masurekar, M., Dowling, P.C., Billeter, M.A., Udem, S.A., 1995. Rescue of synthetic measles virus minireplicons: measles genomic termini direct efficient expression and propagation of a reporter gene. Virology 208, 800 – 807. Stone, D.M., Way, K., Dixon, P.F., 1997. Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia virus (VHS) viruses from different geographical areas: a link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.). J. Gen. Virol. 78, 1319 – 1326. Yunus, A.S., Krishnamurthy, S., Pastey, M.K., Huang, Z., Khattar, S.K., Collins, P.L., Samai, S.K., 1999. Rescue of a bovine respiratory syncytial virus genomic RNA analogue by bovine, human and ovine respiratory viruses confirms the ‘functional integrity’ and ‘cross-recognition’ of BRSV cis-acting elements by HRSV and ORSV. Arch. Virol. 144, 1977 – 1990.
Johnson, M.C., Simon, B.E., Kim, C.H., Leong, J.C., 2000. Production of recombinant snakehead rhabdovirus: The Nv protein is not required for viral replication, J. Virol. 2343 – 2350 Peeters, B.P.H., Leeuw, G.K., Gielkens, A.L.J., 1999. Rescue of Newcastle disease virus from cloned cDNA: Evidence that cleavability of the fusion protein is a major determinant of virulence. J. Virol. 73, 5001 –5009. Peosz, A., He, B., Lamb, R.A., 1999. Reverse genetics of negative-strand RNA viruses: closing the circle. Proc. Natl. Acad. Sci. USA 96, 8804 – 8806. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York. Schutze, H., Mundt, E., Mettenleiter, T.C., 1999. Complete genomic sequence of viral haemorrhagic septicaemia virus, a fish rhabdovirus. Virus Genes 19, 59 –65.
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Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus Alan M. Betts *, David M. Stone The Centre for the En6ironment, Fisheries and Aquaculture Science, Weymouth Laboratory, Barrack Road, Weymouth, Dorset DT4 8UB, UK Received 20 July 2000; received in revised form 16 October 2000; accepted 16 October 2000
Abstract A mammalian expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used to demonstrate that CAT could be successfully used as a reporter system in fish cells growing at low temperatures. We then constructed a viral haemorrhagic septicaemia virus (VHSV) minigenome by cloning the CAT reporter gene between the viral leader and trailer sequences. This construct was used in transfection experiments with helper VHSV to demonstrate that the minigenome can be encapsidated and transcribed by helper virus proteins. In addition, passaging of viruses collected from cells expressing the minigenome showed that the minigenome was being packaged and replicated in the presence of helper virus. These experiments provide the initiating steps for a reverse genetics system for VHSV. © 2001 Elsevier Science B.V. All rights reserved. Keywords: VHSV; Minigenome; Helper virus; CAT
Viral haemorrhagic septicaemia virus (VHSV) is a fish rhabdovirus and the causative agent of viral haemorrhagic septicaemia, a disease causing considerable losses in farmed trout throughout Europe. The virus usually causes skin haemorrhages and haemorrhaging of the kidney and liver, with mortality rates as high as 90%. Initial indications are that the Pacific and Atlantic marine strains of VHSV are considerably less virulent for rainbow trout than those isolated from freshwater species. However, the mutation * Corresponding author. Tel.: + 44-130-5206647; fax: + 44130-5206601. E-mail address: [email protected] (A.M. Betts).
rate of RNA viruses is known to be high and it is possible therefore, that intensive farming conditions could provide the selective pressures that favour the virulent strains of VHSV that would subsequently lead to the development of a VHS outbreak. VHSV has a non-segmented negative-sense RNA genome of 11,161 nucleotides coding for five structural proteins: the nucleocapsid (N), the RNA polymerase associated protein (P), the matrix protein (M), the glycoprotein (G) and an RNA polymerase (L). In addition, a non-structural protein termed the Nv protein is encoded between the G and L genes. The complete nucleotide sequence of VHSV has been determined
0168-1702/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0168-1702(01)00261-1
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A.M. Betts, D.M. Stone / Virus Research 77 (2001) 19–23
(Schutze et al., 1999) and several strains of VHSV have recently been sequenced (Betts and Stone, 2000). However, coding regions involved in the determination of virulence have not yet been identified and discriminating between virulent and avirulent strains other than by experimental challenge remains problematic. There have been significant advances recently in the recovery of non-segmented negative-strand RNA viruses entirely from cDNA (reviewed by Conzelmann, 1998; Peosz et al., 1999). Such systems allow for manipulation of viral RNA genomes and can provide an invaluable tool for
the investigation of viral pathogenicity. Therefore we have carried out the initial stages in the development of a reverse genetics system for VHSV by constructing a VHSV minigenome and demonstrating replication and rescue of this construct by helper virus at the low temperatures required for VHSV replication. In this study we have used VHSV strain 96-43, isolated from an Atlantic herring caught in the English Channel that has been shown to be avirulent for rainbow trout under experimental conditions (Dixon et al., 1997). This virus strain shares \ 98.6% amino acid sequence identity with the
Fig. 1. (A) Schematic representation of the VHSV minigenome construct. Genetic element abbreviations are: T7 promoter (T7-P), VHSV 3% leader (Leader), chloramphenicol acetyltransferase (CAT), VHSV 5% trailer (Trailer), hepatitis delta virus ribozyme (HDV) and T7 terminator (T7-T). (B) Nucleotide sequence of the T7 promoter, 3% and 5% terminal ends of VHSV and the start of the hepatitis delta ribozyme sequence are shown. Arrows indicate the transcription start site of T7 RNA polymerase and the ribozyme cleavage site.
Fig. 2. The effect of temperature on CAT production. EPC cells were transfected with plasmid pcDNA3CAT at various temperatures and incubated for 48 h before CAT production was analysed.
A.M. Betts, D.M. Stone / Virus Research 77 (2001) 19–23
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Fig. 3. Replication and transcription of a VHSV minigenome by helper virus. EPC cells were transfected with pVHSVCAT or infected with VHSV as negative controls. Cells were also transfected with pcDNA3CAT as a positive control. Cells were then infected with VHSV and subsequently transfected with in vitro transcribed pVHSVCAT and CAT production was assayed. Virus was harvested from minigenome transfected plates and passaged onto fresh EPC cells which were then assayed for CAT production.
virulent isolates 14-58 and Hededam (Betts and Stone, 2000) making it an ideal candidate for determining which amino acids are involved in the determination of virulence for rainbow trout. Viruses were grown in EPC cells and genomic RNA extracted as described previously (Stone et al., 1997). A minigenome was then constructed. Primers were designed for RT-PCR of nucleotides 1-168 of the 3%-leader sequence and nucleotides 11010– 11161 of the 5%-trailer sequence of the VHSV genome (Schutze et al., 1999; Betts and Stone, 2000). First strand cDNA synthesis and PCR were carried out in duplicate using standard protocols (Sambrook et al., 1989). Products were cloned into the pGEM-T vector to create pGEMT-LEADER and pGEMT-TRAILER according to the manufacturer’s protocol (Promega) and several clones were sequenced in both directions using an Applied Biosystems 310 automated sequencer. A primer containing the T7 promoter and two additional G residues and the first 15 nucleotides of the leader sequence and a primer containing the final 15 nucleotides of the leader sequence and the first 15 nucleotides of the chloramphenicol acetyltransferase (CAT) gene were designed. These primers were used in a PCR reaction with pfu DNA polymerase (Promega) and plasmid pGEMT-LEADER to amplify a T7leader PCR product containing 15 nucleotides of
the CAT gene. The full open reading frame of CAT was then PCR amplified from plasmid pcDNA3CAT and joined to the T7-leader product by overlapping PCR. Primers were then designed to amplify the trailer region of VHSV from pGEMT-TRAILER containing the final 15 nucleotides of the CAT gene and the first 15 nucleotides of the hepatitis delta ribozyme (HDV) sequence. This product was joined to the T7leader-CAT construct using overlapping PCR to produce a T7-leader-CAT-trailer product. DNA fragments corresponding to the HDV ribozyme and the T7 terminator were amplified from plasmid pOLTV5 (Peeters et al., 1999) and joined to the T7-leader-CAT-trailer construct as described previously. The resulting construct was ligated into the mammalian expression vector pCI (Promega) creating pVHSVCAT (Fig. 1). Constructs were sequenced in duplicate to confirm that errors had not been introduced during PCR. Minigenome constructs were linearised with Sma1 and 1 mg was transcribed in vitro using the Ribomax T7 transcription kit (Promega). EPC cell monolayers grown in six well plates were infected at a m.o.i of 1 with VHSV and incubated at 18°C for 1 h. The cells were then washed with PBS and transfected with 5 mg of in vitro transcribed RNA using Transfast transfection reagent (Promega). The cells were incubated at 18°C for 48 h and
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then assayed for CAT production using a CAT ELISA (Roche Biochemicals). As a positive control for transfections, EPC cells were transfected with 1 mg of the mammalian expression vector pcDNA3CAT (Invitrogen) which contains the CAT gene under control of the CMV promoter. Initial experiments were carried out using pcDNA3CAT to optimise the transfection conditions. In addition, a range of temperatures from 17 to 28°C were tested to check that a functional CAT protein could be expressed in fish cells at the low temperatures required for VHSV replication. The CAT activity increased from 90 pg/well at 17°C to 1590 pg/well at 28°C (Fig. 2) and probably reflects an increase in the efficiency of the CMV promoter in pcDNA3CAT at higher temperatures. Although CAT production was 18-fold lower at 17°C than at 28°C the results demonstrate that the CAT reporter system can be used in fish cells grown at low temperatures. As VHSV strain 96–43 replicates most efficiently between 14 and 18°C and does not produce a productive infection at temperatures \ 20°C (Betts, unpublished observations), subsequent VHSV minigenome experiments were carried out at 18°C. To confirm that the leader and trailer sequences of the VHSV minigenome would allow replication and packaging by helper virus, EPC cells were infected with VHSV and then transfected with in vitro transcribed minigenome RNA. A similar approach has been used for other single-stranded negative-sense RNA viruses (Baron and Barrett, 1997; Sidhu et al., 1995; Peeters et al., 1999; Yunus et al., 1999) and this strategy relies upon the transfected minigenome RNA becoming encapsidated and then transcribed and replicated by helper virus proteins. The results are illustrated in Fig. 3. There was no background production of CAT in EPC cells or in cells infected with VHSV alone. Cells infected with helper VHSV and then transfected with in vitro transcribed pVHSVCAT expressed CAT, albeit at relatively low levels (Fig. 3, ‘pVHSVCAT +VHSV’). Identical experiments were carried out with a minigenome construct that lacked the two additional G residues from the T7 promoter, however this construct produced very low levels of CAT activity (data not shown).
This suggests that the two extra G residues are tolerated by the virus and suggests both transcription and encapsidation of the model genome by helper virus. Viruses were then harvested from the medium of infected and transfected cells, passaged onto fresh cells and assayed for CAT activity (Fig. 3, ‘passaged pVHSVCAT’). These cells showed an increased CAT production compared to minigenome plus helper virus and demonstrates that the minigenome is being replicated. Plasmids encoding the VHSV N, P and L genes have been constructed in the expression vector pcDNA3.1 under the control of the T7 promoter and we are currently constructing a vector containing a cDNA copy of the entire VHSV genome. These constructs will be used in a vaccinia virus driven rescue system similar to that used for recovery of recombinant snakehead rhabdovirus (Johnson et al., 2000). However, the low temperature required by fish viruses to replicate efficiently may be problematic as the T7 vaccinia virus expression system is less efficient at low temperatures. We are currently optimising our minigenome recovery system using the viral N, P and L constructs prior to the recovery of infectious VHSV entirely from cDNA. Acknowledgements We would like to thank Dr Ben Peeters and Dr Michael Baron for their kind gifts of plasmids pOLTV5 and pMDB1, respectively. This work was funded by MAFF contract number F1118. References Baron, M.D., Barrett, T., 1997. Rescue of rinderpest virus from cloned cDNA. J. Virol. 71, 1265 – 1271. Betts, A.M., Stone, D.M., 2000. Nucleotide sequence analysis of the entire coding regions of virulent and avirulent strains of viral haemorrhagic septicaemia virus. Virus Genes 20, 259 – 262. Conzelmann, K.K., 1998. Nonsegmented negative-strand viruses: genetics and manipulation of viral genomes. Ann. Rev. Genet. 132, 123 – 162. Dixon, P.F., Feist, S., Kehoe, E., Parry, L., Stone, D.M., Way, K., 1997. Isolation of viral haemorrhagic septicaemia virus from Atlantic herring, Clupea harengus from the English channel. Dis. Aquat. Org. 30, 81 – 89.
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Sidhu, M.S., Chan, J., Kaelin, K., Speilhofer, P., Schneider, H., Masurekar, M., Dowling, P.C., Billeter, M.A., Udem, S.A., 1995. Rescue of synthetic measles virus minireplicons: measles genomic termini direct efficient expression and propagation of a reporter gene. Virology 208, 800 – 807. Stone, D.M., Way, K., Dixon, P.F., 1997. Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia virus (VHS) viruses from different geographical areas: a link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.). J. Gen. Virol. 78, 1319 – 1326. Yunus, A.S., Krishnamurthy, S., Pastey, M.K., Huang, Z., Khattar, S.K., Collins, P.L., Samai, S.K., 1999. Rescue of a bovine respiratory syncytial virus genomic RNA analogue by bovine, human and ovine respiratory viruses confirms the ‘functional integrity’ and ‘cross-recognition’ of BRSV cis-acting elements by HRSV and ORSV. Arch. Virol. 144, 1977 – 1990.
Johnson, M.C., Simon, B.E., Kim, C.H., Leong, J.C., 2000. Production of recombinant snakehead rhabdovirus: The Nv protein is not required for viral replication, J. Virol. 2343 – 2350 Peeters, B.P.H., Leeuw, G.K., Gielkens, A.L.J., 1999. Rescue of Newcastle disease virus from cloned cDNA: Evidence that cleavability of the fusion protein is a major determinant of virulence. J. Virol. 73, 5001 –5009. Peosz, A., He, B., Lamb, R.A., 1999. Reverse genetics of negative-strand RNA viruses: closing the circle. Proc. Natl. Acad. Sci. USA 96, 8804 – 8806. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York. Schutze, H., Mundt, E., Mettenleiter, T.C., 1999. Complete genomic sequence of viral haemorrhagic septicaemia virus, a fish rhabdovirus. Virus Genes 19, 59 –65.
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