Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a, D-17498 Insel Riems, Germany1
Intervet International B.V., Wim de Körverstraat 35, NL-5830 AA Boxmeer, The Netherlands2
Author for correspondence: Angela Römer-Oberd örfer.Fax +49 38351 7219. e-mail angela.oberdoerfer{at}rie.bfav.de
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Abstract |
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Introduction |
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Since several live vaccines currently available are associated with mild disease signs and weight loss, introduction of an improved live vaccine is greatly desired by the poultry industry. Development of improved attenuated vaccines is currently facilitated by the availability of powerful recombinant DNA technologies. These techniques were initially applicable only to DNA viruses and later to positive- strand RNA viruses. Very recently, however, the large group of negative- strand RNA viruses, to which NDV belongs, also became amenable to genetic engineering (reviewed by Palese et al., 1996 ; Conzelmann, 1998
).
NDV is a member of the genus Rubulavirus in the family Paramyxoviridae (Rima et al., 1995 ) and contains a nonsegmented single-stranded RNA genome of negative polarity. The NDV genome encodes six structural proteins, namely, the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), haemagglutininneuraminidase (HN) and the RNA- dependent RNA polymerase (L). Apart from the six structural proteins, NDV was also shown to edit its P gene mRNA and to encode two additional gene products, designated V and W proteins (Steward et al., 1993
). As for all other members of the order Mononegavirales, the genomic RNA of NDV, together with the NP, P and L proteins, forms the ribonucleoprotein (RNP) complex, a structure that serves as template for the viral RNA polymerase. Therefore, in contrast to many DNA viruses and positive-strand RNA viruses, it is not possible to initiate an infectious cycle of negative-strand RNA viruses by simple transfection of cells with naked genomic nucleic acid. Only after intracellular reconstitution of an RNP complex entirely from cDNA is recovery of recombinant viruses possible. A system based on cotransfection of a plasmid expressing full-length antigenomic RNA together with three other plasmids encoding viral N, P, and L proteins under control of the phage T7 RNA polymerase promoter, which resulted in the recovery of recombinant viruses, was first developed for rabies virus (Schnell et al., 1994
), and subsequently for other members of the Mononegavirales (for a recent review see Conzelmann, 1998
). Most of the recovery systems described to date utilize recombinant vaccinia virus as a source of bacteriophage T7 RNA polymerase (Schnell et al., 1994
; Collins et al., 1995
; Garcin et al., 1995
; Lawson et al., 1995
; Whelan et al., 1995
; Baron & Barrett, 1997
; Durbin et al., 1997
; He et al., 1997
; Hoffmann & Banerjee, 1997
). A modification of the initial protocol was introduced by replacing vaccinia virus with cell lines stably expressing T7 RNA polymerase. This approach was successfully applied to recover attenuated or slowly growing recombinant measles virus and bovine respiratory syncytial virus (Radecke et al., 1995
; Buchholz et al., 1999
), thus circumventing difficult and time-consuming purification steps of a newly generated recombinant virus.
Here, we report the cDNA cloning and sequencing of the entire NDV genome as well as the establishment of a system that allows the genetic manipulation of NDV. We show that the growth characteristics and pathogenicity of the recombinant NDV are indistinguishable from parental virus, confirming its authenticity. This recovery system, which is based on a vaccine strain of NDV, not only provides the possibility for experimental investigation of NDV molecular biology, but also serves as a basis for the development of improved marker/vector vaccines.
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Methods |
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Sequencing.
Initial PCR fragments of Clone-30 and the assembled full-length cDNA clone as well as support plasmids encoding NDV NP, P and L proteins were sequenced using an automated sequencer (LiCor, MWG Biotech). Sequences were analysed using Wisconsin package version 9.1 (Genetic Computer Group, Madison, WI, USA).
Transfection and recovery of recombinant NDV.
Transfection experiments were done using BHK 21 cells, clone BSR T7/5, stably expressing the phage T7 RNA polymerase (Buchholz et al. , 1999 ). Cells grown to 80% confluency in 32 mm diameter dishes were transfected with a total amount of 10 µg DNA (5 µg pflNDV-1, 2 µg pCiteNP, 2 µg pCiteP and 1 µg pCiteL) (Superfect transfection kit, Qiagen). Supernatants and cell monolayers were harvested at various times after transfection, cleared by centrifugation, and a volume of 200 µl was inoculated into the allantoic cavity of 10-day-old embryonated specific-pathogen-free (SPF) chicken eggs to amplify the recovered recombinant virus. The allantoic fluid was harvested 5 days after inoculation and tested for haemagglutinating activity (HA) (CEC, 1992
).
Indirect immunofluorescence assay (IFA).
Transfected cells were fixed with 3% paraformaldehyde at various times after transfection. To screen for the presence of infectious NDV, an IFA was performed, using monoclonal antibody HN-10 (Werner et al., 1999 ) directed to NDV HN protein and FITC-conjugated goat anti-mouse IgG F(ab)2 (DAKO).
Virus growth.
QM7 cells (3·5x105 per 35 mm dish) were infected with wt Clone-30 or recombinant NDV at an m.o.i. of 1 and incubated at 37 °C in minimum essential medium supplemented with 10% foetal calf serum in a 5% CO2 atmosphere. Supernatants and infected cells were frozen at the indicated times. Titrations were done in duplicate in microwell plates. Briefly, 100 µl of tenfold serial virus dilutions were added to 104 cells per well. After 48 h of incubation, the cells were fixed with 3% paraformaldehyde, and the 50% tissue culture infective dose (TCID50) was determined by IFA using rabbit hyperimmune serum to NDV and FITC-conjugated swine anti-rabbit IgG F(ab)2 (DAKO). To analyse virus growth in embryonated eggs, the allantoic cavity of 10-day-old embryonated SPF chicken eggs was inoculated with 7x104 TCID50/0·2 ml recombinant and parental Clone-30 virus, respectively. Allantoic fluid was harvested and pooled at the indicated times. Titrations were done in duplicate on QM7 cells as described above.
RTPCR.
RNA from allantoic fluid was prepared using the Purescript RNA isolation kit (Biozym). RTPCR was performed using primers P1 (Clone-30 nt 126) and P10R (Clone-30 nt 639620) to amplify a fragment including the newly introduced MluI site in the noncoding region of the NP gene. Similarly, a fragment containing the MluI site in the noncoding region of the L gene was amplified using primers P10F (Clone-30 nt 1475714775) and P9R (see above). The RTPCR was essentially performed as described (Oberdörfer & Werner, 1998 ).
Determination of the intracerebral pathogenicity index (ICPI).
The ICPI of the recombinant NDV as well as of the parental wild- type virus was determined following intracerebral inoculation of 1-day- old SPF chickens with 8·0 log10 EID50 virus per chicken. The appearance of clinical signs and mortality was scored for 8 days as described by Alexander (1989) .
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Results |
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Recovery of infectious recombinant NDV from cDNA
For the generation of infectious NDV from cDNA, we used a vaccinia- virus-free system in which the T7 RNA polymerase was expressed in a cell line (BHK-21, clone BSR T7/5) stably transfected with the phage T7 RNA polymerase gene (Buchholz et al., 1999 ). Dishes of subconfluent cells were transfected with plasmids pflNDV-1, pCiteNP, pCiteP and pCiteL, to achieve simultaneous intracytoplasmic expression of RNP proteins and full-length antigenome RNA. To test whether the process led to intracellular reconstitution of RNPs and initiation of an infectious cycle, transfected cells were first examined for expression of HN protein. Forty-eight hours post-transfection, very few single cells exhibited weak fluorescence, whereas at 72 h post- transfection few but clearly stained cells were detected (Fig. 4
a). No positive signal was observed in untransfected controls. Since most host cells are not permissive for lentogenic NDV in cell-to-cell spread (Rott & Klenk, 1988
) because they lack a trypsin-like protease necessary for cleavage of the F0 protein, 200 µl of supernatants that were collected 24, 48 and 72 h after transfection were inoculated into the allantoic cavity of 10-day-old embryonated SPF chicken eggs. The allantoic fluid was harvested 5 days after inoculation and tested for HA. No HA was found in allantoic fluid from eggs inoculated with material harvested 24 and 48 h post-transfection, whereas after inoculation of supernatant harvested 72 h post-transfection we reproducibly found eggs positive with HA titres ranging from 2 to 2048. IFA was performed 48 h after inoculation of 200 µl of a 10 -1 dilution of HA-positive allantoic fluid into QM7 cells using monoclonal antibody HN-10 (Werner et al., 1999
), yielding a strong immunofluorescence staining of single cells, which is typical for lentogenic NDV (Fig. 4b
), thus proving the recovery of NDV entirely from cDNA.
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Discussion |
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Recovery systems for recombinant negative-strand RNA viruses are based on intracytoplasmic reconstitution of the RNP complex, which represents the template for the viral polymerase, and is the prerequisite needed to start an infectious cycle (reviewed by Palese et al., 1996 ; Conzelmann, 1998
). The expression systems most widely used depend on infection of cells with recombinant vaccinia virus (vTF-7-3 or MVA) (reviewed by Palese et al., 1996
; Conzelmann, 1998
), providing T7 RNA polymerase needed for expression of RNA and proteins from transfected plasmids. However, this entails several problems: (i) the cytopathic effect caused by vaccinia virus interferes with recovery of slow growing viruses; (ii) newly recovered virus has to be separated from vaccinia virus by filtration (Schnell et al., 1994
), by passage on cells that are not permissive for the vaccinia virus strain (Collins et al., 1995
) or by vaccinia virus inhibitors (Whelan et al., 1995
); (iii) vaccinia virus may cause RNA recombination during recovery (Garcin et al., 1995
). For the successful generation of recombinant negative-strand RNA viruses of slow-growing species like measles virus (Radecke et al., 1995
) and bovine respiratory syncytial virus (BRSV) (Buchholz et al. , 1999
), a vaccinia-virus-free recovery system is crucial. We therefore used a cell line stably expressing T7 RNA polymerase, which has proven to be suited for recovery of attenuated recombinant BRSV mutants (Buchholz et al., 1999
).
Lentogenic NDV strains possess an amino acid motif at the cleavage site of the precursor glycoprotein F0 which requires cleavage by a trypsin-like host cell protease to be transformed into the fusogenic active form. The cell line BSR T7/5 used for recovery is derived from BHK-21 cells, which do not support efficient propagation of lentogenic NDV. Like its biologically derived parent, recombinant Clone-30 cannot be propagated to high titres in this and other cell lines because virions containing uncleaved F glycoprotein lack full infection capacity (Rott & Klenk, 1988 ). Plasmid- derived intracellular reconstitution of Clone-30 RNPs led to the onset of a replicative cycle in BSR T7/5 cells, with few infected cells that could be demonstrated in IFA. Clarified supernatants of transfected cells were inoculated into the allantoic cavity of embryonated chicken eggs, possessing proteases of trypsin-like specificity, which reproducibly led to amplification of recombinant Clone-30. Thus, a system for recovery of recombinant lentogenic NDV was established that combines a cellular T7 expression system and a virus propagation step allowing amplification of few primary infectious recombinant virions originating from single recovery events.
Genome analogues of members of the paramyxovirus genus replicate efficiently only if the total genome length is a multiple of six, a requirement which is called the `rule of six' (Calain & Roux, 1993 ; reviewed by Kolakofsky et al., 1998
). All NDV genomes determined to date (Krishnamurthy & Samal, 1998
; de Leeuw & Peeters, 1999
; Phillips et al., 1998
) comprise 15186 nts, which is a multiple of six. This feature strongly suggests that the paramyxovirus `rule of six' also applies to NDV. Recombinant NDV could reproducibly be recovered using the approach described above, though the recovery events, as determined by IFA, were more than tenfold lower as compared to recovery events of recombinant BRSV when using the identical system (U. Buchholz, unpublished results). Like other negative-strand RNA virus rescue systems, full- length NDV and BRSV antigenome expression plasmids contain three additional G residues adjacent to the T7 promoter. These three G residues were shown to enhance the T7 polymerase-driven transcription (Pattnaik et al., 1992
). BRSV is a member of the pneumovirus genus, and thus does not obey the `rule of six'. One reason for the fewer recovery events observed in transfected dishes might be the need for trimming the extra G residues from the 5' antigenome end of NDV genome, so that the genome length remains a multiple of six. Trimming of extra G residues from the end of the genome is well documented for vesicular stomatitis virus, a rhabdovirus, although genome length in this case does not follow the `rule of six' (Pattnaik et al., 1992
). In the case of Sendai virus, it was reported that minigenomes containing the P editing site were constructed that did not obey the rule of six. After passage, they contained corrective nucleotide insertions or deletions in the P editing site (Kolakofsky et al. , 1998
). Therefore, RTPCR of the P editing site was done from egg-passaged recombinant NDV; subsequent sequencing revealed that the P editing site was unaltered (not shown). Thus, antigenomic RNAs or RNPs that should undergo a trimming process might give rise to lower rescue efficiency compared to RNAs that might be encapsidated and maintained as biologically active RNPs even with extra G residues.
The complete sequence of Clone-30 was highly similar to the sequence of La Sota (de Leeuw & Peeters, 1999 ), with the exception of the L gene which contains a double frameshift resulting in a 28 aa difference located in block V (Poch et al., 1990
). Block V represents a cluster of highest amino acid conservation among the members of the Mononegavirales, which includes one stretch of five highly conserved amino acids (Poch et al., 1990
). In contrast to the published NDV sequences of La Sota (de Leeuw & Peeters, 1999
) and Beaudette C (Krishnamurthy & Samal, 1998
), the sequences determined for Clone-30, for our La Sota isolate, and for strain Komarov contain the conserved block V motif common to members of Mononegavirales (Poch et al., 1990
; Fig. 3
). Moreover, recombinant Clone-30 containing the determined sequence was indistinguishable from its biologically derived parent, and similar in its properties to NDV strain La Sota.
The in vitro and in vivo characterization of recombinant NDV revealed a phenotype identical to parental virus, thus proving that the mutations artificially introduced had no biological effect, and moreover, that the recombinant system now available for NDV yields a faithful copy of the biologically derived virus.
By using the NDV recombinant system, defined mutants can be designed enabling the experimental investigation of virushost interactions and of the molecular basis of NDV pathogenesis. Characterization of recombinant NDVs with altered F protein cleavage sites will be helpful to elucidate the molecular basis of pathogenicity. Determination of the function of individual NDV proteins as well as of additional gene products, particularly V and W proteins that are synthesized as a result of P gene mRNA editing, will certainly be facilitated by the availability of this system. The evidence showing a correlation between Sendai virus pathogenicity and V protein expression (Kato et al., 1997 ) makes investigation in this area of NDV research appealing. Apart from giving insight into NDV pathogenesis, the above approaches will be useful for the design of safe and effective vaccines against this devastating poultry disease. Moreover, as shown for other negative-strand RNA viruses, it will be possible to use lentogenic NDV as a vaccine vector by expressing additional proteins to confer protection against other respiratory and intestinal pathogens of poultry.
Note added during revision. We acknowledge the successful recovery of another strain of NDV (Peeters et al., 1999 ) which was reported after submission of this article.
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Acknowledgments |
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Footnotes |
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References |
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Received 13 April 1999;
accepted 15 July 1999.