1 Department of Infectious Disease & Food Chain Quality, Institute for Animal Science and Health, PO Box 65, NL-8200 AB Lelystad, The Netherlands
2 Central Institute for Animal Disease Control, PO Box 2004, NL-8203 AA Lelystad, The Netherlands
Correspondence
Ben Peeters
b.p.h.peeters{at}id.wag-ur.nl
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ABSTRACT |
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Introduction |
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NDV contains a non-segmented single-stranded RNA genome of negative polarity with a size of 15186 nucleotides (Krishnamurthy & Samal, 1998; de Leeuw & Peeters, 1999
; Phillips et al., 1998
; Römer-Oberdorfer et al., 1999
) and contains six genes encoding the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), haemagglutininneuraminidase protein (HN) and large polymerase protein (L) (Lamb & Kolakofsky, 1996
).
NDV is an enveloped virus with two membrane proteins, one of which is involved in cell attachment and release (HN protein) and the other in mediating fusion of the viral envelope with cellular membranes (F protein). The F protein is synthesized as a precursor, F0, and is only fusogenic after cleavage into disulfide-linked F1 and F2 polypeptides. This cleavage is accomplished by host-cell proteases (Nagai et al., 1976; Morrison et al., 1985
; Ogasawara et al., 1992
). Thus, NDV becomes infective only when the precursor glycoprotein F0 is cleaved into F1 and F2. The ability to cleave F0 varies among different strains of NDV and is the main determinant of mortality and morbidity in infected chickens. Using mean death time (MDT) determined in chicken eggs, NDV strains can be classified as highly virulent (velogenic), intermediate (mesogenic) or non-virulent (lentogenic) (Beard & Hanson, 1984
). Within the highly virulent NDV strains, two types of virus can be distinguished. These strains are termed neurotropic velogenic or viscerotropic velogenic, based on the clinical signs induced (Alexander, 2000
).
Phylogenetic studies at the molecular level of the NDV F0 cleavage site have determined the consensus amino acid sequence 112R/K-R-Q-R/K-R-F117 for virulent and mesogenic strains and 112G/E-K/R-Q-G/E-R-L117 for NDV strains of low virulence (Collins et al., 1993). However, two exceptions to the consensus amino acid sequence for virulent strains have been observed for the so-called pigeon paramyxovirus type 1 (PPMV-1) isolates. These virulent isolates show fusion protein cleavage sites of 112GRQKRF117 (Collins et al., 1993
) and 112RRKKRF117 (Werner et al., 1999
).
The different F0 protein cleavage sites are the substrates for different kinds of cellular proteases (Kawahara et al., 1992; Sakaguchi et al., 1991
). The F0 of lentogenic viruses can be cleaved only by trypsin-like enzymes, as found in the respiratory and intestinal tracts, whereas F0 of virulent viruses can be cleaved by a host protease (or proteases) found in a wide range of cells and tissues. Consequently, infection with a virulent strain results in fatal systemic infection (Garten et al., 1980
; Nagai et al., 1976
). Until now, it has not been clear which specific host-cell protease is responsible for cleavage of the different F0 proteins. However, velogenic and mesogenic strains show a clear dibasic amino acid motif (K/R residues at amino acid positions 112/113 and 115/116) with a phenylalanine (F117) at the N terminus of the F1 protein after cleavage (Glickman et al., 1988
; Morrison et al., 1993
; Toyoda et al., 1987
). This sequence motif is most comparable with the motif of the furin family of proteases (Fujii et al., 1999
; Sakaguchi et al., 1994
). In contrast to the velogenic and mesogenic strains, lentogenic strains show a monobasic motif and a leucine (L117) at the N terminus of the F1 protein. This sequence motif is recognized by a factor X-like protease (vitamin K-dependent serine protease of the prothrombin family), which is present in the allantoic fluid and the amniotic fluid of the chicken egg (Gotoh et al., 1990
; Ogasawara et al., 1992
). In conclusion, there is a correlation between virulence or pathogenicity and a high content of basic amino acid residues at the F0 cleavage site.
The F0 protein is able to induce cell fusion only when the F0 protein is cleaved into F1 and F2. The HN protein is also needed in the promotion of fusion to co-operate with the F protein (Deng et al., 1997). Syncytium formation by cells can be observed when the F0 protein and HN protein are co-expressed after co-transfection of plasmids containing the F and HN genes (Li et al., 1998
; Peeters et al., 2001
). This assay can be used to examine the effect of specific amino acid mutations on cleavability of the F0 protein.
Mutagenesis of the amino acids of a velogenic F0 cleavage site has recently been carried out to investigate which amino acid mutation influences syncytium formation (cell fusion) after co-transfection of cultured cells with F and HN genes (Fujii et al., 1999; Li et al., 1998
). These data clearly showed that the dibasic structure as well as the phenylalanine at position 117 is necessary for the host-cell protease to cleave F0 and to form a biologically active complex with the HN protein. Using reverse genetics techniques, we recently established an infectious clone of NDV and we showed that modification of the lentogenic cleavage site 112GRQGR
L117 into the consensus sequence of virulent strain 112RRQRR
F117 increased the intracerebral pathogenicity index (ICPI) from 0 to 1·3 (Peeters et al., 1999
). These results confirmed that the presence of one or both arginines at positions 112 and 115 and/or the phenylalanine at residue 117 are necessary for efficient cleavage of the F0 protein and result in an increase in virulence.
With the already available data on the amino acid sequences of the F0 cleavage site, we wanted to examine different F0 cleavage site mutations at the whole-virus level. Li et al. (1998) showed that mutations of the F0 cleavage site such as K115G, F117L and R113G/K115G were fusion-negative. However, all mutant F0 proteins activated fusion after the addition of exogenous trypsin. Thus, it should be possible to generate viruses with different F0 cleavage sites that can grow in an environment with trypsin-like proteases (e.g. allantoic fluid and the amniotic fluid of the embryonated chicken egg). If it is possible to generate viruses with mutations of the F0 cleavage site, then the next step is to investigate the effect of those mutations on virulence and pathogenesis.
In this study, we have used our full-length NDV cDNA clone to generate infectious NDV with defined mutations in the F0 protease cleavage site. The following F0 cleavage site mutants were generated: RRQRRL, GRQGR
F, RRQGR
F, RGQRR
F and RKQKR
F. Here, we show that all the F0 cleavage site mutants were viable and that the mutations were maintained after virus propagation in embryonated eggs. The F0 cleavage site mutants were non-virulent based on plaque assays in cell culture. However, after intracerebral inoculation in 1-day-old chickens, virulent revertants were isolated from infected animals.
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Methods |
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The strains NDFL and NDFLtag were obtained from the NDV strain La Sota (ATCC VR-699), as previously described (Peeters et al., 1999).
Modification of the protease cleavage site of the F0 protein.
All standard cloning procedures were performed as described in Sambrook et al. (1989), unless otherwise noted. Mutagenesis reactions on the sequence encoding the protease cleavage site of the F0 protein were carried out using PCR as described previously (Peeters et al., 1999
). The same procedure was used for the new F0 cleavage mutants, which are listed in Table 1
.
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Co-transfections.
DNA transfections using Fugene6 were carried out essentially as recommended by the manufacturer (Roche). QM5 cells were grown in six-well culture dishes and after 24 h the cells were infected at an m.o.i. of 1 with FPV-T7 for 1 h at 37 °C. The cells were washed with PBS and co-transfected with 1 µg of the various pCIneo-F constucts and 1 µg pCIneo-HN in QM5 medium containing 5 % allantoic fluid, 5 % FCS and antibiotics. After 48 h, expression of the F0 protein was verified in an immunoperoxidase monolayer assay (Wensvoort et al., 1986) using mAb 8E12A8C3 (ID-Lelystad).
Fusion activity of NDV F0 cleavage site mutants was determined microscopically 48 h post-transfection by examining syncytium formation of QM5 cells. Before observation, the QM5 cells were washed with PBS, stained for 30 min with Giemsa (Merck; 1 : 30 dilution in water) and washed again with PBS.
Rescue of infectious virus.
To rescue viable virus from the various full-length pNDFL-F constucts, QM5 cells were infected with FPV-T7 and co-transfected as describe above. One µg pNDFL-F, 0·8 µg pCIneo-NP, 0·4 µg pCIneo-P and 0·4 µg pCIneo-L were used for each co-transfection. After 3 days, the culture supernatant was harvested and inoculated into the allantoic cavities of 811-day-old embryonated specific pathogen-free (SPF) eggs (Peeters et al., 1999).
Haemagglutination (HA) assay and pathogenicity tests.
The HA assay and determination of the intracerebral pathogenicity index (ICPI) in 1-day-old chickens was performed as described in the European Community Council Directive 92/66/EEC (14 July 1992).
Re-isolation of virulent virus from dead chickens.
Brains, livers and lungs were collected from three dead chickens and homogenized with a mortar and sterile sand in tryptose/phosphate broth (Invitrogen) with antibiotics. After incubation at room temperature for 2 h, the organ suspensions of three chickens were pooled and centrifuged for 10 min at 2500 r.p.m. The supernatant was used for further virus propagation.
RT-PCR and sequence analysis.
To sequence the region of the F gene that encodes the protease cleavage site, an RT-PCR fragment was obtained from the viral genome using primer 3UIT (5'-ACCAAACAGAGAATCCGTGAGTTA-3') for reverse transcription and primers NDV5Fxho and NDV3Fmlu for the PCR. The resulting PCR fragment was purified using a High Pure PCR purification kit (Roche) and sequenced with primer p4731+ (5'-AAGCTCCTCCCGAATCTGCC-3') using a PRISM kit (Perkin-Elmer) and an Applied Biosystems AB310 automatic sequencer.
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Results |
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Mutagenesis of the fusion cleavage site of F0 was performed by PCR using plasmid pCIneo-FWT (RRQRRF) as the template (see Methods). In FWT, the following cleavage site mutations were introduced: F117L (FFM), R115G (FFM2), R113G (FFM3), R112G+R115G (FFM1) and R113K+R115K (FFM4). These five mutant F0 genes were cloned in plasmid pCIneo.
Expression of the different F0 proteins was controlled by transfection into QM5 cells followed by immunological staining of the fixed monolayer with mAb 8E12A8C3 directed against the F protein. Immunological staining of the expressed (mutant) F0 proteins did not show any significant differences in the number of positive cells (data not shown).
To determine whether the F0 mutants were able to induce syncytium formation, QM5 cells were co-transfected with the fusion cleavage site mutants and pCIneo-HNLS (La Sota). None of the F0 cleavage site mutants showed syncytium formation after 48 h on QM5 cells (Table 1). Only the positive control pCIneo-FWT showed syncytium formation.
Rescue of infectious virus containing the F0 cleavage site mutations
The pCIneo plasmids with the five different F0 cleavage site mutations were used to introduce these mutations into full-length NDV cDNA in plasmid pNDFL (see Methods). The resulting plasmids were named pNDFL-FFM, pNDFL-FFM1, pNDFL-FFM2, pNDFL-FFM3 and pNDFL-FFM4.
These plasmids were used to generate infectious NDV with the different F0 cleavage site mutations (see Methods). The supernatants of transfected monolayers were used to inoculate the allantoic cavities of 811-day-old embryonated SPF eggs. The allantoic fluid was harvested 4 days after infection and analysed in a HA assay. Allantoic fluid with a positive HA titre was used for the isolation of the viral RNA genome followed by sequence analysis of an RT-PCR fragment that covered the F0 cleavage site. The results showed that all NDFL F0 cleavage site mutants could be recovered from cloned full-length cDNA. The mutants grew to similar titres compared with the parent strain, and the sequences of the F0 cleavage sites were still intact after two passages in embryonated eggs (data not shown).
The F0 cleavage site mutants were used to examine plaque formation on CEF cells. None of the mutants produced plaques 3 days post-inoculation except for the positive controls NDFLtag (RRQRRF) (Peeters et al., 1999
) and NDFL-FFM5 (RRQKR
F) (for details of FFM5, see below) (Fig. 1
; data for FFM1 are not shown). All other mutations gave rise to single-cell infections, indicating that the F0 protein was not cleaved.
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Increase in virulence of NDV after one passage in chicken brains
The revertant NDV strains, which were called NDFL-FFMR, NDFL-FFM2R, NDFL-FFM3R and NDFL-FFM4R, were again used to determine the ICPI. The resulting ICPI values varied from 1·4 to 1·7 (Table 2b), well above the value of the parent strain NDFLtag, which has the same cleavage site (RRQRR
F) but which has an ICPI value of 1·3. The revertant NDV strains were propagated in embryonated eggs and again used for sequence analysis of the F0 cleavage site. These sequence analyses showed no further alteration of the F0 cleavage sites.
The complete sequence of the F0 gene of strain NDFL-FFM4R was determined and compared with the sequence of the F0 gene of the parent strain NDFL-FFM4 and of the cDNA of pNDFL-FFM4. This analysis showed that no other mutations were present in the F gene except for the one in the cleavage site. This means that the increase in virulence of the revertant NDV strains is not caused by another mutation in the F0 gene but probably by a mutation(s) elsewhere in the NDV genome.
To confirm that the virulence indeed increases after one passage in chicken brains, we determined the ICPI of isolate NDFLtagPass, which was recovered after one passage of strain NDFLtag in chicken brains. The ICPI value increased from 1·3 (NDFLtag) to 1·5 (NDFLtagPass) (Table 2a, b). Nucleotide sequence analysis showed that the F0 cleavage site of strain NDFLtagPass was identical to that of NDFLtag. Additional proof for increase in virulence was obtained by generating NDFL-FFM5, which has the same cleavage site as NDFL-FFM4R (RRQKR
F). The ICPI value of NDFL-FFM5 was found to be 1·3, which is equal to that of NDFLtag. This means that the lysine (K) instead of the arginine (R) at position 115 does not have any effect on the virulence. However, after one passage of NDFL-FFM5 in chicken brain, the ICPI of the re-isolate (NDFL-FFM5Pass) also showed an increase in the ICPI from 1·3 to 1·5. In this case also, nucleotide sequence analysis showed that the F0 cleavage site of strain NDFL-FFM5Pass was similar to that of NDFL-FFM5 (Table 2a, b
). Although the virulence of NDV increased after a passage in chicken brains, significant differences in plaque size, for example between NDFLtag and NDFLtagPass, on CEF cells could not be observed.
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Discussion |
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Compared with the parent strain, all the newly generated NDV F0 mutants grew to similar titres in the allantoic cavity of embryonated eggs. Sequence analysis of the cleavage sites of the F0 mutants showed that no change occurred after two egg passages. This indicated that the allantoic fluid of the chicken egg contains a trypsine-like protease that is able to cleave the mutant F0 proteins shown in Table 1. However, transient expression of the mutant F0 proteins together with the HN protein did not result in syncytium formation of QM5 cells. This suggests that the mutant cleavage sites behaved like the lentogenic cleavage site of FLS (La Sota). Only FWT protein induced syncytium formation, indicating that it was cleaved by the host-cell protease (Table 1
). The same lentogenic behaviour of the F0 cleavage site mutants was observed at the whole-virus level after examining plaque formation on CEF cells (Fig. 1A
).
To analyse the effect of the mutant cleavage sites on virulence, the ICPI was determined (Table 2a). Interestingly, even though the ICPI values were all below the value of the parent strain NDFLtag (ICPI=1·3), they were much higher than expected based on the lentogenic behaviour of the F0 cleavage site mutants. These high ICPI values were caused by a dramatic increase in morbidity and mortality after day 5 for most of the NDV F0 cleavage site mutants (Table 2a
). This increase in morbidity and mortality was the result of the presence of revertants with an F0 cleavage site RRQ(K/R)R
F. These revertants are the result of replication errors and were probably already present after virus propagation in eggs. Apparently the cells in chicken brain constitute a highly selective environment for NDV viruses with the velogenic F0 cleavage site RRQ(K/R)R
F. Initially, the ICPI values indicated that the cleavage site mutants were either lentogenic or mesogenic. But in fact these values were influenced by the presence of virulent revertants. Therefore, precautions must be taken when the ICPI of strains containing cleavage site mutations is determined. It is sensible to sequence the F0 cleavage site of the re-isolate from dead chickens after the ICPI test to be sure that the amino acid sequence of the cleavage site is identical before and after the ICPI test.
The ICPIs of the velogenic revertants were all above the ICPI of the parent NDFLtag (ICPI=1·3). However, the fusion cleavage site of NDFLtag is the same as the fusion cleavage site of NDFL-FFM2R (ICPI=1·7) and NDFL-FFM3R (ICPI=1·5). Sequence analysis of the entire F0 gene of NDFL-FFM4, NDFL-FFM4R and pNDFL-FFM4 did not show any mutations other than the mutations of the cleavage site. This suggests that the revertants have somehow adapted to the host cells after one passage in the chicken brain. This might be due to another mutation somewhere in the NDV genome that results in an increase of the ICPI. NDFLtagPass and NDFL-FFM5Pass indeed showed an increase in ICPI compared with NDFLtag and NDFL-FFM5, respectively, whereas the cleavage sites remained unaltered. Thus, the pathogenicity of NDV is not only related to the F0 cleavage site. A similar increase in virulence has been observed by Collins et al. (1994) for a variant of NDV, PPMV-1. Similarly, in this case the F0 cleavage site of PPMV-1 isolates did not change after passage in chickens.
From our results, it is clear that the amino acid sequence 112R-R-Q-K/R-R-F117 is required for efficient cleavage of the velogenic/mesogenic F0 protein by a furin-like protease in chicken tissues. This amino acid sequence is identical to the consensus amino acid sequence 112R/K-R-Q-R/K-R-F117 of virulent NDV strains. Interestingly, a basic lysine (K) at amino acid position 113 is not accepted by this protease. In agreement with this observation, a K at position 113 has never been observed in field isolates. Thus, whereas the dibasic combination K-R at amino acid positions 112 and 113 does exist, the combination R-K does not. The dibasic structure at amino acid positions 112 and 113 was not required for some velogenic strains that were isolated from pigeons (PPMV-1) and had the fusion cleavage site GRQ(K/R)RF (Collins et al., 1994
). Surprisingly, the F0 cleavage mutant NDFL-FFM1 (GRQGR
F) did not revert to GRQ(K/R)R
F, even though just a single nucleotide mutation is needed to change the amino acid at position 115 from a G (codon= GGG) to an R (codon=CGG). Fujii et al. (1999)
demonstrated with F0 cleavage site mutants expressed by vaccinia virus vectors in CEF cells that the R residue at position 112 was only required for maximal cleavage by the host-cell protease. This also suggested that the revertant of NDFL-FFM1 could exist. The fact that the revertant did not appear after intracerebral inoculation could indicate that the cleavage site GRQ(K/R)R
F of NDFL-FFM1, in contrast to the pigeon isolate with the same cleavage site, is not virulent.
It is clear that our NDV F0 cleavage site mutants give rise to revertants with a velogenic cleavage site by point mutations. After just one passage in chicken brains, we were able to isolate such revertants. This evolution of the NDV F0 cleavage site is reminiscent of the emergence of virulent strains, which were responsible for the outbreaks of Newcastle disease in Ireland in 1990 (Alexander et al., 1992) and in Australia in the years 19982000 (Gould et al., 2001
; Westbury, 2001
). Analysis of these virulent NDV isolates showed that they were closely related to a lentogenic strain (antigenically and genetically) that was considered to be the precursor of the virulent virus. The Australian NDV isolate Peats Ridge with the cleavage site RRQGR
L probably required two mutations to evolve via RRQRR
L (Somersby), RRQKR
L (MP-2000) or RRQGR
F (PR-32) to the velogenic site RRQ(K/R)R
F. Toyoda et al. (1989)
have already concluded that different strains of NDV have evolved through various degrees of accumulation of point mutations rather than gene exchange by recombination.
We have shown in this study that NDV can evolve from a non-virulent type to a virulent type by point mutations. At this moment, there is no evidence that this frequently happens in the field. One reason for this is that the chance of accumulating three mutations from GRQGRL to (K/R)RQ(R/K)R
F is probably very low. This is confirmed by NDFL-FFM1 (GRQGR
F), which needs just two mutations but did not revert to a virulent strain. A second reason is the fact that mutations to virulence have only been observed in a relatively small non-vaccinated chicken population (in a country like Australia) and never in an immune population. This might be explained by the low transmission of mutants in vaccinated populations. Furthermore, it is not clear where mutations to virulence occur in the field. Do the mutations take place in feral birds and are then passed to poultry or do they occur once the virus has been introduced in poultry? In this study, the NDV F0 cleavage site mutants were propagated in embryonated eggs followed by a passage in chicken brains where the virulent revertants appeared. This is far from the natural infection route of NDV and therefore it is of interest to know whether NDV F0 cleavage site mutants give rise to revertants when the natural infection route, i.e. the respiratory tract, is used.
Nevertheless, attention must be paid when a non-virulent NDV strain is isolated from the field that has just one or two amino acid differences in the F0 cleavage site compared with a virulent strain. Such NDV strains can evolve to a virulent strain, which can cause devastating disease in poultry.
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Received 15 July 2002;
accepted 13 September 2002.