Institute for Animal Health, Pirbright, Surrey GU24 0NF, UK
Correspondence
Ashley C. Banyard
ashley.banyard{at}bbsrc.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Rinderpest virus (RPV) has a single-strand, non-segmented, negative-polarity RNA genome and is thus classified within the order Mononegavirales, family Paramyxoviridae, subfamily Paramyxovirinae, genus Morbillivirus, which also includes human Measles virus, Canine and Phocine distemper viruses, Peste-des-petits-ruminants virus and the more recently isolated cetacean morbilliviruses (Barrett, 2001). The negative-strand genome contains six tandemly arranged transcription units encoding eight proteins. The genome is flanked by extragenic sequences at the 3' and 5' ends, often referred to as the leader and trailer, respectively. Recent efforts to define the extent of sequence required at the genome termini have led to a new terminology being used, that of genome promoter (GP) and antigenome promoter (AGP). The more-studied promoter regions have been shown to contain distinct domains that include sequences required for binding of the RNA polymerase to the nucleocapsid template, nucleation sequences to direct encapsidation of the nascent RNA by the viral nucleoprotein and leader RNA termination sequences that dissociate the encapsidation sequences from the viral mRNA transcripts. For RPV, the GP is currently recognized as the sequence at the negative-sense 3' genome terminus that includes the 55 nt leader region as well as the complement of sequence present within the 5' untranslated region (UTR) of the first gene, the N gene. The RPV AGP is similarly located at the 3' end of the antigenome and includes sequences in the 3' UTR of the L gene as well as the 37 nt trailer region, although the exact requirements for sequence at the 3' end of the antigenome have not been extensively studied and are essentially undefined. The limits of sequence required within the promoter regions of a number of paramyxoviruses have been partially defined using artificial replication systems including naturally occurring defective-interfering particles and artificially constructed genome analogues or minigenomes' expressing reporter genes (Harty & Palese, 1995
; Hoffman & Banerjee, 2000
; Mioulet et al., 2001
; Tapparel et al., 1998
; Tapparel & Roux, 1996
; Whelan & Wertz, 1999a
, b
).
Recent attempts to define critical residues within various GPs and AGPs suggest that these promoters are at least bipartite in nature. The first element consists of 2231 often highly conserved nucleotides at the genome termini, whilst a series of three-hexamer motifs, that span nt 7996 form a second promoter element. These hexamer motifs are strikingly conserved within the morbilliviruses and respiroviruses and have been discussed in detail elsewhere (Mioulet et al., 2001; Tapparel et al., 1998
).
The availability of a full-length genome cDNA of the vaccine RBOK strain of RPV (Baron & Barrett, 1997) as well as the virulent parental Kabete O (KO) strain (Baron et al., 2005
; accompanying paper) has allowed artificial genetic recombination of these two viruses to study the effects of these alterations in vitro and in vivo using reverse-genetics techniques. Here, we describe the construction and rescue of full-length cDNAs containing altered virus promoters and studies on their growth in vitro and pathogenesis in vivo.
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METHODS |
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Cell lines.
B95a cells (Kobune et al., 1991) were maintained in RPMI 1640 supplemented with 5 % fetal calf serum (FCS), 100 U penicillin ml1 and 100 µg streptomycin sulphate ml1. Vero cells were maintained in Dulbecco's modified Eagle's medium with 25 mM HEPES buffer and 2 mM glutamine supplemented with 5 % FCS, 100 U penicillin ml1 and 100 streptomycin sulphate µg ml1.
Virus strains.
Both virulent and avirulent viruses were analysed in this study, with recombinant viruses being generated that swapped the GP and part of the AGP between the virulent and avirulent strains. The RBOK (R) strain of RPV is the vaccine strain and as such is avirulent. The KO (K) strain of RPV is the field strain from which the vaccine was derived and is highly virulent in nature, as is the Saudi/81 (S) field strain, the promoter regions of which were used in this study. Where sequences derived from virulent viruses were used to replace regions in the vaccine strain, the virulent component of the recombinant virus is underlined: S GP and AGP with K genome fragments=SPK; S GP and AGP with R genome fragments=SPR; K GP and AGP with R genome fragments=KPR; and R GP and AGP with K genome fragments=RPK.
Amplification and cloning of the GP and AGP of the S strain of RPV.
The pMDB1 vector, used in the production of the K and R full-length genome clones (Baron & Barrett, 1997), was used as the plasmid backbone for the production of all recombinant full-length viruses. The RPV S strain GP and AGP were amplified using RACE techniques (Loh et al., 1989
; Schuster et al., 1992
) and were cloned into the pMDB1 vector as previously described for the R strain (Baron & Barrett, 1997
) to produce pSGPAGP. This construct contained 107 nt of GP sequence and 76 nt of AGP sequence. A ClaI site was introduced at the end of the N gene 5' UTR (nt 102) and a SalI site at nt 15807. The SalI site was present at 76 nt from the genome terminus and as such the region used here as the AGP was not complete. It did not include all of promoter element II, but did cover all of the trailer region and part of the UTR of the L gene. The ClaI and SalI sites were already present in these positions in the full-length R and K cDNAs and so were also used in this study to produce pSGPAGP. The 3'-terminal 107 nt of the negative-sense morbillivirus genome have been shown to be necessary and sufficient for promoter activity, but an exact length requirement for the AGP has not been determined.
Construction of recombinant viruses.
To construct recombinant full-length viruses, restriction digestion was carried out on the full-length R and K genome cDNAs. The series of manipulations to produce the required full-length chimeras is described below and illustrated in Fig. 1(a). The full-length K and R clones contained a number of, mostly unique, restriction sites that were inserted during their construction (Baron & Barrett, 1997
; Baron et al., 2005
; accompanying paper). The ClaI and SalI sites were used to define the GP and AGP as described above. However, the SalI site was not a unique restriction site within the full-length clones. As a result, the full-length genomes had to be cut at other unique restriction sites to generate smaller fragments that together spanned the region from the end of the GP (ClaI site) to the SalI site at the start of the AGP region used (nt 15807). The full-length cDNA constructs were used to generate a fragment that ran from the ClaI site at the end of the GP (nt 102) to the AscI site at nt 7195 in the F gene (Fig. 1a
, region I), a fragment that extended from the AscI site in the F gene to the AatII site in the L gene (nt 12833) (Fig. 1a
, region II) and a fragment that contained the remainder of the L gene to the SalI site in that gene (Fig. 1a
, region III).
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Virus rescue.
All viruses described were rescued from plasmid DNA on B95a cells using a recombinant vaccinia virus, vTF7-3, which expresses the bacteriophage T7 polymerase (Fuerst et al., 1986). Briefly, B95a cells at 70 % confluency in six-well plates were infected with vTF7-3 (m.o.i. of approx. 0·2) and subsequently transfected with the full-length recombinant plasmid construct along with pKS-N, pKS-P and pGEM-L, all under the transcriptional control of the T7 polymerase promoter, to form the ribonucleoprotein complex and initiate the RPV virus replication cycle (Baron & Barrett, 1997
). All viruses, including the parental strains, were rescued from full-length clones using R-based N, P and L helper plasmids. Cells were checked daily for the appearance of viral cytopathic effect (CPE) and once a significant level was observed, the virus was passaged into 75 cm2 flasks and grown to prepare stocks. CPE was generally seen 23 days after transfection. Each rescued clone was taken to virus passage 3 on B95a cells and stored at 70 °C until required.
Analysis of virus growth in vitro.
Growth curves were carried out in 35 mm2 six-well plates seeded with an appropriate number of cells to ensure 6070 % confluence after overnight incubation at 37 °C. Both B95a cells (5x105 cells ml1) and Vero cells (5x104 cells ml1) were used. Virus was inoculated on to the cells at an m.o.i. of 0·01 and the plates incubated at 37 °C for 1 h. After this time, the virus inoculum was removed and the cells washed once with serum-free medium before 3 ml of the appropriate growth medium was added. The time 0 plate was immediately stored at 70 °C. The remaining plates were left at 37 °C for the required period after which time they were placed at 70 °C. TCID50 was determined at each time point using standard techniques (Reed & Muench, 1938).
Animal experimentation.
Six-month-old outbred Fresian Holstein bullocks were used for the animal experiments. They were fed on commercial pelleted food concentrates and hay. Throughout the experiment, rectal temperatures were recorded daily and blood was usually taken at 23-day intervals post-infection (p.i.) to be processed for white cell counts, virus isolation on cell culture and serum antibody analysis. Isolation of peripheral blood mononuclear cells (PBMCs) was carried out using techniques described previously (Lund et al., 2000). Animals were also checked daily after infection for rinderpest-specific disease signs, such as pyrexia, mucosal lesions, excessive salivation, dehydration and diarrhoea. Ocular and nasal discharges were also noted, as was the general demeanour of the animals (depressed, anorexic, etc). At completion of the experiments, the animals were euthanized. A full post-mortem examination was carried out on any animal that was euthanized with severe clinical disease.
Leukopenia, a characteristic of morbillivirus infection, was determined by measuring white cell counts. For this, 10 µl whole blood was diluted in 190 µl 1 % acetic acid and the non-lysed white cells were counted using an improved Neubauer haemocytometer. Leukopenia was considered severe if the white blood cell count (WBC) fell below 50 % of the day 0 level (Anderson et al., 1996). The normal WBC for cattle is between 7000 and 10 000 leukocytes (mm3 whole blood)1 (Swenson, 1970
).
Virus isolation.
Isolation of virus from blood and eye swabs was attempted to determine viraemia and virus excretion, respectively. To detect circulating virus, 500 µl PBMCs derived from 10 ml whole blood was inoculated on to a monolayer of B95a cells and the appearance of viral CPE monitored over a period of 18 days. Cells were passaged when necessary and the presence of virus syncytia was taken as a positive indication of viraemia. To detect virus excretion, eye-swab material was extracted in 200 µl PBS and inoculated on to B95a cells. The monolayers were monitored for CPE and passaged as described above. Samples were considered negative if no CPE was observed after 18 days.
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RESULTS |
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Growth of recombinant viruses in vitro
To establish the effects of swapping the GP and the terminal portion of the AGP of each of these viruses, their growth in vitro was assessed on two cell types, Vero and B95a cells. Virus at passage 3 was used to determine their relative growth rates in these cells. Multi-step growth curves were carried out for each of the four recombinant viruses and the parental K and R viruses. The growth of the recombinants in vitro in both cell lines was comparable with the growth rate of the parental viruses. The final titres reached in B95a cells were similar for the R-based recombinants, KPR and SPR, and the unmodified parental R (105·55105·8 TCID50 ml1). Similarly, the K-based recombinants, RPK and SPK, reached similar titres to the parental K in B95a cells (Fig. 2a). In Vero cells, the R-based recombinants KPR and SPR grew to a similar titre to the unmodified R (106·05106·8 TCID50 ml1). Wild-type K is known to grow to lower titres in Vero cells (between 102 and 104·5 TCID50 ml1) and, as expected, the rescued K also grew to a relatively low titre of 104·05 TCID50 ml1, as did the two K-based recombinant viruses, RPK and SPK (Fig. 2b
).
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The two cattle inoculated with the parental K virus developed typical rinderpest. Both animals were pyrexic 2 days p.i. and their temperatures continued to increase until 6 days p.i. when both animals reached a peak fever of 41·6 °C. They also developed mucopurulent ocular and nasal discharge, reddened nasal mucosa and severely eroded oral papillae. Small necrotic lesions were seen on the gums and under the tongue of both animals. As these symptoms progressed, the rectal temperatures began to drop, often a sign that the animals are about to succumb to infection, and they developed diarrhoea, anorexia and dehydration and were euthanized on either day 8 or day 9 p.i. to prevent suffering.
The four cattle inoculated with RPK developed much less severe clinical disease than those inoculated with K and developed a delayed pyrexic response that was short lived (Fig. 3) except for UZ73, which was feverish from day 5 to 11 (Table 1
and Fig. 3
). Two animals also developed mild ocular and nasal discharge as well as oral lesions during infection. At no time did the animals develop even mild diarrhoea and they continued to feed normally during the experiment. All four animals survived infection with RPK and developed RPV-specific antibodies as determined by competitive ELISA (Anderson et al., 1996
; data not shown).
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None of the animals infected with the R-based recombinants displayed any RPV-specific clinical signs during the 2 weeks of observation. The R-infected animals, as well as those infected with KPR or SPR, maintained normal body temperatures throughout the experimental period and this lack of pyrexia was consistent with the absence of clinical disease (Fig. 3). The sera from all animals developed RPV-specific antibodies (Anderson et al., 1996
; data not shown). The clinical data observed for all of the animal infections are summarized in Table 1
, whilst rectal temperatures are shown in Fig. 3
.
Development of leukopenia
Leukopenia was monitored for 2 weeks p.i. to assess the effects of virus infection on the immunological status of the animals. The increase or decrease in WBC was expressed as a percentage of WBC on day 0 (100 %) and the results are shown in Fig. 4. The WBC for the two animals infected with K decreased from 2 days p.i., with both animals showing a greater than 60 % decrease in WBC by day 5. These counts remained low until the animals were euthanized. Similarly, animals infected with SPK showed a severe decrease in WBC following infection. UN21 initially showed a 60 % increase in WBC at 2 days p.i., but by 5 days p.i. this had dropped to less than 40 % of its value on day 0. The WBC for animals UN21 and UN22 then appeared to increase to levels comparable with day 0 counts before the animals were euthanized at 7 days p.i. This apparent increase in WBC resulted from the greatly increased packed-cell volume of the blood due to the severe dehydration seen in these two animals and was not indicative of a recovery in WBC (Anderson et al., 1996
).
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The WBC for the R-infected animals, as well as the KPR- and SPR-infected animals, fluctuated within normal levels for healthy cattle (Swenson, 1970) until day 12 p.i. However, for the R-infected cattle and two of the SPR-infected animals the WBC started to decrease 12 days p.i. and by day 14 all four animals had unusually low WBC (Fig. 4
). This drop in WBC may have been due to a mild infection with some other pathogen, a problem associated with working with outbred animals obtained under natural conditions and not specifically bred for research. Morbilliviruses are known to be immunosuppressive (Heaney et al., 2002
) and, as the drop was seen after the period when vaccine virus was expected to replicate and no RPV could be detected in their PBMCs, the effect could not be attributed to rinderpest disease.
Development of viraemia and virus excretion
The results of attempts to isolate live virus from infected PBMCs from each animal are shown in Table 2. The presence of virus in PBMCs was noted over a period of several days for all animals infected with K-based virus recombinants. Virus was detected in PBMCs derived from SPK- and K-infected animals from day 2 through to the day when the animals were euthanized, whilst the PBMCs from RPK-infected animals were positive from day 4 (US39), 5 (UZ73 and UZ74) or 7 (US40) to day 9 or 14 (Table 2
). For animals infected with the R-based recombinants, positive isolation of virus was only made for the SPR-infected animals and not the R- or KPR-infected animals (Table 2
).
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Confirmation of the identity of the infecting virus
RNA derived from PBMCs of SPK- and RPK-infected animals was checked to ensure that no mutations had occurred within the promoter regions that might have affected pathogenesis. For this, RACE was carried out, as described previously (Baron & Barrett, 1997; Loh et al., 1989
; Schuster et al., 1992
), on material derived from infected animals and the identity of the correct promoters confirmed by sequence analysis (data not shown).
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DISCUSSION |
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Two other areas within the GP where the virulent and vaccine strains of RPV differed were at nt 90 and 95 (Fig. 1). These differences lie within the last two hexamers of the recognized series of three conserved hexamer motifs present at positions 7984, 8590 and 9196 in the N gene UTR (Tapparel et al., 1998
). For Sendai virus (SeV), the exact positioning or phase context of these hexamers relative to the genome terminus has been shown to be critical for promoter function, indicating that they are an essential feature of GPs and cannot be altered, even in vaccine strains (Iseni et al., 2002
). The significance of transversional differences at nt 90 and 95 within the RPV GP remain to be investigated.
Sequence alignments of the AGP regions of RPV, shown in antigenome (positive-sense) in Fig. 1, showed a much greater degree of sequence identity between S, K and R than that seen in the GP. The defined trailer region, nt 1584515882, spanned the final 37 nt of the genome, and within this region only one transversional difference, at nt 15852, occurred that may be of significance. Several residues within the more extensive AGP showed further lineage-specific differences between the Asian S strain and the African R and K strains. Such lineage differences may be compensating mutations related to evolutionary changes in polymerases and/or they may have a significant effect on AGP function. As expected, the further away from the 3' end of the antigenome sequence (i.e. the further into the L gene UTR), the more lineage-specific base changes are seen that may also be of importance in determining promoter efficiency.
In the present investigation, complete recombinant viruses with altered promoters were rescued and the affect of these changes on growth in cell culture was assessed in vitro. In each case, the only alterations were to the GP and part of the AGP and all viruses were rescued in B95a cells to minimize the effect of cell-culture attenuation. The decision to use B95a cells was based on studies that have shown that virus passage in non-lymphoid cell lines attenuates virulent strains of RPV (Kobune et al., 1991). All recombinant viruses were rescued using R-based N, P and L helper plasmids in a vaccinia-based rescue system (Baron & Barrett, 1997
). Other studies have reported that recombination can occur between helper plasmids and the full-length genome plasmids in a different virus rescue-system (Garcin et al., 1995
). However, we have not observed such recombination in previous studies where such a recombinogenic event would have given a significant advantage to highly debilitated viruses (Das et al., 2000
; Baron & Barrett, 2000
).
Altering the viral promoters had no effect on growth of the recombinant viruses in vitro, as each grew to a titre similar to the respective parental R or K virus (Fig. 2). We have found that the K strain is unable to infect fibroblast cells such as Vero cells efficiently due to the absence of the wild-type virus receptor (M. D. Baron, unpublished data). Interestingly, comparable studies with rescued SeV have shown that alterations to a virus promoter may not affect growth in vitro, but can greatly influence pathogenesis in the host. Passage of the virulent Hamamatsu strain of SeV in vitro results in attenuation of its virulence for mice, and sequence comparisons between the virulent and the derived attenuated viruses identified only four mutations across the entire 15384 nt genome. Two of these mutations were in the L gene (nt 9346, silent; nt 12174, Ser to Cys), whilst the other two were present at nt 24 and 28 within the GP. Furthermore, the two SeV GP mutations alone were shown to be able to attenuate virulence 25-fold in vivo (Fujii et al., 2002
). Recent studies with the rhabdovirus Vesicular stomatitis virus have also suggested that in vitro analyses do not necessarily highlight alterations to virus pathogenicity that are seen in vivo (Novella et al., 2004
). Thus, despite the lack of an appreciable effect on growth in vitro, it was decided to test the ability of these recombinant viruses to cause disease in cattle. The nucleotide differences required to have an effect in vivo may be quite subtle and immunological factors, along with the myriad of other host factors that may affect virus replication in vivo that may not be relevant in vitro, could have an effect. Animals infected with the rescued K strain developed the classical disease signs of rinderpest, which were indistinguishable from those observed with a wild-type K infection (Ngichabe et al., 2002
). Two of the animals infected with SPK (UN21 and UN22) showed a faster and more severe progression of clinical disease, while the other two (UW73 and UW74) showed very similar disease patterns to the K controls. It was not possible to state, therefore, whether or not the S promoters increased K virus virulence. It was interesting to observe, however, that the presence of the S promoter sequences increased the secretion of virus for both SPK and SPR (Table 2
). This may indicate a change in virus replication or packaging efficiency in epithelial cells.
When the GP and part of the AGP were derived from the avirulent vaccine (R) strain, the opposite effect was seen. Animals infected with RPK exhibited greatly attenuated disease and recovered after showing only mild clinical signs. The four animals infected with this virus showed a delayed onset of pyrexia that lasted only 1 or 2 days, and one (US39) developed flat lesions on the palate, which were not seen in the other three animals. Another (US40) developed a few pinprick lesions, which resolved quickly. This mild disease, rather than the classical disease symptoms typical of K infection, clearly demonstrated that the vaccine promoters had a significant attenuating effect on the virulence. Although inter-animal variation occurred and it has been shown that K infection is not always 100 % fatal, in cases of wild-type infection, disease was always much more severe than that seen with RPK virus (Anderson et al., 1996; Ngichabe et al., 2002
).
Animals infected with rescued R, KPR and SPR showed no clinical disease, indicating that the promoters are not the only factors governing pathogenesis. Sequence alignments of the genomes of the R vaccine and the parental K strain of RPV showed that they differed by only 0·54 %, there being only 87 nt that differ between the two viruses (Baron et al., 1996). Of these, seven were found in the GP region, whilst two more were seen in the AGP region, representing 10·34 % of all observed differences seen across the total genome length of 15882 nt. This is a high percentage considering that the GP and AGP cover less than 1·5 % of the total genome length. These data predict a multi-gene involvement in attenuation of the RPV vaccine and show that changes to viral promoters alone do not account for the complete attenuation of the vaccine strain; rather, they are one element within the genome that contributes to the avirulent phenotype (Baron et al., 2005
; accompanying paper).
The full-length recombinants generated in this study showed that, although growth in vitro was not noticeably affected, altering the viral GP and AGP can have a profound effect in vivo. Further studies should indicate whether all changes in the GP and AGP are required for attenuation, or if, as in the case of SeV, only one or two are critical (Fujii et al., 2002).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 November 2004;
accepted 16 December 2004.