1 Department of Veterinary Pathology, University of Liverpool, Leahurst, Neston, Cheshire CH64 7TE, UK
2 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Correspondence
Clive J. Naylor
cnaylor{at}liv.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the APV genome sequence described in this study is AY640317.
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INTRODUCTION |
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Manipulation of the genome and study of genome function in APV have been limited, due to the absence of a methodology whereby precise changes can be made. Reverse-genetics systems have been developed for other viruses of the family Paramyxoviridae, which have enabled specific mutations to be introduced into the virus genome and the subsequent phenotypic consequences determined. These have included Human parainfluenza virus 3, Human respiratory syncytial virus (HRSV), Bovine respiratory syncytial virus, Newcastle disease virus, Sendai virus, Rinderpest virus and Simian virus 5 (Marriott & Easton, 1999). These analyses have identified many important common features of the molecular biology of these viruses. However, analysis of HRSV by using reverse genetics has identified several aspects that differentiate it from other paramyxoviruses. Rescue studies with HRSV have shown that the minimal replicative unit is comprised of the nucleocapsid protein (N), phosphoprotein (P), M2 protein, RNA polymerase (L) and full-length viral genome in the antigenome sense (Collins et al., 1995
). This paper describes the development of a reverse-genetics system for APV. The system was used to produce APV that entirely lacked the SH and G genes, indicating that these genes are not essential for viability in tissue culture.
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METHODS |
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APV1T7 and APV9+10HDVR were cloned into the low-copy plasmid pCTPE (a modified version of pOLTV5 with the HDVR, T7 terminator and lacZ sequences removed to improve cloning efficiency; Fig. 1d). The remaining PCR products were cloned into a modified form of pUC18 with the SalI site changed to an EcoRI site by site-directed mutagenesis (Quikchange; Stratagene) (Fig. 1d
). Site-directed mutagenesis was also used to correct coding changes that were identified by sequencing (Imperial College London Medical School service, London, UK) and to introduce an SstII restriction site into APV3. The latter introduced conservative (lysine to arginine) changes at aa 299 and 300 of the predicted F protein.
Sequential ligation of SalI/XhoI-cut fragments generated clones containing APV1T72+3 or APV610HDVR (Fig. 1e). Attempts to clone APV4+5 into these constructs were not successful. PCRs on APV1T72+3/APV4+5 and APV4+5/APV610 ligations carried out in the presence of SalI and XhoI were performed by using primers CTPE 240 and G11 or M2 mid and CTPE 190, respectively (Fig. 1f
). Exonuclease I (USB) was used to remove remaining oligonucleotides and an overlap PCR was used to join the two products, with T7 APVLead2 and CTPE 110 being added after two cycles (Fig. 1g
). No primers were present in the first two cycles, in order to allow the fragments to anneal and copy each other from the overlap prior to the addition of the PCR primers. A 15·5 kb product was obtained, which was circularized by ligation and transformed into Escherichia coli (Fig. 1h
). DNA from the resulting colonies was screened by multiplex PCR and restriction-enzyme digestion. One of two sequences of midiprep-purified DNA (Qiagen) lacked mutations in intergenic regions or coding changes.
After cloning, the full-length cDNA copy of the genome was modified between positions 3828 and 3831 (antigenome sense) by site-directed mutagenesis (QuikChange; Stratagene) to introduce a convenient SstII restriction site. This also resulted in a conservative substitution at aa 299 and 300 of the F gene, with lysines being converted to arginine residues. Confirmation of the presence of the mutation after passage of the recombinant virus was obtained by RT-PCR amplification of the region of the genome between nt 3625 and 4063 using primers F6+ and F7-B (Table 1).
A defined deletion mutant of APV was generated by high-fidelity PCR (10 cycles) amplification of the full-length clone to introduce a deletion in the region of the SH gene. The amplicon was intended to terminate at genome position 5363, 1 nt prior to the SH gene transcription start signal, and to restart at position 5965, 2 nt prior to the G gene transcription start signal. Long-distance PCR (Thiel et al., 2003) was employed to amplify the full-length genome clone so as to remove the entire SH gene by using primers SH omit+ and SH omit. The PCR mixture was run on an agarose gel to reveal solely the expected band (approx. 15·0 kb). This was self-ligated and used to transform competent cells. Resulting colonies were screened by multiplex PCR and restriction-enzyme analysis and a single clone from almost 1000 was identified as intact. This was sequenced across the M2G gene junction region and it was found that several bases had been lost from the start of the G gene. The amplicon terminated at position 5363, as expected. However, the sequence unexpectedly continued from a point 12 nt into the ORF and hence the G gene lacked its transcription start, translation start and a small section of coding region. The entire sequence of the mutant genome was sequenced to confirm that this deletion was the only mutation present.
Preparation of plasmids expressing the virus replication proteins.
N, P and M2 gene sequences from strain APV-A were amplified by PCR and cloned. RNA was extracted (RNeasy; Qiagen) from infected cells, reverse-transcribed (Superscript II; Invitrogen) and amplified by PCR (Pfu polymerase; Stratagene) using primers that amplified the gene to include the start codon (N and P) or introduced the T7 promoter sequence (M2 and L) immediately prior to the start codon of each gene, as shown in Table 1. The downstream primer in all cases terminated beyond the gene's stop codon. N and P genes were cloned into the SmaI site of pCI (Promega) downstream of a bacteriophage T7 promoter, allowing gene expression under the control of the cytomegalovirus promoter. T7M2 was cloned into the SmaI site of the modified pUC18 mentioned above.
The L gene was cloned in sections into the EcoRV site of pCTPE by using the sequential approach used for the complete viral genome. In order, APV9+10, APV8, APV7 and APV6 were ligated into pCTPE. It proved impossible to add the final section of the gene (APVT7Lstart), representing approximately the first 400 bp of the gene, in a similar manner. However, the full L gene, together with the pCTPE plasmid, was prepared as a single PCR product (Pfu polymerase; Stratagene) following their ligation. The blunt-ended product was circularized by ligation and the mixture was treated with the restriction endonuclease DpnI to remove the original methylated plasmid and leave only the desired product for transformation into E. coli. Three resultant clones were sequenced fully and were found to be free of coding errors.
Testing of support proteins in a minigenome.
The four support genes were tested functionally by using a cloned APV minigenome in which the virus genome had been modified so that all genes were replaced by a single copy of the chloramphenicol acetyltransferase (CAT) reporter gene, flanked by the virus leader and trailer regions. This adopted the method of Randhawa et al. (1997) except that, in this instance, bacteriophage T7 polymerase was generated by a Fowlpox virus recombinant (FPT7; Britton et al., 1996
) at an m.o.i. of 1 p.f.u. per cell and lipofectamine 2000 (Invitrogen) was used to transfect DNA into Vero cells. CAT reporter-gene expression was measured 48 h after transfection by ELISA, as described previously (Ahmadian et al., 2000
).
Virus rescue.
The method adopted followed that used for the minigenome, except that full-length antigenome (wild-type or deletion) replaced the minigenome and incubation times were increased to allow time for any virus to produce detectable cytopathic effect (CPE). Fowlpox virus will destroy most avian cells before this time, but it is unable to package in mammalian Vero cells; hence, additional means of limiting Fowlpox virus replication were unnecessary (Britton et al., 1996).
Control transfections of two types were also prepared whereby some cell sheets, containing 106 cells, were inoculated with Fowlpox virus (at an m.o.i. of 1 p.f.u. per cell) and transfection reagent, whereas others received solely the latter. The transfected cell sheet was viewed, freezethawed and filtered (0·2 µm) to remove cellular debris and to eliminate any possibility of effect from residual Fowlpox virus. The clarified material was used to infect new cells. Resultant cell sheets were examined daily for signs of CPE typical of APV.
If CPE was not evident, material was passaged further. When CPE was detected, RNA was extracted for RT-PCR by using an RNeasy kit (Qiagen) with the optional DNase digestion step. RNA was isolated 4 days post-infection. The region of the genome encoding the F gene containing the novel SstII mutation was amplified in the case of both full-length and deleted recombinant genome-derived material and a further PCR was used to amplify between the M2 and G genes in the case of the latter. All RT-PCR products were generated by using primers that were designed to anneal initially to genome RNA and were sequenced. Standard PCR conditions used were 10 s at 94 °C, 20 s at 50 °C and 60 s at 72 °C for 30 cycles using Taq DNA polymerase (Promega). The F gene-derived products were incubated with the restriction endonuclease SstII to assess their cleavability.
Confluent Vero cells were inoculated with the passaged material. After 4 days, these were incubated with APV-specific polyclonal antiserum that was raised in turkeys. After washing, this was further incubated with fluorescein isothiocyanate (FITC)-linked anti-turkey conjugate to detect infected cells (Baxter-Jones et al., 1986) and then viewed by using fluorescence microscopy and photographed.
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RESULTS AND DISCUSSION |
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RNA was extracted from the passaged virus in the presence of DNase I and a 438 bp region of the F gene containing the altered sequence was amplified by RT-PCR and sequenced to confirm the presence of the altered bases between positions 3828 and 3831. In addition, the product was digested with SstII and the two expected cleavage products were clear on an agarose gel, as shown in Fig. 3. A control PCR in which the reverse transcriptase was not included did not produce any DNA fragments (data not shown). These data indicate clearly that the passaged virus was derived from the cloned cDNA and that the amplified fragment was not generated from contaminating plasmid DNA. This represents the first description of rescue of a metapneumovirus genome from cDNA.
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By analogy with HRSV, the G gene of APV is anticipated to encode the attachment protein that mediates the initial interaction between the virus and host cell (Levine et al., 1987). Despite its central role in the virus life cycle, the HRSV G protein has been shown to be dispensable for virus growth in cell culture. Karron et al. (1997)
reported the characterization of an HRSV vaccine candidate that was shown to contain a deletion that removed both the SH and G genes. Reverse-genetics approaches subsequently confirmed that loss of the G gene in HRSV, either singly or together with removal of the SH gene, did not result in loss of infectivity in vitro (Techaarpornkul et al., 2001
; Teng et al., 2001
). This is consistent with the observations reported here for
SH/G-APV and further strengthens the similarity of the two systems. However, deletion of the HRSV G gene was shown to affect the efficiency of replication in certain cell types (Teng et al., 2001
). It was suggested that this effect was primarily at the level of attachment and entry, although the precise reasons for the host cell-dependent differences are not yet clear. As with the SH- and G-deleted vaccine candidate, recombinant virus analysis showed that the G protein is important for infection in vivo. It will be of interest to study the replication characteristics of
SH/G-APV in various cell types and in vivo.
The new ability to manipulate the APV genome will lead to fundamental questions being addressed concerning gene function. Several recombinant HRSV viruses have been shown to be attenuated (Marriott & Easton, 1999). Commercially derived, live APV vaccines for administration to poultry have been available for over 10 years in Europe and these have led to major improvements in disease control (Cook, 2000
). However, doubts remain about their performance under certain conditions, especially where vaccine administration may not be optimal and significant numbers remain unvaccinated (Jones, 1996
; Cook, 2000
). APV vaccines are known to revert to a virulent state under experimental conditions (Naylor & Jones, 1994
) and there is evidence to suggest that this also occurs in the field, especially where vaccine administration is poor (Naylor et al., 2003
). It is widely believed that the current vaccines may represent the maximum stability that is achievable by empirical cell passage (Naylor et al., 2003
) and attempts to protect by using recombinant and DNA vaccines have to date conferred protection markedly inferior to that conferred by the current products (Yu et al., 1994
; Kapczynski & Sellers, 2003
). Killed vaccines have been shown to be poorly effective unless preceded by an initial live vaccination (Cook, 2000
). Current work in our laboratory, involving the study of previously derived empirical vaccines, together with associated progenitor strains and virulent revertants (Naylor & Jones, 1994
), is starting to identify genome regions where specific mutations may significantly alter pathogenesis. It is anticipated that the reverse-genetics system described here will open new avenues for the development of live vaccines to improve on the empirical types that are currently in use. It is also probable that such developments may have directly useful applications in studies of the recently discovered human metapneumovirus (van den Hoogen et al., 2001
).
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ACKNOWLEDGEMENTS |
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Received 27 April 2004;
accepted 29 July 2004.