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Short Communication |
Division of Avian and Fish Diseases, Kimron Veterinary Institute, PO Box 12, Bet Dagan 50250, Israel
Correspondence
Irit Davidson
davidsoni{at}int.gov.il
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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Two supplementary tables are available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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).
Vaccination with various live vaccines is widely practised and was thought to be effective in preventing the disease, at least until 10 years ago; since then, the vaccination efficacy has been doubted (Fatunmbi & Reed, 1996; Tripathy et al., 1998; Singh et al., 2000). The avian retrovirus reticuloendotheliosis virus (REV) (Witter & Fadly, 2003), which has inserted various genomic fragments into FWPV (Garcia et al., 2003; Hertig et al., 1997; Kim & Tripathy, 2001; Moore et al., 2000; Singh et al., 2003), was implicated as being responsible for the FWPV virulence alteration (Garcia et al., 2003; Fatunmbi & Reed, 1996). Subsequently, a field FWPV isolate containing an REV insert was used to create a modified FWPV in which the REV inserts were eliminated (Singh et al., 2005). By comparing the lesions induced by the two versions of the same FWPV, the contribution of the REV insert to the increased virulence of the progenitor FWPV was confirmed. REV-containing FWPV isolates were also found to be responsible for immunosuppression and even dissemination of REV, if a full infective provirus is integrated into the FWPV (Singh et al., 2000).
REV integration also occurred in FWPV vaccine strains, leading to their immediate elimination from use (Hertig et al., 1997). Initial observations from commercial flocks suggested that FWPV vaccine contamination with REV caused tumours in the vaccinated birds (Bendheim, 1973; Bagust & Dennett, 1977; Fadly et al., 1996). The link between FWPV and REV was evident from the wide prevalence of REV antibodies detected in conjunction with FWPV lesions. The REV envelope gene functions as the immunodominant protein (Davidson et al., 1996) and is responsible for eliciting production of REV antibodies. However, two simultaneous and independent studies deciphered the link between DNA viruses and retroviruses. In the first study, Hertig et al. (1997) demonstrated integration of intact or partial REV provirus into FWPV field isolates and Australian vaccine strains. In the second study, Davidson & Borenshtain (2001) (recently reviewed by Davidson & Silva, 2008) demonstrated that REV may integrate into another avian DNA virus, Marek's disease virus (MDV), in dually virus-infected chickens. This event occurred less in vivo than was reported in studies describing in vitro virus replication (Brunovskis & Kung, 1996). Both phenomena result from the replication requirements of retroviruses; synthesis of the retroviral RNA leading to the promotion of virion assembly necessitates the integration of reverse-transcribed viral RNA into double stranded (ds) DNA. In MDV or FWPV co-infected avian cells, the retroviral integration process can occur in either the cellular or the ds viral DNA, and recombinant FWPV or MDV genomes can be created.
Molecular descriptions of FWPV isolates with REV genomic insertions have accumulated from various parts of the world (Garcia et al., 2003; Hertig et al., 1997; Kim & Tripathy, 2001; Singh et al., 2000; Prukner-Radovcic et al., 2006). These studies demonstrated that REV-inserted fragments in wild-type field FWPV isolates were variable in content, although they included REV long-terminal repeats (LTRs) of various lengths and additional sections of the proviral genome. Although genomic integration of REV into the FWPV genome has only been demonstrated in the last 10 years, the analysis of previous field isolates indicates that such events had occurred 50 years ago (Kim & Tripathy, 2001). In contrast to FWPV field isolates, most FWPV vaccines include only partial REV-LTR insertions of various sizes, with the exception of the Australian vaccine strain FWPV-S, which carries a nearly full-length REV provirus (Diallo et al., 1998; Hertig et al., 1997; Moore et al., 2000; Singh et al., 2000). According to the currently available information, all field FWPV isolates contain the REV-LTR and additional variable provirus fragments; the only known exception is an FWPV isolated in 1956, in which only LTR remnants and not additional provirus genes were found (Boulanger et al., 1998).
Analysis of the REV integration site in wild-type field FWPV isolates and in FWPV vaccine strains revealed that all insertion events occurred in a hot spot of the FWPV genome, located between the FWPV open reading frame (ORF) 201 and FWPV ORF 203 (Garcia et al., 2003; Moore et al., 2000; Afonso et al., 2000). The REV fragment that was inserted into the FWPV genome differed in composition between wild-type FWPV and FWPV vaccine strains. While all the wild-type isolates contained complete REV provirus or various fragments of the 3' and 5' REV-LTRs in addition to other REV fragments, including the REV env gene, recent FWPV vaccine strains contained only the REV-LTR (Garcia et al., 2003; Moore et al., 2000; Singh et al., 2003).
REV genome integration into FWPV has so far been described for all FWPVs of domestic poultry, as other poxviruses (pigeon pox, quail pox and canary pox) were negative for REV-LTR (Moore et al., 2000; Prukner-Radovcic et al., 2006). In the present study, we sought to identify the molecular characteristics of a wide collection of isolates which were obtained directly from poultry and wild birds over the last 10 years, in order to determine the universality of REV integration in vivo in poultry.
We analysed 243 DNA samples, originating from 108 chicken and turkey flocks and from individual wild birds cases, by PCR. The collection originated from two sources; 51 samples were from the Tzfat Regional Poultry Laboratory and 57 were from flocks which were submitted directly to Kimron Veterinary Institute (KVI) that were gathered from cases occurring from 1997 to 2002. The Tzfat samples were collected from the northern region of Israel, while the KVI samples were from all areas of the country. The Tzfat samples were submitted as lesion tissue homogenates in PBS, thus one sample represented one individual flock. The KVI samples were received from cases that have occurred since 2003; these were prepared from individual birds and lesions, thus each flock is represented by one or more samples.
The first step in the molecular analysis was screening by the PER-PCR (PER stands for pox, envelope, REV) (see Supplementary Tables S1 and S2 for primer sequences and PCR conditions; Garcia et al., 2003); this amplified the FWPV junction at ORF203 with the 3' REV-LTR and a fragment of the REV env gene. From the total of 243 samples, 129 samples were positive for FWPV PER-PCR (Table 1), indicating that they possess the REVenv insertion. In the second step, all samples were subjected to the pGen-PCR (see Supplementary Tables S1 and S2; Luschow et al., 2004). This was a general PCR that amplified DNA from all FWPV isolates, as the primers were based on the conserved FWPV 4b gene.
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and Supplementary Table S2). All seven isolates were negative, indicating that they lack the REV insert from both the 5' and 3' junctions of the REV provirus with the FWPV ORF 201 and 203. The seven isolates were identified as FWPV as they were positive in the pGen-PCR (Supplementary Table S2).
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shows the REV insert content of the FWPV isolates that were collected from the Tzfat and KVI laboratories, both as separate and as cumulative data of individual samples. Following the initial amplification using the PER-PCR, in which all DNA samples were positive for the 3' LTR and REV env gene, all samples were analysed for the presence of various fragments of the REV insert, including the 3' LTRs and the REV gag (system 201+gag) and REV pol (system 201+pol) genes. The pol gene amplification yielded three types of product, according to the insert size, which reflected the variability in the virus population within one bird. The same variability was observed for the PCR products obtained with primer FWPV ORF 201 paired with the 5' LTR primers, R4 and R2 (Supplementary Table S2 and Fig. 1). While the Tzfat laboratory isolates were almost uniform in the REV insert content, the KVI laboratory isolates were variable. Possible explanations might include (i) the limited geographical distribution of the Tzfat isolates in the northern region of Israel, compared with the broad distribution of the KVI isolates that were collected from all over the country; and (ii) that while all Tzfat isolates were examined in parallel by their inoculation in embryonated eggs and their ability to cause the production of typical pocks on the chorioallantoic membrane (CAM), most KVI isolates were not examined in embryonated eggs, thus they were not selected to have the common property of forming CAM pocks. This might provide a filter for FWPV uniformity. The consistency of the the sequence of the REV gag gene inserts among the Tzfat FWPV isolates was also verified. DNA from twelve FWPV isolates (eleven from layers and one from a turkey) was amplified using PCR system 201+gag and aligned to FWPV and REV (GenBank accession numbers AF006065 [GenBank] and NC_006934 [GenBank] , respectively). The FWPV genomic fragment of these isolates was 100 % identical to FWPV AF006065 [GenBank] [over a 500 nucleotide (nt) sequence]. Compared to each other, all FWPV isolates were 100 % identical in their REV sequences; however, this sequence was 3 % divergent from the published sequence of REV (over a 660 nt sequence).
The vaccine virus strains of three companies were also verified for their REV insert content using three PCR systems (Supplementary Tables S1 and S2). These were (i) two PCRs directed to amplify the REV genes only [the REV-LTR (using systems 201+R2 and 201+R4; Supplementary Table S2) and the REV env gene fragment (using system REVenv)], both of these were negative for the three vaccine virus strains; (ii) two PCRs to amplify the FWPVenv (using system FWPVenv) and FWPV 4b gene fragments (using system pGen-PCR); both PCRs were positive for the three vaccine virus strains; and (iii) three PCRs to amplify the FWPV and the REV virus genome fragments at the junctions (systems J-I, J-II and PER-PCR). Amplification of the 3' LTR junction fragments with the primers FPRJ1 and FPRJ2 and PG-1 and PG-2 (systems JI and JII; Supplementary Table S2) (Kim & Tripathy, 2001; Moore et al., 2000) was positive and the PER-PCR was negative, indicating that the REV env gene was not present in the insert.
To ascertain whether the REV inserts were absent in the FWPV vaccine strains, excluding the remnants of partial REV-LTR, the vaccine DNAs were amplified with the FWPVORF203 and FWPVORF201 yielding the expected product size of about 1300 bp, indicating that the REV inserts were absent in the FWPV vaccine strains.
In parallel, to ascertain the presence of REV-LTR and REVenv sequences in the product obtained using the PER-PCR system, the products from two FWPV isolates were sequenced and aligned to FWPV and REV-LTR (GenBank accession numbers AF198100 [GenBank] and M22224 [GenBank] , respectively), revealing fragments of the FWPV ORF 203 (nt 8137–8221), and REV-LTR and REVenv fragments (nt 7718–8078).
Finally, we analysed the viability of FWPV isolates with and without REV inserts for their replication and creation of pocks on CAM of embryonated eggs. The original homogenates of FWPV isolates from three wild birds with fowlpox (owl, dove and crane) without a REV insert (positive PCR for pGen and negative for PER), three FWPV isolates from turkeys (one with a REV insert and the other two without inserts) and seven isolates from chickens (two of them without inserts) created similar CAM pocks.
In summary, the present survey presents the first evidence not only that FWPV from wild birds lack REV inserts, but also that FWPV from poultry can be deficient in that genomic fragment, while retaining viability and producing disease. Therefore, it appears that the present concept that the REV insertion is universal in FWPV isolated from chickens and turkeys (Garcia et al., 2003; Hertig et al., 1997; Kim & Tripathy, 2001; Singh et al., 2000) no longer holds. Our report provides evidence that recent poultry FWPV isolates can appear in vivo without REV inserts. Until the present report, only one isolate, FP9, from 1956, was shown to lack REV inserts. As previous studies demonstrated, REV inserts into FWPV at a unique FWPV genomic site only, between ORFs 203 and 201 (Garcia et al., 2003), so we focused our study at that site. Nevertheless, we cannot exclude the presence of REV inserts in additional genomic sites of FWPV, which were not explored here. Future studies might elaborate the presence of retrovirus insertions in other sites of the whole FWPV genome.
The molecular analysis presented in here confirms the findings of Singh et al. (2003) who demonstrated the variability of the REV insert in various isolates. However, the present analysis of isolates from two sources revealed that within a restricted area, FWPV are more similar to each other compared with the wide collection that was available by direct submission to the KVI laboratory. Similarly to previous studies, we demonstrated that FWPV vaccine virus strains and FWPV from wild birds do not contain REV env gene inserts (Garcia et al., 2003; Moore et al., 2000; Afonso et al., 2000).
The mechanism of REV insertion at a unique FWPV ORF 201/203 site (Garcia et al., 2003; Moore et al., 2000; Afonso et al., 2000; Prukner-Radovcic et al., 2006) has two consequences; the relatively unproblematic REV genomic fragment insertion into the poultry FWPV, considered until the present report to be universal, can revert to non-universal for many FWPV poultry isolates, but REV insertion can also occur suddenly and remain undetected in FWPV vaccine strain viruses under field conditions. Careful examination using a panel of PCR systems, as presented in this study, will allow monitoring of leakage of REV into vaccine viruses. The spontaneous genomic changeability of FWPVs from poultry and wild birds is a natural example of recombination between viruses, which contributes to the creation of virus variability, leading in turn to many biological advantages and disadvantages (Davidson & Silva, 2008).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Received 14 February 2008;
accepted 13 May 2008.
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