| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Viral Oncogenesis Group, Division of Microbiology, Institute for Animal Health, Compton, Berkshire RG20 7NN, UK
Correspondence
Venugopal Nair
venu.gopal{at}bbsrc.ac.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and is one of the six envelope subgroups of ALV affecting chickens, the others being subgroups A, B, C, D and E (Fadly & Payne, 2003). ALV-J has been differentiated from the other subgroups on the basis of neutralization and interference assays (Payne et al., 1991b, 1992) and by genomic sequence analysis of the prototype strain HPRS-103 (Bai et al., 1995). ALV subgroups A and B, including the RCAS-BP replication-competent retroviral vectors derived from them, primarily induce lymphoid leukosis (LL) and erythroblastosis (EB) through insertional activation of the c-myc (Hayward et al., 1981) and the c-erbB (Fung et al., 1983) proto-oncogenes, respectively. In comparison, experimental infection of leghorn and meat-type strains of chickens with HPRS-103 mainly induced ML (Payne et al., 1993), although acutely transforming viruses that induce EB have also been isolated from ALV-J-induced tumours (Venugopal et al., 2000). We have shown previously that ALV-J-induced ML also occurs through insertional activation of c-myc (Chesters et al., 2001), demonstrating that the molecular mechanisms of induction of LL and ML are the same (Venugopal, 1999), although other factors, such as levels of c-myc expression and co-operating oncogenes/tumour-suppressor genes, may also be important.
The oncogenic specificity of ALV subgroups has been ascribed to differences in env or the long terminal repeat (LTR) of the virus (Brown et al., 1988), reflecting the two fundamental aspects of retroviral infection, attachment and replication. Virus attachment and entry are mediated by the viral envelope glycoprotein through specific binding to cell-surface receptors. Virus replication is regulated by the LTR, which drives viral gene expression in association with various cis-acting elements in eukaryotic cells (Ruddell et al., 1989). Studies carried out by using chimeric viruses have demonstrated that the viral envelope is a major determinant of cell tropism (Brown & Robinson, 1988; Chesters et al., 2002).
The genome structure of ALV-J shows overall similarity to that of other subgroups, with the gag and pol genes showing over 96 % sequence identity to those of subgroups A–E (Bai et al., 1995). The LTR region of ALV-J also showed >90 % sequence identity to that of other ALV subgroups. However, the sequence of ALV-J env showed much less similarity, although it showed a close relationship to that of a novel group of chicken endogenous retroviral elements designated EAV-HP (Benson et al., 1998; Smith et al., 1999; Sacco et al., 2000). This unique structure of ALV-J, consisting of a divergent env gene and a conserved ALV backbone, suggests that it has emerged by recombination (Bai et al., 1995). As retroviral envelopes mainly act as ligands for receptor binding, the tropism of ALV subgroups for cells of different lineages is thought to be related to the distribution of the specific virus receptors on different cell types. Recently, the chicken Na+/H+ exchanger type 1 has been identified as the specific receptor for ALV-J (Chai & Bates, 2006). As this receptor is distributed over a wide range of cell types, including some that are not transformed by ALV-J, it is unlikely that the envelope–receptor interaction alone is the single determinant in the lineage-specific oncogenicity of these viruses.
Yet another feature unique to the ALV-J sequence is the presence of a hairpin stem–loop structure (Fig. 1) called the E element in the 3' untranslated region (UTR) (Bai et al., 1995). The function of the E element, homologous to the XSR (exogenous virus-specific region) found in certain strains of Rous sarcoma virus (RSV) (Bizub et al., 1984; Laimins et al., 1984), is not known. In RSV, it has been suggested that the XSR contributes to the differences in disease spectra, possibly through an enhancer function (Laimins et al., 1984), but its absence in many other oncogenic sarcoma viruses indicates that it may not be essential for the induction of sarcomas (Bizub et al., 1984). However, the role of the E element in the oncogenicity of ALV-J strains is far from clear. Isolation of natural ALV-J strains with deletions in the 3' UTR from cases of ML would suggest that the E element may not be essential for oncogenicity (Cui et al., 2003). Conversely, the presence or absence of the E element was thought to account for the differences in the induction of tumours between two naturally occurring recombinant ALV-J/A chimeric viruses (Lupiani et al., 2003). Both of these studies used field virus stocks, which may contain viruses with multiple sequence changes that may be associated with the differences in pathogenicity. Hence, for a more precise analysis of the role of the E element in ALV-J oncogenesis, we chose to use virus derived from the molecular clone of HPRS-103 from which the E element was deleted.
|
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The propagation of viruses was initiated by transfection of 1–2 µg proviral DNA into CEFs, and culture supernatant containing virus stocks was harvested 7 days later (Chesters et al., 2002). An ALV group-specific antigen (p27) ELISA (Smith et al., 1979) was used for the titration of virus stocks and for detecting virus shedding in cloacal swabs. Virus-specific antibodies were detected by a microneutralization test (Fadly & Witter, 1998).
Strategy for deletion of the E element.
For deletion of the E element, the ClaI–SpeI fragment from the full-length HPRS-103 provirus clone (Bai et al., 1995) was replaced with a fusion product of two PCR fragments containing overlapping sequences spanning the deleted region. The principle of the procedure has been described in detail elsewhere (Higuchi, 1989) and is shown in Fig. 2. Briefly, two PCR products spanning the region to be deleted were produced by using primer pairs F1/R1 and F2/R2. Primer R1 included sequences immediately upstream of the first nucleotide of the E element and sequences immediately downstream of the last nucleotide. Primer F2 similarly spanned the region to be deleted by overlapping the 5' end of R1. The two products with overlapping sequences at their termini were combined in equimolar amounts and amplified with primers F1 and R2 to obtain the PCR product with the deleted E element. The ClaI–SpeI fragment derived from the PCR product was cloned into the equivalent region of the HPRS-103 full-length proviral clone to generate the pHPRS-103
E deletion mutant.
|
. PCRs were carried out by the ‘touchdown’ procedure (Don et al., 1991) using the Expand Long Template PCR system (Roche Diagnostics). Cycling conditions for the F1/R1 PCR were one cycle of 92 °C for 5 min; 16 cycles of 92 °C for 10 s, 68 °C (–1 °C per cycle) for 30 s and 68 °C for 1 min; 30 cycles of 92 °C for 10 s, 53 °C for 30 s and 68 °C for 1 min; and one cycle of 68 °C for 10 min. Cycling conditions for the F2/R2 and F1/R2 PCRs were one cycle of 92 °C for 5 min; 16 cycles of 92 °C for 10 s, 65 °C (–1 °C per cycle) for 30 s and 68 °C for 1 min; 30 cycles of 92 °C for 10 s, 50 °C for 30 s and 68 °C for 1 min; and one cycle of 68 °C for 10 min. The PCR using primer pair F3/R3, designed from the region flanking the E element, was used as a diagnostic test to detect deletion of the E element. This was carried out with Taq polymerase using cycling conditions of one cycle of 92 °C for 5 min; 13 cycles of 92 °C for 10 s, 60 °C (–1 °C per cycle) for 30 s and 72 °C for 45 s; 30 cycles of 92 °C for 10 s, 48 °C for 30 s and 72 °C for 1 min; and one cycle of 72 °C for 10 min.
|
E virus stock through a vein in the chorioallantoic membrane. Infection of hatched chicks was carried out by intra-abdominal inoculation of 5x104 TCIU of the two viruses. Control birds were injected with uninfected tissue-culture fluid. Cloacal swabs were taken from all embryo-infected birds and tested for infection status by direct p27 ELISA. Serum samples collected from post-hatch-infected birds 7 weeks after infection were tested by using a microneutralization test (Fadly & Witter, 1998). The birds were observed for 141 days and examined post-mortem for any gross or microscopic tumours.
Analysis of tumour DNA.
Frozen tumour tissues collected during post-mortem examination of infected birds were used for DNA extraction. Homogenized tissues were digested with proteinase K and ribonuclease A (Sigma-Aldrich), and high-molecular-mass DNA was obtained by phenol/chloroform extraction and ethanol precipitation using standard methods (Sambrook & Russell, 2001). DNA samples were initially analysed by F3/R3 PCR to confirm deletion of the E element. DNA was also used to detect virus integration junctions within the c-myc or c-erbB loci by using a nested PCR (Gong et al., 1998). PCR was carried out with primers L1/L2 derived from the U5 LTR and M1/M2 from exon 2 of c-myc or E1/E2 from c-erbB exon 15 as described previously (Chesters et al., 2001). Nucleotide sequences of the PCR products were obtained directly or after cloning into the pGEM-T vector (Promega).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
E was constructed as described in Fig. 2
. The precise deletion of the 147 bp E element (nt 7359–7505 of HPRS-103) in the HPRS-103E construct was confirmed by sequence analysis. DNA from the HPRS-103 and HPRS-103E clones was transfected into CEFs to produce the virus stocks. HPRS-103E virus obtained from the culture supernatants of transfected CEFs gave titres (105 TCIU ml–1) similar to those of HPRS-103 virus, indicating that deletion of the E element did not have a major effect on in vitro replication.
Birds from lines 0 and 15I infected as 1-day-old chicks with either of the viruses did not show any gross or histological lesions. However, evidence of virus replication was apparent in these birds from examination of serum samples 7 weeks after infection. These tests detected neutralizing antibodies in 5/32 (15.6 %) HPRS-103-infected and 12/32 (37.5 %) HPRS-103E-infected line 0 birds. In comparison, none of the line 15I birds infected with HPRS-103 (0/29) or HPRS-103E (0/29) developed neutralizing antibodies during this time (Table 2). The infection status of birds infected as embryos was determined from the results of a p27 ELISA on cloacal swabs collected 16 days after hatching. There was no significant difference in the numbers of p27 ELISA-positive birds in line 0 (29/30) and line 15I (36/36) infected with HPRS-103. However, for HPRS-103E infection, 30/30 line 0 birds were positive by p27 ELISA (Table 2), whilst only approximately half (15/27) of the line 15I birds were positive (P
0.05).
|
1.0). In comparison, the levels of oncogenicity were marginally lower (23.3 %) in line 0 birds infected with HPRS-103E virus (P1.0). Significantly, however, none (0/27) of the line 15I birds (P0.01) infected as embryos with HPRS-103E virus developed tumours during the course of the experiment. Histologically, the tumours were diagnosed as either EB with immature intravascular blast cells or ML with well-differentiated myeloid cells (Fig. 3). In a few cases, mixed ML/EB tumours were also present (Table 3). Uninfected control birds showed no evidence of tumours.
|
|
E virus was tested initially by using the diagnostic F3/R3 PCR to identify proviruses with an intact or deleted E region (Fig. 4). As expected, these tests showed a smaller (377 bp) product in the three samples from birds infected with HPRS-103E (Fig. 4, lanes 1–3) and a larger (523 bp) product in the eight tumours from birds infected with HPRS-103 (Fig. 4, lanes 4–11). Further analysis to determine the provirus integration site in these tumour DNA samples demonstrated virus integration in both the c-myc and the c-erbB loci (Fig. 5). The presence of multiple bands in these tests also indicated the oligoclonal nature of these tumours. All three tumours derived from HPRS-103E-infected birds and four of the eight tumours derived from HPRS-103-infected birds showed evidence of integration within the c-myc locus (Fig. 5, lanes 1–7). The four remaining tumour DNAs derived from HPRS-103-infected birds showed evidence only of c-erbB integration (Fig. 5, lanes 9–12). One tumour sample each from an HPRS-103E-infected (no. 390) and an HPRS-103-infected (no. 1999) bird also showed evidence of integration in both the c-myc and c-erbB loci (Fig. 5, lanes 8 and 13), suggesting involvement of either multiple cell types or multiple integrations within the same cell type (Table 3).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). By using chimeric ALV constructs substituted with env genes from the A or J subgroups, we have shown previously that env is the major determinant of cell type (lymphoid or myeloid)-specific oncogenicity of ALV (Chesters et al., 2002). However, this study suggested that env alone did not account for all of the variations in the incidence of tumours induced by the two chimeric viruses in two different chicken lines and that other viral/host factors are also important. This prompted us to examine the role of the other unique ALV-J sequence, the 147 bp hairpin stem–loop-structured E element within the 3' UTR (Fig. 1). The function of the E element in ALV-J is unclear, although the closely related XSR element in RSV (Bizub et al., 1984) shows an enhancer function (Laimins et al., 1984). Reports on the involvement of the E element in the pathogenicity of ALV-J based on natural isolates have been inconclusive. For example, the isolation from myeloid tumours of ALV-J strains with deletions in the 3' UTR including the E element suggests a non-essential role for the E element in ALV-J pathogenesis (Sung et al., 2002; Cui et al., 2003). However, in another study using two naturally occurring recombinant ALV-J/A chimeric viruses with or without the 3' UTR containing the E element, there were differences in the incidence of tumours, indicating a potential function in oncogenesis (Lupiani et al., 2003). These studies used virus stocks of field isolates and thus may show heterogeneity and sequence variations in the virus pool. In order to examine the precise role of the E element in ALV-J oncogenicity, we examined the oncogenic properties of virus stocks derived from molecular clones of HPRS-103 and HPRS-103E in two lines of birds.
Deletion of the E element did not affect in vitro replication, as the titres of virus stocks generated by transfection of the provirus clones were identical. Deletion of the E element also did not appear to affect virus replication in line 0 birds, as demonstrated by the similar proportion of infected birds detected either by cloacal swab ELISA or by neutralizing-antibody response at 7 weeks of age (Table 2). However, in line 15I, only 55.6 % of the birds infected with HPRS-103E virus showed evidence of infection by cloacal swab ELISA (compared with 100 % of birds infected with the HPRS-103 virus), demonstrating that deletion of the E element interferes with virus replication in this line. As the line 15I birds are immunologically tolerant to ALV-J infection (Sacco et al., 2004), the failure to detect neutralizing antibodies in these birds 7 weeks after infection with either virus was not unexpected.
In addition to reduced in vivo replication of HPRS-103E virus in line 15I, there was also reduced oncogenicity. Whilst infection with HPRS-103 did not show any significant difference in the incidence of tumours between line 0 and line 15I (33.3 %), this was not the case for HPRS-103E virus (Table 2). In line 0 birds, the incidence of tumours induced by HPRS-103E virus (23.3 %) was slightly lower than that induced by HPRS-103. The oncogenic spectrum of the two viruses was very similar and consisted predominantly of EB or ML (Fig. 3). As we have shown previously (Chesters et al., 2001), the DNA samples extracted from these tumours showed viral integration in the c-erbB and c-myc loci (Table 3 and Fig. 5), suggesting that these tumours are induced by insertional activation of these oncogenes.
In line 15I birds, deletion of the E element had a dramatic negative effect on oncogenicity, as none of the birds infected with HPRS-103E virus developed tumours during the experimental period. This was not due to any specific resistance of line 15I, as the parental HPRS-103 virus induced tumours in 33.3 % of the infected birds. Thus, the loss of oncogenicity in line 15I could be attributed directly to deletion of the E element. Inbred line 15I is known for its increased susceptibility to lymphoid leukosis, Marek's disease (Bacon et al., 2000) and ALV-J tumours (Payne et al., 1991b). However, the study presented here shows that this susceptibility to ALV-J tumours is dependent specifically on the presence of an intact E element. This would also perhaps explain the absence of oncogenicity in line 15I birds that we observed in an earlier study, where we used a chimeric RCAS-J construct lacking the E element for infection (Chesters et al., 2002). Despite these observations, the unique role of the E element in the oncogenicity of ALV-J in line 15I remains unclear. One of the well-known differences between lines 0 and 15I is the absence of any of the ev loci in the former. Also, we have recently identified an intact EAV-HP element with over 99 % sequence identity to HPRS-103 env in the line 15I genome (Sacco et al., 2004). Based on its expression and distinct high similarity to the HPRS-103 envelope, it was suggested that this novel locus may have contributed to the emergence of ALV-J by recombination (Venugopal, 1999). Further studies are needed to examine any possible relationships in the distribution of endogenous retroviruses, the HPRS-103 E element and differences in oncogenicity between these lines. The molecular basis for modulation of oncogenicity by the E element remains to be identified. Some indication of the E element function is given by the predicted secondary hairpin stem–loop structure (Fig. 1), which provides a potential nucleation site for RNA folding and multiple interactions with some of the host factors. In RSV, the XSR has been suggested to have an enhancer function (Laimins et al., 1984). If these functions are attributable to the HPRS-103 E element, our study shows that it is only essential and unique in certain lines, and further studies are needed to unravel the molecular mechanisms and differences between chicken lines.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bai, J., Payne, L. N. & Skinner, M. A. (1995). HPRS-103 (exogenous avian leukosis virus, subgroup J) has an env gene related to those of endogenous elements EAV-0 and E51 and an E element found previously only in sarcoma viruses. J Virol 69, 779–784.[Abstract]
Benson, S. J., Ruis, B. L., Fadly, A. M. & Conklin, K. F. (1998). The unique envelope gene of the subgroup J avian leukosis virus derives from ev/J proviruses, a novel family of avian endogenous viruses. J Virol 72, 10157–10164.
Bizub, D., Katz, R. A. & Skalka, A. M. (1984). Nucleotide sequence of noncoding regions in Rous-associated virus-2: comparisons delineate conserved regions important in replication and oncogenesis. J Virol 49, 557–565.
Brown, D. W. & Robinson, H. L. (1988). Influence of env and long terminal repeat sequences on the tissue tropism of avian leukosis viruses. J Virol 62, 4828–4831.
Brown, D. W., Blais, B. P. & Robinson, H. L. (1988). Long terminal repeat (LTR) sequences, env, and a region near the 5' LTR influence the pathogenic potential of recombinants between Rous-associated virus types 0 and 1. J Virol 62, 3431–3437.
Chai, N. & Bates, P. (2006). Na+/H+ exchanger type 1 is a receptor for pathogenic subgroup J avian leukosis virus. Proc Natl Acad Sci U S A 103, 5531–5536.
Chesters, P. M., Howes, K., McKay, J. C., Payne, L. N. & Venugopal, K. (2001). Acutely transforming avian leukosis virus subgroup J strain 966: defective genome encodes a 72-kilodalton Gag-Myc fusion protein. J Virol 75, 4219–4225.
Chesters, P. M., Howes, K., Petherbridge, L., Evans, S., Payne, L. N. & Venugopal, K. (2002). The viral envelope is a major determinant for the induction of lymphoid and myeloid tumours by avian leukosis virus subgroups A and J, respectively. J Gen Virol 83, 2553–2561.
Cui, Z., Du, Y., Zhang, Z. & Silva, R. F. (2003). Comparison of Chinese field strains of avian leukosis subgroup J viruses with prototype strain HPRS-103 and United States strains. Avian Dis 47, 1321–1330.[CrossRef][Medline]
Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K. & Mattick, J. S. (1991). ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19, 4008.
Fadly, A. M. & Payne, L. N. (2003). Leukosis/sarcoma group. In Diseases of Poultry, 11th edn, pp. 465–516. Edited by Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald & D. E. Swayne. Ames, IA: Iowa State Press.
Fadly, A. M. & Witter, R. L. (1998). Oncornaviruses: leukosis/sarcoma and reticuloendotheliosis. In A Laboratory Manual for the Isolation and Identification of Avian Pathogens, 4th edn, pp. 185–196. Kennett Square, PA: American Association of Avian Pathologists.
Fung, Y.-K. T., Lewis, W. G., Crittenden, L. B. & Kung, H.-J. (1983). Activation of the cellular oncogene c-erbB by LTR insertion: molecular basis for induction of erythroblastosis by avian leukosis virus. Cell 33, 357–368.[CrossRef][Medline]
Gong, M., Semus, H. L., Bird, K. J., Stramer, B. J. & Ruddell, A. (1998). Differential selection of cells with proviral c-myc and c-erbB integrations after avian leukosis virus infection. J Virol 72, 5517–5525.
Hayward, W. S., Neel, B. G. & Astrin, S. M. (1981). Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature 290, 475–480.[CrossRef][Medline]
Higuchi, R. (1989). Using PCR to engineer DNA. In PCR Technology: Principles and Applications for DNA Amplification, pp. 61–70. Edited by H. A. Erlich. New York: Stockton Press.
Laimins, L. A., Tsichlis, P. & Khoury, G. (1984). Multiple enhancer domains in the 3' terminus of the Prague strain of Rous sarcoma virus. Nucleic Acids Res 12, 6427–6442.
Lupiani, B., Williams, S. M., Silva, R. F., Hunt, H. D. & Fadly, A. M. (2003). Pathogenicity of two recombinant avian leukosis viruses. Avian Dis 47, 425–432.[CrossRef][Medline]
Payne, L. N., Gillespie, A. M. & Howes, K. (1991a). Induction of myeloid leukosis and other tumours with the HPRS-103 strain of ALV. Vet Rec 129, 447–448.[Medline]
Payne, L. N., Brown, S. R., Bumstead, N., Howes, K., Frazier, J. A. & Thouless, M. E. (1991b). A novel subgroup of exogenous avian leukosis virus in chickens. J Gen Virol 72, 801–807.
Payne, L. N., Howes, K., Gillespie, A. M. & Smith, L. M. (1992). Host range of Rous sarcoma virus pseudotype RSV(HPRS-103) in 12 avian species: support for a new avian retrovirus envelope subgroup, designated J. J Gen Virol 73, 2995–2997.
Payne, L. N., Gillespie, A. M. & Howes, K. (1993). Recovery of acutely transforming viruses from myeloid leukosis induced by the HPRS-103 strain of avian leukosis virus. Avian Dis 37, 438–450.[CrossRef][Medline]
Ruddell, A., Linial, M. L. & Groudine, M. (1989). Tissue-specific lability and expression of avian leukosis virus long terminal repeat enhancer-binding proteins. Mol Cell Biol 9, 5660–5668.
Sacco, M. A., Flannery, D. M. J., Howes, K. & Venugopal, K. (2000). Avian endogenous retrovirus EAV-HP shares regions of identity with avian leukosis virus subgroup J and the avian retrotransposon ART-CH. J Virol 74, 1296–1306.
Sacco, M. A., Howes, K., Smith, L. P. & Nair, V. K. (2004). Assessing the roles of endogenous retrovirus EAV-HP in avian leukosis virus subgroup J emergence and tolerance. J Virol 78, 10525–10535.
Sambrook, J. & Russell, D. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Smith, E. J., Fadly, A. & Okazaki, W. (1979). An enzyme-linked immunosorbent assay for detecting avian leukosis-sarcoma viruses. Avian Dis 23, 698–707.[CrossRef][Medline]
Smith, L. M., Toye, A. A., Howes, K., Bumstead, N., Payne, L. N. & Venugopal, K. (1999). Novel endogenous retroviral sequences in the chicken genome closely related to HPRS-103 (subgroup J) avian leukosis virus. J Gen Virol 80, 261–268.[Abstract]
Sung, H. W., Reddy, S. M. & Fadly, A. M. (2002). High virus titer in feather pulp of chickens infected with subgroup J avian leukosis virus. Avian Dis 46, 281–286.[CrossRef][Medline]
Venugopal, K. (1999). Avian leukosis virus subgroup J: a rapidly evolving group of oncogenic retroviruses. Res Vet Sci 67, 113–119.[CrossRef][Medline]
Venugopal, K., Howes, K., Flannery, D. M. J. & Payne, L. N. (2000). Isolation of acutely transforming subgroup J avian leukosis viruses that induce erythroblastosis and myelocytomatosis. Avian Pathol 29, 327–332.[CrossRef]
Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 3406–3415.
Received 30 January 2006;
accepted 29 April 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
