In this study, the whole genomic sequences of three ALV-J isolates associated with hemangioma from layers were determined and compared with the published sequences of ML and hemangioma ALV-J strains. Although the three isolates were collected from different sick layers in three separate provinces of China, they all contained special sequences such as modifications of the LTRs and 5' UTRs that seem to be molecular markers of some hemangioma strains.
Consistent with other ALV-J strains, gag and pol genes of the three isolates were highly conserved. However, env genes of hemangioma strains seem to differentiate from ML strains, since the fact that all hemangioma strains were clustered in the same branch while ML strains belonged to the other branch in the phylogenetic tree (Figure 1C). The env glycoprotein of ALV, as in other retroviruses, functions mainly as a ligand for receptor binding for virus entry into susceptible cells , and was also demonstrated to be a major determinant of the lineage-specific oncogenicity and host range [30–32]. Whether the tumor spectrum is determined by the env genes of ALV-J or not needs further study.
Hemangioma ALV-Js seem to contain special U3 region in the LTR and 5' UTR sequences. Phylogenetic analyses based on the U3 LTR and the 5' UTR revealed that all hemangioma isolates examined and some other retroviruses belonged to the same branch, whereas the published ML ALV-J sequences belonged to another branch (Figure 1A, 1B), implying that these U3 LTR and the 5' UTR sequences of hemangioma ALV-Js were most likely derived from some other retroviruses. To our knowledge, the ALV-J strains reported to date have been recognized as slowly transforming viruses, while related retroviruses such as HBI and RSVs quickly induce tumors in chickens and have altered biological properties [33, 34]. For example, HBI, which was derived from the myc-containing virus MC29, was demonstrated to be capable of inducing a variety of tumors when injected into newborn chickens . Hence, the acute-like sequences of those hemangioma strains may influence the tumor spectrum and pathogenicity of ALV-J in layers. The three isolates in this study can replicate to a high titer in DF-1 cells, a characteristic that is shared with some other hemangioma viruses . This new biological property may have a close relationship with the U3 of the 5' LTR and the 5' UTR contained in the genome of ALV-Js.
It is noteworthy that there were two 19 bp insertions in the U3 LTR and 5' UTR in our three isolates that have not been previously reported. The U3 LTR region of the avian retroviruses has been extensively characterized as a model of a strong transcription regulatory unit. This compact enhancer and promoter drives high levels of viral and cellular gene transcription in many cell types in birds and in mammals [35–37]. According to our analysis, parts of the transcriptional regulatory elements in the U3 region of the three isolates have changed. The binding sites for NFAP-1 and AIB REP1 were not observed in our three isolates but exist in all ML ALV-Js. The NFAP-1 site is recognized by activator protein 1 (AP-1). AP-1 is a heterodimeric protein that regulates gene expression in response to stimuli such as cytokines, growth factors, stress, and bacterial and viral infections . AIB REP1, known as repair of chromatin damage 1, was recently reported to be a regulatory factor of human gene Apo-AI. Apo-AI is closely related to the vascular invasion of hepatocellular carcinoma in humans . The absence of the NFAP-1 and AIB REP1 binding sites may influence the infection abilities of the ALV-J virus in chickens or the development of hemangioma. Moreover, the insertion in the U3 introduced an E2BP binding site. E2BP was first found in hepatitis B virus (HBV) and influences the liver specificity of HBV replication through the combination with an enhancer, EII . The additional DNA binding site for E2BP introduced in the U3 may be related to viral tropism . The insertion or deletion of transcription regulatory elements may severely impact the transcriptional activity of the LTR in hemangioma isolates, thereby affecting the replication or infection capability of this virus.
The 5' UTR played an important role in viral replication through potential intra- and intermolecular interactions . One RNA polymerase II transcription factorIIB element and core promoter motif ten elements were introduced via a 19-bp insertion in the 5' UTR of all three isolates; this insertion is also found in the hemangioma strains SCAU-HN06 and JS09GY6 [13, 14]. However, the influence of the inserted sequence in viral replication is still unknown.
Elements in the 3' UTR play a key role in virion assembly and the ability to induce tumors . Early studies found that the rTM exists in the majority ALV-J trains in China; however, deletion of the rTM has been observed in a large number of ALV-J strains in recent years [13, 14, 40]. The loss of the rTM seems to be a trend in the sequence variation of the 3' UTR in current ALV-Js in China (unpublished data). Therefore, the rTM is suspected to be related to the evolution and virulence of this virus, and its role should not be ignored. The E element, containing a biding site for the transcription factor c/EBP and acting as an enhancer, was previously only found in the gene of sarcoma viruses . Currently, the E element is present in many ALV-J strains in addition to our isolates (unpublished data). The existence of the E element in the 3'UTR does increase the occurrence rate of tumors in chickens infected with ALV-J, although this element is not the decisive factor in the induction of tumors . For the hemangioma ALV-Js examined, a binding site for c-Ets-1, introduced in JL093-1 and SD09DP03 due to a 1-bp deletion, was associated with the development of hemangiomas. Consistent with other reports, DR 1 of all hemangioma ALV-Js was very well conserved. This high level of conservation is consistent with the fact that DR 1 plays an important role in the assembly of the genomic RNA of ALV-Js .
In conclusion, our study is the first to discovery the coexistence of two novel insertions in the U3 region in the LTR and the 5' UTR of ALV-J associated with hemangioma symptoms, and the transcriptional regulatory elements introduced should be taken into consideration in the occurrence of hemangioma.