A comprehensive analysis of avian lymphoid leukosis-like lymphoma transcriptomes including identification of LncRNAs and the expression profiles.

Avian lymphoid leukosis-like (LL-like) lymphoma has been observed in some experimental and commercial lines of chickens that are free of exogenous avian leukosis virus. Reported cases of avian lymphoid leukosis-like lymphoma incidences in the susceptible chickens are relatively low, but the apathogenic subgroup E avian leukosis virus (ALV-E) and the Marek's disease vaccine, SB-1, significantly escalate the disease incidence in the susceptible chickens. However, the underlying mechanism of tumorigenesis is poorly understood. In this study, we bioinformatically analyzed the deep RNA sequences of 6 lymphoid leukosis-like lymphoma samples, collected from susceptible chickens post both ALV-E and SB-1 inoculation, and identified a total of 1,692 novel long non-coding RNAs (lncRNAs). Thirty-nine of those novel lncRNAs were detected with altered expression in the LL-like tumors. In addition, 13 lncRNAs whose neighboring genes also showed differentially expression and 2 conserved novel lncRNAs, XLOC_001407 and XLOC_022595, may have previously un-appreciated roles in tumor development in human. Furthermore, 14 lncRNAs, especially XLOC_004542, exhibited strong potential as competing endogenous RNAs via sponging miRNAs. The analysis also showed that ALV subgroup E viral gene Gag/Gag-pol and the MD vaccine SB-1 viral gene R-LORF1 and ORF413 were particularly detectable in the LL-like tumor samples. In addition, we discovered 982 novel lncRNAs that were absent in the current annotation of chicken genome and 39 of them were aberrantly expressed in the tumors. This is the first time that lncRNA signature is identified in avian lymphoid leukosis-like lymphoma and suggests the epigenetic factor, lncRNA, is involved with the avian lymphoid leukosis-like lymphoma formation and development in susceptible chickens. Further studies to elucidate the genetic and epigenetic mechanisms underlying the avian lymphoid leukosis-like lymphoma is indeed warranted.

The expression level of chicken telomerase reverse transcriptase in tumors induced by ALV-J is positively correlated with methylation and mutation of its promoter region.

Avian leukosis virus subgroup J (ALV-J) can cause neoplastic diseases in poultry and is still widely prevalent in China. Chicken telomerase reverse transcriptase (chTERT) is the core component of telomerase, which is closely related to the occurrence and development of tumors. Our previous studies showed that chTERT is overexpressed in ALV-J tumors, but the mechanism is still not completely clear. Therefore, this study aims to analyze the possible molecular mechanism of chTERT overexpression in ALV-J tumors from the perspective of DNA methylation and promoter mutation. Methylation sequencing of the chTERT amplicon showed that ALV-J replication promoted the methylation level of the chTERT promoter. And the methylation level of the chTERT promoter in ALV-J tumors was significantly higher than that in tumor-adjacent and normal tissues. Compared with the tumor-adjacent and normal tissues, the chTERT promoter in each ALV-J tumors tested had a mutation of -183 bp C > T, and 36.0% (9/25) of the tumors also had mutations of -184 bp T > C, -73 bp::GGCCC and -56 bp A > T in the chTERT promoter, which formed the binding sites for the transcription factors NFAT5, TFAP2A and ZEB1, respectively. The results of RT-qPCR and Western blotting showed that the occurrence of these mutations significantly increased the expression level of chTERT. In conclusion, this study demonstrated that the high expression of chTERT in ALV-J tumors is positively correlated with the level of hypermethylation and mutation in its promoter, which provides a new perspective for further research on the molecular mechanism of chTERT in ALV-J tumorigenesis.

Semen extracellular vesicles mediate vertical transmission of subgroup J avian leukosis virus.

Subgroup J avian leukosis virus (ALV-J) is a highly oncogenic retrovirus that has been devastating the global poultry industry since the late 1990s. The major infection model of ALV-J is vertical transmission, which is responsible for the congenital infection of progeny from generation to generation. Increasing evidence has suggested that extracellular vesicles (EVs) derived from virus-infected cells or biological fluids have been thought to be vehicles of transmission for viruses. However, the role of EVs in infection and transmission of ALV-J remains obscure. In the present study, semen extracellular vesicles (SE) were isolated and purified from ALV-J-infected rooster seminal plasma (SE-ALV-J), which was shown to contain ALV-J genomic RNA and partial viral proteins, as determined by RNA sequencing, reverse transcription-quantitative PCR and Western blotting. Furthermore, SE-ALV-J was proved to be able to transmit ALV-J infection to host cells and establish productive infection. More importantly, artificial insemination experiments showed that SE-ALV-J transmitted ALV-J infection to SPF hens, and subsequently mediated vertical transmission of ALV-J from the SPF hens to the progeny chicks. Taken together, the results of the present study suggested that ALV-J utilized host semen extracellular vesicles as a novel means for vertical transmission, enhancing our understanding on mechanisms underlying ALV-J transmission.

Transcriptome-Wide Dynamics of m6A Methylation in Tumor Livers Induced by ALV-J Infection in Chickens.

Avian Leukosis Virus Subgroup J (ALV-J) is a tumorigenic virus with high morbidity and rapid transmission. N6-methyladenosine (m6A) is a common epigenetic modification that may be closely related to the pathogenicity of ALV-J. Currently, there are no reports on whether m6A modification is related to ALV-J induced tumor formation. In this study, we used methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to examine the differences in m6A methylation and gene expression in normal livers and ALV-J-induced tumor livers systematically, with functional enrichment and co-expression analysis. The results identified 6,541 m6A methylated peaks, mainly enriched in CDS, and more than 83% of the transcripts contained 1-2 m6A peaks. For RNA-seq, 1,896 and 1,757 differentially expressed mRNAs and lncRNAs were identified, respectively. Gene enrichment analysis indicated that they may be involved in biological processes and pathways such as immunology-related and apoptosis. Moreover, we identified 17 lncRNAs, commonly existing in differently expressed methylome and transcriptome. Through co-expression analysis, 126 differentially expressed lncRNAs, and 18 potentially m6A-related methyltransferases were finally identified and connected, suggesting that m6A modifications might affect gene expression of lncRNAs and play a role in ALV-J induced tumor formation. This study provides the first comprehensive description of the m6A expression profile in tumor livers induced by ALV-J infection in chickens, which provides a basis for studying the role of m6A modification in ALV-J induced tumorigenesis. This study provides clues for studying the epigenetic etiology and pathogenesis of ALV-J.

Endogenous retroviruses transcriptomes in response to four avian pathogenic microorganisms infection in chicken.

The impact of Endogenous retroviruses (ERVs) on chicken disease is not well understood. Here, we systematically identified 436 relatively complete ChERVs from the chicken genome. Subsequently, ChERV transcriptomes were analyzed in chicken after subgroup J avian leukosis virus (ALV-J), avian influenza virus (AIV), Marek's disease virus (MDV) and avian pathogenic Escherichia coli (APEC) infection. We found that about 50%-68% of ChERVs were transcriptionally active in infected and uninfected-samples, although the abundance of most ChERVs is relatively low. Moreover, compared to uninfected-samples, 49, 18, 66 and 17 ChERVs were significantly differentially expressed in ALV-J, AIV, MDV and APEC infected-samples, respectively. These findings may be of significance for understanding the role and function of ChERVs to response the pathogenic microorganism infection.

Identification, biogenesis and function prediction of novel circRNA during the chicken ALV-J infection.

Circular RNA (circRNA) is a new non-coding RNA with a highly conserved and stable covalently closed loop structure, and it plays an important role in a variety of biological processes and the occurrence of diseases. Based on the sequencing results, circRNA_3079 had the most significant difference between the infected group and normal group, up to about 8 times. It has attracted our attention and was selected for further verification and analysis. Though the characteristics analysis of circRNA_3079 in chicken, we found circRNA_3079 is a stable, circular transcript, which mainly exists in the cytoplasm. And it is widely expressed in various tissues of chickens, and highly expressed in lung, spleen, lymph and bursa of fabricius. Bioinformatics analysis results showed that circRNA_3079 and the predicted target genes are enriched in many pathways related to immunity or tumors, such as p53 signaling pathway, Jak-STAT signaling pathway and NOD-like receptor signaling pathway, which revealed that circRNA_3079 may indirectly regulate the ALV-J infection process through the regulation of target genes.HIGHLIGHTSCircRNA_3079 is an abundant and stable circular RNA expressed in different tissues and cells in chicken.The circularization of circRNA_3079 does not depend on the reverse complementary repetitive sequence of the flanking sequence.CircRNA_3079 may indirectly regulate the ALV-J infection process.

The impact of endogenous Avian Leukosis Viruses (ALVE) on production traits in elite layer lines.

Avian Leukosis Virus subgroup E (ALVE) integrations are endogenous retroviral elements found in the chicken genome. The presence of ALVE has been reported to have negative impacts on multiple traits, including egg production and body weight. The recent development of rapid, inexpensive and specific ALVE detection methods has facilitated their characterization in elite commercial egg production lines across multiple generations. The presence of 20 ALVE was examined in 8 elite lines, from 3 different breeds. Seventeen of these ALVE (85%) were informative and found to be segregating in at least one of the lines. To test for an association between specific ALVE inserts and traits, a large genotype by phenotype study was undertaken. Genotypes were obtained for 500 to 1500 males per line, and the phenotypes used were sire-daughter averages. Phenotype data were analyzed by line with a linear model that included the effects of generation, ALVE genotype and their interaction. If genotype effect was significant, the number of ALVE copies was fitted as a regression to estimate additive ALVE gene substitution effect. Significant associations between the presence of specific ALVE inserts and 18 commercially relevant performance and egg quality traits, including egg production, egg weight and albumen height, were observed. When an ALVE was segregating in more than one line, these associations did not always have the same impact (negative, positive or none) in each line. It is hypothesized that the presence of ALVE in the chicken genome may influence production traits by 3 mechanisms: viral protein production may modulate the immune system and impact overall production performance (virus effect); insertional mutagenesis caused by viral integration may cause direct gene alterations or affect gene regulation (gene effect); or the integration site may be within or adjacent to a quantitative trait region which impacts a performance trait (linkage disequilibrium, marker effect).

Falling fowl of the chicken reference genome: pitfalls of studying polymorphic endogenous retroviruses.

High quality reference genomes have facilitated the study of endogenous retroviruses (ERVs). However, there are an increasing number of published works which assume the ERVs in reference genomes are universal; even those of evolutionarily recent integrations. Consequently, these studies fail to properly characterise polymorphic ERVs, and even propose biological functions for ERVs that may not actually be present in the genomes of interest. Here, I outline the pitfalls of three studies of chicken endogenous Avian Leukosis Viruses (ALVEs or "ev genes": the "original" ERVs), all confounded by the assumption that the reference genome provides a representative ALVE baseline.

Gp37 Regulates the Pathogenesis of Avian Leukosis Virus Subgroup J via Its C Terminus.

Different from other subgroups of avian leukosis viruses (ALVs), ALV-J is highly pathogenic. It is the main culprit causing myeloid leukemia and hemangioma in chickens. The distinctiveness of the env gene of ALV-J, with low homology to those of other ALVs, is linked to its unique pathogenesis, but the underlying mechanism remains unclear. Previous studies show that env of ALV-J can be grouped into three species based on the tyrosine motifs in the cytoplasmic domain (CTD) of Gp37, i.e., the inhibitory, bifunctional, and active groups. To explore whether the C terminus or the tyrosine motifs in the CTD of Gp37 affect the pathogenicity of ALV-J, a set of ALV-J infectious clones containing different C termini of Gp37 or the mutants at the tyrosine sites were tested in vitro and in vivo Viral growth kinetics indicated not only that ALV-J with active env is the fastest in replication and ALV-J with inhibitory env is the lowest but also that the tyrosine sites essentially affected the replication of ALV-J. Moreover, in vivo studies demonstrated that chickens infected by ALV-J with active or bifunctional env showed higher viremia, cloacal viral shedding, and viral tissue load than those infected by ALV-J with inhibitory env Notably, the chickens infected by ALV-J with active or bifunctional env showed significant loss of body weight compared with the control chickens. Taken together, these findings reveal that the C terminus of Gp37 plays a vital role in ALV-J pathogenesis, and change from inhibitory env to bifunctional or active env increases the pathogenesis of ALV-J.IMPORTANCE ALV-J can cause severe immunosuppression and myeloid leukemia in infected chickens. However, no vaccine or antiviral drug is available against ALV-J, and the mechanism for ALV-J pathogenesis needs to be elucidated. It is generally believed that gp85 and LTR of ALV contribute to its pathogenesis. Here, we found that the C terminus and the tyrosine motifs (YxxM, ITIM, and ITAM-like) in the CTD of Gp37 of ALV-J could affect the pathogenicity of ALV-J in vitro and in vivo The pathogenicity of ALV-J with Gp37 containing ITIM only was significantly less than ALV-J with Gp37 containing both YxxM and ITIM and ALV-J with Gp37 containing both YxxM and ITAM-like. This study highlights the vital role of the C terminus of Gp37 in the pathogenesis of ALV-J and thus provides a new perspective to elucidate the interaction between ALV-J and its host and a molecular basis to develop efficient strategies against ALV-J.

Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus.

Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.

Long non-coding RNA and MicroRNA profiling provides comprehensive insight into non-coding RNA involved host immune responses in ALV-J-infected chicken primary macrophage.

Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a crucial role in host defense against invading pathogens. In the present study, whole transcriptome analysis was performed to analyze the host factors including genes, microRNA (miRNA), long non-coding RNA (lncRNA) and their regulatory network in chicken primary monocyte-derived macrophages (MDMs). In total, 128 differentially expressed (DE) lncRNAs and 15 DE miRNAs were identified in MDMs at 3 h post infection (hpi), and 30 DE lncRNAs and 8 DE miRNAs were identified in MDMs at 36 hpi during ALV-J infection. We further constructed the DE lncRNAs-mRNAs, miRNA-mRNAs and lncRNAs-miRNA-mRNAs interaction networks. The results suggested that DE lncRNAs and miRNAs are involved in the regulation of CCND3 and SOCS5 in Jak-STAT signaling pathway via ceRNA network in ALV-J-infected MDMs at 3 hpi. In addition, lncRNAs including XLOC_672329, ALDBGALG0000001429, XLOC_016500 and ALDBGALG0000000253 cis-regulating CH25H, CISH, IL-1ß and CD80 respectively in MDMs at 3 hpi participated in host antiviral responses. Our findings give a comprehensive view of the connection between non-coding RNA and ALV-J in chicken primary macrophages, and provide an excellent resource for further studies of epigenetic effects on ALV-J disease resistance breeding as well as immune system and genomic researches.

The Novel Avian Leukosis Virus Subgroup K Shares Its Cellular Receptor with Subgroup A.

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.

Expression patterns of novel circular RNAs in chicken cells after avian leukosis virus subgroup J infection.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that causes severe economic losses to the poultry industry worldwide. Circular RNAs (circRNAs) are a class of non-coding RNAs that has been described in various biological systems and pathogenic processes. However, the immune mechanisms in response to circRNAs remain unknown. In this study, high-throughput transcriptome sequencing was used to detect circRNAs present in chicken macrophage (HD11) and chick embryo fibroblast (CEF) cells infected with ALV-J. We identified 7684 circRNAs from diverse genomic locations in CEF and HD11 after ALV-J infection, these RNAs showed complex expression patterns that differed based on the cells type and infection time. In total, 302 differentially expressed (DE) circRNAs and 164 DE circRNAs were identified in CEF and HD11 after ALV-J infection, respectively. CircRNA7419-associated with KDM4C- and circRNA6679 and circRNA6680-associated with TNFAIP6- were involved in the immune response upon ALV-J infection in CEF. Host genes were analyzed through further bioinformatics analysis. The result confirmed that a large number of DE circRNAs corresponded to several immune-associated or tumor-associated terms and pathways, such as Mucin type O-Glycan biosynthesis, MAPK signaling pathway, B cell receptor signaling, and Wnt signaling pathway in CEF, as well as Jak-STAT signaling pathway, apoptosis, and MAPK signaling pathway in HD11. CircRNAs related to the B cell receptor signaling pathway in CEF, and the Jak-STAT signaling pathway in HD11, were selected for circRNA-miRNA interaction network analyses. Our study indicates that circRNAs expression was altered by ALV-J infection in both CEF and HD11, and may play a key role in the progression of ALV-J infection.

Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells.

Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.

The high conserved cellular receptors of avian leukosis virus subgroup J in Chinese local chickens contributes to its wide host range.

Avian leukosis virus (ALV) is a tumor-inducing virus that spreads among most chicken species, causing serious financial losses for the poultry industry. Subgroup J avian leukosis virus (ALV-J) is a recombinant exogenous ALV, which shows more extensive host range in comparison with other subgroups, especially in Chinese local chickens. To identify the relationship between ALV-J host range and the polymorphism of its cellular receptors, we performed a wide range epidemiological investigation of current ALV-J infection in Chinese local chickens, and discovered that all the 18 local chicken breeds being investigated from main local chicken breeding provinces were ALV-J positive. Furthermore, we cloned ALV-J cellular receptor genes of chNHE1 and chANXA2 of these 18 chicken breeds. Sequence alignment demonstrated that despite several regular mutations at the nucleotide level, there were no corresponding amino acid mutations for either chNHE1 gene or chANXA2 gene. Additionally, virus entry assay indicated that the level of viral enter into cells is stable among different chicken breeds. Results of this study indicated that the wide host range of ALV-J in Chinese local chickens was partially due to the high conservatism of its cellular receptors, and also provide target sites for drug design of resistance to ALV-J infection.

Reticuloendotheliosis virus and avian leukosis virus subgroup J synergistically increase the accumulation of exosomal miRNAs.

BACKGROUND: Co-infection with avian leukosis virus subgroup J and reticuloendotheliosis virus induces synergistic pathogenic effects and increases mortality. However, the role of exosomal miRNAs in the molecular mechanism of the synergistic infection of the two viruses remains unknown. RESULTS: In this study, exosomal RNAs from CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time were analysed by Illumina RNA deep sequencing. A total of 54 (23 upregulated and 31 downregulated) and 16 (7 upregulated and 9 downregulated) miRNAs were identified by comparing co-infection with two viruses, single-infected ALV-J and REV, respectively. Moreover, five key miRNAs, including miR-184-3p, miR-146a-3p, miR-146a-5p, miR-3538 and miR-155, were validated in both exosomes and CEF cells by qRT-PCR. GO annotation and KEGG pathway analysis of the miRNA target genes showed that the five differentially expressed miRNAs participated in virus-vector interaction, oxidative phosphorylation, energy metabolism and cell growth. CONCLUSIONS: We demonstrated that REV and ALV-J synergistically increased the accumulation of exosomal miRNAs, which sheds light on the synergistic molecular mechanism of ALV-J and REV.

Identification of key genes fluctuated induced by avian leukemia virus (ALV-J) infection in chicken cells.

Avian leukemia subgroup J (ALV-J) is one of the most detrimental neoplastic diseases in poultry production. However, the differences between somatic cells and immune cells post-infection remain poorly understood. The aim of our study was to detect the different responses in chicken to infection with ALV-J in different cell lines. In this study, we detected transcriptome expression changes during infection with ALV-J in chicken embryo fibroblast (CEF) and HD11 cell lines. RNA-Seq was used to determine the expression levels of mRNA transcripts from the two cell types after infection with ALV-J at 1, 4, and 7 dpi, and gene ontology analyses were used to cluster differentially expressed genes into pathways. Quantitative real-time PCR confirmed the expression of 336 and 269 differentially expressed genes in CEF and HD11 lines, respectively, involved in innate immunity (OASL, CCL4), adaptive immunity (LYZ, CD72), apoptosis and autophagy (WISP2, COMP), inflammation (JSC, IL8), and tumorgenesis (PCNA, GPX3). The notable signal transduction pathways included the PPARs signaling pathway and ECM-receptor interactions in CEF, and the Toll-like receptor, NOD-like receptor, and RIG-I-like receptor signaling pathways in HD11. To our knowledge, this is the first study to use high-throughput sequencing methods to investigate viral infection in different cell types. The results of the present study form a foundation for developing potential biological markers for viral infection.

Naturally Occurring Frameshift Mutations in the tvb Receptor Gene Are Responsible for Decreased Susceptibility of Chicken to Infection with Avian Leukosis Virus Subgroups B, D, and E.

The group of highly related avian leukosis viruses (ALVs) in chickens are thought to have evolved from a common retroviral ancestor into six subgroups, A to E and J. These ALV subgroups use diverse cellular proteins encoded by four genetic loci in chickens as receptors to gain entry into host cells. Hosts exposed to ALVs might be under selective pressure to develop resistance to ALV infection. Indeed, resistance alleles have previously been identified in all four receptor loci in chickens. The tvb gene encodes a receptor, which determines the susceptibility of host cells to ALV subgroup B (ALV-B), ALV-D, and ALV-E. Here we describe the identification of two novel alleles of the tvb receptor gene, which possess independent insertions each within exon 4. The insertions resulted in frameshift mutations that reveal a premature stop codon that causes nonsense-mediated decay of the mutant mRNA and the production of truncated Tvb protein. As a result, we observed that the frameshift mutations in the tvb gene significantly lower the binding affinity of the truncated Tvb receptors for the ALV-B, ALV-D, and ALV-E envelope glycoproteins and significantly reduce susceptibility to infection by ALV-B, ALV-D and ALV-E in vitro and in vivo Taken together, these findings suggest that frameshift mutation can be a molecular mechanism of reducing susceptibility to ALV and enhance our understanding of virus-host coevolution.IMPORTANCE Avian leukosis virus (ALV) once caused devastating economic loss to the U.S. poultry industry prior the current eradication schemes in place, and it continues to cause severe calamity to the poultry industry in China and Southeast Asia, where deployment of a complete eradication scheme remains a challenge. The tvb gene encodes the cellular receptor necessary for subgroup B, D, and E ALV infection. Two tvb allelic variants that resulted from frameshift mutations have been identified in this study, which have been shown to have significantly reduced functionality in mediating subgroup B, D, and E ALV infection. Unlike the control of herpesvirus-induced diseases by vaccination, the control of avian leukosis in chickens has relied totally on virus eradication measures and host genetic resistance. This finding enriches the allelic pool of the tvb gene and expands the potential for genetic improvement of ALV resistance in varied chicken populations by selection.

Transcription analysis of the interaction between chicken thymus and recombinant avian leukosis virus isolate FJ15HT0.

Avian leukosis virus (ALV) induces multiple avian tumors, growth decrease and immune suppression. Previously, a novel natural recombinant ALV isolate FJ15HT0 was proven to be associated with significant body weight decrease, immune suppression and lymphocytoma in infected SPF chickens. In order to uncover the interaction between virus and host, we compared differences in the transcriptomes of the thymuses from the mock chickens and simulated congenitally infected chickens at 5days (d), 13d and 21d of age by RNA-seq analysis of the thymuses. Signaling pathways including cytokine-cytokine receptor interactions, peroxisome proliferator-activated receptor (PPAR) signaling pathway, Janus tyrosine kinase/signal transducers and activators of transcription (Jak-STAT) signaling pathway and fatty acid degradation were involved in the interaction between FJ15HT0 and SPF chickens. Interestingly, fold change of ciliary neurotrophic factor receptor α (CNTFRα) in infected donor collected from 2d to 21d showed a significant positive correlation with the corresponding expression of the viral gp85 gene in thymuses (r=0.656, P<0.01) and in livers (r=0.525, P<0.05). It will provide new insights for the molecular pathogenesis of ALV infection.

Acquisition of resistance to avian leukosis virus subgroup B through mutations on tvb cysteine-rich domains in DF-1 chicken fibroblasts.

Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection.