Effect of oral administration of microcin Y on growth performance, intestinal barrier function and gut microbiota of chicks challenged with Salmonella Pullorum.

The lasso peptide microcin Y (MccY) effectively inhibits various serotypes of Salmonella in vitro, but the antibacterial effect against S. Pullorum in poultry is still unclear. This study was the first to evaluate the safety and anti-S. Pullorum infection of MccY in specific pathogen-free (SPF) chicks. The safety test showed that the body weight, IgA and IgM levels of serum, and cecal microbiota structure of 3 groups of chicks orally administrated with different doses of MccY (5 mg/kg, 10 mg/kg, 20 mg/kg) for 14 days were not significantly different from those of the control group. Then, the chicks were randomized into 3 groups for the experiment of anti-S. Pullorum infection: (I) negative control group (NC), (II) S. Pullorum-challenged group (SP, 5 × 108 CFU/bird), (III) MccY-treated group (MccY, 20 mg/kg). The results indicated that compared to the SP group, treatment of MccY increased body weight and average daily gain (P < 0.05), reduced S. Pullorum burden in feces, liver, and cecum (P < 0.05), enhanced the thymus, and decreased the spleen and liver index (P < 0.05). Additionally, MccY increased the jejunal villus height, lowered the jejunal and ileal crypt depth (P < 0.05), and upregulated the expression of IL-4, IL-10, ZO-1 in the jejunum and ileum, as well as CLDN-1 in the jejunum (P < 0.05) compared to the SP group. Furthermore, MccY increased probiotic flora (Barnesiella, etc.), while decreasing (P < 0.05) the relative abundance of pathogenic flora (Escherichia and Salmonella, etc.) compared to the SP group.

The key amino acid sites 199-205, 269, 319, 321 and 324 of ALV-K env contribute to the weaker replication capacity of ALV-K than ALV-A.

BACKGROUND: Avian leukosis virus (ALV) is an infectious retrovirus, that mainly causes various forms of tumours, immunosuppression, a decreased egg production rate and slow weight gain in poultry. ALV consists of 11 subgroups, A-K, among which ALV-K is an emerging subgroup that has become prevalent in the past 10 years. Most ALV-K isolates showed weak replication ability and pathogenicity. In this study, the weak replication ability of ALV-K was explored from the perspective of the interaction between ALV-K gp85 and the Tva receptor. METHODS: Fourteen soluble recombinant ALV-A/K gp85 chimeric proteins were constructed by substituting the sequence difference regions (hr1, hr2 and vr3) of the ALV-A gp85 protein with the skeleton ALV-K gp85 protein for co-IP and competitive blocking tests. RESULTS: The binding capacity of ALV-K gp85 to Tva was significantly weaker than that of ALV-A gp85 (P < 0.05) and the key amino acid sites 199-205, 269, 319, 321 and 324 of ALV-K env contributed to the weaker replication capacity of ALV-K than ALV-A. CONCLUSIONS: This is the first study to reveal the molecular factors of the weak replication ability of ALV-K from the perspective of the interaction of ALV-K gp85 to Tva, providing a basis for further elucidation of the infection mechanism of ALV-K.

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.

Chicken telomerase reverse transcriptase promotes the tumorigenicity of avian leukosis virus subgroup J by regulating the Wnt/ß-catenin signaling pathway.

This research aimed to analyze the regulatory effect of chicken telomerase reverse transcriptase (chTERT) on the Wnt/ß-catenin signaling pathway and its effect on the tumorigenicity of avian leukosis virus subgroup J (ALV-J) through in vivo experiments. The chTERT eukaryotic expression plasmid and its recombinant lentivirus particles were constructed for in vivo transfection of chTERT to analyze the effect of chTERT continuously overexpressed in chickens on the tumorigenicity of ALV-J. During 156 days of the artificial ALV-J tumor-inducing process, 7 solid tumors developed in 3 chickens in the chTERT-overexpression group (n = 26*2) and no tumors developed in the control group (n = 26*2). Another 18 tumors induced by ALV-J were confirmed and collected from breeding poultry farms. And we confirmed that chTERT was significantly highly expressed in ALV-J tumors. The ELISA data suggested that the protein levels of ß-catenin and c-Myc in the chicken plasma of the chTERT-overexpressing group with ALV-J infected were consistently and significantly higher than those of the control group. Compared with that of the tumor-adjacent tissues, the activity of the Wnt/ß-catenin signaling pathway and expression of the c-Myc was significantly increased in ALV-J tumors. And the percentage of apoptosis in ALV-J tumors significantly lower than that in tumor-adjacent tissues. Immunohistochemistry, Western blot and RT-qPCR suggested that the replication level of ALV-J in tumors was significantly higher than that in tumor-adjacent tissues. This study suggests that chTERT plays a critical role in the tumorigenicity of ALV-J by enhancing the Wnt/ß-catenin signaling pathway, which will contribute to further elucidating the tumor-inducing mechanism of ALV-J.

Mutual regulation between chicken telomerase reverse transcriptase and the Wnt/ß-catenin signalling pathway inhibits apoptosis and promotes the replication of ALV-J in LMH cells.

This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/ß-catenin signalling pathway and its effects on cell growth and avian leukosis virus subgroup J (ALV-J) replication in LMH cells. First, LMH cells stably overexpressing the chTERT gene (LMH-chTERT cells) and corresponding control cells (LMH-NC cells) were successfully constructed with a lentiviral vector expression system. The results showed that chTERT upregulated the expression of ß-catenin, Cyclin D1, TCF4 and c-Myc. chTERT expression level and telomerase activity were increased when cells were treated with LiCl. When the cells were treated with ICG001 or IWP-2, the activity of the Wnt/ß-catenin signalling pathway was significantly inhibited, and chTERT expression and telomerase activity were also inhibited. However, when the ß-catenin gene was knocked down by small interfering RNA (siRNA), the changes in chTERT expression and telomerase activity were consistent with those in cells treated with ICG001 or IWP-2. These results indicated that chTERT and the Wnt/ß-catenin signalling pathway can be mutually regulated. Subsequently, we found that chTERT not only shortened the cell cycle to promote proliferation but also inhibited apoptosis by downregulating the expression of Caspase 3, Caspase 9 and BAX; upregulating BCL-2 and BCL-X expression; and promoting autophagy. Moreover, chTERT significantly enhanced the migration ability of LMH cells, upregulated the protein and mRNA expression of ALV-J and increased the virus titre. ALV-J replication promoted chTERT expression and telomerase activity.

Ubiquitination of non-lysine residues in the retroviral integrase.

Retroviral integrase catalyzes the integration of retroviral genome into host chromosomal DNA, which is a prerequisite of effective viral replication and infection. The human immunodeficiency virus type 1 (HIV-1) integrase has previously been reported to be regulated by the ubiquitination, but the molecular characterization of integrase ubiquitination is still unclear. In this study, we analyzed the ubiquitination of avian leukosis virus (ALV) integrase in detail. The ubiquitination assay showed that, like HIV-1, ALV integrase could also be modified by ubiquitination when expressed in 293 T and DF-1 cells. Domain mapping analysis revealed that the ubiquitination of ALV integrase might mainly occurred in the catalytic core and the N-terminal zinc-binding domains. Both lysine and non-lysine residues within integrase of ALV and HIV-1 were responsible for the ubiquitin conjugation, and the N-terminal HHCC zinc-binding motif might play an important role in mediating integrase ubiquitination. Interestingly, mass spectrometry analysis identified the Thr10 and Cys37 residues in the HHCC zinc-binding motif as the ubiquitination sites, indicating that ubiquitin may be conjugated to ALV integrase through direct interaction with the non-lysine residues. These findings revealed the detailed features of retroviral integrase ubiquitination and found a novel mechanism of ubiquitination mediated by the non-lysine residues within the N-terminal zinc-binding domain of integrase.

Inhibition of ERK/MAPK suppresses avian leukosis virus subgroup A and B replication.

We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway contributes to subgroup J avian leukosis virus (ALV-J) replication and tumorigenicity. However, a role for ERK/MAPK signaling in ALV-A and ALV-B replication is unknown. In this study we successfully constructed and recovered a recombinant form of ALV-A strain GD13-1 which showed similarities in growth to the parental wild type virus in vitro. ALV subgroups J, A or B all triggered ERK2 activation in primary CEF cells. ERK/MAPK inhibition markedly suppressed ALV-A and ALV-B replication as shown by extremely low levels of viral transcription and virus protein production. This finding provides evidence that ERK/MAPK signaling responses play important roles in ALV replication and may represent novel drug targets for therapeutic intervention strategies.

The MYC, TERT, and ZIC1 genes are common targets of viral integration and transcriptional deregulation in avian leukosis virus subgroup J-induced myeloid leukosis.

UNLABELLED: The integration of retroviruses into the host genome following nonrandom genome-wide patterns may lead to the deregulation of gene expression and oncogene activation near the integration sites. Slow-transforming retroviruses have been widely used to perform genetic screens for the identification of genes involved in cancer. To investigate the involvement of avian leukosis virus subgroup J (ALV-J) integration in myeloid leukosis (ML) in chickens, we utilized an ALV-J insertional identification platform based on hybrid capture target enrichment and next-generation sequencing (NGS). Using high-definition mapping of the viral integration sites in the chicken genome, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. On the basis of previous statistical definitions, MYC, TERT, and ZIC1 genes were identified as common insertion sites (CIS) of provirus integration in tumor cells; these three genes have previously been shown to be involved in the malignant transformation of different human cell types. Compared to control samples, the expression levels of all three CIS genes were significantly upregulated in chicken ML samples. Furthermore, they were frequently, but not in all field ML cases, deregulated at the mRNA level as a result of ALV-J infection. Our findings contribute to the understanding of the relationship between multipathotypes associated with ALV-J infection and the molecular background of tumorigenesis. IMPORTANCE: ALV-Js have been successfully eradicated from chicken breeding flocks in the poultry industries of developed countries, and the control and eradication of ALV-J in China are now progressing steadily. To further study the pathogenesis of ALV-J infections, it will be necessary to elucidate the in vivo viral integration and tumorigenesis mechanism. In this study, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. In addition, MYC, TERT, and ZIC1 genes were identified as the CIS of ALV-J in tumor cells, which might be a putative "driver" for the activation of the oncogene. In addition, the CIS genes showed deregulated expression compared to nontumor samples. These results have potentially important implications for the mechanism of viral carcinogenesis.

Development and application of SYBR Green I real-time PCR assay for the separate detection of subgroup J Avian leukosis virus and multiplex detection of avian leukosis virus subgroups A and B.

BACKGROUND: Subgroup A, B, and J ALVs are the most prevalent avian leukosis virus (ALV). Our study attempted to develop two SYBR Green I-based real-time PCR (RT-PCR) assays for specific detection of ALV subgroup J (ALV-J) and multiplex detection of ALV subgroups A and B (ALV-A/B), respectively. RESULTS: The two assays showed high specificity for ALV-J and ALV-A/B and the sensitivity of the two assays was at least 100 times higher than that of the routine PCR assay. The minimum virus detection limit of virus culture, routine PCR and real-time PCR for detection of ALV-A strain was 10(3) TCID50 units, 10(2) TCID50 units and fewer than 10 TCID50 units, respectively. In addition, the coefficients of variation for intra- and inter-assay were both less than 5%. Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively. When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection. Except in lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues. CONCLUSIONS: The SYBR Green I-based real-time RT-PCR assay provides a powerful tool for the detection of ALV and study of virus replication and infection.

Further observations on serotype 2 Marek's disease virus-induced enhancement of spontaneous avian leukosis virus-like bursal lymphomas in ALVA6 transgenic chickens.

Breeders of the 2009 generation of Avian Disease and Oncology Laboratory transgenic chicken line ALVA6, known to be resistant to infection with subgroups A and E avian leukosis virus (ALV), were vaccinated at hatch with a trivalent Marek's disease (MD) vaccine containing serotypes 1, 2, and 3 Marek's disease virus (MDV) and were maintained under pathogen-free conditions from the day of hatch until 75 weeks of age. Spontaneous ALV-like bursal lymphomas, also termed lymphoid leukosis (LL)-like lymphomas, were detected in 7% of the ALVA6 breeders. There was no evidence of infection with exogenous and endogenous ALV as determined by virus isolation tests of plasma and tumour tissue homogenates. For the next three generations, serotype 2 MDV was eliminated from the trivalent MD vaccine used. Results show, for the first time, that removal of serotype 2 MDV from MD vaccines eliminated spontaneous LL-like lymphomas within 50 to 72 weeks of age for at least three consecutive generations. Two experiments were also conducted to determine the influence of in ovo vaccination with serotype 2 MD vaccines on enhancement of spontaneous LL-like lymphomas in ALVA6 chickens. Chickens from the 2012 generation were each inoculated in ovo or at hatch with 5000 plaque-forming units of serotype 2 MDV. Results indicate that by 50 weeks of age the incidence of spontaneous LL-like lymphomas in chickens inoculated in ovo with serotype 2 MDV was comparable with that in chickens inoculated with virus at hatch, suggesting that the augmentation effect of serotype 2 MDV is independent of age of vaccination.

COP9 signalosome subunit 6 binds and inhibits avian leukosis virus integrase.

The retroviral integrase plays an essential role in the integration of reverse-transcribed retroviral cDNA into the host cell genome, and serves as an important target for anti-viral therapeutics. In this study, we identified the COP9 signalosome subunit 6 (CSN6) as a novel avian leukosis virus (ALV) integrase binding protein. Co-immunoprecipitation and GST pull-down assays showed that CSN6 bound to ALV integrase likely through direct interaction of CSN6 to the catalytic core of the integrase. We further demonstrated CSN6 inhibited integrase activity in vitro; knockdown of CSN6 in DF-1 promoted ALV production. These results indicated that CSN6 may be a negative regulator of ALV replication by binding to and inhibiting integrase. Our findings provided the insight into the integrase-based host defense system and may have implications in the development of integrase-based anti-viral strategies.

Microcin Y utilizes its stable structure and biological activity to regulate the metabolism of intestinal probiotics and effectively clear gut Salmonella.

MccY is a novel, structurally stable microcin with antibacterial activity against Enterobacteriaceae. However, the bioavailability of orally administrated MccY is unknown. This study evaluated the effects of MccY as a antimicrobial on pre-digestion in vitro and its intake, digestion and gut metabolism in vivo. The result of pre-digestion results that MccY maintained its biological activity and was resistant to decomposition. The study established a safe threshold of 4.46-9.92 mg/kg for the MccY dosage-body weight relationship in BALB/c mice. Mice fed with MccY demonstrated improved body weight and intestinal barrier function, accompanied with increased IgM immunogenicity and decreased levels of TNF-α, IL-6, and IL-10 in the intestine. MccY significantly facilitates the growth and activity of probiotics including Lactobacillus, Prevotella, and Bacteroides, and leading to the production of SCFAs and MCFAs during bacterial interactions. Furthermore, MccY effectively protects against the inflammatory response caused by Salmonella Typhimurium infection and effectively clears the Salmonella bacteria from the gut. In conclusion, MccY is seen as a promising new therapeutic target drug for enhancing the intestinal microbe-barrier axis and preventing enteritis.

Subgroup J avian leukosis virus infection inhibits autophagy in DF-1 cells.

BACKGROUND: Subgroup J avian leukosis virus (ALV-J) infection can induce tumor-related diseases in chickens. Previous studies by our laboratory demonstrated that ALV-J infection of DF-1 cells resulted in altered activity and phosphorylation of AKT. However, little is known about the subsequent activation of host DF-1 cells. RESULTS: In the current study, autophagy inhibition was observed for ALV-J infected DF-1 cells. Our data showed that the autophagosome protein, microtubule-associated protein 1 light chain 3-II (LC3-II), was reduced considerably in DF-1 cells infected with active ALV-J, while no change was observed for cells infected with inactivated ALV-J. Autophagy inhibition was also confirmed by fluorescence microscopy and transmission electron microscopy. Interestingly, when autophagy was promoted by rapamycin, the titers of ALV-J replication were decreased, and the replication level of ALV-J was significantly enhanced when atg5 (autophagy-related gene 5) was knocked out. CONCLUSIONS: These results suggested that ALV-J infection could down-regulate autophagy in DF-1 cells during viral replication. This study is the first to report on the relationship between ALV-J infection and autophagy in DF-1 cells.

Use of polymerase chain reaction in detection of Marek's disease and reticuloendotheliosis viruses in formalin-fixed, paraffin-embedded tumorous tissues.

A simple PCR method was developed for the detection of Marek's disease (MD) and reticuloendotheliosis (RE) in formalin-fixed paraffin-embedded (FFPE) tissues, and for the detection of MD in tissues only preserved in 10% neutral buffered formalin. MD virus (MDV) and RE virus proviral DNA were detected in FFPE tissues stored for over 20 yr. MDV was also detected in tissues only preserved in formalin for up to 6 mo. The data indicate that PCR of formalin-fixed and FFPE tissues is a simple and valuable tool that can be used to identify MD and RE infection. The method described in this paper is a good alternative to any biologic or immunohistochemical assay to confirm the detection of MD and RE, as it does not require shipping frozen tissues to the diagnostic laboratory.

Lasso peptide MccY alleviates non-typhoidal salmonellae-induced mouse gut inflammation via regulation of intestinal barrier function and gut microbiota.

IMPORTANCE: Diseases caused by Enterobacteriaceae multidrug-resistant strains have become increasingly difficult to manage. It is necessary to verify the new antibacterial drug MccY effect on non-typhoid Salmonella infection in mice since it is regarded as a promising microcin. The results demonstrated that MccY has a potential therapeutic application value in the protection against Salmonella-induced intestinal damage and alleviating related intestinal dysbiosis and metabolic disorders. MccY could be a promising candidate as an antimicrobial or anti-inflammatory agent for treating infectious diseases.

Chicken telomerase reverse transcriptase mediates LMH cell pyroptosis by regulating the nuclear factor-kappa B signaling pathway.

The activation of human telomerase reverse transcriptase is regulated by the nuclear factor kappa B (NF-κB) signaling pathway to various degrees to promote the occurrence and development of tumors. However, the regulatory roles of chicken telomerase reverse transcriptase (chTERT) and the NF-κB signaling pathway in chickens are still elusive, particularly in respect to the regulation of cell pyroptosis. In this study, we found that chTERT upregulated the expression of p65 and p50, downregulated the expression of IκBα, promoted the phosphorylation of p65, p50, and IκBα, and significantly increased the transcript levels of the inflammatory cytokines IFNγ, TNFα, and IL-6 in LMH cells. The activity of NF-κB was significantly decreased after siRNA-mediated chTERT silencing. The expression of chTERT and telomerase activity were also significantly decreased when the NF-κB signaling pathway was blocked by p65 siRNA, MG132 or BAY 11-7082. In cells treated with LPS, the activity of NF-κB signaling pathway and the expression of chTERT were significantly upregulated. All of the results suggested that chTERT and the NF-κB pathway could regulate each other, reciprocally. Moreover, the expression of Caspase-1, NLRP3, GSDMA, IL-18, and IL-1ß and caused membrane perforation, suggesting the development of pyroptosis by chTERT in LMH cells. And the expression of caspase-11 did not significantly increased in chTERT overexpression group. Genetic silence of NF-κB p65 or chTERT gene by siRNA suppressed the expression of these proinflammatory cytokines, indicating that chTERT mediates pyroptosis by regulating the NF-κB signaling pathway in LMH cells.

Resistance Mechanism and Physiological Effects of Microcin Y in Salmonella enterica subsp. enterica Serovar Typhimurium.

Salmonella bacteria pose a significant threat to animal husbandry and human health due to their virulence and multidrug resistance. The lasso peptide MccY is a recently discovered antimicrobial peptide that acts against various serotypes of Salmonella. In this study, we further explore the resistance mechanism and activity of MccY. Mutants of Ton system genes, including tonB, exbB, and exbD, in Salmonella enterica subsp. enterica serovar Typhimurium were constructed, and the MICs to MccY exhibited significant increases in these deletion mutants compared to the MIC of the parent strain. Subsequently, MccY resistance was quantitatively analyzed, and these mutants also showed greatly reduced rates of killing, even with a high concentration of MccY. In addition, a minimal medium with low iron environment enhanced the sensitivity of these mutants to MccY. Measurements of a series of physiological indicators, including iron utilization, biofilm formation, and motility, demonstrated that MccY may decrease the virulence of S. Typhimurium. Transcriptomic analysis showed that iron utilization, biofilm formation, flagellar assembly, and virulence-related genes were downregulated to varying degrees when S. Typhimurium was treated with MccY. In conclusion, deletion of Ton system genes resulted in resistance to MccY and the susceptibility of these mutants to MccY was increased and differed under a low-iron condition. This lasso peptide can alter multiple physiological properties of S. Typhimurium. Our study will contribute to improve the knowledge and understanding of the mechanism of MccY resistance in Salmonella strains. IMPORTANCE The resistance of Salmonella to traditional antibiotics remains a serious challenge. Novel anti-Salmonella drugs are urgently needed to address the looming crisis. The newly identified antimicrobial peptide MccY shows broad prospects for development and application because of its obvious antagonistic effect on various serotypes of Salmonella. However, our previous study showed that the peptide could confer resistance to Salmonella by disrupting the receptor gene fhuA. In this study, we further explored the potential resistance mechanism of MccY and demonstrated the importance of the Salmonella Ton complex for MccY transport. Disruption in Ton system genes resulted in S. Typhimurium resistance to this peptide, and MccY could alter multiple bacterial physiological properties. In summary, this study further explored the resistance mechanism and antibacterial effect of MccY in S. Typhimurium and provided a scientific basis for its development and application.

Residues 140-142, 199-200, 222-223, and 262 in the Surface Glycoprotein of Subgroup A Avian Leukosis Virus Are the Key Sites Determining Tva Receptor Binding Affinity and Infectivity.

Subgroup A avian leukosis virus (ALV-A) invades cells through gp85-encoded surface glycoprotein (SU) via specifically recognizing the cellular receptor Tva. To identify the key residues of ALV-A SU that determine the Tva binding affinity and infectivity in DF-1 cells, a strategy of substituting corresponding residues of SU between ALV-A RSA and ALV-E ev-1 (using Tvb as the receptor) was adopted. A series of chimeric soluble gp85 proteins were expressed for co-immunoprecipitation (co-IP) analysis and blocking analysis of viral entry, and various recombinant viruses based on replication-competent avian retrovirus vectors containing Bryan polymerase (RCASBP) were constructed for transfection into DF-1 cells and measurement of the percentage of GFP-positive cells. The results revealed that the substitution of residues V138, W140, Y141, L142, S145, and L154 of host range region 1 (hr1), residues V199, G200, Q202, R222, and R223 of host range region 2 (hr2), and residue G262 of variable region 3 (vr3) reduced the viral infectivity and Tva binding affinity, which was similar to the effects of the -139S, -151N, -155PWVNPF, -201NFD, Δ214-215, and -266S mutations. Our study indicated that hr1 and hr2 contain the principal receptor interaction determinants, with new identified-vr3 also playing a key role in the receptor binding affinity of ALV-A.

The PI3K/Akt pathway is involved in early infection of some exogenous avian leukosis viruses.

Avian leukosis virus (ALV) is an enveloped and oncogenic retrovirus. Avian leukosis caused by the members of ALV subgroups A, B and J has become one of the major problems challenging the poultry industry in China. However, the cellular factors such as signal transduction pathways involved in ALV infection are not well defined. In this study, our data demonstrated that ALV-J strain NX0101 infection in primary chicken embryo fibroblasts or DF-1 cells was correlated with the activity and phosphorylation of Akt. Akt activation was initiated at a very early stage of infection independently of NX0101 replication. The specific phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin could suppress Akt phosphorylation, indicating that NX0101-induced Akt phosphorylation is PI3K-dependent. ALV-A strain GD08 or ALV-B strain CD08 infection also demonstrated a similar profile of PI3K/Akt activation. Treatment of DF-1 cells with the drug 5-(N, N-hexamethylene) amiloride that inhibits the activity of chicken Na(+)/H(+) exchanger type 1 significantly reduced Akt activation induced by NX0101, but not by GD08 and CD08. Akt activation triggered by GD08 or CD08 was abolished by clathrin-mediated endocytosis inhibitor chlorpromazine. Receptor-mediated endocytosis inhibitor dansylcadaverine had a negligible effect on all ALV-induced Akt phosphorylation. Moreover, viral replication of ALV was suppressed by LY294002 in a dose-dependent manner, which was due to the inhibition of virus infection by LY294002. These data suggest that the activation of the PI3K/Akt signalling pathway by exogenous ALV infection plays an important role in viral entry, yet the precise mechanism remains under further investigation.

An ALV-J isolate is responsible for spontaneous haemangiomas in layer chickens in China.

Subgroup J avian leukosis virus (ALV-J), first isolated in 1989, mainly induces tumours of myeloid leukosis (ML) in meat-type chickens. In 2006, ALV-J strain SCAU-HN06 was isolated from commercial layer hens with spontaneous haemangiomas in China. To confirm its role in the induction of haemangioma, we constructed a full-length copy of the proviral genome from SCAU-HN06, recovered virus from DF-1 cells detected by enzyme-linked immunosorbent assay, characterized its growth property and investigated its pathogenicity. The recovered virus appeared to be identical to SCAU-HN06 analysed by both blast gene sequences and indirect immunofluorescence assay. It also showed similarities in growth to the parental wild-type virus in vitro. The pathogenicity of the rescued and parental virus in specific-pathogen-free White Leghorn chickens was investigated. Both SCAU-HN06 and rSCAU-HN06 could induce haemangioma, with incidence of 52% and 42.8% respectively. Overall, our findings indicated that the ALV-J strain SCAU-HN06 was the causal agent inducing haemangiomas rather than ML in certain layer chickens.