The Impact of Early-Life Cecal Microbiota Transplantation on Social Stress and Injurious Behaviors in Egg-Laying Chickens.

Injurious behaviors (i.e., aggressive pecking, feather pecking, and cannibalism) in laying hens are a critical issue facing the egg industry due to increased social stress and related health and welfare issues as well as economic losses. In humans, stress-induced dysbiosis increases gut permeability, releasing various neuroactive factors, causing neuroinflammation and related neuropsychiatric disorders via the microbiota-gut-brain axis, and consequently increasing the frequency and intensity of aggression and violent behaviors. Restoration of the imbalanced gut microbial composition has become a novel treatment strategy for mental illnesses, such as depression, anxiety, bipolar disorder, schizophrenia, impulsivity, and compulsivity. A similar function of modulating gut microbial composition following stress challenge may be present in egg-laying chickens. The avian cecum, as a multi-purpose organ, has the greatest bacterial biodiversity (bacterial diversity, richness, and species composition) along the gastrointestinal tract, with vitally important functions in maintaining physiological and behavioral homeostasis, especially during the periods of stress. To identify the effects of the gut microbiome on injurious behaviors in egg-laying chickens, we have designed and tested the effects of transferring cecal contents from two divergently selected inbred chicken lines on social stress and stress-related injurious behaviors in recipient chicks of a commercial layer strain. This article reports the outcomes from a multi-year study on the modification of gut microbiota composition to reduce injurious behaviors in egg-laying chickens. An important discovery of this corpus of experiments is that injurious behaviors in chickens can be reduced or inhibited through modifying the gut microbiota composition and brain serotonergic activities via the gut-brain axis, without donor-recipient genetic effects.

Transgenerational epigenetic inheritance and immunity in chickens that vary in Marek's disease resistance.

Marek's disease virus (MDV), a naturally oncogenic, highly contagious alpha herpesvirus, induces a T cell lymphoma in chickens that causes severe economic loss. Marek's disease (MD) outcome in an individual is attributed to genetic and environmental factors. Further investigation of the host-virus interaction mechanisms that impact MD resistance is needed to achieve greater MD control. This study analyzed genome-wide DNA methylation patterns in 2 highly inbred parental lines 63 and 72 and 5 recombinant congenic strains (RCS) C, L, M, N, and X strains from those parents. Lines 63 and 72, are MD resistant and susceptible, respectively, whereas the RCS have different combinations of 87.5% Line 63 and 12.5% Line 72. Our DNA methylation cluster showed a strong association with MD incidence. Differentially methylated regions (DMRs) between the parental lines and the 5 RCS were captured. MD-resistant and MD-susceptible markers of DNA methylation were identified as transgenerational epigenetic inheritable. In addition, the growth of v-src DNA tumors and antibody response against sheep red blood cells differed among the 2 parental lines and the RCS. Overall, our results provide very solid evidence that DNA methylation patterns are transgenerational epigenetic inheritance (TEI) in chickens and also play a vital role in MD tumorigenesis and other immune responses; the specific methylated regions may be important modulators of general immunity.

Cecal microbiota transplantation: unique influence of cecal microbiota from divergently selected inbred donor lines on cecal microbial profile, serotonergic activity, and aggressive behavior of recipient chickens.

BACKGROUND: Accumulating evidence from human trials and rodent studies has indicated that modulation of gut microbiota affects host physiological homeostasis and behavioral characteristics. Similarly, alterations in gut microbiota could be a feasible strategy for reducing aggressive behavior and improving health in chickens. The study was conducted to determine the effects of early-life cecal microbiota transplantation (CMT) on cecal microbial composition, brain serotonergic activity, and aggressive behavior of recipient chickens. METHODS: Chicken lines 63 and 72 with nonaggressive and aggressive behavior, respectively, were used as donors and a commercial strain Dekalb XL was used as recipients for CMT. Eighty-four 1-d-old male chicks were randomly assigned to 1 of 3 treatments with 7 cages per treatment and 4 chickens per cage (n = 7): saline (control, CTRL), cecal solution of line 63 (63-CMT), and cecal solution of line 72 (72-CMT). Transplantation was conducted via oral gavage once daily from d 1 to 10, and then boosted once weekly from week 3 to 5. At weeks 5 and 16, home-cage behavior was recorded, and chickens with similar body weights were assigned to paired aggression tests between the treatments. Samples of blood, brain, and cecal content were collected from the post-tested chickens to detect CMT-induced biological and microbiota changes. RESULTS: 63-CMT chickens displayed less aggressive behavior with a higher hypothalamic serotonergic activity at week 5. Correspondingly, two amplicon sequence variants (ASVs) belonging to Lachnospiraceae and one Ruminococcaceae UCG-005 ASV were positively correlated with the levels of brain tryptophan and serotonin, respectively. 72-CMT chickens had lower levels of brain norepinephrine and dopamine at week 5 with higher levels of plasma serotonin and tryptophan at week 16. ASVs belonging to Mollicutes RF39 and GCA-900066225 in 72-CMT chickens were negatively correlated with the brain 5-hydroxyindoleacetic acid (5-HIAA) at week 5, and one Bacteroides ASV was negatively correlated with plasma serotonin at week 16. CONCLUSION: Results indicate that CMT at an early age could regulate aggressive behavior via modulating the cecal microbial composition, together with central serotonergic and catecholaminergic systems in recipient chickens. The selected CMT could be a novel strategy for reducing aggressive behavior through regulating signaling along the microbiota-gut-brain axis.

Role of T Cells in Vaccine-Mediated Immunity against Marek's Disease.

Marek's disease virus (MDV), a highly cell-associated oncogenic α-herpesvirus, is the etiological agent of T cell lymphomas and neuropathic disease in chickens known as Marek's disease (MD). Clinical signs of MD include neurological disorders, immunosuppression, and lymphoproliferative lymphomas in viscera, peripheral nerves, and skin. Although vaccination has greatly reduced the economic losses from MD, the molecular mechanism of vaccine-induced protection is largely unknown. To shed light on the possible role of T cells in immunity induced by vaccination, we vaccinated birds after the depletion of circulating T cells through the IP/IV injection of anti-chicken CD4 and CD8 monoclonal antibodies, and challenged them post-vaccination after the recovery of T cell populations post-treatment. There were no clinical signs or tumor development in vaccinated/challenged birds with depleted CD4+ or CD8+ T cells. The vaccinated birds with a combined depletion of CD4+ and CD8+ T cells, however, were severely emaciated, with atrophied spleens and bursas. These birds were also tumor-free at termination, with no virus particles detected in the collected tissues. Our data indicated that CD4+ and CD8+ T lymphocytes did not play a critical role in vaccine-mediated protection against MDV-induced tumor development.

Small RNA deep sequencing revealed microRNAs' involvement in modulating cellular senescence and immortalization state.

Unlike rodent cells, spontaneous immortalization of avian cells and human cells is a very rare event. According to patent publications and current literature, there are no more than 4 spontaneously immortalized chicken embryo fibroblast (CEF) cell lines established up to date. One of those cell lines is ADOL (Avian Disease and Oncology Laboratory) ZS-1 cell line, which was established by continuous passaging of the CEFs derived from the specific pathogen free (SPF) 0.TVB*S1 (commonly known as rapid feathering susceptible or RFS) genetic line of chickens. The RFS genetic line of chickens was developed and has been maintained on the SPF chicken farm of USDA-ARS facility, ADOL, in East Lansing, Michigan, which is known as one of a few lines of chickens that are free of any known avian endogenous virus genes. To explore potential roles that epigenetic factors may play in modulating cellular senescence processes and spontaneous immortalization state, total RNAs extracted from samples of the RFS primary CEFs, RFS CEFs reached the 21st passage, and the ZS-1 cells were subjected to small RNA sequencing. Collectively, a total of 531 miRNAs was identified in the 3 types of samples. In contrast to the primary CEF samples, 50 miRNAs were identified with significantly differential expression only in the 21st passage samples; a different subset of 63 differentially expressed miRNAs was identified only in the ZS-1 samples; the majority of differentially expressed miRNAs identified in both the 21st passage CEF and the ZS-1 samples were more or less directionally consistent. Gene Ontology analysis results suggested that the epigenetic factor, miRNAs, plays a role in modulating the cellular senescence and spontaneous immortalization processes through various bioprocesses and key pathways including ErbB and MAPK signaling pathways. These findings provided the experimental and bioinformatic evidence for a better understanding on the epigenetic factor of miRNAs in association with cellular senescence and spontaneous immortalization process in avian cells.

MDV-induced differential microRNA expression in the primary lymphoid organ of thymus.

Marek's disease virus (MDV), a highly contagious cell associated virus, is the etiological agent of Marek's disease (MD), a lymphoproliferative and neuropathic disease of domestic chickens. Clinical signs of MD include transient paralysis, bursal/thymic atrophy, and T cell lymphomas. MicroRNAs (miRNAs) are short single-stranded non-coding RNAs that regulate gene expression by transcriptional suppression or mRNA degradation. Herpesviruses, including MDV, encode for miRNAs that are known to play essential roles in viral pathogenicity, oncogenesis, and evasion of immune responses. In this study, we performed miRNA sequencing in thymuses of control and MDV-infected chickens of MD-resistant (63) and susceptible (72) lines at 21 days post infection (dpi). The thymus is a lymphoid organ that undergoes severe atrophy due to MDV-induced apoptotic mediated destruction of T cells. Sequence analysis identified 658 total chicken miRNAs in the thymuses of control and MDV-infected birds of both lines. Of these, 453 were novel and 205 were known microRNAs. All novel miRNAs mapped to chicken genome with no sequence homology to existing miRNAs in the chicken miRbase. Comparative analysis between the thymuses of control and infected birds of resistant and susceptible lines identified 78 differentially expressed microRNAs that might provide insights into mechanisms of thymus atrophy.

The Emergence, Diversification, and Transmission of Subgroup J Avian Leukosis Virus Reveals that the Live Chicken Trade Plays a Critical Role in the Adaption and Endemicity of Viruses to the Yellow-Chickens.

The geographical spread and inter-host transmission of the subgroup J avian leukosis virus (ALV-J) may be the most important issues for epidemiology. An integrated analysis, including phylogenetic trees, homology modeling, evolutionary dynamics, selection analysis and viral transmission, based on the gp85 gene sequences of the 665 worldwide ALV-J isolates during 1988-2020, was performed. A new Clade 3 has been emerging and was evolved from the dominating Clade 1.3 of the Chinese Yellow-chicken, and the loss of a α-helix or ß-sheet of the gp85 protein monomer was found by the homology modeling. The rapid evolution found in Clades 1.3 and 3 may be closely associated with the adaption and endemicity of viruses to the Yellow-chickens. The early U.S. strains from Clade 1.1 acted as an important source for the global spread of ALV-J and the earliest introduction into China was closely associated with the imported chicken breeders in the 1990s. The dominant outward migrations of Clades 1.1 and 1.2, respectively, from the Chinese northern White-chickens and layers to the Chinese southern Yellow-chickens, and the dominating migration of Clade 1.3 from the Chinese southern Yellow-chickens to other regions and hosts, indicated that the long-distance movement of these viruses between regions in China was associated with the live chicken trade. Furthermore, Yellow-chickens have been facing the risk of infections of the emerging Clades 2 and 3. Our findings provide new insights for the epidemiology and help to understand the critical factors involved in ALV-J dissemination. IMPORTANCE Although the general epidemiology of ALV-J is well studied, the ongoing evolutionary and transmission dynamics of the virus remain poorly investigated. The phylogenetic differences and relationship of the clades and subclades were characterized, and the epidemics and factors driving the geographical spread and inter-host transmission of different ALV-J clades were explored for the first time. The results indicated that the earliest ALV-J (Clade 1.1) from the United States, acted as the source for global spreads, and Clades 1.2, 1.3 and 3 were all subsequently evolved. Also the epidemiological investigation showed that the early imported breeders and the inter-region movements of live chickens facilitated the ALV-J dispersal throughout China and highlighted the needs to implement more effective containment measures.

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 Microbiota-Gut-Brain Axis: Gut Microbiota Modulates Conspecific Aggression in Diversely Selected Laying Hens.

The gut microbiota plays an important role in regulating brain function, influencing psychological and emotional stability. The correlations between conspecific aggression, gut microbiota, and physiological homeostasis were further studied in inbred laying chicken lines, 63 and 72, which were diversely selected for Marek's disease, and they also behave differently in aggression. Ten sixty-week-old hens from each line were sampled for blood, brain, and cecal content. Neurotransmitters, cytokines, corticosterone, and heterophil/lymphocyte ratios were determined. Cecal microbiota compositions were determined by bacterial 16s rRNA sequencing, and functional predictions were performed. Our data showed that the central serotonin and tryptophan levels were higher in line 63 compared to line 72 (p < 0.05). Plasma corticosterone, heterophil/lymphocyte ratios, and central norepinephrine were lower in line 63 (p < 0.05). The level of tumor necrosis factor α tended to be higher in line 63. Faecalibacterium, Oscillibacter, Butyricicoccus, and Bacteriodes were enriched in line 63 birds, while Clostridiales vadin BB60, Alistipes, Mollicutes RF39 were dominated in line 72. From the predicted bacterial functional genes, the kynurenine pathway was upregulated in line 72. These results suggested a functional linkage of the line differences in serotonergic activity, stress response, innate immunity, and gut microbiota populations.

Sex-specific extracellular matrix remodeling during early adipogenic differentiation by gestational bisphenol A exposure.

Bisphenol A (BPA) is an endocrine disrupting chemical known to promote adipose tissue mass in vivo and adipogenesis in vitro. Whether BPA can affect and reprogram early adipogenic differentiation signals that trigger adipogenic differentiation, remains unknown. We hypothesized that gestational BPA exposure results in a preadipocyte phenotype that leads to accelerated adipogenic differentiation, and that this phenotype is sex specific. Primary ovine fetal preadipocytes were derived from control (C) and BPA-exposed during pregnancy and differentiated in vitro. Gestational BPA enhanced lipid accumulation at early stages of differentiation (48 h) and this was evident in females but not male-derived fetal preadipocytes. After an RNA sequencing approach, samples were compared as follows: 2 groups (C vs. BPA); 2 sexes (female (F) vs. male (M)); and 2 time points (0 h vs. 48 h). Before differentiation, 15 genes were differentially expressed between the C and the BPA-exposed preadipocytes within sex. In BPA-F, extracellular matrix remodeling genes cathepsin K and collagen 5α3 were upregulated compared to C-F. At 48 h, BPA-F had 154 genes differentially expressed vs. C-F and BPA-M had 487 genes differentially expressed vs. C-M. Triglyceride and glycerophospholipid metabolism were the most upregulated pathways in BPA-F. Downregulated pathways were associated with extracellular matrix organization in BPA-exposed preadipocytes. These findings are among the first to demonstrate that gestational BPA can modify the fate of adipocyte precursors by altering pathways associated to extracellular matrix components, an often-disregarded, but required aspect of adipogenic differentiation. This work highlights the need to investigate early adipogenic differentiation changes in other obesogenic chemicals.

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.

ALV-miRNA-p19-01 Promotes Viral Replication via Targeting Dual Specificity Phosphatase 6.

MicroRNAs (miRNAs) are a group of regulatory noncoding RNAs, serving as major regulators with a sequence-specific manner in multifarious biological processes. Although a series of viral families have been proved to encode miRNAs, few reports were available regarding the function of ALV-J-encoded miRNA. Here, we reported a novel miRNA (designated ALV-miRNA-p19-01) in ALV-J-infected DF-1 cells. We found that ALV-miRNA-p19-01 is encoded by the genome of the ALV-J SCAU1903 strain (located at nucleotides site 779 to 801) in a classic miRNA biogenesis manner. The transfection of DF-1 cells with ALV-miRNA-p19-01 enhanced ALV-J replication, while the blockage of ALV-miRNA-p19-01 suppressed ALV-J replication. Furthermore, our data showed that ALV-miRNA-p19-01 promotes ALV-J replication by directly targeting the cellular gene dual specificity phosphatase 6 through regulating ERK2 activity.

B cells do not play a role in vaccine-mediated immunity against Marek's disease.

BACKGROUND: Marek's disease virus (MDV), a highly oncogenic α-herpesvirus, is the etiological agent of Marek's disease (MD) in chickens. The antiviral activity of vaccine-induced immunity against MD reduces the level of early cytolytic infection, production of cell-free virions in the feather follicle epithelial cells (FFE), and lymphoma formation. Despite the success of several vaccines that have greatly reduced the economic losses from MD, the mechanism of vaccine-induced immunity is poorly understood. METHODS: To provide insight into possible role of B cells in vaccine-mediated protection, we bursectomized birds on day of hatch and vaccinated them eight days later. The birds were challenged 10 days post vaccination with or without receiving adoptive lymphocytes from age-matched control birds prior to inoculation. The study also included vaccinated/challenged and non-vaccinated challenged intact birds. Flowcytometric analysis of PBMN cells were conducted twice post bursectomy to confirm B cell depletion and assess the effect of surgery on T cell population. Immunohistochemical analysis and viral genome copy number assessment in the skin samples at termination was performed to measure the replication rate of MDV in the FFE of the skin tissues of the challenged birds. RESULTS: The non-vaccinated/challenged birds developed typical clinical signs of MD while the vaccinated/challenged and bursectomized, vaccinated/challenged groups with or without adoptive lymphocyte transfer, were fully protected with no sign of transient paralysis, weight loss, or T cell lymphomas. Immunohistochemical analysis and viral genome copy number evaluation in the skin samples revealed that unlike the vaccinated/challenged birds a significant number of virus particles were produced in the FFE of the non-vaccinated/challenged birds at termination. In the bursectomized, vaccinated/challenged groups, only a few replicating virions were detected in the skin of birds that received adoptive lymphocytes prior to challenge. CONCLUSIONS: The study shows that B cells do not play a critical role in MD vaccine-mediated immunity.

Chromosome-level genome assembly of goose provides insight into the adaptation and growth of local goose breeds.

BACKGROUND: Anatidae contains numerous waterfowl species with great economic value, but the genetic diversity basis remains insufficiently investigated. Here, we report a chromosome-level genome assembly of Lion-head goose (Anser cygnoides), a native breed in South China, through the combination of PacBio, Bionano, and Hi-C technologies. FINDINGS: The assembly had a total genome size of 1.19 Gb, consisting of 1,859 contigs with an N50 length of 20.59 Mb, generating 40 pseudochromosomes, representing 97.27% of the assembled genome, and identifying 21,208 protein-coding genes. Comparative genomic analysis revealed that geese and ducks diverged approximately 28.42 million years ago, and geese have undergone massive gene family expansion and contraction. To identify genetic markers associated with body weight in different geese breeds, including Wuzong goose, Huangzong goose, Magang goose, and Lion-head goose, a genome-wide association study was performed, yielding an average of 1,520.6 Mb of raw data that detected 44,858 single-mucleotide polymorphisms (SNPs). Genome-wide association study showed that 6 SNPs were significantly associated with body weight and 25 were potentially associated. The significantly associated SNPs were annotated as LDLRAD4, GPR180, and OR, enriching in growth factor receptor regulation pathways. CONCLUSIONS: We present the first chromosome-level assembly of the Lion-head goose genome, which will expand the genomic resources of the Anatidae family, providing a basis for adaptation and evolution. Candidate genes significantly associated with different goose breeds may serve to understand the underlying mechanisms of weight differences.

Anti-CD81 antibody blocks vertical transmission of avian leukosis virus subgroup J.

Control of ALV-J in breed of chicken is still a serious issue that need more attention to be paid. Vertical transmission of ALV-J often give rise to more adverse pathogenicity. However, the way to elimination of ALV-J underlying vertical transmission remains not-well understood. In addition, effective vaccines or drugs have not been developed to prevent and control the transmission of ALV-J so far. CD81, a member of the tetraspanins superfamily, plays important roles in regulating membrane proteins, facilitating cells adhesion or fusion, and also participates in viral infection. The purpose of this study was to investigate whether antibodies against certain tetraspanins affect infection of ALV-J. Here, we showed that anti-CD81 antibody could inhibit viral RNA and protein level. We also found that anti-CD81 antibody interacts with viral protein p27, p32 and gp37. Moreover, treatment with antibody to CD81 can effectively prevent the vertical transmission of ALV-J in animal model. Collectively, current study provides new avenues for the control of ALV-J transmission.

Viral deubiquitinases and innate antiviral immune response in livestock and poultry.

Among many of the pathogens, virus is the main cause of diseases in livestock and poultry. A host infected with the virus triggers a series of innate and adaptive immunity. The realization of innate immune responses involves the participation of a series of protein molecules in host cells, including receptors, signal molecules and antiviral molecules. Post-translational modification of cellular proteins by ubiquitin regulates numerous cellular processes, including innate immune responses. Ubiquitin-mediated control over these processes can be reversed by cellular or viral deubiquitinases (DUBs). DUBs have now been identified in diverse viral lineages, and their characterization is providing valuable insights into virus biology and the role of the ubiquitin system in host antiviral mechanisms. In this review, we briefly introduce the mechanisms of ubiquitination and deubiquitination, present antiviral innate immune response and its regulation by ubiquitin, and summarize the prevalence of DUBs encoded by viruses (Arteriviridae, Asfarviridae, Nairoviridae, Coronaviridae, Herpesviridae, and Picornaviridae) infecting domestic animals and poultry. It is found that these DUBs suppress the innate immune responses mainly by affecting the production of type I interferon (IFN), which causes immune evasion of the viruses and promotes their replication. These findings have important reference significance for understanding the virulence and immune evasion mechanisms of the relevant viruses, and thus for the development of more effective prevention and treatment measures.

Genetic Diversity of MHC B-F/B-L Region in 21 Chicken Populations.

The chicken major histocompatibility complex (MHC) on chromosome 16 is the most polymorphic region across the whole genome, and also an ideal model for genetic diversity investigation. The MHC B-F/B-L region is 92 kb in length with high GC content consisting of 18 genes and one pseudogene (Blec4), which plays important roles in immune response. To evaluate polymorphism of the Chinese indigenous chickens as well as to analyze the effect of selection to genetic diversity, we used WaferGen platform to identify sequence variants of the B-F/B-L region in 21 chicken populations, including the Red Jungle Fowl (RJF), Cornish (CS), White Leghorns (WLs), 16 Chinese domestic breeds, and two well-known inbred lines 63 and 72. A total of 3,319 single nucleotide polymorphism (SNPs) and 181 INDELs in the B-F/B-L region were identified among 21 populations, of which 2,057 SNPs (62%) and 159 INDELs (88%) were novel. Most of the variants were within the intron and the flanking regions. The average variation density was 36 SNPs and 2 INDELs per kb, indicating dramatical high diversity of this region. Furthermore, BF2 was identified as the hypervariable genes with 67 SNPs per kb. Chinese domestic populations showed higher diversity than the WLs and CS. The indigenous breeds, Nandan Yao (NY), Xishuangbanna Game (XG), Gushi (GS), and Xiayan (XY) chickens, were the top four with the highest density of SNPs and INDELs. The highly inbred lines 63 and 72 have the lowest diversity, which might be resulted from a long-term intense selection for decades. Collectively, we refined the genetic map of chicken MHC B-F/B-L region, and illustrated genetic diversity of 21 chicken populations. Abundant genetic variants were identified, which not only strikingly expanded the current Ensembl SNP database, but also provided comprehensive data for researchers to further investigate association between variants in MHC and immune traits.

The Efficacy of a Live Attenuated TW I-Type Infectious Bronchitis Virus Vaccine Candidate.

Infectious bronchitis (IB) is a highly contagious avian disease caused by infection with infectious bronchitis virus (IBV), which seriously affects the development of the global poultry industry. The distribution of TW I-type IBV in China has increased in recent years, becoming a widespread genotype. We previously isolated a TW I-type IBV strain termed CK/CH/GD/GZ14 in 2014, but its pathogenicity and possibility for vaccine development were not explored. Therefore, this research aimed to develop a live-attenuated virus vaccine based on the CK/CH/GD/GZ14 strain. The wild type IBV CK/CH/GD/GZ14 strain was serially passaged in SPF embryos for 145 generations. The morbidity and mortality rate of wild-type strain in 14 day-old chickens is 100% and 80% respectively, while the morbidity rate in the attenuated strain was 20% in the 95th and 105th generations and there was no death. Histopathological observations showed that the pathogenicity of the 95th and 105th generations in chickens was significantly weakened. Further challenge experiments confirmed that the attenuated CK/CH/GD/GZ14 strain in the 95th and 105th generations could resist CK/CH/GD/GZ14 (5th generation) infection and the protection rate was 80%. Tracheal cilia stagnation, virus shedding, and viral load experiments confirmed that the 95th and 105th generations provide good immune protection in chickens, and the immunogenicity of the 105th generation is better than that of the 95th generation. These data suggest that the attenuated CK/CH/GD/GZ14 strain in the 105th generation may be applied as a vaccine candidate against TW I-type IBV.

Differential DNA Methylation and Gene Expression Between ALV-J-Positive and ALV-J-Negative Chickens.

Background: Avian leukosis virus subgroup J (ALV-J) is an oncogenic virus that causes serious economic losses in the poultry industry; unfortunately, there is no effective vaccine against ALV-J. DNA methylation plays a crucial role in several biological processes, and an increasing number of diseases have been proven to be related to alterations in DNA methylation. In this study, we screened ALV-J-positive and -negative chickens. Subsequently, we generated and provided the genome-wide gene expression and DNA methylation profiles by MeDIP-seq and RNA-seq of ALV-J-positive and -negative chicken samples; 8,304 differentially methylated regions (DMRs) were identified by MeDIP-seq analysis (p ≤ 0.005) and 515 differentially expressed genes were identified by RNA-seq analysis (p ≤ 0.05). As a result of an integration analysis, we screened six candidate genes to identify ALV-J-negative chickens that possessed differential methylation in the promoter region. Furthermore, TGFB2 played an important role in tumorigenesis and cancer progression, which suggested TGFB2 may be an indicator for identifying ALV-J infections.