10× single-cell sequencing revealed cellular composition heterogeneity in cardiac myxoma with malignant glandular properties.

Cardiac myxoma is the most common primary cardiac tumor in adults. The histogenesis and cellular composition of myxoma are still unclear. This study aims to reveal the role of myxoma cell components and their gene expression in tumor development. We obtained single living cells by enzymatic digestion of tissues from 4 cases of surgically resected cardiac myxoma. Of course, there was 1 case of glandular myxoma and 3 cases of nonglandular myxoma. Then, 10× single-cell sequencing was performed. We identified 12 types and 11 types of cell populations in glandular myxoma and nonglandular myxoma, respectively. Heterogeneous epithelial cells are the main components of glandular myxoma. The similarities and differences in T cells in both glandular and nonglandular myxoma were analyzed by KEGG and GO. The most important finding was that there was active communication between T cells and epithelial cells. These results clarify the possible tissue occurrence and heterogeneity of cardiac myxoma and provide a theoretical basis and guidance for clinical diagnosis and treatment.

Metabolomics analysis of CEF cells infected with avian leukosis virus subgroup J based on UHPLC-QE-MS.

Avian leukosis virus subgroup J (ALV-J) is a retrovirus that can cause immunosuppression and tumors in chicken. However, relative pathogenesis is still not clear. At present, metabolomics has shown great potential in the screening of tumor metabolic markers, prognostic evaluation, and drug target design. In this study, we utilize an untargeted metabolomics approach based on ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) to analyze the metabolic changes in chicken embryo fibroblast (CEF) cells infected by ALV-J. We found that ALV-J infection significantly altered a wealth of metabolites compared with control group. Additionally, most of the differentially expressed metabolites belonged to lipid metabolism, purine nucleotide metabolism and amino acid metabolism. Among them, the proportion of lipid metabolites account for the highest proportion (around 31%). Results suggest that these changes may be conductive to the formation of virion, thereby promoting the replication of ALV-J. These data provided metabolic evidence and potential biomarkers for the cellular metabolic changes induced by ALV-J, and provided important insight for further understanding the replication needs and pathogenesis of ALV-J.

Efficient cross-protection against serotype 4/8a fowl adenoviruses (FAdVs): recombinant FAdV-4 with FAdV-8a Fiber.

IMPORTANCE: Epidemiological data reveal that FAdV-4 and FAdV-8a are the dominant serotypes of FAdVs in the poultry industry in China. Although three commercial inactivated vaccines against FAdV-4 have been licensed in China, the bivalent vaccine against both FAdV-4 and FAdV-8a is not available. Here, we used CRISPR-Cas9 and Cre-LoxP system to generate a recombinant virus FAdV4-F/8a-rF2 expressing the Fiber of FAdV-8a. Notably, FAdV4-F/8a-rF2 was highly attenuated and could provide efficient protection against both FAdV-4 and FAdV-8a in the chicken infection model, highlighting the applaudable application of FAdV4-F/8a-rF2 as a novel live-attenuated bivalent vaccine against the diseases caused by the infection of FAdV-4 and FAdV-8a.

Metabolomic profiling of Marek's disease virus infection in host cell based on untargeted LC-MS.

Marek's disease (MD) caused by Marek's disease virus (MDV), poses a serious threat to the poultry industry by inducing neurological disease and malignant lymphoma in infected chickens. However, the underlying mechanisms how MDV disrupts host cells and causes damage still remain elusive. Recently, the application of metabolomics has shown great potential for uncovering the complex mechanisms during virus-host interactions. In this study, chicken embryo fibroblasts (CEFs) infected with MDV were subjected to ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) and multivariate statistical analysis. The results showed that 261 metabolites were significantly altered upon MDV infection, with most changes occurring in amino acid metabolism, energy metabolism, nucleotide metabolism, and lipid metabolism. Notably, MDV infection induces an up-regulation of amino acids in host cells during the early stages of infection to provide the energy and intermediary metabolites necessary for efficient multiplication of its own replication. Taken together, these data not only hold promise in identifying the biochemical molecules utilized by MDV replication in host cells, but also provides a new insight into understanding MDV-host interactions.

3'UTR of ALV-J can affect viral replication through promoting transcription and mRNA nuclear export.

IMPORTANCE: 3'UTRs can affect gene transcription and post-transcriptional regulation in multiple ways, further influencing the function of proteins in a unique manner. Recently, ALV-J has been mutating and evolving rapidly, especially the 3'UTR of viral genome. Meanwhile, clinical symptoms caused by ALV-J have changed significantly. In this study, we found that the ALV-J strains containing △-r-TM-type 3'UTR are the most abundant. By constructing ALV-J infectious clones and subgenomic vectors containing different 3'UTRs, we prove that 3'UTRs directly affect viral tissue preference and can promote virus replication as an enhancer. ALV-J strain containing 3'UTR of △-r-TM proliferated fastest in primary cells. All five forms of 3'UTRs can assist intron-containing viral mRNA nuclear export, with similar efficiency. ALV-J mRNA half-life is not influenced by different 3'UTRs. Our results dissect the roles of 3'UTR on regulating viral replication and pathogenicity, providing novel insights into potential anti-viral strategies.

Identification of novel B cell epitopes in Fiber-2 protein of duck adenovirus 3 and their application.

Duck adenovirus 3 (DAdV-3), a newly emerged duck adenovirus, has resulted in significant economic losses to the duck industry across China since 2014. However, little is known about the B cell epitopes in major antigen of DAdV-3 and the serological approach for detection of DAdV-3 is not available. In this study, four monoclonal antibodies (mAbs) specific to Fiber-2 protein of DAdV-3 were first generated and designated as 2G10, 3D9, 5E6, and 6B12. Indirect immunofluorescence assay (IFA) showed that all of the mAbs reacted with the Fiber-2. Moreover, mAbs 2G10, 5E6, and 6B12 demonstrated good activity with Fiber-2 in Western blot. Notably, the Fiber-2 could be immunoprecipitated efficiently by mAb 3D9. Epitope mapping revealed that mAbs 2G10, 3D9, 5E6, and 6B12 recognized 397-429aa, 463-481aa, 67-99aa, and 1-66aa of Fiber-2, respectively. Besides, a novel sandwich ELISA for efficient detection of DAdV-3 was developed based on mAb 3D9 and horseradish peroxidase (HRP) conjugated mAb 3D9. The sandwich ELISA only reacted with DAdV-3 but not with other duck-associated viruses. The limit of detection of the ELISA was 6.25 × 103 TCID50/mL. Overall, the mAbs generated laid the foundation for elucidating the critical role of Fiber-2 in mediating infection and pathogenesis, and the sandwich ELISA approach established here provided efficient and rapid serological diagnostic tool for DAdV-3.

An efficient double-fluorescence approach for generating fiber-2-edited recombinant serotype 4 fowl adenovirus expressing foreign gene.

Recently, the infection of serotype 4 fowl adenovirus (FAdV-4) in chicken flocks has become endemic in China, which greatly threatens the sustainable development of poultry industry. The development of recombinant FAdV-4 expressing foreign genes is an efficient strategy for controlling both FAdV-4 and other important poultry pathogens. Previous reverse genetic technique for generating the recombinant fowl adenovirus is generally inefficient. In this study, a recombinant FAdV-4 expressing enhanced green fluorescence protein (EGFP), FA4-EGFP, was used as a template virus and directly edited fiber-2 gene to develop an efficient double-fluorescence approach to generate recombinant FAdV-4 through CRISPR/Cas9 and Cre-Loxp system. Moreover, using this strategy, a recombinant virus FAdV4-HA(H9) stably expressing the HA gene of H9N2 influenza virus was generated. Chicken infection study revealed that the recombinant virus FAdV4-HA(H9) was attenuated, and could induce haemagglutination inhibition (HI) titer against H9N2 influenza virus at early time points and inhibit the viral replication in oropharynx. All these demonstrate that the novel strategy for constructing recombinant FAdV-4 expressing foreign genes developed here paves the way for rapidly developing attenuated FAdV-4-based recombinant vaccines for fighting the diseases caused by both FAdV-4 and other pathogens.

A novel recombinant serotype 4 fowl adenovirus expressing fiber-2 protein of duck adenovirus 3.

Recently, the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and duck adenovirus 3 (DAdV-3) were outbroken and widespread, causing substantial economic losses to the duck industry. Therefore, there is an urgent need to generate a recombinant genetic engineering vaccine candidate against both FAdV-4 and DAdV-3. In this study, a novel recombinant FAdV-4 expressing the Fiber-2 protein of DAdV-3, designated as rFAdV-4-Fiber-2/DAdV-3, was generated based on CRISPR/Cas9 and Cre-LoxP systems. Indirect immunofluorescence assay (IFA) and western blot (WB) showed that the Fiber-2 protein of DAdV-3 in rFAdV-4-Fiber-2/DAdV-3 was expressed successfully. Moreover, the growth curve revealed that rFAdV-4-Fiber-2/DAdV-3 replicated efficiently in LMH cells and even showed a stronger replication ability compared to the wild type FAdV-4. The generation of the recombinant rFAdV-4-Fiber-2/DAdV-3 provides a potential vaccine candidate against both FAdV-4 and DAdV-3.

A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

Chicken Glycogen Synthase Kinase 3ß Suppresses Innate Immune Responses and Enhances Avian Leukosis Virus Replication in DF-1 Cells.

Glycogen synthase kinase 3ß (GSK3ß) is a widely distributed multifunctional serine/threonine kinase. In mammals, GSK3ß regulates important life activities such as proinflammatory response, anti-inflammatory response, immunity, and cancer development. However, the biological functions of chicken GSK3ß (chGSK3ß) are still unknown. In the present study, the full-length cDNA of chGSK3ß was first cloned and analyzed. Absolute quantification of chicken chGSK3ß in 1-day-old specific-pathogen-free birds has shown that it is widely expressed in all tissues, with the highest level in brain and the lowest level in pancreas. Overexpression of chGSK3ß in DF-1 cells significantly decreased the gene expression levels of interferon beta (IFN-ß), IFN regulatory factor 7 (IRF7), Toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA5), MX-1, protein kinase R (PKR), and oligoadenylate synthase-like (OASL), while promoting the replication of avian leukosis virus subgroup J (ALV-J). Conversely, levels of most of the genes detected in this study were increased when chGSK3ß expression was knocked down using small interfering RNA (siRNA), which also inhibited the replication of ALV-J. These results suggest that chGSK3ß plays an important role in the antiviral innate immune response in DF-1 cells, and it will be valuable to carry out further studies on the biological functions of chGSK3ß. IMPORTANCE GSK3ß regulates many life activities in mammals. Recent studies revealed that chGSK3ß was involved in regulating antiviral innate immunity in DF-1 cells and also could positively regulate ALV-J replication. These results provide new insights into the biofunction of chGSK3ß and the virus-host interactions of ALV-J. In addition, this study provides a basis for further research on the function of GSK3 in poultry.

A novel monoclonal antibody efficiently blocks the infection of duck adenovirus 3 by targeting Fiber-2.

Duck adenovirus 3 (DAdV-3), identified as the causative agent of a disease characterized by swelling and hemorrhage of liver and kidney, has caused substantial economic losses to duck industry in China. However, the neutralizing epitopes and the infection mechanism of DAdV-3 have not been extensively elucidated. In this study, a novel monoclonal antibody (mAb) targeting Fiber-2 protein of DAdV-3 was generated and designated as mAb 3E7. Indirect immunofluorescence assay showed that mAb 3E7 specifically reacted with the Fiber-2 in LMH cells transfected with pcDNA3.1-Fiber-2 or infected with DAdV-3. Moreover, mAb 3E7 could immunoprecipitate the Fiber-2 and efficiently inhibit the infection of DAdV-3 in vitro. Further epitope mapping revealed mAb 3E7 recognized the epitope 108LALGDGLE115 in Fiber-2, which was highly conserved among DAdV-3 strains. These findings not only identified a novel neutralizing epitope in Fiber-2, but also paved the way for further elucidating the vital roles of Fiber-2 in the infection and pathogenesis of DAdV-3.

Research Note: A novel peptide-based ELISA for efficient detection of antibody against chicken infectious anemia virus.

Chicken infectious anemia virus (CIAV) is the pathogen of chicken infectious anemia. Currently, due to the lack of effective diagnostics technology and prevention approach, CIAV has spread globally and caused huge economic losses to poultry industry. In this study, a novel peptide-based ELISA (pELISA) for efficient detection of antibody against CIAV was developed. The peptide (25CRLRRRYKFRHRRRQRYRRRAF45) used in pELISA was highly conserved in VP1 protein of different CIAV isolates. The specificity and reproducibility showed that the pELISA only reacted with sera against CIAV, not with sera against other pathogens tested, and the CV of the intra-/inter-assay of the pELISA was 6.8 to 9.22%. Moreover, the comparison assay using 56 clinical samples showed that the positive rate of the pELISA and the commercial ELISA kit (IDEXX) was 85.7 and 80.4%, respectively. The pELISA generated here provides a rapid and efficient serological detection method for diagnosis of CIAV infection and evaluation of the efficacy of CIAV vaccination.

A peptide-based ELISA for detection of antibodies against novel goose astrovirus type 1.

Goose gout disease is a high morbidity and mortality disease caused by novel serotype 1 goose astrovirus (GAstV-1), which has resulted in huge economic loss to the goose industry of China. However, few diagnostic methods have been developed for serological surveillance of GAstV-1. In our previous study, several novel B cell epitopes were identified in the ORF2 protein of GAstV-1. In this study, one novel peptide of 627-646 aa in the ORF2 recognized by monoclonal antibody (mAb) 6C6 was used as an antigen to develop an efficient peptide-based ELISA (pELISA) for detection of antibodies against GAstV-1. Specificity analysis showed that the pELISA only reacted with sera against GAstV-1, but not with sera against other pathogens tested. The sensitivity of the pELISA in detecting positive sera was higher than that of the IFA (Indirect immunofluorescence assay). The coefficients of variation (CV) of the intra-assay and inter-assay were both < 10%, indicating that the reproducibility of pELISA was good. For detection of clinical samples, the pELISA had 87.5% concordance with the IFA. Our data demonstrate that the pELISA generated here provides an accurate, rapid, and economical method for the detection antibodies against GAstV-1 for serological surveillance.

Novel monoclonal antibodies against Fiber-1 of duck adenovirus 3 and their B cell epitopes.

Recently, the outbreak of the infection of Duck adenovirus 3 (DAdV-3) characterized by swelling and hemorrhagic liver and kidney has caused huge economic losses to duck industry since 2014 in China. To date, the B cell epitopes in the Fiber-1 protein and the underlying infection mechanism of DAdV-3 have not been investigated. In this study, the recombinant Fiber-1 protein was first expressed in E. coli and six novel monoclonal antibodies (mAbs) against Fiber-1 were generated, designated as 1D8, 1E6, 3G6, 4G1, 4G2, and 6F10, respectively. Moreover, mAbs 3G6 and 6F10 could efficiently immunoprecipitate the Fiber-1 in LMH cells infected with DAdV-3 or transfected with pcDNA3.1-Fiber-1. Notably, mAbs 3G6 and 4G2 also showed certain neutralizing activity against DAdV-3 infection in vitro. Epitopes mapping revealed that the B cell epitope recognized by 6F10, 3G6, 4G1, 1D8, 4G2, and 1E6 was located in 34-66aa, 67-99aa, 64-296aa, 297-329aa, 330-362aa, and 363-395aa, respectively. Sequence alignments further found that the six epitopes recognized by these mAbs were highly conserved among different DAdV-3 isolates. The generated mAbs specific to Fiber-1 and their defined epitopes provide powerful tools for establishing rapid and efficient diagnostics for the detection of DAdV-3 and pave the way for further studying on the critical role of Fiber-1 in mediating the infection of DAdV-3.

CD4+ T-cell epitope-based heterologous prime-boost vaccination potentiates anti-tumor immunity and PD-1/PD-L1 immunotherapy.

BACKGROUND: Antitumor therapeutic vaccines are generally based on antigenic epitopes presented by major histocompatibility complex (MHC-I) molecules to induce tumor-specific CD8+ T cells. Paradoxically, continuous T cell receptor (TCR) stimulation from tumor-derived CD8+ T-cell epitopes can drive the functional exhaustion of tumor-specific CD8+ T cells. Tumor-specific type-I helper CD4+ T (TH1) cells play an important role in the population maintenance and cytotoxic function of exhausted tumor-specific CD8+ T cells in the tumor microenvironment. Nonetheless, whether the vaccination strategy targeting MHC-II-restricted CD4+ T-cell epitopes to induce tumor-specific TH1 responses can confer effective antitumor immunity to restrain tumor growth is not well studied. Here, we developed a heterologous prime-boost vaccination strategy to effectively induce tumor-specific TH1 cells and evaluated its antitumor efficacy and its capacity to potentiate PD-1/PD-L1 immunotherapy. METHODS: Listeria monocytogenes vector and influenza A virus (PR8 strain) vector stably expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein-specific I-Ab-restricted CD4+ T cell epitope (GP61-80) or ovalbumin-specific CD4+ T cell epitope (OVA323-339) were constructed and evaluated their efficacy against mouse models of melanoma and colorectal adenocarcinoma expressing lymphocytic choriomeningitis virus glycoprotein and ovalbumin. The impact of CD4+ T cell epitope-based heterologous prime-boost vaccination was detected by flow-cytometer, single-cell RNA sequencing and single-cell TCR sequencing. RESULTS: CD4+ T cell epitope-based heterologous prime-boost vaccination efficiently suppressed both mouse melanoma and colorectal adenocarcinoma. This vaccination primarily induced tumor-specific TH1 response, which in turn enhanced the expansion, effector function and clonal breadth of tumor-specific CD8+ T cells. Furthermore, this vaccination strategy synergized PD-L1 blockade mediated tumor suppression. Notably, prime-boost vaccination extended the duration of PD-L1 blockade induced antitumor effects by preventing the re-exhaustion of tumor-specific CD8+ T cells. CONCLUSION: CD4+ T cell epitope-based heterologous prime-boost vaccination elicited potent both tumor-specific TH1 and CTL response, leading to the efficient tumor control. This strategy can also potentiate PD-1/PD-L1 immune checkpoint blockade (ICB) against cancer.

An Efficient and Rapid Assay for Detecting Neutralizing Antibodies Against Serotype 4 Fowl Adenovirus.

Currently, the outbreak of serotype 4 fowl adenovirus (FAdV-4) has spread worldwide and caused tremendous economic loss to the poultry industry. Although inactivated vaccines have been licensed against FAdV-4 in China, a rapid and efficient serological method for measuring the titer of neutralizing antibodies (NAbs) specific for FAdV-4 post-infection or vaccination is rarely reported. Classical virus neutralization test (VNT) is superior in sensitivity and specificity for detecting NAbs but is either time-consuming or laborious. In this study, a recombinant virus FA4-EGFP expressing EGFP-fiber-2 fusion protein, rather than wild type (WT) FAdV-4 was used to develop a novel VNT for detecting FAdV-4 NAbs. Specificity analysis showed that the approach only reacted with the sera against FAdV-4, not with the sera against other avian pathogens tested. The novel VNT was effective in the detection of NAbs against FAdV-4 in sera from both experimentally infected and clinically vaccinated chickens, and had good linear correlation with the classical VNT. Moreover, the novel VNT not only significantly simplifies the procedure for detection of NAbs, but also shortens the timeline to 24 h in comparison with the classical VNT with 3-4 d. All these data demonstrate that the FA4-EGFP based VNT developed here provides an efficient diagnostic method for monitoring the immunological state of the vaccination or diagnosing the clinical infection of FAdV-4 in a quick and funding-saving manner.

A Novel Recombinant FAdV-4 Virus with Fiber of FAdV-8b Provides Efficient Protection against Both FAdV-4 and FAdV-8b.

Since 2015, the outbreaks of hydropericardium-hepatitis syndrome (HHS) and inclusion body hepatitis (IBH) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and serotype 8 fowl adenovirus (FAdV-8), respectively, have caused huge economic losses to the poultry industry. Although several vaccines have been developed to control HHS or IBH, a recombinant genetic engineering vaccine against both FAdV-4 and FAdV-8 has not been reported. In this study, recombinant FAdV-4 expressing the fiber of FAdV-8b, designated as FA4-F8b, expressing fiber of FAdV-8b was generated by the CRISPR-Cas9 and homologous recombinant techniques. Infection studies in vitro and in vivo revealed that the FA4-F8b replicated efficiently in LMH cells and was also highly pathogenic to 2-week-old SPF chickens. Moreover, the inoculation of inactivated the FA4-F8b in chickens could not only induce highly neutralizing antibodies, but also provide efficient protection against both FAdV-4 and FAdV-8b. All these demonstrate that the inactivated recombinant FA4-F8b generated here can act as a vaccine candidate to control HHS and IBH, and FAdV-4 can be an efficient vaccine vector to deliver foreign antigens.

FAdV-4 without Fiber-2 Is a Highly Attenuated and Protective Vaccine Candidate.

Hepatitis-hydropericardium syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) has resulted in huge economic losses to the poultry industry globally. The fiber-2 gene, as a major virulence determiner, is also an important vaccine target against FAdV-4. In this study, we used a CRISPR/Cas9-based homology-dependent recombinant technique to replace the fiber-2 gene with egfp and generate a novel recombinant virus, designated FAdV4-EGFP-rF2. Although FAdV4-EGFP-rF2 showed low replication ability compared to the wild-type FAdV-4 in LMH cells, FAdV4-EGFP-rF2 could effectively replicate in LMH-F2 cells with the expression of Fiber-2. Moreover, FAdV4-EGFP-rF2 was not only highly attenuated in chickens, but also could provide efficient protection against a lethal challenge of FAdV-4. Moreover, FAdV4-EGFP-rF2 without fiber-2 could induce neutralizing antibodies at the same level as FA4-EGFP with fiber-2. These results clearly demonstrate that although fiber-2 affects the viral replication and pathogenesis of FAdV-4, it is not necessary for virus replication and induction of neutralizing antibodies; these findings provide novel insights into the roles of fiber-2 and highlight fiber-2 as an insertion site for generating live-attenuated FAdV-4 vaccines against FAdV-4 and other pathogens. IMPORTANCE Among all serotypes of fowl adenovirus, serotypes FAdV-1, FAdV-4, and FAdV-10 are unique members with two fiber genes (fiber-1 and fiber-2). Recent studies reveal that Fiber-1, not Fiber-2, directly triggers viral infection of FAdV-4, whereas Fiber-2, but not Fiber-1, has been identified as the major virulence determiner and an efficient protective immunogen for subunit vaccines. Here, we replaced fiber-2 with egfp to generate a novel recombinant virus, designated FAdV4-EGFP-rF2. In vitro and in vivo studies on FAdV4-EGFP-rF2 revealed that fiber-2 was not necessary for either virus replication or efficient protection for FAdV-4; these results not only provide a novel live-attenuated vaccine candidate against HHS, but also give new ideas for generating a FAdV-4 based vaccine vector against other pathogens.

Chicken hepatomegaly and splenomegaly associated with novel subgroup J avian leukosis virus infection.

BACKGROUND: Subgroup J avian leukosis virus (ALV-J) is an oncovirus which can induce multiple types of tumors in chicken. In this report, we found novel ALV-J infection is closely associated with serious hepatomegaly and splenomegaly in chicken. CASE PRESENTATION: The layer chickens from six flocks in Jiangsu province, China, showed serious hemoperitoneum, hepatomegaly and splenomegaly. Histopathological results indicated focal lymphocytic infiltration, cell edema and congestion in the liver, atrophy and depletion of lymphocyte in the spleen. Tumor cells were not detected in all the organs. avian hepatitis E virus (aHEV), which is thought to be the cause of a very similar disease, big liver and spleen disease (BLS), was not detected. Other viruses causing tumors or liver damage including Marek's disease virus (MDV), reticuloendotheliosis virus (REV), fowl adenovirus (FAdV) and chicken infectious anemia virus (CIAV) were also proved negative by either PCR or RT-PCR. However, we did detect ALV-J in those chickens using PCR. Only novel ALV-J strains were efficiently isolated from these chicken livers. CONCLUSIONS: This is the first report that chicken hepatomegaly and splenomegaly disease was closely associated with novel ALV-J, highlighting the importance of ALV-J eradication program in China.

Fiber-1 of serotype 4 fowl adenovirus mediates superinfection resistance against serotype 8b fowl adenovirus.

In recent years, hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH) caused by serotype 4 fowl adenovirus (FAdV-4) and serotype 8b fowl adenovirus (FAdV-8b), respectively, are widely prevalent in China, causing huge economic losses to the poultry industry. Numerous studies have revealed the mechanism of the infection and pathogenesis of FAdV-4. However, little is known about the mechanism of infection with FAdV-8b. Among the major structural proteins of fowl adenoviruses, fiber is characterized by the ability to recognize and bind to cellular receptors to mediate the infection of host cells. In this study, through superinfection resistance analysis and an interfering assay, we found that Fiber-1 of FAdV-4, rather than hexon, penton, and fiber of FAdV-8b, conferred efficient superinfection resistance against the infection FAdV-8b in LMH cells. Moreover, truncation analysis depicted that the shaft and knob domains of FAdV-4 Fiber-1 were responsible for the inhibition. However, knockout of the coxsackie and adenovirus receptor (CAR) in LMH cells inhibited the replication of FAdV-8b only at early time points, indicating that CAR might not be the key cell receptor for FAdV-8b. Overall, our findings give novel insights into the infection mechanism of FAdV-8b and provide a new target for the prevention and control of both FAdV-4 and FAdV-8b.