Exosomal circ_CCDC7/gga-miR-6568-3p/Pax7 axis accelerates the differentiation of chicken embryonic stem cells infected with subgroup J avian leukosis virus.

Exosome-mediated horizontal and vertical transmission of subgroup J avian leukosis virus (ALV-J) in poultry flocks can lead to growth inhibition and severe immunosuppression. However, there are few reports on the early infection of chicken embryonic stem cells (cESCs) with ALV-J. In this study, we confirmed that early infection with ALV-J can accelerate the differentiation of cESCs and promote the secretion of exosomes. To investigate the modulation strategy of ALV-J in cESCs, circRNA sequencing was performed for further analysis. A total of 305 differentially expressed circRNAs (DECs) were obtained, including 71 upregulated DECs. Circ-CCDC7 was found to be the most upregulated DEC and was assessed by qRT-PCR, with the result consistent with the result of circRNA-seq. Based on qRT-PCR, gga-miR-6568-3p was found to be the target of the top 3 DECs, including circ-CCDC7, and the stem cell marker gene Pax7 was identified as the target gene of gga-miR-6568-3p. This study demonstrated that exosomal circ-CCDC7/gga-miR-6568-3p/Pax7 accelerates the differentiation of cESCs after early infection with ALV-J.

Synergy of Subgroup J Avian Leukosis Virus and Chicken Infectious Anemia Virus Enhances the Pathogenicity in Chickens.

Subgroup J avian leukemia virus (ALV-J) and chicken infectious anemia virus (CIAV) are widely acknowledged as significant immunosuppressive pathogens that commonly co-infect chickens, causing substantial economic losses in the poultry industry. However, whether co-infection of ALV-J and CIAV have synergistic pathogenicity remains uncertain. To explore their synergistic pathogenesis, we established a co-infection model of ALV-J and CIAV in HD11 cells and specific-pathogen-free (SPF) chickens. We discovered that ALV-J and CIAV can synergistically promote the secretion of IL-6, IL-10, IFN-α, and IFN-γ and apoptosis in HD11 cells. In vivo, compared to the ALV-J and CIAV mono-infected group, the mortality increased significantly by 27% (20 to 47%) and 14% (33 to 47%) in the co-infected group, respectively. We also discovered that ALV-J and CIAV synergistically inhibited weight gain and exhibited more severe organ damage in co-infected chickens. Furthermore, we found that CIAV can promote the replication of ALV-J in HD11 cells and significantly enhance ALV-J viral load in blood and tissues of co-infected chickens, but ALV-J cannot promote the replication of CIAV. Moreover, by measuring the immune organ indexes and proportions of blood CD3+CD4+ and CD3+CD8+ lymphocytes, more serious instances of immunosuppression were observed in ALV-J and CIAV co-infected chickens than in mono-infected chickens. Taken together, our findings demonstrate that ALV-J and CIAV synergistically enhance pathogenicity and immunosuppression.

Establishment and Application Prospect of Reverse Transcriptase Recombinase-Aided Amplification Assay for Subgroup C Avian Metapneumovirus.

Among broilers, the main pathogen that leads to swollen head syndrome (SHS) is the subgroup C avian metapneumovirus (aMPV-C). The aMPV-C infection can lead to an upsurge in the rate of soft-shell eggs, resulting in reduced egg production and seriously affecting the economy of the livestock industry. Therefore, a rapid method for aMPV-C detection needs to be invented. According to the N gene of aMPV-C, we designed the specific probe and primer and created a reverse transcription recombinase-aided amplification assay (RT-RAA) for the detection of aMPV-C. aMPV-C could be detected quickly and specifically by this method at 41 °C for 30 min. The sensitivity assay inferred that the minimum detection threshold of RT-RAA was 3.38 × 101 copies/µL. A specificity assay showed that the RT-RAA method did not cross-react with other subgroups (aMPV-A, aMPV-B, aMPV-D) or other viruses (H9N2, NDV, IBV, IBDV). Forty samples of known clinical background were tested by RT-RAA and RT-qPCR. The two approaches had a 100% correlation rate. In conclusion, this research successfully created an RT-RAA assay for aMPV-C.

A review of the influence of environmental pollutants (microplastics, pesticides, antibiotics, air pollutants, viruses, bacteria) on animal viruses.

Microorganisms, especially viruses, cause disease in both humans and animals. Environmental chemical pollutants including microplastics, pesticides, antibiotics sand air pollutants arisen from human activities affect both animal and human health. This review assesses the impact of chemical and biological contaminants (virus and bacteria) on viruses including its life cycle, survival, mutations, loads and titers, shedding, transmission, infection, re-assortment, interference, abundance, viral transfer between cells, and the susceptibility of the host to viruses. It summarizes the sources of environmental contaminants, interactions between contaminants and viruses, and methods used to mitigate such interactions. Overall, this review provides a perspective of environmentally co-occurring contaminants on animal viruses that would be useful for future research on virus-animal-human-ecosystem harmony studies to safeguard human and animal health.

Non-electrocardiogram-gated, free-breathing, off-resonance reduced, high-resolution, whole-heart myocardial T2 * mapping at 3 T within 5 min.

PURPOSE: Widely used conventional 2D T2 * approaches that are based on breath-held, electrocardiogram (ECG)-gated, multi-gradient-echo sequences are prone to motion artifacts in the presence of incomplete breath holding or arrhythmias, which is common in cardiac patients. To address these limitations, a 3D, non-ECG-gated, free-breathing T2 * technique that enables rapid whole-heart coverage was developed and validated. METHODS: A continuous random Gaussian 3D k-space sampling was implemented using a low-rank tensor framework for motion-resolved 3D T2 * imaging. This approach was tested in healthy human volunteers and in swine before and after intravenous administration of ferumoxytol. RESULTS: Spatial-resolution matched T2 * images were acquired with 2-3-fold reduction in scan time using the proposed T2 * mapping approach relative to conventional T2 * mapping. Compared with the conventional approach, T2 * images acquired with the proposed method demonstrated reduced off-resonance and flow artifacts, leading to higher image quality and lower coefficient of variation in T2 *-weighted images of the myocardium of swine and humans. Mean myocardial T2 * values determined using the proposed and conventional approaches were highly correlated and showed minimal bias. CONCLUSION: The proposed non-ECG-gated, free-breathing, 3D T2 * imaging approach can be performed within 5 min or less. It can overcome critical image artifacts from undesirable cardiac and respiratory motion and bulk off-resonance shifts at the heart-lung interface. The proposed approach is expected to facilitate faster and improved cardiac T2 * mapping in those with limited breath-holding capacity or arrhythmias.

On the loss of image contrast in double-inversion-recovery prepared T2* MRI of Intramyocardial hemorrhage.

PURPOSE: Studies have shown that double-inversion-recovery (DIR) prepared dark-blood T2*-weighted images result in lower SNR, CNR and diagnostic accuracy for intramyocardial hemorrhage (IMH) detection compared to non-DIR-prepared (bright-blood) T2*-weighted images; however, the mechanism contributing to this observation has not been investigated and explained in detail. This work tests the hypothesis that the loss of SNR on dark-blood cardiac T2*-weighted images of IMH stems from spin-relaxation during the long RF pulses in double inversion preparation, as a result, compromising image contrast for intramyocardial hemorrhage detection. METHODS: Phantom and in-vivo animal studies were performed to test the hypothesis of the study. An agar phantom was imaged with multi-gradient-echo T2* imaging protocols with and without double-inversion-recovery (DIR) preparation. Image acquisitions were placed at different delay times (TD) after DIR preparation. SNR, T2* and Coefficient of Variation (COV) were measured and compared between DIR-prepared and non-DIR-prepared images. Canines with hemorrhagic myocardial infarctions were scanned at 3.0 T with DIR-prepared (dark-blood) and non-DIR-prepared (bright-blood) T2* imaging protocols. DIR-prepared T2* images were acquired with short, medium, and long delay times (TD). SNR, CNR, intramyocardial hemorrhage (IMH) extent, T2* and COV were measured and compared between DIR-prepared T2* images with short, medium, and long delay times (TD) to non-DIR-prepared bright-blood T2* images. RESULTS: Phantom studies confirmed the hypothesis that the SNR loss on DIR-prepared T2* images originated from signal loss during DIR preparation. SNR followed T1 recovery curve with increased delay times (TD) indicating that SNR can be recovered with longer time delay between DIR and image acquisition. Myocardial T2* values were not affected by DIR preparation but COV of T2* was elevated. Animal studies supported the hypothesis and showed that DIR-prepared T2* images with insufficient delay time (TD) had impaired sensitivity for IMH detection due to lower SNR and CNR, and higher COV. CONCLUSION: We conclude that lower SNR and CNR on DIR-prepared T2* images originate from signal loss during DIR preparation and insufficient recovery between DIR preparation and image acquisition. Although, the impaired sensitivity can be recovered by extending delay time (TD), it will extend the scan time. Bright-blood T2* imaging protocols should remain the optimal choice for assessment of intramyocardial hemorrhage. DIR-prepared dark-blood T2* imaging protocols should be performed with extra attention on image signal-to-noise ratio when used for intramyocardial hemorrhage detection.

Exploring the role of miRNAs in early chicken embryonic development and their significance.

In the early stages of embryonic development, a precise and strictly controlled hierarchy of gene expression is essential to ensure proper development of all cell types and organs. To better understand this gene control process, we constructed a small RNA library from 1- to 5-day-old chick embryos, and identified 2,459 miRNAs including 827 existing, 695 known, and 937 novel miRNAs with bioinformatic analysis. There was absolute high expression of a number of miRNAs in each stage, including gga-miR-363-3p (Em1d), gga-miR-26a-5p (Em2d and Em3d), gga-miR-10a-5p (Em4d), and gga-miR-199-5p (Em5d). We evaluated enriched miRNA profiles, identifying VEGF, Insulin, ErbB, MAPK, Hedgehog, TLR and Hippo signaling pathways as primary regulatory mechanisms enabling complex morphogenetic transformations within tight temporal constraints. Pathway analysis revealed miRNAs as pivotal nodes of interaction, coordinating cascades of gene expression critical for cell fate determination, proliferation, migration, and differentiation across germ layers and developing organ systems. Weighted Gene Co-Expression Network Analysis (WGCNA) generated hub miRNAs whose modular connections spanned regulatory networks, including: gga-miR-181a-3p (blue module), coordinating immunegenesis and myogenesis; gga-miR-126-3p (brown module), regulating vasculogenesis and angiogenesis; gga-miR-302c-5p (turquoise module), enabling pluripotency and self-renew; and gga-miR-429-3p (yellow module), modulating neurogenesis and osteogenesis. The findings of this study extend the knowledge of miRNA expression in early embryonic development of chickens, providing insights into the intricate gene control process that helps ensure proper development.

Construction and immune evaluation of the recombinant duck adenovirus type 3 delivering capsid protein VP1 of the type 1 duck hepatitis virus.

Adenovirus serves as an excellent viral vector and is employed in vector vaccine research. Duck hepatitis A virus type 1 (DHAV1) and duck adenovirus type 3 (DAdV3) cause significant economic losses in the Chinese duck industry. In this study, we found an excellent exogenous gene insertion site in DAdV3 genome of CH-GD-12-2014 strain, within 3 intergenic regions (IGR). Subsequently, we generated a recombinant duck adenovirus named rDAdV3-VP1-188, which exhibits excellent replication characteristics and immunogenicity of DAdV3 and DHAV1. Animal experiments showed that rDAdV3-VP1-188 can provide 100% protection against the DAdV3 and 80% protection against DHAV1. These results showed that rDAdV3-VP1-188 could induce protection against DAdV3 and DHAV1 in ducks, thus indicating the feasibility of DAdV3 as a vector for the development of avian vector vaccines. These insights contribute to the further development of DAdV3 vectors and other adenovirus vectors.

Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/ß-catenin signaling via interaction with P27.

Avian leukemia virus subgroup J (ALV-J) causes various diseases associated with tumor formation and decreased fertility and induced immunosuppressive disease, resulting in significant economic losses in the poultry industry globally. Virus usually exploits the host cellular machinery for their replication. Although there are increasing evidences for the cellular proteins involving viral replication, the interaction between ALV-J and host proteins leading to the pivotal steps of viral life cycle are still unclear. Here, we reported that ribonucleoside-diphosphate reductase subunit M2 (RRM2) plays a critical role during ALV-J infection by interacting with capsid protein P27 and activating Wnt/ß-catenin signaling. We found that the expression of RRM2 is effectively increased during ALV-J infection, and that RRM2 facilitates ALV-J replication by interacting with viral capsid protein P27. Furthermore, ALV-J P27 activated Wnt/ß-catenin signaling by promoting ß-catenin entry into the nucleus, and RRM2 activated Wnt/ß-catenin signaling by enhancing its phosphorylation at Ser18 during ALV-J infection. These data suggest that the upregulation of RRM2 expression by ALV-J infection favors viral replication in host cells via activating Wnt/ß-catenin signaling. IMPORTANCE Our results revealed a novel mechanism by which RRM2 facilitates ALV-J growth. That is, the upregulation of RRM2 expression by ALV-J infection favors viral replication by interacting with capsid protein P27 and activating Wnt/ß-catenin pathway in host cells. Furthermore, the phosphorylation of serine at position 18 of RRM2 was verified to be the important factor regulating the activation of Wnt/ß-catenin signaling. This study provides insights for further studies of the molecular mechanism of ALV-J infection.

Proteomic profiling of purified avian leukosis virus subgroup J particles.

While the presence of host cell proteins in virions and their role in viral life cycles have been demonstrated in various viruses, such characteristics have remained largely unknown in avian leukosis virus (ALV). To investigate whether this is the case in ALV, we purified high-integrity and high-purity virions from the avian leukosis virus subgroup J (ALV-J) and subjected them to proteome analysis using nano LC-MS/MS. This analysis identified 53 cellular proteins that are incorporated into mature ALV-J virions, and we verified the reliability of the packaged cellular proteins through subtilisin digestion and immunoblot analysis. Functional annotation revealed the potential functions of these proteins in the viral life cycle and tumorigenesis. Overall, our findings have important implications for understanding the interaction between ALV-J and its host, and provide new insights into the cellular requirements that define ALV-J infection.

Molecular characterization, complete genome sequencing, and pathogenicity of Novel Duck Reovirus from South Coastal Area in China.

Novel Duck Reovirus (NDRV) that has been found throughout the world in waterfowl, and it has been extensively described. Here, we report the complete genome sequence of a NDRV strain isolated in China called NDRV YF10. This strain was collected from 87 samples with infected ducks in South Coastal Area. The NDRV genome consists of 23,419 bp. With the assistance of computer analysis, the promoter and terminator of each gene segment and 10 viral genes segments were identified, which encode polypeptides ranging from 98 to 1,294 amino acids. All gene fragments of this virus strain were determined and compared to previously reported strains, revealing genetic variation with similarity rates ranging from 96 to 99% for each gene segment. Each gene segment formed 2 host-associated groups, the waterfowl-derived reovirus and the avian-derived reovirus, except for the S1 gene segment, which was closely related to ARV evolution and formed a host-independent subcluster. This difference may be due to Avian Reovirus (ARV) evolving in a host-dependent manner. In order to evaluate the pathogenicity of YF10, a novel isolated strain of NDRV was tested in 2 types of ducks. It was observed that the YF10 isolated strain exhibits varying degrees of virulence, highlighting the potential risk posed to different types of ducks. In conclusion, our findings emphasize the importance of epidemiology studies, molecular characterization, and prevention of NDRV in waterfowl.

Whole genome characterization of a multidrug-resistant hypervirulent Pasteurella multocida with a new drug-resistant plasmid.

Pasteurella multocida (P. multocida) is a zoonotic bacterium that can cause diseases in a variety of animals. It was divided into 5 serogroups, and serogroup A is mainly prevalent in avian hosts. We isolated a virulent and multidrug-resistant P. multocida strain from Guangdong duck liver and named it PMWSG-4 (GenBank accession no. CP077723.1). To understand the pathogenicity of this strain, the pathogenicity test was carried out with mice and ducks. The results showed that PMSWG-4 was highly pathogenic to ducks and mice, and the LD50 is 4.5 and 73 CFU, respectively. In order to study its genetic characteristics, pathogenicity, and relationship with the host, we performed a whole genome sequencing. The genome size of the isolated PMWSG-4 was 2.38 Mbp, with a G+C content of 40.3%, and coding 2,313 Coding DNA Sequence (CDS). The genome carries 162 potential virulence-associated genes, 32 different drug resistance phenotypes, 102 genes possibly involved in pathogen-host interaction, 2 gene island groups, and 4 prophages. In addition, we also found a new drug-resistant plasmid from strain PMWSG-4, named pXL001 (GenBank accession no. CP077724.1). After verified, the plasmid is a new plasmid carrying the floR florfenicol resistance gene. The whole genome is of great significance for further studying the pathogenesis and genetic characteristics of duck-derived P. multocida.

Duck plague virus infection alter the microbiota composition and intestinal functional activity in Muscovy ducks.

Intestinal damage from the duck plague virus (DPV) infection affects intestinal inflammation factors expression and barrier dysfunction. Here we report findings from the pathogenicity of the intestinal tract, intestinal morphological, intestinal permeability, inflammatory cytokines, and tight junction gene expression in 72 two-wk-old Muscovy ducks exposed to DPV. The characterization of intestinal metabolites and their classification were examined using 16-sequencing technology. The primary outcomes of the study evaluated the correlation between intestinal microbiota characteristics and the degree of infected tissue. The secondary outcomes were to determine whether the biosignatures that defined the microbiota were positively or negatively correlated with viral infection. The tissue was infected accompanied a mild damage of liver and spleen, and severe intestinal bleeding. Two inoculation routes were constructed with susceptible animals to assess the pathogenicity of the DPV in order to enrich the status of infection in Muscovy ducks. High levels of virus titer from Muscovy ducks were found being in the intestine. The expression of INF-α and IL-ß with viral infection increased at 4, and 6 dpi, respectively, after detecting of the inflammatory factor and barrier function genes. At 4 and 6 dpi, barrier function gene of ZO-1 and Occludin reduced. The severity of viral infection was significantly correlated with the characteristics of the intestinal microbiota. Ducks infected with the DPV had an increase in the phylum Firmicutes, a decrease in the phylum Actinobacteriota, and differential enrichment with the genus Bacteroides, Tyzzerella, Enterococcus, and Escherchia-Shigella, while the genus Rothia, Streptococcus, and Ralstonia were differentially enriched in the control group. The findings from the current study demonstrated that DPV infection leads to an imbalance of the intestinal microbiota and disruption of the microbial homeostasis in the intestinal tissue in ducks, which might be one of the mechanisms whereby DPV infection might be established in Muscovy ducks. Na+/K+-ATPase and Ca2+/Mg2+-ATPase activity monitoring also showed that viral infection reduced these activities. These findings imply that changes in intestinal microbiota, intestinal barrier gene expression, and inflammatory factor are related to viral infection. When taken as a whole, this work provides fresh perspectives on the characteristics of intestinal microbiota and the infection damage caused by the DPV.

Transcriptomic analysis reveals the dynamic changes of transcription factors during early development of chicken embryo.

BACKGROUND: The transition from fertilized egg to embryo in chicken requires activation of hundreds of genes that were mostly inactivated before fertilization, which is accompanied with various biological processes. Undoubtedly, transcription factors (TFs) play important roles in regulating the changes in gene expression pattern observed at early development. However, the contribution of TFs during early embryo development of chicken still remains largely unknown that need to be investigated. Therefore, an understanding of the development of vertebrates would be greatly facilitated by study of the dynamic changes in transcription factors during early chicken embryo. RESULTS: In the current study, we selected five early developmental stages in White Leghorn chicken, gallus gallus, for transcriptome analysis, cover 17,478 genes with about 807 million clean reads of RNA-sequencing. We have compared global gene expression patterns of consecutive stages and noted the differences. Comparative analysis of differentially expressed TFs (FDR < 0.05) profiles between neighboring developmental timepoints revealed significantly enriched biological categories associated with differentiation, development and morphogenesis. We also found that Zf-C2H2, Homeobox and bHLH were three dominant transcription factor families that appeared in early embryogenesis. More importantly, a TFs co-expression network was constructed and 16 critical TFs were identified. CONCLUSION: Our findings provide a comprehensive regulatory framework of TFs in chicken early embryo, revealing new insights into alterations of chicken embryonic TF expression and broadening better understanding of TF function in chicken embryogenesis.

Pathogenicity and transmissibility studies on live attenuated duck enteritis virus vaccine in non-target species.

In the second half of 2021, a highly pathogenic case occurred in a mixed chicken and duck family farm in Guangdong, China. After the duck flocks were immunized with live attenuated duck enteritis virus vaccine (live attenuated DEV vaccine), the chickens of the same farm showed clinical symptoms similar to duck enteritis, such as pericardial effusion, hepatic hemorrhagic spots, kidney enlargement, and intestinal bleeding, with mass mortality. The infection model of target animal tested, as well as the non-target species, was established according to the risk of live attenuated DEV vaccine and transmission in chickens. Live attenuated DEV vaccine was initially replicated in host animals, released the virus, and effectively colonized in the common environment, according to birds challenged experiments. There was evidence to suggest the mode of transmission of duck enteritis virus, and horizontal transmission is the main route of DEV transmission. In addition, high levels of virus titer were detected in chicken embryos and different tissues of SPF chickens. Different degrees of pathological damage occurred in the tissue of chickens. After the SPF chickens were inoculated with live attenuated DEV vaccine, different degrees of virulence were exhibited, pointing to a potential risk to other domestic bird species.

A new rapid and sensitive method for detecting chicken infectious anemia virus.

Since the chicken infectious anemia virus (CIAV) was discovered in 1979, which has been reported as an economically significant and immunosuppressive poultry disease in the world. A novel clinical detection method for the prevention and control of CIAV in the poultry sector is urgently needed. Here, we established a real-time recombinase-aided amplification assay (RAA) for CIAV on-site with a rapid, highly sensitive, strongly specific, low-cost, and simple operational molecular diagnosis detection method. The primers and probe were developed using the CIAV VP2 gene sequence, which has a 117-bp specific band. This assay, which could be carried out at 41°C and completed in 30 min without cross-reactivity with other viruses, had the lowest detection limit of 10 copies of CIAV DNA molecules per reaction. Furthermore, the kappa value of this assay was 0.947, the sensitivity was 93.33%, and the specificity was 100% when compared to the real-time quantitative polymerase chain reaction assay (real-time qPCR). These results indicate that using a real-time RAA assay to detect CIAV on-site could be beneficial. In the future, the real-time RAA test may be a regular assay for the prevention and control of CIAV, as well as help the reduction of economic losses in the poultry business.

Immunogenicity and protective efficacy of the recombinant Pasteurella multocida lipoproteins VacJ and PlpE, and outer membrane protein H from P. multocida A:1 in ducks.

Duck cholera (duck hemorrhagic septicemia) is a highly contagious disease caused by Pasteurella multocida, and is one of the major bacterial diseases currently affecting the duck industry. Type A is the predominant pathogenic serotype. In this study, the genes encoding the lipoproteins VacJ, PlpE, and the outer membrane protein OmpH of P. multocida strain PMWSG-4 were cloned and expressed as proteins in E. coli. The recombinant VacJ (84.4 kDa), PlpE (94.8 kDa), and OmpH (96.7 kDa) proteins were purified, and subunit vaccines were formulated with a single water-in-oil adjuvant, while killed vaccines were prepared using a single oil-coated adjuvant. Antibody responses in ducks vaccinated with recombinant VacJ, PlpE, and OmpH proteins formulated with adjuvants were significantly antigenic (p<0.005). Protectivity of the vaccines was evaluated via the intraperitoneal challenge of ducks with 20 LD50 doses of P. multocida A: 1. The vaccine formulation consisting of rVacJ, rPlpE, rOmpH, and adjuvant provided 33.3%, 83.33%, and 83.33% protection, respectively, the vaccine formulation consisting of three recombinant proteins, rVacJ, rPlpE, rOmpH and adjuvant, was 100% protective, and the killed vaccine was 50% protective. In addition, it was shown through histopathological examination and tissue bacterial load detection that all vaccines could reduce tissue damage and bacterial colonization to varying (p<0.001). These findings indicated that recombinant PlpE or OmpH fusion proteins formulated with oil adjuvants have the potential to be used as vaccine candidates against duck cholera subunits.

Efficacy of recombinant Newcastle disease virus expressing HA protein of H9N2 Avian influenza virus in respiratory and intestinal tract.

H9N2 subtype avian influenza virus (AIV) is a low pathogenic AIV, which is widely prevalent all over the world. The infection of H9N2 AIV often leads to secondary infection with other pathogens, causing serious economic losses to poultry industry. Up to now, several recombinant Newcastle disease viruses (NDV) expressing H9N2 AIV hemagglutinin (HA) protein had been developed. However, the efficacy of recombinant virus on tracheal and intestinal injury caused by H9N2 AIV was rarely reported. The aim of this study was to evaluate the efficacy of recombinant NDV expressing H9N2 AIV HA protein in respiratory and intestinal tract. In this study, based on Red/ET homologous recombination technology, H9N2 AIV HA gene was embedded into the genome of NDV LaSota vaccine strain to obtain the recombinant virus rNDV-H9. The recombinant virus rNDV-H9 showed similar replication kinetic characteristics with the parent LaSota strain and had good genetic stability. The immunization result showed that rNDV-H9 induced high HI antibody titer against H9N2 AIV. In the H9N2 AIV challenge experiment, rNDV-H9 could significantly reduce the virus shedding in trachea and cloaca. In addition, rNDV-H9 protected the barrier function of chicken intestinal mucosal epithelial cells and reduced the virus-induced inflammatory response to a certain extent, so as to inhibit the abnormal proliferation of E. coli. This study suggests that rNDV-H9 is a promising vaccine candidate against H9N2 AIV.

Synergistic Immunosuppression of Avian Leukosis Virus Subgroup J and Infectious Bursal Disease Virus Is Responsible for Enhanced Pathogenicity.

In recent years, superinfections of avian leukosis virus subgroup J (ALV-J) and infectious bursal disease virus (IBDV) have been frequently observed in nature, which has led to the increasing virulence in infected chickens. However, the reason for the enhanced pathogenicity has remained unclear. In this study, we demonstrated an effective candidate model for studying the outcome of superinfections with ALV-J and IBDV in cells and specific-pathogen-free (SPF) chicks. Through in vitro experiments, we found that ALV-J and IBDV can establish the superinfection models and synergistically promote the expression of IL-6, IL-10, IFN-α, and IFN-γ in DF-1 and CEF cells. In vivo, the weight loss, survival rate, and histopathological observations showed that more severe pathogenicity was present in the superinfected chickens. In addition, we found that superinfections of ALV-J and IBDV synergistically increased the viral replication of the two viruses and inflammatory mediator secretions in vitro and in vivo. Moreover, by measuring the immune organ indexes and blood proportions of CD3+, CD4+, and CD8α+ cells, our results showed that the more severe instances of immunosuppression were observed in the superinfected chickens. In the present study, we concluded that the more severe immunosuppression induced by the synergistic viral replication of ALV-J and IBDV is responsible for the enhanced pathogenicity.