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The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density lipoprotein (LDL-A) module and is involved in the uptake of transcobalamin bound vitamin B(12)/cobalamin (Cbl). To map the functional determinants of Tva responsible for ALV-A receptor activity, a series of chimeric receptors were created by swapping the LDL-A module fragments between huCD320 and Tva. These chimeric receptors were then used for virus entry and binding assays to map the minimal ALV-A functional domain of Tva. The results showed that Tva residues 49 to 71 constituted the minimal functional domain that directly interacted with the ALV-A gp85 protein to mediate ALV-A entry. Single-residue substitution analysis revealed that L55 and W69, which were spatially adjacent on the surface of the Tva structure, were key residues that mediate ALV-A entry. Structural alignment results indicated that L55 and W69 substitutions did not affect the Tva protein structure but abolished the interaction force between Tva and gp85. Furthermore, substituting the corresponding residues of huCD320 with L55 and W69 of Tva converted huCD320 into a functional receptor of ALV-A. Importantly, soluble huCD320 harboring Tva L55 and W69 blocked ALV-A entry. Finally, we constructed a Tva gene-edited cell line with L55R and W69L substitutions that could fully resist ALV-A entry, while Cbl uptake was not affected. Collectively, our findings suggested that amino acids L55 and W69 of Tva were key for mediating virus entry. IMPORTANCE Retroviruses bind to cellular receptors through their envelope proteins, which is a crucial step in infection. While most retroviruses require two receptors for entry, ALV-A requires only one. Various Tva alleles conferring resistance to ALV-A, including Tva(r1) (C40W substitution), Tva(r2) (frame-shifting four-nucleotide insertion), Tva(r3), Tva(r4), Tva(r5), and Tva(r6) (deletion in the first intron), are known. However, the detailed entry mechanism of ALV-A in chickens remains to be explored. We demonstrated that Tva residues L55 and W69 were key for ALV-A entry and were important for correct interaction with ALV-A gp85. Soluble Tva and huCD320 harboring the Tva residues L55 and W69 effectively blocked ALV-A infection. Additionally, we constructed gene-edited cell lines targeting these two amino acids, which completely restricted ALV-A entry without affecting Cbl uptake. These findings contribute to a better understanding of the infection mechanism of ALV-A and provided novel insights into the prevention and control of ALV-A.

Inclusion body hepatitis (IBH), hydropericardium syndrome, and gizzard erosion associated with fowl adenovirus (FAdV) infection have caused notable economic losses worldwide. In 2020, severe IBH was observed in a layer chicken farm in Hebei Province, China. Liver samples were collected from layer chickens with severe IBH and virus isolation was performed in LMH cells. DNA sequence and bioinformatics analyses were conducted to determine the phylogenetic relationship and the pathogenicity assay was conducted in specific-pathogen-free (SPF) chickens. HeB20 strain was isolated and identified as FAdV-8b, and the complete genome was successfully sequenced (GenBank No. OK188966). Although widespread recombination in clinical strains has been reported within FAdVs, HeB20 showed some novel characteristics, and did not show any recombination, highlighting that recombinant and non-recombinant FAdV-8b coexist in the clinic poultry industry. Finally, pathogenicity animal model of HeB20 was developed and showed severe IBH and 10% mortality. Collectively, a new FAdV-8b strain (HeB20) was isolated and responsible for the severe IBH in layer chickens. Complete genome of HeB20 was sequenced and valuable for future epidemiological investigations. HeB20 was capable of inducing severe IBH and 10% mortality in SPF chickens; this animal model provides a powerful tool for the future vaccine development.

Infectious bursal disease virus (IBDV) is one of the main threats to the poultry industry worldwide. Very virulent IBDV (vvIBDV) is a fatal virus strain that causes heavy mortality in young chicken flocks. Ca

Fowl adenovirus (FAdV) was first reported in Angara Goth, Pakistan, in 1987. For this reason, it is also known as “Angara disease.” It was later reported in China, Japan, South Korea, India, the United States, Canada, and other countries and regions, causing huge economic losses in the poultry industry worldwide. Notably, since June 2015, a natural outbreak of severe hydropericardium hepatitis syndrome (HHS), associated with a hypervirulent novel genotype FAdV-4 infection, has emerged in most provinces of China. The novel virus FAdV-4 spread rapidly and induced a 30-100% mortality rate, causing huge economic losses and threatening the green and healthy poultry breeding industry. Vaccines against FAdV-4, especially the emerging novel genotype, play a critical role and will be the most efficient tool for preventing and controlling HHS. Various types of FAdV-4 vaccines have been developed and evaluated, such as inactivated, live-attenuated, subunit, and combined vaccines. They have made great contributions to the control of HHS, but the details of cross-protection within FAdVs and the immunogenicity of different vaccines require further investigation. This review highlights the recent advances in developing the FAdV-4 vaccine and promising new vaccines for future research.

Newcastle disease (ND) is one of the most important avian diseases that seriously threaten the poultry industry worldwide. Sometimes vaccination might not effectively reduce ND occurrence in some poultry farms for unclear reasons. Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases, and the novel variant IBD virus (IBDV) is threatening chicken farms in China. This study evaluated the influence of the novel variant IBDV (SHG19 strain) on immunization of ND vaccine (LaSota strain) in broiler and layer chickens. In commercial broilers, the hemagglutination inhibition antibody titers against LaSota strain were decreased by the prior infection of the novel variant IBDV. Pathological examination revealed that the novel variant IBDV severely damaged the key immune organs of the bursa and spleen, and the B lymphocytes in the bursa were severely destroyed, which was the primary reason involved in the immunosuppression on ND vaccination. Moreover, the novel variant IBDV dramatically reduced the BW of infected broilers by about 16% compared to that of control, which indicated huge economic losses. Furthermore, this study confirmed the immunosuppression induced by the novel variant IBDV in specific pathogen-free layer chickens. In this study, it was discovered that the novel variant IBDV could interfere with ND vaccination in both broilers and layers, which was one important factor involved in immune failure in poultry farms. This study therefore suggests the urgency to control the novel variant IBDV infection for healthy breeding.

Marek's disease virus (MDV), an oncogenic virus belonging to the subfamily Alphaherpesvirinae, causes Marek's disease (MD). Vaccines can control MD but cannot block the viral infection; they are considered imperfect vaccines, which carry the risk of recombination. In this study, six natural recombinant MDV strains were isolated from infected chickens in commercial flocks in China. We sequenced and analysed the genetic characteristics of the isolates (HC/0803, CH/10, SY/1219, DH/1307, DH/1504 and Hrb/1504). We found that the six strains resulted from recombination between the vaccine CVI988/Rispens (CVI988) strain skeleton and the virulence strain's partial unique short region. Additionally, a pathogenicity study was performed on recombinant strains (HC/0803 and DH/1307) and reference strains (CVI988 and LHC2) to evaluate their virulence. LHC2 induced 84.6% mortality in infected chickens; however, no mortality was recorded in chickens inoculated with HC/0803, DH/1307 or CVI988. However, HC/0803 and DH/1307 induced a notable spleen enlargement, and mild thymus and bursa atrophy at 11-17 days post-challenge (dpc). The viral genome load in the HC/0803- and DH/1307-challenged chickens peaked at approximately 10

Avian leukosis virus subgroup J (ALV-J) is an important pathogen for various neoplasms and causes significant economic losses in the poultry industry. Serological detection of specific antibodies against ALV-J infection is important for successful clinical diagnosis. Here, a 293F stable cell line was established to stably express gp85 protein. In this cell line, gp85 protein was expressed at approximately 30 mg/L. A subgroup-specific indirect enzyme-linked immunosorbent assay (iELISA) was developed using ALV-J gp85 protein as coated antigen to detect antibodies against ALV-J. The sensitivity of the iELISA (1:51200 diluted in serum) was 16 times more than that of indirect immunofluorescence assay (IFA; 1:3200 diluted in serum). Moreover, there was no crossreactivity with antibodies against other common avian viruses and other avian leukosis virus subgroups, such as subgroups A and B. The practicality of the iELISA was further evaluated by experimental infection and clinical samples. The results from experimental infection indicated that anti-ALV-J antibodies were readily detected by iELISA as early as 4 weeks after ALV-J infection, and positive antibodies were detected until 20 weeks, with an antibody-positive rate of 11.1% to 33.3%. Moreover, analysis of clinical samples showed that 9.49% of samples were positive for anti-ALV-J antibodies, and the concordance rate of iELISA and IFA was 99.24%. Overall, these results suggested that the subgroup-specific iELISA developed in this study had good sensitivity, specificity, and feasibility. This iELISA will be very useful for epidemiological surveillance, diagnosis, and eradication of ALV-J in poultry farms.

Since 2015, outbreaks of hepatitis-hydropericardium syndrome (HPS) caused by a novel genotype of fowl adenovirus 4 (FAdV-4) infection have created serious economic losses in China. Given that other serotypes of hypervirulent FAdVs have also been reported in poultry around the world, a common ELISA for all serotypes within the group I fowl adenoviruses (FAdV-I) is urgently needed, especially for clinical epidemic serotypes. In this study, we used high purity and concentration virions of FAdV-4 and developed a common ELISA for detecting antibodies against 12 FAdV-I serotypes. The developed ELISA was able to distinguish between antibodies against FAdV-I, FAdV-III, and other heterologous viruses without any cross-reaction. Furthermore, the ELISA showed higher sensitivity than the FAdV-1-based ELISA to the novel FAdV-4 found in China. Moreover, since there are no commercial vaccines against FAdVs in China, the ELISA was applied to detect sera samples from specific pathogen-free chickens inoculated with inactivated FAdV-1, FAdV-4, and FAdV-8a. The assay showed high sensitivities for all three detected serotypes within FAdV-I. In conclusion, a novel, common ELISA for FAdV-I was developed in this study and could be a powerful tool for seroepidemiological investigations and FAdVs vaccine development.

The emerging hepatitis-hydropericardium syndrome (HHS) caused by the novel genotype of fowl adenovirus 4 (FAdV-4) and the infectious bursal disease (IBD) caused by the infectious bursal disease virus (IBDV) are important avian diseases, both cause huge economic losses to the poultry industry. Therefore, it is necessary to develop an efficient and convenient FAdV-4/IBDV bivalent vaccine to prevent the spread of FAdV-4 and IBDV infections. Given that VP2 is the main structural protein and protective antigen of IBDV, we constructed a recombinant FAdV-4 expressing IBDV VP2 in our previous study. In the current study, an inactivated bivalent FAdV-4/IBDV vaccine was developed from the recombinant strain. The inactivated bivalent vaccine elicited effective and specific neutralizing antibodies against both FAdV-4 and IBDV in specific-pathogen-free chickens. Furthermore, the novel vaccine not only protected chickens from death caused by FAdV-4 and the very virulent IBDV (vvIBDV) infection, but also ameliorated target organ damage and reduced viral load. The FAdV-4-vectored vaccine developed in this study provides new options for the development of avian polyvalent inactivated vaccines and is a powerful tool for the prevention of both emerging HHS and IBD.

In recent years, hepatitis-hydropericardium syndrome (HHS), caused by novel fowl adenovirus 4 (FAdV-4), has caused serious economic losses to the poultry industry. Vaccines are important for preventing and controlling HHS. Current FAdV-4 vaccine research and development are mainly focuses on inactivated vaccines and relatively fewer live vaccines. We previously demonstrated that the hexon gene is the key gene responsible for the high pathogenicity of FAdV-4 and constructed a non-pathogenic chimeric virus rHN20 strain based on the emerging FAdV-4. In this study, the immunogenicity of artificially rescued rHN20 was evaluated in chickens using different routes and doses as a live vaccine. The live rHN20 vaccine induced high titers of neutralizing antibodies against FAdV-4 and fully protected the immunized chickens against a lethal dose of FAdV-4. Furthermore, immunized chickens showed no clinical symptoms or histopathological changes in the FAdV-4-targeted liver, and the viral load in the tissues of immunized chickens was significantly lower than that of chickens in the challenge control group. Collectively, the live rHN20 vaccine effectively protected our sample against FAdV-4 infection and can be considered a live vaccine candidate for preventing HHS in the poultry industry.

Hepatitis-hydropericardium syndrome (HHS) in birds is mainly caused by virulent fowl adenovirus 4 (FAdV-4). A novel genotype, hypervirulent FAdV-4, emerged in 2015 with a high mortality rate ranging from 30 % to 100 % in chickens. Vaccination is an economically feasible method to control HHS. Although there have been various reports of inactivated vaccines from virulent wild-type FAdV-4 against HHS, biosafety threats of inactivated vaccines from potential pathogenic components have been presented to the poultry industry, and safer vaccines are urgently needed. A non-pathogenic recombinant FAdV-4 strain, designated as rHN20, was generated based on the hypervirulent strain in our previous study. Here, we developed a novel inactivated oil-adjuvanted vaccine derived from rHN20 strain and evaluated its immunogenicity in specific-pathogen-free chickens. Chickens subcutaneously or intramuscularly immunized with the inactivated vaccine produced high titers of neutralizing antibodies and were protected from a lethal dose of virulent wild-type FAdV-4 challenge. Collectively, an inactivated vaccine was developed, which was capable of providing full protection for chickens against HHS, and significantly reduced the potential biosafety threats.

In this study, we expressed rAvBD1-2–6-13 protein throughLactococcus lactisNZ3900, and the effects of the recombinantL. lactisNZ3900 as an immune enhancer and immune adjuvant were verified using in vivo and in vitro tests. In vitro tests revealed that recombinantL. lactisNZ3900 significantly activated the NF-κB signaling pathway and IRF signaling pathway in J774-Dual™ report cells and significantly increased the transcript levels of IL-10, IL-12p70, CD80, and CD86 in chicken PBMCs and chicken HD11 cells. In vivo experiments revealed that the immunized group supplemented with recombinantL. lactisNZ3900 as an adjuvant had significantly higher serum antibody titers and higher proliferative activity of PBMCs in the blood of the chickens immunized with NDV live and inactivated vaccines. Our study shows that the recombinantL. lactisNZ3900 has strong immunomodulatory activity both in vivo and in vitro and is a potential immune enhancer. Our work lays the foundation for the research and development of new animal immune enhancers for application in the poultry industry.

Infectious bursal disease virus (IBDV) is a non-enveloped, bi-segmented double-stranded RNA virus and the causative agent of a poultry immunosuppressive disease known as infectious bursal disease (IBD). The novel variant IBDV (nVarIBDV) recently posed a great threat to the development of the poultry industry. In this study, we identified a novel segment-reassortant IBDV strain, IBDV-JS19-14701 (Genotype A2dB3). Phylogenic analysis showed that Segments A and B of IBDV-JS19-14701 were derived from emerging nVarIBDV (Genotype A2dB1) and long-prevalent HLJ0504-like strains (Genotype A3B3) in China, respectively. The pathogenicity of IBDV-JS19-14701 was further evaluated via animal experiments. IBDV-JS19-14701 exhibited a similar virulence to chickens with the nVarIBDV. The identification of this reassortment event is beneficial for understanding the epidemiology of nVarIBDV and will contribute to the efficient prevention and control of IBD.

Since 2015, severe hydropericardium-hepatitis syndrome (HHS) associated with a novel fowl adenovirus 4 (FAdV-4) has emerged in China, representing a new challenge for the poultry industry. Although various highly pathogenic FAdV-4 strains have been isolated, the virulence factor and the pathogenesis of novel FAdV-4 are unclear. In our previous studies, we reported that a large genomic deletion (1,966 bp) is not related to increased virulence. Here, two recombinant chimeric viruses, rHN20 strain and rFB2 strain, were generated from a highly pathogenic FAdV-4 strain by replacing the hexon or fiber-2 gene of a nonpathogenic FAdV-4, respectively. Both chimeric strains showed similar titers to the wild-type strain in vitro. Notably, rFB2 and the wild-type strain induced 100% mortality, while no mortality or clinical signs appeared in chickens inoculated with rHN20, indicating that hexon, but not fiber-2, determines the novel FAdV-4 virulence. Furthermore, an R188I mutation in the hexon protein identified residue 188 as the key amino acid for the reduced pathogenicity. The rR188I mutant strain was significantly neutralized by chicken serum in vitro and in vivo, whereas the wild-type strain was able to replicate efficiently. Finally, the immunogenicity of the rescued rR188I was investigated. Nonpathogenic rR188I provided full protection against lethal FAdV-4 challenge. Collectively, these findings provide an in-depth understanding of the molecular basis of novel FAdV-4 pathogenicity and present rR188I as a potential live attenuated vaccine candidate or a novel vaccine vector for HHS vaccines. IMPORTANCE HHS associated with a novel FAdV-4 infection in chickens has caused huge economic losses to the poultry industry in China since 2015. The molecular basis for the increased virulence remains largely unknown. Here, we demonstrate that the hexon gene is vital for FAdV-4 pathogenicity. Furthermore, we show that the amino acid residue at position 188 of the hexon protein is responsible for pathogenicity. Importantly, the rR188I mutant strain was neutralized by chicken serum in vitro and in vivo, whereas the wild-type strain was not. Further, the rR188I mutant strain provided complete protection against FAdV-4 challenge. Our results provide a molecular basis of the increased virulence of novel FAdV-4. We propose that the rR188I mutant is a potential live attenuated vaccine against HHS and a new vaccine vector for HHS-combined vaccines.

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, causes immunosuppression and high mortality in 3–6-week-old chickens. Innate immune defense is a physical barrier to restrict viral replication. After viral infection, the host shows crucial defense responses, such as stimulation of antiviral effectors to restrict viral replication. Here, we conducted RNA-seq in avian cells infected by IBDV and identified TRIM25 as a host restriction factor. Specifically, TRIM25 deficiency dramatically increased viral yields, whereas overexpression of TRIM25 significantly inhibited IBDV replication. Immunoprecipitation assays indicated that TRIM25 only interacted with VP3 among all viral proteins, mediating its K27-linked polyubiquitination and subsequent proteasomal degradation. Moreover, the Lys854 residue of VP3 was identified as the key target site for the ubiquitination catalyzed by TRIM25. The ubiquitination site destroyed enhanced the replication ability of IBDV in vitro and in vivo. These findings demonstrated that TRIM25 inhibited IBDV replication by specifically ubiquitinating and degrading the structural protein VP3., Author summary Owing to the immunosuppression and high mortality caused by infectious bursal disease virus (IBDV) in chickens, hosts urgently boost their antiviral immune responses to resist IBDV infection. Here, we identified the host antiviral factor TRIM25, which defended against IBDV infection by direct targeting the viral protein VP3 for subsequent ubiquitination and degradation. To our knowledge, this is the first report of host TRIM25 restricting IBDV replication. Additionally, we found that Lys854 of VP3 was the target site ubiquitinated by TRIM25. Mutation in the ubiquitination site of VP3 abrogated the degradation of VP3 and further enhanced IBDV replication, but did not influence its virulence. Our work not only uncovers an all-important molecular mechanism of chicken TRIM25 against IBDV but also provides new insights for developing high-titer live IBDV vaccines.

Recent reports of infectious bursal disease virus (IBDV) infections in China, Japan, and North America have indicated the presence of variant, and the current conventional IBDV vaccine cannot completely protect against variant IBDV. In this study, we constructed recombinant Lactococcus lactis (r-L. lactis) expressing a novel variant of IBDV VP2 (avVP2) protein along with the Salmonella resistance to complement killing (RCK) protein, and Western blotting analysis confirmed that r-L. lactis successfully expressed avVP2-RCK fusion protein. We immunized chickens with this vaccine and subsequently challenged them with the very virulent IBDV (vvIBDV) and a novel variant wild IBDV (avIBDV) to evaluate the immune effect of the vaccine. The results show that the r-L. lactis-avVP2-RCK-immunized group exhibited a 100% protection rate when challenged with avIBDV and 100% survival rate to vvIBDV. Furthermore, this immunization resulted in the production of unique neutralizing antibodies that cannot be detected by conventional ELISA. These results indicate that r-L. lactis-avVP2-RCK is a promising candidate vaccine against IBDV infections, which can produce unique neutralizing antibodies that cannot be produced by other vaccines and protect against IBDV infection, especially against the variant strain.

Chicken infectious anemia (CIA), caused by chicken anemia virus (CAV), is an important immunosuppressive disease that seriously threatens the global poultry industry. Here, we isolated and identified 30 new CAV strains from CAV-positive flocks. The VP1 genes of these strains were sequenced and analyzed at the nucleotide and amino acid levels and were found to have very similar nucleotide sequences ( 97% identity); however, they showed 93.9-100.0% sequence identity to the VP1 genes of 55 reference strains. Furthermore, alignment of the deduced amino acid sequences revealed some unique mutations. Phylogenetic analysis indicated the division of VP1 amino acid sequences into two groups (A and B) and four subgroups (A1, A2, A3 and A4). Interestingly, 22 of the newly isolated strains and some Asian reference strains belonged to the A1 group, whereas the remaining eight new isolates belonged to the A3 group. To evaluate the pathogenicity of the epidemic CAV strains from China, the representative strains CAV-JL16/8901 and CAV-HeN19/3001 and the reference strain Cux-1 were selected for animal experiments. Chickens infected with the isolates and reference strain all showed thymus atrophy and bone marrow yellowing. The mortality rates for CAV-JL16/8901, CAV-HeN19/3001, and the reference strain was 30%, 20%, and 0%, respectively, indicating that the epidemic strains pose a more serious threat to chickens. We not only analyzed the molecular evolution of the epidemic strains but also showed for the first time that the epidemic strains in China are more pathogenic than reference strain Cux-1. Effective measures should be established to prevent the spread of CIA in China.

Subgroup J avian leukemia virus (ALV-J), belonging to the genus Alpharetrovirus, enters cells through its envelope surface unit (gp85) via specifically recognizing the cellular receptor chicken Na(+)/H(+) exchanger type I (chNHE1), the 28 to 39 N-terminal residues of which were characterized as the minimal receptor functional domain in our previous studies. In this study, to further clarify the precise organization and properties of the interaction between ALV-J gp85 and chNHE1, we identified the chNHE1-binding domain of ALV-J gp85 using a series of gp85 mutants with segment substitutions and evaluating their effects on chNHE1 binding in protein-cell binding assays. Our results showed that hemagglutinin (HA) substitutions of amino acids (aa) 38 to 131 (N terminus of gp85) and aa 159 to 283 (C terminus of gp85) significantly inhibited the interaction between gp85 and chNHE1/chNHE1 loop 1. In addition, these HA-substituted chimeric gp85 proteins could not effectively block the entry of ALV-J into chNHE1-expressing cells. Furthermore, analysis of various N-linked glycosylation sites and cysteine mutants in gp85 revealed that glycosylation sites (N6 and N11) and cysteines (C3 and C9) were directly involved in receptor-gp85 binding and important for the entry of ALV-J into cells. Taken together, our findings indicated that the bipartite sequence motif, spanning aa 38 to 131 and aa 159 to 283, of ALV-J gp85 was essential for binding to chNHE1, with its two N-linked glycosylation sites and two cysteines being important for its receptor-binding function and subsequent viral infection steps. IMPORTANCE Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of envelope (Env) surface proteins to cell receptors is a key step in triggering infections and has been the target of antiviral drug screening. ALV-J is an economically important avian pathogen that belongs to the genus Alpharetrovirus and has a wider host range than other ALV subgroups. Our results showed that the amino acids 38 to 131 of the N terminus and 159 to 283 of the C terminus of ALV-J gp85 controlled the efficiency of gp85 binding to chNHE1 and were critical for viral infection. In addition, the glycosylation sites (N6 and N11) and cysteines (C3 and C9) of gp85 played a crucial role in the receptor binding and viral entry. These findings might help elucidate the mechanism of the entry of ALV-J into host cells and provide antiviral targets for the control of ALV-J.

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is the most important immunosuppressive disease threatening the poultry industry worldwide. Recently, the novel variant IBDV has been emerging in large-scale in Asia including China and is becoming a new threat to the healthy development of the poultry industry, but no ideal vaccine is available. Therefore, it is necessary and urgent to develop a new vaccine against the novel variant IBDV. In this study, based on the skeleton of an attenuated vaccine strain Gt, a reassortment virus strain rGtVarVP2 was constructed for the first time, which could express the main protective antigen VP2 of the novel variant IBDV and replicate well in cell culture. Subsequently, the safety and effectiveness of rGtVarVP2 were further evaluated using animal experiments. The rGtVarVP2 is nonpathogenic to specific-pathogen-free (SPF) chicken. The immunization of rGtVarVP2 could induce the specific neutralizing antibodies against the novel variant IBDV. The challenge protection tests further confirmed the effectiveness of the IBDV reassortment virus rGtVarVP2. No atrophy and obvious lesions were observed in the immunization group while the bursae of non-immunization control group were severely destroyed after challenge, which showed that rGtVarVP2 could provide complete protection against the novel variant IBDV. These data indicate that the vaccine candidate (rGtVarVP2 strain) is safe and effective, which is of great significance for comprehensive control of IBD and healthy breeding.

Reticuloendotheliosis is an important immunosuppressive disease, associated with avian reticuloendotheliosis virus (REV) infection, and causes notable economic losses worldwide. Glycoprotein gp90 is an important structural protein of REV, and considered to be the most important immunogenic antigen, which can induce neutralizing antibodies against REV. In this study, an optimized suspension culture system was developed and applied to secretory express the immunogenic surface antigen gp90. To achieve an optimal glycosylation, the gp90 was designed to secretory expressed into the supernatant of the cell culture, which also occurs in the natural protein maturation procedure of REV. Serum-free culture medium was introduced to simplify the purification process and reduce the production costs. Based on the purified glycosylated gp90, an oil-emulsion subunit REV vaccine candidate was developed and evaluated in chickens. The subunit gp90-based vaccine induced fast immune responses, high levels of antibodies (REV-specific antibody, gp90-specific antibody, and neutralizing antibody against REV), and preferential T helper 2 (Th2) (interleukin-4 secretion) not Th1 (interferon-γ secretion) response. Furthermore, the viremia induced by REV infection was significantly reduced in chickens immunized with the glycosylated gp90. Overall, an optimized secretory expression system for glycosylated gp90 was developed, and the glycosylated gp90 obtained in this study retained good immunogenicity and could be an attractive vaccine candidate to protect chickens against REV horizonal infection.

Infectious bursal disease (IBD) is an immunosuppressive, highly contagious, and lethal disease of young chickens caused by IBD virus (IBDV). It results in huge economic loss to the poultry industry worldwide. Infection caused by very virulent IBDV (vvIBDV) strains results in high mortality in young chicken flocks. However, the replication characteristics of vvIBDV are not well studied. Publications have shown that virus protein 3 (VP3) binds to VP1 and viral double-stranded RNA, and together they form a ribonucleoprotein complex that plays a key role in virus replication. In this study, vvIBDV VP3 was used to identify host proteins potentially involved in modulating vvIBDV replication. Chicken eukaryotic translation elongation factor 1&alpha, (cheEF1&alpha, ) was chosen to further investigate effects on vvIBDV replication. By small interfering RNA-mediated cheEF1&alpha, knockdown, we demonstrated the possibility of significantly reducing viral polymerase activity, with a subsequent reduction in virus yields. Conversely, over-expression of cheEF1&alpha, significantly increased viral polymerase activity and virus replication. Further study confirmed that cheEF1&alpha, interacted only with vvIBDV VP3 but not with attenuated IBDV (aIBDV) VP3. Furthermore, the amino acids at the N- and C-termini were important in the interaction between vvIBDV VP3 and cheEF1&alpha, Domain III was essential for interactions between cheEF1&alpha, and vvIBDV VP3. In summary, cheEF1&alpha, enhances vvIBDV replication by promoting the activity of virus polymerase. Our study indicates cheEF1&alpha, is a potential target for limiting vvIBDV infection.

Since subgroup J avian leukosis virus (ALV-J) was first isolated in the United Kingdom in 1988, it has seriously hindered the development of the poultry industry worldwide. Although cases of ALV-J infection have been reported as early as 2001 in Pakistan, there was no further research on the isolation and molecular characteristics of ALVs. In the present study, we first isolated two ALVs from suspicious clinical samples that were collected from a desi chicken farm in Pakistan. The results of multiplex PCR and indirect immunofluorescent antibody assays confirmed that the two isolates (PK19FA01 and PK19SA01) belonged to ALV-J. The complete genomes of the two isolates were amplified, sequenced, and systematically analyzed. We found that gp85 of PK19FA01 was more similar to that of the prototype strain HPRS103, whereas gp85 of PK19SA01 was more similar to that of American strains. The two isolates contained an intact E element of 147 residues and had a unique 135 bp deletion in the redundant transmembrane of the 3′ untranslated region. The U3 region of the two isolates was highly homologous to that of American ALV-J strains. To our knowledge, this is the first report of the isolation, complete genome sequencing, and systematic molecular epidemiological investigation of ALV-J in Pakistan. Our findings could enrich epidemiological data and might contributed to more effective measures to prevent and control avian leukosis in Pakistan.

Avian leukosis virus subgroup J (ALV-J) was first isolated from broiler chickens in China in 1999; subsequently, it was rapidly introduced into layer chickens and Chinese local chickens. Recently, the incidence of ALV-J in broiler and layer chickens has significantly decreased. However, it has caused substantial damage to Chinese local chickens, resulting in immense challenges to their production performance and breeding safety. To systematically analyze the molecular characteristics and the epidemic trend of ALV-J in Chinese local chickens, 260 clinical samples were collected for the period of 2013–2018; 18 ALV-J local chicken isolates were identified by antigen-capture enzyme-linked immunosorbent assay and subgroup A-, B-, and J-specific multiplex PCR. The whole genomic sequences of 18 isolates were amplified with PCR and submitted to GenBank. Approximately, 55.5% (10/18) of the 18 isolates demonstrated a relatively high homology (92.3–95.4%) with 20 ALV-J early-isolated local strains (genome sequences obtained from GenBank) in gp85 genes clustering in a separated branch. The 3ʹ untranslated region (3ʹ UTR) of the 18 isolates showed a 195–210 and 16–28 base pair deletion in the redundant transmembrane region and in direct repeat 1, respectively; 55.5% (10/18) of the 18 isolates retained the 147 residue E element. The U3 gene of 61.1% (11/18) of the 18 isolates shared high identity (94.6–97.3%) with ALV-J early-isolated local strains. These results implied that the gp85 and U3 of ALV-J local chicken isolates have rapidly evolved and formed a unique local chicken branch. In addition, it was determined that the gene deletion in the 3′UTR region currently serves as a unique molecular characteristic of ALV-J in China. Hence, the obtained results built on the existing ALV-J molecular epidemiological data and further elucidated the genetic evolution trend of ALV-J in Chinese local chickens.

The CRISPR/CRISPR-associated protein 9 (Cas9) system is a powerful gene-editing tool originally discovered as an integral mediator of bacterial adaptive immunity. Recently, this technology has been explored for its potential utility in providing new and unique treatments for viral infection. Marek's disease virus (MDV) and avian leukosis virus subgroup J (ALV-J), major immunosuppressive viruses, cause significant economic losses to the chicken industry. Here, we evaluated the efficacy of using MDV as a CRISPR/Cas9-delivery system to directly target and disrupt the reverse-transcribed products of the ALV-J RNA genome during its infection cycle in vitro and in vivo. We first screened multiple potential guide RNA (gRNA) target sites in the ALV-J genome and identified several optimized targets capable of effectively disrupting the latently integrated viral genome and providing efficient defense against new infection by ALV-J in cells. The optimal single-gRNAs and Cas9-expression cassettes were inserted into the genome of an MDV vaccine strain. The results indicated that engineered MDV stably expressing ALV-J-targeting CRISPR/Cas9 efficiently resisted ALV-J challenge in host cells. These findings demonstrated the CRISPR/Cas9 system as an effective treatment strategy against ALV-J infection. Furthermore, the results highlighted the potential of MDV as an effective delivery system for CRISPR/Cas9 in chickens.

Infectious bursal disease virus (IBDV), the etiological agent of infectious bursal disease (IBD), is a variable RNA virus of Avibirnavirus. Some artificially attenuated vaccine strains of IBDV can adapt to cell culture of chicken embryo fibroblast (CEF) cell or its immortalized cell line DF1 in vitro while wild-type IBDV cannot. In this study, for the first time, a naturally occurring cell-adapted classic strain (genogroup 1) of IBDV named IBD17JL01 was identified in China. Animal experiments showed that IBD17JL01 could severely damage the central immune organ of infected chickens. Sequence analysis of the full-length genome revealed the peculiar molecular characteristics of IBD17JL01 with a few amino acid substitutions that might be involved in cell-tropism, antigenicity, and virulence of IBDV. Identification of this novel strain is beneficial to our understanding of the complexity of the epidemiology of IBDV. And the expansion of viral cell-tropism might increase the potential risk of the reassortment of different IBDVs including the live vaccines.

Infectious bursal disease virus (IBDV) is an important member of the Birnaviridae family, causing severe immunosuppressive disease in chickens. The major capsid protein VP2 is responsible for the binding of IBDV to the host cell and its cellular tropism. In order to find proteins that potentially interact with IBDV VP2, a liquid chromatography-mass spectrometry (LC-MS) assay was conducted, and the host chicken CD74 protein was identified. Here, we investigate the role of chicken CD74 in IBDV attachment. Coimmunoprecipitation assays indicated that the extracellular domain of CD74 interacted with the VP2 proteins of multiple IBDV strains. Knockdown and overexpression experiments showed that CD74 promotes viral infectivity. Confocal assays showed that CD74 overexpression allows the attachment of IBDV and subvirus-like particles (SVPs) to the cell surface of nonpermissive cells, and quantitative PCR (qPCR) analysis further confirmed the attachment function of CD74. Anti-CD74 antibody, soluble CD74, depletion of CD74 by small interfering RNA (siRNA), and CD74 knockdown in the IBDV-susceptible DT40 cell line significantly inhibited IBDV binding, suggesting a pivotal role of this protein in virus attachment. These findings demonstrate that CD74 is a novel important receptor for IBDV attachment to the chicken B lymphocyte cell line DT40. IMPORTANCE CD74 plays a pivotal role in the correct folding and functional stability of major histocompatibility complex class II (MHC-II) molecules and in the presentation of antigenic peptides, acting as a regulatory factor in the antigen presentation process. In our study, we demonstrate a novel role of CD74 during IBDV infection, showing that chicken CD74 plays a significant role in IBDV binding to target B cells by interacting with the viral VP2 protein. This is the first report demonstrating that CD74 is involved as a novel attachment receptor in the IBDV life cycle in target B cells, thus contributing new insight into host-pathogen interactions.

Avian leukosis virus subgroup J (ALV-J), a highly oncogenic retrovirus, causes leukemia-like proliferative diseases in chickens. microRNAs post-transcriptionally suppress targets and are involved in the development of various tumors. We previously showed that miR-221 is upregulated in ALV-J-induced tumors. In this study, we analyzed the possible function of miR-221 in ALV-J tumorigenesis. The target validation system showed that CDKN1B is a target of miR-221 and is downregulated in ALV-J infection. As CDKN1B arrests the cell cycle and regulates its progression, we analyzed the proliferation of ALV-J-infected DF-1 cells. ALV-J-infection-induced DF1 cell derepression of G1/S transition and overproliferation required high miR-221 expression followed by CDKN1B downregulation. Cell cycle pathway analysis showed that ALV-J infection induced DF-1 cell overproliferation via the CDKN1B-CDK2/CDK6 pathway. Thus, miR-221 may play an important role in ALV-J-induced aggressive growth of DF-1 cells; these findings have expanded our insights into the mechanism underlying ALV-J infection and tumorigenesis.

Infectious bursal disease virus (IBDV), the causative agent of infectious bursal disease (IBD), mainly damages the bursa of Fabricius, which is a central immune organ of birds. As an RNA virus, IBDV is prone to mutation owing to a combination of factors including natural selection pressure. In this study, a naturally occurring mutated IBDV associated with bursa damage was identified, IBDV-HeN20-7103 strain, in an infected chicken flock in central China. Its full-length genome was cloned, and sequence analysis showed that the IBDV-HeN20-7103 strain was located along with the attenuated IBDV, which corresponds to genotype A8B1 of the recently proposed classification scheme, on the branch of the phylogenetic tree. The amino acid sequence comparisons further highlighted the specific characteristics of IBDV-HeN20-7103 with mutation H253Q compared to the attenuated strain. Animal experiments showed that IBDV-HeN20-7103 could induce serious bursal lesions without mortality, which revealed a unique cause of disease in this flock. The identification of such a strain reaffirms the complexity of IBDV evolution and prevalence.

In recent years, atypical infectious bursal disease (IBD) with severe immunosuppression has brought new threats to the poultry industry and has caused considerable economic losses. Novel variant infectious bursal disease virus (IBDV) has been identified as the etiological pathogen and for unknown reasons is widespread in poultry on many chicken farms in China that have been immunized with vaccines against very virulent IBDV (vvIBDV). Using immunoprotection experiments in specific-pathogen-free chickens, we first verified that novel variant IBDV could severely damage the bursa of Fabricius of the important immune organ of immunized chicken in the presence of antibodies induced by three types of vvIBDV vaccines, which is a primary reason for the current epidemic of atypical IBD. Monoclonal antibody reactivity patterns and cross-neutralization assays further confirmed the obvious antigenic mismatch between novel variant IBDV and vvIBDV. Sequence analysis of the genome of novel variant IBDV (SHG19 strain) was performed and the key amino acid residues that might be involved in antigenicity and virulence differences of novel variant IBDV compared to vvIBDV were further analyzed. This study not only determined the primary reason for the atypical IBD epidemic, but also remind us of the urgency for developing new vaccines against novel variant IBDV.

Marek's disease (MD) is a highly contagious lymphoproliferative poultry disease caused by the oncogenic herpesvirus, Marek's disease virus (MDV). MDV strains have shown a continued evolution of virulence leading to immune failure, and MD cases continue to occur. Co-infection of virulent MDV strains is an important factor leading to viral evolution and host immune failure. This study conducted a laboratory diagnosis and analysis of a MDV infected flock. Testing showed that all samples were MDV positive. PCR detection identified a variable 132-base pair repeat (132-bpr) sequence copy number. This indicated that two virulent strains of MDV were co-infecting the flock. Therefore, we performed homology, sequence alignment, and phylogenetic tree analysis of MDV variant genes including meq, pp38, and RLORF4. Two MDV strains had co-infected the flock; one was the 132bpr two-copy characteristic strain (AH2C) and the other was a 132bpr three-copy characteristic strain (AH3C). Specific mutations in AH3C were found, suggesting that it is a new variant strain. Furthermore, the viral load of the two strains in vivo indicated that both strains had high and similar replication ability. There was no significant difference in the proportion of positive samples of the two strains causing disease. In the whole flock, neither strain displayed an obvious advantage. However, there was a dominant strain in individual chickens, with the exception of one sample. This study reported the co-infection regularity of two virulent MDV strains in the same flock, and even in the same chicken in field conditions. In the context of overall epidemiology, this study is a useful reference.

Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3&prime, untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis.

Background Infectious bursal disease (IBD) is an acute contagious immunosuppressive disease which lead to acute bursal injury and immune dysfunction in poultry. It has caused heavy economic losses in the commercial poultry industry for many years in worldwide. Attenuated live vaccine has widely used in poultry showing some promising signs against IBDV infection. But it has defects such as generating enhanced virulence and immunosuppression prohibits. Therefore, the development of mucosal vaccines using the food-grade lactic acid bacterium is necessary. Here, we construct a recombinant Lactococcus co-expressing the major IBDV antigens VP2 and RCK protein of Salmonella enterica to prevent IBD. Results The recombinant fusion protein VP2-RCK was expressed in a soluble and stable form in the cytoplasm of the recombinant Lactococcus lactis. Animal experiments showed that: (1) the survival rates of the injected immunization inactivated recombinant LAB group and oral immunization live recombinant LAB group were 100% and 80%, respectively; (2) ELISA titers of all serum samples from all experimental groups were negative, but high amounts of specific neutralizing antibodies were detected (1:210 to 1:212); and (3) the bursas of the injected immunization inactivated recombinant LAB group did not suffer damage, as confirmed by clinical observation and bursal histopathological examination. Our results indicate that r-L. lactis-OptiVP2-RCK induces a specific neutralizing-antibody-mediated immune response that confers full protection against very-virulent IBDV (vvIBDV) challenge. Conclusion Lactococcus lactis NZ3900 strain and its matching plasmid pNZ8149 could express the recombinant fusion protein VP2-RCK in a soluble form in the cytoplasm. The protective efficacy of r-L. lactis-OptiVP2-RCK (100%) was better than r-L. lactis-OptiVP2 (0%) which prove RCK protein played its unique role. The neutralizing antibodies titers against infectious bursal disease virus via one-time vaccination with inactivated r-L. lactis-OptiVP2-RCK could reach 1:210 to 1:212, but ELISA titers of all serum samples were negative. For this phenomenon, perhaps because of the change of delivery pathway or the spatial structure of fusion protein. We need further study to test these hypotheses.

Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases that seriously threaten poultry farming and food safety worldwide. The variant strain of infectious bursal disease virus (IBDV) has been greatly neglected for more than 30 years. Recently, the subclinical infection of suspected IBD, causing considerable economic losses, occurred in the main chicken-farming regions of China. Through RT-PCR, sequencing, and phylogenic analyses, novel variant IBDVs were first identified in six provinces of eastern China. Immunological detection further confirmed the antigenic variation of the Chinese variant IBDVs. The Chinese IBDV variants were obviously different from the American IBDV variants, with less than a 97.7% (VP1) or 98.7% (VP2) amino acid sequence identity. Animal experiments further confirmed the serious threat of the variant IBDVs to chickens, demonstrating irreversible damage to the central immune organ, obvious immunosuppression, and growth retardation. This study not only identified the pandemic nature of the novel variant IBDVs for the first time but also discovered the distinct molecular epidemiological characteristics of these viruses, which will contribute more to the control of the disease.

Infectious bursal disease virus (IBDV) causes immunosuppression in young chickens, leading to increased susceptibility to other diseases and a reduction in the immune response to other vaccines. Thus, IBDV results in great economic losses to the poultry industry. The most effective method of prevention is vaccination. However, medium-virulence vaccines can cause bursal pathological damage and immunosuppression. Here, we describe a safer, self-assembled, subvirus-like particle (sVP) vaccine without a complex purification process. The IBD-VP2 gene was cloned into Pichia pastoris, and the expressed protein self-assembled into T=1 sVPs (∼23nm). Immunization experiments showed that the sVP vaccine elicited high IBDV-neutralizing antibodies in each group, and all birds survived challenge with very virulent IBDV (vvIBDV). Additionally, IBDV RNA was not detected, and sterile immunity was achieved. In conclusion, the IBD-sVP is a suitable candidate for a recombinant subunit vaccine against IBDV.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease (IBD), an important immunosuppressive disease seriously threatening poultry farming worldwide. Since the identification of the classic strain in 1957, variant IBDV, very virulent IBDV, and novel variant IBDV have successively emerged brought severe challenges. Over the years, attenuated, intermediate, and intermediate-plus live vaccines have been developed to control the disease. The coexistence of various strains in flocks increases the probability of homologous recombination, and in this study, a naturally occurring homologous recombination between a novel variant strain and an intermediate vaccine strain of IBDV was first identified. Sequence analyses demonstrated that the IBD16HeN01 strain was a recombinant IBDV incorporating the skeleton of the novel variant IBDV (SHG19-like strain), where the 3' region of segment A (nt 1539-3260) was replaced by an intermediate vaccine strain (W2512-like strain). Pathogenicity experiments indicated that IBD16HeN01 could cause severe bursal lesions and the recombination increased viral pathogenicity to chick embryos compared with the novel variant IBDV. Homologous recombination in IBDV has increased the complexity of disease prevention and control and reminds us that we should use live vaccines more scientifically and cautiously.

Inclusion body hepatitis (IBH), hydropericardium syndrome, and gizzard erosion associated with fowl adenovirus (FAdV) infections are reported globally and resulted in significant poultry industry economic losses. In 2018, severe IBH appeared in Pakistan in a 17-week-old layer flock. Subsequently, a FAdV-11 strain (designated as PKFAd18) was isolated from liver samples and identified based on phylogenetic analyses of the serotype-specific L1 region of the capsid hexon gene. There is no complete genome sequence of the Pakistani FAdV-11. This study successfully sequenced the complete genome of PKFAd18. The full genome of PKFAd18 contains 43 840 base pairs (bp) with a G + C content of 53.9 %, which is comparable to other FAdV serotypes. Similar to other FAdV-11 strains, PKFAd18 has only one fiber, while FAdV-1 and FAdV-4 have two fibers. Notably, PKFAd18 showed unique characteristics compared to other FAdV-11 strains. A natural large genomic deletion (1215 bp) appeared in tandem repeat region two, relative to the ON-NP2 strain. Phylogenetic analyses of the PKFAd18 penton gene showed higher homology with FAdV-9, highlighting potential natural recombination between FAdV-11 and FAdV-9. Moreover, the pathogenicity of PKFAd18 studied in specific-pathogen-free chickens showed that PKFAd18 is capable of inducing severe IBH and could be responsible for IBH in Pakistan. Thus, the first complete genome of FAdV-11 in Pakistan was sequenced in this study, which enriches the diversity of knowledge about FAdV-11 and is useful for developing diagnostics and vaccines for IBH induced by FAdV-11 in Pakistan.

Avian leukosis virus (ALV) is an avian oncogenic retrovirus that induces leukemia-like proliferative diseases in chickens. ALV infection can result in the development of immunological tolerance and persistent viremia. Since effective vaccines against ALV are not yet available, its current prevention primarily depends on detection and eradication to establish exogenous ALV-free poultry flocks. In this study, a rapid and simple colloidal gold test strip method, specific for the group-specific antigen, p27 protein, was developed and systematically evaluated for the detection of ALV from different samples. The detection limit of this assay was as low as 6.25 ng/ml for p27 protein and 80 TCID50/ml for different subgroups of ALV. Besides, the test strip showed high specificity in the detection of different subgroups of ALV, including ALV-A, ALV-B, ALV-J, and ALV-K, with no cross-reaction with other avian pathogens. Furthermore, we artificially infected specific pathogen-free (SPF) chickens with ALV-J, collected cloacal swabs, and examined viral shedding using both test strips and ELISA. Results from the test strip were highly consistent with that from ELISA. In addition, 1104 virus isolates from anti-coagulant blood samples, 645 albumen samples, and 4312 meconium samples were tested, and the test strip results agreed with those of ELISA kit up to 97.1%. All the results indicated that the colloidal gold test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for eradication of ALV in poultry farms.

Marek's disease virus (MDV), an α-herpesvirus targeting avian species, causes fatal Marek's disease (MD) in chickens. The host interferon (IFN) responses play a key role in resisting viral infection. However, host IFN responses following MDV infection in the chicken central immune organs (thymus and bursa of Fabricius), which contain numerous MDV target cells, is poorly understood. In this study, we performed animal experiments in specific pathogen-free chickens infected with two virulent MDV strains (BS/15 and Md5) or without infection as negative controls. Specifically, the type I IFN (IFN-α and IFN-β) transcriptional and proteomic expression levels at 7, 10, 14, 17, and 21 days post infection (dpi) were detected and analyzed. Our results indicated that the mRNA and protein expression levels of IFN-α and IFN-β in the thymus and bursa of Fabricius were mainly downregulated in cytolytic infection (such as 10 dpi) and reactivation (such as 17 dpi) stages, but not the latent (such as 14 dpi) stage of MDV infection, which was determined by comprehensively analyzing the MDV viral load and immune organ damage caused by MDV infection. These data suggest that MDV could inhibit the expression of host type I IFNs, which may be involved in the MDV-induced host immunosuppression and contribute to the immune escape of MDV from host immunity. Furthermore, we found that the downregulated expression of the host type I IFNs induced by BS/15 and Md5 infection was significantly different, which we speculated may be related to the diverse virulence and pathogenicity of MDV strains. In conclusion, our study demonstrated that MDV mostly inhibited the expression of type I IFNs in infected hosts, which may be associated to its pathogenesis.

The prevalence of Marek’s disease (MD) caused by Gallid herpesvirus-2 (GaHV-2) has been increasing in chickens in China despite universal vaccination. Among the possible reasons for this trend, of Reticuloendotheliosis virus (REV) contamination in vaccines could lead to co-infection and reduce the vaccine efficacy. Here, we report the epidemiological findings of our continuous surveillance of MD, and an examination of the effects of REV and/or GaHV-2 co-infection. A total of 1230 samples were collected between 2011 and 2015 from 305 flocks covering many of the chicken-raising regions of China. Among these, 606 samples were determined to be GaHV-2-positive, 13.0% of which were found to be co-infected with REV from 18.8% of the flocks. One GaHV-2 strain (HS/1412), a REV strain (HS/1412R), and a GaHV-2 and REV-co-infected strain (HS/1412 GR) were isolated from different chickens of a GaHV-2 and REV co-infected flock. Pathogenicity tests showed that HS/1412 and HS/1412 GR caused disease in all chickens and that HS/1412R induced morbidity in 84.6% of the infected chickens. HS/1412 GR induced 100% mortality and 76.9% tumor formation, which were significantly higher frequencies than those observed with strain HS/1412 (38.5% and 15.4%, respectively) and HS/1412R (0% and 0%). These results indicate that co-infection with GaHV-2 and REV might explain the persistent, sporadic outbreaks of neoplastic disease in some commercial flocks, resulting in a significant economic burden to the poultry industry of China.

Chicken Na + /H + exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry. IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are located in the membrane-proximal region of the N terminus of chECL1, suggesting that the binding site of ALV-J gp85 on chNHE1 is probably located on the apex of the molecule; the receptor-binding mode might be different from that of retroviruses. We also found that soluble chECL1, as well as huECL1 harboring chNHE1 residues 28 to 39, effectively blocked ALV-J infection. These findings contribute to a better understanding of the ALV-J infection mechanism and also provide new insights into the control strategies for ALV-J infection.

Infectious bursal disease virus (IBDV) is a highly contagious disease that results in enormous economic losses in the global poultry sector. Lactic acid bacteria are an appealing vehicle for the safe and effective delivery of heterologous protein antigens. Oral administration of the commensal bacterium Lactococcus lactis expressing recombinant fusion proteins has been used to elicit mucosal and systemic immune responses. In this study, a Lactococcus lactis NZ3900 strain co-expressing the outer membrane protein (Omp) H of the microfold (M) cell-targeting ligand and the viral capsid protein (VP)2 antigen of IBDV was genetically engineered, and its immunopotentiating capacity as an oral and injected vaccine in chickens was evaluated. Western blotting analysis demonstrated that VP2-OmpH was expressed in the cytoplasm of cells and had high immunoreactivity. An in vivo study showed that in the absence of any adjuvant, the recombinant L. lactis VP2-OmpH strain stimulated the immune response and protected against very virulent IBDV challenge in 100% and 80% of chickens immunized by injection and oral administration, respectively. Moreover, the antiviral neutralizing antibody titers induced by injection administration were higher than those induced by oral administration. Mucosal secretory IgA titers induced by oral administration were higher than those induced by injection administration. These results suggested that the recombinant L. lactis VP2-OmpH strain is a promising candidate vaccine to prevent IBDV infection.

Since 2015, China has experienced outbreaks of severe hydropericardium syndrome (HPS), associated with a novel genotype and hypervirulent fowl adenovirus serotype 4 (FAdV-4) infection, with a prevalence in various provinces of the country. This has resulted in huge economic losses in the poultry industry. The novel FAdV-4 showed new genome characters, such as the natural deletion of open reading frame (ORF) 19 and ORF 27 (1966 bp), and high pathogenicity toward chickens. These are coupled with severe hydropericardium, inclusion body hepatitis, and mortality rates ranging from 30% to 90%. Although several inactivated and subunit vaccines against the traditional FAdV-4 have been developed, no commercial vaccine against the emerged disease caused by the novel strain has been available until now. The potential risks of infection with this novel hypervirulent FAdV-4 urgently require an effective vaccine. Thus, an inactivated oil-emulsion FAdV-4 vaccine formulated with the novel genotype virus was developed in this study. The vaccine provided a high level of antibody, preferential T helper 2 (Th2) (interleukin-4 secretion) not Th1 (interferon-γ secretion) response, and full protection against a lethal dose of the novel hypervirulent FAdV-4. Therefore, the novel genotype FAdV-4 vaccine is proposed as an attractive candidate to prevent and reduce the spread of HPS in the poultry industry of China.

Gallid herpesvirus 2 (GaHV-2), which causes Marek's disease in chickens and has caused extensive economic losses, has recently evolved increased virulence in China. To better understand the genetic basis of the pathogenic characteristics changed and increased virulence, we sequenced the genomes of six new GaHV-2 strains (LCC, LTS, WC/1203, JL/1404, CC/1409, and HS/1412) isolated from chickens with failed immunisation as well as one previously isolated Chinese GaHV-2 strain, J-1. Based on a multiple sequence alignment, several characteristic point mutations were detected in the open reading frames of the Chinese isolates. In addition, two deletions and an insertion were identified at the unique short region and terminal repeat short region junctions in Chinese isolates, and the insertion was a characteristic of the new Chinese isolates. According to a phylogenetic analysis, the GaHV-2 genome diverged substantially over the last two decades in China. Based on the internal repeat long region, the new isolates were closely related to very virulent or very virulent plus strains. Additionally, the new Chinese isolates diverged from the previously isolated strains J-1 and 814. In conclusion, our results provide evidence that Chinese GaHV-2 strains contain characteristic sequences, especially the new isolates. The observed genetic divergence in the new Chinese GaHV-2 strains over the last two decades may be related to observed changes in pathogenic characteristics and virulence.

Marek’s disease (MD) virus (MDV) has been evolving continuously, leading to increasing vaccination failure. Here, the MDV field strain BS/15 was isolated from a severely diseased Chinese chicken flock previously vaccinated with CVI988. To explore the causes of vaccination failure, specific-pathogen free (SPF) chickens vaccinated with CVI988 or 814 and unvaccinated controls were challenged with either BS/15 or the reference strain Md5. Both strains induced MD lesions in unvaccinated chickens with similar mortality rates of 85.7% and 80.0% during the experimental period, respectively. However, unvaccinated chickens inoculated with BS/15 exhibited a higher tumor development rate (64.3% vs. 40.0%), but prolonged survival and diminished immune defects compared to Md5-challenged counterparts. These results suggest that BS/15 and Md5 show a similar virulence but manifest with different pathogenic characteristics. Moreover, the protective indices of CVI988 and 814 were 33.3 and 66.7 for BS/15, and 92.9 and 100 for Md5, respectively, indicating that neither vaccine could provide efficient protection against BS/15. Taken together, these data suggest that MD vaccination failure is probably due to the existence of variant MDV strains with known virulence and unexpected vaccine resistance. Our findings should be helpful for understanding the pathogenicity and evolution of MDV strains prevalent in China.

An outbreak of bronchial embolization with 50%-70% morbidity and 30% mortality occurred in broilers in northeast China. This highly contagious disease is characterized by the sudden onset of clinical symptoms, including dyspnea, hemorrhagic tracheal discharge, and bronchial obstruction. Subsequently, six strains of Ornithobacterium rhinotracheale (ORT) and three strains of Streptococcus zooepidemicus were isolated from the various organs and identified using biochemical tests and PCR methods. The pathogenesis of embolization in chickens is poorly understood. The current experimental study confirmed that ORT infection alone could induce a significantly fatal hemorrhagic pneumonia and high mortality in comparison with S. zooepidemicus infection. Moreover, co-infection of ORT with S. zooepidemicus could induce even higher mortality, with severe bronchial obstruction, than that observed in chickens infected with S. zooepidemicus or ORT alone. Therefore, the combination of ORT and S. zooepidemicus may be associated with the outbreak of chicken bronchial embolization. Further investigation of the pathogenesis of ORT and Streptococcus is urgently needed.