Regions of Bovine Adenovirus-3 Protein VII Involved in Interactions with Viral and Cellular Proteins.

The L 1 region of bovine adenovirus (BAdV)-3 encodes a multifunctional protein named protein VII. Anti-protein VII sera detected a protein of 26 kDa in transfected or BAdV-3-infected cells, which localizes to nucleus and nucleolus of infected/transfected cells. Analysis of mutant protein VII identified four redundant overlapping nuclear/nucleolar localization signals as deletion of all four potential nuclear/nucleolar localization signals localizes protein VII predominantly to the cytoplasm. The nuclear import of protein VII appears to use importin α (α-1), importin-ß (ß-1) and transportin-3 nuclear transport receptors. In addition, different nuclear transport receptors also require part of protein VII outside nuclear localization sequences for efficient interaction. Proteomic analysis of protein complexes purified from recombinant BAdV-3 expressing protein VII containing Strep Tag II identified potential viral and cellular proteins interacting with protein VII. Here, we confirm that protein VII interacts with IVa2 and protein VIII in BAdV-3-infected cells. Moreover, amino acids 91-101 and 126-137, parts of non-conserved region of protein VII, are required for interaction with IVa2 and protein VIII, respectively.

Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model.

BACKGROUND: Necrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations. METHODS: This study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches. RESULTS: Our findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens. CONCLUSION: Our study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.

Development of a multivalent adjuvanted inactivated vaccine against variant arthrotropic avian reoviruses.

Variant avian reoviruses (ARVs) are economically important emerging pathogens of poultry, which mainly affect young broiler chickens and cause significant production losses. Currently, there are no effective commercial vaccines available for control and prevention of emerging variant ARVs. In this study, monovalent inactivated adjuvated (20% Emulsigen D) broiler breeder vaccines containing antigens from ARV genotype cluster (C) group -2, -4, -5, or -6, and a multivalent vaccine containing antigens from all the four indicated genotypic cluster groups were developed and evaluated for their efficacy in protecting broiler progenies against homologous or heterologous ARV challenge. The use of monovalent or multivalent inactivated vaccines in a prime-boost immunization strategy induced the production of ARV specific antibodies in broiler breeders. The maternal antibodies were effectively transferred to broiler progenies. Broiler progenies obtained from immunized breeders demonstrated milder clinical symptoms and reduced gross and histopathological lesions after homologous ARV challenge. More severe gross and histological lesions were observed in challenged progenies from unvaccinated broiler breeders. However, cross protection was not observed when either of the monovalent-vaccine groups were challenged with a heterologous virus. In addition, the progenies from the unvaccinated ARV challenged control or heterologous ARV challenged vaccinated groups had significantly reduced body weight gain (p < 0.01) than the unchallenged-control, challenged-multivalent, or homologous ARV-challenged monovalent vaccine groups. However, homologous ARV challenged progenies in the multivalent or monovalent vaccine groups had similar body weight gain as the control unchallenged group with significantly reduced viral load (p < 0.01) in the gastrocnemius tendon tissue. This study indicates that broad-spectrum protection of broiler progenies from variant ARV infections is feasible through the development of multivalent vaccines after proper characterization, selection and incorporation of multiple antigens based on circulating ARV genotypes in targeted regions.

Bovine Herpesvirus-1 Glycoprotein M Mediates the Translocation to the Golgi Apparatus and Packaging of VP8.

VP8, the most abundant tegument protein of bovine herpesvirus-1 (BoHV-1), plays an important role in viral replication. According to our previous studies, VP8 localizes to the Golgi apparatus of BoHV-1-infected cells where it can be packaged into the virus; however, Golgi localization of VP8 does not occur outside of the context of infection. The goal of this study was to identify the viral factor(s) involved in the tropism of VP8 towards the Golgi. VP8 was found to interact with glycoprotein M (gM), and the VP8 and gM domains that are essential for this interaction were identified. VP8 and gM colocalized to the Golgi apparatus in BoHV-1-infected cells. In cells co-transfected with VP8- and gM-encoding plasmids, VP8 was also found to be localized to the Golgi, suggesting gM to be sufficient. The localization of VP8 to the Golgi was lost in cells infected with a gM deletion mutant, and the amount of VP8 incorporated into mature virus was significantly reduced. However, with the restoration of gM in a revertant virus, the localization to the Golgi and the amount of VP8 incorporated in the virions were restored. These results indicate that gM plays a critical role in VP8 subcellular localization to the Golgi and packaging into mature virions.

Evaluation of five circulating strains of variant infectious bursal disease virus (varIBDV) for their immunogenicity as broiler breeder vaccines and protective efficacy in neonatal broiler chicks.

The majority of infectious bursal disease virus (IBDV) strains circulating in the broiler chicken industry in Canada are variant strains (varIBDV). Despite high levels of maternally derived antibodies (MtAb), the circulating varIBDVs can establish infection and cause severe immunosuppression in broiler chicks. The objective of this study was to evaluate circulating varIBDVs as broiler breeder vaccine candidates and investigate their protective efficacy against varIBDV challenge in their progeny chicks. Six groups of breeders (20 females/group) were vaccinated with varIBDV strains, SK09, SK10, SK11, SK12, and SK13 or saline at the age of 13 weeks and antibody response was determined by ELISA at 3-7-, and 20- weeks post-vaccination. We also included commercial chicks for the comparison. Results showed that SK-09 is the most antigenic strain, followed by SK-10, SK-12, and SK-13. In contrast, SK-11 showed the lowest antibody response, and over time, antibody titers steadily decreased. Eggs from breeders were collected at 21-week post-vaccination and incubated to produce their respective progenies. The serum antibody titer in day-old chicks showed a successful MtAb transfer. Progeny chicks (n = 40/group) were orally challenged with varIBDV-SK-09 strain at 6 days of age and serum antibody titer (19 d and 35 d of age), bursa to body weight ratio (19 d and 35 d of age), bursal viral load (9 d and 19 d of age) was examined to assess the protection against IBDV. Following the challenge, we found a significant increase in the antibody titers in MtAb-free and commercial vaccine groups than in the varIBDV groups, both at 19 d and 35 d of age. The BBW ratio and viral load data indicated a significant homologous and heterologous protection against varIBDV-SK-09 challenge by SK-09 and SK-10 MtAbs, respectively. Overall, this study demonstrated the feasibility of developing breeder vaccines using circulating varIBDV as candidate vaccine antigens.

Comparison of Therapeutic Antibiotics, Probiotics, and Synthetic CpG-ODNs for Protective Efficacy Against Escherichia coli Lethal Infection and Impact on the Immune System in Neonatal Broiler Chickens.

The poultry industry needs alternatives to antibiotics, as there are growing public concerns about the emergence of antimicrobial resistance owing to antimicrobial use in animal production. We have reported that the administration of neonatal chicks with synthetic DNA oligodeoxynucleotides containing unmethylated cytosine guanine dinucleotide (CpG) motifs (CpG-ODN) can protect against bacterial pathogens in chickens. The objective of this study was to compare the immunoprotective effects of CpG-ODN and probiotics against Escherichia coli infection vs. commonly used therapeutic antibiotics. Day-old broiler chicks were divided into five groups (n = 35/group; 30 for the challenge experiment and 5 for the flow cytometry analysis). The chicks in Group 1 received a single dose of CpG-ODN by the intramuscular route on day 4 (D4) posthatch (PH), and Group 2 received drinking water (DW) with a probiotic product (D1-D15 PH, DW). The Group 3 chicks received tetracycline antibiotics during D9-D13 in DW; the Group 4 chicks got sodium sulfamethazine on D9, D10, and D15 PH in DW; and the Group 5 chicks were administered intramuscular (IM) saline D4 PH, DW. We challenged all the groups (n = 30/group) with E. coli (1 × 105 or 1 × 106 colony-forming units/bird) on D8 PH through the subcutaneous route. Our data demonstrated that the CpG-ODNs, but not the probiotics, could protect neonatal broiler chickens against lethal E. coli septicemia, as would the tetracycline or sodium sulfamethazine. The flow cytometry analysis (n = 5/group) revealed enrichment of immune cells in the CpG-ODN group and a marked decrease in macrophages and T-cell numbers in antibiotics-treated groups, indicating immunosuppressive effects. Our data showed that, like therapeutic antibiotics, CpG-ODNs reduced clinical signs, decreased bacterial loads, and induced protection in chicks against E. coli septicemia. Unlike therapeutic antibiotics-induced immunosuppressive effects, CpG-ODN caused immune enrichment by increasing chicken immune cells recruitment. Furthermore, this study highlights that, although therapeutic antibiotics can treat bacterial infections, the ensuing immunosuppressive effects may negatively impact the overall chicken health.

Comparación de antibióticos terapéuticos, probióticos y CpG-ODN sintéticos en su eficacia protectora contra la infección letal por Escherichia coli y el impacto en el sistema inmunológico en pollos de engorde recién eclosionados. La industria avícola necesita alternativas a los antibióticos ya que existe una creciente preocupación pública sobre la aparición de resistencia a los antimicrobianos debido a su uso en la producción animal. Se ha informado que la administración de oligodesoxinucleótidos de ADN sintético que contienen motivos de dinucleótidos de citosina guanina (CpG) no metilados (CpG-ODN) a pollitos recién eclosionados puede proteger contra patógenos bacterianos en pollos. El objetivo de este estudio fue comparar los efectos inmunoprotectores de CpG-ODN y de los probióticos contra la infección por Escherichia coli frente a los antibióticos terapéuticos de uso común. Los pollos de engorde de un día se dividieron en cinco grupos (n = 35/grupo; 30 para el experimento de desafío y 5 para análisis de citometría de flujo). Los pollitos del Grupo 1 recibieron una dosis única de CpG-ODN por vía intramuscular el día 4 (D4) después de la eclosión (PH), y el Grupo 2 recibió agua potable (DW) con un producto probiótico del día uno al quince después de la eclosion en agua de bebida. Los pollitos del Grupo 3 recibieron tetraciclina durante los días nueve a trece (D9­D13) en agua de bebida (DW9; los pollitos del Grupo 4 recibieron sulfametazina de sodio en los días nueve, diez y 15 (D9, D10 y D15) después de la eclosion en agua de bebida; ya los pollitos del Grupo 5 se les administró solución salina intramuscular (IM) al día cuatro después de la eclosión en agua de bebida. Se desafiaron todos los grupos (n = 30/grupo) con E. coli (1 × 105 o 1 × 106 unidades formadoras de colonias/ave) en el día ocho después de la eclosión por vía subcutánea. Nuestros datos demostraron que los CpG-ODN, pero no los probióticos, pudieron proteger a los pollos de engorde recién eclosionados contra la septicemia letal por E. coli, al igual que la tetraciclina o la sulfametazina sódica. El análisis de citometría de flujo (n = 5/grupo) reveló un enriquecimiento de células inmunes en el grupo CpG-ODN y una marcada disminución en el número de macrófagos y células T en los grupos tratados con antibióticos, lo que indica efectos inmunosupresores. Nuestros datos mostraron que, al igual que los antibióticos terapéuticos, los CpG-ODN redujeron los signos clínicos, disminuyeron las cargas bacterianas e indujeron protección en los pollitos contra la septicemia por E. coli. A diferencia de los efectos inmunosupresores inducidos por antibióticos terapéuticos, los CpG-ODN provocaron un enriquecimiento inmunitario al aumentar el reclutamiento de células inmunitarias de pollo. Además, este estudio destaca que, aunque los antibióticos terapéuticos pueden tratar las infecciones bacterianas, los efectos inmunosupresores resultantes pueden tener un impacto negativo en la salud general de los pollos.

Virulence of Emerging Arthrotropic Avian Reoviruses Correlates With Their Ability to Activate and Traffic Interferon-γ Producing Cytotoxic CD8+ T Cells Into Gastrocnemius Tendon.

Newly emerging arthrotropic avian reoviruses (ARVs) are genetically divergent, antigenically heterogeneous, and economically costly. Nevertheless, the mechanism of emerging ARV-induced disease pathogenesis and potential differences in virulence between virus genotypes have not been adequately addressed. In this study, the life cycle of ARV, including the formation of cytoplasmic ARV neo-organelles, paracrystalline structures, and virus release mechanisms, were characterized in the infected host cell by transmission electron microscopy (TEM). In addition, progressive changes in the structure of infected cells were investigated by time-lapse and field emission scanning electron (FE-SE) microscopy. ARVs from the four genotypic cluster groups included in the study caused gross and microscopic lesions in the infected birds. Marked infiltration of γδT cells, CD4+ and CD8+ T lymphocytes were observed in ARV infected tendon tissues starting day 3 post-infection. The ARV variant from genotype cluster-2 triggered significantly high trafficking of IFN-γ producing CD8+ T lymphocytes in tendon tissues and concomitantly showed high morbidity and severe disease manifestations. In contrast, the ARV variant from genotype cluster-4 was less virulent, caused milder disease, and accompanied less infiltration of IFN-γ producing CD8+ T cells. Interestingly, when we blunted antiviral immune responses using clodronate liposomes (which depletes antigen-presenting cells) or cyclosporin (which inhibits cytokine production that regulates T-cell proliferation), significantly lower IFN-γ producing CD8+ T cells infiltrated into tendon tissues, resulting in reduced tendon tissues apoptosis and milder disease manifestations. In summary, these data suggest that the degree of ARV virulence and tenosynovitis/arthritis are potentially directly associated with the ability of the virus to traffic massive infiltration of cytotoxic CD8+ T cells into the infected tissues. Moreover, the ability to traffic cytotoxic CD8+ T cells into infected tendon tissues and the severity of tenosynovitis differ between variants from different ARV genotype cluster groups. However, more than one virus isolate per genotype group needs to be tested to further confirm the association of pathogenicity with genotype. These findings can be used to further examine the interaction of viral and cellular pathways which are essential for the pathogenesis of the disease at the molecular level and to develop effective disease control strategies.

CpG-ODN induced antimicrobial immunity in neonatal chicks involves a substantial shift in serum metabolic profiles.

Synthetic CpG-ODNs can promote antimicrobial immunity in neonatal chicks by enriching immune compartments and activating immune cells. Activated immune cells undergo profound metabolic changes to meet cellular biosynthesis and energy demands and facilitate the signaling processes. We hypothesize that CpG-ODNs induced immune activation can change the host's metabolic demands in neonatal chicks. Here, we used NMR-based metabolomics to explore the potential of immuno-metabolic interactions in the orchestration of CpG-ODN-induced antimicrobial immunity. We administered CpG-ODNs to day-old broiler chicks via intrapulmonary (IPL) and intramuscular (IM) routes. A negative control group was administered IPL distilled water (DW). In each group (n = 60), chicks (n = 40) were challenged with a lethal dose of Escherichia coli, two days post-CpG-ODN administration. CpG-ODN administered chicks had significantly higher survival (P < 0.05), significantly lower cumulative clinical scores (P < 0.05), and lower bacterial loads (P < 0.05) compared to the DW control group. In parallel experiments, we compared NMR-based serum metabolomic profiles in neonatal chicks (n = 20/group, 24 h post-treatment) treated with IM versus IPL CpG-ODNs or distilled water (DW) control. Serum metabolomics revealed that IM administration of CpG-ODN resulted in a highly significant and consistent decrease in amino acids, purines, betaine, choline, acetate, and a slight decrease in glucose. IPL CpG-ODN treatment resulted in a similar decrease in purines and choline but less extensive decrease in amino acids, a stronger decrease in acetate, and a considerable increase in 2-hydroxybutyrate, 3-hydroxybutyrate, formic acid and a mild increase in TCA cycle intermediates (all P < 0.05 after FDR adjustment). These perturbations in pathways associated with energy production, amino acid metabolism and nucleotide synthesis, most probably reflect increased uptake of nutrients to the cells, to support cell proliferation triggered by the innate immune response. Our study revealed for the first time that CpG-ODNs change the metabolomic landscape to establish antimicrobial immunity in neonatal chicks. The metabolites highlighted in the present study can help future targeted studies to better understand immunometabolic interactions and pinpoint the key molecules or pathways contributing to immunity.

Adenovirus Core Proteins: Structure and Function.

Adenoviruses have served as a model for investigating viral-cell interactions and discovering different cellular processes, such as RNA splicing and DNA replication. In addition, the development and evaluation of adenoviruses as the viral vectors for vaccination and gene therapy has led to detailed investigations about adenovirus biology, including the structure and function of the adenovirus encoded proteins. While the determination of the structure and function of the viral capsid proteins in adenovirus biology has been the subject of numerous reports, the last few years have seen increased interest in elucidating the structure and function of the adenovirus core proteins. Here, we provide a review of research about the structure and function of the adenovirus core proteins in adenovirus biology.

Bovine Adenovirus-3 Tropism for Bovine Leukocyte Sub-Populations.

A number of characteristics including lack of virulence and the ability to grow to high titers, have made bovine adenovirus-3 (BAdV-3) a vector of choice for further development as a vaccine-delivery vehicle for cattle. Despite the importance of blood leukocytes, including dendritic cells (DC), in the induction of protective immune responses, little is known about the interaction between BAdV-3 and bovine blood leukocytes. Here, we demonstrate that compared to other leukocytes, bovine blood monocytes and neutrophils are significantly transduced by BAdV404a (BAdV-3, expressing enhanced yellow green fluorescent protein [EYFP]) at a MOI of 1-5 without a significant difference in the mean fluorescence of EYFP expression. Moreover, though expression of some BAdV-3-specific proteins was observed, no progeny virions were detected in the transduced monocytes or neutrophils. Interestingly, addition of the "RGD" motif at the C-terminus of BAdV-3 minor capsid protein pIX (BAV888) enhanced the ability of the virus to enter the monocytes without altering the tropism of BAdV-3. The increased uptake of BAV888 by monocytes was associated with a significant increase in viral genome copies and the abundance of EYFP and BAdV-3 19K transcripts compared to BAdV404a-transduced monocytes. Our results suggest that BAdV-3 efficiently transduces monocytes and neutrophils in the absence of viral replication. Moreover, RGD-modified capsid significantly increases vector uptake without affecting the initial interaction with monocytes.

The dynamics of molecular evolution of emerging avian reoviruses through accumulation of point mutations and genetic re-assortment.

In the last decade, the emergence of variant strains of avian reovirus (ARV) has caused enormous economic impact in the poultry industry across Canada and USA. ARVs are non-enveloped viruses with ten segments of double-stranded RNA genome. So far, only six genotyping cluster groups are identified worldwide based on sequence analysis of the σC protein encoded by the S1 segment. In this study, we performed deep next generation whole-genome sequencing and analysis of twelve purified ARVs isolated from Saskatchewan, Canada. The viruses represent different genotyping cluster. A genome-wide sequence divergence of up to 25 per cent was observed between the virus isolates with a comparable and contrasting evolutionary history. The proportion of synonymous single-nucleotide variations (sSNVs) was higher than the non-synonymous (ns) SNVs across all the genomic segments. Genomic segment S1 was the most variable as compared with the other genes followed by segment M2. Evidence of positive episodic/diversifying selection was observed at different codon positions in the σC protein sequence, which is the genetic marker for the classification of ARV genotypes. In addition, the N-terminus of σC protein had a persuasive diversifying selection, which was not detected in other genomic segments. We identified only four ARV genotypes based on the most variable σC gene sequence. However, a different pattern of phylogenetic clustering was observed with concatenated whole-genome sequences. Together with the accumulation of point mutations, multiple re-assortment events appeared as mechanisms of ARV evolution. For the first time, we determined the mean rate of molecular evolution of ARVs, which was computed as 2.3 × 10-3 substitution/site/year. In addition, widespread geographic intermixing of ARVs was observed between Canada and USA, and between different countries of the world. In conclusion, the study provides a comprehensive analysis of the complete genome of different genotyping clusters of ARVs including their molecular rate of evolution and spatial distribution. The new findings in this study can be utilized for the development of effective vaccines and other control strategies against ARV-induced arthritis/tenosynovitis in the poultry industry worldwide.

CpG-ODN Induces a Dose-Dependent Enrichment of Immunological Niches in the Spleen and Lungs of Neonatal Chicks That Correlates with the Protective Immunity against Escherichia coli.

Immunoprotective function of oligodeoxynucleotides containing CpG motifs (CpG-ODN) has been demonstrated in neonatal chickens against common bacterial pathogens such as E.coli and Salmonella sp. Our recent study reported that CpG-ODN administration enriches immune compartments in neonatal chicks. However, a causal relationship between CpG-ODN-induced immune enrichment and protective mechanisms remains unestablished. In this study, we investigated in ovo administered CpG-ODN-mediated immune cell recruitment in the immunological niches in lymphoid (spleen) and nonlymphoid (lungs) organs using various doses of CpG-ODN and examined whether the immunological profiles have any correlation with immunoprotection against E.coli infection. Eighteen-day-old embryonated eggs were injected with either 5, 10, 25, and 50 µg of CpG-ODN or saline (n = ~40 per group). On the day of hatch (72 hr after CpG-ODN treatment), we collected the spleen and lungs (n = 3-4 per group) and examined the recruitment of macrophages/monocytes, their expression of MHCII and CD40, and the number of CD4+ and CD8+ T-cell subsets in the immunological niches in the spleen and lungs using flow cytometry. We observed the dose-dependent recruitment of immune cells, wherein 25 µg and 50 µg of CpG-ODN induced significant enrichment of immunological niches in both the spleen and the lungs. Four days after the CpG-ODN treatment (1-day after hatch), chicks were challenged with a virulent strain of E. coli (1 × 104 or 1 × 105 cfu, subcutaneously). Clinical outcome and mortality were monitored for 8 days postchallenge. We found that both 25 µg and 50 µg of CpG-ODN provided significant protection and reduced clinical scores compared to saline controls against E. coli infection. Overall, the present study revealed that CpG-ODNs orchestrate immunological niches in neonatal chickens in a dose-dependent manner that resulted in differential protection against E. coli infection, thus supporting a cause and effect relationship between CpG-ODN-induced immune enrichment and the antibacterial immunity.

Regions of bovine adenovirus-3 IVa2 involved in nuclear/nucleolar localization and interaction with pV.

Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24-48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1-25 and 373-448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4-18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1-25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1-25 and 120-140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.

Mucosal delivery of CpG-ODN mimicking bacterial DNA via the intrapulmonary route induces systemic antimicrobial immune responses in neonatal chicks.

The transition to antibiotic-free poultry production in the face of pathogenic threats is a very challenging task. We recently demonstrated that mucosal delivery of CpG-ODN alone by the intrapulmonary route (IPL) has potential as an effective alternative to antibiotics in neonatal chicks against Escherichia coli septicemia. How exactly mucosal delivery of CpG-ODN elicits, protective antibacterial immunity remained poorly understood. In this study, CpG-ODN or saline was delivered via the intrapulmonary route to day-old chicks (n = 80/group) using a compressor nebulizer in an acrylic chamber (1 mg/mL CpG-ODN for 15 minutes). In the first part of the study, two days after mucosal CpG-ODN delivery, 40 chicks from each group were challenged subcutaneously with 1 × 105 cfu (n = 20) or 1 × 106 cfu (n = 20) of E. coli and the mortality pattern was monitored for seven days. We found significantly higher survival, better clinical conditions and lower bacterial loads in chicks that received mucosal CpG-ODN. To explore the mechanisms behind this protective immunity, we first looked at the kinetics of the cytokine gene expression (three birds/ group/ time for 10 time-points) in the lungs and spleens. Multiplex gene analysis demonstrated a significant elevation of pro-inflammatory cytokine genes mRNA in the CpG-ODN group. Interleukin (IL)-1ß robustly upregulated many folds in the lung after CpG-ODN delivery. Lipopolysaccharide-induced tumor necrosis factor (LITAF) and IL-18 showed expression for an extended period in the lungs. Anti-inflammatory cytokine IL-10 was upregulated in both lungs and spleen, whereas IL-4 showed upregulation in the lungs. To investigate the kinetics of immune enrichment in the lungs and spleens, we performed flow cytometry, histology, and immunohistochemistry at 24, 48 and 72 hrs after CpG-ODN delivery. CpG-ODN treated lungs showed a significant enrichment with monocytes/macrophages and CD4+ and CD8+ T-cell subsets. Macrophages in CpG-ODN treated group demonstrated mature phenotypes (higher CD40 and MHCII expression). Importantly, mucosal delivery of CpG-ODN via the intrapulmonary route significantly enriched immune compartment in the spleen as well, suggesting a systemic effect in neonatal chicks. Altogether, intrapulmonary delivery of aerosolized CpG-ODN orchestrates protective immunity against E. coli septicemia by not only enhancing mucosal immunity but also the systemic immune responses.

Nuclear and Nucleolar Localization of Bovine Adenovirus-3 Protein V.

The L2 region of bovine adenovirus-3 (BAdV-3) encodes a Mastadenovirus genus-specific protein, designated as pV, which is important for the production of progeny viruses. Here, we demonstrate that BAdV-3 pV, expressed as 55 kDa protein, localizes to the nucleus and specifically targets nucleolus of the infected cells. Analysis of deletion mutants of pV suggested that amino acids 81-120, 190-210, and 380-389 act as multiple nuclear localization signals (NLS), which also appear to serve as the binding sites for importin α-3 protein, a member of the importin α/ß nuclear import receptor pathway. Moreover, pV amino acids 21-50 and 380-389 appear to act as nucleolar localization signals (NoLs). Interestingly, amino acids 380-389 appear to act both as NLS and as NoLS. The presence of NoLS is essential for the production of infectious progeny virions, as deletion of both NoLs are lethal for the production of infectious BAdV-3. Analysis of mutant BAV.pVd1d3 (isolated in pV completing CRL cells) containing deletion/mutation of both NoLS in non-complementing CRL cells not only revealed the altered intracellular localization of mutant pV but also reduced the expression of some late proteins. However, it does not appear to affect the incorporation of viral proteins, including mutant pV, in BAV.pVd1d3 virions. Further analysis of CsCl purified BAV.pVd1d3 suggested the presence of thermo-labile virions with disrupted capsids, which appear to affect the infectivity of the progeny virions. Our results suggest that pV contains overlapping and non-overlapping NoLS/NLS. Moreover, the presence of both NoLS appear essential for the production of stable and infectious progeny BAV.pVd1d3 virions.

Synthetic CpG-ODN rapidly enriches immune compartments in neonatal chicks to induce protective immunity against bacterial infections.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) induce innate immunity against bacterial infections. Despite recent advances, how CpG-ODN alone protects against bacterial infections remained elusive. Here, we report for the first time, to our knowledge, that CpG-ODN orchestrates anti-microbial protective immunity by inducing a rapid enrichment of various immune compartments in chickens. In this study, eighteen-day-old embryonated eggs were injected with either 50 µg of CpG-ODN or saline (~n = 90 per group). In the first experiment, four days after CpG-ODN treatment, chicks were challenged subcutaneously with a virulent strain of Escherichia coli (E. coli) and mortality was monitored for 8 days. We found significant protection, and reduced clinical scores in CpG-ODN treated chicks. To gain insights into mechanisms of protection induced by CpG-ODN, first we investigated cytokine expression kinetics elicited by CpG-ODN. The spleen and lung were collected from embryos or chicks (n = 3-4 per group) at 10 time points post-CpG-ODN inoculation. Multiplex gene analysis (interleukin (IL)-1, IL-4, IL-6, IL-10, IL-18, interferon (IFN)-γ, IFN-α, and lipopolysaccharide induced tumor necrosis factor (LITAF), revealed a significantly higher expression of pro-inflammatory cytokines following CpG-ODN treatment compared to the saline controls. In our study, LITAF stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN-induced protection. The third experiment was designed to examine the effects of CpG-ODN on immune cell populations in spleen, lungs, and thymus. Flow cytometry analysis was conducted at 24, 48 and 72 hrs (thymus only collected at 72 hr) after CpG-ODN administration to examine the changes in CD4+ and CD8+ T-cell subsets, monocyte/macrophage cell populations and their expression of maturation markers (CD40 and CD86). Flow cytometry data indicated a significant enrichment of macrophages, CD4+ and CD8+ T-cell subsets in both spleen and lungs of CpG-ODN treated embryos and chicks. Macrophages in spleen and lungs showed an upregulation of CD40 but not CD86, whereas thymocytes revealed significantly high CD4 and CD8 expression. Overall, the present study has demonstrated that CpG-ODN provides protection in neonatal chicks against E. coli infection not only by eliciting cytokine responses and stimulating immune cells but also through enriching immunological niches in spleen and lungs.

US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells.

Bovine herpesvirus 1 (BoHV-1) infects bovine species, causing respiratory infections, genital disorders and abortions. VP8 is the most abundant tegument protein of BoHV-1 and is critical for virus replication in cattle. In this study, the cellular transport of VP8 in BoHV-1-infected cells and its ability to alter the cellular lipid metabolism were investigated. A viral kinase, US3, was found to be involved in regulating these processes. In the early stages of infection VP8 was localized in the nucleus. Subsequently, presumably after completion of its role in the nucleus, VP8 was translocated to the cytoplasm. When US3 was deleted or the essential US3 phosphorylation site of VP8 was mutated in BoHV-1, the majority of VP8 was localized in the nuclei of infected cells. This suggests that phosphorylation by US3 may be critical for cytoplasmic localization of VP8. Eventually, the cytoplasmic VP8 was accumulated in the cis-Golgi apparatus but not in the trans-Golgi network, implying that VP8 was not involved in virion transport toward and budding from the cell membrane. VP8 caused lipid droplet (LD) formation in the nuclei of transfected cells and increased cellular cholesterol levels. Lipid droplets were not found in the nuclei of BoHV-1-infected cells when VP8 was cytoplasmic in the presence of US3. However, when US3 was deleted or phosphorylation residues in VP8 were mutated, nuclear VP8 and LDs appeared in BoHV-1-infected cells. The total cholesterol level was increased in BoHV-1-infected cells but not in ΔUL47-BoHV-1-infected cells, further supporting a role for VP8 in altering the cellular lipid metabolism during infection.IMPORTANCE Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important elements directing VP8 to the desired locations in the BoHV-1-infected cell. In this study, a critical regulator that switches the nuclear and cytoplasmic localization of VP8 in BoHV-1-infected cells was identified. BoHV-1 used viral kinase US3 to regulate the cellular localization of VP8. Early during BoHV-1 infection VP8 was localized in the nucleus, where it performs various functions; once US3 was expressed, phosphorylated VP8 was cytoplasmic and ultimately accumulated in the cis-Golgi apparatus, presumably to be incorporated into virions. The Golgi localization of VP8 was only observed in virus-infected cells and not in US3-cotransfected cells, suggesting that this is mediated by other viral factors. Interestingly, VP8 was shown to cause increased cholesterol levels, which is a novel function for VP8 and a potential strategy to supply lipid for viral replication.

Generation of infectious clone of bovine adenovirus type I expressing a visible marker gene.

BACKGROUND AND OBJECTIVE: Bovine adenovirus type 3 (BAdV3) has been widely used as a vector for vaccine research and development, whereas BAdV1 biology and BAdV1-based vectored vaccine have been less frequently reported. We aimed to construct an infectious BAdV1 clone and explore the functions of BAdV1 genes. METHODS: First, the infectious clone of pUCBAdV1 containing the full-length BAdV1 DNA and the recombinant plasmid pUCBAV1-EYFP expressing the marker gene EYFP were constructed. Then, the recombinant viruses BAdV101 and rBAdV1-EYFP were rescued. The stability of the exogenous EYFP gene was analyzed by continuous passage, PCR, and western blotting. Finally, the virus neutralization titer of the rescued viruses was evaluated. RESULTS: The infectious clones of pUCBAdV1 and pUCBAV1-EYFP were constructed and the recombinant viruses BAdV101 and rBAdV1-EYFP were rescued successfully. Moreover, the results showed that the EYFP gene could be expressed continuously. In addition, the replication of rBAdV1-EYFP was less efficient than that of the wild-type virus wtBAdV1 in vitro, while the efficacy of BAdV101 replication was almost the same as that of wtBAdV1. Furthermore, the neutralization test showed that the neutralization titer of rBAdV1-EYFP was consistent with that of wtBAdV1. CONCLUSION: To our knowledge, the infectious genome of pUCBAV1-EYFP expressing a visible marker gene EYFP was constructed for the first time, and the finding forms a basis for the development of BAdV1-based efficient vectored vaccine.

Porcine Adenovirus Type 3 E3 Encodes a Structural Protein Essential for Capsid Stability and Production of Infectious Progeny Virions.

The adenovirus E3 region encodes proteins that are not essential for viral replication in vitro The porcine adenovirus type 3 (PAdV-3) E3 region encodes three proteins, including 13.7K. Here, we report that 13.7K is expressed as an early protein, which localizes to the nucleus of infected cells. The 13.7K protein is a structural protein, as it is incorporated in CsCl-purified virions. The 13.7K protein appears to be essential for PAdV-3 replication, as mutant PAV13.73A expressing a mutated 13.7K could be isolated only in VIDO AS2 cells expressing the 13.7K protein. Analysis of PAV13.73A suggested that even in the presence of reduced levels of some late viral proteins, there appeared to be no effect on virus assembly and production of mature virions. Further analysis of CsCl-purified PAV13.73A by transmission electron microscopy revealed the presence of disrupted/broken capsids, suggesting that inactivation of 13.7K protein expression may produce fragile capsids. Our results suggest that the PAdV-3 E3 region-encoded 13.7K protein is a capsid protein, which appears to be essential for the formation of stable capsids and production of infectious progeny virions.IMPORTANCE Although E3 region-encoded proteins are involved in the modulation of leukocyte functions (N. Arnberg, Proc Natl Acad Sci U S A 110:19976-19977, 2013) and inducing a lytic infection of lymphocytes (V. K. Murali, D. A. Ornelles, L. R. Gooding, H. T. Wilms, W. Huang, A. E. Tollefson, W. S. Wold, and C. Garnett-Benson, J Virol 88:903-912, 2014), none of the E3 proteins appear to be a component of virion capsid or required for replication of adenovirus. Here, we demonstrate that the 13.7K protein encoded by the E3 region of porcine adenovirus type 3 is a component of progeny virion capsids and appears to be essential for maintaining the integrity of virion capsid and production of infectious progeny virions. To our knowledge, this is the first report to suggest that an adenovirus E3-encoded protein is an essential structural protein.