Search Results (15)

Biosecurity measures applied on poultry farms, with a recent history of highly pathogenic avian influenza virus infection, were monitored using 24 h/7 days-per-week video monitoring. Definition of biosecurity breaches were based on internationally acknowledged norms. Farms of four different production types (two broiler, two layer, two breeder broiler, and one duck farm) were selected. Observations of entry to and exit from the anteroom revealed a high degree of biosecurity breaches in six poultry farms and good biosecurity practices in one farm in strictly maintaining the separation between clean and potentially contaminated areas in the anteroom. Hand washing with soap and water and/or using disinfectant lotion was rarely observed at entry to the anteroom and was almost absent at exit. Egg transporters did not disinfect fork-lift wheels when entering the egg-storage room nor change or properly disinfect footwear. The egg-storage room was not cleaned and disinfected after egg transport by the farmer. Similarly, footwear and trolley wheels were not disinfected when introducing young broilers or ducklings to the poultry unit. Biosecurity breaches were observed when introducing bedding material in the duck farm. This study shows a need for an engaging awareness and training campaign for poultry farmers and their co-workers as well as for transporters to promote good biosecurity practices. Full article

Duck enteritis virus (DEV), goose parvovirus (GPV), and muscovy duck parvovirus (MDPV) all have similar symptoms after infection, such as severe diarrhea, which seriously affects the healthy development of the waterfowl industry. Hence, it is important to devise a rapid and precise assay for the detection of these three viruses. In this study, a TaqMan probe-based multi-quantitative polymerase chain reaction (qPCR) assay was developed and optimized. Three specific primers and probes were designed according to the conserved regions of UL6 of DEV, REP of GPV, and VP1 of MDPV, respectively. DEV demonstrated a detection limit of 11.6 copies, GPV detected a limit of 95 copies, and MDPV showcased a detection limit of 14.8 copies. The correlation coefficient is greater than 0.99, and the amplification efficiency is 89% to 93%. These results indicate that the multiplex qPCR assay has high sensitivity, specificity, and stability. Of the 215 clinical samples used in this study, 33 tested DEV positive, 25 tested GPV positive, and 24 tested MDPV positive. Overall, the assay established in the current study presents a rapid, efficient, specific, and sensitive tool for of detecting DEV, GPV, and MDPV. Full article

Background: Avian influenza (AI), caused by orthomyxoviruses, is a globally significant disease affecting avian and non-avian species. It manifests in two variants, according to the two biovariants of the virus differentiated as highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) strains, both of which compromise animal welfare, reduce productivity, and cause substantial economic loss. The zoonotic potential of HPAI strains, particularly the currently dominant clade 2.3.4.4b, raises concerns about public health and epidemic risks. This review assesses the results of current vaccine trials targeting HPAI clade 2.3.4.4b, emphasizing these studies because most outbreak strains in domestic poultry currently belong to this dominant clade. Methods: Multiple scientific databases comprised reports of research trials on vaccine efficacy against HPAI clade 2.3.4.4b. The Boolean term “Clade 2.3.4.4b AND vaccine” was entered into the following databases: PubMed, PubAg, Scopus, Cochrane Library, and ScienceDirect. Results: The resulting papers were analyzed. Studies revealed that antigenic similarity between vaccine and field strains enhances protective efficacy (PE), reduces viral shedding, and improves hemagglutination inhibition titers. While multivalent vaccines showed potential, results were inconsistent and varied depending on strain compatibility. Single-dose vaccines may provide sufficient PE for poultry, though ducks and geese often require multiple doses, and long-term PE is yet unknown. It was discovered that vector vaccines can provide appropriate PE against clade 2.3.4.4.b. Conclusions: Further analysis is needed as their effects may be short-lived, and subsequent doses may be required. Limited research exists on the long-term efficacy of these vaccines and their effectiveness in many avian species. Addressing these gaps is crucial for optimizing vaccination strategies. A re-evaluation of vaccination strategies is recommended but essential to implement adequate biosecurity measures on in poultry farms. This review synthesizes current evidence and may assist veterinarians and authorities in deciding whether to apply or license vaccines to reduce economic losses caused by AI. Full article

Goose parvovirus (GPV), duck enteritis virus (DEV), Muscovy duck parvovirus (MDPV), duck hepatitis A virus type 1 (DHAV-1), duck hepatitis A virus type 3 (DHAV-3), duck Tembusu virus (DTMUV), and novel duck reovirus (NDRV) are significant pathogens that spread extensively among waterfowl populations, causing economic losses for the waterfowl industry. In order to detect seven pathogens simultaneously, a visual gene chip for the detection of multiple waterfowl disease pathogens was developed in this study. The gene chip was capable of specifically amplifying GPV, DEV, MDPV, DHAV-1, –DHAV-3, DTMUV, and NDRV. The sensitivity results showed that the lowest detection limit of the gene chip was 1 copy/μL for single and mixed samples. The reproducibility and stability tests demonstrated that the gene chip developed in this experiment exhibited not only excellent reproducibility but also remarkable stability, remaining functional for a minimum of 180 days. Compared to qPCR methods, the results showed that the sensitivity of the gene chip was slightly better than that of the qPCR method in detecting both single and mixed pathogens of the seven viruses. In this study, a total of 210 clinical samples were detected by the gene chip and qPCR, respectively, and the results of the two methods had a concordance rate of 98.1~100%, with a kappa value of 0.952, indicating that the consistency of the two detection methods was good. Full article

Herpesvirus of turkey (HVT) recombinant vector vaccines are widely used in the poultry industry. However, due to limitations in loading multiple foreign antigens into a single HVT vector, other viral vectors are urgently needed. Since chickens lack maternal immunity to duck enteritis virus (DEV), vector vaccines using DEV as a backbone are currently under study. Even though a recently developed DEV vector vaccine expressing the influenza hemagglutinin H5 of highly pathogenic avian influenza (DEV-H5) induces highly detectable anti-HA antibodies, safety issues hamper further vaccine development. In this work, tissue affinity and horizontal transmission in 1-day-old chickens were systematically evaluated after DEV-H5 vector vaccine inoculation. Sixty percent of DEV-H5-inoculated chickens died between day 2 and day 7 post-inoculation. The displayed clinical signs consisted of lethargy, anorexia, and diarrhea, and virus was shed in feces. Gross and/or histological lesions were recorded in the kidney, heart, intestine, liver, lung, and spleen. Moreover, DEV-H5 replication in intestinal cells caused an increment in interferon-α expression, while occluding junction proteins and ZO-1 expression were significantly upregulated. As a control, birds inoculated with a commercial recombinant turkey herpesvirus expressing the VP2 protein of the infectious bursal disease virus (HVT-VP2) vector vaccine showed neither clinical signs nor mortality. Overall, while the HVT-VP2 vaccine demonstrated complete safety in 1-day-old chickens, our potential DEV-H5 vaccine requires further attenuation for consideration as a vector vaccine candidate in chickens. Full article

Duck hepatitis B virus (DHBV) is widely prevalent in global ducks and has been identified in Chinese geese with a high prevalence; the available detection techniques are time-consuming and require sophisticated equipment. In this study, an assay combining multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) was developed for the efficient and rapid detection of DHBV. The primary reaction condition of the MIRA assay for DHBV detection was 10 min at 38 °C without a temperature cycler. Combined with the LFD assay, the complete procedure of the newly developed MIRA assay for DHBV detection required only 15 min, which is about one-fourth of the reaction time for routine polymerase chain reaction assay. And electrophoresis and gel imaging equipment were not required for detection and to read the results. Furthermore, the detection limit of MIRA was 45.6 copies per reaction, which is approximately 10 times lower than that of a routine polymerase chain reaction assay. The primer set and probe had much simpler designs than loop-mediated isothermal amplification, and they were only specific to DHBV, with no cross-reactivity with duck hepatitis A virus subtype 1 and duck hepatitis A virus subtype 3, goose parvovirus, duck enteritis virus, duck circovirus, or Riemerella anatipestifer. In this study, we offer a simple, fast, and accurate assay method to identify DHBV in clinical serum samples of ducks and geese, which would be suitable for widespread application in field clinics. Full article

Duck enteritis virus and Pasteurella multocida are major duck pathogens that induce duck plague and fowl cholera, respectively, in ducks and other waterfowl populations, leading to high levels of morbidity and mortality. Immunization with live attenuated DEV vaccine containing P. multocida outer membrane protein H (OmpH) can provide the most effective protection against these two infectious diseases in ducks. We have recently reported the construction of recombinant DEV expressing P. multocida ompH gene using the CRISPR/Cas9 gene editing strategy with the goal of using it as a bivalent vaccine that can simultaneously protect against both infections. Here we describe the findings of our investigation into the systemic immune responses, potency and clinical protection induced by the two recombinant DEV-ompH vaccine constructs, where one copy each of the ompH gene was inserted into the DEV genome at the UL55-LORF11 and UL44-44.5 intergenic regions, respectively. Our study demonstrated that the insertion of the ompH gene exerted no adverse effect on the DEV parental virus. Moreover, ducklings immunized with the rDEV-ompH-UL55 and rDEV-ompH-UL44 vaccines induced promising levels of P. multocida OmpH-specific as well as DEV-specific antibodies and were completely protected from both diseases. Analysis of the humoral and cellular immunity confirmed the immunogenicity of both recombinant vaccines, which provided strong immune responses against DEV and P. multocida. This study not only provides insights into understanding the immune responses of ducks to recombinant DEV-ompH vaccines but also demonstrates the potential for simultaneous prevention of viral and bacterial infections using viral vectors expressing bacterial immunogens. Full article

Duck enteritis virus (DEV) can infect several types of waterfowl can cause high mortality and huge economic losses to the global waterfowl industry. Type I interferons (IFN) are important for host defense against virus infection through induction of antiviral effector molecules. TANK-binding kinase 1 (TBK1) is a key kinase required for the induction of type I IFNs; however, the role of TBK1 on DEV infection remains unclear. Here, we observed that the expression levels of TBK1 and IFN-β were upregulated during DEV infection in vivo and in vitro. Thus, the function of TBK1 on DEV infection was determined. The results showed that overexpression of TBK1 reduced DEV infection and knockdown of TBK1 resulted in the increased of DEV infection. Additionally, TBK1 overexpression upregulated the expression of IFN-β and a few interferon-stimulated genes (ISGs), which thus inhibited the synthesis of DEV glycoprotein B. On the other hand, the TBK1 inhibitor Amlexanox down-regulated the expression levels of IFN-β and IRF3. Interestingly, the expression levels of MAVS and GSK-3β were decreased in the cells treated with Amlexanox. Furthermore, overexpression of TBK1 activated the expression of upstream molecules MAVS and GSK-3β. Whereas, the expression of TBK1, IRF3 and IFN-β was inhibited by the GSK-3β inhibitor SB216763. Our findings suggest that DEV–stimulated TBK1 may be involved in defense against DEV infection. Full article

Duck enteritis virus (DEV) and Pasteurella multocida, the causative agent of duck plague and fowl cholera, are acute contagious diseases and leading causes of morbidity and mortality in duck. The NHEJ-CRISPR/Cas9-mediated gene editing strategy, accompanied with the Cre–Lox system, have been employed in the present study to show that two new sites at UL55-LORF11 and UL44-44.5 loci in the genome of the attenuated Jansen strain of DEV can be used for the stable expression of the outer membrane protein H (ompH) gene of P. multocida that could be used as a bivalent vaccine candidate with the potential of protecting ducks simultaneously against major viral and bacterial pathogens. The two recombinant viruses, DEV-OmpH-V5-UL55-LORF11 and DEV-OmpH-V5-UL44-44.5, with the insertion of ompH-V5 gene at the UL55-LORF11 and UL44-44.5 loci respectively, showed similar growth kinetics and plaque size, compared to the wildtype virus, confirming that the insertion of the foreign gene into these did not have any detrimental effects on DEV. This is the first time the CRISPR/Cas9 system has been applied to insert a highly immunogenic gene from bacteria into the DEV genome rapidly and efficiently. This approach offers an efficient way to introduce other antigens into the DEV genome for multivalent vector. Full article

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 10³ TCID₅₀/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions. Full article

Duck Tembusu virus (DTMUV), a pathogenic member of the Flavivirus family, was first discovered in the coastal provinces of South-Eastern China in 2010. Many previous reports have clearly shown that some Flaviviruses utilize several endocytic pathways to enter the host cells, however, the detailed mechanism of DTMUV entry into BHK-21 cells, which is usually employed to produce commercial veterinary vaccines for DTMUV, as well as of other Flaviviruses by serial passages, is still unknown. In this study, DTMUV entry into BHK-21 cells was found to be inhibited by noncytotoxic concentrations of the agents chloroquine, NH₄Cl, and Bafilomycin A1, which blocked the acidification of the endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of DTMUV virions to pH 5.0 in the absence of host cell membranes decreased entry into cells by 65%. Furthermore, DTMUV infection was significantly decreased by chlorpromazine treatment, or by knockdown of the clathrin heavy chain (CHC) through RNA interference, which suggested that DTMUV entry depends on clathrin. Taken together, these findings highlight that a low endosomal pH is an important route of entry for DTMUV. Full article

Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system. Full article

Duck Tembusu virus (DTMUV) is a recently emerging pathogenic flavivirus that has resulted in a huge economic loss in the duck industry. However, no vaccine is currently available to control this pathogen. Consequently, a practical strategy to construct a vaccine against this pathogen should be determined. In this study, duck enteritis virus (DEV) was examined as a candidate vaccine vector to deliver the envelope (E) of DTMUV. A modified mini-F vector was inserted into the SORF3 and US2 gene junctions of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The envelope (E) gene of DTMUV was inserted into the C-KCE genome through the mating-assisted genetically integrated cloning (MAGIC) strategy, resulting in the recombinant vector, pBAC-C-KCE-E. A bivalent vaccine C-KCE-E was generated by eliminating the BAC backbone. Immunofluorescence and western blot analysis results indicated that the E proteins were vigorously expressed in C-KCE-E-infected chicken embryo fibroblasts (CEFs). Duck experiments demonstrated that the insertion of the E gene did not alter the protective efficacy of C-KCE. Moreover, C-KCE-E-immunized ducks induced neutralization antibodies against DTMUV. These results demonstrated, for the first time, that recombinant C-KCE-E can serve as a potential bivalent vaccine against DEV and DTMUV. Full article

With total dependence on the host cell, several viruses have adopted strategies to modulate the host cellular environment, including the modulation of microRNA (miRNA) pathway through virus-encoded miRNAs. Several avian viruses, mostly herpesviruses, have been shown to encode a number of novel miRNAs. These include the highly oncogenic Marek’s disease virus-1 (26 miRNAs), avirulent Marek’s disease virus-2 (36 miRNAs), herpesvirus of turkeys (28 miRNAs), infectious laryngotracheitis virus (10 miRNAs), duck enteritis virus (33 miRNAs) and avian leukosis virus (2 miRNAs). Despite the closer antigenic and phylogenetic relationship among some of the herpesviruses, miRNAs encoded by different viruses showed no sequence conservation, although locations of some of the miRNAs were conserved within the repeat regions of the genomes. However, some of the virus-encoded miRNAs showed significant sequence homology with host miRNAs demonstrating their ability to serve as functional orthologs. For example, mdv1-miR-M4-5p, a functional ortholog of gga-miR-155, is critical for the oncogenicity of Marek’s disease virus. Additionally, we also describe the potential association of the recently described avian leukosis virus subgroup J encoded E (XSR) miRNA in the induction of myeloid tumors in certain genetically-distinct chicken lines. In this review, we describe the advances in our understanding on the role of virus-encoded miRNAs in avian diseases. Full article