Domain in Fiber-2 interacted with KPNA3/4 significantly affects the replication and pathogenicity of the highly pathogenic FAdV-4.

The outbreaks of hepatitis-hydropericardium syndrome (HPS) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) have caused a huge economic loss to the poultry industry globally since 2013. Although the Fiber-2 has been identified as a key virulent related factor for FAdV-4, little is known about its molecular basis. In this study, we identified the efficient interaction of the Fiber-2 with the karyopherin alpha 3/4 (KPNA3/4) protein via its N-terminus of 1-40aa. The analysis of the overexpression and knockout of KPNA3/4 showed that KPNA3/4 could efficiently assist the replication of FAdV-4. Moreover, a fiber-2-edited virus FAV-4_Del with a deletion of 7-40aa in Fiber-2 was rescued through the CRISPR-Cas9 technique. In comparison with the wild type FAdV-4, FAV-4_Del was highly attenuated in vitro and in vivo. Notably, the inoculation of FAV-4_Del in chickens could provide full protection against the lethal challenge with the wild type FAdV-4. All these findings not only give novel insights into the molecular basis for the pathogenesis of Fiber-2 but also provide efficient targets for developing antiviral strategies and live-attenuated vaccine candidates against the highly pathogenic FAdV-4.

Spotlight on avian pathology: fowl adenovirus (FAdV) in chickens and beyond - an unresolved host-pathogen interplay.

Fowl adenovirus (FAdV) infections in chickens have undergone substantial changes in recent decades, driven by host and pathogen factors. Based on the pathogenesis of inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS), modern broilers are much more inclined to have difficulties keeping the metabolic homeostasis, whereas adenoviral gizzard erosion (AGE) is noticed equally in broilers and egg-layers. Defining the importance of certain serotypes for specific FAdV diseases is a major achievement of recent years but the isolation of viruses from clinically healthy birds remains unexplained, as virulence factors are hardly known and continue to be a "black box". Together with further studies on pathogenesis of FAdV-induced diseases, such knowledge on virulence factors would help to improve protection strategies, which presently mainly concentrate on autogenous vaccines of breeders to prevent vertical transmission.

Fowl Adenovirus Serotype 4 Influences Arginine Metabolism to Benefit Replication.

Hydropericardium syndrome (HPS) is caused by fowl adenovirus serotype 4 (FAdV-4). HPS has caused outbreaks in Chinese populations of broiler chickens since 2015. However, little is known about the molecular mechanisms underlying HPS. In this study, we used transcriptomic analysis to screen differentially expressed genes (DEGs) in the livers of FAdV-4-infected and noninfected chicks. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the gene network associated with the arginine metabolism pathway was enriched in livers infected by FAdV-4; 10 genes were downregulated and 8 genes were upregulated in these livers when compared to noninfected livers. The DEGs identified in livers were reanalyzed by real-time fluorescence quantitative PCR (qPCR); results indicated that the mRNA levels of the DEGs concurred with the data derived from KEGG analysis. Next, we used qPCR to detect the DEGs of the arginine metabolism pathway in a hepatocellular carcinoma cell line (LMH) after infection with FAdV-4 for 24 hr; this also indicated that the mRNA levels of the DEGs concurred with that seen in the liver. We also used si-RNA oligonucleotides to knock down the mRNA levels of iNOS in LMH cells infected with FAdV-4 and found that the viral load of FAdV-4 was increased. Further investigation revealed that the addition of 240 µg/ml of arginine into the culture medium of LMH cells infected with FAdV-4 for 24 hr led to a significant increase in the mRNA levels of iNOS but a significant reduction in the viral load of FAdV-4. Therefore, our data indicated that when broiler chickens become infected with FAdV-4, the arginine metabolic pathway in the liver becomes dysfunctional and the iNOS mRNA level decreases. This will add benefit to the replication of FAdV-4 but can be inhibited by the addition of an appropriate amount of arginine.

El adenovirus del pollo serotipo 4 influye en el metabolismo de la arginina para favorecer su replicación. El síndrome de hidropericardio (HPS) es causado por el adenovirus del pollo serotipo 4 (FAdV-4). Este síndrome ha causado brotes en las poblaciones de pollo de engorde en China desde 2015. Sin embargo, se conoce poco sobre los mecanismos moleculares subyacentes al hidropericardio. En este estudio, se utilizó el análisis transcriptómico para seleccionar genes expresados en forma diferencial (DEGs) en los hígados de pollos infectados y no infectados con el adenovirus serotipo 4. El análisis mediante la Enciclopedia de Genes and Genomas de Kyoto (KEGG) mostró que la red de genes asociada con la ruta del metabolismo de la arginina se enriqueció en hígados infectados por el adenovirus serotipo 4. Diez genes fueron regulados a la baja y ocho genes fueron regulados a la alta en estos hígados en comparación con los hígados de aves no infectadas. Los genes expresados en forma diferencial identificados en los hígados se volvieron a analizar mediante un método cuantitativo de PCR de fluorescencia en tiempo real (qPCR). Los resultados indicaron que los niveles de ARNm de los genes expresados en forma diferencial coincidían con los datos derivados del análisis la Enciclopedia de Genes and Genomas de Kyoto. Posteriormente, se utilizó qPCR para detectar los genes expresados en forma diferencial de la vía del metabolismo de la arginina en una línea celular de carcinoma hepatocelular (LMH) infectadas con el adenovirus del pollo serotipo 4 durante 24 horas. Esto también indicó que los niveles de ARNm de los genes expresados en forma diferencial coincidían con los observados en el hígado. También se utilizaron oligonucleótidos de ARN para bloquear los niveles de ARN mensajero de iNOS en células LMH infectadas con el adenovirus del pollo serotipo 4 y se descubrió que la carga viral del adenovirus aumentó. La investigación adicional reveló que la adición de 240 µg/ml de arginina en el medio de cultivo de las células LMH infectadas con el adenovirus serotipo 4 durante 24 horas condujo a un aumento significativo en los niveles de ARN mensajero de iNOS pero con una reducción significativa en la carga viral del adenovirus serotipo 4 Por lo tanto, estos datos indican que cuando los pollos de engorde se infectan con adenovirus del pollo serotipo 4, la vía metabólica de la arginina en el hígado se vuelve disfuncional y el nivel de ARN mensajero de iNOS disminuye. Esto favorecerá la replicación del adenovirus del pollo serotipo 4, pero puede inhibirse mediante la adición de una cantidad adecuada de arginina.

Gizzard Erosion Associated with Fowl Adenovirus Infection in Slaughtered Broiler Chickens in Iran.

Gizzard erosions have been noticed in slaughtered broiler chickens during inspection at a processing plant in Iran. The condition was detected in piled gizzards derived from seven commercial broiler farms brought to slaughter on the same day. In total, 48 gizzards with lesions underwent thorough pathologic and virologic investigation. Perforation, roughening, and discoloration of the koilin layer as well as inflammation of the mucosa were observed macroscopically. Histologic examination showed dissociation of and cellular debris in the koilin layer accompanied by a loss and degeneration of glandular epithelium with mild to marked infiltration of inflammatory cells in the mucosa, submucosa, and muscular layer. Fowl adenovirus serotypes 1 (FAdV-1), 11 (FAdV-11), and 8a (FAdV-8a) were found in 13, 12, and 1 gizzard(s), respectively. Therein included were two gizzards that showed mixed infections with FAdV-1 and FAdV-11. Detailed analysis of the hexon gene revealed that the Iranian FAdV-1 isolates could be divided into two subclusters, more closely related to either the European (CELO) or the Asian (Ote) FAdV-1 reference strains. The present study, for the first time, describes not only the appearance of gizzard erosion but also the isolation of FAdV-1 and FAdV-8a from broilers in Iran and offers insights on the epidemiology of FAdV infection in Iranian flocks.

Erosión de molleja asociada con infección por adenovirus del pollo en pollos de engorde procesados en Irán. Se han observado erosiones de molleja en pollos de engorde sacrificados durante la inspección en una planta de procesamiento en Irán. La condición se detectó en mollejas apiladas derivadas de siete granjas comerciales de pollos de engorde que fueron sacrificados el mismo día. En total, 48 mollejas con lesiones se sometieron a una exhaustiva investigación patológica y virológica. Se observó macroscópicamente perforación, rugosidad y la decoloración de la capa de queratina, así como inflamación de la mucosa. El examen histológico mostró disociación y restos celulares en la capa de queratina acompañada por una pérdida y degeneración del epitelio glandular con infiltración leve a marcada de células inflamatorias en la mucosa, la submucosa y la capa muscular. Se encontraron aviadenovirus del pollo de los serotipos 1 (FAdV-1), 11 (FAdV-11) y 8a (FAdV-8a) en trece, doce y una molleja (s), respectivamente. Se incluyeron dos mollejas que mostraban infecciones mixtas con FAdV-1 y FAdV-11. El análisis detallado del gene de la proteína del hexon reveló que los aislamientos iraníes del serotipo FAdV-1 se dividieron en dos subgrupos, más estrechamente relacionados con las cepas de referencia del serotipo 1 de Europa (CELO), o de Asia (Ote). El presente estudio describe por primera vez, no solo la aparición de la erosión de la molleja, sino también el aislamiento de FAdV-1 y FAdV-8a de los pollos de engorde en Irán y ofrece información sobre la epidemiología de la infección por FAdV en parvadas iraníes.

Epizootiology, Clinical Signs, and Phylogenetic Analysis of Fowl Adenovirus in Chicken Farms in Indonesia from 2018 to 2019.

Fowl adenovirus (FAdV) infection is an emerging problem in the world poultry industry, especially in broilers, as the causal agent of inclusion body hepatitis or hepatitis-hydropericardium syndrome. From December 2017 to January 2019, we recorded 116 cases of suspected hepatitis-hydropericardium syndrome in chicken farms throughout Indonesia. Necropsy was done on each farm site with three to five freshly dead birds per farm. Tissue samples were collected in virus transport medium and frozen at -20 C. The virus was cultivated in 9-day-old fertilized specific-pathogenic-free chicken eggs. FAdV was detected using polymerase chain reaction with a published primer set. The polymerase chain reaction products were sequenced and subjected to a BLAST search. The phylogeny was inferred using the neighbor-joining method and tested using the bootstrap test. FadV-D and -E are present in Indonesia and confirmed in 40 of 116 suspected cases. The affected chicken ages were 27.27 ± 8.94 days. Most affected farms were raising broiler chickens. The only typical clinical sign was unusual daily mortality of >1%, while the three most frequent pathologic lesions were swelling and hemorrhage of kidney and liver, as well as hydropericardium. To reduce economic loss, a vaccine should be developed immediately.

Epizootiología, signos clínicos y análisis filogenético del adenovirus de pollos en granjas avícolas en Indonesia entre los años 2018 a 2019. La infección por adenovirus de aves (FAdV) es un problema emergente en la industria avícola mundial, especialmente en pollos de engorde, como agente causal de la hepatitis por cuerpos de inclusión y del síndrome de hepatitis-hidropericardio. Desde diciembre del año 2017 hasta enero de 2019, se registraron 116 casos sospechosos de síndrome de hepatitis-hidropericardio en granjas avícolas en toda Indonesia. Se realizaron necropsias en los sitios de las granjas con tres a cinco aves recién muertas por granja. Se recogieron muestras de tejido en medio de transporte viral y se congelaron a -20 C. El virus se cultivó en huevos embrionados de aves libres de patógenos específicos de 9 días de edad. Se detectaron adenovirus del pollo usando una reacción en cadena de la polimerasa con un conjunto de iniciadores previamente publicados. Los productos de reacción en cadena de la polimerasa se secuenciaron y se sometieron a una búsqueda mediante la herramienta básica de búsqueda de alineación local (BLAST). La filogenia se infirió usando el método Neighbor-Joining y se evaluó mediante la prueba bootstrap. Se determinó la presencia de adenovirus del pollo D y E en Indonesia y se confirmó su presencia en 40 de 116 casos sospechosos. Las edades de los pollos afectados fueron de 27.27 ± 8.94 días. Las granjas más afectadas fueron de pollos de engorde. El único signo clínico típico fue una mortalidad diaria inusual mayor al 1%, mientras que las tres lesiones patológicas más frecuentes fueron inflamación y hemorragia de riñón e hígado, así como hidropericardio. Para reducir la pérdida económica, se debe desarrollar una vacuna de inmediato.

Pathogenesis of Hypervirulent Fowl Adenovirus Serotype 4: The Contributions of Viral and Host Factors.

Since 2015, severe outbreaks of hepatitis-hydropericardium syndrome (HHS), caused by hypervirulent fowl adenovirus serotype 4 (FAdV-4), have emerged in several provinces in China, posing a great threat to poultry industry. So far, factors contributing to the pathogenesis of hypervirulent FAdV-4 have not been fully uncovered. Elucidation of the pathogenesis of FAdV-4 will facilitate the development of effective FAdV-4 vaccine candidates for the control of HHS and vaccine vector. The interaction between pathogen and host defense system determines the pathogenicity of the pathogen. Therefore, the present review highlights the knowledge of both viral and host factors contributing to the pathogenesis of hypervirulent FAdV-4 strains to facilitate the related further studies.

Immunosuppressive potential of fowl adenovirus serotype 4.

Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium syndrome. To clarify the effects of FAdV-4 on immune organs in birds, we conducted a detailed examination of dynamic morphology and damage mechanisms in chickens randomly divided into 4 groups (FAdV-4, vaccination, FAdV-4 plus vaccination, and control). FAdV-4 caused the depletion of lymphocytes and subsequent growth impairment in the thymus and bursa. Chickens infected with FAdV-4 and subjected to vaccination experienced greater inhibition of antibody responses to inactivated vaccines against Newcastle disease and avian influenza virus subtype H9 than uninfected and vaccinated chickens. The mechanisms underlying adenovirus-mediated lymphoid organ damage were further investigated via transferase-mediated dUTP nick-end labeling and apoptotic genes transcription analyses. Notably, lymphocytes apoptosis in lymphoid organs and expression of specific gene transcripts was significantly upregulated after infection (P < 0.05). Furthermore, increased expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α mRNA was observed (P < 0.05), compared to the control group. Our collective findings suggested that FAdV-4 caused structural and functional damage of immune organs via apoptosis along with induction of a severe inflammatory response.

A novel monoclonal antibody efficiently blocks the infection of serotype 4 fowl adenovirus by targeting fiber-2.

A recent outbreak of hepatitis-hydropericardium syndrome caused by serotype 4 fowl adenovirus (FAdV-4) has resulted in significant economic losses to the poultry industry worldwide. However, little is known about the molecular pathogenesis of FAdV-4. In this study, a novel monoclonal antibody (mAb) targeting the fiber-2 protein of FAdV-4 was generated, mAb 3C2. Indirect immunofluorescence assay showed that mAb 3C2 neither reacted with serotype 8 fowl adenovirus (FAdV-8) nor reacted with the fiber-1 protein of FAdV-4; it specifically reacted with the fiber-2 protein of FAdV-4. Notably, mAb 3C2 could efficiently immunoprecipitate the fiber-2 protein in chicken liver cells either infected with FAdV-4 or transfected with pcDNA3.1-Fiber2. Moreover, mAb 3C2 demonstrated marked neutralizing activity against FAdV-4 and could efficiently inhibit the infection of FAdV-4 in vitro. Using truncated fiber-2 constructs, the epitope recognized by mAb 3C2 was determined to be located between amino acids 416-448 at the C-terminus of fiber-2. Our data not only provide a foundation for the establishment of a rapid fiber-2 peptide-based diagnostic assay for FAdV-4 but also highlight the critical role of the fiber-2 protein in mediating infection by FAdV-4. Furthermore, the epitope recognized by 3C2 might serve as a novel target for the development of a vaccine targeting FAdV-4.

Fowl adenovirus: history, emergence, biology and development of a vaccine against hydropericardium syndrome.

The poultry industry has emerged as one of the largest and fastest growing public sectors in the developed and developing countries. Unfortunately, this industry is under a major threat from diseases that are viral (Newcastle disease, infectious bursal disease, influenza, hydropericardium syndrome), bacterial (colibacillosis, pasteurellosis, salmonellosis, mycoplasmosis), parasitic (coccidiosis, histoplasmosis) or nutritional (dyschondroplasia, osteoporosis). Among these diseases, hydropericardium syndrome (HPS) is one of the important emerging diseases occurring in the specific areas of the world where broilers (chickens) are reared under intensive conditions. HPS was first observed in 1987 at Angara Goth, an area near Karachi, Pakistan, where broilers are raised. Since then, HPS has been reported in many countries of the world. From these reported cases, an adenovirus that was either isolated from or visualized electron microscopically in the liver of affected broilers has been implicated in the syndrome. The syndrome has been reproduced by inoculation of isolated fowl adenovirus (FAdV) strains, and hence, the syndrome is also called infectious hydropericardium syndrome. To our knowledge, HPS has not been observed in humans, so it is not considered a zoonotic disease, but it is of economic importance and causes huge losses to the poultry industry. Efforts have been made to develop conventional vaccines against this disease, which were formulated from infected liver homogenate. Formalin-inactivated liver organ vaccines have failed to protect the poultry industry. Hence, there is a dire need to develop a suitable vaccine to combat this disease. Currently, recombinant vaccine candidates are being developed by using molecular biology and biotechnological approaches for the prevention and control of infectious diseases, including HPS. Therefore, it is suggested that the immunogenicity of these recombinant proteins should be evaluated for their use as subunit vaccines.

Real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses (FAdV-A to FAdV-E).

The present study describes the development of a SYBR Green based real-time polymerase chain reaction (real-time PCR) for detection and quantitation of all fowl adenovirus (FAdV) species. Primers were designed based on conserved nucleotide sequences within the 52K gene. Ten-fold serial dilutions of a vector DNA were used as standard for quantitation. The real-time PCR had an efficiency of 98%, a regression squared value of 0.999 and showed a range of 6.73-6.73×10(8) copies of FAdV DNA per reaction. The assay was highly specific for FAdVs and an exact quantitation of all 5 FAdV species (FAdV-A to FAdV-E) could be demonstrated. It was shown, that twelve FAdV serotypes (FAdV-1 to 8a, and 8b to 11) were detectable and quantifiable. Other viral genomes as well as uninfected chicken embryo liver (CEL) cells did not produce positive signal. Cloacal swabs were taken during the animal experiment, which was performed with all FAdV species. Shedding of FAdVs was investigated in cell culture, by conventional PCR and by the developed real-time PCR. The real-time PCR was found more sensitive than cell culture and conventional PCR. Detection and quantitation of FAdVs in different type of samples was possible by the new real-time PCR.

First step in characterization of cis-acting sequences involved in fowl adenovirus 1 (CELO) packaging and its effect on the development of a helper-dependent vector strategy.

Adenovirus-based vectors are widely developed for potential utilization as vectors in vaccine and gene therapy strategies. We focused on developing a helper-dependent adenoviral (HD-Ad) vector for the potential use of CELO, a member of the Aviadenovirus genus, in avian species vaccination. Our aim was to localize sequences which could play an essential role in CELO genome encapsidation and, when deleted, was unable to produce viruses to develop a helper CELO virus. A panel of 6 mutants with deletions between nt 80 and 350 of the CELO genome was constructed and characterized for its ability to produce viable virus. To develop a helper-dependent adenoviral vector derived from CELO, a helper virus was developed by inserting loxP sequences around the region containing the identified putative packaging sequences. A LMH (Leghorn Male Hepatocarcinoma) cell line expressing Cre recombinase was developed to allow the excision of this region. We demonstrated that the region from nt 200 to 250 was important and the region from nt 250 to 300 at the left end of the CELO genome was essential for virus encapsidation. We also showed that the loxP-flanked region was efficiently removed in a Cre expressing cell line to produce a candidate helper virus.