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.

Crude Inactivated Influenza A Virus Adjuvated with a Bispecific Antibody Complex Targeting Chicken CD40 and AIV M2e Confers Protection Against Lethal HPAI Challenge in Chickens.

In vivo targeting an immunogen to the CD40 receptor expressed on professional antigen-presenting cells (APCs) dramatically enhances speed, magnitude, and quality of the immune response. Our previous evaluation of this strategy in poultry was limited to immunogenicity studies using CD40-targeted synthetic peptides, which demonstrated significant antigen-specific serum IgG and tracheal IgA levels <1 week after primary administration. In this study, this antibody-guided immunization strategy was modified to permit incorporation of inactivated highly pathogenic avian influenza virions (in lieu of short synthetic peptides) as the immunogen by simply mixing a bispecific antibody complex (anti-CD40/M2e) with crude inactivated virus before injection. Adjuvated avian influenza virus (AIV) induced significant hemagglutination inhibition titers up to 6 weeks postimmunization. In efficacy studies, administration of a single vaccine dose yielded 56%-64% survival against challenge with highly pathogenic H5N1, and 100% protection was achieved upon boosting. These results represent a feasible strategy to effectively target whole inactivated influenza A virus to chicken APCs, regardless of AIV clade and without phenotyping or purifying the virus from crude allantoic fluid. The data represent proof of principle for the unique prophylactic efficacy and versatility of a CD40-targeting adjuvation strategy that can in principle also be harnessed in other poultry vaccines.

Identification of avian influenza virus subtype H9N2 in chicken farms in Indonesia.

Avian influenza virus subtype H9N2 (AIV-H9N2) has become established in domestic poultry in Asia and Africa. AIV-H9N2 has not been reported previously in Indonesia. Here we describe the presence of AIV-H9N2 in chicken farms in Indonesia. Ninety-nine cases were observed in various provinces in Indonesia. Clinical signs, pathologic lesions and egg production were recorded. Confirmation was made using virus isolation, reverse transcriptase PCR (RT-PCR), and sequencing. To construct hemaglutinin (HA) phylogeny, the secondary data of Eurasian lineages were downloaded from GenBank. For neuraminidase, five sequences with the highest similarities with every sequence found in this study were downloaded. Phylogeny was inferred using Neighbor-Joining method in MEGA6 package. Forty-nine AIV-H9N2-positive cases were observed, of which 35 were tested positive for AIV-H9N2 only. The age of the infected chickens was 43.17 ± 16.56 weeks, and their egg production was 35.85 ± 17.80% lower than before outbreak. BLAST search revealed that the nucleotide sequence of the HA-encoding gene identified in this study shared 98% sequence identity with that of A/Muscovy duck/Vietnam/LBM719/2014(H9N2), while its neuraminidase-encoding gene sequences shared 94%, 98%, and 100% identities with three different influenza viruses. The phylogeny shows that the HA of AIV-H9N2 found in this study forms distinct cluster with some Vietnam and China's sequence data. The NA sequence data form three distinct clusters. We conclude that AIV-H9N2 is widespread in many provinces in Indonesia. To lessen economic losses to the poultry industry, flock biosecurity and vaccination against this virus subtype should be implemented rapidly. Thorough and rigid AIV surveillance is paramount to prevent further veterinary and public health consequences of the circulation of this virus in Indonesia.

Molecular analysis of hemagglutinin-1 fragment of avian influenza H5N1 viruses isolated from chicken farms in Indonesia from 2008 to 2010.

Highly pathogenic avian influenza virus of subtype H5N1 (AIV-H5N1) has been circulating in Indonesia since 2003. To understand the genetic diversity of these viruses, and to predict vaccine efficacy, the hemaglutinin-1 (HA-1) fragment of viruses isolated from chicken farms in Indonesia from 2008 to 2010 was sequenced and analyzed. The effects of these molecular changes were investigated in challenge experiments and HI assays of homologous and heterologous strains. Molecular analysis showed that these AIV-H5N1 isolates had evolved into three distinct sub-lineages from an ancestor circulating since 2003. Although no significant positive selection of residues was detected, 12 negatively selected sites were identified (p<0.05). Moreover, four sites showed evidence of significant episodic diversifying selection. The findings indicated complete protectivity and high HI titers with homologous strains, compared with protectivity ranging from 40 to 100% and lower HI titers with heterologous strains resulting from polymorphisms at antigenic sites. Our findings provide valuable insight into the molecular evolution of AIV and have important implications for vaccine efficacy and future vaccination strategies.