Fowl adenoviruses (FAdV), detected during routine diagnostic investigations from 38 countries (5 continents) over a decade, were partially sequenced and grouped by phylogenetic analysis. The partial polymerase gene nucleotide sequences of the 365 fowl adenovirus isolates resulted in the following species distribution: 11% FAdV-A; 3% FAdV-B; 2% FAdV-C; 34% FAdV-D; and 50% FAdV-E. Noticeably, only 79 of the detected strains could be associated with adenovirus-specific pathologic conditions: 62 (79%) with inclusion body hepatitis; 9 (11%) with gizzard erosion; and 8 (10%) with hepatitis hydropericardium syndrome. The remainder of the FAdV strains was detected as concomitant infection from other disease conditions almost exclusively in boilers of 27 to 42 d of age: the majority of them was FAdV-E followed by FAdV-D, and to a lesser extent of FAdV-A, B, and C, the latter ones have not been associated with any of the established adenovirus-caused syndromes in our collection. The highest ratio of coinfections was observed for FAdV-B (62%), while it was about 30% for the rest of the FAdV species. The most frequent coinfection, in connection with all FAdV species, was with the avian infectious bronchitis virus. The presented database will serve as the basis for comparative whole genome and cross-neutralization analysis of selected FAdV isolates.

The H9N2 subtype of low-pathogenic avian influenza viruses (LPAIV) is a widespread pathogen of poultry that can also infect humans. The characterization of viral infections is a complex process, involving clinical, pathological, and virological investigations. The aim of this study was to adapt and optimize an immunohistochemical (IHC) technique developed for LPAIVs specifically for the detection of H9N2 virus antigens in infected tissues. Twenty-one-day-old broiler chickens were inoculated with three different strains of H9N2 virus by different infection routes (i.e., intranasal-intratracheal and intravenous) or co-infected with infectious bronchitis virus (IBV) and observed for 11 days post infection. The suggested IHC protocol was modified: (i) DAB (diamino-benzidine) was substituted with AEC (3-amino-9-ethyl carbazole) as chromogen; and (ii) indirect two-step immune reactions of monoclonal primary and peroxidase-labeled anti-mouse secondary antibodies were used instead of avidin–biotin complexes. Avian influenza virus antigen appears as a red precipitate in the nuclei of affected cells but can also be identified in the cytoplasm. Mild hyperemia and congestion were observed in the trachea, air sac, and lungs of the challenged birds, and fibrinous exudate was found at the bifurcation in a few cases. Neither gross pathological nor IHC lesions were found in the control group. Using the optimized protocol and an associated scoring scheme, it was demonstrated that the H9N2 strains tested exhibited respiratory and urinary tract tropism irrespective of the route of inoculation. On day 5, viral antigen was detected in the respiratory tract and kidney in 30–50% of the samples. On day 11, no IHC signal was observed, indicating the lack of viral replication. Slight differences in viral antigen expression were found between the different H9N2 virus strains, but, in contrast to highly pathogenic avian influenza (HPAI), no viral antigen was detected in the brain and pancreas. Thus, IHC can be considered as an informative, visual addition to the toolkit for the characterization of H9N2 LPAIV infections.

A real-time reverse transcriptase (RT)-PCR assay, applying light upon extension (LUX) fluorogenic primers, was developed for rapid and efficient detection of Newcastle disease virus (NDV). The method, which targets the fusion (F) protein gene of the viral genome, gave positive signal with all NDV isolates tested (32/32), while negative results were obtained with heterologous pathogens (35/35), including 13 avian influenza virus isolates. The detection limit of the assay was approximately 10^+1.2 egg infectious dose (EID)₅₀/0.2 ml and 10^+2.2 EID₅₀/0.2 ml for virus suspensions and spiked chicken fecal samples, respectively. As expressed in plasmid copy number, the procedure has a sensitivity of approximately 20 copies of the plasmid harboring the target gene. Due to its high specificity, sensitivity, and relative simplicity, the LUX RT-PCR assay provides a novel, rapid, and practical tool for the detection of NDV.

Infectious bursal disease virus (IBDV) is a major threat to the poultry industry globally, represented by a variety of genetic, pathogenic, and antigenic variants. The recognition of the infection may be challenging due to several factors, as the virulence of the strain, age, and immune status of the birds at infection, to name the most important ones.

Avian infectious bronchitis (IB) is among the major viral respiratory and reproductive diseases of chickens caused by Avian coronavirus. In the African continent, IB was first described in countries located in the Mediterranean basin. In other parts of the continent, the epidemiological situation of IB remains unclear. In this study, the complete genome sequences of five IBV strains, originating from the sub-Saharan area were determined. Phylogenetic analysis based on the full-length S1 sequences identified three lineages (GI-14, GI-16, and GI-19) common in Africa and revealed that a strain, D2334/11/2/13/CI, isolated in Ivory Coast may represent a novel lineage within genotype GI. The maximum inter- and intragenotype sequence identities between this strain and other IBVs were 67.58% and 78.84% (nucleotide) and 64.44% and 78.6% (amino acid), respectively. The whole-genome nucleotide identity of the novel variant shared the highest values with a reference Belgian nephropathogenic strain (B1648, 92.4%) and with another study strain from Ivory Coast (D2334/12/2/13/CI, 94.6%). This study illustrates the importance of epidemiological monitoring of IBV in sub-Saharan Africa, as the area may serve as a focal point for newly emerging viral lineages.

Routine molecular diagnostic testing by our laboratory, based on using a primer pair with conservative binding sites on the spike glycoprotein coding sequence, has indicated the recurring of a unique phylogenetic cluster of chicken infectious bronchitis viruses (IBV) in the Middle East since 2010. The nearly full-length S1 subunit of the spike gene phylogeny of selected strains, however, split up this grouping, suggesting potential recombination in the S1 gene. In order to clarify this, various bioinformatic analyses of the strains were carried out, which confirmed this supposition. Two patterns of recombination were found among the strains, one of which could also be identified in GenBank-deposited IBV sequences from the region. These findings demonstrate that IBV strains of different recombinant patterns occur simultaneously in the same geographic region and could circulate for an extended period of time, thus contributing to the knowledge on IBV evolution.