An Amplicon-Based Application for the Whole-Genome Sequencing of GI-19 Lineage Infectious Bronchitis Virus Directly from Clinical Samples.

Infectious bronchitis virus (IBV) causes a highly contagious respiratory disease in chickens, leading to significant economic losses in the poultry industry worldwide. IBV exhibits a high mutation rate, resulting in the continuous emergence of new variants and strains. A complete genome analysis of IBV is crucial for understanding its characteristics. However, it is challenging to obtain whole-genome sequences from IBV-infected clinical samples due to the low abundance of IBV relative to the host genome. Here, we present a novel approach employing next-generation sequencing (NGS) to directly sequence the complete genome of IBV. Through in silico analysis, six primer pairs were designed to match various genotypes, including the GI-19 lineage of IBV. The primer sets successfully amplified six overlapping fragments by long-range PCR and the size of the amplicons ranged from 3.7 to 6.4 kb, resulting in full coverage of the IBV genome. Furthermore, utilizing Illumina sequencing, we obtained the complete genome sequences of two strains belonging to the GI-19 lineage (QX genotype) from clinical samples, with 100% coverage rates, over 1000 × mean depth coverage, and a high percentage of mapped reads to the reference genomes (96.63% and 97.66%). The reported method significantly improves the whole-genome sequencing of IBVs from clinical samples; thus, it can improve understanding of the epidemiology and evolution of IBVs.

Application of metagenomics for diagnosis of broilers displaying neurological symptoms.

BACKGROUND: Thirty-two-day-old broiler chickens at a farm located in northwestern South Korea displayed adverse neurological symptoms including limping, lying down, and head shaking. Approximately 2.1% of chickens died or were culled due to severe symptoms. Five carcasses were submitted to the Avian Disease Division of the Animal and Plant Quarantine Agency (APQA) for disease diagnosis. RESULTS: Broilers displayed severe pericarditis and perihepatitis associated with gross lesions. Broilers also displayed microscopic lesions in the cerebrum and in the granular layer of the cerebellum, which were associated with multifocal perivascular cuffing and purulent necrosis in the cerebrum, and severe meningitis with heterophil and lymphocyte infiltration. Staphylococcus spp. were identified in the liver and heart using bacteriological culture. PCR/RT-PCR assays revealed that broilers were negative for avian Clostridium botulinum, Newcastle disease virus, and avian encephalomyelitis virus. Bacterial and viral metagenomic analysis of brain sample further revealed the presence of Pseudomonas spp. and Marek's disease virus, which are known etiological agents of chicken meningoencephalitis. CONCLUSIONS: This study reports a diagnostic analysis of gross and histopathological lesions from 32-day-old broilers displaying unique neurological symptoms that revealed the presence of the several neurological diseases including meningoencephalitis. The causative agents associated with meningoencephalitis of broilers that had not been identified by routine diagnostic methods could be diagnosed by metagenomics, which proves the usefulness of metagenomics as a diagnostic tool for unknown neurological diseases in broilers.

The Development of Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assays for the Detection and Differentiation of Virulent Newcastle Disease Virus.

Newcastle disease (ND) is a highly pathogenic viral infection of poultry with significant economic impacts worldwide. Despite the widespread use of vaccines, ND outbreaks continue to occur even within vaccinated poultry farms. Furthermore, novel Newcastle disease virus (NDV) genotypes are emerging in poultry, increasing the need for the development of rapid, accurate, and simple diagnostic methods. We therefore developed two novel sets of visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays based on highly conserved regions of the HN and F genes. The limits of detection of the NDV-Common-LAMP assay, for all the NDV strains, were 103.0 EID50/0.1 mL for Kr005 and 102.0 EID50/0.1 mL for Lasota within 35 min. The sensitivity of the NDV-Patho-LAMP assay, used for the strain differentiation of virulent NDV, was 102.0 EID50/0.1 mL for Kr005. No amplification was detected for the non-NDV templates. Next, we probed 95 clinical strains and 7 reference strains with the RT-LAMP assays to assess the feasibility of their use in diagnostics. We observed no cross-reactivity across the 102 strains. Furthermore, there was 100% congruence between the RT-LAMP assays and full-length sequencing of the target genes, indicating the potential for visual RT-LAMP in the identification and differentiation of NDV. These novel RT-LAMP assays are ideally suited for the field or resource-limited environments to facilitate the faster detection and differentiation of NDV, which can reduce or avoid further spread.

Genetic Diversity of Fowlpox Virus and Putative Genes Involved in Its Pathogenicity.

To determine the genomic variations of fowlpox virus (FPV)-the largest, very ancient, and still harmful avian virus-the complete genomes of 21 FPVs were analyzed. The genomes showed low genetic diversity relative to their overall size. Our studies revealed that FPVs could phylogenetically be divided into two clades, based on their regional distribution, and comparative analysis showed that 40 putative proteins of FPV were associated with geographic differences in viruses, viral pathogenicity, or the onset of diphtheritic lesions. The strain, classified into a subgroup different from others in the genomic analysis, showed relatively low pathogenicity in chickens, and the onset of diphtheritic lesions was observed to be caused only by the specific strain. Despite genetic differences, some commercial vaccines are protective against virulent strains, and intact reticuloendotheliosis virus inserted into field FPV strains was activated but there was no enhancement of the pathogenicity of FPV. These findings will expand our knowledge of the viral proteome and help us understand the pathogenicity of FPV. IMPORTANCE This study aims at determining molecular candidates using comparative genomics to differentiate between the diphtheritic and cutaneous forms of FPV infection, in addition to their association with the pathogenicity of the virus. Full-genomic analyses of multiple fowlpox strains, including field viruses, isolated between 1960s and 2019, and vaccine strains showed the genetic diversity due to regional differences. Comparative genomic analysis offered the clues related to viral virulence. We believe that our study makes a significant contribution to the literature because we are the first to perform such an elaborate study that compares 21 FPVs to study and highlight their diversity, despite the high level of homology between them. Our results shall help provide insights for tackling FPV that has been taking a toll on the poultry for years now.

Histopathological Findings and Metagenomic Analysis of Esophageal Papillary Proliferation Identified in Laying Broiler Breeders.

White or pale-yellow nodules 2-7 mm in length were observed in the esophageal lumen in a number of laying broiler breeders with reduced laying rates. Metaplasia of the mucosal epithelial layer and mucous gland, as well as lymphocyte infiltration under the esophageal mucous gland and epithelial cell layer, were observed, which we found were caused by vitamin A deficiency. In one chicken, however, the stratified squamous epithelial cells of the esophagus were completely replaced by increased numbers of ducts/ductules, lymphocytes, and connective tissue, resulting in a papillary morphology. The ducts were surrounded by a fibrous stroma. Multiple hyperplasia of the esophageal gland was also observed and the esophageal glands were lined by a single layer of columnar cells, and a large number of lymphocytes were infiltrated into the submucosal layer. Based on the gross findings, this papillary proliferation was considered papilloma, but histopathologically, a mass composed of squamous epithelium was not observed. Therefore, the papillary lesion appeared as adenoma with squamous metaplasia of the esophageal gland and ectasia, or mucosal epithelial papillary hyperplasia, associated with chronic esophagitis. A metagenomic analysis of the esophagus samples from this chicken was performed to determine the infectious etiology. No viral cause was identified; however, a contributing role of Bradyrhizobium sp. could not be excluded. In this study, we report the first histopathological examination of a rare case of esophageal papillary proliferation in a chicken and highlight the importance of histopathological results for a definitive diagnosis of such cases.

DNA-dependent protein kinase is involved in heat shock protein-mediated accumulation of hypoxia-inducible factor-1alpha in hypoxic preconditioned HepG2 cells.

Hypoxic preconditioning may afford protection against subsequent lethal hypoxia. As hypoxic tolerance induces changes in the expression of genes involved in DNA damage and repair response pathways, we investigated whether DNA-dependent protein kinase (DNA-PK), one of the DNA double-strand break repair proteins, could be involved in hypoxic preconditioning-induced protective signaling cascades. We showed that induction of hypoxia-inducible factor-1alpha expression during hypoxic preconditioning by repeated hypoxic exposure was associated with increased mRNA and protein levels of DNA-PK catalytic subunit (DNA-PKcs) and Ku70/Ku80, the DNA-PK components, in human hepatoma HepG2 cells, followed by upregulation of Hsp70/Hsp90 and Bcl-2 and concurrent downregulation of Bax. Additionally, loss of DNA-PKcs led to attenuated expression of Hsp70/Hsp90, accelerated hypoxia-inducible factor-1alpha degradation, and increased susceptibility to hypoxia-induced cell death. We also found that the mRNA and protein levels of heat shock factor-1 (HSF1) were progressively increased with DNA-PK activation during hypoxic preconditioning, and inhibition of HSF1 function by KNK437 resulted in a significant decrease in the level of protein kinase Akt as well as of DNA-PKcs, with downregulation of Hsp70/Hsp90 and HIF-1alpha. Our results suggest the possibility that DNA-PK-mediated signaling pathway is required for the increase in HIF-1alpha expression through activation of HSF1 and subsequent upregulation of heat shock proteins after hypoxic reconditioning.

Camptothecin acts synergistically with imatinib and overcomes imatinib resistance through Bcr-Abl independence in human K562 cells.

In this study, we have tried to find new targets and effective drugs for imatinib-resistant chronic myelogenous leukemia (CML) cells displaying loss of Bcr-Abl kinase target dependence. The imatinib-resistant K562/R1, -R2 and -R3 cells showed profound declines of Bcr-Abl level and concurrently exhibited up-regulation of Bcl-2 and Ku70/80, and down-regulation of Bax, DNA-PKcs and BRCA1, suggesting that loss of Bcr-Abl after exposure to imatinib might be accompanied by other cell survival mechanism. K562/R3 cells were more sensitive to camptothecin (CPT)- and radiation-induced apoptosis than K562 cells, indicating hypersensitivity of imatinib-resistant cells to DNA damaging agents. Moreover, when K562 cells were treated with the combination of imatinib with CPT, the level of Bax and the cleavage of PARP-1 and DNA-PK were significantly increased in comparison with the effects of each drug. Therefore, our study suggests that CPT can be used to treat CML with loss of Bcr-Abl expression.