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.

Dynamics of the Emerging Genogroup of Infectious Bursal Disease Virus Infection in Broiler Farms in South Korea: A Nationwide Study.

Infectious bursal disease (IBD), caused by IBD virus (IBDV), threatens the health of the poultry industry. Recently, a subtype of genogroup (G) 2 IBDV named G2d has brought a new threat to the poultry industry. To determine the current status of IBDV prevalence in South Korea, active IBDV surveillance on 167 randomly selected broiler farms in South Korea from August 2020 to July 2021 was conducted. The bursas of Fabricius from five chickens from each farm were independently pooled and screened for IBDV using virus-specific RT-PCR. As a result, 86 farms were found to be infected with the G2d variant, 13 farms with G2b, and 2 farms with G3. Current prevalence estimation of IBDV infection in South Korea was determined as 17.8% at the animal level using pooled sampling methods. G2d IBDV was predominant compared to other genogroups, with a potentially high-risk G2d infection area in southwestern South Korea. The impact of IBDV infection on poultry productivity or Escherichia coli infection susceptibility was also confirmed. A comparative pathogenicity test indicated that G2d IBDV caused severe and persistent damage to infected chickens compared with G2b. This study highlights the importance of implementation of regular surveillance programs and poses challenges for the comprehensive prevention of IBDV infections.

Nationwide Surveillance for Infectious Bronchitis Virus in South Korea from 2020 to 2021.

Infectious bronchitis virus (IBV) is a major pathogen in poultry. The genotypes of IBV vary considerably, and their antigenicity may differ. Nationwide surveillance in South Korea was performed to determine the prevalence and distribution of IBV and its genotypes. By both active and passive surveillance, a total of 939 samples were collected and tested for IBV detection by pathogen-specific reverse transcriptase-PCR. IBV RNA-positive samples were inoculated in embryonated eggs for virus isolation. IBV was genotyped and analyzed phylogenetically based on a partial nucleotide sequence of the S1 gene. A total of 114 IBV strains were isolated; 34 (30.9%) of the 110 samples obtained by passive surveillance, and 80 (9.7%) of the 829 samples obtained by active surveillance, were positive. Most IBVs in both groups were isolated from broilers. Five genotypes (QX-like, B4-like, KM91-like, K40/09-like, and 20AD17-like) were observed in South Korea, with the QX-like genotype being the most common, and the 20AD17-like genotype being a novel genotype. These findings will help to maximize protection against IBV infection by providing a reference for the selection of an avian vaccine for IBV in South Korea.

Vigilancia nacional del virus de la bronquitis infecciosa en Corea del Sur del año 2020 al 2021. El virus de la bronquitis infecciosa (IBV) es un patógeno importante en la avicultura. Los genotipos del virus de la bronquitis varían considerablemente y su antigenicidad puede ser diversa. Se realizó un estudio de vigilancia a nivel nacional en Corea del Sur para determinar la prevalencia y distribución del virus de bronquitis y sus genotipos. Mediante vigilancia activa como pasiva, se recolectaron un total de 939 muestras y se analizaron para la detección del virus de la bronquitis infecciosa mediante transcripción reversa y PCR específica para este patógeno. Se inocularon muestras positivas para ARN del virus de bronquitis en huevos embrionados para el aislamiento del virus. Los virus de bronquitis se genotipificaron y analizaron filogenéticamente basándose en una secuencia parcial de nucleótidos del gene S1. Se aislaron un total de 114 cepas del virus de bronquitis; 34 (30.9%) de las 110 muestras obtenidas por vigilancia pasiva y 80 (9.7%) de las 829 muestras obtenidas por vigilancia activa resultaron positivas. La mayoría de los virus de bronquitis en ambos grupos se aislaron de pollos de engorde. Se observaron cinco genotipos (similares a QX, similares a B4, similares a KM91, similares a K40/09 y similares a 20AD17) en Corea del Sur, siendo el genotipo similar a QX el más común y el genotipo similar a 20AD17 que ha sido un genotipo de nueva aparición. Estos hallazgos ayudarán a maximizar la protección contra la infección por el virus de la bronquitis infecciosa al proporcionar una referencia para la selección de vacunas aviares para bronquitis infecciosa en Corea del Sur.

G protein-coupled estrogen receptor regulates the KLF2-dependent eNOS expression by activating of Ca2+ and EGFR signaling pathway in human endothelial cells.

G protein-coupled estrogen receptor (GPER) is important for maintaining normal blood vessel function by preventing endothelial cell dysfunction. It has been reported that G-1, an agonist of GPER, increases nitric oxide (NO) production through the phosphorylation of endothelial nitric oxide synthase (eNOS). However, the effect of GPER activation on eNOS expression has not been studied. Our results show that G-1 significantly increased the expression of eNOS and Kruppel-like factor 2 (KLF2) in human endothelial EA.hy926 cells. The individual silences of KLF2 and GPER attenuated G-1-induced eNOS expression. In addition, inhibition of the Gαq and Gßγ suppressed G-1-induced the expression of eNOS and KLF2 in EA.hy926 cells. Interestingly, these effects were similar in HUVECs. Furthermore, we found that GPER-mediated Ca2+ signaling increased the phosphorylation of CaMKKß, AMPK, and CaMKIIα in the cells. The phosphorylation of histone deacetylase 5 (HDAC5) by activation of AMPK and CaMKIIα increased the expression of eNOS via transcriptional activity of KLF2. We further demonstrate that GPER activation increased the phosphorylation of Src, EGFR, ERK5, and MEF2C and consequently induced the expression of eNOS and KLF2. Meanwhile, inhibition of ERK5 and HDAC5 suppressed the expression of eNOS and KLF2 induced by G-1 in the cells. These findings suggest that GPER provides a novel mechanism for understanding the regulation of eNOS expression and is an essential therapeutic target in preventing cardiovascular-related endothelial dysfunction.

Characterization of antigenic variant infectious bursal disease virus strains identified in South Korea.

Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases of young chickens, causing considerable economic losses to the poultry industry. More than 30 years ago, an antigenic variant (av) pathotype of the IBD virus (IBDV) was reported to originate in, and subsequently spread among, poultry farms in the USA. Recently, a novel avIBDV lineage was identified in China and was shown to exhibit clear differences in its pathogenicity as well as molecular characteristics compared with the previously isolated variant strains. In this study, we conducted a passive surveillance of chicken carcasses submitted to our research division from June-December 2019, and detected the IBDV strains by reverse transcription PCR. Five avIBDV strains were isolated, and their pathogenicity was determined by necropsy and molecular analysis. Additionally, a coinfection field case involving an avIBDV strain and a very virulent IBDV (vvIBDV) strain was identified. Multiple sequence alignment and phylogenetic analysis of partial viral protein 1 (VP1) and hypervariable region (hv) VP2 genes revealed that those strains originated from two different avIBDV lineages. The co-occurrence of two sub-groups of avIBDVs in South Korea confirms for the first time the evolution of antigenic variant IBDV strains, and highlights the urgency for the development of new strategies for IBDV intervention in South Korea.RESEARCH HIGHLIGHTS Five avIBDV strains were identified in South Korea by passive surveillance test in 2019.A coinfection between two IBDV strains from different genogroups was reported in a field case.By phylogenetic analysis, Korean avIBDVs belonged to two distinct lineages of antigenic variant genogroup.

Betulinic Acid Induces eNOS Expression via the AMPK-Dependent KLF2 Signaling Pathway.

Betulinic acid (BA) is a natural pentacyclic triterpenoid with protective effects against inflammation, metabolic diseases, and cardiovascular diseases. We have previously shown that BA prevents endothelial dysfunction by increasing nitric oxide (NO) synthesis through activating endothelial nitric oxide synthase (eNOS) in human endothelial cells. However, the effect of BA on eNOS expression remains unclear. Thus, the aim of our study was to investigate the intracellular pathways associated with the effect of BA to regulate eNOS expression in human endothelial cells. BA significantly increased eNOS expression in a time- and concentration-dependent manner. Additionally, BA upregulated the expression of the transcription factor KLF2, which is known to regulate eNOS expression. KLF2 silencing in human endothelial cells attenuated the ability of BA to upregulate eNOS. BA also increased levels of intracellular Ca2+, activating CaMKKß, CaMKIIα, and AMPK. Inhibition of the TRPC calcium channel abolished BA-mediated effects on intracellular Ca2+ levels. Moreover, BA increased the phosphorylation levels of ERK5, HDAC5, and MEF2C. Pretreatment of cells with compound C (AMPK inhibitor), LMK235 (HDAC5 inhibitor), and XMD8-92 (ERK5 inhibitor) attenuated the BA-induced eNOS expression. Collectively, these findings suggest that BA induces eNOS expression by activating the HDAC5/ERK5/KLF2 pathway in endothelial cells. The data presented here provide strong evidence supporting the use of BA to prevent endothelial dysfunction and treat vascular diseases, such as atherosclerosis.

Sesamin Induces Endothelial Nitric Oxide Synthase Activation via Transient Receptor Potential Vanilloid Type 1.

Sesamin, the most abundant lignan in sesame seed oil, has many biological activities. However, the underlying molecular mechanisms behind the regulatory effects of sesamin on endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) generation in endothelial cells (ECs) remain unclear. Sesamin induced the intracellular level of NO and eNOS phosphorylation in ECs in a concentration- and time-dependent manner. Additionally, sesamin induced levels of intracellular calcium, leading to the phosphorylation of calmodulin-dependent protein kinase II (CaMKII) at Thr286, calcium/calmodulin-dependent protein kinase kinase beta (CaMKKß) at Ser511, protein kinase A (PKA) at Thr197, Akt at Ser473, and AMP-activated protein kinase (AMPK) at Thr172. In particular, blocking of the transient receptor potential vanilloid type 1 (TRPV1) channel by capsazepine (TRPV1 antagonist), as well as TRPV1 knockdown via TRPV1 silencing RNA, abrogated sesamin-induced PKA, Akt, AMPK, CaMKII, CaMKKß, and eNOS phosphorylation and NO level in ECs. Furthermore, sesamin inhibited TNF-α-induced NF-κB translocation, intercellular adhesion molecule-1 expression, and monocyte adhesion. Sesamin triggered eNOS activity and NO production via activation of TRPV1-calcium signaling, which involved the phosphorylation of PKA, CaMKII, CaMKKß, Akt, and AMPK. Sesamin may be useful for treating or preventing the endothelial dysfunction correlated with cardiovascular diseases.