siRNA treatment targeting integrin α11 overexpressed via EZH2-driven axis inhibits drug-resistant breast cancer progression.

BACKGROUND: Breast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets. METHODS: Transcriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target. RESULTS: Integrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours. CONCLUSION: Our findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.

Epidemiology of fowl adenovirus (FAdV) infections in South Korean chickens during 2013-2019 following introduction of FAdV-4 vaccines.

Fowl adenoviruses (FAdV) are important infectious pathogens responsible for causing substantial economic losses to the poultry industry worldwide. One hundred and forty-six FAdV strains were continuously collected and analysed from 2013 to 2019 to understand the epidemiological change and nature of the virus in South Korea from two different standpoints, before and after the release of multiple commercial FAdV-4 vaccines. Phylogenetic analysis of the hexon loop-1 gene sequences showed that 92 strains belonged to FAdV-C (63%), 35 strains to FAdV-E (24%), 18 strains to FAdV-D (12.3%), and one strain to FAdV-A (0.7%), respectively. We provide evidence that the dominant FAdV serotype has recently changed from FAdV-4 to FAdV-8b, as reflected in the proportion of each serotype in field cases in 2019 (18.5% and 77.8%, respectively). The newly emerged FAdV-8b cluster was significantly noticeable compared to the old FAdV clusters, indicating that the development of a vaccine for FAdV-8b may be necessary. Overall, this new insight into FAdV prevalence provides a foundation for strategic control and the development of efficient vaccines against FAdV cases in chickens in South Korea.RESEARCH HIGHLIGHTS The dominant FAdV serotype in South Korea shifted from FAdV-4 to FAdV-8b in 2013-2019.A new cluster of FAdV-8b has emerged in South Korea, indicating the development of new vaccines.

First report of field cases of Y280-like LPAI H9N2 strains in South Korean poultry farms: pathological findings and genetic characterization.

H9N2 low-pathogenic avian influenza (LPAI) viruses have long been circulating in the world poultry industry, resulting in substantial economic losses. In addition to bird health consequences, viruses from specific lineages such as G1 and Y280 are also known to have the potential to cause a pandemic within the human population. In South Korea, after introducing inactivated H9N2 vaccines in 2007, there were no field outbreaks of H9N2 LPAI since 2009. However, in June 2020, an H9N2 virus was isolated from an outbreak in a Korean chicken farm. This strain was distinct from the predominant Korean/Y439 lineage and was believed to be part of the Y280-like lineage. Since the first case of this new H9N2 LPAI, nine more cases of field infections in poultry farms were documented through July and December of 2020. Phylogenetic analysis of the haemagglutinin (HA) and neuraminidase genes of these case isolates revealed that all strains were grouped with exotic Y280-like strains that did not previously exist in South Korea and were emerging into a new cluster. Serological assays also confirmed the existence of antibodies to Y280-like viruses in field sera collected from infected birds, and that they had seroconverted. Further analysis of the receptor-binding region in the HA protein also revealed that these isolates harboured a human-like motif that could potentially affect mammals and humans, demonstrating a possible public health risk. This is the first report of field cases caused by Y280-like H9N2 LPAI in the Korean poultry industry.RESEARCH HIGHLIGHTS Field outbreaks caused by Y280-like H9N2 avian influenza viruses were confirmed.A human-like motif was found at the HA receptor-binding region of all isolates.

Prediction of highly pathogenic avian influenza vaccine efficacy in chickens by comparison of in vitro and in vivo data: A meta-analysis and systematic review.

Vaccines for avian influenza (AI) can protect poultry against disease, mortality, and virus transmission. Numerous factors, including: vaccine platform, immunogenicity, and relatedness to the field strain, are known to be important to achieving optimal AI vaccine efficacy. To better understand how these factors contribute to vaccine protection, a systematic meta-analysis was conducted to evaluate efficacy data for vaccines in chickens challenged with highly pathogenic (HP) AI. Data from a total of 120 individual trials from 25 publications were selected and evaluated. Two vaccine criteria were evaluated for their effects on two metrics of protection. The vaccine criteria were: 1) the relatedness of the vaccine antigen and challenge strain in the hemagglutinin 1 domain (HA1) protein sequence; 2) vaccine-induced antibody titers to the challenge virus (VIAC). The metrics of protection were: A) survival of vaccinated chickens vs unvaccinated controls; and B) reduction in oral virus-shedding by vaccinated vs unvaccinated controls 2-4 days post challenge. Three vaccine platforms were evaluated: oil-adjuvanted inactivated whole AI virus, recombinant herpes virus of turkeys (rHVT) vectored, and a non-replicating alpha-virus vectored RNA particle (RP) vaccine. Higher VIAC correlated with greater reduction of virus-shed and vaccine efficacy by all vaccine platforms. Both higher HA1 relatedness and higher VIAC using challenge virus as antigen correlated with better survival by inactivated vaccines and rHVT-vectored vaccines. However, rHVT-vectored and RP based vaccines were more tolerant of variation in the HA1; the relatedness of the HA1 of the vaccine and challenge virus did not significantly correlate with survival with rHVT-vectored vaccines. Protection was achieved with the lowest aa similarity for which there was data, 90-93 % for rHVT vaccines and 88 % for the RP vaccine.

The Thermal Stability of Newcastle Disease Virus in Poultry Litter.

Sanitary disposal of contaminated organic material during recovery from an animal disease outbreak is costly and laborious. Characterizing the thermal stability of avian paramyxovirus type 1 (APMV-1; virulent APMV-1 strains cause Newcastle disease in poultry) will help inform risk assessments on the presence of viable virus on infected premises or in organic waste from infected premises. In some environments and housing types, heat may also be used as a decontamination method. Therefore, the objective of this study was to characterize the thermal stability (i.e., decimal reduction values [D values]) of APMV-1 in poultry litter. Virus inactivation was evaluated at seven temperatures from 10.0 C through 43.3 C, at 5.5 C intervals (50-110 F in 10 F intervals), using the I2 isolate of APMV-1, a vaccine strain known to be thermally stable. A high titer of virus (approximately 108 50% egg infectious doses) was added to wood shavings based, soiled chicken litter (poultry litter). Litter with both low and high moisture levels were evaluated. Samples were collected at different time intervals, and infectious virus was titrated in embryonated chicken eggs. At high temperatures (37.8 C-43.3 C), infectious virus could not be detected after 2-7 days, whereas at lower temperatures (10 C-21.1 C), it took up to 112 days for virus to decrease to undetectable levels. Furthermore, the D values were almost always shorter in the high moisture litter.

Estabilidad térmica del virus de la enfermedad de Newcastle en la cama avícola. La eliminación sanitaria de material orgánico contaminado durante la recuperación de un brote de enfermedad en los animales es costosa y laboriosa. La caracterización de la estabilidad térmica del paramixovirus aviar tipo 1 (APMV-1; las cepas virulentas de APMV-1 que causan la enfermedad de Newcastle en avicultura) contribuirá con información para las evaluaciones de riesgo sobre la presencia de virus viables en las instalaciones infectadas o en los desechos orgánicos de las instalaciones infectadas. En algunos entornos y tipos de casetas, el calor también se puede utilizar como método de descontaminación. Por lo tanto, el objetivo de este estudio fue caracterizar la estabilidad térmica (es decir, valores de reducción decimal [valores D]) del APMV-1 en la cama de aves. La inactivación del virus se evaluó a siete temperaturas, desde 10.0 C hasta 43.3 C, en intervalos de 5.5 C (50­110 F en intervalos de 10 F), utilizando el aislamiento I2 de APMV-1, que es una cepa vacunal conocida por ser térmicamente estable. Se añadió el virus con un título alto (aproximadamente 108 dosis infecciosas de embrión de pollo al 50%) a la cama de pollo con base de virutas de madera (cama avícola). Se evaluaron camas con niveles de humedad altos y bajos. Se recolectaron muestras en diferentes intervalos de tiempo y se tituló el virus infeccioso en huevos embrionados de pollo. A altas temperaturas (37.8 C­43.3 C), el virus infeccioso no se pudo detectar después de dos a siete días, mientras que a temperaturas más bajas (10 C­21.1 C), el virus tardó hasta 112 días en disminuir a niveles no detectables. Además, los valores D fueron casi siempre más cortos en la cama con alta humedad.

Quantitative real-time PCR assays for the concurrent diagnosis of infectious laryngotracheitis virus, Newcastle disease virus and avian metapneumovirus in poultry.

Newcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (aMPV) can be similar making it critical to quickly differentiate them. Herein, we adapted pre-existing molecular-based diagnostic assays for NDV and ILTV, and developed new assays for aMPV A and B, for use under synchronized thermocycling conditions. All assays performed equivalently with linearity over a 5 log10 dynamic range, a reproducible (R² > 0.99) limit of detection of ≥ 10 target copies, and amplification efficiencies between 86.8%-98.2%. Using biological specimens for NDV and ILTV showed 100% specificity. Identical amplification conditions will simplify procedures for detection in diagnostic laboratories.

Optimizing sample collection methods for detection of respiratory viruses in poultry housing environments.

Viral respiratory diseases, such as avian influenza, Newcastle disease, infectious bronchitis and infectious laryngotracheitis, have considerable negative economic implications for poultry. Ensuring the virus-free status of premises by environmental sampling after cleaning and disinfection is essential for lifting a quarantine and/or safely restocking the premises following an outbreak. The objectives of this study were to identify optimal sample collection devices and to determine the locations in poultry housing which are best for poultry respiratory virus sample collection. Chickens exposed to infectious bronchitis virus, which was used as a representative virus for enveloped poultry respiratory viruses, were housed in floor-pens in either a curtain-sided wood framed house or a cement block house. Foam swabs, cellulose sponges, polyester swabs, dry cotton gauze and pre-moistened cotton gauze were evaluated for comparative efficiency in recovering viral RNA. Cotton gauze pre-moistened with the viral transport media had the highest sensitivity among the devices (wood-framed house: 78% positive, geometric mean titre [GMT] of 2.6 log10 50% egg infectious doses [EID50 ] equivalents/ml; cement block houses: 55% positive, GMT of 1.7 log10 EID50 equivalents/ml). Targeting virus deposition sites is also crucial for efficient virus elimination procedures and subsequent testing; therefore, 10 locations within the houses were compared for virus detection. In both housing types, the highest viral RNA loads were recovered from the tops of drinker lines within the pen. Places the chickens could contact directly (e.g., feeder rim) or were contacted by caretaker feet (hallway floor) also yielded higher levels of viral RNA more consistently. These results will facilitate the establishment of efficient environmental sampling procedures for respiratory viruses of poultry.

Historical Investigation of Fowl Adenovirus Outbreaks in South Korea from 2007 to 2021: A Comprehensive Review.

Fowl adenoviruses (FAdVs) have long been recognized as critical viral pathogens within the poultry industry, associated with severe economic implications worldwide. This specific group of viruses is responsible for a broad spectrum of diseases in birds, and an increasing occurrence of outbreaks was observed in the last ten years. Since their first discovery forty years ago in South Korea, twelve antigenically distinct serotypes of fowl adenoviruses have been described. This comprehensive review covers the history of fowl adenovirus outbreaks in South Korea and updates the current epidemiological landscape of serotype diversity and replacement as well as challenges in developing effective broadly protective vaccines. In addition, transitions in the prevalence of dominant fowl adenovirus serotypes from 2007 to 2021, alongside the history of intervention strategies, are brought into focus. Finally, future aspects are also discussed.

Identification of optimal sample collection devices and sampling locations for the detection of environmental viral contamination in wire poultry cages.

Environmental testing of poultry premises after an outbreak of an infectious disease like avian influenza (AI) or Newcastle disease is essential to promptly verify virus-free status and subsequently return to normal operations. In an attempt to establish an optimized sampling protocol, a laboratory study simulating an AI virus-contaminated poultry house with wire layer cages was conducted. Three sample collection devices, pre-moistened cotton gauze, dry cotton gauze and a foam swab, were evaluated with each of four sample locations within a cage and when sampling all four locations with one device. Virus was detected with quantitative real-time RT-PCR utilizing a standard curve of a quantified homologous isolate of AI virus to determine titre equivalents of virus. The pre-moistened gauze detected the most virus RNA (100% positive, geometric mean titre [GMT): 3.2 log10 50% embryo infectious doses [EID50 ] equivalents per 25 cm2 ) in all four sample locations compared to dry gauze (93% positive, GMT: 2.6 EID50 equivalents per 25 cm2 ) and foam swabs (95% positive, GMT: 2.8 log10 EID50 equivalents per 25 cm2 ). The highest viral RNA loads were observed from the cage floor, and when the four locations were sampled with the same device. Overall, the pre-moistened gauze performed the best, and sampling multiple locations within a cage with the same device would likely optimize detection of residual virus.

The pathogenicity and transmission of live bird market H2N2 avian influenza viruses in chickens, Pekin ducks, and guinea fowl.

H2N2 subtype low pathogenic avian influenza viruses (LPAIVs) have persisted in live bird markets (LBMs) in the Northeastern United States since 2014. Although unrelated to the 1957 pandemic H2N2 lineage, there is concern that the virus could have animal and public health consequences because of high contact with humans and numerous species in the LBM system. The pathogenicity, infectivity, and transmissibility of six LBM H2N2 viruses isolated from three avian species in LBMs were examined in chickens. Two of these isolates were also tested in Pekin ducks and guinea fowl. Full genome sequence was obtained from all 6 isolates and evaluated for genetic markers for host adaptation and pathogenicity in poultry. Clinical signs were not observed in any host with any of the isolates, however one recent isolate was shed at higher titers than the other isolates and had the lowest bird infectious dose of all the isolates tested in all three species. This isolate, A/chicken/NY/19-012787-1/2019, was also the only isolate with a deletion in the stalk region of the neuraminidase protein (NA). This supports the theory that the NA stalk deletion is evidence of adaptation to gallinaceous poultry.

Validation of specific quantitative real-time RT-PCR assay panel for Infectious Bronchitis using synthetic DNA standards and clinical specimens.

Infectious bronchitis (IB) is a highly contagious upper respiratory tract disease of chickens caused by infectious bronchitis virus (IBV), which has various serotypes that do not cross-protect. Vaccine control strategies for this virus are only effective when designed around the currently circulating serotypes. It is essential to not only rapidly detect IBV but also to identify the type of virus causing disease. Six TaqMan™-based quantitative real-time RT-PCR assays (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. The analytical sensitivity of TaqMan™-based quantitative real-time RT-PCR assays (qRT-PCR) assays was evaluated using synthetic DNA standards that were identical with the target sequence and specificity was further validated using clinical and biological specimens. All developed assays performed equivalently when using synthetic DNA templates as standard material, as it achieved linearity over a 5 log10 dynamic range with a reproducible limit of detection of ≤10 target copies per reaction, with high calculated amplification efficiencies ranging between 90%-115%. Further validation of specificity using clinical and biological specimens was also successful.