Data Sheet 1_Simultaneous detection and partial molecular characterization of five RNA viruses associated with enteric disease in chickens: chicken astrovirus, avian nephritis virus, infectious bronchitis virus, avian rotavirus a and avian orthoreovirus, via multiplex RT–qPCR.pdf

In the poultry industry, intestinal diseases can lead to significant economic losses due to diarrhea, weight loss and mortality, often linked to viral infections. Chicken astrovirus (CAstV), avian nephritis virus (ANV), infection bronchitis virus (IBV), avian rotavirus A (AvRVA) and avian orthoreovirus (ARV) are key pathogens on this disease including feed malabsorption and runting-stunting syndrome (RSS). This study proposes a multiplex RT–qPCR assay for the simultaneous detection of these five viruses in chickens with enteritis in Ecuador. Primers and hydrolysis probes were designed for the five viruses, along with a synthetic gBlock as a positive control. The method was evaluated for sensitivity, repeatability, and specificity, and 200 jejunal samples were tested. Genome regions of each virus were sequenced, and a phylogenetic analysis confirmed their presence in the samples. The optimized RT-qPCR assay showed efficiency between 98.8–105.9%, with a detection limit of 1 copy/μL. It specifically amplified the five target viruses without cross-reactivity. Among 200 chickens tested, 97% were positive for at least one virus, with ANV (89%) and CAstV (53%) being the most prevalent. Coinfections were common, especially between CAstV and ANV, with three samples positive for all viruses. Sequencing and phylogenetic analysis confirmed the circulation of multiple strains in chickens with enteric disease in Ecuador. This study describes a multiplex RT-qPCR assay for detecting key enteric viruses in Ecuadorian poultry highlighting the high prevalence of astroviruses, emphasizing the impact of coinfections, its possible role in the disease and the importance of improving disease control strategies.

在家禽养殖业中，肠道疾病常由病毒感染引发，可导致腹泻、体重下降及死亡，进而造成严重的经济损失。鸡星状病毒（Chicken astrovirus, CAstV）、禽肾炎病毒（avian nephritis virus, ANV）、传染性支气管炎病毒（infectious bronchitis virus, IBV）、A群禽轮状病毒（avian rotavirus A, AvRVA）以及禽正呼肠孤病毒（avian orthoreovirus, ARV）是引发此类疾病（包括饲料吸收不良与矮小综合征（runting-stunting syndrome, RSS））的关键病原体。本研究针对厄瓜多尔肠炎鸡只，开发了一种可同时检测上述五种病毒的多重RT-qPCR检测方法。研究人员为这五种病毒设计了引物与水解探针，并合成gBlock作为阳性对照。本研究对该方法的灵敏度、重复性与特异性进行了评估，并对200份空肠样本开展了检测。对各病毒的基因组区域进行测序，并经系统发育分析确认了样本中上述五种病毒的存在。优化后的RT-qPCR检测方法扩增效率为98.8%~105.9%，检测限可达1拷贝/微升。该方法可特异性扩增五种靶病毒，无交叉反应。在检测的200只鸡只中，97%的样本至少检出一种病毒，其中以ANV（89%）与CAstV（53%）的检出率最高。混合感染现象较为普遍，尤以CAstV与ANV的混合感染最为常见，另有3份样本同时检出全部五种靶病毒。测序与系统发育分析证实，厄瓜多尔患肠道疾病的鸡只中存在多种病毒毒株的流行。本研究开发了用于检测厄瓜多尔家禽关键肠道病毒的多重RT-qPCR方法，明确了星状病毒的高流行率，强调了混合感染的影响、病毒在疾病发生中的潜在作用，以及完善疾病防控策略的重要性。