Liu Zhe, Yang Yupeng, Qi Ruibin, Gu Haorong, Chen Mengru, Feng Kexin, Jiang Qian, Jia Honglin, Kang Hongtao, Liu Jiasen
State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
College of Veterinary Medicine, Northeast Agricultural University, Harbin, China.
Microbiol Spectr. 2025 Jul;13(7):e0183024. doi: 10.1128/spectrum.01830-24. Epub 2025 Jun 2.
Feline coronavirus (FCoV) includes Feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV primarily affects the gastrointestinal system of cats, causing mild and self-limiting gastrointestinal symptoms. In contrast, FIPV infection leads to severe immune-mediated disease with rapid progression and a very high mortality rate. The occurrence of feline infectious peritonitis (FIP) is associated with mutations in the viral genome during FECV infection, which makes diagnosing FECV an effective measure for preventing FIP. This study aims to establish rapid and efficient detection methods to provide reliable diagnostic tools for disease prevention and treatment. In this study, the recombinant plasmid pCold I-FIPV-DF-2-N of the FIPV-DF-2 virus N protein was constructed. It was then expressed prokaryotically and purified. Using this plasmid, BALB/c mice were immunized, and two highly specific and stable monoclonal antibodies, mAbs 2G7 and mAb 3H5, were successfully prepared. mAb 2G7 recognizes the amino acid sequence 18RGRSNSRGRKN28 on the N protein, and mAb 3H5 recognizes the amino acid sequence 295GDQVKVTLTHTYYLPKDDAKTSQFLEQID323. We successfully developed a colloidal gold immunochromatographic test strip for FCoV detection using a double-antibody sandwich method. Experimental results showed that this test strip had a detection limit of 209.8 ng/mL for the recombinant pCold I-FIPV-DF-2-N protein; the overall agreement rate for detecting FCoV in 10 ascites samples, which tested positive by conventional PCR, was 70%; and there was no cross-reaction with feline parvovirus, feline calicivirus, and feline herpesvirus. The detection method can realize the rapid detection on site and provide a convenient detection method for pet clinics, animal shelters, etc.IMPORTANCEIn recent years, pets have become an indispensable part of people's lives. As companion animals, the number of pet cats in domestic families has increased year by year. According to the 2023 Pet White Paper, the number of pet cats in China has reached more than 65 million. Therefore, the prevention and treatment of infectious diseases in cats is becoming more and more important. Therefore, in this study, a double-antigen sandwich colloidal gold immunochromatographic assay was developed to detect feline coronavirus (FCoV). The detection of feline enteric coronavirus (FECV) excreted by cats can effectively implement treatment, reduce the coronavirus load, and further prevent the risk of FECV infection evolving into feline infectious peritonitis virus. This method is simple, rapid, and specific and can be used for the detection of FCoV. Therefore, this method is more suitable for pathogen diagnosis in the field than previous methods.
猫冠状病毒（FCoV）包括猫肠道冠状病毒（FECV）和猫传染性腹膜炎病毒（FIPV）。FECV主要影响猫的胃肠道系统，引起轻微且自限性的胃肠道症状。相比之下，FIPV感染会导致严重的免疫介导疾病，病情进展迅速且死亡率极高。猫传染性腹膜炎（FIP）的发生与FECV感染期间病毒基因组的突变有关，这使得诊断FECV成为预防FIP的有效措施。本研究旨在建立快速高效的检测方法，为疾病的预防和治疗提供可靠的诊断工具。在本研究中，构建了FIPV-DF-2病毒N蛋白的重组质粒pCold I-FIPV-DF-2-N。然后进行原核表达和纯化。用该质粒免疫BALB/c小鼠，成功制备了两种高度特异性和稳定性的单克隆抗体，即单克隆抗体2G7和单克隆抗体3H5。单克隆抗体2G7识别N蛋白上的氨基酸序列18RGRSNSRGRKN28，单克隆抗体3H5识别氨基酸序列295GDQVKVTLTHTYYLPKDDAKTSQFLEQID323。我们成功开发了一种采用双抗体夹心方法检测FCoV的胶体金免疫层析试纸条。实验结果表明，该试纸条对重组pCold I-FIPV-DF-2-N蛋白的检测限为209.8 ng/mL；在10份经常规PCR检测呈阳性的腹水样本中检测FCoV的总体符合率为70%；与猫细小病毒、猫杯状病毒和猫疱疹病毒无交叉反应。该检测方法可实现现场快速检测，为宠物诊所、动物收容所等提供了一种便捷的检测方法。
近年来，宠物已成为人们生活中不可或缺的一部分。作为伴侣动物，家庭养猫的数量逐年增加。根据《2023年宠物白皮书》，中国宠物猫的数量已超过6500万。因此，猫传染病的预防和治疗变得越来越重要。因此，在本研究中，开发了一种双抗原夹心胶体金免疫层析法来检测猫冠状病毒（FCoV）。检测猫排出的猫肠道冠状病毒（FECV）可有效实施治疗，降低冠状病毒载量，并进一步预防FECV感染演变为猫传染性腹膜炎病毒的风险。该方法简单、快速且特异，可用于FCoV的检测。因此，该方法比以往方法更适合现场病原体诊断。
