Table 6_A pan-genotypic indirect competitive ELISA for serological detection of pigeon circovirus antibodies.docx

Pigeon circovirus (PiCV) infection, which causes young pigeon disease syndrome (YPDS) and immunosuppression, significantly impacts both the meat and racing pigeon industries. Currently, no inactivated vaccine exists for PiCV prevention, primarily due to the challenges associated with isolating the PiCV virion, except for some gene subunit vaccines express the Cap protein of PiCV. The development of detection techniques is crucial for the diagnosis of PiCV. This study aimed to develop and validate a specific, sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for detecting PiCV antibodies in pigeons. We identified the cap gene from a group C PiCV strain (PiCV/Shaanxi/China/10/2021, SX10) isolated from racing pigeons. The Cap of SX10, an immunogen, can self-assemble into virus-like particles (VLPs). A mouse monoclonal antibody (mAb) against Cap, 1G6-4C4, was selected to establish an icELISA. This mAb could identify the PiCV Cap of the strains in groups A to E. The pan-genotypic reactivity of mAb 1G6-4C4 might target a conserved conformational epitope, overcoming limitations of PCR and prior serological assays. The icELISA method exhibited no cross-reactivity with antibodies against other common pigeon pathogens, such as pigeon paramyxovirus type 1 (PPMV-1), avian influenza (H9N2), avian adenovirus type 4 (FAdV-4) or rotavirus (RV). Compared with indirect ELISA (iELISA), icELISA demonstrated comparable performance, as testing of 29 clinical serum samples revealed antibody-positive rates of 51.72% (icELISA) and 44.82% (iELISA), with a 93.10% concordance rate. To a certain extent, icELISA has demonstrated good specificity and sensitivity for detecting PiCV-specific antibodies in pigeons. The developed icELISA provides a robust, specific, and sensitive tool for the serological detection of PiCV infection. Complementary to PCR test, icELISA enhances the comprehensive detection of PICV in epidemiological studies by offering a more practical and sensitive alternative for field applications. Its utility for large-scale epidemiological surveillance in PiCV-endemic regions is validated, highlighting its potential to inform targeted biosecurity and control interventions.

鸽圆环病毒（Pigeon circovirus, PiCV）感染可引发幼鸽病综合征（young pigeon disease syndrome, YPDS）并导致免疫抑制，对肉鸽与赛鸽产业均造成显著影响。目前尚无用于预防PiCV的灭活疫苗，这主要源于分离PiCV病毒粒子存在技术难点，目前仅存在部分表达PiCV衣壳蛋白（Cap protein）的基因亚单位疫苗。检测技术的开发对于PiCV的诊断至关重要。本研究旨在开发并验证一种特异性强、灵敏度高的间接竞争性酶联免疫吸附试验（indirect competitive enzyme-linked immunosorbent assay, icELISA），用于检测鸽血清中的PiCV抗体。研究人员从一株分离自赛鸽的C组PiCV毒株（PiCV/Shaanxi/China/10/2021, SX10）中鉴定得到cap基因。SX10株的Cap蛋白作为免疫原，可自发组装为病毒样颗粒（virus-like particles, VLPs）。研究人员筛选获得一株靶向Cap蛋白的小鼠单克隆抗体（mouse monoclonal antibody, mAb）1G6-4C4，并以此建立icELISA检测方法。该单克隆抗体可识别A至E组各毒株的PiCV Cap蛋白。单克隆抗体1G6-4C4的泛基因型反应性可能靶向一处保守构象表位，可弥补聚合酶链式反应（PCR）及现有血清学检测方法的局限。该icELISA方法与鸽源常见病原体——例如1型鸽副粘病毒（pigeon paramyxovirus type 1, PPMV-1）、禽流感病毒（H9N2）、4型禽腺病毒（FAdV-4）及轮状病毒（RV）——的抗体无交叉反应。与间接酶联免疫吸附试验（indirect ELISA, iELISA）相比，icELISA的检测性能相当：对29份临床血清样本的检测结果显示，icELISA与iELISA的抗体阳性率分别为51.72%与44.82%，符合率达93.10%。综上，icELISA在检测鸽源PiCV特异性抗体方面展现出良好的特异性与灵敏度。本研究开发的icELISA为PiCV感染的血清学检测提供了一种稳定、特异且灵敏的工具。作为PCR检测的补充手段，icELISA可为流行病学研究中PiCV的综合检测提供更实用、灵敏的现场检测替代方案。该方法已验证可用于PiCV流行区的大规模流行病学监测，有望为针对性生物安全防控措施的制定提供科学依据。