兽研1. 引言
作为主要的伴侣物种，家猫（Felis catus）与人类群体保持着密切接触，目前全球种群数量已超过6亿[1]。猫传染病具有至关重要的意义。上呼吸道疾病（URTDs）在猫科医学中较为常见，其特征为一系列临床表现，包括浆液性至黏液脓性眼鼻分泌物增多、角膜溃疡、口腔炎及发热[2,3]。在主要病原体中，猫杯状病毒（FCV）是影响家猫和野猫的重要病原体，长期以来被认为是猫上呼吸道疾病和口腔溃疡的主要原因[4,5,6,7]。近年来，FCV在免疫选择压力下持续进化，产生了多种基因型[8]。因此，FCV毒株表现出显著的逃逸宿主免疫清除的能力[8]。目前，尚无针对FCV的特异性抗病毒治疗药物，防控策略主要依赖疫苗接种。然而，现有的灭活疫苗——通常针对猫泛白细胞减少症病毒（FPV）、FCV和猫疱疹病毒1型（FHV-1）——对新兴FCV变异株往往提供不完全的保护[9,10,11,12]。这些发现强调了持续开展FCV研究的必要性，以更好地阐明伴侣动物中的病毒感染动力学[1]。
FCV是一种高度传播性的病原体，在分类学上属于杯状病毒科水疱病毒属。该科包括重要的人类病原体，如诺如病毒，以及多种动物特异性病毒，包括兔出血症病毒和欧洲棕色野兔综合征病毒。杯状病毒科的命名源于病毒粒子结构上观察到的特征性杯状表面凹陷[13]。FCV是一种RNA病毒，具有高突变率，在家猫群体中普遍分布[14]。虽然FCV宿主限于猫科动物且不具有人兽共患潜力，但其表现出广泛的遗传和抗原变异性[15]。该病毒在猫群体中呈地方性流行[16]，临床表现多样，包括上呼吸道疾病、舌溃疡、牙周炎和跛行综合征。在过去二十年中，某些谱系已进化为强毒全身性FCV（VS-FCV）变异株，其特征为增强的复制动力学。VS-FCV的临床表现可包括严重脱毛、皮肤溃疡以及耳廓和鼻孔的坏死性趾皮炎，常伴有浆液性结痂。其他全身性并发症包括皮下水肿、支气管间质性肺炎和多器官坏死（胰腺、肝脏和脾脏），最终导致感染个体高死亡率[13,17,18,19,20,21,22,23,24,25]。
FCV基因组由约7.5千碱基的正 sense 单链RNA组成，有利于快速的进化适应[13]。非节段性基因组RNA包含三个功能性开放阅读框（ORFs），编码六种非结构蛋白（NS1–NS6/7）和两种结构蛋白（VP1和VP2）[26]。ORF1编码一个大 polyprotein，经翻译后切割为非结构组分，如蛋白酶和聚合酶[27]。ORF2编码一个 polyprotein，经蛋白水解加工产生衣壳蛋白前导序列和主要衣壳蛋白（VP1），而ORF3编码次要衣壳蛋白（VP2）[28]。成熟病毒衣壳在结构上由180个拷贝的VP1和12个拷贝的低丰度VP2蛋白组成[29]。
自1957年首次从出现呼吸道症状的猫中分离到FCV以来[30]，该病毒已在全球范围内广泛存在，欧洲、美洲和亚洲均有检测报道[31,32]。1992年，该病毒在非洲狮和东北虎的口腔溃疡性病变中被鉴定；随后[33]，于2000年首次从中国的虎中分离获得。这些发现表明FCV地理分布广泛且宿主范围广，几乎所有猫科动物均易感[34,35]。作为一种RNA病毒，FCV易于频繁突变，导致高毒力变异株的出现。其中，GI基因型的流行毒株与强毒全身性疾病（VSD-FCV）相关[36]。尽管历史上较为罕见，但这些毒株在20世纪90年代美国对VS-FCV进行表征后已在全球传播[37]。中国猫繁育产业的扩张加之高种群密度，为病毒传播提供了有利环境。缺乏强有力的预防措施以及病毒的持续突变削弱了疫苗效力，对家猫和野猫的健康与保护构成重大威胁[19]。尽管对FCV分子流行病学和致病性已有广泛研究，但对中国近期流行FCV毒株的表征仍存在重要空白。特别是，新出现分离株的遗传多样性并非总能伴随对其体外复制特性和体内致病性的系统评估。此外，基因组变异，尤其是VP1衣壳蛋白高变区内的变化，与流行FCV毒株生物学特性之间的关系仍不完全清楚。因此，持续从不同地理区域分离和表征当代FCV毒株对于增进我们对FCV进化和致病多样性的理解十分必要。
在本研究中，我们从中国河南省郑州市的一个猫交易市场分离到一株FCV毒株，命名为HN/ZZ/2025。我们对其完整基因组和系统发育关系进行了表征，并系统评估了其体外复制特性、理化稳定性以及在猫体内的致病性。我们进一步研究了实验感染后的病毒排毒、组织分布和组织病理学变化。这些数据为深入了解中国流行FCV毒株的分子流行病学格局、复制特性和致病动力学提供了有用信息，并为未来FCV遗传多样性和致病机制研究提供了有用的分离株。
2. 材料与方法
2.1. 伦理批准与知情同意
所有涉及动物的实验程序均经河南农业大学实验动物管理与使用委员会审查批准（批准号HNND2024050715）。本研究按照相关机构规定和动物福利原则进行，包括3R原则（替代、减少和优化）。在整个实验期间对动物进行监测，以尽量减少不适和痛苦。预定实验终点为感染后21天，之后所有动物通过静脉注射戊巴比妥钠（100 mg/kg）实施安乐死。随后采集器官用于病毒学、Western blot、组织病理学和免疫组织化学分析，剩余尸体按照机构生物安全程序处理。
2.2. 细胞与抗体
CRFK（ATCC，CCL-94）、F81（Cellosaurus，CVCL_9259）和Fc3Tg（ATCC，CCL-176）细胞在补充有10%胎牛血清（FBS，Gibco，Thermo Fisher Scientific，Waltham，MA，USA）的Eagle最低必需培养基（EMEM，BNCC，北京，中国）中，于37 °C、5% CO2加湿培养箱中培养。靶向FCV VP1蛋白的兔多克隆抗体（pAb）由本实验室制备并表征。针对GAPDH的一抗（10494-1-AP）、CoraLite488标记的山羊抗兔IgG（H+L）（SA00013-2）和HRP标记的山羊抗兔IgG（SA00001-2）二抗购自Proteintech（武汉，中国）。
2.3. 样品采集与检测
2025年1月，在中国河南省郑州市的一个猫交易市场，共选取了22只表现出提示FCV感染临床症状的猫进行采样。从每只猫采集口腔和鼻腔拭子并合并为每只猫的一份临床样本，共获得22份临床样本。采样策略基于是否存在提示FCV感染的临床表现。样本悬浮于磷酸盐缓冲液（PBS，pH 7.2）中，并在冷藏条件下运送至实验室。由于这些猫来自商业猫交易市场，其年龄、性别、品种、疫苗接种史及既往治疗状况的详细信息无法获得或无法可靠核实。使用FastPure Viral DNA/RNA Mini Kit（Vazyme，RC311，南京，中国）提取总病毒DNA/RNA，并使用HiScript IV All-in-One Ultra RT SuperMix（Vazyme，R433-01）进行cDNA合成。采用特异性引物通过PCR测定FCV、FPV、FHV、FIPV、FBoV和FeAstV的存在情况（补充表S1）。
2.4. 病毒分离、培养与纯化
选取FCV阳性标本进行病毒分离。匀浆液涡旋后于4 °C、12,000 rpm离心5 min。所得上清液经0.22 μm滤器（Biosharp，北京，中国）过滤，并用无血清MEM按1:10稀释。用PBS洗涤融合的CRFK细胞单层两次，然后接种补充有1%青霉素-链霉素-庆大霉素（Solarbio，P1410，北京，中国）的悬液。监测细胞病变效应（CPE），并在三次冻融循环后收获细胞。为进行纯化，使用1%低熔点琼脂糖覆盖层（Solarbio，A8350）连续进行三轮噬斑纯化。通过中性红染色（Beyotime，C0125，上海，中国）识别可见噬斑，并挑选单个噬斑进行克隆扩增及后续PCR验证。
2.5. 下一代测序分析
使用NEBNext Ultra II RNA Library Prep Kit（Illumina，New England Biolabs，Ipswich，MA，USA）构建元病毒RNA文库。使用minimap2（版本v2.28）将质量过滤后的reads比对到宿主参考基因组，以消除宿主来源污染。使用MEGAHIT（版本v1.2.9）对clean reads进行从头组装，并通过Prodigal（版本v2.6.3）预测ORF。通过使用MMseqs2（版本v2.0）将序列比对到Swiss-Prot数据库进行功能注释。
2.6. 基因组测序与系统发育分析
基于NGS结果和NCBI参考序列设计引物，进行FCV全长基因组扩增（补充表S2）。基因组分为三个重叠片段进行扩增，使用一步法ZTOPO-Blunt/TA试剂盒（ZOMANBIO，北京庄盟国际生物基因科技有限公司，北京，中国）克隆，并进行Sanger测序。该FCV分离株命名为HN/ZZ/2025，已提交至GenBank（登录号PX408781）。基于General Time Reversible（GTR+Γ+I）替换模型，使用MEGA版本7.0中的最大似然（ML）法进行系统发育重建。Bootstrap值由1000次重复计算得出。
2.7. 病毒鉴定与生长曲线
2.7.1. 病毒增殖与滴定
将纯化的FCV毒株HN/ZZ/2025接种到60 mm培养皿中融合的CRFK、F81和Fc3Tg细胞单层上。每日监测CPE并使用倒置显微镜（Olympus，东京，日本）记录。当出现约80% CPE时，对培养物进行三次冻融循环。于4 °C、8000 rpm离心10 min去除细胞碎片，收获含病毒上清液并保存于−80 °C。
采用Reed-Muench数学模型测定50%组织培养感染剂量（TCID50）。简言之，将细胞以每孔1 × 10⁴个细胞的密度接种于96孔板中。24 h后，用10倍系列稀释的病毒（10⁻¹–10⁻¹²）接种细胞，并于37 °C孵育1 h。吸附后，将接种物替换为100 μL维持培养基（补充有2% FBS的MEM）。将板孵育3–5天，每日监测CPE。每次滴定均设三个重复以计算平均病毒滴度。
2.7.2. 免疫印迹分析
通过免疫印迹验证FCV蛋白的表达。以0.05的感染复数（MOI）用FCV HN/ZZ/2025感染CRFK、F81和Fc3Tg细胞。感染后24 h（hpi），收获细胞并使用RIPA缓冲液（Beyotime，P0013B，上海，中国）裂解。蛋白样品经SDS-PAGE分离并转移至聚偏二氟乙烯膜（Millipore，Billerica，MA，USA）。膜用5%脱脂奶粉于室温封闭1 h，随后与兔抗VP1多克隆抗体于4 °C孵育过夜。洗涤后，膜与相应的HRP偶联二抗于室温孵育1 h。使用Luminata Crescendo Western HRP Substrate（Millipore，WBLUR0500）显示蛋白条带，并使用GE AI600成像系统（GE，Boston，MA，USA）采集图像。
2.7.3. 间接免疫荧光试验（IFA）
对于免疫荧光试验（IFA），将CRFK、F81和Fc3Tg细胞以FCV感染（MOI = 0.05）24小时。细胞用4%多聚甲醛固定10分钟，并用含0.1% Triton X-100的PBS透化10分钟。用含10% FBS的PBS封闭1小时后，细胞与靶向主要衣壳蛋白（VP1）的一抗多克隆抗体在37 °C孵育1小时。用PBS洗涤三次后，细胞与Alexa-Fluor标记的二抗（用10% FBS稀释）在室温孵育1小时。使用Olympus荧光显微镜观察并拍摄病毒定位。
2.7.4. 复制动力学与RT-qPCR分析
为评估FCV复制动力学，将35 mm培养皿中的CRFK、F81和Fc3Tg细胞以MOI 0.05的FCV感染。在37 °C吸附1小时后，细胞用PBS洗涤三次以去除未结合的病毒粒子。在感染后0、6、12、18、24、30、36、48和60小时收集细胞样本并进行总RNA提取。通过靶向VP1基因的实时荧光定量PCR（RT-qPCR）定量病毒载量。使用pET21b-FCV-VP1重组标准质粒的10倍系列稀释液生成标准曲线。RT-qPCR使用以下引物一式三份进行：FCV VP1-Fw：5′-TTGCAAAGAGCATGGGGTGA-3′，FCV VP1-Rv：5′-CCAAGCATTGCCAGTGTGAC-3′。通过将平均循环阈值（Ct）值与建立的标准曲线比对来计算病毒拷贝数。
2.8. 透射电子显微镜（TEM）与理化特性分析
2.8.1. 形态学表征
为表征FCV病毒粒子的形态，在CRFK细胞中增殖病毒并收获所得培养上清液。病毒通过超速离心浓缩，随后使用碘克沙醇（OptiPrep）密度梯度纯化。通过PCR和SDS-PAGE分析确认纯化FCV的存在。对于TEM观察，将纯化病毒悬液施加于碳包覆铜网（支持膜）上，并用1%磷钨酸负染。用滤纸去除多余染液，铜网在室温干燥。使用透射电子显微镜（JEOL，JEM-1400，JEOL Ltd.，东京，日本）在80–100 kV加速电压下检查病毒粒子结构和直径。
2.8.2. 理化稳定性
使用起始滴度为1 × 10^7.5 TCID50/mL的培养物评估FCV HN/ZZ/2025的环境稳定性。病毒悬液经受各种应激条件，包括热处理（50 °C）、酸性条件（pH 3.0）、碱性条件（pH 10.0）以及暴露于有机溶剂（氯仿和乙醚）。处理后，样品按需中和或处理，并接种到汇合的CRFK细胞单层上以监测CPE。接种后48小时收获培养上清液，并使用TCID50试验定量残余病毒感染性。
2.9. 抗VP1蛋白兔多克隆抗体的制备
扩增FCV VP1基因并亚克隆到pET21b和pGEX-4T-1原核表达载体中（图S1）。将重组质粒转化到大肠杆菌BL21（DE3）感受态细胞中。转化细菌在37 °C培养至OD600达到0.6–0.8，此时用0.5 mM异丙基-β-D-硫代半乳糖苷在18 °C诱导蛋白表达12小时。通过离心收获细胞，重悬于30 mL裂解缓冲液中，并通过超声破碎。
使用抗His标签和抗GST标签抗体通过Western blot验证重组VP1蛋白的表达。His标签和GST标签的VP1蛋白分别使用Ni-NTA树脂（GenScript，L00250，南京，中国）和谷胱甘肽树脂（GenScript，L00206）按照制造商方案纯化。纯化的VP1蛋白用作免疫原免疫一只4月龄新西兰白兔。每次免疫时，将100 μg纯化VP1蛋白与Freund佐剂（Sigma，Burlington，MA，USA）乳化并皮下给药。以14天间隔进行免疫，共三次免疫。随后单独使用VP1蛋白进行第四次加强免疫。最终加强免疫后收集血清，抗体滴度约为1:50,000。通过Western blot和IFA验证兔抗VP1血清用于检测FCV的特异性和滴度（图S2）。
2.10. 动物感染研究
选取六只两个月大的家猫（四雌两雄）纳入攻毒试验。这些猫均为普通家猫，实验前经PCR确认FCV、FPV和FHV均为阴性。攻毒前采集血清样本用于ELISA和病毒中和试验。六只猫在攻毒前对FCV、FPV和FHV均未检出可检测抗体，也未检测到针对这些病毒的中和抗体。动物饲养于河南农业大学专用动物设施内，通风良好，整个研究期间接受常规饲养管理，包括自由采食和饮水。经过7天适应期后，猫被随机分为两组（每组n=3）。攻毒组经鼻接种1 mL含1×10^7 TCID50/0.1 mL FCV HN/ZZ/2025的MEM，对照组接种等体积的无病毒MEM。采用标准化临床评分系统每日记录临床健康状态，包括发热、体重波动、精神状态、食欲和黏膜溃疡。每日采集口腔和鼻腔拭子，通过RT-qPCR进行病毒排毒分析。攻毒后第21天，所有猫均被安乐死以进行系统性病理评估。采集心脏、肝脏、脾脏、肺、肾脏和气管组织，用于定量病毒载量和评估组织病理学变化。动物操作按照批准的动物福利方案及替代、减少和优化（3Rs）原则进行。整个实验过程中采取适当措施以尽量减少动物的不适和痛苦。动物通过静脉注射戊巴比妥钠（100 mg/kg）实施安乐死，详见第2.1节。
建立临床评分系统以综合评估实验猫的健康状态。评估八项参数：体温、体重、精神状态、食欲、眼分泌物、鼻分泌物、口腔溃疡和存活状态。体温在38.0至39.5 °C之间计0分，≥39.5 °C计1分，<37.0 °C计2分。体重变化<3%计0分，体重减轻≥3%计2分。精神状态正常计0分，嗜睡计1分。食欲正常计0分，食欲减退计1分，厌食计2分。无眼分泌物计0分，有眼分泌物计1分。无鼻分泌物计0分，轻度分泌物计1分，中度至重度分泌物计2分。无口腔溃疡计0分，存在少量小病灶计1分，存在大量多发病灶计2分。存活动物计0分，死亡计10分。
2.11. 病毒RNA载量测定
实验攻毒后，通过绝对RT-qPCR对采集组织以及感染猫口腔和鼻腔拭子中的病毒载量进行定量。按照制造商说明，使用TIANamp Virus DNA/RNA Kit（TIANGEN，DP315，北京，中国）从200 μL拭子上清液或组织匀浆中提取总病毒RNA。随后使用HiScript IV All-in-One Ultra RT SuperMix（Vazyme，R433-01）进行cDNA合成。
FCV基因组拷贝数的定量按先前所述方法进行。简言之，将FCV VP1基因的179 bp片段克隆至pET21b载体作为标准品。使用重组质粒10倍系列稀释液（10^1–10^12拷贝/μL）生成的标准曲线进行绝对定量。RT-qPCR使用ChamQ SYBR qPCR Master Mix（Vazyme，Q341-02）在实时PCR系统上以三重复进行。所用引物序列为FCV VP1-Fw：5′-TTGCAAAGAGCATGGGGTGA-3′，FCV VP1-Rv：5′-CCAAGCATTGCCAGTGTGAC-3′。
2.12. 组织病理学和免疫组织化学（IHC）分析
2.12.1. 组织病理学
尸检时系统采集心脏、肝脏、脾脏、肺、肾脏和气管的代表性样本。组织于室温下在4%多聚甲醛中固定72 h，随后经梯度乙醇脱水、二甲苯透明并石蜡包埋。使用切片机（Servicebio，武汉，中国）将石蜡包埋块切成5 μm厚切片。组织病理学评估时，切片经脱蜡、复水后行苏木精-伊红染色。在光学显微镜下检查病理变化并拍照。
2.12.2. IHC
为原位检测FCV抗原，对组织切片进行IHC。简言之，玻片经脱蜡、复水后，使用EDTA缓冲液（pH 9.0）进行热诱导抗原修复。淬灭内源性过氧化物酶活性后，切片于室温下用5% BSA封闭30 min。随后切片与兔抗FCV VP1多克隆抗体于4 °C孵育过夜。
对于免疫组化，切片随后与HRP标记的抗兔IgG二抗（1：500，Servicebio）孵育。FCV VP1抗原阳性细胞用3，3‘-二氨基联苯胺显色，产生特征性棕色沉淀，细胞核用苏木精复染。操作流程与第2.7节所述一致，额外增加了EDTA抗原修复步骤。所有切片在光学或荧光显微镜下分析，以评估病毒抗原的分布和强度。
2.13. 统计分析
定量数据使用GraphPad Prism 8.0软件分析。组间统计学显著性采用Student’s t检验确定。数据以平均值 ± 标准差表示，p值 < 0.05视为具有统计学显著性（* p < 0.05，** p < 0.01，*** p < 0.001）。
3. 结果
3.1. FCV样本采集、检测及下一代测序分析
2025年1月，在中国河南省郑州市一个猫交易市场，从22只临床疑似FCV感染的猫采集了口咽拭子和鼻拭子。每只猫的口咽拭子和鼻拭子合并为一份临床样本，共获得22份样本（图1A）。提取总DNA/RNA，后续PCR分析确认标本中FCV阳性。对其他猫病毒的筛查显示，大多数病例存在混合感染，22份样本中仅2份（2/22）为单纯FCV感染。除FCV外，猫泛白细胞减少症病毒（FPV）、猫博卡病毒（FBoV）和猫疱疹病毒（FHV）的阳性率最高（图1B）。为探究潜在的隐匿病原体，对口咽拭子和鼻拭子进行了下一代测序。针对NCBI-nt和RVDB数据库进行综合分析，以鉴定任何相关的微生物特征。在过滤宿主来源序列并进行分类学注释后，发现FCV特异性序列是最丰富的病毒reads，占病毒群体的近100%（图1C）。因此，选择单纯感染病例的FCV阳性样本进行进一步的病毒分离。
3.2. FCV的分离、鉴定与纯化
用处理后的FCV阳性口咽拭子和鼻拭子上清液接种CRFK细胞。接种的CRFK细胞从感染后12小时（hpi）开始出现明显的细胞病变效应（CPE）——以细胞变圆、皱缩和“葡萄串样”形态为特征，而对照细胞保持汇合状态（图2A）。在F81和Fc3Tg细胞中也观察到类似的CPE，但Fc3Tg细胞的细胞病变变化出现延迟，仅在24 hpi后出现。成功分离后进行噬斑纯化，经过连续三轮纯化后可见清晰的噬斑（图2B）。通过在CRFK细胞中连续传代六次确认了FCV的稳定复制，并通过PCR在培养上清液中持续检测到FCV特异性基因组片段（图2C）。CRFK、F81和Fc3Tg细胞中的病毒滴度（TCID50）分别为每0.1 mL 10^7.18、10^7.50和10^5.89（图2D）。使用MOI为0.05评估的复制动力学显示，该分离株在CRFK和F81细胞中于24 hpi达到病毒载量峰值，在Fc3Tg细胞中于36 hpi达到峰值（图2E、F）。这些结果表明，病毒在CRFK和F81细胞中达到复制峰值的时间早于Fc3Tg细胞。为在蛋白质水平验证分离结果，表达VP1蛋白（由ORF2编码）以制备兔源多克隆抗体（pAb）。使用该抗VP1 pAb进行IFA和免疫印迹，确认了所有三种细胞系中强劲的病毒复制和特异性蛋白表达（图2G、H和图S2）。该新分离的FCV毒株被命名为HN/ZZ/2025株（GenBank登录号：PX408781）。
3.3. FCV HN/ZZ/2025株的病毒颗粒及理化特性
使用碘克沙醇密度梯度纯化病毒，病毒颗粒主要分配于30%层，经PCR和SDS-PAGE确认（图3A–C）。随后通过透射电子显微镜（TEM）观察，显示典型的二十面体病毒粒子，直径范围为35至40 nm（图3D）。进一步表征了HN/ZZ/2025分离株在各种环境应激条件下的理化稳定性。与未处理对照组相比，该分离株在50 °C孵育30分钟后完全灭活，在pH 3.0或pH 10.0处理后未检测到残留感染性。相反，病毒在暴露于乙醚或氯仿后仍保持高度感染性并诱导特征性CPE。这些发现表明，HN/ZZ/2025株对热不稳定，对极端pH条件敏感，但对脂溶剂具有抵抗力，与非囊膜病毒的特性一致（图3E、F）。
3.4. FCV HN/ZZ/2025的基因组特征及系统发育分析
菌株HN/ZZ/2025的完整基因组序列已提交至GenBank（登录号：PX408781）。基于HN/ZZ/2025的全基因组和VP1序列，结合33条参考序列重建了系统发育树（图4A、B）。系统发育分析将该分离株鉴定为基因型GI株（依据VP1分类[38]），与中国FCV毒株CH-JL4紧密聚类。该分离株与CH-JL4的核苷酸和氨基酸同源性最高，分别为85.7%和62.8%。与全球FCV分离株比较，核苷酸同源性范围为75.6%至85.7%，氨基酸同源性为46.3%至62.8%（表1）。氨基酸比对将VP1蛋白的E区定位于第427–524位残基。5′端（427–461 aa）和3′端（490–520 aa）高变区保守性低，而中间序列（462–489 aa）相对保守，可能构成E结构域的结构框架。这些基因组特征证实HN/ZZ/2025是基因型GI的代表性变异株（图4C、D）。使用WebLogo（版本v3.）分析了VP1 E区氨基酸的保守性（图4E–G）。
3.5. 感染FCV HN/ZZ/2025株猫的临床症状
为评估致病性，将两月龄猫纳入攻毒研究。实验组经鼻接种1 × 107 TCID50/0.1 mL的HN/ZZ/2025，阴性对照组给予等体积MEM。每日监测体重、直肠温度及症状评分等临床参数；感染后14天（dpi）进行全血细胞计数（图5A）。感染组发热在9 dpi达到峰值39.8–0.2 °C，并在整个观察期内持续显著高于对照组（图5B）。感染猫出现显著体重下降，在8至14 dpi达到峰值，可能继发于厌食、口腔溃疡导致的吞咽困难及全身性炎症（图5C）。临床评分在3 dpi后急剧上升，8–10 dpi达到峰值后逐渐下降（图5D）。值得注意的是，14 dpi时观察到显著白细胞增多（图5E），这可能主要反映与FCV感染相关的全身性炎症反应。尽管不能完全排除继发性细菌因素，但本研究中未进行专门的细菌学检查，因此无法确定其作用。
3.6. FCV HN/ZZ/2025株在猫中的致病性和组织嗜性
在21 dpi时，对猫实施安乐死进行剖检。攻毒猫表现出典型的呼吸道症状，包括流泪、眼分泌物增多及严重的牙龈充血伴相关溃疡。剖检显示感染组弥漫性肺充血伴多灶性点状出血及轻度气管黏膜下水肿（图6A）。组织病理学分析证实间质性肺炎，特征为广泛的炎性细胞浸润和肺水肿，同时伴有气管上皮脱屑和黏膜充血。肾小管上皮细胞显示退行性变化，包括空泡化，脾脏可见局灶性坏死（图6B）。免疫组化和免疫荧光显示，显著的FCV特异性（VP1）信号和荧光定位于肺、气管、肾脏和脾脏（图6C、D），而心脏和肝脏为阴性。这些结果表明，HN/ZZ/2025诱导全身性组织病理学损伤，主要嗜性为呼吸和肾脏组织。
3.7. 病毒排毒、病毒载量及蛋白表达的检测
通过每日采集感染猫的口腔和鼻腔拭子评估病毒排毒动力学，并采用RT-qPCR和TCID50试验进行定量。病毒RNA首次在1 dpi检测到，病毒RNA载量在5至7 dpi达到峰值，并持续可检测至观察期结束（21 dpi）（图7A）。在排毒期间，口腔和鼻腔拭子中也检测到感染性病毒，其趋势与病毒RNA水平相似（图7B）。在21 dpi时，感染猫的多个器官中检测到病毒RNA，而对照猫相应组织中未检测到病毒RNA（图7C）。与RT-qPCR结果一致，通过TCID50试验在感染猫的多个组织中检测到感染性病毒（图7D）。这些结果表明病毒呈全身性分布，其中呼吸道病毒检出最为显著。免疫印迹进一步证实感染组织中存在FCV VP1蛋白表达（图7E）。总之，这些结果表明HN/ZZ/2025在实验感染猫中能够有效复制、持续排毒和全身播散，而在21天观察期内未观察到死亡。
4. 讨论
在社会经济发展的驱动下，心理陪伴需求的日益增长推动了伴侣动物市场的快速扩张。据估计，全球有超过6亿只猫和9亿只狗与人类共同生活，其中猫的饲养尤为突出[1]。“同一健康”框架强调人类健康、伴侣动物及其共享环境之间深刻的相互关联性。对猫病毒性感染的研究不仅有助于提升兽医诊疗水平，还为相关人类病原体诊断工具和疫苗的开发提供信息。
在本研究中，我们对一株基因型GI的FCV毒株进行了鉴定，命名为HN/ZZ/2025，该毒株新分离自中国郑州的一个宠物市场。系统发育分析显示，HN/ZZ/2025与吉林毒株CH-JL4亲缘关系密切。值得注意的是，与某些VS-FCV毒株所报道的严重临床表现和高死亡率相比，以HN/ZZ/2025攻毒的猫在21天观察期内未发生致死性疾病。然而，感染猫出现了发热、体重减轻、口腔溃疡、持续性排毒和組織病理学病变，表明HN/ZZ/2025在本研究所采用的实验条件下可引起临床和病理学上明显的疾病。高密度环境，如收容所和宠物市场——其特征是免疫史不明的动物频繁流入——充当了FCV传播和遗传多样化的催化剂[39]。系统发育聚类表明HN/ZZ/2025与来自不同地理区域的FCV分离株之间存在遗传相关性。然而，本分析未提供足够证据来推断地理传播的方向或历史路径。
FCV和FHV-1仍是猫上呼吸道疾病的主要病毒病原体[5,6,7]。FCV尤其以高传染性和环境稳定性著称。我们对HN/ZZ/2025理化特性的研究表明，该分离株在50 °C下不耐热，对极端pH（3.0和10.0）敏感。然而，作为无囊膜病毒，其缺乏脂质囊膜使其对氯仿和乙醚等有机溶剂具有强抵抗力。这些发现表明，有效的生物安全措施必须优先使用经证实对无囊膜病毒有效的消毒剂，如次氯酸钠、过硫酸钾或高温处理（≥60 °C）[40,41]。
FCV基因组约7500 nt，由于复制过程中缺乏校对活性，具有高突变率[42]。由ORF2编码的VP1蛋白是主要结构蛋白，也是中和抗体的免疫优势靶标。我们观察到，虽然A、B、D和F区域保守，但高变E区（残基427–524）表现出显著多样性，这可能促进免疫逃逸并降低疫苗效力。该区域介导与猫连接黏附分子A（fJAM-A）受体的相互作用；其破坏与口腔溃疡的特征性形成相关。虽然在HN/ZZ/2025的ITTANQY中和表位中鉴定到一个点突变[43]，但PAGDY表位相对保守，提示其有潜力作为广泛交叉反应性诊断检测的靶标[44]。
历史上，FCV表现为低致病性病原体，直至以高死亡率（33–100%）为特征的VS-FCV变异株出现[45]。VS-FCV暴发已在全球范围内被记录，从美国和欧洲传播至亚洲。在中国，VS-FCV于2016年首次在东北虎中报道[35]，此后在家养种群中亦有检出。我们的实验攻毒表明，HN/ZZ/2025引起临床疾病和全身性组织受累，但在观察期内未导致死亡。本研究使用的感染剂量（每只猫1 × 10^7 TCID50/0.1 mL）旨在建立可重复的实验感染模型。该剂量与先前一项FCV致病性研究中使用的剂量相当，该研究中以1 × 10^7 TCID50/0.1 mL的FCV毒株TIG-1、2280和F9攻毒猫[35]。然而，使用受控的高剂量接种可能无法完全再现自然FCV暴露相关的感染动态。因此，本研究中观察到的临床表现、病毒排毒和组织分布应在实验攻毒模型的背景下进行解读。尽管观察到的疾病严重程度似乎不如某些VS-FCV毒株（如2280和TIG-1）所报道的那样明显，但本研究中观察到的全身性播散以及典型的呼吸道受累，凸显了HN/ZZ/2025广泛的组织分布。然而，基因组分析未揭示与高毒力一致相关的特定氨基酸特征，提示致病性可能为多基因决定，值得进一步开展功能基因组学研究。
FCV 在家猫种群中的持续循环为病毒进化提供了有价值的自然模型。有效管理需要临床观察与实验室确认（通过 RT-qPCR、病毒分离和免疫组织化学）之间的诊断协同[16]。尽管 RT-qPCR 具有高灵敏度，但 FCV 的遗传异质性要求对结果进行谨慎解读。目前，临床管理仍以支持性治疗为主，因为尚无明确的抗病毒疗法。虽然疫苗接种仍是预防的基石，但在 HN/ZZ/2025 等毒株中观察到的显著抗原变异凸显了持续优化疫苗的必要性。总之，有效的 FCV 控制需要采取综合策略，结合早期诊断、严格消毒以及开发具有更广泛交叉保护效力的疫苗。
本研究存在若干局限性。首先，动物实验每组仅纳入三只猫，这限制了致病性评估的统计效力和普适性。其次，未纳入毒力表型特征明确的参考 FCV 毒株进行直接体内比较；因此，对 HN/ZZ/2025 的相对致病性应谨慎解读。第三，未评估中和抗体应答和血清转化，因此无法表征感染后的宿主免疫应答。最后，本研究未评估 HN/ZZ/2025 的免疫原性、保护效力、安全性或遗传稳定性。因此，需要进一步研究以确定该分离株的生物学和免疫学意义及其在未来 FCV 研究中的潜在应用。
5. 结论
总之，本研究描述了 FCV 毒株 HN/ZZ/2025 的成功分离和全面表征，该毒株属于优势 GI 基因型。该分离株在免疫优势高变 E 区具有独特的基因组变异，并在 CRFK、F81 和 Fc3Tg 细胞系中表现出强劲的复制动力学和广泛的细胞嗜性。虽然实验性感染引起了临床疾病、全身性病毒播散和持续性病毒排毒，但在 21 天观察期内未观察到死亡。这些发现将 HN/ZZ/2025 表征为一株新分离的 FCV 毒株，具有独特的分子和生物学特征，并为未来 FCV 遗传多样性和致病性研究提供了有用的分离株。
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1. Introduction
As a primary companion species, domestic cats (Felis catus) maintain intimate proximity to human populations, with a global population now exceeding 600 million [1]. Feline infectious diseases are of paramount importance. Upper respiratory tract diseases (URTDs) are frequently encountered in feline medicine and are characterized by a spectrum of clinical manifestations, including increased serous to mucopurulent ocular and nasal discharge, corneal ulceration, oral stomatitis, and pyrexia [2,3]. Among the primary etiological agents, feline calicivirus (FCV) is a major pathogen affecting both domestic and wild felids and has long been recognized as a leading cause of feline URTDs and oral ulceration [4,5,6,7]. In recent years, FCV has undergone continuous evolution, giving rise to diverse genotypes under immune selective pressure [8]. As a result, FCV strains demonstrate a remarkable capacity for escaping host immune clearance [8]. At present, no specific antiviral therapeutics are available for FCV, and control strategies rely primarily on vaccination. However, existing inactivated vaccines—typically targeting feline panleukopenia virus (FPV), FCV, and feline herpesvirus type 1 (FHV-1)—frequently offer incomplete protection against emerging FCV variants [9,10,11,12]. These findings underscore the necessity for continued investigation into FCV to better elucidate viral infection dynamics in companion animals [1].
FCV is a highly transmissible pathogen taxonomically classified within the genus Vesivirus of the family Caliciviridae. This family encompasses significant human pathogens, such as noroviruses, as well as several animal-specific viruses, including rabbit hemorrhagic disease virus and European brown hare syndrome virus. The designation Caliciviridae is derived from the characteristic chalice-shaped surface depressions observed on the virion architecture [13]. FCV is an RNA virus with a high mutation rate that is ubiquitously distributed among domestic feline populations [14]. While host-restricted to felids and lacking zoonotic potential, FCV exhibits extensive genetic and antigenic variability [15]. The virus is endemic in feline populations [16] and presents with diverse clinical signs, including URTDs, lingual ulceration, periodontitis, and limping syndrome. Over the past two decades, certain lineages have evolved into virulent systemic FCV (VS-FCV) variants characterized by enhanced replication kinetics. Clinical manifestations of VS-FCV can include severe alopecia, cutaneous ulceration, and necrotizing pododermatitis of the pinnae and nostrils, often accompanied by serous crusting. Additional systemic complications include subcutaneous edema, bronchointerstitial pneumonia, and multi-organ necrosis (pancreas, liver, and spleen), culminating in high mortality rates among infected individuals [13,17,18,19,20,21,22,23,24,25].
The FCV genome consists of a positive-sense, single-stranded RNA of approximately 7.5 kilobases, which facilitates rapid evolutionary adaptation [13]. The non-segmented genomic RNA contains three functional open reading frames (ORFs) encoding six non-structural proteins (NS1–NS6/7) and two structural proteins (VP1 and VP2) [26]. ORF1 encodes a large polyprotein that undergoes post-translational cleavage into non-structural components, such as protease and polymerase [27]. ORF2 encodes a polyprotein that is proteolytically processed to yield the leader of the capsid protein and the major capsid protein (VP1), while ORF3 encodes the minor capsid protein (VP2) [28]. The mature viral capsid is architecturally composed of 180 copies of VP1 and 12 copies of the low-abundance VP2 protein [29].
Since its initial isolation in 1957 from cats presenting with respiratory symptoms [30], FCV has demonstrated global ubiquity, with detections reported throughout Europe, the Americas, and Asia [31,32]. In 1992, the virus was identified in ulcerative oral lesions of African lions and Siberian tigers; subsequently [33], it was first isolated from tigers in China in 2000. These findings indicate that FCV is geographically widespread and possesses a broad host range, with nearly all felid species being susceptible to infection [34,35]. As an RNA virus, FCV is predisposed to frequent mutations, leading to the emergence of highly virulent variants. Among these, circulating strains of the GI genotype are associated with virulent systemic disease (VSD-FCV) [36]. While historically rare, these strains have disseminated globally following the characterization of VS-FCV in the United States in the 1990s [37]. The expansion of the feline breeding industry in China, combined with high population densities, has provided a fertile environment for viral propagation. The lack of robust preventive measures and the continuous mutation of the virus have compromised vaccine efficacy, posing a significant threat to the health and conservation of both domestic and wild felids [19]. Despite extensive studies on FCV molecular epidemiology and pathogenicity, important gaps remain in the characterization of recently circulating FCV strains in China. In particular, the genetic diversity of newly emerging isolates has not always been accompanied by systematic evaluation of their in vitro replication characteristics and in vivo pathogenicity. Moreover, the relationship between genomic variation, particularly changes within the hypervariable regions of the VP1 capsid protein, and the biological properties of circulating FCV strains remains incompletely understood. Therefore, continuous isolation and characterization of contemporary FCV strains from different geographic regions are necessary to improve our understanding of FCV evolution and pathogenic diversity.
In the present study, we isolated an FCV strain, designated HN/ZZ/2025, from a feline trading market in Zhengzhou, Henan Province, China. We characterized its complete genome and phylogenetic relationships and systematically evaluated its in vitro replication properties, physicochemical stability, and in vivo pathogenicity in cats. We further investigated viral shedding, tissue distribution, and histopathological changes following experimental infection. These data provide useful insights into the molecular epidemiological landscape, replication characteristics, and pathogenic dynamics of circulating FCV strains in China, providing a useful isolate for future studies of FCV genetic diversity and pathogenesis.
2. Materials and Methods
2.1. Ethics Approval and Consent to Participate
All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Henan Agricultural University (Approval No. HNND2024050715). The study was conducted in accordance with relevant institutional regulations and animal welfare principles, including the 3Rs principles (Replacement, Reduction, and Refinement). The animals were monitored throughout the experimental period to minimize discomfort and distress. The scheduled experimental endpoint was 21 days post-infection, after which all animals were humanely euthanized by intravenous administration of pentobarbital sodium (100 mg/kg). The organs were subsequently collected for virological, Western blot, histopathological, and immunohistochemical analyses, and the remaining carcasses were disposed of according to institutional biosafety procedures.
2.2. Cells and Antibodies
CRFK (ATCC, CCL-94), F81 (Cellosaurus, CVCL_9259), and Fc3Tg (ATCC, CCL-176) cells were maintained in Eagle’s Minimum Essential Medium (EMEM, BNCC, Beijing, China) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in a humidified 5% CO2 atmosphere. A rabbit polyclonal antibody (pAb) targeting the VP1 protein of FCV was generated and characterized in our laboratory. Primary antibodies against GAPDH (10494-1-AP), CoraLite488-conjugated Goat Anti-Rabbit IgG (H+L) (SA00013-2) and HRP-conjugated goat anti-rabbit IgG (SA00001-2) secondary antibodies were purchased from Proteintech (Wuhan, China).
2.3. Sample Collection and Detection
In January 2025, a total of 22 cats exhibiting clinical signs suggestive of FCV infection were selected for sampling at a feline trading market in Zhengzhou, Henan Province, China. Oral and nasal swabs were collected from each cat and combined as one clinical sample per cat, resulting in a total of 22 clinical samples. The sampling strategy was based on the presence of clinical manifestations suggestive of FCV infection. The samples were suspended in phosphate-buffered saline (PBS, pH 7.2) and transported to the laboratory under refrigerated conditions. Because the cats were obtained from a commercial feline trading market, detailed information regarding their age, sex, breed, vaccination history, and previous treatment status was not available or could not be reliably verified. Total viral DNA/RNA was extracted using the FastPure Viral DNA/RNA Mini Kit (Vazyme, RC311, Nanjing, China), and cDNA synthesis was performed using HiScript IV All-in-One Ultra RT SuperMix (Vazyme, R433-01). The presence of FCV, FPV, FHV, FIPV, FBoV, and FeAstV was determined by PCR using specific primers (Supplementary Table S1).
2.4. Virus Isolation, Cultivation and Purification
FCV-positive specimens were selected for viral isolation. Homogenates were vortexed and centrifuged at 12,000 rpm for 5 min at 4 °C. The resulting supernatant was filtered (0.22-μm, Biosharp, Beijing, China) and diluted 1:10 in serum-free MEM. Confluent CRFK cell monolayers were washed twice with PBS and inoculated with the suspension supplemented with 1% Penicillin-Streptomycin-Gentamicin (Solarbio, P1410, Beijing, China). Cells were monitored for cytopathic effects (CPE) and harvested after three freeze–thaw cycles. For purification, three consecutive rounds of plaque purification were conducted using a 1% low-melting-point agarose overlay (Solarbio, A8350). Visible plaques were identified via Neutral Red staining (Beyotime, C0125, Shanghai, China) and individual plaques were selected for clonal expansion and subsequent PCR verification.
2.5. Next-Generation Sequencing Analysis
Metaviral RNA libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit (Illumina, New England Biolabs, Ipswich, MA, USA). Quality-filtered reads were aligned to the host reference genome using minimap2 (version v2.28) to eliminate host-derived contamination. De novo assembly of clean reads was performed using MEGAHIT (version v1.2.9), and ORFs were predicted via Prodigal (version v2.6.3). Functional annotation was conducted by aligning sequences against the Swiss-Prot database using MMseqs2 (version v2.0).
2.6. Genome Sequencing and Phylogeny Analysis
Full-length FCV genomic amplification was conducted using primers designed based on NGS results and NCBI reference sequences (Supplementary Table S2). The genome was amplified in three overlapping segments, cloned using the one-step ZTOPO-Blunt/TA kit (ZOMANBIO, Beijing Zoman Biotechnology Co., Ltd., Beijing, China), and subjected to Sanger sequencing. The FCV isolate, designated HN/ZZ/2025, was deposited in GenBank (Accession No. PX408781). Phylogenetic reconstruction was performed using the Maximum-likelihood (ML) method in MEGA version 7.0, based on the General Time Reversible (GTR+Γ+I) substitution model. Bootstrap values were calculated from 1000 replicates.
2.7. Virus Identification and Growth Curve
2.7.1. Viral Propagation and Titration
The purified FCV strain HN/ZZ/2025 was inoculated onto confluent monolayers of CRFK, F81, and Fc3Tg cells in 60 mm culture dishes. CPE were monitored daily and documented using an inverted microscope (Olympus, Tokyo, Japan). Upon the appearance of approximately 80% CPE, the cultures were subjected to three freeze–thaw cycles. Cell debris was removed by centrifugation at 8000 rpm for 10 min at 4 °C, and the virus-containing supernatants were harvested and stored at −80 °C.
The 50% tissue culture infectious dose (TCID50) was determined using the Reed-Muench mathematical model. Briefly, cells were seeded in 96-well plates at a density of 1 × 104 cells per well. After 24 h, the cells were inoculated with 10–fold serial dilutions of the virus (10−1–10−12) and incubated for 1 h at 37 °C. Following adsorption, the inoculum was replaced with 100 μL of maintenance medium (MEM supplemented with 2% FBS). The plates were incubated for 3–5 days and monitored daily for CPE. Each titration was performed in triplicate to calculate the mean viral titer.
2.7.2. Immunoblotting Analysis
Expression of FCV proteins was verified via Immunoblotting. CRFK, F81, and Fc3Tg cells were infected with FCV HN/ZZ/2025 at a multiplicity of infection (MOI) of 0.05. At 24 h post-infection (hpi), cells were harvested and lysed using RIPA buffer (Beyotime, P0013B, Shanghai, China). Protein samples were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and subsequently incubated overnight at 4 °C with a primary rabbit anti-VP1 polyclonal antibody. After washing, the membranes were incubated with a corresponding HRP-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized using Luminata Crescendo Western HRP Substrate (Millipore, WBLUR0500) and captured with a GE AI600 imaging system (GE, Boston, MA, USA).
2.7.3. Indirect Immunofluorescence Assay (IFA)
For IFA, CRFK, F81, and Fc3Tg cells were infected with FCV (MOI = 0.05) for 24 h. Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Following blocking with PBS containing 10% FBS for 1 h, the cells were incubated with a primary polyclonal antibody targeting the major capsid protein (VP1) for 1 h at 37 °C. After three washes with PBS, the cells were incubated with an Alexa-Fluor-conjugated secondary antibody (diluted in 10% FBS) for 1 h at room temperature. Viral localization was visualized and photographed using an Olympus fluorescence microscope.
2.7.4. Replication Kinetics and RT-qPCR Analysis
To evaluate FCV replication kinetics, CRFK, F81, and Fc3Tg cells in 35 mm dishes were infected with FCV at an MOI of 0.05. After 1 h of adsorption at 37 °C, the cells were washed three times with PBS to remove unbound virions. Cell samples were collected at 0, 6, 12, 18, 24, 30, 36, 48, and 60 hpi and subjected to total RNA extraction. Viral loads were quantified by real-time fluorescent quantitative PCR (RT-qPCR) targeting the VP1 gene. A standard curve was generated using 10-fold serial dilutions of a pET21b-FCV-VP1 recombinant standard plasmid. The RT-qPCR was performed in triplicate using the following primers: FCV VP1-Fw: 5′-TTGCAAAGAGCATGGGGTGA-3′, FCV VP1-Rv: 5′-CCAAGCATTGCCAGTGTGAC-3′. The viral copy numbers were calculated by mapping the mean cycle threshold (Ct) values against the established standard curve.
2.8. Transmission Electron Microscopy (TEM) and Physicochemical Characterization
2.8.1. Morphological Characterization
To characterize the morphology of FCV virions, the virus was propagated in CRFK cells, and the resulting culture supernatant was harvested. The virus was concentrated via ultracentrifugation and subsequently purified using an iodixanol (OptiPrep) density gradient. The presence of purified FCV was confirmed by PCR and SDS-PAGE analysis. For TEM visualization, the purified viral suspension was applied to a carbon-coated copper grid (supporting membrane) and negatively stained with 1% phosphotungstic acid. Excess stain was removed using filter paper, and the grids were dried at room temperature. The virion architecture and diameter were examined using a transmission electron microscope (JEOL, JEM-1400, JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 80–100 kV.
2.8.2. Physicochemical Stability
The environmental stability of FCV HN/ZZ/2025 was assessed using cultures with a starting titer of 1 × 107.5 TCID50/mL. The viral suspensions were subjected to various stressors, including thermal treatment (50 °C), acidic conditions (pH 3.0), alkaline conditions (pH 10.0), and exposure to organic solvents (chloroform and ether). Following treatment, the samples were neutralized or processed as required and inoculated onto confluent CRFK cell monolayers to monitor for CPE. Culture supernatants were harvested 48 h post-inoculation, and residual viral infectivity was quantified using the TCID50 assay.
2.9. Preparation of Rabbit Polyclonal Antibody Against VP1 Protein
The FCV VP1 gene was amplified and subcloned into the pET21b and pGEX-4T-1 prokaryotic expression vectors (Figure S1). Recombinant plasmids were transformed into Escherichia coli BL21 (DE3) competent cells. The transformed bacteria were cultured at 37 °C until the OD600 reached 0.6–0.8, at which point protein expression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside for 12 h at 18 °C. The cells were harvested by centrifugation, resuspended in 30 mL of lysis buffer, and disrupted via sonication.
The expression of recombinant VP1 protein was verified by Western blot using anti-His-tag and anti-GST-tag antibodies. The His-tagged and GST-tagged VP1 proteins were purified using Ni-NTA Resin (GenScript, L00250, Nanjing, China) and Glutathione Resin (GenScript, L00206), respectively, according to the manufacturer’s protocols. The purified VP1 protein was used as an immunogen to immunize one 4-month-old New Zealand White rabbit. For each immunization, 100 μg of purified VP1 protein was emulsified with Freund’s adjuvant (Sigma, Burlington, MA, USA) and administered subcutaneously. Immunizations were performed at 14-day intervals for a total of three immunizations. A fourth booster immunization was subsequently administered with VP1 protein alone. Serum was collected after the final booster immunization and the antibody titre was approximately 1:50,000. The specificity and titer of the rabbit anti-VP1 serum were validated via Western blot and IFA for the detection of FCV (Figure S2).
2.10. Animal Infection Studies
Six two-month-old domestic cats, including four females and two males, were enrolled in the challenge study. The cats were common domestic cats, and were confirmed negative for FCV, FPV, and FHV by PCR before the experiment. Prechallenge serum samples were also collected for ELISA and virus neutralization assays. All six cats were negative for detectable antibodies against FCV, FPV, and FHV, and no neutralizing antibodies against these viruses were detected before challenge. The animals were housed in a dedicated animal facility at Henan Agricultural University with adequate ventilation and received routine husbandry, including free access to food and water, throughout the study. Following a 7-day acclimatization period, the cats were randomly assigned to two groups (n = 3/group). The challenge group was intranasally inoculated with 1 mL of MEM containing 1 × 107 TCID50/0.1 mL of FCV HN/ZZ/2025, while the control group received an equal volume of virus-free MEM. Clinical health status—including pyrexia, weight fluctuations, mental status, appetite, and mucosal ulceration—was recorded daily using a standardized clinical scoring system. Oral and nasal swabs were collected daily for viral shedding analysis via RT-qPCR. On day 21 post-challenge, all cats were humanely euthanized for systemic pathological evaluation. Heart, liver, spleen, lung, kidney, and trachea tissues were harvested to quantify viral burden and evaluate histopathological alterations. Animal procedures were conducted in accordance with the approved animal welfare protocol and the principles of Replacement, Reduction, and Refinement (3Rs). Appropriate measures were taken throughout the experiment to minimize animal discomfort and distress. Animals were euthanized by intravenous administration of pentobarbital sodium (100 mg/kg), as described in Section 2.1.
A clinical scoring system was established to comprehensively assess the health status of the experimental cats. Eight parameters were evaluated: body temperature, body weight, mental status, appetite, ocular discharge, nasal discharge, oral ulceration, and survival status. Body temperature was scored as 0 when it ranged from 38.0 to 39.5 °C, 1 when it was ≥39.5 °C, and 2 when it was <37.0 °C. A body weight change of <3% was assigned a score of 0, whereas a weight loss of ≥3% was assigned a score of 2. Mental status was scored as 0 for normal behavior and 1 for lethargy. Appetite was scored as 0 for normal food intake, 1 for reduced appetite, and 2 for anorexia. Ocular discharge was scored as 0 when absent and 1 when present. Nasal discharge was scored as 0 when absent, 1 for mild discharge, and 2 for moderate to severe discharge. Oral ulceration was scored as 0 when absent, 1 when small and few lesions were present, and 2 when large and multiple lesions were present. Survival status was scored as 0 for surviving animals and 10 in the event of death.
2.11. Viral RNA Load Determination
The viral burden in harvested tissues, as well as oral and nasal swabs from infected cats, was quantified via absolute RT-qPCR following experimental challenge. Total viral RNA was extracted from 200 μL of swab supernatants or tissue homogenates using the TIANamp Virus DNA/RNA Kit (TIANGEN, DP315, Beijing, China) according to the manufacturer’s instructions. Subsequent cDNA synthesis was performed using HiScript IV All-in-One Ultra RT SuperMix (Vazyme, R433-01).
Quantification of FCV genomic copy numbers was conducted as previously described. Briefly, a 179 bp fragment of the FCV VP1 gene was cloned into the pET21b vector to serve as a standard. Absolute quantification was achieved using a standard curve generated from 10-fold serial dilutions of the recombinant plasmid (101–1012 copies/μL). The RT-qPCR was performed in triplicate using ChamQ SYBR qPCR Master Mix (Vazyme, Q341-02) on a real-time PCR system. The primer sequences utilized were FCV VP1-Fw: 5′-TTGCAAAGAGCATGGGGTGA-3′, FCV VP1-Rv: 5′-CCAAGCATTGCCAGTGTGAC-3′.
2.12. Histopathological and Immunohistochemical (IHC) Analyses
2.12.1. Histopathology
During necropsy, representative samples of the heart, liver, spleen, lung, kidney, and trachea were systematically collected. Tissues were fixed in 4% paraformaldehyde at room temperature for 72 h, followed by dehydration in a graded ethanol series, clearing in xylene, and embedding in paraffin wax. The paraffin-embedded blocks were sectioned at a thickness of 5 μm using a microtome (Servicebio, Wuhan, China). For histopathological evaluation, the sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin. Pathological alterations were examined and photographed under a light microscope.
2.12.2. IHC
For the in situ detection of FCV antigens, IHC was performed on tissue sections. Briefly, slides were deparaffinized, rehydrated, and subjected to heat-induced epitope retrieval using EDTA buffer (pH 9.0). Endogenous peroxidase activity was quenched, and sections were blocked with 5% BSA for 30 min at room temperature. The sections were then incubated with the primary rabbit anti-FCV VP1 pAb overnight at 4 °C.
For IHC, sections were subsequently incubated with an HRP-labeled anti-rabbit IgG secondary antibody (1:500, Servicebio). FCV VP1 antigen-positive cells were visualized with 3,3′-Diaminobenzidine, yielding a characteristic brown precipitate, and nuclei were counterstained with hematoxylin. The procedures mirrored those described in Section 2.7, with the additional inclusion of the EDTA antigen retrieval step. All slides were analyzed under a light or fluorescence microscope to evaluate the distribution and intensity of the viral antigen.
2.13. Statistical Analysis
Quantitative data were analyzed using GraphPad Prism 8.0 software. Statistical significance between groups was determined using Student’s t-test. Data are presented as mean ± SD, and p-values < 0.05 were considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001).
3. Results
3.1. FCV Sample Collection, Detection and Next-Generation Sequencing Analysis
In January 2025, oral and nasal swabs were collected from 22 cats clinically suspected of FCV infection at a feline trading market in Zhengzhou, Henan Province, China. Oral and nasal swabs from each cat were combined as one clinical sample, resulting in 22 samples in total (Figure 1A). Total DNA/RNA was extracted, and subsequent PCR analysis confirmed FCV positivity in the specimens. Screening for other feline viruses revealed that the majority of cases involved co-infections, with only two of the 22 samples (2/22) identified as FCV-only infections. Aside from FCV, feline panleukopenia virus (FPV), feline bocavirus (FBoV), and feline herpesvirus (FHV) exhibited the highest prevalence rates (Figure 1B). To investigate potential cryptic pathogens, oral and nasal swabs were subjected to next-generation sequencing. Comprehensive analysis against the NCBI-nt and RVDB databases was performed to identify any associated microbial signatures. After filtering host-derived sequences and performing taxonomic annotation, FCV-specific sequences were found to be the most abundant viral reads, comprising nearly 100% of the viral population (Figure 1C). Consequently, FCV-positive samples from single-infection cases were selected for further virus isolation.
3.2. Isolation, Characterization, and Purification of FCV
CRFK cells were inoculated with supernatants from processed FCV-positive oral and nasal swab. Inoculated CRFK cells exhibited significant CPE—characterized by cell rounding, shrinkage, and a “grape-like” morphology—starting at 12 h post-infection (hpi), whereas control cells remained confluent (Figure 2A). Similar CPE was observed in F81 and Fc3Tg cells, though cytopathic changes in Fc3Tg cells were delayed, appearing only after 24 hpi. Successful isolation was followed by plaque purification, with distinct plaques visible after three successive rounds (Figure 2B). Stable FCV replication was confirmed through six consecutive passages in CRFK cells, with FCV-specific genomic fragments consistently detected in the culture supernatants via PCR (Figure 2C). The viral titers (TCID50) in CRFK, F81, and Fc3Tg cells were 107.18, 107.50, and 105.89 per 0.1 mL, respectively (Figure 2D). Replication kinetics, assessed using an MOI of 0.05, demonstrated that the isolate reached peak viral loads at 24 hpi in CRFK and F81 cells, and at 36 hpi in Fc3Tg cells (Figure 2E,F). These results indicate that the virus reached its peak replication level earlier in CRFK and F81 cells than in Fc3Tg cells. To validate the isolation at the protein level, the VP1 protein (encoded by ORF2) was expressed to generate a rabbit-derived polyclonal antibody (pAb). IFA and immunoblotting using this anti-VP1 pAb confirmed robust viral replication and specific protein expression across all three cell lines (Figure 2G,H and Figure S2). This newly isolated FCV strain was designated strain HN/ZZ/2025 (GenBank accession number: PX408781).
3.3. Virus Particles and Physicochemical Properties of the FCV Strain HN/ZZ/2025
The virus was purified using an iodixanol density gradient, with viral particles primarily partitioning into the 30% layer, as confirmed by PCR and SDS-PAGE (Figure 3A–C). Subsequent visualization via transmission electron microscopy (TEM) revealed typical icosahedral virions with diameters ranging from 35 to 40 nm (Figure 3D). The physicochemical stability of the HN/ZZ/2025 isolate was further characterized under various environmental stressors. Compared to the untreated control group, the isolate was completely inactivated after incubation at 50 °C for 30 min, and no residual infectivity was detected following treatment at pH 3.0 or pH 10.0. Conversely, the virus remained highly infectious and induced characteristic CPE after exposure to ether or chloroform. These findings indicate that strain HN/ZZ/2025 is thermolabile and sensitive to extreme pH conditions but remains resistant to lipid solvents, consistent with the properties of a non-enveloped virus (Figure 3E,F).
3.4. Genomic Characteristics and Phylogeny Analysis of FCV HN/ZZ/2025
The complete genome sequence of strain HN/ZZ/2025 was deposited in GenBank (accession number: PX408781). Phylogenetic trees were reconstructed based on the whole-genome and VP1 sequences of HN/ZZ/2025 in conjunction with 33 reference sequences (Figure 4A,B). Phylogenetic analysis identified the isolate as a genotype GI strain (classified by VP1 [38]), clustering closely with the Chinese FCV strain CH-JL4. The isolate shared its highest nucleotide and amino acid identities with CH-JL4 at 85.7% and 62.8%, respectively. Comparison with global FCV isolates revealed nucleotide identities ranging from 75.6% to 85.7% and amino acid identities from 46.3% to 62.8% (Table 1). Amino acid alignment localized the E region of the VP1 protein to residues 427–524. The 5′ (427–461 aa) and 3′ (490–520 aa) hypervariable regions exhibited low conservation, whereas the intervening sequence (462–489 aa) remained relatively conserved and likely constitutes the structural framework of the E domain. These genomic features confirm that HN/ZZ/2025 is a representative genotype GI variant (Figure 4C,D). The conservation of amino acids in the VP1 E region was analyzed using WebLogo (version v3.) (Figure 4E–G).
3.5. Clinical Signs of Cats Infected with the FCV HN/ZZ/2025 Strain
To evaluate pathogenicity, two-month-old cats were enrolled in a challenge study. The experimental group was intranasally inoculated with 1 × 107 TCID50/0.1 mL of HN/ZZ/2025, while the negative control group received an equivalent volume of MEM. Clinical parameters, including body weight, rectal temperature, and symptomatic scores, were monitored daily; a complete blood count was performed at 14 days post-infection (dpi) (Figure 5A). In the infected group, pyrexia peaked at 39.8–0.2 °C by 9 dpi and remained significantly elevated relative to the control group throughout the observation period (Figure 5B). Infected cats exhibited significant weight loss peaking between 8 and 14 dpi, likely secondary to anorexia, dysphagia caused by oral ulceration, and systemic inflammation (Figure 5C). Clinical scores rose sharply after 3 dpi, peaking at 8–10 dpi before gradually declining (Figure 5D). Notably, significant leukocytosis was observed at 14 dpi (Figure 5E), which may primarily reflect a systemic inflammatory response associated with FCV infection. Although secondary bacterial factors cannot be completely excluded, no specific bacteriological investigations were performed in the present study, and therefore their contribution could not be determined.
3.6. Pathogenicity and Tissue Tropism of the FCV Strain HN/ZZ/2025 in Cats
At 21 dpi, cats were euthanized for necropsy. Challenged cats presented with typical respiratory signs, including lacrimation, increased ocular discharge, and severe gingival congestion with associated ulceration. Necropsy revealed diffuse pulmonary hyperemia with multifocal petechial hemorrhages and mild tracheal submucosal edema in the infected group (Figure 6A). Histopathological analysis confirmed interstitial pneumonia, characterized by extensive inflammatory cell infiltration and pulmonary edema, alongside tracheal epithelial desquamation and mucosal congestion. Renal tubular epithelial cells showed degenerative changes, including vacuolization, while focal necrosis was identified in the spleen (Figure 6B). Following IHC and IFA, significant FCV-specific (VP1) signals and fluorescence were localized to the lungs, trachea, kidneys, and spleen (Figure 6C,D), whereas the heart and liver remained negative. These findings demonstrate that HN/ZZ/2025 induces systemic histopathological damage, with a primary tropism for respiratory and renal tissues.
3.7. Detection of Virus Shedding, Viral Load, and Protein Expression
Viral shedding kinetics were evaluated by daily collection of oral and nasal swabs from infected cats and quantified by RT-qPCR and TCID50 assays. Viral RNA was first detected at 1 dpi, with viral RNA loads peaking between 5 and 7 dpi and remaining detectable until the end of the observation period at 21 dpi (Figure 7A). Infectious virus was also detected in oral and nasal swabs during the shedding period, with a similar trend to viral RNA levels (Figure 7B). At 21 dpi, viral RNA was detected in multiple organs of infected cats, whereas no viral RNA was detected in the corresponding tissues of control cats (Figure 7C). Consistent with the RT-qPCR results, infectious virus was detected in several tissues of infected cats by TCID50 assay (Figure 7D). These findings indicate systemic viral distribution, with the respiratory tract showing prominent viral detection. Immunoblotting further confirmed FCV VP1 protein expression within the infected tissues (Figure 7E). Collectively, these findings demonstrate that HN/ZZ/2025 is capable of efficient replication, persistent viral shedding, and systemic dissemination in experimentally infected cats, while no mortality was observed during the 21-day observation period.
4. Discussion
Driven by socio-economic development, the growing demand for psychological companionship has led to a rapid expansion of the companion animal market. It is estimated that over 600 million cats and 900 million dogs live alongside humans worldwide, with feline ownership being particularly prominent [1]. The “One Health” framework emphasizes the profound interconnectivity between human health, companion animals, and their shared environments. Research on feline viral infections not only enhances veterinary care but also informs the development of diagnostic tools and vaccines for related human pathogens.
In the present study, we characterized a genotype GI FCV strain, designated HN/ZZ/2025, newly isolated from a pet market in Zhengzhou, China. Phylogenetic analysis revealed that HN/ZZ/2025 is closely related to the Jilin strain CH-JL4. Notably, compared with the severe clinical manifestations and high mortality reported for some VS-FCV strains, cats challenged with HN/ZZ/2025 did not develop fatal disease during the 21-day observation period. However, the infected cats developed pyrexia, weight loss, oral ulceration, persistent viral shedding, and histopathological lesions, indicating that HN/ZZ/2025 can cause clinically and pathologically evident disease under the experimental conditions used in this study. High-density settings, such as shelters and pet markets–characterized by a frequent influx of animals with undocumented immunological histories—act as catalysts for FCV transmission and genetic diversification [39]. Phylogenetic clustering indicates genetic relatedness between HN/ZZ/2025 and FCV isolates reported from different geographic regions. However, the present analysis does not provide sufficient evidence to infer the direction or historical route of geographic dissemination.
FCV and FHV-1 remain the predominant viral agents of feline URTDs [5,6,7]. FCV is particularly notable for its high contagiousness and environmental stability. Our investigation into the physicochemical properties of HN/ZZ/2025 demonstrated that the isolate is thermolabile at 50 °C and sensitive to extreme pH (3.0 and 10.0). However, as a non-enveloped virus, its lack of a lipid envelope confers robust resilience against organic solvents like chloroform and ether. These findings suggest that effective biosecurity measures must prioritize disinfectants validated against non-enveloped viruses, such as sodium hypochlorite, potassium persulfate, or high-level heat treatment (≥60 °C) [40,41].
The ~7500 nt FCV genome is subject to high mutation rates due to the absence of proofreading activity during replication [42]. The VP1 protein, encoded by ORF2, is the primary structural protein and immunodominant target for neutralizing antibodies. We observed that while regions A, B, D, and F are conserved, the hypervariable E region (residues 427–524) exhibits significant diversity, which likely facilitates immune evasion and reduces vaccine efficacy. This region mediates interaction with the feline junctional adhesion molecule A (fJAM-A) receptor; its disruption is linked to the characteristic formation of oral ulcers. While a point mutation was identified in the ITTANQY neutralizing epitope of HN/ZZ/2025 [43], the PAGDY epitope remained relatively conserved, suggesting its potential utility as a target for broadly cross-reactive diagnostic assays [44].
Historically, FCV manifested as a low-pathogenicity pathogen until the emergence of VS-FCV variants characterized by high mortality (33–100%) [45]. Outbreaks of VS-FCV have been documented globally, spreading from the United States and Europe to Asia. In China, VS-FCV was first reported in 2016 in a Amur tiger [35] and has since been detected in domestic populations. Our experimental challenge demonstrated that HN/ZZ/2025 caused clinical disease and systemic tissue involvement but did not result in mortality during the observation period. The infectious dose used in the present study (1 × 107 TCID50/0.1 mL per cat) was selected to establish a reproducible experimental infection model. This dose is comparable to that used in a previous FCV pathogenicity study, in which cats were challenged with 1 × 107 TCID50/0.1 mL of FCV strains TIG-1, 2280, and F9 [35]. However, the use of a controlled high-dose inoculum may not fully reproduce the infection dynamics associated with natural FCV exposure. Therefore, the clinical manifestations, viral shedding, and tissue distribution observed in the present study should be interpreted in the context of the experimental challenge model. Although the observed disease severity appeared less pronounced than that reported for some VS-FCV strains such as 2280 and TIG-1, the systemic dissemination observed in the present study, together with typical respiratory involvement, highlights the broad tissue distribution of HN/ZZ/2025. However, genomic analysis did not reveal specific amino acid signatures consistently associated with hyper-virulence, suggesting that pathogenicity is likely polygenic and warrants further functional genomic investigation.
The persistent circulation of FCV within domestic cat populations serves as a valuable natural model for viral evolution. Effective management necessitates diagnostic synergy between clinical observation and laboratory confirmation via RT-qPCR, virus isolation, and immunohistochemistry [16]. Although RT-qPCR offers high sensitivity, the genetic heterogeneity of FCV requires cautious interpretation of results. Currently, clinical management remains largely supportive, as definitive antiviral therapies are lacking. While vaccination remains the cornerstone of prevention, the substantial antigenic variation observed in strains like HN/ZZ/2025 underscores the need for continuous vaccine optimization. In conclusion, effective FCV control requires an integrated approach combining early diagnosis, rigorous disinfection, and the development of vaccines with broader cross-protective efficacy.
This study has several limitations. First, the animal experiment included only three cats per group, which limited the statistical power and generalizability of the pathogenicity assessment. Second, no reference FCV strain with a well-characterized virulence phenotype was included for direct in vivo comparison; therefore, the relative pathogenicity of HN/ZZ/2025 should be interpreted with caution. Third, neutralizing antibody responses and seroconversion were not evaluated, and thus the host immune response following infection could not be characterized. Finally, the present study did not evaluate the immunogenicity, protective efficacy, safety, or genetic stability of HN/ZZ/2025. Therefore, further studies are required to determine the biological and immunological significance of this isolate and its potential applications in future FCV research.
5. Conclusions
In conclusion, this study describes the successful isolation and comprehensive characterization of FCV strain HN/ZZ/2025, a member of the dominant GI genotype. The isolate features distinct genomic variations within the immunodominant hypervariable E region and exhibits robust replication kinetics and broad cellular tropism across CRFK, F81, and Fc3Tg cell lines. While experimental infection caused clinical disease, systemic viral dissemination, and persistent viral shedding, no mortality was observed during the 21-day observation period. These findings characterize HN/ZZ/2025 as a newly isolated FCV strain with distinct molecular and biological characteristics and provide a useful isolate for future studies of FCV genetic diversity and pathogenesis.