兽研1. 引言
猫细小病毒（FPV；与猫泛白细胞减少症病毒或猫病毒性肠炎同义）是一种高度传播的病毒性病原体，在国内猫中具有重要临床意义，晚期病例常导致严重疾病和死亡[1,2]。该病毒可感染所有类型的猫，包括猫科物种和某些非猫科宿主，如浣熊和水貂[1]。在受影响群体中，幼猫尤其易感，因为其免疫系统尚未成熟，难以有效防御，导致死亡率较高[3,4]。
该病的临床症状范围广泛，从轻微自限性疾病到严重病症和快速致死性结局不等[2]。感染猫通常表现嗜睡、厌食、呕吐、腹泻和明显脱水等症状[5]。临床上该病分为四种类型，包括超急性、急性、亚急性和围产期型。此外，发病率和死亡率均受感染株毒力的影响[6]。FPV具有高度的环境稳定性和抵抗力，对极低温度和许多常用消毒剂具有抵抗力，使其能在污染环境中长期存活[7]。
FPV属于细小病毒科，是一种无囊膜、单链DNA的小型病毒。其基因组、结构和免疫学特征与犬细小病毒（CPV）高度相似。CPV分为2a、2b和2c等几种类型，也可感染猫。普遍认为FPV是原始病毒，CPV通过自然突变由其进化而来[8]。在细小病毒属中，FPV、CPV和水貂肠炎病毒（MEV）构成一个密切相关的抗原群[8,9,10,11]。
FPV基因组大小约5.2 kb，包含四个编码开放阅读框（ORF）：两种衣壳蛋白（VP1和VP2）和两种非结构蛋白（NS1和NS2）[12,13]。其中，由第四个ORF编码的VP2蛋白约占病毒衣壳的90%，在决定宿主特异性方面起关键作用。它还包含多个B细胞表位，对引发中和性保护抗体反应至关重要[14]。由于其在病毒进化、宿主适应和免疫识别中的重要性，VP2基因已被广泛研究，并常用作亚单位疫苗开发中的靶抗原[14,15]。
FPV和CPV在全球伴侣动物中已被广泛报道。在越南，已有细小病毒的报道[16,17,18]；然而，关于FPV和CPV的可用数据仍然有限，尤其是在越南中部，其遗传特征和进化关系尚未得到充分表征。因此，本研究基于VP2序列对越南中部猫的FPV和CPV分子特征进行了系统分析，这对于填补该地区FPV和CPV分子流行病学的重要空白是必要的。
2. 结果
2.1. 通过PCR鉴定细小病毒基因组
本研究采集了越南中部家猫的样本。在269份检测样本中，25份（9.29%）样本病毒DNA PCR阳性。具体而言，多乐省和嘉莱省的细小病毒DNA阳性率分别为5.16%和25%（表1），表明两省间猫细小病毒DNA检出率存在显著差异（p < 0.001）。有趣的是，本研究中所有猫的疫苗接种状况均不可知，包括25份阳性样本。
此外，进一步分析了采集样本中细小病毒基因组在品种、年龄、性别和健康状况方面的分布（表2）。具体而言，在所有年龄类别中均发现了细小病毒基因组，包括<6月龄、6–12月龄和>12月龄。值得注意的是，6月龄以下猫的感染率（17.65%）显著高于6至12月龄猫（4.24%）（p = 0.003）。>12月龄猫的基因组阳性率为7.58%，低于<6月龄猫，高于6–12月龄猫，但差异无统计学意义（分别为p = 0.09和p = 0.33）。相比之下，品种、性别和健康状况之间未观察到显著差异。简言之，病毒基因组在23/216只本地猫（10.65%）和2/53只外来或杂交猫（3.77%）中检出。此外，在雄性（7.96%）和雌性（10.26%）以及健康猫（8.02%）和患病猫（14.04%）中均鉴定出细小病毒。
2.2. 越南细小病毒株完整VP2基因分析
为研究病毒株的遗传特征，对来自 Daklak 和 Gialai 两省家猫的七份细小病毒 DNA 阳性样本进行了核苷酸测序。测序色谱图显示出对应于各个核苷酸的清晰峰，表明测序数据质量高且可靠。通过组装使用不同引物对获得的三个重叠扩增子，并通过重复测序独立验证，生成了完整的 VP2 基因序列，确保了 VP2 基因的全长覆盖。在本研究中，从 25 份 PCR 阳性样本中，基于 PCR 产物的质量和数量足以进行测序并提供两个相邻省份的代表性，选择了七份样本进行完整 VP2 基因测序。所选样本包括来自 Gialai 的三份样本和来自 Daklak 的四份样本。获得的越南毒株 VP2 基因序列分别命名为 Cat/Vietnam/FPV-334/2024、Cat/Vietnam/FPV-340/2024、Cat/Vietnam/FPV-343/2024、Cat/Vietnam/FPV-351/2025、Cat/Vietnam/FPV-361/2025、Cat/Vietnam/CPV-455/2025 和 Cat/Vietnam/FPV-465/2025。为简洁起见，毒株名称分别缩写为 FPV-334/2024（PZ276925）、FPV-340/2024（PZ276926）、FPV-343/2024（PZ276927）、FPV-351/2025（PZ276928）、FPV-361/2025（PZ276929）、CPV-455/2025（PZ276931）和 FPV-465/2025（PZ276930）。序列分析显示，六株细小病毒被确定为 FPV，而一株细小病毒聚类于 CPV 内。
将七株具有完整 VP2 基因序列（1755 bp）的越南细小病毒株与 GenBank 中的参考序列进行比对和分析。这些毒株之间的核苷酸序列相似性范围为 98.23% 至 100%（表3）。值得注意的是，毒株 FPV-334/2024、FPV-340/2024 和 FPV-361/2025 在核苷酸水平上完全相同（100%），而毒株 FPV-465/2025 和 CPV-455/2025 表现出最低的相似性水平（98.23%）（表3A）。在蛋白质水平上，VP2 氨基酸（aa）序列的同一性范围为 98.22% 至 100%（表3B）。
对七株获得的细小病毒株的完整 VP2 基因序列与 GenBank 参考序列进行比较评估，揭示了高核苷酸相似性。毒株 FPV-343/2024 和 FPV-351/2025 与2020年在中国报道的 JSYZ-122 株（登录号 MW017628.1）表现出99.77%的同一性（表4）。毒株 FPV-334/2024、FPV-340/2024 和 FPV-361/2025 与2019年最初在中国报道的 YZ2019-025 株（登录号 OQ863617.1）共享最高的核苷酸相似性（99.94%）。在当前越南毒株中，FPV-465 株与2008年在意大利报道的 Felocell 株（登录号 EU498681.1）显示出99.85%的核苷酸同一性。CPV-455/2025 株与2022年在中国鉴定的 QD-201 株（登录号 MT394166.1）表现出99.25%的核苷酸同一性（表4）。
2.3. 越南细小病毒株完整 VP2 基因序列的系统发育评估
对完整 VP2 基因序列（包括七株代表性细小病毒株和 GenBank 中的其他可用序列）进行的系统发育分析显示，六株细小病毒分离株在 FPV 组内被分类为三个独立的分支，而 CPV 株聚类于 CPV 组内，特别是属于犬 CPV-2c 谱系（图1）。更具体地说，FPV-343/2024 和 FPV-351/2025 株共聚类于一个独特的分组内，与2018年从中国一只猫中分离的 Beijing-I.3/2018 株密切相关（图1）。此外，这些毒株与先前报道的2018年越南 FPV 株具有高度遗传相似性。有趣的是，四株获得的 FPV 株（FPV-340/2024、FPV-334/2024、-361/2025 和 -465/2025）属于 FPV 组的一个独立分支。它们与2018年至2022年间在中国以及2023年在西班牙的猫和犬宿主中报道的 FPV 株显示出密切的遗传相关性。关于 CPV 组，CPV-455/2025 株位于一个包含同期来自猫和犬的中国及先前报道的越南 CPV 株的分支内（图1）。该株还与2022年报道的一株中国株（登录号 MT392166.1）共享高度相似性。此外，几个内部节点的自举支持率为低至中等；因此，这些关系被解释为遗传相关性的证据，而非确定的进化关系。
2.4. 获得的细小病毒 VP2 蛋白预测氨基酸序列的评估
对翻译序列的分析表明，七个FPV/CPV分离株的长度均为584个氨基酸。与先前报道的来自犬和猫的越南FPV以及疫苗株的比较分析显示，五重β-折叠区域（aa 152–169）在所有分析的FPV/CPV株中高度保守。然而，在N端、Loop 1–Loop 4和C端区域观察到多个氨基酸替换（表5）。此外，第101、122、236、237和299位的残基分别为T、N、T、D和G。值得注意的是，FPV-343/2024、FPV-351/2025、CPV-455/2025和FPV-465/2025在第91位具有A，而FPV-334/2024、FPV-340/2024和FPV-361/2025在该位置保留S。此外，当前四个FPV/CPV株在第232位表现为V，而CPV-455/2025和FPV-465/2025在同一位置含有I。
2.5. 所获得FPV株VP2蛋白序列的选择压力模式评估
选择压力分析在所分析的FPV分离株VP2蛋白内鉴定出26个位点存在负选择证据，每个位点的后验概率值均高于0.9（Prob[α < β] > 0.9）。相比之下，在VP2蛋白中未检测到处于正选择下的位点（表6）。
3. 讨论
细小病毒，尤其是FPV，是全球范围内影响猫的最具传染性和常致死的疾病之一。尽管常规进行疫苗接种，但在越南的家猫中仍持续检测到细小病毒。此外，基因组分析揭示了独特的抗原变异，可将FPV野毒株与疫苗衍生株区分开来[19]。
在本研究中，2023–2025年期间在越南中部的两个省/市的家猫粪便样本中，有9.29%检测到猫细小病毒基因组。有趣的是，该研究涵盖了先前未进行过猫细小病毒调查的地区，从而产生了新的区域数据。这些发现有助于填补流行病学空白并改善整体监测基础设施。此外，结果显示6月龄以下的猫细小病毒基因组检出率显著高于6至12月龄的猫（p < 0.05）。这一观察与先前的研究一致，即三至五月龄的幼猫对FPV特别易感[20]。然而，年龄与FPV/CPV阳性之间的关联基于单变量分析，未针对潜在混杂因素（尤其是本研究中的省份）进行调整。因此，<6月龄猫中观察到的较高阳性率应谨慎解读，并不一定表明年龄的独立效应。未来应开展更大样本量的研究，使用多变量分析来确定FPV感染的独立预测因素。此外，雄性和雌性阳性率分别为7.96%和10.26%，差异无统计学意义（p > 0.05）。该结果表明性别不影响猫细小病毒的检出率[21]。本研究结果与其他报道一致，即疾病频率基于性别或品种无显著差异[21,22,23,24]。需要注意的是，本研究中样本猫的疫苗接种史不可获得。因此，病毒DNA的发现无法明确区分自然FPV/CPV感染与潜在的疫苗株排毒或疫苗失败。在解读本研究的流行率和分子发现时应考虑这一局限性。
既往研究指出，在伊拉克（38%）、伊朗（34%）和孟加拉国（22%）具有腹泻临床症状的家猫粪便中，FPV阳性样本的检出率较高。然而，诊断和流行病学因素（样本类型、研究群体、临床纳入标准、疫苗接种状态等）的差异可显著影响FPV的检出概率，这可能部分解释了已报道检出率的差异。在本研究中，临床样本和非临床样本中均检出了病毒基因组。同时，Luan等（2026）[25]报道，在42份阳性样本中，仅有一份采集自临床健康的猫。相反，有或无猫泛白细胞减少症临床症状的猫中均观察到FPV排毒。作者发现，在阳性样本中，分别有60.3%（44/73）和39.7%（29/73）来自临床健康猫和患病猫。关于亚临床野毒感染，此前报道的排毒率范围为4%至37%不等。在本研究中，我们在越南中部的健康猫（8.02%）和患病猫（14.04%）中均检测到了FPV DNA。尽管FPV在患病猫中的检出频率更高，但在临床健康猫中检测到病毒DNA表明，病毒基因组可能在没有明显临床症状的情况下存在。需要注意的是，本研究是一项基于分子检测的横断面研究；临床健康猫中病毒DNA的存在不能解释为活动性或持续性亚临床排毒的明确证据。应当指出，其他模式，如年龄、感染阶段、病毒载量、疫苗接种史和采样条件，可能对评估的亚临床排毒流行率有所影响。关于PCR检测，本研究未纳入提取阴性对照，限制了对DNA提取过程中污染的评估。尽管如此，这一发现凸显了表观健康的FPV/CPV阳性猫潜在的流行病学重要性，并强调需要实施严格的生物安全措施，以防止越南中部与亚临床病毒排毒相关的感染。
基于VP2的系统发育树表明，6株病毒被归类为FPV，而1株分离株被归入CPV谱系。该结果进一步支持了FPV和CPV均具备感染猫科动物群体并引起临床相似疾病表现的假说[26]。此外，该观察结果与先前记录CPV在犬和猫宿主之间跨物种传播的研究一致[27,28,29]。有趣的是，其他研究提出猫与犬之间的频繁密切接触可能促进FPV和CPV的交叉传播循环[30,31]。值得注意的是，鉴定出的FPV和CPV分离株与中国参考株聚类紧密。然而，尽管完整VP2基因序列与中国分离株具有密切的进化关系，但有限的样本量和缺乏流行病学关联数据使得无法就FPV/CPV可能跨境传入越南作出确定性结论。观察到的遗传相似性可能反映共同祖先、相关病毒的广泛流行、采样偏倚、有限的系统发育分辨率或动物流动。这些结果指出了加强区域监测策略的重要性[32]。就市售FPV疫苗而言，其依赖多剂次免疫程序以实现最佳免疫效果，这可能增加幼猫在保护不完全期间感染的风险。值得注意的是，FPV的持续全球传播和进化已导致流行株在遗传特征和毒力方面出现显著变异[33,34]。在本研究中，Cat/Vietnam/FPV-465/2025分离株在系统发育上与疫苗相关FPV株聚类，而其他检测到的FPV株形成独立于疫苗群的独特谱系。需要指出的是，尽管Cat/Vietnam/FPV-465/2025株与疫苗株亲缘关系密切，但系统发育分析无法确定该株是否来源于疫苗。这也不应被解读为疫苗失败或疫苗效力降低的证据。先前一项研究指出，在中国流行的FPV野毒株与2008年Felocell疫苗株具有高水平核苷酸一致性，全长VP2基因序列一致性范围为98.7%至100%。这种遗传相似性可能是由于犬和猫从多个国家（包括中国和欧洲部分地区如法国和意大利）频繁输入，从而促进了亲缘关系密切毒株的全球传播。另一种可能性是该特定病毒谱系已经进化并在包括中国和越南中部在内的亚洲国家广泛流行[25]。先前已证实，疫苗对变异株的有效性可能受损，因为在接种疫苗的猫中已发现感染，反映出对进化中的病毒变异株的交叉免疫较差[1]。未来研究应优先对流行的FPV野毒株进行分离和全面表征，尤其侧重于阐明病毒遗传变异性对当前许可的市售疫苗免疫原性和保护效力的影响。
近期证据表明，FPV的VP2基因会发生渐进性氨基酸改变。观察到的替换影响了病毒致病性[35]，并通过修饰抗原表位降低了疫苗效力[36]，因而引起广泛关注。与这些发现一致，本研究在VP2蛋白中检测到若干氨基酸改变。值得注意的是，据报道VP2蛋白中的I101T氨基酸改变通过与第99位天冬氨酸介导极性相互作用，影响受体结合区域的结构构象[16]。该替换还可能通过改变表位的空间构象降低中和抗体的效力。在本研究中，所有获得的毒株在第101位均表现为苏氨酸（T）。在结构上，VP2蛋白由四个明确的环（1–4）以及一个柔性环组成[37]。环1、环2和环4构成三重刺突的顶端区域，而环3构成其肩部。柔性环对于确定FPV的宿主范围和血凝能力至关重要[38]。此外，本研究中观察到的A91S替换尤其值得关注。预测建模表明，该突变可使FPV VP2的无规卷曲结构域扩展至涵盖第91–95位残基，导致环1（第89–91位氨基酸）从α-螺旋构象转变为柔性卷曲。这一观察到的改变可能降低中和抗体的相互作用[39,40]。相比之下，本研究中所有分析毒株的环4区域（包括第440位点）表现出高度保守性。既往研究表明，FPV/CPV VP2蛋白的环4区域暴露于病毒粒子表面，参与宿主细胞入侵，并作为中和抗体靶向的主要抗原位点[41,42]。FPV/CPV VP2衣壳蛋白中A91S、I101T和第440位残基的替换可能潜在影响静电表面电位和表位结构，从而损害抗体结合。这些既往发现提示，此类突变可能与免疫逃逸和宿主适应相关。在本研究中，FPV表现出以232V-297V-300A-305A为特征的特异性氨基酸基序，这与CPV-2中观察到的变异基序（232I-297A-300G-305Y）形成明显对比。这种分子水平的差异与既往多项研究高度一致，这些研究共同证明第80、93、103、232、323、564和568位氨基酸残基发挥关键的功能或结构作用。最终，这些特定位点的变异有助于成功区分FPV与CPV-2，我们的数据进一步验证了它们作为可靠诊断或进化标记的实用性。需要针对分子机制和免疫学后果开展进一步研究，以确定这些替换如何影响当前疫苗的效力。
关于自然选择谱的分析，既往研究指出犬细小病毒VP2蛋白在CPV-2c和CPV-2b毒株中分别有50个和41个负选择位点。作者得出结论，这些CPV毒株的完整VP2蛋白处于负选择之下[41]。同样，Luan等（2026）指出，在中国猫细小病毒毒株的VP2蛋白中未检测到正选择位点[25]。这些作者还检测到三条在第51–59、242–250和284–292位表现出强表位信号的肽段。另外，另一个研究组证明第5–33和59–75位氨基酸区间包含线性B细胞表位。此外，四个不同的表面环——环1（第84–94位残基）、环2（第216–235位残基）、环3（第295–306位残基）和环4（第409–444位残基）——包含先前在VP2蛋白中定位的免疫优势结构域[43]。在本研究中，我们在越南中部FPV毒株中共鉴定出26个负（纯化）选择位点。值得注意的是，第57、69、221、291和305位点直接定位于环2和环3、已知的B细胞表位或强信号表位区域。该证据表明，VP2基因及其编码蛋白在这些地区FPV毒株中可能正经历严格的纯化选择。这种进化约束限制了相对于更具可塑性的病毒进化模式的适应性多样化，与先前描述的细小病毒动态一致[44]。然而，FUBAR分析基于有限数量的序列，可能降低其统计效力和稳健性；因此这些发现应视为初步结果并谨慎解读。本分析也无法确定这些位点是否直接影响报告结合、宿主范围、抗原性或病毒适应性。需要进一步研究来评估这些保守位点的确切生物学意义。
本研究存在若干局限性，包括样本量相对较小、地理采样覆盖范围有限。此外，未分离到病毒，因此未研究其生物学特性、抗原特征和致病潜力，也未评估现有商品化疫苗的保护效力。未来研究应扩大样本量和地理采样范围，以便对猫细小病毒的生物学、抗原和致病特征以及全基因组序列进行全面分析。这将有助于更完整地了解该病毒在越南各地的流行病学和分子特征。
4. 材料与方法
4.1. 伦理声明
研究方案（CARE-2023/08）于2023年3月25日获得批准，符合越南国家农业大学关于动物操作和样本采集程序的伦理规定。该批准由大学动物研究与伦理委员会授予。所有采样程序均以尽量减少动物应激和不适的方式实施，符合国际公认的动物福利标准。
4.2. 样本采集
2023年7月至2025年7月期间，从越南中部多乐省和嘉莱省的家猫中共采集269份粪便样本。根据有无呕吐和腹泻对猫进行分类。出现呕吐或腹泻的猫被归类为有胃肠道临床症状，而未报告呕吐或腹泻的猫被归类为无报告的胃肠道临床症状。样本采集自不同家庭，整个采样期间未从同一家庭重复采集样本。然而，研究中所有猫均无疫苗接种信息。样本在10%磷酸盐缓冲盐水（w/v）中匀浆，并保存于−80 °C直至进一步分析。
4.3. DNA提取及猫细小病毒基因组检测的PCR
使用Viral Gene-spin™ Viral DNA/RNA Extraction Kit（iNtRON Biotechnology，首尔，韩国）从匀浆液中分离总DNA，操作按制造商推荐程序进行。
为检测细小病毒DNA，使用Parvo-F/R引物扩增VP2基因的225 bp片段（表7），方法如前所述。PCR扩增包括95 °C初始变性5 min，随后35个循环：95 °C 30 s、60 °C 60 s用于检测、72 °C延伸60 s，最后72 °C终延伸10 min。扩增产物在1.5%琼脂糖凝胶上电泳分离，并在紫外光下观察。阳性样本为从FELOCELL疫苗（Zoetis Inc.，林肯，内布拉斯加州，美国）中提取的DNA，阴性对照为蒸馏水。
4.4. 核苷酸测序、遗传与系统发育分析
使用三对引物进行测序，以获得708 bp、719 bp和569 bp的PCR产物（表7）。这些引物靶向FPV VP2基因的保守区域以及密切相关CPV毒株的同源VP2区域。PCR扩增包括95 °C初始变性5 min，随后40个循环：95 °C 30 s、52 °C 30 s、72 °C延伸60 s，最后72 °C终延伸10 min。扩增产物在1.5%琼脂糖凝胶上电泳分离，并在紫外光下观察。
PCR产物的测序由1st BASE（雪兰莪，马来西亚）完成。每个扩增片段均进行双份测序。所得序列使用GENETYX version 10软件（GENETYX Corp.，东京，日本）和BLAST分析（https://blast.ncbi.nlm.nih.gov/Blast.cgi）与GenBank中可用序列进行比对和比较（表8）。推导的氨基酸序列使用BioEdit version 7.2中的Clustal W算法进行分析[46,47]。系统发育分析使用MEGA 7中的最大似然法进行，采用1000次bootstrap重复[48]。选择包含伽马分布和不定位点的Tamura-3参数模型（T92 + G + I）作为最佳拟合模型。
4.5. 选择压力谱分析
应用Datamonkey（http://www.datamonkey.org/）评估本研究中猫细小病毒间的进化选择谱。分析采用快速无约束贝叶斯近似（FUBAR）方法进行[49]。
4.6. 核苷酸序列登录号
本研究中猫源FPV/CPV毒株的全长VP2序列已提交至GenBank，登录号为PZ276925至PZ276931。
4.7. 统计分析
采用双侧Fisher精确检验评估不同地理区域及其他标准（品种、性别和健康状况）之间猫细小病毒阳性基因组检出率的差异。对于年龄，使用Pearson卡方检验，随后进行双侧两两Fisher精确检验。对于两组比较，计算比值比（ORs）及其95%置信区间（CIs）。p值<0.05被认为具有统计学意义。
5. 结论
总体而言，本研究首次对2025年在越南中部多乐省和嘉莱省采样的家猫中FPV/CPV进行了分子特征分析，并详细描述了该地区流行的FPV/CPV毒株的分子特征。该研究为推进细小病毒进化研究提供了宝贵数据，并强调了遗传学方法在理解病毒多样性方面的关键作用。
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1. Introduction
Feline parvovirus (FPV; synonymous with feline panleukopenia virus or feline viral enteritis) is a highly transmissible viral pathogen of major clinical importance in domestic cats, frequently causing severe disease and death in advanced cases [1,2]. The virus can infect all types of cats, both felid species and certain non-felid hosts such as raccoons and minks [1]. Among affected populations, kittens are particularly susceptible, as their immature immune systems are less capable of mounting an effective defense, resulting in higher mortality rates [3,4].
Clinical signs of the disease range widely, from mild and self-limiting illness to severe conditions and rapidly fatal outcomes [2]. Infected cats typically display symptoms such as lethargy, anorexia, vomiting, diarrhea, and marked dehydration [5]. Clinically, the disease is divided into four forms, including peracute, acute, subacute, and perinatal. Additionally, both morbidity and mortality rates are influenced by the virulence of the infectious strain [6]. FPV exhibits a high level of environmental stability and resistance, demonstrating resistance to extremely low temperatures and many commonly used disinfectants, which enables its prolonged persistence in contaminated environments [7].
FPV, classified within the Parvoviridae family, is a non-enveloped, single-stranded DNA virus of small size. It shows high similarity with canine parvovirus (CPV) in terms of its genome, structure, and immunological characteristics. CPV is divided into several types, 2a, 2b, and 2c, which can also infect cats. It is widely hypothesized that FPV is the original virus from which CPV evolved through natural mutations [8]. In the Parvovirus genus, FPV, CPV, and mink enteritis virus (MEV) constitute a closely related antigenic cluster [8,9,10,11].
With a genome size of about 5.2 kb in size, FPV contains four encoded open reading frames (ORFs): two capsid proteins (VP1 and VP2) and two non-structural proteins (NS1 and NS2) [12,13]. Among them, the VP2 protein, encoded by the fourth ORF, represents about 90% of the viral capsid and plays a critical role in defining host specificity. It also contains multiple B-cell epitopes that are essential for eliciting neutralizing, protective antibody responses [14]. Due to its importance in viral evolution, host adaptation, and immune recognition, the VP2 gene has been extensively investigated and is commonly used as a target antigen in the development of subunit vaccines [14,15].
FPV and CPV have been widely reported in companion animals worldwide. In Vietnam, parvovirus has been reported [16,17,18]; however, the available data on FPV and CPV are still limited, particularly in Central Vietnam, where their genetic characteristics and evolutionary relationships have not been well characterized. Therefore, this study provides a systematic analysis of the molecular features of FPV and CPV in cats from Central Vietnam based on VP2 sequences, which is necessary to address an important gap in the molecular epidemiology of FPV and CPV in the region.
2. Results
2.1. Identification of the Parvovirus Genome by PCR
This study involved the collection of samples from domestic cats in Central Vietnam. Of 269 tested samples, 25 (9.29%) samples were PCR-positive for the viral DNA. Specifically, the parvovirus DNA-positive rates were 5.16% and 25% in the Daklak and Gialai provinces, respectively (Table 1), which indicated a significant difference in the feline parvovirus DNA detection rates between the two provinces (p < 0.001). Interestingly, vaccination status was unavailable for any of the cats in the current study, including the 25 positive samples.
Additionally, the distribution of the Parvovirus genome in the collected samples was further analyzed with respect to breed, age, sex, and health status (Table 2). In detail, the parvovirus genome was found in all age categories, including <6 months, 6–12 months, and >12 months. Notably, the occurrence rate in cats younger than 6 months (17.65%) was significantly higher than that in cats aged 6 to 12 months (4.24%) (p = 0.003). Cats aged >12 months had a genome-positive rate of 7.58%, which was lower than that observed in cats aged <6 months and higher than that in cats aged 6–12 months, although the differences were not statistically significant (p = 0.09 and p = 0.33, respectively). In contrast, no significant differences were observed across breed, sex, and health status. Briefly, the viral genome was detected in 23/216 native cats (10.65%) and 2/53 exotic or cross-bred cats (3.77%). Furthermore, parvovirus was also identified in both male (7.96%) and female (10.26%) as well as in healthy (8.02%) and sick cats (14.04%).
2.2. Analysis of the Complete VP2 Gene of Vietnamese Parvovirus Strains
To investigate the genetic characteristics of the viral strains, seven Parvovirus DNA-positive samples obtained from domestic cats in two provinces, Daklak and Gialai, were subjected to nucleotide sequencing. The sequencing chromatograms exhibited distinct peaks corresponding to individual nucleotides, indicating the high quality and reliability of the sequencing data. The complete VP2 gene sequences were generated by assembling three overlapping amplicons obtained using separate primer pairs and independently verified by duplicate sequencing, ensuring full-length coverage of the VP2 gene. In the current study, among the 25 PCR-positive samples, seven samples were selected for complete VP2 gene sequencing based on the quality and quantity of PCR products sufficient for sequencing and to provide representation from the two neighboring provinces. The selected samples represented three samples from Gialai and four samples from Daklak. The obtained VP2 gene sequences of the Vietnamese strains were named Cat/Vietnam/FPV-334/2024, Cat/Vietnam/FPV-340/2024, Cat/Vietnam/FPV-343/2024 Cat/Vietnam/FPV-351/2025, Cat/Vietnam/FPV-361/2025, Cat/Vietnam/CPV-455/2025, and Cat/Vietnam/FPV-465/2025. For brevity, the strain names were abbreviated as FPV-334/2024 (PZ276925), FPV-340/2024 (PZ276926), FPV-343/2024 (PZ276927), FPV-351/2025 (PZ276928), FPV-361/2025 (PZ276929), CPV-455/2025 (PZ276931), and FPV-465/2025 (PZ276930), respectively. Sequence analysis revealed that six parvovirus strains were determined as FPV, whereas one parvovirus strain was clustered within CPV.
Seven Vietnamese parvovirus strains with complete VP2 gene sequences (1755 bp) were aligned and analyzed alongside reference sequences available in GenBank. The nucleotide sequence similarity among these strains ranged from 98.23% to 100% (Table 3). Notably, strains FPV-334/2024, FPV-340/2024 and FPV-361/2025 were identical at the nucleotide level (100%), whereas strains FPV-465/2025 and CPV-455/2025 exhibited the lowest level of similarity (98.23%) (Table 3A). At the protein level, identities of VP2 amino acid (aa) sequences ranged from 98.22% to 100% (Table 3B).
Comparative evaluation of the complete VP2 gene sequences from the seven obtained parvovirus strains and GenBank reference sequences revealed high nucleotide similarity. Strains FPV-343/2024 and FPV-351/2025 exhibited 99.77% identity with the JSYZ-122 strain (Accession no. MW017628.1) reported in China in 2020 (Table 4). Strains FPV-334/2024, FPV-340/2024, and FPV-361/2025 shared the highest nucleotide similarity (99.94%) with the YZ2019-025 strain (Accession no. OQ863617.1), originally reported in China in 2019. Among the current Vietnamese strains, the FPV-465 strain displayed 99.85% nucleotide identity with the Felocell strain (Accession no. EU498681.1) reported in Italy in 2008. The CPV-455/2025 strain demonstrated 99.25% nucleotide identity with the QD-201 strain (Accession no. MT394166.1) identified in China in 2022 (Table 4).
2.3. Phylogenetic Evaluation of the Completed VP2 Gene Sequences of Vietnamese Parvovirus Strains
Phylogenetic analysis of the complete VP2 gene sequences, including the seven representative parvovirus strains and other sequences available in GenBank, revealed that the six parvovirus isolates were classified into three separate branches within the FPV group, while the CPV strain clustered within the CPV group, especially belonging to the canine CPV-2c lineages (Figure 1). More specifically, the FPV-343/2024 and FPV-351/2025 strains co-clustered within a distinct group and were closely related to the Beijing-I.3/2018 strain isolated from a cat in China in 2018 (Figure 1). Furthermore, these strains shared high genetic similarity with the previously reported Vietnamese FPV strains from 2018. Interestingly, four obtained FPV strains (FPV-340/2024, FPV-334/2024, -361/2025 and -465/2025) belonged to a separate branch of the FPV group. They showed close genetic relatedness to FPV strains reported in feline and canine hosts in China between 2018 and 2022 and Spain in 2023. Regarding CPV group, the CPV-455/2025 strain was positioned within a branch including Chinese and previously reported Vietnamese CPV strains from cats and dogs from the same period (Figure 1). The strain also shared high similarity with a Chinese strain reported in 2022 (Accession no. MT392166.1). In addition, several internal nodes had low-to-moderate bootstrap support; consequently, these relationships were interpreted as evidence of genetic relatedness rather than definitive evolutionary relationships.
2.4. Evaluation of Predicted aa Sequences of the Obtained Parvovirus VP2 Proteins
Analysis of the translated sequences indicated that seven FPV/CPV isolates were each 584 aa in length. Comparative analysis with the previously reported FPV Vietnamese from canines and cats, as well as vaccine strains, showed that the five-fold β-ribbon region (aa 152–169) was highly conserved across all analyzed FPV/CPV strains. However, multiple aa substitutions were observed in the N-terminal, Loop 1–Loop 4, and C-terminal regions (Table 5). In addition, residues at positions 101, 122, 236, 237, and 299 were T, N, T, D, and G, respectively. Notably, the FPV-343/2024, FPV-351/2025, CPV-455/2025, and FPV-465/2025 possessed A at position 91, whereas the FPV-334/2024, FPV-340/2024, and FPV-361/2025 retained S at this position. Furthermore, four of the current FPV/CPV strains exhibited V at position 232, while the CPV-455/2025 and FPV-465/2025 contained I at the same position.
2.5. Evaluation of Selective Pressure Patterns Across VP2 Protein Sequences of the Obtained FPV Strains
Selection pressure analysis identified evidence of negative selection at 26 sites within the VP2 protein of the analyzed FPV isolates, each with posterior probability values above 0.9 (Prob[α < β] > 0.9). In contrast, no sites under positive selection were detected in the VP2 protein (Table 6).
3. Discussion
Parvovirus, especially FPV, is one of the most transmissible and often fatal diseases affecting cats worldwide. Although vaccination is routinely practiced, parvovirus continues to be detected in domestic cats in Vietnam. Furthermore, genomic analyses have uncovered unique antigenic variations that distinguish FPV field strains from vaccine-derived strains [19].
In the present investigation, the genome of feline parvovirus was detected in 9.29% of fecal samples from domestic cats in two provinces/cities in Central Vietnam during 2023–2025. Interestingly, the study encompassed districts that had not previously been surveyed for feline parvovirus, thereby generating novel regional data. These findings help close epidemiological gaps and improve the overall surveillance infrastructure. Furthermore, the results showed that cats younger than 6 months exhibited a significantly greater rate of Parvovirus genome detection than cats aged 6 to 12 months (p < 0.05). This observation is in accordance with a previous research that three- to five-month-old kittens are particularly vulnerable to FPV [20]. However, the association between age and FPV/CPV positivity was based on univariable analysis and was not adjusted for potential confounding factors, particularly province in the current study. Therefore, the higher positivity observed in cats <6 months should be interpreted cautiously and does not necessarily indicate an independent effect of age. Future studies with larger sample sizes should use multivariable analyses to identify independent predictors of FPV infection. In addition, male and female positive rates were 7.96% and 10.26%, respectively, without a statistically validated difference (p > 0.05). This result indicates that sex does not influence the detection rate of feline parvovirus [21]. The present results are concordant with other studies reporting no meaningful differences in disease frequency based on sex or breed [21,22,23,24]. It is noted that vaccination history was unavailable for the sample cats in the present study. Therefore, the finding of viral DNA could not definitively distinguish natural FPV/CPV infection from potential vaccine-strain shedding or vaccine failure. This limitation should be considered when interpreting the prevalence and molecular findings of the present study.
Previous studies noted that a high rate of FPV-positive samples was detected in feces from domestic cats with diarrheal clinical signs in Iraq (38%), Iran (34%), and Bangladesh (22%). However, differences in diagnostic and epidemiological factors (sample types, study populations, clinical inclusion criteria, vaccination status, etc.) can substantially affect the probability of detecting FPV. They may partly explain the variation in reported detection rates. In the present study, the viral genome was detected in both clinical and non-clinical samples. Meanwhile Luan et al. (2026) [25] reported that out of 42 positive samples, only one was collected from a clinically healthy cat. In contrast, FPV shedding was observed in cats with or without clinical signs of feline panleukopenia. The authors found that among the positive samples, 60.3% (44/73) and 39.7% (29/73) were from clinically healthy and sick cats, respectively. Regarding subclinical field virus infections, viral shedding has been previously reported at various rates ranging from 4% to 37%. In the present study, we detected FPV DNA in both healthy (8.02%) and sick (14.04%) cats in Central Vietnam. Although FPV was detected more frequently in sick cats, the detection of viral DNA in clinically healthy cats indicated that the viral genome may be present without apparent clinical signs. It is noted that the current study is a cross-sectional study based on molecular detection; the presence of viral DNA in clinically healthy cats may not be interpreted as definitive evidence of active or prolonged subclinical shedding. It should be noted that other patterns, such as age, infection stage, viral load, vaccination history and sampling conditions, may contribute to an evaluated prevalence of subclinical shedding. Regarding the PCR assay, an extraction negative control was not included in the current study, limiting the assessment of contamination during DNA extraction. Nevertheless, this finding highlights the potential epidemiological importance of apparently healthy FPV/CPV-positive cats and underscores the need to implement stringent biosecurity measures to prevent infection associated with subclinical viral shedding in Central Vietnam.
The VP2-based phylogenetic tree indicated that six viruses were classified as FPV, whereas one isolate was assigned to the CPV lineage. This result reinforces the hypothesis that both FPV and CPV possess the capacity to infect feline populations and induce clinically similar disease manifestations [26]. Additionally, the observation is consistent with previous studies documenting cross-species transmission of CPV between canine and feline hosts [27,28,29]. Interestingly, other studies have proposed that the frequent close contact between cats and dogs may facilitate a cross-transmission cycle of FPV and CPV [30,31]. Notably, the identified FPV and CPV isolates clustered closely with Chinese reference strains. However, although the complete VP2 gene sequences shared close evolutionary relationships with Chinese isolates, the limited sample size and absence of epidemiological linkage data preclude definitive conclusions regarding potential cross-border transmission of FPV/CPV into Vietnam. The observed genetic similarity may reflect shared ancestry, widespread circulation of related viruses, sampling bias, limited phylogenetic resolution, or animal movement. These results point to the importance of strengthened regional surveillance strategies [32]. With respect to commercially available FPV vaccines, they rely on multidose schedules to achieve optimal immunity, which may increase the risk of infection in kittens during the period of incomplete protection. It is noteworthy that the continued global spread and evolution of FPV have resulted in substantial variability in genetic characteristics and virulence among circulating strains [33,34]. In this study, the Cat/Vietnam/FPV-465/2025 isolate clustered phylogenetically with the vaccine-related FPV strains, whereas the other detected FPV strains formed a distinct lineage separate from the vaccine group. It is noted that although the Cat/Vietnam/FPV-465/2025 strain was closely related to vaccine strains, the phylogenetic analysis could not determine whether the strain was of vaccine origin. It also should not be interpreted as evidence of vaccine failure or reduced vaccine efficacy. A previous study noted that FPV field strains circulating in China exhibited a high level of nucleotide identity to the 2008 Felocell vaccine strain, with full-length VP2 gene sequence identities ranging from 98.7% to 100%. This genetic similarity could be due to the frequent importation of dogs and cats from multiple countries, including China and parts of Europe (such as France and Italy), which facilitates the global dissemination of closely related strains. Another possibility is that this specific viral lineage has evolved and is widely circulating across Asian countries, including China and Central Vietnam [25]. It has been previously demonstrated that the vaccine effectiveness may be compromised for variant strains, as infections have been identified in vaccinated cats, reflecting poor cross-immunity against evolving viral variants [1]. Future studies should prioritize the isolation and comprehensive characterization of circulating field FPV strains, with a particular emphasis on elucidating the influence of viral genetic variability on the immunogenicity and protective efficacy of currently licensed commercial vaccines.
Recent evidence indicates that the VP2 gene of FPV is subject to progressive amino acid changes. The observed substitutions have influenced viral pathogenicity [35] and reduced vaccine effectiveness by modifying antigenic epitopes [36], thereby drawing significant attention. In agreement with these findings, the current study detected several amino acid changes in the VP2 protein. Notably, the I101T amino acid change in the VP2 protein is reported to influence structural configuration at the receptor-binding region by mediating polar interactions with Asp at position 99 [16]. This substitution may also reduce the effectiveness of neutralizing antibodies by modifying the spatial configuration of epitope. In the current study, all obtained strains exhibited the amino acid T at position 101. Structurally, the VP2 protein consists of four defined loops (1–4) along with a flexible loop [37]. Loops 1, 2, and 4 form the apical region of the threefold spike, whereas Loop 3 contributes to its shoulder. The flexible loop is essential for defining host range and hemagglutination capacity of FPV [38]. In addition, the A91S substitution observed in the study warrants particular attention. Predictive modeling indicates that this mutation could expand the random coil domain of the VP2 of FPV to encompass residues 91–95, leading to a structural transition of Loop 1 (aa 89–91) from an α-helix configuration to a flexible coil. This observed change may decrease neutralizing antibody interactions [39,40]. In contrast, the Loop 4 region (including site 440) exhibited a high level of conservation across all analyzed strains in the current study. Previous studies have demonstrated that the Loop 4 region of the VP2 protein of FPV/CPV is exposed on the virion surface, where it is implicated in host cell entry and serves as a major antigenic site targeted by neutralizing antibodies [41,42]. Substitutions at A91S, I101T, and residue 440 within the VP2 capsid protein of FPV/CPV may potentially influence electrostatic surface potential and epitope structure, thereby impairing antibody binding. These previous findings suggested that such mutations could be associated with immune evasion and host adaptation. In the present study, FPV exhibited a specific amino acid motif characterized by 232V-297V-300A-305A, which stands in clear contrast to the variant motif (232I-297A-300G-305Y) observed in CPV-2. This divergence at the molecular level is highly consistent with several previous investigations, which have collectively demonstrated that the amino acid residues at positions 80, 93, 103, 232, 323, 564, and 568 serve critical functional or structural roles. Ultimately, variations at these specific coordinates are instrumental in successfully differentiating FPV from CPV-2, and our data further validate their utility as reliable diagnostic or evolutionary markers. Additional studies focusing on both molecular mechanisms and immunological consequences are required to determine how these substitutions influence the efficacy of current vaccines.
Regarding the analysis of the natural selection profile, a previous study noted that the canine parvovirus VP2 protein had 50 and 41 sites of negative selection among CPV-2c and CPV-2b strains, respectively. The authors concluded that the complete VP2 protein of these CPV strains is under negative selection [41]. Similarly, Luan et al. (2026) noted that no positive selection sites were detected within the VP2 protein of feline parvovirus strains in China [25]. These authors also detected three peptides exhibiting strong epitope signals at positions 51–59, 242–250, and 284–292. Separately, another research group demonstrated that amino acid intervals 5–33 and 59–75 encompass linear B-cell epitopes. Furthermore, four distinct surface loops—Loop 1 (residues 84–94), Loop 2 (residues 216–235), Loop 3 (residues 295–306), and Loop 4 (residues 409–444)—contain the immunodominant domains previously mapped within the VP2 protein [43]. In the present study, we identified a total of 26 negative (purifying) selection sites among Central Vietnamese FPV strains. Notably, sites 57, 69, 221, 291, and 305 map directly to Loops 2 and 3, known B-cell epitopes, or strong-signal epitope regions. This evidence reveals that the VP2 gene and its encoded protein may potentially be undergoing strict purifying selection among these regional FPV strains. This evolutionary constraint limits adaptive diversification relative to more plastic viral evolutionary patterns, aligning consistently with previously described parvovirus dynamics [44]. However, the FUBAR analysis was based on a limited number of sequences, which may reduce its statistical power and robustness; therefore these findings should be considered preliminary and interpreted with caution. The present analysis also cannot determine whether these sites directly influence report binding, host range, antigenicity, or viral fitness. Further studies will be needed to evaluate the precise biological significance of these conserved sites.
This study has several limitations, including a relatively small sample size and restricted geographical sampling coverage. In addition, the virus was not isolated; therefore, its biological properties, antigenic characteristics, and pathogenic potential were not investigated, nor was the protective efficacy of currently available commercial vaccines assessed. Future investigations should incorporate larger sample sizes and broader geographic sampling to facilitate comprehensive analyses of the biological, antigenic, and pathogenic characteristics, as well as the complete genome sequences, of feline parvovirus. This will provide a more complete understanding of the epidemiological and molecular features of this virus across Vietnam.
4. Materials and Methods
4.1. Ethics Statement
The study protocol (CARE-2023/08) was approved on 25 March 2023, in accordance with ethical regulations of the Vietnam National University of Agriculture for animal handling and sample collection procedures. Approval was granted by the University’s Committee on Animal Research and Ethics. All sampling procedures were conducted to minimize animal stress and discomfort, consistent with internationally recognized animal welfare standards.
4.2. Sample Collection
A total of 269 fecal samples were obtained from domestic cats in Daklak and Gialai provinces in Central Vietnam over the period from July 2023 to July 2025. The cats were classified according to the presence or absence of vomiting and diarrhea. Cats showing either vomitting or diarrhea wer classified as having gastrointestinal clinical signs, whereas cats without reported vomitting or diarrhea were classified as having no reported gastrointestinal clinical signs. Samples were collected from different households, and no samples were repeatedly collected from the same household throughout the sampling period. However, all cats in the study had no vaccination information. The samples were homogenized in a 10% phosphate-buffered saline (w/v) and stored at −80 °C until further analysis.
4.3. DNA Extraction and PCR for the Feline Parvovirus Genome Detection
The Viral Gene-spin™ Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seoul, Republic of Korea) was utilized to isolated total DNA from homogenates, following the procedures recommended by the manufacturer.
To detect Parvovirus DNA, Parvo-F/R primers were applied to amplify a 225 bp segment of VP2 gene (Table 7), as described previously. PCR amplification involved an initial denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 60 °C for 60 s for detection, and extension at 72 °C for 60 s, with a final extension step at 72 °C for 10 min. Amplicons were resolved on 1.5% agarose gels and visualized using UV illumination. The positive samples were DNA extracted from a FELOCELL vaccine (Zoetis Inc., Lincoln, NE, USA), whereas the negative controls were distilled water.
4.4. Nucleotide Sequencing, Genetic and Phylogenetic Analysis
Sequencing was conducted using three primer pairs to obtain PCR products of 708 bp, 719 bp, and 569 bp (Table 7). The primers target conserved regions of the VP2 genes of FPV and homologous VP2 regions of closely related CPV strains. PCR amplification involved an initial denaturation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 s, 52 °C for 30 s, and extension at 72 °C for 60 s, with a final extension step at 72 °C for 10 min. Amplicons were resolved on 1.5% agarose gels and visualized using UV illumination.
The sequencing of the PCR products was carried out by 1st BASE (Selango, Malaysia). Each amplicon segment was sequenced in duplicate. The sequences obtained were aligned and compared with those available in GenBank (Table 8) by using GENETYX version 10 software (GENETYX Corp., Tokyo, Japan) and BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Deduced amino acid sequences were analyzed using the Clustal W algorithm within the BioEdit version 7.2 [46,47]. Phylogenetic analysis was performed using the maximum likelihood method with 1000 bootstrap replicates in MEGA 7 [48]. The Tamura-3 parameter model incorporating a gamma distribution and invariant sites (T92 + G + I) was selected as the best-fit model.
4.5. Analysis of Selection Profiles
Datamonkey (http://www.datamonkey.org/) was applied to evaluate the evolutionary selection profiles among feline parvoviruses in this study. The analysis was determined using the Fast Unconstrained Bayesian AppRoximation (FUBAR) method [49].
4.6. Nucleotide Sequence Accessions Numbers
The full-length VP2 sequences of feline-derived FPV/CPV strains in this study were deposited into GenBank under the accession numbers PZ276925 to PZ276931.
4.7. Statistical Analysis
A two-sided Fisher’s exact test was applied to assess differences in feline parvovirus-positive genome detection rates between geographical regions and other criteria (breed, sex, and health status). For age, Pearson’s chi-square test followed by two-sided pairwise Fisher’s exact tests were used. For two-group comparison, odds ratios (Ors) with 95% confidence intervals (CIs) were calculated. A p-value < 0.05 was considered statistically significant.
5. Conclusions
Overall, this research provides the first molecular characterization of the FPV/CPV in domestic cats sampled in the Daklak and Gialai provinces in Central Vietnam in 2025 and presents a detailed molecular characterization of FPV/CPV strains circulating in this region. The study provides valuable data for advancing research on parvovirus evolution and underscores the critical role of genetic approaches in understanding viral diversity.