兽研1. 引言
犬细小病毒感染是一种以急性出血性肠炎为特征的严重疾病，常呈致死性，尤其是在未免疫或免疫不良的宿主中[1]。该病的病原体为犬细小病毒（CPV），是一种小型、无囊膜、单链DNA病毒，于1978年首次被描述，目前被归类为原细小病毒属食肉动物1种；自出现以来，CPV原始毒株已迅速演化为分布全球的不同遗传和抗原变异株，并已完全取代祖先毒株[2,3,4]。目前根据VP2衣壳蛋白第426位氨基酸残基对CPV进行分型：CPV-2a、CPV-2b、CPV-2c；此外，基于第297位的变化还提出了两种推测的新类型：new-CPV-2a和new-CPV-2b[5,6,7]。该VP2蛋白是病毒衣壳的主要成分，通过与宿主细胞转铁蛋白受体（TfR）1型的相互作用与宿主亲和性高度相关，充当必须与该受体完美契合才能启动感染的分子“钥匙”[8,9,10]。
尽管为DNA病毒，原细小病毒属食肉动物1种成员具有高替换率，估计约为每站点每年1.09×10⁻⁴–1.79×10⁻⁴个核苷酸替换，这一频率与许多RNA病毒显著相似[10]。尽管缺乏直接证据，但有人提出CPV是作为猫泛白细胞减少症病毒（FPV）的宿主变异株进化而来的，可能通过涉及野生动物的传播动力学实现[11]。事实上，该假说主要得到以下证据的支持：在野生食肉动物群体中检测到中间型FPV样变异株、FPV与CPV之间98–99%的核苷酸相似性以及FPV先验的宿主范围限制[3,10]。在这方面，分析两种病毒宿主范围的研究表明，所有CPV变异株均已在健康和临床猫中鉴定到，因为它们可感染不同的猫科细胞[12,13,14,15,16]。相比之下，犬中FPV或FPV样感染为偶发性且仅限于少数病例[12,17,18,19,20,21,22,23]。历史上，FPV被认为在犬中复制能力高度受限，实验研究显示其复制主要限于胸腺和骨髓组织[16,24]。然而，近期鉴定出能够在犬细胞中高效复制并在犬中引起全身感染的FPV样病毒，提示这种宿主限制可能并非绝对的[25]。因此，当前研究集中于对来自猫和犬的CPV和FPV变异株的VP2序列进行特征分析，以鉴定可能与宿主范围和致病性相关的不同突变[19,20,22,23]。这些发现对于揭示这些病毒的进化途径以及评估FPV作为犬原发致病病原的作用至关重要。
与此相关，由于原细小病毒属食肉动物1种的快速进化，当前免疫策略面临重大挑战。在犬医学中，大多数商业疫苗使用基于原始CPV毒株或CPV-2b变异株的改良活病毒（MLV）制剂。虽然这些制剂通常对所有流行变异株（2a、2b和2c）提供强效交叉保护，但成年犬中“免疫失败”的报道已成为深入研究的热点[15,16]。猫群体和FPV流行的情况也面临类似困难。猫疫苗使用经典Cu-4株；然而，近期一项分子调查显示该疫苗株与当代田间分离株之间存在显著遗传差距，可能降低有效性[23]。此外，猫频繁感染CPV变异株增加了复杂性，引发了对FPV基础疫苗是否在猫中提供充分交叉免疫的担忧[15]。
流行病学格局进一步受到宠物种群密度增加以及犬猫在城市环境和收容所中密切共居的影响。这些环境充当“生物熔炉”，高病毒载量和频繁的种间接触有利于新变异株的出现[16]。在猫中，混合感染——即个体同时感染FPV和CPV——的发生为重叠感染提供了机会，并增加了病毒准种的遗传异质性。这种多样性充当变异株的储存库，其中一些可能具备探索新宿主物种或组织嗜性的必要特征[23]。
鉴于这一背景，本研究旨在对在西班牙一只患有出血性腹泻的犬中检测到的FPV样毒株进行完整VP2序列特征分析，比较其氨基酸序列与参考株及当地FPV和CPV株的差异。
2. 材料与方法
2.1. 病例介绍
2021年6月，一只八周龄、无既往疫苗接种史的雌性犬因严重胃肠道症状被送至西班牙西北部卢戈（Lugo）的一家兽医诊所就诊。患犬精神沉郁，出现呕吐和黄色黏液样腹泻。体格检查发现发热（40.4 °C）、中度脱水（5–8%）及触诊腹痛。血液学分析显示轻度再生性贫血（红细胞4.26 × 106/µL；血细胞比容31%），伴白细胞减少（2940个细胞/µL）和中性粒细胞减少（763个细胞/µL）。血清生化检查显示低白蛋白血症、电解质浓度轻度降低（Na+和Cl−）以及血尿素氮（BUN）和肌酐轻度升高。由于入院后不久腹泻转为出血性，遂进行了针对CPV、CCoV和十二指肠贾第虫的快速免疫层析检测（PARVO-CORONA-GIARDIA，Petia Vet Health，O Porriño，西班牙），结果仅CPV阳性。随后将粪便样本送至卢戈兽医学院传染病科进行分子确认。
基于上述检查结果，患犬被收治入院并开始治疗；简言之，该犬接受了强化支持治疗，包括液体疗法、止吐治疗、镇痛、抗菌治疗及早期肠内营养。根据脱水程度给予静脉乳酸林格氏液（Hartmann Braun，Melsungen，德国），首个24小时处方量按维持需求、估计液体缺失量及持续性胃肠道丢失量计算。使用马罗匹坦（1 mg/kg，皮下注射，每日一次；Cerenia® 10 mg/mL，Zoetis，Louvain-La-Neuve，比利时）控制呕吐，同时给予奥美拉唑（1 mg/kg，静脉注射，每日一次；Omeprazol Normon 40 mg EFG，Laboratorios Normon，马德里，西班牙）预防胃十二指肠溃疡。镇痛使用丁丙诺啡（0.01 mg/kg，静脉注射，每日三次；Bupaq® 0.3 mg/mL，VetViva Richter GmbH，Wels，奥地利）。此外，使用安乃近（25 mg/kg，每日三次；Buscapina Compositum® 500 mg/mL + 4 mg/mL，Boehringer Ingelheim Vetmedica GmbH，Ingelheim am Rhein，德国）控制发热，每两小时监测一次直肠温度以避免继发低体温，直至体温稳定维持至少12–24小时；此外，该产品还含有丁基东莨菪碱，有助于管理腹痛。抗菌治疗采用阿莫西林–克拉维酸（12.5–25 mg/kg，皮下注射，每日一次；Synulox® 140 mg/mL + 35 mg/mL，Zoetis，Louvain-La-Neuve，比利时）。呕吐缓解12小时后开始使用商品化胃肠道液体日粮进行早期肠内营养（Royal Canin GI High Energy Liquid，Royal Canin，Aimargues，法国）；喂养从每日能量需求的25%开始，随后每天逐步增加约30%，直至达到全热量摄入。重组猫干扰素ω（2.5 MU/kg，静脉注射，每日一次，连续3天；Virbagen® 10 MU，Virbac，Carros，法国）作为辅助治疗给药，因其使用与自然感染犬细小病毒犬的死亡率和临床严重程度降低相关[26]。由于低白蛋白血症不严重，未考虑输注冷冻犬血浆。每24小时进行一次临床和实验室监测，包括测量体重、血糖、总蛋白和白蛋白浓度、全血细胞计数及血清电解质，以便进行治疗调整。患犬在整个治疗过程中临床状况逐渐改善，住院七天并进行临床和实验室监测后，被认为临床稳定，状况良好出院回家。
2.2. PCR分析与测序
首先，使用专门优化以去除粪便来源PCR抑制物的商业试剂盒提取和纯化DNA，该试剂盒将机械裂解和化学裂解与硅胶膜纯化相结合（HigherPurity™ Stool DNA Isolation Kit，Canvax，Valladolid，Spain）；提取时使用200 mg粪便样本，每一步均严格按照制造商说明进行操作。随后，按照先前描述的方法[27]，使用引物P1（5′-ATGAGTGATGGAGCAGTTC-3′）和VPR（5′-TTCTAGGTGCTAGTTGAG-3′）进行常规PCR检测，靶向FPV/CPV VP2基因的1745 bp片段。PCR反应在25 μL终体积中进行，包含2.5 U NZYTaq II DNA Polymerase、1×反应缓冲液、1×Optimizer Solution、2 mM MgCl2、0.2 mM dNTPs（所有试剂均由NZYTech Lda.，Lisbon，Portugal提供）、每种引物0.5 μM（Invitrogen Custom Primers，Thermo Fisher Scientific，Carlsbad，CA，USA）以及5 μL模板DNA。扩增在Bio-Rad T100热循环仪（Bio-Rad Laboratories Inc.，Hercules，CA，USA）上进行，热循环设置如下：95 °C初始热启动60 s；10个触减循环（95 °C 20 s、55 °C 30 s、68 °C 60 s），每个循环退火温度降低0.5 °C；25个循环（95 °C 20 s、50 °C 30 s、68 °C 60 s）；68 °C最终延伸3 min。纳入两份阳性对照（先前确认的FPV和CPV样本）和一份阴性对照（HyPure Molecular Biology Grade Water，HyClone Laboratories，South Logan，UT，USA）。PCR产物在1×TAE缓冲液中的1%琼脂糖凝胶上电泳，经RedSafe™（iNtRON Biotechnology Inc.，Boston，MA，USA）染色后观察。最后，PCR产物经纯化后由外部服务提供商（StabVida，Lisbon，Portugal）进行Sanger测序。使用ChromasPro 2.1.4（Technelysium，Brisbane，Australia）对正向和反向链获得的色谱图进行组装和人工校对。所得共识序列首先使用美国国家生物技术信息中心（NCBI）提供的BLAST工具（https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch，于2026年9月7日访问）与GenBank NCBI数据库中收录的序列进行比较。
2.3. 系统发育分析
在构建系统发育树之前，使用MEGA X版本10.2.2 [28]中实现的ClustalW多序列比对算法将所得序列与所选序列进行比对；简言之，分析中纳入了全球范围内先前报道的在犬中检测到的FPV序列，以及先前从研究区域获得的参考和代表性区域FPV和CPV序列（PZ279922-PZ279924和PZ279926-PZ279932）。此外，使用BioEdit 7.2.5 [29]生成包含所有所用序列的核苷酸同源性矩阵。比对中纳入一条水貂肠炎病毒序列（GenBank登录号M23999）作为外群，以推断祖先关系。随后，使用jModelTest v.2.1.10 [30,31]中实现的赤池信息准则（AIC）选择最佳拟合核苷酸替换模型。使用MrBayes 3.2.7 [32]构建系统发育树，采用贝叶斯方法结合马尔可夫链蒙特卡洛采样（20,000,000代，每1000步采样一次）。所得树使用FigTree 1.4.3进行可视化和编辑。最后，核苷酸比对序列还被翻译为氨基酸序列，以鉴定本研究中获得的VP2序列与其他地区先前报道的在犬中检测到的FPV序列之间的氨基酸变化。
3. 结果
来自该临床病例的样本（CP50FPV）经常规PCR检测为阳性，确认存在病毒DNA。将我们的序列与GenBank中收录的序列进行比较后，在来自西班牙一只家猫的FPV毒株（GenBank登录号PQ436979）中观察到最高核苷酸同源性（99.89%）。本研究中获得的新序列已以登录号PZ279925存入GenBank。
系统发育树见图1。根据赤池信息准则（AIC），最佳拟合核苷酸替换模型为带不变位点比例的HKY模型。可以看出，FPV与CPV序列之间的分离界限清晰，尽管一条先前描述的中国来源FPV序列（OQ815873）位于两个分支之间。关于本研究中获得的序列，其明确归入一个与其他从研究地区猫中获得的FPV毒株共同构成的明确分支，提示该毒株可能来源于当地猫群。同样，这组序列也与先前在其他地区犬中描述的大多数FPV序列聚类。这些观察结果进一步得到序列分析结果的支持。因此，当与来自同一研究区域的FPV毒株比较时，CP50FPV分离株显示出99.6%至100%的核苷酸一致性，而与同一地区的CPV毒株比较时同源性较低（98.1–98.6%）。同样，与先前在其他地区犬中报道的其他FPV毒株的核苷酸一致性为99.4%至99.8%。
表1总结了所分析序列中观察到的氨基酸变化。如图所示，与FPV参考株M38246相比，我们的CP50FPV序列在第101位表现出异亮氨酸到苏氨酸的替换（I101T），而其余氨基酸序列完全相同。该I101T突变也存在于先前从世界其他地区犬中报道的所有FPV株中；然而，这些毒株还至少表现出第568位额外的甘氨酸到丙氨酸替换（G568A），而CP50FPV序列中不存在该替换。
4. 讨论
传统上排除FPV感染犬的生物学界限近年来已变得越来越模糊。历史上，FPV被认为是一种显示受限嗜性的病毒，因为其在犬中的复制主要局限于初级淋巴组织，而不会通过粪便途径导致显著的病毒排毒[10,22,24]。然而，20世纪70年代CPV的出现——源自FPV或野生食肉动物中非常密切相关的祖先——证明了该病毒家族通过最小的遗传变化定殖新生态位的潜力[2,10,11]。目前，来自多个国家（包括越南、泰国、中国、日本、巴基斯坦、埃及和意大利）的若干近期研究已在患有临床胃肠炎的犬中鉴定出FPV株，表明FPV正在获得克服物种屏障能力的活跃进化过程[12,17,18,19,20,21,22,23]。尽管存在基于单一病例的局限性，但据作者所知，本研究代表了在西班牙首次报道从具有与细小病毒感染相符临床体征的犬中检测到FPV株，也是欧洲第二次此类报道。然而，应当注意，尽管样本对CCoV和G. duodenalis检测为阴性，但其他潜在肠道病原体，包括Salmonella spp.和Campylobacter spp.，未被专门调查。因此，尽管在犬中观察到的显著白细胞减少和中性粒细胞减少更符合通常与细小病毒感染相关的全身效应，但不能完全排除其他传染原对临床表现的贡献。
大多数研究表明，VP2基因在宿主范围特异性中发挥关键/主要作用，因为它编码病毒衣壳的主要蛋白，从而决定病毒的外部结构[34]。此外，该蛋白在与宿主转铁蛋白受体1型（TfR）的相互作用中也具有决定性作用，这是细胞进入和确定易感物种范围的关键步骤[35,36]。对此，现有数据表明，FPV适应犬不需要完整的基因组大修，而是需要在战略性氨基酸残基上发生点突变以提高对犬TfR受体的亲和力[22,23]。
现有文献已提出若干与FPV宿主特异性相关的分子变化，包括VP2中第101位异亮氨酸替换为苏氨酸（I101T），该变化已被提出为关键事件[37]。虽然原型或较早的FPV株保留异亮氨酸，但当代变异株在该位点几乎普遍采用苏氨酸，如本研究中鉴定的CP50FPV株[19,20,21,22,23,38]。该突变并非简单的随机遗传漂变，因为残基101位于受体结合区域的核心，并与中和抗体识别的表位重叠。生物信息学分析表明，该位点替换为苏氨酸有利于与Asp99残基发生极性相互作用，改变衣壳表面峡谷样结构的静电荷和拓扑结构；因此，有人提出这种结构调整优化了与TfR的相互作用，使病毒能够更有效地探索犬类等新宿主[19]。然而，该VP2构象变化在宿主适应和种间传播中的具体作用仍有待实验验证。
I101T突变在当前分离株中的频率非常高，无论是从猫获得的毒株还是迄今为止报道的犬相关病例中均如此[19,20,21,22,23]。例如，从越南犬中检测到的所有FPV株均呈现该变异[19]。值得注意的是，与原始CPV株相比，CPV-2a、2b和2c变异株中一致鉴定出相同的突变（Thr101），这强化了第101位苏氨酸对于病毒在犬群中持续存在几乎不可或缺的观点[19,22]。该突变在现代FPV群体中的普遍性——甚至在猫中也是如此，因为本研究中的猫FPV序列中也发现了该突变——表明FPV已经具备宿主转换所需的分子变化之一。
然而，I101T突变似乎只是谜题的一部分。近期研究已鉴定出其他可作为宿主嗜性重要决定因素的替换。一项特别有影响力的发现是，在直接从猫体内分离的FPV毒株中鉴定出A300P突变[23]。因此，携带该特定突变的FPV分离株已表现出前所未有的能力，能够在MDCK和A72等犬源细胞系中稳定且高滴度复制，这与传统FPV毒株不同。此外，致病性研究已证实，这些变异体不仅能在体外复制，还能通过口服途径感染犬，引起肠道病变、肠系膜淋巴结炎和粪便排毒，从而模拟CPV的临床行为。然而，如Table 1所示，本研究分析的毒株以及既往研究报告的毒株在VP2蛋白第300位均未观察到氨基酸替换[19,20,21,22,23]。与此类似，该进化过程中另一个值得关注的突变是第93位残基的赖氨酸至天冬酰胺替换（K93N），该突变被认为是使原始FPV进化为CPV的关键变化之一，并且最近在泰国腹泻犬分离的FPV毒株中被检测到[20]。然而，全球范围内从犬体内检测到的大多数FPV毒株，包括本研究中发现的毒株，均缺乏该特定氨基酸替换，提示K93N突变不太可能在适应犬宿主中发挥关键作用[19,21,22,23]。
关于这些FPV犬适应变异体的出现，有人提出猫对FPV和CPV的易感性可促进该物种的自然共感染，从而为基因组重组创造允许性环境[15,16]。尽管本研究的序列未被鉴定为重组毒株，但应注意，该现象可能产生具有嵌合衣壳特性的中间型FPV样变异体，从而促进其进入犬细胞并在其中高效复制[17]。因此，猫可作为重组原细小病毒出现的关键储存宿主，这些病毒具有克服物种屏障的潜力。对现有分子流行病学数据（包括本研究结果）的全面分析表明，FPV并非静态病原体；在这方面，系统发育推断表明，当前从猫体内分离的循环FPV毒株与现有疫苗所基于的经典FPV毒株在遗传上存在距离，这与既往报道一致[23]。这种遗传差距，加之高选择压力以及犬猫之间的密切接触，构成了FPV变异体出现并扩大宿主嗜性的理想场景。因此，在犬体内检测到FPV并非诊断错误，而是由关键VP2突变鉴定所支持的生物学现实。就本研究而言，所描述的病例涉及来自农村地区的动物，该地区家犬与猫之间的互动频繁，从而促进了暴露于物种特异性病毒。此外，在西班牙此类农村地区，FPV暴发通常每年在猫中发生，尤其是在产仔季节，使得病毒存在于环境中的可能性极高。这一事实可能增加犬暴露于病毒的机会，从而提高种间传播的可能性；然而，在该地区开展进一步流行病学研究将有助于描述FPV在当地猫和犬群体中的循环特征，并更好地评估该现象的潜在风险。另一方面，值得注意的是，该患者为八周龄幼犬，这一年龄代表细小病毒感染易感性的关键窗口期。该时期与母源抗体（MDAs）下降同时发生，形成“免疫空白”，使个体易感于野毒株。该动物尚未接受针对核心疾病（包括犬细小病毒病）的初级疫苗系列接种，这一事实进一步加剧了其易感性。因此，该患者固有的宿主相关因素可能使其在FPV感染后易发生明显临床疾病，这是合理的。尽管如此，必须对这些临床病例进行持续监测，以确定这种宿主转换现象的发生频率是否高于先前预期。
5. 结论
本报告构成西班牙首例有文献记载的FPV样毒株引起犬疾病的病例。尽管这是一项基于单一病例且存在若干局限性的初步研究，但犬相关FPV毒株（包括本研究中鉴定的毒株）中存在I101T突变，似乎是全球范围内犬体内检测到的FPV毒株的共同特征，因此可能与犬宿主适应相关。在此背景下，有必要监测这些分子变化，尤其是VP2基因中的变化，并结合旨在确定其生物学意义的功能研究，以更好地评估这些“宿主转换”毒株潜在定植的风险。
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1. Introduction
Canine parvovirosis is a serious disease characterized by acute hemorrhagic enteritis that is frequently fatal, especially in naïve or poorly immunized hosts [1]. The etiological agent of this disease is the canine parvovirus (CPV), a small, non-enveloped, single-stranded DNA virus first described in 1978 currently classified as Protoparvovirus carnivoran1 species; since its emergence, the original strain of CPV has rapidly evolved into different genetic and antigenic variants distributed worldwide, which have completely replaced the ancestral one [2,3,4]. Different types of CPV are currently described based on the amino acid residue located at position 426 of the VP2 capsid protein: CPV-2a, CPV-2b, CPV-2c; in addition, two putative new types has also been proposed based on changes at position 297: new-CPV-2a and new-CPV-2b [5,6,7]. This VP2 protein is the major component of the viral capsid and is highly related to host affinity through the interaction with transferrin receptor (TfR) type 1 in host cells, acting as a molecular “key” that must perfectly fit into this receptor to initiate infection [8,9,10].
Despite being DNA viruses, the members of the Protoparvovirus carnivoran1 species are characterized by a high substitution rate, estimated at approximately 1.09 × 10−4–1.79 × 10−4 nucleotide substitutions per site and year, a frequency remarkably similar to that of many RNA viruses [10]. Although there is a lack of direct evidence, it has been suggested that CPV evolved as a host variant of feline panleukopenia virus (FPV), possibly through transmission dynamics involving wild fauna [11]. In fact, this hypothesis is mainly supported by the detection of intermediate FPV-like variants in wild carnivore populations, the 98–99% nucleotide similarity between FPV and CPV and the a priori host range restriction of FPV [3,10]. In this regard, investigations analyzing the host range of both viruses revealed that all CPV variants have been identified in both healthy and clinical cats since they can infect different feline cells [12,13,14,15,16]. In contrast, FPV or FPV-like infections in dogs are occasional and limited to a small number of cases [12,17,18,19,20,21,22,23]. Historically, FPV was considered to have a highly restricted replication capacity in dogs, with experimental studies showing limited replication primarily in thymic and bone marrow tissues [16,24]. However, the recent identification of FPV-like viruses capable of efficient replication in canine cells and of causing systemic infection in dogs suggests that this host restriction may not be absolute [25]. Thus, current research is focused on the characterization of the VP2 sequence in CPV and FPV variants from both cats and dogs, in order to identify different mutations potentially associated with host range and pathogenicity [19,20,22,23]. These findings are essential for unraveling the evolutionary routes of these viruses and for assessing the role of FPV as a primary causative pathogen for dogs.
Related to this, current immunization strategies face significant challenges due to the rapid evolution of Protoparvovirus carnivoran1. In canine medicine, most commercial vaccines utilize modified live virus (MLV) formulations based on either the original CPV strain or the CPV-2b variant. While these formulations typically provide robust cross-protection against all circulating variants (2a, 2b, and 2c), reports of “vaccination failures” in adult dogs have become a subject of intense research [15,16]. The situation regarding feline populations and FPV circulation presents similar difficulties. Feline vaccines utilize the classic Cu-4 strain; however, a recent molecular survey revealed a significant genetic gap between this vaccine strain and contemporary field isolates, which could potentially reduce effectiveness [23]. Moreover, the frequent infection of cats with CPV variants adds complexity, raising concerns about whether FPV-based vaccines provide adequate cross-immunity in felines [15].
The epidemiological landscape is further influenced by the increasing density of pet populations and the close cohabitation of dogs and cats in urban environments and shelters. These settings act as “biological melting pots” where high viral loads and frequent interspecies contact favor the emergence of novel variants [16]. In cats, the occurrence of mixed infections—where an individual is simultaneously infected with FPV and CPV—provides opportunities for superinfection and increases the genetic heterogeneity of the viral quasispecies. This diversity acts as a reservoir of variants, some of which may possess the necessary traits to explore new host species or tissue tropisms [23].
Considering this background, the present study aims to characterize the complete VP2 sequence of an FPV-like strain detected in a dog with hemorrhagic diarrhea in Spain, analyzing the differences in its amino acid sequence compared with reference and local FPV and CPV strains.
2. Materials and Methods
2.1. Case Presentation
In June 2021, an eight-week-old female dog with no prior vaccination history was presented to a veterinary clinic in Lugo (NW Spain) due to severe gastrointestinal signs. The animal was lethargic and exhibited vomiting and yellowish, mucoid diarrhea. Physical examination revealed fever (40.4 °C), moderate dehydration (5–8%), and abdominal pain on palpation. Hematological analysis showed mild regenerative anemia (4.26 × 106 erythrocytes/µL; hematocrit 31%), accompanied by leukopenia (2940 cells/µL) and neutropenia (763 cells/µL). Serum biochemistry revealed hypoalbuminemia, mildly decreased electrolyte concentrations (Na+ and Cl−), and a slight increase in blood urea nitrogen (BUN) and creatinine. As the diarrhea became hemorrhagic shortly after admission, a rapid immunochromatographic test for CPV, CCoV and Giardia duodenalis (PARVO-CORONA-GIARDIA, Petia Vet Health, O Porriño, Spain) was performed, yielding a positive result only for CPV. A fecal sample was subsequently submitted to the Infectious Diseases Unit of the Veterinary Faculty in Lugo for molecular confirmation.
Based on these findings, the patient was hospitalized and treatment was initiated; briefly, the dog was managed with intensive supportive care, including fluid therapy, antiemetic treatment, analgesia, antimicrobial therapy, and early enteral nutrition. Intravenous Ringer’s lactate solution (Hartmann Braun, Melsungen, Germany) was administered according to the degree of dehydration, with the volume prescribed during the first 24 h calculated to cover maintenance requirements, estimated fluid deficit, and ongoing gastrointestinal losses. Maropitant (1 mg/kg, SC, SID; Cerenia® 10 mg/mL, Zoetis, Louvain-La-Neuve, Belgium) was used for control of vomiting, while omeprazole (1 mg/kg, IV, SID; Omeprazol Normon 40 mg EFG, Laboratorios Normon, Madrid, Spain) was administered for the prevention of gastroduodenal ulceration. Analgesia was provided with buprenorphine (0.01 mg/kg, IV, TID; Bupaq® 0.3 mg/mL, VetViva Richter GmbH, Wels, Austria). In addition, metamizole (25 mg/kg, TID; Buscapina Compositum® 500 mg/mL + 4 mg/mL, Boehringer Ingelheim Vetmedica GmbH, Ingelheim am Rhein, Germany) was administered for control of pyrexia, monitoring rectal temperature every two hours to avoid subsequent hypothermia until thermal stability was maintained for at least 12–24 h; in addition, this product also contains butylscopolamine, which is useful for the management of abdominal pain. Antimicrobial therapy consisted of amoxicillin–clavulanic acid (12.5–25 mg/kg, SC, SID; Synulox® 140 mg/mL + 35 mg/mL, Zoetis, Louvain-La-Neuve, Belgium). Early enteral nutrition with a commercial gastrointestinal liquid diet was initiated 12 h after resolution of vomiting (Royal Canin GI High Energy Liquid, Royal Canin, Aimargues, France); feeding was initiated at 25% of the daily energy requirement and progressively increased by approximately 30% per day until full caloric intake was reached. Recombinant feline interferon omega (2.5 MU/kg, IV, SID for 3 consecutive days; Virbagen® 10 MU, Virbac, Carros, France) was administered as an adjunctive treatment, as its use has been associated with reduced mortality and clinical severity in dogs naturally infected with canine parvovirus [26]. As hypoalbuminemia was not severe, frozen canine plasma transfusion was not considered. Clinical and laboratory monitoring was performed every 24 h and included measurement of body weight, blood glucose, total protein and albumin concentrations, complete blood count, and serum electrolytes, in order to stablish therapeutic adjustments. The dog showed a gradual clinical improvement throughout treatment and, after seven days of hospitalization with clinical and laboratory monitoring, was considered clinically stable and returned home in good condition.
2.2. PCR Analysis and Sequencing
Firstly, DNA was isolated and purified using a commercial kit specifically optimized to remove stool-derived PCR inhibitors, which combines mechanical and chemical lysis with silica membrane-based purification (HigherPurity™ Stool DNA Isolation Kit, Canvax, Valladolid, Spain); the extraction was performed using 200 mg of fecal sample, following the manufacturer’s instructions at every step. Subsequently, a conventional PCR assay targeting a 1745 bp fragment of the VP2 gene of FPV/CPV was performed using primers P1 (5′-ATGAGTGATGGAGCAGTTC-3′) and VPR (5′-TTCTAGGTGCTAGTTGAG-3′) as previously described [27]. PCR reactions were performed in a final volume of 25 μL containing 2.5 U of NZYTaq II DNA Polymerase, 1×x Reaction Buffer, 1× Optimizer Solution, 2 mM of MgCl2, 0.2 mM of dNTPs (all reagents supplied by NZYTech Lda., Lisbon, Portugal), 0.5 μM of each primer (Invitrogen Custom Primers, Thermo Fisher Scientific, Carlsbad, CA, USA), and 5 μL of template DNA. Amplification was carried out in a Bio-Rad T100 thermocycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) under the following thermal set-up: initial hot start at 95 °C for 60 s; 10 touch-down cycles (95 °C for 20 s, 55 °C for 30 s, and 68 °C for 60 s), decreasing the annealing temperature by 0.5 °C per cycle; 25 cycles (95 °C for 20 s, 50 °C for 30 s, and 68 °C for 60 s); and a final extension at 68 °C for 3 min. Two positive controls (previously confirmed FPV and CPV samples) and a negative control (HyPure Molecular Biology Grade Water, HyClone Laboratories, South Logan, UT, USA) were included. PCR products were visualized by electrophoresis on a 1% agarose gel in 1× TAE buffer stained with RedSafe™ (iNtRON Biotechnology Inc., Boston, MA, USA). Finally, PCR products were purified and sequenced by Sanger sequencing at an external service provider (StabVida, Lisbon, Portugal). The chromatograms obtained from both forward and reverse strands were assembled and manually proofread using ChromasPro 2.1.4 (Technelysium, Brisbane, Australia). The consensus sequence obtained was initially compared with those deposited in the GenBank NCBI database using the BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch accessed on 7 September 2026) available at the National Center for Biotechnology Information (NCBI).
2.3. Phylogenetic Analysis
Prior to phylogenetic tree construction, the obtained sequence was aligned with selected sequences using the ClustalW multiple alignment algorithm implemented in MEGA X version 10.2.2 [28]; briefly, previously reported FPV sequences detected in dogs worldwide, as well as both reference and representative regional FPV and CPV sequences previously obtained from the study area (PZ279922-PZ279924 and PZ279926-PZ279932), were included in the analysis. In addition, a nucleotide homology matrix with all the employed sequences was generated using BioEdit 7.2.5 [29]. A mink enteritis virus sequence (GenBank accession no. M23999) was included in the alignment as an outgroup to infer ancestral relationships. Subsequently, the best fitting nucleotide substitution model was selected by using the Akaike Information Criterion (AIC) implemented in jModelTest v.2.1.10 [30,31]. A phylogenetic tree was constructed using MrBayes 3.2.7 [32], applying a Bayesian approach with Markov Chain Monte Carlo sampling (20,000,000 generations with sampling every 1000 steps). The resulting tree was visualized and edited using FigTree 1.4.3. Finally, the nucleotide alignment was also translated into amino acid sequences to identify amino acid changes between the VP2 sequence obtained in this study and previously reported FPV sequences detected in dogs elsewhere.
3. Results
The sample from the clinical case (CP50FPV) tested positive by conventional PCR, confirming the presence of viral DNA. After comparing our sequence with those deposited in GenBank, the highest nucleotide homology (99.89%) was observed in an FPV strain identified in a domestic cat from Spain (GenBank accession no. PQ436979). The novel sequence obtained in the present study was deposited in GenBank under accession number PZ279925.
The phylogenetic tree is shown in Figure 1. According to the Akaike Information Criterion (AIC), the best-fitting nucleotide substitution model was the HKY model with a proportion of invariable sites. As can be observed, the separation between FPV and CPV sequences is clearly defined, although one previously described FPV sequence of Chinese origin (OQ815873) is positioned between both clades. Regarding the sequence obtained in the present study, it was clearly included within a well-defined clade with other FPV strains obtained from cats from the study region, suggesting that this strain likely originated from the local feline population. Similarly, this group of sequences also clusters with most FPV sequences previously described in dogs elsewhere. These observations are further supported by the sequence analysis results. Thus, the CP50FPV isolate showed nucleotide identity values ranging from 99.6% to 100% when compared to FPV strains from the same study region, whereas homology was lower (98.1–98.6%) compared with CPV strains from the same area. Similarly, the nucleotide identity with other FPV strains previously reported in dogs elsewhere ranged from 99.4% to 99.8%.
Table 1 summarizes the amino acid changes observed among the analyzed sequences. As shown, our CP50FPV sequence exhibits a substitution of isoleucine to threonine at position 101 (I101T) relative to the FPV reference strain M38246, while the rest of the amino acid sequence is identical. This mutation I101T is also present in all FPV strains previously reported in dogs from other parts of the world; however, these strains also show at least an additional glycine-to-alanine substitution at position 568 (G568A) which is not present in the CP50FPV sequence.
4. Discussion
The biological boundary that traditionally excluded FPV from infecting dogs has become increasingly blurred in recent years. Historically, FPV was considered a virus showing restricted tropism because its replication in dogs was mainly limited to primary lymphoid tissues without resulting in significant viral shedding via the fecal route [10,22,24]. However, the emergence of CPV in the 1970s—derived from FPV or very closely related ancestors in wild carnivores—demonstrated the potential of this viral family to colonize new niches through minimal genetic changes [2,10,11]. At present, a number of recent studies from various countries, including Vietnam, Thailand, China, Japan, Pakistan, Egypt, and Italy, have identified FPV strains in dogs associated with clinical gastroenteritis, suggesting an active evolutionary process in which FPV is acquiring the capacity to overcome the species barrier [12,17,18,19,20,21,22,23]. Despite the limitation of being based on a single case, to the best of the authors’ knowledge, the present study represents the first report of an FPV strain detected in a dog with clinical signs compatible with parvovirosis in Spain, and the second such report in Europe. Nevertheless, it should be noted that, although the sample tested negative for CCoV and G. duodenalis, other potential enteric pathogens, including Salmonella spp. and Campylobacter spp., were not specifically investigated. Thus, although the marked leukopenia and neutropenia observed in the dog were more consistent with the systemic effects commonly associated with parvoviral infection, the contribution of other infectious agents to the clinical presentation cannot be completely excluded.
Most studies indicate that the VP2 gene plays a key/major role in host range specificity since it encodes the major protein of the viral capsid and, consequently, defines the external architecture of the virus [34]. In addition, this protein also has a decisive role in interacting with the host’s transferrin receptor type 1 (TfR), a critical step for cellular entry and for determining the range of susceptible species [35,36]. Regarding this, available data suggests that a complete genomic overhaul is not required for FPV adaptation to dogs, but rather point mutations in strategic amino acid residues improving affinity for this canine TfR receptor [22,23].
The available literature has proposed a number of molecular changes associated with FPV host specificity, including the substitution of isoleucine for threonine at position 101 (I101T) in VP2, which has been proposed as a critical event [37]. While prototypical or older FPV strains retained isoleucine, contemporary variants have almost universally adopted threonine at this site, such as the strain CP50FPV identified in this study [19,20,21,22,23,38]. This mutation is not simply random genetic drift, since residue 101 lies at the core of the receptor-binding region and overlaps with an epitope recognized by neutralizing antibodies. Bioinformatic analyses have shown that the substitution to threonine at this position facilitates a polar interaction with residue Asp99, modifying the electrostatic charge and topography of a canyon-like structure on the capsid surface; thus, it was suggested that this structural adjustment optimize interaction with TfR, enabling the virus to more efficiently explore new hosts as canine species [19]. Nevertheless, the specific role of this VP2 conformational change in host adaptation and interspecies transmission remains to be experimentally validated.
The frequency of the I101T mutation is remarkably high in current isolates both in strains obtained from cats and in the canine-associated cases reported to date [19,20,21,22,23]. For example, all FPV strains detected in dogs from Vietnam presented this variant [19]. It is worth noting that this same mutation (Thr101) was consistently identified in CPV-2a, 2b and 2c variants when compared to original CPV strains, reinforcing the idea that threonine at position 101 is nearly indispensable for viral persistence in the canine population [19,22]. The ubiquity of this mutation in modern FPV populations—even in cats, as it was also found among the feline FPV sequences in this study—suggests that FPV already possesses one of the molecular changes necessary for host switching.
However, the I101T mutation appears to be only one piece of the puzzle. Recent research has identified other substitutions that could act as powerful determinants of host tropism. A particularly impactful finding has been the identification of the A300P mutation in FPV strains isolated directly from cats [23]. Thus, FPV isolates harboring this specific mutation have demonstrated an unprecedented ability to replicate stably and at high titers in canine cell lines such as MDCK and A72, differently from conventional FPV strains. Moreover, pathogenicity studies have confirmed that these variants not only replicate in vitro but are also capable of infecting dogs via the oral route, causing intestinal lesions, mesenteric lymphadenitis, and viral shedding in feces, and thereby mimicking the clinical behavior of CPV. However, no amino acid substitution was observed at position 300 of the VP2 protein in either the strain analyzed in the present study or the strains reported in previous studies, as shown in Table 1 [19,20,21,22,23]. Similar to this, another mutation of interest in this evolutionary process is the lysine-to-asparagine substitution (K93N) in residue 93, considered one of the key changes that enabled the original FPV to evolve into CPV and recently detected in FPV strains isolated from dogs with diarrhea in Thailand [20]. Nevertheless, most FPV strains detected in dogs worldwide, including the one found in the present study, lack this specific amino acid substitution, suggesting that it is unlikely that K93N mutation plays a critical role in adaptation to canine hosts [19,21,22,23].
Regarding the emergence of these FPV canine-adapted variants, it has been proposed that feline susceptibility to both FPV and CPV can facilitate natural coinfections in this species, creating a permissive environment for genomic recombination [15,16]. Although the sequence of the present study was not characterized as a recombinant strain, it should be noted that this phenomenon may generate intermediate FPV-like variants with chimeric capsid properties facilitating their entry and efficient replication in dog cells [17]. Consequently, cats can represent a crucial reservoir for the emergence of recombinant protoparvoviruses with the potential to overcome species barriers. A thorough analysis of available molecular epidemiological data, including the results of the present investigation, indicates that FPV is not a static agent; in this regard, the phylogenetic inference demonstrates that current circulating FPV strains isolated from cats are genetically distant from those classical FPV strains on which current vaccines are based, agreeing with previous reports [23]. This genetic gap, combined with a high selection pressure and close contact between dogs and cats, represents an ideal scenario for the emergence of FPV variants with expanded host tropism. Thus, detection of FPV in dogs is not a diagnostic error but a biological reality supported by the identification of key VP2 mutations. In relation to the present study, the described case involves an animal from a rural area where interaction between domestic dogs and cats is frequent, thereby facilitating exposure to species-specific viruses. In addition, FPV outbreaks commonly occur every year among cats in these kinds of rural areas in Spain, particularly during the kittening season, making the presence of the virus in the environment highly likely. This fact may increase dogs’ exposure to the virus, thereby increasing the likelihood of interspecies transmission; however, further epidemiological studies in the area would be valuable to characterize FPV circulation in local cat and dog populations and to better assess the potential risk of this phenomenon. In other hand, it is noteworthy that the patient was an eight-week-old puppy, an age that represents a critical window of susceptibility for parvovirus infection. This period coincides with the decline of maternally derived antibodies (MDAs), creating an ‘immunity gap’ that leaves the individual vulnerable to field strains. This susceptibility was further compounded by the fact that the animal had not yet received its primary vaccination series against core diseases, including canine parvovirosis. Consequently, it is plausible that the inherent host-related factors of this patient predisposed the individual to the development of overt clinical disease following FPV infection. Nevertheless, continuous surveillance of these clinical cases is imperative to determine whether this host-switching phenomenon occurs with a greater frequency than previously anticipated.
5. Conclusions
This report constitutes the first documented case of an FPV-like strain causing disease in dogs in Spain. Although this is a preliminary study based on a single case and with several limitations, the presence of the I101T mutation in canine-associated FPV strains, including the strain identified in the present study, appears to be a common feature among FPV strains detected in dogs worldwide and may therefore be associated with adaptation to canine hosts. In this context, monitoring these molecular changes, particularly in the VP2 gene, together with functional studies aimed at determining their biological significance, is warranted to better assess the potential establishment of these “host-switching” strains.