广深地区3种常见猫病毒的分离鉴定及遗传进化分析

    Isolation, identification and genetic evolution analysis of three common feline viruses in Guangzhou-Shenzhen region

    • 摘要:
      目的 猫杯状病毒(Feline calicivirus,FCV)、猫细小病毒(Feline parvovirus,FPV)和猫疱疹病毒1型(Feline herpesvirus-1,FHV-1)是常见的高传染性、高致病性病原体,本文针对广深地区的这3种病毒开展研究,以期明确本地FCV、FPV和FHV-1的流行情况、遗传变异和演化方向。
      方法 本研究于2021年10月至2022年6月收集了广州、深圳地区的动物医院、动物收容所和华南农业大学病猫的眼鼻口拭子、肛拭子样本,通过提取临床样本核酸、one step RT-PCR、常规PCR技术检测FCV、FPV及FHV-1;对阳性样品进行基因扩增及测序,利用DNAStar分析核苷酸序列相似性,使用MEGA 7.0构建系统发育树;使用克兰德尔−里斯猫肾(Crandell-Reese feline kidney,CRFK)细胞接种FCV、FHV-1阳性样品进行病毒分离,连续盲传至出现细胞病变,利用PCR、电子显微镜等技术鉴定分离毒株。
      结果 379例具有临床症状的病猫中,FPV、FCV、FHV-1感染率分别为22.16%、19.00%和3.17%;病毒阳性率与猫只性别无关,6月龄及以下患猫中FCV、FPV检出率最高,分别为63.89%、58.33%,FHV-1在12月龄及以上患猫中阳性占比最高,为66.67%。已免疫“妙三多”疫苗的患猫中FCV、FHV-1、FPV均有检出,阳性率分别为75.00%、75.00%、20.24%。成功分离到7株FCV、1株FHV-1和2株FPV。7株FCV分离株全基因组与参考株的核苷酸相似性为75.0%~87.5%,7株FCV分离株形成了两大分支,GZ-1-249和GZ-1-184属于GI分支,而其他5株分离株均属于GII分支。14条FPV VP2基因序列与参考株的核苷酸相似性为97.4%~100.0%,14条序列与“妙三多”疫苗株M38246.1(FPV USA Cu4)等序列共同归属于一大分支。
      结论 研究结果丰富了FCV、FPV和FHV-1相关流行病学数据,增强了对本地流行毒株的认识,为未来本土疫苗的研发和优化提供了重要支持。

       

      Abstract:
      Objective Feline calicivirus (FCV), feline parvovirus (FPV), and feline herpesvirus-1 (FHV-1) are common highly contagious and pathogenic pathogens. In this study, these three viruses in Guangzhou-Shenzhen region were investigated to elucidate their prevalence, genetic variations and evolutionary trends.
      Method From October 2021 to June 2022, oropharyngeal, conjunctival, and anal swab samples were collected from clinically affected cats in animal hospitals, animal shelters and South China Agricultural University across Guangzhou and Shenzhen. Nucleic acids were extracted from these clinical specimens and subjected to one-step RT-PCR and conventional PCR for the detection of FCV, FPV, and FHV-1. Positive samples were further processed for gene amplification and sequencing. Nucleotide sequence similarity was analyzed using DNAStar, and phylogenetic trees were constructed with MEGA 7.0. Virus isolation was performed by inoculating FCV- and FHV-1-positive samples into Crandell-Rees feline kidney (CRFK) cells, followed by serial blind passages until cytopathic effects were observed. The isolated viral strains were then identified and characterized using PCR and electron microscopy.
      Result Among the 379 symptomatic cats, the infection rates for FPV, FCV, and FHV-1 were 22.16%, 19.00%, and 3.17%, respectively. Viral positivity showed no correlation with cat gender. The highest detection rates for FCV (63.89%) and FPV (58.33%) were observed in cats aged ≤6 months, while FHV-1 infections were most prevalent (66.67%) in cats aged ≥12 months. In cats vaccinated with Felocell 3®, FCV, FHV-1, and FPV were still detected with positive rates of 75.00%, 75.00%, and 20.24%, respectively. A total of seven FCV strains, one FHV-1 strain, and two FPV strains were successfully isolated. Genomic analysis revealed 75.0%−87.5% nucleotide similarities between the seven FCV isolates and reference strains. Phylogenetic analysis classified the seven FCV isolates into two distinct clades: GZ-1-249 and GZ-1-184 clustered within GI, while the remaining five isolates belonged to GII. The 14 FPV VP2 sequences showed 97.4%−100.0% nucleotide similarities to reference strains, and all these sequences were clustered into a major phylogenetic branch with the Felocell 3® vaccine strain M38246.1 (FPV USA Cu4).
      Conclusion The results of this study enrich the epidemiological data of FCV, FPV and FHV-1, and enhance the understanding of the local prevalent variants, which will provide crucial support for the future development and optimization of regional vaccines.

       

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