【摘要】 为鉴定我国河南地区致牛高热、关节肿大、活动能力下降且有严重气喘等症状的病原并分离该病原，分析其遗传演化特征，本研究于2024年从河南省3个地区的规模化养牛场与养殖户共采集24份疑似感染牛流行热病毒（BEFV）的抗凝血样品。将RT-PCR检测为BEFV的阳性样品分离病毒，采用RT-PCR和IFA及中和试验鉴定盲传3代的分离病毒。结果显示，有17份样品扩增到与预期大小相符的目的基因条带，其中13份样品在传至第3代时出现明显的CPE，盲传3代后13份样品均扩增出420 bp的BEFV目的基因条带，且其感染的细胞均出现特异性绿色荧光；中和试验结果显示，分离病毒感染5 d后细胞均未出现CPE，表明从17份阳性样品中分离到13株BEFV。将8株BEFV分离株经高通量测序以获得其全基因组序列，利用SnapGene软件分析8株分离BEFV之间，及与GenBank中登录的BEFV代表株G基因序列的同源性。利用Megalign软件中的Clustal W法，分析BEFV分离株与GenBank中登录的BEFV代表株G蛋白氨基酸序列的同源性。利用MEGA-X软件包构建BEFV G基因及全基因组的系统进化树。同源性分析结果显示，8株BEFV分离株全基因组序列的同源性达99.96%～99.99%，提示这些病毒可能为同一株病毒，因此选其中的HN1/2024株进行后续分析。全基因组测序结果显示，该株病毒全基因组长14 934 bp,GC含量33%。同源性分析结果显示，HN1/2024株与GenBank中登录的BEFV代表株的同源性为86.6%～99.3%，与国内BEFV流行株的同源性达95.9%～99.3%（G基因）；与BEFV代表株的同源性为94.1%～99.8%（G蛋白氨基酸序列），且二者均与重庆分离株CQ1/2022株（OP887034.1）的同源性最高（99.3%），与南非分离株1999-013（MN026892.1）同源性最低（86.6%）。系统进化树结果显示，HN1/2024株与我国重庆分离株CQ1/2022（OP887034.1）处于同一进化分支（G基因与全基因组），但HN1/2024株与云南DLLI29-3株（MW316736.1）的遗传距离最近，均属于东亚分支（G基因）；而其与伊朗分离株BEFV/BA（MZ687779.1）的遗传距离最近（全基因组）。采用MEGA 11软件与MegAlign程序比对分析HN1/2024株与国内BEFV流行株G蛋白4个抗原表位氨基酸序列，结果显示HN1/2024株与河南地区2011与2012年BEFV分离株LYC11（JX564638）、LS11（JX564637）G蛋白4个抗原表位的氨基酸序列均一致，与2011年BEFV山东分离株（JX174661）相比仅存在一个氨基酸变异(E⁴⁷⁶D)，本研究明确了2024年河南地区牛疫情的病原为BEFV，并分离到13株当地流行株，证实河南地区BEFV流行株存在地域来源差异，但G蛋白抗原表位无明显变异，现有商品化疫苗仍具备良好的免疫保护潜力。为我国BEFV的流行病学监测、牛流行热的科学防控及新型疫苗研发提供了重要的物质基础与参考依据。更多还原

【Abstract】 To identify the pathogen causing high fever, joint swelling, impaired mobility, and severe dyspnea in cattle in Henan Province, China, and to isolate virus, analyze the genetic evolutionary characteristics of the isolated virus, this study collected 24 anticoagulant blood samples from cattle suspected of being infected with bovine ephemeral fever virus(BEFV) from large-scale cattle farms and individual breeders in three regions of Henan Province in 2024. RT-PCR was performed for detection, and samples confirmed as BEFV positive were used for virus isolation. The isolates after three blind passages were identified by RT-PCR, indirect immunofluorescence assay(IFA), and virus neutralization test. The results showed that 17samples exhibited a target gene band at the expected 420bp. Virus isolation revealed that 13 of these samples exhibited obvious cytopathic effect(CPE) at the third passage. Identification of the third-passage isolates demonstrated that all 13 samples amplified a 420bp target band for BEFV, and specific green fluorescence was observed in infected cells. The typical CPE was observed in virus-infected cells, indicating that 13 BEFV strains were isolated from the 17 positive samples. Eight BEFV isolates were subjected to high-throughput sequencing to obtain their complete genome sequences. Snap Gene software was used to analyze the homology of the G gene sequences among the eight BEFV isolates and with representative BEFV strains deposited in Gen Bank. The Clustal W method in the Meg Align software was used to analyze the homology of G protein amino acid sequences between the BEFV isolates and representative BEFV strains available in Gen Bank, and phylogenetic trees based on the BEFV G gene and complete genome were constructed using the MEGA-X software package. Homology analysis showed that complete genome sequence homology among the eight BEFV isolates ranged from 99.96% to 99.99%, suggesting that these viruses likely originated from a single strain. Therefore, strain HN1/2024 was selected for subsequent analysis. Complete genome sequencing revealed that the full-length genome of this strain was 14934bp, with a GC content of 33%. Homology analysis showed that HN1/2024 shared 86.6%-99.3% homology with representative BEFV strains in Gen Bank, and 95.9%-99.3% homology with domestic prevalent BEFV strains at the G gene level; at the G protein amino acid level, it shared 94.1%-99.8% homology with representative BEFV strains. Both the G gene and G protein of HN1/2024 exhibited the highest homology(99.3%) with the Chongqing isolate CQ1/2022(OP887034.1) and the lowest homology(86.6%) with the South African isolate 1999-013(MN026892.1). Phylogenetic trees showed that strain HN1/2024 clustered in the same evolutionary branch as the Chinese Chongqing isolate CQ1/2022(OP887034.1) for both the G gene and complete genome. For the G gene, HN1/2024 had the closest genetic distance to the Yunnan isolate DLLI29-3(MW316736.1), both of which belong to the East Asian lineage; for the complete genome, it was most closely related to the Iranian isolate BEFV/BA(MZ687779.1). Amino acid in the four antigenic epitopes of the G protein between HN1/2024 and the domestic prevalent BEFV strains were analyzed using MEGA 11 and Meg Align. The results showed that the four G protein antigenic epitopes of BEFV HN1/2024 were identical to those of the Henan isolates LYC11(JX564638) and LS11(JX564637) from 2011 and 2012, respectively. Compared with the 2011 Shandong isolate(JX174661), only one amino acid mutation(E476D) was identified. This study confirmed that the pathogen responsible for the 2024cattle epidemic in Henan Province was BEFV, and 13 local prevalent strains were isolated, and confirmed although the prevalent BEFV strains in Henan differ in geographical origin, no significant variation was observed in the G protein antigenic epitopes,indicating that the currently available commercial vaccines still retain strong immunoprotective potential. These findings provide an important material basis and reference for the epidemiological surveillance of BEFV, scientific prevention and control of bovine ephemeral fever, and development of novel vaccines in China.更多还原