Isolation and Characterization of Porcine Epidemic Diarrhea Viruses Associated with the 2013 Disease Outbreak among Swine in the United States
Isolation and Characterization of Porcine Epidemic Diarrhea Viruses Associated with the 2013 Disease Outbreak among Swine in the United States
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DOI：
10.1128/jcm.02820-13
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发表时间：
2014-01-01
影响因子：
9.4
通讯作者：
Zhang, Jianqiang
中科院分区：
文献类型：
--
作者：
Chen, Qi;Li, Ganwu;Zhang, Jianqiang
猪流行性腹泻病毒（PEDV）于2013年5月首次在美国猪群中被发现，并造成了重大的经济损失。获得能在细胞培养中有效生长的美国PEDV分离物对于研究发病机制、开发诊断方法和开发疫苗至关重要。另一个目的是确定PEDV的哪个基因最适合研究该病毒的遗传相关性。本文描述了分别从印第安纳州和爱荷华州猪场的仔猪小肠中成功获得的两株PEDV分离株（ISU13-19338E和ISU13-22038）。这两个分离株在细胞培养中连续繁殖了30多代，并对前10代进行了鉴定。通过pedv特异性实时反转录PCR （RT-PCR）、免疫荧光测定和电镜检查证实了细胞培养中病毒的产生。前10代病毒的感染滴度为6 × 10(2) ~ 2 × 10(5) 50%的组织培养感染剂量(TCID50)/ml。此外，还测定了6种病毒（ISU13-19338E匀浆、P3和P9； ISU13-22038匀浆、P3和P9）的全长基因组序列。遗传上，两个PEDV分离株在细胞培养的前10代中相对稳定。并比较了另外4株美国PEDV毒株和23株非美国PEDV毒株的序列。美国压力。所有美国PEDV毒株遗传亲缘关系密切（>= 99.7%核苷酸同源性），与2011 ~ 2012年报道的中国PEDV毒株遗传最相似。利用PEDV不同基因的系统发育分析表明，全长刺突基因或S1部分适合测序以研究这些病毒的遗传亲缘关系。
Porcine epidemic diarrhea virus (PEDV) was detected in May 2013 for the first time in U. S. swine and has since caused significant economic loss. Obtaining a U. S. PEDV isolate that can grow efficiently in cell culture is critical for investigating pathogenesis and developing diagnostic assays and for vaccine development. An additional objective was to determine which gene(s) of PEDV is most suitable for studying the genetic relatedness of the virus. Here we describe two PEDV isolates (ISU13-19338E and ISU13-22038) successfully obtained from the small intestines of piglets from sow farms in Indiana and Iowa, respectively. The two isolates have been serially propagated in cell culture for over 30 passages and were characterized for the first 10 passages. Virus production in cell culture was confirmed by PEDV-specific real-time reverse-transcription PCR (RT-PCR), immunofluorescence assays, and electron microscopy. The infectious titers of the viruses during the first 10 passages ranged from 6 x 10(2) to 2 x 10(5) 50% tissue culture infective doses (TCID50)/ml. In addition, the full-length genome sequences of six viruses (ISU13-19338E homogenate, P3, and P9; ISU13-22038 homogenate, P3, and P9) were determined. Genetically, the two PEDV isolates were relatively stable during the first 10 passages in cell culture. Sequences were also compared to those of 4 additional U. S. PEDV strains and 23 non-U. S. strains. All U. S. PEDV strains were genetically closely related to each other (>= 99.7% nucleotide identity) and were most genetically similar to Chinese strains reported in 2011 to 2012. Phylogenetic analyses using different genes of PEDV suggested that the full-length spike gene or the S1 portion is appropriate for sequencing to study the genetic relatedness of these viruses.