Isolation and Molecular Characterization of Foot and Mouth Disease SAT2 Virus during Outbreak 2012 in Egypt
Isolation and Molecular Characterization of Foot and Mouth Disease SAT2 Virus during Outbreak 2012 in Egypt
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2012 年埃及爆发的手足口病 SAT2 病毒的分离和分子特征
DOI：
10.5455/jva.20130219104353
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
E. Farouk
中科院分区：
文献类型：
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作者：
M. A. Elmoety;M. M. A. El;Hiam Mohamed Fakry;H. Daoud;E. Ibrahim;W. G. Eldin;S. Rizk;Hany abu El;A. Mohamed;Ahmed Salah Abd El;E. Farouk
2012 年 4 月和 5 月期间，埃及各省报告了 6 起 SAT 2 型口蹄疫疫情：EL-Gharbiya（Kafr Qeretna、Al-Mahala Alkobra、Ebshaway Almalak、Qutoor）、Kafr El-Shaykh（Alsalmya、Fouah）的当地牛、水牛和奶牛场、AL-Minya（3 号村、Samaout、Mansafees、 Abo Querkas)、Dumyat (Ezbet 20、Kafr saad)、亚历山大 (Iraq Village、Al Aamerya)、EL minufya (Monshaat Gerges、Ashmoun、Shatanoof、Ezbet Algalayla、Berket Alsabaa、Mahalet Sabak)、卢克索 (Al Gorna) 和 Al-Sharqiya (Al -Telleen、Menya Al kamh)省份。疑似FMD病毒样本（22份舌上皮（TE）、12份水疱液样本（VF）），此外还有10份舌上皮来源的细胞培养病毒、6份水疱液来源的细胞培养病毒。本研究中使用的检测方法包括用于 FMDV 病毒样本分型的 CFT 和间接 ELISA 分型试剂盒； 3ABC-FMD 用于在 2012 年埃及疫情检查血清中任何显示的保护性免疫反应中区分已接种疫苗的动物和受感染的动物。 PCR 作为一种先进的验证技术，在向英国皮尔布赖特的手足口病参考实验室提交 FMDV 疑似临床样本之前进行了。样品在原代牛肾和幼仓鼠肾细胞培养物中分离，所有样品均产生细胞病变效应。此外，接种于瑞士白化乳鼠幼鼠的所有病毒样本均显示出该病毒对小鼠后肢的特征性麻痹作用。使用 FMD-3ABC 检查从疑似感染牛身上采集的血清样本。所有 3ABC 抗体阳性样本均表明动物感染了 FMDV。 FMD 抗体检测显示，从 Al-Gharbiya、Kafr El-Shaykh、Dumyat、Alexandria、Al-Monufya、Luxor 和 Al-Sharqiya 采集的血清分别有 62%、100%、61%、37.5%、45.5%、72.7% 和 66.6% 被感染。随后通过间接ELISA试剂盒证实了3ABC抗体的结果，结果显示此次疫情的病原体是FMDV SAT2。最终的验证技术是逆转录聚合酶链反应（RT-PCR），该技术用于使用目标引物检测临床可疑样本。此外，对当前 FMDV 毒株进行 DNA 测序，结果显示，分离毒株之间不存在显着差异，差异为 0.3% 至 2.2%，并且分离毒株与 FMDV SAT2/EGY/5/2012 和 SAT2/EGY/15/2012 之间也没有显着差异。分离毒株与参考毒株相比，最接近的同一性百分比为 89.4%， FMDV SAT2/LIB/2003。为了得到官方确认，分离出的病毒被送往英国皮尔布赖特的手足口病参考实验室。分离病原体是诊断该疾病的第一步。最后，利用基因库数据对PCR产物进行测序和分析，以确认新的产物
During April and May 2012, six outbreaks of FMD type SAT 2 were reported in Egyptian governorates: EL- Gharbiya (Kafr Qeretna, Al – Mahala Alkobra, Ebshaway Almalak, Qutoor), local cattle, buffaloes and dairy farms in Kafr El-Shaykh (Alsalmya, Fouah), AL - Minya (Village 3, Samalout, Mansafees, Abo Querkas), Dumyat (Ezbet 20, Kafr saad), ALexandria (Iraq Village, Al Aamerya), EL minufya (Monshaat Gerges, Ashmoun, Shatanoof, Ezbet Algalayla, Berket Alsabaa, Mahalet Sabak),Luxor (Al Gorna) and Al- Sharqiya (Al -Telleen, Menya Al kamh) Governorates. Suspected FMD virus samples (22 Tongue epithelium (TE), 12 vesicular fluid samples (VF)), in addition to, 10 cell culture grown virus of tongue epithelium origin, 6 cell culture grown virus of vesicular fluid origin. The assays used in this study involved CFT and Indirect ELISA typing kits for typing of FMDV virus samples; 3ABC-FMD for differentiation between vaccinated and infected animals in any revealed protective immune response in the examined sera from Egypt 2012 outbreak. PCR as an advanced confirmatory technique was done before submitting FMDV suspected clinical samples for Reference Laboratory of Foot and Mouth Disease in Pirbright, United Kingdom. Samples were isolated on primary bovine kidney and Baby Hamster kidney cell cultures, where all samples gave cytopathic effect. Also, all virus samples inoculated in swiss albino suckling baby mice showed pathognomonic paralysis effect of the virus in hind limbs of mice. Serum samples collected from suspected infected cattle were examined using FMD-3ABC. All positive samples to 3ABC antibodies indicated that the animals were infected by FMDV. FMD antibody detection revealed infection of 62%, 100%, 61%, 37.5%, 45.5% , 72.7% and 66.6% of sera collected from Al-Gharbiya, Kafr El-Shaykh, Dumyat, Alexandria, Al-Monufya, Luxor and Al-Sharqiya respectively. The obtained results of 3ABC antibodies were subsequently confirmed by Indirect ELISA Kit, which revealed that the causative agent of the outbreak was FMDV SAT2. The prefinal confirmatory technique was the Reverse transcription polymerase chain reaction (RT-PCR), which was done to detect clinically suspicious samples using target primers. Also, DNA sequencing of the current FMDV strain was performed and revealed that there was no significant divergence among the isolated strains as the divergence was 0.3 to 2.2% among them, and also there was no significant divergence between the isolated strains and FMDV SAT2/EGY/5/2012 and SAT2/EGY/15/2012 The closest percentage of identity of the isolated strains in comparison with reference strains was 89.4% with FMDV SAT2/LIB/2003. For official confirmation, the isolated viruses were sent to the Reference Laboratory for Foot and Mouth Disease in Pirbright, United Kingdom. Isolation of the causative agent is the first step for diagnosis of the disease. Finally, the PCR product was sequenced and analyzed using the gene bank data to confirm that the new out