A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant.
A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant.
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DOI：
10.1128/mbio.00047-14
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发表时间：
2014-03-25
期刊：
影响因子：
6.4
通讯作者：
Baric RS
中科院分区：
文献类型：
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作者：
Agnihothram S;Yount BL Jr;Donaldson EF;Huynh J;Menachery VD;Gralinski LE;Graham RL;Becker MM;Tomar S;Scobey TD;Osswald HL;Whitmore A;Gopal R;Ghosh AK;Mesecar A;Zambon M;Heise M;Denison MR;Baric RS
人畜共患冠状病毒（CoV）的跨物种传播可导致大流行性疾病暴发。中东呼吸综合征冠状病毒（MERS-CoV）于2012年发现，迄今已造成182例病例，死亡率约为43%，尚未报道小动物模型。MERS-CoV和Betacoronavirus（β-CoV）亚组2c的伏翼蝙蝠冠状病毒（BtCoV）株HKU 5在几个区域的氨基酸水平上共享>65%的同一性，包括非结构蛋白5（nsp 5）和核衣壳（N）蛋白，其是重要的药物和疫苗靶标。BtCoV HKU 5已在计算机上进行了描述，但尚未显示在培养中复制，因此阻碍了针对亚群2c β-CoV的药物和疫苗研究。我们报道了含有严重急性呼吸综合征（SARS）-冠状病毒S糖蛋白胞外区的BtCoV HKU 5（BtCoV HKU 5-SE）的合成重建和测试。这种病毒在细胞培养物和年轻和老年小鼠中有效复制，其中病毒靶向气道和肺泡上皮细胞。与一些亚组2b SARS-CoV疫苗在攻击后引起强烈的嗜酸性粒细胞增多不同，我们证明BtCoV HKU 5和MERS-CoV N表达的委内瑞拉马脑炎病毒复制子颗粒（VRP）疫苗在BtCoV HKU 5-SE攻击后不会引起广泛的嗜酸性粒细胞增多。BtCoV HKU 5-SE在年轻小鼠中的传代导致增强的毒力，在老年小鼠中引起20%的体重减轻、弥漫性肺泡损伤和透明膜形成。传代病毒的特征在于nsp 13、nsp 14、开放阅读框5（ORF 5）和M基因中的突变。最后，我们鉴定了对亚组2c β-CoV的nsp 5蛋白酶有活性的抑制剂。合成基因组平台能够重建新兴的人畜共患病病毒病原体或其系统发生的亲属提供了新的策略，以确定广泛的治疗，评估疫苗的结果，并研究病毒的发病机制。2012年中东呼吸综合征冠状病毒的爆发引发了另一场全球流行病的幽灵，类似于2003年的SARS冠状病毒流行病。MERS-CoV与BtCoV HKU 5在药物和疫苗测试所必需的目标区域相关。由于没有小动物模型来评估MERS-CoV的发病机制或测试疫苗，我们构建了表达SARS-CoV刺突（S）糖蛋白区域的重组BtCoV HKU 5，从而允许重组病毒在细胞培养物和小鼠中生长。我们发现这种重组病毒靶向气道上皮细胞，并导致老年小鼠的疾病。我们使用这个平台来（i）确定一种广谱抗病毒药物，可以潜在地抑制与MERS-CoV密切相关的病毒，（ii）证明基于MERS-CoV N蛋白的疫苗没有增加的嗜酸性粒细胞免疫病理学，以及（iii）小鼠适应这种病毒以确定跨物种传播和毒力的病毒遗传决定因素。这项研究作为控制新出现病毒的策略具有重要意义。
Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. The 2012 outbreak of MERS-CoV raises the specter of another global epidemic, similar to the 2003 SARS-CoV epidemic. MERS-CoV is related to BtCoV HKU5 in target regions that are essential for drug and vaccine testing. Because no small animal model exists to evaluate MERS-CoV pathogenesis or to test vaccines, we constructed a recombinant BtCoV HKU5 that expressed a region of the SARS-CoV spike (S) glycoprotein, thereby allowing the recombinant virus to grow in cell culture and in mice. We show that this recombinant virus targets airway epithelial cells and causes disease in aged mice. We use this platform to (i) identify a broad-spectrum antiviral that can potentially inhibit viruses closely related to MERS-CoV, (ii) demonstrate the absence of increased eosinophilic immune pathology for MERS-CoV N protein-based vaccines, and (iii) mouse adapt this virus to identify viral genetic determinants of cross-species transmission and virulence. This study holds significance as a strategy to control newly emerging viruses.