Hou Yixuan, Lin Chun-Ming, Yokoyama Masaru, Yount Boyd L, Marthaler Douglas, Douglas Arianna L, Ghimire Shristi, Qin Yibin, Baric Ralph S, Saif Linda J, Wang Qiuhong
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA.
Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan.
J Virol. 2017 Jun 26;91(14). doi: 10.1128/JVI.00227-17. Print 2017 Jul 15.
We previously isolated a porcine epidemic diarrhea virus (PEDV) strain, PC177, by blind serial passaging of the intestinal contents of a diarrheic piglet in Vero cell culture. Compared with the highly virulent U.S. PEDV strain PC21A, the tissue culture-adapted PC177 (TC-PC177) contains a 197-amino-acid (aa) deletion in the N-terminal domain of the spike (S) protein. We orally inoculated neonatal, conventional suckling piglets with TC-PC177 or PC21A to compare their pathogenicities. Within 7 days postinoculation, TC-PC177 caused mild diarrhea and lower fecal viral RNA shedding, with no mortality, whereas PC21A caused severe clinical signs and 55% mortality. To investigate whether infection with TC-PC177 can induce cross-protection against challenge with a highly virulent PEDV strain, all the surviving piglets were challenged with PC21A at 3 weeks postinoculation. Compared with 100% protection in piglets initially inoculated with PC21A, 88% and 100% TC-PC177- and mock-inoculated piglets had diarrhea following challenge, respectively, indicating incomplete cross-protection. To investigate whether this 197-aa deletion was the determinant for the attenuation of TC-PC177, we generated a mutant (icPC22A-S1Δ197) bearing the 197-aa deletion from an infectious cDNA clone of the highly virulent PEDV PC22A strain (infectious clone PC22A, icPC22A). In neonatal gnotobiotic pigs, the icPC22A-S1Δ197 virus caused mild to moderate diarrhea, lower titers of viral shedding, and no mortality, whereas the icPC22A virus caused severe diarrhea and 100% mortality. Our data indicate that deletion of this 197-aa fragment in the spike protein can attenuate a highly virulent PEDV, but the virus may lose important epitopes for inducing robust protective immunity. The emerging, highly virulent PEDV strains have caused substantial economic losses worldwide. However, the virulence determinants are not established. In this study, we found that a 197-aa deletion in the N-terminal region of the S protein did not alter virus (TC-PC177) tissue tropism but reduced the virulence of the highly virulent PEDV strain PC22A in neonatal piglets. We also demonstrated that the primary infection with TC-PC177 failed to induce complete cross-protection against challenge by the highly virulent PEDV PC21A, suggesting that the 197-aa region may contain important epitopes for inducing protective immunity. Our results provide an insight into the role of this large deletion in virus propagation and pathogenicity. In addition, the reverse genetics platform of the PC22A strain was further optimized for the rescue of recombinant PEDV viruses This breakthrough allows us to investigate other virulence determinants of PEDV strains and will provide knowledge leading to better control PEDV infections.
我们之前通过在Vero细胞培养物中对腹泻仔猪的肠道内容物进行盲传，分离出一株猪流行性腹泻病毒（PEDV），PC177。与高致病性的美国PEDV毒株PC21A相比，适应组织培养的PC177（TC - PC177）在刺突（S）蛋白的N端结构域有一个197个氨基酸（aa）的缺失。我们给新生的、传统哺乳仔猪口服接种TC - PC177或PC21A以比较它们的致病性。接种后7天内，TC - PC177引起轻度腹泻且粪便病毒RNA排出量较低，无死亡情况，而PC21A引起严重的临床症状且死亡率为55%。为了研究感染TC - PC177是否能诱导对高致病性PEDV毒株攻击的交叉保护，所有存活的仔猪在接种后3周用PC21A进行攻击。与最初接种PC21A的仔猪100%得到保护相比，接种TC - PC177和未接种的仔猪在攻击后分别有88%和100%出现腹泻，表明交叉保护不完全。为了研究这个197 - aa缺失是否是TC - PC177减毒的决定因素，我们从高致病性PEDV PC22A毒株的感染性cDNA克隆（感染性克隆PC22A，icPC22A）中构建了一个带有197 - aa缺失的突变体（icPC22A - S1Δ197）。在新生无菌仔猪中，icPC22A - S1Δ197病毒引起轻度至中度腹泻、较低的病毒排出滴度且无死亡，而icPC22A病毒引起严重腹泻且死亡率为100%。我们的数据表明，刺突蛋白中这个197 - aa片段的缺失可使高致病性PEDV减毒，但该病毒可能会失去诱导强大保护性免疫的重要表位。新出现的高致病性PEDV毒株已在全球范围内造成了巨大的经济损失。然而，毒力决定因素尚未明确。在本研究中，我们发现S蛋白N端区域的197 - aa缺失并未改变病毒（TC - PC177）的组织嗜性，但降低了高致病性PEDV毒株PC22A在新生仔猪中的毒力。我们还证明，初次感染TC - PC177未能诱导对高致病性PEDV PC21A攻击的完全交叉保护，这表明197 - aa区域可能包含诱导保护性免疫的重要表位。我们的结果为这一大片段缺失在病毒传播和致病性中的作用提供了见解。此外，PC22A毒株的反向遗传学平台进一步优化用于重组PEDV病毒的拯救。这一突破使我们能够研究PEDV毒株的其他毒力决定因素，并将为更好地控制PEDV感染提供知识。
