Center for Food Animal Health, Department of Animal Sciences, College of Food, Agricultural and Environmental Sciences, The Ohio State Universitygrid.261331.4, Wooster, Ohio, USA.
Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State Universitygrid.261331.4, Columbus, Ohio, USA.
J Virol. 2022 Jun 8;96(11):e0046922. doi: 10.1128/jvi.00469-22. Epub 2022 May 18.
Coronavirus (CoV) nonstructural protein 1 (nsp1) inhibits cellular gene expression and antagonizes interferon (IFN) response. Porcine epidemic diarrhea virus (PEDV) infects pigs and causes high mortality in neonatal piglets. We hypothesized that a recombinant PEDV carrying mutations at the conserved residues N93 and N95 of nsp1 induces higher IFN responses and is more sensitive to IFN responses, leading to virus attenuation. We mutated PEDV nsp1 N93 and N95 to A93 and A95 to generate the recombinant N93/95A virus using the infectious clone of a highly virulent PEDV strain, PC22A (icPC22A), and evaluated N93/95A virus and . Compared with icPC22A, the N93/95A mutant replicated to significantly lower infectious titers, triggered stronger type I and III IFN responses, and was more sensitive to IFN treatment . To evaluate the pathogenicity and immunogenicity, 5-day-old gnotobiotic piglets were orally inoculated with the N93/95A or icPC22A strain or mock inoculated and then challenged at 22 days postinoculation (dpi) with icPC22A. icPC22A in all pigs (100% [5/5]) caused severe diarrhea and death within 6 dpi. Only one pig (25% [1/4]) died in the N93/95A group. Compared with the icPC22A group, significantly delayed and diminished fecal PEDV shedding was detected in the N93/95A group. Postchallenge, all piglets in N93/95A group were protected from severe diarrhea and death, whereas all pigs in the mock-challenged group developed severe diarrhea, and 25% (1/4) of them died. In summary, nsp1 N93A and N95A mutations attenuated PEDV but retained viral immunogenicity and can be targets for the development of live attenuated vaccines for PEDV. PEDV causes porcine epidemic diarrhea (PED) and remains a great threat to the swine industry worldwide because no effective vaccines are available yet. Safe and effective live attenuated vaccines can be designed using reverse genetics to induce lactogenic immunity in pregnant sows to protect piglets from the deadly PED. We found that an engineered PEDV mutant carrying N93A and N95A mutations of nsp1 was partially attenuated and remained immunogenic in neonatal pigs. Our study suggested that nsp1 N93 and N95 can be good targets for the rational design of live attenuated vaccines for PEDV using reverse genetics. Because CoV nsp1 is conserved among alphacoronaviruses (α-CoVs) and betacoronaviruses (β-CoVs), it may be a good target for vaccine development for other α-CoVs or β-CoVs.
冠状病毒（CoV）非结构蛋白 1（nsp1）抑制细胞基因表达并拮抗干扰素（IFN）反应。猪流行性腹泻病毒（PEDV）感染猪，并导致新生仔猪高死亡率。我们假设携带 nsp1 保守残基 N93 和 N95 突变的重组 PEDV 诱导更高的 IFN 反应，并对 IFN 反应更敏感，导致病毒衰减。我们使用高毒力 PEDV 株的感染性克隆（icPC22A）将 PEDV nsp1 N93 和 N95 突变为 A93 和 A95，生成重组 N93/95A 病毒，并评估 N93/95A 病毒和。与 icPC22A 相比，N93/95A 突变体的感染性滴度显著降低，触发更强的 I 型和 III 型 IFN 反应，并且对 IFN 治疗更敏感。为了评估致病性和免疫原性，5 日龄无菌仔猪经口接种 N93/95A 或 icPC22A 株或模拟接种，然后在接种后 22 天（dpi）用 icPC22A 攻毒。icPC22A 在所有猪（100%[5/5]）中引起严重腹泻和 6dpi 内死亡。N93/95A 组仅 1 头猪（25%[1/4]）死亡。与 icPC22A 组相比，N93/95A 组粪便 PEDV 脱落明显延迟和减少。攻毒后，N93/95A 组所有仔猪均免受严重腹泻和死亡的影响，而模拟攻毒组所有仔猪均发生严重腹泻，其中 25%（1/4）死亡。总之，nsp1 N93A 和 N95A 突变使 PEDV 减毒，但保留了病毒的免疫原性，可作为 PEDV 活疫苗开发的靶点。PEDV 引起猪流行性腹泻（PED），并且由于目前尚无有效的疫苗，仍然是全球养猪业的巨大威胁。使用反向遗传学设计安全有效的活疫苗可以诱导妊娠母猪产生乳源性免疫力，以保护仔猪免受致命 PED 的侵害。我们发现，携带 nsp1 N93A 和 N95A 突变的工程化 PEDV 突变体在新生仔猪中部分减毒并保持免疫原性。我们的研究表明，nsp1 N93 和 N95 可以作为使用反向遗传学设计 PEDV 活疫苗的合理靶标。由于冠状病毒（CoV）nsp1 在α-冠状病毒（α-CoVs）和β-冠状病毒（β-CoVs）之间保守，因此它可能是其他α-CoVs 或β-CoVs 疫苗开发的良好靶点。
