猪流行性腹泻病毒(Porcine Epidemic Diarrhea Virus，PEDV)感染可导致不同日龄猪只出现腹泻和脱水等症状，对仔猪的致死率可达100%，严重危害我国养猪业的发展。PEDV属于尼多病毒目、冠状病毒科、α-冠状病毒属成员，病毒基因组容易变异，目前尚无理想疫苗。本研究对苏皖地区猪场的PEDV抗原和抗体开展系统性监测，完成流行毒株的分离鉴定，基于分离毒株研制灭活疫苗，并构建以AH2012/12骨架嵌合S-INDEL S基因的重组PEDV毒株。本研究为PEDV的防控提供重要的毒株资源与疫苗研发基础。主要内容如下： 1．2021-2024年苏皖地区规模化猪场PEDV流行病学调查 2021-2024 年，从江苏和安徽的7个规模化猪场收集了 5177 份临床腹泻样品，采用荧光定量PCR方法检测了PEDV、猪德尔塔冠状病毒(Porcine Deltacoronavirus, PDCoV)、猪轮状病毒(Porcine rotavirus, PoRV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus, TGEV)的感染情况。结果显示，4 种病毒总阳性率为 21.2%(1095/5177)，其中 PEDV单独感染阳性率达14.1%(731/5177)，PEDV与PDCoV、PoRV混合感染率分别为3.2%(168/5177)和2.36%(122/5177)，PEDV、PDCoV、PoRV三重感染占比为 0.09%(5/5177)，TGEV均为阴性。选择PEDV阳性病料进行病毒S基因扩增测序，共获得38条S基因序列，分析结果显示，GIIc 亚型 26 份，S-INDEL 亚型 11 份，GIIb亚型1份。其中，2021-2022 年主要流行 GIIc 亚型，2023 年开始出现 S-INDEL 亚型，占比 50%(7/14)，2024 年 S-INDEL 亚型占比 80%(4/5)。临床调查结果显示，S-INDEL毒株感染可导致产房仔猪腹泻，且存在引发怀孕母猪繁殖障碍的风险。本研究明确了苏皖地区PEDV流行特点及S-INDEL毒株优势流行毒株新趋势，为该病防控提供了科学依据。 2．PEDV疫苗免疫猪群PEDV IgA抗体水平监测与保护作用分析 本研究采集苏皖地区的规模化养殖场母猪分娩前PEDV疫苗免疫猪群血清和初乳样品共计2929份，采用商品化PEDV IgA 抗体检测试剂盒检测PED IgA抗体，观察仔猪腹泻死亡淘汰率。结果为：(1)怀孕15-60d母猪，IgA 抗体阳性率≤20%。分娩前7W进行“活疫苗+灭活疫苗”免疫后，血清IgA抗体阳性率和平均抗体 S/P 值明显升高，分娩前 2W的阳性率达到 100%，平均抗体 S/P 值高达 4.13。母猪分娩当天血清IgA抗体阳性率77.8%-83.7%，断奶阶段抗体阳性率80%-82.9%，说明该免疫程序能够使猪群的IgA抗体快速达到较好的水平。(2)2023-2024年母猪初乳监测结果显示，2023年IgA 抗体合格率50.5%-89.7%，S/P平均值1.39-5.2。2024年1-6月份IgA 抗体合格率82%-100%，S/P平均值4.7-7.23。(3)仔猪 7 日龄时血清 IgA 抗体 S/P 比值较高，分别为 97.7%和4.34，随后逐渐降低。(4)2023年1月和2月腹泻仔猪死淘率分别为6.4%(562/8720)和5.6%(368/6562)，对应月份的初乳IgA阳性率仅为52.3%、50.5%，S/P平均值1.56和1.39。表明初乳PEDV IgA 抗体水平与仔猪发病率存在一定关联性。综上，母猪产前免疫可以显著提高母猪和其后代仔猪血清中IgA水平，提高仔猪对PEDV的抵抗力。猪场通过监测初乳IgA抗体水平，可以预警仔猪PED发生风险，及时调整免疫方案，预防疫病发生。 3．PEDV S-INDEL型毒株分离与致病性分析 本研究从安徽省的一头患病仔猪的肠道组织中分离到一株PEDV S-INDEL基因型毒株AHFY2023。病毒S蛋白基因测序分析显示，该分离毒株S1蛋白在第 138 和 139 位存在两个氨基酸缺失。与 S-INDEL 代表株 oh851 相比，NTD(19-233 aa)结构域中存在多个氨基酸突变。与GII型毒株相比，SAN27-29QLT、55-72 位、84-89 位、114-166 位氨基酸等存在变异。基因重组分析显示，AHFY2023毒株存在潜在重组事件，其亲本毒株可能为 JX2020 毒株(GIIc)，次要毒株为 SM98 毒株(GIa)。AHFY2023毒株攻毒试验表明，1 日龄未吃初乳仔猪在感染后18 h出现腹泻，感染后48 h内全部死亡。妊娠40天和80天的母猪攻毒后出现发热、采食量下降和腹泻症状，并持续 3-4 天。综上，本研究分离获得一株S-INDEL 基因型PEDV，来源于GIIc和GIa亚型毒株重组，对1日龄仔猪具有致死性，而对妊娠母猪的致病性比较轻微。 4．PEDV AHFY2023分离株灭活疫苗制备及其免疫保护效力 将PEDV AHFY2023毒株接种Vero细胞扩大培养，收取培养物，采用0.1% 福尔马林灭活，并与ISA15A VG佐剂乳化制备灭活疫苗。对分娩前4 W和2 W的怀孕母猪分别免疫接种，采集母猪分娩后初乳检测 PEDV IgA 抗体，结果显示：AHFY2023灭活疫苗组IgA抗体阳性率达94.4%，显著高于商品疫苗组(59.6%，1.79)和空白组(40%，0.46)。选取免疫母猪后代的6日龄仔猪，分别用2 mL 105.5 TCID50/mL AHFY2023病毒液口服攻毒。结果显示：AHFY2023免疫组仔猪存活率为 100%，腹泻率为80%，但腹泻程度较轻微(腹泻粪便总评分 4.33±2.56)；商品疫苗组存活率 80%，腹泻率 100%，腹泻严重程度评分 5.5±2.5；非免疫攻毒对照组存活率 40%，腹泻率 100%，腹泻较严重(评分 8±4.67)。粪便排毒量检测，AHFY2023攻毒后第5 天达最高峰104.19copies/mL，明显低于非免疫攻毒组(攻毒后第3天达到峰值 106.05copies/mL)。研究表明，AHFY2023 灭活疫苗对 PEDV S-INDEL 同型毒株攻击具有一定保护作用，有助于防控 PEDV S-INDEL 毒株的流行。 5．PEDV变异毒株与S-INDEL流行毒株S基因嵌合毒株的构建与致病性研究 本研究首先通过靶向设计 2 条 sgRNA 对 AH2012/12 全基因 BAC 重组质粒进行切割，然后将 S-INDEL 毒株的 S 基因片段与 AH2012/12 基因 BAC 质粒共同转化至 NEB 10-beta 大肠杆菌，进而获得重组 BAC 质粒，最后将该重组质粒转染至 Vero 细胞，成功拯救出 S-INDEL 毒株 S 基因替代 AH2012/12 毒株 S 基因的嵌合重组病毒，命名为 rAH2012/12-S。通过嵌合病毒空斑试验和生长曲线测定发现，其在 Vero 细胞上的空斑形成能力介于亲本毒 AH2012/12 和 AHFY2023 之间，且在细胞中的增殖动态曲线与亲本毒无显著差异。5日龄仔猪致病性试验结果显示， rAH2012/12-S嵌合毒株感染未引起仔猪水样腹泻且无死亡发生，肠道病理变化主要集中在十二指肠，病毒载量总体低于亲本毒，表明其致病性减弱，为PEDV新型疫苗研发和疫病防控提供了生物材料支持。 综上所述，本研究通过对苏皖地区猪场的PEDV进行流行病学调查，探明了该区域毒株流行情况；通过养殖场母猪产前疫苗免疫猪群初乳中的PEDV IgA抗体检测和仔猪腹泻死淘率观察，证明母猪产前疫苗免疫可产生较高 IgA抗体水平的初乳，同时初乳IgA抗体水平与仔猪腹泻死淘率之间存在较好相关性；对分离到的一株PEDV S-INDEL株进行了鉴定，证明该毒株为GIIc亚型和GIa亚型的重组株，并明确了其致病性；利用该毒株研制了灭活疫苗，临床免疫试验证明其具有较好免疫保护作用；此外，构建了以AH2012/12骨架嵌合S-INDEL S基因的重组PEDV毒株，为PEDV新疫苗研究奠定了基础。本研究有力推进了PEDV有效防控。

Infection with Porcine Epidemic Diarrhea Virus (PEDV) can cause symptoms such as diarrhea and dehydration in pigs of different ages. The fatality rate of piglets can reach up to 100%, which seriously endangers the development of the pig - farming industry in China. PEDV belongs to the genus Alpha-Coronavirus of the family Coronaviridae within the order Nidovirales. The viral genome is prone to variation, and currently, there is no ideal vaccine available. This study conducted an epidemiological investigation of antigens and antibodies against PEDV variant strains in the Suwan region, isolated and identified the prevalent strains in this region, and developed an inactivated vaccine using the isolated strains. Additionally, this study constructed a recombinant PEDV strain with the AH2012/12 backbone and a chimeric S-INDEL S gene. This study provides important strain resources and a vaccine development foundation for PEDV prevention and control. The main contents are as follows: 1.Epidemiological investigation of PEDV in large-scale pig farms in the Jiangsu-Anhui region from 2021 to 2024 From 2021 to 2024, a total of 5,177 clinical diarrhea samples were collected from seven large - scale pig farms in Jiangsu and Anhui provinces. The infection status of PEDV, Porcine Deltacoronavirus (PDCoV), Porcine rotavirus (PoRV), and Transmissible gastroenteritis virus (TGEV) was detected using the fluorescence-quantitative PCR method. The results showed that the total positive rate of the four viruses was 21.2% (1,095/5,177). The positive rate of PEDV single - infection reached 14.1% (731/5,177). The mixed - infection rates of PEDV with PDCoV and PoRV were 3.2% (168/5,177) and 2.36% (122/5,177), respectively. The proportion of triple - infection of PEDV, PDCoV, and PoRV was 0.09% (5/5,177), and all TGEV tests were negative. The S genes of PEDV in positive samples were amplified and sequenced. A total of 38 S gene sequences were obtained. The analysis results showed that there were 26 samples of the GIIc subtype, 11 samples of the S-INDEL subtype, and 1 sample of the GIIb subtype. Among them, the GIIc subtype was the predominant strain from 2021 to 2022. The S-INDEL subtype began to appear in 2023, accounting for 50% (7/14), and in 2024, the proportion of the S-INDEL subtype reached 80% (4/5). Clinical investigation results showed that infection with S-INDEL strains can cause diarrhea in nursery piglets and poses a risk of inducing reproductive disorders in pregnant sows. This study clarified the prevalence characteristics of PEDV in the Jiangsu - Anhui region and the new trend of the S-INDEL strain becoming the predominant epidemic strain, providing a scientific basis for the prevention and control of this disease. 2.Monitoring of PEDV IgA antibody levels in PEDV vaccinated pig herds and analysis of their protective effects In this study, a total of 2,929 serum and colostrum samples were collected from PEDV-vaccinated pig herds before sow parturition in large-scale farms in the Jiangsu-Anhui region. The PEDV IgA antibody detection kit of IDEXX Company was used to detect PED IgA antibodies, and the diarrhea - related mortality and culling rate of piglets were observed. The results were as follows: (1) For sows at 15-60 days of pregnancy, the positive rate of IgA antibodies was ≤20%. After immunization with a combination of live and inactivated vaccines 7 weeks before parturition, the positive rate of serum IgA antibodies and the average S/P value of antibodies significantly increased. The positive rate reached 100% 2 weeks before parturition, and the average S/P value of antibodies was as high as 4.13. The positive rate of serum IgA antibodies on the day of sows' parturition was 77.8% - 83.7%, and the positive rate of antibodies at the weaning stage was 80%-82.9%, indicating that this immunization program could rapidly raise the antibody levels of the pig herd to a good level. (2) The monitoring results of sows' colostrum from 2023 to 2024 showed that the qualification rate of IgA antibodies in 2023 was 50.5%-89.7%, and the average S/P value was 1.39-5.2. From January to June 2024, the qualification rate of IgA antibodies was 82% - 100%, and the average S/P value was 4.7-7.23. (3) The S/P ratio of serum IgA antibodies in piglets at 7 days of age was relatively high, which were 97.7% and 4.34 respectively, and then gradually decreased. (4) The diarrhea-related mortality and culling rates of piglets in January and February 2023 were 6.4% (562/8,720) and 5.6% (368/6,562), respectively. The positive rates of colostrum IgA antibodies in the corresponding months were only 52.3% and 50.5%, and the average S/P values were 1.56 and 1.39. These results indicate that there is a certain correlation between the PEDV IgA antibody level in colostrum and the incidence rate of piglets. The above results show that immunization of sows before parturition can significantly increase the IgA levels in the maternal antibodies of sows and their offspring piglets, and improve the resistance of piglets to PEDV. By measuring the IgA antibody level in colostrum, pig farms can predict the risk of PED occurrence in piglets, adjust the immunization program in a timely manner, and prevent the occurrence of the disease. 3.Isolation and pathogenicity analysis of PEDV S-INDEL genotype strain In this study, a PEDV S-INDEL genotype strain AHFY2023 was isolated from the intestinal tissue of a diseased piglet in Anhui Province. Sequencing analysis of the viral S - protein gene showed that there were two amino - acid deletions at positions 138 and 139 of the S1 protein of the isolated strain. Compared with the S-INDEL representative strain oh851, there were a large number of amino-acid mutations in the NTD (19-233 aa) domain. Compared with GII-type strains, there were variations in amino acids at positions such as SAN27-29QLT, 55-72, 84-89, and 114-166. Gene recombination analysis showed that there was a potential recombination event in the AHFY2023 strain. Its parental strain was the JX2020 strain of the GIIc subtype, and the secondary strain was the SM98 strain of the GIa subtype. The challenge test of the AHFY2023 strain showed that 1-day-old piglets that had not suckled colostrum showed diarrhea 18 hours after infection and all died within 48 hours after infection. Pregnant sows at 40 days and 80 days of gestation showed symptoms such as fever, decreased feed intake, and diarrhea after the challenge, which lasted for 3-4 days. The above results indicate that a PEDV strain of the S-INDEL genotype was isolated in this study, which originated from the recombination of GIIc and GIa subtype strains. It is lethal to 1-day-old piglets, while its pathogenicity to pregnant sows is relatively mild. 4.Preparation of inactivated vaccine of PEDV AHFY2023 isolate and determination of its immunoprotective efficacy The PEDV AHFY2023 strain was inoculated into Vero cells for expansion culture. The culture was harvested, inactivated with 0.1% formaldehyde, and emulsified with the ISA15A VG adjuvant to prepare an inactivated vaccine. Pregnant sows 4 weeks and 2 weeks before parturition were immunized. The colostrum of sows after parturition was collected to detect PEDV IgA antibodies. The results showed that the positive rate of IgA antibodies in the AHFY2023 inactivated-vaccine group reached 94.4%, which was significantly higher than that in the commercial - vaccine group (59.6%, 1.79) and the blank group (40%, 0.46). Six-day-old piglets from the immunized sows were selected and orally challenged with 2 mL 105.5 TCID50/mL of AHFY2023 virus solution. The results showed that the survival rate of piglets in the AHFY2023 immunization group was 100%, the diarrhea rate was 80%, and the diarrhea was relatively mild (the total diarrhea-feces score was 4.33±2.56). The survival rate of the commercial-vaccine group was 80%, the diarrhea rate was 100%, and the diarrhea-severity score was 5.5±2.5. The survival rate of the non - immunized challenge control group was 40%, the diarrhea rate was 100%, and the diarrhea was relatively severe (the score was 8±4.67). Detection of fecal virus shedding showed that the virus shedding in the AHFY2023 group reached the peak of 104.19 copies/mL on the 5th day after the challenge, which was significantly lower than that in the non-immunized challenge group (the peak of 106.05 copies/mL was reached on the 3rd day after the challenge). The research shows that the prepared AHFY2023 inactivated vaccine has a certain protective effect against the attack of PEDV S-INDEL homotypic strains and is helpful for the prevention and control of the prevalence of PEDV S-INDEL strains. 5.Construction and pathogenicity study of chimeric strains of PEDV variant strains and S-INDEL prevalent strains with the S gene In this study, first, two sgRNAs were designed by targeting to cut the full-length gene BAC recombinant plasmid of AH2012/12. Then, the S gene fragment of the S-INDEL strain and the BAC plasmid of the AH2012/12 gene were co- transformed into NEB 10-beta Escherichia coli to obtain a recombinant BAC plasmid. Finally, the recombinant plasmid was transfected into Vero cells, and a chimeric recombinant virus in which the S gene of the S-INDEL strain replaced the S gene of the AH2012/12 strain was successfully rescued and named rAH2012/12-S. Through the plaque assay and growth-curve determination of the chimeric virus, it was found that its plaque - forming ability on Vero cells was between that of the parental viruses AH2012/12 and AHFY2023, and there was no significant difference in the proliferation-dynamic curve in cells compared with the parental viruses. The pathogenicity-test results of 5-day-old piglets showed that the infection of the rAH2012/12-S chimeric strain did not cause watery diarrhea in piglets and there was no death. The intestinal pathological changes were mainly concentrated in the duodenum, and the overall viral load was lower than that of the parental virus, indicating that its pathogenicity was weakened. This provides biological-material support for the research and development of new PEDV vaccines and the prevention and control of the disease.  Taken together, in this study, an epidemiological investigation of PEDV variant strains in the Jiangsu-Anhui region was carried out to clarify the prevalence of the strains in this area. A PEDV S-INDEL strain was isolated and identified. It was proven to be a recombinant strain of GIIc and GIa subtypes, and its pathogenicity was determined. An inactivated vaccine was developed using this strain, and clinical immunization tests demonstrated that it had a good immunoprotective effect. By detecting PEDV IgA antibodies in the colostrum of vaccinated pig herds before sow parturition on farms and observing the diarrhea - related mortality and culling rate of piglets, it was proven that pre - parturition vaccination of sows can produce colostrum with a high level of IgA antibodies. Moreover, there is a good correlation between the IgA antibody level in colostrum and the diarrhea - related mortality and culling rate of piglets. In addition, a recombinant PEDV strain with the S gene of S-INDEL chimerized on the AHFY2012/12 backbone was constructed, laying a foundation for the research on new PEDV vaccines. This study effectively promoted the effective prevention and control of PEDV.

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