Replication of Attenuated Strain S of Novel Duck Reovirus in Muscovy Ducklings
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摘要:
目的 研究新型鸭呼肠孤病毒（Novel duck reovirus，NDRV）弱毒S株C30在雏番鸭血液中的病毒血症，口咽、泄殖腔的排毒规律及靶器官的带毒时间，了解弱毒S株在雏番鸭体内的增殖规律和致弱机理。 方法 设计特异性引物，建立检测NDRV核酸的RT-qPCR方法；新型鸭呼肠孤病毒弱毒S株第30代（NDRV-S-C30）腿部肌肉注射2日龄雏番鸭，在免疫后1、2、3、4、6、8、10、12、14 d（Days post vaccination，dpv）采集其血液、肝脏、脾脏、泄殖腔分泌液和口咽液，用RT-qPCR 方法检测NDRV在血液、口咽、泄殖腔及靶器官的增殖能力及带毒时间。 结果 建立了检测NDRV 核酸的特异性RT-qPCR方法，使用该方法检测NDRV弱毒攻毒雏鸭的口咽和泄殖腔排毒时间分别为2～8 d和2～10 d，排毒高峰期为4～6 d；病毒血症时间为1～4 d，其中1～2 d 为病毒血症高峰期；弱毒在肝脏的带毒时间为3～6 d，在脾脏的带毒时间为1～8 d。 结论 NDRV-S-C30弱毒株免疫雏番鸭后可通过口腔和泄殖腔向外界排毒，仅能引起较弱的病毒血症反应，且在肝脏中的增殖能力弱，丧失了对易感靶器官造成病理损伤的能力。明确了NDRV 弱毒S株的排毒规律及病毒血症时间，为建立弱毒免疫效力评价方法奠定了基础。 Abstract:Objective Disease symptoms and reproduction of the attenuated strain S C30(NDRV-S-C30) of novel duck reovirus on inoculated Muscovy ducklings were studied. Method Specific primers were designed to establish a RT-qPCR method for detecting the NDRV nucleic acid in two-day-old Muscovy ducklings intramuscularly injected with NDRV-S-C30. Symptoms of viremia in the blood and viral shedding in the throat and cloaca were observed on the infected ducklings. Viral replication in the birds was monitored on samples of sera, liver, and spleen as well as cloaca and throat swabs collected in 1, 2, 3, 4, 6, 8, 10, 12, and 14 d after the vaccination (dpv) for NDRV nucleic acid detection by RT-qPCR. Result A specific RT-qPCR assay developed for the NDRV detection showed the viral shedding in the throat appeared in 2–8 dpv and in the cloaca in 2–10 dpv and peaked in 4–6 dpv, the viremia in 1–4 dpv and peaked in 1–2 dpv, and the NDRV-S in the liver within 3–6 dpv and in the spleen 1–8 dpv. Conclusion NDRV-S-C30, a attenuated strain, showed virus shedding through the oral cavity and cloaca after immunization of young ducks. It only caused a weak viral bloodstream response and had weak replication ability in the liver. It had lost the ability to cause pathological damage to susceptible target organs. The occurrences and time durations of the viral shedding and viremia in the Muscovy ducklings caused by NDRV-S-C30 were determined. The information would aid the evaluation of immunization efficacy in combating the disease caused by the attenuated virus strain. -
Key words:
- Novel duck reovirus (NDRV) /
- attenuated strain /
- RT-qPCR /
- viral shedding /
- viremia
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图 1 qPCR引物特异性验证
A：RT-PCR 扩增电泳图；M为DNA DL 2000 相对分子质量标准，1为pMD18T-NDRV质粒，2为NDRV-S-C30，3～9分别为GPV、MDPV、MDRV、GRV、DHAV-3、DAdV-B2和DTMUV，10：DEPC水。B：RT-qPCR扩增曲线；1为pMD18T-NDRV质粒，2为NDRV-S-C30，3～9分别为GPV、MDPV、MDRV、GRV、DHV-3、DAdV-B2和DTMUV，10：DEPC水。
Figure 1. Specificity of qPCR primers
A: RT-PCR amplification; M: DL2000 DNA marker; 1: pMD18T-NDRV plasmid; 2: NDRV-S-C30; 3–9: GPV, MDPV, MDRV, GRV, DHV-3, DAdV-B2, and DTMUV, respectively; 10: DEPC water; B: RT-qPCR amplification; 1: pMD18T-NDRV plasmid; 2: NDRV-S-C30; 3–9: GPV, MDPV, MDRV, GRV, DHV-3, DAdV-B2, and DTMUV, respectively; 10: DEPC water.
图 2 RT-qPCR和普通RT-PCR的敏感性比较
A：荧光定量RT-PCR；1～9分别为1.89×109～1.89×101 拷贝·μL−1，10为DEPC水；B：普通RT-PCR；M：DNA DL 2000 相对分子质量标准，1～9分别为1.89×109～1.89×101拷贝·μL−1，10为DEPC水。
Figure 2. Sensitivity of conventional PCR and RT-qPCR assays in detecting NDRV
A: RT-qPCR; 1–9: 1.89×109−1.89×101 copies·μL−1; 10: DEPC water; B: conventional RT-PCR; M: DL2000 DNA marker; 1–9: 1.89×109−1.89×101 copies·μL−1, respectively; 10: DEPC water.
表 1 RT-qPCR检测NDRV-S-C30免疫雏番鸭的排毒规律
Table 1. RT-qPCR detection of viral shedding in Muscovy ducklings after NDRV-S-C30 vaccination
采集部位
Sampling site检测结果（阳性样品/检测样品）
Detection result(positive samples/detected samples)1 d 2 d 3 d 4 d 6 d 8 d 10 d 12 d 14 d 喉 Throat 免疫组 Vaccination group 0/3 2/3 2/3 3/3 3/3 1/3 0/3 0/3 0/3 对照组 Control group 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 泄殖腔 Cloaca 免疫组 Vaccination group 0/3 2/3 2/3 3/3 3/3 1/3 1/3 0/3 0/3 对照组 Control group 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 
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表 2 NDRV-S-C30免疫后雏番鸭血液RT-qPCR检测结果
Table 2. RT-qPCR detection on blood of Muscovy ducklings after NDRV-S-C30 vaccination
采集部位
Sampling site处理组
Treatment group检测结果（阳性样品/检测样品）
Detection result(psositive samples/detected samples)1 d 2 d 3 d 4 d 6 d 8 d 10 d 12 d 14 d 血清 Serum 免疫组 Vaccination group 2/3 2/3 1/3 1/3 0/3 0/3 0/3 0/3 0/3 对照组 Control group 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2
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表 3 NDRV-S-C30在雏番鸭肝、脾中的RT-qPCR检测结果
Table 3. RT-qPCR detection on liver and spleen of Muscovy ducklings after NDRV-S-C30 vaccination
采集部位
Sampling site处理组
Treatment group检测结果（阳性样品/检测样品）
Detection result(positive samples/detected samples)1 d 2 d 3 d 4 d 6 d 8 d 10 d 12 d 14 d 肝脏 Liver 免疫组 Vaccination group 0/3 0/3 2/3 1/3 1/3 0/3 0/3 0/3 0/3 对照组 Control group 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 脾脏 Spleen 免疫组 Vaccination group 2/3 1/3 3/3 3/3 1/3 1/3 0/3 0/3 0/3 对照组 Control group 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2
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