Characterization of a recombinant Akabane mutant virus with knockout of a nonstructural protein NSs in a pregnant goat model.
Characterization of a recombinant Akabane mutant virus with knockout of a nonstructural protein NSs in a pregnant goat model.
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在怀孕山羊模型中敲除非结构蛋白 NS 的重组 Akabane 突变病毒的表征。
DOI：
10.1007/s12250-015-3704-2
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
et al.
中科院分区：
文献类型：
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作者：
Takenaka-Uema A;Horimoto T;et al.
赤羽病毒（Akabane virus，AKAV）是一种正布尼亚病毒，主要由蚊虫叮咬传播，除欧洲外，世界各地均有分布。AKAV首先在日本从蚊子中分离（Oya et al.，1961年）。尽管感染AKAV的怀孕母牛、母羊和山羊没有表现出疾病的临床体征，但子宫内感染导致流产、早产、死产和先天性畸形，如关节弯曲-积水性无脑综合征（Kurogi et al. 1976年），造成畜牧业的经济损失。减毒活疫苗株TS-C2来源于作为温度敏感突变体的OBE-1株（Kurogi等人，1979年）。尽管疫苗接种降低了疾病的流行，但已分离出AKAV的抗原性和致病性变体（Lee等人，2002; Ogawa等人，（2007年a）;例如，在日本，从患有非化脓性脑炎和神经症状的小牛中分离出变异的Iriki菌株（Miyazato等，1989），在中和试验中与参考菌株的抗原交叉反应性较低（Akashi和Inaba，1997）。因此，有必要重新考虑疫苗接种策略，以有效控制该病。在此，我们评估了敲除非结构蛋白NSs的突变病毒的特征，所述非结构蛋白NSs充当I型干扰素拮抗剂并参与宿主蛋白质合成的调节（Weber等人，2002年），通过实验感染怀孕的山羊。妊娠山羊模型可用于AKAV研究，如山羊胎儿的实验性经胎盘感染的先前报道所建议的（Kurogi等人，1977年）。在我们先前的研究中，通过反向遗传学产生了重组TS-C2（rTTT）及其具有NS敲除的突变体病毒（rTTT NSs）（Takenaka-Uema et al.，2016）。在细胞培养物中，rTTT CNS以比rTTT更慢的速率生长，尽管其在感染后48小时达到最大产量，这与rTTT的产量相当（Takenaka-Uema et al.，2016）。7只妊娠和5只未妊娠的芝山羊（9个月至30个月）来自东京大学农业和生命科学研究生院动物资源科学中心。驯化3天后，在妊娠37 - 49天，通过颈静脉静脉接种3 mL（2.3-4.6× 107 PFU）野生型（wt）Iriki菌株、rTTT或TTT cDNANS。接种后，测量体重，检查直肠体温和其他一般临床变量。接种后采集血样，分离白色血细胞（WBC）。接种后3周（wpi），从子宫中取出胎仔进行尸检。由于该山羊模型的设计是定性的而不是定量的，并且考虑了动物实验中的替换、减少和改进的原则，因此本研究受到用于评估重组病毒潜力的动物数量的限制。分析在病毒接种时和在接下来的3周内以1周间隔获得的血清中病毒中和（VN）抗体的存在（Ogawa等人，2007年a）。接种病毒的6只山羊中有5只在接种时没有针对病毒的抗体，而1只山羊（#3）由于未知原因具有可检测的抗体滴度（8），并且在该山羊中引发的最高抗体滴度（181）是在2 wpi（表1），可能是由于免疫加强。所有接种的山羊在感染后1周产生可检测的VN抗体，其抗体水平在感染后2或3周达到峰值。为了比较...
Akabane virus (AKAV), an orthobunyavirus, is transmitted primarily by biting midges and is widely distributed throughout the world except the Europe. AKAV was first isolated from mosquitoes in Japan (Oya et al., 1961). Although pregnant cows, ewes, and goats infected with AKAV exhibit no clinical signs of disease, in utero infections result in abortion, premature birth, stillbirth, and congenital deformities such as arthrogryposis-hydranencephaly syndrome (Kurogi et al., 1976), causing economic losses in the livestock industry. The live, attenuated vaccine strain, TS-C2, was derived from the OBE-1 strain as a temperature sensitive mutant (Kurogi et al., 1979). Although vaccination has reduced the prevalence of the disease, antigenic and pathogenic variants of AKAV have been isolated (Lee et al., 2002; Ogawa et al., 2007a); for example, a variant Iriki strain was isolated from a calf with nonsuppurative encephalitis and neurological symptoms in Japan (Miyazato et al., 1989) and it shows low antigenic cross-reactivity with the reference strain in neutralization tests (Akashi and Inaba, 1997). Therefore, it is necessary to reconsider the vaccination strategy to effectively control the disease. Here we evaluated characters of a mutant virus with knockout of a nonstructural protein NSs, which acts as type I interferon antagonist and is involved in the regulation of host protein synthesis (Weber et al., 2002), by experimentally infecting pregnant goats. The pregnant goat model might be useful for AKAV studies, as suggested by previous reports of experimental transplacental infection of caprine fetuses (Kurogi et al., 1977). The recombinant TS-C2 (rTTT) and its mutant virus with knockout of NSs (rTTT∆ NSs) were generated by reverse genetics in our previous study (Takenaka-Uema et al., 2016). rTTT∆ NSs grew at a slower rate than rTTT in cell culture, although it reached a maximal yield at 48 hours post infection, which was equivalent to those of rTTT (Takenaka-Uema et al., 2016). Seven pregnant and five non-pregnant Shiba goats (9 months to 30 months) were obtained from the Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, University of Tokyo. After 3 days of acclimatization, two pregnant goats were inoculated with 3 mL (2.3–4.6× 107PFU) of wild-type (wt) Iriki strain, rTTT, or TTT∆ NSs intravenously via the cervical vein at 37 to 49 days gestation. After inoculation, body weights were measured, and rectal body temperatures and other general clinical variables were examined. Blood samples were collected post inoculation, and white blood cells (WBCs) were isolated. Fetuses were removed from the uteruses for autopsy at 3 weeks post inoculation (wpi). Since the design of this goat model is qualitative rather than quantitative and takes the principles of replacement, reduction, and refinement in animal experiments into account, the present study is limited by the number of animals that were used to assess the potentiality of a recombinant virus.Clinical signs were absent in all inoculated goats during the experimental period. Sera obtained at the time of virus inoculation and at 1-week intervals over the next 3 weeks were analyzed for the presence of virus-neutralizing (VN) antibodies (Ogawa et al., 2007a). Five of the six goats inoculated with virus had no antibodies against the virus at the time of inoculation, whereas one goat (# 3) had a detectable antibody titer (8) due to unknown reasons, and the highest antibody titer (181) elicited in this goat was at 2 wpi (Table 1), possibly because of immune boosting. All inoculated goats developed detectable VN antibodies at 1 wpi, and their antibody levels peaked at 2 or 3 wpi. For comparison …