Preclinical Studies of Bunyaviruses

布尼亚病毒的临床前研究

• 批准号: 8156979
• 负责人: Kanta Subbarao
• 金额: $ 106.49万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8156979
• 关键词: Orthobunyavirus; preclinical study

La Crosse virus (LACV), family Bunyaviridae, is a mosquito-borne virus recognized as a major cause of pediatric encephalitis in North America with 70-130 symptomatic cases each year. The virus was first identified as a human pathogen in 1960. The majority of LACV infections are mild and never reported, however, serologic studies estimate infection rates of 10-30/100,000 in endemic areas. LAC encephalitis has become the most commonly reported pediatric arboviral encephalitis in the US with 70-130 symptomatic cases a year with severe sequelae. For these reasons we are interested in developing a vaccine to prevent this CNS disease. LACV sequence analysis: To better understand the genetic factors contributing to disease, the full genome sequence of the five known human isolates of LACV and of 13 mosquito isolates was analyzed, and a high level of genetic relatedness amongst the 18 viruses was observed. Two major LACV genetic groupings were identified that had allelic differences in the S, M and L genome segments. Group 1 viruses were underrepresented in mosquitoes (1 of 13 isolates) but predominant in humans (4 of 5 isolates), while Group 2 viruses were isolated predominantly from mosquitoes. The presence of Group 1 viruses in 80% of humans with severe central nervous system (CNS) disease suggested that Group 1 viruses possess a higher encephalitogenic potential than Group 2 viruses. To explore this hypothesis, viruses from both genetic groups were analyzed for their neurovirulent phenotype in mice, and a representative virus from each genetic group was analyzed in nonhuman primates. Whereas the viruses from both genetic groups exhibited somewhat similar neuroinvasiveness and neurovirulence in mice, the primate model demonstrated that Group 1 human virus possesses higher neurovirulence, as defined by greater levels of virus replication in the CNS and more severe virus-induced neuroinflammation. This study confirms the observation that Group 1 LACV is more likely to be associated with CNS disease. Therefore, the low incidence of encephalitis due to LACV infection in humans may be partially explained by the observed low frequency of neurovirulent Group 1 LACV among mosquitoes. Conversely, the greater prevalence of less pathogenic Group 2 LACV in mosquito populations may account for the asymptomatic or mild nature of the majority of LACV infections in humans. A detailed study of the neuropathogenesis of LACV for the CNS of non-human primates is ongoing. JCV sequence analysis: Jamestown Canyon virus (JCV), family Bunyaviridae, is a mosquito-borne pathogen endemic in the United States and Canada that can cause encephalitis in humans and is considered an emerging threat to public health. The virus is genetically similar to Inkoo virus circulating in Europe, suggesting that much of the northern hemisphere contains JCV or similar variants. We have completed the sequence of three isolates of JCV collected in geographically diverse locations over a 57 year time span. The nucleotide identify for the three JCV viruses is 90, 83, and 85% for the S, M, and L segments respectively whereas the percent identify for the predicted amino acid sequences of the N, NSS, M poly, GN, NSM, GC, and L proteins was 97, 91, 94, 98, 91, 94, and 97%, respectively. In Swiss Webster mice, each JCV isolate exhibits low neuroinvasiveness but high infectivity. Two of the three JCV isolates were highly neurovirulent after IC inoculation whereas one isolate, JCV/03/CT, exhibited low neurovirulence. In rhesus monkeys, JCV infection is accompanied by a low-titered viremia, lack of clinical disease, but a robust neutralizing antibody response. A relatively high level of amino acid sequence conservation was observed even for viruses isolated 57 years apart indicating that the virus is in relative evolutionary stasis. JCV is highly infectious for mice and monkeys, and these animals, especially mice, represent useful experimental hosts for further study. TAHV sequence analysis: Tahyna virus (TAHV) is a human pathogen of the California encephalitis virus (CEV) serogroup (Bunyaviridae) endemic to Europe, Asia, and Africa. TAHV maintains an enzootic life cycle with several species of mosquito vectors and hares, rabbits, hedgehogs, and rodents serving as small mammal amplifying hosts. Human TAHV infection occurs in summer and early fall with symptoms of fever, headache, malaise, conjunctivitis, pharyngitis, and nausea. TAHV disease can progress to CNS involvement, although unlike related La Crosse virus (LACV), fatalities have not been reported. Human infections are frequent with neutralizing antibodies present in 60-80% of the elderly population in endemic areas. In order to determine the genomic sequence of wild-type TAHV, we chose three geographically distinct TAHV isolates isolated over a 26-year period and are the first to determine the complete sequence of the TAHV S, M, and L segments. TAHV maintains a highly conserved genome with both nucleotide and amino acid sequence identity greater than 99%. In order to determine the extent of genetic relatedness to other members of the CEV serogroup, we compared protein sequences of TAHV with LACV, Snowshoe Hare virus (SSHV), Jamestown Canyon virus (JCV), and Inkoo virus (INKV). By amino acid comparison, TAHV was most similar to SSHV followed by LACV, JCV, and INKV. The sequence of the GN protein is most conserved followed by L, N, GC, NSS, and NSM. In a weanling Swiss Webster mouse model, all three TAHV isolates were uniformly neurovirulent, but only one virus was neuroinvasive. In rhesus monkeys, the virus was highly immunogenic even in the absence of viremia. Cross neutralization studies utilizing monkey immune serum demonstrated that TAHV is antigenically distinct from North American viruses LACV and JCV. However, we have also demonstrated significant cross-neutralization which may complicate virus identification since anti-JCV serum neutralized both LACV and TAHV. These data also suggest that a single vaccine could generate a cross-neutralizing antibody response which may provide protection against CEV serogroup viruses from a wide geographic range. Vaccine development: Given our experience with antigenic chimeric viruses of the various flaviviruses, we sought to explore the possibility that we could use an attenuated LACV as a backbone to create antigenic chimeric viruses in which the coding region of the M segment of LACV is replaced with that of a second bunyavirus. In our efforts to create live-attenuated viral vaccine candidates for this virus group, we have generated a recombinant La Crosse virus expressing the attachment glycoproteins of Jamestown Canyon virus. The JCV/LACV chimeric virus contains full-length S and L segments derived from LACV. In the M segment, the ORF of LACV is replaced with that derived from JCV and is flanked by the non-coding regions of LACV. Chimerization did not affect the growth of the chimeric virus in tissue culture, and the virus remains highly infectious and immunogenic in Swiss Webster mice. Although both LACV and JCV parental viruses are highly neurovirulent in 21 day-old mice, with LD50 values (log10 PFU) of 0.1 and 0.5 respectively, chimeric JCV/LACV is highly attenuated and does not cause disease even after intracerebral inoculation of 3 log10 PFU. Parenteral vaccination of mice with 3 log10 PFU of JCV/LACV protected against lethal challenge with LACV, JCV, and Tahyna virus (TAHV). The chimeric virus was infectious and immunogenic in rhesus monkeys and protected against the development of viremia after JCV challenge. The use of the LACV genetic background to deliver M segment protective antigens from pathogenic Bunyaviruses other than JCV could be an efficient method for rapid development of vaccines effective against pathogenic bunyaviruses.

Bunyaviridae家族的La Crosse病毒（LACV）是一种蚊子传播的病毒，被认为是北美小儿脑炎的主要原因，每年有70-130例有症状的病例。该病毒于1960年首次被鉴定为人类病原体。大多数LACV感染是轻度的，但从未报道，但是，血清学研究估计了流行地区10-30/100,000的感染率。 LAC脑炎已成为美国最常见的小儿弧病毒脑炎，每年有70-130例有症状的病例患有严重的后遗症。由于这些原因，我们有兴趣开发一种疫苗以防止这种CNS疾病。 LACV序列分析：为了更好地理解造成疾病的遗传因素，分析了五个已知的LACV人类分离株和13个蚊子分离株的完整基因组序列，并观察到了18个病毒之间的高遗传相关性。 确定了两个主要的LACV遗传分组，它们在S，M和L基因组段中存在等位基因差异。第1组病毒在蚊子中（13个分离株中的1个）中的人数不足，但主要在人类中（5种分离株中的4种），而主要从蚊子则分离出2组病毒。 80％的患有严重中枢神经系统（CNS）疾病的人类的1组病毒的存在表明，第1组病毒具有比2组病毒更高的脑生成潜力。为了探讨这一假设，分析了两个遗传组的病毒在小鼠中的神经毒素表型中进行了分析，并在非人类灵长类动物中分析了每个遗传组的代表性病毒。尽管两个遗传组的病毒在小鼠中均表现出某种相似的神经性侵袭性和神经毒性，但灵长类动物模型表明，第1组人类病毒具有较高的神经毒物，这是由CNS中的病毒复制水平较高的，以及更严重的病毒诱发的神经诱导的神经诱导的定义。这项研究证实了这样一种观察结果，即1组LACV更可能与中枢神经系统疾病有关。因此，由于观察到的蚊子在蚊子中观察到的低频1 LACV的低频，可能部分解释了由于人类的LACV感染引起的脑炎的低发生率。 相反，蚊子种群中病原2 LACV的较高患病率可能解释了人类大多数LACV感染的无症状或轻度性质。对非人类灵长类动物中枢神经系统的LACV神经发病的详细研究正在进行中。 JCV序列分析：Bunyaviridae家族的Jamestown Canyon病毒（JCV）是美国和加拿大蚊子传播的病原体，可以引起人类的脑炎，被认为是对公共卫生的新兴威胁。该病毒在遗传上与欧洲循环的Inkoo病毒相似，这表明北半球大部分都包含JCV或类似变体。我们已经完成了在57年的时间段内在地理上不同地点收集的三个分离株的序列。 S，M和L段的三种JCV病毒的核苷酸分别为90％，83％和85％，而百分比为N，NSS，M Poly，M Poly，GN，NSM，NSM，GC和L蛋白的预测氨基酸序列的确定百分比为97、94、94、94、94、98、91、94、91、94、94、97％和97％，以及 在瑞士韦伯斯特小鼠中，每种JCV分离株表现出低的神经性侵袭性，但感染率很高。 IC直接进行后，三个JCV分离株中的两个是高度神经性肺部的，而一个分离株JCV/03/CT表现出低神经动脉。 在恒河猴中，JCV感染伴随着低静脉病毒血症，缺乏临床疾病，但具有强大的中和抗体反应。即使是分离出57年的病毒，也观察到相对较高的氨基酸序列保守，表明该病毒处于相对进化的停滞状态。 JCV对小鼠和猴子具有高度感染性，这些动物，尤其是小鼠代表了有用的实验宿主进行进一步研究。 TAHV序列分析：Tahyna病毒（TAHV）是加利福尼亚脑炎病毒（CEV）血清群（Bunyaviridae）的人类病原体，是欧洲，亚洲和非洲的特有。 TAHV保持了一个enzootic生命周期，其中有几种蚊子和野兔，兔子，刺猬和啮齿动物，它们是小型哺乳动物放大宿主。人类TAHV感染发生在夏季和初秋，出现发烧，头痛，不适，结膜炎，咽炎和恶心的症状。尽管与相关的LA Crosse病毒（LACV）不同，但尚未报道TAHV疾病，但尚未报道与中枢神经系统的参与。 人类感染经常发生，中和抗体中有60-80％的中和人口中的抗体。为了确定野生型TAHV的基因组序列，我们选择了在26年内分离出的三个地理上不同的TAHV分离株，并且是第一个确定TAHV S，M和L段的完整序列的序列。 TAHV保持高度保守的基因组，核苷酸和氨基酸序列同一性大于99％。 为了确定与CEV血清群的其他成员的遗传相关性的程度，我们将TAHV的蛋白质序列与LACV，雪鞋野兔病毒（SSHV），Jamestown Canyon Canyon病毒（JCV）和Inkoo Virus（Inkoo病毒）进行了比较。 通过氨基酸比较，TAHV与SSHV最相似，其次是LACV，JCV和INKV。 GN蛋白的序列最保守，其次是L，N，GC，NSS和NSM。 在断奶的瑞士韦伯斯特小鼠模型中，所有三个TAHV分离株都是均匀的神经性肺部，但只有一种病毒是神经侵染的。 在恒河猴中，即使没有病毒血症，该病毒也具有高度免疫原性。利用猴子免疫血清的交叉中和研究表明，TAHV在抗原上与北美病毒LACV和JCV不同。但是，我们还显示出明显的跨性和中和化，这可能使病毒鉴定复杂化，因为抗JCV血清中和lacV和TAHV。 这些数据还表明，单个疫苗可以产生跨中和抗体反应，该抗体反应可以提供针对广泛地理范围的CEV血清群病毒的保护。 疫苗开发：鉴于我们在各种黄病毒的抗原嵌合病毒方面的经验，我们试图探索我们可以将衰减的LACV用作产生抗原嵌合病毒的骨架的可能性，在该骨架中，LACV的M段的编码区域被第二个Bunyyavirus代替。为了为该病毒组创建实时衰减的病毒疫苗候选病毒疫苗，我们产生了一种重组的La Crosse病毒，表达了Jamestown Canyon病毒的附着糖蛋白。 JCV/LACV嵌合病毒包含来自LACV的全长S和L段。 在M段中，LACV的ORF被源自JCV衍生的ORF替换为LACV的非编码区域。 嵌合不影响组织培养中嵌合病毒的生长，并且该病毒在瑞士韦伯斯特小鼠中仍然具有高度感染和免疫原性。 尽管LACV和JCV亲本病毒在21天大的小鼠中均高度神经性肺癌，LD50值（Log10 PFU）分别为0.1和0.5，但嵌合JCV/LACV被高度减弱，即使在脑内接种3 Log10 PFU后也不会引起疾病。 肠胃外疫苗接种3 log10 PFU的JCV/LACV的小鼠，可抵抗LACV，JCV和Tahyna病毒的致命挑战（TAHV）。 嵌合病毒在恒河猴中具有感染性和免疫原性，并在JCV挑战后免受病毒血症的发展。使用LACV遗传背景从JCV以外的致病性Bunyavirus中输送M段保护抗原可能是快速开发有效针对致病性Bunyaviruses的疫苗的有效方法。