Study of Human Bocavirus Infection in Human Airway Epithelia

描述（由申请人提供）：人类bocavavirus 1 (HBoV1)（属，bocavavirus；科，细小病毒科）是一种呼吸道病毒，可引起幼儿，特别是两岁以下婴儿急性呼吸道感染并伴有喘息。它通常与其他呼吸道病毒相关（检出率为12-16%），在因急性喘息而住院治疗的两岁以下婴儿中，它是继呼吸道合胞病毒、腺病毒和鼻病毒之后的第四大常见呼吸道病毒。急性HBoV1感染会导致呼吸道疾病，对儿科患者可能会危及生命。目前，对于HBoV1感染，特别是对患有严重喘息的婴儿，尚无有效的治疗和预防策略。HBoV1具有自主细小病毒的特征，单链DNA基因组约为5.5 kb。它有效地感染极化原发性人气道上皮（HAE）。迄今为止，我们在hbo1感染的研究方面取得了很大进展。我们建立了HBoV1反向遗传系统，通过将HBoV1全长复制型基因组转染到人胚胎肾（HEK） 293细胞中产生HBoV1病毒粒子。这些HBoV1病毒粒子感染极化原发性HAE培养物可导致明显的细胞病变作用，表现为上皮完整性的破坏。我们还证明，当将HBoV1病毒粒子应用于根尖表面时，其后代可以感染极化的原发性HAE培养物，感染的倍数低至0.001个病毒基因组拷贝/细胞。然而，hbov1导致上皮完整性丧失的机制尚未得到研究。此外，HBoV1与其他呼吸道病毒共感染时的协同效应尚不清楚。我们的长期目标是使用极化HAE培养和人类支气管异种移植物（与HBoV1感染生理相关）来确定HBoV1复制和由此导致的上皮完整性破坏的关键分子机制。在过去的几年中，我们已经积累了大量关于宿主细胞如何对细小病毒感染作出反应以及病毒如何诱导细胞死亡的知识。对于本文提出的研究，我们将与爱荷华大学基因治疗中心合作，使用极化原发性HAE培养和人类支气管异种移植物：i)阐明HBoV1破坏气道上皮完整性的机制，揭示感染气道上皮的相关形态学和超微结构变化，确定HBoV1感染气道上皮的细胞类型以及在HBoV1感染过程中介导上皮损伤的病毒蛋白；ii)确定HBoV1在极化HAE培养物和合并其他呼吸道病毒感染的人支气管异种移植物中的协同作用。

DESCRIPTION (provided by applicant): Human bocavirus 1 (HBoV1) (genus, Bocavirus; family, Parvoviridae) is a respiratory virus that causes acute respiratory tract infections with wheezing in young children, particularly in infants under two years old. It is commonly associated with other respiratory viruses (with a detection rate of 12-16%) and is the fourth most common respiratory virus after respiratory syncytial virus, adenovirus and rhinovirus in infants under two years old who are hospitalized for the treatment of acute wheezing. Acute HBoV1 infection causes respiratory illness, which can be life-threatening in pediatric patients. At present, there are no effective treatment and prevention strategies for HBoV1 infection, especially in infants with a severe wheeze. HBoV1 has characteristics of an autonomous parvovirus, with a single-stranded DNA genome of approximately 5.5-kb. It productively infects polarized primary human airway epithelium (HAE). To date, we have made much progress in our studies of HBoV1 infection. We have established an HBoV1 reverse genetics system in which HBoV1 virions are produced by transfecting the full-length HBoV1 replicative-form genome into human embryonic kidney (HEK) 293 cells. Infection of polarized primary HAE cultures with these HBoV1 virions results in clear cytopathogenic effects, manifested by the disruption of epithelial integrity. We also demonstrated that progeny HBoV1 virions can infect polarized primary HAE cultures at a multiplicity of infection as low as 0.001 viral genome copies/cell when applied to the apical surface. However, the mechanisms underlying the HBoV1-caused loss of epithelial integrity have not been studied. In addition, the synergistic effects of HBoV1 when co-infected with other respiratory viruses are unknown. Our long-term goal is to use polarized HAE cultures and human bronchial xenografts (physiologically relevant to HBoV1 infection) to identify the key molecular mechanisms underlying HBoV1 replication and the resulting destruction of epithelial integrity. Over the past few years, we have accumulated a great deal of knowledge regarding how host cells respond to parvovirus infection and how the virus induces cell death. For the study proposed herein, we will collaborate with the Center for Gene Therapy at the University of Iowa and use polarized primary HAE cultures and human bronchial xenografts to: i) elucidate the mechanisms by which HBoV1 destroys airway epithelial integrity, reveal the associated morphological and ultrastructural changes of the infected airway epithelium, and identify which cell types of airway epithelium are infected by HBoV1 and which viral proteins mediate the epithelial damage during HBoV1 infection; and ii) determine the synergetic effects of HBoV1 in both polarized HAE cultures and human bronchial xenografts co-infected with other respiratory viruses.