2018年济南市14株柯萨奇病毒A组6型VP1基因特征分析

目的 分析济南市导致手足口病(hand, foot and mouth disease, HFMD)的柯萨奇病毒A组6型(Coxsackievirus A6, CVA6)分离株VP1基因特征及氨基酸变异。方法 2018年济南市HFMD常规监测病例样本经实时荧光RT-PCR检测,随机选择部分CVA6核酸阳性样本进行细胞分离鉴定,扩增阳性分离株VP1基因序列并进行同源性比较、氨基酸变异及系统进化分析。结果 2018年共检测652份样本,其中CVA6核酸阳性率为47.85%（312/652）,远高于人肠道病毒A组71型（human enterovirus A 71, EV71）（7.06%, 46/652）和柯萨奇病毒A组16型(Coxsackievirus A16, CVA16)（8.44%,55/652 ）,为济南市HFMD最主要病原体。通过细胞分离成功获得14株CVA6病毒,经VP1基因系统进化分析显示,济南市CVA6分离株均为基因D5亚型,VP1基因核苷酸及氨基酸序列同源性分别为94.2%~100%和98.0%~100%,与2017年广东株（GenBank 登录号MG385796、MG385815）、广西株（GenBank 登录号MH018512）、云南株（GenBank 登录号LC413143）及2014年江西株（GenBank 登录号KY913482）等亲缘关系较近。VP1蛋白主要存在V174I等位点变异,与国内流行的D5亚型基本一致。结论 2018年CVA6病毒为导致济南HFMD的主要病原,与广东、广西、云南和江西等省分离株亲缘关系较近,属于我国目前流行的基因D5亚型。     

武汉市儿童医院婴幼儿腹泻轮状病毒的VP4型别分析

目的研究武汉市儿童医院腹泻门诊A组轮状病毒VP4基因的分子流行病学特征. 方法利用聚丙烯酰胺凝胶电泳,将检测出的A组轮状病毒阳性样利用多重RT-PCR技术对VP4基因进行分型研究. 结果武汉地区793份腹泻患儿粪便样本经检测轮状病毒阳性257例,阳性率为32.4%.其中P [8]型232例(90.3%),P [4]型3例(1.2%),P [8]与P [4]混合感染15例(5.8%),尚有7例(2.7%)未能分出型别.对检测结果按采样时间、年龄和性别分布分别进行了分析. 结论武汉地区A组轮状病毒以P[8]型为主要流行基因型,患儿以6月至1岁为主,男女性别差异不大,武汉地区婴幼儿腹泻A组轮状病毒VP4基因分型研究将为轮状病毒疫苗的研制提供基础.     

Bovine Herpesvirus-1 Glycoprotein M Mediates the Translocation to the Golgi Apparatus and Packaging of VP8

VP8, the most abundant tegument protein of bovine herpesvirus-1 (BoHV-1), plays an important role in viral replication. According to our previous studies, VP8 localizes to the Golgi apparatus of BoHV-1-infected cells where it can be packaged into the virus; however, Golgi localization of VP8 does not occur outside of the context of infection. The goal of this study was to identify the viral factor(s) involved in the tropism of VP8 towards the Golgi. VP8 was found to interact with glycoprotein M (gM), and the VP8 and gM domains that are essential for this interaction were identified. VP8 and gM colocalized to the Golgi apparatus in BoHV-1-infected cells. In cells co-transfected with VP8- and gM-encoding plasmids, VP8 was also found to be localized to the Golgi, suggesting gM to be sufficient. The localization of VP8 to the Golgi was lost in cells infected with a gM deletion mutant, and the amount of VP8 incorporated into mature virus was significantly reduced. However, with the restoration of gM in a revertant virus, the localization to the Golgi and the amount of VP8 incorporated in the virions were restored. These results indicate that gM plays a critical role in VP8 subcellular localization to the Golgi and packaging into mature virions.     

Generation of a Soluble African Horse Sickness Virus VP7 Protein Capable of Forming Core-like Particles

A unique characteristic of the African horse sickness virus (AHSV) major core protein VP7 is that it is highly insoluble, and spontaneously forms crystalline particles in AHSV-infected cells and when expressed in vitro. The aggregation of AHSV VP7 into these crystals presents many problems in AHSV vaccine development, and it is unclear whether VP7 aggregation affects AHSV assembly or contributes to AHSV pathogenesis. Here, we set out to abolish VP7 self-assembly by targeting candidate amino acid regions on the surface of the VP7 trimer via site-directed mutagenesis. It was found that the substitution of seven amino acids resulted in the complete disruption of AHSV VP7 self-assembly, which abolished the formation of VP7 crystalline particles and converted VP7 to a fully soluble protein still capable of interacting with VP3 to form core-like particles. This work provides further insight into the formation of AHSV VP7 crystalline particles and the successful development of AHSV vaccines. It also paves the way for future research by drawing comparisons with similar viral phenomena observed in human virology.     

Development of a bivalent conjugate vaccine candidate against rotaviral diarrhea and tuberculosis using polysaccharide from Mycobacterium tuberculosis conjugated to ΔVP8* protein from rotavirus

Conjugation of carbohydrate antigens with a carrier protein is a clinically proven strategy to overcome the poor immunogenicity of bacterial polysaccharide. In addition to its primary role, which is to help generate a T cell-mediate long-lasting immune response directed against the carbohydrate antigen, the carrier protein in a glycoconjugate vaccine can also play an important role as a protective antigen. Among carrier proteins currently used in licensed conjugate vaccines, non-typeable Haemophilus influenzae protein D has been used as an antigenically active carrier protein. Our previous studies also indicate that some carrier proteins provide B cell epitopes, along with T cell helper epitopes.Herein we investigated the dual role of truncated rotavirus spike protein ΔVP8* as a carrier and a protective antigen. Capsular polysaccharide lipoarabinomannan (LAM), purified from Mycobacterium tuberculosis (M.tb), was chemically conjugated with ΔVP8*. Mouse immunization experiments showed that the resultant conjugates elicited strong and specific immune responses against the polysaccharide antigen, and the responses were comparable to those induced by Diphtheria toxoid (DT)-based conjugates. The conjugate vaccine induced enhanced antibody titers and functional antibodies against ΔVP8* when compared to immunization with the unconjugated ΔVP8*. Thus, these results indicate that ΔVP8* can be a relevant carrier protein for glycoconjugate vaccine and the glycoconjugates consisting of ΔVP8* with LAM are effective bivalent vaccine candidates against rotavirus and tuberculosis.     

Human enterovirus D68 in clinical and sewage samples in Israel

BackgroundSince mid-August 2014, North America experienced a wide outbreak of Enterovirus D68 (EV-D68) associated with severe respiratory illness in children. Several other countries also reported cases of EV-D68 in 2014.ObjectivesThe aim of this study was to determine whether EV-D68 circulated in Israel in 2014, caused severe respiratory illness in children and was the causative agent of Acute Flaccid Paralysis.Study designArchived clinical respiratory samples from a cohort of 710 hospitalized pediatric patient’s (<10 years old) with respiratory illness were screened for clade B specific EV-D68 by real-time PCR. The patients were seen at four medical centers covering the entire country between August and November 2014. We also evaluated 49 patient stool samples from 26 AFP cases during 2014 for presence of EV-D68. In addition, RNA from sewage samples collected throughout Israel during the same study period was also tested for EV-D68. Partial VP1 sequencing was performed on all positive samples.ResultsOf the 710 clinical samples evaluated, 7 (1%) were positive for EV-D68. Two patients were from the central part of Israel, while the rest was from the southern part. The majority of the patients did not have any underlying disease. Not only that, but, none of the 26 suspected AFP cases had EV-D68 nucleic acid in their stool samples. EV-D68 RNA was detected in 9 out of 93 sewage samples, mainly from Southern Israel. Sequence analysis of EV-D68 VP1 gene from both sewage and clinical samples indicated that the Israeli EV-D68 RNA belonged to Clade B which was genetically similar to 2014 circulating European and North American EV-D68 virus.ConclusionsEV-D68 circulated in Israel during the 2014 summer-fall season and caused hospitalization of a small percent of the patients with respiratory illness.     

Flagellin is a Th1 polarizing factor for human CD4+ T cells and induces protection in a murine neonatal vaccination model of rotavirus infection

Neonates have an increased susceptibility to infections, particularly those caused by intracellular pathogens, leading to high morbidity and mortality rates. This is partly because of a poor response of neonatal CD4⁺ T cells, leading to deficient antibody production and a low production of IFN-γ, resulting in deficient elimination of intracellular pathogens. The poor memory response of human neonates has underpinned the need for improving vaccine formulations. Molecular adjuvants that improve the response of neonatal lymphocytes, such as the ligands of toll-like receptors (TLRs), are attractive candidates. Among them, flagellin, the TLR5 ligand, is effective at very low doses; prior immunity to flagellin does not impair its adjuvant activity. Human CD4⁺ and CD8⁺ T cells express TLR5. We found that flagellin induces the expression of IFN-γ, IL-1β and IL-12 in mononuclear cells from human neonate and adult donors. When human naïve CD4⁺ T cells were activated in the presence of flagellin, there was high level of expression of IFN-γ in both neonates and adults. Furthermore, flagellin induced IFN-γ production in Th1 cells obtained from adult donors; in the Th2 population, it inhibited IL-4 cytokine production. Flagellin also promoted expression of the IFN-γ receptor in naive CD4⁺ T cells from neonates and adults. To test the adjuvant capacity of flagellin in vivo, we used a murine neonate vaccination model for infection with rotavirus, a pathogen responsible for severe diarrhea in young infants. Using the conserved VP6 antigen, we observed an 80% protection against rotavirus infection in the presence of flagellin, but only in those mice previously primed in the neonatal period. Our data suggest that flagellin could be an attractive adjuvant for achieving a Th1 response.