流行性腮腺炎病毒的分子流行病学研究

流行性腮腺炎(腮腺炎)是由腮腺炎病毒(MuV)引起的急性呼吸道传染病。截止2004年,MuV已发现了12个基因型,分别命名为A~L基因型。不同基因型MuV的分布具有地域性。研究表明,在同一地域的不同时期可能有不同基因型MuV在流行,在一个国家或地区也可能同时有不同基因型的MuV流行。MuV仅一个血清型,不同的MuV基因型之间有抗原交叉性,这种交叉性是至关重要的。可保护接种疫苗后的人群免受不同基因型MuV的感染。但有些体内和体外实验表明,基因型之间的抗原交叉性是有限的。流行病学数据还表明,某些毒株和基因型或基因型内某一组病毒具有神经毒性。近年来,调查了不同MuV的神经毒性,发现C、D、G、HI、、J基因型具有明确的神经毒性。但目前引起神经毒性的遗传学基础还不清楚。虽然,目前还无流行病学证据证明在不同的基因型或毒株之间出现抗原交叉性降低现象。但有数据表明,连续的进化和基因型的再分布在理论上可能导致神经毒力增强或交叉中和能力降低的毒株出现。因此,对MuV进行分子流行病学研究,了解基因型的分布,监测人群对基因型的特异性免疫是非常必要的。     

The molecular epidemiology of mumps virus.

The molecular epidemiology of MuV is characterized by the co-existence of 10 (or more) distinct genotypes named A-J based on the nucleotide sequence of the SH gene. MuV show distinct geographic clustering. More than one genotype may circulate simultaneously in a geographic region. Limited data suggest redistribution of genotypes to occur over time. The selective forces remain speculative. Currently used vaccine strains belong to different genotypes. Some MuV genotypes (C, D, H, J) and vaccine strains (Urabe Am9) have been associated with enhanced neurovirulence. Also, reduced cross-neutralization capacity has been observed between genotype A and genotypes C and D. At present vaccine failure cannot be attributed to this phenomenon. Strain redistribution may lead to the emergence of new MuV strains with enhanced neurovirulence or reduced cross-neutralization capacity with current vaccine strains. Close monitoring of the genotype distribution of MuV and genotype-specific population immunity is needed in the vaccine era.     

不同固定液对大鼠脾石蜡切片HE染色标本的影响

目的:比较经不同固定液处理的大鼠脾石蜡切片HE染色效果,优化制作大鼠脾石蜡切片HE染色标本的最佳方法。方法:用3种不同的固定液处理大鼠脾脏组织,经上行梯度乙醇脱水、二甲苯透明、石蜡包埋、切片、HE染色,显微镜下观察,比较其染色效果,选择最佳的大鼠脾石蜡切片标本的制作方法。结果:不同的固定液处理的大鼠脾石蜡切片HE染色效果各有不同,经Helly固定液处理后的标本的染色效果最佳。结论:Helly固定液是制作大鼠脾石蜡切片HE染色标本的理想固定液。     

谷氨酸钠对胎鼠脑发育影响的实验研究

味精的主要成份谷氨酸钠，经代谢产生的谷氨酸是一典型的兴奋性氨基酸，其神经毒性作用已受到广泛关注。本实验采用健康昆明种小白鼠，从妊娠后的第４天开始腹膜腔注射谷氨酸钠（剂量分别为６０、３０、１０ｍｇ／ｋｇ，隔日一次）。于妊娠后的第１８天检查胎鼠，取脑Ｃａｒｎｏｒｙ＇ｓ液固定，常规石蜡切片，ＨＥ、Ｆｅｕｌｇｅｎ和Ｎｉｓｌ染色。进行形态观察，图像分析仪随机测定大脑皮质１００个神经元的Ｆｅｕｌｇｅｎ与Ｎｉｓｌ染色的灰度值，作定量分析。结果显示：各实验组与对照组均未见形态异常。实验组各项参数与对照组比较Ｐ＞０．０５。本实验结果提示：谷氨酸钠对胎鼠脑发育无不良影响。     

新型超声组织处理仪在病理快速石蜡制片技术中的应用

目的探讨新型的JYCL-2超声组织处理仪快速制作石腊切片的效果。方法应用该仪器的超声波技术结合水浴加温,加快固定组织脱水、透明和浸蜡的速度。结果用本法能快速制作石蜡切片,用这些切片进行的常规HE、组织化学和免疫组化染色,质量优良,组织细胞形态完好,与常规技术制作的切片质量无明显差别。结论使用JYCL-2超声组织处理仪能快速有效完成组织固定、脱水、透明、浸蜡和包埋过程,制作的切片能满足临床病理诊断的需要。该仪器也适用于尚无冰冻切片机的病理科室开展术中快速活体组织病理学检查。     

促红细胞生成素对窒息新生大鼠脑组织Omi/HtrA2表达水平及细胞凋亡的影响

目的 探讨促红细胞生成素(erythropoietin,EPO)对常压窒息新生大鼠脑组织内Omi/HtrA2表达水平及细胞凋亡的影响。 方法 7日龄新生SD大鼠随机分为对照组、窒息组和EPO干预组(每组n=25)。对照组模拟窒息过程但不缺氧,窒息组和EPO干预组制备新生大鼠常压窒息模型,EPO干预组窒息模型制备后即刻腹腔注射重组人促红细胞生成素5 000 U/kg,对照组和窒息组均注射同体积生理盐水。各组于造模后不同时间点(6、12、24、48、72 h)取脑组织行石蜡切片,HE染色观察脑组织病理改变,TUNEL法检测神经细胞凋亡,免疫组化法检测Omi/HtrA2表达。 结果 窒息组脑组织Omi/HtrA2的表达和凋亡细胞数在各时间点均高于对照组(P均<0.05),而EPO干预组同窒息组比较,各时间点凋亡细胞数和Omi/HtrA2表达均显著下降,其差异均有统计学意义(P均<0.05),但均未恢复至对照组水平(P<0.05)。 结论 EPO可能通过抑制脑组织Omi/HtrA2的表达而减少神经细胞的凋亡,从而对窒息脑损伤起到保护作用。     

探讨HE染色在病理诊断中的应用效果

目的分析研究HE染色在病理诊断中的应用效果。方法选取本院360例病理组织石蜡切片开展本次研究,标本纳入时间范围为2018年2月-2019年1月,通过数字表法对其进行平均分组,分别为对照组和研究组,各180例。予以对照组HE染色,结合对照组显露的不足对染色技术进行相应改进后再对研究组实施HE染色,比较两组染色诊断检出率。结果在胃肠肝胆、脂肪组织、子宫内膜以及骨制片的诊断检出率比较上,研究组相较于对照组明显更高(P<0.05)。结论在病理诊断中应用病理技术HE染色可为疾病治疗提供可靠的参考,具有较高的临床应用价值,实际操作中需结合切片特点的差异应用差别化的染色技术,最大限度的保证诊断准确性。     

愤怒对D-gal致脑老化大鼠学习记忆功能的影响

目的观察愤怒情志对D-gal诱导的脑老化模型大鼠学习记忆障碍的影响。方法采用夹尾间接激怒法引入愤怒刺激。Morris水迷宫法检测大鼠学习记忆能力。采用亚硝酸盐法、硫代巴比妥酸比色法、荧光法检测脑组织内SOD、MDA、LPF活性。以HE染色进行脑组织形态学观察。结果愤怒D-gal组大鼠在水迷宫中游泳持续时间明显延长;脑组织内SOD活力明显降低,MDA、LPF含量均明显升高;大脑皮质变薄,其中以颗粒层、锥体细胞层萎缩明显,神经细胞数量减少、体积变小,核浆界限不清,细胞周围可见空泡形成;大鼠海马CA1区神经细胞排列较为松散,细胞结构模糊,胞体肿胀,部分细胞核固缩、深染。结论愤怒情志对D-半乳糖诱导的脑老化大鼠的学习记忆功能减退具有促进作用,可能与其清除氧自由基的能力减退,抗氧化能力下降引起脑组织损伤有关。     

缺氧缺血新生大鼠脑组织超氧化物歧化酶、丙二醛的变化及高压氧的保护作用

【目的】 探讨高压氧对宫内缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)新生大鼠脑组织中超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malonyldiade-hyde,MDA)的影响及病理影响。 【方法】 按Bjelke法制作HIBD新生大鼠动物模型,随机分为HIBD组、HIBD+HBO组、对照鼠+HBO组和对照组4组,每组30只。HBO组大鼠于术后24 h开始给予2ATA的高压氧治疗,1 h/d,持续14 d。于出生后12 h、第15天各组大鼠随机取10只,分别处死取脑,制作脑组织匀浆,检测脑组织中SOD、MDA水平,并于第15天作HE和尼氏染色观察大鼠脑组织病理学变化。 【结果】 经HBO治疗后,HIBD+HBO组大鼠脑组织SOD升高[(90.43±10.83) U/mgprot]、MDA降低[(16.76±3.80) nmol/mgprot]、海马存活神经元数目增多[(122±10.52)个/0.2 mm²],与HIBD组[(74.18±8.52)U/mgprot、(28.05±4.28)nmol/mgprot、(92±6.51)个/0.2 mm²]比较差异均有统计学意义(P<0.05)。 【结论】 高压氧可通过拮抗氧自由基、减轻脑水肿和减少神经细胞死亡,发挥对HIBD新生鼠脑组织的保护作用。     

新生鼠缺血缺氧性脑病中神经干细胞的形态学观察

【目的】建立新生鼠缺血缺氧性脑病动物模型，探讨HIE病程中神经干细胞（neural stem cells，NSCs）的形态学改变。【方法】210只新生7d SD乳鼠随机分为正常对照组、单纯缺氧组及缺血缺氧组。每组70只。每组又根据处死时间点随机分成3h，6h，1d，3d，7d，14d，21d等7个小组。每小组10只。缺血缺氧组结扎新生7d SD大鼠左颈总动脉，置于8％氧浓度的低氧环境中2．5h。单纯缺氧组缺氧2．5h。采用HE染色、免疫组织化学染色以及光镜技术分别对3组SD大鼠脑组织中的NSCs形态学进行检测。【结果】缺血缺氧组缺血缺氧后3h出现轻度脑损伤，1d病变最严重．3d、7d胶质细胞增生．14d、21d出现脑萎缩。3组SD鼠在7个时间点脑组织均存在NSCs，且细胞呈单一的圆形，突起不超过1个．阳性NSCs呈明显的区域性分布．神经球集落样存在的NSCs较多，单纯缺氧组在每一时间点NSCs的表达明显高于缺血缺氧组及正常对照组。在6h时间点，处于增殖状态的NSCs增多，尤其是室下区。1d和3d时间点，坏死脑组织中仍可见NSCs及其神经球。3d时间点后病侧脑组织的NSCs逐渐下降。【结论】成功建立了缺血缺氧性脑病动物模型；HIE发病中早期NSCs增殖；NSCs随着病情的演变开始减少；低氧有利于NSCs的增殖；早期采用NSCs干预治疗有希望成为临床治疗HIE的重要途径。     

Combined peptides of human enterovirus 71 protect against virus infection in mice

Human enterovirus 71 (EV71) is a cause of hand, foot and mouth disease (HMFD) in children under 6 years old, and could cause serious neurological complications in some patients. Numerous large outbreaks of EV71 caused HMFD have occurred recently in Asia, especially in China. The cross-reactivity of EV71 with human brain tissue was observed and the cross-reactivity inducing regions were identified in previously study, which suggested that there were two regions in structural proteins of virus should be avoided in the vaccine. Six peptides without cross-reactivity were selected and combined into three vaccine candidates and applied in further evaluation in neonatal mice. The Vac6 comprising the peptides of P_70–159, P_140–249, P_324–443 and P_746–876 of the structural proteins could provide effective protection on pups against virus infection, as shown in viral copies detection and histopathology examination. Immunohistochemical staining results indicated that Vac6 had no cross-reactivity with human brain tissues. Our results suggested that Vac6 could have potential clinical value against EV71 epidemics caused mainly by C4 strains in the mainland of China.     

布拉氏酵母菌联合早期干预对高胆红素血症仔鼠脑组织NF-kB表达和学习记忆功能的影响

目的 分析布拉氏酵母菌联合早期干预对高胆红素血症仔鼠脑组织NF-kB表达和学习记忆功能的影响。 方法 将足月7日龄SD大鼠88只随机分为对照组(NS组)和试验组(T1、T2、T3组),每组22只。试验组于7日龄和10日龄分别腹腔注射胆红素50 μg/g,对照组注射等量生理盐水。于末次注射12 h后,各组随机选取6只,测定血清胆红素和脑组织胆红素含量。剩余仔鼠,T1组单纯给予布拉氏酵母菌,T2组给予布拉氏酵母菌和早期干预,T3组不干预,至28日龄。HE染色和免疫组化观察NF-kB表达情况,Morris水迷宫评测仔鼠逃避潜伏时间和穿过目标象限次数。 结果 10日龄NS组血清胆红素和脑组织胆红素浓度显著低于试验组(P<0.01),试验组间差异无统计学意义(P>0.05);28日龄NS组显著低于T1、T2、T3组(P<0.05),T2组显著低于T1、T3组(P<0.05)。HE染色:NS组神经元结构完整,T1、T2、T3组均观察到神经元数量减少,不同程度的胆红素沉积。免疫组化:NS组NF-kB阳性细胞数明显少于T1、T2、T3组(P<0.01),T2组明显少于T1组及T3组 (P<0.05) 。NS组逃避潜伏时间和穿过目标象限次数显著优于T1、T2、T3组(P<0.05),T2组显著优于T1组及T3组(P<0.05)。 结论 布拉氏酵母菌联合早期干预可有效地减少高胆红素血症仔鼠脑组织NF-kB表达,改善学习记忆功能。     

Cross-Neutralization between Human and African Bat Mumps Viruses

Recently, a new paramyxovirus closely related to human mumps virus (MuV) was detected in bats. We generated recombinant MuVs carrying either or both of the fusion and hemagglutinin-neuraminidase bat virus glycoproteins. These viruses showed replication kinetics similar to human MuV in cultured cells and were neutralized efficiently by serum from healthy humans.     

Investigation of mumps vaccine failures in Minsk, Belarus, 2001-2003

The purpose of this study was to investigate mumps vaccine failures (VF) in a highly vaccinated population of Minsk, Belarus, and to investigate a possible role for virus strain-specific immunity. During our 3-year study period, 22 adults were admitted to the Infectious Diseases Hospital in Minsk with a diagnosis of mumps. A genotype H1 mumps virus (MuV) strain was identified in all patients. Of 15 patients from whom the paired sera were collected, 9 were confirmed to have been previously vaccinated. Serological examinations indicated primary VF in seven of these cases and secondary VF in two. Despite almost all vaccinated patients possessing MuV specific IgG, few possessed neutralizing antibody to the vaccine strain and titers were nominal. Importantly, none of the sera were able to neutralize a genotype H MuV strain. Our results demonstrate the importance of assaying for neutralizing antibody and support the assertion that antigenic differences between wild type and vaccine MuV strains may play a role in cases of breakthrough infection in vaccinees.     

Immunogenicity of mumps virus vaccine candidates matching circulating genotypes in the United States and China

Mumps virus (MuV) causes acute infection in humans with characteristic swelling of the parotid gland. While vaccination has greatly reduced the incidence of MuV infection, there have been multiple large outbreaks of mumps virus (MuV) in highly vaccinated populations. The most common vaccine strain, Jeryl Lynn, belongs to genotype A, which is no longer a circulating genotype. We have developed two vaccine candidates that match the circulating genotypes in the United States (genotype G) and China (genotype F). We found that there was a significant decrease in the ability of the Jeryl Lynn vaccine to produce neutralizing antibody responses to non-matched viruses, when compared to either of our vaccine candidates. Our data suggests that an updated vaccine may allow for better immunity against the circulating MuV genotypes G and F.     

Complete genome sequence of mumps viruses isolated from patients with parotitis,pancreatitis and encephalitis in India

Limited information is available regarding epidemiology of mumps in India. Mumps vaccine is not included in the Universal Immunization Program of India. The complete genome sequences of Indian mumps virus (MuV) isolates are not available, hence this study was performed. Five isolates from bilateral parotitis and pancreatitis patients from Maharashtra, a MuV isolate from unilateral parotitis patient from Tamil Nadu, and a MuV isolate from encephalitis patient from Uttar Pradesh were genotyped by the standard protocol of the World Health Organization and subsequently complete genomes were sequenced. Indian MuV genomes were compared with published MuV genomes, including reference genotypes and eight vaccine strains for the genetic differences. The SH gene analysis revealed that five MuV isolates belonged to genotype C and two belonged to genotype G strains. The percent nucleotide divergence (PND) was 1.1% amongst five MuV genotype C strains and 2.2% amongst two MuV genotype G strains. A comparison with widely used mumps Jeryl Lynn vaccine strain revealed that Indian mumps isolates had 54, 54, 53, 49, 49, 38, and 49 amino acid substitutions in Chennai-2012, Kushinagar-2013, Pune-2008, Osmanabad-2012a, Osmanabad-2012b, Pune-1986 and Pune-2012, respectively. This study reports the complete genome sequences of Indian MuV strains obtained in years 1986, 2008, 2012 and 2013 that may be useful for further studies in India and globally.     

新生大鼠高胆红素血症模型脑组织中NLRP3小体活化与神经损害的相关性研究

目的 研究新生大鼠高胆红素血症模型脑组织中NOD样受体蛋白3(NLRP3)小体活化与神经损害的相关性。方法 选择3日龄的SPF级新生SD大鼠36只,随机分为对照组、模型组和NLRP3小体抑制剂VX-765组(VX组)各12只,模型组和VX组通过腹腔注射100 μg/g胆红素的方式建立新生大鼠高胆红素血症模型,VX组同时给予NLRP3小体抑制剂VX-765腹腔注射。比较3组间血清及脑组织中胆红素含量、脑组织含水量及ATP含量、Nissl染色及TUNEL染色、血清NSE及S100B含量、NLRP3炎性小体表达的差异。结果 模型组大鼠脑组织中总胆红素含量、含水量、TUNEL阳性染色染色细胞率、NLRP3、ASC、cleaved-caspase-1、active-IL-1β、active-IL-18的表达水平及血清中总胆红素、NSE、S100含量明显高于对照组,脑组织中ATP含量及Nissl阳性染色细胞数目明显低于对照组(P<0.05);VX组大鼠脑组织中含水量、TUNEL阳性染色细胞率、cleaved-caspase-1、active-IL-1β、active-IL-18的表达水平及血清中NSE、S100含量明显低于模型组(P<0.05),脑组织中ATP含量及Nissl阳性细胞数目显著高于对照组(P<0.05)。结论 新生大鼠高胆红素血症模型中NLRP3小体活化参与了神经损伤过程。     

Generation of a recombinant Oka varicella vaccine expressing mumps virus hemagglutinin-neuraminidase protein as a polyvalent live vaccine

We constructed a recombinant varicella-zoster virus (VZV) Oka vaccine strain (vOka) that contained the mumps virus (MuV) hemagglutinin-neuraminidase (HN) gene, inserted into the site of the ORF 13 gene by using the bacterial artificial chromosome (BAC) system in Escherichia coli. Insertion of the HN gene into the VZV genome was confirmed by PCR and Southern blot. The infectious virus reconstituted from the vOka-HN genome (rvOka-HN) had a growth curve similar to the original recombinant vOka without the HN gene. The mumps virus HN protein expressed in rvOka-HN infected cells was expressed diffusely in the cytoplasm, and modification of the protein was similar to that seen in MuV-infected cells. Electron microscopic examination of infected cells revealed that HN was expressed on the plasma membrane of the cells but not in the viral envelope, suggesting that the tropism of rvOka-HN would be unchanged from that of the original vOka strain. Immunization of guinea pigs with rvOka-HN-induced VZV- and HN-specific antibodies. Interestingly, the induced antibodies had a strong neutralizing activity against virus-cell infections of both MuV and VZV. Therefore, the novel varicella vaccine expressing MuV HN protein is suitable as a polyvalent live attenuated vaccine against VZV and MuV infections.     

Genetic characterization of historical epidemic mumps viruses in northern Spain, 1987–1990

The mumps virus (MuV) is genetically diverse and is divided into 12 genotypes. The World Health Organization has recommended expanding virological surveillance for MuV, and therefore molecular characterization of circulating strains (i.e. genotypes) is increasingly performed. Nevertheless, little is known about the genotypes circulating before the massive vaccination of children and adolescents. The present study analyzed the strains causing the 1988–1989 mumps epidemic in the Basque Country, northern Spain, which occurred in the early vaccination period, before the endemic circulation of mumps virus was blocked. The epidemic reached an annual incidence rate of more than 400 cases/100,000 inhabitants, and caused a large number of cases of mumps meningitis. MuV RNA was amplified from the cerebrospinal fluid of 15 infected patients during the epidemic and from three more patients affected shortly before or after this epidemic (1987, early 1988 and 1990). Genotyping of the complete small hydrophobic gene (316 nucleotides), amplified in the 18 strains, as well as of the entire hemagglutinin-neuraminidase gene (1749 nucleotides), amplified in four strains, assigned all strains to genotype K, a genotype infrequently detected at present. Although the putative HN protein sequence differed by 4.8–5.5% in relation to Jeryl Lynn 5 strain (the main strain used in the vaccination program in this region), the vaccine was effective, and dramatically reduced the incidence of mumps over the following years. The presence of genotype K strains in Spain in the 1980s, together with their contemporary detection in Scandinavia, suggests that this genotype could have caused the Spanish epidemic and was also circulating widely in Europe at that time.     

培养细胞的石蜡切片用于DcR3免疫化学染色的探讨

[目的]建立一种培养细胞进行石蜡切片用于免疫细胞化学染色的方法。[方法]分别采用细胞爬片和石蜡切片方法处理细胞，比较HE及免疫化学染色效果。[结果]石蜡切片细胞分布均匀，阳性反应明显，定位明确，背景清晰。[结论]细胞石蜡切片的染色效果明显优于细胞爬片，值得推广。