小儿副轮状病毒的检出

用聚丙烯酰胺凝胶电泳法,从婴幼儿急性腹泻粪便中检出轮状病毒RNA电泳阳性293份,发现一株副轮状病毒(青-27株),此株病毒经电泳观察,呈典型的轮状病毒形态,但易破碎。ELISA检测表明不具有一般轮状病毒的特异性群抗原,病毒RNA基因组由11个片段组成,但电泳图型特殊,吴4:3:2:2排列模式,本文证实,此一小儿副轮状病毒与国外报道的,散发罕见的小儿副轮状病毒RNA电泳图型相同,提示此病毒的重要意义。     

哈尔滨地区婴幼儿腹泻轮状病毒优势株的变化

本文对1988—1989年哈尔滨地区婴幼儿腹泻进行了轮状病毒核酸电泳型调查,并于1984—1985年资料比较,以探明这一地区轮状病毒流行优势株的变化。 标本:婴幼儿腹泻粪便标本采自哈尔滨医科大学附属二院儿科及哈尔滨红十字儿童医院。病毒RNA提取:参考Herring方法。聚丙烯酰胺电泳(PAGE):参考Uaemmli方法。 结果:123份急性婴幼儿腹泻粪便标本PAGE检测,47例呈现轮状病毒RNA图型,阳性率为38.2％。电泳图型的基本模式为4:2:3:2,表明均为A组轮状病毒。根据各片段的迁移位置差异,共可见9种不同的电泳型,其中短型3例,占6.4％,含2个电泳型;长型44例,     

轮状病毒生物学性状的研究

对最常应用的3个轮状病毒(RV)标准株(人的Wa株,牛的NCDV株,猴的SA_(11)株)进行了形态学、培养特性及理化性质的比较观察与检测。它们在MA_(104)细胞上的培养特性基本相同,在电镜下呈现RV特有的形态结构,RNA电泳图具有典型的电泳带分布模式4、2、3、2规律,各项理化性质亦与文献报道一致。表明RV的细胞适应株的生物学性状有相当高的稳定性。     

沙市婴幼儿腹泻中检出3株C群轮状病毒

1987～1988年在沙市165份婴幼儿急性腹泻标本中,用PAGE法检出轮状病毒37株(22.4％),其中3株为少见的轮状病毒,此种病毒经电镜观察,具有典型轮状病毒的形态结构,ELISA证实该病毒不具有A群和B群轮状病毒的群特异性抗原。RNA电泳分析表明,其基因组由11个双链RNA片段组成,电泳图型特殊,呈4:3:2:2的排列模式。上述试验表明,该病毒为世界上罕见的C群轮状病毒。免疫电镜证实,该病毒能被病人恢复期血清所凝集,提示该病毒是腹泻病儿的致病因子。     

人轮状病毒的细胞培养分离

用CV-1细胞从急性腹泻病儿7份粪便标本中直接分离出4株人轮状病毒(HRV),并适应传代14代。感染细胞出现特征性细胞病变,经电镜、ELISA、免疫荧光染色及病毒RNA电泳等试验证实HRV毒株在CV-1细胞中的繁殖及抗原特异性。病毒滴度为10~(6.0)TCID_(60)。分离的4株HRV毒株均为RNA电泳型长型,经CV-1细胞传7～14代后,RNA图型与原粪样相比未见变异。     

轮状病毒新敏感细胞的筛选

轮状病毒（RV）的细胞培养问题的尚未完全解决,其原因之一可能与敏感细胞较少有关。为此,我们进行了RV新敏感细胞的筛选,发现恒河猴胚肾传代细胞Frhk-4感染不同血清型的人和动物RV标准株Wa、DS－1、YO、ST－3、SA－11和UK,37℃旋转或静止培养均能产生明显CPE。培养物经ELISA测定含RV抗原,用PAGE检测有特征性RV核酸图型,电镜超萍切片检查感染细胞可见RV颗粒,证实Rrhk-4是RV的敏感细胞,这在国内外尚未见报道。同时与MA104－CV－1细胞的比较研究证明,三种细胞对Wa和DS－1的敏感性及增殖滴度,以CV－1最高,Frhk-4次之,MA－104最低。     

人轮状病毒HRB-02株的分离鉴定

从哈尔滨医科大学第二临床医学院发生腹泻患儿粪便中取得标本 ,用MA10 4细胞传代培养 ,传至第4代时 ,细胞出现明显病变 ;电镜观察见到典型的轮状病毒颗粒 ;其RNA电泳型为 4∶2∶3∶2型 ,属A群轮状病毒。对其蚀斑特性和血凝特性进行了初步研究 ,把这株轮状病毒地方株命名为HRB 0 2株。为研究该株轮状病毒的分子生物学特性 ,从而研制诊断性抗原和疫苗奠定基础。     

从一次新生小牛腹泻流行中分离到牛轮状病毒

1985年3月下旬,合肥市某牛奶场发生了一次新生牛非细菌性腹泻流行。用MA104细胞从牛_1粪便标本中分离到一株牛轮状病毒。电镜检查为典型轮状病声形态。在MA104细胞上第三代开始呈现胞浆内颗粒状荧光并产生细胞病变(CPE)。PAGE核酸电泳图型与牛轮状病毒NCDV株完全一致。经ELISA鉴定属A群轮状病毒。     

1984年上海市第一人民医院新生儿病房急性胃肠炎流行

用ELISA和RNA PAGE银染法检测了上海市第一人民医院新生儿病房及隔壁婴儿病房8份急性腹泻粪便标本;3份粪标本呈轮状病毒特异抗原阳性,3份粪标本能提供RNA进行电泳分析。6份病人恢复期血清抗体效价均>1:1280,较正常配对组的抗体水平高4倍以上,说明新生儿病房及隔壁婴儿病房急性腹泻流行为轮状病毒感染所引起。分析其RNA迁移图谱,均为电泳型2L,第3电泳亚型。经混合电泳显示3例RNA无差异,提示病人感染来自同一传染源;并探讨了轮状病毒传播的可能途径,为今后改进医疗质量提供了资料。     

被动免疫轮状病毒NSP4&VP7双抗原重组腺病毒对感染乳鼠的排毒保护作用

为建立小鼠轮状病毒(Rotavirus,RV)感染动物模型,研究可同时表达轮状病毒NSP4 (Nonstructural protein 4)和VP7(Viral protein 7)的重组腺病毒疫苗免疫孕鼠后对新生乳鼠感染RV的被动保护作用.新生乳鼠口服异源株轮状病毒Wa、ZTR-68或SA11株后(分2次给予,每次含5×104 CCID50的RV),观察乳鼠是否有腹泻症状、肠道病理变化,检测乳鼠粪便排毒百分率;另以重组腺病毒rAd-NSP4-VP7免疫孕鼠后,检测母鼠血清抗体产生情况,并对比乳鼠粪便中RV抗原检出率初步评价疫苗的被动免疫保护作用.发现口服异源株RV的乳鼠未出现类似人类婴幼儿感染后的明显腹泻症状,但在粪便中可检测到RV抗原的存在(Wa、ZTR-68攻毒组均超过80％).经rAd-NSP4-VP7被动免疫的乳鼠接受Wa和ZTR-68攻毒后其粪便中的RV检出率比未受到被动免疫保护的对照组降低(P＜0.05).rAd-NSP4-VP7重组腺病毒免疫母鼠可显示出对孕鼠感染RV的被动免疫保护作用.     

Evidence for natural reassortants of human rotaviruses belonging to different genogroups.

Of 335 rotavirus isolates associated with diarrheal disease in Bangladesh that were culture adapted and subsequently characterized for electropherotype, subgroup, and serotype, 9 had properties that suggested they may be natural reassortants between human rotaviruses belonging to different "genogroups." Two of these were examined in greater detail by RNA-RNA hybridization with prototype strains representative of each of the three proposed human rotavirus genogroups. One subgroup II isolate, 248, with a "long" electrophoretic pattern was neutralized by hyperimmune antisera to both serotype 2 and 4 strains. Consistent with these results, seven RNA segments of this isolate formed hybrids with human strains belonging to the Wa genogroup and four segments hybridized with strains belonging to the DS-1 genogroup. The second isolate examined, 456, belonged to subgroup II and had a long electrophoretic pattern but was found to be a serotype 2 strain. This isolate also appeared to be an intergenogroup reassortant because three of its segments formed hybrids with strains belonging to the Wa genogroup and eight hybridized with viruses of the DS-1 genogroup. On the basis of the relative migration rates of these RNA-RNA hybrids during gel electrophoresis, a suggested origin for each gene segment was proposed which was consistent with the results expected from electrophoretic, subgroup, and serotypic analyses.     

Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge.

A baculovirus-expressed VP4 protein derived from the simian rhesus rotavirus (RRV) was used to parenterally immunize murine dams. VP4-immunized dams developed high levels of neutralizing antibodies against RRV and low levels of cross-reactive neutralizing antibodies against human strains Wa, ST3, and S2 and animal strains SA-11, NCDV, and Eb. Newborn mice suckled on VP4-immunized dams were protected against a virulent challenge dose of the simian strain RRV and against murine rotavirus Eb. The cross-reactive nature of the serum-neutralizing response generated by VP4 immunization and the protective efficacy of the immunization suggest that recombinant-expressed VP4 proteins should be considered as viable vaccine candidates.     

High-Pressure Inactivation of Rotaviruses: Role of Treatment Temperature and Strain Diversity in Virus Inactivation

Rotavirus (RV) is the major etiological agent of acute gastroenteritis in infants worldwide. Although high-pressure processing (HPP) is a popular method to inactivate enteric pathogens in food, the sensitivity of different virus strains within same species and serotype to HPP is variable. This study aimed to compare the barosensitivities of seven RV strains derived from four serotypes (serotype G1, strains Wa, Ku, and K8; serotype G2, strain S2; serotype G3, strains SA-11 and YO; and serotype G4, strain ST3) following high-pressure treatment. RV strains showed various responses to HPP based on the initial temperature and had different inactivation profiles. Ku, K8, S2, SA-11, YO, and ST3 showed enhanced inactivation at 4°C compared to 20°C. In contrast, strain Wa was not significantly impacted by the initial treatment temperature. Within serotype G1, strain Wa was significantly (P < 0.05) more resistant to HPP than strains Ku and K8. Overall, the resistance of the human RV strains to HPP at 4°C can be ranked as Wa > Ku = K8 > S2 > YO > ST3, and in terms of serotype the ranking is G1 > G2 > G3 > G4. In addition, pressure treatment of 400 MPa for 2 min was sufficient to eliminate the Wa strain, the most pressure-resistant RV, from oyster tissues. HPP disrupted virion structure but did not degrade viral protein or RNA, providing insight into the mechanism of viral inactivation by HPP. In conclusion, HPP is capable of inactivating RV at commercially acceptable pressures, and the efficacy of inactivation is strain dependent.     

A synthetic peptide corresponding to the cleavage region of VP3 from rotavirus SA11 induces neutralizing antibodies.

Antibodies were elicited in rabbits by immunization with the synthetic tetradecapeptide Gln-Asn-Thr-Arg-Asn-Ile-Val-Pro-Val-Ser-Ile-Val-Ser-Arg, corresponding to amino acids 228 to 241 of SA11-VP3. Protein specificity of the antipeptide serum is demonstrated. The antipeptide serum revealed neutralizing activity directed against SA11 in a neutralization assay. Human rotavirus strains Wa, S2, and Hochi and bovine strains NCDV and UK were not neutralized, demonstrating the strain-specific neutralizing activity of the raised antipeptide serum. Upon immune electron microscopy, aggregation of SA11 particles was observed.     

Expression and purification of polyhistidine-tagged rotavirus NSP4 proteins in insect cells

The rotavirus nonstructural NSP4 protein, a transmembrane endoplasmic reticulum-specific glycoprotein, has been described as the first viral enterotoxin. Purified NSP4 or a peptide corresponding to NSP4 residues 114-135 induces diarrhea in young mice. NSP4 has a membrane-destabilizing activity and causes an increase in intracellular calcium levels and chloride secretion by a calcium-dependent signalling pathway in eucaryotic cells. In this study, four recombinant baculoviruses were generated expressing the rotavirus NSP4 glycoprotein from the human strains Wa and Ito, the porcine strain OSU, and the simian strain SA11, which belong to two different NSP4 genotypes, A and B. The recombinant glycoproteins, expressed as polyhistidine-tagged molecules, were analyzed by Western blotting and immunoprecipitation. Newborn mice responded with diarrhea after inoculation with each of the recombinant NSP4 proteins.