轮状病毒腹泻患儿检测结果分析

目的:探讨轮状病毒感染与天气因素之间的关系,以指导临床采取合理的防治策略.方法:采用免疫胶体金法对1155例腹泻患儿进行轮状病毒检测;收集2005年到2007年的天气资料;分别分析轮状病毒阳性患儿人次与温度、湿度之间变化的规律.结果:501份标本轮状病毒阳性,6m-24m年龄段婴幼儿阳性检出率最高.每年秋季(第33周时)随着周平均气温缓慢下降患儿人次开始逐渐上升,到第41周患儿人数达到最高峰,之后回落,达高峰前几周湿度也略有下降,表明天气因素与轮状病毒的传播有密切的关系.结论:秋季周平均气温、相对湿度是影响轮状病毒传播流行的自然因素.     

轮状病毒所致幼儿腹泻中肠道双歧杆菌的动态变化

测定36例轮状病毒腹泻幼儿的粪便双歧杆菌数量,与15例健康小儿对照,并对5例轮状病毒腹泻患儿自发病早期至愈后一周作双歧杆菌的动态定量分析,结果提示双歧杆菌在腹泻早期(3天以内)未表现明显脱水时数量不减,腹泻过程中则减少,减少的程度与脱水程度有关。对双歧杆菌在轮状病毒腹泻过程中的作用作了讨论。     

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。     

IgY Antibodies Protect against Human Rotavirus Induced Diarrhea in the Neonatal Gnotobiotic Piglet Disease Model

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization –VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV–specific IgA ASC in the duodenum. We further analyzed the pigś immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea.     

Group A Rotavirus Infection and Age-Dependent Diarrheal Disease in Rats: a New Animal Model To Study the Pathophysiology of Rotavirus Infection

Group A rotaviruses are major pathogens causing acute gastroenteritis in children and animals. To determine if group A rotavirus replicates and induces disease in rats, antibody-negative Lewis neonatal or adult rats were inoculated orally with tissue culture-adapted human (Wa, WI61, and HAL1166), simian (rhesus rotavirus [RRV] and SA11), bovine (WC3), lapine (ALA), or porcine (OSU) rotavirus strains, wild-type murine (EC(wt)) rotavirus strain, or phosphate-buffered saline (PBS). Rotavirus infection in rats was evaluated by (i) clinical findings, (ii) virus antigen shedding or infectious virus titers in the feces or intestinal contents measured by enzyme-linked immunosorbent assay or fluorescent-focus assay, (iii) histopathological changes in the small intestine, (iv) distribution of rotavirus antigen in small-intestine sections by immunofluorescence, and (v) growth rate. Rotavirus infection of 5-day-old but not > or =21-day-old rats resulted in diarrhea that lasted from 1 to 10 days postinoculation. The severity of disease and spread of infection to naIve littermates differed depending on the virus strain used for inoculation. The duration of virus antigen shedding following infection was considerably prolonged (up to 10 days) in neonatal rats compared to that in 21-day-old rats (1 or 2 days). Based on lack of virus antigen shedding and disease induction, the murine EC(wt) rotavirus was the only strain tested that did not infect rats. Histopathological changes in the small-intestine mucosa of 5-day-old RRV-inoculated rats but not of PBS-inoculated rats was limited to extensive enterocyte vacuolation in the ileum. In RRV-inoculated neonatal rats, rotavirus antigen was detected in the epithelial cells on the upper half of the intestinal villi of the jejunum and ileum. In addition, infection of neonatal rats with RRV but not with PBS resulted in reduced weight gain. Rats infected with group A rotaviruses provide a new animal model with unique features amenable to investigate rotavirus pathogenesis and the molecular mechanisms of intestinal development, including physiological factors that may regulate age-dependent rotavirus-induced diarrhea.     

Complete Genome Sequence of Porcine Epidemic Diarrhea Virus Strain AJ1102 Isolated from a Suckling Piglet with Acute Diarrhea in China

A diarrhea outbreak caused by porcine epidemic diarrhea virus (PEDV) has been observed in China since December 2010. We report here the complete genome sequence of PEDV strain AJ1102 isolated from a suckling piglet with acute diarrhea, which will help toward understanding the molecular and evolutionary characteristics of the epidemic PEDV in China.     

Analysis of Host Range Restriction Determinants in the Rabbit Model: Comparison of Homologous and Heterologous Rotavirus Infections

The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 10³ 50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction. Also, our results suggest that VP4 may be involved in, but is not exclusively responsible for, host range restriction in the rabbit model. The replication efficiency of rotavirus in rabbits also is not controlled by the product of gene 5 (NSP1) alone, since a reassortant rotavirus with ALA gene 5 and all other genes from SA11 was more severely replication restricted than either parental rotavirus strain.Rotaviruses are the leading cause of acute viral gastroenteritis in humans and animals throughout the world. Rotaviruses belong to the Reoviridae family and are characterized by a genome consisting of 11 segments of double-stranded RNA (dsRNA), enclosed in a triple-layered protein capsid (28). Serotype designations are based on independent neutralization determinants on the two outer capsid proteins VP4 (P serotypes, for protease-sensitive protein) and VP7 (G serotypes, for glycoprotein) (28). Serotype specificity determined by cross-neutralization assays using hyperimmune sera against the whole virus is mainly defined by VP7, and 14 G serotypes have been identified (28). Recently, antisera or monoclonal antibodies raised to VP4 and sequence analysis of VP4 identified 12 P serotypes and 20 P genotypes, respectively (28, 39). Rotavirus VP4 protein is responsible for a number of important biological functions, such as the enhancement of infectivity by proteolytic cleavage of VP4 into VP8* and VP5*, hemagglutination, restricted growth in cell culture, virulence, initial virus attachment to cells, and protease sensitivity associated with plaque formation (1, 4, 25, 34, 40, 51).The use of animal models, including the rabbit and mouse models, has been essential to the understanding of rotavirus infection, pathology, disease, immunity, and testing of prospective vaccines in children (21). The limitations of the rabbit and adult mouse models of rotavirus infection for vaccine testing are as follows: (i) human rotavirus (HRV) strains do not efficiently replicate in either animal, (ii) clinical disease is not observed, and (iii) only homologous virus strains (isolated from the same species) replicate efficiently and spread horizontally to uninoculated control animals, whereas heterologous virus strains (isolated from a different species) do not (6, 15, 16, 29, 31, 35, 37, 44, 50, 55). We and others developed a rabbit model of rotavirus infection that is useful for defining basic parameters of active immunity, immunogenicity, and protective efficacy of vaccines (12, 15–21, 36, 61). Rabbits are productively infected with homologous lapine rotavirus strains up to at least the age of 5 years, which allows examination of active and long-term immunity for vaccine studies (13, 15–17, 36, 61). Group A lapine rotavirus strains have been isolated in Canada, Japan, Italy, and the United States, and those that have been characterized are serotype G3 (8, 11, 15, 53, 56, 61). Recently, the P type of four different strains was identified as genotype P[14] (11). Previously, limited infection of rabbits with a heterologous strain had been obtained only with SA11 Cl3 (P[2], G3) (15).Attempts to identify host range and virulence determinants for rotavirus have implicated different constellations of genes, including genes 2, 3, 4, 5, 8, 9, 10, and 11 (5, 23, 30, 33, 37, 38, 41, 43, 44, 60, 62, 65). Although host range restriction and virulence may be multigenic, two genes, 4 and 5, are of interest because they cluster according to species of origin, suggesting a role in host range restriction. The finding that genome segment 5 (NSP1) sequences cluster according to species of origin (24, 39, 65) and that, in the mouse model, gene 5 segregates with transmission of virus among littermates (5), led to the hypothesis that NSP1 is involved in host range restriction. VP4 sequence analyses of rotavirus strains isolated from different species revealed that specific VP4 types also generally correlate with the species of origin of each rotavirus strain (43, 60). Therefore, once we identified the P type of four lapine rotaviruses as P[14], we tested two P[14] HRV strains, PA169 (G6) and HAL1166 (G8) (32) to determine if VP4 is involved in host range restriction. We also tested several other HRV strains, live attenuated reassortant candidate vaccine strains [rhesus rotavirus (RRV) × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4)], and animal rotavirus strains of different P and G types to determine if they could productively infect rabbits. In addition, to evaluate whether the single rotavirus gene 5 is responsible for replication efficiency in rabbits, rabbits were inoculated with a reassortant rotavirus with the lapine ALA gene 5 and all the other genes from the simian rotavirus SA11 Cl3 strain.     

Results of Intracecal Inoculation of Germfree and Conventional Guinea Pigs and Germfree Rats with Axenically Cultivated Entamoeba histolytica

SYNOPSIS. Germfree and conventional guinea pigs and germfree rats were inoculated with large numbers of axenically cultivated Entamoeba histolytica. Amebic lesions were not found in any of the animals, and there was no indication that the ameba had established lumen infections in any instance. The axenic amebae appeared to have lost their pathogenicity. This loss was believed to depend, in part at least, upon the fact that they did not encyst and thus did not complete their life cycle in axenic culture.     

Viral Determinants of Rotavirus Pathogenicity in Pigs: Evidence that the Fourth Gene of a Porcine Rotavirus Confers Diarrhea in the Homologous Host

A porcine rotavirus (prv) monoreassortant, S-F4, which carries RNA segment 4 of the pig-pathogenic variant prv 4F in the genetic background of the pig-apathogenic variant prv 4S (G. I. Tauscher and U. Desselberger, J. Virol. 71:853–857, 1997), was found to be pathogenic in gnotobiotic piglets. This indicates that RNA segment 4 of the pig-pathogenic variant prv 4F is a major determinant of pathogenicity in its homologous host.     

成人腹泻轮状病毒蛋白的免疫印迹分析

我们从腹泻病人便样中纯化了病毒,其结构蛋白组分按分子量大小分别为VP1(136K),VP2(113K),VP3(92K),VP4(84K),VP5(64K),VP6(47K),VP7(41K)。所有这些蛋白皆具有抗原性。WP6是B组轮状病毒的共同抗原。B组轮状病毒的每一结构蛋白与A组轮状病毒都无交叉免疫反应。另外注意到二例不同病人对VP6和VP7刺激产生的抗体水平不同。     

乳鼠与成年大鼠晶状体中某些代谢的比较

对乳鼠与10月龄成年大鼠的晶状体代谢进行比较。结果表明,年成大鼠晶状体脂类过氧化作用较乳鼠显著高(P<0.05),而非蛋白质就基含量则较乳鼠低约22％(P<0.01),谷胱甘肽过氧化物酶(GSH-Px)活性随鼠龄增长而逐渐下降,成年大鼠GSH-Px活性较18日龄乳鼠降低62％(P<0.01),成年大鼠的谷胱甘肽还原酶活性较18日龄乳鼠低47％(P<0.01),而谷胱甘肽硫转移酶则高14％(P<0.01)。据此结果推测,去年性白内障的发生可能与晶状体抗氧化遗伤的防御机制减弱与脂类过氧化作用增加有关。而乳鼠晶状体谷胱甘肽硫转移酶(GST)活性低于成年大鼠或许是某些中毒性白内障只可在幼年动物诱发成功的影响因素之一。     

轮状病毒致腹泻机制的研究进展

轮状病毒(Rotavirus,RV)是发展中国家致5岁内婴幼儿胃肠道感染,引起感染性腹泻的重要原因,每年因此而死亡的病例高达600,000多人.[1]近年来对此的研究也越来越多,但是其引起腹泻的具体病理生理机制始终未达成统一的说法.目前主要是有两种假说,即轮状病毒非结构蛋白4 (NSP4)假说和肠道神经系统(ENS)假说.对于轮状病毒腹泻的预防及治疗,现阶段主要是进行减毒活疫苗预防,但其疗效不甚令人满意,并且有可能导致肠梗阻.[2-3]本文将对轮状病毒致婴幼儿腹泻的可能机制及其研究进展进行综述,此外,针对其独特的致病机制将讨论轮状病毒感染可能的预防策略.     

中国河北省卢龙县儿童轮状病毒腹泻研究

轮状病毒是我国儿童重症腹泻的主要病原。按照WHO轮状病毒监测方案,于1999年7月至2003年6月,在河北省卢龙县开展了医院和社区为基础<5岁儿童轮状病毒腹泻的监测。结果表明:卢龙县<5岁儿童腹泻的发病率为1 3次/人/年。4年中全县共有2350名<5岁急性腹泻患儿住院,占所有住院儿童的38%(2350/6213)。住院的腹泻患儿每年有两个高峰,一个是夏季(6～8月),占全年腹泻住院病例的22%;另一个在冬季(12月至次年2月),占58%。住院的轮状病毒腹泻只有一个高峰,是在冬季(12月至次年2月),高峰期的轮状病毒腹泻住院患儿数占全年轮状病毒腹泻患儿住院数的86%。按全年统计,轮状病毒腹泻占住院腹泻患儿的46%,轮状病毒腹泻的住院率为11/1000儿童/年。在门诊腹泻患儿中轮状病毒腹泻患儿占28%,在社区腹泻患儿中轮状病毒腹泻占10%。轮状病毒毒株的分布,G3型(45%)最常见,其次为G1型(35%)、G2型(8%)、G4型(3%)、G9型(0 6%),混合感染较少(1%)。还有8%的毒株未能分出型别。在轮状病毒腹泻患者中,9～11月龄的儿童检出率最高(53%),其次是12～17月龄(51%)、18～23月龄(36%)和6～8月龄(30%)。在4年研究期间共有5～10名1～59月龄儿童可能因为轮状病毒腹泻死亡,其中有1例确诊为P[8]G1型毒株感染。初步估计,该县1～59月龄儿童轮状病毒腹泻的死     

空心莲子草口服治疗乳鼠流行性出血热病毒感染的研究

空心莲子草经口服治疗流行性出血热病毒（ＥＨＦＶ）感染的乳鼠。结果显示,５、７．５、１０．０ｍｇ三个剂量组的存活率各为８０％、７２．２％和４０．０％,平均存活天数（ＭＴＤ）为５６．５±０．９、５３．５±１．１和４１．５±２．７ｄ,而病毒对照组的存活率为０．０％,ＭＴＤ为２６．３±０．８ｄ,经该药治疗的感染鼠体内的ＥＨＦＶ抗原表达减少,而未经治疗的感染乳鼠体内的ＥＨＦＶ抗原则在全病程中都显示出较强的表述。治疗组抗病毒效果类似于病毒唑,且口服病毒唑毒性小。说明空心莲子草在体内对ＥＨＦＶ感染鼠有治疗作用。     

Changing Characteristics of Rotavirus Diarrhea in Children Younger than Five Years in Urban Bangladesh

Background

Childhood rotavirus diarrhea is still one of the major public health challenges. The present study aimed to determine changing characteristics of rotavirus diarrhea in under-5 children at two periods of time.

Methods

We enrolled 5,357 under-5 children with rotavirus positive in two different time periods; i) 1993-1997 (n = 2,493), and ii) 2008–2012 (n = 2,864) considering beginning and ending of two decades. These children were enrolled in the urban Dhaka Hospital of icddr,b.

Results

Overall, proportion of rotavirus was about 25% in 1993–97, which was 42% in 2008–12 (68% rise; p<0.001). Significant higher proportion of children were stunted [38% vs. 22%; aOR-1.33 (95% CI-1.09-1.62)], had vomiting [87% vs. 74%; aOR-2.58 (95% CI-2.02-3.28)], fever [10% vs. 8%; aOR-1.31 (95% CI-0.96-1.78)], family members >5 [38% vs. 35%; aOR-1.32 (95% CI-1.10-1.58)] required more intravenous fluid [9% vs. 3%; aOR-4.93 (95% CI-3.19-7.63)], had higher co-infection with Shigella [3% vs. 1%; aOR-3.36 (95% CI-1.61-7.03)], Vibrio cholerae [4% vs. 1%; aOR-3.70 (95% CI-2.12-6.46)]; and ETEC [13% vs. 7%; aOR-2.21 (95% CI-1.65-2.97)]; however, significantly lower proportion of them used sanitary toilets [54% vs. 78%; aOR-0.66 (95% CI-0.54-0.80)], boiled drinking water [16% vs. 38%; aOR-0.60 (95% CI-0.48-0.74)], used antimicrobial at home [63% vs. 82%; aOR-0.56 (95% CI-0.46-0.69)] and had some or severe dehydration [18% vs. 34%; aOR-0.15 (95% CI-0.12-0.20)] in 1^st observation period compared to that of 2^nd.

Conclusion

Proportion of episodes of under-5 rotavirus diarrhea increased over the period. Concomitant changes in host, socio-demographic and clinical characteristics, and co-infections were also observed. Thus, vaccination campaign which is prevailing in private sector should also be introduced in public sector.     

Kinetic Model Development for Accelerated Stability Studies

Accelerated stability coupled with modeling to predict the stability of compounds, blends, and products at long-term storage conditions provides significant benefits in science-based decision-making throughout drug substance and drug product development. The study can often be completed, including data analysis in the space of three working weeks, and the information gathered and learning made in this time period can rival years of traditional analysis. The speed of the studies allows an earlier assessment of risk to quality enabling appropriate risk mitigation strategies to be implemented in a timely manner. The scientific foundation is based upon Arrhenius kinetic equations that can be linear or nonlinear in time, and can be based upon water vapor pressure or liquid water activity (relative humidity). A variety of kinetic models are evaluated, and the best model is chosen based upon both Bayesian information criteria and an automated assessment of kinetic model parameters fitting within acceptable ranges. Confidence intervals are estimated based upon a bootstrapping approach. Moisture vapor transmission rate models are applied on top of the resulting kinetic models in order to simulate different packaging types and the use of desiccant. The kinetic models are integrated with the prediction of packaging humidity over time to create a long-term prediction of impurities and other phenomena. The resulting models have been shown to be useful for not only the prediction of drug product impurities in long-term storage but other physical phenomena as well such as hydrate development and solvate loss.     

Trivalent Combination Vaccine Induces Broad Heterologous Immune Responses to Norovirus and Rotavirus in Mice

Rotavirus (RV) and norovirus (NoV) are the two major causes of viral gastroenteritis (GE) in children worldwide. We have developed an injectable vaccine design to prevent infection or GE induced with these enteric viruses. The trivalent combination vaccine consists of NoV capsid (VP1) derived virus-like particles (VLPs) of GI-3 and GII-4 representing the two major NoV genogroups and tubular RV recombinant VP6 (rVP6), the most conserved and abundant RV protein. Each component was produced in insect cells by a recombinant baculovirus expression system and combined in vitro. The vaccine components were administered intramuscularly to BALB/c mice either separately or in the trivalent combination. High levels of NoV and RV type specific serum IgGs with high avidity (>50%) as well as intestinal IgGs were detected in the immunized mice. Cross-reactive IgG antibodies were also elicited against heterologous NoV VLPs not used for immunization (GII-4 NO, GII-12 and GI-1 VLPs) and to different RVs from cell cultures. NoV-specific serum antibodies blocked binding of homologous and heterologous VLPs to the putative receptors, histo-blood group antigens, suggesting broad NoV neutralizing activity of the sera. Mucosal antibodies of mice immunized with the trivalent combination vaccine inhibited RV infection in vitro. In addition, cross-reactive T cell immune responses to NoV and RV-specific antigens were detected. All the responses were sustained for up to six months. No mutual inhibition of the components in the trivalent vaccine combination was observed. In conclusion, the NoV GI and GII VLPs combination induced broader cross-reactive and potentially neutralizing immune responses than either of the VLPs alone. Therefore, trivalent vaccine might induce protective immune responses to the vast majority of circulating NoV and RV genotypes.     

补骨脂素对乳鼠颅骨成骨细胞分化成熟的影响

骨质疏松症是一种全身性骨骼疾病,其特点是骨量低、骨微结构恶化、骨脆性增加、易骨折。为了改善骨质疏松,需要寻找合成代谢和口服药物的副作用最小的替代药物。补骨脂素是从中草药中提取的香豆素衍生物。然而,补骨脂素在成骨细胞功能中的作用及其分子机制尚不清楚。本研究发现,补骨脂素通过上调成骨细胞特异性标志基因(包括icollagen,骨钙素和骨唾液蛋白)的表达,增强碱性磷酸酶的活性,以剂量依赖的方式促进小鼠原代成骨细胞的成骨分化。本研究同时证明补骨脂素能上调BMP2和BMP4基因的表达,提高磷酸化smad1/5/8蛋白水平,激活BMP报告基因(12xbe-oc-luc)的活性以及增强BMP信号直接靶基因osx的表达。BMP2和BMP4基因的缺失消除了补骨脂素对成骨细胞标志基因col1、alp、oc和bsp表达的促进作用。结果表明,补骨脂素通过激活BMP信号促进成骨细胞分化,提示补骨脂素可能是治疗骨质疏松等骨丢失相关疾病的一种潜在的合成代谢剂。