胆道闭锁与病毒感染的动物实验研究
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摘要
研究背景与目的：胆道闭锁是发生于婴幼儿的进行性炎症性胆管病,最终导致肝外、肝内胆管的梗阻及肝硬化。胆道闭锁的病因和发病机制仍不清楚。来自临床和动物实验的证据提示围生期的某种病毒感染诱发的宿主炎性反应很可能是致病原因之一。其中,巨细胞病毒和轮状病毒是胆道闭锁研究中最受关注的两种病毒。尽管研究发现胆道闭锁患儿有很高的巨细胞病毒感染率,但是临床上仍缺乏足够的证据表明这样的感染与肝外胆道损伤继而闭锁存在直接的因果联系。因此我们的研究目的之一是在原有豚鼠先天性感染模型的基础上,根据国内高致病力毒株无法通过近交系传代获得的现状,摸索围产期远交系豚鼠巨细胞病毒(Guinea pig cytomegalovirus, gpCMV)感染并导致幼鼠肝胆损伤的合适病毒滴度,建立感染模型,并在此基础上分析围产期CMV感染后肝脏实质和胆道系统的病变情况,进一步了解CMV在胆道闭锁发病中的作用；恒河猴轮状病毒感染(Rhesus Rotavirus, RRV)的胆道闭锁动物模型是目前胆道闭锁研究中最为常用的工具。由于轮状病毒对反转录基因基因技术的抵抗,使得对该病毒致病分子生物学机制研究受到极大限制。本研究的第二部分拟利用同组轮状病毒混合感染后可发生基因重排的特性,筛选单基因重排病毒株,通过病毒株的表现型分析确定RRV致胆道损伤的关键致病基因节段及其致病机制。
材料与方法：一,豚鼠围生期巨细胞病毒感染模型的建立及肝胆损伤机制研究：gpCMV于豚鼠胚胎肺成纤维细胞上传代适应。按病毒接种时间将动物分组为：1,孕晚期组,孕晚期豚鼠于孕40-43天腹腔接种1×109TCID50病毒悬液,同时设生理盐水或空白对照,活产子豚鼠出生后不同时间点处死取标本。2,新生鼠组,随机取新生豚鼠23只,于生后12-24小时腹腔注射1×108TCID50病毒悬液,于接种后不同时间点处死取标本。3,幼鼠组,随机取新生豚鼠16只,于生后10天腹腔注射1×108TCID50病毒悬液,于接种后不同时间点处死取标本。观察生长情况及黄疸症状。所有血液标本常规肝功能检测。肝脏及肝外胆道标本石蜡切片常规HE染色,原位检测细胞凋亡情况。部分标本冰冻切片原位杂交检测gpCMVmRNA在肝胆系统的表达。胆汁标本双抗酶标ELISA法检测胆汁肝细胞生长因子(Hepatocyte growth factor, HGF)和白介素6(Interleukin 6, IL-6)表达情况。免疫组化检测CD8+细胞的表达。二,轮状病毒致小鼠胆管损伤关键基因的研究：将毒性病毒株RRV与非毒性病毒株EDIM(Epizootic Diarrhea ofInfant Mice)(体内)或TUCH(Tulane University and Cincinnati Childen's Hospital,新分离出的猴轮状病毒株)(体外)共感染。所得部分双基因或三基因重排子代与亲代,或其他子代进行回交或杂交以施加选择性压力。将培养所得病毒悬液接种MA104细胞系后琼脂糖凝胶铺板,挑选病毒斑。病毒纯化后抽提RNA,聚丙烯酰胺凝胶电泳进行基因型分析。将所筛选出的单基因重排病毒株接种新生BALB/C小鼠,进行病毒致病性分析,部分小鼠接种后7天杀死取肝脏及肝外胆道标本进行组织病理分析。此外,将单基因重排各株接种胆管细胞系,4℃共孵育1小时后计算结合病毒量与总接种量的比例进行病毒细胞结合力分析；重排病毒株接种胆管上皮细胞系,感染剂量MOI (Multiplicity Of Infection)为1。48小时后对病毒进行滴定,了解增殖情况。各重排克隆接种新生鼠后7天后取肝外胆道标本制成匀浆,于MA104细胞系上进行滴定,了解病毒负荷量。
结果：
一,围生期豚鼠巨细胞病毒感染模型建立及分析：
孕晚期及出生时腹腔接种巨细胞病毒可引起子代豚鼠肝胆系统损伤,表现为：
1,孕晚期感染豚鼠出生体重明显低于盐水及空白对照(P=0.0029,P=0027)。生后20天,体重增长也明显落后于盐水及空白对照。新生鼠及幼鼠组病毒接种后豚鼠体重增长较对照组相比无明显差异。
2,孕晚期感染子豚鼠与空白及盐水对照组相比,出生时及生后10天TB、DB、ALT、AST水平均明显高于对照组(P＜0.05),出生时及生后10天两组比较各指标无明显差异(P>0.05),生后20天各指标均明显低于前两组,较对照无明显差别(P>0.05)。部分子豚鼠肉眼可见大便变白,伴TB和DB水平的升高。新生鼠组病毒接种后10天AST水平较20天组和空白对照升高(P=0.027,P=0.043)。
3,63.6%的孕晚期感染子豚鼠肝脏存在单核淋巴细胞浸润为主的炎症／病理改变,炎性细胞浸润以汇管区为主。可见小叶间汇管区的破坏和纤维化改变,合并小胆管增生。伴有肝细胞气球样变性和坏死,肝内胆汁淤积。偶可见肝内微脓肿形成。新生鼠接种后10天出现肝脏病变(3／8),但病变较孕晚期组明显减轻。幼鼠组未见明显肝脏病变。
4,有肝胆损伤者肝内细胞凋亡信号密度分布基本与炎症浸润程度一致,主要表达于肝脏实质细胞和汇管区间质细胞,血管和胆管上皮偶见信号表达。孕晚期组出生时肝脏细胞凋亡指数均值6.7±1.322,汇管区细胞凋亡指数均值5.13±2.112,与对照相比有明显增加(P<0.05),与新生鼠组肝损伤者凋亡指数无明显差别。
5,病毒gpCMV阳性杂交信号表达于胆管上皮及血管内皮细胞及汇管区基质内,但肝细胞内未见表达。孕晚期组豚鼠gpCMV阳性率17.27%,而新生鼠及幼鼠组豚鼠肝胆系gpCMV-mRNA阳性率(55%,50%)均明显高于孕晚期组(P=0.011,0.018<0.05)。
6,新生鼠感染后10天,20天胆汁中可见HGF表达增加(P=0.0292,P=0.0461)。各组胆汁标本中未见IL-6的表达。
7,孕晚期组(8／14)和新生鼠组(3／3)肝胆损伤者有CD8+细胞表达,主要分布于肝脏实质和汇管区间质。
二,轮状病毒致小鼠胆管损伤关键基因的研究：
1,在RRV对EDIM的体内杂交中,得到单基因重排株4株,将EDIM背景单基因重排株D6／2接种新生鼠后(第4基因节段来自RRV),诱导出与RRV类似的胆管损伤症状。RRV对TUCH的体外混合感染中(包括回交及子代杂交),分离出所有22个RRV或TUCH背景下单基因重排病毒株。分别命名为RTn(RRV背景重排株,第n个基因节段来自TUCH)或TRn(TUCH背景重排株,第n个基因节段来自RRV)。n代表第1至11个基因节段。
2,致病性分析发现：重排株RT4没有致病性。相反,重排株TR4诱导出与RRV相似的黄疸症状,致病率(94.2%vs.100%,P=1.000)及致死率(88.24%vs.80.96%,P=1.000)无明显差别。58.33%的RT3感染小鼠表现胆道梗阻症状,其致病率和致死率明显低于RRV(p=0.001,0.000<0.05)。相对应的TR3尽管诱导出与RRV相似的致病率(88.89%,P=0.218),77.78%的小鼠最终黄疸症状消退并存活。其余RRV背景重排病毒株疾病的表现型与RRV类似。TR7、TR1和TR2一样未诱导出任何肝胆损伤症状。其余大多数TUCH背景单基因重排株诱导—过性肝胆损伤症状,几乎所有小鼠最终存活。
3,病理分析证实各重排株的疾病表现型与汇管区及肝外胆道病理改变密切相关。
4,细胞结合能力：重排株RT4的细胞结合率(3.97%±0.93%)明显低于RRV(16.03%±1.31%,P=0.003＜0.05)及其他所有RRV背景重排株(P＜0.05)。其余RRV背景重排株细胞结合率与RRV无明显差别(P＞0.05)。相反,重排株TR4(12.43%±2.25%)表现出明显高于TUCH：5.33%±1.27%,p=0.001)及所有其他TUCH背景重排株的细胞结合率(P<0.05)。除了TR8和TR2外,其它TUCH背景重排株细胞结合率与TUCH相似(P>0.05)。
5,感染性分析：克隆RT4在胆管上皮细胞系和肝外胆道的滴度均明显低于RRV(P=0.013,0.000)。相对应的克隆TR4的滴度无论在胆管细胞系还是小鼠肝外胆道均明显高于TUCH(P=0.004,0.0446)。在胆管细胞系上,所有其它RRV背景重排株的滴度与RRV类似(P＞0.05),在肝外胆道大部分低于RRV(P＜0.05)。TR7、TR1和TR2在胆管细胞系及肝外胆道滴度均低于TUCH(P=0.001,0.012,0.0069)。其余引起症状TUCH背景病重排株在胆管细胞系的滴度均高于TUCH(P＜0.05)。等级相关分析提示TUCH背景重排株在肝外胆道平均滴度与病毒致病率相关。
结论：
一,围生期豚鼠巨细胞病毒感染模型建立及分析：
1,孕晚期和出生后即刻接种gpCMV病毒可导致子代豚鼠的感染和肝胆系统炎症损伤。
2,肝内和汇管区浸润炎性细胞以单核淋巴细胞为主,伴有CD8+T细胞的表达,提示Thl抗病毒免疫在此病理过程中发挥重要作用。
3,病毒mRNA主要表达于汇管区及内皮系统,证实了CMV病毒对胆管上皮的亲和性。其攻击胆管上皮标本并引起炎症损伤的机制待进一步研究。
4,损伤主要发生于围产期,与接种时间直接相关,表明宿主免疫状态不成熟可能是致病的重要背景因素。
二,轮状病毒致小鼠胆管损伤关键基因的研究：
1,基因节段4是决定小鼠胆道模型RRV对胆管上皮致病性的关键基因,也决定了RRV对胆管上皮感染的特异性,其机制在于影响病毒与胆管上皮细胞的结合能力,继而影响病毒在细胞内复制,其蛋白产物VP4与胆管上皮相关受体的相互作用很可能是决定胆管细胞对病毒易感性的关键环节。
2,基因节段7被替代后,病毒的致病性、细胞结合能力和胆管上皮复制均未受影响,其蛋白产物VP7相关细胞表面受体的表达可能对胆管上皮的易感性影响不大。
3,基因节段3对RRV致病性有重要影响,其被替代所带来的“中间型”可能是包括宿主因素在内的多因素相互作用的结果。
4,基因节段1,2对病毒致病性无明显影响,但两者之间可能在病毒复制中存在协同作用。
Backgrounds and Purpose:Biliary atresia (BA) is a progressive, inflammatory cholangiopathy of infancy that leads to obstruction of extra-and intrahepatic bile ducts with eventual biliary cirrhosis. Despite its importance in child health, the cause of biliary atresia remains unclear. Evidence accumulated from both human and animal studies indicated that one possible pathological mechanism is the host immune response triggered by perinatal viral infection. Among the suspected viral agents, cytomegalovirus (CMV) and rotavirus drew more attentions. High incidence of cytomegalovirus CMV infection was found in patients with BA, but no solid evidence supported that the infection was the causing factor of the biliary system obstruction. So one purpose of our study is to set up a perinatal CMV infection induced hepatobiliary injury model, analyze the pathological change in hepatobiliary system and systemic inflammatory or immune response in the host.The study would provide important information on understanding the role of CMV in the inducing of the bile injury or even obstruction. The rhesus rotavirus (RRV)-induced murine model shares many similarities to the disease process found in children affected with BA and were used as a vital tool in analyzing various aspect of the infectious process in biliary atresia. As rotavirus families are refractory to the reverse genetic technique, the studies aimed to understand the molecular mechanism that characterizes the rotavirus life cycle and the pathogenesis of viral infection was hampered. Take advantage of a well-known property of rotavirus that they undergo genetic reassortment after mixed infection in cell culture or in vivo, we aim to create virus with single gene segment reassortment. Administrate these reassortants to neonatal mice, characterize their disease phenotype, we will define the key gene segment which determine the viral virulent to biliary tract. We also aim to define the role of other gene segments in this model. Cell binding assay and infectivity analysis were also employed to verify the mechanism by which the gene segments or its protein products worked. The studies will contribute to the understanding the initial stage that virus attact target cell, shadow light on a potential mechanism by which biliary atresia occurs.
Material and methods:A, Creation of perinatal CMV infection induced hepatobiliary injury model and injury evaluation:Guinea pig cytomegalovirus (gpCMV) were maintained in fetal guinea pig pulmonary fibroblast cells.The experiment has 3 groups:Prenatal group:Female guinea pigs at the 40th to 43th pregnant day (3rd trimester) were introperitoneally injected with virus supernates at a dose of 1×109TCID50 per dam. Two other subsets of dams severed as saline or blank control. Alive pups were sacrificed at different time point after birth. Neonatal group:A subset of healthy pups was introperitoneally injected with virus supernates within 24 hours of birth at a dose of 1×108 TCID50 per pup, Pups were sacrificed at different time point after inoculation. Infantile Group:Another subset of healthy pups was inoculated with a same dose of virus supernates at day 10 after birth. Pups were sacrificed at different time point after inoculation. Samples of the liver and proximal extrohepatic bile duct, blood, bile were collected. Weight gain, clinical sign of hepatobiliary injury and survival were recorded. Hepatic biochemical analysis was performed. A portion of the liver and extrohepatic bile duct were preserved in formalin. Serial sections underwent hematoxylin and eosin (HE) staining or in situ apoptosis analysis or immunohistochemical analysis for the presence of CD8+T cells. The expression of hepatic growth Factor (HGF), interleukin-6 (IL-6) in bile was examined with enzyme linked immunosorbent assay (ELISA). Other portion of liver and extrohepatic bile duct were iced and underwent in situ hybridization for the distribution of viral mRNA. B. Determine the specific RRV gene domain responsible for the injury bile duct injury in murine. The Virulent strain RRV was matched with avirulent strain EDIM (in vivo) or TCUH (in vitro). A subset of reassortants with double or triple gene segment reassortment was adapted to back cross with parental strain or cross with other progeny clones. MA 104 cells was infected with virus supernants generated from matching, the cell plates were overlayed with 0.2% agarose in EBSS, the plaque purified virus were freeze-thawed once and genotyped by polyacrylamide gel electrophoresis. Newborn balb/c mice were inoculated with reassortants, monitored for 21 days. A subset of pups was sacrificed at the 7th day after injection for histological analysis. The reassortant's binding ability was analyzed: immortalized cholangiocytes were inoculated with reassortant clones at 4℃for 1 hour; the amount of attached virus was expressed as a percentage of the total amount of virus used to inoculate the cells. In the infectivity essay, immortalized cholangiocytes were infected with reassortant at MOI of 1.the viral titer was measured after 48 hours. The extrohepatic bile ducts collected from mice at 7 days post inoculation were homogenized, tiered on the MA 104 cells.
Results:A.In guinea pigs, perinatal inoculation of gpCMV induced signs of hepatobiliary system injury.
1. Birth weight of pups in prenatal group was lower than saline or blank controls (P=0.0029, P=0027, respectively). These pups developed growth retardation versus that of saline and blank controls. Pups in neonatal and infantile group showed no sign of growth retardation.
2. TB, DB, ALT, AST levels in Pups of prenatal group were higher at day 1 or day 10 after birth as compared to controls (P<0.05), while they were not significantly different between these two time points (P>0.05). Those parameters at day 20 decreased as compared to those at day 1 or day 10. A few pups in prenatal showed sign of jaundice within 10 days after birth, which were associated with increased TB and DB level. In neonatal group, a temporary increased AST level was observed at day 10-post injection.
3. Histological analysis of the livers revealed a notable infiltration of mononuclear cells in 63.6%(14/22) of pups in prenatal group. Inflammatory cells were mainly found in portal tract area. There was interlobular portal tract damage and fibrosis, bile ductular proliferation. Hepatocytes balloon-like degeneration, necrosis and ductular cholestasis were often found. Micro-abscesses were seen (2/14). Pathological changes were also found in pups of neonatal group (3/8, but not as severe as those in prenatal group. No notable pathologic change was found in infantile group.
4. Apoptosis signals were mainly seen in hepatocytes and mesenchymal cells in portal tract, coincident with the distribution of inflammatory cells, no definite signal was observed in vascular or ductular epithelial cells.
5. Viral distribution:gpCMV mRNA signal were found in endothelial and epithelial cells and portal tract stoma.No signal was found on hepatocytes. Positive rate of gpCMV was 17.27%(4/22) in pups of prenatal group, significantly lower than those of neonatal or infantile groups (55%,50%respectively).
6. HGF level increased in bile from pups of neonatal group and infantile group (P=0.0292, P=0.0461). No IL-6 was found in any sample with this method.
7. CD8+T cells were often found in samples of prenatal group (8/14) and neonatal group (3/3), distributed mainly in liver parenchyma and portal area.
B. In the murine biliary atresia model,
1. Reassortant creation and pathogenecity assessment:21 reassortants were generated in the matching of RRV with EDIM, among of them only 4 were clones with single gene reassortment (4/170). Only one of them was on EDIM background. The reassortant D6/2(EDIM background strain with the 4th gene segment derived from RRV) caused signs of biliary injury that was similar with that of RRV. In the crossing of RRV with TUCH (back crossing or progeny crossing included), all the 22 single-segment reassortants were generated and selected to inoculate newborn pups. They were named as RTn (RRV background clone with the nth gene segment from TUCH) TRn (TUCH background clone with the nth gene segment from RRV).
2.Clone RT4 did not induce disease, on the contrary, clone TR4 carried on signs of biliary obstruction in 94.12% of pups and produced a mortality rate of 88.24%, which is of no difference with that of pups injected with RRV (P=1.000＞0.05). An intermediate phenotype was seen in RT3 (RRV background strain with the 3rd gene segment deriving from TUCH) infected mice, although 58.33 percent of pups developed sign of biliary obstruction, the rate was significantly lower than that of mice injected with RRV (P=0.001＜0.05), so was the mortality rate (20.83%, P=0.000<0.05). Pups injected with other RRV background reassortants developed manifestation of biliary obstruction, which were similar with that in RRV induced model. Reassortant clone TR7as well as TR1 and TR2, did not elicit any sign of hepatobiliary injury nor did they cause mortality. Most of other TUCH background reassortants brought on temporary sign of hepatobiliary injury, and most of the pups survived.
3. The histological appearances of the portal area as well as the extrohepatic bile ducts from different strain infected mice were consistent with the mice's symptoms.
4. Viral binding ability to cholangiocyte was significantly changed by manipulating the 4th gene segment. The clone RT4's binding ratio (3.97%±0.93%) was much lower than that of the parental strain RRV (16.03%±1.31%) (P=0.003<0.05) and that of other RRV background strains (P＜0.05). Other RRV background reassortants'binding ratios were not significantly different from that of parental strains (P＜0.05). As we prospected, replacement of the 4th gene segment of non-pathogenic TUCH strain with corresponding gene from RRV enhanced the viral attachment, clone TR4 exhibited a significantly higher binding percentage (12.43%±2.25%) as compared with that of parental strain TUCH (5.33%±1.27%, p=0.001). Besides TR8 and TR2 other TUCH background clones showed a similar binding ratio as compared with that of TUCH (P>0.05)
5.Consistent with the finding in binding assay, clone RT's titer in cholangiocytes as well as in bile duct at day 7 post inoculation was significantly lower than that of RRV(P= 0.013,0.000 accordingly).On the other hand, clone TR4's titer were definitely higher than that of TUCH both in vitro and in vivo (P=0.046,0.004 accordingly). On the cholangiocytes, all the other RRV background clones'titer were similar with that of parental strain RRV (P>0.05), but most of those (except for RT8, RT9) were lower than that of RRV in vivo (P<0.05). Consistent with the observation that mice injected with R1,R2 and R7 did not show any symptoms of the disease, the viral titer both in chlangiocytes and in bile duct were even lower than TUCH (P＜0.05). Alternatively, most of the other symptoms inducing strains titer were higher than that of parental strain TUCH (P<0.05) in vitro. Spearman correlation analysis indicated the mean titers were related to the morbidity of the disease among TUCH background reassortants.
Conclusion:
A.In the guinea pigs model,
1 Introperitoneally inoculating the third trimester pregnant dams or neonatal pups with gpCMV could induce inflammation and injury in hepatobiliary system.
2 Intra-hepatic infiltrated inflammatory cells were mainly mononuclear cells, parts of which were CD8+T cells. Those indicated Thl immune response played an important role in the phenotypic progression.
3 Viral mRNA signals were mainly found in portal tract and endothelial system, the tropism of gpCMV to bile duct epithelial cells and the corresponding damage to bile duct system were proved.
4 All injury phenotypes were from pups of prenatal and neonatal groups, closely related to the inoculation time, which indicated immature host immune system might play a key role in the process.
B. In the murine biliary atresia model:
1 Rhesus rotavirus gene segment 4 was the major determinants of pathogenecity. It also determined the RRV's specific tropism to cholangiocytes. Manipulation of gene segment 4 greatly changed the viral binding ability and infectivity, may in turn, and determined the viral phenotype. Expression of related cell surface receptor with binding site for VP4 (product of gene segment 4) might determine the susceptibility of changiocyte to viral insults.
2 Gene segment 7 did not contribute to the viral pathogenecity. Replacement of the 7th segment did not affect the viral binding; either did it affect its replication on cholangiocytes. Indicated the gene (encoding VP7) did not contribute to the virulence, the cell surface binding receptor related to VP7 might not play an important role in cholangiocyte susceptibility.
3 Gene segment 3 (encoding VP3) played an important role in RRV's pathogenecity, the intermediated virulent phenotype induced by reassortant TR3 probably was the result of interaction of multiple-factors, among them, host immune response may take an important role.
4 Gene segment 1,2 did not contribute to the viral pathogenecity, but they might coordinate in the process of viral replication.
材料与方法：一,豚鼠围生期巨细胞病毒感染模型的建立及肝胆损伤机制研究：gpCMV于豚鼠胚胎肺成纤维细胞上传代适应。按病毒接种时间将动物分组为：1,孕晚期组,孕晚期豚鼠于孕40-43天腹腔接种1×109TCID50病毒悬液,同时设生理盐水或空白对照,活产子豚鼠出生后不同时间点处死取标本。2,新生鼠组,随机取新生豚鼠23只,于生后12-24小时腹腔注射1×108TCID50病毒悬液,于接种后不同时间点处死取标本。3,幼鼠组,随机取新生豚鼠16只,于生后10天腹腔注射1×108TCID50病毒悬液,于接种后不同时间点处死取标本。观察生长情况及黄疸症状。所有血液标本常规肝功能检测。肝脏及肝外胆道标本石蜡切片常规HE染色,原位检测细胞凋亡情况。部分标本冰冻切片原位杂交检测gpCMVmRNA在肝胆系统的表达。胆汁标本双抗酶标ELISA法检测胆汁肝细胞生长因子(Hepatocyte growth factor, HGF)和白介素6(Interleukin 6, IL-6)表达情况。免疫组化检测CD8+细胞的表达。二,轮状病毒致小鼠胆管损伤关键基因的研究：将毒性病毒株RRV与非毒性病毒株EDIM(Epizootic Diarrhea ofInfant Mice)(体内)或TUCH(Tulane University and Cincinnati Childen's Hospital,新分离出的猴轮状病毒株)(体外)共感染。所得部分双基因或三基因重排子代与亲代,或其他子代进行回交或杂交以施加选择性压力。将培养所得病毒悬液接种MA104细胞系后琼脂糖凝胶铺板,挑选病毒斑。病毒纯化后抽提RNA,聚丙烯酰胺凝胶电泳进行基因型分析。将所筛选出的单基因重排病毒株接种新生BALB/C小鼠,进行病毒致病性分析,部分小鼠接种后7天杀死取肝脏及肝外胆道标本进行组织病理分析。此外,将单基因重排各株接种胆管细胞系,4℃共孵育1小时后计算结合病毒量与总接种量的比例进行病毒细胞结合力分析；重排病毒株接种胆管上皮细胞系,感染剂量MOI (Multiplicity Of Infection)为1。48小时后对病毒进行滴定,了解增殖情况。各重排克隆接种新生鼠后7天后取肝外胆道标本制成匀浆,于MA104细胞系上进行滴定,了解病毒负荷量。
结果：
一,围生期豚鼠巨细胞病毒感染模型建立及分析：
孕晚期及出生时腹腔接种巨细胞病毒可引起子代豚鼠肝胆系统损伤,表现为：
1,孕晚期感染豚鼠出生体重明显低于盐水及空白对照(P=0.0029,P=0027)。生后20天,体重增长也明显落后于盐水及空白对照。新生鼠及幼鼠组病毒接种后豚鼠体重增长较对照组相比无明显差异。
2,孕晚期感染子豚鼠与空白及盐水对照组相比,出生时及生后10天TB、DB、ALT、AST水平均明显高于对照组(P＜0.05),出生时及生后10天两组比较各指标无明显差异(P>0.05),生后20天各指标均明显低于前两组,较对照无明显差别(P>0.05)。部分子豚鼠肉眼可见大便变白,伴TB和DB水平的升高。新生鼠组病毒接种后10天AST水平较20天组和空白对照升高(P=0.027,P=0.043)。
3,63.6%的孕晚期感染子豚鼠肝脏存在单核淋巴细胞浸润为主的炎症／病理改变,炎性细胞浸润以汇管区为主。可见小叶间汇管区的破坏和纤维化改变,合并小胆管增生。伴有肝细胞气球样变性和坏死,肝内胆汁淤积。偶可见肝内微脓肿形成。新生鼠接种后10天出现肝脏病变(3／8),但病变较孕晚期组明显减轻。幼鼠组未见明显肝脏病变。
4,有肝胆损伤者肝内细胞凋亡信号密度分布基本与炎症浸润程度一致,主要表达于肝脏实质细胞和汇管区间质细胞,血管和胆管上皮偶见信号表达。孕晚期组出生时肝脏细胞凋亡指数均值6.7±1.322,汇管区细胞凋亡指数均值5.13±2.112,与对照相比有明显增加(P<0.05),与新生鼠组肝损伤者凋亡指数无明显差别。
5,病毒gpCMV阳性杂交信号表达于胆管上皮及血管内皮细胞及汇管区基质内,但肝细胞内未见表达。孕晚期组豚鼠gpCMV阳性率17.27%,而新生鼠及幼鼠组豚鼠肝胆系gpCMV-mRNA阳性率(55%,50%)均明显高于孕晚期组(P=0.011,0.018<0.05)。
6,新生鼠感染后10天,20天胆汁中可见HGF表达增加(P=0.0292,P=0.0461)。各组胆汁标本中未见IL-6的表达。
7,孕晚期组(8／14)和新生鼠组(3／3)肝胆损伤者有CD8+细胞表达,主要分布于肝脏实质和汇管区间质。
二,轮状病毒致小鼠胆管损伤关键基因的研究：
1,在RRV对EDIM的体内杂交中,得到单基因重排株4株,将EDIM背景单基因重排株D6／2接种新生鼠后(第4基因节段来自RRV),诱导出与RRV类似的胆管损伤症状。RRV对TUCH的体外混合感染中(包括回交及子代杂交),分离出所有22个RRV或TUCH背景下单基因重排病毒株。分别命名为RTn(RRV背景重排株,第n个基因节段来自TUCH)或TRn(TUCH背景重排株,第n个基因节段来自RRV)。n代表第1至11个基因节段。
2,致病性分析发现：重排株RT4没有致病性。相反,重排株TR4诱导出与RRV相似的黄疸症状,致病率(94.2%vs.100%,P=1.000)及致死率(88.24%vs.80.96%,P=1.000)无明显差别。58.33%的RT3感染小鼠表现胆道梗阻症状,其致病率和致死率明显低于RRV(p=0.001,0.000<0.05)。相对应的TR3尽管诱导出与RRV相似的致病率(88.89%,P=0.218),77.78%的小鼠最终黄疸症状消退并存活。其余RRV背景重排病毒株疾病的表现型与RRV类似。TR7、TR1和TR2一样未诱导出任何肝胆损伤症状。其余大多数TUCH背景单基因重排株诱导—过性肝胆损伤症状,几乎所有小鼠最终存活。
3,病理分析证实各重排株的疾病表现型与汇管区及肝外胆道病理改变密切相关。
4,细胞结合能力：重排株RT4的细胞结合率(3.97%±0.93%)明显低于RRV(16.03%±1.31%,P=0.003＜0.05)及其他所有RRV背景重排株(P＜0.05)。其余RRV背景重排株细胞结合率与RRV无明显差别(P＞0.05)。相反,重排株TR4(12.43%±2.25%)表现出明显高于TUCH：5.33%±1.27%,p=0.001)及所有其他TUCH背景重排株的细胞结合率(P<0.05)。除了TR8和TR2外,其它TUCH背景重排株细胞结合率与TUCH相似(P>0.05)。
5,感染性分析：克隆RT4在胆管上皮细胞系和肝外胆道的滴度均明显低于RRV(P=0.013,0.000)。相对应的克隆TR4的滴度无论在胆管细胞系还是小鼠肝外胆道均明显高于TUCH(P=0.004,0.0446)。在胆管细胞系上,所有其它RRV背景重排株的滴度与RRV类似(P＞0.05),在肝外胆道大部分低于RRV(P＜0.05)。TR7、TR1和TR2在胆管细胞系及肝外胆道滴度均低于TUCH(P=0.001,0.012,0.0069)。其余引起症状TUCH背景病重排株在胆管细胞系的滴度均高于TUCH(P＜0.05)。等级相关分析提示TUCH背景重排株在肝外胆道平均滴度与病毒致病率相关。
结论：
一,围生期豚鼠巨细胞病毒感染模型建立及分析：
1,孕晚期和出生后即刻接种gpCMV病毒可导致子代豚鼠的感染和肝胆系统炎症损伤。
2,肝内和汇管区浸润炎性细胞以单核淋巴细胞为主,伴有CD8+T细胞的表达,提示Thl抗病毒免疫在此病理过程中发挥重要作用。
3,病毒mRNA主要表达于汇管区及内皮系统,证实了CMV病毒对胆管上皮的亲和性。其攻击胆管上皮标本并引起炎症损伤的机制待进一步研究。
4,损伤主要发生于围产期,与接种时间直接相关,表明宿主免疫状态不成熟可能是致病的重要背景因素。
二,轮状病毒致小鼠胆管损伤关键基因的研究：
1,基因节段4是决定小鼠胆道模型RRV对胆管上皮致病性的关键基因,也决定了RRV对胆管上皮感染的特异性,其机制在于影响病毒与胆管上皮细胞的结合能力,继而影响病毒在细胞内复制,其蛋白产物VP4与胆管上皮相关受体的相互作用很可能是决定胆管细胞对病毒易感性的关键环节。
2,基因节段7被替代后,病毒的致病性、细胞结合能力和胆管上皮复制均未受影响,其蛋白产物VP7相关细胞表面受体的表达可能对胆管上皮的易感性影响不大。
3,基因节段3对RRV致病性有重要影响,其被替代所带来的“中间型”可能是包括宿主因素在内的多因素相互作用的结果。
4,基因节段1,2对病毒致病性无明显影响,但两者之间可能在病毒复制中存在协同作用。
Backgrounds and Purpose:Biliary atresia (BA) is a progressive, inflammatory cholangiopathy of infancy that leads to obstruction of extra-and intrahepatic bile ducts with eventual biliary cirrhosis. Despite its importance in child health, the cause of biliary atresia remains unclear. Evidence accumulated from both human and animal studies indicated that one possible pathological mechanism is the host immune response triggered by perinatal viral infection. Among the suspected viral agents, cytomegalovirus (CMV) and rotavirus drew more attentions. High incidence of cytomegalovirus CMV infection was found in patients with BA, but no solid evidence supported that the infection was the causing factor of the biliary system obstruction. So one purpose of our study is to set up a perinatal CMV infection induced hepatobiliary injury model, analyze the pathological change in hepatobiliary system and systemic inflammatory or immune response in the host.The study would provide important information on understanding the role of CMV in the inducing of the bile injury or even obstruction. The rhesus rotavirus (RRV)-induced murine model shares many similarities to the disease process found in children affected with BA and were used as a vital tool in analyzing various aspect of the infectious process in biliary atresia. As rotavirus families are refractory to the reverse genetic technique, the studies aimed to understand the molecular mechanism that characterizes the rotavirus life cycle and the pathogenesis of viral infection was hampered. Take advantage of a well-known property of rotavirus that they undergo genetic reassortment after mixed infection in cell culture or in vivo, we aim to create virus with single gene segment reassortment. Administrate these reassortants to neonatal mice, characterize their disease phenotype, we will define the key gene segment which determine the viral virulent to biliary tract. We also aim to define the role of other gene segments in this model. Cell binding assay and infectivity analysis were also employed to verify the mechanism by which the gene segments or its protein products worked. The studies will contribute to the understanding the initial stage that virus attact target cell, shadow light on a potential mechanism by which biliary atresia occurs.
Material and methods:A, Creation of perinatal CMV infection induced hepatobiliary injury model and injury evaluation:Guinea pig cytomegalovirus (gpCMV) were maintained in fetal guinea pig pulmonary fibroblast cells.The experiment has 3 groups:Prenatal group:Female guinea pigs at the 40th to 43th pregnant day (3rd trimester) were introperitoneally injected with virus supernates at a dose of 1×109TCID50 per dam. Two other subsets of dams severed as saline or blank control. Alive pups were sacrificed at different time point after birth. Neonatal group:A subset of healthy pups was introperitoneally injected with virus supernates within 24 hours of birth at a dose of 1×108 TCID50 per pup, Pups were sacrificed at different time point after inoculation. Infantile Group:Another subset of healthy pups was inoculated with a same dose of virus supernates at day 10 after birth. Pups were sacrificed at different time point after inoculation. Samples of the liver and proximal extrohepatic bile duct, blood, bile were collected. Weight gain, clinical sign of hepatobiliary injury and survival were recorded. Hepatic biochemical analysis was performed. A portion of the liver and extrohepatic bile duct were preserved in formalin. Serial sections underwent hematoxylin and eosin (HE) staining or in situ apoptosis analysis or immunohistochemical analysis for the presence of CD8+T cells. The expression of hepatic growth Factor (HGF), interleukin-6 (IL-6) in bile was examined with enzyme linked immunosorbent assay (ELISA). Other portion of liver and extrohepatic bile duct were iced and underwent in situ hybridization for the distribution of viral mRNA. B. Determine the specific RRV gene domain responsible for the injury bile duct injury in murine. The Virulent strain RRV was matched with avirulent strain EDIM (in vivo) or TCUH (in vitro). A subset of reassortants with double or triple gene segment reassortment was adapted to back cross with parental strain or cross with other progeny clones. MA 104 cells was infected with virus supernants generated from matching, the cell plates were overlayed with 0.2% agarose in EBSS, the plaque purified virus were freeze-thawed once and genotyped by polyacrylamide gel electrophoresis. Newborn balb/c mice were inoculated with reassortants, monitored for 21 days. A subset of pups was sacrificed at the 7th day after injection for histological analysis. The reassortant's binding ability was analyzed: immortalized cholangiocytes were inoculated with reassortant clones at 4℃for 1 hour; the amount of attached virus was expressed as a percentage of the total amount of virus used to inoculate the cells. In the infectivity essay, immortalized cholangiocytes were infected with reassortant at MOI of 1.the viral titer was measured after 48 hours. The extrohepatic bile ducts collected from mice at 7 days post inoculation were homogenized, tiered on the MA 104 cells.
Results:A.In guinea pigs, perinatal inoculation of gpCMV induced signs of hepatobiliary system injury.
1. Birth weight of pups in prenatal group was lower than saline or blank controls (P=0.0029, P=0027, respectively). These pups developed growth retardation versus that of saline and blank controls. Pups in neonatal and infantile group showed no sign of growth retardation.
2. TB, DB, ALT, AST levels in Pups of prenatal group were higher at day 1 or day 10 after birth as compared to controls (P<0.05), while they were not significantly different between these two time points (P>0.05). Those parameters at day 20 decreased as compared to those at day 1 or day 10. A few pups in prenatal showed sign of jaundice within 10 days after birth, which were associated with increased TB and DB level. In neonatal group, a temporary increased AST level was observed at day 10-post injection.
3. Histological analysis of the livers revealed a notable infiltration of mononuclear cells in 63.6%(14/22) of pups in prenatal group. Inflammatory cells were mainly found in portal tract area. There was interlobular portal tract damage and fibrosis, bile ductular proliferation. Hepatocytes balloon-like degeneration, necrosis and ductular cholestasis were often found. Micro-abscesses were seen (2/14). Pathological changes were also found in pups of neonatal group (3/8, but not as severe as those in prenatal group. No notable pathologic change was found in infantile group.
4. Apoptosis signals were mainly seen in hepatocytes and mesenchymal cells in portal tract, coincident with the distribution of inflammatory cells, no definite signal was observed in vascular or ductular epithelial cells.
5. Viral distribution:gpCMV mRNA signal were found in endothelial and epithelial cells and portal tract stoma.No signal was found on hepatocytes. Positive rate of gpCMV was 17.27%(4/22) in pups of prenatal group, significantly lower than those of neonatal or infantile groups (55%,50%respectively).
6. HGF level increased in bile from pups of neonatal group and infantile group (P=0.0292, P=0.0461). No IL-6 was found in any sample with this method.
7. CD8+T cells were often found in samples of prenatal group (8/14) and neonatal group (3/3), distributed mainly in liver parenchyma and portal area.
B. In the murine biliary atresia model,
1. Reassortant creation and pathogenecity assessment:21 reassortants were generated in the matching of RRV with EDIM, among of them only 4 were clones with single gene reassortment (4/170). Only one of them was on EDIM background. The reassortant D6/2(EDIM background strain with the 4th gene segment derived from RRV) caused signs of biliary injury that was similar with that of RRV. In the crossing of RRV with TUCH (back crossing or progeny crossing included), all the 22 single-segment reassortants were generated and selected to inoculate newborn pups. They were named as RTn (RRV background clone with the nth gene segment from TUCH) TRn (TUCH background clone with the nth gene segment from RRV).
2.Clone RT4 did not induce disease, on the contrary, clone TR4 carried on signs of biliary obstruction in 94.12% of pups and produced a mortality rate of 88.24%, which is of no difference with that of pups injected with RRV (P=1.000＞0.05). An intermediate phenotype was seen in RT3 (RRV background strain with the 3rd gene segment deriving from TUCH) infected mice, although 58.33 percent of pups developed sign of biliary obstruction, the rate was significantly lower than that of mice injected with RRV (P=0.001＜0.05), so was the mortality rate (20.83%, P=0.000<0.05). Pups injected with other RRV background reassortants developed manifestation of biliary obstruction, which were similar with that in RRV induced model. Reassortant clone TR7as well as TR1 and TR2, did not elicit any sign of hepatobiliary injury nor did they cause mortality. Most of other TUCH background reassortants brought on temporary sign of hepatobiliary injury, and most of the pups survived.
3. The histological appearances of the portal area as well as the extrohepatic bile ducts from different strain infected mice were consistent with the mice's symptoms.
4. Viral binding ability to cholangiocyte was significantly changed by manipulating the 4th gene segment. The clone RT4's binding ratio (3.97%±0.93%) was much lower than that of the parental strain RRV (16.03%±1.31%) (P=0.003<0.05) and that of other RRV background strains (P＜0.05). Other RRV background reassortants'binding ratios were not significantly different from that of parental strains (P＜0.05). As we prospected, replacement of the 4th gene segment of non-pathogenic TUCH strain with corresponding gene from RRV enhanced the viral attachment, clone TR4 exhibited a significantly higher binding percentage (12.43%±2.25%) as compared with that of parental strain TUCH (5.33%±1.27%, p=0.001). Besides TR8 and TR2 other TUCH background clones showed a similar binding ratio as compared with that of TUCH (P>0.05)
5.Consistent with the finding in binding assay, clone RT's titer in cholangiocytes as well as in bile duct at day 7 post inoculation was significantly lower than that of RRV(P= 0.013,0.000 accordingly).On the other hand, clone TR4's titer were definitely higher than that of TUCH both in vitro and in vivo (P=0.046,0.004 accordingly). On the cholangiocytes, all the other RRV background clones'titer were similar with that of parental strain RRV (P>0.05), but most of those (except for RT8, RT9) were lower than that of RRV in vivo (P<0.05). Consistent with the observation that mice injected with R1,R2 and R7 did not show any symptoms of the disease, the viral titer both in chlangiocytes and in bile duct were even lower than TUCH (P＜0.05). Alternatively, most of the other symptoms inducing strains titer were higher than that of parental strain TUCH (P<0.05) in vitro. Spearman correlation analysis indicated the mean titers were related to the morbidity of the disease among TUCH background reassortants.
Conclusion:
A.In the guinea pigs model,
1 Introperitoneally inoculating the third trimester pregnant dams or neonatal pups with gpCMV could induce inflammation and injury in hepatobiliary system.
2 Intra-hepatic infiltrated inflammatory cells were mainly mononuclear cells, parts of which were CD8+T cells. Those indicated Thl immune response played an important role in the phenotypic progression.
3 Viral mRNA signals were mainly found in portal tract and endothelial system, the tropism of gpCMV to bile duct epithelial cells and the corresponding damage to bile duct system were proved.
4 All injury phenotypes were from pups of prenatal and neonatal groups, closely related to the inoculation time, which indicated immature host immune system might play a key role in the process.
B. In the murine biliary atresia model:
1 Rhesus rotavirus gene segment 4 was the major determinants of pathogenecity. It also determined the RRV's specific tropism to cholangiocytes. Manipulation of gene segment 4 greatly changed the viral binding ability and infectivity, may in turn, and determined the viral phenotype. Expression of related cell surface receptor with binding site for VP4 (product of gene segment 4) might determine the susceptibility of changiocyte to viral insults.
2 Gene segment 7 did not contribute to the viral pathogenecity. Replacement of the 7th segment did not affect the viral binding; either did it affect its replication on cholangiocytes. Indicated the gene (encoding VP7) did not contribute to the virulence, the cell surface binding receptor related to VP7 might not play an important role in cholangiocyte susceptibility.
3 Gene segment 3 (encoding VP3) played an important role in RRV's pathogenecity, the intermediated virulent phenotype induced by reassortant TR3 probably was the result of interaction of multiple-factors, among them, host immune response may take an important role.
4 Gene segment 1,2 did not contribute to the viral pathogenecity, but they might coordinate in the process of viral replication.
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[30]Schleiss MR. Nonprimate Models of Congenital Cytomegalovirus (CMV) Infection:Gaining Insight into Pathog enesis and Prevention of Disease in Newborns.ILAR Journal.47(1)
[31]王听荣,陈素华,乔福元等.子宫巨细胞病毒感染与妊娠结局的相关性研究.中华医学杂志[J].2004,84(17)：1445-1446
[32]Morotti RA. Tata M, Drut R,Liver Pathology In Chinldren With AIDS:A Comparison Between The South American And North American Population.Pediatric Pathology and Molecular Medicine [J].2001,20: 537-545
[33]Lautenschlager I. Halme L. H ckerstedt K. Krogerus L. Cytomegalovirus infection of the liver transplant:virological, histological,immunological, and clinical observations Transpl Infect Dis [J].2006:8:21-30
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[12]Petersen C, Bruns E, Kuske M, et al. Treatment of extrahepatic biliary atresia with interferon-alpha in a murine infectious model. Pediatr Res [J].1997,42:623-8.
[13]Qiao H, De Vincentes A, Alashari M, et al. Pathogenesis of rotavirus-induced bile duct obstruction (a model for human biliary atresia) in normal Balb/c and CB17 scid mice with severe combined immunodeficiency (SCID). Pediatr Res [J].1999,45:116A.
[14]Allen, S. R., M. Jafri, B. Donnelly, M. McNeal, D. Witte, J. Bezerra, R. Ward, and G. M. Tiao.2007. Effect of rotavirus strain on the murine model of biliary atresia. J. Virol [J].81:1671-1679.
[15]Estes, M. K. Rotaviruses and their replication.In D. M. Knipe and P. M. Howley (ed.), Fields virology[M],4th ed. Lippincott-Raven Publishers, Philadelphia, Pa.2001:1747-1785.
[16]Ball, J. M., P. Tian, C. Q.-Y. Zeng, A.Morris, and M. K. Estes. Age-dependent diarrhea is induced by a viral nonstructural glycoprotein. Science [J].1996,272:101-104.
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[19]Hoshino, Y, L. J. Saif, S. Y Kang, M. M. Sereno, W. K. Chen, and A. Z. Kapikian. Identification of group A rotavirus genes associated with virulence of a porcine rotavirus and host range restriction of a human rotavirus in the gnotobiotic piglet model. Virology [J].1995,209:274-2
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[21]Broome, R. L., P. T. V, R. L. Ward, H. F. Clark, and H. B. Greenberg. Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restriction and virulence. J. Virol [J].1993,67:2448-2455.
[22]Arias CF, Isa P, Guerrero CA, Mendez E, Zarate S, Lopez T, Espinosa R, Romero P, Lopez S. Molecular biology of rotavirus cell entry. Arch. Med. Res [J].2002,33 (4):356-61.
[23]Schleiss MR.Animal models of congenital cytomeglovirus infection:an overview of progress in the characteristic of guinea pig cytomeglovirus.J Clin Virol [J].2002(Suppl.2),25:37-49
[24]Schleiss MR, Bourne N, Bravo FJ,et al.Quantitive-competitive PCR monitoring of Viral load following experimental guinea pig cytomegalovirus
infection.J Virol Meth 2003 [J].108:103-110
[25]傅继华.病毒学实用实验技术[M].济南：山东科学技术出版社,2001：60-64
[26]Jafri M, Donnelly B, McNeal M, Ward R, Tiao G. MAPK signaling pathway contributes to rotavirual induced cholangiocyte injury and viral replication.Surgery [J].2007,142:192-201.
[27]熊锦文,陈素华,闻良珍.套式聚合酶链反应检测豚鼠巨细胞病毒宫内感染.中华实验和临床病毒学杂志[J].2004,18(1)：34
[28]Schleiss MR, Lacayo JC. The guinea pig model of congenital CMV infection. In:Reddehase MJ, ed. Cytomegaloviruses:Pathogenesis, Molecular Biology and Infection Control [M]. Wymondham Norfolk UK:Horizon Scientific Press Ltd.2005, p 525-550
[29]Schleiss MR, Heineman TC. Progress toward an elusive goal:Current status of cytomegalovirus vaccines. Expert Rev Vaccines [J].2005,4:381-406
[30]Schleiss MR. Nonprimate Models of Congenital Cytomegalovirus (CMV) Infection:Gaining Insight into Pathog enesis and Prevention of Disease in Newborns.ILAR Journal.47(1)
[31]王听荣,陈素华,乔福元等.子宫巨细胞病毒感染与妊娠结局的相关性研究.中华医学杂志[J].2004,84(17)：1445-1446
[32]Morotti RA. Tata M, Drut R,Liver Pathology In Chinldren With AIDS:A Comparison Between The South American And North American Population.Pediatric Pathology and Molecular Medicine [J].2001,20: 537-545
[33]Lautenschlager I. Halme L. H ckerstedt K. Krogerus L. Cytomegalovirus infection of the liver transplant:virological, histological,immunological, and clinical observations Transpl Infect Dis [J].2006:8:21-30
[34]Muraji T, Hosaka N, Irie N,et al.Maternal Microchimerism in Underlying Pathogenesis of Biliary Atresia:Quantification and Phenotypes of Maternal Cells in the Liver Pediatrics [J].2008,121:517-521
[35]M. Lurie, I. Elmalamch, L. Schuger, Z. Weintraub Liver findings in Infantile cytomegalovirus infection:similarity to extrah epatic biliary obstruction.Histopathology[J].1987,11 (11),1171-1180.28
[36]Kristiansen TZ,Bunkenborg J,Gronborg M,et al.A proteomic analysis of human bile.Mol cell proteomics [J].2004,3:715-728
[37]A J Demetris, John G Lunz III, Susan Specht, Isao Nozaki.Biliary wound healing, ductular reactions, and IL-6/gp130 signaling in the development of liver disease.World J Gastroenterol [J].2006, June 14; 12(22):3512-3522
[38]Strazzabosco M, Fabris L, Spirli C.Pathophysiology of Cholangiopathies.J Clin Gastroenterol [J]2005,39:S90-S102
[39]Ahmad M, Jan M,Ali M,et al.Neonatal cholestasis in Kashmiri children.JK Pract[J].2000,7(2):125-6
[40]Holt P.GFunctionally mature virus-specific CD8+ T memory cells in congenitally infected newborns:proof of principle for neonatal vaccination? J Clin Invest[J].2003 June 1; 111(11):1645-1647
[41]Elbou Ould, M. A., Luton, D., Yadini, M., et al. Cellular Immune Response of Fetuses to Cytomegalovirus. Pediatric Research[J].2004, (55):280-286
[42]Ramig, R. F. Genetics of the rotaviruses. Annu. Rev. Microbiol [J].1997, 51:225-255
[43]T Vesikari, AZ Kapikian, A Delem and G Zissis, A comparative trial of rhesus monkey (RRV-1) and bovine (RIT 4237) oral rotavirus vaccines in young children.J Infect Dis [J].1986,153:832-839.
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