猴免疫缺陷病毒（SIV）在艾滋病模型恒河猴组织中的分布和载量测定

目的研究当艾滋病恒河猴模型的血浆病毒载量处于低水平或阴性时,猴免疫缺陷病毒（simian immunodeficiency viruses,SIV）在宿主组织中的分布情况。方法SIVmac251感染恒河猴10只,定期检测其血浆载量,感染病毒平均高峰时间第14天时,活检取淋巴结。选取感染18个月后病毒载量最低水平和阴性的2只艾滋病猴（SAIDS）,经安死术后取淋巴结、脾、肝、肺、肾、脑等组织,用原位杂交和实时荧光定量PCR的方法检测病毒在组织中的分布和组织中的病毒载量。结果感染后14d,10只猴血浆病毒载量达到10^7copies/mL,淋巴结组织病毒载量为10^5-10^8copies/g,原位杂交方法在腹股沟淋巴结中检测到强阳性斑点。感染后第18个月的2只猴,血浆病毒载量下降并维持不高于10^2copies/mL水平或阴性,但组织分布不尽相同,在肠系膜淋巴结、肾上腺、海马回、空肠、脾脏等组织中检测到10^5-10^6copies/g的病毒载量,于一只猴的脑积液中检测到10^3copies/mL的病毒载量。用原位杂交的方法在肠系膜淋巴结和空肠中检测到强阳性斑点,其它组织中未检测到阳性斑点。结论实验证实SAIDS猴在血浆病毒载量低甚至阴性时,病毒在不同组织中仍有分布,有些组织中甚至出现高病毒载量,提示在制备SIV/SAIDS模型中,尤其在药物筛选和疫苗评价时,应考虑组织病毒载量指标的测定和药物、疫苗对组织病毒的治疗清除作用的评价。     

裸鼠呼吸道合胞病毒感染的动物模型

呼吸道合胞病毒(RSV)是全世界婴幼儿下呼吸道感染的首位病毒病原体,免疫缺陷个体容易发生严重感染,目前尚无理想RSV感染动物模型用于研究。我们用细胞免疫缺陷裸鼠感染RSV,旨在建立理想的动物模型,为RSV感染的防治研究奠定基础。裸鼠滴鼻感染RSV后肺组织分离到病毒,直接免疫荧光检测到支气管肺泡灌洗液RSV抗原阳性,空斑形成实验检测肺组织病毒滴度在感染后第3天达高峰,并持续到第9天仍能检测到病毒。免疫组化检测RSV抗原主要分布在细支气管、毛细支气管和肺泡上皮细胞胞浆内。肺组织病理学显示RSV感染导致裸鼠淋巴细胞浸润为主的肺间质性炎症,电镜分析超微结构可见到细胞内病毒颗粒和气血屏障的破坏。支气管肺泡灌洗液白细胞计数显示裸鼠RSV感染炎症高峰在感染后第9天。裸鼠RSV感染的病毒复制和病理改变特点与人相似,病毒持续高水平复制,是客观而实用的评价抗RSV制剂效果的小鼠模型。     

裸鼠呼吸道合胞病毒感染的动物模型

呼吸道合胞病毒(RSV)是全世界婴幼儿下呼吸道感染的首位病毒病原体,免疫缺陷个体容易发生严重感染,目前尚无理想RSV感染动物模型用于研究.我们用细胞免疫缺陷裸鼠感染RSV,旨在建立理想的动物模型,为RSV感染的防治研究奠定基础.裸鼠滴鼻感染RSV后肺组织分离到病毒,直接免疫荧光检测到支气管肺泡灌洗液RSV抗原阳性,空斑形成实验检测肺组织病毒滴度在感染后第3天达高峰,并持续到第9天仍能检测到病毒.免疫组化检测RSV抗原主要分布在细支气管、毛细支气管和肺泡上皮细胞胞浆内.肺组织病理学显示RSV感染导致裸鼠淋巴细胞浸润为主的肺间质性炎症,电镜分析超微结构可见到细胞内病毒颗粒和气血屏障的破坏.支气管肺泡灌洗液白细胞计数显示裸鼠RSV感染炎症高峰在感染后第9天.裸鼠RSV感染的病毒复制和病理改变特点与人相似,病毒持续高水平复制,是客观而实用的评价抗RSV制剂效果的小鼠模型.     

减蛋综合征病毒在不同品系小鼠体内的组织分布

目的通过人工感染减蛋综合征病毒(egg drop syndrome virus,EDSV),观察病毒在不同品系小鼠体内增殖情况以及动态变化规律,为EDSV构建载体提供理论依据与数据支持。方法选取免疫系统正常的BALB/c小鼠、T细胞免疫缺陷裸鼠(Nu)以及高度免疫缺陷小鼠(NSG)为研究对象,每品系32只,雌性,5～6周龄,经腹腔注射人工感染EDSV,分别于攻毒后1、3、5、7、14、21、28、35 d采集血清,应用间接ELISA方法进行抗体监测;选择攻毒后1、7、14、21、28 d小鼠,采集心脏、肺、肝、脾、肾、小肠、子宫、气管、食管、脑10种组织,应用荧光定量PCR相对定量比较Ct法(△△CT)进行各组织内病毒载量的检测。结果 BALB/c小鼠于攻毒后3 d即可在血清内检测到抗体的表达,14 d抗体水平达到最高,并一直维持至监测期内35 d;Nu小鼠也可于攻毒后3 d检测到抗体,表达水平较BALB/c小鼠有所降低,攻毒14 d后,Nu小鼠血清中抗体水平出现下降,至35 d抗体一直维持在较低的水平;NSG小鼠在整个监测过程中,抗体水平一直处于阴性状态。核酸相对定量结果显示,BALB/c小鼠感染后1 d,肝组织中的病毒表达量最高,达到5.45个数量级,其次由高到低依次是脾、食管、子宫、小肠、肺、气管、肾、心脏,脑组织中病毒含量最低,随感染时间的延长,各组织内病毒表达量较感染1 d均有所下降,至攻毒后28 d,肝、脾病毒表达量依然维持着较高的水平;Nu小鼠和NSG小鼠感染1 d表现为脾中病毒表达量最高,分别为3.95和4.05个数量级,其次为肝,攻毒28 d,两种小鼠体内各器官内仍可以检出阳性信号,肝、脾病毒表达量较高。结论 EDSV可刺激小鼠产生免疫应答,在免疫缺陷小鼠体内抗体水平表达量较低。该病毒在小鼠体内有肝、脾等组织嗜性,为EDSV开发成为载体以及在实验动物模型上的进一步研究与应用提供了参考数据。     

雪貂感染流感病毒H3N2动物模型的建立

目的建立甲型流感病毒H3N2感染的雪貂动物模型。方法按实验要求筛选出流感抗体反应阴性的雪貂,经兽用氯胺酮轻度麻醉后进行滴鼻感染H3N2流感病毒株A/Brisbane/10/07,设立两个稀释度106和107 TCID50,每个稀释度接种3只雪貂,感染后第5天安乐处死。感染前采集鼻甲骨活检,感染后1～5 d鼻甲骨活检检测病毒载量,每天记录雪貂一般临床变化。处死时取雪貂肺、肝、脾、小肠、脑组织作病毒滴度检测,肺组织做病理检查。结果 106和107TCID50的H3N2病毒分别感染雪貂,没有雪貂死亡。雪貂感染后都出现一过性的体温升高,体重的下降,流涕、打喷嚏等症状。在鼻甲骨活检物中可测到病毒载量,肠组织可分离到病毒。肺组织以轻度性间质性肺炎为主要病理变化。结论雪貂感染H3N2病毒株A/Brisbane/10/07后,临床表现、病毒学、分子生物学、病理学方面的检测都可以证实雪貂感染H3N2病毒动物模型已建立,其中106 TCID50病毒滴度的是一个建立感染动物模型比较合适的剂量。     

H7N9禽流感病毒小鼠感染动物模型的建立

目的建立H7N9禽流感病毒小鼠感染模型。方法 1×108,1×107或1×106TCID50H7N9禽流感病毒原液(A/Anhui/1/2013)滴鼻感染BALB/c小鼠。主要观测指标:临床症状、死亡率、病理变化、病毒载量和血清抗体检测。结果被感染的小鼠表现为竖毛、弓背、体重下降;病理表现为间质性肺炎,感染后第2天开始在呼吸道脱落细胞中检测到病毒;免疫组化或病毒分离方法在肺、肾、脑、肠、脾等组织检测到病毒;感染后14 d在小鼠血清中血凝抑制试验特异性抗体效价达到160;淋巴细胞减少,中性粒细胞增多。结论 H7N9感染BALB/c小鼠模型与人类禽流感感染疾病的基本特征相似,为研究该病的发病机制及药物疫苗的研发提供了工作基础。     

登革Ⅱ型病毒在白纹伊蚊体内分布的研究

利用蚊虫连续石蜡切片免疫组织化学技术,对登革Ⅱ型病毒(DEN-2)感染白纹伊蚊Aedes albopictus后的散播时间、程度及组织器官的感染顺序进行监测,以了解DEN-2在媒介白纹伊蚊体内的分布规律。结果表明：大剂量感染登革Ⅱ型病毒后,在蚊虫消化道的主要部位以及大多数组织器官包括神经及内分泌系统在内,如涎腺、脑、神经节等亦检测到病毒抗原。登革Ⅱ型病毒一旦感染并逸出中肠会迅速侵染其它组织。从各组织感染率的高低推断,病毒逸出中肠后通过血淋巴传播到其它组织的顺序通常为：前肠、涎腺、咽部神经节、脑及食管下神经节、后肠及复眼的小眼等。     

气管插管法接种H5N1病毒感染恒河猴及系统组织病毒检测

目的测试气管插管法接种高致病性禽流感病毒H5N1感染恒河猴的优势效果及疾病分析,为有效感染恒河猴、制备H5N1疾病模型提供实验依据。方法使用人源H5N1病毒液经气管插管滴入恒河猴上呼吸道进行感染,观察感染恒河猴的临床表现,每天采集咽拭子、鼻灌洗液,在感染前2d感染后第3、5、7天采血,感染后第3和7天分别解剖1只恒河猴,取支气管淋巴结、肠淋巴结、鼻甲、心、肝、脾、肺、肾、肠、气管、脑及血液进行病毒分离、核酸载量检测和血常规测定。结果感染后第2天恒河猴出现食欲下降,活动减少,并伴有一过性体温升高,白细胞数和淋巴细胞数下降。咽拭子、鼻灌洗液、肺、心、气管、脑、肝、肾、肠和血液中都能分离到H5N1病毒。结论气管插管法接种H5N1病毒能有效感染恒河猴,并在猴体内多组织中分离、检测到病毒,为制备完善的H5N1模型和检测指标确定、进一步研究H5N1病毒的致病机制等奠定了基础。     

血液通路在H5N1高致病性禽流感病毒入侵小鼠中枢神经系统中的作用

【目的】研究血液通路在H5N1高致病性禽流感病毒入侵小鼠中枢神经系统中的作用。【方法】用3株H5N1病毒滴鼻感染BALB/c小鼠,研究小鼠肺、脑、血中的病毒在感染后不同时间点的复制动态及病理进展,通过免疫组化和免疫荧光染色显示病毒在脑部血管内皮细胞及血管周围神经组织的感染情况。【结果】小鼠感染后病毒迅速在肺中高效复制,随即形成病毒血症;感染后第6天病毒在肺中的滴度和在血液样本中的检出率达到峰值,此时小鼠脑部才开始检测到病毒;小鼠脑内血管内皮细胞、脑血管周围神经组织的神经元和神经胶质细胞中可检测到流感病毒NP蛋白。【结论】血液播散可能是高致病性H5N1禽流感病毒进入中枢神经系统的途径之一。     

人H7N9禽流感病毒与H1N1流感病毒感染小鼠病理学损伤的比较

目的比较分析H7N9病毒与H1N1病毒感染小鼠病理学损伤特点,初步探讨两种病毒感染致小鼠急性肺损伤的致病机制。方法 H7N9病毒与H1N1病毒分别感染小鼠,观察不同病毒感染后小鼠生存率,并于不同时间点取心、肝、脾、肺、肾、脑、肠等组织,伊红-苏木素染色并进行组织病理学分析,免疫组化检测病毒抗原分布及中性粒细胞浸润。综合分析肺组织病理损伤与病毒复制、宿主免疫反应之间的关系。结果 H7N9病毒感染小鼠肺及脾脏损伤较轻,存活率较高。H1N1病毒感染的小鼠肺及脾脏损伤较重,感染后9 d全部死亡;两种病毒抗原主要分布于支气管上皮细胞、少量间质细胞和肺泡上皮细胞,病毒复制水平无明显差异。但H1N1病毒感染后肺及脾脏中均有大量中性粒细胞浸润,小鼠机体炎症反应明显强于H7N9病毒感染后小鼠炎症反应。结论 H7N9病毒与H1N1病毒感染后小鼠病理学损伤特点及程度均不同,病毒复制是小鼠肺损伤的诱发因素但并非决定因素,宿主针对病毒感染产生的免疫反应程度与急性肺损伤密切相关。     

Experimental challenge and clinical cases of Bohle iridovirus (BIV) in native Australian anurans

Ranaviruses have been observed with increasing frequency amongst poikilothermic vertebrate hosts. The impact of ranaviruses upon amphibian populations has remained largely unknown. A gene probe for Bohle iridovirus (BIV) based upon primers designed to detect epizootic haematopoietic necrosis virus (EHNV) was constructed. A PCR and dot-blot system was used successfully in screening for the presence of BIV nucleic acid in digested formalin-fixed, paraffin-embedded amphibian tissues. Juvenile frogs were more susceptible to BIV than adults. In experimental challenges and epizootics in captive frogs, juvenile Litoria caerulea, L. alboguttata, Cyclorana brevipes and Pseudophryne coriacea were acutely susceptible. High mortality (at or near 100%) resulted, usually occurring within 5 to 25 d depending on dose and method of exposure. Histopathological changes included mainly hepatic, renal and splenic necroses. Significant haemosiderosis was encountered in more chronically infected frogs. BIV could be reisolated from juvenile L. caerulea >40 d after inoculation, and >200 d after the first mortalities occurred in an epizootic in L. alboguttata. Adult L. rubella, L. inermis, L. caerulea, Cophixalus ornatus and Taudactylus acutirostris were less susceptible in trials ranging from 30 to > 100 d. There was some evidence of chronic infection, and BIV could be detected by PCR. Wild moribund adult L. caerulea from Townsville and captive juvenile Pseudophryne corieacea from Sydney undergoing mortality tested positive with the BIV PCR. PCR and dot blot was more sensitive than viral isolation. PCR could detect BIV in amphibians long after BIV challenge, and in amphibians which appeared healthy. Ranaviruses could be having an impact on Australian herpetofauna.     

Quantitation of mRNAs specific for the mixed-function oxidase system in rat liver and extrahepatic tissues during development

Evaluation of ontogenetic expression of the cytochrome P450PCN and cytochrome P450b gene families as well as the NADPH-cytochrome P450 oxidoreductase and epoxide hydrolase genes in Holtzmann rats showed that basal levels of mRNAs encoding these enzymes could be detected in most tissues. Distinct developmental patterns of mRNA expression are evident for these four proteins in liver and extrahepatic tissues. Levels of cytochrome P450b-like mRNA were comparable in adult lung and liver, while cytochrome P450PCN-homologous mRNA exhibited low levels in lung and approximately 100-fold higher levels in liver. Cytochrome P450PCN-homologous mRNA also reached substantial levels in adult intestine, and was also present in placenta, where it increased approximately 4-fold 24 h before birth. Epoxide hydrolase mRNA was demonstrated to be highest in liver followed by kidney, lung, and intestine but was extremely low in brain. NADPH-cytochrome P450 oxidoreductase mRNA in kidney, lung, prostate, adrenal, and intestine exhibited levels comparable to that found in liver; however, the pattern of expression for oxidoreductase mRNA was unique in that levels declined at maturity in liver, kidney, and intestine but not in lung and brain. Development of mixed-function oxidase and epoxide hydrolase activities in liver was distinct from that in other tissues in that mRNAs for all four proteins rose dramatically after parturition. Testis from immature males demonstrated low levels of all the mRNAs assayed, which ranged from 20% (oxidoreductase) to less than 1% (cytochrome P450PCN and epoxide hydrolase) of the levels found in liver.     

Endohelminth fauna of the marsh frog Rana ridibunda from Lake Hazar, Turkey

In this study, 236 marsh frogs Rana ridibunda collected from Lake Hazar (Elazig, Turkey) at 15 d intervals between March 2001 and February 2002 were examined for endohelminths; of these, 148 (62.71%) frogs were found to be infected with helminths. In total, 9 helminth species (3 trematodes, 5 nematodes and 1 acanthocephalan) were identified. We observed Gorgoderina vitelliloba (prevalence 2.97%) in the urinary bladder, Haematoloechus variegatus (4.66%) and Rhabdias bufonis (8.90%) in the lung, Pleurogenoides medians (1.69%), Oswaldocruzia filiformis (3.81 %) and Acanthocephalus ranae (26.27 %) in the small intestine, Neoxysomatium brevicaudatum (16.95%) and Cosmocercoides sp. (3.39%) in the large intestine, and Eustrongylides excisus (14.41%) in the body cavity and on,the stomach. No helminth was found in the spleen, kidney, gall bladder, liver, heart or muscle. Of the 9 helminth species identified, Acanthocephalus ranae (26.27 %) had the highest prevalence and abundance and Oswaldocruzia filiformis (8.33+/-4.09) had the highest mean intensity.     

不同日龄大白猪视黄酸受体α基因组织表达

研究采用RT-PCR方法对大白猪的视黄酸受体α基因在1日龄、90日龄、180日龄、270日龄和360日龄的心、肝、胃、脾、肾、肺、大肠、小肠、肌肉、子宫、卵巢共11个组织的表达情况进行了研究。结果表明,RARαmRNA在肝、脾、肾、大肠、小肠、子宫和卵巢中持续表达,其中脾、大肠和小肠是持续高表达;180日龄时,所有组织的RARαmRNA的表达量普遍降低;360日龄时,所检的11个组织均高水平表达该基因。     

Patterns of infection by lungworms, Rhabdias ranae and Haematoloechus spp., in northern leopard frogs: a relationship between sex and parasitism

We examined a population of northern leopard frogs to determine whether sex biases in investment in immunity, previously reported for this host species under controlled exposures to lung nematodes, is predictive of patterns of parasitism in nature. We examined Rhabdias ranae and Haematoloechus spp. infections in 74 breeding adult, 28 non-breeding adult, and 53 juvenile frogs. Contrary to our predictions, R. ranae prevalence and mean abundance were higher in breeding female frogs (prevalence: 39.4%, abundance: 3.05 +/- 0.85) than on breeding males (prevalence: 26.0%, abundance: 1.17 +/- 0.52), although no sex bias was observed among non-breeding adults or juvenile frogs. Female frogs also carried larger R. ranae worms, on average, than did males (females: 6407.38 microm +/- 153.80; males: 5198 microm +/- 131.09), regardless of age or breeding condition. We observed no sex-linked patterns of parasitism by Haematoloechus spp. worms in either adult or juvenile frogs. Alternative hypotheses, such as differences among sexes in the selection of thermal clines for hibernation, may explain the observed female bias in parasitism by nematode lungworms in nature and, thus, need to be considered.     

Cydia pomonella granulovirus Genotypes Overcome Virus Resistance in the Codling Moth and Improve Virus Efficiency by Selection against Resistant Hosts

Cydia pomonella granulovirus (CpGV) has been used for 15 years as a bioinsecticide in codling moth (Cydia pomonella) control. In 2004, some insect populations with low susceptibility to the virus were detected for the first time in southeast France. RGV, a laboratory colony of codling moths resistant to the CpGV-M isolate used in the field, was established with collection of resistant insects in the field followed by an introgression of the resistant trait into a susceptible colony (Sv). The resistance level (based on the 50% lethal concentrations [LC₅₀s]) of the RGV colony to the CpGV-M isolate, the active ingredient in all commercial virus formulations in Europe, appeared to be over 60,000-fold compared to the Sv colony. The efficiency of CpGV isolates from various other regions was tested on RGV. Among them, two isolates (I12 and NPP-R1) presented an increased pathogenicity on RGV. I12 had already been identified as effective against a resistant C. pomonella colony in Germany and was observed to partially overcome the resistance in the RGV colony. The recently identified isolate NPP-R1 showed an even higher pathogenicity on RGV than other isolates, with an LC₅₀ of 166 occlusion bodies (OBs)/μl, compared to 1.36 × 10⁶ OBs/μl for CpGV-M. Genetic characterization showed that NPP-R1 is a mixture of at least two genotypes, one of which is similar to CpGV-M. The 2016-r4 isolate obtained from four successive passages of NPP-R1 in RGV larvae had a sharply reduced proportion of the CpGV-M-like genotype and an increased pathogenicity against insects from the RGV colony.     

Field trials of CpGV virus isolates overcoming resistance to CpGV-M

The Cydia pomonella granulovirus (CpGV) has been used for many years as biological agent for codling moth control in apple orchards. Resistance to the Mexican strain of CpGV was detected in orchards in Germany, France and Italy. A laboratory insect colony was started from insects collected in a French resistant orchard. It was named RGV. Various virus isolates were identified as active against this resistant insect colony. Field tests were carried out in 2007 to test if the two virus isolates CpGV-I12 and NPP-R1 were effective in the field. Although these virus isolates were not able to reduce insect caused fruit damages, they significantly reduced the overwintering insect populations. NPP-R1 was subjected to eight passages on RGV larvae (NPP-R1.8) that improved its biological activity on RGV larvae. 2008 field trials were set up to test this improved virus strain, compared to CpGV-I12 and Madex plus active on RGV. These tests confirmed the ability to control both in susceptible and resistant insect populations.     

Apoptosis in the digestive tract of herbivorous Rana pipiens larvae and carnivorous Ceratophrys ornata larvae: An immunohistochemical study

The lifespan of herbivorous Rana pipiens larvae is ～3 months, while that of carnivorous Ceratophrys ornata larvae is only about 2 weeks. During metamorphic climax, the larval gut shortens dramatically, especially in R. pipiens, and its luminal epithelium is replaced by adult‐type epithelium. To determine when programmed cell death occurs during the metamorphic restructuring of the gut, we prepared cross‐sections of the stomach, small intestine, and large intestine from representative larval stages and from juvenile frogs of both species. The sections were incubated with monoclonal antibody against active caspase‐3, one of the key enzymes in the apoptotic cascade. We observed apoptosis in some luminal epithelial cells in each of the three regions of the larval gastrointestinal tract of both species. However, apoptotic cells appeared earlier in larval stages of R. pipiens than C. ornata and few were seen in juvenile frogs of either species. The results demonstrate the occurrence of apoptosis in the metamorphic remodeling of the gut of both R. pipiens larvae and C. ornata larvae. J. Morphol., 2011. © 2011 Wiley Periodicals, Inc.     

Canopy Closure and Emigration by Juvenile Gopher Frogs

ABSTRACT Although studies have addressed effects of abrupt transitions in habitat type (e.g., forest-clear-cut or forest-field edges) on amphibian movements, little is known about effects of more subtle habitat transitions on patterns of migration and habitat use in amphibians. We used radiotelemetry to study movement patterns of juvenile gopher frogs (Rana capito) emigrating from ponds that were surrounded by longleaf pine (Pinus palustris) forest that varied in structure as a result of fire suppression. Our primary purpose was to determine if frogs emigrate directionally from their natal ponds and select habitat at random during their first month following metamorphosis. We found that frogs emigrated in nonrandom directions from ponds that were surrounded by heterogeneous habitat and selected fire-maintained habitat that was associated with an open canopy, few hardwood trees, small amounts of leaf litter, and large amounts of wiregrass (Aristida beyrichiana). Fire-maintained habitat contained higher densities of burrows excavated by gopher tortoises (Gopherus polyphemus) and small mammals, which are the priamry refuge sites for both juvenile and adult gopher frogs. Frogs moved up to 691 m from their natal ponds, frequently crossed dirt roads, and even seemed to use these roads as migration corridors. To maintain suitable terrestrial habitat for gopher frogs, including habitat used by migrating individuals, it is important to apply frequent prescribed fire to uplands surrounding breeding ponds that lead all the way to the edges of breeding ponds, as well as through ponds during periodic droughts.