肠道病毒71型感染C57BL/6小鼠模型的初步建立

以1日龄C57BL/6乳鼠作为试验对象,分别通过颅内和腹腔注射肠道病毒71型(EV71)福建株感染,观察记录小鼠体重和体征,定期采集小鼠大脑、小肠、肺、肌肉4种组织,分析病毒载量,通过HE染色和免疫组化观察各种组织病理变化。结果:2种感染途径的C57BL/6乳鼠均于不同时间出现竖毛、弓背和消瘦症状。经腹腔注射感染的乳鼠能在肌肉组织中检测到病毒RNA,经颅内注射感染的乳鼠可在肌肉和肺组织中检测到病毒RNA。病理学及免疫组化结果显示:EV71福建株在骨骼肌中大量增殖,并可导致肌肉组织水肿和坏死、肺组织水肿充血以及神经元坏死等多种组织器官炎症反应。综上,初步建立通过颅内和腹腔注射感染EV71的C57BL/6乳鼠模型,为研究EV71病毒感染机制、建立更优的动物模型提供参考。     

鸭坦布苏病毒在雏麻鸭体内的分布及排毒

为了解鸭坦布苏病毒感（Duck Tembusu virus, DTMUV）染麻鸭的组织嗜性和排毒情况,应用RT-PCR方法检测鸭坦布苏病毒在麻鸭体内各组织的动态分布及和泄殖腔棉拭子的排毒情况。结果显示最早在感染第1 d于心脏、脾脏、肺脏、气管、胰腺、直肠可检测到鸭坦布苏病毒核酸的存在;至第7、9 d时,在各组织脏器中均仍能检测到该病毒核酸;至第21 d时,仅能在脑、胰腺和直肠中可检测到病毒核酸。病毒在脑和直肠中时间最长（25 dpi）,胰腺（21 dpi）和肝脏（21 dpi）次之。对不同时间采集的泄殖腔棉拭子检测结果显示,于感染第1 d即能检测到该病毒核酸,25 d后即检测不到该病毒的存在。以上检测结果表明鸭坦布苏病毒感染雏麻鸭后能迅速入侵麻鸭各组织脏器,主要侵染肝、肺、肾、胰腺和直肠,并可通过粪便等途径向外界排毒。     

应用qRT-PCR方法检测鼠痘病毒在小鼠不同组织中的差异分布

鼠痘(mouse pox)是由鼠痘病毒(Ectromelia virus,ECTV)感染实验小鼠引起的一种毁灭性、烈性传染病,为SPF小鼠必须排除的病原之一。本研究基于ECTV主要核蛋白P4b基因片段建立了实时定量PCR方法(real-time quantitative PCR,qRTPCR),用于检测C57BL/6小鼠感染ECTV后心脏、肝脏、脾脏、肺脏、肾脏和皮肤中的病毒载量,并用病理组织学观察验证了qRT-PCR方法的特异性和可靠性。结果表明,所建立的qRT-PCR方法能够准确检测小鼠感染后d5和d7不同组织的病毒载量,d5不同组织中病毒载量低于d7,与病理组织学检测结果一致。本研究建立的qRT-PCR检测方法且具有较高的灵敏性、特异性和可靠性,可用于ECTV感染细胞和组织器官中病毒载量、分布和消长动态的定性定量检测。     

猪乙型脑炎病毒GZ0409-31分离株E基因遗传进化分析

本研究采用RT-PCR方法对分离自贵州省发病猪睾丸组织中的流行性乙型脑炎病毒GZ0409-31株的E基因进行克隆和序列测定,利用生物信息学软件对E基因核苷酸序列和推导的氨基酸序列进行分子遗传进化关系分析。结果表明,乙型脑炎病毒GZ0409-31毒株与基因Ⅲ型的乙脑病毒毒株属同一分支,遗传进化关系较近。     

C57BL/6NcGAS基因敲除鼠的构建及对猪伪狂犬病病毒增殖的影响

胞质DNA感受器环鸟苷酸-腺苷酸合成酶(cyclic GMP-AMP synthase, cGAS)能够识别胞质中的双链DNA(double stranded DNA,dsDNA),从而激活Ⅰ型干扰素信号通路抵御外界病原微生物的感染。为研究cGAS基因对猪伪狂犬病病毒(PRV)增殖的影响，本试验首先利用CRISPR/Cas9基因编辑技术构建cGAS基因敲除小鼠，通过H&E染色检测C57BL/6N小鼠和C57BL/6N-cGAS^-/-小鼠肺脏和脑组织形态差异及感染PRV后肺脏组织中炎性浸润的影响；Q-PCR检测C57BL/6N小鼠和C57BL/6N-cGAS^-/-小鼠感染PRV后对PRV-gB、PRV-TK、IFN-β、ISG15和ISG20基因mRNA表达水平的差异；Western blot及免疫组化检测C57BL/6N小鼠和C57BL/6N-cGAS^-/-小鼠感染PRV后PRV-gB和PRV-gE蛋白表达水平；最后利用病毒PFU法检测cGAS敲除鼠对PRV子代病毒滴度的影响。Western blot检测结果显示...     

单纯疱疹病毒脑炎模型的研究

对单纯疱疹病毒(HSV)感染小鼠致病特点进行了观察,小鼠感染HSV第3天后开始发病,感染后第2天脑内可检测出病毒核酸,第3天脑内可分离出病毒,第4天病毒滴度达到高峰,发病2天内死亡。电镜观察感染鼠脑内组织出现超微结构改变,并可找到病毒颗粒。结果说明所建立的HSV脑炎模型可用于评价抗HSV药物。     

日本脑炎病毒感染 ICR小鼠模型的初步建立

为建立可表现重症日本脑炎病毒(Japanese encephalitis virus, JEV)感染中枢神经系统损害症状及病理表现的小鼠模型,以1日龄ICR乳鼠为试验对象,通过皮下注射及腹腔注射方式感染JEV SA14株,观察记录乳鼠体重、临床表现、存活情况。感染第5天处死所有乳鼠,采集脑、肺、脾、肝等4种组织,荧光定量PCR(qRT-PCR)结合核酸电泳分析病毒感染情况,通过苏木精-伊红染色及免疫组化检测组织病理变化。结果发现,皮下感染和腹腔感染乳鼠均于注射后第3天出现精神萎靡、嗜睡、运动功能障碍,症状逐渐加重,皮下注射感染模型临床状更明显。病理结果显示,乳鼠脑组织出现神经元坏死、炎症浸润现象,肺组织存在水肿充血、结构破坏。qRT-PCR产物核酸电泳显示,两种模型脑、肺、脾存在JEV复制,皮下注射感染模型肝组织存在病毒复制。本试验成功建立通过皮下注射感染JEV的ICR乳鼠模型,为研究JEV感染途径及发病机制奠定基础。     

猪群混合感染伪狂犬病毒及乙型脑炎病毒的鉴定与分离

云南省文山市某规模化猪场2015年7~8月频繁发生以种公猪一侧睾丸肿大,母猪流产、产死胎、木乃伊胎及弱仔为主要临床症状的繁殖障碍疫情。为了查明病因,对送检流产胎猪进行病理解剖,采集胎猪及胎盘病料进行猪瘟病毒、猪繁殖与呼吸综合征病毒欧洲型及美洲型毒株、猪细小病毒、伪狂犬病毒、乙型脑炎病毒、猪流产衣原体、布氏杆菌及弓形虫PCR和realtime PCR检测,同时应用猪肾原代细胞及BHK-21传代细胞进行病毒分离鉴定以及细菌分离鉴定。检测结果显示,从流产胎猪内脏器官、大脑及胎盘组织扩增出乙型脑炎病毒pr M基因及伪狂犬病毒g E基因目的条带。序列分析结果显示,扩增的乙型脑炎病毒pr M基因序列与NCBI Gen Bank中的参考毒株(Accession no.AB594829.1)序列同源性达99%;伪狂犬病毒g E基因核苷酸序列与NCBI Gen Bank中的参考毒株(Accession no.KT936475.1)序列同源性达100%。应用BHK-21传代细胞盲传分离获得1株可致细胞病变(cytopathic effect,CPE)的病毒株,鉴定为乙型脑炎病毒,并命名为JEV-2015-WS-YN。由此推测,引起此次猪群繁殖障碍极有可能为日本乙型脑炎病毒和伪狂犬病毒共同感染造成。     

免疫组化法检测番鸭呼肠孤病毒在番鸭体内的分布

选取10日龄健康番鸭84只,人工感染番鸭呼肠孤病毒,于攻毒后6、12、24、48、60、72、84、108、144、204、312、384 h取材,应用免疫组化方法对病毒在番鸭体内的分布进行检测,从而探索番鸭呼肠孤病毒感染的细胞及发病机理。检测结果表明,感染后6、12 h时各脏器均呈阴性反应;感染后24 h,从肝脏、肠道、大脑中最先检测到阳性细胞;感染后144 h,各脏器均呈强阳性;感染后204 h,脾脏、肺脏及胸腺的阳性反应强度仍持续,其他脏器的阳性反应强度都有所下降。番鸭呼肠孤病毒对肝脏、脾脏、盲肠的组织嗜性最强,表明这3种组织为该病毒的主要靶器官。番鸭呼肠孤病毒感染的细胞如下：各组织器官的血管内皮细胞;肝脏的肝细胞、枯否氏细胞;脾脏的淋巴细胞、巨噬细胞;肾脏的肾小管上皮细胞、足细胞;心脏的心肌细胞;肺脏的肺泡上皮细胞、尘细胞;胸腺的淋巴细胞、巨噬细胞;法氏囊的淋巴细胞;肠道的上皮细胞、杯状细胞、淋巴细胞;大脑和小脑内的少突胶质细胞。在肝脏、肾脏、心脏、肺脏等组织内可见炎性浸润的淋巴细胞呈阳性。     

免疫胶体金试纸膜和AGP检测vvIBDV GX 株在SPF鸡体内的侵染性变化规律

对28日龄无特定病原（SPF）鸡通过点眼、滴鼻方式人工感染了传染性法氏囊病病毒（IBDV）超强毒株GX;自感染第2天至第9天,每天用免疫胶体金试纸膜和琼脂扩散试验（AGP）两种方法检测九种组织（法氏囊、脾脏、胸腺、肾脏、肝脏、盲肠、扁桃体、肺脏、心脏、直肠内容物）的病毒含量变化,初步探索出传染性法氏囊病病毒在鸡体内各组织的侵染性变化规律。     

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.     

Effects of experimental inoculation of bovine respiratory syncytial virus in different inbred mice lineages: establishment of a murine model for BRSV infection

Bovine respiratory syncytial virus (BRSV), a member of the subfamily Pneumovirinae, family Paramyxoviridae, is a major cause of respiratory disorders in young cattle. A number of studies were conducted to validate a reliable animal model for the infection, since BRSV inoculation on the natural host is costly and often unsuccessful. Unfortunately, after inoculation of BRSV in Balb/C mice, viral replication may be detected; however, evident pathological alterations are absent on the experimentally infected animals. In order to establish a mice model that could be used further for preliminary studies of pathological and immunological aspects of BRSV infection, three mice inbred lineages (Balb/C, A/J and C57BL6), possessing different genetic backgrounds, were tested about its susceptibility to the inoculation with BRSV. Animals were inoculated through the nasal and ocular routes and were observed after inoculation. At 7 days post-inoculation (dpi) animals were necropsied and virological (virus isolation and viral nucleic acid amplification) as well as histopathological examinations were performed. A/J and C57BL6 showed interstitial pneumonia, when compared to the Balb/C group. These findings shows that mice may constitute a suitable model for the study of BRSV infections, depending on the mice strain used for experimental inoculations.     

Viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b

The viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b (PCV-2b) were investigated. Seventy special pathogen free mice were divided into 2 groups with 35 mice in each group. The test group (TG) was infected with PCV-2b, the control group (CG) was inoculated with sterile cell cultures. Five mice in each group were sacrificed at 3, 7, 14, 21, 28, 35 and 42 dpi (day post infection), respectively. Necropsies were performed on all mice and tissues were collected for testing by histopathology, immunohistochemistry, transmission electron microscope (TEM) and polymerase chain reaction (PCR). Apoptosis and necrosis in lymphoid organs were observed in virus-infected mice, and became severe from 14 to 28 dpi. The proportion of PCV-2b antigen-positive cells was moderate in lung, heart, thymus, liver or kidney, and low in brain from TG. In spleen and cervical lymph node, the proportions of PCV-2b antigen-positive cells were low to high from 7 to 28 dpi, and moderate from 35 to 42 dpi. PCV-2b DNA was detected in all tissues examined in TG from 7 to 42 dpi. Viral inclusion bodies presented in the cytoplasm of lymphocytes, macrophages, hepatocytes, podocytes, neurocytes, spermatids and uterine epithelial cells in TG. In CG, no viruses and viral lesions were detected. PCV-2b could replicate in mice, and PCV-2b associated lesions in mice were similar to those observed in pigs. The present results indicate that it is possible to use Kunming mouse as an animal model for PMWS research.     

Detection of Histomonas meleagridis DNA in different organs after natural and experimental infections of meat turkeys

Histomonas meleagridis infection of turkeys is usually accompanied by a severe disease with unspecific clinical symptoms but with distinct pathological lesions in the ceca and liver. In the literature some macro- and microscopic evidence of the spread of histomonads to the other organs has been provided. The aim of the present investigations was to use real-time polymerase chain reaction (PCR) to demonstrate the dissemination of H. meleagridis DNA to different organs after natural and experimental infection of meat turkeys. Samples from several organs were collected from a meat-turkey flock, which proved to be naturally infected with histomoniasis, and examined for histomonad DNA by real-time PCR. Histomonad DNA was detected in all investigated ceca, livers, spleens, kidneys, and pooled brain swabs. Additionally it was found in 75% of investigated samples from bursae of Fabricius, in 50% of investigated duodenums, and in 40% of investigated jejunum samples. After experimental intracloacal infection of 3-wk-old turkey poults with 147,500 histomonads, similar samples were collected from all turkeys that died. After a 3-wk observation period the surviving birds, as well as the noninfected control group, were euthanatized and samples were taken. During the entire experimental period, 10 birds out the 20 infected birds died. Histomonad DNA was detected in all investigated ceca, livers, lungs, and hearts (100%) and almost all kidneys (90%) and bursae of Fabricius (80%). On the other hand, only 30% of examined spleens and 10% of brain samples revealed positive results. Surviving infected birds were euthanatized and necropsied; histomonad DNA was found in one out of 10 livers but not in any ceca. Also, histomonad DNA could not be detected in examined cecal and lung samples from the noninfected control group.     

两株不同J亚群禽白血病毒株诱发组织增生病变和细胞因子表达的比较

旨在比较ALV-J-SD1005毒株和ALV-J-NX0101毒株感染鸡只致病性、诱发先天性免疫因子和致肿瘤因子表达的差异,用感染剂量为10³TCID50的两株病毒分别颈部皮下接种75只1日龄海兰褐鸡,感染后7、14、21 d检测体重、肿瘤病变、死亡率、血液和皮下纤维组织中病毒含量以及肝中鸡TRIM25、MDA5、IRF7、IFN-α/β、14-3-3σ、P53、STAT1等免疫因子或肿瘤因子的mRNA表达量。结果显示,雏鸡感染ALV-J-SD1005毒株后最早于第10天出现纤维组织增生,14 d致纤维组织增生率为100%(18/18),死亡率为5.2%(1/19);随着感染日龄的增加,增生组织指数和死亡率不断升高,21 d时分别达34.4%(74.5/209.5)和58.3%(7/12)。血液和皮下纤维组织的病毒载量显著升高;同时,显著上调鸡肝中MDA5、IRF7、P53等基因的表达量,下调IFN-α/β和14-3-3σ基因的表达量;而鸡TRIM25基因呈现感染早期(7 d)表达显著下调,后期(14~21 d)表达显著上调。ALV-J-NX0101毒株感染后21 d未检测到肿瘤发生,也没有鸡只死亡,但见鸡血液等组织中病毒载量显著增多,鸡TRIM25、MDA5、IRF7、IFN-β、IFN-α基因表达显著下降,STAT1基因表达显著上调。上述结果可以看出,ALV-J-SD1005毒株与ALV-J-NX0101毒株在感染鸡体内诱发不同的抗病毒反应和抗肿瘤反应,导致产生明显不同的病毒增殖和致病特点。本研究为深入理解两株ALV-J病毒致肿瘤机制、探索新诊断标识提供重要科学依据。     

猪繁殖-呼吸综合征活疫苗对仔猪的安全性试验

本试验用猪繁殖-呼吸综合征（PRRS）活疫苗和国内分离的PRRS强毒CH—1a株接种PRRS阴性的断奶仔猪，分别在接种后的3、7、14d各剖杀1头，取各脏器分别做冰冻切片和病理切片观察。用间接免疫荧光法检测各脏器PRRS病毒的分布。结果表明，PRRS活疫苗在免疫初期，抗原主要分布在脾脏、淋巴结，其次是肾脏和肺脏，少见于肝脏和心脏，第14d时在脾、淋巴结和肾脏有一定量的抗原，而肺脏相比则数量很少，肝脏和心脏未检到PRRS病毒抗原的存在，表明接种PRRS活疫苗随着时间的推移抗原分布呈下降趋势。而强毒抗原分布以脾脏最多，依次是肾脏、肺脏、淋巴结、肝脏、心脏，接种后第14d仍能在各脏器检到PRRS病毒抗原。病理组织学检测结果表明，活疫苗产生以下颌淋巴结、脾脏增生为特征的免疫应答，组织损伤轻微，对肺的病变较少，且仔猪生长良好。强毒则引起以大面积的肺泡隔增宽为特点的间质性肺炎和微循环障碍的病理变化，淋巴小结、脾脏滤泡发生崩解与周围界限不清，个别淋巴细胞核浓缩，组织损伤严重。本试验表明弱毒疫苗对仔猪是安全的。     

Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge (Alectoris rufa)

ABSTRACT: An experimental infection with highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) was carried out in red-legged partridges (Alectoris rufa) in order to study clinical signs, gross and microscopic lesions, and viral distribution in tissues and viral shedding. Birds were infected with a HPAIV subtype H7N1 (A/Chicken/Italy/5093/1999) and a LPAIV subtype H7N9 (A/Anas crecca/Spain/1460/2008). Uninoculated birds were included as contacts in both groups. In HPAIV infected birds, the first clinical signs were observed at 3 dpi, and mortality started at 4 dpi, reaching 100% at 8 dpi. The presence of viral antigen in tissues and viral shedding were confirmed by immunohistochemistry and quantitative real time RT-PCR (qRRT-PCR), respectively, in all birds infected with HPAIV. However, neither clinical signs nor histopathological findings were observed in LPAIV infected partridges. In addition, only short-term viral shedding together with seroconversion was detected in some LPAIV inoculated animals. The present study demonstrates that the red-legged partridge is highly susceptible to the H7N1 HPAIV strain, causing severe disease, mortality and abundant viral shedding and thus contributing to the spread of a potential local outbreak of this virus. In contrast, our results concerning H7N9 LPAIV suggest that the red-legged partridge is not a reservoir species for this virus.     

Distribution of viral antigen and tissue lesions in persistent and acute infection with the homologous strain of noncytopathic bovine viral diarrhea virus.

Viral distribution and lesions were compared between calves born with persistent infection (PI) and calves acutely infected with the same bovine viral diarrhea virus (BVDV) isolate. Two PI calves from 1 dairy herd were necropsied. The PI viruses from these calves were isolated, characterized by sequencing, and found to be identical. This virus strain, designated BVDV2-RS886, was characterized as a noncytopathic (ncp) type 2 BVDV. To establish acute infections, BVDV2-RS886 was used to inoculate clinically healthy, seronegative calves which were 3 weeks to 3 months old. Nine calves received 10(6)-10(7) tissue culture infective dose of BVDV2-RS886 intranasally. Four additional age-matched animals served as noninfected controls. Infected calves were necropsied at 3, 6, 9, or 13 days postinoculation (dpi). Viral antigen was detected by immunohistochemistry in frozen sections, and lesions were evaluated in hematoxylin eosin-stained paraplast sections. In the PI calves, a wide distribution of viral antigen was found in all tissues and was not associated with lesions. In the acutely infected calves, viral antigen was widespread in lymphoid tissues at 6 dpi but had been mostly eliminated at 9 and 13 dpi. Depletion of lymphoid tissues was seen at 6, 9, and 13 dpi and repopulation at 9 and 13 dpi. In 1 of the calves at 13 dpi, severe arteritis was present in lymph nodes and myocardium. This comparison shows that an ncp BVDV strain that causes no lesions in PI animals is able to induce marked depletion of lymphoid tissues in calves with acute infection. Therefore, the failure to eliminate PI cattle from a herd causes problems not only in pregnant cattle but may also affect other age groups.     

Concurrent highly pathogenic porcine reproductive and respiratory syndrome virus infection accelerates Haemophilus parasuis infection in conventional pigs

This study was aimed at determining the effect of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) on Haemophilus parasuis (HPS) in co-infection. A quantitative real-time PCR targeting infB gene, which is conserved among different HPS serotypes, was developed to improve the accuracy and speed of the detection of HPS. A total of 32 four-week-old conventional pigs were distributed randomly into four groups: pigs in group I were intranasally infected with HP-PRRSV first, and were then intraperitoneally inoculated with HPS on 5 days after HP-PRRSV infection; pigs in group II were intranasally inoculated with HP-PRRSV alone; pigs in group III were intraperitoneally inoculated with HPS alone; pigs in group IV were intraperitoneally inoculated with physiological saline. The amount of HPS in serum on 0, 3, 6, 9 and 12 days post-inoculation (dpi) with HPS were detected using the established quantitative real-time PCR. Clinical signs, pathological changes and histopathological lesions were observed. The amount of HPS in serum reached 10(6)copies/μl at 3 dpi with HPS in pigs of group I, while it arrived 10(5.7)copies/μl at 9 dpi with HPS in pigs of group III. The HPS loads in hearts and lungs were much higher than in other tissues. The study showed that HP-PRRSV was able to accelerate HPS infection and loads.