水泡性口炎病毒血清型特异LUX实时荧光RT-PCR检测方法的建立

采用LUX荧光核酸扩增技术原理,以水泡性口炎病毒（VSV）NJ、IND型NS基因为模板分别设计并合成单标记LUX荧光引物,建立血清型特异的VSV荧光RT—PCR检测方法。试验表明,两种型特异的LUX荧光RT-PCR能分别特异地鉴定VSV血清型,对口蹄疫、猪水泡病等病毒以及对照细胞、健康动物组织RNA样品的检测结果均为阴性。对比检测试验表明,LUX荧光RT—PCR的检测敏感性比常规RT-PCR提高达10倍以上,对VSV细胞增殖病毒液的检测灵敏度可达1 TCID50。对人工感染豚鼠样品以及临床样品的检测试验证实,该LUX荧光RT—PCR可有效检测到人工感染动物组织以及进口牛临床样品中的水泡性口炎病毒,并能鉴定感染病毒血清型。所报道的检测方法,包括样品核酸提取、LUX荧光RT—PCR以及熔解曲线分析,可在3h内完成。     

莱斯顿型埃博拉病毒GP蛋白嵌合型水泡性口炎病毒的构建与免疫原性研究

莱斯顿型埃博拉病毒(REBOV)可以感染猪和非人类灵长类动物并引起动物发病和死亡,为制备安全、有效的REBOV重组病毒后备疫苗,本研究利用表达绿色荧光蛋白的水泡性口炎病毒(r VSV-EGFP)印第安纳弱毒疫苗株反向遗传操作系统,拯救出了REBOV GP蛋白嵌合型VSV(r VSV△G*GFP-REBOV-GP),并对其生物学特性进行分析,评估其作为REBOV活疫苗的安全性和有效性。激光共聚焦和western blot试验显示REBOV GP蛋白在嵌合病毒中获得正确表达;病毒生长动力学结果显示,r VSV△G*GFP-REBOV-GP与亲本株具有不同的生长特性;将r VSV△G*GFP-REBOV-GP接种小鼠,未引起小鼠发病,体重增长趋势与对照组一致,表明嵌合病毒具有良好的安全性;中和试验结果表明该嵌合病毒能够诱导小鼠产生针对REBOV GP的高滴度中和抗体。本研究制备的r VSV△G*GFP-REBOV-GP为进一步的动物免疫实验奠定了基础。     

埃博拉和马尔堡病毒囊膜糖蛋白嵌合型重组水泡性口炎病毒的构建及鉴定

埃博拉病毒(EBOV)和马尔堡病毒(MARV)属于丝状病毒科,能够导致人和动物发生致命的埃博拉出血热和马尔堡出血热,这两种病是重要的尚未传入我国的烈性人兽共患病。为研制安全有效的储备疫苗,本研究利用水泡性口炎病毒(VSV)反向遗传操作技术,将VSV自身糖蛋白(GP)分别替换为EBOV或MARV的GP,构建了表达扎伊尔型EBOV、苏丹型EBOV及MARV GP的嵌合VSV重组病毒r VSV△G/ZEBOVGP、r VSV△G/SEBOVGP及r VSV△G/MARVGP。Western blot和间接免疫荧光试验表明这3种GP蛋白在重组病毒感染的细胞中均能够正确表达。电镜观察结果显示,3种重组病毒均与亲本病毒具有相同形态特征,表明这3种外源GP蛋白均能够嵌合在重组病毒粒子表面。本研究构建的这3种重组病毒为进一步的动物免疫试验并评价其免疫效果奠定了基础。     

Matrix蛋白和G蛋白双位点突变型水泡性口炎病毒的构建及生物学活性观察

重组水泡性口炎病毒(Vesiclar stomatitis virs,VSV)是一种具有良好应用前景的病毒载体,可应用于制备疫苗和治疗肿瘤,但该载体仍存在安全性问题,高剂量注射实验动物可导致产生神经毒性.为减低乃至去除野生型VSV的致病性,作者针对VSV毒力因子,构建了Matrix蛋白和G蛋白双位点突变型重组VSV,该病毒敲除了原来Matrix蛋白的第51位精氨酸和G蛋白C末端的28个氨基酸.相比野生型VSV,新病毒的致病性显著降低.试验也证实这个致弱的病毒是由于2种不同弱化机理共同作用所致,即Ⅰ型干扰素作用和复制能力减低.新型VSV病毒载体有希望成为一个更安全、有效的疫苗载体.     

水泡性口炎病毒感染犊牛口腔黏膜上皮细胞的超微结构变化

将水泡性口炎病毒(VSV)接种于原代培养的犊牛口腔黏膜上皮细胞(CPMBCs),通过对不同时间收集的感染细胞制作超薄切片,观察病毒在该细胞上的形态发生。结果表明,VSV能导致CPMBCs出现典型的CPE和超微结构变化,主要表现为细胞圆缩、线粒体肿胀、胞浆空泡化,病毒游离于胞浆内或以出芽方式进入胞浆空泡内,病毒主要在细胞突起处进行出芽增殖,获得囊膜而成熟。感染24h后,细胞出现严重的纤维化,细胞骨架明显。该研究结果为VSV的嗜上皮机制研究提供了重要的依据。     

TaqMan(R) RT-PCR对水疱性口炎病毒的鉴定检测

水疱性口炎分为印第安那型(Indiana)和新泽西型(New Jersey)。这两种血清型病毒的快速和可靠的鉴别对该病的诊断、检疫、分子流行病学调查和监测至关重要。文章按照VSV核蛋白基因序列,设计了一对两型通用引物和两型各自特异性探针。研究建立了VSV实时荧光定量PCR检测方法,对VSV细胞培养物、人工感染实验动物组织、血清样品,以及系列稀释的不同TCID50样品、其它相关或相似病毒进行鉴定,同时与常规PCR、病毒分离试验作了比较。TaqMan RT-PCR的特异性和敏感性相当于或优于对照方法。重复性和稳定性试验证实,该方法可靠。每个试验中设立阳性、阴性对照和标准稀释度对照,使试验结果可对病毒RNA作准确定量,并可在4h内获得结果。研究结果表明,TaqMan RT-PCR方法是一种特异性强、敏感性高、快速安全的定量检测方法。因此,可以作为VSV的快速检测和定型。     

TaqMan~T-PCR对水疱性口炎病毒的鉴定检测

水疱性口炎分为印第安那型 ( Indiana)和新泽西型 ( New Jersey)。这两种血清型病毒的快速和可靠的鉴别对该病的诊断、检疫、分子流行病学调查和监测至关重要。文章按照 VSV核蛋白基因序列 ,设计了一对两型通用引物和两型各自特异性探针。研究建立了 VSV实时荧光定量 PCR检测方法 ,对 VSV细胞培养物、人工感染实验动物组织、血清样品 ,以及系列稀释的不同 TCID50 样品、其它相关或相似病毒进行鉴定 ,同时与常规 PCR、病毒分离试验作了比较。Taq Man○R RT-PCR的特异性和敏感性相当于或优于对照方法。重复性和稳定性试验证实 ,该方法可靠。每个试验中设立阳性、阴性对照和标准稀释度对照 ,使试验结果可对病毒 RNA作准确定量 ,并可在 4h内获得结果。研究结果表明 ,Taq Man○R RT-PCR方法是一种特异性强、敏感性高、快速安全的定量检测方法。因此 ,可以作为 VSV的快速检测和定型。     

蠓携带并传播水泡性口膜炎病毒

根据美国农业部农业研究院(ARS)微生物学家Barbara Drolet的研究结果表明，一种被称作刺蠓的可在恶劣条件下生存的小型昆虫在水泡性口膜炎病毒(VSV)的传播过程中扮演重要角色，该病毒可以感染包括猪、马、牛等在内的多种动物。     

表达扎伊尔型埃博拉病毒囊膜糖蛋白重组水泡性口炎病毒的构建

埃博拉病毒(EBOV)能够引起一种人畜共患急性出血性传染病,即埃博拉出血热.研制安全、有效的抗病毒疫苗具有重要意义.本研究利用水泡性口炎病毒(VSV)印第安纳株反向遗传操作系统,构建并拯救得到表达扎伊尔型埃博拉病毒(ZEBOV)囊膜糖蛋白GP的重组VSV (rVSV-ZEBOV-GP),通过westemblot和免疫荧光试验证明在重组病毒中ZEBOV GP蛋白获得正确表达;动物试验显示重组病毒对小鼠高度安全;中和试验结果表明重组病毒能诱导小鼠产生针对ZEBOV囊膜糖蛋白GP嵌合VSV假病毒粒的特异性中和抗体.本研究表明rVSV-ZEBOV-GP作为防控ZEBOV的储备性疫苗具有潜在的应用价值.     

水疱性口炎病毒经AKT/Bax信号通路诱导神经细胞凋亡

VSV神经嗜性是该病毒作为活病毒疫苗载体广泛应用的最大障碍,因此研究VSV诱导神经细胞的损伤对深入探讨VSV嗜神经性的分子机制尤为重要。本研究通过直接免疫荧光检测,可见VSV感染N2a细胞呈细胞核碎裂、染色质致密浓染等细胞凋亡的形态学变化;Annexin-V FITC/PI流式细胞术检测表明,接种VSV 8 h后N2a细胞凋亡率接近50%;Western blot结果显示在VSV感染过程中,磷酸化AKT表达量降低,Bax表达量升高。上述结果表明,VSV可通过AKT/Bax信号通路诱导神经细胞凋亡。     

仔猪先天性震颤的病理组织学诊断

利用病理组织学方法对仔猪先天性震颠病病例进行诊断。病理组织学主要变化为大脑软脑膜血管充血,脑膜下水肿;大脑白质和脊髓部位的有髓神经髓鞘发育不全,横切面可见神经髓鞘相对数量少,疏密、大小不均,形态差别大。其他脏器见继发性细菌感染病变。     

Meningoencephalomyelitis in a foal due to Salmonella agona infection

CASE HISTORY: A neonatal Thoroughbred foal was presented with rib fractures and left forelimb lameness secondary to dystocia. CLINICAL FINDINGS: The foal developed a head tilt, seizures and watery diarrhoea during hospitalisation and died at 7 days of age. Histological examination of the brain and spinal cord revealed a suppurative meningoencephalomyelitis with vasculitis, and numerous intralesional, gram-negative bacilli. Similar microscopic lesions were noted in the lungs, renal medullary interstitium, and umbilicus. Bacilli in the brain, spinal cord and umbilicus were identified immunohistochemically as Salmonella group B. Salmonella agona was isolated in pure culture from the brain, lung, liver, kidney, and intestine. CONCLUSION: This is the first report of meningoencephalomyelitis and septicaemia due to Salmonella infection in an equine neonate.     

Bovine progressive degenerative myeloencephalopathy (weaver syndrome) in brown swiss cattle in Canada: a literature review and case report

A 15-month-old purebred Brown Swiss heifer was presented because of posterior paresis and ataxia. Histopathological examination of the brain and spinal cord showed evidence of a mild diffuse degenerative myeloencephalopathy. The most severe degenerative lesions were located in the white matter of the thoracic spinal cord. We believe this to be the first documented case of bovine progressive degenerative myeloencephalopathy (“weaver syndrome”) in Canada.     

Pathologic and immunohistochemical findings in naturally occuring West Nile virus infection in horses

The pathologic and peroxidase immunohistochemical features of West Nile flavivirus (WNV) infection were compared in four horses from the northeastern United States and six horses from central Italy. In all 10 animals, there were mild to severe polioencephalomyelitis with small T lymphocyte and lesser macrophage perivascular infiltrate, multifocal glial nodules, neutrophils, and occasional neuronophagia. Perivascular hemorrhages, also noted macroscopically in two animals, were observed in 50% of the horses. In the four American horses, lesions extended from the basal nuclei through the brain stem and to the sacral spinal cord and were more severe than the lesions observed in the six Italian horses, which had moderate to severe lesions mainly in the thoracolumbar spinal cord and mild rhombencephalic lesions. WNV antigen was scant and was identified within the cytoplasm of a few neurons, fibers, glial cells, and macrophages. WNV infection in horses is characterized by lesions with little associated antigen when compared with WNV infection in birds and some fatal human infections and with other important viral encephalitides of horses, such as alphavirus infections and rabies.     

Pathology of natural infections by H5N1 highly pathogenic avian influenza virus in mute (Cygnus olor) and whooper (Cygnus cygnus) swans

Mortality in wild aquatic birds due to infection with highly pathogenic avian influenza viruses (HPAIV) is a rare event. During the recent outbreak of highly pathogenic avian influenza in Germany, mortality due to H5N1 HPAIV was observed among mute and whooper swans as part of a rapid spread of this virus. In contrast to earlier reports, swans appeared to be highly susceptible and represented the mainly affected species. We report gross and histopathology and distribution of influenza virus antigen in mute and whooper swans that died after natural infection with H5N1 HPAIV. At necropsy, the most reliable lesions were multifocal hemorrhagic necrosis in the pancreas, pulmonary congestion and edema, and subepicardial hemorrhages. Major histologic lesions were acute pancreatic necrosis, multifocal necrotizing hepatitis, and lymphoplasmacytic encephalitis with neuronal necrosis. Adrenals displayed consistently scattered cortical and medullary necrosis. In spleen and Peyer's patches, mild lymphocyte necrosis was present. Immunohistochemical demonstration of HPAIV nucleoprotein in pancreas, adrenals, liver, and brain was strongly consistent with histologic lesions. In the brain, a large number of neurons and glial cells, especially Purkinje cells, showed immunostaining. Occasionally, ependymal cells of the spinal cord were also positive. In the lungs, influenza virus antigen was identified in a few endothelial cells but not within pneumocytes. The infection of the central nervous system supports the view that the neurotropism of H5N1 HPAIV leads to nervous disturbances with loss of orientation. More investigations are necessary to clarify the mechanisms of the final circulatory failure, lung edema, and rapid death of the swans.     

Meningoencephalomyelitis in a foal due to Salmonella agona infection

CASE HISTORY: A neonatal Thoroughbred foal was presented with rib fractures and left forelimb lameness secondary to dystocia.

CLINICAL FINDINGS: The foal developed a head tilt, seizures and watery diarrhoea during hospitalisation and died at 7 days of age. Histological examination of the brain and spinal cord revealed a suppurative meningoencephalomyelitis with vasculitis, and numerous intralesional, gram-negative bacilli. Similar microscopic lesions were noted in the lungs, renal medullary interstitium, and umbilicus. Bacilli in the brain, spinal cord and umbilicus were identified immunohistochemically as Salmonella group B. Salmonella agona was isolated in pure culture from the brain, lung, liver, kidney, and intestine.

CONCLUSION: This is the first report of meningoencephalomyelitis and septicaemia due to Salmonella infection in an equine neonate.     

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus)

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.     

A Spontaneous Oligodendroglioma in the Lumbar Portion of the Spinal Cord in a Young BrlHan:WIST@Jcl (GALAS) Rat

Oligodendroglioma is a rare tumor originating from oligodendrocytes found mainly in the cerebrum in aged rats. Only a few reports have shown spontaneous occurrence of this tumor in the spinal cord, and no report has mentioned its occurrence in young rats. We encountered a case of spontaneous oligodendroglioma in the lumbar portion of the spinal cord in a young (9 weeks old) female BrlHan:WIST@Jcl (GALAS) rat. Here we report the detailed histopathological and immunohistochemical characteristics of this case. No clinical signs, no gross lesions at necropsy, and no specific changes in hematology or blood biochemistry were observed. The tumor was located in the dorsal funiculus in the lumbar portion of the spinal cord and widely spread to the dorsal root nerve. The neoplastic cells showed a sheet-like growth pattern and had small round nuclei, clear cytoplasm and distinct cell borders that resulted in a honeycomb pattern. No mitotic figures or other histological lesions were observed. The neoplastic cells were positively stained for Olig2 and PCNA. The present case was considered to be a low-grade oligodendroglioma based on the histological and immunohistochemical features. To our knowledge, our case is considered to be extremely rare and the first report in a young rat.     

SPINAL SUB ARACHNOID CYSTS IN 13 DOGS

Thirteen dogs, including 6 Rottweiler dogs, exhibiting clinical signs of spinal cord dysfunction and myelographically confirmed subarachnoid space enlargement were investigated. To characterize the lesions and to get a better understanding of their pathogenesis, different imaging techniques were used in association with explorative surgical procedures (12 dogs) and histopathologic techniques (5 dogs). All subjects underwent preoperative myelography, five of which were examined by computed tomography (CT) scanning and one by magnetic resonance imaging (MRI) as well as cerebrospinal fluid (CSF) flow measurement (velocimetry). Most animals were <12 months old (7/13 dogs) and Rottweilers were over-represented (6/13 dogs). The lesions were mainly located dorsally with respect to the spinal cord (10/13 dogs) and in the cranial cervical area (8/13 dogs). MRI suggested spinal cord deviation with signs of ventral leptomeningeal adhesion opposite the enlarged space. In one dog, velocimetry confirmed that the "cyst" was freely communicating with the surrounding CSF space. Surgical investigation confirmed leptomeninges-induced ventral adhesion in 4/5 dogs. Follow-up studies, carried out from 6 months to 2.5 years postoperatively, showed there was full recovery in 8/13 dogs. This study suggests that the compression of the spinal cord is possibly not caused by a cyst. Adhesion resulting from a combination of microtrauma and chronic inflammatory processes induces a secondary enlargement of the subarachnoid space and may be a significant causative factor in spinal cord compression and dysfunction. The over-representation of Rottweilers and the young age of the animals in the study suggest a possible genetic predisposition and an inherited etiology.