Hendra (equine morbillivirus)

Hendra has been recognized in Australia as a new zoonotic disease of horses since 1994/5 and subsequent work has shown that the viral agent is endemic in certain species of fruit bat. The Hendra virus is the type species of a new genus within the sub-family Paramyxovirinae, which also contains another newly identified zoonotic bat virus, namely Nipah. It is assumed that contact with bats has led to the Hendra virus being transferred to horses on each of the three separate incidents that have been reported in the last five years. No evidence has been found for widespread subclinical infection of horses.Infected horses can develop a severe and often fatal respiratory disease characterized by dyspnoea, vascular endothelial damage and pulmonary oedema. Nervous signs may also occur. Fatal respiratory disease has been seen in cats and guinea pigs following experimentally induced infections. Transmission of the virus from horses to other horses or man seems to have taken place, but very close contact was required. Three human cases have been recognized, all in association with equine cases. There have been two human fatalities, one due to respiratory failure and the other from a delayed-onset encephalitis. A number of diagnostic methods have been developed, but great care must be taken in obtaining samples from suspected cases.     

MAGNETIC RESONANCE IMAGING CHARACTERISTICS OF THE FOOT IN HORSES WITH PALMAR FOOT PAIN AND CONTROL HORSES

Palmar foot pain is a common cause of lameness. Magnetic resonance imaging (MRI) has the potential to detect damage in all tissues of the equine foot, but an understanding of the differences in magnetic resonance (MR) images between feet from horses with and without palmar foot pain is required. This study aimed to describe MR findings in feet from horses with no history of foot-related lameness, and to compare these with MR findings in horses with lameness improved by palmar digital local analgesia. Thirty-four limbs from horses euthanized with a clinical diagnosis of navicular syndrome (ameness >2 months duration, positive response to palmar digital nerve blocks and absence of other forelimb problems) (Group L), and 25 feet from age-matched horses with no history of foot pain (Group N) were examined. For each anatomic structure, MR signal intensity and homogeneity, size, definition of margins, and relationships with other structures were described. Alterations in MR signal intensity and homogeneity were graded as mild, moderate, or severe and compared between Groups L and N. Results revealed that there were significant differences in MR images between Groups N and L. Multiple moderate-severe MR signal changes were present in 91% of limbs from Group L and moderate (none were graded severe) in 27% of limbs from Group N. In most Group L limbs, more than three structures and frequently six to eight structures were abnormal. Concomitant abnormalities involved most frequently the deep digital flexor tendon, distal sesamoidean impar ligament, navicular bone, collateral sesamoidean ligament, and navicular bursa (with significant associations in severity grade between these structures), sometimes with involvement of the distal interphalangeal joint and/or its collateral ligaments. It was concluded that findings on MR images were different between horses with and without foot pain, and that pain localized to the foot was associated with MR changes in a variety of structures, indicating that damage to several structures may occur concurrently and that MR imaging was useful for evaluation of foot pain.     

马传染性贫血病毒驴白细胞弱毒疫苗株囊膜基因的优化及其DNA疫苗体外瞬时表达研究

马传染性贫血病毒(Equine infectious anemia virus,EIAV)的密码子使用频率与哺乳动物间存在着明显差异。为此,对马传染性贫血病毒驴白细胞弱毒疫苗株(DLA-EIAV)囊膜全长基因按照哺乳动物优势密码子的使用原则进行了重新设计和合成,并以此为基础通过重叠延伸PCR、限制酶切等方法得到结合型和分泌型囊膜基因,将其插入含有鸡beta-actin/兔beta-globin复合启动子(AG)的高效表达载体pCAGGS中,构建了EIAV驴白细胞弱毒疫苗株结合型和分泌型囊膜基因的DNA疫苗质粒pCAGGS-opti-bou-env、pCAGGS- opti-sec-env。将构建的质粒纯化后分别转染293T细胞,以间接免疫荧光和Western blot方法检测转染48 h后细胞及上清中囊膜蛋白的表达。结果显示,两种表达质粒均可正确表达EIAV囊膜蛋白,与相对应未优化的表达载体pCAGGS-wt-bou-env和pCAGGS-wt-sec—env相比,密码子优化的基因体外瞬时表达水平有极为显著的提高,而且蛋白表达部位也与预期的结果符合。这一结果为EIAV囊膜蛋白的单抗制备、表位鉴定、免疫试验、新疫苗的开发等奠定了基础。     

马γ-干扰素成熟蛋白基因在大肠埃希氏茵中表达及其多克隆抗体制备

采用RT-PCR从ConA刺激的马外周血单核细胞（PBMC）中扩增获得含信号肽的马γ-干扰素（Equine interferon-γ，EIFN-γ）基因，将其克隆至载体pCR2．1-TOPO中。通过PCR方法从重组质粒（pCR-EIFN-γ）中扩增马干扰素成熟蛋白（MatureEIFN-γ，mEIFN-γ）基因，并将其亚克隆至原核表达载体pET-28a（＋）。重组子经测序鉴定正确后转化至大肠杆菌BL21（DE3），经IPTG诱导，对其产物进行SDS-PAGE分析和Western blot鉴定，将纯化后的表达产物免疫新西兰白兔制备多克隆抗体。结果表明，mEIFN-γ基因全长为441b口，含一个开放阅读框，编码146个氨基酸的成熟蛋白；对其表达产物以可溶性和包涵体两种形式存在，重组蛋白相对分子量约为18Ku，且具有免疫反应活性。mEIFN-γ基因在大肠杆菌中高效表达并在非变性和变性条件下，采用Ni^＋亲和层析纯化方法均可获得高纯度的重组马γ-干扰素制备的多克隆抗体，为建立马γ-干扰素检测方法、监测机体免疫状态和研究机体免疫机制奠定了基础。     

长蒴母草黄脉病毒——一个双生病毒的新种

 从采集于海南儋州地区表现黄脉症状的长蒴母草(Lindernia anagallis)上分离到病毒分离物L2, DNA-A全序列分析结果表明, 全长2739个核苷酸(nt)(GenBank登录号:AY795900), 共编码6个ORF, 其中病毒链编码AV1(CP)、AV2, 互补链编码AC1、AC2、AC3、AC4。利用BLAST程序对DNA-A进行分析表明, 与L2 DNA-A有同源关系的病毒均为双生病毒科(Geminiviridae)菜豆金色黄花叶病毒属(Begomovirus)成员。进一步比较发现, L2 DNA-A与我国广东报道的广东番茄曲叶病毒(Tomato leaf curl Guangdong virus, ToLCGuV)(AY602165)全基因组核苷酸序列的同源性最近, 仅为77.0%, 说明L2为Begomovirus中的一个新种, 命名为长蒴母草黄脉病毒(Lindernia anagallis yellow vein virus, LAYVV)。与L2的IR区及各基因编码的氨基酸序列有最高同源性的病毒均来源于亚洲。利用DNA-B特异引物和DNA-β的特异引物, 均未检测到DNA-B和卫星DNA-β的存在。     

ULTRASONOGRAPHY OF THE FEMORAL ARTERY IN SIX NORMAL HORSES AND THREE HORSES WITH THROMBOSIS

Ultrasonography of the femoral artery in the horse, including color, power and spectral Doppler, provides morphologic and dynamic information. This paper describes the use of the techniques in six clinically normal horses and three with femoral artery thrombosis. Useful landmarks for orientation are the saphenous artery and the medial saphenous vein. The lateral circumflex femoral artery can not be visualized. Recognition of the genus descendens artery is complicated due to the presence of multiple distal caudal femoral arteries. The femoral artery feeds a high resistance bed. In normal horses the peak systolic velocity varies between 50–90 cm/sec. Echoic tissue in the femoral artery lumen with stenosis, occlusion and collateral blood flow formation are features encountered in the patients.     

Suspected venous air embolism in a Finnhorse

Vascular air embolism (VAE) is the entrainment of air from the operative field or other communication with the environment into the venous or arterial vasculature, producing systemic effects. The horse described in this report, after having been discovered to have dislodged the extension set from the jugular vein catheter, demonstrated clinical signs considered most likely to have resulted from a VAE, and represents a case of primarily neurological signs resolving spontaneously. Emphasis is given to the prevention and prompt recognition of this event and to the use of all available tools in the management of cardiovascular complications.     

马传染性贫血病毒弱毒株LTR的克隆及序列分析

从马传染性贫血病毒（ＥＩＡＶ）弱毒株感染的驴胎皮肤（ＦＤＤ）细胞中提取前病毒ＤＮＡ，以其为模板，通过ＰＣＲ扩增出ＥＩＡＶ弱毒株的ＬＴＲ，并将其克隆到载体质粒ｐＵＣ１９中。经酶切分析，ＰＣＲ及Ｓｏｕｔｈｅｒｎ杂交筛选、鉴定，获得含有ＥＩＡＶ弱毒株ＬＴＲ片段的重组质粒ｐＬＴＲ。对该质粒的序列分析发现，在中国ＥＩＡＶ弱毒株ＬＴＲ的Ｕ３区含有４个与细胞转录因子结合的位点，其中有３个ＰＵ．１位点和１个ＡＰ－１位点，Ｕ３区还存在１个ＴＡＴＡＴＡＡ启动子序列；Ｒ区长为８１ｂｐ，含有１个帽位点ＧＧ和１个Ｐｏｌｙ（Ａ）信号序列ＡＡＴＡＡＡ；在帽位点下游是１个病毒Ｔａｔ蛋白结合位点，即ＴＡＲ位点；Ｕ５区长为３９ｂｐ。中国ＥＩＡＶ弱毒株ＬＴＲ序列与国外发表的其他毒株序列相比，在ＬＴＲ的一些调控部位有变异发生，中国ＥＩＡＶ弱毒株与美国ＥＩＡＶ原型株（Ｗｙｏｍｉｎｇ株）ＬＴＲ区核苷酸序列有１８．７２％的差异。     

应用荧光抗体检测马鼻肺炎病毒的实验研究

用自然感染马阳性血清及兔免疫血清制备的马鼻肺炎荧光抗体(EHV_1·FA),和马鼻肺炎病毒(HEV_1)不同毒株以及从匈牙利引进的Bartha株标准毒均产生特异性荧光。通过对不同滴度地鼠肾细胞培养液内抗原的测定,表明EHV_1·FA法比补结反应及细胞病变法敏感,高1～2个滴度,应用冰冻切片直接荧光染色检查乳地鼠31份,肝阳性率为83.8％,肺阳性率为77.4％,对照10份均为阴性。检查人工感染HEV_1流产胎儿5份,肝阳性率为100％,肺阳性为80％,检查2份健康驴胎儿,均为阴性。表明应用冰冻切片、直接荧光染色检查马鼻肺炎病毒具有较高特异性和检出率。