鸡传染性支气管炎病毒LH2/01/10的分离鉴定

2001年在我国黑龙江省某鸡场疑似鸡传染性支气管炎的发病鸡群中，分离到一株病毒，将该病毒接种10日龄SPF鸡胚，取72h的尿囊液进行电镜观察并用1％胰酶处理，结果发现尿囊液中存在典型的冠状病毒，用胰酶处理过的尿囊液能凝集SPF鸡的红细胞，初步鉴定为鸡的传染性支气管炎病毒。将该病毒尿囊液再次接种10日龄SPF’鸡胚，通过病毒对鸡胚的致病作用、病毒超微形态特征以及病毒凝集鸡红细胞的特性等对该毒株进行研究，结果表明：经胰酶处理后的病毒尿囊液可凝集鸡红细胞。鸡胚的第二代病毒尿囊液(命名为LH2,／01／10)分别接种1日龄和15日龄的SPF鸡，发现对不同日龄的鸡表现不同的致病性，对1日龄接种鸡和同笼饲养的同居对照鸡致病力高，发病率为11／11，致死率分别为4／6和2／5；15日龄接种和同居感染鸡发病率为9／9，致死率分别为1／5和1／4。实验应用反转录一聚合酶链式反应技术对LH2／01／10的膜蛋白基因进行扩增、克隆和序列测定，结果表明该基因具有IBVM基因的共有分子特征．与IBV标准株M41的M基因核苷酸的同源性为90％，氨基酸的同源性为91％。这从分子水平进一步证实引起鸡群死亡的病毒为鸡传染性支气管炎病毒。     

金刚烷胺在禽流感病毒M2基因原核表达中的作用

禽流感病毒(Avian Influenza virus，AIV)为正粘病毒科A型流感病毒，其基因组是由8个分节段的单股负链RNA组成，分别编码8个不同功能的蛋白，PB1、PB2、PA、HA、NP、NA、M1和M2及非结构蛋白NSl1和NS2。M2基因位于AIV基因组的第7个节段上，包含14～39和728～995两个部分，其初始转录产物在剪切40～727位的内含子后，拼接成成熟的mRNA后翻译成M2蛋白。M2蛋白是禽流感病毒的跨膜蛋白，具有H^ 离子通道的功能，     

Sporadic congenital transmission of avian leukosis virus in hens discharging the virus into the oviducts.

The efficacy of the albumen test for infectious avian leukosis virus (ALV) was examined in detecting congenitally transmitting hens. Seventy-three White Leghorn non-viremic hens with antibody to ALV were used. Eleven of the hens shed infectious ALV into their egg albumen, whereas only 7 of the 11 ALV-positive hens shed ALV antigens. The egg albumen test for infectious ALV was shown to be more effective in detecting the congenitally transmitting hens than that for ALV antigens. Then, twenty of the 62 hens which shed no infectious ALV into the albumen were studied for transmission of ALV to their embryos and for discharging ALV into the oviduct and vagina. Six of the 50 embryos from 4 hens were found to be infected with ALV but all of the 227 embryos from remaining 16 hens were free from the infection. Discharge of the virus into the oviduct and vagina was found both in the 4 transmitting hens and in 6 of the 16 non-transmitting hens. These results suggest that the hens discharging ALV into the oviduct, even though they do not shed ALV into egg albumen, may transmit the virus sporadically to their embryos.     

Seroprevalence of avian influenza virus, infectious bronchitis virus, reovirus, avian pneumovirus, infectious laryngotracheitis virus, and avian leukosis virus in Nigerian poultry

Eight poultry farms in Nigeria, including chickens from nine breeder, 14 broiler, 28 pullet, 11 layer, and three cockerel flocks, were tested for antibody seroprevalence to the following poultry viruses of potential economic importance: infectious bronchitis virus (IBV), avian reovirus, avian pneumovirus (APV), infectious laryngotracheitis virus (ILTV), avian influenza virus (AIV), and avian leukosis virus (ALV). Serum samples were collected between 1999 and 2004 and were tested for antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits. Seroprevalence was very high for IBV (84%); intermediate for reovirus (41%), APV (40%), and ILTV (20%); and very low for ALV (<5%) antibodies. By commercial ELISA, the seroprevalence of antibodies against AIV was, in some flocks, up to 63%. However, more specific assays did not confirm AIV antibodies, indicating that all flocks tested were free of avian influenza antibodies. Birds seemed to be first infected by IBV (at about 7 wk of age), then by reovirus at 12 wk, before they became infected by APV (week 25) and ILTV (week 30). This is the first report of serological evidence of the above viruses in West Africa. Further studies are necessary to assess economic losses due to these avian viruses and the costs and benefits of countermeasures.     

Attenuation of avian infectious bronchitis virus by cold-adaptation.

Avian infectious bronchitis virus (IBV) Arkansas-type DPI strain was passaged 10 times in specific-pathogen-free (SPF) chicken embryos incubated at 28 C and 37 C. Virus grown at 28 C acquired cold-adapted (CA) and temperature-sensitive (TS) characteristics based on more-rapid growth at 28 C and a reduced ability to grown at 41 C, respectively, compared with non-cold-adapted (non-CA) virus grown at 37 C. The pathogenicity and immunogenicity were determined for CA and non-CA IBV in 1-day-old SPF chickens following intratracheal inoculation. The percentage of CA IBV-vaccinated chicks exhibiting respiratory disease exceeded 30% on only 1 day postinoculation (PI) (day 5 PI), compared with 8 days (days 2-9 PI) for birds given non-CA IBV. Mortality was 0% for CA IBV-vaccinated chickens and 6% for non-CA virus-vaccinated chickens. Microscopically, both CA and non-CA IBV caused diffuse tracheal deciliation, although mucosal hyperplasia, necrosis, and heterophil infiltration were more severe with non-CA IBV. Virus was reisolated from kidneys of chickens given CA IBV, suggesting the loss of the TS property. The instability of the TS property was confirmed by growth of the reisolated virus at 41 C. Both CA and non-CA viruses induced complete protection against homologous challenge virus infection of the upper respiratory tract.     

Antiviral efficacy of oseltamivir against avian influenza virus in avian species

Avian influenza is one of the most contagious viral diseases in bird species and, increasingly, interspecies transmission to mammalian species has been reported. Prevention and eradication of avian influenza virus (AIV) infection in birds may require vaccines as part of a comprehensive program including biosecurity, culling, diagnostics, and surveillance. However, for valuable bird species in zoos, novel eradication strategies are needed, including antiviral treatments. The present study evaluated the anti-influenza efficacy of the potent neuraminidase inhibitor oseltamivir in avian species using the orders Galliformes (chickens) and Anseriformes (ducks). Viral replication of low pathogenic AIV was significantly reduced in the chicken model and completely reduced in the duck model. Anti-influenza drug administration to valuable bird species with an appropriate extrapolation approach could be useful for control of AIV in combination with active surveillance and vaccination strategies. Further, evaluation of oseltamivir against highly pathogenic avian influenza (HPAI) using avian models would be needed to optimize the oseltamivir application guideline for HPAI control.     

Demonstration of avian influenza-A virus in Iran by immunodiffusion technique.

A total of 1000 chicken serum samples (CSS) and 235 turkey serum samples (TSS) were tested by an immunodiffusion procedure against soluble antigen (S-antigen) prepared from avian influenza-A virus (AIAV), T/Calif/5142/66. None of the CSS tested developed any precipitin line, whereas 8.9% of the TSS tested developed well-defined precipitin lines against S-antigen. This observation confirmed the presence of AIAV in Iran.     

禽流感病毒PB2亚单位抗血清的制备

采用RT-PCR方法分段扩增PB2基因,将目的基因定向克隆至含HisTAG的原核表达载体pET-32a,经序列验证正确后,转化BL21大肠杆菌,诱导表达重组蛋白。靶蛋白经Ni+柱纯化后与弗氏免疫佐剂混合免疫新西兰大耳白兔,获得高效价的抗PB2的抗血清。Western-blot检测结果证实制备的抗血清可与PB2蛋白发生特异性反应。进一步将PB2全长基因准确插入真核表达载体pCAGGS,转染293T细胞表达PB2蛋白,通过IFA检测发现,抗血清与细胞表达的PB2蛋白发生反应,出现特异性荧光,经激光共聚焦检测PB2亚单位只在细胞核中出现荧光,说明PB2亚单位单独表达仅限于细胞核内。上述研究为进一步探索PB2亚单位的生物学特性及其结构与功能研究打下基础。     

禽白血病p27抗原检测阳性与病毒分离的相关性比较

本试验以80只300日龄的A品系蛋鸡为试验对象,分5个日龄段按翅号采集蛋清、泄殖腔棉拭子,无菌抗凝血和血清.用ALV p27抗原检测试剂盒检测蛋清和泄殖腔棉拭子.将无菌抗凝血分离血浆接种DF-1细胞,培养一周后用同样方法检测上清收集液,分析该群鸡只在不同日龄段泄殖腔棉拭子阳性、蛋清样本阳性和病毒分离阳性之间的相关性.用ALV-Ab抗体试剂盒检测各日龄段血清的抗体水平.此外,选取某一日龄段蛋清和泄殖腔拭子样本用4个不同厂家的ALV p27抗原检测试剂盒进行检测比较.结果表明,5个日龄段泄殖腔棉拭子平均阳性率为61％,蛋清样本平均阳性率为72.6％,病毒分离平均阳性率为48.8％.5个日龄段ALV的抗体阳性率一直为零;4个厂家的ELISA试剂盒对同一批样本的检测结果表明,IDEXX试剂盒的敏感度最高.本试验为外源性鸡白血病病毒检测及鸡白血病净化其方法的应用、试剂盒的选择、减少判定的误差、提高净化效果提供了一定的科学依据.     

J-亚群禽白血病JL-2株的分离鉴定

近年来，J-亚群禽自血病的暴发流行在国内时有报道，在本研究中，自吉林某患病鸡群分离出一株病毒，采用特异性引物，经RT-PCR扩增出长度为545bp的J-亚群禽自血病病毒特异性核苷酸片段；将病毒经SPF鸡胚成纤维细胞增殖，获取其前病毒DNA，依据原型毒株RPRS-103 cDNA序列设计并合成一对引物，经PCR扩增得到包括gp85、gp37、E-element基因在内的近1．8kb的DNA片段，将其连到pMD18-T载体上，转化大肠杆菌JM109，培养后提取质粒分别用Hind Ⅲ，BamHI进行单酶切和双酶切鉴定，得到了阳性重组质粒pMD18-T-JL2／env，核苷酸序列测定结果表明，该片段为J-亚群禽白血病病毒囊膜基因，其中亚型特异性片段gp85和标准对照毒株HPRS-103的同源率为94％，所编码氨基酸的同源率为87％。     

利用鸡胚成纤维细胞培养禽脑脊髓炎病毒的研究

利用鸡胚成纤维细胞 (CEF)培养禽脑脊髓炎病毒 (AEV) ,经过六次盲传发现 :AEV在 CEF上无细胞病变 (CPE) ,但利用 CEF细胞上清接种 SPF鸡胚 ,可产生不同程度的 AE鸡胚病变。分别取不同时间的感染细胞上清 ,测定 AEV浓度 ,结合培养条件 ,进而确定 AEV培养的最佳时机。结果表明 :以 AEV在 CEF上培养 7天最好 ,病毒滴度可达 10 2 .8EID50 / 0 .2 ml。将经 CEF培养的 AEV差速离心 (浓缩约 5 0 0倍 ) ,接种 SPF鸡胚 ,可产生典型的鸡胚病变 ,其滴度为 8× 10 5.0 EID50 / 0 .2 m l。通过 Cs Cl密度梯度离心提纯病毒 ,在电镜下观察到了大小基本一致的病毒粒子 ,病毒直径约为 2 5 nm。利用 AEV感染的 CEF或通过“细胞飞片”制备荧光片 ,建立了间接免疫荧光快速检验 CEF是否感染 AEV的方法。     

Influenza virus surveillance in waterfowl in Pennsylvania after the H5N2 avian outbreak

During the latter stages of the lethal H5N2 influenza eradication program in domestic poultry in Pennsylvania in 1983-84, surveillance of waterfowl was done to determine if these birds harbored influenza viruses that might subsequently appear in poultry. From late June to November 1984, 182 hemagglutinating viruses were isolated from 2043 wild birds, primarily ducks, in the same geographical area as the earlier lethal H5N2 avian influenza outbreak. The virus isolates from waterfowl included paramyxoviruses (PMV-1, -4, and -6) and influenza viruses of 13 antigenic combinations. There was only one H5N2 isolate from a duck. Although this virus was antigenically related to the lethal H5N2 virus, genetic and antigenic analysis indicated that it could be discriminated from the virulent family of H5N2 viruses, and it did not originate from chickens. Many of the influenza viruses obtained from wild ducks were capable of replicating in chickens after experimental inoculation but did not cause disease. These studies show that many influenza A virus strains circulating in waterfowl in the vicinity of domestic poultry in Pennsylvania did not originate from domestic poultry. These influenza viruses from wild ducks were capable of infecting poultry; however, transmission of these viruses to poultry apparently was avoided by good husbandry and control measures.     

Pathogenicity of an avian influenza virus serotype H9N2 in chickens

A low pathogenic avian influenza virus (AIV) serotype H9N2 affected many commercial flocks in the Middle East in late 1990s and early 2000s. Due to the varying pathogenicity ofAIV H9N2 reported in previous studies, this study was carried out to determine the pathogenicity of a Jordanian isolate of H9N2 in broiler and specific-pathogen-free (SPF) chickens. Mild tracheal rales were observed in the broilers but not in the SPF birds starting 3 days postinfection (DPI) and until the end of the experiment at 16 DPI. Infected chickens had gross and histologic changes limited to the respiratory system (sinuses, trachea, lungs, and air sacs) characterized by congestion and lymphoplasmacytic inflammation. However, the lesions in the broiler chickens were more severe than those in the SPF chicks. Furthermore, the virus caused significant (P = 0.004) reduction (230 g) in average body weight of the infected broiler group compared with the uninfected broiler group. Both broiler and SPF-infected groups seroconverted, and they had a geometric mean titer of 2(8.2) and 2(9.3), respectively, on the hemagglutination inhibition test at 16 DPI. Cloacal virus shedding was not detected by 9 DPI and 15 DPI in broiler and SPF-infected groups, respectively. This study demonstrated the pathogenic nature of the local Jordanian H9N2 isolate and the variation from what it has been reported in other countries of the region. Regional effort should be directed to start an eradication program of this disease because of its pathogenicity for chickens, wide distribution, and possible interference with surveillance for H5N1 serotype.     

Isolation of avian influenza virus in Texas

An avian influenza virus with surface antigens similar to those of fowl plague virus (Hav 1 Nav 2) was isolated in 1979 from 2 commercial turkey flocks in Central Texas. Two flocks in contact with these infected flocks developed clinical signs, gross lesions, and seroconversion but yielded no virus. This was the first recorded incidence of clinical avian influenza in Texas turkeys and only the second time that an agent with these surface antigens was isolated from turkeys in U.S.     

Induction of chicken interferon by avian infectious bronchitis virus.

The ability of nine strains of avian infectious bronchitis virus (IBV) to induce chicken interferon has been investigated using Semliki Forest virus for the tests. The Beaudette, H120 and Connecticut 46 strains induced interferon in the allantoic fluids of embryonated hens' eggs, the highest titre (1 : 30) being associated with Beaudette; but these as well as the Massachusetts M-41 and H52 strains failed to yield interferon in primary monolayer cultures of chick kidney cells as did all nine strains in organ cultures of chick embryo trachea. None of six strains of IBV investigated was susceptible to the inhibitory effects of chicken interferon.     

Immunofluorescent study on egg-adapted avian encephalomyelitis virus infection in chickens.

Fluorescent antibody study showed persistent infection of egg-adapted avian encephalomyelitis virus in the central nervous system and pancreatic tissues of infected embryos and chickens hatched from them. The limited organ tropism of the egg-adapted virus in hatched chickens was in striking contrast to the systemic infection that occurs with a field strain. In chidkens orally infected with egg-adapted virus strains, transient infection of a few organs was found despite occurrence of viremia.