Phylogenetic analysis of avian paramyxovirus serotype-1 in pigeons in Japan

To understand the epidemiology of Avian paramyxovirus serotype-1 (APMV-1) in pigeons in Japan, phylogenetic analysis was comprehensively conducted based on partial fusion protein gene using isolate from the surveillance of this virus with previously known Japanese pigeon strains. This surveillance was conducted using feces obtained from domestic pigeons collected in 40 prefectures throughout Japan from June 2011 to March 2013. From a total of 1,021 samples, a single virus (APMV1/pigeon/Japan/Kanagawa/2013: JP/Kanagawa-pg/2013) was isolated. All Japanese pigeon APMV-1 strains were clustered into a single genetic lineage, which was termed VIb/1 by phylogenetic analysis based on the F gene including the sequence of the cleavage site. These APMV-1 strains were further subdivided into four subgroups identified over 4 separate timeframes: 1984–1995 (group 1), 1995–2000 (group 2), 2001–2007 (group 3) and the novel subgroup isolated in 2013 (group 4). Each subgroup has specific amino acid motifs at a cleavage site of the F protein, namely, ¹¹²GRQKR-F¹¹⁷(except for one strain), ¹¹²RRKKR-F¹¹⁷, ¹¹²RRQKR-F¹¹⁷ and ¹¹²RRQKR-F¹¹⁷, respectively. Our data suggest that Japanese APMV-1 strains from pigeons were diverse and reinforced the possibility that there were multiple introduction routes from foreign countries into Japan.     

Experimental infection of chickens, ducks and quails with the highly pathogenic H5N1 avian influenza virus

Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype have spread since 2003 in poultry and wild birds in Asia, Europe and Africa. In Korea, the highly pathogenic H5N1 avian influenza outbreaks took place in 2003/2004, 2006/2007 and 2008. As the 2006/2007 isolates differ phylogenetically from the 2003/2004 isolates, we assessed the clinical responses of chickens, ducks and quails to intranasal inoculation of the 2006/2007 index case virus, A/chicken/Korea/IS/06. All the chickens and quails died on 3 days and 3-6 days post-inoculation (DPI), respectively, whilst the ducks only showed signs of mild depression. The uninoculated chickens and quails placed soon after with the inoculated flock died on 5.3 and 7.5 DPI, respectively. Both oropharyngeal and cloacal swabs were taken for all three species during various time intervals after inoculation. It was found that oropharyngeal swabs showed higher viral titers than in cloacal swabs applicable to all three avian species. The chickens and quails shed the virus until they died (up to 3 to 6 days after inoculation, respectively) whilst the ducks shed the virus on 2-4 DPI. The postmortem tissues collected from the chickens and quails on day 3 and days 4-5 and from clinically normal ducks that were euthanized on day 4 contained the virus. However, the ducks had significantly lower viral titers than the chickens or quails. Thus, the three avian species varied significantly in their clinical signs, mortality, tissue virus titers, and duration of virus shedding. Our observations suggest that duck and quail farms should be monitored particularly closely for the presence of HPAIV so that further virus transmission to other avian or mammalian hosts can be prevented.     

鸭Ⅰ型禽副粘病毒YH99V株HN基因的克隆和序列分析

在浙江地区进行鸭病病因的调查过程中，从患病鸭群中分离到一株引起鸭产蛋锐减而不死亡的病毒，经鉴定该病毒属于禽副粘病毒Ⅰ型，命名为YH99V株。以YH99V株的基因组RNA为模板，通过RT—PCR一步法扩增出其HN基因的cDNA片段，然后将其克隆至pMD18-T载体中，对其进行序列测定。测序后拼接出HN基因的序列长度为1785bp，该基因的ORF总长为1734bp，编码577个氨基酸。将YH99V株HN基因序列和推导的氨基酸序列与新城疫毒株的HN基因相应序列比较后发现，它们的核苷酸序列同源性分别在82．1％～99．7％，氨基酸序列同源性为87．2％～99．5％。在同源性比较的基础上，进一步绘制了Ⅰ型禽副粘病毒株HN基因的系统发育树。这对于Ⅰ型禽副粘病毒毒力基因的功能分析和该病的分子流行病调查有着重要的意义。     

动物副黏病毒感染与对策思考

尼帕病毒(Ni V)和亨德拉病毒(HeV)属于副黏病毒亚科的亨尼帕病毒属的成员。Ni V和HeV感染引起新的两种重要的人兽共患传染病。有关APMV-1(NDV)的发病和流行以及病原学研究认为APMV-1不断发生演化,新的基因型不断产生,对不同宿主的致病力不断变化,病毒感染的宿主谱不断扩大。本文简要介绍两种新的人兽共患传染病和APMV-1对鹅、鸭、猪的感染研究现状,就APMV-1宿主感染谱与演化加以分析,思考新的动物副黏病毒病防控对策。     

鸭源副黏病毒的致病性试验

利用鸭源副黏病毒(DPMV)SDFC株分别人工感染21日龄健康非免疫樱桃谷肉鸭和SPF雏鸡,观察试验鸡、鸭的发病情况及临床症状,于感染后不同时间剖杀,观察各组织器官的病理变化,并进行病理组织学研究。结果显示,鸭感染DPMV后发病率为100%,死亡率为43.3%;而鸡感染后的发病率、死亡率均为100%。病理组织学研究结果显示,鸭感染DPMV后,以心脏、肺脏、肝脏、肾脏、脾脏、法氏囊广泛性出血、变性为特征,消化道病变较轻微;而鸡感染DPMV后,各组织器官广泛性出血,脾脏、胸腺、法氏囊、肝脏、胰腺、肾脏、心肌组织细胞出现不同程度的变性、坏死;食管、腺胃和各段肠管广泛性出血,并伴有黏膜脱落。结果表明,DPMV对鸡、鸭均有较强的致病性,而与鸡相比,对鸭的致病性较弱。     

B亚型禽偏肺病毒对SPF鸡的致病性

利用分离的1株B亚型禽偏肺病毒,采用静脉注射和点眼滴鼻两种途径接种SPF鸡,并在接种同时免疫新城疫弱毒疫苗。在攻毒后3、6、9、12、15d,每组随机抽取3只翅下采血,检测血液生化指标,利用血凝和血凝抑制试验检测新城疫HI抗体效价;利用流式细胞术分析外周血CD40／CD8比值,ELISA试剂盒测定血清中IL-2、IFN7的含量;同时观察静脉注射组和点眼滴鼻组鸡的临床症状、剖检病变和病理组织学变化。结果表明,攻毒后9～15d,静脉注射组和点眼滴鼻组外周血CD4＋/CD8＋比值和血清II-2、IFN-γ的含量均显著低于对照组（P〈0．05）,而静脉注射组与点眼滴鼻组间无显著差异（P〉0．05）;静脉注射组和点眼滴鼻组的免疫器官指数低于对照组,但各组间差异不显著（P〉0．05）;各组间新城疫HI抗体水平无显著差异（P〉0．05）;血液生化指标仪谷丙转氨酶（ALT）和谷草转氨酶（AST）在攻毒后显著升高（P〈0．05）,其他指标在各组间无显著差异（P〉0．05）;静脉注射组和点眼滴鼻组在感染后3～10d出现轻微临床症状;病理组织学检测结果显示,静脉注射组和点眼滴鼻组的呼吸道和肝脏病变最明显。综上所述,B亚型禽偏肺病毒感染SPF鸡后,在一段时间内抑制机体细胞免疫,导致免疫机能下降,并引起轻微的组织病变;B亚型禽偏肺病毒通过两种不同的攻毒途径对SPF鸡的致病性无显著差异。     

B亚型禽白血病病毒所致免疫抑制条件下共感染禽波氏杆菌对雏鸡的影响

选择B亚型禽白血病病毒（ALV-B）感染雏鸡造成免疫机能低下,建立免疫抑制模型,以探索免疫抑制条件下共感染禽波氏杆菌后对雏鸡致病性的影响。设计了ALV-B单独感染组,B.avium单独感染组,ALV-B和B.avium共感染组,空白对照组,并且比较相互之间的差异。通过对雏鸡免疫学指标、生长性能及病毒血症和排毒状况的检测,对二者混合感染雏鸡的影响进行了研究。结果显示,ALV-B和B.avium共感染能引起雏鸡的免疫力低下,表现为免疫器官的发育、抗体的产生、淋巴细胞的转化、IL-2和IFN-γ的合成和分泌等受到明显抑制;混合感染组延长了B.avium的病程,增强其对雏鸡的致病性;同时机体出现明显的生长迟滞现象,尤其是第5周龄,共感染组平均体质量仅相当于对照组的40%。结果表明,ALV-B和B.avium在雏鸡体内的增殖方面起协同作用,从而促进了病原在鸡群间的散播;而B.avium对ALV-B导致的病毒血症有一定的抑制作用。在ALV-B免疫抑制作用下,B.avium对雏鸡的致病性显著增强。     

禽呼肠孤病毒鸡源分离株的鉴定与人工感染试验

从临床患病鸡关节病料中分离到一株病毒，在电镜下观察到病毒粒子无囊膜，有双层衣壳，外衣壳直径约75nm，内核约50nm；分离病毒株对酸、热有较强的抵抗力，对乙醚不敏感；病毒感染鸡胚成纤维细胞上能产生以融合细胞为特征的CPE；爪垫接种1日龄雏鸡表现出明显的病毒性关节炎临床症状，并伴有吸收障碍型和坏死型的病理变化；血清中和交叉试验表明，该病毒分离株与国内的2株番鸭源呼肠孤病毒（YH和YB）分离株和国外S1133呈不同程度的交叉反应。通过对分离病毒株进行电镜观察、理化特性试验、病毒感染力测定、致病性试验、血清学试验，可以确定该分离病毒为禽呼肠孤病毒。     

Experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9

ABSTRACT: Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (APMV-1 to -9). Only in the case of APMV-1, the infection of non-avian species has been investigated. The APMVs presently are being considered as human vaccine vectors. In this study, we evaluated the replication and pathogenicity of all nine APMV serotypes in hamsters. The hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. On the basis of one or more of these criteria, each of the APMV serotypes was found to replicate in hamsters. The APMVs produced mild or inapparent clinical signs in hamsters except for APMV-9, which produced moderate disease. Gross lesions were observed over the pulmonary surface of hamsters infected with APMV-2 & -3, which showed petechial and ecchymotic hemorrhages, respectively. Replication of all of the APMVs except APMV-5 was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. Histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. The majority of APMV-infected hamsters exhibited transient histological lesions that self resolved by 14 days post infection (dpi). All of the hamsters infected with the APMVs produced serotype-specific HI or neutralizing antibodies, confirming virus replication. Taken together, these results demonstrate that all nine known APMV serotypes are capable of replicating in hamsters with minimal disease and pathology.     

麻鸡副黏病毒ZH-1株HN基因真核表达载体的构建及其免疫试验

应用RT-PCR方法从麻鸡副黏病毒ZH-1株中扩增HN基因并克隆人pGEM-T载体,经序列测定、分析,结果显示ZH-1株HN基因与NDV国家标准强毒F48E9株的核苷酸、氨基酸序列同源性均达到92％.将HN基因克隆入真核表达载体pCI-neo,构建真核表达质粒pCI-HN,转染Vero细胞,能在Vero细胞中表达,利用pCI-HN质粒进行动物试验,结果表明,雏鸡免疫14d后在体内可检测到特异性抗体,pCI-HN质粒二次免疫雏鸡后,对NDV强毒的攻毒保护率为67％,这一结果提示,利用HN基因构建的核酸疫苗具有重要的开发应用价值.     

禽多杀性巴氏杆菌C48-3株hexABC基因缺失株的构建及其生物学特性分析

为研究荚膜在禽多杀性巴氏杆菌(Pasteurella multocida)致病过程中的作用,本研究利用同源重组基因敲除技术构建禽P.multocida C48-3株荚膜多糖输出蛋白基因hexABC的缺失突变株,并以小鼠为动物模型检测荚膜缺陷对细菌毒力的影响。PCR、RT-PCR和DNA测序结果均表明253 bp的hexC基因下游序列、798 bp的hexB基因全序列和290 bp的hexA基因上游序列完全被四环素抗性基因替代,表明构建了基因敲除突变株C48-3ΔhexABC。电镜观察结果表明突变株荚膜合成能力缺失,对小鼠的致病性试验结果显示突变株毒力基本丧失。本研究获得的无荚膜突变株为进一步研究禽P.multocida的致病机理奠定基础。     

不同H9N2亚型禽流感病毒株致病力的比较试验

参照NDV评价毒力的方法对1998—2008年间收集的25株H9N2AIV的致病性进行了比较研究。结果显示,25株H9N2AIV不同毒株间致病性有较大差异,大致分为3类：致病性偏强、致病性中等和致病性较弱。从中选出致病力有差异的8个毒株进行了1日龄雏鸡脑内接种指数（ICPI）、6周龄鸡静脉接种指数（IVPI）以及8周龄SPF鸡人工感染排毒试验,结果证明大部分毒株ICPI和IVPI基本上为0,排毒散毒的高峰期在攻毒后第5天到第6天。在25个毒株中,3#和12#表现出了比较高的致病性,不仅其EID50值最高（分别为10-8.8/0.2mL和10-8.8/0.2mL）、ELD50值最高（分别为10-7.9/0.2mL和10-8.7/0.2mL）,而且鸡胚平均死亡时间也最短（分别为66.5,69h）。3#、12#还出现了1日龄雏鸡脑内接种指数（分别为0.238,0.437）和6周鸡静脉内接种指数（分别为0.34,0.51）。在8周龄SPF鸡人工感染排毒试验中3#和12#毒株的排毒量大,排毒时间也明显长于其他毒株（第2～9天）。以上试验结果表明我国H9N2AIV不同毒株致病性有明显差异,呈多态性,致病性偏强的毒株造成鸡群较高的死亡率。     

鸭源H10亚型禽流感病毒血凝素基因的序列分析及其致病特性

采用血清学试验和RT-PCR方法从华东地区家养水禽中分离鉴定出多株H10亚型禽流感病毒，对其中1株A／Duck／Yangzhou／502／2003（简称Dk／YZ／502／03）的血凝素（HA）基因进行了序列测定，并与GenBank中登录的其他序列进行了比较。结果显示，Dk／YZ／502／03株血凝素基因与哺乳动物水貂分离株A／Mink／Sweden／84（H10N4）的同源性最高；其推导的氨基酸剪切位点序列为P-E-I-M-Q-G-R，为典型低致病性禽流感病毒的特征序列，这与该毒株对SPF鸡和BALB／c小鼠的低致病特性相吻合。     

Experimental infection of turkeys with avian pneumovirus and either Newcastle disease virus or Escherichia coli

Avian pneumoviruses (APVs) are RNA viruses responsible for upper respiratory disease in poultry. Experimental infections are typically less severe than those observed in field cases. Previous studies with APV and Escherichia coli suggest this discrepancy is due to secondary agents. Field observations indicate APV infections are more severe with concurrent infection by Newcastle disease virus (NDV). In the current study, we examined the role of lentogenic NDV in the APV disease process. Two-week-old commercial turkey poults were infected with the Colorado strain of APV. Three days later, these poults received an additional inoculation of either NDV or E. coli. Dual infection of APV with either NDV or E. coli resulted in increased morbidity rates, with poults receiving APV/NDV having the highest morbidity rates and displaying lesions of swollen infraorbital sinuses. These lesions were not present in the single APV, NDV, or E coli groups. These results demonstrate that coinfection with APV and NDV can result in clinical signs and lesions similar to those in field outbreaks of APV.     

Experimental infection of specific-pathogen-free chickens with serotype-1 fowl adenovirus isolated from a broiler chicken with gizzard erosions

Gizzard lesions were formed in specific-pathogen-free (SPF) white leghorn chickens inoculated with fowl adenovirus (FAV). The virus, serotype 1 FAV 99ZH strain (FAV-99ZH), was originally isolated from the gizzard mucosa of commercial broiler chickens exhibiting gizzard erosion with intranuclear inclusion bodies. Five-day-old and 53-day-old SPF white leghorn chickens were inoculated with FAV-99ZH by both oral and ocular routes and then examined at necropsy on days 3, 5, 7, 10, 14, and 21 postinoculation (PI). There were no clinical signs in any of the chickens after the inoculation. Focal gizzard lesions occurred macroscopically, however, in inoculated chickens at several experimental periods. FAV was recovered from tissue samples of the proventriculus, gizzard, pancreas, and rectum by day 10 or 7 PI but was not recovered from liver samples of any of the chickens. These results indicate that FAV isolated from gizzard erosion is able to reproduce gizzard lesions as necrosis and erosion in SPF white leghorn chickens and that it may have a greater degree of tissue tropism in gizzards and other digestive organs than in the liver.