Evaluation of immune effects of fowlpox vaccine strains and field isolates

The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2-3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression.     

Clinical effects, virological and serological responses in chickens following in-ovo inoculation of reticuloendotheliosis virus

Six-day-old embryonated specific pathogen free chicken eggs were inoculated with reticuloendotheliosis virus (REV) into the yolk sac and were incubated until they hatched. The hatchability of eggs inoculated with REV was significantly less (P less than 0.025) than that of media-inoculated controls. Although there were no significant differences in the body weights of these chickens at hatching, there were differences (P less than 0.001) at 6, 25 and 51 days of age between the infected and control chickens. Six of 10 chickens hatched from eggs inoculated with REV had feathering defects at 6 days of age. All chickens hatched from infected eggs had cell-free viraemia and antigenaemia, but not precipitating antibodies. Some of these chickens had very low neutralising antibody titres (less than 45) when examined at 25 and 37 days of age, as did all 10 chickens at 51 days of age. A low rate of horizontal transmission was indicated by the detection of antibodies at 37 and 51 days of age in chickens running in contact with the chickens hatched from eggs inoculated with REV.     

Influence of reticuloendotheliosis on the severity of Eimeria tenella infection in broiler chickens

Broiler chickens free of maternal immunity to reticuloendotheliosis virus (REV) were used in the experiment. Two groups of 25 chickens were inoculated with REV at one day of age. One of these groups and another group of 25 chickens were inoculated with Eimeria tenella sporulated oocysts at 7 days of age. Chickens inoculated with E. tenella showed bloody diarrhoea from 12 to 14 days of age. Six out of 25 chickens died (P less than 0.05) at 13 and 14 days of age in the dual infected group. At 14 days of age, when chickens were killed, the lesion score in the combined infection group, was statistically different from that in the chickens inoculated with E. tenella alone. Also the weights of the bursa of Fabricius and thymus were lower in the two REV infected groups than in the controls. Although REV infection alone adversely affected the weight gain and feed conversion, with combined infection this effect was much greater. Following REV inoculation most of the chickens showed feathering defects and all the examined chickens were viraemic at 21 days of age. At the same age, all but one chicken failed to show precipitating antigenaemia and about one-half of these chickens showed a very low serum neutralisation titre. None of these chickens showed precipitating antibodies.     

用单抗介导的免疫荧光试验检测组织切片中的禽网状内皮组织增生病病毒

以禽网状内皮组织增生病病毒(Reticuloendotheliosis virus，REV)的单克隆抗体11B154作为一抗，建立了从组织病理切片中检测REV的免疫荧光技术(Mc-IFA)。运用该技术对人工接种REV的肉鸡和疑似REV病鸡的肝脏、脾脏、心脏和肾脏等器官的组织切片进行了检测，并与病毒分离和斑点杂交试验的敏感性和特异性进行了比较。结果表明，在人工接种的感染肉鸡中，3种方法均为阳性；在30份疑似REV的病料中，用Mc-IFA可以检测出10份阳性，而病毒分离只有8份样品为阳性。由此表明，Mc-IFA的特异性和敏感性均较高，完全可以用于REV的检测。     

从表现腺胃炎的病鸡中分离到1株网状内皮增生症病毒

从表现传染性腺胃炎的病鸡中分离到 １ 株病毒。该病毒呈球形,真径 ８０～１００ ｎｍ ,有囊膜。将病料接种于鸡胚成纤维细胞,分别用单克隆抗体免疫荧光试验、酶联免疫吸附试验,证明该病毒为网状内皮组织增生症病毒。用细胞培养物接种于 １ 日龄健康鸡,复制出与临床完全相同的症状。     

整合禽网状内皮组织增殖病病毒基因的鸡痘病毒野毒株致病性研究

以分离保存的整合禽网状内皮组织增殖病病毒（REV）基因的鸡痘病毒（FPV）野毒株接种CEF细胞,间接免疫荧光试验检测REV,结果未检测到游离REV病毒粒子;以该毒株人工感染7日龄SPF雏鸡,同时设阴性对照,常规免疫新城瘦弱毒苗（ND）。接毒后每周采血动态检测NDV抗体、REV抗体、REV病毒血症;同时对试验鸡免疫器官指数、组织学变化动态观察,并对免疫器官的细胞凋亡动态检测。结果表明,整合REV基因的FPV感染后2周可引起鸡REV病毒血症,并产生REV抗体;导致中枢免疫器官发育分化不良,尤其对胸腺的影响最为明显,攻毒2周后,试验组与对照组相比,胸腺指数明显下降（P〈o．05）;免疫器官主要表现实质细胞数量少,正常结构发育不良,间质结缔组织增生而发生纤维化;细胞凋亡检测证实免疫器官的淋巴细胞和巨噬细胞均有明显的凋亡现象,细胞凋亡是引起免疫器官萎缩的主要原因。对新城疫疫苗免疫反应呈显著的抑制作用,攻毒3周后,试验组血清新城疫病毒抗体滴度显著低于对照组（P〈0．05）,其抗体滴度峰值低,维持时间短,下降速度快。本试验证实FPV整合REV前病毒基因组会明显改变FPV的致病性,表现某些REV的致病特征,使感染鸡REV抗体转为阳性并出现病毒血症,进一步证实REV可以通过基因组整合于FPV而进行传播的推测。     

免疫组织化学法与间接免疫荧光法对REV抗原定位的比较

本研究分别用免疫组织化学(IHC)法、间接免疫荧光(IFA)法对肿瘤病鸡不同脏器中的禽网状内皮增生病病毒(REV)进行了检测,旨在为两种REV抗原定位方法的应用提供实验依据.采用REV SD1005分离株人工接种1日龄海兰褐鸡,分别采取28日龄肿瘤病鸡的肿瘤、法氏囊、骨髓、肝脏、心脏、脾脏、腺胃的新鲜切面在洁净盖玻片上触片,同时制备其病理组织连续切片,分别用IHC法、IFA法对触片和切片进行REV检测.结果显示,两种用于抗原定位的染色方法检测结果完全一致,均可对病鸡的肿瘤、法氏囊、骨髓、肝脏、心脏、脾脏、腺胃等组织中存在的REV进行定位,依据染色后的阳性细胞感染率可判断REV的组织嗜性.本研究表明,IHC法与IFA法均可用于检测不同脏器中的REV,并具有良好的特异性、低背景和简便快速的特点.但相对于IFA法,IHC法更经济实用并且实验材料易于保存,因此可以在REV抗原定位研究中推广应用.     

禽网状内皮组织增生症病毒的分离鉴定、全基因序列分析及致病性试验

本试验采集广东某鸡场疑似禽网状内皮组织增生症患病鸡病料并进行处理,经接种DF1细胞、聚合酶链式反应（PCR）及特异性间接免疫荧光试验（IFA）,分离鉴定出1株禽网状内皮组织增生症病毒（REV）,命名为 GD1210。依据已发表的REV前病毒全基因组序列设计并合成6对引物,采用分段扩增的方法完成了分离株的全基因组序列测定。结果显示,该分离株基因组序列全长8443 bp。将该分离株的基因序列与国内外各参考株相应序列进行同源性比对分析,与SNV株亲缘关系最近,核苷酸同源性最高。将该分离株感染1日龄SPF鸡以鉴定其致病性,结果显示该分离株使SPF鸡产生严重的免疫抑制作用。     

CAV与REV共感染SPF鸡对疫苗免疫反应的抑制作用

用1日龄SPF鸡人工感染鸡贫血病毒(CAV)和禽网状内皮增生病病毒(REV),探讨病毒感染对鸡体疫苗免疫反应的影响。结果表明,在用禽流感病毒(AIV,H5和H9)疫苗免疫后,CAV与REV单独感染均显著抑制了鸡体对H5和H9亚型禽流感病毒灭活疫苗的HI抗体反应,在CAV与REV共感染后,这种抑制作用更为明显。CAV单独感染后鸡体对新城疫病毒(NDV)和传染性法氏囊病病毒(IBDV)疫苗的免疫反应受到抑制,但与对照组在统计学上的差异不显著,然而,CAV可以显著加重REV感染对鸡体在NDV和IBDV疫苗免疫后抗体反应的抑制作用。从而证实CAV与REV共感染在疫苗免疫抑制上有协同作用。     

不同感染量REV对鸡免疫反应和细胞毒性作用的影响

用不同剂量网状内皮增生症病毒（REV）感染肉鸡和SPF鸡后，检测血液中T淋巴细胞对ConA的反应和NK细胞、细胞毒T细胞（CTL）的细胞毒性作用以及NDV抗体生成变化等，观察REV感染对机体非特异性、特异性细胞免疫反应和体液免疫反应的影响。结果表明无论高剂量还是低剂量REV感染均造成体液免疫和非特异性细胞免疫抑制，而且高剂量比低剂量对免疫功能的抑制作用更强，但对NK细胞和细胞毒T细胞的细胞杀伤活性却有升高趋势，在抗肿瘤方面发挥一定的作用。这种结果说明REV感染对机体的免疫抑制是有选择性的，且抑制程度与感染病毒量有关。     

Effect of reticuloendotheliosis virus on the response of chickens to Salmonella typhimurium infection

Groups of 25 chickens free of maternal antibody to reticuloendotheliosis virus (REV) were inoculated with either third or seventh passage REV at either one or seven days of age. Some of the birds inoculated at day 1 with REV were inoculated with Salmonella typhimurium either concurrently or six or 13 days later while some of those inoculated with REV at day 7 were inoculated concurrently with S typhimurium. At day old, infection with S typhimurium alone caused the death of 12 of 25 chicks whereas in the dual infection, using the third passage REV, 18 of 25 birds died. Similarly no seven or 14 day old chickens died when challenged with S typhimurium alone, but previous day-old infection with REV caused a respective mortality of eight of 25 and five of 25 birds. With the seventh passage REV a similar pattern was seen. At day old S typhimurium infection alone killed seven of 25 birds whereas combined with virus the mortality was 14 of 25 and while S typhimurium alone killed none of 25 chicks infected at seven days old, the mortality in birds also infected with REV was 14 of 25. Combined virus and bacterial infections did not increase the proportion of feathering defects in birds surviving S typhimurium infections. There was a significantly higher proportion of feathering defects in birds infected with third passage virus compared with seventh passage virus. Although a higher proportion of birds had antibody responses to REV in the seventh than in the third passage group, there was no discernible difference in the effect the different viruses had on chickens' susceptibility to S typhimurium.     

用斑点杂交法同时检测鸡群中的CAV MDV和REV

为研究鸡传染性贫血病毒(CAV)在鸡群中的感染状况以及马立克氏病病毒(MDV)和网状内皮细胞增生病病毒(REV)在鸡群中的发病率，用斑点杂交法对山东省4个肉鸡场和2个肉种鸡场进行CAV、MDV和REV的检测，结果表明除一个肉种鸡场没有检测出REV以外，其他鸡场均同时检测出CAV、MDV和REV，并且发现直接从病科中检测CAV的阳性率(20％)远远低于将病科接种SPF鸡胚后的检出率(80％)。     

禽网状内皮组织增生病病毒和马立克氏病病毒共感染对鸡的致肿瘤作用

用禽网状内皮组织增生病病毒(REV)和马立克氏病病毒(MDV)共感染肉鸡，研究这2种病毒对鸡的致瘤作用，结果表明肉鸡共感染MDV和REV后13d即可发生死亡．接种后100d死亡率达84％。死亡鸡的肝脏、脾脏、肾脏和心脏等可以形成2种外观明显不同的散在的大肿瘤结节和弥漫性的小肿瘤结节。取发病鸡的肝脏、脾脏、肾脏、心脏和腺胃等组织样品做连续石蜡切片，HE染色后发现这些脏器均存在2种不同类型的肿瘤细胞增生。对这些连续切片分别用MDV和REV的单克隆抗体进行间接荧光试验，则同1份样品存在可以与REV和MDV分别呈现阳性反应的肿瘤细胞团，结果表明REV和MDV可以在感染鸡的体内分别诱发形成肿瘤。在接种后14、21、28和42d随机采集3只鸡的全血分离MDV和REV，均可以同时分离到MDV和REV。表明REV和MDV的共感染延长了病毒血症的时间。     

SPF雏鸡感染REV后免疫器官免疫功能的动态变化

试验通过对 1日龄 SPF雏鸡人工感染网状内皮组织增殖病病毒 ( REV) ,观察了免疫器官胸腺、法氏囊和脾脏 T、B淋巴细胞增殖反应的动态变化。结果表明 ,雏鸡感染 REV后胸腺 T淋巴细胞的增殖反应于7、2 1、4 2和 4 9日龄明显降低 ( P<0 .0 5) ,于感染后 1 4、2 8和 3 5日龄极显著减弱 ( P<0 .0 1 ) ;脾脏 T淋巴细胞的增殖反应分别于感染后 1 4～ 2 8d和 3 5～ 4 9d较对照组雏鸡明显或极明显降低 ( P<0 .0 5,P<0 .0 1 )。法氏囊和脾脏 B淋巴细胞增殖反应分别在感染后 7～ 4 9d和 1 4～ 4 9d极显著减弱 ( P<0 .0 1 )。免疫器官胸腺重量和脾脏重量与体重比值及体重分别于感染后 7～ 4 9、7～ 3 5、7～ 4 9d明显降低 ( P<0 .0 5,P<0 .0 1 )。说明SPF雏鸡感染 REV后 ,免疫器官 (胸腺、法氏囊和脾脏 )淋巴细胞发生变性坏死、数量减少和功能降低 ,机体的细胞免疫和体液免疫功能均发生明显降低或抑制     

Construction and characterization of a fowlpox virus field isolate whose genome lacks reticuloendotheliosis provirus nucleotide sequences

Fowlpox virus (FWPV) has been isolated from vaccinated chicken flocks during subsequent fowlpox outbreaks that were characterized by a high degree of mortality and significant economic losses. This inability of current vaccines to induce adequate immunity in poultry could be reflective of an antigenic and/or biologic distinctiveness of FWPV field isolates. In this regard, whereas an infectious reticuloendotheliosis virus (REV) provirus is present in the majority of the field viruses' genomes, only remnants of REV long terminal repeats (LTR) have been retained in the DNAs of each vaccine strain. Although it has not been demonstrated whether the partial LTRs can provide an avenue for FWPV to reacquire the REV provirus by homologous recombination, utilizing viruses of which genomes lack any known integrated retroviral sequences could resolve concern over this issue. Therefore, such an entity was created by genetically modifying a recently isolated field strain of FWPV. This selection, in lieu of a commercial vaccine virus, as the progenitor was based on the probability that a virus circulating in the environment would be more antigenically similar to others in this locale and thus might be a better candidate for vaccine development. A comparison in vivo of the pathogenic traits of the parental wild-type field isolate, its genetically modified progeny, and a rescue mutant in whose genome the REV provirus was inserted at its previous location, indicated that elimination of the provirus sequence correlated with reduced virulence. However, even with elimination of the parasitic REV, the modified FWPV was still slightly more invasive than a commercial vaccine virus. Interestingly, both types of attenuated FWPV elicited a similar degree of antibody production in inoculated chickens and afforded them protection against a subsequent challenge by a field virus, the origin of which was temporally and geographically distinct from that of the progenitor strain. Due to its antigenicity being retained despite a decrease in virulence, this REV-less FWPV could potentially be developed as a vaccine against fowlpox.     

鸡胚中人工感染网状内皮增殖病病毒的检测

为了模仿禽网状内皮增生病毒(REV)垂直感染鸡胚,在1和3日龄的发育SPF鸡胚人工接种不同剂量的REV,并继续孵化至12日龄.在将发育鸡胚制成成纤维细胞单层后,再用抗REV单克隆抗体11B118作间接荧光抗体反应检测REV.结果表明,从所有人工感染REV的鸡胚12日龄制备的成纤维细胞中均能在培养48h检测出REV.该方法可用于检测生产弱毒疫苗的SPF鸡胚及其相应细胞有无REV的污染.     

禽痘病毒活疫苗中网状内皮组织增生病病毒的分离鉴定及其序列比较

为了从怀疑污染禽网状内皮组织增生病病毒（Reficuloendotheliosisvirus,REV）的禽痘病毒（Fcwlpoxvirus,FPV）活疫苗中分离病毒,将市场上购买的某些厂家的禽痘弱毒疫苗接种7日龄SPF雏鸡。在5d后采其抗凝血浆接种鸡胚成纤维细胞（chickenembryofibroblast,CEF）,在连续传2代后,用REV特异性单抗做间接免疫荧光试验,确认REV感染,命名为JS0809。提取感染细胞的DNA,采用PCR扩增env基因。测序分析表明,该毒株的env基因开放阅读框同大部分REV-毒株一致,为176lbp。JS0809的囊膜蛋白基因（envelopeproteingene,env）与国内已发表株的同源性高达96．0％-99．％。相对而言,它与早年分离到的毒株的同源性只有96％-97．3％,但与近几年的分离珠的同源性则均高达99．5％-99．8％。它与整合进FPV野毒株中的全基因组REV的env基因同源性很高为99．8％,而与整合进FPV疫苗株中全基因组REV的env基因同源性较低仅为97．3％。本研究证实了在我国生产的某些FPV疫苗中存在REV污染,所污染的REV的env基因更接近最近的流行野毒,这是国内第一次报道从禽痘弱毒疫苗中分离~qREV活毒。     

母源抗体对同源及异源禽网状内皮增生病病毒感染诱发免疫抑制的预防作用

蛋用型海兰褐母鸡在用网状内皮增生病病毒（REV）细胞适应毒REV-C99（p30）免疫接种后,均在2周内产生REV特异性抗体。来自免疫种鸡的雏鸡在7日龄内100%（10/10）REV母源抗体阳性。分别在有母源抗体和没有母源抗体的1日龄鸡接种与疫苗株同源的低传代毒REV-C99（p3）或异源的REV中国野毒株HA9901（p5）,以此比较母源抗体对同源和异源REV病毒感染造成的免疫抑制的预防作用。结果表明,REV母源抗体能同等有效地预防同源和异源REV感染造成的对H5和H9禽流感灭活疫苗HI抗体反应的抑制作用。     

Occurrence of reticuloendotheliosis in Chinese partridge

Two commercial flocks of Chinese partridge experienced increased mortality associated with a wasting disease at 120-day old in June 2006. Postmortem examination of dead chickens consistently showed visceral tissues mainly proventriculus, liver and spleen were diffuse enlargement. Microscopic examination revealed masses of immature lymphocytes with frequent mitotic figures were seen in various tissues including proventriculus, liver, spleen, kidney, heart, lung, thymus and intestine. Reticuloendotheliosis virus (REV) was isolated from each of four blood samples. Viral antigens were observed in cultured CEF (SPF embryos came from the Ji-nan poultry institute) inoculated blood samples via on indirect immunofluorescent assay. Three hundred bp fragments of LTR of REV obtained from liver samples of six chickens by PCR. This disease has not previously been reported in Chinese partridge. Chinese partridge may represent a potential reservoir of infection for other Chinese local chickens.     

从自然发病鸡中分离出一株新城疫病毒

应用电镜技术、血清学检查、生物学试验等方法,从未注射过疫苗的某自然发病鸡群分离出一株新城疫病毒。该分离株对１０日龄鸡胚的平均致死时间为１１６小时,静脉接种致病指数为０．０６,其毒力介于新城疫病毒的ＬａＳｏｔａ株与Ｖ４株之间。免疫原性试验结果表明,该分离株具有免疫后无不良反应、免疫后６天开始产生抗体、产生的抗体效价高、对新城疫强毒的攻击能１００％保护等特点,是一株良好的新城疫候选疫苗株