Homidium (ethidium) bromide inhibition of avian reovirus replication.

Homidium bromide inhibited replication of avian reovirus in cell culture. Inhibition was dose dependent, and the critical event required that the dye be present during the replicative viral cycle and was not attributable to a cellular function.     

Characterization of avian reovirus strain-specific polymorphisms

Avian reoviruses have been associated with several pathologic conditions, but correlative relationships between genotypes and specific diseases have not been demonstrated. Six avian reoviruses (883, 176, 81-5, S1133, FC, and TX) were selected for this study, and a comparative study of the pathogenic properties of the viruses in chickens, following peroral and footpad inoculation, was carried out, along with a comparison of the electrophoretic mobility of viral genomic segments and viral proteins encoded by the gene segments. The pathogenic properties of the viruses were shown to be diverse, with three distinct pathotypes being defined: Pathotype I (883) caused only a syndrome that we have termed "transient digestive system disorder" (TDSD); Pathotype II (FC, TX, and S1133) caused only "viral arthritis syndrome" (VAS), whereas Pathotype III (176 and 81-5) caused both TDSD and VAS. Likewise, the genomes of the viruses were shown to be extremely polymorphic, with a maximum of five segments co-migrating between any two strains. Considerable variation in the electrophoretic mobility of the encoded proteins also was demonstrated with pronounced variation in the molecular size of the sigma 4 protein, the purported viral attachment protein, being evident. These results show that the genomes of avian reoviruses were extremely polymorphic, preventing correlation between genotypes and pathotypes. But these studies have provided us with the genetic elements needed to characterize the gene functions involved in viral pathogenesis.     

鸭源呼肠孤病毒对雏鸭致病性研究

30只10日龄樱桃谷肉鸭随机平均分为试验组和对照组,试验组静脉接种分离的鸭源呼肠孤病毒(DRV)SDWF株,每只0.2mL(ELD50=10-2.36/0.2mL),对照组每只静脉接种0.2mL生理盐水。于感染后3、6、9、12、15d每组随机抽取3只采血,利用流式细胞术分析外周血CD4+/CD8+比值变化,并测定血清中IL-6及INF-γ含量以及免疫器官指数,同时观察试验组鸭临床症状、剖检变化及病理组织学变化。结果显示,攻毒后3d,试验组雏鸭表现精神沉郁,食欲不振,生长发育缓慢,体质量下降。剖检变化表现为脾脏出血、肿大、坏死,肝脏坏死。病理组织学变化表现为脾脏、法氏囊淋巴细胞流失严重,网状纤维显现;脾脏坏死,形成肉芽肿,血管动脉管壁疏松、增厚。攻毒后3d,外周血CD4+/CD8+比值升高,随后于攻毒后6、9d开始迅速下降,显著低于对照组(P0.05),后期虽有所回升,但是CD4+/CD8+比值仍然低于对照组;血清中IL-6及IFN-γ含量变化规律与外周血CD4+/CD8+比值变化规律相似,在攻毒后9d均显著低于对照组(P0.05);胸腺、法氏囊指数均在攻毒后6d显著低于对照组(P0.05),脾脏指数在整个试验观察期间高于对照组。结果表明,脾脏、法氏囊是雏鸭感染DRV的主要靶器官,免疫器官淋巴细胞流失严重,T细胞数量减少,IL-6、IFN-γ分泌量降低,从而导致机体免疫抑制。     

Yeast-derived sigma C protein-induced immunity against avian reovirus

Avian reoviruses (ARVs) can result in disease and economic losses in the poultry industry. Vaccines against ARV may not provide full protection and can cause adverse reactions. The coding sequence of the sigma C protein from strain S1133 of avian reovirus was expressed in Schizasaccharomyces pombe. Sigma C protein expression was demonstrated by Western blotting, and the protein was evaluated for its ability to protect specific-pathogen-free (SPF) chickens against challenge with the virulent S1133 strain. Serologic and challenge-infection data showed the efficacy of the recombinant vaccine administered orally each week for 3 consecutive wk. Sigma C protein induced antibody, as determined by enzyme-linked immunosorbent assay. Percentage (%) protection induced by the low dose (125 microg purified yeast-expressed sigma C protein/chicken) or the high dose (250 microg purified yeast-expressed sigma C protein/chicken) was 64 and 91, respectively. The commercial vaccine administered once or twice provided 82% protection. Results supported the feasibility of a plant-derived vaccine for use in poultry immunization schemes.     

In vitro and in vivo characterization of avian reoviruses. I. Pathogenicity and antigenic relatedness of several avian reovirus isolates

Pathogenicity, pathogenesis, and antigenic relatedness of four avian reovirus isolates obtained from commercially reared broilers were investigated. Chickens of various ages were inoculated both orally and intratracheally with reovirus. Based on disease signs, mortality, weight depression, tissue lesions, invasiveness, and viral persistence in chickens inoculated at 1 day of age, the isolates were classified as being of low, intermediate, or high pathogenicity. The low-pathogenicity isolate (2177) did not cause mortality, weight depression, or clinical disease. The isolate of intermediate pathogenicity (2035) produced low mortality rates (8%), some weight reduction by 7 weeks postinoculation, and microscopic lesions in the intestine and gastrocnemius tendons. The pathogenic isolates, 2408 and 1733, caused severe clinical disease characterized by stunting, feathering abnormalities, mortality as high as 84%, and microscopic lesions in the liver, intestine, pancreas, and/or gastrocnemius tendon. Highly pathogenic isolates also persisted longer in tissues of infected birds and elicited a more prompt and prolonged antibody response. Birds inoculated at 1 day or 1 week of age were more susceptible to reovirus-induced disease than birds inoculated at 2 weeks, suggesting an age-associated resistance. All isolates produced mortality with equal frequency in embryos. The isolates characterized were found to be antigenically similar based on cross-neutralization and cross-protection studies.     

In vitro and in vivo characterization of avian reoviruses. II. Clinical evaluation of chickens infected with two avian reovirus pathotypes

The effect of two avian reovirus isolates (2408 and 1733) on digestion and nutrient metabolism in infected chickens was assessed by an in vitro absorption assay and clinical blood chemistry analysis. Birds of various ages were inoculated orally and intratracheally with reovirus and sampled periodically for the respective assays. Transitory malabsorption was observed in the duodenum of birds infected with reovirus 2408. Conversely, increased absorption was detected in the ileum of these same birds. Clinical blood chemistry analyses of birds infected with both isolates revealed that severely affected birds had abnormally elevated plasma total protein, plasma albumin, and calcium levels. Decreases were found in percent bone ash and, due to abnormally high globulin levels, in albumin:globulin (A:G) ratios. A significant (P less than 0.05) correlation between body weights and total protein, albumin, A:G ratio, and bone ash was found in infected birds. The most pronounced metabolic and physiologic changes occurred in the severely affected birds, and, in general, pathogenicity of the isolates was reflected by the degree of metabolic change.     

Nuclear lobe index in avian eosinophilia

The nuclear lobe index was calculated for eosinophils from adult fowl, ducks and quail with spontaneous or experimentally induced eosinophilia. Appropriate control birds were also examined. The nuclear lobe index in normal fowl was 1.95 compared with 1.91 in birds with eosinophilia. Although these results (in the fowl) are almost identical, further analysis revealed a distinct 'shift to the right' in those birds with counts greater than 20 per cent eosinophils (P less than 0.01). The normal nuclear lobe index in ducks was 2.68 compared with 2.60 in those with eosinophilia and thus no 'shift' was recognised. The quail, on the other hand, demonstrated a slight 'shift to the left' in which the normal nuclear lobe index was 2.46 compared with 2.20 lobes per cell in eosinophilic quail.     

Characteristics and analysis of electropherotypes of avian reovirus field isolates

Genomic segments of 10 selected isolates of avian reoviruses recovered from the intestine of birds affected with malabsorption syndrome or runting/stunting syndrome were separated by polyacrylamide gel electrophoresis. Different electropherotypes were observed and analysed, depending on the period of recovery and particular geographic locations. The analysis showed great variability in the dsRNA profiles of the isolates and higher mobility of the segments L1, S1, S2, S3 and S4. There was no correlation between electropherotype and geographic origin of the isolate. The analysis also showed the emergence of electropherotypically distinct strains since the introduction of modified live reovirus vaccines.     

Preparation and characterization of monoclonal antibodies against an avian reovirus

Thirteen monoclonal antibodies against avian reovirus strain Uchida were derived. Of the 13 antibodies, three (MAb1, MAb2, and MAb3) had the ability to neutralize the infectivity of the virus. MAb1 neutralized strains Uchida, CS-108, and TS-142 equally. MAb2 neutralized the same three strains, but the activity of neutralization was 10 times higher against Uchida than against CS-108 and TS-142. MAb3 neutralized only strain Uchida. It seems that MAb1 and MAb2 have a rather broad neutralization activity and that MAb3 has a type-specific activity. These data indicate that there are both type-specific and more broadly specific neutralization epitopes in avian reovirus particles, as in mammalian reoviruses. In Western blot analysis, MAb1 bound to a sigma protein of strain Uchida, so it was suggested that this protein carries the epitope of the less specific neutralizing activity.     

Association of avian reovirus M and S genes with viral behavior in vivo. I. Viral persistence

Persistent infections were initiated in chickens with four different avian reovirus strains of varying virulence. Chickens 1 day old, 1 week old, or 2 weeks old were inoculated with each. Eight weeks later, isolates from all four parent strains were obtained; all isolates but one were from the tendons, and that was from the pancreas. Biochemical characterization of the isolates showed their genomes to be similar to those of the parent strains, although the proteins of the persistent isolates occasionally appeared to migrate differently from those of the original strains. Hybridization studies of the genes of the isolates indicated that at least two genes, S2 and S4, consistently seemed to undergo the greatest degree of mutation in the most virulent strain. These data suggest that the S2 and S4 genes may be associated with initiation and maintenance of persistent infection in vivo, and that changes in these genes may be noted by 56 days postinfection.     

Immune response to avian reovirus in chickens and protection against experimental infection

Objectives To assess the efficacy of the vaccination procedure and the effect of the transfer of maternal antibodies to progeny chickens on reovirus pathogenicity.
Design To vaccinate chickens and challenge progeny chickens with high doses of homologous and heterologous viruses.
Procedure High doses of reovirus strains RAM-1, 1091 and 724 were used to induce tenosynovitis lesions. High doses were produced by concentration of viruses grown in cell culture. Then similar doses of viruses were used to challenge immunised chickens progeny.
Result Vaccination of breeding hens with the RAM-1 strain of avian reovirus, which resulted in the passive transfer of neutralising antibody to progeny chickens, completely prevented the development of tenosynovitis in 80% of progeny chickens infected with the homologous virus. Even though multiple injection of hens resulted in broadening of the normal type-specificity of the neutralising antibody response against heterologous serotypes of avian reovirus, only marginal protection against strains of two heterologous serotypes of avian reovirus was obtained.
Conclusions A model for assessing the efficacy of vaccination against avian reovirus strains on clinical signs such as tenosynovitis was developed that overcome the normal low virulence of Australian strains of avian reovirus. Breeding hens can be immunised with Australian strain of avian reovirus with passive transfer of antibody via the yolk to the progeny chickens. Although the neutralising antibody response to three injections of inactivated virus decreased the specificity of the neutralising antibody response against antigenically heterologous strains of avian reovirus, the protective immunity appeared to retain type-specificity.     

Association of avian reovirus M and S genes with viral behavior in vivo. II. Viral pathogenicity

A group of avian reoviruses comprising serially passaged S1133 strains and their vaccine derivatives was examined biochemically to study the temporal evolution of the viruses and biologically to assess their relative pathogenicities. The strains fell into three groups of differing virulence, the viruses becoming less pathogenic the longer they were passaged. Protein and RNA profiles of the strains showed no distinct patterns of evolution nor any trend that could be correlated with pathogenicity. Nucleic acid hybridization studies of the strains indicated that all the genes were altered to some extent during passage. The S1 and M3 genes appeared to change the most during the first half of passage history, but later, as the virus was cold-adapted or passaged extensively, the M2, S2, and S3 genes also appeared to vary. When viruses were grouped according to virulence, the greatest changes were seen in the S1, M2, and M3 genes, suggesting that these may be associated with the virulence of a given avian reovirus strain.     

A comparison of avian and mammalian cell cultures for the propagation of avian reovirus WVU 2937

Two avian and seven mammalian cell lines were evaluated for their application in propagating avian reovirus WVU 2937. Cultures were compared for monolayer-formation time, support of viral replication, passages and postinfection time required for expression of cytopathic effect (CPE), type of CPE, and virus yield. CPE was observed on the first passage with infected egg yolk in primary chicken embryo kidney cells, primary through tertiary chicken embryo liver (CEL) cells, and African green monkey kidney (VERO) cells; on the third blind passage of infected supernatant in Georgia bovine kidney cells, Crandall feline kidney cells, and baby hamster cells; on the fifth blind passage in rabbit kidney cells; and on the tenth blind passage in porcine kidney cells. CPE was not observed after 10 viral passages in rabbit bone-marrow cells. Monolayer formation time and postinfection time for CPE expression occurred sooner, and virus yield was greater, with CEL and VERO cells than with other cell lines.     

中国鸡群禽呼肠孤病毒抗体的检测与分析

为了解我国鸡群中禽呼肠孤病毒(Avian reovirus,ARV)的抗体水平,本研究利用ELISA方法对2010-2013年来自全国19个省市的鸡血清样品进行了ARV抗体检测。在检测的17 058份鸡血清样品中共检出ARV抗体阳性血清156 80份,阳性率为92.83%,其中免疫鸡群的阳性率为98.50%,非免疫鸡群的阳性率为74.72%。除山东省和广东省外,其他各个省市的样本阳性率均在90%以上。商品肉鸡、肉种鸡、商品蛋鸡和蛋种鸡的ARV抗体阳性率分别为72.66%,98.48%,93.40%和95.53%。部分地方品种鸡群的ARV抗体水平也较高。结果表明,我国鸡群ARV感染情况普遍存在,主要表现为一定比例的免疫鸡群抗体过高、未免疫鸡群存在较高的抗体阳性率。     

禽呼肠孤病毒σNS蛋白的原核表达及纯化

对重组表达质粒σNS-pGEX-4T-1 IPTG的诱导浓度、诱导时间和诱导温度等条件进行了试验。当细菌的D600值为0.55～0.65时,IPTG最佳诱导浓度为0.8mmol//L,最佳诱导时间为4h,最适诱导温度为28～37℃,表达的目的蛋白相对分子质量约为66200,约占菌体总量的31.5%～33.3%。37℃及32℃诱导时,目的蛋白σNS主要以包涵体的形式存在;28℃诱导时目的蛋白主要以可溶性的方式表达。利用28℃诱导表达目的蛋白,用谷胱苷肽活化的Sepharose 4B亲和层析柱纯化,测定纯化蛋白的纯度与浓度,并对纯化的蛋白进行Western-blot分析。结果显示,纯化后的目的蛋白纯度达93%,质量浓度为0.91g/L,并能与禽呼肠孤病毒阳性血清发生特异性反应。     

禽呼肠孤病毒C-98分离株S3基因的克隆及序列分析

参考GenBank中禽呼肠孤病毒S3基因序列设计一对引物,以禽呼肠孤病毒内蒙古分离株(C-98)基因组RNA为模板,应用RT-PCR方法获得病毒的S3基因,并克隆到pMD19-T载体后测序。应用计算机软件将所测定序列与参考毒株ARV 176、ARV S1133、ARV 138的S3基因序列进行比较,核苷酸同源性分别为99.8%,99.6%,87.7%,推导其氨基酸同源性分别为99.5%,98.9%,94.8%。应用PHD程序和DNAStar软件,对S3基因所编码的σB蛋白结构进行预测,结果表明,σB蛋白为结构紧密的球状蛋白分子。