番鸭呼肠孤病毒的鉴定

从以软脚为主要临床症状,以肝、脾表面有多量灰白色坏死点,肾肿大及出血为主要病变的病番鸭肝、脾组织中分离到5株病毒(MW9710、MW9803、MW9806、MW9809和MW9810).雏番鸭和雏鹅经人工感染后出现与自然病例相同的临床症状和病理变化,并能回收到病毒.樱桃谷鸭、麻鸭和鸡人工感染不发病.经电镜观察,病毒呈球形,正二十面体,立体对称,无囊膜,有双层衣壳,直径为60nm～73nm,病毒粒子在胞浆中增殖.病毒对乙醚、氯仿、胰蛋白酶、50℃处理1h和3%甲醛处理不敏感.对pH3处理2h、60℃处理30min和紫外线照射敏感.1mol/L Mg-Cl2不能增强病毒的感染力.病毒核酸型为dsRNA,病毒核酸具有禽呼肠孤病毒的特征:有10条带,按其大小可分为三类:大片段(L1～L3)、中片段(M1～M3)和小片段(S1～S4).但是MW9710株的M2和S1～S4片段的迁移率明显不同于鸡关节炎病毒(ARV)S1133株,而与番鸭呼肠孤病毒法国株(89330、89026株)的核酸电泳图谱极为相似.在血清中和试验中,ARV S1133和MW9710株互不交叉.并且,以针对ARV S1基因的特异性引物XZ11、XZ12对MW9710株等进行RT-PCR扩增,只能从ARV中扩增出特异性条带;而以MDRV89026株S1基因的特异引物HP11、HP12进行RT-PCR扩增,只能从MW9710等分离毒株中扩增出特异性条带.上述结果表明,MW9710株等5株病毒是上述疫病(番鸭"肝白点病")的病原,属呼肠孤病毒科正呼肠孤病毒属番鸭呼肠孤病毒.     

番鸭呼肠孤病毒YB株σB蛋白的基因序列分析及其原核表达

番鸭呼肠孤病毒(Muscovy duck reovirus,DRV)是造成雏番鸭高死亡率的重要病原体,深入其检测与免疫研究对于防控DRV感染意义重大。利用RT-PCR和测序技术,对3株福建DRV分离株的S3基因进行序列分析,发现DRV-YH、YJL株与禽呼肠孤病毒(Avian reovirus,ARV)遗传距离较近,同源性高达94.6%～98.9%,而DRV-YB株与ARV同源性仅为60.6%～61.7%。构建和鉴定DRV YB株重组原核表达质粒pET-30a-S3,并转化大肠杆菌BL21,SDS-PAGE表明,表达的目的蛋白分子量约为42ku,IPTG最适诱导浓度为0.1mM,最适诱导时间为5h,最适诱导温度为37℃,以包涵体形式存在。薄层扫描显示重组DRVσB蛋白占菌体总量的67.7%。以Ni 2+柱亲和层析纯化蛋白,纯化后的目的蛋白纯度为93%,质量浓度为0.86g/L。Western blot分析该融合蛋白能与抗DRV阳性血清发生特异性反应,表明重组DRVσB蛋白具有良好的免疫反应性。     

基于高通量测序技术分析番鸭呼肠孤病毒感染雏番鸭肠道转录组学的变化

番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)感染雏番鸭可导致肠道黏膜受损,造成肠黏膜免疫功能低下甚至丧失。为寻找番鸭呼肠孤病毒感染雏番鸭肠道组织免疫相关的差异表达基因,本研究建立番鸭呼肠孤病毒自然发病模型,在感染后3d、6d、21d采集同居感染组和对照组试验雏番鸭的空肠,对其进行高通量测序,对差异表达基因进行GO功能分类和KEGG数据库分析。结果显示,感染后3d、6d和21d分别筛选出2 297、5 860和3 721个差异表达基因;GO分子功能结果显示,免疫相关差异表达基因主要和免疫球蛋白的产生、T细胞活化和单核细胞趋化性等有关;KEGG通路富集结果显示,差异表达基因主要涉及在吞噬体、细胞溶质DNA传感途径、细胞因子-细胞因子受体相互作用、Toll样受体信号通路、Jak-STAT信号通路和RIG-I受体信号通路;荧光定量RT-PCR对差异表达基因进行验证,其结果与转录组学结果基本一致。本研究为进一步阐明MDRV感染导致雏番鸭肠道损伤和黏膜免疫抑制的机制奠定基础。     

草鱼呼肠孤病毒HZ08株S4基因序列分析

草鱼呼肠孤病毒HZ08株是本实验室从患出血病草鱼体内分离到的一个新毒株,已完成部分基因序列的分析,其氨基酸序列的同源性和873株相比,仅为20%~30%之间.因序列差异较大,无法通过设计特异性引物来扩增和分析其基因序列,采用单引物扩增技术,对HZ08株S4基因进行序列分析表明:S4全长为2263 bp,最大的ORF编码717个氨基酸,推导出其表达的蛋白约为79 kDa.正如其他基因节段,基因末端也含有保守碱基序列5′(GUAAUUU…UUCAUC),3′.S4基因推导的氨基酸序列与同宿主的其他呼肠孤病毒的非结构蛋白NS1同源性最大,其次是和哺乳动物正呼肠孤病毒的非结构蛋白mu-NS以及禽呼肠孤病毒非结构蛋白NS1同源性较大,表明S4可能表达细胞骨架相关蛋白.基于S4推导出的氨基酸序列构建的系统进化树HZ08株单独作为一个分支,与同宿主的其他呼肠孤病毒亲缘关系比较近,而与其他呼肠孤病毒则相对较远.这提示HZ08株可能是多个毒株的遗传信息经长期的遗传进化而得,综合其它已知序列信息,推测HZ08株可能为呼肠孤病毒的一个新成员.     

鸭细小病毒04Nb株的分离鉴定与rep基因测序与分析

采集浙江宁波地区以腹泻、呼吸困难为主要症状的病鸭肝组织,接种正常鸭胚尿囊腔增殖病毒.雏鸭感染试验显示发病症状及病理变化明显,死亡率为75%.电镜下可见纯化病毒直径约20nm左右的球形病毒粒子.免疫琼脂扩散实验结果显示与鸭细小病毒(duckparvovirus DPV)标准株阳性血清有明显沉淀线.经SDS-PAGE呈现3条结构蛋白带,与DPV标准株一致;参照GenBankDPV非结构蛋白基因序列设计引物,PCR扩增反应获得目的条带,克隆测序后,与DPV代表株序列同源性达98%.根据上述实验结果,确定引起本次鸭场疫病的病原为DPV.为进一步研究该分离株rep基因的序列特征,对其rep基因克隆测序,与GenBank中两株DPV、两株鹅细小病毒(GPV)进行序列比对,结果显示rep基因核苷酸序列与DPV参考毒株同源性为98%以上,与GPV同源性为80%左右.     

鸭细小病毒04Nb株的分离鉴定与rep基因测序与分析

采集浙江宁波地区以腹泻、呼吸困难为主要症状的病鸭肝组织,接种正常鸭胚尿囊腔增殖病毒。雏鸭感染试验显示发病症状及病理变化明显,死亡率为75%。电镜下可见纯化病毒直径约20nm左右的球形病毒粒子。免疫琼脂扩散实验结果显示与鸭细小病毒(duckparvovirusDPV)标准株阳性血清有明显沉淀线。经SDS-PAGE呈现3条结构蛋白带,与DPV标准株一致;参照GenBankDPV非结构蛋白基因序列设计引物,PCR扩增反应获得目的条带,克隆测序后,与DPV代表株序列同源性达98%。根据上述实验结果,确定引起本次鸭场疫病的病原为DPV。为进一步研究该分离株rep基因的序列特征,对其rep基因克隆测序,与GenBank中两株DPV、两株鹅细小病毒(GPV)进行序列比对,结果显示rep基因核苷酸序列与DPV参考毒株同源性为98%以上,与GPV同源性为80%左右。     

二株不同基因型树鼩呼肠孤病毒的分离和鉴定

目的从腹泻树鼩的粪便样本中分离和鉴定病毒。方法树鼩腹泻粪便样本分别接种Vero、LLCMK2和KMB17细胞,经连续传代,观察记录细胞病变,并对培养上清进行透射电镜检查、病毒RNA-PAGE电泳分析、轮状病毒鉴别筛查、S1全长基因片段扩增和生物信息学分析。结果树鼩腹泻粪便样品在KMB17、Vero和LLC-MK2细胞上经连续3代次传代后,均能产生细胞病变。经电镜检查、病毒RNA-PAGE电泳分析和轮状病毒鉴别筛查,推测其为呼肠孤病毒。病毒基因组全长S1基因扩增、序列测定和分析结果表明,KMB17培养上清中获得的病毒与I型原型株T1L同源性最高,核苷酸和氨基酸同源性分别为85%和90%,因此该病毒定义为呼肠孤病毒I型。而LLC-MK2和Vero细胞上清中的病毒S1基因与III型原型株T3D核苷酸和氨基酸同源性分别为85%和92%,因此为呼肠孤病毒III型。结论对今后树鼩和其他宿主呼肠孤病毒的分离鉴定有一定的指导意义。     

安徽省禽呼肠孤病毒感染的血清学调查

目的了解禽呼肠孤病毒(Avian reovirus,ARV)在安徽省鸡和鸭群中的感染情况。方法应用ELISA方法对采自安徽省205份鸡血清和218份鸭血清进行ARV抗体检测。结果肉鸡阳性率为55.88%,蛋鸡阳性率为92.23%,鸭群感染率为43.12%。结论禽呼肠孤病毒感染在安徽省鸡群和鸭群中感染较为普遍,应引起高度重视。     

番鸭呼肠孤病毒病病原学研究

番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)为番鸭"肝白点病"的病原体.该病毒自1972首次从发病番鸭中分离出来后,国内外各学者相继对各自分离株进行了研究,并在病毒特性、病毒基因组及病毒编码蛋白等方面取得了一定的进展.就该病毒的特性、基因组及其编码蛋白的研究作一综述,并指出了今后的研究重点.     

番鸭呼肠孤病毒和H9 亚型禽流感病毒共感染对番鸭胸腺免疫抑制作用

【目的】探讨番鸭呼肠孤病毒(muscovy duck reovirus,MDRV)和H9亚型禽流感病毒(H9 avian influenzavirus,AIV)共感染对番鸭胸腺免疫功能的影响。【方法】8日龄番鸭人工感染MDRV或/和H9 AIV,观察番鸭感染后发生率和死亡率、胸腺形态和显微结构变化,淋巴细胞增殖试验检测胸腺细胞增殖功能,RT-PCR检测MDRV或H9 AIV在番鸭胸腺的分布。【结果】H9 AIV感染后番鸭发病率低,无死亡;不影响胸腺的发育,胸腺病理变化不明显,但能显著抑制胸腺淋巴细胞增殖反应。MDRV单独感染番鸭生长迟缓,发病率80%,死亡率50%;胸腺萎缩,出现局限性坏死灶;对番鸭胸腺细胞增殖反应的有抑制作用,差异显著。共感染组番鸭生长迟缓,发病率90%,死亡率70%;胸腺萎缩,淋巴细胞减少,出现局限性坏死灶;对番鸭胸腺细胞增殖反应的有抑制作用,差异极显著。共感染组在病毒检出时间和检出率上均大于单一病毒感染组。【结论】H9AIV感染对胸腺的免疫抑制作用较弱,MDRV感染后对胸腺的免疫抑制作用较强,MDRV与H9AIV共感染在番鸭免疫反应抑制上有协同作用。     

禽呼肠孤病毒感染对鸡法氏囊及免疫应答的影响

研究LY株禽呼肠孤病毒(ARV)感染1日龄SPF鸡后对法氏囊发育影响,对传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、新城疫病毒(NDV)疫苗免疫诱发的抗体的影响,及对强毒株IBDV致病作用的影响。结果表明,LY株ARV感染1日龄SPF鸡可引起法氏囊萎缩和部分淋巴细胞减少,但对增重及AIV和NDV疫苗免疫后抗体滴度却没有显著影响。ARV感染可降低弱毒IBDV疫苗免疫后的抗体反应,但对随后IBDV强毒株攻毒的抵抗力却与对照鸡无显著差异。经IBDV弱毒疫苗免疫后,再接种强毒株IBDV,不会引起死亡,但却仍能显著抑制对AIV、NDV疫苗免疫后的抗体滴度。然而,对于1~7日龄经ARV感染的鸡,IBDV强毒的这种免疫抑制作用又显著低于未经ARV感染的对照鸡。     

Complete Genomic Sequence of a New Muscovy Duck-Origin Reovirus from China

The complete genomic sequence of a new Muscovy duck-origin reovirus (N-MDRV), strain J18 from China, was determined. The virus has a tricistronic S1 genome segment that is distinct from the originally described MDRV, which possesses a bicistronic S4 genome segment. Pairwise comparisons and phylogenetic analyses suggest that N-MDRV J18 is a new isolate within the species Avian orthoreovirus.     

Avian reovirus temperature-sensitive mutant tsA12 has a lesion in major core protein sigmaA and is defective in assembly

Members of our laboratory previously generated and described a set of avian reovirus (ARV) temperature-sensitive (ts) mutants and assigned 11 of them to 7 of the 10 expected recombination groups, named A through G (M. Patrick, R. Duncan, and K. M. Coombs, Virology 284:113-122, 2001). This report presents a more detailed analysis of two of these mutants (tsA12 and tsA146), which were previously assigned to recombination group A. The capacities of tsA12 and tsA146 to replicate at a variety of temperatures were determined. Morphological analyses indicated that cells infected with tsA12 at a nonpermissive temperature produced approximately 100-fold fewer particles than cells infected at a permissive temperature and accumulated core particles. Cells infected with tsA146 at a nonpermissive temperature also produced approximately 100-fold fewer particles, a larger proportion of which were intact virions. We crossed tsA12 with ARV strain 176 to generate reassortant clones and used them to map the temperature-sensitive lesion in tsA12 to the S2 gene. S2 encodes the major core protein sigmaA. Sequence analysis of the tsA12 S2 gene showed a single alteration, a cytosine-to-uracil transition, at nucleotide position 488. This alteration leads to a predicted amino acid change from proline to leucine at amino acid position 158 in the sigmaA protein. An analysis of the core crystal structure of the closely related mammalian reovirus suggested that the Leu(158) substitution in ARV sigmaA lies directly under the outer face of the sigmaA protein. This may cause a perturbation in sigmaA such that outer capsid proteins are incapable of condensing onto nascent cores. Thus, the ARV tsA12 mutant represents a novel assembly-defective orthoreovirus clone that may prove useful for delineating virus assembly.     

Avian reoviruses cause apoptosis in cultured cells: viral uncoating,but not viral gene expression,is required for apoptosis induction

The cytopathic effect evidenced by cells infected with avian reovirus S1133 suggests that this virus may induce apoptosis in primary cultures of chicken embryo fibroblasts. In this report we present evidence that avian reovirus infection of cultured cells causes activation of the intracellular apoptotic program and that this activation takes place during an early stage of the viral life cycle. The ability of avian reoviruses to induce apoptosis is not restricted to a particular virus strain or to a specific cell type, since different avian reovirus isolates were able to induce apoptosis in several avian and mammalian cell lines. Apoptosis was also provoked in ribavirin-treated avian reovirus-infected cells and in cells infected with UV-irradiated reovirions, indicating that viral mRNA synthesis and subsequent steps in viral replication are not needed for apoptosis induction in avian reovirus-infected cells and that the number of inoculated virus particles, not their infectivity, is the critical factor for apoptosis induction by avian reovirus. Our finding that apoptosis is no longer induced when intracellular viral uncoating is blocked indicates that intraendosomal virion disassembly is required for apoptosis induction and that attachment and uptake of parental reovirions are not sufficient to cause apoptosis. Taken together, our results suggest that apoptosis is triggered from within the infected cell by viral products generated after intraendosomal uncoating of parental reovirions.     

Antigenic Analysis of Monoclonal Antibodies against Different Epitopes of σB Protein of Avian Reovirus

Background

Avian reovirus (ARV) causes arthritis, tenosynovitis, runting-stunting syndrome (RSS), malabsorption syndrome (MAS) and immunosuppression in chickens. σB is one of the major structural proteins of ARV, which is able to induce group-specific antibodies against the virus.

Methods and Results

The present study described the identification of two linear B-cell epitopes in ARV σB through expressing a set of partially overlapping and consecutive truncated peptides spanning σB screened with two monoclonal antibodies (mAbs) 1F4 and 1H3-1.The data indicated that ²¹KTPACW²⁶ (epitope A) and ³²WDTVTFH³⁸ (epitope B) were minimal determinants of the linear B cell epitopes. Antibodies present in the serum of ARV-positive chickens recognized the minimal linear epitopes in Western blot analyses. By sequence alignment analysis, we determined that the epitopes A and B were not conserved among ARV, duck reovirus (DRV) and turkey reovirus (TRV) strains. Western blot assays, confirmed that epitopes A and B were ARV-specific epitopes, and they could not react with the corresponding peptides of DRV and TRV.

Conclusions and Significance

We identified ²¹KTPACW²⁶ and ³²WDTVTFH³⁸ as σB -specific epitopes recognized by mAbs 1F4 and 1H3-1, respectively. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against ARV groups.     

Comparison of enzyme-linked immunosorbent and virus neutralization assays for the serology of reovirus infections in laboratory animals

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of reovirus-specific IgM and IgG serum antibodies in rodents, detecting reovirus group reactive antibodies, was compared with reovirus types 1, 2 or 3 specific virus neutralization (VN) assays. To this end, serum samples were collected from specific pathogen-free (SPF) BALB/c RIVM mice, after experimental infection with any of the three mammalian reovirus serotypes. The majority (seven out of twelve) of the reovirus type 1-infected and one third (two out of six) of the reovirus type 3-infected mice died within 15 days after infection, whereas all (six out of six) of the reovirus type 2-infected animals survived. Using reovirus type 2 antigen in the ELISA, reovirus-specific IgM antibodies could be demonstrated within 1 week after infection in all the mice infected with reovirus types 2 or 3 and in the majority of the mice infected with type 1. Similarly, reovirus-specific IgG could be detected within 3 weeks in all the surviving mice infected with reovirus types 2 or 3 and within 5 weeks in all surviving mice infected with reovirus type 1. These results coincided well with the data obtained in the VN assays, in which all the infected animals also could be detected within 1 week after infection. As expected, titers were detected most rapidly and to the highest levels in the homologous VN assays. Given the sensitivity and the specificity of the ELISA system demonstrated in this paper and its suitability for incorporation in automated systems, the ELISA system should be considered valuable for the routine serologic diagnosis of reovirus infections in rodents.     

Apoptosis induction by avian reovirus through p53 and mitochondria-mediated pathway

Although induction of apoptosis by avian reovirus has been demonstrated in primary chicken embryonic fibroblast and several cell lines, to date, the potential significance of avian reovirus (ARV)-induced apoptosis and its pathways in cultured cells are still largely unknown. We now provide the first evidence of upregulation of p53 and Bax and specifically for Bax translocation from cytosol to mitochondria following infection with a cytoplasmically replicating RNA virus. Bax translocation to the mitochondria led to the release of mitochondrial proapoptic factors cytochrome c and Smac/DIABLO from mitochondria to the cytosol, but not the release of apoptosis-inducting factor. Activation of caspases-9 and -3 which cleaves the enzyme poly(ADP-ribose) polymerase in ARV-infected BHK-21 cells was also detected. Internucleosomal DNA cleavage was prevented by caspase inhibitors, further demonstrating that ARV-induced apoptosis was executed through caspase-dependent mechanisms. Stable expression of human bcl-2 in BHK-21 cells not only blocked ARV-induced apoptosis and DNA fragmentation but also reduced the level of infectious virus production and its spread in BHK-21 cells infected with ARV at a low multiplicity of infection. All our data suggest that p53 and the mitochondria-mediated pathway played an important regulatory role in ARV-induced apoptosis in BHK-21 cells. To further study the pathogenesis of ARV infection, a dual-labeling assay was used for the simultaneous detection of cells containing viral antigen and apoptotic cells. Dual-labeling assay revealed that the majority of antigen-expressing cells were not apoptotic. Remarkably, some apoptotic but non-antigen-expressing cells were frequently located in the vicinity of antigen-expressing cells. Syncytium formation in ARV-infected BHK-21 cells undergoing apoptosis, was apparent in large syncytia at late infection times, indicating a correlation between virus replication and apoptosis in cultured cells.     

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.