鸡传染性贫血病毒vp1基因的克隆表达及抗原性分析

从组织病料中提取CAV核酸,根据GenBank上发表的序列设计两对引物,分别扩增出vp1的139 bp～83 bp和547 bp～1337 bp两个基因片段,然后分别将其充隆到原核表达载体PMXB10上,经PCR和酶切鉴定,证明成功构建了重组表达载体PMXB10-vp1C和PMXB10-vp1D。在0.3 mM的IPTG诱导下,融合蛋白在大肠杆菌ER（2566）以分泌型得到大量表达,经大量表达后,用几丁质柱挂柱切割纯化后,得到切割蛋白E、F,在Western blot免疫分析印迹中,两组融合蛋白和切割蛋白与CAV阳性血清均能发生特异性反应。用ELISA方法检验,纯化的两组蛋白与CAV阳性血清均能发生特异性反应。用两组蛋白免疫SPF鸡后,用全病毒ELISA试剂盒检测血清呈阳性,表明两组蛋白均可诱发机体产生抗CAV的抗体。     

Biological and molecular characterization of chicken anemia virus isolates from Slovenia

The presence of chicken anemia virus (CAV) in Slovenia was confirmed by inoculation of 1-day-old chickens without antibodies against CAV and isolation of the virus on the Marek's disease chicken cell-MSB1 line and by polymerase chain reaction (PCR). Experimental inoculation of 1-day-old chickens resulted in lower hematocrit values, atrophy of the thymus, and atrophy of bone marrow. CAV was confirmed by PCR in the thymus, bone marrow, bursa of Fabricius, liver, spleen, ileocecal tonsils, duodenum, and proventriculus. The nucleotide sequence of the whole viral protein (VP)1 gene was determined by direct sequencing. Alignment of VP1 nucleotide sequences of Slovenian CAV isolates (CAV-69/00, CAV-469/01, and CAV-130/03) showed 99.4% to 99.9% homology. The VP1 nucleotide sequence alignment of Slovenian isolates with 19 other CAV strains demonstrated 94.4% to 99.4% homology. Slovenian isolates shared highest homology with the BD-3 isolate from Bangladesh. Alignment of the deduced VP1 amino acids showed that the Slovenian isolates shared 100% homology and had an amino acid sequence most similar to the BD-3 strain from Bangladesh (99.6%) and were 99.1% similar to the G6 strain from Japan and the L-028 strain from the United States. The Slovenian isolates were least similar (96.6%) to the 82-2 strain from Japan. A phylogeneric analysis on the basis of the alignment of the VP1 amino acids showed that CAV isolates used in the study formed three groups that indicated the possible existence of genetic groups among CAV strains. The CAV isolates were grouped together independent of their geographic origin and pathogenicity.     

我国部分地区鸡贫血病病毒全基因组核苷酸序列的比较与分析

本研究测定了3个鸡贫血病病毒（Chinken anemia virus,CAV）国内分离株及12个直接来源于临床样品的CAV毒株全基因组序列,结果表明15个CAV毒株的全基因组长度和结构与已报道的其它CAV毒株一致,长度均为2298nt,包含有3个重叠的ORFs。与GenBank发表的13个毒株序列进行序列比较发现,所有毒株核苷酸序列的相似性在95．2％～99．5％之间,CAV全基因组序列有2个高度变异区（HVR）,分别位于1033nt～1470nt和1858nt～2193nt。CAVORF3变异度最高,其5’端2／5处和3’端2／5～1／5处为2个高度变异区。ORF2变异度也较高,主要出现在5’端1／4处,ORFI最为保守。以CAV全基因组核苷酸序列和ORF3核苷酸序列为基础分别构建的CAV毒株分子进化树,均可以将全部CAV毒株划分为2个基因群。我国部分地区分离的大多数毒株,属基因Ⅰ群,与德国Cux-1等主要流行毒株有较为相近的亲缘关系;其它CAV国内毒株,属基因Ⅱ群,与日本G6株和TR20株有着更为相近的关系。     

鸡贫血病毒哈尔滨分离株VP2基因克隆测序和比较

通过PCR方法克隆了从我国哈尔滨分离的一株鸡贫血病毒（CAV）的VP2基因，并对之进行了测序，该基因的开放读码框由65bp组成，编码216个氨基酸。通过将本次克隆的基因与GenBank收录的CAV的VP2基因比较，同源性至少为99％，未发现与本次克隆的VP2蛋白完全一致的CAV分离株，与之同源性最好的是CIA－1的VP2蛋白，相差1个氨基酸，与Cux-1也仅相差2个氨基酸，因此从该蛋白看CAV哈尔滨分离株的变异程度不很高。比较这27株病毒的VP2及其基因序列，发现共有31处核苷酸发生变异，这些变异将导致VP2的12个氨基酸变化，尽管他们散布于整个VP2，但在186到191号氨基酸区域存在一个没有报道过的变异程度相对较高的区域，CAV的VP2是相对较保守的蛋白。     

Comparative susceptibility of Marek's disease cell lines to chicken infectious anemia virus

Chicken infectious anemia virus (CIAV) is known to infect and replicate in various Marek's disease chicken cell lines (MDCCs) derived from Marek's disease (MD) tumors. One line, MDCC-MSB1, has been the substrate used in most studies. We compared a total of 26 MDCCs, including two sublines of MDCC-MSB1, MSB1 (L) and MSB1 (S), four other MD tumor-derived lines, and 20 lines derived from MD virus-induced local lesions, for susceptibility to the Cux-1 and CIA-1 strains of CIAV. The cell lines represented six phenotypic groups of T cells based on the expression of CD4, CD8, and TCR-2 and -3 surface markers. Susceptibility was measured by the number of cells positive for viral antigen in immunofluorescence (IF) tests at 3-10 days postinfection. No clear-cut differences were found in susceptibility related to phenotype, although CD4-/8+ lines and CD4-/8- lines might be more susceptible than CD4+/8- lines. However, several individual lines were more susceptible to Cux-1 than the two MSB1 sublines tested. Contrary to an earlier report, cells of MDCC-CU147, a CD8+, TCR3+, local-lesion derived line, were found to be susceptible to CIA-1. In fact, CU147 was distinguished by very high susceptibility to both CIAV strains. In direct comparisons with MSB1, CU147 detected approximately 10-fold lower doses of virus. Also, virus spread was faster (P < 0.05) in CU147 than in MSB1 and other lines. Results from polymerase chain reaction (PCR) tests to detect infection in titrations were in general agreement with IF test results although PCR detected infection in a few terminal dilution cultures that were negative by IF.     

广东省鸡传染性贫血病毒编码区基因的克隆与分子进化分析

为了解鸡传染性贫血病毒(chicken anemia virus,CAV)广东分离株变异情况,通过PCR方法克隆了于2012年分离到的10株CAV的编码区序列,并对其进行了测序及分子进化分析。结果显示,广东CAV分离株编码区全长1 823bp,含有3个互相重叠的开放阅读框(ORF)。序列分析表明,广东省CAV分离株与GenBank上已发表的其他36株CAV比较,VP1核苷酸的同源性在94.1%⁹9.2%之间,推导出的氨基酸的同源性在95.3%99.2%之间,推导出的氨基酸的同源性在95.3%⁹9.3%之间;VP2的核苷酸同源性在98.2%99.3%之间;VP2的核苷酸同源性在98.2%¹00%之间,推导出的氨基酸的同源性在95.9%100%之间,推导出的氨基酸的同源性在95.9%⁹9.5%之间;VP3的核苷酸同源性在97.8%99.5%之间;VP3的核苷酸同源性在97.8%¹00%之间,推导出的氨基酸的同源性在95.1%100%之间,推导出的氨基酸的同源性在95.1%⁹9.2%之间;VP1蛋白共有17个位点发生了变异,VP2蛋白共有11个位点发生了变异,VP3蛋白共有6个位点发生了变异。分子进化分析显示,10株CAV广东分离株主要位于两个分支,其中9株CAV分离株与GenBank上已发表的35株CAV处于一个大分支,而GDN-12与分离株KC414026处于另外一个小分支,由此得出其中9株广东CAV分离株是目前主要的流行毒株。     

Molecular characterization of Brazilian isolates of orf virus

Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modified methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed field gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was confirmed by these hybridizations.     

Partial sequencing of env gene of bovine leukaemia virus from Brazilian samples and phylogenetic analysis

Analysis of the partial bovine leukaemia virus (BLV) env gp51 gene sequences obtained from three BLV strains isolated in three different regions of Brazil was carried out. The Brazilian BLV env gp51 sequences were compared with seven other corresponding sequences of BLV strains isolated in different countries and with consensus sequence as well. The obtained data point on qualitative and quantitative differences among the analysed strains as far as the occurrence of single point mutations is concerned. Two Brazilian strains show significantly higher mutation rate than other analysed strains. Amino acid analysis did not show, however, any substantial changes of the primary protein structure coded by well conserved region of BLV env gp51 gene. Based on the obtained data, the putative dendogram image of possible phylogenetic relations among the studied BLV strains is presented as well.     

Tissue tropism of three cloacal isolates and Massachusetts strain of infectious bronchitis virus

Three virus isolates (ECV-1, -2, and -3) recovered from cloacae of chickens in flocks that experienced drops in egg production were identified as infectious bronchitis virus (IBV), based on characteristic embryo lesions, chloroform sensitivity, coronavirus morphology, and serology. Because these isolates were recovered from the cloacae of the hens, their tissue tropism was compared with the prototype strain of IBV, Massachusetts-41 (M-41), in experimentally inoculated chickens. During the 39-day period postinoculation (PI), virus isolation was attempted from digestive and respiratory tracts, kidney, and cloacal swabs. ECV-1, ECV-2, and M-41 were more frequently recovered from the cecal tonsils than from other tissues. ECV-1, ECV-3, and M-41 were also recovered from kidney for up to 39 days PI. ECV-2 and ECV-3 had a limited distribution in respiratory tissues, being isolated only sporadically from trachea, bronchus, and lung. Surprisingly, ECV-2 was isolated from esophagus at 2, 16, 30, and 39 days PI; otherwise, its distribution in other tissues was sporadic. Results confirmed that IBV, including M-41, can infect a variety of tissues and that some isolates may be recovered frequently from digestive tract tissues, particularly from the cecal tonsils.     

鸡贫血病毒vp1和vp2基因在毕赤酵母中的表达及产物免疫学特性的初步研究

利用特异性引物从pBluemCAV中PCR扩增得到鸡贫血病病毒（CAV）vp1、vp2基因,经EcoRⅠ和NotⅠ双酶切处理,纯化后,克隆至EcoRⅠ和NotⅠ双酶切处理的表达载体pPIC9K中,构建了真核表达质粒pPIC9K-VP1和pPIC9K-VP2。将pPIC9K-VP1和pPIC9K-VP2转化至毕赤酵母SMD1168中,在0.5%甲醇诱导下表达CAV-VP1、CAV-VP2蛋白,运用Westernblot和Dot-ELISA鉴定表达蛋白。结果表明,重组酵母菌株表达出约54ku和24ku的目的蛋白,与针对鸡贫血病毒的单克隆抗体发生特异性反应。将表达产物乳化后免疫6周龄BALB/c小鼠,用ELISA、IFA检测免疫小鼠血清,均检测到抗体。     

Oral infection with chicken anemia virus in 4-wk broiler breeders: lack of effect of major histocompatibility B complex genotype

The pathologic consequences of chicken anemia virus (CAV) oral inoculation in 4-wk-old broiler breeders of different major histocompatibility B complex (MHC) genotypes were evaluated. MHC B complex was determined by hemagglutination and sequence-based typing. Clinical signs, serology, gross lesions, histopathologic analysis, and CAV genome quantification were used to evaluate disease progression. Clinical disease was not apparent in the inoculated broilers throughout the experimental period. At 14 days postinoculation, antibodies against CAV were detected in 26.4% (29/110) of the inoculated birds. The distribution of percent positive was 34.6% (9/26) and 32.3% (10/31) of the chickens with B A9/A9 and B A9/A4 MHC genotypes, respectively, and seroconversion in six other genotypes was 19% (10/53). These differences among MHC genotypes for specific seroconversion rate were not statistically significant. CAV genomes were detected in the thymus of 87.7% (93/110) of the inoculated birds with no statistically significant differences between MHC genotypes. Mild thymic lymphocytolysis, lymphedema, and medullary hemorrhage were observed in the inoculated chickens. Histomorphometric analysis showed that cortical lymphocyte-to-parenchyma ratios did not differ between inoculated and uninoculated groups or among MHC genotypes. Similar findings have been reported previously in white-leghorn chickens of similar age, suggesting that broilers show a similar resistance to the effects of CAV infection at this age. The absence of significant clinical and pathological changes in the orally inoculated broilers at this age contrasts with CAV-associated thymus damage seen frequently in condemned commercial broilers at harvest.     

鸡传染性贫血病毒VP1、VP2基因的表达及免疫原性研究

为探索鸡传染性贫血重组蛋白亚单位疫苗的可行性,将鸡传染性贫血病毒基因分别克隆到转移载体p Fast Bac HTA中,再将其分别转化DH10Bac感受态细胞得到相应的重组表达载体,转染昆虫细胞Sf21获得含有VP1、VP2基因的重组杆状病毒v Bac-VP1、v Bac-VP2,应用悬浮培养的Sf21细胞表达重组蛋白。SDS-PAGE及Western blotting结果证明,VP1、VP2基因在昆虫细胞中得到了表达,动物试验进一步证实,重组蛋白免疫SPF鸡可产生ELISA抗体,提示杆状病毒表达的VP1、VP2具有良好的免疫原性。     

竞争性PCR定量检测鸡传染性贫血病毒核酸

将删除33个碱基序列的CAVDNA克隆进质粒pSBII,提取质粒DNA,经过纯化、定量后作为竞争性模板,建立了一种定量检测鸡传染性贫血病毒核酸方法。该方法能够检测出的最低CAVDNA为103.5Molecules/μL,其精确度可达到100.5Molecules/μL。该方法与传统方法相比,具有节省时间、节约试剂和受其它因素干扰小等特点。     

Pathogenicity of CL-1 chicken anemia agent.

Twelve-day-old broiler-type chickens had hemorrhagic necrotic wing tips. After 10 blind subcultures in an MDCC-MSB1 cell line, a virus (so-called chick anemia agent [CAA]) was isolated and designated CL-1 CAA. Five-day-old specific-pathogen-free chicken embryos from a commercial breeder flock that were found not to possess antibody against CAA were infected with CL-1 virus via yolk-sac injection. Many (49%) infected embryos were small and apparently had died from severe systemic hemorrhage. Hatched chicks were small and had pale feathers, skin, skeletal muscles, bone marrow, and viscera. All infected chicks had small thymuses. These thymuses often were so small that they could not be found grossly (P = 0.002). Anemia occurred within 4 days post-hatch. Microscopically, all hematopoietic organs were markedly atrophic. Septic necrotizing lesions were seen only in organs from CL-1-injected chicks. Physicochemical and pathological characteristics of this virus indicate that it is similar to other isolates of CAA found in Europe and Japan.     

Organ culture of chicken bursa as a model to study the pathogenicity of infectious bursal disease virus isolates.

In vitro study with chicken bursal organ culture was attempted to assess the pathogenicity of locally isolated infectious bursal disease virus (IBDV) initially isolated from the bursa of naturally infected birds. In bursal organ culture, lymphoblastic transformation was noticed as early as 24 hr postinoculation and reached maximum at 72 hr postinoculation. The other microscopic changes were increased number of macrophages and formation of plasma cells. The IBDV antigen was detected 24 hr onward by coagglutination test with antibody coated Staphylococcus aureus strain Cowan I. On the basis of lesion score, the three isolates of IBDV (A, B, and C) were graded as virulent (B isolate) and moderately virulent (A and C isolates). A similar pattern of pathogenicity was also observed in the in vivo pathogenicity studies in chicken based on bursa: body weight ratio and histopathologic lesion score. The bursal organ culture thus provides a useful experimental model to differentiate the IBDV isolates on the basis of their virulence.     

Genome analysis of Marek's disease virus strain CVI-988: effect of cell culture passage on the inverted repeat regions.

The genomes of different derivatives of Marek's disease virus (MDV) strain CVI-988, a low oncogenic isolate of a serotype 1 MDV, were analyzed by restriction enzyme analyses to detect whether alterations occurred after passages in cell culture. DNA molecules of strain 988 isolated directly from blood cells contained mainly two copies of the 132-bp repeat sequence previously reported within BamH1-H and -D fragment as previously reported for more virulent MDV strains. Although a minority of virus particles showed repeat amplification was already at the fifth passage level, amplification mainly occurred between passages 17 and 34 in cell culture. In addition, a 400-bp deletion was detected within the BamH1-A fragment of two derivatives of CVI-988, 988C and 988C/R6.