Isolation and molecular biological investigations of avian poxviruses from chickens, a turkey, and a pigeon in Croatia

In the last 3 yr, several outbreaks of avian poxviruses (APVs) have been observed in different parts of Croatia. Four strains of APVs, from chickens, a pigeon, and a turkey, were isolated from cutaneous lesions by inoculation onto the chorioallantoic membranes (CAM) of 12-day-old specific-pathogen-free chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies. The characteristic viral particles of poxvirus were detected in the infected CAM and also in the infected tissues by transmission electron microscopy. Further identification and differentiation of the four various APVs were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis. Using one primer set, which framed a region within the APV 4b core protein gene, it was possible to detect APV-specific DNA from all four tested isolates. PCR results revealed no recognizable differences in size of amplified fragments between the different APVs from chickens, turkey, and pigeon. Restriction enzyme analysis of PCR products using NlaIII showed the same cleavage pattern for turkey and chicken isolates and a different one for the pigeon isolate. Multiplex PCR for direct detection of APV and reticuloendotheliosis virus (REV) was carried out to determine the possible integration of REV in the genome of isolated APVs. The obtained results revealed that REV was present in chicken and turkey strains of poxviruses, whereas the pigeon isolate was negative. It is not known whether the avipoxvirus vaccine strain used in Croatia is contaminated with REV or if the REV is naturally contaminating Croatian field strains of fowl poxvirus. The latter is indicated by the negative REV finding in the pigeon, which was not vaccinated. The results of the present study indicate the reemergence of fowlpox in Croatia, where infections have not been recorded since 1963 and never confirmed etiologically.     

Development of PCR-based tests for the identification of North American isolates of epizootic haemorrhagic disease virus.

A serogroup-specific polymerase chain reaction (PCR) assay and isolate identification strategies (restriction endonuclease analysis (REA) and nucleotide sequencing) were developed for the detection of North American isolates of epizootic haemorrhagic disease virus (EHDV). PCR primers (EHDV-pr4, EHDV-pr5) were designed to hybridize to the L3 gene of a North American isolate of EHDV serotype 1. Total nucleic acid was extracted from preparations of infected tissue culture and PCR was performed using a cDNA-PCR kit, according to the manufacturer's specifications. The PCR assay generated a 459 base pair product from North American isolates of EHDV serotypes 1 and 2, while bluetongue virus (BTV) serotypes 10, 11, 13, and 17, and cell controls, failed to demonstrate PCR products. Slight modifications allowed for the PCR detection of EHDV-1 and -2 in white-tailed deer blood (Odocoileus virginiatus); PCR fragments were not amplified from uninfected deer blood. A number of restriction endonucleases and sequencing primers were evaluated for their utility in isolate identification experiments. Specifically, REA employing HincII and cycle sequencing with an internal primer (EHDV-1-pr3) proved most successful for identifying isolate-specific genome markers. The techniques presented are expected to prove valuable for rapid and specific detection of possible future EHDV incursions in wild and domestic animal species.     

A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus.

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.     

猪巨细胞病毒福建株DPOL基因的克隆及序列分析

为了明确猪巨细胞病毒（porcine cytomegalovirus,PCMV）福建株（PCMV-FJ01株）DPOL基因特征,本研究根据GenBank数据库中PCMV DPOL基因序列特征,利用引物设计软件Oligo 7.0设计针对DPOL基因的引物,以PCMV-FJ01株核酸DNA为模板,对其进行分段PCR扩增。将分段扩增的产物经胶回收试剂盒回收后进行克隆测序,对测序结果进行拼接后获得PCMV-FJ01株DPOL基因序列,并进行生物信息学分析。结果表明,PCMV-FJ01株DPOL基因全长为3 021 bp,编码1 006个氨基酸;其与GenBank中的PCMV分离株DPOL基因核苷酸和氨基酸同源性分别在98.7%和99.1%以上。遗传进化分析发现,相对于巨细胞病毒属其他种代表株,PCMV分离株DPOL基因在遗传进化上和玫瑰疱病毒属代表株更近。建议根据PCMV DPOL基因遗传进化特征,将其划归为玫瑰疱病毒属的一个病毒新种--猪玫瑰疹病毒（porcine roseolovirus）。     

Cloning and Sequencing Analysis of DPOL Gene of Porcine Cytomegalovirus

In order to know the DPOL gene characterization of porcine cytomegalovirus (PCMV) (PCMV-FJ01 strain),three pairs of specific primer were designed by Oligo 7.0 software based on comparison the characterization of DPOL gene retrieved from GenBank.The target DPOL gene fragments were amplified using PCR methods with the strain PCMV-FJ01 genomic DNA. The target PCR fragments were cloned and sequenced. The assembly sequences were bioinformatics analysis. The DPOL gene of PCMV-FJ01 strain was 3 021 bp in length, coding 1 006 amino acids. The results showed that the homology of the nucleotide sequence and amino acid sequence were above 98.7% and 99.1%, respectively. Phylogenetic analysis revealed that the PCMV was closer with genus Roseolovirus rather than genus Cytomegalovirus. The results suggested that PCMV should be new species under genus Roseolovirus base on the phylogenetic relationship.     

Nucleotide sequence and polymerase chain reaction/restriction fragment length polymorphism analyses of Aleutian disease virus in ferrets in Japan.

Two ferrets with spontaneous Aleutian disease (AD) were found in Japan. The diagnosis was verified by polymerase chain reaction (PCR) amplification of part of the capsid gene specific to AD virus (ADV). The nucleotide sequences (365 bp in length) of the amplified fragments from the 2 ferrets differed by a single nucleotide, producing an amino acid alteration. Compared with other types of ADV, these isolates had 96% sequence similarity to a published ferret ADV (FADV) in contrast to <91% homology to various types of mink ADV (MADV). The phylogenetic tree of ADVs indicates that these 2 isolates and the published FADV belong to the same genetic group and definitely are divergent from MADVs. The predicted amino acid sequence of the hypervariable segment in the capsid gene was conserved among the 3 types of FADV. These results indicated that the 2 isolates found in Japan were new DNA types of FADV and could have been derived from FADV(s). A restriction fragment length polymorphism (RFLP) method to distinguish the ferret types of ADV from the mink types of ADV was developed on the basis of differences in their nucleotide sequences. Digestion of the PCR products with Afal or ScaI provided different cleavage patterns for FADV and MADV. This PCR/RFLP analysis of the ADV capsid gene will be a valuable asset for diagnosis of this virus infection in ferrets.     

The Use of PCR Combined with Restriction Enzyme Analysis to Characterize Fowl Adenovirus Field Isolates from Northern India

Ten fowl adenoviruses (FAVs), isolated from suspected cases of inclusion body hepatitis (IBH) in quails and broilers, were characterized by a hexon-based polymerase chain reaction (PCR) combined with restriction enzyme analysis (REA) of the amplified DNA fragments. All the isolates could be detected using H1/H2 and H3/H4 primer sets. Amplification of DNA with H1/H2 and H3/H4 primer sets resulted in fragments of approximately 1219 bp and 1319 bp, respectively. HaeII digestion of the H1/H2 PCR products and HpaII digestion of the H3/H4 PCR products characterized all the isolates in FAV groups, known from genomic typing using the whole DNA. For some of the isolates, neutralization tests were used to confirm these results. The results revealed that, as well as FAV serotype 1, which is the sole member of DNA group A, FAVs of DNA group E are also associated with IBH in poultry in northern India. The FAV specific PCR combined with REA was found to be very useful in investigating the epidemiological situation in the field. It was even possible to define mixed infections with more than one FAV.     

2株猪圆环病毒2型ORF2的克隆及序列分析

根据GenBank中猪圆环病毒2型(PCV-2)的核酸序列,设计一对引物,采用PCR方法从疑似断奶仔猪多系统衰竭综合征(PMWS)的死亡仔猪组织病料中扩增出ORF2基因,将其克隆到pMD18-T载体上,筛选获得重组质粒pMD18T-ORF2。序列分析表明,克隆的ORF2基因与其他PCV-2ORF2的分离株的同源性在91.5%～99.4%,与杭州分离株的同源性达到99.4%,表明我国各地区之间PCV-2的分离株存在较大的差异。     

Cloning and Sequence Analysis of M Gene of Avian Infectious Bronchitis Virus Guangxi Strain

According to the M gene nucleotide sequence of avian infectious bronchitis virus (IBV) published in GenBank,one pair of primers were designed,the M gene fragments of IBV isolated from Guangxi province were amplified by PCR.Then the amplified fragments were cloned into pMD18-T vector and the positive recombinant plasmids were sequenced.The results showed that M gene from all of the IBV isolates consisted of 678 bp,coding for 225 amino acids.Two glycosylated sites were located nearby the N-terminal,three transmembrane domains were located in the 23 to 98 peptide region.Variations within the hydrophilicity region were easier than that in the hydrophobicity region.Compared with that of other published IBV strains,the homologies of nucleotide and amino acid sequences of the isolates were 83.6% to 92.5% and 82.7% to 95.1%,respectively.The phylogenetic tree analysis showed that it was closely related to SAIB20 and LX4,and clustered into one group;But it belonged to different branches with other reference strains,and had a distant relationship.These results suggested that the isolate was a new variant of IBV.     

鸡传染性支气管炎病毒广西株M基因的克隆与序列分析

参照GenBank中鸡传染性支气管炎病毒(IBV)的核苷酸序列设计1对引物,利用 PCR 扩增IBV广西株的M基因片段,将其克隆到pMD18-T载体中.序列分析结果表明,M基因全长为678 bp,编码225个氨基酸,近N端含有2个潜在的N-糖基化位点,3个跨膜区位于23—98肽段区,亲水区较疏水区更易变异.IBV广西株与国内外IBV参考毒株相比,核苷酸序列同源性为83.6%~92.5%,氨基酸序列同源性为82.7%~95.1%.系统进化分析结果显示IBV广西株与SAIB20和LX4两参考株位于同一个分支上,它们的亲缘关系较近,而与其他参考株属于不同的分支,亲缘关系较远.结果表明IBV广西株是1株新的IBV变异株.     

布鲁氏菌内蒙古分离株omp31基因的克隆与序列分析

对三株布鲁氏菌内蒙古分离株的omp31基因进行克隆与序列分析.参照GenBank中布鲁氏菌的omp31基因序列,设计一对特异性引物,用PCR方法扩增布鲁氏菌omp31基因,对PCR产物进行克隆、测序,将测定序列拼接后与GenBank中获得的参考毒株omp31基因序列进行了比较.结果显示,三株布鲁氏菌分离株的omp31基因开放阅读框核苷酸序列同源性均为100.0%;与Brucella ceti B14/94、Brucella neotomae 5K33、Brucella suis 1330三个参考毒株omp31基因核苷酸序列同源性最高,为100.0%;与Brucella canis RM6/66、Brucella melitensis 183、Brucella melitensis 16M三个参考毒株omp31基因核苷酸序列同源性为99.9%;与Brucella melitensis Rev1参考毒株omp31基因核苷酸序列同源性为99.6%;与Brucella ovis ATCC 25840参考毒株omp31基因核苷酸序列同源性最低,为98.8%.     

3株O型FMDV VP1基因的克隆与序列分析

以3株国内分离的O型口蹄疫病毒(FMDV)(分别命名F1、F2、F3)为研究目标,根据GenBank中注册的FMDV VP1基因的序列设计2对引物,采用RT-PCR方法成功地扩增出含有VP1全基因的cD-NA片段,将3个cDNA片段分别克隆到pMD20-T Vector载体中进行序列测定,得到3个毒株VP1基因的序列。结果表明,3个O型FMDV毒株VP1基因cDNA长度均为639 bp,编码213个氨基酸。3株O型毒株彼此之间的核苷酸序列同源性在92.3%~94.2%之间,推导氨基酸序列同源性在97.2%~98.6%之间。与3个毒株同源性高的主要为香港和台湾的毒株。     

A detection assay forCampylobacter fetus in bovine semen by restriction analysis of PCR amplified DNA

A rapid screening assay forCampylobacter fetus in bull semen was developed using the polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) to complement isolation by culture. An oligonucleotide primer pair (C₁/C₂) from the hypervariable region of 16S rRNA ofC. fetus was used to amplify a 362 base pair fragment by PCR. The PCR/REA assay, which is completed in 10 hours, detected as few as threeC. fetus subsp.venerealis cells in experimentally infected raw bull semen and in semen diluted with milk or egg yolk Tris (EYT). All the strains tested, of both subspecies ofC. fetus, were amplified, as were some otherCampylobacter species. Restricting the amplified products byAluI differentiatedC. fetus from the other organisms. There was no visible product generated by PCR fromC. sputorum subsp.bubulus, a saprophytic organism found in the prepuce of bulls, or from seven other species of bacteria found in semen. A modification of the PCR assay, using another primer pair (C₃/C₂) and two temperature PCR cycling conditions, increased the probability of detectingC. fetus subsp.venerealis. PCR amplification followed by REA could be used to screen bovine semen rapidly forC. fetus. In most cases, sequencing of C₁/C₂ PCR generated products would be preferable for distinguishing between the two subspecies ofC. fetus.Abbreviations AI artificial insemination - bp base pair - Cff Campylobacter fetus subsp.fetus - Cfv Campylobacter fetus subsp.venerealis - EYT egg yolk Tris - MH Mueller-Hinton - PCR polymerase chain reaction - REA restriction endonuclease analysis - TE Tris-EDTA     

Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb

Strains of hepatitis E virus (HEV) genotypes 1 and 4 have been detected on the Chinese mainland although there have been no previous reports of zoonotic genotype 3 HEV. In the present study, 65 swine fecal specimens were collected from five pig farms located in different Shanghai suburbs. RT-PCR and nested PCR were undertaken using partial nucleotide sequences of Open Reading Frame 2 (ORF2) of HEV to detect HEV RNA. Genetic analysis was based on alignments of an amplified 150-nt ORF2 sequence. RT-PCR revealed 15 HEV positive samples among 65-pig fecal specimens examined. Phylogenetic analysis of the amplified sequences indicated seven HEV strains belonged to genotype 3 and eight strains to genotype 4. This is the first time that genotype 3 hepatitis E virus has been identified on the Chinese mainland.     

云南猪屎豆属遗传多样性的ISSR分析

以云南41份野生猪屎豆属种质为材料,用ISSR进行分析,结果表明, 100个ISSR引物中共筛选出16个多态性明显、反应稳定的引物, 41份材料DNA共扩增出164条条带,平均每个引物的扩增条带数为10.3条,其中多态性条带总数为159条,多态性比率为99.3%。材料间遗传相似系数范围在0.481 7~1.000 0,表现出丰富的遗传多样性;通过正交设计优化,得出适合猪屎豆属野生植物的ISSR-PCR反应体系为:40 ng模板DNA、引物0.2 μmol/L、200 μmol/L dNTP、1×Buffer (Mg²⁺ plus)、0.5 U Taq酶(TaKaRa);ISSR引物最佳退火温度为50或53℃;通过聚类分析,可将41份猪屎豆属种质分为5大类,同一种的种质可以很好的聚成一类;通过对思茅猪屎豆ISSR鉴定,得出它与猪屎豆属的遗传相似系数接近,然而确定该物种属于猪屎豆属需要做更多的鉴定。     

辽宁新品系绒山羊ZFX、ZFY基因片段的进化研究与性别鉴定

根据摩弗伦羊（Mouflon）和人（Human）的ZFX、ZFY基因序列以及相关文献设计引物,以雌雄辽宁新品系绒山羊的基因组DNA为模板,用PCR方法扩增并克隆其ZFX、ZFY基因第11号外显子的部分片段,进行序列测序.结果用NCBI的BLAST,生物软件ClustalX.exe、Mega、BioEdit.exe、DNAClub等进行分析.对辽宁新品系绒山羊（New-breeding cashmere goat）、藏系绵羊（Tibetan sheep）、摩弗伦羊（Mouflon）、马（Horse）、山狗（Coyote）、猪（Pig）、鲸（Whale）、人（Human）和牛（Cow）的ZFX、ZFY基因的外显子同源性进行序列对比分析.用N-J方法构建分子系统树.分析结果表明：ZFX、ZFY基因两者核苷酸同源性达92%,推导的氨基酸同源性为95%.经分析可知,辽宁新品系绒山羊与摩弗伦羊亲缘关系最近,与其他物种的亲缘关系较远.利用限制性内切酶AvaII对辽宁新品系绒山羊ZFX、ZFY基因的PCR产物进行酶切,确定AvaII为ZFX基因上的特异酶切位点,以此对辽宁新品系绒山羊性别进行鉴定.     

大肠杆菌肠毒素ST1b基因的亚克隆及其核苷酸序列分析

以大肠杆菌pSLM004菌株为始发菌株,利用合成的一对与ST_1基因特定序列相匹配的引物,通过PCR扩增出了ST_(1b)基因。该基因片段扩增物经适当纯化,与克隆载体pUC18的HincⅡ切点平端连接,转化到受体菌JM101中。利用Ap抗性作为压力选择标志以及LacZ基因插入失活显色反应,在Ap/Xgal-IPTG培养基上,筛选出转化子。将白色菌落转化子经酚/氯仿法抽提重组质粒,利用所扩增ST_(1b)基因片段上所设计的BglⅡ位点及pUC18载体具有的PvuⅡ切点,经酶切反应鉴定重组子,得到了理想重组子pBST2-6。该重组质粒经双脱氧法双向核苷酸序列分析,确定了插入的ST_(1b)基因与载体的连接向位及其全部核苷酸序列。     

基因芯片检测5种动物冠状病毒的初步研究

采用蔗糖密度梯度离心,纯化浓缩犬冠状病毒(CCV)、猫冠状病毒(FCV)、猫传染性腹膜炎病毒(FIPV)、猪传染性胃肠炎病毒(TGEV)、猪呼吸道冠状病毒(PRCV)的细胞培养物,分别设计7,17,11,10和4对引物,构建了49个基因片段的克隆。煮沸裂解法制备质粒DNA,回收PCR扩增产物,点制冠状病毒基因芯片。抽提病毒总RNA,利用Cy3-dCTP随机渗入反转录PCR标记,与芯片进行杂交检测,淘汰交叉的克隆片段。结果表明:克隆CCV1,CCV2,CCV5和CCV7可特异诊断CCV,克隆FCV6,FCV7,FCV8和FCV9可特异诊断FCV,克隆FIPV2,FIPV7,FIPV8和FIPV9可特异诊断FIPV,克隆PRCV1,PRCV2和PRCV3可特异诊断PRCV,克隆TGEV3,TGEV4,TGEV5和TGEV6可特异诊断TGEV。将这些特异克隆扩增片段重新点制基因芯片,与病毒PCR产物杂交,未发现交叉现象。基因芯片检测比传统PCR敏感1000倍,可有效应用于这5种动物冠状病毒的检测与区分。     

猪圆环病毒3型PCR检测方法的建立与Cap基因序列分析

建立一种检测PCV3流行毒株感染的PCR方法,并分析Cap基因序列变异情况,为PCV3流行病学调查提供技术支持.根据GenBank中公开的PCV3全基因序列,分别设计了质粒构建引物和检测引物,通过构建阳性质粒、灵敏度试验、特异性试验、临床样品检测试验以及测序分析,建立PCR检测方法.结果显示:建立的PCR方法检测的极限...