Clinical and molecular characterization of a transmitted reciprocal translocation t(1;12)(p32.1;q21.3) in a family co-segregating with mental retardation, language delay, and microcephaly

<p>Abstract</p> <p>Background</p> <p>Chromosome translocation associated with neurodevelopmental disorders provides an opportunity to identify new disease-associated genes and gain new insight into their function. During chromosome analysis, we identified a reciprocal translocation between chromosomes 1p and 12q, t(1; 12)(p32.1; q21.3), co-segregating with microcephaly, language delay, and severe psychomotor retardation in a mother and her two affected boys.</p> <p>Methods</p> <p>Fluorescence in situ hybridization (FISH), long-range PCR, and direct sequencing were used to map the breakpoints on chromosomes 1p and 12q. A reporter gene assay was conducted in human neuroblastoma (SKNSH) and Chinese hamster ovary (CHO) cell lines to assess the functional implication of the fusion sequences between chromosomes 12 and 1.</p> <p>Results</p> <p>We determined both breakpoints at the nucleotide level. Neither breakpoint disrupted any known gene directly. The breakpoint on chromosome 1p was located amid a gene-poor region of ~ 1.1 Mb, while the breakpoint on chromosome 12q was located ~ 3.4 kb downstream of the ALX1 gene, a homeobox gene. In the reporter gene assay, we discovered that the fusion sequences construct between chromosomes 12 and 1 had a ~ 1.5 to 2-fold increased reporter gene activity compared with the corresponding normal chromosome 12 sequences construct.</p> <p>Conclusion</p> <p>Our findings imply that the translocation may enhance the expression of the ALX1 gene via the position effect and result in the clinical symptoms of this family. Our findings may also expand the clinical phenotype spectrum of ALX1-related human diseases as loss of the ALX1 function was recently reported to result in abnormal craniofacial development.</p

Studies on a CMV Isolate From of Spinach Yellow Mosaic Disease

緒言 前人研究 材料與方法 甲、材料 一、試驗植物來源 二、育苗準備 乙、方法 一、汁液接種與單斑分離 二、不同離心力病毒之分離與接種試驗 三、還原劑對本病毒病原性之影響 四、病毒之純化與汁液接種試驗 五、物理性質測定 六、罹病材料超薄切片與病毒在組織內分佈 七、蛋白鞘分子量大小之電泳動測定 八、抗血清製備與抗體IgG 抽取 九、力價測定及純度測定 結果 一、汁液接種後寄主反應與單斑分離 二、不同離心力之分離試驗 三、還原劑對病原性之影響 四、病毒之純化與電子顯微鏡觀察 五、物理性質測定 六、超薄切片與病毒在組織內分佈 七、蛋白鞘分子量之電泳動測定 八、抗血清製備與抗體IgG 抽取 九、力價測定與純度判

感染冬瓜的南瓜捲葉病毒之鑑定

During a disease survey of wax gourd (Benincasa hispida Cogn.) in Taiwan, virus-like symptoms of yellowing, vein enation, rugose mosaic and leaf curling were observed and further characterized. A naturally infected wax gourd sample (Wg1) was collected from experimental field at Beidou, Changhua County, Central Taiwan. The plant was tested negative with indirect ELISA using the antisera against eight RNA viruses commonly found in cucurbits, including Cucurbit aphid-borne yellows virus, Cucumber green mottle mosaic virus, Cucumber mosaic virus, Melon vein-banding mosaic virus, Papaya ringspot virus-watermelon type, Watermelon silver mottle virus and Zucchini yellow mosaic virus. However, it showed positive to a geminivirus, tentatively named isolate of Squash leaf curl virus Wg1 (SqLCV-Wg1), with polymerase chain reaction (PCR) using primer pair that is specific to begomovirus genomes. After cloning and sequencing, the PCR product was identified to be 1116 nucleotides (nt) in length, encompassing 126, 771, and 219 nt of the AV2, CP, and AC3 gene coding regions, respectively. Sequence alignment with the corresponding regions of other begomoviruses revealed that SqLCV-Wg1 is most closely related to Squash leaf curl Philippines virus, with percent nucleotide identities of 97.6, 97.7, and 93.8 for AV1, CP, and AC3 genes, respectively. The CP gene of SqLV-Wg1 was further cloned into the vector pET21d(+), which was then used to express proteins in Escherichia coli BL21(DE3) and to produce specific antiserum for future diagnosis purposes. This is the first record of cucurbitaceous plants infected with Squash leaf curl virus in Taiwan. 冬瓜(Benincasa hispida Cogn.)上發現一種新病害病徵，葉部褪綠、葉脈突起及褪色、葉面皺縮、甚至捲葉之現象，使得植株生長勢衰弱。採自彰化縣北斗鎮之冬瓜疑似病毒罹病株(Wg1)，經瓜類作物的八種病毒血清檢測，包括瓜類蚜媒黃化病毒(Cucurbit aphid-borne yellows virus, CABYV)，胡瓜綠斑嵌紋病毒(Cucumber green mottle mosaic virus, CGMMV)，胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)，甜瓜脈綠嵌紋病毒(Melon vein-banding mosaic virus, MVbMV)，木瓜輪點病毒-西瓜系統(Papaya ringspot virus W type, PRSV-W)，番茄斑點萎凋病毒（(Tomato spotted wilt virus, TSWV)，西瓜銀斑病毒(Watermelon silver mottle virus, WSMoV)，及矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV)，均早陰性反應。針對begomovirus外鞘蛋白之高度保留區域核酸序列，經比對設計引子對，以PCR增幅之。經由勝任細胞的轉形作用與核酸解序，獲得116個核甘酸，分為AV2 (126個核苷酸)、CP (771個核苷酸)及AC3 (219個核苷酸)。經與其他begomoviruses比對，得知Wgl病毒分離株與Squash leaf curl Philippines virus最有親源關係，其核酸相同度在AV1, CP, and AC3基因分別為97.6, 97.7, and 93.8%。利用載體pET21d (+)夾帶SqLV-Wg1的CP基因，在Escherichia coli BL21 (DE3)中表現病毒鞘蛋白，並用以注射紐西蘭白兔製備專一性的抗血清，供進一步診斷鑑定用。本文為台灣瓜類感染Squash leaf curl virus的首次紀錄

Molecular characterization of economically important viruses infecting fruit and energy crop in Taiwan

台灣向日葵葉片退綠斑點病，其葉片粗汁液接種奎藜會形成局部性病斑，經三次單斑分離後得到一個病毒分離株（SF-1）。經電子顯微鏡陰染法觀察其外型病原為長絲狀病毒，大小約770 x 13 nm，罹病組織超薄切片可觀察到細胞質束狀及風車狀圓柱內含體。以奎藜接種葉為材料進行病毒純化，經硫酸銫等密度平衡離心，所得病毒試料以膠體電泳分析測定鞘蛋白之分子量，估計為35kDa。以純化之病毒注射白兔製備抗血清，結果以瓊脂擴散反應測定，其抗血清只與同源反應。再經西方轉漬反應及間接法免疫酵素分析證實其血清有專一性，但本病毒以Agdia 之potyvirus group test kit 檢測亦呈正反應，因而將本病毒暫時命名為向日葵退綠斑點病毒(Sunflower chlorotic spot virus, SCSV)。抽取SCSV-SF-1全長的potyvirus核酸，將之定序並分析。 SCSV-SF-1核酸長度為9,741個核苷酸，可以轉譯成potyviruses的保留基本模組3,071胺基酸的polyprotein。 序列比較顯示3’端SCSV-SF-1與Bidens mottle virus (BiMoV)有96%相同性。 然而，SCSV-SF-1有狹窄的寄主範圍，不包括BiMoV的診斷寄主種類（鬼針草屬Bidens pilosa.和百日菊屬 Zinnia elegans）。 所以SCSV-SF-1是BiMoV一個分離株。 這是感染臺灣向日葵病毒病原BiMoV全長核苷酸序列的第一個報告。 冬瓜（Benincasa hispida Cogn.）上發現一種新病害病徵，葉部褪綠、葉脈突起及褪色、葉面皺縮、甚至捲葉之現象，使植株生長勢衰弱。採自彰化縣北斗鎮之冬瓜疑似病毒罹病株（Wg1），經瓜類作物的八種病毒血清檢測，包括瓜類蚜媒黃化病毒(Cucurbit aphid-borne yellows virus, CABYV)，胡瓜綠斑嵌紋病毒(Cucumber green mottle mosaic virus, CGMMV)，胡瓜嵌紋病毒（Cucumber mosaic virus, CMV），甜瓜脈綠嵌紋病毒(Melon vein-banding mosaic virus, MVbMV)，木瓜輪點病毒-西瓜系統（Papaya ringspot virus W type, PRSV-W），番茄斑點萎凋病毒(Tomato spotted wilt virus, TSWV)，西瓜銀斑病毒（Watermelon silver mottle virus, WSMoV），及矮南瓜黃化嵌紋病毒（Zucchini yellow mosaic virus, ZYMV），均呈陰性反應。針對begomovirus外鞘蛋白之高度保留區域核酸序列，經比對設計引子對，以PCR增幅之。經由DNA產物之選殖與核酸解序，獲得1116個核苷酸，分為AV2(126個核苷酸)、CP(771個核苷酸) 及AC3(219個核苷酸)。經與其他begomoviruses比對，得知Wg1病毒分離株與Squash leaf curl Philippines virus 最有親源關係，其核酸相同度在AV1, CP, and AC3 基因分別為97.6, 97.7, and 93.8％。利用載體pET21d(+)夾帶SqLCV-Wg1的CP基因，在Escherichia coli BL21(DE3)中表現病毒鞘蛋白，並用以注射紐西蘭白兔製備專一性的抗血清，供進一步診斷鑑定用。本文為台灣瓜類感染Squash leaf curl Philippines virus 的首次紀錄。 洋香瓜等瓜類作物極具經濟價值，然近年來迭遭數種新興病毒病害之大規模感染，造成農民的重大損失。其中由菸草粉蝨(Bemisia tabaci species complex)所傳播的南瓜捲葉病毒Squash leaf curl Philippines virus (SqLCV，雙生病毒科)於台灣中、南部地區洋香瓜上造成捲葉病的肆虐。在2008下半年造成洋香瓜捲葉病害的大流行，甚而導致全園廢耕的嚴重經濟損失，成為洋香瓜產業的主要限制因子之一。因此急需開發方便有效的方法，以監測SqLCV於粉蝨與作物中的帶毒與感染狀況，預作適當之防治工作。本研究研發利用滾環式擴增法(Rolling Circle Amplification，RCA) 檢測雙生病毒環狀DNA的技術，持續於雲林、嘉義、與台南等縣市之主要瓜類作物栽培區進行SqLCV之監測。發現SqLCV普遍存在於田間瓜類作物與粉蝨族群中，但其基因體變異性極大。且帶毒率與感染率隨作物種類而不同，其中以西瓜最低，洋香瓜最高。為進一步了解雙生病毒基因體在不同時空環境中的變異性，本研究分析比對自2007年至2010年不同時間點，於台灣中南部分屬不同縣市行政區的地理位置洋香瓜專業栽培區所採集的SqLCV基因體序列，並分別比對來自於洋香瓜葉片或粉蝨體內所選殖之SqLCV基因體序列，發現在不同時空環境下經常發生變異的核苷酸位置較常出現於基因間隙區域(intergenic region)，而其他基因體區域相對較為穩定。不同的地理環境與瓜類作物對於雙生病毒的基因體序列亦有影響，因此基因體序列之變異性亦可作為追蹤雙生病毒來源之用。A disease of sunflower (Helianthus annuus) showing symptoms of chlorotic spot ,enation, yellow of leaves and stunted was collected from Puli, and from which a pure line of virus isolate (SF-1) was obtained through series of single lesion isolation. Before correctly identify the pathogen, A filamentous potyvirus about 770 x 13 nm in size was isolated from the infected plant and proved to be the causal agent of the disease. The molecular size of the coat protein of the pathogen was found to 35 kDa. The pathogen induced cylindrical inclusion body including the shapes of scrolls、laminated aggregated and bundles in host cytoplasm. According to preliminary test including westing blotting test、ELISA test、nucleic acid sequencing and amino acid sequencing, the 3’end region and analysis of identical relationship suggests this sunflower virus could belong to PVY subgroup and the name of “sunflower chlorotic spot virus”(SCSV) is tentatively porposed. The full-length genome of a potyvirus, previously known as sunflower chlorotic spot virus isolate SF-1 (SCSV-SF-1) which causes novel symptoms on sunflowers was sequenced and analyzed. The genome of SCSV-SF-1 is 9,741 nucleotides long, encoding a polyprotein of 3,071 amino acids containing the consensus motifs of potyviruses. Sequence comparison revealed that the 3’-terminus of SCSV-SF-1 shared over 96% similarities with isolates of Bidens mottle virus (BiMoV). However, SCSV-SF-1 has a very narrow host range, excluding the diagnostic host species for BiMoV, Bidens pilosa and Zinnia elegans. Therefore, SCSV-SF-1 is a distinct isolate of BiMoV. This is the first report of the full-length nucleotide sequence of BiMoV infecting sunflower in Taiwan. During a disease survey of wax gourd (Benincasa hispida Cogn.) in Taiwan, virus-like symptoms of yellowing, vein enation, rugose mosaic and leaf curling were observed and further characterized. A naturally infected wax gourd sample (Wg1) was collected from experimental field at Beidou, Changhua County, Central Taiwan. The plant was tested negative with indirect ELISA using the antisera against eight RNA viruses commonly found in cucurbits, including Cucurbit aphid-borne yellows virus, Cucumber green mottle mosaic virus, Cucumber mosaic virus , Melon vein-banding mosaic virus , Papaya ringspot virus-watermelon type , Watermelon silver mottle virus and Zucchini yellow mosaic virus. However, it showed positive to a geminivirus, tentatively named isolate of Squash leaf curl virus Wg1 (SqLCV-Wg1), with polymerase chain reaction (PCR) using primer pair that is specific to begomovirus genomes. After cloning and sequencing, the PCR product was identified to be 1116 nucleotides (nt) in length, encompassing 126, 771, and 219 nt of the AV2, CP, and AC3 gene coding regions, respectively. Sequence alignment with the corresponding regions of other begomoviruses revealed that SqLCV-Wg1 is most closely related to Squash leaf curl Philippines virus, with percent nucleotide identities of 97.6, 97.7, and 93.8 for AV1, CP, and AC3 genes, respectively. The CP gene of SqLV-Wg1 was further cloned into the vector pET21d(+), which was then used to express proteins in Escherichia coli BL21(DE3) and to produce specific antiserum for future diagnosis purposes. This is the first record of cucurbitaceous plants infected with Squash leaf curl Philippines virus in Taiwan. Cucurbits are economically important crops in Taiwan. However, several emerging viral diseases have devastated the production of cucurbits and caused great losses in recent years. Squash leaf curl Philippines virus (SqLCV, Geus Begomovirus , Family Geminiviridae), transmitted by whiteflies (Bemisia tabaci species complex) is among the most severe viral pathogens in cucurbits. The development of efficient and convenient methods for the monitoring of SqLCV in cucurbits is in need. In this study, modified rolling circle amplification (RCA) method to detect the circular DNA genome of SqLCV was developed and applied in the survey of SqLCV in whiteflies and cucurbit crops in the major production areas in southern and central Taiwan. The results revealed the common and persistent presence of SqLCV among various cucurbit crops and whitefly populations. The disease incidences and viruliferous ratios varied widely among different crops and whitefly populations, with watermelon and muskmelon being the lowest and highest, respectively. The spatial and temporal effects on the variations of SqLCV genomic sequences were further analyzed using samples collected from various localities belonged to different territorial district in Taiwan over the period of 2007 to 2010. Most genomic variations were identified in intergenic region sequences, while the others coding sequences were highly conserved. These results provide applicable references for the tracing of origins of SqLCV isolates and the design of effective disease management measures for SqLCV.摘 要 i ABSTRACT iii Literature review 1 Background: 1 Source of genetic variations of viruses 1 Significance of genetic variations of viruses 2 The scope of genetic variations investigated in this study 3 A. Economical significance of viral diseases 3 A1. Bidens mottle virus (BiMoV): 3 B. Taxonomy: 5 C. Genetic variations and evolutions 7 C1. Genetic variations in RNA viruses (potyviruses): 8 C2. Genetic variations in DNA virus (geminiviruses): 9 The Objectives of This Study 11 References: 12 Part I 19 Full-length sequence analysis of a distinct isolate of Bidens mottle virus infecting sunflower in Taiwan 19 Abstract 20 Introduction 21 Provenance of virus material 22 Sequence properties 22 Discussion 23 Reference 25 Table 26 Fig 27 Part II 32 Identification of Squash leaf curl Philippines virus on Benincasa hispida in Taiwan 32 Abstract 33 Introduction 34 MATERIALS AND METHODS 35 Field observation and virus source 35 Polymerase chain reaction (PCR) 35 Cloning and sequence analyses 35 Expression of coat protein (CP) gene 36 Preparation of CP-enriched fractions 36 Antiserum production 36 Indirect enzyme-linked immunosorbent assay(Indirect ELISA) 36 Result 37 Field observation. 37 Host range and ELISA test. 37 Polymerase chain reaction(PCR). 37 Sequence analysis. 38 Expression and purification of Wg1-CP gene. 38 Indirect ELISA. 38 Discussion 38 Reference 40 Table 1. 42 Fig. 43 Part III 49 Spatial and Temporal Analysis on the Genetic Diversity of Squash leaf curl virus in Muskmelons and Whiteflies in Central and Southern Taiwan 49 Abstract 50 Introduction 51 Materials and methods 53 Cucurbit leaf and whitefly samples 53 Rolling circle amplification (RCA) 53 Southern blot hybridization 53 DNA sequence analysis of viral genome 53 Results and discussion 54 Detection of geminivirus DNA by RCA 54 Southern blot hybridization to confirm the RCA method of DNA amplification 55 The surveys on SqLCV disease incidences in cucurbits and viruliferous ratios of whitefly populations by using RCA method 56 The analysis of Genetic relationship of SqLCV genome 57 Analysis of variability of SqLCV genome 57 Conclusion and prospectives 59 References 61 Table 64 Fig 6

A simplified method of constructing infectious clones of begomovirus employing limited restriction enzyme digestion of products of rolling circle amplification

Most infectious clones of geminiviruses consist of (partial) tandem repeats of viral genomes in the vectors, which usually involve tedious, multi-step assemblies of genomic fragments in the construction process. A simplified procedure was devised to circumvent these problems, which employs limited restriction digestion of multimeric viral genomes produced by rolling circle amplification (RCA), followed by direct cloning into appropriate vectors. The efficiency of the procedure, and infectivity of the dimeric constructs it produced, were demonstrated using three different geminiviruses, namely ageratum yellow vein virus, tomato leaf curl virus, and squash leaf curl virus

Molecular and serological characterization of a distinct potyvirus causing latent infection in calla lilies

A virus (isolate: Ca-M19) capable of inducing local lesions on Chenopodium quinoa Willd. was isolated from calla lilies (Zantedeschia spp.). Subculture of Ca-M19 was easily maintained in C. quinoa, but a back inoculation from single lesion of C. quinoa to calla lilies has so far not been successful. Typical potyvirus-like flexuous particles were consistently detected in Ca-M19 infected plants, and a 1.3-kb DNA fragment was amplifiedfrom these plants by reverse-transcription polymerase chain reaction (RT-PCR) using potyvirus degenerate primers. The PCR product was cloned and its sequence analyzed (AF469171). The amplicon was revealed to correspond to the 3’ terminal region of a potyviral genome. After comparing this sequence with known potyvirus sequences in the GenBank, we considered the virus a new species of Potyvirus based on the uniqueness in its coat protein gene (CP) and the 3’ non-coding region (NCR). Comparative studies showed that Soybean mosaic virus (SMV) and Watermelon mosaic virus 2 (WMV 2) were the two most similar potyviruses with Ca-M19, but they shared only 80% of nucleotide identities in CP and NCR with Ca-M19. Attempts to purify a sufficient quantity of Ca-M19 from C. quinoa for preparation of antibodies were unsuccessful. Alternatively, Ca-M19 CP was expressed by the vector pET28b and puri-fied from E. coli culture, and polyclonal antibodies were prepared in rabbits. The antibody was applied in ELISA, Western blotting, SDS-immunodiffusion and immuno-specific electron microscopy for the detection of Ca-M19 in calla lilies. It did not react with at least five calla lily infecting potyviruses, including Dasheen mosaic virus, Bean yellow mosaic virus, Konjak mosaic virus, Turnip mosaic virus, and Zantedeschia mild mosaic virus. Indirect ELISA and SDS-immunodiffusion tests showed that Ca-M19 was serologically related, but distinct from Bean common mosaic virus (BCMV), Black cowpea mosaic virus (BlCMV), Melon vein banding mosaic virus (MVbMV), Passionfruit mottle virus (PaMV), Passionfruit crinkle virus (PCV), Pas-sionfruit woodness virus (PWV), Soybean mosaic virus (SMV), Watermelon mosaic virus 2 (WMV 2), and Zucchini yellow mosaic virus (ZYMV). Besides serological techniques, a primer pair (M19u/M19d) and a DNA probe were designed which could also specifically detect and differentiate Ca-M19 from other viruses. By the use of specific antibodies in ELISA, Ca-M19 was frequently detected in calla lily plants collected from several major calla lily production townships in Taiwan. Among 86 field samples positively reacting to the antibody, 77 of them exhibited evident systemic mosaic symptoms, but these symptomatic plants were confirmed to be infected simultaneously by other viruses. Nine plants were found to be infected by Ca-M19 alone. These plants were confirmed to have remained symptomless throughout a 6-month observation period. Therefore, we propose naming this isolate Calla lily latent virus (CLLV) for its inability to develop any visible symptoms on calla lily