Mitochondrial DNAs of Suillus: three fold size change in molecules that share a common gene order

Summary We constructed restriction-site and gene maps for mitochondrial DNAs from seven isolates of five species of Suillus (Boletaceae, Basidiomycotina). Each mitochondrial genome exists as a single circular chromosome, ranging in size from 36 to 121 kb. Comparisons within species and between two closely related species revealed that insertions and deletions are the major form of genome change, whereas most restriction sites are conserved. Among more distantly related species, size and restriction-site differences were too great to allow precise alignments of maps, but small clusters of putatively homologous restriction sites were found. Two mitochondrial gene orders exist in the five species. These orders differ only by the relative positions of the genes for ATPase subunit 9 and the small ribosomal RNA and are interconvertible by a single transposition. One of the two gene arrangements is shared by four species whose mitochondrial DNAs span the entire size range of 36 to 121 kb. The conservation of gene order in molecules that vary over three-fold in size and share few restriction sites demonstrates a low frequency of rearrangements relative to insertions, deletions, and base substitutions.Abbreviations COI, COII, COIII Cytochrome oxidase subunits I, II, and III - APT6, ATP9; large and small ribosomal RNA gene apocytochrome b, COB; ATP synthase subunits 6, 9 - mtDNA LrRNA, SrRNA; mitochondrial DNA     

Full genomic sequence analysis of swine genotype 3 hepatitis E virus isolated from Shanghai

The full genomic nucleotide sequence of a previously identified genotype 3 hepatitis E virus (HEV), strain SAAS-JDY5, was obtained using RT-PCR and rapid amplification of cDNA ends (RACE). The genome consisted of 7225 nucleotides, excluding a poly-A tail at the 3′ terminus, and contained three open reading frames (ORFs), ORF-1, ORF-2 and ORF-3, encoding 1702, 660 and 113 amino acids, respectively. Phylogenetic analysis confirmed that SAAS-JDY5 belonged to genotype 3 HEV and was most closely related to the Japanese isolate wbJYG1 (AB222184). SAAS-JDY5 shared approximately 87% nucleotide similarity to human and swine strains from the United States, compared with 74–75% similarity to Asian (genotype 4) and Mexican strains (genotype 2). Alignment of the SAAS-JDY5 genomic sequence with reference sequences of the same genotype revealed one nucleotide substitution and one deletion at positions 5145 and 7189 (3′ UTR), respectively. Moreover, SAAS-JDY5 contained two additional nucleotides (AC) at the very end of the 3′-terminus preceding the poly-A tail of the genome. Comparison of the putative amino acid sequence encoded by the SAAS-JDY5 genome with sequences of other genotype 3 isolates revealed 15 unique amino acid substitutions and one deletion in ORF-1, and three substitutions in ORF-2.     

浙江省基因Ⅰ型乙脑病毒全基因序列的测定及分析

目的 为了获得浙江省乙脑病毒基因组详尽的资料,研究基因Ⅰ型乙脑病毒的分子特征及变异程度,为乙脑病毒分子流行病学及其基因研究提供科学依据.方法 设计特异性引物、RT-PCR分段扩增XJ69和XJP613株全基因,PER产物纯化后克隆于T载体并进行序列测定.通过生物学软件进行核苷酸序列和氨基酸序列分析和病毒的系统进化分析.结果 新分离乙脑病毒XJ69和NJP613株全基因组全长均为10 964个核苷酸,含有一个开放阅读框架,编码3432个氨基酸.与GenBank中选择的32株乙脑病毒全基因序列比较发现,其核苷酸同源为83.5%～99.2%,氨基酸总体同源性为97.5%～99.7%.通过PrM/C区段、E区段及全基因序列进行系统进化分析均显示该毒株属于基因Ⅰ型乙脑病毒.结论 新分离的乙脑病毒XJ69和XJP613株属于基因Ⅰ型,与上海三带喙库蚊分离株SH17M-07关系最为接近.     

Nucleotide sequence of a circular single-stranded DNA associated with coconut foliar decay virus

A circular single-stranded (ss) covalently closed (ccc) DNA associated with coconut foliar decay virus (CFDV) was purified, amplified by the polymerase chain reaction, and subcloned and its sequence established by analysis of overlapping subgenomic cDNA clones. The complete CFDV sequence comprised 1291 nucleotides and contained open reading frames for six proteins of molecular weight larger than 5 kDa. One of these (ORF1, 33.4 kDa) codes for a leucine-rich protein with the nucleoside triphosphate-binding motif GXGKS and may possibly participate in virus replication. The putative viral protein encoded by ORF3 (6.4 kDa) is a positively charged arginine-rich protein with homology to the capsid protein of nuclear polyhedrosis virus, and may represent the CFDV coat protein. CFDV DNA can form a stable stem structure of 10 GC base pairs subtending a loop sequence which in one orientation closely resembles the motif TAATATTAC conserved in a similar structural arrangement within the geminivirus group. Otherwise no sequence homology to DNA-containing plant viruses of the gemini- or caulimovirus groups was found. CFDV therefore represents a new taxonomic group of plant viruses.     

Closed circular DNAs with tandem repeats of a sequence from polyoma virus

The R₁ restriction endonuclease from bacteria containing the RTF-1 plasmid cleaves polyoma virus DNA at a single site. Virus stocks which have been passaged through cells several times at moderate or high multiplicities contain closed circular DNAs which are not cleaved by the R₁ endonuclease. One such population of DNAs, is not infectious, and when sheared and denatured, reassociates at a rate approximately four times faster than polyoma DNA. The predominant DNA in the population is 10% smaller than polyoma DNA and contains a fraction of the polyoma genome tandemly repeated three to four times. There is no significant contamination of the covalently closed circular DNAs by repetitive host sequences. A model explaining how such trandemly repeated molecules could arise by abortive rounds of polyoma DNA replication is discussed.     

Discovery of a novel circular single-stranded DNA virus from porcine faeces

A large number of novel single-stranded DNA (ssDNA) viruses have been characterised from various environmental sources in the last 5 years. The bulk of these have been from faecal sources, and faecal sampling is an ideal non-invasive pathogen sampling method. We characterised a novel ssDNA from a porcine faecal sample from Cass Basin of the South Island of New Zealand. The novel viral genome has two large open reading frames (ORFs), which are bidirectionally transcribed and separated by intergenic regions. The largest ORF has some degree of similarity (<30 %) to the putative capsid protein of chimpanzee stool-associated circular ssDNA virus (ChiSCV) and pig stool-associated single-stranded DNA virus (PigSCV), whereas the second-largest ORF has high similarity to the putative replication-associated protein (Rep) of ChiSCV (~50 %) and bovine stool-associated circular DNA virus (BoSCV; ~30 %). Based on genome architecture, location of putative stem-loop like elements, and maximum-likelihood phylogenetic analysis of the gene encoding the Rep protein, the novel isolate belongs to the same family of ssDNA viruses as ChiSCV and BoSCV.     

Complete genomic sequence of the coronavirus transmissible gastroenteritis virus SHXB isolated in China

A strain of transmissible gastroenteritis virus (TGEV), SHXB, was isolated in Shanghai, China. The complete genome of strain SHXB was sequenced, and its sequence was compared those of other TGEV strains in the GenBank database. The comparison showed that there were no insertions or deletions in the 5′ and 3′- non-translated regions, in the nonstructural genes ORF1, ORF3, and ORF7, or in the genes encoding the structural proteins envelope (E), membrane (M) and nucleoprotein (N). A phenomenon in common with other strains was that nucleotide (nt) 655 of the spike (S) gene was G, and a common change in nt 1753 of the S gene was a T-to-G mutation that caused a serine-to-alanine mutation at amino acid 585, which is in the region of the main major antigenic sites A and B of the TGEV S protein. A 6-nt deletion was also found at nt 1123-1128 in all Purdue strains except the strain Virulent Purdue. Phylogenetic analysis showed that TGEV SHXB was closely related to the Purdue strains and shared a common ancestor with the Miller strains as well as strain PRCV-ISU-1.     

Complete genomic sequence of a reovirus isolated from grass carp in China

A reovirus was isolated from sick grass carp in Guangdong, China in 2009, and tentatively named ‘grass carp reovirus Guangdong 108 strain’ (GCRV-GD108). This reovirus was propagated in grass carp snout fibroblast cell line PSF with no obvious cytopathic effects. Its genome was 24,703 bp in length with a 50% G + C content and 11 dsRNA segments encoding 11 proteins instead of 12 proteins. It has been classified as an Aquareovirus (AQRV). Sequence comparisons showed that it possessed only 7 homologous proteins to grass carp reovirus (GCRV) (with 17.6-45.8% identities), but 9 homologous proteins to mammalian orthoreoviruses (MRV) (with 15-46% identities). GCRV-GD108 lacked homology to VP7, NS4&NS5 and NS3 of GCRV, while it had sigma1 and sigma NS homology to MRV. VP2 of GCRV-GD108 shared high amino acid sequence identity (44-47%) with AQRVs, whereas VP5 did not exhibit much identity (24-25%) to AQRVs. Conserved terminal sequences, 5′-GUAAUUU and UUCAUC-3′, were found in all of the 11 genomic segments of GCRV-GD108 at the 5′ and 3′ non-coding regions (NCRs) of the segments. The 5′ NCRs of GCRV-GD108 was similar to GCRV, but differed from other species of AQRV or Orthoreoviruses (ORV). Phylogenetic analysis of coat proteins belonging to Reoviridae, VP1-VP6, showed that GCRV-GD108 clustered with AQRVs and grouped with ORVs, suggesting that GCRV-GD108 belonged to the genus Aquareovirus but was distinctive from any known species of AQRV. Morphological and pathological analyses, and genetic characterization of GCRV-GD108 suggested that it may be a new species of AQRV and it was more closely related with ORVs than other AQRVs. In addition, RT-PCR analysis of diseased grass carp samples collected from different regions of China indicated that these viruses displayed high similarities to each other (95.3-99.4%). They also shared high sequence similarities to GCRV-GD108 (96.7-99.4%), indicating that GCRV-GD108 is representative of the prevalence strain in southern China.     

Complete genome sequence analysis of Japanese encephalitis virus isolated from a horse in India

The complete genome of the Japanese encephalitis virus (JEV) strain JEV/eq/India/H225/2009(H225), isolated from an infected horse in India, was sequenced and compared to previously published JEV genomes. H225 genome was 10,977-nucleotides long, comprising a single ORF of 10,299-nucleotides, a 5′-UTR of 95 nucleotides and a 3′-UTR of 582 nucleotides. The H225 genome showed high levels of sequence identity with 47 fully sequenced JEV genomes, ranging from 99.3 % to 75.5 % for nucleotides and 99.2 % to 91.5 % for amino acid sequences. Phylogenetic analysis of the full-length sequence indicated that the H225 strain belongs to genotype III and is closely related to the Indian JEV strain Vellore P20778. A comparison of amino acids associated with neurovirulence in the E proteins and non-structural proteins of known virulent and attenuated JEV strains suggested H225 to be a highly virulent strain. This is the first report of whole-genome sequencing of a genotype III JEV genome isolated from equines.     

Analysis of the genetic information of a DNA segment of a new virus from silkworm

Summary In 1983, a parvo-like virus (Yamanashi isolate) was newly isolated from silkworm. However, unlike parvovirus, two DNA molecules (VD1 and 2) were always extracted from purified virions. To investigate the structure and organization of the virus genomes, we determined the complete nucleotide sequence of VD2. The sequence consisted of 6031 nucleotides (nts) and contained a large open reading frame (ORF1) with 3513 nts. A smaller open reading frame (ORF2) with 702 nts was found in the complementary sequence. Computer analysis revealed that both ORFs did not code for the major structural proteins (VP1, 2, 3, and 4). These results suggest that VD2 has not enough information to produce progeny virions by itself. Further, the structural importance of the terminal sequence (CTS) common to both VD1 and VD2 was also predicted by a computer analysis.     

Structure and nucleotide sequence of the terminal regions of adeno-associated virus DNA.

Both of the ends of adeno-associated virus (AAV) DNA contain regions which are capable of rapidly reforming duplex structures after denaturation, suggesting the presence of a terminal symmetrical sequence. These regions are estimated to involve at most the terminal 4% of the DNA. The nucleotide sequence of each 5′ end is found to be predominately either TTGGCCA (35%) or TGGCCA (50%). Sequence data for the 3′ terminal consistent with the 5′ data are also presented.     

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20–25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3′ terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.     

Relationship between single-stranded DNA isolated from mouse cells transformed by simian virus 40 and transcription of cellular and virus genes.

A minor fraction of single-stranded DNA (ssDNA) was isolated by an improved method of hydroxylapatite chromatography (HAC) from the native nuclear DNA (nDNA) of SV--3T3 cells, non-productively transformed by SV40. Molecular hybridization, monitored by the use of S1 nuclease, HAC, isopycnic centrifugation and thermal melting showed that ssDNA from SV-3T3 cells (which amounts to 1.5 to 2% of the total nDNA) has the same characteristics as ssDNA previously isolated from other cell species. Only 27 to 28% of ssDNA can be self-hybridized but the greatest part can be reassociated to the non-repetitive portion of nDNA and up to 38% hybridized to homologous RNAs, as compared with 7 to 8% for bulk nDNA. Highly radioactive virus probes (SV40-3H-cRNA synthesized in a cell-free system and the separated 'early' and 'late' strands of SV40 DNA labelled with 125I) were annealed to different excess amounts of cellular DNA. Both the quantities of each probe hybridized at saturation levels and the various reaction kinetics indicated that ssDNA is greatly enriched for virus sequences, mainly originating from the 'early' DNA strand which is predominantly expressed in SV-3T3 cells. The mode of formation of ssDNA is discussed in the light of other findings on the effects of DNA untwisting proteins and susceptibility of active animal genes to selective enzymic attacks.     

一起引发脑膜炎流行的ECHO病毒的序列分析

目的 测定山东省招远县1987年2－8月发生的无菌性脑膜炎大流行的病因病毒之W株病毒（Zhaoyuan/W/87）的部分序列,分析其与其他肠道病毒的关系。方法 病毒用酚－氯仿抽提RNA,经逆转录合成cDNA,经聚合酶链反应（PCR）扩增,产物纯化,采用双脱氧链末端终止法在AB1377自动核酸测序仪上测定序列。结果 W株5′端非区、2C和3D区的部分序列长度分别为244、275和353个核苷酸;VP1区全基因为876个核苷酸,编码292个氨基酸,与检索到的2株ECH029核苷酸序列的同源性分别为82．1％和81．7％,氨基酸序列的同源性分别为95．1％和94．4％,与检索到的1株ECH02核革苷酸序列的同源性为62．3％。种系发生树分析,W株在单独的一个分支上,与2株ECH029所在分支较近,与ECHO2所在分歧较远。结论 目前无法判定W株与ECHO2的关系,但可以说W株是ECHO029的变异味。     

SARS患者咽拭标本中新分离呼肠病毒部分基因组序列分析

目的 研究SARS患者中多病原感染状况，测定并分析新分离呼肠病毒基因组序列，从病毒的分子生物学角度研究其分类学位置。方法 SARS患者咽拭子标本经处理后接种Hep-2细胞，提取分离到的病毒RNA，以随机引物逆转录PCR扩增，PCR产物经克隆、测序，将其核苷酸序列及推断的氨基酸序列与GenBank（数据库中基因进行比较分析并建立系统进化树。结果 从4份SARS患者咽拭子标本中分离到3株呼肠病毒，部分基因片段测序结果显示为同一病毒，其病毒基因与呼肠病毒1、2、3型有较高的同源性，尤其是与呼肠病毒1、3型，相应片段的核苷酸序列同源性达82％～92％，推断相应氨基酸序列同源性高达94％～99％，而与其他病毒不存在有意义的同源性；系统进化树分析显示新分离呼肠病毒属于呼肠病毒科呼肠病毒属，且可能为哺乳动物呼肠病毒中的新血清型。结论 SARS患者咽拭子标本中存在呼肠病毒，新分离呼肠病毒核苷酸和氨基酸序列与呼肠病毒1、3型同源性最高，由于尚未得到决定病毒血清型的S1片段基因序列，故该病毒的确切分型有待进一步确定，与SARS患者病情的关系有待进一步研究。