人偏肺病毒广州分离株全基因组序列测定及分析

目的 探讨广州地区儿童感染的偏肺病毒基因组分子结构特点和基因型类型.方法 参考GenBank上的偏肺病毒00-1株(AF371337)基因组设计分段扩增引物,进行RT-PCR分段扩增偏肺病毒基因组,克隆于T载体上,序列测定,用Clustal W/X、DNASTAR、MEGA4.1等软件分析基因组序列.结果 偏肺病毒hMPVgz01全基因组为13 327 bp,提交到GenBank上的序列号为GQ153651,有8个开放阅读框(open reading frames,ORFs),基因组结构为:3-N-P-M-F-M2-SH-G-L-5;将hMPVgz01株全基因组核苷酸序列与GenBank上的偏肺病毒全基因组序列进行Clustal W比较,发现与偏肺病毒的A组相似性较高,为92%～97%,与A2b组的BJ1887相似性为最高,而与B组相似性为81%,与C组的禽偏肺病毒为71%.将hMPVgz01株的N、F、G基因与偏肺病毒的A1、A2、B1、B2的相对应基因进行相似性比较,同样也是与A2b型的相似性为最高,因此确认广州地区的hMPVgz01株为A2b型.结论 广州地区儿童感染的偏肺病毒hMPVgz01株全基因组序列为13 327 bp,GenBank 序列号为GQ153651,hMPVgz01与偏肺病毒的A2b型相似性最高,确认hMPVgz01株属于偏肺病毒A2b型.     

Genetic analysis of a human rotavirus that belongs to subgroup I but has an RNA pattern typical of subgroup II human rotaviruses.

We have previously found (O. Nakagomi, T. Nakagomi, H. Oyamada, and T. Suto, J. Med. Virol. 17:29-34, 1985), during an epidemiological study in Japan, a novel human rotavirus that belongs to subgroup I but has a long RNA pattern typical of subgroup II human rotaviruses. From the stool specimen containing this virus, we successfully isolated in MA104 cells a rotavirus, designated AU-1, which possesses these novel characteristics. The possibility that strain AU-1 was a laboratory contaminant of an animal rotavirus previously adapted to tissue culture cells was ruled out, and the identity of the AU-1 strain was established. Genetic analysis by RNA-RNA hybridization revealed that the AU-1 strain is not a simple reassortant between subgroup I and II human rotaviruses but that it shares a high level of sequence homology only with the gene encoding VP7 (the major neutralization protein) of serotype 3 human rotaviruses. Weak homology of the genomic RNA segments was also observed between the AU-1 strain and animal rotavirus strains, including rhesus rotavirus strain RRV and bovine rotavirus strain NCDV. These results suggest that the AU-1 strain may be an animal rotavirus that infected a human.     

Molecular characterization of an atypical Old World strain of Peanut stunt virus

Summary.  Northern blot analyses of RNAs from an Iranian strain of Peanut stunt virus (PSV-I Cucumovirus) using cloned cDNA probes to the genomic RNAs from two PSV strains, PSV-ER (subgroup I) and PSV-W (subgroup II), indicated that PSV-I RNA-3 is derived from a subgroup II strain. No hybridization signals, however, were detected with PSV-I RNAs 1 and 2 suggesting they are distinct from both subgroups I and II. Nucleotide (nt) sequence analysis of cloned cDNA fragments (1–1.5 kbp in size) representing PSV-I RNAs 1 and 2 showed that PSV-I is more closely related to subgroup II strains than to any other sequenced cucumovirus. The percent nt identity between RNA-1 sequences from PSV-I and PSV-ER or between PSV-I and PSV-W were 79.1% and 88.8%, respectively. The corresponding values for RNA-2 were 77.5% and 86.7%. Sequence comparison analyses of deduced amino acid sequence of cloned partial sequences of PSV-I RNAs 1 and 2 indicated that PSV-I 1a and 2a proteins are most closely related to those of subgroup II strains (93% identity). PSV-I supported the replication and encapsidation of PSV G-satellite RNA (G-satRNA), but not cucumber mosaic virus WL1-satRNA. PSV-I may be perceived as an Old World derivative of subgroup II strains, or it may represent a natural reassortment between a subgroup II (RNA-3) and an as yet uncharacterized subgroup of PSV strains (RNAs 1 and 2). Received November 11, 1998 Accepted March 15, 1999     

Molecular characterization of a strain of cucumber mosaic virus based on coat protein and movement protein genes

Cucumber mosaic virus (CMV) A strain (CMV-A) isolated from Amaranthus tricolor was partially characterized at molecular level. Complete coat protein (CP) and movement protein (MP) ORFs were cloned and sequenced. The 657 bp region of CP gene and the 840 bp region of MP gene encode 218 and 276 amino acids, respectively. CP, at nucleotide level, showed 90-98% sequence identity with the CMV subgroup I and less than 80% with the CMV subgroup II, it showed at amino acid level 92-96% identity with the subgroup I and 74-87% with the subgroup II. The nucleotide and amino acid sequence identities of MP ranged in 91-94% and 92-96%, respectively with the subgroup I but in 81-83% with the subgroup II. Phylogenetic trees generated from nucleotide and amino acid sequences of both CP and MP genes identified the virus strain as a member of the subgroup IB. CMV-A CP also displayed a remarkably higher homology with Indian strains of CMV than with other CMV strains and formed a separate cluster within the subgroup IB.     

新分离登革2型病毒福建株基因组全序列的测定

目的 对新近分离的导致1999年福建省登热流行的登革2型病毒FJ－10株进行基因组全序列测定及系统发生树分析。方法 利用RT－PCR和5′、3′RACE法扩增FJ－10株cDNA,并进行克隆测序,利用DNASTAR折Clustal方法绘制系统发生树。结果 JF－10株基因组全长10723个核苷酸,有1个单一开放读码框架（ORF,第97－10269nt）,编码3391个氨基酸,5′和3′非编码区长度分别为96和454个核苷酸,通过与标准株NGC株和我国其他地区分离株DEN2－04、43、44株比较,核苷酸同源性分别为94.0%、92.8%、93.9%和93.9%,氨基酸同源性分别为97.9%、97.2%、97.7%和97.9%。以47株登革2型病毒E／NS1连接区240个核苷酸序列进行发生树分析,福建株与印度尼西亚和斯里兰卡分离株亲缘关系较近,同属第Ⅳ基因型。结论 FJ－10株基因组全序列一级结构与其他登革2型病毒类似,其基因型不同于我国其他地区分离株DEN2－04、43和44株。     

The complete genomic sequence analysis of genotype 4 human astrovirus HASTVgz01 strain in Guangzhou

To analyze the genomic molecular structure and genotype of human astrovirus isolated from infant in Guangzhou of China, the primers were designed based on the genomic sequence of astrovirus from the GenBank and the target sequence were amplified by RT-PCR. Then the PCR-products were cloned to T vector and sequenced. The genomic nucleotide sequences were analyzed by the programs CLUSTAL W and DNASTAR. It was found that the full genomic length of HASTVgz01 strain was 6721 bp and the ORFs were 6558 bp. The 5' and 3' UTR were 82 and 81 nucleotides. The genome included 3 open reading frames (ORFs): ORF1a, ORF1b and ORF2. The 5'-terminal ORF1a started at nucleotide 83 and extended to nucleotide 2845. ORFlb (nt 2785 to nt 4332) overlaped ORFla by 61 nucleotides. The 3'-terminal ORF2 began at nucleotide 4325 and terminated at nucleotide 6640. ORF2 had 2316 nucleotides. Compared with other astrovirus sequences in GenBank, the homology of the amino acid sequence of ORF2 of HASTVgz01 strain with that of serotype 4 was 93% . Homology with other serotypes ranged from 61% to 70% . The complete nucleotide sequence of astrovirus HASTVgz01 strain isolated from Guangzhou in China was 6721 bp in length, GenBank accession NO. DQ344027. Comparing the ORF2 of astrovirus HASTVgz01 with the known sequences of types 1-8 the highest homology was serotype 4 (93%). Comparative sequence analysis of the HASTVgz01 ORF2 with the reported human astrovirus sequences revealed that the isolated astrovirus belongs to genotype (serotype) 4.     

Full genomic analysis of human rotavirus strain TB-Chen isolated in China

A G2P[4]/NSP4[A] rotavirus strain TB-Chen was isolated from a 2-year-old patient hospitalized with acute gastroenteritis in Kunming, China. The strain TB-Chen was demonstrated having group A-specific antigenicity, a “short” (subgroup II) electropherotype. To investigate its overall genomic relatedness and to determine which group it belonged, the complete genome of strain TB-Chen was determined. Genomic comparison based on amino acid sequence identity and phylogenetic analysis revealed that all 11 gene segments of strain TB-Chen were highly identical (> 91.80%) with the representative G2P[4]/NSP4[A] human strains DS-1, S2, NR1 and IS2, suggesting that this rotavirus strain was derived from human host. Besides, almost all the available representative rotavirus gene segments among group A were analyzed and identified within 15 G-types, 28 P-types, and 6 NSP4 genotypes. This is the first report of group A rotavirus genomic analyses in China and the findings have important implications for rotavirus vaccine development.     

Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses

We have determined the complete sequence of the RNA of dengue 2 virus (S1 candidate vaccine strain derived from the PR-159 isolate) with the exception of about 15 nucleotides at the 5' end. The genome organization is the same as that deduced earlier for other flaviviruses and the amino acid sequences of the encoded dengue 2 proteins show striking homology to those of other flaviviruses. The overall amino acid sequence similarity between dengue 2 and yellow fever virus is 44.7%, whereas that between dengue 2 and West Nile virus is 50.7%. These viruses represent three different serological subgroups of mosquito-borne flaviviruses. Comparison of the amino acid sequences shows that amino acid sequence homology is not uniformly distributed among the proteins; highest homology is found in some domains of nonstructural protein NS5 and lowest homology in the hydrophobic polypeptides ns2a and 2b. In general the structural proteins are less well conserved than the nonstructural proteins. Hydrophobicity profiles, however, are remarkably similar throughout the translated region. Comparison of the dengue 2 PR-159 sequence to partial sequence data from dengue 4 and another strain of dengue 2 virus reveals amino acid sequence homologies of about 64 and 96%, respectively, in the structural protein region. Thus as a general rule for flaviviruses examined to date, members of different serological subgroups demonstrate 50% or less amino acid sequence homology, members of the same subgroup average 65-75% homology, and strains of the same virus demonstrate greater than 95% amino acid sequence similarity.     

Molecular evidence and sequence analysis of a natural reassortant between <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and II strains

Cucumber mosaic virus (CMV) is a tripartite RNA virus and has been divided into three subgroups, named IA, IB, and II. Some studies have found a few natural reassortants between CMV subgroups, although reassortment between CMV subgroups is infrequent. In our present work, a CMV reassortant, named CMV-Tsh, was obtained from a tomato plant. The complete sequence of CMV-Tsh genomic RNAs has been determined and analyzed. The results of sequence comparisons and phylogenetic analyses revealed that CMV-Tsh RNAs 1 and 3 are derived from one or two CMV subgroup II strain(s), while RNA2 is derived from a CMV subgroup IA strain. A PCR and restriction enzyme analysis-based method was developed to analyze the possibility of mixed infection by CMV strains of different subgroup in the CMV-Tsh-infected tomato plant. The results of the restriction enzyme analysis proved that CMV-Tsh is the unique strain in the tomato plant. Taken together, CMV-Tsh is a natural reassortant having CMV subgroup IA RNA2 and subgroup II RNAs 1 and 3. The GenBank Accession numbers of the sequences reported in this paper are EF202595- EF202597.     

甲肝减毒活疫苗L-A-1疫苗株的核酸序列测定与比较分析

目的 测定甲型肝炎(甲肝)减毒活疫苗龙甲-1株(L-A-1)核酸序列，了解其减毒和适应二倍体细胞的分子机制，并与其他甲肝病毒株进行比较，得出L-A-1株的一些特点。方法 通过抗原捕获聚合酶链反应(AC-PCR)扩增甲肝病毒减毒活疫苗L-A-1株23代的片段，产物纯化后克隆至T载体并进行序列测定，应用生物信息学软件分析比较。结果 得到了甲肝减毒活疫苗L-A-1株的基因序列(nt25-7418)。L-A-1株开放读码框架(ORF)长6675个核苷酸，编码2225个氨基酸。比较分析表明，该株与国际代表株MBB株和HM175野毒株在核苷酸水平上同源性分别为98．00％和94．00％，在氨基酸水平上同源性分别为98．51％和98．65％。通过与其他细胞适应株、细胞病变株以及减毒株的比较得出：L-A-1株5′非编码区(5′NTR)nt-152、591、646、687处的突变和180-181处插入11个核苷酸以及2B区的m3889(aa1052-Val)处的突变是病毒适应宿主细胞的分子基础；2C区的m4185(aa1152-Lys)处的突变可能参与病毒的减毒过程；由于3A区缺失6个核苷酸(nt5020-5025)从而导致两个氨基酸(Asn、Asp)缺失是病毒适应细胞快速增殖的分子基础。结论 分析了LA-1株的基因序列。得出其适应细胞和减毒的核苷酸位点。     

Nucleotide sequence of RNA segment 5, encoding the nucleoprotein, of influenza A/FPV/Rostock/34

The nucleotide sequence of a cloned DNA copy of genome RNA segment 5 of fowl plague virus (influenza A/FPV/Rostock/34) has been determined. Segment 5 is 1565 nucleotides in length and codes for a nucleoprotein of 498 amino acids. Comparison of the sequence with the nucleoproteins of two human influenza A strains reveals 84–85% homology with the avian strain at the nucleotide level but 91–93% homology at the amino acid level. This presumably reflects common functional requirements of influenza A nucleoproteins.     

The Taiwanese hepatitis C virus genome: sequence determination and mapping the 5' termini of viral genomic and antigenomic RNA.

The complete nucleotide sequence of hepatitis C virus (HCV) cloned from the liver tissue of a Taiwanese patient with post-transfusion type C hepatitis was determined. The 5' end of HCV genomic RNA was located 341 nucleotides upstream from the initiation codon for the viral polyprotein open reading frame. The 5' end of the viral antigenomic RNA was shown to have 13 consecutive As. Thus the 3' terminus of the viral genome is a stretch of U which ends about 50 nucleotides downstream from the stop codon of the large open reading frame. The nucleotide sequence homology between this HCV strain and two Japanese isolates was 90.5 and 90.7%, respectively. Homology with the United States strain, however, was only 77.8%. Accordingly, the indigenous Taiwanese HCV strain is of the same subtype as the Japanese isolates. Novel features of the viral genome termini are possibly relevant to HCV genome replication.     

The Genome-linked Protein (VPg) of Southern Bean Mosaic Virus is Encoded by the ORF2

The sequence of the 20 N-terminal amino acids of the viral protein (VPg) which is covalently attached to the genomic RNA of the bean strain of Southern bean mosaic virus (SBMV-B) has been determined. The obtained VPg sequence mapped to position 327 to 346 of the SBMV-B ORF2 product, downstream of the putative protease domain and in front of the RNA-dependent RNA polymerase. Thus indicating that the sobemovirus genomic arrangement is similar to that of subgroup II luteoviruses. Comparison with other viral sequences revealed a high similarity with the sequence of the ORF2-product of the cowpea strain of SBMV (SBMV-C). No significant similarities were detected with amino acid sequences derived of other sobemoviruses or non-related viruses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequence analysis of cDNA for the VP6 protein of group A avian rotavirus: a comparison with group A mammalian rotaviruses

Summary cDNA corresponding to the genomic segment 6 of avian rotavirus strain PO-13, which has group A common and subgroup I antigens, but does not hybridize in Northern blots with RNA probes from group A mammalian rotaviruses, was cloned and sequenced. When the deduced amino acid sequence was compared between strain PO-13 and eight group A mammalian rotaviruses, the extent of homology ranged from 73–75%. An alignment of the amino acid sequences allowed us to identify three amino acids (Positions 120, 317 and 350) that may contribute to determining the subgroup epitopes. A phylogenetic tree constructed on the basis of nucleotide substitutions in the VP6 gene of nine rotaviruses strongly suggests that the avian rotavirus is an ancestral prototype of mammalian rotaviruses.     

7型腺病毒疫苗株DNA左侧17.5 mu片段克隆及4.8 mu片段核苷酸序列分析

目的获得７型腺病毒疫苗株ＤＮＡ左侧０～１７５ｍｕ片段克隆,分析０～４８ｍｕ片段（末端倒置重复序列、包装信号位点和Ｅｌａ区）核苷酸序列。方法从Ａｄ７疫苗株感染的Ａ５４９细胞提取Ａｄ７ＤＮＡ,将其０３～１７５ｍｕ片段克隆到质粒ｐＡｄ７Ｔ,并用自动和银染方法测序。结果获得了Ａｄ７疫苗株左侧１７５ｍｕ片段克隆,测定了左侧１７３７ｂｐ核苷酸序列,其中Ｅｌａ区所编码的６３００、２４０００、２８０００等３个蛋白的ＤＮＡ序列,与Ｇｏｍｅｎ株的同源性分别为９８９％、９７３％和９７５％,推导编码氨基酸的同源性分别为９６６％、９６５％和９６９％。这３个蛋白与Ｇｒｉｄｅｒ株的同源性分别为１００％、９９７％、和９９７％;推导编码氨基酸的同源性分别为１００％、９９１％和９９２％。结论Ａｄ７疫苗株左侧１７３８ｂｐ核苷酸与Ａｄ７Ｇｏｍｅｎ株和Ｇｒｉｄｅｒ株相应片段,具有高度的同源性。     

1型星状病毒上海分离株基因组序列测定及分析

目的 了解上海地区腹泻患儿感染的星状病毒基因组分子结构特点和基因型.方法 用RT-PCR方法分段扩增星状病毒基因组,克隆于pMD18-T载体上,测定序列并拼接,用MEGA、DNAStar软件对所得序列进行分析.结果 本次分离株基因组全长6807 bp,命名为HAstV-SH,提交到GenBank上的序列号为FJ375759,有3个开放阅读框架,非结构基因ORF1a、ORF1b共长4290 bp,位于基因组83～4372 nt之间;编码结构蛋白的ORF2基因全长2364 bp,位于基因组4764～6727 nt之间.与GenBank中星状病毒ORF2基因组序列同源性比较发现,HAstV-SH与1型星状病毒核苷酸同源性最高(97%),与其他基因型的同源性为63%～70%.结论 上海地区儿童感染的星状病毒HAstV-SH属于1型星状病毒,与日本分离株AB009985亲缘关系较近,氨基酸同源性为97.5%.