猪Ⅱ型圆环病毒四川分离株鉴定及其ORF2基因序列分析

猪圆环病毒(Porcine circovirus,PCV),属于圆环病毒科(Circoviridae),圆环病毒属(Circovirus),血清型为PCV-1和PCV-2[1]。PCV-1首先由Tis-cher[2]于1974年从PK-15猪肾传代细胞系中分离获得,PCV-2首先由Clark[3]报道了是断奶仔猪多系统衰竭综合征(PMWS)的主要病原,随即相继报道了PCV-2与PDNSS(猪皮炎与肾炎综合症)、NP(增生性坏死性肿炎)、PRDC(猪呼吸道综合征)、繁殖障碍、先天性颤抖、肠炎等疾病亦有重要关联[4,5];它常与猪呼吸与繁殖综合征病毒(PRRSV)或猪细小病毒(PPV)并发感染或继发细菌感染[6]。我国自从2000年郎…     

Low Genetic Diversity of the Coat Protein Gene among Blueberry Red Ringspot Virus Isolates

Blueberry red ringspot virus (BRRSV) isolates have been investigated for genetic diversity. Nucleotide sequences of the coat protein (CP) gene of 19 isolates from Poland, Czech Republic, Slovenia and the United States were analysed. The nucleotide and amino acid sequence identity were 92–100% and 89–100%, respectively. Estimations of the distribution of synonymous and non‐synonymous changes indicated negative selection within the analysed CP gene and confirmed the genetic stability of the virus. At a capsid protein level, our results revealed BRRSV to be distinct from other, recombination‐prone pararetroviruses.     

The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism.

Sequence analysis of a substantial part of the polymerase gene of the murine coronavirus MHV-A59 revealed the 3' end of an open reading frame (ORF1a) overlapping with a large ORF (ORF1b; 2733 amino acids) which covers the 3' half of the polymerase gene. The expression of ORF1b occurs by a ribosomal frameshifting mechanism since the ORF1a/ORF1b overlapping nucleotide sequence is capable of inducing ribosomal frameshifting in vitro as well as in vivo. A stem-loop structure and a pseudoknot are predicted in the nucleotide sequence involved in ribosomal frameshifting. Comparison of the predicted amino acid sequence of MHV ORF1b with the amino acid sequence deduced from the corresponding gene of the avian coronavirus IBV demonstrated that in contrast to the other viral genes this ORF is extremely conserved. Detailed analysis of the predicted amino acid sequence revealed sequence elements which are conserved in many DNA and RNA polymerases.     

Molecular characterization of human immunodeficiency virus from Zaire: nucleotide sequence analysis identifies conserved and variable domains in the envelope gene

To examine the genetic relatedness of human immunodeficiency viruses (HIV) from different geographic locations, we molecularly cloned the genome of HIV isolated from a Zairian AIDS patient. Restriction mapping of the recombinant clone, designated HIV-Zr6, revealed both common (as observed in other HIV isolates) and unique restriction sites. The DNA clone of HIV-Zr6, shown to give rise to infectious cytopathic virus after transfection of cultured lymphoid cells, was sequenced in several regions. The long terminal repeat (LTR), open reading frame 1 (ORF1), C-terminal envelope (env) gene domain, and ORF2 showed less than 6% difference in nucleotide sequence when compared to other HIV isolates including human T-lymphotropic virus-type III (HTLV-III) clone B10, lymphadenopathy-associated virus-1 (LAV-1), and AIDS-associated retrovirus-2 (ARV-2). About 15% difference in nucleotide sequences was noted in the N-terminal env gene domain. Alignments of env gene sequences revealed conserved, moderately variable, and hypervariable stretches in the predicted amino acid sequences. This model provides a basis for assessing the significance of sequence variation on properties controlled by the viral Env glycoproteins such as cell tropism and immunogenicity.     

Sequence Analysis of Segment 8 of Five Chinese Isolates of Rice Gall Dwarf Virus and Expression of a Main Outer Capsid Protein in Escherichia coli

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.     

Phylogenetic relationships of aquatic birnaviruses based on deduced amino acid sequences of genome segment A cDNA.

Aquatic birnaviruses, such as infectious pancreatic necrosis virus (IPNV), cause serious diseases in a variety of fish species used worldwide in aquaculture and have also been isolated from a variety of healthy fish and shellfish species. These viruses exhibit a high degree of antigenic heterogeneity and variation in biological properties such as pathogenicity, host range, and temperature of replication. To better understand genetic and biological diversity among these viruses, the nucleotide and deduced amino acid sequences were determined from cDNA of the large open reading frame (ORF) of genome segment A of the 9 type strains of Serogroup A and 4 other representative strains of Serotype A1, the predominant serotype in the United States. In addition, nucleotide and deduced amino acid sequences were determined for the VP2 coding region of a variety of isolates representing 5 of the 9 serotypes. VP2 is the major outer capsid protein of aquatic birnaviruses. RT-PCR was used to amplify a 2904 bp cDNA fragment including all but a few bp of the large ORF of genome segment A or a 1611 bp fragment representing the entire VP2 coding region. Nucleotide and deduced amino acid sequences were determined from the PCR products. Pairwise comparisons were made among our data and 2 other aquatic birnavirus sequences previously published. Several hypervariable regions were identified within the large ORF. The most divergent pair of viruses exhibited a similarity of 80.1% in the deduced amino acid sequence encoded by the large ORF. Genomic relationships revealed in a phylogenetic tree constructed from comparison of the deduced amino acid sequences of the large ORF demonstrated that these viruses were clustered into several genogroups. Phylogenetic comparison of the deduced amino acid sequences of the VP2 coding region of 28 aquatic birnavirus isolates, including the type strains of all 9 serotypes, demonstrated 6 genogroups, some of which were comprised of several genotypes. The most divergent pair of viruses exhibited a similarity of 81.2% in the deduced amino acid sequence from the VP2 coding region. In contrast to previous studies of much shorter genomic sequences within the C-terminus-pVP2/NS junction coding region, these genogroups based on the entire large ORF or the VP2 coding region generally correlated with geographical origin and serological classification. Isolates from the major Canadian serotypes were more closely related to the European isolates than to isolates from the United States.     

Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.     

Identification of a consistent pattern of mutations in neurovirulent variants derived from the sabin vaccine strain of poliovirus type 2.

Complete nucleotide sequencing of the RNAs of two unrelated neurovirulent isolates of Sabin-related poliovirus type 2 revealed that two nucleotides and one amino acid (amino acid 143 in the major capsid protein VP1) consistently departed from the sequences of the nonneurovirulent poliovirus type 2 712 and Sabin vaccine strains. This pattern of mutation appeared to be a feature common to all neurovirulent variants of poliovirus type 2.     

Sequence analysis of segment 8 of five Chinese isolates of Rice gall dwarf virus and expression of a main outer capsid protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8 (S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%–98.8% and 97.3%–99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%–95.6% and 95.0%–96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta (DE3) II cells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression. Foundation item: National natural science foundation of China (30370929) and Guangdong province natural science foundation (C036845)     

Mapping of the Feline Calicivirus Proteinase Responsible for Autocatalytic Processing of the Nonstructural Polyprotein and Identification of a Stable Proteinase-Polymerase Precursor Protein

Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.     

中华蜜蜂囊状幼虫病毒北京分离株VP1蛋白基因序列特征及原核表达

【目的】研究中华蜜蜂囊状幼虫病毒（Chinese sacbrood virus, CSBV）VP1蛋白的分子进化特征及遗传多样性。【方法】利用RT-PCR方法,克隆了8株CSBV北京分离株VP1蛋白的基因编码区。【结果】序列分析表明,VP1蛋白基因编码区开放阅读框长945 bp,编码315个氨基酸,推测编码蛋白的相对分子量和等电点分别为35.42 kDa和9.23,具有亲水性和免疫原性。序列同源性分析表明,不同年份CSBV北京分离株VP1蛋白氨基酸序列间差异较小,仅个别氨基酸存在差异。北京分离株与辽宁分离株及越南分离株VP1核苷酸序列一致性达93%,与印度及韩国分离株VP1核苷酸序列一致性达92%,与英国分离株VP1核苷酸序列一致性最低,为88%。序列分析同时表明,CSBV北京分离株VP1蛋白序列存在特有的序列特征,同其他地区分离株比较,北京分离株VP1蛋白序列中存在着氨基酸的插入突变。序列替换率分析表明,亚洲型分离株间序列替换率低于亚洲分离株与欧洲分离株间的替换率。构建原核表达载体pEASY-E1-VP1,经IPTG诱导,CSBV VP1蛋白在大肠杆菌Escherichia coli BL21（DE3）pLysS菌株中表达。【结论】本研究提示CSBV不同分离株基因序列存在变异,结果为进一步研究CSBV致病性分化的分子机理奠定了基础。     

Human Adeno-Associated Virus Type 5 Is Only Distantly Related to Other Known Primate Helper-Dependent Parvoviruses

We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy.     

Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T = 3 capsid protein leads to larger T = 4 capsids

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.     

Sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the S, 3, and 3-1 genes.

Four new porcine respiratory coronavirus (PRCV) isolates were genetically characterized. Subgenomic mRNA patterns and the nucleotide sequences of the 5' ends of the S genes, the open reading frame (ORF) 3/3a genes, and the ORF 3-1/3b genes of these PRCV isolates were determined and compared with those of other PRCV and transmissible gastroenteritis virus (TGEV) isolates. The S, ORF 3/3a, and ORF 3-1/3b genes are under intense study because of their possible roles in determining tissue tropism and virulence. Northern (RNA) blot analysis of subgenomic mRNAs revealed that mRNA 2, which encodes for the S gene, of the PRCV isolates migrated faster than the mRNA 2 of TGEV. The PRCV isolates AR310 and LEPP produced eight subgenomic mRNA species, the same number as produced by the virulent Miller strain of TGEV. However, the PRCV isolates IA1894 and ISU-1 produced only seven subgenomic mRNA species. All four of the PRCV isolates were found to have a large in-frame deletion in the 5' end of the S gene; however, the size and location of the deletion varied. Analysis of the ORF 3/3a gene nucleotide sequences from the four PRCV isolates also showed a high degree of variability in this area. The ORF 3 gene of the PRCV isolates AR310 and LEPP was preceded by a CTAAAC leader RNA-binding site, and the ORF 3 gene was predicted to yield a protein of 72 amino acids, the same size as that of the virulent Miller strain of TGEV. The PRCV isolates AR310 and LEPP are the first PRCV isolates found to have an intact ORF 3 gene. The ORF 3a gene of the PRCV isolate IA1894 was preceded by a CTAAAC leader RNA-binding site and was predicted to yield a truncated protein of 54 amino acids due to a 23-nucleotide deletion. The CTAAAC leader RNA-binding site and ATG start codon of ORF 3 gene of the PRCV isolate ISU-1 were removed because of a 168-nucleotide deletion. Analysis of the ORF 3-1/3b gene nucleotide sequences from the four PRCV nucleotides isolates also showed variability.     

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.     

我国蚊虫分离的一种新型胞质多形体病毒

0507BS3是从中国新疆喀什地区采集的库蚊和按蚊混合蚊标本分离的病毒株,对C6/36细胞致病变而对Ve-ro和BHK-21细胞不致病变。电镜观察显示病毒颗粒呈圆球形,直径约60nm(n=20),无包膜,单层衣壳,衣壳内有中央核。基因组核酸电泳显示基因组包括10条双链RNA(double stranded RNA,dsRNA)片段。病毒第10基因片段核酸序列测定显示该片段全长964bp(GenBankID:FJ150869),具有单一开放读码框,编码长度为275个氨基酸的蛋白,分子量约30.8kD。病毒第10基因片段核酸序列比对未发现相似的病毒核酸序列,氨基酸序列与胞质多形体病毒(Cytoplasmic polyhedrosis virus,CPV)第10基因片段编码的多形体蛋白部分区段匹配。病毒第10基因片段和已知各型CPV第10基因片段核酸序列共同进行系统进化分析显示该病毒位于独立的进化分枝,提示0507BS3病毒可能是一种新型CPV病毒。     

Molecular characterization of Indian Sugarcane streak mosaic virus isolates reveals recombination and negative selection in the P1 gene

Sugarcane streak mosaic virus (SCSMV), a member of the genus Poacevirus is an important viral pathogen affecting sugarcane production in India. The P1 gene of ten Indian isolates was sequenced and compared with previously reported SCSMV isolates. Comparative sequence analysis revealed a high level of diversity in the P1 gene (83–98% nucleotide sequence identity; 87–100% amino acid sequence identity), and the Indian SCSMV isolates were found to be the most variable (up to 9% diversity at the amino acid level). Phylogenetic tree analysis showed clustering of 17 SCSMV isolates into two groups: group I included isolates from India (except SCSMV-TPT) and Pakistan, and group II consisted of isolates from Japan, Indonesia, Thailand and SCSMV-TPT. The results obtained from phylogenetic study were further supported by the different in silico analysis viz. SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. A significant proportion of recombination sites were observed at the N terminal region of P1 gene. Analysis of selection pressure indicated that the P1 gene of the Indian SCSMV isolates is under strong negative or purifying selection. It is likely that recombination identified in Indian SCSMV isolates, along with strong purifying selection, enhances the speed of elimination of deleterious mutations in the P1 gene. The evolutionary processes (recombination and selection pressure) together contributed to the observed genetic diversity and population structure of Indian SCSMV isolates.