Epstein-Barr virus stimulates torque teno virus replication: a possible relationship to multiple sclerosis

Viral infections have been implicated in the pathogenesis of multiple sclerosis. Epstein-Barr virus (EBV) has frequently been investigated as a possible candidate and torque teno virus (TTV) has also been discussed in this context. Nevertheless, mechanistic aspects remain unresolved. We report viral replication, as measured by genome amplification, as well as quantitative PCR of two TTV-HD14 isolates isolated from multiple sclerosis brain in a series of EBV-positive and -negative lymphoblastoid and Burkitt's lymphoma cell lines. Our results demonstrate the replication of both transfected TTV genomes up to day 21 post transfection in all the evaluated cell lines. Quantitative amplification indicates statistically significant enhanced TTV replication in the EBV-positive cell lines, including the EBV-converted BJAB line, in comparison to the EBV-negative Burkitt's lymphoma cell line BJAB. This suggests a helper effect of EBV infections in the replication of TTV. The present study provides information on a possible interaction of EBV and TTV in the etiology and progression of multiple sclerosis.     

In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons

Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.     

Genomic characterization of a Brazilian TT virus isolate closely related to SEN virus-F

SEN virus (SENV) is a circular, single stranded DNA virus that has been first characterized in the serum of a human immunodeficiency virus type 1 (HIV-1)-infected patient. Eight genotypes of SENV (A-H) have been identified and further recognized as variants of TT virus (TTV) in the family Circoviridae. Here we describe the first genomic characterization of a SENV isolate (5-A) from South America. Using 'universal' primers, able to amplify most, if not all, TTV/SENV genotypes, a segment of > 3 kb was amplified by polymerase chain reaction from the serum of an HIV-1 infected patient. The amplicon was cloned and a 3087-nucleotide sequence was determined, that showed a high (85%) homology with the sequence of the Italian isolate SENV-F. Proteins encoded by open reading frames (ORFs) 1 to 4 consisted of 758, 129, 276, and 267 amino acids, respectively. By phylogenetic analysis, isolate 5-A was classified into TTV genotype 19 (phylogenetic group 3), together with SENV-F and TTV isolate SAa-38.     

TT型病毒基因组的克隆与进化分析

利用PCR方法从输血传播性病毒 (transfusiontransmittedvirus,TTV)阳性标本中获得不同长度且重叠覆盖TTV基因组的DNA片段。将PCR扩增片段克隆到pT Adv载体中 ,筛选获得阳性克隆。DNA序列测定结果表明所克隆的片段为TTV基因组序列。利用DNA片段中特有的限制性内切酶位点将TTV的DNA片段首尾相连 ,得到近全长的基因组克隆 ,命名为TTV0 2 1。对TTV0 2 1的核酸序列进行分析 ,TTV0 2 1长 3472nt,存在 2个阅读框架ORF1和ORF2 ,分别编码 785和 1 46个氨基酸。将TTV0 2 1与其它已知的TTV基因组全序列进行了同源性比较 ,并进行进化分析。结果表明 ,TTV0 2 1序列与TTV分离株CHN2、BDH1的遗传距离较近 ,而与其它分离株相对较远。     

Isolate KAV: a new genotype of the TT-virus family.

A novel DNA sequence belonging to a new genotype of TT virus (TTV) was detected by long-distance PCR in the serum of a chronically HCV-infected patient. The isolate was designated KAV according to the patient's initials. Extending the sequence to full length revealed a 3705-nt viral genome, which is about 100 nucleotides shorter than the other TT-viruses. KAV showed common features with the TTV family, such as the organization of open reading frames and conserved noncoding regions. The largest open reading frame of KAV (ORF 1) was about 40 aa shorter than that of other TT-viruses. Overall sequence homology with known TTV isolates was less than 66%. Phylogenetic analysis poses KAV in one major group with three recently published TTV sequences. So KAV can be considered as a new genotype of the TTV family (provisionally designated genotype 28).     

Multiple infection of TT virus (TTV) with different genotypes in Japanese hemophiliacs

To clarify the persistent TT virus (TTV) infection, we studied a possibility of multiple TTV infection by genotype analysis of isolated TTV obtained from seven Japanese hemophiliacs. The nucleotide sequences including 222 bp in the open reading frame 1 (ORF1) region of 10 TTV isolates from each patient were analyzed and classified into various TTV genotypes such as G1 to G6 by phylogenetic analysis using a N-J method. Multiple TTV genotypes were observed in all the hemophiliacs: three different TTV genotypes were found in three patients, whereas four different TTV genotypes were observed in the other three patients. The remaining patient was also infected with TTV of five different genotypes. Moreover, new TTV genotypes were found in these seven patients and tentatively designated as G7. The present findings indicate that multiple TTV infection with different TTV genotypes has occurred in Japanese hemophiliacs. They also provide valuable information to understand persistent TTV infection.     

Phosphorylation of serine-rich protein encoded by open reading frame 3 of the TT virus genome

TT virus (TTV) is a newly discovered human virus with a single-stranded, circular DNA genome. The TTV DNA sequence includes two major open reading frames (ORFs), ORF1 and ORF2. Recently, spliced TTV mRNAs were detected and revealed two additional coding regions, ORF3 and ORF4. We found sequence similarity between the TTV ORF3 protein and hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, which is a phosphoprotein and is thought to associate with various cellular proteins. To test whether the TTV ORF3 protein is phosphorylated, the state of phosphorylation was analyzed with a transient protein production system. The TTV ORF3 protein was phosphorylated at the serine residues in its C-terminal portion. Furthermore, the TTV ORF3 gene generated two forms of proteins with a different phosphorylation state, similar to the HCV NS5A region, suggesting that TTV ORF3 protein has function(s) similar to phosphorylated viral proteins such as the HCV NS5A protein.     

Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans.

BACKGROUND: Endogenous retroviruses contribute to the evolution of the host genome and can be associated with disease. Human endogenous retrovirus K (HERV-K) is related to the mouse mammary tumor virus and is present in the genomes of humans, apes and cercopithecoids (Old World monkeys). It is unknown how long ago in primate evolution the full-length HERV-K proviruses that are in the human genome today were formed. RESULTS: Ten full-length HERV-K proviruses were cloned from the human genome. Using provirus-specific probes, eight of the ten were found to be present in a genetically diverse set of humans but not in other extant hominoids. Intact preintegration sites for each of these eight proviruses were present in the apes. A ninth provirus was detected in the human, chimpanzee, bonobo and gorilla genomes, but not in the orang-utan genome. The tenth was found only in humans, chimpanzees and bonobos. Complete sequencing of six of the human-specific proviruses showed that full-length open reading frames for the retroviral protein precursors Gag-Pro-Pol or Env were each present in multiple proviruses. CONCLUSIONS: At least eight full-length HERV-K genomes that are in the human germline today integrated after humans diverged from chimpanzees. All of the viral open reading frames and cis-acting sequences necessary for HERV-K replication must have been intact during the recent time when these proviruses formed. Multiple full-length open reading frames for all HERV-K proteins are present in the human genome today.     

TT病毒ORF2在Cos7细胞中的表达及蛋白质定位

应用PCR方法从含有1TrVirusORF2的质粒pET-His-TTV2中扩增出606bp的蛋白质编码区,并将其克隆到真核表达载体pEGFP．NI中以表达成GFP—VP2融合蛋白。构建出的重组质粒pEGFPTFV2经过酶切分析和PCR鉴定。用脂质体介导法将pEGFPTTV2质粒DNA转染Cos7细胞,通过RT-PCR分析,证实细胞中存在ORF2基因的转录产物。用共聚焦显微镜结合PI染色技术研究1TTV P2蛋白在细胞中的分布情况。结果表明,1TrVVP2分布在细胞质中和细胞核膜内侧。因此推测VP2作为一种非结构蛋白,功能可能是参与病毒DNA的复制或转录。     

New Phylogenetic Groups of Torque Teno Virus Identified in Eastern Taiwan Indigenes

Torque teno virus (TTV) is a single-stranded DNA virus highly prevalent in the world. It has been detected in eastern Taiwan indigenes with a low prevalence of 11% by using N22 region of which known to underestimate TTV prevalence excessively. In order to clarify their realistic epidemiology, we re-analyzed TTV prevalence with UTR region. One hundred and forty serum samples from eastern Taiwanese indigenous population were collected and TTV DNA was detected in 133 (95%) samples. Direct sequencing revealed an extensive mix-infection of different TTV strains within the infected individual. Entire TTV open reading frame 1 was amplified and cloned from a TTV positive individual to distinguish mix-infected strains. Phylogenetic analysis showed eleven isolates were clustered into a monophyletic group that is distinct from all known groups. In addition, another our isolate was clustered with recently described Hebei-1 strain and formed an independent clade. Based on the distribution pattern of pairwise distances, both new clusters were placed at phylogenetic group level, designed as the 6th and 7th phylogenetic group. In present study, we showed a very high prevalence of TTV infection in eastern Taiwan indigenes and indentified new phylogenetic groups from the infected individual. Both intra- and inter-phylogenetic group mix-infections can be found from one healthy person. Our study has further broadened the field of human TTVs and proposed a robust criterion for classification of the major TTV phylogenetic groups.     

The product of the bovine papillomavirus type 1 modulator gene (M) is a phosphoprotein.

The M gene of bovine papillomavirus type 1 has been genetically defined as encoding a trans-acting product which negatively regulates bovine papillomavirus type 1 replication and is important for establishment of stable plasmids in transformed cells. The gene for this regulatory protein has been mapped in part to the 5' portion of the largest open reading frame (E1) in the virus. We constructed a trpE-E1 fusion gene and expressed this gene in Escherichia coli. Rabbits were immunized with purified fusion protein, and antisera directed against the product were used to identify the M gene product in virus-transformed cells. In this way a polypeptide with an apparent molecular mass of 23 kilodaltons was detected. The virus-encoded product is phosphorylated and can be readily detected by immunoprecipitation assays from cells transformed by the virus. Cells that harbor viral DNA without M as integrated copies do not produce this protein, whereas cells that harbor integrated viral genomes which are defective for another E1 viral gene important for plasmid replication, R, do produce this protein. The protein has an anomalously low electrophoretic mobility. An in vitro translation product of an SP6 RNA product of a sequenced cDNA predicts a molecular mass of 16 kilodaltons for the protein, and this in vitro translation product has an electrophoretic mobility identical to that of the in vivo immunoprecipitated protein. The results of these studies confirm our previous genetic studies which indicated that part of the E1 open reading frame defined a discrete gene product distinct from other putative products which may be encoded by this open reading frame.     

Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus

Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.     

Complete nucleotide sequence of tobacco streak virus RNA 3

Double-stranded cDNA of in vitro polyadenylated tobacco streak virus (TSV) RNA 3 has been cloned and sequenced. The complete primary structure of 2,205 nucleotides reveals two open reading frames flanked by a leader sequence of 210 bases, an intercistronic region of 123 nucleotides and a 3'-extracistronic sequence of 288 nucleotides. The 5'-terminal open reading frame codes for a Mr 31,742 protein, which probably corresponds to the only in vitro translation product of TSV RNA 3. The 3'-terminal coding region predicts a Mr 26,346 protein, probably the viral coat protein, which is the translation product of the subgenomic messenger, RNA 4. Although the coat proteins of alfalfa mosaic virus (A1MV) and TSV are functionally equivalent in activating their own and each others genomes, no homology between the primary structures of those two proteins is detectable.     

Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus.

To determine the minimal requirements for autonomous RNA replication of classical swine fever virus (CSFV), genomes having in-frame deletions within the genes for structural and flanking nonstructural proteins were constructed, based on an infectious cDNA clone of CSFV Alfort/187. RNA was transcribed in vitro from the respective plasmids and transfected into SK-6 swine kidney cells. The replication competence of the RNA was determined by immunostaining transfected cells for CSFV NS3 protein and by analysis of cell extracts for viral RNA, as well as protein synthesis at different times after transfection. The genes encoding N(pro), C, E(rns), E1, E2, p7, and NS2 proved to be dispensable for RNA replication, but the efficiency of replication varied strongly between individual constructs. RNA replicons containing the complete NS2-NS3 gene persisted in transfected cells and continued to replicate without causing any obvious morphological or functional damage to the cells, whereas genomes lacking the NS2 gene replicated more efficiently and induced a cytopathic effect. These findings suggest that NS2, although it is not essential for pestivirus RNA replication, has a regulatory function therein. Both cytopathogenic and noncytopathogenic replicons were packaged into virus particles provided in trans by a cotransfected full-length helper virus genome.     

TT virus infection in nonhuman primates and characterization of the viral genome: identification of simian TT virus isolates

Newly discovered TT virus (TTV) is widely distributed in human populations. To understand more about the relationship between TTV and its hosts, we tested 400 sera from various nonhuman primates for the presence of TTV DNA by PCR assay. We collected serum samples from 24 different species of nonhuman primates. TTV DNA was determined by PCR with primers designed from the 5'-end region of the TTV genome. Nucleotide sequencing and phylogenetic analysis of viral genomes were also performed. TTV DNA was detected in 87 of 98 (89%) chimpanzees and 3 of 21 (14%) crab-eating macaques. Nucleotide sequences of the PCR products obtained from both animals were 80 to 100% identical between two species. In contrast, the sequences differed from TTV isolates in humans by 24 to 33% at the nucleotide level and 36 to 50% at the amino acid level. Phylogenetic analysis demonstrated that all TTV isolates obtained from simians were distinct from the human TTV isolates. Furthermore, TTV in simians, but not in humans, was classified into three different genotypes. Our results indicate that TTV in simians represents a group different from, but closely related to, TTV in humans. From these results, we tentatively named this TTV simian TTV (s-TTV). The existence of the s-TTV will be important in determining the origin, nature, and transmission of human TTV and may provide useful animal models for studies of the infection and pathogenesis of this new DNA virus.