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).     

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.     

Sequence heterogeneity of TT virus and closely related viruses

TT virus (TTV) is a recently discovered infectious agent originally obtained from transfusion-related hepatitis. However, the causative link between the TTV infection and liver disease remains uncertain. Recent studies demonstrated that genome sequences of different TTV strains are significantly divergent. To assess genetic heterogeneity of the TTV genome in more detail, a sequence analysis of PCR fragments (271 bp) amplified from open reading frame 1 (ORF1) was performed. PCR fragments were amplified from 5 to 40% of serum specimens obtained from patients with different forms of hepatitis who reside in different countries (e.g., China, Egypt, Vietnam, and the United States) and from normal human specimens obtained from U.S. residents. A total of 170 PCR fragments were sequenced and compared to sequences derived from the corresponding TTV genome region deposited in GenBank. Genotypes 2 and 3 were found to be significantly more genetically related than any other TTV genotype. Moreover, three sequences were shown to be almost equally related to both genotypes 2 and 3. These observations suggest a merger of genotypes 2 and 3 into one genotype, 2/3. Additionally, five new groups of TTV sequences were identified. One group represents a new genotype, whereas the other four groups were shown to be more evolutionary distant from all known TTV sequences. The evolutionary distances between these four groups were also shown to be greater than between TTV genotypes. The phylogenetic analysis suggested that these four new genetic groups represent closely related yet different viral species. Thus, TTV exists as a "swarm" of at least five closely related but different viruses. These observations suggest a high degree of genetic complexity within the TTV population. The finding of the additional TTV-related species should be taken into consideration when the association between TTV infections and human diseases of unknown etiology is studied.     

Identification of a novel GC-rich 113-nucleotide region to complete the circular, single-stranded DNA genome of TT virus, the first human circovirus

The sequence data (H. Okamoto et al., Hepatol. Res. 10:1-16, 1998) of a newly discovered single-stranded DNA virus, TT virus (TTV), showed that it did not have the terminal structure typical of a parvovirus. Elucidation of the complete genome structure was necessary to understand the nature of TTV. We obtained a 1.0-kb amplified product from serum samples of four TTV carriers by an inverted, nested long PCR targeted for nucleotides (nt) 3025 to 3739 and 1 to 216 of TTV. The sequence of a clone obtained from serum sample TA278 was compared with those registered in GenBank. The complete circular TTV genome contained a novel sequence of 113 nt (nt 3740 to 3852 [=0]) in between the known 3'- and 5'-end arms, forming a 117-nt GC-rich stretch (GC content, 90.6% at nt 3736 to 3852). We found a 36-nt stretch (nt 3816 to 3851) with an 80.6% similarity to chicken anemia virus (CAV) (nt 2237 to 2272 of M55918), a vertebrate circovirus. A putative SP-1 site was located at nt 3834 to 3839, followed by a TATA box at nt 85 to 90, the first initiation codon of a putative VP2 at nt 107 to 109, the termination codon of a putative VP1 at nt 2899 to 2901, and a poly(A) signal at nt 3073 to 3078. The arrangement was similar to that of CAV. Furthermore, several AP-2 and ATF/CREB binding sites and an NF-kappaB site were arranged around the GC-rich region in both TTV and CAV. The data suggested that TTV is circular and similar to CAV in its genomic organization, implying that TTV is the first human circovirus.     

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。     

Cell culture replication of a genotype 1b hepatitis C virus isolate cloned from a patient who underwent liver transplantation

The introduction of the genotype 2a isolate JFH1 was a major breakthrough in the field of hepatitis C virus (HCV), allowing researchers to study the complete life cycle of the virus in cell culture. However, fully competent culture systems encompassing the most therapeutically relevant HCV genotypes are still lacking, especially for the highly drug-resistant genotype 1b. For most isolated HCV clones, efficient replication in cultured hepatoma cells requires the introduction of replication-enhancing mutations. However, such mutations may interfere with viral assembly, as occurs in the case of the genotype 1b isolate Con1. In this study, we show that a clinical serum carrying a genotype 1b virus with an exceptionally high viral load was able to infect Huh7.5 cells. Similar to previous reports, inoculation of Huh7.5 cells by natural virus is very inefficient compared to infection by cell culture HCV. A consensus sequence of a new genotype 1b HCV isolate was cloned from the clinical serum (designated Barcelona HCV1), and then subjected to replication studies. This virus replicated poorly in a transient fashion in Huh7.5 cells after electroporation with in vitro transcribed RNA. Nonetheless, approximately 3 weeks post electroporation and thereafter, core protein-positive cells were detected by immunofluorescence. Surprisingly, small amounts of core protein were also measurable in the supernatant of electroporated cells, suggesting that HCV particles might be assembled and released. Our findings not only enhance the current method of cloning in vitro HCV replication-competent isolates, but also offer valuable insights for the realization of fully competent culture systems for HCV.     

Complete genome of a dengue virus serotype 4 strain from Amazonas, Brazil

Dengue virus (DENV) infections represent a significant concern for public health worldwide, being considered as the most prevalent arthropod-borne virus regarding the number of reported cases. In this study, we report the complete genome sequencing of a DENV serotype 4 isolate, genotype II, obtained in the city of Manaus, directly from the serum sample, applying Ion Torrent sequencing technology. The use of a massive sequencing technology allowed the detection of two variable sites, one in the coding region for the viral envelope protein and the other in the nonstructural 1 coding region within viral populations.     

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.     

Torque teno virus 10 isolated by genome amplification techniques from a patient with concomitant chronic lymphocytic leukemia and polycythemia vera

An infectious etiology has been proposed for many human cancers, but rarely have specific agents been identified. One difficulty has been the need to propagate cancer cells in vitro to produce the infectious agent in detectable quantity. We hypothesized that genome amplification from small numbers of cells could be adapted to circumvent this difficulty. A patient with concomitant chronic lymphocytic leukemia (CLL) and polycythemia vera (PV) requiring therapeutic phlebotomy donated a large amount of phlebotomized blood to test this possibility. Using genome amplification methods, we identified a new isolate (BIS8-17) of torque teno virus (TTV) 10. The presence of blood isolate sequence 8-17 (BIS8-17) in the original plasma was confirmed by polymerase chain reaction (PCR), validating the approach, since TTV is a known plasma virus. Subsequent PCR testing of plasmas from additional patients showed that BIS8-17 had a similar incidence (~20%) in CLL (n = 48) or PV (n = 10) compared with healthy controls (n = 52). CLL cells do not harbor BIS8-17; PCR did not detect it in CLL peripheral blood genomic deoxyribonucleic acid (DNA) (n = 20). CLL patient clinical outcome or prognostic markers (immunoglobulin heavy chain variable region [IGHV ] mutation, CD38 or zeta-chain associated protein kinase 70 kDa [ZAP-70]) did not correlate with BIS8-17 infection. Although not causative to our knowledge, this is the first reported isolation and detection of TTV in either CLL or PV. TTV could serve as a covirus with another infectious agent or TTV variant with rearranged genetic components that contribute to disease pathogenesis. These results prove that this method identifies infectious agents and provides an experimental methodology to test correlation with disease.     

Isolation and complete nucleotide sequence of the measles virus IMB-1 strain in China

The complete nucleotide sequence of the measles virus strain IMB-1, which was isolated in China, was determined. As in other measles viruses, its genome is 15,894 nucleotides in length and encodes six proteins. The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4%–5% at the nucleotide sequence level. This isolate has amino acid variations over the full genome, including in the hemagglutinin and fusion genes. This report is the first to describe the full-length genome of a genotype H1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.     

Full-length genomic sequence of hepatitis B virus genotype C2 isolated from a native Brazilian patient

The hepatitis B virus (HBV) is among the leading causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma. In Brazil, genotype A is the most frequent, followed by genotypes D and F. Genotypes B and C are found in Brazil exclusively among Asian patients and their descendants. The aim of this study was to sequence the entire HBV genome of a Caucasian patient infected with HBV/C2 and to infer the origin of the virus based on sequencing analysis. The sequence of this Brazilian isolate was grouped with four other sequences described in China. The sequence of this patient is the first complete genome of HBV/C2 reported in Brazil.     

Transmission of human TT virus of genotype 1a to chimpanzees with fecal supernatant or serum from patients with acute TTV infection

Fecal supernatant or serum containing TT virus (TTV) of genotype 1a (10(5) copies/ml) from patients with acute TTV infection was inoculated intravenously into two naive chimpanzees. Serum samples were obtained weekly and tested for TTV DNA by genotype 1-specific polymerase chain reaction. TTV DNA was detected in chimpanzee 228 at weeks 5-15 after inoculation with 0.5 ml of serum, and in chimpanzee 234 at weeks 7-19 after inoculation with 1 ml of fecal supernatant. The TTV DNA titer peaked at weeks 12 and 13 in chimpanzee 228 and at weeks 14-16 in chimpanzee 234. Mild biochemical and histological changes in biopsied liver samples were observed in both chimpanzees in association with the reduction in TTV titer. TTV DNA was transient in chimpanzee 228, but in chimpanzee 234 it reappeared at week 21 and persisted through week 30. These results indicate that TTV in feces is infectious and suggest that TTV has hepatitis-inducing capacity.     

RNA/cDNA Hybridization and Infectivity Tests Suggest that Barley Yellow Mosaic Virus Isolate M has a Bipartite Genome

Northern blot analysis with random-primed cDNAs revealed that RNAs 1 and 2 of barley yellow mosaic virus (Ba YMV) isolate M have no extensive base sequence homologies. Both RNAs are needed for infection. RNA 2 is therefore neither a subgenomic nor a, satellite RNA, but rather an essential part of the viral genome. Ba YMV isolate M has thus a bipartite genome.     

Complete nucleotide sequence of a mumps virus SP strain isolated in China

The complete nucleotide sequence of the mumps virus SP, which was isolated in China, was determined. As with other mumps viruses, its genome was 15 384 nucleotides (nts) in length and encoded seven proteins. The full-length nucleotide sequence of the SP isolate differed from other strains by 4%-6.8% at the nucleotide sequence level. Due to variations of amino acids over the full genome (including the HN and N genes), this isolate exhibited significant variations in the antigenic sites. This report is the first to describe the full-length genome of a genotype F strain and provide an overview of the diversity of genetic characteristics of a circulating mumps virus.     

15株中国TT病毒基因型鉴定和部分序列分析

ＴＴ病毒（ＴＴＶ）为一种新鉴定的输血后肝炎相关的病毒。用聚合酶链式反应（ＰＣＲ）扩增ＴＴＶ ＤＮＡ序列核苷酸１９１５到２１８５之间的片段,并将该片段克隆入ｐＧＥＭ－Ｔ Ｅａｓｙ载体。重组克隆经酶切鉴定后,用全自动测序仪测序,发现本组病例中１５侏ＴＴＶ毒株间的同源性在６６．８％到９９．３％之间,与ＴＴＶ日本株比较同源性在６６．１％到９７．４％之间,分析结果显示在我国不仅有国外已报告的不同基因亚型毒株     

新型冠状病毒上海株(nCoV-SH01)的分离和鉴定

新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)疫情对人类生命健康构成极大威胁。病毒的分离是构建新型冠状病毒(2019 novel coronavirus, 2019-nCoV)细胞感染模型和动物感染模型的基础。本研究利用冠状病毒易感的Vero E6细胞,从1例上海感染者的咽拭子中分离到一株2019-nCoV,命名为nCoV-SH01。对该毒株全基因组采用一代Sanger和二代Illumina法测序,发现该毒株与GenBank MN908947的同源性＞99.99%。免疫荧光检测显示,该毒株与COVID-19康复者的血清呈阳性反应。当nCoV-SH01感染Vero E6细胞后,导致典型的合胞体病变,细胞病变效应明显且进展迅速,提示nCoV-SH01可用于进一步建立2019-nCoV的细胞感染和动物感染模型,为开展致病性研究以及抗病毒药物和疫苗研发奠定了基础。     

New DNA viruses identified in patients with acute viral infection syndrome

A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing.     

家养野猪源猪圆环病毒2型安徽株的分离鉴定及其全基因组序列分析

目的对安徽省临诊疑似PMW S家养野猪病例进行猪圆环病毒2型(PCV2)分离鉴定,并对分离株的全基因组进行克隆与序列分析。方法应用PK-15细胞进行PCV2的分离与增殖,根据PCR、IFA、电镜技术进行PCV2分离株的鉴定,克隆分离株的全基因组,并对序列进行分析。结果获得1株来自安徽家养野猪源PCV2分离株,命名为YZ0901。该毒株全基因组长为1 767 bp,属于PCV2b基因型。与GenBank已发表的国内外参考毒株的同源性介于93.9%~99.2%,与安徽分离毒株彼此之间的同源性介于93.4%~99.5%。结论安徽省家养野猪中存在PCV2感染,分离毒株与家猪源病毒差异不大,PCV2核苷酸序列比较稳定,其进化不存在明显的地域相关性,家养野猪源PCV2的基因型与当地PCV2流行株的基因型密切相关。     

TT virus infection in children and adults who visited a general hospital in the south of Brazil for routine procedure

TT virus (TTV) is a newly described nonenveloped human virus, with a circular, negative-stranded DNA genome, that was first identified in the blood of a patient with posttransfusion hepatitis of unknown etiology. PCR primers and conditions used for TTV DNA amplification may greatly influence the level of TTV detection in serum. Three PCR assays, with different regions of the genome as targets, were used to test TTV DNA in 130 sera from children and adults visiting a hospital in the south of Brazil, most of them for routine procedure. Forty-four percent of adult sera and 73% of sera from children aged 0-10 years were TTV positive with at least one PCR assay. However, the three assays were able to detect only 33%, 35%, and 70% of the total positive samples. Our results showed a high prevalence of TTV infection in the south of Brazil, particularly among young children, and confirmed the necessity of performing several PCR assays to assess the true TTV prevalence in a determined population.