Distinct genotypes of a nonenveloped DNA virus associated with posttransfusion non-A to G hepatitis (TT virus) in plasma and peripheral blood mononuclear cells

TT virus (TTV) is a nonenveloped, single-stranded DNA virus with little sequence homology to known viruses, and associated with elevated transaminase levels in the patients with posttransfusion hepatitis of unknown etiology. The DNA of TTV was detected, by semi-nested polymerase chain reaction, in peripheral blood mononuclear cells (PBMC) from the 30 healthy individuals with circulating virus in plasma. A sequence of 222 bases was determined on 6-10 TTV DNA clones each from plasma and 6 clones -each from PBMC from eight individuals selected at random from this group. TTV can be classified into genotypes separated by an evolutionary distance > 0.30, which can be divided further into subtypes separated by that of 0.15. Three individuals possessed two different TTV variants of distinct genotypes, with predominant genotypes different between plasma and PBMC. Another possessed TTV of the same genotype in both the plasma and PBMC, but clones with a subtype not seen in plasma were observed in PBMC. A third individual had TTV variants with or without a deletion mutation, and those with the deletion mutation abounded only in PBMC. The remaining three individuals were infected with TTV with the same sequence both in plasma and PBMC. These results indicate that TTV variants with phylogenetic differences could infect the same individual, and that some variants would have a predilection for PBMC. It remains to be seen, however, if TTV replicates in PBMC or whether it has been sequestered before its evolution in the host.     

TT virus: a study of molecular epidemiology and transmission of genotypes 1, 2 and 3.

BACKGROUND: TT virus (TTV) is a recently discovered virus, which is not related to any other known virus infecting humans. OBJECTIVES: To investigate: (i) the world-wide distribution of the three major TTV genotypes; and (ii) the possible routes of viral transmission. STUDY DESIGN: (i) The phylogenetic distribution of 494 TTV isolates originating from 31 countries was analysed, using partial ORF1 sequences. (ii) Faeces samples (n=22) and saliva samples (n=72) from French individuals were tested for the presence of TTV DNA. (iii) Viral titres in paired serum and saliva samples were compared. RESULTS: (i) Genotypes 1, 2 and 3 were distributed world-wide, with a high proportion of type 1 in Asia (71%) and no type 3 identified in Africa to date. In the USA, 77% of isolates were grouped in four clusters only (genetic distances <10%). This was also the case of 76% of French isolates, 76% of Japanese isolates, and 89% of Hong Kong isolates. (ii) TTV DNA was detected in 18% of faeces samples and 68% of saliva samples tested. (iii) Viral titre in saliva samples was 100-1000 times higher than that of the corresponding serum. CONCLUSIONS: (i) The observed epidemiological distribution of TTV isolates is compatible with an ancient dissemination of viral ancestors belonging to the different genotypes and a slow genetic evolution in sedentary populations. (ii) Besides the possible transmission of TTV by the parental and oral-faecal routes, the high titre of TTV DNA observed in saliva raises the hypothesis of the viral transmission by saliva droplets. This route of transmission could explain the high degree of exposure to viral infection observed in the general population.     

Effect of interferon on a nonenveloped DNA virus (TT virus) associated with acute and chronic hepatitis of unknown etiology.

An unenveloped DNA virus named TT virus (TTV) has been reported in association with acute and chronic hepatitis of unknown etiology. The effect of interferon on TTV was evaluated in the patients with chronic hepatitis C who were coinfected with TTV. TTV DNA was determined by a polymerase chain reaction with heminested primers in the 96 patients with chronic hepatitis C who received interferon-alpha (516 million units in 26 weeks) and followed for 24 months thereafter. TTV DNA was detected in 31 (32%) patients before therapy. TTV DNA became undetectable during interferon therapy and remained absent in 14 (45% of the 31 patients) through 24 months thereafter. The four patients with pretreatment TTV DNA titer > or =10(3)/ml did not respond. These results indicate that TTV is sensitive to interferon, and the response would be inversely correlated with pretreatment viral titers.     

Complete genome sequence analysis of a predominant infectious bronchitis virus (IBV) strain in China

Infectious bronchitis (IB) is one of the major diseases in poultry flocks all over the world caused by infectious bronchitis virus (IBV). In the study, the complete genome sequence of strain A2 was sequenced and analyzed, which was a predominant IBV strain in China. The results indicated that there were mutations, insertions, and deletions distributed in the whole genome. The A2 virus had the highest identity to S14 and BJ in terms of full genome, whereas had a further distance to Massachusetts strains. Phylogenetic analysis showed that A2 isolate clustered together with most Chinese strains. The results of this study suggest that strain A2 may play an important role in IBV’s evolution and A2-like IBVs are predominant strains in China.     

Whole-comparative genomic hybridization (W-CGH): 1. The quick overview of repetitive DNA sequences on a genome

We present a technique (W-CGH) based on Comparative Genomic Hybridization (CGH), but using whole DNA probes, which permits the identification of chromosomal polymorphisms related to highly repetitive DNA sequences that exist between the two genomes compared. The procedure employs two differently colored whole DNA probes from two different individuals that are mixed and hybridized to metaphase chromosomes. The method provides a simple way to map whole genome differences for highly repetitive DNA sequences between two individuals, since it does not require chromosome-specific probes. This revised version was published online in July 2006 with corrections to the Cover Date.     

The entire nucleotide sequence of a TT virus isolate from the United States (TUS01): comparison with reported isolates and phylogenetic analysis.

A nonenveloped and single-stranded DNA virus designated TT virus (TTV) has been reported from Japan in association with hepatitis of unknown etiology. Very recently, the prototype TTV isolate (TA278) of genotype 1 is proven to have a circular genome with 3852 nucleotides. A TTV isolate (TUS01) was recovered from a blood donor in the United States, and its entire circular nucleotide sequence of 3818 nucleotides was determined. It possessed two open reading frames coding for 761 and 156 amino acids, respectively. TUS01 shared 60.5% of the nucleotide sequence with the TA278 isolate from Japan that was longer by 35 nt. The sequence of the noncoding region of 1203 nt was conserved with a similarity of 83.4%. Sequence preservation was much lower for the two open reading frames; nucleotide and amino acid sequences were 54.8 and 37.0% similar, respectively, for one and 55.5 and 38.8% similar for the other. By comparison of a partial sequence of 222 nucleotides among 239 TTV isolates available from various countries, at least 11 genotypes with sequence divergence of >30% were recognized. TUS01 was deduced to be of genotype 11, which has not been reported before. Conserved sequences in the noncoding region could be used as primers for sensitively detecting TTV DNA by polymerase chain reaction. Divergent sequences in coding regions would be useful as primers for distinguishing various TTV genotypes.     

Characterization of DNA complementary to nucleotide sequences adjacent to poly(A) at the 3'-terminus of the avian sarcoma virus genome.

The temperature-sensitive influenza virus A/Hong Kong/68 (H3N2) ts-1[E] has been used as a prototype live attenuated influenza virus vaccine. Using recently developed techniques to map the genome of influenza viruses and to “genotype” influenza virus recombinants, the temperature-sensitive lesions in the virus were identified. These defects, responsible for the attenuation of the virus, are located in the genes for the P3 protein and the nucleoprotein and are associated with virus-specific RNA synthesis. Hong Kong/68 (H3N2) ts-1[E] virus can also serve as a donor of “attenuation characteristics” for the selection of recombinant strains which have different surface antigens and may be used as vaccine strains in the future. The temperature-sensitive mutations of Hong Kong/68 (H3N2) ts-1[E] virus were previously transferred to recombinant viruses carrying the HO hemagglutinin. The RNAs of 8 of these temperature-sensitive recombinants were analyzed. One of these viruses, R1, classified in group 1 of the Hong Kong mutant virus set was found to possess a ts defect only in the P3 protein. R8, a member of group 2 of the Hong Kong mutant virus set had a ts mutation in the nucleoprotein.     

A study of the sequences of chicken embryo lethal orphan (CELO) virus DNA present in a transformed hamster cell line with use of specific fragments of the virus genome.

Chicken embryo lethal orphan (CELO) virus DNA (molecular weight 28.3 × 10⁶) was cleaved by the restriction enzyme EcoR1 into seven fragments whose molecular weights ranged from 1.8 × 10⁶ to 10.2 × 10⁶ as measured by gel electrophoresis and electron microscopy. ³²P-labeled CELO virus DNA was treated with EcoR1 and the resulting fragments were separated by gel electrophoresis. The kinetics of renaturation of the isolated fragments was measured in the presence of DNA extracted from a line of CELO virus-transformed hamster skin (THS) cells and from control hamster cells. THS cell DNA contained sequences homologous to only six of the seven EcoR1-produced CELO virus DNA fragments. No sequence homologous to a 2.0 × 10⁶ molecular weight fragment was detected indicating that the DNA of THS cells did not contain a complete copy of the virus genome.     

Complete genome sequence of Habenaria mosaic virus, a new potyvirus infecting a terrestrial orchid (Habenaria radiata) in Japan

The complete genomic sequence of Habenaria mosaic virus (HaMV), which infects terrestrial orchids (Habenaria radiata), has been determined. The genome is composed of 9,499 nucleotides excluding the 3′-terminal poly(A) tail, encoding a large polyprotein of 3,054 amino acids with the genomic features typical of a potyvirus. Putative proteolytic cleavage sites were identified by sequence comparison to those of known potyviruses. The HaMV polyprotein showed 58 % amino acid sequence identity to that encoded by the most closely related potyvirus, tobacco vein banding mosaic virus. Phylogenetic analysis of the polyprotein amino acid sequence and its coding sequences confirmed that HaMV formed a cluster with the chilli veinal mottle virus group, most of which infect solanaceous plants. These results suggest that HaMV is a distinct member of the genus Potyvirus.     

Genotypic distribution of TT virus (TTV) in a Czech population: evidence for sexual transmission of the virus.

BACKGROUND: TTV is a new DNA virus distinguished by its high degree of strain heterogeneity. The geographic clustering of viral genotypes suggests frequent community transmission. While no specific human disease has yet been linked to it, a transmission mechanism that facilitates strain diversity may eventually select for a strain that will become pathogenic. OBJECTIVE: This study was performed to examine the prevalence, genotypic distribution, and mode of transmission of TTV in detail. STUDY DESIGN: Three groups of study subjects were recruited between October 1998 and January 2000 in Prague, Czech Republic. Group 1 included 152 injection drug users with liver disease; group 2 included 102 persons with liver disease who denied ever using injection drugs; group 3 included 111 prospective blood donors. TTV DNA was detected from blood by a semi-nested PCR assay, and a selected set of PCR products was genotyped by direct sequencing. Factors associated with TTV prevalence in groups 1 and 2 subjects were compared. RESULTS: TTV was detected in 15.8, 13.7, and 13.5% of Groups 1, 2, and 3 subjects, respectively (P>0.05). The most common genotype was 2 (54%), followed by 1 (13%). The prevalence of TTV viremia was nearly three times higher in persons with a present or past history of hepatitis B compared to those without (P<0.05). TTV prevalence increased proportionately with the number of lifetime sex partners in both groups (P<0.05); it was highest (32%) among non-users of injection drugs who had five or more lifetime sex partners. CONCLUSION: TTV prevalence in the Czech population is similar among blood donors, persons with liver disease, as well as in a high-risk population of injection drug users. TTV appears to be sexually transmitted.     

Complete genome sequence of an Ebola virus (Sudan species) responsible for a 2000 outbreak of human disease in Uganda

The entire genomic RNA of the Gulu (Uganda 2000) strain of Ebola virus was sequenced and compared to the genomes of other filoviruses. This data represents the first comprehensive genetic analysis for a representative isolate of the Sudan species of Ebola virus. The genome organization of the Sudan species is nearly identical to that of the Zaire species, but the presence of a gene overlap (between GP and VP30 genes) and a longer trailer sequence distinguish it from that of the Reston species. As has been observed with other filoviruses, stemloop structures were predicted to form at the 5' end of Ebola Sudan mRNA molecules, and the genomic RNA termini showed a high degree of sequence complimentarity. Comparisons of the amino acid sequences of encoded gene products shows that there is a comparable level of identity or similarity between Ebola virus species, with Sudan and Zaire actually showing a slightly closer relationship to the Reston species than to one another. These comparisons also indicated that the VP24 is the most conserved Ebola virus protein (followed closely by the VP40 and L proteins), while the GP is the least conserved gene product. The most divergent regions were seen in the C-terminus of GP1 (mucin-like region) and within the C-terminal third of the nucleoprotein sequence.     

Complete nucleotide and deduced amino acid sequence of genome segment 5 encoding the outer capsid protein, VP5, of a U.S. isolate of bluetongue virus serotype 11.

The complete nucleotide sequence of the RNA genome segment coding for the outer capsid protein, VP5, of the United States prototypic strain of bluetongue virus (BTV) serotype 11 was determined from two overlapping cDNA clones. The genome segment was found to be 1638 nucleotides in length with a single open reading frame coding for a 526 amino acid protein of MW 59,278 and having a net charge of -4.0 at neutral pH. Comparisons of the predicted amino acid sequence of VP5 of BTV 11 with those of the United States serotypes 2, 10, and 13 and two isolates of BTV 1 from Australia and South Africa confirmed earlier reports that VP5 is a conserved protein with no clear regions of variability. A computer generated consensus sequence suggested VP5 of BTV 2 to be representative of the average VP5 sequences reported thus far.     

Sequence analysis of open reading frames (ORFs) 2 to 4 of a U.S. isolate of porcine reproductive and respiratory syndrome virus

Summary The sequence of ORFs 2 to 4 of a U.S. isolate of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was determined by analysis of a cDNA library. The cDNA clones containing PRRSV specific sequences were selected using a VR2385 ORF 4 specific PCR probe and sequenced. The ORFs 2, 3 and 4 overlapped each other and encoded polypeptides with predicted M _r of 29.5 kDa (ORF 2), 28.7 kDa (ORF 3) and 19.5 kDa (ORF 4), respectively. No overlap was found between ORFs 4 and 5, and instead there was a 10 bp sequence which separated these two ORFs. The nucleic acid homology with corresponding ORFs of the European PRRSV isolate Lelystad virus (LV) was 65% for ORF 2, 64% for ORF 3 and 66% for ORF 4. Comparison of the ORF 4 sequences of VR2385 with that of another U.S. isolate MN-1b revealed only 86% amino acid sequence homology and the presence of deletions in the ORF 4 of MN-1b. Our results further strengthen the observation that there is sequence variation between US and European PRRSV isolates.     

High prevalence of TT virus (TTV) infection in patients on maintenance hemodialysis: frequent mixed infections with different genotypes and lack of evidence of associated liver disease.

Recently, a novel DNA virus, TT virus (TTV), was identified in patients with post-transfusion non-A-G hepatitis. We analyzed the prevalence and clinical implications of TTV infection in a cohort of 96 Spanish patients on long-term hemodialysis. TTV DNA was detected by nested PCR in 51 (53%) of 96 patients, a prevalence significantly higher than that found in healthy blood donors. Persistent liver test abnormalities were found in only 2 (7.7%) of 26 patients infected with TTV alone, compared with 12 (75%) of 16 patients infected with hepatitis C or hepatitis B virus, or both (P < 0.01). Mixed infections with multiple strains of TTV, including different major genotypes, were common in patients on hemodialysis. These patients had received a significantly greater number of blood units (22.7 +/- 20) compared with patients apparently infected with a single strain of TTV (8.9 +/- 11) (P = 0.01). Phylogenetic analyses of TTV from infected patients identified strains of genotypes 1, 2, 3, and 4. In summary, TTV infection was common in patients on hemodialysis but was not associated with liver disease     

Enhancement of herpes simplex virus type 2 (HSV-2) DNA synthesis in infected cells that constitutively express the BglII-N region of the HSV-2 genome

The BglII-N fragment of the herpes simplex virus type-2 (HSV-2) genome encodes one of two known transforming regions of this DNA virus. In this study, we report the derivation of HeLa S3 cells (2DC₄) that stably express the HSV-2 BglII-N region, including the small subunit of HSV-2 ribonucleotide reductase (RR). Superinfection of the 2DC₄ cells with wild-type HSV-2 resulted in the efficient induction of HSV-2-encoded ICP10, DNA polymerase, and thymidine kinase. The amount of HSV-2 DNA synthesis in 8-hr HSV-2-infected 2DC₄ cells was enhanced 2.6±0.6-fold relative to infected control cells. Furthermore, the replication kinetics of HSV-2 DNA in 2DC₄ cells were accelerated relative to HeLa S3 cells; HSV-2 DNA synthesis was detectable as early as 3 hr postinfection in 2DC₄ cells as compared to 6 hr postinfection in HeLa S3 cells. These results suggest that the BglII-N region of HSV-2 encodes function(s) that activate the viral DNA synthesis apparatus and that this activation could relate to the transforming ability of this DNA region.     

Replicating Marek's disease virus (MDV) serotype 2 DNA with inserted MDV serotype 1 DNA sequences in a Marek's disease lymphoblastoid cell line MSB1-41C

Summary We characterized the properties of herpes-type viruses which grew well in a Marek's disease lymphoblastoid cell line, MSB1-41C, inducing cytopathic effect characterized by the formation of syncytial giant cells. Examination of the infectious virus by field inversion gel electrophoresis revealed the presence of DNA of about 180 kbp in both the culture fluid and cell fractions of the infected MSB-41C cells. The DNA was found to consist of Marek's disease virus (MDV) serotype 2 (MDV2) and MDV serotype 1 (MDV1) DNA by Southern blot hybridization. The MDV1 DNA consisted of sequences mainly from the long inverted repeats including multiple copies of 132 bp direct tandem repeats. Molecular cloning of BamHI digests of the MDV2 DNA revealed a fragment of MDV1 DNA and MDV2 DNA fused together, indicating that the recombinant MDV2 DNA had been generated by genetic recombination with the latent MDV1 DNA.     

First genome sequence of St. Louis encephalitis virus (SLEV) isolated from a human in Brazil

St. Louis encephalitis virus (SLEV), a member of the family Flaviviridae, genus Flavivirus, is a causative agent of encephalitis in the Americas. In Brazil, sporadic cases of SLEV infection have been reported since 1953, but the first outbreak of SLEV in Brazil was identified only in 2007, concomitant with an outbreak of dengue virus (DENV) serotype 3. This finding, along with other reports, indicates that SLEV circulation in Brazil is largely unknown, and there may be epidemiological implications of the co-circulation of SLEV, DENV and other flaviviruses in Brazil. Here, we describe the first complete genome sequence of an SLEV strain isolated from a human patient in Brazil, strain BeH 355964. Phylogenetic analysis was performed to determine the genotype of BeH 355964 using the full-length genome and envelope (E) gene sequences separately. Both analyses showed that BeH 355964 could be classified as genotype V. Although the number of single gene sequences available is greater (such as for the E gene), the phylogenetic tree based on the complete genome sequence was better supported and provided further information about the virus.