Analytical approximations to inhomogeneously broadened, radiation damped free precession and echo signals.

The Dirac-Frenkel-Maclachlan (DFM) variation of parameters approach to approximately solving the time dependent Schrödinger equation is used to generate free precession and echo signals from the Bloch equations corrected for the effects of radiation damping and inhomogeneous broadening. Following a brief description of how the DFM method can be applied to the non-linear Bloch equations, two figures of merit designed to evaluate how a DFM optimized approximation compares with the exact solution is provided. This framework is used to optimize and evaluate the performance of six trial functions describing inhomogeneously broadened, radiation damped free precession and echo signals. The trial functions are then used to analyze the resolution enhancement and signal attenuation produced by pulse sequences that suppress radiation damping.

A single coil radio frequency gradient probe for nuclear magnetic resonance applications.

A single coil nuclear magnetic resonance (NMR) probehead and associated electronics capable of asynchronously applying both homogeneous and inhomogeneous radio frequency (rf) pulses to solid, liquid, or gaseous samples is described. This equipment can be interfaced with a conventional single channel NMR spectrometer. Carefully placed PIN diodes on the NMR probehead are used to switch the coil between a homogeneous end tapped configuration and an inhomogeneous center tapped rf gradient configuration. This approach dramatically improves channel isolation in comparison to existing two coil designs. Descriptions of the new probehead, the transistor-transistor logic (TTL) controlled dc pulser for PIN diode gating, and the high power rf switch are provided. Several NMR pulse sequences are used to test the channel isolation and probe performance. Finally an application to liquid phase solvent suppression is provided.

Rapid detection of Hepatitis E virus RNA by reverse transcription-polymerase chain reaction using universal oligonucleotide primers.

A rapid reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of Hepatitis E virus (HEV) RNA in serum is described. Total nucleic acids are extracted from a small volume of human serum and reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in nested PCR employing degenerate HEV consensus primers. These primers are designed to sequences conserved between the Burma, Mexico, and US HEV strains, generating amplicons within each of the three open reading frames. Reactions are analyzed by agarose gel electrophoresis and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated as positive. This protocol allows for the rapid and sensitive detection of HEV infection in human serum.

Analyses of TT virus full-length genomic sequences.

Since the identification of TT virus, only one full-length and two near full-length sequences representing a single subtype of the virus have been reported. In order to understand further the nature of the TT virus genome, nine of the most divergent TT virus sequences have been extended to full-length or near full-length. Phylogenetic analysis demonstrated that these sequences represent three distinct TT virus genotypes and two subtypes. A high degree of nucleotide sequence variability (approximately 30%) was observed across the genomes with several significantly more divergent regions. Three conserved ORFs were identified, none of which shared significant amino acid sequence identity to sequences present in public databases. Additionally, sequence motifs, such as those necessary for protein translation and for rolling circle replication, were found to be partially conserved between all TT virus isolates.

Prevalence of TT virus infection in US blood donors and populations at risk for acquiring parenterally transmitted viruses.

Two overlapping sets of TT virus (TTV)-specific polymerase chain reaction primers were used to test for presence of TTV, which was found in approximately 10% of US volunteer blood donors, 13% of commercial blood donors, and 17% of intravenous drug abusers. The rate of TTV infection among US non-A, non-B, non-C, non-D, non-E hepatitis patients was only 2%. Among commercial blood donors and intravenous drug abusers, only 1%-3% of the TTV-positive individuals were coinfected with GB virus C (GBV-C), a parenterally transmitted virus. This suggests that GBV-C and TTV may have different routes of transmission. Comparison of the sensitivities of 2 TTV polymerase chain reaction (PCR) primer sets showed that the majority of samples were detected with only 1 of the 2 sets. Therefore, previous studies in which only a single PCR primer pair was used may have significantly underestimated the true prevalence of TTV.

Molecular and biophysical characterization of TT virus: evidence for a new virus family infecting humans.

The recent isolation of a novel DNA virus from the serum of a Japanese patient (T.T.) has provided the latest possible candidate virus associated with cryptogenic hepatitis. In the present study, we report the complete nucleotide sequence of this virus (TTV) isolated from the serum of a West African. Based on PCR studies designed to amplify overlapping regions of the viral genome and sensitivity to digestion with mung bean nuclease, the viral genome is circular and negative stranded, and comprises 3,852 nt, which is 113 nt longer than the prototype isolate from Japan. Cesium chloride density gradient centrifugation demonstrated banding of the virus at 1.31-1.34 g/ml; filtration studies indicated that TTV had a particle size of 30-50 nm. These results suggest that the virus is similar to the Circoviridae, viruses known to infect plants and vertebrates (e. g., birds and swine); however, sequence similarity searches of available databases did not reveal identity between TTV and other viruses. Phylogenetic analyses of a 260-nt region from 151 globally distributed isolates demonstrated the existence of three major TTV genotypes. Several individuals at high risk for infection with parenterally transmitted viruses were infected with more than one genotype. There was no correlation between genotype and geographic origin. Finally, intravenous inoculation of TTV-positive human serum into chimpanzees demonstrated that TTV can be transmitted to primates; no biochemical or histological evidence for hepatitis was obtained. The distinct biophysical and molecular characteristics of TTV suggest that it is a member of a new family of viruses, which we have tentatively named the Circinoviridae.

A hepatitis E virus variant from the United States: molecular characterization and transmission in cynomolgus macaques.

The partial sequence of a hepatitis E virus (HEV-US1) isolated from a patient in the United States (US), suffering from acute viral hepatitis with no known risk factors for acquiring HEV, has been reported. These sequences were significantly different from previously characterized HEV isolates, alluding to the existence of a distinct human variant. In this paper, we report the near full-length sequences of HEV-US1 and a second US isolate (HEV-US2). HEV-US2 was identified in a US patient suffering from acute viral hepatitis. These sequences verify the presence of a new HEV strain in North America and provide information as to the degree of variability between variants. The HEV-US nucleotide sequences are 92% identical to each other and only 74% identical to the Burmese and Mexican strains. Amino acid and phylogenetic analyses also demonstrate that the US isolates are genetically distinct, suggesting the presence of three genotypes of HEV. Serum from the second US patient induced hepatitis following inoculation into a cynomolgus macaque. Within 2-4 weeks, HEV-US2 RNA was detectable in both the serum and faecal material coinciding with elevated serum alanine transaminase levels. Infection resolved as antibody titres increased 8 weeks post-inoculation.

A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis.

Recent studies have reported and provided nucleotide sequence data from divergent isolates of hepatitis E virus (HEV), including isolates from North America and Africa. Sera were investigated from 29 Chinese patients with a diagnosis of acute hepatitis and who were negative for hepatitis viruses A-E by serology (HEV was excluded by testing for IgG antibody only). To determine whether some patients were infected with HEV but had yet to seroconvert to antibody positivity, RT-PCR was carried out with primers designed within conserved sequences of the HEV open reading frame (ORF) 1 and ORF2 regions. Fifteen patients were found to harbour sequences related to HEV. Analysis of the HEV products revealed that nucleotide sequences from nine of the sera closely matched Burmese-like HEV sequences (more than 92% nucleotide identity across ORF1 and 88% in ORF2). The remaining six HEV isolates were similar to each other but divergent from all other known HEV sequences (74 to 83% nucleotide identity in ORF1 or ORF2). Phylogenetic analysis suggests that the six divergent isolates represent a fourth genotype of HEV, distinct from the previously described Burmese, Mexican and United States variants (genotypes 1, 2 and 3). This novel variant, referred to here as the Chinese genotype (genotype 4), may be responsible for a significant proportion of cases of acute hepatitis in China, as seen by the fact that 40% of the HEV-infected patients in this study were genotype 4 positive.

Optimized PCR assay for the detection of TT virus.

A polymerase chain reaction (PCR)-based procedure for the detection of TT virus DNA is described. In this method. total nucleic acid extracted from a small volume of serum or plasma is utilized as a template in PCR employing TT virus specific primers designed to highly conserved regions of the virus genome. Additional sensitivity is obtained by carrying out a second round of amplification. Reactions are analyzed by agarose gel electrophoresis, and samples having an ethidium bromide stainable fragment of the appropriate size in the first and/or second amplification are designated as positive. This protocol allows for the rapid and sensitive detection of TT virus in human plasma or serum.

The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States.

A variant of hepatitis E virus (HEV), designated HEV US-1, was identified in a hepatitis patient in the United States (US); the patient had no history of travel to areas where HEV is endemic. Nucleotide sequences were obtained from the 5' end of open reading frame (ORF) 1 (1418 nt), the 3' end of ORF1 (1359 nt), the entire ORF2 and ORF3 regions, and the 3'-untranslated region (2127 nt). The HEV US-1 strain is significantly divergent from other human HEV isolates with nucleotide identities ranging from 76.8 to 77.5%. Phylogenetic analyses indicate that HEV US-1 and a recently discovered HEV variant from swine may represent separate isolates of a new strain of HEV, significantly divergent from the Mexican and Burmese strains. Synthetic peptides derived from the carboxyl amino acids of ORF2 and ORF3 were shown to be useful for detecting exposure to HEV. In addition, IgM class antibodies directed against HEV US-1 synthetic peptides were detected in the US patient infected with HEV US-1, but were absent using synthetic peptides from the Burmese or Mexican strains of HEV. A preferential reactivity to HEV US-1 specific peptides has lead to the identification of a second isolate of this virus also from a patient with acute hepatitis from the US. The discovery of these HEV variants may be important in understanding the worldwide distribution of HEV infection.

Rapid detection of GB virus C RNA by reverse transcription-polymerase chain reaction (RT-PCR) using primers derived from the 5'nontranslated region.

A simple reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of GB virus C (GBV-C) RNA in serum or plasma is described. In this method, total nucleic acid, extracted from a small volume of human plasma, is reverse transcribed using random hexamers. An aliquot of cDNA is then utilized in PCR employing GBV-C specific primers designed to highly conserved regions of the 5'nontranslated region (NTR). For additional sensitivity, a second round of nested amplification is performed. Reactions are analyzed on an agarose gel and samples showing an ethidium bromide stained band of the appropriate size in the first and second amplification, or in the second amplification only, are designated to be positive. This protocol allows for the rapid and sensitive detection of GBV-C infection in human plasma or serum.

Genomic analysis of two GB virus A variants isolated from captive monkeys.

The recent isolation of GB viruses A and B from GB agent infected tamarins and their lack of involvement in human hepatitis has sparked interest in the origin of these viruses. Several healthy non-human primate species have been shown to harbour sequences 52-79% identical to the GBV-A 5' nontranslated region. In this paper we report the near genome length sequence of GBV-Amx 70047 and GBV-Atri 1122. These sequences support previous observations about the genomic organization of GBV-A and provide insight into the genomic variability within this virus genus. Although the GBV-A variant polyproteins possess many motifs conserved between other members of the Flaviviridae, they do not encode a basic core-like protein. Amino acid sequence comparisons and phylogenetic analysis demonstrate variability within the GBV-A genus similar to that observed between hepatitis C virus (HCV) types. However, genomic organization and disease association demonstrate a closer evolutionary relationship to GBV-C than to HCV.

Identification of GB virus C variants by phylogenetic analysis of 5'-untranslated and coding region sequences.

Phylogenetic analysis of 44 GB virus C (GBV-C) 5'-untranslated region (5'-UTR) sequences from 37 individuals suggested the presence of GBV-C genotypes (A. S. Muerhoff, J. N. Simons, T. P. Leary, J. C. Erker, M. L. Chalmers, T. J. Pilot-Matias, G. J. Dawson, S. M. Desai, and I. K. Mushahwar, J. Hepatol. 25:379-384, 1996) that correlated with geographic origin: type 1, 2a and 2b, and 3 isolates are found predominantly in West Africa, the United States and Europe, and Japan, respectively. We have extended our analysis to include 5'-UTR sequences from 129 globally distributed GBV-C isolates and sequences from the second envelope protein (E2) gene and nonstructural (NS) regions 3 and 5b from a subset of these isolates. Bootstrap analysis of a 157-nucleotide segment of the 5'-UTR from 129 sequences provided weak support for the existence of the four major groups of GBV-C isolates previously described, although phylogenetic analysis of a 374-nucleotide segment of the 5'-UTR from 83 isolates provided stronger support. Thus, the groups of GBV-C variants previously identified upon analysis of the entire 5'-UTR can be distinguished by analysis of the shorter, 374-nucleotide region from the 5'-UTR. In contrast, independent analysis of the E2, NS3, or NS5b region sequences does not identify groups of GBV-C variants that correlate with geographic origin. However, bootstrap analysis of these coding sequences, when linked to form colinear sequences, demonstrates that longer coding regions can produce GBV-C groupings that are similar to that determined from 5'-UTR sequence analysis. The inability to distinguish between GBV-C variants by using small segments of coding sequence suggests that the GBV-C genome is constrained. As a result of these constraints, there is a high degree of nucleotide and amino acid sequence conservation between isolates from widely separated geographic areas. Hence, substitutions at many nucleotide positions are not tolerated, so that substitutions at the positions which can change are saturated, thereby obscuring the evolutionary relationships.

The sequence and genomic organization of a GB virus A variant isolated from captive tamarins.

Recently, gene fragments of several novel variants of GB virus A were isolated from the serum of distinct monkey species that had not been experimentally inoculated with an infectious agent. These variants appeared to be species-specific in that sequences isolated within a species were virtually identical, though sequences were strikingly different when compared between each species. In the present study, the nucleotide sequence of one of these variants, GBV-Alab, was extended to near-genome length. Similar to the other GB viruses, GBV-Alab appears to encode a single large polyprotein of 2967 amino acids that is post-translationally cleaved by cellular and viral proteases into the individual viral proteins. The structural proteins are found at the N-terminal end of the polyprotein, while the nonstructural proteins are found at the C teminus. Amino acid sequence comparisons of the large polyprotein demonstrate that GBV-Alab is 74% identical to GBV-A and 48% identical to GBV-C, sharing only marginal identity with GBV-B and HCV-1 at 27%. Examination of the GBV-Alab polyprotein reveals that structural motifs are conserved for a protease, a helicase and a replicase. Phylogenetic analysis of the polyprotein confirms previous results that GBV-Alab is a member of the Flaviviridae, distinct from GBV-B and HCV, though more closely related to GBV-A and GBV-C.

Characterization of genes for a second Mo-dependent nitrogenase in the cyanobacterium Anabaena variabilis.

Anabaena variabilis ATCC 29413 is a filamentous heterocystous cyanobacterium that fixes nitrogen under a variety of environmental conditions. Under aerobic growth conditions, nitrogen fixation depends upon differentiation of heterocysts and expression of either a Mo-dependent nitrogenase or a V-dependent nitrogenase in those specialized cells. Under anaerobic conditions, a second Mo-dependent nitrogenase gene cluster, nifII, was expressed in vegetative cells long before heterocysts formed. A strain carrying a mutant gene in the nifII cluster did not fix nitrogen under anaerobic conditions until after heterocysts differentiated. The nifII cluster was similar in organization to the nifI cluster that is expressed in heterocysts and that includes nifBSUHDKENXW as well as three open reading frames that are conserved in both cyanobacterial nif clusters.

Molecular cloning and characterization of a GB virus C isolate from a patient with non-A-E hepatitis.

Recently, the isolation of a novel virus, GB virus C (GBV-C), associated with cryptogenic hepatitis has been reported. Following the molecular cloning of this virus genome, it became apparent that the genomic sequence did not encode a protein resembling a nucleocapsid or core-like protein similar to those observed in other flaviviruses, pestiviruses, hepatitis C virus (HCV) and GB virus B. Similar findings were subsequently observed in the cloning of two viral genomes representing isolates of GBV-C, namely hepatitis G virus (HGV). To verify the presence or absence of a viral nucleocapsid protein, identify conserved protein motifs and determine the overall genomic variability, an additional virus isolate has been characterized. Here we report the full-length genomic sequence of GBV-C(EA), isolated from an East African suffering from acute non-A-E hepatitis. GBV-C(EA) was compared with the prototype West African isolate (GBV-C) and the two HGV isolates from the United States. The analyses demonstrate several characteristics of these novel viruses. (1) The degree of variability within the 5' nontranslated region (NTR) approximates that observed between HCV isolates. (2) The nucleotide sequence of the coding region and the 3' NTR is highly conserved between these isolates, in contrast to the extensive variability observed between HCV isolates from distinct geographical locations. (3) There is a high degree of amino acid conservation across the precursor polyproteins of these isolates; most striking is the lack of 'hypervariable' regions within the envelope proteins. (4) There appears to be no nucleocapsid protein near the amino terminus of the GBV-C/HGV polyproteins.

Identification of conserved nucleotide sequences within the GB virus C 5'-untranslated region: design of PCR primers for detection of viral RNA.

Recently, the discovery of a new human RNA virus, GB virus C (GBV-C), was reported. GBV-C was isolated from the serum of a West African individual using degenerate oligonucleotide PCR primers designed from a consensus sequence of the NS3 helicase genes of hepatitis C virus (HCV), GBV-A, and GBV-B. Seven other individuals were shown to be infected with GBV-C via RT-PCR using these primers. Subsequently, degenerate PCR primers based upon a consensus sequence of the eight original isolates were designed. These primers were shown to be superior to the original set. However, since they were derived from a region of the viral genome exhibiting up to 17% nucleotide sequence divergence, mismatch between the primers and template may result in an underestimation of the true GBV-C prevalence. To overcome this potential problem, we obtained the sequences at the 5'-untranslated region (UTR) of the GBV-C genome from 35 infected individuals and identified regions of high sequence conservation among the isolates. We describe the design and testing of PCR primers derived from conserved sequences within the 5'-UTR of the GBV-C genome. These primers were shown to be as effective as the helicase-derived primers in detecting GBV-C RNA in human sera.

Sequence heterogeneity within the 5'-terminal region of the hepatitis GB virus C genome and evidence for genotypes.

BACKGROUND: GB virus C is a positive-strand RNA virus that is associated with hepatitis in humans. GB virus C bears some resemblance to hepatitis C virus in its genomic sequence and organization. However, unlike hepatitis C virus, an open reading frame possessing a complete core protein was not identified in the original isolate. METHODS: To verify the sequence at the 5'-end of the GB virus C genome, we amplified approximately 600 nucleotides from this region from 35 globally distributed individuals. The nucleotide sequences were translated in all possible reading frames and then examined for conserved motifs indicative of nucleocapsid or core-like peptides. RESULTS: Forty-two unique GB virus C sequences were obtained from the 35 individuals. The deduced amino acid sequences upstream of the putative E1 gene from each isolate varied in length and composition, such that a conserved core-like sequence was not apparent. No core-like sequences were evident in the other reading frames. There was, however, a single methionine codon held in common among all isolates, although it was located very near the presumed amino-terminus of the putative E1 protein. Further analysis of the sequences for their evolutionary relatedness demonstrated the existence of five GB virus C subtypes that demonstrated a significant correlation with geographic distribution. CONCLUSIONS: GB virus C differs from hepatitis C virus and GB virus B in that it does not encode a nucleocapsid or core protein. The existence of GB virus C subtypes emphasizes the importance of investigating the correlation between infecting subtype and the severity of liver disease and/or responsiveness to treatment of GB virus C-associated hepatitis.

Identification of antigenic regions in the GB hepatitis viruses GBV-A, GBV-B, and GBV-C.

The genomes of two novel members of the Flaviviridae associated with GB agent hepatitis (GB viruses A and B) were cloned and sequenced recently. The genome of a third novel virus (GB virus C), related to but distinct from GB viruses A and B, has also been identified and characterized. Overlapping clones encompassing the large open reading frames of these three viruses have been expressed in E. coli as CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CKS) fusion proteins. Bacterial lysates were subjected to Western blot analyses using sera from GB agent-infected tamarins and human sera from various individuals with or "at risk" for non-A, non-B, non-C, non-D, non-E hepatitis. Antigenic regions were identified in the putative NS3, NS4, and NS5 proteins from all three viruses. An antigenic region was also identified in the putative core protein of GB virus B. Many of the clones identified originally as encoding antigenic proteins were quite large. To map these regions more narrowly, smaller overlapping clones were generated by polymerase chain reaction (PCR), expressed as recombinant CKS fusion proteins and tested by Western blot. Additionally, a lambda gt11 expression library was generated from infectious tamarin sera and immunoscreened. These studies have identified at least three epitopes in GB virus A, five epitopes in GB virus B and four epitopes in GB virus C.

Sequence and genomic organization of GBV-C: a novel member of the flaviviridae associated with human non-A-E hepatitis.

Recently, sequences from a novel virus, termed GB virus C (GBV-C), were identified in serum from several patients with cryptogenic hepatitis. In the present study, the nucleotide sequence of this virus has been extended to near-genome length. GBV-C encodes a putative single large polyprotein in which the structural proteins are positioned at the N-terminal end, with the non-structural proteins located at the C-terminal end. Amino acid sequence analysis of this large polyprotein reveals the presence of protease, helicase, and replicase motifs. Sequence alignments of the polyprotein followed by phylogenetic analyses suggest that GBV-C is a member of the Flaviviridae, most closely related to the recently described GB virus A.