Imaging findings using a combined MRI/CT protocol to identify the "entire iceberg" in post-COVID-19 mucormycosis presenting clinically as only "the tip".

AIM: To analyse combined computed tomography (CT) and magnetic resonance imaging (MRI) characteristics of invasive rhino-orbital mucormycosis (IROM) in post-COVID-19 infection patients for accurate diagnosis and delineation of the extent of involvement. MATERIALS AND METHODS: A retrospective analysis was undertaken of 50 patients who developed IROM post-COVID-19 infection who underwent combined CT/MRI evaluation. RESULTS: The age range of the 50 affected patients was 23-73 years. Out of these, 41 were diabetic. CT/MRI showed predominant involvement of the maxillary (n=26) and ethmoid (n=19) sinuses. Extension of disease to the orbit (n=35), cavernous sinus (n=18), hard palate (n=15), skull base (n=8), and intracranial involvement (n=3) was seen. Perineural spread of the disease was analysed along all divisions of the trigeminal nerve and its branches. MRI showed T2-hypointense soft-tissue thickening with heterogeneous contrast enhancement with corresponding hyperdensities on CT diagnosing the presence of fungal elements. CONCLUSION: Clinicians should be aware of the possibility of IROM post-COVID-19 infection. Conjunctive use of CT, which depicts bone destruction and other reactive bony changes along with MRI, which reveals characteristic findings of soft-tissue thickening of the involved sinuses with extension of disease to the orbits, cavernous sinus, dura, hard palate, skull base, and intracranial structures. Accurate diagnosis and early recognition of the disease and its extension with appropriate use of these techniques helps to initiate appropriate and timely treatment, which is vital to prevent a fatal outcome.

Emergent Premedication for Contrast Allergy Prior to Endovascular Treatment of Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Management of contrast media allergies may lead to treatment delays in patients with acute ischemic stroke undergoing endovascular therapy. The optimal premedication strategy remains unclear. The aim of this report was to analyze our experience with emergent administration of premedication regimens before endovascular therapy. MATERIALS AND METHODS: We retrospectively reviewed prospective data for all patients undergoing endovascular therapy from 2012 to 2019 at an academic comprehensive stroke center. Records of patients with documented contrast allergy were reviewed and analyzed. Data collected included stroke risk factors and characteristics, historical contrast reaction details, premedication regimens administered, and signs or symptoms of allergic reaction developing post-endovascular therapy. Hospital arrival time to endovascular therapy was compared with that in those who did not have a history of contrast allergy. RESULTS: We analyzed 1521 patients undergoing endovascular therapy; 60 (4%) had documented contrast allergies and constituted the study cohort. The median age was 73 years (interquartile range, 66-81 years), and 65% were women. The median time from premedication to contrast was 24 minutes (interquartile range, 0-36 minutes). Forty-three patients (72%) proceeded directly to endovascular therapy; in 17 patients, the first contrast exposure was CTA. Time from hospital arrival to endovascular therapy was not slower for patients with documented allergies (96 versus 134 minutes, P = .32). No patients experienced a contrast media reaction. CONCLUSIONS: In a single-institution cohort study of 60 consecutive patients with documented contrast allergies undergoing endovascular therapy with emergent premedication en route to (or in) the neuroangiography suite, no patients experienced allergic symptoms. This pragmatic approach may be safe for patients who have documented contrast media allergies.

Predicting outcomes after acute reperfusion therapy for basilar artery occlusion.

BACKGROUND AND PURPOSE: Basilar artery occlusion (BAO) leads to high rates of morbidity and mortality, despite successful recanalization. The discordance between flow restoration and long-term functional status clouds clinical decision-making regarding further aggressive care. We sought to develop and validate a practical, prognostic tool for the prediction of 3-month favorable outcome after acute reperfusion therapy for BAO. METHODS: This retrospective, multicenter, observational study was conducted at four high-volume stroke centers in the USA and Europe. Multivariate regression analysis was performed to identify predictors of favorable outcome (90-day modified Rankin scale scores 0-2) and derive a clinically applicable prognostic model (the Pittsburgh Outcomes after Stroke Thrombectomy-Vertebrobasilar (POST-VB) score). The POST-VB score was evaluated and internally validated with regard to calibration and discriminatory ability. External validity was assessed in patient cohorts at three separate centers. RESULTS: In the derivation cohort of 59 patients, independent predictors of favorable outcome included smaller brainstem infarct volume on post-procedure magnetic resonance imaging (P < 0.01) and younger age (P = 0.01). POST-VB score was calculated as: age + (10 × brainstem infarct volume). POST-VB score demonstrated excellent discriminatory ability [area under the receiver-operating characteristic curve (AUC) = 0.91] and adequate calibration (P = 0.88) in the derivation cohort (Center A). It performed equally well across the three external validation cohorts (Center B, AUC = 0.89; Center C, AUC = 0.78; Center D, AUC = 0.80). Overall, a POST-VB score < 49 was associated with an 88% likelihood of favorable outcome, as compared to 4% with a score ≥ 125. CONCLUSIONS: The POST-VB score effectively predicts 3-month functional outcome following acute reperfusion therapy for BAO and may aid in guiding post-procedural care.

Draft Genome Sequence of Arthrobacter crystallopoietes Strain BAB-32, Revealing Genes for Bioremediation.

Arthrobacter crystallopoietes strain BAB-32, a Gram-positive obligate aerobic actinobacterium having potential application in bioremediation and bioreduction of a few metals, was isolated from rhizosphere soil of Gandhinagar, Gujarat, India. The draft genome (4.3 Mb) of the strain revealed a few vital gene clusters involved in the metabolism of aromatic compounds, zinc, and sulfur.

A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma.

Hepatitis C virus (HCV) exposure in blood donors is determined serologically by the detection of anti-HCV antibodies in serum or plasma. However, a "window" period of 30-70 days after exposure exists where specific antibodies to HCV antigens are not detected. The use of nucleic acid testing for the detection of HCV RNA or antigen testing for the detection of HCV core protein have resulted in dramatic reductions in the pre-seroconversion window period. In this study, an automated HCV core antigen detection test was developed. This magnetic microparticle-based assay utilizes anti-HCV core monoclonal antibody to capture antigen present in human serum or plasma. Captured antigen is then detected using an anti-HCV core monoclonal antibody conjugated with a chemiluminescent compound. The specificity of this assay was established at 99% upon testing a population of normal volunteer blood donors. Sensitivity was determined by testing 16 commercially available HCV seroconversion panels representing genotypes 1a, 1b, 2b, and 3a. In each panel tested, HCV core antigen was detected prior to anti-HCV antibody, resulting in a reduction of the window period by greater than 23 days on average, and greater than 34 days on panels initially NAT negative. In addition, HCV core antigen was detected in >97% of HCV RNA positive/antibody negative specimens, exhibiting sensitivity nearly equivalent to nucleic acid testing in the pre-seroconversion window period for the panels examined.

Recently discovered blood-borne viruses.

In recent years, molecular biology advances have enabled many investigators to discover a number of viruses that have been difficult to characterise by cell culture techniques. Two blood-borne viruses have been identified. These are GB virus C (GBV-C) and TT virus (TTV). GBV-C was discovered in 1995. It is a flavivirus-like enveloped particle measuring 50-100 nm in diameter with a density of 1.08-1.13 g/cm3. The genome of GBV-C is a single-stranded, positive strand ribonucleic acid of approximately 8600 nucleotides. The TTV was discovered in 1997. It is a circular single-stranded deoxyribonucleic acid virus, non-enveloped of approximately 3900 nucleotides. It has a density of 1.31-1.34 g/cm3 and a particle size of 30-50 nm. Both viruses are distributed widely throughout the world. Most GBV-C infections are asymptomatic, transient and self-limiting. To date, solid evidence for any association of TTV with disease has not been demonstrated.

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.

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.

Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV.

Hepatitis E infection is associated with areas in which hepatitis E virus (HEV) infection is endemic. Acute infections in industrialized nations are usually linked to travel to endemic areas. Recently, an acute hepatitis infection in a patient from the United States (US), with no recent foreign travel history, was linked to a novel strain of HEV. Although a few additional cases have been reported from patients who have not traveled to endemic areas, the source of these infections has not been determined. The objective of this study was to identify additional HEV isolates from patients with acute infection who had no recent history of travel to areas where HEV is considered endemic, and to determine the genetic relationship between these and other HEV isolates. Viral RNA was isolated from serum and polymerase chain reaction (PCR) was performed using consensus primers based on a number of HEV isolates. HEV sequence in open reading frame (ORF) 1 and ORF2 was identified in three patients from nonendemic areas, one from Italy and two from Greece. Comparative and phylogenetic analyses were performed. The Greek and Italian isolates were significantly divergent from two isolates from the US and isolates identified previously from HEV-endemic regions. The Italian isolate was distinct from the two Greek isolates. In addition, the two Greek isolates were significantly divergent from each other. Phylogenetic analysis indicated that the Italian and two Greek isolates represent three new genotypes of HEV, distinct from the Burmese, Mexican, and US genotypes.

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.

Isolation of a GB virus-related genome from a chimpanzee.

Recently, two new flaviviruses, GB virus A (GBV-A) and GB virus B (GBV-B), were identified in the plasma of a tamarin infected with the hepatitis GB agent. A third virus, GB virus C (GBV-C), was subsequently identified in humans. In the current study, representational difference analysis (RDA) was used to search for a new virus in the serum of a chimpanzee that developed acute resolving hepatitis following inoculation with a pool of chimpanzee plasma. The plasma pool originated from serial passages of a human sample containing virus-like particles. Numerous cDNA clones were obtained that exhibited 62-80% identity with GBV-C. With the exception of the extreme 5' and 3' ends, the complete viral genome was sequenced, revealing a single large open reading frame encoding a 2833 amino acid polyprotein that contains two envelope proteins, two proteases, a helicase, and an RNA-dependent RNA polymerase. Phylogenetic analysis of the new virus indicates that it is closely related to GBV-C, yet still sufficiently divergent as to be placed in a separate group, tentatively labeled GB virus Ctroglodytes (GBV-Ctro). Numerous human samples were screened by reverse transcriptase-polymerase chain reaction (RT-PCR), but GBV-Ctro sequence was not detected. However, a second chimpanzee inoculated with the same plasma pool was shown to develop a GBV-Ctro infection. Although isolated from an Old World primate with hepatitis, the primary host of GBV-Ctro and any association with disease remains to be determined.

Predictors of GBV-C infection among patients referred for renal transplantation.

The etiology of liver disease remains unknown in about 4 to 23% of dialysis patients and 10 to 16% of renal transplant recipients. A search for other causative agents of liver disease led to the discovery of the GB group of viruses. We studied the association between the presence of GB virus C (GBV-C) infection, known risk factors for parenterally-transmitted infections and history or laboratory evidence of liver disease among end-stage renal disease (ESRD) patients referred for renal transplantation to the New England Organ Bank, MA. Stored sera from patients on the renal transplantation waiting list between November 1986 and June 1990 were tested for antibody to hepatitis C virus (HCV). Sera were available in 1544 of 3243 (48%) patients, and anti-HCV was detected by ELISA3 in 287 (19%). All 287 anti-HCV positive patients formed the anti-HCV positive cohort and 286 randomly selected anti-HCV negative patients formed the anti-HCV negative cohort (573 patients overall). Additional sera were available for GBV-C RNA testing in 465 of 573 (81%) patients, and GBV-C RNA was detected by RT-PCR in 146. The overall extrapolated prevalence of serum GBV-C RNA was 29%. The prevalence of serum GBV-C RNa among anti-HCV positive patients (35%) was not significantly different from that among anti-HCV negative patients (29%; P = 0.22). In a univariate analysis, compared to patients without GBV-C RNA, patients with serum GBV-C RNA were younger [odds ratio (OR) 0.98 per year of age, P = 0.01], had a lower proportion of males (OR 0.64, P = 0.03), lower proportion of patients with diabetes mellitus (OR 0.44, P = 0.01), higher proportion of patients with a previous transplantation (OR 1.53, P = 0.04), longer duration of dialysis at the time of enrollment (OR 1.004 per month on dialysis, P = 0.03), and a higher proportion of patients with history of transfusions (OR 4.58, P = 0.01). Serum GBV-C RNA was not associated with a significantly increased OR for history of liver disease or non-A, non-B hepatitis, or elevated serum alanine aminotransferase levels. In a step-wise multivariate regression analysis, a younger age (OR 0.98 per year of age, P = 0.03), and history of blood transfusions (OR 3.89, P = 0.03) were associated with an increased OR for serum GBV-C RNA, while diabetes mellitus was associated with a decreased OR for GBV-C RNA (OR 0.47, P = 0.01). Anti-HCV was not a predictor of serum GBV-C RNA (OR 1.07, P = 0.77). The results of this study support the fact that GBV-C is a parenterally transmitted virus and shed light on the modes of transmission of GBV-C among ESRD patients. However, the association with liver disease remains to be established.

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.

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.

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.

GB virus C (GBV-C) infection in patients with chronic hepatitis C. Influence on liver disease and on hepatitis virus behaviour: effect of interferon alfa therapy.

The aim of this study was to evaluate, in patients with chronic hepatitis C, 1) the prevalence and the epidemiological characteristics of GB virus C (GBV-C) infection, 2) the influence of GBV-C on hepatitis C virus (HCV) infection, 3) the pathogenicity of GBV-C in the absence of treatment and under interferon therapy, and 4) the effect of interferon alfa on GBV-C and HCV replications. One hundred fifteen patients with chronic hepatitis C were studied. Before treatment, they were tested for GBV-C RNA by PCR and GBV-C genotype was determined for positive samples. Pretreatment information was collected, including age, gender, source of HCV, estimated duration of HCV infection, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score on liver biopsy, HCV genotype, HCV viral burden and anti-HCV core IgM antibodies. The genetic complexity of the hypervariable region 1 (HVR1) of HCV was studied by PCR-Single Strand Conformation Polymorphism. All patients were treated with 3 to 9 mega units of interferon alfa-2a three times per week for 3 to 6 months. The influence of GBV-C on the evolution of ALT and HCV replication during and after treatment was studied, and GBV-C and HCV RNA were monitored monthly by PCR during this period. Eighteen patients (16%) were GBV-C RNA-positive. Among 11 samples studied, GBV-C genotype 2a was present in 9 cases, 2b in one case and type 3 in one case. GBV-C RNA-positive patients were significantly younger than GBV-C RNA-negative ones (38.4 +/- 11.5 vs. 47.4 +/- 14.0, P = 0.012), a result independent of the route of transmission and the disease duration. No difference between GBV-C RNA-positive and -negative patients was found for other epidemiological parameters (e.g. gender, risk factor for parenteral viral infections, disease duration and HCV genotypes), or for the characteristics of HCV infection and related liver disease (e.g. HCV RNA level, genetic complexity of the HVR1, anti-HCV core IgM, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score). GBV-C did not influence the rates of ALT normalization at months 3, 6 and 12 and of sustained hepatitis C virological response at month 12 of treatment follow-up. During treatment, GBV-C viremia became undetectable in 12 patients (67%) but relapse occurred after treatment withdrawal in all the nine patients with sufficient follow-up. In the remaining six patients (33%), GBV-C resisted interferon. Whatever the effect of interferon on GBV-C replication, the ALT levels correlated with the presence of HCV RNA. In conclusion, GBV-C infection is frequent in patients with chronic hepatitis C, who are mainly, but not exclusively, infected by GBV-C genotype 2a. GBV-C positive patients are significantly younger than GBV-C negative ones. GBV-C does not seem to affect HCV replication, liver disease and responses of HCV infection and liver disease to interferon therapy. GBV-C is sensitive to 3 mega units of interferon alfa administered three times per week in two-thirds of the patients, but relapse is constant with this dosage after treatment withdrawal.

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.