A. Scott Muerhoff

Prevalence studies of GB virus-C infection using reverse transcriptase-polymerase chain reaction

Among the three recently described GB viruses (GBV‐A, GBV‐B, and GBV‐C), only GBV‐C has been linked to cryptogenic hepatitis in man. Because of the limited utility of currently available research tests to determine antibody response to GBV‐C proteins, the prevalence of GBV‐C RNA in human sera was studied using reverse transcription‐polymerase chain reaction (RT‐PCR). The prevalence of GBV‐C is higher among volunteer blood donors with elevated serum alanine aminotransferase (ALT) levels (3.9%) than among volunteer blood donors with normal ALT levels (0.8%). Higher rates were also noted among commercial blood donors (12.9%) and intravenous drug users (16.0%). GBV‐C was frequently detected in residents of West Africa, where the prevalence was >10% in most age groups. Approximately 20% of patients diagnosed with either acute or chronic hepatitis C virus (HCV) were found to be positive for GBV‐C RNA. In addition, GBV‐C RNA sequences were detected in individuals diagnosed with non‐A‐E hepatitis, with clinical courses ranging from mild disease to fulminant hepatitis. Fourteen of sixteen subjects with or without clinically apparent hepatitis were positive for GBV‐C RNA more than 1 year after the initial positive result.

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.

Consensus oligonucleotide primers for the detection of GB virus C in human cryptogenic hepatitis

Recently, sequences from a putative member of the Flaviviridae, GB virus C (GBV-C), were isolated from the serum of patients with cryptogenic hepatitis. These sequences were 83-99% identical at the nucleotide level. Because of the divergence between these GBV-C isolates, it is likely that the PCR-based detection assay yields false negatives, underestimating dramatically the true prevalence of GBV-C in human hepatitis. We report the design of a GBV-C consensus oligonucleotide primer pair that is superior to those originally described. These primers identify GBV-C sequences in cases of cryptogenic hepatitis, allowing a better estimation of the prevalence of this virus in human populations.

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. J. Med. Virol. 56:44–51, 1998.

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 λgt11 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.

Detection of HCV core antigen in human serum and plasma with an automated chemiluminescent immunoassay.

BACKGROUND: Currently, the detection of HCV infection in blood donors relies on the ability of immunoassays to detect circulating HCV antibodies. However, a significant delay exists between the time of infection and the development of antibodies. This delay (window period) can last up to 70 days. The introduction of NAT for the detection of HCV RNA has reduced this window period dramatically. However, NAT is labor intensive, prone to contamination, and expensive as compared with standard serologic tests.

Combination HCV core antigen and antibody assay on a fully automated chemiluminescence analyzer

BACKGROUND: HCV exposure among blood donors is serologically determined by detection of antibodies to HCV (anti‐HCV); however, the recent development of an assay for the detection of HCV core antigen identifies infection before anti‐HCV development. Simultaneous detection of HCV core antigen and anti‐HCV would shorten the window period before seroconversion over conventional HCV antibody screening assays.

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 Knodells 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 Knodells 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. J. Med. Virol. 54:26–37, 1998.

Isolation and Characterization of the MSP1 Genes from Plasmodium malariae and Plasmodium ovale

The merozoite surface protein 1 (MSP1) is the principal surface antigen of the blood stage form of the Plasmodium parasite. Antibodies recognizing MSP1 are frequently detected following Plasmodium infection, making this protein a significant component of malaria vaccines and diagnostic tests. Although the MSP1 gene sequence has been reported for Plasmodium falciparum and Plasmodium vivax, this gene has not been identified for the other two major human-infectious species, Plasmodium malariae and Plasmodium ovale. MSP1 genes from these two species were isolated from Cameroon blood donor samples. The genes are similar in size to known MSP1 genes and encode proteins with interspecies conserved domains homologous to those identified in other Plasmodium species. Sequence and phylogenetic analysis of all available Plasmodium MSP1 amino acid sequences clearly shows that the Po and Pm MSP1 sequences are truly unique within the Plasmodium genus and not simply Pf or Pv variants.

Enzyme-Linked Immunosorbent Assays Using Recombinant Envelope Protein Expressed in COS-1 and Drosophila S2 Cells for Detection of West Nile Virus Immunoglobulin M in Serum or Cerebrospinal Fluid

ABSTRACT Humans infected with West Nile virus (WNV) develop immunoglobulin M (IgM) antibodies soon after infection. The microtiter-based assays for WNV IgM antibody detection used by most state public health and reference laboratories utilize WNV antigen isolated from infected Vero cells or recombinant envelope protein produced in COS-1 cells. Recombinant antigen produced in COS-1 cells was used to develop a WNV IgM capture enzyme immunoassay (EIA). A supplementary EIA using WNV envelope protein expressed in Drosophila melanogaster S2 cells was also developed. Both assays detected WNV IgM in the sera of experimentally infected rhesus monkeys within approximately 10 days postinfection. Human sera previously tested for WNV IgM at a state public health laboratory (SPHL) were evaluated using both EIAs. Among the sera from 20 individuals with laboratory-confirmed WNV infection (i.e., IgM-positive cerebrospinal fluid [CSF]) that were categorized as equivocal for WNV IgM at the SPHL, 19 were IgM positive and one was negative by the new EIAs. Of the 19 IgM-positive patients, 15 were diagnosed with meningitis or encephalitis; the IgM-negative patient was not diagnosed with neurological disease. There was 100% agreement between the EIAs for the detection of WNV IgM. CSF samples from 21 individuals tested equivocal for WNV IgM at the SPHL; all 21 were positive in both bead assays, and 16 of these patients were diagnosed with neurological disease. These findings demonstrate that the new EIAs accurately identify WNV infection in individuals with confirmed WNV encephalitis and that they exhibit enhanced sensitivity over that of the microtiter assay format.