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.

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.

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.

Amplification of Chlamydia trachomatis DNA by ligase chain reaction.

Amplification of Chlamydia trachomatis DNA by polymerase chain reaction was compared with amplification by ligase chain reaction (LCR). Both amplification procedures were able to consistently amplify amounts of DNA equivalent to three C. trachomatis elementary bodies. All 15 C. trachomatis serovars were amplified to detectable levels by LCR, and no DNA from 16 organisms potentially found in clinical specimens or from Chlamydia psittaci and Chlamydia pneumoniae was amplified by LCR.

Saccharomyces cerevisiae CDC8 gene and its product.

The product of the Saccharomyces cerevisiae CDC8 gene is essential for normal cellular DNA replication; the determination of the structure of the gene and the identification of its product would facilitate the examination of its role in this process. We have cloned a 1,000-base-pair fragment of the S. cerevisiae genome carrying the functional gene. The nucleotide sequence includes one long open reading frame; it is flanked by sequences typical of other S. cerevisiae genes. This sequence predicts a polypeptide chain product of 216 amino acids with a molecular weight of 24,600. A polyadenylated RNA transcript of this sequence was identified by hybridization; in vitro translation of RNA samples enriched for this transcript produced a specific polypeptide chain of apparent molecular weight between 24,000 and 25,000. Thus the reading frame identified represents the authentic CDC8 gene, and the amino acid sequence of its product has been deduced. Our observations differ from two previous reports of the identification of the putative CDC8 protein based upon in vitro complementation assays.