Analytical and clinical sensitivity of West Nile virus RNA screening and supplemental assays available in 2003.

BACKGROUND: Transfusion-transmitted West Nile virus (WNV) infections were first reported in 2002, which led to rapid development of investigational nucleic acid amplification tests (NAT). A study was conducted to evaluate sensitivities of WNV screening and supplemental NAT assays first employed in 2003. STUDY DESIGN AND METHODS: Twenty-five member-coded panels were distributed to NAT assay manufacturers. Panels included five pedigreed WNV standards (1, 3, 10, 30, and 100 copies/mL), 15 or 16 donor units with very-low-level viremia identified through 2003 screening, and four or five negative control samples. Samples were tested neat in 10 replicates by all assays; for NAT screening assays, 10 replicates were also performed on dilutions consistent with minipool (MP)-NAT. The viral load distribution for 142 MP-NAT yield donations was characterized, relative to the analytical sensitivity of MP-NAT systems. RESULTS: Analytical sensitivities (50% limits of detection [LoD] based on Poisson model of detection of WNV standards) for screening NAT assays ranged from 3.4 to 29 copies per mL; when diluted consistent with MP pool sizes, the 50 percent LoD of screening NAT assays was reduced to 43 to 309 copies per mL. Analytical sensitivity of supplemental assays ranged from 1.5 to 7.7 copies per mL (50% LoD). Detection of RNA in donor units varied consistent with analytical LoD of assays. Detection of low-level viremia after MP dilutions was particularly compromised for seropositive units, probably reflecting lower viral loads in the postseroconversion phase. Based on the viral load distribution of MP-NAT yield donations (median, 3519 copies/mL; range, < 50-690,000), 13 to 24 percent of units had viral loads below the 50 percent LoD of screening NAT assays run in MP-NAT format. CONCLUSION: WNV screening and supplemental assays had generally excellent analytical sensitivity, comparable to human immunodeficiency virus-1 and hepatitis C virus NAT assays. The presence of low-level viremic units during epidemic periods and the impact of MP dilutions on sensitivity, however, suggest the need for further improvements in sensitivity as well as a role for targeted individual-donation NAT in epidemic regions.

Highly sensitive multiplex assay for detection of human immunodeficiency virus type 1 and hepatitis C virus RNA.

Various nucleic acid assays have been developed and implemented for diagnostics and therapeutic monitoring of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections. The high-throughput, semiautomated assays described here were developed to provide a method suitable for screening plasma specimens for the presence of HIV-1 and HCV RNAs. Three assays were developed: a multiplex HIV-1/HCV assay for simultaneous detection of HIV-1 and HCV, and discriminatory assays for specific detection of HIV-1 and HCV. The assay systems utilize three proprietary technologies: (i) target capture-based sample preparation, (ii) transcription-mediated amplification (TMA), and (iii) hybridization protection assay (HPA). An internal control is incorporated into each reaction to control for every step of the assay and identify random false-negative reactions. The assays demonstrated a sensitivity of at least 100 copies/ml for each target, and they detected with similar sensitivity all major variants of HCV and HIV-1, including HIV-1 group O strains. Assay sensitivity for one virus was not affected by the presence of the other. The specificity of these TMA-driven assays was >or=99.5% in both normal donor specimens and plasma containing potentially interfering substances or other blood-borne pathogens. Statistical receiver operating characteristic plots of 1 - specificity versus sensitivity data determined very wide analyte cutoff values for each assay at the point at which the assay specificity and sensitivity were both >or=99.5%. The sensitivity, specificity, and throughput capability predict that these assays will be valuable for large-volume plasma screening, either in a blood bank setting or in other diagnostic applications.

Sequence variation and phylogenetic analysis of the 5' terminus of hepatitis G virus.

We determined the nucleotide and deduced amino acid sequence of the 5' terminus of the hepatitis G virus (HGV) genome from isolates of varied geographical origins. Our analysis showed that the putative 5' non-coding region (NCR) contains several blocks of highly conserved sequences that may be useful for the development of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of HGV RNA. Overall, the degree of conservation within the 669-nucleotide (nt) 5'terminal sequence was found to range from 99.5% to 86% sequence identity. We also showed that the HGV NCR from some isolates contained conserved insertions or deletions that altered the translational reading frames at the 5'-end of the genome, resulting in different sizes of predicted polyproteins encoded by genomes of individual isolates. Specifically, the insertions/deletions affected the size of the peptide preceding the putative first envelope (E1) protein. Phylogenetic analysis of the nucleotide sequences suggested that the isolates examined can be classified into distinct groups that may be useful for studying the molecular evolution of HGV and possible relationships between isolate sequence characteristics and infection patterns.

Two related localized mRNAs from Xenopus laevis encode ubiquitin-like fusion proteins.

The uneven distribution of maternal mRNAs in unfertilized eggs and the unequal inheritance of these molecules by dividing blastomeres may be one mechanism for determining cell fate during embryogenesis. Complementary DNA (cDNA) clones corresponding to maternal mRNAs localized to specific regions of the Xenopus laevis egg have been previously identified and cloned [Rebagliati et al., Cell 42(1985) 769-777]. The maternal mRNA, An1, was originally identified as being localized to the animal hemisphere of X. laevis eggs and early embryos. We describe here the two proteins encoded by two An1 mRNA isoforms which we designate An1a and An1b. These mRNAs are both approximately 3.0 kb long and are concentrated in the animal hemisphere of unfertilized eggs. The predicted amino acid (aa) sequences encoded by An1a and An1b correspond to 76.9 and 78.6 kDa, respectively, and are 88% identical. Both proteins contain a single N-terminal ubiquitin (Ub)-like domain (50% identical to X. laevis Ub) and a putative Zn(2+)-binding region near the C terminus. Unlike Ub polyproteins and most Ub fusion proteins, the N-terminal Ub-like domain found in the An1 proteins does not undergo proteolytic processing. In contrast to earlier studies showing that the An1 mRNA represents a strictly maternal transcript, we report that both related An1 transcripts are found in later embryonic stages and in all adult tissues tested.