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.

Complete nucleotide sequence of Kashmir bee virus and comparison with acute bee paralysis virus.

The complete nucleotide sequence of a novel virus is presented here together with serological evidence that it belongs to Kashmir bee virus (KBV). Analysis reveals that KBV is a cricket paralysis-like virus (family Dicistroviridae: genus Cripavirus), with a non-structural polyprotein open reading frame in the 5' portion of the genome separated by an intergenic region from a structural polyprotein open reading frame in the 3' part of the genome. The genome also has a polyadenylated tail at the 3' terminus. KBV is one of several related viruses that also includes acute bee paralysis virus (ABPV). Although KBV and ABPV are about 70 % identical over the entire genome, there are considerable differences between them in significant areas of the genome, such as the 5' non-translated region (42 % nucleotide identity), between the helicase and 3C-protease domains of the non-structural polyprotein (57 % amino acid identity) and in a 90 aa stretch of the structural polyprotein (33 % amino acid identity). Phylogenetic analyses show that KBV and ABPV isolates fall into clearly separated clades with moderate evolutionary distance between them. Whether these genomic and evolutionary differences are sufficient to classify KBV and ABPV as separate species remains to be determined.

Genetic and serotypic characterization of Sin Nombre-like viruses in Canadian Peromyscus maniculatus mice.

In Canada, hantavirus infected deer mice (Peromyscus maniculatus) have been collected from British Columbia to Newfoundland. Partial sequencing of G1 and N protein encoding regions from Canadian Peromyscus maniculatus-borne hantaviruses demonstrated the existence of significant genotypic divergence among strains. Phylogenetic analysis showed that Sin Nombre (SN)-like viruses from eastern and western Canadian deer mice can be divided into at least two broad-based genogroups. Sequencing of mitochondrial DNA from infected deer mice originating from various eastern and western provinces showed that SN-like virus genogroups appeared to be associated with distinct haplotypes of mice. Sera from deer mice infected with eastern and western viral genotypes neutralized the Sin Nombre virus strain, Convict Creek 107, but not the New York 1 hantavirus. Despite the genetic heterogeneity of Canadian SN-like strains these hantaviruses do not appear to define unique hantavirus serotypes.

A member of a novel family of yeast 'zn-finger' proteins mediates the transition from stationary phase to cell proliferation.

The cloning and molecular characterization of the GCS1 gene from the budding yeast Saccharomyces cerevisiae show that stationary phase is in fact a unique developmental state, with requirements to resume cell proliferation that can be distinct from those for maintenance of proliferation. Deletion of the GCS1 gene products a novel phenotype: stationary-phase mutant cells do not resume proliferation at a restrictive temperature of 15 degrees C, but mutant cells lacking Gcs1p that are proliferating at the permissive temperature of 29 degrees C continue to proliferate after transfer to 15 degrees C as long as nutrients are available. The GCS1 gene sequence predicts a 39 kDa polypeptide with a novel 'Zn-finger' motif. A point mutation within the finger motif produces a phenotype that mimics that of deletion of the GCS1 gene, showing that the finger motif is essential for full Gcs1p activity. Gcs1p and the products of two newly identified genes, SPS18 and GLO3, constitute a family of novel Zn-finger proteins.

Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae.

Starvation of cells of the yeast Saccharomyces cerevisiae causes cessation of proliferation and acquisition of characteristic physiological properties. The stationary-phase state that results represents a unique developmental state, as shown by a novel conditional phenotype (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987): mutant cells cannot proliferate at the restrictive temperature when stimulated to reenter the mitotic cell cycle from stationary phase but are unaffected and continue proliferation indefinitely if transferred to the restrictive temperature during exponential growth. We have exploited this reentry mutant phenotype to demonstrate that the same stationary-phase state is generated by nitrogen, sulfur, or carbon starvation and by the cdc25-1 mutation, which conditionally impairs the cyclic AMP-mediated signal transduction pathway. We also show that heat shock, a treatment that elicits physiological perturbations associated with stationary phase, does not cause cells to enter stationary phase. The physiological properties associated with stationary phase therefore do not result from residence in stationary phase but from the stress conditions that bring about stationary phase.

Differential effect of interferon on glycoprotein and membrane protein of vesicular stomatitis virus released from murine and simian cells.

Previous studies by Maheshwari et al. have indicated that vesicular stomatitis virus (VSV) released from interferon (IFN)-treated mouse L-929 (L) cells was structurally defective. Such virions had significantly smaller amounts of glycoprotein (G) and membrane protein (M). Olden et al. recently reported, however, that they were not able to repeat the findings of Maheshwari et al. We have examined the effect of IFN on VSV released from three different cell lines and observed that treatment of L-cells and secondary mouse embryo (ME) cells with an amount of mouse IFN that reduced infectious virus yield 100-fold, led to the release of VSV with reduced amounts of G and M proteins. However, at concentrations of IFN less than this concentration, this effect was not observed. In contrast, VSV released from human (Hu)IFN-treated primate BSC-1, cells showed no reduction in their G and M protein even at concentrations resulting in 400-fold decreases in infectious virus yield.