Detection of West Nile virus RNA and antibody in frozen plasma components from a voluntary market withdrawal during the 2002 peak epidemic.

BACKGROUND: The US West Nile virus (WNV) epidemic in the summer and fall of 2002 included the first documented cases of transfusion-transmitted WNV infection. In December 2002, the FDA supported a voluntary market withdrawal by the blood banking community of frozen blood components collected in WNV high-activity areas. At the time, the prevalence of viremia and serologic markers for WNV in the blood supply was undefined. STUDY DESIGN AND METHODS: In collaboration with America's Blood Centers, 1468 frozen plasma components (of approx. 60,000 frozen units voluntarily withdrawn from the market) were selectively retrieved from the peak epidemic regions and season (June 23, 2002-September 28, 2002). These units were unlinked, subaliquoted, and tested by WNV enzyme immunoassays (EIAs; Focus Technologies and Abbott Laboratories) and nucleic acid amplification tests (NATs; Gen-Probe Inc. and Roche Molecular Systems). RESULTS: Of the 1468 EIA results from Abbott and Focus, 7 were anti-immunoglobulin M (IgM)- and anti-immunoglobulin G (IgG)-reactive by both assays, 8 and 1 were IgM-only-reactive, and 8 and 23 were IgG-only-reactive, respectively. NAT by Gen-Probe and Roche Molecular Systems yielded one RNA-positive, antibody-negative unit containing approximately 440 RNA copies per mL. An additional 10-fold replicate NAT testing by Gen-Probe on 14 of 15 IgM-reactive specimens yielded 2 additional IgM- and IgG-reactive units with low-level viremia (i.e., 7/10 and 2/10 replicates tested reactive). CONCLUSION: The prevalence of acute (RNA-positive) and recent (IgM-seroreactive) WNV infections indicates that transfusion risk in high-risk areas could have been considerable and that voluntary market withdrawal of frozen components likely averted some WNV transfusion transmissions. The existence of very-low-level viremic units raises concerns, because WNV minipool NAT screening will miss such units and individual NAT may not completely correct this situation.

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.

Seroprevalence of GB virus C and persistence of RNA and antibody.

Exposure to GB virus C (GBV-C) was determined in several U.S. populations by both reverse-transcription-polymerase chain reaction (RT-PCR) and by an enzyme linked immunosorbent assay (ELISA) for antibodies to mammalian cell-expressed GBV-C envelope protein, E2 (GBV-C E2). Most individuals exposed to GBV-C were either RNA positive/ELISA negative or ELISA positive/RNA negative. Exposure, therefore, was measured as the sum of GBV-C RNA positive and GBV-C E2 antibody positive specimens, and was higher in commercial plasmapheresis donors (40.5%) than in volunteer blood donors (5.5%). In intravenous drug users (IVDUs), GBV-C exposure was 89.2%. Serial bleed specimens tested for GBV-C RNA indicate that some patients remain viremic for at least 3 years and fail to produce detectable antibodies to GBV-C E2. In other exposed individuals who tested negative for GBV-C RNA, antibodies to E2 appear to be similarly long-lived (greater than 3 years) with a fairly constant titer (ranging in reciprocal endpoint dilution from 336 to 21,504). Since the detection of GBV-C RNA and GBV-C E2 antibody are mutually exclusive in most exposed individuals, studies pertaining to incidence and prevalence of GBV-C infection require both antibody and nucleic acid detection.

An ELISA for detection of antibodies to the E2 protein of GB virus C.

An ELISA was developed for detection of antibodies to GB virus C (GBV-C) using a recombinant E2 protein expressed in CHO cells. Seroconversion to anti-E2 positivity was noted among several persons infected with GBV-C RNA-positive blood through transfusion. Of 6 blood recipients infected by GBV-C RNA-positive donors, 4 (67%) became anti-E2 positive and cleared their viremia. Thus, anti-E2 seroconversion is associated with viral clearance. The prevalence of antibodies to E2 was relatively low (3.0%-8.1%) in volunteer blood donors but was higher in several other groups, including plasmapheresis donors (34.0%), intravenous drug users (85.2%), and West African subjects (13.3%), all of whom tested negative by GBV-C reverse-transcription polymerase chain reaction (RT-PCR). These data demonstrate that testing for anti-E2 should greatly extend the ability of RT-PCR to define the epidemiology and clinical significance of GBV-C.

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.

Prevalence of specific antibodies to herpes simplex virus type 2 as revealed by an enzyme-linked immunoassay and western blot analysis.

A solid-phase ELISA for the detection of antibodies to gG-2 was developed. The assay utilizes a recombinant DNA-derived gG-2 as a solid-phase "capture" reagent and goat anti-human IgG (gamma) conjugate to horseradish peroxidase as a probe (detector) reagent. A total of 229 serum samples collected from various populations were tested by ELISA and western blot analysis. On comparison with confirmed HSV-2 infection, the sensitivity and specificity of the ELISA were 92.9% and 98.7%, respectively. Western blot had a sensitivity of 83.9% and a specificity similar to the ELISA. The ELISA is fast and easy to perform and may be used to diagnose previous exposure to genital herpes and to monitor human response to future HSV-2 vaccines.

Inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine.

Addition of 50 micrograms/ml chloroquine to neuroblastoma cells 1 h before infection with temperature-sensitive mutant ts G31 (III) of vesicular stomatitis virus (VSV) prevented virus-induced cell fusion from occurring. Interestingly, addition of chloroquine after infection still inhibited cell fusion. Based on the number of fusion events required to produce the polykaryocytes observed, cell fusion was inhibited 92% when chloroquine was added 1 h post-infection and 77% when chloroquine was added 2 h post-infection. The inhibition of virus-induced cell fusion could not be accounted for by an inhibition of virus protein synthesis because the virus protein synthesis measured 6 h post-infection was 90% of that in untreated, infected cells with chloroquine added 1 h post-infection, and the same as untreated, infected cells when chloroquine was added 2 h post-infection. No virus proteins were made, however, when chloroquine was added before infection, which is consistent with a chloroquine-mediated inhibition of virus uncoating. The release of infectious virions was completely inhibited when chloroquine was added before infection or 1 or 2 h post-infection, which indicated an inhibition of virus maturation in the later stages of virus assembly. By indirect immunofluorescence the virus glycoprotein (G protein) could not be detected on the surface of chloroquine-treated, infected cells, but the G protein was present inside the treated cells. With 125I-labelled anti-G protein IgG, 16% of the G protein found on the surface of untreated, infected cells was on the cell surface when chloroquine was added 2 h post-infection. When chloroquine was removed from infected cells, the G protein accumulated at the cell surface, and this accumulation could not be prevented by tunicamycin, an inhibitor of glycosylation. Furthermore, galactose was incorporated into the G protein in the presence of chloroquine. Therefore, the VSV G protein was being synthesized and glycosylated in the presence of chloroquine but the drug prevented the expression of the glycoprotein at the cell surface during the final stages of G protein assembly.U

Neuroblastoma cell membranes: specificity for cell fusion mediated by a temperature-sensitive mutant of vesicular stomatitis virus.

Temperature-sensitive mutant G31 of vesicular stomatitis virus induces mouse neuroblastoma N-18 cells to fuse during infections that are nonpermissive for virus replication, but BHK-21 cells do not undergo the viral glycoprotein-mediated cell fusion. The viral glycoprotein was expressed at the cell surface of both N-18 and BHK-21 cells; therefore, the host cell specificity did not stem from an absence of the viral glycoprotein at the surface of BHK-21 cells. Cell fusion readily occurred between infected and uninfected N-18 cells in mixed cultures, demonstrating that the viral glycoprotein was interacting with an uninfected cell for the initial cell-cell interaction of the cell fusion. Mixing infected BHK-21 cells with uninfected N-18 cells resulted in cell fusion initiated by BHK-21 cell-synthesized viral glycoprotein, but 88% of the nuclei in polykaryocytes were N-18 nuclei. The N-18 cell fusion specificity was readily apparent when infected N-18 cells were mixed with uninfected BHK-21 cells; 98% of the nuclei in polykaryocytes were N-18 nuclei. Similar results also were obtained with mixed cultures of N-18 cells and primary astroglial cells. Thus, the viral glycoprotein synthesized in any of the cell types could initiate cell fusion, but the properties of plasma membranes of neuroblastoma cells appeared to be much more suitable for cell-cell fusion.

Cytopathic effects in mouse neuroblastoma cells during a non-permissive infection with a mutant of vesicular stomatitis virus.

Morphological changes were extensive following infection of murine neuroblastoma N-18 cells with a temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), G31 (complementation group III), and incubation at 39 degrees C, a non-permissive condition for virion maturation. Incubation for 24 h after infection resulted in extensive morphological degeneration of mitochondria with over 80% from that in uninfected cells. Janus green B supravital staining, was reduced by 81% from that in uninfected cells. Cellular ATP levels were reduced by 50% 12 h after infection. Mitochondrial degeneration still occurred in infected cells after the inactivation of lysosomes with chloroquine. Extensive cell fusion and cytoplasmic vacuole formation also occurred during the non-permissive infection with ts G31. Loss of plasma membrane integrity was not the cause of vacuole formation since 90% of the cells were able to exclude trypan blue 24 h after infection, nor were the vacuoles the result of inactivation of the mitochondria since cyanide-poisoned cells did not form vacuoles. The cytopathic alterations observed in N-18 cells during the non-permissive infection of N-18 cells with ts G31 did not occur during the non-permissive infection of N-18 cells with ts G11 (I), ts G41 (IV), or u.v.-inactivated ts G31. However, the non-permissive infection with ts O45 (V) led to mitochondrial degeneration and cytoplasmic vacuole formation, but no cell fusion occurred. These results are discussed in light of the ultrastructural features previously observed in the central nervous system of mice infected with ts G31 and cells in culture infected with wild-type VSV.

Neuroblastoma cell fusion by a temperature-sensitive mutant of vesicular stomatitis virus.

A temperature sensitive mutant of vesicular stomatitis virus which does not mature properly when grown at 39 degrees C promoted extensive fusion of murine neuroblastoma cells at this nonpermissive temperature. Polykaryocytes apparently formed as a result of fusion from within the cells that requires low doses of infectious virions for its promotion and is dependent on viral protein synthesis. Although 90% of infected N-18 neuroblastoma cells were fused by 15 h after infection, larger polykaryocytes continued to form, leading to an average of 28 nuclei per polykaryocyte as a result of polykaryocytes fusing to each other. Two neuroblastoma cell lines have been observed to undergo fusion, whereas three other cell lines (BHK-21, CHO, and 3T3) were incapable of forming polykaryocytes, suggesting that nervous system-derived cells are particularly susceptible to vesicular stomatitis virus-induced fusion. Although the normal assembly of the protein components of this virus is deficient at 39 degrees C, the G glycoprotein was inserted into the infected cell membranes at this temperature. Two lines of evidence suggest that the expression of G at the cell surface promotes this polykaryocyte formation: (i) inhibition of glycosylation, which may be involved in the migration of the G protein to the cellular plasma membranes, will inhibit the cell fusion reaction; (ii) addition of antiserum, directed toward the purified G glycoprotein, will also inhibit cell fusion.