Determination of CD4 and CD8 lymphocyte subsets by a new alternative fluorescence immunoassay.

The purpose of this study was to evaluate a new alternative fluorescence immunoassay method (Zymmune CD4/CD8 Cell Monitoring Kit; Zynaxis, Inc., Malvern, Pa.) for determining the absolute CD4+ and CD8+ T-lymphocyte concentrations in whole blood. The investigation was performed as a two-site comparison of the reference whole blood flow cytometric method with the Zymmune method. In this investigation, a total of 166 patient samples were evaluated of which approximately 20% were from human immunodeficiency virus-positive individuals. The mean value for samples performed by the Zymmune CD4 assay was 1,094 (range, 74 to 2,586) cells per microliters, while the reference method yielded a mean of 890 (range, 35 to 2,033) cells per microliter. The correlation coefficient for regression analysis was 0.940. The mean value for samples performed by the Zymmune CD8 assay was 700 (range, 212 to 1,813) cells per microliter, while the reference method yielded a mean of 546 (range, 82 to 2,158) cells per microliter. The correlation coefficient for regression analysis was 0.921. No site-specific differences or trends in CD4 or CD8 values were seen when the data were analyzed by site of collection. The average precision of the CD4 assay varied from 6 to 14%, corresponding to the high and low concentration ranges. For CD8, the average precision varied from 8.3 to 16% over the respective high to low concentration ranges. We conclude that the Zymmune CD4/CD8 Cell Monitoring Kit method provides absolute CD4+ and CD8+ T-lymphocyte concentrations which are equivalent to those given by the reference flow cytometric method.

Characterization of murine Caraparu Bunyavirus liver infection and immunomodulator-mediated antiviral protection.

A rapid, peripheral disease model utilizing the Bunyavirus, Caraparu, was established in mice for the evaluation of antiviral therapy with immunomodulators. 4-6-week-old B6C3F1 female mice, inoculated intraperitoneally with virus, developed coagulative liver necrosis and died between 4-6 days after infection. This Caraparu disease model was relatively resistant to treatment with immunomodulators, such as ABMP, Ampligen, alpha-interferon (IFN-alpha) or beta-interferon (IFN-beta). However, a significant increase in median survival time (MST) was consistently observed upon treatment with gamma-interferon (IFN-gamma). The nucleoside analog--ribavirin--was highly effective against Caraparu virus in repeated treatment schedules begun on either day -1, day 0, or day +1 of infection. Ribavirin gave little protection when initiation of treatment was delayed until day +2. However, combined treatment with IFN-gamma, starting on day 0 and ribavirin starting on day +2, significantly reduced mortality.

Detection of viral-specific nucleic acid and intracellular virions in ventral horn neurons of lactate dehydrogenase-elevating virus infected C58 mice.

C58 mice which have been immunosuppressed by treatment with cyclophosphamide (200 mg/kg) one day prior to infection with the C strain of lactate dehydrogenase-elevating virus (LDV-C) develop poliomyelitis. Using in situ hybridisation, we found that some ventral horn neurons in these mice contain cytoplasmic viral-specific nucleic acid. Viral-specific nucleic acid was also found within a few small cells located near inflammatory foci. In addition, mature virus particles were observed by electron microscopy in some ventral horn neurons, indicating that these cells are productively infected in C58 mice. Neither viral nucleic acid nor virions were found in the ventral horn neurons of poliomyelitis-resistant mouse strains or C58 mice that were not immunosuppressed prior to infection. Ventral horn neurons which contained viral nucleic acid or virions within cytoplasmic vesicles generally were normal in appearance and were not located within poliomyelitis inflammatory foci. Our data are consistent with the hypothesis that infected neurons first replicate virus and subsequently are attacked and cleared by inflammatory cells.

Genotypic variation among six isolates of lactate dehydrogenase-elevating virus.

In the present study, six lactate dehydrogenase-elevating virus (LDV) isolates obtained independently from inbred mice were compared by RNA oligonucleotide fingerprint analysis. The genome RNAs of four of the isolates gave unique fingerprint patterns. The patterns obtained for the other two isolates were similar, but not identical to one of the four unique patterns. These results indicate that more than one genotype of LDV exists and that virus isolates can be grouped by genotype. We have also demonstrated the presence of a 3'-terminal poly(A) tract by direct sequencing of 3'-32P-labelled LDV genome RNA. The presence of a 3'-terminal poly(A) tract distinguishes LDV from the members of the family Flaviviridae, which lack a 3'-poly(A), and justifies the current classification of LDV within the family Togaviridae.