Pathology of experimental aerosol Zaire ebolavirus infection in rhesus macaques.

There is limited knowledge of the pathogenesis of human ebolavirus infections and no reported human cases acquired by the aerosol route. There is a threat of ebolavirus as an aerosolized biological weapon, and this study evaluated the pathogenesis of aerosol infection in 18 rhesus macaques. Important and unique findings include early infection of the respiratory lymphoid tissues, early fibrin deposition in the splenic white pulp, and perivasculitis and vasculitis in superficial dermal blood vessels of haired skin with rash. Initial infection occurred in the respiratory lymphoid tissues, fibroblastic reticular cells, dendritic cells, alveolar macrophages, and blood monocytes. Virus spread to regional lymph nodes, where significant viral replication occurred. Virus secondarily infected many additional blood monocytes and spread from the respiratory tissues to multiple organs, including the liver and spleen. Viremia, increased temperature, lymphocytopenia, neutrophilia, thrombocytopenia, and increased alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transpeptidase, total bilirubin, serum urea nitrogen, creatinine, and hypoalbuminemia were measurable mid to late infection. Infection progressed rapidly with whole-body destruction of lymphoid tissues, hepatic necrosis, vasculitis, hemorrhage, and extravascular fibrin accumulation. Hypothermia and thrombocytopenia were noted in late stages with the development of disseminated intravascular coagulation and shock. This study provides unprecedented insight into pathogenesis of human aerosol Zaire ebolavirus infection and suggests development of a medical countermeasure to aerosol infection will be a great challenge due to massive early infection of respiratory lymphoid tissues. Rhesus macaques may be used as a model of aerosol infection that will allow the development of lifesaving medical countermeasures under the Food and Drug Administration's animal rule.

Advances in medical biological defense technology.

No single medical countermeasure will meet the needs for defense against all biological threats in all possible scenarios (civilian, military, clinical, and environmental). As the threat of genetically engineered organisms rises and the risk increases that these organisms might escape detection and successful treatment, it will be necessary to use advances in bioengineering to combat these new threats. This article presents several novel approaches taken by the DoD in collaboration with industry partners and other federal laboratories to produce improved biowarfare vaccines, diagnostics, and treatments. In the future, the United States must remain on the cutting edge of biotechnology and continue to predict the next areas of research necessary to maintain protection for the state-of-the-art warfighter on the battlefield.

Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice.

A candidate live-attenuated virus vaccine for protection against Venezuelan equine encephalitis (VEE) (designated V3526) was tested in mice to measure the magnitude, duration, and kinetics of virus replication in the blood and the central nervous system and its phenotypic stability after multiple passages in mice and cell culture. All results were compared to parallel experiments with parental virus and the existing VEE virus vaccine, TC-83. Maximum virus titers in the brains of V3526-inoculated mice were between 10- and 100-fold less than those observed in brains of mice inoculated intracranially (i.c.) with either the parental virus or TC-83. Neither V3526 nor TC-83 was lethal in BALB/c mice inoculated i.c.. However, mice inoculated with TC-83 developed acute symptoms lasting at least 14 days. In contrast, i.c. inoculation of TC-83 was uniformly lethal for C3H/HeN mice. V3526 was avirulent in both BALB/c and C3H/HeN mice after i.c. inoculation. The virulence characteristics of V3526 remained unchanged after five serial i.c. passages in mouse brains or after five cell culture passages. Finally, pathologic changes induced after i.c. inoculation of V3526 were consistently less severe and of shorter duration than those observed in TC-83-inoculated mice. Based on these results, V3526 is stable and appears to be significantly less neurovirulent in mice than TC-83.

Use of telemetry to assess vaccine-induced protection against parenteral and aerosol infections of Venezuelan equine encephalitis virus in non-human primates.

Two investigational vaccines, TC-83 (live-attenuated) and C-84 (formalin-inactivated), are currently available to immunize at-risk individuals against Venezuelan equine encephalitis virus (VEE). Ideally, such vaccines should protect against both the natural mosquito-borne route of infection and from aerosol, the most common route of laboratory infection. Whereas considerable data on vaccine efficacy following parenteral challenge are available, the efficacy of these vaccines against disease caused by aerosol exposure is not well established in primates. We compared the immunogenicity and protective capacity of TC-83 and C-84 against either subcutaneous or aerosol routes of infection in cynomolgus monkeys implanted with temperature-monitoring radiotelemetry devices. A single s.c. dose of TC-83, or three s.c. doses (days 0, 7, 28) of C-84, elicited similar serum virus-neutralizing antibody responses. Animals immunized with either TC-83 or C-84 were protected against s.c. infection. In contrast, after aerosol infection, 40% of the animals vaccinated with either TC-83 or C-84 developed signs nearly as severe as those seen in unvaccinated animals. Protection was not entirely consistent with the measured preinfection immune responses: unprotected animals had serum virus-neutralizing antibody titers and lymphoproliferative responses similar to those seen in protected animals. In this study, C-84 (three doses) protected monkeys as well as TC-83 (one dose) against either a s.c. or aerosol VEE challenge.

Experimental infection of nonhuman primates with sandfly fever virus.

Due to the lack of an animal model, previous studies of sandfly fever have relied upon human challenge trials. We examined the infectivity and potential pathogenicity of sandfly fever virus in cynomolgus monkeys (Macaca fascicularis). Three different preparations of sandfly fever virus. Sicilian strain, and a placebo were compared by different routes of administration. The most notable postchallenge clinical event was a decrease in lymphocytes in the intramuscularly challenged monkeys. Plaque-reduction neutralization responses peaked earlier in animals challenged intravenously as compared with those in animals challenged intramuscularly. There was no evidence for neurotropism or meningeal inflammation. Sandfly fever virus was infectious for cynomolgus monkeys, but produced no detectable clinical disease that might serve as a marker for animal modeling studies. On the other hand, the preclinical data provide supportive evidence for safe parenteral administration of a Sicilian strain of sandfly fever virus inoculum to humans as a challenge model for sandfly fever disease.

Venezuelan equine encephalitis in BALB/c mice: kinetic analysis of central nervous system infection following aerosol or subcutaneous inoculation.

OBJECTIVE: To investigate the routes of entry of Venezuelan equine encephalitis (VEE) virus into the brain, we infected BALB/c mice with a virulent strain (V3000) by aerosol or subcutaneous inoculation. METHODS: Immunohistochemistry and in situ hybridization methods were used to detect VEE virus in tissues taken at daily intervals postinfection. RESULTS: In both groups, virus in the brain first appeared in olfactory regions. Aerosol exposure caused early massive infection of olfactory epithelium, which developed into bilaterally symmetrical infection of the olfactory nerves, olfactory bulbs, and lateral olfactory tracts by day 2 postinfection. After subcutaneous inoculation, VEE in the brain also appeared first in olfactory regions, but was not detected until day 3 postinfection. By day 4 postinfection, VEE viral infection had spread throughout the brain in both groups. Vascular endothelium and the choroid plexus remained uninfected during the entire study. CONCLUSIONS: Our findings suggest that VEE virus, whether given by aerosol or subcutaneously, first enters the brain through the olfactory tract.

Syngeneic lysis of reticuloendotheliosis virus-transformed cell lines transfected with Marek's disease virus genes by virus-specific cytotoxic T cells.

Cell-mediated immune responses against Marek's disease virus (MDV) antigens were examined using reticuloendotheliosis virus (REV)-transformed cell lines of two haplotypes (B19B19 and B13B13). These cell lines were stably transfected with cloned fragments of MDV DNA resulting in the expression of the MDV-specific phosphoprotein pp38. Effector cells were obtained from P2a (B19B19) and S13 (B13B13) chickens at 7 days post inoculation with REV, oncogenic or attenuated serotype 1 MDV (JM-16/O and JM-16/A, respectively), serotype 2 MDV (SB-1), or herpesvirus of turkeys (HVT). Transfection of MDV genes did not influence the expression of Class I major histocompatibility complex antigens. The optimal effector to target cell ratio was determined to be 100:1. REV-sensitized effector cells lysed REV cell lines and REV cell lines transfected with MDV DNA in a syngeneic fashion. Effector cells from chickens inoculated with JM-16/O, JM-16/A, SB-1 or HVT lysed only the syngeneic, transfected cell lines, but not the parent REV cell lines. The percentage specific release caused by the MDV-sensitized effector cells was low, but statistically significant.

Enhanced expression of the Marek's disease virus-specific phosphoproteins after stable transfection of MSB-1 cells with the Marek's disease virus homologue of ICP4.

Phosphoprotein pp38, coded for by the BamHI-H fragment of the Marek's disease herpesvirus (MDV) genome is expressed in tumor cells and tumor cell lines. pp38 is associated with two other phosphoproteins, pp41 and pp24, and can be detected in a small percentage of tumor cells by indirect immunofluorescence assays (IIFA). The importance of MDV ICP4 for the regulation of pp38 expression was examined in the following MSB-1-derived cell lines stably transfected with the selection plasmid pNL1 [MDCC-CU221 (CU221)], pNL1 and the BamHI-A fragment of MDV DNA containing ICP4 (CU224), MDV ICP4 inserted in antisense direction in the eukaryotic expression vector pXT1 (CU222), or ICP4 in sense direction in pXT1 (CU223) or cotransfected with pNL1 and EcoRI-linearized BamHI-A MDV DNA (CU225, -237, -243, -244). IIFA analysis showed that CU223 had a markedly increased expression of pp38, while CU224 had a slightly increased expression. No changes were noted in CU221 or CU222, while expression of pp38 was decreased in CU225, -237, -243, and -244. Radioimmunoprecipitation assays demonstrated that the expression of all three phosphoproteins was enhanced in CU223. Steady-state transcriptional analysis showed that CU223 had increased levels of pp38-specific (1.9 and 3.3 kb) and ICP4-specific (10.0 kb) transcripts.

Characterization of reticuloendotheliosis virus-transformed avian T-lymphoblastoid cell lines infected with Marek's disease virus.

The expression of Marek's disease virus (MDV) transcripts and protein products was investigated in reticuloendotheliosis virus-transformed avian T-lymphoblastoid cell line RECC-CU91, which was superinfected with MDV. The presence of MDV in the superinfected cell line, renamed RECC-CU210, was demonstrated by Southern hybridization with 32P-labeled BamHI-H and -B fragments of the BamHI MDV DNA library. Examination of RECC-CU210 for the expression of MDV-specific RNA transcripts encoded by the internal repeat long (IRL), internal repeat short (IRS), and unique short (US) regions of the MDV genome revealed two small transcripts of 0.6 and 0.7 kb. These transcripts were mapped to the IRL and IRS regions, respectively. In contrast, RECC-CU211, which was developed through transfection of CU210 with the BamHI-A fragment of MDV, expressed an additional nine transcripts from the IRL, IRS, and US regions. CU211 but not CU210 also expressed a complex of polypeptides of 40, 38, and 24 kDa, identified by monoclonal antibodies as MDV-specific phosphoproteins. The 38-kDa phosphoprotein is likely to be pp38, an early viral protein that maps within the IRL region of the MDV genome. These findings suggest that genes located within the transfected BamHI-A fragment transactivated a number of genes located in the IRL region of the MDV genome.

Cell-mediated cytolysis of lymphoblastoid cells expressing Marek's disease virus-specific phosphorylated polypeptides.

Cell-mediated immune responses against Marek's disease virus (MDV)-antigens were examined using reticuloendotheliosis virus (REV)-transformed lymphoblastoid cell line CU91 and three cell lines derived from CU91. CU210 was established by establishing a latent MDV infection in CU91. Transfection of CU210 with pNL1, a selectable plasmid or with pNL1 and the cloned BamHI A fragment of MDV DNA resulted in the establishment of CU212 and CU211, respectively. CU211 expressed a MDV-specific phosphorylated polypeptide, while CU210 and CU212 were negative for MDV antigens. Only CU211 was lysed by MDV-specific effector cells. All cell lines were lysed by syngeneic REV-specific effector cells, although high levels of expression of the phosphorylated protein reduced the level of REV-specific lysis.

Stable transfection of reticuloendotheliosis virus-transformed lymphoblastoid cell lines.

Lymphoblastoid T cell lines were established by infection of chicken splenocytes with reticuloendotheliosis virus (REV). The target cells first were cultured in interleukin-containing conditioned medium or were stimulated by concanavalin A, or both. Most cell lines were T cells expressing CD3 and one of the T cell receptors, and all cell lines were positive for major histocompatibility complex (MHC) class II antigens. Several REV-transformed cell lines were stably transfected using electroporation with a selectable plasmid, pNL1, containing the neor gene. Transfected cell lines were selected using G418 and were maintained for periods up to 137 days. Transfected cell lines were susceptible to MHC class-I restricted lysis by cytotoxic T lymphocytes from REV-infected chickens.

Effects of selenium supplementation on bull sperm metabolism in vitro.

An in vitro study was conducted to examine the effects of direct supplementation of diluted semen with sodium selenite on the metabolism of bovine sperm. Selenium (Se) supplementation increased the percent motility yet did not affect the percent viability of the sperm. An increase in the oxygen consumed by the sperm was associated with the increase in sperm motility. Both the adenosine triphosphate (ATP) and total adenyl nucleotide (TN) concentrations were lowered by supplementing the sperm with Se. Although changes occurred in the adenyl nucleotide pool of the Se-supplemented sperm, these changes were not reflected in the energy charge. There was no difference in the energy charge between the Se-supplemented and unsupplemented sperm. The metabolic changes caused by Se were in vitro and occurred in a short interval of time, suggesting a catalytic effect as opposed to an enzymatic effect.