Gregory H. Tignor

Ribavirin efficacy in an in vivo model of Crimean-Congo hemorrhagic fever virus (CCHF) infection.

Abstract After intraperitoneal (i.p.) infection of infant mice with CCHF virus, virus titers in liver remained significantly higher than in other organs except blood (serum). Within the liver, virus antigen was first found by immunofluorescence (IFA) in Kupffer cells followed by more extensive hepatic spread. Later, virus was found in other organs including brain and heart. Ribavirin treatment significantly reduced infant mouse mortality and extended the geometric mean time to death. Ribavirin treatment reduced CCHF virus growth in liver and significantly decreased, but did not prevent, viremia. Despite a substantial viremia, infection of other organs including brain and heart was not detected in ribavirin-treated mice. A hepatotropic virus subpopulation with less neurovirulence than the parent was isolated from liver of ribavirin-treated mice (single dose, 100 mg/kg). After serial passage in placebo-treated mice, the exclusive hepatotropism was lost.

Isolation and assay of rabies serogroup viruses in CER cells.

Infection of CER cell cultures with field strains of rabies virus, ranging from 0 to 5 mouse brain passages, was detected by immunoflurescence within 2-4 days after infection. A fluorescent focus assay for measuring infectivity of seven rabies serogroup viruses was rapid and reproducible. Rabies field strians and other rabies serogroup viruses also induced cytopathic effect, usually on initial passage. The hemadsorption-negative (HAD-) plaque test in BSC-1 cells was successfully applied to laboratory-adapted rabies strains. HAD- test attempts were unsuccessful with CER cells and with field isolates of rabies virus in both cell lines. CER cells are heteroploid and are antigenically related to BHK-21 cells by fluorsecent antibody tests.

The Nairovirus Genus: Serological Relationships

Serological relationships among viruses of the Nairovirus genus, family Bunyaviridae, were investigated. Relationships were found among viruses of the following serogroups: Congo-Cr

Entry of rabies virus into the peripheral nerves of mice.

Young adult mice were inoculated in the hind limb with rabies virus or Sindbis virus. Rabies 1820B virus antigen was detected in leg sections by immuno-fluorescence at 1 h post-inoculation at sites comparable in form and distribution to cholinesterase-positive sites, which represent motor end-plates (MEPs). Sites which were rabies virus antigen-positive by immunofluorescence were also cholinesterase- positive on double-stained slides. Rabies CVS virus detected by autoradiography was similarly distributed at 6 h post-inoculation. Uptake of rabies virus at motor nerve endings was confirmed by the detection of rabies antigen by immunofluorescence in ventral horn cells in the spinal cord at 20 h post-inoculation before involvement of dorsal root ganglia. Rabies virus antigen could not be detected at MEPs if the virus had been inactivated by beta propiolactone or mixed with antibody prior to injection or if the sciatic nerve had been cut 7 days earlier, similarly treated groups of mice survived for the observation period of 6 weeks. Rabies virus antigen was found at MEPs in mice given antibody 24 h before virus injection, but virus antigen was not found in the spinal cord, and mice similarly treated survived. Sindbis virus strain Ar86, which like rabies virus is neurotropic in adult mice, was also found at MEPs and in peripheral nerves by autoradiography at 6 h post-inoculation. In contrast to results with rabies virus-infected mice, stimulation of the sciatic nerve for the first hour post-inoculation prevented mortality. Sindbis virus strain Ar339, which is not neurotropic in adult mice, could not be detected at MEPs by immunofluorescence or autoradiography and mice injected with virus survived. The results presented here suggest that rabies virus and perhaps other neurotropic viruses can use the motor axon terminal at the neuromuscular junction as a site of entry into the nervous system.

Rabies virus binding at neuromuscular junctions

Morphological, immunocytochemical, biochemical, and immunological techniques have been used to describe rabies virus binding to a sub-cellular unit and molecular complex at the neuromuscular junction (NMJ). Early after infection in vivo, virus antigen and virus particles were found by immunofluorescence, electron microscopy and immunoelectron microscopy in regions of high density acetylcholine receptors (AChR) at NMJs. One monoclonal antibody (alpha-Mab) to the alpha subunit of the AChR blocked attachment of radio-labeled rabies virus to cultured muscle cells bearing high density patches of AChR. A sub-cellular structure, resembling an array of AChR monomers, bound both rabies virus antigens and alpha-Mab. By immunoblotting with electrophoretically transferred motor endplate proteins, rabies virus proteins and alpha-Mab bound to two proteins of 43 000 and 110 000 daltons. A rabies virus glycoprotein antibody detected virus antigen bound to the 110 000 dalton protein. An auto-immune (anti-idiotypic) response followed immunization of mice with rabies virus glycoprotein antigen; the antibody was directed to the 110 000 dalton protein. This auto-antibody altered the kinetics of neutralization by rabies virus antibody and induced the formation of rabies virus antibody after inoculation of mice. These results define, at the neuromuscular junction, a rabies virus receptor which may be part of the acetylcholine receptor complex.

Duvenhage virus: morphological, biochemical, histopathological and antigenic relationships to the rabies serogroup.

Summary Duvenhage virus was originally isolated in South Africa from the brain of a man who had been bitten by a bat and died after a rabies-like illness. Previous immunofluorescence tests indicated that the virus was distinct from rabies virus. In the present study an antigenic relationship of this virus to rabies is defined, and pathological, morphological and further serological characterization is presented. Duvenhage virus-infected mice developed a central nervous system disease characterized by a short incubation period, a moderate degree of inflammatory infiltration of brain parenchyma and by small intraneuronal inclusion bodies. By electron microscopy typical rhabdovirus particles were found budding upon endoplasmic reticulum and plasma membranes of brain neurons. In these characteristics Duvenhage virus resembled laboratory or ‘fixed’ strains of rabies virus. The structural polypeptide composition of Duvenhage virus was very similar to that of rabies virus. Duvenhage virus could be distinguished from rabies by in vivo neutralization and cross-challenge tests in mice, and to a lesser extent by complement-fixation and fluorescent antibody tests. Antibody to purified ribonucleoproteins used in indirect immunofluorescence tests did not distinguish between rabies and Duvenhage virus. In vitro neutralization tests using antisera against whole virus, and against purified virus glycoprotein, confirmed the distinction; consequently Duvenhage virus should be considered a new member of the rabies serogroup (i.e. Lyssavirus genus, Rhabdoviridae family).

Device-specific sharps injury usage rates: An analysis by hospital department

BACKGROUND Whether universal precautions training has reduced percutaneous sharps injuries is questioned. Prevention programs directed to specific problem areas are required to further reduce injury. Our purpose was to identify target areas. METHODS Device-specific sharps injury rates per 100,000 devices purchased were determined by department at Yale New Haven Hospital (1993 to 1994). Usage per full-time equivalent was calculated by department. Rates were modelled using Poisson regression. RESULTS Three epidemiologic patterns resulted: (1) injury rates were independent of usage (butterfly needles); (2) injury rates varied directly with usage (lancets); (3) injury rates varied inversely with usage (intravenous catheters, sutures, and scalpels). Device-specific usage and injury rates varied by department. Devices used little (9/full-time equivalent) but under difficult circumstances, such as intravenous catheters in pediatric patients, were associated with high injury rates (67.7/100,000). Devices, sometimes disassembled, such as blood collecting tubes, caused significantly more injury in departments where health care professionals work under time constraints, such as in the emergency department and nursing. Unconventional use of devices (Luer-Lok syringes and scalpels) resulted in higher rates of injury (nursing and laboratories). Building services appeared to be at risk for injury. CONCLUSIONS With device-specific injury and usage rates by department, injury prevention programs can now focus on specific devices and departments.

Host cell receptors for two strains of sindbis virus

SummaryExperiments were performed to determine whether neuronal cells have different numbers of receptors for a neurovirulent and an avirulent strain of the same virus and whether neurotropic strains of different viruses share the same cellular receptors. Attempts were also made to characterize the receptors for the two strains of Sindbis virus on viable cells by studying their enzyme sensitivity.The number of cellular receptors available for Sindbis virus attachment to several cell lines was determined by saturation studies using two virus strains differing in their pathogenicity for adult mice. Cultured neuronal cells had 1.3×106 receptors for a neurovirulent strain (SaAr86) and only 5×104 for the avirulent prototype strain (EgAr339) of Sindbis virus. A refractory Lepidopteran cell type possessed 1×105 surface receptors for the neurovirulent variant while the average number of receptors on five permissive cell types was 1.5×106. Cellular receptors for the two strains of Sindbis virus on rat glioma cells were found to be distinct.The cellular receptors for EgAr339 on viable mammalian cells were sensitive to proteolytic cleavage, while those on living mosquito cells were insensitive to proteases, phospholipases and neuraminidase. The receptors for the neurovirulent variant on three mammalian cell types were less sensitive to enzymatic inactivation than those for the avirulent counterpart. After cleavage, the receptors for EgAr339 reappeared rapidly at 37° and 4° C, apparently in the absence of cellular synthesis.

Isolation of Field Rabies Virus Strains in CER and Murine Neuroblastoma Cell Cultures

Infection of CER and murine neuroblastoma (clone N18) cell cultures by inoculation of brain tissue from rabid skunks, dogs, equines, foxes, bats and cows was detected by immunofluorescence 2--5 days after inoculation.

A new isolate of Lagos bat virus from the Republic of South Africa

Abstract In 1980, a virus isolated from a bat in Natal, Republic of South Africa, was initially identified in the fluorescent antibody test as rabies virus. We have now shown this isolate to be Lagos bat virus. The possible epizootiological significance of this finding is discussed.