Hantavirus pulmonary syndrome: the first 100 US cases.

In the spring of 1993, hantavirus pulmonary syndrome (HPS) "emerged" in the southwestern United States, where a multiagency investigation led to the rapid description of this new clinical entity and its etiology. Analysis of the first 100 US cases identified showed that the disease was distributed in 21 states, had gone unrecognized since at least 1959, and had a distinct spring-early summer seasonality. Of the infected persons, 54% were male; 63% were Caucasian, 35% were Native American, and 2% were African American. The average age of case-patients was 34.9 years, and 8 were children or adolescents aged < or = 16 years. The overall case-fatality rate was 52%. There was a 91% concordance among serologic, immunohistochemical, and molecular results. HPS in the United States is caused by at least three newly described pathogenic hantaviruses, each of which has a distinct rodent host, and cases of HPS have been recently recognized in Canada and South America. National surveillance of this sporadic disease remains essential for further defining the epidemiology and clinical spectrum.

Improved specificity of testing methods for filovirus antibodies.

An epizootic among monkeys imported into the United States created an immediate need for detection of antibodies to filoviruses. Thousands of samples were submitted to the Centers for Disease Control and Prevention for testing. Problems of sensitivity and specificity existed in the methods available for these assays. The experiments described in this report resulted in improved methods for the detection of antibodies to filoviruses, both for indirect fluorescent antibody assays (IFA) by standardizing methods and the Western blot (WB) by minimizing antigen load and by incorporating skim milk in diluents.

Protection of guinea pigs from Lassa fever by vaccinia virus recombinants expressing the nucleoprotein or the envelope glycoproteins of Lassa virus.

A recombinant vaccinia virus that expresses the nucleoprotein gene of Lassa virus (Josiah strain) under the control of the P7.5 promoter was constructed using the lacZ coexpression transfer vector pSC11. Southern blot analysis demonstrated that recombination of the sequences inserted within the thymidine kinase gene of the transfer vector into the HindIII J fragment of vaccina virus genomic DNA occurred properly. A 63-kDa protein identical in electrophoretic mobility to authentic Lassa nucleoprotein was observed in recombinant virus-infected cell lysates. The reactivity of vaccinia-expressed Lassa proteins to a panel of monoclonal antibodies representing multiple epitopes on each of the N, G1, and G2 proteins was determined by indirect immunofluorescence. Lassa proteins expressed in recombinant vaccinia virus-infected cells reacted in a manner indistinguishable from that of the proteins expressed in Lassa virus-infected cells, indicating that there are no significant differences between authentic and recombinant virus-expressed proteins. Vaccine efficacy trials in guinea pigs indicated that both the nucleoprotein and the envelope glycoproteins are capable of eliciting a protective immune response against a lethal dose of Lassa virus. Ninety-four percent of the animals vaccinated with V-LSN, 79% vaccinated with V-LSGPC, and 58% vaccinated with both recombinant viruses survived a Lassa virus challenge in which only 14% of unvaccinated animals and 39% of animals vaccinated with the New York Board of Health (NYBH) strain of vaccinia virus survived. The protection resulting from vaccination with the recombinant virus vaccines did not correlate with the levels of prechallenge serum antibodies, suggesting that a cell-mediated immune response is a critical component of protective immunity to Lassa fever.

Construction of a recombinant vaccinia virus expressing the Lassa virus glycoprotein gene and protection of guinea pigs from a lethal Lassa virus infection.

A cloned cDNA (1.65 kb) containing the complete glycoprotein gene of the Josiah strain of Lassa virus was inserted into the thymidine kinase (TK) gene of the New York Board of Health (WYETH) strain of vaccinia virus. The Lassa virus glycoprotein precursor, GPC, and the posttranslational cleavage products, G1 and G2, were shown by Western blot analysis to be properly expressed in cells infected with the recombinant virus. Northern blot hybridization of total cytoplasmic RNA extracted from recombinant virus infected cells demonstrated the presence of RNA transcripts of appropriate size considering the site of transcription initiation from the vaccinia P7.5 promoter, the size of the Lassa glycoprotein gene, and the presumed location of the transcription terminator in the vaccinia thymidine kinase gene. All guinea pigs vaccinated with the recombinant virus survived a lethal challenge infection with Lassa virus, whereas 80% of control animals died. The vaccinated guinea pigs did, however, develop transient, low-grade, fevers and detectable viremias following infection with Lassa virus, indicating that protection was not complete.

Antibody to hemorrhagic fever with renal syndrome viruses (Hantaviruses) in the United States.

Hantaviruses similar to those which cause hemorrhagic fever with renal syndrome have been isolated from rodents in the United States. Serologic evidence suggests that these viruses infect humans. Clinical disease has not, however, been associated with infection in the United States. To expand knowledge of the distribution of Hantaviruses in the United States and attempt to identify a clinical syndrome associated with infection, a serologic survey was undertaken of US Forestry Service personnel and US Geological Survey personnel in Mississippi, Virginia, and Alaska. In addition, sera from persons with unidentified illnesses were collected from state public health laboratories in Washington state and Virginia. One of 85 sera (1.2%) from Forestry Service personnel in Mississippi and one of 79 sera (1.3%) from Forestry Service personnel in Virginia, and nine of 360 sera (2.5%) from Forestry Service and Geological Service personnel in Alaska were tested by immunofluorescent assay and were found to have antibody to Hantaan, Tchoupitoulas, or Prospect Hill viruses, ranging in titer from 1:32 to 1:512. Those persons questioned revealed no renal disease, hemorrhagic phenomenon, or unidentified febrile illnesses. Sera from two persons in Virginia, collected at the time of an illness, had antibody titers of 1:32 and 1:64, respectively, to Prospect Hill virus. An etiologic role for Prospect Hill virus could not be confirmed. Current information would suggest that Hantaviruses do not present a public health problem in the United States.

Serological and virological evidence of a Hantaan virus-related enzootic in the United States.

The first isolation of a Hantaan-related virus from a feral rat in the United States was made from a Rattus norvegicus caught in New Orleans. The strain, designated Tchoupitoulas virus, is antigenically related to, but distinct from, the prototype strain 76-118 of Hantaan virus and is the first Hantaan-like virus isolated from the pancreas of a naturally infected animal. Serosurveys of wild rodents from urban and rural areas in the United States indicated that Hantaan-related viruses infected urban rats in coastal and inland cities and infected five species of New World rodents in the western United States (Peromyscus maniculatus, Peromyscus difficilis, Peromyscus californicus, Neotoma mexicana, and Neotoma cinerea). Serosurveys disclosed no evidence of Hantaan-virus infection in rats in large-scale breeding colonies.

Immunofluorescence studies of disseminated Hantaan virus infection of suckling mice.

Hantaan virus, the etiological agent of Korean hemorrhagic fever, was inoculated intracerebrally or intraperitoneally into suckling mice, and the course of the infection was followed by infectivity titration and immunofluorescence studies. Mice became ill and were moribund by 13 to 14 days postinfection. In mice inoculated either intracerebrally or intraperitoneally, virus antigen was present in brain, heart, lungs, liver, and kidney. Less consistently, specific fluorescence was observed in spleen, pituitary gland, thymus, lymph nodes, adrenal, pancreas, salivary glands, trigeminal ganglia, adipose tissue, intestine, and muscle. In all of these tissues, the primary target of infection was the capillary endothelium. In mice inoculated intracerebrally, virus antigen was present mainly in choroid plexus, hippocampal nuclei, and meninges, but in mice inoculated intraperitoneally, central nervous system infection was marked by antigen accumulation in cortical nuclei and thalamus.

The effect of acute and chronic renal failure on theophylline clearance.

Theophylline total body clearance was measured in 29 anuric, chronic obstructive pulmonary disease patients with acute or chronic renal failure during a continuous intravenous infusion. They were divided into two groups depending on the absence (group 1, N = 16) or presence (group 2, N = 13) of congestive heart failure and compared to normal renal function control patients with similar disease states. All study and control patients smoked cigarettes. The theophylline mean total body clearance values (+/- S.D.) for group 1 were 68.0 +/- 14.5, 64.5 +/- 12.9, and 62.6 +/- 17.3 ml/kg.hr for acute renal failure patients, uremic chronic renal failure patients, and control patients, respectively. For group 2, the corresponding values were 25.6 +/- 5.1, 28.6 +/- 8.7, and 27.4 +/- 12.9 ml/kg.hr. There was no significant difference between study and control patients in either group 1 or group 2 (P greater than 0.05, one-way analysis of variance). Since total body clearance determines the steady-state concentration of a drug after repeated administration, theophylline doses do not need to be reduced in acute renal failure or uremia patients.

Multisite intradermal antirabies vaccination. Immune responses in man and protection of rabbits against death from street virus by postexposure administration of human diploid-cell-strain rabies vaccine.

Lymphocyte transformation, production of neutralising antibody, and the development of antirabies IgG antibody were studied in ten healthy volunteers in response to 0.8 ml of human diploid-cell strain (HDCS) rabies vaccine administered on one occasion in divided doses in 8 intradermal (i.d.) sites. All ten volunteers rapidly developed substantial titres of rabies antibody, and eight of the ten had T lymphocytes that were immunologically stimulated by HDCS rabies-virus antigen. Postexposure treatment with 0.8 ml of HDCS vaccine given at 4 i.d. sites completely protected fourteen rabbits from death by street virus. The results suggest that in developing countries patients could be protected with small volumes of potent tissue-culture vaccine administered intradermally shortly after exposure.

Interference induced in GL-V3 monkey kidney cells by rabies virus strains.

Resistance to superinfection with vesicular stomatitis virus (VSV) occurred in GL-V3 monkey kidney cells infected with the CVS-11, Pitman Moore, LEP Flury, but not the ERA strain of rabies virus. Specific immunofluorescent staining of intracellular rabies antigen showed that the number and size of fluorescent foci increased after the onset of interference, and that this was paralleled by increasing yields of infectious virus. Although CVS-11 and ERA differed in their ability to induce interference, the virus yields from monolayers infected with either strain were similar. Interference apparently had no effect on the replication or dissemination of the inducing virus, and seems unrelated to the long incubation period or aberrant forms of infection in vivo.

Enteric inoculation with ERA rabies virus: evaluation of a candidate wildlife vaccine in laboratory rodents.

A series of experiments on the safety and efficacy of enteric vaccination was carried out in laboratory rodents using the ERA strain of rabies virus both live and inactivated. In the first and second experiments in white Norway rats, several methods of inoculation were compared for the development of rabies neutralizing antibody. In later experiments, the potential for disease transmission through salivary excretion of ERA strain, or through scavenging, or cannibalism were evaluated. Enteric inoculation of rats with high doses of live ERA virus often failed to give an antibody response yet would occasionally kill adult animals. Rabies antigen was demonstrable in the trigeminal ganglion and tongue of one of these rats, and 14 of 65 (21.5 percent) adult mice died from rabies after eating infants infected with the ERA strain.

Everglades virus infection in man, 1975.

Within 72 hours of visiting the Everglades National Park in south Florida, a 43-year-old man became ill with fever, malaise, myalgia, severe headache, pharyngitis, and enlarged, tender lymph nodes. Everglades (Venezuelan equine encephalomyelitis, subtype II) virus was isolated from a blood sample drawn from the patient five days after onset of symptoms.

Experimental chronic rabies in the cat.

Two cats, inoculated with a street rabies virus strain, survived with only some progressive debility and atrophy of musculature in the injected limb for 136 weeks. They had continuously increasing titers of neutralizing antibody in serum and in cerebrospinal fluid, and terminally they had high antibody titers in the brain. Virus was isolated from two brain specimens of one cat obtained at necropsy; isolation was successful only by explant culture and inoculation of explanted tissue into mice. Virus antigen was detected in eight sites in the brain and spinal cord of the same cat by frozen-section immunofluorescence. Lesions in the central nervous system consisted of neuronal degeneration and neuronophagia, associated with the prescence of inclusion bodies and widespread inflammatory cell inflitration into brain and spinal cord parenchyma, perineuronal sites, and perivascular spaces. The inflitrates contained lymphocytes, monocytes-macrophages, and a high proportion of plasma cells. These experimental cases of chronic progressive rabies resembled more closely subacute sclerosing panecephalitis of man than the usual subacute fatal rabies encephalitis of man and other mammalian species.

Review of gentamicin therapy based on pharmacokinetics.

The feasibility of using pharmacokinetic estimations of drug serum levels to evaluate gentamicin use was explored. A consecutive series of 40 adult patients who received nonprophylactic "piggyback" gentamicin therapy was retrospectively evaluated. Traditional factors in the evaluation of antibiotic use, such as identity of microbe and sensitivity to antibiotic, were monitored. Additionally, peak and trough drug serum levels were computed for each patient. These were compared with toxic serum levels and minimum inhibitory concentrations as reported in the literature. Certain factors, such as duration of therapy and existence of alternate antibiotic therapy, suggested less than optimal therapy in the study patients. However, computed serum levels revealed that the patients experienced both safe and effective peak and trough gentamicin levels. Only with the use of pharmacokinetic estimations of drug serum levels could a comprehensive conclusion be reached regarding the appropriateness of therapy. Gentamicin therapy was generally rational in the study group. The use of pharmacokinetic principles in the calculation of serum levels may be a useful tool in drug use studies.

Relationship of two arthropod-borne rhabdoviruses (kotonkan and Obodhiang) to the rabies serogroup.

Indirect immunofluorescence confirmed the antigenic relationship between kotankan and Obodhiang viruses and Mokola virus that had originally been shown by complement fixation test. This relationship suggests inclusion of these two arthropod isolates in the rabies subgroup of the Rhabdoviridae family. Cross-reactivity with Mokola virus was also demonstrated by direct immunofluorescence but was easily eliminated when conjugates were diluted. No crossreactivities were found by neutralization tests or by surface immunofluorescence. Other than these immunological ties to the rabies serogroup, other biological characteristics of kotonkan and Obodhiang viruses were distinct. Maximum yield of infectivity of kotonkan and Obodhiang in cell culture was at 30 C, antigen usually filled the cytoplasm of infected cells diffusely, and syncytia were formed before severe cytonecrosis. By electron microscopy, virus particles and their nucleocapsids appeared cone shaped (mean lengths: kotonkan, 182 nm; Obodhiang, 170 nm). Viral morphogenesis took place on plasma membranes of cells in culture, mouse brain neurons, and inflammatory cells (macrophages) in brain lesions. All of these characteristics of the two viruses, and the known association of kotonkan virus with an acute, febrile illness of cattle in Nigeria, suggest a biological relationship with bovine ephemeral fever virus. The latter is known to exist in the same geographic area but exhibits no serological cross-reaction with either kotonkan or Obodhiang virus. The question of whether these two viruses deserve placement in an expanded rabies subgroup (at the cost of a less precise definition of the subgroup) or in a separate subgroup (which would include bovine ephemeral fever virus) of the Rhabdoviridae family will only be answered by further physicochemical characterization and comparison.