Sylvia G. Whitfield

Epidemiology of Human Rabies in the United States, 1980 to 1996

One of the oldest recognized zoonotic diseases, rabies continues to plague humankind and causes more than 35 000 deaths annually [1]. These potentially preventable deaths occur primarily in Asia, Africa, and Latin America, where animal control, vaccination programs, and effective human postexposure prophylaxis are not widely available. In contrast, in the United States, deaths in humans caused by rabies totaled 99 in the 1950s, 15 in the 1960s, 23 in the 1970s, 10 in the 1980s, and 22 from 1990 through 1996 [2, 3]. The epidemiology of human rabies is ultimately linked to cycles of rabies virus transmission in animals. With the interruption of dog-to-dog transmission in most regions, the incidence of human rabies in the United States has reached a level that cannot be further reduced without targeting wildlife. An understanding of epidemiologic patterns of rabies virus maintenance in natural populations has emerged in the past 20 years, largely because of advances in immunology and molecular biology. Monoclonal antibody and genetic sequence analyses of rabies virus variants permit detailed descriptions of enzootic maintenance cycles of specific virus variants in the United States [4, 5]. These analyses have led to an understanding of how variants of rabies virus are maintained in natural reservoirs within geographic regions and have provided information on variability of the virus itself. Current epidemiologic patterns of rabies in the United States can be summarized as follows: The annual reports of rabies in wildlife exceed those of rabies in domestic animals [6]; rabies variants in bats are associated with a disproportionate number of infections in humans, although bats constitute only about 10% of all reported rabies cases in animals annually; most other cases of human rabies diagnosed in the United States can be attributed to infections acquired in areas of enzootic canine rabies outside of the United States; most persons with a case of rabies that originated in the United States have no history of an animal bite; and rabies is diagnosed after death in more than one third of the latter group. The last published summary of cases of human rabies in the United States covered the period from 1960 to 1979 [3]. This review discusses the clinical and epidemiologic features of cases of human rabies in the United States from 1980 to 1996. Methods Case Definition This report includes all laboratory-confirmed cases of human rabies in the United States or its territories from 1980 to 1996 [7-31]. All of the cases were reported to the Centers for Disease Control and Prevention (CDC) by health authorities as part of ongoing national surveillance. Variable Definitions Onset of illness was defined as either the first day of reported symptoms attributable to rabies or the date of initial presentation for medical care before confirmation of rabies. Clinical signs attributable to rabies included paresthesia, anxiety, agitation, confusion, disorientation, hydrophobia, aerophobia, hypersalivation, dysphagia, paresis, paralysis, and fluctuating levels of consciousness [32, 33]. The type of transmitting animal and the geographic location of exposure were listed if the case history included a definite animal bite. The reliability of information that linked rabies exposure to a human was assessed by subsequent laboratory typing of the rabies virus variant. All other exposures were defined as unknown. The diagnosis of rabies was considered antemortem if it was tentatively made and samples were obtained specifically for rabies testing before the patients death. Laboratory Tests The diagnosis of rabies was confirmed by using standard tests [34] conducted at the CDC or at a state laboratory. Serology Two tests were used to detect rabies antibody: the rapid fluorescent focus inhibition test and the indirect immunofluorescence assay. The rapid fluorescent focus inhibition test measures neutralizing antibody. An antibody titer of 1:5 or more, as defined by the reciprocal of the serum or cerebrospinal fluid dilution that reduces the challenge virus by 50%, was considered positive. An indirect immunofluorescence assay, using patient serum or cerebrospinal fluid diluted 1:4 or more, detects serum reactive with rabies antigen in infected cell cultures. The presence of antibody in serum was considered diagnostic if no vaccine or antirabies serum was given to the patient. Antibody in the cerebrospinal fluid, regardless of the rabies immunization history, was considered indicative of rabies virus infection. Virus Isolation Suspensions of brain or saliva specimens were added to mouse neuroblastoma cells and cultured for 24 and 48 hours. Culture slides were fixed and examined by direct immunofluorescence assay for antigen. Samples that were initially negative were maintained for an additional 3 to 4 days and retested. The negative result was considered definitive if it occurred both times. Antigen Detection Antigen detection was performed by direct immunofluorescence of assay serial frozen sections of nuchal skin biopsy specimens, touch impressions of corneal epithelial cells, or fresh brain matter. Paraffin-embedded fixed brain matter was sectioned and enzyme-digested before direct immunofluorescence. RNA Detection Standard extraction procedures and reagents were used to obtain nucleic acids from samples of undiluted saliva; from fresh or paraffin-embedded fixed samples of the brain; or, occasionally, from other tissues. Reverse transcription of RNA and complementary DNA amplification were performed by polymerase chain reaction (PCR) with primers derived from the sequence of the N protein gene. The nucleotide sequence of all PCR products was obtained by standard dideoxynucleotide sequencing methods. Rabies virus variants were identified by comparing samples of rabies virus obtained from all known reservoirs for rabies in the United States [5] with samples of rabies virus obtained from dogs in Asia, Africa, and Latin America [35]. Statistical Analysis Data analyses were performed by using EPI INFO 6 (Centers for Disease Control and Prevention, Atlanta, Georgia) or SPSS 6.0 for Windows (SPSS Inc., Chicago, Illinois) [36, 37]. Specific tests are identified in the text. Some variables were dichotomized before statistical comparisons for determination of odds ratios and 95% CIs. All reported P values are for two-tailed tests of significance. Results Demographic Information Thirty-two persons died of rabies in the United States from 1980 through 1996. Patients ranged in age from 4 to 82 years (median, 27 years) and 20 (63%) were male (Table 1). Cases were reported from 20 states; 7 cases (22%) were reported in California and 6 in Texas. Eleven patients were exposed to rabies in eight foreign countries on the basis of variant typing. The onset of illness occurred in all months and had no apparent seasonal pattern. Dates of exposure, based on the history of an animal bite, were obtained for 7 patients (22%) (Table 1). Table 1. Human Rabies in the United States, 1980-1996 Exposure History A definite history of animal exposure was identified in 7 of the 32 patients (22%), and 25 remained unknown or indefinite (Table 1). Of the 7 cases of definite exposure, 6 resulted from a dog bite received in a foreign country and 1 was from a bat bite received in the United States. Although rabies was not diagnosed in any of the animals that inflicted bites, in each case the rabies virus variant identified in the human sample was consistent with that in the animal species implicated as the source of infection (Table 1). Contact with an animal, thereby suggesting the source for infection, was identified in 12 persons (8 with a bat, 2 with a dog, 1 with a cow, and 1 with a cat). This human-animal contact, however, could not be linked to a bite or mucous membrane contact with the saliva of an animal potentially infected with rabies virus. The remaining 13 patients did not report animal contact; thus, a potential source of exposure was not identified. Histories were obtained before death from friends or relatives in 9 cases and from 4 children aged 11 to 13 years. Prophylaxis None of the 32 patients received a complete series of rabies prophylaxis after exposure; patient 7 reported receiving a single injection of an unknown type after a dog bite in Guatemala, and patients 15, 29, and 30 received human rabies immune globulin during the course of their clinical illness. Clinical Presentation For the 7 patients in which a definite animal bite occurred, the median incubation period was 85 days (range, 53 to 150 days). The first signs and symptoms of rabies were often nonspecific, including fever, sore throat, chills, malaise, anorexia, headache, nausea, vomiting, dyspnea, cough, and weakness. Specific symptoms, such as paresthesias at or near the presumed exposure site, were also reported early in the clinical course, and 19 of the 32 patients (59%) had three or more clinical findings suggestive of rabies during the course of their illness (Table 2). The 32 patients were seen by physicians on an outpatient basis a median of one time (range, 0 to 5 times) before hospitalization, and the median length of time from the onset of illness attributable to rabies to hospitalization was 4 days (range, 1 to 10 days). Table 2. Clinical Findings Suggestive of Rabies in 32 Patients* On admission, 21 of the 32 patients (66%) were febrile (oral temperature > 37.8C), including 12 patients with temperatures greater than 39.5C. Of the 11 patients who were afebrile on admission, 5 reported being febrile before admission, 2 became febrile within 2 days of admission, and 4 had no additional temperatures recorded. The antemortem diagnosis of rabies was first considered at the time of hospitalization in 5 patients, within 1 day of hospitalization in 5 patients, and after a median of 6 days of hospitalization (range, 2 to 12 days) in 10 patients. In 12 patients, rabies was diagnosed after death. Th

Bunyaviridae: morphologic and morphogenetic similarities of Bunyamwera serologic supergroup viruses and several other arthropod-borne viruses.

Thin section electron microscopic examination of several viruses of the Bunyamwera serologic supergroup of arbo viruses confirmed their precise similarities in morphology and morphogenesis and their differences from viruses of other groups. Several viruses that are serologically unrelated to the supergroup were indistinguishable from Bunyamwera virus when observed in the same way. A separate taxonomic group or ‘family’, ultimately to be based upon multiple common physicochemical virion properties, is proposed to encompass the more than 130 viruses listed; the name Bunyaviridae is proposed for this taxon.

Agent of Disease Contracted from Green Monkeys

An infectious agent obtained from patients who became ill after exposure to tissues of African green monkeys is viral in character. By electron microscopy, the agent appeared cylindrical, 90 to 100 nanometers in diameter, and 130 to 2600 nanometers in length. Cross-striations at 5-nanometer intervals and a core diameter of 45 nanometers were observed. The agent was completely resistant to the effects of the metabolic inhibitor 5-bromodeoxyuridine, which may mean that RNA is the genetic material. It was sensitive to ether and relatively sensitive to destruction by heat.

St. Louis encephalitis virus: an ultrastructural study of infection in a mosquito vector.

Abstract The course of St. Louis encephalitis virus infection of Culex pipiens pipiens Linn, mosquitoes was followed sequentially by electron microscopy. At the site of initial viral invasion of mosquito parenchyma in the midgut, epithelial infection involved a rather constant proportion of cells that yielded only moderate numbers of virus particles. Virus was observed in the midgut at locations where spread via the hemolymph could occur. Tissues in intimate contact with hemolymph (abdominal muscles, malpighian tubules, ovarian sheath) became infected, but only modest numbers of virus particles were ever produced. In sharp contrast, an ever increasing number of of virus particles were formed in the epithelial cells of the salivary glands. Virus was primarily yielded into the cisternae of endoplasmic reticulum and then shed from the apical end of cells into the lumen of the glands. Very few particles were associated with lateral or basal margins of salivary gland epithelium, indicating a directional “preference” of virus for shedding through apical plasma membrane. So much virus was shed into the limited space of the glandular lumen and its diverticula that dispersed particles formed into crystalline arrays from day 25 onward; one of the larger of these crystals was estimated to contain more than 50,000 virus particles. Changes in the infected cells of all the mosquito organs examined were interpreted as physiologic variances relative to feeding time and not as specifically due to viral cytopathology.

Protein composition of coronavirus OC 43

Abstract A human coronavirus, strain OC 43, was propagated in suckling mouse brain and purified 5000-fold with, a 90% yield. Purity of the virus was confirmed by electrophoretic, ultracentrifugal, and electron microscopic procedures. Immunodiffusion and immunoelectrophoresis tests revealed one precipitin line with normal mouse brain, three with purified virus, and four with crude virus when tested against anti-pure virus or anti-crude virus animal serums. The association of a host cell antigen with the virion was confirmed by standard HI and CF tests. Polyacrylamide gel electrophoresis of solubilized purified virus revealed a minimum of six polypeptides with apparent molecular weights of 191,000 (No. 1), 104,000 (No. 2), 60,000 (No. 3), 47,000 (No. 4), 30,000 (No. 5), and 15,000 daltons (No. 6). A seventh band was occasionally found in the 165,000-dalton region of the gels. Four polypeptides contained carbohydrate and one contained lipid. Polypeptide No. 5 comprised 26% of the total viral protein and glycopolypeptide No. 3 comprised 23%. Three other components accounted for most of the remaining protein: polypeptide No. 4 (16%), glycopolypeptide No. 6 (14%), and glycolipopolypeptide No. 1 (13%). Glycopolypeptide No. 2 was 8% of the total protein. Bromelin digestion of the viral projections (spikes) removed glycopolypeptides No. 2 and No. 6. Association of the remaining polypeptides with structural components of the virion is only tentatively postulated. The buoyant density in potassium tartrate of the bromelin-treated virus was 1.15 g/cm3 and of the intact OC 43 virion was 1.18 g/cm3. By analytical ultracentrifugation the corrected sedimentation coefficient (s 0 20w) of the OC 43 virion was determined to be 390 ± 16 S, and the apparent molecular weight (MW a ) was calculated to be 112 ± 5 × 106 daltons.

Eastern equine encephalomyelitis virus: an electron microscopic study of Aedes triseriatus (Say) salivary gland infection.

Abstract Aedes triseriatus (Say) mosquitoes were infected with eastern equine encephalomyelitis virus from a blood meal on viremic chicks. Individual mosquitoes that transmitted virus by bite were dissected at intervals for light and thin section electron microscopy of salivary glands. Viral nucleocapsids were found in the cytoplasm of salivary gland epithelial cells from day 13 onward. Nucleocapsids envelopment occurred during passage through membranes of the acinar cells. Subsequently virus particles accumulated within cisternae of cytoplasmic organelles and in extracellular spaces. After 21 days of incubation, the endoplasmic reticulum of many cells was distended by masses of trapped virus particles. Budding from apical plasma membranes of acinar cells deposited virus directly into secretory products within gland lumina; the striking number of particles in luminal saliva was consistent with the transmission efficiency of arthropods in nature. Viral morphogenesis in mosquito salivary glands was similar in character to that in mammalian tissues and cell cultures, although tubular structures and accumulations of nucleocapsids were not found. No cytopathic effect in virus-infected salivary gland cells was observed during the 31 days of the experiment.

Procedures for reproducible detection of rabies virus antigen mRNA and genome in situ in formalin-fixed tissues

Procedures allowing the reproducible in situ detection of rabies virus antigen and RNAs (both genome and message) in formalin-fixed tissue are described. These procedures can be used on sequential tissue sections and thereby permit comparison of results from tests detecting both antigen and RNA in the same tissue. This antigen-detecting procedure has also been used to identify both the phylogenetically distant rabies viruses from silver-haired bat and vampire bat and the rabies-related viruses Mokola, Duvenhage, and Lagos bat. One of the critical steps in these procedures is the digestion (and the resulting exposure of the target molecules) with proteinase K. These methods may be useful for the identification of other viruses of public health importance. Because in many situations only formalin-fixed tissue is available for postmortem diagnosis, the technical ability to identify a virus antigen and nucleic acid in such tissues greatly extends potential diagnostic capabilities.

Bovine ephemeral fever virus in cell culture and mice

Light, immunofluorescent and electron microscopic observations were carried out sequentially on mice and VERO cell cultures infected with bovine ephemeral fever (BEF) virus. In early harvests from cell culture, 185×73 nm cone-shaped particles with nearly parallel sides predominated; these particles had all other features typical of the Rhabdoviruses (surface projections, envelope, axial channel, precisely coiled helical nucleocapsid with 35 cross-striations at a 4.8 nm interval). Identical particles were the most frequent form in mouse brain. Variation in shape toward more broadly based cone-shapes occurred late in infection and reflected anomalous morphogenesis or particle breakdown. T (truncated) particles were common at all stages of infection. It was concluded that the reported variation in length and shape of particles in various BEF virus isolates is not necessarily releated to “strain variation” but more likely to varying growth rates and T particle interference associated with varying degrees of adaptation to a host system. BEF viral morphogenesis took place primarily upon plasma membranes in vivo and in cell culture in association with small accumulations of intracytoplasmic matrix. Fusion of viral envelopes was observed, and an early syncytium formation occurred in infected cell cultures. Cytopathology was similarin vivo and in cell culture; a protracted stage of cell rounding was followed by extreme cytoplasmic vacuolation and condensation and then by lysis. Necrotic encephalomyelitis was severe in moribund mice but extraneural sites of viral propagation and damage were not found with any of the techniques employed. Since this strict neurotropism is most likelynot characteristic of naturally acquired BEF virus infection in nature, it was concluded that the mouse was an unsatisfactory host for the further study of pathogenesis.

California group arboviruses: Electron microscopic studies

Abstract Electron microscopic examination of mouse brains infected with five California group arboviruses revealed similar changes consisting of perineuronal edema formation, minimal inflammatory cell infiltration and neuronal degeneration. Virus particles were observed in the cytoplasm of neurons in association with organelle membranes; particles were round or oval, with a moderately electron dense core surrounded by a ragged halo. Mean virus diameter was 98 mμ. Virus particles also accumulated in extracellular spaces. Similar virus morphology and cellular degeneration were observed in cell cultures infected with one of the viruses.

A comparative study of the fluorescent antibody test for rabies diagnosis in fresh and formalin-fixed brain tissue specimens

Many diagnostic methods have been used to detect rabies virus antigen. The preferred method for routine diagnosis of rabies in fresh or frozen brain tissues is the fluorescent antibody test (FAT). In this study, the FAT was used to evaluate the rabies status of fresh/frozen brain specimens from more than 800 rabies-suspected cases, in more than 14 different species of animals. A comparable brain specimen from each case was fixed in 10% buffered formalin and examined by the FAT. The evaluation of rabies status between fresh and formalin-fixed tissues was in agreement in more than 99.8% of the cases. When fresh tissue is not available for testing, these results validate the use of this procedure for routine diagnosis of rabies in formalin-fixed brain tissues.