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The surviving members of 27 households in which someone had been infected with Ebola virus were interviewed in order to define the modes of transmission of Ebola hemorrhagic fever (EHF). Of 173 household contacts of the primary cases, 28 (16%) developed EHF. All secondary cases had direct physical contact with the ill person (rate ratio [RR], undefined; P.001), and among those with direct contact, exposure to body fluids conferred additional risk (RR, 3.6; 95% confidence interval [CI], 1.9-6.8). After adjusting for direct contact and exposure to body fluids, adult family members, those who touched the cadaver, and those who were exposed during the late hospital phase were at additional risk. None of the 78 household members who had no physical contact with the case during the clinical illness were infected (upper 95% CI, 4%). EHF is transmitted principally by direct physical contact with an ill person or their body fluids during the later stages of illness.

Laboratory diagnosis of Ebola hemorrhagic fever (EHF) is currently performed by virus isolation and serology and can be done only in a few high-containment laboratories worldwide. In 1995, during the EHF outbreak in the Democratic Republic of Congo, the possibility of using immunohistochemistry (IHC) testing of formalin-fixed postmortem skin specimens was investigated as an alternative diagnostic method for EHF. Fourteen of 19 cases of suspected EHF met the surveillance definition for EHF and were positive by IHC. IHC, serologic, and virus isolation results were concordant for all EHF and non-EHF cases. IHC and electron microscopic examination showed that endothelial cells, mononuclear phagocytes, and hepatocytes are main targets of infection, and IHC showed an association of cellular damage with viral infection. The finding of abundant viral antigens and particles in the skin of EHF patients suggests an epidemiologic role for contact transmission. IHC testing of formalin-fixed skin specimens is a safe, sensitive, and specific method for laboratory diagnosis of EHF and should be useful for EHF surveillance and prevention.

A cohort of convalescent Ebola hemorrhagic fever (EHF) patients and their household contacts (HHCs) were studied prospectively to determine if convalescent body fluids contain Ebola virus and if secondary transmission occurs during convalescence. Twenty-nine EHF convalescents and 152 HHCs were monitored for up to 21 months. Blood specimens were obtained and symptom information was collected from convalescents and their HHCs; other body fluid specimens were also obtained from convalescents. Arthralgias and myalgia were reported significantly more often by convalescents than HHCs. Evidence of Ebola virus was detected by reverse transcription-polymerase chain reaction in semen specimens up to 91 days after disease onset; however, these and all other non-blood body fluids tested negative by virus isolation. Among 81 initially antibody negative HHCs, none became antibody positive. Blood specimens of 5 HHCs not identified as EHF patients were initially antibody positive. No direct evidence of convalescent-to-HHC transmission of EHF was found, although the semen of convalescents may be infectious. The existence of initially antibody-positive HHCs suggests that mild cases of Ebola virus infection occurred and that the full extent of the EHF epidemic was probably underestimated.

In April 1996, laboratory testing of imported nonhuman primates (as mandated by quarantine regulations) identified 2 cynomolgus macaques (Macaca fascicularis) infected with Ebola (subtype Reston) virus in a US-registered quarantine facility. The animals were part of a shipment of 100 nonhuman primates recently imported from the Philippines. Two additional infected animals, who were thought to be in the incubation phase, were identified among the remaining 48 animals in the affected quarantine room. The other 50 macaques, who had been held in a separate isolation room, remained asymptomatic, and none of these animals seroconverted during an extended quarantine period. Due to the rigorous routine safety precautions, the facility personnel had no unprotected exposures and remained asymptomatic, and no one seroconverted. The mandatory quarantine and laboratory testing requirements, put in place after the original Reston outbreak in 1989-1990, were effective for detecting and containing Ebola virus infection in newly imported nonhuman primates and minimizing potential human transmission.

Argentine hemorrhagic fever (AHF), caused by the arenavirus Junin, is a major public health problem among agricultural workers in Argentina. A prospective, randomized, double-blind, placebo-controlled, efficacy trial of Candid 1, a live attenuated Junin virus vaccine, was conducted over two consecutive epidemic seasons among 6500 male agricultural workers in the AHF-endemic region. Twenty-three men developed laboratory-confirmed AHF during the study; 22 received placebo and 1 received vaccine (vaccine efficacy 95%; 95% confidence interval [CI], 82%-99%). Three additional subjects in each group developed laboratory-confirmed Junin virus infection associated with mild illnesses that did not fulfill the clinical case definition for AHF, yielding a protective efficacy for prevention of any illness associated with Junin virus infection of 84% (95% CI, 60%-94%). No serious adverse events were attributed to vaccination. Candid 1, the first vaccine for the prevention of illness caused by an arenavirus, is safe and highly efficacious.

To examine safety and efficacy of a mutagen attenuated Rift Valley fever virus (RVFV) vaccine (RVF MP-12) in cattle.38 pregnant cows, 14 steers, and 10 lactating dairy cows.Pregnant cows in their third, fifth, or eighth month of gestation were vaccinated (1 ml of RVF MP-12 containing 5 log10 plaque-forming units [PFU] of virus) and were monitored daily through parturition for signs of disease, viremia, and immunologic response. Additionally, 10 vaccinated pregnant cows were challenge inoculated with virulent RVFV at post-vaccination day (PVD) 30 and were monitored daily for untoward effects. Ten unvaccinated pregnant cows also were challenge inoculated with virulent RVFV and served as challenge controls. Vaccinated lactating dairy cows were monitored for viremia and virus shedding in the milk through PVD 14. Yearling steers were vaccinated to assess their immunologic response to various doses of vaccine and were challenge inoculated with virulent RVFV at PVD 28 to assess protection.10 of 38 (26.3%) cows vaccinated during pregnancy developed transient postvaccination viremia titeror = 2.5 log10 PFU/ml of serum. All vaccinated cows delivered live, healthy calves that were RVFV seronegative at birth, but which quickly acquired colostral antibodies. Vaccinated cows and their fetuses were protected when challenge exposed with virulent RVFV at PVD 30, whereas unvaccinated pregnant cows inoculated with RVFV became febrile and viremic, and aborted. Vaccine virus was unsuccessfully sought from milk of lactating dairy cows after vaccination, suggesting that shedding of vaccine virus through milk should not be a concern. Steers, inoculated with tenfold escalating vaccine doses, beginning with 1.0 log10 PFU, were protected against virulent RVFV challenge exposure.RVF MP-12 may be safe and efficacious for use in pregnant or lactating bovids, and a minimal dose of vaccine may provide suitable protection against viremia.

To examine effects of in utero inoculation with a mutagen-attenuated Rift Valley fever virus (RVFV) vaccine (RVF MP-12) on fetal bovids and to assess the safety and efficacy of calfhood vaccination with RVF MP-12.18 pregnant Hereford and Hereford-type cows in the third or fifth month of gestation, their progeny, and 25 calves from cows immunized with RVF MP-12 during pregnancy.Bovine fetuses were inoculated, via laparotomy, with 1 ml of RVF MP-12 containing 5 log10 plaque-forming units (PFU) of virus. Blood was obtained from newborn calves prior to their ingestion of colostrum. Immune-naive calves and calves born to RVF MP-12-vaccinated dams, ranging in age from 2 to 45 days, were vaccinated with RVF MP-12, and some were later challenge exposed with 1 ml of 5.7 log10 PFU of virulent RVFV strain ZH-501. Cows were monitored for viremia and antibody responses and for hematologic and serum biochemical alterations through parturition or abortion.Surviving in utero-vaccinated calves were healthy, with no noticeable defects. Except for 1 vaccine-inoculated fetus that died on postinoculation day 21, all in utero-vaccinated fetuses had serum neutralizing antibody titeror = 1:20 at the time of delivery. All dams of in utero-vaccinated fetuses also developed neutralizing antibody titer. Calves born to cows vaccinated during gestation did not have antibody at birth, and all but 1 quickly acquired colostral antibody. Postparturient inoculation of immune-naive calves and calves with colostral antibodies resulted in no untoward effects, and all calves with detectable neutralizing antibodies were protected against virulent virus challenge exposure.Fetal death and abortion would be rare even if fetuses were exposed to RVF MP-12. The trauma and complications associated with in utero inoculation do not make this a practical method of immunization. RVF MP-12 was safe, immunogenic, and protective in calves as young as 2 days of age.

Hantavirus pulmonary syndrome is a rodent-borne zoonosis first recognized in the United States in 1993. Person-to-person transmission has not been reported; however, in the outbreak of 20 cases reported here, epidemiologic evidence strongly suggests this route of transmission.

The objective of this report is to summarize the medical literature relevant to the epidemiology, diagnosis, treatment, and control of hantavirus pulmonary syndrome (HPS). The English language literature was searched from January 1993 through March 1995, manually and using Medline, including conference proceedings and reference lists. We selected relevant articles on clinical aspects of HPS. HPS is a newly described illness that progresses through three phases: prodromal, cardiopulmonary, and convalescent. Its hallmark is the rapid progression from nonspecific prodromal symptoms to respiratory insufficiency caused by noncardiogenic pulmonary edema. Unlike other hantavirus infections, renal involvement and hemorrhagic manifestations are not dominant features in HPS. Treatment is based on close intensive care monitoring, oxygen, and cardiovascular support with inotropic and vasopressor drugs. Preventive measures include avoiding contact with rodents and their excrement. Peromyscus maniculatus is the primary rodent vector for the Sin Nombre virus, which caused the epidemic of HPS in the southwest United States in May 1993. HPS has a unique set of clinical findings and is caused by a genetically distinct hantavirus. Physicians and other health professionals should consider this disease when evaluating patients with unexplained respiratory insufficiency after a febrile prodrome, especially when history suggests possible rodent contact. Sporadic cases and possibly epidemics of HPS are likely in the future, particularly when ecological changes increase the population of infected rodent vectors in an area.

Lymphocytic choriomeningitis virus (LCMV), which is one of several arenaviruses that are pathogenic for humans, causes encephalitis and meningitis in man. In this study, single-stage and nested reverse transcription-polymerase chain reaction (RT-PCR) assays were developed that targeted the GPC and N genes of LCMV. Both assays detected1 TCID50 unit of LCMV. These assays were used to measure the incidence of LCMV infection by testing cerebrospinal fluid (CSF) samples withor = 10 leukocytes/microl collected over 1 year from patients undergoing lumbar puncture for diagnostic reasons at two Birmingham hospitals. Samples were tested for the presence of LCMV RNA by using the RT- PCR assay and for LCMV-specific IgM antibody by using an ELISA assay. None of the specimens collected from 813 patients was positive by either assay. Although no cases of acute infection were detected, 4% (11/272) of serum collected from a subset of patients was positive for LCMV-specific IgG. A significantly greater rate of seropositivity was found among subjects over 60 years of age (9.4%; P0.025) than was found in younger subjects (2.4% at 30-59 years of age; 0% at30 years of age). These data suggest that serious central nervous system disease due to LCMV infection is not common in this population. The high rate of seropositivity in those over 60 years of age suggest that infection was once more common.

This study describes the pathogenesis of the Ebola-Reston (EBO-R) subtype of Ebola virus for experimentally infected cynomolgus monkeys. The disease course of EBO-R in macaques was very similar to human disease and to experimental diseases in macaques following EBO-Zaire and EBO-Sudan infections. Cynomolgus monkeys infected with EBO-R in this experiment developed anorexia, occasional nasal discharge, and splenomegaly, petechial facial hemorrhages and severe subcutaneous hemorrhages in venipuncture sites, similar to human Ebola fever. Five of the six EBO-R infected monkeys died, 8 to 14 days after inoculation. One survived and developed high titered neutralizing antibodies specific for EBO-R. The five acutely ill monkeys shed infectious virus in various bodily secretions. Further, abundant virus was visualized in alveolar interstitial cells and free in the alveoli suggesting the potential for generating infectious aerosols. Thus, taking precautions against aerosol exposures to filovirus infected primates, including humans, seems prudent. This experiment demonstrated that EBO-R was lethal for macaques and was capable of initiating and sustaining the monkey epizootic. Further investigation of this animal model should facilitate development of effective immunization, treatment, and control strategies for Ebola hemorrhagic fever.

Most of the viral hemorrhagic fevers (VHFs) are caused by viruses that are handled in high containment laboratories in Europe and the United States because of their high pathogenicity and their aerosol infectivity. Special precautions should be taken when caring for patients infected with these viruses, but most hospitals can safely provide high-quality care. The major danger is parenteral inoculation of a staff member. Fomites and droplets must be considered as well. The role of small particle aerosols in inter-human transmission continues to be controversial. We believe that the aerosol infectivity observed for these viruses in the laboratory and the rare clinical situations that suggest aerosol spread dictate caution, but the many instances in which no transmission occurs provide a framework in which a measured approach is possible. The major challenge is in early recognition by an educated medical staff and rapid specific etiological diagnosis.

A commercially available immunoglobulin G (IgG) from horses, hyperimmunized to Ebola virus, was evaluated for its ability to protect cynomolgus monkeys against disease following i.m. inoculation with 1 000 PFU Ebola virus (Zaire '95 strain). Six monkeys were treated immediately after infection by i.m. infection of 6.0 ml IgG; these animals developed passive ELISA titers of 1:160 to 1:320 to Ebola, two days afer inoculation. However, the beneficial effects of IgG treatment were limited to a delay in onset of viremia and clinical signs, in comparison with untreated controls. The six IgG recipients had no detectable viremia day 5, in contrast with three virus infected controls whose viremias exceeded 7.0 log10 PFU/ml that day. The controls died on days 6, 6, and 7, while two IgG recipients died day 7 and the remaining 4 died day 8, all with high viremias. These results document that passively acquired antibody can have a beneficial effect in reducing the viral burden in Ebola-infected primates; however, effective treatment of human patients may require antibodies with higher specific activities and more favorable pharmacokinetic properties than the presently available equine IgG.

Plaque-reduction neutralization tests were done with eight of nine known representative hantaviruses and immune sera from experimentally infected animals and from patients with hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). Results obtained with animal sera demonstrated each virus to be antigenically unique. Neutralization with the HPS patient sera was highest with Sin Nombre (SN) virus and to a lesser extent with Black Creek Canal (BCC) virus. Sera from Korean HFRS patients reacted best with Hantaan virus, but cross-reactivity with all other viruses except Thottapalayam (TPM) virus was also observed. Sera from Swedish HFRS patients reacted best with Puumala virus but cross-reacted with Prospect Hill, SN, and BCC viruses and to a lesser extent with all of the other viruses except TPM virus.

The residual ion damage due to low‐energy ion implantation during molecular beam epitaxy growth was investigated by measuring the minority carrier lifetime in the base of a silicon bipolar transistor. The base was doped with As+ ions at 200 eV to a concentration of 1019 cm−3. Three samples were grown at temperatures of 500, 650, and 800 °C. The 500 °C sample had a minority carrier lifetime in the base ∼1/6 that of the samples grown at the higher temperatures. On annealing at 650 °C the lifetimes of all samples were essentially equal. The results indicate that at this dopant concentration the collision cascades caused by ion bombardment do not overlap.

Puumala (PUU) virus (Bunyaviridae: Hantavirus), the etiologic agent of nephropathia epidemica (NE), the mild form of hemorrhagic fever with renal syndrome, is enzootic in Europe and has been known to occur in France since 1983. We report the first isolation of PUU virus in France and western Europe from a case of NE acquired in France. The virus was isolated from a serum collected in the acute phase of the clinical course by successive blind passages in Vero E6 cells. Serologic typing using monoclonal antibodies confirmed the identity of the virus as PUU. The sequence of an 832-nucleotide fragment of the virus medium RNA segment obtained by the polymerase chain reaction (PCR) also classified it as a PUU virus. The sequence of this isolate from a human case in France is closely related to the sequence of a PUU virus obtained by the PCR from a German patient.

It has been observed that the sheet resistance of a Ti-salicided polysilicon-gate electrode or source/drain region increases significantly as the dimension reaches the lower sub-micron range. The resistance of platinum and nickel silicide (PtSi and NiSi), however, does not increase with reduced linewidth. We have studied PtSi and NiSi films with deep sub-micron linewidths on single crystal or poly-Si substrates. In this study, the material properties such as sheet resistance, grain structure and surface morphology of these silicide films in confined geometries are reviewed and compared with TiSi2. Process windows for forming and maintaining these silicides are explored.

In fatal human Lassa fever, severe hypotension, circulatory shock, and pulmonary edema develop as terminal events. We examined cardiovascular and respiratory functions in strain 13 guinea pigs infected with Pichinde virus, an animal model for studying human Lassa fever. Cardiovascular functions were studied in anesthetized and conscious guinea pigs, whereas pulmonary functions were measured only on animals under anesthesia. In anesthetized animals, cardiovascular disturbances were severe and progressive from postinoculation day (PID) 10. Cardiac output, measured by thermodilution, decreased 28 to 53% below baseline values from PID 10 to 12 and was accompanied by a gradual reduction of mean arterial blood pressure and heart rate. Although left ventricular systolic pressure decreased significantly, the left ventricular +dp/dtmax and -dp/dtmax decreased only slightly on PID 12. Similar depressed cardiovascular responses were observed in conscious animals infected with Pichinde virus. Changes included decreased cardiac output, heart rate, cardiac work, cardiac power, and stroke volume, as well as increased total peripheral resistance and prolonged mean transit time. We postulate that a global cardiovascular dysfunction with the involvement of right and left sides of the heart may be the main cause of irreversible circulatory deterioration and death during Pichinde virus infection in strain 13 guinea pigs.

We report the results of indirect fluorescent antibody screening for antibody to Junin virus in 1,101 sera from small mammals captured on two mark-recapture grids in the epidemic area of Argentine hemorrhagic fever. Twenty-six of 29 seropositive animals were the cricetid rodent Calomys musculinus, for a 30-month prevalence of 7.9% in that species. Combining these data with previously published data on antigen detection provided an estimated total prevalence of infection of 10.9% for this, the principal reservoir species. Other infected species included two cricetids, C. laucha and Bolomys obscurus, and a predatory carnivore, Galictis cuja. Approximately half of infected animals simultaneously carried serum antibody and antigen in blood and saliva, some for 29-61 days. Except for C. laucha, which was associated with crop habitats, seropositive animals were strongly associated with the relatively rare roadside and fence-line habitats. Seropositive C. musculinus were predominantly males in the oldest age and heaviest body mass classes, and seropositive males were twice as likely to have body scars as seronegative males. These observations suggest that most infections were acquired through horizontal transmission and that aggressive encounters among adult, male C. musculinus in relatively densely populated roadside and fence-line habitats are an important mechanism of transmission of Junin virus within reservoir populations.

Summary After travelling in subSaharan Africa, an area known for sporadic cases of Marburg virus infection, a young Swedish man presented with a classical picture of severe viral haemorrhagic fever complicated by disseminated intravascular coagulation and septicaemia. Serum samples examined by electron microscopy revealed particles of a size compatible with filovirions. Indirect fluorescent antibody tests indicated transient seroconversion to Marburg virus. In lymphocyte transformation assays of cells isolated from the patient 11 months after the onset of acute disease, Marburg viral antigen was able to stimulate lymphocyte proliferation 3.9-fold; however, exhaustive attempts to isolate virus from acute phase blood cultured in vitro or in vivo from guinea pigs and monkeys failed. Data suggest that this patient may have been infected with a filovirus. This case demonstrates the difficulties that may occur in laboratory diagnosis of viral haemorrhagic fevers.

In May 1993 an outbreak of severe respiratory illness occurred in the southwestern United States. A previously unknown hantavirus was identified as the cause. In Asia hantaviruses are associated with hemorrhagic fever and renal disease. They have not been known as a cause of human disease in North America.We analyzed clinical, laboratory, and autopsy data on the first 17 persons with confirmed infection from this newly recognized strain of hantavirus.The mean age of the patients was 32.2 years (range, 13 to 64); 61 percent were women, 72 percent were Native American, 22 percent white, and 6 percent Hispanic. The most common prodromal symptoms were fever and myalgia (100 percent), cough or dyspnea (76 percent), gastrointestinal symptoms (76 percent), and headache (71 percent). The most common physical findings were tachypnea (100 percent), tachycardia (94 percent), and hypotension (50 percent). The laboratory findings included leukocytosis (median peak cell count, 26,000 per cubic millimeter), often with myeloid precursors, an increased hematocrit, thrombocytopenia (median lowest platelet count, 64,000 per cubic millimeter), prolonged prothrombin and partial-thromboplastin times, an elevated serum lactate dehydrogenase concentration, decreased serum protein concentrations, and proteinuria. Rapidly progressive acute pulmonary edema developed in 15 of the 17 patients (88 percent), and 13 patients, all of whom had profound hypotension, died (case fatality rate, 76 percent). Increases in the hematocrit and partial-thromboplastin time were predictive of death.Infection with a newly described hantavirus causes the hantavirus pulmonary syndrome, which is characterized by a brief prodromal illness followed by rapidly progressive, noncardiogenic pulmonary edema.

In supercritical fluid applications, the solute usually consists of larger, much more complex molecules than the solvent. In binary mixtures of very dissimilar components, multiphase behavior occurs near the critical point of the more volatile component, the solvent. This phase behavior undergoes further complications by the occurrence of solid phases. For applications, it is essential to know the character of the phase separation, and the location of the phase boundaries. The types of phase diagrams that may occur are described, and illustrated with many examples, drawn from experimental data obtained for the solvent class of the hydrocarbons methane through butane, and ethylene; and from the solute classes of n-alkanes, n- alkanols, n-carboxylic acids, as well as triglycerides and polyaromatic compounds.

Brain concentrations of platelet-activating factor (PAF), catecholamines, and serotonin were measured in control and Pichinde virus-infected strain 13 guinea pigs on postinoculation day (PID) 12. After virus inoculation, PAF concentrations increased 81% in cerebrum, 147% in diencephalon-brain stem, and 110% in cerebellum from baseline values of 2.6 +/- 0.3, 4.3 +/- 0.2, and 6.1 +/- 0.5 (ng/g wet tissue), respectively. Dopamine concentrations in the infected cerebrum and diencephalon-brain stem increased significantly, whereas norepinephrine concentration increased only in cerebrum. However, serotonin concentrations in all three regions of infected brain decreased significantly as compared with control values. There were no significant changes in epinephrine concentrations of infected brain. Norepinephrine and epinephrine concentrations in plasma and cerebrospinal fluid on PID 7 and 12 increased significantly as compared with control values, while plasma dopamine concentration increased significantly on PID 7. Increased brain PAF, dopamine, and and norepinephrine concentrations with decreased brain serotonin concentrations may mediate the hyperactivity of the hypothalamic-pituitary-adrenal axis and involve some unknown pathophysiologic processes of arenaviral infection. Furthermore, increased plasma catecholamine concentrations are associated with stress and may be partially responsible for the development of cardiovascular dysfunction and pulmonary edema during this viral disease.

This report provides interim recommendations for prevention and control of hantavirus infections associated with rodents in the southwestern United States. It is based on principles of rodent and infection control and contains specific recommendations for reducing rodent shelter and food sources in and around the home, recommendations for eliminating rodents inside the home and preventing them from entering the home, precautions for preventing hantavirus infection while rodent-contaminated areas are being cleaned up, prevention measures for persons who have occupational exposure to wild rodents, and precautions for campers and hikers.

Cardiovascular and respiratory responses to a 2 h intravenous constant infusion of PAF (5 and 10 ng/kg per min) were studied in strain 13 guinea pigs. PAF decreased arterial blood pressure, left systolic ventricular pressure, and cardiac output (CO). These cardiovascular changes were dose-dependent. The PAF-induced hypotension returned to a pre-infusion level spontaneously with increased total peripheral resistance despite continuous infusion of PAF. The decreased CO was most striking, and did not recover to pre-infusion levels due to depressed cardiac contractility and impaired ventricular relaxation. Respiratory responses to PAF infusion at these doses were mild and only occurred after serious cardiovascular dysfunctions developed. A higher dose of PAF (20 ng/kg per min) produced drastically decreased CO and dynamic lung compliance (Cdyn), increased pulmonary airway resistance, hypoventilation and apnea within 10-40 min. BN 52021, a PAF receptor antagonist, administered as a single i.v. dose (6 mg/kg) 15 min after PAF infusion, reversed most of cardiopulmonary dysfunctions and prevented death by increasing cardiac contractility, CO, and minute volume from extremely low values. The data suggest that marked cardiopulmonary disturbances induced by intravenous PAF infusion reflects certain pathophysiological mechanisms of diseases that may involve the cellular release of PAF. The administration of BN 52021 or other potent PAF antagonists may be beneficial under these circumstances.

Arenavirus epidemiology must be understood on three levels: (1) The most fundamental and scientifically interesting concerns the distribution of virus in rodent populations. The dominant characteristic of the known, well-characterized arenaviruses is their ability to establish chronic viremic infections in specific rodent hosts. At the population level, this complex problem depends on the interaction of virus, rodent, and ecological variables that mutually determine the abundance of infected rodents, which may in turn contaminate humans with virus. (2) The variables that bring humans in contact with these rodents and their excreta in such a way as to lead to human infection. This process depends on human ecology and habits, as well as the dynamics of infected rodents. (3) The situations in which infected humans may be responsible for secondary infections of humans.

Venezuelan equine encephalitis (VEEI-specific immunoglobulin responses to the two vaccines, TC-83 (a live attenuated vaccine) and C-84 (a formalin inactivated vaccine derived from the TC-83 strain of virus) were evaluated using an antigen and isotype-specific enzyme-linked immunoadsorbent assay (ELISA). The VEE-specific ELISA for IgG, IgG subclasses, IgA and IgM were developed and standardized using sera from vaccine- exposed and unexposed human subjects. Paired human sera (before and 28 days after immunization) were tested from laboratory workers vaccinated with either TC-83 (Group A: 20 paired sera from subjects receiving a single TC-83 vaccine and with no prior history of vaccination) or C-84 in varying schedules (Group B: 19 paired sera from subjects who had a distant vaccination history to TC-83 but no evidence of neutralizing antibody; Group C: 19 paired sera from subjects receiving their first C-84 vaccination and no prior documented history of vaccination; Group D: 15 paired sera from subjects receiving a C-84 booster vaccination with prior history of C-84 but no TC-83 exposure). Sera were all tested for viral neutralization in vitro using a Vero cell monolayer for culturing virus and establishing 80% plaque reduction for each serum tested. All pre-sera tested demonstrated no plaque reduction neutralization at a level of 80% for a dilution of 1 :10. ELSA antibody titers for all pre-sera with no prior VEE exposure through vaccination or possible environmental factors were negative at a titer of 1 :160 for IgM, 1 :80 for IgG, IgA, and G subclasses. All vaccine types and strategies generated a significant IgG response postvaccination (P < 0.0001) and this response correlated with the 80% plaque reduction neutralization titer (PRN-80) for VEE-specific IgG, GI, G3 and IgA at a P value of < 0.001 for both Group A and B. No such correlation was observed for G2 and no G4 responses to immunization were noted in any of the groups tested. There was a significant difference between geometric mean (GM) titers postvaccination for Group A or Group B versus Group C (P< 0.001) and for Group C versus Group D (P < 0.001) for IgG. Neither group C or D (1 or 2 doses of C-84 alone) demonstrated an IgA response in contrast to the TC-83 exposed groups (Groups A and B). C-84 was an effective booster vaccine in subjects previously exposed to the live attenuated vaccine and generated a significant neutralization antibody response mirrored in the IgG, G1, G3 and IgA titer increases by ELISA. © 1992 Wiley-Liss, Inc.

Platelet-activating factor (PAF) has been implicated as a cause of cardiopulmonary disturbances in certain diseases. In the present study, concentrations of PAF in hearts, lungs, whole blood, and other organs of control and Pichinde virus-infected guinea pigs on post-inoculation days (PID) 10 and 14 were measured by radioimmunoassay. Results were further confirmed by bioassay after separation and purification with thin-layer chromatography. PAF concentration in the hearts and lungs of virus-infected animals increased significantly on PID 10 and 14, as compared with control levels. PAF level in the blood of infected guinea pigs also significantly increased on PID 14. There was little change of PAF levels in liver and kidney after viral inoculation. Increased PAF concentrations in both hearts and lungs of infected strain 13 guinea pigs suggest that this lipid mediator may play an important role in the development of cardiopulmonary disturbances.

Previously, we demonstrated that naloxone, an opiate antagonist, prolonged survival of strain 13 guinea pigs infected with Pichinde virus. Thus, endogenous opiates may be involved in the pathogenesis of this viral disease. To determine whether endogenous opiate levels were affected by Pichinde viral infection, Beta-endorphin concentrations in plasma and cerebrospinal fluid (CSF) of normal and infected strain 13 guinea pigs were measured by radioimmunoassay. Cerebrospinal fluid Beta-endorphin concentrations were 78.0 +/- 13.2 pg/mi on postinoculation day (PID) 7, 59.0 +/- 5.6 pg/mi on PID 12, and 58.8 +/- 5.4 pg/mi on PID 14. These values were significantly higher than baseline levels of CSF Beta-endorphin: 30.8 +/- 1.9 pg/mi. Plasma Beta-endorphin concentrations of infected animals increased significantly to 202.1 +/- 17.9 pg/ml on PID 7 and to 154.2 +/- 21.4 pg/mi on PID 12 from a mean baseline value of 84.2 +/- 13.1 pg/mi. After a primer intravenous injection of Beta-endorphin (10, 15, or 30 jig/kg), followed by constant infusion of Beta-endorphin (15, 45, or 90 microg/kg(dot)hr) to control noninfected guinea pigs, heart rate (except with the lowest dose) and mean blood pressure decreased markedly. Under these experimental conditions, concentrations of plasma and CSF Beta-endorphin increased simultaneously with different magnitude. Because both Pichinde viral infection and Beta-endorphin administration produced a similar trend of cardiovascular disturbances, leading to hypotension and bradycardia, increased concentrations of plasma and CSF Beta-endorphin may play a partial role in the pathophysiological mechanisms of Pichinde virus infection.

Following the detection of an Ebola-like virus in cynomolgus macaques recently imported into the United States from The Philippines, studies were initiated to document transmission at export facilities located in the latter country. At one export facility, 52.8% of 161 monkeys that died over a 2.5-month period were shown to be infected with this virus using an enzyme-linked immunosorbent assay to detect antigen in liver homogenates. A case fatality rate of 82.4% was documented for the infected monkeys. The initial anti-viral antibody prevalence among the captive macaques at this facility was 25.9% (indirect fluorescent antibody titer greater than or equal to 1:16). Followup documented infection of 24.4% of initially seronegative animals and 8.7% of initially seropositive monkeys. Being held in a gang cage versus a single cage was found to be a significant risk factor for subsequent virus infection, and the presence of IFA antibody was shown to predict protection. This study documents unequivocally for the first time the presence of an Ebola-related filovirus in Asia.

Simian hemorrhagic fever (SHF) virus and a new strain of Ebola virus were isolated concurrently in recently imported cynomolgus monkeys (Macaca fascicularis) being maintained in a quarantine facility. Ebola virus had never been isolated in the U.S. previously and was presumed to be highly pathogenic for humans. A chronology of events including measures taken to address the public health concerns is presented. The clinicopathologic features of the disease were abrupt anorexia, splenomegaly, marked elevations of lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase, with less prominent elevations of blood urea nitrogen, creatinine, and other serum chemistry parameters. Histologically, fibrin deposition, hemorrhage, and necrosis of lymphoid cells and reticular mononuclear phagocytes were present in the spleens of SHF and of Ebola virus-infected animals. Intravascular fibrin thrombi and hemorrhage were also present in the renal medulla and multifocally in the gastrointestinal tract. Necrosis of lymphoid and epithelial cells was occasionally noted in the gastrointestinal tract. The histopathologic findings considered specific for Ebola virus infection include hepatocellular necrosis, necrosis of the zona glomerulosa of the adrenal cortex, and interstitial pneumonia, all of which were generally associated with the presence of 1 to 4 mu intracytoplasmic amphophilic inclusion bodies. The disease spread within rooms despite discontinuation of all direct contact with animals, and droplet or aerosol transmission was suspected. Antibody to Ebola virus developed in animal handlers but no clinical disease was noted, suggesting a less virulent strain of virus.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of our work was to determine if aerosols of Junin virus can infect rhesus macaques and if the disease is the same as that produced by virus inoculated parenterally. The 6 macaques exposed to the virus by aerosol became acutely ill during the 3rd week after exposure, and all died. Three died by day 21, while the remainder died after 1 month. Junin virus was found primarily in visceral organs of those animals dying before 21 days after infection and in the central nervous system tissues from animals dying later. Histological changes were similar to those reported in rhesus monkeys after parenteral Junin viral infection. Gastrointestinal necrosis, however, was less severe in aerosol-infected animals and the associated septicemia was not seen. High levels of alpha interferon were detected by the 3rd day in all infected macaques. Experimental Argentine hemorrhagic fever induced by aerosol infection in rhesus macaques was similar to that seen after parenteral challenge and mimicked closely the clinical syndrome observed in humans.

The filoviruses Marburg and Ebola comprise a newly recognized family of viruses. The first filovirus to be isolated was Marburg virus in 1967. This virus was imported in shipments of African green monkeys from Uganda and infected several cell-culture technicians, with serious illness resulting. The rarity of Marburg and Ebola virus transmission, decreasing use of imported African monkeys, and quarantine efforts have presumably been responsible for the lack of additional episodes until 1989, when a new filovirus related to Ebola was isolated from quarantined monkeys in Reston, Virginia. This virus was imported on multiple occasions from a Philippine supplier of cynomolgus macaques as a consequence of an epidemic of acute infections in the foreign holding facility. While quarantine procedures prevented the use of any of these animals in research and the three human infections that occurred were asymptomatic, this episode emphasizes that these little understood viruses have considerable potential for mischief. The finding of antibodies reacting with Ebola viruses in many biomedically important Old World primates, including colonized monkeys in the U.S., emphasizes the need for more research to understand the specificity of the antibodies, spectrum of filovirus strains in nature, potential hosts, and true distribution of the family. The filoviruses grow well in primary and established cell strains and cell lines, and cytopathogenic effects may be absent or require several days to be manifest, leading to the possibility of occult contamination. The known viruses are readily detected by polyclonal and monoclonal antibody staining of cells and by electron microscopy; nucleic acid probes exist to develop more sensitive techniques if warranted.

Prophylactic treatment of rhesus macaques with 10(4)-10(6) U/kg of recombinant human interferon-gamma (rHuIFN-gamma) modulated Rift Valley fever (RVF) virus infection. IFN was given intramuscularly at 24 h prior to infection and daily thereafter for a total of five doses. After infection, treated monkeys showed no evidence of clinical disease; some had no detectable viremia; when viremia was observed, peak virus titers were decreased compared to control infected monkeys; and only minor and transient perturbations in hematologic and clinical chemistry values were seen. Untreated infected control monkeys developed high-titered viremia, mild to severe clinical disease, and moderate to severe changes in hemostatic parameters and clinical laboratory measurements. No evidence of synergism was noted when RVF virus-infected monkeys were treated prophylactically with combined low doses of rHuIFN-gamma and rHuIFN-alpha A.

To assess a possible role of ticks as the maintenance host for epizootic strains of Venezuelan equine encephalomyelitis (VEE) virus, laboratory experiments were conducted to determine if ticks could become infected, maintain, and transmit the virus. Larval and nymphal Amblyomma cajennense (F.) and larval Dermacentor nitens Neumann ticks were exposed to epizootic VEE virus (Trinidad donkey strain) by allowing them to feed on viremic guinea pigs (strain 13). In A. cajennense, transstadial transmission was observed from larvae to nymphs and adults. Horizontal viral transmission to a mammalian host was accomplished by nymphs. Infection rates in nymphs and adults were 2% (42/2,750) and 4% (9/244), respectively, after ingestion of virus as larvae. Virus was detected in A. cajennense adult ticks for up to 171 d after infection in the larval stage. A. cajennense, exposed as nymphs, ingested virus but did not become infected (0/164 after 10 d after taking an infective bloodmeal). No virus was detected in D. nitens 7 d after exposure. These findings suggest that A. cajennense potentially could be involved in an interepizootic maintenance cycle of epizootic VEE viral strains.

The first recognized case in Scandinavia with potential man to man transmission of viral haemorrhagic fever occurred in Linkoping, Sweden, in January 1990. Following a visit to Kenya a 21-year-old male student suffered a very severe illness including extremely prolonged high grade fever, rash, disseminated intravascular coagulation with thrombocytopenia and severe bleedings. This necessitated one month of intensive care support including respirator treatment. The patient was discharged after 2 1/2 months in good condition, with a partial femoral nerve paresis. About 100 medical personnel were exposed to aerosol or blood before a strict containment regimen was established. No secondary cases occurred.

An epizootic caused by an Ebola-related filovirus and by simian haemorrhagic fever virus began among cynomolgus monkeys in a US quarantine facility after introduction of monkeys from the Philippines. This incident, the first in which a filovirus has been isolated from non-human primates without deliberate infection, raises the possibility that cynomolgus monkeys could be a reservoir of Ebola virus infection.

Rhesus monkeys inoculated intravenously with Rift Valley fever (RVF) virus presented clinical disease syndromes similar to human cases of RVF. All 17 infected monkeys had high-titered viremias but disease ranged from clinically inapparent to death. Three (18%) RVF virus-infected monkeys developed signs of hemorrhagic fever characterized by epistaxis, petechial to purpuric cutaneous lesions, anorexia, and vomiting prior to death. The 14 remaining monkeys survived RVF viral infection but, 7 showed clinical signs of illness characterized by diminished food intake, cutaneous petechiae, and occasional vomiting. The other 7 monkeys showed no evidence of clinical disease. All monkeys had detectable serum interferon 24-30 h after infection, but 4 of 7 monkeys that did not develop clinical illness had serum interferon titers within 12 h after infection. In lethally infected macaques, indices of hepatic function and blood coagulation were abnormal within 2 days, implicating early pathogenetic events as critical determinants of survival. Serum transferase values were elevated in proportion to severity of clinical disease and outcome of infection. Both myocardial damage and laboratory evidence consistent with disseminated intravascular coagulation were present in fatal infections. All surviving monkeys developed neutralizing antibodies to RVF virus 4-7 days after infection, and this coincided with termination of viremia. Two fatally infected monkeys were viremic until death on days 6 and 8, and the third cleared viremia on day 5 and developed antibody on day 6 but died on day 15. There was a significant correlation between a delayed interferon response and mortality, suggesting that the early appearance of interferon was influential in limiting the severity of disease.

Plaque reduction neutralization (PRN) and indirect immunofluorescence (IFA) tests were used to detect human antibodies to certain viruses of the families Flaviviridae and Bunyaviridae. Blood samples for the serosurveys were mainly collected from healthy farmers, wood cutters and shepherds. By PRN test antibodies were found to West Nile, sandfly fever Naples, and sandfly fever Sicilian viruses with seropositives 1.2%, 16.7%, and 2.0%, respectively. Antibodies to Rift Valley fever virus were not detected. By IFA tests, antibodies to the phlebovirus Corfu, tick-borne encephalitis, Crimean-Congo hemorrhagic fever, and Hantaan viruses were found with seropositives 4.0%, 1.7%, 1.0%, and 3.4%, respectively. Epidemiological data concerning the geographic and occupational distributions of the infected individuals are discussed.