Quantification of regional aerosol deposition patterns as a function of aerodynamic particle size in rhesus macaques using PET/CT imaging.

Aerosol aerodynamic particle size is known to affect deposition patterns of inhaled aerosol particles, as well as the virulence of inhaled bioaerosol particles. While a significant amount of work has been performed to describe the deposition of aerosol particles in the human respiratory tract, only a limited amount of work has been performed to describe the deposition of aerosol particles in the respiratory tract of nonhuman primates, an animal model commonly utilized in pharmacological and toxicological studies, especially in the biodefense field. In this study, anesthetized rhesus macaques inhaled radiolabeled aerosols with MMADs of 1.7, 3.6, 7.4 and 11.8 µm to characterize regional deposition patterns. The results demonstrate that the regional deposition pattern shifts as particle size increases, with greater deposition in more proximal regions of the respiratory tract and decreased deposition in the pulmonary region. The results of this study extend the findings of previous studies which demonstrated a similar shift in the deposition pattern as a function of particle size by providing greater resolution of deposition patterns. These data on regional deposition patterns provide a starting point to begin to explore potential mechanisms responsible for the differences in virulence of infectious bioaerosols as a function of particle size and deposition pattern reported in previous studies. Additionally, the data are useful to assess the performance of various deposition models that have been published in the literature.

Virus-induced pancreatic disease by Venezuelan encephalitis virus. Alterations in glucose tolerance and insulin release.

Viral infections have been implicated in the induction of diabetes mellitus in man and laboratory animals. Since virus-specific immunofluorescence (FA) is detectable in hamster pancreas during the acute phase of Venezuelan encephalitis (VE), experiments were designed to correlate pathologic and virologic events with metabolic studies in VE-infected hamsters. Golden Syrian hamsters were inoculated s.c. in groups of four to 12 with 100,000 plaque-forming units (PFU) of the vaccine strain (TC-83) of VE or 1,000 PFU of the virulent Trinidad strain of VE. Ultrastructurally, during Trinidad infection, mature virions were associated with the cell surfaces and within pancreatic beta cells in contrast to absence of virus-related changes in TC-83-infected hamsters. Virus-specific-FA was noted in islet cells and acinar cells of Trinidad-infected hamsters. VE growth curves demonstrated viral replication in pancreas with both strains. Although ultrastructural and FA changes were much more prominent in Trinidad-infected hamsters in contrast to TC-83-infected hamsters during the first few days of illness, the rapid lethality of the Trinidad-infected group necessitated performing all metabolic studies in TC-83-strain-infected hamsters. Accordingly, for the metabolic studies, glucose tolerance tests (GTT) using 2 mg. or 5 gm./kg. glucose i.p. were performed in groups of hamsters acutely infected two days earlier with the TC-83 vaccine strain and in 24-day and 90-day convalescent hamsters after TC-83 vaccine strain. Samples were obtained for glucose and immunoreactive insulin (IRI) determinations. Glucose intolerance occurred in hamsters in each of the infected groups given 5 gm./kg. glucose except for the 90-day convalescent TC-83 group. Severely decreased IRI responses occurred in the 24-day and 90-day convalescent TC-83 hamsters following both 2- and 5-gm./kg. glucose. Pancreatic IRI content in 24-day convalescent TC-83 hamsters was within normal limits, suggesting a defect in IRI release from the beta cells at this stage of convalescence.

A probable endocrine basis for the depression of ketone bodies during infectious or inflammatory state in rats.

The effects of infection with Streptococcus pneumoniae, Francisella tularensis, and Venezuelan equine encephalitis virus as well as inflammatory stress induced by the administration of turpentine and endotoxin on plasma ketone bodies and insulin were studied in white rats. All of the infectious/inflammatory stresses caused a significant decrease in the ketonemia of fasting and an elevation of plasma insulin. When a pneumococcal infection was initiated in a diabetic rat, inhibition of fasting ketonemia did not occur. Similarly, pneumococcal infection in the hypophysectomized rat did not result in a noticeable depression of either fasting ketonemia or plasma FFA. The increase in circulating insulin appears to be closely correlated with the inhibition of fasting ketonemia noted in the infectious/inflammatory stress.

Human monkeypox and smallpox viruses: genomic comparison.

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person-to-person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197-kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host-range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

Differentiation of filoviruses by electron microscopy.

Cultured monolayers of MA-104, Vero 76, SW-13, and DBS-FRhL-2 cells were infected with Marburg (MBG), Ebola-Sudan (EBO-S), Ebola-Zaire (EBO-Z), and Ebola-Reston (EBO-R) viruses (Filoviridae, Filovirus) and examined by electron microscopy to provide ultrastructural details of morphology and morphogenesis of these potential human pathogens. Replication of each filovirus was seen in all cell systems employed. Filoviral particles appeared to enter host cells by endocytosis. Filoviruses showed a similar progression of morphogenic events, from the appearance of nascent intracytoplasmic viral inclusions to formation of mature virions budded through plasma membranes, regardless of serotype or host cell. However, ultrastructural differences were demonstrated between MBG and other filoviruses. MBG virions recovered from culture fluids were uniformly shorter in mean unit length than EBO-S, EBO-Z, or EBO-R particles. Examination of filovirus-infected cells revealed that intermediate MBG inclusions were morphologically distinct from EBO-S, EBO-Z, and EBO-R inclusions. No structural difference of viral inclusion material was observed among EBO-S, EBO-Z, and EBO-R. Immunoelectron microscopy showed that the filoviral matrix protein (VP40) and nucleoprotein (NP) accumulated in EBO-Z inclusions, and were closely associated during viral morphogenesis. These details facilitate the efficient and definitive diagnosis of filoviral infections by electron microscopy.

Infection of rhesus and cynomolgus macaques with a rapidly fatal SIV (SIVSMM/PBj) isolate from sooty mangabeys.

A variant of simian immunodeficiency virus (SIVSMM/PBj), isolated from a chronically infected pig-tailed macaque has been shown in previous studies to produce acutely fatal disease uniformly in pig-tailed macaques and in some rhesus macaques. The present study extends investigation of SIVSMM/PBj pathogenesis in rhesus and cynomolgus monkeys. Cynomolgus and rhesus macaques were found to be uniformly susceptible to infection, but as previously reported, the rhesus were found to not be uniform in their response during the acute disease. Homogenized tissues from a rhesus that died acutely from SIVSMM/PBj were passaged to 6 rhesus monkeys in an attempt to increase lethality. Five of 6 rhesus monkeys receiving intravenous inoculation of either spleen (10(3) TCID50) or lymph node (10(5) TCID50) homogenate developed acute disease; 4 died (days 8-10), 1 recovered, and one rhesus remained asymptomatic. Three of 3 cynomolgus macaques and 4 of 4 pig-tailed macaques receiving the same inoculum died acutely within 9 days. Clinical disease in macaques that died was characterized by diffuse lymphadenopathy within 5 days of inoculation and severe diarrhea beginning 1 to 3 days before death. Anorexia, lymphopenia (< 1000 cells/mm3), and mild hypoalbuminemia preceded onset of diarrhea by 24 h. Viral p27 was detected in circulation by day 6 postinfection, with all animals dying acutely having detectable serum p27 and no detectable humoral response. Acute lethality was attributed to severe metabolic acidosis (pH < 7.20) which was observed 24-48 h prior to death in the pig-tailed and cynomolgus macaques. Immunohistochemistry revealed numerous SIV antigen-positive lymphocytes and macrophages in the lymph nodes, spleen, gut-associated lymphoid tissues and gastrointestinal lamina propria. Histopathologic lesions included marked to severe hyperplasia of the T-cell-dependent areas in lymphoid tissues and diffuse nonulcerative lymphohistiocytic gastroenteritis. Surviving rhesus developed strong humoral immune responses to the major SIV proteins.

Association of interleukin-6 in the pathogenesis of acutely fatal SIVsmm/PBj-14 in pigtailed macaques.

Infection with a variant of simian immunodeficiency virus (SIVsmm/PBj-14) causes death in juvenile pigtailed macaques within 8 days of infection. The primary pathology is localized to the lymphoid tissues of the gut and spleen. Although the virus is present, the lesions are most consistent with acute reactive inflammation. We studied the serum and tissues for evidence of acute cytokine production often associated with acute inflammation. One factor, IL-6, was found to be significantly increased (> 1000-fold) over all other measured cytokines in all the pigtailed macaques who died acutely. Increased levels of IL-6 were found both in the serum and in the inflamed tissues. mRNA for IL-6 was found in the tissues with the highest protein levels of IL-6. The marked increase in IL-6 and IL-6 mRNA correlated with the virus levels in the tissues and serum as determined by viral isolation, immunohistochemistry, and Northern blot analysis. These findings suggest that the underlying pathogenesis of primary tissue damage, necrosis, and death by PBj-14 is the induction of cytokine production. Although the presence of the virus may be critical for the initiation of these events, the intense inflammatory reaction is associated with the cause of death.

Antiviral efficacy of pyrazofurin against selected RNA viruses.

The antibiotic pyrazofurin, 3-(beta-D-ribofuranosyl)-4-hydroxypyrazole-5-carboxamide, markedly inhibited the in vitro replication of a number of RNA viruses including Rift Valley fever (RVF), Venezuelan equine encephalomyelitis (VEE), Sandfly, Pichinde, Lassa and LCM virus. Plaque formation was reduced by 80% or more with 2-10 micrograms/ml of pyrazofurin while 2 micrograms/ml reduced by 1000-fold the yield of Lassa and LCM virus in a yield reduction assay. In vivo, pyrazofurin failed to protect mice and guinea pigs against a lethal challenge with VEE and Pichinde virus, respectively. On the other hand, pyrazofurin caused a slight increase in the mean time to death of mice infected with RVF virus.

Use of immunoelectron microscopy to show Ebola virus during the 1989 United States epizootic.

A filovirus, serologically related to Ebola virus, was detected by "post-embedment" immunoelectron microscopical examination of MA-104 cells. These had been infected by inoculation with serum samples obtained during the 1989 epizootic in cynomolgus monkeys (Macaca fascicularis), imported from the Philippines and maintained at Reston, Virginia, USA, a primate holding facility. The immunoelectron microscopy method, when used in conjunction with standard transmission electron microscopy (TEM) of infected cells, provided consistent results and was simple to perform in this epizootic. It is concluded that immunoelectron microscopy is potentially useful in the direct immunological diagnosis of Ebola and related filoviral infections (such as Marburg) in clinical samples obtained from those with acute infection.

Rapid identification of Ebola virus and related filoviruses in fluid specimens using indirect immunoelectron microscopy.

Recent filoviral outbreaks in animal primates have raised public awareness of the potential for filoviruses to become a public health concern; methods that efficiently identify these viruses are therefore of high priority. An indirect immunoelectron microscopy method, which uses homologous guinea pig polyclonal antiserum, successfully identified Ebola-related (Reston) virus particles in serum and tissue culture fluid specimens with infectivity titres of 300 plaque forming units (pfu) per ml or more. The sensitivity of this procedure is sufficient to show virus in most acute phase sera, and is equal to that of the antigen capture enzyme linked immunosorbent assay (ELISA). The immunoelectron microscopy fluid technique can differentiate among antigenically distinct filoviruses in less than three hours. It should be valuable in the rapid diagnosis of potential filoviral infections.

Patients infected with high-hazard viruses: scientific basis for infection control.

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.

Characterization of a new Marburg virus isolated from a 1987 fatal case in Kenya.

In 1987, an isolated case of fatal Marburg disease was recognized during routine clinical haemorrhagic fever virus surveillance conducted in Kenya. This report describes the isolation and partial characterization of the new Marburg virus (strain Ravn) isolated from this case. The Ravn isolate was indistinguishable from reference Marburg virus strains by cross-neutralization testing. Virus particles and aggregates of Marburg nucleocapsid matrix in Ravn-infected vero cells, were visualized by immunoelectron microscopic techniques, and also in tissues obtained from the patient and from inoculated monkeys. The cell culture isolate produced a haemorrhagic disease typical of Marburg virus infection when inoculated into rhesus monkeys. Disease was characterized by the sudden appearance of fever and anorexia within 4 to 7 days, and death by day 11. Comparison of nucleotide sequences for portions of the glycoprotein genes of Marburg-Ravn were compared with Marburg reference strains Musoki (MUS) and Popp (POP). Nucleotide identity in this alignment between RAV and MUS is 72.3%, RAV and POP is 71%, and MUS and POP is 91.7%. Amino acid identity between RAV and MUS is 72%, RAV and POP is 67%, and MUS and POP is 93%. These data suggest that Ravn is another subtype of Marburg virus, analogous to the emerging picture of a spectrum of Ebola geographic isolates and subtypes.

Experimental infection of cynomolgus macaques with Ebola-Reston filoviruses from the 1989-1990 U.S. epizootic.

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.

Passive immunization of Ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses.

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.

Pathology of Lassa virus infection in the rhesus monkey.

The clinical signs and gross and microscopic lesions of Lassa virus infection in the rhesus monkey are described. Of 17 monkeys infected with Lassa virus, nine died or were killed when moribund. The clinical signs were lethargy, aphagia, constipation, fever, conjunctivitis, and skin rash. Pulmonary congestion, pleural effusion, pericardial edema, hydropericardium, and a few visceral hemorrhages were present grossly. Major microscopic lesions were necrotizing hepatitis and interstitial pneumonia. Other microscopic changes were present in the heart, small intestine, spleen, lymph nodes, kidney, urinary bladder, adrenal glands, and central nervous system; however, most of these lesions were mild. In fact, death could not always be attributed to the morphologic changes; therefore, function alterations must be examined.

Description of Guanarito virus (Arenaviridae: Arenavirus), the etiologic agent of Venezuelan hemorrhagic fever.

This paper characterizes Guanarito virus, the etiologic agent of Venezuelan hemorrhagic fever. Based on its morphology and antigenic properties, Guanarito virus appears to be a new member of the Tacaribe complex of the genus Arenavirus, family Arenaviridae. Complement fixation and indirect fluorescent antibody tests showed that Guanarito virus and its antiserum are broadly cross-reactive with other members of the Tacaribe complex, but it can be differentiated from other members of the complex by neutralization test. Guanarito virus causes mortality in suckling mice and adult guinea pigs, but not in adult mice. Inoculated rhesus monkeys developed viremia and became ill; however, they subsequently recovered and responded with production of antibody. To date, all isolates of Guanarito virus have come from sick persons or wild rodents living within a single geographic focus in the central plains of Venezuela.

Experimental infection of guinea pigs with Venezuelan hemorrhagic fever virus (Guanarito): a model of human disease.

Venezuelan hemorrhagic fever (VHF), a newly described disease caused by an arenavirus (Guanarito), has resulted in multiple human deaths in Venezuela. To develop an animal model of this disease, strain 13 and Hartley strain guinea pigs were inoculated subcutaneously with Guananto strain 95551 of arenavirus in a pilot study to determine susceptibility of the species to the virus. All animals were killed when moribund 12-14 days following inoculation. Animals were necropsied and tissues were fixed and examined by both light and electron microscopy. Viral antigen was demonstrated in the tissues by immunohistochemistry at both the light and electron microscopic levels. Lesions were characterized by single cell necrosis of epithelium of the gastrointestinal tract, interstitial pneumonia, lymphoid and hematopoietic cell necrosis, and the presence of platelet thrombi in occasional blood vessels associated with hemorrhage. Viral antigen was demonstrated in lymphoid tissues and macrophages, endothelial cells of multiple organs, pulmonary epithelium, epithelium of the gastrointestinal tract, and in miscellaneous other tissues and cells. Intact virions and typical arenavirus inclusions were demonstrated by immunoelectron microscopy in these tissues. Based on these findings, the guinea pig appears to be a valid animal model of the human disease.

Pediatric Lassa fever: a review of 33 Liberian cases.

Thirty-three cases of pediatric Lassa fever were identified at Curran Lutheran Hospital and Phebe Hospital in Liberia between January 1980 and March 1984. All 18 fetal cases died and the case-fatality rate for 15 childhood cases was 27%. We identified four clinical presentations according to age, including a case of congenital Lassa fever, a condition not reported previously. Two cases of Lassa fever were found serologically during a one-month survey of all pediatric admissions at Curran Lutheran Hospital, 2.4% of those children who had serum pairs collected. We also identified a "swollen baby syndrome" consisting of widespread edema, abdominal distention, and bleeding. This distinctive clinical presentation of Lassa fever ended in death in three of four cases and was present in three of the four childhood deaths in this series. Its absence seems to be a good prognostic indicator in children.

Prevalence of infection with Junin virus in rodent populations in the epidemic area of Argentine hemorrhagic fever.

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.

Studies of the coagulation system in arenaviral hemorrhagic fever: experimental infection of strain 13 guinea pigs with Pichinde virus.

Significant coagulation abnormalities were associated with experimental infection of strain 13 guinea pigs with Pichinde virus, an arenavirus related to the virulent human pathogens Junin, Machupo, and Lassa viruses. Infected animals developed decreased activity of multiple coagulation factors, decreased antithrombin III levels, high levels of fibrin-fibrinogen degradation products, impaired platelet function, and thrombocytopenia. Testing for the presence of a coagulation inhibitor revealed a pattern consistent with factor deficiency. Fibrin thrombi were not found at necropsy. The findings of high fibrin-fibrinogen degradation product levels and decreased antithrombin III levels, in association with decreased activity of multiple coagulation factors and thrombocytopenia, suggest that intravascular coagulation is a feature of this experimental infection. The demonstration of abnormal platelet function is also significant, as this could contribute to defective hemostasis despite the moderate thrombocytopenia which usually occurs in arenaviral disease.