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.

Comparative pathology of smallpox and monkeypox in man and macaques.

In the three decades since the eradication of smallpox and cessation of routine vaccination, the collective memory of the devastating epidemics caused by this orthopoxvirus has waned, and the human population has become increasingly susceptible to a disease that remains high on the list of possible bioterrorism agents. Research using surrogate orthopoxviruses in their natural hosts, as well as limited variola virus research in animal models, continues worldwide; however, interpretation of findings is often limited by our relative lack of knowledge about the naturally occurring disease. For modern comparative pathologists, many of whom have no first-hand knowledge of naturally occurring smallpox, this work provides a contemporary review of this historical disease, as well as discussion of how it compares with human monkeypox and the corresponding diseases in macaques.

Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox.

Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurring smallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaks of human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear that naturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious health risk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizing antibodies developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible.

Analysis of the monkeypox virus genome.

Monkeypox virus (MPV) belongs to the orthopoxvirus genus of the family Poxviridae, is endemic in parts of Africa, and causes a human disease that resembles smallpox. The 196,858-bp MPV genome was analyzed with regard to structural features and open reading frames. Each end of the genome contains an identical but oppositely oriented 6379-bp terminal inverted repetition, which similar to that of other orthopoxviruses, includes a putative telomere resolution sequence and short tandem repeats. Computer-assisted analysis was used to identify 190 open reading frames containing >/=60 amino acid residues. Of these, four were present within the inverted terminal repetition. MPV contained the known essential orthopoxvirus genes but only a subset of the putative immunomodulatory and host range genes. Sequence comparisons confirmed the assignment of MPV as a distinct species of orthopoxvirus that is not a direct ancestor or a direct descendent of variola virus, the causative agent of smallpox.

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.

The pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (Macaca fascicularis).

Cynomolgus monkeys (Macaca fascicularis) were exposed by fine-particle aerosol to lethal doses of monkeypox virus, Zaire strain. Death, attributable to fibrinonecrotic bronchopneumonia, occurred 9 to 17 days postexposure. Lower airway epithelium served as the principal target for primary infection. The relative degree of involvement among lymphoid tissues suggested that tonsil, mediastinal, and mandibular lymph nodes were also infected early in the course of the disease, and may have served as additional, although subordinate, sites of primary replication. The distribution of lesions was consistent with lymphatogenous spread to the mediastinal lymph nodes and systemic dissemination of the virus through a monocytic cell-associated viremia. This resulted in lesions affecting other lymph nodes, the thymus, spleen, skin, oral mucosa, gastrointestinal tract, and reproductive system. The mononuclear phagocyte/dendritic cell system was the principal target within lymphoid tissues and may also have provided the means of entry into other systemic sites. Hepatic involvement was uncommon. Lesions at all affected sites were characterized morphologically as necrotizing. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining of select lesions suggested that cell death within lymphoid and epithelial tissues was due in large part to apoptosis. Skin and mucosal surfaces of the respiratory and gastrointestinal tracts also exhibited variable proliferation of epithelial cells and subjacent fibroblasts. Epithelial intracytoplasmic inclusion bodies, consistent with Guarnieri bodies, were usually inconspicuous by light microscopy, but when present, were most readily apparent in the stratified squamous epithelium of the oral mucosa and epidermis. Multinucleated syncytial cells were also occasionally observed in the stratified squamous epithelium of the tongue, tonsil, and skin, and in the intestinal mucosa. Monkeypox virus antigen was readily demonstrated by immunohistochemistry using anti-vaccinia mouse polyclonal antibodies as well as anti-monkeypox rabbit polyclonal antibodies. Detectable poxviral antigen was limited to sites exhibiting obvious morphologic involvement and was most prominent within epithelial cells, macrophages, dendritic cells, and fibroblasts of affected tissues. The presence of poxviral antigen, as determined by immunohistochemistry, correlated with ultrastructural identification of replicating virus. Concurrent bacterial septicemia, present in one monkey, was associated with increased dissemination of the virus to the liver, spleen, and bone marrow and resulted in a more rapidly fatal clinical course.

Individual and bivalent vaccines based on alphavirus replicons protect guinea pigs against infection with Lassa and Ebola viruses.

Lassa and Ebola viruses cause acute, often fatal, hemorrhagic fever diseases, for which no effective vaccines are currently available. Although lethal human disease outbreaks have been confined so far to sub-Saharan Africa, they also pose significant epidemiological concern worldwide as demonstrated by several instances of accidental importation of the viruses into North America and Europe. In the present study, we developed experimental individual vaccines for Lassa virus and bivalent vaccines for Lassa and Ebola viruses that are based on an RNA replicon vector derived from an attenuated strain of Venezuelan equine encephalitis virus. The Lassa and Ebola virus genes were expressed from recombinant replicon RNAs that also encoded the replicase function and were capable of efficient intracellular self-amplification. For vaccinations, the recombinant replicons were incorporated into virus-like replicon particles. Guinea pigs vaccinated with particles expressing Lassa virus nucleoprotein or glycoprotein genes were protected from lethal challenge with Lassa virus. Vaccination with particles expressing Ebola virus glycoprotein gene also protected the animals from lethal challenge with Ebola virus. In order to evaluate a single vaccine protecting against both Lassa and Ebola viruses, we developed dual-expression particles that expressed glycoprotein genes of both Ebola and Lassa viruses. Vaccination of guinea pigs with either dual-expression particles or with a mixture of particles expressing Ebola and Lassa virus glycoprotein genes protected the animals against challenges with Ebola and Lassa viruses. The results showed that immune responses can be induced against multiple vaccine antigens coexpressed from an alphavirus replicon and suggested the possibility of engineering multivalent vaccines based upon alphavirus vectors for arenaviruses, filoviruses, and possibly other emerging pathogens.

Marburg virus vaccines: comparing classical and new approaches.

An effort to develop a safe and effective vaccine for Marburg virus (MBGV), one of the filoviruses known to cause high mortality rates in humans, led us to compare directly some of the merits of modern versus classical vaccine approaches for this agent. Prior work had established the MBGV-glycoprotein (GP), the only known virion surface antigen, as a candidate for inclusion in a vaccine. In this study, we vaccinated groups of Hartley guinea pigs with killed MBGV, live attenuated MBGV, soluble MBGV-GP expressed by baculovirus recombinants, MBGV-GP delivered as a DNA vaccine, or MBGV-GP delivered via an alphavirus RNA replicon. Serological responses were evaluated, and animals were challenged with a lethal dose of MBGV given either subcutaneously or via aerosol. Killed MBGV and replicon-delivered MBGV-GP were notably immunogenic and protective against MBGV, but results did not exclude any approach and suggested a role for DNA vaccines in immunological priming.

Clinical recognition and management of patients exposed to biological warfare agents.

Concern regarding the use of biological agents (bacteria, viruses, or toxins) as tools of warfare or terrorism has led to measures to deter their use or, failing that, to deal with the consequences. Unlike chemical agents, which typically lead to severe disease syndromes within minutes at the site of exposure, diseases resulting from biological agents have incubation periods of days. Rather than a paramedic, it will likely be a physician who is first faced with evidence of the results of a biological attack. Provided here is an updated primer on 11 classic BW and potential terrorist agents to increase the likelihood of their being considered in a differential diagnosis. Although the resultant diseases are rarely seen in many countries today, accepted diagnostic and epidemiologic principles apply; if the cause is identified quickly, appropriate therapy can be initiated and the impact of a terrorist attack greatly reduced.

Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus.

RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP-VRP and GP-VRP, respectively) using a bipartite helper system that provided the VEE structural proteins in trans and prevented the regeneration of replication-competent VEE during packaging. The immunogenicity of NP-VRP and GP-VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP-VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP-VRP and GP-VRP in primates as candidate vaccines against Ebola hemorrhagic fever.

Apoptosis induced in vitro and in vivo during infection by Ebola and Marburg viruses.

Induction of apoptosis has been documented during infection with a number of different viruses. In this study, we used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling to investigate the effects of Ebola and Marburg viruses on apoptosis of different cell populations during in vitro and in vivo infections. Tissues from 18 filovirus-infected nonhuman primates killed in extremis were evaluated. Apoptotic lymphocytes were seen in all tissues examined. Filoviral replication occurred in cells of the mononuclear phagocyte system and other well-documented cellular targets by TEM and immunohistochemistry, but there was no evidence of replication in lymphocytes. With the exception of intracytoplasmic viral inclusions, filovirus-infected cells were morphologically normal or necrotic, but did not exhibit ultrastructural changes characteristic of apoptosis. In lymph nodes, filoviral antigen was co-localized with apoptotic lymphocytes. Examination of cell populations in lymph nodes showed increased numbers of macrophages and concomitant depletion of CD8+ T cells and plasma cells in filovirus-infected animals. This depletion was particularly striking in animals infected with the Zaire subtype of Ebola virus. In addition, apoptosis was demonstrated in vitro in lymphocytes of filovirus-infected human peripheral blood mononuclear cells by TEM. These findings suggest that lymphopenia and lymphoid depletion associated with filoviral infections result from lymphocyte apoptosis induced by a number of factors that may include release of various chemical mediators from filovirus-infected or activated cells, damage to the fibroblastic reticular cell conduit system, and possibly stimulation by a viral protein.

Cytotoxic T lymphocytes to Ebola Zaire virus are induced in mice by immunization with liposomes containing lipid A.

An eight amino acid sequence (TELRTFSI) present in the carboxy terminal end (aa 577-584) of membrane-anchored GP, the major structural protein of Ebola virus, was identified as an H-2k-specific murine cytotoxic T cell epitope. Cytotoxic T lymphocytes (CTLs) to this epitope were induced by immunizing B10.BR mice intravenously with either irradiated Ebola virus or with irradiated Ebola virus encapsulated in liposomes containing lipid A. The CTL response induced by irradiated Ebola virus could not be sustained after the second round of in vitro stimulation of immune splenocytes with the peptide, unless the irradiated virus was encapsulated in liposomes containing lipid A. The identification of an Ebola GP-specific CTL epitope and the requirement of liposomal lipid A for CTL memory recall responses could prove to be a promising approach for developing a vaccine against Ebola virus infection.

Smallpox as a biological weapon: medical and public health management. Working Group on Civilian Biodefense.

OBJECTIVE: To develop consensus-based recommendations for measures to be taken by medical and public health professionals following the use of smallpox as a biological weapon against a civilian population. PARTICIPANTS: The working group included 21 representatives from staff of major medical centers and research, government, military, public health, and emergency management institutions and agencies. Evidence The first author (D.A.H.) conducted a literature search in conjunction with the preparation of another publication on smallpox as well as this article. The literature identified was reviewed and opinions were sought from experts in the diagnosis and management of smallpox, including members of the working group. CONSENSUS PROCESS: The first draft of the consensus statement was a synthesis of information obtained in the evidence-gathering process. Members of the working group provided formal written comments that were incorporated into the second draft of the statement. The working group reviewed the second draft on October 30, 1998. No significant disagreements existed and comments were incorporated into a third draft. The fourth and final statement incorporates all relevant evidence obtained by the literature search in conjunction with final consensus recommendations supported by all working group members. CONCLUSIONS: Specific recommendations are made regarding smallpox vaccination, therapy, postexposure isolation and infection control, hospital epidemiology and infection control, home care, decontamination of the environment, and additional research needs. In the event of an actual release of smallpox and subsequent epidemic, early detection, isolation of infected individuals, surveillance of contacts, and a focused selective vaccination program will be the essential items of an effective control program.

Ebola virus can be effectively neutralized by antibody produced in natural human infection.

The activity of antibodies against filoviruses is poorly understood but has important consequences for vaccine design and passive prophylaxis. To investigate this activity, a panel of recombinant human monoclonal antibodies to Ebola virus antigens was isolated from phage display libraries constructed from RNA from donors who recovered from infection in the 1995 Ebola virus outbreak in Kikwit, Democratic Republic of Congo. Antibodies reactive with nucleoprotein (NP), envelope glycoprotein (GP), and secreted envelope glycoprotein (sGP) were characterized by immunofluorescence and radioimmunoprecipitation assays. Four antibodies reacting strongly with sGP and weakly with GP and two antibodies reacting with NP were not neutralizing. An antibody specific for GP neutralized Ebola virus to 50% at 0.4 microgram/ml as the recombinant Fab fragment and to 50% at 0.3 microgram/ml (90% at 2.6 microgram/ml) as the corresponding whole immunoglobulin G1 molecule. The studies indicate that neutralizing antibodies are produced in infection by Ebola virus although probably at a relatively low frequency. The neutralizing antibody may be useful in vaccine design and as a prophylactic agent against Ebola virus infection.

Ebola (subtype Reston) virus among quarantined nonhuman primates recently imported from the Philippines to the United States.

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.

Field investigations of an outbreak of Ebola hemorrhagic fever, Kikwit, Democratic Republic of the Congo, 1995: arthropod studies.

During the final weeks of a 6-month epidemic of Ebola hemorrhagic fever in Kikwit, Democratic Republic of the Congo, an extensive collection of arthropods was made in an attempt to learn more of the natural history of the disease. A reconstruction of the activities of the likely primary case, a 42-year-old man who lived in the city, indicated that he probably acquired his infection in a partly forested area 15 km from his home. Collections were made throughout this area, along the route he followed from the city, and at various sites in the city itself. No Ebola virus was isolated, but a description of the collections and the ecotopes involved is given for comparison with future studies of other outbreaks.

ELISA for the detection of antibodies to Ebola viruses.

EIAs for IgG and IgM antibodies directed against Ebola (EBO) viral antigens have been developed and evaluated using sera of animals and humans surviving infection with EBO viruses. The IgM capture assay detected anti-EBO (subtype Reston) antibodies in the sera of 5 of 5 experimentally infected animals at the time they succumbed to lethal infections. IgM antibodies were also detected in the serum of a human who was infected with EBO (subtype Reston) during a postmortem examination of an infected monkey. The antibody was detectable as early as day 6 after infection in experimentally infected animals and persisted for <90 days. The IgG response was less rapid; however, it persisted for >400 days in 3 animals who survived infection, and it persisted for approximately 10 years after infection in the sera of 2 humans. Although these data are limited by the number of sera available for verification, the IgM assay seems to have great promise as a diagnostic tool. Furthermore the long-term persistence of the IgG antibodies measured by this test strongly suggests that the ELISA will be useful in field investigations of EBO virus.

Pathogenesis of experimental Ebola virus infection in guinea pigs.

The subtype Zaire of Ebola (EBO) virus (Mayinga strain) was adapted to produce lethal infections in guinea pigs. In many ways, the disease was similar to EBO infections in nonhuman primates and humans. The guinea pig model was used to investigate the pathologic events in EBO infection that lead to death. Analytical methods included immunohistochemistry, in situ hybridization, and electron microscopy. Cells of the mononuclear phagocyte system, primarily macrophages, were identified as the early and sustained targets of EBO virus. During later stages of infection, interstitial fibroblasts in various tissues were infected, and there was evidence of endothelial cell infection and fibrin deposition. The distribution of lesions, hematologic profiles, and increases in serum biochemical enzymes associated with EBO virus infection in guinea pigs was similar to reported findings in experimentally infected nonhuman primates and naturally infected humans.