SUMMARY: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 ϩ 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. (Lab Invest 2000, 80:171-186).I nfections with filoviruses cause severe and often fatal hemorrhagic disease in humans and nonhuman primates, killing up to 90% of infected individuals. Ebola (EBO) and Marburg (MBG) viruses comprise the family Filoviridae (Murphy et al, 1995). The EBO species consists of the Zaire and Sudan subtypes first isolated during separate outbreaks in 1976, the Reston subtype initially isolated from monkeys imported from the Philippines to the United States in 1989, and the Cô te d'Ivoire subtype discovered in the Tai Forest of the Ivory Coast in 1994. The recent re-emergence of EBO subtype Zaire (EBO-Z) in former Zaire and Gabon, and subtype Reston (EBO-R) in the United States, are but the last of a succession of filoviral outbreaks that have occurred sporadically since the initial zoonotic outbreak of MBG in 1967. Although outbreaks have been self-limiting, the lack of proven effective prophylactic or therapeutic regimens to treat filoviral infections has heightened concerns about the public health threat of these pathogens.The factors contributing to the development of severe lesions in EBO and MBG hemorrhagic fevers are unknown. Filoviral infections of humans and nonhuman primates are characterized by a failure o...

African swine fever is a significant disease of domestic swine, with mortality rates approaching 100%. No vaccine is currently available, making quarantine and slaughter the only effective control strategy (44).African swine fever virus (ASFV), the causative agent of African swine fever, is a unique and complex DNA virus that infects cells of the mononuclear-phagocytic system, including fixed-tissue macrophages and specific lineages of reticular cells. Affected tissues show extensive necrosis following infection with highly virulent viral strains (13,36,38). Moderately virulent ASFV strains also appear to infect these cell types, but the degree of tissue involvement and the resulting tissue damage are much less severe (13,36,38). The abilities of ASFV to replicate and efficiently induce marked cytopathology in monocytes-macrophages in vivo appear to be critical factors for ASFV virulence.ASFV is the sole member of the family Asfarviridae and the only known DNA arbovirus (14,38). ASFV is a large, icosahedral virus that contains a linear double-stranded DNA genome (170 to 190 kbp) encoding approximately 165 genes (50; C. A. Balinsky et al., unpublished data). The availability of complete ASFV genome sequences has revealed that, similar to poxviruses, ASFVs encode proteins with functions essential for viral replication, including those involving structure and assembly of the virion and those responsible for biogenesis of mRNA and DNA. A large number of ASFV genes are of unknown function and may be involved in aspects of viral virulence and host range (46,50; Balinsky et al., unpublished).Pathogenic ASFV genomes contain 11 to 15 multigene family 360 (MGF360) genes and either 9 or 10 multigene family 530 (MGF530) genes (Balinsky et al., unpublished). Recently, we have identified MGF360 and MGF530 genes as novel macrophage host range determinants necessary for efficient growth in macrophages (54). Infection of macrophage cell cultures with MGF360-MGF530 (MGF360/530) gene deletion mutant Pr4⌬35 (six MGF360 and two MGF530 genes deleted) resulted in a 2-to 3-log reduction in virus titers and early cell death, suggesting a direct or indirect role for these genes in some aspect of infected-cell survival (54) (L. Zsak, unpublished data). In addition, a swine virulence determinant (VAD) containing MGF360/530 genes was mapped by using in vivo marker rescue to the left variable region of the ASFV genome (37).The mode of action of the ASFV MGF360/530 genes is unknown. Homology searches reveal no homology to other known genes. MGF360/530 genes have a conserved motif of 100 amino acids (28% amino acid identity) at the amino ter-

No abstract

The ability to rapidly recognize Ebola virus infections is critical to quickly limit further spread of the disease. A rapid, sensitive, and specific laboratory diagnostic test is needed to confirm outbreaks of Ebola virus infection and to distinguish it from other diseases that can cause similar clinical symptoms. A one-tube reverse transcription-PCR assay for the identification of Ebola virus subtype Zaire (Ebola Zaire) and Ebola virus subtype Sudan (Ebola Sudan) was developed and evaluated by using the ABI PRISM 7700 sequence detection system. This assay uses one common primer set and two differentially labeled fluorescent probes to simultaneously detect and differentiate these two subtypes of Ebola virus. The sensitivity of the primer set was comparable to that of previously designed primer sets, as determined by limit-of-detection experiments. This assay is unique in its ability to simultaneously detect and differentiate Ebola Zaire and Ebola Sudan. In addition, this assay is compatible with emerging rapid nucleic acid analysis platforms and therefore may prove to be a very useful diagnostic tool for the control and management of future outbreaks.

The ability to rapidly recognize Marburg virus infections is critical to quickly institute proper barrier nursing precautions and limit further spread of the disease. A rapid, sensitive, and specific laboratory diagnostic test is necessary to confirm outbreaks of Marburg virus and to distinguish it from other diseases that can present with similar clinical symptoms.A one-tube reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the identification of Marburg virus was developed and evaluated using the ABI PRISM TM 7700 Sequence Detection System and TaqMan  chemistry. The sensitivity and specificity of the newly designed primer/probe set (MBGGP3) was evaluated. MBGGP3 was equivalent to or 10-100-fold more sensitive than previously designed primer sets as determined by limit of detection experiments. In addition, the MBGGP3 assay was able to detect all strains of Marburg virus tested, but gave negative results with other haemorrhagic fever and genetically related viruses. The results of this study indicate that the MBGGP3 primer/probe set is both sensitive and specific. In addition, this assay is compatible with emerging rapid nucleic acid analysis platforms and therefore may prove to be a useful diagnostic tool for the control and management of future outbreaks.