PAirwise Sequence Comparison (PASC) and Its Application in the Classification of Filoviruses

PAirwise Sequence Comparison (PASC) is a tool that uses genome sequence similarity to help with virus classification. The PASC tool at NCBI uses two methods: local alignment based on BLAST and global alignment based on Needleman-Wunsch algorithm. It works for complete genomes of viruses of several families/groups, and for the family of Filoviridae, it currently includes 52 complete genomes available in GenBank. It has been shown that BLAST-based alignment approach works better for filoviruses, and therefore is recommended for establishing taxon demarcation criteria. When more genome sequences with high divergence become available, these demarcations will most likely become more precise. The tool can compare new genome sequences of filoviruses with the ones already in the database, and propose their taxonomic classification.     

Use of the Syrian Hamster as a New Model of Ebola Virus Disease and Other Viral Hemorrhagic Fevers

Historically, mice and guinea pigs have been the rodent models of choice for therapeutic and prophylactic countermeasure testing against Ebola virus disease (EVD). Recently, hamsters have emerged as a novel animal model for the in vivo study of EVD. In this review, we discuss the history of the hamster as a research laboratory animal, as well as current benefits and challenges of this model. Availability of immunological reagents is addressed. Salient features of EVD in hamsters, including relevant pathology and coagulation parameters, are compared directly with the mouse, guinea pig and nonhuman primate models.     

Ebola Virus Antibodies in Fruit Bats,Bangladesh

To determine geographic range for Ebola virus, we tested 276 bats in Bangladesh. Five (3.5%) bats were positive for antibodies against Ebola Zaire and Reston viruses; no virus was detected by PCR. These bats might be a reservoir for Ebola or Ebola-like viruses, and extend the range of filoviruses to mainland Asia.     

Correlates of immunity to filovirus infection

Filoviruses can cause severe, often fatal hemorrhagic fever in humans. Recent advances in vaccine and therapeutic drug development have provided encouraging data concerning treatment of these infections. However, relatively little is known about immune responses in fatal versus non-fatal filovirus infection. This review summarizes the published literature on correlates of immunity to filovirus infection, and highlights deficiencies in our knowledge on this topic. It is likely that there are several types of successful immune responses, depending on the type of filovirus, and the presence and timing of vaccination or drug treatment.     

A Characterization of Aerosolized Sudan Virus Infection in African Green Monkeys,Cynomolgus Macaques,and Rhesus Macaques

Filoviruses are members of the genera Ebolavirus, Marburgvirus, and “Cuevavirus”. Because they cause human disease with high lethality and could potentially be used as a bioweapon, these viruses are classified as CDC Category A Bioterrorism Agents. Filoviruses are relatively stable in aerosols, retain virulence after lyophilization, and can be present on contaminated surfaces for extended periods of time. This study explores the characteristics of aerosolized Sudan virus (SUDV) Boniface in non-human primates (NHP) belonging to three different species. Groups of cynomolgus macaques (cyno), rhesus macaques (rhesus), and African green monkeys (AGM) were challenged with target doses of 50 or 500 plaque-forming units (pfu) of aerosolized SUDV. Exposure to either viral dose resulted in increased body temperatures in all three NHP species beginning on days 4–5 post-exposure. Other clinical findings for all three NHP species included leukocytosis, thrombocytopenia, anorexia, dehydration, and lymphadenopathy. Disease in all of the NHPs was severe beginning on day 6 post-exposure, and all animals except one surviving rhesus macaque were euthanized by day 14. Serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were elevated during the course of disease in all three species; however, AGMs had significantly higher ALT and AST concentrations than cynos and rhesus. While all three species had detectable viral load by days 3-4 post exposure, Rhesus had lower average peak viral load than cynos or AGMs. Overall, the results indicate that the disease course after exposure to aerosolized SUDV is similar for all three species of NHP.     

From the Cover: Vaccinating captive chimpanzees to save wild chimpanzees

Infectious disease has only recently been recognized as a major threat to the survival of Endangered chimpanzees and Critically Endangered gorillas in the wild. One potentially powerful tool, vaccination, has not been deployed in fighting this disease threat, in good part because of fears about vaccine safety. Here we report on what is, to our knowledge, the first trial in which captive chimpanzees were used to test a vaccine intended for use on wild apes rather than humans. We tested a virus-like particle vaccine against Ebola virus, a leading source of death in wild gorillas and chimpanzees. The vaccine was safe and immunogenic. Captive trials of other vaccines and of methods for vaccine delivery hold great potential as weapons in the fight against wild ape extinction.There is growing recognition that infectious diseases pose a threat to the survival of African apes: a threat on par with poaching and habitat loss. Heightened awareness is due both to better data on rates of disease mortality in gorillas and chimpanzees and to new molecular diagnostic assays that pinpoint the cause of death. These assays tell us that wild apes are regularly infected by a variety of virulent pathogens, including simian immunodeficiency virus (SIV) (1), anthrax (2), and malaria (3). The ethical finger has been pointed squarely and quantitatively at researchers and conservationists with the discovery that “spillover” of human respiratory viruses cause about half of deaths among chimpanzees (4, 5) and gorillas (6) habituated to human approach for research or tourism. Even more widely recognized have been massive Ebola virus (EBOV) outbreaks in gorillas and chimpanzees (7, 8), which have killed roughly one-third of the world gorilla population and led to the 2007 upgrading of western gorillas to Critically Endangered status on the World Conservation Union’s Red List of Threatened Species (9).The ability to accurately diagnose diseases that afflict wild apes has opened the door to an active management response: vaccination (10). The door has been pushed further open by recent advances in vaccinology, including experimental vaccines against several previously unpreventable disease threats to wild apes, new vaccine platforms that reduce or eliminate the risk of infection or vaccine spillover into nontarget species, and new adjuvants that enhance vaccine efficacy (11). Although recognition of the magnitude of the disease threat has made a historically noninterventionist ape conservation community increasingly receptive to vaccination, park managers are still adamant that any experimental vaccine be tested for safety and immunogenicity in captive apes before being used on apes in the wild.Both to address a salient disease threat and to evaluate whether captive testing of vaccines is feasible with the meager budgets typically available to ape conservationists, we decided to test an experimental vaccine against EBOV. During a consultative process lasting several years, vaccine experts, veterinarians, and park managers persistently expressed concerns about the safety of using live (replicating) vaccines on immunologically stressed wild animals. Some cited the case of SIV, which is typically benign in well-cared-for captive chimpanzees but virulent in environmentally challenged wild chimpanzees (1). Therefore, we chose to test a new virus-like particle (VLP) that does not contain an entire replicating virus but only a fragment of viral coat protein. Because they do not cause infection, VLP-based vaccines are particularly safe and several have been recently approved for human use (12). We tested a VLP vaccine based on the virulent Zaire species of EBOV that previously has been given to more than 80 captive macaques without serious health complications (13). In the present study, we did not challenge vaccinated chimpanzees with EBOV. Rather, we simply evaluated whether the vaccine caused health complications that had not been observed in macaques and whether the vaccine induced immune responses comparable to those observed in macaques who survived Ebola challenge. We also tested whether antibodies harvested from vaccinated chimpanzees could protect mice against EBOV challenge.     

Cell adhesion-dependent membrane trafficking of a binding partner for the ebolavirus glycoprotein is a determinant of viral entry

Ebolavirus is a hemorrhagic fever virus associated with high mortality. Although much has been learned about the viral lifecycle and pathogenesis, many questions remain about virus entry. We recently showed that binding of the receptor binding region (RBR) of the ebolavirus glycoprotein (GP) and infection by GP pseudovirions increase on cell adhesion independently of mRNA or protein synthesis. One model to explain these observations is that, on cell adhesion, an RBR binding partner translocates from an intracellular vesicle to the cell surface. Here, we provide evidence for this model by showing that suspension 293F cells contain an RBR binding site within a membrane-bound compartment associated with the trans-Golgi network and microtubule-organizing center. Consistently, trafficking of the RBR binding partner to the cell surface depends on microtubules, and the RBR binding partner is internalized when adherent cells are placed in suspension. Based on these observations, we reexamined the claim that lymphocytes, which are critical for ebolavirus pathogenesis, are refractory to infection because they lack an RBR binding partner. We found that both cultured and primary human lymphocytes (in suspension) contain an intracellular pool of an RBR binding partner. Moreover, we identified two adherent primate lymphocytic cell lines that bind RBR at their surface and strikingly, support GP-mediated entry and infection. In summary, our results reveal a mode of determining viral entry by a membrane-trafficking event that translocates an RBR binding partner to the cell surface, and they suggest that this process may be operative in cells important for ebolavirus pathogenesis (e.g., lymphocytes and macrophages).