Tacaribe virus causes fatal infection of an ostensible reservoir host, the Jamaican fruit bat.

Tacaribe virus (TCRV) was first isolated from 11 Artibeus species bats captured in Trinidad in the 1950s during a rabies virus surveillance program. Despite significant effort, no evidence of infection of other mammals, mostly rodents, was found, suggesting that no other vertebrates harbored TCRV. For this reason, it was hypothesized that TCRV was naturally hosted by artibeus bats. This is in stark contrast to other arenaviruses with known hosts, all of which are rodents. To examine this hypothesis, we conducted experimental infections of Jamaican fruit bats (Artibeus jamaicensis) to determine whether they could be persistently infected without substantial pathology. We subcutaneously or intranasally infected bats with TCRV strain TRVL-11573, the only remaining strain of TCRV, and found that low-dose (10(4) 50% tissue culture infective dose [TCID(50)]) inoculations resulted in asymptomatic and apathogenic infection and virus clearance, while high-dose (10(6) TCID(50)) inoculations caused substantial morbidity and mortality as early as 10 days postinfection. Uninoculated cage mates failed to seroconvert, and viral RNA was not detected in their tissues, suggesting that transmission did not occur. Together, these data suggest that A. jamaicensis bats may not be a reservoir host for TCRV.

Progress in Anti-Mammarenavirus Drug Development.

Mammarenaviruses are prevalent pathogens distributed worldwide, and several strains cause severe cases of human infections with high morbidity and significant mortality. Currently, there is no FDA-approved antiviral drugs and vaccines against mammarenavirus and the potential treatment option is limited to an off-label use of ribavirin that shows only partial protective effect and associates with side effects. For the past few decades, extensive research has reported potential anti-mammarenaviral drugs and their mechanisms of action in host as well as vaccine candidates. This review describes current knowledge about mammarenavirus virology, progress of antiviral drug development, and technical strategies of drug screening.

Prevalence of inclusion body disease and associated comorbidity in captive collections of boid and pythonid snakes in Belgium.

Inclusion body disease (IBD) is caused by reptarenaviruses and constitutes one of the most notorious viral diseases in snakes. Although central nervous system disease and various other clinical signs have been attributed to IBD in boid and pythonid snakes, studies that unambiguously reveal the clinical course of natural IBD and reptarenavirus infection are scarce. In the present study, the prevalence of IBD and reptarenaviruses in captive snake collections and the correlation of IBD and reptarenavirus infection with the clinical status of the sampled snakes were investigated. In three IBD positive collections, long-term follow-up during a three- to seven-year period was performed. A total of 292 snakes (178 boas and 114 pythons) from 40 collections in Belgium were sampled. In each snake, blood and buffy coat smears were evaluated for the presence of IBD inclusion bodies (IB) and whole blood was tested for reptarenavirus RNA by RT-PCR. Of all tested snakes, 16.5% (48/292) were positive for IBD of which all were boa constrictors (34.0%; 48/141) and 17.1% (50/292) were reptarenavirus RT-PCR positive. The presence of IB could not be demonstrated in any of the tested pythons, while 5.3% (6/114) were reptarenavirus positive. In contrast to pythons, the presence of IB in peripheral blood cells in boa constrictors is strongly correlated with reptarenavirus detection by RT-PCR (P<0.0001). Although boa constrictors often show persistent subclinical infection, long-term follow-up indicated that a considerable number (22.2%; 6/27) of IBD/reptarenavirus positive boas eventually develop IBD associated comorbidities.

Animal models for viral haemorrhagic fever.

Viral haemorrhagic fever can be caused by one of a diverse group of viruses that come from four different families of RNA viruses. Disease severity can vary from mild self-limiting febrile illness to severe disease characterized by high fever, high-level viraemia, increased vascular permeability that can progress to shock, multi-organ failure and death. Despite the urgent need, effective treatments and preventative vaccines are currently lacking for the majority of these viruses. A number of factors preclude the effective study of these diseases in humans including the high virulence of the agents involved, the sporadic nature of outbreaks of these viruses, which are typically in geographically isolated areas with underserviced diagnostic capabilities, and the requirements for high level bio-containment. As a result, animal models that accurately mimic human disease are essential for advancing our understanding of the pathogenesis of viral haemorrhagic fevers. Moreover, animal models for viral haemorrhagic fevers are necessary to test vaccines and therapeutic intervention strategies. Here, we present an overview of the animal models that have been established for each of the haemorrhagic fever viruses and identify which aspects of human disease are modelled. Furthermore, we discuss how experimental design considerations, such as choice of species and virus strain as well as route and dose of inoculation, have an influence on animal model development. We also bring attention to some of the pitfalls that need to be avoided when extrapolating results from animal models.

[Selected immunological processes in viral haemorrhagic fevers infections]. / Wybrane procesy immunologiczne zwiazane z wirusami goraczek krwotocznych.

The paper reviews the known facts on the immunological response in infections with viral haemorrhagic fevers--dangerous pathogens for life and health of people. Immunological process registered in infections with viruses from Arenaviridae, Bunyaviridae, Filoviridae and Flaviviridae have been described. Moreover, the immunological response in infection with the RHD (rabbit haemorrhagic disease) virus from Caliciviridae family have been shown as a potential model for laboratory research on the duration and pathogenesis of viral haemorrhagic fevers.

Entry Studies of New World Arenaviruses.

Identification of cell moieties involved in viral binding and internalization is essential since their expression would render a cell susceptible. Further steps that allow the uncoating of the viral particle at the right subcellular localization have been intensively studied. These "entry" steps could determine cell permissiveness and often define tissue and host tropism. Therefore applying the right and, when possible, straightforward experimental approaches would shorten avenues to the complete knowledge of this first and key step of any viral life cycle. Mammarenaviruses are enveloped viruses that enter the host cell via receptor-mediated endocytosis. In this chapter we present a set of customized experimental approaches and tools that were used to describe the entry of Junín virus (JUNV), and other New World mammarenavirus members, into mammalian cells.

Ancient Evolution of Mammarenaviruses: Adaptation via Changes in the L Protein and No Evidence for Host-Virus Codivergence.

The Mammarenavirus genus includes several pathogenic species of rodent-borne viruses. Old World (OW) mammarenaviruses infect rodents in the Murinae subfamily and are mainly transmitted in Africa and Asia; New World (NW) mammarenaviruses are found in rodents of the Cricetidae subfamily in the Americas. We applied a selection-informed method to estimate that OW and NW mammarenaviruses diverged less than ∼45,000 years ago (ya). By incorporating phylogeographic inference, we show that NW mammarenaviruses emerged in the Latin America-Caribbean region ∼41,400-3,300 ya, whereas OW mammarenaviruses originated ∼23,100-1,880 ya, most likely in Southern Africa. Cophylogenetic analysis indicated that cospeciation did not contribute significantly to mammarenavirus-host associations. Finally, we show that extremely strong selective pressure on the viral polymerase accompanied the speciation of NW viruses. These data suggest that the evolutionary history of mammarenaviruses was not driven by codivergence with their hosts. The viral polymerase should be regarded as a major determinant of mammarenavirus adaptation.

Effects of mammarenavirus infection (Wenzhou virus) on the morphology of Rattus exulans.

The circulation of mammarenaviruses in rodent populations of the Mekong region has recently been established, with a genetic variant of Wenzhou virus, Cardamones virus, detected in two Rattus species. This study tests the potential teratogenic effects of Wenzhou infection on the development of a Murid rodent, Rattus exulans. Using direct virus detection, morphological records and comparative analyses, a link was demonstrated between host infection status and host morphologies (the spleen irrespective of weight, the skull shape and the cranial cavity volume) at the level of the individual (females only). This study demonstrates that mammarenavirus infections can impact natural host physiology and/or affect developmental processes. The presence of an infecting micro-parasite during the development of the rat may lead to a physiological trade-off between immunity and brain size. Alternatively, replication of virus in specialized organs can result in selective morphologic abnormalities and lesions.

[Haemorrhagic fever viruses, possible bioterrorist use]. / Fièvres hémorragiques virales, possibilité d'utilisation bioterroriste.

The majority of haemorrhagic fever viruses are responsible for various clinical manifestations, the mutual characteristics of which are fever and haemorrhage in 5 to 70% of cases. All degrees of severity can be observed, ranging from isolated fever to multi-organ failure and death. These viruses belong to one of the following families: filoviridae, arenaviridae, bunyaviridae, and flaviviridae. They must be considered as dangerous biological weapons that could potentially be used. Most of the viruses responsible for haemorrhagic fever can be transmitted to humans through the air in spray form, except the dengue virus and the agents of haemorrhagic fever from the Congo Crimea and the haemorrhagic fever with renal syndrome that are difficult to handle in cell culture. In the event of a bioterrorist act, the management of persons infected or suspected of being so will be made by the referent departments of infectious diseases, defined by the French Biotox plan. Management includes isolation, confirmation or invalidation of the diagnosis and rapid initiation of treatment with ribavirin. Ribavirin is recommended for the treatment and prophylaxis of arenavirus and bunyavirus infections; it is not effective for the other families of virus. Except for yellow fever, there is no vaccination for the other forms of viral haemorrhagic fever.

Reduced virulence of a Junin virus mutant is associated with restricted multiplication in murine cells.

C167, a mutant derived from the XJC13 strain of Junin virus, is highly attenuated in its pathogenic properties for newborn mice. Whereas 10(2).PFU of XJC13 injected intracerebrally killed 100% of two-day-old mice, the mutant showed no detectable lethality. Survival of mice infected with C167 was associated with a reduced and delayed virus replication in brain and a defective spread of virus from the site of inoculation to the other tissues, including spleen, kidney, thymus, liver, peritoneal cells and serum. As an apparent consequence of the restricted replication of C167 in mice, no detectable interferon induction and low levels of neutralizing antibodies were observed. Analysis of multiplication kinetics of C167 and XJC13 in different cell cultures in vitro has confirmed that the attenuated phenotype of C167 was related to a specific inefficient replication in murine cells. This host-range restriction was due to a combination of adsorption and penetration blockage.

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.

Modification of Junin virus neutropism in the guinea pig model.

Virulent and attenuated Junin virus (JV) strains have been employed to study the influence of virus passage history on the neurotropism for guinea pigs. Five i.p. successive passages (P1-P5) of the pathogenic JV-XJ strain and of the attenuated XJO variant were performed in guinea pig spleen. Viral titrations of organ suspensions were made through P1-P5 passages. The XJ strain produced a widespread infection in P1 guinea pigs with viral dissemination to all organs except brain, in P5 animals the brain has been involved as well. XJO-infected P1 guinea pigs showed lower viral titres than XJ-infected P1 animals, and again, the virus reached the CNS in P5 only. The passaging by i.p. route was shown to enhance CNS invasivity of the XJ strain as well as to maintain the XJO neurotropism for guinea pigs. Neurotropism of both strains seemed somewhat affected by the passage history of the virus and the inoculation route appeared critical for its expression. In addition, the neurotropic potential of the attenuated strains has apparently remained unaltered.

Early protection to Junín virus of guinea pig with an attenuated Junín virus strain.

Inoculation of guinea pigs with attenuated XJO Junín virus (JV) strain confers protection against challenge with pathogenic XJ-JV strain starting as early as 3 days post infection (p.i.). The protection increased continuously up to 100% by 30 days p.i. Neither stimulation of non-specific cell mediated mechanisms by previous BCG sensitization nor circulating interferon (IFN) seemed essential for such protection. The early detection of the virus in guinea pig organs considered the site of primary JV multiplication suggests that early resistance phenomenon was attributed mainly to direct viral interference.

[pH-dependent fusion of eukaryotic cells, caused by arenaviruses, pathogenic and non-pathogenic for humans]. / pH-zavisimoe sliianie éukarioticheskikh kletok, vyzyvaemoe patogennymii nepatogennymi dlia cheloveka arenavirusami.

The conditions necessary for fusion from inside (FFWI) of the BHK-21 cell culture affected by the Lassa and Mopeya arenaviruses were studied. The fusion was shown to occur only in the slightly acid medium and at lower pH meanings for the Mopeya virus, than for the Lassa virus.

[Attenuation in the virulence of a mutant of Junin virus in suckling mice]. / Atenuación en la virulencia para ratón lactante de una mutante de virus Junín.

The virulence in neonatal mice of a temperature-sensitive mutant of Junin virus, named C167, was studied. The thermosensitive properties of this mutant were tested by titration on Vero cells at 37 and 40 degrees C. The ratio of infectivity 40/37 was approximately 100-fold lower for C167 with respect to XJC13 (Table 1). The attenuation of C167 was determined by measurement of mean survival time and 50% lethal dose after intracerebral injection of 2 and 11 day old mice. For C167 the lethality index (expressed ad the ratio TCID50/LD50) was greater than 580, while for XJC13 the index was 4.4 (Table 2). The lack of virulence of C167 was correlated with a restricted ability to replicate in suckling mouse brains. By contrast, the mutant and the parental virus grew to a similar titre in Vero cells (Figure 2). Both viruses were indistinguishable in cross-neutralization tests using hyperimmune antisera.

Attenuation parameters for Junin virus in the newborn rat.

To characterize a virus strain as attenuated, both biologic and biochemical criteria are necessary. In the case of Junin virus, the 2-day-old rat has proved to be a biologic attenuation marker as regards mortality. Here we studied the behaviour of the prototype XJ vs the attenuated XJC13 strain inoculated by either ic or ip route to determine differential hematologic and splenic parameters. Humoral immune response against SRBC was also investigated. By either route XJ caused significant leucocytosis, while the other hematologic parameters remained unchanged. No alterations were found following XJC13 infection. XJ produced significant splenomegaly whereas XJC13 had no effect. Similarly PFC anti-SRBC count was decreased during XJ infection but not after XJC13 infection. These differences between the pathogenic XJ and the attenuated XJC13 strain may be attributed to the former's greater spread. The drop in PFC could be due to spleen dysfunction and/or viral effects on the cell subpopulation involved.

[Pathogenicity of the Lassa virus for laboratory mice]. / Patogennost' virusa Lassa dlia laboratornykh myshei.

Pathogenicity for randombred and inbred mice of various age groups of the standard Lassa virus and the virus enriched with defective interfering particles (DIP) was studied. The standard Lassa virus inoculated intracerebrally caused 100% death of C3H/Sn mice aged up to 4 weeks and 60%-70% death of randombred white mice aged 3-4 weeks. BALB/C mice were found to be nonsusceptible to the virus, and its lethality for C57BL/6 and AKR mice varied within the range of 30%-60%. Lassa virus enriched with DIP caused no death of the susceptible animals and showed poor protective activity against the standard virus.

[Experimental evaluation of the pathogenicity of Lassa virus variants]. / Eksperimental'naia otsenka patogennosti variantov virusa Lassa.

Pathogenicity for guinea pigs and white mice of various Lassa virus variants: native, having had 1 passage in Vero cell culture and 4 passages in newborn white mouse brain; a virus having gone through 10 passages in Vero cells and 8 mouse brain passages (variant No. 10); a small-plaque clone derived from variant No. 10 by the method of plaque-to-plaque cloning (variant No. 11k), was studied. Both the native virus and variant No. 10 were found to be similarly pathogenic for susceptible laboratory animals, while the small-plaque variant of Lassa virus became non-fatal for guinea pigs and white mice. The decline of pathogenic properties in variant No. 11k was shown not to be associated with the presence of temperature-sensitive mutants or defective interfering particles.

[The isolation and characteristics of reassortants between the Lassa and Mopeia arenaviruses]. / Poluchenie i kharakteristika reassortantov mezhdu arenavirusami Lassa i Mopeiia.

Reassortants with a mixed phenotype were produced by combined inoculation of Vero cells with Lassa and Mopeya viruses. These reassortants produced small plaques (Mopeya virus phenotype) and were not pathogenic for newborn mice (Lassa virus phenotype). The genotype of the reassortants was studied by dot hybridization experiments on filters using cDNA-probes differentiating genome segments of these viruses. The reassortants were shown to have Mopeya virus L-RNA and Lassa virus S-RNA.

[Factors affecting plaque formation by Lassa virus in Vero cells]. / Faktory, vliiaiushchie na obrazovanie bliashek virusom Lassa na kletkakh Vero.

The method of Porterfield and Allison was adapted for titration of the infectious activity of Lassa virus by the plaque formation in Vero cells. The virus was cloned, and the effect of the time of adsorption, pH, temperature, as well as polycations (DEAD-dextran, protamine sulphate) dimethylsuphoxide (DMSO), and trypsin added during adsorption or into the agar overlay on the effectiveness of plaque production by Lassa virus (virus titres, plaque size) were studied. The optimal adsorption time was found to be 1 1/2-2 hours, pH 8.0. The number of plaques produced by the virus was approximately similar at 35 degrees C. The substances under study did not enhance the efficacy of plaque formation, on the contrary, DMSO and high concentrations of polycations decreased plaque size.