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.

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.

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.

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.

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.

Molecular and cell biology of the prototypic arenavirus LCMV: implications for understanding and combating hemorrhagic fever arenaviruses.

Arenaviruses merit interest as experimental model systems to study virus-host interactions and as clinically important human pathogens. Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) in humans. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen. Moreover, arenaviruses pose a biodefense threat. No licensed arenavirus vaccines are available, and current therapy is limited to the use of ribavirin, which is only partially effective and associated with significant side effects. The development of arenavirus reverse genetics systems has made it possible to manipulate the arenavirus genome, which is contributing to significant progress in understanding arenavirus molecular and cell biology, as well as arenavirus-host interactions underlying arenavirus-induced HF disease in humans. This, in turn, should facilitate the development of novel both vaccines and antiviral drugs to combat the dual threats of naturally occurring and intentionally introduced arenavirus infections.

[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.

Receptor use by pathogenic arenaviruses.

The arenavirus family contains several important human pathogens including Lassa fever virus (LASV), lymphocytic choriomeningitis virus (LCMV) and the New World clade B viruses Junin (JUNV) and Machupo (MACV). Previously, alpha-dystroglycan (alpha-DG) was identified as a receptor recognized by LASV and certain strains of LCMV. However, other studies have suggested that alpha-DG is probably not used by the clade B viruses, and the receptor(s) for these pathogens is currently unknown. Using pseudotyped retroviral vectors displaying arenavirus glycoproteins (GPs), we are able to explore the role played by the GP in viral entry in the absence of other viral proteins. By examining the ability of the vectors to transduce DG knockout murine embryonic stem (ES) cells, we have confirmed that LASV has an absolute requirement for alpha-DG in these cells. However, the LCMV GP can still direct substantial entry into murine ES cells in the absence of alpha-DG, even when the GP from the clone 13 variant is used that has previously been reported to be highly dependent on alpha-DG for entry. We also found that neither LASV or LCMV pseudotyped vectors were able to transduce human or murine lymphocytes, presumably due to the glycosylation state of alpha-DG in these cells. In contrast, the JUNV and MACV GPs displayed broad tropism on human, murine and avian cell types, including lymphocytes, and showed no requirement for alpha-DG in murine ES cells. These findings highlight the importance of molecules other than alpha-DG for arenavirus entry. An alternate receptor is present on murine ES cells that can be used by LCMV but not by LASV, and which is not available on human or murine lymphocytes, while a distinct and widely expressed receptor(s) is used by the clade B viruses.

[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.

Hemorrhagic fever viruses as biological weapons: medical and public health management.

OBJECTIVE: To develop consensus-based recommendations for measures to be taken by medical and public health professionals if hemorrhagic fever viruses (HFVs) are used as biological weapons against a civilian population. PARTICIPANTS: The Working Group on Civilian Biodefense included 26 representatives from academic medical centers, public health, military services, governmental agencies, and other emergency management institutions. EVIDENCE: MEDLINE was searched from January 1966 to January 2002. Retrieved references, relevant material published prior to 1966, and additional sources identified by participants were reviewed. CONSENSUS PROCESS: Three formal drafts of the statement that synthesized information obtained in the evidence-gathering process were reviewed by the working group. Each draft incorporated comments and judgments of the members. All members approved the final draft. CONCLUSIONS: Weapons disseminating a number of HFVs could cause an outbreak of an undifferentiated febrile illness 2 to 21 days later, associated with clinical manifestations that could include rash, hemorrhagic diathesis, and shock. The mode of transmission and clinical course would vary depending on the specific pathogen. Diagnosis may be delayed given clinicians' unfamiliarity with these diseases, heterogeneous clinical presentation within an infected cohort, and lack of widely available diagnostic tests. Initiation of ribavirin therapy in the early phases of illness may be useful in treatment of some of these viruses, although extensive experience is lacking. There are no licensed vaccines to treat the diseases caused by HFVs.

Experimental infection of the cane mouse Zygodontomys brevicauda (family Muridae) with guanarito virus (Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever.

Chronic infections in specific rodents appear to be crucial to the long-term persistence of arenaviruses in nature. The cane mouse, Zygodontomys brevicauda, is a natural host of Guanarito virus (family Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever. The purpose of this study was to elucidate the natural history of Guanarito virus infection in Z. brevicauda. Thirty-nine laboratory-reared cane mice each were inoculated subcutaneously with 3.0 log10 plaque-forming units of the Guanarito virus prototype strain INH-95551. No lethality was associated with infection in any animal, regardless of age at inoculation. The 13 newborn, 14 weanling, and 8 of the 12 adult animals developed chronic viremic infections characterized by persistent shedding of infectious virus in oropharyngeal secretions and urine. These findings indicate that Guanarito virus infection in Z. brevicauda can be chronic and thus support the concept that this rodent species is the natural reservoir of Guanarito virus.

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.

Infeccao humana adquirida em laboratorio causada pelo virus SPH 114202 (Arenavirus: familia Arenaviridae): aspectos clinicos e laboratoriais / Laboratory acquired infection by the virus SP H 114202 (Arenavirus : Arenaviridae) : clinical and laboratory findings

Sao descritos os achados clinico-laboratoriais da infeccao acidental pelo virus SP H 114202 (Arenavirus, familia Arenaviridae), um virus novo causador de febre hemorragica humana. O paciente, tecnico de laboratorio, apresentou quadro febril por 13 dias. A doenca cursou com febre elevada (39ºC) diaria, cefaleia, calefrios e mialgias por 8 dias. A partir do 3§ dia surgiram nauseas, vomitos alimentares e anorexia e no 10§ dia, epigastralgia, diarreia e gengivorragia....

[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.

Fiebre hemorrágica en Guanarito / Hemorrhagic fever in Guanarito

En los últimos cuatro meses de 1989, comienzan a consultar al Hospital Universitario "Dr. Miguel Oráa", en Guanare, Estado Portuguesa, Venezuela, pacientes agricultores en su mayoría procedentes del Municipio Guanarito, con cuadro clínico caracterizado por fiebre, postración deshidratación, malestar general, cefalea, odinofagia, manifestaciones hemorrágicas (gingivorragias, hematemesis, epistaxis), alteraciones de laboratorio compatibles con leucopenia y trombocitopenia, los cuales fallecieron a los pocos días de ingresados en estado de shock sépticos o a consecuencia de severas manifestaciones neurológicas. A lo largo de 1990 se continuaron observando nuevos casos y en septiembre de 1990 fue aislado el virus de un caso fatal, en el Departamento de Virología del Instituto Nacional de Higiene "Rafael Rangel" de Caracas-Venezuela y posteriormente identificado por la Unidad de Investigación de Arbovirus de Yale y el Instituto de Investigación de Enfermedades Infecciosas de las Fuerzas Armadas de los E.E.U.U., indicando que este Virus es un nuevo miembro de la familia Arenaviridae perteneciente al complejo Tacaribe. Con el interés de conocer aspectos epidemiológicos, clínicos, anatomopatológicos y de laboratorio de esta nueva entidad, analizamos todos aquellos casos que ingresaron al Hospital Universitario "Dr. Miguel Oráa" en el lapso comprendido entre el 1- de enero de 1989 al 31 de diembre de 1991, que cumplieran con los siguientes criterios: 1) manifestaciones clínicas de fiebre, cefalea, malestar general, odionofagia, manifestaciones hemorrágicas por mucosas u orificios naturales alteraciones neurológicas como temblor y convulsiones; 2) alteraciones de laboratorio como leucocitos menor de 4000 xmm3 y plaquetas menor de 140.000 xmm3.; 3) procedencia de la zona geográfica correspondiente al Municipio Guanarito; 4) presencia de cultivos de tejido o sangre o serología para Arenavirus. Se recopila una muestra de 188 casos, 62% del sexo masculino, dedicados en su gran mayoría a la actividad agricola 43.6%. el grupo etario más afectado estaba comprendido entre 15 y 44 años (67%). Hubo 41 muertos (21%), las cuales en su gran mayoría presentaron en la autopsia hemorrágica pulmonar, edema renal, hepatomegalia, hemorragia gastrointestinal, neumoní, esplenomegalia, y cardiomegalia. Hallazgos que semejan reportes de la literatura de la fiebre de Lassa, la Argentina y la Boliviana

[The Mopeia virus induces pH-dependent "fusion from within" in a BHK-21 cell culture]. / Virus Mopeiia indutsiruet pH-zavisimoe "sliianie iznutri" kul'tury kletok BHK-21.

The capacity of BHK-21 cell culture to produce Mopeia virus (Arenaviridae family) to form syncytium upon acidification of the culture medium to pH 5.5 and lower was demonstrated. The cell fusion requires their active virus production: a virus titre in the culture medium must be at least 10(5) PEU/ml. The inhibition of virus multiplication with ammonium chloride as well as treatment of the cells before the medium acidification with immune serum reduced syncytium formation markedly. No cell fusion was observed upon acidification of the medium immediately after virus adsorption to cells. Thus, the observed cell fusion under the influence of the virus is an "internal fusion" and confirms our previous data on the endocytosis mode of arenavirus penetration into the cell.

[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.