Arenavirus Research Articles

An attenuated Machupo virus with a disrupted L-segment intergenic region protects guinea pigs against lethal Guanarito virus infection

Machupo virus (MACV) is a New World (NW) arenavirus and causative agent of Bolivian hemorrhagic fever (HF). Here, we identified a variant of MACV strain Carvallo termed Car91 that was attenuated in guinea pigs. Infection of guinea pigs with an earlier passage of Carvallo, termed Car68, resulted in a lethal disease with a 63% mortality rate. Sequencing analysis revealed that compared to Car68, Car91 had a 35 nucleotide (nt) deletion and a point mutation within the L-segment intergenic region (IGR), and three silent changes in the polymerase gene that did not impact amino acid coding. No changes were found on the S-segment. Because it was apathogenic, we determined if Car91 could protect guinea pigs against Guanarito virus (GTOV), a distantly related NW arenavirus. While naïve animals succumbed to GTOV infection, 88% of the Car91-exposed guinea pigs were protected. These findings indicate that attenuated MACV vaccines can provide heterologous protection against NW arenaviruses. The disruption in the L-segment IGR, including a single point mutant and 35 nt partial deletion, were the only major variance detected between virulent and avirulent isolates, implicating its role in attenuation. Overall, our data support the development of live-attenuated arenaviruses as broadly protective pan-arenavirus vaccines.

Assembly of Arena virus an Ultra structural Perspective

The Arenaviredae are a family of viruses whose members are generally associated with rodent transmitted disease in humans which currently comprises 24 viral species. Arenavirus infections are relatively common in humans in some areas of the world and can cause severe illnesses including several haemorrhagic fevers. The virus particles vary in diameter from 60 to more than 300 nm. They are spherical and have a reported average diameter of 92 nanometres. All are enveloped in a lipid bilayer and have a bisegmented ambisense RNA genome, but relatively little is known about how virions are assembled and how virion structure relates to transmissibility. To investigate the role of each viral structural protein in forming and maintaining the structure of the virion, we have imaged particles of arenaviruses LCMV, PICV and TCRV, and compared their shape and structural characteristics to similar sized phospholipid vesicles. A very strong association between particle size and shape was found for all arenavirus particles: small virions were significantly rounder than vesicles of similar size, while large particles tended to be more elliptical in appearance. The natural variation in surface glycoprotein decoration and ribonucleoprotein incorporation was then measured. From this data it was concluded that there is no strong evidence relating particles size to decoration for arenaviruses as a group, but we did detect significant correlations between internal density and virion shape. Overall, we are able to conclude that small virions are round and relatively rigid compared to vesicles of the same size, while large virions are not. By comparing relative density of the membrane- proximal region it was discovered that arenavirus shape is controlled by complexes containing GPC, Z and NP at the surface of the virion, and that an unbroken inner shell of NP is essential for maintaining a rigid spherical shape. Furthermore, it was revealed that the inner leaflet of intact arenaviruses has a lower density than the inner leaflet of vesicles consistent with the interpretation that viral proteins are displaying lipid molecules from the inner leaflet of the viral membrane. These data provide a new way of assessing the function of viral protein interactions on virion structure and may be of use in designing antiviral drugs that act at the level of virion structure.

Activation of the RLR/MAVS Signaling Pathway by the L Protein of Mopeia Virus.

Distinct innate immune responses are observed when hosts are infected with different arenaviruses. It has been widely accepted that NP and certain Z proteins of arenaviruses inhibit the RLR/MAVS signaling pathway. The viral components responsible for the activation of the RLR/MAVS signaling pathway remain to be determined. In the current study, we demonstrate for the first time that the Lp of MOPV, an OW arenavirus, can activate the RLR/MAVS signaling pathway and thus induce the production of IFN-I. Based on our results, we proposed that dynamic interactions exist among Lp-produced RNA, NP, and the RLR/MAVS signaling pathway, and the outcome of these interactions may determine the final IFN-I response pattern: elevated or reduced. Our study provides a possible explanation for how IFN-I can become activated during arenavirus infection and may help us gain insights into the interactions that form between different arenavirus components and the innate immune system.

Role of Epidemiology of Arena Viruses

Role of Epidemiology of Arena Viruses

The Role of Arena Virus Protein and Their Molar Ratio

The Role of Arena Virus Protein and Their Molar Ratio

Highly Pathogenic New World and Old World Human Arenaviruses Induce Distinct Interferon Responses in Human Cells.

The arenavirus family includes several important pathogens that cause severe and sometimes fatal diseases in humans. The highly pathogenic Old World (OW) arenavirus Lassa fever virus (LASV) is the causative agent of Lassa fever (LF) disease in humans. LASV infections in severe cases are generally immunosuppressive without stimulating interferon (IFN) induction, a proinflammatory response, or T cell activation. However, the host innate immune responses to highly pathogenic New World (NW) arenaviruses are not well understood. We have previously shown that the highly pathogenic NW arenavirus, Junin virus (JUNV), induced an IFN response in human A549 cells. Here, we report that Machupo virus (MACV), another highly pathogenic NW arenavirus, also induces an IFN response. Importantly, both pathogenic NW arenaviruses, in contrast to the OW highly pathogenic arenavirus LASV, readily elicited an IFN response in human primary dendritic cells and A549 cells. Coinfection experiments revealed that LASV could potently inhibit MACV-activated IFN responses even at 6 h after MACV infection, while the replication levels of MACV and LASV were not affected by virus coinfection. Our results clearly demonstrated that although all viruses studied herein are highly pathogenic to humans, the host IFN responses toward infections with the NW arenaviruses JUNV and MACV are quite different from responses to infections with the OW arenavirus LASV, a discovery that needs to be further investigated in relevant animal models. This finding might help us better understand various interplays between the host immune system and highly pathogenic arenaviruses as well as distinct mechanisms underlying viral pathogenesis. Infections of humans with the highly pathogenic OW LASV are accompanied by potent suppression of interferon or proinflammatory cytokine production. In contrast, infections with the highly pathogenic NW arenavirus JUNV are associated with high levels of IFNs and cytokines in severe and fatal cases. Arenaviruses initially target macrophages and dendritic cells, which are potent IFN/cytokine-producers. In human macrophages, JUNV reportedly does not trigger IFN responses. We here demonstrated that JUNV activated IFN responses in human dendritic cells. MACV, another highly pathogenic NW arenavirus, also activated IFN responses. LASV did not induce detectable IFN responses, in spite of higher replication levels, and blocked the MACV-triggered IFN response in a coinfection assay. Although these viruses are highly pathogenic to humans, our study highlights distinct innate immune responses to infections with the NW arenaviruses JUNV and MACV and to infection with the OW arenavirus LASV and provides important insights into the virus-host interaction and pathogenesis.

Analysis of Two Different Sequences of Old Arena Viruses by Decision Tree, Apriori Algorithm, and Shannon Entropy

Analysis of Two Different Sequences of Old Arena Viruses by Decision Tree, Apriori Algorithm, and Shannon Entropy

Human and host species transferrin receptor 1 use by North American arenaviruses.

At least five New World (NW) arenaviruses cause hemorrhagic fevers in South America. These pathogenic clade B viruses, as well as nonpathogenic arenaviruses of the same clade, use transferrin receptor 1 (TfR1) of their host species to enter cells. Pathogenic viruses are distinguished from closely related nonpathogenic ones by their additional ability to utilize human TfR1 (hTfR1). Here, we investigate the receptor usage of North American arenaviruses, whose entry proteins share greatest similarity with those of the clade B viruses. We show that all six North American arenaviruses investigated utilize host species TfR1 orthologs and present evidence consistent with arenavirus-mediated selection pressure on the TfR1 of the North American arenavirus host species. Notably, one of these viruses, AV96010151, closely related to the prototype Whitewater Arroyo virus (WWAV), entered cells using hTfR1, consistent with a role for a WWAV-like virus in three fatal human infections whose causative agent has not been identified. In addition, modest changes were sufficient to convert hTfR1 into a functional receptor for most of these viruses, suggesting that a minor alteration in virus entry protein may allow these viruses to use hTfR1. Our data establish TfR1 as a cellular receptor for North American arenaviruses, highlight an "arms race" between these viruses and their host species, support the association of North American arenavirus with fatal human infections, and suggest that these viruses have a higher potential to emerge and cause human diseases than has previously been appreciated. hTfR1 use is a key determinant for a NW arenavirus to cause hemorrhagic fevers in humans. All known pathogenic NW arenaviruses are transmitted in South America by their host rodents. North American arenaviruses are generally considered nonpathogenic, but some of these viruses have been tentatively implicated in human fatalities. We show that these North American arenaviruses use the TfR1 orthologs of their rodent host species and identify TfR1 polymorphisms suggesting an ongoing "arms race" between these viruses and their hosts. We also show that a close relative of a North American arenavirus suggested to have caused human fatalities, the Whitewater Arroyo species complex virus AV96010151, uses human TfR1. Moreover, we present data that imply that modest changes in other North American arenaviruses might allow these viruses to infect humans. Collectively, our data suggest that North American arenaviruses have a higher potential to cause human disease than previously assumed.

A Kinase Inhibitor Cocktail as a Prophylaxis in the Syrian Golden Hamster Pirital Virus Model

Arena viruses are negative strand RNA viruses that have the potential to cause a wide spectrum of disease including hemorrhagic fever in humans and experimental animals. There are currently limited therapeutic treatments available for arena virus infection, which includes ribavirin and in some cases immune plasma. Therapeutic research testing the efficacy of products on arena virus infection can be difficult because work with the arena viruses that cause hemorrhagic fever require Biosafety level (BSL)-4 containment. However, surrogate arena virus animal models have been developed that can be utilized as a screening tool to test product efficacy of antivirals that may have broad acting anti-arena viral activity. Because there is a strong need to develop novel prophylaxis and therapeutics, we have utilized the Syrian golden hamster Pirital virus (PIRV) surrogate model to test antiviral efficacy in a BSL-3 environment. PIRV is a New World arena virus that is not known to cause human disease, but it is capable of causing hemorrhagic fever, morbidity, and complete mortality in the Syrian golden hamster. Thus, we infected hamsters with PIRV and treated with two different dosages of a kinase inhibitor cocktail consisting of genistein and tyrphostin AG1478. Drug efficacy was determined by evaluating physical signs of disease, mortality, viral titers in specific tissues, and terminal viremia. In this study, treating PIRV-infected hamsters with a kinase inhibitor cocktail led to a significant survival rate, lower viral titers, and an absence of viremia in survivors. In all, the results demonstrate the potential of the cocktail as an antiviral against arena virus infection.

La fièvre de Lassa: réponses immunes et pathogenèse.

Lassa fever is a hemorrhagic fever endemic to West Africa and caused by Lassa virus, an Old-World arenavirus. It may be fatal, but most patients recover from acute disease and some experience asymptomatic infection. The immune mechanisms associated with these different outcomes have not yet been fully elucidated, but considerable progress has recently been made, through the use of in vitro human models and non-human primates, the only relevant animal model that mimics the pathophysiology and immune responses induced in patients. We discuss here the roles of the various components of the innate and adaptive immune systems in Lassa virus infection and in the control of viral replication and pathogenesis.

Complex patterns of host switching in New World arenaviruses

We empirically tested the long-standing hypothesis of codivergence of New World arenaviruses (NWA) with their hosts. We constructed phylogenies for NWA and all known hosts and used them in reconciliation analyses. We also constructed a phylogenetic tree of all Sigmodontinae and Neotominae rodents and tested whether viral-host associations were phylogenetically clustered. We determined host geographical overlap to determine to what extent opportunity to switch hosts was limited by host relatedness or physical proximity. With the exception of viruses from North America, no phylogenetically codivergent pattern between NWA and their hosts was found. We found that different virus clades were clustered differently and that Clade B with members pathogenic to humans was randomly distributed across the rodent phylogeny. Furthermore, viral relatedness within Clade B was significantly explained by the geographic overlap of their hosts' ranges rather than host relatedness, indicating that they are capable of host switching opportunistically. This has important bearings on their potential to become panzootic. Together, these analyses suggest that NWA have not codiverged with their hosts and instead have evolved predominantly via host switching.

Development of a New Tacaribe Arenavirus Infection Model and Its Use to Explore Antiviral Activity of a Novel Aristeromycin Analog

BackgroundA growing number of arenaviruses can cause a devastating viral hemorrhagic fever (VHF) syndrome. They pose a public health threat as emerging viruses and because of their potential use as bioterror agents. All of the highly pathogenic New World arenaviruses (NWA) phylogenetically segregate into clade B and require maximum biosafety containment facilities for their study. Tacaribe virus (TCRV) is a nonpathogenic member of clade B that is closely related to the VHF arenaviruses at the amino acid level. Despite this relatedness, TCRV lacks the ability to antagonize the host interferon (IFN) response, which likely contributes to its inability to cause disease in animals other than newborn mice.Methodology/Principal FindingsHere we describe a new mouse model based on TCRV challenge of AG129 IFN-α/β and -γ receptor-deficient mice. Titration of the virus by intraperitoneal (i.p.) challenge of AG129 mice resulted in an LD50 of ∼100 fifty percent cell culture infectious doses. Virus replication was evident in the serum, liver, lung, spleen, and brain 4–8 days after inoculation. MY-24, an aristeromycin derivative active against TCRV in cell culture at 0.9 µM, administered i.p. once daily for 7 days, offered highly significant (P<0.001) protection against mortality in the AG129 mouse TCRV infection model, without appreciably reducing viral burden. In contrast, in a hamster model of arenaviral hemorrhagic fever based on challenge with clade A Pichinde arenavirus, MY-24 did not offer significant protection against mortality.Conclusions/SignificanceMY-24 is believed to act as an inhibitor of S-adenosyl-L-homocysteine hydrolase, but our findings suggest that it may ameliorate disease by blunting the effects of the host response that play a role in disease pathogenesis. The new AG129 mouse TCRV infection model provides a safe and cost-effective means to conduct early-stage pre-clinical evaluations of candidate antiviral therapies that target clade B arenaviruses.

Z Proteins of New World Arenaviruses Bind RIG-I and Interfere with Type I Interferon Induction

The retinoic acid-inducible gene I product (RIG-I) is a cellular sensor of RNA virus infection that regulates the cellular beta interferon (IFN-beta) response. The nucleoproteins (NP) of arenaviruses are reported to antagonize the IFN response by inhibiting interferon regulatory factor 3 (IRF-3). Here, we demonstrate that the Z proteins of four New World (NW) arenaviruses, Guanarito virus (GTOV), Junin virus (JUNV), Machupo virus (MAVC), and Sabia virus (SABV), bind to RIG-I, resulting in downregulation of the IFN-beta response. We show that expression of the four NW arenavirus Z proteins inhibits IFN-beta mRNA induction in A549 cells in response to RNA bearing 5' phosphates (5'pppRNA). NW arenavirus Z proteins interact with RIG-I in coimmunoprecipitation studies and colocalize with RIG-I. Furthermore, expression of Z proteins interferes with the interaction between RIG-I and MAVS. Z expression also impedes the nuclear factor kappa light chain enhancer of activated B cells (NF-kappaB) and IRF-3 activation. Our results indicate that NW arenavirus Z proteins, but not Z protein of the Old World (OW) arenavirus lymphocytic choriomeningitis virus (LCMV) or Lassa virus, bind to RIG-I and inhibit downstream activation of the RIG-I signaling pathway, preventing the transcriptional induction of IFN-beta.

Change in blood test after irradiation with high doses for inactivation of Lassa virus

In 2006 we had a patient with Lassa fever in the University Hospital of Frankfurt. To insure a short turn-around time from the laboratory, it was necessary to have blood which was not contaminated with viruses. One method of achieving this is by irradiating the blood with high doses of ionising radiation. Inactivation of arena viruses requires doses between 12 kGy and 20 kGy, depending on temperature. In this study we investigated if plasma and serum parameters are changed by irradiation with 5 kGy, 10 kGy, 15 kGy, 20 kGy or 40 kGy of 10 MeV electrons. For the 22 serum parameters measured there was no influence of radiation up to 20 kGy. Only at 40 kGy was a significant decrease noted. For the six plasma parameters the values were significantly dose dependent. To correct this a mathematical function was defined. It is possible to inactivate Lassa virus with high doses of radiation. Most of the measured blood values don't change. For those which were influenced it was possible to define a mathematical function.

Host-species transferrin receptor 1 orthologs are cellular receptors for nonpathogenic new world clade B arenaviruses.

The ability of a New World (NW) clade B arenavirus to enter cells using human transferrin receptor 1 (TfR1) strictly correlates with its ability to cause hemorrhagic fever. Amapari (AMAV) and Tacaribe (TCRV), two nonpathogenic NW clade B arenaviruses that do not use human TfR1, are closely related to the NW arenaviruses that cause hemorrhagic fevers. Here we show that pseudotyped viruses bearing the surface glycoprotein (GP) of AMAV or TCRV can infect cells using the TfR1 orthologs of several mammalian species, including those of their respective natural hosts, the small rodent Neacomys spinosus and the fruit bat Artibeus jamaicensis. Mutation of one residue in human TfR1 makes it a functional receptor for TCRV, and mutation of four residues makes it a functional receptor for AMAV. Our data support an in vivo role for TfR1 in the replication of most, if not all, NW clade B arenaviruses, and suggest that with modest changes in their GPs the nonpathogenic arenaviruses could use human TfR1 and emerge as human pathogens.

Arenavirus deaths--emergency air services tighten up.

Arenavirus deaths--emergency air services tighten up.

A New Arenavirus in Transplantation

A New Arenavirus in Transplantation

Synthesis and Evaluation of Octadecyloxyethyl Esters of Five 3-Hydroxy-2-(Phosphonomethoxy)Propyl Nucleoside Phosphonates in HIV-1 Infected Cells

Several arenaviruses, including Lassa and Lujo viruses in Africa and five New World arenavirus (NWA) species in the Americas, cause life-threatening viral hemorrhagic fevers. In the absence of licensed antiviral therapies, these viruses pose a significant public health risk. The envelope glycoprotein complex (GPC) mediates arenavirus entry through a pH-dependent fusion of the viral and host endosomal membranes. It thus is recognized as a viable target for small-molecule fusion inhibitors. Here, we report on the antiviral activity and pre-clinical development of the novel broad-spectrum arenavirus fusion inhibitors, ARN-75039 and ARN-75041. In Tacaribe virus (TCRV) pseudotyped and native virus assays, the ARN compounds were active in the low to sub-nanomolar range with selectivity indices exceeding 1000. Pharmacokinetic analysis of the orally administered compounds revealed an extended half-life in mice supporting once-daily dosing, and the compounds were well tolerated at the highest tested dose of 100 mg/kg. In a proof-of-concept prophylactic efficacy study, doses of 10 and 35 mg/kg of either compound dramatically improved survival outcome and potently inhibited TCRV replication in serum and various tissues. Additionally, in contrast to surviving mice that received ribavirin or placebo, animals treated with ARN-75039 or ARN-75041 were cured of TCRV infection. In a follow-up study with ARN-75039, impressive therapeutic efficacy was demonstrated under conditions where treatment was withheld until after the onset of disease. Taken together, the data strongly support the continued development of ARN-75039 as a candidate therapeutic for the treatment of severe arenaviral diseases.

Lymphocytic Choriomeningitis Virus (LCMV): Propagation, Quantitation, and Storage

AbstractLymphocytic choriomeningitis virus (LCMV) is an enveloped, ambisense RNA virus and the prototypic virus of the arenavirus group. It can cause viral meningitis and other ailments in humans, but its natural host is the mouse. The LCMV/mouse model has been useful for examining mechanisms of viral persistence and the basic concepts of virus‐induced immunity and immunopathology. This unit discusses strain differences and biosafety containment issues for LCMV. Recommendations are made for techniques for propagating LCMV to high titers to quantify it by plaque assay and PCR techniques and to preserve its infectivity by appropriate storage. Curr. Protoc. Microbiol. 8:15A.1.1‐15A.1.11. © 2008 by John Wiley &amp; Sons, Inc.

Immune Response to Vaccination Against Argentine Hemorrhagic Fever in an Area Where Different Arenaviruses Coexist

Neutralizing antibody (NT Ab) titers to Candid #1 (C#1) vaccine against Argentine hemorrhagic fever were studied for 2 years post-vaccination in 330 volunteers, to assess whether the kinetics and/or magnitude of this immune response is modified by previous infection with the arena viruses Junin (JUN) and lymphocytic choriomeningitis (LCM). A total of 160 volunteers received C#1, distributed as follows: without detectable pre-infection with arenaviruses (n = 54); with pre-existing antibodies to JUN (n = 55); with pre-existing antibodies to LCM (n = 51). The remaining 170 individuals received placebo. Levels of anti-JUN NT Ab displayed a trend in which titers increased with the virulence of the infecting strain, from C#1 (X = 49), through subclinical JUN infection (X = 229), vaccination following subclinical infection (X = 367) to JUN clinical infection (X =773). It was also found that the mean titer of NT Ab to C#1 did not vary significantly during 2 years of study and was: a) significantly lower than that elicited by wild strains of JUN, both clinical and subclinical infections (p < 0.01); b) significantly increased the titers of pre-existing anti-JUN Ab (p < 0.01); and c) was not modified by pre-existing anti-LCM Ab (p > 0.05). These data indicate that the immune response to C#1 boosts pre-existing immunity to JUN virus and is not changed by previous experience with LCM virus.