Past, present and future therapeutic and prophylactic strategies against arenaviruses responsible of human hemorrhagic fever.

For most viral hemorrhagic fevers caused by arenaviruses, no prophylactic vaccine is available yet. Only one therapeutic treatment is currently available and should be administered at the early stages of the infection. This is particularly problematic as these diseases are difficult to diagnose and cure. Lassa fever is the most important pathology caused by arenaviruses, including millions of people at risk in West Africa. For decades, promising studies focusing on the development of vaccine candidates targeting Lassa virus have been published, but no vaccine candidate had reached the clinical phase. The second arenavirus in terms of number of human infections is the Junín virus in Argentina. The Junín infected case number has drastically decreased since the use of the Candid #1 vaccine. This review summarizes past and present experimental studies regarding treatments against arenaviruses responsible for human hemorrhagic fevers from a prophylactic and therapeutic point of view. It also discusses future breakthroughs to get available and effective treatments.

Increased Immune Response Variability during Simultaneous Viral Coinfection Leads to Unpredictability in CD8 T Cell Immunity and Pathogenesis.

UNLABELLED: T cell memory is usually studied in the context of infection with a single pathogen in naive mice, but how memory develops during a coinfection with two pathogens, as frequently occurs in nature or after vaccination, is far less studied. Here, we questioned how the competition between immune responses to two viruses in the same naive host would influence the development of CD8 T cell memory and subsequent disease outcome upon challenge. Using two different models of coinfection, including the well-studied lymphocytic choriomeningitis (LCMV) and Pichinde (PICV) viruses, several differences were observed within the CD8 T cell responses to either virus. Compared to single-virus infection, coinfection resulted in substantial variation among mice in the size of epitope-specific T cell responses to each virus. Some mice had an overall reduced number of virus-specific cells to either one of the viruses, and other mice developed an immunodominant response to a normally subdominant, cross-reactive epitope (nucleoprotein residues 205 to 212, or NP205). These changes led to decreased protective immunity and enhanced pathology in some mice upon challenge with either of the original coinfecting viruses. In mice with PICV-dominant responses, during a high-dose challenge with LCMV clone 13, increased immunopathology was associated with a reduced number of LCMV-specific effector memory CD8 T cells. In mice with dominant cross-reactive memory responses, during challenge with PICV increased immunopathology was directly associated with these cross-reactive NP205-specific CD8 memory cells. In conclusion, the inherent competition between two simultaneous immune responses results in significant alterations in T cell immunity and subsequent disease outcome upon reexposure. IMPORTANCE: Combination vaccines and simultaneous administration of vaccines are necessary to accommodate required immunizations and maintain vaccination rates. Antibody responses generally correlate with protection and vaccine efficacy. However, live attenuated vaccines also induce strong CD8 T cell responses, and the impact of these cells on subsequent immunity, whether beneficial or detrimental, has seldom been studied, in part due to the lack of known T cell epitopes to vaccine viruses. We questioned if the inherent increased competition and stochasticity between two immune responses during a simultaneous coinfection would significantly alter CD8 T cell memory in a mouse model where CD8 T cell epitopes are clearly defined. We show that some of the coinfected mice have sufficiently altered memory T cell responses that they have decreased protection and enhanced immunopathology when reexposed to one of the two viruses. These data suggest that a better understanding of human T cell responses to vaccines is needed to optimize immunization strategies.

Past, present, and future of arenavirus taxonomy.

Until recently, members of the monogeneric family Arenaviridae (arenaviruses) have been known to infect only muroid rodents and, in one case, possibly phyllostomid bats. The paradigm of arenaviruses exclusively infecting small mammals shifted dramatically when several groups independently published the detection and isolation of a divergent group of arenaviruses in captive alethinophidian snakes. Preliminary phylogenetic analyses suggest that these reptilian arenaviruses constitute a sister clade to mammalian arenaviruses. Here, the members of the International Committee on Taxonomy of Viruses (ICTV) Arenaviridae Study Group, together with other experts, outline the taxonomic reorganization of the family Arenaviridae to accommodate reptilian arenaviruses and other recently discovered mammalian arenaviruses and to improve compliance with the Rules of the International Code of Virus Classification and Nomenclature (ICVCN). PAirwise Sequence Comparison (PASC) of arenavirus genomes and NP amino acid pairwise distances support the modification of the present classification. As a result, the current genus Arenavirus is replaced by two genera, Mammarenavirus and Reptarenavirus, which are established to accommodate mammalian and reptilian arenaviruses, respectively, in the same family. The current species landscape among mammalian arenaviruses is upheld, with two new species added for Lunk and Merino Walk viruses and minor corrections to the spelling of some names. The published snake arenaviruses are distributed among three new separate reptarenavirus species. Finally, a non-Latinized binomial species name scheme is adopted for all arenavirus species. In addition, the current virus abbreviations have been evaluated, and some changes are introduced to unequivocally identify each virus in electronic databases, manuscripts, and oral proceedings.

Loss of anti-viral immunity by infection with a virus encoding a cross-reactive pathogenic epitope.

T cell cross-reactivity between different strains of the same virus, between different members of the same virus group, and even between unrelated viruses is a common occurrence. We questioned here how an intervening infection with a virus containing a sub-dominant cross-reactive T cell epitope would affect protective immunity to a previously encountered virus. Pichinde virus (PV) and lymphocytic choriomeningitis virus (LCMV) encode subdominant cross-reactive NP205₋212 CD8 T cell epitopes sharing 6 of 8 amino acids, differing only in the MHC anchoring regions. These pMHC epitopes induce cross-reactive but non-identical T cell receptor (TCR) repertoires, and structural studies showed that the differing anchoring amino acids altered the conformation of the MHC landscape presented to the TCR. PV-immune mice receiving an intervening infection with wild type but not NP205-mutant LCMV developed severe immunopathology in the form of acute fatty necrosis on re-challenge with PV, and this pathology could be predicted by the ratio of NP205-specific to the normally immunodominant PV NP38₋45-specific T cells. Thus, cross-reactive epitopes can exert pathogenic properties that compromise protective immunity by impairing more protective T cell responses.

Point mutation in the glycoprotein of lymphocytic choriomeningitis virus is necessary for receptor binding, dendritic cell infection, and long-term persistence.

Arenaviruses are a major cause of hemorrhagic fevers endemic to Sub-Saharan Africa and South America, and thus a major public health and medical concern. The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is widely used as a model system for studying persistent and acute infections, as well as for gaining an understanding of mammalian immune function. When originally characterized three decades ago, the LCMV isolate, Armstrong, which causes an acute infection in adult mice, was found to differ from the LCMV Clone 13 strain that causes a persistent infection by two amino acid changes, one within the virus surface glycoprotein (GP1: F260L) and the other within the virus L polymerase (K1076Q). Mutation F260L was considered solely responsible for the exceptionally strong binding affinity of Clone 13 (L at GP1 260) to its cellular receptor, α-dystroglycan, which among cells of the immune system is preferentially expressed on dendritic cells, and consequently, alters dendritic cell function leading to viral persistence. Recently, we noted a previously overlooked nucleotide difference between these two strains that results in an additional amino acid change in GP1, N176D. To investigate the potential contribution of this newly identified mutation to the Clone 13 phenotype, we used reverse-genetics approaches to generate recombinant LCM viruses with each of these individual mutations. Phenotypic characterization of these rLCMV showed that mutation F260L, but not N176D, in the GP1 of LCMV is essential for mediating the long-term persistence of Clone 13 infections. This work emphasizes the importance of subtle differences in viral strains that determine disease outcomes.

Expanded potential for recombinant trisegmented lymphocytic choriomeningitis viruses: protein production, antibody production, and in vivo assessment of biological function of genes of interest.

The recombinant engineering of trisegmented lymphocytic choriomeningitis virus (LCMV) to express two genes of interest was recently reported. We used this technology to efficiently express green fluorescent protein (GFP) and the immunoregulatory gene product interleukin-10 (IL-10) in vitro, assess IL-10 function in vivo during viral meningitis, and generate specific, robust monoclonal antibody responses to IL-10. Tripartite viruses were attenuated in wild-type and TLR7(-/-) mice. However, IFNAR1(-/-) mice sustained systemic viral replication when 2 nucleotide substitutions from a persistent LCMV variant were present. These findings demonstrate the utility of tripartite LCMV in vitro and in vivo to study genes in the context of a well-defined model system.

Rescue from cloned cDNAs and in vivo characterization of recombinant pathogenic Romero and live-attenuated Candid #1 strains of Junin virus, the causative agent of Argentine hemorrhagic fever disease.

The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), which is associated with high morbidity and significant mortality. Several pathogenic strains of JUNV have been documented, and a highly attenuated vaccine strain (Candid #1) was generated and used to vaccinate the human population at risk. The identification and functional characterization of viral genetic determinants associated with AHF and Candid #1 attenuation would contribute to the elucidation of the mechanisms contributing to AHF and the development of better vaccines and therapeutics. To this end, we used reverse genetics to rescue the pathogenic Romero and the attenuated Candid #1 strains of JUNV from cloned cDNAs. Both recombinant Candid #1 (rCandid #1) and Romero (rRomero) had the same growth properties and phenotypic features in cultured cells and in vivo as their corresponding parental viruses. Infection with rRomero caused 100% lethality in guinea pigs, whereas rCandid #1 infection was asymptomatic and provided protection against a lethal challenge with Romero. Notably, Romero and Candid #1 trans-acting proteins, L and NP, required for virus RNA replication and gene expression were exchangeable in a minigenome rescue assay. These findings support the feasibility of studies aimed at determining the contribution of each viral gene to JUNV pathogenesis and attenuation. In addition, we rescued Candid #1 viruses with three segments that efficiently expressed foreign genes introduced into their genomes. This finding opens the way for the development of a safe multivalent arenavirus vaccine.

Generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest.

Several arenaviruses cause hemorrhagic fever disease in humans for which no licensed vaccines are available and current therapeutic intervention is limited to the off-label use of the wide-spectrum antiviral ribavirin. However, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) has proven to be a Rosetta stone for the investigation of virus-host interactions. Arenaviruses have a bisegmented negative-strand RNA genome. The S segment encodes for the virus nucleoprotein and glycoprotein, whereas the L segment encodes for the virus polymerase (L) and Z protein. The ability to generate recombinant LCMV (rLCMV) expressing additional foreign genes of interest would open novel avenues for the study of virus-host interactions and the development of novel vaccine strategies and high-throughput screens to identify antiarenaviral molecules. To this end, we have developed a trisegmented (1L + 2S) rLCMV-based approach (r3LCMV). Each of the two S segments in r3LCMV was altered to replace one of the viral genes by a gene of interest. All r3LCMVs examined expressing different reported genes were stable both genetically and phenotypically and exhibited wild-type growth properties in cultured cells. Reporter gene expression in r3LCMV-infected cells provided an accurate surrogate of levels of virus multiplication. Notably, some r3LCMVs displayed highly attenuated virulence in mice but induced protective immunity against a subsequent lethal challenge with wild-type LCMV, supporting the potential development of r3LCMV-based vaccines.

Identification of amino acid residues critical for the anti-interferon activity of the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus.

Lymphocytic choriomeningitis virus (LCVM) nucleoprotein (NP) counteracts the host type I interferon (IFN) response by inhibiting activation of the IFN regulatory factor 3 (IRF3). In this study, we have mapped the regions and specific amino acid residues within NP involved in its anti-IFN activity. We identified a region spanning residues 382 to 386 as playing a critical role in the IFN-counteracting activity of NP. Alanine substitutions at several positions within this region resulted in NP mutants that lacked the IFN-counteracting activity but retained their functions in virus RNA synthesis and assembly of infectious particles. We used reverse genetics to rescue a recombinant LCMV strain carrying mutation D382A in its NP [rLCMV/NP*(D382A)]. Compared to wild-type (WT) LCMV, rLCMV/NP*(D382A) exhibited a higher level of attenuation in IFN-competent than IFN-deficient cells. In addition, A549 cells infected with rLCMV/NP*(D382A), but not with WT LCMV, produced IFN and failed to rescue replication of the IFN-sensitive Newcastle disease virus.

Phylogeny of the genus Arenavirus.

The family Arenaviridae consists of a unique genus (Arenavirus) that currently comprises 22 viral species, as recognized by the International Committee for Taxonomy of Viruses. Seven newly discovered represent putative new species. Here, our aims were to provide the most comprehensive phylogenetic analysis of members and putative members of the family Arenaviridae to date, and to investigate the genetic diversity observed within and between recognized species of New world arenaviruses to determine whether the genetic criteria previously proposed to define arenavirus species for Old world arenaviruses should be retained or are more widely applicable to the whole genus.

Mammarenaviruses deleted from their Z gene are replicative and produce an infectious progeny in BHK-21 cells.

Mammarenaviruses bud out of infected cells via the recruitment of the endosomal sorting complex required for transport through late domain motifs localized into their Z protein. Here, we demonstrated that mammarenaviruses lacking this protein can be rescued and are replicative, despite a 3-log reduction in virion production, in BHK-21 cells, but not in five other cell lines. Mutations of putative late domain motifs identified into the viral nucleoprotein resulted in the almost complete abolition of infectious virion production by Z-deleted mammarenaviruses. This result strongly suggested that the nucleoprotein may compensate for the deletion of Z. These observations were primarily obtained using the Lymphocytic choriomeningitis virus, and further confirmed using the Old World Lassa and New World Machupo viruses, responsible of human hemorrhagic fevers. Z-deleted viruses should prove very useful tools to investigate the biology of Mammarenaviruses.

A rapid and specific real time RT-PCR assay for diagnosis of Toscana virus infection.

BACKGROUND: To scan a virus (TOSV) belongs to the Phlebovirus genus within the Bunyaviridae family. TOSV is an arbovirus transmitted by sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis. OBJECTIVES: Development and assessment of a new sensitive and specific real-time RT-PCR assay for TOSV diagnosis. STUDY DESIGN: TOSV-specific primers and probe targeting the S-segment of the genome were designed, based on recent TOSV sequences available in public databases. Sensitivity was assessed using 10-fold serial dilutions of a RNA transcript and serial dilutions of TOSV strains isolated from infected human beings. Specificity was determined by testing RNA extracts from closely related Phleboviruses. The assay was then used for TOSV infection diagnosis in 971 clinical samples and for TOSV detection in 2000 sandflies. RESULTS: The real-time RT-PCR assay exhibited a sensitivity of under 257 copies per reaction for the RNA transcripts and 0.0056 and 0.014 TCID50 of Italian and Spanish TOSV genotypes per reaction, respectively. No other close Phleboviruses were detected. TOSV was identified in 17 clinical samples and in 3 sandflies. CONCLUSIONS: The assay described is a rapid, robust and reliable real-time RT-PCR test for accurate diagnosis of human TOSV infection as well as for the surveillance of TOSV in vector populations.

Toscana virus inhibits the interferon beta response in cell cultures.

Toscana virus (TOSV) is an emerging pathogen in the Mediterranean basin where it causes summertime outbreaks of aseptic meningitis and meningoencephalitis. Many aspects of TOSV biology remain unknown including the possible implication of an amplifying mammalian host besides its vector. The three experiments described here were designed to assess the relationship between TOSV and type-I interferon (IFN) response. The main findings were as follows. First, TOSV growth in Vero cells is sensitive to an antiviral state induced by low-dose addition of exogenous IFN beta (IFN-ß) (10IU/ml). Second, no IFN-ß mRNA or IFN-ß was detectable after infection of HeLa and 293T cells by TOSV. Finally, TOSV inhibits IFN-ß production induced by Sendaï virus, a well known inducer of IFN-ß production. In addition to showing that TOSV can inhibit the IFN-ß response, these findings suggest that anti-IFN capability is maintained by regular contact with that of a mammalian host.

Arenavirus reverse genetics: new approaches for the investigation of arenavirus biology and development of antiviral strategies.

Several arenaviruses, chiefly Lassa virus, cause hemorrhagic fever disease in humans and pose a significant public health problem in their endemic regions. On the other hand the prototypic arenavirus LCMV is a superb workhorse for the investigation of virus-host interactions and associated disease. The development of novel antiviral strategies to combat pathogenic arenaviruses would be facilitated by a detailed understanding of the arenavirus molecular and cell biology. To this end, the development of reverse genetic systems for several arenaviruses has provided investigators with novel and powerful approaches to dissect the functions of arenavirus proteins and their interactions with host factors required to complete each of the steps of the virus life cycle, as well as to cause disease.

Arenavirus genetic diversity and its biological implications.

The Arenaviridae family currently comprises 22 viral species, each of them associated with a rodent species. This viral family is important both as tractable experimental model systems to study acute and persistent infections and as clinically important human pathogens. Arenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. The interaction with the cellular receptor and subsequent entry into the host cell differs between Old World and New World arenavirus that use alpha-dystoglycan or human transferring receptor 1, respectively, as main receptors. The recent development of reverse genetic systems for several arenaviruses has facilitated progress in understanding the molecular biology and cell biology of this viral family, as well as opening new approaches for the development of novel strategies to combat human pathogenic arenaviruses. On the other hand, increased availability of genetic data has allowed more detailed studies on the phylogeny and evolution of arenaviruses. As with other riboviruses, arenaviruses exist as viral quasispecies, which allow virus adaptation to rapidly changing environments. The large number of different arenavirus host reservoirs and great genetic diversity among virus species provide the bases for the emergence of new arenaviruses potentially pathogenic for humans.

Mouse-to-human transmission of variant lymphocytic choriomeningitis virus.

A case of lymphocytic choriomeningitis virus (LCMV) infection led to investigation of the reservoir. LCMV was detected in mice trapped at the patient's home, and 12 isolates were recovered. Genetic analysis showed that human and mouse LCMVs were identical and that this LCMV strain was highly divergent from previously characterized LCMV.

Long PCR Product Sequencing (LoPPS): a shotgun-based approach to sequence long PCR products.

Here we describe a practical procedure for sequencing long PCR products. The method relies on ultrasonic shearing of PCR products, resulting in fragments 700-1,000 nt long. Termini are subsequently repaired to obtain blunt ends and 3' A-overhangs are added before TA cloning. A predetermined number of clones are sequenced using an insert-independent primer to obtain an overlapping contig covering the full length of the PCR product. This method is cost effective and enables the complete sequencing of any large PCR product in a high-throughput format. Processing of amplified DNA requires 3 h handling time prior to the ligation step, and the clone library is available 2 d later. The complete sequence information is obtained approximately 5 d after the PCR step, depending on the sequencing procedure adopted.

Phylogeny and evolution of old world arenaviruses.

The intention of this study was to investigate the genomics, phylogeny and evolution of the Old World arenaviruses based on sequence data representing the four viral genes. To achieve this aim, we sequenced the complete S and L RNA segments of Ippy virus (IPPYV), Mobala virus (MOBV) and Mopeia virus (MOPV). Full-length sequences of the NP, GPC, Z and L genes were used to reconstruct phylogenetic relationships and to compare resulting tree topologies. Each of the five Old World arenavirus species (namely Lassa virus [LASV], IPPYV, MOBV, MOPV and Lymphocytic choriomeningitis virus [LCMV]) are monophyletic; seven selected strains of LASV showed a similar topology regardless of the gene under analysis; IPPYV rooted the three other African arenaviruses; the four African arenaviruses are rooted by the ubiquitous LCMV; and the tree topologies of the three African arenaviruses other than LASV are identical regardless of the gene used for analysis. No evidence for significant evolutionary events such as intra- or intersegmental recombination was obtained.

LoPPS: a long PCR product sequencing method for rapid characterisation of long amplicons.

Here, we propose an optimised protocol (LoPPS, long PCR product sequencing) which allows the fast, cost-attractive, and high-throughput sequencing of long PCR products. LoPPS constitutes an alternative to the primer-walking technology which is expensive and time consuming but remains the current standard procedure. It is based on the ultrasonic shearing, polishing, and cloning of PCR or RT-PCR products and is compatible with 96- or 384-well microplate systems in which bacterial growth, preparation of plasmid DNA, and sequencing can be automated. We present results obtained from 24 different RT-PCR products (2.5-4.8 kbp long) obtained from various RNA viruses and fully sequenced using LoPPS. The method proved to be robust and fast. It was successfully used on a low amount of DNA and allowed each target nucleotide position to be controlled twice or more, with a final cost which is one-third of that of primer-walking.