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.

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.

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.

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.

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.

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.

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.

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.