Distinct mechanisms govern populations of myeloid-derived suppressor cells in chronic viral infection and cancer.

Myeloid-derived suppressor cells (MDSCs) are major negative regulators of immune responses in cancer and chronic infections. It remains unclear if regulation of MDSC activity in different conditions is controlled by similar mechanisms. We compared MDSCs in mice with cancer and lymphocytic choriomeningitis virus (LCMV) infection. Chronic LCMV infection caused the development of monocytic MDSCs (M-MDSCs) but did not induce polymorphonuclear MDSCs (PMN-MDSCs). In contrast, both MDSC populations were present in cancer models. An acquisition of immune-suppressive activity by PMN-MDSCs in cancer was controlled by IRE1α and ATF6 pathways of the endoplasmic reticulum (ER) stress response. Abrogation of PMN-MDSC activity by blockade of the ER stress response resulted in an increase in tumor-specific immune response and reduced tumor progression. In contrast, the ER stress response was dispensable for suppressive activity of M-MDSCs in cancer and LCMV infection. Acquisition of immune-suppressive activity by M-MDSCs in spleens was mediated by IFN-γ signaling. However, it was dispensable for suppressive activity of M-MDSCs in tumor tissues. Suppressive activity of M-MDSCs in tumors was retained due to the effect of IL-6 present at high concentrations in the tumor site. These results demonstrate disease- and population-specific mechanisms of MDSC accumulation and the need for targeting different pathways to achieve inactivation of these cells.

Heterologous arenavirus vector prime-boost overrules self-tolerance for efficient tumor-specific CD8 T cell attack.

Therapeutic vaccination regimens inducing clinically effective tumor-specific CD8+ T lymphocyte (CTL) responses are an unmet medical need. We engineer two distantly related arenaviruses, Pichinde virus and lymphocytic choriomeningitis virus, for therapeutic cancer vaccination. In mice, life-replicating vector formats of these two viruses delivering a self-antigen in a heterologous prime-boost regimen induce tumor-specific CTL responses up to 50% of the circulating CD8 T cell pool. This CTL attack eliminates established solid tumors in a significant proportion of animals, accompanied by protection against tumor rechallenge. The magnitude of CTL responses is alarmin driven and requires combining two genealogically distantly related arenaviruses. Vector-neutralizing antibodies do not inhibit booster immunizations by the same vector or by closely related vectors. Rather, CTL immunodominance hierarchies favor vector backbone-targeted responses at the expense of self-reactive CTLs. These findings establish an arenavirus-based immunotherapy regimen that allows reshuffling of immunodominance hierarchies and breaking self-directed tolerance for efficient tumor control.

Effect of Strain Variations on Lassa Virus Z Protein-Mediated Human RIG-I Inhibition.

Mammarenaviruses include several known human pathogens, such as the prototypic lymphocytic choriomeningitis virus (LCMV) that can cause neurological diseases and Lassa virus (LASV) that causes endemic hemorrhagic fever infection. LASV-infected patients show diverse clinical manifestations ranging from asymptomatic infection to hemorrhage, multi-organ failures and death, the mechanisms of which have not been well characterized. We have previously shown that the matrix protein Z of pathogenic arenaviruses, including LASV and LCMV, can strongly inhibit the ability of the innate immune protein RIG-I to suppress type I interferon (IFN-I) expression, which serves as a mechanism of viral immune evasion and virulence. Here, we show that Z proteins of diverse LASV isolates derived from rodents and humans have a high degree of sequence variations at their N- and C-terminal regions and produce variable degrees of inhibition of human RIG-I (hRIG-I) function in an established IFN-ß promoter-driven luciferase (LUC) reporter assay. Additionally, we show that Z proteins of four known LCMV strains can also inhibit hRIG-I at variable degrees of efficiency. Collectively, our results confirm that Z proteins of pathogenic LASV and LCMV can inhibit hRIG-I and suggest that strain variations of the Z proteins can influence their efficiency to suppress host innate immunity that might contribute to viral virulence and disease heterogeneity.

Absence of keratin 1 restricts the course of infection with lymphocytic choriomeningitis virus strain MX.

A rodent-transmitted enveloped lymphocytic choriomeningitis virus (LCMV) is an RNA virus causing persistent infection. During persistent infection, a unique strain MX of LCMV does not yield infectious virions, therefore it is not able to use a receptor for its dissemination, and spreads by cell-to-cell contacts. Virus can be transported to the neighboring cell by different cellular structures such as tunneling nanotubes or cytonemes. Using q-PCR, immunofluorescence, siRNA and western blot, we show that keratin 1 (K1) is essential for the persistent infection caused by LCMV strain MX, and its absence very effectively slows down the course of infection. In contrast, other LCMV strains, namely Clone 13 and Armstrong, which produce expression of K1, desmosomes in cells expressing K1 (42-MG-BA) but not in cells without K1 expression (NIH/3T3). We conclude that the presence of the virus enhances the K1 expression, while the presence of K1 protein potentiates the viral spread in persistently infected cells. Keywords: lymphocytic choriomeningitis virus; keratin 1; persistent infection; desmosomes; virus transport.

Sequence-specific detection of different strains of LCMV in a single sample using tentacle probes.

BACKGROUND: Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems. METHODS: We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe. RESULTS: Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA. CONCLUSIONS: Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.

Difference of two new LCMV strains in lethality and viral genome load in tissues.

More than 30 strains of lymphocytic choriomeningitis virus (LCMV) have been isolated from mice, hamsters and humans in the United States, Europe and Japan. Experimentally infected mice exhibit different clinical signs and lethality depending on a combination of LCMV epitope peptides and host major histocompatibility complex (MHC) class I molecules. This study examined the pathogenicity, clinical signs and lethality, of two new LCMV strains (BRC and OQ28) using three inbred mouse strains with different genetic backgrounds having different H-2D haplotypes. Strain OQ28 (OQ28) infected mice exhibited clinical signs and lethality, whereas strain BRC (BRC) infected mice showed no clinical signs of infection. The viral genome load in tissues of C57BL/6 mice infected with two strains was determined using one-step real time RT-PCR. In C57BL/6 mice, higher levels of OQ28 viral genome load were detected in all tissues rather than were present in BRC infected mice. The viral genome load in lungs of both virus strains remained higher levels than in other tissues at 28 days post infection. Comparing sequences of the three LCMV epitope peptide regions revealed one non-conservative amino acid substitution codon in OQ28 and two amino acid differences in BRC. These results suggest that the varied pathogenicity and viral genome load of LCMV strains are not based only on differences in the host MHC class I molecule.

Identification of lymphocytic choriomeningitis mammarenavirus in house mouse (Mus musculus, Rodentia) in French Guiana.

Thirty-seven house mice (Mus musculus, Rodentia) caught in different localities in French Guiana were screened to investigate the presence of lymphocytic choriomeningitis mammarenavirus (LCMV). Two animals trapped in an urban area were found positive, hosting a new strain of LCMV, that we tentatively named LCMV "Comou". The complete sequence was determined using a metagenomic approach. Phylogenetic analyses revealed that this strain is related to genetic lineage I composed of strains inducing severe disease in humans. These results emphasize the need for active surveillance in humans as well as in house mouse populations, which is a rather common rodent in French Guianese cities and settlements.

Evidence of lymphocytic choriomeningitis virus (LCMV) in domestic mice in Gabon: risk of emergence of LCMV encephalitis in Central Africa.

Lymphocytic choriomeningitis virus (LCMV) can cause acute fatal disease on all continents but was never detected in Africa. We report the first detection of LCMV RNA in a common European house mouse (Mus musculus domesticus) in Africa. Phylogenetic analyses show a close relationship with North American strains. These findings suggest that there is a risk of the appearance of LCMV acute encephalitis cases. This is a perfect example of virus dissemination by its natural host that may have dramatic public health consequences.

Lymphocytic choriomeningitis virus in employees and mice at multipremises feeder-rodent operation, United States, 2012.

We investigated the extent of lymphocytic choriomeningitis virus (LCMV) infection in employees and rodents at 3 commercial breeding facilities. Of 97 employees tested, 31 (32%) had IgM and/or IgG to LCMV, and aseptic meningitis was diagnosed in 4 employees. Of 1,820 rodents tested in 1 facility, 382 (21%) mice (Mus musculus) had detectable IgG, and 13 (0.7%) were positive by reverse transcription PCR; LCMV was isolated from 8. Rats (Rattus norvegicus) were not found to be infected. S-segment RNA sequence was similar to strains previously isolated in North America. Contact by wild mice with colony mice was the likely source for LCMV, and shipments of infected mice among facilities spread the infection. The breeding colonies were depopulated to prevent further human infections. Future outbreaks can be prevented with monitoring and management, and employees should be made aware of LCMV risks and prevention.

Genomic analysis and pathogenic characteristics of lymphocytic choriomeningitis virus strains isolated in Japan.

Lymphocytic choriomeningitis virus (LCMV) is a zoonotic pathogen of which mice are the natural reservoir. Different strains and clones of LCMV show different pathogenicity in mice. Here we determined the complete genomic sequences of 3 LCMV strains (OQ28 and BRC which were isolated from mice in Japan and WE(ngs) which was derived from strain WE). Strains OQ28 and BRC showed high sequence homology with other LCMV strains. Although phylogenetic analyses placed these 2 Japanese strains in different subclusters, they belonged to same cluster of LCMV isolates. WE(ngs) and WE had many sequence substitutions between them but fell into same subcluster. The pathogenicity of the 3 new LCMV isolates was examined by inoculating ICR mice with 10² and 104 TCID50 of virus. ICR mice infected with OQ28 or WE(ngs) exhibited severe clinical signs, and some of the infected mice died. In contrast, all ICR mice infected with BRC showed no clinical signs and survived infection. Virus was detected in the blood, organs, or both of most of the surviving ICR mice inoculated with either OQ28 or WE(ngs). However, virus was below the level of detection in all ICR mice surviving infection with strain BRC. Therefore, LCMV strains OQ28 and BRC were genetically classified in the same cluster of LCMV strains but exhibited very different pathogenicity.

Lymphocytic choriomeningitis virus-associated meningitis, southern Spain.

Lymphocytic choriomeningitis virus (LCMV) was detected in 2 patients with acute meningitis in southern Spain within a 3-year period. Although the prevalence of LCMV infection was low (2 [1.3%] of 159 meningitis patients), it represents 2.9% of all pathogens detected. LCMV is a noteworthy agent of neurologic illness in immunocompetent persons.

High diversity and ancient common ancestry of lymphocytic choriomeningitis virus.

Lymphocytic choriomeningitis virus (LCMV) is the prototype of the family Arenaviridae. LCMV can be associated with severe disease in humans, and its global distribution reflects the broad dispersion of the primary rodent reservoir, the house mouse (Mus musculus). Recent interest in the natural history of the virus has been stimulated by increasing recognition of LCMV infections during pregnancy, and in clusters of LCMV-associated fatal illness among tissue transplant recipients. Despite its public health importance, little is known regarding the genetic diversity or distribution of virus variants. Genomic analysis of 29 LCMV strains collected from a variety of geographic and temporal sources showed these viruses to be highly diverse. Several distinct lineages exist, but there is little correlation with time or place of isolation. Bayesian analysis estimates the most recent common ancestor to be 1,000-5,000 years old, and this long history is consistent with complex phylogeographic relationships of the extant virus isolates.

No evidence of selection for mutational robustness during lethal mutagenesis of lymphocytic choriomeningitis virus.

Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication. Theoretical studies suggest that viruses can evolve towards regions of their fitness landscapes at which they display resistance to the deleterious effects of mutations. It has been suggested that such mutational robustness could jeopardize lethal mutagenesis. We have used the Arenavirus lymphocytic choriomeningitis virus (LCMV) to explore whether treatment with the mutagenic base analogue 5-fluorouracil (FU) selected for viral populations displaying resistance to lethal mutagenesis. Neither average LCMV populations with a history of FU mutagenesis, nor individual biological LCMV clones derived from those populations, displayed any resistance to lethal mutagenesis by FU. They were as sensitive to FU-induced extinction as LCMV populations and clones treated in parallel, but without a history of FU mutagenesis. Current evidence of the molecular events affecting quasispecies dynamics suggests that it is unlikely that a viral population can acquire mutational robustness under the increased mutation rates associated with mutagenic treatments. We consider mechanisms by which viruses could escape extinction by lethal mutagenesis, and provide evidence that mutational robustness is unlikely to be one of them.

Molecular characterization of the genes coding for glycoprotein and L protein of lymphocytic choriomeningitis virus strain MX.

Lymphocytic choriomeningitis virus (LCMV) is the prototype Arenavirus with ambisense coding strategy. We have previously described a new MX strain LCMV and determined the primary structure of the genes coding for the nucleoprotein and RING finger Z protein. In this report, we describe amplification and sequencing of the entire coding sequences of additional MX genes, the glycoprotein precursor (GPC) and L protein. The obtained MX GPC cDNA sequence was 1,615 nucleotides long and contained an ORF, which encodes the GPC precursor of 498 amino acids. MX L polymerase cDNA sequence was 6,668 nucleotides long and predicted ORF encodes the L polymerase of 2,209 amino acids. Nucleotide and deduced amino acid sequences were compared with the known GPC and L sequences and the comparison revealed that both genes shared the highest amino acid identity with Armstrong strain. Phylogenetic analysis confirmed that MX represents a separate LCMV strain. The GPC and L genes products contained several characteristic conserved regions. On the other hand, we have observed numerous differences in predicted protein sequences, which distinguish MX LCMV from other LCMV strains and might be of potential biological significance.

Pet rodents and fatal lymphocytic choriomeningitis in transplant patients.

In April 2005, 4 transplant recipients became ill after receiving organs infected with lymphocytic choriomeningitis virus (LCMV); 3 subsequently died. All organs came from a donor who had been exposed to a hamster infected with LCMV. The hamster was traced back through a Rhode Island pet store to a distribution center in Ohio, and more LCMV-infected hamsters were discovered in both. Rodents from the Ohio facility and its parent facility in Arkansas were tested for the same LCMV strain as the 1 involved in the transplant-associated deaths. Phylogenetic analysis of virus sequences linked the rodents from the Ohio facility to the Rhode Island pet store, the index hamster, and the transplant recipients. This report details the animal traceback and the supporting laboratory investigations.

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.

Genetic identification of Kodoko virus, a novel arenavirus of the African pigmy mouse (Mus Nannomys minutoides) in West Africa.

Genetic evidence of a novel arenavirus species related to but distinct from lymphocytic choriomeningitis virus (LCMV) was obtained from a rodent belonging to an endemic African subgenus of Mus (Nannomys). The phylogenetic position among Old World arenaviruses of this new arenavirus, named Kodoko virus, was reconstructed based on L and NP genes sequences. The finding of an Old World arenavirus related to LCMV outside the known geographical range of LCMV in a novel rodent species reveals new insights about the evolutionary history of arenaviruses.

Transmission of lymphocytic choriomeningitis virus by organ transplantation.

BACKGROUND: In December 2003 and April 2005, signs and symptoms suggestive of infection developed in two groups of recipients of solid-organ transplants. Each cluster was investigated because diagnostic evaluations were unrevealing, and in each a common donor was recognized. METHODS: We examined clinical specimens from the two donors and eight recipients, using viral culture, electron microscopy, serologic testing, molecular analysis, and histopathological examination with immunohistochemical staining to identify a cause. Epidemiologic investigations, including interviews, environmental assessments, and medical-record reviews, were performed to characterize clinical courses and to determine the cause of the illnesses. RESULTS: Laboratory testing revealed lymphocytic choriomeningitis virus (LCMV) in all the recipients, with a single, unique strain of LCMV identified in each cluster. In both investigations, LCMV could not be detected in the organ donor. In the 2005 cluster, the donor had had contact in her home with a pet hamster infected with an LCMV strain identical to that detected in the organ recipients; no source of LCMV infection was found in the 2003 cluster. The transplant recipients had abdominal pain, altered mental status, thrombocytopenia, elevated aminotransferase levels, coagulopathy, graft dysfunction, and either fever or leukocytosis within three weeks after transplantation. Diarrhea, peri-incisional rash, renal failure, and seizures were variably present. Seven of the eight recipients died, 9 to 76 days after transplantation. One recipient, who received ribavirin and reduced levels of immunosuppressive therapy, survived. CONCLUSIONS: We document two clusters of LCMV infection transmitted through organ transplantation.

Rescue of the prototypic Arenavirus LCMV entirely from plasmid.

We document a helper-independent reverse genetics system for rescuing infectious arenaviruses from cloned cDNAs. We constructed plasmids containing full-length cDNAs of the antigenomic (ag) L and S segments of the Armstrong (ARM) strain of the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) flanked at their 5'- and 3'-termini by the T7 RNA polymerase (T7RP) promoter and ribozyme sequences, respectively. These plasmids directed intracellular synthesis of viral L and S ag RNA species in cells expressing plasmid-supplied T7RP. Co-expression of plasmid-supplied LCMV trans-acting factors, nucleoprotein (NP) and polymerase (L), resulted in replication and expression of L and S ag and genome RNA species, and generation of LCMV infectious progeny termed rT7/LCMV. The recombinant rT7/LCMV was unequivocally identified based on a genetic tag introduced in the recombinant S segment. In addition, rT7/LCMV exhibited growth and biological properties predicted for an ARM-like LCMV. To our knowledge, this is the first documented Arenavirus rescue, as well as of an ambisense negative strand (NS) RNA virus, entirely from cloned cDNAs. Our results extend the use of reverse genetic approaches for DNA-mediated virus rescue to all known virus families with NS RNA genome.

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.