Chapare Hemorrhagic Fever and Virus Detection in Rodents in Bolivia in 2019.

BACKGROUND: In June 2019, the Bolivian Ministry of Health reported a cluster of cases of hemorrhagic fever that started in the municipality of Caranavi and expanded to La Paz. The cause of these cases was unknown. METHODS: We obtained samples for next-generation sequencing and virus isolation. Human and rodent specimens were tested by means of virus-specific real-time quantitative reverse-transcriptase-polymerase-chain-reaction assays, next-generation sequencing, and virus isolation. RESULTS: Nine cases of hemorrhagic fever were identified; four of the patients with this illness died. The etiologic agent was identified as Mammarenavirus Chapare mammarenavirus, or Chapare virus (CHAPV), which causes Chapare hemorrhagic fever (CHHF). Probable nosocomial transmission among health care workers was identified. Some patients with CHHF had neurologic manifestations, and those who survived had a prolonged recovery period. CHAPV RNA was detected in a variety of human body fluids (including blood; urine; nasopharyngeal, oropharyngeal, and bronchoalveolar-lavage fluid; conjunctiva; and semen) and in specimens obtained from captured small-eared pygmy rice rats (Oligoryzomys microtis). In survivors of CHHF, viral RNA was detected up to 170 days after symptom onset; CHAPV was isolated from a semen sample obtained 86 days after symptom onset. CONCLUSIONS: M. Chapare mammarenavirus was identified as the etiologic agent of CHHF. Both spillover from a zoonotic reservoir and possible person-to-person transmission were identified. This virus was detected in a rodent species, O. microtis. (Funded by the Bolivian Ministry of Health and others.).

The interplay between viperin antiviral activity, lipid droplets and Junín mammarenavirus multiplication.

Junín arenavirus infections are associated with high levels of interferons in both severe and fatal cases. Upon Junín virus (JUNV) infection a cell signaling cascade initiates, that ultimately attempts to limit viral replication and prevent infection progression through the expression of host antiviral proteins. The interferon stimulated gene (ISG) viperin has drawn our attention as it has been highlighted as an important antiviral protein against several viral infections. The studies of the mechanistic actions of viperin have described important functional domains relating its antiviral and immune-modulating actions through cellular lipid structures. In line with this, through silencing and overexpression approaches, we have identified viperin as an antiviral ISG against JUNV. In addition, we found that lipid droplet structures are modulated during JUNV infection, suggesting its relevance for proper virus multiplication. Furthermore, our confocal microscopy images, bioinformatics and functional results also revealed viperin-JUNV protein interactions that might be participating in this antiviral pathway at lipid droplet level. Altogether, these results will help to better understand the factors mediating innate immunity in arenavirus infection and may lead to the development of pharmacological agents that can boost their effectiveness thereby leading to new treatments for this viral disease.

Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor.

UNLABELLED: Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCE: The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.

Superinfection exclusion is absent during acute Junin virus infection of Vero and A549 cells.

Many viruses have evolved strategies of so-called "superinfection exclusion" to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.

Reactive astrogliosis in response to hemorrhagic fever virus: microarray profile of Junin virus-infected human astrocytes.

BACKGROUND: Arenavirus Junin is the causative agent of Argentine hemorrhagic fever. Limited information is available concerning the pathogenesis of this human disease, especially the pathogenesis of acute and late neurological symptoms. METHODS: In our study we present for the first time cDNA microarray profile of human astrocytes infected with the virulent strain of Junin virus. Transcriptional profiling was confirmed by quantitative real-time RT-PCR and cytokine/chemokine/growth factor assay. RESULTS: We demonstrated the impact of virus infection on immune/inflammatory response/interferon signaling and apoptosis. Pro-apoptotic response and amplification with time of pro-inflammatory cascade of human astrocytes suggested neurodegenerative dysfunctional reactive astrogliosis in response to Junin virus infection. CONCLUSION: Our results suggest potential pathogenic role of astroglial cells in the development of neurological symptoms and late neurological syndrome during Argentine hemorrhagic fever.

RIG-I enhanced interferon independent apoptosis upon Junin virus infection.

Junin virus (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF), a human disease with a high case-fatality rate. It is widely accepted that arenaviral infections, including JUNV infections, are generally non-cytopathic. In contrast, here we demonstrated apoptosis induction in human lung epithelial carcinoma (A549), human hepatocarcinoma and Vero cells upon infection with the attenuated Candid#1 strain of, JUNV as determined by phosphatidylserine (PS) translocation, Caspase 3 (CASP3) activation, Poly (ADP-ribose) polymerase (PARP) cleavage and/or chromosomal DNA fragmentation. Moreover, as determined by DNA fragmentation, we found that the pathogenic Romero strain of JUNV was less cytopathic than Candid#1 in human hepatocarcinoma and Vero, but more apoptotic in A549 and Vero E6 cells. Additionally, we found that JUNV-induced apoptosis was enhanced by RIG-I signaling. Consistent with the previously reported role of RIG-I like helicase (RLH) signaling in initiating programmed cell death, we showed that cell death or DNA fragmentation of Candid#1-infected A549 cells was decreased upon siRNA or shRNA silencing of components of RIG-I pathway in spite of increased virus production. Similarly, we observed decreased DNA fragmentation in JUNV-infected human hepatocarcinoma cells deficient for RIG-I when compared with that of RIG-I-competent cells. In addition, DNA fragmentation detected upon Candid#1 infection of type I interferon (IFN)-deficient Vero cells suggested a type I IFN-independent mechanism of apoptosis induction in response to JUNV. Our work demonstrated for the first time apoptosis induction in various cells of mammalian origin in response to JUNV infection and partial mechanism of this cell death.

Rescue of a recombinant Machupo virus from cloned cDNAs and in vivo characterization in interferon (αß/γ) receptor double knockout mice.

Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.

[Argentine hemorrhagic fever].

Features of the Argentine hemorrhagic fever are described in the review. Epidemiology, etiology, clinical presentation and pathogenesis of the disease are examined. Special consideration is given to the features of the pathological agent of Argentine hemorrhagic fever--the Junin virus. Features of the disease diagnostics are indicated--virological and serological studies, immunochemical and molecular-biological methods of identification of the pathological agent and antibodies against it. Approaches to etiotropic therapy of this disease and vaccination are examined. Based on the foreign experience perspective guidance for the creation of the system of protection of the population of the Russian Federation against Argentine hemorrhagic fever are presented.

Junin virus infection impairs stress-granule formation in Vero cells treated with arsenite via inhibition of eIF2α phosphorylation.

Stress granules (SGs) are ephemeral cytoplasmic aggregates containing stalled translation preinitiation complexes involved in mRNA storage and triage during the cellular stress response. SG formation is triggered by the phosphorylation of the alpha subunit of eIF2 (eIF2α), which provokes a dramatic blockage of protein translation. Our results demonstrate that acute infection of Vero cells with the arenavirus Junín (JUNV), aetiological agent of Argentine haemorrhagic fever, does not induce the formation of SGs. Moreover, JUNV negatively modulates SG formation in infected cells stressed with arsenite, and this inhibition correlates with low levels of eIF2α phosphorylation. Transient expression of JUNV nucleoprotein (N) or the glycoprotein precursor (GPC), but not of the matrix protein (Z), inhibits SG formation in a similar manner, comparable to infectious virus. Expression of N and GPC also impaired eIF2α phosphorylation triggered by arsenite. A moderate inhibition of SG formation was also observed when DTT and thapsigargin were employed as stress inducers. In contrast, no inhibition was observed when infected cells were treated with hippuristanol, a translational inhibitor and inducer of SGs that bypasses the requirement for eIF2α phosphorylation. Finally, we analysed SG formation in persistently JUNV-infected cells, where N and GPC are virtually absent and truncated N products are expressed abundantly. We found that persistently infected cells show a quite normal response to arsenite, with SG formation comparable to that of uninfected cells. This suggests that the presence of GPC and/or N is crucial to control the stress response upon JUNV infection of Vero cells.

Association of reduced interleukin-2 production with genetic susceptibility to Pichinde virus in inbred strains of hamsters.

Adult inbred MHA hamsters are susceptible to lethal infections with Pichinde virus while inbred LSH hamsters resist such infections. Previous studies demonstrated higher levels of endogenous and induced natural killer (NK) activity in MHA splenocytes than in LSH splenocytes. Preferential replication of Pichinde virus in cells with NK activity was suggested by showing that the greater numbers of infected spleen cells observed in MHA hamsters could be accounted for by a cell population that cosedimented with a peak of NK activity. Increased cellularity of thymi and spleens as well as increased cells sensitive to lymphokines was also found in MHA hamsters as compared to LSH hamsters. In the present study we found that injection of anti-asialo GM 1 serum reduced NK activity but did not alter susceptibility to virus infection. However, MHA hamsters were found to be relatively deficient in the production of interleukin 2 and injection of interleukin 2 altered the mortality of hamsters infected with Pichinde virus. These findings suggest that susceptibility to lethal infection by Pichinde virus is associated with reduced ability to produce interleukin 2 in MHA hamsters.

Junin virus-induced chromosomal aberrations in the guinea pig. Synergism between the attenuated strain XJ-clone 3 and caffeine.

The frequency of chromosomal aberrations in bone marrow cells of guinea pigs inoculated with the pathogenic XJ strain of Junin virus increased significantly at 6, 9, and 11 days postinoculation (p.i.). Animals inoculated with the attenuated XJ-clone 3 strain only showed significant increments of achromatic lesions (gaps) at 9 days p.i. Guinea pigs inoculated with the XJ-clone 3 strain and then treated with two doses of caffeine 24 and 12 h before killing at 9 days p.i. exhibited a significant increase of chromatid breaks and a parallel decrease of gaps. Because caffeine acts as an inhibitor of repair mechanisms of genetic damage, these results suggest a mutagenic effect of the attenuated strain.

Cytogenetic effect of two strains of Junin virus in the guinea pig.

The cytogenetic effect of two strains of Junin virus on bone marrow chromosomes of the guinea pig was studied. Animals infected with the attenuated strain XJ-Cl3 showed no differences from control animals. Guinea pigs inoculated with the pathogenic strain XJ exhibited a significant increase of abnormal cells, chromatid breaks, and chromosome fragments. The clastogenic ability of the XJ strain is similar to the reported effect of other viruses, while the XJ-Cl3 strain does not appear to be clastogenic.