Comparison of the Innate Immune Responses to Pathogenic and Nonpathogenic Clade B New World Arenaviruses.

The New World (NW) arenaviruses are a diverse group of zoonotic viruses, including several causative agents of severe hemorrhagic fevers in humans. All known human-pathogenic NW arenaviruses belong to clade B, where they group into sublineages with phylogenetically closely related nonpathogenic viruses, e.g., the highly pathogenic Junin (JUNV) and Machupo viruses with the nonpathogenic Tacaribe virus (TCRV). Considering the close genetic relationship of nonpathogenic and pathogenic NW arenaviruses, the identification of molecular determinants of virulence is of great importance. The host cell's innate antiviral defense represents a major barrier for zoonotic infection. Here, we performed a side-by-side comparison of the innate immune responses against JUNV and TCRV in human cells. Despite similar levels of viral replication, infection with TCRV consistently induced a stronger type I interferon (IFN-I) response than JUNV infection did. Transcriptome profiling revealed upregulation of a largely overlapping set of interferon-stimulated genes in cells infected with TCRV and JUNV. Both viruses were relatively insensitive to IFN-I treatment of human cells and induced similar levels of apoptosis in the presence or absence of an IFN-I response. However, in comparison to JUNV, TCRV induced stronger activation of the innate sensor double-strand RNA-dependent protein kinase R (PKR), resulting in phosphorylation of eukaryotic translation initiation factor eIF2α. Confocal microscopy studies revealed similar subcellular colocalizations of the JUNV and TCRV viral replication-transcription complexes with PKR. However, deletion of PKR by CRISPR/Cas9 hardly affected JUNV but promoted TCRV multiplication, providing the first evidence for differential innate recognition and control of pathogenic and nonpathogenic NW arenaviruses by PKR.IMPORTANCE New World (NW) arenaviruses are a diverse family of emerging zoonotic viruses that merit significant attention as important public health problems. The close genetic relationship of nonpathogenic NW arenaviruses with their highly pathogenic cousins suggests that few mutations may be sufficient to enhance virulence. The identification of molecular determinants of virulence of NW arenaviruses is therefore of great importance. Here we undertook a side-by-side comparison of the innate immune responses against the highly pathogenic Junin virus (JUNV) and the related nonpathogenic Tacaribe virus (TCRV) in human cells. We consistently found that TCRV induces a stronger type I interferon (IFN-I) response than JUNV. Transcriptome profiling revealed an overlapping pattern of IFN-induced gene expression and similar low sensitivities to IFN-I treatment. However, the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) contributed to the control of TCRV, but not JUNV, providing the first evidence for differential innate recognition and control of JUNV and TCRV.

TRIM2, a novel member of the antiviral family, limits New World arenavirus entry.

Tripartite motif (TRIM) proteins belong to a large family with many roles in host biology, including restricting virus infection. Here, we found that TRIM2, which has been implicated in cases of Charcot-Marie-Tooth disease (CMTD) in humans, acts by blocking hemorrhagic fever New World arenavirus (NWA) entry into cells. We show that Trim2-knockout mice, as well as primary fibroblasts from a CMTD patient with mutations in TRIM2, are more highly infected by the NWAs Junín and Tacaribe virus than wild-type mice or cells are. Using mice with different Trim2 gene deletions and TRIM2 mutant constructs, we demonstrate that its antiviral activity is uniquely independent of the RING domain encoding ubiquitin ligase activity. Finally, we show that one member of the TRIM2 interactome, signal regulatory protein α (SIRPA), a known inhibitor of phagocytosis, also restricts NWA infection and conversely that TRIM2 limits phagocytosis of apoptotic cells. In addition to demonstrating a novel antiviral mechanism for TRIM proteins, these studies suggest that the NWA entry and phagocytosis pathways overlap.

Machupo virus glycoprotein determinants for human transferrin receptor 1 binding and cell entry.

Machupo virus (MACV) is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1). TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors.

Cell entry by human pathogenic arenaviruses.

The arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15-35%. The present review focuses on the first steps of infection with human pathogenic arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World arenaviruses these pathogens use different primary receptors. The Old World arenaviruses employ alpha-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World arenavirus lymphocytic choriomeningitis virus. Upon internalization, arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for arenaviruses with important implications for viral entry.

Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. AHF Study Group.

Argentine hemorrhagic fever (AHF), caused by the arenavirus Junin, is a major public health problem among agricultural workers in Argentina. A prospective, randomized, double-blind, placebo-controlled, efficacy trial of Candid 1, a live attenuated Junin virus vaccine, was conducted over two consecutive epidemic seasons among 6500 male agricultural workers in the AHF-endemic region. Twenty-three men developed laboratory-confirmed AHF during the study; 22 received placebo and 1 received vaccine (vaccine efficacy 95%; 95% confidence interval [CI], 82%-99%). Three additional subjects in each group developed laboratory-confirmed Junin virus infection associated with mild illnesses that did not fulfill the clinical case definition for AHF, yielding a protective efficacy for prevention of any illness associated with Junin virus infection of 84% (95% CI, 60%-94%). No serious adverse events were attributed to vaccination. Candid 1, the first vaccine for the prevention of illness caused by an arenavirus, is safe and highly efficacious.

Tacaribe virus gene expression in cytopathic and non-cytopathic infections.

Tacaribe virus (TV) replication was compared in Vero cells infected under conditions leading either to cell death (c.p.e.(+) infection) or to the establishment of persistence (c.p.e.(-) infection). To this end, two virus preparations were employed: one containing a ratio of standard (plaque-forming) viruses to interfering particles (IP) that would induce a distinct lytic response in Vero cells infected at multiplicities giving synchronous infection and another virus stock enriched in IP that would block the cell-killing potential of the cytolytic virus stock. The following results were obtained: (1) No qualitative differences were observed in the species of intracellular viral RNAs in the lytic infection in comparison with infections leading to persistence or during the early stages of persistence. (2) Levels of viral RNAs were severely reduced when the cells were infected with IP in addition to standard viruses, the RNA accumulation being inversely proportional to the ratio of IP to standard viruses used in the infections. (3) Accumulation of the three measurable mRNAs (those corresponding to the glycoprotein precursor [GPC], to the nucleoprotein [N], and to the p11Z protein) ended earlier in the c.p.e.(-) infections (around 18 hr p.i.) than in the c.p.e.(+) infection (45-68 hr p.i.). (4) The rates of synthesis of the GPC, N, and p11Z proteins were largely determined in both the c.p.e.(+) and c.p.e.(-) infections by the amounts of their corresponding mRNAs. (5) The kinetics of accumulation of the S genomes and also the ratios of the S genome to S antigenome were similar in the different infections (accumulation ending at 45-68 hr p.i.). (6) L genome accumulation proceeded for longer time (until 92 hr p.i.) in the c.p.e.(+) infection than in the c.p.e.(-) infections. In the latter accumulation ended at around 45 hr p.i. Until this time ratios of L genome to L antigenome were similar in the different infections. It is concluded that IP affect virus mRNA synthesis early after infection reducing in this way the rate of viral protein synthesis. Low levels of viral proteins might then limit virus replication. In addition, the results support the idea that in TV infections transcription and replication are independently regulated. The implications of these results with regards to the nature and mode of action of TV IP are discussed.

Treatment of lethal Pichinde virus infections in weanling LVG/Lak hamsters with ribavirin, ribamidine, selenazofurin, and ampligen.

A lethal Pichinde (An 4763 strain) virus infection was produced in 3-week-old random-bred Golden Syrian (LVG/Lak strain) hamsters inoculated intraperitoneally with virus, causing mortality in 6-9 days. High virus titers (> or = 10(7.5) cell culture infectious doses/g) were present in visceral organs, serum, brain and salivary glands near the time of death. Intraperitoneal treatments with ribavirin (10 and 32 mg/kg) and ribamidine (32, 100, and 320 mg/kg) for 10 days starting 24 h after virus challenge significantly decreased mortality and reduced virus titers by 100- to > 10,000-fold in liver, spleen, brain, and serum. Serum alanine aminotransferase (an indicator of liver damage) was also reduced in animals treated with the two compounds (ribavirin at 32 mg/kg; ribamidine at 100 and 320 mg/kg). Intraperitoneal selenazofurin (1-100 mg/kg per day for 10 days) and ampligen (0.5 and 5 mg/kg every other day for 5 injections) treatments provided neither protection from the lethal infection nor increased mean survival times. In fact, selenazofurin was overtly toxic, causing death of uninfected hamsters at 32 and 100 mg/kg. The random-bred LVG/Lak hamster appears to be a viable and cost-effective model for evaluating new therapies for arenavirus infections.

Experimental neuroinvasiveness of wild and laboratory Junin virus strains.

The neuroinvasiveness of Candid 1 and XJCL3 laboratory strains and CbalV4454 and CbaFHA5069 wild strains of Junin virus was studied in albino mice, guinea pigs, and a South American wild rodent, Calomys musculinus (Cm), of different ages inoculated by a non-neural route. Infectivity in brain, blood and organs, as well as lethality, were determined. The results with the 3 hosts indicate that Junin virus neuroinvasiveness is virus-strain-dependent, host species- and age-dependent, with the Candid 1 strain proving to be the least neuroinvasive of the strains studied. The lethal efficiency index (log PFU/LD50) in 2-day old albino mice and the neuroinvasiveness index (Log PFU/ND50) in 6 +/- 1 day-old Cm of the various strains using the intraperitoneal (ip) route could therefore be useful markers of Junin virus neuroinvasiveness. Moreover, different patterns of infection were established using the results of the presence of infectious virus in brain and viraemia in the 3 hosts. In nearly all cases, virus neuroinvasion was present without detectable viraemia (virus in plasma). Current evidence leads to the assumption that virus might reach the brain associated with the white cells in blood (undetectable by conventional isolation methods) or by another possible mechanism of neuroinvasion which is not haematogenous.

Experimental infection of suckling mice with a host range mutant of Junin virus.

Experimental infection of three mouse strains with a non-pathogenic mutant of Junin virus named Cl67 was compared with respect to the parental XJCl3 strain. After intracerebral (ic) or intraperitoneal inoculation, XJCl3 was highly virulent for 2 day-old C3H/HeJ, OF1, and BALB/cJ mouse strains, whereas its derivative Cl67 was attenuated. Survival of the Cl67-infected mouse was associated with a restricted replication at the site of inoculation which would impair spread of virus. Thus, the reduced virulence of Cl67 for suckling mice is independent of the mouse strain and the route of viral entry. When Cl67 was preinoculated ic 10 days before the challenge inoculation with XJCl3 by the same route, mice were partially protected from lethal infection. Since neutralizing antibodies were first detected at 30 days post-infection, an interference mechanism is postulated as a mechanism of protection of the mice.

Method for improving accuracy of virus titration: standardization of plaque assay for Junin virus.

Titrating infective virus is one of the most important and common techniques in virology. However, after many years of widespread use, the parameters governing the accuracy of titration values are still not well understood. It was found that under conditions currently used for virus titration, only a small percentage of virus in the inoculum is adsorbed onto the cells and thereby detected in the titration assay. The objective of our work was to establish the conditions for a plaque assay which could estimate more accurately the titer of Junin virus. Two different stain methods were compared and several parameters governing plaque formation were studied. The volume of the inoculum appeared as the most important factor affecting observed titer. A linear relationship between the volume of inoculum and the reciprocal apparent titer allowed us to estimate an absolute titer by extrapolation. The approach described here is likely to be applicable to the more accurate estimation of the titer of a wide range of virus.

A comparison of Junin virus strains: growth characteristics, cytopathogenicity and viral polypeptides.

The growth characteristics, cytopathogenicity and viral polypeptides of the virulent strain XJ of Junin virus (JV), its attenuated derivative XJC13 and another naturally attenuated JV strain, IV4454, were comparatively studied. IV4454 and XJC13 viruses showed the highest and lowest cytopathology for Vero cells, respectively, as measured by plaque morphology, cell viability and inhibition of host cell protein synthesis. The kinetics and electrophoretic patterns of viral polypeptides in infected cell extracts were very similar among the three strains, whereas differences were detected in the surface glycoprotein GP38 by peptide mapping after limited proteolysis.

Protective effect of a low-dose of cyclophosphamide in experimental infection of guinea pigs with Junin virus.

Administration of cyclophosphamide (CY) to guinea pigs infected with a lethal strain of Junin virus (JV) delayed the time of death, with survival of a small number of animals. Virological studies showed a temporary decrease of virus concentration in blood and viscera shortly after the CY injection. In the pathological study no differences were found in the organic lesions present in CY-treated and nontreated animals, with the exception of the pulmonary alterations. In CY-treated guinea pigs the lungs appeared almost normal, but in the control, nontreated animals severe alterations with the pattern of the "respiratory distress syndrome of the adult" were consistently present. In in vitro experiments, incorporation of serum collected from guinea pigs injected 30 minutes before exsanguination with CY to cell cultures, infected with JV, prevented virus replication. On the basis of these results it is suggested that the delay of time of death and eventual survival of CY-treated guinea pigs after JV infection depends on a direct antiviral effect of the drug rather than on its known immunosuppressive action. In addition, the absence of pulmonary alterations in CY-treated animals was tentatively considered to be dependent on the marked polymorphonuclear leukocyte depletion induced by the drug.

Is vertical transmission sufficient to maintain Junin virus in nature?

The quantitative contribution of vertical transmission to the prevalence rate of Junin virus infection in subsequent generations of its natural reservoir, Calomys musculinus, was analysed. Data on mortality and reproduction of C. musculinus infected at birth with a wild strain of Junin virus were used to estimate the infection-dependent relative survival rate (beta = 0.4849) and relative fertility of the infected host (alpha = 0.2088). Prevalence rates of infection, obtained by mathematical simulation in optimal conditions of vertical transfer, dropped steadily to zero in a few generations. Vertical transmission was found to be insufficient to overcome the effect of highly depressed survival and fertility of the infected host and maintain a stabilized prevalence of Junin virus infection in successive generations; this suggested that viral maintenance is mainly dependent upon horizontal transmission.

Effect of polymorphonuclear depletion on experimental Argentine hemorrhagic fever in guinea pigs.

The role that polymorphonuclear leukocytes (PMN) may play in Argentine hemorrhagic fever (AHF), an endemo-epidemic disease caused by Junín virus (JV), was investigated in experimentally infected guinea pigs depleted of PMN by means of specific antiserum. In leucopenic animals the evolution of the infection with a highly pathogenic strain of JV was more severe, with earlier mortality and higher virus yields in blood and viscera. The pathological study showed similar lesions in both the control and PMN-depleted animals with the exception of the lung, which showed the pathological picture of the human "pulmonary distress syndrome of the adult" in nontreated guinea pigs and appeared histologically unaltered in the PMN-depleted animals. On the basis of these results it is suggested that in AHF, PMN play a dual role. In the first stage of infection they display a defensive antiviral action, but later on they participate in the pathogenesis of tissue damage.

Partial characterization of two temperature-sensitive mutants of junin virus.

Two temperature sensitive (ts) mutants of Junin virus, a member of Arenaviridae, have been partially characterized. Both mutants, named ts-32 and ts-40, had a relative plating efficiency 40 degrees C/34 degrees C lower than 10(-3) an exhibited a leak yield below 10(-4). Standard growth curves showed that at 34 degrees C the viral mutants multiplied slower than wt virus and at high multiplicity did not display autointerference. No differences in thermolability were observed between wt and ts mutants. By contrast, when the pathogenic properties of the mutants were investigated they were significantly attenuated for mice. At the restrictive temperature both mutants were unable to synthesize viral-specific polypeptides, while at the permissive temperature the pattern was similar to wt virus. Shift-up and down experiments suggested that ts defect is expressed between 2 and 4 hours post-infection. It is concluded that ts-32 and ts-40 are early function mutants. The possible nature of their defect is discussed.

[Production of infectious virus and the accumulation in Vero cells of Pichinde virus antigens detectable by an indirect fluorescent antibody method]. / Produktsiia infektsionnogo virusa i nakoplenie v kletkakh Vero antigenov virusa Pichinda, vyiavliaemykh nepriamym metodom fliuorestsiruiushchikh antitel.

The dynamics of accumulation of infectious Pichinde virus in the culture medium and virus-specific antigens in cells was studied in relation to multiplicity of infection in a multicycle experiment. Differences in the fluorescence pattern of Pichinde virus antigens in IFAT were found to depend on the use of acetone or formaldehyde for fixation of the infected cells.

Junin virus access to CNS by extraneural rat inoculation.

This study was carried out to determine the pathways along which two strains of Junin virus (JV), the pathogenic XJV and the attenuated XJC13V, reach the CNS following IP inoculation of 2-day-old rats. A sequential study of infectivity and antigen distribution in peritoneal macrophages, spleen, and brain was performed. Mortality was 85% with the former strain, but only 15% with the latter. At 4-7 days PI, XJV-infected animals had viral antigen in 10% of peritoneal macrophages. Viremia and spleen virus lasted for 10-15 days. Low brain titers were detected at day 7, with a peak at day 15. Brain antigen correlated with virus titers. In contrast, XJC13V-infected rats, macrophage antigen appeared later and to a lesser degree (1% of cells). Viremia and spleen virus were transient, while both the titer of brain virus and the viral antigen proved lower. Antibody titers were over twofold higher for XJ-infected animals. It is suggested that the different replication rate at the inoculation site could account for the greater ability of the XJV strain to reach the CNS. A greater antigen mass and/or more numerous antigenic determinants presented by the macrophage could explain the higher antibody titers found in XJ-injected rats, which were unable, however, to prevent viral spread.

Antigenic variants of Junín virus isolated from infected Calomys musculinus. Brief report.

Junín virus establishes a long-term persistent infection in its natural host, Calomys musculinus. Virus recovered from blood of infected animals from 14 to 61 days p.i. behaved antigenically distinct to parental virus, as shown by cross neutralization assays. The emergence of antigenic viral variants occurs during both the acute and persistent state of infection.

Administration of antithymocyte serum modifies the response of Calomys musculinus to junin virus infection.

Approximately 80% of Calomys musculinus inoculated with an attenuated strain of Junin virus (JV) developed a lethal encephalitis. Antithymocyte serum, a potent suppressor of T-cell-mediated immunity, was studied for its effect on JV pathogenicity. Early administration of an anti-C. musculinus thymocyte serum (ACTS) to neonatal animals significantly diminished clinical disease and death and abrogated brain damage, which is usually associated with viral presence in the brain. Late ACTS administration did not modify the pattern of JV infection. These results suggest that immune mechanisms participate in the pathogenesis of JV infection for its main natural host.