Enhanced protection against experimental Junin virus infection through the use of a modified favipiravir loading dose strategy.

A collection of Old and New World arenaviruses are etiologic agents of viral hemorrhagic fever, a syndrome that features hematologic abnormalities, vascular leak, hypovolemia, and multi-organ failure. Treatment is limited to ribavirin for Lassa fever and immune plasma for Argentine hemorrhagic fever. Improved therapeutic options that are safe, more effective and widely available are needed. Here, we show that modification of favipiravir treatment to include a high-dose loading period achieves complete protection in a guinea pig model of Argentine hemorrhagic fever when treatment was initiated two days following challenge with Junin virus (JUNV). This loading dose strategy also protected 50% of animals from lethal disease when treatment was delayed until 5 days post-infection and extended the survival time in those that succumbed. Consistent with the survival data, dramatic reductions in serum and tissue virus loads were observed in animals treated with favipiravir. This is the first report demonstrating complete protection against uniformly lethal JUNV infection in guinea pigs by administration of a small molecule antiviral drug.

Calomys musculinus, the natural reservoir of Argentine haemorrhagic fever, is a semipermissive host for a mouse-attenuated mutant of Junin virus.

C167, a mouse-attenuated strain of Junin virus derived from the XJC13 strain, also displayed reduced virulence for the South American cricetid Calomys musculinus, a natural reservoir of this virus in nature. Intracerebral inoculation of C. musculinus with 500 PFU of C167 produced only 25% mortality, whereas the parental XJC13 killed 85% of the animals. The attenuation of C167 for this cricetid was lower than for albino mice. The multiplication of C167 in C. musculinus-derived embryo or kidney fibroblasts was diminished with respect to XJC13, allowing us to define C. musculinus cells as a semipermissive system for C167, whereas murine and Vero cells were restrictive and permissive cultures, respectively. As a consequence, C167 as well as XJC13 were able to establish a persistent infection in C. musculinus embryo fibroblasts and Vero cells, but the mutant could not induce a carrier state in murine cells. Thus, the degree of susceptibility of C. musculinus to C167 was linked to the semipermissiveness of cricetid cells to virus multiplication.

Pathological and virological features of arenavirus disease in guinea pigs. Comparison of two Pichinde virus strains.

A guinea pig passage-adapted strain of the arena-virus Pichinde (adPIC) is highly virulent in inbred guinea pigs, whereas the related strain PIC3739 is attenuated. Both viruses were macrophage tropic and infected peritoneal, splenic, liver, and alveolar macrophages during experimental Pichinde virus infection. Infection with the virulent strain was associated with unlimited viral replication in the face of exaggerated delayed-type hypersensitivity response, manifested by the macrophage disappearance reaction. Histopathological lesions unique to adPIC-infected guinea pigs included intestinal villus blunting with mucosal infiltration by pyknotic debris-laden macrophages and apoptosis of crypt epithelial cells. Splenic red pulp necrosis was also significantly associated with adPIC infection but not PIC3739 infection. These findings may provide clues to the pathogenesis of a group of poorly understood human viral hemorrhagic fevers.

Antiviral treatment of Argentine hemorrhagic fever.

Argentine hemorrhagic fever is a systemic viral disease caused by Junin virus, with a mortality of 15-30% in untreated individuals. Current specific therapy is highly effective in reducing mortality, and consists of the early administration of immune plasma in defined doses of specific neutralizing antibodies per kg of body weight. However, several reasons suggest the need to investigate alternative therapies. Ribavirin, a broad spectrum antiviral agent, is effective in the treatment of other viral hemorrhagic fevers, and the studies done with Junin virus infections to date indicate that this drug may also have a beneficial effect in Argentine hemorrhagic fever.

Protection of guinea pigs against experimental Argentine hemorrhagic fever by purified human IgG: importance of elimination of infected cells.

Antibody-containing plasma from patients recovered from Argentine hemorrhagic fever (AHF) is of proven value in treatment of the acute disease, but the possibility of transmitting blood-borne organisms such as HIV and hepatitis virus detracts from this approach. Purified human immune plasma fractions IgG1,2,4, IgG1,2,3,4 and F(ab')2 neutralized Junin virus in vitro. IgG1,2,3,4 and IgG1,2,4 lysed (in the presence of complement) cells infected with Junin virus, and protected infected guinea pigs from AHF. However, large quantities of the immune F(ab')2 fraction from the same plasma pool failed to protect guinea pigs from death, to increase the mean time to death, and to diminish virus load in target organs of infected guinea pigs. This suggests that elimination of infected cells rather than virus neutralization may play a critical role in protection against Junin virus.

[Attenuation in the virulence of a mutant of Junin virus in suckling mice]. / Atenuación en la virulencia para ratón lactante de una mutante de virus Junín.

The virulence in neonatal mice of a temperature-sensitive mutant of Junin virus, named C167, was studied. The thermosensitive properties of this mutant were tested by titration on Vero cells at 37 and 40 degrees C. The ratio of infectivity 40/37 was approximately 100-fold lower for C167 with respect to XJC13 (Table 1). The attenuation of C167 was determined by measurement of mean survival time and 50% lethal dose after intracerebral injection of 2 and 11 day old mice. For C167 the lethality index (expressed ad the ratio TCID50/LD50) was greater than 580, while for XJC13 the index was 4.4 (Table 2). The lack of virulence of C167 was correlated with a restricted ability to replicate in suckling mouse brains. By contrast, the mutant and the parental virus grew to a similar titre in Vero cells (Figure 2). Both viruses were indistinguishable in cross-neutralization tests using hyperimmune antisera.

[Treatment of Argentine hemorrhagic fever with convalescent's plasma. 4433 cases]. / Traitement de la fièvre hémorragique argentine par le plasma de convalescent. 4,433 cas.

During the 23 consecutive annual epidemics of Argentine haemorrhagic fever observed from 1959 to 1983, a group of 4,433 patients were treated at Junin (Argentina) with convalescent's plasma; the overall mortality rate was 3.29%. In 1958, before convalescent's plasma was used, the mortality rate in 448 patients who received the conventional treatment was 42.85%. The difference between the two groups is highly significant and demonstrates the value of convalescent's plasma in the treatment of the disease.

Role of Calomys musculinus peritoneal macrophages in age-related resistance to Junin virus infection.

In nature, the cricetid Calomys musculinus is the principal host of Junin virus, the etiological agent of Argentine hemorrhagic fever. In the experimental infection, adult C. musculinus survived whereas newborns died after intraperitoneal inoculation with the XJ.Cl3 strain of Junin virus. The role of peritoneal macrophages in this age-related resistance was studied. Junin virus multiplied in cultivated macrophages from either neonatal or adult animals and, therefore, it was not possible to correlate the susceptibility of peritoneal macrophages to Junin virus infection with the age-dependent resistance. When adult and neonatal animals were treated with silica prior to Junin virus infection, deaths occurred in the adults, while a delay and decrease in the mortality rate were observed in neonatals. These results suggest that in neonatal C. musculinus macrophages could be permissive cells for Junin virus multiplication, whereas in adult cricetids, these cells would act as a barrier against viral infection by means of an extrinsic antiviral activity.

[Susceptibility of 11-day-old mice to infection with the Junin virus grown in different hosts]. / Susceptibilidad del ratón de 11 días a la infección con virus Junín propagado en distintos huéspedes.

Twelve clones derived from a stock of Junin virus grown in baby mouse brain were isolated in Vero cells. Some properties of those viral clones were determined and compared with parental virus in order to ascertain the degree of heterogenicity of the original population. No differences were observed among clones and parental virus when the degree of thermolability was measured by heating them at 50 degrees C for 30 min. (Table 1). Similarly no ts phenotype character was present among all viral isolates tested since ratios 40 degrees C/37 degrees C, alike parental virus, oscillated around 0.48 (Table 1). Also virulence for 2 days old mice, expressed as the ratio of PFU/LD50 varied between 5.27 to 25 while parental virus ratio was 4.56. A completely different picture was observed when virulence ratio was determined in 11 days old mice. The values found for viral clones were all above 47 being the maximum 210 (Table 1) whereas the ratio of parental virus was 2.65. Considering that the difference observed could be due to the last host where the virus multiplied, parental virus stock was passed once or two times in Vero or BHK21 cells before assaying virulence. Results quoted in Table 2 show that after one or two passages in cells, the ratio of virulence decreased, at least, 60 times independently of the virus stock or the type of cells used. Furthermore, the appearance of a viral population with an intermediate virulence index was detected by a passage through mouse embryo cells (Table 2).(ABSTRACT TRUNCATED AT 250 WORDS)

Infection of pregnant guinea pigs with attenuated Junin virus strains.

The effect of the attenuated XJC13 and XJ0 strains of Junin virus (JV) was studied in guinea pigs infected before and during pregnancy. The 58% mortality rate in animals infected during gestation and the 16.7% mortality rate in chronically infected animals were attributed to a viral effect. An abortion rate of 33% occurred in animals infected before the 7th week of gestation. Regardless of the time of infection, JV was isolated from central nervous system tissue, placentas, and fetuses of animals killed just before parturition, even when circulating neutralizing antibodies were present. Results confirmed that transplacental infection is a regular event and showed that guinea pigs are more susceptible to attenuated JV strains during pregnancy, most probably due to immunosuppression, hormonal changes, or both.

Correlation between endogenous interferon and the clinical evolution of patients with Argentine hemorrhagic fever.

To explore the endogenous interferon levels in patients of Argentine hemorrhagic fever (AHF) with different clinical evolution of the disease, 29 fatal and 33 surviving cases of AHF were analyzed. As previously reported, the titers of endogenous alpha-IFN in patients with AHF are very high, generally between 2,000 and 64,000 IU/ml. Thus far, these are the highest levels of circulating interferon detected in any human viral disease. In this study it was found that during the second week of evolution the titers of interferon were significantly higher in fatal cases than in survivors. Therefore, very high levels of interferon have a prognostic value in AHF.

Importance of dose of neutralising antibodies in treatment of Argentine haemorrhagic fever with immune plasma.

P6 a retrospective study outcome in patients with Argentine haemorrhagic fever was associated with the amount of neutralising antibodies against Junin virus present in the transfused units of immune plasma. Low doses of neutralising antibodies were associated with higher mortality. A prospective study gave comparable results. A dose of no less than 3000 therapeutic units of neutralising antibodies per kg body weight is recommended. It is also suggested that the lack of effectiveness of immune plasma in the treatment of other viral haemorrhagic fevers, such as Lassa fever and Crimean-Congo haemorrhagic fever, may be due to a low dose of the specific neutralising antibodies.

[Effect of various schedules of cyclophosphamide administration on the mortality of the adult mouse infected with Junín virus]. / Efecto de distintos esquemas de administración de ciclofosfamida en la mortalidad del ratón adulto infectado con virus Junín.

The object of this paper was to determine the influence of cyclophosphamide immunosuppression on the mortality of 40-45 day old Balb/c mice infected intracerebrally with a pathogenic strain of Junin virus, using different administration schedules. Up to 200 mg/kg of cyclophosphamide were not toxic. Results show that, unlike in other experimental models, three or four 50 mg/kg cyclophosphamide doses given both before and after viral infection were required to break-down resistance to Junin virus (90-96.5% mortality vs. 8% in controls). Taking into account the effect of cyclophosphamide on the cell populations involved in the immune response, causes likely to lead to the greater susceptibility of the suppressed adult mouse to Junin virus are discussed.

[Effect of cyclophosphamide on experimental infection of rats with 2 strains of the Junin virus]. / Acción de la ciclofosfamida en la infección experimental de la rata con dos cepas de virus Junín.

The course of viral infection in rats of several ages after intracerebral inoculation with two strains of Junin virus, as well as the effect of an immunosuppressor was studied. The survival rate in 2-day-old rats was 95%, which fell to 45% in cyclophosphamide-treated infected animals (Figure 1a). However, barely 5% of these rats inoculated with the XJCl3 strain survived, while the cyclophosphamide suppressive treatment increased the rate to 36% (Figure 1b). This contrasting behaviour suggested that the XJ strain produces a subclinical infection in 2-day-old rats and the host immune mechanisms are responsible for recovery from the viral infection. Adequate immunosuppression converts sublethal experimental infections into lethal infection, accompanied by persistent viral replication in the target organ (brain) and suppression of anti-viral antibodies (Figure 2a). On the other hand, the inoculation of 2-day-old rats with the XJCl3 Junin virus may give rise to an immunopathology avoidable by CY treatment. In 10-day-old rats both strains of Junin virus caused a direct pathology, not modified by CY treatment (Table 3). This treatment failed to change susceptibility or virus concentration in the brain, but specific antibodies were considerably reduced. In the case of 26-day-old rats, there was total resistance to viral infection which remained unchanged after CY treatment.