Contribution of GPC Ectodomain of Candid#1

Machupo virus (MACV), a New World arenavirus, is the etiological agent of Bolivian hemorrhagic fever (BHF). Junin virus (JUNV), a close relative, causes Argentine hemorrhagic fever (AHF). Previously, we reported that a recombinant, chimeric MACV (rMACV/Cd#1-GPC) expressing glycoprotein from the Candid#1 (Cd#1) vaccine strain of JUNV is completely attenuated in a murine model and protects animals from lethal challenge with MACV. A rMACV with a single F438I substitution in the transmembrane domain (TMD) of GPC, which is equivalent to the F427I attenuating mutation in Cd#1 GPC, was attenuated in a murine model but genetically unstable. In addition, the TMD mutation alone was not sufficient to fully attenuate JUNV, indicating that other domains of the GPC may also contribute to the attenuation. To investigate the requirement of different domains of Cd#1 GPC for successful attenuation of MACV, we rescued several rMACVs expressing the ectodomain of GPC from Cd#1 either alone (MCg1), along with the TMD F438I substitution (MCg2), or with the TMD of Cd#1 (MCg3). All rMACVs exhibited similar growth curves in cultured cells. In mice, the MCg1 displayed significant reduction in lethality as compared with rMACV. The MCg1 was detected in brains and spleens of MCg1-infected mice and the infection was associated with tissue inflammation. On the other hand, all animals survived MCg2 and MCg3 infection without detectable levels of virus in various organs while producing neutralizing antibody against Cd#1. Overall our data suggest the indispensable role of each GPC domain in the full attenuation and immunogenicity of rMACV/Cd#1 GPC

Zika virus infection elicits auto-antibodies to C1q

Zika virus (ZIKV) causes mostly asymptomatic infection or mild febrile illness. However, with an increasing number of patients, various clinical features such as microcephaly, Guillain-Barré syndrome and thrombocytopenia have also been reported. To determine which host factors are related to pathogenesis, the E protein of ZIKV was analyzed with the Informational Spectrum Method, which identifies common information encoded by primary structures of the virus and the respective host protein. The data showed that the ZIKV E protein and the complement component C1q cross-spectra are characterized by a single dominant peak at the frequency F = 0.338, suggesting similar biological properties. Indeed, C1q-specific antibodies were detected in sera obtained from mice and monkeys infected with ZIKV. As C1q has been known to be involved not only in immunity, but also in synaptic organization and different autoimmune diseases, a ZIKV-induced anti-C1q antibody response may contribute to the neurological complications. These findings might also be exploited for the design of safe and efficacious vaccines in the future

STAT-1 Knockout Mice as a Model for Wild-Type Sudan Virus (SUDV)

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014–2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 101 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures

Filovirus Virulence in Interferon α/β and γ Double Knockout Mice, and Treatment with Favipiravir

The 2014 Ebolavirus outbreak in West Africa highlighted the need for vaccines and therapeutics to prevent and treat filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would facilitate the screening of anti-filovirus agents. To that end, we characterized knockout mice lacking α/β and γ interferon receptors (IFNAGR KO) as a model for wild-type filovirus infection. Intraperitoneal challenge of IFNAGR KO mice with several known human pathogenic species from the genus Ebolavirus and Marburgvirus, except Bundibugyo ebolavirus and Taï Forest ebolavirus, caused variable mortality rate. Further characterization of the prototype Ebola virus Kikwit isolate infection in this KO mouse model showed 100% lethality down to a dilution equivalent to 1.0 × 10−1 pfu with all deaths occurring between 7 and 9 days post-challenge. Viral RNA was detectable in serum after challenge with 1.0 × 102 pfu as early as one day after infection. Changes in hematology and serum chemistry became pronounced as the disease progressed and mirrored the histological changes in the spleen and liver that were also consistent with those described for patients with Ebola virus disease. In a proof-of-principle study, treatment of Ebola virus infected IFNAGR KO mice with favipiravir resulted in 83% protection. Taken together, the data suggest that IFNAGR KO mice may be a useful model for early screening of anti-filovirus medical countermeasures

The Ectodomain of Glycoprotein from the Candid#1 Vaccine Strain of Junin Virus Rendered Machupo Virus Partially Attenuated in Mice Lacking IFN-αβ/γ Receptor

<div><p>Machupo virus (MACV), a New World arenavirus, is the etiological agent of Bolivian hemorrhagic fever (BHF). Junin virus (JUNV), a close relative, causes Argentine hemorrhagic fever (AHF). Previously, we reported that a recombinant, chimeric MACV (rMACV/Cd#1-GPC) expressing glycoprotein from the Candid#1 (Cd#1) vaccine strain of JUNV is completely attenuated in a murine model and protects animals from lethal challenge with MACV. A rMACV with a single F438I substitution in the transmembrane domain (TMD) of GPC, which is equivalent to the F427I attenuating mutation in Cd#1 GPC, was attenuated in a murine model but genetically unstable. In addition, the TMD mutation alone was not sufficient to fully attenuate JUNV, indicating that other domains of the GPC may also contribute to the attenuation. To investigate the requirement of different domains of Cd#1 GPC for successful attenuation of MACV, we rescued several rMACVs expressing the ectodomain of GPC from Cd#1 either alone (MCg1), along with the TMD F438I substitution (MCg2), or with the TMD of Cd#1 (MCg3). All rMACVs exhibited similar growth curves in cultured cells. In mice, the MCg1 displayed significant reduction in lethality as compared with rMACV. The MCg1 was detected in brains and spleens of MCg1-infected mice and the infection was associated with tissue inflammation. On the other hand, all animals survived MCg2 and MCg3 infection without detectable levels of virus in various organs while producing neutralizing antibody against Cd#1. Overall our data suggest the indispensable role of each GPC domain in the full attenuation and immunogenicity of rMACV/Cd#1 GPC.</p></div

Mice Lacking Alpha/Beta and Gamma Interferon Receptors Are Susceptible to Junin Virus Infection▿

Junin virus (JUNV) causes a highly lethal human disease, Argentine hemorrhagic fever. Previous work has demonstrated the requirement for human transferrin receptor 1 for virus entry, and the absence of the receptor was proposed to be a major cause for the resistance of laboratory mice to JUNV infection. In this study, we present for the first time in vivo evidence that the disruption of interferon signaling is sufficient to generate a disease-susceptible mouse model for JUNV infection. After peripheral inoculation with virulent JUNV, adult mice lacking alpha/beta and gamma interferon receptors developed disseminated infection and severe disease

Frequency of histopathological observation.

<p>Frequency of histopathological observation.</p

IFN-αβ/γ R^-/- mice were infected with 10,000 PFU of rMACV, rCd#1, MCg1, MCg2 and MCg3 by the intraperitoneal route.

<p>(A) Survival rate of the IFN-αβ/γ R^-/- mice after rMACV, rCd#1, MCg1, MCg2 and MCg3 infections (N = 7 per group except rCd#1, for which N = 6). Statistically significant differences between rMACV- and MCg3-infected groups are indicated by asterisks (**, <i>P<</i>0.01 by log rank test). The survival rate of the MCg1-infected group was significantly increased (**, <i>P<</i>0.01 for rMACV-infected group versus the MCg1-infected group). No significant difference was observed between MCg1- and MCg3-infectred groups (<i>P</i> = 0.06). (B) Clinical symptoms were monitored daily. All rMACV-infected animals showed disease such as scruffy coats and hunched postures at 11 to 15 dpi, while MCg1-infected animals showed delayed onset of the symptoms by 2–12 days. Two of mice infected MCg2 showed mild scruffy coats at 26 dpi to 28 dpi. (C) Body weight changes were monitored on the indicated days. Error bars indicate the SEM (N = 7 per group except rCd#1, for which N = 6). Loss of 5% of body weight in MCg1-infected mice was delayed by 6 to 13 days than in rMACV group. (D) Body temperatures were monitored on the indicated days. While six of seven rMACV-infected animals showed hypothermia (below 34°C) at 1 to 3 days prior to death, no exhibited hypothermia in groups of rCd#1-, MCg1-, MCg2- and MCg3-infected animals. The dots line indicates 34°C. The rMACV and rCd#1 were inoculated into IFN-αβ/γ R^-/- mice as a positive control and negative control, respectively, at the same time with MCg1, MCg2 and MCg3. The data of rMACV and rCd#1 in the Fig 2A, 2C and 2D has been published in Fig 2A, 2B and 2C respectively, of [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004969#pntd.0004969.ref016" target="_blank">16</a>].</p

Schematic diagrams and the growth curve of MCg1, MCg2 and MCg3.

<p>(A) The ectodomain of GPC alone of rMACV was replaced by the Cd#1 counterpart in rMACV backbone (MCg1), ectodomain of rMACV was replaced by the Cd#1 GPC ectodomain with F427I TMD mutation (MCg2), and the ectodomain and TMD of rMACV GPC were replaced by those of Cd#1 GPC (MCg3). (B) Growth curve of the MCg1, MCg2 and MCg3 were determined in Vero cells (rMACV: N = 3, rCd#1: N = 4, MCg1-3: N = 7). The virus titer of MCg1 was significantly higher than those of rCd#1, MCg2 and MCg3 at 48 hpi in Vero cells (**, <i>P</i><0.01, one-way ANOVA followed by Dunnett's test for MCg1 versus rCd#1, MCg1 versus MCg2 and MCg1 versus MCg3). (C) Growth curve of the MCg1, MCg2 and MCg3 were determined in A549 cells (rMACV: N = 3, rCd#1: N = 4, MCg1-3: N = 6). The virus titer of MCg1 was significantly higher than those of rCd#1, MCg2 and MCg3 at 72 hpi and 96 hpi in A549 cells (**, <i>P</i><0.01, one-way ANOVA followed by Dunnett's test for MCg1 versus rCd#1, MCg1 versus MCg2 and MCg1 versus MCg3). The dashed lines indicate the detection limit. The data of rCd#1 in the Fig 1B and 1C has been published in Fig 1B and 1C respectively, of [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004969#pntd.0004969.ref016" target="_blank">16</a>].</p