Maporal virus as a surrogate for pathogenic New World hantaviruses and its inhibition by favipiravir

Abstract

Background: Pathogenic hantaviruses geographically distributed in the Old World cause haemorrhagic fever with renal syndrome (HFRS), whereas New World hantaviruses are the aetiological agents of hantavirus cardiopulmonary syndrome (HCPS). Ribavirin, a drug associated with toxicities, is presently indicated for treatment of HFRS, whereas treatment of the more frequently lethal HCPS is limited to supportive care. Because of the need for safe and effective antivirals to treat severe hantaviral infections, we evaluated favipiravir (T-705) against Dobrava and Maporal viruses as representative Old World and New World hantaviruses, respectively. Dobrava virus causes HFRS in Europe. Maporal virus (MPRLV), recently isolated from western Venezuela, is phylogenetically similar to Andes virus, the principal cause of HCPS in Argentina.

Methods: Hantavirus replication in the presence of various inhibitors was measured by focus-forming unit assays and quantitative reverse transcriptase PCR. Phylogenetic relationships were assessed by the neighbour-joining and bootstrap consensus methods.

Results: Here, we show that infection of Vero E6 cells with MPRLV is dependent on β3 integrins, similar to that reported for pathogenic hantaviruses. Furthermore, by analysis of molecular determinants associated with the G1 glycoprotein cytoplasmic tail, we show the close genetic proximity of MPRLV to other HCPS-causing hantaviruses in these regions predictive of pathogenicity. We also demonstrate anti-hantavirus activity by favipiravir with inhibitory concentrations ranging from 65 to 93 μM and selectivity indices>50.

Conclusions: Our data suggest that MPRLV may serve as a safer alternative to modelling infection caused by the highly lethal Andes virus and that hantaviruses are sensitive to the effects of favipiravir in cell culture.

Figure 1. MPRLV infection is mediated by β3 integrins

Duplicate wells of Vero E6 cells were pretreated with A) integrin-specific antibodies (1 μg/ml) or B) vitronectin and fibronectin (1 and 20 μg/ml, respectively) for 1 hour prior to viral infection. After infection and incubation for 1-5 days (depending on virus) cell monolayers were fixed and stained as described in the methods. Data are presented as percent FFU of infected 1% BSA PBS-treated controls. Data are representative of three independent experiments. *P < 0.05 compared to respective controls by two-way analysis of variance with Bonferroni posttest.

Figure 2. Amino acid comparison and phylogenetic relationships among the ITAM/degron sequences of fourteen different hantaviruses

A) Alignment of the G1 protein cytoplasmic tail region containing the ITAM/degron residues. The asterisks indicate amino acids important in the proteosomal degradation of G1 tails. The gray columns represent the ITAM motif residues. B) Phylogentic analysis based on ITAM/degron sequence. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. The tree is not drawn to scale, but indicates branch lengths (next to the branches) in the same units as those of the evolutionary distances used to infer the phylogenetic tree. BAYV, Bayou virus; BMJV, Bermejo virus; CHOV, Choclo virus, HTNV, Hantaan virus; LNV, Laguna Negra virus; NYV, New York-1 virus, PUUV, Puumula virus; SEOV, Seoul virus; TULV, Tula virus.

Figure 3. Favipiravir reduces MPRLV RNA during infection

Viral RNA was measured by qRT-PCR from total RNA extracted from infected Vero E6 cells treated with varying concentrations of favipiravir or ribavirin. Data are presented as percent of virus-infected, untreated controls and are representative of 2-3 experiments.