Phospholipase A₂ Isolated from the Venom of <i>Crotalus durissus terrificus</i> Inactivates <i>Dengue virus</i> and Other Enveloped Viruses by Disrupting the Viral Envelope

<div><p>The <i>Flaviviridae</i> family includes several virus pathogens associated with human diseases worldwide. Within this family, <i>Dengue virus</i> is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against <i>Dengue virus</i> or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A₂ isolated from the venom of <i>Crotalus durissus terrificus</i> was able to inhibit <i>Dengue virus</i> and <i>Yellow fever virus</i> infection in Vero cells. Here, we present evidence that phospholipase A₂ has a direct effect on <i>Dengue virus</i> particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A₂ might gain access to the <i>Dengue virus</i> lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A₂ is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.</p></div

Analysis of the exposure of DENV-2 RNA.

<p>DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p<0.01).</p

Analysis of the exposure of DENV-2 genomic RNA.

<p>DENV-2 was first treated with PLA₂-CB, crotoxin (8 ng/µL each) or PBS at 37°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p<0.05).</p

Virucidal assay.

<p>DENV-2 was treated with different concentrations of PLA₂-CB (a) and crotoxin (b) and then used to infect Vero cells for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (*<i>p</i><0.05, **p<0.01).</p

Pre-treatment assay.

<p>Vero cells were treated with different concentrations of PLA₂-CB (a) and crotoxin (b) and then infected with DENV-2 for 72 h. The antiviral effect of the toxins was evaluated by determining the virus titer in the cell culture supernatant by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated cells (*p<0.05, **p<0.01).</p

Susceptibility of Balb/c and C57BL/6 mice to ROCV infection.

<p>Mice were infected with different doses of ROCV (n = 10 mice per group). Survival rate and body weight loss for C57BL/6 (A and B) and for Balb/c (C and D). Comparison of mortality rate between C57BL/6 and Balb/c (Fig 1E). Survival rate and body weight loss were measured for up to 21 days post-infection. Statistically significant differences in B and D: ****p<0.0001 was determined by one-way ANOVA with Dunnett's multiple comparisons test, in E: *p<0.01 by Log-rank (Mantel-Cox) test.</p

Evaluation of cross-protective immunity against ROCV after prior infections with different flaviviruses.

<p>Immunization and challenge regime (A). Mice (n = 40 per group) were infected twice with the different flaviviruses known to circulate in Brazil, and were then challenged (n = 20 per group) with 2.76x10⁷ PFU of ROCV and survival rates (B and E), clinical scores (C and F) and body weight loss (D and G) were determined up to 21 days post-infection. Statistically significant differences in B and E: *p<0.01, **p<0.001 and ****p<0.0001 was determined by Log-rank (Mantel-Cox) test. In F: ****p<0.0001 was analyzed by a student t-test. In D-G *p<0.01, **p<0.001 and ****p<0.0001 was determined by One-way ANOVA with Dunnett's multiple comparisons test. All statistically significant differences were with group control (Mock).</p

Amino acid identity between the E proteins of Brazilian flaviviruses used in this study.

<p>Amino acid identity between the E proteins of Brazilian flaviviruses used in this study.</p

Ilheus and Saint Louis encephalitis viruses elicit cross-protection against a lethal Rocio virus challenge in mice

<div><p>Rocio virus (ROCV) was the causative agent of an unprecedented outbreak of encephalitis during the 1970s in the Vale do Ribeira, Sao Paulo State, in the Southeast region of Brazil. Surprisingly, no further cases of ROCV infection were identified after this outbreak; however, serological surveys have suggested the circulation of ROCV among humans and animals in different regions of Brazil. Cross-protective immunity among flaviviruses is well documented; consequently, immunity induced by infections with other flaviviruses endemic to Brazil could potentially be responsible for the lack of ROCV infections. Herein, we evaluated the cross-protection mediated by other flaviviruses against ROCV infection using an experimental C57BL/6 mouse model. Cross-protection against ROCV infection was observed when animals had prior exposure to Ilheus virus or Saint Louis encephalitis virus, suggesting that cross-reactive anti-flavivirus antibodies may limit ROCV disease outbreaks.</p></div