Broad-spectrum antiviral JL122 blocks infection and inhibits transmission of aquatic rhabdoviruses

Abstract

The aquaculture industry is growing rapidly to meet the needs for global protein consumption. Viral diseases in aquaculture are quite challenging due to lack of treatment options as well as limited injection-delivery vaccines, which are costly. Thus, water-immersion antiviral treatments are highly desirable. This study focused on broad-spectrum, light-activated antivirals that target the viral membrane (envelope) of viruses to prevent viral-cell membrane fusion, ultimately blocking viral entry into cells. Of the tested small-molecules, JL122, a new broad-spectrum antiviral previously unexplored against aquatic viruses, blocked infection of three aquatic rhabdoviruses (IHNV, VHSV and SVCV) in cell culture and in two live fish challenge models. Importantly, JL122 inhibited transmission of IHNV from infected to uninfected rainbow trout. Further, the effective antiviral concentrations were not toxic to cells or susceptible fish. These results show promise for JL122 to become an immersion treatment option for outbreaks of aquatic enveloped viral infections.

Keywords: Antivirals; Enveloped; Fish viruses; Immersion; JL122; Rhabdovirus.

Fig. 1.. In vitro efficacy of three antiviral compounds.

(A) Chemical structures of LJ001, JL122, and JL118 (Vigant et al., 2013). (B) Concentrations up to 1 μM of each compound (LJ001, JL122 and JL118) were pre-incubated with 1×10⁴ PFU/mL IHNV, VHSV or SVCV for 30 min while exposed to light. Viral titer was determined by plaque assay. Antiviral inhibition was compared between compounds. For all three viruses, JL122 completely blocked infection at 1.0 μM concentrations. JL122 was the most efficacious antiviral, followed by LJ001, and JL118 was least effective. Positive controls (PC) were virus pre-incubated with 0.01% DMSO (vehicle control). Data represents mean viral titer ± SE (n=3; experiments repeated 3 times). *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, ANOVA, Bonferroni’s Multiple Comparison Test.

Fig. 2.. EPC cell viability at 24 and 48 h post-exposure to JL122.

EPC cells were exposed to up to 10 μM JL122, vehicle control only (0.1% DMSO; 0 μM JL122), or hydrogen peroxide (40 mM final concentration) for 24 or 72 h in the presence of light. Formazan dye absorbance was measured at 450 nm and used to assess % cell viability. Negative control [-] = no compound. DMSO concentration varied for each JL122 dose depending on dilution: 10 μM JL122 had 0.1% DMSO; 1 μM JL122 had 0.01% DMSO. Cytotoxicity was absent when JL122 concentrations were ≤ 5 μM. Toxicity to cells was present when exposed to ≥7 μM JL122. A hyperplastic response occurred with 3 μM JL122 at 24 h post-exposure. Data represents mean cell viability ± SE (n=5; triplicate samples are shown and a similar trend was observed in 5 repeated experiments) when normalized to no treatment (negative control). *: P<0.001, ANOVA, Tukey’s Multiple Comparison Test.

Fig. 3.. In vivo efficacy of JL122 against IHNV and VHSV.

(A) 1×10⁴ PFU/mL IHNV was pre-incubated with up to 1.0 μM JL122 or LJ001 for 15 min, while exposed to light. Eight naïve rainbow trout (5/8 fish used for viral titer) were added to each challenge container and held for 72 h. Homogenate supernatant from whole trout was used for plaque assay to determine IHNV titer. Positive control: IHNV and vehicle control (0.0001% DMSO, final concentration). IHNV infection was completely blocked with 0.1 and 1.0 μM JL122. JL122 inhibition is displayed with previously published LJ001 data (Balmer et al., 2017) that showed significant (but not complete) inhibition of IHNV infection with 0.1 and 1.0 μM LJ001. (B) Largemouth bass were similarly immersion infected with VHSV pre-incubated with up to 1.0 μM JL122. Homogenate supernatant from pooled tissues (liver, kidney, heart and spleen = TP) and brain from bass was used for RT-qPCR to quantify VHSV RNA. Positive control: VHSV and 0.0001% DMSO. VHSV infection was also completely blocked with 0.1 μM and 1.0 μM JL122. Plaque assay data from the pooled tissues (not shown) confirms and correlates with the qPCR data. There was a significant decrease in brain viral load even at the 0.01 μM JL122 dose. Data represents mean viral titer ± SE (n=5 fish per treatment group). Mock controls had MEM and vehicle control only; all mock infected fish had negative titers (data not shown). *: P<0.05, **: P<0.01, ***: P<0.001, ANOVA, Dunnett’s post-test.

Fig. 4.. Horizontal transmission inhibition of IHNV by JL122.

Rainbow trout were immersion infected with 2×10⁵ PFU/mL IHNV (donor fish) or MEM medium (mock) and remained in flow-through for 24 h. Experimental groups: IHNV- or mock-infected donor fish (three) were placed in static challenge containers, followed by addition of JL122 (5 μM final concentration) or 0.005% DMSO (vehicle/positive control); after 15 min, nine naïve recipient fish were added to each challenge container for cohabitation (n=1 mock-DMSO group [12 fish]; n=1 mock-JL122 group [12 fish], n=4 IHNV-DMSO groups [48 fish] and n=4 IHNV-JL122 groups [48 fish]). A total of 3 doses of JL122 or DMSO (dosed every 24 h) were delivered during the 72 h cohabitation period. All mock-infected fish (not shown) had negative titers (below detection). (A) Comparison of DMSO (control; black bar) vs. JL122-treated fish (gray bar). There was a significantly (***: P=0.0003, Student’s t-test) lower mean IHNV titer in the JL122-treated groups compared to the positive control fish. (B) Separation of donor (fin-clipped for identification) and recipient fish to determine inhibition of transmission. For immersion infected IHNV donor fish, there was a slight and non-significant decrease in viral load (P=0.3308) for the JL122-treated donor fish (left gray dots; 12 fish) compared to DMSO-treated (control) donor fish (left black dots; 12 fish). There was a highly significant decrease in viral load for JL122-treated IHNV recipient fish (right gray dots; 36 fish) with 8 fish below detection (negative titers; gray triangles) compared to DMSO-treated (control) IHNV recipient fish (right black dots; 36 fish). LDL = lower detection limit. ***: P<0.001, ANOVA, Tukey’s Multiple Comparison Test. Data represents mean IHNV titer ± SE.