Schirtzinger, E. E. | Search bioRxiv

bioRxiv2021

Improving comparability of studies using HCoV-OC43 as a surrogate for SARS-CoV-2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of [~]107 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of [~]106.5 TCID50/ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts.

microbiology↗

bioRxiv2020

Old drugs with new tricks: Efficacy of fluoroquinolones to suppress replication of flaviviruses

Antiviral therapies are urgently needed to treat infections with flaviviruses such as Zika (ZIKV) and dengue (DENV) virus. Repurposing FDA-approved compounds could provide the fastest route to alleviate the burden of flaviviral diseases. In this study, three fluoroquinolones, enoxacin, difloxacin and ciprofloxacin, curtailed replication of flaviviruses ZIKV, DENV, Langat (LGTV) and Modoc (MODV) in HEK-293 cells at low micromolar concentrations. Time-of-addition assays revealed that enoxacin suppressed ZIKV replication when added at 6 hours post-infection, suggesting inhibition of an intermediate step in the virus life cycle, whereas ciprofloxacin and difloxacin had a wider window of efficacy of 2, 6, and 8 hours post-infection for difloxacin and 2 to 8 hours post-infection for ciprofloxacin. The efficacy of enoxacin to suppress ZIKV replication in 5-week-old A129 mice was evaluated in two experiments. First, mice were infected with 1x105 plaque-forming units (pfu) ZIKV FSS13025 (n=20) or PBS (n=11) on day 0 and subsets were treated with enoxacin at 10mg/kg or 15mg/kg or diluent orally twice daily on days 1-5. Treated and control mice did not differ in weight change or virus titer in serum or brain. Mice treated with enoxacin showed a significant, 5-fold decrease in ZIKV titer in testes relative to controls. Second, mice were infected with 1x102 pfu ZIKV (n=13) or PBS (n=13) on day 0 and subsets were treated with 15mg/kg oral enoxacin or diluent twice daily on days 0 (pre-treatment) and 1-5. Mice treated with enoxacin showed a significant, 2.5-fold decrease in ZIKV titer in testes relative to controls, while weight and viral load in the serum, brain, and liver did not differ between treated and control mice. Enoxacin efficacy in cultured murine Sertoli cells was not enhanced compared to efficacy in HEK-293 cells. ZIKV can be sexually transmitted, so reduction of titer in the testes by enoxacin should be further investigated. Author SummaryFlaviviruses such as Zika and dengue virus pose a significant threat to public health worldwide, and there are currently no antiviral therapies to treat any flaviviral infection. Repurposing FDA-approved drugs as anti-flaviviral therapies can accelerate clinical use. We demonstrated that fluoroquinolone antibiotics exhibit anti-flaviviral efficacy, suppressing flavivirus replication in cultured human cells. Additionally, we found that the fluoroquinolone enoxacin suppressed Zika virus replication in mouse testes. While Zika virus is primarily transmitted via mosquitoes, the virus also undergoes sexual transmission. The importance of sexual transmission for the overall epidemiology of the virus remains unclear; nonetheless all routes of potential transmission to pregnant women are of concern as fetal infection in utero can have devastating effects. Thus, our data indicate that fluoroquinolones hold promise for treatment of flaviviral infections, particularly infection of the testes by Zika virus, and that this class of drugs warrants further study.

microbiology↗