key: cord-0963533-s1hv2ra9 authors: Antony, Ferrin; Vashi, Yoya; Morla, Sudhir; Vandna,; Mohan Saini, Hari; Kumar, Sachin title: Therapeutic potential of Nitazoxanide against Newcastle disease virus: A possible modulation of host cytokines date: 2020-05-03 journal: Cytokine DOI: 10.1016/j.cyto.2020.155115 sha: 32ba7839296537e72e19f0f651573968dc8fb608 doc_id: 963533 cord_uid: s1hv2ra9 Abstract Newcastle disease (ND) is prevalent among the domesticated and the wild birds and is caused by the avian paramyxovirus serotype-I (APMV-I). It is commonly known to affect chicken, pheasant, ostrich, pigeon and waterfowl. Depending on the virulence, the velogenic NDV strains cause severe respiratory and nervous disorders with a high mortality rate. The live and killed vaccines are available for the prevention of infection in the market, but the drug for the treatment is not available. Nitazoxanide (NTZ), a member of thiazolides, is an antiparasitic drug. In the present study, the effect of NTZ on the NDV replication was explored. The experiments were conducted in chicken fibroblast cells (DF-1), PBMC, embryonated chicken eggs, and two-week old chickens. The inhibition of the NDV was observed upon post-treatment of NTZ at a concentration of ∼12.5 μM. Cytokine profiling of the DF-1, PBMC, and chicken embryonic tissue treated with NTZ revealed significant upregulation in all the cytokines studied except for IL-1β in DF-1 cells. It is plausible that NTZ is involved in causing immune-modulatory effects in poultry. NTZ treatment in two weeks old chicken showed significant reduction in NDV replication in trachea, and lungs, respectively, at 72 h post-infection. Encouraging results from the present study warrants repurposing NTZ as a drug for the treatment of viral infection in poultry. It will also pave the way towards understanding of similar effect against other animal pathogens. Newcastle Disease is a panzootic disease which affects the avian species. It is caused by Newcastle disease virus (NDV) belonging to the Avulavirus genus of the family Paramyxoviridae. It is an enveloped virus having linear, non-segmented single-stranded RNA of negative polarity [1] . Based on the genome size, strains of NDV are classified into 3 groups having 15186, 15192, and 15198 nucleotides [2] [3] [4] [5] . The genome of NDV has six transcriptional units, comprising nucleocapsid protein (N), matrix protein (M), phosphoprotein (P), fusion protein (F), haemagglutinin-neuraminidase protein (HN), and large polymerase protein (L) from 3′ to 5′ direction. The N and P, along with L, forms an RNA polymerase complex for the viral genome replication [6] . Depending on the severity of the infection, NDV strains are classified into lentogenic (less virulent), mesogenic (moderate virulent), and velogenic (high virulent) [7] . Furthermore, based on the tissue tropism, it can be further classified as viscerotropic velogenic, which produces lethal haemorrhagic lesions in the viscera and neurotropic velogenic that cause respiratory and neurological disorders [8] . The clinical signs of the infection vary depending on the virus, host species, the age of the host, infection with other organisms, environmental stress, and immune status. The average incubation time of the virus is 15-21 days and can be transmitted easily, causing significant loss to the poultry industry [9] . It can cause a substantial economic loss by reduced egg production and the death of the ailing birds. There are live, inactivated, and vectored vaccines that are available for the prevention of the disease in poultry [10, 11] . Among these, the traditional live vaccines such as LaSota, B1, and VG/GA strains are commonly used in the endemic areas [12, 13] . However, vaccine failures have been frequently reported due to the emergence of its variant strains [14] . There is no available drug against NDV; the reason why the development of the antiviral drug is of prime importance. Nitazoxanide (NTZ), also known as 2-(acetyloxy)-N-(5-nitro-2-thiazolyl) benzamide is a commercially available antiprotozoal drug which was initially developed for the cure of cryptosporidiosis and giardiasis [15] . NTZ has also been classified as a broad-spectrum firstclass antiviral compound against the respiratory syncytial virus, parainfluenza virus, Sendai virus, coronavirus, rotavirus, and norovirus infection [16] [17] [18] [19] [20] . Additionally, NTZ has been shown to inhibit hepatitis viruses [21, 22] , flaviviruses [23] [24] [25] , chikungunya virus [26] , human immunodeficiency virus [27] and vaccinia virus [28] . Clinical trials are under progress for the use of NTZ against influenza and its subtypes [29, 30] . Several mechanisms of action have been suggested for NTZ mediated viral inhibition. It can curtail influenza virus replication by inhibiting the maturation of hemagglutinin protein [29] . In the Sendai virus, the active metabolite of NTZ acts as a non-competitive inhibitor of ER-resident proteins to misfold the F protein and halts its trafficking towards the plasma membrane [17] . The reduction in the size of viroplasm was observed against rotavirus, which led to a decrease in the formation of dsRNA [31] . As an anti-hepatitis C compound, NTZ was shown to activate protein kinase R (PKR), resulting in the phosphorylation of eukaryotic initiation factor 2α (eIF2-α) [32] . NTZ blocks the replication of bovine viral diarrhoea virus by causing stress in the endoplasmic reticulum leading to depletion of ATP sensitive intracellular Ca 2+ stores [33] . However, the effect of NTZ on NDV biology has not been addressed to date. The present study reports the inhibition of NDV upon NTZ treatment on chicken fibroblast cells (DF-1), embryonated chicken eggs, and two-week old chickens. The chicken embryo fibroblast cells (DF-1) cells used in the experiments were obtained from ATCC (Manassas, VA, USA). The DF-1 cells were maintained in Dulbecco's minimum essential medium (DMEM) with 10% fetal bovine serum (FBS) (HiMedia Laboratories, India) at 37°C with 5% CO 2 . The stock solution (10 mg/mL) of NTZ (Sigma Aldrich, USA) was made by dissolving it in dimethyl sulfoxide (DMSO). Further dilutions of NTZ for treatment were made in DMEM with 2% FBS, unless differently specified. The cytotoxicity of the compound was found by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide formazan conversion assay, as described previously [34] . DF-1 cells were seeded in a 96 well plate with a seeding density of 10 5 cells/well prior to the experiment. Further, the cells were incubated with NTZ (0.3 μM to 100 μM) for 24 h and 48 h. The absorbance was taken at 570 nm using the microplate reader, and the percentage cell viability was calculated. DMSO was used as vehicle control. The NDV strain of Bareilly (virulent), previously sequenced in our lab [35] , was propagated in 9-day-old specific pathogen free (SPF) embryonated chicken eggs. Recombinant NDV expressing the green fluorescent protein (rNDV-GFP) available in the lab was used to visualize the infection in the cells [36] . The virus titer was calculated by hemagglutination assay (HA) with 1% chicken RBC and by plaque assay. Plaque assay was performed by infecting the DF-1 cells with NDV and incubating it at 37°C for an hour [37] . Muench algorithm [38] . Additionally, the virus was also quantified by plaque assay and compared with the controls. cDNA was prepared using high capacity cDNA reverse transcription kit (Thermo Fischer Scientific, USA), and qPCR was performed using chicken specific primers to check the expression of host cytokines (Table 1) . qPCR results are an average of three independent experiments. Normalization was done with the mock-treated controls. GAPDH was used as an endogenous gene expression control. Two weeks old specific pathogen free (SPF) chickens were used for animal studies. The chickens were randomly divided into four groups, namely untreated-uninfected control, Experimental results were analyzed using the ANOVA for multiple comparisons (GraphPad Prism 8). All data were obtained from at least three independent experiments, and a p-value of < 0.05 was considered significant. The MTT assay determined the cytotoxicity of NTZ in DF-1 cells. A decrease in cell viability was observed in the concentrations higher than 50 μM (Figure 1a ). The cell toxicity was linearly related to the NTZ when treated from 25 μM to 100 μM concentrations. The IC 50 (50% inhibitory concentration) value of NTZ was calculated as ~71.6 μM (Figure 1b) , whereas it decreased to ~63.3 μM at 48 h (Figures 1c and 1d ). Its toxicity was temporal and dose-dependent, as evident in the cell images visualized under bright field microscopy ( Figure 1e ). In The treatment of NTZ showed ~700-fold downregulation of N gene of NDV as compared to the untreated control at 48 h post-treatment ( Figure 4 ). The analysis of P and L genes of NDV post-NTZ treatment at different time intervals showed a significant reduction as compared to untreated DF-1 cells (Figure 4) . The mock infected DF-1 cells were used as a baseline control for all the experiments. The 9-day-old embryo showed lesions after infection with NDV; however, the treatment of NTZ after virus infection did not show any visible lesion of infection ( Figure 5a ). NDV quantification of the allantoic fluid and tissue sample by plaque assay showed a 1.5-fold reduction in its titer upon NTZ treatment (Figure 5b and 5c) . Similarly, an about 2fold reduction in the kinetics of NDV was observed in the tissue isolated from the NTZ treated embryo (Figure 5c ). control. Maximum regulation was observed in TLR7 and TNF-α, while the minimum was observed in IL-1β and NLRP3. All the observed changes were found to be significant. Replication of the virus was observed in the trachea and lungs, whereas no virus was found in the spleen for virus control group ( Figure 7) . The pre-treatment group showed a complete reduction of the virus in all the three organs studied. Co and post-treatment groups showed no virus in the lungs and 1-log reduction of the virus in the trachea as compared to virus control. NTZ treated groups had no gross pathological lesions on organs. Newcastle disease is an economically important disease, causing substantial loss to the poultry industry. The recommended strategy for the prevention of the disease is by using killed or live attenuated vaccines. Vaccination failure is a common problem and has been reported from places where the velogenic strains of NDV are endemic [10, 39, 40] . An alternative to this problem is the use of antiviral drugs against NDV, which is not commercially available. NTZ is a licensed drug for the treatment of diarrhoea induced by Cryptosporidium parvum and Giardia intestinalis [15, 41] . NTZ has also been used as a broad-spectrum antiviral drug [16, 42] . The present study is an attempt to repurpose NTZ as an antiviral against NDV infection. The IC 50 value of NTZ in the chicken fibroblast cells was found to be ~60 μM in 48 h. The cytotoxic concentration that reduced the exponential growth in baby hamster kidney (BHK-21) cells was earlier reported as 18.59 μg/ml (60.4 μM) [24] , which is as per the IC 50 value reported in our study. We performed the time of addition assay to identify the stage of NDV replication affected by NTZ. The minimal reduction was observed in the pre-treatment of NTZ, as observed in the rNDV-GFP infected cells. No reduction was observed in the co-treatment, while the most significant reduction was visible in the post-treatment of NTZ. Our results were similar to the results reported earlier [17, 29, 43] . It was concluded that the drug does not directly affect the virus binding or entry into target cells. In previous reports, concentration ranging from 3-25 μM was used to show the antiviral activity of NTZ [26, 33] . A peak concentration of up to 200 μM has been detected in the blood plasma of humans after the oral dosage of NTZ (4g) and is safe [44] . In order to minimize the cytotoxic effect of NTZ, concentrations ranging from 3.125-25μM was chosen for checking it's effect against NDV. Our study showed a reduction of NDV by two-fold at NTZ concentration of 12.5 μM, suggesting that the drug is significantly effective far below the calculated IC 50 value, which further suggests the efficacy of NTZ as anti-NDV. The expression of NDV non-structural genes (N, P, and L), which make up the polymerase complex, was analyzed in our study. The significant reduction in the viral genes further confirmed the blocking of viral replication. Effect of NTZ against rubella virus replication has been previously reported showing the reduction of RNA transcription of viral genes [43] . We found a reduction in the expression of viral genes in accordance with the earlier report; however, further experimentation is required to understand whether NTZ blocks the transcription of mRNA or whether the reduced gene expression is due to lower infectivity. Our data suggested a decrease in viral structural and non-structural proteins post-NTZ treatment, which could perhaps have perturbed the effective assembly of the NDV virion in the cytoplasm. Earlier reports suggested inhibition of enveloped viruses by downregulating the structural and non-structural proteins [24, 42] . However, we did not get an appreciated protein level post 24 h of NDV infection, which could be due to a lesser viral protein that falls below the detection threshold. We also wanted to explore other possible mechanisms. NTZ has been reported to stimulate innate immunity and reduce HIV replication [45] . Our results showed significant up-regulation in TLR7 and type I interferons, along with few other cytokines such as IL18 and IL-1β. Out of all reported TLRs in birds, TLR7 has been reported to reduce the avian influenza replication [46] . TLR7, which induces the type I interferon production, might play a role in reducing the NDV infection [47] . Type I interferons and pro-inflammatory cytokines are shown to modulate virus replication [48] . Our results showed significant upregulation of pro-inflammatory genes such as IL-1β, NLRP3, and IL18 at the transcription level. GCTGACCCGCTTCATCTTCTA CGCCCACTTAGCTTGCAGGT NLRP3 GCTGCAAGGGAGGAGTATTT CATCTGTGTGGTGGTCTTAGAG Table 1 . Real-time primers used in the host gene expression post-NDV and/or nitazoxanide treatment. 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Yoya Vashi: Conceptualization, Data curation, Formal analysis, Methodology, Software, Visualization, Writing-original draft Vandna: Data curation, Formal analysis, Methodology. Hari Mohan Saini: Formal analysis, Methodology, Resources. Sachin Kumar: Conceptualization, Funding acquisition, Investigation We are thankful to all lab members for their constant support and help. The NDV research work is supported by grants from the department of biotechnology BT/PR16147/NER/95/83/2015 and BT/562/NE/U-Excel/2016. The authors declare no conflict of interest. ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: