key: cord-0820638-d0ibaxc4
authors: Zhang, Yunfei; Xia, Lu; Yuan, Yixin; Li, Qianqian; Han, Li; Yang, Guoyu; Hu, Hui
title: Rhodanine derivative LJ001 inhibits TGEV and PDCoV replication in vitro
date: 2020-09-19
journal: Virus Res
DOI: 10.1016/j.virusres.2020.198167
sha: c83addd3f6980ca78cc03576e969c9874f26f499
doc_id: 820638
cord_uid: d0ibaxc4

Transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) are members of the family coronaviridae and mainly cause acute diarrhea/vomiting, dehydration and mortality in piglets, which lead to huge economic losses to the swine industry. Rhodanine derivative LJ001 has been verified to be effective against some enveloped virus infections in vitro. In this study, we evaluated the antiviral activity of LJ001 towards TGEV and PDCoV replication on swine testicular(ST) cells. Our results showed the 50% cellular cytotoxicity (CC(50)) value of LJ001 was 146.4 μM on ST cell. The virus titers of TGEV and PDCoV were obviously decreased in the presence of LJ001 with the concentrations of 3.125 and 12.5 μM, and LJ001 potently inhibited TGEV and PDCoV infection at the replication stages of viral life cycle. Further study indicated that LJ001 inhibited TGEV and PDCoV replication by inhibition of viral RNA and protein synthesis, and reducing virus yields at 12 and 24 h post-inoculation. These data indicated that LJ001 had antiviral activities on TGEV and PDCoV replications in vitro, which may serve as a new candidate for treatment of coronaviruses infections.

Coronaviruses are enveloped, single-stranded and positive-sense RNA viruses that can infect and cause diseases in avian and mammal species .

According to the genome and antigenicity features, coronaviridae is divided into four genera: Alphacoronavirus (α-CoV), Betacoronavirus (β-CoV), Gammacoronavirus (γ-CoV) and Deltacoronavirus (δ-CoV) (International Committee on Taxonomy of Viruses and King, 2012; Lu et al., 2015) . To date, there are five diff erent species of CoV that cause diseases in pigs : α-CoV: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), swine enteric alphacoronavirus (SeACoV) and porcine respiratory coronarivurs (PRCV); β-CoV: porcine hemagglutinating encephalomyelitis virus (PHEV); and porcine deltacoronavirus (PDCoV). TGEV, PEDV, PDCoV and SeACoV can cause swine enteric coronavirus disease and significant economic losses for the pork industry Yang et al., 2020; Huang et al., 2013) .

TGEV, a member of the genus Alphacoronavirus in the family coronaviridae, was first identified as an etiological agent of transmissible gastroenteritis in swine in 1946 in the United States, and since then, it has become one of the most common viral causes of diarrhea in pig herds (Niederwerder et al., 2018) . TGEV causes severe watery diarrhea, vomiting, and dehydration in suckling piglets less than 2 weeks old, resulting in a high rate of mortality (up to 100%) (Niederwerder et al., 2018) .

Inactivated and live-attenuated vaccines against TGEV have been proven effective in preventing TGEV infections in some extent, however, protection of TGEV infections J o u r n a l P r e -p r o o f is not complete in clinic Jin et al., 2018) . To solve this problem, enhancing the immune effects of TGEV vaccines and development of new effective antiviral agents are the effective ways.

PDCoV, belonging to the Deltacoronavirus genus of the Coronaviridae family, is a novel swine enteropathogenic coronavirus with worldwide distribution . PDCoV was first identified in pigs in Hong Kong in 2012 (Woo et al., 2012) , then the detection of PDCoV in swine herds were reported in the US, Canada, China, South Korea, and Thailand (Wand et al., 2014b; Ma et al., 2015; Janetanakit et al., 2016; Lee et al., 2016) . Similar to TGEV, PDCoV causes severe diarrhea, vomiting, and dehydration in piglets . There are currently no approved treatments or vaccines available for PDCoV. Therefore, screening drugs that are effectively against PDCoV infections are needed. LJ001, a novel small-molecule rhodanine derivative, has been shown to have broad-spectrum antiviral activities in vitro (Balmer et al., 2011) . LJ001 can inhibit the entry and spread of some enveloped viruses, including human immunodeficiency virus, hepatitis C virus, influenza, Ebola, arenaviruses and poxvi ruses (Balmer et al., 2011) . LJ001 could inhibit enveloped virus infection at the stage of virus entry, because it mainly acts on viruses, and not the cell (Wolf et al., 2010) . Further research showed the antiviral activity of LJ001 was light-dependent, and required the presence of molecular oxygen. The exact mechanism was that the LJ001-generated singlet oxygen ( 1 O 2 ) mediated lipid oxidation, which then negatively affects on the biophysical properties of viral membranes (membrane curvature and fluidity), and J o u r n a l P r e -p r o o f then result in the virus-cell membrane fusion (Vigant et al., 2013) . The virion structures and the viral envelope functions remain intact during LJ001 treatment, so the virions treated with LJ001 are able to bind to their receptors (Wolf et al., 2010) .

Previous research confirms that even though LJ001 was lipophilic, it still could bind to both viral and cellular membranes, and it inhibit virus-cell but not cell-cell fusion (Balmer et al., 2017; Wolf et al., 2010) . Although LJ001 has shown the antiviral activities on many viruses, the effects of LJ001 on coronaviruses replication have yet to be demonstrated. So in current study we conducted experiments about LJ001 antiviral activity on TGEV and PDCoV replication on swine testicular (ST) cells. The following report described the LJ001 antiviral activities on TGEV and PDCoV replication. University, was dissolved in DMEM containing 0.1% (v/v) dimethylsulfoxide (DMSO, Solarbio) , and stored at 4C and protected from light.

Cell viability assay was performed with ST cells in 96-well plates. Briefly, cells were seeded into 96-well plates and grown to 100% confluence after 24 h. Eight wells containing a monolayer of cells were added with the different concentrations of LJ001 (0.782, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 μM) , and the cells that treated with DMEM containing 0.1% DMSO were served as mock controls. After 24 h, cells were washed twice with D-Hanks, and then incubated with 100 μl of 3-(4,5-

for 4 h. The reaction was stopped by adding 150 μl of DMSO, and the absorbance was measured at 570 nm. The CC 50 was calculated using GraphPad Prism software.

To performed in three independent experiments. Virus titer was determined by TCID 50 assay as previously described (Hu et al., 2015) .

RNA was extracted from ST cells using TRIzol reagent (TaKaRa, China) and cDNA was synthesized by using SuperQuick RT MasterMix (TaKaRa, China) according to the manufacturer's instructions. RT-qPCR was performed using SYBR Green PCR Master (TaKaRa, China) and the specific primers are shown in Table 1 .

Data were normalized against β-actin expression and are expressed as fold differences between control and treated cells using the 2 -ΔΔCT method.

To determine whether LJ001 could influence the viral proteins synthesis, the expression levels of viral N proteins of TGEV and PDCoV were tested by western blot at 12 and 24 h post-infection. Protein lysates were obtained from ST cells using J o u r n a l P r e -p r o o f ice-cold lysis RIPA buffer containing 10 Mm phenylmethylsulfonyl fluoride (PMSF).

Total protein concentration was determined by BCA protein assay kit (Beyotime, China). Equal amounts of protein were subjected to SDS-PAGE analysis and transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA).

Membranes were blocked with 5% nonfat milk for 3 h at room temperature, and then incubated with primary antibody at 4C overnight. The following primary antibodies were used: β-actin (Abcam, USA), TGEV N and PDCoV N polyclonal antibody (prepared in our laboratory using standard methods). After incubated with the primary antibody, HRP-conjugated secondary antibody were added and incubated for 2 h. Protein bands were detected by enhanced chemioluminescence reagents (Thermo, USA), and analyzed by ImageJ software.

Results are expressed as the means ± standard deviation (SD) from three independent experiments. Statistical analyses were performed using Student's t-test.

Differences were considered significant at p < 0.05. Statistical significance is indicated in figures as follows: * 0.01 < p < 0.05, ** p < 0.01.

The chemical structure of LJ001 is (5Z)-3-Allyl-5-[(5-phényl-2-furyl) méthylène]-2-thioxo-1, 3-thiazolidin-4-one. It's a rhodanine derivative of thiazolidines (Fig.1A) . (Fig. 1C) .

To determine whether LJ001 has the negative effect on the production of TGEV and PDCoV, ST cells were treated with various concentrations of LJ001 (ranging from 0.782 μM to 12.5 μM) before (pre-treatment), during (co-treatment), and after (post-treatment) the virus inoculation, (Fig. 2A) , all experiments were performed 24 h after infection. In the pre-treatment group,LJ001 had only marginal effect on TGEV infection with slight reduction of viral titers (Fig. 2B ). In the co-treatment and posttreatment groups, no obvious antiviral effects were observed on the TGEV replication when 0.782 μM of LJ001 was added. While LJ001 at the concentrations of 3.125 and 12.5 μM obviously suppressed TGEV replication, and the virus titers ( TCID 50 ) decreased approximately by 1.35 to 2.0 log 10 respectively ( Fig. 2C and 2D ).

The effect of LJ001 on PDCoV infection was also evaluated, the result showed only the 12.5 μM of LJ001 pretreated group induced a slight decrease in the PDCoV virus titer (Fig. 3A) . In the co-treatment group, exceeding 24% and 36% of decrease of the viral titers were observed in the 3.125 and 12.5 µM of LJ001 treated-groups as compared to the virus only groups, respectively (Fig. 3B) . The similar phenomena were also observed in the case of LJ001 post-treated groups (Fig. 3C) , which resulted in 1.3 and 2.1-log 10 decrease of PDCoV viral titers in the 3.125 and 12.5 µM of J o u r n a l P r e -p r o o f LJ001-treated groups, respectively.

To further investigate which steps of the viral life cycle were affected by LJ001, was similar to that in the virus only group at 1 hpi (p>0.05), while the inhibitory effects were prominent at 6, 12 and 24 hpi when compared to the virus only groups (Fig. 4A) . The TCID 50 titers of TGEV in the cultural supernatants in the LJ001 treated group were markedly reduced at 12 and 24 hpi when compared with the virus only group, there was over 2 log10 viral-titer decrease in the LJ001-treated groups at 24 hpi (Fig. 4B) . Western blot analysis showed that about 50% of TGEV N protein expression was reduced at 24 hpi in the infected cells with 12.5 μM of LJ001 treatment, comparing to the TGEV-infected cells (Fig. 4C) .

Similar results were found in the PDCoV groups, with the evident decrease of intracellular viral RNA titers at 12 and 24 hpi (p<0.01) (Fig. 4D) , the obvious J o u r n a l P r e -p r o o f reduction of extracellular viral titers at 6, 12 and 24 hpi (Fig. 4E) , and significant suppression of the expression of PDCoV N-protein at 24 hpi (Fig. 4F) . These results indicated that LJ001 inhibited TGEV and PDCoV infection at the replication stage of the virus life cycle.

The threat of emerging and re-emerging coronaviruses highlights the need to develop broad-spectrum antivirals. Rhodamine derivative LJ001 has previously reported to inhibit the entry of numerous lipid-enveloped viruses at non-cytotoxic concentrations. LJ001 exerts antiviral activities by binding to both viral and cellular membranes and inhibits virus-cell fusion (Wolf et al., 2010) . In this study, LJ001 was shown to have antiviral activities against two swine enteropapthogenic coronaviruses (TGEV and PDCoV) infection in vitro. which can serve as a new candidate for treatment of swine enteric coronaviruses infections. Moreover, there is an urgent need for targeted and effective COVID-19 treatments during the COVID-19 pandemic, we deduced the SARS-CoV-2 also needs to be tested under similar in vitro conditions with or without LJ001 treatment, especially in comparison to those positive control groups treated with known antivirals, such as remdesivir.

Our cellular toxicity assays revealed that the cytotoxicity of LJ001 to ST cells was as high as 146.4 μM, at the LJ001 of 12.5 μm has no obvious influence on ST cell viability and morphology (Fig. 1) . Wolf et al. also demonstrated no effect on active cell metabolismin LJ001-treated Vero cells (Wolf et al., 2010) . In the pretreatment group (Fig.2B and 3A) , different concentration of LJ001 (12.5, 3.125 and J o u r n a l P r e -p r o o f 0.782 μM) was added to ST cells and incubated for 1h, then cells were infected with TGEV or PDCoV, the miscible liquids were detected at 24hpi. The results showed that LJ001 had no obvious effect on cells. Wolf et al. also demonstrated that LJ001 does not act on the cells, there is no obvious inhibitory effects for VSV infection after the cell pretreated with LJ001 (Wolf et al., 2010) .

A, HIV, Rift Valley Fever Virus (RVFV) and HCV (Wolf et al., 2010; Vigant et al., 2013) . In the co-treatment group, the cells were infected with virus in the presence of different concentration of LJ001 (0.782, 3.125 and 12.5 μM) for 1 h, then the presence of different concentration of LJ001 (0.782, 3.125 and 12.5 μM) were added, the miscible liquids were detected at 24 hpi. The results showed that the LJ001 have obvious inhibitory effect for TGEV and PDCoV ( Fig.2C and 3B) . Furthermore, the cells infected with TGEV or PDCoV for 1 h, 12.5 μM of LJ001 were added to ST cells, the miscible liquids were detected after infected for 1, 6, 12 and 24h. The results showed that LJ001 had no obvious effect at the early stages of TGEV and PDCoV replication, while its inhibitory effect was apparent at the viral replication cycle (Fig. 4) . Previous research showed that LJ001 acts on VSV during the viralentry stage, further studies indicated LJ001 targeted the virus-cell fusion binding to lipid membranes of VSV in an irreversible manner (Wolf et al., 2010; Vigant et al., 2013) . These previous results differ with our observations of the LJ001 effects on the viral replication stage, which may be because of the difference of the lipid membrane of TGEV and PDCoV with other enveloped viruses in biophysical and physiological J o u r n a l P r e -p r o o f properties. However, the specific mechanism needs further investigation. Future experiments will determine if LJ001 can be used not only as a treatment but also for preventing TGEV and PDCoV infection. TGEV and PDCoV belong to different genera of coronavirus, encoding distinct nucleotide sequence and amino acid sequence . The clinical symptoms of PDCoV are similar to that of TGEV. Histologic lesions are observed in all sections of small intestine, while the pathogenesis mechanism behind TGEV and PDCoV infections remain largely unknown (Koonpaew et al., 2019) . No treatments or vaccines are available for PDCoV currently. In this study, LJ001 was described to have the antiviral activity against TGEV or PDCoV at a micromolar range. Recent years, the emerging and re-emerging coronaviruses cause severe threat to global public health, and new drugs were constantly discovered to anti-coronaviruses. Prior research showed saracatinib can inhibit middle east respiratory syndrome-coronavirus (MERS-CoV) at the early stages of the viral life cycle by suppressing the Src-family of tyrosine kinases (SFK) signaling pathways (Shin et al., 2018) . Agostini' reports showed that antiviral remdesivir (GS-5734) could inhibit several coronaviruses replication, including severe acute respiratory syndrome coronavirus (SARS-CoV), murine hepatitis virus (MHV) and MERS-CoV. They further demonstrated that this antivial activity was mediated by the viral polymerase and the proofreading exoribonuclease (Agostini et al., 2018) . However, the exact mechanism of the antiviral activity of LJ001 on TGEV or PDCoV replication need further study.

In summary, this study suggested that LJ001 showed antiviral activity at the 

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