GS 0840

Adefovir dipivoXil efficiently inhibits the proliferation of pseudorabies virus in vitro and in vivo

Guosong Wang, Ruiqi Chen, Pengfei Huang, Junping Hong, Jiali Cao, Qian Wu, Wei Zheng, Lina Lin, Qiangyuan Han, YiXin Chen, Ningshao Xia
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, School of Public Health, Xiamen University, Xiamen, 361102, Fujian Province, China

A B S T R A C T
Since 2011, highly pathogenic pseudorabies virus (PRV) variants that emerged on many farms in China have posed major economic burdens to the animal industry and have even recently caused several human cases of viral encephalitis. Currently, there are no approved effective drugs to treat PRV associated diseases in humans or pigs. Thus, it is important to develop a new effective drug for the treatment of PRV infection. To this end, we established a novel rapid method to screen drugs against PRV from 1818 kinds of small molecular drugs approved by the FDA. Using this method, we identified 21 kinds of them that can strongly suppress the prolif- eration of PRV. MitoXantrone, puromycin dihydrochloride, mitoXantrone hydrochloride and adefovir dipivoXil effectively inhibited PRV in vitro. Of them, only adefovir dipivoXil could potently protect mice against lethal PRV infection. Our work identifies several kinds of potential therapeutics against PRV and may offer important guidance for controlling PRV epidemics and treating associated diseases in humans and animals.
PRV, a member of the Alphaherpesvirinae subfamily and the genus Varicellovirus, is the causative agent of pseudorabies or Aujeszkys’ dis- ease (Muller et al., 2011; Taka´cs et al., 2013). Infected pigs manifest various clinical symptoms, such as a high mortality rate in newborn pigs, respiratory distress and growth retardation in growing pigs, and repro- ductive failure in adults, which is a devastating threat to the pig industry (Hu et al., 2015). Importantly, several cases of human infectious endophthalmitis caused by PRV have been reported (Ai et al., 2018; Liu et al., 2020; Yang et al., 2019; Zheng et al., 2019). Thus, the threat of PRV should not be ignored.
There are no currently approved effective drugs against PRV, and there are few studies examining inhibitory molecules for the treatment of PRV. Some natural compounds have been found to be effective in vitro against various herpesviruses but failed against PRV (Zouharova et al., 2016). Acyclovir, the drug used to treat HSV-1, was slightly active against PRV in BHK21 cells. The combination of acyclovir and ribavirin could inhibit PRV proliferation in vitro (Pancheva, 1991). It has also been reported that a series of synthetic diaminopurine-based acyclic nucleoside phosphonate analogs have anti-PRV activities (Lisov et al., 2015; Zouharova et al., 2016). Natural plant extracts, including Kumazasa extract (Iwata et al., 2010), Duabanga grandiflora leaf extract (Malik et al., 2016), and Houttuynia cordata (Ren et al., 2011), can suppress the proliferation of PRV in vitro at high concentrations. Iso- bavachalcone inhibited PRV by impairing virus-induced cell-to-cell fusion in vitro (Wang et al., 2020b). Resveratrol was shown to act against PRV in vitro and in vivo (Zhao et al., 2017, 2018). However, it effectively inhibited PRV only at high doses.
In cases of human viral encephalitis caused by PRV infection, the clinical symptoms of the patients showed slight improvement after acyclovir or acyclovir-like drug treatment (Liu et al., 2020). We evalu- ated the inhibition of acyclovir, valacyclovir and famciclovir against PRV in vitro at drug concentrations of 1.25, 2.5 and 5 μmol/L. These drugs did not suppress the proliferation of PRV in vitro (Fig. 1A). The virus titer in the supernatant was not significantly different from that in the control group (Fig. 1B). Acyclovir or acyclovir-like drugs may not significantly inhibit PRV in vitro. Thus, there is an urgent need to develop more efficient drugs to treat animals and humans infected by PRV.
To develop more effective anti-PRV drugs, we attempted to establish a detection method for inhibitory effects against PRV using a PRV-specific antibody to label PRV-infected cells. A detailed material and methods section is provided as supplementary content. The gB of PRV has an important function in promoting viral entry in the cell(Li et al.,2017; Vallbracht et al., 2018). Thus, the gB protein of PRV was horseradish peroXidase. After the tetramethylbenzidine (TMB) substrate was added, the infected cells were specifically labeled by the antibody H5H8 and dyed blue (Fig. 2C).
The FDA-approved small molecule library has approXimately 1818 kinds of drugs that have been approved on the market to address various human diseases. Thus, most of them are safe and reliable. We utilized the previous method to screen the drugs against PRV (Fig. 3A). To screen the more effective drugs, we diluted each kind of drug to the lower con-centration, 5 μmol/L. Then, these samples were added to the PK-15 cells of 96-microwell plates before 100 pfu of PRV virus was added and infected. Twenty-four hours later, these cells were fiXed, permeated and blocked. With the antibody H5H8, the number of infected cells can be obtained, which can help us calculate the inhibition ratio against PRV (Fig. 3B). The Z′ factor throughout the screening campaign was 0.91910.0203. The drugs were selected for further research only when their inhibition ratio was no less than 85%. These drugs had their own characteristics (Table S1). To evaluate the efficiency of these drugs, we measured the inhibitory ratio against PRV at lower concentrations. Thus, these drugs were seri- ally diluted twice, and then, the inhibitory ratio was measured by a previous method (Fig. 4A). We found that most drugs possessed strong effects against PRV. To compare their efficiency in an objective way, we calculated the IC50 values against PRV (Fig. 4B–E). It was confirmed that mitoXantrone, puromycin dihydrochloride, mitoXantrone hydro- chloride and adefovir dipivoXil can still inhibit the proliferation of PRV at nanomolar concentrations. Thus, they may be the potential anti-PRV drugs and require further research.
We tried to measure the effects of these drugs against the prolifera- tion of PRV by immunofluorescence assays. Four drugs revealed strong effects against PRV (Fig. 5A). Moreover, the content of gB in the lysates of PK-15 cells was measured by Western blotting. At high concentrations of mitoXantrone, puromycin dihydrochloride and mitoXantrone hydro- chloride, the expression of gB was completely suppressed. This phe- nomenon disappeared as the drug concentration decreased. However, adefovir dipivoXil exhibited a limited activity to suppress the expression of gB. (Fig. 5B). The cell supernatants of various concentrations of drugs were detected by PFU assays (Fig. 5C). According to the virus titer, we found that these 4 kinds of drugs can strongly inhibit the proliferation of PRV in vitro. Adefovir dipivoXil showed the strongest activity to restrain the proliferation of viruses. The mechanism of adefovir dipivoXil against PRV may be associated with virus assembly, release, or chain termi- nator. Further study is needed.
To confirm the therapeutic activity of candidate drugs against PRV, we established a lethal mouse model by intraperitoneal PRV injection. In this model, we evaluated the therapeutic activity of adefovir dipivoXil, acyclovir, mitoXantrone, mitoXantrone hydrochloride and puromycin dihydrochloride. All drugs (5 mg/kg) were given by intraperitoneal in- jection 2 h after PRV injection once a day for five days (Fig. 6A). Mice in expressed by the baculovirus expression system and utilized to immu-each group except adefovir dipivoXil treatment exhibited severe nize mice. Then, we screened many antibodies against gB by the monoclonal antibody technique. One antibody H5H8 had a strong binding ability with gB in enzyme-linked immunosorbent assays (Fig. 2A). The H5H8 antibody can detect the gB of PRV in PK-15 cells after they have been infected with PRV for 24 h H5H8 could label the cells infected by PRV Fa strain, as shown by immunofluorescence assay (Fig. 2B). However, the negative control antibody 19C10 against influ- enza virus could not bind with PK-15 cells or PK-15 cells infected by PRV (Gui et al., 2014; Wang et al., 2020a). To determine the number of infected cells, we performed an enzyme-linked immunospot assay (ELISPOT). The antibodies H5H8 and 19C10 were conjugated with neurotoXic symptoms at 3 days after the PRV injection (Fig. 6B). The body weight curves and survival curves demonstrated that adefovir dipivoXil inhibited the proliferation of PRV in vivo (Fig. 6C and D). It was confirmed that acyclovir, which has been used to treat patients infected by PRV, had no obvious therapeutic activity against PRV in a mouse model. MitoXantrone, mitoXantrone hydrochloride and puro- mycin dihydrochloride did not suppress PRV proliferation in vivo.
The late therapeutic activity of adefovir dipivoXil was evaluated by intraperitoneal injection one day after PRV injection (Fig. 7A). The mice in the adefovir dipivoXil treatment group had an obvious reduction in the clinical score versus the mice in the PBS treatment group (Fig. 7B). The major organs of each group were harvested at 3 days post infection.
Fig. 2. Cells infected by PRV can be specifically labeled by the anti-gB antibody H5H8. (A) The antibody H5H8 has strong binding activity with gB expressed by the baculovirus system. The negative control antibody 19C10 against the NP of influenza A virus cannot bind with gB. (B) PK-15 cells infected by PRV can be labeled by the antibody H5H8, as shown by immunofluorescence assays. Under the same conditions, the antibody 19C10 cannot mark the infected cells that have been infected by PRV for 24 h. Green, infected cells positive for gB protein of PRV; blue, cell nucleus. This experiment was repeated three times; one representative dataset is shown. (C) The antibody H5H8 can specifically dye PK-15 cells infected by PRV in an enzyme-linked immunospot assay. The negative antibody 19C10 can only label the cells infected by the influenza A strain (A/California/04/2009).
Fig. 3. Screening of small molecule drugs against PRV. (A) An outline of the drug screening protocol. PK-15 cells were seeded in 96-well plates and treated with escalating doses of each of the 1818 compounds in the drug screen in combination with 100 pfu PRV. Infected cells can be labeled by antibody H5H8. By ELISPOT, the number of infected cells can be counted.
(B) The inhibitory ratio against PRV of 1818 drugs. The inhibitory ratio was generated for PRV in the absence or presence of drugs, and the inhibitory ratio was calculated according to the following formula:
(spotsvirus-spotsvirus+drug)/spotsvirus.
The Z-factor was calculated for each microplate.
Fig. 4. Evaluating the inhibitory activity of candidate drugs against PRV. (A) The inhibitory activity of 21 candidate drugs was evaluated. Serial 2-fold dilutions of 21 candidate drugs and 100 pfu PRV were added to PK-15 cells. Twenty-four hours later, the inhibitory ratio was calculated according to the previous formula. (B) Fifty percent inhibitory concentrations (IC50) of the 4 indicated drugs against PRV were determined. MitoXantrone, puromycin dihydrochloride, mitoXantrone hydro- chloride and adefovir dipivoXil could inhibit the proliferation of PRV at nanomolar concentrations.
Fig. 5. The mechanism of 4 candidate drugs against PRV. (A) The inhibitory activity of 4 candidate drugs against PRV was determined by immunofluorescence assays. PK-15 cells were inoculated with PRV and 4 candidate drugs. The expression of the PRV gB protein in PK-15 cell monolayers 24 h after inoculation was detected by immunofluorescence using the antibody H5H8. Green, infected cells positive for gB protein of PRV; blue, cell nucleus. This experiment was repeated three times; one representative dataset is shown. (B) Immunoblots of gB detected in the lysates of PK-15 cells infected with PRV and incubated with serial 2-fold dilutions of 4 candidate drugs. Twenty-four hours later, the lysates of cells were detected by the H5H8 antibody. (C) The viral titer of the PRV virus detected in the super- natants of the PK-15 cells infected by PRV and incubated with serial 2-fold dilutions of 4 candidate drugs.
Fig. 6. The early (2 h postinfection) therapeutic activity of candidate drugs. (A to D) Timeline of the experimental setup (A), The percent clinical score level of the mice in different groups at day 3 postinfection (B), the body weight change (C), and survival curves (D) for BALB/c mice (n = 6 per group) treated intraperitoneally with the candidate drugs (5 mg/kg/day) and PBS, shown 2 h after lethal challenge with PRV. AD, adefovir dipivoXil; MT, mitoXantrone; MT HY, mitoXantrone hydrochloride; Puro, puromycin dihydrochloride. The black bars indicate mean values. The body weight curves represent the mean ± SD. For (C), comparisons were performed by area under the curve (AUC) analysis. For (D), statistical analysis was performed by log-rank test. ***: P < 0.001, compared to the control PBS-treated group. Fig. 7. The late (1 day postinfection) therapeutic activity of adefovir dipivoXil. (A to D) Timeline of the experimental setup (A), the percent clinical score level of the mice in different groups at day 3 postinfection (B), the body weight change (C), and the survival curves (D) for BALB/c mice (n = 6 per group) treated intraperi- toneally with adefovir dipivoXil (5 mg/kg/day) and PBS, indicated 1 day after lethal challenge with PRV. The black bars indicate mean values. The body weight curves represent the mean ± SD. For (C), comparisons were performed by area under the curve (AUC) analysis. For (D), statistical analysis was performed by log-rank test. ***: P < 0.001, compared to the control PBS-treated group. Fig. 8. Mice cured by adefovir dipivoXil treatment can protect completely from PRV rechallenge. (A to D) Timeline of the experimental setup (A), the percent clinical score of the mice in different groups at day 3 postinfection (B), body weight change (C), survival curves (D) for BALB/c mice (n = 6 per group) rechallenged with PRV. The black bars indicate mean values. The body weight curves represent the mean ± SD. For (C), comparisons were performed by area under the curve (AUC) analysis. For (D), statistical analysis was performed by log-rank test. The major organs of the PBS treatment group had obvious pathological lesions by H&E staining: the brains showed tubular infiltration, the lungs showed severe pulmonary abscesses, the kidneys showed severe capillary hyperemia, and the livers showed granular degeneration of hepatocytes. Adefovir dipivoXil treatment reduced the pathological le- sions of the PRV-infected mice (Fig. S1A). There were significantly fewer PRV particles in the major organs of the mice treated with adefovir dipivoXil than in the mice in the PBS treatment group (Fig. S1B). The body weight curves and survival curves demonstrated that two of siX mice survived after adefovir dipivoXil treatment. All siX mice in the PBS treatment group died at 5 days post PRV infection (Fig. 7C and D). These results suggested that adefovir dipivoXil had late therapeutic activity against PRV. The cured mice in the adefovir dipivoXil treatment group were rechallenged with PRV by intraperitoneal (i.p.) injection 14 days after initial infection according to the previously described method (Hu et al., 2019) (Fig. 8A). They exhibited no neurotoXic symptoms (Fig. 8B). Their body weights had no obvious reductions, and all mice survived (Fig. 8C and D). These results suggested that the cured mice may induce effective immunity against PRV after adefovir dipivoXil treatment. Although adefovir dipivoXil exhibits the therapeutic activity against PRV in mouse model, whether it has the same function in humans or pigs is a question needed to be addressed by further investigation. Adefovir dipivoXil is also known to be nephrotoXic at certain doses (Lin et al., 2017), the development for an anti-PRV drug would require extensive work to establish its treatment dose and duration in humans or pigs. In summary, our work identifies 21 drugs that can significantly inhibit the proliferation of PRV at a drug concentration of 5 μmol/L. Among them, mitoXantrone, mitoXantrone hydrochloride, puromycin dihydrochloride and adefovir dipivoXil were effective against PRV, and their IC50 values can reach the nanomolar concentrations in vitro. Only adefovir dipivoXil can protect mice from death after they are infected with a lethal dose of PRV. These findings may offer important guidance for controlling PRV epidemics and treating associated diseases. Declaration of competing 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. 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