Feline infectious peritonitis virus (FIPV) is the etiologic agent of feline infectious peritonitis (FIP) and causes fatal disease in cats of almost all ages. Currently, there are no clinically approved drugs or effective vaccines for FIP. Furthermore, the pathogenesis of FIP is still not fully understood. There is an urgent need for an effective infection model of feline infectious peritonitis induced by FIPV. Here, we constructed a field type I FIPV full-length cDNA clone, pBAC-QS, corresponding to the isolated FIPV QS. By replacing the FIPV QS spike gene with the commercially available type II FIPV 79-1146 (79-1146_CA) spike gene, we established and rescued a recombinant virus, designated rQS-79. Moreover, we constructed 79-1146_CA infectious full-length cDNA pBAC-79-1146_CA, corresponding to recombinant FCoV 79-1146_CA (r79-1146_CA). In animal experiments with one- to two-year-old adult cats orally infected with the recombinant virus, rQS-79 induced typical FIP signs and 100% mortality. In contrast to cats infected with rQS-79, cats infected with 79-1146_CA did not show obvious signs. Furthermore, by rechallenging rQS-79 in surviving cats previously infected with 79-1146_CA, we found that there was no protection against rQS-79 with different titers of neutralizing antibodies. However, high titers of neutralizing antibodies may help prolong the cat survival time. Overall, we report the first reverse genetics of virulent recombinant FCoV (causing 100% mortality in adult cats) and attenuated FCoV (causing no mortality in adult cats), which will be powerful tools to study the pathogenesis, antiviral drugs and vaccines for FCoV. Importance Tissue- or cell culture-adapted feline infectious peritonitis virus (FIPV) usually loses pathogenicity. To develop a highly virulent FIPV, we constructed a field isolate type I FIPV full-length clone with the spike gene replaced by the 79-1146 spike gene, corresponding to a virus named rQS-79, which induces high mortality in adult cats. rQS-79 represents the first described reverse genetics system for highly pathogenic FCoV. By further constructing the cell culture-adapted FCoV 79-1146_CA, we obtained infectious clones of virulent and attenuated FCoV. By in vitro and in vivo experiments, we established a model that can serve to study the pathogenic mechanisms of FIPV. Importantly, the wild-type FIPV replicase skeleton of serotype I will greatly facilitate the screening of antiviral drugs, both in vivo and in vitro.

Antimicrobial resistance (AMR) poses a huge threat to public health. The development of novel antibiotics is an effective strategy to tackle AMR. Cyclic diadenylate monophosphate (c-di-AMP) has recently been identified as an essential signal molecule for some important bacterial pathogens involved in various bacterial physiological processes, leading to its synthase diadenylate cyclase becoming an attractive antimicrobial drug target. In this study, based on the enzymatic activity of diadenylate cyclase of Streptococcus suis (ssDacA), we established a high-throughput method of screening for ssDacA inhibitors. Primary screening with a compound library containing 1133 compounds identified IPA-3 (2,2′-dihydroxy-1,1′-dinapthyldisulfide) as an ssDacA inhibitor. High-performance liquid chromatography (HPLC) analysis further indicated that IPA-3 could inhibit the production of c-di-AMP by ssDacA in vitro in a dose-dependent manner. Notably, it was demonstrated that IPA-3 could significantly inhibit the growth of several Gram-positive bacteria which harbor an essential diadenylate cyclase but not E. coli, which is devoid of the enzyme, or Streptococcus mutans, in which the diadenylate cyclase is not essential. Additionally, the binding site in ssDacA for IPA-3 was predicted by molecular docking, and contains residues that are relatively conserved in diadenylate cyclase of Gram-positive bacteria. Collectively, our results illustrate the feasibility of ssDacA as an antimicrobial target and consider IPA-3 as a promising starting point for the development of a novel antibacterial.

Aim Antimicrobial resistance (AMR) has become a global concern. Developing novel antimicrobials is one of the most effective approaches in tackling AMR. Considering its relatively low cost and risk, drug repurposing has been proposed as a valuable approach for novel antimicrobial discovery. The aim of this study was to screen for antimicrobial compounds against Streptococcus suis, an important zoonotic bacterial pathogen, from an Food and Drug Administration (FDA)‐approved drug library. Methods and Results In this study, we tested the antimicrobial activity of 1815 FDA‐approved drugs against S. suis. Sixty‐seven hits were obtained that showed a growth inhibition of more than 98%. After excluding already known antibiotics and antiseptics, 12 compounds were subjected to minimal inhibition concentration (MIC) assessment against S. suis. This showed that pralatrexate, daunorubicin (hydrochloride), teniposide, aclacinomycin A hydrochloride and floxuridine gave a relatively low MIC, ranging from 0.85 to 5.25 μg/ml. Apart from pralatrexate, the remaining four drugs could also inhibit the growth of antimicrobial‐resistant S. suis. It was also demonstrated that these four drugs had better efficacy against Gram‐positive bacteria than Gram‐negative bacteria. Cytotoxicity assays showed that floxuridine and teniposide had a relatively high 50% cytotoxic concentration (CC50). The in vivo efficacy of floxuridine was analysed using a Galleria mellonella larvae infection model, and the results showed that floxuridine was effective in treating S. suis infection in vivo. Conclusions Five compounds from the FDA‐approved drug library showed high antimicrobial activity against S. suis, among which floxuridine displayed potent in vivo efficacy that is worth further development. Significance and Impact of Study Our study identified several antimicrobial compounds that are effective against S. suis, which provides a valuable starting point for further antimicrobial development.

Defensins are a major class of antimicrobial peptides that facilitate the immune system to resist pathogen infection. To date, only β-defensins have been identified in pigs. In our previous studies, porcine β-defensin 2 (PBD-2) was shown to have both bactericidal activity and modulatory roles on inflammation. PBD-2 can interact with the cell surface TLR4 and interfere with the NF-κB signaling pathway to suppress the inflammatory response. In this study, the intracellular functions of PBD-2 were investigated. The fluorescently labeled PBD-2 could actively enter mouse macrophage cells. Proteomic analysis indicated that 37 proteins potentially interacted with PBD-2, among which vasohibin-1 (VASH1) was further tested. LPS, an inflammation inducer, suppressed the expression of VASH1, whereas PBD-2 inhibited this effect. PBD-2 inhibited LPS-induced activation of Akt, expression and release of the inflammatory mediators vascular endothelial growth factor and NO, and cell damage. A follow-up VASH1 knockdown assay validated the specificity of the above observations. In addition, PBD-2 inhibited LPS-induced NF-κB activation via Akt. The inhibition effects of PBD-2 on LPS triggered suppression of VASH1 and activation of Akt, and NF-κB and inflammatory cytokines were also confirmed using pig alveolar macrophage 3D4/21 cells. Therefore, the data indicate that PBD-2 interacts with intracellular VASH1, which inhibits the LPS-induced Akt/NF-κB signaling pathway, resulting in suppression of inflammatory responses. Together with our previous findings, we conclude that PBD-2 interacts with both the cell surface receptor (TLR4) and also with the intracellular receptor (VASH1) to control inflammation, thereby providing insights into the immunomodulatory roles of defensins.