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Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause significant economic losses to the global swine industry, yet effective prevention and control measures remain elusive. The development of novel antivirals is thus urgently needed. Rifampicin (RFP), a semisynthetic derivative of rifamycin, has been previously reported to inhibit the replication of certain mammalian DNA viruses as well as RNA viruses. In this study, we unveil RFP as a potent inhibitor of PRRSV both in Marc-145 cells (half-maximal inhibitory concentration 61.26 μM) and porcine alveolar macrophages (half-maximal inhibitory concentration 53.09 μM). The inhibitory effect of RFP occurred during viral replication rather than binding, internalization and release. We also demonstrated that RFP inhibits PRRSV proteins production in the early stage of infection, without inhibiting host protein synthesis. Moreover, RFP effectively restricted porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV) infection in Vero cells. In summary, these findings indicate the promising potential of RFP as a therapeutic agent for PRRSV, PEDV and PEAV infection in pig farms.

African swine fever (ASF) is a highly contagious disease and provokes severe economic losses and health threats. At present no effective vaccine or treatment is available to prevent or cure ASF. Consequently, there is an urgent need to develop effective drugs against ASF virus (ASFV). Chlorine dioxide (ClO2), an ideal biocide, has broad-spectrum antibacterial activity and no drug resistance. Here, we found that ClO2 strongly inhibited ASFV replication in porcine alveolar macrophages (PAMs). The inhibitory effect of ClO2 occurred during viral attachment rather than entry, indicating that ClO2 suppressed the early stage of virus life cycle. ClO2 showed a potent anti-ASFV effect when added either before, simultaneously with, or after virus infection. Furthermore, ClO2 could destroy viral nucleic acids and proteins, which may contribute to its capacity of inactivating ASFV virions. The minimum concentration of degradation of ASFV nucleic acids by ClO2 is 1.2 μg/mL, and the degradation is a temperature-dependent manner. These have guiding significance for ClO2 prevention and control of ASFV infection in pig farms. In addition, ClO2 decreased the expression of ASFV-induced inflammatory cytokines. Overall, our findings suggest that ClO2 may be an ideal candidate for the development of novel anti-ASFV prophylactic and therapeutic drugs in swine industry.

Porcine alveolar macrophages without the CD163 SRCR5 domain are resistant to porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, whether the deletion of CD163 SRCR5 in MARC-145 cells confers resistance to PRRSV and interaction of which of the host proteins with CD163 is involved in virus uncoating remain unclear. Here we deleted the SRCR5 domain of CD163 in MARC-145 cells using CRISPR/Cas9 to generate a CD163ΔSRCR5 MARC-145 cell line. The modification of CD163 had no impact on CD163 expression. CD163ΔSRCR5 cells were completely resistant to infection by PRRSV-2 strains Li11, CHR6, TJM, and VR2332. The modified cells showed no cytokine response to PRRSV-2 infection and maintained normal cell vitality comparable with the WT cells. The resistant phenotype of the cells was stably maintained through cell passages. There were no replication transcription complexes in the CD163ΔSRCR5 cells. SRCR5 deletion did not disturb the colocalization of CD163 and PRRSV-N in early endosomes (EE). However, the interaction of the viral proteins GP2a, GP3, or GP5 with CD163, which is involved in virus uncoating was affected. Furthermore, 77 CD163-binding cellular proteins affected by the SRCR5 deletion were identified by LC–MS/MS. Inhibition of calpain 1 trapped the virions in EE and forced then into late endosomes but did not block viral attachment and internalization, suggesting that calpain 1 is involved in the uncoating. Overall, CD163ΔSRCR5 MARC-145 cells are fully resistant to PRRSV-2 infection and calpain 1 is identified as a novel host protein that interacts with CD163 to facilitate PRRSV uncoating.