Differential beta-coronavirus infection dynamics in human bronchial epithelial organoids.

The lower respiratory system serves as the target and barrier for beta-coronavirus (beta-CoV) infections. In this study, we explored beta-CoV infection dynamics in human bronchial epithelial (HBE) organoids, focusing on HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2. Utilizing advanced organoid culture techniques, we observed robust replication for all beta-CoVs, particularly noting that SARS-CoV-2 reached peak viral RNA levels at 72 h postinfection. Through comprehensive transcriptomic analysis, we identified significant shifts in cell population dynamics, marked by an increase in goblet cells and a concurrent decrease in ciliated cells. Furthermore, our cell tropism analysis unveiled distinct preferences in viral targeting: HCoV-OC43 predominantly infected club cells, while SARS-CoV had a dual tropism for goblet and ciliated cells. In contrast, SARS-CoV-2 primarily infected ciliated cells, and MERS-CoV showed a marked affinity for goblet cells. Host factor analysis revealed the upregulation of genes encoding viral receptors and proteases. Notably, HCoV-OC43 induced the unfolded protein response pathway, which may facilitate viral replication. Our study also reveals a complex interplay between inflammatory pathways and the suppression of interferon responses during beta-CoV infections. These findings provide insights into host-virus interactions and antiviral defense mechanisms, contributing to our understanding of beta-CoV infections in the respiratory tract.

Production of a chimeric porcine reproductive and respiratory syndrome virus (PRRSV)-2 vaccine using a lab-scale packed-bed bioreactor CelCradle.

BACKGROUND: We developed a MARC-145 cell culture and porcine reproductive and respiratory syndrome (PRRS) vaccine production using a novel CelCradle bioreactor. CelCradle is a packed-bed bioreactor capable of both batch and perfusion culture, and the operating parameters are easy to optimize. RESULTS: In this study, CelCradle reached a maximum cell density of 8.94 × 105 cells/mL at 5 days post-seeding when seeded at 8.60 × 104 cells/mL (doubling time = 35.52 h). Inoculation of PRRS vaccine candidate, K418DM1.1, was performed at a multiplicity of infection (MOI) of 0.01 at 5 days post-seeding, which resulted in a high viral titer of 2.04 × 108 TCID50/mL and total viral load of 1.02 × 1011 TCID50/500 mL at 2 days post-infection (dpi). The multilayer cultivation system, BioFactory culture, yielded a higher doubling time (37.14 h) and lower viral titer (i.e., 8.15 × 107 TCID50/mL) compared to the CelCradle culture. Thus, the culture medium productivity of the CelCradle culture was 2-fold higher than that of the BioFactory culture. In the animal experiment, the CelCradle-produced vaccine induced high levels of neutralizing antibodies and effectively protected pigs against homologous challenge, as shown by the significantly lower levels of viremia at 1- and 7-days post-challenge (dpc) compared to the non-vaccinated pigs. CONCLUSIONS: Overall, this study demonstrates that the CelCradle system is an economical platform for PRRS vaccine production.

Rottlerin-Liposome Inhibits the Endocytosis of Feline Coronavirus Infection.

Rottlerin (R) is a natural extract from Mallotus philippensis with antiviral properties. Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) that is characterized by systemic granulomatous inflammation and high mortality. We investigated the antiviral effect of liposome-loaded R, i.e., rottlerin-liposome (RL), against FCoV. We demonstrated that RL inhibited FCoV replication in a dose-dependent manner, not only in the early endocytosis stage but also in the late stage of replication. RL resolved the low solubility issue of rottlerin and improved its inhibition efficacy at the cellular level. Based on these findings, we suggest that RL is worth further investigation as a potential treatment for FCoV.

Porcine Reproductive and Respiratory Syndrome Virus Engineered by Serine Substitution on the 44th Amino Acid of GP5 Resulted in a Potential Vaccine Candidate with the Ability to Produce High Levels of Neutralizing Antibody.

N-linked glycans covering GP5 neutralizing epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) have been proposed to act as a sheath blocking the production of neutralizing antibodies. Herein, we genetically engineered PRRSV with serine (S) substitution on the 44th asparagine (N) on the GP5 ectodomain of PRRSV-2 lineage-1. To evaluate the recombinant PRRSV, in vivo experiments were performed in piglets. The recombinant virus group showed no viremia until 42 days post-inoculation (dpi), and the rectal temperature and average daily weight gain were in the normal range at the same time point as the negative control group. On the 42 dpi, both groups were challenged with the wild-type virus. The recombinant PRRSV group showed lower rectal temperature, viremia, and the lung lesions than that of the negative control group for 19 days post-challenge (dpc). Additionally, the recombinant virus induced 4.50 ± 3.00 (log2) and 8.25 ± 0.96 (log2) of neutralizing antibody before and after challenge, respectively. Taken together, this study confirmed that N44S substitution can create an infectious PRRSV that strongly induces neutralizing antibodies. In addition, the vCSL1-GP5-N44S mutant that we produced was confirmed to have potential as a vaccine candidate, showing good safety and protective effects in pigs.

Development of a Chimeric Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-2 Vaccine Candidate Expressing Hypo-Glycosylated Glycoprotein-5 Ectodomain of Korean Lineage-1 Strain.

Vaccination is a practical method to provide protection against porcine reproductive and respiratory syndrome virus (PRRSV), but current PRRSV vaccines show limited efficacy against divergent field strains. Lineage 1 PRRSV includes virulent strains such as NADC30 and MN184 and now has become one of the most prevalent viruses in Korea. Accordingly, there is an urgent need to develop a new vaccine for Korean lineage-1 strains. In this study, a vaccine candidate against Korean lineage-1 PRRSV, vCSL1-GP5-N33D, was developed by reverse genetics technology. vCSL1-GP5-N33D was designed as a hypo-glycosylated chimeric virus containing the glycoprotein 5 ectodomain region of the Korean lineage-1 wild-type strain. An inactivated vaccine of vCSL1-GP5-N33D was applied to a PRRS-endemic farm and elicited high serum virus neutralization (SVN) antibody titers. The vaccinated group induced SVN antibody titers of 4.40 (log2) ± 2.46, which were approximately 2-fold higher than those of the negative control at 8-weeks post-vaccination. Moreover, 60% of pigs in the vaccinated group displayed SVN antibody titers of ≥5 (log2), while none of the pigs in the negative control exhibited SVN antibody titers of ≥5 (log2). The overall results of the animal experiment suggest that the vCSL1-GP5-N33D inactivated vaccine is a promising vaccine candidate.

Inhibition of endocytosis of porcine reproductive and respiratory syndrome virus by rottlerin and its potential prophylactic administration in piglets.

Owing to several limitations of porcine reproductive and respiratory syndrome virus (PRRSV) control procedures, the importance of antiviral agents is increasing; however, limited studies have been done on the development of anti-PRRSV agents. Herein, we explored the antiviral effect and mechanism of rottlerin against PRRSV. We demonstrated that treatment of rottlerin at an early stage of PRRSV infection significantly inhibited the viral replication. PRRSV infection induced protein kinase C-δ phosphorylation, which was specifically downregulated by rottlerin. The treatment of rottlerin led to disrupting the PRRSV entry pathway by blocking endocytosis of the virions. Further, to evaluate the anti-PRRSV effect of the rottlerin in vivo, we administrated rottlerin loaded liposome to pigs infected with PRRSV LMY or FL12 strain. The treatment of rottlerin-liposome reduced the blood viral load, interstitial pneumonia and clinical scores compared to untreated pigs. These results provide an evidence of anti-PRRSV effect of rottlerin in vitro via inhibiting PRRSV internalization and in vivo, all of which strongly suggest the applicability of rottlerin as a potential PRRSV prophylactic treatment.

A chimeric porcine reproductive and respiratory syndrome virus (PRRSV)-2 vaccine is safe under international guidelines and effective both in experimental and field conditions.

Vaccination is currently the most effective strategy to control porcine reproductive and respiratory syndrome (PRRS). New-generation PRRS vaccines are required to be safe and broadly cross-protective. We have recently created the chimeric PRRS virus K418DM which proved to be a good vaccine candidate under field conditions. In the present study, we designed safety and efficacy tests under experimental and field conditions for further evaluation of K418DM1.1, a plaque-purified K418DM. In the homologous challenge study, K418DM1.1 induced high serum virus neutralization (SVN) antibody titers (i.e., 4.2 log2 ± 1.7) at 21 days post-challenge (dpc) and provided protection as demonstrated by the significantly lower levels of viremia at 3 and 7 dpc and significantly lower microscopic lung lesion scores compared to the unvaccinated group. K418DM1.1 was also protective in the heterologous challenge study, with vaccinated pigs showing significantly lower levels of viremia at 14 dpc compared to the unvaccinated pigs. A field study was performed to evaluate the efficacy of K418DM1.1 against heterologous exposure and vaccinated pigs presented significantly lower viremia than unvaccinated pigs. According to the safety test for the examination of virulence reversion, no infectivity was observed in tissue homogenate filtrate both in the vaccinated and comingled groups. Thus, the risk of virulence, as well as transmission, appeared negligible. These overall results indicate that K418DM1.1 is a good vaccine candidate based on its safety and protective efficacy.

Evaluation and optimization of a conventional SPCE for FMD post-vaccination monitoring.

BACKGROUND: Foot-and-mouth disease (FMD) can be controlled by either stamping out or vaccination, a choice which depends on both the economic importance of the livestock sector as well as the disease status. In FMD-free countries with vaccination, such as Korea, vaccination programs should guarantee prevention against transmission of FMD. Monitoring of vaccination programs is also essential for ensuring sufficient coverage that will limit the transmission of FMDV. There are several methods to screen FMD virus (FMDV) structural protein (SP) antibodies including SPCE (Solid-phase competitive ELISA), LPBE (Liquid-phase blocking ELISA), and VNT (Virus neutralization test). Among these, SPCE is widely used for serological monitoring since VNT-the gold standard method-has certain practical limitations, such as high costs in terms of time and labor. However, whether SPCE can ensure the vaccination status of individual animals and whole farms is unclear. In this study, SPCE, LPBE and VNT were compared with respect to correlation with each other and sensitivity at commercial pig farms. RESULTS: The positive results obtained by PrioCHECK SPCE differed from those obtained by LPBE and VNT. The sensitivity of SPCE relative to those of the other tests was fairly low. The raw data of SPCE were most highly correlated with those of VNT with XJ strain, while their positivity and negativity were most highly correlated with LPBE. The results of ROC analysis proposed new cut-off for PrioCHECK SPCE higher than the previous 50% inhibition. CONCLUSIONS: The high false positive rate of PrioCHECK SPCE suggested that high seropositivity by SPCE may not guarantee a true vaccination coverage. Adjusting the cut-off percentage (%) inhibition value for SPCE is needed to address this problem, and it is highly recommended that routine FMDV serological monitoring programs using PrioCHECK SPCE should be combined with alternative methods such as LPBE or VNT.