Zili Li

Porcine reproductive and respiratory syndrome virus (PRRSV) has the characteristics of a fast evolution rate and high genetic heterogeneity. PRRSV-1 and PRRSV-2 are co-circulating, and the threat they pose to the health of pigs worldwide continues to rise. In this study, two PRRSV strains with significantly different genetic backgrounds, namely SHEU-2022 (PRRSV-1, full length 15,050 nt) and SHCH-2023 (PRRSV-2, full length 15,019 nt), were simultaneously isolated from a single serum sample of a co-infected sow in Shanghai, China, and their whole-genome characteristics were analyzed. SHEU-2022 clustered with indigenous PRRSV-1 isolates in China, harbored a unique 15-nt deletion in the ORF3-ORF4 overlapping region, carried an additional N-glycosylation site on GP5, and replicated only in porcine alveolar macrophages (PAMs) under the conditions used. SHCH-2023 is a novel recombinant strain with a CH-1a-like (lineage 8) genome backbone and two NADC30-derived fragments—one in Nsp2 (sublineage 1.8) carrying the characteristic 131-amino acid deletion and one in the GP2/E region; it replicated in both PAMs and Marc-145 cells, though this may partly reflect the enrichment procedure. No PRRSV-1/PRRSV-2 recombination was detected between the two co-infected strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes substantial losses to the swine industry, creating a need for complementary antiviral interventions. This study evaluated the antiviral and protective effects of baicalin against PRRSV in vitro and in vivo. Cytotoxicity and antiviral activity were assessed in porcine alveolar macrophages (PAMs), and stage-specific assays were used to identify the affected phase of infection. Twenty piglets were assigned to Mock, baicalin, PRRSV, and PRRSV plus baicalin groups to evaluate clinical outcomes, viremia, respiratory shedding, tissue lesions, and inflammatory responses. Baicalin did not markedly affect PAM viability at concentrations up to 80 μg/mL. Within the tested non-cytotoxic concentration range, baicalin was associated with concentration-dependent reductions in infectious virus titers and PRRSV nsp9 mRNA levels. Among the stage-specific conditions examined, the greatest reduction in progeny virus production was observed when baicalin was present during the viral attachment phase, whereas no significant effects were detected in the direct virucidal, entry, intracellular replication, or progeny-release assays under the tested conditions. Compared with the PRRSV group, the PRRSV plus baicalin group showed lower viral loads in serum, oropharyngeal swabs, and nasal swabs at multiple sampling time points, together with less pronounced fever and clinical signs and partial preservation of body-weight gain. By the end of the experiment, four of five piglets survived in the PRRSV plus baicalin group, compared with one of five piglets in the PRRSV group. Serum IL-1β, IL-6, IL-8, and TNF-α concentrations were also lower in the PRRSV plus baicalin group at several sampling time points. Collectively, baicalin showed antiviral activity against PRRSV in PAMs, with the greatest activity observed when it was present during the viral attachment phase. In piglets, prophylactic dietary baicalin supplementation was associated with lower viral burden and less severe clinical, inflammatory, and pathological changes.

In the post-antibiotic era, bacteriophages have emerged as viable alternatives for combating antibiotic-resistant infections. Analogous to the emergence of antibiotic resistance, bacteria can also develop resistance to phages, a process often accompanied by a reduction in bacterial fitness. In this study, we investigated the mechanisms behind the development of phage resistance in Salmonella enterica serovar Enteritidis strain WJ48 during laboratory coevolution with phage GRNsp8, as well as the associated fitness costs and community dynamics. Three types of phage-resistant mutants, VP81, VP82, and VP84, were isolated. VP81 exhibited a frameshift mutation in the gene encoding glycosyltransferase and displayed partial resistance to GRNsp8. Both VP82 and VP84 carried frameshift mutations in btuB , conferring complete resistance to GRNsp8. Deletion of btuB abolished phage adsorption and conferred complete resistance. Meanwhile, complementation with btuB restored susceptibility. In vitro competition assays showed that the btuB -deletion strain was competitively disadvantaged relative to the wild-type strain within 24 h, exhibiting a relative fitness value of in vivo experiments in mice showed a marked attenuation of virulence in the mutant strain. Specifically, the LD₅₀ of the btuB -deficient strain in mice was 6.8 × 10⁸ CFU, 121 times higher than the wild-type strain. During the 9-day coevolution experiment, a single-point mutation in btuB consistently dominated and represented the primary mode of resistance. These findings shed light on the adaptive trade-offs that bacteria undergo to evade phage infection and provide valuable insights for designing more rational and effective phage therapy strategies that exploit these trade-offs. IMPORTANCE: As emerging antibacterial agents in the post-antibiotic era, Bacteriophages also face the challenge of bacterial resistance. However, phage resistance development by bacteria is frequently accompanied by a reduction in bacterial fitness. To elucidate the adaptive trade-offs associated with resistance, in this study, we used Salmonella enterica serovar Enteritidis strain WJ48 and the broad-host-range bacteriophage GRNsp8 as a model system. We found that the acquisition of phage resistance by bacteria was significantly associated with a reduction in virulence. These findings deepen our understanding of bacteria–phage coevolution but also offer key insights into leveraging the resistance–fitness trade-off to inform the strategic design of more effective phage therapies. The results highlight the potential for improving the application of phages in agriculture and animal husbandry, supporting the sustainable development of phage-based antimicrobial strategies.

Background: Enteropathogenic Escherichia coli (EPEC) disrupts intestinal barrier integrity by adhering to epithelial cells, leading to diarrhea, impaired nutrient absorption, oxidative stress, and intestinal inflammation in young animals. This study aimed to isolate and characterize a neutral exopolysaccharide (EPS-T1) from Enterococcus faecium HDRsEf1, evaluate its functional activities in vitro, and assess its protective effects against EPEC-induced enteritis in vivo. Results: EPS-T1, with a molecular weight of 81.21 ± 1.28 kDa, was mainly composed of glucose, galactose, rhamnose, and mannose, and exhibited a porous, sheet structure with relatively high thermal stability. In vitro, EPS-T1 (200 μg/mL) significantly inhibited EPEC growth and biofilm formation, reduced bacterial adhesion to intestinal epithelial cells, and exhibited broad-spectrum free radical scavenging activity. In vivo, EPS-T1 treatment alleviated EPEC-induced weight loss and intestinal tissue damage, reduced the intestinal EPEC load, downregulated pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), upregulated the anti-inflammatory cytokine IL-10, and improved serum antioxidant indices (T-AOC, SOD, and GSH-PX) while decreasing MDA levels. Conclusions: These results demonstrate that EPS-T1 derived from Enterococcus effectively mitigates EPEC-induced intestinal inflammation and oxidative stress, highlighting its potential as an immunobiotic functional candidate.

Porcine epidemic diarrhoea virus (PEDV) is a highly pathogenic porcine enteric coronavirus that causes severe watery diarrhoea and mortality in piglets. The nucleocapsid protein (N) is the most abundant viral protein and is highly phosphorylated, with the phosphorylation level directly affecting infection and proliferation. Here, we characterized the phosphorylation level of the N protein and found that its SR (Ser and Arg) motif was highly phosphorylated. The phosphorylation level significantly decreased after mutation of threonine (Thr) to serine (Ser). Through screening, it was determined that GSK3α/β plays a major role in phosphorylating the SR motif. Using GSK3α/β inhibitors or directly knocking out the GSK3α/β gene significantly inhibit PEDV proliferation. Finally, we used yeast recombination technology to develop a reverse genetics system for assessing PEDV and confirmed that no differences existed between the wild-type strain and the rescued virus. Using this platform, we generated a PEDV N protein SR motif mutant strain. We found that, compared to the wild-type strain, the proliferation of the mutant strain was significantly weakened, confirming that the phosphorylation of the SR motif is crucial for PEDV proliferation. In summary, we verified the phosphorylation sites of the PEDV N protein and the associated protein kinases, providing new insights into the development of relevant therapeutic strategies.

Duck infectious serositis is a septicemic disease caused by the bacterium Riemerella anatipestifer (R. anatipestifer), which affects ducks, geese, turkeys, and other poultry. While outbreaks have been reported worldwide, the exact mechanisms of infection and disease progression remain unclear. Our previous research identified the two-component system PhoPR within the genome of R. anatipestifer and demonstrated its association with the bacterium’s pathogenicity. Through multi-omics analysis, we found that PhoP directly regulates the expression of several genes, including moxR, within the Bacteroides aerotolerance (Bat) operon. However, the function of MoxR in R. anatipestifer has not yet been reported. To investigate the impact of MoxR on the expression of the bat operon and the pathogenicity of R. anatipestifer, we constructed ΔmoxR and other derivative strains. Our findings revealed that overexpression of MoxR inhibits the transcription of the bat operon. Conversely, deletion of moxR, along with exposure of R. anatipestifer to thermal or oxidative stress, results in increased transcription levels of the bat operon. By measuring the survival ability of each strain under stress, we discovered that MoxR is closely associated with the resistance of R. anatipestifer to thermal and oxidative stress by influencing the expression of the bat operon. Duckling infection experiments, along with adhesion and invasion assays, showed that deletion of moxR in R. anatipestifer led to decreased pathogenicity, and lower bacterial load in various tissues. Collectively, our findings collectively demonstrate the significant role of MoxR in the anti-stress and pathogenicity of R. anatipestifer, providing new insights into its pathogenic mechanisms.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Previous studies from other groups showed that CD163 is required for PRRSV uncoating and genome release. However, CD163 does not interact with nucleocapsid (N) protein. In this study, the neonatal Fc receptor (FcRn) was demonstrated to be irreplaceable for PRRSV infection by knockdown, overexpression, antibodies or IgG blocking, knockout, and replenishment assays. FcRn was further revealed to be involved in PRRSV uncoating for the first time rather than viral attachment and internalization. In detail, FcRn was determined to colocalize with CD163 and PRRSV virions in early endosomes and specially interact with N protein in early endosomes. Taken together, these results provide evidence that FcRn is an essential cellular factor for PRRSV uncoating, which will be a promising target to interfere with the viral infection. IMPORTANCE: PRRSV infection results in a severe swine disease affecting pig farming in the world. Although CD163 has been implicated as the uncoating receptor for PRRSV but the uncoating mechanism of PRRSV remains unclear. Here, we identified that FcRn facilitated virion uncoating via interaction with viral N protein in early endosomes. Our work actually expands the knowledge of PRRSV infection and provides an attractive therapeutic target for the prevention and control of PRRS.