Microbiology and Immunology最新文献

The spike (S) glycoprotein of porcine respiratory coronavirus (PRCV) plays a critical role in viral entry, with its receptor binding domain (RBD) on the S1 subunit responsible for interacting with the host receptor porcine aminopeptidase N. In this study, the PRCV RBD protein was successfully expressed, and a monoclonal antibody (mAb), 2E6, was produced using hybridoma technology. The specificity of 2E6 was confirmed by western blot and indirect immunofluorescence assay (IFA). To identify the linear B-cell epitope recognized by 2E6, the RBD was initially divided into three overlapping fragments, cloned into pET-32a vectors, and expressed in Escherichia coli BL21 (DE3). Dot blot analysis revealed that 2E6 reacted with the RBD-1 fragment (amino acids 299–357). Further subdivision into RBD1-1, RBD1-2, and RBD1-3, followed by expression using both pET-32a and pEGFP-C1 vectors, enabled Dot blot and IFA validation of specific recognition of RBD1–3 (amino acids 329–347). Subsequent peptide mapping using synthetic overlapping peptides (RBD-P1, P2, P3) confirmed that the minimal linear epitope lies within RBD-P2 (amino acids 334–343). This epitope is surface-exposed and conserved among PRCV strains, making it a promising candidate for diagnostic assay development and epitope-based vaccine design.

The emergence of drug-resistant viruses, particularly in immunocompromised individuals, has necessitated the development of novel antiviral drugs. We previously identified compound 147B3 as an inhibitor of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). Although 147B3 exhibits cytotoxicity, it inhibits the function of infected cell protein 4 (ICP4), a viral transcription factor essential for HSV-1 replication. In this study, we evaluated five commercially available compounds (1C6, 1C6L, 1H6, 1H6L, and 2D10) that had structural similarity to 147B3, exhibited potent anti-HSV-1 activity with reduced cytotoxicity. Among these, 1C6L and 1H6L are structural analogs of 1C6 and 1H6, respectively. An HSV-1 strain resistant to 147B3 carrying a mutation in ICP4 exhibited resistance to all the five compounds, suggesting a shared mechanism of action involving ICP4. Among these compounds, 1H6L had the highest selective index against HSV-1. Notably, these compounds did not reduce early or late protein expression of HSV-1, unlike the parent compound 147B3. Although viral genome replication occurred in the presence of 1H6L, it prevented virion release into the culture supernatant. The cell fraction analysis revealed that 1H6L reduced the size of the cytoplasmic HSV-1 genome. These findings suggest that 1H6L shares certain aspects of its mechanism of action with the parent compound 147B3 but may also inhibit multiple steps in the HSV-1 life cycle.

Herpesvirus nucleocapsids are transported from the nucleus to the cytoplasm via a conserved process known as nuclear egress, which is mediated by the nuclear egress complex (NEC) consisting of two core viral proteins. Although the NEC structure is conserved among herpesviruses, functional divergence may exist. Human herpesvirus 6A (HHV-6A) and HHV-6B are genetically similar members of the Roseolovirus genus within the Betaherpesvirinae subfamily, yet they differ in their pathogenic profiles. In this study, we examined and compared the functions of NEC components U37 and U34 from HHV-6A and HHV-6B. We demonstrated that HHV-6A U34 localizes to the nuclear envelope via its C-terminal transmembrane domain and is essential for viral replication. Moreover, NEC components from HHV-6A and HHV-6B colocalize at the nuclear rim and share a high degree of sequence similarity. These findings suggest that the nuclear egress mechanism is highly conserved within roseoloviruses, despite their distinct biological properties.

Bacterial vaginosis (BV) is characterized by reduced vaginal lactobacilli and pathogenic bacteria proliferation, leading to symptoms such as vaginal epithelial cell detachment and unpleasant odor, along with various complications. Most Lactobacillus species play a crucial role in not only inhibiting BV, but also maintaining vaginal health. We aimed to determine whether Lactobacillus plantarum ATG-K2 has therapeutic and preventive effects against Gardnerella vaginalis (GV), a representative pathogen of BV. In vitro cell assays confirmed that ATG-K2 clusters within cells without cytotoxic effects and inhibits both the cytotoxicity and GV adhesion. Furthermore, biofilm formation assays using crystal violet revealed that ATG-K2 disrupts GV-formed biofilms. In GV-induced BV mouse models, ATG-K2 reduced clue cell formation and sialidase activity, thereby addressing the key aspects of BV. Additionally, when ATG-K2 was administered, the measurement of GV DNA within the vaginal tissue revealed a reduction in GV. In conclusion, ATG-K2 holds promise as a therapeutic agent for BV by inhibiting GV colonization and the associated pathology, potentially offering an alternative to antibiotics.

To investigate the mechanisms underlying pneumococcal infection, a proteomic analysis was previously conducted to identify pneumococcal proteins in infected mouse samples. In the present study, we characterized three proteins, ATP synthase subunit beta (AtpD), ABC transporter transmembrane protein (Vex3), and fructose bisphosphate aldolase (Fba), which bind to human plasminogen and subsequently facilitate its conversion to plasmin by tissue-type plasminogen activator. These findings suggest that Streptococcus pneumoniae might exploit the proteolytic activity of plasmin to promote infection and highlights the potential importance of plasminogen-binding capacity in the pathogenesis of pneumococcal infection.

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis worldwide, and its pathogenic strains typically harbor thermostable direct hemolysin (TDH) and type III secretion system 2 (T3SS2). Although these virulence factors are associated primarily with clinical isolates, their presence in nonclinical environmental and food isolates raises concerns about their potential infection risk. In this study, we investigated the pathogenic potential of nonclinical V. parahaemolyticus isolates from Vietnam, which share serotypic and genotypic characteristics with pandemic strains. Serotyping and genetic analysis of 56 isolates (35 clinical and 21 nonclinical) revealed that two nonclinical isolates from shrimp and environmental water carried the tdh gene, T3SS2α genes, and pandemic markers that clustered phylogenetically with the pandemic strains. Protein expression assays confirmed that these isolates secreted TDH and the T3SS2 translocator (VopD2) at levels similar to those in the clinical reference strain. Bile exposure induced T3SS2-related gene expression, which suggests a conserved gene regulatory mechanism. Enterotoxicity evaluated using a rabbit ileal loop assay showed that two nonclinical isolates induced significant fluid accumulation. Genetic deletion and complementation experiments confirmed that T3SS2 was essential for enterotoxicity. These findings provide the first experimental evidence that nonclinical pandemic strains of V. parahaemolyticus possess functional enteric virulence mechanisms and suggest their potential as infection sources in endemic regions.