Microbiology and Immunology最新文献

Surveillance of SARS-CoV-2 in white-tailed deer (WTD, Odocoileus virginianus) has revealed its widespread and sustained transmission across North America, with evidence suggesting possible transmission from deer to humans. In the following surveillance studies in other deer species, however, little evidence of infection spread was found, including in sika deer (Cervus nippon) in our previous study. Differences in the structure of the virus entry receptor angiotensin-converting enzyme 2 (ACE2) are known to act as one of the functional barriers to SARS-CoV-2 infection. To investigate the molecular basis of the lack of SARS-CoV-2 transmission to sika deer, we performed structural and functional analyses of the sika deer ACE2 in comparison with WTD ACE2. Comparison of sika deer ACE2 sequence and those of cervid species with WTD ACE2, followed by in silico molecular dynamics analysis, revealed a substitution of lysine to asparagine in position 31 commonly found in cervid ACE2s can potentially alter binding to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). Functional assays in cells expressing sika deer and WTD ACE2s showed minimal differences in viral binding and replication, demonstrating that SARS-CoV-2 can similarly utilize ACE2 from both species. These findings suggest that sika deer and possibly other cervids may be highly susceptible to SARS-CoV-2 and highlight the need to investigate other factors impacting virus spread in deer populations.

Previous studies have revealed various treatment modalities for community-acquired pneumonia (CAP), but there is still a lack of in-depth research on inflammation-related genes (IRGs) in CAP. In this analysis, we collected clinical data from CAP patients and identified CAP differentially expressed IRGs (CAP-DE-IRGs) between healthy individuals and CAP patients. Subsequently, functional and pathway enrichment analyses were carried out on CAP-DE-IRGs. Furthermore, hub genes in IRGs were identified, with their diagnostic ability and function validated. Enrichment analyses demonstrated that these CAP-DE-IRGs were abundant in biological processes such as cytokine-cytokine receptor interaction and JAK-STAT signaling pathway. Five hub genes were selected from IRGs. Quantitative Real-time PCR (qRT-PCR) results showed that CCL5, IL7R, IL2RB, and IL10RA were significantly downregulated in CAP patients, and IL18R1 was significantly upregulated in CAP patients (p < 0.05). In the validation of the diagnostic ability of hub genes, most of the genes exhibited area under curve (AUC) values exceeding 0.7, indicating the excellent diagnostic ability of hub genes. The function prediction of hub genes unraveled that hub genes had the functions of cytokine receptor binding, immune receptor activity, response to interleukin-7, and so on. The immune infiltration analysis demonstrated that immune cell Neutrophils exhibited higher infiltration in CAP patients than in healthy individuals. The correlation analysis of hub genes and CAP-related genes manifested that the hub gene IL18R1 was positively correlated with CAP-related genes. In summary, our analysis identified a connection between CAP patients and IRGs, which is beneficial for further research on CAP.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in young children in low- and middle-income countries and the most common cause of diarrhea in international travelers. Currently, there are no vaccines licensed to prevent ETEC-associated diarrhea. MecVax, a protein-based, multivalent ETEC vaccine candidate, has been demonstrated to be broadly immunogenic and cross-protective in preclinical studies. However, the two recombinant proteins of MecVax were prepared from 2× Yeast Extract Tryptone medium (2× YT, which contains animal materials) and are therefore unsuitable for a human vaccine. In this study, we prepared MecVax recombinant proteins using two animal material-free media (Terrific Broth Complete with animal-free Soytone and 2× YT with animal-free Soytone) and comparatively evaluated the vaccine products for immunogenicity and antibody functions against ETEC bacterial adherence and toxin enterotoxicity. Data showed that MecVax protein antigens prepared from a culture medium without or with animal materials showed no differences in protein yield, reactivity with specific antibodies, and induction of antigen-specific IgG responses in mice. Moreover, the mouse serum antibodies exhibited the same level of inhibition of adherence from ETEC bacteria expressing CFA/I, CS1–CS6, as well as neutralization of ETEC heat-stable toxin or heat-labile toxin. These results indicate that MecVax can be prepared using an animal-free Soytone medium, thereby accelerating the development of this vaccine product against ETEC-associated diarrhea in children and travelers. Additionally, we conducted an additional MecVax dosage study in mouse immunization and refined the minimum effective dose to 2 μg of each protein antigen.

Sepsis is a life-threatening condition caused by infection-induced immune dysregulation. Clinically distinguishing sepsis from infection remains to be a challenge due to overlapping clinical features. Although miR-186 regulates cell proliferation and apoptosis, and was predicted to target immune-related genes, its role in sepsis is unclear. We retrospectively enrolled 21 infected patients and 20 sepsis patients. The miR-186 level in blood cells was detected using real-time PCR. Cytokine concentrations and lymphocyte subpopulation proportions were determined using flow cytometry. Clinical data were retrieved from medical records. The diagnostic ability of miR-186 was compared with procalcitonin and lactate using the receiver operating characteristic (ROC) curve. miR-186 was inhibited in human umbilical vein endothelial cells (HUVECs) and mice, followed by measurement of cytokine expression using real-time PCR and flow cytometry. The expression level of miR-186 was significantly higher in septic patients than in infected patients. miR-186 showed relatively better diagnostic performance for sepsis than procalcitonin and lactate. The in vitro assay showed that LPS enhanced miR-186 expression under a dose-dependent manner. In vitro miR-186 inhibition in HUVECs inhibited IL-1β, IL-6, and IL-8 expression. In vivo miR-186 inhibition significantly lowered IL-1β concentration and natural killer cell ratio. In this study, we found that miR-186 is significantly upregulated in sepsis and plays a regulatory role in cytokine expression, highlighting its potential as a diagnostic biomarker for sepsis.

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.