Microbiology and Immunology最新文献

Reverse genetics enables the generation of recombinant viruses; however, conventional approaches using full-length cDNA cloned into bacterial artificial chromosomes or plasmids are time-consuming and technically challenging. While the circular polymerase extension reaction (CPER) has been applied to RNA viruses, it has not been used for DNA viruses. Here, we successfully applied CPER to generate infectious recombinant adenoviruses (AdVs) of two serotypes. The resulting viruses replicated comparably to parental strains, and replication was confirmed by immunostaining. These findings demonstrate that CPER is a rapid and efficient platform for reverse genetics of DNA viruses and may accelerate AdV research and therapeutic development.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a highly contagious respiratory disease. It is classified into two biovars on the basis of NAD requirement for growth: biovar 1 (NAD-dependent) and biovar 2 (NAD-independent). Biovar 1 consists of 17 serovars (1−12 and 15−19) and biovar 2 has 8 serovars (2, 4, 7, 9, 11, 13, 14, and 17). Several virulence factors are responsible for the pathogenesis of A. pleuropneumoniae, particularly Apx toxins, capsular polysaccharides, lipopolysaccharides, and membrane proteins. In this review, the current knowledge of biovars, serovars, epidemiology, virulence factors of A. pleuropneumoniae as well as laboratory diagnosis and vaccines are summarized.

Dengue fever poses a grave threat to public health worldwide, claiming numerous fatalities each year with tropical regions being particularly hard hit by dengue outbreaks. The multiple-epitope construct is tailored to the dengue virus's geographical prevalence and the genetics of the Pakistani population. 14 experimentally validated MHC-I and MHC-II epitope sequences, were employed to generate the variants by taking into account the conservancy in all serotypes. Subsequently, the binding affinities of each derived variant against the human leukocyte antigen alleles most common among the Pakistani population were analyzed. A total of three epitopes, two Class-I (GTSGSPIINR and RSWNTGFDW), and one Class-II (ILAPTRVVAAEMEEA), with a combined Pakistani population coverage of more than 73%, together with five linear B cell epitopes were used to create six possible multi-epitope fusion constructs. The molecular docking analysis indicated that two constructs demonstrated notable binding affinities for the most abundant HLA-A*11:01 in Pakistan. Additionally, molecular dynamics (MD) simulations identified one of the constructs as a promising therapeutic candidate. The vaccine construct selected from this analysis could aid in future vaccine design for the dengue virus following further in vitro test validation and in vivo studies to investigate its immune protection capacity.

We investigated the receptor usage of the hepatitis C virus (HCV) genotype 3a S310 strain using cell culture–derived HCV (HCVcc) and trans-complemented HCV particles (HCVtcp). Infection by HCV strains of genotypes 1, 2, and 3 was inhibited by anti-CD81 antibody. By contrast, anti–claudin (CLDN)1 antibody reduced infection by the genotype 1b TH strain and genotype 2a JFH-1 strain but had no effect on the S310 strain (genotype 3a). Moreover, CLDN1-knockout cells remained permissive to infection with a chimeric HCVcc bearing the S310 envelope in a JFH-1 backbone. In CLDN1-deficient cells, infection by HCVtcp derived from the S310 strain was significantly reduced by treatment with anti–claudin-6 (CLDN6) antibody or knockdown of CLDN6 mRNA, suggesting that the S310 strain utilizes CLDN6 as an alternate entry factor. Further analyses revealed that HCVtcp of genotype 4a (ED43 strain) and genotype 6a (HK6a strain) also infected CLDN1-deficient cells. These findings provide new insights into CLDN usage by diverse HCV genotypes and raise the possibility that CLDN tropism may affect viral entry, infection efficiency, and pathogenesis.

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.