Disruption of Bacterial Biofilms by a Green Synthesized Artemisinin Nano-copper Nanomaterial.

Bacterial biofilms are associated with antibiotic resistance and account for approximately 80% of all bacterial infections. In this study, we explored novel nanomaterials for combating bacteria and their biofilms. Artemisinin nano-copper (ANC) was synthesised using a green synthesis strategy, and its shape, size, structure, elemental composition, chemical valence, zeta potential, and conductivity were characterised using transmission electron microscopy, X-ray diffractometer, X-ray photoelectron spectroscopy, zeta potential, and dynamic light scattering (DLS). The results showed that ANC was successfully synthesised utilizing a liquid-phase chemical reduction method using chitosan as a modified protectant and l-ascorbic acid as a green reducing agent. The stability of ANC was evaluated using DLS. The results showed that the particle size of the ANC at different concentrations was comparable to that of the original solution after 7 days of storage, and there was no significant change in PDI (P > 0.05). The antibacterial effects of ANC on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were determined by Disk diffusion and broth dilution methods. The results demonstrated that ANC inhibited and killed E. coli and S. aureus. The effect of ANC on bacterial biofilms was investigated using Crystal Violet staining, scanning electron microscopy, laser confocal microscope, and quantitative PCR. The results showed that ANC treatment was able to destroy bacterial biofilms and downregulate biofilm- and virulence-related genes in E. coli (HlyA, gyrA, and F17) and S. aureus (cna, PVL, ClfA, and femB). Green-synthesised ANC possesses excellent anti-biofilm properties and is expected to exhibit antibacterial and anti-biofilm properties.

The Effects of Bivalirudin and Ordinary Heparin on the Incidence of Bleeding Events and the Level of Inflammation after Interventional Therapy for Acute Myocardial Infarction.

Objective: To evaluate and compare the efficacy, bleeding events, and inflammation levels of optimized bivalirudin versus ordinary heparin in the context of percutaneous coronary intervention (PCI) for patients with acute myocardial infarction. This approach will underscore the comprehensive scope of the study, addressing multiple dimensions of clinical outcomes. Methods: This study involved 120 acute myocardial infarction patients treated from January 2022 to January 2023, randomly allocated into two groups: the control group received ordinary heparin, and the observation group received bivalirudin. Both groups underwent percutaneous coronary intervention (PCI). The study specifically measured coagulation indexes such as prothrombin time (PT) and activated partial thromboplastin time (aPTT), and inflammatory markers including C-reactive protein (CRP) and interleukin-6 (IL-6). Additionally, the incidence of bleeding events and major adverse cardiovascular events (MACE) within 30 days post-PCI were recorded, with bleeding events categorized according to the Bleeding Academic Research Consortium (BARC) criteria and MACE defined by the occurrence of death, non-fatal myocardial infarction, or stroke. Results: No significant differences were observed in coagulation indexes and pre-operation inflammation levels between the two groups (P > .05). However, at 7 days post-operation, despite both groups showing reduced inflammation-NLR decreased by 25%, hs-CRP by 30%, and IL-10 increased by 20%-the bivalirudin group exhibited notably lower incidence rates of various bleeding events (mucosal 2% vs 6%, gingival 1% vs 4%, puncture site 3% vs 8%, and hematuria 1% vs 5%) within 30 days post-PCI compared to the heparin group. TIMI blood flow grades 3 (indicating normal flow) were achieved in 85% of the bivalirudin group compared to 70% in the heparin group. The incidence of MACE was comparable between groups with both reporting a 5% occurrence rate (P > .05). Conclusion: The study reveals that while both bivalirudin and ordinary heparin effectively prevent MACE post-acute myocardial infarction intervention, bivalirudin significantly reduces postoperative bleeding events and maintains comparable anti-inflammatory effects. This suggests its preferable use in clinical settings, particularly in patient populations at high risk for bleeding. Future research could further explore the specific patient characteristics that optimize bivalirudin's benefits over heparin, enhancing tailored therapeutic approaches. This could potentially include randomized trials focusing on patients with different baseline bleeding risks.

Tick-borne Rickettsia, Anaplasma, Theileria, and enzootic nasal tumor virus in ruminant, PET, and poultry animals in Pakistan.

Introduction: Pakistan is an agricultural country; most of its income is based on livestock rearing. The increasing prevalence of tick-borne pathogens among animals may affect the animal production and livelihood of owners, which eventually derange the economy of a country. Methodology: To further comprehend TBPs, 213 ticks were collected from different animals, including ruminants, pets, and poultry. After molecular and phylogenetic analysis identification, ticks were managed into different pools based on their species level (Hyalomma anatolicum = 80, Rhipicephalus microplus = 35, Hyalomma scupense = 23, Rhipicephalus turanicus = 70, and Rhipicephalus sanguineus = 5). Results and discussion: After tick species identification, further molecular PCR amplification was carried out to screen out the pathogens for the presence of Theileria, Rickettsia, Anaplasma, and enzootic nasal tumor virus (ENTV). The following pathogens were detected: 11 (5.16%) for Anaplasma, 1 (0.47%) for Rickettsia, and 9 (4.23%) for Theileria. Nevertheless, other TBPs that had not been reported so far in Pakistan 3 (1.41%), were positive for enzootic nasal tumor virus (ENTV). Besides, phylogenetic analysis of the enzootic nasal tumor virus (ENTV) strain confirmed its resemblance to the Chinese strain, while Anaplasma has comparability with Pakistan and China, Rickettsia with Pakistan, China, and Iran, and Theileria with India, South Africa, United States, Japan, and Spain. Conclusion: This study reveals that there is a considerably wider range of TBPs held in Pakistan that take in various contagious zoonotic pathogens than was previously thought. This information advances TBP epidemiology and will contribute to upgrade future control measure.

Spatial risk of Haemaphysalis longicornis borne Dabieshan tick virus (DBTV) in China.

The uncertainty and unknowability of emerging infectious diseases have caused many major public health and security incidents in recent years. As a new tick-borne disease, Dabieshan tick virus (DBTV) necessitate systematic epidemiological and spatial distribution analysis. In this study, tick samples from Liaoning Province were collected and used to evaluate distribution of DBTV in ticks. Outbreak points of DBTV and the records of the vector Haemaphysalis longicornis in China were collected and used to establish a prediction model using niche model combined with environmental factors. We found that H. longicornis and DBTV were widely distributed in Liaoning Province. The risk analysis results showed that the DBTV in the eastern provinces of China has a high risk, and the risk is greatly influenced by elevation, land cover, and meteorological factors. The risk geographical area predicted by the model is significantly larger than the detected positive areas, indicating that the etiological survey is seriously insufficient. This study provided molecular and important epidemiological evidence for etiological ecology of DBTV. The predicted high-risk areas indicated the insufficient monitoring and risk evaluation and the necessity of future monitoring and control work.

A modified recombinant adenovirus vector containing dual rabies virus G expression cassettes confers robust and long-lasting humoral immunity in mice, cats, and dogs.

During the COVID-19 epidemic, the incidence of rabies has increased in several countries, especially in remote and disadvantaged areas, due to inadequate surveillance and declining immunization coverage. Multiple vaccinations with inactivated rabies virus vaccines for pre- or post-exposure prophylaxis are considered inefficient, expensive and impractical in developing countries. Herein, three modified human recombinant adenoviruses type 5 designated Adv-RVG, Adv-E1-RVG, and Adv-RVDG, carrying rabies virus G (RVG) expression cassettes in various combinations within E1 or E3 genomic regions, were constructed to serve as rabies vaccine candidates. Adv-RVDG mediated greater RVG expression both in vitro and in vivo and induced a more robust and durable humoral immune response than the rabies vaccine strain SAD-L16, Adv-RVG, and Adv-E1-RVG by more effectively activating the dendritic cells (DCs) - follicular helper T (Tfh) cells - germinal centre (GC) / memory B cells (MBCs) - long-lived plasma cells (LLPCs) axis with 100% survival after a lethal RABV challenge in mice during the 24-week study period. Similarly, dogs and cats immunized with Adv-RVDG showed stronger and longer-lasting antibody responses than those vaccinated with a commercial inactivated rabies vaccine and showed good tolerance to Adv-RVDG. In conclusion, our study demonstrated that simultaneous insertion of protective antigens into the E1 and E3 genomic regions of adenovirus vector can significantly enhance the immunogenicity of adenoviral-vectored vaccines, providing a theoretical and practical basis for the subsequent development of multivalent and multi-conjugated vaccines using recombinant adenovirus platform. Meanwhile, our data suggest Adv-RVDG is a safe, efficient, and economical vaccine for mass-coverage immunization.

Analysis of the diversity of tick-borne viruses at the border areas in Liaoning Province, China.

Ticks play a significant role in transmitting arboviruses, which pose a risk to human and animal health. The region of Liaoning Province, China, with abundant plant resources with multiple tick populations, has reported several tick-borne diseases. However, there remains a scarcity of research on the composition and evolution of the tick virome. In this study, we conducted the metagenomic analysis of 561 ticks in the border area of Liaoning Province in China and identified viruses related to known diseases in humans and animals, including severe fever with thrombocytopenia syndrome virus (SFTSV) and nairobi sheep disease virus (NSDV). Moreover, the groups of tick viruses were also closely related to the families of Flaviviridae, Parvoviridae, Phenuiviridae, and Rhabdoviridae. Notably, the Dabieshan tick virus (DBTV) of the family Phenuiviridae was prevalent in these ticks, with the minimum infection rate (MIR) of 9.09%, higher than previously reported in numerous provinces in China. In addition, sequences of tick-borne viruses of the family Rhabdoviridae have first been reported from the border area of Liaoning Province, China, after being described from Hubei Province, China. This research furthered the insight into pathogens carried by ticks in the northeastern border areas of China, offering epidemiological information for possible forthcoming outbreaks of infectious diseases. Meanwhile, we provided an essential reference for assessing the risk of tick bite infection in humans and animals, as well as for exploring into the evolution of the virus and the mechanisms of species transmission.

Metavirome of 31 tick species provides a compendium of 1,801 RNA virus genomes.

The increasing prevalence and expanding distribution of tick-borne viruses globally have raised health concerns, but the full repertoire of the tick virome has not been assessed. We sequenced the meta-transcriptomes of 31 different tick species in the Ixodidae and Argasidae families from across mainland China, and identified 724 RNA viruses with distinctive virome compositions among genera. A total of 1,801 assembled and complete or nearly complete viral genomes revealed an extensive diversity of genome architectures of tick-associated viruses, highlighting ticks as a reservoir of RNA viruses. We examined the phylogenies of different virus families to investigate virome evolution and found that the most diverse tick-associated viruses are positive-strand RNA virus families that demonstrate more ancient divergence than other arboviruses. Tick-specific viruses are often associated with only a few tick species, whereas virus clades that can infect vertebrates are found in a wider range of tick species. We hypothesize that tick viruses can exhibit both 'specialist' and 'generalist' evolutionary trends. We hope that our virome dataset will enable much-needed research on vertebrate-pathogenic tick-associated viruses.

A Phi29-based unbiased exponential amplification and genotyping approach improves pathogen detection in tick samples.

Ticks are vectors for many infectious diseases, such as spotted fever group (SFG) rickettsioses and borreliosis, and are valuable in the study of pathogen ecology. Ticks have several growth stages that vary considerably in size; therefore, in most cases, DNA extracted from ticks is insufficient for subsequent studies, particularly for multiple pathogen screening and genotyping. Unbiased amplification of DNA from tick samples before analysis is a major requirement for subsequent ecological surveys and other studies. Phi29 DNA polymerase, an enzyme that exhibits strand displacement activity, can exponentially amplify DNA randomly, generating large quantities of DNA. In the present study, we developed a Phi29-based unbiased exponential amplification (PEA) assay to obtain sufficient tick DNA for genetic analysis. By using tick-borne pathogen detection and genotyping as a model, we tested and evaluated the feasibility of the assay. DNA was extracted from single ticks and subjected to PEA. The results showed that tick DNA could be amplified up to 105 fold. The amplified products were successfully used for pathogen screening and genotyping. Rickettsia was successfully detected and genotyped in samples with amplified DNA from single ticks. Furthermore, we identified a new genotype of Rickettsia from ticks collected from Dandong city, Liaoning province, Northeast China. This PEA assay is universal and can be extended to other applications where the quantity of DNA is greatly limited.

Identification of severe fever with thrombocytopenia syndrome virus genotypes in patients and ticks in Liaoning Province, China.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS), caused by the SFTS virus (SFTSV), is an acute infectious disease transmitted by ticks that has recently been identified. There are no reports of epidemic serotypes in Liaoning Province, PR China. The aim of this study was, therefore, to identify genotypes of SFTSV in this province. METHODS: In 2019, quantitative PCR testing was performed on 17 patients suspected of being infected with SFTS in Liaoning Province and on 492 ticks from the counties and cities surrounding the patients' residences. Four samples were subjected to virus isolation and whole-genome amplification. RESULTS: Molecular diagnostic results confirmed SFTSV infection in five of the 17 suspected cases of SFTS and in 12 of the 492 ticks, with a prevalence of 2.4%. Four strains of SFTSV were successfully isolated from patients' blood and ticks. Phylogenetic analysis after whole-genome amplification and sequencing showed that they all belonged to genotype A of SFTSV. CONCLUSIONS: This study is the first to determine the genotype of SFTSV in patients and ticks in Liaoning Province, PR China. The results deepen our understanding of the SFTS epidemic and provide information on the variability in mortality rate among genotypes.

Rapid Spread of Severe Fever with Thrombocytopenia Syndrome Virus by Parthenogenetic Asian Longhorned Ticks.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is spreading rapidly in Asia. This virus is transmitted by the Asian longhorned tick (Haemaphysalis longicornis), which has parthenogenetically and sexually reproducing populations. Parthenogenetic populations were found in ≥15 provinces in China and strongly correlated with the distribution of severe fever with thrombocytopenia syndrome cases. However, distribution of these cases was poorly correlated with the distribution of populations of bisexual ticks. Phylogeographic analysis suggested that the parthenogenetic population spread much faster than bisexual population because colonization is independent of sexual reproduction. A higher proportion of parthenogenetic ticks was collected from migratory birds captured at an SFTSV-endemic area, implicating the contribution to the long-range movement of these ticks in China. The SFTSV susceptibility of parthenogenetic females was similar to that of bisexual females under laboratory conditions. These results suggest that parthenogenetic Asian longhorned ticks, probably transported by migratory birds, play a major role in the rapid spread of SFTSV.

Evaluation and Differential Diagnosis of a Genetic Marked Brucella Vaccine A19ΔvirB12 for Cattle.

Brucella abortus is an important zoonotic pathogen that causes severe economic loss to husbandry and poses a threat to human health. The B. abortus A19 live vaccine has been extensively used to prevent bovine brucellosis in China. However, it is difficult to distinguish the serological response induced by A19 from that induced by natural infection. In this study, a novel genetically marked vaccine, A19ΔvirB12, was generated and evaluated. The results indicated that A19ΔvirB12 was able to provide effective protection against B. abortus 2308 (S2308) challenge in mice. Furthermore, the safety and protective efficacy of A19ΔvirB12 have been confirmed in natural host cattle. Additionally, the VirB12 protein allowed for serological differentiation between the S2308 challenge/natural infection and A19ΔvirB12 vaccination. However, previous studies have found that the accuracy of the serological detection based on VirB12 needs to be improved. Therefore, we attempted to identify potential supplementary antigens with differential diagnostic functions by combining label-free quantitative proteomics and protein chip technology. Twenty-six proteins identified only in S2308 were screened; among them, five proteins were considered as potential supplementary antigens. Thus, the accuracy of the differential diagnosis between A19ΔvirB12 immunization and field infection may be improved through multi-antigen detection. In addition, we explored the possible attenuation factors of Brucella vaccine strain. Nine virulence factors were downregulated in A19ΔvirB12. The downregulation pathways of A19ΔvirB12 were significantly enriched in quorum sensing, ATP-binding cassette transporter, and metabolism. Several proteins related to cell division were significantly downregulated, while some proteins involved in transcription were upregulated in S2308. In conclusion, our results contribute to the control and eradication of brucellosis and provide insights into the mechanisms underlying the attenuation of A19ΔvirB12.

Disulfiram inhibits inflammation and fibrosis in a rat unilateral ureteral obstruction model by inhibiting gasdermin D cleavage and pyroptosis.

BACKGROUND: As an inhibitor of GSDMD, Disulfiram (DSL) can significantly inhibit cell pyroptosis. Cell pyroptosis plays an important role in renal fibrosis. METHODS: HK-2 cells were induced by Lps and ATP to form a pyroptosis model, and the cells were treated by DSL. CCK-8 detected the cell activity. Immunofluorescence (IF) detected the GSDMD. ELISA detected the expression of inflammatory cytokines. Flow cytometry and Western blot detected cell apoptosis and pyroptosis. Collagen type I kit detected collagen secretion, and western blot detected fibrosis marker protein expression. Then, a rat model of unilateral ureteral obstruction (UUO) was established. HE staining detected the degree of renal tissue injury, and Masson staining detected the degree of fibrosis. What's more, the apoptosis level of tissue cells was detected by TUNEL. And the inflammatory factors in peripheral blood and renal tissue were detected by ELISA. Furthermore, the expression of GSDMD was detected by immunohistochemistry (IHC), and Western blot was used to detect the expression levels of apoptosis and pyroptosis-related proteins in tissues. RESULTS: It was found that DSL can inhibit the cell membrane perforation of GSDMD-N by inhibiting the cleavage of GSDMD, hence, it inhibited the occurrence of inflammation, cell pyroptosis, and the fibrosis of HK-2 cells. But if the cell has already undergone pyroptosis, DSL does not provide significant prevention. In vivo experiment, it further verified that pretreated DSL had inhibited renal fibrosis injury. CONCLUSION: Disulfiram can inhibit inflammation and fibrosis in renal fibrosis rats by inhibiting GSDMD.

Ubiquitin-Modified Proteome of SARS-CoV-2-Infected Host Cells Reveals Insights into Virus-Host Interaction and Pathogenesis.

The outbreak of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a serious threat to global public health. The mechanism of pathogenesis and the host immune response to SARS-CoV-2 infection are largely unknown. In the present study, we applied a quantitative proteomic technology to identify and quantify the ubiquitination changes that occur in both the virus and the Vero E6 cells during SARS-CoV-2 infection. By applying label-free, quantitative liquid chromatography with tandem mass spectrometry proteomics, 8943 lysine ubiquitination sites on 3086 proteins were identified, of which 138 sites on 104 proteins were quantified as significantly upregulated, while 828 sites on 447 proteins were downregulated at 72 h post-infection. Bioinformatics analysis suggested that SARS-CoV-2 infection might modulate host immune responses through the ubiquitination of important proteins, including USP5, IQGAP1, TRIM28, and Hsp90. Ubiquitination modification was also observed on 11 SAR-CoV-2 proteins, including proteins involved in virus replication and inhibition of the host innate immune response. Our study provides new insights into the interaction between SARS-CoV-2 and the host as well as potential targets for the prevention and treatment of COVID-19.

Combined nucleic acid assays for diagnosis of A19 vaccine-caused human brucellosis.

Brucellosis is a common zoonotic disease caused by Brucella and is an epidemic worldwide. Currently, the most effective way to prevent and control the disease in animals is to use live, attenuated vaccines A19 strain. In China, the live attenuated Brucella abortus vaccine is widely used in animal immunization. To detect and confirm which vaccine strain caused the infection, we developed a new method to distinguish A19 strain from non-A19 strains. By comparing the genomic sequences of A19 and wild strain 2,308, we identified signature sequences that are unique to A19. A PCR assay for specific A19 identification was developed based on the genetic marker ABC transporter permease gene. Samples from the outbreak patients were then analysed using the universal quantitative PCR and A19-specific PCR assay, and the A19 strain was successfully identified in them, providing pathogenic evidence of the vaccine-derived infection outbreak. This combined A19-specific differential diagnosis method can provide a means to distinguish between animal vaccine immunization, natural infection and human infection by the vaccine strain. This strategy also has applications in diagnosis, epidemiology and surveillance of A19-related immunizations or infections.

Temporal and spatial distribution trends of human brucellosis in Liaoning Province, China.

Brucellosis is a natural epidemic zoonotic disease. Liaoning province, north-east of China, has been among the top 10 provinces with highest brucellosis incidence. In this study, the spatial and temporal distribution of brucellosis in Liaoning Province from 2006 through 2017 was analysed using the Bayesian theory of space-time modelling. The study found that in Liaoning Province, (a) all regions of the entire study area were stable counties; (b) the risk of brucellosis declined slowly with time without an obvious trend; (c) the declining trend of disease risk in three sub-hot-spot counties was faster than the overall trend, whereas in other counties, the trend was similar to the overall trend. Furthermore, the time and spatial trends of brucellosis incidence in Liaoning Province were calculated and analysed. These results may provide a theoretical and scientific basis for the public health department to develop targeted effective prevention and control measures for the disease.

Large-Scale Comparative Analyses of Tick Genomes Elucidate Their Genetic Diversity and Vector Capacities.

Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases.

Distribution of the COVID-19 epidemic and correlation with population emigration from Wuhan, China.

BACKGROUND: The ongoing new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) outbreak is spreading in China, but it has not yet reached its peak. Five million people emigrated from Wuhan before lockdown, potentially representing a source of virus infection. Determining case distribution and its correlation with population emigration from Wuhan in the early stage of the epidemic is of great importance for early warning and for the prevention of future outbreaks. METHODS: The official case report on the COVID-19 epidemic was collected as of January 30, 2020. Time and location information on COVID-19 cases was extracted and analyzed using ArcGIS and WinBUGS software. Data on population migration from Wuhan city and Hubei province were extracted from Baidu Qianxi, and their correlation with the number of cases was analyzed. RESULTS: The COVID-19 confirmed and death cases in Hubei province accounted for 59.91% (5806/9692) and 95.77% (204/213) of the total cases in China, respectively. Hot spot provinces included Sichuan and Yunnan, which are adjacent to Hubei. The time risk of Hubei province on the following day was 1.960 times that on the previous day. The number of cases in some cities was relatively low, but the time risk appeared to be continuously rising. The correlation coefficient between the provincial number of cases and emigration from Wuhan was up to 0.943. The lockdown of 17 cities in Hubei province and the implementation of nationwide control measures efficiently prevented an exponential growth in the number of cases. CONCLUSIONS: The population that emigrated from Wuhan was the main infection source in other cities and provinces. Some cities with a low number of cases showed a rapid increase in case load. Owing to the upcoming Spring Festival return wave, understanding the risk trends in different regions is crucial to ensure preparedness at both the individual and organization levels and to prevent new outbreaks.

High Incidence of a Novel Rickettsia Genotype in Parasitic Haemaphysalis longicornis from China-North Korea Border.

Ticks are notorious vectors for various pathogens that cause infections in animals and humans worldwide. Rickettsia spp., a zoonotic tick-borne pathogen that could be used as a weapon agent, is widely spread in China. In the present study, ticks were collected for species identification and Rickettsia screening. PCR amplification targeting the tick 18s rRNA gene was first conducted for species validation, and then, amplification was conducted for the Rickettsia housekeeping gene for the infection rate and phylogenetic analysis. The collected ticks were identified as Haemaphysalis longicornis, 7.36% of which were Rickettsia-positive. The phylogenetic analysis showed that the Rickettsia in the parasitic ticks belonged to a novel genotype, whose closest genetic relationship was with Rickettsia heilongjiangenesis. The samples were collected in Dandong, a city on the border between China and North Korea. Considering the geographical and biological situations of the sampling sites, more extensive surveillance and risk evaluation of the tick species and tick-borne diseases are required.