Objective:To investigate the mosquito-borne viruses carried by mosquito specimens collected from Huachuan county and Huanan county in Heilongjiang province.Methods:Mosquito samples were collected locally in 2023 and processed in the laboratory. Homogenates of the mosquitoes were inoculated into cells for virus isolation, followed by molecular and bioinformatics analyses of the viral isolates.Results:In 2023, ten viral isolates were obtained from Anopheles sinensis specimens collected in Heilongjiang province, China. Among these isolates, one was identified as Culex flavivirus (CxFV), one as Menghai rhabdovirus (MRV), and eight as Nam Dinh virus (NDiV). The phylogenetic analysis showed that CxFV belongs to genotype I and is clustered with the strains isolated from Liaoning province in 2011 and Ningxia Hui autonomous Region in 2019 in the same evolutionary branch, with amino acid similarity ranging from 98.2% to 99.2% and nucleotide similarity ranging from 98.8% to 99.2%. The MRV strain belongs to the same evolutionary subclade as the strain detected in Guangdong, with both nucleotide and amino acid similarity of 98.0%. Eight NDiV isolates clustered with the South Korean isolates on the same evolutionary branch, forming an independent evolutionary sub-branch. The nucleotide similarity among these eight isolates ranged from 98.5% to 99.7%, while the amino acid similarity ranged from 98.1% to 99.7%. In comparison, when matched with other NDiV isolates from China, the nucleotide similarity of these eight isolates ranged from 94.1% to 97.8%, and the amino acid similarity ranged from 93.5% to 97.7%.Conclusions:This study represents the first isolation of CxFV, MRV, and NDiV in Heilongjiang province, China, and the findings provide fundamental data for the prevention and control of mosquito-borne viral diseases in this region.

Objective:To establish a method for the rapid detection of varicella-zoster virus (VZV) by recombinase-aid amplification (RAA) combined with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system.Methods:Clinical samples of suspected herpes zoster in Shandong province and Shanghai from 2023 to 2024 were collected, nucleic acids of positive samples were extracted, RAA-specific primers and crRNA (CRISPR RNA, crRNA) were designed for the conserved region of VZV, and the fluorescence intensity generated by Cas12a non-specific cleavage of single-stranded fluorescent probes was used to screen highly sensitive crRNAs and optimize the concentrations of crRNA, Cas12a and ssDNA probes. The sensitivity and reproducibility of the RAA-CRISPR/Cas12a detection method were evaluated by using synthesized plasmids and clinical samples, and the specificity of the method was evaluated by using other viral nucleic acids. The method was used to detect clinical samples by using the method and quantitative real-time PCR (qPCR) method, and the detection rate and consistency of the two method were compared.Results:The highly sensitive crRNA-4 was screened from the four crRNAs designed, and a VZV detection method for RAA-CRISPR/CAS12a based on fluorescence intensity measurement was established, which could be detected at 37℃ in 45 min, and the sensitivity of the detection could reach 10 copies/μL, a minimum clinical sample with a Ct value of 38.980 can be detected. It has high specificity and no cross-reactivity with Adenovirus 7, Herpes simplex virus type I, Herpes simplex virus type II, Coxsackieviruses A16, Cytomegalovirus, Epstein-Barr virus, Measles virus, Mumps virus, Enterovirus 71, Japanese encephalitis virus genotype 5. It has good stability, and can be successfully detected in low, medium and high concentrations of viral positive plasmids with good consistency. The detection rate of the clinically positive samples was 100%, which was completely consistent with the qPCR test result.Conclusions:RAA isothermal amplification technology combined with CRISPR-CAS12a technology was used to establish an accurate method for the detection of VZV virus, which was highly sensitive, specific, and had low requirements for experimental conditions, and could be completed within 45 min, which could provide strong technical support for the early detection of VZV.

Objective:To investigate the mosquito-borne viruses carried by mosquito specimens collected from Huachuan county and Huanan county in Heilongjiang province.Methods:Mosquito samples were collected locally in 2023 and processed in the laboratory. Homogenates of the mosquitoes were inoculated into cells for virus isolation, followed by molecular and bioinformatics analyses of the viral isolates.Results:In 2023, ten viral isolates were obtained from Anopheles sinensis specimens collected in Heilongjiang province, China. Among these isolates, one was identified as Culex flavivirus (CxFV), one as Menghai rhabdovirus (MRV), and eight as Nam Dinh virus (NDiV). The phylogenetic analysis showed that CxFV belongs to genotype I and is clustered with the strains isolated from Liaoning province in 2011 and Ningxia Hui autonomous Region in 2019 in the same evolutionary branch, with amino acid similarity ranging from 98.2% to 99.2% and nucleotide similarity ranging from 98.8% to 99.2%. The MRV strain belongs to the same evolutionary subclade as the strain detected in Guangdong, with both nucleotide and amino acid similarity of 98.0%. Eight NDiV isolates clustered with the South Korean isolates on the same evolutionary branch, forming an independent evolutionary sub-branch. The nucleotide similarity among these eight isolates ranged from 98.5% to 99.7%, while the amino acid similarity ranged from 98.1% to 99.7%. In comparison, when matched with other NDiV isolates from China, the nucleotide similarity of these eight isolates ranged from 94.1% to 97.8%, and the amino acid similarity ranged from 93.5% to 97.7%.Conclusions:This study represents the first isolation of CxFV, MRV, and NDiV in Heilongjiang province, China, and the findings provide fundamental data for the prevention and control of mosquito-borne viral diseases in this region.

Objective:To establish a method for the rapid detection of varicella-zoster virus (VZV) by recombinase-aid amplification (RAA) combined with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system.Methods:Clinical samples of suspected herpes zoster in Shandong province and Shanghai from 2023 to 2024 were collected, nucleic acids of positive samples were extracted, RAA-specific primers and crRNA (CRISPR RNA, crRNA) were designed for the conserved region of VZV, and the fluorescence intensity generated by Cas12a non-specific cleavage of single-stranded fluorescent probes was used to screen highly sensitive crRNAs and optimize the concentrations of crRNA, Cas12a and ssDNA probes. The sensitivity and reproducibility of the RAA-CRISPR/Cas12a detection method were evaluated by using synthesized plasmids and clinical samples, and the specificity of the method was evaluated by using other viral nucleic acids. The method was used to detect clinical samples by using the method and quantitative real-time PCR (qPCR) method, and the detection rate and consistency of the two method were compared.Results:The highly sensitive crRNA-4 was screened from the four crRNAs designed, and a VZV detection method for RAA-CRISPR/CAS12a based on fluorescence intensity measurement was established, which could be detected at 37℃ in 45 min, and the sensitivity of the detection could reach 10 copies/μL, a minimum clinical sample with a Ct value of 38.980 can be detected. It has high specificity and no cross-reactivity with Adenovirus 7, Herpes simplex virus type I, Herpes simplex virus type II, Coxsackieviruses A16, Cytomegalovirus, Epstein-Barr virus, Measles virus, Mumps virus, Enterovirus 71, Japanese encephalitis virus genotype 5. It has good stability, and can be successfully detected in low, medium and high concentrations of viral positive plasmids with good consistency. The detection rate of the clinically positive samples was 100%, which was completely consistent with the qPCR test result.Conclusions:RAA isothermal amplification technology combined with CRISPR-CAS12a technology was used to establish an accurate method for the detection of VZV virus, which was highly sensitive, specific, and had low requirements for experimental conditions, and could be completed within 45 min, which could provide strong technical support for the early detection of VZV.

Objective　To establish a dual droplet digital PCR (ddPCR) assay for herpes simplex virus type I (HSV-1) and varicella-zoster virus (VZV). Methods The specific primers and probes were derived based on the conserved regions of HSV-1 and VZV genome. The primer-probe combinations were screened, and the annealing temperatures and primer-probe concentration ratios of the dual-droplet digital PCR reaction were optimized to establish a dual-droplet digital PCR reaction system for HSV-1 and VZV, which was tested for other viruses and validated for clinical samples. The sensitivity, specificity, and reproducibility of the established dual microtiter digital PCR method were analyzed. Results The optimal concentrations of primers and probes for the dual ddPCR detection method of HSV-I and VZV were determined to be 800 nmol/L and 250 nmol/L, respectively, with an optimal annealing temperature of 56 ℃. The correlation coefficient (R2) of the standard curve of the dual ddPCR assay was 0.99, showing a clear linear relationship. The method showed high sensitivity, with the lowest detection limit of herpes simplex virus type I being 2.97 copies/μL, and for VZV being 2.73 copies/μL. The repeatability was high with a small coefficient of variation and stable detection results; the specificity was excellent, and no cross-reaction was found with herpes simplex virus type Ⅱ, Epstein-Barr virus, Adenovirus, Coxsackievirus (CA6/CA10/CA16), Cytomegalovirus, Human Cytomegalovirus, Human enterovirus 71, Japanese Encephalitis virus, West Nile virus, Measles virus, Mumps virus, and human nucleic acids. Conclusions The dual droplet digital PCR assay for herpes simplex virus type I and varicella-zoster virus established in this experiment has strong sensitivity, specificity, and high repeatability, and can provide a solution for rapid quantitative detection of the two viruses in different scenarios.

Objective:This study conducted mosquito-borne viruses RNA screening and analysis of virus evolution characteristics on mosquito specimens collected in 2023 from Hunchun city, Jilin province, China.Methods:Firstly, morphological method were employed for mosquito specimen classification. Then, real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the RNA of six mosquito-borne viruses in the collected mosquitos, i. e., Banna virus (BAV), Kadipiro virus (KDV), Liaoning virus (LNV), Tahyna virus (TAHV), Getah virus (GETV) and Japanese encephalitis virus (JEV). And by sequencing, the viral genome sequence of positive samples was obtained.Results:A total of 5490 mosquito specimens were collected from Hunchun city, Jilin province, included 4400 Aedes vexans (80.15%), 1090 Anopheles sinensis (19.85%). A total of 41 groups were obtained by mixing samples according to the time, location, and mosquito species collected. qRT-PCR result showed that only the Aedes vexans sample with the number JLHC2321 was tested positive for LNV, while the remaining samples were tested negative for the detected viruses. According to the phylogenetic analysis of the segment 10 gene, this LNV strain had the closest genetic relationship with NE9731 and belonged to the type II branch. Meanwhile, the amino acid sequence analysis based on the coding sequence (CDS) in the segment 10 showed that JLHC2321 only had 2 amino acid differential sites with the GII reference strain NE9731.Conclusions:This study detected LNV for the first time in Aedes vexans in Hunchun city, and our result provide important basic data for the monitoring and prevention strategies of mosquito-borne viruses in the region.

Objective:To investigate the species and distribution of mosquito-borne arboviruses in the southeastern part of Gansu province.Methods:In 2023, mosquitoes were collected using ultraviolet lights in Longnan and Tianshui regions of Gansu province. After classification by morphology, about 50 are only 1 batch. RT-qPCR was used to detect the RNA of Japanese encephalitis virus (JEV), Banna virus (BAV), Getah virus (GETV), Culex flavivirus (CxFV) and Tahyna virus (TAHV). The genomes of the positive samples were sequenced and the genetic evolution of the virus genomes were analyzed by bioinformatics software.Results:In total, there were 8 176 mosquitoes from 4 genera and 9 species collected from Longnan and Tianshui from June to August 2023. Culex tritaeniorhynchus was the most common species with 55.80% (4 562/8 176) of the total mosquitoes collected, followed by 16.43% (1 343/8 176) of Culex pipiens pallens. A total of 263 batches of samples were obtained, and the nucleic acid test showed that 1 batch was positive for JEV, 2 batches were positive for BAV, 3 batches were positive for GETV and 25 batches were positive for CxFV. Conclusions:Culex tritaeniorhynchus was the dominant mosquito species in the southeastern part of Gansu province, and local mosquitoes carried a variety of arboviruses.

Objective:To understand the situation of arboviruses carried by mosquito specimens in Daozhen county, Guizhou province.Methods:In July 2023, mosquito specimens were collected in Daozhen county, Guizhou province, using photocatalytic mosquito trap method. Reverse transcription polymerase chain reaction (RT-PCR) technology was used to detect the types of viruses carried by mosquitoes, and phylogenetic trees were constructed using the neighbor-joining method to analyze the phylogenetic position of the detected viruses.Results:A total of 5 313 mosquito specimens belonging to 2 genera and 2 species were collected in Daozhen county, Guizhou province, including 3 953 Culex tritaeniorhynchus, 1 355 Armigeres subalbatus, and 5 other mosquito species. The specimens were divided into 54 batches according to their species for grinding, nucleic acid extraction, and testing. Among them, 6 batches of Cx. tritaeniorhynchus specimens tested positive for flavivirus genus using universal primers, and further identification revealed that 5 batches were positive for Japanese encephalitis virus (JEV) nucleic acid, and 1 batch was positive for Tembusu virus (TMUV) nucleic acid. Phylogenetic analysis showed that the detected JEV strains were all genotype I and fell within the same evolutionary branch; the detected TMUV was located on the third branch of the evolutionary tree, with the main host source of the strains on this branch being mosquitoes. Conclusions:In the mosquito specimens collected in Daozhen county, Guizhou province in 2023, genotype I JEV and TMUV were detected, with TMUV being detected for the first time in mosquitoes in Guizhou province. It is essential to enhance the surveillance of JEV and TMUV to mitigate the potential public health risks they pose.

ObjectiveTo support intelligent clinical decision-making in traditional Chinese medicine（TCM）， this study utilized ontology and knowledge graph construction techniques to achieve the IT application of clinical practice guidelines. MethodBased on the principles of findability， accessibility， interoperability， and reusability （FAIR principles）， this study employed ontology techniques to construct an ontology for TCM clinical practice guidelines and built a knowledge graph using coronary heart disease as an example. Based on the Checklist for Reporting Practice Guidelines in Traditional Chinese Medicine and Recommendation Grading in TCM Clinical Guidelines/Consensus （T/CAS 530—2021），the ontology of TCM clinical practice guidelines was constructed using the seven-step ontology construction method. On this basis，the TCM diagnosis and treatment data from the Guidelines for the Diagnosis and Treatment of Stable Angina Pectoris in Coronary Heart Disease were stored in Neo4j in the form of triples through knowledge extraction，integration，and storage. ResultThe information in the clinical practice guidelines was divided into three categories： onset and prevention information， diagnosis information， and treatment information， and the TCM clinical practice guideline ontology was constructed. A total of 27 concepts related to TCM clinical diagnosis and treatment and 14 data attributes were obtained， and 12 conceptual relationships including hierarchical relationships and object attributes were established. By taking coronary heart disease as an example and the TCM clinical practice guideline ontology as the model layer， the knowledge map of TCM diagnosis and treatment guidelines for stable angina pectoris in coronary heart disease with 276 nodes and 336 relationships was constructed， realizing the visual display and query of the guideline content. ConclusionThe ontology of TCM clinical practice guidelines and the knowledge graph of stable angina pectoris in coronary heart disease constructed by combining the seven-step ontology construction method and Neo4j graph database technology are efficient and flexible，providing an intelligent TCM diagnosis and treatment scheme and promoting the standardization and objectification of TCM diagnosis and treatment.

The West Nile virus (WNV), a mosquito-borne arbovirus, is also a zoonotic pathogen first isolated in the 1930s in Africa, followed by the identification of the prevalence of febrile illness caused by West Nile virus infections. In 1999, the West Nile virus was first introduced into New York City of the United States, and caused the outbreak of viral encephalitis in adults. This marked the first reported outbreak of mass adult viral encephalitis caused by West Nile virus. Subsequently, West Nile virus and its infections in humans and animals spread rapidly throughout the United States, causing a worldwide sensation. West Nile virus is currently considered the most widely distributed emerging mosquito-borne arboviruses worldwide. Humans or animals infected by mosquito bites can develop symptoms such as fever, encephalitis (meningitis), and in rare cases, present with severe pancreatitis, hepatitis, myocarditis, miscarriage, or even death, posing a huge global public health burden. This review introduces China's progress in the isolation and identification of West Nile virus, the prevalence of adult viral encephalitis, and the field surveys and laboratory investigations of the coinfection of West Nile virus and typhoid bacteria, aiming to promote the research work and control and prevention of West Nile virus and its infection in China.