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 To compare the effects of angular and unilateral vertebroplasty on spinal compression fractures in the elderly.Methods A total of 122 elderly patients with spinal compression fractures admitted to the Department of Orthopedics,Handan First Hospital from January 2018 to January 2021 were selected as the research subjects.They were divided into a control group and an observation group according to the surgical method,with 61 patients in each group.Patients in the control group were treated with unilateral vertebroplasty,while patients in the observation group were treated with angular vertebroplasty.The operation time,intraoperative blood loss and bone cement injection volume,bone cement distribution,bone cement leakage,spinal-pelvic parameters,visual analogue score(VAS),Barthel index score,and the occurrence of complications were compared between the two groups.Results There was no statistically significant different in the operation time,intraoperative blood loss and bone cement injection volume between the two groups(P＞0.05).The proportions of grade Ⅰ,grade Ⅱ,grade Ⅲ and grade Ⅳ bone cement distribution also showed no significant difference between the two groups(P＞0.05).The total proportion of grade Ⅰ and grade Ⅱ bone cement distribution in the observation group was significantly higher than that in the control group(P＜0.05).The incidence of bone cement leakage in the observation group was significantly lower than that in the control group(P＜0.05).Before operation,there was no statistically significant difference in sagittal balance,thoracic kyphosis angle,pel vic tilt angle,lumbar lordosis angle,and sacral slope angle between the two groups(P＞0.05).After operation,the sagittal balance,thoracic kyphosis angle and pelvic tilt angle significantly decreased in the two groups compared to before operation,and the sagittal balance,thoracic kyphosis angle and pelvic tilt angle of patients in the observation group were significantly smaller than those in the control group(P＜0.05);the lumbar lordosis angle and sacral slope angle significantly increased in the two groups compared to before operation,and the lumbar lordosis angle and sacral slope angle of patients in the observation group were significantly greater than those in the control group(P＜0.05).Before operation,there was no statistically significant difference in the VAS score and Barthel index between the two groups(P＞0.05).Three months after operation,the VAS score of patients in the two groups decreased,while the Barthel index increased compared to before operation(P＜0.05);the Barthel index of patients in the observation group was significantly higher than that in the control group(P＜0.05),while the VAS score showed no significant difference between the two groups(P＞0.05).During treatment,1 case of extraspinal hematoma occurred in the observation group,and the complication rate was 1.64％(1/61);in the control group,2 cases of extraspinal hematoma and 1 case of incision infection occurred,and the complication rate was 4.92％(3/61).There was no statistically significant difference in the incidence of complications between the two groups(x2=1.034,P=0.309).Conclusion Compared with unilateral vertebroplasty,angular vertebroplasty has better effect of bone cement distribution in treating elderly patients with spinal compression fractures,and it can improve the spiral balance and enhance the postoperative quality of life.

Objective Genotypes(G)1,3,and 5 of the Japanese encephalitis virus(JEV)have been isolated in China,but the dominant genotype circulating in Chinese coastal areas remains unknown.We searched for G5 JEV-infected cases and attempted to elucidate which JEV genotype was most closely related to human Japanese encephalitis(JE)in the coastal provinces of China. Methods In this study,we collected serum specimens from patients with JE in three coastal provinces of China(Guangdong,Zhejiang,and Shandong)from 2018 to 2020 and conducted JEV cross-neutralization tests against G1,G3,and G5. Results Acute serum specimens from clinically reported JE cases were obtained for laboratory confirmation from hospitals in Shandong(92 patients),Zhejiang(192 patients),and Guangdong(77 patients),China,from 2018 to 2020.Seventy of the 361 serum specimens were laboratory-confirmed to be infected with JEV.Two cases were confirmed to be infected with G1 JEV,32 with G3 JEV,and two with G5 JEV. Conclusion G3 was the primary infection genotype among JE cases with a definite infection genotype,and the infection caused by G5 JEV was confirmed serologically in China.

Objective:To explore the relationship between the expressions of P21,P27 and proliferating cell nuclear antigen(PCNA)in glomerular mesangial tissue and poor renal prognosis in patients with immunoglobulin A(IgA)nephropathy.Methods:A total of 145 patients with IgA nephropathy treated in Xiaogan Central Hospital from April 2017 to August 2019 were selected as the research object.The expressions of P21,P27 and PCNA in glomerular mesangial tissue were detected by immunohistochemistry.All patients were followed up for 24 months,and the prognosis were counted.The expressions of P21,P27 and PCNA in glomerular mesangial tissue of patients with different prognosis were compared and the influencing factors of poor prognosis in patients with IgA nephropathy were analyzed by Logistic regression analysis.Results:The expression rates of P21,P27 and PCNA positive cells in glomerular mesangial tissue of patients with IgA nephropathy were(38.69±6.83)%,(55.94±8.08)%,(33.47±5.72)%,respectively.The incidence rete of poor prognosis in patients with IgA nephropathy was 17.24%,and the expression rates of P21 and PCNA positive cells in glomerular mesangial tissue of patients with poor prognosis were higher than those in good prognosis group(P<0.05),while the expression rate of P27 positive cells was lower than that in good prognosis group(P<0.05).Logistic multiple regression analysis showed that elevated diastolic blood pressure,increased 24 h proteinuria,mesangial cell proliferation,segmental glomerulosclerosis,renal tubular atrophy/interstitial fibrosis,crescentic body,increased expression rates of P21 and PCNA positive cells and decreased expression rate of P27 positive cells were all risk factors affecting the poor prognosis of patients with IgA nephropathy(P<0.05).Conclusion:There are positive expressions of P21,P27 and PCNA in glomerular mesangial tissue of IgA nephropathy.The expression rates of P21 and PCNA positive cells in glomerular mesangial tissue of of patients with poor prognosis of IgA nephropathy are higher than those with good prognosis,while the expression rate of P27 protein positive cells is lower than those with good prognosis,which are risk factors for poor prognosis of patients with IgA nephropathy.

Objective:To analyze the growth characteristics of different genotypes of Japanese encephalitis virus (JEV) in different cell lines, and to provide scientific basis for the selection of cell lines in the study of JEV.Methods:BHK-21, Vero, C6/36, PK-15, DF-1, N2a, SH-sy5y and MDCK cell lines were selected. The proliferation ability of genotype 1 (NX1889 strain), genotype 3 (P3 strain) and genotype 5 (XZ0934 strain) JEV in these cell lines was evaluated by plaque assay and RT-qPCR.Results:Significant cytopathogenic effects (CPE) were observed in BHK-21, Vero, C6/36, DF-1, N2a and PK-15 cell lines across all three JEV genotypes. However, no significant differences in CPE characteristics were observed within the same cell line. SH-sy5y and MDCK cell lines did not show significant CPE, but virus proliferation was detected in SH-sy5y cell line, while MDCK cell line were found to be insensitive to JEV. No significant difference was observed in the proliferation curves of G1, G3 and G5 JEV in BHK-21, Vero and SH-sy5y cell lines. In C6/36 and PK-15 cell lines, the titer of G1 JEV was higher than that of G3 and G5. In DF-1 cell line, G5 demonstrated a higher titer than the other two genotypes, whereas in N2a cell line, G5 showed a lower titer than the other two.Conclusions:There are differences in the proliferation of three different genotypes of JEV in different cell lines, which can provide reference for the study of JEV in different directions.

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.