Objective:To investigate the inhibitory effect of tripartite motif-containing 25 (TRIM25) on the replication of Japanese encephalitis virus (JEV) in cells and its molecular mechanism.Methods:Human glioma cells (U251 cells) and Kunming mice were infected with JEV, and then the cells and brain tissue samples were collected. The transcription levels of six TRIM genes were detected by real-time PCR, and the expression of TRIM25 in cells was detected by Western blot. U251 and A549 cells overexpressed with TRIM25 and U251 cells knocked out with TRIM25 gene were constructed. Cells were infected with JEV, and the replication of JEV was detected by viral plaque assay, real-time PCR and Western blot. The interaction of TRIM25 with viral proteins was investigated by co-immunoprecipitation (Co-IP) and indirect immunofluorescence assay. The expression of IFN-β in overexpressed TRIM25 cells was detected by real-time PCR and ELISA.Results:JEV infection promoted the expression of TRIM25 in cells and mouse brain tissues. TRIM25 overexpression restricted JEV replication in U251 and A549 cells, while TRIM25 knockout enhanced JEV replication. TRIM25 overexpression upregulated the level of IFN-β in cells. TRIM25 interacted with JEV capsid protein and promoted the degradation of capsid protein.Conclusion:TRIM25 can inhibit the replication of JEV in cells by upregulating IFN-β and promoting the degradation of JEV C protein.

Objective:To study the genotype and molecular characteristics of Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) in Shenzhen so as to provide basis for the prevention and control.Methods:The serum samples of HFRS patients at acute stage were collected from hospitals, and viral RNA from the sera was extracted as a template for typing detection by real-time fluorescent PCR. The nucleotide sequences of M fragment (G2 segment) and S fragment were amplified by reverse transcription-nested polymerase chain reaction (RT-nested PCR). The PCR products were then sequenced and homology and phylogenetic tree analysis were conducted.Results:In a total of 123 HV IgM antibody positive serum samples were collected in Shenzhen from 2015 to 2023, including 97 males and 26 females. Most of patients were young and middle-aged men. Cases were found throughout the year, with high incidence in spring and early summer. The result of fluorescence PCR showed that among 92 clinical specimens, 63 cases (68.5%) were tested positive for hantavirus genotypes, 59 cases (93.7% of positives) were identified as SEOV and 4 cases (6.3% of positives) as HTNV. Notably, all HTNV-positive patients were male. Analysis on nucleotide homology and phylogenetic tree showed that the difference in nucleotide sequences among SEO viruses in Shenzhen was small, and most of them were S2 subtype except for two cases of S3 subtype. The mutation rate of HTN viruses was high, with two cases of H5 subtype and two cases of H8 subtype.Conclusions:The major genotype of hantavirus causing HFRS in Shenzhen is SEO genotype, especially S2 subtype, which is closely related to some strains in Guangzhou, and has high homology with L99, Z37 and other vaccine strains. The existence of S3 subtype cases was detected for the first time in Shenzhen, while some cases of HTNV were still imported.

Objective:This study aimed to investigate the regulatory role and mechanism of myosin heavy chain 9 (MYH9) in the invasion of Zika virus (ZIKV) into human glioma cells (U251).Methods:Utilizing CRISPR/Cas9 technology, MYH9-knockout U251 cells (U251-MYH9 KD) were constructed. Following ZIKV infection, the protein expression levels, RNA load, and viral titer of ZIKV were detected through western blot (WB), Real-time fluorescence quantitative polymerase chain reaction (qPCR), and plaque formation assays, respectively. The infection efficiency of ZIKV in U251 cells treated with the MYH9 inhibitor blebbistatin was assessed. The binding and internalization efficiency of ZIKV were measured in U251-MYH9 KD cells. The interaction between MYH9 and the ZIKV envelope protein (E) was studied using co-immunoprecipitation (Co-IP). The effects of soluble MYH9 recombinant protein and anti-human MYH9 antibodies on ZIKV infection were evaluated by qPCR and plaque formation assays. Results:It was found that knockout or inhibition of MYH9 significantly suppressed ZIKV infection in U251 cells. MYH9 knockout notably inhibited the binding and internalization of ZIKV in U251 cells. MYH9 interacted with the ZIKV E protein, and both MYH9 recombinant protein and anti-human MYH9 antibodies, by blocking the binding of ZIKV E protein to cell surface MYH9, inhibited ZIKV infection in U251 cells in a dose-dependent manner.Conclusions:MYH9 facilitates ZIKV invasion into U251 cells through interaction with the ZIKV E protein.

Objective:To investigate the inhibitory effect of tripartite motif-containing 25 (TRIM25) on the replication of Japanese encephalitis virus (JEV) in cells and its molecular mechanism.Methods:Human glioma cells (U251 cells) and Kunming mice were infected with JEV, and then the cells and brain tissue samples were collected. The transcription levels of six TRIM genes were detected by real-time PCR, and the expression of TRIM25 in cells was detected by Western blot. U251 and A549 cells overexpressed with TRIM25 and U251 cells knocked out with TRIM25 gene were constructed. Cells were infected with JEV, and the replication of JEV was detected by viral plaque assay, real-time PCR and Western blot. The interaction of TRIM25 with viral proteins was investigated by co-immunoprecipitation (Co-IP) and indirect immunofluorescence assay. The expression of IFN-β in overexpressed TRIM25 cells was detected by real-time PCR and ELISA.Results:JEV infection promoted the expression of TRIM25 in cells and mouse brain tissues. TRIM25 overexpression restricted JEV replication in U251 and A549 cells, while TRIM25 knockout enhanced JEV replication. TRIM25 overexpression upregulated the level of IFN-β in cells. TRIM25 interacted with JEV capsid protein and promoted the degradation of capsid protein.Conclusion:TRIM25 can inhibit the replication of JEV in cells by upregulating IFN-β and promoting the degradation of JEV C protein.

Objective:To study the genotype and molecular characteristics of Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) in Shenzhen so as to provide basis for the prevention and control.Methods:The serum samples of HFRS patients at acute stage were collected from hospitals, and viral RNA from the sera was extracted as a template for typing detection by real-time fluorescent PCR. The nucleotide sequences of M fragment (G2 segment) and S fragment were amplified by reverse transcription-nested polymerase chain reaction (RT-nested PCR). The PCR products were then sequenced and homology and phylogenetic tree analysis were conducted.Results:In a total of 123 HV IgM antibody positive serum samples were collected in Shenzhen from 2015 to 2023, including 97 males and 26 females. Most of patients were young and middle-aged men. Cases were found throughout the year, with high incidence in spring and early summer. The result of fluorescence PCR showed that among 92 clinical specimens, 63 cases (68.5%) were tested positive for hantavirus genotypes, 59 cases (93.7% of positives) were identified as SEOV and 4 cases (6.3% of positives) as HTNV. Notably, all HTNV-positive patients were male. Analysis on nucleotide homology and phylogenetic tree showed that the difference in nucleotide sequences among SEO viruses in Shenzhen was small, and most of them were S2 subtype except for two cases of S3 subtype. The mutation rate of HTN viruses was high, with two cases of H5 subtype and two cases of H8 subtype.Conclusions:The major genotype of hantavirus causing HFRS in Shenzhen is SEO genotype, especially S2 subtype, which is closely related to some strains in Guangzhou, and has high homology with L99, Z37 and other vaccine strains. The existence of S3 subtype cases was detected for the first time in Shenzhen, while some cases of HTNV were still imported.

Objective:This study aimed to investigate the regulatory role and mechanism of myosin heavy chain 9 (MYH9) in the invasion of Zika virus (ZIKV) into human glioma cells (U251).Methods:Utilizing CRISPR/Cas9 technology, MYH9-knockout U251 cells (U251-MYH9 KD) were constructed. Following ZIKV infection, the protein expression levels, RNA load, and viral titer of ZIKV were detected through western blot (WB), Real-time fluorescence quantitative polymerase chain reaction (qPCR), and plaque formation assays, respectively. The infection efficiency of ZIKV in U251 cells treated with the MYH9 inhibitor blebbistatin was assessed. The binding and internalization efficiency of ZIKV were measured in U251-MYH9 KD cells. The interaction between MYH9 and the ZIKV envelope protein (E) was studied using co-immunoprecipitation (Co-IP). The effects of soluble MYH9 recombinant protein and anti-human MYH9 antibodies on ZIKV infection were evaluated by qPCR and plaque formation assays. Results:It was found that knockout or inhibition of MYH9 significantly suppressed ZIKV infection in U251 cells. MYH9 knockout notably inhibited the binding and internalization of ZIKV in U251 cells. MYH9 interacted with the ZIKV E protein, and both MYH9 recombinant protein and anti-human MYH9 antibodies, by blocking the binding of ZIKV E protein to cell surface MYH9, inhibited ZIKV infection in U251 cells in a dose-dependent manner.Conclusions:MYH9 facilitates ZIKV invasion into U251 cells through interaction with the ZIKV E protein.

Objective:To investigate the necessity of adaptive re-planning during radiotherapy for nasopharyngeal carcinoma (NPC) and its impact on dose improvement.Methods:Clinical data of 89 NPC patients admitted to Sun Yat-sen University Cancer Center from July 2014 to December 2017 were retrospectively analyzed. All patients received 25+7 rounds of adaptive re-planning during radiotherapy. Plan-A was defined as the initial CT scan-based 25-fraction radiotherapy plan, while plan-B was defined as the re-planned 7-fraction radiotherapy plan based on a subsequent CT scan. The changes in the target and parotid gland volumes were compared between plan-A and plan-B. Plan-I was a one-time simulation of plan-A extended to 32 fraction radiotherapy plan, and plan-II was generated through registration and fusion of the plan-A and plan-B for adaptive re-planning. The differences in dose metrics, homogeneity index (HI), conformity index (CI), and dose to organs at risk (OAR) were compared between plan-I and plan-II. Statistical analysis was performed by using paired t-test. Results:Compared with plan-A, the gross tumor volume of massive bleeding lesions (GTV nx) and parotid gland volume of plan-B were decreased by 13.14% and 11.12%, respectively (both P<0.001). While planning clinical target volume of metastatic lymph nodes (PCTV nd) of plan-B was increased by 7.75%( P<0.001). There were significant changes in the lymph nodes of plan-A and plan-B. The D mean, D 5%, D 95% of massive bleeding lesions planning target volume (PTV nx) and D 5% of high risk planning target volume (PTV1) in plan-II were all significantly higher than those in plan-I (all P<0.05). The CI of PTV nx and PTV1 in plan-II was closer to 1 than that in plan-I. In all assessed OAR, the D mean, D 50%, and D max of plan-II were significantly lower than those of plan-I (all P<0.05). Conclusions:During radiotherapy, NPC patients may experience varying degrees of primary tumor shrinkage, parotid gland atrophy, and lymph node changes. It is necessary to deliver re-planning and significantly improve the dose of target areas and OAR.

Objective:To understand the epidemiological characteristics of human respiratory syncytial virus (HRSV) among cases presenting with influenza-like illness (ILI) in Shenzhen City from 2019 to 2023.Methods:Respiratory specimens were collected from two national sentinel hospitals in Shenzhen from March 2019 to December 2023, specifically targeting cases of ILI. The real-time PCR method was used for the detection and genotyping of HRSV. Basic demographic information was collected and used for the epidemiological analysis.Results:A total of 9 278 respiratory specimens of influenza-like cases were collected and detected, with a total positive rate of 4.77% (443/9 278) for HRSV. In 2021 (8.48%, 167/1 970), the positive rate of HRSV was significantly higher than in 2019 (3.35%, 52/1 552), 2022 (1.80%, 39/2 169), and 2023 (4.49%, 133/2 960), and the difference was statistically significant ( χ 2=102.395, P<0.001). The prevalence of HRSV was mainly in summer and early autumn (September), and there was an abnormal increase in the positive rate of HRSV in winter 2022. The highest positive rate of HRSV was in children under five years old (9.84%, 330/335). The typing results showed that in 2022, the prevalence of HRSV-A was predominant (71.79%, 28/39), and in 2023, HRSV-A and HRSV-B subtypes coexisted. Conclusions:The prevalence of HRSV in Shenzhen from 2019 to 2023 has obvious seasonality, mainly in summer and early autumn. Children under five years old are the main population of HRSV infections.

Objective:To understand the epidemiological characteristics of human respiratory syncytial virus (HRSV) among cases presenting with influenza-like illness (ILI) in Shenzhen City from 2019 to 2023.Methods:Respiratory specimens were collected from two national sentinel hospitals in Shenzhen from March 2019 to December 2023, specifically targeting cases of ILI. The real-time PCR method was used for the detection and genotyping of HRSV. Basic demographic information was collected and used for the epidemiological analysis.Results:A total of 9 278 respiratory specimens of influenza-like cases were collected and detected, with a total positive rate of 4.77% (443/9 278) for HRSV. In 2021 (8.48%, 167/1 970), the positive rate of HRSV was significantly higher than in 2019 (3.35%, 52/1 552), 2022 (1.80%, 39/2 169), and 2023 (4.49%, 133/2 960), and the difference was statistically significant ( χ 2=102.395, P<0.001). The prevalence of HRSV was mainly in summer and early autumn (September), and there was an abnormal increase in the positive rate of HRSV in winter 2022. The highest positive rate of HRSV was in children under five years old (9.84%, 330/335). The typing results showed that in 2022, the prevalence of HRSV-A was predominant (71.79%, 28/39), and in 2023, HRSV-A and HRSV-B subtypes coexisted. Conclusions:The prevalence of HRSV in Shenzhen from 2019 to 2023 has obvious seasonality, mainly in summer and early autumn. Children under five years old are the main population of HRSV infections.

Objective:To quantify the associations between periconceptional maternal homocysteine (HCY) and offspring′s birth weight and risk of small for gestational age (SGA) infant.Methods:The 19 984 mother-child pairs in this prospective cohort study were recruited from the Shanghai preconception cohort; the infants were delivered from 1 st September 2016 to 11 th November 2022. A standardized questionnaire was used to collect the mothers′ demographic information, medical history, dietary supplement use, and maternal complications during pregnancy, and their serum samples were collected. Serum HCY, folate, and vitamin B 12 were measured using chemiluminescent microparticle immunoassay based on serum sample drawn at enrollment. Birth weight data were obtained from medical records. Multiple imputation methods were applied to handle missing data in key variables. Multivariable linear regression and Poisson regression models were used to analyze the relationship between maternal HCY concentration during the periconceptional period and the birth weight and SGA risk of the offspring. Results:A total of 9 452 pairs were enrolled preconceptionally and the remaining 10 532 pairs were enrolled in early pregnancy. The proportion of mothers whose pregnancy age was greater than 35 years was 9.2% (1 832/19 984), the proportion of primiparous women was 76.5% (15 283/19 984), the proportion of pre-pregnancy overweight and obesity was 14.0% (2 804/19 984), the proportion of using folic acid supplements before pregnancy was 21.4% (4 272/19 984), and the proportion of those who supplemented with folic acid during early pregnancy was 85.2% (8 976/10 532); gestational diabetes mellitus was in 6.2% (1 245/19 984), gestational hypertensive syndrome in 3.6% (711/19 984). The birth weight of the offspring was (3 297±468) g, and there were 1 962 SGA children (9.8%). The HCY concentration in the overall population in appropriate for gestational age during the periconceptional period was (7.9±3.2) μmol/L, with (8.3±3.7) μmol/L in the preconception subgroup and (7.3±2.4) μmol/L in the early pregnancy subgroup. After adjustment for the covariates of perinatal demographic information, adverse pregnancy outcomes, serum folate and vitamin B 12, increased maternal periconceptional HCY was significantly associated with lower offspring birth weight ( β=-2.30, 95% CI -4.43--0.16, P=0.035). Only the early pregnancy subgroup was significantly associated with lower offspring birth weight ( β=-7.39, 95% CI-11.50--3.21, P<0.001). No association was found between peripregnancy HCY and offspring SGA risk. However, elevated HCY in early pregnancy was associated with an increased risk of SGA in the offspring ( RR=1.05, 95% CI 1.01-1.08, P=0.002). Periconceptional vitamin B 12 was a mediator of the association between HCY and offspring birth weight, accounting for 16.5%, 41.2% and 5.4% of its total effect in the overall periconceptional population, the pre-pregnancy subgroup and the early pregnancy subgroup, respectively. Conclusions:Maternal periconceptional HCY level is associated with lower birth weight in offspring, but not with the risk of SGA. Elevated maternal HCY in early pregnancy subgroup may be associated with increased risk of SGA in offspring.