Objective: To investigate the molecular transmission network characteristics of HIV-1 among men who have sex with men (MSM) in Shaoxing City, Zhejiang Province, so as to provide insights into AIDS prevention and control among MSM. Methods: Newly reported HIV/AIDS cases among MSM in Shaoxing City were selected from January 2021 to September 2023. Plasma samples before the antiviral treatment were collected. The HIV-1 pol gene was amplified using reverse transcription PCR and nested PCR to construct phylogenetic trees for gene subtype analysis. The HIV-TRACE method was used to construct a molecular transmission network with a genetic distance of 1.5% to analyze clustering and the characteristics of cases within molecular clusters. Results: A total of 216 HIV/AIDS cases among MSM were included, and 179 qualified sequences were obtained. The predominant HIV-1 subtypes were CRF07_BC and CRF01_AE, with 95 and 66 cases, respectively. At 1.5% genetic distance, 20 molecular clusters were formed, with 61 nodes and 58 edges. A total of 61 sequences were connected to the transmission network (34.08%). HIV/AIDS cases among MSM from all the counties (cities, districts) in Shaoxing City were included in the network. There was the largest molecular cluster with ≥10 nodes, involving 12 cases from five counties (cities, districts), 3 medium-sized molecular clusters with 4 to 5 nodes, and the 16 small-sized molecular clusters with 2 or 3 nodes. Seven cases with high risk of transmission, each with ≥4 edges, were all CRF07_BC subtypes. Among them, two cases were from the large molecular cluster, and five cases were from the same molecular cluster composed of cases from Shengzhou City and Xinchang County. Conclusions The predominant HIV-1 subtypes among MSM in Shaoxing City were CRF07_BC and CRF01_AE. There was cross-regional HIV transmission, and potential transmission risk might exist in Shengzhou City and Xinchang County.

Objective:To screen host cell proteins interacting with rotavirus nonstructural protein 2(NSP2) and provide a theoretical basis for the discovery of antiviral targets.Methods:E. coli BL21(DE3) was transformed with a recombinant plasmid NSP2-pGEX-6P-1 and induced by IPTG to express NSP2-GST. GST affinity chromatography was used to purify NSP2-GST and Western blot was performed for verification. NSP2-GST and GST proteins were used as targets to capture interacting proteins in MA104 cells via GST pull-down. After silver staining, differential proteins were screened by in-gel enzymatic hydrolysis and mass spectrometry. Protein pilot platform was used to filter peptides, and the names and the biological functions of the proteins were obtained by Paragon algorithm. The potential connection between the interacting proteins was demonstrated by protein interaction network diagrams and GO functional annotation analysis. Moreover, the molecular docking of the top three proteins with NPS2 was predicted using HDOCK server and verified by docking and confidence scores, so as to investigate the visual docking model between interacting proteins. Results:SDS-PAGE and Western blot showed the successful purification of the recombinant protein NSP2. Ten host proteins including PKM2, which might interact with NSP2, were identified by GST pull-down and protein profiling. GO analysis and interaction diagrams revealed that RPS4X, EZR, SUPT16H and EIF2S3 mediated molecular expression; PKM2, LDHA and ATP5A1 participated in energy metabolism; HSP90, ACTB and ANXA2 were involved in biological movement. Besides, there were functional connections and interaction networks among them. Molecular docking further verified PKM2, HSP90 and RPS4X did interact with NSP2, and the interaction force were strong enough to form a stable structure.Conclusions:This study successfully discovered several host proteins including PKM2, HSP90 and RPS4X that could interact with NSP2, providing reference for investigating the process of rotavirus infection and making related prevention and control strategies.

OBJECTIVE: To explore the serological feature and molecular mechanism for a case with A307 subgroup of the ABO blood group system. METHODS: Serological assay was carried out to determine the ABO blood group of the proband and his family members. Genotypes for exons 1 to 7 of the ABO gene were determined with sequence-specific primer polymerase chain reaction (SSP-PCR) and direct sequencing. The impact of the variant on the stability of alpha-1,3-N-acetylgalactosaminyltransferase (GTA) was predicted through construction of a 3D molecular model. RESULTS: The proband, his brother and daughter were diagnosed with Aend phenotype by serological analysis. Their ABO genotype was determined as A307/O02, with heterozygous c.467C>T (p.P156L) and c.745C>T (p.R249W) variants identified in exon 7 of the ABO gene. Molecular modeling suggested that the p.R249W variant may alter the number of hydrogen bonds between the amino acids. The protein was predicted to have a decreased Δ Δ G value of thermodynamic stability. CONCLUSION The p.R249W variant may give rise to the A307 subgroup by reducing the stability of the GTA enzyme, leading to serological features of Aend phenotype.

Objective:To investigate the characteristics of subtype diversity and transmission on HIV-1 among 12 to 30 years old student MSM in Zhejiang province.Methods:A total of 290 newly diagnosed HIV infected student MSM were selected as the research objects for molecular studies on HIV, in Zhejiang province during 2013 to 2015. Data on epidemiology and plasma samples of these people were collected. HIV-1 nucleotide sequences of pol gene regions were amplified using the RT-PCR/nested PCR method and sequenced. Phylogenetic analysis was performed to determine the HIV-1 genotypes. Characteristics of transmission mode among these cases were also analyzed. Results:A total of 290 cases, 50.3 % were diagnosed in Hangzhou and 81.0 % had college or above degrees. 178 sequences including 10 subtypes, were obtained, with the main subtypes as CRF01_AE (49.4 %, 88/178) and CRF07_BC (39.3 %, 70/178). A total of 18 molecular transmission clusters were formed (42 cases, cluster size from 2 to 4), with the proportions of clusters as 23.6 % (42/178). 61.9 % (26/42) of student MSM with their schools located in the same district within the transmission clusters. Their sexual partners would include both student MSM and non-student MSM. The proportion of clusters among middle school students was 38.2 % (13/34), higher than that of college students (20.1 %, 29/144) ( χ2=4.996, P<0.05). Conclusions:The HIV-1 subtypes of student MSM in Zhejiang province appeared diversity, which indicated with the diversity of sources of infection. The geographical distribution of cluster cases is relatively centralized. In order to effectively control the spread of AIDS, more attention should be paid to the sexual partners involved and to specific programs on intervention.

Objective:To evaluate the applicability of limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA, LAg for short) in determining the new HIV-1 infection status of MSM population with seroconversion and make correlation analysis of other biological indicators.Methods:The 15 cases with HIV seroconversion were found in a MSM observation cohort for calculating the HIV prevalence in Zhejiang.The subjects were conducted epidemiological investigation and sampled.The interval of infection time was estimated according to the exposure history and the time of HIV-positive confirmation.LAg, immunoblotting, CD4 cell counting and viral load test were applied in the testing of the related blood samples. McNermar test was conducted for consistency of the two methods.Results:Of 15 cases, the average age was (31.5±8.0) years old, ranging from 24 to 57 years old. The interval of infection time ranged from 40 days to 366 days, and the median was 134 days, with inter-quartile range from 89 to 180 days. A total of 7 cases were classified as new HIV-1 infection by LAg, and 8 cases were classified as chronic infection.The consistent rate was high to 86.67%, and kappa value was 0.73.The samples lacking at least two bands in p31, p51, p66 and gp120 by immunoblotting were determined as recent infection, of which the new infection proportion was significantly higher than that of other samples ( P=0.029).There was no statistical difference in the distribution of CD4 counts ( P=0.533) and viral loads ( P=0.467) between the new infection and chronic infection groups that divided by LAg. Conclusion:By combining with exposure history, the limiting antigen avidity enzyme immunoassay can be used to estimate the new HIV-1 infection.The other biological indicators such as immunoblotting bands, CD4 cell counts and viral loads, can be used as accessory indicators in evaluating the status of new HIV-1 infection.

Objective:To evaluate the applicability of limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA, LAg for short) in determining the new HIV-1 infection status of MSM population with seroconversion and make correlation analysis of other biological indicators.Methods:The 15 cases with HIV seroconversion were found in a MSM observation cohort for calculating the HIV prevalence in Zhejiang.The subjects were conducted epidemiological investigation and sampled.The interval of infection time was estimated according to the exposure history and the time of HIV-positive confirmation.LAg, immunoblotting, CD4 cell counting and viral load test were applied in the testing of the related blood samples. McNermar test was conducted for consistency of the two methods.Results:Of 15 cases, the average age was (31.5±8.0) years old, ranging from 24 to 57 years old. The interval of infection time ranged from 40 days to 366 days, and the median was 134 days, with inter-quartile range from 89 to 180 days. A total of 7 cases were classified as new HIV-1 infection by LAg, and 8 cases were classified as chronic infection.The consistent rate was high to 86.67%, and kappa value was 0.73.The samples lacking at least two bands in p31, p51, p66 and gp120 by immunoblotting were determined as recent infection, of which the new infection proportion was significantly higher than that of other samples ( P=0.029).There was no statistical difference in the distribution of CD4 counts ( P=0.533) and viral loads ( P=0.467) between the new infection and chronic infection groups that divided by LAg. Conclusion:By combining with exposure history, the limiting antigen avidity enzyme immunoassay can be used to estimate the new HIV-1 infection.The other biological indicators such as immunoblotting bands, CD4 cell counts and viral loads, can be used as accessory indicators in evaluating the status of new HIV-1 infection.

Objective: To observe the clinical efficacy of inverted internal limiting membrane (ILM) flap technique with autologous blood for myopic macular hole. Methods: A retrospective, case-controlled study. Twenty-nine eyes of 29 patients with myopic macular hole who received treatment in Eye Hospital of Wenzhou Medical University from January 2017 to May 2019 were enrolled in this study. There were 5 males(5 eyes) and 24 females (24 eyes), with an average age of 55.28±11.40 years. According to the surgical method, the patients were divided into inverted ILM flap group (12 eyes) and ILM peeling group (17 eyes). All patients underwent BCVA, spectral domain OCT and axial length measurement before surgery. There was no significant difference between the two groups in age, gender, course of disease, hole diameter, BCVA and other baseline data. Follow-up was over 4 months after surgery. The BCVA, macular hole closure and continuity of outer retina after surgery were observed. Results: In ILM peeling group, 11 eyes were closed (64.7%) and 6 eyes were not closed at 3 months after surgery. In ILM flap group, 12 eyes were closed (100.0%). The difference of closure rate between two groups was statistically significant (χ²=5.34, P=0.028). The BCVA of inverted ILM flap group was significantly improved at 1, 3 months after surgery compared with preoperative measurements, and the difference was statistically significant (F=3.813, 4.667; P=0.003, 0.001). The BCVA of ILM peeling group was improved at 1 month after surgery, but the difference was not statistically significant (F=1.556, P=0.139). And the BCVA was significantly improved at 3 month after surgery compared with preoperative measurements, and the difference was statistically significant (F=2.453, P=0.026). But there was no significant difference in BCVA between the two groups at 1 and 3 months after surgery (F=0.647, 0.535; P=0.551, 0.612). There was no significant difference in the recovery of outer structure (ELM and EZ continuity) between the two groups at 3 month after surgery (F=0.008, P=0.631). Conclusions Inverted ILM flap technique with autologous blood is a safe and effective method to treat myopic macular hole. The closure rate of the hole can be improved significantly.

OBJECTIVETo explore the molecular mechanism of a case of ABO discrepancies based on the results of blood group serology.

METHODSFive cases of the two-generation pedigrees were analyzed. ABO genotypes were determined using serological tests. DNA sequence analysis was performed on exon 6, exon 7 and intron 3 of the 5 cases to confirm the genotypes of a proband with B subgroup and 4 family members.

RESULTSThere were 3 cases of subgroup AB3 and 1 case of subgroup B3 among the 5 family members. The genotypes were identified as A102/B303 and O02/B303, respectively. B303 differed from B101 by intron 3 point mutation (intron3 + 5G>A).

CONCLUSIONThe point mutation of intron 3 (intron 3+5G>A) is specific in B303.

Objective: To understand the molecular subtypes and recombinant structure of HIV-1 infection in Zhejiang Province. Methods: According to the method of stratified random sampling method, a total of 302 newly HIV infected people (excluding AIDS patients) were selected as the subjects of the investigation from April to June, 2015, who were all newly HIV positive patients reported in Zhejiang Province and were diagnosed as HIV positive and had not received antiretroviral treatment. Questionnaires were used to collect information such as gender, residency, marital status, route of transmission and case reporting area. At least 8 ml of venous blood was collected from HIV infected people, reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR were used to amplify the pol gene sequence of HIV-1 strains and circulating recombination forms (CRF) and unique recombinant form (URF) strains were screened using phylogenetic tree, and molecular subtype analysis and recombinant analysis were performed. Results: Of the 302 HIV infected people, 18 cases were infected with new CRFs or URFs, among which 15 were male (83.3%), 3 were heterosexual transmission (17.7%); 8 cases (44.4%) were infected with new CRFs, which included CRF55_01B 5(27.8%), CRF67_01B 2 (11.1%), CRF69_01B 1 (5.6%); 10 cases (55.6%) were infected with URFs, in which 6 cases were infected with two groups of recombinant strains composed of CRF01_AE subtype and CRF07_BC subtypes in pol region, 3 cases were infected with three groups of recombinant strains of the two subtypes in pol region, and 1 case could not be accurately determined of the recombinant structure. The results of recombinant source analysis showed that the newly recombinant strains found from Zhejiang province were highly homologous to the strains from Guangdong, Anhui, Hebei, Yunnan, Liaoning province, and the sequence similarity was 94.4%-98.0%. Conclusion The new recombinant strains of Zhejiang province included CRFs strains and URFs strains with CRF01_AE and CRF07_BC as the main recombinant structure mode, and all of them were introduced from other places and formed a trans-regional chain of HIV transmission.

Object: To explore the electrocardiographic characteristics of ventricular arrhythmias (VAs) originating from tricuspid annulus region. Methods: Present study included 169 consecutive patients undergoing catheter ablation of VAs from tricuspid annulus origin in our department from August 2007 to September 2016. Based on the origin sites, the patients were divided into two subgroups, the free wall group (81 cases) and septal wall group (88 cases). Based on the location, patients in the free wall group were classified into anterolateral (22 cases), lateral (26 cases) and posterolateral (33 cases) subgroups. Patients in the septal group were classified into anteroseptal (10 cases), midseptal (71 cases) and posteroseptal (7 cases) subgroups. We analyzed the electrocardiographic features of these patients and in 87 patients with PVCs/VT originating from right ventricular outflow tract. Results: (1) A positive R wave inⅠ, aVL, V₅-V₆ leads were found among most of patients, only few cases originating from tricuspid annulus anteroseptum group and tricuspid annulus anterolateral group demonstrated qr or qs pattern in aVL lead. 97.53% (79/81) patients demonstrated rS pattern in V₁-V₃ leads with VAs originating from tricuspid annulus free wall, and 9/10 patients demonstrated rS pattern in V₁ lead with VAs originating from anteroseptum, and 97.44% (76/78) patients demonstrated QS pattern in V₁ lead with VAs originating from midseptum and posteroseptum. Precordial lead transition zone was on or behind V₃ for tricuspid annulus free wall group (96.3%, 78/81), but in front of V₃ for tricuspid annulus septum wall group (47.73%, 42/88) (P<0.01). The S wave's amplitude smaller than-1.81 mV in lead V₂ can be used as a cutoff value to identify if PVC/VT is originating from free wall or septum of TA. R wave in inferior wall leads was found among 98.85% (86/87) patients with PVCs/VT originating from right ventricular outflow tract. Conclusion A positive R wave in Ⅰ, aVL, V₅-V₆ leads was found among most of patients with idiopathic ventricular arrhythmias originating from the tricuspid annulus regions, but VAs originating from different portions of tricuspid annulus area have distinct electrocardiographic characteristics.