Objective: To learn the population,virus status and viral types of hantavirus（HV）hosts in Tiantai County of Zhejiang Province from 2011 to 2018,and to provide evidence for hemorrhagic fever with renal syndrome（HFRS）control. Methods: Rodents in Tiantai County were captured by night trapping method. After the species and age of rodents were identified,the composition of rodent species,dominant species and density of rodents were analyzed. The lungs and blood of rodents were sampled to detect the antigen and antibody of HV by immunofluorescence method. The HV antigen-positive lung samples were detected by RT-PCR with specific primers of HV S fragment,then HV was isolated and identified by inoculating Vero-E6 cells. Results: The average rodent density in Tiantai County from 2011 to 2018 was 4.44%. The rodent density in the field and residential areas were 4.94% and 2.23%,respectively. Ten species of rodents were identified,with Apodemus agrarius dominant in the field and Rattus norvegicus in the residential areas. Sixty-seven lung samples were HV antigen positive（4.13%）,one from Rattus norvegicus and the other sixty-six from Apodemus agrarius. Seventy-nine blood samples were HV antibody positive（4.86%）,all from Apodemus agrarius. Thirty-four HV antigen-positive lung samples were positive（50.75%）after RT-PCR amplification. Twenty-two strains of virus were isolated and all of them were from Apodemus agrarius,including twenty-one strains of Hantaan type（HTN）and one strain of Seoul type（SEO）. Conclusion In Tiantai County,Apodemus agrarius is the main source of HFRS infection;the main epidemic type of HV is HTN and SEO is first found in Apodemus agrarius.

Objective:The epidemiological and host animal pathogen data of leptospirosis in the population of Zhejiang Province in 2023 were analyzed, providing scientific basis for formulating prevention and control strategies of leptospirosis.Methods:The data of human leptospirosis in the population were collected from the China Information System for Disease Control and Prevention, and analyzed using descriptive epidemiological methods. The data on isolation, culture, and nucleic acid testing of Leptospira pathogens from mouse kidneys, frog kidneys, pig kidneys, and duck kidneys as well as duck serum antibody data were collected from Zhejiang Provincial Center for Disease Control and Prevention "Leptospirosis Surveillance Project of Zhejiang Province". The carrying and changing status of Leptospira epidemic microbiota in populations and host animals were analyzed. Results:In 2023, a total of 83 cases of leptospirosis were reported in Zhejiang Province, with a incidence rate of 0.126 2/ 100 000, aged (62.66 ± 11.31) years, including 68 males and 15 females. Leptospirosis cases were reported in 11 cities, mainly concentrated in the southern cities of Wenzhou City, Lishui City and Taizhou City(a total of 68 cases), accounting for 81.93% of the total number of cases. August to October were high incidence months for leptospirosis (a total of 70 cases), accounting for 84.34% of the total number cases. The male to female ratio of patients was 4.53 ∶ 1.00, and all were adults ≥20 years old, the middle and old people aged 45 - 79 years were the high-risk population (a total of 77 cases), accounting for 92.77% of the total number of cases. The patient's occupation was mainly farmers, with a total of 54 cases, accounting for 65.06% of the total number cases. The shortest time from onset to initial diagnosis for patients with leptospirosis was 0 day, and the longest was 13 days. The shortest time from initial diagnosis to confirmed diagnosis was 0 day, and the longest was 16 days. The 72.29% of the leptospirosis cases (60 cases) had a history of field labor or suspected contact with contaminated water within one month before the onset of the disease, and 18.07% of the leptospirosis cases (15 cases) had a history of contact with animals such as mice, frogs, pigs, cows, dogs, fish or ducks, or their excreta within one month before the onset of the disease. The average nucleic acid positive rate of host animals with leptospirosis was 5.92% (31/524) in mouse kidney, 6.74% (36/534) in frog kidney, and 0.66% (1/151) in pig kidney. The isolation and culture of leptospirosis from duck kidney, nucleic acid detection, and antibody detection in duck blood were all negative. The leptospirosis bacteria detected in human population were serogroup Icterohaemorrhagiae (3 samples) and Hebdomadis (4 samples), and the bacteria group detected in host animals was serogroup Icterohaemorrhagiae (3 samples). Conclusions:The outbreak of leptospirosis in Zhejiang Province mainly occurs in the summer and autumn, with the affected areas mainly concentrated in the southern region of Zhejiang Province. The affected population is mainly middle-aged and elderly males, and the population carrying Leptospira is still mainly composed of the serogroup Icterohaemorrhagiae and the Hebdomadis, with the host animal being the serogroup Icterohaemorrhagiae.

Objective:To identify the phosphodiesterase (PDE) activity of the gene product encoded by LA_RS06960 of Leptospira interrogans ( L. interrogans), and analyze whether dinucleotides that can be degraded by PDE can activate macrophages to express innate immune factors. Methods:The LA_RS06960 gene in L. interrogans strain 56601 was amplified by PCR, and the prokaryotic expression system was constructed for the protein expression. The expressed rPDE was purified by Ni-NTA affinity chromatography. High performance liquid chromatography (HPLC) was used to measure the degration of c-di-AMP or 5′-pApA to AMP by rPDE. Real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to detect the changes in the expression of target genes in Leptospira or THP-1 cells associated with innate immune factors during infection. qRT-PCR and ELISA were used to detect the changes in the expression and secretion level of the innate immune factors in macrophages treated with bacterial dinucleotide. Results:The prokaryotic expression system for LA_RS06960 gene of L. interrogans was constracted successfully, and the purified rPDE could degrate 5′-pApA and c-di-AMP into AMP in vitro. The mRNA level of leptospiral LA_RS06960 gene was significantly down-regulated, while IFN-β, TNF-α, IL-6 and IL-1β encoding genes of macrophages were significantly up-regulated during infection. The mRNA level or the secretion level of IFN-β and IL-6 of macrophages were increased after treated with the bacterial dinucleotide substrate of PDE. Conclusions:PDE encoded by LA_RS06960 gene has phosphodiesterase activity, and the bacterial dinucleotide substrate of the PDE could activate the innate immune response of macrophages.

Objective:To understand the in vitro antimicrobial resistance and resistance phenotypes profile of Streptococcus suis strains isolated from human in Zhejiang Province. Methods:The strains of sporadic Streptococcus suis infections were isolated during 2005 to 2021 in Zhejiang Province, and were subjected to antimicrobial resistance analysis using agar dilution method. Polymerase chain reaction (PCR) technology was also used to detect 70 resistance genes including tetracyclines, macrolides and aminoglycosides. Results:The results of antimicrobial resistance analysis showed that these strains were sensitive to eleven kinds of antimicrobial agents with a sensitivity rate ≥96.8%, including cefepime, cefotaxime, ceftriaxone, chloramphenicol, daptomycin, ertapenem, levofloxacin, linezolid, meropenem, penicillin and vancomycin. These strains were mainly resistant to tetracycline, clindamycin, azithromycin and erythromycin, especially resistant to tetracycline with a rate of 93.5%(29/31). Fourteen strains (45.2%) exhibited multidrug resistant patterns. The PCR analysis of 70 drug resistance genes showed that 14(20.0%) different resistance genes were detected. The highest detection rate of resistant genes came from tetracycline, including tet ( O) gene (58.1%, 18/31), tet ( M) gene (48.4%, 15/31), tet ( 40) gene (35.5%, 11/31), followed by ermB gene (41.9%, 13/31) in the class of macrolide. Fourteen strains (45.2%) with more than three drug resistance genes were detected, of which eight strains (25.8%) detected 10 drug resistance genes. The analysis of antibiotic resistance and resistance phenotypes profile showed that tet ( M)+ ST7 accounted for 35.5%(11/31), tet( O)+ tet( 40)+ ermB+ mef( A)+ mef( A/ E)+ msrD+ Ant( 6)- Ⅰb+ aph( 3′)- Ⅲa+ aadB+ sat4+ ST7 accounted for 25.8%(8/31). Conclusions:The antimicrobial resistance and resistance phenotypes profile of sporadic Streptococcus suis strains isolated from human in Zhejiang Province are endemic, with mainly two types of characteristic genetic cloning of drug resistance genes.

Objective:To investigate the positive rate and isolate Bordetella pertussis in children with suspected whooping cough and their close family members in Zhejiang Province, and further explore the susceptibility and resistant mechanism of Bordetella pertussis to antibiotics. Methods:A total of 273 nasopharynx swabs specimens from children with suspected whooping cough in Hangzhou Children′s Hospital from May 2022 to October 2022 were collected. The strains were isolated and cultured using charcoal select agar plate. Pertussis target genes were detected by RT-PCR. E-test method was used to detect the sensitivity of Bordetella pertussis strains to different antibiotics. The mechanism of resistance of Bordetella pertussis to macrolides was analyzed by whole genome sequencing. The phylogenetic analysis of isolated strains was based on core genome multilocus sequence typing(cgMLST). Results:Among 273 clinical samples of children with suspected pertussis and their close family members, 168 samples were positive by fluorescence quantitative PCR, accounting for 61.54%, and 30 pertussis strains were successfully isolated with a positive rate of 10.98%. In addition, among the 143 samples of close family members, 54.55% (78/143) samples were positive by RT-PCR and 9.79% (14/143) samples were positive by culture, suggesting that the close family member are important in family transmission of pertussis. Besides, most of the positive samples were from mothers. The results of E-test showed that 96.67%(29/30) strains showed high resistance to azithromycin with MIC value>256 mg/L, and the resistant mechanism of azithromycin was A2047G mutation in 23S rRNA. The phylogenetic analysis based on the cgMSLT showed that the isolated strains were clustered into two new different clades.Conclusions:The positive rate of Bordetella pertussis in close family members is at a high level and the mother may be the main source of infection, which is of great significance for monitoring and laboratory detection of suspected children′s family members. Bordetella pertussis shows high resistance to macrolides in Zhejiang Province, so monitoring of the antimicrobial resistance should be further strengthened to provide theoretical basis for clinical treatment and drug guidance.

Objective: By investigating the genotype and evolutionary variation of hantavirus (HV) in Tiantai county, a national surveillance site for hemorrhagic fever with renal syndrome (HFRS) was set in Zhejiang province, from 2011 to 2018, to reveal the molecular epidemiological characteristics of hantavirus (HV) in Tiantai. Methods: Total RNA was extracted from ultrasound treated HV antigen- positive rat lung samples in Tiantai from 2011 to 2018. After cDNA was prepared, nested PCR was used to amplify partial sequence of M fragments by using specific primers of HV. The sequences of HV in Tiantai from 2011 to 2018 were compared with other known HV sequences in order to identify the genotype and analyze the evolution and variation of the virus. Results: In 67 HV antigen-positive lung specimens, 31 were positive in nested PCR amplification with type-specific primers, including 30 Hantaan virus (HTNV) positive samples, 1 Seoul virus (SEOV) positive sample, and all the 31 samples were from Apodemus agrarius. The phylogenetic tree based on partial M segment was divided into monophyletic group, 30 strains were distributed in HTNV group and 1 was in SEOV group. The HTNV strain Tiantai T2018-130 was independently in one branch, sharing 84.8%-87.9% homology with other strains both at home and abroad, including 29 strains in HTNV group in Tiantai. The other 29 HTNV strains in Tiantai showed closer relationship. The SEOV strain T2016-31 from Apodemus agrarius showed closer relationship with previous strains of SEOV, Tiantai ZT71, ZT10 and Z37 strains of Wenzhou, Zhejiang province. Conclusions HTNV, the main genotype of HV in Tiantai of Zhejiang province, showed obvious geographic clustering, but the strain T2018-130 was distinct from the others in Tiantai. Meanwhile, by sequence analysis, we confirmed that The SEOV strain T2016-31 existed in in Apodemus agrarius, indicating there was a phenomenon of "spillover" between virus and host in SEOV evolution.