[Genetic characteristics of hantavirus from rodents in port area of Ningde , Fujian province in the summer of 2020].

Objective: To explore the genetic characteristics and evolution of hantavirus carried by rodents in port area of Ningde in Fujian province in the summer of 2020. Methods: Rodents were captured in the port area of Ningde, the RNA was extracted from rodent lung tissues and detected by using specific kit. The positive samples were used for whole-genome sequencing of the virus. Bioinformatics software was used for the analysis on the similarity and genetic variation of the sequences. Results: A total of 112 rodents were captured, including 5 Rattus norvegicus and 2 Rattus flavipectus, the positive rate of hantavirus was 6.25% (7/112). By virus gene sequencing, two hantavirus complete genome sequences were obtained (named as FJ35 and FJ36, GenBank accession numbers: MW449188-MW449193). The genetic analysis results showed that the hantavirus detected in positive samples were SEOV and shared 99% nucleotide similarity with hantavirus strains LZSF21 and JX20140581 isolated from Shandong province. Phylogenetic analysis using the maximum likelihood method showed that the hantavirus detected in positive samples belonged to S3 subtype, sharing the same subtype with hantavirus strains Z37 from Zhejiang province, LZSF21 from Shandong province, and zy27 and Gongzhuling 415 from northeastern China. Compared with FJ372, the amino acid variation of N259S was observed at sites 251-264 of nucleoprotein, which might be related to antigenicity. Another variation of Q81R was observed in glycoprotein compared with SEOV 80-39 segment of coded amino acid of international reference strain, which might also cause the change in antigenicity. Conclusion: The high positive rate of hantavirus in rodents in the port area of Ningde- would increase the risk of natural human infection and epidemic in local area. The hantavirus positive rodents in this focus might be from an endemic area in Shandong. It is necessary to strengthen the imported rodent control in the port area of Ningde. The virus detected in 2 positive samples belonged to SEOV subtype Ⅲ and shared high homologies of nucleotides and amino acid sequences with the hantavirus strains in surrounding area. However, some slight variations occurred in glycoprotein and nucleoprotein amino acid sequences, which might cause changes in its antigeniity.

[Genotype and evolution of hantavirus in Tiantai of Zhejiang province, 2011-2018].

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.

[Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody].

Objective: S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology. The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum. Methods: Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z10S-TN was constructed by using the routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP. WB assay was established to detect the serum samples from 95 confirmed HFRS patients. Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method. Results: rBAC-Z10S-TN was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV IgG could efficiently recognize rNP and hybridize with the recombinant protein. 97.67% of the serum samples from the HFRS patients were positive confirmed by WB. Conclusions: We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10. WB assay which was established in this study could be used as a new serological test for HFRS diagnosis, thanks to the simplicity, safety, sensitivity and specificity of this method.

Biological monitoring of deltamethrin in sprayers by HPLC method.

Using HPLC, the authors had investigated the three metabolites of deltamethrin (DM) in the urine of spraymen and one suicide, namely: dibromovinyl-dimethylcyclopropane carboxylic acid (Br2A), 3-phenoxybenzyl-hydroxy-ethyl acetate (PHE) and 3-phenoxyl-benzoic acid (BA). Br2A was chosen as the biological monitoring parameter for DM exposed people, and the urine samples of one suicide and 11 farmers sprayed DM or DM plus methamidophos were examined for Br2A quantitatively which was detected in 8 of 11 sprayers and in the suicide case.

An epidemiological study on occupational acute pyrethroid poisoning in cotton farmers.

A cross sectional survey on the prevalence of acute pyrethroid poisoning in cotton farmers was conducted in 1987 and 1988. A total of 3113 pyrethroid spraymen (2230 men (71.6%) and 883 women (28.4%] were interviewed after spraying and followed up for 72 hours. Adverse effects of pyrethroid exposure were found in 834 of them (26.8%) manifested as abnormal facial sensations, dizziness, headache, fatigue, nausea, or loss of appetite. Only 10 subjects, who developed significant systemic symptoms and had signs of listlessness or muscular fasciculation, were diagnosed as having mild occupational acute pyrethroid poisoning with a prevalence of 0.31% in subjects exposed to pure pyrethroids and 0.38% in subjects exposed to pyrethroid organophosphate mixtures. Measurements of pyrethroid concentrations in the air of the breathing zone, in skin pads, and in urine samples showed that dermal contamination is the main route of exposure to pyrethroids in cotton growers. Preventive measures are recommended.

Changes of nerve excitability and urinary deltamethrin in sprayers.

The nerve excitability of median nerve as well as the urinary deltamethrin and its metabolite dibromovinyl-dimethyl-cyclopropane carboxylic acid (Br2A) were detected in 24 deltamethrin sprayers in an assessment of the exposure and effect of deltamethrin. Twenty-nine male, unexposed referents of the same age range were also selected. The urinary deltamethrin and its metabolite Br2A were detectable by GC and HPLC in the sprayers after exposure. The nerve excitability detected with an electromyograph and pairs of stimuli at variable intervals showed that there was a prolongation of supernormal period in median nerve of the exposed group after a 3-d spraying compared with that before spraying which became more significant 2 d after cessation of exposure. In the mean time, no change of supernormal period in the median nerve of reference group was found at the 3-d interval. Nearly half of the sprayers had a supernormal period prolonged by more than 4 ms after spraying, whereas nearly none of the reference group showed similar changes after repeated examinations. Although there was neither correlation between the nerve excitability changes and the urinary deltamethrin or Br2A excretion, nor was a case of acute deltamethrin poisoning diagnosed, the non invasive technique used for nerve excitability detection in this study seems to be valuable in studying deltamethrin toxicity on human.

Phenolic metabolites of amitriptyline and nortriptyline in rat bile.

Anesthetized bile fistula rats received amitriptyline (AT), its N-oxide (AT-NO), or nortriptyline (NT) at doses of 72 mumol/kg ip, and bile was collected for 6-9 hr. Isolation of metabolites was achieved by enzymic deconjugation and repeated TLC of extracted aglycones. Purified compounds were characterized by UV, NMR, and mass spectrometry, by color reactions, and by chemical interconversions. Besides the alcohols E-10-hydroxy-AT and 10,11-dihydroxy-AT, the phenol E-2-hydroxy-AT occurred as a major AT metabolite, while 2,10- and 2,11-dihydroxy-AT, 2,10,11-trihydroxy-AT, and 2-hydroxy-3-methoxy (or 3-hydroxy-2-methoxy)-AT were present in smaller quantities. Further minor metabolites were 2-hydroxy-11-oxo-AT, 3-hydroxy-AT, 3,11-dihydroxy-AT, and its dehydration product 3-hydroxy-10,11-dehydro-AT. The exact position of the functional groups was elucidated by the nuclear Overhauser effect (NOE) in NMR spectroscopy and by analyzing metabolite patterns in the bile of rats given E- or Z-10-hydroxy-AT. Administration of AT-NO led to a larger proportion of methylated catechols and a smaller one of 10-hydroxy metabolites. Besides the tertiary amines, rats given AT or AT-NO excreted the demethylated analogues of some of the hydroxylation products. The latter also occurred as metabolites of NT, the ratio of aromatic and aliphatic hydroxylation being lower than with AT or AT-NO. Urine of rabbits treated orally with AT contained mono- and dihydroxylated metabolites resulting from attack at positions 2, 3, 10, and/or 11 and the same methylated catechols as rat bile.