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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.
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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.
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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-Z 10S-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.
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Objective To compare the long term effectiveness of tonsillotomy(TT)vs. tonsillectomy (TE)by hypercator in treatment of obstructive sleep apnea hypopnea syndrome(OSAHS)in children. Methods 119 children with tonsil hypertrophy were diagnosed with OSAHS and included in the study. Among them,57 children underwent TT by hypercator and 62 TE only after preoperative laboratory polysomnography(PSG). All participants were interviewed 5 years after operation to assess the satisfaction of their caregivers,investigate the frequency of respiratory infections,the rate of tonsil regrowth and evaluate their quality of life by Obstructive Sleep Apnea-18(OSA-18). Results Some of them received postoperative PSG. All parents reported alleviation of breathing obstruction,but the postoperative satisfaction in TT group was higher than TE group mainly because of the shorter time return to normal diet. The rate of the respiratory tract infections was not increased in all cases after surgery. Four cases of children were observed with tonsillar regrowth,but no surgery was needed for further treatment. The rate of the dry throat symptoms in TT group was lower than TE group. Conclusions Tonsillotomy by hypercator can effectively eliminate the obstruction symptoms in children with OSAHS,superior to TE for its shorter recovery period and smaller oral mucosal injury and lower rate of tonsils regrowth and it is worth to clinical promotion.
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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-Z 10S-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.
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To explore the molecular characteristics of Streptococcus suis serotype 2(ss2) isolated in Zhejiang province by deciding the variation loci and its variation frequency of Cps2J gene.The total DNA of 9 strains of ss2 isolated in Zhejiang province were extracted and amplifed by PCR. Then,the Cps2J fragments were cloned into plasmid carrier and completely sequenced after purification.Finally,the sequence results of all 9 ss2 isolates were compared with those obtained by other studies around the world.It was found that the open reading fragments of Cps2J in 9 SS2 isolates encoding 333 amino acids were 999 bp in length.Comparisons of this region among ss2 isolates revealed a similarity of between 98.8% and 99.9%, while the homology to ss1 strains varied between 56.8% and 57.0%.Our study shows the sequences of complete Cps2J segment are fairly stable and all these 9 ss2 strains of different sources possibly have the same evolutionary origin.
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The M and S segment cDNAs of hantavirus Z5 strain was amplified by RT-PCR,and the purified PCR products were cloned into vector pGEM-T and then sequenced.It was demonstrated that the M genome segment of Z5 was found to be 3 616 nucleotides in length with a single open reading frame encoding 1 135 amino acids.And the S genome segment was 1700 nucleotides in length with a single open reading frame encoding 429 amino acids.As demonstrated by the homologous analysis of nucleotides and amino acids,it was showed that the Z5 strain belonged to hantaan viruses HTN type and was the same subtype of the Z10 strain.It is conclouded that difference in nucleotide sequence exists between Z5 strain with other Hantavirus strains but high level of homology in amino acid sequences is still present.
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One strain of Hantavirus(HV) was isolated from patients with hemorrhagic fever with renal syndrome (HFRS) in Zhejiang province and its S-segment was cloned and submitted to nucleotide sequence analysis in order to determine the type of strain and extent of genetic variation for further study on its evolution and mutation .The HV antigen was detected from mouse lungs in endemic area by direct immunofluorescene test and the HV-positive sample of mouse lung was inoculated to Vero-E6 cells to isolate virus. The total cellular RNA was extracted from infected cell culture and the S segment gene was amplicated by RT-PCR. Then, the purified PCR product was cloned into T vector for sequencing. The result showed that the full-length sequence of the S segment was 1 700 bp with one open reading frame (ORF) encoding 429 amino acids. Comparison with Hantavirus HTN type showed 85.7%-91.9% homology at the nucleotide level. In comparison with the SEO type of viruses, the homology at nucleotide level was shown to be 71.2%-75%. This new HV strain may be typed as to HTN virus and may be a new subtype of this virus.
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OBJECTIVE@#To explore the method and effect of the midfacial degloving approach and modified hemifacial degloving approach associated with nasal endoscopic surgery in the treatment of the nasal diseases.@*METHOD@#Thirty patients with nasal diseases were treated with nasal endoscopic surgery by midfacial degloving approach and modified hemifacial degloving approach. Four cases underwent midfacial degloving approach with standard method, three cases were underwent by hemifacial degloving approach and our modified hemifacial degloving approach associated with nasal endoscopic surgery were performed in twenty-three cases. We used Caldwell-Luc's approaches which located mainly in affected-side, and modified bilateral intercartilaginous incision, which at first peeled off integrality healthy-side cutis and mucosa of nasal septum as well as periosteum of basis nasi. With preserving the integrality of the healthy-side nasal cavity parenchyma, the pyriform aperture incisions extending to the healthy-side vestibule wasn't been cut. With the incisions of septal cartilage of nasal and disease- side cutis and mucosa of nasal septum as well as the pyriform aperture incisions extending to the affected-side vestibule, the lesion were cleared away completely by modified midfacial degloving approach associated with nasal endoscopic surgery.@*RESULT@#All cases cuts achieved primary healing. One of four cases with midfacial degloving approach suffered from straightness of nasal vestibule. One of three cases with hemifacial degloving approach was led to perforation of nasal septum. In 23 cases operated hy modified hemifacial degloving approach, no straightness of nasal vestibule and no perforation of nasal septum was happened.@*CONCLUSION@#The midfacial degloving approach and modified hemifacial degloving associated with endoscopic surgery can achieve the advantages of a widely exposed field for operation, no facial scar, making tumour resection easier, and also no nasal- stuffed in healthy nasal cavity as well as no straightness of nasal vestibule after modified approach.
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Adolescent , Adult , Aged , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Young Adult , Endoscopy , Methods , Face , General Surgery , Nasal Septum , General Surgery , Nose , General Surgery , Nose Diseases , General Surgery , Otorhinolaryngologic Surgical Procedures , MethodsABSTRACT
Objective To compare the molecular characteristics of hantanvirus strains isolated from 1980 to 2002 in Zhejiang Province by PCR and nucleotide sequencing.Methods Total RNA was extracted from hantanvirus infected Vero-E6 cells. The M segment cDNA of hantaan ZJ4 and ZJ7 strains were obtained by reverse transcription and polymerase chain reaction, subsequently cloned into pUCm-T vector and sequencing.Results Two strains ZJ4 and ZJ7 of hantanvirus isolated from Rottus in Zhejiang province were HTN viruses. HTN virus strain Z10 was isolated in 1980. Comparison ZJ4 and ZJ7 with Z10 strains indicated that there were 88.3% and 92.3% homology at the nucleotide level.Conclusion The HTN virus were very conserved. The homology of HTN virus isolated from Zhejiang Province was high nucleotide level, though they were isolated at more than 20-year intervals.
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<p><b>OBJECTIVE</b>To study the severe acute respiratory syndrome (SARS)-associated coronavirus genotype and its characteristics.</p><p><b>METHODS</b>A SARS-associated coronavirus isolate named ZJ01 was obtained from throat swab samples taken from a patient in Hangzhou, Zhejing province. The complete genome sequence of ZJ01 consisted of 29,715 bp (GenBank accession: AY297028, version: gi: 30910859). Seventeen SARS-associated coronavirus genome sequences in GenBank were compared to analyze the common sequence variations and the probability of co-occurrence of multiple polymorphisms or mutations. Phylogenetic analysis of those sequences was done.</p><p><b>RESULTS</b>By bioinformatics processing and analysis, the 5 loci nucleotides at ZJ01 genome were found being T, T, G, T and T, respectively. Compared with other SARS-associated coronavirus genomes in the GenBank database, an A/G mutation was detected besides the other 4 mutation loci (C:G:C:C/T:T:T:T) involved in this genetic signature. Therefore a new definition was put forward according to the 5 mutation loci. SARS-associated coronavirus strains would be grouped into two genotypes (C:G:A:C:C/T:T:G:T:T), and abbreviated as SARS coronavirus C genotype and T genotype. On the basis of this new definition, the ZJ01 isolate belongs to SARS-associated coronavirus T genotype, first discovered and reported in mainland China. Phylogenetic analysis of the spike protein gene fragments of these SARS-associated coronavirus strains showed that the GZ01 isolate was phylogenetically distinct from other isolates, and compared with groups F1 and F2 of the T genotype, the isolates of BJ01 and CUHK-W1 were more closely related to the GZ01 isolate. It was interesting to find that two (A/G and C/T) of the five mutation loci occurred in the spike protein gene, which caused changes of Asp to Gly and Thr to Ile in the protein, respectively.</p><p><b>CONCLUSION</b>Attention should be paid to whether these genotype and mutation patterns are related to the virus's biological activities,epidemic characteristics and host clinical symptoms.</p>
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Humans , Middle Aged , Genotype , Mutation , Severe acute respiratory syndrome-related coronavirus , GeneticsABSTRACT
Objective To construct hantavirus Z37 envelope glycoprotein genes G1 and G2 recombinant and express in eukaryocyte. Methods Using three pairs of primers based on hantavirus Z37 M gene sequences. PCR products F1, F2 and G2 were obtained by PCR from the Plasmid pGEMZ37(containing partial cDNA of G1) and Plasmid pCUMZ37(containing partial cDNA of G1 and whole cDNA of G2). G1 PCR products were also obtained by fusing F1 and F2 PCR products. The G1 and G2 PCR products were then digested by BamHⅠ and XhoⅠ and cloned into the corresponding sites of expression vector pcDNA3.1 respectively. The recombinants pcDNA3.1 G1 and pcDNA3.1 G2 were identified by digestion with endonuclease BamHⅠ and XhoⅠ and confirmed by sequencing. After transfecting COS 7 cells by Calcium phosphate/DNA precipitating method, indirect immunofluorescence assay (IFA) was used to verify the transient expression of HV Z37 G1 or G2 protein in COS 7 cells. Results The recombinant expression plasmids pcDNA3.1 G1 and pcDNA3.1 G2 were constructed. After transfection with the above recombinant expression plasmids, specific antigens were detected within cells by IFA. Conclusions The reombinant expression plasmids pcDNA3.1 G1、pcDNA3.1 G2 were constructed successfully and expressed transiently in eukaryocyte.