Molecular epidemiological characteristics of human rhinovirus in patients with upper respiratory tract infection in Qingdao in the winter of 2020 / 中华微生物学和免疫学杂志

Objective:To analyze the epidemiological characteristics and genotypes of human rhinovirus (HRV) in patients with upper respiratory tract infection in Qingdao in the winter of 2020.Methods:Throat swab samples were collected from 101 patients with upper respiratory tract infection in Qingdao from November 2020 to January 2021. Quantitative PCR was used to detect 15 common respiratory viruses in the samples. HRV-positive samples were further analyzed with RT-PCR to amplify and sequence HRV VP4/VP2 gene. A phylogenetic tree was constructed based on the sequencing results and homology analysis was conducted.Results:Six common respiratory viruses were detected in the 101 patients. Thirty-four cases (34/101, 33.66%) were single pathogen infection and two cases were multiple infection (2/101, 1.98%). The positive rate of HRV was the highest (21.78%, 22/101). Twenty HRV VP4/VP2 sequences were successfully amplified. Phylogenetic analysis showed that there were 16 strains of HRV-A subtype and four strains of HRV-C subtype and 14 serotypes were involved.Conclusions:HRV was one of the leading viral pathogens causing upper respiratory tract infection in Qingdao in the winter of 2020 and the predominant subtype was HRV-A.

Pathogen identification and genotyping of two cluster outbreaks of herpangina in children in Dapeng New District， Shenzhen / 上海预防医学

ObjectiveTo determine the pathogenic characteristics and genotype of two outbreaks of herpangina in children in Dapeng New District， Shenzhen， in May 2021. MethodsA total of five throat swabs from children in the two outbreaks of herpangina were collected and examined for common enteroviruses by real-time PCR. The VP1 region was further amplified by nested RT-PCR. The CLUSTAL W program in MEGA7 software was used to conduct the alignment and reconstruct a phylogenetic tree. ResultsThe pathogen causing the 2 cluster outbreaks of herpangina was coxsackievirus A4 （CVA4）. The sequences of CVA4 VP1 genes revealed that a nucleotide identity of 92% between the strains in the two outbreaks. The three CVA4 strains isolated in kindergarten A had the closest phylogenetic relationship with that isolated in Shenzhen in 2018（MN840533）， with the nucleotide identity of 98.11%. The two strains in kindergarten B had the closest phylogenetic relationship with CVA4 strain isolated in Sichuan in 2018（MW178763）， with the nucleotide identity of 97.88%. The phylogenetic tree showed that all five CVA4 strains in this study belonged to the C2 genotype. ConclusionThe C2 genotype of CVA4 is the causative agent in both outbreaks of herpangina.

Genomic Epidemiology of Imported Cases of COVID-19 in Guangdong Province, China, October 2020 - May 2021 / 生物医学与环境科学（英文）

Objective@#The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been engendering enormous hazards to the world. We obtained the complete genome sequences of SARS-CoV-2 from imported cases admitted to the Guangzhou Eighth People's Hospital, which was appointed by the Guangdong provincial government to treat coronavirus disease 2019 (COVID-19). The SARS-CoV-2 diversity was analyzed, and the mutation characteristics, time, and regional trend of variant emergence were evaluated.@*Methods@#In total, 177 throat swab samples were obtained from COVID-19 patients (from October 2020 to May 2021). High-throughput sequencing technology was used to detect the viral sequences of patients infected with SARS-CoV-2. Phylogenetic and molecular evolutionary analyses were used to evaluate the mutation characteristics and the time and regional trends of variants.@*Results@#We observed that the imported cases mainly occurred after January 2021, peaking in May 2021, with the highest proportion observed from cases originating from the United States. The main lineages were found in Europe, Africa, and North America, and B.1.1.7 and B.1.351 were the two major sublineages. Sublineage B.1.618 was the Asian lineage (Indian) found in this study, and B.1.1.228 was not included in the lineage list of the Pangolin web. A reasonably high homology was observed among all samples. The total frequency of mutations showed that the open reading frame 1a (ORF1a) protein had the highest mutation density at the nucleotide level, and the D614G mutation in the spike protein was the commonest at the amino acid level. Most importantly, we identified some amino acid mutations in positions S, ORF7b, and ORF9b, and they have neither been reported on the Global Initiative of Sharing All Influenza Data nor published in PubMed among all missense mutations.@*Conclusion@#These results suggested the diversity of lineages and sublineages and the high homology at the amino acid level among imported cases infected with SARS-CoV-2 in Guangdong Province, China.

A novel pathogenic Colletotrichum species for rhizome rot disease of Polygonatum kingianum / 中国中药杂志

Rhizome rot disease is one of the main disease of planted Polygonatum kingianum. In this study, six strains of pathogenic fungus was isolated from P. kingianum samples with rhizome rot disease collected from six counties in Yunnan province. Its pathogenicity was confirmed by inoculation to healthy P. kingianum rhizome according to Koch's postulates. The colonies of the isolated fungi on potato dextrose agar(PDA) were orange with abundant crescentic conidia which were eseptate with a mean size of 19. 3-24. 9 μm×5. 2-5. 9 μm and a L/W ratio of 3. 4-4. 5. There was an oil ball in the center of the conidium. It's easy to see setae on PDA colony.The phylogenetic tree based on ITS, GAPDH, CHS-1, HIS3, ACT, and TUB2 sequences by maximum likelihood(ML) method indicated that the pathogenic fungus for P. kingianum rhizome rot disease was clustered into the clade of Colletotrichum spaethianum species complex, and was close to C. spaethianum. However, there were some differences in morphological and genetic characteristics between the pathogenic fungus and C. spaethianum. Therefore, the pathogenic fungus for rhizome rot disease of P. kingianum was identified as a new Colletotrichum species named C. kingianum. The disease spreads primarily due to the plantation of infected seedlings of P. kingianum. It is necessary to choose healthy seedlings and take rigorous disinfection measures for the disease prevention.

Identification of Chinese medicine sea snake based on cytochrome B (Cytb) / 中国中药杂志

The identification of species primordium has been one of the hot issues in the identification of traditional Chinese medicine. Sea snake is one of the most valuable Chinese medicinal materials in China. In order to understand the origin and varieties of sea snake in the market, we studied the molecular identification of 46 sea snakes by cytochrome B(Cytb). After comparison and manual correction, the sequence length was 582 bp, and the content of A+T(58.9%) was higher than that of G+C(41.1%). There exist 197 variable sites and 179 parsimony-informative sites of the sequence. There are 44 kinds of sequence alignment with consistency equal to 100%, and 2 kinds equal to 96%. A total of 408 Cytb effective sequences were downloaded from GenBank database, with a total of 68 species. Phylogenetic tree of a total of 454 sea snake sequences with the samples in this study were constructed by neighbor-joining trees and Bayesian inference method, respectively, which can identify 42 samples of medicinal materials, while 4 samples can not be identified because of their low node support. The results showed that the species of the sea snake medicine were at least from 2 genera and 5 species, namely, Aipysurus eydouxii, Hydrophis curtus, H. caerulescen, H. curtus, H. ornatus and H. spiralis. This study suggested that the original species of commercial sea snake are very complex and can provide insight into the identification of sea snakes.

Isolation and identification of phosphatolytic bacteria in Paris polyphylla var. yunnanensis / 中国中药杂志

The wild resources of Paris polyphylla var. yunnanensis, a secondary endangered medicinal plant, are severely scarce. Introduction and cultivation can alleviate market demand. To screen phosphatolytic bacteria in the rhizosphere soil of P. polyphylla var. yunnanensis and provide data support for the development of high-efficiency microbial fertilizer, in this study, the dilution plate coating method was used to isolate and screen the phosphorus solubilizing bacteria with the ability of mineralizing organic phosphorus from the rhizosphere soil of wild and transplanted varieties of P. polyphylla var. yunnanensis in 10 different locations in Yunnan, Sichuan and Guizhou. After separation and purification, the phosphatolytic capacity was analyzed by qualitative and quantitative analysis. Combined with physiological and biochemical experiments, the strains were identified using 16 S rDNA sequencing analysis. Forty one strains were selected from the rhizosphere soil of P. polyphylla var. yunnanensis from 10 different habitats. Among them, 21 strains were obtained from the rhizosphere soil of the wild variety P. polyphylla var. yunnanensis and 20 strains were obtained from the rhizosphere soil of the transplanted variety. And significance analysis found that 41 organophosphate solubilizing strains had significant differences in their ability to solubilize phosphorus. The amount of phosphate solubilizing was 0.08-67.61 mg·L~(-1), the pH value was between 4.27 and 6.82. The phosphatolytic amount of strain Y3-5 was 67.61 mg·L~(-1), and the phosphorus increase amount was 57.57 mg·L~(-1). All 41 strains were identified as Gram-positive Bacillus. Combining physiological characteristic and phylogenetic trees, Bacillus mobilis Y3-5 was finally selected as the candidate rhizosphere phosphatolytic bacteria of P. polyphylla var. yunnanensis. The distribution of phosphorus solubilizing bacteria in the rhizosphere soil of P. polyphylla var. yunnanensis was different, and there were significant diffe-rences in phosphorus solubility. Organophosphate-dissolving strain Y3-5 is expected to be a candidate strain of P. polyphylla var. yunnanensis microbial fertilizer.

Molecular analysis of the first dengue fever case in Dapeng New District of Shenzhen City / 上海预防医学

Objective:To analyze the first suspected dengue fever case in Dapeng New District by characterizing the virus and exploring its origin. Methods:Dengue virus nucleic acid in blood sample was identified by real-time fluorescent RT-PCR. The E gene of dengue virus was amplified and sequenced. Homology and phylogenetic tree of this dengue virus were compared with the isolates from other regions. Results:Nucleic acid of dengue virus type 1 was detected in the serum sample from the suspected dengue fever patient. Phylogenetic tree showed that the homology based on nucleotide sequence of E gene of DP-DGR19001 was very similar with the isolates of MG894965/TW/CHN/2016 （99.87%）， LC428079/VNM/2017（99.80%）， MG894867/TW/CHN/2015 （99.60%） and MN512242/GX/CHN/2014 （99.46%）. The deduced amino acid sequence of 5 isolates was 100% identical. The patient had traveled to high-incidence country Cambodia in 14 days before infection. Conclusions:The first suspected dengue fever case in Dapeng New District is caused by dengue virus type 1. The case is an imported dengue fever .

Screening and identification of fibrinolytic enzyme-producing microbes in fermentation process of Sojae Semen Praeparatum / 中草药

Objective: To screen and identify the dominant strains which produce fibrinolytic enzyme during the processing of Sojae Semen Praeparatum (SSP, Dandouchi in Chinese). Methods: SSP was prepared according to the Chinese Pharmacopoeia (2020 edition), and samples were taken at different time points during the fermenting process of SSP.The casein plate method and fibrin plate method were used to screen the fibrinolytic enzyme-producing microorganisms in samples at different time points. The fibrinolytic enzyme-producing microorganisms were inoculated in the designated liquid medium to obtain single strain fermentation broth, and fibrin plate method was used to measure the fibrinolytic activity of the fermentation broth. The DNA sequences of fibrinolytic enzyme-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA universal primer by PCR respectively.The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was done by phylogenetic tree constructed by MEGA 4.1 software. Results: Three types of fibrinolytic enzyme-producing bacteria were screened out and identified in this study. They were Bacillus subtilis, Stenotrophomonas maltophilia and Micrococcus, respectively. The result of fibrin plate method showed that the fermentation broth of S. maltophilia had the highest fibrinolytic activity, reaching 527.49 IU/mL. Conclusion: There are fibrinolytic enzyme-producing dominant microorganisms existing in the fermenting process of SSP and the thrombolytic effect of SSP is worthy of further study. This study lays the foundation for revealing the formation mechanism of fibrinolytic enzyme in the fermentation process of SSP.

Identification of hepatitis C virus genotypes in volunteer blood donors from blood transfusion center of Tuban, Indonesia

@#Aims: The most common transmission route of hepatitis C virus (HCV) is via blood transfusion. Therefore, the screening of HCV is necessary to be performed regularly for all the volunteer blood donors. The prevalence of HCV subtypes varies in different geographical areas. The aim of this study is to identify the HCV genotypes of the HCV-RNA positive samples and performed serological and molecular characterization of HCV among blood donors from Blood Transfusion Center of Tuban, East Java, Indonesia collected during the year of 2015. Methodology and results: All blood donor samples were screened by enzyme-linked immunosorbent assay (ELISA) for anti-HCV. Reverse Transcription - Polymerase Chain Reaction (RT-PCR) was performed to detect the HCV-RNA. Subsequently, the HCV-RNA positive samples were genotyped using direct sequencing followed by subtype/genotype and phylogenetic analysis. Of the 500 blood samples, 7 were positive for anti-HCV antibody (1.4%) and 6 out of 7 (85.71%) were determined to be HCV-RNA positive. Among HCV-RNA carriers, genotyping showed genotypes 1 was the most prevalent. HCV subtypes 1a and 1b were detected in total of 4 out of 6 individuals (66.67%), two individuals for each. HCV subtypes 2a and genotype 1 were the least frequent among blood donors (each counted for 16.67%). Conclusion, significance and impact for study: The prevalence of HCV found in this study is considerably low. The identification of genotypes 1a and 1b as major HCV genotypes circulating in blood donors in the Tuban city of East Java. This result may contribute in a better medical management towards HCV carriers.

Chloroplast Genomic Analysis of Pyrrosia assimilis / 中国实验方剂学杂志

Objective::The complete chloroplast genome of Pyrrosia assimilis was sequenced, its sequence characteristics was analyzed and herbgenomics of P. assimilis was discussed. Method::Its complete chloroplast genome sequence was determined through high-throughput sequencing technology, and its structural characteristics and phylogenetic relationships were analyzed by bioinformatics. Result::The chloroplast genome of P. assimilis was a circular double-chain structure with a total length of 154 964 bp, and the total content of guanine and cytosine (GC) was 41.2%. A total of 131 genes were annotated, including 88 protein-coding genes, 35 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes. A total of 43 dispersed repetitive sequences and 56 simple sequence repeats (SSRs) were detected. The frequency of codon encoding leucine was the highest, while the number of codon encoding tryptophan was the lowest. Five highly divergent regions (psbA, rrn16, petA-psbJ, ndhC-trnM, and psbM-petN) were screened, phylogenetic analysis showed that P. assimilis was closely related to P. bonii. Conclusion::Comparative analysis of the complete chloroplast genome of P. assimilis reveals that non-coding regions exhibited a higher divergence than the coding regions, the large single copy region (LSC) and small single copy region (SSC) are more divergent than the reverse repeat region (IR), the selected five highly variable regions can be used as specific DNA barcodes for identification of Pyrrosia species. Study on the chloroplast genome of P. assimilis can provide a reference for the molecular identification, genetic transformation, expression of resistance protein and secondary metabolism pathway analysis of other Pyrrosia medicinal plants.

Deciphering species relationships and evolution in Chenopodium through sequence variations in nuclear internal transcribed spacer region and amplified fragment-length polymorphism in nuclear DNA

Evaluation of sequence variations in the internal transcribed spacer (ITS) region of 19 accessions, comprising of 11 accessions of Chenopodium quinoa, eight accessions of Chenopodium album and 165 retrieved sequences of different species of Chenopodium belonging to subfamily Chenopodioideae revealed a higher intraspecific genetic diversity in Himalayan C. album than that in C. quinoa. ITS and amplified fragment-length profiles of the accessions suggest the existence of accessions of Himalayan C. album as heteromorphs of the same species rather than a heterogenous assemblage of taxa. While the evolutionary relationship reconstructed from variations in 184 sequences of ITS region from species belonging to Chenopodiaceae, Amaranthaceae, Polygonaceae and Nelumbonaceae established a paraphyletic evolution of family Chenopodiaceae, it also revealed a monophyletic evolution of Chenopodieae I. The reconstruction also established five independent lineages of the subfamily Chenopodioideae with C. album as a sister clade of C. quinoa within the tribe Chenopodieae I. The results also indicate a much younger age for Himalayan chenopods (C. album) than the reported crown age of Chenopodieae I.

Screening and identification of GABA-producing microbes in fermentation process of Sojae Semen Praeparatum / 中国中药杂志

A high-content GABA was found in Sojae Semen Praeparatum(SSP), which is a famous traditional Chinese medicine and officially listed in Chinese Pharmacopoeia. To screen out and identify GABA-producing microbes from samples at different time points during the fermenting process of SSP, traditional microbiological methods combined with molecular biological methods were used to study the predominant GABA-producing microorganisms existing in the fermenting process of SSP. This study would lay a foundation for further studying the processing mechanism of SSP. The fermenting process of SSP was based on Chinese Pharmacopoeia(2010 edition), and samples were taken at different time points during the fermenting process of SSP. The bacteria and fungi from samples at different time points in the fermenting process of SSP were cultured, isolated and purified by selective medium, and dominant strains were selected. The dominant bacteria were cultured in the designated liquid medium to prepare the fermentation broths, and GABA in the fermentation broth was qualitatively screened out by thin-layer chromatography. The microbial fermentation broth with GABA spots in the primary screening was quantitatively detected by online pre-column derivatization and high performance liquid chromatography established in our laboratory. GABA-producing microorganisms were screened out from predominant strains, and their GABA contents in fermentation broth were determined. The DNA sequences of GABA-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA sequences by PCR respectively. The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was made by phylogenetic tree constructed by MEGA 7.0 software. Through the homology comparison of NCBI and the construction of phylogenetic tree by MEGA 7.0 software, nine GABA-producing microorganisms were screened out and identified in this study. They were Bacillus subtilis, Enterococcus faecium, E. avium, Aspergillus tamarii, A. flavus, A. niger, Cladosporium tenuissimum, Penicillium citrinum and Phanerochaete sordida respectively. For the first time, nine GABA-producing microorganisms were screened out and identified in the samples at different time points during the fermenting process of SSP in this study. The results indicated that multiple predominant GABA-producing microorganisms exist in the fermenting process of SSP and may play an important role in the formation of GABA.

Genetic analysis of norovirus strains detected from sporadic cases in Anhui province, 2016-2017 / 中华实验和临床病毒学杂志

Objective@#To analyze the genotype diversity and phylogenetic characterization of norovirus(NoV) in patients with diarrhea from Anhui province.@*Methods@#NoV positive fecal specimens from sentinel hospitals were collected from January, 2016 to December, 2017. The samples were detected by Real-time fluorescent quantitative PCR. Positive samples were of randomly selected and amplified by RT-PCR and the products were sequenced. Phylogenetic tree was constructed by the Neighbor-Joining method based on partial VP1 gene regions of NoV to perform phylogenetic analysis.@*Results@#A total of 263 NoV positive samples were genotyped, of which 239 belonged to genogroup II, 24 belonged to genogroup I. Fifty-five positive samples were successfully sequenced. There were 6 NoV GII genotypes, which included GII.2, 3, 4/Sydney_2012, 13, 17 and 21, while NV GII.17 and GII.4 were the dominant genotypes from 2016 to 2017. The predominant genotype was GII.4/Sydney 2012 (47.27%, 26/55), followed by GII.17 (23.64%, 13/55) and GII.2 (14.55%, 8/55). Phylogenetic tree showed that 26 strains belonged to genotype GII.4/Sydney 2012, NoV. The nucleotide homology among the 26 VP1 genes was 97.8% to 100%. Analysis of the partial VP1 genes of 26 strains showed that it shared the highest homology of 98.9% with the strain of GII.4Sydney2012 (GenBank ID: KU720515). However, the prevailing genotype in the Anhui province has shifted on two separate occasions, the GII.17 strain was dominant in 2016, and the GII.4/Sydney 2012 strain was dominant in 2017.@*Conclusions@#NoV GII was the major pathogen causing sporadic diarrhea in Anhui province during from 2016 to 2017, the genotypes are widely distributed, and shifted into the two predominant strains.

Characteristics of Coxsackievirus A16 VP1 gene in Jinan, 2017 / 中华实验和临床病毒学杂志

Objective@#To analyze the VP1 gene characteristics of Coxsackievirus A16 (CV-A16) isolated in(hand, foot and mouth disease, HFMD) patients of Jinan 2017.@*Methods@#The samples collected from patients with HFMD in Jinan city in 2017 were analyzed and some of the CV-A16 nucleic acid positive samples were choosen with simple random sampling method and pretreated to inoculate RD cells. The whole VP1 gene sequences of CV-A16 stains which had obvious cytopathic effect in RD cells were amplified and sequenced. MEGA5.2 software was used to constructed phylogenetic tree and Lasergene software was used to analysis the homology consistency of nucleotide and amino acid of VP1 gene.@*Results@#All the 10 CV-A16 strains isolated from Jinan were located 3 clusters belonged to B1b genotypes, with a nucleotide similarity of 94.9%~100% and deduced amino acid similarity of 99.3%~100%. The Jinan CV-A16 strains were nearest related with Shanghai strains KX871333(nucleotide similarity: 99.7%), Henan strains KM260134(99.0%), Jiangsu strains KP751580、KR138327(98.7%) and Guangdong strains MH004016(98.5%). Compared with some reference strains from our country, amino acid mutation were identified at positions 14, 59, and 145 in VP1 proteins of Jinan CV-A16 strains.@*Conclusions@#CV-A16 strains B1b genotypes were the strains prevailed in Jinan city, and 3 amino acid substitutions in VP1 proteins were happened in CV-A16 strains isolated from Jinan.

Hemagglutinin genetic analysis of a Yamagata influenza B virus infected in a military camp in Chengdu / 第二军医大学学报

Objective: To analyze the genetic evolution and hemagglutinin (HA) gene mutation sites of influenza B virus in Chengdu. Methods: Influenza virus was isolated from patient's throat swab samples using Madin-Darby canine kidney (MDCK) cells in vitro. The HA gene of influenza B virus was obtained by PCR and was sequenced. The phylogenetic tree was constructed and mutation sites were analyzed by online comparison with NCBI database and MEGA 6.06 software. Results: One strain of influenza B virus was isolated by MDCK cells, and 1 755 bp full-length HA gene was obtained by PCR amplification using the nucleic acids of the throat swab and the isolated strain as templates. The obtained sequence was submitted to the GenBank database, and the gene accession number was MH236281. By online alignment and phylogenetic tree construction, the virus infected in this case was confrmed to be Yamagata type B influenza virus. There were 57 HA point mutation bases as compared with Influenza B/Yamagata/16/88 (GenBank No. M36105). There were 20 mutated bases in HA as compared with the World Health Organization (WHO)-recommended vaccine strain Influenza B/Utah/08/2014 (GenBank No. KU592766). HA1 amino acid mutation site was further analyzed. Compared with the epidemic strains in Sichuan in recent years, HA1 amino acid sites have undergone varying degrees of mutations, of which there were only 4 point mutations compared with Wenjiang/2010 isolate (GenBank No. KP461138). Compared with the WHO-recommended vaccine strain Influenza B/Utah/08/2014, two amino acid sites mutated (L176Q and M255V), but the mutation was not located in the antigenic determinant region of HA1. Among them, site 176 was a new mutation. The amino acid was leucine (L) at site 176 of HA1 epidemic strains in Sichuan, Influenza B/Yamagata/16/88, and the WHO-recommended vaccine strain Influenza B/Utah/08/2014, whereas the amino acid was glutamine (Q) in the influenza B strain isolated in this study. Conclusion: Mutations have occurred in the HA gene of influenza B virus infected in Chengdu during 2017 - 2018, while these mutations have not yet caused antigenic changes.

Evolution of VP1 gene of coxsackievirus A16 in hand, foot, and mouth disease / 中华传染病杂志

Objective To investigate the genetic evolution of VP1 gene of pathogenic coxsackievirus A16 (CV-A16) strain isolated from clinical hand,foot,and mouth disease (HFMD) patients.Methods A total of 160 HFMD cases with CV-A16-positive results were collected from hospitals in Kunming during January 2015 to June 2017.Fecal samples were collected.Real-time polymerase chain reaction (PCR) was used to detect the CV-A16 virus nucleic acid.The VP1 genes of CV-A16-positive samples were amplified by reverse transcription-PCR.The amplified positive products were sequenced and aligned.The homologies were identified and their subgenotypes were determined.The phylogenetic tree was constructed and homology modeling was conducted.Results All the 160 CV-A16 isolates were B2 subtypes.The genetic distance between detected strains of CV-A16 and the strains in Fujian,Beijing,Nanjing was 0.76.The genetic distance to the strains in Malaysia was 0.78,and to the strains in Australia was 1.86.Homologous modeling revealed that the amino acid sequence of the VP1 gene of the strain had a G227R mutation.Conclusions There is no major genetic variation in the CV-A16 strains during 3 years.CV-A16 isolates are close to those of epidemic strains in Beijing,Fujian and Malaysia,but are far fram the strains from Australia.

Characteristics of gene sequence changes of hemorrhagic fever with renal syndrome virus in Heilongjiang Province / 中国实用内科杂志

OBJECTIVE: To study the gene sequence characteristics of hantavirus in Heilongjiang Province in order to find out the reasons for the changes of clinical characteristics of hemorrhagic fever with renal syndrome in Heilongjiang Province in recent years.METHODS: Totally 110 rat lung specimens,121 blood specimens from patients diagnosed with mild or atypical HFRS and 100 blood specimens from patients diagnosed with typical HFRS in the First Affiliated Hospital of Harbin Medical University and the Seventh Hospital of Qiqihar City from 2005 to 2015 were collected. The gene sequences were obtained by nucleic acid extraction, RT-NestPCR, and gene sequencing. Explore the possible reasons for the changes in clinical characteristics of HFRS by comparing the obtained sequences with previous strains, homology analysis, building phylogenetic trees of M gene, and finding out the law of nucleotide and amino acid loci changes. RESULTS: TM gene of twenty-six mild or atypical HFRS patients were successfully amplified, including 14 cases of HTN type and 12 cases of SEO type; M gene of twenty-two typical HFRS patients were amplified, including 19 cases of HTN type and 3 cases of SEO type. Compared with the standard strain 76-118, the nucleotide homology of hantavirus from mild or atypical HFRS patients, typical HFRS patients and mice was 74.4%-89.2%, 87.4%-90.3% and 88.1%-88.5%. Comparing hantavirus gene sequence from mice and from patients, the nucleotide homology was 79.7-99.1%. Hljh38 and Hljh39 from patients were significantly different from the other strains. They were the same subtype as Amur virus because they had high homology with Amur strains H5 and H8205(94.9%-97.6%). The deduced amino acids showed some variations compared with the standard strains, but no obvious variation law was observed. CONCLUSION: The reason for the changes of clinical characteristics of hemorrhagic fever with renal syndrome in Heilongjiang province is related to the change of viral type. There are also variations of hantavirus and amino acid, but the relationship between specific variation law and clinical manifestations needs to be further verified.

Screening of DNA Barcoding Sequences for Identification of Multiple Species of Aquilaria L / 中国药学杂志

OBJECTIVE: To screen out efficient sequences from DNA barcoding sequences in order to rapidly identify multiple species of Aquilaria. METHODS: Five species of Aquilaria were collected from eight countries as test materials. The ITS2 sequence in nuclear genome as well as matK, rbcL and trnH-psbA sequences from chloroplast genome were analyzed and evaluated. Phylogenetic trees based on different sequence combinations were built to evaluate the identification efficiency of Aquilaria species. RESULTS: All four kinds of sequences exhibited relatively high success rate of PCR amplification and sequencing. matK had the most variation sites and SNPs while trnH-psbA had the fewest. Barcoding gap test demonstrated that the identification effect of sequences from high to low was matK>ITS2>rbcL>trnH-psbA. According to the homogeneity test and average node supporting rate, phylogenetic trees based on the combination of matK+ITS2+rbcL could clearly divide different species into separated clusters with high node supporting rate. The matK showed the best identification effect when combined with other sequences as shown by the BBA method of Taxon DNA. CONCLUSION: When using DNA barcoding technique to identify Aquilaria species, matK has the highest identification rate among single barcoding candidate sequences. And the combination of matK+ITS2+rbcL can precisely distinguish multiple Aquilaria species.

Characterization and phylogenetic analysis of a Aeromonas veronii clinical strain isolated from blood culture / 中华微生物学和免疫学杂志

Objective To identify an unknown pathogenic strain (X1) isolated from blood culture of an inpatient and to conduct a phylogenetic analysis on it. Methods The X1 strain was isolated from blood culture of an inpatient in the Second Xiangya Hospital of Central South University in 2016. It was identified by morphology biochemical testing and 16S rRNA gene sequencing. Based on genetic distances, a phylogenetic tree was constructed to compare the X1 strain with its homologous species in GenBank in order to reveal its genetic evolution. Results The X1 strain was gram-negative. Biochemical testing for oxidase, glucose and V-P reaction were positive (+) , while the result of citrate was negative (-) . Sequence align-ment analysis of 16S rRNA showed that the X1 strain (1466 bp) was highly homologous with Aeromonas veronii X71120. 1 with a similarity of more than 99％. Genetic distances between the X1 strain and its ho-mologous species, including Aeromonas veronii, Aeromonas hydrophila and Aeromonas sobria, were calculat-ed by MEGA5. 0 and the results revealed that the genetic distance (0. 014) between it and Aeromonas veronii X71120. 1 was the shortest, but the subspecies of it was unable to identify. Cluster analysis also indicated that the X1 strain and Aeromonas veronii were clustered to a small branch. Conclusions The X1 isolate was an Aeromonas veronii strain. Besides, Aeromonas veronii and Aeromonas sobria were similar in genetic char-acteristics.

Genotyping-by-sequencing based single nucleotide polymorphisms enabled Kompetitive Allele Specific PCR marker development in mutant Rubus genotypes

Background: Rubus is an economically important fruit crop across the globe. Recently, several Rubus mutant genotypes with improved agronomic traits have been developed using gamma ray irradiation. This study investigated genetic diversity and variations in Rubus mutant genotypes using single nucleotide polymorphism (SNP) markers generated from genotyping-by-sequencing (GBS) analysis. A GBS library of 14 Rubus genotypes, consisting of seven boysenberry mutant lines, four blackberry mutant lines, and three original varieties, were sequenced on the Illumina Hiseq2000 platform. A set of SNPs were analyzed by Kompetitive Allele Specific PCR (KASP) assay in order to discriminate the Rubus genotypes. Results: A total of 50,831,040 (86.4%) reads of clean data were generated, and the trimmed length ranged from 116,380,840 to 509,806,521 bp, with an average of 228,087,333 bp per line. A total of 19,634 high-quality SNPs were detected, which contained 11,328 homozygous SNPs and 8306 heterozygous SNPs. A set of 1504 SNPs was used to perform a phylogenetic analysis, which showed that there were clear differences among the Rubus genotypes based on their origin. A total of 25 SNPs were used for the KASP assays, of which six KASP primer sets were successfully distinguished among the Rubus genotypes. Conclusions: This study demonstrated that the SNP and KASP method is an economically efficient tool for mutant screening in Rubus breeding programs.