Objective:To understand the spectrum of pathogens and epidemic characteristics of respiratory infectious diseases in influenza virus-negative influenza-like cases in Yantai, and provide reference for disease prevention and control and clinical diagnosis and treatment.Methods:From March 2020 to February 2021, nasopharyngeal swab samples of 233 influenza virus-negative influenza like cases were collected in all sentinel hospitals monitored by Yantai National Influenza Network Laboratory, and 22 respiratory pathogens were detected by multiple fluorescence quantitative polymerase chain reaction to analyze epidemiological characteristics.Results:The total pathogen detection rate of 233 samples was 69.96% (163/233). A total of 17 respiratory pathogens were detected. The top three pathogens were human coronavirus (HCoV, 32.62%), rhinovirus/enterovirus (RhV/EV, 17.17%) and Legionella pneumophila (LP, 16.74%). The detection rates in different age groups were 80.28% (57/71) in the 0-15 years old group, 62.65% (52/83) in the 16-30 years old group, 68.18% (30/44) in the 31-45 years old group, 64.28% (9/14) in the 46-60 years old group, and 71.43% (15/21) in the >60 years old group. There was no significant difference among the groups. Respiratory pathogens were detected throughout the year, mainly in a single pathogen carrying mode (44.21%), and there was no significant difference in the physical examination rate of respiratory pathogens in different seasons. The seasonal prevalence of various pathogens was different, and the detection rate of HCoV 229E was the highest in spring (68.75%); the detection rate of rhinovirus/enterovirus was higher in autumn (26.98%) and winter (23.08%); the detection rate of LP was high in spring (19.05%) and summer (27.27%); the detection rate of human parainfluenza virus (HPIV) in spring (22.22%) was significantly higher than that in summer (3.64%). The number of HPIV and Bordetella pertussis (Bp) detected in the 0-15 year old group was the highest, and the detection rate was statistically significant among different age groups. Conclusions:The continuous monitoring of respiratory pathogens such as HCoV, RhV, EV, LP, HPIV should be strengthened to understand their epidemiologic characteristics and the standardization of pathogenicity, which provides data support and reference for epidemiological investigation of outbreaks that may be caused by other pathogens.

Objective:To investigate the pathogenic spectrum of enteroviruses associated with hand, foot and mouth disease (HFMD) in the Yantai region of Shandong province in 2016, and analyze the evolution of epidemic strains of coxsackie virus group A type 6 (CV-A6) in the pathogenic spectrum of HFMD enteroviruses and the variations of important amino acid sites in the VP1 region.Methods:A total of 738 samples were collected from the patients with HFMD in Yantai region in 2016 to conduct DNA and serotype tests of enterovirus (EV) by real-time RT-PCR and further count the number and proportion of each type of enterovirus positive specimens. Based on the predominant serotype of enteroviruses, eight serotypes of the CV-A6 strains were selected to carry out VP1 regions amplification for the determination and analysis of nucleotide sequencing and phylogenetic analysis.Results:A total of 460 enteroviruses strains were isolated from 738 samples, including pathogens strains: 258 CV-A16 (56.09%), 62 EV-A71 (13.48%), 49 CV-A10 (10.65%), 44 CV-A6 (9.57%) and 9 CV-A4 (1.96%). Eight CV-A6 positive specimens were isolated from the viruses and the nucleotide-sequence analysis of the whole VP1 region was conducted. The sequence analysis of eight CV-A6 strains demonstrated that the homologies of nucleotide and amino acid were 96.12% - 100% and 97.78% - 100% respectively. The phylogenetic analysis indicated that the eight CV-A6 strains were subdivided into the genotype D subtype D3. Compared with the reference strain, CVA6-Gdula-AY421764, amino acids of CV-A6 strains in Yantai city observed at sites 10, 14, 174, 194, 279, 283 and 305 in VP1 region appeared mutant.Conclusions:CV-A16, EV-A71, CV-A10 and CV-A6 were the main common pathogens of HFMD in Yantai region in 2016. All the CV-A6 strains isolated in this study belonged to subtype D3 in genotype D.

In this study, the swabs were collected among patients with an influenza-like illness (ILI) admitted to 2 sentinel surveillance hospitals of Yantai from April 2014 to August 2017. All specimen were cultured and identified by hemagglutination inhibition assay. Complete sequences of Hemagglutinin (HA) of influenza A were amplified, sequenced and analyzed using molecular and phylogenetic methods. The potential vaccine efficacy were calculated using Pepitope model. The results showed that the antigenicity of A (H3N2) had changed greatly. 8 strains of influenza A (H1N1) pdm09 belonged to subclade 6B.1 and 14 strains clustered in 6B.2. 12 strains of influenza A (H3N2) fell into subgroup 3C.3a and 33 strains clustered in 3C.2a. Several residues at antigen sites and potential glycosylation sites had changed in influenza A strains. Vaccine efficacy of influenza A (H1N1) pdm09 in 2015/2016 and 2016/2017 seasons were 77.29% and 79.11% of that of a perfect match with vaccine strain, meanwhile vaccine efficacy of influenza A (H3N2) in 2014/2015, 2015/2016 and 2016/2017 were-5.18%, 16.97% and 42.05% separately. In conclusion, the influenza A virus circulated in Yantai from 2014 to 2017 presented continual genetic variation. The recommended vaccine strains still afforded protection against influenza A (H1N1) pdm09 strains and provided suboptimal protection against influenza A (H3N2) strains.

In this study, the swabs were collected among patients with an influenza?like illness (ILI) admitted to 2 sentinel surveillance hospitals of Yantai from April 2014 to August 2017. All specimen were cultured and identified by hemagglutination inhibition assay. Complete sequences of Hemagglutinin (HA) of influenza A were amplified, sequenced and analyzed using molecular and phylogenetic methods. The potential vaccine efficacy were calculated using Pepitope model. The results showed that the antigenicity of A (H3N2) had changed greatly. 8 strains of influenza A (H1N1) pdm09 belonged to subclade 6B.1 and 14 strains clustered in 6B.2. 12 strains of influenza A (H3N2) fell into subgroup 3C.3a and 33 strains clustered in 3C.2a. Several residues at antigen sites and potential glycosylation sites had changed in influenza A strains. Vaccine efficacy of influenza A (H1N1) pdm09 in 2015/2016 and 2016/2017 seasons were 77.29% and 79.11% of that of a perfect match with vaccine strain, meanwhile vaccine efficacy of influenza A (H3N2) in 2014/2015, 2015/2016 and 2016/2017 were-5.18%, 16.97% and 42.05% separately. In conclusion, the influenza A virus circulated in Yantai from 2014 to 2017 presented continual genetic variation. The recommended vaccine strains still afforded protection against influenza A (H1N1) pdm09 strains and provided suboptimal protection against influenza A (H3N2) strains.

In this study, the swabs were collected among patients with an influenza?like illness (ILI) admitted to 2 sentinel surveillance hospitals of Yantai from April 2014 to August 2017. All specimen were cultured and identified by hemagglutination inhibition assay. Complete sequences of Hemagglutinin (HA) of influenza A were amplified, sequenced and analyzed using molecular and phylogenetic methods. The potential vaccine efficacy were calculated using Pepitope model. The results showed that the antigenicity of A (H3N2) had changed greatly. 8 strains of influenza A (H1N1) pdm09 belonged to subclade 6B.1 and 14 strains clustered in 6B.2. 12 strains of influenza A (H3N2) fell into subgroup 3C.3a and 33 strains clustered in 3C.2a. Several residues at antigen sites and potential glycosylation sites had changed in influenza A strains. Vaccine efficacy of influenza A (H1N1) pdm09 in 2015/2016 and 2016/2017 seasons were 77.29% and 79.11% of that of a perfect match with vaccine strain, meanwhile vaccine efficacy of influenza A (H3N2) in 2014/2015, 2015/2016 and 2016/2017 were-5.18%, 16.97% and 42.05% separately. In conclusion, the influenza A virus circulated in Yantai from 2014 to 2017 presented continual genetic variation. The recommended vaccine strains still afforded protection against influenza A (H1N1) pdm09 strains and provided suboptimal protection against influenza A (H3N2) strains.

Objective To knock out the Abcb1 gene of rat,and establish the Abcb1 humanized rat model based on the Abcb1 knock out rat.Methods The animal model was established using BAC and CRISPR/Cas9 technology,and was analyzed by PCR, RT-PCR and real-time PCR.Results Establishing a rat model expressing human Abcb1 stably by transfer the 153 kb BAC containing human Abcb1 promoter and cDNA into rat genome, and establishing the Abcb1 knock out rat at the same time.Establishing the Abcb1 humanized model by crossing these two strains together.The expression pattern of Abcb1 in Abcb1 humanized rat is different from the wild type rat.The Abcb1 humanized model express not only the human Abcb1 gene but also has similar expression pattern as human.Conclusions The Abcb1 knock out rat and the Abcb1 humanized rat were successfully established, and this model is close to human concerning about the drug metabolism related to Abcb1.

Objective To obtain more physiological data of Leptin knockout SD rats available for the user , the long-term observation of fasting blood glucose and pathological phenotypes were performed .Methods The protein expression levels in liver tissues were determined by western blot .Body weight of Leptin knockout rats ( Leptin-/-) and littermate lean rats (Leptin+/+) were weighed up at 1,3,6,8 months of age.Fasting blood glucose of Leptin +/+rats and Leptin-/-rats at 1,3,6,8 months of age were measured using One Touch? brand blood glucose monitoring systems.Pathological changes of pancreas and livers of Leptin -/-rats were observed by the method of HE staining and Immunohistochemistry (IHC).Results Short null Lepin proteins were expressed in liver tissues from Leptin -/-rats. Leptin-/-rats become heavier than Leptin +/+rats since they were one month old .The body weight of Leptin -/-rats at 8 months of age was twice as heavy as Leptin +/+rats, female Leptin-/-rats weighing 884g, and male Leptin-/-rats weighing 1200g.Overt hyperglycemia was observed during the first month after birth .Compared with Leptin+/+female rats,the fasting blood glucose of Leptin -/-female rats was increased by 40%-26%from 1 to 6 months old. After that, blood glucose values decreased and eventually become nearly normal at 8 months of age.Pathological examination indicated that Leptin -/-rats at 8 months of age had a fatty liver , more pancreas islets with lager volume and more beta cells with increased insulin secretion .Conclusion Leptin-/-rat were characterized by obesity , fatty liver, islet cell hyperplasia and early hyperglycemia .

Objective To study the relationship of insulin receptor substrate-1 (Irs1) and metabolic disease, we generated Irs1 gene knockout rat by CRISPR/Cas9 system.Methods Two sgRNA targeting sites were designed for Irs1 targeting.The Cas9 and sgRNAs were transcribed by T7 RNA polymerase in vitro.Cas9 mRNA and sgRNA mixtures were pooled and microinjected into one-cell fertilized eggs of SD rats to generate rats with targeted mutation .Results Five rats with the mutations were detected with the efficiency of 83%.Conclusion The Irs1 gene knockout rats generated in this study can be transmitted by germline .

Objective To investigate the functional role of 25 microRNAs in breast cancer ,and to find new tumor suppressor microRNAs that may serve as specific targets of new gene therapies . Methods Twenty-five microRNAs expression vectors were constructed and stably transfected into mouse mammary tumor cells 4To7 by Lipofectamine2000. Cells were selected with G418 and sorted by Flow cytometry.The cells in logarithmic phase were collected and 2 ×105 cells/mouse was inoculated into BALB/c mice via tail vein .Lungs were harvested 14 days after tumor cell inoculation , and the number of metastasis foci was counted .Results Mice inoculated with mir-449a-expressing 4To7 cells via tail vein developed reduced lung metastases compared with mice inoculated with negative control cells .Mice inoculated with mir-1935-expressing 4To7 cells via tail vein developed increased lung metastases compared with mice inoculated with negative control cells .Other twenty-three microRNAs neither promoted nor inhibited lung metastases of breast cancer .Conclusions Two of twenty-five microRNA were identified to be associated with breast cancer metastasis .MiR-449a may play a tumour suppressor role in the regulation of migration and metastasis in breast cancer .miR-1935 transgenic over-expression promoted tumor growth and metastasis .

Objective To establish the sperm specific Sleeping Beauty ( SB ) transposase-expression transgenic mouse for the study of the genetic modification mediated by transposon system in mouse .Methods Prm1 promoter was cloned from mouse genomic DNA to drive the expression of SB transposase .The transgenic mice were generated by microinjection .The gene type of transgenic line was identified by PCR .The expressing level in testis was determined by western blot and immunohistochemistry (IHC) staining.Results Five lines of transposase transgenic mice were obtained by microinjection and three can be germline .One mouse line with higher expression level of transposase in the testis was obtained.Conclusion One transgenic mouse model with Sleeping Beauty transposase - expression was successfully established .This model will greatly contribute to the research of genetic modification mediated by transposon in mouse.