Objective:To study the complete genome characterization of Human Astrovirus (HAstV) in Shandong Province.Methods:Stool samples from acute flaccid paralysis (AFP) surveillance in Shandong Province from 2020 to 2022 were collected, and HAstV nucleic acid was examined by real-time quantitative PCR (qPCR). Next-generation sequencing (NGS) was conducted for the positive samples to obtain complete genome sequences and identify the genotype. Homology comparison and phylogenetic analysis were performed by using BioEdit and Mega software.Results:A total of 667 samples were examined by qPCR, of which 14 were HAstV-positive (2.1%), including HAstV-1 ( n=6), MLB1 ( n=6), MLB2 ( n=1), and VA2 ( n=1). The complete genome sequences were obtained from 11 samples. The six HAstV-1 sequences of this study had 98.2% to 99.9% nt similarities with each other and 87.6% to 98.6% with those from other regions. The four MLB1 sequences of this study had 99.1% to 99.9% nt similarities with each other and 92.2% to 99.4% with those from other regions. The VA2 sequence of this study had 96.0% to 96.3% nt similarities with those from other regions. Phylogenetic analysis based on ORF2 region showed that the local HAstV-1 sequences were most closely related to Japanese strains, and had distinct topology with phylogenies based on ORF1a and ORF1b regions. Conclusion:The complete genome sequences of 11 HAstV strains are obtained, and the VA2 complete genome is found.

Objective:To investigate the complicated virus infection of infants with pertussis and its effect on the disease.Methods:From January 2019 to March 2020, a total of 100 hospitalized infants with pertussis were admitted to the Second Affiliated Hospital of Medical College of Shantou University, nasopharyngeal swabs were collected for detection of ten pathogens including pertussis, namely respiratory syncytial virus(RSV), parainfluenza virus(PIV), bordetella pertussis (BP), human rhinovirus(HRV), human bocavirus(HBoV), human metapneumovirus(hMPV), influenza B virus (INF-B), adenovirus, influenza A virus and cytomegalovirus(CMV). According to the results of pathogen detection, all infants were divided into single detection group of BP(single detection group) and co-detection group of BP combined with viruses(co-detection group). The clinical data of the two groups were retrospectively analyzed and compared to explore the differences of clinical characteristics and its impact on the course of disease.Results:Among 100 cases, there were 54(54.0%) boys and 46(46.0%)girls.The age ranged from 28 days to 2 years and 5 months, with a median age of 3.5 months.Fifty-six cases were classified as single detection group, while 44 cases were included into co-detection group.Among infants in co-detection group, fourteen cases were co-infected with CMV(31.8%, 14/44), seven cases with HRV(15.9%, 7/44), seven cases with PIV(15.9%, 7/44), four cases with RSV(9.1%, 4/44), one case with hMPV(2.2%, 1/44), eight cases with CMV+ HRV(18.2%, 8/44), one case with HRV+ HBoV (2.2%, 1/44), one case with CMV+ PIV(2.2%, 1/44)and one case with CMV+ PIV+ INF-B(2.2%, 1/44). The number of infants in the single detection group who had cyanosis before treatment, requiring repiratory support, PICU admission, severe pneumonia or abnormal myocardial enzymes were higher than those in the co-detection group( P<0.05), while the months of age were lower than that in the co-detection group( P<0.05). When comparing the clinical characteristics of infants over three months of age, only the number of cases of combined cyanosis before treatment and the number of days in hospital were higher in the single detection group than those in the co-detection group ( P<0.05), no statistically significant differences were found in the other clinical characteristics between the two groups( P>0.05). Conclusion:The cases of infants requiring repiratory support, complicated with severe pneumonia or abnormal myocardial enzymes in the single detection group are higher than those in the co-detection group, which may be attributed to the small age of months.

Objective:The investigation and research on the application status of Hepatic Venous Pressure Gradient (HVPG) is very important to understand the real situation and future development of this technology in China.Methods:This study comprehensively investigated the basic situation of HVPG technology in China, including hospital distribution, hospital level, annual number of cases, catheters used, average cost, indications and existing problems.Results:According to the survey, there were 70 hospitals in China carrying out HVPG technology in 2021, distributed in 28 provinces (autonomous regions and municipalities directly under the central Government). A total of 4 398 cases of HVPG were performed in all the surveyed hospitals in 2021, of which 2 291 cases (52.1%) were tested by HVPG alone. The average cost of HVPG detection was (5 617.2±2 079.4) yuan. 96.3% of the teams completed HVPG detection with balloon method, and most of the teams used thrombectomy balloon catheter (80.3%).Conclusion:Through this investigation, the status of domestic clinical application of HVPG has been clarified, and it has been confirmed that many domestic medical institutions have mastered this technology, but it still needs to continue to promote and popularize HVPG technology in the future.

Objective:To explore newer enteroviruses (EVs) circulating in China and research their genetic characterization.Methods:Sewage samples were collected in Jinan in January and July of 2018, respectively. PCR amplification of the P1 coding region of EV and next-generation sequencing (NGS) were conducted. Homologous and phylogenetic analyses were performed on the newly identified newer EVs.Results:The EV-A89 (n=2) and EV-C96 (n=1) nucleic acid sequences were detected in the sewage in January and July, respectively and obtained complete P1 coding sequence. The two EV-A89 sequences had 6.1% nucleotide divergence among themselves, and had 88.2%-95.3% homologies with other strains. No close genetic relationship was obtained with the sequences from Xinjiang, China and those from other countries. The EV-C96 sequence in this study had 76.2%-89.4% nucleotide similarities with other isolates throughout the world. Phylogenetic analysis showed that global EV-C96 strains were divided into 5 branches, i. e, A to E. Except for the Shandong 1991 isolate, the other Shandong and domestic strains were all located in branch D.Conclusions:This study added the molecular epidemiologic data of newer enteroviruses EV-A89 and EV-C96, and provides basic data for future research on the epidemic trend of EV-A89 and EV-C96 as well as their association with diseases.

Objective:To explore the effects of deleted in lung and esophageal cancer 1 (DLEC1) on osteosarcoma cells and the underlying mechanism.Methods:Immunohistochemical staining for DLEC1 was scored in sixteen paired osteosarcoma tissues and adjacent normal tissues obtained. The present study was conducted on human osteosarcoma 143B cells which were randomly divided into two groups, pDC316-DLEC1 transfection group and pDC316-Null transfection group. Differences in the proportion of EdU-positive cells, cell cycle distribution, proportion of apoptosis cells, number of migrating and invasive cells, expression of epithelial-mesenchymal transformation (EMT) markers (E-cadherin and vimentin), relative protein expression levels of NF-κB, AKT and ERK signaling pathways were assessed between the pDC316-DLEC1 and pDC316-Null transfection groups in in vitro study. The subcutaneous inoculation model and tail vein injection model were developed to evaluate the differences in subcutaneous tumor volume, subcutaneous tumor weight and pulmonary tumor nodules between the above two groups in in vivo study.Results:The DLEC1 immunostaining scores for osteosarcoma tissues and adjacent normal tissues were 2.88±1.15 and 4.25±1.06, respectively. The proportions of EdU-positive cells (36.47%±1.90% vs 51.47%±2.89%) and S phase cells (33.31%±0.61 vs 43.77%±1.47%) were decreased, while G0/G1 phase cells (46.87%±0.73% vs 35.47%±1.14%) and apoptotic cells (13.83%±1.01% vs 3.30%±0.26%) were increased in the pDC316-DLEC1 transfection group compared to those in the pDC316-Null transfection group. Decreased number of migrating cells (199.00±12.53 vs 369.67±10.02) and invasive cells (104.67±9.07 vs 299.67±12.06) and relative expression of vimentin mRNA (0.59±0.02 vs 1.00±0.02) and protein (0.54±0.08 vs 1.00±0.00) were observed in the pDC316-DLEC1 transfection group, while relative expression of E-cadherin mRNA (2.40±0.05 vs 1.00±0.02) and protein(1.98±0.10 vs 1.00±0.00) in the pDC316-DLEC1 transfection group were higher than those in the pDC316-Null transfection group. The relative protein expression of NF-κB (p65), p-AKT (Ser473) and p-ERK (Thr202/Tyr204) in the pDC316-DLEC1 transfection group were decreased by 51.67%±4.04%, 64.67%±5.51% and 48.67%±4.73% compared to those in the pDC316-Null transfection group. In in vivo study, 143B cells in the pDC316-DLEC1 transfection group formed smaller (320.00±145.22 mm 3vs 798.00±221.94 mm 3) and lighter (0.49±0.17 g vs 0.88±0.14 g) subcutaneous tumors and less metastatic lung nodules (7.71±1.80 vs 20.86±3.53) compared with those in the pDC316-Null transfection group. Conclusion:Overexpression of DLEC1 could suppress the NF-κB/AKT/ERK signaling pathways in 143B cells, which further induces G0/G1 arrest and apoptosis that ultimately inhibits cell proliferation and reduces the metastatic potential through reversing EMT.

Objective： ：To investigatetheeffectofsalidroside(SAL)onthephenotype of dendritic cells (DCs) and the antitumor ability of cytotoxic T lymphocytes (CTL). Methods： ：Lewis lung cancer cell line 3LL, wild type (WT) C57BL/6 mice and TLR4-/- C57BL/6 mice were chosen for this study. Mice bone marrow derived DC precursor cells were obtained to differentiate into immature DCs, which were harvested on the sixth day of culture. CD11c+ DCs were obtained by magnetic beads screening, and further divided into PBS group, SAL group and lipopolysaccharide (LPS) group.After being cultured for 48 h, the effects of SAL on surface molecules and phagocytosis of DCs as well as the efffect of TLR4 pathway on the killing effect of T cells were detected by Fow cytometry. Results： ： Compared with PBS group, expressions of DC surface molecules CD80, CD86 and MHC Ⅱ significantly increased (all P<0.05), phagocytosis significantly decreased (P<0.05), and TLR4 expression level significantly increased (P<0.01) in SAL group; Compared with WT group, after being treated with SAL or LPS, the expressions of DC surface molecules CD80, CD86 and MHC Ⅱ decreased significantly in TLR4-/- group (all P<0.05); ComparedwithPBSgroup,theactivatedCTLinSALgroupexhibited a significantly elevated killing effect against lung cancer 3LLcells (P<0.05). Conclusion：SAL can induce DC maturation by regulating TLR4, thus improving the killing ability of T cells.

Objective:To understand the detection of group A rotavirus (RVA) in domestic sewage and its molecular epidemiological characteristics, and further explore the feasibility and necessity of RVA environmental surveillance.Methods:From 2016 to 2018, we collected domestic sewage samples monthly in Jinan city, and concentrated them via anion membrane adsorption-elution method. Then RNA extraction and RVA VP7 and VP4 coding region RT-PCR amplification were performed. After purification, TA cloning and sequencing, homology analysis and phylogenetic analysis were conducted on the obtained sequences.Results:RVA G gene was detected in 31 of the 36 sewage samples (86.1% detection rate); RVA P genotype was detected in 33 samples (91.7% detection rate). A total of 536 RVA sequences were obtained, of which 225 G-type sequences belonged to 6 genotypes, and the G9 accounted for 92.4% (208/225); 311 P-type sequences were obtained, which belonged to 4 genotypes. The dominant P[8] accounted for 50.1% (156/311), followed by P[4] with 41.8% (130/311). Phylogenetic analysis shows that there were multiple transmission chains circulating in the dominant genotypes G9 and P[8].Conclusion:The genotype, homology, and phylogenetic characteristics of sequences obtained from domestic sewage in Jinan area were described, which further confirmeing that RVA environmental surveillance is not feasible but also necessary.

Objective:To understand the detection of group A rotavirus (RVA) in domestic sewage and its molecular epidemiological characteristics, and further explore the feasibility and necessity of RVA environmental surveillance.Methods:From 2016 to 2018, we collected domestic sewage samples monthly in Jinan city, and concentrated them via anion membrane adsorption-elution method. Then RNA extraction and RVA VP7 and VP4 coding region RT-PCR amplification were performed. After purification, TA cloning and sequencing, homology analysis and phylogenetic analysis were conducted on the obtained sequences.Results:RVA G gene was detected in 31 of the 36 sewage samples (86.1% detection rate); RVA P genotype was detected in 33 samples (91.7% detection rate). A total of 536 RVA sequences were obtained, of which 225 G-type sequences belonged to 6 genotypes, and the G9 accounted for 92.4% (208/225); 311 P-type sequences were obtained, which belonged to 4 genotypes. The dominant P[8] accounted for 50.1% (156/311), followed by P[4] with 41.8% (130/311). Phylogenetic analysis shows that there were multiple transmission chains circulating in the dominant genotypes G9 and P[8].Conclusion:The genotype, homology, and phylogenetic characteristics of sequences obtained from domestic sewage in Jinan area were described, which further confirmeing that RVA environmental surveillance is not feasible but also necessary.

Objective: To understand the genotype distribution and molecular epidemiological characteristics of the group A rotavirus (RVA) in domestic sewage, and further explore the importance of environmental surveillance in investigating RVA regional circulation. Methods: Sewage samples were collected monthly in the city of Yantai from January 2014 to December 2016. After concentration, total RNA was extracted, and RVA VP7 and VP4 coding regions were amplified via RT-PCR. PCR products were purified, cloned and Sanger sequenced. Genotyping and phylogenetic analysis was conducted based on the sequences. Results: Thirty-six sewage samples were collected and 86.1% was positive with VP7 and VP4 sequences. A total of 205 VP7 and 239 VP4 nucleotide sequences were obtained, belonging to 4 G genotypes and 6 P genotypes. Among these, G9 (95.6%, 196/205), P[8] (58.6%, 140/239) and P[4] (28.0%, 67/239) were the most common genotypes. Phylogenetic analysis for G9, P[8] and P[4] sequences revealed co-circulation of multiple transmission chains in local population. Conclusions This study describes the genotype distribution and sequence characteristics of local RVA in Shandong province, and the result demonstrate that surveillance on environmental sewage is an effective way in investigating RVA molecular epidemiology.