Objective:To characterize and analyze the genetic variation of hemagglutinin (HA) of influenza A (H1N1) pdm09 subtype virus in Shandong Province, and explore the genetic variation patterns for providing reference for influenza monitoring, epidemic prevention and control, and vaccine strain selection.Methods:HA gene sequences of the recommended strains of influenza vaccine from 2009 to 2024 and the representative strains of each branch were downloaded from the GISAID Influenza Data Platform, and were phylogenetically analyzed and characterized in terms of amino acid site variation with the HA gene sequences of 298 influenza A (H1N1) virus strains isolated from Shandong Province. A phylogenetic tree was constructed using the maximum likelihood (ML) method of the IQ-TREE online tool, and the amino acid site variants were viewed using MegAlign software. The potential glycosylation sites of the HA gene were predicted using the NetNGlyc 1.0 online software.Results:The HA gene homology of the 298 influenza A (H1N1) viruses isolated in Shandong Province ranged from 91.2% to 100.0%. The evolutionary branches were gradually distantly related over time, but the direction of evolution was roughly the same as that in other provinces. Amino acid mutations in the HA occurred every year and most were found in the antigenic determinants.Conclusions:The HA genes of influenza viruses isolated in Shandong Province from 2009 to 2024 are still in the process of continuous evolution, and continuous monitoring of the epidemiological trends and the evolutionary directions of influenza viruses is essential for early warning of influenza virus pandemics.

Objective:To analyze the cause and epidemiological characteristics of an outbreak of cutaneous anthrax in Caoxian County, Heze City, Shandong Province, and to provide scientific basis for anthrax prevention and control.Methods:Using on-site epidemiological investigation methods and the "Anthrax Epidemiological Case Investigation Form", case investigations were conducted based on the epidemiological contact history and close contacts of suspected anthrax cases reported by the national health care system ( n = 83). Scorched skin smears, diseased cattle tissues, soil samples from the slaughter site and smears from slaughter utensils were collected from cases for Real-time PCR testing and pathogenic bacteria isolation and culture, respectively. Anthrax determination criteria were carried out with reference to "Anthrax Diagnosis" (WS 283-2020). Results:A total of 13 cases of cutaneous anthrax were found in this outbreak, including 12 clinically diagnosed cases and one confirmed case (positive Real-time PCR test and isolation of a strain of Bacillus anthracis). The epidemiological investigation determined that the source of infection in this outbreak was diseased cattle, the transmission route was through slaughter of diseased cattle, contact with contaminated utensils and related cattle products, and the patients were mainly engaged in occupations related to cattle slaughter or cattle product collection and sale. A total of 84 samples were collected, including 13 skin scabs, 64 environmental samples and 7 beef samples. Thirty-six positive PCR tests were performed, with a positive rate of 42.86% (36/84). Among them, 100.00% (13/13) were positive for skin scab smear specimens, 29.69% (19/64) for environmental samples and 4/7 for beef samples. A total of 8 strains of Bacillus anthracis were isolated, including 6 environmental specimens, 1 suspected case and 1 beef strain, with an overall detection rate of 9.52% (8/84). Eighty-three close contacts were investigated. Thirteen households involved in the epidemic were disinfected by spraying (200 ml/m 2) with chlorine-containing disinfectant (5 000 mg/L), and a total of 40 households involved in the epidemic were disinfected, covering an area of about 10 765 m 2. Forty-five pieces of suspected contaminated clothing were burned and disposed of, and 152 pieces of kitchenware were soaked. Conclusions:Slaughter of infected cattle, contact with contaminated utensils and related cattle products are the main causes of this skin anthrax outbreak. Strengthening market supervision, deepening inter-animal epidemic prevention, carrying out publicity and education on anthrax prevention and control, and enhancing practitioners' awareness of disease prevention is the key to prevent anthrax from occurring.

Objective:To investigate the form of programmed cell death (PCD) induced by severe fever with thrombocytopenia syndrome virus (SFTSV) infection in Vero cells and further explore the existence of pyroptosis, so as to provide new ideas for studying the pathogenic mechanism of SFTSV.Methods:Vero cells were infected with SFTSV at different multiplicity of infection (MOI), cytopathic effect (CPE) was observed daily, cell viability was detected by CCK-8 method, and cell membrane damage was detected by LDH release test to determine the optimal amount of virus infection and cell death time. Vero cells were pretreated with different PCD inhibitors and infected with SFTSV. CCK-8 kit was used to detect the cell viability and determine the death form of PCD caused by SFTSV. The expression of pyroptosis related proteins was detected by Western blotting to further explore the existence of pyroptosis.Results:At 48 h and 72 h after SFTSV infected Vero cells with MOI=10, the optimal infection amount and time of subsequent experiments were observed. At this time, the CPE of cells was obvious, the cell viability decreased to 51% and 41% of the control group ( P<0.001, P<0.001), and the LDH release amount reached 24% and 37% of the maximum enzyme activity release wells, were 3.8, 3.4 times of LDH release in the control group ( P<0.001, P<0.001). The inhibition of SFTSV-induced cell death by different PCD inhibitors showed that pan-caspase inhibitor and receptor-interacting serine/threonine-protein kinase 3 (RIP3) inhibitor had inhibitory effects at 48 h and 72 h. The cell viability was 2.1 and 1.6 times of the viral control group at 48 h, 2.3 and 1.7 times of the viral control group at 72 h, and the effect of pan-caspase inhibitor was significantly higher than that of the other inhibitor groups. Caspase-1 and caspase-3 inhibitors only had inhibitory effect at 48 h, and the cell viability was 1.5 and 1.3 times of the viral control group. After SFTSV infection of Vero cells, the expressions of caspase-1 and IL-1β increased gradually with the prolongation of time, and reached 3.4 and 9.5 times of the control group at 48 h, respectively ( P<0.001, P<0.001). Conclusions:SFTSV infection of Vero cells can lead to various forms of PCD, including apoptosis, pyroptosis and programmed necrosis, and pyroptosis related protein activation can be detected in the process of PCD, which further explored the existence of pyroptosis.

Objective:To explore the relationship between severe fever with thrombocytopenia syndrome virus (SFTSV) Gc and its N-glycosylation site and viral infectivity, a recombinant pseudovirus containing SFTSV Gc glycosylation site mutant was constructed.Methods:The eukaryotic expression vectors pcDNA3.1(+ )-GC, PCDNA3.1(+ )-GC(N291Q), PCDNA3.1(+ )-GC(N352Q) and PCDNA3.1(+ )-GC (N374Q) were constructed by site-directed mutagenesis and homologous recombination. After their successful expression in 293T cells, we infected VSVΔG-Fluc*G pseudovirus, constructed four recombinant pseudoviruses and tested their effects on the cell force of infection.Results:Double digestion identification and sequence determination confirmed the successful construction of eukaryotic expression vectors pcDNA3.1 (+ )-Gc, pcDNA3.1 (+ )-Gc(N291Q), pcDNA3.1 (+ )-Gc(N352Q) and pcDNA3.1 (+ )-Gc(N374Q). Indirect immunofluorescence and Western Blotting result indicated the successful expression of all the four recombinant plasmids. SFTSV Gc recombinant pseudoviruses are specific for infecting Vero cells. Pseudovirus infection capacity was decreased significantly after the glycosylation site mutation, and the mutant strain with the glycosylation site at position 352 had the lowest level of infectivity ( P<0.001, P=0.001). Conclusions:The glycosylation site of SFTSV Gc may be associated with the infectious effect of the viral infection, and the amino acid mutation at position 352 has the greatest effect on the viral infectivity.

Objective:To explore the effect of key amino acid mutations of 3D protein in enterovirus 71 on viral proliferation, and infer the mechanism of mutation affecting viral proliferation according to the tertiary structure model of 3D protein.Methods:The proliferation characteristics of the mutant strain that was constructed by site-directed mutagenesis and reverse genetics using the pMD19T-SDLY107-EGFP constructed from the fatal strain SDLY107 as a template were measured. Meanwhile the tertiary structure of the 3D protein was predicted, and then the possible mechanism of the mutation affecting the proliferation ability of the virus was speculated according to the functional characteristics of the domain of the 3D protein.Results:Two mutant viruses, eGFP-EV-A71 (S37N) and eGFP-EV-A71 (R142K), were successfully constructed by double enzyme digestion and viral gene sequencing. The proliferation rate of the two mutant strains in RD cells was significantly lower than that of the parent strains. The 3D protein tertiary structure prediction model showed that the 3D protein consisted of three domains: "Finger" , "thumb" and "Palm" , constituting a cupped right-handed structure. S37N and R142K are located in the "thumb" domain and " finger" domain, respectively. The "thumb" and "finger" domains have important effects on the activity of 3D polymerase and the stability of protein.Conclusions:Mutations at S37N and R142K sites of EV71 3D protein decrease the replication ability of EV71, and these two mutations may affect the proliferation of EV71 virus by changing the 3D protein polymerase activity and the interactions between multiple domains.

Objective:To analyze the risk factors and human papillomavirus (HPV) genotypes distribution in patients with condyloma acuminatum (CA) in two regions of Shandong province.Methods:From August 2019 to December 2020, an anonymous questionnaire survey of CA patients was conducted in three hospitals in Jinan City and Jining City, Shandong Province, and samples were collected for HPV typing. Multivariate binary logistic regression was used to analyze the risk factors of CA recurrence. HPV typing was detected by PCR-reverse dot blot hybridization.Results:A total of 653 questionnaires were collected, and the valid questionnaires accounted for 98.77% (645/653). Recurrence of the disease occurred in 174 patients, with a recurrence rate of 26.98%. Univariate analysis showed that there were statistically significant differences in the distribution of CA recurrence among residence time at current address, sexual frequency, genitalia cleaning, and knowledge of preventing HPV infection ( P<0.05). Multivariate binary logistic regression showed that knowing how to prevent HPV infection was a significant factor that influences CA recurrence. A total of 428 patients underwent HPV typing, and the positive detection rate of HPV was 98.60% (422/428). The top three positive rates were HPV6 (57.58%), HPV11 (36.49%) and HPV16 (11.37%). The main type of infection was low-risk HPV, accounting for 51.42% (217/422). Conclusions:CA patients have the phenomenon of "separation of knowledge and action" , so it is necessary to strengthen health education and behavioral intervention, guide the population to correctly treat sexual behavior, and improve self-prevention awareness and risk awareness.

Objective:We try to screen out predictive indicators with higher value by analyzing the differences in clinical and laboratory indicators between severe fever with thrombocytopenia syndrome (SFTS) patients in the intensive care unit (ICU) group and non-ICU group.Methods:The clinical and laboratory index data of 69 SFTS patients diagnosed in the laboratory in a hospital from June to December 2019 were retrospectively collected. According to the clinical outcome of the patients, they were divided into ICU and non-ICU groups. The differences in clinical manifestations and laboratory indicators between the two groups were analyzed. The receiver operating characteristic curve (ROC) was used to screen the more valuable predictive indicators.Results:Compared with the non-ICU group, ICU group SFTS patients had significantly higher procalcitonin (PCT), C-reactive protein (CRP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), glutamyl transpeptidase (GGT), leucine aminopeptidase (LAP), glutamate dehydrogenase (GDH), adenosine deaminase (ADA), cystatin C (Cys C), α-hydroxybutyrate dehydrogenase (α-HBDH), creatine kinase (CK), lactate dehydrogenase (LDH) levels ( W=530.0, P=0.003; W=496.5, P=0.015; W=496.0, P=0.015; W=535.5, P=0.002; W=545.5, P=0.001; W=498.5, P=0.013; W=537.0, P=0.002; W=523.0, P=0.004; W=512.0, P=0.007; W=502.0, P=0.012; W=486.0, P=0.023; W=509.0, P=0.008; W=541.0, P=0.002) and significantly lower platelet count (PLT), indirect bilirubin (IBIL), albumin/globulin ratio(A/G) and superoxide dismutase (SOD) levels ( W=199.0, P=0.024; W=175.5.5, P=0.009; t=-2.9, P=0.004; W=209.5, P=0.036; t=-3.0, P=0.004). ROC result showed that ALP [area under the curve (AUC)=0.804, 95% confidence interval ( CI) (0.679~0.929)] and LDH [AUC=0.805, 95% CI (0.680~ 0.930)] have a higher value for predicting the risk of severe illness. Conclusions:Abnormal liver function, heart function, and renal function indicators in SFTS patients indicate that patients are at risk of exacerbation. Among them, ALP and LDH levels have higher predictive value for risk of severe disease, suggesting that the monitoring of patients with the above symptoms should be strengthened in the clinical nursing process.

Objective:To rescue enterovirus group A type 71 (EV-A71) VP1 protein mutant strain (Val147→Ala) and explore the effect of this locus on viral virulence.Methods:The SDLY107 constructed plasmid pMD19-T-EGFP-107 was used as template, the recombinant plasmid pMD19-T-EGFP-107 (VP1-1) was constructed by site-directed mutation and reverse genetics. The recombinant plasmid was transfected into BSR-T7/5 cells, and the transfection products were subcultured in RD cells for three times. The mutant strain SDLY-EGFP-107(VP1-1) was successfully saved. The viral replication was detected by qRT-PCR, and the cell damage caused by the virus was detected by cell proliferation (CCK-8) and lactate dehydrogenase (LDH) tests.Results:The target fragment was amplified by overlapping fusion PCR, and the size was about 3.4 kb. The recombinant plasmid was identified by double enzyme digestion and the mutation was verified by sequencing. Obvious fluorescence was observed 24 hours after transfection of BSR-T7/5 cells with recombinant plasmid and 24 hours after inoculation into RD cells. The replication ability of mutant strain SDLY107 (VP1-1) in RD cells was weaker than that of virulent strain SDLY107 ( t=9.58, P<0.001) by qRT-PCR. CCK-8 and LDH tests showed that the cytotoxicity of mutant strain SDLY-EGFP-107(VP1-1) in RD cells ( t=106.60, P<0.001; t=39.88, P<0.001), SH-SY5Y cells ( t=18.72, P<0.001; t=19.09, P<0.001) were significantly weaker than that of virulent strain SDLY107. Conclusions:The mutant strain SDLY-EGFP-107(VP1-1) was successfully saved, which confirmed that the mutation of the 147th amino acid site of VP1 protein could reduce the replication and cell damage of the virus, providing a basis for further study of the role of VP1 protein in the pathogenesis of EV-A71.

OBJECTIVE: To analyze the clinical and genetic features of three patient diagnosed with Kleefstra syndrome. METHODS: Whole exome sequencing (WES) was carried out for the probands and their parents. Suspected variants were validated by Sanger sequencing. Copy number variations (CNV) were detected by CNV-seq and validated by real-time PCR. RESULTS: Proband 1 was found to carry a de novo heterogeneous variant (c.823+1G>T) of the EHMT1 gene, which may affect its expression. Based on the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic (PVS1+PS2+PM2). Proband 2 was found to carry a de novo missense variant c.439C>G (p.L147V) of the EHMT1 gene, which was predicted to be likely pathogenic (PS2+PM1+PM2+PP3). Proband 3 was found to carry a heterozygous 520 kb deletion at 9q34.3 by CNV-seq. The deletion has encompassed the whole of the EHMT1 gene. Real-time PCR has detected no CNV of this region in her parents. CONCLUSION Variants of the EHMT1 gene probably underlay the disease in these patients. Genetic testing has provided a basis for their clinical diagnosis.
Chromosome Deletion ; Chromosomes, Human, Pair 9 ; Craniofacial Abnormalities ; DNA Copy Number Variations ; Female ; Genetic Testing ; Heart Defects, Congenital ; Humans ; Intellectual Disability/genetics* ; Mutation

Objective:To study the amino acid site variation of HIV-1 Tat protein from different parts of AIDS patients with HAD and non HAD and its effect on oxidative stress of U87 cells.Methods:HIV-1 Tat amino acid sequences were analyzed by BLAST and MEGA6 software to study the variation of amino acid sites in four parts of central nervous tissue basal ganglia(BG) and peripheral spleen (SPL) of an HIV-associated dementia (HAD) patient(H) and a non-HAD patient(N), The HIV-1 tat genes were transfected into U87 cell. The green fluorescent protein was observed under microscope to determine the Tat protein expression. The expression of Tat protein in U87 cells was detected by Western blotting. CK-8 method , Western blotting and malondialdehyde (MDA) detection kit were used to study the effect of Tat protein on cell activity, oxidative stress index glutathione peroxidase (GPX), MDA level. Results:Amino acid sequence analysis showed that the key amino acid sites of HIV-1 Tat protein from N-BG, N-SPL, H-BG and H-SPL were different; Tat protein could inhibit the activity of U87 cells, which could be reversed by antioxidant N-acetyl-L-cysteine (NAC). Compared with the control group, the levels of MDA were increased and the expression of GPX protein was decreased in the four experimental groups ( P<0.05). And different sources of Tat protein had different ability to induce oxidative stress, the level of MDA in H-BG group was higher than that in N-BG group( P<0.05). The expression of GPX protein in BG group of both HAD and non-HAD patients was lower than that of SPL group( P<0.05). Conclusions:There are differences in the key amino acid sites of Tat protein in peripheral and central nervous system between HAD and non-HAD patients, and their effects on oxidative stress were also different.