Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O₂ ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.
Animals ; Humans ; L-Lactate Dehydrogenase/genetics* ; Lactic Acid ; Adenoviruses, Human ; Ammonia ; HEK293 Cells ; Glucose/metabolism* ; Adenosine Triphosphate/metabolism* ; Kidney/metabolism* ; Mammals/metabolism*

Objective: To investigate the epidemiological characteristics of human adenovirus (HADV) 2, 3 and 7 in hospitalized children with respiratory infection. Methods: A total of 25 686 children with respiratory infection hospitalized at Children's Hospital of Hebei Province from January 2018 to December 2020 were retrospectively included.Deep sputum or nasopharyngeal aspirates of those children were collected. Then thirteen common respiratory pathogens were detected by multiplex PCR. 510 HADV positive specimens were randomly selected via random number and classified for type 2, 3 and 7 using a multiplex real-time quantitative PCR. SPSS 21.0 software was used to perform all of the statistical analyses. Enumeration data were expressed by frequency and percentage. χ² test was used for comparison between groups. Results: The HADV-positive rate was 7.99% (2 052/25 686). Children at age 3-<6 years had the highest HADV-positive rate (11.44%). The HADV-positive rate in 2019 was highest (10.64%). Among the 510 HADV-positive specimens, the proportion of type 3 was the highest (31.16%), followed by type 7 (21.37%) and type 2 (11.18%). The rate of type 2 in 2019 was significantly lower than that in 2018 and 2020 (χ²=8.954 and 16.354; P=0.003 and <0.01), while the rate of type 3 was significantly higher than that in 2018 and 2020 (χ²=5.248 and 4.811; P=0.022 and 0.028). The rate of type 2, type 3 and type 7 were lowest in winter, spring and autumn, respectively. The rate of type 2 increased significantly in autumn and the rate of type 3 and type 7 increased significantly in winter.The co-detection rate of HADV with other respiratory pathogens was 43.33%(221/510). Among, the co-detection rate of type 3 was highest (47.32%), and the co-detection rate of type 2, 3 and 7 was significantly higher than the alone detection rate (χ²=20.438, P<0.01; χ²=42.105, P<0.01; χ²=27.573, P<0.01).The proportion of severe pneumonia in children with type 7 positive (15.89%) was higher than that in children with non-type 7 positive (8.23%) (χ²=5.260, P=0.022). Conclusion: HADV is one of the important pathogens of children with respiratory infection in Children's Hospital of Hebei Province. The susceptible population of HADV is preschool children aged 3 to 6 years. HADV often co-detects with other respiratory pathogens. Type 3 and 7 is likely to be the dominant genotypes in this region, and type 7 may be one of the risk factors of severe pneumonia in children.
Child, Preschool ; Child ; Humans ; Infant ; Adenoviruses, Human/genetics* ; Child, Hospitalized ; Retrospective Studies ; Adenovirus Infections, Human/epidemiology* ; Respiratory Tract Infections/epidemiology* ; Pneumonia ; Hospitals

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.
Animals ; Mice ; Humans ; Adenoviruses, Human/genetics* ; Escherichia coli/genetics* ; HEK293 Cells ; Isopropyl Thiogalactoside ; Blotting, Western ; Immunoglobulin G ; Antibodies, Monoclonal ; Antibody Specificity ; Mice, Inbred BALB C

As a single-center retrospective study, we analyzed the results of rotavirus and human adenovirus antigens in stool samples with colloidal gold immunochromatography method in children with acute gastroenteritis under the age of five who were treated in our hospital from 2019 to 2022. After excluding nonconforming cases and duplicate cases, 2 896 cases were included, of which 559 cases were detected with at least one viral antigen. According to the test results, they were divided into RV positive group, HAdV positive group and RV & HAdV double positive group. The gender, age, seasonal distribution, clinical symptoms and related laboratory tests were compared and analyzed with χ² test, analysis of variance and nonparametric test. Among the single samples from 2 896 children, the positive rate of RV antigen was 6.21% (180/2 896), the positive rate of HAdV antigen was 10.91% (316/2 896), and the double positive rate of RV & HAdV was 2.18% (63/2 896). The positive rate of HAdV antigen in 2021 was 16.11%, a significant increase compared with 6.20% in 2020. RV infection has obvious seasonality, and spring and winter are the seasons with high incidence of infection (χ²=74.018, P<0.001), while HAdV infection has no obvious seasonality (χ²=2.110, P=0.550), showing sporadic infection throughout the year. The proportions of fever and vomiting symptoms in children with RV infection were significantly higher than those in the HAdV infection group (χ²=40.401, P<0.001; χ²=32.593, P<0.001), but the positive rate of white blood cells in the stool was significantly lower than that in the HAdV infection group (χ²=13.741,P<0.01). In summary, paying attention to the epidemiological changes of RV and HAdV is of great significance for clinical diagnosis and treatment and disease prevention and control.
Child ; Humans ; Infant ; Rotavirus ; Retrospective Studies ; Gastroenteritis/epidemiology* ; Hospitals ; Feces ; Adenoviruses, Human ; Adenovirus Infections, Human/epidemiology*

Objective: To express DNA-binding protein (DBP) of human adenovirus (HAdV) type 7 using the prokaryotic expression system, and product anti-HAdV-7 DBP rabbit polyclonal antibody. Methods: The HAdV-7 DBP gene was synthesized and cloned into prokaryotic expressing vector pET30a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein DBP was expressed by induced Isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with Ni-NTA affinity column. The titer of anti-DBP polyclonal antibody produced in immunized rabbit was measured by indirect ELISA, and the specificity of the antibody was identified by Western blotting and indirect immunofluorescence assay (IFA). In addition, purified rDBP was used as coating antigen for indirect ELISA assay to detect specific IgM and IgG antibodies against DBP in the serum of children infected with HAdV. Results: The HAdV-7 DBP plasmid was constructed successfully. The purified recombinant DBP was more than 95% after purification. The titer of polyclonal antibody was 1∶1 024 000. The polyclonal antibody showed high specificity in vitro using Western blotting and IFA. The positive rate of specific anti-DBP IgM and IgG antibody in acute-phase serum samples collected from children infected with HAdV were 50.0% (19/38) and 63.2% (24/38), respectively, using indirect ELISA. Conclusion: In summary, the HAdV-7 rDBP is expressed using prokaryotic expression system, and the recombinant HAdV-7 DBP protein and the anti-DBP rabbit polyclonal antibody with high titer are prepared.
Adenoviruses, Human/genetics* ; Animals ; Antibody Specificity ; Blotting, Western ; DNA-Binding Proteins/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli/genetics* ; Immunoglobulin G ; Rabbits

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×10^9.1 TCID₅₀/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.
Adenoviridae/genetics* ; Adenoviruses, Human/genetics* ; Animals ; Antibodies, Viral ; Capsid/metabolism* ; Capsid Proteins ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins/genetics* ; Serogroup ; Swine ; Viral Proteins ; Viral Vaccines/genetics*

Objective: To compare the clinical characteristics of different types of human adenovirus (HAdV) infection in hospitalized children with acute respiratory infection in Beijing, and to clarify the clinical necessity of adenovirus typing. Methods: In a cross-sectional study, 9 022 respiratory tract specimens collected from hospitalized children with acute respiratory infection from November 2017 to October 2019 in Affiliated Children's Hospital, Capital Institute of Pediatrics were screened for HAdV by direct immunofluorescence (DFA) and (or) nucleic acid detection. Then the Penton base, Hexon and Fiber gene of HAdV were amplified from HAdV positive specimens to confirm their HAdV types by phylogenetic tree construction. Clinical data such as laboratory results and imaging data were analyzed for children with predominate type HAdV infection using t, U, or χ² test. Results: There were 392 cases (4.34%) positive for HAdV among 9 022 specimens from hospitalized children with acute respiratory infection. Among those 205 cases who were successfully typed, 131 were male and 74 were female, age of 22.6 (6.7, 52.5) months,102 cases (49.76%) were positive for HAdV-3 and 86 cases (41.95%), HAdV-7, respectively, while 17 cases were confirmed as HAdV-1, 2, 4, 6, 14 or 21. In comparison of clinical characteristics between the predominate HAdV type 7 and 3 infection, significant differences were shown in proportions of children with wheezing (10 cases (11.63%) vs. 25 cases (24.51%)), white blood cell count >15 ×10⁹/L (4 cases (4.65%) vs.14 cases (13.73%)), white blood cell count <5×10⁹/L (26 cases (30.23%) vs.11 cases (10.78%)), procalcitonin level>0.5 mg/L (43 cases (50.00%) vs. 29 cases (28.43%)), multilobar infiltration (45 cases (52.33%) vs.38 cases (37.25%)), pleural effusion (23 cases (26.74%) vs. 10 cases (9.80%)), and severe adenovirus pneumonia (7 cases (8.14%) vs. 2 cases (1.96%)) with χ²=5.11, 4.44, 11.16, 9.19, 4.30, 9.25, 3.91 and P=0.024, 0.035, 0.001, 0.002, 0.038, 0.002, 0.048, respectively, and also in length of hospital stay (11 (8, 15) vs. 7 (5, 13) d, Z=3.73, P<0.001). Conclusions: HAdV-3 and 7 were the predominate types of HAdV infection in hospitalized children with acute respiratory tract infection in Beijing. Compared with HAdV-3 infection, HAdV-7 infection caused more obvious inflammatory reaction, more severe pulmonary symptoms, longer length of hospital stay, suggesting the clinical necessity of further typing of HAdVs.
Adenovirus Infections, Human/epidemiology* ; Adenoviruses, Human/genetics* ; Beijing/epidemiology* ; Child ; Child, Hospitalized ; Cross-Sectional Studies ; Female ; Humans ; Infant ; Male ; Phylogeny ; Respiratory Tract Infections/epidemiology*

OBJECTIVE: To investigate the effect of silencing CD46 and desmoglein 2 (DSG2) in host A549 cells on the entry of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and host cell secretion of inflammatory cytokines. METHODS: RNA interference technique was use to silence the expression of CD46 or DSG2 in human epithelial alveolar A549 cells as the host cells of HAdV-3 or HAdV-7. The binding of the viruses with CD46 and DSG2 were observed with immunofluorescence staining at 0.5 and 1 h after viral infection. The viral load in the host cells was determined with qRT-PCR, and IL-8 secretion level was measured using ELISA. RESULTS: In infected A549 cells, immunofluorescent staining revealed colocalization of HAdV-3 and HAdV-37 with their receptors CD46 and DSG2 at 0.5 h and 2 h after infection, and the copy number of the viruses increased progressively after the infection in a time-dependent manner. In A549 cells with CD46 silencing, the virus titers were significantly lower at 2, 6, 12 and 24 h postinfection in comparison with the cells without gene silencing; the virus titers were also significantly decreased in the cells with DSG2 silencing. The secretion level of IL-8 increased significantly in A549 cells without siRNA transfection following infection with HAdV-3 and HAdV-7 (P < 0.0001), but decreased significantly in cells with CD46 and DSG2 silencing (P < 0.0001). CONCLUSION HAdV-3 and HAdV-7 enter host cells by binding to their receptors CD46 and DSG2, and virus titer and cytokines release increase with infection time. Silencing CD46 and DSG2 can inhibit virus entry and cytokine IL-8 production in host cells.
A549 Cells ; Adenoviruses, Human/metabolism* ; Desmoglein 2/metabolism* ; Humans ; Interleukin-8 ; Membrane Cofactor Protein/genetics* ; RNA, Small Interfering

Human adenoviruses are widespread causative agent that induces respiratory diseases, epidemic keratoconjunctivitis and other related diseases. Adenoviruses are commonly used in experimental and clinical areas. It is one of the most commonly used virus vectors in gene therapy, and it has attracted a lot of attention and has a high research potential in tumor gene therapy and virus oncolytic. Here, we summarize the biological characteristics, epidemiology and current application of adenovirus, in order to provide reference for engineering application of adenovirus.
Adenovirus Infections, Human ; epidemiology ; virology ; Adenoviruses, Human ; genetics ; Genetic Engineering ; methods ; trends ; Genetic Vectors ; Humans ; Oncolytic Virotherapy ; trends ; Oncolytic Viruses ; genetics ; Virus Replication

Acute Disease ; epidemiology ; Adenovirus Infections, Human ; epidemiology ; virology ; Adenoviruses, Human ; genetics ; physiology ; Child, Preschool ; China ; epidemiology ; Feces ; virology ; Female ; Gastroenteritis ; epidemiology ; virology ; Genotype ; Humans ; Infant ; Infant, Newborn ; Male ; Prevalence ; Rotavirus ; genetics ; physiology ; Rotavirus Infections ; epidemiology ; virology