| Human adenovirus(HAdV)belongs to the family of adenoviridae and the genus of mast adenovirus,and charactered as non-enveloped,non-segmented,double-stranded DNA virus with a genome length of 3.4-3.6 kb.HAdV is an important pathogen of respiratory tract infections.In addition to infecting the respiratory tract,it also has an extensive tissue tropismmay,induce infection of the corneal conjunctiva,digestive tract,urinary system,myocardial,nervous system and so on.There are widely distributed in nature.New types of HAdV have emerged frequently.To date.with 113 types named and belonging to seven species(A-G).Among them,the HAdV that causes respiratory tract infections are HAdV-B,C and E,with the main types being HAdV-1～7,-14,-21,and-B55.In China,HAdV-B3/7 are the dominant types causing infections among children,and HAdV-B55 is a key type of pathogen inducing community-acquired pneumonia in adults.Existing bioinformatics analysis has documented that this type is developed by homologous recombination of two different types of HAdV.Notably,HAdV can escape the host’s immune surveillance through recombination.producing highly virulent and transmissible recombinants that pose a threat to human health and safety.Consequently,it highlights the great significance of investigating the epidemic patterns of HAdV,clarifying the phylogenetic characteristics,and exploring recombination mechanisms for the prevention and treatment of HAdV.Firstly,16,097 respiratory samples collected from the children with acute respiratory infections from Affiliated Children’s Hospital,Capital Institute of Pediatrics duing 2015 to 2021,PCR was employed to amplify the genome sequence of 466 cases of HAdV-positive.Then.HAdV was accurately classified through phylogenetic analysis of hexon,penton base,and fiber genes.To analyze the HAdV epidemiological data,the implementation of non-pharmaceutical interventions(NPIs)on January 24,2020 in Beijing was selected as the boundary.The results showed that HAdV could be detected throughout the year,with a total positive rate of 2.89%(466/16.097).Multiple types of HAdV were prevalent in Beijing.with HAdV-B3(51.56%,198/384)and HAdV-B7(29.17%,112/384)accounting for the highest proportion.followed closely by HAdV-C1(5.47%.21/384).Among them,HAdV-B3 and HAdV-B7 were the dominant epidemic types in Beijing,showing the characteristics of co-epidemic or alternating epidemic patterns.After NPIs launched on Jan,2020,the positive rate of HAdV dropped sharply from 3.19%to 1.41%,with a drop rate of 55.80%.Before NPIs launched,the dominant epidemic types were HAdV-B3(54.78%)and HAdV-B7(31.46%),while after NPIs launched,HAdV-C1 and HAdV-B3 accounted for 39.28%and 10.71%,respectively,with the disappearance of HAdV-B7.These data suggest that the original dominant epidemic types HAdV-B3/B7 have gradually been replaced by HAdV-C1 after NPIs launched.After NPIs launched,due to the different types of hexon,penton base,and fiber genes,it was unable to be determined,with the increase from 6.46%in before NPIs launched to 39.28%after NPIs launched.Meanwhile,there were significant differences in clinical manifestations caused by infection with dominant epidemic types HAdV-3 and HAdV-B7,with statistical significance observed in the occurrence of wheezing,increased white blood cell count(>15×109/L),and decreased white blood cell coun(<5×109/L)t,procalcitonin level>0.5 mg/L,multilobar infiltration,pleural effusion,severe adenovirus pneumonia(χ2=5.11、4.44、11.16、9.19、4.29、9.25、3.91,P=0.024、0.035、0.001、0.002、0.038、0.002、0.048),as well as the length of stay in the hospital(Z=3.728,P<0.01).In addition,there was more severe condition caused by infection with HAdV-7 according to the clinical symptoms,imaging results and laboratory indicators.In this study,we found that the cases where the type cannot be determined due to the different types of hexon,penton base and fiber gene sequences,which might be a coinfection or recombinant.We collected the undetermind respiratory specimens through the different three HAdV coat protein genes sequencing from 2010 to 2023.Through multiple-PCR methods,different types of HAdV co-infections are excluded according to the multiple-PCR methods and sequence analysis.The specimen of the recombinant viruses are determined by the whole genome sequence,with the development of three coat protein ML trees is constructed.And the RDP4 and Simplot software are used for the recombination analysis.A total of five HAdV-C and one HAdV-D recombinants were identified.Further isolation and purification of these viruses obtained four HAdV-C and one HAdV-D recombinants,but one HAdV-C strains failed to isolate.Subsequently,Next Generation Sequencing(NGS)and in-depth analysis were performed on the sequences of these five recombinants.The results of the evolutionary tree showed that HAdV-C was divided into five main branches,with frequent recombination events occurred among different branches.Specifically,the strain 1 w5060 obtained in this study might be a recombination of HAdV-C2 and HAdV-C1,strain 86413 was recombined by HAdV-C2 and HAdV-C89,95031 was recombined with HAdV-C5 as the skeleton,and the penton base came from KF268129;while strain 51383 was restructured by HAdV-C5 and HAdV-C89.Meanwhile,S8130 was identified as HAdV-D,and its entire genome was closest to HAdV-D53/85.Hexon was from HAdV-D8,the penton base from HAdV-D64/42/22,and Fiber showed a relatively closer genetic relationship with HAdV-D85/8/53,indicating a novel recombinant possibly.Collectively,it suggests that homologous recombination can effectively drive the evolution of HAdV-C and HAdV-D.Finally,in order to explore the differences in virulence between the recombinant and its parent virus as well as the potential recombination mechanism of HAdV,this study further compared the biological characteristics of the recombinant.HAdV-55 with its parent strains HAdV-B11 and HAdV-B14.Comparison of plaque size revealed that the size of HAdV-B55 and HAdV-B14 was significantly larger than that of HAdV-11(0.1 8±0.012vs 0.16±0.009vs 0.14±0.005 mm),with significant difference(P<0.05).Furthermore,there was no significant difference in the proliferation of A549 cells among the three strains.However,in Hep-2 cells cell proliferation of HAdV-55 and HAdV-B14 was weaker than that of HAdV-11.After infecting A549 cells with high MOI(10 MOI),the cytotoxicity of HAdV-B55 and HAdV-B14 was weaker than that of HAdV-11.Collectively,compared with the parent virus HAdV-B11/14,HAdV-B55 has closer in vitro biological characteristics to HAdV-B14.A cell model of HAdV-B11 and-B14 co-infecting host cells was further constructed to monitor the variation of the viral genome through cloning sequencing.According to the established co-infection model,HAdV recombination was prone to occur after co-infection with HAdV-1 1/14.The A549 cells were then infected with viruses at different doses,and the highest recombination rate was 16%in the co-infection group with 5 MOI.After infection with 1 MOI in different time sequences,HAdV-B14 was first infected,followed by HAdV-B11 at 8h,with a recombination rate of 13%,showing no significant difference from that of simultaneous infection at the same dose.However,the recombination rate was 9%only after infection with HAdV-B11 first and then HAdV-B14.While regardless of the exact type of virus that was infected firstly,the recombination rate would be decreased after infection w ith another virus 16h later.These data suggest that the recombination rate is dependent on the dose and interval of infection.This study discovered that the genome of HAdV took one of the genomes of HAdV-B11/14 as the skeleton,respectively,and presented recombination at hypervariable region(HVR)of hexon,with the highest recombination frequently observed at HVR7.Moreover,the resulting recombinant virus could be passaged continuously.In conclusion,the present study systematically uncovers the epidemic characteristics of HAdV in China from 2015 to 2021,with preliminary revealing of its phylogenetic characteristics simultaneously.Gene recombination is a significant approach for HAdV-C and HAdV-D to achieve dominant type switching and continue to be prevalent.In addition,this study also constructs a cell model of co-infection with heterologous types of HAdV to clarify the recombination pattern of the hexon gene region and reveal the complexity of virus recombination in nature.Eventually,findings in our study may provide a scientific reference for the prevention and control of HAdV infections,vaccine development,and prediction of possible recombinants. |
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