Genetic Diversity of the Human Adenovirus C Isolated from Hospitalized Children in Russia (2019–2022)

The human adenovirus (HAdV) is a common pathogen in children that can cause acute respiratory virus infection (ARVI). However, the molecular epidemiological and clinical information relating to HAdV among hospitalized children with ARVI is rarely reported in Russia. A 4-year longitudinal (2019–2022) study among hospitalized children (0–17 years old) with ARVI in Novosibirsk, Russia, was conducted to evaluate the epidemiological and molecular characteristics of HAdV. Statistically significant differences in the detection rates of epidemiological and virological data of all positive viral detections of HAdV were analyzed using a two-tailed Chi-square test. The incidence of HAdV and other respiratory viruses such as human influenza A and B viruses, respiratory syncytial virus, coronavirus, parainfluenza virus, metapneumovirus, rhinovirus, bocavirus, and SARS-CoV-2 was investigated among 3190 hospitalized children using real-time polymerase chain reaction. At least one of these respiratory viruses was detected in 74.4% of hospitalized cases, among which HAdV accounted for 4%. A total of 1.3% co-infections with HAdV were also registered. We obtained full-genome sequences of 12 HAdVs, which were isolated in cell cultures. Genetic analysis revealed the circulation of adenovirus of genotypes C1, C2, C5, C89, and 108 among hospitalized children in the period from 2019–2022.


Introduction
The human adenovirus (HAdV) has double-stranded DNA and a non-enveloped icosahedral structure, which includes three viral capsid proteins: hexon, penton base, and fiber [1].HAdV belongs to the genus Mastadenovirus (family Adenoviridae) [2].Currently, seven different species of HAdV (from A to G) have been identified [2], while, according to the classification criteria of the Human Adenovirus Working Group, 115 HAdV genotypes have been assigned [3].

Virus Isolation
After receiving the positive result for HAdV in real-time PCR, virus isolation on HeLa, HEp-2, Vero, SPEV, and Caco-2 cells was carried out.In total, 96-well plates with a daily monolayer of HeLa, Hep-2, Vero, SPEV, and Caco-2 cells in a growth medium were washed with a Henks solution and then inoculated with 30 µL aliquots of the clinical sample.After 1 h of incubation at 37 • C, 5% CO 2 supernatant was removed, and 200 µL/well of DMEM (Capricorn Scientific, Germany) with 2% of FBS (Capricorn Scientific, Germany) and 100 µg/mL of gentamicin sulfate (BioloT, Russia) were added.Intact HeLa, HEp-2, Vero, SPEV, and Caco-2 cells were used as controls in each plate.Infected plates were incubated at 37 • C, 5% CO 2 for up to 7 days, and were checked daily under the microscope (Micromed I, Russia) by the presence of a cytopathogenic effect (CPE) manifested by cell death.

Light and Electronic Microscopy and Immunocytochemical Analysis
Inoculated cells were fixed with a 4% formalin solution on days 4-5 post infection.After that, the cells were stained with azur and eosin for 30 s (Minimed, Russia).Evaluation of CPE was estimated under an inverted AxioVert 40 microscope (Carl Zeiss, Germany) with AxioCam ICc 3 camera (Carl Zeiss, Germany), Bright Field mode.The CPE score was assessed by the presence of cytopathic changes in the cell monolayer, the destruction of the cell monolayer, the formation of clusters of rounded apoptotic cells, the loss of intercellular contacts, and the rounding of the cell shape.Intact HEp-2 and Vero cells were used as controls to compare the morphological state of inoculated and intact cells monolayer.
Localization of HAdV in the infected cells was revealed by immunofluorescence (IF) assay [13]: cells on the coverslips were fixed with a 4% formalin solution for 10 min and were treated by diagnostic kit with FITC-labeled fluorescent antibodies to HAdV (LLC "Enterprise for the production of diagnostic drugs", St. Petersburg, Russia).The presence of HadV was identified by a green immunofluorescent signal in the cells.The cell nuclei were contrasted with 5µM 4,6-diamino-2phenyl indole (DAPI), briefly washed with deionized water, and mounted in the Prolong Antifade/Slow Fade mounting medium.Cells were imaged using an LSM710/NLO confocal microscope (Carl Zeiss, Germany), and images were processed with Adobe Photoshop CS6 software.The presence of HAdV was identified by a green immunofluorescent signal in the cells.
For electronic microscopy, a 200-mesh copper TEM grid (SPI Supplies, West Chester, PA, USA) with ultrathin (invisible on the water surface) [14] formvar support film was placed on a 10 µL droplet of Hela cell suspension infected with HAdV for 30 s and then dried using filter paper.A 10 µL droplet of uranyl acetate water solution (1% w/v) was then added to the grid surface for 15 s.After drying, using filter paper again, the grid was examined with a TEM (JEM-1400; Jeol, Japan) at an accelerating voltage of 80 kV.

Sequence Analysis
Human adenovirus sequences being investigated were combined with sequences retrieved from the GenBank database.For multiple alignments, a MUSCLE software was used [17].Comparative pairwise sequence alignment of 12 investigated viruses was performed via BioEdit [18].Phylogenetic trees were built via MEGA 11 using maximum likelihood following results of the best-fit model test in MEGA (complete genome sequence -GTR + G + I; penton -T92 + G + I; hexon -TN93 + G; fiber -HKY + I) [19].Bootstrap support values were generated using 1000 bootstrap replicates.SimPlot analysis was performed with SimPlot++ v.1.3[20].Features of the 8.171V strain genome were visualized with DNA Feature Viewer.

Statistical Analysis
Data analysis was performed using Microsoft Excel 16.16.2for MacOS and GraphPad 9.1.1(GraphPad Software).Descriptive statistics included mean and standard error (for quantitative traits) and absolute and relative proportion (for qualitative traits).Categorical data were analyzed using a two-tailed Chi-square test undertaken to compare infection rates for respiratory viruses among different age groups.p-value < 0.05 was considered to be statistically significant.

Virus Isolation and Virological Characteristics
We randomly selected 30 samples out of 127 PCR positives with the lowest Ct values (18)(19)(20)(21)(22)(23), representing different ages and sexes, which we attempted to isolate in cell cultures.In order to study virological characteristics, five cell cultures of HeLa, HEp-2, Vero, SPEV, and Caco-2 were infected with the first human adenovirus (strain HAdVC/Novosibirsk/7.134V/2019) isolated at the beginning of the research period-in December 2019.Cells were evaluated daily, and on the 4th or 5th day of the appearance of CPE, they were fixed and stained for further analysis under light or confocal microscopes.The results showed the same morphological changes in studied cell cultures, characterized by an apoptotic type of destruction on the 4th or 5th day post infection (Figures 2 and 3).

Virus Isolation and Virological Characteristics
We randomly selected 30 samples out of 127 PCR positives with the lowest Ct values (18)(19)(20)(21)(22)(23), representing different ages and sexes, which we attempted to isolate in cell cultures.In order to study virological characteristics, five cell cultures of HeLa, HEp-2, Vero, SPEV, and Caco-2 were infected with the first human adenovirus (strain HAdVC/Novosibirsk/7.134V/2019) isolated at the beginning of the research period-in December 2019.Cells were evaluated daily, and on the 4th or 5th day of the appearance of CPE, they were fixed and stained for further analysis under light or confocal microscopes.The results showed the same morphological changes in studied cell cultures, characterized by an apoptotic type of destruction on the 4th or 5th day post infection (Figures 2 and 3).Because the observed cytopathic effect of human adenovirus was similar in all five studied cell cultures, we decided to use cell cultures most commonly employed as laboratory tools: Vero, HEp-2, or HeLa cell cultures-for the further isolation of adenoviruses.
The identification of human adenovirus by isolation on cell cultures was confirmed by PCR and immunofluorescence.IF analysis of cells infected with HAdVs showed  Because the observed cytopathic effect of human adenovirus was similar in all five studied cell cultures, we decided to use cell cultures most commonly employed as laboratory tools: Vero, HEp-2, or HeLa cell cultures-for the further isolation of adenoviruses.
The identification of human adenovirus by isolation on cell cultures was confirmed by PCR and immunofluorescence.IF analysis of cells infected with HAdVs showed Because the observed cytopathic effect of human adenovirus was similar in all five studied cell cultures, we decided to use cell cultures most commonly employed as laboratory tools: Vero, HEp-2, or HeLa cell cultures-for the further isolation of adenoviruses.
The identification of human adenovirus by isolation on cell cultures was confirmed by PCR and immunofluorescence.IF analysis of cells infected with HAdVs showed specific fluorescence of the cells (Figure 4).The morphology of virions was confirmed by electron microscopy.The morphology typical of adenovirus-icosahedral non-enveloped capsid (diameter 70-90 nm)-was observed (Figure 5).
Viruses 2024, 16, x FOR PEER REVIEW 8 of specific fluorescence of the cells (Figure 4).The morphology of virions was confirmed b electron microscopy.The morphology typical of adenovirus-icosahedral non-envelope capsid (diameter 70-90 nm)-was observed (Figure 5).Thus, 12 isolates showed active replication with extensive CPE and were processed for NGS with de novo assembly.
In addition, we found two strains of the twelve isolates studied to be of interest.Strain HAdVC/Novosibirsk/8.234V/2020 was determined to belong to the C89 genotype, according to the phylogenetic tree referenced and based on the complete genome, and was closely related to a strain isolated from China (Accession number MH121097.1)(Figure 6).This strain had fiber and hexon genes belonging to the C2 genotype and pentone base genes of the C89 clade.
Another strain, HAdVC/Novosibirsk/8.171V/2020, had the highest nucleotide identity with C108 genotype HAdV sequences (OQ108498, 99.61%; ON054624, 99.89%) (Table 3, N4).Its complete genome sequence formed a large clade with other C108 genotype strains isolated mainly in Asia.The penton base of this genotype strain belongs to the C1 genotype, while hexon and fiber genes relate to the C2 genotype.SimPlot analysis showed that its nucleotide sequence has higher identities for the C108 sequence than C2; hence, Thus, 12 isolates showed active replication with extensive CPE and were processed for NGS with de novo assembly.

Phylogenetic and Genomic Analyses
The complete genomes of twelve HAdV isolates were successfully sequenced and deposited in the NCBI Genbank (accession numbers are indicated in the Section 2).Then, we conducted the phylogenetic analysis of these HAdV isolates and other representative HAdVs available from the Genbank database.Phylogenetic analysis based on the complete genome revealed that our isolates belonged to five genotypes: C1, C2, C5, C89, and C108 (Figure 6; Tables 2 and 3; Supplementary Table S1).According to the phylogenetic analysis of the complete genome, as well as additional analyses undertaken separately made for the fiber gene, hexon gene, and penton base gene  2 and 3; Supplementary Table S1).
In addition, we found two strains of the twelve isolates studied to be of interest.Strain HAdVC/Novosibirsk/8.234V/2020 was determined to belong to the C89 genotype, according to the phylogenetic tree referenced and based on the complete genome, and was closely related to a strain isolated from China (Accession number MH121097.1)(Figure 6).This strain had fiber and hexon genes belonging to the C2 genotype and pentone base genes of the C89 clade.
Another strain, HAdVC/Novosibirsk/8.171V/2020, had the highest nucleotide identity with C108 genotype HAdV sequences (OQ108498, 99.61%; ON054624, 99.89%) (Table 3, N4).Its complete genome sequence formed a large clade with other C108 genotype strains isolated mainly in Asia.The penton base of this genotype strain belongs to the C1 genotype, while hexon and fiber genes relate to the C2 genotype.SimPlot analysis showed that its nucleotide sequence has higher identities for the C108 sequence than C2; hence, the 1-8000 region has a lower identity score with the C108_1 reference sequence than with the most related C108_2 sequence (Figure 7A,B).
In general, the strains of human adenovirus C studied formed several groups according to the pairwise distances of full-genomic nucleotide sequences, as confirmed by the similarity of genotypes 1, 2, 5, 89, and 108 (Table 2; Supplementary Table S1).
All strains studied had similar virus titers ranging from 3.25 to 3.75 lgTCID50/mL (Table 3).Thus, we did not observe differences in virus growth between the different genotypes.

Discussion
In our study, we analyzed certain molecular, epidemiological, and virological characteristics of HAdV with C1, C2, C5, C89, and C108 detected from children 0-17 years old.The human adenovirus is a pathogen commonly associated with acute respiratory infections among hospitalized children.In conformity with the officially determined epidemic season in the Russian Federation, all samples for this study were collected from October-November to March-April during the period 2019-2022.The incidence of ARVs (HAdV, HIFV, HRSV, HCoV, HPIV, HMPV, HRV, HBoV, and SARS-CoV-2) was analyzed by realtime PCR among 3190 hospitalized patients 0-17 years old with acute respiratory infections in the city of Novosibirsk, Russia.At least one of these respiratory viruses was detected in 74.4% (2372/3190) of the hospitalized children.The lowest incidence of ARIs was registered among patients older than seven (Table 1), as also determined in earlier works [21][22][23].Those children 0-2 years old were the most sensitive to viral infections, with a 68% positive rate (1248/1836) (Chi-square test, p < 0.01).The most common etiological agent of ARI among children during the 2019-2020 period was HIFV 28.7% (312/1088); during the 2020-2021 period was HMPV 28% (316/1130); and during the 2021-2022 period In general, the strains of human adenovirus C studied formed several groups according to the pairwise distances of full-genomic nucleotide sequences, as confirmed by the similarity of genotypes 1, 2, 5, 89, and 108 (Table 2; Supplementary Table S1).
All strains studied had similar virus titers ranging from 3.25 to 3.75 lgTCID 50 / mL (Table 3).Thus, we did not observe differences in virus growth between the different genotypes.

Discussion
In our study, we analyzed certain molecular, epidemiological, and virological characteristics of HAdV with C1, C2, C5, C89, and C108 detected from children 0-17 years old.The human adenovirus is a pathogen commonly associated with acute respiratory infections among hospitalized children.In conformity with the officially determined epidemic season in the Russian Federation, all samples for this study were collected from October-November to March-April during the period 2019-2022.The incidence of ARVs (HAdV, HIFV, HRSV, HCoV, HPIV, HMPV, HRV, HBoV, and SARS-CoV-2) was analyzed by real-time PCR among 3190 hospitalized patients 0-17 years old with acute respiratory infections in the city of Novosibirsk, Russia.At least one of these respiratory viruses was detected in 74.4% (2372/3190) of the hospitalized children.The lowest incidence of ARIs was registered among patients older than seven (Table 1), as also determined in earlier works [21][22][23].Those children 0-2 years old were the most sensitive to viral infections, with a 68% positive rate (1248/1836) (Chi-square test, p < 0.01).The most common etiological agent of ARI among children during the 2019-2020 period was HIFV 28.7% (312/1088); during the 2020-2021 period was HMPV 28% (316/1130); and during the 2021-2022 period was HRSV 20.3% (197/972).The most common etiological agent of ARI among children during the period 2019-2020 was HIFV 28.7% (312/1088); during the period 2020-2021 was HMPV 28% (316/1130); and during the period 2021-2022 was HRSV 20.3% (197/972).In our previous study, the absence of HIFV among circulated viruses during the period 2020-2021 was shown, as well as the absence of HMPV among other respiratory viruses during the subsequent period (2021-2022) [21].In total, as shown in our previous work, the assessment reports about etiological agents of ARVI during the period 2019-2022 that HAdV presented at a lower rate, following HIFV, SARS-CoV-2, HRSV, and HMPV [21,22].
Initially, we set the goal to identify isolates using various cell cultures.We randomly selected 30 samples with the lowest Ct values (18)(19)(20)(21)(22)(23), which represented different age groups and sexes, and attempted to isolate them.As a result, 12 isolates showed active replication in cell cultures, and as a consequence, it was decided to conduct NGS sequencing and undertake a genome analysis since such data did not exist for this region of Russia.
It is interesting to note that human adenovirus C may be isolated at least on five cell cultures-HeLa, HEp-2, Vero, SPEV, and Caco-2-studied in this research.The average viral titers shown on different cell cultures were in diapason 3.25-3.75lgTCID 50 /mL.Virological characteristics did not differ between the five genotypes of HAdVs detected.In comparison with the results of Biliavska et al. [28], the viral activity of our isolated HAdVs isolated on cell cultures was lower.Through electron microscopy, we confirmed the standard icosahedral form characteristic of adenoviruses.
The complete genome sequences and phylogenetic analysis of the twelve HAdV isolates revealed that all of them belonged to the C species.In this study, the maximum likelihood trees and pairwise differences in human adenovirus sequences were used to evaluate genotypic relationships and paired with the epidemiological data from routine infection prevention and control records and hospital activity data.Here, we have shown the results of the sequences and phylogenetic analysis of the complete genome, fiber, hexon, and penton base genes of the HAdVs isolated.The research indicates that various evolutionary variants of HAdVs were circulating in Novosibirsk during the period of this research.We detected several genotypes of HAdV-C1, C2, C5, C89, and C108-that were circulating during the period 2019-2022.However, such data do not allow us to make conclusions concerning the genotype prevalence, as we have not yet determined the complete genotype for all PCR-positive samples.Such work in the future will allow us to determine in more detail the epidemiology of adenoviruses and to assess whether there is a difference in clinical manifestations and disease severity.This will be of great importance for practical medicine.
Despite the fact that we cannot support the conclusions about the genotypes prevalent in our study because of the limited number of genotyped samples, we showed evidence of the circulation of various HAdV variants.For the first time, the circulation of the HAdV-C89 and C108 genotypes in Russia was shown, according to the results of phylogenetic analysis based on penton base, fiber, hexon nucleotide sequences, and complete genomes.The phylogenetic clade of HAdVC/Novosibirsk/8.234V/2020 includes strains 29C2 and 47C2, according to a recent study in Germany [10], where these strains were assigned to the new C-89 genotype, based on differences in penton base gene sequences.Phylogenetic analyses of other isolates through fiber and hexon gene sequencing showed seven strains clustered with HAdV-C2, three strains clustered with HAdV-C1, and two with HAdV-C5.It is noteworthy that the presence of the HAdV-C108 genotype strain (HAdVC/Novosibirsk/8.171V/2020) has also been described for the first time in Russia in our study.According to phylogenetic analysis, it has the typical genetic features of this particular genotype-P1H2F2.

Limitations
It should be noted that there are some limitations in our study.Our study sample was obtained only from hospitalized patients.It is also known that the state of virus infection of a carrier can be asymptomatic.Accordingly, the distribution of viruses among the Russian population may differ from our sample results.We also did not analyze social and behavioral factors that may serve as potential drivers of viral interference, and we cannot exclude the possibility of surveillance artifacts.The strength of our data is based on sequential observations from both pre-pandemic and pandemic periods through research spanning three years and the collection of samples from 3190 patients 0-17 years old.Annual correct monitoring of seasonal fluctuations of common respiratory viruses has the potential to improve general knowledge about viral interactions.However, as global surveillance systems for non-influenza respiratory viruses and for non-COVID-19 research remain limited in both scope and funding, the ability to undertake such investigations remains challenging.

Conclusions
The study of the incidence of HAdV and other respiratory viruses, namely HIFV, HRSV, HCoV, HPIV, HMPV, HRV, HBoV, and SARS-CoV-2 among 3190 hospitalized children showed that at least one of these respiratory viruses was detected in 74.4% of hospitalized cases, among which HAdV accounted for 4%, whereas co-infections with HAdV among 1.3 %.We obtained full-genome sequences of twelve HAdVs isolated in cell cultures.Genetic analysis has demonstrated that at least five genotypes (C1, C2, C5, C89, and C108) of the HAdV virus were circulating among children (0-17 years old) with ARVI in Novosibirsk, Russia, during the period 2019-2022.We have revealed the circulation of 89 and 108 genotypes for the first time in Russia.Further comprehensive and systematic monitoring, detection, and characterization of HAdV are necessary for future research and clinical practice.A future integrated approach that includes clinical data and obligatory full-genome analysis will provide the opportunity to explore the clinical significance of different genotypes, understand the implications of co-infections, and further investigate yet unassigned strains.Data on differences in clinical manifestation and disease severity for certain genotypes will be of great importance for practical medicine.

Figure 1 .
Figure 1.Acute respiratory virus infections and their combinations detected in samples from patients aged 0-17 years hospitalized during the period 2019-2022.Note: Dots-combinations or individual infections with dark grey bars visualizing number of cases in different combinations.Only cases ≥ 2 included.

Figure 1 .
Figure 1.Acute respiratory virus infections and their combinations detected in samples from patients aged 0-17 years hospitalized during the period 2019-2022.Note: Dots-combinations or individual infections with dark grey bars visualizing number of cases in different combinations.Only cases ≥ 2 included.

Figure 4 .Figure 4 . 17 Figure 5 .
Figure 4. Immunofluorescent analysis of HeLa cell cultures infected with human adenovirus.No HeLa cells were infected with the human mastadenovirus C (strain HAdVC/Novo birsk/7.134V/2019),incubated for 5 days, then fixed and stained using a diagnostic kit with fluore cent FITC-labeled antibodies to HAdV (Enterprise for the Production of Diagnostic Drugs LLC, Petersburg, Russia) and 4,6-diamino-2phenyl indole (DAPI) for nuclei labeling.Cells were image using an LSM710/NLO confocal microscope (Carl Zeiss, Germany).Magnification ×4000.(a) Repr sentative confocal images of infected HeLa cell nuclei labeled with DAPI (blue labeling) in fluore cent FDAPI-channel mode.(b) Representative confocal images of adenovirus localization (gre labeling) in infected HeLa cells.HeLa cells were stained using a diagnostic kit with fluorescent FIT labeled antibodies to HAdV (Enterprise for the Production of Diagnostic Drugs LLC, St Petersbur Russia), in fluorescent FITC-channel mode; (c) Representative confocal images of infected HeLa ce in phase contrast mode.(d) Representative confocal images of adenovirus localization (green labe ing) in infected HeLa cells with DAPI-stained nuclei (blue labeling).HeLa cells were stained usin a diagnostic kit with fluorescent FITC-labeled antibodies to HAdV (Enterprise for the Production Diagnostic Drugs LLC, St Petersburg, Russia) in merge mode.

Figure 6 .
Figure 6.Phylogenetic relationship between our human adenovirus C genotypes and other representative genotypes based on complete genome (A), penton base (B), hexon (C), and fiber (D).Maximum likelihood trees were constructed using the best-fit nucleotide substitution model, GTR + G + I (complete genome), K2 + G (penton), TN93 + G (hexon), and HKY + G (fiber) in MEGA X.Our sequences are indicated in red.

Figure 6 .
Figure 6.Phylogenetic relationship between our human adenovirus C genotypes and other representative genotypes based on complete genome (A), penton base (B), hexon (C), and fiber (D).Maximum likelihood trees were constructed using the best-fit nucleotide substitution model, GTR + G + I (complete genome), K2 + G (penton), TN93 + G (hexon), and HKY + G (fiber) in MEGA X.Our sequences are indicated in red.

Table 1 .
Data on acute respiratory viruses collected from patients aged 0-17 years hospitalized during the period 2019-2022.

Table 2 .
Pairwise distances of full-genomic nucleotide sequences of human adenovirus obtained in the period 2019-2022.

Table 2 .
Pairwise distances of full-genomic nucleotide sequences of human adenovirus obtained in the period 2019-2022.

Table 3 .
Molecular biological and virologic data about human adenoviruses C collected during the period 2019-2022 in Novosibirsk, Russia.
Note: genotype C1 is colored in pale pinkViruses 2024, 16, x FOR PEER REVIEW 12 of 17

Table 3 .
Molecular biological and virologic data about human adenoviruses C collected during the period 2019-2022 in Novosibirsk, Russia.
Note: genotype C1 is colored in pale pink , genotype C2 is colored in yellow , genotype C5 is colored in green , genotype C89 is colored in brown , and genotype C108 is colored in red ., genotype C2 is colored in yellow iruses 2024, 16, x FOR PEER REVIEW 12 of 17

Table 3 .
Molecular biological and virologic data about human adenoviruses C collected during the period 2019-2022 in Novosibirsk, Russia.
Note: genotype C1 is colored in pale pink , genotype C2 is colored in yellow , genotype C5 is colored in green , genotype C89 is colored in brown , and genotype C108 is colored in red ., genotype C5 is colored in green es 2024, 16, x FOR PEER REVIEW 12 of 17

Table 3 .
Molecular biological and virologic data about human adenoviruses C collected during the period 2019-2022 in Novosibirsk, Russia.
Note: genotype C1 is colored in pale pink , genotype C2 is colored in yellow , genotype C5 is colored in green , genotype C89 is colored in brown , and genotype C108 is colored in red ., genotype C89 is colored in brownViruses 2024, 16, x FOR PEER REVIEW

Table 3 .
Molecular biological and virologic data about human adenoviruses C collected during the period 2019-2022 in Novosibirsk, Russia.