Exploring the Correlation between HAdV Infection and Respiratory Tract Infection Symptoms: A Study in Nanning, China

doi:10.21203/rs.3.rs-2839165/v1

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Exploring the Correlation between HAdV Infection and Respiratory Tract Infection Symptoms: A Study in Nanning, China

https://doi.org/10.21203/rs.3.rs-2839165/v1

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Journal Publication

published 02 Aug, 2023

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Background

Human adenoviruses (HAdV) can cause various diseases, but there is limited information about the genotype diversity and epidemiology of HAdV associated with respiratory tract infections (RTIs) in Nanning.

Methods

From Jun 2019 to December 2021, throat swab, nasal swab, or nasopharyngeal swab samples were collected from hospitalized patients with RTIs, and Epidemiological data were analyzed using statistical software. HAdV were detected using a qPCR assay targeting a highly conserved 132-bp gene region of the HAdV hexon, and phylogenetic analysis was performed on the 875-bp hexon gene fragment.

Results

The age and sex distribution of HAdV-positive patients showed significant differences (P = 0.014 and P = 0.025, respectively). The majority of HAdV-positive patients were in the 0–6 age group (67.89%). The HAdV detection rate showed the highest prevalence in spring and autumn, peaking in February. In addition, a total of 37 HAdV-positive samples with 875-bp fragments were successfully genotyped and found to be circulating in Nanning from 2019 to 2021 with HAdV species B, C, and E.

Conclusions

HAdV prevalence is associated with age and season, with HAdV-B, HAdV-C, and HAdV-E co-circulating among hospitalized patients with RTIs in Nanning. The most frequent genotypes were HAdV-C1, HAdV-C6, and HAdV-E4.

Human adenoviruses

respiratory infections

Nanning

prevalence

hexon gene

Human adenoviruses (HAdV) are non-enveloped, double-stranded DNA viruses belonging to the Adenoviridae family. Their genome is approximately 36Kb in size and encodes around 40 proteins, including Penton base, Hexon, and Fiber [1]. HAdV can infect various tissues and organs, causing a range of illnesses from mild to severe, such as sore throat, rhinitis, fever, pneumonia, and bronchitis [2, 3]. Although people of all ages can be affected, those with weakened immune systems, cardiovascular disease, and children are at higher risk of developing severe disease[4, 5].

HAdV infections are often underreported worldwide, and different geographical regions and human populations may have different distributions of adenovirus types. Currently, there are approximately 110 recognized HAdV genotypes that have been classified into seven species (A-G) in the Mastadenovirus genus based on their physical, chemical, and biological properties [6]. Among all the HAdV, genotypes 3, 4, 7, 14, 21, and 55 are known to cause severe infections and have been associated with global outbreaks [7–9]. In recent years, new HAdV genotypes with new pathogenicity and epidemic characteristics have emerged, posing new threats and challenges to human health [10]. Therefore, it is crucial to study the molecular epidemiological characteristics of HAdV-induced acute respiratory tract infections.

HAdV particles consist of 252 putamens, comprising 240 hexons, 12 pentons, and 12 fibers. Hexons are crucial type-specific and species-specific antigens that are highly vulnerable to immune selection. The hypervariable regions (HVR1-7) within hexons contain numerous antigen epitopes, making them the most sensitive to immune selection. A recent study conducted in Nanning, China, from June 2019 to December 2021, aimed to evaluate the epidemiological, clinical, and molecular characteristics of HAdV infections among patients with respiratory tract infections (RTIs) who did not have influenza. The study also aimed to explore the correlation between HAdV infection and RTI symptoms to provide insight into controlling and preventing HAdV infection in China.

Patient specimens

In this study, a total of 8,315 samples were collected from patients with RTIs at Nanning First People's Hospital and Nanning Maternal and Child Health Care Hospital in Nanning, China, between June 2019 and December 2021. Informed consent was obtained from all patients or their guardians who participated in the study. RTI illness was defined as the presence of at least two of the following influenza-like symptoms within three days: fever, cough, nasal obstruction, expectoration, sneezing, and dyspnea. The collected specimens were stored in Yocon virus preservation solution (MT0301), which contained Hank's medium with streptomycin, antifungal antibiotics, BSA (V), cryoprotectant, biological buffer, and amino acids. The samples were transported to the laboratory on ice and stored at -80℃ until further processing. Besides, clinical data of the patients were obtained from the hospital database.

The Detection Of Influenza Viruses In The Enrolling Sample

The study employed the Prefill Viral Total NA Kit 4x48 preps for KingFisher FLEX (Fisher Scientific LabServ) to extract total viral nucleic acids from 200 µL of each clinical specimen, following the manufacturer’s instructions. The detection of influenza viruses was carried out using a qPCR assay that targeted specific primers for A type (Forward: 5'-GAC CRA TCC TGT CAC CTC TGA C-3’; Reverse: 5'-GGG CAT TYT GGA CAA AKC GTC TAC G-3’) and B type (Forward: 5'-TCC TCA ACT CAC TCT TCG AGC G-3’; Reverse: 5'-CGG TGC TCT TGA CCA AAT TGG-3’), with a common probe for A and B types (5'HEX-CCA ATT CGA GCA GCT GAA ACT GCG GTG-BHQ2 3’). The qPCR cycling program included 50°C for 30 min, 95°C for 15 min, 5 cycles of 95°C for 15 s, and 50°C for 30 s, 72°C for 1 min, followed by 40 cycles of 95°C for 15 s, and 55°C for 45 s. Samples with a cycle threshold (Ct) value < 38 were considered positive for influenza virus.

The Inspection Method Of Hadv In Influenza-negative Specimens

The total nucleic acid of each clinical specimen was extracted by using the Prefill Viral Total NA Kit 4x48 preps for KingFisher FLEX(Fisher Scientific LabServ)according to the manufacturer’s instructions. HAdV detection was performed using qPCR assay targeting the highly conserved gene region (132-bp) of the HAdV hexon. QuantiTect Probe RT-PCR Kit (200)༈QIAGEN༉ (AppliedBiosystems, USA) was used to amplify HAdV hexon DNA using specific primers (Forward:5’-CAGGACGCCTCGGRGTAYCTSAG-3’; Reverse: 5’-GGAGCCACVGTGGGRTT-3’) and probe (5’FAM-CCGGGTCTGGTGCAGTTTGCCCGC-BHQ1-3’). The qPCR cycling program was as follows: 50°C for 30 min, 95°C for 15min, followed by 5 cycles of 95°C for 15 s, 50°C for 30 min, and 72°C for 1 min, follow by 40 cycles of 95°C for 15 s, and 60°C for 45s. Samples with a cycle threshold (Ct) < 38 were regarded as positive.

Hadv Genotyping And Phylogenetic Analysis (Dup: Abstract ?)

HAdV genotyping was performed using nested PCR targeting the hypervariable region [11]. The primers used were forward 5’-TTCCCCATGGCNCACAACAC-3’ and reverse 5’-GCCTCGATGACGCCGCGGTG-3’. Samples that failed to amplify were defined as untyped. The nested-PCR products were sequenced by TaKaRa LA Taq with GC Buffer(Takara Code No.RR02) and the acquired hexon gene sequences were used together with 26 HAdV strain sequences downloaded from GenBank to construct the phylogenetic tree. The Maximum Likelihood (ML) method in MEGA 7.0 software was used for phylogenetic analysis. The evolutionary tree was evaluated using 1000 bootstrap replicates to verify the HAdV genotypes.

Statistical analysis

The detection rates of HAdV among different populations and seasons were evaluated using the X2 test, with P-values < 0.05 considered statistically significant. Sequence alignment and phylogenetic analysis were conducted using MEGA7.0. Epidemiological data analysis and graph drawing were performed using SPSS26.0 and R 4.2.0 software.

Characteristics of the enrolling samples

During the period from June 2019 to December 2021, 8,315 hospitalized patients were enrolled. Among them, 4,645 (55.86%) were males, and 3,670 (44.14%) were females, resulting in a sex ratio of 1.27:1. Out of all patients, 218 (2.62%) tested positive for HAdV infection, and there was a significantly different detection rate among age groups (P = 0.014). Among the 218 HAdV-positive patients, 138 (63.30%) were male, and 80 (36.70%) were female, with a sex ratio of 1.725:1 (Table 1). The age range of the HAdV-positive patients depicted in Fig. 1A was from 1 month to 95 years. The highest number of cases were observed in the 0–6 years age group, which accounted for 67.89% (148/218) of all cases. The number of cases gradually declined with increasing age, with the 7–20 years and 21–40 years age groups having the next highest number of cases.

Table 1

Age and sex differences among hospitalized patients with HAdV infection
Variable	Number of patient	Number of patient positive for HAdV	Percentage patient positive for HAdV (%)	P value
Age (years)
0 ~ 6	4888	148	3.44	0.004
~ 20	1385	40	2.89
~ 40	1512	22	1.46
~ 60	324	7	2.23
~ 95	206	1	0.49
Gender
Male	4645	138	2.97	0.025
Female	3670	80	2.18
Total	8315	218	2.62

Epidemiology Of Hadv

The prevalence of HAdV infection varied significantly over the course of the year (P = 3×10 − 6), with the highest detection rate occurring from August to December (63.30%, 138 out of 218 cases), and a peak in December (15.60%, 34 out of 218 cases). Similar detection rates were observed from February to April (17.89% of 218 cases). The prevalence of HAdV infection across the four age groups (0–6, 7–20, 21–40, and 41–60) mirrored the overall prevalence over the 12-month period. Specifically, the highest rates of infection were observed in the 21–40 and 41–60 age groups, followed by the 7–20 age group, and the lowest rates were observed in the 0–6 age group. These findings suggest that HAdV infection is seasonal and may be influenced by age.

Hadv Genotyping And Phylogenetic Analysis

After the hexon gene amplified by nested-PCR, 37 HAdV-positive samples with 875-bp fragment were obtained and successfully genotyped. In our research project, we conducted an analysis of 37 samples using phylogenetic methods based on the hexon gene sequence. Our findings indicate the presence of three distinct HAdV genotypes. The phylogenetic analyses showed that 10 cases belonged to species B (HAdV-B1, HAdV-B7, HAdV-B14), 22 belonged to species C (HAdV-C1, HAdV-C2, HAdV-C5, HAdV-C57, HAdV-C6), and 5 belonged to species E (HAdV-E4). HAdV-C1 was the most prevalent genotype (10/37), followed by HAdV-C6 and HAdV-E4 (Fig. 2). These results indicate that species B, species C and species E was circulated in Nanning from 2019–2021, making up 27.03%, 59.46% and 13.51% of the isolates, respectively.

In this study, we aimed to investigate the prevalence and characteristics of HAdV infections out of avian influenza in Nanning, China, over a three-year period from June 2019 to December 2021. Specifically, our objectives were to determine the age and sex distribution of HAdV infections in these hospitals, as well as to track the prevalence of HAdV over time. In addition, we sought to analyze the genetic diversity of the HAdV detected in different age groups and months.

According to reports, clinical symptoms of RTIs caused by HAdV can resemble those caused by other respiratory pathogens such as influenza and parainfluenza viruses, making it challenging to diagnose HAdV infections based solely on symptoms [12]. As a result, it's crucial to have rapid and effective diagnostic methods to identify and genotype HAdV. In this study, a qPCR assay was developed to detect and quantify HAdV in nasal and pharyngeal aspirate (NPA) specimens collected from 8,315 individuals. Out of the total specimens screened, 218 (2.62%) tested positive for HAdV, which is lower than the reported prevalence of HAdV in conjunction with avian influenza (ranging from 1.70–13.90%) [11, 13, 14]. In addition, the detection rate of HAdV varied across different regions in China. For instance, in Zhejiang province from 2006 to 2012, the detection rate of HAdV in hospitalized children with acute lower respiratory tract infections was 0.63%, while in Shenzhen city from 2012 to 2015, it was 2.24% [15, 16]. This study also found differences in the detection rate based on sex composition, age distribution, and seasonal effects.

Our study found that the highest prevalence of HAdV occurred during the winter and autumn seasons (63.30% from August to December), and in the spring season (17.89% from February to April). The peak prevalence was observed in December. Similar seasonal patterns have been observed in other studies conducted in Northern China and Mexico [17, 18]. However, some previous studies have suggested that the detection rates of HAdV are positively associated with the monthly mean temperature and sunshine duration, while being negatively correlated with wind speed. In fact, higher air temperatures are often associated with higher detection rates [7]. It is important to note that Nanning experiences high temperatures and humidity in almost all years. This may explain the discrepancy in our findings compared to other cities.

This study indicates that HAdV infections are predominantly observed in children under the age of 6, accounting for 67.89% of cases, highlighting the importance of HAdV as a pediatric pathogen. These findings are consistent with previous studies that have also reported HAdV infections to be common in young children [14, 19, 20]. HAdV can be easily transmitted through fomites contaminated with infectious bodily fluids. Interestingly, our study found that the detection rate of HAdV was lowest among individuals over the age of 60 (0.45%). However, these findings need to be interpreted with caution as the sample size was small, and longer observation periods may be required to confirm the results.

Our study employed nested-PCR to genotype 218 HAdV samples, resulting in 37 hexon 875-bp sequence. Phylogenetic analysis of these sequences revealed that 10 cases belonged to species B(B1, B7 and B14), 22 cases belonged to species C(C1, C2, C5, C6,C57), and 5 cases belonged to species E(E4). The most commonly found genotype was HAdV-C1 (10/37), followed by HAdV-C6 and HAdV-E4. Several studies have previously reported that the most common HAdV species causing RTIs in children worldwide are B (B3, B7, B21), C (C1, C2, C5, C6), and E (E4) [21–23]. In addition, in China, HAdV species B, C, and E are associated with severe pneumonia and are also the most prevalent species. In our study, we identified and analyzed 37 samples phylogenetically based on the hexon gene sequence, revealing three HAdV genotypes. The results showed that HAdV species B and C were the most commonly found, accounting for 27.03% and 59.46% of isolates, respectively.

This three-year study investigated the prevalence, age distribution, seasonality, and molecular epidemiology of HAdV infections associated with RTIs in Nanning, China, excluding avian influenza. The findings suggest recent changes in the circulating HAdV genotypes associated with RTIs in Nanning. These changes could aid in improving the diagnosis of HAdV-related diseases and developing new detection, treatment, and prevention strategies for clinical settings. However, only 37 sequences of the hexon gene were amplified from 218 HAdV samples in this study. Therefore, further analysis of the full-length genome is necessary to understand the extent of genetic variation and any potential recombination that occurred in the strains identified in this study.

Ct	cycle threshold
HAdV	Human adenoviruses
ML	Maximum Likelihood
MEGA	Molecular evolutionary genetics analysis
NPA	nasal and pharyngeal aspirate
RTIs	respiratory tract infections
qPCR	quantitative real-time polymerase chain reaction
SPSS	Statistical product and service solutions

Author contributions

YLH, BX, HQ, CY, and JQQ: designed the study and provided the correlative knowledge. LJY, HQ, LW, BX and JQQ: collected and provided the data. JQQ and TYY extracted data and cleaned data. LJY and TYY generated the figures and tables. YLH, BX, JQQ and TYY: wrote and edited the manuscript. All of the authors read and approved the final manuscript.

Funding

This work was supported by Nanning science and technology development program（20206124） and Guangxi Key Research and Development Program (GuiKeAB22035027).

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No competing interests reported.

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Journal Publication

published 02 Aug, 2023

Read the published version in Virology Journal →

Editorial decision: Major revision
10 May, 2023
Reviews received at journal
03 May, 2023
Reviewers agreed at journal
27 Apr, 2023
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24 Apr, 2023
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Submission checks completed at journal
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You are reading this latest preprint version

Exploring the Correlation between HAdV Infection and Respiratory Tract Infection Symptoms: A Study in Nanning, China

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Materials And Methods

Patient specimens

The Detection Of Influenza Viruses In The Enrolling Sample

The Inspection Method Of Hadv In Influenza-negative Specimens

Hadv Genotyping And Phylogenetic Analysis (Dup: Abstract ?)

Statistical analysis

Results

Characteristics of the enrolling samples

Epidemiology Of Hadv

Hadv Genotyping And Phylogenetic Analysis

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1