High-Incidence of Human Adenoviral Co-Infections in Taiwan

Background Respiratory infections caused by adenovirus (HAdV) are common year round. Recently, a significant increase of adenoviral infections was observed in Taiwan. Objective To understand the prevalence and molecular epidemiology of respiratory adenovirus circulating in Taiwan for the past decade. Study Design One hundred and twenty-six human adenoviruses, isolated between 2002 to 2011, were characterized via DNA sequencing of the hexon and fiber genes. The nucleotide sequences were then compared by phylogenetic analysis. Results HAdV-B3 accounted for 64.3% (81/126) and peaked almost every year, whereas the sequences of hexon and fiber genes of HAdV-B3 were highly conserved in different years. A high incidence of co-infection of adenoviruses was observed (19.0%, 24/126); HAdV-B3 co-infected with HAdV-C2 was the most common combination (58.3%, 14/24). An additional interesting finding of repeated infection was noted in 10 children, all of whom showed first infection with adenovirus species HAdV-C, followed by species HAdV-B or HAdV-E. Conclusions HAdV-B3 was the predominant type of respiratory adenovirus circulating in Taiwan over the past ten years. This merits further attention for vaccine development. Furthermore, the observed high-incidence of adenoviral co-infections along with repeated infections found in our study provides important epidemiological insights into adenovirus infections.


Ethics Statement
This study was conducted in Taiwan only, and Institutional Review Board (IRB) approval was obtained from National Cheng Kung University Hospital (No. A-BR-101-020). This was a retrospective study without intervention or obtaining extra clinical specimens. Human specimens were not directly used in this research and informed consent was waived. The waiving of informed consent was also approved by the Institutional Review Board of National Cheng Kung University Hospital.

Viral culture
Viral cultures of laboratory-confirmed adenovirus infection were processed as described previously [9]. This study used adenovirus isolated from either nasopharyngeal aspirate or throat swabs; and stored at -80°C at the Virology Laboratory of National Cheng Kung University Hospital before use. The study population was children from either hospitalized patients or outpatients of Cheng Kung University Hospital. Viruses used in this study were sub-cultured in A549 cells with Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. A549 cells in 15-ml culture tubes were infected with viral suspension, and then incubated at 35°C with 5% CO 2 . When 85% cytopathic effect was observed, cells were harvested for DNA extraction.

Viral genome extraction and genotyping
Viral DNA was extracted by the phenol/chloroform method according to a modified procedure from a previous report [23]. Volumes of reagents used in the procedure were reduced, and DNA pellets were finally resuspended in 50 μl of ddH 2 O.
To type the adenovirus, the Loop 1 region of the hexon gene was amplified with generic primer pair HXL1F (5′-CGTGTGCAGTTYGCCCG) and HXL1R (5′-ACAGCCTGATTCCACAT). The Loop 2 region of the hexon gene was amplified with generic primer pair BL (5′-TTGACTTGCAGGACAGAAA) and BR (5′-CTTGTATGTGGAAAGGCAC) (Table S1) [24]. To identify the hexon gene in co-infected isolates, type-specific primer pairs were designed. For the Loop 2 region, primers AdC2F and HXL2R were used for HAdV-C2; AdC5F and HXL2R for HAdV-C5; CDL and HXL2R-2 for HAdV-C6. In addition, AdBL1F and BR were used to amplify the region containing both Loop 1 and 2 for HAdV-B3. To amplify the fiber gene, primers FiBL and FiBR were used for HAdV-B3, HAdV-E4, HAdV-B7 and HAdV-B11; FiCL-2 and FiCR-2 for HAdV-C1; FiCL and FiCR for HAdV-C2; FiCL-2 and FiCR-3 for HAdV-C5. PCR mixtures consisted of 1 U of DNA polymerase (KOD plus polymerase, Toyobo), 1 mM MgSO 4 , 0.2 mM dNTP, 300 pM of each primer and 1 to 2 μl of template from the original purified DNA solution in a 50-μl reaction volume. PCR cycling consisted of initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 94°C for 20 seconds, 54°C or 56°C for 20 seconds, depending on primers used, and 72°C for 40 or 80 seconds, depending on the length of PCR products. DNA sequencing of PCR products was carried out with the primers used in PCR by Sanger's method.

Phylogenetic analysis
Phylogenetic trees were constructed by the neighbor-joining method based on PAUP software [25]. About 1500 base pairs of hexon gene (nt 139-1612 based on HAdV-B3) and 700 base pairs of fiber gene (nt 224-888 based on HAdV-B3) were analyzed. Sequences of reference strains were obtained from GenBank (for hexon gene, accession number for HAdV-C1

DNA sequencing of hexon and fiber genes of respiratory adenovirus circulating between 2002 and 2011
To analyze the molecular epidemiology of respiratory adenoviruses circulating in Taiwan, adenovirus-positive isolates collected at National Cheng Kung University Hospital (NCKUH) during 2002-2011 were examined. There were 2,001 adenovirus-positive isolates associated with respiratory diseases (Figure 1). One to two isolates per month over a decade were randomly selected for our investigation. A total of 106 adenovirus isolates were successfully subcultured. In addition, 20 isolates from repeated infections were also examined. The repeated infections were identified from our adenovirus database by computerized system according to patient identification number. In total, there were 126 isolates

Multiple infections of adenovirus
Sequencing results showed superimposed peaks in the chromatograms in 26 isolates using the generic primers BL and BR for the hexon gene ( Figure 3). Multiple templates indicated a mix of multiple adenoviral strains in one specimen. To confirm this probability, PCR products amplified by BL and BR for hexon Loop 2 region in two co-infected isolates were cloned and sequenced; distinct hexon genes of different types were verified (data not shown). The other co-infected isolates were confirmed by direct evidence of sequencing results of the hexon and/or fiber genes using type-specific primers developed in this study for different hexon (Loop 1 or 2) or fiber regions (Table S1). Co-infections were confirmed when two types of hexon genes and/or fiber genes from the same region were identified by sequencing (Table 1). In all, 24 samples (19.0%, 24/126) with more than one adenoviral strain in each were identified: 14 samples (58.3%) containing HAdV-C2 and HAdV-B3; 5 (20.8%) containing HAdV-C1 and HAdV-B3; 2 (8.3%) containing HAdV-B3 and HAdV-C5; 1 each (4.1%) containing HAdV-B3 and HAdV-C6; HAdV-C2 and HAdV-B11; and HAdV-C2, HAdV-B3 and HAdV-B11. Interestingly, all co-infected strains involved different species of adenovirus, including species HAdV-B and HAdV-C.

Repeated infections of adenoviruses
From our database, we noted that there were some children with repeated adenoviral infections from 2002 to 2011. Repeated infections may be caused by a variant strain of adenovirus. Therefore, these twenty isolates from three females and seven males were examined for genotypes ( Table  2). Intervals between infections in each patient ranged from three months to five years. Intriguingly, all these children were first infected with species HAdV-C adenoviruses (HAdV-C1, HAdV-C2 or HAdV-C5), followed by species HAdV-B (HAdV-B3) or species HAdV-E (HAdV-E4). This is the first report tracking repeated adenoviral infections over a decade.

Phylogenetic analysis of the hexon and fiber genes
Phylogenetic analysis of hexon and fiber genes identified 152 strains clustering with reference strains from the GenBank (Figure 4 and Figure S1). Strains of most genotypes from diverse years were grouped into only one cluster in both hexon and fiber genes, indicating sequences conserved throughout. Interestingly, only HAdV-C5 strains revealed two phylogenetic  (Table S2); by contrast, their fiber gene regions were grouped in one cluster. Differences in the fiber gene of HAdV-C1 strains of 2.2% and 6.3% were also found in nucleotide and amino acid sequences, respectively. Overall, the results indicate higher nucleotide variability in species HAdV-C adenoviruses than in species HAdV-B and HAdV-E.

Discussion
This molecular epidemiology study of adenovirus in Taiwan found a high incidence of adenoviral co-infection along with repeated infections. Adenoviral co-infections found in the present study were based on DNA sequencing of hexon and fiber genes, thus providing a high level of confidence in the validity of our data. Furthermore, unique infection patterns involving different species and type order were revealed in repeated adenoviral infections. Taken together, our investigation affords interesting insights into the respiratory adenoviral epidemiology.
Multiple-infection (co-infection) of adenoviruses was detected in 19.0% of clinical isolates analyzed. Adenoviral co-infection has also been reported in other countries, with a range of 1.5-19.0% [14,[26][27][28][29][30]. Variance in co-infection rate may arise from different communities where samples were taken or methodologies used. Primers used in PCR may also play a critical role in detecting co-infection. In this study, the phenomenon of multiple templates was detected in 26 isolates with the generic primer pair of hexon Loop 2, but only in two isolates with the generic primer pair of hexon Loop 1 (data not shown). This indicates that multiple primer sets may be required to detect co-infection.
This study discovered adenoviral co-infection via multiple PCR-sequencing templates, which were amplified using generic primers of the hexon Loop 2 region. Distinct hexon genes in co-infected isolates were then verified with typespecific primers and followed by nucleotide sequencing. In other studies, most adenoviral co-infections were determined with type-specific primers without DNA sequencing [14,26,27,[29][30][31]. For detecting co-infection, the application of generic primers is more convenient than that of type-specific primers in a community with diverse adenovirus populations; nucleotide sequencing must be determined subsequently. Genotypes in co-infected isolates still must be identified with type-specific primers, since multiple templates may appear in nucleotide sequencing. Furthermore, direct sequencing of the same genomic region should be the required criterion for the determination of co-infection since recombinants between different adenoviruses may exist. In addition, our evidence for co-infections may give rise to potential recombinants which can lead to more potent HAdVs [32].
Adenoviral co-infection has never been reported in previous epidemiology studies in Taiwan, which used genomic-RFLP or PCR-RFLP for adenovirus typing [21,22]. Our study revealed only dominant type (species HAdV-C) in the genomic-RFLP pattern (data not shown). Bias in titer in co-infected isolates was also found by plaque purification in another study [31], which suggests that PCR-sequencing for genotyping may be   period examined. Most common co-infections of adenoviruses in other nations also correlated with the predominant types in their community [14,26,30,31], suggesting adenovirus coinfection is not type-dependent. Nevertheless, we noted that strains in co-infected isolates always came from different species (HAdV-B and HAdV-C), with the exception of one coinfected case with three strains. These findings suggest a requirement for different species involved in co-infection and warrant further investigation.
Notably, repeated infections of adenovirus from 10 children were found and analyzed; all 10 were first infected with species HAdV-C, followed by species HAdV-B or HAdV-E. This suggests that primary infection with species HAdV-C may be able to protect against subsequent HAdV-C infections, but not against subsequent infection by species HAdV-B or HAdV-E. This may also explain why the proportion of species HAdV-C adenovirus is lower than species HAdV-B in many countries [15][16][17][18]. Adenoviral infection of vaccinated persons has been reported in American military recruits [31]. Infections in vaccinated persons were usually caused by a variant of the vaccine-targeted strain HAdV-E4 or HAdV-B7, indicating that adaptive immunity induced by species HAdV-B or HAdV-E is insufficient to safeguard against infection by variant strains of species HAdV-B or HAdV-E. Repeated detection of adenovirus in upper respiratory infections has also been reported by Kula   et al [33]. Three types of repeated infections were identified including 1) adenovirus of same strain, 2) adenovirus of same type but variant strains, 3) adenovirus of different types which were infected with species HAdV-B or HAdV-E (HAdV-B3 or HAdV-E4) and followed by species HAdV-C (HAdV-C1) of adenovirus in an 8-month or 24-month old child, respectively. The difference between this study and our results may be due to the younger age of patients and shorter interval of repeated infections in the study by Kula et al [33]. Taken together, these results suggest that further serologic study is required to understand the phenomenon of repeated infections of adenovirus. This study showed HAdV-B3 as the most common respiratory adenovirus strain circulating in Taiwan during 2002-2011, except in 2007. It was likewise the most common in America, Germany, Palestine and China during 1999-2010 [15][16][17][18]. This indicates that the most prevalent type of respiratory adenovirus in Taiwan is similar to that in many other countries. Sequences of hexon and fiber genes of HAdV-B3 were highly conserved between outbreaks, suggesting HAdV-B3 outbreaks may not be caused by changes in antigenicity. Conservation of the hexon gene evident in this study is consistent with a previous report that the hexon gene was conserved in most types circulating in Japan from 1988 to 2007 [34]. This study found two phylogenic clusters of HAdV-C5 in the hexon gene. Variation within HAdV-C5 was also discovered in Germany and Japan [15,34]. It is not clear whether different HAdV-C5 clusters are prevalent in different years, since only 10 isolates of HAdV-B5 were identified here.
In summary, this 10-year surveillance study showed that HAdV-B3 accounted for 64.3% of respiratory adenoviral infections, with strong nucleotide sequence conservation of the hexon and fiber genes of HAdV-B3. The results suggest development of HAdV-B3 vaccine is warranted. In addition, a high incidence of adenoviral co-infection was revealed. Most co-infections were HAdV-B3 co-infected with HAdV-C2. Finally, variant antigenic specificity among types of adenovirus was observed in children with repeated infections. Figure S1. Analysis of nucleotide sequences of a portion of the fiber gene (nucleotides 224-888/HAdV-B3).