Human adenoviruses in respiratory infections: Sequencing of the hexon hypervariable region reveals high sequence variability

Journal of Clinical Virology 47 (2010) 366–371

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Human adenoviruses in respiratory infections: Sequencing of the hexon hypervariable region reveals high sequence variability Barbara Biere ∗ , Brunhilde Schweiger Robert Koch-Institut, Nationales Referenzzentrum für Influenza, Nordufer 20, 13353 Berlin, Germany

a r t i c l e

i n f o

Article history: Received 28 October 2009 Received in revised form 8 January 2010 Accepted 9 January 2010 Keywords: Adenovirus Hexon genotype Hypervariable region PCR

a b s t r a c t Background: In respiratory infections, human adenoviruses (hAdV) of species B1 and C are frequently detected, but severe or even fatal disease outbreaks are predominantly caused by only few serotypes. The molecular typing of hAdV hexon sequences can help to speed up the discrimination of serotypes, thus improving on-time epidemiological examinations and patient care. Objectives: We aimed to develop a molecular method for the rapid species B1 and C serotype identification in respiratory samples based on sequence generation of the hexon hypervariable region (HVR). Study design: We developed two PCR-based genotyping systems for a generic HVR amplification and sequence determination of species B1 and C viruses. The assays were applied to hAdV prototypes and 106 samples. Results: The primer sets proved to be capable of amplifying all B1 and C serotypes. The viruses detected in clinical samples belong to serotypes 1, 2, 3, 5 and 6. The obtained sequences of serotypes 2, 3 and 5 form 2–3 phylogenetic clusters that are based on the characteristic amino acid changes within the variable HVR sites. Conclusions: Our assay can significantly speed up the time-span needed for serotype identification and will improve epidemiological surveillance and patient care. The obtained hexon sequences of field viruses vary significantly and form multiple genetic lineages. The variability is focussed on the HVR sites and can be interpreted as the ongoing evolutionary process. Further research is needed on the hexon sequence variability of other (respiratory) hAdV serotypes. © 2010 Elsevier B.V. All rights reserved.

1. Background The 51 serotypes of human adenoviruses (hAdV) known to date were grouped into six species (A–F), based on biological and molecular criteria. Infections of the respiratory tract are predominantly caused by serotypes of species B1, C or E, but severe acute respiratory disease is mainly caused by only few serotypes, namely types 7, 3, 4 and 21.1–3 The knowledge about the differences among hAdV types has increased the medical value of typing. Recent studies furthermore suggest that in vitro susceptibility to some antivirals is species-specific.4 The determination of the viral serotype can thus contribute to decisions on patient care if achieved early in infection.4

The gold standard for serotyping is virus neutralization by specific immune sera,5 which is time-consuming, tedious and needs virus isolation as a prerequisite. Genotyping was suggested as alternative, based on the identification of a hypervariable region (HVR) in the main capsid component.6 The HVR consists of seven sites located on loops 1 (HVR1–6 ) and 2 (HVR7 ) of the hexon protein on the outer surface of the virion.7,8 It is conserved for a given serotype, but varies between serotypes, thus enabling serotype discrimination.5,6 2. Objectives We aimed to develop a genotyping system for the rapid identification of respiratory hAdV serotypes to complement serotyping by neutralization testing. 3. Study design

Abbreviations: hAdV, human adenovirus; HVR, hypervariable region; ORF, open reading frame; PCR, polymerase chain reaction. ∗ Corresponding author at: Robert Koch-Institut, Nationales Referenzzentrum für Influenza, FG 17 Influenza/Respiratorische Viren, Nordufer 20, 13353 Berlin, Germany. Tel.: +49 3018 754 2383; fax: +49 3018 754 2699. E-mail address: [email protected] (B. Biere). 1386-6532/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2010.01.005

3.1. Clinical samples Samples were taken from German patients with influenza-like illness between the years 2001 and 2007. Nasal and throat swabs

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Table 1 Oligonucleotide sequences. Species B1

C

Oligonucleotide sequence (5 –3 orientation)

Positiona

Amplicon length

Ta

F1 R1 F2 R2

gCATACATgCACATCgCCg AgAACggTgTACgCAggTAgAC gACAggATgCTTCggRgTACC gCTgATgCACTCTgACCACg

31–49 2812–2791 50–70 2767–2748

2793 bp

60 ◦ C

2729 bp

60 ◦ C

AdV HVR Seq B1

F571 F1043 R1662 R1116 R679

CCAgARCCTCARgTKggA TgAATgCDgTggTTgACTT CACTTgWATgTggAAAggCAC gTCHCCCAgAgARTCAAgC ACCCRTAgCAKggYTTCAT

571–588 1043–1061 1662–1642 1116–1088 679–661

Seq

55 ◦ C

AdV HVR PCR C

F1 R1 F2 R2

ATgATgCCgCAgTggTCTTAC ATTAAAggACTggTCgTTggTgTC ACgACgTRACCACAgACCg gCCACCACTCgCTTgTTCAT

16–36 1980–1957 161–179 1577–1558

1965 bp

60 ◦ C

1417 bp

60 ◦ C

F652 F1183 R1148 R744

ggMgAATCTCAgTggWAYgAA TAYTTTTCYATgTggAAKCAggC TgRTAKgAMAgCTCTgTgTTTCTg ATANgAWCCRTARCATggTTTCAT

652–672 1183–1205 1148–1125 744–721

Seq

55 ◦ C

Name b

AdV HVR PCR B

AdV HVR Seq C

Ta : annealing temperature; Seq: primer for sequencing. a Position in the hexon ORF of serotype 3 (AF542104) or serotype 2 (AY224391). b Slightly modified from primers P-060 s/as and P-061 s/as as published in [10].

were washed in cell culture medium, which was filter-sterilized and stored at −40 ◦ C until processed further.

3.5. Sequence determination and analyses

Prototypes of hAdV serotypes 1, 2, 3, 5, 6, 7, 16 and 50 were kindly provided by Albert Heim (Medizinische Hochschule Hannover, Germany). Serotype 21 was purchased from the American Type Cell Culture Collection (ATCC). Viral stock preparation was performed as described elsewhere.9

Amplicons were sequenced after purification (PCR Purification Kit, QIAGEN) using the dye terminator chemistry (ABI-Prism Big Dye Terminators v3.1 Cycle Sequencing Kit, Applied Biosystems) in a 3130xl Genetic Analyzer (Applied Biosystems). The sequences were processed using the DNA STAR Software Package. Alignments and phylogenetic analyses were done with the MEGA 4.02 software. All sequences were deposited at the GenBank database (Accession Numbers EU867450–EU867493 and FJ943581–FJ943637).

3.3. Preparation of nucleic acids

4. Results

3.2. Preparation of viral stocks

DNA was extracted from 300 ␮l sample material using the MagAttract DNA Blood M48 Mini Kit (QIAGEN) and eluted in 80 ␮l elution buffer. Alternatively, the RTP DNA/RNA Virus Mini Kit (Invitek) was used to extract from a volume of 400 ␮l, and nucleic acids were eluted in 60 ␮l elution buffer. 3.4. PCR analysis PCR reactions were set up in a total volume of 25 ␮l containing 1× PCR buffer, 0.8 mM dNTPs, 500 nM of each primer (listed in Table 1), 1 U PfuTurbo HotStart DNA Polymerase (Stratagene) and 5 ␮l template DNA. After an initial denaturation step at 95 ◦ C for 2 min, 40 cycles consisted of 95 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 1 min per 1000 bp amplicon length in a Mastercycler ep gradient (Eppendorf). For nested amplification, 1 ␮l of the first round product served as template for second round PCR with again 40 cycles. The PCR product was visualized with ethidium bromide on a 1% agarose gel under ultraviolet light. Real-time PCR and Fluorescence Melting Curve Analysis (FMCA) for pre-typing was carried out as described elsewhere.9

To amplify the hexon hypervariable region of species C viruses, a nested PCR system was designed yielding an amplicon of approximately 1.9 kb/1.4 kb in first/second round PCR. For the amplification of species B1 viruses, a published PCR system10 was slightly modified to optimize the amplification of species B1 viruses. Additional primers for sequence generation were designed for both species (Fig. 1). To test for the generic potential of these primers, amplicons of adenovirus prototypes were sequenced, yielding sequences that could be interpreted easily (data not shown). Applying our assays, 106 samples from patients with respiratory disease were examined in which hAdV of species B1 (38 samples) or C (70 samples) had been detected by real-time PCR (Ct values 18–35),9 including two samples containing both species. For 13 samples the amplification was done from virus isolates, while for 93 samples primary material was used. 101 of the 108 nested PCR reactions yielded a strong PCR product of correct size (36 of species B1, 65 of species C; Fig. 2) which could subsequently be sequenced without difficulties (data not shown). For 18 primary samples also a virus isolate was obtained and sequenced, yielding identical sequences for both materials.

Fig. 1. Primer concept. The PCR amplicon is generated by nested PCR with primers F1 + R1 (first round PCR) and F2 + R2 (second round PCR). The obtained amplicon then is sequenced with the PCR primers (depicted in black) as well as additional sequencing primers (depicted in grey). For sequencing of species B1 amplicons, the reverse PCR primer (R2) is omitted.

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Table 2 Analyzed sequence region and sample material. Species

Type



nta

Sequence divergenceb (%) nt

Yearc (#)

Material (#)

aa

B1

3

36

103–1603

0–1.3

0–2.0

2001 (3) 2002 (5) 2003 (1) 2005 (1) 2006 (3) 2007 (23)

Throat swab (16) Nasal swab (16) Nasal/throat swab (1) Nasal/throat washing (1) Throat secretion (1) Cell culture (1)

C

1

24

221–1495

0–0.5

0–0.5

2001 (2) 2002 (2) 2003 (2) 2004 (3) 2005 (2) 2006 (2) 2007 (11)

Throat swab (9) Nasal swab (8) Throat secretion (1) Throat washing (1) Cell culture (5)

2

29

216–1520

0–2.8

0–0.9

2001 (5) 2002 (7) 2003 (3) 2004(1) 2005 (2) 2006 (4) 2007 (7)

Throat swab (14) Nasal swab (8) Throat secretion (1) Cell culture (6)

5

11

210–1481

0–5.7

0–3.1

2002 (1) 2003 (1) 2005 (1) 2006 (1) 2007 (7)

Throat swab (5) Nasal swab (5) Cell culture (1)

6

1

231–1521

0.2

0

2003 (1)

Throat swab (1)

nt = nucleotides; aa = amino acids. a Nucleotide position in hexon ORF of the corresponding serotype (reference sequences hAdV1: AF534906, hAdV2: J01917, hAdV3: AB330084, hAdV5: M73260, hAdV6: DQ149613.1). b Sequence divergence was calculated with the DNA Star Software Package. c Year of sample collection.

Table 3 Amino acid changes of sample sequences in comparison to the corresponding reference sequences.

Reference sequences are the same as in Fig. 3 (Accession Numbers hAdV1: AF534906, hAdV2: J01917, hAdV3: AB330084, hAdV4: AB330085, hAdV5: M73260, hAdV6: DQ149613.1). Amino acid changes that occur only in 1–2 sequences are shaded in grey and were disregarded for lineage discrimination. a HVR genotypes defined in this study are named alphabetically (␣, ␤, and ␥), while prototype lineages are named ␲.

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Fig. 2. Amplification results of species B1 and species C PCRs. For both assays, PCR gel electrophoresis results (second round PCR of six samples) are depicted. 3 ␮l of PCR product was analyzed on a 1% agarose gel and stained with ethidium bromide (size marker: 300 ng of 250 bp DNA Ladder (Invitrogen)).

The analyses of the obtained sequences revealed that all 36 B1 hAdV belonged to serotype 3. The species C results were more heterogeneous with 24 sequences of serotype 1, 29 sequences of type 2, 11 sequences of type 5 and a single type 6 sequence (Table 2). A phylogenetic analysis including database reference sequences allowed an unequivocal classification of the amplified sequences to a certain serotype (Fig. 3). Serotype 2 splits into two genetic clusters: on the amino acid level, four amino acids in HVR1 , HVR7 and inter-HVR are divergent, including a glutamate insertion at AA 152 (Table 3). The overall sequence divergence between the two clusters is up to 2.8%/0.9% on the nucleotide/amino acid level (Table 2). An even higher sequence divergence of up to 5.7% was observed for the two serotype 5 phylogenetic clusters. The nucleotide substitutions account for 11 amino acid differences in HVR1–3 , HVR5 and HVR7 (Table 3). Three genetic lineages were identified for serotype 3: only four sequences were nearly identical to the reference sequence, while the majority differed in seven amino acids. These viruses could furthermore be differentiated into two separate lineages due to four additional amino acid changes in HVR3 and HVR7 . On the nucleotide level, the overall sequence divergence is 0–1.3% (0–2.0% amino acid level). For serotype 1, only low sequence variability was found (sequence divergence 0.5% on both the nucleotide/amino acid level), and the serotype 6 sequences proved to be identical to the reference sequence on the amino acid level (Table 2). 5. Discussion In this study we applied a method for the rapid and accurate identification of hAdV species B1 and C serotypes to 106 clinical samples. This method is based on sequence determination of the hexon hypervariable region, which has been described as suitable for serotype identification.5,6,11–13 Several PCR assays have been published that aim to genotype hAdV by hexon HVR sequencing.6,14–16 Although these assays proved to be suitable tools for serotype determination, they give only limited information for strain identification due to the short PCR product length including either HVR1–6 or HVR7 . We therefore chose to amplify the complete HVR, accepting the need for additional primers for sequence generation of the complete amplicon.

Two sets of primers were developed that comprise nested primer pairs for the amplification of the complete (B1, ca. 2700 bp) or partial (C, ca. 1400 bp) hexon gene, as well as additional primers that serve as sequencing primers. Initially, the assays were tested on hAdV prototypes. Both PCR assays yielded high amounts of PCR product for all targeted virus serotypes even if virus genome copy numbers were close to the detection limit, thus allowing unproblematic sequence determination. The assays were then applied to a total of 93 clinical samples and 13 field virus isolates that were pre-typed by application of a realtime PCR assay that allows for species determination by FMCA,9 but also other methods might be applied (e.g.17 ). 101 amplicons were obtained and sequences determined. Additionally, for 18 samples both a virus isolate and the clinical material were sequenced, yielding identical sequence results. We therefore conclude that the designed primers are suitable for sequence determination of viruses in the field. The results of our sequencing efforts were in concordance with the current knowledge on serotype and age distribution among patients with hAdV respiratory disease. Only nine samples were derived from patients older than 14 years, while 51 samples (50.5%) were derived from children ≤3 years. Although only a limited spectrum of hAdV serotypes was detected, our results indicate a continuous circulation of these types in Germany during the observed time-span. Apart from the samples presented, only one species D and one species E viruses were amplified from all hAdVpositive respiratory samples by real-time PCR. The detection of a species B2 virus has been described earlier.10 The obtained sequences showed a surprisingly high heterogeneity for serotypes 2, 3 and 5 that mainly affected the hypervariable regions and resulted in up to 13 amino acid substitutions when compared to database reference sequences. The amino acid changes were localized in several HVR sites, and often single HVRs contained multiple substitutions. The highest degree of variability was found in HVR7 . The majority of amino acid changes were conserved among several sequences, thus enabling a definition of genetic virus lineages (hexon genotypes) that cocirculated during the examined time-span. For serotype 3 this finding has been described before: based on the hexon sequence, Japanese hAdV-3 isolates

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were classified into three genotypes 3x, 3y and 3z.18 Applying that classification scheme, the German hAdV3 viruses belong to genotypes 3x and 3z (␤ and ␥ lineage in this study), while no counterpart is described for our ␣ lineage. A phylogenetic separation into two genetic lineages has also been described for Korean hAdV-3 isolates.19 The authors of this study hypothesize that these sequence variations account for an alteration of antigenic characteristics and therefore might be causative for the occurrence of hAdV-3 epidemics in the Korean population. Although a multitude of genome types has been defined for serotypes 2 and 5,20,21 only little is known about their HVR variability. A recent report on Japanese isolates includes data that are in part congruent with our observations, but not all amino acid changes are identical.22 However, the Japanese isolates of serotype 5 also form two clusters in phylogenetic analyses, but a separation of serotype 2 in different genetic lineages was not clearly demonstrated. For both serotypes, the distinction of genetic lineages can phylogenetically be verified by the inclusion of database sequences into our data set. We therefore suggest the definition of hexon genotypes also for types 2 and 5. In total, our data and the data of others indicate that the evolution of hAdV into separate genetic lineages is ongoing. This theory is supported by the fact that both loops 1 and 2 and thus the complete antigenic determinant “epsilon” underly sequence variability which thus may result in viruses with altered antigenic features. Moreover, the cocirculation of the different hexon genotype viruses indicates a separate evolution from a common ancestor. The acquisition of additional mutations in the future could therefore lead to an antigenic drift that might even result in the generation of separate serotypes in the long term. However, the hexon genotypes do not correlate to the adenovirus genome types that are defined by restriction enzyme analyses of viral DNA, as has been reported before.23,24 Regarding the clinical disease, only one of the 106 patients examined was reported to suffer from pneumonia. Interestingly, the patient was not infected with one of the hAdV serotypes known to be most frequently isolated from lower respiratory tract infections,1–3,25 but with serotype 2. This underlines the necessity to further clarify the role that the multitude of hAdV serotypes – including the different hexon genotypes – plays within differing disease settings. Our assay can greatly contribute to these examinations, as it helps to circumvent the laborious methods for hAdV serotyping and yields additional information on the hexon genotype. The amplification of the complete hypervariable region (HVR1–7 ) furthermore gives an overview of all antigenically relevant domains of the hexon protein and thus enables a more detailed analysis of antigenic changes than the previously published genotyping strategies. The data are available within 2 days instead of several weeks, enabling short-term decisions on patient care as well as on-time epidemiological examinations. Furthermore, the identification of virus serotypes and hexon genotypes in different clinical diseases will give further insight into hAdV pathogenesis.

Conflict of interest declaration

Fig. 3. Phylogenetic analysis of deduced amino acid sequences from 101 clinical samples. The MEGA software package was used to generate a phylogenetic tree applying the Neighbor joining method (Kimura 2 parameter) and 1000 replicates for bootstrap analysis. Reference sequences are highlighted in grey (Accession Numbers: hAdV1: AF534906, hAdV2: J01917, hAdV3: AB330084, hAdV4: AB330085, hAdV5: M73260, hAdV6: DQ149613.1, hAdV7: AF515814.1, hAdV8: DQ149614.1, hAdV11p: AF532578, hAdV12: X73487, hAdV14: DQ149612.1, hAdV16: X74662.1, hAdV21: AY008279.1, hAdV34: AB052911.1, hAdV35p: AY271307, hAdV40: X51782.1, hAdV50: DQ149643.1). Serotype clusters are framed in black, genetic virus lineages are framed in grey and assigned with lineage nomenclature according to Table 3.

Funding: None. Competing interests: None declared. Ethical approval: Not required.

Acknowledgements We thank Pia Henselmann and Florian Weigend for excellent technical assistance.

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