Journal of Medical Virology 87:117–124 (2015)

Genome Stability of Adenovirus Types 3 and 7 During a Simultaneous Outbreak in Greater Manchester, UK Moustafa Alissa Alkhalaf,1 Malcolm Guiver,2* and Robert J. Cooper1 1 2

Virology Unit, Institute of Inflammation and Repair, The University of Manchester, Manchester, United Kingdom Public Health England, Manchester, United Kingdom

A total of 96 isolates of species B adenovirus collected in Greater Manchester, UK and typed previously by serum neutralization were analyzed in five genome regions. Of these, 62 isolates were HAdV-B3 and HAdV-B7 collected during a simultaneous 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 and other species B adenovirus types collected in different years following the outbreak. The phylogenetic analysis results of all the isolates in the structural regions hexon L2, penton, and fiber knob were found to be consistent and no mismatches were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable even when different types are circulating in a limited geographical area simultaneously. J. Med. Virol. 87:117–124, 2015. # 2014 Wiley Periodicals, Inc.

KEY WORDS:

sequence typing; phylogenetic analysis; adenovirus species B

INTRODUCTION Adenovirus can undergo antigenic shifts (recombination) and antigenic drifts (mutations) [CrawfordMiksza and Schnurr, 1996] and recombination has been well documented [Wigand and Adrian, 1989; Robinson et al., 2009; Walsh et al., 2009]. Historically, C 2014 WILEY PERIODICALS, INC. 

the intermediate strains, which have hexon of one type and fiber of a different type, used to be detected by the conflicting results of the serum neutralization and hemagglutination inhibition tests [Hierholzer, 1992]. Limited sequencing of the hexon and fiber has been used to detect this kind of recombination [Madisch et al., 2005, 2006]. In addition, sequencing of other regions of the adenovirus genome revealed hot spots of recombination within some genes such as hexon and penton [Robinson et al., 2009; Walsh et al., 2009]. Moreover, recombination events in other coding and non-coding regions have been detected using full genome sequencing and analysis [Walsh et al., 2009]. Many mismatches were observed in the DNA polymerase and E1A genes of species C adenoviruses which indicated recombination events in these nonstructural regions [Lukashev et al., 2008]. Coinfections of more than one type of adenovirus have been reported which gives these types the opportunity to interact and for recombination to occur [Echavarria et al., 2006; Vora et al., 2006]. This could be observed in special cases such as AIDS patients from whom many types of adenovirus were identified especially from species D [De Jong et al., 1999; Al Qurashi et al., 2012]. All these findings suggest that recombination plays an important role in adenovirus evolution. The changes in the adenovirus genome may result in more virulent variants [Walsh et al., 2010]. In addition, it could result in the emergence of new types of adenovirus such as HAdV-D53 [Walsh et al., The present address of Moustafa Alissa Alkhalaf is Department of Microbiology, Faculty of Pharmacy, University of Aleppo, Aleppo, Syria  Correspondence to: Malcolm Guiver, Public Health England, 3rd Floor, Clinical Sciences Building II, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, United Kingdom. E-mail: [email protected] Accepted 4 April 2014 DOI 10.1002/jmv.23969 Published online 7 May 2014 in Wiley Online Library (wileyonlinelibrary.com).

J. Med. Virol. DOI 10.1002/jmv

P1BR P2BL P2BR

CGAACTTAACACCAATGTCAC GTGCAGAACAATGACTTTACC AGGAACGTTTTCACTGACGG

1,196

14541–14562 14190–14211 15366–15386

Madisch et al. (2007) 597 Penton AdE1B1-R AdE1B-F AdE1B2-R P1BL

CTGGGAGCAGCTGAAATGGA CTGYGGGGTKAACGGYTA TTGRGTTCCGTGGACTGTGA TACGAGAGCGTGATGCAGC

822

896–916 87–107 888–908 13965–13984

Modified from Lukashev et al. (2008) 830 E1A AdPOL1R AdPOL2F AdPOL2R AdE1B-F

GTGGAAACCTACACCTGGATGGG TGGCGAGGTCATAGGCGAT GGTAGCGCCCAAGGGRCAC CTGYGGGGTKAACGGYTA

683

7772–7795 7684–7703 8347–8366 87–105

Modified from Lukashev et al. (2008) 32343–32363 7143–7166 653 GGAGGCAAAATAACTACTCG GCAAAAGTGTCCCTGACCATGAC DNA polymerase FiBR AdPOL1F

Madisch et al. (2005) 20470–20507 31362–31384 987 GCGTTGCGGTGGTGGTTAAATGGGTTTACGTTGTCCAT TACCCCTATGAAGATGAAAGCA Fiber HSAd2 FiBL

19879–19910 629 ATGTACTACAACAGCACTGGCAACATGGG

Primers position (HAdV-B3: AY599834) Amplicon size (bp)

Hexon L2 (HVR7)

DNA was extracted using the QIAamp DNA mini kit according to the manufacturer’s instructions. Fragments from different genome regions of the species B clinical isolates where amplified by PCR using different sets of primers (Table I). Mobyle@Pasture portal [Rice et al., 2000, http://mobyle.pasteur.fr/ cgi-bin/portal.py?#forms::merger] was used to merge the two overlapping sequences when two sets of primers were used (penton, E1A, DNA polymerase). The PCR mixture contained 10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each primer, 0.2 mM dNTPs, 1.25 Units AmpliTaq Gold DNA polymerase and 5 ml of extract or control in a final volume of 50 ml. Every PCR run included an extraction

HSAd1

Polymerase Chain Reaction (PCR)

Sequence 50 –30

A total of 96 isolates of HAdV-B were kindly supplied by Mr. Andrew Bailey (Central Manchester University Hospitals NHS Foundation Trust) who typed them by serum neutralization at the time of isolation. Of these, 27 HAdV-B3 and 35 HAdV-B7 isolates were collected during a 15-month outbreak between December 1983 and February 1985 and a further 23 isolates of HAdV-B3 and HAdV-B7 were collected from 1986 to 1997. In addition, 11 isolates of other types of HAdVB (HAdV-B11, HAdV-B16, HAdV-B21, and HAdV-B35) were included in this study. All samples were collected in Manchester, UK.

Target region

Species B Clinical Isolates

Primer

METHODS

TABLE I. Primers Used for the Amplification of Genome Regions of the HAdV-B Isolates

2009]. It could also change the neutralizing epitopes such as HAdV-B55 [Walsh et al., 2010] or the tropism such as HAdV-D56 [Robinson et al., 2010]. However, other studies suggest that the adenovirus genome can be stable over long periods of time. These reports used the full genome sequencing to compare prototype strains, which were isolated in the 1950s and recently isolated field strains. Full genome sequencing of three strains of HAdV-C5 [Seto et al., 2010] compared the prototype from 1953 with a strain circulating in laboratories for 39 years and a field strain from 1998 revealed very limited changes and no recombination events, which indicate that HAdV-C5 genome is stable over a long period of time. Similar studies of HAdV-B3 [Mahadevan et al., 2010], HAdV-B7 and HAdV-E4 [Purkayastha et al., 2005] revealed genomic stability for up to 50 years. In this study, different genome regions of HAdV-B3 and B7 isolated from patients during a simultaneous 15-month outbreak of both types in Greater Manchester, UK have been compared together with those of species B isolates made up to 11 years after the outbreak. This affords the opportunity to look for recombination events among viruses circulating in a localized geographical area over a short period of time, circumstances that might be expected to favor such events.

Sarantis et al. (2004)

Alkhalaf et al. Reference

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Genome Stability of Adenovirus Types 3 and 7

control, negative control (distilled H2O) and a positive DNA control. Positive controls are DNA extracts from isolates typed previously using both serum neutralization (SN) and sequencing of the hexon L2 region. All amplifications (GeneAmp1PCR System 9700, Applied Biosystems, Warrington, UK) were for 40 cycles and began with a single denaturation cycle of 95˚C for 15 min and ended with a final step of 72˚C for 5 min. Cycling was 95˚C (1 min), 51˚C (1 min) and 72˚C (1 min) for all except the penton, which was 94˚C (20 sec), 56˚C (20 sec), and 72˚C (40 sec). Sequencing and Phylogenetic Analysis Both strands of the amplicons were sequenced separately and analyzed as described previously [Al Qurashi et al., 2012]. The accession numbers of the control sequences used to create the phylogenetic trees were as follows: HAdV-B3 GB (AY599834); HAdV-B3 NHRC 1276 (AY599836); HAdV-B7 Gomen (AY594255); HAdV-B7 NHRC 1315 (AY601634); HAdV-B11 (AY163756); HAdV-B14 (AY803294); HAdV-B16 (AY601636); HAdV-B21 (AY601633); HAdV-B34 (AY737797); HAdV-B35 (AC000019); HAdV-B50 (AY737798); HAdV-B55 (FJ643676). The accession numbers of the sequences from isolates in this study are Hexon L2: HAdV-B7 84-E1 (KF279615), 92-59 (KF279616), 95-13 (KF279617), 84-C3 (KF279618), HAdV-B3 97-03 (KF279619), 84D6 (KF279620), 87-91 (KF279621), 84-J1 (KF279622), 92-54 (KF279623), HAdV-B35 84-05 (KF279624), HAdV-B11 87-15 (KF279625), HAdV-B16 84-44 (KF279626), 84-22 (KF279627), HAdV-B21 85-65 (KF279628); Fiber Knob: HAdV-B7 84-C1 (KF279597), 84-D4 (KF279598), 92-59 (KF279599), HAdV-B11 87-15 (KF279600), HAdV-B3 88-49 (KF279601), 92-54 (KF279602), 84-J1 (KF279603), 87-91 (KF279604), 84-A10 (KF279605), 84-E2 (KF279606), 84-E3 (KF279607), 84-B3 (KF279608), 97-03 (KF279609), HAdV-B16 84-44 (KF279610), 8422 (KF279611), HAdV-B21 85-65 (KF279612), HAdVB35 94-03 (KF279613), 84-05 (KF279614); Penton: HAdV-B7 92-59 (KF279581), 84-D4 (KF279582), 84E1 (KF279583), HAdV-B3 84-A8 (KF279584), 92-54 (KF279585), 83-A10 (KF279586), 84-E3 (KF279587), 97-03 (KF279588), HAdV-B16 84-44 (KF279589), 8422 (KF279590), HAdV-B21 88-11 (KF279591), 85-65 (KF279592), HAdV-B35 85-15 (KF279593), 94-03 (KF279594), 84-05 (KF279595), HAdV-B11 87-15 (KF279596); E1a: HAdV-B3 84-E2 (KF279566), 84-D6 (KF279567), 97-03 (KF279568), 84-A1 (KF279569), 92-54 (KF279570), 84-J1 (KF279571), 83-A10 (KF279572), HAdV-B7 84-A6 (KF279573), 92-59 (KF279574), HAdV-B21 85-65 (KF279575), HAdVB16 84-22 (KF279576), 84-44 (KF279577), HAdV-B35 84-05 (KF279578), 85-15 (KF279579), HAdV-B11 8715 (KF279580); DNA Polymerase: HAdV-B7 84-A9 (KF279546), 84-C1 (KF279547), 84-E1 (KF279548), 84-F6 (KF279549), 95-13 (KF279550), 92-59 (KF279551), HAdV-B3 84-J1 (KF279552), 83-A10

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(KF279553), 84-A8 (KF279554), 84-E3 (KF279555), 88-49 (KF279556), 92-54 (KF279557), 97-03 (KF279558), HAdV-B16 84-22 (KF279559), 84-44 (KF279560), HAdV-B21 85-74 (KF279561), 85-65 (KF279562), HAdV-B35 85-15 (KF279563), 84-05 (KF279564), HAdV-B11 87-15 (KF279565). RESULTS Molecular Analysis of HAdV-B Clinical Isolates DNA extracts of 96 clinical isolates of HAdV-B species were amplified, sequenced and phylogenetic analysis of the hexon L2, fiber knob, penton, E1A, and DNA polymerase regions was performed. Sixtytwo of the isolates (27 HAdV-B3 and 35 HAdV-B7) were collected between December 1983 and February 1985 during a 15-month simultaneous outbreak in the Manchester area. These isolates were chosen because they were circulating at the same time and in the same geographical area providing an opportunity for recombination events. Analysis was also carried out on a further 23 isolates of HAdV-B3 and HAdV-B7 collected in different years following the outbreak in addition to 11 isolates of other types of HAdV-B (HAdV-B11, HAdV-B16, HAdV-B21, and HAdV-B35). In addition to the prototype strains HAdV-B3 (GB) and HAdV-B7 (Gomen), the reference strains HAdV-B3 NHRC 1267 and HAdV-B7 NHRC 1315 were included in the phylogenetic analysis because they are among the main reference strains that have been used elsewhere in the phylogenetic analysis of HAdV-B and because most of the isolates in this analysis clustered close to them in all genome regions. Sequencing and Phylogenetic Analysis of HAdV-B3 and HAdV-B7 Clinical Isolates From the 1983 to 1985 Outbreak The phylogenetic analysis of the hexon L2 region (Fig. 1a) showed that HAdV-B3 clinical isolates grouped in two major clusters I and II. The first cluster (I) contained 11 isolates which were identical to the prototype HAdV-B3 (GB) while the second cluster (II) contained 16 isolates identical to the reference strain HAdV-B3 NHRC 1276. HAdV-B7 isolates grouped in one cluster and divided into two variants, which differ from the prototype HAdV-B7 (Gomen) by 4.56 and 5.05%. However, these isolates were very close to the reference strain HAdV-B7 NHRC 1315. Only one isolate belonged to v2 and differed from the other 34 v1 isolates by one nucleotide. The phylogenetic analysis of the clinical isolates in the fiber knob region showed the same clustering patterns as in the hexon L2 region and no evidence of recombination between the isolates in these two regions (Fig. 1b). The isolates of HAdV-B3 grouped in two clusters and most of cluster I isolates had the same sequences as the prototype. The second cluster J. Med. Virol. DOI 10.1002/jmv

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Fig. 1. Phylogenetic analysis of nucleotide sequences of the species B isolates. The number in square brackets represents the number of isolates of each variant. Variants in the boxes were isolated during the simultaneous outbreak of HAdV-B3 and HAdV-B7 between December 1983 and February 1985. Variants underlined in d and e have a different clustering pattern to that of their structural genes. a: hexon L2 region, b: fiber knob region, c: penton region, d: E1a region, e: DNA polymerase region.

contains two variants, which were very close to the reference strain NHRC 1276. HAdV-B7 isolates were identical or very close to the reference strain NHRC 1315. The clustering patterns of the HAdV-B clinical isolates in the penton region were the same as in the hexon L2 and fiber knob regions (Fig. 1c). Two clusters of HAdV-B3 were detected as with the hexon L2 and fiber knob regions. All of the isolates in J. Med. Virol. DOI 10.1002/jmv

cluster I were identical to the prototype. Two variants of cluster II isolates were detected and all of them were very close to HAdV-B3 NHRC 1276 reference isolate. All HAdV-B7 isolates nucleotide sequences were closer to the reference strain HAdVB7 NHRC 1315 than the prototype. In the E1A phylogenetic tree, HAdV-B3 and HAdVB7 clinical isolates grouped in similar clusters to the hexon L2, fiber knob and penton regions trees

Genome Stability of Adenovirus Types 3 and 7

(Fig. 1d). HAdV-B3 cluster I isolates were identical or very similar to HAdV-B3 prototype. Most of the isolates in cluster II were identical or very close to reference strain HAdV-B3 NHRC 1276. HAdV-B7 isolates were identical or very close to the reference strain HAdV-B7 NHRC 1315. Most of the clinical isolates in the DNA polymerase region grouped in the same clusters as the previous four genome regions (Fig. 1e). Two clusters of HAdVB3 isolates were detected. The isolates in the first cluster were identical to the prototype HAdV-B3 while the second cluster isolates were very close to the reference strain NHRC 1276. HAdV-B7 clustered close to the reference strain NHRC 1315 and can be divided into different variants which differed from the reference strain by 0.1– 0.3% nucleotide divergence. One isolate (84-F6; v4) was clustered with the rest of HAdV-B7 isolates and the reference strain NHRC 1315 in the hexon L2, fiber knob, penton and E1A regions. However, this isolate shifted in the DNA polymerase region to a different cluster and became closer to HAdV-B3 prototype with (8 nucleotides; 0.75%) divergence comparing to (10 nucleotides; 1.03%) and (18 nucleotides; 1.58%) divergence from NHRC1315 reference strain and HAdV-B7 prototype, respectively. However, at the amino acid level, isolate 84-F6 was closer to HAdV-B7 NHRC 1315 reference strain and differed from it by 4 amino acids comparing to 6 and 11 amino acids from HAdV-B3 and HAdV-B7 prototypes. Sequencing and Phylogenetic Analysis of HAdVB3 and HAdV-B7 Clinical Isolates From Later Years and Isolates From Other HAdV-B Types A total of 23 isolates of HAdV-B3 and HAdV-B7 collected in different years following the outbreak (1986–1997) in addition to 11 isolates of HAdV-B types 11, 16, 21, and 35 were sequenced and analyzed in different genome regions. The phylogenetic analysis of the hexon L2 region (Fig. 1a) showed that HAdV-B3 and HAdV-B7 isolates from different years had the same clustering patterns as the outbreak isolates. Of the 12 HAdV-B3 isolates, which were collected between 1986 and 1996, 11 clustered with the reference strain NHRC 1276 and only one isolate from 1997 clustered with the HAdV-B3 prototype. All 10 HAdV-B7 isolates clustered with the reference strain NHRC 1315. Similar results were found by the phylogenetic analysis of these isolates in the fiber knob, penton (Fig. 1b, c), E1A and DNA polymerase regions (Fig. 1d,e) with no mismatches between the isolates. The isolates of other HAdV-B types clustered with their prototype in the hexon L2, fiber knob, and penton regions. In the E1A region, all clinical isolates from different types of species B grouped close to their prototype except HAdV-B16 isolates (Fig. 1d). One of these isolates (v1) was closer to HAdV-B50 with 0.4%

121

nucleotide divergence comparing to 1.57% with HAdV-B16 and the other two isolates (v2) were closer to HAdV-B21 with 0.27% nucleotide divergence comparing to 0.4% divergence with HAdV-B16. In the DNA polymerase region, HAdV-B21 and HAdV-B35 isolates clustered close to their prototypes as in the previously analysed regions (Fig. 1e). One isolate which clustered close to the HAdV-B11 prototype in the previously analyzed regions aligned close to HAdV-B34 in the DNA polymerase region with 0.93% divergence comparing to 1.3% with HAdV-B11. In addition, HAdV-B16 isolates also clustered differently in the DNA polymerase region and had the same clustering patterns as HAdV-B16 isolates in the E1A region. The closest type to one of the isolates (v1) was HAdV-B50 with 0.65% nucleotide and 0.84% amino acid divergence comparing to 0.93% nucleotide and 1.4% amino acid divergence with HAdV-B16. The other two isolates (v2) clustered with HAdV-B21 which was the closest type to it with 0.1% nucleotide and 0.28% amino acid divergences comparing to 1.21% nucleotide and 1.95% amino acid divergence with HAdV-B16. Gene Stability of HAdV-B Clinical Isolates The phylogenetic analysis of the clinical isolates from the outbreak in different genome regions showed no mismatches between HAdV-B3, HAdV-B7 and other HAdV-B types in the hexon L2, fiber, and penton regions which indicate that the genome is stable in these regions. The same results were found with HAdV-B3 and HAdV-B7 from different years following the outbreak, which indicates that genome of these two types remained stable for over 13 years. No mismatches were detected in HAdV-B3 and HAdV-B7 isolates in the E1A region unlike 3 HAdV-B16 isolates that clustered with different HAdV-B types. In the DNA polymerase region, one HAdV-B7 isolate from the outbreak was clustered with HAdV-B3. In addition, HAdV-16 and HAdV-B11 isolates were clustered with different HAdV-B types in this region (Table II). This indicates that HAdV-B genome might be less stable in DNA polymerase and E1A regions than in the structural regions (hexon, fiber, and penton). DISCUSSION Genome stability of HAdV-B3 and B7 has been investigated previously by comparing the full genome sequence of the prototype strain, which was isolated in 1953 to recently isolated strains and has provided evidence for genome stability for up to 50 years [Purkayastha et al., 2005; Mahadevan et al., 2010]. There have, however, been reports of recombination between hexon and fiber of HAdV-B [Hierholzer & Pumarola, 1976; Adrian and Wigand, 1986; Hierholzer et al., 1992]. Intermediate strains of species B adenoviruses have been associated with outbreaks and more severe disease. For example, HAdV-B7h (HAdV-BH7/F3), which has a HAdV-B7 hexon but J. Med. Virol. DOI 10.1002/jmv

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Alkhalaf et al. TABLE II. Typing Results of Isolates Which Changed Their Clustering Patterns in Different Genome Regions

Hexon L2

Fiber knob

Penton

E1A

DNA polymerase

Isolate

SN serotype

Type

Var

Type

Var

Type

Var

Type

Var

Type

Var

84-F6 87-15 84-22 84-44 85-95

7 11 16 16 16

7 11 16 16 16

v1 v1 v1 v2 v2

7 11 16 16 16

v1 v1 v1 v2 v2

7 11 16 16 16

v2 v1 v1 v2 v2

7 11 50 21 21

v1 v1 v1 v2 v2

3 (I) 34 50 21 21

v5 v1 v1 v2 v2

The table summarizes the isolates, serum neutralization (SN) typing, types and variants (Var) in different genome regions. The changes in the clustering patterns are indicated in bold.

HAdV-B3 fiber and E3 regions has been associated with epidemics of severe respiratory disease in South America [Kajon and Wadell, 1996; Videla et al., 1999] and Japan [Hashido et al., 1999]. Similarly, HAdVBH3/F16 has been detected in cases of acute respiratory disease in Argentina [Kajon et al., 2010]. Also, HAdV-BH11/F14 has been isolated from an outbreak of respiratory tract disease in China [Yang et al., 2009]. Most of the isolates in this study were HAdVB3 and HAdV-B7 circulating at the same time and in the same area, which gave them the opportunity to interact and undergo recombination in vivo. However, the phylogenetic grouping results of all the isolates in the hexon L2, penton, fiber knob, and E1A regions were found to be consistent and no mismatches were observed between types 3 and 7 isolates or between them and any other HAdV-B types which indicates that adenovirus genome is stable in these regions. In addition, most of the HAdV-B3 and HAdV-B7 isolates in the DNA polymerase region had the same clustering patterns as the other regions. An exception was one isolate that clustered with HAdV-B7 NHRC1315 reference strain in all the regions analyzed but appeared closer to HAdV-B3 prototype in the DNA polymerase region. However, this change of clustering was the result of three nucleotide substitutions only which suggest a natural variation rather than recombination. The types 3 and 7 isolates from different years also had the same clustering patterns, which indicate the stability of the genome over long periods of time (more than 13 years). The same result was found for isolates from HAdVB types other than 3 and 7 in the hexon L2, fiber knob, and penton regions. However, different clustering patterns were observed for HAdV-B16 isolates in the E1A and DNA polymerase regions (one isolate clustered with HAdV-B50 and two with HAdV-B21). In addition, HAdV-B11 isolate became closer to HAdV-B34 in the DNA polymerase region. These results indicate that E1A and DNA polymerase region might be less stable than the other three structural regions: hexon L2, penton, and fiber knob. Only a few sequences of HAdV-B16 are available in the GenBank which is not enough to understand the J. Med. Virol. DOI 10.1002/jmv

natural variation of this type. The divergences between the prototype of HAdV-B16 and the prototypes of HAdV-B21 and HAdV-B50 in DNA polymerase region are 1.2% (11 nucleotides) and 0.7% (7 nucleotides), respectively and these divergences in E1A region are 0.4% (3 nucleotides) and 1.4% (10 nucleotides), respectively. These divergences are very low in comparison with other regions of HAdV-B16 with HAdV-B21 and HAdV-B50 which are as follows: hexon L1: 30.7 and 41%, hexon L2: 25.5 and 24.4%, fiber knob: 42.3% and 41.7%, penton: 19.3% and 19%, respectively. So, the difference in the clustering patterns of HAdV-B16 isolates in the structural and non-structural region could be because of the natural variation of this type rather than recombination. Fifteen isolates of HAdV-B collected in Portugal during a pediatric outbreak were typed by phylogenetic analysis of the hexon and fiber regions [Rebelode-Andrade et al., 2010]. Most of the isolates belonged to HAdV-B3 (10 isolates) and the rest to HAdV-B7. None of HAdV-B3 isolates clustered with the prototype but they clustered with the HAdV-B3 NHRC1276 reference strain, which is the same as cluster II in this study. The rest of the isolates, which belonged to type 7 did not cluster with the prototype but with HAdV-B7 NHRC1315 reference strain. The phylogenetic analysis for the fiber region revealed the same clustering patterns as in the hexon region for all but one isolate. All the isolates of HAdV-B3 cluster I were from 1983 and 1984 (outbreak isolates) except isolate 9703 which was isolated in 1997. The remaining isolates collected in different years outside the outbreak (1986–1996) in addition to some outbreak isolates belonged to cluster II. This may indicate that both clusters dominated during 1983–1984, then cluster II became predominant until 1997 when cluster I appeared again although the number of analyzed isolates outside the outbreak was small. It was reported previously that a specific genotype of species B could dominate in a specific time or a particular geographical area [Erdman et al., 2002; Kim et al., 2003]. For example, phylogenetic analysis of the full hexon of HAdV-B3 isolates from Taiwan collected between 1983 and 2005 revealed two

Genome Stability of Adenovirus Types 3 and 7

clusters, one of them contained most of the isolates collected in 1983–1999 and the second one contained most of the isolates collected between 2001 and 2005 [Lin et al., 2010]. The stability of 16 HAdV-C isolates collected from different geographical areas was studied by Lukashev et al. (2008). These isolates were analyzed in four genome regions: hexon, fiber, DNA polymerase and E1A. All of the isolates clustered with their prototypes in the hexon and fiber regions except one. However, in the other two regions the clustering patterns were different and most of the isolates did not cluster with their prototypes and some of them had identical sequences to different types. In contrast, of the 96 isolates collected from the Greater Manchester area examined in our study, only four of them changed their clustering patterns in the DNA polymerase and E1A regions but none of them became identical to a different type. This indicates that HAdV-B types may be more stable than HAdV-C types in these two regions. The results obtained in this study indicate the HAdV-B types and especially HAdV-B3 and HAdVB7 are stable even when circulating in the densely populated (2.7 million people) Greater Manchester area (1,277 km2) which supports the earlier mentioned reports about adenovirus genome stability over long periods of time. It may be that intermediate strains have not been detected in this study because they occurred in patients who did not require hospitalization. When such strains have been isolated elsewhere they have been associated with more severe disease suggesting recombination has conferred a selective advantage that may not occur in all outbreaks making their detection less likely. REFERENCES Adrian T, Wigand R. 1986. Adenovirus 3-7, an intermediate strain of subgenus B. Intervirology 26:202–206. Al Qurashi YMA, Alkhalaf MA, Lim L, Guiver M, Cooper RJ. 2012. Sequencing and phylogenetic analysis of the hexon, fiber, and penton regions of adenoviruses isolated from AIDS patients. J Med Virol 84:1157–1165. Crawford-Miksza LK, Schnurr DP. 1996. Adenovirus serotype evolution is driven by illegitimate recombination in the hypervariable regions of the hexon protein. Virology 224:357–367. De Jong JC, Wermenbol AG, Verweij-Uijterwaal MW, Slaterus KW, Wertheim-van Dillen P, van Doornum GJ, Khoo SH, Hierholzer JC. 1999. Adenoviruses from human immunodeficiency virusinfected individuals, including two strains that represent new candidate serotypes Ad50 and Ad51 of species B1 and D, respectively. J Clin Microbiol 37:3940–3945. Echavarria M, Maldonado D, Elbert G, Videla C, Rappaport R, Carballal G. 2006. Use of PCR to demonstrate presence of adenovirus species B, C, or F as well as coinfection with two adenovirus species in children with flu-like symptoms. J Clin Microbiol 44:625–627. Erdman DD, Xu W, Gerber SI. 2002. Molecular epidemiology of adenovirus type 7 in the United States, 1966–2000. Emerg Infect Dis 8:269–277. Hashido M, Mukouyama A, Sakae K, Tsuzuki H, Yamashita T, Inada T, Inouye S. 1999. Molecular and serological characterization of adenovirus genome type 7h isolated in Japan. Epidemiol Infect 122:281–286.

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J. Med. Virol. DOI 10.1002/jmv

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J. Med. Virol. DOI 10.1002/jmv

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Genome stability of adenovirus types 3 and 7 during a simultaneous outbreak in Greater Manchester, UK.

A total of 96 isolates of species B adenovirus collected in Greater Manchester, UK and typed previously by serum neutralization were analyzed in five ...
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