Arch Virol DOI 10.1007/s00705-016-2798-7
BRIEF REPORT
Temporal variation in the distribution of type-1 human astrovirus lineages in a settled population over 14 years ´ kos Gelle´rt3 Simona De Grazia1 • Floriana Bonura1 • Krisztia´n Ba´nyai2 • A 1 • 4 • Sandra Marineo Vito Martella Giovanni M. Giammanco1
•
Received: 7 December 2015 / Accepted: 14 February 2016 Ó Springer-Verlag Wien 2016
Abstract Human astroviruses (HAstVs) are important enteric pathogens that are genetically and antigenically heterogeneous and can be classified into eight sero/genotypes (HAstV-1 to -8) and different lineages within each HAstV type. This study describes the genetic diversity of HAstVs circulating in southern Italy over 14 years. Molecular analysis of HAstV-1 strains showed that three different lineages (1a, 1b and 1d) of the predominant genotype were circulating during the study period. The study of an archival collection of HAstV strains offers a unique opportunity to evaluate the patterns of variation of HAstV infections over the years and to correlate the observed epidemiological changes to the genetic variability of HAstVs.
Astroviruses (AstVs) are important enteric pathogens of mammals and birds [1]. They belong to the family Astroviridae and have a single-stranded positive-sense RNA genome containing three open reading frames (ORFs): ORF1a, ORF1b and ORF2. ORF1a and ORF1b, at the 5’ end of the genome, encode the non-structural viral proteins,
& Simona De Grazia [email protected] 1
Department of Health Promotion Sciences and Mother and Child Care ‘‘G. D’Alessandro’’, University of Palermo, via del Vespro 133, 90127 Palermo, Italy
2
Veterinary Medical Research Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary
3
Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary
4
Department of Veterinary Medicine, University Aldo Moro of Bari, Valenzano, Italy
including the RNA-dependent RNA polymerase, while ORF2, at the 3’ end, encodes the capsid protein precursor. Human AstVs (HAstVs) are genetically and antigenically heterogeneous [2]. HAstVs have been classified into eight types (HAstV -1 to -8) by sequence analysis of short fragments localized at the 5’ end or the 3’ end of ORF2. Importantly, a correlation has been found between genetic and antigenic characteristics [3]. Genotyping surveys have shown that HAstV-1 is the most common type identified in children, followed by HAstV-2, -3, -4 and -5, whereas HAstV-6, -7, and -8 have been detected more rarely [4, 5]. The incidence and distribution of HAstV sero/genotypes varies seasonally and geographically. Recent studies have identified novel HAstVs that are more closely related to animal astroviruses than to classical HAstVs [6–9]. The greatest sequence variability in the AstV genome is found in ORF2, characterised by a highly conserved N-terminal domain (aa 1-424), a hypervariable domain (HVR) (aa 425-688) and a highly acidic C-terminal domain [1, 5]. Interestingly, upon molecular analysis of ORF2, discrete sequence variation has been observed with different lineages within each HAstV type. Both HAstV-1 (HAstV-1a-1d) and HAstV-2 (HAstV-2a-2d) have been divided into four lineages, whereas HAstV-3 (HAstV-3a3b) and HAstV-4 (HAstV-4a-4b-4c) have been classified into two and three lineages, respectively [4, 10–12]. In Palermo, Sicily, in southern Italy, a documented uninterrupted HAstV surveillance has been conducted since 1999 [11–15]. The resulting archival collection, spanning nearly 15 years, offers an opportunity to evaluate the patterns of variation of HAstV infections over the years and to correlate the observed epidemiological changes to the genetic variability of HAstVs. This study describes the genetic diversity of HAstVs circulating in a settled population of Palermo from August
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Table 1 Yearly prevalence of astrovirus infection in relation to the number of strains sequenced Year 2000
Number of sample tested 135
Number (%) of HAstVpositive samples
other studies conducted in different parts of the world, such as Spain, Hungary, Brazil, China, India and Bangladesh [4, 18–21, 22]. In this study, the yearly detection rates of HAstVs rates ranged from 0.53 % in 2005 to 10.14 % in 2006. No HAstV was detected in 2001. Mixed infections with rotavirus and norovirus were observed in[50 % of all HAstV-positive cases (39/78) and comparable rates of mixed infections were reported in other epidemiological studies [4, 18–20, 23]. A genotype was determined for 65 (83.3 %) out of 78 HAstV strains detected during the study (Table 1). In Sicily, HAstV circulation occurred throughout the year with wide seasonal fluctuations. In temperate areas, HAstV infections have been reported more frequently in the winter and rainy seasons [18, 23–25]. Sequence analysis of the 5’ portion of ORF 2 revealed the presence of five genotypes (HAstV-1 to -5) circulating during the study period. HAstV-1 represented the predominant genotype, accounting for 84.6 % of all cases positive for HAstV. The other four genotypes (HAstV-2, -3 -4 and -5) were detected at low frequency, indicating a minor epidemiological role (Fig. 1A). Novel AstVs (VA1, VA2, MLB1, MLB2, VA3, HMO-A-B-C) were not detected in this study, as we used primers that were highly specific for classical HAstVs. To overcome this great limitation, it would be useful in the future to also screen the study population for the presence of these unusual novel HAstVs. The predominance of HAstV-1 is in agreement
A
10 8
N° samples
1999 to December 2014. A total of 2966 stool samples were collected from children under 5 years of age hospitalised with acute gastroenteritis at the ‘‘G. Di Cristina’’ Children’s Hospital of Palermo. Viral RNA was extracted from 140 ll of stool suspension using a QIAmp Viral RNA Kit (QIAGEN, GmbH, Hilden, Germany). The samples were screened for the presence of HAstVs by RT-PCR with HAstV-specific primers Mon269 and Mon270, amplifying a 348-nucleotide (nt) portion of the 5’ end of ORF 2 [3]. Strains representative of the whole study period (65/78) were selected for sequence analysis. Sequence alignment was performed using CLUSTAL W [16]. Phylogenetic analysis was carried out using MEGA software version 6.0 [17], with the Kimura 2-parameter model as the method of substitution and the maximum-likelihood method to construct phylogenetic trees. The statistical significance of the phylogenies inferred was estimated by bootstrap analysis with 1,000 pseudoreplicate datasets. The nucleotide sequences of 10 Italian HAstV strains representative of each sublineage were deposited in GenBank under accession numbers KU508607 to KU508616. This study reports the epidemiology and genetic diversity of HAstV circulating in Sicily, southern Italy, during a 14-year surveillance period from August 1999 to December 2014. In the 2966 stool samples collected from children hospitalised with diarrhoea at the ‘‘G. Di Cristina‘‘ Children’s Hospital of Palermo, HAstV showed a low overall prevalence (2.63 %). The number of samples tested each year and the percentage of HAstV positivity are shown in Table 1. This prevalence is in concordance with many
6 4
5 (3.7)
2
0 (0)
0
2001
48
2002
106
5 (4.72)
2003
215
4 (1.86)
2004
199
5 (2.51)
2005
188
1 (0.53)
10 8
69
7 (10.14)
2007
171
6 (3.51)
2008
153
4 (2.61)
2009
309
5 (1.62)
2010 2011
438 360
9 (2.05) 9 (2.5)
2012
194
12 (6.19)
2013
187
2 (1.07)
2014
194
4 (2.06)
Total
2966
78 (2.63)
123
N° samples
2006
HAstV1
B
HAstV2
HAstV3
HAstV4
HAstV5
6 4 2 0
HAstV-1a
HAstV-1b
HAstV-1d
Fig. 1 A. Prevalence and temporal distribution of HAstV genotypes. B. HAstV-1 lineages circulating during the study period
14 years of astrovirus circulation in southern Italy Fig. 2 Phylogenetic analysis of nucleotide sequences of a portion (348 bp) of the HAstV ORF2 capsid region of 65 strains collected in Palermo from 1999 to 2014. The Kimura two-parameter model of substitution and the maximumlikelihood method were used to construct the phylogenetic tree. Bootstrap values above 70 % obtained from 1000 pseudoreplicate datasets are indicated at each node. *, Nucleotide sequences representative of each sublineage deposited in GenBank
MAstV-1/Hu/ITA/2010/PA180/10/type1a MAstV-1/Hu/ITA/2010/PA188/10/type1a MAstV-1/Hu/ITA/2010/PA173/10/type1a MAstV-1/Hu/ITA/2009/PA408/09/type1a MAstV-1/Hu/ITA/2009/PA407/09/type1a MAstV-1/Hu/THA/2011/CMHS60/type1a MAstV-1/Hu/ITA/2010/PA189/10/type1a MAstV-1/Hu/ITA/2010/PA219/10/type1a MAstV-1/Hu/ITA/2010/PA215/10/type1a MAstV-1/Hu/ITA/2010/PA204/10/type1a MAstV-1/Hu/ITA/2011/PA45/11/type1a* MAstV-1/Hu/ITA/2010/PA241/10/type1a MAstV-1/Hu/ITA/2009/PA418/09/type1a MAstV-1/Hu/ITA/2009/PA429/09/type1a MAstV-1/Hu/ITA/2009/PA430/09/type1a MAstV-1/Hu/ITA/2014/PA588/14/type1a* MAstV-1/Hu/ITA/2012/PA30/12/type1a* MAstV-1/Hu/ITA/2012/PA38/12/type1a MAstV-1/Hu/ITA/2012/PA44/12/type1a MAstV-1/Hu/ITA/2012/PA51/12/type1a MAstV-1/Hu/ITA/2011/PA399/11/type1a 71 MAstV-1/Hu/ITA/2010/PA281/10/type1a* MAstV-1/Hu/ITA/2011/PA165/11/type1a MAstV-1/Hu/ITA/2011/PA356/11/type1a MAstV-1/Hu/ITA/2011/PA387/11/type1a MAstV-1/Hu/ITA/2011/PA408/11/type1a MAstV-1/Hu/ITA/2012/PA25/12/type1a MAstV-1/Hu/ITA/2012/PA52/12/type1a MAstV-1/Hu/ITA/2014/PA96/14/Type1a MAstV-1/Hu/ITA/2014/PA118/14/type1a MAstV-1/Hu/ITA/2008/PA148/08/type1a* MAstV-1/Hu/VEN/1995/Ven835/type1c MAstV-1/Hu/ITA/2007/PA150/07/type1a* MAstV-1/Hu/ITA/2008/PA36/08/type1a MAstV-1/Hu/ITA/2007/PA157/07/type1a MAstV-1/Hu/ITA/2007/PA161/07/type1a MAstV-1/Hu/GBR/Oxford-1/type1a MAstV-1/Hu/ITA/2000/PA631/00/type1b MAstV-1/Hu/ITA/2000/PA657/00/type1b MAstV-1/Hu/ITA/2000/PA762/00/type1b MAstV-1/Hu/ESP/1999-007Bcn1.177type1b MAstV-1/Hu/ITA/2007/PA64/07/type1b* MAstV-1/Hu/ITA/2007/PA67/07/type1b* 97 MAstV-1/Hu/ITA/2007/PA76/07/type/1b 78 MAstV-1/Hu/ITA/2006/PA11/06/type1d* MAstV-1/Hu/ITA/2006/PA69/06/type1d MAstV-1/Hu/ITA/2006/PA53/06/type1d MAstV-1/Hu/ITA/2006/PA52/06/type1d MAstV-1/Hu/ITA/2008/PA47/08/type1d MAstV-1/Hu/ITA/2006/PA44/06/type1d* MAstV-1/Hu/ITA/2004/PA17R-/04/type1d 82 MAstV-1/Hu/ITA/2004/PA70R-/04/type1d MAstV-1/Hu/DEU/2004/Dresden-1/type1d MAstV-1/Hu/ITA/2005/PA124/05/type1d MAstV-1/Hu/ITA/1999/PA364/99/type1d MAstV-1/Hu/ITA/2003/PA60R-/03/type/1d MAstV-1/Hu/ITA/2006/PA60/06/type1d MAstV-1/Hu/ITA/2004/PA30R-/04/type1d* MAstV-1/Hu/ITA/2004/PA45R-/04/type1d MAstV-1/Hu/ITA/2006/PA10/06/type1d MAstV-1/Hu/ESP/1998-99/Bcn2.1/Type2a 93MAstV-1/Hu/ITA/2009/PR2346/type2d MAstV-1/Hu/BRA/2004/MG043/type2d 72 MAstV-1/Hu/GBR/L13745/type2b 93 MAstV-1/Hu/AUS/1996/Melb/96/Type2c MAstV-1/Hu/ITA/2008/PA100/08/type2c 97 MAstV-1/Hu/ITA/2002/PA54R-/02/type2c MAstV-1/Hu/ITA/2002/PA65/02/type2c MAstV-1/Hu/GBR/L38507/type6 MAstV-1/Hu/ITA/2002/PA120/02/type4b 91 MAstV-1/Hu/ITA/2002/PA73/02/type4b MAstV-1/Hu/ITA/2002/PA110/02/type4b MAstV-1/Hu/BRA/2004/RJ7987/Type4b MAstV-1/Hu/GBR/L38506/type4a MAstV-1/Hu/GBR/Z66541/type8 MAstV-1/Hu/GBR/L38508/type7 MAstV-1/Hu/GBR/U15136/type5 MAstV-1/Hu/ITA/2012/PA484/12/type5 89 MAstV-1/Hu/ITA/2013/PA292/13/type5 MAstV-1/Hu/ITA/2013/PA683/13/type5 MAstV-1/Hu/ITA/2000/PA737/00/type3 85 MAstV-1/Hu/GBR/L38505/type3 MAst-3/Po/AB0377272/W-1
HAstV-1
HAstV-2
HAstV-4
HAstV-5 HAstV-3
0.1
123
S. De Grazia et al.
with other studies demonstrating that this genotype is mostly prevalent worldwide [23, 25, 26]. Sequence and phylogenetic analysis of the HAstV strains detected in Sicily revealed high heterogeneity when lineages of the same genotype co-circulating in the study site were compared. Molecular analysis of the HAstV-1 strains showed significant sequence variation, with three different HAstV-1 lineages (1a, 1b and 1d) being observed over the study period. In particular, HAstV-1a, first detected in 2007, continued to circulate until 2012 and again in 2014. HAstV-1b was observed in 2000, then disappeared and re-emerged in 2007. HAstV-1d sporadically appeared in 1999/2000, 2003-2006 and in 2008 (Fig. 1B). No HAstV-1c strains were identified. Of the other HAstV genotypes detected in this survey, HAstV-2c was identified in 2002 and in 2008, HAstV-3 in 2000, HAstV-4b in 2002, and HAstV-5 in 2012 and 2013 (Fig. 2). The prolonged circulation of HAstV-1 could be explained by the emergence and re-emergence of different HAstV-1 lineages over time coupled with molecular recombination and/or rearrangement [27]. Prolonged circulation of HAstV-1 of different lineages was shown in other geographical areas. HAstV-1a was detected in Brazil and Australia during a period of more than 10 consecutive years, while HAstV-1d circulated largely in Spain, Brazil and Vietnam approximately in the same time periods observed in Palermo [4, 25, 28]. The fact that HAstV-1a and HAstV-1d persisted for many consecutive years (6 and 4 years, respectively) could be accounted for by a gradual slow increase in the immunity of the local paediatric population due to the low prevalence of the pathogen in the community. Many seasons of continuous circulation of the same antigenic type might be necessary to observe the effects of the acquisition of protective immunity. Neutralising antibodies against HAstV-1 have been reported in many seroprevalence studies, and it has been hypothesized that population immunity against HAstV-1 could exert continual pressure on the viruses, driving the emergence and re-emergence of strains belonging to different lineages over time [1]. A high level of genetic and antigenic variability is the likely reason for the continuous circulation of the prevalent G1P[8] rotavirus and GII.4 norovirus genotypes in the same study population. Different combinations of lineages/sublineages of the antigenic proteins VP7 and VP4 of rotavirus G1P[8] and continuous introduction of new variations into the polymerase and the capsid genes of norovirus GII.4 are believed to allow these viruses to escape immunity induced by previous waves of circulation of these major human gastroenteric pathogens [29, 30]. Sequence analysis of the relatively conserved 348-nt diagnostic region C, located on the ORF2 (capsid protein precursor gene) revealed very few amino acid changes among the different HAstV-1 lineages [4, 13, 26, 31]. The
123
diagnostic region C was found to be a good proxy for lineage characterization, although this region does not span the hypervariable region of the capsid protein that forms the capsid spike [27]. The identification of specific amino acid changes involved in the antigenic structure should be useful for better understanding of the mechanisms of HAstV evolution and could also be used for finer and more standardized strain characterization. Attempts have been made to define criteria for classification of HAstV lineages on the basis of the complete ORF2 sequence [27]. Analysis of the whole ORF2 might give a better overview of the genomic diversity among HAstVs-1 strains and provide clues for understanding the reasons for the continuous predominance of HAstV-1 in the population. A systematic investigation of the genetic variability of HAstVs will be important for understanding if periodic genotype shifts and genetic/antigenic drifts occur and if these changes fit a model of evolution analogous to those observed for other antigenically/genetically heterogeneous viruses. ´ kos Gelle´rt was the recipient of a Ja´nos Bolyai Acknowledgments A fellowship from the Hungarian Academy of Sciences. Krisztia´n Ba´nyai was supported by the Momentum program awarded by the Hungarian Academy of Sciences (OTKA, T100727). Compliance with ethical standards Conflict of interest of interest.
The authors declare that they have no conflict
Ethical approval Although the collection of faecal samples was part of the general process of diagnosis of acute gastroenteritis affecting minors/children, we obtained verbal informed consent from the family or caretakers.
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14 years of astrovirus circulation in southern Italy 6. Banyai K, Meleg E, Moschidou P, Martella V (2010) Detection of newly described astrovirus MLB1 in stool samples from children. Emerg Infect Dis 16:169–170 7. Finkbeiner SR, Le BM, Holtz LR, Storch GA, Wang D (2009) Detection of newly described astrovirus MLB1 in stool samples from children. Emerg Infect Dis 15:441–444 8. Finkbeiner SR, Li Y, Ruone S, Conrardy C, Gregoricus N, Toney D, Virgin HW, Anderson LJ, Vinje J et al (2009) Identification of a novel astrovirus (astrovirus VA1) associated with an outbreak of acute gastroenteritis. J Virol 83:10836–10839 9. Kapoor A, Li L, Victoria J, Oderinde B, Mason C, Pandey P, Zaidi SZ, Delwart E (2009) Multiple novel astrovirus species in human stool. J Gen Virol 90:2965–2972 10. Belliot G, Laveran H, Monroe SS (1997) Detection and genetic differentiation of human astroviruses: phylogenetic grouping varies by coding region. Arch Virol 142:1323–1334 11. De Grazia S, Martella V, Chironna M, Bonura F, Tummolo F, Calderaro A, Moschidou P, Giammanco GM, Medici MC (2012) Nationwide surveillance study of human astrovirus infections in an Italian paediatric population. Epidemiol Infect 141:524–528 12. De Grazia S, Platia MA, Rotolo V, Colomba C, Martella V, Giammanco GM (2012) Surveillance of human astrovirus circulation in Italy 2002–2005: emergence of lineage 2c strains. Clin Microbiol Infect 17:97–101 13. De Grazia S, Giammanco GM, Colomba C, Cascio A, Arista S (2004) Molecular epidemiology of astrovirus infection in Italian children with gastroenteritis. Clin Microbiol Infect 10:1025–1029 14. De Grazia S, Ramirez S, Giammanco GM, Colomba C, Martella V, Lo Biundo C, Mazzola R, Arista S (2007) Diversity of human rotaviruses detected in Sicily, Italy, over a 5-year period (2001–2005). Arch Virol 152:833–837 15. Colomba C, De Grazia S, Giammanco GM, Saporito L, Scarlata F, Titone L, Arista S (2006) Viral gastroenteritis in children hospitalised in Sicily, Italy. Eur J Clin Microbiol Infect Dis 25:570–575 16. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 17. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729 18. Afrad MH, Karmakar PC, Das SK, Matthijnssens J, Ahmed F, Nahar S, Faruque AS, Rahman MZ, Rahman M, Azim T (2013) Epidemiology and genetic diversity of human astrovirus infection among hospitalized patients with acute diarrhea in Bangladesh from 2010 to 2012. J Clin Virol 58:612–618 19. Guix S, Bosch A, Pinto RM (2005) Human astrovirus diagnosis and typing: current and future prospects. Lett Appl Microbiol 41:103–105 20. Jakab F, Meleg E, Banyai K, Melegh B, Timar L, Peterfai J, Szucs G (2004) One-year survey of astrovirus infection in
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BRIEF REPORT
Temporal variation in the distribution of type-1 human astrovirus lineages in a settled population over 14 years ´ kos Gelle´rt3 Simona De Grazia1 • Floriana Bonura1 • Krisztia´n Ba´nyai2 • A 1 • 4 • Sandra Marineo Vito Martella Giovanni M. Giammanco1
•
Received: 7 December 2015 / Accepted: 14 February 2016 Ó Springer-Verlag Wien 2016
Abstract Human astroviruses (HAstVs) are important enteric pathogens that are genetically and antigenically heterogeneous and can be classified into eight sero/genotypes (HAstV-1 to -8) and different lineages within each HAstV type. This study describes the genetic diversity of HAstVs circulating in southern Italy over 14 years. Molecular analysis of HAstV-1 strains showed that three different lineages (1a, 1b and 1d) of the predominant genotype were circulating during the study period. The study of an archival collection of HAstV strains offers a unique opportunity to evaluate the patterns of variation of HAstV infections over the years and to correlate the observed epidemiological changes to the genetic variability of HAstVs.
Astroviruses (AstVs) are important enteric pathogens of mammals and birds [1]. They belong to the family Astroviridae and have a single-stranded positive-sense RNA genome containing three open reading frames (ORFs): ORF1a, ORF1b and ORF2. ORF1a and ORF1b, at the 5’ end of the genome, encode the non-structural viral proteins,
& Simona De Grazia [email protected] 1
Department of Health Promotion Sciences and Mother and Child Care ‘‘G. D’Alessandro’’, University of Palermo, via del Vespro 133, 90127 Palermo, Italy
2
Veterinary Medical Research Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary
3
Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary
4
Department of Veterinary Medicine, University Aldo Moro of Bari, Valenzano, Italy
including the RNA-dependent RNA polymerase, while ORF2, at the 3’ end, encodes the capsid protein precursor. Human AstVs (HAstVs) are genetically and antigenically heterogeneous [2]. HAstVs have been classified into eight types (HAstV -1 to -8) by sequence analysis of short fragments localized at the 5’ end or the 3’ end of ORF2. Importantly, a correlation has been found between genetic and antigenic characteristics [3]. Genotyping surveys have shown that HAstV-1 is the most common type identified in children, followed by HAstV-2, -3, -4 and -5, whereas HAstV-6, -7, and -8 have been detected more rarely [4, 5]. The incidence and distribution of HAstV sero/genotypes varies seasonally and geographically. Recent studies have identified novel HAstVs that are more closely related to animal astroviruses than to classical HAstVs [6–9]. The greatest sequence variability in the AstV genome is found in ORF2, characterised by a highly conserved N-terminal domain (aa 1-424), a hypervariable domain (HVR) (aa 425-688) and a highly acidic C-terminal domain [1, 5]. Interestingly, upon molecular analysis of ORF2, discrete sequence variation has been observed with different lineages within each HAstV type. Both HAstV-1 (HAstV-1a-1d) and HAstV-2 (HAstV-2a-2d) have been divided into four lineages, whereas HAstV-3 (HAstV-3a3b) and HAstV-4 (HAstV-4a-4b-4c) have been classified into two and three lineages, respectively [4, 10–12]. In Palermo, Sicily, in southern Italy, a documented uninterrupted HAstV surveillance has been conducted since 1999 [11–15]. The resulting archival collection, spanning nearly 15 years, offers an opportunity to evaluate the patterns of variation of HAstV infections over the years and to correlate the observed epidemiological changes to the genetic variability of HAstVs. This study describes the genetic diversity of HAstVs circulating in a settled population of Palermo from August
123
S. De Grazia et al.
Table 1 Yearly prevalence of astrovirus infection in relation to the number of strains sequenced Year 2000
Number of sample tested 135
Number (%) of HAstVpositive samples
other studies conducted in different parts of the world, such as Spain, Hungary, Brazil, China, India and Bangladesh [4, 18–21, 22]. In this study, the yearly detection rates of HAstVs rates ranged from 0.53 % in 2005 to 10.14 % in 2006. No HAstV was detected in 2001. Mixed infections with rotavirus and norovirus were observed in[50 % of all HAstV-positive cases (39/78) and comparable rates of mixed infections were reported in other epidemiological studies [4, 18–20, 23]. A genotype was determined for 65 (83.3 %) out of 78 HAstV strains detected during the study (Table 1). In Sicily, HAstV circulation occurred throughout the year with wide seasonal fluctuations. In temperate areas, HAstV infections have been reported more frequently in the winter and rainy seasons [18, 23–25]. Sequence analysis of the 5’ portion of ORF 2 revealed the presence of five genotypes (HAstV-1 to -5) circulating during the study period. HAstV-1 represented the predominant genotype, accounting for 84.6 % of all cases positive for HAstV. The other four genotypes (HAstV-2, -3 -4 and -5) were detected at low frequency, indicating a minor epidemiological role (Fig. 1A). Novel AstVs (VA1, VA2, MLB1, MLB2, VA3, HMO-A-B-C) were not detected in this study, as we used primers that were highly specific for classical HAstVs. To overcome this great limitation, it would be useful in the future to also screen the study population for the presence of these unusual novel HAstVs. The predominance of HAstV-1 is in agreement
A
10 8
N° samples
1999 to December 2014. A total of 2966 stool samples were collected from children under 5 years of age hospitalised with acute gastroenteritis at the ‘‘G. Di Cristina’’ Children’s Hospital of Palermo. Viral RNA was extracted from 140 ll of stool suspension using a QIAmp Viral RNA Kit (QIAGEN, GmbH, Hilden, Germany). The samples were screened for the presence of HAstVs by RT-PCR with HAstV-specific primers Mon269 and Mon270, amplifying a 348-nucleotide (nt) portion of the 5’ end of ORF 2 [3]. Strains representative of the whole study period (65/78) were selected for sequence analysis. Sequence alignment was performed using CLUSTAL W [16]. Phylogenetic analysis was carried out using MEGA software version 6.0 [17], with the Kimura 2-parameter model as the method of substitution and the maximum-likelihood method to construct phylogenetic trees. The statistical significance of the phylogenies inferred was estimated by bootstrap analysis with 1,000 pseudoreplicate datasets. The nucleotide sequences of 10 Italian HAstV strains representative of each sublineage were deposited in GenBank under accession numbers KU508607 to KU508616. This study reports the epidemiology and genetic diversity of HAstV circulating in Sicily, southern Italy, during a 14-year surveillance period from August 1999 to December 2014. In the 2966 stool samples collected from children hospitalised with diarrhoea at the ‘‘G. Di Cristina‘‘ Children’s Hospital of Palermo, HAstV showed a low overall prevalence (2.63 %). The number of samples tested each year and the percentage of HAstV positivity are shown in Table 1. This prevalence is in concordance with many
6 4
5 (3.7)
2
0 (0)
0
2001
48
2002
106
5 (4.72)
2003
215
4 (1.86)
2004
199
5 (2.51)
2005
188
1 (0.53)
10 8
69
7 (10.14)
2007
171
6 (3.51)
2008
153
4 (2.61)
2009
309
5 (1.62)
2010 2011
438 360
9 (2.05) 9 (2.5)
2012
194
12 (6.19)
2013
187
2 (1.07)
2014
194
4 (2.06)
Total
2966
78 (2.63)
123
N° samples
2006
HAstV1
B
HAstV2
HAstV3
HAstV4
HAstV5
6 4 2 0
HAstV-1a
HAstV-1b
HAstV-1d
Fig. 1 A. Prevalence and temporal distribution of HAstV genotypes. B. HAstV-1 lineages circulating during the study period
14 years of astrovirus circulation in southern Italy Fig. 2 Phylogenetic analysis of nucleotide sequences of a portion (348 bp) of the HAstV ORF2 capsid region of 65 strains collected in Palermo from 1999 to 2014. The Kimura two-parameter model of substitution and the maximumlikelihood method were used to construct the phylogenetic tree. Bootstrap values above 70 % obtained from 1000 pseudoreplicate datasets are indicated at each node. *, Nucleotide sequences representative of each sublineage deposited in GenBank
MAstV-1/Hu/ITA/2010/PA180/10/type1a MAstV-1/Hu/ITA/2010/PA188/10/type1a MAstV-1/Hu/ITA/2010/PA173/10/type1a MAstV-1/Hu/ITA/2009/PA408/09/type1a MAstV-1/Hu/ITA/2009/PA407/09/type1a MAstV-1/Hu/THA/2011/CMHS60/type1a MAstV-1/Hu/ITA/2010/PA189/10/type1a MAstV-1/Hu/ITA/2010/PA219/10/type1a MAstV-1/Hu/ITA/2010/PA215/10/type1a MAstV-1/Hu/ITA/2010/PA204/10/type1a MAstV-1/Hu/ITA/2011/PA45/11/type1a* MAstV-1/Hu/ITA/2010/PA241/10/type1a MAstV-1/Hu/ITA/2009/PA418/09/type1a MAstV-1/Hu/ITA/2009/PA429/09/type1a MAstV-1/Hu/ITA/2009/PA430/09/type1a MAstV-1/Hu/ITA/2014/PA588/14/type1a* MAstV-1/Hu/ITA/2012/PA30/12/type1a* MAstV-1/Hu/ITA/2012/PA38/12/type1a MAstV-1/Hu/ITA/2012/PA44/12/type1a MAstV-1/Hu/ITA/2012/PA51/12/type1a MAstV-1/Hu/ITA/2011/PA399/11/type1a 71 MAstV-1/Hu/ITA/2010/PA281/10/type1a* MAstV-1/Hu/ITA/2011/PA165/11/type1a MAstV-1/Hu/ITA/2011/PA356/11/type1a MAstV-1/Hu/ITA/2011/PA387/11/type1a MAstV-1/Hu/ITA/2011/PA408/11/type1a MAstV-1/Hu/ITA/2012/PA25/12/type1a MAstV-1/Hu/ITA/2012/PA52/12/type1a MAstV-1/Hu/ITA/2014/PA96/14/Type1a MAstV-1/Hu/ITA/2014/PA118/14/type1a MAstV-1/Hu/ITA/2008/PA148/08/type1a* MAstV-1/Hu/VEN/1995/Ven835/type1c MAstV-1/Hu/ITA/2007/PA150/07/type1a* MAstV-1/Hu/ITA/2008/PA36/08/type1a MAstV-1/Hu/ITA/2007/PA157/07/type1a MAstV-1/Hu/ITA/2007/PA161/07/type1a MAstV-1/Hu/GBR/Oxford-1/type1a MAstV-1/Hu/ITA/2000/PA631/00/type1b MAstV-1/Hu/ITA/2000/PA657/00/type1b MAstV-1/Hu/ITA/2000/PA762/00/type1b MAstV-1/Hu/ESP/1999-007Bcn1.177type1b MAstV-1/Hu/ITA/2007/PA64/07/type1b* MAstV-1/Hu/ITA/2007/PA67/07/type1b* 97 MAstV-1/Hu/ITA/2007/PA76/07/type/1b 78 MAstV-1/Hu/ITA/2006/PA11/06/type1d* MAstV-1/Hu/ITA/2006/PA69/06/type1d MAstV-1/Hu/ITA/2006/PA53/06/type1d MAstV-1/Hu/ITA/2006/PA52/06/type1d MAstV-1/Hu/ITA/2008/PA47/08/type1d MAstV-1/Hu/ITA/2006/PA44/06/type1d* MAstV-1/Hu/ITA/2004/PA17R-/04/type1d 82 MAstV-1/Hu/ITA/2004/PA70R-/04/type1d MAstV-1/Hu/DEU/2004/Dresden-1/type1d MAstV-1/Hu/ITA/2005/PA124/05/type1d MAstV-1/Hu/ITA/1999/PA364/99/type1d MAstV-1/Hu/ITA/2003/PA60R-/03/type/1d MAstV-1/Hu/ITA/2006/PA60/06/type1d MAstV-1/Hu/ITA/2004/PA30R-/04/type1d* MAstV-1/Hu/ITA/2004/PA45R-/04/type1d MAstV-1/Hu/ITA/2006/PA10/06/type1d MAstV-1/Hu/ESP/1998-99/Bcn2.1/Type2a 93MAstV-1/Hu/ITA/2009/PR2346/type2d MAstV-1/Hu/BRA/2004/MG043/type2d 72 MAstV-1/Hu/GBR/L13745/type2b 93 MAstV-1/Hu/AUS/1996/Melb/96/Type2c MAstV-1/Hu/ITA/2008/PA100/08/type2c 97 MAstV-1/Hu/ITA/2002/PA54R-/02/type2c MAstV-1/Hu/ITA/2002/PA65/02/type2c MAstV-1/Hu/GBR/L38507/type6 MAstV-1/Hu/ITA/2002/PA120/02/type4b 91 MAstV-1/Hu/ITA/2002/PA73/02/type4b MAstV-1/Hu/ITA/2002/PA110/02/type4b MAstV-1/Hu/BRA/2004/RJ7987/Type4b MAstV-1/Hu/GBR/L38506/type4a MAstV-1/Hu/GBR/Z66541/type8 MAstV-1/Hu/GBR/L38508/type7 MAstV-1/Hu/GBR/U15136/type5 MAstV-1/Hu/ITA/2012/PA484/12/type5 89 MAstV-1/Hu/ITA/2013/PA292/13/type5 MAstV-1/Hu/ITA/2013/PA683/13/type5 MAstV-1/Hu/ITA/2000/PA737/00/type3 85 MAstV-1/Hu/GBR/L38505/type3 MAst-3/Po/AB0377272/W-1
HAstV-1
HAstV-2
HAstV-4
HAstV-5 HAstV-3
0.1
123
S. De Grazia et al.
with other studies demonstrating that this genotype is mostly prevalent worldwide [23, 25, 26]. Sequence and phylogenetic analysis of the HAstV strains detected in Sicily revealed high heterogeneity when lineages of the same genotype co-circulating in the study site were compared. Molecular analysis of the HAstV-1 strains showed significant sequence variation, with three different HAstV-1 lineages (1a, 1b and 1d) being observed over the study period. In particular, HAstV-1a, first detected in 2007, continued to circulate until 2012 and again in 2014. HAstV-1b was observed in 2000, then disappeared and re-emerged in 2007. HAstV-1d sporadically appeared in 1999/2000, 2003-2006 and in 2008 (Fig. 1B). No HAstV-1c strains were identified. Of the other HAstV genotypes detected in this survey, HAstV-2c was identified in 2002 and in 2008, HAstV-3 in 2000, HAstV-4b in 2002, and HAstV-5 in 2012 and 2013 (Fig. 2). The prolonged circulation of HAstV-1 could be explained by the emergence and re-emergence of different HAstV-1 lineages over time coupled with molecular recombination and/or rearrangement [27]. Prolonged circulation of HAstV-1 of different lineages was shown in other geographical areas. HAstV-1a was detected in Brazil and Australia during a period of more than 10 consecutive years, while HAstV-1d circulated largely in Spain, Brazil and Vietnam approximately in the same time periods observed in Palermo [4, 25, 28]. The fact that HAstV-1a and HAstV-1d persisted for many consecutive years (6 and 4 years, respectively) could be accounted for by a gradual slow increase in the immunity of the local paediatric population due to the low prevalence of the pathogen in the community. Many seasons of continuous circulation of the same antigenic type might be necessary to observe the effects of the acquisition of protective immunity. Neutralising antibodies against HAstV-1 have been reported in many seroprevalence studies, and it has been hypothesized that population immunity against HAstV-1 could exert continual pressure on the viruses, driving the emergence and re-emergence of strains belonging to different lineages over time [1]. A high level of genetic and antigenic variability is the likely reason for the continuous circulation of the prevalent G1P[8] rotavirus and GII.4 norovirus genotypes in the same study population. Different combinations of lineages/sublineages of the antigenic proteins VP7 and VP4 of rotavirus G1P[8] and continuous introduction of new variations into the polymerase and the capsid genes of norovirus GII.4 are believed to allow these viruses to escape immunity induced by previous waves of circulation of these major human gastroenteric pathogens [29, 30]. Sequence analysis of the relatively conserved 348-nt diagnostic region C, located on the ORF2 (capsid protein precursor gene) revealed very few amino acid changes among the different HAstV-1 lineages [4, 13, 26, 31]. The
123
diagnostic region C was found to be a good proxy for lineage characterization, although this region does not span the hypervariable region of the capsid protein that forms the capsid spike [27]. The identification of specific amino acid changes involved in the antigenic structure should be useful for better understanding of the mechanisms of HAstV evolution and could also be used for finer and more standardized strain characterization. Attempts have been made to define criteria for classification of HAstV lineages on the basis of the complete ORF2 sequence [27]. Analysis of the whole ORF2 might give a better overview of the genomic diversity among HAstVs-1 strains and provide clues for understanding the reasons for the continuous predominance of HAstV-1 in the population. A systematic investigation of the genetic variability of HAstVs will be important for understanding if periodic genotype shifts and genetic/antigenic drifts occur and if these changes fit a model of evolution analogous to those observed for other antigenically/genetically heterogeneous viruses. ´ kos Gelle´rt was the recipient of a Ja´nos Bolyai Acknowledgments A fellowship from the Hungarian Academy of Sciences. Krisztia´n Ba´nyai was supported by the Momentum program awarded by the Hungarian Academy of Sciences (OTKA, T100727). Compliance with ethical standards Conflict of interest of interest.
The authors declare that they have no conflict
Ethical approval Although the collection of faecal samples was part of the general process of diagnosis of acute gastroenteritis affecting minors/children, we obtained verbal informed consent from the family or caretakers.
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