Circulation of human astroviruses genotypes in Central West of Brazil
P.A. Silva1, R.A.T. Santos1, P.S.S. Costa1, J.M.S. Teixeira2, L.G. Giugliano3, M.S.A. Andreasi4, J.P.G. Leite5, E. Schreier6, D.D.P. Cardoso1
1Laboratório de Virologia, Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, Goiânia, Go, Brasil
2Instituto de Saúde do Distrito Federal, Brasília, DF, Brasil
3Laboratório de Microbiologia, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brasil
4Centro de Ciências Biológicas e da Saúde, Universidade Federal do Mato Grosso do Sul, Campo Grande, MS, Brasil
5Laboratório de Virologia Comparada, Departamento de Virologia, Instituto Oswaldo Cruz-FIOCRUZ, Rio de Janeiro, RJ, Brasil
6Robert Koch-Institute, Berlin, Germany
Research supported by CNPq nº 471968/03-0 and 520729/99-4 Correspondence: D.D.P. Cardoso
IPTSP/UFG – Rua 235, esquina com 1ª Avenida, Setor Universitário 74605-050 Goiânia, Goiás, Brasil
Fax: +55-62-3521-1839
E-mail: dcardoso@iptsp.ufg.br
Abstract
Human astroviruses (HAstV) are recognized as a common cause of diarrhea in young children worldwide. Fifty seven astrovirus samples from children less than five years old with (n=56) and without diarrhea (n=1) from three locations of the Central West region of Brazil (Brasília, Goiânia and Campo Grande) were detected by RT-PCR. These samples were genotyped by nested RT-PCR and/or genomic sequencing of the ORF 2 5’end region. It was detected HAstV-1 (42.8%), HAstV-2 (23.2%), HAstV-3 (3.6%), HAstV-4 (14.3%) and HAstV-5, -6, -7 and -8 (1.8% each), in diarrheic samples. Five samples (8.9%) were untypeable. The positive non-diarrheic sample was classified as HAstV-2. In Goiânia and Campo Grande, HAstV-1 was the most frequent genotype detected, while in Brasília it was HAstV-2. Additionally, data from Brasília and Campo Grande showed the occurrence of shift in astroviruses genotypes circulation from HAstV-4 to HAstV-2 and from HAstV-2 to HAstV-1, respectively. The astroviruses genotypes were detected in all age groups and there was no correlation between genotype and age group. The results of this study reinforced data about the role of astroviruses in diarrhea and provided information about viral genotypes circulation in Central West region of Brazil.
Introduction
Human astroviruses (HAstV) were first described in 1975 in fecal samples from children with diarrhea, by electron microscopy [1]. Since then, the medical importance of these viruses has been established and nowadays, in some studies, HAstV has been shown to be the second most common cause of viral diarrhea in young children [2-5]. Epidemiological data have demonstrated astrovirus prevalence rates between 2 and 26% in children with diarrhea [6, 7].
The astroviruses, family Astroviridae, genera Mamastrovirus, are non-enveloped spherical particles with 29-30 nm of diameter [1]. Their genome is a single-stranded polyadenylated positive-sense RNA of approximately 6.8 kb long [8, 9], which presents three open reading frames (ORFs), 1a, 1b and 2 [10]. The ORF 1a and 1b encode non-structural proteins including a serine protease and a RNA-dependent RNA polymerase, respectively, while the ORF 2 encodes the viral capsid protein precursor [10, 11].
HAstV have been classified in eight serotypes/genotypes, according to their antigenic and genomic characteristics [12, 13]. Previous studies have shown that HAstV-1 is predominant [12, 14, 15], however simultaneous circulation of different astrovirus types in the same geographical area have been observed [15, 16].
In Brazil, there are few reports about human astroviruses genotypes circulation [17, 18]. This information is important concerning the future development of preventive therapies. In this study, we describe the occurrence of genotypes of astroviruses isolated from children with and without acute gastroenteritis in Central West region of Brazil, including previous data [17, 19].
Material and Methods
Fecal Samples
A total of 1,588 fecal samples (1,374 diarrheic and 214 non-diarrheic) were collected from children under five years old from three location of Central West region of Brazil. This region comprises by four states and in this study children from three of them were involved [Brasília-DF (n=501), Goiânia-Go (n=743) and Campo Grande-MS (n=344)]. All samples were screened for astroviruses by RT-PCR and 56 (4.1%) from diarrheic and 1 (0.4%) from non-diarrheic samples were positive for this virus. Samples from Brasília were collected in two periods, 1994-1996 and 2000-2002. In Goiânia, samples were collected between 1998 and 2002, while in Campo Grande it were between 2000 and 2003. The samples from Brasília, collected between 2000 and 2002, were the majority (297/343) obtained by rectal swabs. Fecal suspensions (20%) were prepared in PBS, pH 7.2 and stored at -20ºC until analysis.
The fecal samples were collected after written agreement of children’s parents or guardians. This study was approved by the Ethics Committee on Human and Animal Research of the Federal University of Goiás (004/2000).
Detection of astroviruses
Astroviruses were detected by RT-PCR assay using specific primers Mon269 (4526-4545 nt) and Mon270 (4955-4974 nt) directed towards the ORF 2 5’ end region, as described previously [20]. The viral RNA was extracted from 200 µL of the 20 % fecal suspension by glass powder method, using guanidinium thiocyanate as described by Boom et al [21]. The RT-PCR and first amplification conditions were the same as described previously for astrovirus [17].
Genotyping of astroviruses
Nested RT-PCR - The astroviruses genotyping was carried out in 44 positive samples by
nested RT-PCR using primers that amplified the ORF 2 3’ end region, as described by Sakamoto et al [12]. The RNA extraction, RT-PCR, first and second amplification were the same as described previously [17].
Nucleotide sequencing and astroviruses genotyping - For determination of astroviruses
genotypes by genomic sequencing, phylogenetic analysis over the 348 bp of ORF 2 5’ end region from 27 positive samples were performed. 14 samples were genotyped by both methodologies. Total viral RNA was extracted from 140 µL of a 20% fecal suspension using the QIAamp viral RNA Kit (Qiagen, Hilden, Germany). The products of nested RT-PCR, which were obtained using the primer pair Mon269/Mon270 [20] in the first amplification and AV11as/AV12s1 in the second amplification [19], were sequenced directly in both directions using the same pair of primer as in the nested-PCR, utilizing ABI Prism 3100 Genetic Analyzer and Big Dye Terminator Cycle Sequencing Mix (Applied Biosystems). The RT-PCR and amplifications assays conditions were the same as described previously for astrovirus [22]. Sequence alignments and phylogenetic analysis were carried out using the CLUSTAL W program and the Phylogeny Inference Package (PHYLIP), version 3.57c [23, 24].
Nucleotide sequences of astrovirus detected in this study have been submitted to GenBank under accession number: DQ139825 to DQ139832, DQ070852 and DQ028633.
Results
The genotyping of each astrovirus sample, carried out by nested RT-PCR and/or genomic sequencing, showed that all known viral genotypes (HAstV-1 to HAstV-8) were detected in Central West region of Brazil. Samples genotyped by both methodologies (n=14) presented coincident results. Five samples (two from Brasília, two from Goiânia and one from Campo Grande) submitted only to nested RT-PCR genotyping were untypeable.
HAstV-1, -2, -3 and -4 were detected in 42.8%, 23.2%, 3.6% and 14.3%, respectively, while the HAstV-5, -6, -7 and -8 were detected each one in 1.8% of diarrheic samples. Considering the origin location of the samples, it was observed that in Brasília HAstV-2 was the most frequent genotype (37.5%), followed by HAstV-4 (25%), HAstV-1 (12.5%), HAstV-3 (8.3%), HAstV-5 and HAstV-6 (4.2% each). In Goiânia, HAstV-1 (66.6%) was the predominant genotype, followed by HAstV-4 (9.5%), HAstV-2, -7 and -8 (4.8% each). In Campo Grande, only two genotypes were detected, HAstV-1 (63.6%) and HAstV-2 (27.3%) (Table 1). The only one non-diarrheic positive sample was collected in Brasília in 2001 and it was characterized as HAstV-2.
Although it was not observed statistical significance between predominant genotype and years of the collect, in Brasília, 4 was the most frequent in 1994 and 1995, while HAstV-2 was detected in 71.4% of the samples in 1996. Between HAstV-2000 and HAstV-200HAstV-2 astroviruses were not detected in diarrheic samples from Brasília. In Goiânia, HAstV-1 was the predominant genotype in all years of the study, but in 1999, only two positive samples were detected and one was classified as HAstV-1 and the other HAstV-8. In Campo Grande, HAstV-2 predominated in 2000 and 2002, while HAstV-1 was the only one genotype detected in 2003 (Figure 1).
The different astroviruses genotypes were detected in children from all age groups up to five years old and it was not observed correlation between genotype and age group (p> 0.1). Discussion
Besides the knowledge about the genetic variability of the virus circulating in different regions of the world, the large use of molecular techniques in epidemiological studies has reinforced the importance of human astroviruses as cause of acute gastroenteritis [5, 9, 15]. The present study reports the occurrence and distribution of astrovirus genotypes in Central West region of Brazil.
Our data showed that HAstV-1 was the most frequent genotype in the studied region. This finding corroborates data of other authors from several regions of the world, including Brazil [9, 17, 25, 26]. However, other genotypes considered less common (HAstV-2 to HAstV-5) or rare (HAstV-6 to HAstV-8) were also detected. The circulation of different genotypes in the same region is not uncommon and has also been reported in countries such as Germany, Spain, Argentina, Mexico, and Egypt [9, 15, 16, 27, 28]. In Brasília and Goiânia the occurrence of at least five astrovirus genotypes was observed, while in Campo Grande only two genotypes were detected. This data could be explained by the fact that Brasília is a national and international tourist destination beyond the high emigration rate that makes possible the occasional introduction of different astroviruses genotypes. Similarly, Goiânia also received a significant high number of tourists and emigrants that could introduce different viral genotypes in the native population.
Considering the origin of the samples, in Goiânia and Campo Grande the most frequent genotype was HAstV-1, while in Brasília it was HAstV-2. This variation in genotype predominance from one location to another could suggest the occurrence of a regional predominance of astroviruses genotypes. This fact was also observed in others studies [15, 29].
Analysis of data obtained from positive samples from Brasília and Campo Grande in different years, showed a shift of astroviruses genotypes circulation from HAstV-4 to HAstV-2 and from HAstV-2 to HAstV-1, respectively. This event has been observed in some studies with
other viruses, including gastrointestinal viruses, and some authors suggested that this viral genotypes shift could reflect a natural selection due to a lack of heterotypic immunity among the different viral antigenic types [12, 27, 30-32].
From 44 positive samples that were submitted to nested RT-PCR genotyping, five were untypeable. This fact has been reported in other study, which suggested that the failure of nested RT-PCR typing is not related to sequence variation but could be due to mismatches, inhibitors, or low RNA yield [33].
Enteric viruses usually have been detected directly from fecal samples in natura but other collection methods, as rectal swabs also have been used [34]. In this study, we consider that the absence of positive astrovirus samples from Brasília collected between 2000 and 2002 is due to the method of fecal collection utilized because the majority of samples collected in this period were obtained from rectal swabs that could impair the viral detection, fact also observed for caliciviruses [Cardoso DDP et al., unpublished data].
In this study there was no correlation between age groups and astrovirus genotypes, although previous data in Spain had indicated a predominance of HAstV-1 and -3 in children up to 2 years old, and HAStV-4 and -8 in children older than 3 years [27].
In conclusion, the results of this study reinforce data about the role of astroviruses as an important agent of diarrhea in children and provide the first data about viral genotypes circulation in three location of Central West region of Brazil. This information is relevant for future implementation of preventive strategies for the HAstV infection.
P.A. Silva was supported by a grant from DAAD (German Academic Exchange Service), CNPq (Brazilian National Council for Scientific and Technological Development) and CAPES (Coordination of Improvement of High Degree People).
We would like to thank Dr.Wilia Brito for the critical reading of the manuscript and colleagues from Laboratory of Virology in Goiânia-Brazil and from FG15 in Robert Koch Institute-Berlin for the constant aid and support.
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Table 1. Distribution of astroviruses genotypes detected in diarrheic samples of children from three locations of Central West region of Brazil.
Genotypes Brasília Nº (%) Goiânia Nº (%) Campo Grande Nº (%) Total Nº (%) HAstV-1 3 (12.5) 14 (66.6) 7 (63.6) 24 (42.8) HAstV-2 9 (37.5) 1 (4.8) 3 (27.3) 13 (23.2) HAstV-3 2 (8.3) - - 2 (3.6) HAstV-4 6 (25) 2 (9.5) - 8 (14.3) HAstV-5 1 (4.2) - - 1 (1.8) HAstV-6 1 (4.2) - - 1 (1.8) HAstV-7 - 1 (4.8) - 1 (1.8) HAstV-8 - 1 (4.8) - 1 (1.8) Untypeable 2 (8.3) 2 (9.5) 1 (9.1) 5 (8.9) Total 24 (100) 21 (100) 11 (100) 56 (100)
Figure 1. Distribution of HAstV genotypes detected in Central West region of Brazil, according to the location and year of collection. (A) Brasília, (B) Goiânia and (C) Campo Grande.
(A) Brasília H A st V -1 H A st V -1 H A st V -2 H A st V -2 H A st V -2 H A st V -4 H A st V -4 H A st V -5 H A st V -6 U nt yp ea bl e U nt yp ea bl e H A st V -3 0 20 40 60 80 100 1994 1995 1996 Years of collection % (B) Goiânia H A st V -1 H A st V -1 H A st V -2 H A st V -4 H A st V -4 H A st V -8 U nt yp ea bl e H A st V -1 H A st V -1 H A st V -7 0 10 20 30 40 50 60 70 80 90 100 1998 1999 2000 2001 Years of collection %
(C) Campo Grande H A st V -1 H A st V -2 H A st V -2 H A st V -2 U nt yp aa bl e 0 10 20 30 40 50 60 70 80 90 100 2000 2001 2002 2003 Years of collection %