Received: 22 September 2016 | Accepted: 26 April 2017

DOI: 10.1002/jmv.24854

RESEARCH ARTICLE

Epidemiology and genetic diversity of classic human

astrovirus among hospitalized children with acute
gastroenteritis in Uruguay

Fernando Lopez1 | Andrés Lizasoain1 | Matías Victoria2 | Cecilia Papalardo2 |

Sebastian Castro2 | Edit Arreseigor3 | Patricia López4 | Rodney Colina1

1 Molecular Virology Laboratory, CENUR

Litoral Norte, Universidad de la República, Classic Human Astrovirus (Classic HAstV) are one of the most important causes of
Salto, Uruguay
pediatric acute gastroenteritis (AGE), after rotaviruses and arguably caliciviruses. The
2 Department of Mathematics and Statistics
CENUR Litoral Norte, Universidad de la
aim of this study was to determine the molecular epidemiology of Classic HAstV from
República, Salto, Uruguay 175 clinical samples, being 153 stools and 22 vomits, collected from pediatric
3 Pediatric Unit, Medical-Surgical Society of hospitalized patients with AGE in Salto city, Uruguay, from January 2011 to
Salto, Salto, Uruguay
4 Pediatric Unit, Saltó s Public Hospital, Salto,
December 2012. Classic HAstV were detected and genotyped by using a qualitative
Uruguay Retro Transcription—Polimerase Chain Reaction (RT-PCR) directed to the Open

Correspondence
Dr. Rodney Colina, Laboratorio de Virología
Molecular, CENUR Litoral Norte, Sede Salto,
Universidad de la República, Salto, Uruguay.
Email: rodneycolina1@gmail.com
Funding information
Universidad de la República Uruguay,
Grant number: PDU Program; Comisión
Sectorial de Investigación Científica,
Grant number: I+D 2010
Reading Frame-2 (ORF2) region C. Amplicons were sequenced and phylogenetic
analyses were carried out in order to determine genotypes and lineages. Classic HAstV
were detected in 18 out of 175 analyzed samples (10.3%) and 14 of them (78.0%) were
successfully sequenced being 6 (42.8%) classified as HAstV-1 (1a lineage), 4 (28.6%) as
HAstV-2 (2c lineage), and 4 (28.6%) as HAstV-3 (3c lineage). A higher detection of
Classic HAstV infections was observed in autumn for both years of surveillance, and
the majority of the positive cases were observed in 2011. The group of children
between 2 and 5 years old presented the higher percentage of infections. To our
knowledge, the present study represents the first report of astrovirus from acute
gastroenteritis cases in Uruguay, evidencing its role as a relevant etiologic agent in
severe cases of this disease.

KEYWORDS
classic human astrovirus (classic HAstV), epidemiology, genetic diversity, hospitalized children,
Uruguay

1 | INTRODUCTION
The Classic Human Astrovirus (Classic HAstV) are formally classified as
Mamastrovirus 1 viral specie (MAstV-1), and belong to the Mamastrovirus
genus in the Astroviridae family. The Classic HAstV are classified in 8
genotypes (HAstV-1 to HAstV-8), and are described as one of the most
important causes of pediatric acute gastroenteritis (AGE).1 The prevalence
of Classic HAstV infections range from 2% to 9% of nonbacterial AGE
cases among children, and the mean incidence worldwide is 11%, being
usually higher in developing countries with reported incidences as high as
61%.2 Recently, the screening of human feces by molecular assays
revealed the existence of novel astroviruses, HAstV-VA/HMO and
HAstV-MLB, that are more closely genetically related to nonhuman
Mamastrovirus than to Classic HAstV. They are actually classified as new
Mamastrovirus species (MAstV-6, -8, and -9), and their role in
gastrointestinal pathogenicity is not yet well established.3

J Med Virol. 2017;89:1775–1781. wileyonlinelibrary.com/journal/jmv © 2017 Wiley Periodicals, Inc. | 1775

1776 | LOPEZ
The virion has a non-enveloped icosahedral capsid with a single
positive-strand RNA genome of 6.8 kb length. This genome comprise
three open reading frames (ORF): ORF1a, ORF1b, and ORF2, which
encode the serine protease, the RNA-dependent RNA-polymerase,
and the capsid protein precursor, respectively.1 Classic HAstV have
been detected and typed based on phylogenetic studies directed to
four regions located along the genome (named A to D), nevertheless,
majority of reported molecular epidemiology studies are based on the
region C, located at the 5′end of the ORF2.4
HAstV-1 has been the predominant circulating genotype
worldwide, followed by HAstV-2 to -5 and HAstV-8 that are
generally detected with less frequency and its prevalence depend on
the region, meanwhile HAstV-6 and -7 are rarely detected.1,2
Additionally, discrete genetic variation in ORF2 small regions used
for diagnostic revealed intra-typic genetic lineages for HAstV-1, (1a,
1b, 1d, 1e, and 1f), HAstV-2 (2a to 2d), HAstV-3 (3a to 3c), HAstV-4
(4a to 4c), HAstV-5 (5a to 5c), HAstV-6 (6a and 6b), and HAstV-8
4–11
(8a to 8c).
In Uruguay, Classic HAstV have been previously detected,
quantified and molecularly characterized from raw sewage samples
collected from the northwestern and eastern regions of the country,
revealing the circulation of HAstV-1 as the prevalent followed by
HAstV-2 and HAstV-5.12–14 In this sense, the aim of this study was to
determine the molecular epidemiology of Classic HAstV in AGE cases
of hospitalized children in Salto, Uruguay, that have been previously
5
studied for rotavirus. To our knowledge, the present study represents
the first report of Astrovirus from acute gastroenteritis cases in
Uruguay.
2 | M ATERIA LS AN D METH ODS
2.1 | Clinical samples
A total of 175 clinical samples: 153 diarrheal stools and 22 vomits,
were collected from 172 pediatric patients, with acute gastroenteritis,
hospitalized at the pediatric units of the Salto’s public hospital and the
Salto’s private hospital “Medical-Surgical Society of Salto,” between
January 2011 and December 2012. The samples were collected
continuously during the two years of the study when AGE with
hospitalization occurred. Voluntary written informed consent was
obtained from the parents or legal guardians at enrollment in
accordance with national ethical regulations and the Declaration of
Helsinki. Nineteen patients only presented vomit as clinical symptom,
while 150 presented only acute diarrhea, in this sense, only three
patients have presented clinical symptoms of acute diarrhea and
vomits at the same time, and both type of samples were collected and
analyzed for these patient. Fecal and vomit samples were collected in
sterile containers 24 h after admission, immediately storage at 4°C at
the hospital, and transported during the same working day at 4°C to
the Molecular Virology Laboratory of the Universidad de la República,
Salto, Uruguay. Ten percent fecal suspensions or diluted vomits were
prepared in phosphate buffered saline at pH 7.2 and stored at
−20°C until processing. These samples were previously analyzed for
ET AL.
Group A Rotavirus (RVA), as described in Ref.15 and no other
pathogens were investigated. The studied children were not vacci-
nated against rotavirus.
2.2 | RNA extraction, cDNA synthesis, and PCR
detection
RNA was extracted from fecal/vomit suspensions using QIAmp viral
RNA mini kit (QIAGEN®, Hilden, Germany) according to the
manufacturer’s instructions. The positive control was a 10% fecal
suspension previously characterized as HAstV-1 (Accession number:
JQ796912), and UltraPure® DNase/RNase-Free distilled water
(Invitrogen™, Carlsbad, CA) was used as a negative control. Extracted
RNA was reverse transcribed into cDNA in a final volume of 50 μL
using the SuperScrip® II Reverse Transcriptase and Random Hexamer
Primers (both from Invitrogen™), according to the manufacturer’s
instructions. Then, the molecular detection of Classic HAstV was
performed by using a qualitative PCR directed to the ORF2 region C,
with the primers Mon269 and Mon270 as described by Noel et al16
Briefly, the PCR was performed with an initial step of 95°C × 4 min,
followed by 40 cycles of 95°C × 30 s; 50°C × 30 s; and 72°C × 60 s;
with a final extension at 72°C × 10 min. The PCR reaction was
performed in a final volume of 25 μL containing final concentration of
0.5 μM of each primer, 20 mM Tris–HCl (pH 8.4), 50 Mm KCl, 1.6 mM
MgCl2, 0.2 mM of each dNTP, 1U of Taq DNA Polymerase
(Invitrogen™), and 5 μL of template cDNA.
All amplifications products were visualized in a 2% agarose gel
electrophoresis (AMRESCO®, Fountain Parkway Solon, OH) stained
with GoodView™ Nucleic Acid Stain (SBS Genetech, China), by using a
trans-illuminator (Cleaver Scientific Ltd., United Kindom) and the
FOTODYNE Incorporated FOTO/Analyst® Express system (Fotodyne
Incorporated, Hartland, WI) for the gel photo documentation.
2.3 | DNA sequencing and phylogenetic analysis
The amplified segment was purified directly from the PCR product
with the QIAquick PCR purification kit (QIAGEN®) and sequenced by
using the dideoxynucleotide chain terminator method with the
BigDye® Terminator Cycle Sequencing Kit on the automated
sequencer ABI3130 Genetic Analyzer (both from Applied Biosys-
tems®, Foster City, CA) by the DNA Sequencing Service of the
Molecular Biology Unit at the Institute Pasteur of Montevideo.
Sequencing reactions were carried out in forward and reverse
directions employing the PCR primers.
Nucleotide sequences of the Classic HAstV Uruguayan strains
were edited and assembled by using the SeqMan program as
implemented in the DNAStar v7.00 package (DNASTAR®, Madison,
WI) and were aligned with sequences retrieved from the NCBI
database by using the Clustal W tool incorporated into MEGA v5.0
software.17 In the alignment were included prototypes sequences of
the eight classic human astrovirus genotypes (MAstV-1) and proto-
types sequences of each genetic lineage. Also were included those
sequences of Classic HAstV strain detected in the South American
LOPEZ ET AL.
region, and sequences of strain detected elsewhere, closely related to
the Uruguayan sequences and resulted from a Basic Local Alignment
Search Tool (BLAST).
The phylogenetic analysis was conducted with MEGA v5.0
version17 by using the Neighbor Joining method and Kimura two
parameters as nucleotide substitution model. The statistical signifi-
cance of the phylogenetic trees constructed was estimated by the
bootstrap method (5000 replicates).
2.4 | DNA sequences accession numbers
Sequences obtained in this study were deposited in the NCBI database
under the following accession numbers: KX595343-KX595356.
2.5 | Statistical analysis
The z-test for the difference in proportions between two independent
binomial populations for large samples and the Chi-Square test of
independence, were used as statistical methods.
3 | RE SULTS
Classic HAstV were detected in 18 (10.3%) out of 175 collected clinical
samples. The vomit samples were all negative for Classic HAstV. This
way, taking into account only the fecal samples, an 11.86% of Classic
HAstV diarrhea positivity was observed. Five RVA and Classic HAstV
co-infection cases were identified (28% of Classic HAstV positive
samples). On the other hand, 50 of which do not presented Classic
HAstV had RVA (37% of Classic HAstV negative samples). A
Chi-Square test was performed to analyze the association between
the presence of RVA and Classic HAstV obtaining a P-value of 0.61.
Therefore, there is insufficient evidence in the data to consider that
there is some kind of association.
The group of children between 2 and 5 years old presented the
higher percentage of infections (25 to 60 months of age, 9/48, 19%),
followed by patients older than 5 years old (>60 months of age, 6/55,
11%). The children between 1 and 2 years old (13 to 24 months of age),
and those between 7 and 12 months of age, presented a similar
number of positive cases, 2/34 (6%) and 1/25 (4%), respectively.
Positives cases were not detected in children between 0 and 6 months
of age (Table 1).
TABLE 1 Age groups distribution of classic human astrovirus (Classic HAstV) infections in hospitalized children in Salto-Uruguay during two
consecutive years (January 2011 to December 2012)
Age group (months) Patients HAstV positive
0-6 10 0 0
7-12 25 1 4
13-24 34 2 6
25-60 48 9 19
>60 55 6 11
ND, non detected.
| 1777
Considering the ages divided in three categories (0 to 24 months,
25 to 60 months, >60 months), a weak evidence of association
between a presence of Classic HAstV and age for this data was
observed (a Chi-Square test gives a P-value of 0.04). Moreover, 19%
(9/48) of children with 2 to 5 years old presented Classic HAstV
compared with 7% (9/124) of children in the other age groups showing
a significant difference between these proportions (z-test with a
P-value of 0.03).
A higher detection of Classic HAstV infections was observed in
autumn for the two consecutive years of surveillance covered by this
study. However, the percentage of positivity verified in the summer of
2011 (2/10, 20%) is not significant different of the corresponding
percentage in autumn (11/48, 23%) of the same year (z-test gives a
P-value of 0.84), neither in 2012 (P-value of 0.16). Most of the positive
cases were observed in autumn of the year 2011, and a higher diversity
of genotypes was detected in this period. HAstV-1 was detected
during the two consecutive years of surveillance covered by this study
(6/14, 43%), while HAstV-2 (4/14, 28,5%) and HAstV-3 (4/14, 28,5%)
were only detected in 2011 (Figure 1).
Fourteen partial nucleotide sequences of C region (ORF2) from
the 18 RT-PCR amplicons (78%) were obtained and submitted to
phylogenetic studies. Phylogenetic analysis of the 14 obtained
sequences permitted us to observe that six strains belonged to the
HAstV-1 genotype. Furthermore, all of them clustered within lineage a
(HAstV-1a) in two separate cluster inside this lineage, along with
a HAstV-1a strain from sewage samples of Uruguay collected in 2011
(Figure 2, upper part). Four samples were classified as HAstV-2 and
clustered within lineage c (HAstV-2c), together with a HAstV-2c strain
detected in sewage from Salto city in March, 2011 (Figure 2, bottom
part). The remaining four Uruguayan strains, were classified as
HAstV-3 and clustered within the emerging lineage c (HAstV-3c),
recently described, along with contemporary HAstV-3c strains
detected in 2008 and 2010 in Brazil, and in 2012 in Italy (Figure 2,
middle part).
4 | DISCUSSION
Classic HAstV were previously detected, quantified and molecularly
characterized from raw sewage samples collected in six cities located
at northwestern and eastern regions of Uruguay (including Salto city),
revealing a high detection frequency (up to 70%) and a great genetic
% positive Lineages/sublineages detected
ND
1a
1a
1a, 2c, 3
1a, 2c, 3
1778 | LOPEZ
FIGURE 1 Detection rates and seasonality of classic human astrovirus (Classic HAstV) in cases of acute gastroenteritis in Salto, Uruguay,
during two consecutive years (January 2011 to December 2012). Southern hemisphere seasons are as follow: summer (January, February, and
March), autumn (April, May, and June), winter (July, August, and September), and spring (October, November, and December)
diversity of this virus in these environmental samples.12–14 Neverthe-
less, the present study represents the first report about the circulation
of Classic HAstV in clinical samples of hospitalized children with AGE in
Salto city, Uruguay, collected during two years (2011, 2012). These
same samples were previously tested for rotavirus with a positivity of
15
37%. Classic HAstV are associated with 2% to 9% of cases of
nonbacterial AGE in children,18 and a mean incidence worldwide of
11%,2 which is similar to the prevalence of 11.86% that was observed
in this study. Mixed infections with RVA were detected in 28% of
Classic HAstV positive samples, which is in agreement with previous
reports from clinical surveys of AGE cases of hospitalized children
describing even higher rates of mixed infections with RVA and others
enteric viruses.19–22 RVA vaccination is not yet part of the National
Immunization Program in Uruguay, and vaccines against this virus are
only available on the local market at an elevated cost for consumer.
However, although effective against RVA, the two most commonly
used RVA vaccines, RV1 and RV5, does not protect against other viral
23,24
or bacterial agents.
Classic HAstV infections have been found worldwide primarily,
but not exclusively, in young children with diarrhea. Detection in
healthy adults, elderly and immunocompromised patients were also
1,2
reported. Classic HAstV has been described predominately affecting
children under the age of 2 years.2,18 However, in this study, the group
of children between 2 and 5 years old presents the higher percentage
of infections, which is in concordance with a recent report of Ref.25 in
which the authors observed that the prevalence increased with respect
to the age in children under 5 years old. On the other hand, Classic
HAstV was not detected in infants younger than 6 months of age. A
possible explanation to this fact could be that generally infants of this
age are exclusively at home under the permanent care of their parents
or guardians, with breastfeeding and protected from contact with
other people and the environment (such as in daycare centers). In the
case of breastfeeding is widely known its benefit to the child,
protecting him against viral and no-viral AGE (among others infectious
disease). This is due to the antibodies transmission from mother to
baby through breast milk.26,27
ET AL.
A seasonal pattern with a higher incidence of infection in the cold-
weather period in temperate regions have been described for Classic
HAstV, although there is a dramatic decline in the number of cases in
the warm-weather period, the virus continues to circulate throughout
the year and infections also occur in summer.2 This seasonality may
be the consequence of a better stability of the virus at lower
temperatures and could be also associated with the fact that in cold-
weather period people tend to be agglomerated in closed environ-
ments, facilitating the person-to-person transmission of the virus.28,29
However, a majority (but not exclusively) of positives cases were
observed in autumn for the two years of this study. This seasonality
could be explained by the fact that the beginning of the school year in
Uruguay is in autumn, bringing many children together, and potentially
causing local AGE outbreaks as previously described for this virus in
this kind of institution.30 Nevertheless, although a little higher
detection was observed in autumn 2011, there were not highly
difference between autumn (22%) and summer (20%). More studies
will needed in the near future with a larger number of samples and in a
more extended time, to be able to draw more solid conclusions about
seasonality in the studied region.
A limitation of the present study was that 22% of the positive
samples could not be sequenced. This could be explained because of
the observation that in these samples a PCR product of the expected
size was obtained, but, unfortunately, the intensity of the electropho-
resis band, that is a reflect of the PCR product concentration, was
always very low despite several attempts to increase their
concentration.
Classic HAstV was not detected in vomit samples analyzed in this
study. This is in accordance with the literature where no reports of
Classic HAstV detection in vomit has been documented.1,2
The molecular characterization of Classic HAstV through phylo-
genetic analysis revealed a genetic diversity in the analyzed clinical
samples in this study with the co-circulation of three genotypes, being
HAstV-1 the most prevalent genotype detected (43%), followed
by HAstV-2 and HAstV-3, at equal prevalence of detection (29%). The
HAstV-1 strains were all classified as lineage “a” (HAstV-1a), and
LOPEZ ET AL.
| 1779

FIGURE 2 Neighbor joining phylogenetic tree of ORF2, region C, nucleotide sequences of classic human astrovirus (Classic HAstV) strains
detected in hospitalized children from Salto, Uruguay, between 2011 and 2012. Strains in the tree are named according to the nomenclature
proposed by Martella et al.4 Classic HAstV strains described elsewhere are also preceded by the accession numbers. Numbers at the nodes in
the tree indicate Bootstrap values (only values above 75% are shown). The scale bar at the bottom represents substitutions per nucleotide
position (nt.subst./site). Strains detected in this study are marked with a filled circle. Uruguayan strains previously detected in sewage,12 are
marked with filled triangle
1780 | LOPEZ
clustered together with contemporary strains of the same lineage
previously detected from raw sewage samples collected at northwest-
ern and eastern regions of Uruguay, including Salto city.13,14 In these
previous studies, as observed here, the HAstV-1a was the genotype
most frequently detected, and its circulation was observed during all
the 2011. Although the majority of the Classic HAstV cases were
observed in autumn 2011, HAstV-1 was the only one of the three
genotypes detected that was also observed in 2012, and considering
2011, was also detected in summer. The high prevalence of HAstV-1
observed in this study, is in agreement with others studies showing
that this genotype is the most prevalent in the Latin American region
and worldwide.5,7,19,20,22,25,31–36
HAstV-2 has been generally described as the second most
prevalent genotype in clinical studies performed worldwide, after
HAstV-1 genotype.19,34,35 Also, this genotype has been sporadically
identified as the most frequent genotype, as observed in the study
performed in Brazil in which this genotype was determined as the
causative agent of an outbreak of gastroenteritis in 2004 in an
25,37–39
indigenous population at the southeast region of the country.
This genotype was previously detected in the northwestern and
eastern regions of Uruguay from raw sewage samples, and two
lineages were identified: HAstV-2c and HAstV-2d. However, in the
present study all the HAstV-2 clinical samples were classified as
belonging to lineage “c” (HAstV-2c) and clustered together with a
contemporary strain of the same lineage previously detected from a
sewage sample of Salto, Uruguay. This result, is in agreement with the
previous observation of the circulation of this specific lineage only in
the sewage samples collected from Salto city of the six cities
analyzed.14,33
The prevalence observed in the present study for HAstV-3 is in
accordance with previous studies performed in the Latin American
region, showing this genotype as the second or third most prevalent
genotype, after HAstV-1.32,37,40 More recently, this genotype has
been reported as the most prevalent in the Northeast region of Brazil
from 2005 to 2011.25 The HAstV-3 strains detected in this study were
classified as lineage “c” (HAstV-3c), considered as an emerging lineage
since many of the recently reported HAstV-3 belong to this
lineage.11,20,25,41,42
This is the first study documenting the prevalence, age related
distribution, seasonality and genetic diversity of Classic HAstV
infections in cases of AGE hospitalized children in Salto city, Uruguay.
A higher detection frequency and genetic diversity was previously
observed in Salto city compared with other five Uruguayan cities,
through the molecular characterization of Classic HAstV strains
detected from raw sewage.13,14 This could be explained because
Salto city is the second most densely populated city nationwide after
Montevideo city (the capital of the country), and, also, is the third
touristic spot of Uruguay for national and international travelers, after
the cities of Montevideo and Punta del Este, according to national data
recorded from 2005 to 2014.43,44 Salto city is a high touristic spot due
to the presence of developed and attractive hot springs water parks,44
and related to this, there are previous reports of recreational
associated water-borne viral gastroenteritis outbreaks in hot spring
ET AL.
parks.45 In this sense, an important approach achieved in this study was
the comparison at sequence level of contemporary Uruguayan clinical
and environmental strains, thus strengthening epidemiological surveil-
lance. The findings of the present study complement those from
elsewhere and fill up a void of information regarding Classic HAstV for
Uruguay. They also demonstrate that this virus is less common than
RVA in cases of AGE in hospitalized children in Salto city,15 but, even
so, a potentially important viral etiological agent with a significant role
in acute gastroenteritis in Uruguay.
ACKNOWLEDGMENTS
We want to thank the financial support by the program “Polo de
Desarrollo Universitario” (PDU) and CSIC I+D 2010 Project, “Universidad
de la República” (UDELAR), Uruguay.
REFERENCES
1. Méndez E, Arias CF. Astroviruses. In: Knipe DM, Howley PM, eds.
Fields Virology, (6th ed.). Philadelphia: Wolters Kluwers Health,
Lippincott Williams & Wilkins; 2013:609–628.
2. Bosch A, Pintó RM, Guix S. Human astroviruses. Clin Microbiol Rev.
2014;27:1048–1074.
3. Vu DL, Cordey S, Brito F, Kaiser L. Novel human astroviruses: novel
human diseases? J Clin Virol. 2016;82:56–63.
4. Martella V, Pinto P, Tummolo F, et al. Analysis of the ORF2 of human
astroviruses reveals lineage diversification, recombination and rear-
rangement and provides the basis for a novel sub-classification
system. Arch Virol. 2014;159:3185–3196.
5. Medina SM, Gutierrez MF, Liprandi F, Ludert JE. Identification and
type distribution of astroviruses among children with gastroenteritis in
Colombia and Venezuela. J Clin Microbiol. 2000;38:3481–3483.
6. Guix S, Caballero S, Villena C, et al. Molecular epidemiology of
astrovirus infection in Barcelona, Spain. J Clin Microbiol. 2002;40:
133–139.
7. Gabbay YB, Leite JP, Oliveira DS, et al. Molecular epidemiology of
astrovirus type 1 in Belém, Brazil, as an agent of infantile
gastroenteritis, over a period of 18 years (1982-2000): identification
of two possible new lineages. Virus Res. 2007a;129:166–174.
8. Gabbay YB, Linhares AC, Oliveira DS, et al. First detection of a human
astrovirus type 8 in a child with diarrhea in Belãm, Brazil: comparison
with other strains worldwide and identification of possible three
lineages. Mem Inst Oswaldo Cruz. 2007b;102:531–534.
9. De Grazia S, Martella V, Chironna M, et al. Nationwide surveillance
study of human astrovirus infections in an Italian paediatric
population. Epidemiol Infect. 2013;141:524–528.
10. Martella V, Medici MC, Terio V, et al. Lineage diversification and
recombination in type-4 human astroviruses. Infect Genet Evol.
2013;20:330–335.
11. Medici MC, Tummolo F, Martella V, et al. Genetic heterogeneity and
recombination in type-3 human astroviruses. Infect Genet Evol.
2015;32:156–160.
12. Victoria M, Tort LF, García M, et al. Assessment of gastroenteric
viruses from wastewater directly discharged into Uruguay River,
Uruguay. Food Environ Virol. 2014;6:116–124.
13. Lizasoain A, Tort LF, García M, et al. Environmental assessment of
classical human astrovirus in Uruguay. Food Environ Virol. 2015a;7:
142–148.
14. Lizasoain A, Tort LF, García M, et al. Environmental assessment reveals
the presence of MLB-1 human astrovirus in Uruguay. J Appl Microbiol.
2015b;119:859–867.
LOPEZ ET AL.
15. Tort LF, Victoria M, Lizasoain AA, et al. Molecular epidemiology of
group A rotavirus among children admitted to hospital in Salto,
Uruguay, 2011-2012: first detection of the emerging genotype G12.
J Med Virol. 2015;87:754–763.
16. Noel JS, Lee TW, Kurtz JB, Glass RI, Monroe SS. Typing of human
astroviruses from clinical isolates by enzyme immunoassay and
nucleotide sequencing. J Clin Microbiol. 1995;33:797–801.
17. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S.
MEGA5: molecular evolutionary genetics analysis using maximum
likelihood, evolutionary distance, and maximum parsimony methods.
Mol Biol Evol. 2011;28:2731–2739.
18. De Benedictis P, Schultz-Cherry S, Burnham A, Cattoli G. Astrovirus
infections in humans and animals-molecular biology, genetic diversity,
and interspecies transmissions. Infect Genet Evol. 2011; 11:
1529–1544.
19. Victoria M, Carvalho-Costa FA, Heinemann MB, Leite JP, Miagosto-
vich MP. Genotypes and molecular epidemiology of human astrovi-
ruses in hospitalized children with acute gastroenteritis in Rio de
Janeiro, Brazil. J Med Virol. 2007;79:939–944.
20. Pativada M, Nataraju SM, Ganesh B, et al. Emerging trends in the
epidemiology of human astrovirus infection among infants, children
and adults hospitalized with acute watery diarrhea in Kolkata, India.
Infect Genet Evol. 2012;12:1685–1693.
21. Afrad MH, Karmakar PC, Das SK, et al. Epidemiology and genetic
diversity of human astrovirus infection among hospitalized patients
with acute diarrhea in Bangladesh from 2010 to 2012. J Clin Virol.
2013;58:612–618.
22. De Grazia S, Bonura F, Bányai K, et al. Temporal variation in the
distribution of type-1 human astrovirus lineages in a settled
population over 14 years. Arch Virol. 2016;161:1633–1637.
23. Ruiz-Palacios GM, Pérez-Schael I, Velázquez FR, et al. Safety and
efficacy of an attenuated vaccine against severe rotavirus gastroen-
teritis. N Engl J Med. 2006;354:11–22.
24. Vesikari T, Matson DO, Dennehy P, et al. Safety and efficacy of a
pentavalent human-bovine (WC3) reassortant rotavirus vaccine.
N Engl J Med. 2006;354:23–33.
25. Xavier Mda P, Carvalho Costa FA, Rocha MS, et al. Surveillance of
human astrovirus infection in Brazil: the first report of MLB1
astrovirus. PLoS ONE. 2015;10:e0135687.
26. Brown KH, Peerson JM, Baker SK, Hess SY. Preventive zinc
supplementation among infants, preschoolers, and older prepubertal
children. Food Nutr Bull. 2009;30:S12–S40.
27. Lamberti LM, Fischer Walker CL, Noiman A, Victora C, Black RE.
Breastfeeding and the risk for diarrhea morbidity and mortality. BMC
Public Health. 2011;11:S15.
28. Abad FX, Pintó RM, Villena C, Gajardo R, Bosch A. Astrovirus survival
in drinking water. Appl Environ Microbiol. 1997;63:3119–3122.
29. Abad FX, Villena C, Guix S, Caballero S, Pintó RM, Bosch A. Potential
role of fomites in the vehicular transmission of human astroviruses.
Appl Environ Microbiol. 2001;67:3904–3907.
30. Oishi I, Yamazaki K, Kimoto T, et al. A large outbreak of acute
gastroenteritis associated with astrovirus among students and
teachers in Osaka, Japan. J Infect Dis. 1994;170:439–443.
31. Gaggero A, O’Ryan M, Noel JS, et al. Prevalence of astrovirus infection
among Chilean children with acute gastroenteritis. J Clin Microbiol.
1998;36:3691–3693.
| 1781
32. Espul C, Martínez N, Noel JS, et al. Prevalence and characterization of
astroviruses in Argentinean children with acute gastroenteritis. J Med
Virol. 2004;72:75–82.
33. Jeong AY, Jeong HS, Jo MY, et al. Molecular epidemiology and genetic
diversity of human astrovirus in South Korea from 2002 to 2007. Clin
Microbiol Infect. 2011;17:404–408.
34. Malasao R, Khamrin P, Chaimongkol N, Ushijima H, Maneekarn N.
Diversity of human astrovirus genotypes circulating in children with
acute gastroenteritis in Thailand during 2000-2011. J Med Virol.
2012;84:1751–1756.
35. Medici MC, Tummolo F, Albonetti V, Abelli LA, Chezzi C, Calderaro A.
Molecular detection and epidemiology of astrovirus, bocavirus, and
sapovirus in Italian children admitted to hospital with acute
gastroenteritis, 2008-2009. J Med Virol. 2012;84:643–650.
36. Tseng WC, Wu FT, Hsiung CA, et al. Astrovirus gastroenteritis in
hospitalized children of less than 5 years of age in Taiwan, 2009.
J Microbiol Immunol Infect. 2012;45:311–317.
37. Walter JE, Mitchell DK, Guerrero ML, et al. Molecular epidemiology of
human astrovirus diarrhea among children from a periurban commu-
nity of Mexico City. J Infect Dis. 2001;183:681–686.
38. Gabbay YB, Chamone CB, Nakamura LS, et al. Characterization of an
astrovirus genotype 2 strain causing an extensive outbreak of
gastroenteritis among Maxakali Indians, Southeast Brazil. J Clin Virol.
2006;37:287–292.
39. Resque HR, Munford V, Castilho JG, Schmich H, Caruzo TA, Rácz
ML. Molecular characterization of astrovirus in stool samples from
children in Sáo Paulo, Brazil. Mem Inst Oswaldo Cruz. 2007;102:
969–974.
40. Méndez-Toss M, Griffin DD, Calva J, et al. Prevalence and genetic
diversity of human astroviruses in Mexican children with symptomatic
and asymptomatic infections. J Clin Microbiol. 2004;42:151–157.
41. Alam MM, Khurshid A, Rana MS, et al. Serotype diversity of
astroviruses in Rawalpindi, Pakistan during 2009-2010. PLoS ONE.
2013;8:e61667.
42. Monastiri A, Aouni M, Guix S, et al. Clinical surveillance for human
astrovirus in Monastir region, Tunisia. BMC Public Health. 2016;
16:57.
43. I.N.E. (Uruguayan National Institute of Statistic). 2017. Available
online at: http://www.ine.gub.uy (Accessed in February of 2017).
44. M.T., 2017. Uruguayan Ministry of Tourism. 2017. Top destinations of
tourist 2004–2014: Available online at: http://www.mintur.gub.uy/
index.php/es/2014/itemlist/category/638-destinos. Hotspring Tour-
ism: Available online at: http://www.turismo.gub.uy/index.php/que-
hacer/turismo-termal (Accessed in February of 2017).
45. Sinclair RG, Jones EL, Gerba CP. Viruses in recreational water-borne
disease outbreaks: a review. J Appl Microbiol. 2009;107:1769–1780.
How to cite this article: Lopez F, Lizasoain A, Victoria M,
et al. Epidemiology and genetic diversity of classic human
astrovirus among hospitalized children with acute
gastroenteritis in Uruguay. J Med Virol. 2017;89:1775–1781.
https://doi.org/10.1002/jmv.24854