Annual incidence, serotype distribution, and genetic diversity of human astrovirus isolates from hospitalized children in Melbourne, Australia.

Abstract

The incidence of astrovirus infection in children under 5 years of age hospitalized for acute gastroenteritis in Melbourne, Australia, during 1995 was determined. Astrovirus was detected in 16 fecal specimens by Northern (RNA) dot blot analysis of RNA isolated from feces with an astrovirus-specific cDNA probe. The incidence of astrovirus infection was determined as 4.2% (16 of 378 total samples) compared with rates of 63.2, 3.7, and 4.2% for rotavirus, adenovirus, and all bacterial pathogens, respectively. Astrovirus was detected during the winter season and mainly in infants between 6 and 12 months of age. Serotyping of samples was carried out by reverse transcriptase PCR and direct sequencing of a 348-bp region of the capsid protein gene. Type 1 strains predominated (11 of 13 typeable samples), although type 4 isolates were also detected. Astrovirus was retrospectively identified in 13 fecal samples collected from hospitalized infants between 1980 and 1985 and shown to contain small viruses by electron microscopy. Type 1 isolates were again the most common, although a type 5 strain was also found. Comparative sequence analysis indicated that type 1 astroviruses exhibited up to 7% sequence divergence over a 15-year period; however, all mutations were silent. The incidence of astrovirus reported here indicates that the virus is a significant cause of severe diarrhea in young children. The genetic analysis also provides important molecular epidemiological information relevant to the development of preventative therapies.

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JOURNAL OF CLINICAL MICROBIOLOGY, July 1996, p. 1750–1753
0095-1137/96/$04.00?0
Copyright ? 1996, American Society for Microbiology
Vol. 34, No. 7
Annual Incidence, Serotype Distribution, and Genetic Diversity
of Human Astrovirus Isolates from Hospitalized Children
in Melbourne, Australia
ENZO A. PALOMBO* AND RUTH F. BISHOP
Department of Gastroenterology, Royal Children’s Hospital, Parkville, Victoria 3052, Australia
The incidence of astrovirus infection in children under 5 years of age hospitalized for acute gastroenteritis
in Melbourne, Australia, during 1995 was determined. Astrovirus was detected in 16 fecal specimens by
Northern (RNA) dot blot analysis of RNA isolated from feces with an astrovirus-specific cDNA probe. The
incidence of astrovirus infection was determined as 4.2% (16 of 378 total samples) compared with rates of 63.2,
3.7, and 4.2% for rotavirus, adenovirus, and all bacterial pathogens, respectively. Astrovirus was detected
during the winter season and mainly in infants between 6 and 12 months of age. Serotyping of samples was
carried out by reverse transcriptase PCR and direct sequencing of a 348-bp region of the capsid protein gene.
Type 1 strains predominated (11 of 13 typeable samples), although type 4 isolates were also detected.
Astrovirus was retrospectively identified in 13 fecal samples collected from hospitalized infants between 1980
and 1985 and shown to contain small viruses by electron microscopy. Type 1 isolates were again the most
common, although a type 5 strain was also found. Comparative sequence analysis indicated that type 1
astroviruses exhibited up to 7% sequence divergence over a 15-year period; however, all mutations were silent.
The incidence of astrovirus reported here indicates that the virus is a significant cause of severe diarrhea in
young children. The genetic analysis also provides important molecular epidemiological information relevant
to the development of preventative therapies.
Astroviruses were first detected in fecal samples from chil-
dren with diarrhea by visualization of virus particles by elec-
tron microscopy (EM) (1, 15). The agent is now clearly asso-
ciated with gastrointestinal illness both in volunteers ingesting
virus filtrates and in natural infection in the community (7).
Although astrovirus is commonly associated with mild disease
of short duration, symptoms in children are often as severe as
those observed with rotavirus diarrhea (7). Nosocomial astro-
virus infections have also been reported (3, 4, 22).
Epidemiological surveys of agents isolated from young chil-
dren with diarrhea attending outpatient clinics, admitted to the
hospital or investigated in longitudinal community surveys, in-
dicate that astroviruses are a significant cause of acute gastro-
enteritis, with prevalence rates of 2.7 to 8.6% (2, 7–9, 19).
Astrovirus was also detected in 6 to 10% of fecal samples from
children with diarrhea in which no bacterial or viral agents
were identified (26), while an attack rate of 89% was recently
determined in an astrovirus-associated diarrhea outbreak in a
day care center (18). The incidence of astrovirus determined in
surveys reflects the diagnostic assay employed, with the enzyme
immunoassay (EIA) exhibiting greater sensitivity than EM.
Genetic methods (hybridization and reverse transcriptase PCR
[RT-PCR]) have been developed and evaluated but not yet
applied to large-scale epidemiological surveys (11, 19, 28).
However, the sensitivity of RT-PCR detection has been shown
to be greater than that of the EIA (18). Surveys of antibody
prevalence indicate that 70% of children are exposed to astro-
virus during the first 5 years of life (13).
The astrovirus virion is composed of a single nonenveloped
capsid layer 28 nm in diameter, while the viral genome is a
single-stranded positive-sense polyadenylated RNA 6.8 to 7.6
kb in length (7, 10, 27). The genome contains three open
reading frames (ORFs): ORF1a and ORF1b, which encode
the viral protease and polymerase; and ORF2, which encodes
the capsid precursor (10, 27). A subgenomic RNA that is
colinear with the 3? end of the genome and contains ORF2 has
been identified in the cytoplasm of infected cells (17, 20, 21).
Human astroviruses are currently classified into seven sero-
types by reactivity of capsid proteins with polyclonal sera and
monoclonal antibodies (12, 14). Recently, sequence analysis of
the capsid gene (ORF2) identified a 348-bp region that is
conserved in viruses of the same serotype but varies between
strains of different serotypes as determined by EIA (23). Thus,
a correlation between genotype and serotype has been estab-
lished.
Limited information is available about the extent of genetic
variation in astrovirus clinical isolates, especially in relation to
seasonal variation occurring in isolates collected from children
hospitalized with gastroenteritis. Such information is impor-
tant in the context of the future development of preventative
therapies. In this report, we describe the incidence, monthly
occurrence, and distribution of serotypes of astroviruses iso-
lated from children under 5 years of age admitted to the Royal
Children’s Hospital in Melbourne during 1995. In addition, the
extent of sequence variation in these isolates was determined
and compared with those of isolates collected 10 to 15 years
earlier and isolates causing outbreaks of gastroenteritis in the
Melbourne community in 1995.
MATERIALS AND METHODS
Viruses and fecal samples. Standard human astrovirus (HAstV) types 1 to 7
grown in CaCo-2 cells were kindly provided by John Kurtz, John Radcliffe
Hospital, Oxford, United Kingdom, and stored at ?70?C. Fecal samples were
collected from children under 5 years of age within 48 h of admission with acute
gastroenteritis to the infectious diseases ward at the Royal Children’s Hospital in
Melbourne, Australia, in 1995. Astrovirus-positive samples isolated from chil-
dren admitted to the Royal Children’s Hospital between 1980 and 1985 or from
children involved in gastroenteritis outbreaks in Melbourne in 1995 (provided by
John Marshall, Victorian Infectious Diseases Laboratory, Fairfield Hospital,
Melbourne, Australia) had been detected by EM examination of negatively
* Corresponding author. Mailing address: Department of Gastroen-
terology, Royal Children’s Hospital, Flemington Rd., Parkville, Victo-
ria 3052, Australia. Phone: 61 3 9345 5060. Fax: 61 3 9345 6240.
Electronic mail address: palomboe@cryptic.rch.unimelb.edu.au.
1750

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stained fecal extracts. Fecal samples, as 10% (wt/vol) homogenates or ultracen-
trifuged pellets (resuspended in Tris buffer) for the outbreak isolates, were
stored at ?70?C.
Isolation of astrovirus RNA, RT-PCR, and sequencing. RNA was isolated by
phenol-chloroform extraction of tissue culture supernatants, fecal homogenates,
or ultracentrifuged pellets; purified by adsorption to hydroxyapatite; and eluted
in potassium-phosphate buffer (6). RT-PCR was carried out by the method of
Gentsch et al. (5) and with the astrovirus-specific primers Mon269 and Mon270
(23). After gel purification of PCR-derived cDNA, direct cycle sequencing was
carried out with the fmol DNA Sequencing System (Promega, Madison, Wis.)
and the same primers.
Preparation of a cross-serotype astrovirus-specific probe. To prepare an as-
trovirus-specific probe to detect all astrovirus serotypes, RNA derived from each
of the type 1 to 7 HAstVs was used to generate a 449-bp cDNA product by
RT-PCR as described above. Each of the cDNAs was used as a template for
second-round PCR with the same primers and with incorporation of digoxigenin
(DIG)-11-dUTP (Boehringer Mannheim, Mannheim, Germany). The seven in-
dividual DIG-labeled cDNAs were gel purified, quantified, and blended so that
the final concentration of each in hybridization (described below) was 10 ng
ml?1.
Northern RNA hybridization. Northern hybridization was carried out under
stringent conditions (50% formamide, 5? SSC [1? SSC is 0.15 M NaCl plus
0.015 M sodium citrate], 50?C). Total RNA from virus or fecal samples (5 ?l) was
denatured by being boiled for 3 min and spotted onto a positively charged nylon
membrane (Boehringer Mannheim). RNA was fixed to the membrane by UV
cross-linking for 5 min before prehybridization at 50?C for 4 h in a buffer
consisting of 50% formamide, 7% sodium dodecyl sulfate (SDS), 50 mM sodium
phosphate (pH 7), 2% blocking reagent (Boehringer Mannheim), 5? SSC, and
0.1% N-laurylsarcosine. Hybridization was carried out at 50?C in the same buffer,
which also contained 10 ng of each DIG-labeled probe ml?1. After being washed
in 2? SSC–0.1% SDS at room temperature (twice) and 0.1? SSC–0.1% SDS at
50?C (twice), the bound probe was detected with anti-DIG antibody conjugated
to alkaline phosphatase (Boehringer Mannheim) and the chemiluminescent sub-
strate CDP-Star (Boehringer Mannheim).
Nucleotide sequence accession number. The nucleotide sequences determined
in this study have been deposited in the GenBank sequence database and have
been assigned the accession numbers U49212 to U49220.
RESULTS
Epidemiology of astrovirus infection. Between January and
December 1995, fecal specimens were collected within 48 h of
admission from 378 children under 5 years of age admitted to
the Royal Children’s Hospital with acute gastroenteritis. Sam-
ples were examined by routine techniques for the presence of
bacterial and parasitic pathogens and by EIA for the presence
of rotavirus and adenovirus. A total of 239 samples were found
to be positive for rotavirus and excluded from further investi-
gation. Of the remainder, 113 were screened for the presence
of astrovirus by Northern hybridization of RNA extracted from
fecal homogenates with a cross-serotype astrovirus-specific
probe. The specificity of the probe was determined with RNA
derived from HAstV 1 to 7, an astrovirus-positive fecal sample,
rotavirus-positive stool, and negative control stool. The probe
hybridized only to astrovirus RNA and was equally sensitive in
detecting all standard serotypes (Fig. 1). This analysis indi-
cated the presence of astrovirus RNA in 16 fecal samples,
representing 4.2% of the total number of samples. The rate of
astrovirus detection was second only to that for rotavirus
(63.2%) and greater than those for adenovirus (3.7%) and
bacterial pathogens (4.2% for all pathogens).
Subsequent review of patient records suggested that three
patients were likely to have acquired astrovirus nosocomially,
having been readmitted with diarrhea after recent discharge
from the hospital where they had been treated for illness un-
related to gastroenteritis. In addition, one patient was subse-
quently found to have had simultaneous infection with astro-
virus and Clostridium difficile. The remaining 15 astrovirus-
positive samples had no other pathogen identified. Astrovirus
was detected between May and August (Fig. 2) and was de-
tected mainly in children between 6 and 12 months of age (7 of
16). Three of the patients were less than 6 months of age, three
were between 1 and 2 years of age, and three were older than
2 years. During the major winter period (June to August),
astrovirus was identified in 8% of samples.
Genotyping of 1995 astrovirus isolates. RNAs from the 16
astrovirus isolates identified above were subjected to RT-PCR
with the astrovirus-specific primers Mon269 and Mon270 (23).
Thirteen samples yielded the expected 449-bp cDNA, thereby
confirming the hybridization results. Determination of the se-
quence of an internal 348-bp region was carried out with the 13
amplified specimens with the same primers. Comparisons with
the sequences of standard astrovirus serotypes described in the
literature indicated that the 13 Melbourne samples could be
classified as either type 1 (11 isolates) or type 4 (2 isolates). On
the basis of nucleotide sequence variation within these types,
four subtypes of type 1 (designated RCH1A, RCH1B,
RCH1C, and RCH1D) and two subtypes of type 4 (designated
RCH4A and RCH4B4B) were identified (Table 1). Strain
RCH1A was represented by six isolates, RCH1B and RCH1C
were represented by two isolates each, and RCH1D was rep-
resented by a single isolate. The two type 4 isolates exhibited
1.1% sequence variation (4 bp differences), while the type 1
strains showed 0.3 to 0.9% sequence variation (1 to 3 bp
differences [Table 2]). However, none of the nucleotide se-
quence changes translated into amino acid substitutions within
each group. Strains RCH1A and RCH1B were associated with
the three nosocomial infections.
The sequences of two astrovirus isolates collected from in-
fants (6 and 12 months of age) during independent community
outbreaks of gastroenteritis in Melbourne in 1995 were deter-
mined. Both isolates exhibited sequences identical to that of
RCH1A.
Sequence comparisons with historical astrovirus isolates.
Thirteen astrovirus isolates collected from children admitted
to the hospital in Melbourne during 1980 and between 1982
and 1985 were investigated. Previous routine EM of fecal sam-
ples had indicated the presence of small viruses. The charac-
FIG. 1. Northern hybridization of RNA extracted from HAstV types 1 to 7
(A1 to A5 and B1 and B2), an astrovirus-positive fecal sample (B3), a rotavirus-
positive fecal sample (B4), and a negative fecal sample (B5) with a cross-serotype
DIG-labeled probe derived from HAstV 1 to 7.
FIG. 2. Monthly occurrence of astrovirus in children under 5 years of age
hospitalized with gastroenteritis at the Royal Children’s Hospital, Melbourne,
Australia, 1995.
VOL. 34, 1996INCIDENCE OF HUMAN ASTROVIRUS1751

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teristic star-like appearance was not detected by EM, but all
isolates were confirmed as astrovirus by RT-PCR. Nucleotide
sequence analysis, as described above, indicated that this col-
lection comprised three individual strains: two type 1 strains
(designated RCH1E and RCH1F) and a type 5 strain (RCH5)
(Table 1). Strain RCH1E was represented by the single 1980
isolate, while RCH1F was recovered from 11 patients between
1982 and 1985. RCH5 was isolated from a patient with severe
combined immunodeficiency who developed diarrhea after ad-
mission to the hospital for a bone-marrow transplant and is
therefore likely to have been acquired nosocomially.
Sequence comparisons between the type 1 astroviruses iso-
lated in 1980 to 1985 and those circulating in 1995 indicated
that significant temporal diversity existed. Strains RCH1E and
RCH1F (isolated 2 years apart) exhibited 1.4% sequence vari-
ation; however, the degree of variation between RCH1E and
the 1995 strains was between 5.8 and 7% (Table 2). Similarly,
strain RCH1F exhibited 5.5 to 6.6% diversity compared with
the 1995 strains (Table 2). However, although as many as 24
nucleotide differences were observed, none of the changes
resulted in amino acid substitutions. Hence, astrovirus strains
isolated from the Melbourne pediatric community appear to
exhibit the steady accumulation of silent mutations. Neverthe-
less, diversity in the distribution of serotypes was apparent.
Interestingly, only a single type 1 strain was identified in the
3-year period 1982 to 1985, while four type 1 strains appear to
have cocirculated in 1995.
DISCUSSION
In this report, we have described the incidence, monthly
occurrence, and serotype distribution of astroviruses isolated
in Melbourne during 1995. Our data indicate that astrovirus
was responsible for 4.2% (16 of 378) of the total number of
gastroenteritis infections requiring admission of children un-
der 5 years of age to the hospital investigated during 1995.
Astroviruses were the sole enteric pathogen identified in 15 of
the 16 positive samples (94%), and the virus was identified
during the winter season, mainly in children between 6 and 12
months of age. The detection of astrovirus in children with
diarrhea requiring hospitalization indicates that the virus can
cause severe disease.
The incidence determined here concurs with that deter-
mined by a survey of infectious agents causing diarrhea in
Australian children in 1978 to 1983 which showed an overall
incidence of astrovirus of 2.17% and an incidence of 4.3% in
children under 5 years of age (8). In contrast, a survey of
specimens collected from the United States, Korea, and Peru
indicated a rate of occurrence of 3.5% for astrovirus (19),
while surveys from Guatemala (2) and Thailand (9) detected
astrovirus in 7.3 and 8.6% of samples, respectively. Results
from the earlier Australian survey, carried out by examination
of fecal samples by EM (8), imply that detection by hybridiza-
tion and EM can be equally sensitive techniques. However,
comparison of the sensitivities of EM and hybridization in the
same laboratory suggests that hybridization is more sensitive
than EM for the detection of astrovirus in stools (28). Hybrid-
ization, however, appears to be more sensitive than RT-PCR,
since only 13 of 16 samples positive by dot blot analysis were
able to be amplified. In addition, the dot blot method proved
to be highly specific, since none of the hybridization-negative
samples tested yielded a positive RT-PCR result. The reason
for RT-PCR’s inability to amplify all positive samples may
reside in the presence of inhibitors of RT-PCR in the fecal
sample-derived RNA.
The distribution of serotypes of astrovirus isolates in Mel-
bourne in 1995 (as determined from nucleotide sequence data)
showed a predominance of type 1 strains (85%). This is the
dominant serotype worldwide (12, 23). Nucleotide sequence
differences in a 348-bp region of the capsid gene permitted
subdivision of type 1 isolates into four subtypes which are likely
to be distinct genetic strains. In addition to the cocirculation of
genetically different type 1 strains, type 4 strains were simul-
taneously present. The genetic complexity of cocirculating
strains differs from the findings of Noel et al. (23), who dem-
onstrated that there was a clustering of strains with the same
sequence circulating in various locations worldwide. Clustering
of genetically identical strains was apparent, however, in
strains collected in Melbourne during 1982 to 1985 which ex-
hibited identical type 1 sequences. Whether this represents a
change in the genetic epidemiology of the virus over time or is
due to the restricted sample size examined in 1982 to 1985 is
unknown.
Of particular interest was the identification of the first Aus-
tralian strains belonging to the rare types 4 and 5. These
serotypes exhibited a prevalence of 10 and 2%, respectively, in
a survey which included samples from various locations world-
wide (23). A survey of samples collected over 10 years in the
Oxford region showed incidences of 6 and 5%, respectively
(12), while a 17-year survey in the same region indicated a low
incidence of these types (14). In contrast, type 4 had an inci-
dence of 15% of typeable isolates in the 1995 survey described
here, and the type 5 isolate was one of five randomly selected
samples from 1984.
The rate of temporal genetic variation observed in astrovirus
was similar to that seen in the VP7 capsid protein gene of
rotavirus isolates collected in Melbourne over a particular win-
ter epidemic season, i.e., ?1% (24). However, astroviruses
exhibited a greater rate of accumulation of mutations over
TABLE 1. Percentage identity in 348-bp region of ORF2 of
astrovirus clinical strains and HAstV types 1 to 7a
Strain (Yr [s])
% Identity with HAstV typeb
1234567
RCH1A (1995)
RCH1B (1995)
RCH1C (1995)
RCH1D (1995)
RCH1E (1980)
RCH1F (1982–1985)
RCH4A (1995)
RCH4B (1995)
RCH5 (1984)
90.5
90.5
91.1
91.2
96.8
96.5
77.9
77.9
77.3
77.6
77.0
78.2
78.2
79.3
79.3
79.6
79.9
73.3
77.0
77.0
77.6
78.2
79.6
79.9
79.0
78.2
78.2
77.0
77.0
78.2
78.2
78.2
78.7
96.0
96.6
78.2
78.4
78.4
79.6
79.6
78.4
78.7
77.3
77.3
95.1
83.9
83.3
84.5
84.5
84.5
84.8
80.5
81.3
76.7
79.0
79.0
80.2
80.2
79.6
79.9
79.9
80.5
81.3
aSequences of standard strains were obtained from the GenBank database
(accession numbers L06802, L23513, L38505, L38506, L38507, L38508, and
U15136).
bNumbers in boldface indicate greatest identity with the specified strain.
TABLE 2. Percentage identity in 348-bp region of ORF2 of type 1
astrovirus clinical strains
Strain (Yr [s])
% Identity with:
RCH1A RCH1B RCH1C RCH1D RCH1E RCH1F
RCH1A (1995)
RCH1B (1995)
RCH1C (1995)
RCH1D (1995)
RCH1E (1980)
RCH1F (1982–1985)
100
99.4
99.7
99.7
93.0
93.4
100
99.1
99.7
93.0
93.4
100
99.4
93.7
94.0
100
94.2
94.5
100
98.6 100
1752 PALOMBO AND BISHOPJ. CLIN. MICROBIOL.

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extended periods of time (10 years or more) than did rotavirus
(maximum of 7% sequence variation for astrovirus, maximum
of 3.4% for rotavirus) without any accompanying amino acid
substitutions. Similar temporal accumulation of mainly silent
mutations has been observed for the capsid protein genes of
foot-and-mouth disease virus over a 6-decade period (16). The
rate of accumulation of mutations in astrovirus also appears to
be greater than that recently determined for Ross River virus
(25), in which isolates collected over a 33-year period exhibited
a maximum of 4.9% sequence divergence. The region of the
astrovirus capsid gene investigated in this study is not known to
contain any immunoreactive epitopes of the virus (17), so no
conclusion about the degree of antigenic stability of the virus
can be reached. Therefore, further sequence information
about the astrovirus capsid gene is required before a detailed
analysis of mutation rates and antigenic variation can be made.
The limited survey of astrovirus isolates responsible for com-
munity outbreaks of gastroenteritis carried out here indicated
that the same strain caused sporadic cases resulting in hospi-
talization of children. Further analysis of outbreak isolates may
yield information about the extent of strain variation in the
total pediatric community of Melbourne.
In this report, we have determined that astrovirus has an
incidence of 4.2% in children admitted to the hospital for acute
gastroenteritis. This rate was second to that of rotavirus but
greater than those of enteric adenoviruses and common bac-
terial pathogens during 1995. However, this rate is probably an
underestimate of the incidence of astrovirus infection, since
not all rotavirus-negative fecal samples were tested because of
limited fecal material. In addition, simultaneous infection with
rotavirus and astrovirus is possible, and rotavirus-positive spec-
imens were excluded from analysis. Since a pathogen remains
unidentified in a large proportion of samples collected
(?20%), further investigation of possible etiologic agents, e.g.,
calicivirus, is warranted.
ACKNOWLEDGMENTS
We thank Paul Masendycz for identifying the rotavirus-negative
fecal samples used in this study, John Kurtz for standard human
astroviruses, and John Marshall for outbreak-associated astrovirus
specimens.
We are supported by the National Health and Medical Research
Council of Australia and the Royal Children’s Hospital Research
Foundation.
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