Astrovirus MLB1 Is Not Associated with Diarrhea in a
Cohort of Indian Children
Lori R. Holtz1, Irma K. Bauer2, Priya Rajendran3, Gagandeep Kang3, David Wang2*
1Washington University School of Medicine, Department of Pediatrics, St. Louis, Missouri, United States of America, 2Washington University School of Medicine,
Departments of Molecular Microbiology and Pathology and Immunology, St. Louis, Missouri, United States of America, 3Christian Medical College, Department of
Gastrointestinal Sciences, Vellore, India
Astroviruses are a known cause of human diarrhea. Recently the highly divergent astrovirus MLB1 (MLB1) was identified in a
stool sample from a patient with diarrhea. It has subsequently been detected in stool from individuals with and without
diarrhea. To determine whether MLB1 is associated with diarrhea, we conducted a case control study of MLB1. In parallel,
the prevalence of the classic human astroviruses (HAstVs) was also determined in the same case control cohort. 400 cases
and 400 paired controls from a longitudinal birth cohort in Vellore, India were analyzed by RT-PCR. While HAstVs were
associated with diarrhea (p=0.029) in this cohort, MLB1 was not; 14 of the controls and 4 cases were positive for MLB1.
Furthermore, MLB1 viral load did not differ significantly between the cases and controls. The role of MLB1 in human health
still remains unknown and future studies are needed.
Citation: Holtz LR, Bauer IK, Rajendran P, Kang G, Wang D (2011) Astrovirus MLB1 Is Not Associated with Diarrhea in a Cohort of Indian Children. PLoS ONE 6(12):
Editor: Leo L. M. Poon, University of Hong Kong, Hong Kong
Received October 25, 2011; Accepted November 11, 2011; Published December 9, 2011
Copyright: ? 2011 Holtz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by National Institutes of Health (NIH)/National Center for Research Resources Washington University-ICTS grant
number KL2 RR024994 and by NIH grant R21 AI090199. DW holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome
Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The first astrovirus infecting humans was described in 1975 .
Since then, a total of 8 serotypes closely related to this original
astrovirus (‘‘classic human astroviruses’’ (HAstVs)) have been
identified, all of which are believed to cause diarrhea. Diarrhea
symptoms typically last 2–4 days following a 3–4 day incubation
period . These infections most commonly affect children, the
elderly, and the immunocompromised . HAstVs account for up
to ,10% of sporadic cases of non-bacterial diarrhea in children
[4,5,6,7]. Since 2008, five highly divergent astroviruses have been
discovered in human diarrhea specimens: MLB1 , astrovirus
MLB2 (MLB2) , astrovirus VA1 (VA1) , astrovirus VA2
(VA2) , and astrovirus VA3 (VA3) . Of these viruses, MLB1
has been detected at the highest frequency. MLB1 was first
identified in the stool of child with unexplained diarrhea.
Subsequently, it has been found in stools collected from around
the world [9,11,12,13,14,15] from patients with and without
The finding of the novel astrovirus MLB1 in stool specimens
from patients with diarrhea raises the question of whether or not,
like all other human astroviruses, it also causes diarrhea.
Historically, proof of causality for diarrheagenic viruses relied
upon ingestion of fecal filtrates by human volunteers [2,16,
17,18]. Such studies are, however, no longer feasible for novel
viruses of unknown pathogenicity. Thus, assigning pathogenicity
to a newly identified virus requires indirect approaches such
as infection of surrogate animal models or epidemiologic analyses
of human populations. Here we describe a case control study
aimed at determining whether or not MLB1 is associated with
Materials and Methods
Description of Samples
The institutional review boards of Christian Medical College,
Vellore, India and Washington University School of Medicine, St.
Louis, USA approved this study. 400 case and 400 control stool
samples were selected from a previously described longitudinal
birth cohort in Vellore, India [19,20,21]. Children were followed
for 3 years with twice-weekly home visits and collection of stool
every two weeks and during every diarrheal episode. 373 children
completed the three year study. A total of 1955 diarrhea and
,27,000 control stools were collected from 2002–2006. The
severity of each diarrheal episode was recorded using the 20 point
Vesikari scale developed for rotaviral gastroenteritis, which
includes number and duration of diarrhea and vomiting episodes,
presence of fever and dehydration and classifies gastroenteritis as
mild, moderate, severe and very severe . 400 stool samples
from acute diarrhea episodes that were negative for rotavirus by
enzyme immunoassay and PCR, for norovirus by PCR, for
bacterial pathogens (Vibrio cholerae, enteropathogenic Escherichia coli,
Salmonella, Shigella, Aeromonas and Plesiomonas) by culture, biochem-
ical reactions and serogrouping where appropriate and for
parasites by routine saline and iodine preparations and modified
acid fast stain were chosen as cases . To obtain paired control
samples, an asymptomatic surveillance stool sample collected at
least 6 weeks prior to the acute diarrhea sample was selected from
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the same child. The 400 paired samples were collected from 249
200 mL of a ,20% fecal suspension were extracted using the
Boom method  and the extracted total nucleic acid was eluted
into 40 mL of water. As previously described, a two stage screening
strategy was used to detect astroviruses . In brief, astrovirus
consensus primers SF0073 (59-GATTGGACTCGATTTGAT-
GG-39) and SF0076 (59-CTGGCTTAACCCACATTCC-39),
designed to the RNA polymerase (ORF 1b) were used to screen
all samples in the first stage. Samples that were positive in the first
phase of screening were then subjected to additional RT-PCR
screenings with primers specific for classic human astroviruses
[Mon269 (59-CAACTCAGGAAACAGGGTGT-39) and Mon270
(59-TCAGATGCATTGTCATTGGT-39)]  and primers spe-
cific for MLB1 [SF0053 (59-CTGTAGCTCGTGTTAGTCT-
TAACA-39) and SF0061 (59-GTTCATTGGCACCATCAGAAC-
39)], both of which target the capsid region (ORF 2). PCR
amplicons were cloned into pCR4 (Invitrogen) and sequenced using
standard Sanger sequencing technology.
Sequences of the capsid region amplicons from HAstV and
MLB1 positive samples were aligned using ClustalX1.83. PAUP
was then used to generate maximum parsimony trees with 1,000
MLB1 consensus primers ( LG0189 59- AAGTGTGCA-
TATGTTGGGACC -39 and LG0190 59- CTACACCTCTC-
CAATTCATG -93) targeting a 131 nt segment of the ORF1a
region of the MLB1 genome were designed by alignment of the 4
MLB1 sequences present in GenBank (NC_011400.1, FJ402983.1,
HM450380.1, and HM989952.1) containing the region of interest
as of 12/23/2010; these primers were used in a SYBR green qRT-
PCR assay. QRT-PCR was performed using qScript One-Step kit
(Quanta) as follows: 50uC for 10 min, 95uC for 5 min, 45 cycles of
95uC for 10 sec and 60uC for 30 sec followed by a melt curve. To
establish a standard curve for this assay, in-vitro transcribed RNA
was generated from a plasmid containing nt 1 to 846 of MLB1
(GenBank NC_011400.1) using MAXIscript (Ambion) per man-
ufacturer’s protocol. Serial dilutions of this in-vitro transcribed
RNA from 56106copies to 5 copies were used for the standard
RT-PCR case control study
For HAstVs, 14 of the 400 cases were positive and 4 of the 400
controls were positive. By contrast, for MLB1 only four of the
cases were positive while 14 of the controls were positive. To take
into account the fact that some participants were sampled more
than once, logistic regression models were fit using generalized
estimating equations. HAstVs were more likely to be present in the
diarrheal samples than in the asymptomatic samples (OR 3.59,
95% CI 1.14–11.31, p=0.029), consistent with results of previous
studies . By contrast MLB1 was less likely to be present in the
diarrheal samples than in the asymptomatic samples (OR 0.28,
95% CI 0.09–0.89, p=0.033).
All RT-PCR positive samples were cloned and sequenced
(GenBank JN871233–JN871268). Amplicons from the capsid
region were chosen for sequencing, as this is the least conserved
region of the genome and might reveal the most divergence. The
MLB1 capsid amplicons shared 96–99% nucleotide identity to
each other while the HAstV capsid amplicons shared 77–100%
nucleotide identity to each other. Phylogenetic analysis of the
HAstV and MLB1 amplicons obtained in the capsid regions
showed no clustering of the cases vs controls (data not shown). Five
samples (4 controls and 1 case) were positive for both HAstV and
Of the 249 children studied 107 were sampled at multiple time
points. One subject had two diarrhea samples positive for HAstVs
separated by 14 months. The intervening control stool, collected
12 months after the 1stdiarrhea sample and 7 weeks before the 2nd
diarrhea sample, from this subject was negative for HAstVs. The
capsid-derived amplicons from these two diarrhea samples shared
78% identity, suggesting that this subject was infected by two
different HAstV serotypes at the two time points. Two subjects
had two asymptomatic stools positive for MLB1 with an
intervening diarrhea sample that was negative for MLB1. For
one subject these asymptomatic samples were separated by 18
months. The capsid-derived amplicons from these two samples
shared 98% identity. For the other subject, these asymptomatic
samples were separated by 8 months and shared 99% identity.
One interpretation of this observation is that reinfection by MLB1
qRT-PCR of positive samples
Samples positive for MLB1 by RT-PCR were then subjected to
qRT-PCR to establish if viral load differed between cases and
controls as has been described for norovirus . There was no
significant difference in the RNA copy number/ml of fecal
suspension between cases and controls. Specifically, for the 4 cases
the average RNA copy number/ml fecal suspension was 76103
and for the 14 positive control samples the average copy number/
ml of fecal suspension was 46104. Using the Mann-Whitney test,
there was not a significant difference between cases and controls
In this study, we performed a case control study of MLB1 and
HAstVs. We demonstrated in this cohort that HAstVs are
associated with diarrhea. By contrast, MLB1 was not associated
with diarrhea. These results suggest that MLB1 may not play an
etiologic role in human diarrhea. However, a single case control
study is not sufficient to definitively answer this question as
evidenced by the fact that there are examples from the literature of
bona fide diarrhea pathogens for which case control studies did
not yield positive associations. For example, a case control study of
Campylobacter found that it was present in 22% of cases vs. 25% of
controls . Furthermore, Giardia lamblia was found more
commonly in controls than in cases in two studies [28,29]. A case
control study from Vietnam found diarrheagenic E.coli in 23% of
cases and 23% of controls . These studies highlight the
challenges of determining association of a microbe with a given
disease strictly by a case control study. The role of MLB1 in
human health remains uncertain. It remains possible that MLB1 is
an agent of diarrhea. One formal possibility is that it is present in
stool simply as a result of dietary ingestion and it has no
pathogenic role. Another possibility is that MLB1’s pathogenic
effects are outside of the enteric system and that its detection in
stool simply reflects its mode of transmission, much like poliovirus.
In support of this possibility, we have recently described a febrile
child with MLB2 viremia, demonstrating that astroviruses can
access the circulatory system and thus could have broader tropism
outside the GI tract . In addition, another astrovirus was
Astrovirus MLB1 Case Control Study
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recently found in the brain tissue of an immunocompromised
patient with encephalitis . Further studies are still needed to
establish the role of MLB1 in human health and disease.
Conceived and designed the experiments: DW GK LRH. Performed the
experiments: LRH IKB PR. Analyzed the data: LRH IKB DW.
Contributed reagents/materials/analysis tools: GK. Wrote the paper:
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