Astrovirus MLB1 Is Not Associated with Diarrhea in a Cohort of Indian Children

Article (PDF Available)inPLoS ONE 6(12):e28647 · December 2011with25 Reads
DOI: 10.1371/journal.pone.0028647 · Source: PubMed
  • 26.32 · Washington University in St. Louis
  • 17.96 · Washington University in St. Louis
  • 19.06 · National Institute for Research in Tuberculosis
  • Washington University in St. Louis
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.
Astrovirus MLB1 Is Not Associated with Diarrhea in a
Cohort of Indian Children
Lori R. Holtz
, Irma K. Bauer
, Priya Rajendran
, Gagandeep Kang
, David Wang
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):
e28647. doi:10.1371/journal.pone.0028647
Editor: Leo L. M. Poon, University of Hong Kong, Hong Kong
Received October 25, 2011; Accepted November 11, 2011; Published December 9, 2011
Copyright: ß2011 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:
The first astrovirus infecting humans was described in 1975 [1].
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 [2]. These infections most commonly affect children, the
elderly, and the immunocompromised [3]. 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 [8], astrovirus
MLB2 (MLB2) [9], astrovirus VA1 (VA1) [10], astrovirus VA2
(VA2) [9], and astrovirus VA3 (VA3) [9]. 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 [22]. 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 [23]. To obtain paired control
samples, an asymptomatic surveillance stool sample collected at
least 6 weeks prior to the acute diarrhea sample was selected from
PLoS ONE | 1 December 2011 | Volume 6 | Issue 12 | e28647
the same child. The 400 paired samples were collected from 249
200 mLofa,20% fecal suspension were extracted using the
Boom method [24] 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 [9]. 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)] [25] and primers spe-
39)], both of which target the capsid region (ORF 2). PCR
amplicons were cloned into pCR4 (Invitrogen) and sequenced using
standard Sanger sequencing technology.
Phylogenetic Analysis
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
bootstrap replicates.
MLB1 consensus primers ( LG0189 59- AAGTGTGCA-
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 5610
copies 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 [26]. 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 1
diarrhea sample and 7 weeks before the 2
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
is possible.
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 [27]. 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 7610
and for the 14 positive control samples the average copy number/
ml of fecal suspension was 4610
. Using the Mann-Whitney test,
there was not a significant difference between cases and controls
(p = 0.51).
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 [28]. 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 [30]. 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 [31]. In addition, another astrovirus was
Astrovirus MLB1 Case Control Study
PLoS ONE | 2 December 2011 | Volume 6 | Issue 12 | e28647
recently found in the brain tissue of an immunocompromised
patient with encephalitis [32]. Further studies are still needed to
establish the role of MLB1 in human health and disease.
Author Contributions
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:
1. Madeley CR, Cosgrove BP (1975) Letter: 28 nm particles in faeces in infantile
gastroenteritis. Lancet 2: 451–452.
2. Midthun K, Greenberg HB, Kurtz JB, Gary GW, Lin FY, et al. (1993)
Characterization and seroepidemiology of a type 5 astrovirus associated with an
outbreak of gastroenteritis in Marin County, California. Journal of clinical
microbiology 31: 955–962.
3. Mendez E, Arias CF (2007) Astroviruses. In: Knipe D, Howley PM, eds. Fields
Virology. 5th ed. Philadelphia: Lippincott Williams &Wilkins. pp 981–1000.
4. Glass RI, Noel J, Mitchell D, Herrmann JE, Blacklow NR, et al. (1996) The
changing epidemiology of astrovirus-associated gastroenteritis: a review.
Archives of virology Supplementum 12: 287–300.
5. Klein EJ, Boster DR, Stapp JR, Wells JG, Qin X, et al. (2006) Diarrhea etiology
in a Children’s Hospital Emergency Department: a prospective cohort study.
Clinical infectious diseases : an official publication of the Infectious Diseases
Society of America 43: 807–813.
6. Nguyen TA, Hoang L, Pham le D, Hoang KT, Mizuguchi M, et al. (2008)
Identification of human astrovirus infections among children with acute
gastroenteritis in the Southern Part of Vietnam during 2005–2006. Journal of
medical virology 80: 298–305.
7. Soares CC, Maciel de Albuquerque MC, Maranhao AG, Rocha LN,
Ramirez ML, et al. (2008) Astrovirus detection in sporadic cases of diarrhea
among hospitalized and non-hospitalized children in Rio De Janeiro, Brazil,
from 1998 to 2004. Journal of medical virology 80: 113–117.
8. Finkbeiner SR, Allred AF, Tarr PI, Klein EJ, Kirkwood CD, et al. (2008)
Metagenomic analysis of human diarrhea: viral detection and discovery. PLoS
pathogens 4: e1000011.
9. Finkbeiner SR, Holtz LR, Jiang Y, Rajendran P, Franz CJ, et al. (2009) Human
stool contains a previously unrecognized diversity of novel astroviruses. Virology
journal 6: 161.
10. Finkbeiner SR, Li Y, Ruone S, Conrardy C, Gregoricus N, et al. (2009)
Identification of a novel astrovirus (astrovirus VA1) associated with an outbreak
of acute gastroenteritis. Journal of virology 83: 10836–10839.
11. Ahmed S, Sebeny PJ, Klena JD, Pimentel G, Mansour A, et al. Novel
Astroviruses in Children, Egypt. In press.
12. Banyai K, Meleg E, Moschidou P, Martella V (2010) Detection of newly
described astrovirus MLB1 in stool samples from children. Emerging infectious
diseases 16: 169; author reply 169–170.
13. Chu DK, Chin AW, Smith GJ, Chan KH, Guan Y, et al. (2010) Detection of
novel astroviruses in urban brown rats and previously known astroviruses in
humans. The Journal of general virology 91: 2457–2462.
14. Finkbeiner SR, Le BM, Holtz LR, Storch GA, Wang D (2009) Detection of
newly described astrovirus MLB1 in stool samples from children. Emerging
infectious diseases 15: 441–444.
15. Kapoor A, Li L, Victoria J, Oderinde B, Mason C, et al. (2009) Multiple novel
astrovirus species in human stool. The Journal of general virol ogy 90:
16. Kapikian AZ, Wyatt RG, Levine MM, Yolken RH, VanKirk DH, et al. (1983)
Oral administration of human rotavirus to volunteers: induction of illness and
correlates of resistance. J Infect Dis 147: 95–106.
17. Thornhill TS, Kalica AR, Wyatt RG, Kapikian AZ, Chanock RM (1975)
Pattern of shedding of the Norwalk particle in stools during experimentally
induced gastroenteritis in volunteers as determined by immune electron
microscopy. J Infect Dis 132: 28–34.
18. Atmar RL, Opekun AR, Gilger MA, Estes MK, Crawford SE, et al. (2008)
Norwalk virus shedding after experimental human infection. Emerging
infectious diseases 14: 1553–1557.
19. Gladstone BP, Das AR, Rehman AM, Jaffar S, Estes MK, et al. (2010) Burden of
illness in the first 3 years of life in an Indian slum. Journal of tropical pediatrics
56: 221–226.
20. Gladstone BP, Muliyil JP, Jaffar S, Wheeler JG, Le Fevre A, et al. (2008) Infant
morbidity in an Indian slum birth cohort. Archives of disease in childhood 93:
21. Gladstone BP, Ramani S, Mukhopadhya I, Muliyil J, Sarkar R, et al. (2011)
Protective effect of natural rotavirus infection in an Indian birth cohort. The
New England journal of medicine 365: 337–346.
22. Ruuska T, Vesikari T (1990) Rotavirus disease in Finnish children: use of
numerical scores for clinical severity of diarrhoeal episodes. Scandinavian
journal of infectious diseases 22: 259–267.
23. Ajjampur SS, Rajendran P, Ramani S, Banerjee I, Monica B, et al. (2008)
Closing the diarrhoea diagnostic gap in Indian children by the application of
molecular techniques. Journal of medical microbiology 57: 1364–1368.
24. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, et al.
(1990) Rapid and simple method for purification of nucleic acids. Journal of
clinical microbiology 28: 495–503.
25. Noel JS, Lee TW, Kurtz JB, Glass RI, Monroe SS (1995) Typing of human
astroviruses from clinical isolates by enzyme immunoassay and nucleotide
sequencing. Journal of clinical microbiology 33: 797–801.
26. Dennehy PH, Nelson SM, Spangenberger S, Noel JS, Monroe SS, et al. (2001) A
prospective case-control study of the role of astrovirus in acute diarrhea among
hospitalized young children. The Journal of infectious diseases 184: 10–15.
27. Phillips G, Lopman B, Tam CC, Iturriza-Gomara M, Brown D, et al. (2009)
Diagnosing norovirus-associated infectious intestinal disease using viral load.
BMC infectious diseases 9: 63.
28. Bodhidatta L, McDaniel P, Sornsakrin S, Srijan A, Serichantalergs O, et al.
(2010) Case-control study of diarrheal disease etiology in a remote rural area in
Western Thailand. The American journal of tropical medicine and hygiene 83:
29. Albert MJ, Faruque AS, Faruque SM, Sack RB, Mahalanabis D (1999) Case-
control study of enteropathogens associated with childhood diarrhea in Dhaka,
Bangladesh. Journal of clinical microbiology 37: 3458–3464.
30. Hien BT, Trang do T, Scheutz F, Cam PD, Molbak K, et al. (2007)
Diarrhoeagenic Escherichia coli and other causes of childhood diarrhoea: a case-
control study in children living in a wastewater-use area in Hanoi, Vietnam.
Journal of medical microbiology 56: 1086–1096.
31. Holtz LR, Wylie KM, Sodergren E, Jiang Y, Franz CJ, et al. (2011) Astrovirus
MLB2 Viremia in a Febrile Child. Emerging infectious diseases.
32. Quan PL, Wagner TA, Briese T, Torgerson TR, Hornig M, et al. (2010)
Astrovirus encephalitis in boy with X-linked agammaglobulinemia. Emerging
infectious diseases 16: 918–925.
Astrovirus MLB1 Case Control Study
PLoS ONE | 3 December 2011 | Volume 6 | Issue 12 | e28647
    • "The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster [55,57,7475767778. For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found555657. In addition an antibody response was observed against some but not all novel astrovirus types [54,58]. "
    [Show abstract] [Hide abstract] ABSTRACT: The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease.
    Full-text · Article · Feb 2016
    • "A study of children in India failed to associate MLB1 with diarrhea [97]. Meanwhile, MLB2 was also initially detected in fecal samples from Indian children with diarrhea [98] and in the plasma of a child with undifferentiated fever [97], suggesting replication beyond the digestive tract. Other astroviruses in human and animal tissues have also indicated likely neurological involvement99100101, therefore a wide range of diseases may be associated with astrovirus infections. "
    [Show abstract] [Hide abstract] ABSTRACT: Hand, foot, and mouth disease (HFMD) affects infant and young children. A viral metagenomic approach was used to identify the eukaryotic viruses in fecal samples from 29 Thai children with clinical diagnosis of HFMD collected during the 2012 outbreak. These children had previously tested negative by PCR for enterovirus 71 and coxsackievirus A16 and A6. Deep sequencing revealed nine virus families: Picornaviridae, Astroviridae, Parvoviridae, Caliciviridae, Paramyxoviridae, Adenoviridae, Reoviridae, Picobirnaviridae, and Polyomaviridae. The highest number of viral sequences belonged to human rhinovirus C, astrovirus-MLB2, and coxsackievirus A21. Our study provides an overview of virus community and highlights a broad diversity of viruses found in feces from children with HFMD.
    Full-text · Article · Aug 2015
    • "This strategy allowed the identification of AstV-MLB1 in two patients. The unusual AstV-MLB1 strain has been identified in fecal samples of patients in Australia and in countries in North America, Asia, Africa and Europe [9, 10,42434445. Temporal and geographical relationships were revealed following phylogenetic ORF2 nucleotide analyzes homology among AstV-MLB1 strains (Fig 4). "
    [Show abstract] [Hide abstract] ABSTRACT: Human astrovirus (HAstV) represents the third most common virus associated with acute diarrhea (AD). This study aimed to estimate the prevalence of HAstV infection in Brazilian children under 5 years of age with AD, investigate the presence of recently described HAstV strains, through extensive laboratory-based surveillance of enteric viral agents in three Brazilian coastal regions between 2005 and 2011. Using reverse transcription-poly-merase chain reaction (RT-PCR), the overall HAstV detection rate reached 7.1% (207/ 2.913) with percentage varying according to the geographic region: 3.9% (36/921) in the northeast, 7.9% in the south (71/903) and 9.2% in the southeast (100/1.089) (p < 0.001). HAstV were detected in cases of all age groups. Detection rates were slightly higher during the spring. Nucleotide sequence analysis of a 320-bp ORF2 fragment revealed that HAstV-1 was the predominant genotype throughout the seven years of the study. The novel AstV-MLB1 was detected in two children with AD from a subset of 200 samples tested, demonstrating the circulation of this virus both the in northeastern and southeastern regions of Brazil. These results provide additional epidemiological and molecular data on HAstV circulation in three Brazilian coastal regions, highlighting its potential to cause infantile AD.
    Full-text · Article · Aug 2015
Show more