An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea

Rao, Durga C and Reddy, Harikrishna and Naidu, Jagadish R and Raghavendra, A and Radhika, NS and Karande, Anjali (2015) An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea. In: JOURNAL OF VIROLOGICAL METHODS, 224 . pp. 47-52.

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Abstract

We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18-21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4-7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea. The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12 h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9-12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease. (C) 2015 Elsevier B.V. All rights reserved.

Item Type:	Journal Article
Publication:	JOURNAL OF VIROLOGICAL METHODS
Publisher:	ELSEVIER SCIENCE BV
Additional Information:	Copy right for this article belongs to the ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS
Keywords:	Enterovirus; Echovirus; Coxsackievirus; Acute diarrhea; Newborn mouse model; Koch's postulates
Department/Centre:	Division of Biological Sciences > Biochemistry Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	30 Oct 2015 05:52
Last Modified:	30 Oct 2015 05:52
URI:	http://eprints.iisc.ac.in/id/eprint/52577

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