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Open Access Research

Identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified VIDISCA

Shahzad Shaukat12, Mehar Angez1, Muhammad Masroor Alam1, Maarten F Jebbink3, Martin Deijs3, Marta Canuti3, Salmaan Sharif1, Michel de Vries34, Adnan Khurshid1, Tariq Mahmood5, Lia van der Hoek3 and Syed Sohail Zahoor Zaidi1*

Author Affiliations

1 Department of Virology, National Institute of Health, Chak Shahzad, Park Road, Islamabad 45500, Pakistan

2 Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan

3 Department of Medical Microbiology, Laboratory of Experimental Virology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

4 Current address: CBS-KNAW Fungal Biodiversity Center, Utrecht, The Netherlands

5 Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan

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Virology Journal 2014, 11:146  doi:10.1186/1743-422X-11-146

Published: 12 August 2014

Abstract

Background

The use of sequence independent methods combined with next generation sequencing for identification purposes in clinical samples appears promising and exciting results have been achieved to understand unexplained infections. One sequence independent method, Virus Discovery based on cDNA Amplified Fragment Length Polymorphism (VIDISCA) is capable of identifying viruses that would have remained unidentified in standard diagnostics or cell cultures.

Methods

VIDISCA is normally combined with next generation sequencing, however, we set up a simplified VIDISCA which can be used in case next generation sequencing is not possible. Stool samples of 10 patients with unexplained acute flaccid paralysis showing cytopathic effect in rhabdomyosarcoma cells and/or mouse cells were used to test the efficiency of this method. To further characterize the viruses, VIDISCA-positive samples were amplified and sequenced with gene specific primers.

Results

Simplified VIDISCA detected seven viruses (70%) and the proportion of eukaryotic viral sequences from each sample ranged from 8.3 to 45.8%. Human enterovirus EV-B97, EV-B100, echovirus-9 and echovirus-21, human parechovirus type-3, human astrovirus probably a type-3/5 recombinant, and tetnovirus-1 were identified. Phylogenetic analysis based on the VP1 region demonstrated that the human enteroviruses are more divergent isolates circulating in the community.

Conclusion

Our data support that a simplified VIDISCA protocol can efficiently identify unrecognized viruses grown in cell culture with low cost, limited time without need of advanced technical expertise. Also complex data interpretation is avoided thus the method can be used as a powerful diagnostic tool in limited resources. Redesigning the routine diagnostics might lead to additional detection of previously undiagnosed viruses in clinical samples of patients.

Keywords:
VIDISCA; cDNA-AFLP; Enterovirus; Human parechovirus; Human astrovirus; Tetnovirus; Sequence independent method; Acute flaccid paralysis; Pakistan