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J Med Microbiol 58 (2009), 303-311; DOI: 10.1099/jmm.0.005124-0
© 2009 Society for General Microbiology
ISSN 0022-2615

Assignment of the group A rotavirus NSP4 gene into genotypes using a hemi-nested multiplex PCR assay: a rapid and reproducible assay for strain surveillance studies

Krisztián Bányai1,2, Ágnes Bogdán1, György Szücs1, Serenella Arista3, Simona De Grazia3, Gagandeep Kang4, Indrani Banerjee4, Miren Iturriza-Gómara5, Canio Buonavoglia6 and Vito Martella6

1 Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Pécs, Hungary

2 Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary

3 Department of Hygiene and Microbiology, University of Palermo, Palermo, Italy

4 Department of Gastrointestinal Sciences, Christian Medical College and Hospital, Vellore, India

5 Enteric Virus Unit, Enteric, Respiratory and Neurological Virus Laboratory, Health Protection Agency, London, UK

6 Department of Public Health and Zootecnic, Faculty of Veterinary Medicine, University of Bari, Bari, Italy

Correspondence
Krisztián Bányai
bkrota{at}hotmail.com

Received July 16, 2008
Accepted November 12, 2008

The rotavirus non-structural protein NSP4 has been implicated in a number of biological functions during the rotavirus cellular cycle and pathogenesis, and has been addressed as a target for vaccine development. The NSP4 gene has been classified into six genotypes (A–F). A semi-nested triplex PCR was developed for genotyping the major human NSP4 genotypes (A–C), which are common in human rotavirus strains but are also shared among most mammalian rotavirus strains. A total of 192 previously characterized human strains representing numerous G and P type specificities (such as G1P[8], G1P[4], G2P[4], G3P[3], G3P[8], G3P[9], G4P[6], G4P[8], G6P[4], G6P[9], G6P[14], G8P[10], G8P[14], G9P[8], G9P[11], G10P[11], G12P[6] and G12P[8]) were tested for NSP4 specificity by the collaborating laboratories. An additional 35 animal strains, including the reference laboratory strains SA11 (simian, G3P[2]), NCDV (bovine, G6P[1]), K9 and CU-1 (canine, G3P[3]), together with 31 field isolates (canine, G3P[3]; feline, G3P[9]; porcine, G2P[23], G3P[6], G4P[6], G5P[6], G5P[7], G5P[26], G5P[27], G9P[6] and G9P[7]) were also successfully NSP4-typed. Four human G3P[9] strains and one feline G3P[9] strain were found to possess an NSP4 A genotype, instead of NSP4 C, suggesting a reassortment event between heterologous strains. Routine NSP4 genotyping may help to determine the genomic constellation of rotaviruses of man and livestock, and identify interspecies transmission of heterologous strains.

Abbreviations: E-type, electropherotype; GG, genogroup; SG, subgroup.

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are DQ823416, DQ186199, DQ189230–DQ189240, EU872160–EU872171, EU076914–EU076916, EF442741–EF442743, EF534716, EF554113, EF554146, AM992601, AM992604, AM992606 and AM992607.

The 75 NSP4 human and animal rotavirus sequences used to design the oligonucleotide primers are available with the online version of this paper.






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