DETECTION AND GENETIC TYPING OF ROTAVIRUS IN SOME AVIAN SPECIES FROM SOUTHWESTERN NIGERIA

Abstract

Rotaviruses are leading causes of acute viral gastroenteritis in humans and animals worldwide. The disease causes substantial economic loss to the poultry industry due to decreased weight gain, increased production costs and mortality. Despite its broad distribution among humans and animals, avian rotaviruses have not been detected in Nigeria. This study was aimed at designing primers for avian rotavirus detection, characterising gene segments critical for vaccine development and determining genetic relatedness of detected rotavirus strains. A total of 464 faecal samples from chickens (360), crowned cranes (9), ducks (10), eagles (2), geese (7), guinea fowls (33), parrots (3), pigeons (27) and turkeys (13) from some poultry farms were examined between June 2009 and March 2013. Degenerate primers and one step reverse transcription–polymerase chain reaction protocol were designed for detection of VP4, VP6 and VP7 segments. Four primer pairs were designed for VP4 segment and two pairs each for VP6 and VP7 segments. In addition, two previously published primer pairs were used for detection of VP6 and NSP4 segments for a complete coding sequence of each segment. The segments were characterised by nucleotide (nt) and amino acid (aa) sequence analysis using BioEdit® sequence alignment editor and the online rotavirus classification tool (RotaC®). Phylogenetic analysis was conducted using MEGA version 5. Genes encoding VP6, VP7 and NSP4 segments were detected in 129 faecal samples of chickens (124), crowned cranes (2), eagle (1), guinea fowl (1) and parrot (1). The designed primers had specific bands and were efficient for detecting rotavirus in all positive faecal samples than previously published primers due to the inclusion of degenerate nucleotides. All characterised strains possessed VP6, VP7 and NSP4 sequences typical of rotavirus A. Only 3 partial VP4 sequences were characterised from the positive samples. These VP4 sequences had low similarity between 68% and 72% to sequences of other known rotaviruses except Ch-06V0661, which has an encoding sequence of unknown host origin. The VP4 segments of other strains in this study were untypeable. The basic structure of the VP6 encoding gene was 1348 nt long with open reading frame beginning at nt 24, terminated at nt 1214 and coded for 397 aa. This segment also had 88.1% to 95.4% similarity with genotype I11 strains. However, reassortment of chicken VP6 segment occurred in the conserved aa sequence at antigenic site I (aa 45–65) with a change from alanine to threonine at aa 60. The VP7 and NSP4 genes showed aa identities between 89.5% to 96.7% and 91.8% to 93.3% with strains belonging to genotype G19 and E10 respectively. These rotaviruses belong to group A, genotypes I11, G19 and E10 with an untypeable VP4 segment. All characterised segments clustered closely with avian strains except the VP4 segment indicating reassortment and portend a public health hazard based on its zoonotic ability. The reassortment in conserved antigenic site also provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses. This appears to be the first report of avian rotavirus detection in Nigeria. Keywords: Avian rotavirus, Untypeable VP4, Genetic reassortment. Word count: 497