title: Molecular epidemiology and a loop-mediated isothermal amplification method for diagnosis of infection with rabies virus in Zambia.
abstract: The National Livestock Epidemiology and Information Center (NALEIC) in Zambia reported over 132 cases of canine rabies diagnosed by the direct fluorescent antibody test (DFAT) from 2004 to 2009. In this study, the lineage of rabies virus (RABV) in Zambia was determined by phylogenetic analysis of the nucleoprotein (N) and glycoprotein (G) gene sequences. Total RNA was extracted from 87-DFAT brain specimens out of which only 35 (40%) were positive on nested reverse transcription polymerase chain reaction (RT-PCR) for each gene, and 26 being positive for both genes. Positive specimens for the N (n=33) and G (n=35) genes were used for phylogenetic analysis. Phylogenetic analysis of the N gene showed two phylogenetic clusters in Zambia belonging to the Africa 1b lineage present in eastern and southern Africa. While one cluster exclusively comprised Zambian strains, the other was more heterogeneous regarding the RABV origins and included strains from Tanzania, Mozambique and Zambia. Phylogenetic analysis of the G gene revealed similar RABV strains in different hosts and regions of Zambia. We designed primers for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from the consensus sequence of the N gene in an attempt to improve the molecular diagnosis of RABV in Zambia. The specificity and reproducibility of the RT-LAMP assay was confirmed with actual clinical specimens. Therefore, the RT-LAMP assay presented in this study may prove to be useful for routine diagnosis of rabies in Zambia.
authors.affiliation: Department of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Walter
authors.initials: W
authors.lastname: Muleya
authors.forename: Boniface
authors.initials: B
authors.lastname: Namangala
authors.forename: Aaron
authors.initials: A
authors.lastname: Mweene
authors.forename: Luke
authors.initials: L
authors.lastname: Zulu
authors.forename: Paul
authors.initials: P
authors.lastname: Fandamu
authors.forename: Douglas
authors.initials: D
authors.lastname: Banda
authors.forename: Takashi
authors.initials: T
authors.lastname: Kimura
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Sawa
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ishii
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D004283:Dog Diseases / Q000175:diagnosis* / Q000453:epidemiology*; D004285:Dogs; D005838:Genotype; D006023:Glycoproteins / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D017720:Molecular Epidemiology; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019590:Nucleocapsid Proteins / Q000235:genetics; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D011818:Rabies / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary*; D011820:Rabies virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014759:Viral Envelope Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*; D015024:Zambia / Q000453:epidemiology
chemical_list: D000956:Antigens, Viral; D017931:DNA Primers; D006023:Glycoproteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus; C099601:nucleocapsid protein, Rabies virus