Characterisation of a proposed Nucleorhabdovirus new to South Africa

Abstract

A previously uncharacterised plant rhabdovirus, infecting Bermuda grass (Cynodon dactylon) in the North West Province, South Africa, has been found. To determine the morphology and virion size of this virus, embedded ultra-thin sections of infected plant samples were observed under a transmission electron microscope. The virion distribution within the cell, its bullet-shaped morphology and its size (240 × 63 nm) indicated that this might be a rhabdovirus of the genus Nucleorhabdovirus. Degenerate polymerase chain reaction (PCR) primers were designed by alignment of the polymerase gene sequences of several plant rhabdoviruses in order to identify conserved regions. Standard PCR and sequencing protocols were used to determine a partial polymerase gene sequence of this virus sample which was then compared to the most closely related sequences available on Genbank. The analysis indicated that the virus was indeed most closely related to known nucleorhabdoviruses, with the highest nucleotide sequence similarities being to Maize mosaic virus and Taro vein chlorosis virus (70% and 69.7% respectively). Serological testing indicated that the South African Cynodon rhabdovirus had a close serological relationship with the nucleorhabdovirus Cynodon chlorotic streak virus. Abbreviations AMV Avian myoblastosis virus

Partial polymerase gene sequence, phylogeny and RT-PCR diagnostic assay for Datura yellow vein nucleorhabdovirus

“…These degenerate primers were also shown to amplify a corresponding L gene sequence from Lettuce necrotic yellows cytorhabdovirus, one of the viral sequences used in the alignment to design the primers. However, a drawback of using these primers is the short amplicon size, compared to published alternative L gene primers such as those devised by Lamprecht et al (2008Lamprecht et al ( , 2010) that yielded a~1.1 kb amplicon which was more suitable for diagnostic primer design, sequence comparisons and phylogenetic analysis.…”

“…They were designed to amplify conserved blocks I and III of the rhabdovirus L gene polymerase motif with an expected~900 bp amplicon (Lamprecht et al 2008(Lamprecht et al , 2010. Total RNA was reverse transcribed at 37°C for 60 min after incubation with the primers at 55°C for 5 min.…”

Aparición de un aislado estrechamente relacionado con el Maize yellow striate virus en plantas de trigo

“…Total RNA was purified using Nucleospin RNA plant kit (Macherey-Nagel). RT-PCR was performed using degenerate primers targeting a conserved region of plant rhabdovirus polymerase (L) gene (Lamprecht et al, 2009). Amplification products of about 900 bp were cloned into pCR4-TOPO vector (Invitrogen), sequenced and deposited in GenBank database.…”

Bioinformatic Analysis of the L polymerase Gene Leads to Discrimination of New Rhabdoviruses

“…2005). Primers targeting block I and block III of the L gene also have been used for identifying yet unknown plant rhabdovirus in Africa (Lamprecht et al. 2009, 2010).…”

“…However, exploitation of conserved amino acid sequence blocks of RNA-dependent RNA polymerase (L protein) for determining the phylogenetic relationships and identification of new viral species has been described for mammalian and insect rhabdoviruses (Bourhy et al 2005). Primers targeting block I and block III of the L gene also have been used for identifying yet unknown plant rhabdovirus in Africa (Lamprecht et al 2009(Lamprecht et al , 2010.…”