A carlavirus-specific PCR primer and partial nucleotide sequence provides further evidence for the recognition of cowpea mild mottle virus as a whitefly-transmitted carlavirus

Abstract

Cowpea mild mottle virus (CMMV) has physicochemical properties typical of carlaviruses, but has remained unclassified due to a number of unusual properties, including no serological cross-reaction with l 8 carlaviruses; production of brush-like inclusion bodies in vivo; and the ability to be transmitted by whiteflies (Bermisia tabaci).In this paper we report the use of a carlavirus specific PCR primer to identify CMMV as a member of the carlavirus group. This is confirmed by nucleotide sequence (958 nucleotides) from the 3' terminal region of CMMV RNA which contains a partial open reading frame (ORF) having high similarity with the coat proteins of other carlaviruses. The sequence also contains an 11.7K ORF at the 3' terminus, containing a 'zinc-finger' motif which is unique to carlaviruses.Cowpea mild mottle virus (CMMV) has physicochemical properties which resemble those of members of the carlavirus group, namely the presence of filamentous particles c. 650 nm in length, consisting of a coat protein of 31-33K and a single-stranded RNA of Mr 2.5 x 10 6 [Jeyanandarajah and Brunt, 1993]. However, unlike other members of the carlavirus group which are transmitted non-persistently by aphids [Foster, 1992], CMMV is unusual in that it is transmitted in a non-persistent manner by whiteflies (Bemisia tabaci) [Jeyanandarajah and Brunt 1993]. In addition, CMMV has several other unusual properties compared with other carlaviruses, which include, no serological relationships with 18 recognised members of the carlavirus group, and the induction of unusual brush-like intracellular inclusions in infected plants [Brunt et al., 1983]. It has been suggested that CMMV remain unclassified until the taxonomic significance of these differences have been investigated further [Jeyanandarajah and Brunt, 1993].We report here attempts to develop a rapid and specific identification test for carlaviruses which can also be used to determine whether CMMV and similar unclassified viruses are species of the carlavirus genus. The test is based on the polymerase chain reaction (PCR) using primers to conserved and unique sequences in carlavirus RNAs.The overall genome organisation of carlaviruses is very similar to that of potexviruses with the virus genes of both genera being arranged (from 5' to 3') as replicase, a triple gene block (25K, 12K, and 7K), and coat protein open reading frame, with extensive similarity at the amino acid level between the equivalent proteins of potex-and carlaviruses. However, in addition to these proteins, carlavirus genomes contain an additional protein encoded by an open reading frame

First Report of Cowpea Mild Mottle Carlavirus on Yardlong Bean (Vigna unguiculata subsp. sesquipedalis) in Venezuela

“…The semi-flexuous-shaped particles observed in symptomatic yardlong bean plants suggested that the isolated virus was a carlavirus. Moreover, the host range, symptomatology induced in test plants, and transmission via seeds and by whiteflies, all agreed well with what has been reported for CPMMV [1,11,18,22], but the amplification of a ca. 0.9 bp fragment using carlavirus universal primers [13,21] was the most convincing argument for concluding that the virus isolated from yardlong bean is a carlavirus (Figure 3).…”

“…The virus was transmitted by whiteflies to these host species with an average efficiency of 56% and 52%, respectively. The symptoms induced following mechanical and insect transmission tests to host plants resembling those described for Cowpea mild mottle virus (CPMMV), a leguminous seed and whitefly-borne virus [11,18]. …”

Occurrence and incidence of viruses infecting green beans in south-eastern Spain

“…Primers used in this work are described in Table 4. RT-PCR amplifications of cucumovirus, potyviruses, carlaviruses, luteovirures, SBMV and BnYDV, as well as PCR amplifications of TYLCV and geminiviruses, were conducted as previously described (Robertson et al 1991;Badge et al 1996;Wyatt and Brown 1996;Gibbs and Mackenzie 1997;Choi et al 1999;Accotto et al 2000;Verhoeven et al 2003;Segundo et al 2004a). Primers for detecting TSWV and PVuV were designed based on sequences retrieved from GenBank.…”

3'-Terminal sequences of the RNA genomes of narcissus latent and Maclura mosaic viruses suggest that they represent a new genus of the Potyviridae.

“…It was concluded that NLV was neither a potyvirus nor a carlavirus but could be a member of a new genus with MacMV (Mowat et al, 1991). Moreover, a PCR primer designed to a sequence present in 80 % of all carlaviruses sequenced to date failed to produce a product with NLV or MacMV cDNA (Badge et al, 1996).…”