Biological, serological and molecular comparison between isolates of Cowpea severe mosaic virus

Abstract

Cowpea, Vigna unguiculata, can be affected by several diseases, and those caused by viruses are considered of great importance. Cowpea severe mosaic virus (CPSMV), which belongs to the family Comoviridae, genus Comovirus, stands out for its severity and degree of incidence. Its genetic variability can give rise to different strains around the world, including in Brazil. The objective of the present research was to establish a biological, serological and molecular comparison between five CPSMV isolates obtained from naturally infected plants in different States of the Northeast of Brazil. Isolates were obtained from cowpea: CPSMV-AL (State of Alagoas), CPSMV-CE (Ceará); CPSMV-MC (Piauí), CPSMV-PE (Pernambuco) and an isolate obtained from Crotalaria paulinea, in the State of Maranhão -CPSMV-CROT. A host range study evidenced biological differences among the virus isolates, especially in cowpea genotypes. The isolates were showed to be serologically and molecularly related by polymerase chain reaction �� PCR, using degenerated primers which amplified two conserved regions in the coat protein and in the replicase genes. Cloning and sequence of CPSMV-PE made possible its comparison with other CPSMV isolates and other virus species from the genus Comovirus. Keywords: Vigna unguiculata, Comovirus, CPSMV, molecular differentiation. RESUMO Comparação biológica, sorológica e molecular de isolados de Cowpea severe mosaic virusO feijão caupi, Vigna unguiculata, pode ser afetado por várias moléstias, entre as quais, destacam-se as viroses, cuja incidência e severidade variam, dependendo do hospedeiro, do vetor e da fonte do inóculo no campo. O vírus do mosaico severo do caupi (Cowpea severe mosaic virus, CPSMV) é o único vírus pertencente ao gênero Comovirus já identificado no Brasil infetando o feijão caupi. A presente pesquisa teve por objetivo estudar e comparar as características biológicas, sorológicas e moleculares de cinco isolados do CPSMV originários de diferentes estados do Nordeste: Isolados de feijão caupi em Alagoas (CPSMV-AL), no Ceará (CPSMV-CE); no Piauí (CPSMV-MC), em Pernambuco (CPSMV-PE) e o isolado obtido de Crotalaria paulinea no Maranhão (CPSMV-CROT). Os estudos de gama de hospedeiro evidenciaram diferenças biológicas entre os isolados, sobretudo em genótipos de caupi. As análise sorológicas em dupla difusão em agar confirmaram o forte relacionamento entre os isolados, e os estudos moleculares pela reação em cadeia da polimerase -PCR, utilizando oligonucleotídeos degenerados que amplificam seqüências conservadas dos genes do capsídeo e da replicase, não permitiram nenhuma diferenciação entre os isolados. A clonagem e o sequenciamento do CPSMV-PE possibilitaram sua comparação com outros isolados de CPSMV e outras espécies de vírus do gênero Comovirus.

Biological stability of a strain of Cowpea severe mosaic virus over 20 years

“…Despite the low variability of the CP gene among the isolates, there is a notable difference in their symptoms on cowpea genotypes (CAMARÇO et al, 2009), which indicates that, probably, other parts of the viral genome may be more variable, and the virus symptom determinants could be linked to these regions. 2.…”

“…Viral fragments corresponding to a portion of the CP gene were amplified by PCR using 3 µl of the cDNA, 5 µl of buffer, 5 µl of MgCl 2 25 mM, 1 µl from the mixture of desoxinucleotideos (0.01 M), 1 unit of Taq DNA polymerase and 25 pml of each one of the universal primers for the genus Comovirus: F, 5'-GCA TGG TCC ACW CAG GT-3' and R, 5'-YTC RAA WCC VYT RTT KGG MCC ACA-3' (CAMARÇO et al, 2009). PCR amplification was performed with an initial heating at 94 ºC for 5 min followed by 30 cycles of the program: denaturizing (94 ºC/1 min), annealing (41 ºC/2 min) and extension (72 ºC/1 min), followed by a final extension at 72 ºC for 5 min.…”

Sequence variability in the coat protein gene of Cowpea severe mosaic virus isolates from northeastern Brazil

“…The PR, MC and CE isolates presented the largest number of substitutions: four, three and three, respectively (data not shown). Despite the low variability of the CP gene among the CPSMV isolates, there is a notable difference in their symptomatology on cowpea varieties (Camarço et al, 2009). Therefore, while the analyzed region of CP genes is conserved, other parts of the CP genes or of the viral genome may be more variable, and the viral determinants of symptomatology could be linked to these more variable regions.…”

“…Currently, four CPSMV serotypes are recognized in Brazil, and these can also be differentiated by host range (Lin et al, 1984). However, few studies at the molecular level have been conducted in Brazil and in other countries (Chen & Bruening 1992;Brioso et al, 1996;Souto et al, 2002;Nogueira et al, 2003;Camarço et al, 2009). Nucleotide sequence information is available for only two CPSMV isolates: one complete genome sequence of an isolate (CPSMV-DG) obtained from cowpea in the USA (Chen & Bruening, 1992), and a partial sequence from a Brazilian isolate (CPSMV-PR) obtained from soybean (Glycine max L.) (Souto et al, 2002).…”

“…CPSMV isolates used in this study are part of the virus collection of the Plant Virus Laboratory of the Universidade Federal do Ceará (Camarço et al, 2009). The isolates were obtained from cowpea plants collected in different States of Northeastern Brazil: CPSMV-AL (Alagoas State), CPSMV-CE (Ceará State), CPSMV-MC (Piauí State), an isolate that infects cowpea cv.…”