Purification of carrot red leaf virus and evidence from four serological tests for its relationship to luteoviruses

Abstract

SUMMARYCarrot red leaf virus (CRLV) was purified from infected chervil by centrifuging whole plant extracts at low speed and incubating the resuspended pellets with Driselase; the digest was then treated with 1% (v/v) Triton X‐100 and the virus concentrated by centrifugation twice at high speed through a layer of 20% sucrose. The preparations (about 1 μg virus/g tissue) contained isometric particles c. 25 nm in diameter which formed a single u.v.‐absorbing component in sucrose density gradients. Chervil seedlings exposed to aphids (Cavariella aegopodii) that had been injected with or had fed on fractions from the u.v.‐absorbing zone developed typical symptoms of infection with CRLV. CRLV particles had a sedimentation coefficient (s20,w) of 104 S, buoyant density in CsCl of 1.403 g/cm3 and A260/A280 of 1.62. Antiserum with a gel‐diffusion titre of 1/512 was obtained from a rabbit injected intradermally with 100 μg purified virus. CRLV was detected by immunosorbent electron microscopy and enzyme‐linked immunosorbent assay in extracts of the petioles and leaf midribs of infected chervil and in groups of five to 20 viruliferous C. aegopodii.Analysis of antiserum/virus reactions by density gradient centrifugation showed that CRLV is distantly related to all luteoviruses tested; its relationships were closest to barley yellow dwarf virus (RPV strain), and perhaps also to beet western yellows virus, more distant to tobacco necrotic dwarf, potato leafroll and bean leafroll viruses, and very distant to barley yellow dwarf (MAV strain) and soybean dwarf viruses. Some of these relationships were detected by double diffusion in agarose gels and by electron microscopy of antiserum/virus mixtures. Immunosorbent electron microscopy detected all these relationships but suggested that CRLV was more closely related to tobacco necrotic dwarf and potato leafroll viruses than to barley yellow dwarf virus (RPV strain).The results show that CRLV should be considered a definitive member of the luteovirus group, and provide confirmation of recent evidence that potato leafroll virus is a luteovirus.