Generation of Recombinant Porcine Parvovirus Virus-Like Particles inSaccharomyces cerevisiaeand Development of Virus-Specific Monoclonal Antibodies

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeastSaccharomyces cerevisiaeexpression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeastS. cerevisiaerepresents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

First Report of Petunia vein clearing virus in Israel

Petunia vein clearing virus (PVCV), a possible member of the cauli-movirus group, was detected in several cultivars of vegetatively propagated petunias grown in commercial nurseries in Israel. Symptoms associated with PVCV infection in petunia cv. Chaplin consisted of stunted young shoots, leaf yellowing, and severe vein clearing. Virus incidence within this cultivar was 40 to 60%. Symptoms were most frequently expressed when plants were subjected to temperatures above 20°C and nutrient deficiencies. The virus was transmitted to petunia only by grafting, however, not by aphids or mechanical inoculation. Leaf dip preparations and thin sections of leaf tissue were analyzed by transmission electron microscopy. Spherical virus particles (45 to 50 nm diameter) were observed in samples from symptomatic petunia plants. In immunoelectron microscopy, the virus particles produced a distinct pattern with the specific antiserum against PVCV provided by D. Lesemann (Biologische Bundesanstalt, Braunschweig, Germany). Infection with PVCV also was confirmed by polymerase chain reaction with total nucleic acid extract preparations. Two primer pairs—5′-GAGGTCAGAGCAAGTCAGAGG-3′ (nucleotides 4339 to 4359) and 5′-GTAATGATTTGACTTGTTGAG-3′ (nucleotides 5055 to 5075)—were designed to flank a 736-bp sequence in the RNA-dependent RNA polymerase gene of the PVCV genome (GenBank Accession no. U95208). A DNA fragment of the predicted size was visualized in agarose gels. Authenticity of the amplified DNA fragment was confirmed by restriction analysis. The virus has been detected in the past in Germany (1) and recently in the United States (2). This is the first report of PVCV in commercial nurseries in Israel. References: (1) D. Lesemann and R. Casper. Phytopathology 63:1118, 1973. (2) B. Lockhart and D. Lesemann. Plant Dis. 82:262, 1998.

Comparison of Detection Methods for Citrus Tristeza Virus in Field Trees During Months of Nonoptimal Titer

Enzyme-linked immunosorbent assay (ELISA) can reliably detect citrus tristeza virus (CTV) in samples collected during approximately 6 months of a typical year. Two reverse transcriptase polymerase chain reaction (RT-PCR) methods (total nucleic acid extract and immunocapture based) were evaluated and compared to ELISA in order to develop a more sensitive assay for CTV. From May 1994 to October 1995, 6 sweet orange trees infected with CTV from each of 2 geographic areas (Riverside and the San Joaquin Valley) were tested monthly by each method. In the months of August (San Joaquin Valley samples) and September (Riverside and San Joaquin Valley samples) several of the trees had a significant loss of virus titer such that CTV was not reliably detected by ELISA. By contrast, the 2 PCR methods gave definitive positive results for CTV in samples collected during these months. Different tissue types were analyzed by each of the above assays. Petioles and midribs, both phloem-rich tissues, were each satisfactory for ELISA, while distal leaf tips did not always produce a positive result. All tissue types were equally efficient in producing a positive result in both PCR-based assays. The results of this study provide a basis for CTV testing by PCR in months when virus titer drops to a level generally unacceptable for using ELISA.