First Report on the Occurrence of Grapevine leafroll-associated viruses 5 and 9 in Tunisian Grapevines.

Grapevine leafroll disease is one of the most important diseases that occurs in cultivated grapevines in the world. So far, nine serologically distinct viruses of the family Closteroviridae have been isolated from diseased vines (3). A previous study (4) has shown that Grapevine leafroll-associated viruses (GLRaV) -1, -2, and -3 are present in Tunisian grapevines and GLRaV-3 is the predominant virus associated with leafroll disease. A survey was conducted in table grapes to identify other viruses associated with this disease. Samples of dormant canes were collected and screened by indirect Biotin Steptavidin ELISA with specific antibodies to GLRaV-5 (Bio-Rad, Sanofi, France) according to the manufacturer's instructions. Serological analysis revealed that nearly 47% of the samples were infected with GLRaV-5. To confirm GLRaV-5 identification and identify other leafroll viruses, vines with severe leafroll symptoms were collected and total RNA extracts were obtained from six samples and tested at Waite Diagnostics (University of Adelaide, Australia) by reverse transcription (RT)-PCR using primers for GLRaV-5 (2), LR5-1F 5'-CCCGTGATACAAGGTAGGACA-3' and LR5-1R 5'-CAGACTTCACCTCCTGTTAC-3' with a resulting amplicon size of 690 bp and primers for GLRaV-9, LR9F 5'-ACAGTGGTCGGCATAAGAAAAG-3' and LR9R 5'-ACACAAACATGCAGGCCAAAG-3' with a resulting amplicon size of 250 bp. Results showed that 1 of 6 and 5 of 6 of the samples were infected with GLRaV-5 and GLRaV-9, respectively, by RT-PCR and comparable results were obtained by ELISA. Amplicons were cloned and sequenced to confirm the identification of GLRaV-5 and GLRaV-9. The obtained sequences showed 99.1% nt identity and 94.8% amino acid similarity with an isolate of GLRaV-5 (GenBank Accession No. AF233934) and 97.6% nt identity and 94.8% amino acid similarity with an isolate of GLRaV-9 (GenBank Accession No. AY297819). The occurrence of GLRaV-9 has previously been reported in California and Australia (1). To our knowledge, this is the first report on the occurrence of GLRaV-5 and -9 in Tunisian grapevines. The widespread occurrence of GLRaV-5 and -9 is probably due either to the presence of their putative vectors, Planococcus ficus (Signoret) and Planococcus citri (Risso), or by propagation using infected local source material. Further studies are in progress to verify the implication of indigenous mealybugs in the spread of these viruses. References: (1) R. Alkowni et al. J. Plant Pathol. 86:123, 2004. (2) X. Good and J. Monis. Phytopathology 91:247, 2001. (3) P. Gugerli. ICVG, Extended Abstracts 14:23, 2003. (4) N. Mahfoudhi et al. EPPO Bulletin 28:197, 1998.

Antigenic and molecular characterization of a herpesvirus isolated from a North American elk.

OBJECTIVE: To determine whether a herpesvirus isolated from the semen of a North American elk was related to bovine herpesvirus 1 (BHV-1). SAMPLE POPULATION: Semen from 1 healthy bull elk and 2 subtypes of BHV-1 (BHV-1.1 and BHV-1.2). PROCEDURES: A virus with cytopathic and electron microscopic characteristics consistent with an alpha-herpesvirus was isolated from elk semen, using fetal bovine kidney cells. Cross-neutralization assays were performed with antisera against BHV-1 and the elk herpesvirus (EIkHV). Restriction endonuclease digests of EIkHV DNA were compared with digests of BHV-1.1 and BHV-1.2 DNA. A portion of the ElkHV DNA polymerase gene was amplified with consensus primers by use of the polymerase chain reaction and sequenced. Sequence was compared with known sequences of other herpesviruses. An immunoperoxidase monolayer assay was used to determine reactivities of 22 BHV-1-specific monoclonal antibodies (mAb) against ElkHV. In vitro neutralizing activities of the reactive mAb were determined by use of a microneutralization assay. RESULTS: Results of cross-neutralization assays indicated that ElkHV was serologically related to BHV-1. Endonuclease digestion of ElkHV DNA generated fragments that were distinct from those of BHV-1. Nucleotide sequencing confirmed that ElkHV is an alphaherpesvirus closely related to but distinct from BHV-1. Six of 22 BHV-1-specific mAb reacted against ElkHV; 2 of these 6 also neutralized in vitro infectivity of ElkHV. CONCLUSIONS AND CLINICAL RELEVANCE: ElkHV is antigenically and genetically distinguishable from BHV-1. However, the viruses are serologically related and share at least 6 antigenic determinants, one of which is a major neutralizing determinant.

Construction and characterization of a novel recombinant single-chain variable fragment antibody against Western equine encephalitis virus.

A novel recombinant single-chain fragment variable (scFv) antibody against Western equine encephalitis virus (WEE) was constructed and characterized. Using antibody phage display technology, a scFv was generated from the WEE specific hybridoma, 10B5 E7E2. The scFv was fused to a human heavy chain IgG1 constant region (CH1-CH3) and contained an intact 6 His tag and enterokinase recognition site (RS10B5huFc). The RS10B5huFc antibody was expressed in E. coli and purified by affinity chromatography as a 70-kDa protein. The RS10B5huFc antibody was functional in binding to WEE antigen in indirect enzyme-linked immunosorbent assays (ELISAs). Furthermore, the RS10B5huFc antibody was purified in proper conformation and formed multimers. The addition of the human heavy chain to the scFv replaced effector functions of the mouse antibody. The Fc domain was capable of binding to protein G and human complement. The above properties of the RS10B5huFc antibody make it an excellent candidate for immunodetection and immunotherapy studies.

A polymerase chain reaction assay for the detection of Leptospira spp. in bovine semen.

A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.

Rapid detection of bovine herpesvirus 1 in the semen of infected bulls by a nested polymerase chain reaction assay.

A nested polymerase chain reaction (PCR) assay was developed for the detection of bovine herpesvirus 1 (BHV-1) in bovine semen and compared with the virus isolation method. When extended semen, commonly used in the bovine artificial insemination industry, was inoculated with BHV-1, the PCR assay detected BHV-1 DNA in semen inoculated at 0.25-2.5 TCID50 per 0.5 mL. In contrast, the lower limit of detection for virus isolation was 250 TCID50 of BHV-1 inoculated in 0.5 mL of extended semen. These methods were also used to detect BHV-1 in the semen of four bulls which were experimentally infected with BHV-1. All infected bulls demonstrated balanitis at 3 d post-inoculation (DPI) and severe balanoposthitis at 4 DPI. BHV-1 was detected in raw semen by virus isolation and PCR at 2 DPI, before balanitis was evident. For virus isolation, the last day that BHV-1 was detected during primary infection was 7 DPI for two bulls and 9 and 11 DPI for the other two bulls. In contrast, PCR detected BHV-1 in the bulls' semen until 14 or 18 DPI. For individual animals, PCR detected BHV-1 during primary infection for at least 1-10 d longer than virus isolation. Reactivation of BHV-1 from latency without the presence of visible lesions was promoted twice by two series of 5 d dexamethasone injections. For the first series of dexamethasone treatments, a positive virus isolation result was obtained on the 5th d of treatment for only one bull. In contrast, two bulls demonstrated evidence of viral reactivation on this day by PCR. All bulls shed BHV-1 in semen on d 4 after dexamethasone treatment, as evidenced by positive virus isolation and PCR results. One bull was still PCR positive 13 d later. For the second series of dexamethasone treatments, a small amount of virus was isolated from semen collected on d 3 or 4 after treatment for two bulls but not from the other two bulls. In contrast, semen samples from all bulls were PCR positive for either or both of these 2 d. In total, from 80 semen samples, 45 were PCR positive and 26 were virus isolation positive. Thus, the PCR assay detected BHV-1 shedding in bulls earlier, more often, and for a longer duration, than did the virus isolation method.

Bovine viral diarrhea virus proteins: relatedness of p175 with p80 and p125 and evidence of glycoprotein processing.

Three monoclonal antibodies, which recognized two nonoverlapping antigenic domains and were reactive to the bovine viral diarrhea virus (BVDV) p80 protein, were found to cross react with the p125 protein of both cytopathic and noncytopathic BVDVs and a molecular weight 175,000 BVDV protein (p175). Results from limited proteolysis and chemical cleavage experiments confirmed the relatedness of these three proteins. In pulse-chase experiments it was apparent that p175 was a transient protein, as it was diminished during the chase, with a half-life of about 30 min. However, both p125 and p80 were also observed in short-pulsed lysates. Furthermore, during the chase, radiolabel was not found to accumulate into p125 or p80. Rather, these two proteins were stable with half-lives greater than 2 h. A fourth nonglycosylated protein, p37, increased during the chase. Processing of several glycoproteins was evident in these experiments. A glycoprotein of molecular weight 75,000 (gp75) diminished during the chase period, while glycoproteins gp62, gp48, and gp25 appeared or increased during the chase period. In contrast, the glycoprotein gp53 was a major protein in pulse-labeled cell lysates and remained constant throughout the chase period. In further experiments two stable forms of p80 differing in intramolecular disulphide bonding were observed.

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of bovine viral diarrhea virus antibody in cattle.

A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of greater than or equal to 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of less than 1.50, 1.50-1.99 and greater than or equal to 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.

Molecular cloning and nucleotide sequence of another variant of the Escherichia coli Shiga-like toxin II family.

Escherichia coli strain H.I.8 (O128:B12) produces low levels of a Shiga-like toxin (SLT) which we have called SLTIIva because of its close relationship with SLTIIv. The Vero cell cytotoxicity of SLTIIva is neutralized by antisera against SLTII and SLTIIv but not by antisera against SLTI. These data indicate that the SLT of strain H.I.8 is a member of the SLTII family. Since SLTIIva shares with SLTIIv the property of having low cytotoxicity to HeLa cells compared with Vero cells, it is appropriate to consider both toxins as variants of SLTII. SLTIIva differs from SLTIIv in that it is more heat-stable. Further, SLTIIv-producing strains of E. coli have only been isolated from pigs while the SLTIIva-producing E. coli strain examined in this study was isolated from a human infant with diarrhoea. The genes for this SLT were cloned from a cosmid library of total cellular DNA by screening recombinants for Vero cell toxicity and with a DNA probe derived from SLTIIv structural genes. Nucleotide sequence analysis was performed on a 2.0 kb AvaII-HincII fragment which encodes the toxin gene. The nucleotide sequence data confirm the close relationship between SLTIIva and SLTIIv: they have 98% nucleotide sequence homology in the B subunit gene and 70.6% homology in the A subunit gene. Comparison of DNA sequences indicated that SLTIIva was most closely related to SLTIIv, closely related to SLTII and less closely related to SLTI.

Monoclonal antibodies to the p80/125 gp53 proteins of bovine viral diarrhea virus: their potential use as diagnostic reagents.

Monoclonal antibodies reactive to the bovine viral diarrhea virus (BVDV) protein gp53 were produced and characterized. These antibodies and our panel of anti-p80/125 monoclonal antibodies were tested for their cross-reactivity with 11 different North American and European (Danish) BVDV strains and isolates including viruses of both cytopathic and noncytopathic biotypes. The four anti-gp53 monoclonal antibodies were neutralizing for the homologous Danish cytopathic isolate and cross-reacted with all BVDV strains examined except for the Draper strain. Further, anti-gp53 monoclonal antibodies neutralized the majority of BVDV strains examined. The anti-p80/125 monoclonal antibodies cross-reacted with all eleven strains and isolates tested. This indicated that various strains of BVDV have common epitopes. The broad cross-reactivities demonstrated by these monoclonal antibodies suggest that a pool of these antibodies may be used for detection of BVDV cellular contamination or for virus isolation, in place of polyclonal antiserum.

Diagnostic specificity, sensitivity and cross-reactivity of an enzyme-linked immunosorbent assay for the detection of antibody against Leptospira interrogans serovars pomona, sejroe and hardjo in cattle.

Outer sheath antigen was prepared from Leptospira interrogans serovars pomona, sejroe and hardjo by treating the organisms with 1.0M NaC1 followed by 0.04% sodium dodecyl sulfate (SDS). Sodium dodecyl sulfate was removed from the SDS-protein complexes by the extraction of dodecyl sulfate anions as ion pairs with triethylammonium cations into an organic solvent. The outer sheath antigen was recovered from the organic solvent as a precipitate and used as the source of leptospiral enzyme-linked immunosorbent assay (ELISA) antigen. Utilizing this antigen, ELISA was adapted to detect bovine serum antibody to L. interrogans serovars pomona, sejroe and hardjo. The specificity of this assay in 344 bovine sera, which were negative in the microscopic agglutination test (MAT) for seven serovars, was 99.4%. In sera from 37 and 87 cattle which revealed MAT titers greater than or equal to 1:50 for L. interrogans serovars pomona and sejroe, the relative sensitivity of the test was 100%. The ELISA also showed a considerable degree of low level cross-reactivity with other serovars. Sixty-six (75.9%) out of 87 bovine sera which were MAT-positive (MAT titer of greater than or equal to 1:50) with serovars sejroe and hardjo only were ELISA positive with heterologous pomona antigen; 16 (43.2%) and six 16.2%) out of 37 bovine sera which were MAT positive MAT titer of greater than or equal to 1:50) with serovar pomona only were ELISA positive with heterologous sejroe and hardjo ELISA antigen respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of functional domains on reovirus cell attachment protein sigma 1 using cloned S1 gene deletion mutants.

Previously a reovirus (serotype 3) S1 gene cDNA was inserted into the lac cloning site of pUC13 and expressed in Escherichia coli to yield a sigma 1 fusion protein (F-sigma 1) capable of binding to mouse L fibroblasts and of agglutinating human red blood cells (S.A. Masri, L. Nagata, D. C. W. Mah, and P. W. K. Lee, 1986, Virology 149, 83-90). To probe the functional domains on the sigma 1 protein, restriction enzymes which divide the S1 gene into four segments (5'-I-II-III-IV-3') of similar size were used to generate five in-frame deletion mutants (D1-D5). Corresponding mutant forms of sigma 1 were expressed in E. coli and were assayed for (i) host cell (mouse L fibroblasts) binding activity; (ii) glycophorin (reovirus erythrocyte receptor) binding activity (R. W. Paul and P. W. K. Lee, 1987. Virology 159, 94-101 and (iii) recognizability by a library of neutralizing monoclonal anti-sigma 1 antibodies. It was found that mutant sigma 1 forms with segment III or segment IV deleted did not exhibit any detectable L-cell binding activity, whereas mutants with these two segments intact (but lacking segment II or segments I and II) were capable of attaching to L-cell receptors, albeit with reduced efficiencies. On the other hand, only F-sigma 1, but none of the mutants, could bind immobilized glycophorin. These data clearly suggest that the host cell binding domain of sigma 1 is distinct from its hemagglutination domain. Also, the five neutralizing anti-sigma 1 monoclonal antibodies tested were all found to recognize epitopes on either the middle segments or the carboxy-terminal half of sigma 1.

Functional expression in Escherichia coli of cloned reovirus S1 gene encoding the viral cell attachment protein sigma 1.

A cDNA clone encompassing the complete reovirus (serotype 3) S1 gene was constructed using two partial clones containing overlapping sequences. The gene (with the first 15 bases at the 5' end up to and including the first ATG removed) was then inserted in frame into the lac cloning site of the pUC13 plasmid and expressed in Escherichia coli as a fusion product under control of the lac promoter. The expressed product can be immunoprecipitated as a 47,000-mol wt (47K) protein using several monoclonal anti-sigma 1 antibodies. Like authentic soluble sigma 1 from reovirus-infected cells, the expressed protein is capable of attaching to mammalian cells (mouse L fibroblasts) in a specific manner and of competing with reovirus particles for cell surface receptors. Lysates prepared from the recombinant plasmid-transformed, but not those from pUC13-transformed E. coli cells, were also found to exhibit hemagglutinating (HA) activity. Such hemagglutination was inhibited by a monoclonal anti-sigma 1 antibody previously shown to inhibit reovirus HA activity. It is concluded that both the host cell attachment domain and the hemagglutination domain on the expressed protein are functionally intact.

Molecular cloning and sequencing of the reovirus (serotype 3) S1 gene which encodes the viral cell attachment protein sigma 1.

The complete sequence of the reovirus (serotype 3) S1 gene was obtained by using cloned cDNA derived from the RNA segment. This gene is 1416 nucleotides in length and contains two open reading frames. The first reading frame has a coding capacity of 455 amino acids, sufficient to account for the known S1 product, protein sigma 1 (42,000 MW). It possesses a signal peptide as well as three possible glycosylation sites. No homology could be detected when this gene sequence and the deduced amino acid sequence were compared to published sequences of the corresponding gene of a human rotavirus. The second reading frame (not in phase with the first) starts at the second ATG recently shown to be a functional initiation site. It has a coding capacity of 120 amino acids. Its outstanding feature is the highly basic amino-terminal region, a characteristic apparently shared by a number of DNA binding proteins. It is speculated that this protein, hitherto undetected, may play a role in mediating viral and/or host nucleic acid transcription.

Isolation o human fibrinogen of high purity and in high yield using polyethylene glycol 1000.

Fibrinogen was isolated from human plasma using a polyethylene glycol 1000 fractionation procedure that eliminates problems of denaturation, degradation and contamination encountered with other procedures (1). The technique is simple, rapid, gives a high yield and gram amounts of fibrinogen can be readily obtained.