Production and characterization of a monoclonal antibody against the beta-adrenergic agonist ractopamine.

A monoclonal antibody was generated toward the beta-adrenergic agonist ractopamine hydrochloride ¿(1R,3R),(1R, 3S)-4-hydroxy-alpha-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methy l]benzenemethanol hydrochloride¿. Ractopamine-glutarate-keyhole limpet hemocyanin (KLH) was used as the antigen for antibody generation in mice. Clone 5G10, secreted antibody with isotype IgG1kappa, was used for the development of an immunoassay. The selected antibody was specific for racemic ractopamine with an IC(50) of 2.69 +/- 0.36 ng/mL (n = 15). Antibody binding toward ractopamine was stereoselective with (1R,3R)-ractopamine having an IC(50) of 0.55 +/- 0.09 ng/mL (n = 3). IC(50) values for the (1S, 3R)-, (1S,3S)-, and (1R,3S)-ractopamine stereoisomers were 2.00 +/- 0.37, 140 +/- 23, and 291+/- 32 ng/mL (n = 3), respectively. Phenethanolamine beta-agonists showed low cross-reactivity. Studies using a series of ractopamine metabolites and ractopamine analogues demonstrated structural requirements for the antibody binding. A free phenolic group on the N-butylphenol moiety was required for high-affinity binding because methoxylated analogues and metabolites glucuronidated at this phenol generally had IC(50) values greater than 200 ng/mL. Ractopamine analogues methoxylated or glucuronidated at the ethanolamine phenol had IC(50) values of 0.7-2.6 ng/mL. Lack of a benzylic hydroxyl group was of less importance to antibody binding than was the correct stereochemical orientation (3R) of ractopamine's N-phenylalkyl group. In conclusion, a highly specific monoclonal antibody to ractopamine hydrochloride was developed that could be of potential utility in screening assays.

Insertion of a bovine SMT3B gene in NS4B and duplication of NS3 in a bovine viral diarrhea virus genome correlate with the cytopathogenicity of the virus.

Bovine viral diarrhea virus (BVDV) infection in cattle can lead to the development of a fatal syndrome called mucosal disease (MD). The induction of MD requires co-existence of two biotypes of BVDV: cytopathic (cp) and non-cytopathic (ncp). Studies have shown that in some cases, cp viruses are generated from ncp viruses by cellular gene insertions, duplications or rearrangements in the viral genome. Cellular ubiquitin gene is the most frequently reported insert in BVDV 1. Here we report the finding of a novel cellular gene insertion (termed cSNCINS) in the NS4B gene accompanied by the duplication of NS3 in a cpBVDV genome. The amino acid sequence of the insert is identical to that of the human SMT3B protein and is 98% similar to that of the human SMT3A protein. Both SMT3B and SMT3A proteins are homologues of the yeast SMT3 protein. The cSNCINS protein is encoded by an open reading frame located on a 1150-bp bovine endometrial cDNA fragment. Our results indicate that the cSNCINS is a bovine homologue of the human SMT3B gene and that insertion of the BSMT3B gene together with duplication of NS3 in the viral genome may account for the cytopathogenicity of this virus.

Isolation of reptilian calicivirus Crotalus type 1 from feral pinnipeds.

Ten virus isolates were obtained from three species of marine mammals sampled on San Miguel Island (California, USA) and 1,200 km north on Rogue Reef (Oregon, USA) during tagging operations in 1986-87. Seven of these 10 were derived from 30 sampled Steller sea lion (Eumetopias jubatus pups, while two of 10 were isolated from one of 19 sampled California sea lion (Zalophus californianus californianus pups, and the remaining isolate was derived from 30 sampled northern fur seal (Callorhinus ursinus) pups. All 10 isolates were identified as belonging to a single serotype, reptilian calicivirus Crotalus type 1 (RCV Cro-1), previously isolated from both healthy and diseased snakes and frogs in a California zoologic collection. The marine samples also showed that nine of 30 Steller sea lion pups, one of 19 California sea lion pups and zero of 30 fur seal pups were producing type specific neutralizing antibodies to RCV Cro-1. This represents the first reported instance of the isolation from marine sources of calicivirus originally isolated from a terrestrial species.

In vitro isolation and characterization of a calicivirus causing a vesicular disease of the hands and feet.

We report that a calicivirus of oceanic origin, San Miguel sea lion virus serotype 5 (SMSV-5), is a human pathogen. This biotype was isolated originally from blisters on the flippers of northern fur seals (Callorhinus ursinus) and replicates readily in primate and human cell lines. It infects a phylogenetically diverse array of hosts (poikilotherms to primates) and induces type-specific neutralizing antibodies in exposed humans. Group antibody against a pooled antigen of SMSV-5 and two other serotypes was also observed in 18% of 300 blood donors from a population in the northwestern United States. The human calicivirus isolate designated SMSV-5 Homosapien-1 (SMSV-5 Hom-1) was recovered from a laboratory worker with systemic illness, including vesicular lesions on all four extremities. We believe this newly described human disease represents a paradigmatic shift in calicivirus disease recognition.

Ribosomal RNA-based oligonucleotide probes to identify marine green ultraphytoplankton.

Oligonucleotide probes based on small subunit 18S rRNA variable region sequences, were designed and used to discriminate groups of marine green ultraphytoplankton of similar size and morphology. Hybridization of probes was visualized using a combination of biotinylated oligonucleotides and fluorescein labeled avidin. Increased fluorescent signal was generated by building a complex of biotinylated probe, fluorescein labeled avidin and biotinylated anti-avidin. Cells of one phylogenetic group were easily differentiated from morphologically similar cells of a different phylogenetic group. Design of specific probes allowed identification of Mantoniella squamata, Micromonas pusilla, Pseudoscourfieldia marina and the micromonadophyte clone CCMP1194. This technique should be readily adaptable to open ocean samples and should greatly facilitate the description and quantification of ultraphytoplankton in the open ocean.

Natural recombination in bovine viral diarrhea viruses.

BVDV isolates exist as two biotypes differentiated at the molecular level by production of a p80 polypeptide. Insertions consisting of host cell sequences and/or duplicated and rearranged viral sequences have been observed in the portion of the genome coding for the p80 polypeptide in some, but not all, cytopathic BVDV. The significance of these insertions to biotypic expression has yet to be demonstrated. It has been hypothesized that recombination results in the production of the p80 polypeptide by introduction of a cleavage site into a precursor polypeptide or the introduction of a second copy of the p80 gene. Because inserts have not been identified in all cytopathic BVDV examined, it appears that recombination may not be the only mechanism involved in biotypic determination.

The nucleotide sequence of the 5'-untranslated region of bovine viral diarrhoea virus: its use as a probe in rapid detection of bovine viral diarrhoea viruses and border disease viruses.

A 289 bp cDNA fragment from the 5'-untranslated region (UTR) of 16 bovine viral diarrhoea virus (BVDV) isolates was amplified by reverse transcription and polymerase chain reaction, and sequenced by dideoxy DNA sequencing. The sequence showed greater than 90% homology between the isolates and BVDV NADL in this region, and greater than 97% homology within a 72 base sub-region (nt 314-386). The 289 bp fragment was then used as a probe for rapid detection of BVDV and border disease virus (BDV) from cell culture samples by dot-blot hybridization. This probe hybridized to 100% of BVDV isolates (n = 78) and 100% of BDV isolates (n = 9), but not to the uninfected BT cells or other bovine infectious agents. A shorter probe from the more conserved sub-region also was tested for hybridization with some of the isolates, and the results were similar to those using the longer probe. These results suggest that the 5'-UTR is highly conserved among BVDV and BDV isolates, and may be used as a potential probe for rapid detection of BVDV and BDV in clinical and cell culture samples from cattle and sheep.

Analysis of the bovine viral diarrhea virus genome for possible cellular insertions.

Mucosal disease is the most severe disease resulting from bovine viral diarrhea virus (BVDV) infection in cattle. Two biotypes of BVDV may be isolated from animals with mucosal disease: cytopathic (cp) and noncytopathic (ncp). These "pairs" of cp/ncp viruses are often closely related and it has been suggested that the cp virus arises from a ncp virus by insertion of cellular RNA in the p125 region of the BVDV genome. We have used four pairs of cp/ncp BVDV isolated from cattle with mucosal disease, to examine the genomic sequence of the region of the genome coding for the nonstructural protein p125 (processed to p54/p80 in cp viruses) by PCR analysis and sequencing. We did not detect any cellular gene insertions in any of the four ncp viruses; however, we found a large duplication of the p80 gene and a ubiquitin gene insertion in three of the four cp isolates. Our results suggest that cellular RNA insertions in the p125 region may contribute significantly to the cytopathogenicity of BVDV. However, this does not appear to be the only mechanism of cytopathogenicity as we did not detect any insertions or duplications in one of the cp viruses. Comparison of the DNA sequence in the p80 region revealed greater homology within the "pairs" than to NADL, which lend further support to the hypothesis that a cp virus is originated from a ncp virus.

Antigenic and genomic comparison between non-cytopathic and cytopathic bovine viral diarrhoea viruses isolated from cattle that had spontaneous mucosal disease.

Antigenic and genomic relationships between five pairs of non-cytopathic and cytopathic bovine viral diarrhoea (BVD) viruses isolated from cattle with mucosal disease were examined. Antigenic similarity was evaluated by studying the binding characteristics of 10 monoclonal antibodies (MAbs) directed against the gp53 BVD virus glycoprotein. The MAb binding match between members of the same virus pair ranged from 10/10 to 7/10. The genomic relationships was evaluated by studying the hybridization characteristics of two cDNA probes and six complementary 20 base oligomer probes with virus DNA, selected on the basis of conservation between published BVD virus sequences; cDNA probes were hybridized at two stringencies (60 degrees C and 45 degrees C). The match of hybridization results between members of the same virus pair ranged from 4/4 to 1/4 with the cDNA probes and from 6/6 to 3/6 with the oligomer probes. The results indicate that members of the same virus pair can differ at both the antigenic and genomic levels.

Detection of BVD viruses using synthetic oligonucleotides.

This study examined synthetic oligonucleotide probes as potential diagnostic tools for bovine viral diarrhea virus (BVDV). Six 20-base sequences from across the genome were selected by homology analysis of the published genomic sequences of the NADL and Osloss isolates of BVDV. RNA was extracted from 22 BVDV isolates propagated in bovine turbinate (BT) cells, blotted, and probed with 32P end-labeled oligonucleotides. The stringency conditions used were such that more than a single base mismatch would result in no hybridization. The probe originating nearest the 5' end of the viral RNA, ND001, detected 86% of the viral isolates while the other probes detected from 19% to 57%. Both cytopathic and noncytopathic isolates were detected by these synthetic probes. A cocktail of these probes were used to specifically detect BVDV RNA extracted directly from tissues of cattle either persistently or acutely infected.

New marine calicivirus serotype infective for swine.

A new serotype of calicivirus was isolated from California sea lions (Zalophus californianus) with severe vesicular disease. Neutralizing antibodies were found in 27 of 82 (32.9%) serum samples from California sea lions and in 15 of 146 (10.3%) serum samples from Steller sea lions (Eumetopias jubatus) tested. The seropositive animals were widely dispersed along the margins of the eastern Pacific basin, from the Bering Sea to the Santa Barbara Channel. Seropositive samples were found from as early as 1976 through the present time. This new calicivirus serotype, San Miguel sea lion virus type 13, was inoculated into weaned pigs, resulting in induction of severe vesicular disease, which spread to all pigs, including uninoculated pen contacts. Virus was continually shed by most of the pigs throughout the 2-week duration of the experiment.

First isolation of a calicivirus from the Steller sea lion (Eumetopias jubatus).

A calicivirus was isolated from the rectum of a Steller sea lion (Eumetopias jubatus) pup on Rogue Reef, off the southern Oregon coast. Based on the results of neutralization tests with specific typing antisera, the isolate was identified as San Miguel sea lion virus serotype 6 (SMSV-6). Blood obtained from nine of 37 pups (24%) during virus sample collection procedures had specific neutralizing antibodies to SMSV-6. The isolation of SMSV-6 from a Steller sea lion represents, to our knowledge, the first isolation of any virus from this widely distributed marine mammal species, and serves to reconfirm the host-nonspecificity of yet another calicivirus of marine origin.

Antibodies to marine caliciviruses in the Steller sea lion (Eumetopias jubatus Schreber).

Sera from 145 Steller sea lions (76 adults, three subadults, 37 pups, and 29 fetuses) were tested for neutralizing antibodies to nine marine calicivirus serotypes. Antibodies were found to San Miguel sea lion virus (SMSV) types 1, 5, 6, 7, 8, 10 and 13, and to Tillamook (bovine) calicivirus, but no antibodies were found to the walrus calicivirus. Titers (microtiter neutralization assay) ranged from 1:20 to 1:320, with many positive reactions at the higher dilutions (greater than or equal to 1:80). Antibodies to SMSV's 5 and 10 were most common among animals sampled in Alaskan waters, while antibodies to SMSV-6 were most common among pups from the southern Oregon coast. These data provide evidence that Steller sea lions, like their California sea lion (Zalophus c. californianus Lesson) counterparts, have experienced widespread exposure to multiple serotypes of marine caliciviruses.

Prevalence and distribution of serum neutralizing antibodies to Tillamook (bovine) calicivirus in selected populations of marine mammals.

Neutralizing antibodies to Tillamook calicivirus (TCV) were found in sera collected from California sea lions (Zalophus c. californianus Lesson) in 1983 and 1984 and in sera collected from Steller sea lions (Eumetopias jubatus Schreber) in 1976 and 1985. The combined prevalence of antibodies for these two species was 10/228 = 4.38%. Titers ranged from 1:20 (five animals), to 1:40 (four animals), to 1:80 (one animal) by standard microtiter neutralization assay. The seropositive pinnipeds were dispersed widely along the margins of the eastern Pacific rim, from the Bering Sea to the Santa Barbara Channel. Antibodies to TCV were not found in sera collected from northern fur seals (Callorhinus ursinus L.), Pacific walruses (Odobenus rosmarus divergens Illiger), seals of the family Phocidae, or several cetacean species. Tillamook calicivirus was isolated originally in 1981 from dairy calves in Oregon; the finding of neutralizing antibodies in two widely distributed species of sea lions suggests the possibility of a marine origin for this agent.

Antibodies to marine caliciviruses in the Pacific walrus (Odobenus rosmarus divergens Illiger).

Sera from 155 Pacific walruses (Odobenus rosmarus divergens Illiger), sampled in the Chukchi Sea during the summer of 1983, were tested for serum neutralizing (SN) antibodies to six marine calicivirus serotypes. Serotypes tested included San Miguel sea lion virus (SMSV) types 1, 5, 8, and 10, previously isolated from northern fur seals (Callorhinus ursinus Linné) in the Bering Sea; walrus calicivirus (WCV), previously isolated from walrus feces collected off sea ice in the Chukchi Sea; and Tillamook calicivirus (TCV), a bovine isolate from Oregon of suspected marine origin. No antibodies were found to SMSV-1, SMSV-10, or TCV. Antibodies to SMSV-5 were found in two animals (titers 1:20 and 1:160); antibodies to SMSV-8 were found in four animals (all 1:20); and antibodies to WCV were found in one animal (titer 1:40). Antibodies to WCV have been found in the Pacific walrus previously; however, this represents the first report of antibodies to any of the SMSV serotypes in this marine mammal.

A simple, rapid method for preparation of virus isolates from cell culture for electron microscopy.

A simple procedure for the rapid preparation of virus isolates from cell culture for negative-contrast electron microscopy was devised. Using only conventional centrifugation steps (i.e. without ultracentrifugation), the procedure produced consistent, fine-quality preparations of a variety of virus types differing in size/shape and buoyant density.

Immunisation of cattle with a recombinant togavirus-vaccinia virus strain.

Genetic engineering techniques have been used to construct a vaccinia virus recombinant which contains and expresses togavirus (Sindbis) genetic information. Intradermal inoculation of this recombinant strain into calves caused a transient pock-type lesion at the site of inoculation and elicited the production of substantial levels of anti-Sindbis virus neutralising antibodies. These results suggest that recombinant vaccinia virus vaccines may have potential for use in veterinary medicine.

Suppression of interferon synthesis by the pesticide carbaryl as a mechanism for enhancement of goldfish virus-2 replication.

Interferon production was demonstrated by the goldfish-derived CAR cell line in response to infection by goldfish virus-2. Supernatants of infected cultures provided antiviral protection to CAR cells and another cell line derived from goldfish, ABIII. The protective factor retained activity after ultracentrifugation, dialysis, freezing and thawing, acid treatment (pH 2), or heating to 56 degrees C but was sensitive to trypsin. Supernatants of infected cultures did not affect adsorption of virus. Previous studies have shown that replication of goldfish virus type 2 is enhanced by pretreatment of cultures with subcytotoxic concentrations of carbaryl. In the present study, pesticide-treated cultures were found to synthesize reduced levels of interferon.