The core protein and the transmembrane protein, encoded for the structural genes gag and env, respectively, of caprine arthritis-encephalitis virus were amplified by use of polymerase chain reaction, cloned into a pGEX-2T vector, and expressed in Escherichia coli as fusion proteins with the glutathione S-transferase at their C-terminus. The recombinant proteins were purified and evaluated by use of an ELISA. Sera from 269 goats were tested, and the results were compared with those obtained by use of immunoblot analysis. When results from both recombinant ELISA (r-ELISA) were compared, it appeared that the transmembrane glycoprotein was more immunoreactive than the core protein, because it was recognized by a higher percentage of sera from infected goats. When results of the 2 ELISA (p28 r-ELISA and p40 r-ELISA) were combined in parallel, they were comparable to those of the immunoblot test, with sensitivity of 100% and specificity of 98.3%. It was also found that use of both r-ELISA makes it possible to compare the variable immunoreactivity against gag and env viral antigens, which may be correlated with the disease state. The r-ELISA, using core and transmembrane proteins, appears to be highly sensitive and specific for detection of antibodies against caprine arthritis-encephalitis virus.

A double-antibody sandwich ELISA (DAS-ELISA) was developed for detection of avian influenza virus (AIV) antigen. A monoclonal antibody to the viral nucleoprotein (NP) was used to coat the ELISA plates. A direct DAS-ELISA and an indirect DAS-ELISA were evaluated. In the direct DAS-ELISA, monoclonal antibody to the AIV NP conjugated with horseradish peroxidase was used. The direct DAS-ELISA was evaluated for its sensitivity to detect purified NP; this procedure detected as little as 0.1 ng. In the indirect DAS-ELISA, rabbit NP antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobin were used as primary and secondary antibodies, respectively. The indirect DAS-ELISA was evaluated for its ability to detect the AIV antigen in tracheal and cloacal specimens from turkeys inoculated with AIV. Results of indirect DAS-ELISA were compared with those of conventional virus isolation. Percentage agreement between indirect DAS-ELISA and virus isolation in AIV-positive samples was found to be 76.1% and, in AIV-negative samples, it was found to be 82.1%. These results indicate that the DAS-ELISA might be a viable alternative to virus isolation because of its rapidity, compared with virus isolation.

Two African swine fever virus (ASFV) antigens were tested for use in an ELISA to detect antibody to ASFV. Antigens used were the cytoplasmic soluble fraction (CS-P) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (SVP73). Both antigens were tested by ELISA against 72 sera obtained during several ASF field episodes and from ASFV-inapparent carriers. Of the 72 sera, only 2.8% had positive results by ELISA against CS-P antigen; 60% of positive-reacting sera (to both antigens) had higher ELISA values when the CS-P antigen was used. Samples (with positive results) that reacted only to CS-P antigen had results confirmed by immunoblot analysis. Such sera reacted against ASFV-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of ASFV-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (PID) 8. At that time, only the polypeptides IP25, IP25.5, IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, ASFV antibodies were detected by ELISA, using CS-P or SVP73 antigens, on PID 7 and 9, respectively. These results could explain the percentage of sera not having positive results by ELISA using SVP73 antigen, if the sera were obtained from ASFV-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of CS-P antigen advantageous in early serologic detection of AFSV-infected pigs.

A panel of 40 monoclonal antibodies (MAb) specific for bovine viral diarrhea virus (BVDV) was produced, and each MAb was characterized and grouped according to its viral protein specificity, immunoglobulin subclass, virus-neutralizing activity, and immunoreactivity with a large collection of BVDV isolates. The MAb were found to be specific for 1 of 3 sets of related viral-induced proteins found in cells infected with the Singer strain of BVDV. Group-1 MAb were specific for the 80- and 118-kilodalton (kD) proteins of BVDV. Group-2 MAb recognized 3 proteins with molecular sizes of 54, 56, and 58 kD. Group-3 MAb recognized a 43- and a 65-kD protein. The MAb belonged to either the IgG1, IgG2a, IgG2b, IgG3 subclasses or the IgE class of mouse immunoglobulin. All MAb in group 2 were able to neutralize BVDV and had neutralization titers that ranged from 24 to 1,600,000. The reactivity of the MAb with numerous field isolates of BVDV was highly variable. Both cytopathic and noncytopathic biotypes of BVDV were examined and had the same degree of antigenic variation. The greatest degree of variation was detected with group-2 MAb. The data demonstrate that BVDV isolates have a high degree of antigenic variation that is largely confined to the envelope glycoproteins associated with virus neutralization. The results also suggest that antigenic variability of this virus is important in the development and severity of the disease it causes.

Pig epizootic diarrhea virus cannot be grown in cell culture; for its characterization, intestinal perfusate material from a pig infected with the strain CV777 had to be used. In isopyknic sucrose gradients, a peak of virus-specific ELISA activity was detected at a density of 1.17 g/ml. Using immunoprecipitation of radioiodinated-purified virus material followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 3 proteins of low molecular weight (20,000 to 32,000 daltons [D] were found; after blotting nitrocellulose and glycoprotein identification with concanavalin A and horseradish peroxidase, 1 of the proteins (23,000 D) gave a signal. Another protein of 58,000 D was encountered, which was the only protein binding an RNA probe. Finally, a protein of 85,000 D was visible, associated with minor bands of about 110,000 and 135,000 D in most experiments. Using the concanavalin A-blotting technique, the same bands were visualized. The demonstration of a polydisperse cluster of proteins from 20,000 to 32,000 D (of which at least 1 is glycosylated), of glycosylated proteins from 85,000 to 135,000 D, and of an RNA-binding protein of 58,000 D is taken as structural evidence that pig epizootic diarrhea virus should be classified with the Coronaviridae, irrespective of the apparent lack of an antigenic relationship with other members of that family.

Neutralizing and nonneutralizing antibodies to bovine viral diarrhae (BVD) virus were detected in 3 cows persistently infected with noncytopathic BVD virus after vaccination with modified-live cytopathic BVD virus. Neutralizing antibodies detected in serum samples from each persistently infected cow at 3 weeks after vaccination were highly specific for certain isolates of cytopathic BVD virus and reacted only with a viral protein with a molecular weight of 53,000. Neutralizing antibodies to 1 of 3 isolates of noncytopathic BVD virus were detected in a serum sample obtained at 12 weeks after vaccination from 1 of 3 persistently infected cows. Nonneutralizing antibodies were detected in all cows at 7 to 12 weeks after vaccination.The nonneutralizing antibodies were less specific for isolates of BVD virus and reacted with viral proteins with molecular weights of 115,000, 80,000, 53,000, and 47,000.

Epizootic hemorrhagic disease virus was isolated from cattle and sheep in northeastern Colorado during July and August 1984. The isolates were identified as serotype 2 by plaque-inhibition serotyping, genome electropherotyping, and protein analysis.

Objective-To analyze the 7a7b genes of the feline coronavirus (FCoV) of cheetahs, which are believed to play a role in virulence of this virus. Sample Population-Biologic samples collected during a 4-year period from 5 cheetahs at the same institution and at 1 time point from 4 cheetahs at different institutions. Procedures-Samples were first screened for FCoV via a reverse transcription-PCR procedure involving primers that encompassed the 3'-untranslated region. Samples that yielded positive assay results were analyzed by use of primers that targeted the 7a7b open reading frames. The nucleotide sequences of the 7a7b amplification products were determined and analyzed. Results-In most isolates, substantial deletional mutations in the 7a gene were detected that would result in aberrant or no expression of the 7a product because of altered reading frames. Although the 7b gene was also found to contain mutations, these were primarily point mutations resulting in minor amino acid changes. The coronavirus associated with 1 cheetah with feline infectious peritonitis had intact 7a and 7b genes. Conclusions and Clinical Relevance-The data suggest that mutations arise readily in the 7a region and may remain stable in FCoV of cheetahs. In contrast, an intact 7b gene may be necessary for in vivo virus infection and replication. Persistent infection with FCoV in a cheetah population results in continued virus circulation and may lead to a quasispecies of virus variants.

Hemagglutinins HA) of H1N1 swine influenza viruses isolated in the United States have remained antigenically and genetically conserved for many years. In contrast to such conservation, the RA of A/Swine/Nebraska/1/92 (Sw/Neb) could readily be distinguished from those of contemporary porcine viruses. Twenty-eight amino acid mutations differentiated the HA of Sw/Neb and A/Swine/Indiana/1726/88, the most recent H1N1 swine influenza virus for which HA sequence data were available. Among these differences were mutations at potential asparagine-linked glycosylation sites and charge changes at many residues. The Sw/Neb virus also could be differentiated from other swine influenza viruses in hemagglutination-inhibition assays with monoclonal antibodies to recent H1 swine HA. Nonetheless, overall sequence analysis of the HA and the nucleoprotein genes of Sw/Neb indicated that this virus was more closely related genetically to classic H1N1 swine influenza viruses than to H1N1 avian or human viruses. Infection of swine with Sw/Neb under experimental conditions induced clinical signs and lesions typical of swine influenza. However, affected swine in the field had high, persistent fevers, but relatively mild signs of respiratory tract disease. This study indicated that an antigenically and genetically novel variant of swine influenza virus was detected in the United States.

Ten colostrum-deprived calves were assigned to 1 of 2 treatment groups (5 calves/group), and fed colostrum that had either low (naturally infected cows) or high (immunized cows) antibody titers to bovine coronavirus (BCV). All calves were inoculated orally and intranasally with virulent BCV when they were 24 to 48 hours old and challenge exposed 21 days later. Blood, feces, nasal secretions, tears, saliva, and bronchoalveolar lavage (BAL) fluids were collected weekly from each calf for 5 weeks after inoculation. The titers to whole BCV or the relative amounts of isotype-specific antibodies to BCV structural proteins were evaluated in these samples by ELISA or immunoblotting, respectively. Both pools of colostrum contained primarily IgG1, IgG2, and IgA antibodies to the E2 and E3 BCV proteins. Calves fed the high-titer colostrum had correspondingly higher amounts of passive IgG1 and IgA antibodies to whole BCV and to the E2 and E3 BCV proteins in serum, feces, and BAL fluid at postinoculation week 1 than those calves fed low-titer colostrum. Active IgG1, IgA and IgM antibody responses in serum and active IgA and IgM antibody responses in most mucosal secretions to whole BCV and to the E2 and E3 proteins were lower or delayed in calves fed high-titer colostrum, compared with responses in calves fed low-titer colostrum. In contrast, increased responses to the BCV N protein were observed in all samples (except in serum and BAL fluid) in the calves fed high-titer colostrum, compared with calves fed low-titer colostrum. Upon challenge exposure, responses to E2 and E3 BCV proteins in serum and BAL fluid were lower in the group fed high-titer colostrum, compared with those in the group fed low-titer colostrum. Our findings indicate that the level of passive immunity in calves at the time of BCV inoculation can influence the development of active antibody responses in serum, feces, and mucosal secretions to whole BCV and to some BCV proteins individually.