Development of an enzyme-linked immunosorbent assay for caprine arthritis-encephalitis virus.

Because relatively few caprine arthritis-encephalitis virus (CAEV)-infected animals exhibit clinical signs of illness, efforts to control and eradicate this virus will depend heavily on a sensitive diagnostic test that can be easily carried out. The currently utilized tests are of limited usefulness because of relatively low sensitivity or because of incomplete cross-reactivity of goat sera with heterologous test antigens. An enzyme-linked immunosorbent assay (ELISA) with purified CAEV antigen and biotin-avidin amplification steps was therefore developed and compared with a radioimmunoassay (RIA) against CAEV p28. Of over 500 sera tested, there was 99% concordance between the two tests. On the other hand, 23 of 24 sera obtained from animals with clinical signs of disease that were negative by agar gel immunodiffusion test (with ovine progressive pneumonia virus antigen) were positive by ELISA and RIA. These results suggest that an ELISA with CAEV antigen is superior to the agar gel immunodiffusion test and is easier and faster than an RIA, and therefore may be the method of choice for diagnosing CAEV infection.     

False-positive sera do not react with human immunodeficiency virus (HIV) gag-encoded recombinant antigen

Ten sera from healthy blood donors positive by enzyme-linked immunoadsorbent assay (ELISA) were studied by immunoblot assay using natural and recombinant proteins. They interacted only with p17 or p24 proteins but were nonreactive with a recombinant protein (RP 50), which carries antigenic determinants to p17 and p24. Reactions were not blocked by preincubation of sera with genetically engineered p17 and p24 or purified viral p24, indicating that some new epitopes were formed during the Western blot procedure. Recombinant gag-encoded protein is required for confirmation of human immunodeficiency virus (HIV) seropositivity.     

Double-nested polymerase chain reaction for detection of caprine arthritis-encephalitis virus proviral DNA in blood, milk, and tissues of infected goats

A nested polymerase chain reaction (PCR) for detecting proviral DNA of caprine arthritis-encephalitis virus (CAEV) in biological samples was developed. Primers for both gag and pol sequences of the CAEV genome were included in a single tube for simultaneous amplification (‘double’ PCR), and the resulting bands were resolved visually in ethidium bromide-stained agarose gels. Internal gag and pol probes were used to verify the identity of the amplified products by non-radioactive Southern hybridization. Final confirmation of the identity of representative PCR bands was provided by DNA sequence analysis. A comparison between the PCR and an antibody ELISA (with recombinant CAEV p28 as target) using 141 caprine blood samples indicated very strong agreement between the two assays (κ = 0.912). Four of 7 goats with indeterminate ELISA results were PCR-positive as were 5 of 40 (12.5%) seronegative goats, most probably indicating delayed seroconversion. Eleven of 27 goats (41%) PCR-positive on blood had detectable CAEV proviral DNA in milk. Proviral DNA was also detected in lung, mesenteric lymph node, bone marrow, synovial membrane, and mammary gland of a seropositive, clinically affected goat, but not in equivalent tissues of a healthy seronegative goat.     

Adaptation of an indirect enzyme-linked immunosorbent assay for seroepidemiological studies of enzootic bovine leukosis

The aim of this study was to compare a domestic indirect enzyme-linked immunosorbent assay (DI-ELISA) and an in-house agar gel immunodiffusion (AGID) test with a commercial indirect ELISA (CI-ELISA) test for the detection of antibodies to bovine leukaemia virus (BLV). BLV proteins were harvested as described by OIE and used in both the DI-ELISA and AGID tests. Analysis of negative sera showed that consideration of a cutoff equivalent to three times the standard deviation value above the mean value of the negative control sera provided an acceptable specificity and reduced the risk of false positive results for the DI-ELISA test. From 460 serum samples, 425 (92%), 416 (90%) and 435 (94%) sera were found to be negative when using either the CI-ELISA, DI-ELISA or AGID test. Of the six serum samples which yielded suspicious results with the CI-ELISA, four were found to be positive by the DI-ELISA, but all of them were negative by the AGID test. DI-ELISA and AGID tests’ relative (to CI-ELISA) sensitivities were 97% and 86%, respectively. DI-ELISA and AGID tests’ relative (to CI-ELISA) specificities were 84% and 100%, respectively. Comparison of the results from a native breed, Sarabi, with Holstein showed that there is no significant (p?<?0.05) difference in the frequency of enzootic bovine leukosis between the two.     

Serologische AIDS-Diagnostik mit gentechnisch gewonnenen Polypeptiden des menschlichen Immundefizienz-Virus (HIV-1)

Summary Serologic testing for human immunodeficiency virus type 1 (HIV-1) is currently based on enzyme linked immunosorbent assay (ELISA) as screening method. Positive ELISA-results have to be confirmed by at least one second procedure such as Western blotting or immunofluorescence. To obtain new diagnostic reagents for confirmatory testing, we expressed viral antigens in procaryotic systems. Peptides representing epitopes of structural core (gag)- and envelope (env)-proteins of HIV were produced inE. coli as stable immunogenic -galactosidase fusion proteins. Recombinant proteins were taken for immunoblot-assays. The results of Western blotting with those fusion proteins were in general comparable with conventional ELISA, immunofluorescence, immunoblot with cell-culture derived virus and commercially available ELISA tests based on recombinant proteins. Immunoblots using recombinant transmembrane protein (gp41) derived polypeptide were more sensitive than the conventional procedure with purified virion proteins. Western blotting with recombinant fusionproteins provide reliable and inexpensive serodiagnostics without handling of infectious cell cultures.

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Abkürzungsverzeichnis core- (gag-) Proteine Innere Virus-Strukturproteine - HIV-I Humanes Immundefizienz-Virus Typ 1 - ELISA Enzyme linked immunosorbent assay - enve- lope- (env-) Proteine Äußere Virus-Hüllproteine - gp 41 Glykoprotein 41 - p 24 Protein 24     

Detection of Serum Antibodies to Ovine Progressive Pneumonia Virus in Sheep by Using a Caprine Arthritis-Encephalitis Virus Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [³⁵S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.     

Evaluation of a Caprine Arthritis-Encephalitis Virus/Maedi-Visna Virus Indirect Enzyme-Linked Immunosorbent Assay in the Serological Diagnosis of Ovine Progressive Pneumonia Virus in U.S. Sheep

A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect enzyme-linked immunosorbent assay (iELISA) was validated with samples from U.S. sheep and by the use of radioimmunoprecipitation as the standard for comparison. The sensitivity and the specificity were 86.0% (±5.8%) and 95.9% (±2.9%), respectively. The iELISA format and phylogenetic differences based on the MVV gag sequence contribute to the reduced sensitivity.The diagnosis of small ruminant lentivirus (SRLV) infections in sheep and goats is most commonly determined by the detection of anti-SRLV antibodies in serum by an enzyme-linked immunosorbent assay (ELISA) that is typically created by the use of maedi-visna virus (MVV) or caprine arthritis-encephalitis virus (CAEV) isolates from sheep or goats of a given region or country (1). ELISA formats are typically validated against reference standard tests, including the agar gel immunodiffusion (AGID) assay, the radioimmunoprecipitation (IP) assay, or Western blot analysis. Although most seropositive sheep and goats do not show clinical signs of SRLV disease, they are persistent and potential reservoirs for transmission. Therefore, highly specific and sensitive serological diagnostic assays are essential for the early detection of SRLV.Three hundred ten of 332 serum samples from U.S. sheep from a previous CAEV competitive ELISA (cELISA) validation study (4) were tested in duplicate by using a Chekit CAEV/MVV antibody test kit (IDEXX Laboratories, The Netherlands), according to the manufacturer''s instructions. The CAEV/MVV indirect ELISA (iELISA) results were compared with those of the ovine progressive pneumonia virus (OPPV) WLC1 radio-IP assay, which has been described previously (4). The CAEV/MVV iELISA utilizes whole virus from Swiss MVV strain OLV as the antigen (15, 16). With a value of ≥60% being defined as a CAEV/MVV iELISA-positive serum sample, the sensitivity and the specificity of the CAEV/MVV iELISA were 74.0% (±7.6%) (95% confidence interval) and 98.3% (±2.0%), respectively, compared to the results of the radio-IP assay. Since the sensitivity was less than adequate, we reassessed the cutoff by calculating the mean value (in percent) ± 2 standard deviations for the radio-IP assay-negative serum samples. The results of that analysis placed the cutoff mean value at 33.1%. By using the new cutoff value, the sensitivity of the iELISA improved to 86.0% (±5.8%) and the specificity decreased slightly to 95.9% (±2.9%) compared to the results of the radio-IP assay. However, compared to the CAEV cELISA, which has a sensitivity of 98.6% and a specificity of 96.9% when the results of the radio-IP assay are used as the reference standard, the iELISA had a reduced sensitivity.Since the sera were taken from a number of different U.S. sheep kept under different husbandry and management conditions, we also wanted to test the performance of the CAEV/MVV iELISA with sera from one flock in which the sheep are exposed to the same husbandry and management conditions. Sera from an Idaho sheep flock (n = 405) consisting of sheep of the Rambouillet, Polypay, and Columbia breeds ages 3, 4, 5, and 6 years were tested by the iELISA. The results were compared to those of the CAEV cELISA by using the new iELISA cutoff value of 33.1%, and the discrepant samples were analyzed by Western blotting with OPPV WLC1 and by previously published methods (2). The positive and negative concordances of the CAEV cELISA and the CAEV/MVV iELISA were 92.5% (±3.1%) and 99.3% (±1.4%), respectively. Eighteen of 20 CAEV/MVV iELISA-negative and CAEV cELISA-positive serum samples tested positive by Western blot analysis, and the 2 remaining discrepant serum samples tested negative by Western blot analysis. One CAEV/MVV iELISA-positive and CAEV cELISA-negative sample tested negative by Western blot analysis. The 95% confidence interval for the positive and the negative concordances of the results of the CAEV/MVV iELISA relative to those of the CAEV cELISA for Idaho sheep and U.S. sheep overlapped (data not shown).A difference in the limit of detection between the CAEV/MVV iELISA and the CAEV cELISA may be a major reason for the reduced sensitivity of the CAEV/MVV iELISA (86%) compared to that of the CAEV cELISA (98.6%) with sera from U.S. sheep. Sera require dilution 1:10 for testing by the CAEV/MVV iELISA, whereas undiluted sera are used for the CAEV cELISA. To test whether the limit of detection is greater for the CAEV cELISA than the CAEV/MVV iELISA, 15 Western blot analysis-positive, CAEV cELISA-positive, and CAEV/MVV iELISA-negative serum samples from the Idaho flock were diluted 1:10 and 1:50 with 1× phosphate-buffered saline, pH 7.5, and retested by the CAEV cELISA. Twelve of these 15 serum samples tested positive by the CAEV cELISA at a 1:10 dilution, and 7 of 15 tested positive by the CAEV cELISA at a 1:50 dilution. This indicates that the CAEV cELISA has a higher dilution limit for the detection of anti-SRLV antibodies than the CAEV/MVV iELISA with sera from U.S. sheep. This high dilution limit of detection is likely due to the format of the CAEV cELISA, in which this assay is reliant upon anti-OPPV serum antibodies to inhibit the binding of a peroxidase-labeled monoclonal antibody (monoclonal antibody 74A) to a single, specific epitope on the CAEV-63 surface envelope glycoprotein.A previous report of 95.5% sensitivity and 97.2% specificity has been reported for the iELISA with sera from Swiss sheep (15). Large SRLV strain differences between Swiss MVV and U.S. OPPV strains may account for the lower sensitivity of the iELISA with sera from U.S. sheep. Therefore, gag, which encodes the capsid protein, was evaluated because it is a B-cell-immunodominant viral antigen in sheep naturally infected with MVV and OPPV and is more conserved than other viral genes (2, 7, 17). Unfortunately, no gag sequence is available for the Swiss MVV OLV (the strain used in the iELISA); however, several representative gag sequences from SRLV strains from Swiss goats and sheep that were previously reported for SRLV clades A1, A3, A4, A5, B1, and B2 were utilized (13). Genomic DNA was isolated from peripheral blood leukocytes from nine cELISA- and iELISA-positive Idaho sheep by previously described methods (6). The gag gene encoding the capsid was amplified by PCR with the following primers: primer GAGPSf (5′-TGG-CGA-CGC-AAG-GCT-CAA-A-3′) and primer GAGPSr (5′-GCG-GAC-GGC-ACC-ACA-CG-3′) (Integrated DNA Technology, Coralville, IA). The PCR mixture consisted of 100 ng of genomic DNA from sheep peripheral blood leukocytes or goat synovial membrane cells infected with WLC1, 2.5 mM_f (final concentration) MgCl₂, 0.2 mM_f deoxynucleoside triphosphates, and 1 U of Taq polymerase (Fisher Scientific, Pittsburg, PA). Primers GAGPSf and GAGPSr bind to nucleotides 734 to 752 and 2038 to 2054, respectively, of the sequence with GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY101611","term_id":"21539308","term_text":"AY101611"}}AY101611. The amplification conditions for the gag PCR were as follows: 95°C for 4 min, followed by 25 cycles of 95°C for 30 s, 58.1°C for 30 s, and 72°C for 2 min, followed by 72°C for 7 min and 4°C indefinitely. The gag PCR products were analyzed, cloned, and sequenced by previously described methods (5). The gag sequences were further refined by hand by using the Se-Al (version 2.0) program (http://tree.bio.edu.ac.uk/software/seal/), and a consensus gag sequence was generated from gag sequences from four sheep and OPPV WLC1.For phylogenetic analysis, the appropriate model of nucleotide substitution was selected by using hierarchical likelihood ratio testing with the MrModelTest2 command block (11) executed in the PAUP program (14). A general time-reversible model (also called the GTR or REV model) (12) with invariant sites and a measure of the rate of heterogeneity determined by use of the gamma distribution was chosen for full Bayesian analysis by using the program MrBayes (8). A 50%-majority-rule consensus tree based on the gag sequences was constructed by using the Mesquite software package (10). The resulting Bayesian phylogenetic tree showed that the sequences from the Idaho sheep formed one clade with OPPV WLC1, OPPV 85/34, and a single Swiss goat SRLV strain (strain 5692 A3) with high posterior probabilities (0.99 to 1.0) (Fig. ​(Fig.1).1). The finding that three gag sequences from Swiss sheep are found in other clades aside from the U.S. sheep OPPV clade (6247 A1, 5720 B2, and 5776 B1) suggests that Swiss sheep SRLV strains are quite different from U.S. OPPV strains, and these differences contribute to the reduced sensitivity of the iELISA with sera from U.S. sheep compared to that achieved with sera from Swiss sheep.Open in a separate windowFIG. 1.A 50%-majority-rule consensus tree of SRLV gag nucleotide sequences obtained by Bayesian phylogenetic analyses with the general time-reversible model with invariant sites and a measure of the rate of heterogeneity. Consensus gag nucleotide sequences from Idaho sheep (sheep 28Oct76, 29Oct10, 28Oct101, 28Oct27, 28Oct83, 28Oct18, 28Oct89, 28Oct68, and 28Oct99) and a consensus gag nucleotide sequence for WLC1 were utilized and compared to the gag nucleotide sequences from other U.S. OPPV strains (85-34), MVV strains, and CAEV strains. The GenBank accession numbers are given in parentheses next to the name or identification number given in previous studies. The numbers on the branches represent the Bayesian posterior probabilities.This combination of information suggests that U.S. sheep maintain strains of small ruminant lentiviruses phylogenetically different (determined on the basis of the gag sequences) from those from most other parts of the world. This suggests that the creation and validation of one serological assay that is based on one viral strain and that is accepted worldwide might not be successful due to the diversity of SRLV strains worldwide (3, 9).     

Expression and evaluation of Chikungunya virus E1 and E2 envelope proteins for serodiagnosis of Chikungunya virus infection

Purpose

Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIKV fever, which is a mosquitoe-transmitted viral disease in Africa, India, South-East Asia, and recently Southern Europe. Currently, serological diagnostic tests such as hemagglutination inhibition test (HI test), in-house IgM capture enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence test were used for diagnosis of chikungunya fever, which are based on whole virus antigens.

Materials and Methods

CHIKV E1, and E2 envelope proteins for the CHIKV-specific serodiagnostic reagents for chikungunya fever were expressed in baculovirus expression system. The seroreactivity of recombinant CHIKV E1 and E2 envelope proteins were evaluated using sera panels of patients from Laboratoire Marcel Merieux by indirect IgM capture ELISA.

Results

The recombinant CHIKV E1 and E2 envelope protein showed sensitivity of 77.5% and 90%, respectively. The specificities of both CHIKV E1 and E2 envelope proteins were 100%.

Conclusion

The recombinant CHIKV E1 and E2 envelope proteins could be a useful diagnostic reagent for CHIKV infection.     

Detection of serum antibodies to ovine progressive pneumonia virus in sheep by using a caprine arthritis-encephalitis virus competitive-inhibition enzyme-linked immunosorbent assay

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.     

Recombinant M protein-based ELISA test for detection of antibodies to canine coronavirus

The membrane (M) protein of canine coronavirus (CCoV) was cloned and expressed in E. coli. The purified recombinant protein was then evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for detection of CCoV antibodies in dog sera. Fifty serum samples, screened previously by whole virus ELISA and Western blotting, were tested. When the performance of the new test was compared with those of whole virus ELISA and Western blotting, an excellent correlation was found with the latter two assays. The ELISA based on recombinant M protein represents an alternative and valid test for detection of antibodies to CCoV in dog sera.     

Evaluation of agar gel immunodiffusion serology using caprine and ovine lentiviral antigens for detection of antibody to caprine arthritis-encephalitis virus.

The sensitivity of the agar gel immunodiffusion (AGID) test for the detection of antibody to caprine arthritis-encephalitis virus (CAEV) was investigated with CAEV or ovine progressive pneumonia virus (OPPV) as the source of antigen. A total of 218 goat serum specimens were tested for anti-CAEV antibody by AGID and immunoprecipitation of [35S]methionine-labeled CAEV. In comparison with that of immunoprecipitation, the sensitivity of the CAEV AGID test was 0.91, and that of the OPPV AGID test was 0.56. The AGID test with either antigen was 100% specific. The lower sensitivity of the OPPV AGID test in detecting caprine antibody to CAEV indicates that OPPV antigen is of limited value for use in CAEV diagnosis and control programs.     

Recombinant Antigen Targets for Serodiagnosis of African Swine Fever

African swine fever (ASF) is an infectious and economically important disease of domestic pigs. There is no vaccine, and so reliable diagnosis is essential for control strategies. The performance of four recombinant ASF virus (ASFV) protein (pK205R, pB602L, p104R, and p54)-based enzyme-linked immunosorbent assays (ELISAs) was evaluated with European porcine field sera that had been established by Office International des Epizooties (OIE)-approved tests to be ASFV negative (n = 119) and ASFV positive (n = 80). The κ values showed that there was almost perfect agreement between the results of the “gold standard” test (immunoblotting) and the results obtained by the p54-specific ELISA (κ = 0.95; 95% confidence interval [CI], 0.90 to 0.99) and the pK205R-specific ELISA or the pB602L-specific ELISA (κ = 0.92; 95% CI, 0.86 to 0.97). For the pA104R-specific ELISA, there was substantial to almost perfect agreement (κ = 0.81; 95% CI, 0.72 to 0.89). Similar results were observed by the OIE-approved ELISA (κ = 0.89; 95% CI, 0.82 to 0.95). Importantly, antibodies against these proteins were detectable early after infection of domestic pigs. Preliminary testing of 9 positive and 17 negative serum samples from pigs from West Africa showed identical results by the recombinant protein-based ELISA and the OIE-approved tests. In contrast, there was a high degree of specificity but a surprisingly a low level of sensitivity with 7 positive and 342 negative serum samples from pigs from East Africa. With poorly preserved sera, only the p104R-specific ELISA showed a significant reduction in sensitivity compared to that of the OIE-approved ELISA. Finally, these recombinant proteins also detected antibodies in the sera of the majority of infected warthogs. Thus, recombinant ASFV proteins p54, pB602L, and pK205R provide sensitive and specific targets for the detection of antibodies in European and West African domestic pigs and warthogs.African swine fever (ASF) virus (ASFV) is an icosahedral cytoplasmic DNA virus that infects pigs and soft ticks of the Ornithodoros genus. This virus is the sole member of the family Asfarviridae (6). ASFV has variable pathogenicity in domestic pigs, with infections ranging from being highly lethal to subclinical. Infection of wildlife mammalian hosts, the warthog and the bushpig, on the other hand, results in an unapparent, nonpathogenic infection, which provides a potentially dangerous reservoir of virus. There is no vaccine. Therefore, rapid and specific diagnostic procedures are an essential component of any control strategy. In addition, the presence of virus strains with reduced virulence and the resulting presence of asymptomatic infected animals (4, 11) make the serological diagnosis the only realistic basis for the control of the disease in affected countries. As a general rule, pigs that survive natural infection develop antibodies against ASFV from 7 to 10 days after infection. These antibodies persist for long periods of time (16), perhaps due to continuous antigenic stimulation by the frequent occurrence of persistent infection. Thus, antibody detection is a rational approach to the detection of the subacute and chronic forms of the disease.The role of specific antibodies in immunity to ASFV infection in pigs has been controversial. The passive transfer of anti-ASFV antibodies delays the onset of clinical signs but does not consistently protect animals from eventual death (25, 26, 17). Similarly, vaccination with the putative protective proteins p30 and p54 conferred protection to only 50% of the tested animals (10). In a different study, in which no protection was observed after immunization against p54, p30, and p72, the only effects detected were a delay in the onset of clinical disease and a reduction in the level of viremia (15). Such observations emphasize the role of cell-mediated immune responses during ASFV infection. Indeed, a positive correlation was observed between the stimulation of NK cell activity and the absence of clinical symptoms after experimental infection, suggesting that NK cells play an important role in protective immunity (13). In addition to NK cells, CD8⁺ T cells may also play a role, as their depletion in vivo abrogates protective immunity to ASFV infection (18). Therefore, immunity to ASFV is likely to be due to a combination of both serological and cellular mechanisms. This complexity of the porcine immune response to ASFV has impaired the development of an effective vaccine but does justify diagnosis on the basis of the detection of antibodies.Current Office International des Epizooties (OIE)-approved assays for ASFV-specific antibody determination consist of an initial screening of sera by enzyme-linked immunosorbent assay (OIE-ELISA), followed by an immunoblotting assay to confirm the results for samples with doubtful and positive results. These OIE-approved tests are based on the use of live virus as the antigen and involve the requirement of level 3 biosafety facilities for the production and handling of the pathogen (16, 20, 21). The risk associated with the handling of live virus, together with the lack of reliability of the OIE-ELISA for the analysis of poorly preserved samples so often encountered in sera of African origin (1, 3), provides the stimulus for the development of alternative and more robust systems for the detection of anti-ASFV antibodies. Indeed, previous studies have demonstrated that recombinant viral proteins can give improved specificity and sensitivity when they are applied to the analysis of European field sera (9, 19, 22).In previous studies, 12 serological immunodeterminants of ASFV were characterized by exhaustive screening of a representative lambda phage cDNA expression library of the tissue culture-adapted Ba71V isolate of ASFV for antibodies (12). These included four proteins encoded by previously unassigned open reading frames (ORFs) (B602L, C44L, CP312R, and K205R), as well as some of the more well studied structural proteins (pA104R, p10, p32, p54, and p73) and three enzymes (RNA reductase, DNA ligase, and thymidine kinase). The complete sequence of each of these proteins was then recloned into pGEX for expression in Escherichia coli, followed by purification of the recombinant proteins and testing against sera from experimentally infected animals. Four of these proteins (p54/E183L, histone-like/pA104R, pB602L, and pK205R) were revealed to be promising targets for both immunoglobulin G (IgG) and IgM antibody responses (23), and further validation of these proteins as targets is the focus of this work. The results obtained by the analysis of a large collection of serum samples from susceptible animals from Europe and Africa were comparable to the results obtained by the OIE-ELISA prescribed for use for international trade.     

Comparison of serological tests for detection of antibodies to infectious laryngotracheitis virus

Four serological tests i.e. ELISA, serum neutralisation (SN), fluorescent antibody (FA), and agar gel immunodiffusion (AGID) were compared for sensitivity using several criteria, for detection and titration of infectious laryngotracheitis (ILT) virus antibodies in chicken sera. In the ELISA test, sera were tested in parallel on virus positive and negative control antigens with results expressed as positive-negative difference. Non-specific binding was not a problem in this test. SN tests were performed in microtitre plates using chick embryo liver cells, while sera for FA tests were titrated on multispot slides on which were fixed ILT virus-infected chick embryo liver cell cultures. The AGID test was the standard test still widely used for serological diagnosis of ILT. The four tests were compared using (1) sera from experimentally inoculated birds bled regularly at intervals from 4 to 35 days post-inoculation, (2) convalescent sera from a natural outbreak of ILT, and (3) serial dilutions of an ILT positive serum. In all experiments the ELISA test was of slightly greater sensitivity than SN and was comparable to the FA test. In the experimentally infected birds ELISA and FA test detected sero-conversion in more birds at 7 days than SN. In tests with the serially diluted hyperimmune ILT serum, ELISA, FA and SN tests were comparable. SN however was the most useful test for quantification of ILT antibodies. ILT-SN titres in birds were never high, the highest titre recorded in experimental birds and in convalescent sera was 1/48. AGID was found to be less sensitive than ELISA, FA or SN test but was considered useful for detection of antibodies on a flock basis, since, with the convalescent sera AGID detected a significant proportion of positives.     

Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates.

Monoclonal antibodies (MAbs) directed against two phenotypically distinct ovine lentivirus (OvLV) strains were generated by fusion of BALB/c SP2/0-Ag 14 myeloma cells with spleen cells from mice immunized with purified OvLV. Hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and analysis of reactivity on immunoblots. The majority (17 of 21) of the MAbs recognized the gag-encoded capsid protein, CA p27, of both strains. Four other MAbs recognized a smaller structural protein, presumably a matrix protein, MA p17. Three distinct epitopes on CA p27 and one on MA p17 were distinguished by the MAbs with competition ELISA. MAbs from each epitope group were able to recognize 17 North American field isolates of OvLV and the closely related caprine arthritis-encephalitis virus (CAEV). Analysis of the data indicated that these epitopes were highly conserved among naturally occurring isolates. A representative MAb from each epitope group of anti-CA p27 MAbs reacted with four field strains of OvLV and CAEV on immunoblots. An anti-MA p17 MAb recognized the same OvLV strains on immunoblots but failed to recognize CAEV. MAbs which recognize conserved epitopes of gag-encoded lentivirus proteins (CA p27 and MA p17) are valuable tools. These MAbs can be used to develop sensitive diagnostic assays and to study the pathogenesis of lentivirus infections in sheep and goats.     

A viral transmembrane recombinant protein-based enzyme-linked immunosorbent assay for the detection of bovine immunodeficiency virus infection

The expression of bovine immunodeficiency virus (BIV) truncated transmembrane envelope protein (designated hereafter tTM) in insect cells has been described previously (Abed, Y., St-Laurent, G., Zhang, H., Jacobs, R.M., Archambault, D., 1999. Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane proteins expressed from recombinant baculovirus. Clin. Diagn. Lab. Immunol. 6, 168-172). In this study, a tTM-based enzyme-linked immunosorbent assay (ELISA) was developed for the serodetection of BIV infection. A total of 109 bovine sera including 86 BIV-negative and 23 BIV-positive serum samples were tested. The ELISA results were compared with those of three Western blot assays using, as test antigens, cell culture-derived whole virus proteins (WB1), and the tTM (WB2) and p26 (WB3) fusion proteins expressed from recombinant baculovirus in insect cells, respectively. The concordances of the ELISA results with those of the WB1, WB2, and WB3 were 97.2, 100 and 97.2%, respectively. The tTM protein-based ELISA and Western blot permitted the detection of BIV infection in cattle whose sera failed to react with the p26 fusion protein and the whole virus protein preparation. The tTM recombinant protein was also used to study the kinetics of appearance of antibodies against BIV transmembrane envelope protein in rabbits infected experimentally with BIV. Antibodies to tTM were detected at 28 days post-infection and persisted through the entire 36-39.5 months experimental time period. The results of this study showed that the tTM-ELISA might be useful for the serodetection of BIV-infected animals, and for basic studies on BIV replication life cycle.     

Development of TaqMan-based qPCR method for detection of caprine arthritis–encephalitis virus (CAEV) infection

A specific and sensitive two-step TaqMan real-time PCR has been developed for rapid diagnosis of caprine arthritis-encephalitis virus (CAEV) infection by using a set of specific primers and a TaqMan probe targeting a highly conserved region within the gene encoding the viral capsid protein (CA). The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and isolated PBMCs, with a lower detection limit of 10² copies and a linear dynamic range of 10⁵ to 10¹⁰ copies/ml. There was no cross-reaction with other animal viruses (e.g., goat pox virus, bovine leukemia virus, bovine mucosal disease virus, swine influenza virus and Nipah virus). When applied in parallel with serological AGID and conventional PCR for detection of CAEV in field samples, this assay exhibited a higher sensitivity than these traditional methods, and 7.8 % of the 308 specimens collected in the Shanxi and Tianjin regions of China from 1993 to 2011 were found to be positive. Thus, the TaqMan qPCR assay provides a fast, specific and sensitive means for detecting CAEV proviral DNA in goat specimens and should be useful for large-scale detection in eradication programs and epidemiological studies.     

Development of a recombinant ELISA using yeast (Pichia pastoris)-expressed polypeptides for detection of antibodies against avian influenza A subtype H5

Two truncated sequences (designated P1 and rHA1) of influenza A virus subtype H5 haemagglutinin (HA) were cloned and expressed in yeast Pichia pastoris (P. pastoris). These polypeptides were used in an indirect recombinant ELISA (rELISA) for detection of H5 antibodies in poultry. Serum samples obtained from broiler chickens vaccinated with commercial inactivated vaccine (H5N2) and control negative sera from non-vaccinated chickens against influenza were tested using rP1-ELISA, rHA1-ELISA, whole H5N1-ELISA, Western blot, agar gel immunodiffusion (AGID) and haemagglutination inhibition (HI) tests. The rHA1-ELISA proved to be highly sensitive and specific. To study the validity of rHA1-ELISA, a total of 179 serum samples obtained from commercial broiler chickens vaccinated previously with commercial H5N2 inactivated vaccines, were tested by rHA1-ELISA, commercial ELISA (cELISA) and HI. The relative sensitivity and specificity between rHA1-ELISA, and HI tests were 100% and 70%, respectively, and between cELISA and HI were 100% and 57%, respectively. The agreement ratio between rHA1-ELISA and HI was 84.9% and between cELISA and HI tests was 76.5%. Serum samples obtained from ducks vaccinated with commercial inactivated H5N2 were tested by rHA1-ELISA and the results showed significant reactivity with duck sera. In conclusion, the results demonstrate the potential applicability of the rELISA for the determination of antibodies to H5 influenza virus in chickens and ducks.     

Presence of circulating antibodies against gag-gene MuLV proteins in patients with autoimmune connective tissue disorders

An immunoblotting procedure using viral proteins from purified murine sarcoma virus or MSV-(MLV) has been developed to characterize antiviral antibodies in sera from patients with autoimmune connective tissue disorders. Fifty-eight sera with anti-Sm, anti-RNP, anti-SS-B (La), and other undefined specificities were found to react with several major viral polypeptide bands. Most of them corresponded to gag-gene-encoded products: pr65^gag, p40^gag, p30, p15, p12 and p10. Other bands with molecular weights averaging 90K, 60K, 45K, and 28K were recognized by a few sera. Immunological specificity of the reaction was assessed by reproducing the tests with IgG purified from sera and from corresponding F(ab′)2 fragments. Moreover, the specificity of the reaction with gag proteins was confirmed by repeating the tests with p30 and p15 prior purified by immunoprecipitation with anti-p30 and anti-p15 goat sera. Furthermore, the gag polypeptides were recognized by human sera by replacing MSV-(MLV) by three other murine retroviruses of different origin. An indirect confirmation of these results was obtained by applying this method to sera of MRL lpr/lpr mice which develop an autoimmune syndrome comparable to that of human systemic lupus erythematosus. In agreement with previously published results (C. Rordorf, C. Gambke, and J. Gordon (1983), J. Immunol. Methods59, 105–112), we found that anti-gag-gene antibodies were present in the sera of individual mice. Patterns of reactivity were found to vary with the age of the animals. No retroviral polypeptide was significantly detected in the great majority (80%) of sera from normal donors. However, 5 out of 25 sera showed faint bands although to a lesser extent than pathological sera. These five sera also reacted with HeLa cell purified HnRNPs, suggesting that their normal status should be reconsidered.     

Development of a highly sensitive and specific enzyme-linked immunosorbent assay based on recombinant matrix protein for detection of avian pneumovirus antibodies

The matrix (M) protein of avian pneumovirus (APV) was evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for diagnosis of APV infection, a newly emergent disease of turkeys in United States. Sera from APV-infected turkeys consistently contained antibodies to a 30-kDa protein (M protein). An ELISA based on recombinant M protein generated in Escherichia coli was compared with the routine APV ELISA that utilizes inactivated virus as antigen. Of 34 experimentally infected turkeys, 33 (97.1%) were positive by M protein ELISA whereas only 18 (52.9%) were positive by routine APV ELISA 28 days after infection. None of the serum samples from 41 uninfected experimental turkeys were positive by M protein ELISA. Of 184 field sera from turkey flocks suspected of having APV infection, 133 (72.3%) were positive by M protein ELISA whereas only 99 (53.8%) were positive by routine APV ELISA. Twelve serum samples, which were negative by M protein ELISA but positive by routine APV ELISA, were not reactive with either recombinant M protein or denatured purified APV proteins by Western analysis. This indicates that the samples had given false-positive results by routine APV ELISA. The M protein ELISA was over six times more sensitive than virus isolation (11.5%) in detecting infections from samples obtained from birds showing clinical signs of APV infection. Taken together, these results show that ELISA based on recombinant M protein is a highly sensitive and specific test for detecting antibodies to APV.