Epitopes based diagnostic of small ruminant lentivirus infetions

• Forschungsstelle: BLV
• Projektnummer: 1.01.19
• Projekttitel Englisch: Epitopes based diagnostic of small ruminant lentivirus infetions

Schlüsselwörter (Englisch)

caprine arthritis encephalitis virus, CAEV, Maedi-Visna, MVV, epitope, synthetic peptide, ELISA, serology, epitope mapping, epidemiology

Kurzbeschreibung (Englisch)

The Swiss caprine arthritis encephalitis virus (CAEV) eradication program which has reduced the seroprevalence from 60-80% to less than 2% and eliminated clinical cases in the goat population, has been a success story. However, in this last phase of the eradication program, the detection and elimination of the remaining virus carriers appears to be a very demanding task. An additional aspect has come to light which complicates the situation: so far CAEV and Maedi-Visna virus (MVV) have been considered to be specific pathogens of goats and sheep, respectively. Several observations however, indicate that these viruses may circulate between goats and sheep, a species where an eradication campaign has not yet been started. The occurrence of cross-species infections, as for instance may have happened at the Interlaken market, has lead to a new definition of CAEV and MVV, which are no longer viewed as species-specific viruses but are now referred to as small ruminant lentiviruses (SRLV). A failure to complete the eradication campaign would inevitably jeopardize the financial and working resources invested so far.
To improve our diagnostic tools and make them equal to the challenges posed by this difficult phase of the eradication campaign, the adoption of a broadly based strategy is of paramount importance. Both new tools aimed at detecting the viral genome such as MEGA-PCR (Schüpbach and Böni: Development of a high-input PCR-based method for the detection of small ruminant lentivirus sequences) and new serological tools able to detect early seroconversions and to define the source of infection by discriminating between antibodies of different specificity should be developed. Only a combined strategy will minimize the risk of missing virus carriers and of selecting animals with a tendency to slow-low seroconversions and/or viruses able to escape detection by the selected assays.

Projektziele (Englisch)

The development of new methods für serotyping field isolates of small ruminant lentiviruses.

Abstract (Deutsch)

Die serologische Analyse von experimentell infizierten Tieren hat gezeigt, dass Antikörper gegen die SU5 Region sehr früh detektiert werden können und dass diese Antikörper eine markante Affinitätsreifung zeigen. Dies alles sind positive Eigenschaften im Hinblick auf einen diagnostischen Einsatz dieser Peptide. Die Analyse von Seren, die aus PCR positiven Tieren stammen, hat bestätigt, dass die SU5 Region immunodominant und für diagnostische Zwecke geeignet ist, und dass unsere SU5 Peptid Kollektion einen beträchtlichen Teil der zur Zeit in der Schweiz bekannten SRLV Stämme abdeckt. Die Suche nach einem geeigneten Cutoff Wert für die Peptid -ELISAs hat sich als sehr schwierig erwiesen, weil ein Goldstandard für die SRLV Diagnostik fehlt. Viele der nach herkömmlichen Kriterien als seronegativ eingestuften Ziegen- und Schafseren haben eindeutig positive Reaktionen in unseren ELISAs gezeigt. Vor allem Peptide, die aus Virussequenzen abgeleitet wurden, welche phylogenetisch MVV nahestehen, haben viele positive Ergebnisse erbracht. Die Feinanalyse von drei konkreten Feldfällen hat jedoch gezeigt, dass die SU5-Peptide infizierte Tiere sehr effizient nachgewiesen haben. Diese Ergebnisse haben praktische Konsequenzen für die Weiterführung der schweizerischen CAEV Eradikationskampagne sowohl im Hinblick auf die epidemiologische Rolle der Schafe als auch derjenigen der inapparent infizierten Ziegen. Die SU5 Peptid-ELISAs könnten ein unentbehrliches Werkzeug für das effiziente Monitoring der Ziegen- und Schafpopulation werden.

Abstract (Englisch)

Part I
Compilation and characterization of a panel of synthetic peptides encompassing the immunodominant SU5 epitope of the envelope glycoprotein of Swiss small ruminant lentiviruses.

The envelope glycoprotein (Env) of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response. SU5, a linear epitope of Env, was mapped previously and found to be partly type specific and most likely immunodominant. To confirm these preliminary results and explore the possibility of applying SU5 peptides as a novel diagnostic tool, we have amplified by PCR and sequenced the SU5 encoding regions of several SRLV strains circulating in the Swiss goat and sheep populations. Synthetic peptides based on the derived amino acid sequence of different SU5 were used in ELISA to test a large panel of goat sera. With few exceptions, all goats infected with SRLV develop antibodies to at least one of these SU5 peptides. In spite of the limited size (25 to 26 amino acids depending on the strain) SU5 contains at least three distinct antibody-binding sites. The first is located exclusively in the conserved region of SU5 and is not immunodominant. The second and third binding regions encompass the most variable region and the junction between variable and constant part, respectively. Experimentally infected animals show rapid seroconversions to SU5 peptides with a distinct two-peaks kinetic. After a seroconversion the antibody titers fall in the weak positive to negative range to rapidly rise again and reach unambiguously positive and durable titers. Seroconversion is accompanied by a characteristic affinity maturation of the antibody demonstrated by the appearance and persistence of high-avidity antibody.
In contrast to the common assumption that variable epitopes are not suitable for serological diagnosis, we show that anti-SU5 antibody are produced early after infection, maturate to high avidity antibody and that by using a representative panel of SU5 peptides it is possible to detect the majority of seropositive animals.


Part II
SU5 epitopes based diagnostic of small ruminant lentivirus infections
in the context of the Swiss CAEV eradication program.

The Swiss CAEV eradication program has reduced the seroprevalence from 60-80% to less than 2% and eliminated clinical cases in the goat population. However, in this last phase of the eradication program, the detection and elimination of the remaining virus carriers appears to be a very demanding task.
In this work we analyzed 321 goats and 250 sheep randomly sampled sera using four different serological tests. The results obtained with standard tools such as immunoblot, Chekit- and Gag-GST ELISA were compared to those obtained with the novel SU5-core ELISA. Each serum was tested in parallel on 7 synthetic SU5 peptides covering a wide spectrum of viruses circulating in Switzerland (1163-, Zn-, NT-, IS-, 1355-, 615- and 4663-SU5-core). Depending on the cutoff chosen, up to 29% of the goats and 40% of the sheep showed positive reactions with at least one SU5-core peptide. Interestingly, few sera reacted to the SU5-core peptide corresponding to the CAEV strain 615, which is closely related to the virus used in Chekit ELISA, suggesting that the eradication program was very successful in eliminating animals infected with SRLV related to strain 615 but less so with other strains.
A generally accepted gold standard for SRLV diagnostic is still missing and a direct validation of the SU5-core ELISA was not possible, even using a specifically designed software (Pouillot et al., 2001). However, we found strong evidence supporting the hypothesis that SU5 seropositive animals are infected and not false positive in SU5 serology. Analyzing a flock of seronegative sheep, according to Chekit ELISA and immunoblot, but seropositive in SU5-core ELISA, we demonstrated by PCR analysis that these animals were infected. Additionally, the analysis of the deduced acid sequences of the amplified SU5 regions revealed that the infecting virus was closely related to the SRLV strain from which the SU5 peptide used to detect these animals was derived. Recently, several cases of seroconversions in flock considered free of SRLV were detected. The analysis of two such cases using SU5 peptides backed by PCR analysis showed that goats and sheep living on the same farm were infected with almost identical viruses clustering in the MVV prototype group. The horizontal spread of SRLV between sheep and goats is the most likely explanation for re-infections in sanitized flocks. Considering that the eradication campaign is targeted only to goats and not sheep, the latter must be considered a potentially dangerous reservoir for the re-infection of SRLV free goat flocks. In conclusion we have shown that SU5 synthetic peptides may become a very useful tool to monitor flocks for circulating SRLV strains that tend to evade detection by routine tests such as Chekit ELISA and even immunoblot.

Publikationen / Ergebnisse (Deutsch)

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• Analysis of the antibody response to an immunodomi [PDF] 428 kB

Publikationen / Ergebnisse (Englisch)

Mordasini F., Maeschli A., Vogt H.-R., Zahno M.-L., Peterhans E., Zanoni R., Bertoni G. (2002) Synthetic peptides derived from the envelope glycoprotein of the caprine arthritis encephalitis viruses as a tool to monitor virus heterogeneity in the field. Oral presentation, in SGM-SSM Annual Meeting 2002, Luzern.

Maeschli A., Mordasini F., Vogt H.-R., Zahno M.-L., Peterhans E., Zanoni R., Bertoni G. (2003) Detection of small ruminant lentivirus infection by molecular serology. Oral presentation, in SGM-SSM Annual Meeting

Mordasini F, Vogt HR, Zahno ML, Maeschli A, Nenci C, Zanoni R, Peterhans E, Bertoni G. J Clin Microbiol. 2006 Mar;44(3):981-91 Analysis of the Antibody Response to an Immunodominant Epitope of the Envelope Glycoprotein of a Lentivirus and Its Diagnostic Potential.