In this study, characterization of the gag gene of small ruminant lentiviruses was carried out in Italian mixed flocks. The nearly complete gag gene was amplified and sequenced. Within genotype A, subtype A1 and a novel subtype, A8, were found in goats, and another novel subtype, A9, was found in both sheep and goats. Subtype B1 was found in both host species and subtype B2 was identified only in sheep. A novel, highly divergent sequence was obtained from goats in two epidemiologically related flocks and is proposed to represent a novel genotype, E. Major epitopes of matrix and capsid antigen were highly divergent, suggesting that serological identification of animals infected with genotype E may have been missed by using currently available diagnostic tests. A recombinant subunit ELISA, based on genotype E-specific epitopes, was developed and a third independent flock carrying this genotype was identified, based on serology.
The pol and gag gene fragments of small ruminant lentivirus field isolates collected in the last decade in Italy were amplified, sequenced, and analyzed. Phylogenetic analysis revealed that the majority of ovine isolates form a distinct cluster more similar to caprine lentivirus prototypes than to the visna virus prototype. These findings confirm and extend those reported by Leroux et al. (Arch. Virol., 142:1125-1137, 1997). Moreover, we observed that a variable region of Gag, included in the fragment analyzed, corresponded to one of the three major capsid antigen epitopes, which suggests that the antibody response to this epitope may be type specific. To test this hypothesis, two recombinant peptides, derived from the Icelandic prototype K1514 and this novel genotype, were expressed and used in an enzyme-linked immunosorbent assay to screen a panel of ovine and caprine sera collected from different geographical locations in Italy. Several sera reacted in a type-specific manner, indicating that in a diagnostic setting the combination of at least these two type-specific peptides is necessary to cover a wide range of infections. Additionally, these results support the hypothesis of cross-species transmission based on the phylogenetic analysis described above. This has implications for the control and eradication of small ruminant lentivirus infections.To date, maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are considered to be two antigenically related and genetically distinct lentiviruses of the Retroviridae family (3). Since the cross-reactivity between MVV and CAEV involves the major structural proteins (7), a number of serological tests have been proposed, based on ovine strains, to detect specific antibodies in both species. Ovine lentiviruses are usually easier to grow in tissue culture, and well-adapted strains have been extensively used to develop native-based immunoassays (9, 19). Furthermore, since sequence information was first produced from ovine strains, recombinant antigens have been largely employed and characterized from ovine isolates (12,13,14,25,33). The development of a diagnostic test capable of detecting the widest range of infection is obvious from a practical point of view. The current concept of the universality of a single-strain-based immunoassay is based on the finding that the gag-encoded capsid antigen (CA) and the env-encoded transmembrane (TM) protein are conserved among small ruminant lentiviruses (7, 21), despite the variability of the env-encoded surface antigen (1, 31). However, further characterization of the immunodominant epitopes of the major CA has shown that these epitopes are at least in part quite variable, questioning the use of single-strain-based immunoassays for diagnostic purposes. In particular, the immunodominant region of the CA involved in the cross-reaction between ovine and caprine infection was recently identified (22). Partial mapping studies suggested that at least two consecutive linear epitopes, located in the N-terminal half of t...
17The highly divergent SRLV genotype E has recently been characterized in Italy as 18 a low pathogenic caprine lentivirus in the Roccaverano breed. The availability of 19 a genotype specific diagnostic test based on a comparative assay, using a 20 combination of genotype specific recombinant antigens allows a wide serosurvey 21 in other goat populations. The island of Sardinia still has the highest small 22 ruminant population of any Italian region and crossbreeding has been limited to 23 goats, mainly with the Maltese breed. 24A serological survey was carried out on sheep flocks and goat herds, using 25 individual sera as well as a bulk milk-adapted procedure. Genotype E was 26 identified in more than 50% of goat herds and none of the sheep flocks thus 27 supporting the idea that this genotype is specifically associated with the goat 28 species. The full length proviral sequence of a Sardinian isolate revealed and 29 confirmed the deletion of dUTPase subunit and the absence of both vpr gene and 30 the 71 bp repeat of the LTR. Genetic similarity of this isolate with the prototype 31 strain Roccaverano was no more than 84%, supporting the designation of two 32 subtypes within genotype E. Nevertheless, in vitro properties of the Sardinian 33 strain were different from those of the Roccaverano strain in terms of ability to 34 infect synovial membrane and produce syncitia. Remarkable differences in the 35 HV1 and HV2 of the env gene were recorded, with the Sardinian isolate 36 displaying sequence motif more similar to arthritic strains. Data presented suggest 37 diffusion of genotype E is wider than previously thought. 38 39
Two groups of sheep were experimentally infected by intratracheal route with two small ruminant lentivirus (SRLV) isolates belonging to different genotypes (It-561 genotype A3 and It-Pi1 genotype B2). Seroconversion was evaluated using recombinant homologous and heterologous matrix protein/capsid antigen fusion protein. Results clearly indicate that seroconversion against homologous antigen was detected well in advance as regards heterologous antigen in both groups, although the advantage of using homologous antigen was less evident in detecting seroconversion against the caprine arthritis encephalitis virus (CAEV)-like strain, compared with the maedi-visna virus (MVV)-like infection. Commercially available ELISAs detect CAEV-like seroconversion earlier than MVV-like infection suggesting a closer relationship between CAEV-like isolate and the antigen used in the latter ELISA tests. Seven recombinant subunits developed from matrix protein and capsid antigen of strain K1514 (prototype A1) were used to better define the antibody response in sheep infected with It-561 isolate. Two animals clearly reacted against type specific epitopes in the early stage of infection.This study highlights the relative insensitivity of gag encoded cross-reacting epitopes during the early stage of infection and suggests the development of novel diagnostic tests based on both genotype specific antigens. #
Leishmaniases comprise a broad spectrum of human and animal diseases caused by infection of the mononuclear phagocyte system with protozoan parasites of the genus Leishmania. These agents are transmitted by the bite of phlebotomine sandflies in tropical, subtropical, and temperate zones of the world. Although humans are the sole reservoir hosts for some Leishmania species, in most cases other animals play a major role in the maintenance of infections. In countries of the Mediterranean basin, Middle East, and Latin America, wild canids and domestic dogs are the main reservoirs of zoonotic visceral leishmaniasis (ZVL), a severe disease caused by Leishmania infantum (synonym: Leishmania chagasi) (9). Over the past decade, the frequency of ZVL increased due to climate changes and human factors (5, 10).The diagnosis of L. infantum infection in dogs (canine leishmaniasis) is important in veterinary practice and in surveillance of ZVL. The immunofluorescent antibody test (IFAT) is routinely used for the detection of specific antibodies. IFAT is difficult to standardize and to interpret, however, and it is too laborious for screening large numbers of sera.Immunoenzymatic assays such as the enzyme-linked immunosorbent assay (ELISA) are easier to standardize and more practical as routine laboratory tools. The performance of ELISA tests is greatly affected by the quality of the antigens used, however, and test specificity limitations are the main drawback when crude antigen preparations are used.Recombinant technology, together with the characterization of specific immunodominant antigens at the genetic level, allowed the development of a second generation of diagnostic immunoassays, and the recent validation of a recombinant K39 ELISA as a diagnostic marker for canine leishmaniasis represents a good example (11). The rK39 (recombinant K39) antigen is a repetitive immunodominant B-cell epitope of the 230-kDa kinesin-related protein of L. chagasi (3, 13). In Leishmania spp., K39 antigen is mainly expressed in the amastigote stage and elicits a strong immunoresponse in both asymptomatic and clinically infected dogs.In addition to K39, two other antigens of L. chagasi (K9 and K26) have been genetically characterized (1): K26 shows a central 14-amino-acid repeat region which is specific to L. chagasi and L. donovani and a flanking region homologous to K9. Since these two antigens had not been evaluated as diagnostic markers for canine leishmaniasis, in this work we characterized the recombinant K9 and the recombinant repeat region of K26 expressed in Escherichia coli. In addition, since it was not clear whether the single repetition unit of K39 antigen carried an immunodominant epitope, the 39-aminoacid subunit of this kinesin-related protein was expressed in a similar way. The three recombinant antigens were then employed in a multiple-well ELISA to screen a panel of wellcharacterized dog sera.DNA was extracted from promastigotes of the L. infantum reference strain MHOM/TN/80/IPT1 (zymodeme MON-1) by conventional procedures. ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.