An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections.

A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen. Five of the six highly reactive (57-62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.

Visna/Maedi virus genetic characterization and serological diagnosis of infection in sheep from a neurological outbreak.

An extensive outbreak characterized by the appearance of neurological symptoms in small ruminant lentivirus (SRLV) infected sheep has been identified in Spain, but the genetic characteristics of the strain involved and differential diagnostic tools for this outbreak remain unexplored. In this work, 23 Visna-affected naturally infected animals from the outbreak, 11 arthritic animals (both groups presenting anti-Visna/Maedi virus serum antibodies), and 100 seronegative animals were used. Eight of the Visna-affected animals were further studied post-mortem by immunohistochemistry. All had lesions in spinal cord, being the most affected part of the central nervous system in six of them. A representative strain of the outbreak was isolated. Together with other proviral sequences from the outbreak the virus was assigned to genotype A2/A3. In vitro culture of the isolate revealed that viral production was slow/low in fibroblast-like cells but it was high in blood monocyte-derived macrophages. The long terminal repeat (LTR) of the viral genome of this isolate lacked an U3-duplication, but its promoter activity in fibroblast-like cells was normal compared to other strains. Thus, viral production could not be inferred from the LTR promoter activity in this isolate. Analysis of the viral immunodominant epitopes among SRLV sequences of the outbreak and other known sequences allowed the design of a synthetic SU peptide ELISA that detected the Visna affected animals, representing a tool of epidemiological interest to control viral spread of this highly pathogenic strain.

Anticardiolipin antibodies in chronic hepatitis C: implication of hepatitis C virus as the cause of the antiphospholipid syndrome.

Antiphospholipid antibodies are a type of autoantibodies that have been implicated in the occurrence of thrombocytopenia and thrombotic events and have been described in autoimmune disorders and diverse viral diseases. In this study anticardiolipin antibodies (immunoglobulin G [IgG] isotype) were determined in serum from 100 patients with chronic hepatitis C and 52 healthy controls. In addition, hepatitis C virus (HCV) markers (anti-HCV and HCV RNA) were investigated in 73 patients with thrombotic disorders and no clinical evidence of liver disease; of these patients 37 cases tested negatively for anticardiolipin antibodies and 36 positively. Anticardiolipin test was positive more frequently (22%) in the group of patients with chronic hepatitis C than in healthy controls (1.9%; P < .001). Using conditional logistic-regression analysis we found that in hepatitis C patients the presence of thrombocytopenia, portal hypertension and the existence of prior thrombotic episodes were significantly related to positivity for anticardiolipin antibodies (P < .05 in all cases). In patients with no evidence of liver disease and a history of thrombotic events, hepatitis C markers were absent in all cases who tested negatively for anticardiolipin antibodies (n = 37), but were present in 16.7% of those positive for anticardiolipin (n = 36) (P = .01). In conclusion, anticardiolipin antibodies are frequently found in patients with chronic hepatitis C and in these patients they may be implicated in the occurrence of thrombosis and in the development of thrombocytopenia. Occult HCV infection is present in a significant proportion of patients with thrombotic disorders and positive for anticardiolipin (the antiphospholipid syndrome).