Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing.

Persistence of lymphocytic choriomeningitis virus at very low levels in immune mice.

Lymphocytic choriomeningitis virus (LCMV), strain WE, is a non-cytopathic RNA virus that is highly adapted to its natural host, the mouse. Acute infection of adult mice leads to generalized virus spread, followed by cytotoxic T lymphocyte-mediated virus clearance below the detection levels of conventional assays within 2-3 weeks. Indirect evidence had suggested that virus or viral antigen might persist in the immune mouse. Here we demonstrate LCMV-WE persistence at low levels after infection with 10(2) or 10(6) plaque-forming units, shown as viral genome, viral antigen, and replicative virus using sensitive in vitro and in vivo assays. The finding that LCMV-WE persists in the face of apparently intact immune responses resembles the situation in some viral (hepatitis B and C, HIV) and bacterial (tuberculosis, leprosy) infections in humans; the results are relevant to the understanding not only of other murine and human persistent viral infections but also of protective immunological memory by "infection immunity."

Antibodies to Mycobacterium tuberculosis-specific epitopes in lepromatous leprosy.

Sera from patients with leprosy or tuberculosis and healthy subjects have been analysed for the presence of antibodies to four species-specific mycobacterial epitopes, four different viruses and five autoantigens. Antibodies to the Mycobacterium leprae-specific 35-kD protein and phenolic glycolipid I epitopes were not present in patients with active pulmonary tuberculosis. In contrast, antibody levels to species-specific epitopes of the 38-kD and 14-kD antigens M. tuberculosis were significantly elevated in patients with lepromatous leprosy. Neither of the two antigens is cross-reactive with M. leprae at the B cell level. However, it was considered that cross-reactive helper T cells could recall the response of M. tuberculosis-specific memory B cells, which had been primed through prior self-healing tuberculous infection. As an alternative explanation, the possible role of polyclonal B cell stimulation was considered. This seemed unlikely, however, since: (i) antibody levels to autoantigens, except anti-smooth muscle, were not elevated, and (ii) antibody levels to four distinct viruses, unlike those to all mycobacterial epitopes, showed no correlation with titres, to M. tuberculosis-specific epitopes.

Host responses to Epstein-Barr virus and cytomegalovirus infection in leprosy.

A study was undertaken in patients with leprosy to assess the contribution of cell-mediated immunity to the host response to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection. Sixteen of 72 patients (22%) with lepromatous leprosy, with impaired cellular immunity, had anti-EBV titers of 1,640 or higher. Only 4 of 49 patients (8%) with tuberculoid leprosy, with intact cell-mediated immunity, attained the level of 1:640. The anti-EBV antibody titers were significantly higher in patients with lepromatous leprosy (P approximately 0.025). No significant differences were found in the level of anti-CMV antibody titers in patients with the two types of leprosy. The presence of high anti-EBV antibody titers in lepromatous leprosy suggests that cell-mediated immunity is a significant factor in host response to EBV infection. Host immune responses should be taken into consideration when assignment of an etiological role to EBV is based upon seroepidemiological data.