TaqMan Real-Time Reverse Transcription-PCR and JDVp26 Antigen Capture Enzyme-Linked Immunosorbent Assay To Quantify Jembrana Disease Virus Load during the Acute Phase of In Vivo Infection

Abstract

Jembrana disease virus (JDV) is an acutely pathogenic lentivirus that affects Bali cattle in Indonesia. The inability to propagate the virus in vitro has made it difficult to quantitate JDV and determine the kinetics of virus replication during the acute phase of the disease process. We report for the first time two techniques that enable quantification of the virus and the use of these techniques to quantify the virus load during the acute phase of the disease process. A one-step JDV pol TaqMan Jembrana disease virus (JDV) causes an atypical lentivirus infection that results in an acute severe disease that affects Bali cattle (Bos javanicus) within Indonesia (4,20,36). JDV has a short incubation period of 4 to 12 days, and the disease is characterized by a febrile response, lethargy, anorexia, leukopenia, and enlargement of superficial lymph nodes and a mortality rate of about 17% (36). The majority of animals develop detectable antibodies to the virus only 6 weeks or more after recovery from the acute phase of the disease (17, 41). Surviving animals are resistant to reinfection, remain infectious for at least 2 years, and do not appear to suffer further episodes of disease (35).Attempts to cultivate JDV in vitro have been unsuccessful (42), and this has restricted the development of assays for the quantification of infectious virus. A series of animal bioassay experiments reported by Soeharsono et al. (35) provide the only data available regarding virus replication during the acute phase of disease process. These studies revealed that there was a high titer of infectious virus in plasma of about 10 8 50% cattle infectious doses (ID 50 )/ml during the acute febrile period, decreasing to low levels and persisting at low levels in recovered animals. A strong correlation between the initial dose of virus in a sample and the time between infection and onset of the acute febrile disease period was observed (35). Although this bioassay provided a method for titration of infectious virus, it was time-consuming and expensive. Techniques that do not require the use of animals for assay of virus are needed for further studies on the kinetics of virus replication during the acute phase of the disease process and for understanding the persistence of virus in recovered animals. Additionally, the development of an inactivated virus vaccine for JDV which relied on clinical observations to determine the efficacy of different vaccine formulations has been reported (16), and further studies of vaccine development would be greatly facilitated by the capacity to quantify the virus load in vaccinated animals.In this paper, we describe the development and correlation of two techniques for the detection and quantification of the JDV load in plasma: the quantification of virus RNA and the quantification of virus antigen by enzyme-linked immunosorbent assay (ELISA). The quantification of circulating viral RNA is a widely accepted method for monitoring the levels of human immunodeficiency virus (HIV) RNA, and the quantity of circulati...

Vaccine against Jembrana Disease Virus Infection: A Summary Findings

“…Laboratorically, the notified major changes in haematological condition included a normocytic normochronic anaemia, leucopenia (particularly lymphopenia, eosinopenia and a slight neutropenia), a mild thrombocytopenia, elevated blood urea concentrations and reduced total plasma protein (Soesanto et al, 1990). Viruses are also detected in a high titer (up to 10 8 ID 50 /mL, equaling to 10 10 to 10 11 viral genome copies/ml) in the plasma fraction of the blood during the febrile state Soesanto et al, 1990;Stewart et al, 2005). The majority infected animals cultivate possible amount of antibodies to be detected only 6 weeks or more after recovery from the acute phase of the natural disease (Hartaningsih et al, 1994).…”

“…It has been shown that disease transmission possibly occurs when the virus titer is above a threshold below which the risk of disease transmission is highly reduced. In Jembrana disease, the threshold is thought to be 10 6 genome copies/mL in plasma, equivalent to 10 4 ID50/mL in plasma and a titer of 10 10 -10 11 genome copies/ml is currently observed during the febrile state Soesanto et al, 1990;Stewart et al, 2005). Due to the high titers of infectious viral particles, virus transmission is most likely to occur during the acute phase.…”