Isolation of elephant endotheliotropic herpesviruses in Asian Elephants (Elephas maximus)

Abstract

Elephant endotheliotropic herpesviruses (EEHV) infection causes an acute fatal hemorrhagic disease, which brought terribly lost in young Asian elephants (Elephas maximus). Recently, in vitro isolation and propagation of the virus is not successful. This study aims to isolate and propagate the EEHV using the continuous cell lines, derived from human or animal origins. The human cell lines, including A549, U-937, RKO, SW620, HCT-116, HT-29, and animal cell lines, including MARC-145, Vero, MDCK, CT26.CL25, sp2/0-Ag14, were used in this study. Mixed frozen tissue samples from the heart, lung, liver, spleen and kidney of the EEHV1A or EEHV4 cases were homogenized and used for cell inoculation. At 24, 48 and 72 hour-post infection (hpi), EEHV-inoculated cells were observed for the cytopathic effects (CPEs). Then, EEHV infection was determined by utilization of rabbit anti EEHV-gB polyclonal antibody via either immunoperoxidase monolayer assay (IPMA), indirect immunofluorescence assay (IFA),or immunohistochemistry (IHC). The results showed that after EEHV inoculation the immunolabeling positive signals were demonstrated only in U937 cells. Next, the cell supernatant fluid from infected U937 was used as an inoculum for further viral passaging. After, the viral replication in each passage of EEHV inoculated U937 cells were then validated in IHC using rabbit anti EEHV-DNA Polymerase polyclonal antibody. However, the EEHV DNA Polymerase was predominantly detected in early passages of U937 cells. In addition, the EEHV genomes were quantified using absolute quantitative polymerase chain reaction (qPCR). The genome of both EEHV1A and EEHV4 in U937 cell supernatant were gradually decreased at 1, 2, and 3 days post inoculation. This study demonstrated that, despite poorly adaptation in the U937 cells in higher passage, this cell line is promising to be used for isolation of EEHV1 and EEHV4 in vitro.