In vivo characterization of target cells for acute elephant endotheliotropic herpesvirus (EEHV) infection in Asian elephants (Elephas maximus)

Varankpicha Kochagul,Thunyamas Guntawang,Kornravee Photichai,Saralee Srivorakul,Wei-Li Hsu,Kidsadagon Pringproa,Tidaratt Sittisak,Chatchote Thitaram,Nattawooti Sthitmatee

doi:10.1038/s41598-020-68413-4

Abstract

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.

Highlights

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD), caused by EEHV, is a dangerous viral hemorrhagic disease affecting Asian elephants (Elephas maximus) worldwide[1,2,3]
The predicted structures from the I-TASSER software revealed that EEHV1A DNAPol had a template modeling (TM) score of 0.80 ± 0.09, while EEHV4 DNAPol had a TM score of 0.74 ± 0.11, which was similar to the structure of herpes simplex virus-1 (HSV-1) DNA polymerase
SDS-PAGE and western blot analysis showed that candidate EEHV DNAPol proteins were induced by isopropyl β-d thiogalactopyranoside (IPTG) (0.8 mM) treatment; they were present in the insoluble form (Fig. 1B,C)

Summary

Introduction

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD), caused by EEHV, is a dangerous viral hemorrhagic disease affecting Asian elephants (Elephas maximus) worldwide[1,2,3]. EEHV1A has 37 conserved core genes that are similar to other herpesviruses; 15 genes that are similar to either both betaherpesviruses and gammaherpesviruses or just betaherpesviruses; 3 genes that are lost from the betaherpesviruses; and 60 new genes absent from all h erpesviruses[8] Despite this extensive analysis of EEHV’s genome, much of what underlies EEHV-HD remains uncertain or controversial, including which tissues and cell types are permissive for EEHV infection and replication. Antibodies against the envelope glycoprotein B (gB) of EEHV have been generated and used to determine EEHV gB antigens in elephant tissues, including the salivary glands, gastrointestinal epithelia and peripheral blood monocytes[22] These antibodies, derived from structural proteins, are not appropriate as indicators of viral replication, since the presence of EEHV gB in the cytoplasm of monocytes/macrophages could be due to their phagocytic a ctivity[23]. This study aimed to develop antibodies against the non-structural proteins of EEHV (i.e. DNA polymerase); and use these to investigate the target cells for EEHV replication in fatal cases of EEHV-HD in Asian elephants

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Conclusion