Lethal Hemorrhagic Disease and Clinical Illness Associated with Elephant Endotheliotropic Herpesvirus 1 Are Caused by Primary Infection: Implications for the Detection of Diagnostic Proteins

Abstract

Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants, both in captivity and in the wild. Most deaths associated with the virus are caused by two chimeric variants of EEHV1 (EEHV1A and EEHV1B), while two other EEHVs endemic within Asian elephants (EEHV4 and EEHV5) have been recognized but cause death less often. Whether lethal EEHV infections are due to primary infection or reactivation of latent virus remains unknown, and knowledge of the anti-EEHV antibody levels in young elephants is limited. To close these gaps, we sought to develop a serologic assay capable of distinguishing among infections with different EEHVs using a luciferase immunoprecipitation system (LIPS) for antibody profiling and a panel of conserved EEHV recombinant proteins and proteins unique to EEHV1. The results showed that elephants dying from EEHV1 hemorrhagic disease or ill from EEHV infection were seronegative for the EEHV species that caused the disease or illness, indicating that the events were associated with primary infection rather than reactivation of latent virus. We also demonstrated that waning of EEHV1-specific antibodies can occur in the first 2 years of life, when a threshold protective level of antibody may be needed to prevent severe EEHV1-related disease. Use of the LIPS assay to identify putative “diagnostic” proteins would be a valuable asset in determining the EEHV immune status of young elephants and responses to candidate EEHV vaccines in the future. IMPORTANCE Whether clinical illness and deaths associated with elephant endotheliotropic herpesvirus (EEHV) infection result from primary infection or reactivation of latent virus is a longstanding question in the field. By applying a relatively new assay, the luciferase immunoprecipitation system (LIPS), combined with the genomic sequences of the viruses, we gained the insights and tools needed to resolve this issue. Our EEHV1-specific LIPS assay should be useful for assessing the vulnerability of elephant calves to infection with different EEHVs and evaluating antibody responses to anti-EEHV vaccines. A significant proportion of the Asian elephant population is under some form of human care. Hence, the ability to screen for EEHV immune status in elephant calves should have a major impact on the management of these animals worldwide.

Elephant endotheliotropic herpesvirus is omnipresent in elephants in European zoos and an Asian elephant range country

“…Furthermore, all three EEHV-HD cases tested had low or non-detectable EEHV-specific antibody titres, indicating that these animals experienced primary infections as previously suggested (8).…”

“…Both gB and the gH/gL dimer are important targets of the herpesvirus-specific humoral immune response (17)(18)(19)(20), hence interesting for use in EEHV-specific serological assays. Although expression of recombinant EEHV gB has been reported (8,12), purification has only been achieved from bacterial cultures. Expression of recombinant EEHV gH/gL has not been described thus far.…”

Use of immunostimulants in the successful treatment of a clinical EEHV1A infection in an Asian elephant (Elephas maximus)

“…Furthermore, no EEHV shedding was detected in case 2’s weekly, routine trunk samples or in blood swabs in the 5 months between this potential exposure and the observed clinical case. Virus shedding would be expected after initial infection,8 47 as was observed in case 2, and her 11‐month‐old half‐sibling, for several months after case 2’s clinical episode, which might suggest it was a primary infection. The previous serological status of case 2 is not known, so an existing degree of immunity cannot be ruled out.…”

“…Although EEHVs are likely to possess IFN‐evasion strategies as described in other herpesviruses,42–44 the concept of head‐start immunity has been proposed,45 whereby these viral defences can be pre‐empted and, with sufficient and timely activation of the IFN system, can overcome infection, as highlighted by IFN‐based treatment strategies for chronic hepatitis B virus 46. EEHV‐HD is thought to be the result of primary infection in immunologically naive animals, following the decline of maternally derived antibodies 47. In such individuals, the peracute disease course would seem to preclude the involvement of the calf’s own adaptive immune system, relying mainly on the innate response, likely a key mechanism in the disease process still to be explored.…”

“…Pre‐existing immunity from previous exposure is possible but unknown. Case 1 was potentially exposed to EEHV‐1A at the age of three months during an outbreak which resulted in the death of her two herdmates, but at this young age, maternal antibodies may have prevented the development of immunity 47. Case 2 was similarly potentially exposed to EEHV‐1A at 23 months of age, when case 1 and another half‐sibling died of EEHV‐HD.…”