Skip to main content
Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Patterns of serum immune biomarkers during elephant endotheliotropic herpesvirus viremia in Asian and African elephants

  • Katie L. Edwards ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft

    k.edwards@chesterzoo.org

    Current address: North of England Zoological Society, Chester Zoo, Upton-by-Chester, Cheshire, United Kingdom

    Affiliation Center for Species Survival, Smithsonian’s National Zoological Park and Conservation Biology Institute, Front Royal, VA, United States of America

  • Erin M. Latimer,

    Roles Data curation, Funding acquisition, Investigation, Project administration, Resources, Writing – review & editing

    Affiliation National Elephant Herpesvirus Laboratory, Smithsonian’s National Zoological Park and Conservation Biology Institute, Washington, DC, United States of America

  • Jessica Siegal-Willott,

    Roles Funding acquisition, Writing – review & editing

    Current address: USDA, APHIS, Animal Care, Riverdale, MD, United States of America

    Affiliation Department of Wildlife Health Sciences, Smithsonian’s National Zoological Park and Conservation Biology Institute, Washington, DC, United States of America

  • Wendy Kiso,

    Roles Resources, Writing – review & editing

    Affiliation White Oak Conservation Foundation, Yulee, FL, United States of America

  • Luis R. Padilla,

    Roles Resources, Writing – review & editing

    Affiliation Department of Animal Health, Saint Louis Zoo, Saint Louis, MO, United States of America

  • Carlos R. Sanchez,

    Roles Resources, Writing – review & editing

    Affiliation Oregon Zoo, Portland, OR, United States of America

  • Dennis Schmitt,

    Roles Resources, Writing – review & editing

    Affiliations White Oak Conservation Foundation, Yulee, FL, United States of America, William H. Darr College of Agriculture, Missouri State University, Springfield, MO, United States of America

  • Janine L. Brown

    Roles Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing

    Affiliation Center for Species Survival, Smithsonian’s National Zoological Park and Conservation Biology Institute, Front Royal, VA, United States of America

Abstract

Hemorrhagic disease (HD) caused by a group of elephant endotheliotropic herpesviruses (EEHV) is one of the leading causes of death for young elephants in human care. These viruses are widespread and typically persist latently in adult elephants with no negative effects; however, in juvenile Asian and more recently young African elephants, the onset of disease can be rapid and the mortality rate high. Measuring biomarkers associated with the immune response could be beneficial to understanding underlying disease processes, as well as the management of infection and HD. The goal of this study was to measure acute phase proteins and cytokines in serum collected from elephants infected with EEHV (13 Asian and 1 African) and compare concentrations according to presence, severity and outcome of disease. Serum amyloid A (SAA) and haptoglobin (HP) were higher in elephants with EEHV viremia than those without; concentrations increased with increasing viral load, and were higher in fatal cases compared to those that survived. In Asian elephants, SAA was also higher during EEHV1 viremia compared to EEHV5. Cytokine concentrations were typically low, and no statistical differences existed between groups. However, in individuals with detectable levels, longitudinal profiles indicated changes in tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) that may reflect an immune response to EEHV infection. However, the overall low concentrations detected using previously validated assays do not support the presence of a ‘cytokine storm’ and suggest more work is needed to understand if sub-optimal immune responses could be involved in disease progression. These results highlight the potential benefit of measuring circulating biomarker concentrations, such as APPs and cytokines, to improve our understanding of EEHV viremia and HD, assist with monitoring the progression of disease and determining the impact of interventions.

Introduction

Over the past two decades, an acute hemorrhagic disease (HD) caused by a group of elephant endotheliotropic herpesviruses (EEHV) has contributed to 65% of deaths of young, captive-born Asian elephants in North American and European zoos [1], affecting almost one in four globally [2]. EEHV-HD primarily occurs in Asian elephants under 10 years of age, but has also contributed to the death of older individuals, as well as African elephants [36]. Once thought to affect only western ex situ collections, cases of EEHV HD have now been reported in captive and wild populations in India [1,79], Thailand [1014], Laos [15], Cambodia [16], Myanmar [17], Nepal, and Sumatra [18,19].

Zoo studies demonstrate that many Asian and African elephants intermittently shed EEHV DNA via oronasal mucosa and perhaps through ocular and urogenital secretions [2025], with serological investigations finding widespread latent or subclinical infection [11,18,26]. Of the seven major sub-types, EEHV1, 4 and 5 are endemic to Asian elephants, whereas EEHV2, 3, 6, and 7 are found in African elephants [19]. Although low-level viremia may be fairly ubiquitous across both species [5,27], in Asian elephant calves between 1–8 years of age, increasing viremia and the onset of EEHV HD can be rapid, resulting in death within a few hours to days as widespread endothelial cell necrosis occurs [28,29]. Most of the deaths to-date have resulted from EEHV1A and EEHV1B infection in Asian elephants, with fewer fatalities associated with EEHV4 and 5. Recent deaths of young African elephants (6 to 11 years of age) have been associated with EEHV3A [46], with HD also associated with EEHV3B [30]. Early detection through routine testing and immediate intervention with supportive care and anti-viral therapies are critical to successful outcomes [18]. However, what remains unclear is why some individuals quickly succumb to EEHV HD while others that develop high viral loads survive [3032]. Recent evidence suggests that severe cases of EEHV HD result from primary infection, as individuals that died were seronegative for EEHV-specific antibodies [33], and that T-cells likely are key to fighting EEHV infection [34]. Certainly, more detailed knowledge of how the host immune system responds to infection, and any differences between fatal and surviving cases of EEHV viremia and HD would be beneficial to understanding disease pathogenesis, the development of targeted treatments, and ultimately reducing morbidity and mortality from this disease.

The typical immune response to a viral infection includes both innate and adaptive components [3537]. Cytokines play an important role in recruiting, activating and otherwise moderating immune cell function to facilitate the initiation and development of the immune response. They are involved in both innate and adaptive immunity, which results in the body developing pathogen-specific T-cells and antibodies, generally over the course of several days post-infection. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 [38], interferon (IFN)-γ [39], interleukin-2 [40] and interleukin-10 [39] all have been associated with herpesvirus infections in other species. In Asian elephants, increased expression of cytokine mRNA, including TNF- α, IFN-γ and several interleukins, has recently been described in peripheral blood mononuclear cells [41] and in various tissues [42] of elephants infected with EEHV1 and EEHV4. Although circulating cytokine concentrations have not been previously investigated with respect to the development of EEHV HD specifically, they play a role in the elephant immune response to tuberculosis [4345] and other infectious and non-infectious pathologies [46]. Acute phase proteins (APPs) form part of the innate acute phase response against infection or tissue injury and as such are among the first signs of inflammation. Two APPs, serum amyloid A (SAA) and haptoglobin (HP) [47], have previously been investigated in response to EEHV1 infection [29]. Although the exact role of these proteins in elephants has yet to be determined, SAA is one of the most conserved proteins among mammals [48], and is typically involved in chemotaxis of lymphocytes, induction of pro-inflammatory cytokines and tissue repair [4951]. In multiple species, haptoglobin binds free hemoglobin, and is thought to reduce oxidative damage associated with hemolysis [52].

This study will add to existing knowledge by analyzing serum samples from Asian and African elephants during EEHV viremia to compare concentrations of APPs SAA and HP, and cytokines TNF-α and IL-2, which are pro-inflammatory and associated with T-cell growth and differentiation, respectively. We investigated how these measures differ during viremia and non-viremia, among different types of EEHV, with increasing viral load, and between fatal and surviving cases of HD.

Materials and methods

Subjects and sample collection

Serum and whole blood samples submitted to the National Elephant Herpesvirus Laboratory (NEHL) at the National Zoological Park, Washington D.C. for routine EEHV testing were utilized in this study. Blood samples were collected according to approved phlebotomy protocols at each institution, typically from an ear vein through behavioral conditioning, or as part of a medical intervention. Whole blood was collected into EDTA anticoagulant tubes, refrigerated and shipped overnight to the NEHL. A second blood sample collected into serum tubes was allowed to clot at room temperature, before serum was separated, frozen at -20 to -80°C and shipped overnight to the NEHL or the Smithsonian Conservation Biology Institute. The presence of EEHV in whole blood was determined by conventional PCR (cPCR) and/or quantified by real-time PCR (qPCR) according to methodology already described by Latimer et al. [53], Stanton et al. [22] and Bauer et al. [54]. Viral load is presented as viral genome equivalent per milliliter (vge/ml).

This study included serum samples from 14 elephants (13 Asian and 1 African), aged 1 year 2 months to 12 years and 1 months at the time of collection (Table 1). For some individuals, whole blood and serum samples were submitted weekly to the NEHL for routine EEHV surveillance, allowing longitudinal analysis of biomarkers prior to and following the detection of viremia (Day 0); in others, serum was submitted opportunistically. This research was approved by the management at each participating institution, and where applicable, was reviewed and approved by zoo research committees. The study protocol was also reviewed and approved by the Smithsonian’s National Zoo and Conservation Biology Institute (NZP-ACUC #15–03 and #18–18).

thumbnail
Table 1. Subjects and serum samples included in the study.

The age range incorporates the period of sample collection and may encompass multiple viremia episodes over the study period.

https://doi.org/10.1371/journal.pone.0252175.t001

Serum biomarker quantification

SAA and HP concentrations were measured using a RX Daytona automated clinical chemistry analyzer (Randox Industries-US Ltd., Kearneysville, WV, USA). Commercially available reagents, calibrators, and two-level controls were used for each assay (Eiken Chemical Co. Ltd, Tokyo, Japan and Tridelta Tri-DD, Boonton, NJ, USA, respectively), which have previously been validated for elephants [29,46,47]. The technical ranges were 0.1–500 mg/l and 0.01–2.5 mg/ml, respectively. The analyzer was subject to routine quality control measurements throughout the study, with normal and elevated controls for each analyte maintained within two standard deviations (SD) of the respective manufacturer-defined target value. Samples were typically run neat, but those with HP above the technical range were diluted 1:5 in calibrator diluent.

Cytokines were measured by enzyme immunoassay as described in Edwards et al. [46]. In brief, TNF-α was measured using a commercially available equine TNF-α ELISA reagent kit (ESS0017; Thermo Fisher Scientific, Frederick, MD, USA), with anti-equine TNF-α coating antibody and biotinylated anti-equine TNF-α detection antibody both diluted 1:100, and recombinant equine TNF-α standards (3.9 to 1000 pg/ml). Interleukin 2 (IL-2) was measured using a commercially available equine Duoset ELISA development kit (DY1613) with modifications, incorporating anti-equine IL-2 coating (2.0 μg/ml) and biotinylated detection (0.2 μg/ml) antibodies and recombinant equine IL-2 standards (15.6 to 4000 pg/ml). Preliminary testing of samples from three individuals on other cytokine assays that have been validated for elephants (IFN-γ, IL-6, IL-10; [46]) were all below assay detection limits, and so were excluded from the remainder of the study. High and low controls within the standard range for each assay were maintained within an inter-assay CV of 15%. Serum was analyzed undiluted.

Statistical analyses

Serum samples were categorized as viremia positive or negative, by EEHV type, and for viral load based on qPCR testing of whole blood collected on the same day. Data were then analyzed using generalized linear mixed models constructed using the package ‘lmer’ [55] in R [56], using a Gamma distribution with a log-link to model biomarkers concentrations. Due to longitudinal sampling, sample date and animal ID were included as random effects; viremia (positive or negative), EEHV type (EEHV1 or EEHV5) and viral load (0, 102, 103, 104, 105 and 106 or above) were included as categorical fixed effects. Samples from the one African elephant with EEHV3B were excluded from the comparison of EEHV type. In addition, including only samples from cases of higher EEHV viremia (> 5000 vge/ml), concentrations of each biomarker were compared between cases that resulted in either survival or fatality. Animal ID and EEHV type were included as random effects, and data were modelled either using all samples between the start and end of the case of viremia, or using the peak concentration for each biomarker within each case of viremia. All results are presented as the mean prediction ± standard error from the GLMM to account for repeated sampling over time within individuals.

Results

Of 353 serum samples analyzed, 233 were categorized as positive by either cPCR or qPCR of whole blood collected on the same day. These contributed to 46 separate cases of viremia, where viremia was detected in at least one consecutive sample. Six cases were categorized as positive through cPCR (3 EEHV1 and 3 EEHV5) although viral load was not quantified, 17 were transient low concentrations (< 1000 vge/ml; 5 EEHV and 12 EEHV5), six were intermediate (1000–5000 vge/ml; 3 EEHV1 and 3 EEHV5), and 17 were higher level viremia (> 5000 vge/ml;10 EEHV1, 1 EEHV3B and 6 EEHV5; Table 2). Of these 46 cases, all EEHV1 and EEHV5 cases were detected in Asian elephants, and the one African elephant case was EEHV3B.

thumbnail
Table 2. Range (minimum-maximum) of serum biomarker concentrations during cases of viremia exceeding 5000 vge/ml whole blood in 1 African and 13 Asian elephants.

https://doi.org/10.1371/journal.pone.0252175.t002

Comparing biomarker concentrations between groups

Both SAA (P < 0.001) and HP (P = 0.009) were increased in EEHV-positive samples (38.1 ± 4.4 mg/l and 1.0 ± 0.1 mg/ml, respectively) compared to those that did not contain detectable EEHV (14.8 ± 4.0 mg/l and 0.6 ± 0.1 mg/ml, respectively); TNF-α and IL-2 did not differ between positive and negative samples (Fig 1). SAA was also higher (P = 0.002) in EEHV1 (55.2 ± 7.2 mg/l) viremia compared to EEHV5 (19.9 ± 4.9 mg/l); the other biomarkers did not differ by EEHV type in Asian elephants (Fig 2). Both APPs increased with increasing viral load (Fig 3); SAA was elevated in samples with viral load at 103 vge/ml and above, compared to 0 vge/ml, and HP was elevated in samples with a viral load of 104 and 105 compared to 0 vge/ml. TNF-α did not differ significantly with increasing viral load; IL-2 concentrations were lower at 106 vge/ml compared to 0 vge/ml (P = 0.042), although this relationship was driven by a single elevated IL-2 value in an Asian elephant calf with a swollen front limb that was not associated with EEHV viremia. Of the 17 cases of higher-level viremia (i.e. those that included peak viral load of > 5000 vge/ml; Table 2), an observable increase in SAA was detected in 15 cases, HP in 14 cases, TNF-α in five cases and IL-2 in two cases. Of the elephants with high viral loads, overall concentrations of SAA were higher in fatal cases of EEHV HD (P < 0.001), with a tendency for higher concentrations of HP (P = 0.088). Furthermore, peak concentrations (i.e. the maximum concentration observed within a bout of viremia) were higher for HP (P < 0.001), and lower for IL-2 (P < 0.001) in fatal cases of EEHV HD compared to those that survived, with a tendency for higher peak SAA (P = 0.063).

thumbnail
Fig 1. Acute phase protein and cytokine concentrations with positive or negative viremia.

Serum concentrations of serum amyloid A (a), haptoglobin (b), tumor necrosis factor alpha (c) and interleukin 2 (d) on days when whole blood was positive or negative for EEHV viremia by PCR. Bars represent the mean ± sem of the prediction from the GLMM; asterisks denote significant differences between categories (** P < 0.01; *** P < 0.001).

https://doi.org/10.1371/journal.pone.0252175.g001

thumbnail
Fig 2. Acute phase protein and cytokine concentrations with EEHV1 or EEHV5.

Serum concentrations of serum amyloid A (a), haptoglobin (b), tumor necrosis factor alpha (c) and interleukin 2 (d) on days when EEHV1 or EEHV5 was detected in whole blood by PCR. Bars represent the mean ± sem of the prediction from the GLMM; asterisks denote significant differences between categories (** P < 0.01; *** P < 0.001).

https://doi.org/10.1371/journal.pone.0252175.g002

thumbnail
Fig 3. Acute phase protein concentrations with increasing viral load.

Serum amyloid A (a) and haptoglobin (b) in elephants with differing EEHV viral loads (EEHV types combined). Box and whisker plots represent the prediction from the GLMM; asterisks denote significant differences compared to 0 vge/ml (** P < 0.01; *** P < 0.001).

https://doi.org/10.1371/journal.pone.0252175.g003

Temporal changes in biomarkers during individual cases of EEHV viremia

Longitudinal profiles of serum biomarkers during several cases of EEHV1, EEHV5 and EEHV3B viremia, are presented in Figs 46, respectively. A female Asian elephant aged 4 years 8 months (Fig 4A) exhibited EEHV1A viremia increasing from 11,000 vge/ml on Day 0 to 500,000 vge/ml by 7 days following initial detection of viral material in whole blood, returning to undetectable levels on Day 25. SAA and HP were both elevated on Day 0 and peaked on Day 1 (203.2 mg/l) and Day 4 (2.7 mg/ml), respectively. SAA then decreased gradually over the next 24 days, whereas HP only remained elevated for 14 days. A second female (Fig 4B) with peak EEHV1A viremia on Day 7 of 10,350 vge/ml showed a similar magnitude increase in SAA (185.7 mg/l; Day 3), but a smaller increase in HP (1.5 mg/ml, Days 9 and 10). Both APPs had decreased to non-viremia levels by Day 21, at which time viremia had reduced to 350 vge/ml. Three females with EEHV5 viremia exhibited lower peak APP concentrations (Fig 5). These three cases with maximum viremia of 23,000 vge/ml, 30,000 vge/ml and 90,000 vge/ml were associated with SAA concentrations below 100 mg/l and HP concentrations below 1.0 mg/ml. In the case presented in Fig 5B, APP concentrations were relatively low during EEHV5 viremia, but elevated SAA and HP concentrations around Day 38 coincided with increased EEHV1 viremia concurrent with EEHV5. Interleukin-2 was below assay detection for all five cases represented in Figs 4 and 5, and TNF-α was only detected in two cases. An 11-year old female with EEHV1 exhibited TNF-α concentrations averaging 32.8 pg/ml that remained fairly consistent throughout the viremia (Fig 4B). Another 11-year-old female with EEHV5 exhibited a steady increase in TNF-α from Day 5 to 14 (Fig 5C); concentrations then remained elevated for the remainder of the sample collection period. The case of EEHV3B in a male African elephant was described by Bronson et al. [30], and here daily samples were additionally analyzed for APP and cytokine concentrations (Fig 6) to provide a longitudinal profile of each biomarker. TNF-α concentrations increased on Day 3 of detected viremia, peaked at 85.1 pg/ml on Day 4 and then gradually declined in parallel with decreasing viremia to below assay detection on Day 30 (Fig 6C). IL-2 was also elevated on Day 3, peaked on Day 4 (134.0 pg/ml) and began to decrease by Day 9, after which concentrations remained relatively stable until the end of the collection period (Fig 6D).

thumbnail
Fig 4.

Biomarker concentrations during EEHV1 viremia in two female Asian elephants aged 4 y 8 m (a) and 11 y 11 m (b).

https://doi.org/10.1371/journal.pone.0252175.g004

thumbnail
Fig 5.

Biomarker concentrations during EEHV5 viremia in three female Asian elephants aged 4 y 10 m (a), 7 y 2 m (b) and 11 y 1 m (c).

https://doi.org/10.1371/journal.pone.0252175.g005

thumbnail
Fig 6.

Biomarker concentrations during EEHV3B viremia in a male African elephant aged 4 y 11 m for serum amyloid A (a), haptoglobin (b), tumor necrosis factor alpha (c) and interleukin 2 (d).

https://doi.org/10.1371/journal.pone.0252175.g006

Discussion

This study measured APP and cytokine biomarkers associated with the immune response during EEHV infection in elephants and found several that could be useful as indicators of disease severity or prognosis. The APPs SAA and HP were both higher in serum from elephants with viremia than those without, and increasing concentrations were associated with increasing viral load. SAA was also higher in cases of EEHV1 compared to EEHV5, and both APPs were higher during fatal cases than in individuals that survived a bout of viremia exceeding 5000 vge/ml. Although concentrations for three (IFN-γ, IL-6 and IL-10) of the five cytokines validated for elephants [46] were undetectable in longitudinal samples, results for TNF-α and IL-2 may show potential as diagnostic tools for studying the immune response to EEHV. Continued studies are needed to determine if increasing assay sensitivity would be beneficial, or if the low-level responses actually are indicative of sub-optimal immune activation.

APPs are useful indicators of inflammation across species [48], and are increasingly used in veterinary medicine [5759]. As well as being indicative of existing pathology, concentrations can be used as indicators of disease etiology [60,61], severity [58,62,63], and prognosis [59,64,65]. A previous study by Stanton and colleagues [29] reported elevated SAA during EEHV1 viremia in Asian elephants, and in samples with greater than 10,000 vge/ml. Our study built upon this by indicating that concentrations of both SAA and HP are impacted by the presence and magnitude of viremia, and correlate with disease outcome. Overall, APPs were elevated in elephants with viral loads of 103 vge/ml or more, and increased with increasing viremia. Visible clinical signs of EEHV HD are often absent until viremia is quite advanced; in the cases reported here viral loads of 19,000 vge/ml EEHV1 and 90,000 vge/ml EEHV5 were detected by qPCR without accompanying clinical signs of illness. Although clinical signs may not always be apparent, increased APP concentrations indicate that inflammatory processes are underway, and that supportive care should be initiated. We found concentrations of SAA to be greater in EEHV1 infection compared to EEHV5; indeed, APP responses were reduced or even absent in some cases of EEHV5 viremia exceeding 10,000 vge/ml. Historically, EEHV5 has only caused one known death [66] compared to dozens from the two subtypes of EEHV1 [9,19,67], suggesting that EEHV5 may be less virulent, and so may be associated with reduced inflammatory responses.

In general, APPs are non-specific, so elevated concentrations may occur in the absence of EEHV viremia. Indeed, some of the samples utilized for this study had been submitted for analysis in response to non-specific clinical signs (e.g. lameness and temporal swelling) that upon testing were not associated with EEHV. We recently published reference intervals for several biomarkers in African and Asian elephants and found SAA to be elevated in 83% of active clinical cases, including both infectious and traumatic etiologies, and in 50% of deaths studied [46]. The lack of specificity to EEHV viremia should not preclude the use of APPs as diagnostic tools, however. The rapid and high magnitude increases in SAA in particular are a good indicator of significant inflammatory processes and could be used in combination with other blood parameters, such as lymphocyte and platelet counts [30,6871] to ascertain underlying pathology, including EEHV [30,6872], and assist with guiding veterinary interventions. Additional blood parameter data were lacking in this retrospective study, so it remains to be determined how they correlate with biomarkers of inflammation. Although changes in HP were of lower magnitude, higher peak concentrations in fatal cases of EEHV HD compared to survivors suggest that this biomarker may also be useful for predicting disease outcomes.

Cytokines including TNF- α, IFN- γ, IL-1β, IL-2, IL-6 and IL-10 have been linked to the pathogenesis of other viral hemorrhagic diseases, such as dengue [7377], and Ebola [78,79]. However, data have so far been lacking as to whether cytokines facilitate the immune response in fighting infection, suppression of the immune response is part of EEHV pathogenesis, or whether over activation contributes to disease progression through a ‘cytokine storm’ that often occurs in the terminal stages of Ebola and other viral diseases [80]. Here we report for the first-time circulating concentrations of cytokines, specifically TNF-α and IL-2, during EEHV viremia. TNF-α is a pro-inflammatory cytokine that plays multiple roles in the immune response, with powerful immunoregulatory, antiviral, cytotoxic, and pro-coagulatory properties [81]. IL-2 supports the growth and differentiation of antigen-activated T lymphocytes, and the development of T-cell immunologic memory. T-cells play an important role in the immune response to viral hemorrhagic disease [82], and are considered to be key in fighting EEHV infection [34]. TNF-α and IL-2 concentrations in this study were low compared to recently published reference intervals [46], and below detection in five individuals. However, they did correlate well with EEHV3B viremia in the ~5 year old African calf that successfully overcame EEHV HD; TNF-α concentrations were closely associated with viremia, and IL-2 was elevated during at least Days 3–8 of illness, supporting the proposed involvement of T-cells in fighting EEHV infection [34]. In humans, cytokines are significantly lower in children and increase with age [83]. It is possible, therefore, that our EIAs were unable to quantify circulating concentrations of cytokines in the younger calves. However, data from a 2-year old calf following routine rabies and tetanus vaccination showed that both TNF-a and IL-2 responses could be successfully quantified at this age (Edwards, unpublished), suggesting that assay sensitivity may not be limiting the ability to detect immune responses. One potential explanation for overall low concentrations is that a reduced immune response could be involved with the pathogenicity of this disease. Other herpesviruses have been documented to exert a suppressive effect on the immune system, often via functional impairment of immune cells [84], including decreasing the stability of cytokine mRNAs [85], reducing the body’s ability to cope with infection. Although the exact way in which EEHV impacts the elephant immune system is still unknown, a similar suppressive effect, especially in the most virulent strains, could explain the speed and severity of HD outcomes and the low circulating concentrations detected here.

Srivorakul and colleagues [41] recently assessed the expression of cytokines from stimulated PBMCs, and found increased TNF-α expression in isolated blood cells from an Asian elephant with persistent EEHV4 infection. They proposed that TNF-α may contribute to apoptosis of EEHV-infected cells and to leukocyte migration into affected tissues. Although we cannot determine exact mechanisms, the reduction of TNF-α in parallel with decreasing viremia in the case of EEHV3B perhaps supports an association with fighting infection. Conversely, Guntawang et al. [42] found increased cytokine expression in EEHV1A HD and EEHV4 infected tissues, and suggested this was evidence of inflammatory dysregulation contributing to tissue damage. In other viral hemorrhagic diseases, over-expression of certain cytokines, known as a ‘cytokine storm,’ has been associated with damage, including hypotension and hemorrhage [80], and involvement of TNF-α and IL-2 with vascular leakage [82,86]. We cannot yet rule out such pathological involvement, but our inability to detect cytokine responses in several of the fatal cases of EEHV HD suggests that aberrant over-expression of cytokines, at least systemically, may not be involved in progression of EEHV HD. Indeed, it remains a possibility that an inadequate immune response may be involved in more severe cases of EEHV HD, so more sensitive tools for quantifying cytokines, such as mass spectrometry techniques [87], and comparison of tissue-specific and circulating concentrations would be beneficial to investigating differential responses between cases. Further investigation into changes in these biomarkers throughout additional cases of EEHV viremia, including between African and Asian elephants, would be beneficial to understand differential responses and increase our knowledge of disease pathogenesis before immune moderating treatments can be explored. To conclude, these results highlight the potential benefit of measuring circulating biomarker concentrations, such as APPs and cytokines, to better our understanding of EEHV viremia and HD.

Acknowledgments

The authors would like to thank the Albuquerque Biological Park, the Maryland Zoo in Baltimore, the Oklahoma City Zoo, the Oregon Zoo, the Ringling Brothers Center for Elephant Conservation, White Oak Conservation Foundation, and the Saint Louis Zoo for contributing samples and clinical information for use in this study, and Hannah Johnson and Shiling Zhao for laboratory assistance.

References

  1. 1. Zachariah A, Zong JC, Long SY, Latimer EM, Heaggans SY, Richman LK, et al. Fatal herpesvirus hemorrhagic disease in wild and orphan Asian elephants in southern India. J Wildl Dis. 2013;49(2):381–93. Epub 2013/04/10. pmid:23568914; PubMed Central PMCID: PMC3707512.
  2. 2. Hayward GS. Conservation: clarifying the risk from herpesvirus to captive Asian elephants. Vet Rec. 2012;170(8):202–3. Epub 2012/03/01. pmid:22368209; PubMed Central PMCID: PMC3587150.
  3. 3. Richman LK, Hayward GS. Elephant Herpesviruses. In: Fowler M, editor. Fowler’s Zoo and Wild Animal Medicine. Saint Louis: W.B. Saunders, Elsevier; 2012. p. 496–502.
  4. 4. Fayette M. Fatal elephant endotheliotropic herpesvirus 3 infection in two captive African elephants (Loxodonta africana). 16th International Elephant Conservation and Research Symposium; October 21–25, 2019; Limpopo, South Africa: International Elephant Foundation; 2019.
  5. 5. Latimer E. Current knowledge of EEHV in African elephants (Loxodonta africana). 16th International Elephant Conservation and Research Symposium; October 21–25, 2019; Limpopo, South Africa: International Elephant Foundation; 2019.
  6. 6. Fayette MA, Brenner EE, Garner MM, Bowman MR, Latimer E, Proudfoot JS. Acute hemorrhagic disease due to elephant endotheliotropic herpesvirus 3A infection in five African elephants (Loxodonta africana) at one North American zoological institution. J Zoo Wildl Med. 2021;52(1):357–65. pmid:33827199
  7. 7. Barman NN, Choudhury B, Kumar V, Koul M, Gogoi SM, Khatoon E, et al. Incidence of elephant endotheliotropic herpesvirus in Asian elephants in India. Vet Microbiol. 2017;208:159–63. Epub 2017/09/11. pmid:28888631.
  8. 8. Mahato G, Sarma KK, Pathak DC, Barman NN, Gogoi P, Dutta M, et al. Endotheliotropic herpesvirus infection in Asian elephants (Elephas maximus) of Assam, India. Vet World. 2019;12(11):1790–6. Epub 2020/02/06. pmid:32009758; PubMed Central PMCID: PMC6925033.
  9. 9. Zachariah A, Sajesh PK, Santhosh S, Bathrachalam C, Megha M, Pandiyan J, et al. Extended genotypic evaluation and comparison of twenty-two cases of lethal EEHV1 hemorrhagic disease in wild and captive Asian elephants in India. PLoS One. 2018;13(8):e0202438. Epub 2018/08/23. pmid:30133540; PubMed Central PMCID: PMC6105008.
  10. 10. Sripiboon S, Tankaew P, Lungka G, Thitaram C. The occurrence of elephant endotheliotropic herpesvirus in captive Asian elephants (Elephas maximus): first case of EEHV4 in Asia. J Zoo Wildl Med. 2013;44(1):100–4. Epub 2013/03/20. pmid:23505709.
  11. 11. Angkawanish T, Nielen M, Vernooij H, Brown JL, van Kooten PJS, van den Doel PB, et al. Evidence of high EEHV antibody seroprevalence and spatial variation among captive Asian elephants (Elephas maximus) in Thailand. Virol J. 2019;16(1):33. Epub 2019/03/15. pmid:30866975; PubMed Central PMCID: PMC6415343.
  12. 12. Bhusri B, Suksai P, Mongkolphan C, Tiyanun E, Ratanakorn P, Chaichoun K, et al. Detection of elephant endotheliotropic herpesvirus 4 in captive Asian elephants in Thailand. Thai J Vet Med. 2017;47(1):97–102.
  13. 13. Boonprasert K, Punyapornwithaya V, Tankaew P, Angkawanish T, Sriphiboon S, Titharam C, et al. Survival analysis of confirmed elephant endotheliotropic herpes virus cases in Thailand from 2006–2018. PLoS One. 2019;14(7):e0219288. Epub 2019/07/06. pmid:31276571; PubMed Central PMCID: PMC6611605.
  14. 14. Lertwatcharasarakul P, Sanyathitiseree P, Thongtip N, Charoenphan P, Boonyasart B, Maneewan N, et al. Genetic variant of elephant endotheliotropic herpesvirus detected from captive Asian elephants (Elephas maximus) in Thailand from 2007 to 2013. Thai J Vet Med. 2015;45(1):73–9.
  15. 15. Bouchard B, Xaymountry B, Thongtip N, Lertwatcharasarakul P, Wajjwalku W. First reported case of elephant endotheliotropic herpes virus infection in Laos. J Zoo Wildl Med. 2014;45(3):704–7. Epub 2014/10/16. pmid:25314848.
  16. 16. Reid CE, Hildebrandt TB, Marx N, Hunt M, Thy N, Reynes JM, et al. Endotheliotropic elephant herpes virus (EEHV) infection. The first PCR-confirmed fatal case in Asia. Vet Q. 2006;28(2):61–4. Epub 2006/07/18. pmid:16841568.
  17. 17. Oo ZM, Aung YH, Aung TT, San N, Tun ZM, Hayward GS, et al. Elephant endotheliotropic herpesvirus hemorrhagic disease in Asian elephant calves in logging camps, Myanmar. Emerg Infect Dis. 2020;26(1):63–9. Epub 2019/12/20. pmid:31855135; PubMed Central PMCID: PMC6924905.
  18. 18. EEHV Advisory Group. eehvinfo.org 2020 [22 July 2020]. Available from: www.eehvinfo.org.
  19. 19. Long SY, Latimer EM, Hayward GS. Review of elephant endotheliotropic herpesviruses and acute hemorrhagic disease. ILAR Journal. 2015;56(3):283–96.
  20. 20. Hardman K, Dastjerdi A, Gurrala R, Routh A, Banks M, Steinbach F, et al. Detection of elephant endotheliotropic herpesvirus type 1 in asymptomatic elephants using TaqMan real-time PCR. Vet Rec. 2012;170(8):205. Epub 2011/12/22. pmid:22186378.
  21. 21. Schaftenaar W, Reid C, Martina B, Fickel J, Osterhaus AD. Nonfatal clinical presentation of elephant endotheliotropic herpes virus discovered in a group of captive Asian elephants (Elephas maximus). J Zoo Wildl Med. 2010;41(4):626–32. Epub 2011/03/05. pmid:21370642.
  22. 22. Stanton JJ, Zong JC, Latimer E, Tan J, Herron A, Hayward GS, et al. Detection of pathogenic elephant endotheliotropic herpesvirus in routine trunk washes from healthy adult Asian elephants (Elephas maximus) by use of a real-time quantitative polymerase chain reaction assay. Am J Vet Res. 2010;71(8):925–33. Epub 2010/08/03. pmid:20673092; PubMed Central PMCID: PMC3725808.
  23. 23. Stanton JJ, Zong JC, Eng C, Howard L, Flanagan J, Stevens M, et al. Kinetics of viral loads and genotypic analysis of elephant endotheliotropic herpesvirus-1 infection in captive Asian elephants (Elephas maximus). J Zoo Wildl Med. 2013;44(1):42–54. Epub 2013/03/20. pmid:23505702; PubMed Central PMCID: PMC3746492.
  24. 24. Ackermann M, Hatt JM, Schetle N, Steinmetz H. Identification of shedders of elephant endotheliotropic herpesviruses among Asian elephants (Elephas maximus) in Switzerland. PLoS One. 2017;12(5):e0176891. Epub 2017/05/04. pmid:28467495; PubMed Central PMCID: PMC5415103.
  25. 25. Bennett L, Dunham S, Yon L, Chapman S, Kenaghan M, Purdie L, et al. Longitudinal study of Asian elephants, Elephas maximus, indicates intermittent shedding of elephant endotheliotropic herpesvirus 1 during pregnancy. Vet Rec Open. 2015;2(1):e000088. Epub 2015/09/24. pmid:26392899; PubMed Central PMCID: PMC4567181.
  26. 26. van den Doel PB, Prieto VR, van Rossum-Fikkert SE, Schaftenaar W, Latimer E, Howard L, et al. A novel antigen capture ELISA for the specific detection of IgG antibodies to elephant endotheliotropic herpes virus. BMC Vet Res. 2015;11(203):203. Epub 2015/08/14. pmid:26268467; PubMed Central PMCID: PMC4535388.
  27. 27. Hoornweg TE, Schaftenaar W, Maurer G, van den Doel PB, Molenaar FM, Chamouard-Galante A, et al. Elephant endotheliotropic herpesvirus is omnipresent in elephants in European zoos and an Asian elephant range country. Viruses. 2021;13(2):283. pmid:33670367
  28. 28. International Elephant Foundation. Elephant Endotheliotropic Herpesvirus Research May 2011. 2011.
  29. 29. Stanton JJ, Cray C, Rodriguez M, Arheart KL, Ling PD, Herron A. Acute phase protein expression during elephant endotheliotropic herpesvirus-1 viremia in Asian elephants (Elephas maximus). J Zoo Wildl Med. 2013;44(3):605–12. Epub 2013/09/26. pmid:24063088.
  30. 30. Bronson E, McClure M, Sohl J, Wiedner E, Cox S, Latimer EM, et al. Epidemiologic evaluation of elephant endotheliotropic herpesvirus 3B infection in an African elephant (Loxodonta africana). J Zoo Wildl Med. 2017;48(2):335–43. Epub 2017/07/28. pmid:28749266.
  31. 31. Atkins L, Zong JC, Tan J, Mejia A, Heaggans SY, Nofs SA, et al. Elephant endotheliotropic herpesvirus 5, a newly recognized elephant herpesvirus associated with clinical and subclinical infections in captive Asian elephants (Elephas maximus). J Zoo Wildl Med. 2013;44(1):136–43. Epub 2013/03/20. pmid:23505714; PubMed Central PMCID: PMC3746547.
  32. 32. Dastjerdi A, Seilern-Moy K, Darpel K, Steinbach F, Molenaar F. Surviving and fatal Elephant Endotheliotropic Herpesvirus-1A infections in juvenile Asian elephants—lessons learned and recommendations on anti-herpesviral therapy. BMC Vet Res. 2016;12(1):178. Epub 2016/08/29. pmid:27567895; PubMed Central PMCID: PMC5002104.
  33. 33. Fuery A, Pursell T, Tan J, Peng R, Burbelo PD, Hayward GS, et al. Lethal hemorrhagic disease and clinical illness associated with elephant endotheliotropic herpesvirus 1 are caused by primary infection: Implications for the detection of diagnostic proteins. J Virol. 2020;94(3). Epub 2019/11/15. pmid:31723022; PubMed Central PMCID: PMC7000966.
  34. 34. Fuery A, Leen AM, Peng R, Wong MC, Liu H, Ling PD. Asian elephant T -cell responses to elephant endotheliotropic herpesvirus. J Virol. 2018;92(6). Epub 2017/12/22. pmid:29263271; PubMed Central PMCID: PMC5827410.
  35. 35. Koyama S, Ishii KJ, Coban C, Akira S. Innate immune response to viral infection. Cytokine. 2008;43(3):336–41. Epub 2008/08/13. pmid:18694646.
  36. 36. Kindt TJ, Osborne BA, Goldsby RA, editors. Kuby Immunology. Sixth ed: W.H. Freeman and Company; 2006.
  37. 37. Aoshi T, Koyama S, Kobiyama K, Akira S, Ishii KJ. Innate and adaptive immune responses to viral infection and vaccination. Curr Opin Virol. 2011;1(4):226–32. Epub 2012/03/24. pmid:22440781.
  38. 38. Gosselin J, Flamand L, D’Addario M, Hiscott J, Stefanescu I, Ablashi DV, et al. Modulatory effects of Epstein-Barr, herpes simplex, and human herpes-6 viral infections and coinfections on cytokine synthesis. A comparative study. J Immunol. 1992;149(1):181–7. Epub 1992/07/01. pmid:1318897.
  39. 39. Halford WP, Gebhardt BM, Carr DJ. Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1. J Immunol. 1996;157(8):3542–9. Epub 1996/10/15. pmid:8871654.
  40. 40. Roffman E, Frenkel N. Interleukin-2 inhibits the replication of human herpesvirus-6 in mature thymocytes. Virology. 1990;175(2):591–4. Epub 1990/04/01. pmid:2158189.
  41. 41. Srivorakul S, Guntawang T, Kochagul V, Photichai K, Sittisak T, Janyamethakul T, et al. Possible roles of monocytes/macrophages in response to elephant endotheliotropic herpesvirus (EEHV) infections in Asian elephants (Elephas maximus). PLoS One. 2019;14(9):e0222158. Epub 2019/09/07. pmid:31491031; PubMed Central PMCID: PMC6730851.
  42. 42. Guntawang T, Sittisak T, Kochagul V, Srivorakul S, Photichai K, Boonsri K, et al. Pathogenesis of hemorrhagic disease caused by elephant endotheliotropic herpesvirus (EEHV) in Asian elephants (Elephas maximus). Sci Rep. 2021;11(1):1–13. pmid:33414495
  43. 43. Landolfi JA, Mikota SK, Chosy J, Lyashchenko KP, Giri K, Gairhe K, et al. Comparison of systemic cytokine levels in Mycobacterium spp. seropositive and seronegative Asian elephants (Elephas maximus). J Zoo Wildl Med. 2010;41(3):445–55. Epub 2010/10/16. pmid:20945642.
  44. 44. Landolfi JA, Miller M, Maddox C, Zuckermann F, Langan JN, Terio KA. Differences in immune cell function between tuberculosis positive and negative Asian elephants. Tuberculosis. 2014;94(4):374–82. Epub 2014/05/20. pmid:24836563.
  45. 45. Landolfi JA, Terio KA, Miller M, Junecko BF, Reinhart T. Pulmonary tuberculosis in Asian elephants (Elephas maximus): histologic lesions with correlation to local immune responses. Vet Pathol. 2015;52(3):535–42. Epub 2014/09/18. pmid:25228055.
  46. 46. Edwards KL, Miller MA, Siegal-Willott J, Brown JL. Serum health biomarkers in African and Asian elephants: Value ranges and clinical values indicative of the immune response. Animals. 2020;10(10):1756. Epub 2020/10/01. pmid:32992555; PubMed Central PMCID: PMC7601509.
  47. 47. Isaza R, Wiedner E, Hiser S, Cray C. Reference intervals for acute phase protein and serum protein electrophoresis values in captive Asian elephants (Elephas maximus). J Vet Diagn Investig. 2014;26(5):616–21. Epub 2014/07/25. pmid:25057161.
  48. 48. Cray C. Acute phase proteins in animals. Prog Mol Biol Transl Sci. 2012;105:113–50. Epub 2011/12/06. pmid:22137431; PubMed Central PMCID: PMC7149966.
  49. 49. Badolato R, Wang JM, Murphy WJ, Lloyd AR, Michiel DF, Bausserman LL, et al. Serum amyloid A is a chemoattractant: induction of migration, adhesion, and tissue infiltration of monocytes and polymorphonuclear leukocytes. The Journal of Experimental Medicine. 1994;180(1):203–9. pmid:7516407
  50. 50. Patel H, Fellowes R, Coade S, Woo P. Human serum amyloid A has cytokine-like properties. Scand J Immunol. 1998;48(4):410–8. pmid:9790312
  51. 51. Uhlar CM, Whitehead AS. Serum amyloid A, the major vertebrate acute‐phase reactant. Eur J Biochem. 1999;265(2):501–23. pmid:10504381
  52. 52. Cray C, Zaias J, Altman NH. Acute phase response in animals: a review. Comp Med. 2009;59(6):517–26. Cray2009. pmid:20034426
  53. 53. Latimer E, Zong JC, Heaggans SY, Richman LK, Hayward GS. Detection and evaluation of novel herpesviruses in routine and pathological samples from Asian and African elephants: identification of two new probosciviruses (EEHV5 and EEHV6) and two new gammaherpesviruses (EGHV3B and EGHV5). Vet Microbiol. 2011;147(1–2):28–41. Epub 2010/06/29. pmid:20579821; PubMed Central PMCID: PMC2976818.
  54. 54. Bauer KL, Latimer E, Finnegan M. Long-term, intermittent, low-level elephant endotheliotropic herpesvirus 1A viremia in a captive Asian elephant calf. J Vet Diagn Investig. 2018;30(6):917–9. Epub 2018/09/29. pmid:30264667; PubMed Central PMCID: PMC6505841.
  55. 55. Bates D, Mächler M, Bolker B, Walker S. Fitting Linear Mixed-Effects Models Usinglme4. J Stat Softw. 2015;67(1):1–48.
  56. 56. R Core Team. R: A language and environment for statistical computing. Vienna, Austria.: R Foundation for Statistical Computing; 2017.
  57. 57. Eckersall PD, Bell R. Acute phase proteins: Biomarkers of infection and inflammation in veterinary medicine. Vet J. 2010;185(1):23–7. Epub 2010/07/14. pmid:20621712.
  58. 58. Horadagoda NU, Knox KMG, Gibbs HA, Reid SWJ, Horadagoda A, Edwards SER, et al. Acute phase proteins in cattle: discrimination between acute and chronic inflammation. Vet Rec. 1999;144(16):437–41. WOS:000080184000003. pmid:10343375
  59. 59. Jacobsen S, Andersen PH. The acute phase protein serum amyloid A (SAA) as a marker of inflammation in horses. Equine Vet Educ. 2007;19(1):38–46. WOS:000244221300010.
  60. 60. Viner M, Mazan M, Bedenice D, Mapes S, Pusterla N. Comparison of serum amyloid A in horses with infectious and noninfectious respiratory diseases. J Equine Vet Sci. 2017;49:11–3. WOS:000395966200002.
  61. 61. Vandenplas ML, Moore JN, Barton MH, Roussel AJ, Cohen ND. Concentrations of serum amyloid A and lipopolysaccharide-binding protein in horses with colic. Am J Vet Res. 2005;66(9):1509–16. Epub 2005/11/03. pmid:16261823.
  62. 62. Heegaard PM, Godson DL, Toussaint MJ, Tjornehoj K, Larsen LE, Viuff B, et al. The acute phase response of haptoglobin and serum amyloid A (SAA) in cattle undergoing experimental infection with bovine respiratory syncytial virus. Vet Immunol Immunopathol. 2000;77(1–2):151–9. Epub 2000/11/09. pmid:11068073; PubMed Central PMCID: PMC7119828.
  63. 63. Kann RK, Seddon JM, Kyaw-Tanner MT, Henning J, Meers J. Association between feline immunodeficiency virus (FIV) plasma viral RNA load, concentration of acute phase proteins and disease severity. Vet J. 2014;201(2):181–3. Epub 2014/04/08. pmid:24703323.
  64. 64. Huangfu XQ, Wang LG, Le ZD, Tao B. Utility of serum amyloid A as a potential prognostic biomarker of acute primary basal ganglia hemorrhage. Clin Chim Acta. 2020;505:43–8. Epub 2020/02/24. pmid:32088210.
  65. 65. Tamamoto T, Ohno K, Takahashi M, Nakashima K, Fujino Y, Tsujimoto H. Serum amyloid A as a prognostic marker in cats with various diseases. J Vet Diagn Investig. 2013;25(3):428–32. Epub 2013/05/02. pmid:23632661.
  66. 66. Wilkie GS, Davison AJ, Kerr K, Stidworthy MF, Redrobe S, Steinbach F, et al. First fatality associated with elephant endotheliotropic herpesvirus 5 in an Asian elephant: pathological findings and complete viral genome sequence. Sci Rep. 2014;4(1):6299. Epub 2014/09/10. pmid:25199796; PubMed Central PMCID: PMC5385831.
  67. 67. Perrin KL, Nielsen SS, Martinussen T, Bertelsen MF. Quantification and risk factor analysis of elephant endotheliotropic herpesvirus-haemorrhagic disease fatalities in Asian elephants (Elephas maximus) in Europe (1985–2017). JZAR. 2021;9(1):8–13.
  68. 68. Richman LK, Montali RJ, Cambre RC, Schmitt D, Hardy D, Hildbrandt T, et al. Clinical and pathological findings of a newly recognized disease of elephants caused by endotheliotropic herpesviruses. J Wildl Dis. 2000;36(1):1–12. Epub 2000/02/22. pmid:10682740.
  69. 69. Fuery A, Browning GR, Tan J, Long S, Hayward GS, Cox SK, et al. Clinical infection of captive Asian elephants (Elephas maximus) with elephant endotheliotropic herpesvirus 4. J Zoo Wildl Med. 2016;47(1):311–8. Epub 2016/03/25. pmid:27010293.
  70. 70. Fuery A, Tan J, Peng R, Flanagan JP, Tocidlowski ME, Howard LL, et al. Clinical infection of two captive Asian elephants (Elephas maximus) with elephant endotheliotropic herpesvirus 1B. J Zoo Wildl Med. 2016;47(1):319–24. Epub 2016/03/25. pmid:27010294.
  71. 71. Weisbrod TC, Isaza R, Cray C, Adler L, Stacy NI. The importance of manual white blood cell differential counts and platelet estimates in elephant hematology: blood film review is essential. Vet Q. 2020:1–6.
  72. 72. Perrin K, Kristensen A, Krogh A, Grøndahl C, Bertelsen M, editors. Thromboelastography-guided diagnosis and therapy in a case of elephant endotheliotropic herpesvirus hemorrhagic disease. Proc Am Assoc Zoo Vet; 2015.
  73. 73. Garcia-Trejo AR, Falcon-Lezama JA, Juarez-Palma L, Granados J, Zuniga-Ramos J, Rangel H, et al. Tumor necrosis factor alpha promoter polymorphisms in Mexican patients with dengue fever. Acta Trop. 2011;120(1–2):67–71. Epub 2011/06/23. pmid:21693096.
  74. 74. Imad HA, Phumratanaprapin W, Phonrat B, Chotivanich K, Charunwatthana P, Muangnoicharoen S, et al. Cytokine Expression in Dengue Fever and Dengue Hemorrhagic Fever Patients with Bleeding and Severe Hepatitis. Am J Trop Med Hyg. 2020;102(5):943–50. Epub 2020/03/04. pmid:32124729; PubMed Central PMCID: PMC7204576.
  75. 75. Braga EL, Moura P, Pinto LM, Ignacio SR, Oliveira MJ, Cordeiro MT, et al. Detection of circulant tumor necrosis factor-alpha, soluble tumor necrosis factor p75 and interferon-gamma in Brazilian patients with dengue fever and dengue hemorrhagic fever. Mem Inst Oswaldo Cruz. 2001;96(2):229–32. Epub 2001/04/04. pmid:11285501.
  76. 76. Lee YH, Leong W-Y, Wilder-Smith A. Markers of dengue severity: a systematic review of cytokines and chemokines. Journal of General Virology. 2016;97(12):3103–19. pmid:27902364
  77. 77. Findlay JS, Ulaeto D, D’Elia RV. Cytokines and viral hemorrhagic fever: potential for therapeutic intervention. Future Virology. 2015;10(5):547–57.
  78. 78. El Sayed SM, Abdelrahman AA, Ozbak HA, Hemeg HA, Kheyami AM, Rezk N, et al. Updates in diagnosis and management of Ebola hemorrhagic fever. J Res Med Sci. 2016;21:84. Epub 2017/02/07. pmid:28163730; PubMed Central PMCID: PMC5244689.
  79. 79. Stroher U, West E, Bugany H, Klenk HD, Schnittler HJ, Feldmann H. Infection and activation of monocytes by Marburg and Ebola viruses. J Virol. 2001;75(22):11025–33. Epub 2001/10/17. pmid:11602743; PubMed Central PMCID: PMC114683.
  80. 80. Sordillo PP, Helson L. Curcumin suppression of cytokine release and cytokine storm. A potential therapy for patients with Ebola and other severe viral infections. In Vivo. 2015;29(1):1–4. Epub 2015/01/21. pmid:25600522.
  81. 81. Cruse JM, Lewis RE. Illustrated Dictionary of Immunology. 3rd Edition ed: CRC Press; 2009.
  82. 82. Perdomo-Celis F, Salvato MS, Medina-Moreno S, Zapata JC. T-cell response to viral hemorrhagic fevers. Vaccines. 2019;7(1):29. Epub 2019/01/27. pmid:30678246; PubMed Central PMCID: PMC6466054.
  83. 83. Decker ML, Gotta V, Wellmann S, Ritz N. Cytokine profiling in healthy children shows association of age with cytokine concentrations. Sci Rep. 2017;7(1):17842. Epub 2017/12/21. pmid:29259216; PubMed Central PMCID: PMC5736560.
  84. 84. Arena A, Liberto MC, Iannello D, Capozza AB, Foca A. Altered cytokine production after human herpes virus type 6 infection. The new microbiologica. 1999;22(4):293–300. Epub 1999/11/11. pmid:10555198.
  85. 85. Mogensen TH, Melchjorsen J, Malmgaard L, Casola A, Paludan SR. Suppression of proinflammatory cytokine expression by herpes simplex virus type 1. Journal of virology. 2004;78(11):5883–90. Epub 2004/05/14. pmid:15140986; PubMed Central PMCID: PMC415838.
  86. 86. Kurane I, Innis BL, Nimmannitya S, Nisalak A, Meager A, Janus J, et al. Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue. J Clin Invest. 1991;88(5):1473–80. Epub 1991/11/01. pmid:1939640; PubMed Central PMCID: PMC295652.
  87. 87. Mendoza‐Porras O, Pires PR, Goswami H, Meirelles FV, Colgrave ML, Wijffels G. Cytokines in the grass, a lesson learnt: Measuring cytokines in plasma using multiple reaction monitoring mass spectrometry. Rapid Commun Mass Sp. 2020;34(9):e8723. pmid:31922636