fatal herpesvirus hemorrhagic disease in wild and …multibriefs.com/briefs/wda/ele_040313.pdf ·...

FATAL HERPESVIRUS HEMORRHAGIC DISEASE IN WILD AND

ORPHAN ASIAN ELEPHANTS IN SOUTHERN INDIA

Arun Zachariah,1 Jian-Chao Zong,2 Simon Y. Long,2 Erin M. Latimer,3 Sarah Y. Heaggans,2

Laura K. Richman,3 and Gary S. Hayward2,4

1 Department of Forests and Wildlife, Government of Kerala, Sultan Battery, Wayanad, India2 Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins School of Medicine,Baltimore, Maryland 21287, USA3 National Elephant Herpesvirus Laboratory, Pathology Department, National Zoological Park, Washington, DC 20013,USA4 Corresponding author (email: [email protected])

ABSTRACT: Up to 65% of deaths of young Asian elephants (Elephas maximus) between 3 mo and15 yr of age in Europe and North America over the past 20 yr have been attributed to hemorrhagicdisease associated with a novel DNA virus called elephant endotheliotropic herpesvirus (EEHV).To evaluate the potential role of EEHV in suspected cases of a similar lethal acute hemorrhagicdisease occurring in southern India, we studied pathologic tissue samples collected from fieldnecropsies. Nine cases among both orphaned camp and wild Asian elephants were identified bydiagnostic PCR. These were subjected to detailed gene subtype DNA sequencing at multiple PCRloci, which revealed seven distinct strains of EEHV1A and one of EEHV1B. Two orphan calvesthat died within 3 days of one another at the same training camp had identical EEHV1A DNAsequences, indicating a common epidemiologic source. However, the high level of EEHV1subtype genetic diversity found among the other Indian strains matches that among over 30EEHV1 strains that have been evaluated from Europe and North America. These results argueagainst the previous suggestions that this is just a disease of captive elephants and that the EEHV1virus has crossed recently from African elephant (Loxodonta africana) hosts to Asian elephants.Instead, both the virus and the disease are evidently widespread in Asia and, despite the diseaseseverity, Asian elephants appear to be the ancient endogenous hosts of both EEHV1A andEEHV1B.

Key words: Calves, EEHV1A, EEHV1B, Elephas maximus, orphans, proboscivirus, wild.

INTRODUCTION

An acute hemorrhagic disease with 85%

fatality rate was originally described incaptive Asian elephants (Elephas maxi-mus) in North American and Europeanzoos (Ossent et al., 1990; Richman et al.,1999, 2000b; Fickel et al., 2003; Zonget al., 2007). Among 52 suspected casessince 1988, high levels of a novel herpes-virus called elephant endotheliotrophicherpesvirus (EEHV) have been confirmedby DNA PCR tests at the NationalElephant Herpesvirus Laboratory (Wash-ington, DC, USA) on blood or necropsytissue from all 28 North American casesand 10 of 24 European cases (Zong et al.,2008; Garner et al., 2009; Latimer et al.,2011; Richman, unpubl. data). However,EEHV viruses have never been detectedin numerous, random, unrelated elephantnecropsy tissue samples or in blood fromhealthy asymptomatic elephants. The PCR

test results show that EEHV diseaseoccurs predominantly in Asian juveniles1–8 yr old, and that there have been only10 known survivors, all of which receivedtimely and aggressive treatment with thehuman antiherpesvirus drugs famcicloviror ganciclovir (Schmitt et al., 2000; Brocket al., 2012). The EEHV disease ischaracterized by initial mild symptoms offacial edema and tongue cyanosis, whichprogresses rapidly to systemic viremiafollowed by hemorrhaging in all majorinternal organs (Richman et al., 2000a).Nuclear inclusion bodies and envelopedvirions can be observed by light andelectron microscopy, respectively, in mi-crovascular endothelial cells, and highlevels of viral DNA are found in whiteblood cells, serum, and necropsy tissue(Latimer et al., 2011; Zong, unpubl. data).

Most herpesviruses have become highlyadapted to the natural hosts in which theyhave evolved and usually cause only mild

Journal of Wildlife Diseases jwdi-49-02-19.3d 15/2/13 21:12:07 381 Cust # 2012-07-193

DOI: 10.7589/2012-07-193 Journal of Wildlife Diseases, 49(2), 2013, pp. 381–393# Wildlife Disease Association 2013

381

localized disease in healthy animals.Therefore, the original description of aherpesvirus associated with rapid acutehemorrhagic disease in juvenile Asianelephants was unexpected (Richmanet al., 1999). The idea that it might involvea cross-species transfer of a virus that wasadapted to African elephants (Loxodontaafricana) into naıve Asian elephantsseemed to make sense of the unexpectedlysevere pathology. Originally, two speciesof EEHV were described: EEHV1 wasassociated with 11 cases in captive Asianelephants, and EEHV2 was associatedwith the deaths of two young Africanelephants (Richman et al., 1999). TheEEHV1 viruses found in those and allsubsequent European and North Ameri-can cases fall into two subgroups, referredto as EEHV1A and EEHV1B (Fickelet al., 2003; Richman, 2003), which arenow known to represent two partiallychimeric subspecies (Zong et al., 2008).EEHV1 and EEHV2 differ by an averageof 25% at the nucleotide level acrosstheir genomes, whereas EEHV1A andEEHV1B differ by 15–55% at severalsmall loci, but by no more than 1–2% overother large segments of their genomes(Richman, 2003; Ehlers et al., 2006; Zonget al., 2007; Richman, unpubl. data). Twomore highly diverged species, EEHV3 andEEHV4 (35% different), were later iden-tified from two lethal cases of hemorrhagicdisease in Asian elephant calves in NorthAmerica (Garner et al., 2009). Togetherwith EEHV5 or EEHV6 (Latimer et al.,2011) and EEHV7 (Zong, unpubl. data),none of which have been associated withlethal disease, these species of elephantherpesviruses form a novel Proboscivirusgenus that is most closely related to theroseoloviruses in the Betaherpesvirinaesubfamily (Davison et al., 2010). Theprobosciviruses evidently diverged fromall other mammalian herpesviruses over100 million years ago when the ancestors ofmodern elephants diverged from all otherplacental mammals. Many of the captiveelephant cases have been subjected to

EEHV gene subtyping analysis, which hasidentified more than 30 strains of EEHV1Aand six of EEHV1B (Zong et al., 2008;Zong, unpubl. data), indicating that this isprimarily a sporadic and not an epidemicdisease. Those studies have also shown thatthere has been no direct chain of transmis-sion among cases at different elephanthousing facilities or, in most cases, evenamong multiple afflicted progeny of a singlebreeding bull or cow elephant. Recentqualitative real-time PCR assays forEEHV1 have revealed that many asymp-tomatic Asian elephant herdmates of calvesthat died or survived viremic disease at thesame North American housing facilitiescarry and periodically shed EEHV1A,EEHV1B, or EEHV5 in trunk secretions(Stanton et al., 2010; Atkins et al., in press;Stanton et al., in press).

We and others originally speculated thatEEHV disease in Asian zoo elephantsmight be associated with opportunities fordirect or indirect contact with and trans-mission from African zoo elephants (Rich-man et al., 1999, 2000b). However, somecases occurred at facilities that had nevercohoused African elephants and therehave been a number of anecdotal sugges-tions of a similar disease occurring inrange countries, including India, Thailand,Cambodia, Myanmar, and Nepal, withone preliminary published report of anEEHV1A-positive case in Cambodia (Reidet al., 2006). Current evidence indicatesthat only EEHV2, EEHV3, EEHV6, andEEHV7 (but not EEHV1) are found inhealthy asymptomatic African elephants(Zong, unpubl data), whereas onlyEEHV1A, EEHV1B, and EEHV5 havebeen found in healthy asymptomatic Asianzoo elephants (Schaftenaar et al., 2010;Stanton et al., 2010; Hardman et al., 2012;Atkins et al., in press). Therefore, wewished 1) to confirm the presence ofEEHV disease in the wild in an Asianrange country, 2) to address the questionof which EEHV species might be causingdisease there, and 3) to reassess theorigins of pathogenic EEHV1.


382 JOURNAL OF WILDLIFE DISEASES, VOL. 49, NO. 2, APRIL 2013

MATERIALS AND METHODS

Tissue and DNA recovery from field necropsies

Among the 15 South India cases examined,two were captive-born, privately owned ele-phants; six were wild-born juveniles beingreared as orphans at working or training campsor (in one case) at the Arigna Anna ZoologicalPark, Chennai; and seven were from free-ranging wild herds. The locations of the wildcases were distributed relatively uniformlyacross the entire South Western Ghats,including both sides of the Palakhad Gap,which separates two major subpopulations ofelephants in southern India, the largestremaining wild population of E. maximus inAsia. In most cases, liver or heart necropsytissue was collected and preserved in alcoholor directly frozen for long-term storage at245 C. In one positive case, only a viremicblood sample was available without anynecropsy tissue and in another only lymph-node tissue was available. DNA was extractedfrom stored frozen necropsy tissue samplesafter mincing with the use of standardprocedures (Latimer et al., 2011).

PCR amplification

PCR amplification and gel purificationprocedures and most of the PCR primers usedfor the U38/POL, U60/TERex3, U71/gM,U77/HEL, and U51/vGPCR loci were de-scribed by Stanton et al. (2010, in press) orLatimer et al. (2011). Primers used for theU48/gH-TK locus encompassing the N-termi-nal part of U48/glyH and the C-terminal endof U48.5 (TK) were as follows:

L1 LGH7981 59-CT[A/G]CATT[T/G][A/C]CCAAAGTATGGAAGTA-39

L2 LGH7982 59-C[G/A]T[C/T]TATATCATCAAA[A/G]AC[C/T]TCACA-39

R2 LGH7984 59-CAGCCTTCAAGCGGCATACACTG-39

R1 LGH7985 59-GGTAGGTTCACCTACATGGAACTTC-39

First round R1/L151,080 base pairs (bp),second round A L1/R25920 bp, second roundB L2/R151,040 bp, third round A R2/L25860 bp.

Gene subtype DNA sequence analysis

All DNA samples were initially subjected toPCR amplification with first- or second-roundprimers for both PAN-POL and EEHV1-specific DNA polymerase (U38/ POL). DNAsamples from the nine cases that consistently

gave correctly sized PCR products werefurther characterized by additional first-,second-, or third-round seminested PCRanalysis at up to five other previously well-characterized EEHV1 gene loci. We considerthat the other six potential cases are stillinconclusive, possibly because the necropsytissue DNA available was degraded and of lowquality, or because those calves instead diedfrom non-EEHV related causes.

Correctly sized PCR products were purifiedafter agarose gel electrophoresis with a QiagenGel Extraction kit (Qiagen, Valencia, Califor-nia, USA) and subjected to direct-cyclesequencing on both strands with the ABIPRISM DigDye Terminator v3.1 cycle se-quencing kit and analyzed on an ABI 310DNA Sequencer (Life Technologies Inc.,Carlsbad, California, USA) or at MacrogenInc. (Rockville, Maryland, USA). All DNAsequence editing, analysis, and manipulationwas performed with the use of Assembly-LIGN, Clustal-W, and distance-based phylo-genetic trees under a neighbor-joining treeprogram with Tamura Nei logic as implement-ed in MacVector v. 7 (Symantec Corp.,Mountain View, California, USA). Similartrees prepared after alignment in MUSCLEand tree building by maximum likelihood inMEGA5 had identical topography (notshown). Graphic alignments comparing thepatterns of sequence polymorphisms wereprepared in Geneious, where deviations fromconsensus are shown in green (A), blue (C),black (G), or red (T).

The Indian EEHV1 PCR DNA sequenceresults described here are deposited in Gen-Bank under accessions JX011032-JX011038(U38/POL), JX011039-JX011046 (U48/gH-TK), JX011047-JX011055 (U51/vGPCR1),JX011056-JX011062 (U60/TERex3), JX011063-JX011071(U71/gM), and JX011072-JX011079(U77/HEL). The comparative data used in thephylogenetic trees and graphic alignments fromprototype North American EEHV cases EEH-V1A(NAP18, NAP11), EEHV1B(NAP14, NAP19),EEHV2(NAP12), and EEHV6(NAP35) come fromGenBank accessions HM568528, HM568515,HM568541, HM568556, HM568564, andJN983113 (U71-gM), HM568525, HM568513,HM568538, HM568552, and JN983120 (vGPCR1),HM568529, HM568515, HM568542, HM568557,HM568564,andJN983126(HEL)plusHM568525,HM568513, HM568538, HM568552, HM568561,and JN983119 (gH-TK), as well as JN633913(NAP33, vGPCR1), JN633921 (NAP45, vGPCR1),and JQ300058 (NAP42-D, gH-TK). Aftermanual sequence read editing, the assembly,Clustal-W or MUSCLE alignments and distance-based neighbor-joining or maximum-likelihood


ZACHARIAH ET AL.—FATAL ELEPHANT HERPESVIRUS HEMORRHAGIC DISEASE IN INDIA 383

phylogenetic tree dendrograms were generatedas implemented in MacVector v. 7.0 or Geneiousv. 5.4.6.

RESULTS

The deaths of up to 20 young Asianelephant calves within south India since 1997have been suspected of being caused by anacute disseminated viral hemorrhagic diseasefirst described in captive zoo and circus ele-phants (Richman et al., 1999). Field necrop-sies were carried out on 15 suspected cases ofsudden hemorrhagic deaths in juveniles orsubadults that came to the attention of theKerala Forest Department in southern Indiabetween 2007 and 2011. Most revealed edemaand tongue cyanosis as well as typicalhemorrhagic features in the heart and liverconsistent with those described previously inyoung captive Asian elephants that had diedsuddenly of EEHV disease (Richman et al.,2000). Representative examples of grosspathologic features are provided (Fig. 1).

The US National Elephant HerpesvirusLaboratory at the National Zoological Park

in Washington, DC, and the Johns Hop-kins University in Baltimore, Maryland,working with the Kerala Forest Depart-ment, have established a diagnostic EEHVlaboratory in Wayanad Wildlife Sanctuaryin Kerala. Initial PCR analysis carried outthere with the use of both PAN POL andspecific EEHV1 POL primers on DNAsamples from stored frozen tissues identi-fied nine of the 15 suspected cases testedthat that were sufficiently well preservedand abundant to yield high levels of PCRproducts that were positive for EEHV1after first- or second-round amplification(Fig. 2). Features and sources of thesenine positive cases are listed in Table 1.From our previous semiquantitative anal-yses and plasmid DNA reconstructionexperiments with limiting dilution stepswith conventional PCR (Latimer et al.,2011) or real-time PCR analysis (Stantonet al., 2010), we determined that strongfirst-round positive bands with PAN-POLEEHV primers from PBMC DNA repre-sent levels of viral DNA ranging from 1


FIGURE 1. Pathologic changes of elephant endotheliotropic herpesvirus (EEHV) –associated diseasefound during field necropsy. (A) Asian elephant (Elephas maximus) with cyanosis of the tongue attributed toEEHV disease; (B) epicardial surface of the heart (apex view) showing severe extensive hemorrhage; (C)ventricular endocardial surface of the heart showing multifocal areas of ecchymotic hemorrhage; (D) serosalmembrane surface of the liver showing diffusely scattered petechial hemorrhage; (E,F) photomicrograph oftwo capillary endothelial cells containing typical basophilic intranuclear viral inclusion bodies from necropsyliver tissue. H&E stain, bar525 mm.


million to 75 million copies or genomeequivalents per milliliter. Blood and nec-ropsy DNA samples from healthy andknown latently infected elephants notundergoing primary or reactivated infec-tions have always been negative, even afterthird-round conventional PCR. Our bestestimates of the detection limits for first-,second-, and third-round PCR as usedhere are 375,000, 15,000, and 600 genomeequivalents per milliliter of blood. Forwhole-blood DNA with strong first-roundPCR products this represents 1–75 milliongenome equivalents per 5 million PBMCs.Although we did not have blood samplesfrom the India cases, the necropsy tissueDNA samples used here averaged thesame 50–75 mg/mL as for the quantitatedUS PBMC DNA samples. Therefore,when combined with the evidence forviral inclusion bodies within a small subsetof the vascular endothelial cells (assume1%) in sufficiently well-preserved Indiancase necropsy tissue, we can estimate thetotal amount of viral DNA per infectedendothelial cell of 20–1,500 viral genomeequivalents per cell, essentially the sameas in the captive elephant cases. This valueis compatible with active acute lytic viralreplication, not latent-state infection.

Detailed gene subtype DNA sequencinganalysis at multiple PCR loci was per-formed yielding unambiguous DNA se-quence data for four to six tested PCR lociin each case (Table 1). The first three PCRloci used, U38/POL, U60/TER, and U76-U77/HEL represent well conserved coregenes that serve primarily to distinguishunambiguously between the EEHV1A andEEHV1B subspecies, which differ at theseloci by 15/480, 11/360 and 20/640 com-mon nucleotides, respectively (about 3%

each), but otherwise display relatively fewadditional characteristic polymorphisms.The primers at these loci and U71-gMalso have the potential to identify DNAfrom most other species of EEHV in theabsence of EEHV1, but only EEHV1 wasdetected.

The positive samples were also analyzedwith EEHV1-specific primer sets for threestrain variable PCR gene loci, encompass-ing parts of the U71/myristylated tegu-ment protein plus U72/glycoprotein Mregion (U71-gM), the G-protein coupledreceptor (U51/vGPCR1), and an overlap-ping glycoprotein-H (U48/gH) plus thy-midine kinase (U48.5/TK) region (gH-TK). These latter three loci are muchfurther diverged than the others and eachclusters into multiple unlinked subtypes.The results obtained were compared tothose from similar extensive analyses ofEEHV genomes carried out previously onmost known European and North Amer-ican hemorrhagic disease cases (Fig. 3).For U71-gM, the A and B subtypes differat the DNA level by an average of 39/750nucleotides (5%), plus some deletions orinsertions, and there is a rare thirdintermediate subtype (C). In the case ofthe vGPCR1 protein, there are five majorEEHV1 subtype clusters known (A, B, C,D, and E) plus several additional chimericforms E/A, D2/A, or other distinctivevariants (A1, A2, B1, B2, B3, D1, andD2) encompassing a hot spot with up tonine adjacent variable amino acids, in-cluding deletions and insertions. Withinthe hypervariable gH coding segment of


FIGURE 2. Representative agarose gel–ethidiumbromide PCR band results obtained after first-round(520 base pairs [bp]) and second-round seminested(250 bp) amplification with elephant endotheliotro-pic herpesvirus PAN-POL primers from 12 samplesof necropsy tissue DNA from Asian elephants(Elephas maximus). Lanes 1, 2, and 12, differenttissues from case IP07; 3, case IP10 (negative); 4–8multiple tissue samples from case IP06; 9, case IP01;10, case IP04 (negative); 11, case IP11.



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the gH-TK locus, all EEHV1B strainsdiffer from the EEHV1As by 35% at theamino-acid level, with the EEHV1Astrains themselves splitting further intofive more subtype clusters (A, C, D, E,and F) that differ by 7–15% at the aminoacid level.

Overall, the DNA sequencing resultsrevealed that eight of the nine positiveIndian cases involved EEHV1A strains,whereas one was an EEHV1B strain.Furthermore, all but two were readilydistinguishable as independent unrelatedstrains (Table 1). The exceptions werefrom two orphan calves at Kodanad Campthat died within 3 days of one another inDecember 2007. These had identicalDNA sequences at all six PCR loci tested(totaling over 4,000 bp), including over

1,500 bp tested from tissue of each of fiveorgans sampled for calf 1 and in both theheart and liver for calf 2. Several otherinstances of two afflicted calves at thesame location being infected almost si-multaneously with identical EEHV1strains have also been observed in Europeand North America. Remarkably, amongthe two most hypervariable PCR locitested, the Indian cases included four ofthe five known major vGPCR1 subtypesand five of the six known major glycopro-tein-H subtypes, thus encompassing al-most all of the overall genetic range ofEEHV1 variants observed to occur amongthe 31 Asian elephant cases analyzed in asimilar way from Europe and NorthAmerica. Phylogenetic comparisons ofthe viruses from all nine Indian cases with


FIGURE 3. Phylogenetic distance–based dendrograms showing patterns of DNA level divergence andsubtyping among Indian elephant endotheliotropic herpesvirus (EEHV1) cases (boxed in orange) incomparison with prototype examples of EEHV1A, EEHV1B, EEHV6, and EEHV2 at the four most variablePCR sequence loci: (A) U71/gM, (B) U77/HEL, (C) U51/vGPCR1, and (D) U48/gH-TK. Bootstrap values areindicated at branch points.



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selected prototype EEHV1A, EEHV1B,EEHV2, and EEHV6 cases at the U71-gM, vGPCR1, HEL, and gH-TK loci arepresented in Figure 3, and graphic align-ment presentations of the nucleotidepolymorphism patterns for the same fourvariable loci are presented in Figure 4.Although a small number of novel poly-morphisms were detected, all nine Indianexamples were closely related to knownsubtypes at each locus examined. Com-parisons of the overall distribution ofEEHV1 subtypes for the vGPCR1 andgH-TK loci between Indian, North Amer-ican, and European cases are illustrated inFigure 5. The actual numbers of nucleo-tide polymorphisms and deletion or inser-tion differences found among the individ-ual Indian EEHV1 strains at all six PCRloci are presented in Figure 6.

DISCUSSION

We conclude that sporadic EEHV1-associated hemorrhagic disease is distrib-uted across the entire southern Indiaelephant population and that it displaysgross and microscopic pathologic featuresthat are indistinguishable from thosefound in the highly lethal disease docu-mented previously in captive Asian ele-phants in Europe and North America.Most importantly, the very wide range ofgenetic diversity observed among thesenine Indian EEHV1 cases is not compat-ible with an earlier model suggesting thatthis disease resulted from cross-speciestransmission of EEHV1 from African toAsian elephants. The latter interpretationwas largely based on the apparent findingof EEHV1 viral DNA in archival paraffinblock specimens of skin nodules fromseveral African elephant calves that wereimported from Zimbabwe to Florida in themid-1980s. However, we have not beenable to reproduce that result upon re-examination of the same archival skinnodule specimens and no other examplesof EEHV1 DNA have been detected innecropsy samples from African elephants.Instead, multiple examples of EEHV2,EEHV3, EEHV6, and EEHV7 have allbeen found in lung and skin nodules fromeight asymptomatic adult African ele-phants from South Africa, Kenya, andthe United States (Zong, unpubl.; V.Pearson, pers. comm.). Similarly, onlyEEHV2 or EEHV6 have been detectedin blood or necropsy samples from justthree known rare cases of hemorrhagic orviremic disease in African elephants inNorth America. We interpret that theearlier result was most likely a contami-nation error and that both EEHV1A andEEHV1B are natural endogenous virusesthat coevolved with Asian elephant hostsrather than in African elephants. Relative-ly recent introduction (even several thou-sand years ago) from just one or a fewexogenous sources would have resulted ina very limited number of strains and a very


FIGURE 5. Comparison of the major elephantendotheliotropic herpesvirus (EEHV1) gene subtypedistribution patterns among Indian, North American,and European hemorrhagic disease cases at the twomost variable PCR loci examined: (A) U51/vGPCR1and (B) U48/gH-TK.


restricted level of genetic variation. Cur-rent evidence suggests that EEHV4 andEEHV5 (Latimer et al., 2011) may alsobe endogenous to Asian elephants, butwhether they are distributed much less

abundantly or are just less pathogenic thanEEHV1A and EEHV1B is not known.Only one death of a zoo Asian elephantcalf with hemorrhagic disease caused byEEHV3 has been documented (Garner


FIGURE 6. Direct side-by-side nucleotide level differences observed among nine Indian elephantendotheliotropic herpesvirus (EEHV1) cases at all six PCR loci: (A) U38/POL, (B) U60/TERex3, (C) U71/gM, (D) U77/HEL, (E) U51/vGPCR1, and (F) U48/gH-TK. Nucleotide insertion or deletion (Ins/Del)differences found at the U71/gM and U51/vGPCR1 loci are also indicated.


et al., 2009), but otherwise EEHV3 has beenfound on multiple occasions in benign lungand skin nodules from African elephants, andcross-species infections thus appear to be rare.

Herpesvirus genomes evolve relativelyslowly and are generally highly stable, yet

individual human cytomegaloviruses(HCMV), for example, display considerablehypervariability in certain genes that mayrepresent relics of ancient genomic diver-gence that has subsequently become scram-bled through extensive recombination. The


FIGURE 6. Continued.


vGPCR1 and glycoprotein-H subtype hy-pervariability in EEHV1 appears to indi-cate a similar ancient species population-drift effect. We had anticipated that themultiple subtypes of EEHV1 originated,and might still be distributed nonuniformly,in Asian elephant subpopulations, especial-ly considering that the Nilgiri Eastern GhatE. maximus population itself displays rela-tively narrow nuclear and mitochondrialDNA haplotype diversity compared to thatof other subpopulations (Vidya et al., 2005).However, this was not the case; insteadmost known subtypes were present, evenwithin this small number of cases fromsouthern India, representing almost theentire known genetic diversity of EEHV1.This result implies that EEHV1 is likely tobe an ancient infection carried by, trans-mitted between, and widespread among allAsian elephant populations.

Evidently, both the A and B chimericvariants of EEHV1, but seemingly notthe other EEHV2 to EEHV7 species ofprobosciviruses, commonly produce severehemorrhagic pathology upon primary in-fection within all present-day Asian ele-phant populations, whether born andreared in the wild or under human care.Since the late 1980s, acute primary EEHV1viremic disease has afflicted more than20% of Asian elephant calves born in NorthAmerica, with an observed fatality rate of.80%. Why a virus that would normally beexpected to be well adapted to and benignin its natural hosts displays such fulminantpathology remains to be explained, and thepossibility that some other agent or cofactorcontributes to the disease cannot be ruledout. The incidence of disease in the wild isunknown, but if it is similar to that inEurope and North America, the effects onfuture reproductive success and survival ofthis endangered species could be great.Additional studies on the prevalence ofinfections by the EEHV1, EEHV4, andEEHV5 viruses and of EEHV-associatedhemorrhagic disease, especially in free-ranging wild elephants and in all Asianrange countries, are needed.

ACKNOWLEDGMENTS

Financial support for these studies, for thesetup and operation of the diagnostic labora-tory in Wayanad, Kerala, and for travel of teammembers from NEHL and Johns HopkinsUniversity to India came from the Interna-tional Elephant Foundation, the SmithsonianInstitution, and the Kerala Forest Depart-ment. Studies at Johns Hopkins Universitywere supported in part by National Institutesof Health research grant R01 AI24576 toG.S.H. Field research by A.Z. was supportedin part by the US Fish and Wildlife ServicesAsian Elephant Conservation Fund. We thankT. V. Anilkumar for the inclusion body pho-tomicrographs and R. Thirumurugan of theArigna Anna Zoological park in Chennai forproviding tissue samples.

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Submitted for publication 19 July 2012.Accepted 20 October 2012.



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