NOVEL MOLECULAR AND GENOMIC ASPECTS OF ELEPHANT ...
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NOVEL MOLECULAR AND GENOMIC ASPECTS OF
ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS (EEHV)
by
Simon Y. Long, MLAS, VMD
A dissertation submitted to John Hopkins University in conformity with the
requirements for the degree of Doctor of Philosophy
Baltimore, Maryland
December, 2016
Simon Y. Long
© All Rights Reserved
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Abstract
Two variants of elephant endotheliotropic herpesviruses (EEHV) 1A and 1B
cause a fatal hemorrhagic disease that affects young Asian elephants (Elephas maximus)
compromising the future long-term survival of this endangered species both in captive
and wild populations worldwide. Since its discovery in 1999, more than 100 young
captive and wild Asian elephants are known to have died from EEHV. For reasons that
are not yet understood, approximately 20% of Asian elephant calves appear to be
susceptible to the disease when primary infections are not controlled by normal innate
cellular and humoral immune response. Recent crossed EEHV1 virus from African
elephant hosts (Loxodonta cyclotis and Loxodonta africana) to Asian elephants in
captivity was previously suggested as the cause of disease. This argument was disproved
by our field work in India demonstrating the high level of EEHV1 subtype genetic
diversity found among the orphan camp and wild Asian elephant calf cases showing the
Indian strains matches that among over 30 EEHV1 strains that have been evaluated from
Europe and North America. Over the years, multiple species of herpesviruses were
discovered with three different lineages forming the Proboscivirus genus (EEHV1/6,
EEHV2/5, and EEHV3/4/7) infecting both Asian and African elephants with lethal
hemorrhagic disease confined to EEHV1 in Asian elephant calves. Milder disease caused
by EEHV5 and EEHV4 were increasingly being recognized during EEHV1 surveillance
in Asian elephants. The complete 180-kb genomes of prototype strains from three AT-
rich branch viruses, EEHV1A, EEHV1B, and EEHV5, have been published with hints of
the second major branch of GC-rich viruses (EEHV3, EEHV4, and EEHV7). The
complete 206-kb genome of EEHV4 was determined directly from a trunk wash sample
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using next-generation sequencing and de novo assembly procedures revealing a genome
composed of 119 genes with an overall collinear organization similar to the AT-rich
EEHV with major features including a family of 26 paralogous 7xTM and vGPCR-like
genes plus 25 novel or missing genes. Many fundamental questions about the mechanism
of pathogenesis and appropriate effective drug treatment for EEHVs have remained
poorly understood because of the inability of scientists to grow the virus in cell culture.
Successfully propagation of EEHV in laboratory conditions will permit critical future
experimental research for progress towards understanding the pathology and cell biology
of this virus, as well as for any hoped for possibility of generating live attenuated
vaccines. I report here the first elephant umbilical venule endothelial cell (EUVEC)
cultures overlay with fresh minced Asian elephant necropsy tissue samples from a
confirmed EEHV1A associated death with persistence EEHV1A viral DNA PCR
detection for several months in spite of multiple cell culture passages and medium
changes. All these aspect of research in EEHV adds to the general knowledge in hopes to
help recover the 20% of all captive bred calves that are currently lost from this disease,
thus increasing the chances of survival of this species considering all of the enduring
conservation associated pressures it is under.
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Thesis advisor: Gary S. Hayward, PhD
Professor, Dept. of Oncology
Second reader: Joseph L. Mankowski, DVM, PhD, Dipl. ACVP
Professor, Dept. of Molecular and Comparative Pathobiology
Committee: Joseph L. Mankowski, DVM, PhD, Dipl. ACVP (Chair)
Wade Gibson, PhD
J. Marie Hardwick, PhD
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Acknowledgements
First and foremost, immense thanks to my thesis advisor Dr. Gary Hayward for
taking me on as a PhD student under a challenging thesis research theme studying
elephant endotheliotropic herpesvirus combining both herpesvirus virology and
veterinary pathology. I would like to sincerely thank my thesis committee chair and
director of Molecular Comparative Pathobiology (MCP), Dr. Joseph Mankowski for
keeping me on track not only for my PhD training but also my veterinary pathology
postdoctoral fellowship under MCP. I am thankful for the rest of my thesis committee
including Dr. Wade Gibson and Dr. Marie Hardwick, for having been a huge help. I
appreciate all the advice and encouragement that they have provided to me over the last
few years. I am also grateful to the Cellular and Molecular Medicine Graduate program
especially to Dr. Rajini Rao, Colleen Graham, and last but not least Leslie Lichter who all
helped me get through some difficult times during my PhD training. Without their
consistent support, I’m not sure I would have completed my training.
The entire elephant community is so supportive of this research and I thank all the
zookeepers, veterinarians, and veterinary pathologists that have contributed time and
samples that have be invaluable to this research. I would also like to thank the folks at
the National Zoo for travel support and especially to Erin Latimer of the National
Elephant Herpesvirus Laboratory for samples and technical support. The following
laboratory members of the Hayward laboratory have been invaluable resources in terms
of advice and technical support: Sarah Heaggans, Colette apRhys, Jianchao Zong, Yong-
Gu Chi, and Chuang-Jiun Chiou. Most importantly, I would like to thank the Morris
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Animal Foundations (D14ZO-411), International Elephant Foundation, and NIH NIAID
for funding this project and fellowship.
I would also like to extend my thanks to all the faculty members and trainees in
the MCP department in playing a major role in my training to become a veterinary
pathologist. Again, I am deeply thankful for Dr. Mankowksi’s advice and guidance both
to me as a PhD student and as a budding veterinary pathologist. To my veterinary
pathology mentor, Dr. Cory Brayton, for keeping me on track, constantly reminding me
about the American College of Veterinary Pathology board exam, and keeping me
focused on my career goals. To the rest of the faculty including Dr. Christine Zink, Dr.
Richard Montali, Dr. Baktiar Famir, Dr. Kathleen Gabrielson, and the late Dr. David
Huso. Many thanks to the pathology postdoctoral fellows Katie Kelly, Gillian Shaw, and
Sarah Beck who trained me when I first started the residency program. Also to all the
junior pathology postdoctoral fellows and residents after me.
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Table of Contents
Title Page…………………………………………………………………………………..i
Abstract …………………………………………………………………………………...ii
Acknowledgments ………………………………………………………………………...v
Table of Contents...………………………………………………………………………vii
List of Tables …………………………………………………………………………...viii
List of Figures ……………………………………………………………………………ix
List of Abbreviations …………………………………………………………………….xi
I. Introduction..............................................................................................................1
II. Fatal herpesvirus hemorrhagic disease in wild and orphan……………………….8
Asian elephants in southern India
III. Complete genome sequence of EEHV4 as the first example……………………35
of a GC-rich branch Proboscivirus.
IV. Propagating elephant endotheliotropic herpesvirus 1A (EEHV1A)……………..83
in primary and immortalized elephant umbilical venule endothelial cells
(EUVEC)
References………………………………………………………………………………105
Curriculum Vitae……………………………………………………………………….109
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List of Tables
Chapter II
Table 2-1. Summary of features and PCR results for nine positive……………...24
cases of EEHV disease from India.
Chapter III
Table 3-1. Gene content and major features of the complete……………………67
205,896-bp EEHV4 (Baylor) genome.
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List of Figures
Chapter II
Figure 2-1. Pathological changes of EEHV-associated disease………………….25
found during field necropsy.
Figure 2-2. Representative agarose gel-ethidium bromide PCR………………...26
band results obtained after first round (520-bp) and second round semi-nested
(250-bp) amplification with EEHV PAN-POL primers from 12 samples of
necropsy tissue DNA.
Figures 2-3. Phylogenetic distance based dendrograms showing………………..27
patterns of DNA level divergence and subtyping amongst the Indian EEHV1
cases.
Figure 2-4. Graphical presentation generated from Geneious……………...……28
of the patterns of DNA sequence polymorphisms across the four most variable
PCR loci.
Figure 2-5. Comparison of the major EEHV1 gene subtype………………….…30
distribution patterns amongst Indian, North American and European hemorrhagic
disease cases at the two most variable PCR loci examined U51/vGPCR1 and
U48/gH-TK.
Figure 2-6. Direct side-by-side nucleotide level differences………………….....31
observed amongst the nine Indian EEHV1 cases at all six PCR loci.
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Chapter III
Figure 3-1. Annotated physical gene map of the complete…………………..….71
EEHV4 (Baylor) genome.
Figure 3-2. Global alignment patterns for the intact EEHV4……………………73
genome compared to EEHV1 and HCMV.
Figure 3-3. EEHV4 encodes a very large family of distantly……………………75
related paralogous 7XTM and vGPCR-like genes.
Figure 3-4. Codon-specific scanning GC-content panels………………………..77
showing the wobble codon GC-bias effect across selected representative segments
of the EEHV4 (Baylor) genome.
Figure 3-5. Complex repeat and inverted repeat patterns………………………..79
within the predicted Ori-Lyt domain of EEHV4.
Figure 3-6. Positions and sizes of three identified……………………………….81
EEHV4A-EEHV4B chimeric domains and boundaries relative to those of
EEHV1A-EEHV1B.
Chapter IV
Figure 4-1. Microphotograph of pEUVEC……………………...……………...101
Figure 4-2 Agarose gel-ethidium bromide of PAN-HEL PCR…………...…….102
Figure 4-3. DNA sequence alignment between detected EEHV…………..…...103
HEL from heart iEUVEC and whole blood necropsy sample.
Figure 4-4. Levels of VGE in heart pEUVEC between estimated……………..104
compared to qPCR detected based on the number of passages.
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List of Abbreviations
CD Chimeric domain
CRE Cyclic AMP-response elements
E34 Open reading frame C
EEHV Elephant endotheliotropic herpesvirus
EUVEC Elephant umbilical venule endothelial cells
gB Glycoprotein B
gH Glycoprotein H
gM Glycoprotein M
gN Glycoprotein N
gO Glycoprotein O
HCMV Human cytomegalovirus
Hel Helicase
hTERT Human telomerase reverse transcriptase
HSV1 Herpes simplex virus 1
iEUVEC Immortalized EUVEC
IFA Immunofluorescence assay
KSHV Kaposi’s sarcoma-associated herpesvirus
LANA Latency associated nuclear antigen
LM-PCR Ligation-mediated polymerase chain reaction
MCP Major capsid protein
MDBP Major DNA binding protein
MIE1 Major immediate-early enhancer domain 1
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MIE2 Major immediate-early enhancer domain 2
MTA mRNA transcript accumulation
NAP North American Proboscivirus
nBA n-butyric acid
OBP Origin binding protein
OBS OBP-binding sites
ORF Open reading frame
PBMC Peripheral blood mononuclear cell
PBS Phosphate-buffered saline
pEUVEC Primary EUVEC
PEL Primary effusion lymphoma
PCR Polymerase chain reaction
POL DNA Polymerase
qPCR Quantitative polymerase chain reaction
RAIP3 Retinoic acid induced protein 3
RCMV Rat cytomegalovirus
RRB Ribonucleotide kinase B
SSP Single-stranded DNA-binding protein
SV40TL Simian vacuolating virus 40 large T antigen
TER Terminase
TK Thymidine kinase
TPA Tetradecanoyl phorbol acetate
vCXCL1 Viral C-X-C motif chemokine ligand 1
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vECTL Viral C-type lectin
vFUT9 Viral fucosyl transferase 9
VGE Viral genome equivalent
vGCNT1 Viral Glucosaminyl (N-acetyl) transferase 1
vGPCR Viral G-protein-coupled receptor
vICA Viral inhibitor of caspase-8-induced apoptosis protein
vMIP Viral macrophage inflammatory protein
vOGT Viral O-linked acetyl glucosamine transferase
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I. Introduction
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EEHV Background
Wildlife conservationists are concerned about the critically endangered Asian
(Elephas maximus) and African elephant (Loxodonta cyclotis and Loxodonta africana)
species due to their alarming decline over the last decade. These declines arise from both
old and current challenges to these species as a whole. Each species faces similar yet very
different unique problems including habitat loss and human encroachment into natural
ranges resulting in human-elephant conflict. In the last several years, recent surges of
highly organized crime syndicates poaching for ivory in Africa has severely depleted the
population of African elephants overall. All these factors threaten elephant populations,
forcing wildlife conservationists to look for alternative avenues including captive
breeding programs to promote species survival. Elephant endotheliotropic herpesviruses
(EEHV) that cause a fatal hemorrhagic disease that affects young Asian elephant calves
compromises the survival of this species both in captivity and in the wild populations
worldwide and hinders these conservation efforts.
Since the report of the discovery of the first novel elephant endotheliotropic
herpesvirus 1A (EEHV1A) in 19951, eight different species of EEHV within the novel
Proboscivirus genus have been identified between Asian and one of the two African
elephant species 2-7. The viruses under this genus can be divided based on AT-rich
(EEHV1A/B, EEHV2, EEHV5 and EEHV6) vs. GC-rich (EEHV3, EEHV4, and EEHV7)
sequences. The majority of these virus species appear to be common endemic subclinical
universal infections regardless of location. However, EEHV1A and EEHV1B are highly
pathogenic species that cause over 90% of the 100 known disease cases8 exclusively in
Asian elephants with the original hypothesis suggesting a recent crossing from African
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elephants in captivity. The remaining 10% of EEHV associated disease cases are from
other virus species.
After more than two decades of study, numerous fundamental questions still
remain unanswered. Initially, it was hypothesized that cross-species transmission of
EEHV1 from African elephants to Asian elephants in captivity scenarios may explain the
severe pathology1,9. Herpesviruses generally coevolve with their hosts to be
asymptomatic in immune competent native hosts while cross-species transmission can
result in pathology in the non-native host. This hypothesis offered an explanation for the
severe fatal hemorrhagic pathology exhibited by young Asian elephants. Full genomic
sequence of EEHV1A/B and EEHV5 representing the AT-rich branch of the
Proboscivirus is available yet the full genomic sequence of a GC-rich example is still
lacking and represents a gap in knowledge which may provide further insight into a role
for this unique group of viruses in the pathology. The inability to grow EEHV in cell
culture is another major challenge in EEHV research; past attempts to cultivate the virus
using standard methods in established cell lines have been unsuccessful.
Detection of EEHV1 in free ranging Asian elephants in India.
It was hypothesized that the fatal hemorrhagic disease caused by EEHV1 in Asian
elephants may be due to cross species transmission from African elephants to Asian
elephants in captivity. Using conventional PCR techniques, EEHV1 was previously
detected in an archived nodule from an African elephant trunk, but these results could not
be duplicated and thus were suggestive of a false positive due to contamination. Recent
attempts to detect EEHV1 from additional African elephant samples with PCR have also
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failed to detect EEHV1, arguing against the cross species transmission hypothesis.
Detection of EEHV1 in wild Asian elephants in their natural range would further
disprove the hypothesis that EEHV1 only causes pathology following cross species
transmission. Prior to our published 2012 fieldwork10, there has only been one single
report of a PCR positive EEHV1 associated death in Cambodia by Reid11 in 2006 with
additional anecdotal case reports all throughout the natural range of Asian elephants. The
published work that I present in chapter 2 reports 9 different EEHV1-associated deaths in
free-ranging Asian elephant calves in southern India. These deaths are the result of 8
unique strains of EEHV1A with wide genetic diversity in two hypervariable genetic loci
that is similar to the diversity noted in both North American and European cases of fatal
EEHV1 infection. This finding disproves the cross species transmission hypothesis by
confirming the endemic nature of EEHV1 in wild Asian elephants with an additional later
report in Laos12, and unpublished lethal cases confirmed by PCR DNA sequencing in
Sumatra (two) and Nepal (one).
Complete genome sequence of EEHV4 as the first example of a GC-rich branch
proboscivirus.
Multiple species of herpesviruses from three different lineages of the
Proboscivirus genus (EEHV1/6, EEHV2/5, EEHV3/4/7) infect both Asian and African
elephants, but lethal hemorrhagic disease is largely confined to Asian elephant calves and
is predominantly associated with EEHV1. EEHV2, EEHV3, EEHV6, and EEHV7 have
been identified in African elephants7. EEHV1A, EEHV1B, EEHV3, EEHV4 and EEHV5
have been responsible for the deaths of Asian elephant calves3,4,13. Mild disease caused
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by EEHV52,4,14 or EEHV415 is increasingly identified in Asian elephants, but little is
known about the latter. EEHV4 is estimated to have diverged at least 35 million years
ago from the others within a distinctive GC-rich branch of the Probosciviruses. The
complete 180-kb genomes of prototype strains from three AT-rich branch viruses,
EEHV1A16,17, EEHV1B16, and EEHV514, have been published with hints of the second
major branch of GC-rich viruses (EEHV3, EEHV4, and EEHV7)6,7. The death of a Thai
Asian elephant from EEHV4 hemorrhagic disease18 and disease episodes in the USA
involving EEHV3 and EEHV43, as well as detection of EEHV3B and EEHV7B in skin
nodules, emphasizes the need for a greater understanding of other species within the
highly diverged GC-rich subgroup of EEHVs. The very high level of divergence of both
EEHV3 and EEHV4 compared to the other EEHVs deserves a further study so that we
can gain understanding of this group of viruses.
Recent reports in Asian elephant calves at the Houston Zoo noted mild symptoms
with detection of EEHV4 using PCR from blood and later trunk wash samples15. In
collaboration with Paul Ling from Baylor College of Medicine, next generation Illumina
sequencing data directly yielded contigs representing 80% of the genome from a trunk
wash pellet from two different Asian elephant calves who were infected with the same
strain of EEHV4 at a Houston Zoo in 2014. The remaining 20% of the genome was filled
and joined by me using standard Sanger PCR amplification and cycle sequencing on the
same trunk wash pellet sample. Chapter 3 documents the unique features of this GC-rich
EEHV4 genome includes 119 encoded genes arranged in similar co-linear organization to
that of the AT-rich branch of viruses, a family of 26 paralogous 7Xtransmembrane and
viral G-coupled protein receptor-like genes, 25 novel or missing genes with an unusual
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distribution of tracts of 5-11 successive A or T nucleotides in intergenic domains between
the GC-rich protein coding regions with a high GC-rich bias in the third wobble positions
of codons for many proteins, and two novel captured cellular genes. Comparison of the
sequence of this pathogenic subspecies of EEHV4 with the prototype EEHV1A genome
from the AT-rich branch will lead to important insights into the common proteins shared
between these two anciently diverged lineages of elephant herpesviruses, and may lead to
the discovery of novel virulence factors.
Attempts to propagate elephant endotheliotropic herpesvirus 1A (EEHV1A) in
primary and immortalized elephant umbilical venule endothelial cells (EUVEC).
The inability to grow the virus in cell culture is the major roadblock in EEHV
research. If successful viral propagation in cell culture could be achieved, researchers
would gain the ability to (a) accumulate large quantities of virus for easier molecular
analysis of the novel genes and proteins that these viruses encode, (b) carry out
pharmacologic studies to evaluate the efficacy of human anti-herpesviral drugs in
inhibiting the replication of elephant herpesviruses (whether currently in use or in
development); and finally (c) passage attenuated virus promote a vaccine development.
Within the last few years, our lab has established techniques to culture primary elephant
umbilical venule endothelial cells (pEUVEC) and immortalized EUVEC (iEUVEC) from
Asian elephant umbilical cords. A recent experiment that I performed with fresh necropsy
tissue samples from an elephant who died due to confirmed EEHV1 infection resulted in
detection of EEHV1A DNA in both pEUVEC and iEUVEC cultures that persisted for 54
and 122 days, respectively. This finding is suggestive of viral cellular entry and
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persistence retention of virus in latent phase in culture rather than in lytic replicating
phase. Chapter 4 reports the findings of this attempt to propagate EEHV1A in primary
and immortalized EUVEC cultures.
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II. Fatal herpesvirus (EEHV) hemorrhagic disease in wild and orphan
Asian elephants in southern India.
This chapter has been previous published as:
Zachariah A, Zong JC, Long SY, Latimer EM, Heaggans SY, Richman LR, Hayward
GS. Fatal herpesvirus (EEHV) hemorrhagic disease in wild and orphan Asian elephants
in southern India. Journal of Wildlife Diseases. 2013. 49(2):381-393
Reprint with permission of the publisher, Wildlife Disease Association.
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Abstract
Up to 65% of the deaths of young Asian elephants between 3 months and 15 years
of age in Europe and North America over the past twenty years have been attributed to
hemorrhagic disease 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 hemorrhagic disease occurring in Southern India, we studied
pathological tissue samples collected from field necropsies. Nine convincingly positive
cases amongst both orphaned camp and wild Asian elephants were identified by
diagnostic PCR. These were then subjected to detailed gene subtype DNA sequencing at
multiple PCR loci, which revealed seven distinct strains of EEHV1A and one of
EEHV1B. Two orphan calves that died within three days of one another at the same
training camp proved to have identical EEHV1A DNA sequences to one another
indicating a common epidemiological source. However, the high level of EEHV1 subtype
genetic diversity found amongst the other Indian strains matches that amongst over 30
EEHV1 strains that have been evaluated from Europe and North America. These results
argue strongly against the previously suggested notion that this is just a disease of captive
elephants and that the EEHV1 virus has crossed recently from African elephant
(Loxodonta africana) hosts to Asian elephants (Elephas maximus). Instead, both the virus
and the disease are evidently widespread in Asia and, despite the disease severity, Asian
elephants themselves appear to be the ancient endogenous hosts of both EEHV1A and
EEHV1B.
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Introduction
An acute hemorrhagic disease with 85% fatality rate was originally described in
captive Asian elephants in both North American and European zoos 1,19 ,20-22. Amongst 52
suspected cases since 1988, the presence of high levels of a novel type of herpesvirus
called Elephant Endotheliotropic Herpesvirus or EEHV has been confirmed by DNA
PCR tests carried out at the National Elephant Herpesvirus Laboratory in Washington,
DC. on blood or necropsy tissue from all 28 North American cases and from 10 of the 24
European cases (3,4, Richman LK et al unpublished data). However, these viruses have
never been detected in numerous random unrelated elephant necropsy tissue samples nor
in blood from healthy asymptomatic elephants. The PCR test results show that EEHV
disease occurs predominantly in Asian juveniles between one and eight years of age and
that here have been only ten known survivors, all of which received timely and
aggressive treatment with the human anti-herpesvirus drugs famciclovir or ganciclovir
23,24. Initial mild symptoms of facial edema and tongue cyanosis progress rapidly to
systemic viremia followed by hemorrhaging in all major internal organs 9. Nuclear
inclusion bodies and enveloped virions can be observed by light and electron microscopy
respectively in microvascular endothelial cells and extremely high levels of viral DNA
are found in white blood cells, serum and necropsy tissue (4; Zong J-C et al, unpublished
data).
Most herpesviruses have become highly adapted to the natural hosts in which they
have evolved during mammalian evolution and usually cause only mild localized disease
in healthy animals. Therefore, the original description of a herpesvirus associated with
rapid acute hemorrhagic disease in juvenile Asian elephants was unexpected 1. However,
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the idea that it might involve a cross-species transfer of a virus that was adapted to
African elephants into naïve Asian elephants seemed to make sense of the unexpectedly
severe pathology. Originally, two distinct species of EEHV were described: EEHV1 was
associated with 11 cases in captive Elephas maximus, whereas EEHV2 was associated
with the deaths of two young Loxodonta africana 1. The EEHV1 viruses found in both
those and all subsequent European and North American cases fall into two subgroups
referred to as EEHV1A and EEHV1B 21,25, which are now known to represent two
partially chimeric sub-species 22. EEHV1 and EEHV2 differ by an average of 25% at the
nucleotide level all the way across their genomes, whereas EEHV1A and EEHV1B differ
by between 15 and 55% at several small loci, but by no more than 1 to 2% over other
large segments of their genomes 25,26. Two more highly diverged species EEHV3 and
EEHV4 (35% different) were later identified in two other lethal cases of hemorrhagic
disease in North American Asian elephant calves 3. Together with EEHV5 or EEHV6 4 as
well as EEHV7 (Zong J-C et al, unpublished data), which have not yet been associated
with any lethal disease cases, these multiple species of elephant herpesviruses form a
novel Proboscivirus genus that is most closely related to the Roseoloviruses within the
Betaherpesvirinae sub-family27. The Probosciviruses evidently diverged from all other
mammalian herpesviruses over 100 million years ago when the ancestors of modern
elephants diverged from all other placental mammals. All of the positive captive elephant
cases have been subjected to EEHV gene subtyping analysis, which has identified more
than 30 distinct strains of EEHV1A and six of EEHV1B 22,28 (Zong J-C et al, unpublished
data), indicating that this is primarily a sporadic not epidemic disease. Those studies have
also shown that there has been no direct chain of transmission between cases at different
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elephant housing facilities, nor in most cases even between multiple afflicted progeny of
a single breeding bull or cow elephant. Furthermore, recent qualitative real-time PCR
assays for EEHV1 have revealed that many asymptomatic Asian elephant herdmates of
calves that died or survived viremic disease at the same North American housing
facilities carry and periodically shed either EEHV1A, EEHV1B or EEHV5 in trunk
secretions 29,30.
We and others originally speculated that EEHV disease in Asian zoo elephants
might be associated with opportunities for direct or indirect contact with and transmission
from African zoo elephants 1,20. However, some cases occurred at facilities that had never
co-housed African elephants, and there have also been a number of anecdotal suggestions
of a similar disease occurring in range countries including India, Thailand, Cambodia,
Myanmar and Nepal, with one preliminary published report of an EEHV1A-positive case
in Cambodia 11. Furthermore current evidence indicates that only EEHV2, EEHV3,
EEHV6 and EEHV7, but not EEHV1, are found in healthy asymptomatic African
elephants (Zong JC et al, unpublished data), whereas only EEHV1A, EEHV1B and
EEHV5 have so far been found in healthy asymptomatic Asian zoo elephants2,29,31,32.
Therefore, we wished to (a) Confirm the presence of EEHV disease in the wild in an
Asian range country; (b) Address the question of which EEHV species might be causing
disease there; and (c) Reassess the origins of pathogenic EEHV1.
Materials and Methods
Tissue and DNA Recovery from Field Necropsies
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Amongst the 15 South India cases examined, two were captive-born privately-
owned elephants, six were wild-born juveniles being reared as orphans at working or
training camps or in one case at the Arigna Anna Zoological Park, Chennai, and seven
were from free-ranging wild herds. The physical locations of the wild cases were
distributed relatively uniformly across the entire South Western Ghats, including on both
sides of the Palakhad gap which separates two major sub-populations of the elephants in
Southern India, the largest remaining wild population of Elephas maximus in Asia. In
most cases, liver or heart necropsy tissue was collected and preserved in alcohol or
directly frozen for long-term storage at -45°C. In one positive case, only a viremic blood
sample was available without any necropsy tissue and in another only lymph node tissue
was available. DNA was extracted from stored frozen necropsy tissue samples after
mincing using standard procedures that have been described previously 4.
PCR Amplification
PCR amplification and gel purification procedures and most of the PCR primers
used for the U38/POL, U60/TERex3, U71/gM, U77/HEL and U51/vGPCR loci were
described in Stanton et al 29,30 or Latimer et al 4. Primers used for the U48/gH-TK locus
encompassing the N-terminal part of U48/glyH and the C-terminal end of U48.5 (TK)
were as follows:
L1 LGH7981 5’-CT[A/G]CATT[T/G][A/C]CCAAAGTATGGAAGTA-3’
L2 LGH7982 5’-C[G/A]T[C/T]TATATCATCAAA[A/G]AC[C/T]TCACA-3’
R2 LGH7984 5’-CAGCCTTCAAGCGGCATACACTG-3’
R1 LGH7985 5’-GGTAGGTTCACCTACATGGAACTTC-3’
14
First round R1/L1 = 1080-bp; second round A L1/R2 = 920-bp; second round B L2/R1
= 1040-bp; third round A R2/L2 = 860-bp.
Gene Subtype DNA Sequence Analysis
All DNA samples were initially subjected to PCR amplification with first or
second-round primers for both PAN-POL and EEHV1-specific DNA polymerase (U38/
POL). DNA samples from the nine cases that consistently gave correctly-sized PCR
products were then all further characterized by additional first, second or third-round
semi-nested PCR analysis at up to five other previously well-characterized EEHV1 gene
loci. We consider that the other six potential cases are still inconclusive at present,
possibly because the necropsy tissue DNA available was degraded and of low quality, or
because those calves instead died from non-EEHV related causes. Correct sized PCR
products were purified after agarose gel electrophoresis with a Qiagen Gel Extraction kit
(Qiagen, Valencia, CA) then subjected to direct cycle sequencing on both strands with
the ABI PRISM DigDye Terminator v3.1 cycle sequencing kit and analyzed on an ABI
310 DNA Sequencer (Life technologies Inc, Carlsbad, CA) or at Macrogen, Inc,
Rockville, MD. All DNA sequence editing, analysis and manipulation was performed
using Assemblalign, Clustal-W and distance based phylogenetic trees under a neighbor-
joining tree program with Tamura Nei logic as implemented in MacVector vers 7
(Symantec Corp. Mountain View, CA). Similar trees prepared after alignment in
MUSCLE and tree building by maximum likelihood in MEGA5 had the identical
topography (not shown). Graphical alignments comparing the patterns of sequence
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polymorphisms were prepared in Geneious, where deviations from consensus are shown
in green (A), blue (C), black (G) or red (T).
All of the Indian EEHV1 PCR DNA sequence results described here have been
deposited in Genbank under accession Nos. JX011032-JX011038 (U38/POL), JX011039-
JX011046 (U48/gH-TK), JX011047-JX011055 (U51/vGPCR1), JX011056 -JX011062
(U60/TERex3), JX011063-JX011071 (U71/gM), JX011072-JX011079 (U77/HEL). The
comparative data used in the Figure 2-3 phylogenetic trees and Figure 2-4 graphical
alignments from prototype North American EEHV cases EEHV1A (NAP18, NAP11),
EEHV1B (NAP14, NAP19), EEHV2 (NAP12) and EEHV6 (NAP35) comes from
Genbank Accession Nos HM568528, HM568515, HM568541,HM568556, HM568564
and JN983113 (U71-gM), HM568525, HM568513, HM568538, HM568552 and
JN983120 (vGPCR1), HM568529, HM568515, HM568542, HM568557, HM568564 and
JN983126 (HEL) plus HM568525, HM568513, HM568538, HM568552, HM568561 and
JN983119 (gH-TK) as well as JN633913 (NAP33, vGPCR1), JN633921 (NAP45,
vGPCR1) and JQ300058 (NAP42-D, gH-TK). After manual sequence read editing, the
assembly, Clustal-W or MUSCLE alignments and distance-based Neighbor-Joining or
Maximum Likelihood phylogenetic tree dendrograms were generated as implemented in
MacVector vers 7.0 or Geneious vers 5.4.6.
Result
The deaths of up to twenty young Asian elephant calves within South India since
1997 have been suspected of being caused by an acute disseminated viral hemorrhagic
disease first described in captive zoo and circus elephants 1. Field necropsy examinations
16
were carried out on 15 suspected cases of sudden hemorrhagic deaths in juveniles or sub-
adults that came to the attention of the Kerala Forest Department in Southern India
between 2007 and 2011. Most revealed symptoms of edema and tongue cyanosis as well
as typical hemorrhagic features in the heart and liver consistent with those described
previously in young captive Asian elephants that had died suddenly of EEHV disease 9.
Representative examples of gross pathological features, including (A) tongue cyanosis,
(B, C) cardiac and (D) hepatic hemorrhages and (E, F) vascular endothelial cell inclusion
bodies from free-range cases of Indian elephant hemorrhagic disease are pictured in
Figure 2-1.
The National Elephant Herpesvirus Laboratory at the National Zoological Park in
Washington DC and the Johns Hopkins University in Baltimore, MD working with the
Kerala Forest Department, have established a diagnostic EEHV laboratory in Wayanad
Wildlife Sanctuary in Kerala. Initial PCR analysis carried out there using both PAN POL
and specific EEHV1 POL primers on DNA samples from stored frozen tissues identified
nine of the 15 suspected cases tested that that were sufficiently well-preserved and
abundant to yield high levels of PCR products that were positive for EEHV1 after either
first or second round amplification (Figure 2-2). Features and sources of these nine
positive cases are listed in Table 2-1. From our previous semi-quantitative analyses and
plasmid DNA reconstruction experiments using either limiting dilution steps with
conventional PCR 4 or real-time PCR analysis2010 29, we have determined that strong
first round positive bands with PAN-POL EEHV primers from PBMC DNA represent
levels of viral DNA ranging from at least 1 million on up to 75 million copies or genome
equivalents / ml. Blood and necropsy DNA samples from healthy and known latently
17
infected elephants not undergoing primary or reactivated infections have always been
negative even after third round conventional PCR. Our best estimates of the detection
limits for first, second, and third round PCR as used here are 375,000, 15,000 and 600
genome equivalents per ml of blood). For whole blood DNA with strong first round PCR
products this represents 1 to 75 million genome equivalents per 5 million PBMCs. Whilst
we did not have blood samples from the India cases, the necropsy tissue DNA samples
used here averaged the same 50 to 75 ug/ml as for the quantitated USA PBMC DNA
samples. Therefore, when combined with the evidence for viral inclusion bodies within a
small subset of the vascular endothelial cells (assume 1%) in sufficiently well-preserved
Indian case necropsy tissue, we can estimate the total amount of viral DNA per infected
endothelial cell of between 20 to 1500 viral genome equivalents per cell, essentially the
same as in the captive elephant cases. This value is only compatible with active acute
lytic viral replication not latent state infection.
Detailed gene subtype DNA sequencing analysis at multiple PCR loci was then
performed yielding unambiguous DNA sequence data for at least four and up to six tested
PCR loci in each case as summarized in Table 2-1. Amongst the first three PCR loci
used, U38/POL, U60/TER and U76-U77/HEL all represent well conserved core genes
that serve primarily to distinguish unambiguously between the EEHV1A and EEHV1B
sub-species, which differ at these loci by 15/480, 11/360 and 20/640 common nucleotides
respectively (about 3% each), but otherwise display relatively few additional
characteristic polymorphisms. The primers at these loci and U71-gM do also have the
potential to identify DNA from most other species of EEHV in the absence of EEHV1,
but only EEHV1 was detected.
18
Subsequently, the positive samples were also analyzed with EEHV1-specific
primer sets for three strain variable PCR gene loci, encompassing parts of the
U71/myristylated tegument protein plus U72/glycoprotein M region (U71-gM), the G-
protein coupled receptor (U51/vGPCR1) and an overlapping glycoprotein-H (U48/gH)
plus thymidine kinase (U48.5/TK) region (gH-TK). These latter three loci are much
further diverged than the others and each cluster into multiple unlinked subtypes. The
results obtained were then compared to those from similar extensive analyses of EEHV
genomes carried out previously on most known European and North American
hemorrhagic disease cases (Figure 2-3). For U71-gM, the A and B subtypes differ at the
DNA level by an average of 39/750 nucleotides (= 5.3 %) plus some deletions/insertions
and there is a rare third intermediate subtype (C) as well. In the case of the vGPCR1
protein, there are five major EEHV1 subtype clusters known (A, B, C, D and E) plus
several additional chimeric forms E/A, D2/A or other distinctive variants (A1, A2, B1,
B2, B3, D1 and D2) encompassing a hotspot with up to nine adjacent variable amino
acids, including deletions and insertions. Within the hypervariable gH coding segment of
the gH-TK locus, all EEHV1B strains differ from the EEHV1As by 35% at the amino
acid level, with the EEHV1A strains themselves splitting further into five more subtype
clusters (A, C, D, E and F) that differ by between 7 and 15% at the amino acid level.
Overall, the DNA sequencing results obtained revealed that eight of the nine
positive Indian cases involved EEHV1A strains, whereas one was an EEHV1B strain.
Furthermore, all but two were readily distinguishable as independent unrelated strains
(Table 2-1). The exceptions were from two orphan calves at Kodanad Camp that died
19
within three days of one another in Dec 2007. These proved to have identical DNA
sequences to one another at all six PCR loci tested (totaling over 4000-bp), including
over 1500-bp tested of tissue from each of five different organs sampled for calf-1 and in
both the heart and liver for calf-2. Several other instances of two afflicted calves at the
same location being infected almost simultaneously with identical EEHV1 strains have
also been observed in Europe and North America. Remarkably, amongst the two most
hypervariable PCR loci tested, the Indian cases included four of the five known major
vGPCR1 subtypes and five of the six known major glycoprotein-H subtypes, thus
encompassing almost all of the overall genetic range of EEHV1 variants observed to
occur amongst the 31 Asian elephant cases analyzed in a similar way from Europe and
North America. Phylogenetic comparisons of the viruses from all nine Indian cases with
selected prototype EEHV1A, EEHV1B, EEHV2 and EEHV6 cases at the U71-gM,
vGPCR1, HEL and gH-TK loci are presented in the dendrograms in Figure 2-3, panels
A-D and graphical alignment presentations of the nucleotide polymorphism patterns for
the same four variable loci are presented in Figure 4, panels A-D. Although a small
number of novel polymorphisms were detected, all nine Indian examples proved to be
closely related to already known subtypes at each locus examined. Comparisons of the
overall distribution of EEHV1 subtypes for the vGPCR1 and gH-TK loci between Indian,
North American and European cases are illustrated in Figure 2-5, panels A and B. The
actual numbers of nucleotide polymorphisms and deletion/insertion differences found
amongst the individual Indian EEHV1 strains at all six PCR loci are presented in Figure
2-6, panels A-F.
20
Discussion
We conclude that sporadic EEHV1-associated hemorrhagic disease is distributed
across the entire Southern India elephant population and that it displays gross and
microscopic pathological features that are indistinguishable from those found in the
highly lethal disease documented previously in captive Asian elephants in Europe and
North America. Most importantly, the very wide range of genetic diversity observed
amongst these nine Indian EEHV1 cases is not compatible with an earlier model
suggesting that this disease resulted from cross-species transmission of EEHV1 from
African to Asian elephants. The latter interpretation was largely based on the apparent
finding of EEHV1 viral DNA in archival paraffin block specimens of skin nodules from
several African elephant calves that were imported from Zimbabwe to Florida in the mid-
1980s. However, we have not been able to reproduce that result upon re-examination of
the same archival skin nodule specimens, and no other examples of EEHV1 DNA have
ever been detected in necropsy samples from African elephants. Instead, multiple
examples of EEHV2, EEHV3, EEHV6 and EEHV7 have now all been found in both lung
and skin nodules from eight different asymptomatic adult African elephants examined
from South Africa, Kenya and the USA 7 (Pearson V. et al, pers comm.). Similarly, only
EEHV2 or EEHV6 have been detected in blood or necropsy samples from just three
known rare cases of hemorrhagic or viremic disease in African elephants in North
America. Therefore, we interpret that the earlier result was most likely a contamination
error and that both EEHV1A and EEHV1B are natural endogenous viruses that co-
evolved with Asian elephant hosts rather than in African elephants. Relatively recent
introduction (even several thousand years ago) from just one or a few exogenous sources
21
would have resulted in a very limited number of strains and a very restricted level of
genetic variation. Current evidence suggests that EEHV4 and EEHV5 4 may also be
endogenous to Asian elephants, but whether they are distributed much less abundantly or
are just less pathogenic than EEHV1A and EEHV1B is not yet known. Just a single
example of the death of a zoo Asian elephant calf with hemorrhagic disease caused by
EEHV3 has been documented 3, but otherwise EEHV3 has been found on multiple
occasions only in benign lung and skin nodules obtained from African elephants, and
cross-species infections would thus appear to be extremely rare.
Herpesvirus genomes evolve relatively slowly and are generally highly stable, yet
individual human cytomegaloviruses (HCMV) for example display considerable
hypervariability in certain genes that evidently represent relics of very ancient genomic
divergence that has subsequently become scrambled through extensive recombination
events. The vGPCR1 and glycoprotein-H subtype hypervariability in EEHV1 appears to
indicate a similar ancient species population drift effect. We had anticipated that the
multiple different subtypes of EEHV1 originated in and might still be distributed non-
uniformly in different Asian elephant sub-populations, especially considering that the
Nilgiri Eastern Ghat Elephas maximus population itself displays relatively narrow nuclear
and mitochondrial DNA haplotype diversity compared to that of other Asian elephant
sub-populations 33. However, this was not the case; instead most known subtypes proved
to be present, even within just this small number of cases from Southern India,
representing almost the entire known genetic diversity range of EEHV1. This result
implies that EEHV1 is likely to be an ancient infection carried by, transmitted between,
and widespread amongst all Asian elephant populations.
22
Evidently, both the A and B chimeric variants of EEHV1, but seemingly not the
other EEHV2 to EEHV7 species of Probosciviruses, commonly produce severe
hemorrhagic pathology upon primary infection within all present day Asian elephant
populations, whether born and reared in the wild or under human care. Since the late
1980s, acute primary EEHV1viremic disease has afflicted more than 20% of all Asian
elephant calves born in North America, with an observed mortality rate of over 80%.
Why a virus that would normally be expected to be well-adapted to and benign in its
natural hosts displays such devastating fulminant pathology remains to be explained, and
the possibility that some other agent or co-factor contributes to the disease cannot be
ruled out. The rate of disease in the wild is unknown, but if it is anywhere near as high as
observed in Europe and North America, then the effects on future reproductive success
and survival of this highly endangered species could be devastating. Additional studies
on the prevalence of infections by the EEHV1, EEHV4 and EEHV5 viruses and of
EEHV-associated hemorrhagic disease, especially in free-ranging wild elephants, and in
all Asian range countries would appear to be urgently needed.
Acknowledgements
Financial support for these studies, the setting-up and operation of the diagnostic
laboratory in Wayanad, Kerala and for travel of team members from NEHL and Johns
Hopkins University to India came from the International Elephant Foundation, the
Smithsonian Institution and the Kerala Forest Department. Studies at Johns Hopkins
University were supported in part by NIH research grant R01 AI24576 to GSH. Field
research by AZ was supported in part by the U.S. Fish and Wildlife Services Asian
Elephant Conservation Fund. We thank Dr Anilkumar for the inclusion body
23
photomicrographs and Dr R Thirumurugan of the Arigna Anna Zoological park in
Chennai for providing tissue samples.
24
Tables
25
Figures
Figure 2-1. Pathological changes of EEHV-associated disease found during field
necropsy. (A) Asian elephant with cyanosis of the tongue attributed to EEHV disease; (B)
Epicardial surface of the heart (apex view) from an Asian elephant showing severe
extensive hemorrhage attributed to EEHV disease; (C) Ventricular endocardial surface of
the heart from an Asian elephant showing multifocal areas of ecchymotic hemorrhage
attributed to EEHV disease; (D) Serosal membrane surface of the liver showing diffusely
scattered petechial hemorrhage attributed to EEHV disease; (E, F) Photomicrograph of
two capillary endothelial cells containing typical basophilic intranuclear viral inclusion
bodies from necropsy liver tissue. Hematoxylin and eosin stain, bar = 25 µm.
Figure1.(A-D)PathologicchangesofEEHV-associateddiseasefoundduringfieldnecropsy.(A)Asianelephantwithcyanosisofthetonguea ributedtoEEHVdisease;(B)Epicardialsurfaceoftheheart(apexview)fromanAsianelephantshowingsevereextensivehemorrhagea ributedtoEEHVdisease;(C)VentricularendocardialsurfaceoftheheartfromanAsianelephantshowingmul focalareasofecchymo chemorrhagea ributedtoEEHVdisease;(D)Serosalmembranesurfaceofthelivershowingdiffuselysca eredpetechialhemorrhagea ributedtoEEHVdisease;(E,F)Photomicrographoftwocapillaryendothelialcellscontainingtypicalbasophilicintranuclearviralinclusionbodiesfromnecropsyliver ssue.Hematoxylinandeosinstain,bar=25µm.
25 µm
F
25 µm
E
A B C
D
15
26
Figure 2-2. Representative agarose gel-ethidium bromide PCR band results obtained
after first round (520-bp) and second round semi-nested (250-bp) amplification with
EEHV PAN-POL primers from 12 samples of necropsy tissue DNA. Lanes 1, 2 and 12,
different tissues from case IP07; 3, case IP10 (negative); 4, 5, 6, 7 & 8 multiple tissue
samples from case IP06; 9, case IP01; 10, case IP04 (negative); 11, case IP11.
27
Figure 2-3. Phylogenetic distance based dendrograms showing patterns of DNA level
divergence and subtyping amongst the Indian EEHV1 cases (boxed in orange) in
comparison with prototype examples of EEHV1A, EEHV1B, EEHV6 and EEHV2 at the
four most variable PCR sequence loci (A) U71/gM; (B) U77/HEL; (C) U51/vGPCR1 and
(D) U48/gH-TK. Bootstrap values are indicated at branch points.
28
(A
)
(B
)
(C
)
(D
)
29
Figure 2-4. Graphical presentation generated from Geneious of the patterns of DNA
sequence polymorphisms (including gaps) across the four most variable PCR loci (A)
U71-gM; (B) U77/HEL; (C) U51/vGPCR and (D) U48/gH-TK for all Indian cases (IP#)
compared to aligned prototype examples of EEHV1A, EEHV1B, EEHV6 and EEHV2.
30
Figure 2-5. Comparison of the major EEHV1 gene subtype distribution patterns amongst
Indian, North American and European hemorrhagic disease cases at the two most
variable PCR loci examined (A) U51/vGPCR1 and (B) U48/gH-TK.
31
(A) U38/POL Locus (480-bp) IP01 IP05 IP06 IP07 IP11 IP91 IP93 Subtype
IP01 - A IP05 1 - A IP06 1 0 - A IP07 1 0 0 - A IP11 0 1 1 1 - A IP91 0 1 1 1 0 - A IP93 15 14 14 14 15 15 - B
Polymorphisms
(B) U60/TERex3 Locus (360-bp) IP01 IP05 IP06 IP07 IP11 IP91 IP93 Subtype
IP01 - A1 IP05 6 - B2/A IP06 4 8 - A2 IP07 4 8 0 - A2 IP11 0 4 4 4 - A1 IP91 0 4 4 4 0 - A1 IP93 11 15 15 15 11 11 - B1
Polymorphisms (C) U71/gM Locus (640-bp)
32
IP01
IP05
IP06
IP07
IP11
IP43
IP60
IP91
IP93
Subtype
IP01
- 6 3 3 0 0 3 0 12 A
IP05
1 - 9 9 6 6 9 6 6 A
IP06
1 2 - 0 3 3 0 3 9 A
IP07
1 2 0 - 3 3 0 3 9 A
IP11
0 1 1 1 - 0 3 0 12 A Ins/Del
IP43
0 1 1 1 0 - 3 0 12 A
IP60
2 3 3 3 2 2 - 3 9 A
IP91
0 1 1 1 0 0 2 - 12 A
IP93
36 37 37 37 36 36 38 36 - B
Polymorphisms
33
(D) U77/HEL Locus (952-bp) IP01 IP05 IP06 IP07 IP11 IP43 IP60 IP91 Subtype
IP01 - A IP05 3 - A IP06 0 3 - A IP07 0 3 0 - A IP11 3 2 3 3 - A IP43 0 3 0 0 3 - A IP60 2 1 2 2 1 2 - A IP91 3 2 3 3 2 3 1 - A
Polymorphisms (E) U51/vGPCR1 Locus (885-bp) IP0
1 IP05
IP06
IP07
IP11
IP43
IP60
IP91
IP93
Subtype
IP01
- 3 3 3 3 0 0 0 12 A2
IP05
31 - 0 0 0 3 3 3 9 E
IP06
31 0 - 0 0 3 3 3 9 E
IP07
31 0 0 - 0 3 3 3 9 E Ins/Del
IP11
23 6 6 6 - 3 3 3 9 E/A
IP43
45 27 27 27 29 - 0 0 12 D1
IP60
26 28 28 28 26 25 - 0 12 D2/A
IP91
2 31 31 31 25 46 28 - 12 A2
IP93
29 38 38 38 39 45 33 29 - B1
Polymorphisms
(F) U48/gH-TK Locus (807-bp)
34
IP01 IP05 IP06 IP07 IP11 IP43 IP60 IP91 Subtype
IP01 - A1 IP05 26 - F IP06 53 56 - C3 IP07 58 56 0 - C3 IP11 57 64 74 74 - E IP43 51 52 14 14 70 - C1 IP60 65 66 74 74 48 71 - D** IP91 63 66 72 72 48 67 4 - D*
Polymorphisms
Figure 2-6. Direct side-by-side nucleotide level differences observed amongst the nine
Indian 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/deletion
differences found at the U71/gM and U51/vGPCR1 loci are also indicated.
35
III. Complete genome sequence of elephant endotheliotropic
herpesvirus 4 (EEHV4) the first example of a GC-rich branch
Proboscivirus
This chapter has been previous published as:
Ling PD, Long SY, Fuery A, Peng RS, Heaggans SY, Qin X, Worley KC, Dugan S,
Hayward GS. Complete genome sequence of elephant endotheliotropic herpesvirus 4
(EEHV4) the first example of a GC-rich branch proboscivirus. mSphere. 2016. June
15;1(3) . pii: e00081-15. doi: 10.1128/mSphere.00081-15
Reprint with permission of the publisher, American Society for Microbiology.
Abstract
36
A novel group of mammalian DNA viruses called Elephant Endotheliotropic
Herpesviruses (EEHVs) belonging to the Proboscivirus genus has been associated with
nearly 100 cases of highly lethal acute hemorrhagic disease in young Asian elephants
worldwide. The complete 180-kb genomes of prototype strains from three AT-rich
branch viruses EEHV1A, EEHV1B and EEHV5 have been published. However, less than
6-kb of DNA sequence each from EEHV3, EEHV4 and EEHV7 showed them to be a
hugely diverged second major branch with GC-rich characteristics. Here we determined
the complete 206-kb genome of EEHV4 (Baylor) directly from trunk wash DNA by next
generation sequencing and de novo assembly procedures. Amongst a total of 119 encoded
genes with a similar overall co-linear organization to those of the AT-rich EEHVs, major
features of EEHV4 include a family of 26 paralogous 7xTM and vGPCR-like genes plus
25 novel or missing genes. It also contains an unusual distribution of tracts of 5 to 11
successive A or T nucleotides in intergenic domains between the mostly much higher
GC-content protein coding regions. Furthermore, an extremely high GC-rich bias in the
third wobble position of codons clearly delineates the coding regions for many but not all
proteins. There are also two novel captured cellular genes, including a C-type lectin
(vECTL) and an O-linked acetyl glucosamine transferase (vOGT) as well as an unusual
large and complex Ori-Lyt dyad symmetry domain. Finally, 30-kb from a second strain
proved to include three small chimeric domains indicating the existence of distinct
EEHV4A and EEHV4B subtypes.
Introduction
37
The deaths of 62 young Asian elephants with acute hemorrhagic disease in North
America and Europe have been attributed to rapidly developing uncontrolled primary
systemic infections by members of a novel genus of mammalian herpesviruses,
designated Elephant Endotheliotropic Herpesviruses or EEHVs 1,3,8,19,34. At least 43
additional lethal cases have also been confirmed by DNA tests within Asian range
countries, including 16 published examples from India, Thailand, Cambodia and Laos 10-
12. Seven major genotypes named EEHV1 to EEHV7 that all qualify as distinct species
within the Proboscivirus genus have been identified within North American elephants
3,4,6,20,34,35, although EEHV1A has been associated with the majority of lethal cases.
Overall, some 46 highly divergent strains of EEHV1A have been identified by selective
PCR sequencing based gene subtyping at multiple loci. Only ten examples of EEHV1B
plus a smaller number of EEHV4 and EEHV5 viruses have also all been associated with
systemic disease in Asian elephants (Elephas maximus), whereas three others EEHV2,
EEHV3 and EEHV6 were detected in the few rare disease cases in zoo African elephant
(Loxodonta africana) calves 4,6,20. The latter three viruses as well as EEHV7 have also all
been detected as quiescent infections in lung nodules from healthy adult African
elephants 35. Just 12 young captive Asian elephants with confirmed EEHV1 DNA-
positive systemic disease that had been treated with human anti-herpesvirus drugs are
considered survivors 9,20,23.
Until recently, it seemed that EEHV1A and its less common partially chimeric
variant EEHV1B 5,21,25,26 were the predominant viruses that Asian elephants might be
encountering. However, routine testing between 2011 and 2015 within the most closely
monitored USA zoo herd (which consists of just eight individual Asian adults and calves)
38
have now also detected episodes in which multiple herdmates underwent sequential mild
primary viremic infections with subsequent trunk wash shedding by first EEHV5 2 and
then later EEHV4 15. Because at least four different EEHV1A strains had been
documented in lethal cases at this same facility within the previous 15 years, and most of
this herd had already been observed to undergo sub-clinical infections by strains of
EEHV1A or EEHV1B or both either several years earlier or later 15,29,30, we conclude that
it is likely that most Asian elephants eventually become infected with multiple EEHV
species and subtypes. The relative timing and order of these primary EEHV infections are
expected to have major impacts on the levels of immune protection to disease caused by
the others.
Although no Probosciviruses have yet been grown in cell culture, the complete
genomes of four reference strains of AT-rich branch Asian elephant EEHVs have been
determined previously directly from necropsy tissue, including two of EEHV1A and one
each of EEHV1B and EEHV5A 14,16 ,17. Therefore, we wished to take the opportunity that
this latest EEHV4 episode provided to learn more about the genetic relationships amongst
the different EEHV lineages and species by determining the complete genomic DNA
sequence of EEHV4 strain Baylor as the prototype example of a GC-rich branch
Proboscivirus. In the accompanying paper 36 we compare and contrast the genomes of
these two major branches of the Proboscivirus as well as describe a number of additional
characteristic novel features of the entire group.
Materials and Methods
Source
39
The sequenced DNA came from a trunk wash sample collected from a four-year
old Asian elephant that had experienced a mildly symptomatic episode of EEHV4 PCR-
positive viremia starting Sept 2nd 2014, then presented with a transient high level trunk
wash shedding beginning in Oct 2014. Clinical details of the diagnosis, case history and
treatment of the case have been published under animal no. 2 in Feury et al 15.
Library Preparation and DNA Sequencing
Illumina paired-end libraries were prepared according to the manufacturer’s
protocol (Illumina Multiplexing_SamplePrep_Guide_1005361_D) with modifications as
described in the BCM-HGSC protocol (https://www.hgsc.bcm.edu/content/protocols-
sequencing-library-construction). Briefly, 190 ng of DNA was sheared into fragments of
approximately 200-300 base pairs with the Covaris E210 system followed by end-repair,
A-tailing, and ligation of the Illumina multiplexing PE adaptors. Ligation-mediated PCR
(LM-PCR) was performed for 6 to 8 cycles using the Library Amplification Readymix,
which contains KAPA HiFi DNA Polymerase, and universal primer IMUX-P1.0 and
IMUX-P3.0. Purification was performed with Agencourt AMPure XP beads after
enzymatic reactions. Size distribution of the LM-PCR products was determined using the
LabChip GX electrophoresis system (Perkin Elmer), and quantification was performed by
gel analysis using AlphaView SA Version 3.4 software.
Library templates were prepared for sequencing using Illumina TruSeq reagents
and protocols. Briefly, the libraries (or library pools) were denatured with sodium
hydroxide, diluted to 6-9 pM in hybridization buffer, and then loaded on a lane of a
HiSeq flow cell. The libraries then underwent bridge amplification to form clonal
40
clusters, followed by hybridization with the sequencing primer. Sequencing runs were
performed on the Illumina HiSeq 2000 platform using the 2x100 run format.
Illumina Read Processing and Assembly
A total of 416 million reads (42 Gb) were produced using the Illumina HiSeq
sequencing technology. These raw reads were processed using SeqPrep program
(https://github.com/jstjohn/SeqPrep) to remove adapter sequences. Low quality
sequences (with quality less than or equal to 2) at the ends of the reads were trimmed
using an in-house script. The trimmed reads were mapped to the African elephant
reference genome (Loxafr3.0) using BWA 37 to detect sequences with greater than 95%
match, and such mapped reads were assumed to be of host origin and omitted from later
assembly steps. The 212 million reads remaining after these processes were assembled
using Velvet 38 with various kmer sizes of either 29, 45, 63 or 75 and these parameters: -
exp_cov auto -cov_cutoff 20 -min_contig_lgth 400. The processed reads were also sub-
sampled into smaller data sets of 20, 60 and 100 million reads. In the end, Velvet
assemblies using all processed reads or using 100 million reads with the highest kmer
sizes each yielded a total of between two and five contigs that aligned to EEHV1A
(Kimba) genomic DNA sequences in BLAST-N searches and added up to between 205
and 206-kb.
DNA Sequence Analysis and Comparisons
After filling and joining across the several remaining small gaps between adjacent
contigs by standard Sanger PCR amplification and cycle sequencing approaches, a single
final consensus contig file of 205,896-bp was constructed. The assigned gene
41
nomenclature and annotations were generated initially by BLASTP and BLASTX
searches of the Genbank database for every potential ORF of greater than 80-aa that did
not substantially overlap with another already identified ORF. There was just a single
exception to the overlap rule (E44A) within the final assignments. Subsequently, each
ORF was confirmed directly for both orthologues and paralogues by amino acid identity
comparisons with the corresponding ORFs in the EEHV1A (Kimba), EEHV1B (Emelia)
and EEHV5A(Vijay) files, as well as with BLASTX searches of the intact
EEHV4(Baylor) genome file itself. Clustal alignments and phylogenetic trees were
generated in MEGA5 based on MUSCLE alignments or in MacVector 12 as described
previously 5. Dot matrix diagrams showing global nucleotide alignments were generated
as implemented at http://blast.ncbi.nlm.nih.gov/Blast.cgi. Simplot software 5,39 was used
to display nucleotide identity and divergence comparisons.
Database Accession Numbers
The final completed annotated 205,896-bp genomic DNA sequence file of
EEHV4 (Baylor) is deposited at NCBI GenBank under accession number KT832477.
Previous PCR data for this strain totaling 1,828-bp of unique sequence has accession
numbers KR781023-KR781037 15. For comparative purposes an amended version of the
178-kb EEHV1A (Kimba) genome with updated annotations has accession number
KC618427 17. Thirteen new DNA sequence files totaling close to 24.3-kb of extended
data from Sanger cycle sequencing of amplified PCR loci from the prototype
EEHV4(NAP22) genome have accession numbers KT832478-KT832490 and
KU147235.
42
Result
Assembly of the Complete 206-kb EEHV4 (Baylor) Genome Sequence
The intact genomes of four prototype Asian strains have all been assembled by
random high throughput and de novo assembly approaches directly from high quality
necropsy tissue DNA obtained from young elephants that died of acute hemorrhagic
disease 14,16,17. For EEHV4 (Baylor), we instead used a trunk wash pellet DNA sample
with a high measured ratio of viral to host cell DNA. From a total of 420 million 100-bp
long runs of raw data, close to half resembled African elephant DNA and were filtered
out before the remainder were assembled de novo by the Velvet program using a variety
of different K-mer size parameters. Four independent assembled contig libraries that were
searched for matches to EEHV1 (Kimba) genomic DNA produced results of between one
and four contigs each with the largest being 202,155-bp. There were three small gaps in
the data overall each of which was repaired by Sanger PCR sequencing and proved to be
located in internal repetitive regions either within E34 (ORF-C) or in the predicted Ori-
Lyt locus between U41(MDBP) and U42(MTA) or close to the right-hand-side terminus.
Because of this being intracellular and not virion derived DNA, the final results in each
case were identical contiguous circular genomes of 205,896-bp (average coverage of 110-
fold).
To generate a linear version, we arbitrarily defined a G10-tract at the beginning of
the original largest contig as the left-hand-side end of the EEHV4 (Baylor) genome.
Importantly, two regions mapping very close to the right-hand-side end contained distinct
sets of potential packaging signal motifs of which the first matched at 45 out of 54
43
nucleotides (83%) to both copies of the terminal repeats of EEHV1A (Emelia) and
EEHV1A (Raman), and the second matched at 103 of 144 positions (72%) to all three
copies of the terminal repeat “a” sequence of HSV1 (KOS). The extreme left-hand-side
of the genome (outside gene E1) contains several segments including a set of 17-bp
tandem-repeats as well as a cluster of 8-bp palindromic cyclic AMP-response elements
(CREs) that have high level homology to repetitive sequences present within the terminal
repeats of the other EEHV genomes. Therefore, there is clear evidence that both ends of
our assembled EEHV4 genome map within the predicted terminal repeat and lie close to
the legitimate physical ends of the EEHV1A, EEHV1B and EEHV5 genomes as
determined by Wilkie et al 14,16. Furthermore, on the far right-hand-side of the EEHV4
(Baylor) genome there is a 5.3-kb non-coding segment lying outside and immediately
upstream of the gene encoding the U44 (ORF-L) transcription factor-like protein. This
region is similar in size to the nearly 7-kb of non-coding DNA (which includes all of one
copy of the 2.9-kb terminal repeat) at that position in the linear AT-rich branch genomes
of Wilkie et al 14,16. Based on the fact that we could assemble a single intact contig with
terminal repeat sequences near both expected ends, we deduced that this must represent
the entire EEHV4 (Baylor) genome.
Global Features and Initial Comparisons with Other EEHV Genomes
The genome of EEHV4 (Baylor) contains 119 open reading frames (ORFs)
arranged as shown in the map in Figure 3-1. A full listing of the names, sizes and map
coordinate positions for all designated protein coding ORFs arranged from left to right
across the EEHV4 (Baylor) genome oriented in the same direction as in our prototype
44
EEHV1 (Kimba) is presented in Table 3-1. This data includes comparative information
relative to the intact genomes of EEHV1A (Kimba and Raman), EEHV1B (Emelia) and
EEHV5A (Vijay) to indicate all of the genes present in EEHV4 that are not found in the
others, as well as all genes present within the others that are missing from EEHV4. Table
3-1 also shows the GC-content of each ORF in EEHV4 and whether the gene is assigned
to a gene family and its status as a common core herpesvirus gene or is shared between
subsets of the Alpha (α), Beta (β), Gamma (γ) sub-families or is unique to the
Proboscivirus genus. Because we are proposing here that the Probosciviruses have
numerous novel biological properties and genetic and evolutionary features that may
justify their future designation as a new Delta (δ) subfamily of mammalian herpesviruses,
to reduce possible confusion later on we have adopted an interim dual nomenclature of
either p or δ when referring to unique features of the Proboscivirus gene and protein sets
in a phylogenetic context.
The gene-ORF-protein nomenclature used here for EEHV4 (Baylor) is also based
on that used originally for EEHV1B (Kiba) by Ehlers et al 26 and expanded upon for
EEHV1A (Kimba) by Ling et al 17 to include an E-series numbering system for all novel
Proboscivirus-specific proteins. All proteins with identifiable orthologues in EEHV1A
(Kimba) retain those same numbers, whereas all newly assigned proteins that are unique
to EEHV4 have been given distinctive E number descriptors.
The most obvious feature about the EEHV4 genome is that the overall orientation
and gene content (especially within the central core gene segment) are essentially
conserved and collinear with those of the three AT-rich branch genomes, although with
considerable divergence towards both ends as revealed in a full length genomic dot
45
matrix comparison (Figure 3-2a). In particular, the large 40-kb inversion of the
conserved core blocks I, II and III between U27 and U44 in Betaherpesviruses, as well as
a second smaller inversion of a weakly conserved 24-kb gene block, are both retained in
common with the organization found in EEHV1A, EEHV1B and EEHV5. Part of the
core gene region involved in the first of these two genomic inversions is clearly revealed
on the left-hand side within a dot matrix comparison with the prototype human
Betaherpesvirus genome HCMV (Merlin) (Figure 3-2b), although the second inverted
region (further towards the left-hand side) is not sufficiently conserved to be detectable in
the diagram. The other half of the conserved core segment (blocks IV, V, VI and VII)
produces the largest visible signal located further towards the right-hand-side, but is not
inverted. No other mammalian herpesviruses have this same type of overall gene
organization as found within both major branches of the EEHVs.
In common with the pattern found for the other Proboscivirus genomes, the
central core segment of EEHV4 (Figure 3-1) retains the three signature optional shared
Alpha-Gamma (αδ) and Alpha-Beta2 (αβ2) class genes U27.5 (RRB), U48.5 (TK) and
U73 (OBP), plus two other Betaherpesvirus-like genes U47 (gO) and U51 (vGPCR1)
mapping together with the 35 obligatory true core genes found in all mammalian
herpesviruses, and the six other shared Beta-Gamma (βγ) class genes that are absent from
all Alphaherpesviruses. Although 15 other genes in the HCMV US22, vMIP, vICA and
mIE gene block from UL23 through to UL43 are clearly absent and the whole 24-kb
genome segment from E32 (U14.5) to E35 in EEHV4 (Baylor) at coordinates 60.5 to
81.6 lies in an inverted orientation compared to that of the adjacent blocks in all
Betaherpesvirus genomes, some of the genes designated U14.5, U14, U13.5, U12
46
(vGPCR2), U4 and U4.5 (ORF-B) in EEHV1A (Kimba) have low level resemblance to a
subset of their Betaherpesvirus orthologues in this region. Because of the evidently
shared common evolutionary origin within a predicted ancestor of both sub-families,
these six genes in both EEHV1A and EEHV4 have been assigned Beta-Proboscivirus
(βp) or Beta-delta (βδ) class status 17 (Table 3-1).
A dramatic feature of the EEHV4 (Baylor) genome is the presence of 26
paralogous members of an ancient and highly diverged family of 7xTM anchored
transmembrane proteins (designated the p3 or δ3 family) within the left-hand side novel
segment of the genome. The linear distance-based protein level phylogenetic tree given in
Figure 3-3 shows relationships and sub-grouping amongst this protein family, of which
the closest viral or cellular homologue is Retinoic acid induced protein 3 (RAIP3) an
orphan group G-protein coupled receptor or GPCR. Although they are very highly
diverged, a combination of the phylogenetic tree branching patterns together with
PBLAST domain-match identity values (not shown) allowed us to loosely classify them
into five subfamilies, based on closer relationships to the prototype examples of E3, E6,
E14, E15 and E18, of which there are seven (E1, E3, E3.1, E3.1, E3.3, E3.4 and E26), six
(E6, E7, E9, E11, E12, E13), four (E14, E14.1, E14.2. and E16), three (E15, E20 and
E21) and two (E18 and E28) paralogous copies respectively in EEHV4. Only the adjacent
pair of E14.1 and E14.2 display a much closer identity than any others, suggesting that
they are the most recently duplicated and most likely both arose from the next adjacent
gene E14. More detailed descriptions and comparisons of the multiple vGPCR-like
proteins as well as several other smaller gene families and some captured cellular genes
47
from both major branches of the Probosciviruses and related mammalian herpesvirus
proteins are presented in the accompanying manuscript by Ling et al 36.
Another striking feature of the EEHV4 (Baylor) genome assembly is the evident
absence of the entire 10.5-kb block encoding the ten to 12 highly variable genes mapping
between E47 to E55 of EEHV1A (Kimba), or of the rearranged versions of this block
found in EEHV1B (Emelia) and EEHV5 (Vijay). Those genes include between three and
five members of the CD48-related vIgFam gene family plus several vGPCRs, as well as
the E47 (vFUT9) and E54(vOX2-1) genes. All six copies of the signature 10- to 35-bp
long alternating CA-repeats found in EEHV1A (Kimba) are also missing in EEHV4
(Baylor), although that was already known to be the case for EEHV5 (Vijay) as well.
Although the intact genomes of EEHV4 (Baylor) and EEHV1A (Kimba) are too
far diverged for accurate global determinations of the overall DNA sequence identity
level between them, a numerical value of 49.6% was measured by the Emboss DNA
Stretcher program for the most conserved 97-kb core genomic segment from U44
(82,500) to U77 (179,500). Distortions to the linearity of the alignments preclude
obtaining meaningful results for the other non-core outer parts of the EEHV4 genome.
This result compares to values of 63% identity for the intact EEHV1B (Emelia) genome
versus EEHV5 (Vijay) and 92.3% for EEHV1B (Emelia) versus EEHV1A (Raman),
which increase to 64.2% and 94.2% respectively when the equivalents of the 10.5-kb
non-linear vIgFam plus vGPCR block between E47 and E55 in Kimba are omitted.
Unusual Patterns and Distribution of GC-Rich Sequence Blocks and A or T-Tracts
48
The several small previously analyzed PCR segments of EEHV3 and EEHV4
DNA (totaling 4 to 5-kb each) were recognized to have a distinctive much higher overall
GC-content of 63% compared to 43% for EEHV1A, 1B and 5 6,15. However, the overall
base composition of the intact EEHV4 genomic DNA proved to be just 57% GC-content.
Much of this difference results from the curious finding that, unlike the adjacent protein
coding regions, the majority of the interspersed non-protein coding regions in EEHV4 are
highly conspicuous by the inclusion of numerous successive A or T-tracts of between
five and 11 nucleotides in length. These features also apply within the relatively small
number of clearly identifiable introns.
Despite the lower than expected overall GC-content, the level does increase to an
average of 62% (range 57 to 66%) for the 17 largest protein coding regions, which are all
over 2,500-bp in length and occupy 67.6-kb. In fact, all but 11 of the 119 recognized
ORFs display a GC-content of greater than 48% (Table 3-1), whether they map within
the novel left-hand-side part of the genome or within the core conserved region. The only
exceptions to this pattern occur across six small dispersed locations (bolded in Table 3-1)
encompassing just 10.8-kb in total length and encoding genes that are either very highly
diverged from their counterparts in EEHV1 and EEHV5 or entirely novel. Five of these
genes, including the additional second captured acetyl glucosamine transferase E9A
(vOGT), lie adjacent to one another between map coordinates 20.8 to 25.2-kb. The
second largest such locus covers the E37 (ORF-O) and adjacent novel E39A (ORF-R)
glycoprotein between map coordinates 186 to 189.7. The third locus encodes two genes,
including a captured E16D (vECTL) protein mapping between map coordinates 35.6 to
36.8. Finally, E4A at coordinates 15.2 to 15.7 and E30A mapping between coordinates
49
55.5 to 56.2 also fall into this category. Perhaps reflecting a relatively more recent
acquisition of at least one of the genes at each of these five AT-rich ORF locations, they
appear to be much more similar to host cell DNA than to the rest of the viral genome,
with ORF base compositions ranging from only 27 to 45% GC-content.
Novel EEHV4 Type-Specific Genes
The currently updated annotation of the EEHV1A (Kimba) genome contains 118
identified genes. Amongst these, a total of 26 are evidently missing within the EEHV4
(Baylor) genome), whereas a total of 25 additional apparently novel genes are present
(Table 3-1). The missing genes in EEHV4 (Baylor) relative to EEHV1 (Kimba) include,
in addition to the entire 10.5-kb ten gene block from E47 to E55, those designated E2,
E5A, E7A, E8, E10, E16A/B, E17, E18A, E18B, E24, E25, E31, E36A (vCXCL1),
E38(ORF-P) and E39(ORF-Q). The extra genes in EEHV4 (Baylor) compared to
EEHV1A (Kimba) include E3.1, E3.2, E2A, E3.3, E3.4, E4A, E4B, E4C, E6B, E7B,
E9A (vOGT), E9B, E9C, E10A, E12A, E14.1, E14.2, E16D (vECTL), E17A, E18C,
E20B, E24B, E27ex2, E30Aex1/2 and E39A (ORF-R). There are also two genes EE23A
(vOX2-V) and EE44A that are unique to EEHV5 (Vijay) and that are absent from both
EEHV1 and EEHV4, and E39 (ORF-Q) is unique to EEHV1A and EEHV1B, but absent
from EEHV2, EEHV4 and EEHV5, whereas E33A and E36A (vCXCL1) are unique to
EEHV1A although absent from EEHV1B, EEHV2, EEHV4 and EEHV5.
Six of the new genes in EEHV4 represent recognizable highly-diverged
duplicated paralogues of genes already present in both EEHV1 and EEHV4 that could
presumably have been deleted from some ancestral genome as the AT-rich branch
50
evolved separately from the GC-rich branch. All of the latter are members of the p3 (or
δ3) 7xTM family described above (E3.1, E3.2, E3.3, E3.4 and E14.1 and E14.2). E2A
which maps amongst the new duplicated versions of the vGPCR6 cluster, is also clearly a
member of this 7xTM family, but this protein has no recognizable orthologues or
paralogues in either EEHV1 or EEHV5. A similar situation applies for E7A and E10A. In
contrast, the following remaining 16 members of the 7xTM family E1, E3, E7, E9, E11,
E12, E13, E14, E15, E16, E18, E20, E21, E26, E28 and E29 all have direct orthologues
in EEHV4 as recognized by both positional and protein homology criteria.
EEHV4 E37 (ORF-O) and E39A (ORF-R) are both predicted to be spliced
potential envelope glycoproteins mapping between gL and ORF-K at coordinates 186 to
189.6 at the same location and orientation as the similarly spliced E37 (ORF-O) plus E38
(ORF-P) and E39 (ORF-Q) ser plus thr-rich glycoproteins of EEHV1A (Kimba).
However, although quite plausibly these proteins all had common evolutionary origins,
ORF-R now lacks sufficient residual homology to be designated as an unambiguous
orthologue of either ORF-P or ORF-Q. Interestingly, all three proteins have two exons
and residual identity between ORF-P and ORF-Q (especially within exon1) suggests that
ORF-Q is a highly diverged tandem-duplicated version of ORF-P that is present only in
EEHV1A and EEHV6, but is missing from EEHV1B, EEHV2 and EEHV5. However,
both ORF-P and ORF-Q are absent from EEHV4 and have seemingly been replaced by
(or evolved into) the much shorter ORF-R glycoprotein that now displays no detectable
identity to either of them. In contrast, the adjacent predicted to be three exon ORF-O
glycoprotein of EEHV4 still retains significant identity (37% across all of exon-3,
51
although barely detectable in exons 1 and 2) to its orthologues in all of the AT-rich
Probosciviruses.
Varying Patterns of Base Composition Bias within Wobble Codon Positions
We previously pointed out that the coding regions within each of the four to five
small segments sequenced by Sanger PCR approaches from both prototype genomes
EEHV3 (NAP27) and EEHV4 (NAP22) within the GC-rich branch of the
Probosciviruses displayed extraordinarily high GC-content bias at the third nucleotide or
wobble codon position 6. For five of the seven short ORF fragments analyzed there in
each virus the wobble position GC-content was between 86 and 99%. This compares with
codon wobble position GC-contents ranging from just 41 to 50% for the orthologous
ORF segments in EEHV1 and EEHV5.
Evaluation of the complete 206-kb EEHV4 (Baylor) genome shows that a very
high level of GC-content bias at the third nucleotide or wobble codon position applies
across close to 90% of the entire genome involving as many as 105 of the 119 total
annotated genes. Remarkably, a global plot comparison of the GC-content of each of the
three EEHV4 reading frames as an indirect measure of the wobble position effect
produces such a dramatic pattern that the position of virtually every ORF (except those in
the six minority AT-rich regions) are easily recognized and defined. However, there is
very little similar frame-specific demarkation when this type of plot is applied to
EEHV1A (Kimba). Four selected examples of these codon-specific plot panels scanning
18-kb segments each of the EEHV4 (Baylor) genome are shown in Figure 3-4a-d, two
from novel areas of the genome on the left-hand side, one showing a conserved core gene
52
segment surrounding Ori-Lyt near the center of the genome and one encompassing the
presumed transcriptional regulators ORF-K and ORF-L at the extreme right-hand-side.
Typical examples of large genes with a high GC-wobble bias in EEHV4 (Baylor) include
U57 encoding the major capsid protein (MCP), plus U41 encoding the major single-
stranded DNA-binding protein (MDBP or SSP) (Figure 3-4c) and also the novel EEHV-
specific gene E4 encoding the captured vGCNT1 protein (Figure 3-4a). These proved to
have wobble-position GC-contents compared to overall ORF GC-contents of 96.6(63)%,
89(65)% and 86(61)% respectively. Omitting 318-aa at the ends of the U57 (MCP)
protein from the analysis leaves a central 3,000-bp segment with an extreme wobble
position GC-content of 99.5%. The other two proteins also have a typical tendency
towards deviations from the wobble position GC-bias near one or both ends. Almost all
of the well-conserved common herpesvirus core proteins, as well as a subset of the more
novel and diverged group-specific proteins in EEHV4 also follow this trend.
The major exceptions to this very high wobble position GC-content bias pattern
include all 14 genes mentioned above that map within the six small well-dispersed loci
with 48% or less overall GC-content (bolded in Table 3-1). Ten of these genes display
wobble position GC-contents very close to their overall GC-content as follows: E4A =
46(44)%, E7B = 29(27)%, E9 = 31(27)%, E9A (vOGT) = 46(42)%, E9B = 26(23)%, E9C
= 25(27)%, E16D (vECTL) = 45(45)%, E17 = 53(45)%, E30A = 39(42)% and E37
(ORF-O) = 37 (41)%. In contrast, the E39A (ORF-R) glycoprotein shows an apparently
enhanced bias in the opposite direction (ie towards even lower GC-richness) of 26%
wobble GC-content compared to 41% overall GC-content. The first of those aberrant
53
genes is included in the three-frame GC-scan diagram shown in Figure 3-4a and the next
seven in Figure 3-4b.
Interestingly, although 11 genes that do not follow the typical EEHV4 pattern of
high-GC bias neither represent core genes nor are shared with any other virus groups,
they do include a curious mixture of both obvious novel captured cell genes (vOGT and
vECTL), plus the unique ORF-R glycoprotein and several other novel genes that are also
not shared with EEHV1 and EEHV5 (E4A, E7B, E9C and E30A), together with some
adjacent but highly diverged genes that are shared (E9, E9B, E17 and ORF-O). They
even include two apparent members of the p3 or δ3 7xTM multi-gene family (E7B, E9),
although the first of these has no direct matching identity to any other EEHV4 7xTM
protein. Therefore, although some of the AT-rich genes may indeed be relatively newly-
captured cellular genes, it would be hard to argue that they were all acquired by any kind
of common mechanism or event.
The two adjacent large 1,465-aa and 2,065-aa leftwards-oriented potential
transcriptional regulatory proteins E40 (ORF-K) and E44 (ORF-L) of EEHV4 are
especially intriguing in this regard with unusual mixed bias patterns (Fig 3-4d, inverted
orientation). Both have a total wobble position GC-content of just 69 or 63% matching
closely to their overall GC-contents of 66 and 64%. However, this is not distributed
uniformly, with just small 230-aa and 244-aa C-terminal domains in both (matching the
segments that are conserved within the otherwise highly-diverged EEHV1 and EEHV5
orthologues) having wobble position GC-contents of 86% and 90%. In contrast, another
segment of ORF-L that overlaps a potential second much smaller 296-aa coding region
within a different reading frame here 14, known as ORF-EE1A, E44A or ORF-S, has a
54
wobble position GC-content of just 43%. However, this novel internal overlapping ORF-
S protein of EEHV4 itself (which is conserved at the 35% identity level in EEHV1 and
EEHV5) has a typical wobble position GC-content of 89% (overall 65%). Unexpectedly,
in contrast to the situation in the AT-rich branch Probosciviruses EEHV1A (Kimba),
EEHV1A (Raman), EEHV1B (Emelia) and EEHV5 (Vijay) 5,16, where the entire ORF-L
coding region and adjacent upstream region display highly localized CpG-suppression
similar to that seen in the MIE1 (UL123) gene region of Cytomegalovirus and many
other Betaherpesviruses 40, this feature is completely absent from the EEHV4 version of
ORF-L.
Complex Enlarged Ori-Lyt Dyad-Symmetry Domain
Similar to the other EEHVs, EEHV4 encodes a U73 orthologue of the
alphaherpesvirus HSV UL09-type of origin-binding protein (OBP), but the expected
matching dyad-symmetry Ori-Lyt region mapping between the U41 (MDBP) and
U42(MTA) genes of EEHV4 is much larger and more complex than those of EEHV1 and
EEHV5 and was initially very difficult to sequence because of unusual features attributed
to several internal stem-loop structures. The entire combined inverted and direct repeat
arrangement encompassing the predicted EEHV4A (Baylor) 1,180-kb Ori-Lyt domain
from map coordinates 92,120 to 93,324 is illustrated in the dot matrix self-comparison
plot presented in Figure 3-5a. A cartoon representation of the structure is also shown in
Figure 3-5b for comparison with the just 75 and 192-bp versions from HSV Ori-S, HSV
Ori-L, HHV6 Ori-Lyt and EEHV1/5 Ori-Lyt. The EEHV4 locus has a total of seven
copies of the consensus OBP-binding sites (OBS) sequences (GAG)GGTGGAACG
55
present compared to just three to four in the other viruses. Four of these OBS motifs in
EEHV4 are arranged as pairs of direct head-to-head palindromic copies, whereas in the
other viruses (as well as in a third pair in EEHV4) they mostly have five to seven bp gaps
between the head-to-head binding site pairs.
Most of the other herpesvirus Ori-Lyt regions of this type 5,16,17,41 have a single
central very AT-rich loop between the inverted repeated stems containing the OBP-
binding sites (although this is duplicated in EEHV1 and EEHV5 compared to in HHV6),
but the EEHV4 version instead consists predominantly of two large loops with 22 and 15
multimerized copies of 20 to 22-bp long AT-rich direct tandem-repeat elements bounded
by three nearly identical 60 to 70-bp inverted repeats of the binding site containing stem-
loops. There is also an additional pair of 40-bp inverted repeats encompassing this whole
800-bp block, with a few additional repeated elements lying within an apparent
degenerate area beyond that on the left-hand-side.
Clustered Palindromic CREB-Binding Site Motifs
A very characteristic feature of primate cytomegaloviruses is the large and
complex major immediate-early (MIE) enhancer domain mapping directly upstream from
the genes encoding the spliced overlapping MIE1 (UL121) and MIE2 (UL123)
transcriptional regulatory proteins. In particular, the 750-bp core HCMV MIE enhancer
contains several sets of dispersed multimerized cis-acting elements encompassing
consensus binding sites for the cellular transcription factors AP1, NFkB and CREB
located upstream of the TATATAA-box motif. In particular, there are eight copies of the
very high-affinity doubled-up 8-bp palindromic versions of cyclic AMP response
56
elements (CREs) that have in isolation been shown to provide both powerful basal
transcriptional effects and responsiveness to cAMP, Ca2+-forskolin and TPA 42. These
same multimerized cis-acting elements are also present in rearranged patterns within
AGM, rhesus and baboon CMV MIE enhancers, together with additional accessory
control elements including tandemly-repeated further upstream NF1-binding site clusters
and in some cases a large stretch of bent DNA 43-45. Well characterized repetitive control
elements of this type are not present upstream of the equivalent MIE genes of
Muromegaloviruses nor Roseoloviruses, therefore it was surprising to find a closely
spaced cluster of both consensus 8-bp (TGACGTCA) and lower affinity 5-bp (TGACG)
half-site CRE motifs within the terminal repeat regions in all three of the EEHV1,
EEHV5 and EEHV4 genomes (Table 3-1). In EEHV1 there are six copies of the
palindromic 8-bp CREs within a 153-bp segment and eight within an extended 590-bp
segment, but no others at all within the rest of the entire 180-kb genome, whereas in
EEHV4 whilst four of the total of six 8-bp CREs occur within this cluster there are also
six more of the 5-bp CREs within the same 540-bp block. By stochastic chance, a single
8-bp CRE motif should occur just once every 64,000-bp of average GC-content DNA and
a second one within the same 540-bp region would occur about 150-fold less frequently.
Therefore, these are hardly random occurrences. Intriguingly, in the circularized form of
the EEHV genomes the CRE clusters would all lie within a large non-coding region
between 2.5-kb and 6.5-kb directly upstream of the candidate E44 (ORF-L)
transcriptional transactivator protein gene at one end, as well as in EEHV1 and EEHV5
only just a few hundred bp upstream from the E47 (vFUT9) gene at the other end of the
genome. A pair of these same 8-bp CRE motifs are also key functional elements in the
57
LTR enhancers of both HTLV1 and HTLV2. Therefore, based on the astronomical odds
against them occurring by chance, together with the apparent strategical location, it seems
reasonable to speculate that these clusters could play some important role in either
packaging events or in transcriptional regulation in the EEHVs.
Chimeric Subtype Level Divergence Patterns from the Prototype EEHV4 (NAP22)
Genome
To address whether or not there may be chimeric domains of the CD-I, CD-II and
CD-III type described previously that distinguish EEHV1A from EEHV1B 5 and
EEHV5A from EEHV5B 5,9 selectively targeted PCR loci totaling 24.3-kb were Sanger
sequenced from necropsy heart tissue DNA of the prototype EEHV4(NAP22) genome in
addition to the 5.7-kb already available from that strain 3-5. Whilst most loci showed
minimal differences at the nucleotide level (0.3 to 2.5%), three gene blocks
encompassing glycoproteins U39 (gB), plus U46 (gN)-U47 (gO)-U48 (gH) as well as the
O-linked acetyl glucosamine transferase E9A (vOGT) instead proved to be highly
diverged. The results across a contiguous 4.9-kb segment including gN to gH revealed a
clearly defined contiguous chimeric domain of nearly 3.7-kb at Baylor equivalent
coordinates 131,750 to 135,400, which almost exactly matches the same position and size
as CD-II of EEHV1B compared to EEHV1A 5. This complete CD-II-like block displayed
26.3% total nucleotide divergence with the individual protein (and DNA) level
differences being 9(11)%, 23(31)%, 32(37)%, 26(27)% and 13(13)% respectively for the
intact E35A (ORF-J), U46 (gN), U47 (gO), U48 (gH) and nearly intact U48.5 (TK)
genes. For the 1.6-kb segment of the U39 (gB) gene evaluated, the results for the EEHV4
58
(NAP22) version revealed a much smaller version of CD-I than in EEHV1 with 189
nucleotide polymorphisms over an internal 1,150-bp segment mapping between EEHV4
(Baylor) coordinates 99,993 to 101,150 representing 16.4% DNA level and 14% aa
divergence with sharp chimeric boundaries on both sides. Similarly, because we could
not amplify any of the expected gene block between UDG and the C-terminus of ORF-K
for EEHV4 (NAP22) using EEHV4 (Baylor) based primers, we deduce that this latter
region encompassing UDG, gL, ORF-O and ORF-R most likely represents a highly-
diverged chimeric domain of a similar size and location as CD-III of EEHV1. Finally, a
6.6-kb sequenced block surrounding the novel captured vOGT gene proved to encompass
a new fourth 4,660-bp chimeric domain designated CD-IV with sharp chimeric
boundaries mapping at coordinates 20,541 to 25,200. This domain encompasses six genes
including part of E7 plus all of E7B, E9, E9A (vOGT), E9B and E9C and displays 741-
bp (16.0%) overall nucleotide polymorphism. The five intact proteins involved diverge
by 28%, 22%, 13%, 18% and 28% respectively, whereas the values for the adjacent E7
ORF, all of E10A and part of E11 analyzed are instead just 3%, 2% and 0%. Intriguingly,
in addition, the E9C and E9B ORFs have become fused in-frame in EEHV4A (NAP22)
rather than occupying two different reading frames as in EEHV4B (Baylor).
A pictorial illustration of the high (= species-level) patterns of divergence
between these first two known examples of EEHV4 strains within the equivalents of the
CD-I and CD-II chimeric blocks are presented in the Simplot comparisons given in
Figures 3-6a and 3-6b. These diagrams include matching to-scale comparisons with the
equivalent CD-I and CD-II chimeric blocks of EEHV1B (Emelia) versus EEHV1A
(Kimba) as described by Richman et al 5. The apparent new CD-IV chimeric domain in
59
EEHV4 across the E7 to E9C gene block and including the novel captured E9A (vOGT)
gene is also shown as a Simplot comparison in Figure 3-6c, but there is no known
equivalent at this position in EEHV1A-1B or in EEHV5A-5B. In addition, an
intermediate level of nucleotide variability of 11 to 12% was found at three other smaller
loci for E4A, E31B and U50-U51, although 40% of the latter occurs within non-coding
regions, whereas for the vGPCR1 ORF itself the DNA and protein variability levels only
reach 7.8% and 6.7%, respectively. Some additional EEHV4 (NAP22) PCR sequencing
was also carried out to confirm predicted ORFs and consensus splicing patterns at loci
such as E4A, E4B, E4C, E6B, E12A, E16D (vECTL), E17, E17A, E18C, E20B, E23B
and E31B that were somewhat ambiguous from the EEHV (Baylor) data alone. Overall, it
seems amply justified to refer to EEHV4 (NAP22) as the prototype example of an
EEHV4A subtype, whereas EEHV4 (Baylor) would be the prototype of an EEHV4B
subtype. The total of 30-kb of DNA sequence now available for EEHV4 (NAP22)
represents nearly 15% of the total expected genome size, and gives an overall measured
strain divergence from EEHV4B (Baylor) of 8.8% (2,684-bp polymorphisms). However,
that value drops to just 1.9% when the 8.4-kb of data from the three observed CD-like
chimeric domains and the variable vGPCR1 locus are omitted. Finally, it is also very
evident when comparing the two chimeric subtypes of EEHV4 that the intergenic
domains represent rapidly changing sequences.
Accuracy of the Predicted Spicing Patterns
Of necessity, because of the inability to grow EEHVs in cell culture, all predicted
splicing patterns in EEHV genes are provisional. However there are multiple levels of
60
expected accuracy involved. All of our predictions are based on finding typical patterns
of herpesvirus splicing involving strong to medium consensus donor and acceptor sites,
generally small 60 to 150-bp introns and the need to jump across frameshifts in most of
the introns to generate a logical intact protein. In addition, for EEHV1 we had relied on
consistent conserved splicing signal locations and patterns across numerous distinct
strains with sometimes otherwise quite variable sequences. For four of the predicted
spliced proteins of EEHV4, namely U12 (vGPCR2ex1,2), U60-66(TERex1,2,3),
U42(MTAex1,2) and U37(ORF-Oex1,2,3), not only do the consensus motifs and patterns
match those in multiple strains of EEHV1A5,17, but they each also agree with the
predictions made by at least one other group for EEHV1A, EEHV1B or EEHV514,16,26.
The U12 pattern also matches that found in HCMV and the U42 (MTA) does so for the
EBV orthologue. For EEHV4 (ORF-Rex1,2) although it lacks homology with the
positional equivalents ORF-P and ORF-Q of EEHV1A, EEHV1B or EEHV5, the
predicted splicing is consistent with the similar interpretations for them by both our
group5,17 and the Wilkie et al group14,16. Several other genes including E17 and E27 also
match our predicted splicing patterns from multiple strains of the EEHV1 versions.
However, the situation for EEHV4 E24B (vOX2-B) just as for the highly diverged
equivalents in EEHV1A, EEHV1B and EEHV5 is much more complex and speculative.
In all three of the latter, the authors of the complete genome papers made logical
predictions of multiple spliced exons and proteins, which are conserved across many
highly diverged strains (and even include a partial match to a host OX2 splicing motif),
but they have no predictive value for the EEHV4 version. The simplest plausible
interpretation that we could make was the presence here of two partially overlapping
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proteins namely E24Bex1, 2 and E23B of which only the former has amino acid identity
to viral and cellular OX2 proteins. However, the presence of multiple additional
consensus splicing signals here could also be interpreted to generate four separate
alternatively spliced proteins, including one that joins parts of both E24 and E23 from a
total of six different exons plus four donor sites and four acceptor sites mapping in frame
across the region between positions 40,982 and 50,001 in EEHV4B (Baylor).
Furthermore, all of those same signals and putative alternative forms are fully conserved
in EEHV4A (NAP22).
Discussion
There were three major goals in determining the complete genome DNA sequence
of EEHV4. The first was to learn more about the nature of this novel class of elephant
herpesviruses and the range of genes and genetic variation that the different EEHV
species and subtypes display. The second was to further address the question of whether
the entire Proboscivirus genus would be best classified as just an outlier member of the
Betaherpesviruses or instead as the prototype of a distinct sub-family of the mammalian
herpesviruses (the Deltaherpesvirinae) separate from the Alpha, Beta and
Gammaherpesvirus sub-families 5,6,17,46. Thirdly, questions had arisen about not only
whether the AT-rich and GC-rich branches of the EEHVs might also be sufficiently
diverged to justify separate genus status, but also more generically about the nature,
origins and significance of this commonly encountered tendency amongst some other
herpesvirus groups as well of trending towards extremely high GC-content.
62
The intact EEHV4 genome did prove to have many differences from the AT-rich
branch viruses in a manner that is fully consistent with the predicted 30 to 35 million
years since their last common ancestor as judged from the initial 5-kb of data 6. Some
major features of these differences, especially relating to the extraordinarily high GC-bias
found in many coding regions and the novel enlarged repetitive Ori-Lyt domain are
described in detail here, whereas several other unique features relating especially to the
enriched A plus T tracts in non-coding domains and the genus or sub-family specific sets
of novel gene families and captured host genes, some of which are common to both the
AT-rich and GC-rich branches whereas others represent characteristic differences
between them, will be described in detail in the accompanying paper by Ling et al 36.
Finally, we also showed here that even just the first two independent strains of EEHV4
examined display a significant level of localized EEHV4A-4B chimerism, with at least
two of the apparent four such domains (CD-I, CD-II, CDIII and CD-IV) being quite
similar to the patterns described previously for both EEHV1A-1B and EEHV5A-5B
subtype pairs. Understanding such subtype and strain divergence patterns will be a
critical factor if vaccination by non-pathogenic strains and routine monitoring of active
infections are to become useful future management tools for EEHV hemorrhagic disease.
Our findings here of an extraordinary high GC-rich bias within the wobble codon
positions of most but not all EEHV4 genes and the contrasting feature of numerous AT-
rich sequence tracts lying within the intergenic domains in all of the EEHVs, raises the
question of whether other herpesvirus genomes with similar overall GC-contents might
show similar features. In fact HCMV, MCMV and the two highly diverged RCMV(M)
and RCMV(E) genome types do all have a similar overall base composition of around 57
63
to 60% GC-content. The only previous attempt that we are aware of to evaluate GC-
content patterns in those genomes was the work of Brocchieri at al 47, which used a
measure referred to as S-content differences in the three different reading frames to
attempt to identify or clarify additional potential coding genes in MCMV and RCMV
(M). As shown by Geyer et al 48 a small sub-set of those proposed corrected annotations
were indeed later found to also be conserved in RCMV(E).
However, the major features of the S-profiles shown in the Brocchieri paper show
similarity to the GC-wobble position biases that we described here for the EEHV4
genome. In fact, although those authors did not expressly say so, the S-values do serve as
a measure of wobble position GC-biases, which are also fairly common in many of the
core genes of those Muromegalovirus genomes. For example, in MCMV, a total of 40
genes mostly located within the central core domains between M33 to M105, plus M23,
M27, M115 and M139 to M143 show this feature with wobble position GC-biases
approaching or exceeding 90%, and up to a dozen more genes either do so over at least
half of their protein coding regions or exceed 75 to 80% bias. On the other hand, more
than 70 other genes mostly mapping towards the ends of the genome instead show much
less or no evidence for wobble-position bias. Notable examples of the latter include
M122 (IE1), M123 (IE2), M74 (gO) and the N-terminal half of M55 (gB).
The MCMV genome is also very similar to HCMV by having at least three large
AT-rich non-coding domains located around the Ori-Lyt, 5-kb stable intron and upstream
MIE loci, but with no more than five or so smaller intergenic domains that resemble the
very large set in EEHV4 that have GC-contents below 50% with a surfeit of AT-rich
tracts. Overall, these results engender some suspicion that in high-GC herpesvirus
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genomes, the genes with high GC-rich wobble position bias generally tend to be well-
established lytic cycle viral genes that function during late-lytic stages of virus infection,
whereas those genes without any significant GC-bias may tend to be those that function
at immediate-early times and during latency for example and therefore need to more
closely resemble host genes, or alternatively may be genes that have been acquired
relatively recently or evolved unusually rapidly.
A significant issue about EEHV pathogenicity that is not yet resolved is knowing
whether the apparent observed much greater involvement of EEHV1A rather than
EEHV1B, EEHV4 or EEHV5 in lethal disease in Asian elephant calves reflects different
pathogenesis mechanisms or efficiency per se, or perhaps simply reflects a much greater
prevalence and universality of the former over the latter. It is also possible that the other
EEHVs including EEHV4 are just as ubiquitous in Asian elephant hosts as is EEHV1A,
but instead exhibit a tighter and less frequently reactivated shedding relationship (thus
appearing to be less abundant overall). Furthermore, whereas the overall detection rates
for EEHV1B and especially EEHV4 and EEHV5 in asymptomatic Asian elephants have
been quite low in random trunk wash shedding studies, the fact that all four viruses have
nevertheless swept through most of both the adults and juveniles present in the closely
monitored Texas zoo herd over a five-year testing period does tend to imply that infection
by EEHV1B, EEHV4 and EEHV5 might also be just as ubiquitous in all Asian elephant
populations as is EEHV1A. Once the differences or otherwise in pathogenesis of EEHV1,
EEHV5 and EEHV4 and their respective A and B subtypes are better understood,
detailed comparisons of the sequences and gene content of all six genome types will
hopefully provide important and useful information for future diagnosis, serological
65
evaluation and potential vaccine-based or other antiviral approaches to monitoring and
controlling EEHV-associated hemorrhagic disease.
Acknowledgment
We thank Lauren Howard of the Houston Zoo for coordinating the routine blood
and trunk wash screening of the herd, as well as the clinical care and treatment of the
case. PDL received research funding from the Houston Zoo. SYL is the recipient of a
Morris Animal Foundation Postdoctoral Fellowship (D14ZO-411). Partial funding
support for the studies at Johns Hopkins University was obtained by research grants to
GSH from the International Elephant Foundation. Both the PDL and GSH groups were
also supported by subcontracts within a Leadership Grant (MG-30-130086-13) under the
Collections Stewardship Program awarded to Lauren Howard at the Houston Zoo by the
Institute of Museum and Library Services. The authors are grateful for the sequencing
and analysis contributions of the production, Next-Gen, LIMS, Library, and systems
teams in the Human Genome Sequencing Center (HGSC) led by Operations Director
Donna M. Muzny and Directed by Richard A. Gibbs. The HGSC is funded by the
National Human Genome Research Institute, National Institutes of Health grant U54
HG003273 to Richard Gibbs.
Author Contributors
AF carried out initial diagnostic real-time PCR analyses, and identified and
prepared the best DNA sample for analysis. SYL carried out initial PCR sequencing at
selected EEHV4B (Baylor) gene loci and to bridge the gaps between contigs, as well as
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all of the EEHV4A (NAP22) PCR sequencing work. RSP carried out PCR sequencing to
complete the terminal repeat regions of EEHV4B (Baylor). XQ, SD and KW coordinated
Illumina library preparation and sequencing. XQ performed the de novo genome
assembly and analysis to identify viral contigs. GSH and SYH carried out all of the DNA
sequence difference analyses and comparisons for identifying ORFs and generated the
gene annotations for Genbank as well as the phylogenetic trees. GSH wrote several drafts
and revisions of the manuscript. PDL coordinated the overall project, including the
approaches to identifying the most suitable sample for the work and plans for generation
of the sequence data. All authors read and provided input to the manuscript.
67
Tables
68
69
70
71
Figures
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Figure 3-1. Annotated Physical Gene Map of the Complete EEHV4(Baylor)
Genome. The intact 206-kb EEHV4B (Baylor) genome determined here (Genbank
Accession No. KT832774) is depicted to scale. The relative sizes and orientations of all
predicted open reading frames (ORFs) are indicated by horizontal arrows. Gene
nomenclature is shown below each of the ORFs. The color key indicates groups of ORFs
shared between all herpesviruses or subsets of herpesvirus sub-families or multiple
paralogues of repetitive gene families. Grey arrows indicate captured cellular genes and
white arrows denote novel genes that do not have obvious orthologues in EEHV1 or
EEHV5. Thin lines connecting arrows indicate introns. The position of the putative lytic
replication origin is marked by a black rectangle.
73
Figure 3-2. Global Alignment Patterns For the Intact EEHV4 Genome Compared to
EEHV1 and HCMV. The dot matrix diagrams showing direct linear nucleotide
alignments were generated as implemented at http://blast.ncbi. nlm.gov./Blast.cgi. Panel
74
(a): Comparison across the intact 206-kb genome of EEHV4 (Baylor, KT832477) from
the GC-rich branch of the Proboscivirus genus versus the intact 180-kb genome of
EEHV1A (Kimba, KC618527) from the AT-rich branch of the Proboscivirus genus
derived from Ling et al 17 when aligned in the same orientation. Panel (b): Comparison
across the intact 206-kb genome of EEHV4 (Baylor, KT832477) versus the intact 235-kb
genome of HCMV (Merlin, AY446834.2) in the Cytomegalovirus genus of the
mammalian Betaherpesvirus sub-family derived from Dolan et al 49, with the latter
aligned in the standard orientation.
75
76
Figure 3-3. EEHV4 Encodes a Very Large Family of Distantly Related Paralogous
7XTM and vGPCR-like Genes. Linear distance-based Bayesian bootstrap phylogenetic
tree comparisons for all 26 members of the 7xTM containing multigene family from the
EEHV4 (Baylor) GC-rich branch Proboscivirus compared to their nearest host cell
analogue Lox RAIP3 as the outgroup. The entire family is loosely divided into five
subgroups whose designated prototypes of E15, E3, E18, E14 and E6 are indicated. Note
that as indicated by the distance values all of these paralogues are very highly diverged
from one another, with the exception of E14.1 and E14.2 which most likely represent the
most recent duplication event. A subset of the genes in this family (p3 or δ3) exhibit
features of GPCR genes as described in greater detail in the accompanying paper 16.
77
Figure 3-4. Codon-Specific Scanning GC-Content Panels Showing the Wobble
Codon GC-Bias Effect Across Selected Representative Segments of the EEHV4
(Baylor) Genome. Diagrams showing the percentage G plus C-content of each of the
three potential translated codon frames across four selected 18-kb segments of the
EEHV4B (Baylor) genomic DNA sequence as implemented under the codon-specific G
plus C% toolbox item in MacVector 12. Short vertical bars indicate forwards direction
terminators. Annotated ORF positions and sizes are denoted by open arrows. Highly GC-
78
biased wobble position blocks with average values between 80 to 100% are marked with
solid bars. For a hypothetical ORF with an initiator codon beginning in frame 1 at
position x in the diagram the wobble position codons are represented by the succeeding
frame 3 line (or frame 1 for a frame 2 initiator and frame 2 for a frame 3 initiator). Panel
(a): Forwards-directed strand across coordinates 1 to 18,000 at the extreme left-hand side
encompassing 10 out of 11 rightwards-oriented genes from E1 to E4C including E4
(vGCNT1) with high wobble position GC-bias. Panel (b): Inverted segment of the
complementary strand across coordinates 37,000 to 19,000 encompassing predominantly
leftwards-oriented genes (16x between E6 and E16D) and two rightwards oriented genes
(E9A and E17), including 11 genes displaying uniformly high wobble codon GC-bias
plus seven genes in two blocks E7B-E9 (vOGT)-E9A-E9B-E9C and E16D (vECTL)-
E17-E17A that do not display wobble GC-bias (all of the latter are labeled with
asterisks*). Panel (c): Forwards strand across coordinates 86,000 to 104,000
encompassing seven rightwards-oriented core region genes U43 (PRI) to U38 (POL) with
high wobble position GC-bias on either side of the predicted novel Ori-Lyt domain.
Panel (d): Inverted segment of the complementary strand from the extreme right-hand-
side across coordinates 187,867-205,894 encompassing U44A (ORF-S), U44 (ORF-L)
and U40 (ORF-K). The only three high GC-bias wobble codon blocks found within this
region occur in ORF-S and in the conserved C-terminal domains of ORF-L and ORF-K
(marked with solid bars).
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80
Figure 3-5. Complex Repeat and Inverted Repeat Patterns within the Predicted Ori-
Lyt Domain of EEHV4. Panel (a): Dot matrix self-comparison of the DNA from
coordinates 92,021 to 93,860 encompassing the entire intergenic region between the N-
terminus of U41 (MDBP) and the C-terminus of U42 (MTA) from EEHV4 (Baylor). The
proposed 1.2-kb Ori-Lyt domain spans from coordinates 92,120 to 93,325. Direct
tandemly-repeated structure is indicated by additional lines parallel to the main diagonal,
whereas inverted repeats are indicated by additional lines perpendicular to the main
diagonal. Panel (b): Features of the Unusual Expanded Dyad Symmetry Ori-Lyt Region
of EEHV4 Compared to those of EEHV1 and Other Alphaherpesvirus-like Dyad-
Symmetry Type Origins. Cartoon diagram comparing the sizes and major structural
features of the predicted dyad symmetry domains of EEHV4 (Baylor) and EEHV1A
(Kimba) with those of the HHV6 version and with both Ori-L and Ori-S of HSV1. Filled
circles denote alternating A plus T-dinucleotide runs. Short horizontal pointed bars
represent copies of the consensus OBP binding site motif. Other larger sets of arrows
designate various types of inverted repeats as well as the 30x and 20x copies of the 20-bp
AT-rich direct tandem-repeats in the EEHV4 (Baylor) version.
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Figure 3-6: Positions and Sizes of Three Identified EEHV4A-EEHV4B Chimeric
Domains and Boundaries Relative to Those of EEHV1A-EEHV1B Chimeric
Domains. The diagrams show SimPlot (30) comparisons of the nucleotide identity
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patterns between EEHV4A (NAP22) and EEHV4B (Baylor) across three mapped
chimeric domains CD-I (1.1-kb), CD-II (3.7-kb) and CD-IV (4.7-kb) shown as blue lines
in comparison to superimposed data for CD-I (3.2-kb) and CD-II (3.7-kb) of EEHV1A
(Kimba) versus EEHV1B (Emelia) shown as red lines. CD-IV of EEHV4 has no
equivalent in EEHV1 and there is no data available for the presumed region CD-III of
EEHV4. Panel (a): CD-I Chimeric Region within U39 (gB) of EEHV4A versus
EEHV4B at EEHV4 (Baylor) map coordinates 99,993 to 101,150 compared to the much
larger overlapping CD-II of EEHV1A versus EEHV1B which encompasses part of U40,
all of U39 (gB) and part of U38 (POL). Vertical arrows denote the positions of the
EEHV1A-1B chimeric domain boundaries. Panel (b): CD-II Chimeric region
encompassing part of ORF-J, all of gN-gO-gH and part of TK of EEHV4A versus
EEHV4B at EEHV4 (Baylor) map coordinates 131,750 to 135,400 compared to the
nearly equivalent superimposed CD-II of EEHV1A versus EEHV1B. Vertical arrows
denote the positions of the EEHV1A-1B chimeric domain boundaries. Panel (c): CD-IV
chimeric domain of EEHV4A versus EEHV4B mapping between EEHV4 (Baylor)
coordinates at map coordinates 20,541 to 25,210. Vertical arrows denote the positions of
the EEHV4A-4B chimeric domain boundaries that encompass part of E7 and all of E7B,
E9, E9A (vOGT), E9B and E9-C, but end before E10A.
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IV. Attempts to propagate elephant endotheliotropic herpesvirus 1A
(EEHV1A) in elephant umbilical venule endothelial cells (EUVEC)
84
Abstract
A highly lethal bleeding disease caused by a novel herpesvirus species designated as
elephant endotheliotropic herpesvirus 1A (EEHV1A) is posing a major threat to the
successful breeding of future generations of highly endangered Asian elephants (Elephas
maximus) worldwide both in the wild and in ex situ breeding programs. Efforts to
understand and control these viruses depend greatly upon scientists being able to generate
large quantities of virus in the laboratory. The ability to grow and propagate this virus in
the laboratory conditions is critical for the progress of future research towards
understanding the biology and pathology of this virus, as well as developing assays to test
drug inhibitors and the possibility of generating live attenuated vaccines, making this the
top priority goal of EEHV research. Here we report overlaying of fresh minced up
necropsy heart, tongue and lung tissues from a 5-year-old female Asian elephant that
succumbed to an EEHV1A infection onto both primary EUVEC (pEUVEC) and
immortalized EUVEC (iEUVEC) cultures for three days. After the removal of necropsy
tissue and despite the routine cell culture medium replacement and passaging of cell
cultures, EEHV1A DNA polymerase (POL) and/or helicase (HEL) gene DNA was
persistently detectable with first round conventional polymerase chain reaction (PCR) up
to 54 and 122 days in the iEUVEC and pEUVEC cultures, respectively. Quantitative
PCR (qPCR) for EEHV1A DNA in the pEUVEC cultures overlay with heart tissue
revealed decreasing viral DNA over time. These findings suggest viral entry into both the
EUVEC cultures followed by a decrease of viral DNA attributed to passaging and/or die
off of EUVEC with no evidence of viral DNA increase. Further work is needed to
confirm viral entry and latency after viral introduction to the EUVEC cultures.
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Introduction
The lethal acute hemorrhagic disease caused by elephant endotheliotropic
herpesviruses (EEHV) has been responsible for 60% of all deaths among Asian elephant
calves between 4 months and 15 years of age that were born in zoological parks in North
America8. More than 100 cases with an 80% mortality rate have been confirmed in
Europe and North America (representing 20% of all live births)8 and as many as 20 plus
cases have also been confirmed or suspected among both orphan and free ranging calves
in Asia. Despite some Asian elephant calves surviving after clinical intervention, there
were still 14 Asian elephant calf deaths in Europe caused by EEHV within the last 6
years and 4 recent confirmed North America EEHV1 associated deaths within the last 2
years.
Several previous attempts to propagate EEHV in laboratory cell culture by
standard virological approaches using clinical tissue or blood samples collected from
Asian elephant calves with systemic hemorrhagic disease have not been successful,
despite being effective with other well-characterized herpesviruses. Previous attempts in
our group to propagate virus from infected samples included using and passaging
standard primary (HFF, MEF, REF), established human (Hela, K562, U373, BJAB, 143),
mouse (NIH-and BALB/c3T3, Ltk-), and hamster (BHK) cell lines, as well as obtaining
primary cultures of both Asian and African elephant fibroblast cells from the San Diego
Zoo’s collection. Attempts to grow EEHV in embryonated chicken eggs and inoculated
laboratory mice were also unsuccessful (Laura Richman, unpublished data), although this
is a well-established technique for propagating human herpes simplex virus (HSV)50-52.
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Our lab previously found some success using an alternative approach that was
first used to demonstrate infectious Kaposi’s sarcoma-associated herpesvirus (KSHV)
derived from primary effusion lymphoma (PEL) cells lines in contact-inhibited primary
human DMVEC cultures53. Using this technique, we successfully produced latency
associated nuclear antigen (LANA) positive colonies that later turned lytic either
spontaneously or after tetradecanoyl phorbal acetate (TPA) or n-butyric acid (nBA)
induction treatment, as demonstrated by immunoflurorescence assay (IFA) with rabbit
antibodies against viral lytic cycle proteins. Nevertheless, using similar techniques for
EEHV propagation was still not achieved within a laboratory setting.
Further experiments were also conducted in primary elephant peripheral blood
mononuclear cell (PBMC) cultures by adding supernatant or live cells from EEHV
positive blood, including attempts to induce with TPA and other agents and after isolating
adherent monocytes, differentiating monocytes into macrophages and after several weeks
of propagation of T-cells and or B-cells in the presence of IL-2, IL-4, TNF alpha and
other agents. None of these approaches succeeded in producing lytic cycle EEHV
progression at the level of visible cytopathic effects or positive IFA with antibodies to
early lytic cycle nuclear antigens MDBP or OBP. Nevertheless, the ability to grow and
propagate this virus is so critical for future progress towards understanding the biology
and pathology of this virus, as well as for the possibility of generating live attenuated
vaccines, that additional attempts are planned and necessary.
If successful viral propagation in cell culture can be achieved, researchers will
then gain the much needed ability to (a) accumulate large quantities of virus for easier
molecular analysis of all the novel genes and proteins these viruses encode, (b) study
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which cell types these viruses can infect, (c) study the virus life cycle including how
EEHV establishes and reactivates from latency, (d) make progress towards understanding
the basis and mechanisms involved in the unusual pathology of EEHVs in vascular
endothelial and white blood cells, (e) carry out pharmacological studies evaluating the
effectiveness of the human anti-herpesvirus inhibitory drugs and/or screen for additional
drug candidates, and finally, (f) develop virologic methods to passage attenuated virus for
potential vaccine development. Currently, there has been great progress in our ability to
monitor and quantitate EEHV infections in blood and trunk wash secretions, as well as to
assess the genetic relationships between the multiple different species and strains of
EEHV. However, without the ability to successfully cultivate and propagate the virus in
the research laboratory, all progress in understanding the nature of these viruses and
ultimately gaining ability to control disease progression will be distressingly slow.
Here we report persistence EEHV1A viral DNA PCR detection in EUVEC
cultures overlay with fresh minced Asian elephant necropsy tissue samples from a
confirmed EEHV1A associated death for several months in spite of multiple cell culture
passages and medium changes.
Materials and Methods
pEUVEC cultures
pEUVEC were established from fresh unfrozen Asian elephant umbilical cord
harvested immediately after birth. Umbilical cord was collected in 20 to 30 cm segments
stored in sterile phosphate-buffered saline (PBS) with 1X concentration of Antibiotic
Antimycotic solution (Sigma-Aldrich Co., St. Louis, MO), and was transported on
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ice/refrigerator packs within 24-48 hours as listed in the EEHV Research and Tissue
Request Protocol.
EUVEC were isolated following the protocol for “Isolation and culture of human
umbilical vein endothelial cells” 54 with the following modifications. All procedures were
performed in a biological safety level 2 cabinet under sterile and aseptic techniques.
Umbilical cord veins were cannulated with a blunt 10-gauge needle and perfused with 50
ml of sterile PBS twice at both ends to wash out blood. Each vein was then infused with
10 mL warm (37°C) PBS with 0.1% collagenase (Invitrogen, Carlsbad, CA) with the
other end clamped off with a hemostat. Umbilical veins were incubated for 10 min at
37°C. Immediately following incubation, the clamped umbilical cords were briefly
massaged to loosen the cells, the cord was unclamped, and the collagenase solution was
flushed from the cord with 30 ml of PBS. The recovered umbilical extract containing
umbilical vein endothelial cells were collected into a 50 ml conical polypropylene tube
containing Clonetics EBM-2 medium (Lonza, Walkesville, MD) supplemented with
EGM-2 singleQuots (Lonza, Walkersville, MD), 1X concentration of Antibiotic
Antimycotic, and 2% fetal bovine serum. The cells were pelleted at 250 x g for 10
minutes at room temperature. The supernatant was decanted and the pellet was
resuspended in 5 ml of Clonetics EBM-2 medium supplemented with EGM-2
singleQuots, 1X concentration of Antibiotic Antimycotic solution, and 2% fetal bovine
serum and plated into a T25 cell culture flask. Cells were observed daily for 1 week or
until confluent. 1X Antibiotic Antimycotic PBS solution was used to rinse cells with
fungal or bacterial contamination. All persistently contaminated cultures were discarded
after a week. pEUVEC cultures were maintained in T25 cell culture flasks with Clonetics
89
EBM-2 medium supplemented with EGM-2 singleQuots and 2% fetal bovine serum at
37°C with 5% CO2. pEUVEC retained the ability to divide and form cobblestone-like
contact-inhibited monolayers (Figure 4-1).
Creation of iEUVEC cultures
Proviral vectors were received from Addgene (Cambridge, MA). The lentiviral
transfer plasmid pBABE-zero largeTcDNA (Addgene plasmid # 1779) and pBABE-neo-
hTERT (Addgene plasmid # 1774) were gifts from Bob Weinberg. pEUVEC were
transduced with plasmids for both large T antigen (SV40TL) and human telomerase
(hTERT) using a vesicular stomatitis virus-G protein pseudotyped retrovirus. Transduced
cells were selected for drug resistance with 200-250 μg/ml of gentamycin and 50 μg/ml
of zeocin. iEUVEC were maintained in T25 cell culture flasks with Clonetics EBM-2
medium (Lonza, Walkersville, MD) supplemented with EGM-2 singleQuots (Lonza,
Walkersville, MD) and 2% fetal bovine serum. iEUVEC retained the ability to divide and
form cobblestone-like contact-inhibited monolayers.
Necropsy samples
A 5-year-old female Asian elephant (Diazy) from the Albuquerque BioPark, New
Mexico died from an acute hemorrhagic disease on May 9, 2015. PCR confirmed
EEHV1A detected in blood, serum, and other visceral organs. The necropsy was
performed on May 11, 2015 by the zoological staff. Samples were collected according to
the EEHV Research and Tissue Request Protocol. Received samples included frozen
whole blood, serum, and fresh organ tissue samples. 2 ml of frozen whole blood and 2 ml
of serum were shipped in an EDTA tube and screw-top tube, respectively. Fresh unfrozen
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heart, liver, tongue, spleen, and lung tissue samples were aseptically placed into 50 ml
conical polypropylene tubes with sterile PBS with 1X concentration of Antibiotic
Antimycotic solution shipped on ice. All samples were received at Johns Hopkins
University School of Medicine on May 12, 2015.
All fresh tissue samples were grossly normal. Tissue samples were minced
following the standard procedure reported by Latimer 2011 4. Approximately 20 mg of
heart, liver, and spleen were individually minced then resuspended in 1 ml of cold sterile
PBS. Approximately 10 mg of tongue and 10 mg of lung tissue samples were minced
together then resuspended in 1ml of cold sterile PBS. 200-μl of each fresh tissue sample
suspension were overlay into each T25 cell culture flasks with either pEUVEC or
iEUVEC for 3 days with Clonetics EBM-2 medium with supplemented with EGM-2
singleQuots, 1X concentration of Antibiotic Antimycotic, and 2% fetal bovine serum.
After 3 days, medium and fresh tissue sample suspension mixture were removed
followed by a 5 ml sterile 37°C PBS wash and replenished with fresh medium.
After overlay with fresh tissue samples, both the infected pEUVEC and iEUVEC
culture flasks were allowed to become confluent with no passaging for one month
maintaining a monolayer from contact inhibition. Further culture steps include multiple
successive passaging with added fresh uninfected cells.
DNA extraction from fresh initial samples
200-μl of each initial fresh tissue PBS suspensions were used for DNA extractions
using the Qiagen Generation Capture kit (Qiagen, Valenica, CA) with the DNA eluted
into 100-μl elution buffer.
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DNA extraction from EUVEC cultures
When passaging pEUVEC and iEUVEC with trypsin/EDTA (Lonza,
Walkersville, MD), a third (6.6x105 EUVEC) of a confluent T25 cell culture flask (2x106
EUVEC) of EUVEC were collected for DNA extraction. DNA extractions were
performed using the Qiagen Generation Capture kit listed above. The only exception
from this procedure was the first passaging (32 days later) of the iEUVEC cultures with
either heart or tongue/lung where two-thirds of a confluent T25 cell culture flask (1.3x106
EUVEC) were used for DNA extraction. On daily observation of cultures, medium with
noted cellular debris or cloudiness was collected, spun down to separate cellular pellet
and supernatant for DNA extraction using the same procedure stated above.
PCR amplification
PCR amplification and the PCR primers for EEHV1 POL and HEL were as
previously described 4, 29.
DNA sequencing
All DNA sequencing was carried out by direct cycle sequencing on both strands
of purified PCR DNA products from first round PCR amplification. The correct sized
PCR product bands were purified after agarose gel electrophoresis with a Qiagen II Gel
Extraction kit (Valenica, CA). Purified PCR products were sequenced at the Johns
Hopkins University School of Medicine Genetic Resources Core facility. All DNA
sequence editing, analysis and manipulation was performed using Assmeblialign and
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CLustal-W nearest neighbor joining as implemented for MacVector vers 7, together with
BLASTX or TBLASTX comparison programs provided online at NCBI.
qPCR for EEHV1
qPCR for viral DNA levels of EEHV1 based on the EEHV1 major DNA-binding
protein (MDBP) were performed as previously reported 29 and performed at the National
Elephant Herpesvirus Laboratory at the Smithsonian National Zoological Park. 5-μl of
template DNA was used for each qPCR reaction performed in duplicates with the
exception of the heart necropsy DNA extraction using only 1-ul per reaction. The limit of
detection is established as 10 copies/reaction with 100% sensitivity as previously
reported 29.
Results
EUVEC cultures with fungal contamination or nonviable EUVEC
The pEUVEC heart, iEUVEC heart, and iEUVEC tongue/lung cultures were the
only viable or uncontaminated EUVEC cultures after the overlay with fresh necropsy
samples. All other cultures were disposed of due to nonviable EUVEC cells or due to
persistent fungal contamination within the 3 days overlay period. Source of the fungal
contamination is attributed to the non-sterile tissue samples collected during the necropsy
process.
Detection of viral EEHV1A DNA in iEUVEC for 54 days after overlay with fresh heart
and tongue/lung necropsy tissue
93
The iEUVEC tongue/lung and heart cultures were first passaged for 32 days
keeping a third of the confluent T25 cell culture EUVEC to reseed the cultures while
using two-thirds of the remaining cells (approximately 1.3x106 EUVEC) for DNA
extraction. Subsequent passages of cultures only utilized a third of a confluent T25 cell
culture flask (6.6x105 EUVEC) for DNA extraction. First round conventional PCR for
EEHV1 POL yielded the proper-estimated sized 520 bp band for both the tongue/lung
and heart cultures. First round PCR for EEHV1A HEL amplified the estimated sized 980
bp band (Figure 4-2) and was submitted for sequencing with results consistent with the
same EEHV1A strain detected in necropsy samples containing the unique polymorphism
(Figure 4-3). Detection of EEHV1A POL continued until day 54 with failure to detect on
first round conventional PCR in iEUVEC heart and tongue/lung cultures at 61 days. A
total of 6 passages were performed after the overlay with both fresh heart and
tongue/lung by the last PCR detection (32 days, 40 days, 45 days, 47 days, 52 days, and
54 days). Each passaging resulting in an approximately 1/3 dilution with an estimated
calculated 1/729 or a 1.3x10-3 overall dilution of the original input viral DNA amount of
the overlay even by the last passage on 64 days.
Detection of EEHV1 DNA in pEUVEC for 122 days after overlay with fresh heart tissue
The pEUVEC heart culture was maintained for 47 days then split into two T25
cell culture flasks with the remaining third of the EUVEC collected for DNA extraction.
First round PCR for EEHV1A HEL DNA was amplified from this DNA extraction. Last
detection with first round PCR for EEHV1 POL was on pEUVEC heart passaged at 122
days but failed to amplify on the following passage at 126 days. A total of 7 passages of
94
the pEUVEC heart flask (54 days, 66 days, 78 days, 91 days, 119 days, 122 days) were
performed. Each passaging step resulted in a 1/3 dilution giving in a 1/2187 or a 4x10-4
dilution of the total original input of viral DNA amount of the overlay event by the last
passage 122 days later (Figure 4-4).
Initial qPCR measurement in pEUVEC heart cultures
qPCR detected 1.3 X 105 viral genome equivalents (VGE) in 1-μl of extracted
DNA from the fresh heart PBS suspension with calculations estimating approximately
1.3x107 VGE overlay onto 2x106 EUVEC cells per flask with a viral to cellular ratio of
6:1. qPCR quantitation of DNA extracts for heart pEUVEC culture passaged for 47 days,
54 days, 66 days and 73 days yielded 100, 250, 60, and lesser than 60 VGE/reaction
respectively with total VGE in the final culture estimated to be 6x103, 1.5x104, 3.6x103,
and lesser than 60 VGE per T25 cm2 flask respectively (Figure 4-4).
Discussion
The continued detection of persisting EEHV1A DNA using either cPCR or qPCR
in a laboratory cell culture system has not been reported previously to our knowledge.
Sequencing of the amplified EEHV1A DNA from both iEUVEC and pEUVEC using
conventional PCR revealed the same novel HEL gene polymorphism as detected in the
original necropsy tissue samples confirming that it did not represent contamination from
any other viral genome source. First round cPCR for EEHV1A POL revealed persistence
of viral DNA in the pEUVEC heart culture for 122 days and similarly for 54 days in the
iEUVEC heart and lung/tongue culture after overlay.
95
With the removal of the tissue sample PBS suspension overlay after 3 days, plus
routine medium changing (2-3 days intervals), and with continuous passaging of cultures
over the course of maintenance and monitoring these EUVEC cultures, viral EEHV1A
DNA was detected at multiple successive passaging steps. After all these procedures for
several weeks to months, the source of viral DNA would be assumed limited to within
cells as a possible circularized viral genome into a nuclear episome. The basic
maintenance of cell culture procedures should remove any free virus or viral DNA within
the supernatant. Surface viral attachment to EUVEC should be considered but it seems
highly unlikely that the viability would remain for such an extended duration.
Although it is not known how much of the input viral DNA was lost at the first
wash step, the simple direct 3X dilution factor occurring during each of the subsequent
passaging steps would be expected to reduce the overall DNA level (if it just persisted
throughout) by 2,500x fold (i.e. down to 1x103 VGEs). Therefore, there was clearly no
overall increase in total viral DNA over the course of the experiment. However, at 4th
passage, the residual amount of viral DNA present appears to exactly match the expected
dilution factor if all the input DNA was adsorbed or attached to the EUVEC monolayer
with no overall gain or loss. Therefore, theoretically some combination of the following
scenarios might have occurred: (1) All of the input DNA was in live tissue cells (likely
either macrophages or vascular endothelial cells) that became attached and survived
without dividing throughout the full three months; (2) Just a small fraction of the input
cells containing viral DNA became attached and either these cells divided or the viral
genomes replicated to some extent to maintain the levels seen; (3) The same occurred as
in (1) and (2) above except that the viral DNA was transferred to the new uninfected
96
cultured EUVEC and replicated at low levels in them; or (4) Free virions possibly present
within the input hemorrhagic tissue truly established new low level infections in the
cultured EUVEC cells. Nevertheless, because no cytopathic effects nor positive IFA with
any of the three different rabbit antibodies against EEHV lytic cycle nuclear antigens
were ever detected (as done in other similar experiments), we concluded that the
persisting viral DNA genomes whatever cells they had entered (or attached to) were
probably maintained in some form of latent rather than lytic cycle state but that even this
was not permanent.
qPCR quantification of DNA from limited passages from pEUVEC heart cultures
(47 days, 54 days, 66 days and 73 days) revealed a trend for decreasing amounts of
EEHV1A DNA based on the EEHV1A MDBP real-time assay described above (Figure
4-4). A decrease in any persisting original input viral DNA levels should be assumed
based on each passaging event initially reducing the number of cells in the culture to
approximately a third of those in the previous flask (although the overall number of live
EUVEC cells clearly increased about three-fold again before the next passage). Estimated
calculations of 1.3x107 VGE in the heart tissue PBS suspension overlay was determined
or a viral to cellular ratio of 6:1. A measurement of VGE in the removed tissue sample
PBS and medium mixture after the initial 3 day overlay period was not obtained but
would have given an accurate number of the amount of VGE/virus remaining in the flask
and possible levels of cellular entry. Without knowing this remaining VGE amount after
overlay, we used the beginning heart input 1.3x107 VGE as a starting point and applied a
rough calculation that only a maximum of one third of the input unreplicated VGE could
remain after every passage event yielding 5.9x103 and 1.8x104 VGE for pEUVEC (7 total
97
passages) (Figure 4-4) and iEUVEC (6 total passages). Two qPCR measurement from
the pEUVEC heart culture from passage 54 days and 66 days extrapolated to be 1.5x104
and 3.6x103 VGE, respectively showing a four fold decrease compared to a three fold
decrease. Cell death should be considered as a possible cause for the additional decrease.
These numbers indicate that there was no overall increase in viral DNA copy number or
that of there was replication of input genomes that was only enough to replace the
genome lost because of to presumed cell death.
The inconsistency between first round cPCR with the qPCR results puts into
question if EEHV1A DNA truly persisted till 122 days in the pEUVEC heart culture or
was it more similar to iEUVEC cultures which were limited to 54 days consistent with
the lack of detection of viral DNA at 73 days. The lowest limit for first round
conventional PCR band detection is 1000 VGE with the ability to perform additional
second and third rounds for lower limit detection. qPCR measurement is the preferred
total method for quantitation with lower limits of 10 viral copies/reaction 29 or 600 viral
copies per T25 flask. Limited qPCR measurements (47 days - 73 days) failed to detect
any viral copies from passaged iEUVEC heart passaged DNA after 73 days in contrast to
the last conventional PCR detection at 122 days in the iEUVEC heart culture. Additional
qPCR measurements of both iEUVEC and pEUVEC from multiple passage dates with
multiple replicates are needed to obtain an accurate measurement of the viral levels and
degree of decrease to attribute to passage dilution, and/or die off.
At this point, we can only assume that EEHV1A, like all herpesviruses, exhibits
two distinct phases of infection: latency and lytic replication. During primary infection,
herpesviruses access the cell followed by entering a latency phase where the viral
98
genome is circularized and exist as a nuclear episome through multiple host cell divisions
and expressing a subset of latent specific viral genes. Shifting from latency to lytic
replication producing infectious virion particles generally requires chemical agents or
environmental stress. Detection of viral EEHV1A DNA persisting over time and even
after routine cell culture maintenance (changing medium, washing with PBS, passaging,
etc) is suggestive of viral entry of EEHV1A into the EUVEC. Virus or viral DNA is
unlikely to remain stable without cellular entry over this period of time. EEHV1A latent
in the EUVEC cells would explain the persistence of detection of viral DNA after
removal of tissue sample PBS suspension removal and routine cell culture maintenance.
In spite of the decreasing trend of viral EEHV1A DNA directly due to passage dilution
and possible die off, the PCR data does not display any increase in viral DNA suggesting
EEHV1A latency in these EUVEC cells. Future work is needed to definitively confirm
the presence of the nuclear episome within cells.
Potential sources of EEHV1A viral DNA in the cultures are viable cells that
adhered to the cell culture flask during the overlay period. We believe this is unlikely, as
we received these necropsy tissue samples 3 day postmortem, well beyond point of
viability of these tissue cells. The only EUVEC cultures that either were viable with
detectable EEVH1A viral DNA with no fungal contaminations were the pEUVEC heart,
iEUVEC heart, and iEUVEC lung/tongue cultures. Daily visual inspection of the cultures
revealed a monolayer of cobblestone EUVEC with no visual detection of primary cells
from the tissue sample suspensions. We believe the potential source of EEHV1A DNA
from viable cells from necropsy tissue is small to none.
99
For all except the first passaging step for both the “infect” pEUVEC and iEUVEC
cultures that were positive for viral EEHV1A DNA, a third of the EUVEC were
cryopreserved and stored in -80°C. However, multiple attempts to resurrect confirmed
positive cells at 91 and 119 days for pEUVEC failed to detect EEHV1A DNA based on
conventional PCR once cultures reached confluence. This is consistent with the qPCR
results where iEUVEC heart cells after 73 days later did not contain any EEHV1A DNA.
Future directions include additional quantifications of viral EEHV1 DNA with
qPCR for both pEUVEC and iEUVEC, for different passage dates, and from the removed
tissue sample/medium mixture after overlay period to measure an accurate viral DNA
input into each culture. Detection of viral nuclear episome with EUVECs positive for
viral EEHV1A DNA confirming viral entry and latency is also planned. Frozen samples
of the same original heart tissue suspensions have been stored at -80°C for additional cell
culture experiments, which should include adding chemical activators such as TPA or
nBA to induce lytic replication of herpesvirus in endothelial cell cultures 53.
In summary, this is the first report of persistence EEHV1A DNA PCR detection
within a cell culture system in a laboratory setting infected with necropsy tissue samples
from a confirmed EEHV1A associated death. Additional work is needed to confirm viral
entry and to progress into viral propagation to generate large quantities of virus for
research to develop successful treatment methods for EEHV1A disease.
Acknowledgement
I would like to thank our colleague Sarah Y. Heaggans for assisting me with the
cell culture work and for carrying out the PCR phylogenetic analyses. I would also like to
100
thank Colette apRhys for helping me establish the pEUVEC and aiding me in creating the
iEUVEC. The qPCR analysis were performed at the NEHL and were supported by
research grants to Erin Latimer and Timothy Walsh from the Smithsonian Institution,
National Zoo, the International Elephant Foundation, the Morris Animal Foundation, and
the Ringling Bros and Barnum and Bailey Center for Elephant Conservation. We are
especially grateful to the zoo veterinarians and staff of the Albuquerque BioPark who
provided clinical and pathological samples for this research. I would like to thank the
Morris Animal Foundation for the postdoctoral fellowship D14ZO-411. Studies at Johns
Hopkins University were supported at various times by research grant R01 AI24576 to
G.S.H. from NIAID, National Institutes of Health, DHEW, the International Elephant
Foundation, and a subcontract to Lauren Howard at the Houston Zoo under an IMLS
National Leadership Collections Stewardship Program. And special thanks to Sarah Beck
from the Molecular and Comparative Pathobiology department at Johns Hopkins
University School of Medicine for initial revisions.
101
Figures
Figure 4-1. Microphotograph of pEUVEC. 10X microphotograph of pEUVEC
retaining the cobblestone-like contact-inhibited monolayers within a T25 flask.
102
Figure 4-2. Agarose gel-ethidium bromide of PAN-HEL PCR. Agarose gel-ethidium
bromide PCR band results obtained after first-round (980 bp) amplification with elephant
endotheliotropic herpesvirus PAN-HEL primers from DNA extracted from iEVUEC, cell
culture supernatant and necropsy blood. Lane 1, tongue/lung iEUVEC; Lane 2, heart
iEUVEC; Lane 3, supernatant from tongue/lung iEUVEC culture; Lane 4, supernantant
from heart iEUVEC culture; Lane 5, Diazy whole blood (positive control); Lane 6,
iEUVEC (tissue negative control); Lane 7, blanket (negative control).
980 bp
1 2 3 4 5 6 7
103
Figure 4-3. DNA sequence alignment between detected EEHV HEL from heart
iEUVEC and whole blood necropsy sample. DNA sequence alignment for first-round
EEHV PAN-HEL (980 bp) between EEHVSLDAIZYINFHEL719-2.SEQ (heart
iEUVEC) vs. EEHVSLDAIZYWBHEL719-5.SEQ (Diazy whole blood) generated using
MacVector with Assember v12.0.6.
104
Figure 4-4. Levels of VGE in heart pEUVEC between estimated compared to qPCR
detected based on the number of passages. For the estimated pEUVEC VGE, flask was
reseeded with 1/3 of the total flask cells resulting in a 2/3-fold decrease in VGE for each
consecutively passage.
105
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Simon Y. Long 108 South Wolfe Street,
Baltimore, MD 21231
Email address: [email protected]
Mobile phone: (610) 299-6637
PERSONAL STATEMENT: Research veterinary pathologist seeking a position with an
emphasis on molecular and pathology techniques utilizing animal models and molecular
biology to develop therapeutic approaches for patients with unmet medical needs.
PROFILE
Strong research and laboratory animal pathology background that includes
phenotyping/comparative pathology study experience. Research experience
includes academic and pharmaceutical settings.
Extensive research experience including molecular virology investigating disease
pathogenesis, molecular cellular pathways in cell cycling and toxicological
evaluations in both academic and pharmaceutical settings.
Strong laboratory animal pathology and science background in laboratory animal
species including rodents, rabbits, canines, and nonhuman primates in both
academic and pharmaceutical settings for both basic research and safety
assessment including GLP standards.
EDUCATION JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE Baltimore, MD
PhD Candidate- Cellular and Molecular Medicine PhD Program Sept. 2010- June 2016
JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE Baltimore, MD
Pathology Postdoctoral Fellow-Pathology Residency July 2009- June 2013
UNIVERSITY OF PENNSYLVANIA, SCHOOL OF VETERINARY MEDICINE Philadelphia, PA
Veterinary Medicine Doctorate (VMD)-MIXED ANIMAL MAJOR Graduated May 2009
DREXEL UNIVERSITY, BIOSCIENCE AND BIOTECHNOLOGY Philadelphia, PA
Graduate Student Sept. 2003-June 2004
DREXEL UNIVERSITY COLLEGE OF MEDICINE MCP HAHNEMANN Philadelphia, PA
Master in Laboratory Animal Science (MLAS) Graduated May 2003
SUNY COLLEGE OF ENVIRONMENTAL SCIENCE AND FORESTRY Syracuse, NY
Bachelor of Science Environmental Forest Biology/Pre-veterinary Medicine Graduated May 2001
PROFESSIONAL CERTIFICATIONS NATIONAL VETERINARY BOARD EXAMINATION: passed December 2008
Pennsylvania State Veterinary Board Licensed: 2009-present
Eligible for the board certification examination for the American College of Veterinary Pathologists with
passing Phase I General Pathology in March 2015 and continuation with Phase II.
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RESEARCH AND PROFESSIONAL EXPERIENCE Johns Hopkins University: Dr. Gary S. Hayward’s Herpesvirus Laboratory
PhD Graduate Student January 2011-Present
Thesis project on dissecting the molecular pathogenesis of the Elephant Endotheliotropic
Herpesvirus (EEHV) in Asian elephant (Elephas maximus).
Developed a primary and immortalized elephant umbilical vascular endothelial cell lines for
EEHV viral propagation.
Genetic analysis/comparison between different GC-rich and AT-rich EEHV viruses obtained from
clinical and necropsy samples.
Development of a next generation proteomic chip serology assay using cloned GST fusion EEHV
viral proteins.
Detecting viral genomic copies using PCR to determine viral levels and virus subtypes in necropsy
tissues blood samples, and biopsy samples.
Familiar with mammalian cell culture, PCR, western blot, proteinomic cloning and serological
cHip assay development.
Johns Hopkins University: Department of Molecular and Comparative Pathobiology
Postdoctoral Pathology Fellow July 2009- June 2013
Performed necropsies and histopathological evaluation on small animals, lab animals and wildlife
species.
Performed histopathological evaluation on biopsy specimens.
Performed mouse phenotyping studies for principle investigators under the mentorship of Dr.
Cory Brayton, DVM, Dipl. ACLAM, Dipl. ACVP.
Trained and supervised veterinary students in summer veterinary pathology externships.
Attended weekly slide conference and board training sessions.
Performed human autopsy rotation with completion of 6 human autopsy cases from June to July
2010.
Passed American College of Veterinary Pathologist Board Certification Phase I General
Pathology section in March 2015.
Children’s Hospital of Philadelphia: Dr. Jake Kushner’s Diabetes Cell Cycling Laboratory Senior Research Technician II/Laboratory Manager March 2004-March 2008
Hired to start up a diabetes cell cycling laboratory and managed a molecular laboratory, animal
colony and 2-3 junior research technicians.
Involved in leading two major research projects through animals experiments, molecular,
biochemistry techniques plus guiding and assisting other research projects.
Responsibilities included genotyping, characterize mice for diabetic phenotypes, cell culture, gene
expression analysis, immunohistochemistical slide development, fluorescent microscopy, and islet
morphometrics.
NIH/Merck Summer Research Program: Dr. Susan Weiss’s Coronavirus Laboratory Summer Research Veterinary Student Summer of 2006
Researched the role of the N protein in coronaviruses in pathogenesis through using PCR to design
and create chimeric viruses using constructs and cell cultures and observing their affects in mice.
Bristol-Myers Squibb: Drug Metabolism and Pharmacokinetics
Biological Technician August 11- October 2003
Biological technician in the Technical Support Unit of Drug Metabolism and Pharmacokinetics
Department, Lawrenceville New Jersey.
Assisted and lead pharmacokinetic studies on mice, rats, dogs and primates. Assisted in
cannulation surgery on rodents.
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Neuronyx, Inc.
Assistant Researcher December- May 2003
Assisted in a GLP FDA rodent model of spinal cord injury recovery facilitated by transplant of
adult bone marrow stem cells as a potential therapeutic.
Responsibilities included basic husbandry, quality assurance, veterinary care and surgical
preparation.
Medical College of Pennsylvania Hahnemann University: Dr. Libby Blankerhorn’s Laboratory
Assistant Molecular Biologist December - April 2002
Assisted graduate students in daily gel electrophoresis and PCR techniques of genetic materials of
colony of inbred rats for insulin study.
Responsibilities include gel electrophoresis, PCR, proper usage of radioactive material and rodent
handling.
SUNY College of Environmental Science and Forestry: Dr. Chun June Wang’s Laboratory
Assistant Molecular Biologist and Mycologist September- May 2001
Involved in isolating and genetically sequencing a new species of Exophila sp. fungi pathogenic to
lumpfish.
Responsibilities included mycological techniques (aseptic techniques), DNA extraction,
sequencing and PCR.
SUNY College of Environment Science and Forestry: Dr. Scott O. Rogers’ Laboratory
Assistant Molecular/Mycological Biologist September 1997-May1999
Assisted Dr. Scott O. Rogers and Dr. Chun June Wang with numerous molecular projects.
Responsibilities included DNA extraction, PCR, gel electrophoresis, maintaining fungi colonies
with aseptic mycological techniques, micro-cross-section techniques, slide preparation processes
and basic photo-development processes.
SUNY College of Environment Science and Forestry: Dr. Larry Smart’s Laboratory Assistant Molecular Biologist September 1998-May1999
Involved in a two-year project with Dr. James Gibbs and Eriko Motegi in College of
Environmental Science and Forestry as a laboratory assistant in the “UROP Project: The Genetic
Diversity of Sugar Maples”.
Responsibilities included DNA extraction, PCR and gel electrophoresis.
EXTERNSHIP Wildlife Conservation Society, Bronx Zoo: Department of Pathology
Student Externship January 2008
Performed a 4 week externship at the Department of Pathology.
Responsibilities included performing daily necropsies, writing necropsy reports, attending weekly
pathology rounds, weekly Armed Forces Institute of Pathology (AFIP) slide conferences and
presentation of zoonotic diseases of non-human primates.
Merck & Co., Inc. West Point, PA: Laboratory Animal Resource Department
Student Externship February 2008
Performed a 4 week externship at the Laboratory Animal Resource Department.
Daily rotations through the different groups and services of LAR providing veterinary services to
investigators and working on rodents, canines, guinea pigs, and non-human primates models.
Merck & Co., Inc. West Point, PA: Safety Assessment-Pathology
Merck/Merial Veterinary Summer Externship Summer of 2007
Performed a 10 week pathology internship in the Merck West Point Safety Assessment
Department.
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Training included weekly AFIP slide conferences, weekly peer-review case studies, necropsy
training, tissue trimming, histo-technology, attending pathology rounds, presentation of summer
research project on hyperplasia in rat pituitaries.
MERCK & Co., Inc. Rahway, NJ: Laboratory Animal Resource Department Co-op Intern January 13- May 2, 2003
Intern in the comparative medicine department.
Trained in all the basic functions of running and maintaining an animal facility.
Training includes management skills, operations of both barrier and conventional animal facilities,
clinical care of laboratory animals, pharmacokinetic and transgenic studies.
TEACHING EXPERIENCE Johns Hopkins University School of Medicine: Mouse Pathobiology & Phenotyping Short Course
Necropsy Instructor 2010, 2011, and 2015
Introduced and guided participants in performing a full mouse necropsy for phenotyping analysis.
Introduced and guided participants in trimming mouse tissues for phenotyping analysis.
Johns Hopkins University School of Medicine: Toxicology Pathology
Endocrinology Lecturer April 2013
Lectured on the toxicological principals centered on the endocrine system.
Drexel University: Developmental Biology Laboratory
Teaching Assistant March- June 2004
Taught college students the basic concepts of applied biology under Dr. Jeremy Lee.
Responsibilities included teaching and managing undergraduate level laboratory course.
Drexel University: Applied Biology
Teaching Assistant January-March 2004
Taught undergraduate students the basic concepts of applied biology under Dr. Jennifer Quinlan.
Responsibilities included teaching and managing undergraduate level laboratory course.
Drexel university: Organismal Physiology
Teaching Assistant Sept. -December 2003
Taught undergraduate students the basic concepts of organismal physiology under Dr. Jeremy Lee.
Responsibilities included teaching and managing undergraduate level laboratory course.
SUNY College of Environmental Science and Forestry Teaching Assistant January 1999- May 2000
Taught undergraduate students the basic concepts of herpetology under the guidance of Dr. James
Gibbs.
Responsibilities included teaching and managing undergraduate level college laboratory course.
Teaching included lecturing, managing laboratories, setting up a laboratory, and lead field trips.
COMMITTEE Johns Hopkins School of Medicine: Molecular and Comparative Pathobiology Department
Admissions Committee Member 2009-2014
Served as an admissions committee member for screening of candidates applying for residency
and postdoctoral fellow positions for both the anatomic pathology and laboratory animal positions.
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Drexel College of Medicine: Institutional Animal Care and Use Committee
Alternative IACUC Member January- December 2002
Served as an alternative IACUC member to review animal protocols for IACUC board approval
for animal studies at Drexel University, Drexel College of Medicine Hahnemann University.
PUBLICATIONS Ling PD, Long SY, Zong JC, Heaggans SY, Qin X, Hayward GS. Comparison of the gene coding context
and other unusual features of the the GC-rich and AT-rich branch probosciviruses. mSphere. 2016; 1(3):
e00091-16. doi:10.1128/mSphere.00091-16.
Ling PD, Long SY, Fuery A, Peng RS, Heaggans SY, Qin X, Worley KC, Dugan S, Hayward GS.
Complete genome sequence of elephant enodtheliotropic herpesvirus 4 (EEHV4) the first example of a GC-
rich branch proboscivirus. mSphere. 2016; 1(3): e00081-15. doi:10.1128/mSphere.00081-15.
Long SY. Approach to reptile emergency medicine. Vet Clin North Am Exot Anim Pract. 19(2): 567-90.
May 2016. PMID:27131162
Fuery A, Browning GR, Tan J, Long SY, Hayward GS, Cox SK, Flanagan JP, Tocidlowski ME, Howard
LL, Ling PD. Clinical infections of captive asian elephant (Elephas mamimus) with elephant
endotheliotropic herpesvirus 4. Journal of zoo and wildlife medicine. 47(1): 311-318. March 2016. PMID:
27010293
Long SY, Latimer EM, Hayward GS. Review of elephant endotheliotropic herpesviruses and acute
hemorrhagic disease. Institue for Laboratory Animal Research Journal 56(3): 283-96. Feburary 2016.
PMID: 26912715
Zong JC, Heaggans SY, Long SY, Latimer EM, Nofs SA, Bronson E, Casares M, Fouraker MD, Pearson
VR, Richman LK, Hayward GS. Detection of quiescent infections with multiple elephant endotheliotropic
herpesviruses EEHV2, EEHV3, EEHV6 and EEHV7 within lymphoid lung nodules or lung and spleen
tissue samples from five asymptomatic adult African elephants. Journal of Virology 90(6): 3028-43.
December 2015. PMID: 26719245
Zong JC, Latimer EM, Long SY, Richman LK, Heaggans SY, Hayward GS. Comparative genome
anaylysis of four elephant endotheliotropic herpesvirus, EEHV3, EEHV4, EEHV5, and EEHV6, from cases
of hemorrhagic disease or viremia. Journal of Virology 88(23):13547-69. December 2014. PMID:
25231309
Sarotir DJ, Wilbur CJ, Long SY, Rankin MM, Li C, Bradfield JP, Hakonarson H, Grant SF, PU WT,
Kushner JA. GATA factors promte ER integrity and beta-cell survivial and contribute to type 1 diabetes
risk. Molecular Endocrinology 28(1):28-39. January 2014. PMID: 24284823
Zachariah A, Zong JC, Long SY, Latimer EM, Heaggans SY, Richman LK, Hayward GS. (2013). Fatal
Herpesvirus (EEHV) Hemorrhagic Disease in Wild and Orphan Asian Elephants in India. J. Wildl. Dis. 49,
381-393. April 2013 PMID: 23568914
Tuttle AH, Rankin MM, Teta M, Sartori DJ, Stein GM, Kim GJ, Virgillio C, Granger A, Zhou D, Long
SY, Schiffman AB, Kushner JA. Immunofluorescent detection of two thymidine analogues (CldU and IdU)
in primary tissue. Journal of Visualized Experiments (46). December 2010 PMID: 21178965
Cowley TJ, Long SY, Weiss SR: The murine coronavirus nucleocapsid gene is a determinant of virulence.
Journal of Virology 84(4):1752-63. February 2010 PMID: 20007284
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He LM, Sartori DJ, Teta M, Opare-Addo LM, Rankin MM, Long SY, Diehl JA, Kushner JA: Cyclin D2
protein stability is regulated in pancreatic beta-cells. Molecular Endocrinology 23(11): 1865-1875.
November 2009 PMID: 19628581
Teta M, Rankin MM, Long SY, Stein GM, Kushner JA: Growth and regeneration of adult beta cells does
not involve specialized progenitors. Developmental Cell 12(5): 817-826, May 2007 PMID: 17488631
Teta M, Long SY, Wartschow LM, Rankin MM, Kushner JA: Very slow turnover of beta cells in aged
adult mice. Diabetes 54:2557-2567, September 2005 PMID: 16123343
Kushner JA, Ciemerych MA, Sicinska E, Wartschow LM, Teta M, Long SY, Sicinski P, White MF:
Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth. Molecular and Cellular
Biology 25(9436): 3752-3762, May 2005 PMID: 15831479
PRESENTATIONS Long SY, Zachariah A, Zong JC, Latimer EM, Heaggans SY, and Hayward GS. High level genetic
variability amongst nine cases of fatal elephant endotheliotropic herpesvirus hemorrhagic disease in wild
and orphan Asian elephants in southern India. Oral presentation at the 9th Annual International Elephant
Endotheliotropic Herpes virus (EEHV) workshop. January 2013
Long SY, Ap Rhys CJ, Heaggans SY, Hayward GS. Immortalization of elephant umbilical vascular
endothelial cells (EUVEC) for elephant endotheliotropic herpesviruses (EEHV) infection. Poster
presentation at the American College of Veterinary Pathologist Annual Meeting. December 2011.
Long SY, Hadfield CA, Clayton LA, Huso DL, Montali RJ. Disseminated chromoblastomycosis in White’s
Tree frog (Litoria caerulea). Poster presentation at the American College of Veterinary Pathologist Annual
Meeting. December 2010.
Long SY, Li C, Matschinsky FM, Stanley CA, Kushner JA. GATA-4 is essential for normal beta cell
function. Oral presentation at the Mid-Atlantic Diabetes Research Session at National Institute for Health.
October 1, 2005
Long SY, Cowley TJ, Weiss SR. Understanding the role of the nucleocapsid gene in the mouse hepatitis
virus using chimeric viruses. Poster presentation at the 7th Annual Merck-Merial/NIH Veterinary Scholars
Symposium at Louisiana State University. August 3-6, 2006.
Long SY, Cowley TJ, Weiss SR. Understanding the role of the nucleocapsid gene in the mouse hepatitis
virus using chimeric viruses. Poster presentation at the Penn Vet Student Research Day. University of
Pennsylvania School of Veterinary Medicine March 22, 2007.
EXTRAMURAL FUNDING Morris Animal Foundation: Wildlife Studies Fellowship D14ZO-411 (S.LONG)
Fellowship Award June 2014-June 2016
Wildlife fellowship salary funding support for 2 years for “Innovative attempts to propagate
elephant endotheliotropic herpesvirus in cell culture”.
National Institution of Health: 2T32RR007002-35 (C.ZINK)
Postdoctoral training June 2009-June 2013
Training Veterinarians for Careers in Biomedical research
PI: Christine Zink, DVM, PhD, Dipl. ACVP
Mentor: Cory F. Brayton, DVM, Dipl. ACVP, Dipl. ACLAM
Role: Veterinary Pathology Postdoctoral fellow
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AWARDS Delaware Valley Branch of American Association of Laboratory Animal Science
Scholarship Award 2002
Recipient of the 2002 J.J Noonan Scholarship award for laboratory animal science.
PROFESSIONAL ASSOCIATIONS Member of the Wildlife Disease Association
Member of Society of Toxicologic Pathology
Member of Society of Study of Amphibians and Reptiles