We detected herpesvirus infection in a male yellow-footed antechinus (Antechinus flavipes) and male agile antechinus (Antechinus agilis) during the period of postmating male antechinus immunosuppression and mortality. Histopathologic examination of tissues revealed lesions consistent with herpesvirus infection in the prostate of both animals. Herpesvirus virions were observed by transmission electron microscopy in the prostate tissue collected from the male yellow-footed antechinus. Herpesvirus DNA was detected in prostate, liver, lung, kidney, spleen, and ocular/nasal tissues using a pan-herpesvirus PCR targeting the viral DNA polymerase. Nucleotide sequencing identified a novel herpesvirus from the Gammaherpesvirinae subfamily that we have tentatively designated dasyurid herpesvirus 1 (DaHV-1).

Antechinus (Antechinus spp.) are small insectivorous Australian marsupials of the family Dasyuridae. They follow the “big bang breeder” pattern of reproduction (Bradley 2003; Tyndale-Biscoe 2005; Naylor et al. 2008) involving a frenzied 2-wk mating period in late winter, followed by the stress-related synchronous annual mortality of the entire male population at approximately 11.5 mo of age (Barker et al. 1978; Bradley 2003; Tyndale-Biscoe 2005; Ladds 2009). High circulating levels of testosterone and free cortisol are believed to dominate the physiology of mature male antechinus and contribute significantly to postmating male mortality (semelparity) via terminal depression of the immune system (Bradley 2003; Naylor et al. 2008). The high concentration of plasma cortisol mobilizes glucose to fuel fighting and sexual activity (Bradley 2003; Tyndale-Biscoe 2005; Naylor et al. 2008), but ultimately leads to a cascade of metabolic disturbances. These culminate in the programmed postreproductive senescence, truncated natural lifespan, and ultimate demise of all semelparous dasyurid males at the end of their only breeding period (Bradley 2003).

In their investigations into postmating male mortality, Barker et al. (1981) observed evidence of cytomegaloviral infection in the prostate of two small dasyurid species: the brush-tailed phascogale (Phascogale tapoatafa) and the agile antechinus (Antechinus stuartii; now known as Antechinus agilis). Electron microscopy showed viral particles consistent with herpesvirus virions, and the virus was described as a cytomegalovirus based on the observation of enlarged cells containing intranuclear inclusions, characteristic of cytomegalovirus infection. Cytomegaly was also observed in the kidneys of some male agile antechinus, and small eosinophilic intranuclear inclusion bodies were also rarely observed within hepatocytes (Barker et al. 1978, 1981). No inflammatory response was observed, and apart from displacement of normal glandular prostatic tissue, the authors concluded that infection did not appear to be detrimental to the host (Barker et al. 1981). Similar lesions have been observed in the prostate of the dusky antechinus (Antechinus swainsonii) by Munday and Obendorf (1983), and it was hypothesized that stress and hyperadrenocorticism in postbreeding male antechinus may lead to enhanced reactivation and replication of latent viral infections (Munday and Obendorf 1983). We expand upon this knowledge by applying molecular techniques to detect and identify herpesvirus infections in tissues harvested from male yellow-footed antechinus and agile antechinus (hereafter referred to as YFA and AA, respectively).

We captured two adult males toward the end of the breeding period in August 2011. The YFA was captured using an Elliott trap (Elliott Scientific, Upwey, Victoria, Australia) from a free-ranging population in Inman Valley, South Australia, Australia (35°28.290′S, 138°29.288′E). Following euthanasia with an overdose of isoflurane, samples of liver, lung, kidney, spleen, nasal mucosa, and prostate were collected and stored at −20 C. Heart, lung, kidneys, adrenal glands, spleen, liver, pancreas, bladder, diaphragm, hind leg skeletal muscle, brain, skin punch biopsies, testes, penis, prostate, and bulbourethral glands were fixed in 10% phosphate-buffered formalin.

The AA was found weak and in poor body condition near Waratah Bay in Victoria, Australia. The animal died shortly afterward, and samples of spleen, lung, liver, kidney, and prostate tissue, as well as an ocular/nasal swab, were collected and stored at 4 C. Brain, lung, liver, heart, testicle, prostate, kidney, spleen, and adrenal gland samples were fixed in 10% phosphate-buffered formalin.

Formalin-fixed tissues were processed and stained with H&E for light microscopy (both animals) or were processed for transmission electron microscopy (prostate tissue from the YFA). Samples stored at −70 C were homogenized in phosphate-buffered saline using a multidirectional stopcock (B. Braun, Melsungen AG, Melsungen, Germany). DNA was extracted using QIAamp Viral RNA Extraction Mini Kit (Qiagen, Hilden, Germany) and used as template in a generic pan-herpesvirus PCR using degenerate primers targeting a highly conserved region of the viral DNA polymerase gene (Chmielewicz et al. 2003). This nested PCR uses three primers in the first round (forward primers DFA and ILK, reverse primer KG1) and two primers in the second round (forward primer TGV, reverse primer IYG). To obtain additional downstream sequence, further PCRs were performed using the primer pair KG1 and TGV. The PCR products were separated by agarose gel electrophoresis and visualized using SYBR Safe stain (Invitrogen, Carlsbad, California, USA). Each amplicon was purified and sequenced using BigDye Terminator version 3.1 chemistry (Applied Biosystems, Carlsbad, California, USA).

Nucleotide sequence and phylogenetic analysis was performed using ClustalW2 (Larkin et al. 2007) and GENEious software (Biomatters Ltd., Auckland, New Zealand). The predicted amino acid sequence was compared with publicly available sequences in the GenBank database (NCBI 2013) using the blastx online algorithm and subsequently aligned with representative members from the three Herpesviridae subfamilies (Davison et al. 2009; Vaz et al. 2011) from a range of host species. An unrooted maximum-likelihood phylogenetic tree was generated from this sequence alignment using the Jones–Taylor–Thornton model of amino acid replacement.

Herpesvirus DNA was detected by PCR in the lung, liver, and prostate of the YFA and in the lung, liver, prostate, kidney, spleen, and ocular/nasal sample of the AA. Nucleotide sequence (approximately 150 nucleotides) was successfully obtained from PCR products amplified from the liver of the YFA and the liver, lung, and kidney of the AA. The sequence of all amplicons shared 100% nucleotide identity, suggesting that the same virus, or viruses that were genetically very similar, were present in both animals. Alignment of the nucleotide sequence to those of other marsupial herpesviruses showed that the detected virus was distinct from all other known marsupial herpesviruses.

The KG1 and TGV primer pair amplified PCR products of extended length from the liver sample of the YFA and the liver, lung, and kidney samples from the AA. The nucleotide sequence obtained overlapped with the existing sequence with 100% identity and extended the sequence available for analysis to 347 nucleotides. The first 100 matches with the deduced amino acid sequence generated from this nucleotide sequence in blastx (NCBI 2013) were to the DNA polymerase gene of herpesviruses within the subfamily Gammaherpesvirinae. Phylogenetic analyses confirmed that the detected virus grouped with other gammaherpesviruses (Fig. 1).

Figure 1.

The relationship between dasyurid herpesvirus 1 and herpesviruses from the Alphaherpesvirinae (α), Betaherpesvirinae (β), and Gammaherpesvirinae (γ) subfamilies. (A) Alignment of predicted amino acid sequences of all available DNA polymerase gene sequence data from dasyurid herpesvirus 1 (DaHV-1) and the corresponding regions of DNA polymerase gene sequences from other herpesviruses. Dots (.) indicate amino acid identity. Hyphens (-) indicate alignment gaps or a sequence that is not available. (B) Unrooted maximum-likelihood phylogenetic tree, generated using the above amino acid alignment. Divergences between pairs of aligned sequences were calculated and distance trees derived using GENEious software and the Jones–Taylor–Thornton model of amino acid replacement. The reliability of each tree branch was tested using 100 replicates in a bootstrapping analysis. Bootstrapping values (n = 100) are shown for each branch. Abbreviations and GenBank accession details: macropodid herpesvirus 3 (MaHV3: EF467663.1), phascolarctid herpesvirus 1 (PhaHV1: JN585829), phascolarctid herpesvirus 2 (PhaHV2: JQ996387), alcelaphine herpesvirus 1 (AlHV1: AF005370), caprine herpesvirus 2 (CpHV2: HQ116812), bovine herpesvirus 4 (BHV4: NP_076501.1), saimirine herpesvirus 2 (SaHV2: NP_040211.1), ateline herpesvirus 3 (AtHV-3: NP_047983.1), Atlantic bottlenose dolphin (AbdHV: ABC33906.1) equine herpesvirus 2 (EHV2: NC_001650.1), human herpesvirus 4 (HHV4: NC_007605.1), cercopithecine herpesvirus 15 (CeHV15: NC_006146.1), callitrichine herpesvirus 3 (CalHV3: NC_004367.1), human herpesvirus 1 (HHV1: HQ123098), feline herpesvirus 1 (FHV1: NC_013590.2), porcine cytomegalovirus (PCMV: AF268042.1), and human herpesvirus 6 (HHV6: NP_042931.1).

Figure 1.

The relationship between dasyurid herpesvirus 1 and herpesviruses from the Alphaherpesvirinae (α), Betaherpesvirinae (β), and Gammaherpesvirinae (γ) subfamilies. (A) Alignment of predicted amino acid sequences of all available DNA polymerase gene sequence data from dasyurid herpesvirus 1 (DaHV-1) and the corresponding regions of DNA polymerase gene sequences from other herpesviruses. Dots (.) indicate amino acid identity. Hyphens (-) indicate alignment gaps or a sequence that is not available. (B) Unrooted maximum-likelihood phylogenetic tree, generated using the above amino acid alignment. Divergences between pairs of aligned sequences were calculated and distance trees derived using GENEious software and the Jones–Taylor–Thornton model of amino acid replacement. The reliability of each tree branch was tested using 100 replicates in a bootstrapping analysis. Bootstrapping values (n = 100) are shown for each branch. Abbreviations and GenBank accession details: macropodid herpesvirus 3 (MaHV3: EF467663.1), phascolarctid herpesvirus 1 (PhaHV1: JN585829), phascolarctid herpesvirus 2 (PhaHV2: JQ996387), alcelaphine herpesvirus 1 (AlHV1: AF005370), caprine herpesvirus 2 (CpHV2: HQ116812), bovine herpesvirus 4 (BHV4: NP_076501.1), saimirine herpesvirus 2 (SaHV2: NP_040211.1), ateline herpesvirus 3 (AtHV-3: NP_047983.1), Atlantic bottlenose dolphin (AbdHV: ABC33906.1) equine herpesvirus 2 (EHV2: NC_001650.1), human herpesvirus 4 (HHV4: NC_007605.1), cercopithecine herpesvirus 15 (CeHV15: NC_006146.1), callitrichine herpesvirus 3 (CalHV3: NC_004367.1), human herpesvirus 1 (HHV1: HQ123098), feline herpesvirus 1 (FHV1: NC_013590.2), porcine cytomegalovirus (PCMV: AF268042.1), and human herpesvirus 6 (HHV6: NP_042931.1).

Close modal

Histologic evidence of herpesvirus infection, without associated inflammation, was observed in the prostate of both animals (Fig. 2). Additionally, the prostate of the AA showed a focal area of necrosis containing bacterial colonies and degenerating neutrophils. The prostate of the YFA contained sporulating oocysts and gamonts of a novel coccidian species, often in close association with the zones of herpesvirus expression (Fig. 2). Transmission electron microscopy of prostatic epithelial cells from the YFA revealed herpesvirus particles (Fig. 3). Histologic evidence of herpesvirus infection was also observed in the liver of both animals and in the spleen of the AA. The spleen of the AA showed almost complete lymphocyte depletion, consistent with glucocorticoid-mediated immunosuppression. Lungworm infestation, most likely a Marsupostrongylus sp. (Ian Beveridge pers. comm., 2012) without inflammatory cell infiltration, was observed in the AA. Marked vacuolation of hepatocytes, consistent with steroid hepatopathy, was observed in the YFA.

Figure 2.

Prostate of a male agile antechinus (Antechinus agilis) from Victoria, Australia, showing cytomegalic lesions with irregular and distorted epithelial cells, nuclear enlargement, large basophilic intranuclear inclusions (arrows), and basophilic material within tubule lumina, contrasting with normal prostatic tubules containing eosinophilic prostatic secretions to the right. H&E, 400×.

Figure 2.

Prostate of a male agile antechinus (Antechinus agilis) from Victoria, Australia, showing cytomegalic lesions with irregular and distorted epithelial cells, nuclear enlargement, large basophilic intranuclear inclusions (arrows), and basophilic material within tubule lumina, contrasting with normal prostatic tubules containing eosinophilic prostatic secretions to the right. H&E, 400×.

Close modal
Figure 3.

Transmission electron micrographs of herpesvirus particles in prostatic tissue from a South Australian yellow-footed antechinus (Antechinus flavipes). (A) Herpesvirus particles within the cytoplasm of a prostatic epithelial cell. Most of these cytoplasmic virions are enclosed within a second outer enveloping membrane. This pattern suggests that the herpesviral virions may be transported through the host cell cytoplasm inside a small enveloping vesicle, likely derived from host nuclear membrane components. 88,000×. (B) Herpesvirus particles being shed into the lumen of a prostatic tubule at the surface of a prostatic epithelial. These herpesviral virions appear to escape their second outer enveloping membrane in the process of being shed from the host cell. 25,600×. (C) Virus particles within the lumen of a prostatic tubule. These viral particles are morphologically consistent with herpesvirus virions, consisting of a dense central core surrounded by a thick membrane. 20,800×.

Figure 3.

Transmission electron micrographs of herpesvirus particles in prostatic tissue from a South Australian yellow-footed antechinus (Antechinus flavipes). (A) Herpesvirus particles within the cytoplasm of a prostatic epithelial cell. Most of these cytoplasmic virions are enclosed within a second outer enveloping membrane. This pattern suggests that the herpesviral virions may be transported through the host cell cytoplasm inside a small enveloping vesicle, likely derived from host nuclear membrane components. 88,000×. (B) Herpesvirus particles being shed into the lumen of a prostatic tubule at the surface of a prostatic epithelial. These herpesviral virions appear to escape their second outer enveloping membrane in the process of being shed from the host cell. 25,600×. (C) Virus particles within the lumen of a prostatic tubule. These viral particles are morphologically consistent with herpesvirus virions, consisting of a dense central core surrounded by a thick membrane. 20,800×.

Close modal

It is probable that the novel herpesvirus identified in this study is the same virus as the presumed cytomegalovirus described by Barker et al. (1981) in an AA (then called A. stuartii) and possibly the same virus observed by Munday and Obendorf (1983) in a dusky antechinus. The histopathologic observations in our study closely match the descriptions of cytomegalic lesions provided in those earlier reports. The ultrastructural observations also concur with the earlier descriptions and electron micrographs. In the absence of molecular techniques, the earlier studies referred to the virus as a cytomegalovirus, which is classified within the Betaherpesvirinae subfamily (Davison et al. 2009). Our sequencing results, however, identify the virus as a gammaherpesvirus, and so it would be appropriate to derive an alternative designation. Following current conventions for herpesvirus nomenclature, we have tentatively designated it dasyurid herpesvirus 1 (DaHV-1). Gammaherpesviruses generally exhibit a narrow host range (Roizman and Pellet 2001). The detection of this gammaherpesvirus in free-living animals suggests that species of the genus Antechinus are natural hosts of this virus.

Herpesviruses are well known for establishing latent infections that reactivate during periods of stress and immunosuppression (Guliani et al. 1999; Roizman and Pellet 2001). It is likely that this gammaherpesvirus recrudesced during the breeding season, when male Antechinus spp. are profoundly immunosuppressed by high circulating glucocorticoid concentrations (Barker et al. 1978; Bradley 2003; Naylor et al. 2008; Ladds 2009). Herpesvirus-induced pathology was expressed most overtly in the prostates of both males. It is possible that this gland is the site of productive infection, with virus particles being shed in prostatic secretions and hence semen. It is also possible that the liver, lungs, kidneys, and spleen represent sites of infection, or that blood cells provide a reservoir for infection and thus herpesvirus DNA is present in the circulation.

We observed distortion and displacement of normal prostatic tissue architecture in both animals, and although some of the prostatic tubules were likely to be nonfunctional, this was unlikely to be detrimental to the host. The pathogenicity and biological significance of infection with this gammaherpesvirus in Antechinus spp. populations, including any possible role it may play in postmating male mortality, remains to be elucidated.

We thank the Ian Potter Foundation for providing funding for some of this work and the Vizard Foundation for its support of Wildlife Health Surveillance Victoria. We thank Professor Ian Beveridge for advice on parasitology.

Barker
IK
,
Beveridge
I
,
Bradley
AJ
,
Lee
AK
.
1978
.
Observations on spontaneous stress-related mortality among males of the dasyurid marsupial Antechinus stuartii Macleay
.
Aust J Zool
26
:
435
447
.
Barker
IK
,
Carbonell
PL
,
Bradley
AJ
.
1981
.
Cytomegalovirus infection of the prostate in the dasyurid marsupials, Phascogale tapoatafa and Antechinus stuartii
.
J Wildl Dis
17
:
433
441
.
Bradley
AJ
.
2003
.
Stress, hormones and mortality in small carnivorous marsupials
.
In:
Predators with pouches: The biology of carnivorous marsupials
,
Jones
ME
,
Dickman
CR
,
Archer
M
,
editors
.
CSIRO Publishing
,
Melbourne, Australia
, pp.
254
267
.
Chmielewicz
B
,
Goltz
M
,
Lahrmann
KH
,
Ehlers
B
.
2003
.
Approaching virus safety in xenotransplantation: A search for unrecognized herpesviruses in pigs
.
Xenotransplantation
10
:
349
356
.
Davison
AJ
,
Eberle
R
,
Ehlers
B
,
Hayward
GS
,
McGeoch
DJ
,
Minson
AC
,
Pellett
PE
,
Roizman
B
,
Studdert
MJ
,
Thiry
E
.
2009
.
The order Herpesvirales
.
Arch Virol
154
:
171
177
.
Guliani
S
,
Smith
GA
,
Young
PL
,
Mattick
JS
,
Mahony
TJ
.
1999
.
Reactivation of a macropodid herpesvirus from the eastern grey kangaroo (Macropus giganteus) following corticosteroid treatment
.
Vet Microbiol
68
:
59
69
.
Ladds
P
.
2009
.
Pathology of Australian native wildlife
.
CSIRO Publishing
,
Melbourne, Australia
,
640
pp.
Larkin
MA
,
Blackshields
G
,
Brown
NP
,
Chenna
R
,
McGettigan
PA
,
McWilliam
H
,
Valentin
F
,
Wallace
IM
,
Wilm
A
,
Lopez
R
,
et al.
2007
.
Clustal W and Clustal X version 2.0
.
Bioinformatics
23
:
2947
2948
.
Munday
BL
,
Obendorf
DL
.
1983
.
Cytomegalic lesions in Australian marsupials
.
J Wildl Dis
19
:
132
135
.
Naylor
R
,
Richardson
SJ
,
McAllan
BM
.
2008
.
Boom and bust: A review of the physiology of the marsupial genus Antechinus
.
J Comp Physiol B
178
:
545
562
.
NCBI (National Center for Biotechnology Information)
.
2013
.
GenBank
.
http://www.ncbi.nlm.nih.gov/genbank/. Accessed December 2013
.
Roizman
B
,
Pellet
P
.
2001
.
The family Herpesviridae: A brief introduction
.
In:
Fields virology
,
Knipe
D
,
Howley
P
,
editors
.
Lippincott Williams, and Wilkins
,
Philadelphia, Pennsylvania
, pp.
2381
2398
.
Tyndale-Biscoe
H
.
2005
.
Life of marsupials
.
CSIRO Publishing
,
Melbourne, Australia
,
464
pp.
Vaz
P
,
Whiteley
PL
,
Wilks
CR
,
Duignan
PJ
,
Ficorilli
N
,
Gilkerson
JR
,
Browning
GF
,
Devlin
JM
.
2011
.
Detection of a novel gammaherpesvirus in koalas (Phascolarctos cinereus)
.
J Wildl Dis
47
:
787
791
.