Detection of a novel bat gammaherpesvirus in Hungary

Acta Veterinaria Hungarica 56 (4), pp. 529–538 (2008) DOI: 10.1556/AVet.56.2008.4.10

0236-6290/$ 20.00 © 2008 Akadémiai Kiadó, Budapest

DETECTION OF A NOVEL BAT GAMMAHERPESVIRUS IN HUNGARY

Viktor MOLNÁR1*, Máté JÁNOSKA2, Balázs HARRACH2, Róbert GLÁVITS3, Nimród PÁLMAI3, Dóra RIGÓ3, Endre SÓS1 and Mátyás LIPTOVSZKY1

1Budapest Zoo and Botanical Garden, H-1146 Budapest, Állatkerti krt. 6–12, Hungary; 2Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary; 3Central Agricultural Office, Veterinary Diagnostic Directorate, Budapest,

Hungary

(Received 18 December 2007; accepted 7 February 2008)

This paper describes the detection of a novel herpesvirus in a Serotine bat (Eptesicus serotinus) in Hungary. The rescued animal showed signs of icterus and anorexia and died within a day, in spite of immediate supportive therapy. Autopsy confirmed the clinical picture by the major lesions observed in the liver. Histopa-thology revealed vacuolar degeneration in the hepatocytes and leukocytosis in the sinusoidal lumina. By electron microscopy, hydropic degeneration and apoptotic cells with a pycnotic nucleus were found in the liver. Bacteriological examina-tions gave negative results. As part of a routine screening project, detection of adeno- and herpesviruses from homogenised samples of the liver, lungs and small intestines was attempted by nested polymerase chain reaction (PCR) assays. The adenovirus PCR ended with negative results. The herpesvirus PCR resulted in an amplification product of specific size. The nucleotide sequence of the amplicon was determined and analysed by homology search and phylogenetic analysis. A novel herpesvirus was identified, which seemed to be most closely related to members of the genus Rhadinovirus within the subfamily Gammaherpesvirinae. The causative role of the detected rhadinovirus in the fatal condition of the Se-rotine bat could not be proven, but it is most likely that reactivation from a latent infection allowed the detection of the virus by PCR.

Key words: Herpesvirus, Gammaherpesvirinae, Eptesicus serotinus, Chiroptera, nested PCR

*Corresponding author; E-mail: [email protected]; Phone: 0036 (1) 273-4908; Fax: 0036 (1) 273-4917

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Acta Veterinaria Hungarica 56, 2008

Herpesviruses are large, enveloped, double-stranded DNA viruses infect-ing a wide variety of vertebrate hosts from fish to man. Herpesvirus-like particles have been described even in invertebrate hosts such as oysters (Davison et al., 2005). There are many different herpesvirus types, currently regarded as herpes-virus species, classified into three (Alpha-, Beta- and Gammaherpesvirinae) sub-families within the family Herpesviridae. Herpesvirus types normally have strict host specificity; however, the same host species or individual may harbour mul-tiple serotypes belonging to different subfamilies (Davison, 2002). A striking char-acteristic of herpesviruses is the capability of establishing latency (Minárovits et al., 2007). After primary infection and disease, herpesviruses, as a rule, remain in the host for a lifetime. During prolonged latency periods, the presence of the virus cannot be detected by conventional serological methods. Reactivation of latent herpesviruses can be triggered by stress or by any event, such as aging or concomi-tant infections, that diminishes the host’s immunocompetence (Ackermann, 2006).

For isolation and cultivation, herpesviruses generally require homologous cell lines, or cells from a closely related host animal, although there are a number of exceptions to this rule. With the development of molecular-based diagnostic methods such as polymerase chain reaction (PCR) and DNA sequencing, the recognition of uncultivable herpesviruses has become possible. Consequently, the number of serotypes especially in the genus Rhadinovirus has dramatically increased in the past decade.

In the course of a PCR survey on the occurrence of adeno- and herpesvi-ruses in randomly collected animal samples, a novel herpesvirus was detected in a bat. Analysis of the sequence of the PCR product revealed a herpesvirus clus-tering together with the members of genus Rhadinovirus within the subfamily Gammaherpesvirinae.

Materials and methods

Case description

In January 2006, an adult, female Serotine bat (Eptesicus serotinus), found in Budapest, was handed in the Rescue Centre of the Budapest Zoo and Botani-cal Garden. The rescued animal was in very weak body condition and exhibited signs of jaundice (icterus) on all visible mucous membranes. It was anorectic but consumed water. The day after its arrival, the bat died in spite of immediate sup-portive therapy with infusion solution containing amino acids, vitamins, electro-lytes and dextrose (Duphalyte inf. A.U.V., Fort Dodge Animal Health, UK), and silymarin (Legalon 70 mg drg., Madaus, Germany).

Gross pathology, histopathology and electron microscopy

Detailed postmortem examination was carried out. For histopathology, samples from the liver, kidney, heart, lung and spleen were fixed in 8% buffered

NOVEL BAT GAMMAHERPESVIRUS IN HUNGARY 531


formaldehyde solution and embedded into paraffin. Tissue sections were cut by microtome and stained with haematoxylin and eosin. For transmission electron microscopic (EM) examination, ca. 3-mm3 small pieces were cut from the liver and lung samples previously fixed in formaldehyde solution, and prepared as de-scribed elsewhere (Ivanics et al., 2001).

Bacteriology

Bacterium culturing was performed from liver samples by inoculation on conventional agar plates containing 10% sheep blood. The incubation was car-ried out under aerobic conditions at 37 °C.

Virus detection by PCR

Detection of the presence of adeno- or herpesviruses in the samples of in-ternal organs was attempted by PCR. DNA was extracted from a pool of liver, lung and small intestine samples, as described elsewhere (Zsivanovits et al., 2006). For each virus, a nested PCR, with highly degenerate, consensus primers targeting the viral DNA polymerase gene, was used. For adenoviruses, a very sensitive system has recently been described (Wellehan et al., 2004), which seems to be a pan-adenovirus detection method. For herpesviruses, besides the nested PCR aiming at the detection of the viral DNA polymerase gene, another semi-nested system with three primers complementary to the gene of membrane glycoprotein B (gB) was used. The primers and PCR conditions for the general detection of herpesviruses have been described by VanDevanter et al. (1996). Af-ter amplification, 10 µl from the PCR mixture was analysed on 1% agarose gel containing ethidium bromide, and captured by Kodak camera and gel documen-tation system.

DNA sequencing and sequence analysis

PCR products from positive reactions were purified with QIAquick® Gel Extraction Kit (QUIAGEN) after gel electrophoresis. With the PCR primers, di-rect DNA sequencing was performed using the Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The electrophoresis was done on ABI prism 3100® genetic analyser (Applied Biosystems).

For identification of the nucleotide sequences, the BLASTX program was used on-line at the website (www.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information (NCBI). Multiple alignments with the deduced amino acid sequences and those retrieved from the GenBank were made with the use of the MultAlin program (Corpet, 1988) on-line. Phylogenetic calculations were performed with the programs of the PHYLIP package version 3.6 (Felsen-stein, 1989).

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Results

Gross pathology

Gross pathology revealed medium-level jaundice affecting the mucous membranes (Fig. 1), the subcutis and the thoracic serous membrane. Slight an-thracosis in the lungs was also found. The liver was swollen and showed signs of slight fatty degeneration (Fig. 2). Gastric meteorism and subsequent change in the position of the spleen were considered as likely postmortem alterations.

Fig. 1. Postmortem photograph of the Serotine bat showing signs of moderate icterus by yellow

discoloration of the oral mucous membrane

Histopathology

The histopathological examination of the liver revealed leukocytosis, vacuolar degeneration in the cytoplasm of hepatocytes, and swelling of the cells belonging to the mononuclear phagocyte system (Fig. 3). There was no evidence of typical nuclear or cytoplasmic inclusion bodies in the liver. In the sarcoplasm of a number of heart muscle fibres, vacuolisation was detected. In the lungs, leu-kocytosis, focal alveolar oedema, and mononuclear cell infiltration around some blood vessels were seen (Fig. 4). Desquamative catarrhal enteritis and intestinal



parasites were found in the mucous membranes of the small intestines (Fig. 5). In the spleen, accumulation of a moderate amount of haemosiderin and the signs of lymphocyte depletion were seen. No histological lesions were seen in the other organs examined.

Fig. 2. Major gross pathological findings including yellowish discoloration of the thoracic serous membranes and slightly swollen liver with pale colour pattern characteristic of fatty degeneration

Electron microscopy

Electron microscopy indicated hydropic degeneration and fatty infiltration of groups of liver cells. A number of apoptotic liver cells with a pycnotic nucleus were seen. No clear signs of virus-induced nuclear inclusion bodies were seen in the liver or lung preparations. However, a couple of solitary particles with a morphology consistent with the EM appearance of herpesvirions could be ob-served in the liver sample by this method.

Bacteriology

The bacteriological examination did not reveal the presence of significant pathogenic agents in the liver sample.

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Fig. 3. Histopathological slide of the liver, haematoxylin and eosin stain. Leukocytosis and

vacuolar degeneration in the cytoplasm of hepatocytes. Bar = 30 µm

Fig. 4. Histopathological slide of the lung, haematoxylin and eosin stain. Leukocytosis, focal alveolar oedema and mononuclear cell infiltration around some blood vessels. Bar = 140 µm



Fig. 5. Histopathological slide of the small intestine, haematoxylin and eosin stain. Desquamative

catarrhal enteritis, and intestinal parasites in the mucous membrane. Bar = 120 µm

PCR for virus detection

PCRs aimed at demonstrating the presence of adenoviruses gave negative results. However, a PCR product of the expected size (of approximately 220 base pairs) was obtained after the nested PCR specific for the general detection of herpesviruses. The DNA band corresponding to the amplicon was excised from the gel, then purified and sequenced.

Sequence analysis

The nucleotide sequences from the putative herpesvirus, found in the liver sample, were submitted to the GenBank and assigned accession numbers EU363820 (glycoprotein B gene) and EU363821 (DNA polymerase gene).

With the BLASTX homology search, gammaherpesviruses from the genus Rhadinovirus were identified as the genetically closest relatives of the virus de-tected in the present case. On a phylogenetic tree constructed on the basis of dis-tance matrix calculations with a number of homologous amino acid sequences, retrieved from the GenBank, the putative novel bat virus belonged to the genus Rhadinovirus within the subfamily Gammaherpesvirinae.

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Discussion

The study of viruses occurring in members of the genus Chiroptera (flying foxes and bats) has recently become an important field since the reservoir and transmitter role of these creatures was documented or hypothesised in the spread of a considerable number of important human pathogens such as rabies-related lyssaviruses, henipaviruses, Menangle, Tioman, SARS and Ebola viruses, etc. (Calisher et al., 2006).

Prior to the beginning of our investigations, reports about herpesviral in-fection of Chiropteran species have been based almost exclusively on morpho-logical observations or experimental infections. Reagan et al. (1953) used little brown bats (formerly called cave bats, Myotis lucifugus) for experimental infec-tions with a strain of herpes simplex virus, originally isolated in rabbits. In the course of a large-scale study on the ultrastructure of the salivary gland of Chirop-terans, enlarged acinar cells containing numerous virus particles were encoun-tered in the principal submandibular glands of two out of 34 little brown bats (Myotis lucifugus) (Tandler, 1996). The characteristic morphology of the viral particles, together with the cytomegaly that they induced, led to their identifica-tion as cytomegalovirus.

Due to the highly improved efficiency of PCR-based detection methods, the number of known viruses including gammaherpesviruses increased rapidly in the past decade (Chmielewicz et al., 2003; Pagamjav et al., 2005). Nested PCR systems targeting the viral DNA polymerase gene have been proven exception-ally feasible for the recognition of hitherto unknown new adeno- (Wellehan et al., 2004) and herpesviruses (VanDevanter et al., 1996). In gammaherpesviruses, the gene of glycoprotein B is adjacent to the gene of the DNA polymerase, and a sensitive nested PCR has also been elaborated and published for this latter gene (Chmielewicz et al., 2001). After the acquisition of the partial sequence of the DNA polymerase gene, we performed a successful PCR for the amplification of a fragment from the glycoprotein B gene, too. The nucleotide sequence of this amplicon had just been obtained when a comprehensive study on screening dif-ferent Chiropteran species with the same PCR methods as used by us came out of print (Wibbelt et al., 2007).

As part of a broader study concerning the occurrence of histopathological changes and associated infectious pathogens in European bats, Wibbelt et al. (2007) examined 25 moribund or dead individuals, belonging to 8 bat species and including four Serotine bats, found in Germany. The presence of seven new gammaherpesviruses and one new betaherpesvirus has been demonstrated. The new gammaherpesviruses, named as Bat gammaherpesvirus (BatGHV) 1 to 7, have been found in eight true bat species (Microchiroptera suborder), whereas the new betaherpesvirus (Bat betaherpesvirus 1) has been detected in two spe-cies. Certain bat gammaherpesviruses have also occurred in multiple bat species,



and some bat species and even individuals have been found to harbour more than one herpesvirus types.

Interestingly, only one out of the four Serotine bats examined in Germany has been found positive, and the detected virus (BatGHV-1) had identical nu-cleotide sequence, in both PCR products, with the virus detected by us. The cor-responding region of the phylogenetic tree published by Wibbelt et al. (2007) showed almost identical topology with the tree resulting from our calculations (data not shown), therefore further characterisation, such as joining the two PCR-amplified fragments with a 3000 base pair long PCR fragment, with primers spe-cific to BatGHV-1, was not carried out.

The direct pathogenic role of BatGHV-1 detected by us as a causal agent in the death of the Hungarian Serotine bat could not be confirmed. It is more likely that reactivation from a latent infection was triggered by some idiopathic chronic or subacute disease. Nevertheless, our findings support the recent de-scription of a novel virus, and strongly indicate that Serotine bats are common hosts for this virus type.

Wibbelt et al. (2007) have demonstrated BatGHV-1 in three additional bat species, each of which was also shown to possess at least one more herpesvirus type. Herpesviruses are thought to have a long co-speciation history with their natural host, and are generally host species specific (Davison, 2002). We assume that the original herpesvirus of Serotine bat is BatGHV-1, which is capable of in-fecting other hosts such as Natterer’s bat (Myotis nattereri), Common and Nathusius’ pipistrelles (Pipistrellus pipistrellus and P. nathusii). Confirmation of this hypothesis, however, requires further studies.

Additional PCR investigations are in progress to further explore the diver-sity and host range of herpesviruses occurring in Chiropteran species in Hungary.

Acknowledgements

The authors thank Éva Orbán, Ágnes Mészáros and Zoltán Lajos for their excel-lent technical assistance. Part of this work was supported by the Hungarian Scientific Re-search Fund grant OTKA K 61317 and established by the support (Öveges József Pályázat) of the National Office for Research and Technology (NKTH).

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Detection of a novel bat gammaherpesvirus in Hungary

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