Seal gammaherpesviruses: identification, characterisation andepidemiology

B.E.E. Martina a, G.M.G.M. Verjans b, T.C. Harder c, T. Kuiken a,b,M.W.G. van de Bildt a, P.A.C. van Bergen b, A.D.M.E. Osterhaus a,b,*

a Seal Rehabilitation and Research Centre, Hoofdstraat 94a, 9968 AG Pieterburen, Netherlandsb Institute of Virology, Erasmus MC Rotterdam, Dr Molewaterplein 50, P.O. Box 1738, 3015 GE Rotterdam, Netherlands

c Food, Veterinary and Environmental Diagnostic Laboratory of Schleswig-Holstein, Max-Eyth Str. 5, D-24537 Neumuenster, Germany

Received 5 December 2002; received in revised form 10 April 2003; accepted 12 April 2003

Abstract

Phocid herpesvirus type 2 (PhHV-2), tentatively classified as a gammaherpesvirus, has been isolated from European and

American harbour seals (Phoca vitulina ). Here we describe the isolation and the molecular as well as biological characterisation of

different PhHV-2 isolates from harbour seals and grey seals (Halichoerus grypus ). Of 522 harbour seals and 231 grey seals that had

been admitted to the seal research and rehabilitation centre in Pieterburen, The Netherlands, between 1992 and 2000, 38 and 18%,

respectively, proved to have PhHV-2 neutralising antibodies. PhHV-2 was isolated from peripheral blood mononuclear cells

(PBMCs) of 12 and 28% of these seropositive animals, respectively, and 26 and 56% of these cell samples, respectively, were positive

by PCR analysis. Analysis of amino acid sequences of PCR products and of the growth characteristics of different PhHV-2 isolates

indicated that harbour and grey seals are infected with distinct g-herpesviruses, which however, may co-circulate between the two

species.

# 2003 Published by Elsevier Science B.V.

Keywords: Phocid herpesvirus type 2; Seal gammaherpesviruses; Grey seals; Harbour seals; Replication rate; UL52 sequence

1. Introduction

To date two herpesviruses of seals, phocid herpesvirus

type 1 (PhHV-1) and phocid herpesvirus type 2 (PhHV-

2), have been described (Harder et al., 1996; Osterhaus

et al., 1985). PhHV-2 was first isolated from peripheral

blood mononuclear cells (PBMCs) of a harbour seal

(Phoca vitulina) suffering from severe respiratory dis-

ease (Lebich et al., 1994) and subsequently from

harbour seals in the USA (Harder et al., 1996). Based

on nucleotide sequence data, PhHV-2 has been identi-

fied as a putative member of the gamma (g)-Herpesvir-

inae sub-family (Harder et al., 1996). Limited data are

available about the prevalence of g-herpesvirus infec-

tions in pinnipeds. The prevalence of PhHV-2 specific

antibodies in free-ranging marine mammals along the

coasts of Alaska and Russia varied between 17 and 42%,

with the highest prevalence among harbour seals

(Zarnke et al., 1997). Grey seals (Halichorus grypus )

were not included in that study. The present paper

describes the isolation, partial genetic and biological

characterisation of g-herpesviruses of harbour seals (PV)

and grey seals (HG) admitted to the Seal Research andRehabilitation Centre (SRRC) in Pieterburen the Neth-

erlands, from 1992 to 2000. In addition, the prevalence

of PhHV-2 neutralising serum antibodies in these

animals is presented.

2. Materials and methods

2.1. Virus isolation

In December of 1999, a sick harbour seal (PV 9912)

with severe respiratory illness found at the coast of

France, south of the Seine mouth, was admitted to the

* Corresponding author. Tel.: �/31-10-408-8066/8067; fax: �/31-10-

408-9485.

E-mail address: a.osterhaus@erasmusmc.nl (A.D.M.E. Osterhaus).

Virus Research 94 (2003) 25�/31

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Dutch Seal Research and Rehabilitation centre at

Pieterburen (SSRC; The Netherlands) where it died

with a pneumonia. Approximately 500 ml pleural

effusion as well as different organs was collected forroutine virological investigations. A lung homogenate

(10%) was made and inoculated on Crandell Rees feline

kidney (CrFK), Vero, and seal kidney (SeKC; from PV

origin) cultures. The pleural fluid was centrifuged for 10

min at 1000�/g and the supernatant was inoculated on

CrFK, SeKC and concanavalin A (ConA, Sigma, St.

Louis, CA) stimulated PV PBMCs. Cultures displaying

cytopathic effect (CPE) were analysed by negativecontrast electron microscopy. Further confirmation for

the isolation of a herpesvirus was achieved by perfor-

mance of immunofluorescence (IFA), using a panel of

discriminatory monoclonal antibodies (moAb) raised

against PhHV-1 strain PB 84 (2F2, 1H3) and PhHV-2

strain 7848 (3E5, 6C5 and 7C8) (Harder et al., 1996).

PBMC, isolated from heparinised blood samples of

different PV and HG admitted to the SSRC as describedpreviously (de Swart et al., 1993), were stored at �/

135 8C. In order to isolate virus from PBMC of seals,

PBMC were cultured in RPMI 1640 medium (BioWhit-

taker, Belgium) supplemented with antibiotics and 5%

heat inactivated foetal bovine serum (FBS), or stimu-

lated with 5 mg/ml ConA, or alternatively with a mixture

of 20 ng/ml phorbol myristate acetate (PMA; Sigma�/

Aldrich), 30 mM n-butyrate (Sigma�/Aldrich) and 1 mg/ml cyclosporin A (FK506; Sigma�/Aldrich) (PB-FK).

Fourteen days after stimulation, 106 cells were co-

cultivated with CrFK and SeKC monolayer cultures

for 6 weeks in Dulbecco’s Modified Eagle’s Medium

(DMEM) containing 5% FBS. Cultures that did not

show CPE were confirmed as being PhHV-2 negative by

IFA using the moAb 6C5 and by a PhHV-2-specific

PCR as described below.

2.2. PhHV-2-specific polymerase chain reaction (PCR)

DNA was extracted with silica from PBMC samples

used for virus isolation as described previously (Boom et

al., 1990). The primers used for the PhHV-2-specific

PCR, based on the PhHV-2 strain 7848 UL52 gene,

were: 5?-TGTTCAGATGCCAGTTCC-3? (forward pri-mer) and 5?-TTCATCCAAGTCCCACTC-3? (reverse

primer). PCR reactions were performed for 40 cycles

comprising 30 s at 94 8C, 1 min at 55 8C and 1 min at

72 8C. A final elongation phase of 10 min at 72 8C was

allowed. PCR products were size-fractionated on 2%

agarose gel, blotted onto a hybond N�/ membrane

(Amersham, Stad, UK) and subsequently hybridised

with a PhHV-2-specific biotin-conjugated probe (5?-CTTGGTGTGAAAGGTCTCACTC-3?) and visualised

using chemiluminescence. The PhHV-2 strain 7848 and

the PhHV-1 strain PB84 (Harder et al., 1996) or

uninfected cell cultures, were used as positive and

negative control samples, respectively.

2.3. Sequence analyses of UL52 amplicons of different

PhHV-2 isolates

The UL52-specific amplicons, purified from gel using

the Qiagene kit (Westburg, Stad, Belgium), were

sequenced directly with the aforementioned PCR pri-

mers on a 373A-automated sequencer (Applied Biosys-

tems, Stad, Land) according to the manufacturer’s

instructions. The UL52 DNA and corresponding pro-tein sequences were aligned with ‘‘CLUSTALW’’ (Bioedit

software package). Phylogenetic trees were constructed

using the ‘‘DNAML’’ software program of ‘‘PHYLIP’’

(version 3.5, department of Genetics, University of

Washington, State) using 100 bootstraps. The ‘‘CON-

SENSE’’ package of the program was used to generate the

tree.

2.4. Serological analyses

2.4.1. Serum samples

A serum panel of 753 seals, collected from 522 PV and

231 HG between 1992 and 2000 in the Dutch Wadden

Sea, was used to determine the prevalence of PhHV-2

neutralising serum antibodies in seals admitted to the

SRRC. All sera were taken approximately 6�/8 weeks

after admission to the SRRC.Sequential serum samples were available from 80

PhHV-2 seropositive PVs and 42 PhHV-2 seropositive

HG (see below) starting with a sample obtained at the

day of admission and ending with samples just before

release (12�/24 weeks after admission). Serial titration

analyses of these samples were performed to discrimi-

nate between maternal antibodies, past, or active PhHV-

2 infection. Animals PhHV-2 seropositive at admissionthat became seronegative during rehabilitation were

considered to have maternal antibodies. Active infection

during rehabilitation was defined by a 4-fold increase in

neutralisation antibody titres.

2.4.2. PhHV-2-specific serum IgG ELISA

The PhHV-2 strains PV 7848 and PV 9912 were used

to develop a whole virus-based ELISA system to detectserum IgG against PhHV-2. Both virus strains were

propagated in CrFK cells at a moi of 10�2. When more

than 90% of the monolayer showed CPE, cells and

supernatants were harvested and freeze-thawed twice.

Virus was purified by ultra-centrifugation through a

sucrose gradient. Virus containing bands were collected,

dialysed, pooled, and treated with 4% (w/v) Mega-10

(Sigma). The preparation was sonicated twice for 15 s at9 mM. Microtitre plates (96-well flatbottom) with high

protein binding capacities (Costar, USA) were coated

with 50 ng/well viral proteins for 1 h at 37 8C. Sera were

B.E.E. Martina et al. / Virus Research 94 (2003) 25�/3126

tested in a single dilution of 1:30. Further processing

was by means of a protein-A ELISA as previously

described (Martina et al., 2001).

2.4.3. Virus neutralisation assay

A virus neutralisation (VN) assay was performed as

previously described (Martina et al., 2001). Briefly, four

2-fold serum dilutions (starting dilution 1:20) were made

and incubated with 100 TCID50 of PhHV-2 strains PV

7848 or PV 9912. In some cases higher dilutions were

tested. Neutralising titres were determined after 14 days

microscopically on the basis of suppression of CPE.Titres were expressed as the reciprocal of the highest

dilution still giving 100% suppression of CPE.

3. Results

3.1. Isolation of a g-herpesvirus like virus from a harbour

seal

Four to six weeks after inoculation of lung homo-

genate and pleural fluid, obtained from the diseased

harbour seal (PV 9912), in CrFK, Vero, and SeKC

cultures, plaques of rounded and syncytial cells were

observed. The pleural fluid was also inoculated in PV-

PBMC cultures. Approximately 5 weeks after inocula-

tion, ballooning and extensive cell death was observed in

PBMC. Examination of supernatant from these cell-

cultures by negative contrast EM revealed the presence

herpesvirus like particles (results not shown). IFA

performed with a panel of phocine herpesvirus-specific

moAbs showed that the isolate did not react with the

PhHV-1-specific moAb 1H3, whereas a strong fluores-

cence was seen on viral infected cells with the PhHV-2-

specific moAbs 6C5 and 7C8. Interestingly, moAb 3E5,

generated against PhHV-2 strain PV 7848, did not

recognise cells infected with the PhHV-2 like strain PV

9912. These results indicated that the isolate is a PhHV-

2 like virus and is antigenically distinct from the PhHV-

2 strain PV 7848. To further confirm that PV 9912 was a

Table 1

Seals with g-herpesvirus (PhHV-2) specific serum antibodies or PhHV-

2 infection in the Netherlands between 1992 and 2000

Species Protein-A ELISA

(%)a

PV 7848 VN

(%)

PV 9912 VN

(%)

(a ) PhHV-2 seropositive seals

Harbour seal 208/522 (40) 198/522 (38) 154/522 (30)

Grey seal 74/231 (32) 34/231 (15) 42/231 (18)

(b ) PhHV-2 in PBMC

Virus isolation

(%)

PCR analysis (%)

6/50 (12) 13/50 (26)

5/18 (28) 10/18 (56)

a ELISA was based on an antigen mixture from both isolates.

Table 2

Age and clinical signs of PhHV-2 infected sealsa

Isolate Age (month) Clinical signs Virus isolation (yes/no)

PV 1 3 Cough and nasal discharge Yes

PV 2 6 Healthy Yes

PV 3 4 Nasal discharge No

PV 4 5 Severe respiratory disease Yes

PV 5 6 Data not available No

PV 6 12 Respiratory disease Yes

PV 7 8 Healthy Yes

PV 8 4 Data not available No

PV 9 3 Healthy No

PV 10 8 Healthy No

PV 11 5 Lethargy No

PV 12 5 Healthy No

PV 13 4 Data not available Yes

HG 1 1 Healthy Yes

HG 2 3 Cough Yes

HG 3 2 Healthy No

HG 4 2 Data not available No

HG 5 1 Cough Yes

HG 6 B/1 Healthy No

HG 7 B/1 Healthy Yes

HG 8 B/1 Epistaxis and coughing No

HG 9 3 Healthy No

HG 10 3 Data not available Yes

PV 7848 �/12 Moribund seal with pneumonia Yes

PV 9912 6 Pneumonia Yes

a Infection defined as PBMC positive in UL52 PCR.

B.E.E. Martina et al. / Virus Research 94 (2003) 25�/31 27

PhHV-2 like virus, PCR analysis was carried out using

PhHV-2 specific primers. An amplicon of the expected

size was obtained and was recognised by UL52-specific

probe, indicating that PV 9912 was indeed a PhHV-2

like virus.

3.2. Seroprevalence of PhHV-2-like viruses in seals

The prevalence of serum antibodies reacting with

PhHV-2 were determined by using a protein-A basedELISA system. In 208 out of 522 PV (40%) and 74 out

of 231 HG (32%) sera were positive when tested against

an antigenic protein preparation of both isolates (Table

1a). To determine whether seroprevalences against the

PhHV-2 strains PV 7848 and PV 9912 differed between

the seal populations, all sera were subjected to VN

assays using the respective viruses. Among the 522 PV

sera, 198 (38%) and 154 (30%) had VN antibodies (titre]/20) against PhHV-2 strain PV 7848 and PV 9912,

respectively (Table 1a). The 44 PV that did not

neutralise PV 9912 all had a PV 7848-specific VN titre

of 20. Among the 231 HG sera, 34 (15%) and 42 (18%)

neutralised PV 7848 and PV 9912, respectively (Table

1a). The 8 HG sera, not neutralising PV 7848, had a PV

9912-specific VN titre of 40. Overall, serum VN titresranged from 20 to 80 in both seal populations. These

sera were all obtained 6�/8 weeks post-admission. To

investigate whether the observed antibodies could have

been from maternal origin, serial VN assays performed

on sequential serum samples of PhHV-2 seropositive

animals showed that 43% (34 out of 80) of PV and 88%

(37 out of 42) of HG seal sera were positive on the day

of admission. Analysis of sequentially obtained sera ofthe same animals indicated that seven PV (21%) and five

HG (14%) had maternal antibodies rather than a past or

active PhHV-2 infection, as shown by disappearance of

antibodies 8�/10 weeks after admission. Based on a more

than 4-fold antibody titre rise, active infection was

detected in ten out of 26 (38%) PV and in 12 out of 32

(38%) HG tested, with neutralising titres rising from 20

up to 320.

3.3. Isolation and genetic characterisation of PhHV-2-

like viruses in seals

PhHV-2 has tentatively been classified as a lympho-

tropic g-herpesvirus (Harder et al., 1996). Therefore,

PBMC from 50 PV and 18 HG seals seropositive at

admission were cultured in presence of ConA, PB-FK,or plain culture medium. Viruses were isolated from

cultured PBMC, obtained from six PV (12%) (PV isolate

1, 2, 4, 6, 7, 13) and five HG (28%) (HG isolate 1, 2, 5, 7,

10). All these seals showed serological evidence of active

infection (see above). The HG 7 isolate was recovered

from PBMC cultured in PB-FK. No virus was recovered

after ConA stimulation of seal PBMC fractions. Cul-

tures of the PBMC samples that did not show CPE after3 weeks maintenance were co-cultivated for 6 weeks

with CrFK and SeK cells. IFA analyses conducted on

these cells showed no evidence for virus production in

these PBMC cultures.

However, when PCR was performed directly on all

unstimulated PBMC, using a PhHV-2 UL52-specific

PCR, 13 (26%) PV and 10 (56%) HG were found

positive (Table 1b). All PBMC from which virus wasisolated proved to be positive in this PCR. All UL52

PCR positive samples, including the reference PhHV-2

isolates PV 9912 and PV 7848 (see Table 2), were

sequenced and a phylogenetic tree was constructed. The

phylogenetic relationship between the 430 nucleotides of

the different UL52 amplicons was determined on

aligned sequences and maximum likelihood analysis

was performed. Four clusters of viruses were distin-guishable (Fig. 1). When the deduced amino acid

sequences were compared, two isolates were identical

to PV 7848, 11 were identical to PV 9912, three identical

Fig. 1. Unrooted phylogenetic tree of UL52 equivalent gene sequences

of different PhHV-2 isolates. Nucleotide sequences (430 bp long) were

aligned and subjected to bootstrapped (n�/100) maximum likelihood

analysis using the PHYLIP package. The consensus tree is shown with

bootstrap values indicated at tree branchings.

B.E.E. Martina et al. / Virus Research 94 (2003) 25�/3128

to HG 7 and seven had a sequence identical to that of

PV 13 (Figs. 1 and 2).

3.4. Growth characteristics of different isolates

CrFK cells infected with HG like viruses showed large

syncytial cells and ballooning, whereas slow-replicating

viruses induced plaques of rounded cells but did not

result in large syncytia formation and ballooning.

Interestingly, the isolates PV 2, 6, and 9912 (all isolates

recovered from harbour seal PBMC) had the same

growth characteristics and induced the same cytopathic

changes as the HG viruses, in agreement with their

genetic similarity to the HG viruses.

Fig. 2. Nucleotide (a) and deduced amino acid (b) sequence alignment of UL52 PCR fragments of different PhHV-2 isolates. Alignment was

performed with the CLUSTALW program of bioedit software package. Identical amino acids are represented on dark background and boxes indicate

similar amino acids (BLOSUM62 matrix).

B.E.E. Martina et al. / Virus Research 94 (2003) 25�/31 29

4. Discussion

In the present paper, we described the isolation and

characterisation of a series of g-herpesviruses (PhHV-2)

recovered from both harbour and grey seals. We showed

that both species could be infected with similar g-

herpesviruses. However, PV isolates differ both biologi-

cally (replication kinetics (results not shown) and

induction of cytopathic changes) and genetically from

the HG isolates.

PhHV-2-specific serum antibody prevalence for

PhHV-2 in harbour seals from the Dutch Wadden Sea

was similar to that previously described for PV along the

coast of Alaska and Russia (Zarnke et al., 1997). Virus

isolation and UL52 sequence data suggest that PV 7848

is more prevalent within the PV group, whereas the PV

9912-like isolates seem to be more prevalent within grey

seals. Therefore, it is tempting to speculate that the

natural host for PV 7848-like viruses is the harbour seal,

whereas grey seals are the natural host for PV 9912-like

viruses. The most likely explanation for the presence of

HG-like viruses in harbour seals would be cross-species

transmission. The observation that identical UL52

sequences were detected among the virus isolates from

different HG as well as from some PV, kept at the SSRC

during different time periods, suggests that PV 9912 is a

stable genotype circulating preferentially in HG. In

general, HG like viruses were shown to replicate at a

faster rate (average replication rate of 5 h) than the PV

like isolates (results not shown). However, no correla-

tion between virus isolate phenotype (slow vs. fast

replicating) and clinical disease was observed (Table

2). This lack of correlation may be explained by the

small amount of harbour seals that were infected with

the ‘‘fast replicating’’ isolates. In addition, most of the

seals investigated in this study had immunity against the

seal alphaherpesvirus PhHV-1, which has been shown to

cross-react with PhHV-2 at antibody level and possibly

also on cellular immunity level (Harder et al., 1996).

Although there was no correlation between in vitro

growth kinetics and phylogenetic clusters based on the

UL52 nucleotide sequences, phylogenetic analyses of the

amino acid sequences of the different isolates resulted in

two clusters of viruses that correlates with their in vitro

growth kinetics (not shown).

While no clear disease association has been found in

seals naturally infected with PhHV-2 viruses, circum-

stantial evidence suggests that the virus might be linked

to respiratory disease (Table 2). However, these symp-

toms might also be attributable to lung worm infections,

which are a common cause of respiratory disease in

harbour seals (Measures, 2001). It is interesting to note

that replication of PhHV-2 is highly cytolytic for seal

PBMC(s) in vitro. Although the leukocyte target

population(s) of PhHV-2 remain to be elucidated, the

cytolytic activity of PhHV-2 in seal PBMCs in vitro,

may point towards immunosuppressive effects in pro-

ductively infected seals that would predispose them for

secondary infections.

Further studies are needed to determine the suscep-

tible cell types of PhHV-2 in their natural host and its

role in the development of respiratory disease. As for the

bovine g-herpesvirus BHV-4 (Egyed, 1998), PhHV-2

also had an extended tropism for cell lines of other

mammalian species (CrFK and Vero). Therefore, the

possibility of interspecies transmission beyond seals

should be considered. In addition, some g-herpesviruses

have been associated with development of lymphopro-

liferative disorders or other forms of cancer in their

immunocompromised host. Since PhHV-2 has been

shown to group with members of the g-herpesviruses,

it would be important to know if PhHV-2 also has an

oncogenic potential.

Fig. 2 (Continued)

B.E.E. Martina et al. / Virus Research 94 (2003) 25�/3130

Acknowledgements

We thank Dr Ron Fouchier helpful suggestions and

Theo Bestebroer for technical assistance.

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