Novel rat models to study primary genital herpes simplex virus-2 infection

ORIGINAL ARTICLE

Novel rat models to study primary genital herpes simplex virus-2infection

Karin Onnheim • Maria Ekblad • Staffan Gorander •

Stefan Lange • Eva Jennische • Tomas Bergstrom •

Sheryl Wildt • Jan-Ake Liljeqvist

Received: 3 October 2014 / Accepted: 9 February 2015 / Published online: 21 February 2015

� Springer-Verlag Wien 2015

Abstract In this study we describe that six rat models

(SD, WIST, LEW, BN, F344 and DA) are susceptible to

intravaginal herpes simplex virus-2 (HSV-2) infection after

pre-treatment with progesterone. At a virus dose of

5 9 106 PFU of HSV-2, all rat models were infected pre-

senting anti-HSV-2 antibodies, infectious virus in vaginal

washes, and HSV-2 DNA genome copies in lumbosacral

dorsal root ganglia and the spinal cord. Most of the LEW,

BN, F344, and DA rats succumbed in systemic progressive

symptoms at day 8-14 post infection, but presented no or

mild genital inflammation while SD and WIST rats were

mostly infected asymptomatically. Infected SD rats did not

reactivate HSV-2 spontaneously or after cortisone treat-

ment. In an HSV-2 virus dose reduction study, F344 rats

were shown to be most susceptible. We also investigated

whether an attenuated HSV-1 strain (KOS321) given

intravaginally, could protect from a subsequent HSV-2

infection. All LEW, BN, and F344 rats survived a primary

HSV-1 infection and no neuronal infection was established.

In BN and F344 rats, anti-HSV-1 antibodies were readily

detected while LEW rats were seronegative. In contrast to

naıve LEW, BN, and F344 rats where only 3 of 18 animals

survived 5 9 106 PFU of HSV-2, 23 of 25 previously

HSV-1 infected rats survived a challenge with HSV-2. The

described models provide a new approach to investigate

protective effects of anti-viral microbicides and vaccine

candidates, as well as to study asymptomatic primary

genital HSV-2 infection.

Introduction

The species herpes simplex virus-2 (HSV-2) belongs to the

Herpesviridae family, genus Simplexvirus. HSV-2 infects

the genital mucosa and establishes latency into the sensory

lumbosacral dorsal root ganglia (DRG). Frequent reacti-

vation may induce clinical disease with genital lesions or,

more often, asymptomatic shedding of HSV-2 [1, 2]. HSV-

2 is a common sexually transmitted virus and more than

500 million persons are infected globally [3]. A

K. Onnheim � M. Ekblad � S. Gorander � T. Bergstrom �J.-A. Liljeqvist (&)

Section of Virology, Department of Infectious Medicine,

Institute of Biomedicine, Sahlgrenska Academy at University of

Gothenburg, Gothenburg, Sweden

e-mail: [email protected]

K. Onnheim


M. Ekblad


S. Gorander


T. Bergstrom


S. Lange

Section of Bacteriology, Department of Infectious Medicine,

Institute of Biomedicine, Sahlgrenska Academy at University

of Gothenburg, Gothenburg, Sweden


E. Jennische

Department of Medical Biochemistry and Cell Biology,

Institute of Biomedicine, Sahlgrenska Academy at University

of Gothenburg, Gothenburg, Sweden


S. Wildt

Harlan Laboratories, 8520 Allison Pointe Boulevard,

Suite 400, Indianapolis, IN 46250, USA


123

Arch Virol (2015) 160:1153–1161

DOI 10.1007/s00705-015-2365-7

prophylactic vaccine that can reduce the spread of HSV-2

addresses the increased risk for HSV-2 infected individuals

to also acquire an HIV infection [4]. Earlier clinical vac-

cine trials, based on the HSV-2 proteins gB-2 and/or gD-2,

have failed to show protective effects against HSV-2

infection or disease [5, 6]. The clinical studies were initi-

ated after promising results in extensive preclinical eva-

luations using the genital mouse and guinea pig models.

For several years, the genital model of young mice

has been the first alternative to study effects of pro-

phylactic HSV-2 vaccine candidates. Advantages to this

model include low costs of animals and housing, a

variety of inbred and genetic knock-out strains, and a

broad range of immunological reagents. Limitations are

that the murine genital mucosa is, in contrast the female

mucosa, susceptible to HSV-2 infection only in dies-

trous phase. Synchronization of the mucosa therefore

requires progesterone treatment before infection or

challenge [7]. Moreover, the mouse model cannot be

used to study latency and spontaneous reactivation and

genital shedding.

Guinea pigs are frequently used as an alternative

model to evaluate prophylactic and therapeutic HSV-2

vaccine candidates. This model has several advantages as

compared with the mouse model. For example, the gen-

ital mucosa can be infected with HSV-2 without pro-

gesterone treatment, and HSV-2 can establish latency in

lumbosacral DRG, reactivate and induce recurrent dis-

ease, but also shed virus asymptomatically [8]. The gui-

nea pig model is also useful to study the effect of HSV-2

vaccine candidates in already HSV-1 infected animals [9,

10]. This is an advantage as the model recapitulates the

human infection in that HSV-1 often infects early in life

followed by a subsequent HSV-2 infection. The draw-

backs with the model are the scarcity of inbred and

genetically modified animals, and available immunologi-

cal reagents.

Rats, like mice, belong to the Muridae family. In the first

two to three weeks of age, HSV-1 and HSV-2 are equally

virulent after intracranial inoculation, while in adult Spra-

gue Dawley (SD) rats HSV-1 is more virulent than HSV-2

[11]. The susceptibility of HSV-1 induced acute encepha-

litis differs significantly depending on which rat model is

used. For example, Dark Agouti (DA) and Lewis (LEW)

rats develop a lethal CNS disease while the Piebald Virol

Glaxo rat model is resistant after peripheral inoculation of

virus [12, 13]. To our knowledge no information is avail-

able whether laboratory rats are susceptible to genital

HSV-2 infection. We here describe six rat models that are

susceptible to genital HSV-2 infection after progesterone

treatment and that two of the models develop mostly an

asymptomatic primary infection even at a high infective

dose of HSV-2.

Materials and methods

Cells and viruses

African green monkey kidney cells (GMK AH1) were

cultured in Eagle’s minimal essential medium supple-

mented with 2 % calf serum and antibiotics (penicillin and

streptomycin). The HSV-1 KOS321 strain with a defect in

neural spread [14], and the wild type HSV-2 333 strain

were used for intravaginal (i.vag.) infection or challenge.

Rat models of genital infection

Female 6 to 8 weeks old Hsd:Sprague Dawley� SD� (SD),

RccHan:WIST (WIST), LEW/HanHsd (LEW), BN/SsNO-

laHsd (BN), F344/NHsd (F344), and DA/OlaHsd (DA) rats

were purchased from Harlan Laboratories, Inc., Nether-

lands. A rat ‘‘strain’’ includes inbred animals and a rat

‘‘stock’’ includes outbred animals. In this study we use rat

‘‘models’’ which include both inbred and outbred rats. The

susceptibility of the genital mucosa to HSV-2 infection was

initially evaluated without pre-treatment with progesterone

by i.vag. installation with 1 9 106 PFU of HSV-2. In the

following experiments, a subcutaneous injection of 3 mg

Depo-Provera (Pfizer) was given seven days before infec-

tion/challenge with HSV-1 and/or HSV-2. To define when

maximal replication of virus occurred after i.vag. HSV-2

infection, vaginal washes were collected at different time

points and virus doses in SD, LEW, BN, and F344 rats.

Vaginal secretions were collected by gently washing the

vagina with 100 ll Hank’s medium ten times. This pro-

cedure was repeated once and the washes were pooled in a

total volume of 1 mL medium.

Survival and genital and systemic disease were followed

21 days post infection and graded as described earlier for

mice [15]: healthy (score, 0), genital erythema (score, 1),

moderate genital inflammation with blisters (score, 2),

severe and purulent genital lesions with loss of hair (score,

3), and hind-limb paralysis and/or general bad condition

(score, 4). The rats were anesthetized with isoflurane

(Baxter) for all interventions. The animal studies were

approved by the ethical board in Gothenburg.

Genital HSV-2 infection

HSV-2 was installed i.vag. with 5 9 106 PFU of HSV-2 in

50 ll Hank’s medium. Survival and disease progression

were followed, and vaginal washes were collected at day 2

post infection. Blood and lumbosacral DRG and entire

spinal cords were prepared when rats were euthanized due

to severe symptoms at day 8-14 post infection, or for

asymptomatically infected rats, at day 21 post infection

when the scoring was completed. In an HSV-2 virus-dose

1154 K. Onnheim et al.

123

titration study, SD, LEW, BN, and F344 rats were infected

with virus doses with 1 9 102 or 1 9 103 to 1 9 106 PFU

of HSV-2. The outcome was followed and vaginal washes

and blood were collected.

Genital HSV-1 infection followed by a subsequent

HSV-2 infection

One group each of LEW, BN, and F344 rats were infected

i.vag. with 1 9 106 of HSV-1, and another group of ani-

mals were infected i.vag. with 5 9 106 PFU of HSV-1.

Vaginal washes were collected at day 2 post infection and

serum was collected at day 21 after HSV-1 infection for

detection of anti-HSV-1 IgG-antibodies. One month later

the rats were challenged i.vag. with 5 9 106 PFU of HSV-

2. At day 28 after HSV-2 infection, or when animals were

euthanized, blood was collected for detection of anti-HSV-

2 IgG-antibodies. One month after the HSV-2 infection, or

when animals were euthanized, lumbosacral DRG and the

spinal cords were prepared.

Plaque assay

Infectious HSV-1 and HSV-2 (PFU) were estimated in the

vaginal washes by a plaque assay as described earlier [15].

The detection limit was 20 PFU/ml.

Detection of antibodies in serum in ELISA

Sodium deoxycholate-solubilized HSV-1-infected cell

membrane antigen [16] was coated overnight in carbonate

buffer (pH 9.6) on Maxisorp 96 well plates for detection of

HSV-1 or type-common HSV-2 antibodies. Helix pomatia

lectin-purified mgG-2 antigen was used for detection of

type-specific HSV-2 antibodies (17). The antibody titer

was defined as the reciprocal value of the highest serum

dilution giving an optical density (OD) greater than nega-

tive rat sera plus 0.2 OD units. An end-point titer\50 was

considered as negative.

Quantification of HSV-1 and HSV-2 DNA genome

copies in neuronal tissue (qPCR)

HSV DNA was purified from tissue samples using Mag-

naLyser kit, DNA purification kit and Magnapure LC

Robot (Roche) according to manufacturers protocol. The

amount of HSV DNA in the samples was quantified by

Real-time PCR in an ABI Prism 7000 Sequence Detection

System (Applied Biosystems). Type-discriminating pri-

mers and probes, amplifying a segment of the HSV-1 or

HSV-2 gB gene, are described elsewhere [18]. Standard

curves were based on dilutions of a plasmid containing the

gB gene. The spinal cord homogenates were diluted 1:4 in

phosphate-buffered saline (PBS) before extraction of HSV

DNA due to inhibition of the PCR reaction at high cellular

DNA content. Three lumbosacral ganglia were pooled in

PBS. Calculated from the standard curves and the dilution

factors the detection limits were defined to be 50 HSV-1 or

HSV-2 DNA genome copies per ganglion and 600 copies

per entire spinal cord.

Immunohistochemistry of mucosa and neurons

In total, four SD and four F344 rats were infected i.vag.

with 1 9 106 PFU of HSV-2 in two separate experiments.

The SD rat model was selected as they mostly presented an

asymptomatic infection, and the F344 rat model as they

mostly presented a symptomatic infection. At day 3 or at

day 6 post infection (two animals of each model per day),

the animals were deeply anesthetized with isoflurane, and

fixed by transcardial perfusion via the left ventricle with

PBS followed by 4 % formaldehyde. The time points were

selected based on the genital mouse model and the

assumption that maximal replication of HSV-2 precedes

detection of HSV-2 antigen in the vagina (day 3 post

infection) and resolution of genital infection is followed by

spread of HSV-2 to lumbosacral DRG and the spinal cord

(day 6 post infection).

At day 3 post infection vaginas were removed, and at

day 6 post infection vaginas and DRG as well as spinal

cords were collected followed by immersion fixation for

24 h. The entire spinal cords including the DRG were

decalcified, using 10 % EDTA in 0.2 M Tris buffer, pH

7.4, for four weeks. After decalcification, 3-4 mm thick

horizontal slices were cut from the lumbosacral and thor-

acic levels. The samples were dehydrated and embedded in

paraffin using a Leica TP1020 Automatic Tissue Processer.

Sections cut at 4 lm were prepared. After high temperature

antigen retrieval, using a 0.01 M citrate buffer, pH 6.0, the

sections were incubated separately with anti-HSV-2 immune

serum from antibodies-online.com (ABIN387093). Anti-

rabbit Impress Reagent HRP (Vector laboratories) was

used as conjugate and the immunoreactions were visua-

lized using liquid DAB? substrate (DAKO). Nuclei were

counter-stained with hematoxylin. Finally, the sections

were dehydrated and mounted using DPX (Merck). A Zeiss

Axio Image M1 microscope and AxioVision software was

used for documentation.

Reactivation of HSV-2

To investigate whether HSV-2 can reactivate and shed

virus after a primary genital infection, five SD rats were

infected with 1 9 106 PFU of HSV-2. Vaginal washes

were analyzed at day 2 post infection and serum was col-

lected at day 21 post infection to establish that the animals

Rat models to study genital HSV-2 infection 1155

123

were infected. After one month, vaginal washes were col-

lected 3 days per week for 4 weeks, in total 12 samples per

rat. After this time-period betamethasone, at a dose of

4 mg/kg, was administrated intra-muscularly in an attempt

to induce reactivation. Vaginal washes were sampled for

another 2 week period where after lumbosacral DRG and

the spinal cords were collected. Vaginal washes were

analyzed for infectious virus (PFU) and for HSV-2 DNA.

The detection limit was 100 HSV-2 DNA genome copies

per vaginal sample (1 mL). Neuronal tissue was analyzed

for HSV-2 DNA.

Statistics

Two-tailed Fisher’s exact test was used for survival data.

The Mann-Whitney Rank Sum test was used to compare

HSV-2 DNA genome copies between different models. P

values of \0.05 were considered statistically significant.

Results

Rats are susceptible to intravaginal HSV-2 infection

after progesterone treatment

Initially we investigated whether genital infection was

possible without hormone treatment. All six rat models

survived an i.vag. installation with 1 9 106 PFU of HSV-2

and showed no genital or systemic symptoms. No infec-

tious virus was detected in vaginal secretions and no anti-

HSV-2 IgG-antibodies were detected in serum (data not

shown). We conclude that the vaginal mucosa in rats is

only susceptible to HSV-2 infection after progesterone

treatment (Depo-Provera).

To define an optimal time point to detect infectious

HSV-2 in vaginal washes, SD, LEW, BN, and F344 rats

were infected with 1 9 103 to 1 9 106 PFU. Vaginal

secretions were collected at 12 h and daily between day 1

to day 6 post infection. Using the highest virus doses of

1 9 106 or 1 9 105 PFU, the peak titers were detected

between day 1-2 post infection, and using the lower virus

doses of 1 9 104 or 1 9 103 PFU at day 2-3 post infection.

Most vaginal washes were negative at 12 h post infection.

When HSV-2 was detected in the vaginal washes at 12 h

post infection, infectious HSV-2 (PFU) was significantly

lower as compared with day 1 or day 2 post infection

indicating that replicated HSV-2 was measured and not

solely inoculated virus (data not shown). We decided to

collect the vaginal washes at day 2 post infection.

One week after treatment with Depo-Provera, all rat

models were susceptible to i.vag. infection with 5 9 106

PFU of HSV-2, (Table 1). Anti-HSV-2 antibodies in

serum, infectious virus in vaginal washes, and HSV-2 DNA

genome copies in lumbosacral DRG and spinal cord were

detected in all rats. For the SD rats 6 of 8 survived 5 9 106

PFU of HSV-2. Although few animals were investigated,

we observed obvious differences in HSV-2 DNA genome

copies in lumbosacral DRG (D 3.2 9 103) and in the spinal

cord (D 4.1 9 104) between asymptomatic surviving SD

rats as compared with symptomatic non-survivors. For the

WIST rats 4 of 6 survived 5 9 106 PFU of HSV-2, for the

LEW rats 1 of 6 survived, for the F344 rats none of the

animals survived, and for the BN and the DA rats 2 of 6

survived (Table 1). For all models, the symptoms of non-

surviving rats were similar and described as follows; no or

a mild genital inflammation (erythema) was visible around

introitus at day 8-10 post infection. In contrast to the

genital mouse model, genital inflammation in the rats did

not develop further into disease scores 2 or 3. There after

the rats developed progressive systemic symptoms such as

ruffled fur, hunched posture, swollen abdomen, and hind-

limb paralysis in some animals (disease score 4). At this

stage of disease, the animals were immediately euthanized.

At autopsy fecal and urine retention were observed as well

as swollen and inflamed appendix. For surviving rats, no

genital or systemic symptoms were observed.

HSV-2 virus-dose titration

To further evaluate the susceptibility after i.vag. HSV-2

infection, SD, LEW, BN, and F344 rats were infected with

virus doses of 1 9 102 or 1 9 103 to 1 9 106 PFU of

HSV-2. The SD rats were mostly infected asymptomati-

cally and 13 of 15 rats survived 1 9 105 or 1 9 106 PFU of

HSV-2 (Fig. 1A). The SD rats presented infectious HSV-2

in vaginal washes and anti-HSV-2 antibodies at the lowest

dose of 1 9 104 PFU (Fig. 1 A-B). For the LEW rat model,

2 of 9 rats survived 1 9 105 or 1 9 106 PFU of HSV-2.

These rats presented the highest mean values of PFU/

sample, while low levels of anti-HSV-2 antibodies were

detected. For the BN rat model, 5 of 8 rats survived

1 9 105 or 1 9 106 PFU of HSV-2. Infectious virus in

vaginal washes and anti-HSV-2 antibodies were detected at

doses of 1 9 103 to 1 9 106 PFU. The F344 rat model was

most susceptible and could control the infection only when

1 9 102 or 1 9 103 PFU of HSV-2 was given.

Immunohistochemistry of genital mucosa and neurons

after HSV-2 infection

SD rats and F344 rats were infected i.vag. with 1 9 106

PFU of HSV-2 in two separate experiments. Vagina,

lumbosacral DRG and spinal cords were prepared for im-

munohistochemistry at day 3 or day 6 post infection.

Similarly as described for the mouse genital mucosa [7],

the progesterone treatment induced usually a thin (3-7


123

layers) stratified epithelium including stratum basale and

stratum spinosum [19]. However, in the SD and F344 rat

models the epithelial cells close to introitus were often

arranged in 20 - 30 cell layers.

In F344 rats, at day 3 post infection in a first experiment,

HSV-2 antigen was detected close to the introitus in fused

epithelial cells localized at the apical cell surface with

spread to the basal cell layers (Fig. 2A). This is in accor-

dance with the vaginal infection in mice where a produc-

tive infection involves the basal cell layers before the

epithelial cells are sloughed off [7, 20]. At day 6 post

infection most of the lesions were resolved (data not

shown). In a second experiment of F344 rats, a more de-

layed infection was observed with sporadic lesions de-

tected at day 3 post infection (data not shown), while

micro-vesicles were detected close to the introitus at day 6

post infection (Fig. 2B-C). The micro-vesicles were

200-300 lm in length and covered by a cornified layer.

Within the micro-vesicles HSV-2 antigen was mostly de-

tected in the apical cell layers while no antigen was ob-

served in the basal cell layers or in the lamina propria.

In both experiments of HSV-2 infected F344 rats, HSV-

2 antigen was detected in a few neurons within ventrally

localized autonomic ganglia (Fig. 2E), and in the small

sensory neurons in lumbosacral DRG (Fig. 2F). Despite

that both the lumbosacral and thoracic segments of the

spinal cords were examined no HSV-2 antigen was

detected.

In SD rats, in a first experiment, no HSV-2 antigen was

visible in the genital mucosa at day 3 or day 6 post in-

fection, although the epithelial cells were swollen with

numerous vacuoles within the epithelial cell layers (data

not shown). In a second experiment, sporadic HSV-2 in-

fected cells were detected at the apical epithelial cell layers

Vagi

nalw

ash

(PFU

/ml)

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0200400600800

1000

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12000 LewisBNFischer344

SD

Survival

Infectivedose (PFU)

Anti-

HSV-

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Gtit

er

106

105

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0100200300400500

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Infectivedose (PFU)

6/7 5/5 0/4 4/47/8 3/3 2/5 4/4

2/4 4/5 4/4 1/4 3/7 4/43/4 5/5 0/4 4/4

A

B

Fig. 1 Susceptibility of four rat models after intravaginal HSV-2

infection. Animals were infected with 1 9 102 or 1 9 103 to

1 9 106 PFU. Infectious virus was measured in vaginal washes at

day 2 post infection (PFU/ml), (A). Anti-HSV-2 IgG-antibodies were

measured in serum collected when rats were euthanized at day 8-14

post infection or in surviving rats at day 21 post infection (B). Data

are presented as mean values from two separate experiments. Error

bars represent ± SEM

Table 1 Outcome after intravaginal infection of six rat models with 5 9 106 PFU of HSV-2

Rat model Serum IgGa Vaginal wash PFU/mlb Spinal cordc Ganglionc Survival

HSV-2 DNAc

SD 1506 (50-6400) 1567 (20-4540) 4.0 9 108 (NS) 2.0 9 106 (NS) 6/8

9.8 9 103 (S) 6.2 9 102 (S)

WIST 325 (50-800) 1247 (220-4680) 2.6 9 104 3.2 9 103 4/6

LEW 908 (50-3200) 4790 (120-1.4 9 104) 6.2 9 105 3.0 9 104 1/6

BN 4000 (1600-6400) 1784 (40-3960) 1.6 9 107 8.0 9 103 2/6

F344 1942 (50-3200) 348 (80-1100) 9.1 9 106 4.9 9 105 0/6

DA 3467 (1600-6400) 117 (40-220) 2.3 9 105 9.6 9 104 2/6

For the SD rat model, HSV-2 DNA genome copies in lumbosacral DRG and spinal cord were compared between survivors (S) and non-survivors

(NS). Data are from two separate experiments and presented as mean values with ranges within parenthesis for serum IgG and PFU/mla Anti-HSV-2 IgG titer. Serum was collected at day 21 post infection or when animals were euthanizedb PFU in vaginal washes at day 2 post infectionc Spinal cord and lumbosacral DRG were collected at day 21 post infection or when animals were euthanized and analyzed by qPCR


123

close to the introitus (Fig. 2D). No HSV-2 antigen was

detected in autonomic ganglia, lumbosacral DRG or spinal

cords.

No reactivation of HSV-2 in SD rats

Five rats were infected i.vag. with 1 9 106 PFU of HSV-2.

All rats survived without genital or systemic disease. All

animals presented infectious virus in vaginal washes at day

2 post infection and anti-HSV-2 antibodies at day 21 post

infection (data not shown) indicating that the rats were

infected. One month after the primary HSV-2 infection, in

total, 60 vaginal washes were collected during the next four

weeks. There after cortisone treatment was given and 30

additional vaginal washes were collected. All vaginal

washes were negative i.e., no PFU or HSV-2 DNA genome

copies could be detected (data not shown). Ten weeks after

the primary HSV-2 infection the study was terminated, and

the viral loads in lumbosacral DRG and spinal cords were

analyzed with qPCR. Three rats harbored low numbers of

HSV-2 genome copies per ganglion (mean value of 620

HSV-2 DNA genome copies) but were negative in the

spinal cords, one rat harbored 32637 HSV-2 DNA genome

copies per ganglion and 6900 HSV-2 DNA genome copies

in the spinal cord, while one rat was negative in both

lumbosacral DRG and the spinal cord (data not shown).

Genital HSV-1 infection

To evaluate if a genital infection, using an attenuated HSV-

1 strain, could protect against a subsequent HSV-2 infec-

tion, the LEW, BN, and F344 rat models were selected as

most of the animals developed symptoms after an HSV-2

infection. The rats were initially infected i.vag. with

1 9 106 PFU or 5 9 106 PFU of HSV-1. As the results

were similar between the two groups data were combined.

All rats (n = 25) survived and no genital or systemic

symptoms were observed (disease score 0), (Table 2). In 8

of 9 LEW rats, infectious HSV-1 was detected in the

vaginal washes but no anti-HSV-1 IgG antibodies in serum.

In 4 of 8 BN rats, and in 2 of 8 F344 rats, infectious HSV-1

was detected, while all rats presented anti-HSV-1 IgG an-

tibodies in serum. Two months after the HSV-1 infection,

and one month after the subsequent HSV-2 infection,

lumbosacral DRG and entire spinal cords were prepared

and subjected to type-discriminating qPCR. No HSV-1

DNA was detected in lumbosacral DRG or in the spinal

cord (Table 2).

Fig. 2 Immunohistochemical analyses of vaginal epithelial cells,

autonomic ganglia and lumbosacral DRG in F344 and SD rats after

intravaginal infection with 1 9 106 PFU of HSV-2. In F344 rats,

HSV-2 infected cells were detected by an immune serum in the apical

layers with spread into the basal cell layers at day 3 post infection (A),

or as micro-vesicles in the apical cell layers close to the introitus

(indicated with an asterix), at day 6 post infection (B) with

enlargement of the vesicle marked with an arrow (C). In SD rats,

sporadic HSV-2 infected epithelial cells, at the apical cell layers, were

only detected at day 6 post infection (D). In F344 rats, HSV-2 antigen

was detected in the neurons in autonomic ganglia (E), and in small

sensory neurons in lumbosacral DRG (F), at day 6 post infection.

Results are from two experiments


123

Genital challenge of HSV-1 infected rats with HSV-2

Rats were infected with 1 9 106 PFU of HSV-1 (n = 12)

or 5 9 106 PFU of HSV-1 (n = 13). Data from the two

HSV-1 infected groups were combined. One month after

the HSV-1 infection, rats were challenged i.vag. with

5 9 106 PFU of HSV-2. In contrast to the outcome after

primary HSV-2 infection, where only 3 of 18 naıve LEW,

BN, and F344 rats survived (Table 1), 23 of 25 HSV-1

infected LEW, BN, and F344 rats survived a challenge

with the same virus dose of HSV-2 (Table 2). This dif-

ference which was highly significant (P = \0.0001).

Seven of nine LEW rats developed no genital or sys-

temic disease after challenge with HSV-2, while two rats

succumbed late after infection (at day 19 and at day 21 post

infection). After challenge with HSV-2, the majority of

LEW rats presented both anti-HSV-2 antibodies and in-

fectious HSV-2 in the genital tract. All LEW rats harbored

HSV-2 DNA genome copies in the spinal cord and 7 of 9

rats in lumbosacral DRG, implying that they were infected

in the neuronal tissue. The mean HSV-2 DNA genome

copies in lumbosacral DRG (P = 0.012) and spinal cord

(P = 0.007) were significantly higher in LEW rats as

compared with F344 rats but not as compared with BN rats.

A single BN rat and a single F344 rat presented no HSV-2

in vaginal washes, no anti-HSV-2 antibodies, and no HSV-

2 DNA in lumbosacral DRG and the spinal cord, implying

that HSV-2 infection was not established.

Discussion

In this study we show that six rat models are susceptible to

i.vag. infection with HSV-2. The development of lethal

systemic disease symptoms suggested uncontrolled virus

spread to lumbosacral DRG, to the spinal cord and to au-

tonomic ganglia. We confirmed that HSV-2 DNA genome

copies were detected in lumbosacral DRG and in the spinal

cord when sick animals were euthanized at day 8-14 post

infection. In symptomatic rats, spread of HSV-2 to the

autonomic ganglia with faecal and urine retention most

likely contributed to the lethal outcome. The spread of

HSV-2 to autonomic ganglia has also been described for

BALB/c mice [21], and in guinea pigs, HSV-2 was pro-

posed to reach the spinal cord via infection of autonomic

neurons and not via lumbosacral DRG [22]. In the im-

munohistochemistry experiments HSV-2 antigens were

detected in the peripheral nerve system of F344 rats but not

in the spinal cord. As compared with the mouse model

[21], the symptoms in the rat models develop later and

more slowly after infection. Too early sampling at day 6

post infection could therefore explain the lack of HSV-2

antigen in the spinal cords of F344 rats.

With reservation that a small number of animals were

examined there was no obvious correlation between sur-

vival and the levels of infectious HSV-2 in vaginal washes

or anti-HSV-2 IgG antibodies for any of the models.

Similar lack of correlation between PFU in vaginal washes

and survival has been described earlier in the mouse model

after i.vag. infection with HSV-1 or HSV-2 strains [23, 24].

The susceptibility and outcome for the different rat models

after HSV- 2 infection presented in Table 1 were evaluated

simultaneously while the experiments presented in the

HSV-2 titration study (Fig. 1) were run separately. The

results after i.vag. infection with 5 9 106 PFU of HSV-2

(Table 1) are therefore not always comparable with infec-

tion with the lower doses with 1 9 106 to 1 9 102 PFU of

HSV-2 (Fig. 1). In the titration study we observed an

Table 2 Outcome after intravaginal infection of three rat models with 1 9 106 PFU or 5 9 106 PFU of HSV-1 followed by challenge with

5 9 106 PFU of HSV-2 one month later

Rat model Virus Serum IgG Vaginal wash

PFU/mlbSpinal cordc Ganglionc Survival

LEW HSV-1 Nega 2692 (8/9 pos.) Neg Neg 9/9

BN HSV-1 175a 1495 (4/8 pos.) Neg Neg 8/8

F344 HSV-1 880a 20 (2/8 pos.) Neg Neg 8/8

LEW HSV-2 545d (6/9 pos.) 1249 (8/9 pos.) 33648 12955 (7/9 pos.) 7/9

BN HSV-2 132d (4/8 pos.) 296 (5/8 pos.) 4550 (5/8 pos.) 4225 (4/8 pos.) 8/8

F344 HSV-2 88d (5/8 pos.) 90 (4/8 pos.) 1408 (3/8 pos.) 195 (3/8 pos.) 8/8

Data from the two HSV-1 infected groups were combined. When not all animals were positive, the number of positive rats is indicated. Data are

from two separate experiments and presented as mean values including all animalsa Anti-HSV-1 IgG titer. Serum was collected 21 days after HSV-1 infectionb PFU in vaginal washes at day 2 post infectionc Spinal cord and lumbosacral DRG were collected one month after HSV-2 infection or when animals were euthanized and analyzed by qPCRd Anti-HSV-2 IgG titer. Serum was collected 28 days after HSV-2 infection or when sick animals were euthanized


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inverse relation between virus dose and levels of anti-HSV-

2 antibodies in F344 infected rats which could be explained

by the fact that animals given higher virus doses became

sick and euthanized earlier and before antibodies were

elicited.

In the guinea pig model, it has been suggested that the

genital symptoms after a primary HSV-2 infection is de-

pendent on a neural pathway via sensory ganglia [25–27].

The requirement of HSV-1 returning from infected neu-

ronal tissue to induce peripheral lesions in a flank infection

model has also been described in mice [28]. If a similar

mechanism also operates after genital HSV-2 infection in

rats, the lack of genital symptoms in for example, asymp-

tomatically infected SD rats infected with 5 9 106 PFU of

HSV-2 (Table 1) may be explained by low grade of neu-

ronal infection reflected by low HSV-2 DNA genome

copies in lumbosacral DRG. Similarly, the low HSV-2

DNA genome copies in lumbosacral DRG of SD rats 10

weeks after the primary HSV-2 infection, may also explain

that HSV-2 did not reactivate spontaneously or after cor-

tisone treatment. However, although significant levels of

HSV-2 DNA were detected in lumbosacral DRG in

symptomatic and deceased rats in all models (Table 1), the

animals presented no or only mild genital inflammation.

These findings may suggest that a neuronal circuit for de-

velopment of clinical symptoms is of minor importance in

rats, or alternatively, that for example, anterograde trans-

port to the epithelial cells or activation of the local in-

flammatory responses is impaired.

The majority of primary genital HSV-1 or HSV-2 in-

fections in humans are asymptomatic presenting no clinical

disease symptoms [1, 29, 30]. As a consequence, it is

difficult to study molecular and immune mechanisms in-

volved in viral-host interactions in the epithelial cells and

spread of virus into the autonomic and sensory neurons.

Using the mouse or guinea pig models, earlier studies have

shown that HSV-2 mutants with inactivation of a specific

viral gene, as for example the thymidine kinase gene, in-

duce mild or no genital symptoms after a primary i.vag.

infection [31, 32]. In the SD and WIST rat models, an

asymptomatic primary infection was observed despite that

a wild type and a fully virulent HSV-2 strain was used for

infection. The SD rat model can therefore be proposed to

explore initial interactions in the asymptomatic genital

infection. The WIST rat model can also be considered but

was not thoroughly characterized in this study.

We also investigated if rats previously infected with

HSV-1 were susceptible to genital HSV-2. An HSV-1

strain which is defect in neuroinvasion was selected to

study protective immunity. Although no neuronal infection

was established, the HSV-1 infected rats were significantly

more protected against a subsequent genital HSV-2

infection as compared with naıve HSV-2 infected rats

(Table 1 and Table 2). In LEW rats local replication of

HSV-1 was readily detected but no anti-HSV-1 IgG-anti-

bodies were elicited. These data suggest that other arms of

the immune system than antibodies were involved in pro-

tection in this model. Although antibodies usually are used

as a reliable marker of infection, seronegative HSV-2 ex-

posed individuals with T cell responses to several HSV-2

antigens have been described earlier [33, 34]. Thus, T cell

responses to cross reactive HSV-1 antigen might be inter-

esting to evaluate in the LEW rat model after a genital

HSV-1 infection. Alternatively, the HSV-1 infection was

cleared by mucosal innate immune responses.

In the BN and F344 rat models anti-HSV IgG-antibodies

were detected in all animals, suggesting that antigen pre-

senting cells in the mucosa migrated to lymph nodes and

presented HSV-2 antigen to naıve B cells. This migration

reasonably requires replication of HSV-2 in the vagina.

However, we were unable to detect infectious HSV-2 in 4

of 8 of BN rats and in 6 of 8 F344 rats, observations which

may be explained by non-optimal sampling of vaginal

washes at day 2 after the HSV-1 infection.

In summary, we have shown that six rat models are

susceptible to genital HSV-2 infection but the outcome

varies between the different models. The models can be

suitable to evaluate prophylactic strategies to decrease the

transmission of HSV-2 and to study primary asymptomatic

infections. Fine mapping of the genetic basis associated

with symptomatic lethal HSV-2 infection (e.g. F344 rat

model) and asymptomatic infection (e.g. SD rat model) is

now possible. In recent years new technology improve-

ments have facilitated the development of transgenic rat

strains. Genetically defined new rat models can contribute

to explore interactions between virus and the host and the

study of specific gene functions [35] after genital HSV-

infection. Future studies can also address if the rat models

can recapitulate important aspects of a human genital HSV-

1 infection followed by an HSV-2 infection. In such

studies, a more neurovirulent HSV-1 strain can preferably

be used for genital infection.

Acknowledgements This work was supported by grants from the

ALF Foundation at Sahlgrenska University Hospital, the Gothenburg

Medical Society (GLS), the Adlerbertska Foundation, the Clas

Groschinsky Foundation, the Medical Research Council and Torsten

Soderbergs Foundation. We thank Carolina Gustafsson for technical

assistance.

Conflict of interest None declared.

Ethical standard This study was carried out in accordance to the

rules stated by the Swedish board of agriculture. All animal ex-

periments were approved by the ethical board in Gothenburg (Dnr

171-2013).


123

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Novel rat models to study primary genital herpes simplex virus-2 infection

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