* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Virological and pathological features of mice infected with murine
Hospital-acquired infection wikipedia , lookup
Herpes simplex wikipedia , lookup
Trichinosis wikipedia , lookup
2015–16 Zika virus epidemic wikipedia , lookup
Sarcocystis wikipedia , lookup
Schistosomiasis wikipedia , lookup
Influenza A virus wikipedia , lookup
Oesophagostomum wikipedia , lookup
Neonatal infection wikipedia , lookup
Orthohantavirus wikipedia , lookup
Middle East respiratory syndrome wikipedia , lookup
Ebola virus disease wikipedia , lookup
Hepatitis C wikipedia , lookup
West Nile fever wikipedia , lookup
Antiviral drug wikipedia , lookup
Human cytomegalovirus wikipedia , lookup
Marburg virus disease wikipedia , lookup
Herpes simplex virus wikipedia , lookup
Infectious mononucleosis wikipedia , lookup
Henipavirus wikipedia , lookup
2347 Journal of General Virology (1992), 73, 2347 2356. Printed in Great Britain Virological and pathological features of mice infected with murine gammaherpesvirus 68 N. P. Sunil-Chandra,* S. Efstathiou,t J. Arno and A. A. Nash Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K. The primary infection of BALB/c mice with murine herpesvirus 68 (MHV-68) was investigated. When the virus was introduced intranasally, the lung was the main tissue infected, the virus being associated with alveolar epithelium and mononuclear cells. A productive infection lasted for 10 days, after which viral DNA could be detected by in situ hybridization up to 30 days after infection. At that time lymphoproliferative accumulations were also observed in the lung, with formation of germinal centres. Virus could also be recovered from the heart, kidney, adrenal gland and spleen during the primary infection. In addition, the spleen appeared to be the major site of virus persistence, with latently infected cells detected up to 90 days post-infection. During the primary infection, there was atrophy of the thymus and spleen of clinically sick animals. In contrast, lymphoproliferative responses, typified by splenomegaly, were frequently seen in asymptomatic animals. The pattern of infection observed in MHV-68-infected mice is similar to that seen in infectious mononucleosis of man following EpsteinBarr virus infection. The model described in this paper may prove to be useful in studying natural gammaherpesvirus infections of man and domestic animals. Introduction valuable for studying the oncogenic potential of EBV and for vaccine development (Epstein et al., 1985), but the route of infection used is not the natural one and the outcome of infection does not mimic that observed in man. We are interested in developing an amenable animal model with which to study permissive natural infection and latency of gammaherpesviruses. In this paper we present our initial studies on the pathogenesis of murine herpesvirus 68 (MHV-68). This virus was originally isolated from Clethrionomys glareolus (bank vole) in Czechoslovakia (Blaskovic et al., 1980). Based on the c.p.e, observed in infected BHK-21 and rabbit lung cell lines, and on the virion architecture observed by electron microscopy of ultrathin sections of infected rabbit embryo fibroblasts, this virus was classified as a member of the herpesvirus family (Blaskovic et al., 1980; Ciampor et al., 1981). More recently, analysis of the viral genome has revealed that MHV-68 is closely related to the gammaherpesviruses of primates, EBV and herpesvirus saimiri (HVS) (Efstathiou et al., 1990a, b). Studies carried out in newborn mice with an outbred background have revealed a virus-induced pneumonia and viraemia during which virus can be isolated from many tissues (Rajcani et al., 1985). In this paper, we show that MHV68 infection of BALB/c mice has pathogenic features in common with those observed in acute EBV infection of man, and that lung and lymphoid tissues are the major sites of pathology and virus persistence. Herpesviruses are linear dsDNA viruses of eukaryotes which, on the basis of their distinct biological properties, are divided into three major subgroups: the alpha-, betaand gammaherpesviruses (Roizman, 1982; Honess, 1984; Minson, 1989). The gammaherpesviruses, typified by Epstein-Barr virus (EBV), have the capacity to establish a latent infection within their target lymphocyte population, can induce a lymphoproliferative disease in the infected host and can efficiently immortalize lymphocytes infected in vitro. Although we know a great deal about the relationship between EBV and B cells in vitro, and the immune response to EBV has been well studied (Rickinson et al., 1989), studies of gammaherpesvirus infections have been limited almost entirely to clinically apparent EBV infection in man and experimental inoculation of non-human primates with either EBV or related simian viruses. Studies of acute infection with EBV are limited to infectious mononucleosis patients, who present some weeks after infection has occurred and in whom the pathogenesis may differ from the subclinical infection experienced by the majority of the population (Rickinson et al., 1985). The cotton top marmoset is susceptible to EBV-induced lymphoproliferative disease and this model has proved ]-Present address: Herpesvirus Laboratory, Medical Virology, Institute of Medicaland Veterinary Science, Adelaide, Australia. 0001-0946 © 1992 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59 2348 N. P. Sunil-Chandra and others Methods Mice. Female BALB/c mice were obtained from Bantin & Kingman and were infected at 3 to 4 weeks of age. Viruses and cells. Virus working stocks were prepared by infection of BHK-21 cells at a low multiplicity (0-1 p.f.u./cell) with MHV-68 clone G2.4 (Blaskovic et al., 1980; Efstathiou et al., 1990a, b). Virus suspensions in Eagle's MEM were clarified by centrifugation, and the supernatant was dispensed in 0.2 ml volumes and stored at - 7 0 °C until required. Infectious virus was measured by plaque titration using BHK-21 cells grown in Eagle's MEM containing 10~ (v/v) tryptose phosphate broth and 10 % (v/v) newborn calf serum at 37 °C in 5 ~ CO2 for 4 days. Intranasal and intravenous infection o f mice. Groups of 3- to 4-week-old female mice were inoculated intranasally or intravenously with 4 x 105 p.f.u. MHV-68. Approximately 40p.1 of virus was administered intranasally to lightly anaesthetized (ether anaesthesia) mice. For intravenous inoculation, 0.1 ml of the virus dilution was injected into the tail vein. Assay of infectious virus. Mice were killed at different times following infection by injection of Euthatal BP (Vet) intraperitoneally. Blood samples were obtained from the retro-orbital plexus, and each mouse was perfused with PBS (20 ml per mouse). The lung, heart, kidney, liver, spleen, thymus, two mesenteric lymph nodes, adrenal glands, brain and trigeminal ganglia were removed and stored at - 7 0 °C until required. Blood clot and sera were obtained from 100 ktl of blood. Specimens were homogenized separately in 2 ml Eagle's MEM supplemented with 10~ (v/v) tryptose phosphate broth and 10~ (v/v) calf serum. The presence of infectious virus was determined by assay on BHK-21 cells, on which plaques could be detected after 3 to 4 days. Co-cultivation assay for virus during acute and persistent infection. Spleen, thymus, lymph nodes and peripheral blood cells were used for co-cultivation. Cells were obtained from each of these tissues and suspended in RPMI 1640 containing 20~ (v/v) foetal calf serum (FCS) (Hunt, 1987). In the case of peripheral blood, total blood cells were obtained from 100 lal blood immediately after bleeding. In addition, at 90 days post-infection, peripheral blood lymphocytes were obtained by Ficoll gradient purification for the co-cultivation assay. For the nonlymphoid organs, 1 to 3 mm pieces of tissue were co-cultivated with BHK-21 cells. Lymphoid cells or tissue explants were co-cultivated with 2 x 106 BHK-21 cells in RPMI 1640 containing 20~ (v/v) FCS at 37 °C in 5 ~ CO2 for 5 days. The monolayers were fixed and stained, and the number of plaques were counted. The results were recorded as numbers of infectious centres per organ or tissue at each time. Preparation of hyperimmune serum. MHV-68 was grown in RK13 cells; cell-associated virus was disrupted by sonication and the virus suspension was clarified by centrifugation to remove cell debris. This preparation was inoculated intramuscularly into rabbits in Freund's complete adjuvant. Booster injections in Freund's incomplete adjuvant were given to these rabbits at 2 and 3 week intervals. The sera obtained from the final bleed were tested for the presence of antibody by immunofluorescence staining of MHV-68-infected BHK-21 cells. A 1:200 dilution of hyperimmune rabbit serum resulted in specific staining of virus antigen. For the detection of virus antigen in tissue sections of infected organs, a 1 : 250 dilution of final bleed rabbit serum gave optimal specific staining by the indirect immunoperoxidase technique. Hyperimmune sera were also raised in mice by using a similar technique. Histopathological and immunohistochemical studies. Heart, kidney, lung, liver, spleen, thymus, mesenteric lymph nodes and adrenal gland were carefully removed from animals at various times after infection and immediately fixed in 10~ buffered formal saline. Tissues were embedded in paraffin and 5 ~tm sections were prepared for histopathological examination. Sections were stained using haematoxylin and eosin, and indirect immunoperoxidase antibody labelling was used to detect virus antigen. Tissue sections were treated with 0-1 ~ trypsin for 5 min to avoid problems associated with over-fixation of tissue specimens in formalin. Non-specific binding of antibodies was minimized by the use of Tris-buffered saline as the diluent and rinsing, and by the addition of a blocking step which included 5 % normal goat serum and 5 ~ BSA. Endogenous peroxidase activity was minimized by immersing the slides in a solution of 0.75% hydrogen peroxide in methanol for 30 min before trypsin treatment of the tissue sections. Immunoperoxidase staining with an avidin/biotin detection system was performed using the Vectastain ABC Kit (Vector Laboratories). Detection of MHV-68 DNA in tissue sections. Sections from several tissues taken at different times after infection were prepared as described above and cut onto poly-L-lysine-coated slides. The method for detecting viral DNA was a modification of the technique described by Fazakerley et al. (1991). Briefly, this involved treating tissue sections with proteinase K, followed by prehybridization (1 h at 37 °C) and then hybridization with a 1.2 kb DNA probe (boiled for 5 min and rapidly chilled on ice before addition to the hybridization solution) from the terminal region of the MHV-68 genome (Efstathiou et at., 1990a, b). Before the overnight (18 h) hybridization stage, the tissue sections containing DNA probe were heated to 80 °C for 10 min. The sections were then washed twice for 15 min with 2 x SSC at room temperature, twice for 15 min with 0.1 x SSC at room temperature, once for 10 min with 0.1 × SSC preheated to 60 °C and kept at 37 °C, followed by a 15 min wash with 2 x SSC at room temperature. The sections were then dehydrated via a series of graded alcohols, air-dried, and then dipped in photographic emulsion (prepared in a darkroom by adding 20 ml of Amersham LM-1 emulsion to 20 ml of 0-66 M-ammonium acetate at 42 °C), allowed to dry at room temperature in a light-tight box for 2 h and autoradiographed for 1 to 4 weeks at 4 °C. The slides were developed (Ilford Phenisol developer) in a darkroom at 15 °C and fixed, counterstained with haematoxylin and eosin, dehydrated and mounted with DePeX. Results Determination of infectious virus titres from various organs during acute infection Two groups of 25 mice each were inoculated either intranasally or intravenously. Of the intranasally infected mice, 48 ~ developed clinical signs and symptoms 7 to 9 days post-infection. Severe clinical disease was characterized by ruffled fur, hunched back, inactivity, severe weakness and emaciation. Mice showing mild clinical signs and symptoms recovered at 10 to 12 days, whereas severely sick mice died during the same period following intranasal infection. None of the intravenously infected mice developed a severe clinical illness or died during this period, indicating that infection by the intranasal route is more efficient in producing disease than intravenous administration of virus. Three mice from each of the intranasally or intravenously inoculated groups were killed on days 1, 3, 5, 10 and 30 post-infection, and samples of lung, spleen, thymus, mesenteric lymph nodes, peripheral blood, liver, heart, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59 MHV-68 infection of mice 2349 Table 1. Virus titres in various organs* of BALB/c mice infected intranasally with MHV-68 Infectious virus titre (log~0 p.f.u./organ)J Time post-infection (days) Thymus Mesenteric lymph node <1 <0§ <0 1.7 <1 <1 <1 0.9 0.5 <0 0.5 <0 <0 <0 <0 Lung Spleen 1 4-7 3 5 10 30 6.7 4.7 <1 <1 Whole:~ blood Adrenal gland Heart Kidney <0 <1 <1 <0 <0 <0 0.5 <0 <1 <1 5.3 <1 <1 <1 5-1 <1 0.1 0.7 <0 <0 * Infectious virus could not be detected in liver, serum, brain or trigeminal ganglia. t Mean infectious virus titre for three mice at each time following inoculation of 4 x l0 s p.f.u, of virus/mouse. $ One-hundred microlitres of whole blood was used. § <0, Indicates that no virus was detectable. The limit of detection for adrenal gland, ganglia, blood, thymus and lymph node was 1 p.f.u./organ; liver, 100 p.f.u.; other organs, 10 p.f.u. Table 2. Virus titres in various organs* of BALB,/c mice infected intravenously with MHV-68 Infectious virus titre (log10 p.f.u./organ)t Time post-infection (days) Lung Spleen 1 <1 <1 3 5 10 30 3-1 2.5 2.1 <1 3.4 2.6 <1 <1 Thymus Mesenteric lymph node Whole:~ blood Heart Kidney Adrenal gland Liver <0§ 0.8 0.8 <0 <0 <0 1.3 <0 <0 <0 <0 <0 <0 <0 <0 2.0 3-0 1-8 5-3 <1 <1 <1 <1 5.3 <l 1.9 2-9 4-0 <0 <0 4-6 <2 <2 <2 <2 * In the course of the experiment, 0.3 and 0.7 loglo p.f.u, virus were detected in the trigeminal ganglia and brain respectively (at 5 days post-infection) of one of 15 mice sampled. t Mean infectious virus titre for one of three mice at each time following inoculation of 4 x 105 p.f.u, of virus/mouse. :~One-hundred microlitres of whole blood was used. § <0, Indicates that no virus was detectable. The limit of detection for adrenal gland, ganglia, blood, thymus and lymph node was 1 p.f.u./organ; liver, 100 p.f.u.; other organs, 10 p.f.u. k i d n e y , b r a i n a n d t r i g e m i n a l g a n g l i a were r e m o v e d a n d infectious virus was t i t r a t e d ( T a b l e s 1 a n d 2). I n a s e c o n d e x p e r i m e n t , 70 B A L B / c m i c e were i n f e c t e d i n t r a n a s a l l y w i t h 4 × 105 p.f.u. M H V - 6 8 a n d at d a i l y i n t e r v a l s up to 10 days, a n d on d a y s 15 a n d 20 five a n i m a l s were killed a n d v a r i o u s o r g a n s were collected for infectious virus assay. F o l l o w i n g i n t r a n a s a l inoculation, high titres o f infectious virus could be d e t e c t e d in lung tissue b e t w e e n 1 a n d 7 days post-infection, a n d t h e r e a f t e r d e c l i n e d to u n d e t e c t a b l e levels b e t w e e n days 10 a n d 15 (Fig. 1 a n d T a b l e 1). T h e increase in virus titre b e t w e e n d a y s 1 a n d 3 i n d i c a t e s active r e p l i c a t i o n o f the initial M H V - 6 8 i n o c u l u m in this tissue. A l t h o u g h c l e a r a n c e o f infectious virus was o b s e r v e d in the lung at 10 d a y s post-infection, 5-3 a n d 5.1 log~o p.f.u, o f virus was d e t e c t e d in t h e h e a r t a n d k i d n e y o f i n f e c t e d a n i m a l s respectively at this t i m e ( T a b l e 1). H o w e v e r , the d e t e c t i o n o f infectious virus f r o m h e a r t a n d k i d n e y is n o t a c o n s i s t e n t feature. L o w titres o f infectious virus could be d e t e c t e d s p o r a d i c a l l y in the m a j o r l y m p h o i d o r g a n s (spleen, t h y m u s a n d m e s e n teric l y m p h nodes), a d r e n a l g l a n d a n d whole b l o o d d u r i n g the course o f the e x p e r i m e n t . O u r i n a b i l i t y to d e t e c t infectious virus in the b r a i n or t r i g e m i n a l g a n g l i a d u r i n g the acute stage o f infection, a n d the a b s e n c e o f neurological disease, suggest t h a t M H V - 6 8 is n o t n e u r o i n v a s i v e following i n t r a n a s a l inoculation. A s i m i l a r d i s t r i b u t i o n o f infectious virus was o b s e r v e d in the tissues o f a n i m a l s i n f e c t e d i n t r a v e n o u s l y ( T a b l e 2), a l t h o u g h i n v o l v e m e n t o f the liver was a p p a r e n t 3 d a y s p o s t - i n f e c t i o n a n d h i g h e r infectious virus titres were d e t e c t e d in b o t h the m a j o r l y m p h o i d o r g a n s a n d the a d r e n a l gland. Since i n t r a n a s a l i n f e c t i o n p r o v e d m o r e efficient in the g e n e r a t i o n o f clinical d i s e a s e a n d r e p r e s e n t s a m o r e n a t u r a l infectious route t h a n intravenous a d m i n i s t r a t i o n o f virus it was c h o s e n as the m o d e l for further study. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59 N. P. Sunil-Chandra and others 2350 Day 3 Day 5 Day 10 (a) ~6 & / ~5 ~4 ! ~ 3 0 .~ 2 (b) w 0 10 Time post-infection (days) 20 Fig. 1. Virus replication in mice during acute MHV-68 infection. Three- to 4-week-old female BALB/c mice were inoculated with 4 x l0 s p.f.u, virus intranasally. Mean infectious virus titre/organ + S.D. for five mice/group is shown for each time for lung (H) and spleen (.). The limit of detection was 10 p.f.u./organ. Gross pathological and histopathological changes Thirty female BALB/c mice were infected intranasally with 4 × 105 p.f.u, of MHV-68. Three mice were killed at 3, 5, 30 and 170 days post-infection. In addition, six mice, including three clinically normal but infected and three showing signs of clinical disease, were killed 10 days post-infection. Tissues were examined for gross pathological changes before fixation in formal buffered saline. Haematoxylin- and eosin-stained sections were used to study histopathologicat changes, and immunoperoxidase specificity was used to study the location and distribution of virus antigen. Gross pathological and histopathological changes in lymphoid organs were a major feature of MHV-68 infection of BALB/c mice. Animals suffering from severe clinical symptoms 10 days post-infection exhibited marked splenic and thymic atrophy. No infectious virus could be detected in these organs at this time (Table 1). In contrast, animals showing no clinical signs exhibited marked splenomegaly, and moderate mesenteric lymph node and thymic enlargement (Fig. 2). The cause of the splenic atrophy was lymphoid cell depletion; the atrophic thymus was attributable to loss of thymocytes (Fig. 3). The splenomegaly associated with the clinically inapparent infection was characterized by an increase in the number of germinal centres (Fig. 3). In the lungs, a dramatic peribronchiolar, perivascular and interstitial infiltration of lymphoid cells occurred. Necrosis within the cellular infiltrate was observed as early as 3 days post-infection, which is consistent with the lung being a major site of virus replication during Fig. 2. Gross pathologicalchanges of the spleen followingacute MHV68 infection. Three- ta.4rweek-oldBALB/c mice were inoculated with 4 x l0 s p.f.u, virus intranasally~ Haematoxylin- and eosin-stained, formalin-fixed longitudinal sections of spleen dissected at 3, 5 and 10 days post-infection are shown to demonstrate relative splenic enlargement or atrophy during acute infection. Clinically sick mice developed atrophy of the spleen (a); splenomegaly was seen in some clinically normal mice at 10 days post-infection (b). For reference, a normal 4- to 5-week-old BALB/c mouse spleen was similar in size to that shown for day 3 post-infection. Bar marker represents 5 mm. acute infection (Fig. 4, Table 1). At 5 days post-infection, an exudate of cellular debris was seen in the bronchiolar lumen and the infiltration of lymphoid cells in the peribronchiolar, perivascular and interstitial areas was more extensive. Resolution of the inflammation began at 10 days post-infection in both clinically normal and sick animals. This is consistent with the clearance of infectious virus at this stage. However, in the lungs of mice which had recovered from the primary infection, localized areas of lymphoid infiltrate persisted at these same sites, both at 30 and even 170 days post-infection. In places, germinal centre formation was observed (Fig. 4e). In addition, during and after the acute infection occasional small clusters of lymphoid cells were seen in the liver and kidney of the majority of mice. Immunohistochemical studies of virus antigen distribution in the tissues of infected mice Virus antigen was readily detected in the lung during acute infection (Fig. 5). Foci of positive cells were observed in the peribronchiolar and perivascular cellular Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59 MHV-68 infection of mice 2351 Fig. 3. Histopathology of spleen and thymus at 10 days after MHV-68 infection. (a) Atrophied spleen of a clinicallysick infected mouse showing marked lymphoid cell depletion. WP, White pulp; RP; red pulp. (b) Spleen from a clinicallyinapparent infection. Note the size of the spleen (splenomegaly) and the increased number of germinal centres. WP, White pulp; RP, red pulp. (c) Atrophied thymus of a clinicallysick mouse showing almost complete depletion of thymocytes in the thymic cortex and a dense cellular infiltrate in the medulla with interspersed phagocytic macrophages (P). M, Thymic medulla; C, thymic cortex. (d) Thymus of a clinically normal infected mouse. The distribution of lymphoid cells is similar to that in uninfected mice (not shown). C, Thymic cortex composed of densely packed thymocytes; M, thymic medulla, which is paler with fewer thymocytes and more conspicuous epithelial cells. Bar markers represent (a) and (b) 0.5 mm, (c) and (d) 0.25 mm. infiltrates at 3 d a y s post-infection. By 5 days, e n l a r g e d e p i t h e l i a l cells lining t h e alveoli a n d large m o n o n u c l e a r cells w i t h i n the cellular infiltrates were a n t i g e n - p o s i t i v e . W h a t a p p e a r e d to be l y m p h o c y t e s w i t h i n those infilt r a t e s were o n t h e whole n e g a t i v e , b u t a few ceils d i d a p p e a r to c o n t a i n a n t i g e n (Fig. 5), as d i d o c c a s i o n a l h e p a t o c y t e s a n d m o n o n u c l e a r cells w i t h i n t h e spleen. T h e p r e s e n c e o f strong s t a i n i n g o f the nucleus a n d also a p p a r e n t l y o f the c y t o p l a s m o f b o t h e p i t h e l i a l a n d m o n o n u c l e a r cells strongly suggests t h a t a c t i v e transla- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59 2352 N. P. Sunil-Chandra and others Table 3. Recovery of MHV-68 from primary and secondary lymphoid tissues by a co-cultivation assay Infectiouscentres/organ* Time post-infection Mouse Mesenteric Peripheral (days) no. Spleen Thymus lymph node blood1" 1 2 1 2 1 2 30 240 300 400 150 200 2 l 0 1 0 4 0 0 0 0 0 0 0 0 0 0 0 3 1 2 3 620 254 178 ND~ ND ND ND ND ND ND ND ND ! 2 3 4 5 220 0 40 260 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Experiment 1 3 5 10 Experiment 2 36 Experiment 3 90 0 * Number of infectious centres from the entire organ from each mouse. t In the case of peripheral blood, 100 lal whole blood was used. "~ND, Not determined. tion of viral proteins was taking place during the acute infection (Fig. 5). Immunostaining of the columnar epithelium of the bronchioles was negative at every stage. At 10 days post-infection, the amount of viral antigen recognized by this method was much reduced, a finding which is consistent with the inability to detect infectious virus in the lungs of animals at the same stage of the disease (Table 2). No antigen-positive cells were seen in any of the tissues of animals which recovered from the clinical disease, the majority of which were examined at least 30 days after primary infection. Recovery of virus and the detection of MHV-68 DNA from the spleens of infected mice A common biological feature of herpesviruses is their ability to persist in the host and to reactivate periodically, resulting in the release of infectious virus. Gamma herpesviruses characteristically establish a latent infection within lymphocytes. In an attempt to determine the site of persistence of MHV-68, a variety of tissues were co-cultivated with permissive cells to isolate latent/persistent virus. In three separate experiments, 3- to 4-weekold BALB/c mice were inoculated intranasally with 4 × 105 p.f.u. MHV-68. In the first experiment, two mice were killed on days 3, 5 and 10 post-infection, and cells from the spleen, thymus, mesenteric lymph nodes and blood were co-cultivated with BHK-21 cells. By using this technique, virus was readily recovered from the dissociated spleens of acutely infected animals after 5 days in culture (Table 3). These results contrast with those obtained by direct homogenization and assay of spleens, in which only low levels of infectious virus could be detected at similar times post-infection (Table 1). That the spleen is a site of persistent/latent virus was confirmed in two additional experiments. In experiment 2, spleen cells from mice killed 36 days after infection were found to carry persistent/latent virus. In the third experiment, five mice were killed 90 days post-infection, and the organs were removed immediately and divided into two equal parts. One part of each organ was used for direct tissue homogenization and assay for the presence of infectious virus, and the other part for co-cultivation of lymphoid cells or tissue explants to detect persistent/latent virus. Infectious virus could not be detected in any of the splenic tissues assayed by direct homogenization, whereas the spleens from three of five mice assayed following dissociation of the tissue and co-cultivation with permissive BHK-21 cells led to the recovery of virus (Table 3), implicating this organ as a major site of virus persistence. These data are in accord with the detection of MHV-68 DNA in the spleens of mice 30 days after infection (Fig. 6a). It is noted that few positive cells are present in the germinal centres of the spleen; this location tentatively suggests that the infected cells may be of B lymphocyte origin. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59 MHV-68 infection of mice Fig. 4. Histopathology of lung following infection with MHV-68. Formalin- fixed, haematoxylin-stained sections of the lung at various times after infection. (a) Lung of an uninfected mouse (3 to 4 weeks old). A, Alveoli; B, bronchiole. (b) Lung 3 days post-infection with MHV-68. B, Bronchiole; I, infiltration of mononuclear cells in peribronchiolar and interstitial areas of the lung. (c) Lung of infected mouse at 5 days post-infection. B, Bronchiole in which lumen contains exudate of cellular debris; V, blood vessel (artery); I, an infiltrate of mononuclear cells within the intima. (d) Lung of a clinically normal infected mouse at 10 days post-infection. A, Alveoli; V, blood vessel (artery); I, infiltration of mononuclear cells in perivascular and interstitial areas. (e) Lung of a mouse 30 days post-infection. L, Abnormal lymphoid accumulations can be observed in a subpleural area. Similar lymphoid proliferations/accumulations could also be observed in peribronchiolar and perivascular areas of the lung. Bar marker represents 25 ~tm. 2353 Fig. 5. Immunoperoxidase staining of MHV-68 antigens in lung tissue 5 days after infection. Formalin-fixed lung sections were reacted with a hyperimmune anti-MHV-68 serum and stained by the immunoperoxidase method. (a) Peribronchiolar and perivascular areas of the lung. Arrows indicate that the peribronchial and perivascular cellular infiltrate includes positively staining cells containing viral antigen. A, Alveoli; B, bronchiole; V, blood vessel (vein). (b) Lung parenchyma. Positive staining of enlarged alveolar epithelial cells can be seen (AE). (c) A magnified view of the lung parenchyma shows positively stained large mononuclear cells (MN) containing viral antigen. A, Alveolar space. Bar markers represent (a) and (b) 20 ktm, (c) 8 ~tm. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59 2354 N. P. Sunil-Chandra and others Fig. 6. D•t•cti•n•fMH•-68DNAinsp••enand•ung3•daysp•st-inf•cti•n.A•.2kbPstIr•p•atfragment•fMHV-68DNAwasused to prepare a 3sS-labelled DNA probe for detection of latent viral DNA by in situ hybridization. (a) A few cells containing viral nucleic acid could be detected within the lymphoid tissue of the spleen. The arrow indicates two positive cells located at the edge of a germinal centre (G). The inset shows a magnified view which suggests that positive cells have just divided. (b) Positive cells were also seen in the peribronchial tissue of the lung. Bar markers represent (a) 20 ~tm, inset 8 ~tm and (b) 40 ~tm. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59 MHV-68 infection of mice Detection of viral DNA and the recovery of infectious virus from the lungs of latently infected mice Earlier studies on the pathogenesis of MHV-68 in both newborn and 21-day-old outbred mice (Rajcani et al., 1985, 1986) have identified the lung, trigeminal ganglia and spleen as tissues in which virus can be detected by direct homogenization and assay many days after infection, thus implicating these organs as sites of dynamic virus persistence. In our studies using inbred BALB/c mice, virus could not be recovered either by direct homogenization and assay or by co-cultivation of tissue explants from non-lymphoid organs such as the lung, heart, kidney, adrenal glands, brain and trigeminal ganglia from five animals sampled 90 days postinfection. To increase the sensitivity of the assay procedure, homogenization and assay of co-cultured tissue explants was employed. This sensitive assay failed to detect virus in explanted brain, trigeminal ganglia, heart, kidney and adrenal glands removed from animals 90 days post-infection but virus was recovered from the lungs of two of these five mice. This suggests that the lung, in addition to the spleen, is a site of virus persistence and is in agreement with the results of Rajcani et al. (1985). Further evidence to support this idea comes from a single observation of viral D N A detection by in situ hybridization in the lungs of mice 30 days after infection (Fig. 6b). A small focus of positive cells was observed, although these were not positive for viral antigen by the immunohistochemical method. Discussion MHV-68 was originally isolated from free-living rodents (bank vole) in Czechoslovakia and passaged in the brain of newborn mice. In view of this neurological association and the failure of the virus to react with sera specific for murine cytomegalovirus, it was tentatively classified as a member of the alphaherpesvirus subgroup (Svobodova et al., 1982). However, analysis of the structure of the viral genome and limited sequencing of viral genes have revealed that MHV-68 is closely related to the gammaherpesviruses EBV and HVS (Efstathiou et al., 1990a, b). As this is currently the only known gammaherpesvirus naturally infecting mice, it provides an important opportunity to study the properties of the virus in inbred mice, and to compare and contrast these with the properties of the primate gammaherpesviruses. In this paper we have studied two routes of infection of BALB/c mice with MHV-68, focusing on tissue tropism, pathological features of the infection and virus persistence. Intranasal administration of MHV-68 to 3- to 4-weekold BALB/c mice resulted in the lung becoming the main 2355 tissue infected. Peak virus titres were detected 1 week after infection, with viral antigens found in the alveolar epithelium and a small number of mononuclear cells. During this period sections of the lung revealed peribronchiolar, perivascular and interstitial infiltration of mononuclear cells among which there were foci of necrosis and exudates containing cellular debris within bronchioles. By day 10 post-infection, there was a decrease in the amount of viral antigen detected, which corresponds with the failure to isolate infectious virus from the lung at this stage. These observations are consistent with the findings of Rajcani et al. (1985), who studied intranasal infection of 5-, 10- and 21-day-old outbred mice. Following recovery from the primary infection, MHV-68 DNA, but not viral antigen, was detected in lung sections from some animals. In addition, a pathological feature of the lungs at 30 days after infection was the accumulation of lymphoid cells in the sub-pleural and peribronchiolar regions, the architecture of which resembled germinal centres. Lymphoproliferative responses of this type are common features of gammaherpesvirus infections. In addition to the lung, MHV-68 also infects the kidney and heart, and to a lesser extent the spleen, during the primary infection. However, we were unable to isolate virus from the central or peripheral nervous system, indicating that the nervous system is not a primary target for infection, nor a site of virus persistence. In contrast, the adrenal gland appears to be heavily infected when the virus is introduced via the bloodstream. This is a particularly interesting observation because both herpes simplex virus (an alphaherpesvirus) and murine cytomegalovirus (a betaherpesvirus) are known to infect this tissue. The significance of virus infecting the adrenal gland and the consequences for the host are currently under investigation. A major strategy employed by herpesviruses for persisting in their host is the establishment of a latent infection. In the sensory ganglia of mice infected with herpes simplex virus, the presence of latent virus is detected by co-cultivating the tissue with BHK-21 cells. A similar approach was used to identify latent/persistent MHV-68 in the spleen and lungs of BALB/c mice (Table 3). This observation, in conjunction with the detection of the viral genome in the lung and spleen, suggests that these tissues are the main sites of virus persistence. The identification of latent virus in lymphoid cells by this procedure is consistent with the properties of other gammaherpesviruses, notably HVS and herpesvirus sylvilagus (Falk et al., 1972; Rabson et al., 1971 ; Kramp et al., 1985; Medveczy et al., 1984 ). There are two pathological features of MHV-68 infection in mice that are similar to events in man infected with EBV. Lymphoproliferation is observed in Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59 2356 N. P. Sunil-Chandra and others the spleen of some animals and causes splenomegaly, a condition that occurs in man during infectious mononucleosis. Furthermore, atrophy of the spleen and thymus, seen in some BALB/c mice during the primary infection, have also been observed in severe cases of infectious mononucleosis. The cause of the latter is unknown, but it is unlikely to arise from the cytopathic effects of the virus because neither significant infectious virus nor viral antigens are observed in these tissues. From these initial studies on MHV-68 infection of BALB/c mice, it would appear that certain features of the primary and persistent infection resemble EBV infection of man. We have obtained evidence (unpublished data), that the cell harbouring latent virus in the spleen is a B lymphocyte. These observations suggest that mouse gammaherpesvirus is likely to be a useful/ important animal model for understanding the detailed virological and immunological processes in a natural, permissive gammaherpesvirus infection. The authors wish to thank the Medical Research Council of Great Britain for supporting this work and A. C. Minson for helpful discussions. N. P. S.-C. was supported by a grant from the Cambridge Commonwealth Trust. References BLASKOVIC, D., STANCEKOVA, M., SVOBODOVA,J. & MISTRIKOVA, J. (1980). Isolation of five strains of herpes viruses from two species of free living rodents. Acta virologica 24, 468. CIAMPOR, F., STANCEKOVA, M. 8/; BLASKOVlC, D. (1981). Electron microscopy of rabbit embryo fibroblasts infected with herpesvirus isolates from Clethrionomys glareolus and Apodemus flavicollis. Acta virologica 25, 101 107. EFSTATHIOU, S., HO, Y. M. & MINSON, A. C. (1990a). Cloning and molecular characterization of the murine herpesvirus 68 genome. Journal of General Virology 71, 1355-1364. EFSTATHIOU, S., HO, Y. M., HALL, S., STYLES, C. J., SCOTT, S. D. & GOMr'ELS,U. A. (1990 b). Murine herpesvirus 68 is genetically related to the gammaherpesviruses Epstein-Barr virus and herpesvirus saimiri. Journal of Gencral Virology 71, 1365-1372. EPSTEIN, M. A., MORGAN, A. J., FINERTY, S., RANDLE, B. J. 8/; KIRKWOOD, J. K. (1985). Protection of cotton top tamarins against EB virus-induced malignant lymphoma by a prototype subunit vaccine. Nature, London 318, 287-289. FALK, L. A., WOLFE, L. G. & DEINHARDT, F. (1972). Isolation of herpesvirus saimiri from blood of squirrel monkey (Saimiri sciureus). Journal of the National Cancer Institute 48, 1499-1505. FAZAKERLEY, J. K., SOUTHERN, P., BLOOM, F. & BOCI-IMEtER, M. (1991). High resolution in situ hybridization to determine the cellular distribution of lymphocytic choriomeningitis virus RNA in the tissues of persistently infected mice: relevance to arenavirus disease and mechanisms of viral persistence. Journal of General Virology 72, 1611-1625. HONESS, R. W. (1984). Herpes simplex and 'the herpes complex': diverse observations and a unifying hypothesis. Journal of General Virology 65, 2077-2107. HUNT, S. W. (1987). Preparation oflymphocytes and accessory cells. In Lymphocytes: A Practical Approach. pp. 1-32. Edited by G. G. B. Klaus. Oxford & Washington D.C:. IRL Press. KRAMP, W. J., MEDVECZKY, P., MOLDER, C., HINZE, H. C. & SULLIVAN, J. L. (1985). Herpesvirus sylvilagus infects both B and T lymphocytes in vivo. Journal of Virology 56, 60455. MEDVECZKY, P., KRAMP, W. J. & SULLIVAN, J. L. (1984). Circular herpesvirus sylvilagus DNA in spleen cells of experimentally infected cotton tail rabbits. Journal of Virology 52, 711-714. MINSON, A. C. (1989). Herpesviridae. In Andrewes" Viruses of Vertebrates, pp. 293-332. Edited by J. S. Porterfield. London: Bailli6re-Tindall. RABSON, A. S., O'CONNOR, G. T., LORENZ, D. E., KIRSCHSTEIN, R. L., LEGALLAIS, F. Y. & TRALKA, T. S. (1971). Lymphoid cell culture line derived from lymph node of marmoset infected with herpesvirus saimiri - preliminary report. Journal of the National Cancer Institute 46, 1099-1109. RAJCANI, J., BLASKOVIC,O., SVOBODOVA,J., CIAMPOR, F., HUCKOVA, D. 8£ STANEKOVA,D. (1985). Pathogenesis of acute and persistent murine herpesvirus infection in mice. Acta virologica 29, 51~0. RAJCANI, J., BUSTAMANTEDE CONTRERAS, L. R. & SVOBODOVA, J. (1986). Corneal inoculation of murine herpesvirus in mice: the absence of neural spread. Acta virologica 31, 25 30. RICKINSON, A. B., YAO, Q. Y. 8/; WALLACE, L. E. (1985). Epstein-Barr virus as a model of virus host interactions. British Medical Bulletin 41, 75-79. RICKINSON, A. B., GREGORY, C. n., MURRAY, R. J., ULAETO, D. O. & ROWE, M. (1989). Cell-mediated immunity to Epstein Barr virus and the pathogenesis of virus-associated B cell lymphomas. In Immune Responses, Virus Infections and Disease, pp. 59-83. Edited by N. J. Dimmock & P. D. Minor. Oxford: IRL Press. ROIZMAN, B. (1982). The family Herpesviridae; general description, taxonomy, and classification. In The Herpesviruses, vol. 1, pp. 1-23. Edited by B. Roizman. New York & London: Plenum Press. SVOBODOVA, J., BLASKOVIC, D. & MISTRIKOVA, J. (1982). Growth characteristics of herpes viruses isolated from free living small rodents. Acta virologica 26, 256-263. (Received 27 February 1992; Accepted 15 May 1992) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 18:34:59