Theiler's Murine Encephalomyelitis Virus in Nonbarrier Rat Colonies

Theiler’s Murine Encephalomyelitis Virus in Nonbarrier Rat Colonies

Daniele Masselli Rodrigues,1,* Sandra Soares Martins,2 Rovilson Gilioli,1 Ana Maria Aparecida Guaraldo,1 and Maria Silvia Viccari Gatti2

Theiler’s murine encephalomyelitis virus (TMEV), a member of the genus Cardiovirus, is an enteric pathogen of mice that causes acute encephalomyelitis followed by persistent central nervous system infection with chronic infl ammation and demyelination after intracerebral inoculation. Although TMEV is a mouse pathogen, antibodies against TMEV strain GDVII have been detected in conventional rat colonies. Natural infection of rats by Cardiovirus has not yet been described. The purpose of this study was to demonstrate TMEV infection of rat colonies by using serologic assays, reverse transcription–polymerase chain reaction (RT-PCR) analysis, and clinical characterization. Indirect immunofl uorescence assay of rat serum samples demonstrated antibodies against TMEV-GDVII in 86.3% of samples analyzed, and 77.2% of the antibody-positive samples had neutralizing antibodies. To determine whether rats can be infected experimentally with TMEV-GDVII, specifi c pathogen-free newborn mice and rats were inoculated intracerebrally with intestinal suspensions from seropositive rats. Both species showed the typical clinical signs of TMEV infection in mice, which is characterized by fl accid hindlimb paralysis and tremor. RT-PCR in brain tissue of experimentally infected animals detected RNA sequences corresponding to the 5′ noncoding region of Cardiovirus known as the ‘internal ribosome entry site.’ These results suggest that rats can be naturally infected with TMEV and related Cardiovirus. Therefore, continued health monitoring for TMEV infection should be included in rat colonies mainly because these animals are used for various experimental purposes.

Theiler’s murine encephalomyelitis virus (TMEV) is a nonen- of these fi ndings, the purpose of the present study was to dem- veloped single-stranded RNA virus classifi ed as a member of the onstrate that rats can be infected by mouse TMEV as detected genus Cardiovirus in the family Picornaviridae, along with en- by indirect immunofl uorescent assay (IFA). Reverse transcrip- cephalomyocarditis virus (EMCV) and Mengovirus (21, 22, 24). tase–polymerase chain reaction (RT-PCR) analysis and clinical Transmission is primarily fecal-oral and the virus multiplies in signs also were used to determine experimental infection of rats the gut without causing any symptoms (2, 15). TMEV is divided with TMEV-GDVII. A report in the literature clearly indicated into two subgroups based on neurovirulence in mice after intrace- that TMEV interfered with research that was performed using rebral inoculation. The fi rst subgroup consists of highly neurovir- infected mice (2). Therefore, serologic screening of laboratory rats ulent strains represented by GDVII and FA that cause acute and as well as mice should include testing for TMEV infection. fatal encephalitis. The second subgroup comprises less virulent strains, such as BeAn and DA, that produce persistent central Materials and Methods nervous system infection and infl ammatory demyelination of the Cell culture and virus. Baby hamster kidney (BHK-21) white matter (2, 12). Natural TMEV infection typically has been cells were cultured in minimum essential medium (MEM, Nu- reported in the laboratory mouse (2, 10, 15, 16, 27). The preva- tricell, Campinas, Brazil) containing Earle salt solution sup- lence of TMEV-GDVII infection in conventional Brazilian mouse plemented with 10% fetal bovine serum (Nutricell, Campinas, colonies is around 70.6% (5). Antibodies against TMEV in wild Brazil), 100 U/ml penicillin (Sigma Chemical Company, St. Louis, mice (Mus musculus) (14) and guinea pig were also reported (6, Mo.), and 100 μg/ml streptomycin (Sigma Chemical Company; St. 7). Few reports have been described on the natural TMEV infec- Louis, Mo. (3). The TMEV-GDVII and EMCV prototype strains tion in rats. The fi rst report was in 1964 (17) and the isolated used in all experiments were kindly provided by Dr. Volker Kraft virus showed antigens in common with TMEV (8). Japanese re- (Zentralinstitute für Versuchstierzucht, Hannover, Germany). Vi- searchers recently reported isolating a rat virus they called a ° rus was propagated at 37 C and 5% CO2 in BHK-21 cells grown ‘Theiler-like virus’ (19). According to a previous study in 18 Bra- in MEM–Earle (Nutricell, Campinas, Brazil) containing 2% zilian facilities, antibodies against TMEV-GDVII were detected fetal bovine serum. When cells displayed cytopathic effects at in sera of rats reared in nonbarrier colonies. The prevalence of 24 h postinfection, infected cells were subjected to three freeze– infection in these colonies (54.5%) suggested a large spread in the thaw cycles. Virus suspension was clarifi ed by centrifugation at environment and possible infection between rats (5). On the basis 1200 ×g for 30 min at 4°C and stored at –80°C. Virus titers were determined by the Spearman–Karber method (9) in all experi- Received: 5/2/05. Revision requested: 6/9/05. Accepted: 8/20/05. ments. The clarifi ed suspension had a 50% tissue culture infec- Multidisciplinary Center for Biological Investigation (CEMIB),1 and Laboratory tive dose (TCID ) virus titer of about 105.1 per ml. of Virology,2 Department of Microbiology and Immunology, Institute of Biology, 50 State University of Campinas (UNICAMP), São Paulo, Brazil. Neutralizing anti-TMEV-GDVII serum. BALB/c/UNI mice *Corresponding author. (n = 10, 4 weeks old) and WISTAR/UNI rats (n = 8, 4 weeks old)

459 Vol 55, No 5 Comparative Medicine October 2005 were obtained from the Multidisciplinary Center for Biological Cambridge, Mass.). After each dilution, serum was mixed with Investigation (CEMIB/UNICAMP, Campinas, Brazil). Animals an equal volume of TMEV-GDVII virus suspension containing μ ° came from colonies certifi ed free of the following specifi c patho- 100 TCID50%/100 l. After adsorption for 90 min at 37 C, BHK- gens: mouse parvovirus, rat parvovirus, mice minute virus, mouse 21 cells (2 × 105 cells in 100 μl) were added to each well, and the ° hepatitis virus, rat coronavirus, Sendai virus, pneumonia virus plates were incubated at 37 C in 5% CO2. Positive and negative of mice, Theiler’s murine encephalomyelitis virus, reovirus 3, controls were included in each test performed. Neutralizing anti- mouse rotavirus, ectromelia virus, lymphocytic choriomeningitis body titer was determined 24 to 48 h after infection. virus, mouse adenovirus, polyoma virus, mouse cytomegalovirus, Experimental infection. For this purpose, specifi c pathogen- Kilham’s rat virus, Toolan’s H-1 virus, Mycoplasma pulmonis, free newborn BALB/c/UNI mice and WISTAR/UNI rats were other known pathogenic bacteria, and ecto- and endoparasites. used. All procedures adopted in this experiment were approved All animals were maintained in fl exible polyvinylchloride isola- by the Ethical Committee on Animal Experimentation of the tor units and supplied with sterilized food and water. BALB/c/ Institute of Biology, State University of Campinas (protocol no. UNI mice were inoculated intraperitoneally with a TMEV-GD- 424-1). For the fi rst experiment, mice (n = 5) and rats (n = 5) were VII suspension (50 ng/ml). For WISTAR/UNI rats, the same sus- inoculated intracerebrally with 0.03 ml (mice) or 0.06 ml (rats) pension with complete Freund’s adjuvant (1:1) was inoculated of 1:10-diluted intestinal suspension. Mice and rats (n = 5 each subcutaneously at day 0, followed by a booster with incomplete group) serving as negative controls were inoculated intracere- Freund’s adjuvant at day 7. Sera from rats and mice were har- brally with PBS and maintained in a separate barrier room. Ad- vested 28 days after inoculation, and antibodies against TMEV- ditional groups of newborn mice (n = 9) and rats (n = 9) were used GDVII were analyzed by immunofl uorescence and neutralization as positive controls and inoculated intracerebrally with 0.03 ml assay. Sera obtained from noninoculated mice and rats were used (mice) or 0.06 ml (rats) of TMEV-GDVII (1 × 10-4). Animals were as negative controls. inspected daily for clinical signs of infection and were euthanized

Specimens. Blood samples were obtained by cardiac puncture under CO2 anesthesia when signs of infection became apparent. ° of 22 Wistar rats (age, 4 to 8 months) under carbon dioxide (CO2) Brain tissue was aseptically collected and was stored at –70 C anesthesia; these animals had been reared under conventional until RT-PCR tests. A 10% brain suspension prepared in PBS conditions. After heat inactivation at 56°C for 30 min, serum was used for the second inoculation, and the same numbers of samples were stored at –20°C until used. Fecal samples were rats and mice were used (15). obtained from 48 Wistar rats (age, 3 weeks to 1 year) from four Viral RNA extraction. Total RNA was extracted by using different animal facilities. A fecal extract (10% w/v) was prepared TRIzol reagent (Invitrogen Life Technologies, Carlsbad, Calif.) in phosphate-buffered saline (PBS; 0.01 M, pH 7.2), clarifi ed by according to the manufacturer’s instructions. The protocol was centrifugation at 1000 ×g for 20 min (Beckman Instruments, JC- applied to the following samples: brain samples obtained from 21 rotor), and stored at 4°C until used. controls and experimental inoculated animals, intestinal sus- For experimental infection, 10 Wistar rats (4 and 8 months pensions from seropositive rats, and fecal suspensions from rats old) from a conventional colony were euthanized under CO2 an- from conventional colonies. esthesia. The small intestine was removed and rinsed with PBS. Oligonucleotide primers. Primers 1 (5′ CGA AGC CGC An intestinal suspension (20% w/v) was prepared and clarifi ed TTG GAA TA 3′; nucleotides [nts] 529 through 545 of TMEV, nts by centrifugation at 1000 ×g for 20 min at 4°C. The supernatant 291–307 of EMCV) and 2 (5′ ACG TGG CTT TTG GCC GCA GAG was mixed with an equal volume of chloroform (Merck, Darm- G 3′;nts 790–812 of TMEV, nts 552–575 of EMCV) detect the in- stadt, Germany), vortexed for 5 min, and then centrifuged at ternal ribosome entry site of the cardioviruses TMEV and EMCV 11,000 ×g for 10 min at 4°C. The aqueous phase was recovered, (28) and were synthesized by Invitrogen Life Technologies. fi ltered through a 0.22-μm fi lter (Millipore, Bedford, Mass.), and Reverse transcription (RT) and PCR amplifi cation. The stored at –70°C (20). following procedures were done according to a published method Serologic assays. (i) IFA. IFA was carried out according to (23). Extracted RNA was reverse-transcribed in a fi nal volume of the method previously described (11). Briefl y, a confl uent mono- 20 μl containing 5× fi rst-strand buffer (50 mM Tris-HCl, 75 mM layer of BHK-21 cells was inoculated with a predetermined virus KCl, 3 mM MgCl2), 0.5 mM dNTPs, 10 mM dithiotreitol (DTT), dilution to yield infection of 10 to 30% of the culture. After ad- 125 pmol primer 2, and 100 U of Moloney murine leukemia virus sorption for 90 min at 37°C, each well was supplied with MEM- reverse transcriptase (Invitrogen Life Technologies). The reac- Earle containing 2% bovine fetal serum. After 24 h of infection, tion was performed at 37°C for 1 h, and then the enzyme was the slides were air-dried, fi xed with cold acetone for 10 min, and inactivated by incubating at 70°C for 15 min. Synthesized cDNA stored at –20°C until used. The sera to be tested were titrated (5 μl) was added to 50 μl of a PCR reaction mixture consisting of: at serial twofold dilutions (1:5 to 1:160) in PBS, incubated for 30 10× PCR buffer (20 mM Tris-HCl, 50 mM KCl), 0.5 mM dNTPs, min with the prepared BHK-21 cells, and then incubated with a 80 pmol each of primers 1 and 2, and 2.5 U Taq DNA polymerase fl uorescein isothiocyanate conjugate (anti-rat IgG–FITC, Sigma). (Invitrogen Life Technologies). The PCR reaction was performed The slides were washed twice with PBS for 10 min each wash in a Gene Amp PCR System 9700 thermocycler (Perkin Elmer and read under a fl uorescence microscope (Standard 20, Zeiss, Applied Biosystems, Norwalk, Conn.). The samples were de- Oberkochen, Germany). Positive and negative controls were in- natured at 94°C for 5 min and then underwent 30 cycles of cluded in each test. 94°C for 2 min; 63°C for 2 min; and 72°C for 1 min, with a fi nal (ii) Neutralization assay. Neutralization assays of the 22 rat elongation step of 72°C for 7 min. Positive (TMEV-GDVII and sera were performed as previously described (4). Briefl y, twofold EMCV) and negative (diethyl pyrocarbonate [DEPC]-treated dilutions of the rat serum were prepared with MEM-EARLE and water) controls were included in each reaction. PCR products added to 96-well, fl at-bottom microtiter plates (Costar, Broadway, were electrophoresed on 1.5% (wt/vol) agarose gel, which was 460 Theiler’s murine encephalomyelitis virus in rat colonies

B C

Figure 1. (A) Newborn (3-day-old) BALB/c/UNI mouse inoculated intracerebrally with intestinal suspension of rats seropositive for TMEV-GDVII. Weight loss and hindlimb paralysis, 2 days postinoculation. (B) Newborn (3-day-old) BALB/c/UNi noninoculated mouse. (C) Newborn BALB/c/UNI mouse inoculated intracerebrally with the prototype strain (TMEV-GDVII). Flaccid paralysis and weight loss, 4 days postinoculation. stained with ethidium bromide (0.5 μg/μl) and visualized and testinal or TMEV-GDVII suspension yielded positive results for photographed under UV transillumination (MicroVue, Pharma- viral RNA (lanes 2 through 6). A representative positive rat fecal cia LKB, Pittsburgh, Pa.). DNA molecular-weight markers (100- sample derived from a conventional colony is shown in Fig. 5. bp ladder, Invitrogen Life Technologies) were used for accurate sizing of PCR products. Discussion TMEV infection has been of considerable importance to bio- Results medical research since the virus was first described by Max Serologic analysis. IFA analysis of sera obtained from rats Theiler (25, 26). The frequency of natural infection of mouse and mice used in neutralization assays for TMEV-GDVII revealed colonies with TMEV-GDVII in Brazilian facilities was reported specifi c positive reactions at dilutions of ≤ 1:80 in PBS. Sera from to be 70.6% (5), and similar results have been described for many 19 of 22 conventionally housed rats were positive by IFA at dilu- other countries (10, 27). In addition, anti-TMEV-GDVII antibod- tions of ≤ 1:80 in PBS for antibodies against TMEV-GDVII. Test- ies have been detected in conventional rat colonies in Brazilian ing the same sera by neutralization assay with TMEV-GDVII facilities at a reported frequency of 54.5% (5). Other researchers yielded positive test results in only 17 of the samples. have reported that the prevalence of TMEV-GDVII infection in Experimental inoculation. Mice inoculated with intesti- rat colonies is around 44.4% (27) or 20.0% (10). nal suspensions from rats suspected to be infected with TMEV- In the present study, we aimed to demonstrate that rats can GDVII demonstrated fl accid hindlimb paralysis, tremor, weight be infected by mouse TMEV. We used IFA analysis to evaluate loss, and inactivity on the fi rst and second days after inoculation 22 serum samples from rats without any clinical signs of infec- during the fi rst passage (Fig. 1A). Animals inoculated with PBS tion; 19 of these 22 samples (86.3%) contained anti-TMEV-GDVII lacked clinical signs of infection (Fig. 1B). By the third day pos- antibodies, indicating natural infection by this or other antigeni- tinoculation, all mice had died. In contrast, during the second cally closely related viruses. Positive reactions were detected at passage, when brain tissue from affected fi rst-passage animals low serum dilutions (≤ 1:80), which might imply early infection was used as the inoculum, the clinical signs were the same, but or cross-reaction among viruses belonging to the same genus. the animals died on Day 10. Mice inoculated with TMEV-GDVII When we used a neutralization assay to verify the presence of strain (Fig. 1C) showed the same clinical signs described for rats specifi c antibodies against TMEV-GDVII, we detected neutral- on Day 4, although the mice died on Day 5. During both pas- izing antibodies in 17 of the 22 samples (77.2%) tested. sages, rats inoculated with PBS lacked clinical signs of infection In experimental infections, we intracerebrally inoculated new- (Fig. 2A). Infected rats showed the same clinical signs as those in born SPF mice and rats with an intestinal suspension from se- mice, but paralysis persisted until 10 days postinoculation (Fig. ropositive rats. Clinical signs in mice occurred between the third 2B). Rats inoculated with the prototype virus strain (Fig. 2C) and tenth days postinoculation and were characterized by fl accid showed hindlimb paralysis, weight loss, and debility 3 days after paralysis of the hind limbs, tremor, weight loss and death at Day inoculation and died on the fourth day. 10. Similar clinical signs have been described by other research- RT-PCR analysis. RT-PCR was used to determine the optimal ers, but animal death was related to the virus strain and dose concentrations of primers 1 (P1) and 2 (P2) for the TMEV-GDVII used in those studies (25, 26). Mice inoculated with TMEV-GD- and EMCV prototype strains. These strains yielded bands of 283 VII (i.e., positive controls) died on the third day after inoculation, bp for TMEV-GDVII and 284 bp for EMCV (data not shown). similar to results from other researchers (12, 17, 26). Figure 3 presents the RT-PCR amplifi cation of cDNA transcribed Suggesting TMEV infection of this species, rats inoculated with from viral RNA in the brains of mice inoculated with TMEV-GD- the intestinal suspension showed clinical signs similar to those VII. In Fig. 4, brain tissue of rats inoculated with either the in- in mice but remained alive until the tenth day postinoculation 461 Vol 55, No 5 Comparative Medicine October 2005

Figure 3. PCR products of 283 bp (indicative of TMEV) obtained with primers 1 and 2 from brain samples of mice inoculated with intestinal suspension or the prototype strain TMEV-GDVII. MW, DNA molecular weight marker (100-bp ladder); lane 1, brain from noninoculated control mice; lane 2, brain from mice inoculated with intestinal suspension; lane 3, brain from mice inoculated with TMEV-GDVII; lane 4, negative control B (DEPC-treated water); lane 5: positive control (TMEV-GDVII).

Figure 4. PCR products of 283 bp (TMEV) or 284 bp (EMCV) obtained with primers 1 and 2 from brain tissue harvested 11 days postinoculation of normal rats with intestinal suspension or TMEV-GDVII. MW, DNA molecular weight marker (100-bp ladder); lane 1, noninoculated rat; lanes 2 and 3, rats inoculated with intestinal suspension; lane 4, C rat inoculated with cerebral suspension from symptomatic rats; lanes 5 and 6, rats inoculated with TMEV-GDVII; lanes 7 and 8, negative controls (DEPC-treated water); lane 9, TMEV-GDVII positive control; and lane 10, EMCV positive control.

during both the fi rst and second passages. Unlike our fi ndings, those of a previous study indicated that rats experimentally inoculated with an intestinal suspension from rats seropositive for TMEV did not show clinical signs typical of TMEV infection (19). Further, although our rats that were inoculated with TMEV-GD- VII virus showed the same clinical signs as similarly inoculated mice, the rats died 4 days later, suggesting a less virulent strain for these hosts or fewer virus replicative cycles, as typically is observed for Enterovirus infection (13). Persistence of symptoms and survival can be related to the se- lection of less-virulent strains (18, 29). We performed a RAP test Figure 2. (A) Noninoculated WISTAR/UNi rat (11 days old). (B) (1), which successfully confi rmed that no enteric murine viruses WISTAR/UNI rat (11 days old) inoculated intracerebrally with in- other than TMEV were present in the intestinal suspension. To testinal suspension from rats seropositive for TMEV-GDVII. Ruffl ed associate the observed clinical signs to TMEV infection, we used hair coat and fl accid hind limb paralysis, 9 days postinoculation. (C) RT-PCR to verify the presence of Cardiovirus in the brains of WISTAR/UNI rat (4 day old) inoculated intracerebrally with the prototype strain (TMEV-GDVII). Flaccid paralysis and weight loss, 2 all mice and rats inoculated intracerebrally with TMEV-GDVII days postinoculation. viral preparations and in intestinal suspensions from seroposi-

462 Theiler’s murine encephalomyelitis virus in rat colonies

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