Jpn. J. Infect. Dis., 54, 144‑147, 2001

Short Communication

Immunosuppression in Rabies Virus Infection Mediatedby Lymphocyte Apoptosis
Songsri KasemplmOIpom* , Thaweesak Tirawatnapong I , Wachirapom Saengseesom,
Somboon Nookhai2 and Visith SitprlJa
Queen Saovabha Memorial Institute, World Health Organization
Collaborating Center for Resewch on Rabies Pathogenesis and2AIDS Research Center, Thai Red Cross SocieO!,
1871 Rama lVRoad, Bangkok 10330 andlKing Chulalongkorn Memorial Universio!,Bangkok 10330, Thailand(Received April 26, 2001. Accepted August 22, 2001)

SUMMARY: Previously, We demonstrated a depression of cell‑mediated immunlty ln mice by street rabies
virus infection. In the present study, We investlgated several events during the course of infection and looked foralterations in the host lymphoid cells for evidence of apoptosis. Total cellular RNA was extracted from muscle
tissues at the inoculation site of peripherally infected mice at different intervals after infection. Rabies virus
mRNA was monitored by reverse transcrlptlOn‑PCR. The length of vims localization at the site of exposure inthe muscle was as long as 5 days post‑inoculation befわre the vims entered the central neⅣous system. At thisinoculation site, the virus disappeared transiently between days 7 and 9 after infection but then was restored

thereafter until death. Annexin V‑fluorescein isothiocyanate stainlng Of splenocytes and thymocytes from mice

revealed apoptotic changes in these cells with a marked increase after day 6 0finfection･ Rabies virus antigen in
the brain became detectable 6 days after infection; this occurred parallel to the appearance of apoptosis in thelymphoid cells. There was atrophy of the spleen and thymus, with no evidence ofinfection･ Our results suggestthat the interaction between the rabies virus and infected neurons trlggerS the process of lymphoid cell apoptosis,

which renects the defective operation of the immune system.

One difficultyposed to a better.understanding of the path0‑

Ca2+ Concentration or upon alterations in protein kinase C

genesis of rabies is the identificatlOn Ofthe factors that affectactivation involved in the signal transduction pathway of T‑

lymphocytes, and how such changes lead to the cascade ofcell activation (7).

events that ends in immunosuppression. Host defenses

The overall features of immunosuppression in rabies areiヮvolve humoral and cellular immune responses (1 ,2)･ Infec‑

still not clear. We therefわre investlgated the changes in hosttlOn Of experimental animals with an attenuated strain canlymphoid cells fわr evidence of apoptosis, as well as in theinduce a strong specific immune response which results in anormal sequence of events followlng infection. We attempted

nonllethal infection, whereas mice lethally infected with street

to thereby define the mechanisms involved in the immune

rabies virTs show severe suppression of immunity (3)･suppression which occurs due to street rabies vims in mice

Currently lt remains unclear which factors are responsible

during the course of infection.

for this chain of events. Previous studies of human rabies

In the present study, 6‑ to 8‑week‑old BALB/C mice were

have shown that the process of activation, proliferation, andinfected in the hind leg with 0.03 ml ofa lO% suspension ofsalivary glands from a naturally‑infected dog. The salivary

sequestration ofnatural killer cells may not occur in rabies

patients and may reflect a naive condition (4). Only a minority(25%) of nonvaccinated rabies patients had neutralizing

glands were prepared by hoヮogenization with diluent (phos‑

phate buffered saline containing 10% fetal bovine serum, 500
units of penicillin/ml and 1 rTg of streptomycin/ml) andantibody in their seT.m at the onset of symptoms (5)･ A defectin imune recognltlOn tO rabies vims nucleoprotein (N), in

centrifugation at 600 g for 15 mln tO discard cell debris. The

addition to a depressed cell‑mediatedin1ntunity, may be involved

infectivity titer of the suspension was 1 O5･2 mouse intracerebra1

in the inefficient production of rabies vlruS‑neutralizing anti‑

50% lethal doses (LD5.)/ml･ Hindlimb paralysis is a. major

body (6). This defect also innuences the development ofneurologlCal sign of rabies infection. Signs ofparalysIS Were

specific cytotoxic T‑lymphocytes, mitogep‑induced prolifera‑

observed 10‑ 1 2 days alter infection and all mice died by days

tlOn Of T‑cells, as well as IL‑2 secretion. This has been12‑ 1 5. Five mice were sacrificed daily. At necropsy, the muscle

demonstrated in experimental mice (7)･ Moreover, tr.eatment
with a calciumionophore (A23 1 87) or phorbol 1 2‑mynstate‑ I 3

tissues at the inoculation site, the brains, thymus glands, andspleens were collected.

acetate was shown to have no influence on the unresponsive‑

Brains from mice were examined daily for evidence ofness of T‑cells to mitogens. This latter finding suggests thatinfection. Impression brain smears were fixed in 95% acetone

T‑cell suppression appears to be dependent upon a change inand stained withfluorescein isothiocyanate (FITC)‑conj ugated

T‑cell function, rather than upon changes in the cytosolicfree

antirabies globulin (Thai Red Cross production, Thailand). All

preparations were examined under a m10reSCenCe microscope.
*Corresponding author: Tel: +662‑2520161‑3, Fax: +662‑

The vims antlgen in the brain became detectable a洗er day 6254021 2, E‑mail: qsmiskp@redcross.or.th

post‑infection (data not shown).

144

The time course of rabies virus RNA persistence in theappearance of rabies virus replication in muscle fibers pr10r

muscle tissues at the inoculation site was monitored by

to its invasion of the peripheral and central nervous systems

reverse transcription (RT)‑PCR. Total cellular RNA wasextracted from the muscle tissues by homogenization in a(8,9)･ In cQntrast･ Other studies demonstrated that the virus
can also galn entrance tO the brain wimout pnor local replica‑

monophasic solution of phenol and guanidine iso血iocyanate,tion in the muscle cells, resulting ln a Short incubation period

as specified by the manufacturer (TRIzol@, GibcoBRL,(10, 1 1). The relevance of primary replication in muscle tissue
remains unknown. Nevertheless, Our study supports current

Gaithersberg, Md., USA). One step RT‑PCR was carried out
using a commercial kit (Qiagen GmbH, Hilden, Germany)

recommendations for local infiltration of virus inoculationaccording to the manufacturer's specifications･ The RNA (2sites with huヮan or equine rabies immune globulin (12)･LLg) was used in a 50 LEI reactlOn mixture. The mixture

Apoptosis IS a fわrm of programed cell death and is acontained 50 pmol each orprimers fわr rabies virus N genefundamental event in many biological processes ( 1 3). After(5'‑GACATGTCCGGAAGACTGG‑3', at position 3 1 9‑337,

initiating apoptosis, most cell types translocate the membrane

and 5′‑GTATTGCCTCTCTAGCGGTG‑3', at position 823‑

phospholipid phosphatidylserine (PS) from the inner face of842) or fb∫ β‑actin gene (5'‑ACCGTGAAAAGATGACCC

血e plasma membrane to血e cell su血ce. PS can be detected byAG‑3′, at position 266‑285, and 5㌧TCTCAGCTGTGGTGG

stainlng With an FITC conjugate Of annexin V. Extemalization

TGAAG13', at position 5 1 91538). The nucleotide sequencesofN and a‑actin genes were obtained from the GenBank
database, accession numbers NC 001542 and XO3765,
respectively. Amplificationswere performed in a thermocyclerof PS occurs earlier than the nuclear changes associated with

ApoAlert apoptosis kit (Clontech, Palo Alto, Calif., USA)

(Perkin Elmer Cetus, Norwalk, Conn., USA) for 35 cycles

according to the manufacturer's protocol. Thymus glands orapoptosis ( 14)･ To invest.igate apoptosis of lymphoid cells in

infected mice, apoptotlC Cells were identified uslng the(denaturation: 60 s at 94oC; annealing: 60 s at 55oC fわr β‑spleens from infected mice were gently pressed through 50‑

actin, and 500C for rabies virTs; extension: 60 s at 72oC)･

mmmesh stainless steel screens into Dulbecco modified Eagle

The β ‑actin and rabies vims pnmers led to the ampli丘cation

medium(DMEM). The single‑cell suspension was centrifugedof272 bp and 524 bpfragments, respectively. DNA products

at 1,000 g for 5 min. Erythrocytes were lysed by briefly

were visualized on agarose gels by ethidium bromide stainlng.

suspending the cell pellet in a 0.84% NH4CI solution. After

The β‑actin mRNA was assayed as a control or mRNAtreatment, thymocytes or splenocytes were resuspended in

DMEM and subjected to apoptosis analysis. Lymphoid cells

purification, RT‑PCR amplification, and of equal loading.
The duration of virus localization to the muscles at the site of

prepared in this way were routinely 99% viable, as assessed

exposure was as long as 5 days post‑inoculation. Persistenceby trypan blue exclusion assay. Splenocytes or thymocytesof virus in muscle cells pnor to invasion of the nervous systemwere incubated for 5 min with I LLg/ml annexin V‑FITC in

occurred, as detemined by the intensity Of the amplified

binding buffer (10 mM HEPES‑NaOH; pH 7.4, 150 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaC12), and 30,000

rabies vims N gene on daysト5 posトinoculation. Althoughvirus‑Specific RNA almost disappeared from the site ofinfection at 6 days post‑infection, it was detected again atcells were analyzed by FACScan(Becton‑Dickinson, Paramus,N.J., USA) using a single laser emitting excitation light at

this site in later stages of infection, startlng approximately on

488 nm. To distinguish cells that had lost membrane integrity,

day 10 (Fig･ 1)･ It is difficult to determine whether virus

propidium iodide dye was added to a final concentration of

persistence in muscletissues is the result of primary rq)licat10n
in muscle cells or the result ofa second cycle ofreplication

2 LLg/ml before the analysis. Annexin V‑FITC stainlng Of

initiated by virions transported centrifugallyfrom the infected
neurons back to the muscles. Most previous studies in labora‑changes in these cells, with a marked increase after day 6

splenocytes or thymocytes revealed apoptotic biologlCal
following infection (Fig. 2). The data obtained represent two

separate experiments. There was atrophy or the spleen andtory animal‑fixed rabies virus models have revealed thethymus that was parallel to development of the evidence of

apoptosis (data not shown). To further examine whetherVimsinbrain

一一一++++++

101214lymphoid cells are peripheral targets or the rabies virus,DayM

1

35

67

9splenocytes and thymocytes were assayed by RT‑PCR forrabies virus mRNA. Although murine ly.mphocytes have beenshownto be permissive to flXed rabies viruSinfection in vitro(15), no evidence of virus infection in these cells wasobserved during the cour?e of infection (data not shown)･

h our mouse model uslng Street rabies virus infection, theappearance of virus antlgen Within the brain ran parallel tothe occurrence of lymphoid cell apoptosis; this sequence ofevents began 6 days after infection, when the early slgnS Of

disease appeared. A portion or lymphoid cells underwent
apoptosIS, reaching a rate of50% ofall such cells in the latestage of disease (Fig. 2). In other words, the distribution ofrabies virus in the brain, as well as apoptosis oflymphoidcells, became more prominent as the disease advanced. Therewas also a marked decrease in the size of the spleen andthymus during the course of infection (data not shown). Hiraiet al. (16) demonstrated a marked decrease in the number ofFig. I. The time course of rabies virus RNA persistence inthe muscle

mononuclear cells in the spleen of street rabies virus‑infected

at the site of exposure, Compared to that of rabies vims antlgenmice, whereas that of mice infected with an attenuated rabies

detection inthe brain. A 524 bp fragment ofrabiesvirus N gene wasexpressed. M: molecular size markers.

virus strain increased with the infection. Moreover, the145

ACKNOWLEDGMENTS
The authors are grateful to Prof. Henry Wilde for reviewlng
the manuscrlPt, and to Drs. Tamotsu Satoh and Motohide
Takahashi for their kind encouragement.

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