relationship of two arthropod-borne rhabdoviruses (kotonkan and

INFECTION AND IMMUNITY, Nov. 1975, p. 1157-1172Copyright (© 1975 American Society for Microbiology

Vol. 12, No. 5Printed in U.SA.

Relationship of Two Arthropod-Borne Rhabdoviruses(Kotonkan and Obodhiang) to the Rabies Serogroup

SALLY P. BAUER* AND FREDERICK A. MURPHY

Center for Disease Control, Atlanta, Georgia 30333

Received for publication 30 June 1975

Indirect immunofluorescence confirmed the antigenic relationship betweenkotonkan and Obodhiang viruses and Mokola virus that had originally beenshown by complement fixation test. This relationship suggests inclusion of thesetwo arthropod isolates in the rabies subgroup of the Rhabdoviridae family.Cross-reactivity with Mokola virus was also demonstrated by direct immunofluo-rescence but was easily eliminated when conjugates were diluted. No cross-reactivities were found by neutralization tests or by surface immunofluores-cence. Other than these immunological ties to the rabies serogroup, otherbiological characteristics of kotonkan and Obodhiang viruses were distinct.Maximum yield of infectivity of kotonkan and Obodhiang in cell culture was at30 C, antigen usually filled the cytoplasm of infected cells diffusely, and syncytiawere formed before severe cytonecrosis. By electron microscopy, virus particlesand their nucleocapsids appeared cone shaped (mean lengths: kotonkan, 182 nm;Obodhiang, 170 nm). Viral morphogenesis took place on plasma membranes ofcells in culture, mouse brain neurons, and inflammatory cells (macrophages) inbrain lesions. All of these characteristics of the two viruses, and the knownassociation of kotonkan virus with an acute, febrile illness of cattle in Nigeria,suggest a biological relationship with bovine ephemeral fever virus. The latter isknown to exist in the same geographic area but exhibits no serological cross-reaction with either kotonkan or Obodhiang virus. The question of whetherthese two viruses deserve placement in an expanded rabies subgroup (at the costof a less precise definition of the subgroup) or in a separate subgroup (whichwould include bovine ephemeral fever virus) of the Rhabdoviridae family willonly be answered by further physicochemical characterization and comparison.

Taxonomic subgrouping with the familyRhabdoviridae has been complicated by biologi-cal and physicochemical diversity of memberviruses and by distant and poorly understoodserological cross-reactions among some viruses.Two viruses, kotonkan and Obodhiang, are ofparticular concern because of their demon-strated serological relationship to Mokola vi-rus, and thereby to the rabies serologicalsubgroup (7; R. E. Shope, 1975, in G. M. Baer,ed., The Natural History of Rabies, in press).(Obodhiang is a virus name proposed by J. B.Schmidt. The name has not yet been publishedand its mention here is not intended to consti-tute priority.) Although neither Mokola nor ra-bies virus is known to perpetuate itself in na-ture by way of arthropod vectors, evidence indi-cates that kotonkan and Obodhiang viruses aretrue "arboviruses." (Arboviruses are definedwithout regard to physicochemical propertiesas viruses that are maintained in nature princi-pally through biological transmission betweensusceptible vertebrate hosts by hematophagous

arthropods. An arbovirus replicates in both itsvertebrate and arthropod host [19].) Obodhiangvirus was isolated in the Sudan in 1965 frompools of Mansonia uniformis mosquitoes (16).Kotonkan virus was isolated from Culicoidesspp. in Nigeria (7). Little is known of the natu-ral history of Obodhiang virus, but Kemp et al.(7) showed serologically that large numbers ofcattle in Nigeria had been infected with koton-kan virus and that sero-conversions were associ-ated with an acute, febrile illness. This diseaseof cattle clinically resembled bovine ephemeralfever (BEF), which also is caused by a rhabdovi-rus (5, 12), and is known to exist in Nigeria (8).Despite repeated neutralization and comple-ment fixation testing, no relationship betweenkotonkan and BEF viruses has been estab-lished (7, 8, 12). There is no known associationof these viruses with human disease.The tentative conclusion that kotonkan and

Obodhiang viruses bridge the gap between therabies subgroup and arthropod-borne viruses ofthe Rhabdoviridae family prompted the pres-

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1158 BAUER AND MURPHY

ent study. This bridging involves the most di-verse natural history patterns-from the verte-brate bite-transmitted neurotropic pattern ofrabies to the arthropod bite-transmitted epithe-liotropic pattern of BEF and vesicular stomati-tis viruses. Further characterization of koton-kan and Obodhiang viruses is required, how-ever, before the implications of this bridgingcan be adequately determined.

MATERIALS AND METHODS

Viruses. Kotonkan virus, prototype strain IbAr23380, and Obodhiang virus, prototype strain SudAr1154-64, were kindly provided by R. E. Shope, YaleArbovirus Research Unit, New Haven, Conn. Virusstocks consisted of suckling mouse brain suspen-sions: kotonkan virus had been passaged in sucklingmouse brain 9 or 12 times and had an infectivitytiter of 107.8 median lethal doses (LD50) per g upon

intracerebral inoculation of 1-day-old mice. Obod-hiang virus had been passaged similarly six timesand had a titer of 106.8 LD50/g. Adaptation and prepa-ration of cell culture stocks involved six BHK-21 cellpassages plus four Vero cell passages for kotonkanvirus and 13 Vero cell passages for Obodhiang virus.Cell culture-adapted virus stocks had infectivity ti-ters of 1053 and 1043 sucking mouse intracerebralLD,dml, respectively. Viruses were not cloned orpassaged in arthropod cells (1, 2). Other rhabdovi-ruses and antisera were from the collection of ViralPathology Branch, Center for Disease Control.

Cell cultures and viral growth curves. Monolay-ers of Vero cells (CCL 81) or BHK-21 cells (CCL 10)from the American Type Culture Collection were

inoculated with the cell culture-adapted virus stocksat varying multiplicities of infection (always limitedby low titers of stocks) and maintained with en-

riched Eagle minimal essential medium containingfetal calf serum (5% growth, 2% maintenance).BHK-21 and Vero cell cultures were used for immu-nofluorescence and electron microscopic studies. Forgrowth curve experiments, Vero cells in tubes wereinfected at highest multiplicities possible (multiplici-ties of infection of 1.2 for kotonkan and 0.2 for Obod-hiang), and adsorption and maintenance were car-ried out in water baths at 30, 34, and 37 C. Cultureswere harvested at 10 min and 1, 4, 8, 16, 24, 34, 48,and 72 h after the initial 1-h adsorption period. Thedegree of cytopathology was determined at each har-vest, and cultures were then brought immediately to4 C. Cells were scraped and separated from mediumby centrifugation at 1,000 x g for 15 min and thenresuspended in the original volume of fresh me-

dium. Cell and supernatant specimens were storedin multiple aliquots at -76 C. Titrations of eachspecimen were carried out in mice and by fluores-cence focus assay in Vero cells. Newborn ICR Swissmice were inoculated with 10-fold dilutions, andLD50 values were calculated by the method ofKarber (6). Similar serial dilutions were inoculatedonto Vero cells grown in Lab-Tek slide chambers(eight wells/slide; Miles Laboratories, Naperville,Ill.) and incubated for 24 h under a 3% CO2 atmos-phere. Slides were then acetone fixed and treated.

with homologous fluorescein isothiocyanate (FITC)-conjugated antisera; foci were counted with double-blind coding, and end points were determined.

Immunofluorescence. For direct immunofluores-cence, hyperimmune mouse ascites fluids (MAF)were produced by using a multiple-injection course,Freund complete adjuvant, and sarcoma TG180cells. Immunoglobulins were fractionated by diethyl-aminoethyl-Sephadex chromatography and conju-gated with FITC as described previously (13). Forindirect immunofluorescence, twofold dilutions ofthe same MAF were used as well as goat anti-mouseimmunoglobulin G-FITC conjugate obtained fromHyland Laboratories. Substrates included infectedVero cells, suckling mouse brain impressions, andfrozen sections of whole suckling mice and sucklinghamster organs. For surface immunofluorescence,infected Vero cells were treated with the same conju-gates by the direct method; however, the usual 10min/25 C acetone fixation was omitted, and the via-ble cells were finally mounted in a 1:1 glycerine-buffered saline mixture for immediate examination.Specimens were examined in a Zeiss microscopeequipped with a UG-1/41 filter system and mercuryarc excitation. In all immunofluorescence tests, non-infected substrates were used as controls. The speci-ficity ofthe conjugates was determined by preadsorp-tion of each conjugate with either infected or nonin-fected mouse brain material before staining the sub-strates. Inhibition tests involved pretreatment ofthe substrates with homologous or normal MAF be-fore staining with the conjugate. Normal MAF dilu-tions on substrates were included in the controls forindirect immunofluorescence. All controls were un-remarkable.

Virus concentration and partial purification.The two viruses, propagated in Vero cells, werepurified according to the method of Obijeski et al.(15). Briefly, virus was precipitated from cell culturematerial by treatment with polyethylene glycol(PEG 6000), and the resulting concentrate was puri-fied by viscosity-density gradient equilibrium cen-trifugation. Preparations from visible bands wereexamined by negative-contrast electron microscopyfor which 2% sodium silicotungstate, pH 6.7 (12),was used as well as magnifications calibrated withcatalase crystals (20).

Experimental animals and histopathology. Thetwo viruses were inoculated intracerebrally and in-traperitoneally into newborn ICR Swiss mice andnewborn Syrian hamsters. Only mice inoculated in-tracerebrally developed signs of illness; most ani-mals became ruffled and comatose on days 6 and 7after kotonkan virus inoculation and on days 4 and 5after Obodhiang virus inoculation, although in bothcases death patterns were erratic and some animalsdied as late as 14 days after inoculation. Multipleorgan specimens and whole carcasses were preparedfor tissue titration, frozen-section immunofluores-cence (13), and for routine histology (formalin fixa-tion and hematoxylin and eosin staining of paraffin-embedded sections). Hamster tissues collected fromdays 3 to 26 postinoculation were studied in parallel.

Electron microscopy. For thin-section electronmicroscopy, cell culture pellets and brain tissues

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TWO NEW RHABDOVIRUSES 1159

from moribund mice infected with the two viruseswere cut into 1-mm3 blocks and fixed in 2.5%buffered glutaraldehyde at 4 C for 2 h. Specimenswere treated with 1% osmium tetroxide, dehy-drated, and embedded in an Araldite-Epon mixture(11). Sections were stained with uranyl acetate andlead citrate and examined in a Philips EM-200 micro-scope operating at 60 kV.

RESULTS

Immunological relationships and immuno-fluorescence. A weak antigenic relationshipbetween kotonkan and Obodhiang viruses andMokola virus was detected previously by cross-complement fixation testing (7; Shope, inpress). When suckling mouse brain impressionsor infected cell cultures were used as viral anti-gen substrates and MAF against each of theviruses of the rabies serogroup, the same rela-tionships as found with complement fixationwere reproducibly obtained by indirect immuno-fluorescence (Table 1). All reagents used inthese tests were different from those used previ-ously for complement fixation (7; Shope, inpress). Mokola virus and its antiserum cross-reacted with kotonkan and Obodhiang systems;reactivities with rabies and Lagos bat viruseswere nil. Thus, the concept that Mokola virushas the broadest antigenic reactivity in the ra-bies serogroup was confirmed (S. M. Buckley,Trans. N. Y. Acad. Sci., in press). Additionally,one-way indirect immunofluorescence testingwith BEF virus MAF on cell cultures infectedwith kotonkan and Obodhiang viruses was neg-ative; BEF virus was not available because ofDepartment of Agriculture prohibition.

Direct immunofluorescence studies of thesame relationships were undertaken becausediagnostic and identification ambiguities are

more likely to stem from varying cross-reactivi-ties of different FITC conjugates. When FITCconjugates for each of the viruses of the rabiesserogroup were reacted with viral antigen sub-strates (either suckling mouse brain impres-sions or infected BHK-21 or Vero cells), resultsvaried with conjugate dilutions. Great excesses

of conjugates (e.g., 1:5 or 1:10 dilutions of conju-gates with 4+ brightness end points at 80 to160) produced cross-reactivities that appearedas dim, reproducible labeling with the samepatterns as bright homologous systems (Table2). When working dilutions of the same conju-gates were used (e.g., twofold to fourfold excess

over a 4+ brightness end point dilution), thesecross-reactivities with Mokola virus could notbe discerned (Table 3). In all cases, immunofluo-rescence patterns of BHK-21 and Vero cellsinfected with kotonkan or Obodhiang virusesconsisted of a combination of very fine, homoge-neous antigen smoothly spread through the cy-

toplasm of infected cells and of antigenic aggre-gates of varying size (Fig. 1 and 2). Often thesetwo patterns occurred in different cells, butdespite serial harvests no sequence of develop-ment was discerned. In Vero cells, both virusesalso caused syncytia (Fig. 3), but these occurredcoincident with the development of cytopathicchanges beginning at 40 h.

Neutralization tests in suckling mice previ-ously showed minimal cross-reactions betweenkotonkan and Obodhiang and the other virusesof the rabies serogroup (7; Shope, in press);Buckley confirmed this with plaque reductionneutralization tests in Vero cells (Trans. N.Y.Acad. Sci., in press). In an attempt to studycross-relationships of viral ,antigens expressedon the surface of virus particles and infectedcells, and to supplement uninterpretable data

TABLE 1. Indirect immunofluorescence cross-reactions ofkotonkan and Obodhiang viruses with members ofthe rabies serogroup

Viruses (suckling mouse brain impressionsa)

Immune ascitic fluid Rabies

CVS Arctic Mokola Lagos bat Kotonkan Obodhiangstrain foxb

Rabies (CVS) ................ 1,024c 512 128 16 Od 0Mokola ...................... 128 256 1,024 512 16 64Lagos bat ................... 16 32 128 128 0 0Kotonkan ................... 0 0 16 0 256 16Obodhiang .................. 0 0 64 0 0 1,024

a Substrates shown to be heavily infected via homologous direct immunofluorescence.b An arctic fox street rabies isolate in second mouse brain passage.c Ascitic fluid titer. The end point was determined as that dilution where immunofluorescence diminished

from .3+ to 2+ or less (intensity scale of 4+ to nil).d 0, No reaction at 1:8 ascitic fluid dilution.

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TABLE 2. Direct immunofluorescence cross-reactions of kotonkan and Obodhiang viruses with members ofthe rabies serogroup (excess concentrations of conjugates)

Viruses (46 h of infection in BHK-21 cellsa)

Conjugate Rabies

CVS ERA Mokola Lagos bat Kotonkan Obodhiangestrain strain

Rabies (CVS) ........ 4c 4 2 2 0 0Mokola ... ..... 2 2 4 3 1 1Lagos Bat .... .... 0 0 3 4 0 0Kotonkan .... .... 0 0 0 0 4 0Obodhiang .... .... 0 0 1 0 0 4

a Substrates shown to be 80 to 100% infected via homologous direct immunofluorescence.b Obodhiang virus in Vero cells. Conjugate dilutions were used in amounts greater than fourfold excess

over 4+ brightness end point dilutions (intensity scale of 4+ to nil).c Intensity scale of 4+ to nil. Conjugates were used at dilution amounts greater than fourfold excess over

4+ brightness end point dilutions.

TABLE 3. Direct immunofluorescence cross-reactions of kotonkan and Obodhiang viruses with members ofthe rabies serogroup (working dilutions of conjugates)

Viruses (46 h of infection in BHK-21 cellsa)

Conjugate Rabies

CVS ERA Mokola Lagos bat Kotonkan Obodhiangpstrain strain

Rabies (CVS) ........ 4c 4 1 trd 0 0Mokola ... ..... 1 2 4 3 0 0Lagos Bat .... .... tr 1 2 4 0 0Kotonkan .... .... 0 0 0 0 4 0Obodhiang .... .... 0 0 0 0 0 4

a Substrates shown to be 80 to 100% infected via homologous direct immunofluorescence.b Obodhiang virus in Vero cells.c Intensity scale of 4+ to nil. Conjugates were used at dilution amounts in two- to fourfold excess over 4+

brightness end point dilutions.d tr, Trace.

stemming from low-level cross-neutralization,we developed a surface immunofluorescencetechnique for the viruses of the rabies sero-group (Table 4). BHK-21 and Vero cell cultureswere infected with the viruses, and at 24 and 46h conjugates were applied at concentrationsused in Table 3, but without prior fixation (Fig.4). Identical specimens were acetone fixed andstained with the same conjugates (Fig. 1); by 46h, 80 to 100% of the cells in all cultures wereinfected. Surface immunofluorescence on 10 to80% of the viable'infected cells appeared as finebeaded necklaces of antigen at the periphery ofcells with a lower concentration over the sur-face of cells (Fig. 5 versus Fig. 6). Backgroundswere negative, and no immunofluorescent inclu-sion bodies were seen unless acetone fixationwas used. Interpretations were the same oncells after 24 and 46 h of infection, but becauseof beginning cytopathic changes in cells in-fected with kotonkan and Obodhiang viruses by

40 h, both harvests were evaluated. These im-munofluorescent surface patterns were only ex-hibited by homologous systems; no relationshipbetween kotonkan and Obodhiang viruses.andthe other members of the serogroup was found.Growth in cell culture. Kotonkan and Obod-

hiang viruses were previously shown to behavedifferently from rabies virus in cell cultures; forexample, they were propagated in mosquitocells by Buckley (1) and were shown to be rap-idly cytopathic in Vero and BHK-21 cells. Anattempt to describe the kinetics of growth of thetwo viruses was made for comparison withother rhabdoviruses, particularly BEF virusand rabies virus strains (Fig. 7). Obodhianggrowth curves were similar except that peaktiters were 10-fold lower; infectivity assays bysuckling mouse brain inoculation and by fluo-rescence focus counting agreed closely. Underthe conditions described, single-cycle curveswere not obtained because of the conflicting

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FIG. 1. Homologous specific immunofluorescence in BHK-21 cells infected with kotonkan virus. Antigentypically was aggregated as well as finely dispersed throughout the cytoplasm of infected cells. x520.

FIG. 2. Specific immunofluorescence in Vero cells infected with Obodhiang virus. The fluorescent patternofthis acetone-fixed culture was finely particulate and smoothly dispersed throughout the cytoplasm. x520.

FIG. 3. Vero cells infected with kotonkan virus at 40 h. Syncytia formed in the period before the develop-ment of cytonecrosis. x520.

FIG. 4. Surface immunofluorescence of Vero cells infected with Obodhiang virus at 24 h. Finely beadednecklaces of antigen were present at the periphery and in lesser concentration over the surface of the viablecells. This pattern may be contrasted with that of the acetone-fixed culture illustrated in Fig. 2. x520.

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TABLE 4. Surface immunofluorescence cross-reactions ofkotonkan and Obodhiang viruses with members ofthe rabies serogroup

Viruses (24 and 48 h of infections in BHK-21 cells in situa)

Conjugate RabiesCVS ERA Mokola Lagos bat Kotonkan Obodhiangestrain strain

Rabies (CVS) ........ 4c 4 0 trd 0 0Mokola ... ..... 0 tr 4 tr 0 0Lagos Bat .... .... tr 0 tr 3 0 0Kotonkan .... .... 0 0 0 0 4 0Obodhiang .... .... 0 0 0 0 0 4

a Substrates shown to be 80 to 100% infected via homologous direct immunofluorescence of acetone fixedparallel cell cultures.

b Obodhiang virus in Vero cells.c Intensity of scale 4+ to nil. Conjugates were used at dilution amounts greater than fourfold excess over

4+ brightness end point dilutions.d tr, Trace,

FIG. 5. Immunofluorescence of acetone-fixed BHK-21 cells infected with rabies (ERA strain) virus at 24h. Large intracytoplasmic inclusion bodies were present along with dust-like antigen. x360.

FIG. 6. Surface immunofluorescence ofviable BHK-21 cells infected with rabies (ERA strain) virus at24 h.Fine beaded necklaces of antigen appeared at the periphery of cells, but in contrast to Fig. 5, no inclusionswere visible. x360.

1162 BAUER AND MURPHY INFECT. IMMUN.

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HOURS POST ADSORPTIONFIG. 7. Growth curves of kotonkan virus in Vero cells at three different temperatures. Maximum growth

occurred at 30 C, and virus was primarily cell associated. Symbols: A A, 30 C cells; A- -A, 30 Csupernate; *-*, 34 C cells; U- -U, 34 C supernate; * *, 37 C cells; 0--@, 37 C supernate.

dictates regarding multiplicity of infection andT particle defective interference. However,large populations of cell-associated virus werefound consistently, and maximum titers werereached after only 48 h for kotonkan virus and34 h for Obodhiang virus, and in both cases atthe lowest temperature used (30 C).

Negative-contrast and thin-section elec-tron microscopy. Kotonkan and Obodhiangviruses, propagated in BHK-21 and Vero cellcultures, were examined by negative-contrastelectron microscopy after minimal manipula-tion and after the described concentration-puri-fication scheme. Bullet-shaped virus particleswere observed in all preparations together withT particles that varied in number according tothe usual conditions favoring defective interfer-ence (Fig. 8). Substructural details were simi-lar to those of other rhabdoviruses; an 8-nmsurface projection layer covered the viral enve-lope, and an axial channel was often prominentin the flat base (Fig. 9). Virus particle shapewas usually conical but the cone shapes variedwith the degree of flattening. Virus envelopefusion was commonly observed (Fig. 10); previ-ously this had only been seen with BEF virus.When negative-contrast medium penetrateddisrupted virus particles, helical ribonucleocap-sids were seen (Fig. 11 and 12). These struc-tures, which had a beaded substructure andunraveled into a single strand (Fig. 13), werealso cone shaped, indicating that the variationfrom the typical parallel-sided shape of rhabdo-viruses was not a flattening artifact. A koton-kan virus preparation containing very few Tparticles and an Obodhiang virus preparationwith many T particles were used for illustration

(Fig. 14 and 15). Mean B particle lengths were182 and 170 nm for kotonkan and Obodhiangviruses, respectively. Most T particles wereshort (mean length, 85 nm), but in other prepa-rations large numbers of intermediate particleswere present.

Infected Vero and BHK-21 cells were exam-ined by thin-section electron microscopy at 24and 48 h after infection. The bullet-shaped vi-rus particles budded primarily from plasmamembranes and accumulated in extracellularspaces (Fig. 16). A prodominance of cone-shaped particles similar to those seen by nega-tive-contrast microscopy was noted at both har-vest times (Fig. 17); particles in the process ofbudding also exhibited this shape. At 48 h,some cells also had anomalously broad cone-shaped particles on their surfaces (Fig. 18).These did not appear to contain the usual inter-nal nucleocapsid structure, but rather con-tained cytoplasm within typical viral envelope-projection layers. This breakdown in the syn-chrony ofnucleocapsid coiling and envelope bud-ding has not been observed previously in thislaboratory. Although viral nucleocapsid inclu-sion bodies were seen in infected cell cultures,they were not prominent, and necrotizing cyto-pathic changes evident at 48 h precluded theirfurther development.

In vivo studies. Mouse brain-passagedstocks of both viruses were lethal for mice onlywhen inoculated intracerebrally into newborns.In newborn hamsters, inoculated intracere-brally with large virus doses, kotonkan viruswas never recovered and Obodhiang virus wasonly recovered once from brain tissue on day 3,even though serial titrations were made with a

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't~~~~~~~~~~~~I

FIG. 8. Bullet-shaped kotonkan virus particles, with B particles together with T particles. All specimensfor negative-contrast electron microscopy were stained with sodium silicotungstate.

FIG. 9. Broad, cone-shaped kotonkan particles from an infected BHK-21 cell culture at24 h. Substructuraldetails resembled those of other rhabdoviruses; there were 8-nm surface projections, a unit membraneenvelope, and an axial channel extending prominently from the flat base. X106,1009.

FIG. 10. Kotonkan virus particles purified from a 48-h BHK-21 cell harvest. This randomly orientedenvelope fusion was previously seen only with bovine ephemeral' fever virus. x75,000.

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total of 10 organs between day 3 and day 26.Histopathological changes were only found inthe brains ofsuckling mice inoculated intracere-brally (Fig. 19). Interstitial edema, neuronalnecrosis, and interstitial mononuclear cell infil-tration of all areas of the brain were seen on theday preceding death and were severe in mori-

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bund animals. The smooth eosinophilic appear-ance of brain when stained with hematoxylinand eosin was replaced by chaotic dissolution.The only other lesion found was in the Gasse-rian ganglion of mice infected with Obodhiangvirus where ganglionic neurons exhibited necro-sis (Fig. 20).

KOTONKAN VIRUSB Particles (190 Particles >120nm long)

Mean length 182 nm

80 100 120 140 160 180 200 200 240PARTICLF LENGTH (nm)

OBODHIANG VIRUST Particles (70) Mean Length 85 nmB Particles (97) MeanLength 170nm

60 80 100 120 140 160 180 2(PARTICLE LENGTH (nm)

FIG. 14. Histogram of length distribution ofB particles of kotonkan virus.FIG. 15. Histogram of length distribution ofB and T particles of Obodhiang virus.

FIG. 11. Intact kotonkan virus nucleocapsids with the same conical shape of intact viral particles. Typicalrhabdovirus cross-striations are visible. x178,i000.

FIG. 12. Kotonkan virus particle with partial loss of its envelope. Cross-striations of the helically woundnucleocapsid are beaded, indicating the substructure of the nucleoprotein units. x232,700.

FIG. 13. Kotonkan virus particle that has been disrupted and penetrated by negative-contrast medium.These structures had a beaded substructure and unraveled into a single nucleocapsid strand. x178,000.

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A 17.FIG. 16. BHK-21 cells 24 h after infection with kotonkan virus. Virus particles accumulated in the

extracellular space after budding upon plasma membranes. All thin sections for electron microscopy werestained with uranyl acetate and lead citrate. x67,000.

FIG. 17. Vero cells 48 h after infection with Obodhiang virus. Cone-shaped virus particles budded fromplasma membranes in association with severe cytonecrotic changes. x52,000.

These changes were paralleled by widespreadpresence of immunofluorescent antigen inbrain, but nowhere else in these animals. Thisantigen filled the cytoplasm of neurons, usuallyin large foci at random locations throughout thebrain (Fig. 21); often the centers of these fociwere necrotic (as detected by changes in autoflu-orescence). In individual neurons, antigen wasusually punctate or in small aggregates andconcentrated in the perikaryon (Fig. 22).

Thin-section electron microscopy of thesemouse brains confirmed the widespread occur-rence of interstitial edema and the necrosis ofinfected neurons (Fig. 23). Viral nucleocapsidinclusions were found in the cytoplasm ofmany

neurons, and virus particles budded primarilyfrom plasma membranes of neuronal cell bodiesand neuronal processes (Fig. 24). As in cellculture, most virus particles were cone shapedand a few had parallel sides. Far more inflam-matory cells were evident by electron micros-copy than by light microscopy, primarily be-cause of the widespread distribution of neu-ronal damage. These cells in the brain intersti-tium were mostly monocytes and macrophages,but there were polymorphonuclear leukocytes.Most of the cells identified as macrophages con-tained phagocytized debris, but they were alsoinfected and contained budding virus particlesand typical inclusions (Fig. 25). The numbers of

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FIG. 18. Very broad, cone-shaped kotonkan virus particles budding from the plasma membranes ofBHK-

21 cells at 48 h. Typical viral envelopes contained cytoplasm inside the usual nucleocapsid structure at this

time. x43,00X0.FIG. 19. Edema, neuronal necrosis, and interstitial mononuclear cell infiltration in the brain of a mori-

bund mouse infected with kotonkan virus. Hematoxylin and eosin. x250.

FIG. 20. Necrosis of neurons in the Gasserian ganglion of a mouse infected with Obodhiang virus.

Hematoxylin and eosin. x250.1167

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FIG. 21. Large focus of intracytoplasmic immunofluorescent antigen in a suckling mouse brain infectedwith kotonkan virus. This pattern offocal confluence ofantigen was most like that of bovine ephemeral feverand vesicular stomatitis virus infections. x360.

FIG. 22. At high magnification, punctate and aggregated immunofluorescent antigen concentrated in theperikaryon of neurons infected with kotonkan virus. x1,300.

FIG. 23. Brain of suckling mouse infected with kotonkan virus. The intercellular spaces indicate intersti-tial edema and there was also necrosis of infected neurons. Virus particles are budding from plasmamembranes, and an inclusion body (IB) is present in neuronal cytoplasm. x29,000.

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FIG. 24. Kotonkan virus-infected neuron with an inclusion body (IB) made of massed viral nucleocapsid.Virus particles are budding from the plasma membranes into distended extracellular space. x52,000.

FIG. 25. Macrophage in the brain of a mouse infected with kotonkan virus. There were typical inclusionbodies (IB) and budding virus particles, indicating that phagocytic cells became infected. x22,000.

FIG. 26. Brain macrophage infected with Obodhiang virus. Evidence of infection of inflammatory cells(inclusion body [IB] and budding viral particles) were previously seen in bovine ephemeral fever and vesicularstomatitis virus infection, but never in rabies infections. x34,000.

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virus particles observed in extracellular spacenear these cells indicated that they were asproductive as infected neurons (Fig. 26).

DISCUSSIONThe antigenic relationship between koton-

kan and Obodhiang viruses and Mokola virus,which was initially found by cross-complementfixation testing (7; Shope, in press), was con-firmed by indirect immunofluorescence. It wasthis relationship and the absence of any othercross-reactivity with any animal rhabdovirusthat originally led to the inclusion of these twoarthropod isolates in the Rhabdoviridae familyand the rabies virus subgroup. The capacity ofboth viruses to replicate in Aedes aegypti mos-quitoes (R. E. Shope, S. M. Buckley, T. H. G.Aitken, and G. H. Tignor, Proc. 3rd Int. Congr.Virol., in press) and in Aedes albopictus cells inculture (1) suggested that arthropods are in-volved in their perpetuation in a nature andthat their isolation from Mansonia mosquitoesand Culicoides was not anomalous. Other sero-logical approaches have generally failed to ex-

pand the relationship with Mokola virus. In thepresent study, direct immunofluorescencecross-reactivities were easily diluted out, andbecause this method emphasized reactivities ofnucleocapsid antigen (N polypeptide of inclu-sion bodies), any concept that the rabiessubgroup is based upon qualitatively commonnucleocapsid antigens is contradicted (17). Infact, the varying potency of conjugates as usedin different laboratories would make compari-sons ambiguous and distantly related new vi-ruses impossible to characterize without homol-ogous systems. Minimal cross-reactions in neu-tralization testing, whether in suckling mice orcell cultures (7; Buckley, Trans. N.Y. Acad.Sci., in press), further indicated that no majorcross-reactivity existed between antigens ex-posed on the virion surface (G glycopeptide ofsurface projections; possible M polypeptide ofenvelope). In view ofthe difficulties in interpret-ing low-level cross-neutralization indexes, ourfailure to demonstrate any cross-surface immu-nofluorescence reactions between kotonkan andObodhiang viruses and other members of therabies subgroup must be interpreted as confirm-ing major qualitative distinctions. With the sur-

face immunofluorescence technique, internalantigens of virions and infected cells (beforecytopathic damage) are not exposed to thebroadly reactive conjugate and, therefore, reac-

tivities are expected to resemble those obtainedby neutralization testing. This surface immuno-fluorescence is considered an interim procedureto be used before G glycopeptides are isolated

from each of the viruses of the rabies subgroupand before their quantitative comparison usingantisera raised against each purified product aswas done recently with vesicular stomatitis vi-ruses (3). From available evidence, it seemsclear that these relationships involving koton-kan and Obodhiang viruses are too distant toanticipate either competitive exclusion of a vi-rus from an ecological niche via vertebrate hostcross-immunity, or experimental cross-protec-tion. For the same reasons, a broad efficacy ofrabies vaccine should not be anticipated.

All biological characteristics of kotonkan andObodhiang viruses, other than their immuno-logical reactivities as just described, were foundto be quite similar to those of BEF virus anddistinct from those of rabies and the other ra-bies-like viruses (Mokola, Lagos bat, and Du-venhag6 viruses). For example, growth of thetwo viruses in cell culture was greatly aug-mented by serial passage irrespective of condi-tions for autointerference. The same was foundfor BEF virus (12) and is necessary for streetrabies viruses, but not for Mokola or Lagos batviruses. Adaptation to vertebrate cell culturewas furthered by passage in A. albopictus mos-quito cells (1); BEF virus has not been studiedin this way, and of the rabies subgroup viruses,only Mokola will grow in mosquito cells (2;Buckley, Trans. N. Y. Acad. Sci., in press).Peak titers in Vero cells occurred at low temper-atures (30 C was the lowest temperature used)and were associated with an immunofluores-cent antigen accumulation that was smoothlydispersed and/or finely aggregated in cyto-plasm. BEF virus has been shown to yieldhigher titers at 30 to 34 C than at higher tem-peratures (10), and its antigens are distributedin cytoplasm of infected cells similarly (12);rabies and rabies-like viruses typically growbest at 34 to 35 C, and intracytoplasmic anti-gens are predominantly limited to inclusion bod-ies. Syncytia were formed in monolayers in-fected with kotonkan and Obodhiang virusesbefore total cytopathic destruction. The onlyother instance where we have seen syncytia inrhabdovirus-infected cells was in BEF virus-infected Vero cells (12). Likewise, severe cyto-pathology is not characteristic of viruses of therabies subgroup, but is rapidly progressive incells infected with kotonkan and Obodhiangviruses.

In negative-contrast electron microscopic ob-servations, kotonkan and Obodhiang virus par-ticles and formed nucleocapsids were coneshaped and had mean lengths of 182 and 170nm, respectively. This is the most commonlength distribution among animal rhabdovi-ruses (14), but the conical shape is exceptional

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and has only been seen with BEF virus previ-ously (4, 5, 9, 12). We consider this particleshape a most important viral characteristic be-cause other rhabdoviruses, treated and de-graded in many diverse ways, rarely have acorresponding shape distribution. The sameshape distribution was confirmed in thin-sec-tion electron microscopy of infected cell cul-tures, and virus particles were found to budalmost exclusively upon plasma membranes. Ininfected mouse brain tissue, budding similarlyoccurred upon neuronal plasma membranes,with inclusion bodies of excess viral nucleocap-sid material occupying the cytoplasm of thesame cells. Light, immunofluorescent, and elec-tron microscopy confirmed the coincidence ofsites of development of focalized brain necrosis,viral antigen accumulation, and virus particledevelopment. These in vivo growth characteris-tics closely match those of BEF virus; however,rabies, Mokola, Lagos bat, and Duvenhag6 vi-rus infections are typically not necrotizing andnoninflammatory, and antigen distribution inmouse brain is not focalized. The latter virusesbud primarily upon intracytoplasmic organellemembranes (endoplasmic reticulum) and notplasma membranes of mouse neurons (18). In-fection by kotonkan and Obodhiang viruses ofmany infiltrating macrophages in the brain in-terstitium of moribund mice may be contrastedwith the strict neuronotropism of the rabies-like viruses in the brains of mice and hamsters(13, 18). The only instances in which infectedinflammatory cells have been found in animalsinoculated with a rhabdovirus were in miceinfected with BEF virus (12, 14) or in thoseinfected with vesicular stomatitis virus.

Finally, kotonkan virus was incriminated asthe cause of an acute febrile illness in cattle inNigeria (7). Cattle, imported from Europe andserologically negative, developed neutralizingantibody to kotonkan virus shortly after thisillness. Kotonkan virus is not serologicallycross-reactive with BEF virus, but this illnessclinically resembled ephemeral fever and not aprogressively lethal, neurotropic infection suchas caused by rabies virus.The question of whether kotonkan and Obod-

hian viruses should form, with BEF virus, aseparate subgrouping within the Rhabdoviri-dae family or whether the described serologicalcross-reactivities should be used as the basis foran expanded rabies subgroup is not directlyanswered by these studies. The taxonomicbridging of viruses as different in their naturalhistories as rabies and BEF virus would detractfrom the usefulness of current subgroupings ofanimal rhabdoviruses (14; Shope, in press).Clearly, further physicochemical characteriza-

tion of kotonkan and Obodhiang viruses andcomparison with rabies and BEF viruses areneeded. In conformity with the Department ofAgriculture prohibition of BEF virus, one suchcomparative study of viral polypeptides is beingundertaken with the collaboration of John F.Obijeski, Center for Disease Control, and FredBrown, Animal Virus Research Institute, Pir-bright, England. Ultimately, knowledge of thestructural basis for the described antigeniccross-reactivities may come from the same ap-proaches.

ACKNOWLEDGMENT

The contribution of Alyne K. Harrison to the electronmicroscopic studies is gratefully acknowledged.

LITERATURE CITED

1. Buckley, S. M. 1973. Singh's Aedes albopictus cell cul-tures as helper cells for the adaptation of Obodhiangand kotonkan viruses of the rabies serogroup to somevertebrate cell cultures. Appl. Microbiol. 25:695-696.

2. Buckley, S. M., and G. H. Tignor. 1975. Plaque assayfor rabies serogroup viruses in Vero cells. J. Clin.Microbiol. 2:241-242.

3. Dietzschold, B., L. G. Schneider, and J. A. Cox. 1974.Serological characterization of the three major pro-teins of vesicular stomatitis virus. J. Virol. 14:1-7.

4. Holmes, I. H., and R. L. Doherty. 1970. Morphology anddevelopment of bovine ephemeral fever virus. J. Vi-rol. 5:91-96.

5. Ito, Y., Y. Tanaka, Y. Inaba, and T. Omari. 1969.Electron microscopic observations of bovine epizooticfever virus. Natl. Inst. Anim. Health Q. 9:35-44.

6. Karber, G. 1931. Beitrag zur kollektiven Behandlungpharmakologischer Ruhenversucke. Naunyn-Schmie-debergs. Arch. Exp. Pathol. Pharmakol. 162:480.

7. Kemp, G.E., V. H. Lee, D. L. Moore, R. E. Shope, 0. R.Causey, and F. A. Murphy. 1973. Kotonkan, a newrhabdovirus related to Mokola virus of the rabiesserogroup. Am. J. Epidemiol. 98:4349.

8. Kemp, G. E., E. D. Mann, 0. Tomori, A. Fabiyi, and E.O'Connor. 1973. Isolation of bovine ephemeral fevervirus in Nigeria. Vet. Rec. 93:107-108.

9. Lecatsas, G., A. Theodoridis, and B. J. Erasmus. 1969.Electron microscopic studies of-bovine ephemeral fe-ver virus. Arch. Gesamte Virusforsch. 28:390-398.

10. Matumoto, M., Y. Inaba, Y. Tanaka, H. Ito, and T.Omori. 1970. Behavior of bovine ephemeral fever vi-rus in laboratory animals and cell cultures. Jpn. J.Microbiol. 14:413-421.

11. Mollenhauer, H. H. 1964. Plastic embedding mixturesfor use in electron microscopy. Stain Technol. 39:111.

12. Murphy, F. A., W. P. Taylor, C. A. Mims, and S. G.Whitfield. 1972. Bovine ephemeral fever virus in cellculture and mice. Arch. Gesamte Virusforsch.17:549-559.

13. Murphy, F. A., S. P. Bauer, A. K. Harrison, and W. C.Winn. 1973. Comparative pathogenesis of rabies andrabies-like viruses. Lab. Invest. 28:361-376.

14. Murphy, F. A. 1974. Evolution of rhabdovirus tropisms,p. 699-722. In E. Kurstak and K. Maramorosch (ed.),Viruses, evolution and cancer. Academic Press Inc.,New York.

15. Obijeski, J. F., A. T. Marchenko, D. L. Bishop, B. W.Cann, and F. A. Murphy. 1974. Comparative electro-phoretic analysis of the virus proteins of four rhabdo-viruses. J. Gen. Virol. 22:21-23.

16. Schmidt, J. R., M. C. Williams, M. Lulu, A. Mivule,

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and E. Mujombe. 1975. Viruses isolated from mosqui-toes collected in the Southern Sudan and WesternEthiopia. E. Afr. Virus Res. Inst. Rep. 15:24.

17. Schneider, L. G., B. Dietzschold, R. E. Dierks, W.Matthaeus, P.-J. Enzmann, and K. Strohmaier. 1973.Rabies group-specific ribonucleoprotein antigen anda test system for grouping and typing of rhabdovi-ruses. J. Virol. 11:748-755.

18. Shope, R. E., F. A. Murphy, A. K. Harrison, 0. R.

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Causey, G. E. Kemp, D. I. H. Simpson, and D. L.Moore. 1970. Two African viruses serologically andmorphologically related to rabies virus. J. Virol.6:690-692.

19. WHO Scientific Group. 1967. Arboviruses and humandisease. W.H.O. Tech. Rep. Ser. no. 369.

20. Wrigley, N. G. 1968. The lattice spacing of crystallinecatalase as in internal standard of length in electronmicroscopy. J. Ultrastruct. Res. 24:454-464.

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relationship of two arthropod-borne rhabdoviruses (kotonkan and

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