Vol. 38, No. 2INFECTION AND IMMUNITY, Nov. 1982, p. 731-7380019-9567/82/110731-08$02.00/0Copyright © 1982, American Society for Microbiology

Pathogenicity of Avian Reoviruses: Examination of SixIsolates and a Vaccine Strain

VERA GOUVEA1 AND THOMAS J. SCHNITZER .2*

Department of Epidemiology, School of Public Health,1 and Rackham Arthritis Research Unit, Departmentof Internal Medicine, School of Medicine,2 University of Michigan, Ann Arbor, Michigan 48109

Received 10 May 1982/Accepted 20 July 1982

Six avian reovirus isolates and a vaccine reovirus strain were compared forinvasiveness, virulence, and pathological characteristics upon infection of day-oldspecific-pathogen-free chicks by the footpad, subcutaneous, and oral routes ofinoculation. No significant differences were noted regarding the ability of individ-ual isolates to infect target tissues. However, virulence (measured as the 50%lethal dose) among the isolates varied markedly from 2 x 105 to <10 PFU perchick for the most virulent isolate; between the parental wild-type virus and thederivative vaccine virus strain, a million-fold (106) difference in virulence wasdemonstrated. All strains revealed, with considerable variation, arthrogenicpotential.

Avian viral arthritis-tenosynovitis is a naturaldisease of poultry. Olson described the illnessand isolated a viral arthritis agent from an infect-ed hen in 1959 (13). Several years later, majoroutbreaks of this disease occurred in Englandand in the United States (6, 15), and agents wereisolated and identified as reoviruses. The condi-tion appears to be common and widespread;field outbreaks, especially in broiler breeders,have been reported since then in many parts ofthe world (21). Although arthritis and tenosyno-vitis are the best-recognized manifestations, in-fection by avian reoviruses has been associatedwith respiratory (4) and enteric (3) disease,hepatitis, hydropericardium (1, 7), myocarditis,and pericarditis (12) in chickens and with infec-tious enteritis in young turkeys, occasionallyresulting in high mortality (5). Quite often, reovi-ruses have been found in the tracheae and fecesof apparently healthy chickens (8).A number of different serotypes of avian reo-

virus have been defined (25), and some reportssuggest a relationship between the serotype andthe type of disease produced, presumably on thebasis of tissue tropism. However, others haveshown that isolates from field conditions otherthan arthritis can also produce lesions typical oftenosynovitis (14; J. R. M. Guteratne, Ph.D.thesis, University of Liverpool, Wirral, En-gland). Further studies have suggested that theseverity of disease is different with differentavian reovirus isolates, but no study has system-atically examined in a quantitative manner theability of a range of isolates to initiate infectionand cause disease. The work reported here wastherefore undertaken to compare the pathoge-nicity for 1-day-old chicks of seven strains of

avian reovirus when inoculated subcutaneously(s.c.), orally, or via footpad (f.p.).

MATERIALS AND METHODS

Cells. Chick embryo fibroblasts (CEF) were pre-pared from 10-day-old specific-pathogen-free embry-os, and chick embryo liver cell (CELi) cultures wereprepared from 14-day-old embryos, both by conven-tional tissue culture techniques (10). Specific-patho-gen-free eggs were obtained from SPAFAS, Inc.,Norwich, Conn.

Viruses. Seven avian reoviruses were studied: Reo25, isolated by Deshmukh and Pomeroy in Minnesota(3) from week-old chicks with cloacal pasting;WVU2937 (WVU), isolated by Olson in West Virginia(15) from field cases of severe synovitis in a broilerflock; S1133, originally isolated during an outbreak ofsevere tenosynovitis by van der Heide (20) in Connect-icut; P100, an attenuated vaccine strain of S1133 virusproduced by 235 passages in chorioallantoic mem-brane (CAM) followed by 100 additional passages inCEF cultures (22); Fahey-Crawley (FC), isolated byFahey and Crawley (4) in Toronto, Canada, from casesof chronic respiratory disease in chickens; Lasswade126/75 (Lasswade), isolated from cases of tenosynovi-tis in Scotland (11); and R19, isolated by Jones fromtenosynovitis cases in England (7). The 100th finalCEF passage of strain P100 and the 7th and the 73rdCAM passages of the Reo 25 and S1133 strains,respectively, were kindly supplied by L. van derHeide (Department of Pathobiology, University ofConnecticut, Storrs, Conn.). The fourth passage inCELi of strain R19 and tissue culture supensions of theLasswade, FC, and WVU strains at unknown passagelevels were obtained from R. C. Jones (Subdepart-ment of Avian Medicine, University of Liverpool,England). All the viruses were plaque purified threetimes, and a final pool was made in CEF. The viruspools were titrated both in CEF and in CELi, and a

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single pool of each isolate was used in all assays.Virus titration. The stocks of viruses were titrated

on both CEF and CELi by standard techniques (10).After incubation at 37°C for 2 (CELi) or 3 (CEF) days,1 ml of agar containing neutral red (0.9% agarose,0.0005% neutral red) was added per well to the firstagar overlay to permit the visualization of plaques.Plates were then incubated for 24 h at 37°C, andplaques were counted.

Virus detection from tissues. The tissues removedwere homogenized in mortars with pestles and sterilesand and were clarified by low-speed centrifugation.The supernatants were titrated in CELi monolayers asdescribed above.

Animals. All experiments were conducted in 1-day-old specific-pathogen-free chicks hatched in the labo-ratory facility. All chicks were avian reovirus free anddid not possess antibodies to reovirus.

Determination of lethal dose by s.c. and f.p. inocula-tions. A preliminary assay was performed to assess the50% lethal dose (LD50) of each virus strain. Serial 10-fold dilutions of virus were made in sterile phosphate-buffered saline to give final concentrations of 102 to 106PFU/0.1 ml, and then 0.1-ml doses of virus wereinjected into the right f.p. of chicks, four chicks foreach virus dilution. The chicks were observed daily,mortality was recorded, and the LD50 was estimated.Subsequently, two sets of experiments were conduct-ed with two different routes of infection, s.c. and f.p.,and a larger number of birds was used for a moreaccurate LD50 determination. Four groups of 12 chickseach received 10-2, 10-1, 100, and 10' of the previous-ly estimated LD50 by either s.c. or f.p. inoculations.An additional control group of 12 chicks was inoculat-ed with sterile phosphate-buffered saline. Calculationof the LD50 was done by the Reed-Muench method(16).

Determination of infectious dose by f.p. inoculation.Groups of 12 chicks received 103, 102, 10', 100 or 10-1PFU of the virus to be tested by right f.p. injection.Half of the chicks receiving virus at each dilution werekilled at day 2 postinfection (p.i.), and the other halfwere killed at day 7. The synovia and tendons at, andimmediately above and below, the hock joints wereremoved aseptically, placed in 2 ml each of minimalessential medium, and frozen at -70°C until assayedfor virus.

Examination of temporal spread of virus after f.p.inoculation. Groups of 12 chicks were infected via theright f.p. with doses of 102 to 103 PFU of each virus.The chicks were observed daily, and clinical manifes-tations such as imbalance, lameness, reluctance towalk, and swelling of the joints were carefully moni-tored. Chicks were killed at days 1, 3, and 6 p.i. andevery week thereafter. Additional control chicks wereinoculated with a 102 dilution of a homogenate ofnoninfected CEF culture and were killed at the middleand at the end of the experiment. Tendons of the rightand left hock joints, hearts, and segments (approxi-mately 2 cm) of the lower intestines were excised atnecropsy. The tissues were handled for virus isolationas described above.

Determination of LD50 after oral inoculation. Foreach of the seven viruses, 36 chicks were divided intothree groups receiving 104, 105, or 106 PFU of virus.The inocula (0.2 ml) were administered orally. Equalnumbers of chicks were left as controls. Chicks were

examined daily for symptoms and mortality. Fourchicks per dilution per virus were killed at the end ofthe second week p.i., and two chicks per dilution pervirus were killed every week thereafter up to 6 weeksp.i., at which time the experiment was terminated. Atnecropsy, hearts, livers, lower intestines, and rightand left hock joints were carefully examined for grosspathology, and the tendons of one leg of the chickskilled at 14 days p.i. were removed for virus reisola-tion. Later, an additional set of experiments wascarried out with the S1133 virus, including lower dosesof virus (10' to 106 PFU per chick) for the assessmentof the LD50 of this virus by the oral route.

Histological studies. To assess the temporal se-quence of histopathological events occurring uponjoint infection, one reovirus strain, R19, was chosenfor study and was administered by the f.p. and oralroutes of inoculation. Both hock joints with surround-ing tendons and synovia were removed en bloc atvarious times post-inoculation, fixed in buffered For-malin, and prepared for routine histopathology.

RESULTSDetermination of LD50 and clinical manifesta-

tions after s.c. and f.p. inoculation. The virulenceof the seven avian reoviruses when inoculateds.c. and via the f.p. were compared (Table 1).For any individual strain, the mortality rateswere not significantly different with either routeof infection. However, great differences in viru-lence were found among the strains, with theLD50 ranging from 0.6 to 5.3 loglo PFU perchick. From these results, it is possible to classi-fy the isolates into a high-virulence group con-sisting of strains S1133, FC, and Reo 25, and alow-virulence group including the Lasswade,R19, and WVU strains. The P100 vaccine strainwas the least virulent of all viruses, having anLD50 a million-fold less than its parent strain,S1133. Nevertheless, the P100 vaccine strainwas not completely avirulent; at a high concen-tration it was still capable of killing chicks wheninoculated by either the s.c. or f.p. route.Most infected chicks had swellings at the

inoculation sites by day 2, and these swellingsprogressed to the adjacent hock joints within thenext few days and intensified greatly by the endof 2 weeks. Edema and gross swelling of thehock joint were often seen. By week 3 thecondition of the affected joint improved, butmost chicks still had some swelling. In thecontralateral left leg, only a slight swelling of thehock was detected in some birds infected withstrains S1133, P100, and R19. Diarrhea occurredin many chicks, commonly lasting 2 to 3 days. Agood proportion of the surviving chicks inoculat-ed with doses close to the LD50 of virus showedat the end of the experiment marked growthretardation, appearing significantly smaller thancontrols. With all the viruses, mortality oc-curred between the days 3 and 9 p.i. The time ofdeath was directly related to the dose of virus

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TABLE 1. LD50 after s.c., f.p., and oral inoculations of 1-day-old chickens

Route of LD50 (log1o PFU per chicken) for strain:infection S1133 FC Reo 25 WVU R19 Lasswade P100

S.C. 0.6 2.1 3.0 4.9 5.3 5.1 7.6F.P. 0.9 2.6 2.7 4.7 5.1 5.3 7.7Oral 5.3 >7.0 >7.0 >7.0 >7.0 >7.0 >7.0

received, with earlier death at the higher dosesand later death at lower doses. On necropsy, a

common finding was an overall pale, jaundicedliver. Death was apparently due to overwhelm-ing hepatic necrosis.

Determination of 50% infectious doses of differ-ent isolates after f.p. inoculation. In these experi-ments, infectivity was defined by the recoveryof infectious virus from the hock joint of theinoculated leg. Virus isolation was done in CELimonolayer culture, the most sensitive cell cul-ture for avian reovirus. Nevertheless, this invitro assay was shown to be much less sensitivethan the natural in vivo host, precluding theexact determination of the 50% infectious dose,since many chicks got infected with the smallestdose of virus given (10-1 PFU per chick). Theresults of this experiment (Table 2) indicate thatthe amount of virus necessary to infect a birdsuccessfully by the f.p. route was uniformly low(<1 PFU per chick) and approximately the samefor all viruses. Differences seen at day 2 p.i.largely disappeared by day 7 p.i. and probablyonly reflect the rate of replication of the individ-ual viral strains. These results are probably an

overestimate of the true 50% infectious dosesince in those birds given the lowest viral inocu-lum virus multiplication might take longer than

the 7-day termination point of these experimentsto reach levels detectable in the hock joint.

Viral spread after f.p. inoculation. When 1-day-old chicks were infected via the f.p. with 102to 103 PFU per chick, a great proportion of thoseinfected with strains S1133, FC, and Reo 25were found dead. This result is not surprising,for the dose given was higher than the LD50 forthese viruses as found later in another experi-ment done concomitantly and described above.All dead chicks had high amounts of virus (>106PFU) in all parts tested, except for the left jointof two of the S1133-infected birds that died onday 3 p.i., probably too soon for virus replica-tion in the noninfected leg.The results for virus isolation obtained with

each virus demonstrated extremely similar pat-terns of infectivity, both in the times of appear-ance and clearance of infectious virus at individ-ual sites and in the titers of virus recovered fromeach site (Table 3).

Virus injected into the right f.p. could berecovered in high titers (=106 PFU) from thehock joint, tendons, and synovium of the inocu-lated leg within the first day p.i. By 1 day afterinoculation, virus spread from the infected legand established a generalized infection. Virus,often in high titer, was recovered from the heart

TABLE 2. Infectious dose pattern after f.p. innoculation of 1-day-old chickensRate of infectivitya at inoculum size (PFU per chicken):

Virus strain D0l03 102 101 100 10-, ID50

Day 2 p.i.S1133 NDC' 3/6 1/6 2/6 2/6 60FC 6/6 4/6 3/6 3/6 ND 8Reo 25 ND 4/6 2/5 1/6 0/5 38WVU 5/6 3/6 5/6 2/6 ND 8Lasswade 3/6 4/6 1/6 0/6 0/6 68P100 ND 5/5 2/6 115 1/6 10

Day 7 p.i.S1133 ND 4/4 6/6 4/6 1/6 0.7FC 6/6 6/6 6/6 6/6 ND <1.0Reo 25 ND 6/6 6/6 2/5 3/6 0.6WVU 5/6 6/6 3/6 3/6 ND 1.0Lasswade 6/6 5/6 4/4 4/5 1/6 0.7P100 ND 4/5 3/6 4/6 4/5 0.7

a Number of chickens with positive virus isolation from infected leg/total number infected.b ID50, 50% infectious dose, estimated by the Reed-Muench method (16).c ND, Not done.

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TABLE 3. Temporal localization of virus after f.p. inoculation

Rate of virus recovery from:aChickens

Right hock joint Left hock joint Heart Intestine

Sacrified at days p.i.:1 6/7 (87) 0/7 (0) 5/7 (71) 3/7 (43)3 14/14 (100) 12/14 (86) 12/14 (85) 9/14 (64)6 13/14 (93) 12/14 (87) 7/14 (50) 8/14 (57)14 12/14 (86) 10/14 (71) 3/14 (21) 2/14 (14)21 6/8 (75) 2/8 (25) 1/8 (12) 0/8 (0)28 0/5 (60) 2/5 (40) 0/5 (0) 0/5 (0)

Found dead 3-9 days p.i. 22/22 (100) 20/22 (91) 22/22 (100) 22/22 (100)

a Number of chickens with virus present/number examined; percentages in parentheses.

and intestine from this time until day 6 p.i., afterwhich only very low titers of virus (<102 PFU)could be found in these tissues. Virus appearedin the hock joint of the noninoculated (left) legduring the systemic spread, multiplied to hightiter, and persisted longer in both hock jointsthan it did in other tissues. In no instance wasreovirus isolated from any tissue of the controlgroup, nor did the control group show anyclinical signs of infection.

Determination of virulence, infectivity, andclinical manifestations after oral inoculation.Upon oral challenge with high doses of virus,mortality occurred only among chickens infect-ed with strain S1133. The clinical signs were thesame as seen after f.p. and s.c. inoculation, thetime of death was again between 3 and 9 daysp.i., and virus could be recovered from manytissues. The LD50 for the S1133 virus was 5.3logl0 PFU per chick (Table 1).One chick infected with 104 PFU of Reo 25

virus died at day 1 p.i., and one control chickdied at day 2 p.i., both from nonspecific causessince no virus could be isolated from these birds.No other deaths resulted from oral infections ofup to 106 PFU per chick, making it impossible todetermine the virulence in terms of LD50 of anystrain except S1133 by this route of infection.

Clinical signs of infection appeared earlier andwere more prominent in the group infected withstrain S1133. By 2 weeks post-inoculation, mostbirds had some degree of swelling in their hockjoints, and upon dissection a straw-colored exu-date could be detected. This progressed by 3weeks post-inoculation to a bluish coloring ofthe hock joint which could be seen through theskin. Dissection usually revealed broken bloodvessels and thickening of the flexor tendons.Definite involvement of the joints was apparenton gross examination of chicks infected with allreovirus strains except Lasswade. Swelling ofthe hock joints occurred in some chicks infectedwith strains WVU and Reo 25, but to a signifi-cantly lesser extent than in those infected with

strain S1133. However, on necropsy, birds in-fected with WVU revealed profuse yellow exu-date surrounding the flexor tendon.

Infectious virus could be recovered at 2 weeksp.i. from the joints of over 90% of the chicksinfected with each strain, including Lasswade.Thus, although the Lasswade virus produced aclinically silent infection, it was capable of es-tablishing a productive infection upon oral ad-ministration, spreading systemically, and lodg-ing, at least temporarily, in the hock joints.

Arrested growth was first noticed during week3 post-inoculation with strain S1133. By the timethe experiment was terminated (6 weeks post-inoculation), many chickens infected with S1133virus were approximately half the size of chick-ens the same age in the control and the otherinfected groups. A few birds infected with thehighest dose of the R19 virus also showed somereduction in weight gain, but this was not asevident as with the birds infected with strainS1133. Hepatitis was frequently found and usu-ally showed a diffuse pattern, except for birdsinfected with strain R19, where lesions seemedto be confined to the periphery of the liver.

Histological studies. In general, the histopatho-logical findings in the joints after the oral inocu-lation of chicks were similar to, but less severethan, those seen after f.p. inoculation and tend-ed to appear 3 to 5 days later than in f.p.-inoculated chicks. At 10 days after f.p. in-oculation (Fig. 1), there was a marked acuteinflammatory response involving the synoviumcovering not only the joint surfaces (arthritis)but also the tendon sheaths (tenosynovitis), withinfiltration of heterophils as well as mononuclearcells. Similar changes could be seen at 14 daysafter oral infection (Fig. 2). By 3 weeks after f.p.inoculation, many of the hock joints demonstrat-ed evidence of more chronic inflammation, withmany mononuclear cells and occasional plasmacells appearing in both the synovium and sub-synovium. These changes progressed in somebirds to the formation of germinal centers evi-

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FIG. 1. Histological section from the left hock joint 10 days after f.p. inoculation of 1-day-old chickens withvirus strain R19. There is a marked inflammatory response with accumulation of mononuclear cells throughoutthe synovium and subsynovium. Synovial hypertrophy and thickening are also demonstrated.

dent after 4 to 6 weeks of infection. Synovialhypertrophy and thickening was an early finding(1 to 2 weeks) that often regressed but wouldoccasionally lead to pannus formation and ero-sion of underlying joint cartilage (Fig. 3). Bothhock joints were generally equally involved,even after f.p. inoculation.

DISCUSSIONDifferent avian reovirus isolates are known to

have different abilities to produce disease afterexperimental challenge. Among a number ofstudies on the pathogenicity of avian reovirus tochickens at 2 weeks of age or older, Sahu et al.(17, 18) have compared the severity of thepathological lesions produced by several reovi-rus isolates, including strains S1133, FC, Reo25, and WVU when administered by the f.p.route. In their experiments, 100% of the birdsdeveloped lesions characteristic of acute teno-synovitis, but no deaths were reported.Although a definite age susceptibility of chick-

ens to avian reovirus has been demonstrated (9,26), no previous study has directly compared therelative virulences of a number of different iso-lates for day-old chicks. The results presented

here demonstrate that after f.p. or s.c. infectionof day-old chicks, an extremely wide range ofvirulence exists among the avian reovirus strainsexamined. To a certain extent these resultsagree with the original field conditions: Lass-wade and R19 viruses were isolated during out-breaks of mild disease, whereas strains S1133and WVU were isolated during outbreaks ofsevere disease with some mortality. Our resultsalso confirm the high mortality rate found byothers (23, 26) for the S1133 strain after f.p.inoculation of day-old chicks.The basis for the differences in the virulence

after f.p. inoculation of the avian reovirusstrains is not known. All viruses were able toinitiate infection with approximately the sameefficiency, for the infectious dose patterns ob-tained for the various strains were quite similar.Furthermore, the sequential spread of virus totarget tissues showed no marked differencesamong the strains. They were all able to producesystemic infection within 3 days p.i. No specifictissue tropism was noted, all viruses being ableto infect all tissues examined, and the grosspathology at death was not strikingly different inany of the birds necropsied.

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FIG. 2. Histological section from the left hock joint 14 days after oral inoculation of 1-day-old chickens withvirus strain R19. The marked inflammatory changes are similar to those seen after f.p. inoculation (Fig. 1) andconsist of infiltration of mononuclear cells into the synovial and subsynovial connective tissue as well as markedsynovial hypertrophy.

By the more natural oral route of infection,strain S1133 was still the most pathogenic virus,although with a markedly reduced ability to killchicks. A direct comparison of the LD50 of theother isolates could not be made since no deathsoccurred after oral inoculations. The impossibil-ity of estimating the virulence of these reovirusstrains by the oral route may be attributed to anequally profound reduction in virulence ofstrains that were all less highly pathogenic thanS1133 by the f.p. and s.c. routes of infection.The observed lack of virulence was not due totheir inability to initiate infection; this was con-firmed by the isolation of virus from the jointtissue after oral challenge with each of theisolates. Partial inactivation of virus by gastroin-testinal enzymes reducing the actual size of theinoculum when compared with f.p. or s.c. inocu-lations or a preliminary localization of the virusin the gastrointestinal tract resulting in sensitiza-tion of the immune system of the bird beforesystemic spread, however, are factors that mightaccount for the reduction in virulence of theseviruses when acquired orally.

Additional factors need to be assessed to

understand better the various parameters thatmay be important for viral virulence. There needto be examinations of growth rates and peaktiters of the viruses in vitro, their ability toproduce defective-interfering particles, and thetemperature dependency of their replication be-cause these factors have all been correlated withviral virulence in other systems (2). Further-more, as avian reoviruses can now be genotypi-cally distinguished and have been shown todisplay significant polymorphism at both theRNA and protein levels (5a), a more completeanalysis of the structural proteins of these virus-es may permit certain correlations to be madebetween viral structure(s) and virulence. Suchanalyses have been successfully performed withthe mammalian reoviruses (24) and have alreadyprovided important information.Host factors are also significant in determin-

ing viral virulence (19). Age and strain depen-dencies of reovirus virulence have long beenknown (9). The mechanism by which these fac-tors act is not understood but is believed to berelated to the ability of the animals to mount aneffective immunological response to infection.

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FIG. 3. Histological section from the left hock joint 7 weeks after f.p. inoculation of 1-day-old chickens withvirus strain R19. Chronic inflammatory changes are evident with invasion of the articular cartilage by connectivetissue (pannus), grossly evident as pitting of the cartilage surface.

As a number of different serotypes of reoviruswere used in this study, all may not have beenequally efficacious in eliciting production of neu-tralizing antibodies or specific cytotoxic cells.Therefore, careful examination of the immuneresponses, both humoral and cellular, generatedby these strains is needed and might help explainthe differences in virulence observed. Thesestudies are currently being undertaken in thislaboratory.Of particular interest is the finding that the

P100 vaccine virus strain, although highly atten-uated, was able to cause systemic infection andcould be localized to the joints, occasionallyeven late after infection. These results confirmthe observations of others (R. C. Jones, person-al communication) and raise the question of howthis strain differs from its parent, strain S1133.Although examination of the migration patternof the genomic double-stranded RNA segments(genes) of these two viruses upon polyacryl-amide gel electrophoresis failed to distinguishbetween them (Gouvea and Schnitzer, in press),this does not preclude differences at either theRNA or protein level. Preliminary results fromthis laboratory suggest that although serological-ly identical, these two viruses may demonstrate

differences in some of their structural proteins.The importance of these differences is currentlyunder investigation, and it is hoped that it willlead to a better understanding of the basis ofboth viral attenuation and virulence.

ACKNOWLEDGMENTS

We thank Kenneth Colton and Lori Amsterdam for theirtechnical support during the animal studies.

This work was supported in part by grants from the KrocFoundation, the Michigan Chapter of the Arthritis Founda-tion, and research grant R01 AM 27521-02 and Multi-purposeArthritis Center grant AM 20557-05 from the National Insti-tutes of Health. T.J.S. is a Senior Investigator of the ArthritisFoundation. V.G. was supported by a fellowship fromCAPES, Ministerio de Educaqao e Cultura, Brazil.

LITERATURE CITED

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3. Deshmukh, D. R., and B. S. Pomeroy. 1969. Avian Reovi-ruses. I. Isolation and serological characterization. AvianDis. 13:239-243.

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10. Kruse, P. F., Jr., and M. K. Patterson, Jr. (ed.). 1973.Tissue culture: methods and applications. AcademicPress, Inc., New York.

11. McDonald, J. W., C. J. Randell, M. C. Dagless, D. A.McMartin. 1978. Observations on viral tenosynovitis (vi-ral arthritis) in Scotland. Avian Pathol. 7:471-482.

12. Mustaffa-Babjee, A., and P. B. Spradbrow. 1973. Charac-terization of an avian reovirus isolated in Queensland. J.Comp. Pathol. 83:387-400.

13. Olson, N. 0. 1959. Transmissible synovitis of poultry.Lab. Invest. 8:1384-1393.

14. Olson, N. O., and M. A. Kahn. 1972. The effect of intrana-sal exposure of chickens to Fahey-Crawley virus on thedevelopment of synovial lesions. Avian Dis. 16:1073-1078.

15. Olson, N. O., and D. P. Solomon. 1968. A natural out-break of synovitis caused by the viral arthritis agent.Avian Dis. 12:311-316.

16. Reed, L. J., and H. Muench. 1938. A simple method ofestimating fifty percent end points. Am. J. Hyg. 27:493-497.

17. Sahu, S. P., and N. 0. Olson. 1975. Comparison of thecharacteristics of avian reoviruses isolated from the diges-tive and respiratory tract with viruses isolated from thesynovia. Am. J. Vet. Res. 36:847-850.

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