JOURNAL OF VIROLOGY, Sept. 1989, p. 3934-3943 Vol. 63, No. 90022-538X/89/093934-10$02.00/0Copyright C) 1989, American Society for Microbiology

Severe Thrombocytopenia in Young Calves Experimentally Infectedwith Noncytopathic Bovine Viral Diarrhea Virus

WAYNE V. CORAPI, TRACY W. FRENCH,2 AND EDWARD J. DUBOVI*

Diagnostic Laboratory' and Department of Pathology,2 New York State College of Veterinary Medicine,Cornell University, Ithaca, New York 14851

Received 20 March 1989/Accepted 5 June 1989

Seven calves between 1 week and 2 months of age were infected with a noncytopathic field isolate of bovineviral diarrhea virus (BDV) in order to evaluate the effect of BDV infection on the concentration of circulatingplatelets in the blood. All calves were determined to be free of BDV and neutralizing antibodies to BDV beforeinfection. Platelet counts were performed on a daily basis over a 30-day period beginning at the time ofinfection. By 2 weeks postinfection, all calves showed a significant drop in the number of circulating plateletsand a marked hyperplasia of megakaryocytes in the bone marrow. In three of the seven calves, thrombocy-topenia was severe (<5,000/Il) for 1 to 6 days. In two of these three animals, extensive petechial andecchymotic hemorrhages were observed on all mucosal surfaces and on various internal organs during theperiod of severe thrombocytopenia. BDV was consistently isolated from the platelets during the early phases ofthe infection, and viral antigen was occasionally detected on platelets by a fluorescent-antibody assay. Theresults demonstrate that BDV infection is associated with decreases in platelet numbers and suggest thatplatelets may serve as carriers of circulating virus.

Bovine viral diarrhea virus (BDV) and the antigenicallyrelated hog cholera virus and border disease virus of sheepare the three current members of the genus Pestivirus in thefamily Togaviridae. The recent molecular cloning and se-quencing of BDV have permitted an analysis of its geneticorganization and replication strategy, which suggest that itshould be reclassified as a member of the genus Flavivirus inthe newly established family Flaviviridae (9, 11, 42). BDVinfections in cattle have been reported throughout the world,and the wide spectrum of clinical syndromes associated withthis positive-stranded and enveloped RNA virus make it oneof the most important viral pathogens of cattle.The clinical manifestations of BDV infections are depen-

dent on the age and immune status of the animal at the timeof infection as well as the strain and biotype of the virus.Cytopathic (CP) and noncytopathic (NCP) biotypes of BDVare routinely isolated from infected cattle. Severe infectionsresulting in fatal disease have almost exclusively been attrib-uted to the presence of both biotypes residing within thesame animal. The mechanism behind this dual-infectionmodel has received considerable attention (3, 4, 7, 29, 43).Calves born persistently viremic because of an early trans-placental infection with NCP BDV show a specific immuno-tolerance to that particular strain of BDV and fail to developneutralizing antibodies to it (30). Although such animals areoften clinically normal, a naturally occurring superinfectionwith CP BDV that is antigenically identical or very similar tothe persisting NCP BDV results in fatal disease. The closeantigenic similarity between the CP BDV and NCP BDVrecovered from these animals strongly suggests that the CPBDV is the product of a mutation from the NCP BDV (12,22).

In contrast to this more severe form of bovine virusdiarrhea, the more common outcome is the result of an acuteinfection of seronegative and immunocompetent cattle witheither biotype of the virus. The clinical signs produced byacute BDV infections are extremely variable (36), but the

* Corresponding author.

majority of reported infections are mild and often subclini-cal. Typically, these animals show little more than a mildpyrexia and transient leukopenia of a few days' durationfollowed by the presence of neutralizing antibodies 2 to 3weeks after the onset of infection (8). Three extensiveserologic surveys have demonstrated the presence of neu-tralizing antibodies to BDV in as many as 53 to 72% ofclinically normal cattle in the United States before thewidespread use of vaccines for this virus (24, 31, 34). Such ahigh incidence of exposure attests to the subclinical nature ofthe majority of BDV infections.

In spite of the subclinical nature of most acute BDVinfections, there are a number of instances of acute infec-tions leading to severe disease. Because a major focus ofrecent research efforts with this virus has been work relatedto persistently infected cattle, severe acute infections innaive immunocompetent animals have received little atten-tion in the literature. However, several examples of NCPBDV isolates associated with severe acute infections innonpersistently infected cattle have been isolated in thislaboratory (36; W. V. Corapi and E. J. Dubovi, unpublishedobservation).

Using such an NCP BDV isolate recovered from a fieldoutbreak of bovine virus diarrhea, we have attempted toreproduce severe clinical disease in naive immunocompetentcalves. Two recent reports have also noted a relationshipbetween naturally occurring acute BDV infections andthrombocytopenia, and a major emphasis of our work wasdirected at documenting this relationship under experimentalconditions (36, 41).Seven seronegative and BDV-free calves and one vacci-

nated control calf were infected with NCP BDV. Completehemograms and platelet counts were done on a daily basisfor a period of 30 days postinfection (dpi) or until recoveredanimals were virus free. Aspirates and core biopsies of bonemarrow were taken at periodic intervals throughout thistime. All nonvaccinated animals became severely ill andshowed a significant drop in platelet counts after infection.Recovery was associated with a return to normal numbers of

3934

BDV-INDUCED THROMBOCYTOPENIA IN YOUNG CALVES 3935

circulating platelets. The results of the bone marrow biopsiesand the finding of viral antigen on circulating plateletssuggest that platelets are peripherally consumed after BDVinfection and may be involved in the clearance of circulatingvirus.

MATERIALS AND METHODS

Origin of virus inoculum. The original virus inoculum wasobtained from the blood of an adult cow naturally infectedwith NCP BDV during a severe herd outbreak of bovinevirus diarrhea in Warsaw, New York. The outbreak andsubsequent isolation of BDV occurred in January 1987 andinvolved 50% of a milking herd of 100 Holsteins. Clinicalsigns consisted of high fever, up to 41.7°C (107°F), diarrhea,and an acute drop in milk production. Twenty affectedanimals subsequently died. An NCP BDV isolate, desig-nated CD-87, was recovered from eight of a total of eightblood samples submitted for virus isolation. Six of theseeight animals recovered and developed serum neutralizationtiters to BDV that ranged between 1:3,000 and 1:5,000. TheCD-87 isolate was neutralized with an equal volume ofBDV-specific antiserum (derived from the Singer strain ofBDV) and was tested for the presence of other viral patho-gens of cattle. Bovine testicle cells inoculated with neutral-ized CD-87 showed no evidence of cytopathology after serialpassages. Inoculated cells were found to be free of infectiousbovine rhinotracheitis virus, bovine parainfluenza virus 3,bovine respiratory syncytial virus, bovine herpesvirus 3,bovine coronavirus, bovine rotavirus, bovine parvovirus,bovine adenovirus, and bluetongue virus. In addition, anti-serum from experimentally infected calves that recovereddid not develop antibody to bovine syncytial virus or bovineimmunodeficiency virus, as determined by an indirect immu-nofluorescence assay (IFA).Animal inoculations. All animals used were Holstein or

Holstein-Angus calves and were obtained at birth from damsseronegative for BDV. Mononuclear cells and serum werecollected within 3 to 5 days after birth from each calf andwere determined to be free of BDV and neutralizing anti-bodies to BDV. Calves were housed in individual isolationfacilities for the duration of the experiments. Calf 1 wasinfected intravenously with mononuclear cells obtained froma BDV-positive cow involved in the original outbreak. Allother calves were inoculated intravenously with 2.5 x 106mononuclear cells obtained from the blood of calf 1 at 13 dpi.

Virus isolation. Whole blood was mixed with equalamounts of 0.01 M phosphate-buffered saline (PBS), layeredover Ficoll (Histopaque; Sigma Chemical Co., St. Louis,Mo.), and centrifuged for 1 h at 400 x g. The mononuclearcell fraction was then removed, washed once in PBS, andsuspended in Eagle minimum essential medium. The mono-nuclear cells were then either frozen at -70°C after theaddition of 10% dimethyl sulfoxide or seeded onto monolay-ers of bovine testicle cells and incubated at 37°C for 3 to 4days. After incubation, the bovine testicle cells weretrypsinized and seeded onto multispot Teflon-coated micro-scope slides (Cel-Line Associates, Newfield, N.J.). The cellswere then incubated at 37°C for an additional 4 h and fixed inacetone. The presence of virus was detected by IFA using aBDV-specific fluorescein isothiocyanate-conjugated poly-clonal antiserum (American BioResearch, Milton, Tenn.).

Virus neutralization. Serum from infected calves was usedin a standard virus neutralization assay against 100 50%tissue culture infective doses of the CP Singer type strain ofBDV, with bovine testicle cells as the indicator cells. Neu-

tralization titers were recorded as the highest dilution ofserum that inhibited cytopathology.

Indirect IFA. Bovine testicle cells were inoculated withthe same mononuclear cells used for all calf infections. Aftera 72-h incubation at 37°C, the cells were seeded ontomultispot slides and fixed as described above. Serum takenfrom an infected calf at various times postinfection was usedin an indirect IFA with these infected cells. Immunoreactiv-ity was detected by adding fluorescein-conjugated recombi-nant protein G (Zymed Laboratories, San Francisco, Calif.).

Cell cultures. Bovine testicle cells used for viral isolationwere grown in Eagle minimum essential medium with Earlesalts supplemented with 10% fetal bovine serum, 200 IU ofpenicillin per ml, 200 ,ug of streptomycin per ml, and 2.5 ,ugof amphotericin B (Fungizone; E. R. Squibb & Sons, Prince-ton, N.J.) per ml. All cell cultures and fetal calf serum weredetermined to be free of NCP BDV and mycoplasmas beforeinoculation.Hemograms and platelet counts. Daily blood samples were

taken from all calves beginning on the day of infection andcontinuing for 30 days or until virus could no longer beisolated from the mononuclear cell fraction. A portion ofeach blood sample was used for complete blood counts andplatelet counts. Total white cells and platelets were countedon an S+IV Coulter Counter (Coulter Electronics, Inc.,Hialeah, Fla.). Once platelet numbers dropped below thelevel of detection on the Coulter Counter, they were countedmanually by using a hemacytometer.Bone marrow aspirates and core biopsies. For all biopsy

procedures, calves were sedated with an intravenous injec-tion of between 8 and 12 mg of Xylazine (Rompun; MilesLaboratories, Shawnee, Ky.). Bone marrow aspirates weretaken from the ribs by using a 15-gauge Illinois sternalneedle, and core biopsies were taken from the crest of theilium by using a 13-gauge Jamshidi core biopsy needle(American Pharmaseal Co., Valencia, Calif.). Smears madefrom aspirates were stain( J with Wright-Giemsa stain forroutine examination; other smears were fixed in acetonebefore immunofluorescent staining. A portion of the bonemarrow cells were pelleted before fixation and washed threetimes in PBS containing 7.5% anticoagulant citrate-phos-phate-dextrose solution (Fenwal Laboratories, Deerfield,Ill.). The washed bone marrow cells were then centrifugedonto microscope slides at 175 x g for 10 min in a cytocen-trifuge (Cytospin 2; Shandon Southern Products, Astmoor,England) and fixed in acetone. Core biopsies were fixed inBouin fixative for 48 h and decalcified for 1 h in a solutioncontaining 44% formic acid and 10% sodium citrate. Thefixed biopsy sections were then embedded in paraffin, sec-tioned at 4 ,um, and stained with hematoxylin and eosin.

Isolation and staining of platelets. For each sampling ofplatelets, 20 ml of venous blood was collected into a syringecontaining 2 ml of 3.8% sodium citrate to prevent coagula-tion. The blood was then centrifuged three times at 500 x gfor 4 min. The platelet-rich plasma was collected into poly-carbonate centrifuge tubes and suspended in an equal vol-ume of PBS-citrate-phosphate-dextrose. The mixture wasthen centrifuged at 800 x g for 10 min and washed two moretimes in PBS-citrate-phosphate-dextrose. The platelets werecounted, and a portion of them was diluted to 100 x 103/,ul.These were centrifuged onto microscope slides by using acytocentrifuge as previously described and fixed in eitheracetone or 1% paraformaldehyde. The fixed platelets werethen assayed for the pi -sence of BDV antigen by IFA aspreviously described. Platelets were also tested for thepresence of surface-bound immunoglobulin G (IgG) and IgM

VOL. 63, 1989

3936 CORAPI ET AL.

by IFA using either fluorescein isothiocyanate-conjugatedrabbit anti-bovine IgG (Jackson ImmunoResearch Laborato-ries, West Grove, Pa.) or fluorescein isothiocyanate-conju-gated goat anti-bovine IgM (Kirkegaard & Perry Laborato-ries, Gaithersburg, Md.). The remaining undiluted plateletswere pelleted once again, suspended in Eagle minimumessential medium, and seeded onto monolayers of bovinetesticle cells for virus isolation.

Radioimmunoprecipitation (RIP) and electrophoresis.BDV-infected bovine testicle cells were incubated for 30 minin methionine-free medium and then for 1.5 to 3 h inmethionine-free medium containing 500 ,uCi of [35S]methio-nine (ICN Radiochemicals, Irvine, Calif.) per ml. The radio-labeled cells were washed in PBS and lysed in RIP buffer (50mM Tris, pH 8.0, 100 mM NaCl, 5 mM EDTA, 1% TritonX-100, 1% deoxycholic acid, 0.1% sodium dodecyl sulfate,50 p.M phenylmethylsulfonyl fluoride). The cell lysate wascentrifuged at 10,000 x g for 5 min, and the supernatant wasstored at -70°C until used. Virus-specific proteins wereselected from the cell lysates by adding 10 pL. of the appro-priate antiserum to 200 pl. of cell lysate containing 106 cpmand incubating the mixture overnight at 4°C. Immune com-plexes were adsorbed by adding 25 [1I of a 50% solution ofrecombinant protein G bound to Sepharose 4B (ZymedLaboratories) and incubating it for 1 h at room temperatureon a rocking platform. The immunoprecipitated proteinswere gently pelleted and washed twice in RIP buffer, sus-pended in sample buffer (0.2 M Tris, pH 8.8, 2% sodiumdodecyl sulfate, 4 mM dithiothreitol, 5 mM EDTA, 15%glycerol, 0.01% bromophenol blue), and boiled for 3 min.lodoacetamide was added to a final concentration of 0.1 M,and the mixture was incubated at room temperature for 20min. Samples were electrophoresed in 10% polyacrylamidegels in the presence of sodium dodecyl sulfate using adiscontinuous buffer system and then subjected to fluorog-raphy and autoradiography.

RESULTS

Clinical and pathological findings. A total of eight calves,consisting of seven naive calves and one BDV-vaccinatedcontrol calf and ranging in age from 5 to 65 days, wereinoculated intravenously with 2.5 x 106 BDV-infectedmononuclear cells. Mononuclear cells were chosen as thesource of the inoculum to avoid tissue culture passage andpossible attenuation of the virus. The earliest clinical signs ofdisease were seen at 2 to 4 dpi and consisted of a slight feverranging from 39.5 to 39.88°C (103.1 to 103.8°F) and a drop inthe total leukocyte count. In most instances this initial feverand leukopenia resolved within 24 to 48 h. A second, moreprofound period of clinical illness began anywhere from 6 to12 dpi and consisted of fevers ranging from 39.77 to 41.83°C(103.6 to 107.3°F), watery and sometimes bloody diarrhea,leukopenia, anemia, and severe thrombocytopenia. Thelength of this second phase of illness ranged from 5 to 15days. Two of the eight calves died of their illnesses (Table 1,calves 2 and 3), while the remaining animals recovered.Although the vaccinated control calf (calf 8) developed thehighest fever (41.83°C [107.3°F]), it was the only calf that didnot develop diarrhea. Table 1 summarizes some of theclinical signs seen in the eight calves.Gross pathological findings consisted of petechial and

ecchymotic hemorrhages throughout the body and werefound in two of the eight calves (calves 2 and 6) beginning at14 and 15 dpi, respectively. Visible hemorrhages werepresent in calf 2 for a period of 5 days until the time of death.

TABLE 1. Summary of clinical signs

Calf Age Temp (°C ['FI)" Diarrhea Outcome

1 47 41.38 (106.5) Watery Recovery2 18 40.38 (104.7) Bloody Death3 38 39.77 (103.6) Watery Death4 60 41.16 (106.1) Watery Recovery5 61 41.28 (106.3) Watery Recovery6 7 40.89 (105.6) Bloody Recovery7 5 40.6 (105.0) Bloody Recovery8' 65 41.83 (107.3) None Recovery

"Age of calves at time of infection.^ Temperatures shown are the maximum values recorded during the infec-

tion.' Vaccinated control calf.

The hemorrhages in calf 6 were present for a period of 6 daysand then completely resolved. Hemorrhages were seen inboth calves on the sclera of the eyes, the margins and innersurfaces of the eyelids, the surfaces of the third eyelids, themucosal surfaces of the cheeks, the lower gingiva (Fig. IA),around the base of the molar teeth, on the tip and the ventralsurfaces of the tongue, and on the soft palate. Epistaxis wasalso seen in calf 2, and prolonged bleeding from the sites ofvenipuncture was seen in both calves. At necropsy, addi-tional internal hemorrhages were found in calf 2 in theesophagus, on the serosal surface of the spleen, the mucosalsurface of the abomasum, throughout as much as 30% of thewall of the rumen (Fig. iB), on the epicardium, and in asubperiosteal position on the lateral surface of the calvaria(Fig. 1C).

Calves 6 and 7 developed a cough and a mucopurulentdischarge from the nose at 34 and 40 dpi, respectively. Apure culture of Pasteurella multocida was isolated from atracheal wash done on calf 6 at 34 dpi.

Virus isolation and serum neutralization. BDV was isolatedfrom mononuclear cells taken from all seven nonimmunecalves beginning at 4 dpi. The time of clearance of circulatingvirus varied substantially between calves. Viral clearancehad occurred by as early as 16 dpi in calf 5, but virus couldstill be demonstrated as late as 40 dpi in calf 7. No viruscould be isolated from the vaccinated control calf (calf 8) atany time during the infection.The time of appearance of detectable levels of neutralizing

antibody in the sera of those calves that recovered alsovaried and ranged from 24 to 49 dpi when tested against theCP Singer strain of BDV. The maximum titer seen was 1:512in calf 7 at 57 dpi. When serum was tested in an indirect IFAagainst the homologous NCP BDV used in the infections, thepresence of antibody was first detected by good fluorescenceat about the same time as that seen with the neutralizationassay, although extremely weak staining by IFA was seenmuch earlier (Table 2).

Hematological changes. Although a significant drop in thetotal leukocyte count has been associated with BDV infec-tions since the earliest description of the disease (35) andseveral studies have demonstrated the effect on leukocytefunction in vitro after BDV infection (26, 28, 44), littleinformation is available concerning absolute numbers of thevarious circulating blood cell components over the course ofan acute infection. The principle hematologic changes seenin our study consisted of lymphopenia, neutropenia, anemia,and thrombocytopenia.Lymphocyte counts varied considerably but tended to be

low during the period of clinical illness (Table 3). Lymphope-

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BDV-INDUCED THROMBOCYTOPENIA IN YOUNG CALVES 3937

FIG. 1. Hemorrhages associated with severe thrombocytopeniain calf 2 at 19 dpi. Arrows indicate areas of hemorrhage. (A) Lowerjaw showing multiple hemorrhages on the gingiva. (B) Massive areas

of hemorrhage in the wall of the rumen. (C) Sagittal section of theskull with brain removed showing focal area of hemorrhage in thecalvaria.

nia (s2,500/Ipl) occurred in six of the eight calves and was

most severe in calf 7 when lymphocytes fell to less than 18%of preinoculation values at 6 dpi.

All eight calves developed a transient but severe neutro-penia that was well correlated with the time of illness (Table4). The greatest degree of neutropenia was seen in calf 6 at 14

TABLE 2. Development of antibodies to BDV afterexperimental infection of calf 7

dpi IFA" Neutralizing Virus" Fever andantibody titer" diarrhea

0 - <1:4 -7 - <1:4 + +

12 - <1:4 + +17 - <1:4 + -

20 + <1:4 + -

27 + <1:4 + -

32 + <1:4 + -

40 NDd ND + -

49 + 1:96 -57 + 1:512 -

" IFA results are with antibodies against the homologous NCP virus used inthe infection. ±, Very weak staining by IFA.

"Results are for antibodies against the CP Singer strain of BDV.'Virus isolated from mononuclear cell fraction of whole blood.dND, Not done.

dpi, when the neutrophils fell to less than 3% of preinocula-tion values.The greatest hematological alteration was the effect on

platelet counts. The platelet counts in all seven nonimmunecalves diminished substantially. In three of the calves,thrombocytopenia was severe (.s5,000/Il) for periods rang-ing from 1 to 6 days (Table 5, calves 1, 2, and 6). Thehemorrhagic lesions described previously were seen incalves 2 and 6 when the platelets fell to 2,000/,l. of blood orbelow for a period of 24 h or longer. The time of thethrombocytopenia in all of the calves was also clearlyassociated with the onset of clinical signs. The diarrhea andfever were always worst when the platelet counts were attheir lowest, and the condition of the calves invariablyimproved when the platelets began to rise again. In severalinstances, the thrombocytopenia was followed by a reboundthrombocytosis, with platelet counts reaching levels sub-stantially higher than preinoculation values (Table 5, calves1, 4, 5, and 7).

Platelet counts from calves 1, 2, and 6 are also showngraphically in Fig. 2. The same general pattern of develop-ment and resolution of thrombocytopenia was seen in allthree calves, and the lowest counts were detected at andaround day 16 in all three instances.

Various degrees of anemia were seen in most of thecalves, the most severe of which occurred in calf 6 at 20 dpi,when the hematocrit was 15.8%, the platelet count was2,000/Rl, and visible hemorrhages had been present for 5days. Although prolonged bleeding from venipuncture siteswas seen during this time, prothrombin time, activatedpartial thromboplastin time, and the levels of fibrinogen andfibrinogen degradation products were all within normal limits(data not shown).

Platelet-associated virus. In order to determine whether thethrombocytopenia resulting from BDV infections is relatedto the presence of whole virus or viral antigens on theplatelets, washed platelet preparations were used for bothvirus isolation and detection of viral antigen by IFA. Plate-let-rich plasma recovered from the blood of infected calveswas used to prepare pure preparations of platelets. Theplatelets were washed several times as previously describedand used as a tissue culture inoculum. BDV was consistentlyisolated from such preparations during the early course ofthe infection. In two calves (calves 3 and 6), viral antigenwas detected by IFA with acetone-fixed platelets taken

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3938 CORAPI ET AL.

TABLE 3. Lymphocyte counts after infection with BDV

Lymphocyte counts" in calf:dpi

1 2 3 4 5 6 7 8"

0 4.1 5.8 4.3 7.1 4.6 4.5 6.2 4.81 4.8 4.9 2.5 6.0 4.8 3.4 4.2 2.62 7.7 5.1 2.7 6.1 4.6 3.2 6.1 2.63 5.7 2.4 1.4 3.3 2.0 4.9 2.7 3.84 5.1 2.2 1.6 5.1 3.2 3.2 3.7 2.25 4.3 4.1 3.2 3.9 4.4 3.2 4.5 4.06 NDC 2.9 3.1 2.6 2.9 2.4 1.1 3.07 3.3 3.4 2.4 2.8 2.1 3.6 2.7 2.78 5.7 3.2 2.7 4.4 3.1 3.0 2.7 3.39 4.8 2.3 2.1 5.0 3.6 2.4 ND 5.710 3.6 3.0 3.6 L4.4 3.2 3.7 3.0 4.811 3.5 2.9 2.2 5.1 2.4 3.4 2.4 3.412 3.5 2.8 2.2 5.1 7 3.1 2.0 3.713 2.8 2.4 1.8 6.8 3.6 3.0 2.3 4.214 5.3 3.7 2.4 5.9 3.2 2.6 4.1 3.415 7.0 2.3 1.7 6.4 3.1 3.2 3.5 ND16 . 2.2 2.9 7.2 3.4 2.7 3.2 ND17 4.2 2.6 5.8 6.5 ND 3.9 3.3 ND18 3.7 2.9 4.2 6.0 ND 4.5 ND ND19 3.2 ND 4.2 6.1 ND ND ND ND20 5.0 ND 4.5 6.3 ND 5.7 4.2 ND21 4.7 ND 2.1 6.0 ND 7.2 ND ND

' Values are in thousands per microliter of blood. Normal range is repre-sented by values shown at 0 dpi. Boxes indicate times at which calves showedsigns of fever and/or diarrhea.

b Vaccinated control calf.ND, Not done.

between 12 and 15 dpi (Fig. 3). This was not a consistentfinding, however, and several attempts at demonstrating thepresence of viral antigen on platelets from the remainingcalves by IFA were unsuccessful. Platelets that were eitherpositive or negative for viral antigen by IFA were alsoincubated with anti-bovine IgG and IgM but failed to revealthe presence of any surface-bound immunoglobulin. Thepresence of Fc receptors for IgG on platelets did not appearto influence any of the immunological assays, since plateletpreparations taken from uninfected control calves werenegative for both viral antigen and surface immunoglobulin.To eliminate the possibility of nonspecific adherence of

BDV to platelets, normal platelets isolated from a nonin-fected control calf were mixed with BDV-positive serum.After a 5-min incubation at room temperature, the plateletswere washed as before and put into tissue culture. No viruswas isolated from this preparation.An attempt was also made to extract replicating BDV from

disrupted platelets. BDV-positive platelets were mixed withneutralizing antibody for 1 h at room temperature in order toneutralize all surface-associated virus. The platelets werethen washed as before, disrupted by sonication, and used asa tissue culture inoculum. No virus was isolated from thispreparation either.Megakaryocyte hyperplasia. Bone marrow aspirates and

core biopsies were taken at periodic intervals throughout theinfections and evaluated for the numbers of megakaryocytespresent. In both procedures a marked hyperplasia of themegakaryocytes was seen by 12 dpi. The number of mega-karyocytes present in either procedure before inoculationwith BDV was 1 or 2 per x63 magnification field. By 12 dpi,the number of megakaryocytes increased to as many as 25per x63 magnification field (Fig. 4). IFA staining of washedbone marrow aspirates failed to reveal any convincingevidence of megakaryocyte infection with virus, although a

TABLE 4. Neutrophil counts after infection with BDV

Neutrophil counts" in calf:dpi

1 2 3 4 5 6 7 8"

0 4.4 3.2 1.5 1.4 2.0 3.4 4.3 1.71 4.6 5.7 4.6 2.7 1.8 9.6 6.7 4.12 0.9 3.9 7.0 1.8 1.8 13.2 7.4 3.83 2.7 0.2 2.6 0.1 0.2 3.7 4.1 2.04 3.3 0.4 1.7 1.4 1.3 0.8 4.3 0.75 2.6 2.6 1.1 1.2 1.2 1.5 7.4 0.46 ND' 1.4 2.1 2.1 0.3 7.3 9.3 0.17 0.5 1.2 1.3 1.3 1.2 2.4 3.7 0.88 2.5 2.4 2.6 1.9 2.4 2.8 2.5 1.89 0.7 4.4 3.5 0.7 0.3 1.9 ND 6.810 1.3 0.8 1.2 0 0.8 1.5 0.4 2.411 1.1 1.1 2.6 0.4 1.1 1.0 0.2 2.712 1.1 0.6 1.2 0.9 n.9 1.0 0.4 3.813 1.1 0.9 0.6 0.5 0.7 0.2 0.5 4.214 1.5 1.1 0.5 1.3 1.1 0.1 5.215 0.6 0.4 0.7 2.2 1.1 0.7 0.8 ND16 [.8 1.2 5.7 2.1 2.2 0.7 1.9 ND17 0.6 0.9 2.1 4.2 ND 0.3 1.9 ND18 1.0 2.2 5.1 2.6 ND 0.6 ND ND19 2.2 ND 5.9 3.0 ND ND ND ND20 0.6 ND 3.5 2.0 ND 0.8 1.1 ND21 0.8 ND 7.9 2.6 ND 1.1 ND ND

"Values are in thousands per microliter of blood. Normal range is repre-sented by values shown at 0 dpi. Boxes indicate times at which calves showedsigns of fever and/or diarrhea."Vaccinated control calf.ND, Not done.

small percentage of the megakaryocytes showed some lightcytoplasmic staining at 12 dpi.

Viral proteins and immune response. The major viralpolypeptides we have detected in cells infected with CPBDV have molecular sizes of 118, 80, 65, 58, 56, 54, 43, and37 kilodaltons (kDa) (Fig. 5). Despite slight variations be-tween different strains of BDV, this is in close agreementwith previously published reports (9, 10, 14, 16). The moststriking difference observed between RIP done using CPBDV and NCP BDV is the absence of the 80-kDa protein inNCP BDV (15, 38). In order to characterize the NCP BDVused in this study, mononuclear cells taken from the blood ofcalf 1 at the peak of its infection (13 dpi) were used to infectbovine testicle cell cultures. Immunoprecipitations of aradiolabeled lysate derived from these cells were done byusing the homologous sera taken from calves 1 to 8 bothbefore infection and at least 2 weeks postinfection. Heterol-ogous hyperimmune serum taken from a calf infected withthe Singer strain of BDV was used as a control. The onlyBDV proteins detected with sera from the eight experimentalcalves as well as the control hyperimmune serum were the118- and 58-kDa proteins (Fig. 6). When immune serumtaken from calf 7 at 57 dpi was used to immunoprecipitatethe proteins present in a Singer-infected cell lysate, theremaining proteins of BDV were also detected, with theexception of the 37-kDa protein (Fig. 6B). A lack of detect-able antibodies recognizing the 37-kDa protein in sera takenfrom cattle infected with NCP BDV has been previouslyreported (17).The time course of the immune response of calf 7 was

analyzed further by reacting nine different serum samplestaken between 0 and 57 dpi with a Singer-infected cell lysate.The first proteins detected were the 118- and 80-kDa proteinsat 17 dpi (Fig. 7). The immunological cross-reactivity andstructural relationship between these two proteins has been

J. VIROL.

BDV-INDUCED THROMBOCYTOPENIA IN YOUNG CALVES 3939

1200

o 1000r0

900

o 800

700

,-600

C/) 500-

WJ 400-

L 300-

..J 200-

100

0 ll7l l 7l l T I I

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

DAYS POST INFECTIONFIG. 2. Development of thrombocytopenia in calves 1 (LI), 2 (+), and 6 (O) after infection with BDV. Calf 2 died on day 19.

previously reported (10, 15, 40). Significant amounts ofantibody directed against the remaining viral-induced pro-teins did not appear until 49 dpi, the same time at whichneutralizing antibodies to BDV were first detected in this calf(Table 2).

DISCUSSION

Virus-associated thrombocytopenia has been reported forseveral systemic viral infections in both humans and ani-mals, including infections with dengue virus (33, 39, 49),human immunodeficiency virus (1, 32), Epstein-Barr virus(25), canine distemper virus (2), African swine fever virus(18-20), and hog cholera virus (18).

FIG. 3. IFA of platelets from calf 3. Washed platelets taken at 15dpi were centrifuged onto microscope slides, fixed in acetone, andincubated first with anti-BDV bovine serum and then with fluores-cein isothiocyanate-conjugated rabbit anti-bovine IgG.

The etiology of virus-induced thrombocytopenia is poorlyunderstood and has been attributed to both immune- andnonimmune-mediated mechanisms. Platelets possess Fc re-ceptors for both IgG and IgE on their surfaces (23, 37), andthe coating of platelets with IgG or the deposition of viralantigen-antibody complexes on platelet membranes is capa-ble of triggering the immune-enhanced destruction of plate-lets by the mononuclear phagocyte system. The presence ofcross-reacting antibodies that recognize normal surface an-tigens on platelets has also been suggested as an indirectmechanism of thrombocytopenia after viral infection. Inaddition, platelets themselves may be directly involved inthe immune response against microorganisms. IgE-depen-dent, platelet-mediated cytotoxicity of Schistosoma man-soni has been demonstrated in rats and suggests a uniquerole of platelets in immune-mediated cellular cytotoxicity(5). Nonimmune-mediated mechanisms of thrombocytope-nia involving direct platelet-virus interactions, such as viraladsorption onto platelet membranes followed by agglutina-tion and lysis of platelets, have also been demonstrated withinfluenza virus and Newcastle disease virus (46, 47).Thrombocytopenia associated with BDV infections has

not been reported in the literature until very recently. In aretrospective study involving 146 confirmed clinical cases ofBDV infections in adult cattle, thrombocytopenia and bleed-ing were noted in 15 of them (41). Examination of the bonemarrow in five of these cases was inconclusive but sugges-tive of marrow necrosis. Hemorrhage in the intestinal tract,lymph nodes, and heart following infection with CP BDV hasbeen reported in some of the earlier literature, but the statusof the platelets was not reported (27).

In the present study, we have shown that various degreesof thrombocytopenia are consistently seen in young calvesafter infection with a naturally occurring strain of NCPBDV. Although the degree of thrombocytopenia variedmarkedly between calves, all calves showed a significant

VOL. 63, 1989

3940 CORAPI ET AL.

1 2 3 4 5 6

80-

37- _

FIG. 5. Virus-specific polypeptides in BDV-infected cells. Bo-vine testicle cells were infected with the Singer (lane 2), NADL (lane3), Oregon C24V (lane 4), NY-1 (lane 5), or Draper (lane 6) strain ofBDV. Virus-specific polypeptides were immunoprecipitated from[35S]methionine-labeled cell lysates by using bovine antiserum fol-lowed by protein G. The immunoprecipitated proteins were ana-lyzed by sodium dodecyl sulfate-polyacrylamide gel electrophore-sis. Lane 1 contains a noninfected control cell lysate. The numbersto the left are the estimated molecular sizes in kilodaltons for thevirus-induced proteins found with the Singer strain.

FIG. 4. Core biopsies of bone marrow from calf 6. (A) Control

biopsy taken before infection Average number of megakaryocytes

(arrow) was 1 or 2 per field. (B) Biopsy taken at 12 dpi. Note marked

increase in number of megakaryocytes. Magnification, x63.

drop in platelet counts within 4 to 8 days after infection.

Severe petechial and ecchymotic hemorrhages and ex-

tremely prolonged bleeding times (up to 8 h after venipunc-

ture) were explained by the nearly complete absence of

circulating platelets in calves 2 and 6 beginning at 14 and 15

dpi, respectively (Table 5). Although calf 1 also had very

severe thrombocytopenia at 16 dpi, the platelet count re-

bounded dramatically within 24 h, and no indication of

hemorrhage was ever seen. The rebound thrombocytosis

noted in calves 1, 2, 5, and 7 during the period of clinical

recovery has also been seen after dengue virus infections in

humans (39).

The etiology of BDV-induced thrombocytopenia in the

present study does not appear to be due to failure of platelet

production. The bone marrow in all instances showed a

marked megakaryocyte hyperplasia, and there was no evi-

dence of viral infection in the vast majority of megakaryo-cytes. In addition, the dramatic recovery within 24 to 48 h

from the severe thrombocytopenia seen in calf 1 is more

suggestive of an increased peripheral consumption or se-

questration of platelets than a lack of production. The findingof viral antigen on platelets and the recovery of replicatingvirus in tissue culture from an inoculum of washed plateletsalso suggest a more direct viral effect on the platelets. The

inability to detect IgG or IgM on the surfaces of the plateletswould also argue against an indirect immunological cause of

the thrombocytopenia. Evidence for direct damage to plate-

lets by virus, including ultrastructural changes in plateletmorphology and the appearance of platelet aggregates, haspreviously been used to explain the thrombocytopenia seenwith the closely related hog cholera virus (48).

Although the exact mechanism of BDV-induced thrombo-cytopenia has not been determined, it is clear that bovineplatelets are at least capable of acting as carriers of BDV inthe circulation. During the early stages of infection, BDVwas as readily isolated from washed platelet preparations asit was from mononuclear cells. It is conceivable that theplatelets are damaged or destroyed directly by the virus orthat the platelets are somehow consumed while playing someactive role in the elimination of virus. There was no evidenceof viral infection of platelets, a finding which was expectedsince viral multiplication would be unlikely in a cell thatlacks a nucleus. Viral antigen was detected on the plateletsfrom only two of the calves examined. The inability to detectBDV antigen on the platelets from the remaining calves mayindicate that the amount of antigen necessary for detectionby IFA is present on the platelets only for a relatively shorttime.Other possible mechanisms responsible for inducing

thrombocytopenia unrelated to the direct effect of circulatingvirus were considered. Endotoxins are known to causeplatelet aggregation and complement-dependent lysis ofplatelets, resulting in rapid onset of thrombocytopenia (6,45). The presence of gram-negative bacteria in the intestinaltract could serve as a possible source of endotoxin if theepithelial lining were damaged by the presence of replicatingvirus. However, the thrombocytopenia observed in theseexperiments began somewhere between 4 and 8 dpi, longbefore the development of diarrhea, which usually began atabout 9 to 15 dpi. In addition, no evidence of epithelialdamage to the intestinal lining was ever seen in those animalsthat died, and blood cultures for bacteria were negative.

Disseminated intravascular coagulation has also been re-ported as a possible sequela to viral infections such as thatseen with infectious canine hepatitis virus (50). The throm-bocytopenia and bleeding seen with disseminated intravas-

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BDV-INDUCED THROMBOCYTOPENIA IN YOUNG CALVES 3941

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FIG. 6. Immune responses of calves after infection with NCP BDV. Pre- and postinfection sera from eight calves experimentally infectedwith an NCP field isolate ofBDV were used to immunoprecipitate virus-specific proteins present in bovine testicle cells infected with the samevirus. (A) Radioimmunoprecipitation results obtained by using preinoculation (lanes 3, 5, 7, 9, 11, and 13) and postinfection (lanes 4, 6, 8, 10,12, and 14) sera from calves 1, 2, 3, 4, 5, and 6, respectively. (B) Results obtained by using preinoculation (lanes 5 and 7) and postinfection(lanes 6 and 8) sera from calves 7 and 8. Hyperimmune serum from a calf infected with the Singer strain of BDV was used in lane 4. Calf 7postinfection serum was used with a Singer-infected cell lysate in lane 3. Noninfected (lane 1) and Singer-infected (lane 2) cell lysates were

used as controls in both panels. Procedures and molecular size estimates are as described for Fig. 5.

cular coagulation are the end results of increased consump-tion of platelets, fibrin, and coagulation factors. Althoughdisseminated intravascular coagulation was considered as apossible cause of the thrombocytopenia after BDV infection,the absence of prolonged prothrombin and activated partialthromboplastin times as well as the absence of increasedfibrinogen degradation products in the serum were consid-ered to be sufficient evidence that disseminated intravascularcoagulation was not a factor in these experiments.

Chronic furazolidone poisoning in calves has been re-

ported to cause thrombocytopenia and hemorrhagic lesionsvery similar to those we have observed after BDV infection.However, histologic examination of the bone marrow afterchronic furazolidone poisoning revealed that the hematolog-ical changes were the result of a toxic effect on the bonemarrow which consisted of hypoplasia or aplasia involvingall cell lines, including the megakaryocytes (21). In contrast,

1 2 3 4 5 6 7 8 9 10 11

80-. >

65-..

37- __

FIG. 7. Time course study of the immune response of calf 7.

Serum taken from calf 7 at 0, 7, 12, 17, 20, 27, 32, 49, and 57 dpi

(lanes 3 to 11, respectively) was incubated with a Singer-infected

cell lysate. Noninfected (lane 1) and Singer-infected (lane 2) cell

lysates incubated with an anti-BDV hyperimmune serum were used

as controls. Procedures and molecular size estimates are as de-

scribed for Fig. 5.

infection with BDV leads to a marked hyperplasia of themegakaryocytes and normal to increased numbers of theremaining myeloid cell types; we believe that this is aresponse to the increased consumption of platelets andleukocytes occurring peripherally in the circulation.

In addition to the thrombocytopenia that resulted fromBDV infection, a second major finding in these experiments,which was of no less importance, was the ability of an NCPBDV to cause severe disease and even death in someinstances. Since most natural and experimental infections

TABLE 5. Platelet counts after infection with BDV

Platelet counts" in calf:dpi

1 2 3 4 5 6 7 8b

0 616 726 944 636 622 1,068 769 7741 738 762 932 633 629 1,052 1,076 7702 780 810 947 647 533 1,056 1,200 6863 751 609 883 573 347 1,200 986 6764 788 496 688 715 344 1,034 988 6375 755 560 797 633 320 826 845 6336 ND" 508 843 562 380 826 892 5357 660 487 784 470 310 753 612 6208 528 441 704 415 318 558 388 5699 722 419 718 398 394 436 ND .7210 620 347 560 .1536 448 343 312 50411 512 288 553 581 371 429 562 53512 543 229 395 696 373 483 689 63413 435 205 422 804 511 577 822 74014 300 17 329 865 699 215 909 75515 189 2 704 808 831 25 926 ND16 <1 1 744 1,002 885 <1 995 ND17 2 704 978 ND <1 994 ND18 444 6 660 935 ND 1 ND ND19 751 ND 558 970 ND 1 ND ND20 906 ND 491 986 ND 2 986 ND21 958 ND 559 1,094 ND 5 ND ND

"Values are in thousands per microliter of blood. Normal range is repre-sented by values shown at 0 dpi. Boxes indicate times at which calves showedsigns of fever and/or diarrhea.

h Vaccinated control calf.ND, Not done.

VOL. 63, 1989

3942 CORAPI ET AL.

with BDV do not result in severe disease, this suggests some

significant dissimilarities between isolates. Monoclonal anti-body analyses of numerous BDV isolates have previouslyshown that there is extensive antigenic variation among

them (12). Minor variations between BDV isolates, as de-tected by differences in the molecular sizes and the presence

or absence of specific virus-induced polypeptides in infectedcells, are also commonly seen by RIP. The proteins presentbetween 54 and 58 kDa show considerable variation and may

appear as a triplet (Singer strain), doublet (NADL strain), or

single band (Oregon C24V, NY-1, and Draper strains) (Fig.5). The 54-kDa protein is the only member of this complexthat consistently appears in all five of these strains. Neutral-izing monoclonal antibodies to BDV bound to all threeproteins in this region and failed to differentiate betweenthem (13; W. V. Corapi and E. J. Dubovi, unpublished data).However, when immune sera from the eight experimentallyinfected calves were reacted by RIP with a lysate derivedfrom the homologous NCP BDV, only the 58-kDa protein inthis region was detected (Fig. 6). The most likely explanationfor this is that a low level of viral antigen is expressed in cellcultures infected with this virus, since almost all remainingBDV proteins were detected when serum from calf 7 taken at57 dpi was reacted with a lysate derived from Singer-infectedcells (Fig. 6B). Although the 54- and 56-kDa proteins were

obviously present and recognized in vivo, the inability todetect them in vitro may have some significance. If theneutralizing epitopes residing on these proteins are notexpressed very readily or to a degree that is capable ofgenerating an adequate and timely immune response, it mayplay a major role in the ability of this virus to cause severe

disease. RIP data for calf 7 indicated that detectable levels ofIgG directed against the 54- to 58-kDa proteins did notappear until as late as 49 dpi, long after the period of clinicalillness and thrombocytopenia (Fig. 7). Alternatively, the use

of mononuclear cells from an infected animal as the source

of the inoculum may be an important factor in the develop-ment of severe disease, since it avoids the possibility ofattenuation of the virus through multiple passages throughtissue culture.We believe that BDV-induced thrombocytopenia is an

important clinical syndrome that has been largely unrecog-

nized and is occurring with increasing frequency. The factthat platelet counts can drop to extremely low levels (2,000/pl of blood) before any evidence of hemorrhage is detectedmay explain why it has not been reported previously. Agemay be a predisposing factor in the severity of the thrombo-cytopenia, since all of our experimental animals were 2months of age or less when infected. However, we have seena number of natural cases resulting in hemorrhage andplatelet counts of similarly low levels that have occurred inadult animals as well (unpublished observation).

ACKNOWLEDGMENT

This work was supported in part by funds from the U.S. Depart-ment of Agriculture Animal Health and Diseases Research Program.

LITERATURE CITED

1. Abrams, D. I., D. D. Kiprov, J. J. Goedert, M. G. Sarngadha-ran, R. C. Gallo, and P. A. Volberding. 1986. Antibodies tohuman T-lymphotropic virus type III and development of theacquired immunodeficiency syndrome in homosexual men pre-

senting with immune thrombocytopenia. Ann. Int. Med. 104:47-50.

2. Axthelm, M. K., and S. Krakowka. 1987. Canine distemper

virus-induced thrombocytopenia. Am. J. Vet. Res. 48:1269-1275.

3. Bolin, S. R., A. W. McClurkin, R. C. Cutlip, and M. F. Coria.1985. Severe clinical disease induced in cattle persistentlyinfected with noncytopathic bovine viral diarrhea virus bysuperinfection with cytopathic bovine viral diarrhea virus. Am.J. Vet. Res. 46:573-576.

4. Bolin, S. R., A. W. McClurkin, R. C. Cutlip, and M. F. Coria.1985. Response of cattle persistently infected with noncyto-pathic bovine viral diarrhea virus to vaccination for bovine viraldiarrhea and to subsequent challenge exposure with cytopathicbovine viral diarrhea virus. Am. J. Vet. Res. 46:2467-2470.

5. Bout, D., M. Joseph, M. Pontet, H. Vorng, D. Deslee, and A.Capron. 1986. Rat resistance to schistosomiasis: platelet-medi-ated cytotoxicity induced by C-reactive protein. Science 231:153.

6. Brown, D. L., and P. J. Lachmann. 1973. The behaviour ofcomplement and platelets in lethal endotoxin shock in rabbits.Int. Arch. Allergy 45:193-205.

7. Brownlie, J., M. C. Clarke, and C. J. Howard. 1984. Experi-mental production of fatal mucosal disease in cattle. Vet. Rec.114:535-536.

8. Brownlie, J., M. C. Clarke, C. J. Howard, and D. H. Pocock.1987. Pathogenesis and epidemiology of bovine virus diarrhoeavirus infection of cattle. Ann. Rech. Vet. 18:157-166.

9. Collett, M. S., D. K. Anderson, and E. Retzel. 1988. Compari-sons of the pestivirus bovine viral diarrhoea virus with membersof the flaviviridae. J. Gen. Virol. 69:2637-2643.

10. Collett, M. S., R. Larson, S. K. Belzer, and E. Retzel. 1988.Proteins encoded by bovine viral diarrhea virus: the genomicorganization of a pestivirus. Virology 165:200-208.

11. Collett, M. S., R. Larson, C. Gold, D. Strick, D. K. Anderson,and A. F. Purchio. 1988. Molecular cloning and nucleotidesequence of the pestivirus bovine viral diarrhea virus. Virology165:191-199.

12. Corapi, W. V., R. 0. Donis, and E. J. Dubovi. 1988. Monoclonalantibody analyses of cytopathic and noncytopathic viruses fromfatal bovine viral diarrhea virus infections. J. Virol. 62:2823-2827.

13. Donis, R. O., W. V. Corapi, and E. J. Dubovi. 1988. Neutralizingmonoclonal antibodies to bovine viral diarrhea virus bind to the56-58 k glycoprotein. J. Gen. Virol. 69:77-86.

14. Donis, R. O., and E. J. Dubovi. 1987. Glycoproteins of bovineviral diarrhoea-mucosal disease virus in infected bovine cells. J.Gen. Virol. 68:1607-1616.

15. Donis, R. O., and E. J. Dubovi. 1987. Differences in virus-induced polypeptides in cells infected by cytopathic and noncy-topathic biotypes of bovine virus diarrhea-mucosal diseasevirus. Virology 158:168-173.

16. Donis, R. O., and E. J. Dubovi. 1987. Characterization of bovineviral diarrhoea-mucosal disease virus-specific proteins in bovinecells. J. Gen. Virol. 68:1597-1605.

17. Donis, R. O., and E. J. Dubovi. 1987. Molecular specificity ofthe antibody responses of cattle naturally and experimentallyinfected with cytopathic and noncytopathic bovine viral diar-rhea virus biotypes. Am. J. Vet. Res. 48:1549-1554.

18. Edwards, J. F., W. J. Dodds, and D. 0. Slauson. 1984. Coagu-lation changes in African swine fever virus infection. Am. J.Vet. Res. 45:2414-2420.

19. Edwards, J. F., W. J. Dodds, and D. 0. Slauson. 1985. Mecha-nism of thrombocytopenia in African swine fever. Am. J. Vet.Res. 46:2058-2063.

20. Edwards, J. F., W. J. Dodds, and D. 0. Slauson. 1985. Mega-karyocytic infection and thrombocytopenia in African swinefever. Vet. Pathol. 22:171-176.

21. Hayashi, T., 0. Yamane, M. Sakai, C. Itakura, and M. Goto.1976. Hematological and pathological observations of chronicfurazolidone poisoning in calves. Jpn. J. Vet. Sci. 38:225-233.

22. Howard, C. J., J. Brownlie, and M. C. Clarke. 1987. Compari-son by the neutralisation assay of pairs of non-cytopathogenicand cytopathogenic strains of bovine virus diarrhoea virusisolated from cases of mucosal disease. Vet. Microbiol. 13:361-369.

J. VIROL.

BDV-INDUCED THROMBOCYTOPENIA IN YOUNG CALVES 3943

23. Joseph, M., A. Capron, J. C. Ameisen, M. Capron, H. Vorng, V.Pancre, J. P. Kusnierz, and C. Auriault. 1986. The receptor forIgE on blood platelets. Eur. J. Immunol. 16:306-312.

24. Kahrs, R., G. Atkinson, J. A. Baker, L. Carmichael, L. Coggins,J. Gillespie, P. Langer, V. Marshall, D. Robson, and B. Sheffy.1964. Serological studies on the incidence of bovine virusdiarrhea, infectious bovine rhinotracheitis. bovine myxovirusparainfluenza-3, and Leptospira pomonia in New York State.Cornell Vet. 54:360-369.

25. Kernoff, L. M. 1980. Demonstration of increased platelet-boundIgG in infectious mononucleosis complicated by severe throm-bocytopenia. Scand. J. Infect. Dis. 12:67-69.

26. Ketelsen, A. T., D. W. Johnson, and C. C. Muscoplat. 1979.Depression of bovine monocyte chemotactic responses by bo-vine viral diarrhea virus. Infect. Immun. 25:565-568.

27. Lambert, G., A. L. Fernelius, and N. F. Cheville. 1969. Exper-imental bovine viral diarrhea in neonatal calves. J. Am. Vet.Med. Assoc. 154:181-189.

28. Markham, R. J. F., and M. L. Ramnaraine. 1985. Release ofimmunosuppressive substances from tissue culture cells in-fected with bovine viral diarrhea virus. Am. J. Vet. Res.46:879-883.

29. McClurkin, A. W., S. R. Bolin, and M. F. Coria. 1985. Isolationof cytopathic and noncytopathic bovine viral diarrhea virusfrom the spleen of cattle acutely and chronically affected withbovine viral diarrhea. J. Am. Vet. Med. Assoc. 186:568-569.

30. McClurkin, A. W., E. T. Littledike, R. C. Cutlip, G. H. Frank,M. F. Coria, and S. R. Bolin. 1984. Production of cattleimmunotolerant to bovine viral diarrhea virus. Can. J. Comp.Med. 48:156-161.

31. Mills, J. H. L., and R. E. Luginbuhl. 1965. Incidence of bovinemucosal disease in Connecticut. Cornell Vet. 55:583-590.

32. Murphy, M. F., P. Metcalfe, A. H. Waters, C. A. Carne, 1. V. D.Weller, D. C. Linch, and A. Smith. 1987. Incidence and mech-anism of neutropenia and thrombocytopenia in patients withhuman immunodeficiency virus infection. Br. J. Haematol.66:337-340.

33. Nelson, E. R. 1960. Hemorrhagic fever in children in Thailand.J. Pediatr. 56:101-108.

34. Newberne, J. W., V. B. Robinson, and M. L. Alter. 1961.Incidence of infectious bovine rhinotracheitis and bovine virusdiarrhea. Vet. Med. 56:395-398.

35. Olafson, P., A. D. MacCallum, and F. H. Fox. 1946. Anapparently new transmissible disease of cattle. Cornell Vet.36:205-213.

36. Perdrizet, J. A., W. C. Rebhun, E. J. Dubovi, and R. 0. Donis.

1987. Bovine virus diarrhea-clinical syndromes in dairy herds.Cornell Vet. 77:46-74.

37. Pfueller, S. L., S. Weber, and E. F. Luscher. 1977. Studies of themechanism of the human platelet release reaction induced byimmunologic stimuli. III. Relationship between the binding ofsoluble IgG aggregates to the Fc receptor and cell response inthe presence and absence of plasma. J. Immunol. 118:514-524.

38. Pocock, D. H., C. J. Howard, M. C. Clarke, and J. Brownlie.1987. Variation in the intracellular polypeptide profiles fromdifferent isolates of bovine virus diarrhoea virus. Arch. Virol.94:43-53.

39. Powell, G. M. 1953. Clinical manifestations of epidemic hemor-rhagic fever. J. Am. Med. Assoc. 151:1261-1264.

40. Purchio, A. F., R. Larson, and M. S. Collett. 1984. Characteri-zation of bovine viral diarrhea virus proteins. J. Virol. 50:666-669.

41. Rebhun, W. C., T. W. French, J. A. Perdrizet, E. J. Dubovi,S. G. Dill, and L. F. Karcher. 1989. Thrombocytopenia associ-ated with acute bovine virus diarrhea infection in cattle. J. Vet.Intern. Med. 3:42-46.

42. Renard, A., D. Schmetz, C. Guiot, S. Brown-Shimmer, L.Dagenais, P. P. Pastoret, D. Dina, and J. A. Martial. 1987.Molecular cloning of the bovine viral diarrhea virus genomicRNA. Ann. Rech. Vet. 18:121-125.

43. Roeder, P. L., and T. W. Drew. 1984. Mucosal disease of cattle:a late sequel to fetal infection. Vet. Rec. 114:309-313.

44. Roth, J. A., M. L. Kaeberle, and R. W. Griffith. 1981. Effects ofbovine viral diarrhea virus infection on bovine polymorphonu-clear leukocyte function. Am. J. Vet. Res. 42:244-250.

45. Spielvogel, A. R. 1967. An ultrastructural study of the mecha-nisms of platelet-endotoxin interaction. J. Exp. Med. 126:235-249.

46. Terada, H., M. Baldini, S. Ebbe, and M. A. Madoff. 1966.Interaction of influenza virus with blood platelets. Blood 28:213-228.

47. Turpie, A. G., M. A. Chernesky, R. P. Larke, M. A. Packham,and J. F. Mustard. 1973. Effect of Newcastle disease virus onhuman or rabbit platelets. Lab. Invest. 28:575-583.

48. Weiss, E., A. Teredesai, R. Hoffmann, and G. Hoffmann-Fezer.1973. Volume distribution and ultrastructure of platelets inacute hog cholera. Thromb. Diath. Haemorrh. 30:371-380.

49. Weiss, H. J., and S. B. Halstead. Studies of hemostasis in Thaihemorrhagic fever. J. Pediatr. 66:918-926.

50. Wigton, D. H., G. J. Kociba, and E. A. Hoover. 1976. Infectiouscanine hepatitis: animal model for viral-induced disseminatedintravascular coagulation. Blood 47:287-296.

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