reactivity of Bovine Immunodeficiency-like Virus and Human. Immunodeficiency Virus Type 1 Proteins using Bovineand. Human Sera in a Western Blot Assay.
Detection of Multiple Retroviral Infections in Cattle and Crossreactivity of Bovine Immunodeficiency-like Virus and Human Immunodeficiency Virus Type 1 Proteins using Bovine and Human Sera in a Western Blot Assay Robert M. Jacobs, Helen E. Smith, Brian Gregory, Victor E.O. Valli and Cecilia A. Whetstone
ABSTRACT Bovine antibovine immunodefidencylike virus (BIV) antibodies were detected by Western blot analysis (WBA) using a chemiluminescence protocol. Bovine sera with anti-BIV activity, obtained from cows in two dairy herds, had antibodies directed against a variety of BIV-specific antigens indicating chronic infections. These sera were also tested for serological reactivity against bovine leukemia virus (BLV) and bovine syncytial virus (BSV). Cows most commonly had anti-BSV antibodies (12 of 39). Evidence for infection with BSV and BIV or BSV and BLV occurred with almost equal frequency (5 of 39 and 4 of 39, respectively) while only one instance of BIV and BLV coseropositivity was detected. The high prevalence of BSV seropositivity is consistent with a relatively infectious virus, which, as is known, may be transferred congenitally. Similar rates of coseropositivity of BIV or BLV with BSV in this population suggest that BIV is no more infectious than BLV and probably requires prolonged close contact for transmission. Seven of nine cows with anti-BIV antibodies detected primarily human immunodeficiency virus type 1 (HIV-1) p51 and p63 antigens by WBA using an alkaline phosphatase detection system, suggesting that HIV-1 proteins have potential usefulness in screening cattle for BIV seropositivity. Six
human sera that showed strong reactivity against multiple HIV-1 proteins and the serum from one of three patients considered to be an "indeterminate" HIV-1 reactor, cross-reacted primarily with BIV p26. This is the first report of human sera with antibody to BIV-specific proteins. Individual human sera or pooled human sera used as positive controls in the WBA for the detection of anti-HIV-1 antibodies, sera from two patients considered as "indeterminate" anti-HIV-1 reactors, and single sera from patients with anti-HIV-2 or anti-HTLV-1/2 antibodies did not react against BIV antigens. The cross-reactivities we have found were primarily directed to bands
corresponding to reverse transcriptase and major core proteins, proteins that have been relatively well-conserved in retroviral evolution. The crossreactivities likely represent reactivities to conserved epitopes found on BIV and HIV since humans at risk for BIV exposure have not been shown to seroconvert and human cells are not susceptible to BIV infection.
RESUME Des anticorps bovins contre le virus de l'immunodeficience bovine (VIB) ont ete detectes par immunoempreinte en utilisant un protocole de chimioluminescence. Des serums avec une activite anti-VIB, obtenus de vaches provenant de deux troupeaux laitiers,
contenaient des anticorps diriges contre une variete d'antigenes specifiques du
VIB indiquant des infections chroniques. Ces serums ont egalement ete testes pour une reactivite serologique contre le virus de la leucemie bovine (VLB) et le virus syncytial bovin (VSB). Les vaches avaient plus communement des anticorps anti-VSB (12 de 39). L'evidence d'infection avec le VSB et le VIB ou avec le VSB et le VLB etait presque d'egale frequence (5 de 39 et 4 de 39 respectivement) alors qu'un seul cas de co-seropositivite VIB et VLB etait detecte. La haute prevalence de seropositivite VSB est en accord avec un virus relativement infectieux qui peut etre transmis congenitalement. Des taux similaires de coseropositivite VIB ou VLB avec le VSB dans cette population suggerent que le VIB n'est pas plus infectieux que le VLB et qu'il requiert probablement un contact etroit prolonge pour une transmission. Par immunoempreinte et avec un systeme de detection a la phosphatase alcaline, il a ete determine que des neuf vaches avec des anticorps anti-VIB, sept avaient des anticorps reagissant principalement avec les antigenes p51 et p63 du virus de l'immunodfficience humaine type 1 (VIH-1). Ceci suggere que les prot6ines du VIH-1 auraient une utilite potentielle pour le depistage de bovins seropositifs au VIB. Six serums humains qui demontraient une forte reactivite contre plusieurs proteines du VIH-1 de meme que le serum
Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2WI (Jacobs, Smith, Valli), Ontario Ministry of Health, Laboratory Services Branch, Weston, Ontario (Gregory) and the National Animal Disease Center, Ames, Iowa (Whetstone). Present address of Dr. V.E.O. Valli: College of Veterinary Medicine, University of Illinois, Urbana, Illinois. This work was supported by grants from the Ontario Milk Marketing Board, Ontario Ministry of Agriculture and Food and the Natural Sciences and Engineering Research Council of Canada. Submitted April 16, 1992.
Can J Vet Res 1992; 56: 353-359
353
d'un de trois patients consideres comme reacteurs VIH-1 v indetermines >, ont reagit de facon croisee principalement avec la p26 de VIB. Ceci est le premier rapport de serums humains avec des anticorps contre des proteines sp6cifiques du VIB. Des serums humains individuels ou regroupes utilises comme controles positifs en immunoempreinte pour la detection d'anticorps anti-VIH-1, ainsi que le s6rum de deux patients considre's comme reacteurs anti-VIH-1 o indetermines >> et egalement le serum de patients avec des anticorps anti-VIH-2 ou anti-HTLV-1/2 n'ont pas reagi avec les antigenes du VIB. Les reactivites croisees que nous avons observees 6taient dirigees pnncipalement envers des bandes correspondant a la transcriptase inverse et la proteine majeure du nucleoide, proteines qui ont 6te relativement bien conserv6es dans l'evolution retrovirale. Les reactivites crois6es representent probablement des reactivit6s envers des epitopes conserv6s retrouves sur le VIB et VIH puisqu'aucune seroconversion n'a ete d6montr6e chez les humains A risque pour une exposition au VIB et que les cellules humaines ne sont pas sensibles A I'infection au VIB. (Traduit par Dr Ronald Magar)
INTRODUCTION
The bovine immunodeficiency-like virus (BIV), first isolated in 1972 (1) is genetically, antigenically, and structurally closely related to the human immunodeficiency virus type 1 (HIV-1) (2-4). Antigenic relatedness was shown by cross-reactivity between rabbit antiBIV and anti-HIV-1 antibodies with BIV and HIV-1 antigens in indirect immunofluorescence and Western blot assays (WBA). Although the molecular biology of BIV is known in detail (2-4), little is known about the biology of naturally-occurring BIV infection. There has been no association of BIV infection and naturally-occurring disease, although decreased lymphocyte blastogenesis was demonstrated in experimentally infected calves (5). Bovine immunodeficiency-like virus infection in various coinfections with the other bovine retroviruses, bovine leukemia virus (BLV) (6) and bovine syncytial virus (BSV) (7-9), have been 354
reported and among dairy cows BIV infections alone were not found (10). This lack of progress in discovering the natural history of BIV results directly from the inability to produce the large amounts of BIV antigens required for seroepidemiological studies. Only recently have such culture systems been described (11,12). However, antigen production in tissue culture remains relatively inefficient since BIV infection in tissue culture cells is cytocidal; there are no continuously productive cell lines. Furthermore, the development of automated serological tests using BIV antigens harvested from tissue culture has been hampered by high background reactivity with negative control sera (personal communication, M. Horzinek and A. Herrewegh, Utretcht, The Netherlands). Recombinant BIV proteins, although reported (13) are unavailable commercially, but this technology should result in the rapid development of highly sensitive, specific, and automated seroassays. Knowing the antigenic relatedness among the lentiviruses has caused some concern that humans exposed to BIV-infected cattle, as well as other retrovirus-infected animals, might develop false positive, atypical, or "indeterminate" HIV-1 serological reactions (14). The anti-p24 antibody reactivity in HIV-l infected humans is completely blocked by HIV-1 recombinant p24 antigen while this same cloned protein does not block or only partly blocks the reactivity of anti-p24 reactivity in the sera of humans with "indeterminate" reactivities (15). The potential exposure to other human and nonhuman retroviruses has been suggested as possibly accounting for "indeterminate" reactions (15). One observational study found animal exposure common among humans with "indeterminate" reactions (14). More recently, sera from a series of patients with "indeterminate" reactions to HIV-1 and with histories of a wide range of animal exposure or consumption of raw milk showed no cross-reactivity with BIV antigens (16). Furthermore, 11 personnel at risk for BIV exposure and two persons accidentally injected with BIV-infected cell culture material did not seroconvert to BIV (16). Presently there are no data supporting an association between animal exposure or exposure
to BIV and "indeterminate" reactions on the HIV-1 WBA and no human sera have been detected with antibody to BIV-specific proteins (16). Other
suggestions for "indeterminate" test results are autoantigens, early or variability in the anti-HIV-1 antibody response, the presence of nonspecific comigrating antigens in the viral lysate used to prepare the Western blot strips and the presence of epitopes, posttranscriptionally modified antigens, or new antigenic sites in the viral antigen that are not present on the recombinant p24 antigen (15). Bovine immunodeficiency-like virus is known to infect a variety of bovine and nonbovine cell types (17). Although early research showed that human cells in culture could be productively infected (18), a more recent study failed to show active infection or proviral integration (17). Fetal bovine serum, a widely used tissue culture supplement, commonly contains bovine viruses (19). Recent data showed that a large number of fetal bovine sera showed no serological evidence for BIV infection (16) supporting, but not proving, lack of in utero infection of bovine fetuses. It remains possible, although unlikely because of the lack of positive serological data (16), that biologicals of tissue culture origin for in vivo use in cattle and humans could mediate transfer of infectious BIV or BIV antigens resulting in transient viremia or sensitization. The WBA has been used for the serodiagnosis of BIV infection in cattle; here we report an improved WBA using a chemiluminescence detection system and used it to study the frequency of BIV seropositivity in two selected dairy herds. Associations of BIV seropositivity and seropositivity to the other bovine retroviruses, BLV and BSV, were studied. We present preliminary evidence that BIV appears similar to BLV in infectivity. We reasoned that since BIV and HIV-1 are antigenically related, such crossreactivity might be used for the detection of BIV-infected cattle. Because of the concern for possible sensitization or infection of humans with BIV, we wished to test the reactivity of the serum of known HIV-1-infected humans, particularly those with strong anti-HIV-I reactivity, and some human sera with "indeterminate"
HIV-l reactions against BIV antigens. Most bovine sera with anti-BIV reactivity could be identified using HIV-1 proteins while, generally, only those human sera with strong anti-HIV-1 reactivity identified BIV proteins in WBAs. MATERIALS AND METHODS SERA
Bovine serum samples were randomly obtained from cows five years of age or greater in two dairy herds. Cows were tested for serum biochemical, hematological, and physical abnormalities at the time of sampling and monitored for changes in health status for one year. One herd, from which 31 of 60 lactating cows were sampled, was selected on the basis that there was no history of deaths due to lymphoma in the past five years and subjectively there was an unusually high frequency of morbidity (pneumonia, mastitis, metritis) and mortality (8 deaths in the preceding 18 months) in the postparturient period. The owner of this herd had participated voluntarily in a program to decrease the herd prevalence of BLV infection during the past decade. The second herd, from which 8 of 30 lactating cows were sampled, was selected because of an unusually high mortality rate due to lymphoma (mean of two per year). Selected human sera with negative (n = 2), positive (n = 10), and "indeterminate" (n = 3) test results using the WBA for anti-HIV-1 antibodies and interpreted according to standardized criteria (20) were used. Single human serum samples with antiHIV-2 or anti-HTLV-1/2 antibodies (Ontario Ministry of Health, Laboratory Services Branch, Weston, Ontario) were randomly selected. Human sera were coded and treated in a blinded fashion. SEROLOGY
The agar gel immunodiffusion test (BLV-AGID, Bovi-Leuko test, Institut Armand-Frappier, Laval, Quebec) was used to detect anti-BLV (predominately envelope glycoprotein) antibodies in serum. Bovine sera were tested for anti-BSV antibodies by a previously reported BSV-AGID test that utilizes crude virus preparations (8).
The protocol for the production of BIV and the WBA has been described previously (12). Briefly, BIV was grown in primary fetal bovine lung (BIV-FBL) cells between passages 3 and 6, in Earl's MEM plus 10% fetal bovine serum, 50 mg/L gentamicin, 0.02% L-glutamine, and 2 j&g/mL polybrene. The BIV-infected FBL cells produce large syncytia which release virus into the supernatant as cytolysis occurs. Virus was pelleted from supernatant by ultracentrifugation at 100,000 x g through 40Gb glycerol in Dulbecco's phosphate buffered saline (PBS) at pH 7.2. Pellets were washed once in a solution containing 50 mM Tris (pH 8.0) and 5 mM EDTA. Pellets were resuspended in lysis buffer containing 500 mM NaCI, 50 mM Tris (pH 8.0), 5 mM EDTA, 1% Triton X-100, 50 1&M PMSF. Virus in the cell culture supernatant was concentrated approximately 1,000-fold using this protocol. Sodium dodecyl sulfate (SDS)-treated antigen was electrophoresed through preformed 10-201o gradient polyacrylamide gels (PAG) (Enprotech, Hyde Park, Maryland). Gradient separated proteins were transferred electrophoretically to 0.45 jsm pore size nitrocellulose paper (Bio-Rad Laboratories, Richmond, California) which was subsequently blocked overnight at room temperature with a solution containing 0.05%o Tween 20 in TS (10 mM Tris and 150 mM NaCI [pH 8.6]). Bovine sera were diluted 1:10 with TS solution and incubated with the nitrocellulose paper using a miniblot apparatus (Immunetics, Cambridge, Massachusetts) for 2 h at room temperature. When human sera were tested for anti-BIV activity they were treated as described below. Low molecular weight markers (Bethesda Research Laboratories, Gaithersburg, Maryland) and positive control sera derived from a sheep (3005) or cow (6618) immunized with BIV (Dr. C. Whetstone, NADC, Ames, Iowa) were run on each gel. The nitrocellulose paper was washed three times, 10 min per wash, in TBS (20 mM Tris, 500 mM NaCI, [pH 7.5]) followed by application of 1:1500 dilution in TBS of horse radish peroxidase-conjugated recombinant protein G (GammaBind G-HRP, Genex Corp., Gaithersburg, Maryland) for 1 h at room temperature. The blot was washed as described
66.2 t .
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:
31
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Fig. 1. Western blot assay, developed using a chemiluminescence protocol, showing the reactivity of selected bovine sera against BIV antigens. Numbers to the left of lane a are in kD. Molecular weight standards are in lane a. Arrowheads identify BIV-specific proteins corresponding to (from lowest to highest): p13, p15, p24, p26, p28, gp4O and p60. The foflowing sera were tested: lane 1, BIV positive bovine control; lane 2, BIV negative bovine control; lanes 3-8, unreated bovine sera; les 9-12, sequential sera obtained over two years from a single cow; lanes13 and 14 were sera obtained six months apart from one cow. Sera with at least anti-p26 activity were considered test result positive; sera in lanes 1, 3-5, 8-12 were positive while sera in lanes 2, 6, 13 and 14 were negative. High background staining in lane 7 made interpretation impossible. The sera in lanes 9-12 demonstrates persistent anti-DIV reactivity. Most BIV positive sera showed reactivity against bands migrating at 13, 18, 26 and 28 kD. Several background bands migrated at positions above the 42.6 kD marker; these were not viral specific and are likely due to reactivity against tissue culture antigens. AU sera were negative for antibovine leukemia virus antibodies wbile all sera were positive for antibovine syncytial virus antibodies except the ser in lanes 1 and 3.
above then the reaction visualized using a 4-chloronapthol membrane peroxidase substrate kit (Kirkegaard & Perry, Gaithersburg, Maryland). Recently, we have changed our WBA protocol to include final visualization using a chemiluminescence procedure which reportedly improves sensitivity in model systems about 100-fold compared with traditional enzyme-linked procedures (Amersham, Oakville, Ontario). Bovine sera which contained anti-p26 antibody were considered test result positive for BIV. The HIV-1 proteins of tissue culture origin (Ontario Ministry of Health, Laboratory Services Branch, Weston, Ontario) were separated by SDS PAG electrophoresis through a 6% stacking gel and a 10.8% separating gel of 1.5 mm thickness. Proteins were transferred to 0.45 itm pore size polyester355
TABLE 1. Retroviral serological test results for cows from two Ontario dairy herds
Number of Cows 2 12 2 4 5
Serological test resultsa BLV BSV BIV -
_ +
_ -
-
-
+
+
-
+ +
+ +
+
+
92.5< 621.
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_ + + +
8 22 9 Totals 39 antiand aAntibovine leukemia virus (BLV) bovine syncytial virus (BSV) antibodies were detected by agar gel immunodiffusion tests. Antibovine immunodeficiency-like virus (BIV) antibodies were detected by Western blot analysis using a chemiluminescence protocol
reinforced nitrocellulose paper (Schleicher and Schuell, Keene, New Hampshire) using a Transphor unit (LKB, Piscataway, New Jersey) at 45V for 12 h and then 45 min at 80V. Transfer buffer consisted of 20 mM Tris, 0.15 M glycine, and 20% methanol at pH 8.6. The nitrocellulose paper was blocked using a quenching solution consisting of 10% skim milk powder and 0.001 07 merthiolate in deionized distilled water for a minimum of 2 h, then rinsed with TS, numbered, cut into strips and stored at - 700C until required. These nitrocellulose strips were warmed to room temperature and transferred to incubation trays (BioRad) to which 2.5 mL of 1:100 dilutions of bovine sera in PBS was added to each strip. Human sera were diluted in quenching solution (1:100) to minimize high background staining. Sera were incubated while rocking for 2 h at room temperature, rinsed with TS solution (pH 7.4) and then washed once in TS solution for 10 min and then twice in 0.05%o Nonidet P-40 in TS solution for 10 min followed by a final 10 min wash in TS solution. The secondary antibody, alkaline phosphatase conjugated goat antihuman IgG + IgM (H + L) (Jackson Immunoresearch Laboratories, West Grove, Pennsylvania) was diluted 1:20,000 in 0.05% Nonidet P-40 in TS solution and added to the nitrocellulose paper strips. For bovine sera, alkaline phosphatase conjugated goat antibovine IgG (Kirkegaard & Perry) was diluted to 1:1500. The nitrocellulose paper strips were then incubated for a further 2 h with rocking and then rinsed and washed as 356
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Fig. 2. Western blot assay, developed using an alkaline phosphatase detection system, showing the reactivity of selected bovine sera against HIV-1 antigens. Numbers to the left of lane a are in kD. Molecular weight standards are in lane a. Anti-HIV-1 human positive control serum diluted 1:100 in quenching solution (lane b) and 1:100 in PBS (lane c) were used. Numbers to the left of lane b are in kD. The bands represent the major HIV-1 proteins identified by human immune serum in the Western blot assay. The unlabelled band migrating above 92.5 kD likely represents reactivity against HIV-1 gp12O. Negative control human sera diluted 1:100 in quenching solution (lane d) and 1:100 in PBS (lane e) were used. The PBS negative control (lane e) showed weak staining bands between 33 and 41 kD, slightly greater than 41 kD, and several very weak staining bands were present above 63 kD. Lanes 1-7 are selected examples of Western blots showing reactivity of bovine sera against HIV-1 proteins; lane 10 is a bovine negative control serum. Lanes 8 and 9 are dilutions (1:100,000) of human HIV-1 positive control sera. Bovine sera showed reactivity against bands migrating at 45 kD (lanes 1 and 4) and 51 and 63 kD (lanes 2, 4, 5 and 7). A very weak staining band was present in lane 3 at slightly less than 41 kD; this serum was considered negative for crossreactivity. A very weak nonviral band was present across all lanes with bovine sera at 66 kD. Reactivity against nonvirus specific bands are likely due to reactivity against tissue culture antigens.
before. The reaction was visualized using a BCIP-NBT substrate according to manufacturer's recommendations (Promega, Madison, Wisconsin). The reaction was allowed to continue for approximately 3 to 10 min and then stopped by washing for 10 min in a solution containing 20 mM Tris and 5 mM EDTA (pH 8.0) and then mounted and dried on filter paper. RESULTS Serum biochemical, hematological, or physical abnormalities were not detected at the time of sampling; there were no changes in health status dur-
ing the subsequent year. In the herd with no previous history of deaths due to lymphoma, no cows, among the 31 tested, had anti-BLV antibodies. In contrast, all of the eight cows tested from the herd with multiple deaths due to lymphoma had positive BLV-AGID test results. Selected WBAs for the detection of bovine anti-BIV activity are shown in Fig. 1. Positive control bovine serum showed reactivity against BIV specificproteins that migrated at approximately 13, 15, 24, 26, 28, 40 and 60 kD. These proteins correspond almost exactly to those previously recognized except for the bands at 40 and 60 kD that likely
correspond to gp42 and p50 or p55, respectively (12). Cows had antibodies to a variety of antigens but the most prominent reactions were against proteins migrating at 15, 18, 26 (major internal core protein), and 28 kD. Only occasional bovine sera showed reactivity against bands migrating at 45, 53 and 62 kD which likely represent envelope glycoprotein, gag precursor protein, and reverse transcriptase, respectively. Sequential serum samples taken over a year showed an unchanging pattern of reactivity; this was a typical result when multiple testing was done on individuals. Sera from 3 of the 39 cows tested had initial negative WBA test results, but were repeatedly seropositive when retested by WBA using the chemiluminescence protocol. The distribution of serological test results for BSV, BIV and BLV infection are summarized in Table I. Twelve of 39 cows tested had negative serological test results. Sixteen of 39 cows tested had evidence for single retroviral infections; the most common single positive test result was due to BSV (12 of 39 cows tested). Double infections occurred in 10 of 39 cows tested; these were most often due to BSV with either BLV (4 of 39 cows tested) or BIV (5 of 39 cows tested). Only one cow had a positive test result for BIV and BLV. One of 39 cows tested had evidence of a triple retroviral infection. Of the nine cows that had positive serological test results for BIV, seven had serum antibodies that detected HIV-1 proteins. There were no instances of cows having negative test results for anti-BIV activity and positive test results for anti-HIV-I activity. Selected WBAs for the detection of bovine anti-HIV-1 activity are shown in Fig. 2. Bovine sera reacted against a variety of HIV-1 proteins; most consistent reactions were found against bands migrating at 51 and 63 kD. Reactions against bands migrating at 24, 41, 45 and 63 kD were also found but were very weak apart from one cow with anti-p45 reactivity shown in lane 1 of Fig. 2. Selected examples of reactivity of human sera against BIV antigens by WBA are shown in Fig. 3. An ovine BIV positive control showed reactivity against bands migrating at 13, 15, 18, 24 and 26 kD. Of 17 human sera studied, six showed anti-BIV activity. Only
X : .U, i; Cl 1141-1 1f'50 Fig. 3. Western blot assay, developed using an alkaline phosphatase detection system, showing reactivity of selected human sera against BIV antigens. Molecular weight standards are in lane a. Lane b shows reactivity of a BIV positive ovine control serum; arrowheads identify distinct bands (from lowest to highest) at 13, 15, 18, 24 and 26 kD. The following human or control sera we-tested: lane 1, HIV-1 positive control; lane 2, human negative control; lane 3, sem from lan, 1 diluted 1:10,000; lane 4, conjugate control (no serum); lane 5, serum control (no conjugate); lane 6, initial serum from patient 1 with weak anti-HIV-1 reactivity; lane 7, serum from patient 1 taken one month later; lane 8, initial serum from patient 2 with no anti-HIV-1 reactivity; lane 9, serum from patient 2 with strong anti-HIV-1 reactivity taken one month later; lane 10, HIV-2 positive human serum; lane 11 HTLV-1/2 positive human serum; lane 12, "indeterminate" human serum with strong anti-HIV-1 p24 activity only; lane 13, "indetermnate" human serum with activity against HIV-1 pl8, p24, p33 and p5l; lane 14, HIV-1 strong positive human serum; lane 15, HIV-1 positive human serum with activity against HIV-1 p18 and gp4l; lane 16, initial serum from patient 3 with anti-HIV-1 activity; lane 17, HIV-1 positive serum from patient 3 taken one month later; and lane 18, HIV-1 positive pooled human serum. The human sen in lanes 6, 7, 9, 13 and 14 showed reactivity against a band migmting at a position corresponding to BIV p26. Human sen in lanes 9 and 14 showed reactivity against bands corresponding to BIV proteins p24 and pl8, respectively. A weak-staining nonviral band was present across all lanes with human sen at approximately 18 kD and likely represents reactivity against a tissue culture antigen.
those human sera demonstrating strong anti-HIV-i WBA reactivity showed anti-BIV activity. Strong antiHIV-1 reactors showed reactions against HIV-1 antigens that were at least as intense at a 1:100 dilution than the positive control serum similarly diluted and shown in lanes b and c of Fig. 2. Most human sera that reacted, reacted against BIV p26; occasional human sera reacted against BIV p18 and p24. One of the three human sera considered to have "indeterminate" HIV-1 test results identified a BIVspecific band at 26 kD; this patient's serum reacted against p18, p24, p33 and p51 in the HIV-1 WBA. Serum from the other two "indeterminate" HIV-1 reactors showed only anti-p24 activity. Single human sera with either anti-HTLV-1/2 or anti-HIV-2 antibodies showed no anti-BIV activity. DISCUSSION In two dairy herds, perceived to have chronic disease problems, there
was a high frequency (27 of 39 cows tested) of retroviral infections based on serological testing. Single infections were most commonly found and these were predominately due to BSV. Noncross-reactivity between antibody responses to these retroviruses suggests that the testing protocols had high specificity. Bovine syncytial virus could be more infectious than either BLV or BIV, as suggested by the high prevalence of anti-BSV antibodies, however the relative infectivities of the bovine retroviruses in naive cows and major modes of transmission are unknown. Double infections were most often due to BSV in combination with BLV or BIV, in almost equal frequency. The association of BSV with BLV or BIV could result from the relatively large number of cows that were BSV-infected at an early age which then become exposed to BLV or BIV. This paradigm could be correct since congenital BSV infection is known to occur (21). The data are also 357
consistent with BSV predisposing cows to infections with other viruses. Between 20 and 4007 of cattle are infected with BSV however, there are no data available suggesting that BSV mediates immunosuppression or other events that could possibly enhance infectivity (22, 23). Only one instance each of BLV and BIV in combination and a triple retroviral infection were found. The finding of BLV in association with BIV in only one cow suggests relatively low but equal infectivity for these two viruses. As with BLV, BIV probably requires prolonged close contact for transmission. A larger seroepidemiological study is needed to confirm these preliminary findings. A previous study found BLV to be the single most common retroviral infection of dairy cows and BIV appeared most often in association with BLV; BSV accounted for few singleton reactors but clustered with BLV (10). In this previous study no single BIV infections were found among dairy cows although occasional singleton reactors were found among beef cattle. The discrepancies in the occurrence of single and combination retroviral infections between the earlier study (10) and our data might be accounted for by such uncontrolled variables as genetic, management, or environmental factors enhancing the transmissibility of BLV and methodological differences. The clustering of BSV and BLV along with a relative paucity of BSV singleton reactors in the earlier study (10) suggests that BLV may have predisposed cows to BSV infection. Data concerning BLVassociated immunosuppression, in the absence of lymphoid tumors, are controversial (reviewed in 24). There was no illness among the cows sampled suggesting either no effect or a subclinical effect of retroviral infection. Because of the small sample size and biased nature of this initial observational study, case-control studies are needed to examine the potential role of these viruses in mediating disease. Other studies, with BIV-infected sheep and cattle, have shown an early increase in anti-p26 antibody followed in a variable amount of time by an increase in anti-gp42 antibody (12,25). The patterns of antibody reactivity in the bovine sera tested here did not change over time suggesting that these 358
represented persistent infections. Similarities in nucleotide sequences and serological cross-reactivity between HIV-1 and BIV using heterologous antisera show that the two viruses are closely related and stem from a common ancestor (4). Here we have shown that reciprocal serological crossreactivity, using bovine and human sera, exists between BIV and HIV-1. The BIV-infected cows most consistently reacted against HIV-1 bands migrating at 51 and 63 kD. These bands are associated with HIV-1 reverse transcriptase activity, a critical enzyme in the retroviral lifecycle. Reverse transcriptase is highly conserved throughout retroviral evolution and this conservation likely accounts for the cross-reactivity seen here. These preliminary data show that HIV-l antigens could potentially be used to identify BIV-infected cows (seven of nine BIV-infected cows reacted with HIV-1 proteins) although recently cell cultures that better support the growth of BIV have been discovered (12). More highly productive cell cultures and the wide availability of cloned BIV proteins will obviate the use of heterologous retroviral antigens in BIV detection. A small number of human sera containing anti-HIV-l antibodies were tested for reactivity to BIV antigens. Positive control sera used in the HIV-1 WBA failed to identify BIV antigens. The 6 of 17 human sera that did react against BIV antigens had at least as intense reactions against HIV-1 proteins compared with the similarly diluted HIV-1 positive control serum, i.e. they were strong reactors. Therefore, it appears that only those human sera with relatively strong anti-HIV-l WBA reactivity will react against BIV antigens. The cross-reactivity was directed primarily to the BIV p26 which represents the major core protein, another protein which is relatively well-conserved in retroviral evolution. To date, human sera with anti-BIV activity have not been detected however, the most recent data (16) used human sera whose reactivity did not exceed those of positive controls. Serum from one patient considered to be an "indeterminate" reactor showed weak reactivity against a BIV-band migrating at 26 kD. This patient's serum showed reactivity against a number of bands in the HIV-1 Western
blot, unlike sera from the other two "indeterminate" reactors that failed to show cross-reactivity. Antibodies to a wider variety of HIV-l proteins might have increased the chances of detecting cross-reactive antibodies in this patient. Since the most recent data indicate that human cells are not susceptible to BIV infection and human exposure is unassociated with seroconversion, it is likely that the reactions detected here are due to similarities in epitopes between the two lentiviruses and thus are simply an interesting laboratory phenomenon. At the level of sensitivity of the WBA, there were no cross-reactive anti-HIV-2 and anti-HTLV-1/2 antibodies in human sera against BIV. These results are not unexpected since HIV-2 is significantly more distantly related to BIV than is HIV-1 and HTLV-1 belongs to a different lineage of retroviruses. It is important to be able to detect BIV antigens as well as anti-BIV antibody in bovine serum since bovinebased control reagents and tissue culture supplements are used in quality assessment programs and in the preparation of bovine and human biologicals. Previous data have shown that a variety of bovine viruses were present in about 30% of fetal bovine sera (19). Although it is now a common practice to use irradiated fetal bovine serum in culture systems, the possibility of contamination with viral antigens still exists. Current data suggest that BIV does not infect human cells (17), but until more extensive studies are done BIV remains of concern. Since BIV can infect a variety of bovine and other nonhuman cells in tissue culture, it is important to ensure that cultured cells used as sources of biologicals be screened for BIV infection. Propagation of BIV in such cell lines could inadvertently lead to exposure of vaccinates to BIV antigens or infection. ACKNOWLEDGMENTS
The authors thank Drs. J. W. Black, A. Bouillant, D. Hodgins, R. Johnson, B. McLauglin, S. Nadin-Davis and M. J. Van Der Maaten for their generous support and C. Davies and B. Jefferson for their excellent technical assistance.
REFERENCES 1. VAN DER MAATEN MJ, BOOTHE AD, SEGER CL. Isolation of a virus from cattle with persistent lymphocytosis. J Natl Cancer Inst 1972; 49: 1649-1657. 2. BRAUN MJ, LAHN S, BOYD AL, KOST TA, NAGASHIMA K, GONDA MA. Molecular cloning of biologically active proviruses of bovine immunodeficiency-like virus. Virology 1988; 167: 515-523. 3. GARVEY KJ, OBERSTE MS, ELSER JE, BRAUN MJ, GONDA MA. Nucleotide sequence and genome organization of biologically active proviruses of the bovine immunodeficiency-like virus. Virology 1990; 175: 391-409. 4. GONDA MA, BRAUN MJ, CARTER SJ, KOST TA, BESS JW, ARTHUR LO, VAN DER MAATEN MJ. Characterization and molecular cloning of a bovine lentivirus related to human immunodeficiency virus. Nature 1987; 330: 388-391. 5. MARTIN SJ, O'NEILL PO, BILLELLO JA, EISEMAN JL. Lymphocyte transformation abnormalities in bovine immunodeficiency-like virus infected calves. Immunology Letters 1991; 27: 81-84. 6. BURNY A; CLEUTER Y, KETTMAN R, MAMMERICKX M, MARBAIX G, PORIFIELLE D, VAN DEN BROEKE A, WILLEMS L, THOMAS R. Bovine leukemia: facts and hypotheses derived from the study of an infectious cancer. Cancer Surveys 1987; 6: 139-159. 7. GREIG AS. A syncytium regression test to detect antibodies to bovine syncytial virus. Can J Comp Med 1979; 43: 112-114. 8. MALMQUIST WA, VAN DER MAATEN MJ, BOOTHE AD. Isolation, immunodiffusion, immunofluorescence, and electron microscopy of a syncytial virus of lymphosarcomatous and apparently normal cattle. Cancer Res 1969; 29: 188-200.
9. VAN DER MAATEN MJ, BOOTHE AD, MALMQUIST WA. Further studies on bovine syncytial virus. Bibl Haematol 1969; 36: 446-452. 10. AMBORSKI GF, LO JL, SEGER CL. Serological detection of multiple retroviral infections in cattle: bovine leukemia virus, bovine syncytial virus and bovine visna virus. Vet Microbiol 1989; 20: 247-253. 11. BOUILLANT AMP, RUCKERBAUER GM, NIELSEN KH. Replication of the bovine immunodeficiency-like virus in diploid and aneuploid cells: permanent, latent and virus-productive infections in vitro. Res Virol 1989; 140: 511-529. 12. WHETSTONE CA, VAN DER MAATEN MJ, MILLER JM. A Western blot assay for the detection of antibodies to bovine immunodeficiency-like virus in experimentally inoculated cattle, sheep, and goats. Arch Virol 1991; 116: 119-131. 13. RASMUSSEN L, BATTLES JK, ENNIS WH, NAGASHIMA K, GONDA MA. Characterization of virus-like particles produced by a recombinant baculovirus containing the gag gene of the bovine immunodeficiency-like virus. Virology 1990; 178: 435-451. 14. DOCK NL, LAMBERSON HV, O'BRIEN TA, TRIBE DE, ALEXANDER SS, POIESZ BJ. Evaluation of atypical human immunodeficiency virus immunoblot reactivity in blood donors. Transfusion 1988; 28: 412418. 15. POVOLOTSKY J, GOLD JWM, CHIEN N, BARON P, ARMSTRONG D. Differences in human immunodeficiency virus type 1 (HIV-1) anti-p24 reactivities in serum of HIV-l-infected and uninfected subjects: analysis of indeterminate Western blot reactions. J Infect Dis 1991; 163: 247-251. 16. WHEISONE CA, SAYRE KR, DOCK NL, VAN DER MAATEN MJ, MILLER JM, LILLEHOJ E, ALEXANDER SS. Examination of whether persistently indeter-
minate human immunodeficiency virus type 1 Western immunoblot reactions are due to serological reactivity with bovine immunodeficiency-like virus. J Clin Microbiol 1992; 30: 764-770. 17. KASHANCHI F, LIU ZQ, ATKINSON B, WOOD C. Comparative evaluation of bovine immunodeficiency-like virus infection by reverse transcriptase and polymerase chain reaction. J Virol Methods 1991; 31: 197-210. 18. GEROGIADES JA, BILLIAU A, VAN DER SCHUERER B. Infection of human cell cultures with bovine visna virus. J Gen Virol 1978; 38: 375-381. 19. KNIAZEFF AJ, WOPSCHALL LJ, HOPPS HE, MORRIS CS. Detection of bovine viruses in fetal bovine serum used in cell culture. In Vitro 1973; 44: 100-103. 20. CENTERS FOR DISEASE CONTROL. Interpretation and use of the Western blot assay for serodiagnosis of human immunodeficiency virus type 1 infections. MMWR 1988; 260: 674-679. 21. WCAS MH, WESWOU DGF, EDWARDS S, NEWMAN RH, SWALLOW C. Immunofluorescence and cell culture techniques in the diagnosis of viral infection of aborted bovine fetuses. Vet Rec 1986; 118: 242-243. 22. APPLEBY RC. Antibodies to bovine syncytial virus in dairy cattle. Vet Rec 1979; 105: 80-81. 23. WOODS GT. Bovine parvovirus 1, bovine syncytial virus and bovine respiratory syncytial virus and their infections. Adv Vet Sci Comp Med 1974; 18: 273-286. 24. JACOBS RM. Bovine lymphoma. In: Olsen R, Krakowka S, Blakeslee J, eds. Comparative Pathobiology of Viral Diseases. Baton Rouge: CRC Reviews, 1986: 21-51. 25. WHETSTONE CA, VAN DER MAATEN MJ, BLACK JW. Humoral immune response to the bovine immunodeficiencylike virus in experimentally and naturally infected cattle. J Virol 1990; 64: 3557-3561.
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