Bovine Leukemia Virus Antigen Research Articles

Indirect ELISA (iELISA) standardization for the diagnosis of bovine enzootic leukosis

ABSTRACT: Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.

Expression of bovine leukemia virus antigen in breast cancer tissue

Breast cancer is a serious medical and social problem for most developed countries of the world. The search for new prognostic factors and targets for targeted therapy currently continues to be a pressing issue, as there are forms of breast cancer with uncertain targets for any type of therapy. The purpose of this work is to conduct an immunohistochemical study to detect and evaluate the expression of the antigen of the cow leukemia virus in the tissue of various types of breast cancer.
 Method. 100 cases of breast cancer were investigated, divided into subgroups, the studied parameters: steroid hormone receptors, HER2 / neu, VEGF, BLV expression, molecular genetic research on BRCA1/BRCA2 mutations.
 Results. In the group of triple negative breast cancer, 4 cases were detected expressing a cow leukosis virus antigen. Expression was represented by grains and clumps in the cytoplasm and on the surface of the tumor cell membrane, the wall of microvessels. Also in these cases, increased expression of VEGF was detected.
 Conclusion. The results indicate that expression of the BLV antigen was observed only in cases of poorly differentiated triple negative breast cancer. It is also noteworthy that the same type of BRCA1 gene mutation (5382insC) and increased expression of vascular endothelial growth factor were detected in these tumors. The authors first used the immunohistochemical method for detecting the antigen of the BLV virus, which made it possible to reliably confirm its presence in breast tumors with similar molecular genetic and immunohistochemical profiles.

A novel bovine leukemia virus peptide vaccine targeting susceptible cattle-Production by 3-D modelling and nanotechnology

Bovine leukemia virus (BLV), a retrovirus related to human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2), causes enzootic bovine leukosis (EBL), a disease characterized by a much extended course that often involves persistent lymphocytosis and culminates in B-cell lymphoma the future. Our previously study demonstrated that cattle which had bovine major histocompatibility complex (BoLA)-DRB3*1601/*1601 genotype tend to onset EBL and indicates high proviral load. One of the major problems to develop the BLV vaccine is unstable effect of the vaccine because of the individual difference for disease susceptibility, we here tried to develop the BLV vaccine for disease susceptible cattle. Firstly, we determined the Th1 epitope of BLV antigens against disease susceptible cattle using a total 118 kind of peptides corresponding to GAG p15, p24 and p12, and ENV gp51 and gp30. Furthermore, we performed the optimization of our determined Th1 epitopes by antigen peptide modeling using in silico screening to resolve low affinity binding between peptide and susceptible BoLA DR molecule. Moreover, to resolve low stability of peptide and induce effectively Th1 immunity, we capsulated our optimized peptides, p12-4R1 and gp51R1, by Carbonate apatite (CO3Ap) because out of three kinds of nano-particles, such as CO3Ap, Cholesterol-modified gelatin and MGlu-HPG-Liposome, CO3Ap most strongly induced in all of Dendritic cell incorporation, antibody production and cellular immunity. Finally, we demonstrated the induction of antigen-specific cell-mediated immune response in mice by both of subcutaneous vaccination and intradermal vaccination of p12-4R1/gp51R1 peptide-conjugated CO3Ap. Collectively, we succeeded to produce a novel peptide vaccine targeting disease susceptible cattle which can induce antigen-specific cellmediated immune response in vivo.

Differences in cellular function and viral protein expression between IgMhigh and IgMlow B-cells in bovine leukemia virus-infected cattle.

Bovine leukemia virus (BLV) induces abnormal B-cell proliferation and B-cell lymphoma in cattle, where the BLV provirus is integrated into the host genome. BLV-infected B-cells rarely express viral proteins in vivo, but short-term cultivation augments BLV expression in some, but not all, BLV-infected B-cells. This observation suggests that two subsets, i.e. BLV-silencing cells and BLV-expressing cells, are present among BLV-infected B-cells, although the mechanisms of viral expression have not been determined. In this study, we examined B-cell markers and viral antigen expression in B-cells from BLV-infected cattle to identify markers that may discriminate BLV-expressing cells from BLV-silencing cells. The proportions of IgM(high) B-cells were increased in blood lymphocytes from BLV-infected cattle. IgM(high) B-cells mainly expressed BLV antigens, whereas IgM(low) B-cells did not, although the provirus load was equivalent in both subsets. Several parameters were investigated in these two subsets to characterize their cellular behaviour. Real-time PCR and microarray analyses detected higher expression levels of some proto-oncogenes (e.g. Maf, Jun and Fos) in IgM(low) B-cells than those in IgM(high) B-cells. Moreover, lymphoma cells obtained from the lymph nodes of 14 BLV-infected cattle contained IgM(low) or IgM(-) B-cells but no IgM(high) B-cells. To our knowledge, this is the first study to demonstrate that IgM(high) B-cells mainly comprise BLV-expressing cells, whereas IgM(low) B-cells comprise a high proportion of BLV-silencing B-cells in BLV-infected cattle.

Resonant gravimetric immunosensing based on capacitive micromachined ultrasound transducers

High-frequency (40 MHz) and low-frequency (7 MHz) capacitive micromachined ultrasound transducers (CMUT) were fabricated and tested for use in gravimetric detection of biomolecules. The low-frequency CMUT sensors have a gold-coated surface, while the high-frequency sensors have a silicon nitride surface. Both surfaces were functionalized with bovine leukemia virus antigen gp51 acting as the antigen. On addition of an a specific antibody labeled with horseradish peroxidase (HRP), the antigen/antibody complex is formed on the surface and quantified by HRP-catalyzed oxidation of tetramethylbenzidine. It has been found that a considerably smaller quantity of immuno complex is formed on the high frequency sensor surface. In parallel, the loading of the surface of the CMUT was determined via resonance frequency and electromechanical resistance readings. Following the formation of the immuno complexes, the resonance frequencies of the low-frequency and high-frequency sensors decrease by up to 420 and 440 kHz, respectively. Finite element analysis reveals that the loading of the (gold-coated) low frequency sensors is several times larger than that on high frequency sensors. The formation of the protein film with pronounced elasticity and stress on the gold surface case is discussed. We also discuss the adoption of this method for the detection of DNA using a hybridization assay following polymerase chain reaction.

A QCM-D Study of Reduced Antibody Fragments Immobilized on Planar Gold and Gold Nanoparticle Modified Sensor Surfaces

An immunosensor is an analytical system consisting of specific immune system molecules coupled to a signal transducer. Immunosensor sensitivity depends on the type of immunorecognition ligands used, immobilization influence on their activity and orientation on the surface. Quartz crystal microbalance with dissipation (QCM-D) was employed to investigate the immobilization of antibodies against bovine leukemia virus antigen gp51 (gp51). Disulphide bridges of antibody hinge region were reduced chemically to yield two half antibody fragments (Frag-Ab), each having a single antigen binding site and free sulfhydryl groups that were used for immobilization. Frag-Ab were immobilized on planar gold and gold nanoparticle (AuNP) modified QCM-D sensor surfaces from initial solutions of different concentrations. Higher Frag-Ab surface density values were obtained on AuNP modified surfaces at all tested antibody concentrations. Frag-Ab/gp51 specific interaction was registered and it was determined that the highest sensitivity was exhibited by Frag-Ab immobilized at the lowest surface desities on both types of investigated surfaces. Specific gp51 interaction with Frag-Ab and non-specific binding to bovine serum albumin modified surfaces were compared by employing Δf/ΔD plots, which could serve as fingerprints of different processes.

Magnetic gold nanoparticles in SERS-based sandwich immunoassay for antigen detection by well oriented antibodies

The aim of the study was to develop an indirect, robust and simple in application method for the detection of bovine leukemia virus antigen gp51. Surface-enhanced Raman scattering (SERS) was applied as detection method. Magnetic gold nanoparticles (MNP-Au) modified by antibodies in oriented or random manner were used for the binding of gp51. The high performance liquid chromatography analysis indicated that the best antibody immobilization and antigen capturing efficiency was achieved using fragmented antibodies obtained after reduction of intact antibodies with dithiothreitol. In order to increase efficiency and sensitivity of immunoassay Raman labels consisting of gold nanorods coated by 5-thio-nitrobenzoic acid layer with covalently bounded antibodies have been constructed. The LOD and LOQ of the proposed immunoassay for antigen gp51 detection were found to be 0.95μgmL−1 and 3.14μgmL−1, respectively. This immunoassay was successfully applied for the detection of gp51 in milk samples in a rapid, reliable and selective manner. We believe that the proposed SERS-based immunoassay format can be applied for the detection of other proteins.

Serological Survey to Determine the Occurrence of Blue Tongue Virus, Bovine Leukemia Virus and Herpesvirus Infections in the Japanese Small Ruminant Population from Northern Districts

Ovine sera, collected from the northern prefectures of Hokkaido, Aomori and Iwate in Japan, were examined for the presence of antibodies against Blue tongue virus (BTV), bovine leukemia virus (BLV), bovine Herpesvirus type 1, agent of infectious bovine rhinotracheitis (IBR), ovine Herpesvirus type 2 (OvHV-2), agent of sheep-associated malignant catarrhal fever (SA-MCF), and bovine Herpesvirus type 4 (BoHV-4), using agar immune diffusion, serum neutralisation (SN) and enzyme linked immunosorbent assay (ELISA) tests. No animals were positive to IBR, BoHV- 4 or BLV antigens. Antibodies against BTV have been detected in 3 samples (1.11%) in two flocks from Hokkaido. The seroprevalence of OvHV-2 was observed in twelve flocks from the 3 considered prefectures, in 56 sheep and two goats, with 37.66% of samples giving a positive reaction in the serum neutralization test. The infections did not appear to be related to the reduction in sheep productivity. Immune reaction reported in goats could refer to Caprine Herpesvirus-2 (CpHV-2). These results indicate that sheep are reservoirs for OvHV-2 in the field in Japan.

Comparison of Oriented and Random Antibody Immobilization Techniques on the Efficiency of Immunosensor

An immunosensor for bovine leukemia virus antigen (gp51) determination was developed using surface plasmon resonance (SPR) technique. During this work the SPR-chip coated with gold was used for immunosensor design. Different strategies of antibodies immobilization - oriented immobilization of specific antibodies fragments (frag-anti-gp51) on gold surface obtained after chemical reduction and random immobilization of intact antibodies (anti-gp51) on gold surface, were investigated. The immobilization based on the application of frag-anti-gp51 was found to be the most suitable for the design of SPR-immunosensor for gp51 detection. SPR sensor created using frag-anti-gp51 was tested in a range from 0.01 to 0.5 mg/mL of gp51 antigen concentration. Developed immunosensor exhibited sufficient antigen binding capacity, simplicity and low cost in respect to the currently evaluated techniques.

Agar gel immunodiffusion analysis using baculovirus-expressed recombinant bovine leukemia virus envelope glycoprotein (gp51/gp30(T-)).

Bovine leukemia virus (BLV) envelope glycoprotein (gp51/gp30T-), consisting of BLV gp51 and BLV gp30 that lacked its C-terminal transmembrane domain, was expressed in insect cells under the control of the baculovirus polyhedron promoter. Recombinant BLV gp51/gp30T- secreted from insect cells was determined by immunofluorescence, enzyme-linked immunosorbent and western blot assays using a BLV-specific monoclonal antibody and BLV-positive bovine antibodies. An agar gel immunodiffusion (AGID) test using gp51/gp30T- as the antigen for the detection of BLV antibodies in serum was developed and compared to traditional AGID, which uses wild type BLV antigen derived from fetal lamb kidney cells. AGID with the recombinant BLV gp51/gp30T- was relatively more sensitive than traditional AGID. When the two methods were tested with bovine sera from the field, the recombinant BLV gp51/gp30T- and traditional antigen had a relative sensitivity of 69.8% and 67.4%, respectively, and a relative specificity of 93.3% and 92.3%. These results indicated that the recombinant BLV gp51/gp30T- is an effective alternative antigen for the diagnosis of BLV infection in cattle.

Expression of the bovine leukemia virus envelope glycoprotein (gp51) by recombinant baculovirus and its use in an enzyme-linked immunosorbent assay.

The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH2-terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.

Humans have antibodies reactive with Bovine leukemia virus.

Bovine leukemia virus (BLV) is an oncogenic retrovirus that commonly infects cattle and causes B cell leukosis in 1-5% of infected cattle. BLV-infected cells are present in marketed beef and dairy products. In the decade after the discovery of BLV in 1969, studies using agar gel immunodiffusion and complement fixation assays failed to find antibodies to BLV in human sera. This led to the prevailing opinion that exposure of humans to BLV and/or the potential for infection are not significant and therefore the virus is not a public health hazard. We reexamined this issue using more sensitive immunological techniques available today. Using immunoblotting to test the sera of 257 humans for antibodies of four isotypes (IgG1, IgM, IgA, and IgG4) to the BLV capsid antigen (p24), we detected at least one antibody isotype reactive with BLV in 74% of the human sera tested. The specificity of the reactivity was strongly suggested by competition studies and by ruling out cross-reacting antibodies to other chronic human viruses. Our results suggest that antibodies reactive with the BLV capsid antigen may serve as a biomarker for exposure to BLV and this exposure may be widespread. The results do not necessarily mean that humans are actually infected with BLV; the antibodies could be a response to heat-denatured BLV antigens consumed in food. They do, however, suggest that further studies in this area could be important.

Delayed-type hypersensitivity in sheep induced by synthetic peptides of bovine leukemia virus encapsulated in mannan-coated liposome.

Four sheep were immunized with synthesized peptides, derived from bovine leukemia virus (BLV) envelope glycoprotein, encapsulated in mannan coated liposomes as adjuvant. On the seventh day after the immunization, the sheep were intradermally challenged with BLV antigen, or synthesized peptides. The areas challenged with antigen were increased skin thickness and biopsied sequentially for immunohistological examinations. Strong delayed-type hypersensitivity was induced in sheep immunized and challenged with peptides encapsulated in mannan-coated liposomes. The major phenotype of the infiltrating lymphocytes was CD5(+). The ratio of CD4(+) to CD8(+) cells was about 1:1.

Analysis by Sodium Dodecyl Sulfate Polyacrylamide gel Electrophoresis and Western Blot of Nonspecific and Specific Viral Proteins Frequently Detected in Different Antigen Preparations of Bovine Leukemia Virus

Bovine leukemia virus (BLV) infection in cattle is seldom manifested clinically, and is routinely diagnosed by serologic tests such as enzyme-linked immunosorbent assay or Western blot (WB). Because of the difficulty in interpreting WB results, the aim of the present study was to determine which of the bands observed in WB were specifically produced by BLV and which corresponded to nonspecific proteins, either derived from medium components or of a cellular nature. Five different BLV antigen preparations from 2 cell lines (FLK-BLV and BLV-bat2) frequently used for the production of BLV antigen were compared. The protein profiles of these antigen preparations were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and WB. Fetal calf serum, required for cellular growth and important in induction of viral transcription in vitro, was identified as a source of irrelevant proteins. In this study, 15 nonspecific protein bands in the growth medium were observed. These bands interfered with the interpretation of results. A nonspecific protein (25 kD) that was highly reactive in cell lysate preparation from BLV-bat2 was also detected. The unequivocal identification of protein bands, both specific and nonspecific, seen in WB is important not for understanding the protein profile of antigen preparations but also for determining if an animal is BLV positive or negative.

Up-regulation of tumor necrosis factor α mRNA is associated with bovine-leukemia virus (BLV) elimination in the early phase of infection

Protective immune responses were analyzed in eight sheep vaccinated with BLV envelope peptides and experimentally infected with bovine-leukemia virus (BLV). Five of eight peptide-immunized sheep showed a high T-cell proliferative response to the BLV peptides and all of these were protected from the infection. The other three peptide-immunized sheep showed no T-cell proliferative responses to any BLV antigens similar to control sheep, though they also exhibited resistance to BLV challenge. To investigate other mechanisms which suppress BLV expansion in these non-responding sheep, we measured the levels of the cytokine expressions before, and after, BLV challenge using competitive reverse-transcriptase polymerase chain-reaction systems. It was revealed that the expression of tumor necrosis factor α (TNFα) was higher in BLV-resistant sheep than in BLV-susceptible sheep. Thus, TNFα expression rather than specific T-cell activity may play an important role in the protective mechanism against BLV infection, at least during the primary viremia phase.

Provirus Variants of the Bovine Leukemia Virus and Their Relation to the Serological Status of Naturally Infected Cattle

Infection of cattle with the bovine leukemia virus (BLV) results in a strong permanent antibody response to the BLV antigens some weeks after infection. However, cattle may carry provirus and not have detectable antibody titers. To prove the occurrence of different BLV provirus variants in German cattle and to study the influence of special BLV variants on the immunoreaction, a 444-bp fragment of theenvgene of 35 naturally BLV infected animals was analyzed. Seven different groups of BLV provirus variants were found on the basis of restriction fragment length polymorphism. Three BLV provirus variant groups and five additionally sequenced BLV isolates showed a high similarity to BLV provirus isolates from other geographical areas. The variation in nucleotide sequence of the five BLV isolates compared with nine previously sequenced BLV isolates ranged up to 5.3%. While BLV provirus variant groups A, C, D, E, F, and G were clearly related to agar-gel immunodiffusion test (AGID)- and enzyme-linked immunosorbent assay (ELISA)-positive animals, BLV provirus variant group B was solely found in permanent AGID- and ELISA-negative or in transient ELISA-positive animals. Altogether, these results indicate that special BLV provirus variants may be responsible for atypical forms of BLV infection in cattle.

Progression to persistent lymphocytosis and tumor development in bovine leukemia virus (BLV)-infected cattle correlates with impaired proliferation of CD4+ T cells in response to gag- and env-encoded BLV proteins.

The mechanism of leukemogenesis and persistent lymphocytosis (PL; benign expansion of B lymphocytes) in cattle infected with bovine leukemia virus (BLV; a retrovirus closely related to human T-cell leukemia virus type 1) is unknown; however, the immune system likely plays an important role in controlling the outcome of infection. In this study, we compared T-cell competence in serologically positive alymphocytotic (AL) animals with T-cell functions in animals with progressive stages of infection, PL and tumor bearing (TB). Dramatic differences were observed in lymphocyte proliferation to recombinant proteins encoded by BLV gag (p12, p15, and p24) and env (gp30, and gp51) genes in different disease stages. Lymphocytes from AL cattle recognized an average of three of five recombinant proteins per animal. Expansion of antigen pulsed lymphocytes in interleukin-2 increased protein recognition to almost five per animal. In contrast, lymphocytes from PL and TB animals failed to recognize any BLV recombinant proteins. Short-term T-cell cultures from the PL group expanded in interleukin-2, as well as the PL and TB cells cultured in indomethacin (3 to 6 microg/ml), increased the average of recognized proteins per animal to one. Cells proliferating to BLV antigens were CD4+ T lymphocytes, as shown by cell depletion studies. The positive effect of indomethacin suggests involvement of prostaglandin E2 as a negative regulatory factor in the later stages of disease. Thus, for the first time, advancing stages of BLV infection were correlated with decreased T-cell competence, providing deeper insight into pathogenesis of retroviral infections.

Synthesis of Bluetongue Virus Chimeric VP3 Molecules and Their Interactions with VP7 Protein to Assemble into Virus Core-like Particles

Bluetongue virus (BTV) core-like particles (CLPs) are formed in the cytoplasm of insect cells when only two major proteins (VP3 and VP7) of the BTV core are expressed by baculovirus vectors (T. J. French and P. Roy, 1990,J. Virol.64, 1530–1536). We have recently reported that five small internal deletion mutants of VP3 form CLPs when provided with unmodified VP7 protein (D1–5; S. Tanaka and P. Roy, 1994,J. Virol.68, 2795–2802). To investigate whether foreign sequences can be inserted into VP3 and to determine their effect on CLP formation, three of these internal regions (D1, D2, and D5), as well as the carboxy terminus, were modified to create unique restriction enzyme sites, thereby replacing VP3 coding regions with shorter synthetic sequences. Each modified VP3 gene was used to generate baculovirus expression vectors (D1I, D2I, D5I, and VP3C, respectively). Other than the D5I mutant, the mutants formed CLPs when expressed in the presence of VP7. Subsequently, T7 tag epitopes were inserted into the D1I, D2I, and VP3C restriction sites and recombinant baculoviruses were generated to express chimeric VP3 proteins (VP3D1IT, VP3D2IT, and VP3CT). Each chimeric protein retained the ability to form CLPs when VP7 protein was provided. In another construction an immunogenic sequence representing a bovine leukemia virus (BLV) glycoprotein peptide was incorporated into the carboxy terminus of VP3 and the derived CLPs were used to raise antibodies that reacted with the BLV antigen. The results suggest that the VP3 molecule can accommodate foreign sequences at certain sites without perturbing their ability to form CLPs with VP7.

Circulating immune complexes in bovine leukemia virus (BLV)-infected cattle

Circulating immune complexes (ICs) were detected in the sera of bovine leukemia virus (BLV)-seropositive cattle. Immune complexes were precipitated in 2.5% polyethylene glycol (PEG) and further dissociated. Bovine leukemia virus antigens, IgG and IgM molecules were detected after solubilization in the presence of sodium dodecyl sulphate, and quantitated by enzyme-linked immunosorbent assay (ELISA) assays. Mean values of IgG and IgM in BLV-containing ICs did not significantly differ from those obtained from ICs originating from BLV-seronegative animals. However, differences were found in the composition of ICs from older BLV-positive animals as compared to those obtained from young animals. The ratio of IgG/IgM was 5.02 in animals aged 5–10 years, while this ratio was 11.66 in animals of less than 5 years of age and 10.19 in controls. This might indicate a possible increase in the contribution of IgM molecules to the structural composition of ICs in BLV-infected cattle as related to age or stage of infection.

Suppression of immunological responses in rabbits experimentally infected with bovine leukemia virus

Ten 2- to 4-month-old rabbits were inoculated subcutaneously with bovine leukemia virus (BLV)-infected bovine or sheep cells. By 6 weeks after inoculation all ten rabbits had converted to BLV antibody-positive, and BLV or BLV antigen was detected in lymphocytes from most of the rabbits tested, although there were few antigen-producing cells. Three rabbits showed continuous respiratory symptoms after infection and one died with pneumonia. Humoral immune responses against mouse serum were significantly suppressed in BLV-infected rabbits compared with non-infected control rabbits. The lymphocyte blastogenesis response was also suppressed in BLV-infected rabbits. At the time of necropsy, six rabbits showed pulmonary lesions; however, none of the BLV-infected rabbits had tumors during an observation period of over 1 year.